RU2792447C1 - Method for the manufacture of biological microarrays for the detection of genetic and protein markers - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology. A method for the manufacture of biological microarrays for the detection of genetic and protein markers on an inert substrate activated with a 1% solution of chitosan, m.m., is described. 38 kDa in 1.5% acetic acid, followed by washing in distilled water, drying and further application of biological molecules.

EFFECT: invention can be used for the manufacture of oligonucleotide, protein, carbohydrate biological microarrays (microarrays) intended for the detection of genetic and protein markers in multivariate analysis on a solid surface activated with a biopolymer - chitosan.

1 cl, 2 dwg, 2 ex

Description

The invention relates to the field of biotechnology and can be used for the manufacture of oligonucleotide, protein, carbohydrate biological microarrays (microarrays) intended for the detection of genetic and protein markers in multivariate analysis on a solid surface activated with a biopolymer - chitosan.

Polymer hydrogels are used to fabricate DNA, protein, and carbohydrate micromatrices [1, 2]. A significant disadvantage of three-dimensional hydrogel microarrays is the low rate of diffusion of biological molecules into the gel volume, which requires many hours of exposure, which practically eliminates the advantage obtained as a result of nonspecific immobilization of molecules.

In two-dimensional microarrays, biological molecules are adsorbed on the surface or covalently bonded to the substrate and directly contact the material under study, which significantly reduces the analysis time and simplifies the microchip fabrication technology.

Nonspecific binding of biological molecules by adsorption (for example, when using nitrocellulose membranes) provides unstable binding of molecules to the surface, which leads to their washing out during the analysis and a decrease in sensitivity.

For covalent binding of biological molecules, the microchip substrate surface is subjected to chemical modification to introduce functional groups, which ensures strong binding of biological molecules to the carrier surface.

Modified surfaces activated by organosilicon compounds - silanes with epoxy, amino, and aldehyde groups are used to fabricate DNA, protein, and carbohydrate micromatrices by covalent binding of functional groups of biological molecules [3, 4]. Silicone compounds and the method of their manufacture are characterized by high cost. In this case, covalent bonding occurs, in some cases, due to the presence of amino groups in biological molecules. Protein molecules have exposed amino and carboxyl groups in their structure, so they do not require additional modification. Covalent binding of oligonucleotides and carbohydrate (lipopolysaccharide (LPS)) molecules requires their modification with the addition of a functional amino group [5].

At the same time, it is known that the chitosan biopolymer has a high lipopolysaccharide-binding activity and a high sorption capacity [6–8]. Chitosan is an N-deacetylated derivative of the natural polysaccharide chitin obtained by cleavage of the acetyl group. Chitosan is a linear copolymer of N-acetyl-D-glucosamine and D-glucosamine with an additional reactive amino functional group. Chitosan forms a stable complex with LPS molecules. The interaction between LPS and chitosan occurs with the participation of positively charged amino groups of chitosan and negatively charged phosphate and carboxyl groups of lipid A and oligosaccharide of the LPS core [7, 8]. In addition, chitosan has a low cost, is a biodegradable and environmentally friendly biopolymer.

A method for manufacturing hydrophobic glass coated with chitosan, not intended for the immobilization of biological molecules, is described [RF Patent No. 2494984]. A known method of manufacturing substrates coated with chitosan, where the substrate is cellulose-containing materials - paper, cardboard, etc., also not intended for immobilization of biological molecules [Application for invention No. 2013158317]. A method for manufacturing glass coated with chitosan is described for the manufacture of so-called “cellular” biochips for the analysis of single somatic cells [9]. In this case, chitosan is immobilized on the glass surface using hydroxyls, and chitosan itself is activated by poly(ethylene glycol) diglycidyl ether. A known method of manufacturing a planar optical sensor based on an inert carrier (glass) coated with metal nanoparticles and microporous chitosan, modified with a p-acceptor compound capable of recognizing polyaromatic heterocyclic sulfur-containing compounds in petroleum products [RF Patent No. 2572801].

Also known is a method for manufacturing a planar optical sensor based on an inert carrier (glass) coated with metal nanoparticles and a finely porous polyelectrolyte, in particular, chitosan [Application for invention No. 2016148003]. The sensor is intended for the analysis of protein molecules in a liquid sample using Raman spectroscopy.

The objective of the invention is to develop a simpler and more economical method for the manufacture of biological (DNA, protein and carbohydrate) microarrays.

The technical result of the claimed invention is to simplify the manufacturing technology of biological (DNA-, protein and carbohydrate) micromatrices, reduce costs, increase expressivity, selectivity and sensitivity of detection of genetic and protein markers in multivariate analysis.

