RU2440575C1 - Method of determining physiological concentrations of heparin in analysed liquid samples - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method includes creation of integral DNA microchip, possessing anomalous optic properties when irradiated by flow of circularly polarised irradiation and chromophoric properties when irradiated in visual spectrum range and representing placed in water-salt polymer solution particles of lyotropic cholesteric liquid-crystalline dispersion of rigid molecules of double-stranded nucleic acids, in which adjacent molecules are bound with nanobridges, which contain alternating inert to heparin constructive element and heparin-sensitive element, and integral DNA microchip as heparin-sensitive element contains compound of anthracycline series, which has at least one reaction-able amino group, bound with sugar residue. Solution, containing integral DNA microchips, is mixed with heparin-containing sample. Through sample, resulting from said mixing, by means of optic dichrometre, passed is flow of circularly polarised irradiation at wavelength in region of heparin-sensitive element absorption, optic signal is registered.

EFFECT: application of invention makes it possible to simplify and accelerate process of diagnostics.

8 cl, 2 ex, 8 dwg

Description

Technical field

The invention relates to the field of medical research and nanotechnology based on double-stranded nucleic acids, and in particular to methods for diagnosing pathological conditions accompanied by thrombosis, in particular, to ensure monitoring of the condition of patients whose therapy is associated with the use of heparin.

State of the art

Heparin is an acid mucopolysaccharide produced by mast cells and performing mediator functions in all organs of humans and animals and in many body fluids. It consists of several linear polysaccharide chains with a molecular weight of 5000-30000 Da, consisting of glucuronic acid and glucosamine residues esterified with sulfuric acid.

The heparin molecule contains a significant amount of negatively charged sulfate and carboxyl groups, therefore it is a strong polyanion capable of forming complexes with many natural and synthetic cations.

Structural features and a high degree of chemical heterogeneity of heparin determine its biological significance and functional role in humans and animals. Being a natural anticoagulant factor, heparin is most widely known as a direct type of anticoagulant used for the prevention and treatment of thromboembolic diseases and their complications, in operations on the heart and blood vessels, to maintain the liquid state of the blood in cardiopulmonary bypasses and hemodialysis, as well as to prevent blood coagulation in laboratory studies. The anticoagulant activity of heparin is due to its ability to increase the spectrum of serine proteases inhibited by antithrombin III involved in blood coagulation.

In clinical practice, two types of heparin are used - standard (non-fractional) and low molecular weight.

Non-fractional heparin is used more widely, however, it has a number of significant disadvantages:

- a high degree of binding of heparin to plasma proteins and its rapid inactivation by endothelial cells and macrophages;

- the relative short duration of the action of heparin, which requires constant intravenous infusion during the day (or multiple subcutaneous injections of the drug);

- heparin has no aftereffect, and after the end of its use, the conditions for the formation of a blood clot are restored. Thrombotic process activation is possible, which is expressed in an increase in the number of episodes of myocardial ischemia and the development of myocardial infarction.

To eliminate these undesirable properties, low molecular weight heparin is used - preparations in which the molecular weight of the fragments ranges from 2500 to 6500 Da, synthesized from non-fractional heparin.

Low molecular weight heparins inhibit the blood coagulation cascade at a higher level - at the level of factor Xa, have a high bioavailability - more than 90% after deep subcutaneous injection. However, they inhibit the formation of a certain amount of thrombin, which can lead to the development of thrombocytopenia.

The safety of the clinical use of heparin can only be guaranteed by the rational administration of its antagonists, which are able to quickly and without toxic manifestations eliminate the excessive anticoagulant effect of heparin. Difficulties in the restoration of blood coagulation when using heparin antagonists largely depend on the nature of the distribution of heparin in the patient's body. Therefore, to calculate the neutralizing doses of heparin antagonists, it is required to determine the concentration of heparin in the blood in each case. The rationality of the use of antiheparinates mainly depends on the accuracy and speed of obtaining information on the amount of heparin circulating in the bloodstream, which is the main problem of the safe use of heparin as an anticoagulant.

The effectiveness of methods for determining heparin depends on the principle on which they are based.

Coagulation methods are known based on determining the inhibitory effect of heparin with respect to thrombin or factor Xa, for example, the so-called integral methods based on the ability of heparin to lengthen the blood coagulation time as a result of inhibiting the formation of a fibrin clot.

However, despite its simplicity, integrated methods for determining heparin concentration are characterized by a number of disadvantages, which include:

- lack of specificity (the results of integral methods, in addition to the action of heparin, are affected by various changes in coagulation in vivo);

- low sensitivity (integrated methods are practically not sensitive to low (<0.1 international units of heparin activity in 1 ml (U / ml)) heparin concentrations;

- low reproducibility (the relative error in determining heparin concentration using integral methods is ~ 20-25%).

To determine the concentration of heparin in blood plasma, methods based on the use of chromogenic peptide substrates specific for thrombin or factor Xa are also used. In these tests, chromogenic peptide substrates act as “reporter” molecules, by changing the optical properties of which (colorimetric or fluorometric), one can monitor the rate of heparin-catalyzed enzymatic inactivation of antithrombin or factor Xa by antithrombin III. The test conditions are selected so that the enzymatic reaction rate is linearly related to the heparin level in the sample. However, the results of analyzes carried out using chromogenic peptide substrates are influenced by the binding of heparin to plasma protein components having an antiheparin nature.

Colorimetric and fluorometric methods for the determination of heparin are characterized by higher accuracy and stable sensitivity to heparin (up to 0.01 Units / ml) compared with methods based on the formation of a clot, are more convenient for clinical examination and control, but due to their complexity and quite high cost of their production is possible only in well-equipped specialized laboratories with expensive equipment and highly qualified personnel.

Chemical methods are known by which they mainly measure the amount of heparin as a substance, and not its anticoagulant activity, and are based on the detection of heparin as a mucopolysaccharide.

For this, for example, the carbazole reaction of hexuronic acid or the acetylacetone condensation of hexosamines with the quantification of subsequent pyrrole derivatives (derivatives) using p-dimethylaminobenzaldehyde (Erlich reagent) are used. These methods are unsuitable for determining heparin in blood plasma, are characterized by the duration of the analysis - ~ 1 hour or more, and are not very sensitive: they can be used to detect heparin concentrations of more than 12 μg / ml or 1.6 units / ml.

