WO2017204674A1

WO2017204674A1 - A biochip for multiplex analysis and a method of studying cells in the diagnosis of oncological diseases

Info

Publication number: WO2017204674A1
Application number: PCT/RU2016/000317
Authority: WO
Inventors: Святослав Владимирович ЗИНОВЬЕВ; Виктор Владимирович РАЧКОВ; Олег Владимирович УТКИН; Марина Владимировна САВОСТИКОВА; Елена Юрьевна ФУРМИНСКАЯ
Original assignee: Общество С Ограниченной Ответственностью Научно-Производственное Предприятие "Биочип"
Priority date: 2016-05-26
Filing date: 2016-05-26
Publication date: 2017-11-30
Also published as: EA201800291A1

Abstract

The proposed group of inventions relates to the field of medicine, and more particularly to diagnostics, and may be used for the diagnosis of oncological diseases. A biochip for multiplex analysis comprises a clear glass substrate, onto which a coating has been applied that creates a positive charge on the surface of the glass, and antibodies conjugated with Cy-3 fluorescent markers and an anti-drying reagent are inserted into the test wells. The working surface of the biochip is coated with an airtight sterile film. Cells from serous fluid, cells from hemorrhagic fluid, or cells from urine with a precipitate are pretreated by centrifuging, removing the supernatant, and resuspending the precipitate in a nutrient medium; when studying a suspension of cells obtained by fine needle aspiration biopsy or cells obtained from scrapings from a resected tumour before the tumour is placed in formalin, the biopsy specimen is placed in a 0.8-ml Eppendorf tube with a nutrient medium; the sample is centrifuged for 5 minutes at 1500 rpm, and the precipitate is inserted into the test wells of the biochip, after which the biochip with the inserted cells of heterogeneous biomaterial is placed in a shaker for 2-3 minutes and then in an incubator at 37 °C for 25 minutes, after which the biochip is rinsed in buffer solution, air-dried, and subjected to fluorescence analysis. The proposed group of inventions makes it possible to broaden the possibilities for diagnosis by making it possible to analyse an extensive list of cellular material.

Description

TITLE OF THE INVENTION: BIOCHIP FOR MULTIPLEX ANALYSIS AND METHOD FOR STUDYING CELLS DIAGNOSTIC ONCOLOGICAL DISEASES TECHNICAL FIELD

The proposed group of inventions relates to medicine, namely to diagnosis and can be used for the diagnosis of cancer. In addition, the proposed group of inventions can be used in veterinary medicine, as well as in the field of biology, immunology and cytology.

BACKGROUND OF THE INVENTION

The biochip is one of the latest tools in biology and medicine of the 21st century, using a minimum of material and a minimum of time for research, it allows you to determine a lot of different and reliable information, for example, about various genetic defects, oncogenes, allergens. Biochips make it possible to detect markers in the human body that correspond to specific diseases, certain viruses and cancer cells in almost any analyzed material - blood, saliva, sweat.

The wide diagnostic capabilities of biochips determine the relevance of developing their new species.

Currently produced biochips differ from each other by the manufacturing method, the type of substrate and probe, the method of recording the result of the interaction of the reagent with the test sample. Fluorescence tags, lasers and optical scanners are used to read the reaction results.

Currently, the following types of biochips are known:

- DNA biochips used to identify genes and their mutations associated with various diseases, diagnose infectious diseases, and screen microorganisms;

- protein chips that are used to detect protein markers characteristic of various diseases at different stages of their development;

- cell chips, avoiding the problem of protein instability in protein chips and more accurate analysis of the interaction of proteins within the cell, visualization of the cell itself and its organelles; - tissue chips that allow analysis of thousands of tissue samples on a single glass slide and are used to determine the protein content in healthy and pathologically altered tissues and evaluate potential targets for drugs;

- microchips based on small molecules that allow for simultaneous screening of thousands of potential drugs;

- microfluidic chips - a multifunctional device for exchange, between the components of which electrodes and microfluids are used.

