RU2506586C1 - Method to determine level of toxicants in water, food or physiological liquids and test system - Google Patents

Abstract

FIELD: measurement equipment.

SUBSTANCE: invention relates to the field of biotechnology and may be used to determine level of toxicants in water, food or physiological liquids. The method is carried out by performance of enzyme immunoassay in a column of a test system, and this enzyme immunoassay includes placement of a carrier with inoculated anti-species antibodies in the column, treatment of the carrier with a blocking solution, immobilisation of specific antibodies on the carrier, introduction of tested samples, treatment of the carrier with a conjugate-containing solution and analysis of the treated carrier. The carrier is an activated porous substrate with inoculated anti-species antibodies, and the conjugate-containing solution - the solution of conjugate of antigen - toxicant, chemically linked to luminescent quantum points or with liposomes containing luminescent quantum points. The level of toxicants is determined by intensity of luminescence excited in quantum points during illumination of the treated carrier by exciting radiation. The test system for this method includes a column, which is equipped with a device to measure the level of luminescence including a source of exciting radiation and a photodetector. In front of the photodetector there is additionally a focusing optical system, and the output of the photodetector is electrically connected via a signal amplifier and an analog-digital converter to a controller, to the outlet of which an indication unit and a source of exciting radiation are connected.

EFFECT: invention increases efficiency and validity of determination.

4 cl, 3 ex, 1 dwg

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 a column of a test system, including placement of grafted anti-virus antibodies in the column of the carrier, treatment of the carrier with a blocking solution for closing the non-specific binding sites on the carrier, immobilization of specific antibodies on the carrier, extra test samples, treatment of the carrier with a conjugate-containing solution and analysis of the treated carrier, according to the invention, an activated solid phase of physical sorption is used as a carrier - an activated porous substrate with grafted anti-species antibodies, and as a conjugate-containing solution, a solution of a conjugate of an antigen, a toxicant chemically bound with luminescent quantum dots or with liposomes containing luminescent quantum dots, while the level of toxicants is determined t according to the intensity of the luminescence excited in quantum dots when the treated carrier is illuminated with exciting radiation.

At the same time, the activated porous substrate is prepared by sonication of a pure porous substrate made of polypropylene — frit, placed in ethanol — 96% ethanol, followed by washing — successive passage of 50% ethanol through a porous membrane.

The solution of 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 with grafted antibodies specific for the toxicant is mounted, characterized in that the carrier is made in the form of activated solid phases of physical sorption of an activated porous substrate with inoculated with specific for toxicant antibodies, the column is equipped with a device for measuring the level of luminescence, including a source excitation radiation and a photodetector, in addition to which a focusing optical system is installed in front of the photodetector, 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 an indication unit is connected and through the stabilization unit a source of exciting radiation, while the 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 solution of the conjugate of luminescent quantum dots (or liposomes containing luminescent quantum dots) chemically bound to antigen-toxicant molecules that have the ability to bind to specific antibodies adsorbed on a carrier-activated porous support, which remain free after binding to those in the analyzed medium molecules of the toxicant, and when illuminated with exciting radiation, they luminesce, in the claimed invention, which implements a direct competitive solid ofazny immunosorbent assay, the luminescence efficiency is provided by increasing the signal intensity is inversely proportional to the 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 the carrier is mounted in the form of an activated solid phase of physical sorption - activated porous substrate 2 s grafted with specific antibodies, and a device for measuring the level of luminescence. Moreover, the device for measuring the luminescence level includes a source of exciting radiation 3, made, for example, in the form of a set of LEDs with maximum wavelengths of radiation in the range 395–500 nm, and a photodetector 4 (photodiode), the spectral sensitivity range of which lies in the range 420–675 nm, and in front of the photodetector 4, an additional filter 5 is installed, the transmission spectrum of which corresponds to the luminescence spectrum, and a focusing optical system 6 (for example, a collecting lens F = 5–30 mm). The output of the photodetector 4 is electrically connected through a signal amplifier 7 and an analog-to-digital converter 8 to a control unit 9 — a controller, to the output of which an indication unit 10 is connected and, through the stabilization unit 11, a source of exciting radiation 3.

