ES3038141T3 - Fluid sample processing cartridge - Google Patents

Abstract

The present invention relates to a fluid sample processing cartridge comprising: a substrate; a sample well formed in the substrate configured to receive a fluid sample volume and comprising a closure configured to selectively position itself over the sample well; a deformable fluid chamber supported on the substrate; a mixing well formed in the substrate; and an actuated mixing apparatus disposed within the mixing well and constructed and arranged to mix the contents of said mixing well.

Description

DESCRIPTION

Fluid sample processing cartridge

Field of invention

The subject matter of this disclosure relates to systems and methods for providing clinical and molecular diagnostics in an integrated multi-device that delivers sample-to-response results. Specifically, the disclosure relates to a sample-enabled cartridge containing reagents, buffers, and other procedural materials for performing a diagnostic assay or other procedure on the sample, and an instrument configured to independently process a plurality of such cartridges.

Background of the invention

A major challenge in the area of clinical and molecular diagnostics is the ability to have a "sample-to-answer" system that requires minimal sample handling and preparation and minimal requirements for trained clinical laboratory personnel. While many systems have been proposed, to date virtually no commercial systems adequately meet these requirements. The aspects of this disclosure provide such integrated, multi-system solutions. US 2013/331298 A1 discloses a disposable fluid-processing cartridge comprising a substrate, a sample well formed in the substrate, a lid member, several deformable fluid chambers, etc.

Summary of the invention

The present invention is defined in the appended claims. This disclosure describes molecular diagnostic procedures and compositions based on the detection of target analytes, including nucleic acids. The systems described herein are complete integrated sample-to-response systems, in contrast to current commercial systems that require some off-chip sample manipulation, which generally includes sample extraction (e.g., cell lysis) and sample preparation prior to detection. Accordingly, according to aspects of the current system, a sample is loaded onto a test platform, and the target analyte sample is extracted, amplified as required (e.g., when the target analyte is a nucleic acid, using polymerase chain reaction (PCR) techniques, although isothermal amplification procedures may also be used), and then detected using electrochemical detection, all on a microfluidic platform, generally referred to herein as a "multi-cartridge" or "fluid sample processing cartridge."

A particular advantage of this system is its ease of use and speed. For example, no more than two steps are required before sample introduction, making it user-friendly and eliminating the need for highly trained laboratory personnel. Another significant benefit is the speed of sample-to-results, which, in some implementations, is generally no more than approximately 45–90 minutes from sample introduction to the report of assay results, with most results reported in 60–70 minutes or less. This represents a significant advantage for both laboratories and clinicians who rely on rapid assays for diagnosis and the initiation of appropriate treatments. Furthermore, as explained below, the ability to run not only multiple highly multiplexed tests on a single cartridge but also to analyze multiple cartridges in a completely randomized manner is a significant advantage in a clinical laboratory setting. Another benefit of this system is its suitability for point-of-care (POC) diagnostics.

Accordingly, the aspects of this disclosure are directed to integrated systems that enable the detection of target analytes from samples.

The invention as claimed is a fluid sample processing cartridge comprising a substrate, a sample well formed in the substrate, a closure, a deformable fluid chamber supported on the substrate, a mixing well formed in the substrate, and an actuated mixing apparatus disposed within the mixing well. The sample well is configured to receive a fluid sample volume, and the closure is configured to selectively position itself over the sample well. The deformable fluid chamber is configured to contain a fluid therein when in an undeformed state and to collapse upon the application of an external compressive force to expel at least a portion of the fluid from the fluid chamber. The deformable fluid chamber is in fluid communication with the sample well by means of a channel formed in the substrate. The mixing well is in fluid communication with the sample well via a channel formed in the substrate and comprises a first peripheral wall and a first floor that define a well and a fluid inlet snorkel extending upwards along one side of the first peripheral wall, extending from the channel connecting the mixing well to the sample well, and terminating below an upper edge of the first peripheral wall. The driven mixing apparatus is constructed and arranged to mix the contents of the mixing well.

According to other aspects of the invention, the fluid inlet snorkel extends to an outer surface of the first peripheral wall and terminates in an opening formed in the first peripheral wall.

According to other aspects of the invention, the sample well comprises a second peripheral wall and a second floor defining a well and a fluid inlet snorkel extending upwards along one side of the second peripheral wall and terminating below an upper edge of the second peripheral wall.

According to other aspects of the invention, the mixing well further comprises an outlet port comprising one or more openings formed in the floor of the mixing well, wherein the floor tapers downwards towards the outlet port.

According to other aspects of the invention, the driven mixing apparatus comprises a first impeller rotatably arranged within the mixing well and a gear configured to be driven by a conjugate gear of an instrument into which the liquid sample processing cartridge is inserted and to rotate the first impeller when engaged by the conjugate gear.

According to other aspects of the invention, the sample processing cartridge further comprises a lysis chamber containing a plurality of lysis microspheres, the lysis chamber being formed in the substrate and disposed along the channel connecting the mixing well and the sample well, whereby the fluid flowing from the sample well to the mixing well will flow through the lysis chamber, and a microsphere mixer disposed at least partially within the lysis chamber and constructed and arranged to stir the lysis microspheres and the fluid flowing through the lysis chamber.

According to other aspects of the invention, the sample processing cartridge further comprises a first optical interface comprising an enlarged portion of the channel connecting the lysis chamber to the sample well and a second optical interface comprising an enlarged portion of the channel connecting the lysis chamber to the mixing well.

According to other aspects of the invention, the microsphere mixer comprises a motor mounted within the substrate and a second impeller arranged within the lysis chamber and mounted on an output shaft of the motor.

According to other aspects of the invention, the lysis chamber includes a fluid inlet and a fluid outlet and further comprises a mesh filter disposed over each of the fluid inlet and fluid outlet and configured to retain the lysis microspheres within the lysis chamber.

According to other aspects of the invention, the sample processing cartridge further comprises a pressure port formed in the substrate and configured to couple the substrate to an external fluid pressure source and a channel formed in the substrate connecting the pressure port to the sample well.

According to other aspects of the invention, the sample processing cartridge further comprises a waste chamber formed in the substrate, the waste chamber being in fluid communication with the mixing well by means of a channel formed in the substrate, a fluid outlet port formed in the substrate, the fluid outlet port being in fluid communication with the mixing well by means of a channel formed in the substrate, a first externally actuated control valve disposed within the substrate and constructed and arranged to selectively permit or prevent fluid flow from the mixing well to the waste chamber, and a second externally actuated control valve disposed within the substrate and constructed and arranged to selectively permit or prevent fluid flow from the mixing well to the fluid outlet port.

According to other aspects of the invention, the sample processing cartridge further comprises a capture chamber arranged along a channel connecting the mixing well and the waste chamber

According to other aspects of the invention, the sample processing cartridge further comprises a passive valve assembly disposed within the substrate and a pressure port formed in the substrate and in pressure communication with the passive valve assembly via a pressure conduit formed in the substrate. The passive valve assembly is constructed and arranged to close and prevent fluid flow from the mixing well when the pressure within the mixing well is not greater than a threshold pressure, and to open and permit fluid flow from the mixing well when the pressure within the mixing well rises above the threshold pressure. When the pressure port is closed, the pressure within the mixing well is allowed to reach the threshold pressure, which will open the passive valve assembly and permit fluid flow from the mixing well. When the pressure port is open, the pressure within the mixing well cannot reach the threshold pressure, and therefore the passive valve assembly is closed.

According to other aspects of the invention, the sample processing cartridge further comprises a lancet ampoule associated with the deformable fluid chamber. The lancet ampoule is connected or can be connected to the associated deformable fluid chamber and contains a microsphere retained within the lancet ampoule by a breakable septum. The lancet ampoule is configured to collapse upon the application of an external compressive force, thereby pushing the microsphere through the breakable septum.

According to other aspects of the invention, the sample processing cartridge further comprises an outer envelope that externally encloses at least a portion of the cartridge.

According to other aspects of the invention, the sample processing cartridge further comprises a plurality of deformable fluid chambers, and each of the fluid chambers contains one or more substances selected from the group consisting of a lysis buffer, a washing buffer, an oil, a rehydration buffer, target capture microspheres, and a bonding buffer.

According to other aspects of the invention, the sample processing cartridge further comprises a first fluid outlet port formed in the substrate, the first fluid outlet port being in fluid communication with the mixing well by means of a channel formed in the substrate, a second fluid outlet port formed in the substrate, and at least two deformable fluid chambers. One of the two deformable fluid chambers is in fluid communication with the mixing well by means of a channel formed in the substrate, and the other of the two deformable fluid chambers is in fluid communication with the second fluid outlet port by means of a channel formed in the substrate that is different from the channel that connects the first fluid outlet port to the mixing well.

According to other aspects of the invention, the deformable fluid chamber in fluid communication with the mixing well contains a lysis buffer, a washing buffer, target capture microspheres or a bonding buffer, and the deformable fluid chamber in fluid communication with the second fluid outlet contains an oil or a rehydration buffer.

Other non-claimed aspects of the invention are implemented in a fluid sample processing cartridge comprising a sample preparation module comprising a reaction module. The sample preparation module comprises a substrate, a sample well formed in the substrate and configured to receive a sample volume of fluid, a closure configured to be selectively placed over the sample well, a first deformable fluid chamber supported on the substrate and configured to contain a fluid therein when in an undeformed state and to collapse upon application of an external compressive force to expel at least a portion of the fluid from the first fluid chamber, the first deformable fluid chamber being in fluid communication with the sample well by means of a channel formed in the substrate, a mixing well formed in the substrate, the mixing well being in fluid communication with the sample well by means of a channel formed in the substrate, an actuated mixing apparatus disposed within the mixing well and constructed and arranged to mix the contents of the mixing well, and a first fluid outlet port formed in the substrate, the first fluid outlet port being in fluid communication with the mixing well by means of a channel formed in the substrate. The reaction module is attached to the sample preparation module and configured to receive fluid from the sample preparation module via a fluid outlet port formed in the sample preparation module. The reaction module comprises a top plate with an upper surface, a raised wall that at least partially surrounds the upper surface and is in fluid-sealed contact with a surface of the sample preparation module to form an interstitial space between the upper surface and the surface of the sample preparation module, a sample chamber fluidly coupled to the first fluid outlet port of the sample preparation module, a reagent chamber, and a detection chamber, and a fluidic processing panel coupled to a lower surface of the top plate and defining a reaction and processing space between the fluidic processing panel and the top plate. The reaction and processing space opens, or can be opened, to the sample chamber, the reaction chamber, and the detection chamber.

According to other unclaimed aspects of the invention, the reaction module includes an inlet port through which the fluid sample enters the sample chamber and includes a gap between the first fluid outlet port of the sample preparation module and the inlet port of the sample chamber, the gap being open to the interstitial space.

According to other unclaimed aspects of the invention, the first fluid outlet port of the sample preparation module comprises an outlet channel formed through a truncated conical nozzle.

According to other unclaimed aspects of the invention, the reaction module of the fluid sample processing cartridge further comprises an array of electrosensors arranged in each detection chamber.

According to other unclaimed aspects of the invention, the top plate of the reaction module further comprises one or more bubble traps, each bubble trap comprising a bubble-catching bell open to the reaction and processing space and a vent opening open to the interstitial space.

According to other unclaimed aspects of the invention, the sample preparation module further comprises a second deformable fluid chamber supported on the substrate and configured to contain a fluid therein when in an undeformed state and to collapse upon the application of an external compressive force to expel at least a portion of the fluid from the fluid chamber, and a second fluid outlet port formed in the substrate. The second fluid outlet port is in fluid communication with the second deformable fluid chamber by means of a channel formed in the substrate, and the reaction and processing space is fluidly coupled to the second fluid outlet port of the sample preparation module.

According to other unclaimed aspects of the invention, the mixing well comprises a peripheral wall and a floor defining a well and a fluid inlet snorkel extending upwards along one side of the peripheral wall, extending from the channel connecting the mixing well to the sample well and terminating below an upper edge of the peripheral wall.

According to other unclaimed aspects of the invention, the fluid inlet snorkel extends to an outer surface of the peripheral wall and terminates in an opening formed in the peripheral wall.

According to other unclaimed aspects of the invention, the mixing well further comprises an outlet port comprising one or more openings formed in the floor of the mixing well, and the floor tapers downwards towards the outlet port.

According to other unclaimed aspects of the invention, the powered mixing apparatus comprises a first impeller rotatably arranged within the mixing well and a gear configured to be driven by a conjugate gear of an instrument into which the liquid sample processing cartridge is inserted and to rotate the first impeller when engaged by the conjugate gear.

According to other unclaimed aspects of the invention, the sample preparation module further comprises a lysis chamber comprising a plurality of lysis microspheres, the lysis chamber being formed in the substrate and arranged along the channel connecting the mixing well and the sample well, whereby the fluid flowing from the sample well to the mixing well will flow through the lysis chamber, and a microsphere mixer disposed at least partially within the lysis chamber and constructed and arranged to stir the lysis microspheres and the fluid flowing through the lysis chamber.

According to other unclaimed aspects of the invention, the microsphere mixer comprises a motor mounted within the substrate and a second impeller disposed within the lysis chamber and mounted on an output shaft of the motor.

According to other unclaimed aspects of the invention, the fluid sample processing cartridge further comprises a first optical interface comprising an enlarged portion of the channel connecting the lysis chamber to the sample well and a second optical interface comprising an enlarged portion of the channel connecting the lysis chamber to the mixing well.

According to other unclaimed aspects of the invention, the lysis chamber includes a fluid inlet and a fluid outlet and further comprises a mesh filter disposed over each of the fluid inlet and fluid outlet and configured to retain the lysis microspheres within the lysis chamber.

According to other unclaimed aspects of the invention, the sample preparation module further comprises a pressure port formed in the substrate and configured to couple the substrate to an external fluid pressure source and a channel formed in the substrate connecting the pressure port to the sample well.

According to other unclaimed aspects of the invention, the sample preparation module further comprises a waste chamber formed in the substrate, the waste chamber being in fluid communication with the mixing well by means of a channel formed in the substrate, a first externally actuated control valve disposed within the substrate and constructed and arranged to selectively permit or prevent fluid flow from the mixing well to the waste chamber, and a second externally actuated control valve disposed within the substrate and constructed and arranged to selectively permit or prevent fluid flow from the mixing well to the outlet port.

According to other unclaimed aspects of the invention, the sample preparation module further comprises a capture chamber arranged along a channel connecting the mixing well and the waste chamber.

According to other unclaimed aspects of the invention, the sample preparation module further comprises a passive valve assembly disposed within the substrate and constructed and arranged to close and prevent fluid flow from the mixing well when the pressure within the mixing well is not greater than a threshold pressure, and to open and permit fluid flow from the mixing well when the pressure within the mixing well rises above the threshold pressure, and a pressure port formed in the substrate and in pressure communication with the passive valve assembly via a pressure conduit formed in the substrate. When the pressure port is closed, the pressure within the mixing well is allowed to reach the threshold pressure, which will open the passive valve assembly and permit fluid flow from the mixing well. When the pressure port is open, the pressure within the mixing well cannot reach the threshold pressure, and therefore the passive valve assembly is closed.

According to other unclaimed aspects of the invention, the sample preparation module further comprises a lancet ampoule associated with the deformable fluid chamber. The lancet ampoule is connected or can be connected to the associated deformable fluid chamber and contains a microsphere retained within the lancet ampoule by a breakable septum. The lancet ampoule is configured to collapse upon the application of an external compressive force, thereby pushing the microsphere through the breakable septum.

According to other unclaimed aspects of the invention, an outer envelope externally encloses at least a portion of the cartridge.

According to other unclaimed aspects of the invention, the sample preparation module further comprises a plurality of deformable fluid chambers, and each of the fluid chambers contains a substance selected from the group consisting of a lysis buffer, a washing buffer, an oil, a rehydration buffer, target capture microspheres, and a bonding buffer.

Other unclaimed aspects of the invention are embodied in an instrument configured to process a fluid sample processing cartridge, which includes a deformable fluid chamber supported on a flat substrate and configured to contain a fluid therein when in an undeformed state and to collapse upon the application of an external compressive force to expel at least a portion of the fluid from the fluid chamber. The instrument comprises a cartridge carrier assembly, a cartridge carrier assembly being configured to receive and retain a fluid sample processing cartridge inserted into the instrument. A heating and control assembly is disposed contiguous to the cartridge carrier assembly and is configured to move relative to the cartridge carrier assembly between a first position not in functional contact with the cartridge carried within the cartridge carrier assembly and a second position in functional contact with the cartridge carried within the cartridge carrier assembly. One or more sets of movable magnets are each mounted to move relative to the cartridge independently of the heating and control assembly, between a first position that applies substantially no magnetic force to the cartridge and a second position that applies magnetic force to the corresponding discrete portions of the cartridge. A cam block assembly is configured for driven motion and is functionally coupled to the heating and control assembly to convert the driven motion of the cam block assembly into motion of the heating and control assembly relative to the cartridge carrier assembly between the first and second positions of the heating and control assembly. The cam block assembly is functionally coupled to one or more sets of movable magnets to convert the driven motion of the cam block assembly into motion of each set of magnets relative to the cartridge carrier assembly between the first and second positions of the magnet sets. A deformable chamber compression assembly is configured to selectively apply an external compressive force to the deformable fluid chamber to collapse the deformable chamber and expel at least a portion of the fluid from the fluid chamber.

According to other unclaimed aspects of the invention, the heating and control assembly comprises one or more heater assemblies configured to apply a thermal gradient to corresponding discrete portions of the cartridge when the heating and control assembly is in the second position, and a connector plate including one or more electrical connector elements configured to effect an electrical connection between the instrument and the cartridge when the heating and control assembly is in the second position.

According to other unclaimed aspects of the invention, the deformable chamber compression assembly comprises a cam follower plate configured for driven movement in a first direction that is generally parallel to the substrate plane, and a compression mechanism associated with the deformable cartridge chamber and configured to apply a force that compresses the chamber against the substrate by movement in a second direction having a component that is generally normal to the substrate plane. The cam follower plate is functionally coupled to the compression mechanism to convert the movement of the cam follower plate in the first direction into movement of the compression mechanism in the second direction, thereby applying an external compressive force to the chamber.

According to other unclaimed aspects of the invention, the instrument further comprises a pneumatic pump and a pneumatic port connected to the pneumatic pump, wherein the pneumatic port is configured to couple the pneumatic pump to a pressure port of the fluid sample processing cartridge when the cartridge is inserted into the instrument.

According to other unclaimed aspects of the invention, the instrument further comprises an optical detector configured to detect fluid flow through a portion of the fluid sample processing cartridge.

According to other unclaimed aspects of the invention, the fluid sample processing cartridge includes a driven mixing apparatus comprising a drive gear, and the instrument further comprises a mixing motor assembly comprising a driven drive gear. The mixing motor is movable between a first position in which the drive gear is not engaged with the drive gear of the driven mixing apparatus and a second position in which the drive gear is functionally engaged with the drive gear to drive the driven mixing apparatus. The cam block assembly is functionally engaged with the mixing motor assembly to convert the driven motion of the cam block assembly into motion of the mixing motor assembly between the first position of the mixing motor assembly and the second position of the mixing motor assembly.

According to other unclaimed aspects of the invention, the instrument further comprises a heater cooling assembly comprising a fan and a cooling tube configured to direct airflow from the fan to a portion of one of the heater assemblies.

According to other unclaimed aspects of the invention, the cartridge carrier assembly comprises a cartridge holder configured to hold a cartridge inserted therein, a cartridge latch deflected to a cartridge latching position and configured to latch a cartridge inserted into the cartridge holder to retain the cartridge within the cartridge holder, and a cartridge ejection mechanism configured to automatically push a cartridge at least partially out of the cartridge holder when the cartridge latch is released from a cartridge latching position.

According to other unclaimed aspects of the invention, the heating and control assembly comprises a support plate on which one or more heater assemblies and the connector plate are supported. The support plate is mounted in a restraining configuration that prevents horizontal movement of the support plate but allows vertical movement of the support plate to permit movement of the heating and control assembly between its first and second positions.

According to other unclaimed aspects of the invention, the heating and control assembly heater assemblies comprise a resistive heating element attached to the connector plate and a heat diffuser comprising a thermally conductive material thermally coupled to the resistive heating element.

According to other unclaimed aspects of the invention, one of the heater assemblies of the heating and control assembly comprises a thermoelectric element, a heat diffuser comprising a thermally conductive material thermally coupled to the thermoelectric element, and a thermal diffuser including a panel that is in thermal contact with the thermoelectric element and a plurality of heat dissipation rods.

According to other unclaimed aspects of the invention, the electrical connector elements of the connector plate of the heating and control assembly comprise a plurality of connector pin arrays, each connector pin array comprising a plurality of pogo pins.

According to other unclaimed aspects of the invention, one of the movable magnet assemblies comprises a magnet holder mounted on a spindle so that it can rotate about the spindle between the first position and the second position of the magnet assembly, a magnet supported on the magnet holder, an actuator attachment extending from the magnet holder, and a torsion spring configured to deflect the magnet holder to a rotational position corresponding to the first position of the magnet assembly.

According to other unclaimed aspects of the invention, one of the movable magnet assemblies comprises a magnet support frame mounted on a spindle so that it can rotate about the spindle between the first position and the second position of the magnet assembly, a magnet array disposed within the magnet support frame, a focusing magnet disposed within an opening formed in the magnet support frame and configured to focus the magnetic forces of the magnet array, an actuator attachment extending from the magnet support frame, and a torsion spring configured to deflect the magnet support frame to a rotational position corresponding to the first position of the magnet assembly.

According to other unclaimed aspects of the invention, the cam block assembly is functionally coupled to each movable magnet assembly by means of a magnet actuator coupled in a portion thereof to the cam block assembly so that it can be moved by driven movement of the cam block assembly and which includes a tab configured to engage with the actuator attachment of each magnet assembly as the magnet actuator moves with the cam block assembly to cause the corresponding rotation of the magnet assembly from the first position to the second position.

According to other unclaimed aspects of the invention, the cam block assembly comprises a cam frame, a cam block motor coupled to the cam frame and configured to effect the driven movement of the cam frame, and first and second cam rails attached to the cam frame. Each cam rail has two cam grooves. The cam block assembly is functionally coupled to the heating and control assembly by cam followers extending from the heating and control assembly into the cam grooves, such that the movement of the cam frame and cam rails with respect to the heating and control assembly causes corresponding relative movement between the cam followers and the cam grooves to move the cam followers between the respective first segments of the cam grooves corresponding to the first position of the heating and control assembly and the respective second segments of the cam grooves corresponding to the second position of the heating and control assembly.

According to other unclaimed aspects of the invention, the cam frame comprises a first longitudinal rail extending along one side of the heating and control assembly, a second longitudinal rail extending along an opposite side of the heating and control assembly, and a transverse rail extending between the first and second longitudinal rails. Each cam rail is attached to one of the first and second longitudinal rails.

According to other unclaimed aspects of the invention, the compression mechanism of the deformable chamber compression assembly comprises a cam arm having a cam surface and mounted so that it can pivot about one end of the cam arm and a compression pad disposed at an opposite end of the cam arm, wherein the cam arm can pivot between a first position in which the compression pad does not come into contact with the associated deformable chamber and a second position in which the compression pad applies a compressive force to the associated deformable chamber to at least partially collapse the chamber.

According to other unclaimed aspects of the invention, the deformable chamber compression assembly further comprises a cam arm plate, and the cam arm of the compression mechanism is pivotally mounted within a groove formed in the cam arm plate for pivoting the cam arm relative to the cam arm plate. The cam surface of the cam arm projects out of the groove above a surface of the cam arm plate. The cam follower plate is functionally coupled to the compression mechanism by a cam follower element of the cam follower plate that engages with the cam surface of the compression mechanism during the movement of the cam follower plate relative to the cam arm plate to cause the cam arm to pivot from its first position to its second position.