There is a known group of biochips containing a solid carrier, on the working zone of which oligonucleotide zones are immobilized in clusters and identifier: RF patent for utility model Ν ° 69866 (IPC C12Q1 / 68, publication date 01/10/2008), RF patent for the invention> 2270254 (IPC C12Q1 / 68, publication date 02/20/2006), RF patent for the invention JN ° 2436843 (IPC C12Q1 / 00, publication date 12/27/2008).

Another group of biochips is a solid substrate made of glass (RF patent for utility model Ns 141359, IPC G01N33 / 552, publication date 28.1 01.2013) with a coating applied on its surface, for example, from chalcogenide glass (ibid.) Or polylysine (patent RF for utility model N ° 142470, IPC G01N 33/53, publication date 06/27/2014) or a polymer working layer (RF patent for the invention M> 2298797, IPC G01N33 / 531, publication date 05/10/2007). On the coating are placed test sections of the substrate with immobilized antibodies specific for cell surface antigens.

As the closest analogue of the proposed biochip, the well-known biochip for multiplex analysis was selected, containing a transparent glass substrate, divided into functional and working parts, the functional part serves to fix the biochip in hands, the working part is coated and divided into test cells by means of a plastic grid, each the test cell is equipped with immobilized fluorescent antibodies specific for cell antigens, the biochip contains a film fixed on top of the grating (RF patent for utility model N ° 142470, IPC G01N 33/53, publication date 06/27/2014).

The known biochip contains a transparent substrate made of glass with polylysine deposited on its working surface, the biochip is divided into 9 equal cells by means of a plastic lattice. Each cell contains fluorescent antibodies (0.01 μl). For research use test sites with antibodies to estrogen receptors, progesterone receptors, CA-125 receptors, CEA receptors, SK 7 receptors, SK 20 receptors, Veg-EP4 receptors, TTF1 receptors, and WT1 receptors. To preserve antibodies in a humid environment, PBS reagent in an amount of 0.1 μl was added to each well. The biochip is coated on top with a hydrophobic film fixed to the upper edge of the plastic lattice, creating airtight cells, bounded below by a coated substrate, on top with a film, and on the other sides with plastic walls of the lattice, which also prevents the transit of antibodies from one cell to another. The biochip is stored in a dark place to prevent tag fading.

Known biochip works as follows.

The biochip is taken out of an opaque container. Prepare biomaterial for research. A protective hydrophobic film is removed from the working surface of the biochip, after which the test biofluid is immediately installed in the test cells. Then the biochip is placed on a heating table at a temperature of 37 ° C and closed with an opaque cover, to prevent light from entering, for 30 minutes. Next, the biochip is placed on a slide of a fluorescence microscope and fluorescence analysis in each of the cells is carried out. After the study, the cells are fixed. Next, staining is performed for subsequent morphological examination of cells.

The well-known biochip allows you to examine serous fluid, a fluid with a less rich cellular composition than blood, and determine the nature of ascites (reactive or oncological).

However, the known biochip has the following disadvantages:

- the known device provides the ability to study cells contained in serous fluids. However, today it is known that not only serous effusions are a common symptom of diseases of both an inflammatory nature (acute pancreatitis, cirrhosis of the liver, hepatitis and others), and oncological (ovarian cancer, liver cancer, pancreatic cancer, colon cancer and other) diseases. Hemorrhagic fluid cells, urine cells, cells obtained by the fine-needle aspiration puncture biopsy, as well as scrapings from a resected solid tumor before its fixation in formalin possess unique diagnostic information. However, the well-known biochip does not provide the opportunity to study the above cells;

- polylysine coating of the substrate promotes sorption of both antibodies and cells, which prevents the full binding of antibodies to cells, also it is impossible to analyze the reaction in the spot where the antibodies were applied, since the glow around the cells is background, and not specific;

- the FITC fluorescent label used in the known device is not stable enough, which affects the duration of storage of the biochip;

