RU2846301C2 - Method for conjugating carbon quantum dots with polymer - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to technologies for cytological analysis of a cell sample. Disclosed is a method for conjugating carbon quantum dots with a polymer, which involves the following steps: i) mixing a colloidal solution of carbon quantum dots with a polymer solution, where the polymer is biopolymers selected from the set: protein, carbohydrate, nucleic acid, polyethyleneimine, ii) heating the obtained solution to 50–99 degrees Celsius, iii) increasing the ionic strength of the obtained solution for depositing said polymer on the surface of carbon quantum dots.

EFFECT: performing cytometric analysis using labels which are not subject to photodegradation effects, having small dimensions, which are compatible with standard cytometers and which enable their easy conjugation with biological receptors which recognize the desired targets in a microscopic sample.

17 cl, 8 dwg, 23 ex

Description

Field of technology to which the invention relates

The invention relates to the field of chemical modification of nanoparticles, namely, coating carbon quantum dots with various polymers - proteins, carbohydrates, nucleic acids, various homo- and heteropolymers of chemical, biochemical and biological nature to impart specified properties to them for various applications in the field of disease diagnostics, monitoring various parameters and conditions of the body, molecular and cellular biology, nanotechnology, chemistry, etc. Such technologies are useful for the synthesis of modified nanoparticles, obtaining functional nanomaterials, for diagnostic and therapeutic purposes.

Description of the Prior Art

Carbon quantum dots (CQDs) are a class of carbon nanomaterials that combine unique optical properties of bright fluorescence, excellent photostability, tunable spectral characteristics, as well as low toxicity and biodegradability. CQDs are widely used in biomedicine, for example, for detection, bioimaging and therapeutic applications.

To make UQTs or any other nanoparticles biologically functional, they are typically conjugated with proteins, nucleic acids, carbohydrates, or other polymers. For brevity, throughout this paper, we will refer to all of these molecules as “biopolymers.”

There are currently a number of methods for conjugating carbon quantum dots and biopolymers. One of the most widely used methods is the carbodiimide method, which forms amide bonds between carboxylic acids and amines. Immobilization of nucleic acids, as well as various other polymers such as polyethyleneglycol or chitosan, is accomplished by electrostatic adsorption on the surface of CQDs doped with polyethyleneimine. Some polymers are also immobilized by adding CQDs during synthesis.

Although covalent attachment is used for many types of biomacromolecules, it requires individual selection and optimization of linker molecules for each type of biopolymer, as well as, in some cases, the introduction of additional reactive groups into the desired polymer.

The electrostatic adsorption method is a fast and convenient way to conjugate a biopolymer with UQD. However, for a given type of UQD, which has, for example, a negative surface charge, this method is applicable only to strongly positively charged biopolymers.

Adding a biopolymer to the synthesis of UQT allows for a reduction in the number of synthesis stages and, in some cases, direct modulation of the surface properties of UQT. However, one of the most popular strategies for the synthesis of UQT is the solvothermal route, which involves prolonged heating of the reagents at temperatures of 100-200°C, most often in a non-aqueous solvent. Under such harsh conditions, the choice of biopolymer is determined not by the biological application, but by the thermal stability and solubility of the biopolymer in the solvent used.

The authors have discovered a new universal method for non-covalent conjugation of carbon quantum dots with a wide range of biomacromolecules. The method is based on a simple two-step procedure of short-term heating and subsequent deposition of the polymer on the surface of the CQD by the salting-out method. It is noteworthy that the entire process, including the purification stage, takes less than 10 minutes.

From publications US9233166B2, US20130178603A1, the closest method for obtaining conjugates of nanoparticles with a target molecule is known, in which the carboxyl group of a protein or particle is activated using carbodiimide compounds, and then covalent crosslinking of the activated carboxyl group with the amino group of the second reaction participant is performed.

The disadvantage of the known method is the requirement for the presence of carboxyl or amine groups on the nanoparticle, the use of expensive cross-linkers, as well as the long time of the conjugation reaction, as well as the complexity of the purification process of the resulting conjugates for the case of small nanoparticle sizes.

Thus, the technical result consists in the development of a faster and more accessible method for conjugating carbon quantum dots with polymers, which does not require the presence of certain functional chemical groups on the surface, and is also easy to clean from reaction by-products.

Summary of the invention

The purpose of the present invention was to develop, on the basis of the discovered phenomenon of association of various polymers with carbon quantum dots under the influence of temperature and a salting-out agent: A method for conjugating carbon quantum dots with a polymer. To solve the above and other purposes that are obvious to a qualified specialist, the following method was proposed:

A method for conjugating carbon quantum dots to a polymer, which includes the following steps:

1) mixing a colloidal solution of carbon quantum dots with a polymer solution,

2) heating the resulting solution to 50-99 degrees Celsius,

3) increasing the ionic strength of the resulting solution to precipitate the said polymer onto the surface of carbon quantum dots.

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 a 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 a sample and a "binding zone" (test line) containing an immobilized binding reagent or a binding nucleotide sequence capable of binding and immobilizing the nucleotide sequence to be detected. The binding zone, as a rule, 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 can have the form of a point, line, curve, strip, or a pattern of points, lines, curves, stripes, or a combination thereof. As a rule, the direction of movement of the sample along the test strip intersects the binding zone. For the present invention, it is preferable to form a signal in the binding zone in the form of a clearly defined pattern, sharply different from the signal formed in other parts of the test strip. For example, the binding zone may be in the form of an abbreviation of the name or names of the analyte or analytes in the sample being tested.

3) The term "binding partner" refers to a pair of molecules and/or particles that are able to recognize each other (in whole or in part) and form both covalent and non-covalent bonds. There are a number of commonly used binding pairs and 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. Also known are systems in which the binding partners form covalent bonds due to the presence of amino-reactive groups such as succinimidyl esters, isothiocyanate, sulfonyl halides, or sulfhydryl reactive groups including derivatives of haloacetates and maleimides. Other binding partners may be halogens, steroids, and 2,4-dinitrophenyls. For the purposes of the present description, the term "binding partner" refers to both the "recognition receptor" and its ligand.

4) The term "nucleic acid" means a polymer or oligomer consisting of nucleotides or compounds imitating them. The term also refers to oligonucleotides having a non-naturally occurring portion of the chain, but exhibiting the same properties as oligonucleotides of natural origin. It should be obvious to those skilled in the art that a wide variety of assays for detecting target nucleic acid sequences can be carried out in accordance with the present invention. The term "nucleotide" herein refers to ribonucleotides, deoxyribonucleotides, or nucleotide analogs having a chemical modification in one or more of the nucleotide building blocks (nitrogenous bases - purine or pyrimidine, sugar, phosphates). Modifications can be, for example, carried out at the fifth position of a pyrimidine, or at the eighth position of a purine, in the exocyclic amino group of cytosine, by replacing uracil with 5-bromouracil, replacing the hydroxy group at the 2'-position of a sugar with a halo group (either R, OR, SH, H, SR, NR ₂ , NH ₂ , NHR, CN). The term also applies to ribonucleic acids occurring in living organisms, either with a replaced 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 refer to a single- or double-stranded molecule consisting of monomers (nucleotides) that include fragments 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.

5) The method of analysis and its hardware implementation related to the present invention can detect a target nucleic acid or other substances (collectively, analyte) contained in various samples. The term "sample" herein refers to any liquid potentially containing analytes. The sample can be obtained from human or other animal tissues or liquids (blood/blood serum, urine, saliva, tears, sputum, sweat, mucus, stool), from tissue cell culture samples, histological samples, biopsy samples, etc. The sample can also be obtained from agricultural products, bacteria, viruses, or their waste products and processing. Various garbage and waste, their processing products, drinking and waste water, processed or raw food products, air, etc. can also serve as a sample. If this invention is implemented for detecting 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, and for many other applications.

6) The term “recognition oligonucleotide” refers to an NC containing a sequence complementary to the analyzed (target) nucleic acid (analyte).

7) The term "signal-generating substrate" refers to a substance that can interact with a signaling receptor and generate a detectable signal. For example, ABTS/TMB for peroxidase, BCIP/MBT for alkaline phosphatase, light for fluorescent compounds.

8) The term "signal receptor", "signal-producing reporter", "reporter" means a substance capable of detecting the analyte of interest through the release of energy or by enzymatic activity, such as fluorescent and chemiluminescent fragments, particles, enzymes, radioactive labels, light-emitting domains or molecules. For the purposes of this description, the mention of these examples means the possibility of replacing them with other similar substances-"signal receptors".

9) 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 to allow liquid media to move through it by capillary forces.

10) The term "signal-producing system" denotes a set of reagents necessary for generating a detectable signal under the influence of external influence. As a rule, 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.

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

12) The first/second end of a single-stranded recognition oligonucleotide refers not to the actual end of the molecule, but to one or the other of its sides. That is, 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 outermost base in the oligonucleotide. The nucleotide base of the recognition oligonucleotide corresponding to the binding site of the signal receptor (or nanoparticle) refers to the monomer of the nucleic acid nucleotide (including nucleoside, saccharide, and phosphoric acid residue) to which the signal receptor (or nanoparticle) is bound.

13) In this description, the concepts of "receptor" and "ligand" are interchangeable and can be used to designate individual molecules in the above-described concept of "binding partners". For example, if in the literature in pairs streptavidin-biotin, antibody-antigen, the receptor is often understood to mean streptavidin and antibody, and the ligand is understood to mean biotin and antigen, then in the description of this invention the receptor can be understood to mean both streptavidin with antibody and biotin with antigen (in this case the ligand will be biotin with antigen and streptavidin with antibody, respectively).

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

15) The term "nanoparticles" means supramolecular structures that consist of more than one molecule. In this case, although nanoparticles are often used to denote objects up to 100 nm, 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 of up to 100 μm is also acceptable. Thus, nanoparticles are understood to mean both nanoparticles and microparticles, microspheres, etc. In addition, particles with the mentioned dimensions are meant, both in all dimensions and in at least one.

16) Therapeutic means/molecules/agents. Therapeutic molecules/agents may be understood as substances, substances, molecules, supramolecular agents, nanoparticles, microparticles, nanoagents, microagents, formulations, dosage forms, etc., which are used both for therapy and diagnostics of various diseases or conditions of the body or patient, including labels, contrast agents, controlled-release components, paramagnetic and magnetic agents, cytotoxic agents, etc.

17) 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 (e.g., as commonly used in ELISA), biotin, digoxigenin or haptens and proteins or other entities that can be detected, for example, by incorporating a radioactive label into a peptide or antibody specifically reactive with a target peptide. Any method known in the art for conjugating an antibody to a label can be used, for example, using the methods described in (Hermanson, G. T., Academic press, 2013).

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

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

20) “Disease” or “condition” refers to the health condition of a patient or subject that is treatable by the various drugs, dispersions, or other methods provided herein.

