CN110079450B

CN110079450B - Solid phase carrier chip system for in situ hybridization and application thereof

Info

Publication number: CN110079450B
Application number: CN201910404218.6A
Authority: CN
Inventors: 何瑰; 陈绍宇
Original assignee: Lbp Medicine Science & Technology Co ltd
Current assignee: Lbp Medicine Science & Technology Co ltd
Priority date: 2019-05-10
Filing date: 2019-05-10
Publication date: 2020-05-05
Anticipated expiration: 2039-05-10
Also published as: CN110079450A

Abstract

The invention belongs to the field of molecular detection consumables, and relates to a solid phase carrier chip system for in situ hybridization and application thereof. The invention provides a solid phase carrier chip system for in situ hybridization, which consists of a probe chip, a hybridization buffer solution and a sample glass slide; the solid phase carrier chip system has low preparation cost, is suitable for most of cast-off cells except peripheral blood lymphocytes, such as urine, cervical cast-off, amniotic fluid and the like, is made into two structural components according to the characteristics of a sample, increases the physical separation between the hole rings of the slide, has better anti-pollution effect, and realizes the simultaneous detection of multiple groups of probes of a single slide.

Description

Solid phase carrier chip system for in situ hybridization and application thereof

Technical Field

The invention belongs to the field of molecular detection consumables, and relates to a solid phase carrier chip system for in situ hybridization and application thereof.

Background

The in situ hybridization technique is mainly applied to the abnormal detection of gene segments, including amplification, deletion, rearrangement, fusion and the like. In situ hybridization is based on the detection of abnormalities in a sample, a sample carrier (usually a glass slide), in combination with various probes. Typically one sample for each slide, and one probe for each slide. The technology and product morphology gradually show disadvantages with the rapid development of genomics: the method has the advantages that firstly, a patient sample, a sample glass slide and an applied detection probe which are detected in the traditional in-situ hybridization technology are separated, so that the method is not a complete product system, and the problem of poor convenience exists in clinical application; secondly, with the promotion of accurate diagnosis and treatment, the meaning of the gene abnormality information contained in one sample to diagnosis and treatment is more and more definite, more and more gene targets need to be detected clinically, and the detection probes designed respectively are difficult to meet the requirement of simultaneous detection of multiple genes; and thirdly, standardization requirements. In situ hybridization is mainly performed manually at present, and as targets increase, the load faced by a pathology technician increases after the sample size increases, so that the standardization problem exists in the aspect of ensuring the simultaneous and synchronous operation of the same sample.

The multi-probe chip can obviously improve the detection accuracy and the detection efficiency, and the recent research has proposed the multi-probe chip by the technical scheme, but different technical problems and thresholds exist.

The CYTOCELL corporation, USD413390, USD420452, USD420745, USD420746 and USD436668, disclose a method for preparing a hybrid slide of a solid carrier, which comprises fixing probes on a raised platform to achieve the purpose of physical separation, but the assembly manufacturing process is complicated, requiring glass cutting to manufacture a raised hillock/platform, and then grinding, cleaning, polishing and other processes, which is costly, and the corresponding mold must be customized, and chip products for different applications cannot be prepared according to the detection requirements. The method has higher requirements on cleaning and polishing after grinding, and limits the application prospect of the product.

The Kanglu company provides a method for integrating multiple probes to realize FISH detection on one glass slide, and the preparation process is discussed in detail in CN201711095572.2 and CN201721483366.4, wherein the multiple probe hybridization glass slide consists of a probe attachment glass slide, each probe, a hybridization buffer solution and a sample attachment glass slide; the patent realizes the detection of the bone marrow lymphocytes by presetting a probe (fluorescent group labeling, reading fluorescence after being excited by a microscope); the setting of the use scene is single; the fully hydrophobic coating design can play a role in separation at certain intervals, but when the added liquid exceeds the preset 2 mu L, overflow can happen, and mutual pollution risks exist between the probe and the sample hole.

