RU197467U1 - BIOCHIP FOR MULTIPLEX ANALYSIS - Google Patents

Abstract

The proposed utility model relates to medicine, namely to diagnostics, and can be used for the diagnosis of cancer. The biochip for evaluating tumor markers, consisting of a transparent substrate coated with polylysine and divided into cells by means of a lattice covered with a protective film, characterized in that the cells for introducing and fixing the biomaterial, they are located on the part of the substrate coated with polylysine and separated by a pad from the cells for storing and diluting antibodies of the test system located on the part of the substrate without polylysine and equipped with antibodies specific to certain cell antigens contained in the buffer solution. The use of a utility model is to simplify the analysis procedure using a biochip, as well as to shorten the analysis time by simultaneously evaluating antigen expression and cell morphology. In addition, the inventive biochip is devoid of the disadvantages associated with the use of fluorescently labeled antibodies as its functional basis, and is intended to evaluate tumor markers associated with various oncological diseases. 20 ill.

Description

The proposed utility model relates to the field of medicine, namely to diagnosis, and can be used for the diagnosis of cancer.

Understanding the physiological and pathophysiological processes in cells, tissues and the human body, including for diagnostic purposes, requires the identification and analysis of many biological objects, their mechanisms of action and regulation. The technology of biological chips (biochips) is one of the most effective tools that can solve these problems. Biochips have an optimal balance between performance, sensitivity and specificity, and are able to simultaneously detect in the multiplex tests the presence of many biomolecules contained in microvolumes. Biochips allow parallel analysis of healthy or pathologically altered tissues and cells, perform a comparative analysis, identifying changes associated with a wide range of socially significant human diseases, which is an extremely relevant area of modern biomedical research.

Known multi-parameter diagnostic test system,
designed to detect and monitor cancer therapy
breast and ovarian cancer, and method of analysis with
its use, protected by RF patent No. 2599890, IPC G01N 33/53, publ. 20.10.2016.

The test system (reagent kit) is characterized by the fact that detection and monitoring are carried out by simultaneously quantifying several tumor-associated antigens (tumor markers) in human serum, and includes a biological microchip (biochip) to simultaneously determine the concentrations of several tumor-associated antigens in a sample of human blood serum showing the system, a set of calibration samples, a set of buffer solutions and instructions for analysis. A method for conducting multi-parameter analysis of a sample using this test system is to conduct a sandwich of a sandwich variant of an immunoassay of a human serum sample on a biological microchip with fluorescence detection of signals.

 A disadvantage of the known solution is the use of fluorescent dyes, characterized by relatively low photostability and brightness, as well as the presence of a fluorescent background, which makes it difficult to interpret the results obtained using such a biochip. In addition, the said biochip is intended for the quantitative assessment of tumor markers associated with the development of only breast cancer and ovarian cancer.

A known method for the diagnosis / screening of colorectal cancer, based on the simultaneous quantitative determination of tumor markers of protein nature, antibodies to glycans, immunoglobulins G, A and M in human blood on a biological microchip, is protected by RF patent No. 2625018, IPC G01N 33/543, G01N 33/574 published on 07/11/2017.

The biochip contains hydrogel elements with immobilized antibodies to protein tumor markers, hydrogel elements with immobilized glycans, hydrogel elements with immobilized antibodies against human immunoglobulins of classes G, A, M. A change in the level of the analyzed markers compared to the level of markers in the blood serum of healthy donors indicates colorectal cancer .

A disadvantage of the known solution is the fact of using fluorescent dyes along with a quantitative assessment of tumor markers only in blood serum. This biochip is not designed to work with any other biomaterial that could potentially have higher clinical information content.

Known biochip for diagnosis in the field of medicine, protected by RF patent No. 137188, IPC A61B10 / 00, publ. 02.10.2014. The biochip consists of a plastic substrate, divided into two parts: the working one and for fixing the biochip in the hands. The working part of the biochip is divided into 15 equal cells (3 rows of 5 cells each) by means of a plastic grid. Each cell contains fluorescently labeled antibodies (0.01 μl). The first row (5 cells) contains fluorescently labeled antibodies to estrogen receptors, the second row (5 cells) contains fluorescently labeled antibodies to progesterone receptors, and the last (third) row (5 cells) contains fluorescently labeled antibodies to HER-2neu receptors. To preserve antibodies in a humid environment, 0.1 μl of PBS reagent is added to each cell, and a hydrophobic film is applied on top of the working surface to prevent the fluorescently labeled antibodies from drying out and the antibodies to move from one cell to another.