Another group of chemical methods is based on the ability of heparin as a mucopolysaccharide to cause a metachromatic effect when it interacts with some of the main dyes - azure A, toluidine blue, and carbocyanine dye. The intensity of metachromasia during the formation of the heparin-dye complex at the optimal concentration and volume of the solution of the added dye is proportional to the concentration of heparin, and this value can be determined spectrophotometrically. The minimum concentration of heparin, determined using metachromatic methods, is ~ 10 μg / ml or 1.0 units / ml of its activity. The relative error in determining the concentration of heparin using this group of methods is 10-15%.

However, metachromatic methods, like methods based on the use of chemical reactions specific for heparin, are also unsuitable for determining heparin in blood plasma, are quite expensive, have poor reproducibility and are not accurate enough. The last two drawbacks are due to the precipitation of the resulting heparin-dye complex and the sensitivity of the absorption readings of this complex to temperature.

It is known that the practice of clinical medicine, as a rule, includes the analysis of physiological fluids (blood, urine, cerebrospinal fluid, etc.) of the patient in order to determine the level of biologically active substances (hereinafter referred to as BAS) that determine the state of his body.

Known biotechnological methods for determining the content of biologically active substances in physiological fluids, in particular portable universal biosensor test systems (Biosensors: fundamentals and applications. / Ed. Turner E., Karube I., Wilson J. M.: Mir, 1992, 614 p. .), which consist of two fundamental components: a "sensitive" element - a "biosensor" created on the basis of biological molecules, which has the ability to change its optical properties when interacting with biologically active substances, and a system for detecting and converting an optical signal biosensor generated by interaction with a determined biologically active analyzer (Skuridin S.G., Dubinskaya V.A., Lagutina M.A., Rebrov L.B., Bykov V.A., Evdokimov Yu.M. Biosensors based on liquid crystal structures of DNA: creation and applications in biotechnology and medicine. // Technologies of living systems, 2006, v.3, No. 4, pp.3-27).

Biotechnological methods using biosensors have a number of advantages over traditional u1084 methods of analytical chemistry, namely, high sensitivity, selectivity for determining biologically active substances, the absence of the need for special sample preparation, the absence of special requirements for working areas and the qualifications of the personnel conducting the analysis, and the possibility of obtaining an analysis result in real time (Evdokimov Yu.M., Bundin BC, Ostrovsky MA Biosensors and sensory biology. // Sensory systems Topics, 1997, t.11, №4, s.373-387).

Known principles for the functioning of biosensors based on DNA molecules (Yevdokimov Yu.M., Skuridin SG, Chernukha BA The Background for creating biosensors based on nucleic acid molecules. // Advances in Biosensors (Eds. APFTurner and Yu.M. Yevdokimov); JAI Press Inc., London, 1995, v.3, p. 143-164; Skuridin SG, Yevdokimov Yu.M., Efimov VS, Hall JM, Turner APF A new approach for creating double-stranded DNA biosensors. // Biosens. Bioelectron., 1996, v. 11, No. 9, p. 903-911).

There are studies of the possibilities of constructing biosensors of biosensors for determining biologically active substances based on double-stranded nucleic acids (Evdokimov Yu.M., Salyanov V.I., Mchedlishvili B.V., Bykov V.A., Spener F., Palumbo M. Molecular construction based on double-stranded nucleic acids and synthetic polynucleotides to create an integrated biosensor. // Sensory systems, 1999, v.13, No. 1, p. 82-91).

It has been observed that liquid DNA crystals have a number of distinctive features, in particular:

- high packing density and the ordered nature of the packing of DNA molecules in liquid crystals, which practically coincide with similar parameters characteristic of DNA as a part of biological objects;

- high lability of the spatial structure of liquid crystals in response to changes in external conditions and, finally,

- the anomalous optical activity observed during the formation of liquid crystals of cholesteric type DNA, which is manifested, in particular, in the presence of an intense band located in the absorption region of nitrogenous DNA bases (λ≈260 nm) in the circular dichroism spectrum (hereinafter CD) when these crystals are irradiated a stream of circularly polarized radiation in the ultraviolet region of the spectrum.

The amplitude of this band can be tens or hundreds of times greater than the amplitude of the band in the spectrum of circular dichroism (CD), due to the molecular optical activity characteristic of the initial linear DNA molecules.

Such biosensors are called “integrated microarrays based on DNA nanoconstructions” (Yevdokimov Yu.M., Salyanov VI, Zakharov MA A novel type of microscopic size chips based on double-stranded nucleic acids. // Lab on a Chip, 2001, v. 1, No 1, p. 35-41).

As a promising biomaterial used to create multifunctional biosensors of biosensor devices, particles of lyotropic liquid crystal dispersions of linear double-stranded DNA molecules (hereinafter CLCD) and their complexes with biologically active substances (Skuridin SG, Evdokimov Yu.M. Particles of liquid crystal DNA dispersions as the basis for the creation of sensitive elements of biosensors. // Biophysics, 2004, T. 49, No. 3, S. 468-485).

Moreover, DNA molecules fixed in the spatial structure of CLCD particles are used as “building blocks” between which it is possible to “embed” artificial nanoscale “nanobridges” containing “sensitive” elements that can easily “respond” to the presence of various chemical nature (Yevdokimov Yu.M., Skuridin SG, Nechipurenko D.Yu., Zakharov MA, Salyanov VI, Kurnosov AA, Kuznetsov VD, Nikiforov VN Nanoconstructions based on double-stranded nucleic acids. // Int. J. Biol. Macromol. , 2005, v. 36, No. 1-2, p. 103-115).

Known liquid crystal biosensors for the determination of biologically active substances (US, 6246470, B1; RU, 2032895, C1), which are a dispersed phase of linear double-stranded DNA molecules crosslinked by substrate molecules for a defined biologically active substance distributed in a water-polymer matrix, using a substrate subjected to hydrolysis under the influence of biologically active substances.

The principle of the formation and operation of the sensor is that first a DNA – natural compound complex is formed from DNA and natural compounds, then a dispersed liquid crystalline phase is formed from this complex and molecules of the natural compound are split into DNA – natural compound complexes, which form liquid crystals, using a defined BAS, which is a proteolytic enzyme, for example, stellin B. As a result of the action of the enzyme, cross-linking between DNA molecules formed by natural molecules compounds are destroyed, and the liquid crystals themselves pass from an optically inactive to an optically active state. The change in the anomalous optical activity, in particular, the DNA spectra, observed during the rearrangement of the liquid crystal structure, allows not only to detect the presence of the enzyme in the test solution, but also opens up the possibility of determining its type, class, or group by the nature of the observed changes.

