RU223220U1 - Biosensor for indication of biological particles - Google Patents

Abstract

The utility model relates to a biosensor for indicating nano- and micron-sized fine particles (MPs) in suspension: proteins, viruses, bacteria, and can be used in the field of medicine, virology, and biotechnology. The technical result of the claimed utility model is to increase the reliability and detection speed of the biosensor of bioparticles, as well as simplify its operation. A biosensor for indicating biological particles, including a housing (1) and a silicon crystal (2) installed in it with a grounding electrode (3) located on it to ensure electrical contact from below with the suspension of biological particles under study, transistors, above which a measuring chamber (4) is formed. for studying suspensions of biological particles. The transistors contain drains (5, 6) and sources (7, 8), between which there are sensitive elements made in the form of nanowires (9, 10), respectively. The measuring chamber (4) is cup-shaped and axisymmetric, on the inclined side edges of which and in the gap between the body (1) and the silicon crystal (2) a layer (11) of electrical insulating enamel is applied. The grounding electrode (3) is coated with a gold nanolayer on top and is located in the center of the measuring chamber (4) on the silicon crystal (2). The surface of the measuring chamber (4), in contact with the suspension of biological particles under study, with the exception of the grounding electrode (3), is covered with a nanolayer (10) of hafnium dioxide. 1 salary f-ly, 6 ill.

Description

The utility model relates to a biosensor for indicating nano- and micron-sized fine particles (MPs) in suspension: proteins, viruses, bacteria and can be used in the field of medicine, virology, microbiology, biotechnology, toxicology, biology.

Prior Art

Modern biosensors manufactured using SOI (silicon on insulator) technology allow detection of a wide range of low- and high-molecular compounds. Their main advantage is their manufacturing compatibility with standard CMOS technology (CMOS complementary metal oxide semiconductors). This turns biosensors into a universal platform for large-scale or even mass production of highly sensitive disease detection and diagnostic systems.

The CII biosensor design is based on an insulated gate field-effect transistor. SOI semiconductor devices consist of a three-layer substrate with a silicon-dielectric-silicon structure instead of the commonly used monolithic silicon wafers. The operating principle of the biosensor is based on recording the modulation of the current flowing in the source-drain circuit during the process of adsorption of the desired analyte molecules (viruses, bacteria, cells, proteins, toxins, etc.) on the surface of the transistor gate. An adsorbed molecule at the phase interface in the electrolyte-gate system “opens” its own electrical charges. The appearance of these charges and associated electric fields at the phase interface is a fundamental phenomenon. They are combined with the spatial structure, chemical composition, amount of substance, and therefore determine their physicochemical properties in a particular electrolyte-gate system (Feynman R., Layton R., Sands M. Feynman lectures on physics. Electricity and magnetism. Vol. 5. M.: Mir, 1977. 300 pp.). From the above it follows that the properties of the electrolyte-gate system are an important factor in the design and efficiency of the biosensor.

Currently, the development of methods and systems for express detection based on SOI biosensors is carried out in the direction of improving operating characteristics, simplicity of methodological procedures, minimizing material and time costs, the use of mid-level personnel, as well as accessibility to a wide range of users. Developers and designers, based on operating experience, eliminate shortcomings in known biosensors.

A highly sensitive analytical sensor is known - a field-effect transistor with an insulated gate, made using silicon-on-insulator technology (Naumova O.V., Fomin B.I. Interface-State Density in SOI-FET Sensors // WSEAS Transaction on System and Control. - 2018. - V.13. - pp. 514-519.). The sensor is used for qualitative and quantitative analysis of biological and chemical substances. This article (page 516) shows a cross-sectional diagram of the sensor/electrolyte system. From this diagram it follows that the device uses a special separate Pt ground electrode, which connects the potential of the electrolyte under study to the “ground”, structurally located “on top” over the test suspension.

