RU2808559C1 - Method of solid-phase catalytic analysis of analyte content in sample using silver nanowires - Google Patents

Abstract

FIELD: clinical diagnostics.

SUBSTANCE: following is disclosed: a method of detecting an analyte in a sample including at least the following stages: i) immobilization on the surface of a solid phase of a receptor against the said analyte or a molecular complex including an analogue of the said analyte, ii) incubation with the said solid phase of the said sample, iii) incubation with the said solid phase of a suspension of silver nanowires conjugated with an antibody against the said analyte, iv) removing unbound silver nanowires from the sample, v) incubating with the said solid phase a solution of hydrogen peroxide and a chromogenic substrate selected from the group: tetramethylbenzidine, o-phenylenediamine, diaminobenzidine; vi) measuring absorbance to determine the content of said analyte in the said sample from a calibration curve.

EFFECT: invention provides a simpler and more accessible measurement of the analyte content in a sample without the use of expensive equipment with the possibility of signal amplification due to the catalytic activity of nanoparticles, as well as the use of nanomaterials as registered labels, allowing them to be synthesized on a large scale and characterized by stability during storage.

20 cl, 6 dwg, 11 ex

Description

Field of technology to which the invention relates

The invention relates to the field of clinical diagnostics, namely technologies for the detection of various analytes in a sample using catalytic analysis of the analyte content in the sample using nanotags. Such technologies are useful for carrying out precise measurements in the field of disease diagnosis, monitoring of various parameters and conditions of the body, molecular and cellular biology, nanotechnology, chemistry, etc.

Description of the Prior Art

The exponential development of basic life sciences and biomedical technologies has led to the need for modern methods for the accurate diagnosis of various molecules, consisting of the use of high-throughput assays with miniature labels used for detection [1–3].

Various enzymes are commonly used as detection probes in immunobiological assays due to several advantages such as high sensitivity, versatility, and high availability. Among them, horseradish peroxidase and alkaline phosphatase are the most common enzymes used to produce signal in colorimetric immunoassays [4,5]. These enzymes catalyze the oxidation of several substrates such as 3,3',5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD) or diaminobenzidine (DAB) during a chromogenic reaction along with H ₂ O ₂ [6,7] . However, protein molecules as labels in immunoassays also have a number of disadvantages [8,9]. In particular, their protein origin makes them susceptible to degradation by microorganisms, requiring the use of cryopreservatives such as sodium azide to increase shelf life. At the same time, the use of additional stabilizers and cryopreservatives, such as various sulfides, cyanides and azides, can significantly suppress enzyme activity and/or distort the analysis results. Moreover, expensive technologies for protein isolation and purification necessitate the production of new assay labels that are stable at room temperature, not subject to degradation, and do not require the use of preservatives [10].

Hydrogen peroxide is a highly reactive oxidizing agent and its presence in solutions with protein and/or peptide molecules can have a significant effect on the activity of these molecules. In particular, irreversible chemical modification of various functional groups of enzymes leads to a significant loss of stability and a decrease in the efficiency of enzymes of various groups. Thus, in particular, Stadtman and Levin conducted a detailed analysis of the disruption of the functional activity of proteins under the influence of hydrogen peroxide. It has been shown that among the processes in which the activity of hydrogen peroxide is involved in relation to proteins and their derivatives are hydroxylation and nitration of the side chains of aromatic amino acids, the addition of nitric oxide to sulfhydryl groups, the conversion of a number of amino groups into carbonyl derivatives, sulfoxidation of sulfur-containing amino acids, as well as chlorination alpha and epsilon amino groups of protein chains.

The formation of oxidation products, in particular hydrogen peroxide, often significantly harms the biotechnological process and the removal of hydrogen peroxide is of great biotechnological importance. Thus, in various bioreactors containing oxidase as an active component, it is necessary to remove oxidation products - namely, hydrogen peroxide through its catalytic decomposition from the mixture during the reaction to prevent premature inactivation of the enzyme and thereby increase the service life of bioreactors.

In recent decades, nanostructures of noble metals such as gold, silver and platinum have been the most intensively studied as markers/tags in various analytical detection methods such as enzyme-linked immunosorbent assay (ELISA), fluorescence in situ hybridization [11] – FISH [ 12], flow cytometry [13–16], lateral immunoassays [17], phase-sensitive nanoFourier transforms [18], methods for analyzing the assembly/disassembly of colloidal structures [19–23], chemiluminescent [24] and electrochemical [25] analyzes and many others analytical methods [26–36]. In particular, silver, Ag and gold, Au nanoparticles, which have unusual physical and chemical properties, can serve as labels or signal amplifiers in a wide range of biological applications and are considered effective tools for highly sensitive, rapid and cost-effective applications [37,38]. In particular, the property of localized surface plasmon resonance (LSPR), inherent in nanoparticles of noble metals, determines the intense color and effective scattering of light by such nanoobjects. LSPR is the collective oscillation of conduction band electrons at a metal-dielectric interface that is in resonance with the oscillating electric field of incident light, which produces energetic plasmonic electrons through non-radiative excitation. These fluctuations depend on the properties of nanoparticles, namely on composition, shape, size, surface coverage, stage of aggregation and other parameters. Nanoparticles with LPPR properties are effective tools for immunoassays [39,40] and other biomedical applications [18,41], and the use of such labels can improve the detection capabilities of analytes that are implemented by standard fluorescent or colorimetric methods.

Numerous assays have been developed using spherical nanoparticles possessing LSPR to detect molecular compounds with high sensitivity, especially using gold and silver tags [42]. The difference in relative dielectric constants results in the most efficient scattering to absorption ratio of Ag compared to Au particles [43,44]. However, although silver nanostructures are superior to gold nanostructures in scattering and low ohmic losses, Au particles are generally preferred due to their stability, oxidation resistance, and biocompatibility [45–47].

However, when properly stabilized by external coatings of other noble metals or polymers, Ag particles are widely used for biosensing applications. For example, an enzyme-linked immunosorbent assay method was developed based on the change in the intensity of the LPPR absorption peak that occurs due to the growth of spherical silver nanoparticles [48]. An ELISA analysis was also developed based on surface-enhanced Raman scattering based on the aggregation of functionalized silver particles, which allows recording the shift of the Raman signal [49]. In addition to LPPR properties, silver nanoparticles have been shown to increase the activity of horseradish peroxidase used in ELISA tests [50] or have their own peroxidase-like activity [51].

While spherical plasmonic nanostructures with intrinsic enzymatic activity are most often used for enzyme-linked immunosorbent assay (ELISA), the use of anisotropic structures may be more interesting in terms of exploiting other modalities for detection, not just the uptake of colored substrates. For example, the use of glucose oxidase-catalyzed oxidation of glucose by silver triangular prisms to produce hydrogen peroxide leads to a change in the shape of the plasmonic structure and a shift in the SPR peak [52]. However, round silver nanoparticles usually require various stages in the synthesis process to modify the surface to obtain time-stable structures.

From publication RU2166751C1, the closest solid-phase immunoassay using nanoparticles is known, in which magnetic nanoparticles are used as detectable nanoparticles, the signal from which is read using a special magnetometer.

However, the known method has the following limitations: reading requires the use of expensive equipment; the method does not have signal amplification, which can reduce the possible detection limit of the system.

Thus, the required technical result is a simpler and more accessible (cheap) measurement of the analyte content in a sample without the use of expensive equipment, with the possibility of signal amplification due to the catalytic activity of nanoparticles, as well as the use of nanomaterials as registered labels, allowing them to be synthesized on a large scale and characterized by storage stability.

Summary of the invention

The purpose of the present invention was to develop particles - silver nanowires and implement with their help: A method of solid-phase immunoassay using silver nanowires.

