HK1241315B - Nucleic acid purification cartridge - Google Patents

Description

Nucleic acid purification kit

Technical Field

The invention relates to a nucleic acid purification box.

Background Art

An initial nucleic acid purification method based on the affinity of DNA and RNA for silica surfaces (solid phase adsorption) was introduced by Boom et al. The attraction of nucleic acids to the silica surface is promoted by a high concentration of a chaotropic salt (usually guanidine isothiocyanate or guanidine hydrochloride). Boom's method uses a chaotropic salt solution to denature the biological sample and force it through a filter using centrifugal force to promote the adsorption of DNA and RNA onto the silica surface. Once the nucleic acids are bound to the filter, one or more washes are performed with an ethanol buffer to remove the chaotropic salts and other biological impurities while maintaining nucleic acid binding (chaotropic salts are destructive to most nucleic acids in downstream applications). As a final step, after removing the ethanol (via high-speed spinning), the nucleic acids need to be rehydrated using an elution buffer (water or a low-salt buffer). Rehydration promotes the release of DNA and RNA from the silica surface, and the final spin produces a solution in which the purified nucleic acids are resuspended.

Variations of this approach have been described elsewhere, using centrifugal force or vacuum as the driving force for liquid flow. However, all of these methods are quite complex and time-consuming, involving multiple pipetting steps and the subsequent application of different driving forces to control liquid flow, which often results in high yield variability between replicate purification runs.

For example, when manually implementing a purification protocol using vacuum, the purification process consists of five main steps corresponding to the flow of five different liquids through the silica filter: a sample mixture containing nucleic acids, wash buffer 1, and wash buffer 2, which is used to wash the filter and eliminate any amount of contaminants; air, which is used to dry the filter and eliminate any traces of volatile contaminants; and elution buffer, which is used to release the nucleic acids from the filter so that the NAs can be further used for downstream applications (e.g., qPCR amplification and detection). At the end of each step, the vacuum suction is maintained for 1 or 2 minutes even after a certain volume of liquid has flowed through the filter to ensure that almost no liquid remains in the filter crevices before the next liquid is pipetted.

The reproducibility of nucleic acid production depends on the possibility to replicate the contact time of the sample and buffer with the filter and the amount and distribution of liquid flow, which depends on the skill of the operator.

It is therefore an object of the present invention to provide a purification device which provides reproducible purification results independent of the respective operator.

This is achieved through a microfluidic device and method for purifying biological or chemical analytes from complex biological samples. The microfluidic device comprises a chamber with a filter embedded in it, multiple reservoirs, and a valve. The device can interface with an external pump operated by an automated instrument. Thus, the device and method provide an end-to-end automated implementation of traditional nucleic acid purification methods.

Summary of the Invention

The present invention relates to a microfluidic device having a closed chamber containing a filter for purifying biological or chemical analytes from complex biological samples, wherein the chamber contains, in addition to the filter, a plurality of ports, as follows: a first port enabling the chamber to be in gaseous communication with a vacuum generator via a first flow path; a second port enabling the chamber to be in liquid communication with one or more reservoirs via a second flow path; a third port enabling the chamber to be in gaseous and liquid communication with one or more receiving containers and a vacuum generator via a third flow path; and a filter arranged between the third port and the first and second ports so that fluid entering the chamber via the first and/or second port and leaving the chamber via the third port flows through the filter.

The invention disclosed herein also relates to a method for purifying biological or chemical analytes from complex biological samples using the microfluidic device disclosed herein, the method comprising the following steps: (a) allowing a liquid sample to enter the chamber through the second port by applying a negative pressure differential between the chamber and a first reservoir, and a valve in the flow path opens the first and second ports and closes the third port; (b) allowing the sample to flow through a filter into the first receiving container by applying a negative pressure differential between the first receiving container and the chamber, and the valve in the flow path closes the first port, vents the second port to atmospheric pressure, and opens the third port; and (c) eluting the analyte from the filter by applying a negative pressure differential between the chamber and a receiving container, and the valve in the flow path opens the first and second ports and closes the third port.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a purification column (1) from a commercial kit for manual purification, comprising a plastic body (2) and a membrane filter (3) which is squeezed and held in place by a fixing ring (10). The plastic body comprises a liquid inlet (4) and a liquid outlet (5).

