RU118441U1 - DEVICE FOR DETECTION OF BIOLOGICAL OBJECTS USING SURFACE PLASMA RESONANCE - Google Patents

Abstract

1. A sensor for detecting biological objects by surface plasmon resonance (SPR), which is a SPR electrode with a sensitive sensor element applied to its surface, characterized in that the sensitive element of the sensor consists of an interpolymer matrix 10-180 nm thick, represented by a polyaniline / complex poly (2-acrylamido-2-methyl-1-propane) -sulfonic acid (Pan / PAMPSA), and an immobilized biological agent capable of selectively binding to the analyzed object-analyte contained in the analyzed sample. ! 2. The sensor of claim 1, wherein the immobilized biological agent is an enveloped virus. ! 3. A sensor according to claim 1, wherein the immobilized biological agent is an A.! 4. The sensor of claim 1, wherein the immobilized biological agent is a B virus! 5. The sensor of claim 1, wherein the immobilized biological agent is a nucleic acid fragment. ! 6. The sensor of claim 1, wherein the immobilized biological agent is a DNA fragment. ! 7. The sensor of claim 1, wherein the immobilized biological agent is an enveloped virus antibody. ! 8. A sensor according to claim 1, wherein the immobilized biological agent is an A virus antibody! 9. The sensor of claim 1, wherein the immobilized biological agent is a B virus antibody.

Description

The utility model relates to the field of medical diagnostics, bioanalytical chemistry and biochemical technology and is a device for the detection of biological objects using surface plasmon resonance.

A known method for the detection of viruses (antigens) or antibodies using enzyme-linked immunosorbent assay (ELISA), based on the determination of the antigen-antibody complex using conjugates labeled with an indicator enzyme. For this, one of the components of the serological complex is immobilized on the surface of the solid phase (polystyrene well plates or balls). For example, to indicate viral antigens on the surface of the solid phase, specific immunoglobulins (antibodies) are immobilized, which adsorb the antigen from the test sample.

At present, biosensor diagnostics based on the use of various physicochemical methods of analysis combined with biological methods of indication are successfully used for rapid diagnostics in medicine [1]. The advantages of biosensor diagnostics are the quick registration of ongoing interactions without introducing special labels into the studied molecules, the high specificity of the bio-recognizing element, the ability to recognize without additional energy (temperature increase, etc.). Essentially for biosensor diagnostics, a recognition (sensitive) element, a converter, and a signal recording and processing system are required.

Among a huge number of biosensors, sensors sensitive to changes in mass (piezoelectric quartz microbalances), electrochemical sensors (e.g., amperometric, potentiometric), optical sensors and others are distinguished. So, in clinical diagnostics, piezoelectric quartz immunosensors are used to detect hepatitis A and B virus on antibody / protein sensitive elements [2; 3], tobacco mosaic viruses on sensitive piezoelectric elements modified with polymers with molecular imprints [4]. The main disadvantages of such systems is the long preparation of samples and low specificity.

One of the methods used for biosensor diagnostics is surface plasmon resonance spectroscopy (SPR). NDP is a quantum optoelectric phenomenon that occurs when light interacts with a metal surface. The interaction of light with the surface of a metal leads to the appearance of a quasiparticle — a plasmon, whose vibrations correspond to the frequency of excited electrons, which manifest themselves as a single electrical object. The plasmon, in turn, generates an electric field that extends approximately 300 nm above and below the metal surface. The peculiarity of this phenomenon, which determines the analytical functions of the SPR method, is that any change in the chemical composition of the medium in the region of action of the plasmon field causes changes in the angle of incidence of light at which resonance with the plasmon occurs. Those. the chemical change is expressed in the shift of the resonance angle, and the magnitude of the shift is quantitatively related to the magnitude of the chemical change.

The undoubted advantage of SPR spectroscopy is the ability to determine the resonance angle with high accuracy. The light spot excited by the laser is stationary, and the measurement takes several seconds. A single sensor format (ie, size, storage and use protocol, indications, etc.) can be used for a wide variety of chemical analyzes, including immunological [6], nucleic acid binding [7], and enzymatic [8]. However, in these studies, polyaniline compounds were not used to detect.