The use of a field-effect transistor (FET)-type biosensors is known (Park J., Nguyen N.N., Woubit A., Kim, M. Applications of field-effect transistor (FET)-type biosensors. // Applied Science and Convergence Technology. - 2014 -Vol.23, No. 2. - pp. 61-71). The same article provides a schematic diagram of a detection biochip. The diagram shows an RE ground electrode, which performs a similar function to a separate Pt electrode. It controls the electrolyte potential, as in the previous version, being located structurally “on top”.

The biosensor presented in the work is known (Malsagova K.A., Pleshakova T.O., Kozlov A.F., Galiullin R.A., Popov V.P., Tikhonenko F.V., Glukhov A.V., Ziborov V.S., Shumov I.D., Petrov O.F., Generalov V.M., Cheremiskina A.A. et al Detection of influenza virus using a SOI-nanoribbon chip, based on an n-type field-effect transistor // Biosensors. - 2021. - V. 11, No. 4. - Paper 119). The detector design also includes a Pt grounding electrode, which controls the potential of the electrolyte in the measuring cell and is immersed into it according to the “top” principle. The detector circuit includes a stirrer, a Pt electrode, a measuring cell, a silicon nanowire sensor chip on an insulator, a chip holder, a measuring cell holder, a peristaltic pump, a waste container, and a ten-channel data acquisition and storage system.

A biosensor for indicating biopathogens is known (RF patent No. 2774307, IPC G01N 27/14; B82Y 15/00, published 06/17/2022). A biosensor for indicating biopathogens includes a silicon crystal in the form of a substrate on which conductive electrodes are located, representing the source and first drain of the transistor, a sensitive element, which is the first nanowire, made in a thin-film structure of silicon-on-insulator on a silicon substrate and placed between two conductive source and drain electrodes to form a transistor channel, dielectric coatings that provide insulation of the conductive electrodes. A pair of lateral electrodes are installed on both sides of the nanowire for dielectrophoretic concentration of viral particles, located with a gap relative to the specified nanowire and with an axial displacement relative to each other. The electrical circuit of the SOI-NP transistor of the specified biosensor, together with a silicon crystal substrate, contains an induced conductivity channel (gate) of the transistor located under the nanowire (gate) of the transistor and a grounding reference electrode structurally located “on top” of the suspension under study.

However, in the above analogues, the arrangement of RE or Pt grounding electrodes in biochips according to the principle of their location above the surface of the suspension under study has a number of disadvantages:

Manual installation of an electrode over the surface of a biosensor is a complex procedure that requires certain skills and high qualifications on the part of the operator. The complexity is determined by the millimeter dimensions of the biosensor crystal, as well as the small volume of the suspension being studied;

subsequent manual insertion of the electrode into the volume of the drop is the next even more complex procedure, requiring higher skills and qualifications on the part of the operator: for example, manual installation of the electrode along the depth of the drop with a volume of 2 μm ³ of the test suspension can lead to uncontrolled mechanical contact of the electrode with the surface of the biosensor, the appearance of scratches and, as a result, complete or partial failure of the biosensor;

the use of an additional automated device for positional dropping of the test suspension onto the surface of the biosensor will lead to a significant increase in cost and complexity of the detection device.

The closest analogue (prototype) is a biosensor for indicating biological particles (RF patent for utility model No. 215954, IPC G01N 27/414, published 01/11/2023). The biosensor includes a housing and a silicon crystal located in it in the form of a substrate, on which ten transistors, their conducting electrodes, sources and drains, and sensitive elements (gates) are located, which are nanowires made in a thin-film silicon-on-insulator structure and placed between two conducting source and drain of each transistor. There are two grounding electrode pads on the silicon crystal, which have areas without insulation to ensure electrical contact from below with the liquid suspension sample under study. The overall dimensions of the crystal are 6×6 mm.