To solve the above and other goals that are obvious to a qualified specialist, the following method was proposed:

A method for detecting an analyte in a sample, comprising at least the following steps:

i) immobilizing on the surface of the solid phase a receptor against said analyte or a molecular complex including an analogue of said analyte,

ii) incubating with said solid phase of said sample,

iii) incubating with said solid phase a suspension of silver nanowires conjugated with an antibody against said analyte,

iv) incubating with said solid phase a solution of hydrogen peroxide and a chromogenic substrate selected from the group: tetramethylbenzidine, o-phenylenediamine, diaminobenzidine;

v) measuring the absorbance of the resulting mixture to determine the content of said analyte in said sample from a calibration curve.

Dictionary

Unless otherwise defined, the following terms have the following meanings when interpreting the claims and description of the invention:

1) “immunochromatographic test” - placing a sample, which may contain the target nucleotide sequence, on a test strip made of chromatographic material, followed by lateral movement of the sample along the test strip due to capillary forces and its binding to various reagents placed on the test strip .

2) “test strip” – a chromatographic medium in which the analysis can be carried out. The test strip contains an "application zone" for applying the sample and a "binding zone" (test line) containing an immobilized binding reagent or binding nucleotide sequence capable of binding and immobilizing the detected nucleotide sequence. The binding zone typically has a significantly smaller surface area than the test strip and contains an immobilized binding reagent or an immobilized binding nucleotide sequence. The binding zone may be in the form of a point, line, curve, stripe, or a pattern of points, lines, curves, stripes, or a combination thereof. Typically, the direction of movement of the sample along the test strip crosses the binding zone. For this invention, it is preferable to generate a signal in the binding zone in the form of a clearly defined pattern, sharply different from the signal generated in other parts of the test strip. For example, the binding zone may take the form of an abbreviation of the name or names of the analyte or analytes in the test sample.

3) “complementarity” or “complementary” is used in relation to nucleic acids (for example, nucleotide sequences) and refers to the well-known rule of the formation of paired complexes of the nitrogenous bases adenine-thymine and cytosine-guanine. For example, for the sequence 5′-TCGG-3′, the complementary sequence is 3′-AGCC-5′. If all nucleotides in the sequences comply with the principle of complementarity, then they speak of complete complementarity, as in the example given. In the case when only part of the nucleotides are consistent with the principle, then complementarity is called partial. For example, the sequence 5′-GGCCAATCG-3′ is partially complementary to the sequence 3′-C-GGCTAACC-5′. The number of nucleotides corresponding to the principle of complementarity in sequences determines the degree of their complementarity, which affects the efficiency of nucleic acid hybridization. The term “significant complementarity” refers to a pattern that can hybridize to one or more strands of the target nucleic acid under high stringency conditions (see below), or, for example, in a PCR buffer heated to 95 ^° C and cooled to room temperature.

4) The term “binding partner” refers to a pair of molecules and/or particles capable of recognizing each other (in whole or in part) and forming both covalent and non-covalent bonds. There are a number of commonly used binding pairs, as well as structures based on such pairs. Among them are systems based on the interaction of antigen/antibody, hapten/antihapten, dioxygenin/antidioxygenin F(ab')2, folic acid/folate-binding protein, biotin/streptavidin, biotin/avidin, protein A(G)/immunoglobulins , complementary segments of nucleic acids, virus/cellular receptor, lectin/carbohydrate, enzyme/inhibitor, enzyme/substrate. Systems are also known in which binding partners form covalent bonds due to the presence of amino-reactive groups in such partners, such as succinimidyl ethers, isothiocyanates, sulfonyl halides, or sulfhydryl reactive groups, including derivatives of haloacetates and maleimides. Other binding partners may include halogens, steroids and 2,4-dinitrophenyls.

5) The term “nucleic acid” means a polymer or oligomer consisting of nucleotides or compounds that mimic them. The term also refers to oligonucleotides that have sections of the chain that do not occur in nature, but exhibit the same properties as naturally occurring oligonucleotides. It will be apparent to those skilled in the art that a wide variety of assays for detecting target nucleic acid sequences can be performed in accordance with the present invention. The term "nucleotide" as used herein refers to ribonucleotides, deoxyribonucleotides, or nucleotide analogues having a chemical modification in one or more of the nucleotide building blocks (nitrogen bases - purine or pyrimidine, sugar, phosphates). Modifications can be, for example, carried out in the fifth position of pyrimidine, or the eighth position of purine, in the exocyclic amino group of cytosine, by replacing uracil with 5-bromo-uracil, replacing the hydroxy group in the 2´-position of sugar with a halo group (or with R, OR, SH , H, SR, NR ₂ , NH ₂ , NHR, CN). The term also refers to ribonucleic acids found in living organisms, either with a substituted base (xanthine, ionosine, etc.) or sugar (2´-methoxyribose, etc.). Modification of the phosphodiester bond is also possible, for example chain formation with phosphorothionates, methylphosphonates and peptides. “Nucleic acid” can mean a single- or double-stranded molecule consisting of monomers (nucleotides) containing moieties of sugars, phosphates and purines or pyrimidines. In plants, animals, lower eukaryotes and bacteria, “ribonucleic acid” (RNA) performs translation functions, i.e. serves as a transmitter of genetic information stored in “deoxyribonucleic acid” (DNA). The term "fragment" refers to a specific part of a DNA sequence.

6) The analysis method and its hardware implementation related to this invention can detect the target nucleic acid or other substances (collective term - analyte) contained in various samples. The term "sample" here refers to any liquid potentially containing analytes. The sample may be obtained from human or other animal tissues or fluids (blood/serum, urine, saliva, tears, sputum, sweat, mucus, stool), tissue culture samples, histological samples, biopsy samples, etc. The sample can also be obtained from agricultural products, bacteria, viruses, or their waste and processing products. Also, a sample can be various garbage and waste, their processed products, drinking and waste water, food products in processed or raw form, air, etc. If this invention is implemented to detect the desired nucleic acid sequences, it can be used to analyze genetic abnormalities and many other diseases, serve to monitor the progress of treatment of infectious diseases, as well as for many other applications.

7) The term “recognition oligonucleotide” means an NA containing a sequence complementary to the analyzed (target) nucleic acid (analyte).

8) The term “signal-generating substrate” refers to a substance capable of interacting with a signaling receptor and generating a detectable signal. For example, ABTS/TMB for peroxidase, BCIP/MBT for alkaline phosphatase, light for fluorescent compounds.

9) The term “signal receptor”, “signal-produced reporter”, “reporter” means a substance capable of detecting the target analyte through the release of energy or by enzymatic activity, for example, fluorescent and chemiluminescent fragments, particles, enzymes, radiolabels, light-emitting domains or molecules.

10) The term “porous material” or “chromatographic material” means a material having pores of at least 0.1 μm, preferably at least 10.0 μm, of sufficient size for the movement of liquid media through it due to capillary forces.

11) The term “signal-producing system” denotes a set of reagents necessary to generate a detectable signal under the influence of external influence. Typically, such systems generate a signal that can be detected by external means - for example, by measuring electromagnetic radiation, or by visual observation. Basically, such systems consist of a chromophore substrate and an enzyme that converts the substrate into a dye that absorbs electromagnetic radiation of a certain range, phosphorescent or fluorescent substances.

12) The location of the hairpin from the first or second end of the recognition oligonucleotide at a distance of X nucleotide bases implies the following. The site of binding of a nanoparticle or signal receptor with a recognizing oligonucleotide should be considered the nucleotide with which their bond is formed (preferably covalent). Moreover, if this nucleotide is the first in the hairpin (i.e. it forms a non-covalent bond with another nucleotide), then the distance X = 0. If the hairpin starts from a neighboring nucleotide, then X = 1, etc.

13) By the first/second end of a single-stranded recognition oligonucleotide, we do not mean the actual end of the molecule, but one or the second side of it. Those. the nucleotide base of the recognition oligonucleotide corresponding to the binding site of the signal receptor or nanoparticle is preferably located at the edge of the oligonucleotide, but may not be the extreme base in the oligonucleotide. By the nucleotide base of the recognition oligonucleotide corresponding to the binding site of the signal receptor (or nanoparticle), is meant the nucleic acid monomer nucleotide (including nucleoside, saccharide and phosphoric acid residue) to which the signal receptor (or nanoparticle) is bound.