According to other unclaimed aspects of the invention, the cam follower plate comprises a cam groove receiving the cam surface of the cam arm projecting above the surface of the cam arm plate, and the cam follower element comprises a follower shoulder disposed within the cam groove that makes contact with the cam surface as the cam follower plate moves relative to the cam arm plate to cause the cam arm to pivot from its first position to its second position.

According to other unclaimed aspects of the invention, the instrument further comprises a plurality of compression mechanisms, each comprising a cam arm pivotally mounted within a groove formed in the cam arm plate and a cam arm surface, and the cam follower plate comprising a plurality of cam grooves, each cam groove being associated with at least one of the compression mechanisms and each cam groove including a follower lobe disposed within the cam groove that makes contact with the cam surface of the associated compression mechanism as the cam follower plate moves relative to the cam arm plate to cause the cam arm of the associated compression mechanism to pivot from its first position to its second position.

According to other unclaimed aspects of the invention, the sample processing cartridge includes a plurality of deformable fluid chambers, and the deformable chamber compression assembly comprises a plurality of compression mechanisms. Each compression mechanism is associated with one of the deformable fluid chambers, and the cam follower plate is functionally coupled to the compression mechanisms to convert the movement of the cam follower plate in the first direction into the movement of each of the compression mechanisms in the second direction, thereby applying an external compressive force to each of the associated chambers in a specified sequence.

According to other unclaimed aspects of the invention, the fluid sample processing cartridge includes an externally actuated control valve configured to selectively control fluid flow by permitting fluid flow through the valve when not externally actuated and preventing fluid flow through the valve when externally actuated. The instrument further comprises a valve actuator compression mechanism associated with the externally actuated control valve of the sample processing cartridge and configured to actuate the associated externally actuated control valve by movement in a second direction having a component that is generally normal to the substrate plane. The cam follower plate is functionally coupled to the valve actuator compression mechanism to convert the movement of the cam follower plate in the first direction into movement of the valve actuator compression mechanism in the second direction, thereby actuating the associated externally actuated control valve.

Other characteristic features and features of the subject matter of this disclosure, as well as the operating procedures, the functions of the related structural elements and the combination of parts, and the manufacturing economies, will become more evident after consideration of the following description and any appended claims with reference to the accompanying drawings, all of which form part of this descriptive memorandum.

Brief description of the drawings

The accompanying drawings, which are incorporated herein and form part of the descriptive report, illustrate various embodiments of the subject matter of this disclosure. In the drawings, similar reference numbers indicate identical or functionally similar elements.

FIG. 1 is a top perspective view of a multi-cartridge that performs aspects of the present invention.

FIG. 2 is a top plan view of the multiple cartridge.

Figure 3 is a top plan view of the multi-cartridge with annotated identification markers. Figure 4 is an exploded perspective view of the multi-cartridge.

FIG. 5 is a partial cross-sectional perspective view of a deformable fluid compartment (or ampoule) of the multiple cartridge.

Figure 6 is a perspective detail of a sample well and sample cap of the multi-cartridge. Figure 7 is a perspective cross-sectional view of the sample well along line 7-7 in Figure 2.

FIG. 8A is a perspective detail of a mixing well and mixer of the multi-cartridge.

FIG. 8B is a perspective detail of an alternate mixing well of the multi-cartridge.

FIG. 8C is a plan view from above of the mixing well in FIG. 8C.

FIG. 9A is a cross-sectional view of the mixing well and mixing along line 9-9 in FIG. 2.

FIG. 9B is a cross-sectional view of the reciprocating mixing well of FIGS. 8B and 8C and an reciprocating mixer arranged therein.

FIG. 10 is a perspective detail of a passive multi-cartridge valve.

FIG. 11 is a perspective cross-sectional view of the passive valve along line 11-11 in FIG. 2.

FIG. 12 is a perspective cross-sectional view of a lysis chamber and a microsphere mixer along line 12-12 in FIG. 2.

FIG. 13 is a perspective cross-sectional view of an active valve assembly along line 13-13 in FIG. 2.

FIG. 14 is a perspective cross-sectional view of the active valve, in which the valve is actuated by an external valve actuator.

Figure 15 is a top plan view of a sample preparation module of the multi-cartridge. Figures 16-23 show top plan views of the sample preparation module, each illustrating a different step in a sample preparation procedure performed within the module.

FIG. 24 is a perspective view from above of a top plate of a multi-cartridge reaction module.

FIG. 25 is a perspective view from below of the top plate.

FIG. 26 is a plan view from above of the top plate.

FIG. 27 is a plan view from below of the top plate.

FIG. 28 is a perspective cross-sectional view of the reaction module along line 28-28 in FIG. 24.

FIG. 29 is a perspective cross-sectional view of the reaction module along line 29-29 in FIG. 24.

FIG. 30 is a perspective detail of a fluid inlet of the reaction module.

FIG. 31 is a partial cross-sectional view along line 31-31 in FIG. 26.

FIG. 32 is a front view of an instrument.

FIG.33 is a front perspective view of an instrument control console.

FIG.34 is a front perspective view of an instrument processing module.

FIG. 35 is a front perspective view of the processing module with one side wall of the module removed to show the internal components of the processing module.

FIG. 36 is a rear perspective view of the processing module with one side wall and the rear wall of the module removed to show the internal components of the processing module.

FIG. 37 is a front perspective view of the processing module with one side wall and one rear wall of the module removed and with a processing bay of the processing module in the module's exploded view.

FIG.38 is a front perspective view from the right side of a processing bay.

FIG.39 is a front perspective view from the left side of the processing bay.

FIG.40 is a rear perspective view from the right side of the processing bay.

Figure 41 is a front perspective view from the right side of the processing bay. Figure 42 is an exploded perspective view of the cartridge processing assembly within the processing bay.

FIG. 43 is an exploded perspective view of a heating and control assembly of the cartridge processing assembly.

FIG. 44 is a top plan view of a PCB connector and magnets of the cartridge processing assembly heating and control unit.

FIG. 45 is an exploded perspective view of a Peltier heater assembly for sensing the heating and control unit.

FIG. 46 is an exploded perspective view of a cartridge carrier assembly of the cartridge processing assembly.

FIG. 47 is an exploded perspective view of the cam frame assembly of the cartridge processing assembly.

FIG. 48 is a perspective cross-sectional view of the cam frame and magnet actuator of the cartridge processing assembly.

FIG. 49A is a top perspective view of a sample preparation magnet assembly of the cartridge processing assembly.

FIG. 49B is a top perspective view of a cartridge magnet assembly from the cartridge processing assembly.

FIG. 50A is a perspective view of a mixing motor assembly of the cartridge processing assembly.

FIG. 50B is an exploded perspective view of the mixing motor assembly.

FIG. 51 is an exploded prospective view of a processing bay blister compression mechanism assembly.

FIG. 52 is a partial plan view from below of a cam arm plate showing compression pads of a compression mechanism array.

FIG. 53 is a top perspective view of the matrix compression mechanisms isolated from the cam arm plate.

FIG. 54 is a perspective view from below of the matrix compression mechanisms isolated from the cam arm plate.

Figure 55A is an exploded perspective view of a single fluid ampoule compression mechanism. Figure 55B is an exploded perspective view of a single lancet ampoule compression mechanism. Figure 55C is an exploded perspective view of a single valve actuator compression mechanism.

FIG. 56 is a plan view from below of a cam follower plate of the blister compression mechanism assembly.

FIG. 57 is a perspective view from below of the cam follower plate.

Figure 58 is a plan view from below of a fluidic processing panel of the reaction module. Figure 59 is a plan view from above of the fluidic processing panel.

FIG. 60 is a flow diagram illustrating an exemplary procedure that can be performed on the fluidic processing panel.

Detailed description of the invention

While aspects of the subject matter of this disclosure can be embodied in a variety of forms, the following description and accompanying drawings are intended merely to disclose some of these forms as specific examples of the subject matter. Accordingly, the subject matter of this disclosure is not intended to be limited to the forms or embodiments so described and illustrated.

Unless otherwise defined, all technical terms, notations, and other technical terms or terminology used herein have the same meaning as commonly understood by a person skilled in the art to which this disclosure pertains.

Unless otherwise stated or the context suggests otherwise, as used herein, "a" or "an" means "at least one" or "one or more".

This description may use spatial and/or orientational terms when describing the position and/or orientation of a component, apparatus, location, feature, or portion thereof. Unless specifically stated or otherwise dictated by the context of the description, such terms, including, but not limited to, top, bottom, above, below, under, over, upper, lower, to the left of, to the right of, opposite, behind, beside, adjacent, between, horizontal, vertical, diagonal, longitudinal, transverse, radial, axial, etc., are used for convenience in reference to such component, apparatus, location, feature, or portion thereof in the drawings and are not intended to be limiting.

Furthermore, unless otherwise stated, any specific dimension mentioned in this description is merely representative of an exemplary implementation of a device that performs aspects of the invention and is not intended to be limiting.

Related requests

This application is related to U.S. patent application serial no. 14/062.860 (U.S. patent application publication no. 2014-0322706) and U.S. patent application serial no. 14/062.865 (U.S. patent application publication no. 2014-0194305).

Introduction

In general, the system includes two components: the multi-cartridge, into which the sample is loaded and which contains various reagents, buffers, and other processing materials to perform the desired assay or other procedure, and the processing instrument into which the cartridge is inserted to perform sample processing and final detection of the target analytes.

In various embodiments, the microfluidic platform relies on the formation of micronanoparticles and the ability to transport, fuse, mix, and/or process these nanodroplets independently. In various embodiments, such micronanoparticle operations are performed using electrical control of surface tension (i.e., electrowetting). Generally, liquid samples are contained within a microfluidic device, known as a processing module, between two parallel plates. One plate, called the fluidic processing panel, has drive electrodes etched onto its surface, while the other plate contains either etched electrodes or a single continuous flat electrode that is grounded or tuned to a reference potential ("biplanar electrowetting"). A hydrophobic insulator covers the electrodes, and an electric field is generated between the electrodes on the opposing plates. This electric field creates a surface tension gradient that causes a nanodroplet overlapping the driven electrode to move toward that electrode. In some embodiments, the active electrowetting electrodes can be contiguous and in the same plane as the neighboring ground reference electrode, which is called "coplanar electrowetting." Through appropriate electrode arrangement and control, a nanodroplet can be transported by successively transferring it between contiguous electrodes. Stamped electrodes can be arranged in a two-dimensional array to allow the transport of a nanodroplet to any location covered by that array. The space surrounding the nanodroplets can be filled with a gas such as air or an immiscible fluid such as oil, with immiscible oils being preferred.

As nanodroplets containing the target analytes move across the surface, they can pick up reagents and buffers. For example, when dry reagents are placed on the surface (generally described herein as a printed circuit board, although, as will be appreciated by those skilled in the art, additional surfaces can be used), a nanodroplet moving across that area will pick up and dissolve the reagent for use in a biological procedure, such as PCR amplification. Furthermore, as described in more detail below, the addition of buffers and other reagents, such as wash buffers, from a sample preparation module located above the substrate allows for the targeted addition of these reagents, as well as the preparation of the sample (e.g., lysis, purification, dissolution, etc.) before transferring the sample to the microfluidic platform.

Aspects of the present invention also involve the use of electrochemical detection of analytes of interest. Suitable electrochemical detection systems are described in U.S. Patent Nos. 4,887,455; 5,591,578; 5,705,348; 5,770,365; 5,807,701; 5,824,473; 5,882,497; 6,013,170; 6,013,459; 6,033,601; 6,063,573; 6,090,933; 6,096,273; 6,180,064; 6,190,858; 6,192,351; 6,221,583; 6,232,062; 6,236,951; 6,248,229; 6,264,825; 6,265,155; 6,290,839; 6,361,958; 6,376,232; 6,431,016; 6,432,723; 6,479,240; 6,495,323; 6,518,024; 6,541,617; 6,596,483; 6,600,026; 6,602,400; 6,627,412; 6,642,046; 6,655,010; 6,686,150; 6,740,518; 6,753,143; 6,761,816; 6,824,669; 6,833,267; 6,875,619; 6,942,771; 6,951,759; 6,960,467; 6,977,151; 7,014,992; 7,018,523; 7,045,285; 7,056,669; 7,087,148; 7,090,804; 7,125,668; 7,160,678; 7,172,897; 7,267,939; 7,312,087; 7,381,525; 7,381,533; 7,384,749; 7,393,645; 7,514,228; 7,534,331; 7,560,237; 7,566,534; 7,579,145; 7,582,419; 7,595,153; 7,601,507; 7,655,129; 7,713,711; 7,759,073; 7,820,391; 7,863,035; 7,935,481; 8,012,743; 8,114,661 and U.S. Publication No. 2012/01 81 186.

In various embodiments, the processed target analyte nanodroplets are transported to a detection zone in the fluidic processing panel, where they are specifically captured on individual detection electrodes, using systems described in numerous prior patents with specific reference to U.S. Patent Nos. 7,160,678, 7,393,645 and 7,935,481. This detection system is based on the use of marker probes (in the case of nucleic acids) containing electrochemically active markers, such that the presence of the target analyte results in a positive signal, enabling the detection of the pathogen, disease state, etc.

Samples

The disclosure aspects provide systems and procedures for the detection of target analytes in samples to diagnose a disease or infection caused by pathogens (e.g., bacteria, viruses, fungi, etc.). As those skilled in the art will appreciate, the sample solution can comprise any number of things, including, but not limited to, body fluids (including, but not limited to, blood, urine, serum, plasma, cerebrospinal fluid, lymph, saliva, nasopharyngeal swabs, anal and vaginal secretions, feces, tissue samples including tissues suspected of containing cancer cells, sweat, and semen from virtually any organism, mammalian samples being preferred, and human samples being particularly preferred); environmental samples (including, but not limited to, air, agricultural, water, and soil samples, environmental swabs, and other collection kits); samples of microbiological warfare agents; Food and beverage samples, research samples (i.e., in the case of nucleic acids, the sample may be the product of an amplification reaction, including both target and signal amplification, as generally described in WO/1999/037819, such as a PCR amplification reaction); purified samples, such as purified genomic DNA, RNA, proteins, etc.; raw samples (bacteria, viruses, genomic DNA, etc.); as those skilled in the technique will appreciate, virtually any experimental manipulation may have been performed on the sample.

The multi-cartridge can be used to detect target analytes in patient samples. "Target analyte" or "analyte" or grammatical equivalents herein mean any molecule or compound to be detected that can bind to a binding species, as defined below. Suitable analytes include, but are not limited to, small chemical molecules such as chemicals or contaminants, or environmental or clinical biomolecules, including, but not limited to, pesticides, insecticides, toxins, therapeutic and abused drugs, hormones, antibiotics, antibodies, organic materials, etc. Suitable biomolecules include, but are not limited to, proteins (including enzymes, immunoglobulins, and glycoproteins), nucleic acids, lipids, lectins, carbohydrates, hormones, whole cells (including prokaryotic cells (such as pathogenic bacteria) and eukaryotic cells, including mammalian tumor cells), viruses, spores, etc.

In one embodiment, the target analyte is a protein ("target protein"). As those skilled in the art will appreciate, there are a large number of possible protein target analytes that can be detected using the present invention. "Proteins" or grammatical equivalents herein mean proteins, oligopeptides and peptides, derivatives and analogues, including proteins containing non-natural amino acids and amino acid analogues, and peptidomimetic structures. The side chains may be in the (R) or (S) configuration. In a preferred embodiment, the amino acids are in the (S) or (L) configuration. As discussed below, when the protein is used as a binding ligand, it may be desirable to use protein analogues to slow degradation by sample contaminants. Particularly preferred target proteins include enzymes; drugs; cells; antibodies; antigens; cell membrane antigens and receptors (neural, hormonal, nutrient, and cell surface receptors) or their ligands.

In a preferred embodiment, the target analyte is a nucleic acid ("target nucleic acid"). The present system is used in the diagnosis of specific pathogens exogenous to a patient, such as bacteria and viruses, as well as in the diagnosis of genetic diseases, such as single nucleotide polymorphisms (SNPs) that cause diseases (e.g., cystic fibrosis) or are present in diseases (e.g., tumor mutations).

As those skilled in the art will appreciate, the present invention is based on both target nucleic acids and other nucleic acid components such as capture probes and marker probes used in the detection of target nucleic acids. "Nucleic acid" or "oligonucleotide" or grammatical equivalents herein means at least two nucleotides covalently linked to each other. A nucleic acid will generally contain phosphodiester bonds, although in some cases, as explained below, nucleic acid analogues such as primers or probes may be included, which may have alternative backbones, comprising, for example, phosphoramide (Beaucage et al., Tetrahedron 49(10).T 925 (1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinz et al., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986); Sawai et al., Chem. Left. 805 (1984); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); and Pauwelset al., Chemica Scripta 26: 141 91986)), phosphorothioate (Maget al., Nucleic Acids Res. 19: 1437 (1991); and U.S. patent no. 5,644,048), phosphorodithioate (Briue et al., J. Am. Chem. Soc. 111:2321 (1989), O-methylphosphoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press) and peptidonucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc.

114:1895 (1992); Meieret al.,Chem. Int. Ed. Engl. 31: 1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature 380:207 (1996)). Other analogous nucleic acids include those with positive backbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); those with bicyclic structures, including blocked nucleic acids (Koshki et al., J. Am. Chem. Soc. 120:13252-3 (1998)); and nonionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141, and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988)). al., Nucleoside & Nucleotide 13: 1597 (1994); Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J. Biomolecular NMR 34: 17 (1994); Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones, including those described in U.S. patents 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. Y. S. Sanghui and P. Dan Cook. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp 169-176). Several nucleic acid analogues are described in Rawls, C & E News, June 2, 1997, page 35. These modifications of the ribose-phosphate backbone can be made to facilitate the addition of ETMs or to increase the stability and half-life of such molecules in physiological environments.

As those skilled in the art will appreciate, all these nucleic acid analogues can find use in the present invention, generally as capture probes and markers. Furthermore, mixtures of nucleic acids and natural analogues can be prepared (for example, in general, the marker probes contain a mixture of natural and synthetic nucleotides).

Nucleic acids may be single-stranded or double-stranded, as specified, or contain portions of both double-stranded and single-stranded sequence. Nucleic acids (particularly in the case of target nucleic acids) may be DNA, either genomic or cDNA, RNA, or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribonucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, isoguanine, etc. One embodiment uses isocytosine and isoguanine in nucleic acids designed to be complementary to other probes, rather than target sequences, as this reduces nonspecific hybridization, as generally described in U.S. patent 5,681,702. As used herein, the term "nucleoside" includes nucleotides, as well as nucleoside and nucleotide analogues, and modified nucleosides such as amino-modified nucleosides. In addition, "nucleoside" includes non-naturally occurring analogous structures. Thus, for example, individual units of a peptidonucleic acid, each containing a base, are referred to herein as a nucleoside.

As those skilled in the technique will appreciate, a large number of analytes can be detected using the present procedures; basically, any target analyte for which a binding ligand, described below, can be prepared can be detected using the disclosure procedures.

Therefore, disclosure systems are used in target analyte assays that then allow for disease diagnosis, prognosis, or treatment options based on the presence or absence of the target analytes. For example, disclosure systems find use in the diagnosis or characterization of pathogen infections (including bacteria (both Gram-positive and Gram-negative bacteria, and/or the ability to distinguish between them), viruses (including the presence or absence of viral nucleic acid, as well as viral isotypes, for example, in the case of hepatitis C virus (HCV) or respiratory viruses), fungal infections, antibiotic resistance, and genetic diseases (including cystic fibrosis, sickle cell anemia, etc.). The definition of genetic disease for the purposes of the present invention includes genetic conditions that do not necessarily cause disease but may result in alternative treatment options. For example, single nucleotide polymorphisms (SNPs) in many cytochrome P450 enzymes result in different processing of therapeutic drugs, such as in warfarin testing, where a patient may be diagnosed as a "slow," "normal," or "fast" processor, leading to different treatment options. dosage guidelines, or where a drug may be contraindicated for a particular patient based on the patient's genetics, or where the selection between two or more drugs is favored by knowledge of the patient's genetics.

Multi-cartridge

A multi-cartridge embodying aspects of the present invention is shown in Figures 1-4. As shown in Figure 4, the multi-cartridge comprises an assembly including a sample preparation module 70. The sample preparation module 70 includes various wells, inlet and outlet ports, fluid channels, mixing mechanisms, valves, and other components for receiving, conveying, interlacing, mixing, and performing other procedures on fluid sample materials and processing fluids, such as reagents and buffers, in a manner that will be described in more detail below. The sample preparation module 70 comprises a substrate 72, with an upper seal 56 secured to an upper surface thereof and a lower seal 230 secured to a lower surface thereof. The substrate 72 includes a series of open grooves or channels formed in its upper and lower surfaces. Each of the slits can be connected to one or more inlet ports comprising a blind hole formed in the upper surface of the substrate 72 and/or to one or more outlet ports comprising a blind hole formed in the lower surface of the substrate 72. The upper seal 56 and the lower seal 230 cover the top and bottom, respectively, of the substrate 72 and have openings that align with the inlets and outlets formed in the substrate 72, thereby forming a network of conduits, or enclosed channels, through which a fluid (e.g., liquid, gas, solution, emulsion, liquid-solid suspension, etc.) can flow from one part of the sample preparation module 70 to another, and inlet ports and outlet ports through which fluids can flow into and out of the sample preparation module 70, respectively. In various embodiments, the sample preparation module 70 is transparent or translucent and is made of, for example, polycarbonate, polypropylene, acrylic, Mylar, acrylonitrile-butadiene-styrene ("ABS") or other suitable polymers

A rotary mixer 192 is functionally arranged within a mixing well 90 (described below) formed in the substrate 72. In various embodiments, the rotary mixer 192 can be used, for example, to grind solid samples, maximize sample exposure to capture microspheres, mix the sample with chemical lysis buffer, mix magnetic microspheres with bonding buffer (typically, the magnetic microspheres cannot be stored in their bonding buffer and should therefore be combined only at the time of use), etc.

A sample cap 84 is provided to enclose a sample well 78 (described below) formed in the substrate 72. A plurality of deformable compartments (or blisters) 34a, 36a, 38a, 40a, 42a, and 44 are supported on top of the substrate sample preparation module 70. Each deformable compartment can contain a fluid and can be connected to a fluid channel within the sample preparation module 70, via one of the inlet ports, by means of a breakable connection that is initially closed to prevent fluid from flowing from the blister into the channel. Upon application of a compressive force to the outside of the blister, the increase in pressure inside the blister ruptures, opens, or otherwise disrupts the breakable connection to allow fluid to flow from the blister into an associated inlet port and channel of the sample preparation module 70.

A top shroud 12 is disposed over an upper portion of the cartridge above the sample preparation module 70 and includes openings corresponding in number, size, and shape to the various deformable compartments supported in the sample preparation module 70. As can be seen in FIG. 1, the deformable compartments are recessed within the openings formed in the top shroud 12, thereby providing some protection for the deformable compartments while allowing each compartment to be compressed from above by an actuator. In various embodiments, the top shroud 12 further includes an optical inlet port 14 and an optical outlet port 16 to allow monitoring of fluid movement through a particular portion of the sample preparation module 70, as will be described in more detail below. The top shroud 12 may also include a marker panel 24 on which identification information, such as machine-readable and/or human-readable markers (e.g., a barcode), can be placed.

The upper casing 12 may further include valve actuator tabs, such as a sample valve actuator tab 18 and a waste valve actuator tab 20. The valve actuator tabs 18 and 20 are elastic, flexible tabs formed in the casing that will deflect upon the application of an external compressive force on the tab. Each tab further includes a downward-extending actuator post (see, for example, actuator post 26 in FIG. 1), thereby actuating an active valve within the sample preparation module 70 and located below the respective tab 18 or 20, as will be described in more detail below.

