Apparatus for analysing a liquid sample and method of use thereof

Abstract

Apparatus for analysing a liquid sample containing one or more biomolecules, the apparatus comprising: a reaction chamber comprising a reaction chamber opening, through which the liquid sample may be introduced into the reaction chamber, so that it may be subjected therein to at least one reaction specific to the analysis; and an assay housing comprising an assay housing opening leading to a chromatographic flow assay; wherein the apparatus may be assembled so as to form a liquid-tight seal between the reaction chamber opening and the assay housing opening, the reaction chamber and assay housing thereby being in liquid communication with each other, the apparatus, in its assembled state, having a reaction orientation such that gravity acts to hold the liquid sample within the reaction chamber and an analysis orientation wherein gravity acts to aid the flow of the liquid sample to the chromatographic flow assay for analysis.

Description

[0001] Apparatus for Analysing a Liquid Sample

[0002] and Method of Use thereof

[0003] Technical Field

[0004] The present invention relates to an apparatus for analysing a liquid sample containing one or more biomolecules, and a method of use thereof.

[0005] Background

[0006] Devices that carry out a chromatographic flow assay wherein a liquid sample is introduced, such as a lateral flow or vertical flow assay, are known. These operate to detect the presence of a target biomolecule such as a specific protein, or a nucleic acid (e.g. DNA or RNA) of defined sequence, in a liquid sample. The tests operate by formation of a dye complex in the presence of the target biomolecule, which produces a colour test result in a defined area of the assay. Typically, such assays are one-shot and are intended to be used only once.

[0007] Such tests may be provided with a reaction chamber, to receive the liquid sample and wherein the biomolecule may be subject to one or more reactions specific to the analysis. This reaction chamber may have dried ingredients already present within it as required for the reaction and analysis. The reaction may be a binding reaction, such as an immunoassay wherein an antibody binds directly to the biomolecule of interest, and / or a nucleic acid amplification reaction which produces many copies of a nucleic acid amplicon in the presence of the target biomolecule.

[0008] Nucleic acid amplification may be accomplished by PCR, which uses temperature cycling and typically involves the use of sophisticated equipment that is complex to operate. Other processes do not require temperature cycling, such as so-called isothermal methods, where typically heat is applied to maintain a constant temperature during amplification. Typically, this involves placing the sample in a heating unit and the use of specific DNA polymerases and primer sets to amplify a target sequence. Isothermal techniques provide significant benefits over temperature-cycling methods due to the simplicity of the supporting equipment without the requirement for rapid heating and cooling and precise temperature control.Some examples of isothermal techniques include Loop Mediated Isothermal Amplification (LAMP), Nucleic Acid Sequence Based Amplification (NASBA), Strand Displacement Amplification (SDA), Recombinase Polymerase Amplification (RPA), Helicase Dependent Amplification (HAD), Rolling Circle Amplification (RCA) and Ligase Chain Reaction (LCR).

[0009] Nucleic acid amplification reactions produce many copies of a nucleic acid amplicon, which can then be detected by a chromatographic flow assay, using hybridisation or an affinity tag. Use of a chromatographic flow assay is advantageous compared to the use of the fluorescence-based detection commonly used in PCR instruments, as it produces a simple visual read-out and leads to a low-cost test without requirement for complex instrumentation.

[0010] The process of transferring a liquid sample from the reaction chamber to the chromatographic flow assay in a controlled way in known devices can be cumbersome. It can be accomplished manually, using a pipette in a process that can lead to errors or spillage of the liquid sample, or automatically, using a device or instrument which involves complex mechanisms for fluid transfer. The liquid sample may be hazardous due to the presence of biological agents or toxic chemicals.

[0011] Furthermore, in the case of nucleic acid amplification reactions, the release of target amplicon into the environment is a common problem, leading to potential contamination of the testing location and an increased risk of false positives in subsequent tests.

[0012] Devices that are able to carry out chromatographic flow assays that do not suffer from the above technical difficulties associated with transfer of the liquid sample to the chromatographic flow assay would therefore be desirable. In particular such devices that are designed for an isothermal nucleic acid amplification reaction would offer further advantages, enabling easy to operate, low-cost tests that are superior to known devices.