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

22) "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 described herein. Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goats, sheep, cows, deer and other non-mammalian animals. In particular, a patient may be a human.

23) As used herein, the term "controlled release component" or "externally activated agent" refers to a compound that, when combined with a composition as 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, gel-forming polysaccharides, and finely divided drug carrier substrates, among others. These components are discussed in more detail in U.S. Patents 4,911,920; 5,403,841; 5,212,162; and 4,861,760. The entire contents of these patents are hereby incorporated by reference in their entirety for all purposes. In some embodiments, the "controlled release component" may be a delayed release, sustained action, extended release, timed release, synchronized release, controlled release, modified release, or a sustained release compound. In some embodiments, the compound disintegrates at the site of administration (e.g., subcutaneous, intravenous) or within the digestive tract (e.g., stomach, intestine) 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." Additionally, an externally activated agent may be activated (e.g., release a therapeutic agent or bind to a target) by exposure to an external magnetic/electric field, ultrasound, light, electron beam, radioactive decay of any atoms, interaction with a chemical microenvironment, etc.

24) As used herein, the term "paramagnetic agent"/"paramagnetic means" refers to a paramagnetic compound useful in diagnostic imaging techniques (e.g., magnetic resonance imaging) as a contrast agent. In some embodiments, the paramagnetic agent comprises gadolinium, iron oxide, platinum iron, or manganese.

25) Cytotoxic agent (toxin) options include, but are not limited to, ricin, doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, tenoside, vincristine, vinblastine, colchicine, dihydroxyanthracinatedione, actinomycin D, diphtheria toxin, Pseudomonas exotoxin (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.

26) A non-exhaustive list of immunoassays includes: competitive and non-competitive formats, enzyme-linked immunosorbent assays (ELISA), 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, such as for diagnostic imaging.

It should be noted that throughout the application, alternatives are written in Markush groups, such that each position of the therapeutic agent 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 single 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, the following is proposed:

A method for conjugating carbon quantum dots to a polypeptide , which comprises the following steps:

1) mixing a colloidal solution of carbon quantum dots with a polymer solution,

2) heating the resulting solution to 50-99 degrees Celsius,

3) increasing the ionic strength of the resulting solution to precipitate the said polypeptide onto the surface of carbon quantum dots.

Furthermore, a method in which said receptor or second receptor (recognition) is a lectin, antibody, or other "binding partner" of the analyte.

In addition, a method and/or nanolabel, in which the size or average size of said carbon quantum dots (before coating with a polymer) is 10-15, 10-20, 20-30, 10-30, 10-100, 20-40, 10-50, 50-150 nm.

In addition, a method and/or nanolabel, in which the size or average size of said carbon quantum dots conjugated to the polymer is 20-30, 30-40, 40-50, 20-50, 30-60, 20-70, 50-100, 30-80,30-100,30-150, 20-150, 40-200,20-200,20-300,20-400,100-200,200-300,100-300 nm.

In addition, a method and/or nanolabel in which the size of said carbon quantum dots conjugated to a polymer is 30 - 250 nm.

In addition, a method and/or nanolabel in which the size of said carbon quantum dots conjugated to a polymer is 50 - 150 nm.

In addition, a method and/or nanolabel in which the size of said carbon quantum dots conjugated to a polymer is 100 - 250 nm.

Furthermore, a method and/or nanolabel, wherein said salt is ammonium sulfate.

In addition, the method and/or nanolabel, wherein said salt is NaCl, Na2SO4, MgSO4, (NH4)2SO4, KCl, MgCl2.

In addition, a method and/or nanolabel, in which said carbon quantum dots are conjugated with an antibody against a molecular marker by depositing said antibody against a molecular marker on the surface of the carbon quantum dots by heating and adding salt to a solution containing said antibody against a molecular marker and a suspension of carbon quantum dots.

In addition, a method and/or nanomark, in which the mentioned heating is carried out to 30,40,50,60,70,80,90,95,99,40-50,50-60,60-70,70-80,80-90,90-99,90-95,80-95,70-95,60-95,50-99 degrees Celsius.

In addition, a method and/or nano-label in which the said heating is produced up to 55-65 degrees Celsius.

In addition, a method and/or nano-label in which the said heating is produced up to 85-95 degrees Celsius.

In addition, a method and/or nanolabel, wherein said cell sample is biopsy material.

In addition, a method and/or nanolabel, wherein said cellular sample is a histological section.

Furthermore, a method and/or nanolabel, wherein said cellular sample is a suspended cell sample.

In addition, a method and/or nanolabel in which the said fluorescent signal is detected (signal from the UCT) at emission wavelengths of 360-510 nm.

In addition, a method and/or nanolabel, in which the said fluorescent signal is excited at excitation wavelengths of 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 750-800, 800-850.

In addition, a method and/or nanolabel, in which the said fluorescent signal is detected (emitted) at emission wavelengths of 350-400,400-450,450-500,500-550,550-600,600-650,650-700,750-800,800-850,850-900,900-950.

In addition, a method and/or nanolabel in which said fluorescent signal is excited at excitation wavelengths of 360-510 nm.

In addition, a method and/or nanolabel in which said fluorescent signal is excited at excitation wavelengths of 510-600 nm.

In addition, a method and/or nanolabel in which the said fluorescent signal is detected (emitted) at emission wavelengths of 400-525 nm.

In addition, a method and/or nanolabel in which the said fluorescent signal is detected (emitted) at emission wavelengths of 525-700 nm.

In addition, a method and/or nanolabel in which fluorescence is excited upon irradiation with light in the range of 360-510 nm.

In addition, a method and/or nanolabel in which fluorescence is excited upon irradiation with light in the range of 510-600 nm.

In addition, a method and/or nanolabel in which, upon fluorescence excitation, the emission is in the range of 400-525 nm.

In addition, a method and/or nanolabel in which, upon fluorescence excitation, the emission is in the range of 525-700 nm.

Furthermore, a method and/or nanolabel, wherein said molecular marker is a cellular marker.

In addition, a method and/or nanolabel, wherein said cellular marker is selected from the series: EGFR, HER2, PD-L1, EpCAM, CEA, PSA, CA-125, CA 19-9, CA 15-3, CA 72-4, MUC1, CD19, CD20, CD44, CD133, CD166, CD24, CD47, CD117, DR-5.

In addition, a method and/or nanolabel used in the diagnosis of diseases or conditions of the body.

Furthermore, a method and/or nanolabel, wherein said cellular marker is a molecular marker expressed in the cytoplasm of a cell.

In addition, a method and/or nanolabel, in which an increase in the ionic strength of said solution containing said nucleic acid and a suspension of said carbon quantum dots is achieved by adding a salt to said solution.

In addition, the method and/or nanotag, in which said solution containing said nucleic acid and a suspension of said carbon quantum dots, additionally contains a positively charged polymer.

In addition, a method and/or nanolabel, wherein said positively charged polymer is one of the group: polyethyleneimine, polylysine, D-polylysine, L-polylysine.

In addition, a method and/or nanolabel, in which the said nucleic acid is selected from the series: DNA, RNA, plasmid DNA, messenger RNA, antisense DNA, antisense RNA, small interfering RNA, piwiRNA, microRNA, aptamer.

In addition, a method and/or nanolabel used in gene therapy of diseases or conditions of the body.

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

In addition, a method wherein said analyte is a protein.

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

In addition, the method in which said test strip is an immunochromatographic test strip, a thin layer or an affinity chromatographic test strip.

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

In addition, the method in which the said brightness assessment is carried out using optical registration methods.

In addition, a method in which the detection of the amount of particles of said carbon quantum dots retained on the test line is carried out within less than 2, 3, 5, 7, 10, 15, 20 minutes after the start of the analysis.

In addition, a method and/or nanolabel in which said antibody is monoclonal.

In addition, a method and/or nanolabel in which said antibody is polyclonal.

In addition, a method and/or nanolabel, wherein said molecular analogue of the analyte is a carrier protein conjugated to analyte molecules.

In addition, a method and/or nanolabel in which the registration of the number of carbon quantum dots retained on the test line is carried out by registering their fluorescence.

In addition, a method and/or nanolabel, in which the obtained functional quantum dots with the said polymer are additionally washed from molecules of unbound polymer.

In addition, a method and/or nanotag, in which the said washing of the obtained carbon quantum dots is carried out with a solvent from the set: ethanol, methanol, water, acetone.

In addition, a method and/or nanolabel, in which in the said solution of citric acid and urea the mass ratio of citric acid and urea is in the range from 3:1 to 1:5.

In addition, a method and/or nanolabel, in which in the said solution of citric acid and urea the mass ratio of citric acid and urea is in the range from 1:1 to 1:3.

In addition, a method and/or nanolabel, in which the said polymer is one of a set of: protein, carbohydrate, nucleic acid, polyethyleneimine.

Furthermore, a method and/or nanolabel, wherein said polymer is a gamma-immunoglobulin.

In addition, a method and/or nanolabel in which the said autoclaving is carried out at a temperature of 190-210 degrees Celsius.

In addition, the method and/or nanolabel, in which the mentioned autoclaving is carried out at a temperature of 120-130,130-140,140-150,150-160,160-170,170-180,180-190,190-200,200-210,150-180,180-210,200-230,200-250 degrees Celsius.

In addition, a method and/or nanolabel, in which said increase in the ionic strength of the obtained solution for precipitating said biopolymer onto the surface of carbon quantum dots is carried out by adding a salt solution to said obtained solution.

In addition, the method and/or nanolabel further comprises the step of adding a cross-linking agent.

In addition, a method and/or nanolabel, in which said cross-linking agent is selected from the list: epichlorohydrin, glutaraldehyde.

In addition, a method and/or nanolabel in which the said washing of the obtained conjugates is carried out by ultrafiltration.

In addition, a method and/or nanolabel in which the said washing of the obtained conjugates is carried out by centrifugation.

In addition, a method and/or nanolabel in which said polymer is a mixture of several polymers.

In addition, a method and/or nanolabel, in which the said polymer is one of a set of: protein, carbohydrate, nucleic acid, polyethyleneimine.

Furthermore, a method and/or nanolabel, wherein said polymer is a gamma-immunoglobulin.

In addition, a method and/or nanolabel, wherein said luciferase is one of the following: nanoLuc luciferase, firefly luciferase, Renilla luciferase, Gaussia luciferase.

In addition, a method and/or nanolabel, wherein said luciferase is a mixture of several luciferases.

In addition, a method and/or nanolabel in which fluorescence is excited upon irradiation with light in the range of 360-510 nm.

In addition, a method and/or nanolabel in which fluorescence is excited upon irradiation with light in the range of 510-600 nm.

In addition, a method and/or nanolabel in which, upon fluorescence excitation, the emission is in the range of 400-525 nm.

In addition, a method and/or nanolabel in which, upon fluorescence excitation, the emission is in the range of 525-700 nm.

