RU2538707C1 - Method for determining level of toxicants in water, food products or physiological liquids, and test system - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: carrier is arranged in a column of a test system in the form of a layer of immunoaffinity gel, which is fixed between two porous membranes and provided with grafted - covalently bound - molecules of a toxic agent; the carrier - the gel layer is treated with a blocking solution for closure on the carrier of the rest free places of nonspecific binding; test specimens containing a certain amount of pre-introduced antibodies specific to a toxicant are added; the carrier is treated with a conjugate-containing solution, and namely a conjugate solution of anti-species antibodies chemically bound to luminescent quantum dots or to liposomes containing luminescent quantum dots, and a level of toxicants is determined by illumination of the treated carrier with excitation radiation as to intensity of luminescence excited at quantum dots.

EFFECT: group of inventions allows effective and reliable determination of a level of toxicants in water, food products or physiological liquids.

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Description

The invention relates to the field of biotechnology and can be used to increase the efficiency and reliability of determining the level of toxicants in water, food or physiological fluids by conducting an enzyme-linked immunosorbent assay.

A method is known from the prior art for conducting an enzyme immunoassay, including adsorption of antigens on the solid phase of physical sorption, incubation of test biological samples, incubation of a conjugate-containing solution, spectrophotometric analysis of the reaction of extinction of a chromagent solution (RU 2014610 C1, G01N 33/53, 1994).

Also known is a method for determining the level of toxicants in water, food or physiological fluids by an enzyme-linked immunosorbent assay in a column of a test system, including placing an immunoaffinity gel with grafted antivirus antibodies in the column of the carrier, fixed between two porous membranes, treating the carrier with a blocking solution for closure of non-specific binding sites on the carrier, immobilization of specific antibodies on the carrier, introduction of test samples samples, treatment of the carrier with a conjugate-containing solution and analysis of the treated carrier for a color change (RU 2374649 C1, G01N 33/53, 2009). Despite the sufficient simplicity, the accuracy of visual determination of the level of toxicants in this method is not high enough.

In addition, a known test system for enzyme-linked immunosorbent assay of toxicants, including a column in which the carrier is mounted in the form of a layer of immunoaffinity gel grafted with specific antibodies placed between two porous membranes (RU 2374649 C1, G01N 33/53, 2009). The disadvantage of this device is the lack of tools for measuring the level of toxicants.

The technical result to which the invention is directed is to increase the efficiency and reliability of the enzyme-linked immunosorbent assay for determining the level of toxicants in water, food or physiological fluids carried out in a column of a test system.

The solution of this problem with the achievement of the claimed technical result is ensured by the fact that in the method for determining the level of toxicants in water, food or physiological fluids by means of an enzyme-linked immunosorbent assay carried out in the column of the test system, according to the invention, the carrier is placed in the form of a fixed between two the porous membranes of the immunoaffinity gel layer with grafted - covalently bound - toxicant molecules, the carrier is processed - gel layer - block test solution containing a certain amount of preliminarily introduced toxicant-specific antibodies is introduced into the carrier, the carrier is treated with a conjugate-containing solution, which is used as a solution of a conjugate of antispecies antibodies chemically bound to luminescent quantum dots or liposomes containing luminescent quantum dots, and the level of toxicants is determined by illuminating the treated carrier excitation radiation intensity of luminescence excited in the quantum dots.

At the same time, the immunoaffinity gel is prepared in a container with a porous bottom by treating cyanogen bromide with activated sepharose with hydrochloric acid and injecting a solution of the antigen-toxicant in carbonate buffer after swelling into the resulting gel.

The solution to this problem is also ensured by the fact that in the test system for a method for determining the level of toxicants in water, food or physiological fluids, including a column in which a carrier is mounted in the form of a layer of immunoaffinity gel placed between two porous membranes, according to the invention, the column is equipped with a device for measuring the luminescence level, including the source of the exciting radiation and the photodetector, and in front of the photodetector an additional focusing optical system a, and the output of the photodetector is electrically connected through a signal amplifier and an analog-to-digital converter to a control unit - a controller, to the output of which a display unit is connected and through a stabilization unit a source of exciting radiation, while the side walls of the column are made of a material that is transparent to the exciting and luminescent radiation.

In addition, a light filter can be placed in front of the focusing optical system.

