RU2841951C1 - Method of obtaining sensor coating of biosensor with silicon-on-insulator structure for detecting nucleic acids - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention refers to molecular biology, biotechnology and molecular diagnostics. Disclosed is a method of obtaining a sensor coating of a biosensor with a silicon-on-insulator structure for detecting nucleic acids. Sensor surface of the SOI biosensor is activated with 10% potassium dichromate solution in concentrated sulphuric acid. Further, the activated surface is coated with a bifunctional agent carbonyl diimidazole or thiocarbonyl diimidazole in a concentration range of 0.1 mg/ml to 20 mg/ml in an organic solvent, which leads to formation of a coating containing at least one residue of a modifying agent with thickness of not more than 1 nm. Then, amino-containing phosphorylguanidine oligonucleotide derivatives containing phosphate groups are immobilized on the modified sensor surface, in which a substituted guanidine residue is introduced on the phosphorus atom, in the concentration range of 10^-9 - 10^-6 M in a buffer solution with pH values from 7 to 11, followed by washing with distilled water to form a coating containing at least one oligonucleotide probe, with a thickness of not more than 10 nm.

EFFECT: high sensitivity and specificity of analysis when detecting nucleic acids, as well as high reliability of analysis results.

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Description

The invention relates to the field of molecular biology, biotechnology and molecular diagnostics.

Currently, socially significant diseases are detected mainly by enzyme-linked immunosorbent assay (ELISA) or polymerase chain reaction (PCR) analysis. These systems have a number of significant drawbacks, namely, high requirements for personnel qualifications, the presence of expensive equipment, difficulties in transportation, the duration and labor intensity of the process, and the impossibility of conducting analysis at the point of medical care.

The development of biosensors as an alternative to ELISA and PCR has significantly influenced the development and production of functional biological recognition devices due to the ability to quickly and quantitatively determine biological targets. Biosensors are now considered the gold standard in everyday practice due to their carefully developed manufacturing protocol, the ability to conduct real-time analysis, and a high signal-to-noise ratio. The principle of operation of a biosensor is based on the ability to form a highly specific complex between a probe immobilized on the sensor surface and the analyzed target in the biological sample being studied (saliva, blood, urine, etc.). After that, the biochemical signal is converted into a visually or hardware-recorded signal for subsequent processing. To ensure accurate and reliable signal detection, biosensors must have high sensitivity and specificity to the component being determined. Thus, the sensitivity of a biosensor largely depends on the method used to detect the complex being formed, while the specificity is determined by the nature of the interaction of the immobilized molecules with the detected marker.

The operating principle of modern protein biosensors is based on antigen/antibody interactions. However, their sensitivity is insufficient for use in most analyses, and the creation of monoclonal antibodies and the search for specific receptors are quite difficult. In addition, the use of such biosensors is impossible in early diagnostics, before the onset of an immune response.

In this regard, the development of nucleic acid-based biosensors is the most relevant direction, and the development in the field of genetics and genomics has led to the understanding that biomolecules such as deoxyribonucleic acids (DNA) and ribonucleic acids (RNA) can be used as biomarkers to obtain valuable information in the early diagnosis and monitoring of viral, oncological and genetic diseases (Yong E. Cancer biomarkers: written in blood. Nature. 511 (7511), 524-527 (2014)). Due to the small sample volume and the ability to work with various biological fluids, as well as relatively high sensitivity, nucleic acid (NA)-based biosensors have become popular as tools for the clinical analysis of genetic diseases and various types of tumors, diagnosis of viral and bacterial infections, and for the detection of pathogenic microorganisms.

The development of automated sequencing methods for NC has made it possible to rapidly determine the genome of pathogens, which allows the development of methods for their analysis based on nucleic acids. The automated process of oligonucleotide synthesis provides a fast and inexpensive way to obtain specific nucleotide probes.