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

The activated solid phase of physical sorption is prepared by treating (activating) for 15 minutes a porous substrate made of polypropylene 2 with ultrasound in ethanol — 96% ethanol, followed by washing — sequentially passing through a porous substrate 2 passing 500 μl of an aqueous-alcoholic solution — 50% ethyl alcohol, 500 μl of water and 700 μl of carbonate buffer (0.1 M sodium bicarbonate, 0.1 M sodium chloride, pH 8.3).

After activation, 500 μl of a solution of anti-species antibodies (rabbit anti-mouse antibodies) with a concentration of 5 μg / ml in carbonate buffer (0.1 M sodium bicarbonate, 0.1 M sodium chloride, pH 8.3) is applied to the surface of porous substrate 2 and incubated in for 20 minutes at 37 ° C. Then, the porous substrate 2 with grafted anti-species antibodies is washed with 1 ml of phosphate buffer (pH 7.4-7.6) containing 0.05% Tween 20, and 500 μl of a 3% solution of casein in phosphate buffer (pH 7.4- 7.6) within 15 minutes.

After blocking, the porous substrate 2 is washed with 1 ml of phosphate buffer (pH 7.4-7.6) and 500 μl of a solution of antibodies specific to the toxin, for example Zeralenone (dilution 1/10000), is applied to its surface, incubated for 15 min at a temperature of 37 ° C and washed with 3 ml of phosphate buffer (pH 7.4-7.6).

The thus prepared carrier in the form of an activated solid phase of physical sorption — an activated porous support 2 with grafted specific antibodies, is placed in an empty column of type 1 Bond Elut V = 1 ml, previously washed with phosphate buffer (pH 7.4–7.6), and stored at 4 ° С .

Example 1

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

92 mg of n-hydroxysuccinimide and 124 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide are dissolved in 1 ml of dimethylformamide. The resulting solution was diluted 1000 times in dimethylformamide (the required concentration of 0.8 μmol). Then 20 μl of the resulting solution is poured from 800 μl of a CdSe / ZnS quantum dot solution (1/10 dilution in carbonate buffer (0.1 M sodium hydrogen carbonate, 0.1 M sodium chloride, pH 8.3), the number of CdSe / ZnS quantum dots equal to 3.2 × 10 ^-4 μmol), and stirred for 45 minutes, after which 176 μl of a solution of Zelenone (0.4 mg / ml) chemically bound to egg albumin in phosphate buffer (pH 7.4-7.6) is added dropwise. The reaction mixture is constantly stirred for 12 hours at 4 ° C. Excess low molecular weight substances are removed by dialysis for 2 days at a temperature of 4 ° C. The resulting conjugates are stored at 4 ° C.

Through the column 1 prepared as described above, in which the activated porous support 2 with grafted specific antibodies is mounted, 1 ml of the analyzed medium is passed through and the column 1 is washed with phosphate buffer (pH 7.4 ÷ 7.6) containing 0.05% Tween 20.

Then, 100 μl of a preliminarily prepared solution of the toxicant conjugate, Zeralenone bound to luminescent quantum dots, CdSe / ZnS semiconductor nanoparticles (dilution 1/30 in phosphate buffer (pH 7.4 ÷ 7.6), containing 0.05% Tween 20 and 0, is introduced into column 1 , 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) and illuminate the treated porous substrate 2 containing luminescent quantum dots - semiconductor nanoparticles with the light flux coming from the source 3 of the exciting radiation of the device for measuring the luminescence level.

