A TEST STRIP FOR MELAMINE DETECTION

Abstract

Described here is a test strip for detection of melamine, comprising: a support configured for capillary flow of a fluid sample from a first end of the support to a second end of the support that is downstream from the first end; a conjugation pad disposed adjacent to the first end of the support and including nanoparticles configured for suspension in the sample flowing past the conjugation pad, the nanoparticles configured to produce a colorimetric effect when exposed to melamine; and a test portion downstream of the conjugation pad and including a molecular recognition agent immobilized on the support and having an affinity for melamine.

Claims

1. A test strip for detection of melamine, comprising: a support configured for capillary flow of a fluid sample from a first end of the support to a second end of the support that is downstream from the first end; a conjugation pad disposed adjacent to the first end of the support and comprising nanoparticles configured for suspension in the sample flowing past the conjugation pad, the nanoparticles configured to produce a colorimetric effect when exposed to melamine; and a test portion downstream of the conjugation pad and comprising a molecular recognition agent immobilized on the support and having an affinity for melamine.

2. The test strip of claim 1, further comprising: a control portion downstream of the test portion and comprising melamine immobilized on the support.

3. The test strip of claim 1, wherein the nanoparticles comprise at least one metal selected from the group consisting of Au, Ag, Fe, Pt, Pd, Co, Cu, Ga, Ni, Ti, W, Rh, and Cr.

4. The test strip of claim 1, wherein the nanoparticles comprise gold nanoparticles.

5. The test strip of claim 1, wherein the nanoparticles consist essentially of unmodified metal nanoparticles having an average size in a range of 1 nm to 1000 nm.

6. The test strip of claim 1, wherein the molecular recognition agent comprises at least one compound selected from the group consisting of cyanuric acid, acetic acid, oxalic acid, tannin, thymine, uracil, guanine, uric acid, riboflavin, barbituric acid, maleimide, succinimide, diacetamide, glutarimide, and their derivatives.

7. The test strip of claim 1, wherein the molecular recognition agent comprises cyanuric acid.

8. The test strip of claim 1, wherein the conjugation pad is substantially free of anti-melamine antibody.

9. The test strip of claim 1, wherein the test portion is substantially free of melamine.

10. The test strip of claim 1, wherein the presence of melamine in the sample results in the presence of a visual cue at the test portion, and wherein the absence of melamine in the sample results in the absence of a visual cue at the test portion.

11. A test strip for detection of melamine, comprising: a support configured for capillary flow of a fluid sample from a first end of the support to a second end of the support that is downstream from the first end; nanoparticles disposed on the support and configured for suspension in the flowing sample, the nanoparticles configured to bind to melamine when exposed to melamine; and a molecular recognition agent immobilized on the support and configured to bind to melamine to form a first visual cue indicative of the presence of melamine in the sample.

12. The test strip of claim 11, further comprising: melamine immobilized on the support and configured to bind to the nanoparticles to form a second visual cue indicative of the absence of melamine in the sample.

13. The test strip of claim 11, wherein the nanoparticles comprise at least one metal selected from the group consisting of Au, Ag, Fe, Pt, Pd, Co, Cu, Ga, Ni, Ti, W, Rh, and Cr.

14. The test strip of claim 11, wherein the nanoparticles comprise gold nanoparticles.

15. The test strip of claim 11, wherein the molecular recognition agent comprises at least one compound selected from the group consisting of cyanuric acid, acetic acid, oxalic acid, tannin, thymine, uracil, guanine, uric acid, riboflavin, barbituric acid, maleimide, succinimide, diacetamide, glutarimide, and their derivatives.

16. The test strip of claim 11, wherein the molecular recognition agent comprises cyanuric acid.

17. The test strip of claim 11, wherein the test strip is substantially free of anti-melamine antibody.

18. The test strip of claim 11, wherein the test strip is substantially free of modified nanoparticles.

19. A method for detection of melamine, comprising: providing the test strip of claim 1; and contacting the test strip with a fluid sample to determine the presence or absence of melamine in the sample.

20. A method for detection of melamine, comprising: providing the test strip of claim 1; contacting the test strip with a fluid sample to determine the presence or absence of melamine in the sample; and correlating the presence of a visual cue at the test portion to the presence of melamine in the sample, or correlating the absence of a visual cue at the test portion to the absence of melamine in the sample.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1 illustrates an example lateral flow chromatography for melamine detection with gold nanoparticles (AuNPs) in a conjugation pad, cyanuric acid (CA) on a test line, and melamine (M) on a control line.

[0026] FIG. 2 shows an example melamine test strip based on cyanuric acid and unmodified AuNP. Left: Illustration of the testing procedure. Right: Test results for melamine-negative and melamine-positive samples.

DETAILED DESCRIPTION

[0027] Lateral flow chromatography (LFC) based platform can be used in various analytical and diagnostic applications, including diagnostic kits. To bypass the stability issue of antibodies, a LFC melamine strip test is developed by utilizing the interactions of melamine with non-functionalized or unmodified gold nanoparticles (AuNPs) and cyanuric acid. As shown in FIG. 1, when the strip (1) is immersed in a test sample (2), the fluid will be drawn up via capillary action, sequentially passing through a sample pad (11), a conjugate pad (13) including dispersed AuNPs, a test line (15) or portion including cyanuric acid immobilized or coupled to the strip, and a control line (17) or portion including melamine immobilized or coupled to the strip, and finally reaching an absorbent pad (19). Immobilization of cyanuric acid and melamine to the strip (1) can be through covalent binding, non-covalent binding (e.g., adsorption), or a combination of covalent and non-covalent binding.