One of the modern promising high-tech biosensors is the SOI biosensor - a microwire transistor with a silicon-on-insulator structure (Gao, A., Chen, S., Wang, Y., et al. Silicon Nanowire Field-effect-transistor-based Biosensor for Biomedical Applications. Sens. Mater. 30(8) (2018)). The advantages of such biosensors are high sensitivity, label-free detection, and a small amount of sample during analysis. The sensor surface of the SOI biosensor is a silicon crystal with silicon micro- or nanowires located on its surface. The operating principle of such biosensors is based on a change in the conductivity of the micro- or nanowire when charged molecules are deposited on it, which induce the appearance of compensating charges. The detected molecules in this case play the role of a local virtual gate. As a result of these processes, a current flows between the electrodes located on both sides of the micro- or nanowire. Working with such a supersensitive sensor surface, which responds to any change or appearance of charge, including from low-molecular ions, requires analysis in low-salt or completely salt-free conditions. At the same time, natural nucleic acids are capable of forming stable and specific molecular complexes only in solutions with a certain ionic strength, which complicates their use in biosensors of the silicon-on-insulator type.

Neutral derivatives and analogues of nucleic acids (NA), including those containing a substituted guanidine residue on the internucleotide phosphate, are promising as probes in the development of silicon-on-insulator (SOI) biosensors, in which the formation of a specific signal is associated with a change in the local charge near the sensor surface. The absence of a charge in such NA derivatives allows the formation of hybridization complexes under conditions of low ionic strength of the solution, which leads to an increase in the Debye length, sensitivity and specificity of the analysis on a SOI biosensor compared to native oligonucleotide probes.

The following terms are used in the claimed invention:

Hybridization is the joining of single-stranded complementary nucleic acids (probe/target) into one molecule;

The Debye length is the distance over which the electric field of a single charge extends in a neutral medium consisting of free negatively and positively charged particles;

Probe - a molecule capable of attaching to the substance being analyzed;

Immobilization is the process of fixing the probe to the surface;

SOI biosensor is a biosensor with a silicon-on-insulator structure based on field-effect transistors;

Target - a molecule of the substance being analyzed that is detected using a probe;

Specificity is the ability of a biosensor to correctly detect only a specific target in the presence of other components of the sample;

Sensitivity is the ability of a biosensor to correctly detect the presence of a target in a sample.

A method is known for modifying the surface of a biosensor based on a polysilicon field-effect transistor using 3-aminopropyltriethoxysilane followed by treatment with glutaraldehyde for covalent immobilization of amino-containing phosphate-methylated oligonucleotide probes (Hu, W. P., Tsai, C. C., Yang, Y. S., et al. Synergetic improvements of sensitivity and specificity of nanowire field effect transistor gene chip by designing neutralized DNA as probe. Sci. Rep. 8(1), 12598. (2018)). The known method demonstrates the possibility of detecting only short DNA sequences in 1 mM bis-tris-propane with a detection limit of up to 0.1 fM.

The disadvantage of this method is the covalent immobilization of oligonucleotide probes due to the use of 3-aminopropyltriethoxysilane and glutaraldehyde. When using organofunctional silanes, the process of controlling the coating homogeneity and layer thickness is complicated, which can reach up to 20 nm and not fit into the Debye length, thereby reducing the sensitivity of the analysis. When immobilizing probes on the sensor surface of a nanowire field-effect transistor, it is important to minimize the distance between the analyzed molecule and the surface of the nanowire, since the closest environment of the nanowire has the maximum effect on conductivity. When using 3-aminopropyltriethoxysilane, the probability of charge screening from the target molecule increases. Also, for a fully modified oligonucleotide probe, lower specificity to the complementary target is shown compared to an unmodified probe, and therefore it is necessary to select the optimal number and location of modifications.

A method for label-free DNA detection using peptide-nucleic acid (PNA) probes with a biosensor based on a field-effect transistor made of reduced graphene oxide is known (Cai, B., Wang, S., Huang, L., et al. Ultrasensitive label-free detection of PNA-DNA hybridization by reduced graphene oxide field-effect transistor biosensor. ACS nano. 8(3), 2632-2638 (2014)). The sensor surface of the field-effect transistor is modified by π - π stacking interaction between the pyrene group of 1-pyrenebutanoic acid succinimidyl ester and the graphene surface. Immobilization of PNA probes is carried out by forming an amide bond between the reactive group of the ester, and an ethanolamine solution is used to prevent possible non-specific interactions. The possibility of detecting complementary DNA, DNA with a single nucleotide mismatch and completely non-complementary DNA was investigated. In this technical solution, a detection limit of 100 fM was achieved during hybridization with a short complementary DNA molecule with the possibility of reusing the biosensor.