Figure 2A shows a portion of a microfluidic device (100) with an integrated purification cavity (101) and a membrane filter (3) held in place by a retaining ring (10).

Figure 2B shows a rotational view of a microfluidic device (100) with an integrated purification cavity (101) and a membrane filter (3) held in place by a retaining ring (10). Also shown are a gas port (102), a liquid inlet (103), and an outlet (104) connected to the purification cavity (101).

FIG2C shows a separate purification cavity (200).

Figure 2D shows the purification cavity (200) assembled in the microfluidic device (100). The clamping structure (201) holds the purification cavity (200) in place and applies the correct pressure to the membrane filter (3). The gas port (203), liquid inlet (204), and outlet (205) are also shown.

Figure 2E shows the port arrangement and flow direction during liquid loading (step 1) and washing/elution (step 2). Solid arrows indicate liquid flow; dashed arrows indicate gas flow; X indicates a port closed by a valve.

Figures 3 to 12: Detailed flow diagrams showing the port/valve setup for each step detailed in Table 1.

It should be understood that the apparatus of the present invention may, but need not, include every element shown in the figures. The description and/or claims describe the basic elements. In addition to the elements described, one or more further optional elements may be selected independently of each other. The optional elements are represented as follows. 1: Vacuum generator 1 (e.g., syringe pump); 2: Vacuum generator 2 (optional, e.g., diaphragm pump); 3-7: Valves (e.g., multi-port valves); 8 and 9: Pressure sensors (optional); 10: Chamber; 11: Fixing ring (optional); 12: Filter; 13: Waste receiving container (optional); 14: Eluent receiving container; 15: Sample reservoir; 16-17: Reservoirs (optional); 18: Elution buffer reservoir.

DETAILED DESCRIPTION

In principle, purification can be based on any of the effects widely known in chromatography (e.g., displacement, affinity, cation exchange, anion exchange, size exclusion, reverse phase, and normal phase), and the choice depends primarily on the analyte to be purified. However, size exclusion is less preferred than the other techniques because, with the first techniques, permanent binding cannot be achieved. With the latter, conditions can be found under which the analyte to be purified selectively binds to the medium, while, ideally, other components of the sample pass through the medium without binding.

The microfluidic device of the present invention comprises a closed chamber containing a filter. The filter herein represents a medium that differentially interacts with the different components of a sample. In conventional chromatography, this medium is generally referred to as the stationary phase. As the sample moves through the medium in a suitable buffer (generally referred to as the mobile phase in chromatography), the differential interactions (also referred to as separation) result in differential retention times, thereby leading to a purification effect.

The filter used in the device disclosed herein is a filter suitable for purifying biological or chemical analytes from complex biological samples. The analyte is the substance to be purified. A complex biological sample is a sample that contains, in addition to the analyte, many different components of varying size and chemical nature (e.g., proteins, nucleic acids, hormones, lipids, salts). A preferred sample is a cell lysate.

In a preferred embodiment, the filter is made of or at least includes silica. For example, the filter can be in the form of a silica membrane or a resin containing silica beads or silica-coated beads. Silica surfaces are advantageous for separating or purifying nucleic acids, particularly DNA. Silica is known to adsorb DNA molecules under certain salt and pH conditions, and silica adsorption has become an important technique for purifying DNA.

In one embodiment of the present invention, the filter element is integrated into the purification cavity and secured by a retaining ring. In a preferred embodiment, the purification membrane filter is inserted into the cavity as part of the main body of the microfluidic device, and the membrane filter is held in place by compressing its retaining ring (Figures 2A and D).

In an alternative embodiment, the purification cavity is a separate component that fits into the microfluidic device and does not require a retaining ring to hold the membrane filter in place. The purification cavity itself provides a clamping structure that holds the cavity and membrane filter in place with the membrane properly compressed (Figures 2B and C).

