METHODS AND COMPOSITIONS FOR LATERAL FLOW ANALYTE ASSAYS

Abstract

It is an object of the present invention to provide improved lateral flow test devices that can provide sensitive and accurate quantitative test results, and methods for the manufacture thereof.

Claims

1. A lateral flow analyte test device, comprising: (a) a test strip comprising a first bibulous material forming a sample receiving region, a second bibulous material forming an absorbent region, and a porous membrane fluidly connected to the first bibulous material at a proximal end of the porous membrane, and fluidly connected to the second bibulous material at a distal end of the porous membrane, wherein the proximal and distal ends of the porous membrane define a flow path such that a sample applied to the sample receiving region traverses the porous membrane from the proximal end thereof to the distal end thereof, wherein a longitudinal axis is defined from the proximal end of the porous membrane to the distal end of the porous membrane, and a width axis is defined perpendicular to the longitudinal axis from a first lateral edge of the porous membrane to a second lateral edge of the porous membrane, wherein the dimension of the porous membrane along its longitudinal axis is greater than the dimension of the porous membrane along its width axis, and wherein the porous membrane comprises at least one test zone comprising one or more reagents configured to bind for detection at least one analyte of interest from the sample immobilized at the at least one test zone, the test zone occupying an area of the porous membrane defined by a width (W.sub.T) along the width axis of the porous membrane and a length (L.sub.T) along the longitudinal axis of the porous membrane; (b) a generally rigid base which supports the test strip; (c) a circuit board comprising at least one light source configured to illuminate the at least one test zone with electromagnetic radiation, and at least one photodetector configured to detect an optical signal from the test zone resulting from illumination of the test zone by the electromagnetic radiation; (d) a circuit board support configured to position the at least one light source above the test zone at a height h.sub.1 and at a location that is lateral to the width axis of the porous membrane, and to position the at least one photodetector above the at least one test zone at a height h.sub.2 and at a location that is approximately centered on the width axis of the porous membrane, wherein the circuit board support comprises an elongate first aperture having a first end proximal to the light source and a second end proximal to the at least one test zone, wherein the height h.sub.1 and the dimensions of the elongate first aperture are configured and arranged such that electromagnetic radiation from the light source illuminates the at least one test zone at an angle of incidence θ, measured relative to a surface normal from the porous membrane, that is between about 40° and about 10°, and a second aperture between the at least one test zone and the at least one photodetector, wherein the height h.sub.2 and the dimensions of the second aperture are configured and arranged such that that the angle of view of the photodetector is limited to an area (S.sub.A) of the porous membrane that comprises the test zone that is no more than twice the area occupied by the test zone; (e) a processing component operably connected to the at least one light source and the at least one photodetector to (i) control the illumination of the test zone by the light source, (ii) receive an electrical signal from the photodetector resulting from the optical signal, and (iii) convert the electrical signal into an assay result indicative of the presence or amount of the analyte of interest in the sample; and (f) a display component operably connected to the processing component to display the assay result.

2. A lateral flow analyte test device according to claim 1, wherein the optical signal detected from the test zone is defined by the equation
1−[(run time optical signal)−(offset signal)]/[(reference signal)−(offset signal)], wherein the run-time optical signal is defined by the optical signal measured during the test and after the reference signal has been measured for a given measurement zone; the reference signal is defined by an optical signal measured from a corresponding unreacted test device; and wherein the offset signal is defined by an optical signal measured from a corresponding test device in which the porous membrane is replaced with a material that absorbs at least 90% of the electromagnetic radiation emitted from the light source.

3. A lateral flow analyte test device according to claim 1 or 2, wherein the assay result is a quantitative result.

4. A lateral flow analyte test device according to claim 3, wherein the amount of the at least one analyte of interest in the sample is measured by a change in absorbance or fluorescence due to the emitted light source electromagnetic radiation incident on the test zone.

5. A lateral flow analyte test device according to one of claims 1-4, wherein S.sub.A is defined by a sampling width (W.sub.S) that is no more than 50% larger than W.sub.T, and a sampling length (L.sub.S) that is no more than 50% larger than L.sub.T.