In addition, a method and/or nanolabel, wherein said protein is one of the following: transferrin, insulin, endostatin, hemoglobin, myoglobin, lysozyme, immunoglobulins, α-2-macroglobulin protein, fibronectin, laminin, collagen, gelatin, protein components of lipoproteins, synthetic peptides and proteins, and combinations thereof.

Furthermore, a method and/or nanolabel, wherein said polymer is a polysaccharide.

In addition, a method and/or nanolabel, in which the said polysaccharide is one of the following: dextran, carboxymethyl dextran, chitosan.

In addition, a method and/or nanolabel in which said polymer is a mixture of several polymers.

In addition, a method and/or nanolabel, in which said carbon quantum dots are conjugated with an antibody against said analyte or a molecular analogue of the analyte by precipitating said antibody against said analyte or a molecular analogue of the analyte on the surface of the carbon quantum dots by adding a salt to a solution containing said antibody against said analyte or a molecular analogue of the analyte and a suspension of carbon quantum dots, and heating it.

In addition, a method and/or nanolabel in which the surface of the solid phase is the surface of a multi-well plate.

In addition, a method and/or nanolabel in which the surface of the solid phase is the surface of a 96-, 384-, or 1536-well plate.

In addition, the method and/or nanolabel, in which the said registration of the detected signal is carried out using optical registration methods, using the registration of fluorescence, luminescence, bioluminescence, phosphorescence.

In addition, a method and/or nanolabel, wherein said enzyme is selected from the group: peroxidase, phosphatase, luciferase.

In addition, a method and/or nanolabel, wherein said enzyme is a native form of an enzyme selected from the group: horseradish peroxidase, alkaline phosphatase, acid phosphatase, nanoLuc luciferase, firefly luciferase, thermostable luciferase, Renilla luciferase, Gaussia luciferase.

In addition, a method and/or nanolabel, in which the said enzyme is a genetically engineered mutated form of an enzyme selected from the group: horseradish peroxidase, alkaline phosphatase, acid phosphatase, nanoLuc luciferase, firefly luciferase, thermostable luciferase, Renilla luciferase, Gaussia luciferase.

Furthermore, a method and/or nanolabel, wherein said substrate is a substrate selected from the group: a chromogenic substrate, a fluorogenic substrate, or a chemiluminescent substrate.

Furthermore, a method and/or nanotag, wherein said antibody is selected from the group: amimmunoglobulin, immunoglobulin G1 (IgG1), immunoglobulin G2 (IgG2), immunoglobulin G3 (IgG3), immunoglobulin G4 (IgG4), immunoglobulin M, immunoglobulin A, immunoglobulin E, immunoglobulin D, Fab antibody fragment, Fab' antibody fragment, Fab'2 antibody fragment, F(ab')2 antibody fragment, scFv antibody fragment, nanobody, scFv-Fc fragments, scFv-scFv (diabody), bispecific scFv fragments, Fab-scFv antibody fragment.

In addition, a method and/or nanotag, wherein said antibody is a genetically engineered analogue from the group: avimers, adgyrons, adnectins, monobodies, atrimers, anticalins, affibodies, affilins, affimers, affitins, nanophytins, darpins, knottins, obodis, Kunitz domain-based polypeptides, pronectins, repbodys, phynomers, centirins, ADAPT (ABD-Derived Affinity Proteins), NanoCLAMP (nano-CLostridial Antibody Mimetic Proteins), ARM (Armadillo repeat proteins), PDZ proteins.

In addition, a method and/or nanolabel, wherein said molecular analogue of the analyte is a carrier protein conjugated to analyte molecules.

In addition, a method and/or nanolabel, in which, in order to clarify the accuracy of the analysis, a fluorescent signal from carbon quantum dots bound to the said surface of the solid phase is additionally recorded as a measure of the analyte content in the sample, and then the obtained measures of the analyte content in the sample are averaged for a refined measure of the analyte content in the sample.

In addition, a method and/or nanolabel, in which, in order to clarify the accuracy of the analysis, a fluorescent signal from carbon quantum dots bound to the said surface of the solid phase is additionally recorded as a measure of the analyte content in the sample, and then the obtained measures of the analyte content in the sample are averaged for a refined measure of the analyte content in the sample.

Carbon quantum dots (CQDs) are a unique material for biomedical applications due to their inherent optical properties and biocompatibility. However, to date, there is no universal procedure for conjugating CQDs with biopolymers, which significantly reduces their practical value.

In this work, we discovered a new ultrafast method for conjugation of UQDs with biomacromolecules, including proteins, nucleic acids, and carbohydrates. The method is based on rapid heating of UQDs with a biopolymer solution, followed by a salting-out step that causes the biopolymer to precipitate on the UQD surface and simultaneously serves as a purification phase. Using virtually the same HSO protocol, we successfully used UQDs in immunochromatographic analysis (conjugation with antibodies to chloramphenicol), in enzyme immunoassay (simultaneous conjugation with nLuc or HRP and antichloramphenicol), in specific cell targeting (conjugation with Herceptin), in gene delivery (conjugation with polyethyleneimine and nanoLuc plasmid).

The proposed procedure can significantly increase the value of UCT for a wide range of biomedical applications.

Synthesis and characterization of UCT

The synthesis of UCT was carried out by a solvothermal route. The resulting complex mixture of products after synthesis was first separated by hydrophilicity (by precipitation with ethanol and subsequent centrifugation), as in the original work. However, we then additionally removed large particles by centrifugation; and low molecular weight by-products were removed by ultrafiltration.

The synthesis of UCT was carried out according to the following protocol. 1 g of citric acid and 2 g of urea were dissolved in 8 ml of formamide, and the volume was brought up to 10 ml with formamide. The solution was transferred to a Teflon-coated autoclave and placed in an ED56 Binder thermostat (Tuttlingen, Germany) preheated to 160°C for 4 hours. The autoclave was then immediately cooled under running water.

The resulting dark orange mixture together with the aggregates was transferred to a 50 ml test tube and 20 ml of ethyl alcohol was added. The resulting mixture was centrifuged at 10,000 g for 5 min. The supernatant was discarded and 2 ml of water was added. The test tube was treated with ultrasound until the granule was completely dissolved (~ 1-2 min), and the volume was brought to 10 ml with water. The procedure was repeated once more. The resulting UCT solution in water was centrifuged at 10,000 g for 5 min to remove large aggregates. Then the resulting supernatant was transferred to a new test tube.

The final washing step was ultrafiltration through a regenerated cellulose membrane MWCO 100 kDa. UCT was filtered under a pressure of about 3 bar in the washing mode. Washing was carried out until the dialysate became almost colorless (at the beginning of filtration the dialysate was dark orange) for about 1 hour.

The retentate was then aliquoted and stored at -20°C. After defrosting, the aliquot was centrifuged at 15,000 g for 5 min, and the supernatant was used for further experiments within 1–2 days after defrosting.

Next, we studied the obtained UQDs using transmission electron microscopy (Fig. 1a). From the micrographs, we determined that the UQDs have a near-spherical shape with an average diameter of 31 ± 7 nm.

To study the physicochemical properties of UQD in solution, we conducted a study of UQD using the dynamic light scattering method (Fig. 1 c, d). The hydrodynamic size was (26 ± 4 nm), and the zeta potential was -33 ± 7 mV.

Interestingly, both the hydrodynamic diameter and the size obtained by us from TEM micrographs are larger than the corresponding values described in the original work (1-7 nm). This may be due to the ultrafiltration stage we introduced.

Next, we studied the presence of chemical functional groups on the surface of the CQDs using Fourier transform IR spectroscopy (Fig. 1b).

The broad band at 3122 cm ^-1 can be assigned to the OH stretching vibration in both carboxylic acid and alcohol. The peak at 1657 cm ^-1 can indicate the stretching of the C=O stretching in carboxylic acid, amide or aldehyde. These two peaks confirm the presence of carboxylic acid groups. Next, the narrow band at 1098 cm ^-1 indicates the stretching of the C-O stretching in alcohol or ether. Now we can state the presence of alcohol groups in the UCT sample. Finally, we assign the peaks at 1339 and 1395 cm ^-1 to the OH deformation vibrations, and the weak peak at 2785 to the CH stretching vibrations.

Next, we studied the optical properties of the UQD. The absorption spectrum has two peaks - at 370 nm and at 517 nm (Fig. 1g). The second of these peaks gives the UQD a rich violet color, reminiscent of the color of gold nanoparticles.

The fluorescence excitation/emission heat map (Fig. 1e) reveals two distinct fluorescence regions. The first is located in the 350–450 nm excitation region. In this region, the location of the emission peak depends on the excitation wavelength. In the 470–590 nm excitation region, the emission spectrum is fixed, with the emission peak at 636 nm. The maximum fluorescence intensity is observed at excitation/emission parameters of 530/630.

One of the commonly used methods for coating nanoparticles with polymers is the passive adsorption of the polymer at elevated temperatures. Here, we tried to adapt this strategy to UQT.

We heated the UQD solution with BSA at 90°C for 1 min. As a result of heating, a significant increase in UQD fluorescence was observed, visible to the naked eye. This change in fluorescence indicates that binding of UQD and biopolymer (BSA in this case) has occurred.

Here and below, we use a colon ":" to denote conjugations of UQD with a bipolomer. In this case, UQD:BSA was obtained. If two or more biopolymers are immobilized on the surface of UQD, they are separated by a comma, for example UQD:(biopolymer1, biopolymer2).

However, the obtained coated UQTs had a high degree of colloidal stability - the conjugates did not form a sediment when centrifuged at 15,000 g for 30 min. This means that the purification of such conjugates from by-products and unbound biopolymer is a non-trivial task.
Therefore, we decided to apply the salting-out method, which is, for example, routine for the separation of blood plasma proteins.

The conjugates were mixed abruptly with ammonium sulfate (AS) solution at a concentration of 5-50%. The addition of AS immediately disrupted the colloidal stability of the nanoparticles, and the conjugates were centrifuged at 15,000 g for 5 minutes. The resulting pellet was resuspended in the desired buffer.

Although we conceived of salting out as a purification step, it will be shown below that it plays a key role in the formation of conjugates.

Coating of UCT with polysaccharides

Next, we apply the described strategy to coat the UQTs with polysaccharides. For the proof of concept, we chose chitosan and dextran. Both polysaccharides are popular choices for coating various nanoparticles, as they generally enhance the stability, biocompatibility, and bioavailability of the nanoparticles.

Conjugation of polysaccharides with UCT was carried out under the same conditions as for BSA. To prove the presence of polymers on the surface of the dots, we performed immunochromatographic analysis. The corresponding lectins were used, namely wheat germ agglutinin (WGA), which binds N-acetyl-D-glucosamine, which is an acetylated monomer of chitosan; and concanavalin A, which binds D-glucose, a monomer of dextran.