A separate cavity preferably held in place by a clamping feature has several advantages over a cavity comprising a retaining ring

The clamping structure enables reproducible compression of the membrane filter, which ensures reproducible compression, resulting in a reproducible liquid flow through the membrane filter and thus in reproducible production of purified nucleic acids or other purified analytes.

Preferably, the correct positioning of the purification cavity in the microfluidic device will be ensured by its clamping structure, without the need to control the amount of compression given by the design. This facilitates manufacturing.

The purification cavity does not require a retaining ring, which reduces sample contamination. The retaining ring disrupts the fluid path and collects liquid residues, which can contaminate the buffers and result in a certain amount of contaminants in the final purified eluent, which can hinder downstream analysis, such as PCR. The detachable purification cavity creates smooth transitions on its walls, which reduces the amount of contaminants that can adhere to the walls, typically by 5 to 10 fold.

Preferably, the analyte is a nucleic acid. The term nucleic acid includes mRNA (messenger RNA), tRNA (transfer RNA), hn-RNA (heterologous nuclear RNA), rRNA (ribosomal RNA), LNA (locked nucleic acid), mtRNA (mitochondrial RNA), nRNA (nuclear RNA), siRNA (short interfering RNA), snRNA (small nuclear RNA), snoRNA (small nucleolar RNA), scaRNA (small Cajal body-specific RNA), microRNA, dsRNA (double-stranded RNA), ribozymes, riboswitches, viral RNA, dsDNA (double-stranded DNA), ssDNA (single-stranded DNA), plasmid DNA, cosmid DNA, chromosomal DNA, viral DNA, mtDNA (mitochondrial DNA), nDNA (nuclear DNA), snDNA (small nuclear DNA), etc. or all other conceivable nucleic acids in processed and unprocessed form.

In addition to the filter, the chamber also accommodates the following multiple ports: a first port that enables the chamber to be in gas communication with the vacuum generator through a first flow path; a second port that enables the chamber to be in liquid communication with one or more reservoirs through a second flow path; and a third port that enables the chamber to be in gas and liquid communication with one or more receiving containers and the vacuum generator through a third flow path.

The vacuum generator is located upstream of the chamber. One or more reservoirs are also located upstream of the chamber, but in a separate flow path from the vacuum generator. The one or more receiving vessels are located downstream of the chamber. The vacuum generator of the third flow path is located further downstream of the receiving vessel.

The reservoir typically includes at least one reservoir containing the sample to be purified and optionally one or more reservoirs containing one or more wash buffers and/or elution buffers and/or regeneration buffers. The one or more containers typically include at least one container for receiving the analyte and optionally one or more containers for receiving other liquids, such as a flow-through, wash buffer and/or regeneration buffer.

The filter is positioned between the third port and the first and second ports such that fluid entering the chamber through the first and/or second port and exiting the chamber through the third port flows through the filter. Most preferably, the filter expands across the entire cross-section of the chamber. However, the media does not necessarily fill the entire height of the chamber. Preferably, the filter is positioned directly above the third port.

Preferably, the device is a microfluidic cartridge. A cartridge is a consumable component that can be driven by a larger unit via a suitable interface. Typically, the unit contains expensive and/or durable components that are easy to clean and a software code for automatically controlling the process. The unit may optionally include other components for performing other processes upstream or downstream of the purification unit.

In one embodiment, the device is disposable, which means that the device is designed for a single use and is disposed of after use. In another embodiment, the device is reusable, which generally requires regeneration of the device after each use.

The device may also include a valve, and ideally, the vacuum generator is separate. The vacuum generator evacuates the pressure in the chamber, thereby creating a relative negative pressure. Depending on the configuration of the ports (i.e., open or closed), fluid is drawn from a reservoir into the chamber and/or from the chamber into a receiving container. In a preferred embodiment, the vacuum generator is a syringe pump or a diaphragm pump. In another preferred embodiment, the same vacuum generator is used to apply vacuum to the first port and/or the third port.