However, such a biosensor is not simple and reliable enough to operate. One of the main drawbacks of the prototype is the two aluminum ground electrodes. Aluminum is an amphoteric metal and can react with both acids and bases, depending on the nature of the reagents. Under normal conditions, it is covered with an oxide film of Al ₂ O ₃ , which is a dielectric and prevents the conduction of electric current. The presence of two electrodes increases the size of the measuring chamber and the volume of the test sample. Ten transistors, taking into account the diffusion coefficient of viral particles, provide the required detection time of 3-5 minutes. During this time, the liquid suspension under study can significantly change its volume due to natural evaporation, which will lead to measurement errors.

The technical result of the claimed utility model is to increase the reliability of the biosensor and simplify its operation.

The specified technical result is achieved by the fact that a biosensor for indicating biological particles, including a housing and a silicon crystal installed in it with a grounding electrode located on it to ensure electrical contact from below with the suspension of biological particles under study, transistors, above which a measuring chamber is formed for studying the suspension of biological particles , and which contain drains and sources, between which there are sensitive elements made in the form of nanowires, respectively, according to the utility model, the measuring chamber is cup-shaped and axisymmetric, on the inclined side edges of which and in the gap between the body and the silicon crystal a layer of electrical insulating enamel is applied, the grounding electrode is coated on top with a nanolayer of gold and is located in the center of the measuring chamber on a silicon crystal, and the surface of the measuring chamber in contact with the suspension of biological particles under study, with the exception of the grounding electrode, is covered with a nanolayer of hafnium dioxide. EP-91 enamel is used as electrical insulating enamel.

The shape of the measuring chamber ensures natural rolling of the test suspension into the geometric center of the crystal surface above the gold electrode and its installation in a standard position for performing the measurement procedure. The cup-shaped shape of the measuring chamber also reduces and simplifies sample preparation for testing. Electrical insulating enamel EP-91 provides reliable waterproofing of the coatings on the side walls of the measuring chamber and the space between the crystal and the housing. The use of hafnium oxide increases the sensitivity of biosensors due to the relative high dielectric constant and thermodynamic stability. The dielectric constant of hafnium oxide is ε~16-40 versus 3.9-4.5 for silicon dioxide. Adsorption of the desired antibody+antigen complex (AT+AG) in the suspension under study with electrical charges distributed in its volume forms polarization and redistribution of charges at the phase section “nanowire surface - test liquid”, as well as in the area under the nanowire. If the polarization (P) in the volume is non-uniform, then its divergence determines the resulting density of all charges in the material which includes those appearing both due to inhomogeneous polarization and due to the density of free charges. Newly formed free charges, distributed in some way in the space of the semiconductor, are an integral part in the source-drain circuit section of the biosensor (Feynman R., Layton R., Sands M. Feynman lectures on physics. Electricity and magnetism. T. 5. M.: Mir, 1977. 300 p., Golosov D.A., Zavadsky S.M., Melnikov S.N., Villa N. Dielectric characteristics of hafnium oxide films. Russian Nanotechnologies. 2017; 12: 63-68.).

The utility model is illustrated by the following graphic materials. In fig. Figure 1 shows the appearance of the biosensor, top view. In fig. Figure 2 shows a cross section of the biosensor along line AA. In fig. Figure 3 shows the electrical circuit diagram for connecting the biosensor. In fig. Figure 4 presents data on the interaction of the antibody with fragments of the viral proteins WEF1 and p24, verified by enzyme immunoassay. In fig. Figure 5 shows the current-voltage characteristics of biosensor No. 1 (a) and biosensor No. 2 (b) before and after immobilization of antibodies. In fig. Figure 6 shows the time dependence of the biosensor current during the detection of a recombinant fragment of the West Nile virus envelope protein (WEF1).