The known method demonstrates the possibility of detecting only short synthetic DNA molecules. The disadvantages of this method are the duration and high cost of synthesizing PNA probes, poor solubility in water, and the tendency of probes to form parallel complexes with the target, which leads to a decrease in specificity and false results.

The closest analogue to the claimed method - the prototype, is the method for obtaining a sensor coating of a biosensor with a silicon-on-insulator structure for detecting short RNA sequences (Dmitrienko, E., Naumova, O., Fomin, B., et al. Surface modification of SOI-FET sensors for label-free and specific detection of short RNA analyte. Nanomedicine 11(16), 2073-2082 (2016)). The sensor surface of a biosensor with a silicon-on-insulator structure (SOI biosensor) is a silicon crystal on which silicon micro- or nanowires containing a thin film of silicon oxide are located. The method involves preliminary cleaning and preparation of the sensor surface using a mixture of concentrated sulfuric acid and 30% hydrogen peroxide, modification of the sensor surface using the bifunctional reagent carbonyldiimidazole or 3-glycidoxypropyltrimethoxysilane, followed by immobilization of uncharged phosphorylguanidine oligo(2'-OMe)ribonucleotide probes for detection of short RNA targets. The sensitivity of the analysis on the SOI biosensor with the sensor surface obtained using this method was 10 ^-12 M.

The prototype demonstrates the possibility of detecting only short RNA targets, while specific detection of complementary targets, including discrimination of single-nucleotide polymorphisms, is not shown, which significantly reduces the diagnostic potential of the technical solution. The possibility of detecting specific nucleic acid sequences in biological samples is also not demonstrated, which imposes restrictions on the use of the proposed approach in modern diagnostic systems. The absence of all the above-mentioned technical solutions does not allow simultaneous analysis of several targets. In this regard, the proposed biosensor cannot be considered as a multiplex diagnostic system.

The objective of the invention is to develop a method for producing a sensor coating for a biosensor with a silicon-on-insulator structure for detecting nucleic acids.

The technical result of the claimed invention is an increase in the sensitivity and specificity of the analysis on the KNI biosensor when detecting nucleic acids, as well as an increase in the reliability of the analysis results.

The stated task is achieved by the proposed method, which consists of the following.

First, amine-containing uncharged oligonucleotide probes containing a substituted guanidine residue on the internucleotide phosphate are synthesized using the standard protocol of phosphoramidite automated synthesis (Reese, CB. Oligo- and poly-nucleotides: 50 years of chemical synthesis. Org. Biomol. Chem. 3(21), 3851-3868 (2005)) with a change in the oxidation stage of the given structure: 5' - (NPG _m ) _n -NH ₂ - 3', where N is the nucleoside, P is the phosphate group, G is the modification containing a substituted guanidine residue, m is the number of modified phosphate groups, an is the number of monomer units (nucleotides), NH ₂ is the amino group by which the probe is immobilized. The number of modified units can vary from 1 to a completely modified oligonucleotide.

Fig. 1 shows the structures of azides used in the synthesis of amino-containing uncharged oligonucleotide probes, and Fig. 2 shows a diagram of the oxidation stage in the amidophosphite synthesis of oligonucleotides.

The method for introducing such modifications is fully compatible with standard oligonucleotide synthesis protocols; the modification is incorporated by implementing an alternative oxidation step using highly reactive electron-deficient azides (Fig. 1) (Zhukov S. A, Pyshnyi, D. V., Kupryushkin M. S. Synthesis of Novel Representatives of Phosphoryl Guanidine Oligonucleotides. Russ. J. Org. Chem. 3(21), 3851-3868 (2021)). To obtain amino-containing uncharged oligonucleotide probes, the following main stages are carried out:

1. Addition (condensation) of the amidophosphite monomer according to the standard protocol.

2. Modified oxidation stage: treatment with a solution of the corresponding azide in acetonitrile (Fig. 2)

3. Capping and uncapping stages according to standard protocol.

The essence of the claimed method. First, the sensor surface of the SOI biosensor is activated using a reagent that does not damage the surface of the biosensor and leads to the destruction of organic substances, with the formation of hydroxyl groups on the sensor surface. A 10% solution of potassium dichromate in concentrated sulfuric acid is preferably used as an activating reagent. Then, a bifunctional reagent carbonyldiimidazole or thiocarbonyldiimidazole is applied to the activated surface in a concentration range from 0.1 to 20 mg/ml in an organic solvent, leading to the formation of a coating containing at least one residue of the modifying reagent formed during the reaction with hydroxyl groups on the sensor surface of the SOI biosensor, with a thickness of no more than 1 nm. Then, amino-containing uncharged oligonucleotide probes containing a substituted guanidine residue on an internucleotide phosphate are immobilized onto the modified sensor surface in a concentration range of 10 ^-9 - 10 ^-6 M in a buffer solution with pH values from 7 to 11, followed by washing with distilled water to form a coating containing at least one amino-containing uncharged oligonucleotide probe, with a thickness of no more than 10 nm.

