RU2745511C1 - Method for immobilizing short nucleotide sequences on the surface and end regions of nanomaterials - Google Patents

Abstract

FIELD: biology.SUBSTANCE: invention relates to a method for immobilizing short nucleotide sequences of aminomodified DNA or RNA oligonucleotides on the surface and end regions of nanomaterials and can be used in industry in the production of biocomposites. Disclosed is a method for immobilizing short nucleotide sequences of aminomodified DNA or RNA oligonucleotides on the surface and end regions of nanomaterials containing oxygen-containing functional groups, including preparing a solution of oligonucleotides with a concentration of at least 50 mcM in a buffer solution with a pH in the range of 4-6.9, mixing the specified solution with a solution of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide with a concentration of at least 5 mM, in a buffer based on 2-(N-morpholino)ethanesulfonic acid (MES) and ethanol and applying the final solution to a substrate in the sensitive area of ​​the formed biosensor followed by keeping the solution for at least 12 hours on the substrate in order to maximize the complete reaction of binding DNA or RNA oligonucleotides with carbon nanomaterial containing oxygen-containing functional groups.EFFECT: disclosed is a new effective method that makes it possible to form biological sensors based on carbon nanomaterials.6 cl, 2 dwg, 1 ex

Description

The invention relates to methods for modifying nanostructures by covalently linking short nucleotide DNA or RNA sequences with functional groups on the surface or end regions of nanostructures in order to form functional biocomposite structures.

Known methods of binding low-dimensional biological structures with nanomaterials, in particular with carbon nanotubes and graphene-like materials in order to form a sensitive area of biosensors. In this case, the modification can be carried out both covalently and through other types of bonds (hydrogen bond, van der Waals interaction). It should also be noted a wide range of immobilized biological structures and a variety of specific techniques for performing the immobilization procedure and the formation of the biosensitive layer as a whole.

The patent [US 9127304 B2] describes a method of forming biosensors based on conducting polymers, where a solution of monomer units of a conducting polymer is doped with negatively charged nanoparticles before being applied to a substrate. After deposition, the monomer units are electropolymerized on the surface of the substrate electrode. As monomer-bound biosensitive agents, DNA in the form of dendrimers, DNA / RNA in the form of helices and proteins are used. Polyacetylene, polypyrrole, polythiophene, polyaniline, poly-p-phenylene sulfide and polyparaphenylenevinylene can be used as a conductive polymer.

In this patent, a method for binding biosensitive agents to a conductive polymer consists of the following steps. Monomeric units of a conducting polymer bound to nanoparticles are applied to the surface of the working electrode of the sensor (the sensor electrodes are made in the form of spirals)

The main disadvantage of this invention is the complexity of ensuring the repeatability of the electrophysical parameters of sensors in a large batch, because the distribution of components in this case is random and is controlled only by changes in the concentration of components.

In one aspect, the present invention relates to a method for making a polymer-based biosensor comprising the steps of (a) doping a solution of monomer units of a conducting polymer with negatively charged nanoparticles comprising a capture moiety; and (b) polymerizing the monomer units on the surface of the sensor containing the electrode, thereby trapping the nanoparticles on the SSO surface.

In one embodiment, the method further comprises the step of (c) contacting the polymerized surface with a biopolymer of interest conjugated to a target moiety, wherein the target moiety has an affinity for said capture moiety. In one embodiment, the sensor contains gold. In one embodiment, the conductive polymer is selected from the group consisting of poly (acetylene), poly (pyrrole), poly (thiophene), poly (aniline), poly (p-phenylene sulfide), and poly (paraphenylene vinylene). In one embodiment, the conductive polymer is polypyrrole. In one embodiment, the monomer units are pyrrole. In one embodiment, pyrrole is present at a concentration of about 1 to about 100 mM. In one embodiment, pyrrole is present at a concentration of about 5 to about 20 mM. In one embodiment, the charged nanoparticle contains a negatively charged dendrimer. In one embodiment, the dendrimer is a DNA dendrimer. In one embodiment, the charged nanoparticle is present at a concentration of about 0.1% (v / v) to about 1.0% (v / v). In one embodiment, the surface density of the nanoparticles on the surface of the sensor is from about 0.20 pmol / cm to about 2.0 pmol / cm.In one embodiment, the capture fragment is an antibody or functional fragment thereof, avidin, streptavidin, aptamer, spiegelmer, glutathione or S-peptide. In one embodiment, the capture fragment is an anti-biotin antibody or streptavidin. In one embodiment, polymerization is achieved by electropolymerization or photopolymerization. In one embodiment, a cyclic rectangular electric field is used for electropolymerization. In one embodiment, the biological polymer of interest is a protein. In one embodiment, the biological polymer of interest is a nucleic acid. In one embodiment, the target group is biotin. In another aspect, the present invention provides a polymer-based biosensor made by the method of the present invention. In one embodiment, the conductive polymer of the polymer-based biosensor is polypyrrole and the nanoparticles are DNA dendrimers.