The disadvantage of this biochip is the use of fluorescently labeled antibodies capable of photobleaching, as well as the formation of a fluorescent background, which complicates the interpretation of the results and requires additional procedures aimed at reducing its intensity, which ultimately complicates the analysis procedure. In addition, the test system does not include control materials to assess the adequacy of the results obtained during the study.

Closest to the claimed utility model, selected as a prototype, is a biochip for multiplex analysis, protected by RF patent No. 142470, IPC G01N 33/53, publication date 06/27/2014. The biochip for multiplex analysis contains a transparent glass substrate with a polylysine coating and is divided into 9 cells by means of a plastic lattice. Each cell contains fluorescently labeled antibodies (0.01 μl). For research in the work area, test sites with antibodies to estrogen receptors, progesterone receptors, CA-125 receptors, CEA receptors, CK7 receptors, CK20 receptors, Ber-EP4 receptors, TTF1 receptors and WT1 receptors were used. To preserve antibodies in a humid environment, 0.1 μl PBS reagent was added to each cell, and a hydrophobic film was applied on top of the working surface, preventing the antibodies from passing from one cell to another and drying out. Using the proposed utility model for the study of serous fluids, fluids with a less rich cellular composition than blood, containing immobilized fluorescently labeled antibodies specific for cell antigens, it is possible to determine the nature of ascites (reactive or oncological), as well as the primary lesion that has a tumor producing ascites.

The disadvantage of such a biochip, in addition to the use of fluorescently labeled antibodies, is the complexity and duration of the analysis, since the fluorescence stage of the evaluation of antigen expression is first carried out, and then the morphology of the cell is determined. In addition, the drug is suitable for research for 10 minutes, as a result of which it is impossible to re-view the drug.

The technical problem solved by the proposed utility model is the creation of a biochip for multiplex analysis, which allows qualitatively detecting the expression of tumor markers and simultaneously assessing the morphology of cells.

 The technical result of using the utility model is to simplify the analysis procedure using a biochip, as well as to shorten the analysis time by simultaneously evaluating antigen expression and cell morphology. In addition, the inventive biochip is devoid of the disadvantages associated with the use of fluorescently labeled antibodies as its functional basis, and is intended to evaluate tumor markers associated with various oncological diseases.

The specified technical result is achieved in that in a biochip consisting of a transparent substrate coated with an adhesive composition having a positive charge and divided into cells by means of a grating, a closed protective film, the cells for applying and fixing the biomaterial are located on the part of the substrate coated with an adhesive composition having a positive charge, and separated by a pad from the cells for storage and dilution of antibodies of the test system located on a part of the substrate without an adhesive composition and equipped with those contained in the buffer m solution with antibodies specific for certain cell antigens.

The inventive biochip for multiplex analysis is illustrated by the drawings:

Figure 1 shows the proposed biochip, top view;

figure 2 shows the proposed biochip, side view

figure 3 for example 1 - puncture of the prostate gland. The cytological picture of dysplasia is 2-3 degrees. Coloring according to Romanovsky, magnification x100;

figure 4 for example 1 - positive staining with anti-PSA antibody. Peroxidase method, x200 magnification, biochip test system;

figure 5 for example 2 - flushing from the bladder. In the field of view, a group of urothelial cells with an unsharp polymorphism, architectonics preserved. Coloring according to Romanovsky, magnification x1000;

6 for example 2 shows a negative reaction (lack of brown nuclear staining) in the reaction with an antibody to protein P53. Peroxidase method, x400 magnification, biochip test system;

7 for example 3 presents a smear from the cervix. In the field of view of the squamous cell of all layers, in a part of the cells, enlargement of the nucleus and roughness of the nuclear membrane are noted, the presence of dysplasia of the 1-2 degree is not excluded:

on Fig for example 3 shows a negative reaction with an antibody to protein Ki67 (no nuclear staining) and a positive (cytoplasmic staining) reaction of cells with an antibody to protein P16. Peroxidase method, x1000 magnification, biochip test system;

figure 9 for example 4 presents punctate of the thyroid gland. In the field of view is a pseudopapillary accumulation of follicular epithelium, folding is noted in some cells, nucleols are visible. Coloring according to Romanovsky, magnification x1000;

figure 10 for example 4 shows a positive (nuclear) reaction with an antibody to the protein TTF1. Peroxidase method, x200 magnification, biochip test system;