[0013] Summary of Invention

[0014] In a first aspect, the invention relates to an apparatus for analysing a liquid sample containing one or more biomolecules, the apparatus comprising:a reaction chamber comprising a reaction chamber opening, through which the liquid sample may be introduced into the reaction chamber, so that it may be subjected therein to at least one reaction specific to the analysis; and

[0015] an assay housing comprising an assay housing opening leading to a chromatographic flow assay;

[0016] wherein the apparatus may be assembled so as to form a liquid-tight seal between the reaction chamber opening and the assay housing opening, the reaction chamber and assay housing thereby being in liquid communication with each other,

[0017] the apparatus, in its assembled state, having a reaction orientation such that gravity acts to hold the liquid sample within the reaction chamber and an analysis orientation wherein gravity acts to aid the flow of the liquid sample to the chromatographic flow assay for analysis.

[0018] Preferably when in its assembled state the apparatus may be moved from its reaction orientation to its analysis orientation by rotation of the assembled apparatus. The liquid-tight seal between the reaction chamber opening and the assay housing opening ensures that no liquid can leave the assembled apparatus as liquid flows within. As the reaction chamber and assay housing are in liquid communication with each other, liquid can however flow freely between the reaction chamber and assay housing without requiring the removal of any physical restrictions, such as a valve or an encapsulated liquid compartment.

[0019] In a preferred embodiment, when in its assembled state and in its reaction orientation the assay housing is above the reaction chamber, and when moved to its analysis orientation the assay housing is below the reaction chamber. This relative positioning has the effect of determining where liquid in the assembled apparatus is located.

[0020] The invention has the benefit that the results of the assay can be triggered merely by reorienting the assembled apparatus. The apparatus is also especially convenient for use with a standard laboratory heat block or PCR machine if the reaction requires temperature control. Additionally, the transfer of the liquid sample to the chromatographic flow assay is performed using a sealed connection, ensuring no liquid leakage during use.In preferred embodiments, the chromatographic flow assay can be a lateral flow ora flow through assay. The apparatus is broadly applicable in that it may be used for immunoassays, such as antigen tests or enzyme-linked immunosorbent assays (ELISAs), and / or for nucleic acid amplification tests, including those based on PCR and isothermal techniques.

[0021] In the field of molecular diagnostics the term ‘isothermal’ is generally understood to mean heated to a single temperature ratherthan thermally cycled as is the case with PCR (the most widely known / used method), i.e. ‘isothermal’ means does not undergo thermal cycling ratherthan does not undergo heating of any kind.

[0022] Preferably, when the assembled apparatus is in the reaction orientation the reaction chamber opening is above the level of any liquid sample contained in the reaction chamber, and in the analysis orientation the reaction chamber opening is below the level of any liquid sample contained in the reaction chamber such that any liquid sample contained in the reaction chamber is permitted to flow out of the reaction chamber opening and through the assay housing opening to the chromatographic flow assay for analysis. Thus, the assembled apparatus may be reoriented in space in a convenient and simple manner to control the movement of the liquid sample during the analysis. It is advantageous to accomplish the controlled movement of a liquid sample without any complex mechanisms or device parts, which makes the apparatus of the invention simpler to manufacture, lower cost and easier to operate than known alternatives.

[0023] Preferably the apparatus in its assembled state forms a liquid-tight volume containing any liquid sample and the chromatographic flow assay. This has the advantage that the integrated sealed unit prevents spillage or release of the sample, which is particularly important if the sample is hazardous due to the presence of biological agents or hazardous chemicals. For example, if the analysis using the apparatus is performed for the diagnosis of an infectious disease, the liquid sample may be derived from a biological specimen and contain infectious viruses or bacteria. Furthermore, hazardous chemicals which are toxic to people and the environment are frequently used in the analysis of biomolecules. Since the liquid sample may be completely sealed within the assembled apparatus, a human operator is protected from exposure to potentially hazardous substances that may otherwise be released. Likewise the device protects against environmental contamination following disposal.Furthermore, in the case of nucleic acid amplification reactions, many copies of a nucleic acid amplicon are produced and the formation of a liquid-tight volume when the apparatus is in its assembled state minimises the risk of release of the amplicon. This is desirable as it reduces or eliminates potential contamination of the testing location by amplicon that otherwise can trigger a false positive result in a subsequent test. Such amplicon contamination is an important consideration in the field of molecular diagnostics, given the sensitivity of nucleic acid amplification tests and the fact that such tests often produce billions of copies of a target amplicon. Furthermore, in clinical diagnostics, repeat testing is often performed at a single location, such as a clinic, hospital, laboratory or physician office, thus increasing the chance of amplicon contamination and the subsequent generation of false positive test results.