The technical result is achieved by a method for manufacturing biological microarrays, which involves: manufacturing two-dimensional microchips based on an inert substrate (glass), which is activated in a solution of 1% chitosan with a molecular weight of 38 kDa in 1.5% acetic acid; incubation for 5-10 min at room temperature; subsequent washing with distilled water for 5 min at room temperature, drying in air and further application of biological molecules on a chitosan-activated substrate by contact printing using a miniplotter, with each sample of biological molecules diluted 1:1 in the appropriate printing buffer being applied on the surface in three repetitions in the form of 12-16 identical zones on one slide.

Activation of an inert substrate with chitosan provides simultaneous covalent binding and adsorption of biological molecules of various nature (nucleic acids, proteins, carbohydrates, lipids) without additional stages of their modification with amino groups and chitosan activation. An increase in the rapidity of analysis is achieved through the use of a two-dimensional microarray design. Quantitatively, the increase in the sensitivity of the analysis is associated with an increase in the “signal/background” values. An increase in the "signal" value is achieved due to the strong covalent binding of biological molecules to chitosan. The decrease in the "background" value is achieved due to the transparency of chitosan in the wavelength range of 300-800 nm for the detection of biological molecules labeled with fluorescent dyes. The increase in selectivity is due to the use of specific biological molecules.

The novelty of the claimed invention lies in the fact that an inert substrate coated with chitosan is used for the manufacture of DNA, protein and carbohydrate micromatrices.

Implementation of the invention:

Example 1. Preparation of DNA microarrays

в концентрации 100 пмоль/мкл суспендируют в буфере (3М бетаин, 4^х SSPE-буфер, 0,05% твин-20) в соотношении 1:1, наносят на поверхность микрочипа, активированного хитозаном, в объеме 2-3 нл. Иммобилизацию биологических молекул осуществляют при температуре 80°С в течение 20 мин.Preparations of oligonucleotide probes for specific genes of Yersinia pestis

at a concentration of 100 pmol/µl is suspended in a buffer (3M betaine, 4 ^x SSPE buffer, 0.05% tween-20) in a ratio of 1:1, applied to the surface of a microchip activated with chitosan, in a volume of 2-3 nl. Immobilization of biological molecules is carried out at a temperature of 80°C for 20 minutes.

Before the stage of DNA-DNA hybridization, preliminary blocking of the surface of DNA chips is carried out. To do this, 100 µl of blocking solution is added to the wells of the incubation chamber. Blocking is carried out on a thermoshaker at a temperature of 42°C for 30 min at a platform rotation speed of 200 rpm. Then, the contents from the wells are carefully, without touching the area of the array, taken using a vacuum aspirator. For washing, 100 μl of washing solution 1 is added to the wells, incubated at a temperature of 42°C for 5 min at a platform rotation speed of 250 rpm. Then, the contents from the wells are carefully, without touching the area of the array, taken using a vacuum aspirator.

Fluorescently labeled PCR products obtained at the stage of amplification, in a volume of 10 μl, are mixed with a hybridization buffer in a volume of 70 μl, heated in a solid-state thermostat at a temperature of 95°C for 5 minutes. In the wells of the incubation chamber, 80 μl of the heated hybridization mixture is added and incubated on a thermoshaker at a temperature of 42°C for 1.5-2 hours at a platform rotation speed of 200 rpm. Then, the contents from the wells are carefully, without touching the area of the array, taken using a vacuum aspirator. The slides are washed with washing solution 1 at a temperature of 42°C for 3 min at a platform rotation speed of 250 rpm by adding 100 μl of washing solution to each well. The contents of the wells are carefully removed using a vacuum aspirator. Then, the slides are washed with a wash solution at 42°C for 2 min at a platform rotation speed of 250 rpm by adding 100 µl of wash solution to each well. The contents of the wells are carefully removed using a vacuum aspirator. Next, the glasses are taken out of the frames, rinsed with distilled water, dried by centrifugation at 1000 rpm for 1 min. Registration and accounting of results is carried out using a fluorescent scanner. According to the results of the analysis, the level of “signal/background” values using a chitosan-activated substrate is 1.5 times higher than when using an amino-modified substrate and 20 times higher than when using an epoxy-modified substrate.

Figure 1 fluorescent profiles of the results of the analysis of the detection of genetic markers of Yersinia pestis. 1 - printing buffer (negative control), 2 - oligonucleotide probe 3a, 3 - oligonucleotide probe 45, 4 - oligonucleotide probe irp2, 5 - oligonucleotide probe pla, 6 - oligonucleotide probe cafl, 7 - oligonucleotide probe lcrV. A - amino-modified glass, B - epoxy-modified glass, C - chitosan-modified glass.

