RU2386135C2 - Method for electrochemical detection of specific biomolecules, device for implementation thereof and version thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: there is offered method of electrochemical detection of specific biomolecules in a biological sample, and also a device for implementation thereof. In a measuring cell when stirred an immune complex is formed on an electrode surface; an electrode is washed with a buffer solution; marked antibodies are added. There is also provided contact of a biosensor with a reference biological sample; te cell is washed once more, current is detected on an exploring electrode with controlling temperature of the biological sample. The device for electrochemical detection of specific biomolecules comprises a measurement chamber accommodating an exploring electrode, an auxiliary electrode and a reference electrode, a temperature probe. Alternatively, the device has one more temperature probe mounted in close proximity to the exploring electrode.

EFFECT: method allows improving reliability of diagnostic results, extending range of detected objects.

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Description

The invention relates to the field of medicine, biotechnology, and in particular to methods of laboratory research and analysis of various biological fluids carried out for the purpose of medical diagnosis, in particular for the diagnosis of myocardial infarction.

A known method for the quantitative electrochemical analysis of biomolecules (see patent No. 2161653 from 04/28/98). This method provides for the formation of a biosensor by applying a film of an electrically conductive polymer to the surface of a potentiometric electrode during electrochemical synthesis from a monomer solution, contacting the biosensor with a sample of the studied liquid, compiling an electrochemical measuring cell from a biosensor and a reference electrode connected to the measuring device, recording by measuring device of the biosensor potential relative to the electrode changes in the buffer solution, registration by means of a measuring device of a change in the biosensor potential with a stepwise change in the ionic strength of the buffer solution at a constant value of its pH. In the method, the electrically conductive polymer is doped at the stage of electrochemical synthesis with anions that are most capable of ion exchange with the solution surrounding the polymer, the avidin or streptavidin are immobilized onto the film of the electrically conductive polymer, biotinylated affinity bioreceptors are used to immobilize the electrically conductive polymer film, additional labeled bioreceptors are used, additional labeled bioreceptors are used By compiling an electrochemical cell, the biosensor is contacted with biotinylated affinity bioreceptors Ami, before measuring the biosensor potential in the buffer solution, the biosensor is contacted with additional labeled bioreceptors, the change in the biosensor potential is recorded when the composition of the buffer solution changes.

A significant drawback of all potentiometric systems based on the described principles is their sensitivity to the buffer capacity of the solution, which significantly limits their use (in particular for myocardial infarction, since the enzyme is released into peripheral blood in small quantities, to determine which it is necessary to ensure maximum possible sensitivity with minimal error). The sensitivity of the potentiometric method is low. Also, the disadvantages include the complexity and low adaptability of the biosensor manufacturing process.

The closest adopted for the prototype is a method for the electrochemical determination of specific antibodies in blood serum using proteins labeled with metal (see patent No. 2249217 from 06/18/2003). The method includes the formation of an immunocomplex on the electrode surface, moreover, to form an immunocomplex, an antigen immobilized on the surface of a thick film graphite electrode is incubated in the blood serum, the electrode is washed with a buffer solution and incubated with a solution of a protein of non-immune origin labeled with metal, followed by repeated washing with a buffer solution and placed in a three-electrode cell in which specific antibodies are determined by a metal oxidation reaction.

The disadvantage of this method is the duration and complexity of the measurement process, the need for preliminary processing of the sample under strictly designated temperature conditions.

A device for electrochemical measurements is known (see patent No. 2097754 from 05.25.95). The device comprises a measuring chamber, including a working electrode, an auxiliary electrode and a reference electrode. The device is equipped with a supply and receiving drums, a drive and a current collector, the measuring chamber is made with a flow slot and is equipped with floating rollers, enveloped by a loop-like flexible working electrode.

The disadvantage of this device is that it does not provide for its use for the detection of biologically active substances. Also disadvantages include its relatively large dimensions. That is, it is a stationary device that does not provide for its transportation (mobility). The device is difficult to operate due to the presence of feed drums, as well as in maintenance and repair.

The objective of the invention is to provide a method and device that provides accurate electrochemical determination of biomolecules in a biological sample, in particular for the diagnosis of myocardial infarction in the early stages, with microinfarction, with a form of heart attack with atypical localization of pain or its absence, if it is impossible to diagnose with an ECG (absence device, lack of specialist, atypical heart attack). It is also necessary to create a device that is easy to use and maintain.

The problem is solved in that in the method for the electrochemical determination of specific biomolecules in a biological sample using labeled antibodies, an immunocomplex is formed on the surface of the electrode, the electrode is washed with a buffer solution, and labeled antibodies are added. In this case, the formation of the immunocomplex on the surface of the electrode, washing the electrode with a buffer solution, adding labeled antibodies is carried out directly in the measuring cell. Then, the biosensor is contacted with the sample of the biological sample, the cell is re-washed, the appearance of current on the working electrode is detected, and the temperature of the biological sample is monitored. All actions from the formation of the immunocomplex to the completion of the measurement are performed while the sample is mixed. As a specific biomolecule, troponin can be used.

