RU2315999C2 - Nanodiagnosis test-system for detecting hepatitis b virus - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves using biosensor of resonance mirror with biochip. The biochip has biosensory cell having prisma in its base and waveguide coupled with it. The waveguide is manufactured from high refraction coefficient medium nel and medium layer having low refraction coefficient ncl and thickness values of several hundred nanometers depending on layer material. Oligonucleotide probe is complementary to hepatitis B virus DNA site 5'GACCTTGAGGCATACTTC. Light refraction change is recorded in superficial layer on waveguide boundary during hybridization reaction.

EFFECT: high accuracy of diagnosis.

5 dwg

Description

The invention relates to medical nanodiagnostics, virology, in particular to applied immunology.

Nanodiagnostic medical system using sensitive elements having nanoscale dimensions, i.e. sizes of the order of several hundred nanometers, in which or by which a measurement is made, or sensitive elements in it made using nanotechnology.

Hepatitis B virus surface antigen (HBsAg) - a protein complex that makes up the outer shell of this virus, is found in the blood of people with acute or chronic hepatitis B and with the so-called "healthy" carriage. Therefore, the definition of HBsAg virus has a key role in the diagnosis of diseases caused by hepatitis B.

Almost all biological methods for detecting antigen were used to detect HBsAg, from gel precipitation to radioimmunoassay.

Currently, the main methods for determining markers of infectious diseases, including hepatitis virus, are enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR).

In ELISA, antibodies immobilized on a substrate are incubated in blood serum containing antigens, after which secondary antibodies with covalently “linked” enzyme molecules (peroxidase or alkaline phosphatase) are added to the antigen / antibody complexes formed on the substrate. After completion of the complexation reaction, substrates for the named enzymes are added to the medium, which give a color reaction after hydrolysis in proportion to the amount of secondary antibodies bound, which reflects the antigen content in the test sample.

In PCR, amplification by polymerase of DNA fragments of the disease virus occurs, followed by registration of the oligonucleotides thus obtained.

Despite the high sensitivity, these methods have several disadvantages: the duration and complexity of the formulation, the lack of strict quality control of test systems, the possibility of contamination of the studied DNA or RNA samples (in the case of PCR), and the high cost of reagents and devices.

At present, in pharmacological and proteomic studies, biosensor diagnostic methods have been used based on the analysis of hybridization of a specific DNA sample with the DNA of the studied sample (target) (Quinn JG O`Neill S., Doyle A et al. (2000) Anal. Biochem., 281, 135-143; Welschof M., Terness P., Kipriynov SM Proc. Natl. Acad. Sci USA, 94, 1902-1907), (Koval VV, Gnedenko OV, Ivanov Yu.D., Fedorova OS, Archakov AI , Knorre DG. Real - time oligonucleotide hybridization kinetics monitored by resonant mirror technique. IUBMB LIFE, 1999, 48, 1-4).

The method of recording protein-protein interactions using the optical biosensor, which appeared in the 1990s, is characterized by high sensitivity, the ability to determine the kinetic parameters of the formation and decomposition of complexes, and the speed of analysis. Using the method of an optical biosensor, it is possible to register hybridization of macromolecules directly in real time without additional purification and modification of the target. In time, this analysis takes about 10-30 minutes instead of 1-4 hours (Koval VV, Gnedenko OV, Ivanov Yu.D., Fedorova OS, Archakov AI, Knorre DG. Real - time oligonucleotide hybridization kinetics monitored by resonant mirror technique. IUBMB LIFE, 1999, 48, 1-4).

The closest in essential features to the proposed technical solution is a diagnostic method, in accordance with which the first oligonucleotide is obtained that is complementary to all or part of the oligonucleotide sequence characteristic of the RNA or DNA of the material under study, and immobilized on the surface of the waveguide of the vibration attenuation detector, then the resulting immobilized oligonucleotide is processed a solution of a sample of material or DNA or RNA obtained from this material under conditions allowing DNA or RNA hybridizes with the immobilized oligonucleotide, after which the immobilized oligonucleotide is coupled to a fluorescently detectable agent, the conditions are chosen so that the fluorescently detectable agent binds to the hybridization product, the amount of fluorescent detectable agent is measured, and the the surface of the detector of attenuation of oscillations on an optical waveguide (US Pat. Russia 2116349, 1998).

