CN212533007U

CN212533007U - Microfluidic chip for exosome cracking and detection

Info

Publication number: CN212533007U
Application number: CN202021122949.6U
Authority: CN
Inventors: 韩琳; 褚玉金; 张宇
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2020-06-17
Filing date: 2020-06-17
Publication date: 2021-02-12
Anticipated expiration: 2030-06-17

Abstract

The utility model belongs to the technical field of the micro-fluidic, a micro-fluidic chip and method for exosome schizolysis and detection is related to. The micro-fluidic chip comprises an upper control layer, a middle flow channel layer and a functional substrate layer, wherein the middle flow channel layer is provided with a first sample adding port (4), a cracking cavity (7), a detection cavity (8) and a second sample adding port (5); the first sample adding port (4) is connected with the cracking cavity (7) through a first micro-channel (6); the cracking cavity (7) is connected with the detection cavity (8) through a second micro-channel (14); the second sample adding port (5) is connected with the second micro-channel (14); the functionalized substrate includes a lysis zone, a detection reaction zone, and a detection zone. The utility model provides a collect exosome schizolysis and detect micro-fluidic chip as an organic whole, this chip simple structure, convenient operation. The chip is adopted to crack exosomes, so that the accuracy is high, and the impurities are few; and miRNA detection is carried out immediately after exosome lysis is finished, so that the risk of miRNA pollution and damage is avoided.

Description

Microfluidic chip for exosome cracking and detection

Technical Field

The utility model belongs to the technical field of the micro-fluidic, a micro-fluidic chip for exosome schizolysis and detection is related to.

Background

Exosomes are lipid bilayer vesicles secreted by cells, with sizes ranging from 30-200 nm. Exosomes are mainly composed of proteins, mRNA, miRNA, and lipid bilayers on membranes, and have the function of transmitting information between cells. Many studies have shown that exosomes play both a carcinogenic and antitumor role in malignant tumors. Tumor cells continuously release tumor-specific exosomes to the outside of cells in the process of generation and development of the tumor cells, and take part in a series of physiological activities such as regulation of angiogenesis, immune response and the like through biological substances such as DNA, RNA, protein and the like of the tumor cells carried by the tumor cells.

miRNA is a single-stranded RNA molecule with the length of 19-23 bp, and is widely present in animals, plants and viruses. In recent years, many studies have shown that mirnas are involved in various biological processes in organisms, such as cell development and differentiation, mammalian immune cell function, proliferation and apoptosis, etc., and the expression amount is different in each individual due to individual differences, and because of such differences, mirnas can be used as biomarkers for early detection of cancer.

Currently, research on exosomes mainly focuses on the extraction of exosomes, and research on exosome internal genes mainly focuses on the total concentration of miRNA, quantitative PCR, and the like. In the existing technology for extracting exosome miRNA, although the obtaining method is many, the exosome miRNA extracted by the existing extracting method is often insufficient in purity and has more impurities. If the miRNA cannot be detected in time after extraction, the miRNA is damaged, so that the subsequent detection result is influenced.

SUMMERY OF THE UTILITY MODEL

In order to solve the problem that exists among the prior art, the utility model discloses a micro-fluidic technology designs a chip system that collects exosome schizolysis and detect as an organic whole, realizes on a chip that the target that detects is carried out to miRNA immediately after exosome schizolysis, has improved the accuracy that detects simultaneously greatly.

The utility model provides a technical scheme that its technical problem adopted is: the microfluidic chip comprises an upper control layer, a middle flow channel layer and a functional substrate, wherein the middle flow channel layer is provided with a first sample adding port (4), a cracking cavity (7), a detection cavity (8) and a second sample adding port (5); the first sample adding port (4) is connected with the cracking cavity (7) through a first micro-channel (6); the cracking cavity (7) is connected with the detection cavity (8) through a second micro-channel (14); the second sample adding port (5) is connected with the second micro-channel (14); the functionalized substrate includes a lysis zone, a detection reaction zone, and a detection zone.

As a preferred mode of the present invention, the upper control layer comprises a plurality of valves and sample inlets/outlets; and a flow passage connecting the valve and the sample inlet/outlet.

Further preferably, the valves include a first valve (10) disposed between the first sample addition port (4) and the lysis chamber (7), a second valve (11) and a third valve (12) disposed between the lysis chamber (7) and the detection chamber (8), and a fourth valve (13) disposed in front of the second sample addition port (5).

As the utility model discloses an optimal mode, functional substrate on the detection zone correspond and detect chamber (8) position, decorate polylysine, detect that the reaction zone corresponds second miniflow way (14) position, the pyrolysis zone corresponds pyrolysis chamber (7) position, detect reaction zone and pyrolysis zone modification oxidation graphite alkene material.

Further preferably, the upper control layer and the middle flow channel layer are made of PDMS.

Further preferably, the thickness of the upper control layer is 4-8 mm.

Further preferably, the thickness of the intermediate flow channel layer is 10 to 20 μm.

Further preferably, the functionalized substrate layer is made of glass or other substrates with high reflectivity.

