RU142470U1 - BIOCHIP FOR MULTIPLEX ANALYSIS - Google Patents

Abstract

Biochip for multiplex analysis, containing a transparent substrate made of glass with polylysine deposited on its surface, on which test sections with immobilized antibodies specific for cell antigens are placed, characterized in that the test sections are divided by plastic walls into 9 sealed cells, equipped with 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 plastic walls, creating Tight cells, bounded below by a polylysine coated substrate, on top by a polysiloxane film, and on the other sides by plastic walls.

Description

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

Known biochip (RU No. 2436843, IPC C12Q 1/00, date of publication. 12/27/2008). The biochip consists of a solid carrier, work areas with clusters containing probes and an identifier. A solid carrier, in turn, consists of two parts interconnected by means of a flexible multilayer sheet fixed to their surface with an adhesive layer. Clusters with probes are located symmetrically with respect to the longitudinal or transverse axis of the carrier on a multilayer flexible sheet and / or on the surface of a solid carrier. When bending, the multilayer sheet ensures the combination of clusters with probes located on the first and second parts of the carrier and the formation of reaction volumes either through through holes made in a flexible multilayer sheet, the arrangement of which coincides with the location of the clusters, or by means of gaskets fixed at the edges of the multilayer sheet and provided with an adhesive layer.

A disadvantage of the known biochip is the inability to visualize the reaction results in transmitted light, this device is not applicable for the study of whole cells, because contains primers for nucleic acid fragments. A disadvantage of the known device is the need to combine clusters with probes located on the first and second parts of the carrier to create reaction volumes.

As a prototype, a well-known biochip for multiplex analysis was selected, containing a transparent substrate made of glass with polylysine deposited on its surface, on which test sites with immobilized antibodies specific for cell antigens are placed. (A.V. Shishkin, I.I. Shmyrev, S.A. Kuznetsova, N.G. Ovchinina, A.A. Butylin, F.I. Ataullakhanov, A.I. Vorobyov “Immunological biochips for the study of human red blood cells” Biological Membranes, 2008, Volume 25, 4, pp. 267-276).

The known biochip contains the following antibodies: CD2, CD3, CD4, CD5, CD7, CD8, CD9, CD10, CD11a, CD11b, CD16, CD19, CD20, CD21, CD22, CD23, CD27, CD29, CD31, CD36, CD38, CD41, CD44, CD45, CD45RA, CD56, CD71, CD72, CD95, CD98, HLA-DR, IgM.

Known biochip works as follows.

A biochip containing immobilized antibodies is placed in a flow chamber and incubated with a suspension of cells. Cells having corresponding antigens on their surface bind to antibodies immobilized in strictly defined regions of the substrate. Then, the biochip is washed from cells that do not bind to antibodies with a fluid flow at a given speed. The fluid flow rate can be selected such that only those cells that do not bind to antibodies are detached, and cells that even bind weakly to antibodies remain on the surface of the biochip. The presence of bound cells is established by viewing the biochip spots with a microscope.

The disadvantages of the known device are the inability to study the cells contained in serous effusions, due to the presence of a large number of non-cellular elements, namely the presence of fibroblast cells, connective tissue elements, which does not allow research in the flow chamber, increases the mass of the test sample, which entails insufficient binding strength cells with antibodies. Serous effusions today are a common symptom of inflammatory diseases such as acute pancreatitis, cirrhosis, hepatitis and others, as well as oncology (ovarian cancer, liver cancer, pancreatic cancer, stomach cancer, colon cancer and colon cancer) . For example, in ovarian cancer, ascites occurs in 60% of cases. Establishing the causes of ascites carries a lot of time and financial costs. The main way to determine ascites is cytology, but the error rate according to a number of authors is 25-50%. In addition, using a conventional cytological study, it is impossible to determine the primary lesion in ascites of oncological origin. The antibodies used are not used to study serous fluids due to the absence of the corresponding antigens on the cell membranes. Different localization of the antigen in the cell may interfere with the mobilization of cells on the biochip and make semi-quantitative assessment impossible. The disadvantage of this device is the use of a given flow rate of fluid containing cells necessary to prevent separation of cells.

The objective of the proposed model is to create a biochip for diagnostic tests of serous fluids (effusions).

The problem is solved in that in the well-known biochip for multiplex analysis containing a transparent substrate made of glass with polylysine deposited on its surface, on which test sections with immobilized antibodies specific for cell antigens are placed, the test sections are separated by 0.2-thick plastic walls cm, dividing the biochip into 9 sealed cells and equipped with antibodies to estrogen, progesterone, CA-125, CEA, SK-7, CK-20, Ber-EP4, TTF1 and WT1 receptors, closed on top with a hydrophobic polysiloxane film Fixed to the upper edge of the plastic walls, creating a sealed cell, bounded below with polylysine-coated substrate, the top film of the polysiloxane with the other sides of the plastic walls.

The utility model is illustrated by images (Fig. 1, Fig. 2, Fig. 3).

In FIG. 1 shows a schematic structure of a biochip; in FIG. 2 - the appearance of the biochip for multiplex analysis; in FIG. 3 is an image of antibodies through an eyepiece of a fluorescence microscope.