14) In this specification, the terms “receptor” and “ligand” are used interchangeably and can be used to refer to individual molecules in the above-described concept of “binding partners”. So, for example, if often in the literature in pairs streptavidin-biotin, antibody-antigen, the receptor means streptavidin and antibody, and the ligand means biotin and antigen, then in the description of this invention the receptor can mean both streptavidin with antibody and biotin with antigen (in this case, the ligand will be biotin with antigen and streptavidin with antibody, respectively).

15) Nanoparticle - means a particle in a wide range - from 1 nm to 100 microns, preferably from 1 nm to 10 microns, preferably from 1 nm to 1 microns, preferably from 1 nm to 100 nm. In this case, nanoparticles can be metallic, polymeric, core-shell structures, etc.

16) The term “nanoparticles” refers to supramolecular structures that consist of more than one molecule. At the same time, although objects up to 100 nm are often referred to as nanoparticles, within the framework of the description of this invention objects larger than 100 nm are also meant - preferably up to 5 μm, but in some cases a size up to 100 μm is acceptable. Thus, by nanoparticles we mean both nanoparticles and microparticles, microspheres, etc. In addition, we mean particles with the mentioned dimensions, both in all dimensions and in at least one.

17) Therapeutics/molecules/agents. Therapeutic molecules/agents can be understood as substances, substances, molecules, supramolecular agents, nanoparticles, microparticles, nanoagents, microagents, formulations, dosage forms, etc., which are used both for therapy and diagnosis of various diseases or conditions of the body or patient, incl. include tracers, contrast agents, controlled release agents, paramagnetic and magnetic agents, cytotoxic agents, and the like.

18) A “label” or “detectable element” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical or other physical means. For example, commonly used labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in ELISA), biotin, digoxigenin or haptens and proteins or other entities that can be detected, for example, by incorporating radioactive tags into a peptide or antibody that specifically reacts with the target peptide. Any method known in the art for conjugating an antibody to a tag can be used, for example, using the methods described in (Hermanson, G. T., Academic press, 2013).

19) As used herein, the term “pharmaceutical” refers to a composition that is useful in treating a disease or symptom of a disease.

20) The term "treatment" or "therapy" (treating, treatment, therapy) refers to any sign of success in treating or improving a disease, injury, pathology or condition, including any objective or subjective parameters such as reduction; remission; reducing symptoms or slowing the deterioration of a patient's condition, pathology or condition; slowing down the rate of degeneration; which makes the final stage of the disease less painful; improving the patient's physical or mental well-being. Treatment or improvement of symptoms may be based on objective or subjective parameters; including results of physical examination, neuropsychiatric examinations, and/or psychiatric evaluation. For example, certain techniques presented here can successfully treat cancer by reducing the incidence of cancer and inducing cancer remission. The term “treatment” and its extensions include the prevention of injury, pathology, condition or disease.

21) "Disease" or "condition" refers to the health condition of a patient or subject to be treated with the various drugs, dispersions, or other methods presented herein.

22) As used herein, the term "contrast agent"/"contrast agent" refers to a composition that, when administered to a subject, improves the detection limit or detection ability of a physical medical imaging technique, technique, or device (e.g., radiographic instrument, X-ray, CT, PET, MRI (MRI), ultrasound and other methods). The contrast agent can enhance the magnitude of signals associated with various structures or fluids within the subject.

23) "Patient" or "subject in need thereof" refers to a living organism suffering from or possibly susceptible to a disease or condition that can be treated by administration of a drug, pharmaceutical composition or agent as defined herein. Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goats, sheep, cows, deer and other non-mammalian animals. In particular, the patient may be human.

24) As used herein, the term “controlled release agent” or “externally activated agent” refers to a compound that, when combined with a composition described herein (including embodiments), releases the composition at a controlled rate under conditions of use (in a patient). Such compounds include high molecular weight, anionic mucomimetic polymers, gelling polysaccharides and fine drug carrier substrates, and others. These components are discussed in more detail in US Pat. No. 4,911,920; 5403841; 5212162; and 4,861,760. The entire contents of these patents are incorporated herein by reference in their entirety for all purposes. In some embodiments, the implementation of the "controlled release component" may be a sustained release, sustained release, sustained release, timed release, timed release, controlled release, modified release, or sustained release compound. In some embodiments, the compound disintegrates at the site of administration (eg, subcutaneous, intravenous) or within the digestive tract (eg, stomach, intestines) if the compound and composition are administered orally. In some embodiments, the controlled release component is a polymer and may be referred to as a "controlled release polymer." In addition, an externally activated agent can be activated (for example, release a therapeutic agent or bind to a target) as a result of exposure to an external magnetic/electric field, ultrasound, light, electron beam, as a result of the radioactive decay of any atoms, as a result of interaction with chemical microenvironment, etc.

25) As used herein, the term “paramagnetic agent”/“paramagnetic agent” refers to a paramagnetic compound useful in diagnostic imaging techniques (eg, magnetic resonance imaging) as a contrast agent. In some embodiments, the paramagnetic agent includes gadolinium, iron oxide, iron platinum, or manganese.

26) Variants of cytotoxic agents (agents, toxins) include, but are not limited to: ricin, doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracindione, actinomycin D, diphtheria toxin, exotoxin Pseudomonas (PE) A, PE40, abrin and glucocorticoid and other chemotherapeutic agents, as well as radioisotopes. Suitable detectable markers include, but are not limited to, a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator, or an enzyme.

27) A partial list of immunoassays includes: competitive and noncompetitive formats, enzyme-linked immunosorbent assays (ELISAs), microdot assays, Western blots, gel filtration and chromatography, immunochromatography, immunohistochemistry, flow cytometry or fluorescently labeled cell sorting ( FACS), microarrays, etc. Such methods can also be used in situ, ex vivo, in vitro or in vivo, for example for diagnostic imaging.

It should be noted that throughout the application, alternatives are written in Markush groups, for example, each therapeutic agent position may contain more than one possible therapeutic agent. In particular, it is intended that each member of the Markush group should be considered separately, thereby containing a different embodiment, and the Markush group should not be read as a whole.

Detailed Description of the Invention

Embodiments of the present invention will now be described more fully with reference to the accompanying drawings, which show some, but not all, embodiments of the invention.

Within the framework of this invention it is proposed:

A method for detecting an analyte in a sample, comprising at least the following steps:

i) immobilizing on the surface of the solid phase a receptor against said analyte or a molecular complex including an analogue of said analyte,

ii) incubating with said solid phase of said sample,

iii) incubating with said solid phase a suspension of silver nanowires conjugated with an antibody against said analyte,

iv) removing unbound silver nanowires from the sample,

v) incubating with said solid phase a solution of hydrogen peroxide and a chromogenic substrate selected from the group: tetramethylbenzidine, o-phenylenediamine, diaminobenzidine;

vi) measuring the absorbance of the resulting mixture to determine the content of said analyte in said sample from a calibration curve.

In addition, a method in which said silver nanowires have a length from 1 μm to 20 μm.

In addition, a method in which said silver nanowires have a diameter of 10 nm to 100 nm.

In addition, a method in which said nanowires have the property of localized surface plasmon resonance.

In addition, the method in which said nanowires have the property of colloidal stability.

In addition, the method in which said incubation is carried out from 1 to 150 minutes.

In addition, a method in which said incubation is carried out at a temperature of 4 to 30 degrees Celsius.

In addition, a method in which said calibration curve is measured in parallel for samples with a known pH value.

In addition, a method in which the concentration of hydrogen peroxide in said mixture is up to 9 µmol/liter.

In addition, a method in which the concentration of said chromogenic substrate in said mixture is from 1 to 10 mmol/liter.