The biosensor for indicating biological particles (Fig. 1) includes a housing (1) and a silicon crystal (2) installed in it with a grounding electrode (3) located on it to ensure electrical contact from below with the suspension of biological particles under study and transistors (20 pieces), above which a measuring chamber (4) is formed to study a suspension of biological particles. The transistors contain (Fig. 2) drains (5, 6) and sources (7, 8), between which there are sensitive elements made in the form of nanowires (9, 10), respectively. The measuring chamber (4) is cup-shaped and axisymmetric, on the inclined side edges of which and in the gap between the body (1) and the silicon crystal (2) a layer (11) of electrical insulating enamel is applied. EP-91 enamel is used as electrical insulating enamel. The grounding electrode (3) is coated with a gold nanolayer on top and is located in the center of the measuring chamber (4) on the silicon crystal (2). The surface of the measuring chamber (4), in contact with the suspension of biological particles under study, with the exception of the grounding electrode (3), is covered with a nanolayer (12) of hafnium dioxide. A gate electrode (13) is installed at the bottom of the silicon crystal (2). On top of the crystal (2) there are induced conduction channels (14, 15) of the first, second and all other transistors. Under the transistors there is a layer (16) of hidden dielectric. The suspension under study contains biological particles (17).

The electrical circuit diagram for connecting the biosensor (18) (Fig. 3) contains a power supply (19) U=0.15V, connected by the negative terminal to the “ground”, which has electrical contact with the sources (7, 8) of all twenty transistors. The positive terminal of the power source (19) is connected to the drains (5, 6) of the transistors individually, through a microammeter (20). The microammeter (20) registers the transistor current during the detection of bioparticles (17). Switching on and off the microammeter (20) in the active measurement mode is carried out by software. The power source (21) (U=0-30 V) is connected to the gate (13) of the biosensor (18).

The measuring chamber (4) with inclined side edges coated with enamel and the grounding electrode (3) coated with gold nanofilm allow:

reduce the time it takes to bring the biosensor into working condition;

quickly ensure reliable electrical contact with the suspension under study with biological objects (17) and install the biosensor (18) in the working position to measure the value of the waste current for detection detection.

The principle of operation of the biosensor is as follows.

The gate (13) of the biosensor (18) is supplied with a voltage in the range U=0-30 V (Fig. 3). The voltage is set so that the initial collector current Ids of the transistor in idle mode is approximately 10 ^-6 A. The collector current is measured by a microammeter (20). The initial Ids transistor collector current is set in the complete absence of the test suspension with bioparticles (17). Next, the test suspension with antibodies with a volume of (0.5) μl is introduced into the measuring chamber (4) onto the surface of the transistor array, which covers the nanowires (9, 10). After some time (about 30-60 seconds), the transistor current comes to a resting state. This level is taken as the initial reference level. All further measurements of the Ids value of the transistor current are made relative to it. Next, the test suspension with biological objects (17) with a volume of 0.5 μl is added, which contains viral particles (bioparticles, antigen). If antibodies are specific to the introduced viral particles (antigen), then stable antibody+antigen complexes are formed between them. These complexes at the phase interface between the suspension under study and the nanowire surface (9, 10) form an induced electric charge, which modulates the conductivity of the induced channel (14, 15) and, as a consequence, the drain currents (5, 6) of all transistors change. The total electrical charge of the antibody+antigen complexes reflects the number of viral particles and the amplitude of the change in the current of the transistor as a biosensor. The positive sign of the electric charge of the complexes increases the conductivity of the induced channel (14, 15) and the magnitude of the transistor current. This reaction is determined by the well-known properties of the transistor (Dyakonov V.P., Maksimchuk A.A., Remnev A.M., Smerdov V.Yu. Encyclopedia of devices on field-effect transistors / Dyakonov V.P. - M.: SOLON-R, 2002 . -512 pp.). Similarly, the negative charge of the complexes reduces the magnitude and amplitude of the transistor (biosensor) current.