SPR spectroscopy can be combined with electrochemical measurements, which provides new opportunities for studying the bioelectrocatalytic properties of enzymatic electrodes and creating new optical biosensors based on them [8]. It is known that various conductive polymers are used to create electrochemical sensors that detect biological objects. In particular, for the detection of bovine diarrhea virus, an electrochemical sensor was used, the sensitive element of which was modified with antibodies polyaniline [9]; to determine serum albumin, a sensor with polypyrrole modified with antibodies [10] was used.

PPR studies are carried out using a special device - a spectrometer. Well-known manufacturers of PPR spectrometers: Autolab (Netherlands), XanTec (Germany), Viasoge® (Sweden), Optrel (Germany), Moritex systems (USA), Texas Instruments, Inc. (USA), Analytical-µSystem (Germany), etc. For research, PPP electrodes are required. For example, 111 IP-electrodes of the company Analytical-nSystem, (Germany), are coated with gold glass plates (user manual for the device "Biosuplar-2"). For the detection of biological objects, the manufacturer, as a rule, offers sensors with a ready-made sensitive element deposited on PPP electrodes. For example, HapTes or Biacore® use multilayer structures as a sensitive element, including films of dextran and an immobilized biological object (Sensor Chip CM5, Biacore®).

Polyaniline is a promising material for use as the matrix material of the sensitive element of the PNR sensor, because able to sorb biological objects such as nucleic acids, proteins, viruses, antibodies without loss of activity. Known methods for determining glucose by the method of NDP using the polymer composition polyaniline / polyacrylic acid with covalently sewn coenzymes and immobilized apoenzymes on them [11]. The patent No. 2290444 (RU) demonstrated the possibility of immobilization of influenza virus antibodies on the surface of a polyaniline film. Immobilized antibodies retained the ability to interact with the homologous antigen (virus) of the test sample [12]. In patent No. 2372951 (RU), polyaniline compounds (PAn) in the form of an emeraldine base or an interpolymer complex of polyaniline with poly- (2-acrylamido-2-methyl-1-propane) sulfonic acid (Pan / PAMPSK) are used as sorbents for viruses, proteins, antibodies, and also as the basis of immunosorbents that are stable and retain functional activity, i.e. the ability to selectively bind biological objects in the analyzed solutions. It is also important that Pan / PAMPSK can be used not only in the form of powder, but also in the form of films [13]. In the proposed device, these substances in the form of films are used to prepare a matrix of a sensor element for detecting biological objects using the SPR spectroscopy method.

The technical task of the proposed utility model is to create a sensitive element of the PPR sensor, a device for detecting biological objects using the method of surface plasma resonance spectroscopy. In the work, a PNR sensor means a 1U IP electrode with a sensitive element deposited on its surface, capable of selectively contacting the test sample (analyte). Biological objects can be viruses (antigens), antibodies, nucleic acids and other protein structures.

The technical result achieved in the proposed device for the detection of biological objects using the method of surface plasma resonance is to provide the ability to quickly detect biological objects with high sensitivity and selectivity, the ability to work using small volumes of samples without special preparation, as well as low cost the proposed device is a sensitive element of the sensor. To achieve the desired technical result, a thin polyaniline matrix is deposited on the surface of the PPP electrode with further immobilization of the biological agent. The claimed technical solution eliminates the disadvantages inherent in the closest imported analogues, and ensures the implementation of the task.

The problem is solved by creating on the surface of a 1 U IP electrode a device for detecting biological objects by the method of surface plasma resonance (PPP), which is a sensitive element of the sensor deposited on the surface of the PPP electrode, and consisting of an interpolymer matrix 10-180 nm thick, represented by a complex polyaniline / poly (2-acrylamido-2-methyl-1-propane) sulfonic acid (Pan / PAMPSK), and an immobilized biological agent capable of selectively binding to the test object (analyte), soda rzhaschimsya in the sample.