- the plastic lattice attached to the biochip does not allow analysis using a microscope with a magnification of more than 200 due to the impossibility of approaching the lens as close to the bottom of the cell as possible. In addition, a plastic grill is attached to the substrate using polyacrylate glue, which can contribute to premature antibody denaturation due to the toxic effects of the glue;

- antibodies are in cells in a PBS solution, which does not allow long-term storage for two reasons: premature growth of microorganisms and natural sorption of antibodies to the bottom and walls of the cell;

- the protective film is not sterile, which represents an additional risk factor for the growth of microorganisms and damage to the protein component (antibodies);

- the well-known biochip provides the opportunity to study a limited number of markers (9), which is not always enough for the needs of medical research.

A known method for the study of cells in the diagnosis of cancer using a known biochip, including the preliminary preparation of cells obtained from serous fluid, placing the test material in the cells of the biochip, conducting fluorescence analysis and the subsequent study of the presence and detection of fluorescence in each test cell, while at the preliminary stage the preparation of serous fluid is centrifuged for 5 minutes at a speed of 2000 rpm, selected as the closest analogue (RF patent for gender utility model Ng 142470, IPC G01N 33/53, publication date 06/27/2014).

The known method using a known biochip is as follows.

Serous fluid is taken in several routine ways (intraoperatively in a sterile syringe in an amount of at least 2 ml, with a puncture transvaginally, transabdominally in a sterile syringe). Then the material is placed in a test tube and centrifuged for 5 minutes at a speed of 2000 rpm. The supernatant is removed, the precipitate is pipetted into the biochip cells in an amount of 0.2 ml. Examine the number of stained cells and the brightness of fluorescence. After that, the cells are fixed and subsequently stained with Mayer hematoxylin and a morphometric study is performed.

However, the known method has the following disadvantages.

The known method does not provide the ability to study other biological cellular materials, such as hemorrhagic fluid cells, cells in the urine, cells from the material of a fine-needle aspiration biopsy and cells from scrapings of resected solid tumors until the tumor is fixed in formalin, which narrows the diagnostic capabilities of the method. SUMMARY OF THE INVENTION

The objective of the proposed group of inventions is the creation of a biochip for multiplex analysis and a method for researching cells for the diagnosis of cancer using a biochip for multiplex analysis, which are devoid of the disadvantages of the prototype.

The technical result from the use of the proposed biochip is to enable the analysis of hemorrhagic fluid cells, urine cells, cells from the material of a fine needle aspiration biopsy and cells obtained from scrapings of a resected solid tumor, increasing the reliability of the biochip and increasing its shelf life.

The technical result of the proposed method is to increase the accuracy, specificity and sensitivity of the study, as well as to expand the diagnostic capabilities.

The specified technical result is achieved by the fact that in the proposed biochip for multiplex analysis containing a transparent glass substrate, divided into functional and working parts, the functional part serves to fix the biochip in hands, the working part is coated and divided into test cells by means of a plastic grating, each test cell is equipped with immobilized fluorescent antibodies specific for cell antigens, the biochip contains a film fixed on top of the grating on the substrate of the working part on eseno coating, creating a positive charge on the glass surface, the cell introduced into test antibody conjugated to a fluorescent label Cy-3 and the reactant, which prevents desiccation of antibodies made, the working surface of the biochip coated with an airtight sterile film function portion biochip provided with information about the localization of the antibody included. The specified technical result is achieved in that in the proposed method for the study of cells for the diagnosis of cancer using a biochip for multiplex analysis, including preliminary preparation of cells obtained from serous fluid, placing the test material in the cells of the biochip, conducting fluorescence analysis and subsequent investigation of the presence and detection of fluorescence in each cell, while at the stage of preliminary preparation the serous fluid is centrifuged for 5 minutes at a speed and 2000 rpm, the cells of the study are hemorrhagic fluid cells, cells in the urine, cells obtained from the material of a fine-needle aspiration biopsy and cells obtained from scrapings of a resected solid tumor before its fixation in formalin, while preliminary preparation of cells from a turbid serous fluid, hemorrhagic fluid and urine cells with sediment includes centrifugation in a volume of not more than 20 ml, subsequent removal of the supernatant and resuspension of the sediment with a nutrient medium, in the study of cells from the material of a fine-needle aspiration biopsy and cells obtained from scrapings from a resected tumor before it is fixed in formalin, the punctate is placed in a 0.8 ml Eppendorf tube with a nutrient medium, centrifuged for 5 minutes at a speed of 1500 rpm and introduced into biochip test cells, then a biochip with introduced cells is placed in a shaker for 2-3 minutes, then placed in a thermostat for 25 minutes at a temperature of 37 ° C, after which the biochip is rinsed in a buffer solution, dried and a fluorescence analysis is performed.