A device for the electrochemical determination of biomolecules contains a measuring chamber including a working electrode, an auxiliary electrode and a reference electrode, as well as a temperature sensor. Alternatively, there may be another temperature sensor located in the immediate vicinity of the working electrode. Cellulose acetate, or lavsan, or tracing paper, or cellulose nitrate, or gelatin is used to make the biosensor film.

The proposed method and device allows you to quickly and reliably carry out the electrochemical determination of biomolecules, including for the purpose of determining myocardial infarction.

The proposed method is implemented in the design of the device shown in the following drawings.

Figure 1 - section of a General view of the device.

Figure 2 is a view A of the device.

Figure 3 is a view B of the device.

A device for the electrochemical determination of biomolecules has a housing 1, a group of electrodes, including a working electrode 2, a reference electrode 3 and an auxiliary electrode 4; temperature sensor 5. The working electrode is made of noble metals that are not susceptible to corrosion (platinum, gold), or of graphite. The reference electrode is made, for example, silver chloride in the form of a half ring. The auxiliary electrode is made of titanium. The working electrode 2 is in contact with the film 6, on which antibodies are immobilized. Antibodies can also be immobilized directly on the working electrode 2 (in this case, there is no film 6). The working electrode 2, the reference electrode 3 and the film 6 form a biosensor. Between the film 6 and the electrodes 2 and 3 there is a gap 7, which is filled with a buffer solution to make contact between the film and the electrodes. In the second variant of the device, an additional temperature sensor 9 is installed in the immediate vicinity of the working electrode 2. The electrodes and temperature sensors are located in the working area of the cell into which the biological sample under investigation is placed.

A device for electrochemical determination of biomolecules works as follows.

A film (for example, of cellulose nitrate) 6 with immobilized antibodies (for example, troponin) is mounted on a working electrode 2, on which a buffer solution is previously applied (a drop is dripping) to fill the gap 7. The biosensor is installed in a measuring cell filled with a buffer solution. With constant stirring, the test biological sample (for example, the patient’s blood) is introduced into the cell’s working area. Mixing of the sample is carried out to increase the speed of the chemical reaction. Stirring can be accomplished, for example, by installing a rod, driven by a magnet, mounted on the axis of an electric motor (not shown) in the cell’s working area. In the presence of the desired specific biomolecules in the sample, antigen-antibody complexes are formed on the film surface. Then the measuring cell is washed with a buffer solution, removing unbound antigens, and labeled antibodies are introduced into the cell’s working area (for example, with an enzyme, horseradish peroxidase). On the surface of the film is the formation of complexes of antibody-antigen-labeled antibody. An auxiliary electrode 4 is supplied with an operating voltage from a generator (not shown), the value of which is sufficient for the reaction to take place. The voltage is induced on the working electrode 2. Then, a solution containing a substance is introduced into the working zone, which in the presence of labeled antibodies gives a chemical reaction (for example, hydrogen peroxide), which leads to a change in potential and temperature on the working electrode 2 and generates a current. The potential of reference electrode 3 is used as a reference; it does not depend on a chemical reaction, but varies with temperature. To ensure the accuracy of the measurement, the temperature of the solution is monitored using a temperature sensor 5, which makes it possible to reduce the measurement error. The chemical reaction used is carried out in two stages (immune reaction). When the maximum value of the current taken from the working electrode is reached, the concentration of the studied biomolecules is calculated, for example, according to a calibration curve, then the generator is turned off. The motor is also stopped and sample mixing is stopped. The signal is recorded by amperometry. The measurement can be carried out using a micro converter, the obtained data is output to a computer.

Thus, it is possible, for example, in the patient’s body to detect the substance troponin specific for myocardial infarction.

In the second embodiment, the device provides for the installation of a second temperature sensor 9 in close proximity to the working electrode 2. This allows, on the one hand, to eliminate the measurement error by mathematical correction of the potential of the reference electrode taking into account the dependence of the potential on temperature, thereby increasing the accuracy of determining the concentration, and on the other hand, track the working electrode. Also, if there are two sensors, it is possible to measure the concentration of a substance in two ways at the same time, both by increasing the temperature and by changing the current, which will make it possible to control the serviceability of the electrodes and the correctness of the data being taken.

Depending on the selected calibration curve, an abrupt, linear or unfolded voltage is used. When applying a linear sweep on the graph i = f (E), we first obtain an increase in current, and then its gradual decrease. With a cyclic scan of the potential, we graphically obtain a cyclic voltammogram with two peak values. With an abrupt change in potential graphically, we obtain a sharp increase in current, and then its gradual decrease.

