CN102520171B - Method for detecting pattern code suspended array chip - Google Patents

Abstract

The invention relates to a detection method for a pattern-coded suspension array chip, which is characterized in that the method comprises the following steps: a. constructing a micro-pit array capable of fixing a pattern-coded microcarrier on a detection substrate; b. coding the pattern to be detected The microcarriers are fixed in the micropit array on the detection substrate in such a way that one micropit fixes one pattern-coded microcarrier; c. Imaging the pattern-coded microcarrier array formed in the micropit array on the detection substrate by imaging technology for detection . This method not only solves the problem that the pattern-coded microcarriers are difficult to focus and easily interfere with each other in solution, but also can obtain detection information on-chip with high throughput, clear and cheap, so it provides a good foundation for the practical application of the pattern-coded microcarrier suspension array chip. possible.

Description

Detection method of pattern coded suspension array chip

technical field

The invention relates to a detection method of a pattern-coded suspension array chip, especially when the pattern-coded microcarrier interacts with a sample to be tested and the detection signal can be obtained, the pattern-coded microcarrier is first fixed on the micropit array substrate, and then the Under-device decoding and optical signal reading. This method can not only solve the problem that the microcarriers cannot be detected on the film and the detection throughput is limited when the conventional pattern-coded microcarriers are detected by flow cytometry, but also can solve the problem that the microcarriers are difficult to focus and obtain signals due to movement in the solution. . Therefore, it is expected to promote the development and application of the pattern coded suspension array chip.

Background technique

Suspension array chip technology, also known as microcarrier technology, is a tool for multi-target detection and analysis in fluids by encoding specific interactions between sensing sensitive materials immobilized on microparticles and samples to be tested. It not only has the characteristics of large amount of information, short detection time, small volume of detection samples and low detection cost compared with traditional detection methods, which are usually possessed by multi-target detection and analysis technology, but also it is much more expensive than the other one in both preparation and use. Target detection and analysis technology-planar microarray chip technology has many outstanding advantages: greater output, more flexible detection target arrangement, faster response and higher quality experimental results. Therefore, the research and development of suspended array chips is attracting more and more attention and has been widely used in drug screening, medical and health, food safety, anti-terrorism and other fields.

Pattern-encoded microcarriers in suspension array chips have attracted widespread attention due to the easy identification of patterns and large amount of encoding. However, the current pattern-coded microcarrier suspension array chips are all detected by flow cytometry, which not only cannot be detected on the chip (that is, the microcarriers are all on the detection substrate before and after detection), but also its detection throughput is greatly limited. degree of limitation. The reason why people use flow cytometry for detection is that it is difficult to focus to obtain signals when the microcarriers are moving in the solution, and they are easy to interfere with each other due to problems such as occlusion during the detection process. The detection of pattern-coded microcarriers by flow cytometry is also subject to the difficulty of pattern recognition in motion. Therefore, although pattern-coded microcarriers have many advantages including a large amount of coding, the suspension array chips currently used in practice are still based on Optical codes with a small number of codes are the main ones. This not only limits the application range of the suspension array chip, but also affects the popularization and application of the suspension array chip. Therefore, it is very meaningful to develop a detection technology that can effectively identify pattern-coded microcarriers. For this reason, the present application proposes for the first time a method of detecting pattern-coded microcarriers by imaging technology on the basis of constructing and fixing micropit arrays for pattern-coded microcarriers. This technology can not only clearly read the codes of pattern-coded microcarriers on-chip, but also the low-cost and high-throughput imaging technology enables the pattern-coded microcarrier suspension array chip to be compared with planar microarray chips and other coded suspension array chips. has more advantages.

Contents of the invention

Technical problem: the object of the present invention is to provide a kind of detection method suitable for pattern coded microcarrier suspension array chip, especially in the case of pattern coded microcarriers being constructed on the detection substrate to fix the micropit array that needs to be detected. The microcarriers are fixed on the micropit array, and then the pattern-coded microcarrier array to be detected is imaged for detection by imaging technology.

Technical solution: In order to solve the above technical problems, the present invention provides a detection method for a pattern-coded suspension array chip, which includes the following steps:

a. Construct a micropit array capable of fixing pattern-coded microcarriers on the detection substrate;

b. Fix the pattern-coded microcarriers to be detected in the micropit array on the detection substrate in such a way that one micropit fixes one pattern-coded microcarrier;

c. Imaging the pattern-coded microcarrier array formed in the micropit array on the detection substrate by imaging technology for detection. the

Preferably, the micropit array on the detection substrate is a micropit array fabricated by etching technology or template replication technology according to the shape of the microcarrier. the

Preferably, fixing the pattern-coded microcarriers to be detected in the microwell array in such a way that one microwell fixes one pattern-coded microcarrier refers to fixing the pattern-coded microcarriers into the microwell array by force. the

Preferably, the imaging technique refers to imaging the array of pattern-coded microcarriers using imaging equipment including optical microscopes, fluorescence microscopes, infrared microscopes, Raman microscopes, scanners, and cameras.

