WO2008075193A2

WO2008075193A2 - Assay devices and methods

Info

Publication number: WO2008075193A2
Application number: PCT/IB2007/004021
Authority: WO
Inventors: Peter John Skelly; Raj Balbir; Nadine Kasidas
Original assignee: Inverness Medical Switzerland Gmbh
Priority date: 2006-12-19
Filing date: 2007-12-19
Publication date: 2008-06-26
Also published as: WO2008075193A3

Abstract

A lateral flow assay device can produce a color when a metal ion reacts with a color forming regent, The metal ion can be present in a sample to be tested or in a reagent zone on the device. When the metal ion is present in a reagent zone on the device, the device can be used to measure the metal ion binding capacity of a component of a sample.

Description

ASSAY DEVICES AND METHODS

CLAIM FOR PRIORITY

This application claims priority under 35 U.S.C. §119(e) to U.S. Patent Application No. 60/870,729 filed on 19 December 2006, which is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

This invention relates to assay devices and methods.

BACKGROUND

Assays for species of interest (e.g., analytes) have many applications (e.g., in medicine, industry, and environmental analysis). For example, albumin is an analyte found in mammalian blood, a sample material that includes other components such as red blood cells, ionic species (e.g., various salts and metal ions), gases (e.g., solvated oxygen and nitrogen), and multiple biological compounds (e.g., proteins, lipoproteins, blood triglycerides, fatty acids and cholesterol). The metal-binding capacity of albumin can be related to a patient's health status.

Lateral flow strip formats are popular systems for qualitative and semiquantitative assays which use visual detection schemes. This type of assay involves the application of a liquid test sample suspected of containing the analyte to be detected to an application zone of a test strip. The strip includes a matrix material through which the test fluid and analyte (which is suspended or dissolved in the test fluid) can flow by capillary action from the application zone to a capture zone where a detectable signal (or the absence of such) reveals the presence of the analyte.

SUMMARY

Assays have been used to determine the presence of ischemia, a condition associated with poor oxygen supply to a part of the body due to, for example, a constriction or an obstruction of a blood vessel. Two common forms of ischemia include cardiovascular ischemia and cerebral ischemia. The former can be generally a direct consequence of coronary artery disease, while the latter often can be due to a narrowing of the arteries leading to the brain. When an ischemic event occurs, a portion of the subject's albumin (a blood protein) becomes modified. The modified albumin is referred to as ischemia modified albumin (IMA). Determining the amount of IMA in the blood can be useful in the diagnosis of ischemic events. One difference between IMA and normal albumin is that IMA has a lower capacity to bind certain metal ions. International Patent Publication WO 03/046538 describes electrochemical methods and a device for in vitro detection of an ischemic event in a patient sample. The publication describes adding a known amount of a transition metal ion to a sample and then measuring the current or potential difference of non-sequestered transition metal ion in the sample. The amount of non-sequestered transition metal ion in the sample reflects the degree of modification to albumin that is the result of an ischemic event. The WO 03/046538 publication is incorporated herein by reference in its entirety.

In one aspect an assay device includes a lateral flow matrix including a sample application zone, a detection zone spaced apart from the sample application zone, and a color forming reagent immobilized in the detection zone. The color forming reagent is capable of undergoing a color forming reaction with a metal ion. The color forming reagent can be capable of undergoing a color forming reaction with a divalent metal ion, such as a divalent transition metal ion, such as, for example, Co²⁺, Ni²⁺ or Cu²⁺. The matrix can further include a reagent zone between the sample application zone and the detection zone. The reagent zone includes a predetermined quantity of a reagent capable of binding to a component of a sample liquid. The reagent can be capable of reacting with the color forming reagent to form a colored product.

In another aspect, a method of detecting an analyte includes mixing a sample liquid with a predetermined quantity of a first reagent capable of binding to a component of the sample liquid, applying the sample liquid to a sample application zone of a lateral flow matrix, and allowing the sample liquid to flow from the sample application zone to a detection zone of the lateral flow matrix. The detection zone includes an immobilized color forming reagent, and wherein the color forming reagent is capable of undergoing a color forming reaction with the first reagent.

Mixing the sample liquid with the first reagent can occur after applying the sample liquid to the sample application zone of the lateral flow matrix. The lateral flow matrix can include a reagent zone including the first reagent between the sample application zone and the detection zone. The first reagent can be a divalent transition metal ion, such as, for example, Co ⁺, Ni²⁺ or Cu²⁺.

