STABILIZED TEST STRIP FOR THE DETECTION OF HYDROGEN PEROXIDE

Abstract

A colorimetric dry-reagent test strip for direct detection of the presence of hydrogen peroxide in strongly acidic solutions without the necessity of a pH neutralization step comprises a chromogen responsive to hydrogen peroxide, an enzyme peroxidase catalyst and at least one complexing agent. A solution of the components may be applied to a suitable carrier and allowed to dry. The resulting dry test strip is placed in contact with a solution to be tested. The test strip has particular value in the determination of peroxide-based explosive compounds, such as hexamethylene triperoxide diamine (HMTD) or triacetone triperoxide (TATP), which require a treatment with a strong organic acid to release the hydrogen peroxide to be detected, and typically requires neutralization of the acid before a conventional test strip can be employed. The invention dispenses with the need for such a neutralization step. The invention test strip can be incorporated into a kit for the determination of such peroxide-based explosive compounds. The kit may include, in addition to the test strip, only a solvent to dissolve a sample to be tested and the strong mineral acid to degrade the sample.

Claims

1. A colorimetric dry-reagent test strip for direct detection of the presence of hydrogen peroxide in strongly acidic solutions without the necessity of a neutralization step, comprising a chromogen responsive to hydrogen peroxide, an enzyme peroxidase catalyst and at least one complexing agent.

2. The test strip of claim 1, wherein the enzyme peroxidase is horseradish peroxidase having both acidic and basic isoenzymes.

3. The test strip of claim 1, wherein the at least one complexing agent is chosen from the group consisting of polycarboxylic and polyaminopolycarboxylic compounds.

4. The test strip of claim 3, wherein the polycarboxylic and polyaminopolycarboxylic compounds have three to six carboxylic groups.

5. The test strip of claim 1 including two complexing agents.

6. The test strip of claim 1 wherein the chromogen, enzyme peroxidase and at least one complexing agent; and a chromogen indicator responsive to hydrogen peroxide, the catalyst, complexing agent and chromogen indicator are in dry form.

7. The test strip of claim 6 wherein the dry form components are located in a carrier matrix.

8. The test strip of claim 7 wherein the carrier matrix is mounted to a support.

9. A test kit for detecting a peroxide-based explosive compound, comprising a solvent for the explosive compound, a mineral acid for the degradation of the explosive compound to release hydrogen peroxide; an enzyme peroxidase catalyst; at least one complexing agent; and a chromogen indicator responsive to hydrogen peroxide, the catalyst, complexing agent and chromogen indicator being in dry form.

10. The test kit of claim 6 further including a vial for receiving a sample of an explosive compound to be tested, the solvent and the mineral acid, wherein the enzyme peroxidase catalyst, the at least one complexing agent; and the chromogen indicator are in a carrier matrix constructed to be insertable into the vial.

11. The test kit of claim 7 wherein the carrier matrix is mounted to a supporting handle.

Description

BRIEF DESCRIPTION OF THE DRAWING

[0016] FIG. 1 is a representation of a handled dipstick or dipstrip embodying the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0017] Prior attempts for enzyme stabilization were directed to preserving enzyme activity by preventing enzyme decomposition during storage. For example, U.S. Pat. No. 4,283,491 and EP 0 252 750 disclose the use of polymers to improve storage time. Upon realizing that none of the described and applied methods would achieve the type of enzyme protection desired in a peroxide test strip in strong acidic conditions, it was recognized that a different tack was necessarya focus on the enzyme's molecular structure and the mechanism of its catalytic activity.

[0018] Horseradish peroxidase and other vegetable and plant peroxidases, such as soybeen, turnip, barley, tomato and tobacco peroxidases, are class III peroxidases. They contain an N-terminal peptide, two calcium ions, four disulfide bridges and an extra helical region that plays a role in an access to the edge of the heme cofactor present in the peroxidase.

[0019] Horseradish peroxidase (HRP) is a heme protein with 308 amino acids residues. The N-terminal residue is blocked by a pyrrolidennecarboxyl residue which appears to be buried inside the polypeptide chain. The heme prosthetic group is ferriprotoporphyrin, which is made of four pyrrole rings joined by methene bridges with iron (III) in the center of the molecule. Three different substitutes are found in the pyrrole ringsfour methyl, two vinyl and two propionate side chains.