Example. Experimental testing of biosensor operation

Testing of the proposed biosensor design with improved performance characteristics was carried out using the example of detection of a recombinant fragment of the West Nile virus envelope protein (WEF1) (Shanshin D.V., Borisevich S.S., Bondar A.A., Porozov Y.B., Rukhlova E.A., Protopopova E.V., Ushkalenko N.D., Loktev V.L., Chapoval A.I., Ilyichev A.A., Shcherbakov D.N. Can Modern Molecular Modeling Methods Help Find the Area of Potential Vulnerability of Flaviviruses. International journal of molecular sciences. - 2022. - 23(14). - 7721) Materials:

biosensor No. 1 and No. 2 of the claimed design having similar parameters and current-voltage characteristics (CV characteristics);

24-channel, ±24 V, 50 mA precision measuring unit PXIe 4163 Relative measurement error of voltage 0.05% + 5 mV and current 0.10% + 500 nA (National Instruments, USA) [17];

antibodies (AT) - monoclonal antibodies specific to viruses of the Flaviviridae family, concentration 1.7 mg/ml (State Research Center for Virus and Virus "Vector" of Rospotrebnadzor) [19];

antigen (AG1) - a recombinant fragment of the ophthalmic protein (E) of the West Nile fever virus (WEF1) of the Flaviviridae family, concentration 89 μg/ml (FBUN State Research Center for Virus and Virus "Vector" of Rospotrebnadzor);

antigen (AG2) - recombinant protein p24 of the human immunodeficiency virus, nonspecific to the monoclonal antibodies used, concentration 0.625 mg/ml (FBUN State Research Center for Virus Virus "Vector" of Rospotrebnadzor);

(3-aminopropyl) triethoxysilane (APTES) (Sigma Aldrich, USA);

3,3'-dithio [sulfosuccinimidyl propionate] (DSSP) (Sigma Aldrich, USA);

hydrogen peroxide (H ₂ O ₂ ) (Sigma Aldrich, USA);

96% ethanol (C ₂ H ₅ OH) (Reakhim, Russia);

phosphate-buffered saline (PSB) (Rosmedbio, Russia);

distilled water (DV).

Control testing of specific (AP+AT) and nonspecific (AG2+AT) interaction was performed using an enzyme-linked immunosorbent assay.

The analysis was performed using the following materials:

Antibodies (AT) - monoclonal antibodies specific to viruses of the Flaviviridae family, concentration 1.7 mg/ml (FBUN State Research Center for Virus and Virus "Vector" of Rospotrebnadzor) [19];

Antigen (ATI) - a recombinant fragment of the envelope protein (E) of the West Nile fever virus (WEF1) of the Flaviviridae family, concentration 0.089 mg/ml (FBUN SSC VB "Vector" of Rospotrebnadzor);

Antigen (AG2) - recombinant protein p24 of the human immunodeficiency virus, nonspecific to the monoclonal antibodies used, concentration 0.625 mg/ml (FBUN State Research Center for Virus Virus "Vector" of Rospotrebnadzor;

Phosphate-buffered saline (PBS) (Rosmedbio, Russia);

TWEEN-20 - polyethylene glycol sorbitan monolaurate (C58H114O26) (Sigma Aldrich, USA);

Goat anti-mouse IgG antibodies (Invitrogen, USA);

Substrate based on 3,3',5,5'-tetramethylbenzidine (Thermo Scientific, USA).

Methodology:

In the wells of a polystyrene plate, 100 μl of antigen was adsorbed in working buffer TBS (0.15 M NaCl; 0.02 M Tris-HCl, pH = 7.4) at 4°C for 16-20 hours. Unadsorbed proteins were removed by washes in PBS containing 0.1% TWEEN-20 (PBS-T). Sites of nonspecific binding were blocked with a 1% solution of casein in PBS-T for 1 hour at T=37°C. Next, antibodies were added in several dilutions and left for 1 hour at T=37°C. Unbound antibodies were removed 3 times washing with PBS-T buffer twice. Then 0.5 μg/ml goat anti-mouse IgG antibodies were added to the wells and incubated for 1 hour at T=37°C and washed with PBS-T. The manifestation of the immune reaction was carried out with a liquid substrate based on 3,3',5,5'-tetramethylbenzidine. The reaction was stopped by adding 1 N HC1. The optical density (OD) of the solution in the well was determined on a Thermo Scientific Varioskan LUX multimodal reader (Thermo Scientific, USA) at a wavelength X = 450 nm.