With reference to FIG. 4, a reaction module 240 is disposed below the sample processing module 70 and, in various embodiments, can be configured to receive a processed sample from the sample processing module 70. In various embodiments, the reaction module 240 includes process fluid compartments (containing, for example, reagents, buffers, etc.), means for moving fluid nanodroplets in a specified directed manner along the module, means for incubating reaction mixtures, and means for detecting target analytes (for example, nucleic acids).

The reaction module 240 can be secured to the bottom of the sample preparation module 70 by means of an adhesive gasket 232, which preferably provides a fluid-tight seal between the reaction module 240 and the sample preparation module 70. In various embodiments, the reaction module 240 comprises an upper plate 241 and a lower portion, a fluid processing panel 354 secured to the bottom of the upper plate 241, and which together define a gap between the lower surface of the upper plate 241 and an upper surface of the fluid processing panel 354. This gap defines the reaction and fluid processing spaces within which various stages of the test or other procedure are performed.

A lower wrapper 30 partially encloses a lower portion of the cartridge assembly and cooperates with the upper wrapper 12 to define a relatively hard, ribbed outer casing for the cartridge 10. The upper and lower wrappers can provide the cartridge 10 with an asymmetrical shape to ensure that the cartridge 10 is inserted into a processing instrument in only one orientation. In the illustrated embodiment, the lower wrapper 30 has rounded edges 32, while the upper wrapper 12 has relatively square edges. Therefore, a receiving groove in a processing instrument configured to receive the multi-cartridge 10 and having a shape that matches that of the wrapper will ensure that the wrapper is always inserted with the right side facing upward into the instrument. In addition, the lower wrapper 30 may include characteristic contour features, such as longitudinal side grooves 22 that extend only partially along the length of the lower wrapper 30. Such grooves cooperate with corresponding characteristic features in a receiving groove of a processing instrument to ensure that the cartridge is inserted into the instrument in the appropriate direction.

Deformable fluid compartments (blisters)

In general, ampoules are made of a deformable material that preferentially collapses after the application of appropriate pressure; that is, the materials used to form the ampoules do not return to their initial shape when the pressure is released, as this could cause backflow of the applied reagents. Furthermore, ampoules can be used once (a single pressure application is performed during the test) or multiple times (e.g., multiple reagent aliquots are delivered to a single location or to multiple locations during the test). Each ampoule can contain a single processing material (e.g., buffer, reagent, immiscible liquid, etc.), or two or more ampoules can contain the same processing material. This redundancy can be used to deliver the same processing material to multiple locations within the remaining disposable container.

Although the size, number, arrangement, and contents of the compartments are largely dictated by the test or other procedure to be performed on the multiple cartridge 10, the illustrated embodiment includes six deformable fluid compartments or ampoules: 34a, 36a, 38a, 40a, 42a, and 44. A deformable ampoule may have an associated lancet ampoule. In the illustrated embodiment, each of the deformable fluid ampoules 34a, 36a, 38a, 40a, and 42a has an associated deformable lancet cartridge, or lancet ampoule, 34b, 36b, 38b, 40b, and 42b.

The operation of a deformable compartment embodiment is described with reference to FIG.

Figure 5 shows a cross-section of the deformable compartment 34a. In various embodiments, the deformable compartments of the multiple cartridge 10 incorporate characteristic features described in jointly owned U.S. patent application No. 14/206,867 entitled "Devices and Methods for Manipulating Deformable Fluid Vessels".

When a deformable compartment is compressed to displace its fluid contents, sufficient compressive force must be applied to the ampoule to break, or otherwise open, a breakable seal that is retaining the fluid within the compartment. The amount of force required to break the seal and displace the fluid contents of a compartment typically increases with the volume of the compartment. To limit the amount of compressive force that must be applied to a deformable compartment or ampoule to break or otherwise open a breakable seal retaining the fluid within the compartment, a lancet ampoule 34b is provided in conjunction with the deformable compartment 34a. The deformable compartment 34a and the lancet ampoule 34b can be connected by a channel, which can be initially blocked by a breakable seal. The lancet ampoule 34b contains an opening device, for example a microsphere 46 (such as a steel ball bearing), enclosed within the lancet ampoule 34b and supported above a fluid port 136 formed in the sample preparation module 70 by means of a breakable sheet partition, or septum, that retains the microsphere 46 and the fluid contents within the lancet ampoule 34b and the deformable compartment 34a. Therefore, to open the deformable compartment 34a, a compressive force is first applied externally to the lancet ampoule 34b to compress it and force the microsphere 46 through the foil partition blocking the fluid port 136. After the fluid port 136 is opened, the fluid contents of the deformable compartment 34a can be dispensed into the fluid port 136 with relative ease by applying an external compressive force to the deformable compartment 34a. The amount of pressure required to compress the lancet ampoule 34b and force the microsphere 46 through the foil partition is much less than that required to compress the main compartment 34a and create sufficient pressure to open a burst seal. The fluid flowing towards fluid port 136 will then flow through a horizontal channel 137, defined by a slit formed in a lower surface of the substrate 72 and covered by the lower seal 230, to a vertical channel transition 139 and, from there, to one or more other points within the sample preparation module 70.

Sample preparation module

Various details of a sample preparation module 70 are shown in FIGS. 6-15.

Sample well 78 is configured to receive a fluid sample material to be analyzed or otherwise processed in the multi-cartridge 10. As shown in Figures 6 and 7, sample well 78 can be defined by a vertical peripheral wall 79 (which is circular in the illustrated embodiment) and a lower wall, or floor, 81. Sample well 78 further includes an inlet snorkel 80 extending upward along the peripheral wall 79 of sample well 78 and terminating below the top of the peripheral wall. An outlet port 82 is provided in the floor 81 of well 78, and the floor 81 is preferably tapered downward toward the outlet port 82.

A sample cap 84 can be provided to close the sample well 78 after sample material has been deposited into the sample well 78. In one embodiment, the sample cap 84 comprises a circular cover with an outer peripheral wall that fits over the vertical peripheral wall 79 of the sample well 78. The sample cap 84 can include a pivot post 86 defined by radially elastic locking tabs extending through an opening in the substrate 72, enabling the cap 84 to pivot about an axis defined by the pivot post 86 with respect to the sample well 78. After sample material has been deposited into the sample well 78, the sample cap 84 can pivot over the top of the sample well 78 and be pushed down onto the sample well 78. A clip, or other retainer, 88, extending upwards, can be provided to securely engage and lock the cap. of sample 84 when pushed down onto clip 88 and also to provide tactile confirmation that the cap 84 has been securely closed. In some embodiments, the sample cap 84 may have a downward-tapered lower surface when the sample cap 84 is placed over sample well 78 (not shown). The tapered configuration helps reduce the amount of fluid condensate retained on the inner surface of the sample cap 84 during sample processing in sample well 78.

The sample preparation module 70 also includes a mixing well 90 formed in the substrate 72. As shown in the FIGS. In 8A and 9A, the mixing well 90 can be defined by a vertical peripheral wall 91 (which is circular in the illustrated embodiment) and a lower wall, or floor, 93. In various embodiments, a fluid inlet snorkel 92 extends upward through the peripheral wall 91 of the mixing well 90 and terminates below the top of the wall 91. In various embodiments, a pressure snorkel 94 extends upward through another portion of the peripheral wall 91 of the mixing well 90 and terminates in a position below the top of the wall 91. An outlet port 96 allows fluid to exit the mixing well 90 and may comprise a plurality of openings located near the center of a downwardly tapered portion of the floor 93 of the well 90 and surrounding a spindle seat 98 formed at the lower center of the floor 93.

The rotary mixer 192 is disposed within the mixing well 90 and includes an upper circular disc 194 supported on an upper edge of the peripheral wall 91 of the well 90. Peripheral gear teeth 198 are formed around the periphery of the disc 194, and a portion of the teeth 198 project from an outer edge of the upper and lower shrouds 12, 30 of the multi-cartridge 10 so that they can be engaged by an external drive mechanism of a processing instrument to effect the driven rotation of the rotary mixer 192. An O-ring 196 is disposed within a peripheral O-ring groove around the upper disc 194 beneath the peripheral gear teeth 198. The O-ring 196 provides a seal between the rotary mixer 192 and the peripheral wall 91 of the well 90. A spindle 200 extends downward from the upper disc 194 and seats within the seat. of the central spindle 98 of the mixing well 90. A plurality of impeller vanes 202 extend radially from the spindle 200.

An alternative embodiment of a mixing well 90' is shown in FIGS. 8B and 8C. As shown, the mixing well 90' can be defined by a vertical peripheral wall 91' (which is circular in the illustrated embodiment) and a bottom wall, or floor, 93'. A fluid inlet snorkel 92' extends upward from an outer surface of the peripheral wall 91' of the mixing well 90' and includes an opening 92a below the top of the wall 91'. A pressure snorkel 94' extends upward from the outer surface of the peripheral wall 91' of the mixing well 90' and includes an opening 94a below the top of the wall 91'. An outlet port 96' allows fluid to exit the mixing well 90' and may comprise a plurality of openings located near the center of a downwardly tapered portion of the floor 93' of the well 90' and surrounding a screw seat 98 formed at the lower center of the floor 93'. The outlet port 96' and the screw seat 98' may be substantially identical to the outlet port 96' and the screw seat 98', respectively, of the mixing well 90'.

With the alternative mixing well 90' of FIGS. 8B and 8C, a rotary mixer can be configured within the mixing well 90' with impeller vanes extending radially from a mixer spindle substantially to the inner surface of the peripheral wall 91'. This contrasts with the rotary mixer 192 configured to operate in the mixing well 90, in which the radial impeller vanes 202 cannot extend substantially to the inner surface of the peripheral wall 91 to provide clearance for the snorkels 92, 94 formed on the inner surface of the peripheral wall 91. Having a mixer with impeller vanes extending to the inner surface of the peripheral wall 91' can, in some circumstances, provide more thorough and/or efficient mixing of the contents of the mixing well 90'.

With reference to FIG. 9B, the rotary mixer 192' disposed within the mixing well 90' includes an upper circular disc 194', peripheral gear teeth 198', and an O-ring 196' that may be substantially identical to the circular disc 194', peripheral gear teeth 198', and an O-ring 196' of the rotary mixer 192 shown in FIG. 9A. A spindle 200' extends downward from the upper disc 194'. Two or more drive vanes 202' extend radially from the spindle 200'. The drive vanes 202' extend substantially to the inner surface of the peripheral wall 91'. In various embodiments, the impeller vanes 202' may be skewed with respect to the spindle 200' and may also include openings 203 formed therein to improve the mixing efficiency of the rotary mixer 192'.

With reference again to FIG. 15, which shows a plan view from above of the sample preparation module 70, the sample preparation module 70 may include alignment holes 74 and 76, or other alignment features may be provided on the sample preparation module 70, or some other portion of the multiple cartridge 10, to facilitate alignment of the multiple cartridge 10 with a processing instrument, for example, by means of a pin or other structure within the instrument extending into each alignment hole.

The sample preparation module 70 includes a first inlet port 136 formed on an upper surface of the module through which a process fluid can be introduced from the deformable compartment 34a into the sample preparation module 70. In one embodiment, the deformable compartment 34a contains a lysis buffer, such as hypotonic lysis water, or a commercially available lysis buffer, such as those containing chaotropic salts such as guanidinium salts, and/or high/low pH, and/or surfactants such as sodium dodecyl sulfate (SDS), TWEEN® 20 (polysorbate 20), TRITON™ X-100 (polyoxyethylene octyl phenyl ether), etc. In some cases, the lysis buffer optionally comprises reagents to alter unwanted enzyme activity, such as DNase and RNase activity, which are then removed during the microsphere capture/elution procedure (although these may be separate reagents, either dry or liquid, which can be added as needed depending on the target analytes and assay).

After the cells are lysed from the sample material, at least partial purification is often desirable to remove further cellular and sample debris, facilitating subsequent handling and processing. Research samples in buffer do not necessarily require purification, but even then, purification is typically performed. One well-known technique relies on the use of target capture microbeads (e.g., magnetic capture microbeads) that capture and immobilize the desired target analyte(s) to separate them from cellular and sample debris. In various implementations, the capture microbeads and binding buffer are mixed with the sample in lysis buffer after the cells or viruses have been disturbed by mechanical and/or chemical means. The capture microspheres can be magnetic to facilitate the subsequent immobilization of the microspheres and the target analyte bound to them by the selective application of magnetic forces, although, as will be appreciated by those skilled in the art, other implementations may employ non-magnetic microspheres, such as polystyrene or silica microspheres (e.g., the microspheres can be captured in a size zone or in an affinity column).

Therefore, in various embodiments, the sample preparation module 70 includes a second inlet port 138 through which a process fluid can be introduced from the deformable compartment 36a into the sample preparation module 70. In one embodiment, the deformable compartment 36a contains a bonding buffer to facilitate the bonding of target capture microspheres, such as magnetic microspheres, to one or more target analytes of interest.

In various embodiments, the sample preparation module 70 includes a third inlet port 140 through which a processing material can be introduced from the deformable compartment 44 into the sample preparation module 70. In one embodiment, the deformable compartment 44 contains target capture microspheres that may comprise magnetic particles which, in combination with a binding buffer from the deformable compartment 36a, bind to an analyte or analytes of interest within the sample material to isolate and thereby enable magnetic separation of the analyte(s) of interest from the rest of the sample material.

Capture microspheres can be coated with a material that facilitates the capture of the target analyte(s). For example, for nucleic acid capture, the microspheres can be coated with a negatively charged coating to facilitate the adsorption of positively charged nucleic acids onto the surface. These are then washed with buffer and subsequently treated with elution buffer to remove the purified nucleic acids from the microspheres for further processing. As those skilled in the art will appreciate, several suitable microsphere systems are commercially available, including, for example, MagaZorb® microspheres from Promega, MagMax from Life Tech, or microspheres from Qiagen, MoBio, BioRad, and others.

Therefore, the target capture microspheres that can be contained in the deformable compartment 44 facilitate the purification of the desired target analyte with access of the fluid to a binding buffer, such as the binding buffer that can be contained in the deformable compartment 36a, used in conjunction with the capture microspheres.

In an alternative embodiment, the target capture microspheres can be provided directly within the sample preparation module 70, for example, in the form of a lyophilized pellet located in the mixing well 90 during the assembly of the multi-cartridge 10 and stored in the mixing well as a pellet until it is reconstituted by a fluid added to the mixing well 90 during the use of the multi-cartridge 10. In this alternative embodiment, the deformable ampoule 44 can be omitted.

In alternative implementations, capture microspheres can be functionalized with nucleic acid capture probes to extract specific or non-specific nucleic acids. For example, microspheres can be functionalized with random 6-mers to extract nucleic acids in general, or with capture probes specific to the desired target nucleic acids. In some cases, for example, when mRNA is the target, microspheres coated with poly-T capture probes can be used.

In various embodiments, the sample preparation module 70 further includes a fourth inlet port 142 through which a processing material can be introduced from the deformable compartment 38a into the sample preparation module 70. In one embodiment, the deformable compartment 38a contains an immiscible fluid (e.g., an oil, such as mineral oil, silicone oil, etc., as discussed in detail below).

In various embodiments, the sample preparation module 70 further includes a fifth inlet port 144 through which a processing material can be introduced from the deformable compartment 40a into the substrate 72. In one embodiment, the deformable compartment 40a contains an elution buffer.

In various embodiments, the sample preparation module 70 further includes a sixth inlet port 146 through which a processing material can be introduced from the deformable compartment 42a into the sample preparation module 70. In one embodiment, the deformable compartment 42a contains a washing buffer.

In various embodiments, the sample preparation module 70 includes a first outlet port 182, a second outlet port 188, and a third outlet port 190 formed on a lower surface of the sample preparation module 70 through which the fluid can exit the module 70 and flow into the reaction module 240.

It should be noted here that the designation of input ports or output ports as the first, second, third, fourth, fifth, or sixth port is simply to provide a convenient means of distinguishing one port from another and is not intended to be limiting, such as, for example, specifying a particular order or sequence in which the ports may be used.

A first fluid channel 150 extends from the first inlet port 136 to the sample well 78. In the diagrams, fluid channels are represented by parallel lines extending from one point to another through the sample preparation module 70. Each channel may include one or more channel transition points, represented by a circle in the channel, one of which is indicated by the reference number 151. The channel transition point represents a vertically extending channel section that extends upwards, from a channel section formed in the lower part of the substrate 72 to a channel section formed in the upper part of the substrate 72, or downwards, from a channel section formed in the upper part of the substrate 72 to a channel section formed in the lower part of the substrate 72, so that the channel may pass over or under another channel within the substrate 72.

A second fluid channel 152 extends from sample well 78 to the lysis chamber inlet 122. A third fluid channel 156 extends from the lysis chamber outlet 124 to a fifth fluid channel 162 that extends from the third inlet port 140 to the mixing well inlet snorkel 92. A fourth fluid channel 160 extends from the second inlet port 138 to the third inlet port 140. A sixth fluid channel 164 extends from the fourth inlet port 142 to the first outlet port 182. A seventh fluid channel 166 extends from the fifth inlet port 144 to the second outlet port 188. An eighth fluid channel 168 extends from the mixing well outlet port 96 to a passive valve assembly 220 (described below). A ninth fluid channel 170 extends from a passive valve cavity of the passive valve assembly 220 to a catch compartment 100. A tenth fluid channel 172 extends from an active valve assembly 204 to an active valve assembly 219. An eleventh fluid channel 174 extends from the active valve assembly 219 to a waste chamber 102. A twelfth fluid channel 176 extends from the sixth inlet port 146 to the catch compartment 100. A thirteenth fluid channel 178 extends from the catch compartment 100 to the active valve assembly 204. A fourteenth fluid channel 180 extends from the active valve assembly 204 to the third outlet 190.

It should be noted here that the designation of the various fluid channels as the first, second, third, fourth, fifth, etc. fluid channels is simply to provide a convenient means of distinguishing one port from another and is not intended to be limiting, such as, for example, specifying a particular order or sequence in which the fluid channels may be used or a particular direction in which the fluids flow through the channels.

In various embodiments, the sample preparation module 70 further includes a passive valve assembly 220 adjacent to the mixing well 90. In one embodiment, the passive valve assembly 220 is configured so that it closes if the pressure inside the mixing well 90 falls below a threshold pressure, thereby retaining the fluid within the mixing well 90. Conversely, if the pressure inside the mixing well 90 is allowed to rise to a level sufficient to exceed the threshold pressure, the passive valve assembly 220 will open, allowing the fluid within the mixing well to escape through the outlet port 96 and the eighth fluid channel 168, which connects the outlet port 96 of the mixing well to the passive valve assembly 220.

Details of the passive valve assembly 220 are shown in FIGS. 10 and 11. The valve assembly 220 comprises a valve cavity 222 formed in the substrate 72 and an inlet 224 formed in the substrate 72 and extending upwards into the valve cavity 222. A valve 229, which may comprise a Belleville valve, is disposed within the valve cavity 222 over the inlet 224. A retainer 226 is disposed over the valve 229. An outlet 228 extends radially from the valve cavity 222.

Under a non-pressurized condition, the valve 229 and the retainer 226 are at rest at the bottom of the valve cavity 222, with the valve 229 covering the inlet 224. The retainer 226 can be deflected to a lower position, for example, by a suitable spring or similar. Consequently, the fluid flowing from inlet 224 cannot pass into and through valve cavity 222, and therefore the fluid cannot escape from mixing well 90. On the other hand, if the fluid at inlet 224 is pressurized sufficiently to overcome any force (e.g., spring deflection) holding retainer 226 in a lower position (e.g., approximately 3 to 5 psi), valve 229 and retainer 226 will lift from the bottom of valve cavity 222, thereby opening inlet 224 and allowing fluid to flow into valve cavity 222 and out through outlet 228.

The sample preparation module 70 may further include a pump port 104 through which an external pressure source can be coupled to the sample preparation module 70. The pump port 104 is connected, by means of a pressure conduit 106, to the sample well 78 so that the pressure applied to the pump port 104 will pressurize the sample well 78 to cause the contents of the sample well 78 to flow out of the well.

The sample preparation module 70 may further include a passive valve port 108 connected, via a valve conduit 110, to the pressure snorkel 94 of the mixing well 90. If the passive valve port 108 is open, pressure will not build up inside the mixing well 90, and the passive valve assembly 220 will remain closed. If the passive valve port 108 is closed, pressure will build up inside the mixing well 90, and the passive valve assembly 220 will open, allowing the contents of the mixing well 90 to flow out.

Some organisms, such as viruses and many bacteria, can be chemically lysed by the addition of a lysis buffer with or without elevated temperature or proteolytic enzymes. Some organisms are difficult to lyse using chemical and/or enzymatic procedures and require mechanical disruption or rupture of cell membranes. As such, an optional component of the 10-multicartridge is a driving component, in which the driving element is activated to grind or break up solid components so that individual cells are more accessible to the lysis buffer and more target analytes are released. The driving element imparts turbulent action to the fluid in which the lysis microspheres are contained. The primary lysis action results from collisions of the microspheres with the target organisms, which are thereby lysed, broken down, and exposed to the target nucleic acids. The presence of the lysis buffer inhibits DNases or RNases that can destroy the RNA or DNA targets once the cells are disrupted. In various embodiments, the impeller is like a paddle wheel that rotates very quickly.

Therefore, in various embodiments, the sample preparation module 70 further includes a lysis chamber 120 with a driven agitator, such as a motorized microsphere mixer, disposed therein. The driven agitator is disposed at least partially within the lysis chamber 120 and is constructed and arranged to agitate the fluid flowing through the processing chamber. The fluid flowing through the lysis chamber may comprise a mixture of sample material, lysis buffer, and lysis microspheres. The lysis microspheres may comprise silica (ceramic) microspheres (of, for example, 100 µm in diameter) that are dispensed into the lysis chamber 120 during the assembly of the multi-cartridge 10. The microsphere mixer comprises a motor 128 with an impeller 130 mounted on a motor output shaft (see FIG. 2). The fluid flows into the lysis chamber 120 through an inlet 122 and flows out of the lysis chamber 120 through an outlet 124. A mesh filter may be provided opposite the inlet 122 and/or the outlet 124. The mesh filter(s) has a pore size configured to retain the lysis microspheres within the lysis chamber 120 while allowing the sample fluid to flow into and out of the lysis chamber 120. In operation, the motor 128 rotates the impeller 130 at a high rotational speed (e.g., from approximately 5,000 to approximately 100,000 rpm, preferably from approximately 10,000 to approximately 50,000 rpm, more preferably from approximately 20,000 to approximately 30,000 rpm), so that the fluid within the lysis chamber 120, which may include material The sample and lysis microspheres are vigorously agitated by the rotating impeller, thus helping the lysis microspheres to alter the molecular structure of the sample material. Therefore, the sample mixture flowing out of the lysis chamber 120 is more completely lysed than it would be without the microsphere mixer.

A suitable 128 motor for the microsphere mixer includes Feiying, model FY0610-Q-04170Y from Jinlong Machinery. The motor can be driven by a temporary connection of the multi-cartridge 10 to an external power supply of an instrument in which the cartridge 10 is being processed. Control of the motor 128 can be implemented by means of logic elements provided externally and/or internally to the cartridge 10. In one embodiment, a mixer printed circuit board ("PCB") is provided within the lower housing 30, which controls the operation of the microsphere mixer motor 128. The mixer motor 128 ideally operates only when fluid flows through the lysis chamber 120. The fluid flowing into the lysis chamber 120 can be detected by an optical sensor through the inlet optical port 14 formed in the upper housing 12 (see FIG. 2), which is aligned with an inlet optical detection chamber 154 (see, for example, FIG. 15), so that the microsphere mixer motor 128 can be activated, for example, upon detection of the end. front end of a fluid stream flowing through the inlet optical detection chamber 154 into the lysis chamber 120. Similarly, fluid flowing from the lysis chamber 120 can be detected by an optical sensor through the outlet optical port 16 (see FIG. 2), which is aligned with the outlet optical detection chamber 158 (see FIG. 15), so that the microsphere mixer motor 128 can be switched off, for example, after the detection of the rear end of a fluid stream flowing through the outlet optical detection chamber 158.