Known microfluidic devices do not include a means for tracking pressure in the system. The present invention preferably includes one or more pressure sensors. A pressure sensor is preferably located in the third flow path upstream of the receiving container. Another pressure sensor is preferably located in the first flow path downstream of the vacuum generator. The pressure sensors can be used to determine the pressure drop caused by the filter, which is indicative of the fluid state of the filter. Thus, one can determine (i) when a method step is complete, thereby minimizing time and buffer (e.g., when the filter is completely dried in a drying step; when the filter is fully cleaned of liquid residues in a washing step, which preferably occurs after each liquid flow and before the next liquid flow); (ii) when the liquid has completely flowed through the filter, allowing the system to "on the fly" increase the suction pressure when there is increased resistance to liquid flow due to the density and viscosity of the sample; (iii) when the filter becomes clogged; and (iv) the time required for each liquid to flow through the filter, which can be compared to predetermined thresholds for controlling the purification method.

As described above, the device disclosed herein has three ports for communicating with the chamber: a first port (gas outlet), a second port (liquid inlet), and a third port (liquid/gas outlet). Each port can be individually opened, closed, or vented to the atmosphere by a valve located in the corresponding flow path. Preferably, a multi-port valve is used, and the same multi-port valve is used to drive two or three ports when necessary. Preferably, the dead volume surrounded by the third flow path between its corresponding valve and the filter is between 1 μL and 10 mL. Controlled flow of liquid (including a no-flow state for complete wetting of the filter) is achieved by applying a vacuum to the appropriate ports and by opening and closing the appropriate valves in each step. This gives the device higher reproducibility compared to known devices, regardless of the type of biological sample.

For example, in a common purification kit where the purification protocol is manually performed by vacuum, the purification method includes five main steps corresponding to the flow of five different liquids through the filter, namely: loading the sample mixture containing nucleic acids, washing with wash buffer 1 and wash buffer 2 to rinse the filter and remove any amount of contaminants, air drying the filter and removing any traces of volatile contaminants, and elution to release the nucleic acids from the filter for further use in downstream applications (such as qPCR amplification and detection). At the end of each step, even after a certain volume of liquid has flowed through the filter, vacuum suction is maintained for one or two minutes to ensure that almost no liquid remains in the filter crevices before the next liquid is pipetted. This operation is called "clearing."

To achieve the same effect, the automated protocol comprises essentially the same steps, which are achieved by having each port connected to a suitable reservoir and by having the appropriate pressure differential applied at all times by a pressure source (e.g., a syringe or rotary pump), a set of valves, a set of microfluidic channels, and a microcontroller using software so that all steps are automated.

The microfluidic devices disclosed herein are particularly suitable for use in methods for separating one or more analytes from other components, i.e., in purification methods. Therefore, another object of the present invention is a method for purifying a biological or chemical analyte from a complex biological sample using the microfluidic device described herein, the method comprising the following steps: (a) allowing a liquid sample to enter the chamber through the second port by applying a negative pressure differential between the chamber and a first reservoir, with the valve in the flow path being open to the first and second ports and closed to the third port; (b) allowing the sample to flow through a filter into the first receiving container by applying a negative pressure differential between the first receiving container and the chamber, with the valve in the flow path being closed to the first port, venting the second port to atmospheric pressure, and open to the third port; and (c) eluting the analyte from the filter by applying a negative pressure differential between the chamber and a receiving container, with the valve in the flow path being open to the first and second ports and closed to the third port.

The pressure in step a may be generated by a vacuum generator located in the first flow path. The pressure in step b may be generated by a vacuum generator located in the third flow path.