Moreover, if in the analyzed medium the concentration of the toxicant - zeralenone exceeds the concentration of 2 specific antibodies adsorbed on the porous substrate, which corresponds, for example, to the maximum permissible concentration of the toxicant, then all specific antibodies bind to the molecules of the toxicant - zeralenone, and when passing through the porous substrate 2 a solution of the conjugate of the toxicant - zelenone with fluorescent labels - luminescent quantum dots due to the lack of specific antibodies contained in the conjugate, the toxicant molecules, zeralenone, remain unbound (free) and are removed after washing from column 1 along with luminescent quantum dots — CdSe / ZnS semiconductor nanoparticles. As a result, when the porous substrate 2 is irradiated with a source 3 of exciting radiation, luminescence does not occur.

In the event that in the analyzed medium the concentration of the toxicant - zeralenone does not exceed the concentration of 2 specific antibodies adsorbed on the porous substrate, which corresponds, for example, to the maximum permissible concentration of the toxicant, or the toxicant - zeralenone in the analyzed medium is absent, then only part of the specific antibodies binds to molecules of the toxicant - Zeralenone, and when passing through a porous substrate 2 solutions of the conjugate of the toxicant - Zeralenone with fluorescent labels - luminescent quantum bubbled points occurs remaining free binding with specific antibodies contained in the conjugate molecules of fluorine - zearalenone containing fluorescent quantum dots - semiconducting nanoparticles CdSe / ZnS. As a result, when the porous substrate 2 is irradiated with a source 3 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 porous substrate 2, which consists of the useful luminescence signal and the spurious excitation signal, passes through a filter 5, 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 4 (photo diode ) The electric signal generated at the output of the photodetector 4 (the voltage value of which corresponds to the luminescence level) is amplified by a signal amplifier 7, digitized using an analog-to-digital converter 8 and transmitted in digital form for registration to the input of the control unit 9 - controller for recording the luminescence level where it is processed, by comparing with the calibration constants previously stored in the memory, and the quantitative value of the luminescence level, inversely proportional to the center 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 9 - controller and displayed in the display unit 10.

In addition, the control unit 9 — the controller, in accordance with the inherent software algorithm, automatically performs programmable control of the LEDs of the exciting radiation source 4, normalizing the voltage of the exciting radiation source 1 by means of the stabilization unit 11, 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 radiation and preliminary calibration of column 1 using samples of the analyzed medium containing toxicants, for example, Zeralenone, known concentration.

Example 2

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

In the second stage, the synthesis of Zelenone conjugate with liposomes containing quantum dots is carried out.

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, dropwise, with constant stirring, for 2 hours at room temperature, 98 μg of a solution of zelenone (0.4 mg / ml) chemically bound with egg albumin in phosphate buffer (pH 7.4-7.6) is added. Then 60 μl of 3 M glycine in sodium hydroxide solution (pH 7.2) was added to block the remaining free aldehyde groups of glutaraldehyde on the surface of the liposomes. The resulting mixture was maintained at a temperature of 4 ° C with constant stirring. Excess unreacted components are removed by dialysis within 3 hours. The resulting conjugates were stored at 4 ° C.

Then, analogously to example 1, through column 1 prepared in the manner described above, in which an activated porous substrate 2 with grafted specific antibodies is mounted, 1 ml of the analyzed medium is passed through and the column 1 is washed with phosphate buffer (pH 7.4 ÷ 7.6) containing 0.05% Tween 20.

Then, 100 μl of a previously prepared solution of Zelenone conjugate with liposomes containing water-soluble luminescent quantum dots — CdSe / ZnS semiconductor nanoparticles (dilution 1/20 in phosphate buffer (pH 7.4–7.6)) was introduced into column 1 and incubated for 6 minutes. Excess conjugate is removed by washing column 1 with phosphate buffer (pH 7.4 ÷ 7.6), and illuminate the treated porous septum 2 containing luminescent quantum dots - semiconductor nanoparticles with the light flux coming from the source of exciting radiation 3. In this case, optical excitation of luminescence occurs, the level of which is inversely proportional to the concentration of the toxicant, is recorded by the device for measuring the luminescence level in the manner described above.

