RU182822U1 - Multiplex electrochemical biochip for detection of tumor-associated lung cancer biomarkers - Google Patents

Abstract

The utility model relates to the field of biotechnology, namely, to electrochemical chips for detecting lung cancer biomarker proteins in the plasma of patients. A multiplex electrochemical biochip for detecting tumor associated lung cancer biomarkers contains a substrate with six measuring electrodes deposited on it. In addition to the measuring electrodes, one auxiliary electrode and one reference electrode are made on the substrate. A DNA aptamer specific to one given plasma biomarker protein of patients with lung cancer is deposited on the working surface of each of the measuring electrodes. A multiplex electrochemical biochip can improve diagnostic accuracy by simultaneously identifying six lung cancer biomarkers. 1 ill.

Description

The utility model relates to the field of biotechnology, namely to electrochemical chips for detecting biomarker proteins of lung cancer in the plasma of patients.

A well-known biochip for multiplex analysis (RU 142470 U1 G01N 33/53 06/27/2014) containing a transparent substrate made of glass with polylysine applied to its surface, on which test sections with immobilized antibodies specific for cell antigens are placed, is that the test sections are separated plastic walls for 9 tight cells, equipped with fluorescent antibodies to estrogen, progesterone, CA-125, CEA, SK-7, SK-20, Ber-EP4, TTF1 and WT1 receptors, closed on top with a hydrophobic polysiloxane film fixed to the upper edge of the plas teak walls creating airtight cells. The biochip allows you to examine the patient's serous fluid to determine the nature of ascites (reactive or oncological), and the primary lesion that has a tumor producing ascites. Reading and analysis of the result is carried out using a fluorescence microscope.

Using the described biochip using fluorescence microscopy, it is impossible to accurately quantify the analytes bound to the antibodies. In addition, the antibodies used are expensive and require special storage conditions.

A biochip is also known, which is used in the method of analysis of EML4-ALK translocations associated with the sensitivity of lung cancer to antitumor target therapy, amplified using a set of specific primers, which is a plastic substrate with gel cells containing a set of immobilized probes complementary to the analyzed regions of the EML4 K gene AL (RU 2509153 C1 C12N 15/00, C12Q 1/68, 03/10/2014).

The disadvantage of the described biochip is the complexity and time required for the analysis.

To date, there are also electrochemical chips for detecting targets in the test sample, allowing to obtain results within a few minutes.

Three-electrode biochips are known that make it possible to detect target proteins by measuring an electrochemical signal.

So it is known a device for implementing the method for the detection of specific nucleic acid sequences by the electrochemical method (RU 2509157 C2; C12Q 1/68 B82B 3/00 (2006.01) G01N 27/28, 03/10/2014 Bull. No. 7), which is an electrochemical analyzer consisting of a three-electrode electrochemical cell, the electrodes of which are connected to a recording device containing a power source, for example, a potentiostat, characterized in that the working electrode is made of an electrically conductive silicon or metal substrate with grown on it tic-oriented carbon nanotubes, over the entire surface of which a non-covalently immobilized oligonucleotide probe complementary to the region of the determined NC, or a modified oligonucleotide probe complementary to the region of the determined NC; between the working and auxiliary electrodes is a separator.

Using this electrochemical analyzer, you can determine only one nucleic acid sample, complementary to the oligonucleotide probe, which modified the working electrode. This complicates the procedure for quantifying several analytes in a sample.

Also known is a biosensor used in the method of electrochemical detection of nucleic acid sequences (RU 2470938 C2 C07F 15/00; C12Q 1/68 12/27/2012) developed by UNIVERSIDAD AUTONOMA DE MADRID (ES). A biosensor is a set of oligonucleotides immobilized on a solid support. It contains a gold electrode, which was prepared by immobilization of the DNA probe sequence by direct adsorption, an auxiliary platinum electrode and calomel electrode, a reference electrode.

The known biosensor also does not allow simultaneous quantification of several analytes in a sample.

Also known is the DRP-220AT electrochemical chip (DropSens, Spain, dropsens.com © 2016 All Rights Reserved DropSens), which is a ceramic substrate (3.4 × 1.0 × 0.05 cm) with three electrodes deposited on it. The working electrode is gold; auxiliary electrode - gold; reference electrode - silver. The surface of the working electrode is modified using specific antibody ligands or aptamers specific for the desired analyte. After that, the test sample is applied to the surface of the working electrode and measurements are made using a potentiostat. To evaluate the result, the change in the electrochemical signal on the working electrode is determined before and after binding of the target with the modified surface of the working electrode. Thus, the desired biomarker protein is detected in the plasma of the cancer patient. The well-known electrochemical chip is taken as the closest analogue to the claimed utility model

In the DRP-220AT electrochemical chip consisting of three electrodes, the working electrode is modified with only one ligand, which makes it possible to detect the presence of only one target in the sample. The number of analytes detected can be increased by modifying the working electrode with several ligands; however, in this case, it will be impossible to give either qualitative or quantitative characterization of the presence of each specific analyte in the determination of analytes.

The objective of the claimed utility model is to increase the number of simultaneously detected biomarker proteins in the plasma of patients with lung cancer and thereby increase the accuracy of diagnosis.