It was found that the main factor in the stabilization of integrated DNA microarrays is not the osmotic pressure of the aqueous polymer solution, but the number and “strength” of nanobridges (Skuridin S.G., Dubinskaya V.A., Zakharov M.A., Lagutina M.A. , Mineeva M.F., Rebrov L.B., Bykov V.A., Evdokimov Yu.M. Identification of pharmaceutical substances with complexing properties using a bioanalytical test system based on integrated liquid crystal DNA microarrays // Liquid crystals and their practical use, 2005, No. 3-4, p.64-74; Yevdokimov Yu.M., Salyanov V. I., Lortkipanidze GB, Gedig E., Spener F., Palumbo M. Sensing biological effectors through the response of bridged nucleic acids and polynucleotides fixed in liquid-crystalline dispersions. // Biosens. Bioelectron., 1998, v.13, p .279-291).

Therefore, it is possible to obtain DNA microarrays that are stable over a wide range of conditions and can exist outside the formation of particles of lyotropic CLCD DNA (Evdokimov Yu.M., Salyanov V.I., Skuridin S.G. From liquid crystals to DNA nanostructures. // Molecule. Biology, 2009, vol. 43, No. 2, pp. 309-326).

A known method for the determination of heparin in laboratory solutions, based on differences in the spatial organization of CLCD complex "DNA - heparin antagonist" and complex "CLCD DNA", as well as the ability of heparin to interact with its synthetic antagonist - polyconidine, included in the CLCD complex "DNA - heparin antagonist "(RU, 2123008, C1). This method includes:

- the formation of CLCD complex "DNA - heparin antagonist" in a water-salt solution of moderate (physiological) ionic strength;

- mixing the resulting dispersion with a neutral in relation to DNA, heparin antagonist and heparin aqueous-salt solution of the polymer;

- mixing the resulting mixture with the analyzed liquid containing heparin, under conditions in which the optical properties of the CLCD complex of the "DNA - heparin antagonist" are not violated;

- registration of the spectrum of circular dichroism (anomalous optical activity) in the region of absorption of nitrogenous DNA bases when passing through the sample obtained by said mixing a circularly polarized radiation flux in the ultraviolet region of the spectrum;

- determination of the concentration of heparin in the sample using the previously obtained calibration line;

- adjustment of the concentration of heparin in the analyzed fluid, taking into account the dilution used in the preparation of the sample.

Despite the high accuracy and sensitivity of this method for determining heparin, its practical capabilities are limited by the following factors:

- the need to concentrate heparin in the process of sample preparation of physiological fluid for analysis, so that its mixing with a water-salt polymer solution containing CLCD of the DNA – heparin antagonist complex does not lead to a decrease in the polymer concentration to values below the “critical” concentration at which CLCD of DNA not formed (Yevdokimov Yu.M., Skuridin SG, Lortkipanidze GB Liquid-crystalline dispersions of nucleic acids. // Liq. Crystals, 1992, v. 12, No. 1, p. 1-16);

- the use of expensive stationary commercial dichrographs, providing a circularly polarized radiation flux in the ultraviolet region of the spectrum and recording an anomalous optical signal, for example, Jobin-Yvon Mark III or Mark V; "Jasco", Model-710/720, available, as a rule, only in specialized scientific laboratories. The disadvantage of these devices is not only their high cost, but also the low speed of recording an optical signal.

Thus, the above disadvantages significantly complicate the rapid obtaining of accurate information on the concentration of heparin in the analyzed samples and limit the widespread use of the above groups of methods for conducting such analyzes in the laboratory.

Therefore, the development of modern high-precision, simple and inexpensive express methods for determining the level of heparin is an urgent problem of clinical biochemistry and medical instrumentation.

An integral microchip is known, which is a CLCD DNA particle in which neighboring nucleic acid molecules are “crosslinked” by nanobridges (FIG. 1), consisting of alternating Cu2 + ions and anthracycline compounds — antibiotics that form polymer chelate crosslinks (RU, 2139933, C1), in which copper ions have magnetic properties and contribute to the formation of a spatially oriented structure of CLCD particles of DNA and nanobridges (Nikiforov V.N., Kuznetsov V.D., Nechipurenko Yu.D., Salyanov V.I., Evdokimov Yu. M. Magnit e properties of copper in the composition nanobridges formed in the space between the fixed DNA molecules. // JETP Letters, 2005, t.81, s.327-329). In this case, the nanobridge includes 6 Cu2 + atoms and 5 DAU molecules (Fig. 2). The use of these CLCD DNA particles as an integral biosensor is due to the fact that the molecules of many chemical and biologically active compounds form complex compounds with Cu2 + ions, "pulling" these ions out of the structure of the chelate complex with 4 oxygen atoms in the two subsequent molecules of the anthracycline compound. Such a process is accompanied not only by the violation of the integrity of the entire polymer "crosslinking", but also by a corresponding decrease in the anomalous optical activity of the molecules of the indicated CLCD DNA. The magnitude of the decrease in anomalous optical activity is related to the concentration of a chemical or biologically active compound present in the analyzed fluid and destroying the polymer chelate “crosslinking”. The formation of an integrated microchip is accompanied by the appearance of an anomalous optical signal in the spectrum of circular dichroism (CD) in the absorption region of the antibiotic (λmax ~ 515 nm) when the microchip is irradiated with a circularly polarized radiation flux in the visible spectrum. The presence of this signal allows not only monitoring the state of the secondary structure of DNA molecules forming the microchip, but also the integrity of the nanobridges themselves. Therefore, an abnormal optical signal can be used as an analytical criterion, which allows one to determine analytes whose “target” is the “building blocks” of the microchip, in particular, the structural elements of nanobridges, in particular Cu2 + ions and antibiotic molecules.

However, the selective ability of the integrated biosensors described above (RU, 2139933 C1) to detect the presence of heparin in physiological solutions by changing the amplitude of the anomalous optical signal generated by the biosensor when it was irradiated with a circularly polarized radiation flux in the ultraviolet region of the spectrum was not detected.

Disclosure of invention

The aim of the present invention was to provide a method for determining the physiological concentrations of heparin in the analyzed liquid samples using a bioanalytical test system with such conditions that would allow you to quickly, simply, accurately and reliably determine its concentration, including a very low ~ 0.5 -5.0 μg / ml, in physiological solutions.

When creating the invention, the task was to create a method that provides highly sensitive detection of heparin by detecting and recording changes in the optical activity of a biosensor contacting with heparin when it is irradiated in the visible spectrum, caused by the interaction of heparin only with a specific sensitive element of the biosensor, while eliminating the simultaneous interaction of heparin with other biologically active compounds in the studied physiological saline and excluding the necessary The preliminary preparation of the analyzed sample is related to an increase in the concentration of heparin in it.