Lectins were immobilized on test strips and the strips were immersed in a solution containing the conjugates. A negative control was performed by pre-mixing the conjugates with an excess of the corresponding lectin.

Both conjugates showed strong binding to the test strips, with binding being significantly weaker for the conjugates pre-mixed with lectins (Fig. 2), indicating the presence of polysaccharides on the surface of the UCTs.

Conjugates of UCT with antibodies

Conjugation of an antibody to a nanoparticle allows the latter to specifically recognize a predetermined biomolecule. Such conjugates form the basis of many therapeutic and diagnostic approaches.

In this section, we use HSO to prepare conjugates of UCT with anti-chloramphenicol (ACh) antibodies and with anti-human epidermal growth factor receptor 2 (HER2) antibody trastuzumab and demonstrate their implications for diagnostic and therapeutic applications.

Detection of CAF using immunochromatography and UKT conjugates: (Anti-CAF)

Levomycetin (CAF) is a broad-spectrum antibiotic that is banned for use in food-producing animals, including cows and honeybees. Developing methods to detect CAF in food products at trace levels is an important task for the food industry.

One of the popular methods for detecting CAF is immunochromatographic analysis (ICA), given its speed and sensitivity. The analysis scheme is shown in Fig. 3a.

The UKT:(Anti-CAF) conjugates were able to bind specifically to the test line, a representative photograph of the test strips demonstrating the absence (left, strong binding) or presence (right, no binding) of CAF in the sample (Fig. 3 b, c). The detection limit (3σ criterion) of the test system was 9 ng/ml (2.1 μM).

We believe that subsequent optimization of various conjugation and assay parameters, such as antibody affinity, molar ratio of UCT to antibody, and composition of migration buffer, can significantly reduce the detection limit to meet the high sensitivity requirements of real applications.

Cell targeting with UCT:(Anti-HER2) conjugates

Nanoagents that can selectively bind to a specific cell type are a powerful means of targeting a complex biological system, for example to suppress tumor cells with minimal impact on normal cells. In this example, a commonly used target is HER2, a receptor that is overexpressed by cancer cells in many types of breast cancer and is almost absent from the surface of healthy cells. The FDA-approved humanized monoclonal antibody trastuzumab (Anti-HER2) is often used to bind the receptor.

Using the HSO method, we prepared UCT:(Anti-HER2) conjugates and studied their specific binding ability in vitro using EMT6/P (negative control) and EMT6/P-HER2 cell lines. Cell–nanoparticle interactions were performed using a flow cytometer, and cell binding was quantified using UCT fluorescence (Fig. 5b).

The conjugates bound to HER2-positive cells are more than three times more potent compared to HER2-negative cells, as quantified by the staining index (0.56 vs. 0.18), underpinning their potential in cell targeting applications.

Optimization of the synthesis of UCT:(antibody) conjugates

As with BSA, the fluorescent properties of the conjugates (demonstrated on UCT:(Anti-HER2)) were significantly altered compared to the parent UCT.

The fluorescence intensity (F) at 530/630 nm increased with increasing synthesis temperature (T) (Fig. 6a). The data were fitted by a power function F(T) = F0 +α (T - T ₀ ) ^β, where F0, α, β were treated as unknown parameters and T0 is room temperature (25°C). The best-fitting β was found to be 5.6, indicating a sharp growth pattern with more than threefold increase in fluorescence at T = 90°C compared to uncoated CQDs.

However, the fluorescence intensity upon excitation at 350-450 nm was greatly attenuated regardless of the synthesis temperature. This attenuation is probably due to the coexistence of two UQD subpopulations, one of which is not precipitated by the AS and is lost during the washing stage.

On the other hand, the synthesis temperature also influences the antibody activity. Then, the effect of the synthesis temperature and, additionally, the synthesis pH was studied for the UKT:(Anti-CAF) conjugates (Fig. 6e, g). To quantify the antibody activity, we measured the binding of the conjugates to the test line in MAF. We assume that stronger binding reflects the presence of more active antibody molecules in the conjugate, and vice versa.

Conjugates obtained at 70°C and below bind to the test strip almost equally strongly. However, at higher temperatures, almost no binding was observed. Thus, temperatures in the region of 60-70°C are optimal in terms of enhancing the fluorescence of UCTs and the activity of antibodies.

The effect of pH of the synthesis on the activity of the conjugates was studied. The pH was investigated in the neutral and alkaline ranges, since it is expected that acidic pH will disrupt the colloidal stability of the UCTs, since they are presumably stabilized by carboxylic acid groups. It is curious that for the studied pH range of 7.5 - 10, the behavior of the conjugates in the same ICA test was practically independent of pH.

Addition of enzymatic modality to UCT-antibody conjugates.

We next sought to develop UCT:(antibody) conjugates by adding another, enzymatic modality.

We selected two enzymes: horseradish peroxidase (HRP) and nanoLuc luciferase. By oxidizing the corresponding substrate, HRP produces a colored product that is easily determined colorimetrically, and nanoLuc generates bioluminescence. Both enzymes are widely used in routine clinical diagnostics, such as in enzyme-linked immunosorbent assay (ELISA).

The HSO method was used to obtain UKT:(Anti-KHAF, HRP) and UKT:(Anti-KHAF, nanoLuc) conjugates. Due to the Anti-KHAF antibody, the conjugates are capable of selectively binding the antigen, and at the same time can be detected both by enzymatic activity and by UKT fluorescence.

Such conjugates can be naturally used in ELISA as reporter entities, similar to enzyme-conjugated secondary antibodies. Thus, we performed direct ELISA detection of CAF (Fig. 8a) using UCT:(Anti-CAF, HRP) and UCT:(Anti-CAF, nanoLuc) as reporters (Fig. 8b-d).

The dose-response curve obtained with fluorescence detection generally has the same profile as that obtained with enzymatic detection.

As with the antibodies, the dependence of the activity of the conjugated enzymes on the pH and temperature of synthesis was investigated. Both proteins were most active at a more neutral pH and less active at a more alkaline pH, but did not lose activity completely.

It is interesting that for HRP conjugates the dependence of activity on temperature is not monotonic, but has a peak at 70° C. Probably, higher temperatures promote protein adsorption on the surface of the UCT, but, at the same time, promote protein denaturation and thereby reduce its activity.

Gene delivery (transfection) by UKT conjugates: (PEI, plasmid).

Next, to expand the class of biopolymers to which HSO can be conjugated, we applied the same strategy to conjugate nucleic acids. The choice of a particular nucleic acid was dictated by two factors: (i) it should be a long polymer rather than an oligomer, since we expect HSO to be more efficient for longer polymers; (ii) its presence in a living cell should be easily detected; (iii) the detection channel should be orthogonal to the fluorescence of the NQD. To meet these requirements, plasmid DNA encoding the nanoLuc luciferase protein (3kb, CMV promoter) was chosen.

However, due to the electrostatic repulsion between DNA and UQDs, which are negatively charged, the adsorption process of DNA on UQDs hardly occurs. Therefore, we also introduced positively charged linear polyethyleneimine (PEI) into the system, which will serve as a mediator between the dots and DNA. Thus, we used a two-step procedure: UQDs were first conjugated with PEI, washed, and then conjugated with the plasmid. For either of them, a temperature of 90°C was used to maximize the polymer yield and fluorescence of UQDs, since the plasmid is not expected to lose its functional activity upon heating.

Measurement of DNA content on the surface of UCT

To confirm successful conjugation, we measured DNA yield using SYBR gold dye. In the assay, SYBR gold DNA dye is added to the sample and the dye fluorescence increases >1000-fold upon DNA binding, which is then compared to a calibration curve and the concentration is extracted. However, this approach does not take into account the fact that UQT partially absorbs SYBR fluorescence, leading to an overestimation of the yield.

To solve this problem, each point of the calibration curve was measured in the presence of the UQT:(PEI, plasmid) sample (Fig. 4a). Then, the UQT fluorescence was subtracted, and the intersection with the abscissa axis of the calibration curve was found, which corresponds to the desired concentration. The typical yield of the plasmid was about 10%.

Transfection with UCT: (PEI, plasmid)

We next examined the ability of the conjugates to transfect living cells. HEK293 cells were treated with UCT:(PEI, plasmid) conjugates containing 1, 5, 10, 30, or 50 ng of plasmid per well of a 96-well plate. As a control, conventional transfection with PEI containing the same plasmid content was also performed.

The dependence of transfection efficiency on the plasmid concentration used (Fig. 4b) for the conjugates has an optimum at 10 ng, and at this concentration the UKT:(PEI, plasmid) conjugates exceed the conventional PEI transfection by almost three times. However, at higher concentrations UKT tends to aggregate and form films with large macroscopic agglomerates. Although UKT remains virtually non-toxic at these concentrations, as shown by the resazurin toxicity test (Fig. 4c), the resulting films and aggregates probably prevent the penetration of new portions of the transfectant into the cells, thereby reducing the amount of the produced reporter protein.

In addition, UCTs can help to track conjugates inside cells (Fig. 4d). Cells treated with UCT:(PEI, plasmid) show bright red fluorescence (excitation 560 nm, emission 603 nm). Fluorescence can be used, for example, to independently estimate the percentage of transfected cells.

Formation of conjugates

In the two-step heating process, the salting-out step appears to be the essential step, while the heating step is optional but beneficial due to the significant increase in fluorescence.

Dissolved ammonium sulfate is highly hydrated and at high concentrations competes for water molecules with the biopolymer, which reduces the solubility of the polymer and promotes self-aggregation.

The scalable experimental setup can be used in conjunction with the proposed UQD conjugation technique, for example, a UQD and biopolymer solution can be mixed with an AS solution in a microfluidic chip. This will allow for more precise control over the conjugation structure, coating density, biopolymer conformation on the UQD surface, etc.

Salt removal efficiency

The main drawback of HSO conjugation is its non-covalent nature, meaning that the conjugated biopolymer can potentially desorb from the CQD surface. We were unable to detect the impact of unbound polymer in specific applications. It is also possible that the polymer-CQD interaction may actually be irreversible despite being non-covalent.

Versatility of conjugation technology for biopolymers

However, we have managed to conjugate UQT with different classes of biopolymers, it is obvious that one can encounter a biopolymer that cannot be conjugated with this approach. Below we list potentially problematic cases:

A. It is expected that highly negatively charged biopolymers will not form conjugates in high yield due to electrostatic repulsion with the CQD surface. However, it can be overcome by introducing a positive adaptor (as shown in the example of plasmid conjugation)

B. Polymers that are not susceptible to AS, i.e. soluble even at high concentrations of AS, may also be difficult to conjugate. We expect such behavior, for example, from polymers with a short chain length.

