ENZYME-AMPLIFIED LATERAL FLOW DEVICE

Abstract

The invention concerns a lateral flow assay device for determining the presence of an analyte in a liquid sample; the use of said device to test for the presence of an analyte in a liquid sample; and a method for determining the presence of an analyte in a liquid sample 5 involving the use of said device.

Claims

1. An assay device for determining the presence of an analyte in a liquid sample, the device comprising: a lateral flow membrane comprising a test region disposed between opposite first and second end regions of the membrane; a fluid permeable layer having an area in contact with the first end region of the membrane; and a sample pad in fluid communication with the fluid permeable layer for delivery of a liquid sample to the first region of the membrane via the fluid permeable layer, wherein: the fluid permeable layer comprises a mobile assay component configured to selectively bind the target analyte, the mobile assay component comprising a reporter enzyme which specifically converts an active substrate into an optical reporting molecule; the sample pad comprises a mobile latent substrate, the latent substrate comprising one or more active substrate precursor compounds that is/are not reactive with a reporter enzyme; and the test region comprises: an immobilized assay component for retaining the mobile assay component in the test region in dependence on the binding between the analyte, the mobile assay component and the immobilized assay component; and an immobilized activation enzyme that specifically converts the latent substrate into the active substrate.

2. The device according to claim 1, wherein the activation enzyme is an oxidase enzyme and the latent substrate comprises a precursor compound that undergoes oxidation to hydrogen peroxide upon contact with the activation enzyme

3. The device according to claim 2, wherein the activation enzyme is glucose oxidase and the latent substrate comprises glucose as a precursor compound.

4. The device according to claim 1, wherein the latent substrate further comprises a compound selected from amplex red, o-phenylenediamine dihydrochloride, 3,3,5,5-tetram ethylb enzi dine, 2,2-azino-di-[3-ethylbenzthiazoline-6-sulfonic acid], 3,3-diaminobenzidine, 3-amino-9-ehtylcabazole luminol and combinations thereof.

5. The device according to claim 1, wherein the immobilized assay component comprises a compound selected from amplex red, o-phenylenediamine dihydrochloride, 3,3,5,5-tetram ethylb enzi dine, 2,2-azino-di-[3-ethylbenzthiazoline-6-sulfonic acid], 3,3-diaminobenzidine, 3-amino-9-ehtylcabazole luminol and combinations thereof.

6. The device according to claim 1, wherein the reporter enzyme is a peroxidase enzyme.

7. The device according to claim 6, wherein the reporter enzyme is a horseradish peroxidase or a polymer thereof.

8. The device according to claim 1, wherein the sample pad comprises latent substrate in an amount sufficiently high to ensure that, after delivery of the liquid sample to the sample pad, the concentration of latent substrate in the liquid sample is not the rate limiting factor for the activation enzyme-catalyzed conversion of latent substrate to active substrate.

9. The device according to claim 1, wherein the fluid permeable layer comprises the mobile assay component in an amount sufficiently low to ensure that it will be completely dissolved by the solvent front after delivery of the liquid sample to the sample pad.

10. The device according to claim 1, wherein the mobile assay component comprises a conjugate of an analyte-specific biological receptor and the reporter enzyme.

11. The device according to claim 10, wherein the mobile assay component comprises a conjugate of an analyte-specific antibody or nucleic acid and the reporter enzyme.

12. (canceled)

13. The device according to claim 1, wherein the immobilized assay component comprises an analyte-specific biological receptor that is immobilized in the test region of the lateral flow membrane.

14. The device according to claim 13, wherein the immobilized assay component comprises an analyte-specific antibody or nucleic acid that is immobilized in the test region of the lateral flow membrane.

15. (canceled)

16. The device according to claim 1, wherein the test region comprises a test line on which the activation enzyme and immobilized assay component are co-immobilized.

17. The device according to claim 1, wherein the test region comprises at least two test lines, the activation enzyme being immobilized on a first test line and the immobilized assay component being immobilized on a second test line.

18. The device according to claim 17, wherein the first test line is disposed between the first end region of the lateral flow membrane and the second test line.

19. The device according to claim 1, further comprising an absorptive layer in fluid contact with the second end region of the membrane.