The problem was solved by creating a method for determining the physiological concentrations of heparin in the analyzed liquid samples according to the invention, which includes recording an optical signal of a biosensor placed in the indicated sample, which is particles of a lyotropic cholesteric liquid crystal dispersion of rigid double-stranded nucleic acid molecules placed in an aqueous salt solution of the polymer, in which neighboring nucleic acid molecules are connected by nanobridges containing hepar sensitive an element, and having anomalous optical properties when irradiated with a circularly polarized radiation flux, characterized in that it uses an integrated biosensor containing, as a heparin sensitive element, an anthracycline series compound having at least one reactive amino group associated with a sugar residue, and having chromophore properties when irradiated in the visible region of the spectrum, and the method includes:

a) the formation of an integrated biosensor with chromophore properties when irradiated in the visible region of the spectrum, containing in the nanobridges a structural element that is not sensitive to heparin, and a sensitive element to heparin, while doing this:

a1) the formation of particles of a cholesteric liquid crystal dispersion from nucleic acid molecules having a molecular weight of less than 1 × 10 ⁶ Yes, in a water-salt polymer solution capable of excluding said molecules from its composition;

a2) adding a liquid-crystalline dispersion of nucleic acids to the aqueous salt solution of the polymer obtained in step a1), a solution of the compound selected as the heparin-sensitive element, to obtain a dispersion of the nucleic acid-sensitive element complex, in which u1088 are reactive groups of the sensitive element sensitive to heparin;

a3) processing the dispersion obtained in operation a2) with a solution of a compound selected as a heparin-insensitive structural element to obtain a dispersion in which adjacent nucleic acid molecules are connected by nanobridges, which are flat chelate complexes "structural element - sensitive element - structural element ... structural element - sensitive element - structural element ”;

a4) the subsequent placement of the dispersion obtained in operation a3) in a water-salt solution of a polymer optically isotropic and chemically neutral with respect to nucleic acid, to a structural element that is not sensitive to heparin, and to a sensitive element;

a5) measuring the anomalous optical activity of the obtained integrated biosensor at two wavelengths in the visible region of the spectrum, the first wavelength being selected in the absorption region of the nitrogenous nucleic acid bases and used as the internal standard, and the second wavelength selected in the absorption band of the sensitive element, and a multiple increase in the optical signal in the absorption band of the sensing element determines the initial characteristics of the obtained integrated biosensor;

b) determination of the calibration characteristics of the anomalous optical activity of the obtained integrated biosensor in the visible spectrum upon irradiation of calibration samples containing a mixture of the specified polymer solution of the integrated biosensor obtained in step a4) and a solution of heparin of known concentration, with a circularly polarized radiation flux at the specified wavelength in areas of absorption of the sensing element;

c) mixing the obtained polymer solution of the integrated biosensor obtained in operation a4) with the analyzed liquid sample;

d) when the mixture obtained in operation c) is irradiated with a circularly polarized radiation flux at a specified wavelength in the absorption region of the sensing element in the visible spectrum, an optical signal is recorded and the presence and concentration of heparin in the analyzed liquid sample are determined based on calibration characteristics.

Moreover, according to the invention, it is possible to use double-stranded synthetic or natural polynucleotides as nucleic acid molecules capable of forming a cholesteric liquid crystal dispersion during phase exclusion while maintaining their chemical reactivity and anomalous optical properties.

Moreover, according to the invention, it is desirable to use rigid chain polymer molecules as nucleic acid molecules.

Moreover, according to the invention, it is advisable to use metal ions capable of forming extended flat chelate complexes as structural elements of nanobridges.

Moreover, according to the invention, it is possible to use a copper ion as a structural element of nanobridges.

Moreover, according to the invention, it is possible to use an anthracycline series compound selected from the group consisting of rubomycin, violomycin, adriamycin, carminomycin, premycin, doxorubicin, daunomycin as a heparin-sensitive element of nanobridges.

Moreover, according to the invention, it is advisable to use daunomycin (DAU) as a heparin sensitive element.

Moreover, according to the invention, it is reasonable to use a solution of polyethylene glycol as a water-salt solution of a water-soluble, optically isotropic and chemically neutral with respect to nucleic acid, structural element and sensitive element of the polymer.

Brief Description of the Drawings

The invention is further illustrated by examples of its implementation and the accompanying drawings, in which:

Figure 1 - integrated biosensor according to the invention, a schematic representation of the structure of the nanobridge between two adjacent DNA molecules, fixed in the spatial structure of the particles CLCD;

Figa - absorption spectrum of DAU in the absence of heparin (curve 1) and absorption spectra of the complex "DAU-heparin" (curves 2-5) obtained by processing the DAU with heparin solutions of different concentrations, in coordinates "optical density A - wavelength λ" ;

Fig.2b - absorption spectra of the complex "DAU-heparin" (curve 1) and absorption spectra of the complex "DAU-heparin" after processing them with solutions of a heparin antagonist - polyconidine (curves 2-5) of different concentrations, in coordinates "optical density A - length waves λ ";

Figure 3 - CD spectrum of lyotropic CLCD, formed as a result of phase exclusion of DNA molecules from a water-salt solution of a PEG polymer, in coordinates "change in optical density ΔА = (AL-AR) - wavelength λ";

Figure 4 - CD spectra of lyotropic CLCD DNA before processing (curve 1) and the same dispersion after its sequential processing of DAU and CuCl2 (curve 2) in the coordinates "change in optical density ΔA = (AL-AR) - λ - wavelength";

Figure 5 - CD spectra of the integrated biosensor according to the invention before (curve 1) and after (curve 2) interaction with heparin, and the CD spectrum of lyotropic CLCD complex of DNA-DAE (curve 3), in the coordinates "change in optical density ΔА = (AL- AR) is the wavelength λ ";

6 - dynamics of the destruction of the integrated biosensors according to the invention under the influence of different concentrations of heparin, in the coordinates of the "change in optical density ΔAotn. - time T ";

7 - calibration curves for determining the concentration of heparin in the analyzed sample, when processing integrated biosensors with heparin solutions of known concentration for 9 (curve 1), 12 (curve 2), 15 (curve 3) and 18 min (curve 4), coordinates "change in optical density ΔAot. - the concentration of heparin SGeparin. "

However, the presented examples of the invention do not limit it and do not go beyond the scope of the claims.