11, for example 5 presents a scraping from the conjunctiva of the lower eyelid of the left eye. Mature lymphocytes predominate in the field of vision, fragments of destroyed cells are noted. Coloring according to Romanovsky, magnification x1000;

on Fig for example 5 shows a positive staining with an antibody to the protein CD20. Peroxidase method, x1000 magnification, biochip test system;

on Fig for example 6 shows the puncture of the lymph node. Coloring according to Romanovsky, magnification x1000;

on Fig for example 6 shows a negative reaction with an antibody to EpCAM. Peroxidase method, x1000 magnification, biochip test system;

on Fig for example 7 used pleural fluid. In the field of view are clusters of epithelial cells with signs of polymorphism. Cytological picture of metastatic effusion. Coloring according to Romanovsky, magnification x1000;

on Fig for example 7 shows a positive reaction with an antibody to protein CA125. Peroxidase method, x1000 magnification, biochip test system;

on Fig for example 8 appears ascites fluid. In the field of view are cells, presumably mesothelium, with unsharp polymorphism. Coloring according to Romanovsky, magnification x1000;

on Fig for example 8 shows a negative immunocytochemical reaction with an antibody to the EpCAM protein in ascitic fluid cells. Peroxidase method, x1000 magnification, biochip test system;

19, for example 9, ascitic fluid was used. Cellular clusters with polymorphism signs morphologically similar to epithelial are localized in the field of view. Coloring according to Romanovsky, magnification x1000;

on Fig for example 9 shows an immunocytochemical positive reaction with an antibody to EpCAM. Peroxidase method, x1000 magnification, biochip test system.

The biochip for multiplex analysis (Fig. 1) contains a transparent substrate 1 made of glass with a polylysine coating 2, divided into two equal parts separated by a pad 3. One part of the substrate is coated with an adhesive composition having a positive charge necessary for cell binding, the other the part has no adhesive coating on the bottom. Each part is divided into cells by means of a lattice 4 made of plastic. Cells 5 for storage and dilution of antibodies contain antibodies 6, which are the functional basis of the biochip test system. Cells 7 for introducing and fixing the biomaterial are located on a part of the substrate coated with an adhesive composition having a positive charge. The biochip contains a protective film of polysiloxane 8, fixed on top of the grating 4. Area 3 does not allow the solution to flow over the antibody, and after the introduction of the biomaterial, the cell suspension with the antibodies dissolved in it, from cell to cell.

The test system in the biochip format is designed to determine the degree of damage to the prostate gland, bladder, monitoring during the treatment of malignant tumors of the bladder, to clarify the degree of intraepithelial lesion of the squamous epithelium, typing of tumor lesions of the thyroid gland, primary typing of lymphomas, determining the nature of the damage to the lymph node, organ accessories tumor cells in fluids from serous cavities, the nature of the fluids themselves from serous cavities.

The present utility model is a plastic substrate measuring 25.4 mm × 76.2 mm, 1 mm thick. The test system in the biochip format can be stored in the light for 11 months at a temperature of +2 to + 8 ° C.

The proposed biochip works as follows. At the initial stage, biomaterial is sampled for research, using human biological fluids of various localizations, as well as material obtained as a result of a fine-needle aspiration biopsy (TIAB).

Material preparation:

The material delivered to the laboratory is centrifuged to obtain a cell pellet at 2000g for 5 minutes, after which the supernatant is removed.

Research progress on a test system in biochip format

Remove the protective film from the surface of the test system 8. Add the obtained cell pellet to the cells of the test system free of antibodies 7 in a volume of 20.0 μl per cell and carefully distribute over the entire surface. Dry the biomaterial in cells 7 at room temperature (18-22 ° C) until completely dry, add 30.0 μl of a solution of 3% hydrogen peroxide to cells 7 with biomaterial and incubate for 10 minutes at room temperature. Rinse cells 7 of the test system in two containers with a solution of phosphate buffer (pH 7.2-7.4) for 2 minutes in each tank. Remove the protective film 8 from the cells of the test system containing antibodies. In each cell 5 of the test system containing antibodies 6, add 30.0 μl of solvent (with an individual tip) and carefully resuspend. Dissolved antibodies 6 in a volume of 30.0 μl are transferred to the corresponding cells 7 of the test system with biomaterial and incubated for 30 minutes at room temperature. Rinse cells 7 of the test system in two containers with a solution of phosphate buffer (pH 7.2-7.4) for 2 minutes in each tank. Using an individual tip, add 30.0 μl of a solution of secondary antibodies to cells 7 of the test system with biomaterial and incubate for 15 minutes. Rinse cells 7 of the test system in two containers with a solution of phosphate buffer (pH 7.2-7.4) for 2 minutes in each tank. Prepare a working solution of chromogen (DAB) by diluting in a 1: 1 ratio a concentrated solution of DAB and a solvent (attention! The working solution of DAB is stable for 4 hours). Add 30.0 μl of the working chromogen solution to cells 7 with biomaterial and incubate at room temperature for 5 minutes. Rinse cells 7 of the test system in three containers with water for 2 minutes in the first and second containers and for 10 minutes in the third container. In cells 7 with biomaterial, add 30.0 μl of Mayer hematoxylin solution and incubate for 1 minute, then rinse in a container with water for 2 minutes. Dry the test system in air, and microscope in a light field at a magnification of x100-200.