6. A lateral flow analyte test device according to one of claims 1-4, wherein S.sub.A is defined by a sampling width (W.sub.S) that is no more than 90% of W.sub.T, and a sampling length (L.sub.S) that is no more than 10% larger than L.sub.T.

7. A lateral flow analyte test device according to one of claims 1-6, wherein the second aperture is configured and arranged to shield the photodetector from any direct reflected or fluoresced light emanating from a surface of the test device other than the test strip.

8. A lateral flow analyte test device according to one of claims 1-7, wherein the first aperture, light source, and the second aperture and detector are configured and arranged such that the incident light is not specularly reflected off the test strip first surface into the photodetector.

9. A lateral flow analyte test device according to one of claims 1-8, wherein the first bibulous material or the porous membrane comprises a labeled mobilizable reagent that binds to the at least one analyte of interest, and wherein the labeled mobilizable reagent and the at least one analyte of interest form sandwich complexes with the reagents configured to bind for detection the at least one analyte of interest immobilized at the at least one test zone.

10. A lateral flow analyte test device according to one of claims 1-8, wherein the first bibulous material or the porous membrane comprises a labeled mobilizable reagent that competes with the at least one analyte of interest for binding to the reagents configured to bind for detection the at least one analyte of interest immobilized at the at least one test zone.

11. A lateral flow analyte test device according to one of claim 9 or 10, wherein the labeled mobilizable reagent comprises a metal colloid label.

12. A lateral flow analyte test device according to one of claim 9 or 10, wherein the labeled mobilizable reagent comprises a particulate label.

13. A lateral flow analyte test device according to one of claim 9 or 10, wherein the labeled mobilizable reagent comprises a fluorescent label.

14. A method of manufacturing a lateral flow test device for one or more analytes of interest comprising a lateral flow substrate and a housing comprising a base configured to receive the lateral flow substrate and a top configured to mate with the base, comprising: a first fiducial marker comprising an optically detectable label at a location on the lateral flow substrate, wherein the optically detectable label does not participate in an assay for any analyte of interest; positioning a second fiducial marker at a location on the housing base, wherein the second fiducial marker is a physical structure that is integral to the housing base; inserting the lateral flow substrate into the housing base such that the first and second fiducial markers are aligned relative to one another within +/−250 μm of a predetermined orientation; and mating the housing top with the housing base.

15. A method according to claim 14, wherein the first fiducial marker is detectable to the human eye under light of a visible wavelength.

16. A method according to claim 14, wherein the first fiducial marker is detectable to the human eye under light of an ultraviolet wavelength.

17. A method according to claim 14, wherein the first fiducial marker is detectable to the human eye under light of an infrared wavelength.

18. A method according to one of claims 14-17, wherein the first fiducial marker is applied to the lateral flow substrate as a mixture with one or more reagents participating in an assay for an analyte of interest such that the first fiducial marker and the one or more reagents are colocalized on the lateral flow substrate.

19. A method according to one of claims 14-18, wherein the first fiducial marker is configured to be removable from the lateral flow substrate by washing with an aqueous medium.

20. A method according to one of claims 14-19, wherein the second fiducial marker comprises a groove, pit, or notch in the housing base.

21. A method according to one of claims 14-20, wherein the second fiducial marker comprises a raised ridge in the housing base.

22. A method according to claim 20 or 21, wherein the second fiducial marker is formed during molding of the housing base.

23. A method according to one of claims 14-22, wherein the predetermined orientation is a center-to-center alignment of the first fiducial marker and the second fiducial marker.

24. A method according to one of claims 14-23, wherein the alignment is within +/−125 μm of the predetermined orientation.

25. A method according to claim 24, wherein the alignment is within +/−75 μm of the predetermined orientation.

26. A method according to one of claims 14-25, wherein the alignment is performed using machine vision.

27. A method according to one of claims 14-26, wherein the lateral flow test device is a lateral flow test device according to one of claims 1-13.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0050] FIG. 1 depicts a typical lateral flow test strip configuration.