Objects The objects of research were enveloped viruses, which, as is known, include, for example, the families of paramyxoviruses, orthomyxoviruses (influenza viruses), flaviviruses, etc. Their ability to sorb, for example, on erythrocytes of animals or on the Pan and PAN / PAMPSK complex [13] is known. . Influenza A viruses and influenza B viruses were taken as representatives of enveloped viruses. Tests are presented by examples with a pandemic strain of the influenza virus A / IIV-Moscow / 01/2009 (H1N1) swl [14], reference strains of the influenza A virus / New Caledonia / 20/99 (H1N1) and influenza virus B / Florida / 04/06, which have a different structure of surface proteins, as well as antibodies homologous to influenza viruses, and influenza virus cDNA obtained by polymerase chain reaction, as an example of interaction with a nucleic acid acid. Viruses were cultured on chicken embryos.

A method of forming a matrix of a sensor element in the form of ultrathin films of PAn and its interpolymer complexes.

In this work, PPR electrodes from Analytical-nSystem (Germany) were used. Ultrathin Pan / HCl films or PAN / PAMPSK interpolymer complex can be formed on the surface of a 1 U IP electrode using various methods, for example, aniline electrochemical polymerization (potentiostatic, galvanostatic mode or potential cycling mode), vacuum thermal spraying, from solutions ( modes “cast”, “spincoating”, “layer-by-layer”) or by the Langmuir-Blodgett method. Modified SPR electrodes thus used later on for immobilization of biological agents for the purpose of subsequent x 1 U IP research.

Research methods

For PNR measurements, a Biosuplar-2 spectrophotometer (Analytical-USystem, Germany) with a laser diode with a wavelength of λ = 670 nm and an output power of 0.2 mW was used. NDP data was processed using the Biosuplar-2 software (version 2.2.30) on a PC computer. The experimental SPR spectra of polymer films were processed by mathematical modeling based on five-phase Fresnel calculations using the Nelder-Mead minimization algorithm.

We used a DPC-Compact electrochemical analyzer No. 1705, Cronas Ltd, Russia, connected to a PC computer (IPC-Compact No. 1705 software, version 7.9+).

For testing, a PNR sensor with a polyaniline matrix deposited on a polycrystalline gold film on a glass substrate (20 × 20 mm) was used as a working electrode.

The phenomenon of PPR is not specific. While this may seem like a limitation, it is actually a huge advantage. The specificity depends on the choice of a pair of molecules that react only with each other. Of this pair, one molecule is a detector, and the other is an analyzed object (analyte, i.e., the substance that we want to determine and quantify). Any pair of molecules that has a specific bond can be used for PNR measurements. Such pairs can be an antigen and an antibody, a DNA fragment and DNA complementary to it, an enzyme and its substrate, oil and gas or liquid that are soluble in this oil, or a complexing agent and a metal ion [5]. Below are tests demonstrating the detection of biological objects, using the interaction of viruses and antibodies as an example. In the examples, the SPR sensor is a PNR electrode with a sensitive element deposited on its surface, capable of selectively contacting the test sample (analyte). Schematically, the PNR sensor is presented in figure 2 and figure 3.

Brief Description of the Drawings

For a clearer understanding of the claimed utility model, the essence of which is reflected in the formula of the utility model, as well as to demonstrate its features and advantages, the following is a detailed description with reference to the figures of the drawings.

Figure 1 shows a table in which, when tested by surface plasma resonance, the sorption of cDNA (influenza viruses) on polyaniline matrices of various thicknesses is determined. As can be seen from the table, the sorption value, which is confirmed by a change in the resonance angle □ Θ, depends on the thickness of the polyaniline matrix deposited in the form of a film on the SPR electrode. On a commercial PPR electrode e with gold, indicated in the table "Control for gold" sorption of cDNA does not occur.

Figure 2 schematically shows a sensor for the detection of biological objects by the method of surface plasma resonance. In particular, in cross section there is a sensor used for the selective detection of viruses, in which the sensing element is a polyaniline matrix (3) and an immobilized biological agent, an antibody (4). The sensitive element is deposited on a commercial PPP electrode consisting of glass (1) and a gold film (2).

Figure 3 schematically shows a sensor for the detection of biological objects by the method of surface plasma resonance. In particular, in cross section there is a sensor used for the selective detection of virus antibodies, in which the sensitive element is represented by a polyaniline matrix (3) and an immobilized biological agent, the virus (4). The sensitive element is deposited on a commercial PPP electrode consisting of glass (1) and a gold film (2).