The obtained immobilized oligonucleotide probes with the structure 5'-(N-PG _m ) _n -NH ₂ - 3' form hybridization complexes with the sequences of the analyzed nucleic acids.

The use of a KNI biosensor with a coating on the sensor surface obtained by the claimed method allows for recording more reliable, specific and selective results when conducting nucleic acid analysis, compared to native oligonucleotides, without amplification.

The defining significant differences between the proposed method and the prototype are:

1) Carbonyldiimidazole or thiocarbonyldiimidazole is used as a modifier of the sensor surface of the biosensor in a concentration range from 0.1 to 20 mg/ml, while the thickness of the applied coating is no more than 1 nm.

2) To immobilize amino-containing modified oligonucleotide probes, a buffer solution with a pH of 7 to 11 is used. Under these conditions, the amino group remains unprotonated, which has a favorable effect on the reaction proceeding via the addition-elimination mechanism.

3) To prepare the sensor surface, modified oligonucleotides are used, containing from one to 100% of substituted guanidine residues on the internucleotide phosphate, both near the sensor surface and evenly distributed throughout the probe, which allows minimizing the charge on the surface of the SOI biosensor.

4) To detect extended NC targets, 1 to 10 oligonucleotide probes are used per sensor wire of the SOI biosensor, complementary to different areas of the target for its fixation at several points.

5) For hybridization of modified oligonucleotide probes with the NC target, a hybridization buffer is used that provides a pH from 5 to 8.5 and an ionic strength of the solution from 10 ^-7 to 10 ^-1 M, which leads to a significant increase in the signal-to-noise ratio.

The invention can be used to produce biosensors with a silicon-on-insulator structure with immobilized uncharged oligonucleotide probes used to detect nucleic acids by reverse molecular hybridization, including in the diagnosis of genetic, viral and other diseases.

To demonstrate the possibility of detecting NC targets in biological samples, multicomponent blood serum is used in experiments, which makes it possible to use the proposed method for obtaining a sensor coating for a biosensor with a silicon-on-insulator structure for detecting nucleic acids.

The invention is illustrated by the following figures.

Fig. 1 shows the structures of azides used in the synthesis of amino-containing uncharged oligonucleotide probes, where: 1 - 2-azido-1,3-dimethylimidazolidinium hexafluorophosphate, 2 - azido-N,N,N',N'-tetramethylformamidinium hexafluorophosphate, 3 - azidodipyrrolidinocarbenium hexafluorophosphate, 4 - guanyl azide.

Fig. 2 shows a diagram of the oxidation stage in the phosphoramidite synthesis of oligonucleotides using the Staudinger reaction, where 5-0.1 M azide solution in acetonitrile, 25°C, for 30 min (for 1, 2 Fig. 1); 0.25 M azide solution in acetonitrile, 25°C, for 1 hour (for 3 Fig. 1); 0.1 M azide solution in acetonitrile, 25°C, for 1 hour (for 4 Fig. 1).

Fig. 3 shows the structure of phosphorylguanidine derivatives of oligonucleotides containing phosphate groups in which a residue of substituted guanidine is introduced onto the phosphorus atom.

Fig. 4 shows a diagram of the application of the bifunctional reagent carbonyldiimidazole to the sensor surface of the SOI biosensor, followed by the immobilization of amine-containing uncharged oligonucleotide probes containing a substituted guanidine residue on the internucleotide phosphate, where 6 is the concentration of carbonyldiimidazole 10 mg/ml acetonitrile, 25°C, for 2 hours; 7 is the concentration of the oligonucleotide probe 10 ^-6 M, 50 mM carbonate/bicarbonate buffer, pH 10.4, 25°C, for 20 hours.