[0051] FIG. 2 depicts a cross section along the width axis at the location of the aperture.

[0052] FIG. 3 depicts an exploded view of the test strip, the optic block with circuit board, and the top and bottom cartridge housing.

[0053] FIG. 4 depicts a cross section along the longitudinal axis at the location of the 2.sup.nd aperture and test line along the test strip.

[0054] FIG. 5 depicts a cross section along the width axis at the location of a test line.

[0055] FIG. 6 depicts a cross section along the length axis across multiple aperture 2 locations.

[0056] FIG. 7 depicts the features used to align the test strip to the housing base.

DETAILED DESCRIPTION OF THE INVENTION

[0057] Lateral flow test devices have received wide acceptance in the diagnostic arts. These devices place a complicated set of reagents and manufactured elements into a simple, compact, easy to use package. See, e.g., Lateral Flow Immunoassay, Wong and Tse, eds., Humana Press, 2009. A test strip is shown in FIG. 1 in order to define certain elements of a typical test strip for use in the following discussion. A first bibulous material, often referred to as a sample pad, is provided at the proximal end of the test strip. It is this sample pad which receives the fluid sample to be tested. At the distal end of the test strip is a second bibulous material, often referred to as an absorbent pad, which operates to receive the sample fluid once it has passed through the porous membrane. The porous membrane, which may be a nitrocellulose membrane, is positioned between these two ends. The flow path of the sample is from the sample pad, through the nitrocellulose membrane, and into the absorbent pad. In doing so, the sample typically flows through a “conjugate pad”, which is a portion of the device which contains a labeled reagent that binds to, or competes with, the analyte of interest. This labeled reagent is mobilized in the sample matrix so that it flows with the sample.

[0058] A test line is a location on the nitrocellulose membrane that binds the labeled reagent in an amount related to the presence or amount of the analyte of interest. While depicted as a single test line, a lateral flow test strip may have multiple test lines, each of which is used to measure the presence or amount of a different analyte. As defined herein, the test line has a width dimension W.sub.T which lies on the width axis of the test strip, and a length dimension L.sub.T which lies on the longitudinal axis of the test strip. One or more control lines are often employed to act as an environmental control and to determine if the test has been successfully performed by the test strip.

[0059] The test strip is typically held within a housing in order to provide protection to the test strip and for purposes of handling by the user. When the test device is read by an external reader, limited or no electronics need to be provided within the housing. In the case of an integrated reader, this housing will also provide the reader electronics necessary to read and interpret the test result. As shown in the FIG. 3, such a housing can comprise a top portion 301 having an opening 302 into which a sample may be applied. The top can be mated to a bottom portion 304 which holds the test strip 303. In the exemplary embodiment shown in FIG. 3, an optical block 305 affixed to the circuit board 306, contains the light source and photodetector and light apertures. The system is generally powered by a small battery 307 and the digital readout is displayed through the window in the top housing 308

[0060] By way of example only, a test device may be formed by introducing a moldable material into a mold assembly to form the test device lid and base; removing the test device lid and base from the mold assembly; and mating the test device lid to the test device base such that a sample receiving aperture overlies the first bibulous material and a test aperture overlies the one or more test zones. While the lid and base may be formed with discrete molds, the mold assembly may be configured as a single assembly, wherein the test device base and the test device lid are formed as a unitary part. To facilitate fit of the lid and base, the test device base and the test device lid may be formed as a unitary part connected by one or more flexible hinge regions (e.g. living hinges) configured to allow the test device lid to mate to the test device base.

[0061] The skilled artisan will understand that a number of polymers may be used to form the test device base, including thermoplastics, some thermosets, and elastomers. Common thermoplastics include PMMA, cyclic olefin copolymer, ethylene vinyl acetate, polyacrylate, polyaryletherketone, polybutadiene, polycarbonate, polyester, polyetherimide, polysulfone, nylon, polyethylene, and polystyrene. Common thermosets include polyesters, polyurethanes, duroplast, epoxy resins, and polyimides. This list is not meant to be limiting. Functional filler materials such as talc and carbon fibers can be included for purposes of improving stiffness, working temperatures, and part shrinkage.