Examples of specific use of the proposed biochip for multiplex analysis.

1. Test system "UROp" (determination of the degree of damage to the prostate gland. Figure 3, 4.

 The material obtained with TIAB of the prostate gland was delivered to the cytology laboratory (a biopsy could not be performed). Patient A, aged 57, with a diagnosis of prostatic hyperplasia, suspected cancer. With an additional examination, the PSA level was 12.7 ng / L. In a cytological study, cubic epithelial cells predominantly predominantly appeared in the form of bare nuclei, in some of which there were signs of atypia. The cytological picture of epithelial hyperplasia with areas of intraepithelial neoplasia 2-3 degrees (figure 3). To clarify the degree of damage, a study was performed using a test system in the biochip format (figure 4). As a result of the study, the Ki67 expression level was 48%, PSA +, P53 +. This immunophenotype of cells indicates a high degree of intraepithelial damage. A morphological study of the surgical material confirmed the diagnosis.

2. Test system "UROb" (determination of the degree of damage to the bladder, monitoring during the treatment of malignant tumors of the bladder). 5, 6.

A bladder rinse obtained by cystoscopy in a patient with a diagnosis of "Macrohematuria was delivered to the clinical diagnostic laboratory." Bladder tamponade. Susp. Bladder Cr.? " During cytological examination of the material, small accumulations of urothelial cells against the background of blood elements were observed, and some of the urothelial cells showed signs of unsharp polymorphism (Fig. 5). To clarify the degree of cellular changes, the material was examined using a test system in the biochip format (Fig. 6). The study revealed positive expression of the Ki67 protein in 12% of cells, the reaction with antibodies to proteins P16 and P53 is negative. This result testified in favor of reactive changes. A morphological study of the surgical material confirmed the diagnosis.

3. Test system "GYN" (clarification of the degree of intraepithelial lesion of squamous epithelium). 7, 8.

During a preventive medical examination, patient A was diagnosed with a cytological diagnosis of cervical dysplasia of degree 1-2, and signs of viral damage were revealed. (Fig.7). In the study of viral load and genotype, the human papilloma virus type 31 was detected in the amount of 3.8 lg. To clarify the degree of intraepithelial lesion, the remaining amount of material was investigated using a test system in biochip format. No cells with coexpression of Ki67 proteins (in the nucleus) and P16 (in the cytoplasm) were detected in the preparations; therefore, the diagnosis was not confirmed by cytological examination; reactive changes were noted in the preparation (Fig. 8) against the background of viral load. The morphological study of biopsy material confirmed the diagnosis.

4. Test system "TYR" (typing of tumor lesions of the thyroid gland). Fig.9, 10.

Material from TIAB of the thyroid gland of patient A was delivered to the clinical diagnostic laboratory with a diagnosis of nodular formation of the left lobe of the thyroid gland. Euteriosis. According to ultrasound of the thyroid gland in the left lobe, a nodular formation with a size of 12x7 mm of a solid structure with peripheral blood flow loci was noted (Figure 9). During cytological examination of the material in the preparations, an abundant cellular composition was noted, represented by pseudopapillary accumulations of follicular epithelium, intranuclear cytoplasmic inclusions were noted in some cells, nuclear chromatin had grooves and folding, and single multinuclear cells were noted. The cytological picture of a follicular tumor is probably papillary cancer. To clarify the diagnosis, the remainder of the material was analyzed using a test system in the biochip format (Figure 10). As a result of the study, the following results were obtained: positive staining of the cells was noted in the cell of the test system containing antibodies to SK19, thyroglobulin, HMBE1, TTF1, Galectin 3. The obtained immunophenotype indicated papillary thyroid cancer. A morphological study of the surgical material confirmed the diagnosis.