Fig. 5 shows a scheme for applying the bifunctional reagent thiocarbonyldiimidazole to the sensor surface of the SOI biosensor, followed by immobilization of amine-containing uncharged oligonucleotide probes containing a substituted guanidine residue on the internucleotide phosphate, where 8 is the concentration of carbonyldiimidazole 10 mg/ml acetonitrile, 25°C, for 2 hours; 9 is the concentration of the oligonucleotide probe 10 ^-6 M, 50 mM carbonate/bicarbonate buffer, pH 10.4, 25°C, for 20 hours.

Fig. 6 shows the volt-ampere characteristics of the SOI biosensor with immobilized probes containing a substituted guanidine residue on the internucleotide phosphate, before (left graph) and after (right graph) hybridization with a short DNA target at a concentration of 10 ^-9 M.

Fig. 7 shows the volt-ampere characteristics of the silicon wires of the SOI biosensor with immobilized DNA probes (curves No. 11, 12) and probes containing a substituted guanidine residue on the internucleotide phosphate (curves No. 6, 8), before (left graph) and after (right graph) hybridization with an extended rRNA target at a concentration of 10 ^-9 M. 6, 8, 11, 12 are the numbers of the wires on the SOI biosensor.

Fig. 8 shows the volt-ampere characteristics of the SOI biosensor with immobilized probes containing a substituted guanidine residue on the internucleotide phosphate, before (left graph) and after (right graph) hybridization with an extended RNA target at a concentration of 10 ^-9 M.

Fig. 9 shows the response of the SOI biosensor during the formation of a hybridization complex with an extended RNA target in the concentration range from 10 ^-20 M to 10 ^-7 M at a gate voltage of 15 V.

The method is illustrated in more detail below by the following examples of specific implementation, which do not limit the scope of the invention.

Example 1.

To detect nucleic acid targets, the sensor surface of the SOI biosensor was activated by treatment with 10% potassium dichromate solution in concentrated sulfuric acid for 10 minutes, then washed with water (3×10 ml) and ethanol (1×10 ml), then the sensor surface was modified with bifunctional reagents. Carbonyldiimidazole (10 mg/ml) in acetonitrile (Fig. 4) or thiocarbonyldiimidazole (10 mg/ml) in acetonitrile (Fig. 5) were used as modifying agents and kept for 2 hours, then washed with acetonitrile (2×10 ml) and dried. Immobilization of amino-containing modified oligonucleotide probes was carried out in 50 mM carbonate/bicarbonate buffer, pH 10.4, 0.5 µl at a concentration of 10 ^-6 M per point.

Example 2.

The sensor surface of the SOI biosensor was treated with a 10% solution of potassium dichromate in concentrated sulfuric acid for 10 minutes, then washed with water (3×10 ml) and ethanol (1×10 ml), then modified with carbonyldiimidazole by keeping the sensor surface in a reagent solution with a concentration of 10 mg/ml acetonitrile for 2 hours, then washed with acetonitrile (2×10 ml) and dried. 0.5 μl of 10 ^-6 M per wire of uncharged amine-containing oligonucleotide probes in 50 mM carbonate/bicarbonate buffer, pH 10.4, were immobilized on the modified surface of the SOI biosensor, incubated for 18 hours, followed by washing with water. The detection of a short DNA target (up to 50 nucleotides) was carried out at a concentration of 10 ^-9 M in a 10 mM phosphate buffer solution. The volt-ampere characteristics of the SOI biosensor (Fig. 6) are shown before (left graph) and after (right graph) hybridization with the DNA target.

Example 3.

