HK1249475B - Microarray based sample detection system - Google Patents

Description

Microarray-based sample detection system

This application is a divisional application filed pursuant to a notice of first examination regarding lack of unity issued on November 2, 2016 (Document Serial No.: 2016102800861850). This application is based on a divisional application entitled “Microarray-based sample detection system,” filed with Application No. 201510581766.8, filed on April 13, 2012, and with a priority date of April 13, 2011. The original application is entitled “Microarray-based sample detection system,” filed with Application No. 201280018067.7, filed on April 13, 2012. This application claims priority to U.S. Provisional Patent Application No. 61/475,107, filed on April 13, 2011. The entire text of the aforementioned application is incorporated herein by reference.

field

The technical field is microfluidic systems, and in particular, microfluidic systems having microarrays for sample testing.

background

Microarrays are most popular in research laboratories as a tool for profiling gene expression levels because thousands of samples can be taken to investigate a single sample. Their practicality is not as ubiquitous as in clinical diagnostics, environmental, and agricultural applications, despite their information density, redundancy, embedded controls (positive and negative), and analytical sensitivity. The main obstacles to adopting microarrays as diagnostic tests are the complexity and cost of their operation (typically hundreds of dollars per test) and technical problems associated with microfluidic devices containing microarrays, such as unpredictable fluid flow behavior caused by bubbles in the microfluidic device. For example, bubbles may block channels, interfere with biochemical reactions (especially those requiring surface reactions), cause incorrect dosing, interfere with optical readings, and lead to unpredictable flows. Unpredictable flows are particularly problematic for systems that rely on the stable diffusion of analytes to binding ligands such as oligonucleotides or capture antibodies. Therefore, there is still a need for microfluidic detection systems based on microarrays that are designed to provide predictable fluid flow and can be manufactured at low cost.

Overview

One aspect of the present application relates to a microarray assembly for detecting target molecules in a sample. In one embodiment, the microarray assembly comprises: an array chamber having an inlet at a first end, an outlet at a second end, an inner top surface, an inner bottom surface, a plurality of sidewalls, and a microarray located on the inner bottom surface; and a waste sample chamber in fluid communication with the outlet of the array chamber, wherein the array chamber comprises a hydrophilic inner surface positioned to facilitate complete filling of the array chamber with a water-based fluid and facilitate continuous flow of the fluid from the inlet to the outlet, wherein the cross-sectional area at the first end of the array chamber is greater than the cross-sectional area at the second end of the array chamber.

In other embodiments, the microarray assembly includes: an array chamber having an inlet, an outlet, an inner top surface, an inner bottom surface, multiple side walls, and a microarray located on the inner bottom surface; a waste sample chamber including a waste sample inlet and an absorbent material; and a channel having an expansion section, the expansion section having a first end near the outlet of the array chamber and a second end near the inlet of the waste sample chamber, wherein the inner top surface is a hydrophilic surface that helps the aqueous fluid completely fill the array chamber, and the cross-sectional area at the first end of the expansion section is smaller than the cross-sectional area at the second end of the expansion section.

In other embodiments, the microarray assembly includes: an array chamber having an inlet at a first end, an outlet at a second end, an inner top surface, an inner bottom surface, a plurality of side walls, and a microarray located on the bottom surface; a waste sample chamber connected to the outlet fluid of the array chamber, wherein the array chamber includes a hydrophilic inner surface positioned to help the aqueous fluid completely fill the array chamber and includes a plurality of channels having a rectangular cross-section formed in the inner bottom surface and/or the inner top surface to promote drying.

Another aspect of the present application relates to a method for controlling the manufacturing quality of array elements in a microarray. The method comprises the following steps: irradiating a microarray having a plurality of array spots with light waves to generate fluorescence from each array spot; measuring the intensity of the fluorescence for each array spot, wherein the fluorescence is generated by an internal quality control fluorescent group; generating a fluorescence image of the microarray; determining information about each array spot based on the fluorescence image; and encoding the information in a barcode, a storage device, or an RFID tag, wherein the barcode, the storage device, or the RFID tag corresponds to the microarray.

Another aspect of the present invention relates to a method for manufacturing a microarray assembly. The method comprises the following steps: unwinding a base film from one or more base film reels; printing a microarray onto the unwound base film; laminating a liner film onto the printed base film, wherein the liner film is pre-cut to provide space for the array cavity prior to the placement step and is placed onto the printed base film via one or more liner film reels; laminating a cover film onto the liner film to form a multi-layer microarray structure; and cutting the multi-layer microarray structure into individual microarray assemblies.

A microarray assembly for detecting target molecules in a sample, the microarray assembly comprising: an array chamber having an inlet at a first end, an outlet at a second end, an inner top surface, an inner bottom surface, a plurality of side walls, and a microarray located on the inner bottom surface; and a waste sample chamber in fluid communication with the outlet of the array chamber, wherein the array chamber comprises a hydrophilic inner surface positioned to facilitate complete filling of the array chamber with a water-based fluid and facilitate continuous flow of the fluid from the inlet to the outlet, and

The cross-sectional area at the first end of the array cavity is greater than the cross-sectional area at the second end of the array cavity.

According to a preferred embodiment, the array cavity is in the form of a tubular channel, and the cross-sectional area of the array cavity continuously decreases from the first end toward the second end.

According to a preferred embodiment, the cross-sectional area of the array cavity decreases step by step from the first end toward the second end.

According to a preferred embodiment, the cross-sectional area of the array cavity at the first end is three times greater than the cross-sectional area of the array cavity at the second end.

According to a preferred embodiment, the microarray includes a plurality of array spots arranged in a single row extending from a first end to a second end of the array chamber.

According to a preferred embodiment, the microarray includes a plurality of parallel array strips perpendicular to the sample flow direction in the array chamber.

A microarray assembly for detecting target molecules in a sample, the microarray assembly comprising: an array chamber having a sample inlet, a sample outlet, an inner top surface, an inner bottom surface, a plurality of side walls, and a microarray located on the inner bottom surface; a waste sample chamber including a waste sample inlet and an absorbent material; and a channel having an expansion section, the expansion section having a first end proximate to the outlet of the array chamber and a second end proximate to the inlet of the waste sample chamber.

The inner top surface is a hydrophilic surface that helps the aqueous liquid completely fill the array cavity, and the cross-sectional area at the first end of the expansion section is smaller than the cross-sectional area at the second end of the expansion section.

According to a preferred embodiment, the cross-sectional area of the expansion section of the channel increases step by step from the first end to the second end.

According to a preferred embodiment, the channel further comprises a zigzag section between the expansion section and the inlet of the waste chamber.

According to a preferred embodiment, the meandering section comprises two sharp bends in the channel.

A microarray assembly for detecting target molecules in a sample, the microarray assembly comprising: an array chamber having an inlet at a first end, an outlet at a second end, an inner top surface, an inner bottom surface, a plurality of side walls, and a microarray located on the bottom surface; and a waste sample chamber in fluid communication with the outlet of the array chamber; wherein the array chamber comprises a hydrophilic inner surface and a plurality of channels, the hydrophilic inner surface being positioned to facilitate complete filling of the array chamber with a water-based fluid, the plurality of channels having rectangular cross-sections formed in the inner bottom surface and/or the inner top surface to facilitate drying.

According to a preferred embodiment, the channels with rectangular cross sections are perpendicular to the fluid flow direction in the array cavity, and the fluid in the array cavity flows in a direction from the first end toward the second end of the array cavity.

A microarray assembly for detecting target molecules in a sample, comprising: an array chamber having an inlet, an outlet, a top surface, a bottom surface, and a plurality of side walls; and a gel spot microarray located on the bottom surface of the array chamber; wherein the gel spot array comprises a plurality of gel spots, and each gel spot comprises an internal control fluorescent group and a capture agent that specifically binds to the target molecule.