Figure 4 presents the sensogram of surface plasma resonance. Section 1 is the baseline of the sensogram, corresponding to the 111 IP electrode, with the Pan / PAMPSK interpolymer complex deposited on its surface, in a buffer solution. Section 2 of the sensogram shows the change in the resonance angle Θ upon immobilization of the antibody A / New Caledonia / 20/99 (H1N1) on a polyaniline matrix. Section 3 of the sensogram corresponds to the introduction of influenza virus B / Florida / 04/06 into the solution, which does not interact with non-homologous antibodies, which is confirmed by the absence of changes in the resonance angle Θ. Section 4 of the sensogram corresponds to the sorption of virus A / New Caledonia / 20/99 (NSh1) introduced into the solution, which interacts with a mobilized homologous antibody to form an antigen-antibody complex, which is confirmed by a change in the SPR signal in the form of a change in the resonance angle Θ.

Figure 5 presents the sensogram of surface plasma resonance. Section 1 is the baseline of the sensogram, corresponding to a NIR electrode, with the Pan / PAMPSK interpolymer complex deposited on its surface, in a buffer solution. Segment 2 of the sensogram shows the change in the resonance angle Θ upon immobilization of the A / IIV-Moscow / 01/2009 (H1N1) swl virus on a polyaniline matrix. Section 3 of the sensogram corresponds to the introduction of influenza B virus antibodies into the solution, which do not interact with non-homologous antigens, which is confirmed by the absence of changes in the resonance angle Θ. Section 4 of the sensogram corresponds to the sorption of antibodies of the virus A / IIV-Moscow / 01/2009 (H1N1) swl introduced into the solution, which interact with the immobilized homologous antigen to form an antigen-antibody complex, which is confirmed by a change in the PNR signal in the form of a change in the resonance angle Θ.

Example 1. Creating a device - a sensitive element of the sensor with an immobilized biological agent.

Influenza cDNA was used as a biological agent. As a control, an SPR electrode with polycrystalline gold on a glass substrate (20 × 20 mm) was used.

PAN / PAMPSK films of various thicknesses were used to immobilize influenza cDNA. The interaction of cDNA with a polyaniline matrix was recorded by the change in the resonance angle, indicated in the table as ΔΘ (Figure 1). The table shows the results of a study of sorption of cDNA. It was established that sorption of cDNA occurs on cDNA matrices of various thicknesses, but an increase in the thickness of the PAn / PAMPSK film leads to a decrease in ΔΘ.

Example 2. Creating a device - a sensitive element of the sensor with an immobilized biological agent complementary to the influenza virus.

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To this end, a polyaniline matrix (Pan / PAMPSK) was applied onto the surface of a PPR electrode consisting of a gold film (FIG. 2, element 2) sprayed onto a glass substrate (FIG. 2, element 1) (FIG. 2, element 3). The thickness of the polyaniline matrix was 10-80 nm. Next, the biological active agent was immobilized to the interpolymer complex — antibodies to influenza A virus / New Caledonia / 20/99 (H1Sh) or antibodies to influenza B virus (Figure 2, element 4). The process of immobilization of antibodies on a polyaniline matrix and its completion was controlled by the method of surface plasma resonance (Figure 4, segment 2 sensograms). An SPR sensor with a sensitive element consisting of a polyaniline matrix and an immobilized biological agent represented by influenza virus antibodies, for example, influenza A virus / New Caledonia / 20/99 (NP \ G1), is used for the selective determination of influenza virus (Figure 2) . This structure is stable and retains functional activity.

Example 3. The creation of the device is a sensitive element of the sensor with an immobilized biological agent homologous to an antibody of the influenza virus.

For this purpose, a polyaniline matrix (Pan / PAMPSK) was applied onto the surface of a PPR electrode consisting of a gold film (FIG. 3, element 2) sprayed onto a glass substrate (FIG. 3, element 1) (FIG. 2, element 3). The thickness of the polyaniline matrix was 10-80 nm. Then, a biological active agent, an influenza virus, was immobilized to an interpolymer complex, for example, a / iiv-Moscow / o 1/2009 (HlNl) swl (Figure 2, element 4). The process of immobilization (sorption) of influenza virus on a polyaniline matrix and its completion was controlled by the method of surface plasma resonance (Figure 5, segment 2 of the sensogram). An SPR sensor with a sensitive element consisting of a polyaniline matrix and an immobilized biological agent represented by the virus a / iiv-moscow / o 1/2009 (HlNl) swl is used to selectively detect antibodies to the influenza virus (Fig. 3). This structure is stable and retains functional activity. Example 4. Detection of influenza A (H1N1) virus using the proposed device.