To demonstrate the increase in the sensitivity of the analysis on the SOI biosensor when using amino-containing uncharged oligonucleotide probes containing a substituted guanidine residue on the internucleotide phosphate, compared to native oligonucleotides as specific probes in the detection of extended sequences of NA targets (more than 400 nucleotides), the sensor surface of the SOI biosensor was treated with a 10% solution of potassium dichromate in concentrated sulfuric acid for 10 minutes, then washed with water (3 × 10 ml) and ethanol (1 × 10 ml), then modified with carbonyldiimidazole, keeping the sensor surface in a reagent solution with a concentration of 10 mg / ml acetonitrile for 2 hours, then washed with acetonitrile (2 × 10 ml) and dried. Three uncharged amine-containing oligonucleotide probes (Fig. 7, curves 6, 8) corresponding to different regions of the target RNA were immobilized on the modified surface of the SOI biosensor, 0.5 μl of 10 ^-6 M per wire in 50 mM carbonate/bicarbonate buffer, pH 10.4, and three native amine-containing DNA probes (Fig. 7, curves 11, 12) corresponding to different regions of the target RNA were immobilized on the other wire in 50 mM carbonate/bicarbonate buffer, pH 10.4, and incubated for 18 hours with subsequent washing with water. Detection of the extended target RNA was carried out at a concentration of ^{10 -9 M} in 10 mM phosphate buffer solution. In Fig. Figure 7 shows the current-voltage characteristics of the SOI biosensor before (left graph) and after (right graph) hybridization with an extended RNA target. It is demonstrated that the current values for wires with immobilized uncharged oligonucleotide probes containing a substituted guanidine residue on the internucleotide phosphate are higher than for wires with DNA probes, allowing for an increase in the efficiency of analysis on the SOI biosensor.

Example 4.

The sensor surface of the SOI biosensor was treated with 10% potassium dichromate solution in concentrated sulfuric acid for 10 minutes, then washed with water (3×10 ml) and ethanol (1×10 ml), then modified with carbonyldiimidazole by keeping the sensor surface in a reagent solution with a concentration of 10 mg/ml acetonitrile for 2 hours, then washed with acetonitrile (2×10 ml) and dried. Three uncharged amine-containing oligonucleotide probes corresponding to different regions of the target RNA were immobilized on the modified surface of the SOI biosensor, 0.5 μl of 10 ^-6 M per wire in 50 mM carbonate/bicarbonate buffer, pH 10.4, incubated for 18 hours, followed by washing with water. The detection of the extended RNA target (more than 400 nucleotides) was carried out in the concentration range of 10 ^-20 - 10 ^-7 M in 10 mM phosphate buffer solution. Figure 8 shows the current-voltage characteristics of the SOI biosensor before (left graph) and after (right graph) hybridization with the extended RNA target and the response of the SOI biosensor at a threshold voltage of 15 V (Fig. 9). The sensitivity of the SOI biosensor at the attomolar level of 10 ^-17 M is demonstrated, which corresponds to 15 molecules of the RNA target in the sample.

The proposed method allows for the detection of both short and long (more than 400 nucleotides) nucleic acids (DNA and RNA targets), and also ensures the detection of even single-nucleotide mismatches. The sensitivity of the analysis performed using the SOI biosensor with a coating on the sensor surface obtained by the proposed method is up to 15 molecules of the NC target in the sample. The SOI biosensor can be reused.

The invention can be used to produce biosensors with a silicon-on-insulator structure with immobilized uncharged oligonucleotide probes used to detect nucleic acids (NA targets) by the reverse molecular hybridization method, including in the diagnosis of genetic, viral and other diseases.

Claims (7)

1. A method for producing a sensor coating for a biosensor with a silicon-on-insulator structure for detecting nucleic acids, comprising preliminary cleaning and activation of the sensor surface using a 10% solution of potassium dichromate in concentrated sulfuric acid, modification of the sensor surface using a bifunctional reagent, followed by immobilization of amino-containing uncharged modified oligonucleotide probes in a buffer solution, characterized in that phosphoryl guanidine derivatives of oligonucleotides containing phosphate groups in which a residue of substituted guanidine with a structure corresponding to the general formula (I) is introduced onto the phosphorus atom are used as the probe: where the substituents R ₁ , R ₂ , R ₃ and R ₄ represent either a hydrogen atom or form a substituted guanidine residue represented by the formula: and the immobilization of the probe is carried out in a buffer solution with a pH from 7 to 11, by applying the probe at a concentration of 10 ^-9 - 10 ^-6 M with the formation of a coating containing at least one modified oligonucleotide probe, with a coating thickness of no more than 10 nm. 2. The method according to claim 1, characterized in that carbonyldiimidazole or thiocarbonyldiimidazole is used as the bifunctional reagent in a concentration range from 0.1 to 20 mg/ml, while the coating thickness is no more than 1 nm. 3. The method according to item 1, characterized in that in order to detect extended NC targets, from 1 to 10 oligonucleotide probes are immobilized on one sensor wire, complementary to different areas of the target for its fixation at several points.