Preferably, the pattern-coded microcarrier that needs to be detected refers to the pattern-coded microcarrier immobilized with the sensing sensitive material to complete all steps before signal extraction including the interaction with the sample to be tested for signal extraction. .

Beneficial effects: the invention fixes the pattern-coded microcarriers in the micropit array on the detection substrate for the first time, and then detects the pattern-coded microcarriers by imaging technology, which not only solves the problem that the pattern-coded microcarriers are difficult to focus and easy to interfere with each other in the solution , and the detection information can be obtained in a high-throughput, clear and cheap manner, so it provides the possibility for the practical application of the pattern-coded microcarrier suspension array chip.

Description of drawings

Fig. 1 is a schematic diagram of a method for detecting a pattern-coded suspended array chip;

where a detection substrate containing micropit array; b pattern-coded microcarrier; c fluorescent labeling complex;

Figure 2 is a top view of the micropit array;

Fig. 3a is a kind of dot coding template;

Figure 3b is another point coding template;

Fig. 3c is the third kind of point coding template; Wherein

3-1 first code; 3-2 second code; 3-3 third code;

The direction identifier of the a' direction identification area; the code of b' coding area is 1; the code of c' coding area is 0.

Detailed ways

The present invention will be described below with reference to the accompanying drawings.

The detection method of the pattern-coded suspension array chip of the present invention adopts the following steps:

a. Construct a micropit array capable of fixing pattern-coded microcarriers on the detection substrate;

b. Fix the pattern-coded microcarriers to be detected in the micropit array on the detection substrate in such a way that one micropit fixes one pattern-coded microcarrier;

c. Imaging the pattern-coded microcarrier array formed in the micropit array on the detection substrate by imaging technology for detection. the

The micropit array on the detection substrate is a micropit array manufactured by etching technology or template replication technology according to the shape of the microcarrier. the

The fixing of the pattern-coded microcarriers to be detected in the microwell array in the manner that one microwell fixes one pattern-coded microcarrier refers to fixing the pattern-coded microcarriers into the microwell array by force. the

The imaging technique refers to imaging the pattern-coded microcarrier array by using imaging equipment including optical microscope, fluorescence microscope, infrared microscope, Raman microscope, scanner and camera.

The pattern-coded microcarriers to be detected refer to the pattern-coded microcarriers immobilized with sensing sensitive materials to complete all steps before signal extraction including the interaction with the sample to be tested for signal extraction.

Embodiment one:

Firstly, convex templates of characters A, B, and C with a size of 350 microns, a pitch of 500 microns, and a depth of 10 microns are respectively prepared on a silicon wafer through a microelectronic photolithography process. Then mix the basic components of Dow Corning SYLGARD 184 silicone rubber with the curing agent at a weight ratio of 10:1, pour it into a container containing a silicon wafer template with convex characters, evacuate until there are no bubbles, and store it at 100°C After curing for 1.5 hours, cooling and demolding, the PDMS template containing the array of concave reverse characters A, B and C can be obtained. Then prepare an aqueous solution containing 2% silicon dioxide, 1% polyvinyl alcohol 1750, 10% acrylamide and 5% N,N-methylenebisacrylamide, mix well and add 1% ammonium persulfate (APS) before use ) solution, set aside. Take 0.1 microliter of the above solution with a spotting instrument and spot it on the PDMS templates with arrays of characters A, B and C in reverse. Then polymerize at room temperature for 8 hours under nitrogen protection and a humidity greater than 80%RH to obtain tumbler-shaped microparticles containing convex character patterns A, B, and C codes. The tumbler microparticles encoded with convex characters A, B and C patterns are respectively connected to hepatitis A antibody, hepatitis B antibody, and hepatitis C antibody (these antibodies are sensitive materials) through glutaraldehyde to obtain convex character patterns Encoded polyacrylamide tumbler microcarriers. Take 7-8 polyacrylamide tumbler microcarriers with convex character pattern codes connected with hepatitis A antibody, hepatitis B antibody and hepatitis C antibody respectively, mix and react with human serum samples at 37 degrees Celsius for 2 hours, wash with PBS buffer, and then mix with CY3 The fluorescently labeled hepatitis A antibody, hepatitis B antibody, and hepatitis C antibody solution were reacted for 1 hour and washed to obtain the convex character-coded tumbler microcarrier to be tested.