The proposed group of inventions meets the criterion of "novelty", as the patent information research did not reveal sources of information that would discredit the novelty of the proposed method and device, as well as technical solutions with essential features of the proposed technical solutions, which allows us to conclude that the criterion " inventive step ".

The proposed biochip is a substrate with dimensions of 25.4 x76.2 mm, a thickness of 1 mm. Prior to operation, the biochip is stored in an opaque container. The time spent in the light should not exceed 20 minutes before the start of the reaction. Avoid direct sunlight on the product outside the container. Before starting work, an airtight sterile film is removed from the biochip surface. The proposed biochip provides the ability to analyze hemorrhagic fluid cells, urine cells, cells obtained from the material of a fine-needle aspiration biopsy and cells learned from scrapings from solid tumors. The implementation of the substrate with a coating that creates a positive charge on the glass surface, allows selective sorption of cells due to the negative charge of the membrane of the latter, while preventing the sorption of antibodies due to the positive charge of the latter. In the proposed cells, you can adsorb antibodies to any tumor antigens. The possibility of selective binding of the antibody to the cell, and the cell to the substrate, excluding non-specific deposition of antibodies on the substrate, reduces the time to run the analysis and increase the specificity and reliability of the biochip.

The introduction of antibodies conjugated with a fluorescent label, for example, Su3, into the test cells allows preserving the luminescence of the cells up to 2 months.

Adding a reagent to each test cell containing, for example, PBS, BSA, and 0.06% sodium azide allows prolonging the biochip performance (more than 1 year), since it prevents the drying of antibody droplets due to the formation of a protective membrane at the molecular level, and also prevents propagation microorganisms. Coating the biochip with an airtight sterile film also prevents the growth of microorganisms.

The proposed method allows you to:

- To expand the diagnostic capabilities of the method, by obtaining new, diagnostically significant information about the processes occurring in the body as a whole and in a specific area of research.

- To provide the opportunity to study cells of hemorrhagic fluids, urine cells, punctate cells from a fine-needle aspiration biopsy, cells from scrapings of a resected solid tumor before it is fixed in formalin.

- Placement of cells obtained from the material of a fine-needle aspiration biopsy and cells obtained from scrapings from a resected solid tumor before it is placed in formalin in a nutrient medium, introduction of cells into test cells of the biochip, additional mixing on a shaker and subsequent heating of the biochip with the cells introduced into the cells in the thermostat allows you to increase the interaction of antigens with antibodies, which increases the sensitivity of the method. The increase in sensitivity is also due to the possibility of creating a brighter and a persistent signal and a long effect of luminescence of markers with different localization: nuclear, membrane cytoplasmic in a total of up to 30.

- Conducting fluorescence analysis after rinsing the biochip in Tris-buffer allows you to wash unbound antibodies, which increases the accuracy of diagnosis. The high specificity of the method is due to the selectivity of the connection of antibodies with antigens of cells without a background glow.

As a result of the work, an easy-to-use device was created that allows simultaneous analysis of 30 cell markers at once, with the possibility of assessing the relationship of cell morphology and luminescence, the relationship of antigen and cell, its localization, and qualitative characteristics. The use of the above reagents for analysis provides the possibility of a long retrospective study

DETAILED DESCRIPTION OF THE INVENTION

The proposed group of inventions works as follows.