The ELISA technique is presented in [19].

results

In fig. 4. The interaction of the antibody with fragments of the viral proteins WEF1 and p24, verified by enzyme-linked immunosorbent assay (ELISA), is presented. The results of the analysis show that the antibodies interact well with the recombinant WEF1 protein at various dilutions. As expected, no change in optical density was observed when p24 was used as the antigen, indicating that there was no binding between the recombinant protein and the antibodies. Progress of the experiment

Biosensor No. 1 was used to detect the specific binding signal of monoclonal antibodies specific to viruses of the Flaviviridae family and a recombinant fragment of the envelope protein (E) of the West Nile fever virus (WEF1) of the Flaviviridae family (AT and AG1, respectively).

Biosensor No. 2 was used to detect the nonspecific interaction of monoclonal antibodies specific to viruses of the Flaviviridae family and the recombinant protein p24 of the human immunodeficiency virus (AT and AG2, respectively).

The surfaces of biosensors No. 1 and No. 2 were washed from foreign contaminants using a 10% H ₂ O ₂ solution at room temperature for 10 minutes. After cleaning, their surfaces were washed with distilled water (DV) 3 times. Next, AT was immobilized on the surface of both biosensors by covalent binding. At the first stage, the crystal surface was modified with a 10% solution of APTES in ethanol for 30 minutes at room temperature. After time, excess APTES was removed by washing with ethanol.

The surface amino groups of APTES were activated with 0.1 mM DTSSP in 10 mM phosphate-buffered saline (PBS) for 10 minutes. Next, 10 μl of AT specific for AG1 was applied. After 90 minutes, the surface of biosensor No. 1 and No. 2 was washed with DV and unbound antibodies were removed. After completion of the immobilization process, BAC of biosensors No. 1 and No. 2 was measured, Fig. 4 - current-voltage characteristics of biosensor No. 1 (a) and biosensor No. 2 (b) before and after immobilization of antibodies.

The AX was measured as a function of the drain current (7, 16) of the transistors /dsUb _g ) from the voltage at the gate electrode (2) Ub _g for all transistors of one crystal. The gate voltage was varied in the range Ub _g =0-30 V. As can be seen from Fig. 5, after immobilization of antibodies (curve No. 2), the magnitude of the current Ids is less than before immobilization of antibodies (curve No. 1). This indicates that the antibodies are on the surface of the biosensor. In AX (curve No. 1) made it possible to select the operating point (voltage values supplied to the gate Ub _g ). For both biosensors Ub _g =3 V.

After completion of the immobilization procedure, biosensors No. 1 and No. 2 are ready to conduct targeted experiments for the detection of viral proteins.

Immediately before conducting target experiments, a series of dilutions of suspensions containing antigens were prepared in DV: AG1 - 10 pg/μl, 100 pg/μl and AG2 - 10 pg/μl. Detection of AG1 and AG2 was performed according to the following schemes.

Biosensor No. 1. Detection of a specific biosensor signal during the interaction of AT and AG1.

5 μl of DV was added to the surface of biosensor No. 1 and the initial value of Ids of the drain current was measured to confirm the functionality of the biosensor.

After the current stabilized, the active substance was removed from the surface of biosensor No. 1 and dried. On the prepared and dry surface of biosensor No. 1, a micropipette was used to place 5 μl of an AG1 suspension with a concentration of 100 pg/μl.

