RU2510830C2 - Method for preparing microgravimetric immunosensor - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and microbiology. What is presented is a method for preparing a microgravimetric immunosensor. A surface of a quartz crystal resonator is activated first by plasma spray coating with polyethylene imine of molecular weight less than 10000 Da for 10 s in a vacuum unit at UHF field power 30 Wt. Thereafter, immunoglobulins from a solution with the protein concentration of 0.125 mg/ml are immobilised on the activated surface of the quartz crystal resonator. That is followed by washing in distilled water and drying in an air flow.

EFFECT: method enables preparing the immunosensor used for the purpose of the detection of infectious agents with the sensitivity of 1×10³-1×10⁴ mln. cells/ml and preserving the specific activity for.

3 cl, 3 ex.

Description

The invention relates to biotechnology, microbiology and can be used in the production of immunosensors (biosensors) to identify pathogens of infectious diseases.

The function of a microgravimetric immunosensor is based on the ability of quartz resonators to change the frequency characteristics depending on the insignificant mass of a substance fixed on the surface of a quartz plate and electrodes. Microgravimetric immunosensors allow direct registration of biochemical interactions without additional labeling (fluorescent, enzymatic, etc.), which distinguishes them from similar devices. A unique feature of immunosensors is the combination of high sensitivity provided by the use of a piezoelectric crystal resonator as a physical transducer and selectivity determined by the nature of the used receptor molecules [2].

A known method of producing biosensors, including physical sorption of the biolayer using chemical reagents, contributes to the protection of the functional activity of proteins for a long time [8].

The disadvantage of this method is the low sensitivity of the biolayer due to the decrease in the number of active centers of antibodies when using hard-acting chemicals.

A known method of producing immunosensors by physical sorption of lipopolysaccharides isolated from bacteria Yersinia enterocolitica on the surface of a piezoelectric crystal [4]. The application of the proposed method allows the detection of antibodies using a piezoelectric quartz sensor, providing high detection sensitivity.

The disadvantage of this method is the duration of the bioreceptor layer of the sensor (2-3 hours) and the low resistance of the formed coating as a result of physical sorption, which affects the reproducibility of the results and the life of the sensor.

Closest to the claimed invention is a method for the detection of macromolecules by a biosensor device, including the preliminary activation by pairs of aldehydes of the surface of a piezoelectric crystal in a vacuum in an UHF field followed by immobilization of antibodies [5].

The disadvantage of this method is the use of aldehyde as an activating reagent capable of hydrolyzing in an aqueous medium during immobilization with immunoglobulins, which leads to a decrease in the stability of the sensor and the effectiveness of its immobilization. In addition, the use of aldehydes can lead to a nonspecific reaction.

The purpose of the invention is the creation of microgravimetric immunosensors for indicating causative agents of especially dangerous infections (plague, tularemia, brucellosis), taking into account the requirements of maintaining stability, sensitivity, specificity.

To date, the most studied methods of immobilization of protein molecules (physical adsorption, covalent immobilization of receptor molecules). The disadvantages of the method of physical sorption of quartz resonators is the low resistance of immobilized antibodies, which affects the shelf life of the sensor.

To ensure the stability of immobilized quartz resonators, it is necessary to increase the concentration of surface reaction-functional groups, which is achieved by covalent immobilization. In this case, the biolayer (receptor molecules - immunoglobulins, antibodies) is more firmly fixed on the quartz plate and sensor electrode, which reduces the loss of receptor molecules during prolonged contact with the liquid, provides higher reproducibility of the analytical signal, and helps to preserve the functional activity of protein receptor molecules for a long time .

The technical result of the invention is achieved by the fact that quartz resonators with nickel electrodes RK-12 and a diameter of a quartz plate of 8 mm are used as a sensor (ZAO ETNA Moscow), and for activating the surface of a quartz plate and electrodes, plasma spraying of low molecular weight polyethyleneimine (PEI) [ -CH ₂ CH ₂ NR-], where R = H or a side chain; molecular weight less than 10,000 daltons (Yes); density 1.05 g / cm ³ ). PEI is a polymer with repeating structural elements of the amino group and two carbon aliphatic spacers CH ₂ CH ₂ [3]. Plasma sputtering of PEI on the surface of a quartz plate was carried out in our facility [6].

The features of low-temperature vacuum polymerization of PEI in a glow discharge plasma are most objectively reflected in the monograph by X. Yasuda [7]. On page 122, para. 5 and 116, para. 1 it is noted that "a large number of sequential and parallel reactions in plasma leads, respectively, to the formation of an ensemble of active particles," and "free radicals in polymers obtained in plasma can attach oxygen or water molecules to form hydroxyl and carbonyl groups." In a review by A. Mateescu et al. [9] on page 46 with respect to plasma polymerization processes, plasma polymerization characteristics are given that are consistent with the main points of the monograph by X. Yasuda [7]: “Plasma polymerization processes induce crosslinking besides polymerization, even in the absence of a crosslinker molecule due to the strong ionization conditions and molecular fragmentation. "

The main characteristics of the polymerization of PEI in plasma are the formation of free radicals due to the ultraviolet spectrum of the plasma, which are fixed on inorganic surfaces and are able to form a covalent bond with immunoglobulins due to peptide bonds [1]. As a result of plasma spraying of PEI, a nanostructured uniform coating with accessible reaction-functional groups is formed on the surface of a quartz plate and Raman electrodes for covalent bonding with immunoglobulins, which has high mechanical strength and chemical stability.