[0024] In a preferred embodiment the assay housing comprises first and second compartments, the first compartment incorporating the assay housing opening and the second compartment containing the chromatographic flow assay, the only pathway for fluid to flow from the first compartment to the second compartment being a port which is at least partially covered by the chromatographic flow assay. Common chromatographic flow assays include lateral flow assays and vertical flow assays, in which the liquid sample flows laterally along or vertically through a physical material, respectively. The physical material is typically a thin layer of porous membrane such as nitrocellulose comprising an affinity binding reagent deposited in a particular location that forms the site for the development of a coloured results signal in the presence of the target biomolecules. Such assays may further comprise dried coloured reagents that are resolubilised by the liquid sample and subsequently bind to the said affinity binding reagent in the presence of a target biomolecule to produce the results signal. As such, controlling the pathway of fluid flow from the first compartment to the second compartment helps to ensure that any dried colour reagents are released and that affinity binding and signal development occur correctly.

[0025] Preferably, in use the chromatographic flow assay has a direction of sample flow, and the port is oriented transversely to the direction of sample flow. For example, in the case of a lateral flow assay, the positioning of the port transversely to the direction of sample flow helps to ensure the controlled flow of the liquid sample onto one end of the test strip which ensures it passes into the flow assay material, e.g. nitrocellulose, efficiently anddoes not flow over its surface (overflow) thus helping to ensure the consistent release of any dried colour reagents and subsequent development of the colour results signal.

[0026] Preferably the liquid tight seal is formable by the insertion of the reaction chamber into the assay housing or by insertion of the assay housing into the reaction chamber. More preferably the liquid-tight seal is formed by direct contact between an inner female circumferential surface of one of the assay housing opening and the reaction chamber opening, and an outer male circumferential surface of the other of the assay housing opening and the reaction chamber opening, as insertion takes place. Preferably the circumferential surfaces which form the direct contact are configured with vents so as to allow the egress of air as the said inner circumferential surface is inserted inside the said outer circumferential surface. The egress of air helps to avoid a build-up of pressure which would potentially hamper fluid flow and the correct functioning of the assembled apparatus, particularly in embodiments wherein the apparatus in its assembled state forms a liquid-tight volume containing any liquid sample and the chromatographic flow assay.

[0027] The apparatus may further comprise a locking means which locks the assay housing to the reaction chamber when the assay housing opening is attached to the reaction chamber opening. This helps to maintain the apparatus in its assembled state during a reaction and subsequent analysis to ensure correct transfer of a liquid sample from the reaction chamber to the chromatographic flow assay and prevent a user from easily disassembling the apparatus. As such the locking means is advantageous because it minimises the risk of release of any liquid sample, which is particularly important for samples that may contain hazardous biological agents, toxic chemicals or copies of a nucleic acid amplicon as described above.

[0028] The apparatus may be made from a wide variety of materials, although polymeric plastic materials are preferred. Preferably the reaction chamber is formed of polypropylene. Polypropylene has desirable properties due to its suitability for manufacturing of the apparatus, e.g. by injection moulding, and due to its surface properties as it typically does not interact with biomolecules which may interfere with the analysis. Preferably at least a part of the assay housing is transparent. The transparent part of the assay housing may form a window through which the results of the chromatographic flow assay are observed without exposing the liquid sample to the external environment. In an embodiment wherein the apparatus forms a liquid-tight volume containing any liquidsample and the chromatographic flow assay, the use of a transparent window permits the liquid-tight volume to be maintained which is advantageous because it minimises the risk of release of any sample.