The sample preparation module 70 further includes two active valve assemblies, 204 and 219. Valve assembly 204 is known as the sample valve assembly and is located at the junction of the tenth fluid channel 172, the thirteenth fluid channel 178, and the fourteenth fluid channel 180. It controls the flow from the thirteenth fluid channel 178 to the fourteenth fluid channel 180. Valve assembly 219 is known as the waste valve assembly and is located at the junction of the tenth fluid channel 172 and the eleventh fluid channel 174. It controls the flow from the tenth fluid channel 172 to the eleventh fluid channel 174 and the waste chamber 102.

Details of an active valve assembly, for example, valve assembly 204, are shown in FIGS. 13 and 14. The valve assembly 204 comprises a valve cavity 210 formed in the substrate 72. An inlet conduit 208 leads to the valve cavity 210, and an outlet channel 212 extends out of the cavity 210. An access opening 206 is formed in the upper seal 56 disposed above the substrate 72. A flexible valve diaphragm 216 is secured to a lower side of the upper seal 56 below the access opening 206 by means of an adhesive 214 surrounding the access opening 206. In the non-deflected or non-actuated position, as shown in FIG. 13, the fluid can flow into the valve cavity 210 through the inlet 208 and out of the valve cavity 210 through the outlet 212. Consequently, the flow of fluid through the valve assembly 204 is not obstructed. As shown in FIG. 14, when an external valve actuator 218 presses down through the access opening 206 to divert the valve diaphragm 216 over the outlet 212, the flow of fluid through the valve assembly 204 is blocked. The valve actuator 218 may comprise an actuator post 26 of the actuator tab 20 formed in the upper housing 12 (see FIG. 1). Specifically, the valve actuator tab 18 aligns with the active valve assembly 204, and the valve actuator tab 20 aligns with the active valve assembly 219.

In various embodiments, the sample preparation module 70 further includes a waste chamber 102 (or more than one waste chamber) configured to receive and contain excess or used fluids.

Reaction module: top plate

Details of the reaction module 240, and in particular the top plate 241, are shown in FIGS. 24-31. With reference to FIGS. 24 and 26, which show a perspective view from above and a plan view from above, respectively, of the top plate 241, the top plate 241 includes an upper perimeter wall 256 that projects above a top surface 242 of the top plate 241 and at least partially circumscribes the top surface 240 at a location offset inward from the outer edges of the top plate 241. The upper perimeter wall 256 has a continuous open channel or groove 258 formed along its upper edge that provides a seat for the adhesive gasket 232 securing the reaction module 240 to the sample preparation module 70. See FIG. 4. The upper perimeter wall 256 forms a recessed area 260 surrounded by the upper perimeter wall 256 on the upper surface 242. See also FIGS. 28 and 29.

The 241 top plate can be configured in a variety of ways and manufactured from a range of materials. Suitable materials include, but are not limited to, fiberglass, TEFLON®, ceramic, glass, silicon, mica, plastic (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polycarbonate, polyurethanes and derivatives thereof, etc.). Polycarbonate is a particularly preferred top plate material.

An alignment fork 246 extends from one end of the top plate 241, and an alignment loop 244 extends from an opposite end of the top plate 241. The alignment fork 246 and the alignment loop 244 are configured to receive alignment pins in an instrument for processing the multi-cartridge 10 to ensure proper alignment of the cartridge 10, as described in more detail below.

The top plate 241 also includes a sample compartment 266 with an inlet port 268 that is in fluid communication with the third output port 190 of the sample preparation module 70.

The top plate 241 further includes a rehydration (elution) buffer compartment 276 having an inlet port 278 that is in fluid communication with the second outlet port 188 of the sample preparation module 70. A detection buffer compartment 280 contains an initially dry detection buffer (applied to a portion of the top plate 241 forming the detection buffer compartment 280 or a portion of the fluidic processing panel 354 covering the detection buffer compartment 280) that is reconstituted with an amount of reconstitution buffer dispensed into the rehydration buffer compartment 276 and transferred to the detection buffer compartment 280. In one embodiment, the detection buffer compartment 280 has a capacity of 120-160 pl. In various embodiments, the top plate 241 includes a connection passage 274 between the detection buffer compartment 280 and the rehydration buffer compartment 276. The detection buffer compartment 280 may further include a port 282 for injecting a buffer into the compartment 280 during a manufacturing process and/or for venting the compartment 280.

Figures 25 and 27 show a perspective view from below and a plan view from below, respectively, of the top plate 241. With reference to Figures 25 and 27, in addition to Figures 24 and 26, the top plate 241 further includes a buffer compartment 296, which, in one embodiment, contains a dry PCR buffer/enzyme (applied to a portion of the top plate 241 forming the buffer compartment 296 or to a portion of the fluidic processing panel 354 (see Figures 4 and 58) covering the buffer compartment 296), to be reconstituted (rehydrated) later by a quantity of rehydration buffer from the rehydration buffer compartment 276. In one embodiment, the buffer compartment 296 has a capacity of approximately 20 pl. A port 298 is provided for injecting the PCR buffer/enzyme into the compartment during the manufacturing procedure and/or for venting the buffer compartment 296.

The top plate 241 further includes a second buffer compartment 300 that can contain an exonuclease reagent in dry form (applied to a portion of the top plate 241 forming the second buffer compartment 300 or to a portion of the fluidic processing panel 354 covering the second buffer compartment 300), to be reconstituted later by a quantity of rehydration buffer from the rehydration buffer compartment 276. In one embodiment, the second buffer compartment 300 has a capacity of approximately 20 pl. A port 302 may be provided for injecting buffer into the second buffer compartment 300 during a manufacturing procedure and/or for venting compartment 300. A relief valve 306 may be provided between the rehydration buffer compartment 276 and the second buffer compartment 300 to allow fluid flow from the rehydration buffer compartment 276 to compartment 300.

The top plate 241 further includes a lower perimeter wall 290 that circumscribes the lower portion of the top plate 241. The lower perimeter wall 290 defines a recess surrounded by the perimeter wall 290 configured to receive a panel, such as the fluidic processing panel 354, to enclose the lower half of the top plate 241. A raised panel support 290 surrounds the outer periphery of the lower surface of the top plate 241 just inside the perimeter wall 290. The area 294 inside the panel support 292 is slightly recessed relative to the panel support 292, so that a panel inserted within the perimeter wall 290 is supported on the panel support surface 292, and the recess 294 defines a gap 295 (see FIGS. 28, 29) between the panel and the top plate 241.

The top plate 241 may further include fluid inlet ports 250, 252, at least one of which is in fluid communication with the first outlet port 182 of the sample preparation module 70. The inlet ports 250, 252 provide fluid communication with the gap 295 between the lower surface of the upper reaction plate 241, for example in area 294, and the fluidic processing panel 354 enclosing the lower surface of the top plate 241.

The top plate 241 further includes the sensing compartments 350a, 350b, 350c and 350d, each with an inlet port or vent port 352. The illustrated embodiment includes four sensing compartments 350a-d, although alternative configurations of the top plate 241 comprising a smaller or larger number of sensing compartments 350 can easily be imagined.

Area 304 on the lower surface comprises a processing area that is slightly recessed with respect to area 294, thereby forming a larger gap between the top plate 241 and a lower panel in area 304 than in area 294.

The reaction module 240 may further include one or more bubble traps 340 formed in the top plate 241. Each bubble trap 340 includes a bubble-capture bell 342 formed in the top plate 241 that slopes upward toward a vent opening 344. In one embodiment, rising air bubbles generated by fluid movement beneath the bubble trap are captured in the capture bell 344 and released through the vent opening. The capture bell may be shaped to fit a fluid movement path beneath or adjacent to the bubble trap. In the illustrated embodiment, five bubble traps 340 having elongated capture bells 342 are positioned over four fluid movement paths, each located below and between two adjacent bubble traps 340, as described in more detail below.

Details of fluid inlet 252 are shown in FIG. 30. As highlighted, fluid inlet 252 can be aligned with the first fluid outlet 182 of the sample preparation module 70. Fluid inlet 252 can have an inwardly tapered, truncated conical shape, in which the size of outlet 182 above inlet 252 is narrower than the upper end of inlet 252. This helps ensure that the fluid dispensed through outlet 182 is captured by inlet 252.

Details of sample compartment 266, rehydration buffer compartment 276, and detection buffer compartment 280 are shown in FIG. 31. The sample compartment 266 is configured to receive a quantity (e.g., 200 JL) of magnetic microspheres with attached target analyte (e.g., DNA, nucleic acid) from the sample preparation module 270 via the inlet port 268. The inlet port 268 of the sample compartment 266 is preferably conical and aligned with the third outlet 190 of the sample preparation module 70. In various embodiments, the third outlet 190 passes through a tapered nozzle 322 to minimize dangling nanodroplets at the end of outlet 190. The inlet port 268 is also preferably tapered, with its wider end at the top to ensure that the fluid dispensed through outlet 190 is captured in the inlet port 268. Outlet 190 and inlet port 268 are configured with a small gap. between them. This gap comprises part of an interstitial space 308 between the top of the top plate 241 of the reaction module 240 and the bottom of the sample preparation module 70. This gap provides a trap to collect any air bubbles contained in the fluids within the reaction module 240, especially air bubbles that may be generated when fluid is dispensed from outlet 190 into inlet port 268.

The rehydration buffer compartment 276 is configured to receive a quantity (e.g., 200 J L) of a buffer solution suitable for rehydrating dry reagents and eluting nucleic acid from the microspheres of the sample preparation module 270 via the inlet port 278. The inlet port 278 of the rehydration buffer compartment 276 is aligned with the second outlet 188 of the sample preparation module 70. Outlet 188 preferentially flows through a tapered nozzle 320, the end of which is spaced from the tapered inlet port 278. The space between the end of the nozzle 320 and the inlet port 278 allows gas bubbles within the fluid flowing between outlet 188 and inlet port 278 to escape into the interstitial space 308.

Reaction module: fluidic processing panel

With reference to Figures 58 and 59, in various embodiments, the reaction module 240 of the multi-cartridge 10 includes a fluidic processing panel 354, secured to the underside of the upper plate 241. The fluidic processing panel 354 is peripherally surrounded by the perimeter wall 290 and is supported on and secured to the panel support 292, for example, by means of an oil- and temperature-resistant adhesive. The fluidic processing panel 354 facilitates several functionalities of the multi-cartridge 10, such as fluid movement and analyte detection. Such fluid movements may include transporting one or more fluid nanodroplets along fluid transport pathways, mixing fluids by moving one or more nanodroplets in an oscillatory manner (e.g., linearly back and forth or in a continuous path (e.g., circular, oval, rectangular)), combining fluid nanodroplets that may contain different materials, splitting nanodroplets into two or more smaller nanodroplets, etc.

The fluidic processing panel 354 includes a substrate 356. Suitable substrates include metallic surfaces such as gold, electrodes as defined below, glass and modified or functionalized glass, fiberglass, ceramics, mica, plastics (including acrylics, polystyrene and styrene copolymers and other materials, polypropylene, polyethylene, polybutylene, polyimide, polycarbonate, polyurethanes, TEFLON® and derivatives thereof, etc.), GETEK® (a combination of polypropylene oxide and fiberglass), etc., polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, and a variety of other polymers, with printed circuit board (PCB) materials being particularly preferred.

In various embodiments, the 354 fluidic processing panel can be divided into a number of distinct functional areas or processing zones, which can be spatially overlapping, spatially distinct, or partially spatially separated and partially spatially distinct.

In various embodiments, the fluid reaction processing within reaction module 240 is based, at least partially, on the manipulation of microfluidic fluids using so-called electrowetting techniques to form micronanoparticles that can be manipulated both spatially and biochemically.

In general, electrowetting is the modification of the wetting properties of a hydrophobic surface (such as PCBs) with an applied electric field. In an electrowetting system, the change in the substrate-electrolyte contact angle due to an applied potential difference results in the ability to move the electrolyte across a surface. Essentially, as described in U.S. Patent No. 6,565,727, by applying an electric potential to an electrode (or group of electrodes) adjacent to a polar liquid droplet (e.g., one containing a target analyte), the surface of these electrodes becomes more hydrophilic, and the droplet is drawn by a surface tension gradient to increase the overlap of the area with the charged electrodes. This causes the droplet to spread across the surface, and by subsequently removing the potential or activating different electrodes, the substrate returns to a hydrophobic state, resulting in the droplet moving to a new hydrophilic area of the substrate. In this way, droplets can be physically and discreetly moved across the flat surface of the substrate to different processing zones for further processing, manipulation, and detection. The droplets can move at varying speeds, split (for example, a single droplet can split into two or more droplets), pulse, and/or mix (two or more droplets merge at the same location and then split or move as one). Furthermore, electrowetting can induce mixing within a single nanodroplet. As described in more detail below, the droplets can also be used to rehydrate dried reagents stored in different locations on the PCB substrate. A typical feature of electrowetting is the precise manipulation of very small fluid volumes. For example, the isolated target nucleic acid can be eluted to a very high concentration in less than 10 µL prior to PCR amplification, compared to the elution volumes of 100 µL and much lower target analyte concentrations characteristic of other systems. Furthermore, electrowetting allows fluid paths to be altered during development and in the product by means of a computer program, without the need to make any changes to the physical interface (e.g., new valves, fluid paths, etc.).

Exemplary microfluidic systems using electrowetting techniques are described in the publications. of U.S. patent no. 2013/0252262, 2013/0233712, 2013/0233425, 2013/0230875, 2013/0225452, 2013/0225450, 2013/0217113, 2013/0217103, 2013/0203606, 2013/0178968, 2013/0178374, 2013/0164742, 2013/0146461, 2013/0130936, 2013/01 18901, 2013/0059366, 2013/0018611, 2013/0017544, 2012/0261264, 2012/0165238, 2012/0132528, 2012/0044299, 2012/0018306, 2011/0311980, 2011/0303542, 2011/0209998, 2011/0203930, 2011/0186433, 2011/0180571, 2011/01 14490, 2011/0104816, 2011/0104747, 2011/0104725, 2011/0097763, 2011/0091989, 2011/0086377, 2011/0076692, 2010/0323405, 2010/0307917, 2010/0291578, 2010/0282608, 2010/0279374, 2010/0270156, 2010/0236929, 2010/0236928, 2010/0206094, 2010/0194408, 2010/0190263, 2010/0130369, 2010/0120130, 2010/0116640, 2010/0087012, 2010/0068764, 2010/0048410, 2010/0032293, 2010/0025250, 2009/0304944, 2009/0263834, 2009/0155902, 2008/0274513, 2008/0230386, 2007/0275415, 2007/0242105, 2007/0241068, U.S. patents Nos. 8541176, 8492168, 8481125, 8470606, 8460528, 8454905, 8440392, 8426213, 8394641, 8389297, 8388909, 8364315, 8349276, 8317990, 8313895, 8313698, 8304253, 8268246, 8208146, 8202686, 8137917, 8093062, 8088578, 8048628, 8041463, 8007739, 7998436, 7943030, 7939021, 7919330, 7901947, 7851 184, 7822510, 7816121, 7815871, 7763471, 7727723, 7439014, 7255780, 6773566, and 6565727.

Therefore, in various embodiments, the fluidic processing panel 354 comprises an electrode grid that forms and defines discrete processing zones, including pathways, for fluid nanodroplets as appropriate for the tests or other procedures performed in the reaction module 240. In general, a "spot" or "location" or "pad" (sometimes referred to as an "electrowetting pad" or "EWP") is generally represented in the figures as a rectangle in which the lines forming the sides of the rectangle represent electrodes, such that a nanodroplet moves along a path in discrete steps, from pad to pad. By manipulating the electrode grid, the nanodroplets can be selectively moved in any of four directions as required: forward, backward, left, or right, relative to a current position. Therefore, in various embodiments, the 354 fluidic processing panel includes a grid of etched electrodes forming a network of pads to move sample nanodroplets from sample preparation to target analyte detection.

In the illustrated embodiment, the electrodes formed on the substrate 356 of the fluidic processing panel 354 define a series of discrete functional regions that provide movement and/or collection of fluid nanodroplets. As shown in Figures 26, 27, and 59, these zones include a sample microsphere zone 368 spatially corresponding to sample compartment 266 of the upper plate 241, a hybridization zone 370 spatially corresponding to detection buffer compartment 280 of the upper plate 241, a rehydration buffer zone 372 spatially corresponding to rehydration (elution) buffer compartment 276 of the upper plate 241, an exonuclease reagent zone 374 spatially corresponding to the second buffer compartment 300 of the upper plate 241, and a PCR reagent zone 376 spatially corresponding to buffer compartment 296 of the upper plate 241. Other zones defined on the fluidic processing panel 354 include electrosensor zones 360a, 360b, 360c, and 360d spatially corresponding to the detection compartments 350a, 350b, 350c, and 350d, respectively, and which also include electrosensor arrays 363a, 363b, 363c, and 363d, respectively. Still other pathways defined in the fluidic processing panel 354 include the thermocycling, or PCR, pathways 364a, 364b, 364c, and 364d, each being spatially located below and between two adjacent bubble traps 340 of the upper plate 241.

The fluidic processing panel electrodes 354 can further define an exonuclease zone 384.

The electrodes of the 354 fluidic processing panel can further define detection mixing zones which, in the illustrated embodiment, comprise four groups of nine electrode pads indicated by reference numbers 385a, 385b, 385c and 385d.

The fluidic processing panel may further include a series of connector pad arrays configured to make contact and electrical connections with connector pins (e.g., pogo pins) located within the processing instrument, as described in more detail below. The illustrated embodiment includes seven connector pad arrays: 358a, 358b, 358c, 358d, 358e, 358f, and 358g.

As those skilled in the art will appreciate, there are a wide variety of electrode grid configurations that can be used in the 10-multi-cartridge, including, without limitation, the configurations described herein. Exemplary electrowetting electrode configurations for various applications are shown in U.S. Patent No. 8,541,176.

In general, preferred materials for the fluidic processing panel 354 include printed circuit board materials. In various embodiments, circuit board materials are those comprising an insulating substrate (e.g., substrate 356) coated with a conductive layer and processed using lithographic techniques, particularly photolithography, to form the electrode and interconnection patterns (sometimes referred to in the art as interconnects or wires). The insulating substrate is generally, but not always, a polymer. As is known in the art, one or a plurality of layers can be used to prepare either "two-dimensional" boards (e.g., all electrodes and interconnections in one plane, "board connectors at the edges") or "three-dimensional" boards (in which the electrodes are on one surface and the interconnections can pass through the board to the other side, or in which the electrodes are on a plurality of surfaces). Three-dimensional systems often rely on drilling or etching to create holes, or vias, through the substrate, followed by electroplating with a metal such as copper to create through-the-board interconnections. Circuit board materials are often supplied with a foil already bonded to the substrate, such as a copper foil, with additional copper added as needed (e.g., for interconnections), for example, by electroplating. The copper surface may then need to be thickened, for example, by etching, to allow for bonding of the adhesive layer.

In one embodiment, the electrical connections of both the electrowetting electrode grids and the sensing electrodes—that is, the connector pad arrays 360a-g—extend across the panel to produce a landgrid array that can interface with a pogo pin or similar connector to create connections from the chip to a processing instrument. In various embodiments, the surface of the fluidic processing panel 354 (e.g., the PCB with the electrode grids) is coated with a film of a substance to facilitate the electrowetting mechanism and clean pad-to-pad transport. In various embodiments, the surface is coated with a polyimide film, such as DuPont KAPTON® (e.g., black or yellow KAPTON®), which forms a dielectric layer. The surface properties of the dielectric layer are important for facilitating electrowetting and for attenuating the voltage used to prevent electrolysis in the aqueous nanodroplet. In addition, the surface of Kapton® or similar, such as a welding mask, must be coated with a hydrophobic coating, such as Paralyene, TEFLON® (polytetrafluoroethylene), CYTOP® fluoropolymers, to name a few, to make the surface hydrophobic, which is required for electrowetting to work.

As those skilled in the art will appreciate, the form of the reagent provided in reaction module 240 will depend on the reagent. Some reagents may be dry or in solid form (for example, when using specific buffers), others may be lyophilized, etc. Particularly useful embodiments utilize dry reagents with added stabilizers, such as salts, sugars, polysaccharides, polymers, or proteins such as gelatins, etc., as those skilled in the art will appreciate. For example, Biomatrica produces commercial stabilizers for use in this system.

As those skilled in the technique will appreciate, if used, dry reagents can be rehydrated in one of two general ways. Liquid is introduced from the sample preparation module 70 onto the appropriate pad (or zone), or the sample itself serves as an aqueous solvent to bring the solid reagents into solution. For example, the appropriate resuspension buffer (which may be water in some cases) can be added through the top plate 241 from the sample preparation module 70 to a particular pad to rehydrate the reagent(s), and then the reagent nanodroplet can be fused with the sample nanodroplet.

Alternatively, droplets containing the target analyte (e.g., in the elution buffer used to release the target analytes from the capture microbeads) can be transferred to a pad containing the dry reagent(s), which are then suspended within the droplet itself. One advantage of this embodiment is that the final volume of a nanodroplet does not increase significantly, as it does when a reagent droplet is fused with a sample droplet. This can be particularly useful in situations requiring multiple reagent additions.

The number, type, and quantity of the different reagents will depend on the sample, the target analyte, and the desired reaction. For example, for nucleic acid target sequences in a standard PCR reaction, when the starting sample is DNA, the incorporated dry reagents include RT-PCR buffer, PCR enzyme (e.g., a Taq polymerase), dNTPs, PCR primers, exonuclease, signal probes, signal buffer, and detection buffers (with the lysis buffer, binding buffer, elution buffer, reconstitution buffers (optional), and magnetic microsphere suspension all contained in Sample Preparation Module 70, rather than dry in Fluidic Processing Panel 354). Exemplary embodiments are described herein. However, as will be appreciated by those skilled in the art, any number of dry and liquid reagent configurations can be used in Sample Preparation Module 70.

The compartment within the reaction module 240, formed between the fluidic processing panel 354 and the top plate 241 described above, is generally filled with a fluid in which the target analyte nanodroplets (typically aqueous solutions) are immiscible, and this immiscible fluid is generally less polar than the droplet solution. As described in U.S. Patent No. 8,541,177, there are two general methods for isolating droplets in pads, which include filling the compartment with an immiscible fluid comprising immiscible liquids and immiscible gases, or using the immiscible fluid as a nanodroplet encapsulant, for example, by giving the nanodroplet an oil shell by passing the nanodroplet through an air/oil interface.

Immiscible fluids specifically suitable for use in the nucleic acid detection assays described herein include, but are not limited to, silicone oils, mineral oil, fluorosilicone oils; hydrocarbons, including, for example, alkanes such as decane, undecane, dodecane, tridecane, tetradecane, pentadecane, and hexadecane; aliphatic and aromatic alkanes such as dodecane, hexadecane, and cyclohexane; hydrocarbon oils; mineral oils; paraffin oils; halogenated oils such as fluorocarbons and perfluorocarbons (e.g., 3M Fluorinert liquids); and mixtures thereof. Examples of suitable gas packing fluids include, without limitation, air, argon, nitrogen, carbon dioxide, oxygen, humidified air, and any inert gas. In one embodiment, the main phase is an aqueous solution, and the secondary phase is air or oil that is relatively immiscible with water. In another embodiment, the fill fluid includes a gas that fills the space between the plates surrounding the nanodroplets. A preferred fill fluid is a low-viscosity oil, such as silicone oil. Other suitable fluids are described in U.S. Patent Application No. 60/736,399, entitled "Filler Fluids for Droplet-Based Microfluidics," filed November 14, 2005. The fluid may be selected to prevent any significant evaporation of the nanodroplets.