The disadvantage of this method of using an optical biosensor is the need to modify the oligonucleotides using a fluorescent material or add fluorescent materials to the reaction zones, since the oligonucleotides themselves do not fluoresce in the visible spectrum.

Currently, several designs of biosensors have been developed, of which two types of optical biosensors can be distinguished: the first, based on the use of plasma resonance, and the second, the so-called "optical resonance mirror". In both cases, the detection of intermolecular interaction is based on recording changes in the refractive index of the medium during the formation of a protein complex immobilized in the resonance layer of the measuring cell with its partner with very high sensitivity due to the use of resonance effects. Estimates show that the theoretical sensitivity of an optical resonance mirror biosensor is an order of magnitude higher than that based on plasma resonance due to the optimal selection of optical structures that form this type of structure [Cush, R., Cronin, JM, Stewart, WJ, Maule, CH , Molloy, J., and Goddard, NJ, Biosensor and Bioelectronics 1993, 8, 347-353, IAsys Cuvette System / User's Guide / Fisonss Ahhiied Sensor Technology / 1993].

In the Resonance Mirror biosensor, the measuring cell is a prism coupled to a waveguide made of nanostructures. As a sensitive element, a damped wave is used, which probes the reaction zone for the formation of complexes ranging in size from a monomolecular layer to 200-300 nm, depending on the thickness of the immobilized layer of macromolecular probes. Since the sensitive element probes the reaction medium in the range of 200-300 nm, such a measuring system is a nano-measuring system.

The task of identifying markers of hepatitis B and C disease by creating a nanodiagnostic test system using an optical biosensor “resonance mirror” is being solved for the first time.

To solve this problem, a nanodiagnostic test system has been created for the detection of hepatitis B and C diseases, including the use of a two-channel biosensor “resonant mirror” with a biochip, which is a biosensor cell, at the base of which there is a prism with an associated waveguide formed from a medium layer with a high refractive index - “nel” and a quartz layer with a low refractive index “ncl” with thicknesses of the order of several hundred nanometers, depending on the material of the layer, sensitive to The surface of which, which is also the surface of the bottom of the cuvette, is immobilized by probe molecules that form specific complexes with macromolecular disease markers in the biological fluid in the surface layer of a few hundred nanometers to the waveguide by the interaction of antibody molecules (anti-HBs) and HBsAg (hepatitis B virus antigen ) and / or interaction of hepatitis C surface nucleocapsid antigen and antibodies to it and / or hybridization of oligonucleotides with complementary regions to hepatitis B virus DNA and / Whether hybridization of the oligonucleotides with the complementary regions to HCV RNA with subsequent determination of changes in refractive index in the surface layer at the boundary of the waveguide.

The invention is illustrated in the drawings.

Figure 1 shows a diagram of an optical biosensor with a nanobiochip.

Figure 2 shows a typical interaction (complexation) curve of an antibody immobilized on the biochip surface (anti-HBs) with hepatitis B virus antigen present in blood serum No. 41508 obtained using an optical biosensor. For comparison, the same drawing shows the interaction curve of immobilized antibodies with serum N 2 that does not contain the HBsAg antigen.

Figure 3 shows a typical hybridization curve of an oligonucleotide present in a solution that is identical to the DNA site of the hepatitis B virus with an immobilized oligonucleotide probe, obtained using an optical biosensor and a biochip with an immobilized oligonucleotide probe.

Figure 4 shows the result of the detection of antibodies to alpha-fetoprotein in a solution.

Figure 5 shows the result of detection of antibodies to glycodelin using a biochip with covalently immobilized glycodelin.

The optical biosensor used in the proposed nanodiagnostic test system, shown in figure 1, contains a measuring cell - nanobiochip, the bottom of which consists of a glass prism coupled to a waveguide formed from a layer of a medium with a high refractive index - "net" and a layer of quartz with low refractive index "ncl", with thicknesses of the order of several hundred nanometers, depending on the material of the layer. A layer (this can be a monolayer or a layer up to several hundred nanometers thick) of one of the partners of the interacting pair is immobilized to the sensitive surface of the waveguide, which is also the surface of the bottom of the cell.