Example 2. Production of protein and carbohydrate (lipopolysaccharide) microarrays and their use

Preparations of biological molecules of protein (cholerogen-anatoxin) and carbohydrate nature (O139, O1 Ogava, O1 Inaba) Vibrio cholerae at a concentration of 1 mg/ml are suspended in buffer (1x phosphate-buffered saline (PBS), 5% glycerol, 0.01% tween-20) in a ratio of 1:1 is applied to the surface of a microchip activated with chitosan in a volume of 2-3 nl. Immobilization of biological molecules is carried out at a temperature of 60°C for 20 minutes.

The test sera are added to the wells of the reaction cells of microarrays, incubated on a thermoshaker at a temperature of 37°C for 1 hour with a platform rotation speed of 300 rpm. After incubation, the glass is washed three times with PBS and conjugates of anti-species fluorescent antibodies against IgG, IgM are added. The slides are incubated at 37°C for 1 h, washed three times with PBS, and dried by centrifugation at 1000 rpm for 1 min. Registration and accounting of results is carried out using a fluorescent scanner. According to the results of the analysis, the level of “signal/background” values using a chitosan-activated substrate is 2 times higher than when using an amino-modified substrate and 10 times higher than when using an aldehyde-modified substrate.

In Fig.2, Fluorescent profiles of the results of the analysis of the detection of specific antibodies in the serum of diagnostic cholera 01. 8 - print buffer (negative control), 9 - O139 antigen, 10 - Ogawa O1 antigen, 11 - Inaba O1 antigen, 12 - cholerogen -anatoxin. A - amino-modified glass, B - aldehyde-modified glass, C - chitosan-modified glass.

Thus, the use of chitosan-modified glasses as a substrate for the manufacture of biological micromatrices provides an increase in the level of “signal/background” values by 1.5-2 times compared to a commercial ammine-modified substrate, 10 times - compared to an aldehyde-modified substrate, 20 times - compared to the epixi-modified substrate.

Information sources

1. Gryadunov D.A. Hydrogel biochips - tools for multiparametric analysis of markers of bacterial, viral and plant genomes: Abstract of the thesis. dis. … Dr. Biol. Sciences: 03.01.03 / Gryadunov Dmitry Mikhailovich. - Moscow, 2017. 315 p.

2. Mikhailovich V.M. Identification of infectious agents, genetic determinants of pathogenicity and drug resistance of microorganisms and viruses on biological microchips: Ph.D. thesis .... Dr. Biol. Sciences: 03.00.03, 03.00.07 / Mikhailovich Vladimir Mikhailovich. - Moscow, 2009. 53 p.

3. Wingren C, Borrebaeck C.A.K. Antibody-based microarrays // Methods Mol Biol. 2009; 509:57-84. doi: 10.1007/978-1-59745-372-1_5.

4. Schena, M. Microarray Biochip Technology // Eaton Publishing Company/Biotechniques Books. - 2000. - ISBN: 1881299376. - 298 p.

5. Parthasarathy N., Saksena R., Kovac P. [et al.] Application of carbohydrate microarray technology for the detection of Burkholderia pseudomallei, Bacillus anthracis and Francisella tularensis antibodies // Carbohydr Res. - 2008. - V. 343, N 16. - P. 2783-2788.

6. Gorbach V.I., Davydova V.N., Volodko A.V. [et al.] Sorption of lipopolysaccharides by zeolites modified with chitosan // Actual problems of biological physics and chemistry. - 2016. - N 1-2. - S. 60-64.

7. Davydova V.N., Naberezhnykh G.A., Ermak I.M. [et al.] Determination of binding constants of lipopolysaccharides of various structures with chitosan // Biochemistry. - 2006. - T. 71, N 3. - S. 417-425.

8. Naberezhnykh G.A., Gorbach V.I., Likhatskaya G.N. [et al.] Interaction of chitosans and N-acylated chitosan derivatives with lipopolysaccharides of Gram-negative bacteria // Biochemistry. - 2008. - T. 73, N 4. - S. 530-541.

9. Ferrara V., Zito G., Arrabito G. [et al.] Aqueous Processed Biopolymer Interfaces for Single-Cell Microarrays // ACS Biomater Sci Eng. - 2020 May 11;6(5):3174-3186. doi: 10.1021/acsbiomaterials.9b01871. Epub 2020 Apr 17.

Claims (1)

A method for manufacturing biological microarrays for the detection of genetic and protein markers, involving the manufacture of two-dimensional microchips based on an inert substrate (glass), which is activated in a solution of 1% chitosan with a molecular weight of 38 kDa in 1.5% acetic acid, incubation for 5-10 minutes at room temperature, subsequent washing with distilled water for 5 min at room temperature and drying in air, followed by application of biological molecules on a chitosan-activated substrate by contact printing using a miniplotter, with each sample of biological molecules diluted 1:1 in the appropriate buffer for printing, applied to the surface in three repetitions in the form of 12-16 identical zones on one slide.

Images