As those skilled in the art will understand, the movement of nanodroplets from pad to pad, with the addition of reagents as needed, can be used for any number of sample manipulations. In the case of nucleic acid manipulations for nucleic acid detection, these manipulations generally include the addition of reagents such as PCR enzymes, PCR buffer, primers, exonuclease, reverse transcriptase (RT) enzymes, RT-PCR buffers, signal buffers, signal probes, etc.

In various embodiments, one or more portions, or sections, of the electrode grid pathway of the pads are exposed to heat within discrete thermal zones for, for example, amplification, exonuclease digestion, reverse transcription, target elution, and electrochemical detection. Such thermal zones may comprise a detection region 378, an exonuclease region 380, and thermocycling (PCR) regions (also referred to as thermal zones) 382a, 382b, 382c.

As those skilled in the technique will appreciate, some manipulations, such as PCR amplification, require thermocycling between two and three different temperatures (primer binding, extension, and denaturation), while others require a uniform temperature for optimal results. Examples include enzymatic processes such as the use of exonuclease and reverse transcriptase, specific temperature(s) for improved reagent elution and/or resuspension, and binding/assay temperatures in the case of electrochemical detection. Isothermal amplification techniques and other PCR alternatives typically require precise temperature control.

In various embodiments, the heat applied to different portions of the fluidic processing panel 354 is generated by thermal components, such as resistive heaters or thermoelectric (Peltier) chips, and is located outside the cartridge in the processing bays of the instrument in which the cartridge 10 is placed. Examples of such thermal components are described below.

In one embodiment, the sample handling zones in the 354 reaction panel can optionally include sensors, for example, to monitor and control the temperatures of the thermal zones, particularly where specific temperatures are desired. These sensors may include, but are not limited to, thermocouples and resistance temperature detectors (RTDs). Alternatively, these sensors can also be located outside the cartridge in the bays.

In various embodiments for detecting nucleic acid targets, the 354 fluidic processing panel comprises one or more thermocycling or PCR or amplification pathways, 364a, 364b, 364c, and 364d. The 354 fluidic processing panel may contain 1, 2, 3, or more pad thermocycling pathways. These can be used for single PCR reactions (e.g., a nanodroplet moves up and down one pathway or up one pathway and down another, etc.) or for multiplexing (e.g., for multiple pathways, multiple different nanodroplets can move up and down each pathway).

As those skilled in the art will appreciate, each PCR reaction can be further multiplexed. That is, for target-specific amplification, using multiple primer sets in a single PCR reaction can be cumbersome, and therefore the present invention allows multiple reactions to achieve higher levels of multiplexing. For example, for the evaluation of 21 different target sequences (e.g., in respiratory virus screening), it may be desirable to perform three different reactions of seven primer sets; for example, a first PCR sample nanodroplet in a first lane picks up a first set of seven primer pairs ("Primer Mixture A"), a second nanodroplet in a second lane picks up a second set of seven primer pairs ("Primer Mixture B"), and a third nanodroplet in a third lane picks up a third set ("Primer Mixture C"). In some embodiments, more than one nanodroplet can be processed on each lane, so each lane can include more than one primer set. In some embodiments, the primers will be completely different in each set; in others, redundancy and/or internal controls are integrated into the system by adding the same primer sets to different lanes. The number of multiplexes can be easily varied via software without modifying any physical system components.

In general, the amplification reactions suitable for use in the current systems employ primer sets in which one primer in each set has a blocked end that is insensitive to standard exonucleases. That is, it is desirable to remove one strand from the double-stranded amplicons generated in the PCR reaction to simplify the detection reactions and remove the background signal. Therefore, by performing a first PCR reaction and then adding exonuclease, one strand of the double-stranded amplicon is digested, leaving only the detection strand.

The use of multiple heating zones along the thermocycling pathways 364a-d, as generally depicted in Figure 59, allows the nanodroplets to move through the appropriate thermal zones. As shown in Figure 59, the four thermocycling pathways 364a, 364b, 364c, and 364d extend through the three thermal zones 382a, 382b, and 382c. The heating elements, such as resistive heaters, corresponding to thermal zones 382a, 382b, and 382c, are located outside the cartridge and can be maintained at temperatures of 95°C, 72°C, and 64°C for use in PCR thermocycling. In some embodiments, two different temperature zones (e.g., approximately 95 °C for denaturation and approximately 60 °C for annealing and extension) can be used for a two-step PCR reaction. In other embodiments, a three-zone, two-temperature configuration can be employed, in which an intermediate heater corresponding to the intermediate thermal zone 382b controls the denaturation temperature (e.g., approximately 95 °C), and additional heaters corresponding to thermal zones 382a and 382c on either side of the denaturation heater provide substantially the same annealing and extension temperature (e.g., approximately 60 °C). In this configuration, two-step amplification cycles can be performed with more than one nanodroplet in each thermocycling lane 364a–d. For example, two nanodroplets can be placed in each thermocycling pathway and spaced such that when one nanodroplet is in the denaturation zone 382b, the other is in one of the combined hybridization/extension zones 382a or 382b, and vice versa. Each nanodroplet can pick up amplification reagents (e.g., a primer cocktail) at locations, for example, at each end of a thermocycling pathway, such as locations 366a and 366b of each of the thermocycling pathways 364a–d. By transporting the nanodroplets in tandem back and forth between the denaturation and hybridization/extension zones, both can be amplified in the same amount of time it would normally take to amplify a single nanodroplet. In a four-path configuration, as shown, this means that eight nanodroplets can be amplified simultaneously instead of three.

In various embodiments, the multiple cartridge 10 of the present invention is based on the use of electrodes and electrochemical markers for the detection of target analytes. Generally, the surface of the electrodes within each electrosensor array 363a, 363b, 363c, and 363d (optionally coated with a self-assembled monolayer (SAM)) has capture ligands that bind to the target. A second marker ligand, which also binds to the target, is included, such that in the presence of the target, the marker ligand binds near the electrode surface and can be detected electronically.

Therefore, the detection zone of the fluidic processing panel 354 comprises one or more separate arrays of detection electrodes 363a, 363b, 363c, and 363d within the respective electrosensor zones 360a, 360b, 360c, and 360d. "Electrode," as used herein, means a composition that, when connected to an electronic device, can detect a current or charge and convert it into a signal. Alternatively, an electrode can be defined as a composition that can apply a potential to and/or pass electrons to or from species in solution. Preferred electrodes are known in the art and include, but are not limited to, certain metals and their oxides, including gold, platinum, palladium, silicon, and aluminum. Metal oxide electrodes including platinum oxide, titanium oxide, tin oxide, indium tin oxide, palladium oxide, silicon oxide, aluminum oxide, molybdenum oxide (Mo₂O₆), tungsten oxide (WO₃), and ruthenium oxides; and carbon (including glassy carbon electrodes, graphite, and carbon paste). Preferred electrodes include gold, silicon, carbon, and metal oxide electrodes, with gold being particularly preferred. In one particularly useful embodiment, both the electrowetting electrode grid and the sensing electrodes are made of gold and are manufactured simultaneously on the fluid processing panel 354.

The present system is particularly useful in array formats, i.e., those containing an array of addressable sensing electrodes. "Array," as used herein, refers to a plurality of capture ligands on electrodes in an array format; the size of the array depends on its composition and intended use. Arrays can be prepared containing from approximately two different capture ligands to approximately 50 to 100. In some preferred embodiments, 80 or 100 working sensing electrodes are divided into four or five distinct zones of twenty, each zone having up to sixty capture probes (three different capture probes per electrode).

The detection zone of the fluidic processing panel 354 comprises one or more detection electrode arrays 363a-d, each of which is within an electrosensor zone 360a-d that is in fluid communication with the nanodroplet pathway of an associated detection mixing zone 385a-d. That is, nanodroplets containing amplicons will pick up the required detection reagent, such as the marker probe (e.g., a cocktail of signal probes that may be in dry form, e.g., at locations 362a, 362b, 362c, and 362d) adjacent to the electrosensor detection zones 360a, 360b, 360c, and 360d, respectively, and then disperse into the associated electrosensor detection zones 360a, 360b, 360c, and 360d. The signal probe cocktails can be applied to a portion of the top plate 241 forming locations 362a, 362b, 362c, and 362d or to a portion of the fluidic processing panel 354 covering locations 362a, 362b, 362c, and 362d. In general, each detection zone receives one or more sample nanodroplets, which are generally dispersed across the electrode array, forming what is considered a larger "pad."

In one embodiment, the reaction module 240 includes four (4) electrosensor detection zones, and each electrosensor array includes 20 working electrodes (which may include a reference electrode and an auxiliary electrode). Each detection electrode of each electrosensor array 363a-d comprises an independent cable (interconnect) for transmitting input and electronic response signals for each electrode in the array, so that both the input and electronic response signals can be monitored independently for each electrode. That is, each electrode is independently addressable. Furthermore, the reaction module is preferably configured for the independent control of the electrohumidification pads surrounding each electrode of each electrosensor array 363a, 363b, 363c, and 363d.

In addition to the fluidic processing panel 354 components described above, the fluidic processing panel 354 may also optionally comprise an EPROM, EEPROM, or RFID for identifying the cartridge, for example, containing information about the lot, treatment, or contents of the multiple cartridge 10. This may include information about the assay identification, for example.

Instrument overview

An instrument configured to process the multi-cartridge 10 and embodying aspects of the present invention is designated by reference number 400 in FIG. 32. The instrument comprises a control console 402, one or more processing modules 410 functionally coupled to the control console 402, processing bays within each processing module 410, each of which is configured to receive a multi-cartridge and process the multi-cartridge independently of the other bays, and instrument software (ISW). In various embodiments, the instrument comprises a control console 402 and up to four processing modules 410, each processing module including six processing bays. Each processing module 410 is functionally coupled to the control console 402, for example, to exchange power, input and output data, and control signal transmissions with the control console 402, and can also be physically connected to the control console 402. Each processing bay with the processing module 410 is configured to accept one multi-cartridge 10 at a time and to process the cartridge independently of other processing bays processing other multi-cartridges. In various embodiments, the instrument is configured so that each processing bay completes the processing of one cartridge in 60 minutes or less.

The ISW provides the graphical user interface for the user to initiate runs, receive results, and provide inputs that at least partially control the instrument's operation. In various embodiments, the ISW is configured to run on a Windows® computer with a touchscreen 404 located on the control console 402, which provides the primary functionality for user input. In various embodiments, the instrument is configured to provide connectivity to a local area network (LAN) and a laboratory information system (LIS). The instrument may also include a barcode scanner (not shown) that facilitates ISW login, sample tracking, and positive identification of the instrument.

The instrument's control console 402 includes a touchscreen panel 404, a system computer, a power supply, connectivity to external data systems, and connectivity for the processing module(s) and processing bay(s). In various embodiments, a single power supply in the control console powers the entire instrument. Wiring from the control console provides power transmission and enables data flow to and from the processing bays. In various embodiments, the control console also has a provision for physically attaching one or more processing modules to the control console.

Each processing bay includes software (hardware), firmware, and electronics that run an assay on a multi-cartridge. Each processing bay may include a bay PCB. In various embodiments, the bay PCB includes the processing bay's electronics and firmware (such as microprocessors and firmware on the microprocessors), circuitry that supplies power (for example, up to 300 V to the electrohumidifying pads) to the multi-cartridge, circuitry that performs electronic detection of reaction products in the multi-cartridge, circuitry that controls the heaters in the processing bay that interface with the multi-cartridge, circuitry that measures and controls temperatures in the multi-cartridge, circuitry that controls the movement of various moving components in the processing bay, and circuitry that controls a pump in the processing bay.

Each processing bay may also include a connector PCB. In various embodiments, the connector PCB includes pogo pins configured to make contact with the multi-cartridge and transmit data, control signals, and power between the multi-cartridge and the processing bay PCB, and pogo pins configured to make electrical contact with heating elements within the processing bay.

Each processing bay also includes stepper motors. In various embodiments, the processing bay comprises two stepper motors: one stepper motor that controls the positioning of magnets, heaters, and pogo pins, or other connecting elements, with respect to the multi-cartridge, and one stepper motor that controls a cam follower plate within the processing bay that compresses the ampoules in the multi-cartridge and causes the ampoules to dispense their contents in a predefined sequence.

Each processing bay also includes an ampoule compression assembly configured to compress the ampoules of the multi-cartridge 10 in a specified sequence and actuate the active valves of the multi-cartridge 10, thereby dispensing the contents of the ampoules in the specified sequence. In various embodiments, the ampoule compression mechanism assembly comprises an array of ampoule compression actuators, or compression mechanisms, each comprising a cam arm configured to push a compression pad onto an ampoule. The blister compression mechanism assembly further includes a cam arm plate within which the cam arms and compression pads of the compression mechanisms are functionally mounted above the blisters for movement between a retracted position and an extended blister compression position, a cam follower plate that is movable relative to the cam arm plate and includes grooves with raised protrusions (or other cam follower elements) located and sequenced to engage the cam arms of the actuator array as the cam follower plate moves relative to the cam arm plate to actuate the cam arms to compress the blisters in a sequence determined by the relative locations of the compression mechanisms on the cam arm plate and the grooves and protrusions of the cam follower plate.

Each processing bay may also include a pump coupled to the multi-cartridge 10 by means of pump port 104 and configured to provide a driving force for the reagents and sample in the multi-cartridge sample preparation module.

Each processing bay may also include an LED PCB 466 (see FIGS. 38-41) that provides LED indicators of the processing bay status and optical sensors that detect conditions inside the multi-cartridge, e.g., through optical input hole 14 and optical output port 16.

Each processing bay may also include mounting computer equipment configured to attach the processing bay to the processing module and electrical connectors configured to transmit power and data between the processing bay and the processing module.

Each processing bay may also include a multi-cartridge carrier configured to provide a physical connection and alignment between the upper bay, comprising the blister compression mechanism assembly, and a multi-cartridge processing assembly, or lower bay, comprising a cartridge carrier assembly, a heating and control assembly, and a cam frame assembly configured to effect movement of the heating and control assembly with respect to a multi-cartridge contained in the cartridge carrier assembly.

Control console

A processing instrument that implements aspects of the present invention and is configured to process the multi-cartridge 10 described above is designated by reference number 400 in FIG. 32. As stated above, the instrument 400 includes the control console 402 and one or more processing modules 410 functionally associated with the control console 402. In one embodiment, the control console 402 includes a display panel 404 that provides a graphical user interface and comprises a touchscreen through which a user can input information into the control console 402 and/or through which information can be presented to the user. In various embodiments, the control console 402 may comprise additional or alternative means for data input, such as keyboards, microphones, switches, manually operated scanners, voice-activated input, etc. As further indicated above, the instrument may include a barcode scanner for reading barcodes, for example, one-dimensional or two-dimensional barcodes, or other types of scanners for reading machine-readable codes, such as an RFID scanner. In various embodiments, the 402 control console may comprise additional or alternative means for outputting data (i.e., information and/or results), including hard drives or other storage media, monitors, printers, indicator lights or audible signaling devices (e.g., buzzer, horn, bell, etc.), email, text messaging, etc.

Processing module

As shown in Figures 32 and 34, each processing module 410 includes one or more cartridge doors 412, each cartridge door 412 being associated with a processing bay (described below) within which a cartridge 10 can be processed. In the illustrated embodiment, each processing module 410 includes six (6) cartridge doors 412 and associated processing bays. Each cartridge door 412 is configured to accept a multi-cartridge 10, preferably in a single preferred orientation. Each cartridge door also preferably includes a lockable door (e.g., a pivoting door panel) that is deflected, e.g., by a spring or the like, in a closed position, but can be opened by pushing when a cartridge is inserted therein.

In various embodiments, each processing module 410 is functionally coupled to the control console 402. The processing module 410 can be electronically coupled to the control console 402 to enable electronic transmissions between the control console 402 and the processing module 410. Such electronic transmissions may include power transmissions from the control console to the processing module to power various electronic components within the processing module, control signals, input data, output data, etc.

Each processing module 410 can also be physically connected, for example, in a side-by-side relationship as shown in FIG. 32, to the control console 402. As in the illustrated embodiment, the instrument 400 can include one or more processing modules 410 secured to one or both sides of the control console 402. Additional processing modules can be secured to other processing modules in a side-by-side relationship on one or both sides of the control console 402. In a preferred arrangement, the instrument 400 includes up to two processing modules 410 secured to each side of the control console 402, each processing module 410 comprising six (6) cartridge doors 412 and associated processing bays for processing up to six multi-cartridges 10 per processing module.

It is preferable that the control console 402 and the processing module 410 be provided in a modular manner as shown to facilitate scalability of the instrument, for example, by adding one or more processing modules 410 to or subtracting one or more processing modules 410 from a single control console 402, and also to facilitate troubleshooting of the instrument, whereby a processing module 410 that has one or more malfunctioning processing bays can be removed from the instrument for repair or replacement, and the instrument can still be usable with the remaining functional processing modules 410.

In an alternative embodiment, however, a control console and an associated input display and/or other input means and one or more, preferably a plurality of, cartridge doors and associated processing bays can be provided in a single integral instrument having a single housing.

Further details of the processing module 410 are shown in FIGS. 35, 36, and 37. Each processing module 410 includes a plurality of cartridge doors 412 and associated processing bays 440. The illustrated embodiment includes six (6) processing bays 440. The processing bays 440 are arranged in a stacked arrangement within a housing of the processing module 410. Each processing bay 440 has an associated frame 418 that partially surrounds the processing bay with a horizontal top panel 420 and a vertical rear panel 422 (see FIG. 37). As shown in the figures, a front panel 413 of the processing module 410, within which the cartridge doors 412 are located, is oriented at a rearward angled slope from the bottom of the processing module 410 to the top of the processing module 410. This may be for ergonomic and/or aesthetic reasons. In other embodiments, a front panel of the processing module may be vertical. Due to the angle of the front panel 413 of the processing module 410, each processing bay 440 is offset horizontally (i.e., rearward) with respect to the processing bay immediately below it.

In various embodiments, each processing bay 440 has associated with it a ventilation fan 416 secured to the vertical panel 422 of the housing 418 and a ventilation tube 414 extending from the fan 416 to a rear wall of the processing module housing 410. As shown in the figures, due to the tilt of the front panel 413 and the horizontal displacement of the processing bays 440, the ventilation tubes 414 have decreasing lengths progressing from the lowest processing bay 440 to the highest processing bay.

The 410 processing module may also include additional structural elements to secure each of the 440 processing bays within the processing module housing. The 440 processing bays and the 410 processing module are preferably configured so that each 440 bay can be removed independently from the 410 processing module and replaced to facilitate instrument repair if one or more 440 processing bays malfunction or otherwise require maintenance or repair.

Processing Bay

A 440 processing bay is shown in various views in Figures 38, 39, 40, and 41. In each of Figures 38–41, the 418 frame of the 440 processing bay is omitted from the figure. Figure 38 is a front perspective view from the right side of the 440 processing module with a 10-multi-cartridge inserted. Figure 39 is a front perspective view from the left side of the 440 processing module with a 10-multi-cartridge inserted. Figure 40 is a rear perspective view from the right side of the 440 processing module. Figure 41 is an exploded front perspective view from the right side of the 440 processing module with a 10-multi-cartridge inserted.

Each processing bay 440 has a drip tray 446 that forms the bottom floor of the processing bay 440 and is constructed and arranged to contain fluid leaks that may occur from the manifold cartridge 10 and to provide a support and mounting structure for various components of the processing bay 440. A main PCB (printed circuit board) 442, also called the bay PCB, provides the main control of the processing bay 440, as well as data and power distribution and transmission. A flexible connector 444 connects the bay PCB 442 to a connector PCB (described below, not visible in FIGS. 38-41) inside the processing bay 440, as will be described in more detail below. The processing bay 440 may further include alignment elements, such as two (2) tubular female alignment elements 460, 462, which receive male alignment elements arranged within the processing module 410 to properly align and position the processing bay 440 in a bay mounting location within the processing module 410.

The processing bay 440 can be conceptually divided along functional lines into a cartridge processing assembly 470 (also known as the lower bay) and a blister compression mechanism assembly (or deformable chamber) 750 (also known as the upper bay). The primary function of the cartridge processing assembly 470 is to receive cartridge 10, secure the cartridge within bay 440, apply heat and magnetic forces to the multi-cartridge processing module 240, apply controlled feeding to the multi-cartridge 10, engage the rotary mixer 192 of the cartridge 10 and effect the driven rotation of the rotary mixer 192, and eject cartridge 10 from the processing bay 440 upon completion of a test or other procedure performed within bay 440. The primary function of the blister compression mechanism assembly 750 is to collapse the various deformable chambers of the multi-cartridge 10 in an appropriate sequence. Each of these various components will be analyzed in more detail below.

The processing bay 440 further includes an LED PCB 466 for controlling one or more LEDs that provide information to a user, such as indicating the status of the processing bay 440 and/or whether a cartridge is located within the processing bay 440. The status LEDs can be visible through a light pipe or other optical transmitter that provides an optical indication signal adjacent to the cartridge door 412 associated with the bay 440 on the front panel 413 of the processing module 410. The LED PCB 466 can also control optical sensors constructed and arranged to detect (for example, generate a signal) via the optical input and output ports 14, 16, the flow of fluid through the optical input sensing chamber 154 and the optical output sensing chamber 158 of the sample preparation module 70.

The side walls 472, 474 extend upwards along opposite sides of the processing bay 440 and can be secured to upwardly extending elements 443, 445 of the drip tray 446. A mounting plate 640 includes a generally horizontal blister plate 644 (see FIG. 42) secured to the upper edges of the side walls 472, 474 and generally separating the cartridge processing assembly 470 from the blister compression assembly 750.

In various embodiments, each processing bay 440 further includes a cam follower motor 834 and an associated encoder 838 and a cam frame motor 602. The cam plate motor 834 and the cam frame motor 602 are secured to a motor mount 642 of the mounting plate 640 (see FIG. 42).

A pump 458 provides the pressure that is applied to the manifold cartridge 10 by means of the pump port 104.

As described in more detail below, the cartridge processing assembly 470 includes a Peltier heater assembly for performing thermal procedures within the processing bay 440. To ventilate the processing bay 440 and dissipate excess heat generated in the Peltier heater, the processing bay 440 may include a Peltier ventilation assembly. The ventilation assembly comprises a cooling fan 448 attached to a drip tray fan assembly 450 and positioned opposite an airflow tube extending between the cooling fan 448 and the Peltier heater assembly within the processing bay 440. In various embodiments, the airflow tube may comprise a cooling tube 452 and a tube cover 456 extending between the cooling fan 448 and the beginning of the cooling tube 452. (See FIGS. 39 and 41.)

Cartridge processing assembly (lower bay)

The features of cartridge processing assembly 470 are shown in FIGS. 41 and 42. As noted above, most of the features of cartridge processing assembly 470 are located beneath blister plate 644 of the motor assembly 642. Cartridge processing assembly 470 includes a cartridge carrier assembly 650 configured to receive, hold, and later eject a multi-cartridge 10. The cartridge carrier assembly 650 is secured to a lower surface of blister plate 644 of the mounting plate 640.

A cam block assembly 600 includes a cam frame 606 that surrounds the cartridge carrier assembly 650 on three sides and is mounted for linear forward and backward movement within the processing bay 440 where it is supported on linear cam followers 480a, 480b extending from each of the side walls 472, 474 into a follower groove 612 formed on each side of the cam frame 606.

A mixing motor assembly 700 pivotally connects to the blister plate 644 beneath the blister plate and is configured to pivot with and without functional coupling to the rotary mixer 192 of the multi-cartridge 10 disposed within the cartridge carrier assembly 650.