[0029] Preferably the assay housing comprises two or more parts which are joined together so as to contain the chromatographic flow assay. The use of two or more parts permits the chromatographic flow assay to be easily assembled into the assay housing in a way that is suitable for efficient manufacture. It would otherwise be challenging to insert the chromatographic flow assay into the assay housing in a way that maintains the liquid-tight seal between the reaction chamber opening and the assay housing opening, particularly in embodiments wherein the assembled apparatus further forms a liquid-tight volume containing any liquid sample and the chromatographic flow assay. This construction also allows for the creation of a transverse port to aid with the control of flow of a liquid sample whilst being readily manufacturable.

[0030] The chromatographic flow assay preferably comprises colour particles. Commonly in lateral flow and vertical flow assays such colour particles, which may be gold nanoparticles or polystyrene microspheres, for example, are present in a dried form in a separate conjugate pad. In an embodiment the chromatographic flow assay comprises colour particles of two or more different colours. It may be advantageous for assays that detect and differentiate two or more different biomolecules or that include both test and control assays, to utilise two or more different colour particles in order to make it easier for the operator to interpret the results of the different assays.

[0031] Preferably the chromatographic flow assay is an elongate strip. Lateral flow strips are typically constructed in elongate form with a common construction being such that the sample for analysis flows from one end to the other through a conjugate pad, a nitrocellulose membrane and into an absorbent pad constructed of absorbent material, such as cellulose.

[0032] In a second aspect, the invention relates to a method of use of an apparatus as described herein, the method comprising introducing the liquid sample to the reaction chamber, assembling the apparatus into its assembled state whilst it is in its reaction orientation, followed by subjecting the liquid sample to the at least one reaction, followed by moving the apparatus to its analysis orientation for analysis.Preferably the movement of the apparatus from the reaction orientation to the analysis orientation is by rotation of the assembled apparatus. In particular the method preferably comprises inverting the apparatus.

[0033] Preferably in the reaction orientation the reaction chamber opening is above the level of the liquid sample contained therein. Thus, gravity assists in keeping the liquid sample in place in the reaction chamber.

[0034] Once moved to the analysis orientation the reaction chamber opening is preferably below the level of the liquid sample contained therein, so that the liquid sample may flow from the reaction chamber into the assay housing and into contact with the chromatographic flow assay.

[0035] Preferably the reaction includes at least one reaction comprising heating the liquid sample within the reaction chamber.

[0036] Preferably at least one reaction includes at least a nucleic acid amplification.

[0037] In a third aspect the invention relates to a kit of parts including an apparatus as described herein and a polymerase chain reaction (PCR) machine or a heat block, in which the reaction chamber is sized and configured so as to be receivable within the PCR machine or heat block.

[0038] The invention will now be illustrated, by way of example, and with reference to the following figures, in which:

[0039] Figure 1a is a front view of a reaction chamber for use with an apparatus according to the present invention.

[0040] Figure 1b is a side sectional view through line A-A of the reaction chamber shown in figure 1a.

[0041] Figure 2a is a front view of an assay housing for use with an apparatus according to the present invention.Figure 2b is a side sectional view through line B-B through the assay housing shown in figure 2a.

[0042] Figure 3a is a front view of an assembled apparatus according to the present invention, formed by mechanically attaching the reaction chamber of figures 1a and 1b with the assay housing of figures 2a and 2b.

[0043] Figure 3b is a side sectional view through line C-C through the apparatus shown in figure 3a.

[0044] Figure 4 is a cut-away isometric view of the apparatus shown in figures 3a and 3b.

[0045] Figure 5a is an isometric view from above and one side of the apparatus shown in figures 3a and 3b without the removeable outer protective casing on the assay housing.

[0046] Figure 5b is an alternative isometric view from above and one side of the apparatus shown in figure 5a.

[0047] Turning to the figures, figures 1a and 1b show a reaction chamber 10 for use with an apparatus according to the present invention, comprising an outer wall 12 surrounding an inner reaction chamber 13, in which is placed dried reagents 14. The reaction chamber 10 has a reaction chamber opening 16, which in the drawings is covered by a releasable cover 18. The reaction chamber opening 16 is surrounded by a lip 20 that is intended to interact with the assay housing 50 described below. The reaction chamber has an array of vent channels 19 which allow the egress of air as the outer circumferential surface of the assay housing opening 54 is inserted inside the inner circumferential surface of the reaction chamber opening 16.