A method for controlling the manufacturing quality of array elements in a microarray, the method comprising: illuminating a microarray having a plurality of array spots with a light wave so as to generate fluorescence from each array spot; measuring the intensity of the fluorescence for each array spot, wherein the fluorescence is generated by an internal quality control fluorescent group; and generating a fluorescence image of the microarray;

determining information of each array spot based on the fluorescent image; and encoding the information into a barcode, a storage device, or an RFID tag, wherein the barcode, the storage device, or the RFID tag is associated with the microarray.

According to a preferred embodiment, the information of each array point includes the position of each point.

According to a preferred embodiment, the information of each array point includes the fluorescence intensity of each point.

According to a preferred embodiment, the information of each array point includes the diameter of each point.

According to a preferred embodiment, the information of each array point includes the morphology of each point.

A microarray image analysis method comprising: placing a plurality of fixed spot boundary circles for a plurality of microarray spots on an image of a microarray; and measuring target fluorescence intensity within the plurality of fixed spot boundary circles for the plurality of microarray spots;

The plurality of fixed point boundary circles are placed on the array image based on the above-determined information.

A microarray image analysis method comprises: determining the target fluorescence intensity of a target in a microarray; determining the internal fluorescence intensity of the target in the microarray; and determining the signal intensity of the target in the microarray, wherein the signal intensity is the ratio of the target fluorescence intensity to the internal fluorescence intensity, wherein the internal fluorescence intensity of the target in the microarray is determined using the above method.

A method for manufacturing a microarray assembly comprises: unwinding a base film from one or more base film reels; printing a microarray onto the unwound base film; and laminating a liner film onto the printed base film, wherein the liner film is pre-cut to provide space for an array cavity before the placement step, and the liner film is placed onto the printed base film via one or more liner film reels.

laminating a cover film on the backing film to form a multi-layer microarray structure; and cutting the multi-layer microarray structure into a plurality of individual microarray components.

According to a preferred embodiment, the liner film is pre-cut to provide space for one or more waste chambers.

According to a preferred embodiment, the liner film is pre-cut to provide space for one or more array cavities.

According to a preferred embodiment, the printing step is performed on a moving base film on a production line.

According to a preferred embodiment, the base film is a hydrophobic film, and the cover film is a hydrophilic film.

Brief introduction of the attached figure

The detailed description will refer to the following drawings:

Figure 1A is a schematic diagram of an embodiment of a microarray assembly comprising a reservoir, an array chamber of decreasing cross-section, a spot array, a waste chamber, and absorbent material. Figure 1B is a cross-sectional view of the array assembly of Figure 1A.

2 is an enlarged view of an array chamber showing a linear array of dots printed on a chamber floor having decreasing cross-sectional area.

FIG. 3 is a diagram of a microarray assembly having an expansion channel connecting the array chamber to the waste chamber.

FIG4A is a schematic diagram illustrating an array chamber having small rectangular channels perpendicular to the direction of liquid flow within the chamber. FIG4B is a schematic diagram illustrating an array chamber having small rectangular channels parallel to the direction of liquid flow within the reaction chamber. FIG4C is a schematic diagram illustrating an array chamber having small rectangular channels perpendicular to or parallel to the direction of liquid flow within the reaction chamber. FIG4D is a schematic diagram illustrating an array chamber having small rectangular channels that form an angle relative to the direction of liquid flow within the chamber.

FIG5 shows a schematic diagram of a continuous assembly line for manufacturing an on-membrane test device.

FIG6 shows an array image comprising a dilution series of Cy5 and Cy3 spots.

Figure 7 shows an image of a blunt needle print head.

Figure 8 shows bright field images of an array printed on polyester film with a vacuum manifold before and after polymerization, as well as fluorescent images of a Cy3 array.

Figure 9 shows a picture of a thin film vacuum manifold used for blunt needle printing.

FIG10 shows a fluorescent image after PCR of a material combined with a rollable material comprising a polyester film printed with an array.

11 is a combination of photographs showing a roll-to-roll printing configuration including a BioDot Ultra Non-Contact array printer (top panel) and a video frame of printing with the BioDot Ultra Non-Contact on a moving membrane that has not been chemically treated or modified (bottom panel).

FIG12 shows a red channel fluorescence image of the MRSA array taken in factory QC for extraction point parameters.

Figure 13 shows a green channel fluorescence image of a hybrid array imaged with an end-user imager. The imager software uses the array QC data to place a grid and a circle around each individual spot.

FIG14 shows a fluorescence image of a hybrid array imaged with end-user equipment, but without the use of QC data, making placement of the grid and the circles around each individual point more challenging.

Specific instructions

This specification is to be read in conjunction with the accompanying drawings, which are to be considered part of the complete written description of the invention. The drawings are not necessarily to scale, and certain features of the invention may be shown exaggerated in proportion or slightly schematically for the sake of clarity and conciseness. In the specification, relative terms such as "front", "rear", "upper", "lower", "top", and "bottom" and their derivatives should be interpreted as referring to the orientation described subsequently or as shown in the introduced figures. Relative terms are used for convenience of description and are not generally intended to require a specific orientation. Terms such as "connected" and "attached" with respect to "attachment", "engagement", etc. refer to relationships in which these structures are fastened or attached to each other directly or indirectly through intermediate structures, as well as attachments or relationships that are removable or rigid, unless otherwise expressly stated.

As used herein, the term "microarray" refers to an array of ordered spots representing binding sites of interest. A microarray is composed of at least two spots. Interesting sites include, but are not limited to, nucleic acids (e.g., molecular beacons, aptamers, locked nucleic acids, peptide nucleic acids), proteins, peptides, polysaccharides, antibodies, antigens, viruses, and bacteria.

As used herein, the term "hydrophilic surface" refers to a surface that forms a contact angle of 45° or less with a drop of pure water resting on such surface. As used herein, the term "hydrophobic surface" refers to a surface that forms a contact angle greater than 45° with a drop of pure water resting on such surface. Contact angles can be measured using a contact angle goniometer.

As used herein, the term "array chamber" refers to the enclosed space surrounding a microarray that is in direct or indirect fluid communication with an inlet and an outlet. The array chamber, when filled with a liquid sample, allows the microarray to be immersed in the liquid sample so that target molecules in the liquid sample can maintain close contact with the microarray sample.

Microarray systems designed to facilitate fluid flow within the system

A scheme of the present application relates to a kind of detection system based on microarray, it comprises a microarray assembly, and this microarray assembly comprises an array chamber with an injection port, an injection port and the microarray therein, and a waste sample chamber connected with the array chamber fluid.The array chamber has a hydrophilic surface positioned to help fill the array chamber completely and for fluid to flow from the array chamber to the waste sample chamber.The hydrophilic surface contacts the liquid when the liquid enters the array chamber from the injection port and allows the array chamber to be filled completely.In certain embodiments, the array chamber is in the shape of an elongated channel with variable width, which is directly connected to the waste sample chamber.In other embodiments, the array chamber is connected to the waste sample chamber through a waste sample channel.

The surface tension of a liquid sample or reaction mixture typically prevents it from completely filling a small space, such as the array cavity of a microarray system. Surface tension is the product of the mutual attraction between liquid sample molecules using various intermolecular forces. In this type of liquid sample, each molecule is equally pulled in all directions by adjacent liquid molecules, resulting in a net force of zero. On the surface of the liquid sample, many molecules are pulled inward by other molecules deeper within the liquid, without being equally strongly attracted by molecules in the adjacent medium (assuming it is a vacuum, air, or other fluid). Therefore, all molecules on the surface are subject to inward molecular attraction, which can only be balanced by the liquid sample's resistance to compression. The inward pull tends to reduce the surface area, and in this respect, the liquid surface is like a stretched elastic membrane. Therefore, the liquid squeezes itself together until it has the possible local minimum surface area. The end result is that the liquid sample can remain nearly spherical in a small space and does not fill corners, especially square corners of the small space. The typical small gap separating the cover from the microarray surface in the array cavity usually compresses the liquid into a columnar shape.