Best Mode for Carrying Out the Invention

The authors conducted research on the creation of an integrated biosensor, which may contain a heparin-sensitive element with anomalous optical activity in the formation of its complex with heparin in the composition of nanobridges. As such an element, anthracycline compounds were studied using the antibiotic daunomycin (hereinafter DAU) as an example. The ability of daunomycin to form a DAU-heparin complex was investigated. The studies used:

1. A solution of daunomycin: preparation and concentration.

The commercial preparation of the anthracycline antibiotic DAU (C27H29NO10 × HCl; mol. Mass = 564 Yes; Sigma, USA) was used without further purification.

A portion of DAU (~ 0.4 mg) was placed in a test tube (V = 1.5 ml) and dissolved with vigorous stirring in a mixer in 200 μl of distilled water.

In this way, 200 μl of a DAU solution with a DAU concentration of SDAU of ~ 2 mg / ml was prepared.

To completely dissolve the DAU, the tube containing the indicated DAU solution was placed in a lightproof container, placed in a cool place (~ 4 ° C) for 12 hours, and then the determination of the exact antibiotic concentration was started.

The exact concentration of DAA in the solution was determined spectrophotometrically in the wavelength range of 350-650 nm, using the value of its molar absorption coefficient (λmax = 475 nm; εmax = 12000 M ^-1 · cm ^-1 ).

According to the data obtained, the molar concentration of DAU in the specified solution was 3.15 × 10 ^-3 M.

The DAU solution was stored in an opaque container at 4 ° C.

2. Heparin solution preparation and concentration

A commercial preparation of heparin Na-salt (molar mass = 4000 Da; Serva, Germany) with a specific activity of 186000 U / g was used without additional purification.

A portion of heparin Na-salt (4.0 mg) was placed in a measuring tube (V = 5.0 ml) and dissolved with vigorous stirring on a mixer in 1.5 ml of distilled water; the volume of the solution was adjusted to 2.0 ml.

In this way, 2.0 ml of a heparin solution with a fixed concentration of heparin = 2 mg / ml was prepared.

3. Polyconidine solution: preparation and concentration

A preparation of a heparin antagonist, a quaternary ammonium salt of a monodisperse oligomer of conidine with a polymerization degree of 25, polyconidine (molar mass = 4042 Da), synthesized by a chemical scientist A.V. Nekrasov (Institute of Immunology, Ministry of Health of the Russian Federation), was used without additional purification.

A portion of polyconidine (4.0 mg) was placed in a measuring tube (V = 5.0 ml) and dissolved with vigorous stirring on a mixer in 1.5 ml of distilled water; the volume of the solution was adjusted to 2.0 ml.

In this way, 2.0 ml of a polyconidin solution with a fixed concentration of SPolikonidin = 2.0 mg / ml was prepared.

4. Dichrometer for recording the anomalous optical activity of a biologically active substance in the analyzed liquid

As a dichrometer, a dichrometer can be used, which is capable of generating a circularly polarized radiation flux and acting on the analyzed liquid. For example, a portable dichrometer having the following characteristics shown in Table 1.

Table 1 Some desirable features of a portable dichrometer Spectral region, nm 250 (200) ... 750 Spectral resolution 3 The wavelength tuning time between the extreme points of the spectral range, s 0.5 Range of regulation of temperature of a sample, ° C 12 ... 80 Dimensions of the spectral block, mm 500 × 330 × 170 The mass of the spectral block, kg fourteen Minimum detectable signal CD (ΔA / A) 3 × 10 ^-7

For research, a well-known dichrometer (RU, 92960, U1) was used, containing sequentially placed: a source of broadband light radiation; a selector adapted to generate light fluxes with predetermined wavelengths corresponding to the region of optical activity of the substance being determined, manifested in the spectrum of circular dichroism; a polarizer adapted to form a linearly polarized light flux; a spectral gap adapted to isolate a linearly polarized light flux with a specific direction of the polarization vector; a polarization modulator adapted to convert said linearly polarized light flux into a circularly polarized light flux with a periodically changing direction of rotation of the polarization vector; a device for placing an analyzed sample in an optically permeable cuvette, comprising a thermostatic block for said cuvette; a photoelectronic multiplier adapted for recording optical signals of circular dichroism of the analyzed liquid and converting them into a proportional electrical signal; a digital recording system adapted to isolate and amplify said electrical signal and convert it to digital form; means for processing the received electrical signal and calculating the concentration of the biologically active substance.

The operation of such a dichrometer is controlled using a software package that allows you to implement various modes of its operation. The advantages of the described dichrometer are its low cost compared to stationary commercial counterparts, compactness, mobility, the absence of the need for cooling with water or gaseous nitrogen, the ability to work personnel who are not highly qualified.

Example 1. The study of the chemical activity of heparin against DAU.

A) The formation of the complex "DAU-Heparin"

In a rectangular optical cuvette (optical path length 1 cm; V = 4.0 ml), 2.0 ml of distilled H2O and 18.0 μl of a DAU solution prepared as described above in paragraph 1 were mixed, with: DAU ("Sigma ", USA) in a concentration of SDAU = 2.81 × 10 ^-5 M; heparin (Serva, Germany) - mol. heparin mass = 4000 Yes; the specific activity of the heparin preparation is 186,000 U / g.

The absorption spectrum of the resulting mixture was recorded in the wavelength range of 350-650 nm, the absorption spectrum of DAE in the absence of heparin in the coordinates “optical density A - wavelength λ” is shown in Fig. 2a (curve 1).

After recording the absorption spectrum of DAUs in an optical cuvette containing 2.018 ml of a DAU solution, 1, 3, 5, 9, and 11 μl (11 μl total) of the heparin stock solution prepared as described above in step 2 were added sequentially; after each addition of a heparin solution, the mixture obtained in the cuvette was thoroughly mixed and its absorption spectrum was recorded in the wavelength range of 350-650 nm. The absorption spectra of the complex "DAU-heparin" obtained by processing the DAU with heparin solutions of different concentrations are shown in Fig.2A (curves 2-5):

- curve 2 with SGeparin = 0.99 μg / ml;

- curve 3 with SGeparin = 2.97 μg / ml;

- curve 4 with SGeparin = 8.88 μg / ml;

- curve 5 with SGeparin = 10.84 μg / ml.

The addition of heparin was accompanied by a decrease in optical density at the absorption maximum of DAE (hypochromic effect) and the appearance of an isobestic point at λ ~ 550 nm. These results indicated the formation of the DAU-heparin complex.