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[0020] The activity of enzymes is strongly affected by changes in pH. This is due to the importance of the tertiary structure in the enzyme function and forces, e.g. ionic interactions and hydrogen bridges, in determining the shape of the enzyme molecule. Changes in pH alter the state of ionization of charged amino acids that may play a crucial role in substrate binding and/or catalytic action itself. For enzymatic activity to occur there needs to be a certain degree of conformational flexibility in the enzyme structure, enabling entry of the substrate into the active site and attainment of the transition state structure, where there is a delicate balance between hydrophobic interactions, charge interaction and hydrogen bridges. Changes in pH can break the hydrogen bridges and hydrophobic interactions in the enzyme structure of peroxidase, allowing the rapid unfolding and inactivation of the enzyme. Thus an approach to stabilizing enzyme activity would be the introduction of molecules with the proper polar groups and steric arrangement to negate the effect of pH changes on the enzymatic activity of the peroxidase.

[0021] Accordingly, It has been found that complexing a peroxidase with one or more polycarboxylic or polyaminocarboxylic compounds stabilizes the enzyme sufficiently in a highly acidic condition to allow it to catalyze the needed chromogen oxidation for peroxide identification.

[0022] Of these complexing agents, compounds with three to six carboxylic groups have been found to be particularly suitable for proper protection of the HRP active site in such acidic mediums; these include N-(2-hydroxyethyl)ethylenediamine triacetate; 1,2,3-propanetricarboxylic anion; triglycine; ethylenedinitrilotetraacetate; ethylene glycol-bis(-aminoethyl ether) tetraacetate; cyclohexylenedinitrilotetraacetate; diethylene-triaminepentaacetate; and triethylenetetraaminehexaacetate.

[0023] More efficient enzyme protection at pH 0-1 has been observed when a combination of complexing agents, each with three and four carboxylic groups, was used in a concentration appropriate to the peroxidase activity unit (POD Unit), which is that amount of enzyme decomposing 1 mole of peroxide/minute at 25 C.

[0024] Although the specific mode of action providing the novel peroxidase protection and stabilization is not fully understood, the complexing agent treatment brings protection against strong acidic ambient conditions. Another advantage resulting from the treatment is an improved stability of the enzyme during storage and thus prolongation of test strip shelf life.

[0025] FIG. 1 depicts a peroxide dip and read test strip 10 of the invention utilizing an appropriate support or wand. Carrier matrix 14 is advantageously affixed to insoluble support 12 for easy manipulation. Broadly, such a support may comprise a plastic wand, at one end of which is mounted the carrier matrix, an absorbent material test strip impregnated with the appropriate reactant mixture. The term carrier matrix refers to bibulous or nonbibulous materials which are absorbent and maintain their structural integrity when exposed to liquids. Such materials are known in the art. The carrier matrix is initially immersed in, soaked, sprayed or printed with the reactant composition, and thereafter dried by suitable means, such as by ambient or forced air drying, to leave the dry reagent material suspended in the carrier matrix (e.g. paper). The resultant test strip material (typically in squares 55 mm) is then affixed to the support for easy manipulation. Alternatively, the carrier matrix may be first affixed to the wand and thereafter soaked with the reactant composition and allowed to dry. The wand is used by dipping the end bearing the test strip into the test sample, removing it and evaluating the color formed in the paper portion. This type of technology is well known in the art.

[0026] The test strip of the present invention is well suited for the field determination and detection of the presence of peroxide-based explosive compounds, such as hexamethylene triperoxide diamine (HMTD) or triacetone triperoxide (TATP), which release hydrogen peroxide after an acidic degradation treatment. For such use, it is necessary to at least partially dissolve the HMTD or TATP. Such solvents may include methanol and ethanol. Such a field kit may thus include a dropper vial of solvent, a dropper vial of a strong mineral acid to degrade the explosive, a vial into which the sample is placed and into which the solvent and acid can be added, and a test strip for dipping into the dissolved and degraded sample solution for determination and subsequent color-change observation.

[0027] Typical reactant compositions for peroxide test strips in accordance with the present invention may contain, in addition to the chromogen, enzyme peroxidase, and complexing agent for the peroxidase, a buffer, a polymer and a wetting agent, as well known in the art for similar strips. The following examples illustrate that conventional test strip formulations demonstrate no reactivity in a solution of hydrogen peroxide where solution acidity was adjusted with hydrochloric acid to a pH 2 or lower.

EXAMPLES

[0028] The following examples of this inventive method are presented by way of illustration and not of limitation.

