RU2148259C1 - Method for detecting macromolecules using biosensory device - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves preliminarily activating piezoelectric quartz resonator surface using aldehyde damp under vacuum conditions in ultrahigh frequency field, covering the surface with macromolecules in required number of layers, recording resonance frequencies in turn after every operation when resonator mass changes under conditions of being isolated from electromagnetic field and temperature influence. This is done for drawing conclusions concerning macromolecule availability. EFFECT: enhanced sensitivity and specificity of the method; prolonged service life of the biosensory members. 2 cl, 1 tbl

Description

 The invention relates to microbiology and can be used to control the results of specific biological reactions (antigen-antibody, enzyme-substrate, DNA hybridization, etc.).

 Biosensors (biosensors) are miniature devices that detect specific biological or chemical interactions due to electrical signals.

 By design and operating principles, these devices combine the reactions of biological systems with electronic, optical, thermosensitive or other converters that provide information from sensors in the form of specific signals.

 The practical use of biosensors opens up wide possibilities for quick and accurate determination of the content of a certain substance in a minimum quantity among a multitude of chemical substances or biological components similar in their properties.

 Known biosensor devices of the colorimetric type, the main constructive elements of which are highly sensitive electrodes with immobilized enzymes, during the reaction of which detectable ions are released with substrates (Sceller F., Wallenberg U., Schubert F. et al. / Applied Biochemistry and Microbiology, 1982, vol. 18, N4, pp. 454-470).

 When using this type of biosensor with cell organelles, bacterial cells, and other macromolecules with a set of enzyme systems, the reaction specificity and reproducibility deteriorate.

 Biosensors are known, the main structural element of which are thermosensitive elements and biological systems (antigen-antibody). The principle of operation of these devices is based on a change in the enthalpy in immunobiological, enzymatic, and chemical reactions. The disadvantage of biosensor thermistors is their extremely high sensitivity to temperature changes.

 With the introduction of fiber-optic technology, the development of optical biosensors is actively ongoing. Registration of biological reactions is carried out by changing the optical properties of a fiber with biological materials immobilized at its end (North J.R. // Trends Biothechnol. 1985. V.3. N7. P. 180-186.).

 The practical use of this method requires special equipment.

 The use of MIS transistors with a selective membrane is promising.

 Such matrix-type biosensors are compatible with computer technology. However, the problem of creating highly selective membranes has not yet been solved (Gronow M. // Trends Biochem. Sci. 1984. V.9. N8. P.337-340).

 Closest to the claimed is a method for the detection of macromolecules by a biosensor device (US Pat. US N4735906, 436/806; 324/711; 04/05/88). The method includes modifying the surface of a piezoelectric crystal with the formation of reactive groups, covalent immobilization of macromolecules with the required number of layers, and recording changes in the resonant frequency with a change in the mass of the resonator.

 The disadvantage of this method is the complexity and duration of the modification of the surface of the resonator with the formation of reactive active groups for subsequent covalent immobilization of macromolecules, the process of which is also long and complicated.

 In the chemical treatment of the surface of the resonator, hydrolysis of aldehyde groups in aqueous solutions is not excluded, which reduces the stability of the resonator.

 In addition, piezoelectric quartz resonators related to electrogravitometric electronic elements are very sensitive to external environmental conditions (temperature, humidity, electrical conductivity, electromagnetic field intensity).

 In many respects, the disadvantages are associated with the process of immobilization of biological materials on the surface of a sensor device and are common to all known types of sensors.

 During the physical adsorption of biological macromolecules or structures, an unstable bond is formed on the surface of the sensor, the amount of sorbed material varies widely, and non-specific sorption is possible.

 Improvement of the process of immobilization of biological materials is carried out mainly in the direction of creating conditions for covalent bonding of macromolecules with the surface of the sensor. Various methods are known for modifying the surface of a sensor in order to create reactive groups on it for covalent immobilization.

 For example, fixing on the surface of sensor devices various gels, films based on polyacrylamide, agar, gelatin, in the cells of which include antigens, antibodies, enzymes.

 The electrodes are coated with a protein film resulting from the polycondensation of non-specific active proteins in the presence of glutaraldehyde.

 Known methods for the formation on the surface of sensors of polymer films using vacuum plasma spraying (F. Sceller et al. / Applied Biochemistry and Microbiology, 1982, v. 18, N4, p. 454-470).

 However, all these well-known techniques are technologically and instrumentally complex and, in addition, reduce the sensitivity of the sensors.

 Upon receipt of immunosorbents, the strength of fixing the biomaterial on the surface of the carrier is ensured by creating coatings with functional-reaction groups that form a covalent bond with the biomaterial. These coatings are carried out with various aldehydes, ketones by treating carriers with their solutions. In this case, porous carriers (polyacrylamides, silica gels), which cannot fully realize the functions of the sensor, are mainly used. In addition, the surface modification of the carrier in aqueous solutions is uneven, aldehydes are hydrolyzed in an aqueous medium, all this leads to a decrease in the stability of the sorbent and its immobilization efficiency (A.S. USSR, N1517545, C 01 N 33/53, 15.12.93, Bull. N45-46).