Due to the presence in the conjugate solution of luminescent quantum dots (or liposomes containing luminescent quantum dots) chemically associated with anti-species antibodies (rabbit anti-mouse antibodies), which have the ability to bind to toxicant-specific antibodies, and when illuminated with exciting radiation, luminesce, in the claimed invention that implements indirect competitive enzyme-linked immunosorbent assay, an increase in the intensity of the useful luminescence signal, back about ortsionalnogo concentration of toxins, which increases sensitivity method for determining the level of toxicants in water, food or physiological fluids. In addition, the presence in the claimed test system of a device for measuring the level of luminescence, including a source of exciting radiation and a photodetector that are connected to a control unit - a controller, allows to automatically and easily determine the level of toxicants by the degree of luminescence intensity excited in quantum dots.

In the drawing, a general view of the test system is schematically substituted.

The claimed test system for determining the level of toxicants in water, food or physiological fluids includes a column 1 with side walls made of a material that is transparent to the exciting and luminescent radiation, in which a carrier is mounted in the form of a layer 2 immunoaffinity gel with grafted - covalently bound - molecules a toxicant antigen located between two porous membranes 3, and a device for measuring the level of luminescence, including a source 4 of exciting radiation, made, for example p, in the form of a set of LEDs with maximum emission wavelengths in the range 395–500 nm, and a photodetector 5 (photo diode), the spectral sensitivity range of which lies in the range 420–675 nm, with a focusing optical system 6 installed in front of the photodetector 5 (for example, lens F = 5 ÷ 30 mm), in front of which an additional filter 7 is placed, the transmission spectrum of which corresponds to the luminescence spectrum. The output of the photodetector 5 is electrically connected through a signal amplifier 8 and an analog-to-digital converter 9 to a control unit 10 — a controller, to the output of which an indication unit 11 is connected and, through the stabilization unit 12, a source of exciting radiation 4.

The claimed method for determining the level of toxicants is implemented as follows.

For the preparation of an immunoaffinity gel, for example, 0.5 g of cyanogen bromide activated Sepharose 4B is placed in a container with a porous bottom and washed with 100 ml of 0.001 M hydrochloric acid. After gel swelling, the hydrochloric acid solution is drained and 150 μl of a solution of toxicant antigen with a concentration of 2.5 g / l and 450 μl of carbonate buffer (pH 8.3) containing 0.1 M sodium bicarbonate and 0.1 M sodium chloride are added, after which the mixture is shaken continuously (for 2 hours) at room temperature. The remainder of the unbound toxicant antigen is removed by washing the gel with 5 ml of carbonate buffer (pH 8.3) containing 0.1 M sodium bicarbonate and 0.1 M sodium chloride. To close the remaining free sites of nonspecific binding (active Sepharose groups), a blocking solution is added to the resulting gel with grafted - covalently bound - antigen-toxicant molecules, using a 0.2 M solution of glycine in carbonate buffer (pH 8.3) containing 0.1 M sodium bicarbonate and 0.1 M sodium chloride, and stirred for 2 hours at room temperature. After the blocking procedure, the gel is washed three times sequentially with a five-fold volume of acetate buffer (0.1 M sodium acetate, 0.5 mol sodium chloride, pH 4.0) and a five-fold volume of phosphate buffer (pH 7.4–7.6). The resulting immunoaffinity gel with grafted - covalently bound - toxicant-antigen molecules is diluted in phosphate buffer (pH 7.4 ÷ 7.6) in a ratio of 1: 3 and stored at 4 ° C.

Example 1

To determine the level of toxicant, for example, the concentration of zelenone, in the analyzed medium (natural water), they pre-prepare - synthesize a solution of a conjugate of antispecies antibodies chemically bound to luminescent quantum dots - CdSe / ZnS semiconductor nanoparticles, as follows.

20 μl of a solution of antispecies antibodies in carbonate buffer (0.1 M sodium bicarbonate, 0.1 M sodium chloride, pH 8.3) is drained from 800 μl of a CdSe / ZnS quantum dot solution (1/10 dilution in carbonate buffer (0.1 M sodium bicarbonate, 0.1 M sodium chloride, pH 8.3), the number of CdSe / ZnS quantum dots is 3.2 × 10-4 µmo1) and stirred for 12 hours at 4 ° C.