The problem is solved due to the fact that a multiplex electrochemical biochip for detecting tumor-associated protein-biomarkers of lung cancer, containing a substrate with electrodes deposited on it, one of which is an auxiliary electrode and one reference electrode, according to the claimed utility model contains six measuring electrodes on the working surface Each of the measuring electrodes is coated with a DNA aptamer specific to one given plasma biomarker protein of patients with lung cancer.

Each measuring electrode deposited on a substrate of a multiplex electrochemical biochip can be made three-layer, consisting of copper, nickel and gold layers, with the application of a DNA aptamer specific to a given plasma biomarker protein of patients with lung cancer on the working surface of the gold layer of each of the measuring electrodes .

The substrate of the multiplex electrochemical biochip can be made of textolite.

The technical result of the utility model is expressed in increasing the accuracy of diagnosis of lung cancer.

The inventive utility model is illustrated in the drawing, which shows a diagram of a multiplex electrochemical biochip for detecting tumor-associated protein-biomarkers of lung cancer.

The multiplex electrochemical biochip for detecting tumor-associated protein-biomarkers of lung cancer contains a position 1, six measuring electrodes 2, a reference electrode 3 and an auxiliary electrode 4, conductors 5 connecting the electrodes to the terminal printed contacts 6, the inscription field 7, the orientation groove 8.

The multiplex electrochemical biochip for detecting tumor-associated biomarker proteins is a system of three-layer electrodes based on copper, nickel, gold with linear dimensions of about 11 × 35 mm, deposited using a plotter onto a surface made of textolite. The working surface of each measuring electrode is modified by a biologically functional layer, which is a specific DNA aptamer. One aptamer specific to its tumor marker protein is attached to one measuring electrode. DNA aptamers are immobilized (chemically fixed) on a gold surface using thiol groups. Aptamers are not applied to the auxiliary electrode and the reference electrode; these electrodes remain clean. The system uses them in the analysis. In the inventive electrochemical biochip, the structure of the reference electrode and the auxiliary electrode are the same as that of the measuring electrodes.

In the manufacture of multiplex electrochemical biochips, the technology of manufacturing printed circuit boards is used. The function of a thin gold film chemically applied over a nickel sublayer is to protect the nickel from oxidation. Nickel serves as a barrier to prevent the mutual diffusion of gold and copper. From above, with the exception of the working surfaces of the electrodes in contact with the test bioassay, all are covered with an electrically insulating layer of varnish. Before applying aptamers, the gold surface is restored. To do this, a mixture consisting of 3 parts of concentrated sulfuric acid and 1 part of concentrated hydrogen peroxide is applied to the measuring electrodes, after which the electrode surface is thoroughly washed with distilled water and dried in a stream of nitrogen. After that, aptamers are applied to the surface of the measuring electrodes, each of which is specific to a specific protein-biomarker of the lung cancer plasma, modified with a thiol group and incubated for 12 hours at a temperature of 4 ° C in a humid chamber. Then all the electrodes are washed with distilled water, and 2-mercaptoethanol is applied to the working electrodes and incubated for 15 minutes at room temperature, after which they are washed with 40% ethanol and distilled water and a phosphate buffer is applied. The result is electrodes coated with aptamers specific for lung cancer biomarkers.

A multiplex electrochemical biochip for detecting tumor-associated protein-biomarkers of lung cancer works as follows. A multiplex electrochemical biochip is installed in a potentiostat adapter and immersed in a buffer for measurements containing salts of iron-potassium and iron-potassium potassium, and electrochemical signals are measured on the electrodes. Next, the biochip is removed, washed and applied to the working surface of the measuring electrodes of the plasma of patients with lung cancer previously incubated with yeast RNA, the blood plasma is kept on the working surface of the electrodes for 30 minutes, washed with distilled water and the electrochemical signals are again measured on the electrodes. An auxiliary electrode is necessary to ensure the occurrence of current on the measuring electrodes. The reference electrode is needed to build the baseline. By changing the electrochemical signal, one judges the presence of the desired biomarker proteins in the patient's blood plasma. Each DNA aptamer deposited on a separate measuring electrode allows you to identify a specific biomarker protein. By a combination of identified biomarker proteins, lung cancer is diagnosed.

Thus, the inventive multiplex electrochemical biochip allows you to identify not one protein biomarker, but also several protein biomarkers of lung cancer, the analysis of the combination of which allows to increase the accuracy of diagnosis.

Claims (2)

1. A multiplex electrochemical biochip for detecting tumor-associated protein-biomarkers of lung cancer, containing a substrate with electrodes deposited on it, including one auxiliary electrode and one reference electrode, characterized in that it further comprises six measuring electrodes, where each measuring electrode deposited on a multiplex substrate an electrochemical biochip, made of three layers, consisting of copper, nickel and gold layers, with the application of a DNA aptamer specific to its to a given plasma biomarker protein of patients with lung cancer, on the working surface of the gold layer of each of the measuring electrodes. 2. The multiplex electrochemical biochip according to claim 1, characterized in that the substrate is made of textolite.

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