In addition, a method in which the concentration of said silver nanowires in said mixture is from 10 to 100 mg/liter.

In addition, a method in which said measurement of the optical density of the mixture is carried out at a wavelength of 450 nm to 530 nm.

In addition, a method for the catalytic oxidation of chromogenic substrates in a sample for in vitro diagnostic purposes, comprising:

i) mixing a solution of said sample with a suspension of silver nanowires,

ii) adding hydrogen peroxide solution to the resulting mixture.

In addition, the method in which said silver nanowires have a length of from 1 μm to 3 μm, from 3 μm to 6 μm, from 6 μm to 9 μm, from 9 μm to 12 μm, from 12 μm to 15 μm, from 15 μm to 20 microns.

Moreover, a method in which said silver nanowires have a diameter of 10 nm to 20 nm, 20 nm to 30 nm, 30 nm to 40 nm, 40 nm to 50 nm, 50 nm to 60 nm, 60 nm up to 70 nm, from 70 nm to 80 nm, from 80 nm to 90 nm, from 90 nm to 100 nm.

In addition, the method in which said incubation is carried out from 1 to 15 minutes, from 15

min to 30 min, from 30 min to 60 min, from 60 min to 120 min, from 120 min to 150 min.

Moreover, the method in which said incubation is carried out at a temperature of 4 to 10 degrees Celsius, 10 to 15 degrees Celsius, 15 to 30 degrees Celsius.

In addition, a method in which the concentration of said chromogenic substrate in said mixture is from 1 mmol/liter to 3 mmol/liter, from 3 mmol/liter to 6 mmol/liter, from 6 mmol/liter to 10 mmol/liter.

In addition, a method in which the concentration of said silver nanowires in said mixture is from 10 mg/liter to 20 mg/liter, from 20 mg/liter to 30 mg/liter, from 30 mg/liter to 40 mg/liter, from 40 mg/liter to 50 mg/liter, from 50 mg/liter to 60 mg/liter, from 60 mg/liter to 70 mg/liter, from 70 mg/liter to 80 mg/liter, from 80 mg/liter to 90 mg /liter, from 90 mg/liter to 100 mg/liter.

In addition, a method in which said measurement of the optical density of the mixture is carried out at a wavelength of 450 nm to 470 nm, 470 nm to 490 nm, 490 nm to 510 nm, 510 nm to 530 nm.

In addition, a method in which said calibration curve is measured in parallel for samples with a known content of molecules of said analyte.

In addition, a method in which the concentration of hydrogen peroxide in said mixture is from 0.2 µmol/liter to 300 µmol/liter.

In addition, the method in which the antibody used is distinguished by its ability to displace the analyte from the affinity matrix.

In addition, a method in which said analyte is a small molecule.

In addition, a method in which said analyte is a protein.

In addition, a method in which said analyte is a nucleic acid.

In addition, the method in which said solid phase is a multi-well plate.

In addition, a method in which said test strip contains a nitrocellulose membrane.

In addition, a method in which said brightness estimation is carried out using optical detection methods.

Moreover, a method in which said brightness evaluation is carried out visually.

In addition, a method in which detection of the amount of nanoparticles of said silver nanowires retained on the test line is carried out within less than 20 minutes after the start of the analysis.

In addition, a method in which said antibody is monoclonal.

In addition, the method in which said antibody is polyclonal.

In addition, a method in which the progress of the reaction is further assessed by measuring the optical density of said mixture.

In addition, a method in which said analyte analogue is a carrier protein conjugated to analyte molecules.

Silver nanowires (AgNWs) synthesized through the polyol process do not require any stabilization and can be used as is for various applications without any special preservatives or complex buffer systems [53–56].

Here we show that silver nanowires have pH-dependent peroxidase-like activity toward three commonly used peroxidase substrates—3,3',5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), and 3,3'-diaminobenzidine ( DAB) in both precipitating and non-precipitating modes. The properties of SNPs as enzyme-like detection tags have been demonstrated in the ELISA format for the detection of vitamin folic acid (FA). Moreover, the LSPR property of functionalized silver nanowires (SNPs) allows the use of SNPs as optical labels in immunochromatography without the addition of any chromogenic substrate. This optical property was demonstrated in the detection of folic acid by an immunochromatographic test in the working concentration range of 0.1 µM ÷ 100 µM.

Silver nanowires were synthesized from _AgNO3 salt via a glycerol-mediated polyol process using polyvinylpyrrolidone (PVP) and NaCl as in [57], with some modifications. As a result of the synthesis process, silver nanowires (SNPs) were obtained.

The morphology of the synthesized SNPs was studied using scanning electron microscopy (SEM) with an in-beam detector of secondary electrons at an accelerating voltage of 15 kV. SEM images of SNPs are presented in Fig. 1a-c at different magnifications. The length of the original SNP was found to be 5.5 ± 2.2 μm and the diameter to be 46 ± 8 nm according to SEM image processing using ImageJ software ( Figure 1 d).

It should be emphasized that this synthesis is completely safe as it does not require the use of any toxic reducing agents such as sodium borohydride, CTAB or the commonly used ethylene glycol. Instead, biocompatible glycerol, polyvinylpyrrolidone, and NaCl are used to provide a one-pot, high-yield synthesis (14 g/L SNP) and to obtain a salt-stable (e.g., 1 M NaCl) SNP.

The synthesized SNPs were stable for over two years at room temperature in water without the use of any special preservatives or any light protection, making these structures ideal for the development of various commercial assays that place significant restrictions on label storage conditions and other components of immunoassays.

Peroxidase-like activity of silver nanowires

The peroxidase-like activity of silver nanowires was investigated using standard chromogenic substrates, namely 3,3′,5,5′-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), and 3,3′-diaminobenzidine (DAB). AgNWs were incubated with TMB, OPD, DAB and H ₂ O ₂ and these substrates were shown to produce bright colors consistent with the substrate used in the assay with moderate concentrations of SNP (Figure 2a).

Additionally, it should be noted that both TMB and DAB substrates can serve as precipitating substrates, for example, for Western blot studies, while OPD serves as a non-precipitating soluble substrate for applications, for example, in ELISA or cell-based assays.

H ₂ O ₂ -dependent peroxidase-like activity was studied spectrophotometrically for SNP. Absorbance was recorded at 450 nm for solutions with OPD after 10, 30, 60, 90, 120, 150 min of incubation (Fig. 2b).

H ₂ O ₂ varied from 0.8% to 0.00076%, thus covering three orders of magnitude of concentration. Linear regression curve fitting showed that the dependence of absorbance at 450 nm on H ₂ O ₂ concentration is a linear function with R ² = 0.9757 for a 10-minute curve, R ² = 0.9881 for a 30-minute curve, R ² = 0.9915 for the 60-minute curve, ^R2 = 0.9938 for the 90-minute curve, ^R2 = 0.9960 for the 120-minute curve, ^R2 = 0.9937 for the 150-minute curve.

The OPD*SNP system maintains a linear dependence of optical density on H2O2 concentration for 2.5 hours and does not require the use of stop solutions, unlike the widely used TMB*peroxidase or OPD*peroxidase systems. Thus, SNP together with OPD can serve as a sensor of hydrogen peroxide (H ₂ O ₂ ) to detect, for example, oxidative stress in living systems.

pH-dependent peroxidase-like activity of silver nanowires

SNPs exhibit activity as a pH sensor. Using a citrate-phosphate buffer system with different pH ranging from 2 to 9, we showed that the peroxidase-like activity of SNP increases linearly with both increasing SNP concentration and decreasing hydrogen ion concentration. ions. Fig. 3a illustrates part of a 96-well plate with this experiment, and the graph in FIG. Figure 3b, corresponding to the absorption of samples after 30 min of incubation, quantitatively confirms that the higher the pH, the higher the peroxidase-like activity of SNP. Accordingly, SNP together with OPD is a reliable color pH sensor for various biological applications.