B) The destruction of the complex "DAU-Heparin"

In an optical cuvette containing 2.029 ml of the DAU-heparin complex obtained in step A of Example 1, 9, 11, 14 and 24 μl of the initial solution of polyconidine obtained as described above in step 3 were added 24 μl in total. After each addition of a polyconidine solution, the mixture obtained in the cuvette was thoroughly mixed and its absorption spectrum was recorded in the wavelength range of 350-650 nm.

Figure 2b shows the absorption spectra of the DAU-heparin complex before (curve 1) the addition of a heparin antagonist, polyconidine, in the form of the ammonium salt of conidine oligomer with a degree of polymerization of 25, and after the addition of polyconidine in different concentrations (curves 2-5):

- curve 2 with SPOLICONIDIN = 8.83 μg / ml;

- curve 3 with SPOLICONIDIN = 10.78 μg / ml;

- curve 4 with SPOLICONIDIN = 13.71 μg / ml;

- curve 5 with SPOLICONIDIN = 23.38 μg / ml.

At a polyconidine concentration of ~ 23.38 μg / ml, an almost complete restoration of the absorption spectrum characteristic of free (not complexed) DAU molecules is observed, which can be seen from a comparison of curve 5 in Fig. 2b with curve 1 in Fig. 2a. The restoration of the initial absorption spectrum of DAU is due to the dissociation of the DAU-heparin complex under the action of polyconidine and the formation of a more durable complex “heparin-polyconidine”.

Thus, the data shown in FIGS. 2a and 2b indicate that heparin forms a strong complex with DAU molecules.

Consequently, heparin is able to “extract” DAE molecules from the composition of the “-Cu2 + -DAU-Cu2 + - ... Cu2 + -DAU-Cu2 +” nanostructures of the integrated biosensor, which should lead to its destruction, which will be accompanied by a decrease (until complete disappearance) of the optical signal. This means that the abnormal optical signal generated by the integrated DNA microchip can be used as a criterion for judging the presence of heparin in the analyzed fluid.

Example 2. Determination of physiological concentrations of heparin in the analyzed liquid samples.

A method for determining the physiological concentrations of heparin in the analyzed liquid samples of heparin according to the invention is shown by the example of determining heparin using an integrated biosensor, which is a particle of lyotropic cholesteric liquid crystal dispersion of rigid double-stranded nucleic acid molecules placed in a water-salt solution of the polymer, in which neighboring nucleic acid molecules are connected by nanobridges containing sequentially connected in a flat chelate complexes, in heparin-resistant structural elements and heparin-sensitive elements, while the integrated biosensor as an element sensitive to heparin contains an anthracycline compound having at least one reactive amino group associated with a sugar residue and having anomalous optical and chromophore properties when irradiated with a circular polarized radiation in the visible region of the spectrum.

Moreover, according to the invention, it is possible to use an anthracycline series compound selected from the group consisting of rubomycin, violomycin, adriamycin, carminomycin, premycin, doxorubicin, daunomycin as a heparin-sensitive element of nanobridges.

The studies were carried out using an integrated biosensor containing daunomycin as the most commercially acceptable element as a heparin-sensitive element.

In the method for determining heparin according to the invention, an integrated biosensor was created having in its structure, nanostructures, a DAU sensitive to heparin and a heparin inert structural element using copper ion.

Schematically, FIG. 1 shows the structure of nanobridges between two adjacent DNA molecules fixed in the spatial structure of the CLCD particle u1061.

The method for determining heparin according to the invention was carried out as described below.

2A. Creation of an integrated microchip based on DNA molecules fixed in the structure of lyotropic CLCD particles

2A.1) The formation of particles of lyotropic cholesteric liquid crystal dispersion from nucleic acid molecules having a molecular weight of less than 1 × 10 ⁶ Yes in a water-salt polymer solution capable of excluding these molecules from its composition.

Lyotropic CLCD was prepared from double-stranded DNA molecules from the thymus of cattle (Sigma, USA; molar mass of DNA ~ (0.3-0.6) × 10 ⁶ Yes, CDNA = 5.5 μg / ml) by their phase exceptions from the water-salt (0.3 M NaCl + 0.002 M Na + phosphate buffer) PEG solution (Serva, Germany; molar mass of PEG = 4000 Da; SPEG = 170 mg / ml).

The anomalous optical properties of lyotropic CLCD DNA were monitored by measuring the CD spectrum using the portable dichrometer described above (RU, 92960, U1) in the wavelength range of 250-350 nm.

The appearance of an intense negative band in the CD spectrum (λmax ~ 270 nm) indicated an ordered arrangement of DNA molecules in the structure of the liquid crystal dispersion, which is confirmed by registration, as shown in Figure 3, of the CD spectrum of the lyotropic CLCD formed as a result of phase exclusion of DNA molecules from the aqueous salt solution of the PEG polymer, in the coordinates "change in optical density ΔА = (AL-AR) - wavelength λ" (ΔА × 10 ^-6 - circular dichroism, optical units; SDNK ~ 20 μg / ml; pH ~ 7.0 ; L = 1 cm; temperature - 22 ° C).

2A.2) Obtaining lyotropic CLCD complex "DNA-DAU"

To 3 ml of lyotropic CLCD obtained according to Clause 2A.1), 28.6 μL of a DAU solution obtained similarly to that described above in A.1) was added at a concentration of DAU SDAU = 3.15 × 10 ^-3 M with vigorous stirring.

In this way, a complex was obtained between DNA and DAU molecules in the composition of lyotropic CLCD; the CD spectrum of the resulting mixture was measured.

The appearance of an additional negative band in the CD spectrum in the DAU absorption region indicated the formation of the DNA-DAU complex in the case of lyotropic CLCD DNA.

2A.3) Creation of an integrated DNA microchip

Prepare a 0.001 M solution of CuCl2 × 2H2O (Aldrich, USA).

To 3.0 ml of lyotropic CLCD of the DNA-DAU complex obtained according to p.2A.2), 12.0 μl of the obtained CuCl2 solution was added with stirring. After 40 minutes, the CD spectrum of the resulting mixture was recorded in the wavelength range of 400-650 nm (Fig. 7, curve 2).