 A known method of surface treatment of carriers of heterogeneous solid-phase non-porous materials for subsequent immobilization of ligands (US Pat. RF N2070439, B 01 J 20/30, from 20.12.96, Bull. N35).

 According to this method, the surface treatment of the solid-phase carrier is carried out in aldehyde vapors in a vacuum in an UHF field with a power of 15-30 W for 0.5-2.5 minutes, under a pressure of 0.49-0.98 Pa.

 The method can significantly increase the sorption ability of carriers to biological preparations, increase the stability of the carrier, however, detection is carried out by traditional methods, since the sorbents are not sensitive to the conversion of the energy of biospecific reactions into electrical, thermal, and light.

 The technical result of the invention is to increase the sensitivity and specificity of the detection of macromolecules by increasing the sorption ability of the biosensor, increasing the life of the biosensors and ensuring the versatility of their use in monitoring various biological processes.

 The technical result is achieved by modifying the surface of the piezoelectric crystal with the formation of reaction groups by aldehyde vapor in a vacuum in an UHF field, covalent immobilization of macromolecules with the required number of layers is carried out in a neutral buffer for 3-5 minutes for each layer, followed by treatment with an albumin solution , registration of changes in the resonant frequency when changing the mass of the resonator is carried out under conditions of isolation of the electromagnetic field at a constant temperature.

 The causal relationship of the hallmarks of the method with the technical result of the invention is manifested in the following.

 The preliminary activation of the piezoelectric crystal by aldehyde vapor in a vacuum in the UHF field provides plasma polymerization of aldehydes on the surface of the resonator with a high degree of adhesion and the formation of reaction-functional groups for covalent binding to biological components. Due to this, the sorption ability and the life of the resonator are increased. Processing in the UHF field provides safe and gentle temperature conditions.

 Carrying out layer-by-layer immobilization of macromolecules in a neutral buffer for 3-5 minutes in each layer followed by treatment with an albumin solution provides washing of the biosensor from unbound macromolecules and neutralization of non-specific centers, which increases the sensitivity and specificity of detection.

 Registration of changes in resonant frequencies under conditions of isolation from an electromagnetic field at a constant temperature eliminates negative factors on the sensitivity of the biosensor and environmental conditions and ensures standard detection.

 The possibility of practical application of the proposed method using the full set of essential features is illustrated by examples of specific performance.

 Example 1. We took 2 piezoelectric crystals, one of which is experimental, and the other is a control sample, and measured in an insulating chamber connected to a frequency generator and a frequency meter, the initial frequency of the resonant vibrations of both samples.

Then the prototype was placed in a vacuum chamber with a source of UHF ionization and treated with vapors of benzaldehyde, pre-cooled to -70 ^o C when reaching 0.73 Pa and UHF field power of 15 W until a glow discharge with a characteristic glow. Exposure in the UHF field was continued for 45 s.

 After processing, the samples were placed in an insulating chamber and the resonance frequency was measured.

 The treated surface of the test sample was coated with anti-plague immunoglobulins with a concentration of 20 μg / ml in a volume of 20 μl, exposed for 5 min in a neutral buffer, and unbound reaction-functional groups were treated with a 0.01% albumin solution.

 In the insulating chamber, the resonance frequency was measured, which amounted to ≈6 MHz. Then, 20 μl of Y. pestis EV was applied to the ligand layer as an antigand. with a concentration of 20 MK / ml, was exposed for 5 min, washed in a neutral buffer, and again the resonance frequency was measured in the isolation chamber. Subsequent operations of applying the ligand and antiligand were carried out similarly to the prototype, and the resonance frequency was read after each subsequent operation.

 Example 2. All operations were carried out analogously to example 1, except that F1 was used as the ligand, and antiplague serum was used as the ligand.

 Example 3. All operations were carried out analogously to example 1, except that peroxidase was used as a ligand, confirming its fixation on the sample by the presence of enzymatic activity after thorough washing of the sample with neutral buffer.

 The table shows the results of measurements of the resonant frequencies of the resonators sequentially after each operation of the proposed method.

 A change in the relative resonant frequency is inversely proportional to the mass of the biosensor and its frequency characteristics. These changes make up 1-20% of the frequency of the base samples.

 The magnitude of the total change in the resonant frequencies for all of the examples confirms that the experimental samples have a significantly higher sorption ability, which increases the sensitivity and specificity of the detection of macromolecules.

 Thus, the invention is practicable, its use will expand the scope and effectiveness of biosensors in biotechnology, immunology. A significant advantage of the proposed method over the known is the achievement of high stability of biosensors, their service life reaches 2-3 months.

Claims (2)

 1. A method for detecting macromolecules with a biosensor device, including modifying the surface of a piezoelectric crystal with the formation of reactive groups, covalent immobilization of macromolecules with the required number of layers and recording changes in the resonant frequency with a change in the mass of the resonator, characterized in that the surface of the piezoelectric crystal is modified with aldehyde vapor under conditions vacuum in the UHF field, and layer-by-layer immobilization of macromolecules is carried out in a neutral buffer, for 3 to 5 minutes each layer, followed by treatment with an albumin solution.  2. The method according to claim 1, characterized in that the registration of changes in the resonant frequency is carried out in conditions of isolation from an electromagnetic field at a constant temperature.

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