Commonality of conjugation technology in relation to UCT

In addition to the conjugation of biopolymers with UQT, described in detail above, this conjugation technology allowed conjugation with biopolymers of a wide variety of UQT synthesized by other protocols and methods. From this, it can be concluded that this technology is universal in relation to various UQT.

The developed technology for obtaining functionalized UQDs and conjugating UQDs with biopolymers is a universal approach to the functionalization of carbon quantum dots with proteins, carbohydrates, nucleic acids or other biopolymers. The technology is characterized by the speed of the procedure and the simplicity of the purification stage.

Various aspects

The detailed descriptions described above do not limit the range of possibilities and applications of the invention, but are only given for convenience.

Various biopolymers

In one embodiment of the invention, UCT is conjugated with a polymer from the group: proteins, nucleic acids, polysaccharides, and others. In particular, conjugates were obtained with: immunoglobulins (including IgG, IgM), lectins, peroxidases, luciferases, albumins, DNA, RNA, plasmid DNA, matrix RNA, antisense DNA, antisense RNA, small interfering RNA, piwiRNA, microRNA, aptamer, antisense DNA, dextran, carboxymethyldextran, chitosan, polyethyleneimine, polyethyleneglycol, polyacrylic acid.

In one embodiment of the invention, said protein may be: serum albumin, transferrin, holo-transferrin, enzymes.

In one embodiment of the invention, said antibody may be a gamma immunoglobulin, immunoglobulin G1 (IgG1), immunoglobulin G2 (IgG2), immunoglobulin G3 (IgG3), immunoglobulin G4 (IgG4), immunoglobulin M, immunoglobulin A, immunoglobulin E, immunoglobulin D, Fab antibody fragment, Fab' antibody fragment, Fab'2 antibody fragment, F(ab')2 antibody fragment, scFv antibody fragment, nanobody, scFv-Fc fragments, scFv-scFv (diabody), bispecific scFv fragments, Fab-scFv antibody fragment.

In addition, the mentioned antibody is a genetically engineered analogue from the group: avimers, adgyrons, adnectins, monobodies, atrimers, anticalins, affibodies, affilins, affimers, affitins, nanophytins, darpins, knottins, obodis, polypeptides based on the Kunitz domain, pronectins, repbodys, phynomers, centirins, ADAPT (ABD-Derived Affinity Proteins), NanoCLAMP (nano-CLostridial Antibody Mimetic Proteins), ARM (Armadillo repeat proteins), PDZ proteins.

Proteins useful for this aspect of the invention may include, but are not limited to, albumin, transferrin, insulin, endostatin, hemoglobin, myoglobin, lysozyme, immunoglobulins, α-2-macroglobulin protein, fibronectin, laminin, collagen, gelatin, protein components of lipoproteins, synthetic peptides and proteins, and combinations thereof. Examples of suitable proteins include, but are not limited to, albumin, transferrin, insulin, endostatin, hemoglobin, myosin, lysozyme, immunoglobulins, α-macroglobulin, fibronectin, lamin, collagen, Apo-A1, gelatin, artificial proteins, and combinations thereof.

In addition, blood serum, blood plasma, and whole blood can be used to obtain protein nanoparticles within the framework of the invention.

The created conjugates with these polymers showed high specificity of binding to their target (ligand, binding partner).

Various salts

In one embodiment of the invention, the said polymer salting out on the UCT (precipitation) was carried out using various salts. In addition to the most effective ammonium sulfate, NaCl, Na2SO4, MgSO4, KCl, MgCl2 were used.

Heating

In one embodiment of the invention, said heating is carried out to 45-50 degrees Celsius, 50-60 degrees Celsius, 60-70 degrees Celsius, 70-80 degrees Celsius, 80-90 degrees Celsius, 90-99 degrees Celsius, 90-95 degrees Celsius, 95-99 degrees Celsius.

In one embodiment of the invention, said heating is carried out for 1-5 min, 1-10 min, 1-20 min, 1-30 min, 1-60 min, 1-180 min, 1-500 min, 1-2000 min, 5 sec, 10 sec, 30 sec, 1 min, 2 min, 3 min, 5 min, 7 min, 10 min, 30 min, 60 min, 180 min, 10 hours.

In one embodiment of the invention, said heating is carried out at a temperature increase rate of 0.1, 0.3, 1, 3, 10, 30, 100, 300 degrees/min.

In one embodiment of the invention, said heating is produced in a volume (stationary liquid), in a microfluidic channel (cell), in a flow.

In one embodiment of the invention, said heating is produced by microwave irradiation.

Microscopic and cytometric analysis

The mentioned cellular marker can be any clinically significant marker, in particular, UCTs have been created that specifically recognize EGFR, HER2, PD-L1, EpCAM, CEA, PSA, CA-125, CA 19-9, CA 15-3, CA 72-4, MUC1, CD19, CD20, CD44, CD133, CD166, CD24, CD47, CD117, DR-5.

Signal registration

The detected signal is recorded qualitatively, semi-quantitatively or quantitatively visually or on various equipment: fluorescence microscope, cytometer, cytofluorimeter, fluorimeter, luminometer.

Cross-linking

In one embodiment of the method, there is an additional step of cross-linking nanoparticles (UCT with polymer) to stabilize the composition, prevent desorption, etc.

for cross-linking of nanoparticles, they are mixed with bifunctional cross-linkers. This can be glutaraldehyde, formaldehyde, diglycidyl ether, genipin, etc. In one embodiment of the method, a cross-linker is used that leaves the particles or makes them highly specific in terms of recognition/binding to their biological target. In particular, cross-linking with formaldehyde or diglycidyl ether is preferable to using glutaraldehyde, since it usually allows for more specific particles. At the same time, for such tasks as non-specific transfection of cells with genetic material, it is preferable to use a cross-linking agent that does not lead to selectivity of particles, but on the contrary allows them to bind highly efficiently to various targets. For example, glutaraldehyde leads to the formation of Schiff bases, which have a partial positive charge, and therefore will effectively perform the task of binding DNA (which is negatively charged) for the purpose of delivering genetic material to cells.

Methods for immobilizing binding receptors on the membrane of a test strip are well known. Typically, the test and control binding receptors are applied to the membrane in parallel lines at a distance from each other. Solutions with the required substances in appropriate buffers are applied to the test strips using automated dispensers. After air drying for the required time, the membrane is blocked in a suitable buffer and stored 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 the sensitivity if a longer analysis time is used. In addition, the principle of this invention allows for direct measurement of the determined nucleic acid as an aealite without the need for its amplification, provided that its concentration is above the detection limit, and the signal associated with it can be recorded, for example, with the naked eye.

Measurements using the method within the framework of the present invention can be carried out both under stringent and mild conditions. "Stringent" conditions are understood to mean such values of temperature, ionic strength, and the presence of other compounds, at which hybridization of nucleic acids occurs. Under "high stringency" conditions, hybridization will occur only for nucleic acids having a high content of complementary nitrogenous bases. Thus, mild conditions are required in the case when hybridization or annealing of not completely complementary nucleic acids is necessary. A number of methods for creating mild conditions for hybridization are known to those skilled in the art. Hybridization under stringent conditions is possible only in the case of complete complementarity of sequences. Hybridization under less stringent conditions is possible for sequences with less than 100% identity. Changes in environmental conditions, such as a decrease in the salt concentration or an increase in temperature, can make it possible to reduce the stringency of hybridization conditions. Stricter hybridization conditions can be achieved by decreasing the ionic strength or increasing the hybridization temperature.

Thus, in one of the embodiments of the present invention, instead of using the said antibody as a recognition element, it is possible to use DNA/NK receptors. In this case, it is possible to carry out a single-stage, fully functional 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 NK 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 detecting NC under elevated temperature conditions, which expands the scope of its use in various fields, such as forensic examination. In addition to these advantages, the described method is a simple method for detecting nucleic acids and does not require serious training in this field, which can facilitate the penetration of genomic express analysis methods into the field of diagnostics at the patient's bedside and at the point of medical care.

Analysts

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, matrix, ribosomal, micro RNA; synthetic analogues of nucleic acids, such as peptide NA, morpholine, closed, glycolic and threose-NA; and also, due to the use of special recognition oligonucleotides (for example, aptamers) also molecules of non-nucleotide composition, for example, proteins and peptides, hormones, low-molecular physiological regulators, other haptens, metal ions, etc.

Calibration curve

In one embodiment of the method for quantitatively assessing the content of an analyte in a sample (measuring the concentration), an additional step is taken of comparing the obtained results with a calibration curve measured for samples with a known content of the analyte.

Multiplexity

This method is also applicable for the simultaneous determination of several analytes. In the simplest format, the test strip can be divided into separate strips, where separate binding zones are applied for the detection of different analytes. In addition, it is also possible to implement the simultaneous detection of different analytes in one sample. For this purpose, for example, separate binding zones can be applied to one membrane, but in its different areas.

Specific receptor/ligand pairs

Within the framework of the present invention (including instead of the mentioned antibodies), various specific pairs of the "receptor/ligand" type can be used, applicable for creating signal (reporter) conjugates and receptors binding them, as well as recognizing oligonucleotides or protein receptors with their analyte. The components of such pairs applicable in the present invention can be of both immune and non-immune types. Examples of immune binding can be systems based on the interaction of antigen/antibody or hapten/antihapten. Polyclonal, monoclonal antibodies or their immunoactive fragments can be prepared by standard methods known to those skilled in the art. The terms "immunoactive fragment of an antibody" or "immunoactive fragment" refer to parts 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 "half-molecular" fragments produced by reductive cleavage of the disulfide bonds linking the heavy chains of the parent antibody. If the antigen in the binding pair is not immunogenic, such as a hapten, it may be covalently linked to an additional carrier protein to render it immunogenic.

Non-immune specific pairs include systems in which two components exhibit a natural affinity for each other without being antibody and 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. They 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. (MN Bobrow, et al., J. Immunol. Methods , 1989,125:279).

Immunochromatographic (ICA) test strips

The ICA test strip is the environment in which the analysis is carried out using the present invention. The test strip typically consists of a chromatographic porous material and includes a sample application zone and a binding zone, as indicated above.

The chromatographic porous materials of which the strip consists are generally hydrophilic and/or can in principle be converted into such, and include the following materials, used either individually or in combination with other materials - granules of inorganic nature, such as 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, acetylcellulose, polyvinyl chloride, polyacrylamide, cross-linked dextran, agarose, polyacrylates, etc.; ceramic materials; glass; and similar materials. For the described invention, the preferred material is nitrocellulose.

In a preferred embodiment, the membrane may be either a separate structure in the form of a sheet cut into test strips, or may be fixed to a support or solid surface, such as in thin layer chromatography.