Therefore, the present invention is provided based on the technical deficiencies that the conventional multi-probe chip with fixed carrier still has high requirements on production process, expensive preparation and mutual pollution in detection.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a solid phase carrier chip system for in situ hybridization, which comprises a probe chip (prearranged probes), a hybridization buffer solution and a sample glass slide; the invention is designed to expand the application scene, and the detection sample is expanded, except peripheral blood lymphocytes, the invention is suitable for most of cast-off cells, such as urine, cervical cast-off, amniotic fluid and the like, and two structures are made according to the characteristics of the sample; by improving the design of the glass slide, the physical separation between the glass slide hole rings is increased, and the anti-pollution effect is better.

The invention realizes the technical effects through the following technical scheme:

a solid phase carrier chip system for in situ hybridization comprises one or more groups of double spacers and probe attachment regions, wherein the probe attachment regions are circular hole rings, the double spacers are concave-convex structures formed by combining straight lines and loop lines, and the thickness of the concave-convex structures is 20-50 mu m.

Wherein the dual spacer region and the probe attachment region in the solid support chip system are six, eight, fifteen or thirty-two groups.

The preparation method of the solid phase carrier chip comprises the following steps: and printing the combination of the straight line and the loop line by using a screen printing technology to form a concave-convex structure, wherein the printing thickness is 20-50 mu m, using the prepared double-stranded probe to perform pre-spotting on the sample in the hole loop, and drying the sample in a drying oven at the temperature of 45 ℃ to form a probe attachment area.

The solid-phase carrier chip system is used for cell samples with simple shapes or less cytoplasm, such as bone marrow samples. The concave-convex structure formed by the solid phase carrier chip prepared by the invention can play a role in physical barrier in the subsequent hybridization process, and prevent the pollution between samples or hybridization liquid.

A method for performing probe hybridization using the solid phase carrier chip system comprises the following steps: directly dripping the bone marrow sample subjected to hypotonic treatment into a probe/sample attachment area, and dripping a hybridization buffer solution after simple salt treatment for denaturation and hybridization; and hybridizing for 1 hour or 16 hours or 1-16 hours, and observing a fluorescence signal under a mirror.

The solid phase carrier chip system also comprises a sample glass slide, wherein the front surface of the sample glass slide comprises a double spacer region and a sample attachment region which correspond to the solid phase carrier chip, the double spacer region is a concave-convex structure formed by printing a combination of a straight line and a loop line by a silk-screen technology, and the printing thickness is 20-50 mu m; the formed concave-convex structure can play a role in physical barrier in the subsequent hybridization process, so that pollution among samples is prevented; the sample attachment area is a circular hole ring, the sample is directly spotted in the hole ring, and after natural drying, corresponding pretreatment such as digestion, dehydration and the like can be directly carried out.

The sample slide also comprises a marking area which is used for marking the sample information.

The solid phase carrier chip system is suitable for cell samples with more cytoplasm, such as urine cast-off cells, cervix cast-off cells, bacteria samples and the like.

Compared with the prior art, the invention has the following technical advantages:

1. the solid-phase carrier chip system couples a plurality of groups of existing detection probes with a glass slide through a physicochemical process to form the solid-phase carrier chip system;

2. the solid phase carrier chip system in the prior art mostly uses a freezing and freeze-drying process, and has high cost, and the solid phase carrier chip system solves the threshold problems that the existing solid phase carrier chip has complex preparation process, high cost and can not be flexibly produced;

3. the solid phase carrier chip system solves the problem that different gene markers are difficult to effectively separate and easily cause pollution when the multiple genes of the conventional solid phase carrier chip system are detected in parallel.

Drawings

FIG. 1 is a chip assembly of the solid support chip system of the present invention comprising eight channels.

FIG. 2 is a front view of a sample slide of the present invention comprising an eight channel solid support chip system.

FIG. 3 is a side view of a sample slide of the present invention including an eight channel solid support chip system.

FIG. 4 is a perspective view of a sample slide of the present invention comprising an eight channel solid support chip system.

FIG. 5 is a front view of a sample slide of an eight channel solid support chip system including a label zone.

FIG. 6 is a perspective view of a sample slide of a solid support chip system of the present invention including fifteen channels.

FIG. 7 is a perspective view of a sample slide of the solid support chip system of the present invention including thirty-two channels.

FIG. 8 is a perspective view of a sample slide of a six-channel solid support chip system of the present invention.

Detailed Description

The invention is further described below by means of specific embodiments, which are not intended to limit the scope of the patent protection of the invention in any way.