[0048] Figures 2a and 2b show an assay housing 50 designed for use with the reaction chamber 10 shown in figures 1a and 1b. The assay housing 50 comprises a removeable outer protective casing 52 into which is inserted a sealed assay housing insert 56, into which is placed a chromatographic flow assay 58, in this case a lateral flow strip. The assay housing insert 56 extends out of the containing wall 52 to provide an assay housing opening 54 which leads to a first compartment 62. The assay housing insert 56 provides a second compartment housing the chromatographic flow assay 58 which is sealed on the reverse by a sealing strip 66. The only pathway for liquid to flow from the firstcompartment 62 to the chromatographic flow assay 58 being a port 64 which is at least partially covered by part of the chromatographic flow assay 58, in this case the conjugate pad of the lateral flow strip 58.

[0049] The housing insert 56 therefore only exposes the conjugate pad of the lateral flow strip 58 to the contents of the first compartment 62. Thus, when a liquid sample enters through the assay housing opening 54, it remains in the first compartment 62 and is absorbed into the conjugate pad of the lateral flow strip 58. The results of the testing appear, to be viewed through the transparent window 60.

[0050] Extending downwardly from the outer containing wall 52 is a pair of resilient clips 68, which cooperate with the lip 20 of the reaction chamberto form the assembled apparatus 100 comprising the reaction chamber 10 and the assay housing 50 mechanically attached together, so as to form a liquid-tight seal between the reaction chamber opening 16 and the assay housing opening 54, the reaction chamber 10 and assay housing 50 thereby being in liquid communication with each other as shown in Figures 3a, 3b, 4 (with the protective casing 52) and in 5a and 5b (without protective casing 52).

[0051] The liquid-tight seal is formed through direct contact between the inner circumferential surface of the reaction chamber opening 16, and the outer circumferential surface of the assay housing opening, as the assay housing opening 54 is inserted inside the reaction chamber opening 16.

[0052] Once formed, the reaction chamber 10 and assay housing 50 are in liquid communication with each other, and a liquid sample contained within the reaction chamber 10 remains in the reaction chamber 10 while the apparatus 100 remains in its reaction orientation as shown in figures 3a and 3b. When the apparatus in its assembled state 100 is in the orientation shown in the figures, gravity acts to hold any liquid sample contained within reaction chamber 13. Other forces, such as surface tension effects combine with gravity to hold the liquid sample within the reaction chamber 13. When the apparatus 100 in its assembled state is inverted by rotation to adopt its analysis orientation, so that the reaction chamber 10 is located above the assay housing 50, any liquid sample in the reaction chamber 10 is aided by gravity to flow into the testing chamber 62 of the assay housing 50. However, due to surface tension effects, it is usual to assist the flow of liquid by providing a brief shaking action to the assembled apparatus 100.

Claims

Claims1. An apparatus for analysing a liquid sample containing one or more biomolecules, the apparatus comprising:a reaction chamber comprising a reaction chamber opening, through which the liquid sample may be introduced into the reaction chamber, so that it may be subjected therein to at least one reaction specific to the analysis; andan assay housing comprising an assay housing opening leading to a chromatographic flow assay;wherein the apparatus may be assembled so as to form a liquid-tight seal between the reaction chamber opening and the assay housing opening, the reaction chamber and assay housing thereby being in liquid communication with each other,the apparatus, in its assembled state, having a reaction orientation such that gravity acts to hold the liquid sample within the reaction chamber and an analysis orientation wherein gravity acts to aid the flow of the liquid sample to the chromatographic flow assay for analysis.

2. An apparatus according to claim 1 , wherein in the reaction orientation the reaction chamber opening is above the level of any liquid sample contained in the reaction chamber, and in the analysis orientation the reaction chamber opening is below the level of any liquid sample contained in the reaction chamber such that any liquid sample contained in the reaction chamber is permitted to flow out of the reaction chamber opening and through the assay housing opening to the chromatographic flow assay for analysis.

