RU2364870C1 - Device for test-detection of analyte - Google Patents

Abstract

FIELD: chemistry, biotechnology.

SUBSTANCE: invention relates to analytic chemistry with regard to monitoring of environment pollution, control of food products and medications quality, analysis of biological objects. Device contains, first, transparent reservoir with openings for introduction and removal of sample, inside which, at least, one test layer is placed, second, purifying layer, and it also contains additional reservoir with inlet and outlet openings, with possibility to provide in-cut connection with transparent reservoir, purifying layer being placed in additional reservoir. Increase of analysis reliability is achieved.

EFFECT: increase of analysis reliability.

15 cl, 2 dwg

Description

The invention relates to analytical chemistry and can be used in monitoring environmental pollution, quality control of food products and drugs, analysis of biological objects.

In modern practice, non-instrumental immunochemical test methods are widely used to determine various substances. The most popular immunochromatographic (Collin R., Schneider E., Briggs L., Towers N. // Food and Agricultural Immunology. 1998. V.10. P.91-104; Delmulle B., De Saeger S., Sibanda L ., Barna-Vetro I., Van Peteghem C. // J. of Agricultural and Food Chemistry. 2005. V.53. P.3364-3368; Usleber E., Schneider E., Märtlbauer E., Terplan G. / / J. of Agricultural and Food Chemistry. 1993. V.41. P.2019-2023) and immunofiltration tests (Schneider E., Usleber E., Märtlbauer E. // J. of Agricultural and Food Chemistry. 1995. V. 43. P.2548-2552; De Saeger S., Sibanda L., Desmet A., Van Peteghem C. // International Journal of Food Microbiology. 2002. V.75. P.135-142). Both types of tests are based on the use of membranes containing a test zone, the color of which depends on the concentration of the analyte in the sample.

However, these test systems are highly susceptible to the matrix effect of the analyzed sample, to eliminate which dilution of solutions is used, which often leads to insufficient sensitivity of the method. In addition, the high content of colored substances in the matrix of the analyzed sample may interfere with visual detection.

To increase the sensitivity of test systems, an approach is proposed that consists of using a test layer instead of membranes, which is an affinity gel (Goryacheva I.Yu., Beloglazova NV, Eremin SA, Mikhirev DA, Niessner R., Knopp D. // Talanta 2008. Vol. 75. N2. P.517-522). However, this method is applicable only for unpainted simple objects of analysis (aquatic environments).

Closest to the proposed invention are a device and method for detecting the presence of an analyte, International Patent Application No. PCT / EP02 / 01496 (L. Sibanda, S. De Saeger, C. Van Peteghem 2001)). The device allows to detect the presence of analyte in liquid and semi-liquid media and is a transparent container with holes for input and output of the sample, containing at least two separate layers. The analyzed sample is passed sequentially through the first and second layers. The first layer consists of a sorbent capable of retaining at least part of the interfering substances from the sample, and the second layer contains a sorbent with receptor molecules capable of selectively binding the analyte. As such a sorbent, an immunoaffinity gel with grafted specific antibodies fixed on top and bottom by porous filters can be used.

The indicated format of the test system was used to determine ochratoxin A in spices (Goryacheva IY, De Saeger S., Lobeau M., Eremin SA, Barna-Vetru I., Van Peteghem C. 2006. Approach for ochratoxin A fast screening in spices using clean-up tandem immunoassay columns with confirmation by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) // Analytica Chimica Acta 577: 38-45). The aqueous acetonitrile spice extract was passed through a column, then an enzyme-labeled conjugate was introduced. A washing step is used to remove excess conjugate. Then, a substrate that catalyzes the development of color is added to the column. If the sample is contaminated with ochratoxin A above a permissible level, the test layer turns blue; in the absence of ochratoxin A, the color does not develop in the sample — the test layer remains white.

The disadvantage of this device is the leaching of interfering substances from the cleaning layer when introducing reagents for immunoassay and washing, which leads to contamination of the detection layer and distortion of the analysis results. In particular, this approach was unacceptable for the determination of ochratoxin A in black and white pepper.

The objective of the invention is the development of a device for the test determination of analyte in environmental objects, food, drugs, industrial and biological objects.

The technical result consists in reducing the matrix effect of the sample without significantly complicating the procedure for sample preparation, concentration and determination of analytes.

This object is achieved in that the device for the test determination of the analyte, containing, firstly, a transparent reservoir with holes for input and output of the sample, inside of which at least one test layer is placed, and secondly, a cleaning layer, according to the solution it contains an additional reservoir with inlet and outlet openings with the possibility of providing a detachable connection with a transparent reservoir, while the cleaning layer is located in the additional reservoir.

Liquid and semi-liquid media, as well as solid samples transferred into solution, for example, by extraction or dissolution, can serve as a sample.

The test layer is made in the form of a sorbent with specific receptors capable of retaining the analyte.

Antibodies specific for the analyte were selected as specific receptors.

Sepharose or its derivatives are used as a sorbent.

The device is configured to introduce enzymatic or luminescent or fluorescent or chemiluminescent or colloidal labels.

Horseradish peroxidase was used as an enzyme label.

The transparent reservoir with the test layer contains an additional control layer with antibodies specific to the label.

Polymers with molecular imprints were used as a test layer.

As a test layer, a substance obtained by sol-gel technology was used.

The layers are fixed using porous filters.

There are several test layers in the transparent reservoir to identify different analytes.

The tank is tubular.