The laser beam falls on the prism-quartz boundary at an angle greater than the angle of total internal reflection and is reflected, and a field of a damped light wave is formed, the intensity of which decreases exponentially with distance from the boundary surface. Part of the light then tunnels into the medium with a high refractive index through a quartz layer at a distance of several hundred nanometers and, at a certain resonance angle of incidence, propagates along the waveguide, then tunnels back to the prism and then leaves the prism with registration of the obtained resonance angle. When a second partner, forming a complex with an immobilized component, is added to the cuvette, the refractive index in the sensitive layer with a thickness of several hundred nanometers increases and, accordingly, a shift of the maximum of reflected light in the angular coordinate depending on time is observed.

Thus, monitoring the change in the position of the resonant angle of incidence of the laser beam as a function of time makes it possible to record the kinetic curve of the formation of complexes. By recording these curves for various concentrations, one can calculate the rate constants of association and dissociation.

The complexation reaction of the immobilized partner A with the partners B added to the cuvette can be described by the equation

according to the instructions for the device IAsys + (Affinity Sensors, UK).

The signal R of the optical biosensor for complex formation can be written as

where kon, koff are the rate constants of the formation and dissociation of complexes, R is the biosensor signal.

The R value can be used to determine the number of complexes formed, i.e., the concentration of the probe-marker disease complexes can be quantified, and kon, koff characterizing the kinetic process of their formation and decomposition can also be calculated using equation (2). When a solution with a ligand is replaced in a cuvette with a buffer solution, complex dissociation is observed.

Biochips have been developed to detect HBsAg in human serum using an optical biosensor. For this purpose, in the manufacture of a biochip for the diagnosis of hepatitis B disease by the reaction of formation of the antigen / antibody complex: monoclonal antibodies: NE 2, NE 3 or CEN-24 in the solution were immobilized on the surface of the IAsys optical biosensor cuvette (Affinity Sensors).

In the manufacture of a biochip for the diagnosis of hepatitis B disease by a hybridization reaction: streptavidin (or avidin) was covalently immobilized through the amino groups and carboxy groups of the surface of a carboxymethylated dextran on the surface of the biosensor cell waveguide using oligonucleotides, oligonucleotides, and oligonucleotides were oligonucleotide oligonucleotides this cell for immobilization of the probe through the streptavidin (or avidin) / biotin bond.

For the diagnosis of hepatitis C, the biochip was created as follows. Protein - HCcore antigen covalently immobilized the biosensor through its amino group and the amino group of the aminosilane cell of the optical biosensor.

The obtained biochips with immobilized antibodies against HBsAg were used for control experiments on the registration of HBsAg in standard PBS / Tween 20 buffer. It was found that immobilized monoclonal antibodies NE2 and NE3 form a complex with HBsAg, which was detected by changing the refractive index in the surface sensitive layer using an optical biosensor. The NE2 monoclonal antibodies had such a high affinity for HBsAg that the complexes could not be completely destroyed by using any of the following washing buffers: 1) 1 M NaCl containing 0.5% Na cholate, 2) 10 mM HCl, 3) 50 mM HCl 4) 3 M KNCS. As a result, the NE2-based biochip was insensitive to the re-addition of HBsAg in PBS / Tween 20, which indicated that it could not be used in a reusable test.

Control experiments with a biochip based on NE3 or CEN-24 showed that it can be used to detect the formation of monoclonal antibody / HBsAg complexes, and these complexes can be dissociated with 10 mM HCl. Therefore, such a biochip can be used repeatedly for registration of HBsAg antigen.

Testing patient serum samples for HBsAg was based on the following principle. 5-20 μl of serum was added to a cuvette with immobilized monoclonal anti-HBsAg antibodies (NE3 or CEN-24). In the event that the serum contained HBsAg, complexes formed, as a result of which the refractive index near the surface changed, which was recorded by the IAsys + device (Affinity Sensors, England). To register the processes of dissociation of these complexes, the serum from the cuvette was removed and a standard buffer (PBS / Tween 20) was added there.