Detection of folic acid using SNPs as optical tags in competitive ELISA

The property of intrinsic peroxidase-like activity was used to identify the vitamin folic acid (FA).

Detection was carried out in a standard competitive ELISA assay mode. A protein carrier (BSA) conjugated with FA (BSA-FA) was adsorbed onto the surface of 96-well ELISA plates. Next, the wells were washed from unbound BSA-FA. SNPs conjugated with anti-FK IgG mixed with test samples with different concentrations of FA were applied to the wells. After washing from unbound SNP-anti-PK IgG, chromogenic substrate (CHS) was added to the wells, followed by absorbance measurement at 450 nm. When the concentration of FA in test samples is high enough, it competitively blocks the binding of SNP to BSA-FA, resulting in low uptake. When the FA concentration is low, there is no competitive binding and the absorbance at 450 nm is high. According to the ELISA results, the limit of detection of FA, determined by the 2-sigma criterion, was equal to 0.20 μg/ml.

Detection of folic acid using SNPs as optical labels in a competitive immunoassay in the format of immunochromatography on test strips

SNPs are optical without the addition of any chromogenic substrate. This ability was demonstrated in immunochromatography format on nitrocellulose test strips. This method allows you to study the features of the specific interaction of two components. To demonstrate the possibility of using SNPs as optical labels, a competitive immunoassay with the detection of vitamin folic acid (FA) was implemented.

A carrier protein conjugated to FA, namely bovine serum albumin with CAP (BSA-FC), was applied to the surface of a nitrocellulose immunochromatographic test strip. After drying, the chromatographic test strip was placed in a solution containing SNPs conjugated with anti-PK IgG and the test sample with different concentrations of PK. In the case when the concentration of FA is low, binding of SNP to BSA-FA is possible. In this case, due to effective light scattering and the LSPR properties of the wires, a colored stripe is formed. When the concentration of FA is high, it competitively blocks the binding of SNP to BSA-FA, so no colored band is formed (Fig. 5a). The data presented in Fig. 5b indicate the effective use of SNPs as optical labels for the qualitative detection of FA in a competitive immunoassay on chromatographic test strips. The detection limit of this test is 0.1 µM FA. It should be noted that in the concentration range of 0.04 ÷ 10 µM (about 3 orders of magnitude) a concentration-dependent binding mode can be observed, which allows comparison of different samples, for example, in basic scientific analyzes or in different batch-to-batch tests (Fig. 5b ).

Thus, silver nanowires (SNPs) have peroxidase-like activity and can be used in colorimetric tests for the quantitative analysis of analytes. The method involves the use of SNPs as labels in immunological studies, followed by incubation with a horseradish peroxidase substrate - TMB or DAB in a precipitating mode or with OPD in a non-precipitating mode at room temperature and recording the absorption of the solution at an absorption wavelength corresponding to the maximum absorption of the substrate used.

In addition, SNP together with the substrate can serve as a reliable hydrogen peroxide sensor and pH sensor: the peroxidase-like activity of SNP increases linearly with increasing pH. It should also be noted that the LPPR property of SNPs allows them to be used as optical labels for visualizing antibody-hapten interactions and for other types of interactions.

Excellent stability, reproducible synthesis and antibacterial activity, as well as peroxidase-like activity and LPPR properties make SNPs convenient labels for various immunoassays that do not require sterilization and have a long shelf life. Considering their unique stability, SNPs should be considered as robust, convenient, and robust optical labels for a variety of commercial assays for a wide range of applications in biology and medicine.

This invention is widely applicable for the rapid detection of nucleic acids and their fragments, as well as for the detection of other analytes that may be a binding partner with the recognition oligonucleotide. Possible applications include medicine, veterinary medicine, agricultural technology, and the food industry. This method is particularly applicable for the detection of etiological factors such as bacteria and viruses, for the screening of resistant bacteria and for the detection of malignant cells.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which the present invention pertains. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, some of the suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, this specification, including definitions, will be controlled. In addition, the materials, methods and examples are illustrative and do not limit the present invention.

In particular, the proposed method can be used to identify short specific nucleic acid sequences, which is of significant interest in connection with the study of the physiological functions of microRNAs, early diagnosis of malignant tumors, detection of point mutations, etc. Moreover, the role and physiological functions of such compounds are currently undergoing a stage of intensive study, which guarantees a further significant increase in interest in methods for their detection.

Immunochromatographic (or other solid-phase) analysis was carried out as follows. After a short (10 min) pre-incubation of silver nanoparticles with specific or non-specific input DNA (analyte), a pre-made ICA test strip with a recognition receptor applied as a thin line in the center of the membrane was immersed in the mixture. Analyte binding was assessed by optical recording of the color intensity of the test line caused by the formation of a layer of nanoparticles (wire particles).

Various aspects

Methods for immobilizing binding receptors on a membrane are well known. Typically, test and control binding receptors are applied to the membrane in parallel lines spaced apart. Solutions of the required substances in appropriate buffers are applied to test strips using automated dispensers. After air drying for the required time, block the membrane in a suitable buffer and store in a desiccator before assembling the test strip into a single unit.

The speed of analysis using the proposed invention is significantly higher than standard analysis methods while maintaining the accuracy and reliability of the results. In this case, the measurement takes on average from 10 to 300 seconds. However, it is possible to increase sensitivity by using longer run times. In addition, the principle of the present invention allows direct measurement of the detected nucleic acid as aealite without the need for its amplification, provided that its concentration is above the detection limit and the associated signal can be detected, for example, by the naked eye.

Measurements using the method of this invention can be carried out under both hard and soft conditions. By “harsh” conditions we mean such values of temperature, ionic strength, as well as the presence of other compounds under which hybridization of nucleic acids occurs. Under “high stringency” conditions, hybridization will occur only for nucleic acids that have a high content of complementary nitrogenous bases. Thus, mild conditions are required when hybridization or annealing of incompletely complementary nucleic acids is required. Specialists know a number of methods for creating mild conditions for hybridization to occur. Hybridization under harsh conditions is possible only in the case of complete sequence complementarity. Hybridization under less stringent conditions is possible for sequences with less than 100% identity. Changes in environmental conditions, such as decreasing salt concentration or increasing temperature, can reduce the stringency of hybridization conditions. Tightening the hybridization conditions can be achieved by reducing the ionic strength or increasing the hybridization temperature.

Thus, in one of the embodiments of this invention, it is possible to carry out a one-step, full-function determination of specific DNA or RNA molecules in an immunochromatographic analysis format, in which all the necessary reagents are on the test strip in anhydrous form. On the other hand, this invention makes it possible to directly determine NKs without the need for their amplification and provides a method for their rapid detection.

In addition, in its alternative embodiments, the invention is also applicable for the detection of NK under conditions of elevated temperature, which expands the scope of its use in various fields, for example, for forensic medical examination. In addition to these advantages, the described method is a simple method for detecting nucleic acids and does not require significant training in this field, which may facilitate the penetration of rapid genomic analysis methods into the field of bedside and point-of-care diagnostics.

Analytes

The present invention makes it possible to detect a wide range of molecular analytes, including nucleic acids and polynucleotide sequences, including fragments and whole DNA molecules, as well as transport, template, ribosomal, micro RNA; synthetic nucleic acid analogues such as peptide NAs, morpholine, closed, glycolic and threozo-NAs; and also, through the use of special recognition oligonucleotides (for example, aptamers), also non-nucleotide molecules, for example, proteins and peptides, hormones, low-molecular physiological regulators, other haptens, metal ions, etc.

Multiplexity

This method is also applicable for the determination of several analytes on one membrane. In its simplest format, a test strip can be divided into individual strips where separate binding zones will be plotted to detect different analytes. In addition, it is also possible to realize simultaneous detection of different analytes in the same sample. To do this, for example, separate binding zones can be applied to one membrane, but in different parts of it.