A heating and control assembly 500 is located below the cartridge carrier assembly 650 and is functionally coupled to the cam frame 606 and cam block assembly 600 to convert the longitudinal back-and-forth motion of the cam frame 606 into vertical motion of the heating and control assembly 500 to selectively bring the heating and control assembly 500 into contact with a lower surface of the multi-cartridge 10 when a cartridge is inserted into the cartridge carrier assembly 650.

Cartridge carrier assembly

Other details of the 650 cartridge carrier assembly are shown in FIG. 46, which is an exploded perspective view of the 650 cartridge carrier assembly with other components of the 470 cartridge processing assembly omitted. The cartridge carrier assembly 650 includes a carrier holder 652 comprising a generally rectangular frame secured to the underside of the blister plate 644 of the mounting plate 640. A detector may be provided to detect when a multi-cartridge 10 (not shown in FIG. 46) is inserted into the cartridge holder 652. In various embodiments, the detector comprises an optical detector comprising an emitter 686 and a detector 688, each disposed within a respective pocket on opposite sides of the cartridge holder 652. An optical beam from the emitter 686 to the detector 688 is interrupted when a multi-cartridge is inserted into the cartridge holder 652, thereby generating a signal indicating the presence of the cartridge.

A cartridge hitch 654 is mounted for pivoting motion on a closed end of the cartridge holder 652. The cartridge hitch 654 is pivotally mounted on a hitch pin 660 to rotate about a horizontal axis of rotation. The cartridge hitch 654 further includes a front hook 656 and a rear lever 658. A torsion spring 662 rotatably deflects the hitch 654 so that the hook 656 is in an upward position. When a cartridge 10 is inserted into the cartridge holder 652, the cartridge pushes the hook downward until the hook 656 of the cartridge hitch 654 engages a rebate in a lower portion of the lower shell 30 of the cartridge 10. The deflection of the torsion spring 662 holds the hook 656 in that rebate to retain the cartridge within the cartridge holder 652.

A cartridge ejector assembly 670 includes an ejector rack 672 that sits within an ejector fitting 682 extending outward from a rear end of the cartridge holder 652. The linear gear teeth of the ejector rack 672 engage with a damper pinion gear 674, which engages with a rotary damper 676 and is mounted for rotation in the ejector fitting 682 adjacent to the ejector rack 672. A spring-capture pin 680 extends through the ejector rack 672 and is supported at one end by an end wall of the ejector fitting 682. A compression spring 678 is disposed between one end of the ejector rack 672 and the end of the spring-capture pin 680. Consequently, the ejector rack 672 is deflected longitudinally toward the open end of the cartridge holder 652. To provide a limit stop element to prevent the cartridge rack 672 from being pushed too far by the spring 678. The ejector rack 672 initially extends into the cartridge holder 652 and contacts the end of a multi-cartridge 10 inserted into the cartridge holder 652. As the cartridge is further inserted into the cartridge holder 652, the ejector rack 672 is pushed rearward, thereby compressing the spring 678 and generating a deflecting force that pushes the multi-cartridge 10 longitudinally toward the open end of the cartridge holder 652 and out of the processing bay 440. Because the cartridge catch 654 captures the fully inserted multi-cartridge, the ejector assembly 670 is prevented from pushing the cartridge back out of the cartridge holder 652.

A cartridge latch switch 666 is located at the closed end of the cartridge holder 652 and is configured to signal when the multi-cartridge has been inserted into a position within the cartridge holder 652, so that the cartridge will be engaged by the cartridge latch 654. Upon completion of a test or other procedure performed within the processing bay 440, the cartridge latch 654 pivots (counterclockwise in the illustrated embodiment) against the deflection of the torsion spring 662, in a manner described below, thereby releasing the multi-cartridge contained within the cartridge holder 652. After release, the cartridge is ejected by the energy stored in the compression spring 678, which bears against the ejector rack 672. The damper pinion 674 and the functionally associated rotary damper 676, with which the ejector rack 672 is engaged, ensure controlled release of the ejector rack. 672 so that the multiple cartridge 10 is not ejected too abruptly from the cartridge holder 652.

Heating and control unit

Details of the heating and control assembly 500 are shown in FIG. 43, which is an exploded perspective view of the heating and control assembly 500 with other components of the cartridge processing assembly 470 omitted.

The heating and control assembly 500 includes a support plate 502, a connector PCB 504 supported on the support plate 502, a cover plate 550 partially covering the connector PCB 504, a cartridge magnet assembly 552, a sample preparation magnet assembly 570, and a magnet actuator 584 located beneath the support plate 502. A front alignment pin 416 and a rear alignment pin 414 extend upward from the support plate 502.

A pneumatic connector 518 attaches to the pneumatic ports 519a, 519b of the cover plate 550. The pneumatic connector 518 provides a connection between the pressure source, e.g., the pump 458, and the cartridge 10 by means of the pump port 104 and provides a connection between an external valve within the processing bay 440 and the passive valve assembly 220 of the cartridge 10 by means of the passive valve port 108 (see FIG. 15).

With reference to Figures 43 and 44, which are top-down views of the connector PCB 504, the connector PCB 504 includes an elution heater assembly 506, a sensing Peltier assembly 540, and PCR heater assemblies 520a, 520b, and 520c. In various embodiments, the elution heater assembly 506 comprises a resistive heating element bonded to a dedicated PCB and a heat spreader made of a thermally conductive material bonded to or otherwise thermally coupled with the resistive heating element. Similarly, in various embodiments, each PCR heater assembly element 520a, 520b, and 520c comprises a resistive heating element bonded to a dedicated PCB and a heat spreader made of a thermally conductive material bonded to or otherwise thermally coupled with the resistive heating element.

Details of the sensing Peltier assembly 540 are shown in FIG. 45, which is an exploded perspective view of the Peltier assembly 540. The assembly 540 includes a Peltier device 544 (i.e., a thermoelectric element) coupled to a power and control printed circuit board 546. A heat spreader 542, preferably made of a thermally conductive material, is disposed above the Peltier device 544. A heat sink 548 is disposed below the Peltier chip 544. The heat sink 548 may comprise a panel in surface-to-surface contact with a surface of the Peltier device 544 and a plurality of heat dissipation rods (or fins) extending from it and formed from a thermally conductive material. The sensing Peltier assembly 540 is mounted within, and at least a portion of the heat sink 548 extends through, an associated opening formed in the support plate 542. Heat dissipation rods of the heat sink 548 extend below the support plate 502 and are arranged at one end of the Peltier cooling tube 452 (see FIGS. 39 and 41). In one embodiment, the sensing Peltier is configured to apply a thermal gradient, for example, to lower the temperature of a sensing area, for example, the sensing region 378, of the multi-cartridge 10.

A plurality of connector pin arrays 510a, 510b, 510c, 510d, 510e, 510f, and 510g are arranged around the connector PCB 504 and comprise connector pogo pin arrays that make contact and effect the electrical connection between the connection pads of the associated connector pad arrays 358a–358g of the fluidic processing panel 354 of the multi-cartridge 10 (see FIG. 58). The connections between the connector pin arrays 510a–510g and the connector pad arrays 358a–358g provide connections between the instrument 400 and the multi-cartridge 10 for, for example, power, control signals, and data. For example, the connections between the 510a-510g connector pin arrays and the 358a-358g connector pad arrays provide power and control from the instrument to the electrohumidification grid (e.g., the 364a-364d thermal cycling tracks, the 368 sample microsphere zone, the 370 hybridization zone, the 372 elution buffer zone, the 374 exonuclease reagent zone, the 376 PCR reagent zone, the 385a-385d detection mixing zones, and the 384 exonuclease zone). Additionally, the connections between the 510a-510g connector pin arrays and the 358a-358g connector pad arrays provide power to and receive data from the 363a-363d electrosensor arrays.

As shown in FIG. 43, the 504 connector PCB further includes a series of 512 heater pins, which may comprise pogo pins, connecting to the various 540, 506, and 520a, b, c heater assemblies.

The 500 heating and control assembly also includes a 552 cartridge magnet assembly and a 570 sample preparation magnet assembly.

Details of the sample preparation magnet assembly 570 are shown in FIG. 49A, which is a top perspective view of the sample preparation magnet assembly. The sample preparation magnet assembly 570 comprises a magnet holder 572 mounted on a horizontal spindle 574 so that it can rotate about the spindle 574 with respect to the support plate 502. A torsion spring 576 deflects the sample preparation magnet assembly 570 downwards. An actuator attachment 578 extends below the magnet holder 572, and a magnet 580 is supported above the magnet holder 572 and secured to it, for example, by a suitable adhesive. When unfolded and rotated upward against the deflection of the torsion spring 576, the magnet 580 extends through aligned openings formed in the support plate 502, connector PCB 504, and cover plate 550.

The sample preparation magnet assembly 570, when deployed, is positioned adjacent to the capture chamber 100 of the sample preparation module 70 of the multi-cartridge 10, thereby applying a magnetic force to the fluids contained within and flowing through the capture chamber.

Details of the cartridge magnet assembly 552 are shown in FIG. 49B, which is a top perspective view of the cartridge magnet assembly. The cartridge magnet assembly 552 comprises a magnet support frame 554 and a magnet array 556 disposed within the magnet support frame 554. The magnet array 556 may comprise individual magnets (e.g., three) and may be surrounded on all four sides by the magnet support frame 554 to form a frame surrounding the magnet array 556. The magnet array 556 may be secured within the magnet support frame 554, for example, by a suitable adhesive. A focusing magnet 558 is disposed within an opening in the top of the magnet support frame 554. In one embodiment, the focusing magnet 558 is cylindrical and may comprise neodymium N52. The focusing magnet 558 focuses the magnetic forces of the magnet array 556 onto a relatively small area to attract target capture magnetic microspheres to that small area. The magnet holder 554 is mounted on a horizontal spindle 560 connected to the support plate 502 so that the magnet holder 554 and magnet array 556 can rotate around the spindle 560. A torsion spring 562 deflects the cartridge magnet assembly 552 downward. An actuator attachment 566 extends below the magnet holder 554. When the magnet holder 554 rotates upward against the deflection of the torsion spring 562, the upper portion of the magnet assembly 552 extends through aligned openings formed in the support plate 502, the connector PCB 504, and the cover plate 550.

The cartridge magnet assembly 552, when deployed, is located adjacent to the sample chamber 266 of the reaction module 240, adjacent to a position indicated by reference number 270 (see FIG. 26).

Returning now to FIG. 43, cam followers 590a and 590b extend from opposite sides of the support plate 502 and a groove follower 592 extends from opposite sides of the support plate 502. The groove followers 592 extend and can be moved vertically within a groove 476 formed in each of the side walls 472, 474 (see FIG. 42) and are configured to allow vertical movement of the support plate 502 with respect to the side walls 472, 474 while preventing horizontal movement of the support plate 502 with respect to the side walls 472, 474.

Camshaft frame assembly

Details of a 600 cam frame assembly are shown in FIG. 47, which is an exploded perspective view of the 600 cam frame assembly with other components of the 470 cartridge processing assembly omitted. The cam frame assembly 600 includes the cam frame motor 602 that drives a linear actuator 604. A cam frame 606 includes opposing, generally parallel, longitudinal rails 608, 610 and a cross rail 614 extending between the corresponding ends of each of the longitudinal rails 608, 610. The linear actuator 604 is coupled to the cam frame 606 at a motor connector 618 projecting upward from the cross rail 614. A follower groove, or channel, 612 is formed along the outer side beneath a top surface of each of the longitudinal rails 608, 610. Follower elements 480a, 480b extending from each of the side walls 472, 474 (see FIG. 42) extend to the follower slot 612.

A cam rail 620a is secured to the longitudinal member 608, and a cam rail 620b is secured to the longitudinal member 610. An upper edge of the cam rail 620a cooperates with the follower groove 612 formed in the lower outer edge of the longitudinal member 608 to form a channel that receives the cam followers 480a, 480b, which allow longitudinal movement of the cam frame 606 and the cam rails 620a, 620b with respect to the side walls 472, 474, while preventing vertical movement of the cam frame 606 with respect to the side walls 472, 474.

Each cam rail 620a and 620b includes a front cam groove 622a and a rear cam groove 622b. The cam followers 590a, 590b projecting from the side of the support plate 502 of the heating and control assembly 500 (see FIG. 43) extend to the cam grooves 622a, 622b, respectively. Each cam groove 622a, 622b has a lower horizontal segment (the segment on the right side in FIG.

47), an upper horizontal segment (the left-side segment in FIG. 47) and an angled transition between the lower horizontal segment and the upper horizontal segment.

Before inserting a multi-cartridge 10 into the cartridge carrier assembly 650, the cam frame 606 is in a relatively forward position with respect to the heating and control assembly 500, so that the cam followers 590a, 590b extending from the support plate 502 are in the lower horizontal segment (the right-side segment as shown in FIG. 47) of each of the cam grooves 622a, 622b. Therefore, the support plate 502 and the entire heating and control assembly 500 are in a lower position with respect to the cartridge carrier assembly 650. When a multi-cartridge 10 is inserted into the cartridge carrier assembly 650, the alignment fork 246 of the upper plate 241 (see FIG.

24) It attaches to the rear alignment pin 514, which is longer than the front alignment pin 516 and extends upwards to the cartridge carrier assembly 650 even with the support plate 502 in the lower position, to properly position the cartridge within the carrier assembly 650.

After the multi-cartridge is inserted into the cartridge carrier assembly 650, as indicated, for example, when the cartridge latch switch 666 is actuated by the end of a fully inserted cartridge, the cam-frame motor 602 is actuated to retract the linear actuator 604 and the cam-frame 606 attached to it. This causes the cam rails 620a, 620b to move relative to the support plate 502, thereby moving the cam followers 590a, 590b from the lower horizontal segments on the right side of the cam slots 622a, 622b, upwards through the angled transitions, and to the upper horizontal segments on the left side of the cam slots 622a, 622b, thereby raising the support plate 502 and the heating and control assembly 500 until they come into contact with the multi-cartridge that has been placed in the cartridge carrier assembly 650.

Raising the support plate 502 relative to the cartridge contained in the cartridge carrier assembly 650 causes the front alignment pin 516 of the support plate 502 to extend to the alignment loop 244 extending from the top plate 241 (see FIG. 24). With the rear alignment pin 514 engaged by the alignment fork 246 and the front alignment pin 516 extending to the alignment loop 244, the cartridge is substantially immobilized within the cartridge carrier assembly 650.

Raising the heating and control assembly 500 relative to cartridge 10 contained in cartridge carrier assembly 650 places the connector pin arrays 510a-510g of connector PCB 504 in contact with the respective connector pad arrays 358a-358g of fluidic processing panel 354 of multi-cartridge 10. Additionally, the elution heater assembly 506 of connector PCB 504 is in contact with, or in close proximity to (i.e., to permit thermal energy transfer to), a portion of fluidic processing panel 354 corresponding to the exonuclease region 380. Similarly, the PCR heater assembly components 520a, 520b, and 520c of connector PCB 504 are in contact with, or in close proximity to (i.e., to permit thermal energy transfer to), portions of fluidic processing panel 354 corresponding to The thermocycling regions 382a, 382b, and 382c. The Peltier sensing assembly 540 of the connector PCB 504 is in contact with or in close proximity to (i.e., to allow thermal energy transfer) portions of the fluidic processing panel 354 corresponding to the sensing region 378. In addition, the pneumatic connector 518 is in contact with the pump port 104 and the passive valve port 108 of the sample preparation module 70 of the multi-cartridge 10.

Each cam rail 620a, 620b is secured to the respective longitudinal spar 608, 610 of the cam frame 606 by means of two threaded spring-capture posts 624a, 624b with a compression spring 626a, 626b disposed between the cam rail 620a and a head of each of the posts 624a, 624b. This "damper" configuration allows a certain amount of movement of the cam rails 620a, 620b with respect to the longitudinal spars 608, 610 to prevent the heating and control assembly 500 from being pushed against the underside of the manifold cartridge 10 with excessive force. Consequently, the heating and control assembly 500 will be pushed against the bottom of the multi-cartridge with a force that is no greater than the compressive force of springs 626a, 626b.

With reference to FIGS. 43 and 48, which are perspective cross-sectional views of the cam frame and a magnet actuator 584 of the cartridge processing assembly 470, a magnet actuator 584 is coupled to the cam frame 606 and configured to rotate the cartridge magnet assembly 552 and the sample preparation magnet assembly 570 into their respective functional positions relative to a multi-cartridge when the cartridge is inserted into the cartridge carrier assembly. The magnet actuator 584 includes a spring 587 that deflects the actuator to the left in FIG. 48. The magnet actuator 584 includes a vertical tab 585 configured to engage the actuator attachment 566 of the cartridge magnet assembly 552 and a vertical tab 586 configured to engage the actuator attachment 578 of the sample preparation magnet assembly 570. The magnet actuator 584 is engaged to the cam frame 606 by means of a magnet actuator hook 628 that extends below the crossbar 614 and engages a hook loop 589 formed at one end of the magnet actuator 584.

As stated above, before inserting a multi-cartridge 10 into the cartridge carrier assembly 650, the cam frame 606 is in a forward position. The magnet actuator 584 is deflected forward (to the left) by the spring 587, so that the cartridge magnet assembly 552 and the sample preparation magnet assembly 570 rotate clockwise to their retracted positions due to the force of their respective torsion springs 562 and 576. In the present context, the retracted positions of the cartridge magnet assembly 552 and the sample preparation magnet assembly 570 are positions in which the cartridge magnet assembly 552 and the sample preparation magnet assembly 570 do not apply a significant magnetic force to any portion of the multi-cartridge 10. After inserting the multi-cartridge into the cartridge carrier assembly 650, the cam frame 606 is retracted by the cam frame motor 602 and the linear actuator 604 (to the right in FIG. 48) as described above. The retraction of the cam frame 606 causes the heating and control assembly 500 to rise to make contact with the manifold cartridge 10, as the cam followers 590a, 590b of the support plate 502 move from the lower horizontal segments on the right side of the cam slots 622a, 622b, upward through the angled transitions and to the upper horizontal segments on the left side of the cam slots 622a, 622b.

The magnet actuator 584, coupled to the cam frame 606 by the magnet actuator hook 628, also moves with the cam frame 606 to pull the magnet actuator 584 to the right in FIG. 48 against the spring deflection 587. As the movable cam frame 606 pulls the actuator attachment 584, the vertical tab 585, which engages the actuator attachment 566 of the cartridge magnet assembly 552, rotates the magnet assembly 552 counterclockwise to its upward-deployed position as shown in FIG. 48. Similarly, the vertical tab 586 of the actuator attachment 584, which engages the actuator attachment 578 of the cartridge magnet assembly 570, rotates the magnet assembly 570 counterclockwise to its upward-deployed position as shown in FIG. 49. 48. Due to the longitudinal extension of the upper horizontal segment of each of the cam slots 622a, 622b, the cam frame 606 and the cam rails 620a, 620b can be moved longitudinally with respect to the support plate 502, while the cam followers 590a, 590b are positioned on the upper horizontal segments, without changing the height position of the support plate 502 and the heating and control assembly 500 with respect to the multi-cartridge that has been placed in the cartridge carrier assembly 650. In various embodiments, the magnet actuator attachment 584 is configured with respect to the cartridge magnet assembly 552 and the sample preparation magnet assembly 570 such that, as the cam frame 606 is moved (to the right) to raise the support plate 502 and the heating and control assembly 500, the Magnet assemblies 552, 570 do not initially deploy (or do not fully deploy) when the support plate 502 and the heating and control assembly 500 are first lifted to make contact with the multi-cartridge (i.e., when the cam followers 590a, 590b of the support plate 502 first reach the upper horizontal segments of the cam slots 622a, 622b). The additional movement (to the right) of the cam frame 606 (which, due to the longitudinal extension of the upper horizontal segments of the cam grooves 622a, 622b, will not change the position of the support plate 502 and the heating and control assembly 500 with respect to the cartridge carrier assembly 650 and the multi-cartridge contained therein) will additionally pull the magnet actuator attachment 584 to fully rotate the magnet assemblies 552, 570 (counterclockwise) to their fully deployed positions in contact with or in close proximity to the multi-cartridge. Therefore, with the support plate 502 and the heating and control assembly 500 held in the upper position in contact with the multi-cartridge, the magnet assemblies are configured to move independently of the rest of the heating and control assembly 500, and the cam frame 606 can be moved longitudinally to effect the selective deployment of the magnet assemblies 552, 570 in support of the requirements for selectively applying or removing magnetic forces with respect to the multi-cartridge contained within the cartridge carrier assembly 650.

Furthermore, as can be seen more clearly in FIG. 48, when the cam frame 606 (to the left in FIG. 48) is advanced to lower the heating and control assembly 500 relative to the cartridge, the linear actuator connector 618 extending above the crossbar 614 makes contact with the cartridge latch lever 658, thereby rotating the cartridge latch 654 counterclockwise to lower the hook 656 and disengage the hook 656 from the multi-cartridge so that the multi-cartridge can be ejected from the cartridge holder 652 by the cartridge ejector assembly 670.

Mixing motor assembly

Details of the 700 mixing motor assembly are shown in FIGS. 50A and 50B. FIG. 50A is a perspective view of the 700 mixing motor assembly, and FIG. 50B is an exploded perspective view of the 700 mixing motor assembly.

The 700 mixing motor assembly includes a 702 mixing motor mounting onto which a 706 mixing motor is mounted. Suitable motors include the Pololu Micro Metal gearmotor with a 150:1 gearbox and the Maxon DCX10L EB SL 4.5V model with a 64:1 gearbox. Preferred motor characteristics include 100 reps at 12 oz-in torque, 3000 h service life in a 45°C operating environment, and a compact size (e.g., 10 mm wide (diameter) and less than 25 mm long).

A gear unit 708 is fixed to an output shaft of the motor 706. A spur gear unit 710, rotatably mounted on the mixing motor mounting fixture 702, is functionally meshed with the gear unit 708, with the teeth of the spur gear unit 706 meshing with the teeth of the spur gear unit 708. Therefore, the driven rotation of the gear unit 708 about a horizontal axis of rotation corresponding to the output shaft of the motor 706 is converted into a rotation of the spur gear unit 710 about a vertical axis of rotation.

The mixing motor assembly 700 is pivotally connected to a lower side of the blister plate 644 of the mounting plate 640 by means of a pivot screw 716 extending through the mixing motor attachment 702. A spacer 714 (comprising a threaded screw and a cylindrical sleeve disposed on a portion of the screw shaft) is engaged at one end of the mounting attachment 702. A torsion spring 718 is engaged with the pivot screw 716 and deflects the mixing motor assembly 700 inward with respect to the side wall 474 (see FIG. 42) so that the spur gear unit 710 engages with the peripheral gear teeth 198 of the rotary mixer 192 (see FIG. 8) of the multi-cartridge 10.

As shown in FIG. 48, the longitudinal spar 610 of the cam frame 606 includes a chamfered block 616 that extends inward from the longitudinal spar 610. As noted above, the mixing motor assembly 700 is deflected to pivot inward with respect to the side wall 474 and the longitudinal spar 610 due to the torsion spring 718. The chamfered block 616 is positioned to engage the mixing motor assembly 700 when the cam frame 606 is in the forward position. Therefore, when the cam frame 606 is in the retracted position to raise the heating and control assembly 500 in engagement with the manifold cartridge 10 contained in the cartridge carrier assembly 650, the mixing motor assembly 700 pivots inward under the force of the torsion spring 718 in engagement with the manifold cartridge. As the 606 cam frame moves forward (to the left in FIG.

48) To lower the heating and control assembly 500 away from the multi-cartridge contained in the cartridge carrier assembly 650, the chamfered block 616 contacts the separator 714 of the mixing motor assembly 700 and pivots the mixing motor assembly outwards (towards the longitudinal member 610) against the deflection of the torsion spring 718 to disengage the spur gear unit 710 from the rotary mixer 192 of the multi-cartridge 10. In one embodiment, the chamfered block 616 contacts the separator 714 to pivot the uncoupled mixing motor assembly 700 with the rotary mixer 192 before the actuator connector 618 of the cam frame 606 contacts the lever 658 of the cartridge hook 654 to lower the hook 656 and release the cartridge to be ejected by the cartridge ejector assembly 670.