Selective determination of influenza A virus / New Caledonia / 20/99 (H1N1) was carried out using a sensor having a sensitive element "PAN / PAMPSK antibody to virus A / New Caledonia / 20/99 (H1N1)". Prepared according to the method described in example 2, the SPR sensor was used to determine the influenza virus A / New Caledonia / 20/99 (H1N1) with a concentration of 256 GAE (hemagglutinating units) in physiological saline (0.15 M NaCI). Detection of analyte from test

the sample was carried out by the SPR method. The absence of interaction of influenza B virus with non-homologous antibodies is confirmed by the fact that the resonance angle remains unchanged (Figure 4, segment 3 of the sensogram). The formation of the complex "antigen-antibody" led to a change in the optical characteristics of the sensor element of the sensor, and, consequently, to a change in the resonance angle, which allows the selective detection of influenza virus A / New Caledonia / 20/99 (H1N1) using this method (Figure 4 segment 4 sensograms). Example 5. Detection of antibodies to the virus using the proposed device.

Selective detection of antibodies to the influenza virus a / iiv-moscow / o 1/2009 (HlNl) swl was performed using a sensor whose sensitive element is “PAN / PAMPSK - virus a / iiv-moscow / o 1/2009 (HlNl) swl” . Prepared according to the method described in example 3, the sensor element was used to detect antibodies of the influenza virus A / IIV-Moscow / 01/2009 (HlNl) swl. The analyte was detected from the test sample by the method of surface plasma resonance. The lack of interaction of the immobilized influenza virus A / IIV-Moscow / 01/2009 (HlNl) swl with non-homologous antibodies (influenza B antibodies) is confirmed by the fact that the resonance angle remains unchanged (Figure 5, segment 3 of the sensogram). The formation of the antigen-antibody complex leads to a change in the optical characteristics of the sensor element of the sensor and, consequently, to a change in the resonance angle, which allows the selective detection of antibodies of the influenza virus A / IIV-Moscow / 01/2009 (HlNl) swl using this method ( 5, segment 4 of the sensogram).

The proposed utility model solves the problem of fast and efficient detection of biological objects, for example: nucleic acids, proteins, viruses, antibodies, and other protein structures that can selectively bind. Simplifies the identification procedure, for example viruses, reduces the time required to determine the analyte in solutions. PPR measurements combined with the electrochemical method make it possible to simultaneously control at least two parameters of the system under study, for example, a change in the resonance angle and a change in potential, which greatly expands the potential possibilities of using this device (sensor sensitive element). In addition, the proposed utility model according to preliminary calculations is economically more profitable in comparison with existing commercial PPR sensors of foreign manufacturers.

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Claims (9)

1. A sensor for the detection of biological objects by the method of surface plasmon resonance (SPR), which is an SPR electrode with a sensor element deposited on its surface, characterized in that the sensor element consists of an interpolymer matrix 10-180 nm thick, represented by a polyaniline complex / poly (2-acrylamido-2-methyl-1-propane) sulfonic acid (Pan / PAMPSK), and an immobilized biological agent capable of selectively binding to the analyte being studied contained in ana lysed sample. 2. The sensor according to claim 1, in which the immobilized biological agent is an enveloped virus. 3. The sensor according to claim 1, in which the immobilized biological agent is represented by virus A. 4. The sensor according to claim 1, in which the immobilized biological agent is represented by virus B. 5. The sensor according to claim 1, in which the immobilized biological agent is represented by a nucleic acid fragment. 6. The sensor according to claim 1, in which the immobilized biological agent is a DNA fragment. 7. The sensor according to claim 1, in which the immobilized biological agent is an antibody of the enveloped virus. 8. The sensor according to claim 1, in which the immobilized biological agent is an antibody of virus A. 9. The sensor according to claim 1, in which the immobilized biological agent is an antibody of virus B.