Then, 200 μl of a previously prepared immunoaffinity gel with grafted - covalently bound - antigen-toxicant molecules are placed in the form of a carrier layer 2 onto a porous polyethylene membrane 3 (frit) installed in an empty Bond Elut column 1 (V = 1 ml), previously washed with phosphate buffer (pH 7.4 ÷ 7.6) and stored at 4 ° C. Then, on top of layer 2 of an immunoaffinity gel with grafted - covalently bound - molecules of a toxicant-antigen, a second porous polyethylene membrane 3 (frit) is placed.

To 1 ml of the analyzed medium, add 25 μl of a solution specific for the toxicant - Zeralenone monoclonal mouse antibodies, mix for 5 minutes and pass through column 1 prepared as above, then wash column 1 with phosphate buffer (pH 7.4 ÷ 7.6) containing 0, 05% Tween 20.

Then, 100 μl of a pre-prepared solution of a conjugate of antispecies antibodies chemically bound to luminescent quantum dots — CdSe / ZnS semiconductor nanoparticles (1/30 dilution in phosphate buffer (pH 7.4–7.6) containing 0.05% Tween 20 and 0 , 2% bovine serum albumin), and incubated for 6 minutes. Excess conjugate is removed by washing column 1 with phosphate buffer (pH 7.4–7.6), after which the carrier, layer 2, containing luminescent quantum dots — semiconductor nanoparticles — is illuminated with the light flux coming from the source of exciting radiation 4, and the resulting radiation is recorded the level of luminescence, the value of which is inversely proportional to the concentration of the toxicant - Zeralenone - in the analyzed medium.

Moreover, if in the analyzed medium the concentration of the toxicant - Zeralenone - exceeds the concentration of specific antibodies added to the analyzed sample, which corresponds, for example, to the maximum permissible concentration of the toxicant, then all specific antibodies are bound to the molecules of the toxicant - Zeralenone, and when passed through the carrier, layer 2 - a solution of the conjugate of antispecies antibodies with fluorescent labels - luminescent quantum dots - due to the lack of specific antibodies associated with molecules of zeralenone adsorbed on a carrier - layer 2, the antispecies antibodies contained in the conjugate remain unbound (free) and are removed after washing from column 1 together with luminescent quantum dots - CdSe / ZnS semiconductor nanoparticles. As a result, upon irradiation of the carrier — layer 2 — with the source 4 of exciting radiation, luminescence does not occur.

If in the analyzed medium the concentration of the toxicant - Zeralenone - does not exceed the concentration of specific antibodies added to the analyzed sample, which corresponds, for example, to the maximum permissible concentration of the toxicant, or the toxicant - Zeralenone - in the analyzed medium, there is no binding of excess specific antibodies to molecules of the toxicant - zeralenone adsorbed on the carrier - layer 2 - and when passing through the carrier - layer 2 of a solution of a conjugate of antispecies antibodies with a fluorescent label mi - luminescent quantum dots - the binding of specific antibodies that bind to Zeralenone molecules adsorbed on the carrier layer 2 to antivirus antibodies in the conjugate containing luminescent quantum dots - CdSe / ZnS semiconductor nanoparticles. As a result, upon irradiation of the carrier — layer 2 — with the source 4 of exciting radiation, luminescence arises, the level of which is inversely proportional to the concentration of the toxicant, zeralenone, in the analyzed medium.

In this case, the total light front coming from the carrier, layer 2, consists of a useful luminescence signal and a spurious excitation signal, passes through a filter 7, which attenuates the spurious signal, and the filtered total light front with the extracted useful signal is focused by the optical system 6 onto the photodetector 5 (photodiode). The electric signal generated at the output of the photodetector 5 (the voltage value of which corresponds to the luminescence level) is amplified by a signal amplifier 8, digitized using an analog-to-digital converter 9, and transmitted in digital form for registration to the input of the control unit 10 - controller - for recording the luminescence level, where is processed by comparing with the calibration constants previously stored in the memory, and a quantitative value of the luminescence level proportional to the concentration tion of the toxicant - zeralenone, and / or the corresponding value of the level (concentration) of the toxicant is stored in the memory of the control unit 10 - controller - and displayed in the display unit 11.