[0028] When the sample includes melamine, the melamine-AuNP aggregates will be formed in the conjugate pad (13) and then be captured by cyanuric acid on the test line (15), resulting in a colored test line. Other interferents, even those that could cause a color change of AuNP, will not cause a colored test line (or will not cause a noticeable or detectable colored test line); instead, AuNPs will be captured by melamine on the control line (17), thus resulting in a colored control line.

[0029] To demonstrate the concept, a prototype of the melamine test strip has been constructed and tested with two milk samples: one spiked with a high concentration of melamine and the other without (pure milk). For each sample, a test strip was dipped in the sample for about 10 seconds (FIG. 2, top) and then allowed to lay substantially horizontally for about 1 minute. As shown in the bottom panel of FIG. 2, on the test strip dipped in the sample including a high concentration of melamine, a purple test line appeared, thus indicating the presence of melamine in the sample. On the other test strip, instead of a purple test line, a purple control line appeared, thus indicating that the test was performed successfully, but no melamine was detected in the sample.

[0030] Certain embodiments of a lateral flow test strip for melamine present at least the following advantages: (a) easy-to-use the test strip based platform allows a layperson to perform the test; (b) high sensitivity the AuNP enhanced colorimetric change allows a low level of melamine to be detected; (c) high specificity the highly specific interaction between melamine and cyanuric acid reduces false positives; (d) no requirement for sample pretreatment directly dip the test strip into an untreated, raw sample; (e) low-cost reagents can be mass-produced in a low cost and high stability, thus greatly reducing the costs on manufacturing, packaging, and storage; and/or (f) high robustness and long shelf life antibodies are not required.

[0031] Compared to ELISA or chromatography-spectrometry based methods that are often cost- and time-consuming, and involve expensive equipment and trained personnel, the test strip can be readily accessible to a layperson even in resource-limited settings, such as at home and in the field. Compared to other lateral flow based strip tests that rely on surface-immobilized antibodies for molecular recognition and functionalized nanoparticles for visual readout, the test strip utilizes cyanuric acid for molecular recognition and non-functionalized AuNPs for visual readout. Non-functionalized AuNPs and cyanuric acid can be mass-produced with low cost and high stability. This can greatly reduce the costs on manufacturing, packaging, and storage without sacrificing robustness and shelf life. Therefore, the test strip provides a simple-to-use, quick, and economical solution to melamine screening from raw food ingredients and products in low-resource settings.

[0032] In some embodiments, a test strip includes nanoparticles (e.g., AuNPs) having an affinity for melamine and that produce a colorimetric effect or signal when contacted or exposed to melamine. Nanoparticles useful in some embodiments can range in average size from about 1 nm to about 1 m, such as from about 1 nm to about 900 nm, from about 1 nm to about 800 nm, from about 1 nm to about 700 nm, from about 1 nm to about 600 nm, from about 1 nm to about 500 nm, from about 1 nm to about 400 nm, from about 1 nm to about 300 nm, from about 1 nm to about 200 nm, from about 1 nm to about 100 nm, from about 1 nm to about 50 nm, or from about 1 nm to about 25 nm. As used herein, the term size refers to a characteristic dimension of an object. Thus, for example, a size of an object that is spherical can refer to a diameter of the object. In the case of an object that is non-spherical, a size of the non-spherical object can refer to a diameter of a corresponding spherical object, where the corresponding spherical object exhibits or has a particular set of derivable or measurable characteristics that are substantially the same as those of the non-spherical object. Thus, for example, a size of a non-spherical object can refer to a diameter of a corresponding spherical object that exhibits optical characteristics that are substantially the same as those of the non-spherical object. Alternatively, or in conjunction, a size of a non-spherical object can refer to an average of various orthogonal dimensions of the object. Thus, for example, a size of an object that is a spheroidal can refer to an average of a major axis and a minor axis of the object. A variety of materials can be used to form nanoparticles, including, for example, metals and metalloids such as Au, Ag, Fe, Pt, Pd, Co, Cu, Ga, Ni, Ti, W, Rh, Cr, and alloys and mixtures thereof. In addition, in certain embodiments, the nanoparticles can be mixed with or formed from other materials, such as polymeric materials and semiconducting materials. The nanoparticles can have a variety of shapes such as spheres, ellipsoids, rods, fibers, discs, tubes, and the like.

[0033] As noted above, certain embodiments of a test strip include a molecular recognition agent having an affinity for melamine. As disclosed herein, an example molecular recognition agent is cyanuric acid (1,3,5-triazine-2,4,6-triol). In addition or as an alternative to cyanuric acid, other compounds having a sufficient affinity to bind or capture melamine can be used, such as acetic acid, oxalic acid, tannin, and compounds including certain imide groups such as thymine, uracil, guanine, uric acid, riboflavin, barbituric acid, maleimide, succinimide, diacetamide, glutarimide and their derivatives.

[0034] While the disclosure has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the disclosure as defined by the appended claims. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, method, operation or operations, to the objective, spirit and scope of the disclosure. All such modifications are intended to be within the scope of the claims appended hereto. In particular, while certain methods may have been described with reference to particular operations performed in a particular order, it will be understood that these operations may be combined, sub-divided, or re-ordered to form an equivalent method without departing from the teachings of the disclosure. Accordingly, unless specifically indicated herein, the order and grouping of the operations is not a limitation of the disclosure.