In yet another aspect, the present invention relates to a method for detecting an analyte in a solution comprising the steps of (a) contacting the solution with a polymer-based biosensor; (b) applying voltage to the electrode; and (c) determining the current generated by the biosynthesis.

In one embodiment, the analyte is a protein. In one embodiment, the protein is present at a concentration of about 100 fg / ml to about 2.5 g / ml. In one embodiment, the analyte is nucleic acid. In one embodiment, the nucleic acid is present at a concentration of about 10 to about 10 μM. In one embodiment, the solution contains body fluid. In one embodiment, the body fluid is saliva. In one embodiment, the analyte is an oral cancer biomarker. In one embodiment, the analyte is selected from the group consisting of IL-8 protein, IL-IB protein, and IL-8 mRNA.

Closest to the proposed method is the method described in US patent [US 9494582 B2]. Namely, the patent describes a method for creating a sensor on surface acoustic waves, where aptamers having an amino group at the ends and specifically binding to a target biomolecule are used as a biosensitive layer. In addition, the sensitive layer may include a SAM linker between the support and the amine moiety or 3-mercaptopropionic acid (MPA). Also, the method of creating a sensitive area can include the removal of nonspecifically immobilized aptamers.

The immobilization protocol involves the use of one of the chemicals N-Hydroxysuccinimide (NHS) and 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) or mixtures thereof to immobilize the aptamers through the amino-modified end. Namely, the sequence of the aptamer immobilization process is as follows: the gold film substrates were immersed in a 10 mM 3-mercaptopropionic acid (MPA) solution for 30 minutes and then washed with ethanol and deionized water. After drying the slides, they were immersed in a solution of N-hydroxysuccinimide (NHS) and N- (3-dimethylaminopropyl) -N-ethylcarbodiimide hydrochloride (EDC) with solution concentrations of 0.2 M and 0.05 M, respectively, for 30 minutes. Then the gold film substrates were washed with deionized water and then immersed in a 5 μM aptamer solution. Finally, the slides were washed with phosphate buffered saline (PBS) to wash off nonspecifically adsorbed proteins.

Patent [3 US20140363808A1] describes a method for screening graphene-based aptamers without target immobilization and aptamers obtained by this method, and, in particular, a new GO-SELEX method without target immobilization, in which one nucleic acid pool with strands can react with an unbound target material or target material, after which a single-stranded nucleic acid that has not been associated with the target or counter-target can be separated using graphene. In addition, a particular aptamer obtained by the method described above can be used to diagnose diseases associated with a target.

Disclosure of invention

The technical objective of the present invention is the formation of a sensitive biological sensor by immobilizing biomolecule-sensitive DNA / RNA oligonucleotides. as a result, a transducer is formed, a sensitive area of a biological sensor, which simultaneously acts as a transducer. Immobilization occurs on oxygen-containing groups present in carbon nanotubes including end-face functional groups or poorly reduced graphene oxide containing functional groups on the lattice faces. In this case, the process of immobilization itself does not change, regardless of the material used.

In the proposed patent, DNA or RNA oligonucleotides are applied directly to a layer of carbon nanotubes or reduced graphene oxide containing oxygen-containing functional groups, which provides further improved characteristics of the entire biosensor, in particular, it allows to provide a minimum signal-to-noise ratio and maximum sensor sensitivity due to the fact that the distance of charge transport between the biosensitive and the signal-conducting layer is minimized.

To ensure immobilization, additional reagents are required, namely 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC). In this case, EDC, which in this case is the main reagent, which is used to activate the carboxyl groups contained on the surface of weakly reduced graphene oxide for subsequent binding of amino-modified DNA or RNA. At the same time, in contrast to the above-mentioned prior art sources, a one-stage immobilization process is used, including the process of immobilization of aptamers on poorly reduced graphene oxide using an EDC solution in distilled water with the addition of 2- (N-morpholino) ethanesulfonic acid (MES), which provides increased film wettability carbon nanomaterials and ethanol. Also, the method is suitable for any amino-modified aptamer, regardless of the specific nucleotide sequence.