5. Test system "NOD2" (primary typing of lymphomas). 11, 12.

The material obtained by scraping from the conjunctiva of the lower eyelid of the left eye was delivered to the clinical diagnostic laboratory. Clinical diagnosis: Susp. Lymphoma (Fig. 11). During a cytological examination, a large number of predominantly mature lymphocytes was noted in the preparations. For the purpose of primary typing of lymphocytes, part of the material was studied using a test system in the biochip format. As a result of the study, diffuse staining of cells in cells with an antibody to the CD20 protein was noted; in other cells, staining of cells was absent (Fig. 12). Thus, the identification of the marker CD20 indicates the belonging of cells to b-lymphocytes. For detailed cell typing, further investigation of the immunophenotype is necessary. A morphological study of the surgical material confirmed the diagnosis.

6. Test system "NOD1" (determination of the nature of the lesion of the lymph node). Fig.13, 14.

A puncture puncture of the lymph node of the neck on the left was delivered to the clinical diagnostic laboratory. According to ultrasound in the left cervical region there was a single lymph node size of 7x9 mm with loci of nodular blood flow (Fig.13). During cytological examination, a small number of round-oval-shaped cells with a peripherally spread nucleus without signs of sharp polymorphism were noted in the preparation; there were no elements of the lymph node. Cellular changes must be differentiated between cystic changes and non-epithelial process. To clarify the process, a study was carried out using a test system in the biochip format. As a result of the study, all reactions with the antibodies used were negative (Fig. 14). Thus, cellular changes were most consistent with a cyst. A morphological study of the surgical material confirmed the diagnosis.

7. Test system "SER2" (determination of the organisms of tumor cells in fluids from serous cavities). Fig. 15, 16.

Pleural fluid from patient N was delivered to the clinical diagnostic laboratory with a clinical diagnosis of right-sided pleurisy. Condition after PCT for ovarian cancer T3NxMx and breast cancer in 2012, art. T3N0M0 (Fig. 15). A cytological examination of the delivered material revealed a metastatic nature of the lesion. To establish the organization of the tumor cells, a study was carried out using a test system in the biochip format. As a result of the study, a positive reaction of cells with antibodies to CA125, EpCAM was observed, which indicates that the cells belong to the ovarian tissue (Figure 16). A morphological study of the surgical material confirmed the diagnosis.

8. Test system "SER1" (determination of the nature of fluids from serous cavities). Fig.17, 18.

Patient M, age 38 years. Received for elective laparocentesis. Clinical diagnosis: hepatitis C virus infection, cirrhosis. Ascites (Fig. 17). According to the puncture results, 1.7 liters of ascitic fluid were removed, the material was sent to a clinical diagnostic laboratory for cytological examination. As a result of a cytological study, the preparations showed an abundant cellular composition, represented by separately lying cells, in which signs of polymorphism and degenerative changes were noted. Given the clinical diagnosis, a differential diagnosis should be made between reactive changes and the malignant nature of cellular changes, which required additional research methods. When studying the cellular composition using a test system in the biochip format, a positive immunocytochemical reaction with antibodies to the HMBE1 and Desmin proteins was observed, which indicated the reactive nature of cellular changes in ascitic fluid (Fig. 18). The use of the biochip test system made it possible to clarify the nature of ascitic fluid. A morphological study of the surgical material confirmed the diagnosis.

9. Test system "SER1" (determination of the nature of fluids from serous cavities). Fig.19, 20.

Patient R., aged 43, with complaints of an increase in the abdomen in volume, general weakness, was admitted to the surgical department. According to ultrasound, free fluid in the abdominal cavity, the formation of the left ovary (Fig.19). After laparocentesis, 3.0 L of ascitic fluid was removed. The material was delivered to the clinical diagnostic laboratory. During a cytological study, cell clusters of presumably epithelial origin were found in the preparations. To clarify, a study was conducted using a test system in the biochip format. As a result of the study, a positive immunocytochemical reaction with antibodies to CK7, EpCAM was demonstrated (Fig. 20). The resulting immunophenotype of the cells indicated their epithelial origin. A morphological study of the surgical material confirmed the diagnosis.

Thus, the biochip option proposed in comparison with the prototype allows us to simplify and shorten the analysis time by simultaneously evaluating the antigen expression and cell morphology. There is an opportunity to prepare long-term cytological preparations (according to the literature, cytopreparations of this kind can be stored for more than 20 years).

Claims (1)

Biochip for evaluating tumor markers, consisting of a transparent substrate coated with polylysine and divided into cells by means of a lattice, covered with a protective film, characterized in that the cells for applying and fixing the biomaterial are located on the part of the substrate coated with polylysine and separated by a platform from the cells for storage and dilution antibodies of the test system located on a part of the substrate without polylysine and equipped with antibodies specific to certain cell antigens contained in the buffer solution.

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