In the case of a microarray system, the liquid filling the microarray cavity is most likely an aqueous solution, such as a hybridization buffer or a wash buffer. The surface tension of the aqueous solution can be overcome by coating at least a portion of the inner surface of the array cavity with a hydrophilic material. In certain embodiments, the microarray is located on the bottom surface of the array cavity, and the top surface of the array cavity, or at least a portion of the top surface, is coated with a hydrophilic coating.

Examples of hydrophilic materials include, but are not limited to, hydrophilic polymers such as polyethylene glycol, polyhydroxyethyl methacrylate, Bionite, poly(N-vinyl lactam), poly(vinyl pyrrolidone), poly(ethylene oxide), poly(propylene oxide), polyacrylamide, cellulose, methylcellulose, polyanhydrides, polyacrylic acid, polyvinyl alcohol, polyvinyl ether, alkylphenol polyoxyethylene ether, complex polyol monoesters, polyoxyethylene oleate, polyoxyethylene sorbitan monooleate, and sorbitan fatty acid esters; inorganic hydrophilic materials such as inorganic oxides, gold, zeolites, and diamond-like carbon; and surfactants such as polyethylene glycol octylphenyl ether (Triton X-100), polysorbate (Tween), sodium lauryl sulfate (SDS ), ammonium lauryl sulfate, alkyl sulfates, sodium laureth sulfate (SLES), alkyl benzene sulfonate, soap, fatty acid salts, cetyltrimethylammonium bromide (CTAB) also known as cetyltrimethylammonium bromide, alkyltrimethylammonium salts, cetylpyridinium chloride (CPC), poly-o-ethoxyaniline (POEA), benzalkonium chloride (BAC), benzethonium chloride (BZT), lauryl betaine, lauryl dimethylamine oxide, cocamidopropyl betaine, coconut ampholyglycinate, copolymers of poly(ethylene oxide) and poly(propylene oxide) (commercially known as poloxamers or poloxamines), alkyl polyglycosides, fatty alcohols, coconut monoethanolamide, coconut diethanolamide, cocamide diacetate.

In certain embodiments, at least one surfactant is mixed with a reactive polymer such as polyurethane and epoxy resin to serve as a hydrophilic coating. In other embodiments, the top or bottom surface of the array cavity is made hydrophilic by surface treatment, such as atmospheric plasma treatment, corona treatment, or gas corona treatment.

Examples of hydrophilic tapes include, but are not limited to, Adhesives Research (AR) Tape 90128, AR Tape 90469, AR Tape 90368, AR Tape 90119, AR Tape 92276, and AR Tape 90741 (Adhesives Research, Glen Rock, PA). Examples of hydrophilic membranes include, but are not limited to, Fibrefilm and FilmSpecialties, Inc., Hillsborough, NJ, and Lexan HPFAF (GE Plastics, Pittsfield, MA). Other hydrophilic surfaces are available from Surmodics, Inc. (Eden Prairie, MN), Biocoat, Inc. (Horsham, PA), Advanced Surface Technology (Billerica, MA), and Hydromer, Inc. (Branchburg, NJ).

In certain embodiments, the hydrophilic tape or membrane has a sufficiently high transparency to allow optical interrogation of the microarray from the top surface of the array chamber.

In one embodiment, the microarray is an antibody array, and the microarray system is used to capture and label target antigens. In one embodiment, the microarray is formed by the printing gel point method described in, for example, U.S. Patent Nos. 5,741,700, 5,770,721, 5,981,734, 6,656,725 and U.S. Patent Application Nos. 10/068,474, 11/425,667 and 60/793,176, all of which are incorporated herein by reference. In certain embodiments, the microarray comprises a plurality of array spots printed on the array substrate constituting the array chamber bottom surface. In certain embodiments, the array substrate is glass or plastic.

In certain embodiments, the array spots contain internal control fluorophores whose emission spectrum is different from that of the fluorophores associated with the target molecules (i.e., the target molecules will be labeled with fluorophores whose emission spectrum is different from that of the internal control fluorophores). Internal controls can be analyzed in the field or during the manufacturing process to improve quality. Internal controls will provide a quantitative means of evaluating the fluorescence intensity of the spots (e.g., mean, median, or integral), which may vary due to droplet size, morphology, porosity, or any factor that may change the reproducibility between spots. Factors that affect these properties include UV dose, temperature, surface properties, synthesis, viscosity, concentration, washing (i.e., effects caused by differences in temperature, viscosity, flow rate, pressure, or anything that may affect the removal or distortion of the spots), the immersion depth of the needle in the polymer solution in needle printing techniques, or any properties that affect the morphology of the gel element or the concentration of the sample therein. Field imaging will be subject to liability for: user misuse, gel element degradation due to poor handling and cleaning of the gel element, brightness enhancement due to the presence of salts, thermal cycling, high temperature conditions that reduce fluorescence, low temperature conditions that enhance fluorescence, shelf degradation, and/or anything that contributes to changes in fluorescence signal after initial QA/QC during array manufacturing.

Examples of fluorescent groups include, but are not limited to, pyrene, 7-methoxycoumarin, Cascade Blue, 6-MI, 3-MI, 7-aminocoumarin-X (AMCA-X), 6-MAP, Pacific Blue, Marina Blue, dimethylaminocoumarin, boron dipyrrole (BODIPY) 493/503, BODIPY-FI-X, DTAF (5-DTAF), 6-FAM (fluorescent), dansyl-X, Oregon green 500, Oregon green 488 (5 isomers), Rhodol green, Oregon green 514, rhodamine green-X, NBD-X, TET, 2'4'5'7'-tetrabromosulfonic acid fluorescent yellow, BODIPY-FI BR ₂ , BODIPY-R6G, 6-JOE, BODIPY530/550, HEX, carboxyrhodamine 6G, BODIPY558/568, BODIPY-TMR-X, PyMPO, BODIPY564/570, Cy3, TAMRA-X, Rhodamine Red-X, BODIPY576/589, BODIPY581/591, Texas Red-X, Cy3.5, ROX, BODIPY-TR, Syto-81, Cy5, Naphthalene Fluorescent Yellow, Cy5.5, VIC, SYBR green I and SYBR green II.

In other embodiments, the internal control is a colorimetric signal change that varies from spot to spot. In other embodiments, the internal control is a chemiluminescent signal change that varies from spot to spot. In other embodiments, the internal control is an electrochemical signal change that varies from spot to spot.

In certain embodiments, the array spot is a gel spot containing a first fluorescent group (e.g., Cy5). The target in the sample is labeled with a second fluorescent group (e.g., Cy3) during PCR and subsequently hybridized to a sample covalently bound to the gel drop polymer. The first fluorescent group has a different emission peak than the second fluorescent group. Under these conditions, the first fluorescent group (e.g., Cy5) is used to allow precise positioning of the gel spot using an imaging system capable of detecting the first and second fluorescent groups (e.g., Cy3 and Cy5).

In certain embodiments, the imaging system is an integral part of the microarray sample detection system. In other embodiments, the imaging system is a part of the machine observation system used when manufacturing the microarray assembly, so that the coordinates of each point can be accurately determined during observation. These coordinates are uploaded to a bar code or RFID tag attached to the microarray assembly for future analysis. To effectively implement this approach, the first fluorescent group (i.e., internal control fluorescent group) coordinates require the second fluorescent group (i.e., target fluorescent group) reference benchmark to be added as part of the combined diagram so that the grid can be placed. However, unlike traditional solutions (which either attempt to place the grid based on precisely spaced points, or require two color fluorescent group imagers), the disclosed solution uses the coordinates from the bar code to place a fixed circle for point detection. The position of the first fluorescent group (i.e., internal control fluorescent group) point can be used in conjunction with a threshold algorithm to find the center that is subsequently used for fixed circle placement.