In another aspect, a method of making a device includes immobilizing a color forming reagent in a detection zone of a lateral flow matrix, wherein the color forming reagent is capable of undergoing a color forming reaction with a metal ion.

The method can include depositing a first reagent in a reagent zone of the lateral flow matrix, wherein the first reagent includes a metal ion capable of undergoing a color forming reaction with the color forming reagent.

The color forming reagent can be capable of undergoing a color forming reaction with a divalent transition metal ion, such as, for example, Co²⁺, Ni²⁺ or Cu²⁺.

In some circumstances, the color forming reagent can be capable of undergoing a color forming reaction with Ni . The color forming reagent can be dimethylglyoxime or a salt thereof, such as, for example, a tetraalkylammonium salt of dimethylglyoxime.

In other circumstances, the color forming reagent is capable of undergoing a color forming reaction with Co²⁺. The color forming reagent can be thiocyanate or a salt thereof, such as, for example, a tetraalkylammonium salt of thiocyanate.

The assay device and method can accept small fluid samples in a simple step, and is able to present small fluid samples for immediate testing in a reliable and reproducible fashion. The assay method and device can be used in home testing kits for analyzing species present in bodily fluids, e.g. blood, urine, saliva, serum, interstitial fluid; or in industrial or laboratory settings for testing, e.g., a liquid used in an industrial process. The assay method and device can be used for environmental testing, e.g. testing of waste water for presence of metal ions. The assay device and method can be used in veterinary testing kits for analyzing species present in biological samples. Other features, objects, and advantages will be apparent from the description, the drawings and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of a lateral flow test strip. FIG. 2 is a photograph showing a series of lateral flow test strips after application of samples with varying concentrations of an analyte. DETAILED DESCRIPTION

Generally, lateral flow assay devices are useful for rapidly detecting an analyte in a liquid sample (such as an aqueous sample). The liquid sample can be, for example, a body fluid (such as urine, blood, blood plasma, saliva, and the like) or a liquid produced in an industrial setting (such as a waste stream from an industrial process). The assay devices can be used in monitoring the presence, absence, or amount of one or more analytes in a liquid sample.

A lateral flow assay device typically includes a matrix which is capable of bibulous and/or non-bibulous lateral flow, as generally described in U.S. Pat. Nos. 5,424,193, 4,943,522; 4,861,711; 4,857,453; 4,855,240; 4,775,636; 4,703,017; 4,361,537; 4,235,601; 4,168,146; and 4,094,647; each of which is incorporated herein by reference in its entirety. In some circumstances, the matrix is supported by a substrate or device housing.

One component of blood is human serum albumin (HSA). Exposure of HSA to ischemic tissue produces modifications to the N-terminus, and possibly other sites, on the albumin molecule. The N-terminus of albumin has been well characterized as being the primary binding site for several transition metals such as cobalt, nickel and copper. Once the metal binding sites have been modified by exposure to ischemic tissue, they are rendered unable to bind metals. This altered albumin is referred to as Ischemia Modified Albumin (IMA). Therefore, if a known amount of a transition metal is added to a biological sample (for example, a patient sample of whole blood, serum or plasma, urine, cerebrospinal fluid, or saliva), normal albumin can be distinguished from IMA on the basis of differential metal binding. Metal added to the sample will be bound to a greater extent in a non-ischemic sample than in an ischemic sample, where the presence of IMA reduces the metal binding capacity. The unbound metal can then be detected and quantified, for example by using the Albumin Cobalt Binding (ACB) Test (Ischemia Technologies, Inc., Denver, CO), which uses colorimetric methods to determine the amount of IMA present in the sample. See generally WO 03/046538, WO 00/20840, and U.S. Patent No. 5,227,307, each of which is incorporated by reference in its entirety. Referring to FIG. 1, a lateral flow test strip 100 includes a lateral flow matrix 105 including a sample application zone 110 and a detection zone 120. Detection zone 120 can be configured as a narrow line substantially perpendicular to the general direction of liquid flow from the sample application zone 110. Detection zone 120 includes a color forming reagent. The color forming reagent can be immobilized in detection zone 120; in other words, when a liquid sample travels past detection zone 120, substantially none of the color forming reagent is carried out of detection zone 120.

