RU30005U1 - Device for the selective analysis of biologically active compounds in solutions - Google Patents

Description

200310Q36Q

iBiprapiitiipP ™ ° T2opG

Device for the selective analysis of biologically active compounds in

The utility model relates to the field of analytical biochemistry, more specifically, to devices for the selective analysis of biologically active compounds in solutions and can be used in food, pharmaceutical, biotechnological applications, in medical diagnostics, including the selective analysis of various products and substances for the presence of toxins, pesticides and heavy metals.

A device is known for the selective analysis of elements in aqueous solutions by thin-layer chromatography containing a plate with a thin layer of modified chelating cellulose deposited on it, a sample to be analyzed on the initial part of the plate and a liquid mobile phase that gives a color reaction during the complexation of the element to be determined on the chromatographic layer (see G .. Analytical chemistry, 26, No. 7, 1974, p. 1434).

The disadvantage of this device is the relatively narrow scope, which does not allow for qualitative and quantitative analysis of biologically active compounds, in particular, organophosphorus compounds, carbamates, toxins, etc.

The closest technical solution is a device for the selective analysis of biologically active compounds in solutions, containing a measuring unit, reaction cells, a sampler and means for supplying solutions to the measuring unit and reaction cells (see Till T., et al .. Biosensors & Bioelectronics, 2000, 15, p. 193 - prototype).

The disadvantages of the known device are the relatively low accuracy of the measurements, the lack of sensitivity and reproducibility of the measurement results, as well as the inability to fully automate the selective analysis of biologically active compounds in biological solutions and technological environments.

The problem to be solved is the creation of an automatic device for effective quantitative selective analysis of biologically active compounds in biological and technological solutions of wide application, including in the practice of medical diagnostics, industrial biotechnology and environmental monitoring.

This problem is solved in that in a device for the selective analysis of biologically active compounds in solutions containing a measuring unit, reaction cells, a sampler and means for supplying solutions to the measuring unit and reaction cells, according to a utility model, the device is equipped with a microprocessor for programming the operation of the feeding means solutions into the measuring unit and into the reaction cells, the measuring unit is equipped with a flow biosensor analyzer, the reaction cells are placed in a linear, direct carbonic or annular matrix with pre-immobilized in each cell bioactive species to analyze a particular type of biologically active compounds, means for feeding solutions of the reaction cells in the measurement unit

C12M

solutions

includes a hydraulically connected sampler, a biosensor flow analyzer, a first peristaltic pump and a drain tank, means for supplying solutions to the reaction cells, includes a hydraulically connected buffer solution tank, a second peristaltic pump and a distribution tank with an adjustable solution level, means for positioning the sampler relative to the reaction cells the possibility of vertical and horizontal movement of the sampler and / or matrix of reaction cells program according to the program from the microprocessor, and the electrical output of the measuring unit through the converter and amplifier is connected to the first input of the microprocessor, its second input is connected to the output of the mode switching unit, its third input is connected to the electrical output of the buffer level sensor in the distribution tank, the first, second, the third, fourth and fifth outputs of the microprocessor are respectively connected to the control inputs of the indicator of the measurement results, drive mechanisms of vertical and horizontal emescheniya first and the second peristaltic pumps.

In addition, the flow biosensor analyzer can be made in the form of a biochemical analyzer, the reference electrode of which is made of silver chloride, and the active one is platinum, equipped with a semipermeable film with an immobilized cholinoxidase enzyme.

In addition, a flow biosensor analyzer can be made optical by the degree of absorption of the light source radiation by a solution in the working chamber of the analyzer equipped with a semipermeable film with an immobilized cholinoxidase enzyme.

In addition, for selective analysis in solutions of organophosphorus compounds and carbamates, the matrix can contain 12 reaction cells, of which 1 and 3 cells are partially filled with a buffer solution, 2 and 4 cells contain an immobilized organophosphate hydrolase enzyme, and 5,6,7 and 8 cells contain immobilized butyrylcholinesterase enzyme, and 9,10,11 and 12 cells contain butyrylcholine substrate for sequential transfer of solutions and their selection in the measuring unit in accordance with the microprocessor program for this analysis.

In addition, the buffer solution can be filled with a HEPES-based solution with the addition of inorganic salts and complexones at pH 7.5.

This embodiment of the device allows to solve the task of creating an automatic device for quick qualitative and quantitative selective analysis by staff of ordinary qualifications of a wide class of biologically active compounds in the practice of medical diagnostics, industrial biotechnology and environmental monitoring.