The advantage of using machine observation to identify spots is that the same system can be used to reject multiple spots without rejecting the entire microarray, which improves efficiency. Multiple spots can be rejected based on a number of criteria, such as out-of-bounds internal control fluorescence intensity values, asymmetry, and diameter. Therefore, certain embodiments of the present invention relate to a method for controlling the manufacturing quality of array elements in a microarray, comprising: illuminating a microarray having a plurality of array spots with light waves so as to generate fluorescence from each array spot; measuring the fluorescence intensity for each array spot, wherein the fluorescence is generated by an internal quality control fluorescent group; generating a microarray fluorescence image; determining information about each array spot based on the fluorescence image; and encoding this information in a barcode, memory, or RFID tag, wherein the barcode, memory, or RFID tag is associated with the microarray. The information about each array spot can include the location of each spot, the fluorescence intensity of each spot, the diameter of each spot, and the morphology of each spot. Microarray image analysis can be performed by placing a fixed circle for each microarray spot on the microarray image using spot position information determined based on internal control fluorescence.

In one embodiment, the present application provides a microarray image analysis method. The method includes the following steps: obtaining a microarray image; placing a fixed spot boundary circle around each microarray spot on the microarray image based on array spot position information obtained by internal control fluorescence in the array spot; measuring the target fluorescence intensity within the fixed spot boundary circle for each array spot; and determining the number of target molecules in a sample based on the ratio of the target fluorescence intensity to the internal fluorescence intensity at each array spot.

In another embodiment, a microarray image analysis method includes the following steps: determining the target fluorescence intensity for the target in the microarray; determining the internal fluorescence intensity for the target in the microarray; and determining the signal intensity for the target in the microarray, wherein the signal intensity is the ratio of the target fluorescence intensity to the internal fluorescence intensity, wherein the internal fluorescence intensity of the microarray target is determined as described above.

In another embodiment, the present application provides a method for imaging array elements in a microarray. The method includes the following steps: irradiating a microarray having a plurality of array spots with a light wave of a first wavelength to generate fluorescence from an internal control fluorescent group; determining the position of the array spots of the microarray based on the fluorescence generated by the internal control fluorescent group (control fluorescence); irradiating the microarray with a light wave of a second wavelength to generate fluorescence from a target fluorescent group directly or indirectly associated with a target molecule bound to the array spot; measuring the fluorescence generated by the target fluorescent group (target fluorescence); and determining the number of target molecules in the sample based on the ratio of the control fluorescence intensity to the target fluorescence intensity in the relevant array spot.

The waste chamber can be of any shape and generally has a volume greater than the volume of the array chamber. In one embodiment, the waste chamber is formed in a gasket tape that is then attached to the substrate printed with the microarray. In another embodiment, there is a cutout on the top surface of the substrate. The size and position of the cutout can match the size and position of the waste chamber in the gasket, so that once the waste chamber is formed between the substrate and the gasket, it will have a thickness greater than the thickness of the array chamber. In another embodiment, the substrate is made of plastic so that the cutout can be easily formed on the substrate. In another embodiment, the array chamber and the waste chamber are formed in the substrate without using a gasket. However, the waste chamber can have a depth greater than the depth of the array chamber.

In one embodiment, the waste chamber contains an absorbent material that, upon contact with liquid in the array chamber, wicks the liquid away from the array chamber, thereby allowing the microarray to be primed in a dry state.

Absorbent material can be any material that can keep relatively large amount of liquid. In one embodiment, absorbent material is made of fiber agglomeration. In another embodiment, absorbent material is the nonwoven fabric made by air-through bonding method. The constituent fiber of nonwoven fabric can be natural cellulose fiber or regenerated cellulose fiber of hydrophilic synthetic fiber, slurry etc. Fiber can be coated with or be permeated with surfactant or hydrophilic oil to improve liquid absorbency. Not limited to air-through bonding method, nonwoven fabric used here can be made by any other method, for example spunbond process, air-laid process, spunlace process etc. In one embodiment, absorbent material is deriving from cellulose paper (C048) of Millipore (Billerica, MA).

In certain embodiments, the waste chamber is vented to atmosphere through an exhaust port. In one embodiment, the exhaust port is simply created by punching a hole in the waste chamber cover.

In another embodiment, the liquid in the array cavity is removed by forcing the liquid in the reservoir into the array cavity and establishing contact between the liquid in the array cavity and the absorbent material in the waste chamber. This contact can be established by applying pressure to the liquid in the array cavity to push the liquid out of the array cavity or by applying suction at the waste chamber outlet to pull the liquid out of the array cavity. The pressure acting on the liquid in the array cavity can be generated by applying pressure through a control valve (e.g., using a burette or syringe). If the array cavity is covered only with a hydrophilic tape or hydrophilic film, the pressure on the liquid in the array cavity can be simply generated by pressing the hydrophilic tape or film forming the top surface of the array cavity. Alternatively, contact between the liquid in the array cavity and the absorbent material can be established by placing the absorbent material near the array cavity so that the absorbent material contacts the liquid in the channel.

Once contact is established, the liquid in the array chamber is drawn through the array chamber into the absorbent material in the waste chamber. The liquid flow rate is determined by the array chamber dimensions, surface tension, and viscosity of the liquid, as well as the wicking rate of the absorbent material. Furthermore, the flow rate decreases as the absorbent material becomes more saturated.

In another embodiment, the microarray system also includes a one-way valve for injecting liquid (such as sample, PCR buffer containing target, hybridization buffer or cleaning buffer) into the array chamber. The sample is injected into the array chamber through the one-way valve to prevent environmental contamination, which is an important concern in certain applications such as biological reagent detection. The one-way valve can be a control valve, dome valve or duckbill valve placed at the entrance of the array chamber. Dome valves of various sizes can be commercially available, for example, from Minivalve International (Yellow Springs, OH).

In some embodiments, the sidewalls of the array chamber are hydrophobic to trap bubbles. In other embodiments, the array chamber has a hydrophilic cover designed to form a hydrophilic region near the array chamber outlet. In a related embodiment, the hydrophilic region is created using a hydrophilic gel element.

In another embodiment, the array chamber inlet comprises a pierceable septum/ribbon or a dome valve, control valve, or duckbill valve to allow cleaning to occur without causing the contents of the array chamber to escape from the microarray assembly.

In another embodiment, the microarray system further comprises a reservoir for injecting liquid into the array chamber. In a related embodiment, the reservoir is loosely coupled to the device so that it can be broken off and removed for imaging in a conventional microarray or colorimetric reader. In another embodiment, the array chamber is connected to multiple waste chambers to ensure that wicking occurs at appropriate intervals.

In certain embodiments, the bubble of microarray assembly is arranged in the array chamber.In case bubble enters the array chamber, then bubble can be retained in the array chamber and partially or completely blocks the liquid flow in the array chamber.If bubble is just positioned at the interface of liquid and absorbent material, then bubble also can stop the wicking action of absorbent material.In certain embodiments, the shape of the array chamber of microarray assembly helps bubble to move in the array chamber.In certain embodiments, the cross-sectional area of array chamber decreases continuously or decreases step by step from chamber one end to the other, to help the motion of liquid and bubble from the array chamber entrance to the array chamber outlet.