Examples I-III

[0029] Experiments were performed to compare the reactivity of two sets of peroxide test strips; one prepared in as representing conventional test strip formulation, (the control test strip) and the other with the inventive peroxidase treatment (test strips I, II and III). Both sets of test strips contained the same reactant composition of indicator, peroxidase, buffer, polymer, and wetting agent. The chromogen indicator utilized provides a blue color after the oxidation by hydrogen peroxide. Horseradish peroxidase was used as the catalyst, containing both acidic and basic isoenzymes.

[0030] Test strip I contained as a complexing agent penta(carboxymethyl) diethylenetriamine (penta-anion DTPA.sup.5) in a concentration of 9 mol per POD Unit.

[0031] Test strip II contained as a complexing agent ethylene glycol-bis(-aminoethyl ether) tetraacetic anion in a concentration of 5 mol per POD Unit and 2-hydroxy-1,2,3-popane tricarboxylic anion in a concentration of 8 mol per POD unit.

[0032] Test strip III contained as a complexing agent triglycine in a concentration of 3 mol per POD unit; ethylenedinitrilotetraacetic anion in a concentration of 3 mol per POD unit; and -carboxyglutaric anion in a concentration of 5 mol per POD unit.

[0033] Both the control and test strips were prepared in the same conventional manner as well known in the art using paper as a reactant carrier material. The reactivity of the control and test strips was tested on hydrogen peroxide solutions where acidity was adjusted with 6M hydrochloric acid to values about 0 and 1 pH. The hydrogen peroxide in the solutions was at 20 ppm and 5 ppm levels. The strips were dipped into the solutions for approximately one second. The color of the test pad was evaluated both instantly and within 2 minutes after dipping.

[0034] The results of the tests are presented in the following table.

TABLE-US-00001 pH~0 pH~1 Test strips 20 ppm 5 ppm 20 ppm 5 ppm Control test strips No color No color No color No color Example I test strips Dark Blue Blue Dark Blue Blue Example II test strips Dark Blue Blue Dark Blue Blue Example III test strips Dark Blue Blue Dark Blue Blue

[0035] The results confirm that the test strips with the complexing agents provided clear confirmation of the presence of hydrogen peroxide at both pH levels while conventional strips provided no indication whatsoever.

Example IV

[0036] An experiment was conducted to determine the improvement in hydrogen peroxide detection by the present invention over conventionally-available peroxidase peroxide test strips. Three commercially available peroxide test strips, each from a different manufacturer (TS1, TS2, TS3) were tested together with peroxide test strips formulated in accordance with the present invention (EX) on acidified hydrogen peroxide solutions100 ppm solutions of hydrogen peroxide acidified to pH 0 and 1.

[0037] All strips were dipped into a testing solution for approximately one second. The color reaction was evaluated instantly (and in the case of a negative result also for 2 minutes after dipping).

[0038] The results of the tests are presented in the following table.

TABLE-US-00002 Peroxide Test Strips pH~0 pH~1 TS1 Negative (no color) Negative (no color)* TS2 Negative (no color)* Negative (no color)* TS3 Negative (no color)* Negative (no color)* EX Positive (dark blue color) Positive (dark blue color) *No color development was observed during 2 minutes.

Example V

[0039] An experiment was performed to determine the sensitivity of peroxide detection of the present invention in a strong acidic solution of pH 0. A hydrogen peroxide acidified solution was gradually diluted down and the diluted solutions were tested with the inventive test strips (EX) up to last clear positive color reaction. It was found that test strip is able to detect by a noticeable color change within a few seconds the a quantity as small as 0.5 microgram H.sub.2O.sub.2.

Example VI

[0040] An experiment was performed to demonstrate the potential application of the present invention in the detection of peroxide-based explosive compounds, such as hexamethylene triperoxide diamine (HMTD) or triacetone triperoxide (TATP), which release hydrogen peroxide after an acidic degradation treatment. For such use, it is necessary to at least partially dissolve the HMTD or TATP. Such solvents may include methanol and ethanol. Accordingly, on a virtually imperceptible sample of TATP (without residual hydrogen peroxide) in a vial were dropped methanol (to dissolve the TATP) and 6M hydrochloric acid, followed immediately by a test strip dip. A dark blue color was observed within a few seconds, indicating the presence of hydrogen peroxide. The same results were achieved with a HMTD sample.

[0041] A further experiment was carried out to test the potential application in the trace detection of the same compounds. A plain paper sampling strip was used to swipe a trace amount of a sample. One drop of methanol from a dropping bottle, followed by one drop of hydrochloric acid, was applied to the sampling strip. The sampling strip was immediately touched with a test strip, resulting in an instant color reaction indicating a positive reaction.