When constructing microgravimetric immunosensors, the parameters of the initial quartz resonators were measured, then the surfaces of the quartz plates with PEI electrodes were activated by plasma spraying in a vacuum setup at a pressure of 0.48 pascal (Pa) and exposure to a 30 W UHF field (W = J / second (s )), we varied the duration of treatment in plasma: 10, 20, and 40 s and, thus, changed the value of the specific total energy of the plasma flow from 24 to 96 W / cm ² .

Activation of the inorganic surface of the quartz resonator plate was carried out with the help of our designed installation - “Installation for ion deposition” [6]. The operation of the installation in vacuum (0.48-0.96 Pa), the action of an UHF field with a power of 30 W, and the arrival of PEI vapor provided a low-temperature gas-discharge plasma with the formation of a luminous plasma stream between the quartz cylindrical walls of the chamber with a diameter of about 40 mm and a length of ~ 87 mm (82-92 mm) (section ~ 12.5 cm ² , volume ~ 100 cm ³ ), in the central part of which there was a processed quartz plate with electrodes, the surface area of which was 1.0 cm ² with exposure for 10 s. Moreover, for 10 s, based on the power of the UHF field - 30 W, the size of the plasma flow (12.5 cm ² × 8.7 cm ~ 100 cm ³ ), the surface area of the quartz plate equal to 1.0 cm ² and simple calculations, the specific total energy of the plasma activation of the quartz plate is 24.0 W / cm ² (which is equivalent to 3 values of the solar constant). From the above source [7] it follows that there is no need for preliminary modification of the polymers used for plasma polymerization on surfaces. According to this source (C.16, para. 2): "In the case of plasma polymerization, the polymer is deposited on a substrate to a lesser extent depending on its nature and with equal success can be carried out on the surface of glass, organic polymer and metal."

Subsequent covalent IgG immobilization was carried out in 1.0 ml of an immunoglobulin solution (0.125 mg / ml) for 10 minutes. Each series of IgG solutions was monitored by conventional immunoassay (ELISA) with suspensions of homo- and heterologous strains and taking into account the results using a Multiskan photometer (Flow, Finland) at a wavelength of 492 nm in optically transparent polystyrene plates (Flow Laboratories ", USA).

For covalent immobilization of plague, tularemia or brucellosis immunoglobulins on the surface of the quartz plate and electrodes, we varied the concentrations of their solutions (0.063, 0.125, and 0.3 mg / ml) and the exposure duration (5, 10, and 15 min). Wherein the volume concentration of active sites of macromolecules (IgG) in calculating the largest atomic mass units (a. E. M.), Respectively indicated concentrations in a volume of 1 cm ³ was 0,45 × ^{15 October,} 0,9 × 10 ¹⁵ and 2.2 × 10 ¹⁵ spin / cm ³ . However, the results of experiments and calculations of the surface density of active centers showed the advantages of an IgG concentration of 0.125 mg / ml chosen by the authors (i.e., IgG molecules were a multiple in excess compared to the number of reaction-functional groups).

Unbound immunoglobulins were removed from the surfaces of quartz plates with electrodes by washing with distilled water for 2 min, followed by drying of the Raman in an air stream for 2-3 min at a temperature of 35-40 ° C. Five-fold dilutions of the disinfected cultures of vaccine strains of Yersinia pestis EV, Francisella tularensis ZHTV, Brucella abortus 19-BA (from 1 × 10 ² to 1 × 10 ⁹ m.k./ ml) were tested, where the latter were specifically bound to the plate and electrode immobilized on the surface resonator with immunoglobulin molecules to form an immunocomplex. Unbound cells of the causative agents of plague, tularemia or brucellosis were removed from the plates and electrodes of quartz resonators (RS) by washing with distilled water, the residues of which were then disposed of by drying the RS in an air stream for 2-3 min at a temperature of 35-40 ° C. The results of the analysis were measured on a CPNA-330 setup (ETNA Moscow) for 2 minutes. A positive shift was considered to be a frequency shift of 200 Hz or more.

As a result of the experiments, it was found that the parameters for producing microgravimetric immunosensors necessary for their high-quality work are: activation of the surface of quartz plates with electrodes by plasma spraying PEI for 10 seconds with a specific and total plasma flow energy of 24 W / cm ² in an ion deposition apparatus ; immobilization on quartz plates with immunoglobulin electrodes from a solution with a protein concentration of 0.125 mg / ml for 10 min; washing from unbound immunoglobulins with distilled water for 2 minutes; drying the remaining water for 2-3 minutes in a stream of air at a temperature of 35-40 ° C.