Matrix 105 can be, for example, a xerogel (such as, for example, poly hydro xyl ethyl methacrylate, also known as polyHEMA or HYDRON), or a cellulose-based material, such as cellulose, carboxymethylcellulose, or nitrocellulose. Other matrix materials that can be used with the present invention include methoxy nylons, (commercially available for example as ELVAMIDE, from Du Pont), which form low water content hydrogels on hydration; cellulose acetate butyrate, (some CAB's depending on the degrees of substitution of acetate and butyrate groups take up quite a bit of water). Examples of more conventional hydrogels that can be used as matrix materials include poly vinyl alcohols, (many of which are cold water insoluble), polyelectrolyte complexes, (mixtures of polyelectrolytes of different charges), which take up water, but are crosslinked electrostatically and so form hydrogels on hydration, and other polymethacrylates than PHEMA, such as, PHPMA - poly hydroxy propyl methacrylate and PGIyMA - Poly Glyceryl methacrylate). Further example matrix materials include certain cold water insoluble polyelectrolytes such as LPEI - linear Polyethylene imine.

The color forming reagent, or a salt thereof, can be immobilized on the matrix as an ion pair. For example, a charged color forming reagent can be prepared as an ion pair with a counterion having a hydrophobic character (e.g., a tetraalkylammoniurn ion). Hydrophobic interactions between the counterion and the matrix can immobilize the counterion at a desired location in the matrix; and electrostatic interactions between the color forming reagent and the counterion can immobilize the color forming reagent at the same location. The color forming reagent can be selected to form color upon reaction with a metal ion, such as, for example, a monovalent, divalent, or trivalent metal ion. The color forming reagent can be selective; in other words, it does not react with all metal ions, but with one or more particular metal ions. More precisely, the color forming reagent can react to a greater extent with some metal ions than with others. Some color forming reagents are highly selective. A highly selective color forming reagent can react substantially with only one metal ion and does not react substantially with any other metal ion. In some cases, the a color forming reagent is chosen that is highly selective and substantially reacts with only one divalent metal ion. The color forming reagent can be substantially colorless before reacting with a divalent metal ion. Divalent metal ions include, for example, Mg²⁺, Ca²⁺, Mn²⁺, Fe²⁺, Cu²⁺, Co²⁺, Zn²⁺, Cd²⁺, Hg²⁺, and the like, The divalent metal ion can be a divalent transition metal ion, such as Mn²⁺, Fe²⁺, Cu²⁺, Co²⁺, Zn²⁺, Cd²⁺, Hg²⁺, and the like. The particular color forming reagent used can depend on the particular divalent metal ion that is the desired object of detection. For example, when detection of Ni is desired, dimethylglyoxime (DMG) can be used as the color forming reagent. When DMG reacts with Ni²⁺, a strong red color is formed. In another example, when detection Of Co²⁺ is desired, thiocyanate (e.g., a thiocyanate salt) can be used as the color forming reagent. When Co²⁺ reacts with thiocyanate, a blue color is formed.

The extent of color formation can be measured manually (e.g., by comparing the color formed in detection zone 120 to a set of standard color samples) or by an instrument (e.g., a refiectometer or other instrument for measuring color change).

In some embodiments, test strip 100 also includes reagent zone 130. Reagent zone 130 can include the metal ion that will react with the color forming reagent. The metal ion can be in a dry form yet be water soluble, e.g., as a salt. Reagent zone 130 can be located between sample application zone 110 and detection zone 120. A predetermined quantity of the metal ion is disposed in reagent zone 130. When a liquid sample is applied at the application zone 110, capillary action can carry the liquid to reagent zone 130, where the metal ion becomes dissolved in the liquid and carried along with it as it continues to travel via capillary action. When the dissolved metal ion reaches detection zone 120, it reacts with the color forming reagent.

If all of the metal ion that was originally in the reagent zone 130 reacts with the color forming reagent, the predetermined quantity of divalent sets an upper limit on the extent of color change that can occur. Some sample liquids, however, can include a component that interferes (for example, by binding or complexing the metal ion) with the reaction between the divalent metal ion and the color forming reagent. The presence of such a component can diminish the extent of reaction between the metal ion and the color forming reagent, and consequently diminish the extent of color formation that results in the detection zone. Accordingly, the extent to which the color change is reduced from the upper limit can be correlated with the amount of interfering component in the sample.