The proposed device (Fig. 1) contains a transparent glass substrate with a polylysine coating 1 and is divided into 9 equal parts (cells) by means of a plastic lattice 2. Each cell contains fluorescent antibodies (0.01 μl). For studies in the work area, test sites were used with antibodies to estrogen receptors (1 cell) 3, progesterone receptors (1 cell) 4, CA-125 receptors (1 cell) 5, CEA receptors (1 cell) 6, SK 7 receptors (1 cell) 7, SK 20 receptors (1 cell) 8, Ber-EP4 receptors (1 cell) 9, TTF1 10 receptors and WT1 11 receptors. To keep antibodies in a humid environment, 0.1 μl PBS reagent was added to each cell, and a hydrophobic film 10 is deposited on top of the working surface, preventing the transit of antibodies from one cell to another and their high NIJ. The biochip is stored in a dark place to prevent tag fading.

The present utility model is a plastic substrate measuring 25.4 mm × 76.2 mm, 1 mm thick. The surface is divided into two parts: one has a polylysine coating and is considered a working surface, the other does not have a specific coating and is designed to fix the biochip in hands.

The proposed utility model meets the criterion of “novelty,” since the patent information studies did not reveal sources of information that would give rise to the novelty of the proposed utility model.

The proposed device meets the criterion of "industrial applicability", as it can be used in oncology for the diagnosis of breast cancer.

The proposed biochip allows to obtain the following technical result.

The proposed utility model makes it possible to study a serous fluid, a fluid with a cellular composition less rich than blood, containing immobilized fluorescent antibodies specific for cell antigens, and it is possible to determine the character of ascites (reactive or oncological), as well as the primary lesion that has ascites producing tumor.

Polylysine coating has high adhesive characteristics and distinguishes the proposed biochip from all compared models. The structure of the working surface, namely the demarcation of the isolated sections prevents the transit of antibodies from one cell to another and is equipped with plastic walls that delimit the cells, which eliminates the need for background areas and their blocking. The proposed biochip is stored in a sealed opaque container that prevents the passage of light and thereby fading of the fluorescent label. The proposed biochip does not require a flow chamber to conduct research on it. The proposed biochip is able to conduct research even in the presence of a large number of non-cellular elements. The coating of the proposed biochip is a polysiloxane film that prevents the drying of antibodies and the transit of antibodies from one cell to another. The proposed biochip has the ability to conduct studies of the reaction volume in each cell in transmitted light.

The biochip is incubated with the test liquid, after which the result is read and analyzed using a fluorescence microscope. During the study, staining of bound cells can be carried out. Using a biochip can significantly increase the information content of the study and reduce its cost.

The proposed biochip works as follows.

Work with the proposed biochip begins with the preparation of material 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 cells are placed in a test tube and centrifuged for 5 minutes at a speed of 2000 rpm. Then the supernatant is removed, and the sediment is micropipetted into the biochip cells in an amount of 0.2 ml.

Before depositing the deposited cells into the biochip cells, the device is prepared for operation as follows: a protective hydrophobic film is removed from the working surface of the biochip, and then the test liquid is immediately installed in the cells. Then, the biochip is placed on a heating table at a temperature of 37 degrees Celsius, and closed with an opaque cover, to prevent light from entering, for 30 minutes. After the heating stage, the proposed biochip is placed on a glass slide of a fluorescence microscope and 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 conducting a study of each of the areas under a fluorescence microscope, the cells are fixed. After that, Mayer hematoxylin is stained and morphological examination of the cells is carried out.

An example of a specific use of the proposed biochip for multiplex analysis is given in the form

Patient S. 55 years. Diagnosis of ovarian cancer stage 3c. Ascites. A puncture of the posterior vaginal fornix was performed with a sterile syringe. Received 20 ml of light ascitic fluid. The liquid is placed in a sterile tube. The fluid was centrifuged at a speed of 2000 rpm for 2 minutes. Then, the resulting precipitate was micropipetted into the biochip cells of 0.1 ml in each cell. Previously, an insulating film was removed from the working surface of the biochip. After adding the precipitate 4 into the cells, the biochip was closed with an opaque cover and placed on a heating table moistened with sterile physical solution, and incubated at a working surface temperature of the table of 37 degrees Celsius for 40 minutes. Then, each cell was examined under a fluorescence microscope. The study found: five luminous cell nuclei in a cell with antibodies to CA-125, 3 luminous cell nuclei in a cell with antibodies to estrogen receptors, a conglomerate of luminous cells in a cell with antibodies to progesterone receptors, 4 luminous cell nuclei in a cell with antibodies to SK7, more than 10 luminous elements in the cell with antibodies to Ber-EP4. In cells with antibodies to WT1 CK20 TTF1 CEA. After a qualitative and quantitative assessment of the cells under a fluorescence microscope, the biochip was stained according to May-Grunwald Giemsa and a morphological assessment of the picture in the cells was performed under a light microscope. In the cell with antibodies to CA-125, 8 adenocarcinoma cells were detected, in the cell with antibodies to progesterone receptors more than 10 adenocarcinomas, in the cell with antibodies to progesterone receptors 5 cells of adenocarcinoma, in the cell with antibodies to Ber-EP4 more than 10 adenocarcinomas, in the cell with antibodies to CK7-5 adenocarcinoma cells in the remaining cells from 3 to 10 adenocarcinoma cells. Thus, a serous adenocarcinoma of the ovary was diagnosed. A morphological study of the surgical material confirmed the diagnosis.

Claims (1)

Biochip for multiplex analysis, containing a transparent substrate made of glass with polylysine deposited on its surface, on which test sections with immobilized antibodies specific for cell antigens are placed, characterized in that the test sections are divided by plastic walls into 9 sealed cells, equipped with 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 plastic walls, creating Tight cells, bounded below by a polylysine coated substrate, on top by a polysiloxane film, and on the other sides by plastic walls.

Images