FIG1A illustrates one embodiment of a microarray assembly 100 designed to facilitate the removal of bubbles from an array chamber. The microarray assembly 100 includes a funnel-shaped array chamber 110 extending from an inlet 112 to an outlet 114, which leads to a waste chamber 120 containing absorbent material 122. The microarray chamber 110 is provided with a plurality of microarray sites 130 positioned on a substrate 150, which also forms the bottom surface of the array chamber 110. In certain embodiments, the array chamber 110 is connected to a reservoir 140. In this embodiment, the array chamber 110 has a cross-sectional area that decreases toward the waste chamber 120, so that capillary pressure continuously increases as liquid within the array chamber 110 reaches the waste chamber 120. This pressure differential causes the liquid to move toward the absorbent material 122 in the waste chamber 120. In other words, the shape of the array chamber 110 provides for continuous wicking of liquid within the array chamber 110 toward the waste chamber 120 until the liquid reaches the absorbent material 122 in the waste chamber 120. In some embodiments, the cross-sectional area at the inlet end of the array cavity 110 is 2 times, 3 times, 4 times, or 5 times larger than the cross-sectional area at the outlet end of the array cavity 110 .

In one embodiment, the array chamber 110 is trapezoidal in shape, with the inlet end dimensioned between 0.5 and 20 mm, and the outlet end dimensioned between 0.1 and 5 mm. In another embodiment, the array chamber 110 includes a series of stepped structures with a gradually decreasing cross-sectional area from the inlet end to the outlet end. These features are designed to have a smaller curvature radius at the advancing front than at the receding front, thereby forcing bubbles within the array chamber 110 toward the waste sample chamber 120, preventing the aforementioned problems associated with bubbles.

Fig. 1B is the cross-sectional view of the line AA interception along Fig. 1A of microarray assembly 100.In this embodiment, microarray assembly 100 comprises array bottom 150, liner 160 and coating 170.In one embodiment, liner is the double-sided tape such as inner gasket strip (can obtain from 3M, part number 9087) with 0.25mm thickness.In other embodiments, array bottom 150 is injection molded plastics, has the structural feature that produces array chamber 110 walls and waste sample chamber 120 recessed nests, and does not have liner 160 in these embodiments.

In other embodiments, the hydrophilic film is laminated to the plastic array substrate 150 using heat and/or pressure to form a hydrophilic surface on which the microarray is printed. Lamination can be performed using laser welding or ultrasonic welding.

FIG2 provides an enlarged view of the funnel-shaped array chamber 110 of FIG1A . As shown in FIG2 , the decreasing chamber width or tapered shape of the array chamber allows for increased capillary pressure to act on the waste chamber 120 side. This configuration allows air bubbles to flow through the array chamber and prevents them from clogging the array chamber 110. The narrow funnel-shaped chamber 110 also facilitates diffusion of target molecules in the sample to the array sites 130. In certain embodiments, the sample is loaded into a reservoir 140 and continuously flows through the array chamber 110 to the waste chamber 120.

In certain embodiments, the microarray spots 130 are arranged in a plurality of strips (e.g., a protein strip array) that are perpendicular to the flow within the array chamber 110 to improve the interaction between target molecules in the sample and the array elements. In one embodiment, a protein array or protein strip array is printed within the array chamber 110. Proteins extracted from the sample are loaded into the reservoir 140 and continuously flow through the array spots 130 or strips 30 into the waste chamber 120.

Those of ordinary skill in the art will appreciate that microarray assembly 100 can have many variations.For example whole microarray assembly 100 can be molded into two halves, and the dividing line of generation spans the center line of substrate 150, waste sample chamber 120 and storage tank 140.Dividing line can be the profiling path form to allow the hydrophilic surface treatment of array chamber 110 end surfaces and/or print array points 130 on substrate 150 end surfaces simple and easy.The upper half of array assembly can be processed into hydrophilic, for example, with plasma treatment, surfactant or any above-mentioned technology, and utilize ultrasonic welding, laser welding, card joint design, glue, adhesive tape or any joining mode to be joined in place.In certain embodiment, the size of coating 170 can only cover the chamber area, but does not cover the whole upper surface of microarray assembly 100.

FIG3 illustrates another embodiment of a microarray assembly 100 designed to facilitate the removal of bubbles from the array chamber 110 and maintain sample retention within the array chamber 110 during prolonged exposure to extreme temperatures (up to 95° C.). In this embodiment, the microarray assembly 100 includes an array chamber 110 having an inlet 112, an outlet 114, and a plurality of microarray spots 130 positioned above a substrate 150; a waste chamber 120 having an absorbent material 122, an inlet 116, and an outlet 124; and a channel 118 connecting the outlet 114 of the array chamber 110 with the inlet 116 of the waste chamber 120. In this embodiment, the channel 118 comprises an expanding section 118A and a zigzag section 118B. The expanding section 118A has a cross-sectional area that increases toward the waste chamber 120. This allows bubbles within the array chamber 110 to be trapped against the sidewalls of the expanding section 118A upon entering the channel 118, without disrupting fluid flow within the channel 118. During sample expansion when array chamber 110 is subjected to high temperatures, expansion section 118A helps "pin" the liquid contact line to the convex corner of the section. In one embodiment, the sidewalls of channel 118 are hydrophobic to entrap bubbles. In certain embodiments, the cross-sectional area at the waste chamber end of channel 118A is at least 2 times, 3 times, 4 times, or 5 times larger than the cross-sectional area at the array chamber end of channel 118A. In certain embodiments, zigzag section 118B includes two turns to form an S-shaped or Z-shaped channel section. In one embodiment, the two turns are 90° turns.

In other embodiments, the array chamber 110 is machined to have small rectangular channels 180 (i.e., channels with rectangular cross-sections) that are perpendicular to the flow direction to provide a means for drying the array (see FIG4A ). These channels 180 have acute angles that result in a small curvature radius at the fluid-air interface and provide a high capillary pressure that causes the liquid to advance along the sidewall to the waste sample chamber 120. In another embodiment, a plurality of rectangular channels 180 are parallel to the liquid flow path (see FIG4B ). In another embodiment, a plurality of rectangular channels 180 are both parallel to the fluid flow path and perpendicular to the liquid flow path (see FIG4C ). In another embodiment, a plurality of rectangular channels 180 intersect the liquid flow path at an angle ranging from 30 to 120 degrees (see FIG4D ). In another embodiment, the top surface of the substrate 150 is roughened to provide the same wicking action along surface cracks.

The top surface can also be roughened to create a plurality of square microchannels that can be parallel, intersecting, perpendicular, or some or all of these. The contact angle at the corners should be less than 90 degrees to allow the liquid to flow along these channels toward the waste chamber (absorbent material). This is similar to the tracheids (square capillaries) in conifers, which allow the liquid to overcome the effects of hydrostatic pressure and travel up the length of the tree trunk.

Detection of target molecules using microarray components

Another embodiment of the present application relates to a method for detecting target molecules in a sample using the above-mentioned microarray assembly. The sample can be any biological sample such as a swab, nasopharyngeal aspirate, or whole blood sample. Total nucleic acid can be isolated using techniques known to those of ordinary skill in the art. In one embodiment, total nucleic acid is isolated using commercially available nucleic acid isolation reagents or kits such as Qiagen reagents. In another embodiment, total nucleic acid is isolated using a sample preparation device developed by Akonni Biosystems. The general sequence of events of Akonni's sample preparation method includes denaturing the sample in a lysis buffer; continuously perfusing the lysed sample through the sample preparation device; and washing and eluting the nucleic acid from the sample preparation device.

The isolated nucleic acids are loaded into a microarray system and amplified in the microarray assembly using methods known to those skilled in the art. After amplification, the microarray assembly is incubated at a desired temperature for a period of time (e.g., 10-60 minutes at 50-65°C) to allow the amplicons to hybridize to the microarray. After incubation, the microarray system is cleaned (e.g., with water) and imaged on a microarray reader (e.g., Akonni's portable microarray reader). In one embodiment, the microarray system is dried before imaging. In another embodiment, the drying process is accomplished by injecting acetone into the array chamber and/or heating the array chamber. In another embodiment, amplification of the isolated nucleic acids and labeling of the amplification products occur in an asymmetric PCR master mix that contains a significantly greater amount (e.g., 5-20 times greater) of a fluorescently labeled "reverse" primer than an unlabeled "forward" primer. This approach primarily produces a simple standard target with a single label at its 5' end.