The elution in step c can be performed as described in detail below:

i) applying a negative pressure differential between the chamber and the third reservoir, thereby allowing elution buffer contained in the third reservoir to enter the chamber through the second port, with the valve in the flow path being open to the first and second ports and closed to the third port. The pressure may be generated by a vacuum generator located in the first flow path;

ii) allowing the elution buffer to contact the filter for a predetermined time, with the valve in the flow path open for the first and second ports and closed for the third port. This step allows the filter to be sufficiently wetted to release the appropriate analyte; and

iii) applying a negative pressure differential between the second receiving container and the chamber, thereby allowing the elution buffer (containing the released analyte) to flow through the filter into the second receiving container, while the valve in the flow path is closed at the first port, venting the second port to atmospheric pressure, and opening the third port. The pressure can be generated by a vacuum generator in the third flow path.

Preferably, the method further comprises, between steps a and b, the step of allowing the sample to contact the filter for a predetermined time, while the valve in the flow path remains open with respect to the first and second ports and closed with respect to the third port.

The method may optionally include one or more of the following steps between steps b and c:

(i) cleaning and drying the filter for a predetermined time by applying a negative pressure differential between the third flow path and the chamber, the negative pressure differential being generated by a vacuum generator located in the third flow path, while a valve in the flow path is closed with respect to the first port, venting the second port to atmospheric pressure, and open with respect to the third port; and/or

(ii) allowing wash buffer in the second reservoir to enter the chamber through the second port and flow through the filter and into the receiving container by applying a negative pressure differential between the receiving reservoir and the second reservoir, while the valve in the fluid path is open to the second and third ports and closed to the first port. The pressure may be generated by a vacuum generator located in the third flow path; and/or

iii) Negative pressure is applied between the third flow path and the chamber, thereby allowing gas to flow through the filter for a predetermined time. A valve within the flow path is closed at the first port, venting the second port to atmospheric pressure, and opening the third port. The gas displaces the liquid and dries the filter. The pressure can be generated by a vacuum generator located in the third flow path.

Preferably, the pressure difference is determined in order to determine when to switch one or more valves and thus perform the next method step.The pressure drop across the filter indicates that the valve can be actuated in order to perform the next method step.

More preferably, the air flow is used to dry the filter only when the value of the first derivative of the pressure difference between the first and second pressure sensors is below a predetermined limit value.

An example of a method step for performing the purification process by automated means is detailed in Table 1. The flow diagrams shown in the accompanying drawings illustrate the port/valve arrangements used for each of the steps described.

Table 1. Detailed scheme of an example of a purification process.

Advantages of the present invention are that whole automation can be realized, and manual operation is not required to complete the purification process, thereby obtaining reproducible result.And purification device/process can be associated with other upstream or downstream devices/process (for example, cracking, amplification and detection). Contrary to prior art, liquid moves into the chamber and on the filter by the vacuum being applied to the first oral portion, and the 3rd oral portion closes. This prevents flowing through the filter, while allowing the filter to pre-wet the predetermined incubation time. After opening the valve of this oral portion, by the vacuum being applied to the 3rd oral portion, liquid is passed through the filter. In a word, compared with known methods, equal or higher nucleic acid production has been obtained.

Claims (34)