The general sequence of operations is as follows:

1) Removing a preform of a biosensor structure with a nanomaterial film containing oxygen-containing functional groups from a transport container;

2) Placement of sensor blanks in a chamber with an inert atmosphere at room temperature;

3) Formation of an EDC solution with a buffer and the required number of aptamers (the concentration of EDC must be at least 5 mM, the concentration of aminomodified DNA or RNA must be at least 50 μM). In this case, to optimize the deposition process, 2- (N-morpholino) ethanesulfonic acid (MES) is used at a concentration of 100 mM, at a solution pH in the range of 4 - 6.9, and the addition of 25 wt% ethanol.

4) Applying a 5 μl drop of the solution formed in step 3 to the area of the biosensor blank, which is subsequently used as a sensitive one;

5) Keeping the solution of aptamers on a substrate for at least 12 hours in an inert atmosphere.

6) Removal of residues of aminomodified DNA or RNA solution with buffer solution or water

7) Control of the degree of immobilization by various methods (Fourier-IR, etc.) on specially selected structures.

8) Packing finished structures into shipping containers.

Both solid-state (silicon, sapphire, quartz, etc.) and polymer substrates (polyethylene naphthalate, polyethylene terephthalate, polydimethylsiloxane, etc.) can act as a carrier substrate.

Brief Description of Drawings

Figure 1. Raman spectra on the reduced surface of graphene oxide before and after immobilization of aptamers.

Figure 2. FT-IR spectra of the biosensor in the reduced oxide region with and without aptamers applied.

Execution example

To immobilize aptamers, we used a U-shaped film of reduced graphene oxide deposited on a PET substrate 175 μm thick. The reduction of graphene oxide was carried out locally with the minimum parameters of exposure to coherent radiation in the optical range (442 nm). Raman spectra before and after immobilization of the aptamers are shown in Fig. 1. For immobilization, an aminomodified thrombin aptamer with the nucleotide sequence AmTBA (5'-GGTTGGTGTGGTTGG-3 ') in EDC solution (5 mM) supplemented with 100 mM MES buffer and 25 wt. % ethanol. The aptamer-containing, it is also the sensitive area of the sensor was treated with a solution of aptamers with a concentration of 50 μM and left for 24 hours in an argon atmosphere for the most complete course of the immobilization reaction. Fourier transform infrared spectra of structures with and without an immobilized aptamer were obtained. In structures where the aptamer was not immobilized, a vibrational peak from carboxyl groups is clearly observed (Fig. 2).

Sources of information

1. US patent US 9127304 B2.

2. US patent US 9494582 B2.

3. US patent US20140363808A1.

Claims (6)

1. A method for immobilizing short nucleotide sequences of amino-modified DNA or RNA oligonucleotides on the surface and end regions of nanomaterials containing oxygen-containing functional groups, including preparing a solution of oligonucleotides with a concentration of at least 50 μM in a buffer solution with a pH in the range of 4-6.9, mixing the specified solution with a solution of 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide with a concentration of at least 5 mM, in a buffer based on 2- (N-morpholino) ethanesulfonic acid (MES) and ethanol and applying the final solution to the substrate in the sensitive area of the formed biosensor, followed by keeping the solution for at least 12 hours on the substrate in order to maximize the complete course of the reaction of DNA or RNA binding of oligonucleotides with a carbon nanomaterial containing oxygen-containing functional groups. 2. The method of immobilization according to claim 1, characterized in that during the immobilization the workpieces are placed in an inert atmosphere. 3. The method of immobilization according to claim 1, characterized in that functionalized carbon nanotubes, including end functional groups, are used as a carrier of nucleotide sequences. 4. A method for immobilization according to claim 1, characterized in that a film of poorly reduced graphene oxide containing functional groups on the lattice edges is used as a carrier of nucleotide sequences. 5. The method of immobilization according to claim 1, characterized in that an MES buffer is used, which provides increased wettability of the film of carbon nanomaterials. 6. The method according to p. 1, characterized in that for the preparation of the solution use 25 wt. % ethanol.

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