Array assays can be performed with a variety of variations. In one embodiment, the amplified product is maintained in the reaction chamber after hybridization, and the microarray is not cleaned before imaging. In another embodiment, the amplified product is maintained in the array chamber, and the array spots are imaged in real time during hybridization to display the growth curve described by Khodakov et al. (2008). In yet another embodiment, the array chamber supports a series of cultivation and cleaning steps for multi-step assays such as ELISA. In one embodiment, the cultivation step is performed under periodic vibration or continuous vibration to improve the interaction between the array element and the target protein.

Fabrication of microarray components

Another aspect of the present application relates to a microarray assembly manufacturing method utilizing a reelable film material and a roll-to-roll rewinding device, comprising a bottom layer, a liner, and a cover layer. Simply put, a reelable film material is used for the bottom layer, liner, and cover layer of the microarray assembly. These layers are laminated together by unrolling several rolls one on top of the other to create a sandwich structure consisting of the desired components, which is then cut to size at the end of the production line. Specifically, the reelable base film is advanced onto a manufacturing platform. Array dots are printed onto the film to form a microarray with fixed inter-array spacing. The printed base film is then laminated onto a reelable liner tape that has been pre-cut to create space for the array chamber using a separate roll-to-roll rewinding manufacturing method. The reelable cover film is then laminated onto the liner film to seal the array chamber. In certain embodiments, the reelable liner tape is pre-cut to create space for the array chamber and one or more waste sample chambers. Absorbent material is placed into each waste sample chamber before the cover film is laminated onto the liner film. An advantage of this manufacturing method is that mass production is very cost-effective because assembly of the microarray components can be fully automated at very high speeds using standard production equipment.

Base film can be any film, and its surface has double bond carbon atoms.Base film preferably has hydrophobic surface.The example of base film includes but not limited to polyester film, polyester/polycarbonate film composite film, polytetrafluoroethylene, polyethylene, polyetherimide, polyetheretherketone and polystyrene.In certain embodiments, the thickness of base film is in the 20-200 micron scope, preferably in the 50-125 micron scope.

The backing film can be a double-sided tape of desired thickness. In certain embodiments, the backing film is made of a hydrophobic material and has a thickness in the range of 20-500 microns, preferably 100-300 microns. Examples of backing films include, but are not limited to, polyester film, polyester/polycarbonate blends, polypropylene, polycarbonate, acetal, polymethyl methacrylate, 256M tape film from Adchem, and polytetrafluoroethylene. The covering film can be any film having a hydrophilic surface. Examples of hydrophilic films include, but are not limited to, Fibrefilm and Film (Film Specialties, Hillsborough, NJ) and Lexan HPFAF (GE Plastics, Pittsfield, MA). Other hydrophilic surfaces are available from Surmodics (Eden Prairie, MN), Biocoat (Horsham, PA), Advanced Surface Technology (Billerica, MA), and Hydromer (Branchburg, NJ).

In certain embodiments, the film thickness is in the range of 25-250 microns, preferably 50-150 microns.

In certain embodiments, the microarray is a gel spot microarray printed on a base membrane using a non-contact microarray printer (e.g., a piezoelectric printer) that allows printing on a moving membrane. In certain embodiments, the gel spot comprises a sample such as a protein sample or a nucleotide sample that is covalently cross-linked to a polymer backbone by ultraviolet-induced copolymerization.

Fig. 5 shows that the tape rewinding assembly line is used for the manufacture of microarray apparatus of the present invention. In short, base film 510 is laid on assembly line 500 by base film reel 512. Gel dot printer 514 prints array dots on base film 510. The sampling in the gel dot is covalently cross-linked to the polymer backbone by UV irradiation. In one embodiment, cross-linking is completed in UV chamber 516 by single-step argon atmosphere UV copolymerization process. In one embodiment, the film is kept in place on the system using the inherent tension between the reels. This improves the UV irradiation uniformity on the film surface by keeping the film flat in the UV chamber during copolymerization. The cross-linked microarray is cleaned in cleaning station 518, dried by air knife 520, and inspected by quality control (QC) camera 522. Defective arrays are marked by reject marking machine 524, and liner film 526 is laminated to base film 510 by liner film reel 528. Liner film 526 can be pre-cut to produce space for array chamber and one or more waste sample chambers before lamination. Absorbent material 530 is then added to the waste chamber, whereby a pre-cut absorbent material segment containing an adhesive backing is placed into the open waste chamber via absorbent material reel 532. Cover film 534 is then laminated onto the base film/liner structure via cover film reel 536. The resulting multilayer structure is then cut by guillotine 538 to produce individual microarray components.

example

Example 1

Methods for compensating for microarray printing variations

Gel drop microarrays containing Cy3 and Cy5 fluorophores were printed onto ten separate slides according to the following combination pattern. The following steps were used to print the microarrays: (1) prepare the appropriate Cy3/Cy5 oligo mix and dry it on a CentriVap, (2) prepare the polymer solution (monomer + crosslinker + glycerol + buffer), (3) dissolve the dried oligo mix in the copolymer solution, (4) place the solution into a source plate, and (5) use the source plate for array printing/polymerization/washing.

Combination Chart

Analysis was performed using a GenePix 4000B using the following settings: 100% laser power for both colors, 500 gain for the red channel photomultiplier tube voltage setting, 375 gain for the green channel photomultiplier tube voltage setting, 5 μm resolution, and a 175 μm diameter circle. For each spot, the integrated intensity was calculated using GenePix software, and the relative standard deviation (RSD) was calculated for all 198 Cy5 spots, 198 Cy3 spots, and the Cy3/Cy5 ratio. As shown in Table 1, for all 10 slides, the coefficient of variation (CV) was lower when the ratio of the Cy3/Cy5 integrated intensity was used compared to the Cy3 or Cy5 signal intensity, in some cases by up to 3-fold. This data supports the implementation of internal fluorescence controls, such as Cy5 dye, which are scanned or profiled as part of manufacturing QC to compensate for variations due to UV dose, temperature, surface properties, synthesis, viscosity, coagulation, washing (i.e., due to effects caused by differences in temperature, viscosity, flow rate, contingency, or anything that may affect spot removal or distortion), immersion depth of the needle in the polymer solution for needle printing techniques, or any property that can affect the sampled morphology and/or concentration in a spot.

Table 1

Example 2

Image analysis

On the MRSA microarray of Akonni, internal fluorescence control has been implemented and is effectively used to compensate for fluorescence intensity variation as shown in the figure.Table 2 shows the data of one group of 4 gel drop fluorescence in the MRSA microarray doped with Cy5 fluorescent group and MecA sampling.For all 4 identical droplets obtained in factory QC (red channel) and after hybridization (green channel), comprehensive signal intensity is tabulated.Because the physical damage of identical sample 3, red channel and green channel demonstrate the comprehensive signal intensity of significant reduction for identical sample 3.As the result of the identical sample 3 signal intensity that weakens, relative deviation is respectively 23.8% and 29.5% for red channel and green channel.When green channel data and red channel data are calculated as ratio, this relative deviation is reduced to 12.2%.This shows that internal fluorescence control data (red channel) can be used to reduce the variability of microarray image and/or microarray manufacture.

Table 2

Example 3

Image generation algorithm

Algorithm 1

The algorithm captures a Cy5 QC image of the array before hybridization and generates a data file containing the array QC parameters.

1. Read the Cy5QC image and generate two local copies, one is the unaltered original (CY5_original), and the other will be converted to a binary image in steps 2 and 3 (Cy5_processed).

2. Take a Cy5_processed image, perform digital filtering and pixel arithmetic to produce an image containing a uniform zero-value background.