The experimental curves reflecting antigen-antibody binding were approximated by a polynomial of the first degree and are presented as surface concentrations of HBsAg (K) bound to antibodies calculated using the following formula:

where DR = (R1-R2); R1 and R2 - shift of the resonance angle of the device at Dt = 10 sec; 200 - scale factor; 200 units corresponds to a surface concentration of bound protein of 1 ng / mm ² .

In preparation for diagnosis, an oligonucleotide probe with a sequence corresponding to the DNA site of the hepatitis B virus is covalently immobilized on the surface of the cuvette of the optical biosensor with carboxymethylated dextran, while the oligonucleotide N40 (1.5 mg / ml) added to the biochip is completely complementary to the immobilized zone. Hybridization buffer: 5xssc, 5x Denhard solution, 5 mm sodium phosphate buffer (ph 6.8) and 0.1% (v / v) Tween-20. Regeneration buffer: 50 mm naoh, T = 250 ° C.

If the oligonucleotide added to the cuvette of the optical biosensor is not completely complementary to the immobilized probe, as, for example, in the case of D40 (an oligonucleotide with two missing bases in the complementarity site), then, as can be seen from Fig. 3, a much smaller signal of the optical biosensor to the D40 oligonucleotide additive is observed, compared with the addition of a fully complementary oligonucleotide N-40.

Figure 4 shows that after the addition of a solution of antibodies to alpha-fetoprotein to the biochip with covalently immobilized alpha-fetoprotein, their interaction is observed, which is expressed in the growth of the sinal of the optical biosensor. After washing the cuvette with buffer, a slow dissociation of the complex is observed.

From the formation curves of the complexes and their dissociation curves, we can calculate the rate constants of the formation and decomposition of the complexes, which in our case were the values kon = (2.3 ± 0.3) 104M-1c-1, koff = (5.7 ± 0, 1) × 10-5s-1, and the dissociation constant KD = 5.7 × 10-8M.

According to immunofermet analysis, serum No. 41508 contained HBsAg, while serum No. 2 did not. Figure 2 shows a typical response curve of the immobilized monoclonal antibody NE3 to the addition of sera No. 2 and No. 41508, it is seen that after adding serum to the measuring cell, signal growth was observed associated with both the process of specific and non-specific binding. The results obtained on two types of cuvettes (″ CM-Dextran ″ and "Carboxylate") were similar. After recording the association curve, the serum in the cuvette was replaced with PBS / Tween-20 buffer, and desorption of a non-specifically bound substance was observed. As a result, the curve reached a plateau that characterizes the number of antigen / antibody complexes formed.

The curve of figure 2 shows that for serum No. 2, only nonspecific binding is observed, since the adsorbed substance is almost completely washed off with PBS / Tween - 20 buffer. In the case of serum No. 41508, a high level of formation of antigen / antibody complexes was observed after washing with a buffer of non-specifically adsorbed molecules on the surface of the cuvette.

To diagnose hepatitis C disease with an optical biosensor according to the invention, the interaction between the nucleocapsid antigen molecules forming the surface layer of the biochip of the biosensor cell and anticorrosive antibodies is recorded.

When using a two-channel cell, one of the channels can be used as a control, and the oligonucleotide probe or antibody (or antigen) is immobilized in the working channel. The control channel does not immobilize or immobilize macromolecules that do not form complexes with markers of a detectable disease.

The use of a two-channel cuvette made it possible to use the second channel of the optical biosensor to detect antibodies against the HCV nucleocapsid antigen. The free reaction centers after antigen landing were closed with bovine serum albumin. The landing density of the antigen core was 4 ng / mm ² . To detect the presence of antigen against the HCV nucleocapsid antigen in the biological fluid, this solution was added to the cuvette of the optical biosensor and the formation of the antigen / antibody complex on the surface of the optical biosensor was recorded, and signal growth was observed associated with the formation of antigen / antibody complexes. To register the decomposition processes of these complexes, the solution was removed from the cuvette and standard phosphate-saline buffer with tween-20 (PBS-T) was added.

On the curve of figure 2 it is seen that in the case when the serum contains HBsAg, when serum is added to the biochip, a significantly stronger signal of the optical biosensor is observed. In this case, after washing the biochip with buffer, in the case of serum containing the HbsAg antigen, significantly less dissociation of the complexes formed on the surface of the biochip is observed. Compared with the case when serum is added to the biochip that does not contain the HBsAg antigen. This allows the use of a biochip to detect HbSAg in serum.