Specific receptor/ligand pairs

Within the framework of this invention, various specific pairs of the “receptor/ligand” type can be used, applicable for the creation of signal (reporter) conjugates and the receptors that bind them, as well as recognizing oligonucleotides or protein receptors with their analyte. The components of such pairs useful in this invention can be of either immune or non-immune type. Examples of immune binding include systems based on antigen/antibody or hapten/antihapten interactions. Polyclonal, monoclonal antibodies or immunoactive fragments thereof can be prepared by standard methods known to those skilled in the art. The terms "immunoactive antibody fragment" or "immunoactive fragment" refer to portions of antibody molecules that contain a specific binding region. Such fragments may be Fab fragments, which are defined as fragments lacking the Fc portion, such as Fab, Fab' and F(ab)2 fragments, or may be so-called "semi-molecular" fragments obtained by reductive cleavage of the disulfide bonds connecting the heavy chains of the parent antibody . If the antigen that is part of the binding pair is not immunogenic, such as a hapten, it can be covalently linked to an additional carrier protein to make it immunogenic.

Non-immune specific pairs include systems in which two components exhibit natural affinity for each other without being an antibody and an antigen. Well-known examples of non-immune pairs include biotin and avidin, biotin and streptavidin, folic acid and folate binding proteins, complementary nucleic acids, proteins A, G and immunoglobulins, etc. These may also include pairs that form covalent bonds with each other: for example, pairs of sulfhydryl groups/maleimides and haloacetyl derivatives) and amino groups/isothiocyanates, succinimide esters and sulfonyl halides, etc. (M. N. Bobrow, et al., J. Immunol. Methods, 1989,125:279).

Immunochromatographic (ICA) test strips

The ICA test strip represents the environment in which the assay of the present invention occurs. The test strip typically consists of a chromatographic porous material and includes a sample application area and a binding area as described above.

The chromatographic porous materials that make up the strip are generally hydrophilic and/or in principle can be converted into such, and include the following materials, used either alone or in combination with other materials - granules of an inorganic nature, for example silicon, magnesium sulfate, aluminum; natural polymers, in particular cellulose and materials based on it (for example, fibrous polymers - filter paper, chromatography paper, etc.); synthetic or modified natural polymers, such as nitrocellulose, cellulose acetate, polyvinyl chloride, polyacrylamide, cross-linked dextran, agarose, polyacrylates, etc.; ceramic materials; glass; and similar materials. For the present invention, the preferred material is nitrocellulose.

Preferably, the membrane can be a separate structure in the form of a sheet cut into test strips, or can be fixed to a support or solid surface, such as in thin layer chromatography.

Binding of receptors to the membrane surface can be achieved using well-known methods described in the literature. See for example Immobilized Enzymes, Inchiro Chibata, Halsted Press, New York (1978) and Cuatrecasas, J. Bio. Chem., 1970, 245:3059.

Preparing the test strip

When preparing a test strip for analysis, the necessary reagents are first applied, and then the test strip is incubated in the necessary buffer solutions.

Buffer solutions and solvents used for the described invention are known (see for example Weng et al. (US Patent 4,740,468). Typically, the pH values of the buffer for immunochromatographic analysis are in the range of 4-11, more often 5-10, for the present invention preferably 6 -9. pH values are selected to maintain the desired level of affinity between interacting pairs of molecules and to be optimal for signal generation by the respective system. Various buffers can be used to achieve the desired pH value and maintain it during analysis. Examples include borate, phosphate, carbonate, Tris, diethylbarbituric, etc. The use of a specific buffer solution is not significant for the invention as a whole, however, when conducting specific studies, the use of a specific buffer solution may be preferable.

A constant temperature value is typically used for analysis within the scope of this invention. As a rule, to conduct research and obtain a detected signal, temperature values in the range of 4-50 °C are used, usually in the range of 10-40 °C, and even more often - temperatures close to ambient temperature, i.e. 15-25 °C.

When used in one of the embodiments of this invention, a recognition oligonucleotide and its binding (hybridization) with the target nucleotide sequence requires conditions conducive to NA hybridization. Hybridization is usually carried out in Ficoll buffer solution, which may also contain polyvinylpyrrolidine, BSA, yeast tRNA, non-specific DNA, dextran sulfate, dithiothreitol and formamide. Hybridization can occur both in solution and on the solid phase, with one nucleotide chain immobilized on the test strip in the binding zone.

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Examples

Hereinafter, the present invention will be described in detail in conjunction with the following examples so that those skilled in the art can better understand the present invention, but the present invention is not limited to the following examples.

Example 1. Synthesis of SNP

Silver nanowires were synthesized from _AgNO3 via a glycerol-mediated polyol process using polyvinylpyrrolidone (PVP) and NaCl as described in section [57] with modifications. 12.5 mL of glycerol with 0.5 mM PVP (molecular weight 58,000 Da) and 8 mM NaCl was heated to 80 °C and stirred to dissolve the PVP in a 50 mL round-bottom flask. The mixture was stirred and heated until the solution became clear, and then the solution was cooled to room temperature. Then 50 mM AgNO ₃ was added and stirred until AgNO ₃ was completely dissolved. The mixture was then heated to 200 °C in a round-bottomed heating mantle. A consistent change in color (pink - scarlet - brick red opaque - brown opaque - black-brown - gray-green) of the solution was observed. The reaction was continued until a gray-green color appeared, then the mixture was cooled to room temperature and diluted in 100 ml of water. The nanowires were centrifuged at 25 °C at 8000 g for 10 min. The sediment was resuspended in 3 ml _H2O .

The resulting particles were characterized by optical spectroscopy and scanning electron microscopy. The concentration of nanoparticles was determined by inductively coupled plasma mass spectrometry on a NexION 2000 mass spectrometer (Perkin Elmer, USA) by dissolving SNP in 5% _HNO3 and measuring the concentration of Ag ions using a calibration curve obtained from a series of serially diluted AgNO3 _samples .

Example 2. Measuring SNP parameters using SEM

Scanning electron microscopy (SEM) images of SNPs were obtained on a MAIA3 Tescan microscope (Tescan, Czech Republic) at an accelerating voltage of 15 kV. Samples of SNP in water at a concentration of 10 μg/mL were air-dried on a silicon substrate on carbon tape and immediately analyzed. SEM images were processed using ImageJ software to obtain particle size distribution. It is shown that the resulting particles have the shape of wires (Fig. 1 a-c).

Example 3. Enzyme-like activity of SNP.

20 μg of SNP was added to a 0.6 ml tube in 200 μl (40 mM citric acid, 40 mM Na ₂ HPO ₄ 2H ₂ O, pH 5), then 20 μl of 0.3% H ₂ O ₂ and 5 μl were added OFD, TMB or DAB with a concentration of 10 g/l. The solutions were incubated for 60 min at room temperature and photographed using a smartphone camera. Images in Figs. 2a confirm the formation of a colored solution only in the case of adding a substrate - and in the case of OPD the substrate does not precipitate, but in the case of TMB and DAB it precipitates.

Example 4: Detection of hydrogen peroxide using SNP

The H ₂ O ₂ -dependent enzyme-like activity of SNP was studied as follows. 10 μg of SNP in 50 μl of buffer (40 mM citric acid, 40 mM Na ₂ HPO ₄ 2H ₂ O, pH 5) was mixed with 50 μl of H ₂ O ₂ at various concentrations. Then 50 μl of 0.08% OPD substrate was added. The optical density of each well was measured using an Infinite 1000 Pro microplate analyzer (Tecan, Austria) at a wavelength λ = 450 nm after 10, 30, 60, 90, 120 and 150 min of incubation. Fig. Figure 2b confirms the dependence of the change in the absorption of samples with increasing concentration of hydrogen peroxide: the higher the concentration of hydrogen peroxide, the higher the absorption of the solution, and color development is formed after 30 minutes of incubation.