Binding of receptors to the membrane surface can be achieved by 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). As a rule, the pH values of the buffer for conducting an immunochromatographic assay are in the range of 4-11, more often 5-10, for the present invention, 6-9 is preferable. The pH values are selected so as to maintain the necessary level of affinity between the interacting pairs of molecules and to be optimal for signal formation by the corresponding system. Various buffers can be used to achieve the desired pH value and maintain it during the assay. Borate, phosphate, carbonate, Tris, diethylbarbiturate, etc. can be given as an illustration. 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.

For carrying out the analysis within the framework of the present invention, a constant temperature value is usually used. As a rule, for carrying out the study and obtaining the detectable 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 the ambient temperature, i.e. 15-25°C.

When using in one of the embodiments of the present invention, the recognition oligonucleotide and its binding (hybridization) with the target nucleotide sequence requires conditions that facilitate the hybridization of the NC. Hybridization is usually carried out in a 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 a solid phase, with one nucleotide chain immobilized on the test strip in the binding zone.

Examples

The present invention will now 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 carbon quantum dots.

The synthesis was carried out according to the protocol described in ¹ , with modifications in the purification process. 1 g of citric acid and 2 g of urea were dissolved in 8 ml of formamide, and the volume was brought up to 10 ml with formamide. The solution was transferred to a Teflon-lined autoclave and placed in a thermostat preheated to 160°C for 4 hours. The autoclave was then immediately cooled under running water.

The resulting dark orange mixture together with the aggregates was transferred to a 50 ml test tube and 20 ml of ethyl alcohol was added. The resulting mixture was centrifuged at 10,000 g for 5 min. The supernatant was discarded and 2 ml of water was added. The test tube was treated with ultrasound until the granule was completely dissolved (~ 1-2 min), and the volume was brought to 10 ml with water. The procedure was repeated once more. The resulting UCT solution in water was centrifuged at 10,000 g for 5 min to remove large aggregates. Then the resulting supernatant was transferred to a new test tube.

The final washing step was ultrafiltration through a regenerated cellulose membrane MWCO 100 kDa. UCT was filtered under a pressure of about 3 bar in the washing mode. Washing was carried out until the dialysate became almost colorless (at the beginning of filtration the dialysate was dark orange) for about 1 hour.

The retentate was then aliquoted and stored at -20°C. After defrosting, the aliquot was centrifuged at 15,000 g for 5 min, and the supernatant was used for further experiments within 1-2 days after defrosting.

Example 2. Study of the size and morphology of UCT using transmission electron microscopy.

TEM images of the UQDs were obtained at an accelerating voltage of 200 kV (Fig. 1a). UQD samples in water at a concentration of 10 μg/mL were air-dried on a silicon substrate on carbon tape and immediately analyzed. TEM images were processed using ImageJ software to obtain particle size distribution. It was shown that the UQDs have a near-spherical shape with an average diameter of 31 ± 7 nm.

Example 3. Measurement of the hydrodynamic diameter and zeta potential of the UCT

The UQDs were characterized by dynamic light scattering. A sample of UQDs in HEPES 10 mM pH 7.4 buffer at a concentration of 10 mg/L was analyzed using a nanoparticle size and zeta potential analyzer. The measurements were performed in triplicate and then averaged. The hydrodynamic size was (26 ± 4 nm) and the zeta potential was -33 ± 7 mV, the corresponding distributions are shown in Fig. 1c, d.

Example 4. Study of UCT using IR spectroscopy.

We studied the presence of chemical functional groups on the surface of the UQDs using Fourier transform IR spectroscopy (Fig. 1b). 2 mg of dried UQDs were ground in excess KBr and then formed into a tablet using a press. The spectrum of the resulting tablet was measured on a Fourier transform IR spectrometer using a reduced internal reflection module.

The broad band at 3122 cm ^-1 can be assigned to the OH stretching vibration in both carboxylic acid and alcohol. The peak at 1657 cm ^-1 can indicate the stretching of the C=O stretching in carboxylic acid, amide or aldehyde. These two peaks confirm the presence of carboxylic acid groups. Next, the narrow band at 1098 cm ^-1 indicates the stretching of the C-O stretching in alcohol or ether. Now we can state the presence of alcohol groups in the UCT sample. Finally, we attribute the peaks at 1339 and 1395 cm ^-1 to the OH bending vibrations, and the weak peak at 2785 cm ^-1 to the CH stretching vibrations.

Example 5. Study of optical properties of UCT

We studied the optical properties of UQD. The absorption spectrum has two peaks - at 370 nm and at 517 nm (Fig. 1g). The second of these peaks gives UQD a rich violet color.

The fluorescence excitation/emission heat map (Fig. 1e) reveals two distinct fluorescence regions. The first is located in the 350–450 nm excitation region. In this region, the location of the emission peak depends on the excitation wavelength. In the 470–590 nm excitation region, the emission spectrum is constant, with the emission peak at 636 nm. The maximum fluorescence intensity is observed at excitation/emission parameters of 530/630.

Example 6. Functionalization of UCT using polymers.

In a 200 μl test tube, 10 μl of biopolymer at a concentration of 0.1-1 g/l (bovine serum albumin, chitosan, dextran, anti-HER2 receptor antibody, anti-chloramphenicol antibody Anti-SAR, horseradish peroxidase, a mixture of horseradish peroxidase and anti-chloramphenicol antibody in a 1:1 ratio by weight, a mixture of horseradish peroxidase and anti-troponin antibody Anti-cTn in a 1:1 ratio by weight) were mixed with 20 μl of 0.1 M borate buffer, pH 7-10, and 10 μl of UKT, obtained as described in Example 1. The solution was thoroughly mixed with a pipette tip after each addition.

The tube was inserted for 1 min into the thermocycler well, preheated to 50-90°C, unless otherwise stated, with the lid temperature 5°C higher than the block temperature. The tube was then immediately transferred to a thermostat set at 16°C and held for 1 min.

The solution was then quickly mixed with 40 μl of 5 or 50% ammonium sulfate (AS) and mixed with a pipette tip. The tube was then centrifuged at 15,000 g for 5 min. 80 μl of Milli-Q water was very slowly added to the bottom of the tube and the tube was centrifuged for another 1 min at 15,000 g, and the supernatant was removed. Finally, the conjugates were resuspended in 10 mM borate buffer, pH 9, and sonicated for 3 s.

Obtaining UKT: (PEI, plasmid)

In the first step, the biopolymer was linear PEI at a concentration of 1 g/L. After heating and washing with AS as described above, the pellet was resuspended in the following solution: 18 μL Milli-Q, 2 μL plasmid at a concentration of 1 g/L, 20 μL 0.1 M borate buffer, pH 9. The tube in which the reagents were mixed was heated again and then washed using AS.

Example 7. The effect of synthesis temperature and pH of the working solution on the properties of UCT:protein conjugates.

The UCT:protein conjugates were prepared as described in Example 6, but at different temperatures (25 - 90°C) and at different pH (7.5 - 10) of the working solution.

Next, the fluorescence spectra of the obtained conjugates were studied. The fluorescence intensity (F) at a wavelength of 530/630 nm increased with increasing synthesis temperature (T) (Fig. 6a). The data are approximated by a power function F(T) = F0 +α (T - T ₀ )β ^, where F0, α, β were treated as unknown parameters, and T0 is room temperature (25°C). The most suitable β was found to be equal to 5.6, which indicates a sharp growth pattern with more than a threefold increase in fluorescence at T = 90°C compared to uncoated CQDs (Fig. 6a).

However, the fluorescence intensity upon excitation at 350-450 nm was greatly weakened regardless of the synthesis temperature. This attenuation is probably due to the coexistence of two UQD populations, one of which is not precipitated by the AS and is lost during the washing stage (Fig. 6 b-d).

On the other hand, the synthesis temperature also influences the antibody activity. The effect of the synthesis temperature and, additionally, the synthesis pH was then studied for the UKT:(Anti-CAP) conjugates (Fig. 6e, g). To quantify the antibody activity, we measured the binding of the conjugates to the test line in the immunochromatographic assay described in Example 16. We assume that stronger binding reflects the presence of more active antibody molecules in the conjugate, and vice versa.

Conjugates obtained at 70°C and below bind to the test strip almost equally strongly. However, at higher temperatures, almost no binding was observed. Thus, temperatures in the region of 60-70°C are optimal in terms of enhancing the fluorescence of UCTs and the activity of antibodies.

The effect of pH of the synthesis on the activity of the conjugates was studied. The pH was investigated in the neutral and alkaline ranges, since it is expected that acidic pH will disrupt the colloidal stability of the UCTs, since they are presumably stabilized by carboxylic acid groups. For the studied pH range of 7.5 - 10, the behavior of the conjugates in the immunochromatographic analysis was practically independent of pH.

Example 8. Immunochromatographic analysis for the presence of polysaccharides in conjugates of UCT with polysaccharides.

Lectins were applied to the surface of the test strip membrane, namely wheat germ agglutinin (WGA), which binds N-acetyl-D-glucosamine, which is an acetylated monomer of chitosan; and concanavalin A, which binds D-glucose, a monomer of dextran.

A 1 g/L solution of WGA or concanavalin A in 10 mM HEPES, pH 8.5, 0.1 mM CaCl2 buffer was applied to the membranes using a spray system at a flow rate of 4 μL/cm and then dried overnight. The test strips were cut using a guillotine cutting module and had a width of 2 mm.

Next, to prepare the sample, 10 µl of UCT:(chitosan) conjugates, 5 µl of Tween 20 at 5% concentration (w/v) and either 5 µl of running buffer or 5 µl of lectin at 1 g/l (as a negative control) were mixed.

Next, 20 µl of the sample prepared as described above was applied to the test strips by immersing them in this solution. After the analysis, the strips were scanned using a laser scanner with a resolution of 1200 dpi. The resulting TIFF images were analyzed using ImageJ software to calculate the intensity of the bands.

Both conjugates showed strong binding to the test strips, with binding being significantly weaker for the conjugates pre-mixed with lectins (Fig. 2), indicating the presence of polysaccharides on the surface of the UCTs.

Example 9. Cultivation of cell lines.

In these experiments, the cell lines HEK293, EMT 6/P, EMT-HER2 (a cell line stably expressing the HER2 transmembrane receptor, obtained from the EMT6/P line by transduction with a lentiviral delivery system carrying the gene encoding the HER2 receptor, followed by selection of HER2-positive clones) were used.

The cells were cultured under the following conditions: Dulbecco's modified Eagle Medium/Ham's F12 basic medium (1:1); 100 U/ml penicillin-streptomycin; 10 mg/l gentamicin; 10% fetal bovine serum; 300 mg/l L-glutamine; 5% CO2 at 37°C. Cells were detached from the surface of the culture flask using trypsin-EDTA.

Example 10. Carrying out microscopic analysis of a cell sample.