In addition, the control unit 10 - the controller - in accordance with the laid down program algorithm automatically performs programmable control of the LEDs of the excitation radiation source 4, normalizing the voltage of the excitation radiation source 1 by means of the stabilization unit 12, and ensures that the calibration constant parameters are saved in the memory (calibration curve ), the values of which are determined in the process of preliminary calibration measurements using standard excitation sources about radiation and preliminary calibration of column 1 using samples of the analyzed medium containing toxicants, for example zeralenone, of known concentration.

Example 2

To determine the level of a toxicant, for example, the concentration of Zeralenone, in a test medium (natural water), a solution of a conjugate of antispecies antibodies with liposomes containing water-soluble luminescent quantum dots - CdSe / ZnS semiconductor nanoparticles is preliminarily prepared as follows.

At the first stage, liposomes containing water-soluble luminescent quantum dots - semiconductor CdSe / ZnS nanoparticles are prepared by the method of hydration of thin films. For this, 70 mg (94 μmol) of phospholipids (Lipoid S75) are dissolved in 1 ml of chloroform in a round-bottom flask (V = 10 ml). Then chloroform is evaporated using a rotary evaporator until a phospholipid film is formed on the walls of the flask. The resulting phospholipid film is treated with 6 ml of water containing 5 μmole CdSe / ZnS quantum dots, and stirred for 30 minutes at a temperature of 45 ° C. Then the solution is treated with ultrasound for 5 minutes to achieve an acceptable particle size of liposomes (of the order of 100 nm). The resulting solution containing liposomes with water-soluble CdSe / ZnS quantum dots is stored at a temperature of 45 ° C.

At the second stage, a solution of the conjugate of antispecies antibodies with liposomes containing quantum dots is synthesized.

For this, 0.5 ml of a 2.5% solution of glutaraldehyde in water is added dropwise with constant stirring to 0.4 ml of a solution containing liposomes with incorporated quantum dots - CdSe / ZnS semiconductor nanoparticles, after which the resulting solution is stirred for 4 hours at room temperature. Excess glutaraldehyde is removed by dialysis for 2 days at 4 ° C. Then, 20 μl of a solution of antispecies antibodies in phosphate buffer (pH 7.4-7.6) is added dropwise with constant stirring for 2 hours at room temperature. Then 60 μl of 3M glycine in sodium hydroxide solution (pH 7.2) is added to block the remaining free aldehyde groups of glutaraldehyde on the surface of the liposomes. The resulting mixture was kept at 4 ° C with constant stirring. Excess unreacted components are removed by dialysis within 3 hours. The resulting conjugates were stored at 4 ° C.

To 1 ml of the analyzed medium, add 25 μl of a solution of toxicant specific - Zeralenone - monoclonal mouse antibodies, mix for 5 minutes and pass through column 1 prepared analogously to Example 1, after which column 1 is washed with phosphate buffer (pH 7.4 ÷ 7.6) containing 0 , 05% Tween 20.

Then, 100 μl of a pre-prepared solution of a conjugate of antispecies antibodies chemically coupled to liposomes containing water-soluble luminescent quantum dots — CdSe / ZnS semiconductor nanoparticles (dilution 1/30 in phosphate buffer (pH 7.4–7.6)) are then introduced and incubated for 6 minutes, excess conjugate is removed by washing column 1 with phosphate buffer (pH 7.4–7.6), after which the carrier, layer 2 containing luminescent quantum dots — semiconductor nanoparticles — is illuminated by from the source 4 of exciting radiation, and the level of luminescence arising as a result of exciting radiation is recorded, the value of which is inversely proportional to the concentration of the toxicant - zeralenone - in the analyzed medium.

Example 3

To determine the level of a toxicant, for example, the concentration of Zeralenone, in a test medium (natural water), a solution of Zeralenone conjugate with liposomes containing hydrophobic luminescent quantum dots - CdSe / ZnS semiconductor nanoparticles is preliminarily prepared as follows.

At the first stage, 70 mg (94 μmol) of phospholipids (Lipoid S75) and 30 pmol of hydrophobic quantum dots (λem = 577 nm) in toluene (52 μl) are dissolved in 1 ml of chloroform in a round-bottom flask (V = 10 ml) when exposed to ultrasound at temperature 4 ° C.

Then, 3 ml of water was added to the resulting solution, and chloroform was evaporated using a rotary evaporator. Then add another 3 ml of water and mix the solution with the formed liposomes at 4 ° C for 60 minutes.