A known method for the determination of organophosphorus compounds and carbamates, based on their ability to inhibit cholinesterase, that is, reduce its activity. The determination of cholinesterase activity is carried out by changing the pH of the enzymatic reaction, but this method is ineffective due to the need for manual calibration and poor sensitivity and reproducibility of the analysis (see Reybier K. et al., Talanta, 2002, 56, pp. 1015-1020).

To measure the choline formed during the enzymatic reaction, a flowing electrochemical cell is used, the reference electrode of which is silver-plated and the active electrode is platinum. The latter is equipped with a semi-permeable film with the immobilized cholinoxidase enzyme, which is a key component in the implementation of the enzymatic reaction on the electrode in the analysis of this compound.

The selectivity of the analysis is achieved by the fact that the components of the HEPES-based buffer solution with the addition of inorganic salts and complexones at pH 7.5 exclude the influence of heavy metal ions due to complexation and their removal from the reaction medium. In this case, organophosphate hydrolase provides an additional rapid decomposition of organophosphorus compounds and their conclusion from the reaction.

It is this embodiment of the device that makes it possible to automate the process of selective analysis using a matrix of reaction cells with bioactive components pre-immobilized in them to determine a specific type of biologically active compounds.

The block diagram of a specific embodiment of the device is shown in FIG. 1.

A device for the selective analysis of biologically active compounds in solutions contains a measuring unit 1, reaction cells 2, a sampler 3, and means for supplying solutions to the measuring unit and reaction cells.

The device is equipped with a microprocessor 4 for programming the operation of the means for supplying solutions to the measuring unit 1 and to the reaction cells 2. The latter are placed in the form of a rectangular matrix with bioactive components pre-immobilized in the cells for analysis of a particular type of biologically active compounds.

The means for supplying solutions from the reaction cells to the measuring unit includes a hydraulically connected sampler 3, a flow biosensor analyzer of the measuring unit 1, the first peristaltic pump 5 and the drain tank 6.

The means for supplying solutions to the reaction cells 2 includes a hydraulically connected buffer tank 7, a second peristaltic pump 8 and a distribution tank 9 with an optical level controller 10 of the buffer solution.

As a buffer solution, a solution based on HEPES with the addition of inorganic salts and complexones at pH 7.5 is used.

The means for positioning the sampler 3 relative to the reaction cells 2 is made with the possibility of vertical and horizontal movement of the sampler through mechanisms I, 12 relative to the matrix of the reaction cells 2 according to the program of the microprocessor 4.

the second, third, fourth and fifth outputs of the microprocessor 4 are respectively connected to the control inputs of the liquid crystal display 16, drive mechanisms 11,12 of the sampler 3, the first and second peristaltic pumps 5.8.

For selective analysis of the content of organophosphorus compounds and carbamates in the studied solutions, the matrix should contain 12 reaction cells, of which 1 and 3 cells are partially filled with a buffer solution, 2 and 4 cells contain an immobilized enzyme, organophosphate hydrolase, and 5,6,7 and 8 cells contain an immobilized enzyme butyrylcholinesterase, and 9,10,11 and 12 cells contain butyrylcholine substrate for sequential transfer of solutions and their selection in the measuring unit in accordance with the microprocessor program for this analysis.

Sampler 3 uses a steel needle with a diameter of 0.8 mm, a transducer 13 - AD820AR, a display 16 - a four-line POWER TIP display, pumps 5.8 - step motors with a peristaltic system, mechanisms 11.12 step motors with a belt and worm drive, cell matrix 2 - based on a 12-hole strip from Labsystem. Microprocessor 4 is based on the ADuC824QS.

The device operates as follows.

The measuring cycle begins with flushing all the systems of the device with a buffer solution. Then the operator transfers the control sample with a pipette to the first and second reaction cells of the matrix, and the analyzed water sample to the third and fourth reaction cells,

After pressing the power button on the start of the automatic analysis mode on block 15, the needle of the sampler 3 rises by means of the mechanism 11 from the distribution tank 9 with the buffer solution up to the maximum position. Further, using the horizontal movement mechanism 12, the sampler 3 is moved until it is aligned vertically with the first reaction cell. The mechanism 11 lowers the needle of the sampler 3 into the first reaction cell, and part of the sample at the command of the microprocessor 4 is taken using a peristaltic pump 5.

The specified sample in accordance with the program of microprocessor 4 (according to the scheme indicated below) is transferred to the fifth reaction cell with cholinesterase immobilized in it in dry form. The sample, getting into the cell with cholinesterase, is incubated for a predetermined time with the enzyme and then part of it in the same way is automatically transferred according to the program by sampler 3 to the ninth cell with butyrylcholine. After incubation with the latter, the sample is taken by sampler 3 for supply to the measuring unit 1.