[0062] FIG. 2 depicts a cross section along the width axis (W.sub.T) at the point of a test or control line position along the longitudinal axis (LT) showing the arrangement of the optical block of a test device of the present invention. In this schematic view, the test device lid and base are not shown. A circuit board 201 supports a light source 202 and a photodetector 203. The test strip 204 is shown along its width axis W.sub.T such that light from the light source 202 passes through aperture 206 in optic block 205 (see 305 in FIG. 2) in order to illuminate the test line with an angle of incidence θ. The light source 202 is offset laterally from the center of the test line in order to minimize specular reflection of the incident light from reaching photodetector 203. The area of the test line that is illuminated is determined by the height h.sub.1 and the dimensions of aperture 206, and is configured to limit illumination to the test line area, with minimal overlap onto portions of the lateral flow membrane that do not contain the test line. Preferably, the area of the lateral flow membrane that is illuminated is no more than 50% larger than W.sub.T, and no more than 50% larger than L.sub.T, and is more preferably no more than 25%, or 10% larger than W.sub.T and L.sub.T, respectively. This is in order to minimize reflection of non-relevant incident light received by photodetector 203. Furthermore, the illuminated width, W.sub.T, may be actually be less than W.sub.T in order to avoid illuminating anything but the test strip.

[0063] A second aperture 207 in support 205 and height h.sub.2 define the sampling area S.sub.A, which is the area of the lateral flow test strip that is viewed by photodetector 203. The aperture dimension and h.sub.2 are defined to that sampling area S.sub.A is no more than 2× the area occupied by the test line. Preferably, S.sub.A is defined by a sampling width (W.sub.S) that is no more than 0% larger than W.sub.T, and a sampling length (L.sub.S) that is no more than 50% larger than L.sub.T, and is more preferably no more than 25% greater than L.sub.T, or 90% of W.sub.T.

[0064] FIG. 4 depicts a longitudinal cross section of a single photodetector in alignment with a test line. The purpose of the aperture in this direction is to define the extent of the longitudinal viewing cone. In this figure The aperture width A may be defined by the equation A<(h/H)*(Px−3W)+3W, where:

Px=X dim (width) of Photosensor
W=X dim (width) of developed assay line
h=height from top of strip to limiting stop of aperture
H=height from top of strip to bottom of photosensor
A=width of defining aperture.

[0065] This equation is an approximation and may vary if refractive materials, such as a lens, filter, or plastic coating are in the optical path. FIG. 5 depicts a cross-section along the width axis of an exemplary lateral flow test device of the present invention at the level of a test zone, depicting the light source as a light emitting diode illuminating a test device through an elongate aperture, and the photodetector positioned above the test line to interrogate the test line upon illumination.

[0066] FIG. 6 depicts a cross-section along the longitudinal axis of an exemplary lateral flow test device of the present invention showing multiple separate test zones and multiple photodetectors. The height between the photodetector and light source is defined their relative positions on the optic block. The height of the optic block to the test strip is defined by a set of “legs” that reach down to bottom enclosure. This height is a critical parameter that must be maintained to ensure a consistent illumination and viewing region from one device to the next. The legs may be formed as an integral part of the optic block, but could be separate legs which are affixed to the optic block or circuit board.

[0067] FIG. 7 Depicts the positioning of the lateral flow strip with respect to the base. The test strip 701 is aligned to the housing base 704 by matching the fiducial within the control or test line 702 with a fiducial marked on the housing base 705. The alignment correspondence is shown via the dotted line 703.

[0068] One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

[0069] While the invention has been described and exemplified in sufficient detail for those skilled in this art to make and use it, various alternatives, modifications, and improvements should be apparent without departing from the spirit and scope of the invention. The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention and are defined by the scope of the claims.

[0070] It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0071] All patents and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0072] The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of” and “consisting of” may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

[0073] Other embodiments are set forth within the following claims.