Therefore, when the cam frame 606 is in the forward position, the heating and control panel 500 is in the lowered position without contact with the multi-cartridge, the magnet assemblies 552 and 570 rotate downward to their retracted positions away from the multi-cartridge, the mixing motor assembly 700 pivots outward without engagement with the multi-cartridge, and the multi-cartridge hook 654 pivots so that the hook 656 disengages from the multi-cartridge. Therefore, the multi-cartridge is not in contact with or otherwise engaged by any of the components of the multi-cartridge processing assembly 470, and the multi-cartridge 10 can be ejected by the cartridge ejector assembly 670.

Blister compression mechanism assembly (upper bay)

Details of an ampoule compression mechanism assembly 750 are shown in FIG. 51, which is an exploded view of the ampoule compression mechanism assembly 750. The assembly 750 comprises a cam arm plate 752 and a matrix 754 of functional cam arm compression mechanisms functionally mounted within the cam arm plate 752. The cam arm plate 752 is mounted on top of the ampoule plate 644 of the mounting plate 640. The compression mechanisms of the matrix 754 comprise compression mechanisms configured to compress fluid compartments or collapsible ampoules of the multi-cartridge 10, compression mechanisms configured to compress lancet ampoules of the cartridge, and compression mechanisms configured to press and close the active valve assemblies of the cartridge. The various compression mechanisms of matrix 754 align with the blister holes 646 formed in blister plate 644 so that the compression mechanisms of matrix 754 can access the active ampoules and valves of the multi-cartridge 10 located beneath blister plate 644 within processing bay 440.

In various embodiments, the LED PCB 466 is attached to the cam arm plate 752.

The blister compression mechanism assembly 750 further includes a cam follower plate 820 mounted on the cam arm plate 752 for linear motion with respect to the cam arm plate. In various embodiments, one edge of the cam follower plate 820 is secured to a linear guide rail 822 attached to an upper surface of the cam arm plate 752 by means of linear guide carriers 824a and 824b attached to the cam follower plate 820. An opposite edge of the cam follower plate 820 is secured against vertical movement by a clamping element 826 (or Z-axis restraint) mounted within a recess 753 formed in the cam arm plate 752, for example, by suitable fasteners, and which includes a longitudinal groove 828 along one edge thereof that receives a stepped edge 830 of the cam follower plate 820. Suitable materials for constructing the clamping element include Delrin and brass. Accordingly, the cam follower plate 820 is fixed in the Z direction, or vertical direction, or normal direction with respect to the plane of the cam arm plate 752, at a given space from the cam arm plate 752, and movement is permitted in a longitudinal direction corresponding to the longitudinal direction of the linear guide rail 822 and, in general, parallel to the plane of the cam arm plate 752, but movement is restricted in any direction transverse to the linear guide rail 822.

The driven movement of the cam follower plate 820 with respect to the cam arm plate 752 is effected by a cam follower plate motor 834 connected by means of a linear actuator 836 to a drive attachment 840 that is attached to an edge of the cam follower plate 820. In various embodiments, the motor 834 further includes a rotary encoder 838 to provide precise control and feedback of the motor 834. In various embodiments, the drive attachment 840 has an "L" shape with a first portion extending away from an attachment point to the cam follower plate 820 in a plane that generally corresponds to the plane of the cam follower plate and a second portion extending downward in a direction that is generally normal to the plane of the cam follower plate. The linear actuator 836 is attached to the drive fixture 840 at a lower end of the second downward-extending portion of the drive fixture 840. This configuration of the drive fixture 840 limits the amount of extension of the cam follower plate motor 834 above the cam follower plate 820, thus maintaining a slim profile of the processing bay 440.

In various embodiments, a sensor mechanism is provided to indicate when the cam follower plate 820 is in a particular predefined position with respect to the cam arm plate 752. In one embodiment, the sensor mechanism may comprise a start switch 842 mounted on the cam arm plate 752 and made contact by a start switch contact surface 832 of the cam follower plate 820 when the cam follower plate 820 has moved to a start position with respect to the cam arm plate 752.

In various embodiments, the cam arm plate 752 includes two optical sensors 810, 812 positioned to correspond spatially with the locations of the inlet and outlet optical ports 14, 16, respectively (see FIG. 1). The sensors 810, 812 are constructed and arranged to detect (e.g., generate a signal) the flow of fluid through the inlet optical sensing chamber 154 and the outlet optical sensing chamber 158 of the sample preparation module 70 (see, e.g., FIG. 15). The optical sensors 810, 812 can be connected to and at least partially controlled by the LED PCB 466.

Compression mechanism

Details of the compression mechanisms are shown in Figures 52, 53, and 54. Figure 52 is a partial plan view from below of cam arm plate 752 showing compression pads of the compression mechanism array 754. Figure 53 is a perspective view from above of the compression mechanisms of the array 754 isolated from the cam arm plate 752. Figure 54 is a perspective view from below of the compression mechanisms of the array 754 isolated from the cam arm plate 752.

The array 754 comprises a plurality of fluid ampoule compression mechanisms, each configured so that, when actuated, it applies a compressive force to an associated deformable fluid ampoule and thereby compresses the deformable ampoule. In the illustrated embodiment, there are five fluid ampoule compression mechanisms 756a, 756b, 756c, 756d, and 756e corresponding to the deformable fluid chambers 34a, 36a, 38a, 40a, and 42a, respectively, of the multi-cartridge.

The matrix 754 further includes a plurality of lancet ampoule compression mechanisms, each configured so that, when actuated, it applies a compressive force to an associated lancet ampoule that is associated with one of the deformable fluid ampoules, thereby compressing the lancet ampoule and rupturing the fluid seal within the lancet ampoule. In the illustrated embodiment, there are five lancet ampoule compression mechanisms 760a, 760b, 760c, 760d, and 760e corresponding to lancet ampoules 34b, 36b, 38b, 40b, and 42b, respectively, of the multi-cartridge.

Matrix 754 further includes a compression mechanism 758 which has substantially the same configuration as a lancet ampoule compression mechanism 760a-e and corresponds to ampoule 44 of the multi-cartridge.

Array 754 includes two valve actuator compression mechanisms 762a, 762b associated with sample valve assembly 204 and waste valve assembly 219, respectively (see FIG. 15). Each of the valve actuator compression mechanisms 762a, 762b is configured so that, when actuated, it applies a compressive force on the valve actuator tabs 20, 18 (see FIG. 1), respectively, and thereby actuates and closes the active valves 219 and 204.

Details of the construction of each of the various compression mechanisms are shown in Figures 53 and 54, as well as in Figures 55A, 55B, and 55C. Figure 55A is an exploded perspective view of a single fluid ampoule compression mechanism. Figure 55B is an exploded perspective view of a single lancet ampoule compression mechanism. Figure 55C is an exploded perspective view of a valve actuator compression mechanism.

The ampoule compression mechanism assembly employs the principles and concepts described in U.S. Patent Application No. 14/206,817 entitled "Apparatus and Methods for manipulating deformable fluid vessels." Specifically, the ampoule compression mechanism assembly is constructed and arranged to convert the horizontal motion of the cam follower plate 820 into a vertical, or partially vertical, motion of the compression mechanisms to compress a fluid ampoule, a lancet ampoule, and a valve assembly without requiring pneumatic, electromechanical, or other components at greater distances above and/or below the manifold cartridge 10, thereby maintaining a slim profile of the processing bay 440.

With reference to FIG. 55A, each fluid blister compression mechanism, such as fluid blister compression mechanism 756a, includes a cam arm 764 with a cam surface 766 formed along an upper edge thereof. The cam arm 764 is mounted within the cam arm plate 752 for pivoting about an arm pivot pin 768 that extends through a hole formed in one end of the cam arm 764. The cam arm 764 is disposed within a groove 765 formed in the cam arm plate 752, and the arm pivot pin 768 is mounted within the cam arm plate 752 transversely to that groove (see FIG. 52). A compression pad 772 is pivotally mounted on an opposite end of the cam arm 764 for pivoting movement about a pad pivot pin 774 extending through a hole formed in the opposite end of the cam arm 764. In various embodiments, the compression pad 772 is disposed within a blind recess 773 formed in a lower surface of the cam arm plate 752 in a configuration that generally matches the configuration of the compression pad 772 (see FIG. 52).

The fluid ampoule compression mechanism 756a is configured to pivot relative to the cam arm plate 752 around the arm pivot pin 768 between a retracted position, in which the compression mechanism is not applying pressure to the associated fluid ampoule, and an extended, or deployed, position, in which the compression mechanism is applying a compressive force to the fluid ampoule. A torsion spring 770 deflects the compression mechanism 756a to the retracted position. In the retracted position, the cam arm 764 is disposed substantially within the corresponding groove 765 formed in the cam arm plate 752, and the compression pad 772 is disposed within the pad recess 773 formed in the cam arm plate 752 so that the contact surface with the ampoule of the compression pad 772 is substantially level with a surface of the cam arm plate 752. In the extended position, the cam arm 756 rotates about the cam arm pivot pin 768 so that the compression pad 772 extends below the cam arm plate 752 to compress and collapse the reagent ampoule disposed below the compression pad 772.

The cam surface 766 may include a convex protrusion, or other characteristic feature, which, in various embodiments, extends above an upper surface of the cam arm plate 752 (see FIG. 51, which shows characteristic cam features of the cam arms of the die 754 of compression mechanisms extending above the cam arm plate 752). When the cam surface 766 is engaged by a cam follower element that moves relative to the cam arm 764 over the cam surface 766, it causes the cam arm 764 to pivot from the retracted to the extended position as the cam follower moves over the convex protrusion of the cam surface 766. As the cam follower element moves away from the cam surface 766, the cam arm 764 returns to the retracted position under the force of the torsion spring 770.

The cam arm 764 is preferably made of a material that has sufficient strength to withstand the forces applied to it by a cam follower element that pushes the cam arm 764 against a collapsible fluid blister and that has suitable machinability. Suitable materials include steel for applications in which the cam follower element comprises a roller that rolls on the cam surface 766. For applications in which the cam follower element comprises a sliding (i.e., non-rolling) element that slides on the cam surface 766, suitable materials include low-friction, low-abrasion materials such as nylon or a lubricant-impregnated material such as oil-impregnated bronze.

In various embodiments, the construction and operation of the other fluid blister compression mechanisms, 756b, 756c, 756d and 756e, are substantially the same as those of the fluid blister compression mechanism 756a, although the size and shape of the compression pads (e.g., compression pad 772) may vary from one fluid blister compression mechanism to the next according to the size and shape of the fluid blister to be compressed by the compression mechanism.

With reference to FIG. 55B, each lancet ampoule compression mechanism, such as lancet ampoule compression mechanism 760a, includes a cam arm 780 with a cam surface 782 formed along an upper edge thereof. The cam arm 780 is mounted within the cam arm plate 752 for pivoting about an arm pivot pin 784 that extends through a hole formed in one end of the cam arm 780. The cam arm 780 is disposed within a groove 781 formed in the cam arm plate 752, and the arm pivot pin 784 is mounted within the cam arm plate 752 transversely to that groove (see FIG. 52). A compression pad 788 is formed or positioned at an opposite end of the cam arm 780. In various embodiments, the compression pad 788 is disposed within a blind recess 789 formed in a lower surface of the cam arm plate 752 in a configuration that generally matches the configuration of the compression pad 788 (see FIG. 52).

The lancet ampoule compression mechanism 760a is configured to pivot relative to the cam arm plate 752 around the arm pivot pin 784 between a retracted position, in which the compression mechanism is not applying pressure to the associated lancet ampoule, and an extended, or deployed, position, in which the compression mechanism is applying a compressive force to the lancet ampoule. A torsion spring 786 deflects the compression mechanism 760a to the retracted position. In the retracted position, the cam arm 780 is disposed substantially within the corresponding groove 781 formed in the cam arm plate 752, and the compression pad 788 is disposed within the pad recess 789 formed in the cam arm plate 752 so that the contact surface with the ampoule of the compression pad 788 is substantially level with a surface of the cam arm plate 752. In the extended position, the cam arm 780 rotates about the cam arm pivot pin 784 so that the compression pad 788 extends below the cam arm plate 752 to compress and collapse the lancet ampoule disposed below the compression pad 788.

The cam surface 782 may include a convex protrusion, or other characteristic feature, which, in various embodiments, extends above an upper surface of the cam arm plate 752 (see FIG. 51, which shows characteristic cam features of the cam arms of the matrix 754 of compression mechanisms extending above the cam arm plate 752). When the cam surface 782 is engaged by a cam follower element that moves relative to the cam arm 780 over the cam surface 782, it causes the cam arm 780 to pivot from the retracted to the extended position as the cam follower moves over the convex protrusion of the cam surface 782. As the cam follower element moves away from the cam surface 782, the cam arm 780 returns to the retracted position under the force of the torsion spring 786.

The cam arm 780 is preferably made of a material that has sufficient strength to withstand the forces applied to it by a cam follower element that pushes the cam arm 780 against a collapsible lancet ampoule and that has suitable machinability. Suitable materials include steel for applications in which the cam follower element comprises a roller that rolls on the cam surface 782. For applications in which the cam follower element comprises a sliding (i.e., non-rolling) element that slides on the cam surface 782, suitable materials include low-friction, low-abrasion materials such as nylon or a lubricant-impregnated material such as oil-impregnated bronze.

In various embodiments, the construction and operation of the other lancet ampoule compression mechanisms, 760b, 760c, 760d and 760e, and compression mechanism 758 are substantially the same as those of lancet ampoule compression mechanism 760a.

With reference to FIG. 55C, each valve actuator compression mechanism, such as the valve actuator compression mechanism 762a, includes a cam arm 790 with a cam surface 792 formed along an upper edge thereof. The cam arm 790 is mounted within the cam arm plate 752 for pivoting about a cam arm pivot pin 794 extending through a hole formed at one end of the cam arm 790. The cam arm 790 is disposed within a groove 791 formed in the cam arm plate 752, and the cam arm pivot pin 794 is mounted within the cam arm plate 752 transversely to that groove (see FIG. 52). A contact pad 798 is formed or positioned at an opposite end of the cam arm 790. In various embodiments, the contact pad 798 is arranged within a blind recess 799 formed in a lower surface of the cam arm plate 752 in a configuration that generally matches the configuration of the contact pad 798 (see FIG. 52).

In various embodiments, the contact pad 798 may further include a contact pin, or point, 800 projecting from the contact pad 798. The contact point is configured to engage a small dimple or depression formed on the upper surface of the valve actuator flange 18 or 20 when the valve actuator compression mechanism presses against the flange, preventing the compression mechanism from slipping off the valve actuator flange. Additionally, in various embodiments, a portion of the contact pad 798 and the contact pin 800 may be offset relative to the cam arm 690 to accommodate space and orientation constraints within the compression mechanism array 754.

The compression mechanism of the valve actuator 762a is configured to pivot relative to the cam arm plate 752 around the arm pivot pin 794 between a retracted position, in which the compression mechanism is not applying pressure to the associated valve actuator tab and active valve assembly, and an extended, or deployed, position in which the compression mechanism is applying a compressive force to the actuator tab and valve assembly. A torsion spring 796 deflects the compression mechanism 762a to the retracted position. In the retracted position, the cam arm 790 is disposed substantially within the corresponding groove 791 formed in the cam arm plate 752, and the contact pad 798 is disposed within the pad recess 799 formed in the cam arm plate 752, such that the contact surface of the contact pad 798 is substantially level with a surface of the cam arm plate 752. In the extended position, the cam arm 790 rotates about the cam arm pivot pin 794, such that the contact pad 798 extends below the cam arm plate 752 to deflect the valve actuator tab downward and close the associated valve assembly disposed below the valve actuator tab.

The cam surface 792 may include a convex protrusion, or other characteristic feature, which, in various embodiments, extends above an upper surface of the cam arm plate 752 (see FIG. 51, which shows characteristic cam features of the cam arms of the matrix 754 of compression mechanisms extending above the cam arm plate 752). When cam surface 792 is engaged by a cam follower element that moves relative to cam arm 790 over cam surface 792, it causes cam arm 790 to pivot from the retracted to the extended position as the cam follower moves over the convex protrusion of cam surface 792. As the cam follower element moves away from cam surface 982, cam arm 790 returns to the retracted position under the force of the torsion spring 796.

The cam arm 790 is preferably made of a material that has sufficient strength to withstand the forces applied to it by a cam follower element that pushes the cam arm 790 against a valve assembly and that has suitable machinability. Suitable materials include steel for applications in which the cam follower element comprises a roller that rolls on the cam surface 792. For applications in which the cam follower element comprises a sliding (i.e., non-rolling) element that slides on the cam surface 792, suitable materials include low-friction, low-abrasion materials such as nylon or a lubricant-impregnated material such as oil-impregnated bronze.

In various embodiments, the construction and operation of the other compression mechanism of the valve actuator 762b are substantially the same as those of the compression mechanism of the valve actuator 762a.

Details of the 820 cam follower plate are shown in FIGS. 56 and 57. FIG. 56 is a plan view from below of the 820 cam follower plate, and FIG. 57 is a perspective view from below of the 820 cam follower plate.

The cam follower plate 820 includes a series of generally parallel longitudinal cam grooves 850, 852, 854, 856, 858, and 860. Each of the grooves 850-860 in the cam follower plate 820 receives a portion of one or more of the cam arms 764, 780, 790 from the die compression mechanisms 754. In addition, each groove 850-860 includes one or more cam follower elements, for example, in the form of ribs or rollers formed or positioned at discrete locations along the corresponding groove.

The cam follower plate 820, as stated above, is configured for linear motion relative to the cam arm plate 752 in a plane parallel to the cam arm plate 752. As the cam follower plate 820 moves relative to the cam arm plate 752, when a cam follower element within a cam groove encounters the cam surface of the compression mechanism's cam arm (e.g., cam surface 766, 782, or 792 of cam arms 764, 780, or 790, respectively), the cam arm is pushed down, pivoting about its respective arm pivot pin (e.g., pivot pin 768, 784, or 794) to cause the compression mechanism to compress the ampoule (e.g., the compressible fluid ampoule or blister). (of lancet) or press the active valve assembly arranged below that compression mechanism.

During the movement of the cam follower plate 820 with respect to the cam arm plate 852, the relative locations of the compression mechanisms of the matrix 754 of the compression mechanisms and the cam follower ribs formed in the grooves 850, 852, 854, 856, 858 and 860 define the sequence in which the compression mechanisms are actuated.

Computer program and computer hardware

As described in general and specifically above, disclosure aspects are implemented through computer control and computing equipment components, user-created software, data input components, and data output components. Computer equipment components include computing and control modules (e.g., system controller(s)), such as microprocessors and computers, configured to perform computational and/or control steps by receiving one or more input values, executing one or more algorithms stored on non-transient, machine-readable media (e.g., software) that provide instructions for manipulating or otherwise acting upon the input values, and outputting one or more output values. Such outputs may be displayed or otherwise indicated to a user to provide information, for example, about the status of the instrument or about a procedure being performed, or such outputs may comprise inputs to other control procedures and/or algorithms. Data input components comprise elements through which data is entered for use by the computer control and computing equipment components. These data inputs may include position sensors, motor encoders, and manual input elements such as graphical user interfaces, keyboards, touchscreens, microphones, switches, manually operated scanners, voice-activated input, etc. Data output components may include hard drives or other storage media, graphical user interfaces, monitors, printers, indicator lights, or audible signal elements (e.g., buzzer, horn, bell, etc.).

The computer program comprises instructions stored on non-transitory, computer-readable media that, when executed by the computer control and computing equipment, cause the computer control and computing equipment to perform one or more automated or semi-automated procedures.

Sample preparation procedure

An exemplary sample preparation procedure that can be performed in the sample preparation module 70 is described and illustrated in FIGS. 16-23. Those skilled in the art will recognize that sample preparation procedures other than those described herein, for example, rearranging the steps from what is described herein, omitting certain steps described herein, and/or adding certain steps, can be performed using the sample preparation module or a modified version of the sample preparation module.

In the first step, illustrated in Figure 16, a fluid sample is dispensed into sample well 78. The multi-cartridge 10 is generally designed to process either liquid or solid samples. Liquid samples may include blood, serum, plasma, urine, saliva, cerebrospinal fluid, lymph, sweat, semen, or epithelial samples such as cheek, nasopharyngeal, anal, or vaginal swabs to which lysis buffer has been added to resuspend the cells. Solid samples, such as stool or tissue samples (e.g., tumor biopsies), generally need to be resuspended and diluted in a buffer, such as Cary Blair transport medium. Sample well 78 can then be closed using sample cap 84 (see FIG. 6), and multi-cartridge 10 is then placed in a processing instrument (e.g., in processing bay 440 of processing module 410 of instrument 400).

In a first step performed within the instrument, as illustrated in FIG. 17, the lancet ampoule 34b associated with the deformable compartment 34a is compressed by an external actuator (e.g., compression mechanism 760a) to press a microsphere or other opening device through a sealing seal (i.e., tear the seal with the microsphere or other device), and then the deformable compartment 34a is compressed by an external actuator (e.g., compression mechanism 756a) to force out a process fluid contained therein to the first inlet port 136 formed in the substrate 72. In one embodiment, the process fluid contained in the deformable compartment 34a is a lysis buffer. The fluid is directed by the first fluid channel 150 from the inlet port 136 to the sample well 78, where the fluid enters the sample well 78 through the inlet snorkel 80. In addition, an external pump (e.g., pump 458) connected to the sample preparation module 70 at the pump port 104 generates pressure that is applied to the contents of the sample well 78 by means of the pressure conduit 106.

The pressure generated by compressing the deformable compartment 34a and the pressure applied in the pressure conduit 106 pushes the fluid contents, comprising the fluid sample and the contents of the deformable compartment 34a, from the sample well 78 through the second fluid channel 152 to the inlet of the lysis chamber 122. The fluid continues to flow through the lysis chamber, exiting through outlet 124, where it is directed by the third fluid channel 156 and a portion of the fifth fluid channel 162 to the mixing well 90. As the fluid stream first enters or exits the lysis chamber 120 and passes through the inlet optical detection chamber 154 or the outlet optical sensor chamber 158, it is detected through the associated optical port 14 or 16 formed in the upper envelope 12 (see FIG. 1) by an optical detector (e.g., optical detector(s)). mounted on the LED PCB 466). A signal from the optical detector indicating fluid flow (e.g., an air-fluid interface) through the inlet or outlet optical detection chamber 154 or 158 activates the lysis chamber mixer motor 128 to alter the fluid flow through the lysis chamber 120 with lysis microspheres contained within the lysis chamber 120. The motor 128 continues to operate until a signal from an optical detector indicating the end of fluid flow through the inlet or outlet optical detection chamber 154 or 158, and therefore the end of flow through the lysis chamber 120, deactivates the motor 128.

While the fluid mixture flows into the mixing compartment 90, the passive valve port 108 remains open, so that the pressure inside the mixing well 90 does not rise to a level that opens the passive valve assembly 220. Therefore, at the end of the stage illustrated in FIG. 17, the mixing well 90 will contain a mixture of fluid sample and the contents of the deformable compartment 34a (for example, a lysis buffer) that has been physically lysed by the lysis mixer and the lysis microspheres contained in the lysis chamber 120.

Referring now to FIG. 18, after the step shown in FIG. 17, the pneumatic pump applying pressure to the pressure port 104 is switched off, for example, after a prescribed operating period, and the third deformable compartment 44 is compressed by an external actuator (for example, the compression mechanism 758) to force the contents of the deformable compartment 44 out to the third inlet port 140. In one embodiment, the contents of the deformable compartment 44 comprise magnetic target-capturing microspheres.