In the measuring cell, the choline accumulated in the sample interacts with the choline oxidase enzyme when passing through the first surface of the porous membrane at the platinum electrode of the measuring unit 1 and turns into betaine with the release of hydrogen peroxide.

After diffusion of hydrogen peroxide through the second surface of the porous membrane, the electrochemical cell of the measuring unit 1 measures its amount. An electrical signal from the output of unit 1 is supplied through a voltage converter 13 and an amplifier 14 to the first input of microprocessor 4.

Similarly, the sample transfer cycle is carried out according to scheme 2-6-10 and the choline concentration is measured for other groups of reaction cells according to schemes 3-7-11 and 4-8-12.

In this case, the transfer groups 1-5-9 and 2-6-10 are control, in which the studied toxins are absent, for fixation in the memory of microprocessor 4 and subsequent comparison with the analyzed samples according to schemes 3-6-11 and 4-8-12.

After finishing the measurements, the sampler 3 by means of mechanisms 11, 12 is returned to its original state and the system is washed with a buffer solution in accordance with the program of microprocessor 4.

The concentration of neurotoxin in the test sample is calculated by microprocessor 4 in relation to the signals from the measured and control samples, and the display 16 displays information on the excess of the MPC of the measured toxin of this sample relative to the control sample without toxin.

Organophosphorus compounds and carbamates are widely used in industry, agriculture, and as pesticides. These substances have a strong neurotoxic effect on humans and animals; therefore, the problem of analyzing compounds of this class and the like acquires special significance for protecting public health and the environment.

The proposed device, solving the specified range of problems, has important advantages compared with the known devices of a similar purpose. The operation of this device is simple and reliable, the device allows selective analysis of biologically active compounds with high sensitivity and selectivity, while the measuring unit is designed so that the measurements require a minimum amount of buffer and analyzed solutions.

Claims (5)

1. A device for the selective analysis of biologically active compounds in solutions, containing a measuring unit, reaction cells, a sampler and means for supplying solutions to the measuring unit and their selection from the reaction cells, characterized in that the device is equipped with a microprocessor for programming the operation of means for supplying solutions to the measuring block and their selection from the reaction cells, the measuring unit is equipped with a flow biosensor analyzer, the reaction cells are placed in the form of a linear, rectangular or ring A matrix with bioactive components preliminarily immobilized in each cell for analysis of a specific type of biologically active compounds, the means for supplying solutions from the reaction cells to the measuring unit includes a hydraulically connected sampler, a flow biosensor analyzer, a first peristaltic pump and a drain tank, means for supplying solutions to the reaction the cell includes a hydraulically connected buffer tank, a second peristaltic pump and a distribution tank with level of solution, the means for positioning the sampler relative to the reaction cells is made with the possibility of vertical and horizontal movement of the sampler and / or the matrix of reaction cells according to the program from the microprocessor, and the electrical output of the measuring unit through the converter and amplifier is connected to the first input of the microprocessor, its second input is connected to the output of the mode switching unit, its third input is connected to the electrical output of the buffer level sensor in the distribution capacity, the first, second, third, fourth and fifth outputs of the microprocessor are respectively connected to the control inputs of the indicator of the measurement results, the mechanisms of the drive of vertical and horizontal movement, the first and second peristaltic pumps. 2. The device according to claim 1, characterized in that the flow biosensor analyzer is made in the form of a biochemical analyzer, the reference electrode of which is made of silver chloride, and the active one is platinum, equipped with a semipermeable film with an immobilized choline oxidase enzyme. 3. The device according to claim 1, characterized in that the flow biosensor analyzer is made optical by the degree of absorption of radiation of the light source by a solution in the working chamber of the analyzer equipped with a semipermeable film with an immobilized cholinoxidase enzyme. 4. The device according to claims 1 to 3, characterized in that for selective analysis in solutions of organophosphorus compounds and carbamates, the matrix contains 12 reaction cells, of which 1 and 3 cells are partially filled with a buffer solution, 2 and 4 cells contain an immobilized organophosphate hydrolase enzyme, 5, 6, 7 and 8 cells contain the butyrylcholinesterase immobilized enzyme, and 9, 10, 11 and 12 cells contain the butyrylcholine substrate for sequential transfer of solutions and their selection for analysis in the measuring unit in accordance with the microproject program quarrel on this analysis. 5. The device according to claims 1 to 4, characterized in that the capacity of the buffer solution is filled with a HEPES-based solution with the addition of inorganic salts and complexones at pH 7.5.