3. Convert the image to a binary image and store it as Cy5_Processed.

4. Perform particle analysis on the binary image (Cy5_processed) of the identified filtered objects based on size. The measured and recorded parameters of the objects are: center of mass, bounding box, particle area and ellipticity.

5. Check whether the number of objects identified in step 4 meets the minimum requirement, otherwise reject the slide.

6. Find the grid.

a. Select an object and assume its center of mass is the mesh origin.

b. Form a grid and calculate the pixel position of each grid cell.

c. Apply all objects to the grid and check if at least 80% of the grid cells that should contain Cy3 droplets contain an object. If so, the grid is found and proceed to step 7. If not, repeat 6A-6C with the center of mass of a different object as the grid starting point.

7. Rotate the image so that the angle formed by the Cy3 droplet is less than 0.2 degrees from the horizontal axis.

8. Fine-tune the grid. Because in step 6, the grid origin is determined by the mass center of an object in the binary image, the mass center can be slightly offset from the true object center.

a. Move the grid origin to (0,1), that is, minus 0 pixels on the X coordinate and minus 1 pixel on the Y coordinate.

b. For each Cy3 droplet, calculate:

i. Deviation X: The X coordinate distance between the center of the Cy3 droplet and the center of its grid cell.

ii. Deviation Y: The Y coordinate distance between the center of the Cy3 droplet and the center of its grid cell.

The deviations are summed for all Cy3 droplets, using the value = (abs(deviationX) + abs(deviationY)). Lower values mean better grid placement.

c. Repeat 8A-8C for a total of 24 combinations as shown in the table below.

d.

e. Select the center of the grid so that its value is lowest.

9. Calculate QC data for each point.

a. Deviation X: The X coordinate of the droplet center minus the X coordinate of the grid center.

b. Deviation Y: Y coordinate of the droplet center minus the Y coordinate of the grid cell center.

c. Rejection marks: Rejection points based on diameter, ovality, etc.

d.Point density

e.Diameter

10. Refer to Table 3 and write the QC data to a text file.

Table 3. Exemplary array QC data

Algorithm 2

During this process, two images of the hybridized array were taken: one with normal exposure (Image_Normal Exposure) and one with high exposure to highlight the Cy3 label (Image_High Exposure).

1. Read image_high exposure and image_normal exposure into a storage device.

2. Read from QC text file.

3. Operate on the image_high exposure image to find the grid.

a. Take a Cy5_high exposure image, perform digital filtering and pixel arithmetic to produce an image with a uniform zero-value background.

b. Start converting the image into a binary image.

c. Perform particle analysis on the binary image of the identified filtered objects based on size. Measure and record object parameters: center of mass, bounding box, particle area, and ellipticity.

d. Check to see if the number of objects identified in step 3C meets the minimum requirement, if not reject the slide.

e. Similar to step 6 in Algorithm 1, find the grid.

f. Rotate the image so that the angle formed by the Cy3 droplet is less than 0.2 degrees from the horizontal axis.

g. Fine-tune the grid, similar to step 8 in Algorithm 1.

4. Use the grid found in Image_High Exposure to Image_Normal Exposure.

5. Using the X deviation, Y deviation, diameter and rejection mark from the QC file, determine the relevant point parameters.

a. If the reject flag is true, exclude the point from the analysis.

b. Point X coordinate: X coordinate, grid center + X deviation.

c. Point X coordinate: Y coordinate, grid center + Y deviation.

d. Spot diameter: Spot diameter from QC data.

6. Calculate point density and background.

7. Perform final calculations to confirm the analysis results.

Example 4

A protein microarray assembly was constructed using gel droplet elements containing antibodies. A glass slide printed with the gel element microarray was sealed with 1% BSA in PBS at room temperature for 1 hour. The slide was rinsed with DI water and allowed to air dry in a dust-free environment. The microarray assembly was then assembled with the sealed glass slide and laser-cut into 256M tape, a hydrophilic membrane, and a reservoir from Adchem. Approximately 0.5 mL of SEB (1 μg/mL in PBS containing 0.05% Tween-20 and 1% BSA) was titrated into the microarray system reservoir and continuously aspirated through the array chamber at room temperature. Next, 0.2 mL of a dilution of anti-SEB monoclonal antibody in PBST containing 1% BSA was titrated into the microarray system reservoir, which was continuously aspirated through the array chamber to the waste chamber. Next, 0.2 mL of PBST was titrated into the microarray system reservoir, which was continuously aspirated through the array chamber to the absorbent material in the waste chamber. Subsequently, 0.1 mL of Alexa 568-labeled anti-mouse antibody at 2 μg/mL in PBST containing 1% BSA was titrated into the reservoir of the microarray system, which was continuously aspirated through the array chamber to the absorbent material in the waste chamber. An additional 0.2 mL of PBST wash was titrated into the reservoir of the microarray system, which was continuously aspirated through the array chamber to the absorbent material in the waste chamber. The microarray system was then imaged on an Aurora PortArray 5000 using a green laser (532 nm) with a 605 nm filter.

Example 5

Oligonucleotide mixtures were synthesized for MRSA according to the array diagram shown in Figure 9 . Each sample was synthesized alongside an internal control sample, Cy5. An additional Cy3 control sample, corresponding to the same oligonucleotide sequence, was also mixed with the Cy5 control sample. Cy3/Cy5 spots were printed at densities ranging from 0.1 nM to 10 μM over a 1 log concentration range to create a calibration curve. The imaging system consisted of two optical trains, each comprised of an LED and an uncooled CCD camera. One train was used to detect Cy3 spots (550 nm excitation and 570 nm emission), while the other was used to detect Cy5 spots (650 nm excitation and 670 nm emission). These optical trains were fixed in position relative to the instrument. The stage moved the array to the green channel and acquired 10 images to improve dynamic range; the requirement for multiple images with short exposure times prevented saturation that can occur with materials containing high autofluorescence and also allowed signal averaging to mitigate the effects of random noise. The stage then moved the array to the red channel and acquired 10 images. The process was repeated 5 times to account for any misalignment caused by positioning accuracy, incorrect exposure time, loss of focus and/or any other anomaly that may compromise correct imaging. A calibration curve was drawn for the Cy3/Cy5 serial concentration dilutions, as indicated by the outer boundaries of the array in the following combined figures. The calibration curve obtained from the concentration gradient is to correct for factors affecting the entire assembly such as sample shelf degradation, temperature, UV dose variations, synthesis variations, or any system artifacts that may lead to non-reproducible behavior. The calibration curve for Cy5 was drawn when analyzing using the Cy3 calibration curve.

I _red =m _red ×moles(Cy5)+b _red

I _green =m _green n×moles(Cy3)+b _green

Wherein, I _red and I _green are the integrated intensities eliminating background. The slopes m _red and m _green and the coordinates b _red and b _green are calculated from these calibration curves. The average value of the calibration curve is plotted, and values outside the range are rejected. In order to be responsible for the reproducibility between points, between components and between batches, the Cy5 integrated intensity value minus the background is calculated for each point. During the synthesis process, for each point, the sampling concentration of each point (14, 31, 35, 36, 37, 29, 90, H or dN20) has an equimolar concentration of oligonucleotide sampling and Cy5 fluorescent group. Thus, the following relationship is established:

Cy3 concentration ≈ Cy5 concentration

therefore,

I _{greensaturation} ≈m _green ×moles(Cy5)+b _green

Wherein, 1 _{green, saturation} represents the hypothetical situation, at this time all samples in the gel element are bound to the labeled Cy3 molecules. Note that moles (Cy5) replace moles (Cy3) because of the equimolar equivalents in the above equation. Measure at representative points and calculate the background-corrected integrated intensity of the samples at these points for the presence of MRSA. If the intensity meets the following criteria:

The value is considered positive. That is, more than 0.1% of the possible sampled molecules have bound targets with Cy3 tags. This method can also be used for quantitative interpretation.