Prepare the biomaterial in the form of a suspension of cells for research. Serous fluid is taken in several routine ways (intraoperatively in a sterile syringe in an amount of at least 2 ml, with a puncture transvaginally, transabdominally in a sterile syringe). Then the serous fluid is placed in a test tube and centrifuged for 5 minutes at a speed of 2000 rpm The supernatant is removed, the cell suspension in a volume of not more than 0.7-1 ml is pipetted into the biochip cells.

A serous liquid turbid with sediment is taken no more than 20 ml (two centrifuge tubes) and, after centrifugation, the supernatant is carefully removed with a pipette, bringing to a volume of 0.7-1 ml and resuspended, obtaining a concentrated suspension of cells. The resulting cell suspension is centrifuged and the pellet is pipetted into the biochip cells.

The hemorrhagic fluid is taken by puncture, centrifuged in a large number (6-8 each) of 10 ml centrifuge tubes, then drained over the sedimentary liquids, the precipitate is resuspended and combined into one tube.

If the collected cell suspension exceeds a volume of more than 1 ml, it is centrifuged again and the excess liquid is carefully removed with a pipette, bringing the volume to 0.7-1 ml, resuspended to obtain a concentrated suspension of cells in the liquid, and the material is introduced into the biochip cells with measured micropipettes.

A suspension of cells from a fine-needle aspiration biopsy material is placed in an Eppendorf tube with a culture medium of 0.8 ml. Cover the tube lid and mix thoroughly on a shaker, centrifuged for 5 minutes at a speed of 1500 rpm, then the material is introduced into the biochip cells with measured micropipettes.

To study solid tumors, the following is performed. After receiving a resected tumor, before it is fixed with formaldehyde, the tumor formation is dissected and scraped from its surface with a spatula or the edge of the glass. The resulting scraping is placed in a 0.8 ml Eppendorf tube containing culture medium. Then, closing the test tube with a lid, the suspension is thoroughly mixed on a shaker and centrifuged for 5 minutes at a speed of 1500 rpm, after which the material is introduced into the cell of the chip with measured micropipettes.

The introduced biomaterial is gently resuspended by lightly touching the bottom with a pipette for better dissolution of antibodies and their interaction with tumor cells. Then the biochip is covered with an airtight sterile film and placed on a shaker for 2-3 minutes for better dissolution of antibodies and their interaction with tumor cells. Then placed in a thermostat or in a hybridizer for 25 minutes at a temperature of 37 ° C. After the specified time, observing the regime of darkness, with a slight movement the chip is rinsed in a glass with Tris-buffer and allowed to dry. Conduct a study under a fluorescence microscope. The fluorescence analysis in each of the cells is carried out according to the following parameters: the number of fluorescently stained cells and the fluorescence brightness. After a fluorescence study, cell fixation and Leishman staining are performed. Conduct a morphological study of cells. The results of fluorescence analysis and morphometric are compared. If no luminescence is noted, the biochip is stained according to Leishman and a cytological examination is carried out, cellularity and cell morphology are evaluated. After the analysis, the chip is wrapped in foil and placed in a refrigerator at -2-8 ° C.

INDUSTRIAL APPLICABILITY

Examples of specific implementation are given in the form of research protocols.

Protocol N 1

Patient A., 57 years old, was admitted to the hospital with suspected ovarian cancer, ascites. In a clinic, the patient underwent a puncture of the posterior vaginal fornix with a sterile syringe. Received 20 ml of a light straw-colored liquid. The test liquid was placed in a 20 ml tube and centrifuged for 5 min at 2000 rpm. Then, serous fluid without sediment and any inclusions was centrifuged in a large number (4-5 each) of centrifuge tubes with a capacity of 4 ml, then the supernatant was drained, the sediment was resuspended and combined into one tube. The resulting cell suspension was thoroughly mixed on a shaker.