1. A microfluidic device, comprising: (a) One or more storage units located upstream of the purification cavity; (b) One or more waste receiving containers located downstream of the purification cavity; (c) The purification cavity defines a closed column having a tapered shape and a hollow interior, the purification cavity being separable from the microfluidic device, the purification cavity being separate from and not adjacent to any of the storage container or waste receiving container, the purification cavity comprising: (i) A first opening, the first opening enabling the purification cavity to be in gas communication with a vacuum generator through a first flow path; (ii) A second opening, the second opening enabling the purification cavity to be in liquid communication with the one or more reservoirs via a second flow passage; (iii) A third port, which allows the purification cavity to be in gas and liquid communication with the one or more waste receiving containers and the vacuum generator via a third flow path; wherein the first, second, and third ports are located on the outer peripheral surface of the purification cavity, and (d) A filter spanning a cross-section of the purification cavity perpendicular to the direction of liquid flow, the filter being used to purify biological or chemical analytes from complex biological samples and adjacent to a third port, such that fluid enters the purification cavity through a second port, exits the bottom of the purification cavity through a third port, and flows through the filter. 2. The microfluidic device according to claim 1, wherein: the microfluidic device is a microfluidic cartridge, which is disposable or reusable. 3. The microfluidic device according to claim 1, wherein the analyte to be purified is nucleic acid. 4. The microfluidic device according to claim 1, wherein the filter comprises silicon dioxide. 5. The microfluidic device according to claim 4, wherein: the filter comprises a silica membrane or a resin containing silica beads or silica-coated beads. 6. The microfluidic device according to any one of claims 1 to 5, wherein: the purification cavity includes a clamping structure for fixing the purification cavity in place within the microfluidic device. 7. The microfluidic device according to claim 1, wherein: the vacuum generator is a syringe pump, a diaphragm pump, or a combination thereof. 8. The microfluidic device according to claim 1, wherein: a vacuum can be applied to the first port and/or the third port using the same vacuum generator. 9. The microfluidic device of claim 1, wherein: the pressure sensor is located in a third flow path upstream of the one or more waste receiving containers. 10. The microfluidic device according to claim 9, wherein: another pressure sensor is located in the first flow path between the vacuum generator and the purification cavity. 11. The microfluidic device of claim 1, wherein: the first port, the second port, and the third port are individually openable, closed, or vented to the atmosphere by means of one or more multi-port valves within their flow paths. 12. The microfluidic device according to claim 11, wherein the dead zone volume surrounded by the third flow passage between its respective valve and filter is between 1 μL and 10 mL. 13. A method for purifying a biological or chemical analyte from a complex biological sample using a microfluidic device according to any one of claims 1 to 12, the method comprising the steps of: (a) A liquid sample is allowed to enter the purification cavity through the second port by applying a negative pressure difference between the purification cavity and the first reservoir. (b) The sample is allowed to flow through the filter into the waste receiving container by applying a negative pressure difference between one of the one or more waste receiving containers and the purification cavity; and (c) The analyte is eluted from the filter by applying a negative pressure difference between the purification cavity and an eluent receiving container. 14. The method according to claim 13, wherein the elution in step c is performed as follows: i) By applying a negative pressure difference between the purification cavity and the third reservoir, the elution buffer contained in the third reservoir can enter the purification cavity through the second port; ii) to allow the elution buffer to contact the filter for a predetermined incubation time; and iii) By applying a negative pressure difference between the eluent receiving container and the purification cavity, the eluent buffer can flow through the filter into the eluent receiving container. 15. The method according to claim 13 or 14, further comprising: a step between steps a and b, allowing the sample to contact the filter for a predetermined incubation time. 16. The method of claim 13, further comprising: one or more of the following steps between steps b and c: (i) Cleaning and drying the filter for a predetermined time by applying negative pressure between the third flow passage and the purification cavity; and/or (ii) By applying a negative pressure differential between one of the one or more waste receiving containers and a second reservoir, the washing buffer located in the second reservoir is allowed to enter the purification cavity through a second port, flow through a filter, and enter the one waste receiving container; and/or iii) By applying negative pressure between the third flow path and the purification cavity, gas can flow through the filter for a predetermined time. 17. The method of claim 13, further comprising: activating one or more multi-port valves within the first flow passage, the second flow passage, and the third flow passage by means of a pressure difference. 