Example 6

A soft-touch cut tape reel is manufactured for a liner reel, consisting of a cut liner film with pre-punched inlet and outlet holes and vent holes on a cover reel. As shown in Figure 5, during manufacturing, a base film reel is unwound, and the unwound liner material is collected on an upper reel. A gel element printer prints the gel elements on the base film. The film is then exposed to UV light under an inert atmosphere (e.g., argon). A positive pressure of argon is slowly added to the argon chamber. Because its density is greater than air, the argon settles to the bottom of the chamber where the substrate resides. This allows the argon to flow slowly into the chamber, thus minimizing the need for a composition atmosphere within the chamber. To ensure that unpolymerized polymer remains on the substrate, the substrate moves through a cleaning station positioned to eliminate splashing into the polymerization chamber. The cleaned array is then dried using a conventional air knife assembly. A QC camera ensures that the elements are printed according to specifications, and a reject marker alerts the operator to remove non-compliant elements. The double-sided liner tape defining the microarray cavities is unwound and adhered to the substrate. The openings in the backing tape are designed to allow for relatively loose tolerances when the backing tape is laminated to the substrate, which allows arrays of gel elements of variable shape and complexity to be manufactured without having to modify the assembly line. The rollable absorbent material is unrolled and mounted to the waste sample chamber of the assembly, where it is sealed in place with glue or double-sided tape. An alternative approach to adding the absorbent material is to use a pick-and-place robot to insert the absorbent material into the waste sample chamber. Current flow cell designs use an additional liner to accommodate absorbent material that is twice as thick as the reaction chamber. Finally, a covering film with a number of filling holes is applied. The filling holes can be significantly larger or smaller than the holes in the backing film, allowing for gross errors in alignment. A guillotine then cuts the tape to the appropriate size.

A needle-printing robot typically features a print head, typically equipped with precisely machined needles. This print head is connected to a precise xyz-axis control arm (Figure 7). The control arm is responsible for moving the print head with micron-level precision between a printing solution source plate (e.g., a 384-well microtiter plate), a substrate printing station (printing plate), and a wash station (for cleaning needles between depositions of individual solutions). Alternatively, a high-throughput, non-contact print head can simultaneously deposit multiple solutions into a microarray-PCR reaction chamber.

In certain embodiments, the entire microarray is printed at once. Electrical discharge machining (EDM) can be used to manufacture a printhead with a 125-micron needle and a currently used 300-micron center (currently used needle diameter). Furthermore, a parallel synthesis design provides a 24,576-well plate with a 560-micron center in its wells. This translates to approximately 350 dots per square centimeter. Although this many dots are sufficient for most fault diagnosis applications, experiments requiring additional dots can be completed by arranging multiple printers in sequence on a combination line, with the printing time only extending the travel time from one printer to the next. Another embodiment of printing on a mobile membrane includes the use of a non-contact printer that employs piezoelectric crystals and capillary action, which draws in a microarray printing solution and disperses picoliters of liquid into nanoliters of droplets onto a substrate. The printhead can include multiple capillaries for simultaneously printing a series of unique microarray dots containing independent sampling. Furthermore, the printhead can be a high-density capillary array for increasing the number of unique microarray dots. Alternatively, the print head can cross-scan the base film up and down, left and right to print replicated points, or the print head scanning method can be used to aspirate a separate polymer-sampling suspension in order to print multiple independent points with the same print head. Another option is to reinstall the base film onto the roll rewind system after the first printing process to print additional (independent) points on the base film roll again for each microarray. The replication approach can include a benchmark to correctly align the film during the second, third and nth printings. An example of a relevant benchmark is to use a perforated edge, such as a perforated edge used in conjunction with a 35 mm membrane. Another printing option includes the acoustic jet non-contact method from Labcyte, in which high-frequency sound waves spray nanoliter droplets from a source plate to a target plate.

Example 7

FIG8 shows the results of printing Cy3 gel elements on 0.005" polyester film purchased in roll format from McMaster-Carr (Santa Fe Springs, CA). The film was placed in a vacuum chuck as shown in FIG9 and printed. It was imaged using bright long illumination before and after polymerization. The array was also imaged using an Akonni imager, which consists of an LED and an uncooled camera. After printing, the MRSA array described above was printed onto polyester film and subjected to a Qiagen MasterMix using 300 pg of purified MRSA DNA. Thermal cycling was performed on a Quanta Bioscience slide block thermal cycler. FIG10 shows the results when a rollable membrane was used for the top, bottom, and backing tape, showing positive identification of MRSA. FIG11 shows the roll rewind printing setup using the BioDot Ultra Non-Contact Array Printer (top plate) and the BioDot on a moving membrane. Video frames of non-contact printing of an Ultra, where the membrane has not been chemically treated and modified (base plate). These results show the feasibility of printing microarrays on mobile membranes using a non-contact array printer that allows high-speed, low-cost manufacturing of the microarray assembly of the present invention.

Example 8

Figures 12-14 illustrate the array image analysis process. Figure 12 shows an array image captured by a red channel imager during factory QC to extract array QC data. The QC software algorithm automatically finds each point in the array and assigns each point to its respective position in the array grid. The software displays a 12×12 square grid and draws a circle around each point. The QC software calculates the relative position (deviation X, deviation Y) of each circle within its bounding square grid box and stores this information as part of the QC data to aid post-hybridization image analysis.

Figure 13 shows an image of the array after hybridization, captured using an end-user green channel imager. Image analysis software located the array grid based on the Cy3 fluorescent marker and drew a 12×12 square grid. Image analysis then used relative spot positions (Deviation X and Deviation Y from the array QC file) to locate each spot and draw a circle around it for visual identification. Note that with the help of the array QC data, the software was able to locate each spot on this column and draw a circle completely around each spot.

FIG14 is an image of the array taken after hybridization using an end-user green channel imaging instrument. The image analysis software locates the array grid based on Cy3 fluorescence labeling and draws a 12×12 square grid. For this image, the image analysis software does not obtain information about relative spot positions from the array QC file. Instead, the image analysis software identifies each spot assuming that the center of each square grid cell is the center of that spot. The software incorrectly identifies several spots by drawing circles that do not completely surround the spot. The incorrectly identified spots include those located at row 2, column 2 and row 11, column 6. Note that this image analysis method without array QC data is less robust in spot identification than the image analysis using array QC data as shown in FIG13 .

Claims (7)

1. A microarray assembly for detecting target molecules in a sample, the microarray assembly comprising: An array cavity has an inlet, an outlet, an inner top surface, an inner bottom surface, several sidewalls, and a microarray located on the inner bottom surface. The array cavity is designed to allow nucleic acid amplification reactions within the cavity and the amplified nucleic acids to hybridize into the microarray. The inner top surface is a hydrophilic surface, which facilitates the complete filling of the array cavity by an aqueous sample. Including the waste sample inlet and the waste sample cavity for the absorbent material; and A channel having an expansion section, the expansion section having a first end near the outlet of the array cavity and a second end near the inlet of the waste sample cavity, wherein the expansion section of the channel is in the shape of an inverted funnel from the first end to the second end, and the cross-sectional area of the channel continuously and progressively increases from the first end to the second end of the expansion section; and The waste sample chamber includes an exhaust port. 2. The microarray assembly of claim 1, wherein the channel further includes a tortuous section between the expansion section and the inlet of the waste sample cavity. 3. The microarray assembly of claim 2, wherein the zigzag segment includes two sharp bends in the channel. 4. The microarray component according to claim 1, wherein the microarray is an oligonucleotide microarray. 5. The microarray assembly according to claim 1, wherein the microarray is a protein microarray. 6. The microarray assembly according to claim 1, wherein the microarray is a gel dot microarray. 7. The microarray assembly of claim 6, wherein the gel dot microarray comprises a plurality of gel dots, and each gel dot comprises an internally controlled fluorescent group and a trapping agent specifically binding the target molecule.