20,000 EMT and EMT-HER2 cells were seeded in a 96-well plate in 100 μl of medium and incubated overnight. Then, 100 μl of UKT:(Anti-HER2) conjugates, obtained as described in Example 6, were added to the cells at a concentration of 5 mg/l in the medium and incubated overnight.
Then, the medium was replaced with 200 μl PBS, and microscopic images of the cells were obtained using an epifluorescence microscope in the PerCP channel (excitation 560 nm with a bandwidth of 25 nm, emission 603 nm with a bandwidth of 40 nm) and in the bright field channel. The fluorescent signal from EMT-HER2 cells was significantly higher than that of EMT 6/P cells, confirming the specific interaction of UKT:(Anti-HER2) conjugates with cells via the HER2 receptor.

Example 11. Cytometric analysis of a cellular sample using UCT conjugates.

100,000 EMT 6/P and EMT-HER2 cells in 10 μl DMEM-F12 medium were mixed with 2.5 μl UKT:(Anti-HER2) conjugates prepared as described in Example 6, 5 μl 10% BSA and 80 μl PBS. The resulting mixture was incubated for 10 min, centrifuged at 100 g for 5 min and resuspended in 100 μl PBS.

The cells were then immediately analyzed using a flow cytometer. The UCT fluorescence was recorded in the 572-28 nm emission channel excited by a 488 nm laser. 5000 cells were collected in each sample. The strategy for selecting cytometric events is shown in Fig. 5a.

The staining index for the EMT-HER2 line was three times higher than the staining index for the EMT 6/P line, which confirms the specific interaction of the UKT:(Anti-HER2) conjugates with cells via the HER2 receptor (Fig. 5b)

Example 12. Measurement of nucleic acid concentration in the composition of the conjugate UKT:(PEI, plasmid)

The DNA concentration in the UCT:(PEI, plasmid) conjugate was determined using SYBR gold dye. 10 μl of the conjugates diluted 10-fold with milliQ water, 10 μl of the plasmid at a concentration of 0, 0.5, 2 or 5 mg/l and 80 μl of SYBR gold dye (diluted 20,000-fold in HEPES 0.1M buffer pH 7.4, according to the manufacturer's recommendations) were mixed and fluorescence was measured at excitation/emission parameters of 482-16/530-40. The concentration was determined as the intersection of the calibration curve with the abscissa axis. A representative calibration curve is shown in Fig. 4a; the typical DNA yield was 10%.

Example 13. Delivery of nucleic acid into a cell

20,000 HEK293 cells were seeded in a 96-well plate in 100 µl of medium and incubated overnight.

Then, 100 µl of UKT:(PEI, plasmid), obtained as described in Example 6 with a plasmid content of 1, 5, 10, 30 or 50 ng in the medium, were added to the wells.

As a control, 100 µl of a mixture of PEI and plasmid in a mass ratio of 3:1 with a plasmid content of 1, 5, 10, 30 or 50 ng in the medium were added to the wells.

The amount of nanoLuc protein produced by the cells was then determined as follows. 100 μl of lysis buffer (20 mM Tris HCl (pH 7.5), 150 mM NaCl, 0.5% NP-40, 5% glycerol) was added. After 10 min, 100 μl of the lysate was mixed with 100 μl of 1 mg/l coelenterazine in PBS in a 96-well black plate, and luminescence was immediately recorded without an emission filter.

The dependence of transfection efficiency on the plasmid concentration used for conjugates has an optimum at 10 ng, and at this concentration the UKT:(PEI, plasmid) conjugates exceed the usual PEI transfection by more than two times (Fig. 4b). Microfluorescence photographs of cells transfected with conjugates (at a plasmid concentration of 10 ng) are shown in Fig. 4d.

Example 14. Toxicity analysis of UKT conjugates:(PEI, plasmid)

2000 HEK293 cells were seeded in a 96-well plate in 100 µl of medium and incubated overnight.

Then 100 µl of UKT:(PEI, plasmid) with a plasmid content of 1, 5, 10, 30 or 50 ng in the medium were added to the wells.

As a control, 100 µl of a mixture of PEI and plasmid in a mass ratio of 3:1 with a plasmid content of 1, 5, 10, 30 or 50 ng in the medium were added to the wells.

The cells were incubated for 72 h. The medium was then removed, 100 μl of resazurin solution was added, and after 2 h, fluorescence was measured at excitation/emission parameters of 556-15/594-15 nm.

At higher concentrations, UQTs tend to aggregate and form films with large macroscopic agglomerates. Although UQTs remain virtually non-toxic at these concentrations, as shown by the resazurin toxicity test (Fig. 4c), the resulting films and aggregates likely prevent new transfectant portions from entering the cells, thereby reducing the amount of reporter protein produced.

Example 15. Preparation of BSA-CAP conjugate

CAP succinate was dissolved to a concentration of 100 g/L. EDC was dissolved in 50 mM MES, pH6, and then diluted two-fold with DMSO to achieve a final concentration of 100 g/L. NHS was first dissolved in DMSO and then diluted two-fold with 50 mM MES, pH 6, to achieve a final concentration of 100 g/L.

50 μl of CAP succinate were mixed with 18.3 μl of EDC solution, 13.6 μl of NHS solution, 18.1 μl of a mixture of 50 mM MES, pH 6 and DMSO (1:1 by volume). Activation was carried out for 1 hour at room temperature.

After incubation, 58.5 μl of BSA at a concentration of 100 g/l, 28.7 μl of activated CAP succinate and 42.8 μl of 0.1 M borate buffer, pH 8, were mixed. The tube was centrifuged for 1 min at 10,000 g, and the supernatant was used. The tube was incubated overnight.

To purify the conjugate, it was applied to ZEBA desalting columns (7 kDa MWCO) once according to the manufacturer's recommendations. The conjugate was stored in Milli-Q water.

Example 16. Immunochromatographic assay for detection of chloramphenicol in a sample.

The general scheme of the analysis is presented in Fig. 3a.

A 3 g/L BSA-CAP solution was applied to the membranes using a spray system at a flow rate of 4 μL/cm and then dried overnight. The test strips were cut using a guillotine cutting module and had a width of 2 mm.

Next, 2 μl of the UKT:(Anti-CAP) conjugate obtained as described in Example 6 were mixed with a solution of the following composition to be tested for the presence of the antigen: 2 μl of CAP succinate at the desired concentration, 2 μl of 10% BSA and 12 μl of HEPES 0.1 M pH 8.5 or milk (10%) diluted with the same buffer or blood serum (50%).

Next, 20 μl of the test solution were applied to the test strips by immersing them in this solution. Upon completion of the analysis, the strips were scanned using a laser scanner with a resolution of 1200 dpi or in a LumoTrace FLUO fluorescence tomograph (Abisense, Russia). The resulting images in TIFF format were analyzed using ImageJ software to calculate the intensity of the strips.

The UKT:(Anti-CAP) conjugates were able to bind specifically to the test line, demonstrating strong binding easily visible even to the naked eye (Fig. 3b) in the absence of CAP, and demonstrating no binding in the presence of CAP. The detection limit (3σ-criterion) of the test system was 9 ng/ml (2.1 μM), see Fig. 3c.

Example 17. Study of the circulation time of UKT conjugates in the bloodstream.

Mice were anesthetized by i.p. administration of a mixture of Zoletil (20 mg/kg) and Rometar (1.6 mg/kg). Then, 200 μg of CQD:chitosan conjugates prepared as described in Example 6 were injected retro-orbitally. At 1 min, 3 min, 10 min, 30 min, 3 h, 8 h, 24 h after injection, 20 μl of blood were collected from the retro-orbital sinus and immediately centrifuged (5 min, 500 g), and the plasma was collected and frozen at -20°C until analysis. Fluorescence of blood plasma samples was recorded at excitation/emission parameters of 530-8/630-8 nm. The curves of the CQD:chitosan conjugates from the blood circulation are shown in Fig. 7b.

For a typical experiment, the half-life of the UCT:Chitosan conjugates was 2 min.

Example 18. Ex vivo fluorescence bioimaging and biodistribution

Mice were retro-orbitally injected with 200 μg of UCT:chitosan conjugates in 200 μl of PBS. Then, 3 h after injection, mice were euthanized by cervical dislocation, and organs (liver, lungs, kidneys, spleen, muscles, bones, heart, tumor) and blood were removed and visualized with a 550 nm excitation diode and a 600+ nm emission filter using the LumoTrace FLUO bioimaging system (Abisens, Sirius, Russia). The resulting images in the fluorescence channel and in the bright field channel are shown in Fig. 7a. Preferential accumulation of conjugates is observed in the liver and spleen, as well as in the tumor.

Example 19. In vivo fluorescence bioimaging

Mice were retro-orbitally injected with 200 μg of UCT:chitosan conjugates in 200 μl of PBS. Then, 3 hours after the injection, the mice were anesthetized, shaved, and placed in the LumoTrace FLUO bioimaging system (Abisens, Sirius, Russia). Fluorescent images were taken as in Example 17. The signal in the liver and spleen was recorded, and a signal in the tumor was also recorded.

Example 20. Synthesis of fluorescent-bioluminescent labels.

In a 200 μl test tube, 10 μl of the biopolymer at a concentration of 0.1-1 g/l (nanoLuc luciferase, a mixture of nanoLuc luciferase and anti-chloramphenicol antibodies Anti-SAR in a 1:1 ratio by weight) were mixed with 20 μl of 0.1 M borate buffer, pH 7-10, and 10 μl of UCT, obtained as described in Example 1. The solution was thoroughly mixed with a pipette tip after each addition.

The tube was inserted for 1 min into the thermocycler well, preheated to 50-90°C, unless otherwise stated, with the lid temperature 5°C higher than the block temperature. The tube was then immediately transferred to a thermostat set at 16°C and held for 1 min.

The solution was then quickly mixed with 40 μl of 5 or 50% AS and mixed with a pipette tip. The tube was then centrifuged at 15,000 g for 5 min. 80 μl of Milli-Q water was very slowly added to the bottom of the tube and the tube was centrifuged for another 1 min at 15,000 g, and the supernatant was removed. Finally, the conjugates were resuspended in 10 mM borate buffer, pH 9, and sonicated for 3 s.

The obtained conjugates of UKT:(nanoLuc), UKT:(nanoLuc, Anti-CAP), washed from unbound proteins, had both fluorescent and bioluminescent properties: when adding

Example 21. Competitive enzyme immunoassay for the presence of chloramphenicol in a sample using UKT conjugates

Conjugates UKT:(nanoLuc, Anti-CAP) and UKT:(HRP, Anti-CAP) were synthesized as described in Examples 6 and 20.

To analyze the sample, a plate was prepared: 100 μl of BSA-CAP 0.1 g/l in carbonate-bicarbonate buffer (0.1 M, pH 9.6) were placed in the wells of 96-well ELISA plates and incubated at room temperature overnight. The solution was then removed and the wells were washed once with 100 μl of PBS solution (PBS with 0.05% Tween 20). After that, 100 μl of blocking buffer (1% BSA in PBS) were added to the wells. The plate was incubated for 1 h at room temperature and washed three times with PBST.