For example, albumin (for example, human serum albumin (HSA)) can bind certain divalent metal ions including Co²⁺, Cu²⁺, and Ni²⁺. Thus, when a sample including HSA is applied to a test strip including a reagent zone including Co²⁺, the HSA can bind to the Co²⁺ before the Co²⁺ reaches the detection zone. When the Co²⁺ does reach the detection zone, only that portion of the Co²⁺ that was not bound by HSA contributes to the color forming reaction. The extent of color formation at the detection zone is thus indicative of the Co²⁺ binding capacity of the HSA in the sample.

In cases where the metal ion binding capacity of a sample component is the desired measurement (as opposed to a measurement of the metal ion content of the sample), the metal ion need not be present on the test strip as described above. Instead, the sample liquid (e.g., a known quantity of the sample liquid) can combined with a known quantity of the metal ion before the sample liquid is applied to the test strip.

In operation, an amount of a sample liquid is applied to the sample application zone 110 of the matrix 105. The sample liquid can be, for example, a liquid obtained from a patient, such as blood, serum, plasma, urine, or saliva; a sample obtained from an animal, bird or fish for veterinary analyses; a liquid obtained from an environmental source, e.g. waste water, effluent; or a liquid obtained from an industrial process. In some cases, the liquid can be pretreated before it is applied to the sample application zone. Pretreatment can include, for example, filtration to remove particulate matter or undesired proteins.

The liquid undergoes lateral flow away from the sample application zone. When the liquid reaches reagent zone 130, the reagent becomes dissolved in the liquid. Any components in the liquid that can react with the reagent do so as the reagent travels with the liquid to detection zone 120. At detection zone 120, any available reagent (i.e., reagent that has not reacted with any sample component) reacts with the color forming reagent to form a colored product. The colored product is then detected (e.g., by eye or by instrument) and the extent of color formation can be used to determine the extent of the color forming reaction that occurred.

Examples

Example 1. Nickel Test Strip A DMG complex was prepared as follows: 5g of PVA solution, (13 - 23K hydrolysed) solution, Composition 10 % PVA, 50 % water, 40 % ethanol, (All %'s by weight), with + 10 % of Polymer - Disodium dimethyl glyoxime, (0.0507g), 0.1667 mM.+ 2 times the molar amount of Tetrahexyl ammonium bromide, (0.1449 g) 0,334 mM.

The DMG complex prepared had improved immobilization efficiency such that the reagent remained localized on a capture line on a test strip, in comparison to an initial formulation of DMG (DMG @ 16.67% poly HEMA @ 83.33% plus 80:20 (w/w) ethanol pH10.). The original formulation leached from the deposited zone as sample passed the test line. A known volume of samples of blood or serum, obtained from both healthy volunteers and clinical patients, is mixed with a known volume of a nickel chloride solution of known concentration. After centrifugation through a 10 kDa cut off membrane the supernatant is applied to a lateral flow Nitrocellulose test strip including a capture line of the immobilized DMG reagent. The free Ni²⁺ is quantitated at the DMG capture line by virtue of the color change, and the Ni²⁺ binding capacity of the albumin in the sample is calculated. A correlation (R² 0.73) with the commercially available ACB test was achieved.

Example 2. Cobalt Test Strip

An ion pair complex was prepared by combining equimolar amounts of ammonium thiocyanate and tetrahexyl ammonium bromide. The ion pair was combined with a solvent (80/20 ethanol/water by weight) in a ratio of 40% ion pair to 60% solvent, by weight. To this mixture, a portion of Tween 20 equal to 5% of the ion pair was added as a wetting agent. Once combined, a portion was applied to a nitrocellulose test strip in a capture line.

A known volume of a samples of blood or serum is mixed with a known volume of a cobalt chloride solution of known concentration. After centrifugation through a 10 kDa cut off membrane the supernatant is applied to a lateral flow test strip including a capture line of the immobilized thiocyanate reagent. The free Co²⁺ is quantitated at the thiocyanate capture line by virtue of the color change, and the Co²⁺ binding capacity of the albumin in the sample is calculated. See FIG. 2. A correlation with the commercially available ACB test was achieved. Other embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. An assay device comprising a lateral flow matrix including a sample application zone, a detection zone spaced apart from the sample application zone, and a color forming reagent immobilized in the detection zone, wherein the color forming reagent is capable of undergoing a color forming reaction with a metal ion.

2. The device of claim 1, wherein the color forming reagent is capable of undergoing a color forming reaction with a divalent metal ion.