20. A test kit comprising: a device according to claim 1; and a housing; wherein the device is disposed in the housing.

21. (canceled)

22. The use of the device according to claim 1 or the test kit according to claim 20 to test for the presence of an analyte in a liquid sample.

23. (canceled)

24. A method for determining the presence of an analyte in a liquid sample, the method comprising: providing a device according to claim 1 or a test kit according to claim 20; providing a liquid sample for analysis; and applying the liquid sample to the sample pad of said device.

25. (canceled)

Description

[0035] An embodiment of the present invention will now be described by way of example only with reference to the following wherein:

[0036] FIG. 1 is a schematic representation of an exemplary lateral flow device in accordance with the invention, which utilizes a sandwich assay for the single-step detection of an analyte within a liquid sample.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0037] FIG. 1 provides a specific example of the use of a latent substrate-based test device of the invention in a sandwich assay to determine the presence of an analyte in a liquid test sample.

[0038] Shown is a device comprising: (i) a sample pad, which comprises an excess amount of latent substrate (denoted LS); (ii) a conjugate pad, which is a fluid permeable layer disposed between the sample pad and a lateral flow membrane, and which comprises a mobile assay component (denoted Ab1-RE), specifically a conjugate of reporter enzyme (e.g. horseradish peroxidase) and an analyte specific 1.sup.st antibody; (iii) a lateral flow membrane that contains a test region disposed between opposite end regions of the membrane, the test region comprising a single test line on which the activation enzyme and an immobilized assay component (denoted Ab2), which is an analyte-specific 2.sup.nd antibody; and (iv) an absorptive layer or wicking pad.

[0039] The following sequence of events show how such a device can be used to test for presence if an analyte within a liquid sample: [0040] a liquid test sample suspected of containing the analyte of interest is first applied by the end user to the sample pad of the device, thereby dissolving an excess amount of LS within the liquid test sample; [0041] the liquid sample (containing LS) passes through the conjugate pad, lateral flow membrane and into the wicking pad by capillary flow; [0042] as the liquid sample passes into the conjugate pad, Ab1-RE conjugate is dissolved within the solvent front of the LS containing liquid sample; [0043] if present, analyte within the liquid sample will bind to the Ab1-RE conjugate as the sample passes through the conjugate pad and across the lateral flow membrane towards the test line. No enzymic reactions take place at this stage, as no active substrate or activation enzyme is present within the sample; [0044] as the liquid sample (containing LS, Ab1-RE and, if analyte is present, Ab1-RE/analyte complex) reaches the test line, any Ab1-RE/analyte complexes will bind to Ab2, becoming immobilized at the test line. In contrast, any non-complexed AB1-RE conjugates will continue to flow through the lateral flow membrane before arriving at the wicking pad; [0045] a concerted series of enzymic reactions will take place at the test line, provided that AB1-RE/analyte complexes have bound to the immobilized Ab2 Specifically, LS is converted to active substrate by the immobilized activation enzyme, and the active substrate is then converted to form a fluorescent, coloured or chemiluminescent optical reporting molecule by the reporter enzyme of the immobilized Ab2/analyte/Ab1-RE complex, confirming the presence of analyte within the tested liquid sample.

[0046] As shown above, the present invention provides an enzyme-based lateral flow detection device having low non-specific binding and a high optical signal per analyte ratio, and which can detect the presence of an analyte of interest in a single step, without the need for additional user input beyond standard sample application.

REFERENCES

[0047] 1. Development of Chemiluminescent Lateral Flow Assay for the Detection of Nucleic Acids Wang Y., Fill C. and Nugen, S. R. Biosensors 2012, 2(1), 32-42; doi: 10.3390/bios2010032.

[0048] 2. Moving Enzyme-Linked ImmunoSorbent Assay to the Point-of-Care Dry-Reagent Strip Biosensors

Kawde A. N., Mao X., Xu H., Zeng Q., He Y., and Liu G Am. J. Biomed. Sci. 2010, 2(1), 23-32; doi: 10.5099/aj100100023.

[0049] 3. Chemiluminometric Immunosensor for High-Sensitivity Cardiac Troponin I Employing a Polymerized Enzyme Conjugate as a Tracer.

Lim G. S., Seo S. M., Paek S. H., Kim S. W., Jeon J. W., Kim D. H., Cho I. H., and Paek S. H. Sci Rep. 2015 5:14848; doi: 10.1038/srep14848.