3. An apparatus according to any one of the preceding claims, wherein in its assembled state it may be moved from its reaction orientation to its analysis orientation by rotation of the assembled apparatus.

4. An apparatus according to any one of the preceding claims, wherein in its assembled state and in its reaction orientation the assay housing is above thereaction chamber, and when moved to its analysis orientation the assay housing is below the reaction chamber.

5. An apparatus according to any one of the preceding claims, which in its assembled state forms a liquid-tight volume containing any liquid sample and the chromatographic flow assay.

6. An apparatus according to any one of the preceding claims, in which the assay housing comprises first and second compartments, the first compartment incorporating the assay housing opening and the second compartment containing the chromatographic flow assay, the only pathway for fluid to flow from the first compartment to the second compartment being a port which is at least partially covered by the chromatographic flow assay.

7. An apparatus according to claim 6, in which in use the chromatographic flow assay has a direction of sample flow, and in which the port is oriented transversely to the direction of sample flow.

8. An apparatus according to any one of the preceding claims, wherein when assembled, the reaction chamber does not contain any portion of the chromatographic flow assay.

9. An apparatus according to any one of the preceding claims, wherein when assembled, the chromatographic flow assay is contained entirely within the assay housing.

10. An apparatus according to any one of the preceding claims, wherein the liquid tight seal is formable by the insertion of the reaction chamber into the assay housing or by insertion of the assay housing into the reaction chamber.

11. An apparatus according to claim 10, in which the liquid-tight seal is formed by direct contact between an inner female circumferential surface of one of the assay housing opening and the reaction chamber opening, and an outer male circumferential surface ofthe other ofthe assay housing opening and the reaction chamber opening, as insertion takes place.

12. An apparatus according to claim 11, in which the circumferential surfaces which form the direct contact are configured with vents so as to allow the egress of air as the said inner circumferential surface is inserted inside the said outer circumferential surface.

13. An apparatus according to any one of the preceding claims, further comprising locking means which locks the assay housing to the reaction chamber when the assay housing opening is attached to the reaction chamber opening.

14. An apparatus according to any one of the preceding claims, in which the reaction chamber is formed of polypropylene.

15. An apparatus according to any one of the preceding claims, in which at least a part of the assay housing is transparent.

16. An apparatus according to any one of the preceding claims, in which the assay housing comprises two or more parts which are joined together so as to contain the chromatographic flow assay.

17. An apparatus according to any one of the preceding claims, in which the chromatographic flow assay comprises colour particles.

18. An apparatus according to any one of the preceding claims, in which the chromatographic flow assay is an elongate strip.

19. An apparatus according to any one of the preceding claims, in which the reaction chamber contains one or more reagents specific to the analysis.

20. A method of use of an apparatus according to any one of the preceding claims, the method comprising introducing the liquid sample to the reaction chamber, assembling the apparatus into its assembled state whilst it is in its reaction orientation, followed by subjecting the liquid sample to the at least one reaction, followed by moving the apparatus to its analysis orientation for analysis.

21. A method according to claim 20, wherein the movement from the reaction orientation to the analysis orientation is by rotation of the assembled apparatus.

22. A method according to claim 21 , in which moving the apparatus to the second orientation comprises inverting the apparatus.

23. A method according to any one of claims 20 to 22, in which in the reaction orientation the reaction chamber opening is above the level of the liquid sample contained therein.

24. A method according to any one of claims 20 to 23, in which in the analysis orientation the reaction chamber opening is below the level of the liquid sample contained therein, so that the liquid sample may flow from the reaction chamber into the assay housing and into contact with the chromatographic flow assay.

25. A method according to any one of claims 20 to 24, in which the at least one reaction comprises heating the liquid sample within the reaction chamber.

26. A method according to any one of claims 20 to 24, in which the at least one reaction is a nucleic acid amplification.

27. A kit of parts including an apparatus according to any of claims 1 to 19 and a polymerase chain reaction (PCR) machine or a heat block, in which the reaction chamber is sized and configured so as to be receivable within the PCR machine or heat block.

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