Figure 4 shows the CD spectra of lyotropic CLCD DNA upon completion of the change in the signal amplitude before processing DAU and CuCl2 (curve 1) and the same dispersion after its sequential processing of DAU and CuCl2 (curve 2) in the coordinates "change in optical density ΔА = (AL- AR) - λ - wavelength "(ΔА × 10 ^-6 - circular dichroism (optical units); DNA from the thymus of cattle (" Sigma ", USA); molar mass of DNA ~ (0.3-0, 7) · 10 ⁶ Yes; SDNK ~ 5 μg / ml; SPEG = 170 mg / ml; molar mass of PEG = 4000 Da (Serva, Germany); 0.3 M NaCl + 10 ^-2 M Na + phosphate buffer , pH ~ 7.0; DAU (Sigma, USA); SDAU = 30.08 × 10 ^-6 M; CuCl2 × 2H2O (Aldrich, USA); CCu2 + = 9.91 × 10 ^-6 M; L = 1 cm; temperature - 22 ° C).

A multiple increase in the anomalous optical activity in the DAA absorption region (curve 2 of Figure 4) indicated a significant increase in the concentration of anisotropically located DAU molecules in the composition of lyotropic CLCD nanobridges as a result of the formation of an integrated microchip in which “-Cu2 + -ADA-Cu2 + nanobridges formed” between DNA molecules “... -Cu2 + -DAU-Cu2 +.”

Thus, an integrated DNA microchip was obtained containing a DAU element sensitive to heparin in the composition of nanobridges.

2B.1). Obtaining calibration curves of heparin content in the analyzed sample

Prepared 1.0 ml of a solution of integrated microarrays of DNA obtained according to p.2A.3), placed it in an optical cuvette, added 1.0 ml of distilled H2O, the mixture obtained in the cuvette was thoroughly mixed, and its CD spectrum was recorded in the 400- 650 nm. The magnitude of the optical signal generated by the integrated microarray of DNA in the region of the absorption wavelength of the DAE was then used as the value ΔAmax.

5.0 μl of a heparin solution prepared according to claim 2 was added to an optical cuvette containing 2.0 ml of a solution of integrated microarray DNA prepared as described above; the mixture obtained in the cuvette was vigorously stirred. In this way, a sample was prepared with a heparin concentration of 4.99 μg / ml. After 15 min, the CD spectrum of the resulting mixture was recorded in the wavelength range of 400-650 nm.

Figure 5 shows the CD spectra of the integrated biosensor according to the invention after completion of the change in the signal amplitude before (curve 1) and after (curve 2) interaction with heparin, and, for comparison, the CD spectrum of the lyotropic CLCD DNA-DAE complex (curve 3), in coordinates “change in optical density ΔA = (AL-AR) - wavelength λ” (ΔA × 10 ^-6 - circular dichroism (optical units); DNA from cattle thymus (Sigma, USA); mol. mass DNA ~ (0.3-0.7) · 10 ⁶ Yes; CDNA ~ 5 μg / ml; SPEG = 170 mg / ml; molar mass of PEG = 4000 Da (Serva, Germany); 0.3 M NaCl +10 ^-2 M Na + phosphate buffer, pH ~ 7.0; DAU (Sigma, USA ); SDAU = 30.08 × 10 ^-6 M; CuCl2 × 2H2O (Aldrich, USA); CCu2 + = 9.91 × 10 ^-6 M; heparin (Serva, Germany); molar mass of heparin = 4000 Yes; the specific activity of the heparin preparation was 186000 U / g; СGeparin ~ 10 μg / ml; L = 1 cm; temperature -22 ° С).

With the addition of heparin, the amplitude of the negative band in the CD spectrum (curve 3 in FIG. 5), which reflects the presence of nanobridges between adjacent DNA molecules in the integral microchip, sharply decreased. This meant that treatment of the integrated DNA microchip with heparin led to the destruction of nanobridges. This destruction was due to the formation of a stronger complex between DAU molecules and heparin molecules and, as a consequence, the "extraction" of DAUs from the nanobridge.

As can be seen from the results obtained, the amplitude of the abnormal signal of pure DAE (Figure 2a, curve 1), the signal of the DNA-DAU complex (Figure 5, curve 3) and the integrated DNA microchip obtained in the method according to the invention (Figure 4) differ .

Similar data were obtained in the case of determination of clexane and fraxiparin, which are low molecular weight heparin preparations with an average molecular weight of 4000-5000 Da, used in medical practice for the prevention of thromboembolic complications, for the treatment (resorption) of deep venous thrombosis, for pulmonary embolism, acute coronary syndrome and other pathological conditions accompanied by thrombosis.

2B.2). Obtaining calibration curves for the concentration of heparin in the analyzed sample

A series of heparin samples were prepared from a stock solution of heparin obtained as described above in claim 2 for analysis in H2O at a concentration of 1.0, 2.0, and 4.0 μg / ml.

1.0 ml of the corresponding heparin solution of the above concentration was added to 1.0 ml of the solution containing integrated microarrays of DNA obtained in Section 2A.3) with stirring, and CD spectra were recorded in the DAU absorption region with an interval of 5-10 minutes.

Based on the obtained data, the dependence of the change in the relative amplitude of the band in the CD spectrum of the integrated DNA microchip on the time of its treatment with heparin for different concentrations of heparin in the analyzed sample was constructed.

Figure 6 shows the dynamics of the destruction of the integrated microarrays of DNA according to the invention under the influence of different concentrations of heparin, in the coordinates "relative change in optical density ΔAot. - time T "(ΔArel. = (ΔAmax-ΔAT) / ΔAmax is the ratio of the difference in the amplitudes of the anomalous band in the CD spectrum of the integrated DNA microchip in the DAU absorption band at time T0 and T, respectively, to the initial amplitude of this band at time T0; DNA from bovine thymus (Sigma, USA); molar mass of DNA ~ (0.3-0.7) × 10 ⁶ Yes; CDNC ~ 2.75 μg / ml; SPEG = 85 mg / ml; mol PEG mass = 4000 Da (Serva, Germany); 0.15 M NaCl + 0.001 M Na + phosphate buffer, pH ~ 7.0; DAU (Sigma, USA); SDAU = 15.04 × 10 ^{- 6M;} CuCl2 × 2H2O ( «Aldrich» , USA); CCu2 + = 2,0 × 10 ^-6 M; heparin ( «Serva», Germany), Molecular weight heparin = 4000 daltons. specific activity Reparata heparin was 186,000 U / g;. L = 1 cm; temperature -22 ° C). Moreover, in Fig.6, curve 1 corresponds to the concentration of heparin SGheparin = 1 μg / ml; curve 2 - SGeparin = 2 μg / ml; curve 3 - SGeparin = 4 μg / ml.

The data shown in Fig.6 show that the decrease in the amplitude of the strip in the CD spectrum of the integrated DNA microchip in the presence of heparin depends on its concentration.