Example 3

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

At the first stage, 70 mg (94 μmol) of phospholipids (Lipoid S75) and 30 pmol of hydrophobic quantum dots (lem = 577 nm) in toluene (52 μl) of wisQDs are dissolved in 1 ml of chloroform in a round-bottom flask (V = 10 ml) upon exposure ultrasound at a temperature of 45 ° 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 a temperature of 45 ° 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 a temperature of 45 ° C.

In the second stage, the synthesis of Zelenone conjugate with liposomes containing quantum dots is carried out.

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, dropwise, with constant stirring, for 2 hours at room temperature, 98 μg of a solution of zelenone (0.4 mg / ml) chemically bound with egg albumin in phosphate buffer (pH 7.4-7.6) is added. Then 60 μl of 3 M glycine in sodium hydroxide solution (pH 7.2) was added to block the remaining free aldehyde groups of glutaraldehyde on the surface of the liposomes. The resulting mixture was maintained at a temperature of 4 ° C with constant stirring. Excess unreacted components are removed by dialysis within 3 hours. The resulting conjugates are stored at 4 ° C.

Then, analogously to example 1, through column 1 prepared in the manner described above, in which an activated porous substrate 2 with grafted specific antibodies is mounted, 1 ml of the analyzed medium is passed through and the column 1 is washed with phosphate buffer (pH 7.4 ÷ 7.6) containing 0.05% Tween 20.

Then, 100 μl of a previously prepared solution of Zelenone conjugate with liposomes containing hydrophobic luminescent quantum dots - CdSe / ZnS semiconductor nanoparticles (dilution 1/45 in phosphate buffer (pH 7.4-7.6)) is injected into column 1 and incubated for 6 minutes. Excess conjugate is removed by washing column 1 with phosphate buffer (pH 7.4 ÷ 7.6).

After washing column 1, the treated porous septum 2 containing luminescent quantum dots — semiconductor nanoparticles — is illuminated with the light flux coming from the source of exciting radiation 3, and optical luminescence is excited inversely with the toxicant concentration, which is detected by the device for measuring the luminescence level described above way.

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

Claims (4)

1. A method for determining the level of toxicants in water, food or physiological fluids by means of an enzyme-linked immunosorbent assay performed in a column of a test system, including placing in a column of a carrier grafted with anti-virus antibodies, treating the carrier with a blocking solution to close the remaining non-specific binding sites on the carrier, and immobilizing carrier of specific antibodies, introduction of test samples, treatment of the carrier with a conjugate-containing solution and analysis of the treated carrier, distinguish Take advantage of the fact that an activated solid phase of physical sorption — an activated porous substrate with grafted anti-species antibodies — is used as a carrier, and a solution of an antigen-toxicant conjugate chemically bound to luminescent quantum dots or liposomes containing luminescent quantum dots is used as a conjugate-containing solution. this level of toxicants is determined by the intensity of the luminescence excited in quantum dots when the treated carrier is illuminated by excitation from radiation. 2. The method according to claim 1, characterized in that the activated porous substrate is prepared by sonication of a pure porous substrate made of polypropylene, placed in ethanol - 96% ethanol, followed by washing - sequentially passing through the porous substrate 50% ethyl alcohol. 3. The test system for the method of determining the level of toxicants in water, food or physiological fluids according to claim 1, including a column in which a carrier is mounted with vaccinated specific for a toxicant antibodies, characterized in that the carrier is made in the form of an activated solid phase of physical sorption - activated porous substrate grafted with specific for toxicant antibodies, the column is equipped with a device for measuring the level of luminescence, including a source of exciting radiation and a photodetector, and before The photodetector is additionally equipped with a focusing optical system, 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 an indication unit is connected and through a stabilization unit an exciting radiation source, while the side walls of the column are made of transparent for and luminescent radiation of the 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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