The utility model has the advantages that: the microfluidic chip integrates exosome cracking and detection, and is simple in structure and convenient to operate. The utility model firstly cracks exosome, has high accuracy and less impurities; and miRNA detection is carried out immediately after exosome lysis is finished, so that the risk of miRNA pollution and damage is avoided.

Drawings

Fig. 1 is a schematic diagram of the overall structure of a microfluidic chip for exosome lysis and detection in an embodiment of the present invention;

FIG. 2 is a schematic view of an intermediate flow channel layer structure;

FIG. 3 is a schematic structural diagram of an upper control layer;

FIG. 4 is a schematic view of a functionalized glass substrate structure.

Detailed Description

In order to facilitate understanding of the present invention, the present invention will be described in more detail with reference to the accompanying drawings and specific embodiments. Preferred embodiments of the present invention are shown in the drawings. The invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

The utility model provides one of them embodiment is: a micro-fluidic chip for exosome lysis and detection has a structure shown in figure 1 and mainly comprises an upper control layer 1, a middle flow channel layer 2 and a lower functionalized glass substrate 3. Wherein the thicknesses of the upper control layer 1 and the middle flow channel layer 2 are 5 mm and 10 um PDMS respectively. As shown in FIG. 2, the middle flow channel layer comprises a first sample addition port 4, a second sample addition port 5, a lysis chamber 7 and a detection chamber 8. Wherein the first sample adding port 4 is connected with the cracking cavity 7 through the first micro-channel 6, and the cracking cavity 7 is connected with the detection cavity 8 through the second micro-channel 14. The second sample addition port 5 is connected to the second microchannel 14 so as to communicate with the lysis chamber 7 and the detection chamber 8. The first sample adding port 4 is used for adding lysis solution, an antibody for specifically capturing the exosome surface protein and a sample to be detected into the lysis cavity. The second port 5 is used for introducing a nucleic acid probe into the micro flow channel and drawing out excess liquid in the micro flow channel through the port.

As shown in fig. 3, the upper control layer 1 functions to control the flow direction of the liquid in the lower flow channel layer by valves, including a first valve 10, a second valve 11, a third valve 12, and a fourth valve 13, and an inlet/outlet 9 and a micro flow channel for injecting and pumping water into and out of the respective valves.

The first valve 10 is disposed at a corresponding position between the first sample addition port 4 and the lysis chamber 7, that is, a corresponding position above the first microchannel 6, and is configured to control the on/off of the first microchannel 6. The second valve 11 is arranged at a position corresponding to the upper part of the second micro-channel and at the right side of the cracking cavity 7, and is used for controlling the on-off of the micro-channel between the cracking cavity 7 and the detection cavity 8 or the second sample adding port 5. The third valve 12 is disposed at a position corresponding to the upper side of the second microchannel 14, and on the left side of the detection chamber 8, and is used for controlling the on-off of the microchannels between the detection chamber 8 and the lysis chamber 7 or the second sample addition port 5. The fourth valve 13 is disposed at a position corresponding to the position between the second sample port 5 and the second microchannel 14, and controls the on/off of the second sample port 5 and the second microchannel 14.

On upper control layer 1, still seted up with first sample application port 4, second sample application port 5 and cracking chamber 7 hole that link up from top to bottom, be convenient for carry out corresponding operation from upper control layer top.

As shown in fig. 4, the position on the lower functionalized glass substrate 3 corresponding to the detection cavity 8 is a detection region, and Polylysine (PLL) is modified and used for combining a double strand formed by the immobilized probe and miRNA. The position corresponding to the cracking cavity 7 is a cracking area, the position corresponding to the second micro-channel (14) is a detection reaction area, and the cracking area and the detection reaction area are both modified with nano graphene oxide materials for adsorbing and fixing nucleic acid probes.

The manufacturing method of the microfluidic chip of the embodiment comprises the following steps: firstly, an upper control layer 1 is manufactured, then a middle flow channel layer 2 is manufactured, then two layers of PDMS are butted together, then the butted PDMS is pasted on a functionalized glass substrate 3, a detection cavity 8 is butted with a PLL part on the functionalized glass substrate 3, the rest parts are butted with a graphene oxide part on the functionalized glass substrate, and the manufacture of the microfluidic chip is completed.

The utility model discloses a micro-fluidic chip, its theory of operation and application method are:

in the embodiment, the valve is opened by injecting deionized water into the valve to press the valve down and block a micro-channel below the valve; closing the valve means pumping out the deionized water in the valve, releasing the normal pressure of the valve, and communicating the micro-channel below.