The device further comprises a syringe for supplying solutions under pressure.

The device is equipped with a test kit consisting of one or more reagents: a solvent for extraction or dissolution of solid samples, buffer solutions for dilution of the sample, washing buffers, analyte conjugate with a label and a chromogenic substrate.

The invention is illustrated by drawings, figure 1 schematically shows a device, figure 2 is a diagram of an enzyme-linked immunosorbent determination of substances, where:

1 - tanks;

2 - a cleaning layer;

3 - connecting element (adapter);

4 - test layer;

5 - porous filters (frits);

6 - sample;

7 - wash buffer I;

8 - conjugate;

9 - wash buffer II;

10 - substrate.

The analyte test-determination device comprises a reservoir in the form of a column 1, inside which a cleaning layer 2 is placed, optionally fixed on top and bottom by porous filters 5, connected through an adapter 3 to column 1, inside which a test layer 4 containing an analyte-binding substance is placed, if necessary, fixed on top and bottom by porous filters 5.

The device is intended for determination of analytes in liquid and semi-liquid samples, as well as solid samples, including intensively stained samples, transferred to a solution, for example, by extraction. Toxic agents of various nature (mycotoxins, for example, ochratoxin A, toxin T-2, zearalenone, one of aflatoxins or their group; polycyclic aromatic hydrocarbons), biologically active substances, drugs, explosives, are used as analytes.

The device operates as follows.

The solid sample is transferred into the solution by extraction with aqueous-organic mixtures. The extract obtained, either a liquid or semi-liquid sample 6, optionally diluted with a buffer solution with additives, is passed through column 1 from top to bottom, then washed with solution in washing buffer I 7, conjugate 8, which is an analyte or its analogue covalently bound to the enzyme, is introduced playing the role of tags. To remove excess conjugate 8, a washing step using washing buffer II 9 is used. Then, substrate 10 is added to column 1, the color development of which catalyzes the enzyme, and color development is observed. In the case of staining on test layer 4, which is an immunoaffinity gel grafted with antibodies specific for the analyte, the sample is considered to not contain analyte above the acceptable level; if there is no color, the sample is considered to contain analyte above the acceptable level.

The feasibility and effectiveness of the proposed approach is confirmed by the following example of an enzyme-linked immunosorbent assay for ochratoxin A in red wine.

To determine ochratoxin A, two 1 ml columns were used, in one of which a cleaning layer was placed (200 mg of a sorbent, which is silica gel with grafted trimethylaminopropyl groups), and in the second, a test layer, which is a gel based on bromocyansepharose with grafted (covalently bound ) specific to ochratoxin A antibodies.

To determine the aliquot (1 ml) of the analyzed wine was mixed with 1 ml of a solution containing polyethylene glycol 6000 and sodium bicarbonate. 2 ml of the resulting solution was passed through the columns with the cleaning and test layers using a syringe. Then the column with the cleaning layer was disconnected and work continued only with the column containing the detection layer. A wash solution (phosphate-saline buffer solution containing casein) was passed through it, then a conjugate of ochratoxin A with horseradish peroxidase was passed. The conjugate was left in the column for 1 minute, then the column was removed by washing with 10 ml of phosphate-buffered saline with Tween 20. The last step in the determination was to add a substrate based on tetramethylbenzidine to the column, after which a blue color of the detecting layer was observed for several minutes (in the absence of ochratoxin A in the sample). In the presence of the analyte above the detection limit (2 μg / L), the test layer remains white. Color development is fixed visually.

Claims (15)

1. The device for the test determination of the analyte, containing, firstly, a transparent reservoir with holes for input and output of the sample, inside of which at least one test layer is placed, and secondly, a cleaning layer, characterized in that it contains an additional reservoir with inlet and outlet openings with the possibility of providing a detachable connection with a transparent reservoir, while the cleaning layer is located in the additional reservoir. 2. The device according to claim 1, in which the sample can be liquid or semi-liquid medium, as well as solid samples transferred into solution, for example, by extraction or dissolution. 3. The device according to claim 1, characterized in that the test layer is made in the form of a sorbent with specific receptors capable of retaining the analyte. 4. The device according to claim 3, characterized in that the antibodies specific for the analyte are selected as specific receptors. 5. The device according to claim 3 or 4, characterized in that Sepharose or its derivatives are used as the sorbent. 6. The device according to claim 1, characterized in that it is made with the possibility of introducing enzyme, or luminescent, or fluorescent, or chemiluminescent, or colloidal labels. 7. The device according to claim 6, characterized in that horseradish peroxidase is used as the enzyme label. 8. The device according to claim 6, characterized in that the transparent reservoir with the test layer contains an additional control layer with antibodies specific to the label. 9. The device according to claim 1, characterized in that polymers with molecular imprints are used as a test layer. 10. The device according to claim 1, characterized in that the substance obtained by sol-gel technology is used as a test layer. 11. The device according to claim 1, characterized in that the layers are fixed using porous filters. 12. The device according to claim 1, characterized in that in the transparent reservoir there are several test layers for determining various analytes. 13. The device according to claim 1, characterized in that the reservoir has a tube shape. 14. The device according to claim 1, characterized in that it further comprises a syringe for supplying solutions under pressure. 15. The device according to claim 1, characterized in that it is equipped with a test kit consisting of one or more reagents: a solvent for extraction and dissolution of solid samples, buffer solutions for dilution of the sample, washing buffers, conjugate of analyte with a label and a chromogenic substrate.

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