Then the solution is treated with ultrasound for 5 minutes to achieve an acceptable particle size of liposomes (of the order of 100 nm). The resulting solution containing liposomes with hydrophobic CdSe / ZnS quantum dots is stored at 4 ° C.

At the second stage, a conjugate of antispecies antibodies with liposomes containing hydrophobic quantum dots is synthesized.

For this, 0.5 ml of a 2.5% solution of glutaraldehyde in water is added dropwise with constant stirring to 0.4 ml of a solution containing liposomes with incorporated quantum dots - CdSe / ZnS semiconductor nanoparticles, after which the resulting solution is stirred for 3 hours at room temperature. Excess glutaraldehyde is removed by dialysis for 2 days at 4 ° C. Then, 20 μl of a solution of antispecies antibodies in phosphate buffer (pH 7.4-7.6) is added dropwise with constant stirring for 2 hours at room temperature. Then 60 μl of 3M glycine in sodium hydroxide solution (pH 7.2) is added to block the remaining free aldehyde groups of glutaraldehyde on the surface of the liposomes. The resulting mixture was kept at 4 ° C with constant stirring. Excess unreacted components are removed by dialysis within 3 hours. The resulting conjugates were stored at 4 ° C.

To 1 ml of the analyzed medium, add 25 μl of a solution of toxicant specific - Zeralenone - monoclonal mouse antibodies, mix for 5 minutes and pass through column 1 prepared analogously to Example 1, after which column 1 is washed with phosphate buffer (pH 7.4 ÷ 7.6) containing 0 , 05% Tween 20.

Then, 100 μl of a pre-prepared solution of a conjugate of antispecies antibodies chemically coupled to liposomes containing hydrophobic luminescent quantum dots — CdSe / ZnS semiconductor nanoparticles (dilution 1/30 in phosphate buffer (pH 7.4–7.6)) was added and incubated for 10 minutes, excess conjugate is removed by washing column 1 with phosphate buffer (pH 7.4–7.6), after which the carrier, layer 2, containing luminescent quantum dots — semiconductor nanoparticles — is illuminated by the light flux 4 m from the source of the exciting radiation is detected and the resulting excitation emission level of luminescence, which is proportional to the concentration of fluorine - zearalenone - in assay medium.

The claimed invention provides the ability to determine the level of toxicants in water, food, or physiological fluids by conducting an indirect competitive enzyme-linked immunosorbent assay of a toxicant level in the column of the test system with a detection limit (sensitivity) of 1 ÷ 3 ng / ml.

Claims (4)

1. A method for determining the level of toxicants in water, food products or physiological fluids by means of an enzyme-linked immunosorbent assay carried out in a column of a test system, characterized in that a carrier is placed in the form of a layer of immuno-affinity gel grafted between two porous membranes with grafted covalently bound by toxicant molecules, the carrier, the gel layer, is treated with a blocking solution to close the remaining non-specific binding sites on the carrier, test samples containing a certain amount of previously introduced toxicant-specific antibodies are introduced, the carrier is treated with a conjugate-containing solution, which is used as a solution of a conjugate of antispecies antibodies chemically bound to luminescent quantum dots or to liposomes containing luminescent quantum dots, and the level of toxicants is determined by lighting of the treated carrier by exciting radiation in terms of the luminescence intensity excited in quantum dots . 2. The method according to claim 1, characterized in that the immunoaffinity gel is prepared in a container with a porous bottom by treating cyanogen bromide with activated sepharose with hydrochloric acid and injecting a solution of the antigen-toxicant in the carbonate buffer after swelling into the resulting gel. 3. The test system for a method for determining the level of toxicants in water, food or physiological fluids according to claim 1, including a column in which a carrier is mounted in the form of a layer of immunoaffinity gel placed between two porous membranes, characterized in that the column is equipped with a device for measuring the luminescence level, including the source of the exciting radiation and the photodetector, moreover, a focusing optical system is additionally installed in front of the photodetector, and the output of the photodetector is electrically connected Erez signal amplifier and an analog-digital converter to a control unit - a controller, which are connected to the output display unit and the stabilization unit via a source of exciting radiation, the side walls of the columns are made transparent to the exciting radiation and the luminescent material. 4. The test system according to claim 3, characterized in that a light filter is placed in front of the focusing optical system.

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