Example 1

The structure of the solid phase carrier chip system for in situ hybridization is shown in figure 1 of the invention, and the solid phase carrier chip system comprises eight groups of double spacers 1 and probe attachment regions 2, wherein the probe attachment regions 1 are circular hole circles, the double spacers 2 are concave-convex structures formed by combining straight lines and circle lines, and the thickness of the concave-convex structures is 20-50 mu m.

Example 2

A solid phase carrier chip system for in situ hybridization comprises eight groups of double spacers and probe attachment regions, wherein the probe attachment regions are circular hole rings, the double spacers are concave-convex structures formed by combining straight lines and loop lines, and the thickness of the concave-convex structures is 20-50 mu m.

The sample slide also includes a marking zone 3 for marking sample information.

Example 3

A method of performing hybridization of a cell sample using example 1 or example 2, comprising the steps of:

and (3) quantifying the probe, diluting the probe to 20 ng-60 ng/mu L by using a phosphate buffer solution, and spotting 1 mu L of the probe on a chip carrier according to the pore size. Naturally drying at room temperature (humidity below 65%) or oven drying at temperature below 56 deg.C.

Corresponding to example 1: directly dripping a sample to a solid phase carrier probe sheet, performing denaturation hybridization, and observing a signal under a fluorescence microscope.

Corresponding to example 2: cervical exfoliated cell/urine exfoliated cell flaking, involution with a solid phase carrier probe sheet, denaturation hybridization and signal observation under a fluorescent microscope.

Claims

1. A solid phase carrier chip system for in situ hybridization comprises one or more groups of double spacers and probe attachment regions, wherein the probe attachment regions are circular hole rings, the double spacers are concave-convex structures formed by combining straight lines and loop lines, and the thickness of the concave-convex structures is 20-50 mu m;

the dual spacer region and the probe attachment region in the solid phase carrier chip system are six groups, eight groups, fifteen groups or thirty-two groups.

2. A method for preparing the solid support chip system of claim 1, comprising the steps of: and printing the combination of the straight line and the loop line by using a screen printing technology to form a concave-convex structure, wherein the printing thickness is 20-50 mu m, using the prepared double-stranded probe to perform pre-spotting on the sample in the hole loop, and drying the sample in a drying oven at the temperature of 45 ℃ to form a probe attachment area.

3. Use of the solid support chip system of claim 1 for hybridization of morphologically simple or cytoplasmically poor cell-based samples, such as bone marrow samples.

4. Use according to claim 3, wherein the method of hybridization of a bone marrow sample comprises the steps of: directly dripping the bone marrow sample subjected to hypotonic treatment into a probe/sample attachment area, and dripping a hybridization buffer solution after simple salt treatment for denaturation and hybridization; and hybridizing for 1 hour or 16 hours or 1-16 hours, and observing a fluorescence signal under a mirror.

5. The solid phase carrier chip system of claim 1, comprising a sample slide for use in combination with the solid phase carrier chip, wherein the front surface of the sample slide comprises a dual spacer region and a sample attachment region corresponding to the solid phase carrier chip, the dual spacer region is a concave-convex structure printed with a combination of straight lines and loop lines by a silk-screen printing technique, and the printing thickness is 20-50 μm; the sample attachment area is a circular hole ring; the sample slide also comprises a marking area which is used for marking the sample information.

6. The solid support chip system of claim 5, wherein the sample slide is prepared by a method comprising the steps of: coating a layer of coating with super-hydrophobic property on the surface hydrophobic coating area of the glass slide by adopting a printing or vacuum plating method, and performing hydrophilic coating treatment on the hydrophilic area by using a vacuum sputtering method after the hydrophobic coating is coated.

7. The use of the solid support chip system of claim 6 for hybridization of cytoplasmic-rich cell-based samples, such as exfoliated urine cells, exfoliated cervical cells, and bacterial-based samples.

8. The use of claim 7, wherein the probe hybridization method comprises the steps of: carrying out enzyme digestion, dehydration or immobilization treatment on a sample slide, dripping a hybridization buffer solution on a probe chip, combining the probe chip with the sample slide, and carrying out denaturation and hybridization; and hybridizing for 1 hour or 16 hours or 1-16 hours, and observing a fluorescence signal under a mirror.