Example 5: pH Detection Using SNP

The pH of solutions was studied using the enzyme-like activity of SNP as follows. 20 µl of SNP of different concentrations were mixed with 100 µl of 0.04% OPD substrate and 0.03% H ₂ O ₂ in buffers with different pH - from pH 2 to pH 9. The optical density of each well was measured using an Infinite 1000 Pro microplate analyzer ( Tecan, Austria) at wavelength λ = 450 nm after 15 min of incubation. Data in Fig. 3 a, b indicate pH-dependent coloring of the solution in the pH range from 2 to 9 and the concentration of nanowires from 0.3 to 233 μg/ml. Using this method and a calibration line constructed for a nanowire concentration of 78 μg/ml, the pH of buffers with unknown values before the test was measured, which coincided with the values measured by a pH meter.

Example 6 Chemical Conjugation

Folic acid-labeled bovine serum albumin (BSA-FA) was prepared as follows. Folic acid (FA) with 1-ethyl-3-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) was dissolved in a buffer containing 50% DMSO and 50% 0.1 M morpholinoethanesulfonic acid at a final FA concentration of 10 g/L at mass ratio PC:EDC:NHS = 10:6.5:4.8. The resulting mixture was incubated for 40 min at +20 °C, then 400 μl of BSA at a concentration of 6.7 g/l in water was added to this mixture. The mixture was incubated for 8 h at room temperature. Then the resulting conjugate was purified from reaction byproducts using NAP-5 columns (GE Healthcare Life Sciences, USA) and the conjugate concentration was measured using a BCA test.

FITC-labeled BSA (BSA-FITC) was prepared by rapidly mixing 100 μL of 20 g/L BSA in phosphate buffer, pH 7.4, with 10 μL of 9.4 g/L FITC in DMSO. The solution was incubated for 4 h, and excess unreacted FITC molecules were removed using Zeba Spin Desalting Columns (MWCO 7 kDa) according to the manufacturer's recommendations.

FITC-modified SNPs (SNP-FITC) were prepared by chemical conjugation of SNPs with BSA-FITC. 420 μg of SNP were incubated with 10 mg of 1-ethyl-3-carbodiimide hydrochloride and 10 mg of N-hydroxysulfosuccinimide in 150 μl of 0.1 M MES (morpholinoethanesulfonic acid), pH 5, for 30 min at room temperature. Next, excess unreacted substances were removed by centrifugation, 200 μg of BSA-FITC 2 g/l was added to SNP, and the mixture was thoroughly treated with ultrasound. The sample was incubated for 8 hours at room temperature and washed from unbound BSA-FITC by centrifugation.

SNP modified with anti-FK IgG (SNP-anti-FK IgG) was prepared by chemical conjugation of SNP with anti-FK IgG, clone FA1. 500 μg of SNP were incubated with 5 mg of 1-ethyl-3-carbodiimide hydrochloride (Sigma) and 5 mg of N-hydroxysulfosuccinimide in 200 μl of 0.1 M MES (morpholinoethanesulfonic acid), pH 5.0, for 30 min at room temperature. Next, excess unreacted substances were removed by centrifugation, 200 μg of anti-FK IgG 1 g/l was added to the SNP, and the mixture was thoroughly treated with ultrasound. The sample was incubated for 8 hours at room temperature and washed from unbound anti-PK IgG by triple centrifugation.

Anti-biotin IgG modified SNPs (anti-biotin IgG SNPs) were prepared by chemical conjugation of SNPs to anti-biotin IgG, clone B25. 500 μg of SNP were incubated with 5 mg of 1-ethyl-3-carbodiimide hydrochloride and 5 mg of N-hydroxysulfosuccinimide in 200 μl of 0.1 M MES (morpholinoethanesulfonic acid), pH 5.0, for 30 min at room temperature. Next, excess unreacted substances were removed by centrifugation, 200 μg of anti-biotin IgG 1 g/l was added to the SNP, and the mixture was thoroughly treated with ultrasound. The sample was incubated for 8 hours at room temperature and washed from unbound anti-biotin IgG by triple centrifugation.

Example 7. Use of SNPs as labels in immunochromatography on test strips

The use of SNPs as optical markers was studied using immunochromatography on nitrocellulose test strips (UniSart CN 140 Membrane (Sartorius)). 1 μl of anti-FITC IgG at a concentration of 1 g/l was locally adsorbed onto the membrane of a 2.5 mm wide test strip. SNP, modified by FITC, in colloidal form was added to a test tube in 20 μl of Tris buffer with 1% BSA and 0.05% Tween-20. The lower end of the test strip was placed in a test tube, and SNPs, along with the solvent, began to migrate up the strip. When all the liquid had been absorbed by the test strip, 20 μl of Tris with 1% BSA and 0.05% Tween 20 were added to the tube twice in succession to allow all particles to migrate along the test strip and to wash away unbound particles, if any. In the case of a specific interaction between the first and second components, visible SNPs were detected in the area of IgG localization, which was detected visually by the formation of a brown line on the strip. Data in Fig. 4 a-c indicate the effective use of SNPs in the label format for immunochromagoraphy.

Example 8 Detection of folic acid in immunochromatography on test strips

The possibility of using SNPs as optical markers was studied using immunochromatography on nitrocellulose test strips (UniSart CN 140 Membrane, Sartorius). 1 μl of BSA-FA at a concentration of 1 g/l was locally adsorbed onto the membrane of a test strip 2.0 mm wide.

Modified anti-FK IgG SNPs in colloidal form were added to a test tube in 20 μl of 0.1 M Tris buffer with 1% BSA and 0.05% Tween-20 with different concentrations of folic acid (FA). The lower end of the test strip was placed in a test tube, and SNPs, along with the solvent, began to migrate up the strip. When all the liquid had been absorbed by the test strip, 20 μl of Tris with 1% BSA and 0.05% Tween 20 were added to the tube twice in succession to allow all particles to migrate along the test strip and to wash away unbound particles, if any. In the case when the first and second components interacted specifically, visible SNPs were detected in the area of BSA-PA localization, which was detected visually by the formation of a colored line on the test strip. The data presented in Fig. 5a, b indicate effective detection of folic acid in the concentration range from 0.1 µM to 100 µM.

Example 9. Detection of biotin in immunochromatography on test strips

The properties of SNPs as optical markers were demonstrated using immunochromatography on nitrocellulose test strips (UniSart CN 140 Membrane, Sartorius). 1 μl of BSA-biotin at a concentration of 1 g/l was locally adsorbed onto the membrane of a 2.0 mm wide test strip.

Modified anti-biotin IgG SNPs in colloidal form were added to a test tube in 20 μl of 0.1 M Tris buffer with 1% BSA and 0.05% Tween-20 with different concentrations of biotin. The lower end of the test strip was placed in a test tube, and SNPs, along with the solvent, began to migrate up the strip. When all the liquid had been absorbed by the test strip, 20 μl of Tris with 1% BSA and 0.05% Tween 20 were added to the tube twice in succession to allow all particles to migrate along the test strip and to wash away unbound particles, if any. In the case when the first and second components interacted specifically, visible SNPs were detected in the BSA-biotin localization area, which was detected visually by the formation of a colored line on the test strip. Using this method, biotin was detected in the concentration range from 0.5 µM to 100 µM.

Example 10. Detection of oligonucleotides on test strips.

The properties of SNPs as optical markers were demonstrated using immunochromatography on nitrocellulose test strips (UniSart CN 140 Membrane, Sartorius). 1 μl of a capture 20-mer oligonucleotide at a concentration of 1 μmol/l was locally adsorbed onto the test strip membrane in a 2.0 mm wide test line area and irradiated with ultraviolet light for immobilization.