For the study, a biochip was used to study serous fluids. The following types of antibodies were used in the study: cell 1 marker CA-125, cell 2 - CEA, cell 3 - CK7, cell 4 - CK20, cell 5 - CE, cells 6 - TTF, cell 7 -

WT1, cell 8 - Ersash4, cell 9 - vimentin, cell 10 - Cdx2.

An airtight sterile film was removed from the surface and biomaterial was added to suspend the cells into each cell with a measuring pipette. Then the biochip was again covered with a film and placed on a shaker, incubated in a thermostat in complete darkness for 30 minutes at a temperature of 37 ° C. Next, the biochip was washed in a buffer solution.

Excess moisture was removed with filter paper. Then the lattice was removed and a study was performed under a fluorescence microscope. Luminescence was revealed in the complexes of cells located in the cells: 1 - pronounced luminescence, 8 - pronounced luminescence, 3 - moderately pronounced luminescence. No glow was detected in the remaining cells.

The result of the study: on the biochip - the immunophenotype corresponds to ovarian cancer.

Researcher's conclusion: ascites fluid contains ovarian adenocarcinoma complexes.

After evaluating the fluorescence, the biochip was stained according to Leishman, a morphological assessment of the cells in each cell was performed.

Leishman stain protocol: The smear is air dried. It is fixed in the Leishman fixative-dye for 3 minutes, washed with water. 40 ml of 0.1% dye azure II, 30 ml of 0.1% water-soluble eosin, 70 ml of distilled water were stained. A mixture of dyes is prepared before work (extempore).

Morphological conclusion: in all cells, adenocarcinoma cell complexes.

The patient was hospitalized, the operation of hysterectomy with appendages was performed. Omentectomy. Peritoneum biopsy. The result of a nystological study: serous adenocarcinoma of the ovary. Diagnosis - Stage Ovarian Cancer

Protocol Nb 2

Patient I., 38 years old. A neoplasm of the breast was detected. The puncture is completed. Cytological examination: adenogenic cancer. Surgery was performed in the scope of sectoral resection with axillary lymphadenectomy. The material was selected for research on the biochip EPN (breast cancer). AT cell numbers 1, 6, 11 contain antibodies to estrogen receptors, in cell numbers 2,7,12 antibodies to progesterone receptors, in cell numbers 3,8,13 Her2-neu antibodies are added, in cell numbers 4,9,14 antibodies are added Ki67, EGFR antibodies are introduced in cell numbers 5,10,15. To preserve antibodies in a humid environment, PBS reagent, sodium azide 0.06%, BSA was added.

After receiving a resected tumor before it was fixed with formaldehyde, the tumor node was dissected and scraped with a spatula from the surface of the formation. The resulting scraping was placed in a 1.5 ml Eppendorf tube with culture medium. Then, closing the tube with a cap, the suspension was centrifuged for 5 minutes at 1500 rpm. A protective film was removed from the surface of the biochip and the cell suspension material was introduced into each cell with a measuring pipette.

2 lymph nodes were also selectively taken and dissected, scrapings were made from the surface of each lymph node. Each scraping was placed in its 1.5 ml tube with culture medium. Then, closing the tube with a cap, the suspension was centrifuged at a speed of 1500 rpm for 5 minutes. A protective film was removed from the surface of the biochip and the cell suspension material was introduced into each cell with a measuring pipette.

Tumor material in the cells: 1, 2, 3, 4, 5.

The material of the lymph node N ° 1 - 6,7,8,9,10

Lymph node material No. 2 - 11,12,13,14,15

Then the biochip is covered with a removed film and placed on a shaker for 5 minutes. Then it was placed in a thermostat for 30 minutes at a temperature of 37 ° С. The washing was carried out in a buffer. Excess moisture was removed with filter paper. Then the grating was removed and a study was conducted under a fluorescence microscope.

Conclusion on the biochip:

The tumor has the following phenotype:

Estrogen receptors: negative.

Progesterone receptors: negative.