Next, the sample was analyzed directly: 100 μl of the following mixture was added to the well: 5 μl of the UCT:(HRP, Anti-CAP) or UCT:(nanoLuc, Anti-CAP) conjugates, 10 μl of CAP-succinate at a given concentration as a sample, 10 μl of 10% BSA and 75 μl of PBS. The plate was incubated for 1 h, washed three times with PBST, and PBS was added to the wells. The resulting fluorescent signal was measured at excitation/emission parameters of 530-8/630-8 nm. Finally, the solution was removed and the following substrate solutions were added:

A. For UCT conjugates: (HRP, Anti-CAP): 0.1 g/L TMB, 0.3 g/L H2O2 in acetate buffer (0.1 M pH 5). The plate was incubated for 30 min and 50 µL H2SO4 was added. Absorbance was measured at 450 nm.

B. For UKT: (nanoLuc, Anti-CAP): 1 mg/L coelenterazine in PBS and measured immediately without emission filter.

The dose-response curve obtained with fluorescence detection generally had the same profile as that obtained with enzymatic detection. The detection limit ranged from 1.4 to 29 ng/mL.

Example 22. Enzyme immunoassay (sandwich assay) for the presence of troponin in a sample using UKT conjugates

The UKT:(HRP, Anti-cTn) conjugate was synthesized as described in Examples 6 and 20.

To analyze the sample, a plate was prepared: 100 μl of Anti-cTn 0.1 g/l in carbonate-bicarbonate buffer (0.1 M, pH 9.6) were placed in the wells of 96-well ELISA plates and incubated at +4 degrees Celsius overnight. Then the solution was removed, and the wells were washed once with 100 μl of PBS solution (PBS with 0.05% Tween 20). After that, 100 μl of blocking buffer (1% BSA in PBS) were added to the wells. The plate was incubated for 1 hour at room temperature and washed three times with PBST.

Next, the sample was analyzed directly: 100 μl of serum samples with different troponin concentrations were added to the wells. The plate was incubated for 1 h at room temperature and washed three times with PBST with 1% BSA. Then, 5 μl of UCT conjugates: (HRP, Anti-cTb) and 75 μl of PBS with 1% BSA were added to each well. The plate was incubated for 1 h, washed three times with PBST, and PBS was added to the wells. The resulting fluorescent signal was measured at excitation/emission parameters of 530-8/630-8 nm. Finally, the solution was removed and a substrate solution was added: 0.1 g/l TMB, 0.3 g/l H2O2 in acetate buffer (0.1 M pH 5). The plate was incubated for 30 min and 50 µl H2SO4 was added. Absorbance was measured at 450 nm.

The dose-response curve obtained with fluorescence detection generally had the same profile as that obtained with enzymatic detection. The detection limit was 0.1 ng/mL.

Example 23. Averaging the results of analysis of the presence of an analyte in a sample carried out by multimodal agents.

The analysis is carried out as in Examples 21, 22, then the arithmetic mean value for the analyte concentration in the sample, obtained by the fluorescence of the UCT and by the enzymatic analysis, is calculated as the resulting value of the analyte concentration in the sample. This method allows for a smaller measurement error of the concentration when measuring the same sample several times in a row.

The essence of the invention is explained by the drawings Figs. 1-8.

Fig. 1 shows (A) TEM photographs of UQDs, scale bar 100 nm. The particle size, according to this photograph, is 31 ± 7 nm (distribution mean ± distribution standard deviation). (B) IR spectrum of UQDs. The designated peaks correspond to the characteristic absorption lines of carboxylic acids, alcohols, and amides, indicating their presence in the UQDs. (C) Distribution of hydrodynamic diameters of UQDs obtained by dynamic light scattering. The size is (26 ± 4) nm (distribution mean ± distribution standard deviation). (D) Absorption spectrum of UQDs in the UV and visible regions. Two peaks are observed - at 370 and 517 nm. The second of these peaks imparts a violet color to UQDs. (D) Distribution of zeta potentials of UQDs obtained by dynamic light scattering. The zeta potential is -(33 ± 7) mV (mean of the distribution ± standard deviation of the distribution). (E) Heat map of fluorescence intensity as a function of excitation and emission wavelength. Brighter pixels reflect brighter fluorescence. Two main regions are observed: in the 350-450 nm excitation region, in which the location of the emission peak depends on the excitation wavelength; and in the 470-590 nm excitation region, in which the emission spectrum is constant, with an emission peak at 636 nm. The maximum fluorescence intensity is observed at excitation/emission parameters of 530/630.

Fig. 2 shows photographs of test strips for detecting polysaccharides on the surface of UCT. (A) Concanavalin A, which recognizes glucose, a dextran monomer, is immobilized on the test strip. On the leftmost strip, strong binding of UCT:dextran conjugates to the test line is observed. On the middle strips, the binding of congates pre-mixed with excess Concanavalin A is significantly weaker. On the left strips, BSA is immobilized instead of on the test line; no binding of conjugates to these strips is observed.

(B) The test strip contains immobilized WGA lectin, which recognizes N-acetyl-D-glucosamine, a dextran monomer. The leftmost strip shows strong binding of UCT:chitosan conjugates to the test line. The middle strips show significantly weaker binding of conjugates premixed with excess WGA. The left strips contain immobilized BSA instead of the test line; no binding of conjugates to these strips is observed.

Figure 3 shows (A) Schematic diagram of the immunochromatographic assay for detection of the analyte in a sample (B) Comparison of the intensity of test line staining in the presence (right) and absence (left) of the analyte, in this case, CAP. (C) Dependence of the intensity of test line staining on the concentration of CAP in the sample. The detection limit (3σ criterion) is 9 ng/mL.

Fig. 4 shows (A) Representative calibration curve for measuring plasmid concentration in UKT:(PEI, plasmid) conjugate using SYBR gold dye. Typical plasmid yield is 10%. (B) Transfection efficiency using UKT:(PEI, plasmid) conjugates and conventional transfection using PEI as a function of plasmid concentration. The optimum for the conjugates is observed at a plasmid concentration of 10 ng/well of a 96-well plate. (C) Toxicity study of PEI and UKT:(PEI, plasmid) conjugates using the resazurin test. At all concentrations, no statistically significant difference was observed between the control and test groups (n = 3). (D) Micrographs of cells with added UCT:(PEI, plasmid) at 10 ng per well and control cells, obtained in the bright field channel and in the fluorescence channel.

Figure 5 shows (A) Scheme of cytometric event selection. First, events with too low forward scatter value are rejected (left), then events with too high FSC-A parameter value for a given FSC-H value (center), and then the intensity histogram in the PerCP-H fluorescent channel is analyzed (right). (B) Intensity histograms in the PerCP-H fluorescent channel for EMT and EMT-HER2 cell lines interacted with UKT:(Anti-HER2) conjugates. Significantly more efficient binding of the conjugates to EMT-HER2 cells is observed.

Fig. 6 shows: (A) dependence of fluorescence intensity of UQD:(Anti-HER2) conjugates on synthesis temperature. At synthesis temperature of 90°C more than threefold increase in fluorescence is observed compared to uncoated UQD. (B)-(D) heat maps of fluorescence intensity depending on excitation and emission wavelength of UQD and their conjugates. Significant increase in fluorescence is observed in the excitation region of 470-590 nm during conjugation, with simultaneous decrease in the excitation region of 350-450 nm.

(E, G) Binding efficiency of UKT:(Anti-CAP) conjugates with test strips coated with BSA-CAP, depending on the pH of the working solution during synthesis (E) and on the synthesis temperature (G). pH has little effect, while binding efficiency decreases with increasing temperature.

Figure 7 shows (A) ex vivo biodistribution of UCT:Chitosan conjugates. The upper panel shows fluorescent images of organs (excitation 550 nm, emission 600+ nm), the lower panel shows bright field images of organs. Significant accumulation of conjugates is seen in the liver, spleen and tumor. (B) Kinetics of UCT:Chitosan conjugates elimination from the bloodstream. The half-life is 1.87±0.25 min (n = 4).

Fig. 8 shows the results of the enzyme immunoassay of the sample for the presence of CAP using UQT conjugates. (a) Scheme of the assay. In the presence of CAP in solution, UQT modified with an antibody to CAP do not bind to the stationary phase. (b, c) dose-effect curves obtained using UQT:(Anti-CAP, nanoLuc). (d, e) dose-effect curves obtained using UQT:(Anti-CAP, HRP). For both conjugates, the left graphs (b, d) show the results for fluorescent detection of the signal, and the right ones - for enzymatic detection. The detection limits are 29, 1.4, 11.7, 3.2 ng/ml for (b, c, d, e), respectively.

Claims (20)

1. A method for conjugating carbon quantum dots with a polymer, which comprises the following steps: i) mixing a colloidal solution of carbon quantum dots with a polymer solution, where the polymer is a biopolymer selected from the set: protein, carbohydrate, nucleic acid, polyethyleneimine, ii) heating the resulting solution to 50-99 degrees Celsius, iii) increasing the ionic strength of the resulting solution to precipitate the said polymer onto the surface of the carbon quantum dots. 2. The method according to item 1, in which the obtained conjugates are additionally washed from molecules of unbound polymer. 3. The method according to item 2, wherein said washing of the obtained conjugates is carried out with a solvent from the set: ethanol, methanol, water, acetone. 4. The method according to item 2, wherein said washing of the obtained conjugates is carried out by ultrafiltration. 5. The method according to item 2, wherein said washing of the obtained conjugates is carried out by centrifugation. 6. The method according to claim 1, wherein said polymer is a mixture of several polymers. 7. The method according to claim 1, wherein said polymer is a gamma-immunoglobulin. 8. The method according to claim 1, wherein the size of the resulting conjugates is 30-250 nm. 9. The method according to claim 1, wherein the size of the resulting conjugates is 50-150 nm. 10. The method according to claim 1, wherein the size of the resulting conjugates is 100-250 nm. 11. The method according to item 1, wherein said heating of the resulting solution is carried out to 55-65 degrees Celsius. 12. The method according to item 1, wherein said heating of the resulting solution is carried out to 85-95 degrees Celsius. 13. The method according to claim 1, wherein said increase in the ionic strength of the obtained solution for precipitating said polymer onto the surface of carbon quantum dots is carried out by adding a salt solution to said obtained solution. 14. The method according to claim 13, wherein said salt is ammonium sulfate. 15. The method according to claim 13, wherein said salt is NaCl, Na ₂ SO ₄ , MgSO ₄ , (NH ₄ ) ₂ SO ₄ , KCl, MgCl ₂ . 16. The method according to claim 1, which further comprises the step of adding a cross-linking agent. 17. The method according to claim 16, wherein said cross-linking agent is selected from the list: epichlorohydrin, glutaraldehyde.