SNPs modified with a recognizing 20-mer oligonucleotide (by salt aging method) in colloidal form were added to a 20 μl test tube in 0.1 M Tris buffer with 0.05% Tween-20 and a NaCl concentration of 150 mM to 1 M and/or a MgCl2 concentration with a concentration from 1 mM to 3 mM with different concentrations of the analyte oligonucleotide (complementary at one end to the capturing oligonucleotide, and at the other to the recognition oligonucleotide, to form a sandwich). The lower end of the test strip was placed in a test tube, and SNPs, along with the solvent, began to migrate up the strip. When all the liquid had been absorbed by the test strip, 20 µl of buffer was added to the tube twice in succession to allow all particles to migrate along the test strip and to wash away any unbound particles, if any (this step was optional). In the case when the first and second components interacted specifically, visible SNPs were detected on the test line, which was detected visually by the formation of a colored line on the test strip. Using this method, detection of various oligonucleoids was carried out in the concentration range from 1 nM to 10 μM.

Example 11 Detection of fluorescein isiothiocyanate (FITC) in enzyme immunoassay mode

0.3 μg of bovine serum albumin chemically bound to FITC (BSA-FITC) was adsorbed into the wells of 96-well microplates using 100 μl of carbonate-bicarbonate buffer, pH 9.2, overnight at room temperature. Next, the contents of the wells were removed and 200 μl of water with 3% skim milk was added to the wells. After 1 hour, the solution was removed, and the microplate was washed once with water. Next, 3 μg of anti-FITC antibody-modified SNP, clone FC11, was mixed with various concentrations of FITC in 100 μl of phosphate-buffered saline (PBS) with 1% BSA and added to the wells. The solution was incubated for 1 hour at room temperature with stirring, followed by washing three times with 200 μl of water. Next, 185 μl of citrate-phosphate buffer, pH 6.0, and 15 μl of 1.075% hydrogen peroxide in water and 15 μl of 1.72% o-phenylenediamine (OPD) were added to the wells. Optical density was measured using a ClarioStar microplate analyzer (BMG LABTECH, Ortenberg, Germany) at a wavelength of 450 nm. Using this method, detection of FITC was performed with a detection limit of 0.1 μg/ml.

The essence of the invention is illustrated by the drawings of Figs. 1-6.

In fig. 1 shows: SEM images of SNP; scales: 1 µm (a), 2 µm (b), 5 µm (c). (d) Size (diameter) distribution of SNPs obtained by SEM image processing, with an average diameter of 46 ± 8 nm. (e) UV-Vis-IR absorption spectrum of SNP. The SNP sample has two absorption peaks at wavelengths of 352 and 377 nm, caused by the localized surface plasmon resonance property of the synthesized nanoparticles.

In fig. Figure 2 shows: (a) Images of the color reaction of the oxidation of various substrates - TMB, OPD and DAB H ₂ O ₂ with SNP (from left to right, SNP without substrate, SNP in the presence of TMB, OPD and DAB). (b) The peroxidase-like activity of SNP depends on the concentration of H ₂ O ₂ in a linear range by 3 orders of magnitude. Data are presented as mean ± standard deviation.

In fig. Figure 3 shows the pH-dependent enzyme-like activity of SNP. (a) The peroxidase-like activity of SNP depends on the pH and concentration of SNP. (b) Uptake of OPD oxidized by H ₂ O ₂ with SNP, corrected for baseline uptake (SNP uptake). Data are presented as mean ± standard deviation.

In fig. Figure 4 presents a diagram and results of using SNPs as optical labels for analysis in immunochromatography format; in particular, the interaction of antibodies that bind fluorescein isothiocyanate (FITC) with silver nanowires coated with a carrier protein with fluorescein isothiocyanate is demonstrated. (a) Analysis scheme. (b) Fluorescence intensity of pristine SNP silver nanowires and fluorescein isothiocyanate-modified FITC silver nanowires. The analysis was carried out by fluorescence spectroscopy at excitation wavelength = 492 nm, and fluorescence emission wavelength = 518 nm. (c) Representative images of an immunochromatographic assay on a nitrocellulose membrane: negative control (no anti-FITC antibodies) and positive assay with test strip visualization of results.

In fig. Figure 5 shows the diagram and results of detection of vitamin - folic acid using SNPs as optical tags in lateral flow analysis. (a) Analysis scheme. A protein carrier (BSA) conjugated with folic acid (FA) is applied to the surface of an immunochromatographic test strip. The strip is then immersed in a solution containing the test sample, to which SNPs conjugated with anti-FK IgG are added. When the concentration of FA is high enough, it blocks the antibodies on the SNP, and the SNP does not bind to the carrier protein on the test strip. When FA concentration is low, SNPs bind to the carrier protein. Due to the property of localized surface plasmon resonance, SNPs have color and are therefore effective optical labels, producing a colored band upon binding depending on the CAP concentration. (b) Result of immunological analysis. The intensity of banding on the strip test is inversely proportional to the concentration of folic acid: the higher the concentration of FA, the lower the signal.

In fig. Figure 6 shows a scheme for the detection of low molecular weight analytes (for example, fluorescein isothiocyanate, FITC) using the peroxidase-like properties of SNP in an enzyme-linked immunosorbent assay format. (a) Analysis scheme. A protein carrier (BSA) conjugated to an analyte (FITC) is adsorbed on the solid phase - the surface of a 96-well plate. Next, SNPs conjugated with anti-FITC IgG are applied to the surface and mixed with the test sample. When the concentration of FITC is high enough, it blocks the antibodies to the SNP, and the SNP does not bind to the carrier protein. When FITC concentration is low, SNPs bind to the carrier protein. After adding a chromogenic substrate, a color appears in the solution due to the peroxidase-like properties of SNP. Measuring the optical density of a solution allows you to monitor the concentration of FITC.

Claims (26)

1. A method for detecting an analyte in a sample, including at least the following steps: i) immobilizing on the surface of the solid phase a receptor against said analyte or a molecular complex including an analogue of said analyte, ii) incubating with said solid phase of said sample, iii) incubating with said solid phase a suspension of silver nanowires conjugated with an antibody against said analyte, iv) removing unbound silver nanowires from the sample, v) incubating with said solid phase a solution of hydrogen peroxide and a chromogenic substrate selected from the group: tetramethylbenzidine, o-phenylenediamine, diaminobenzidine; vi) measuring absorbance to determine the content of said analyte in said sample from a calibration curve. 2. The method according to claim 1, wherein said silver nanowires have a length of from 1 to 15 microns. 3. The method according to claim 1, wherein said silver nanowires have a diameter of 10 to 100 nm. 4. The method according to claim 1, in which said incubation is carried out from 1 to 150 minutes. 5. The method according to claim 1, in which said incubation is carried out at a temperature of 4 to 30 degrees Celsius. 6. The method according to claim 1, wherein said calibration curve is measured in parallel for samples with a known content of molecules of said analyte. 7. The method according to claim 1, in which the concentration of hydrogen peroxide in said solution is from 0.2 to 300 µmol/liter. 8. The method according to claim 1, in which the concentration of said chromogenic substrate in said solution is from 1 to 10 mmol/liter. 9. The method according to claim 1, wherein the concentration of said silver nanowires in said solution is from 10 to 100 mg/liter. 10. The method according to claim 1, wherein said measurement of the optical density of said solution is carried out at a wavelength from 450 to 530 nm. 11. The method of claim 1, wherein said analyte is a small molecule. 12. The method of claim 1, wherein said analyte is a protein. 13. The method of claim 1, wherein said analyte is a nucleic acid. 14. The method according to claim 1, wherein said solid phase is a multi-well plate. 15. The method according to claim 1, wherein said solid phase is a test strip. 16. The method according to claim 15, wherein said test strip contains a nitrocellulose membrane. 17. The method according to claim 1, in which said measurement of optical density is carried out using optical recording methods. 18. The method according to claim 15, in which detection of the number of nanoparticles of said silver nanowires retained on the test line of said test strip is carried out within less than 20 minutes after the start of the analysis. 19. The method according to claim 1, in which said antibody is monoclonal. 20. The method according to claim 1, wherein said antibody is polyclonal.