RU2547577C1 - Method of determining level of toxicants in water, food products or body fluids and test system - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method of determining the level of toxicants in water, food products or body fluids by the test system of enzyme-linked immunosorbent assay held in the column, consists in that in the column of the test system a carrier is placed, which is used as an activated solid phase of physisorption - activated porous substrate with grafted covalently bonded molecules of toxicant, the carrier is treated with the blocking solution for closing on the carrier of free places of nonspecific binding, the test samples are added, which contain a certain amount of previously added antibodies specific to toxicants, at that the carrier is treated with conjugate-containing solution, which is used as the conjugate solution of antispecies antibodies chemically bound to luminescent quantum dots or with liposomes containing luminescent quantum dots, and the level of the toxicants is determined by lighting of the treated carrier by excitation radiation on the intensity of luminescence excited in the quantum dots. Test system for the method comprises a column in which a carrier in the form of an activated solid phase of physisorption is mounted - activated porous substrate with grafted covalently bound toxicant molecules, at that the column is provided with a device for measuring the level of luminescence, comprising a source of excitation radiation and a photodetector, at that in front of the photodetector a focusing optical system is additionally mounted, and the output of the photodetector is electrically connected through the signal amplifier and the analogue-digital converter to the control unit - a controller, which output is connected to the display unit and through the stabilisation unit to the excitation radiation source. The side walls of the column are made of material transparent for exciting and luminescent radiation.

EFFECT: invention improves the efficiency and accuracy of determining.

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 carrying out 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 extinction reaction 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 the 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, a carrier is placed in the column of the test system as activated solid phase of physical sorption - an activated porous substrate grafted with covalently bound toxicant molecules in the carrier 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, and the carrier is treated with a conjugate-containing solution, which is used as a solution of a conjugate of antispecies antibodies chemically associated with luminescent quantum points or with liposomes containing luminescent quantum dots, and the level of toxicants is determined by light treated support exciting luminescence intensity radiation, excited in the quantum dots.

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 is installed, according to the invention, the carrier is made in the form of an activated solid phase of physical sorption - an activated porous substrate with grafted with covalently bound toxicant molecules, while the column is equipped with a device for measuring the level of luminescence, including a source of exciting radiation and photoprint a focusing optical system is additionally 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 an excitation radiation source is connected through the stabilization unit, while the side walls of the column are made from transparent material for exciting and luminescent radiation.

In addition, a light filter can be placed between the photodetector and the focusing optical system of the device for measuring the luminescence level.

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, the invention is realized indirect competitive enzyme-linked immunosorbent assay, provides an increase in the intensity of the useful luminescence signal, back prop national concentration of toxins, which increases the sensitivity of the 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.

The drawing schematically shows a General view of the test system.

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 - an activated porous substrate (frit ) 2 with grafted - covalently bound molecules of the toxicant antigen, and a device for measuring the level of luminescence, including a source of 3 exciting radiation, for example, in the form of a set of LEDs with maximum emission wavelengths 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 a focusing optical system 5 is additionally installed in front of the photodetector 4 ( for example, a collecting lens F = 5–30 mm), and between the photodetector 4 and the focusing optical system 5, a light filter and a light filter 6 can be placed, the transmission spectrum of which corresponds to the luminescence spectrum. The output of the photodetector 5 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). Then add 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 and incubated for 2 hours at room temperature. The remaining unbound toxicant antigen is removed by washing the porous support with 5 ml of carbonate buffer (pH = 8.3) containing 0.1 M sodium hydrogen carbonate and 0.1 M sodium chloride. To close the remaining non-specific binding sites, the porous substrate is treated with a blocking solution, which is used as 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 porous substrate is washed sequentially with 5 ml of acetate buffer (0.1 M sodium acetate, 0.5 mol of sodium chloride, pH = 4.0) and 10 ml of phosphate buffer (pH = 7.4–7.6).

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.

Preliminary 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 μmol), and stirred for 12 hours at 4 ° C.

Then, the previously prepared porous substrate 2 with grafted - covalently bound antigen toxicant molecules is placed in an empty Bond Elut type 1 column (V = 1 ml), previously washed with phosphate buffer (pH = 7.4–7.6) and stored at 4 ° C.

25 μl of a solution of toxicant-zeralenone-specific monoclonal murine antibodies is added to 1 ml of the analyzed medium, mixed for 5 minutes and passed through column 1 prepared in the above manner, 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 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) and illuminate carrier 2 containing luminescent quantum dots — semiconductor nanoparticles — with the light flux coming from exciting radiation source 3 and register the luminescence level resulting from exciting radiation, the value of which is proportional to the concentration of the toxicant zeralenone in the analyzed medium.