When testing serum samples for the presence of anti-core antibodies, 6 ml of serum was added to the cuvette. If the serum contained antibodies, complexes were formed on the surface of the biochip, as a result of which the refractive index in the cell changed. To register the decomposition processes of these complexes, the serum from the cuvette was removed and a standard phosphate-saline buffer with tween - 20 (PBS / t) was added.

A comparative analysis of the detection of these antibodies of the optical biosensor and enzyme-linked immunosorbent assay showed high specificity and sensitivity of detection of anti-core antibodies using the optical biosensor.

For the simultaneous registration of markers of two hepatitis diseases B and C in one of the channels of the optical biosensor, a probe is immobilized for one of the diseases and in the other for the other disease. For example, the markers of hepatitis B disease are registered simultaneously by the hybridization reaction, if an oligonucleotide probe is immobilized in this channel with a region identical to that of hepatitis B DNA (or if immunocomplexes are recorded in this channel, then the anti-HBsAg antibody is immobilized) and hepatitis C by antigen / antibody complexation reaction, if immobilized in the second channel of the HCCore antigen.

Examples of the results of using the proposed diagnostic test system for determining disease markers of somatic diseases associated with a change in the level of antibodies / to alpha-fetaprotein or glycodeline are shown in FIGS. 4 and 5.

The curve of figure 4 shows the result of the determination of antibodies to alpha-fetoprotein. Alpha-fetoprotein is immobilized on the surface of the working channel of the aminosilane cell of the optical biosensor. The surface of the control channel is blocked by bovine serum albumin. A solution of polyclonal antibodies (Y-fraction) was added to both channels. The upper curve of figure 4 shows the difference signal between the working and control channels, the middle one is the signal of the working channel, the lower one is the signal of the control channel. In the curves of FIG. Figure 4 shows that, during nanodiagnostics, a difference signal of the interaction of immobilized alpha-fetoprotein with antibodies in solution is recorded.

The curve of figure 5 shows the result of the determination of antibodies to glycodelin. Glycodelin is immobilized on the surface of a carboxylated cell of an optical biosensor. The surface of the control channel is blocked by bovine serum albumin. A solution of polyclonal antibodies (Y-fraction) was added to both channels. The upper curve in Fig. 5 shows the difference signal between the working and control channels, the middle one shows the signal of the working channel, and the lower shows the signal of the control channel. It is seen that the difference signal of the interaction of immobilized glycodelin with antibodies in solution is recorded.

Any biosensor can be used as an optical biosensor in the diagnosis of hepatitis B and C diseases instead of a resonant mirror biosensor, the measurement of which is based on recording changes in the optical properties of the medium (refractive index) during complexation, in particular, for example, a biosensor using surface plasma resonance or colorimetric biosensor,

In the proposed nanodiagnostic test system, it is possible to use a multichannel optical biosensor with more than 2 channels, which will allow more than two diseases to be recorded simultaneously.

Claims (2)

1. Nanodiagnostic test system for the detection of hepatitis B disease, including the use of a "resonant mirror" biosensor with a biochip consisting of a biosensor cell, at the base of which there is a prism with an associated waveguide formed from a layer of a medium with a high refractive index - "nel" and a medium layer with a low refractive index "ncl" with thicknesses of the order of several hundred nanometers, depending on the material of the layer, to the sensitive surface of which is simultaneously the surface of the bottom of the cell, immobilized probe molecules are formed that form specific complexes with biological macromolecular disease markers in the biological fluid in the surface layer of a few hundred nanometers in size, characterized in that an oligonucleotide probe complementary to the hepatitis B DNA fragment, 5 'GACCTTGAGGCATACTTC, is used as a probe and recorded a change in the refraction of light in the surface layer at the boundary of the waveguide during the hybridization reaction. 2. Biochip for the diagnosis of hepatitis B for use in the test system according to claim 1, containing an oligonucleotide probe complementary to the DNA site of hepatitis B virus, -5 'GACCTTGAGGCATACTTC.

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