Next, the lancet ampoule 36b associated with the deformable compartment 36a is compressed by an external actuator (e.g., compression mechanism 760e) to press a microsphere or other opening device through a sealing seal (i.e., tear the seal with the microsphere or other device), and then the deformable compartment 36a is compressed by an external actuator (e.g., compression mechanism 756e) to force out a process fluid contained therein into the second inlet port 138 formed in the substrate 72. The process fluid then flows through the fourth fluid channel 160 and the fifth fluid channel 162 to the mixing well 90. The contents of the deformable compartment 36a may comprise a binding buffer to facilitate the binding of the target capture microspheres to the target analyte(s). The fluid flowing past the third inlet port 140, under the pressure generated by the compression of the deformable compartment 36a, carries the fluid contents of the deformable compartment 36a and the contents of the deformable compartment 44 through the fifth fluid channel 162 to the mixing well 90.

As stated above, in an alternative embodiment, the magnetic microspheres can be provided in the form of a freeze-dried sediment contained within the mixing well 90, and the deformable compartment 44, the associated external actuator (e.g., the compression mechanism 758), and the deformable compartment 44 rupture stage can be omitted.

After the step illustrated in FIG. 18 is completed, the rotary mixer 192 inside the mixing well 90 can be activated (e.g., by the mixing motor assembly 700) to agitate the contents of the mixing well 90. In various embodiments, a lyophilized reagent form or another dry reagent form can be pre-positioned in the mixing well 90 and dissolved or reconstituted by the fluids conveyed to the mixing well 90. The rotary mixer 192 helps facilitate the dissolution or reconstitution of the dry reagent and mixes all the materials contained in the mixing well to form a homogeneous, fluid mixture.

With reference to FIG. 19. A further step comprises collapsing the lancet ampoule 38b (e.g., by compression mechanism 760b) associated with the deformable compartment 38a, thereby opening the compartment to the fourth inlet port 142. The deformable compartment 38a then collapses (e.g., by compression mechanism 756b) to direct its fluid contents to the fourth inlet port 142, through the sixth fluid channel 164, and to the first outlet port 182, where the fluid exits the sample preparation module 70. The first outlet port 182 is in communication with the inlet port 252 of the reaction module 240, as described above. The fluid contained in the deformable compartment 38a may comprise an immiscible fluid, e.g., an oil, which is used to fill a reaction space 295 within the reaction module 240 between the top plate 241 and the processing panel. fluidic 354, as shown in FIG. 30.

Referring now to FIG. 20, the lancet ampoule 40b associated with the deformable compartment 40a collapses by means of an external actuator (e.g., the compression mechanism 760c) to open the compartment to the fifth inlet port 144, and then the deformable compartment 40a collapses by means of an external actuator (e.g., the compression mechanism 756c) to force the fluid contents out to the fifth inlet port 144. The fluid contents flow from the fifth inlet port 144 to a second outlet 188 by means of a seventh fluid channel 166. In one embodiment, the fluid contents of the deformable compartment 40a comprise a rehydration or elution buffer that flows from the second outlet port 188 to the rehydration buffer compartment 276 of the reaction module 240 by means of the inlet 278, as shown in FIG. 31 and is described above. The same buffer solution contained in the deformable compartment 40a can be used both for the rehydration of dry or lyophilized reagents or other substances and for the elution of nucleic acid or other target analyte from a substrate to which it binds.

Referring now to FIG. 21, in a further stage, the active valve assembly 204 is closed by means of an external actuator (for example, the valve actuator 762b) that pushes the valve down. The pneumatic pump coupled to pump port 104 is activated to pressurize mixing well 90 via pressure conduit 106, a portion of the first fluid channel 150, the second fluid channel 152, the third fluid channel 156, and a portion of the fifth fluid channel 162. Simultaneously, passive valve port 108 closes to allow a pressure buildup in mixing well 90, which will actuate passive valve assembly 220. This opens passive valve 220, allowing the fluid contents of mixing well 90 to flow, via channels 92 and 172, through the capture compartment 100. The fluid flowing through capture compartment 100 flows through the thirteenth fluid channel 178, but is prevented by the closed active valve assembly 204 from flowing into the fourteenth fluid channel 180. The active valve 219 remains open, so that the fluid within the thirteenth fluid channel 178 flows to the tenth fluid channel 172 and to the waste chamber 102. While the fluid is flowing through the capture compartment 100, the contents are subjected to a magnetic force, for example, by placing an external magnet (for example, by deploying the sample preparation magnet assembly 570) in proximity to the capture compartment 100. The magnetic force retains the target capture magnetic microspheres and the target analyte(s) (for example, nucleic acid(s)) bound to them within the capture compartment 100, while the remainder of the contents of the mixing well 90 flows through the capture compartment 100 and to the waste chamber 102.

With reference now to FIG. 22, in a further step, the valve assembly 204 remains closed and the valve assembly 219 remains open, and the lancet ampoule 42b associated with the deformable compartment 42a collapses (for example, by compression mechanism 760d) to thereby open the compartment to the sixth inlet port 146. The deformable compartment 42a then partially collapses (for example, by compression mechanism 756d) to dispense a portion (for example, approximately 50%) of its contents into the sixth inlet port 146. In one embodiment, the fluid contents of the deformable compartment 42a comprise a washing buffer that flows from the sixth inlet port 146 via the twelfth fluid channel 176 to the capture compartment 100. The washing fluid flows over the capture microspheres that are immobilized (for example, by a magnet) within the capture compartment 100 and flows to through channels 178, 172 and 174 to waste chamber 102 to thereby transport unbound material and other debris to waste chamber 102.

Referring now to FIG. 23, in a further step, the waste valve assembly 219 is closed by means of an external actuator (for example, the valve actuator 762a), and the sample valve assembly 204 is opened by removing the external actuator. Next, the remainder of the deformable compartment 42a collapses due to the external actuator (e.g., the compression mechanism 756d), thereby forcing the remaining fluid (e.g., a wash buffer) out through the twelfth fluid channel 176 into the capture compartment 100. The magnetic force is removed from the capture compartment 100 (e.g., by retracting the sample preparation magnet assembly 570) so that the magnetic microspheres within the capture compartment 100 are released and can be carried by the fluid flowing through the capture compartment 100, through the thirteenth fluid channel 178, through the sample valve assembly 204, and the fourteenth fluid channel 180 to a third outlet 190. The fluid flowing from the third outlet 190, now comprising at least partially purified target analyte carried on the magnetic microspheres, is dispensed. in the sample compartment 266 of the reaction module 240 by means of inlet 268, as shown in FIG. 31 and described above.

In FIGS. 56 and 57, each of the cam follower ribs formed in the cam grooves 850-860 of the cam follower plate 820 is indicated by a unique number in parentheses (1)–(14). As the cam follower plate 820 moves relative to the cam arm plate 852 in direction "A", the cam follower ribs formed in various cam grooves 850–860 contact the compression mechanisms of the actuator array 754 in a predetermined sequence to open the various reagent chambers and dispense their contents and actuate the various active values in a specified sequence. The numbers in parentheses assigned to the cam follower ribs in FIGS. Figures 56 and 57 indicate the sequence in which each rib comes into contact with an associated cam arm of the die compression mechanisms 754 to actuate the compression mechanisms in a sequence corresponding to the sample preparation procedure performed in the sample preparation module 70 as described above and shown in Figures 16-23. Table 1 below shows the correspondence between each rib of the cam follower of the cam follower plate 820, the procedure stage, the corresponding compression mechanism, and the compression-collapsible chamber or active valve of the manifold cartridge 10 for the procedure shown in Figures 16-23.

Table 1

* Step (2) is optional and can be omitted if the magnetic microspheres are supplied directly, for example, by means of a freeze-dried pellet, in mixing well 90.

Sample reaction procedure

The sample material dispensed from sample processing module 70 into sample compartment 266 of reaction module 268 is subjected to a reaction procedure with reaction module 240. In an exemplary embodiment, that reaction procedure includes PCR amplification and analyte detection.

An exemplary procedure will be described with reference to flowchart 900 in FIG. 60. Although the various elements (steps) of flowchart 900 in FIG. 60 are shown as sequential steps in a prescribed order, it should be understood that procedure 900 as illustrated is exemplary and not intended to be limiting. Those skilled in the art will recognize that many of the various elements (steps) of procedure 900 may be performed in different orders than those shown and described herein, may be performed simultaneously or substantially simultaneously with other elements (steps), or may be omitted altogether. Therefore, the order of the elements (steps) as shown in FIG. 60 should not be viewed as limiting unless a specific order for two or more elements (steps) is specifically prescribed or otherwise suggested by the context of the description (e.g., a mixture must be formed first before that mixture can be incubated or otherwise manipulated).

In step S1, an aliquot of the elution/reconstitution buffer (e.g., 15 ll) is dispensed by electrowetting nanodroplet manipulation from the rehydration buffer zone 372 (FIG. 59) (and the rehydration buffer compartment 376 of the upper plate 241 (FIGS. 26, 27)) to an electrowetting pathway defining the exonuclease zone 384 (FIG. 59).

As stated above, in one mode of implementation of the disclosure, the region of the reaction module 240 between the top plate 241 and the fluidic processing panel 354 can be filled with a process fluid, such as an immiscible fluid such as oil, and the nanodroplets are manipulated through the oil.

In step S2, an aliquot of the sample mixture (comprising magnetic microspheres with DNA material attached thereto and wash solution from sample preparation module 70) is retained by electrowetting manipulation within the sample microsphere zone 368 (FIG. 59) (and the sample compartment 266 of the upper plate 241 (FIGS. 26, 27)), while the magnetic microspheres are extracted from the aqueous solution contained within the sample microsphere zone 368 by means of a magnet focused on position 369 (referred to as the microsphere collection area). The microsphere collection area 369 corresponds to the position of the focusing magnet 558 of the cartridge magnet assembly 552 (see FIG. 49B) adjacent to the fluid processing panel 554 of the multi-cartridge 10 when the cartridge magnet assembly 552 is in the deployed position. During the magnetic microsphere collection procedure in the microsphere collection area 369, the aqueous solution can be moved throughout the sample microsphere zone 368 by selectively activating different electrowetting pads to move the aqueous nanodroplets containing the magnetic microspheres to positions in closer proximity using magnetic force in the microsphere collection area 369.

In step S3, the sample residues (i.e., the wash buffer and other materials from which the magnetic microspheres were removed in step S2) are retained by electrowetting nanodroplet manipulation within the sample microsphere zone 368 (and sample compartment 266), thereby separating the magnetic microspheres, and the target analyte material bound to them, from the other constituent substances of the sample microsphere mixture that was supplied from the sample preparation module 70 to the sample microsphere zone 368.

In step S4, a quantity of the reconstitution buffer dispensed from the rehydration buffer zone 372 in step S1 can be moved by electrowetting nanodroplet manipulation to the PCR reagent zone 376 (FIG. 59) (and the buffer compartment 296 of the upper plate 241 (FIGS. 26, 27)). Resuspension of the dry PCR reagent contained within the PCR reagent zone 376 is achieved by oscillating movements of the nanodroplets between the electrowetting pads within the PCR reagent zone 376.

In step S5, a quantity of the reconstitution buffer dispensed from the rehydration buffer zone 372, which was not conveyed to the PCR reagent zone 376, is conveyed by electrowetting nanodroplet manipulation onto the magnetically held microspheres in the microsphere collection area 369 for a final microsphere wash. After the final microsphere wash, the reconstitution buffer is then moved by electrowetting nanodroplet manipulation to one end of the central pathway corresponding to the exonuclease zone 384, where it is held by electrowetting nanodroplet manipulation separate from the magnetic microspheres contained in the microsphere collection area 369.

In step S6, the reconstituted PCR buffer within PCR reagent zone 376 is distributed by electrowetting nanodroplet manipulation to the primer cocktail positions of each of the thermal cycling tracks 364a, 364b, 364c, and 364d. A primer cocktail position 366a at a proximal end of thermal cycling track 364d is marked in FIG. 59. Each of the other thermal cycling tracks 364a, 364b, and 364c has a similar primer cocktail location. Combining the reconstituted PCR reagent with the dried primer cocktail at the primer cocktail position (e.g., position 366) reconstitutes the primer cocktail at that position. In this configuration, reaction module 240 is configured to perform a PCR reaction on each of the thermal cycling tracks 364a, 364b, 364c, and 364d.

In an alternative embodiment, a primer cocktail can also be provided at the distal end of each thermal cycling track 364a, 364b, 364c, and 364d. A primer cocktail position 366b at a distal end of thermal cycling track 364d is marked in Figure 59. Each of the other thermal cycling tracks 364a, 364b, and 364c can have a similar primer cocktail location. In this configuration, the reaction module 240 is configured to perform two PCR reactions on each of the thermal cycling tracks 364a, 364b, 364c, and 364d.

In step S7, the magnetic force is removed from the microsphere collection area 369 (e.g., by moving the cartridge magnet assembly 552 to its retracted position). The reconstitution/elution buffer is moved by electrowetting nanodroplet manipulation from the central pathway 384 to the microsphere collection area 369, and a mixture of the magnetic microspheres and the reconstitution/elution buffer from the rehydration buffer zone 372 is moved back and forth along pathway 384 by electrowetting nanodroplet manipulation to elute the DNA material (or other target analyte) from the magnetic microspheres.

After a sufficient elution period, in step S8, the cartridge magnet assembly 552 is deployed again to apply a magnetic force (by means of the focusing magnet 558) to the microsphere collection area 369 to attract and retain (immobilize) the magnetic microspheres from which the DNA material has been eluted, and the eluted DNA material is transferred by electrowetting nanodroplet manipulation to a PCR staging area at a proximal end of each of the thermal cycling tracks 364a, 364b, 364c, and 364d. In the embodiment and orientation shown in Fig. 59, the PCR staging area is at the left end of the thermal cycling tracks 364a, 364b, 364c, and 364d.

In step S9, PCR nanodroplets, comprising the eluted DNA material, reconstituted PCR reagent, and reconstituted PCR primer, are formed by electrowetting nanodroplet manipulation in the PCR staging area of each of the thermal cycling tracks 364a, 364b, 364c, and 364d. Each PCR nanodroplet is moved to a corresponding one of the thermal cycling tracks 364a, 364b, 364c, and 364d, and a PCR procedure is performed by moving the nanodroplets between two of the PCR (thermal cycling) regions 382a (at approximately, for example, 60 °C for hybridization and extension) and 382b (at approximately, for example, 95 °C for denaturation) or 382c (at approximately 60 °C for hybridization and extension) and 382b (at approximately, for example, 95 °C for denaturation). In another embodiment, two PCR nanodroplets are delivered to each thermal cycling track 364a, 364b, 364c, and 364d, and one nanodroplet is moved between heater areas 382c and 382b, while the other nanodroplet is moved between heater areas 382a and 382b. The PCR procedure can take approximately 40 minutes or less.

In step S10, an amount of elution/reconstitution buffer is dispensed by electrowetting nanodroplet manipulation from the rehydration buffer zone 372 and transported by electrowetting nanodroplet manipulation to the exonuclease reagent zone 374 (FIG. 59) (and the second buffer compartment 300 of the upper plate 241 (FIG. 26, 27)) for resuspension of the dry exonuclease reagent. The resuspension of the dry exonuclease reagent contained within exonuclease reagent zone 374 is achieved by oscillating movements of the nanodroplets between the electrowetting pads within exonuclease reagent zone 374. The reconstituted exonuclease reagent is then transported by electrowetting nanodroplet manipulation from exonuclease reagent zone 374 to the PCR staging areas of thermal cycling track 364a, 364b, 364c, and 364d.

In step S11, after PCR (step 9), each nanodroplet that has undergone the PCR procedure is combined with an amount of the exonuclease agent resuspended in step S10, transported by electrowetting nanodroplet manipulation to exonuclease zone 384, and held in a separate location within exonuclease zone 384. In various embodiments, an amount of elution/reconstitution buffer from buffer zone 372 is added to each PCR nanodroplet by electrowetting nanodroplet manipulation to bring the total volume of each nanodroplet to a preferred amount.

In step S12, the nanodroplet mixtures formed in step S11, comprising the PCR products and the reconstituted exonuclease reagent, are then incubated within the exonuclease region 380 and the exonuclease zone 384 at a prescribed temperature and for a prescribed time period.

In step S13, the detection reagent within the hybridization zone 370 (FIG. 59) (and the detection buffer compartment 280 of the upper plate 241 (FIGS. 26, 27)) is reconstituted with an amount of rehydration buffer from the rehydration buffer zone 372. In one embodiment, an amount of rehydration buffer from the rehydration buffer zone 372 is moved by means of electrowetting nanodroplet manipulation through the connecting step 274 (FIGS. 26, 27) between the detection buffer compartment 280 and the rehydration buffer compartment 276.

In step S14, a quantity of the reconstituted detection reagent (e.g., 25 l) from hybridization zone 370 is combined by electrowetting nanodroplet manipulation with each of the PCR nanodroplets. Each PCR nanodroplet is then combined with a signal probe cocktail stored in positions 362a, 362b, 362c, and 362d of fluid processing panel 354. To effect mixing of the PCR nanodroplet and the signal probe cocktail, and to resuspend the signal probe cocktail, each nanodroplet can be transported by electrowetting nanodroplet manipulation around or into one of the detection mixing zones 385a, 385b, 385c, and 385d.

In step S15, the nanodroplets are transported by electrowetting manipulation to the electrosensor arrays 363a, 363b, 363c and 363d, where they undergo further incubation within the detection region 378 and various analytes of interest are detected by electrodeception techniques, as described above and/or described in the publications listed above.

Claims (15)

1. A fluid sample processing cartridge (10) comprising: a substrate (72); a sample well (78) formed in the substrate configured to receive a sample volume of fluid and comprising a closure (84) configured to be selectively placed over the sample well; a deformable fluid chamber supported on the substrate; a mixing well (90) formed in the substrate; and a powered mixing apparatus disposed within the mixing well and constructed and arranged to mix the contents of said mixing well, in which The deformable fluid chamber is configured to contain a fluid therein when in an undeformed state and to collapse upon the application of an external compressive force to expel at least a portion of the fluid from the fluid chamber, said deformable fluid chamber being in fluid communication with said sample well (78) by means of a channel formed in said substrate; and in which The mixing well (90) is in fluid communication with said sample well by means of a channel formed in said substrate said mixing well (90) comprising a first peripheral wall (91) and a first floor (93) defining a well and a fluid inlet snorkel (92) extending upwards along one side of the first peripheral wall extending from the channel connecting the mixing well to the sample well and terminating below an upper edge of the first peripheral wall. 2. The fluid sample processing cartridge (10) of claim 1, wherein said fluid inlet snorkel (92) extends to an outer surface of the first peripheral wall (91) and terminates in an opening formed in the first peripheral wall. 3. The fluid sample processing cartridge (10) of claim 1 or claim 2, wherein said sample well (78) comprises a second peripheral wall and a second floor defining a well and a fluid inlet snorkel extending upwards along a side of the second peripheral wall and terminating below an upper edge of the second peripheral wall. 4. The fluid sample processing cartridge (10) of any of claims 1-3, wherein said mixing well (90) further comprises an outlet port (96) comprising one or more openings formed in the second floor (93), wherein said floor tapers downwards towards said outlet port (96). 5. The fluid sample processing cartridge (10) of any of the preceding claims, wherein said driven mixing apparatus comprises a first impeller (202) rotatably arranged within said mixing well (90) and a gear configured to be driven by a mating gear of an instrument into which the liquid sample processing cartridge is inserted and to rotate the first impeller when engaged by the mating gear. 6. The fluid sample processing cartridge (10) of any one of claims 1 to 5, further comprising: a lysis chamber (120) containing a plurality of lysis microspheres, said lysis chamber being formed in said substrate and disposed along the channel connecting said mixing well (90) and said sample well (78), such that the fluid flowing from the sample well to said mixing well flows through said lysis chamber; and a microsphere mixer disposed at least partially within said lysis chamber and constructed and arranged to agitate the lysis microspheres and the fluid flowing through said lysis chamber; preferably, including also: a first optical interface comprising an enlarged portion of the channel connecting said lysis chamber to said sample well; and a second optical interface comprising an enlarged portion of the channel connecting said lysis chamber to said mixing well. 7. The fluid sample processing cartridge (10) of claim 6, wherein said microsphere mixer comprises: a motor (128) mounted within said substrate; and a second impeller arranged within said lysis chamber (120) and mounted on an output shaft of said motor. 8. The fluid sample processing cartridge (10) of claim 6 or 7, wherein said lysis chamber (120) includes a fluid inlet (122) and a fluid outlet (124) and further comprises a mesh filter disposed over each of said fluid inlet and fluid outlet and configured to retain said lysis microspheres within said lysis chamber. 9. The fluid sample processing cartridge (10) of any one of claims 1 to 8, further comprising: a pressure port formed in said substrate and configured to couple said substrate to an external fluid pressure source; and a channel formed in said substrate that connects said pressure port to said sample well (78). 10. The fluid processing cartridge (10) of any one of claims 1 to 9, further comprising: a waste chamber (102) formed in said substrate, said waste chamber being in fluid communication with said mixing well (90) by means of a channel formed in said substrate; a fluid outlet port formed in the substrate, said fluid outlet port being in fluid communication with said mixing well by means of a channel formed in said substrate; a first externally actuatable control valve disposed within said substrate and constructed and arranged to selectively permit or prevent fluid flow from said mixing well to said waste chamber; and a second externally actuatable control valve disposed within said substrate and constructed and arranged to selectively permit or prevent fluid flow from said mixing well to said fluid outlet port; preferably, which further comprises a capture chamber arranged along a channel connecting said mixing well and said waste chamber. 11. The fluid processing cartridge (10) of any one of claims 1 to 10, further comprising: a passive valve assembly (220) disposed within said substrate and constructed and arranged to close and prevent fluid flow from said mixing well when the pressure within said mixing well is not greater than a threshold pressure and to open and permit fluid flow from said mixing well when the pressure within said mixing well rises above the threshold pressure; and a pressure port (104) formed in said substrate and in pressure communication with said passive valve assembly by means of a pressure conduit formed in said substrate, wherein when said pressure port is closed, the pressure inside said mixing well is allowed to reach the threshold pressure which will open said passive valve assembly and allow fluid flow from said mixing well, and when said pressure port is open, the pressure inside said mixing well cannot reach the threshold pressure, so the passive valve assembly is closed. 12. The fluid processing cartridge (10) of any one of claims 1 to 11, further comprising a lancet ampoule (34b, 36b, 38b, 40b, 42b) associated with said deformable fluid chamber; said lancet ampoule being connected or capable of being connected to the associated deformable fluid chamber (34a, 36a, 38a, 40a, 42a) and containing a microsphere retained within the lancet ampoule by means of a breakable septum, wherein said lancet ampoule is configured to collapse upon application of an external compressive force to thereby push the microsphere through the breakable septum. 13. The fluid processing cartridge (10) of any one of claims 1 to 12, further comprising an outer envelope (12) externally enclosing at least a portion of said cartridge. 14. The fluid sample processing cartridge (10) of any one of claims 1 to 13, further comprising: a first fluid outlet port (182) formed in the substrate, said first fluid outlet port being in fluid communication with said mixing well (90) by means of a channel formed in said substrate; a second fluid outlet port (188) formed in the substrate; and at least two deformable fluid chambers, one of said two deformable fluid chambers being in fluid communication with said mixing well by means of a channel formed in said substrate, and the other of said two deformable fluid chambers being in fluid communication with said second fluid outlet port by means of a channel formed in said substrate that is different from the channel that connects the first fluid outlet port with said mixing well. 15. The fluid sample processing cartridge (10) of claim 14, wherein the deformable fluid chamber in fluid communication with the mixing well (90) contains a lysis buffer, a washing buffer, target capture microspheres or a bonding buffer, and the deformable fluid chamber in fluid communication with the second fluid outlet contains an oil or a rehydration buffer.