Her2-neu receptors: 3+ (expressed)

Ki67 -more than 30% (2 points)

EGFR receptors: 1+ (mild)

The lymph node N ° l has the following phenotype:

Estrogen receptors: negative.

Progesterone receptors: negative. Her2-neu receptors: 3+ (expressed)

Ki67 -more than 30% (2 points)

EGFR receptors: 1+ (mild)

The lymph node N ° 2 has the following phenotype:

Estrogen receptors: negative.

Progesterone receptors: negative.

Her2-neu receptors: 3+ (expressed)

67 -more than 30% (2 points)

EGFR receptors: 0 (negative)

Researcher's conclusion: Her2-neu breast cancer: 3+, estrogen receptors 0 points, progesterone receptors 0 points, 67-over 30% of cells, EGFR-1 receptors score.

Leishman stain protocol.

Morphological conclusion: in all cells, the complexes of the cells of the adenocarcinoma of the breast

The result of a planned histological examination: invasive carcinoma of the mammary gland of a scirotic structure. In the lymph nodes metastases of adenocarcinoma.

The result of immunohistochemistry: Her2-neu breast cancer: 3+, estrogen receptors 0 points, progesterone receptors 0 points, Cl67-more than 30% of cells, EGFR-1 receptors score.

The patient is diagnosed with stage III breast cancer.

Protocol No. 3

Patient D. 69 years. Urine taken for cytological analysis. Cytology revealed atypical cell complexes. The patient passed the urine test again. 50 ml straw-yellow urine delivered to the laboratory. The test liquid was placed in 20 ml tubes and centrifuged for 5 minutes at a speed of 2000 rpm. Then, the urine was centrifuged in a large number (4-5 each) of 4 ml centrifuge tubes, after which the supernatants were drained, the sediment was resuspended and combined into one tube.

For research, the biochip NPV (urogenital tract) was used. Antibodies conjugated with Cy3 to p16 receptors - cell 1, to Ki67 receptors - cell 2, to p 63 receptors - cell 3, to cytokeratin 5/6 receptors - cell 4, cytokeratin 7 - cell 5, cytokeratin 20 - are placed in test cells of the biochip cell 6, C A 125 - cell 7, WT1 - cell 8, uroplakine - cell 9, CD 95 cell 10, Cdx2 - cell 11. To preserve antibodies in a humid environment, PBS reagent, sodium azide 0.06%, BSA were added.

An airtight sterile film was removed from the surface of the biochip, and the test material — suspensions of urine cells — was introduced into each cell with a measuring pipette. Then the biochip was covered with a removed film. Treated for 5 minutes on a shaker. Incubated in a thermostat in complete darkness for 30 minutes at a temperature of 37 ° C. Next, washing was carried out in a buffer. Excess moisture was removed with filter paper. Then the grating was removed and a study was performed under a fluorescence microscope. When assessed under a fluorescence microscope, luminescence was revealed in the complexes of cells located in the cells: 1 - pronounced, 8 - moderate, 9 - pronounced. Cells 10 and 4 are slightly expressed. The remaining cells are negative.

Conclusion obtained on the biochip: the immunophenotype corresponds to bladder cancer of a high degree of malignancy.

Researcher's conclusion: in the urine there are cells of squamous cell cancer of the bladder with a high degree of malignancy

After evaluating the fluorescence, the biochip is stained according to Leishman and a morphological assessment of the cells in each cell is performed.

Morphological conclusion: atypical cell complexes in all cells.

The patient is hospitalized in a hospital. Performed a cystoscopy with a biopsy. The result of histological examination: squamous cell carcinoma. The diagnosis of stage I bladder cancer.

JNb Protocol 4

Patient S., 45 years old. Nodular neoplasm of the thyroid gland The patient underwent a puncture of the neoplasm in a clinic with a sterile syringe. 5 ml of hemorrhagic fluid was obtained. The fluid is aimed at research. The study was conducted on the biochip TYR (thyroid gland). The biochip test cells contained the following antibodies labeled with Cy3: thyroglobulin - cell 1, calcitonin - cell 2, TTFI - cell 3, galectin - cell 4, mesothelial antigen HBMEI - cell 5, cytokeratin 19 - cell 6, CD44v6 - cell 7, p53 - cell 8. To preserve antibodies in a humid environment, PBS reagent, sodium azide 0.06%, BSA was added.