(1) Laying nucleic acid probes with fluorescent groups. Deionized water is injected into the first valve 10, the second valve 11 and the third valve 12 through the inlet/outlet 9, and the first valve 10, the second valve 11 and the third valve 12 are opened. Injecting a solution containing a nucleic acid probe into the second sample addition port 5, blowing the nucleic acid probe solution at the second sample addition port 5 into the second micro flow channel 14 by adopting nitrogen, then opening the fourth valve 13, closing other valves, and incubating for a period of time, wherein the nucleic acid probe is adsorbed and fixed by graphene oxide on the functionalized glass substrate 3;

(2) excess unbound probes are aspirated. Closing the fourth valve 13, and sucking out the redundant solution in the second microchannel 14 from the second sample port 5;

(3) an antibody that specifically captures exosome surface proteins was added. Injecting an antibody for specifically capturing the surface protein of the exosome into the lysis cavity 7 through the through hole from the upper control layer 1, and incubating for a period of time, wherein the antibody is adsorbed by the graphene oxide on the functionalized glass substrate 3;

(4) excess non-adsorbed antibody is aspirated. Opening the first valve 10 and the third valve 12 to suck out the surplus unadsorbed antibodies from the second sample addition port 5;

(5) a biological sample containing exosomes is injected. Injecting a biological sample containing exosomes into the first sample port 4, with the first valve 10 closed; the second valve 11, the third valve 12 and the fourth valve 13 are opened; blowing the biological sample in the first sample adding port 4 into the lysis cavity 7 through the first micro flow channel 6 by using nitrogen, opening the first valve 10, and after incubating for a period of time, capturing the exosome by the antibody at the bottom of the lysis cavity 7;

(6) excess biological sample is aspirated. Opening the first valve 10 and the third valve 12, closing the second valve 11 and the fourth valve 13, and sucking out the redundant biological sample from the second sample addition port 5;

(7) add lysis solution. Injecting the lysis solution into the first sample port 4, closing the first valve 10,

opening a second valve 11, a third valve 12 and a fourth valve 13, blowing the lysate into the lysis cavity 7 through the first micro flow channel 6 by adopting nitrogen, opening the first valve 10, incubating for a period of time, then cracking the exosomes, and releasing the miRNA inside;

(8) the nucleic acid probe and miRNA released by exosome are combined into double-stranded. Storing miRNA released by exosome in a lysis cavity 7, closing a first valve 10 and a second valve 11, opening a third valve 12 and a fourth valve 13, blowing the solution containing the miRNA released by exosome in the lysis cavity 7 into a second micro-channel 14 from a first sample port 4 by adopting nitrogen, paving a probe with a fluorescent group in the second micro-channel 14, incubating for a period of time, and combining the miRNA released by exosome with the probe with the fluorescent group on a substrate to form a double chain;

(9) excess liquid in the lysis chamber 7 is aspirated. Opening the first valve 10 and the third valve 12, closing the second valve 11 and the fourth valve 13, and sucking out the redundant liquid in the cracking cavity 7 from the second sample adding port 5;

(10) the bound double strand is injected into the detection chamber 8. Closing the first valve 10, the second valve 11 and the third valve 12, opening the fourth valve 13, blowing the solution in the second micro flow channel 14 into the detection chamber 8 from the first sample addition port 4 by using nitrogen, opening the third valve 12, incubating for a period of time, and detecting the fluorescence in the detection chamber 8 by using a gene chip scanner.

Claims

1. A micro-fluidic chip for exosome lysis and detection, characterized in that: the device comprises an upper control layer, a middle flow channel layer and a functional substrate, wherein the middle flow channel layer is provided with a first sample adding port (4), a cracking cavity (7), a detection cavity (8) and a second sample adding port (5); the first sample adding port (4) is connected with the cracking cavity (7) through a first micro-channel (6); the cracking cavity (7) is connected with the detection cavity (8) through a second micro-channel (14); the second sample adding port (5) is connected with the second micro-channel (14); the functionalized substrate includes a lysis zone, a detection reaction zone, and a detection zone.

2. The microfluidic chip for exosome lysis and detection according to claim 1, characterized in that: the upper control layer comprises a plurality of valves and sample inlets/outlets; and a flow passage connecting the valve and the sample inlet/outlet.

3. The microfluidic chip for exosome lysis and detection according to claim 2, characterized in that: the valve comprises a first valve (10) arranged between the first sample adding port (4) and the cracking cavity (7), a second valve (11) and a third valve (12) arranged between the cracking cavity (7) and the detection cavity (8), and a fourth valve (13) arranged in front of the second sample adding port (5).

4. The microfluidic chip for exosome lysis and detection according to claim 1, characterized in that: polylysine is modified at the position of the detection cavity (8) corresponding to the detection area on the functionalized substrate; the detection reaction area corresponds to the position of the second micro-channel (14), the cracking area corresponds to the position of the cracking cavity (7), and the detection reaction area and the cracking area modify the graphene oxide material.

5. Microfluidic chip for exosome lysis and detection according to any one of claims 1-4, characterized in that: the upper control layer and the middle flow channel layer are made of PDMS.

6. The microfluidic chip for exosome lysis and detection according to claim 5, characterized in that: the thickness of the upper layer control layer is 4-8 mm.

7. The microfluidic chip for exosome lysis and detection according to claim 5, characterized in that: the thickness of the middle flow channel layer is 10-20 μm.

8. Microfluidic chip for exosome lysis and detection according to any one of claims 1-4, characterized in that: the functional substrate layer is made of glass or has high reflectivity.