The time dependence of the current Ids of biosensor No. 1 is shown in Fig. 6 - time dependence of the biosensor current during the detection of a recombinant fragment of the West Nile virus envelope protein (WEF1) (orange line, specific, biosensor No. 1) and recombinant HIV p24 protein (black line, non-specific, biosensor No. 2) in various concentrations: WEF1 - adding protein to the surface of the biosensor with concentrations of 10 pg/μl, 100 pg/μl; p24 - adding protein to the surface of the biosensor with concentrations of 1 pg/μl, 10 pg/μl, HgO - adding distilled water. Each count on the graph is an average of 20 transistors

As can be seen from the results presented in Fig. 6, the first introduction of the antigen - the envelope protein of the West Nile virus (WEF1) (t ~ 100 s) with a concentration of 100 pg/μl leads to a sharp increase in the drain current from Ids ~ 0.97 A to -1.06 A, (orange line ). Next, a smooth drop in the current value is observed to the initial Id _S ~0.97 A in the time interval (t~100-450) s. This behavior is associated with the observed reaction of the specific interaction of AT + AG1. The formed AT+AGT complexes have a negative effective electric charge at the phase interface of the crystal surface and the test sample. A negative charge on the biosensor gate reduces the current Ids in the source-drain circuit. The smooth change and roughness of the Ids current line reflects the dynamics of the interaction of AT and ATI with each other on the gate surface. After washing the surface of the biosensor with DV (t~500 s), a slight decrease in the current is observed, which is explained by the removal of a small part of AG1 and the destruction of part of the AT + AG1 complexes on the surface of the biosensor. The next addition (t~550 s) of recombinant WEF1 protein with a concentration of 10 pg also leads to a sudden increase in the current Ids. However, no further decrease is observed. This is due to the fact that AT+AG1 complexes with a negative charge on the gate surface are no longer formed.

Conclusion - specific binding of AT+AG2 occurs. Biosensor No. 2. Detection of a nonspecific biosensor signal during the interaction of AT and AG2.

5 μl of DV was added to the surface of biosensor No. 2 and the initial value of Ids of the drain current was measured to confirm the functionality of the biosensor. After the current stabilized, the active substance was removed from the surface of biosensors No. 1 and dried. On the prepared and dry surface of biosensor No. 2, a micropipette was used to place 5 μl of an AG2 suspension with a concentration of 1 pg/μl.

The time dependence of the current Ids of biosensor No. 2 is shown in Fig. 5. black line.

As can be seen from the results presented in Fig. 6, first application of AG2 with a concentration of 1 pg/μl. (t~140 s) practically does not lead to an increase in the drain current. It remains stable at Id _S ~0.92 A (black line). In the interval (t~360-420 s), the surface of biosensor No. 2 was washed with DV. The next addition (t~550 s) of an AG2 suspension with a concentration of 10 pg/μl leads to a sudden increase in the current Ids. However, no further decrease is observed. There is a stabilization of the current value at the level of Id _S ~0.95 A. There is no characteristic slight change in the current. The process of dynamic equilibrium of the interaction of AG2 with the gate surface is observed. The influence of AG2 on the current value of biosensor No. 2 is limited by their remote position relative to the surface in a state of equilibrium. Prevent AT.

Conclusion: nonspecific binding of AT+AG2 does not occur.

Claims (2)

1. A biosensor for indicating biological particles, including a housing (1) and a silicon crystal (2) installed in it with a grounding electrode (3) located on it to ensure electrical contact from below with the suspension of biological particles under study, transistors, above which a measuring chamber is formed ( 4) to study a suspension of biological particles, and which contain drains (5, 6) and sources (7, 8), between which there are sensitive elements made in the form of nanowires (9, 10), respectively, characterized in that the measuring chamber ( 4) is made cup-shaped and axisymmetric, on the inclined side edges of which and in the gap between the body (1) and the silicon crystal (2) a layer (11) of electrical insulating enamel is applied, the grounding electrode (3) is covered with a gold nanolayer on top and is located in the center of the measuring chamber ( 4) on a silicon crystal (2), and the surface of the measuring chamber (4), in contact with the suspension of biological particles under study, with the exception of the grounding electrode (3), is covered with a nanolayer (12) of hafnium dioxide. 2. Biosensor according to claim 1, characterized in that EP-91 enamel is used as electrical insulating enamel.