The test liquid was poured into a 10 ml test tube containing a nutrient medium. Centrifuged for 5 minutes at a speed of 2000 rpm. in a test tube with a volume of 10 ml. Then the hemorrhagic fluid without sediment and any inclusions was centrifuged in a large number (4-5) centrifugal tubes with a capacity of 4 ml, after centrifugation, the supernatants were drained, the pellets resuspended and combined into one tube. The resulting cell suspension was thoroughly mixed on a shaker. Then, an airtight sterile film was removed from the biochip surface and the cell suspension material was introduced into each cell with a measuring pipette. Then the biochip was covered with a removed film. Incubated in a thermostat in complete darkness for 30 minutes at a temperature of 37 ° C. Next, placed on a shaker. Next, washing was carried out in buffer. Excess moisture was removed with filter paper. Then the grating was removed and a study was performed under a fluorescence microscope.

Luminescence was revealed in the complexes of cells located in the cells: 1 - pronounced, 2 - pronounced, 7 - moderately expressed, 8 - pronounced. The remaining cells are negative.

Conclusion on the results of the analysis on a biochip: the immunophenotype corresponds to thyroid cancer.

Researcher's conclusion: punctate contains thyroid cancer cells.

After evaluating the fluorescence, the biochip was stained according to Leishman and a morphological evaluation of the cells in each cell was performed.

Morphological conclusion: carcinoma cell complexes in all cells.

The patient is hospitalized in a hospital. Hemistrumectomy operation performed. The result of histological examination: follicular carcinoma of the thyroid gland. The patient is diagnosed with thyroid cancer.

Claims

Claim

1. A biochip for multiplex analysis, containing a transparent glass substrate, divided into functional and working parts, the functional part serves to fix the biochip in the hands, the working part is coated and divided into test cells using a plastic grid, each test cell is equipped with immobilized fluorescent antibodies specific to cell antigens, the biochip contains a film fixed on top of the grating, characterized in that a coating is created on the substrate of the working part, which creates a positive charge on the glass surface, antibodies conjugated with Su-3 fluorescent labels and a reagent that prevents drying of the introduced antibodies are introduced into the test cells, the working part of the biochip is covered with an airtight sterile film, the functional part of the biochip is provided with information about the localization of the introduced antibodies.

2. A method for the study of cells for the diagnosis of cancer using a biochip for multiplex analysis, including the preliminary preparation of cells obtained from serous fluid, placing the test material in the cells of the biochip, conducting a fluorescence analysis and subsequent investigation of the presence and detection of fluorescence in each cell, the preliminary preparation stage, the serous fluid is centrifuged for 5 minutes at a speed of 2000 rpm, characterized in that the biological objects of the study are I have hemorrhagic fluid cells, urine cells, cells obtained from fine-needle aspiration biopsy material and cells obtained from scrapings from a resected solid tumor before it is fixed in formalin, while preliminary preparation of cells from turbid serous fluid, cells from hemorrhagic fluid and cells in urine with sediment includes centrifugation in a volume of not more than 20 ml, subsequent removal over sedimentary fluid and resuspension of sediment with a nutrient medium, in the study of suspension of cells obtained from mother Ala fine-needle aspiration biopsy and cells obtained from scraping the resected tumor before it is fixed in formalin, the punctate is placed in a 0.8 ml Eppendorf tube with nutrient medium, centrifuged for 5 minutes at a speed of 1500 rpm and sediment is introduced into test cells of the biochip, then the biochip with the introduced cells of different biomaterial is placed in a shaker for 2-3 minutes, then placed in a thermostat for 25 minutes at a temperature of 37 ° C, after which the biochip is rinsed in a buffer solution, dried and fluorescent centric analysis.