18. A microfluidic device having a closed chamber, the closed chamber comprising a filter for purifying biological or chemical analytes from complex biological samples, the chamber, in addition to the filter, also accommodating a plurality of ports: a. A first opening, the first opening enabling the chamber to be in gas communication with a vacuum generator via a first flow path; b. A second opening, the second opening enabling the chamber to be in liquid communication with one or more reservoirs via a second flow passage; c. A third opening, which allows the chamber to be in gas and liquid communication with one or more receiving containers and a vacuum generator via a third flow path; wherein the first, second, and third openings are located on the outer peripheral surface of the chamber, and d. A filter located between a third port and a first and a second port, the filter spanning a cross-section of the chamber perpendicular to the direction of liquid flow and near the third port, such that fluid entering the chamber through the second port exits the chamber through the third port and flows through the filter. 19. The apparatus of claim 18, wherein: the apparatus is a microfluidic cartridge, which is disposable or reusable. 20. The apparatus of claim 18, wherein the analyte to be purified is nucleic acid. 21. The apparatus of claim 18, wherein: the filter is a silica membrane or a resin containing silica beads or silica-coated beads. 22. The apparatus of claim 18, wherein: the filter is integrated in the purification cavity and is fixed by a retaining ring. 23. The apparatus according to any one of claims 18 to 21, wherein: the chamber is part of a separable purification cavity defining a column having a tapered shape and a hollow interior, the purification cavity being a separate component located in the apparatus, thereby securing the filter in a removable manner. 24. The apparatus of claim 18, wherein the vacuum generator is a syringe pump, a diaphragm pump, or a combination thereof. 25. The apparatus of claim 18, wherein: a vacuum can be applied to the first port and/or the third port using the same vacuum generator. 26. The apparatus of claim 18, wherein: the pressure sensor is located in a third flow path upstream of the receiving container. 27. The apparatus of claim 26, wherein: another pressure sensor is located in a first flow path downstream of the vacuum generator. 28. The apparatus of claim 18, wherein the first port, the second port, and the third port are individually openable, closed, or vented to the atmosphere by means of one or more multi-port valves within their flow paths. 29. The apparatus of claim 28, wherein the dead zone volume surrounded by the third flow passage between its respective valve and filter is between 1 μL and 10 mL. 30. A method for purifying a biological or chemical analyte from a complex biological sample using a microfluidic device according to any one of claims 18 to 29, the method comprising the steps of: (a) A negative pressure difference is applied between the chamber and the first reservoir to allow a liquid sample to enter the chamber through the second port, wherein the valves in each flow path are open for the first and second ports and closed for the third port; (b) A sample is allowed to flow through the filter into the waste receiving container by applying a negative pressure differential between one of the one or more waste receiving containers and the chamber, wherein valves in each flow path are closed for a first port, allow atmospheric pressure to pass through a second port, and open for a third port; and (c) The analyte is eluted from the filter by applying a negative pressure differential between the chamber and an eluent receiving container, wherein the valves in each flow path are open for the first and second ports and closed for the third port. 31. The method according to claim 30, wherein the elution in step c is performed as follows: i) By applying a negative pressure difference between the chamber and the third reservoir, the elution buffer contained in the third reservoir can enter the chamber through the second port, while the valves in each flow path are open for the first and second ports and closed for the third port; ii) Allowing the elution buffer to contact the filter for a predetermined incubation time, while the valves in each flow path are open for the first and second ports and closed for the third port; and iii) By applying a negative pressure difference between the eluent receiving container and the chamber, the eluent buffer can flow through the filter into the eluent receiving container, while the valves in each flow path are closed for the first port, allowing the second port to be vented to atmospheric pressure, and open for the third port. 32. The method according to claim 30 or 31, further comprising: a step between steps a and b, allowing the sample to contact the filter for a predetermined incubation time, wherein the valves in each flow path are open for the first and second ports and closed for the third port. 33. The method of claim 30, further comprising: one or more of the following steps between steps b and c: (i) The filter is cleaned and dried for a predetermined time by applying negative pressure between the third flow passage and the chamber, while the valves in each flow passage are closed for the first port, allowing the second port to vent to atmospheric pressure, and open for the third port; and/or (ii) By applying a negative pressure differential between one of the one or more waste receiving containers and a second reservoir, the cleaning buffer located in the second reservoir is allowed to enter the chamber through a second port, flow through a filter, and enter the one waste receiving container, while valves in each flow path are open for the second and third ports and closed for the first port; and/or iii) By applying a negative pressure between the third flow passage and the chamber, gas is allowed to flow through the filter for a predetermined time, while the valves in each flow passage are closed for the first port, allowing the second port to vent to atmospheric pressure, and open for the third port. 34. The method of claim 30, wherein one or more of the valves are activated by a pressure difference.