Moreover, if in the analyzed medium the concentration of toxicant-zeralenone exceeds the concentration of specific antibodies added to the analyzed sample, which corresponds, for example, to the maximum permissible concentration of toxicant, then all specific antibodies are bound to the molecules of the toxicant-zelenone, and when 2 solutions are passed through the carrier conjugate of antispecies antibodies with fluorescent labels - luminescent quantum dots due to the absence of specific antibodies associated with Zeralenone molecules adsorbed on carrier 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, when the carrier - layer 2 is irradiated with source 3 of exciting radiation, luminescence does not occur.

If the concentration of the toxicant-zeralenone in the analyzed medium 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 there is no toxicant-zeralenone in the analyzed medium, then binding of specific specific antibodies to the molecules toxicant-zeralenone adsorbed on the carrier 2 and when passing through the carrier 2 a solution of the conjugate of antispecies antibodies with fluorescent labels are luminescent Using quantum dots, specific antibodies bind to Zelenone molecules adsorbed on carrier 2 to antispecies antibodies contained in the conjugate containing luminescent quantum dots — CdSe / ZnS semiconductor nanoparticles. As a result, when the carrier 2 is irradiated with a source 4 of exciting radiation, luminescence occurs, the level of which is inversely proportional to the concentration of toxicant zeralenone in the analyzed medium.

In this case, the total light front coming from the carrier 2, consisting of the useful luminescence signal and the spurious signal of the exciting radiation, is focused by the optical system 5, passes through the filter 6, which attenuates the stray signal, and the filtered total light front with the extracted useful signal arrives at the photodetector 4 ( photodiode). 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 comparisons with pre-stored calibration constants, and a quantitative value of the luminescence level proportional to the concentration and 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 3, normalizing the voltage of the exciting radiation source 3 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 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 μmol of 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 add 60 μl of 3M glycine in sodium hydroxide solution (pH = 7.2) 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.

25 μl of a solution of toxicant-zeralenone-specific monoclonal mouse antibodies is added to 1 ml of the analyzed medium, mixed for 5 minutes and passed 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 — semiconductor CdSe / ZnS 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 light carrier 2 containing luminescent quantum is illuminated with a light stream from source 3 of exciting radiation Points - semiconductor nanoparticles, and register resulting from the excitation radiation luminescence level, the magnitude of which is proportional to the concentration of toxicant-zearalenone in the test environment.

Example 3. To determine the level of toxicant, for example, the concentration of Zeralenone, in the analyzed medium (natural water), a phased 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 add 60 μl of 3M glycine in sodium hydroxide solution (pH = 7.2) 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.

25 μl of a solution of toxicant-zeralenone-specific monoclonal mouse antibodies is added to 1 ml of the analyzed medium, mixed for 5 minutes and passed 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 (1/30 dilution in phosphate buffer (pH = 7.4–7.6)) was added and incubated for 10 minutes The excess conjugate is removed by washing column 1 with phosphate buffer (pH = 7.4–7.6), after which the carrier 2, which contains luminescent quantum dots — semiconductor nanoparticles — is illuminated with the light flux from point 3 of the exciting radiation, and the luminescence level resulting from the exciting radiation is recorded, the value of which is proportional to the concentration of toxicant-zeralenone in the analyzed medium.

The claimed invention provides the ability to determine the level of toxicants in water, food or physiological fluids by conducting a direct 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 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 column of the test system as an activated solid phase of physical sorption — an activated porous substrate with grafted covalently bound toxicant molecules, the carrier is treated with a blocking solution to close the remaining non-specific free spaces on the carrier binding, test samples containing a certain amount of previously introduced toxicant-specific antibodies are introduced, and the carrier is treated with a conjugate-containing solution, which is used as a solution of a conjugate of anti-species antibodies chemically bound to luminescent quantum dots or to liposomes containing luminescent quantum dots, and the level of toxicants is determined by illuminating the treated carrier with excitation radiation according to the luminescence intensity, excitation in quantum dots. 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 50% ethanol through a porous membrane. 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 installed, characterized in that the carrier is made in the form of an activated solid phase of physical sorption — an activated porous grafted substrate covalently bound molecules of the toxicant, while the column is equipped with a device for measuring the level of luminescence, including a source of exciting radiation and a photodetector, and in front of the photodetector A focusing optical system is installed, 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 a source of exciting radiation, while the side walls of the column are made of transparent for exciting and luminescent radiation material. 4. The test system according to claim 3, characterized in that a light filter can be placed between the photodetector and the focusing optical system of the device for measuring the luminescence level.

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