The sensitivity of the immunosensors was 1 × 10 ³ -1 × 10 ⁴ MK./ml Preservation of the specific activity of immunosensors was noted for 3 months (observation period). When controlling for specificity, there were no cross-reactions with heterologous strains.

The results obtained allow us to conclude that the developed technology for creating microgravimetric immunosensors for the detection of pathogens of especially dangerous infections (plague, tularemia, brucellosis) provides an analysis that meets the requirements for sensitivity, specificity, and analysis time.

The possibility of practical application of the invention is illustrated by examples of its specific implementation using a combination of the claimed features.

Example 1. Obtaining microgravimetric immunosensor to indicate a plague microbe.

Preliminarily, the frequency of the quartz resonator was measured, a quartz plate with electrodes was activated by plasma spraying of polyethyleneimine for 10 s in a vacuum unit at a 30 W UHF field with a specific total plasma flow energy consumption of 24 W / cm ² , the resonator frequency was monitored, immobilized on the surface quartz plate and electrodes in 1.0 ml of a plague immunoglobulin solution with a concentration of 0.125 mg / ml for 10 min, washed with distilled water for 2 min, dried residual water for 2-3 min in an air stream at a temperature of 35-40 ° C, incubated with a suspension of the plague microbe in concentrations from 1 × 10 ² m.k. / ml to 1 × 10 ⁹ m.k. / ml for 10 min , washed and dried КР as described above, the frequency was measured. A 0.01 M solution of phosphate-buffered saline (PBS) was used as a negative control. By reducing the biosensor frequency after incubation with the plague pathogen, by 200 Hz or more, the presence of a pathogen was judged. As a result, it was found that the sensitivity of the method was 1 × 10 ³ m.k. / ml (22%) and 1 × 10 ⁴ m.k. / ml (78%).

Example 2. Obtaining microgravimetric immunosensor for indicating tularemia microbe.

Received analogously to example 1, only instead of plague immunoglobulins used tularemia immunoglobulins. The sensitivity of the method with homologous strains was 1 × 10 ³ MK./ml (12%) and 1 × 10 ⁴ MK./ml (88%).

Example 3. Obtaining a microgravimetric biosensor to indicate a brucellosis microbe

Received analogously to example 1, only instead of plague immunoglobulins used brucellosis immunoglobulins. The sensitivity of the method with homologous strains was 1 × 10 ³ MK./ml (10%) and 1 × 10 ⁴ MK./ml (90%).

Used Books

1. Bohinski R. Modern views in biochemistry / Per. from English E.Yu. Krynetsky and N.F. Krynetskoy. - M.: Mir, 1987 .-- 544 p., P. 67.

2. Ermolaeva TP, Kalmykova EP, Shashkanova O.Yu. Piezoelectric quartz biosensors for analysis of environmental objects, foodstuffs and for clinical diagnostics // Russian Chemical Journal (ZhRLO named after D.I. Mendeleev). - 2008 .-- T. III. - No. 2. - S.17-29.

3. Polyethyleneimine. http://www.sigmaaldrich.com/catalog/product/fluka/p3143?lang=en...

4. RF patent No. 2287585. Publ. 11/20/2006. Bull. Number 32.

5. RF patent No. 2148259. Publ. 04/27/2000. Bull. No. 12.

6. Certificate for a utility model of the Russian Federation No. 2464. Publ. 07/16/1996. Bull. Number 7.

7. Yasuda X. Polymerization in plasma / X. Yasuda; Per. from English A.B. Gilman, A.A. Kalacheva; Ed. VK. Potapova. - M .: Mir, 1988 .-- 376 p.

8. Carter R. M., Jacobs M. B., Lubrano G. J., Guilbault G. G. Piezoelectric Detection of Ricin and Affinity-Purified Goat Anti-ricin Antibody // Anal. Lett. - 1995. - V.28. - P.1379-1386.

9. Mateescu A., Wang Y., Dostaiek J., and Jonas U. Thin Hydrogel Films for Optical Biosensor Applications // Membranes 2012, 2, 40-69; doi: 10.3390 / membranes2010040; Membranes ISSN 2077-0375 www.mdpi. com / journal / membranes /.

Claims (1)

A method of producing a microgravimetric immunosensor, including activation of the surface of the quartz resonator with the formation of reaction-functional groups, covalent immobilization of immunoglobulins and frequency registration when the mass of the resonator is changed, characterized in that they activate the surface of the quartz resonator by plasma spraying polyethyleneimine with a molecular weight of less than 10,000 Da for 10 s in a vacuum installation at a UHF field power of 30 W, then immunoglobulins are immobilized from a solution with a protein concentration 0.125 mg / ml, washed with distilled water, dried in a stream of air with registration of the shift of the resonant frequency.