3. The device of claim 2, wherein the color forming reagent is capable of undergoing a color forming reaction with a divalent transition metal ion,

4. The device of claim 3, wherein the color forming reagent is capable of undergoing a color forming reaction with Co²⁺, Ni²⁺ or Cu²⁺.

5. The device of claim 1, wherein the matrix further includes a reagent zone between the sample application zone and the detection zone, wherein the reagent zone includes a predetermined quantity of a reagent capable of binding to a component of a sample liquid.

6. The device of claim 5, wherein the reagent is capable of reacting with the color forming reagent to form a colored product.

7. The device of claim 6, wherein the color forming reagent is capable of undergoing a color forming reaction with a divalent transition metal ion.

8. The device of claim 7, wherein the color forming reagent is capable of undergoing a color forming reaction with Ni²⁺.

9. The device of claim 8, wherein the color forming reagent is dimethylglyoxime or a salt thereof.

10. The device of claim 9, wherein the color forming reagent is a tetraalkylammonium salt of dimethylglyoxime.

11. The device of claim 7, wherein the color forming reagent is capable of undergoing a color forming reaction with Co²⁺.

12. The device of claim 11 , wherein the color forming reagent is thiocyanate or a salt thereof.

13. The device of claim 12, wherein the color forming reagent is a tetraalkylammonium salt of thiocyanate.

14. A method of detecting an analyte comprising: mixing a sample liquid with a predetermined quantity of a first reagent capable of binding to a component of the sample liquid; applying the sample liquid to a sample application zone of a lateral flow matrix; and allowing the sample liquid to flow from the sample application zone to a detection zone of the lateral flow matrix; wherein the detection zone includes an immobilized color forming reagent, and wherein the color forming reagent is capable of undergoing a color forming reaction with the first reagent.

15. The method of claim 14, wherein mixing the sample liquid with the first reagent occurs after applying the sample liquid to the sample application zone of the lateral flow matrix.

16. The method of claim 15, wherein the lateral flow matrix includes a reagent zone between the sample application zone and the detection zone, the reagent zone including the first reagent.

17. The method of claim 14, wherein the first reagent is a divalent transition metal ion.

18. The method of claim 15, wherein the divalent transition metal ion is Co 2+

Ni²⁺ Or Cu²⁺.

19. The method of claim 18, wherein the color forming reagent is capable of undergoing a color forming reaction with Ni²⁺.

20. The method of claim 19, wherein the color forming reagent is dimethylglyoxime or a salt thereof.

21. The method of claim 20, wherein the color forming reagent is a tetraalkylammonium salt of dimethylglyoxime.

22. The method of claim 17, wherein the color forming reagent is capable of undergoing a color forming reaction with Co²⁺.

23. The method of claim 22, wherein the color forming reagent is thiocyanate or a salt thereof.

24. The method of claim 23, wherein the color forming reagent is a tetraalkylammonium salt of thiocyanate.

25. A method of making a device comprising immobilizing a color forming reagent in a detection zone of a lateral flow matrix, wherein the color forming reagent is capable of undergoing a color forming reaction with a metal ion.

26. The method of claim 25, wherein the color forming reagent is capable of undergoing a color forming reaction with a divalent transition metal ion.

27. The method of claim 26, wherein the color forming reagent is capable of undergoing a color forming reaction with Co²⁺, Ni²⁺ or Cu²⁺.

28. The method of claim 27, wherein the color forming reagent is dimethylglyoxime or a salt thereof.

29. The method of claim 28, wherein the color forming reagent is a tetraalkylammonium salt of dimethylglyoxime.

30. The method of claim 27, wherein the color forming reagent is thiocyanate or a salt thereof.

31. The method of claim 30, wherein the color forming reagent is a tetraalkylammonium salt of thiocyanate.

32. The method of claim 25, further comprising depositing a first reagent in a reagent zone of the lateral flow matrix, wherein the first reagent includes a metal ion capable of undergoing a color forming reaction with the color forming reagent.

33. The method of claim 32, wherein the first reagent includes Ni²⁺ and the color forming reagent is dimethylglyoxime or a salt thereof.

34. The method of claim 33, wherein the color forming reagent is a tetraalkylammonium salt of dimethylglyoxime.

35. The method of claim 32, wherein the first reagent includes Co²⁺ and the color forming reagent is thiocyanate or a salt thereof.

36. The method of claim 35, wherein the color forming reagent is a tetraalkylammonium salt of thiocyanate.