The method is illustrated by the following examples.

Example 1

A sensitive membrane 6 is formed by immobilization of antibodies (troponin) on a film carrier (cellulose nitrate). A sensitive membrane 6 is installed on the working surface of electrode 2 (platinum). The obtained biosensor with immobilized antibodies is placed in the measuring cell, where the auxiliary electrode is titanium, the reference electrode is silver chloride, the temperature sensor is a copper-constantan thermocouple, the core of the magnetic stirrer, and the background is a buffer solution. With constant stirring, the test sample (patient's blood) is introduced into the cell’s working area, antigen-antibody complexes are formed on the surface of the sensitive membrane. Then the cell is washed with buffer solution and antibodies labeled with the enzyme (horseradish peroxidase) are added. Then the electrode is washed with a solution containing hydrogen peroxide, and the signal is recorded by amperometry. The measurement is carried out using a micro converter, the obtained data is output to a computer. If the troponin concentration in the patient’s blood sample is higher than 0.1 ng / ml, myocardial infarction is diagnosed.

Example 2

A sensitive membrane 6 is formed by immobilization of antibodies (natriuretic hormone (pro-BNP)) on a film carrier (cellulose acetate). A sensitive membrane is installed on the working surface of electrode 2 (graphite) 6. The obtained biosensor with immobilized antibodies is placed in the measuring cell, where the auxiliary electrode is located - titanium, the reference electrode is silver chloride, the temperature sensor is a copper-constantan thermocouple, the core of the magnetic stirrer, the background is a buffer solution. With constant stirring, the test sample (patient's blood) is introduced into the cell’s working area, antigen-antibody complexes are formed on the surface of the sensitive membrane. The cell is then washed with buffer solution and antibodies labeled with the enzyme (catalase) are added. Then the electrode is washed with a solution containing hydrogen peroxide, and a signal is recorded during a linear sweep of the potential of the working electrode by voltammetry. The measurement is carried out using a microconverter, the obtained data is displayed on a liquid crystal display. At a concentration of natriuretic hormone (pro-BNP) in a patient’s blood sample above 6 pmol / L, myocardial infarction is diagnosed.

Example 3

A sensitive membrane 6 is formed by immobilization of antibodies (IgG) on a film carrier (cellulose nitrate). A sensitive membrane 6 is installed on the working surface of electrode 2 (platinum). The obtained biosensor with immobilized antibodies is placed in the measuring cell, where the auxiliary electrode is titanium, the reference electrode is silver chloride, the temperature sensor is a copper-constantan thermocouple, the core of the magnetic stirrer, and the background is a buffer solution. With constant stirring, the test sample (patient's blood) is introduced into the cell’s working area, antigen-antibody complexes are formed on the surface of the sensitive membrane. The cell is then washed with buffer solution and antibodies labeled with the enzyme (catalase) are added. Then the electrode is washed with a solution containing hydrogen peroxide, and a signal is recorded by cyclic voltammetry. The measurement is carried out using a micro converter, the obtained data is output to a computer. The excess of reference IgG values (above 1.1 U / L) indicates a previous or current infection.

Thus, the present invention allows for accurate electrochemical determination of biomolecules in a biological sample, in particular for the diagnosis of myocardial infarction in the early stages. The invention also implements the possibility of mobile, simplified analysis, reducing its cost, increasing the reliability of diagnostic results, expanding the range of defined objects. The claimed device is easy to use and maintain.

Claims (6)

1. A method for the electrochemical determination of specific biomolecules in a biological sample using labeled antibodies, in which an immunocomplex is formed on the electrode surface, the electrode is washed with a buffer solution, labeled antibodies are added, characterized in that the formation of an immunocomplex on the electrode surface, washing the electrode with a buffer solution, adding labeled antibodies carried out directly in the measuring cell with simultaneous convective mass transfer by mixing the sample, also when mixed AI provide contact of the biosensor with the sample of the biological sample, re-wash the cell, detect the appearance of current on the working electrode, monitor the temperature of the biological sample. 2. The method according to claim 1, characterized in that troponin is used as a specific molecule. 3. A device for the electrochemical determination of specific biomolecules, comprising a measuring chamber including a working electrode, an auxiliary electrode and a reference electrode, characterized in that there is additionally a temperature sensor. 4. The device according to claim 3, characterized in that in the manufacture of the film on the working electrode cellulose acetate, or lavsan, or tracing paper, or cellulose nitrate, or gelatin is used. 5. A device for the electrochemical determination of specific biomolecules, comprising a measuring chamber including a working electrode, an auxiliary electrode and a reference electrode, characterized in that there are additional temperature sensors, one of which is installed in the immediate vicinity of the working electrode. 6. The device according to claim 5, characterized in that in the manufacture of the film on the working electrode cellulose acetate, or lavsan, or tracing paper, or cellulose nitrate, or gelatin is used.

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