Based on the obtained data (Fig.6), choosing fixed time intervals, for example, 9, 12, 15 or 18 min, the dependence of the change in the relative amplitude of the strip in the CD spectrum of the integrated DNA microchip on the concentration of heparin in the analyzed sample was constructed.

7 shows calibration curves for determining the concentration of heparin in the analyzed sample when processing integrated DNA microarrays according to the invention with solutions of heparin of known concentration for 9 (curve 1), 12 (curve 2), 15 (curve 3) and 18 minutes (curve 4 ), in the coordinates "the relative change in optical density ΔAot. is the heparin concentration CGeparin ”((ΔArel. = (ΔAmax-ΔAT) / ΔAmax is the ratio of the difference between the amplitudes of the anomalous band in the CD spectrum of the integrated DNA microchip in the DAU absorption band at time T0 (before analysis) and T (after analysis), respectively to the initial amplitude of this band at time T0 (before analysis); DNA from the thymus of cattle (Sigma, USA); molar mass of DNA ~ (0.3-0.7) × 10 ⁶ Da; CDNA ~ 2 , 75 μg / ml; SPEG = 85 mg / ml; molar mass of PEG = 4000 Da (Serva, Germany); 0.15 M NaCl + 0.001 M Na + phosphate buffer, pH ~ 7.0; DAU (" Sigma ", USA); SDAU = 15.04 × 10 ^-6 M; CuCl2 × 2H2O (" Aldrich ", USA); CCu2 + = 2.0 × 10 ^-6 M; heparin (Serva, Germany), molar mass of heparin = 4000 Yes; the specific activity of the heparin preparation was 186000 U / g; L = 1 cm; temperature -22 ° C).

The presence of a proportional relationship between the change in the relative amplitude of the band in the CD spectrum of the integrated DNA microchip and the concentration of heparin in the sample for analysis, in the range of heparin concentrations from 0 to 4 μg / ml, allows this dependence to be used as a calibration one for determining the concentration of heparin in the sample for analysis.

Thus, using an optical biosensor consisting of a portable dichrometer and an integrated microchip of DNA containing a heparin-sensitive element, one can not only judge the presence of heparin in the analyzed liquid, but also determine its sufficiently low (~ 0.5 μg / ml) concentration.

Claims (8)

1. A method for determining the physiological concentrations of heparin in the analyzed liquid samples, comprising recording an optical signal of a biosensor placed in the indicated sample, which is particles of a lyotropic cholesteric liquid crystal dispersion of rigid double-stranded nucleic acid molecules placed in a water-salt solution of the polymer, in which neighboring nucleic acid molecules are connected by nanobridges containing heparin-sensitive element, and having abnormal optical properties and irradiation with a circularly polarized radiation flux, characterized in that they use an integrated biosensor containing alternating heparin inert structural element and heparin sensitive element in nanobridges, containing an anthracycline compound having at least one reactive amino group as a heparin sensitive element associated with the sugar residue, and having chromophore properties when irradiated in the visible region of the spectrum, and the method includes:
a) the creation of an integrated biosensor with chromophore properties when irradiated in the visible region of the spectrum, containing in the nanobridges a structural element that is not sensitive to heparin, and a sensitive element to heparin, while doing this:
a1) the formation of particles of a cholesteric liquid crystal dispersion from nucleic acid molecules having a molecular weight of less than 1 · 10 ⁶ Yes, in a water-salt polymer solution capable of excluding said molecules from its composition;
a2) adding a liquid crystal dispersion of nucleic acids to the water-salt solution of the polymer obtained in step a1), a solution of the compound selected as the heparin-sensitive element, to obtain a dispersion of the nucleic acid-sensitive element complex in which the reactive groups of the sensitive element are sensitive to heparin;
a3) processing the dispersion obtained in operation a2) with a solution of a compound selected as a heparin-insensitive structural element to obtain a dispersion in which neighboring nucleic acid molecules are connected by nanobridges, which are flat chelate complexes "structural element - sensitive element - structural element ... structural element - sensitive element - structural element ”;
a4) the subsequent placement of the dispersion obtained in operation a3) in a water-salt solution of a polymer optically isotropic and chemically neutral with respect to nucleic acid, to a structural element that is not sensitive to heparin, and to a sensitive element; a5) measuring the anomalous optical activity of the obtained integrated biosensor at two wavelengths in the visible region of the spectrum, the first wavelength being selected in the absorption region of the nitrogenous nucleic acid bases and used as the internal standard, and the second wavelength selected in the absorption band of the sensitive element, and a multiple increase in the optical signal in the absorption band of the sensing element determines the initial characteristics of the obtained integrated biosensor;
b) determination of the calibration characteristics of the anomalous optical activity of the obtained integrated biosensor in the visible spectrum upon irradiation of calibration samples containing a mixture of the specified polymer solution of the integrated biosensor obtained in step a4) and a solution of heparin of known concentration, with a circularly polarized radiation flux at the specified wavelength in areas of absorption of the sensing element;
c) mixing the obtained polymer solution of the integrated biosensor obtained in operation a4) with the analyzed liquid sample;
d) when the mixture obtained in operation c) is irradiated with a circularly polarized radiation flux at a specified wavelength in the absorption region of the sensing element in the visible spectrum, an optical signal is recorded and the presence and concentration of heparin in the analyzed liquid sample are determined based on calibration characteristics. 2. The method according to claim 1, characterized in that the molecules of double-stranded synthetic or natural polynucleotides capable of forming a cholesteric liquid crystal dispersion with phase exclusion while maintaining their chemical reactivity and anomalous optical properties are used as nucleic acid molecules. 3. The method according to claim 2, characterized in that the molecules of hard-chain polymers are used as nucleic acid molecules. 4. The method according to claim 1, characterized in that as the structural elements of nanobridges use metal ions capable of forming extended flat chelate complexes. 5. The method according to claim 4, characterized in that copper ion is used as a structural element of the nanobridges. 6. The method according to claim 1, characterized in that the anthracycline series compound selected from the group consisting of rubomycin, violomycin, adriamycin, carminomycin, premycin, doxorubicin, daunomycin is used as a heparin-sensitive element of the nanobridges. 7. The method according to claim 6, characterized in that daunomycin is used as the heparin sensitive element. 8. The method according to claim 1, characterized in that a solution of polyethylene glycol is used as a water-salt solution of a water-soluble, optically isotropic and chemically neutral with respect to nucleic acid, structural element and sensitive element of the polymer.

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