In addition, micro-pit arrays with a size of 400 microns, a depth of 200 microns and a pitch of 500 microns are etched on the surface of the silicon wafer through a microelectronic process. Add the above-mentioned microcarrier solution to be tested dropwise on the micropit array of the silicon wafer, and rely on the gravity of the microcarrier itself to deposit most of the above-mentioned tumbler microcarriers to be tested into the micropit, and then gently wipe off the excess with a sponge. Tumbler microcarriers outside the solution and micropit. Then observe under a metallographic fluorescence microscope, and judge whether the tested serum sample contains hepatitis A virus, hepatitis B virus or hepatitis C virus according to the presence or absence of fluorescence on the tumbler microcarrier with different character codes.

Embodiment two:

First, the cross-linked polymethyl methacrylate microspheres containing 5% superparamagnetic iron oxide with a size of 10 nanometers and a size ranging from 100 nanometers to 250 nanometers with a concentration of 20 wt% by an Epson R230 inkjet printer The aqueous solution containing 40% ethylene glycol prints circular, square and triangular arrays with a pitch of 500 microns and a size of 400 microns on the projection film base (the contact angle of the solution is 80 degrees). After drying at room temperature, it was treated at 150 degrees Celsius for 1 hour, and then the circular, square and triangular sheet-shaped micro-particles constructed of cross-linked polymethyl methacrylate were rinsed off the transparency base with deionized water and washed and dried. The obtained circular, square and triangular sheet-shaped cross-linked polymethyl methacrylate microparticles were first treated with ammonia plasma for 1 minute, and then placed in 1% glutaraldehyde PB buffer at room temperature for 4 hours, and then Wash three times with PBS buffer for 10 min. Then use 0.2 mg/ml of hepatitis A antibody in PBS buffer solution at 4 degrees Celsius to treat round microparticles for 12 hours, use 0.2 mg/ml of hepatitis B antibody in PBS buffer solution at 4 degrees Celsius to treat square microparticles for 12 hours, and use 0.2 mg/ml of hepatitis B antibody in PBS buffer solution for 12 hours. Treat the triangular microparticles with the hepatitis C antibody in PBS buffer solution at 4 degrees Celsius for 12 hours. Unreacted glutaraldehyde was blocked with 1% BSA in PBS buffer for 2 hours. After washing with PBS buffer, store 3-4 pieces/10 microliters in PBS buffer at 4 degrees Celsius to obtain circular pattern-coded sheet-shaped microcarriers capable of detecting hepatitis A, and square-pattern-coded sheet-shaped microcarriers for detecting hepatitis B. Carrier and triangular-pattern-coded sheet microcarrier for detection of hepatitis C. Before testing, take 20 microliters of circular coded microcarrier stock solution, square coded microcarrier stock solution and triangular code microcarrier stock solution, mix and incubate with the serum sample to be tested at 37 degrees Celsius in a shaker at 180 rpm for 2 hours, and then washed three times with PBST solution. Add 4 μg/ml of CY3-labeled hepatitis A antibody, hepatitis B antibody and hepatitis C antibody in PBS buffer solution, incubate at 37 degrees Celsius at a speed of 60 RPM for 1 hour, and then wash three times with PBS to obtain different pattern-coded microcarriers to be tested.

In addition, a circular micropit array with a size of 450 microns, a depth of 150 microns and a pitch of 500 microns is etched on the surface of the silicon wafer through a microelectronic process. Drop the above-mentioned microcarrier solution to be tested on the micropit array of the silicon wafer, and use the gravity of the microcarrier itself and the magnetic force of the magnet on the back of the micropit array to make most of the above-mentioned circular, square and triangular flakes to be tested The microcarriers are deposited into the microwells, and then the excess solution and the microcarriers outside the microwells are gently wiped off with a sponge. Then observe under an inverted fluorescence microscope, and judge whether the measured serum sample contains hepatitis A virus, hepatitis B virus or hepatitis C virus according to the presence or absence of fluorescence on the microcarriers with different shape codes.

Embodiment three:

First, 2M acrylamide containing 5wt% superparamagnetic iron oxide colloid with a size of about 10 nanometers, 2mM methylenebisacrylamide and 1.5% v/v 2-hydroxy-2-methylbenzene were prepared under dark conditions Acetone in water. The solution was kept stationary in a square UV-transparent flow cell approximately 50 microns thick and 500 microns on a side. Then take out the mask plate with a rectangular dot code array with a side length of about 300 microns*150 microns as shown in Figure 3-1. Wherein, a' is a dot-coded orientation identification mark to provide the starting position and direction of micro-carrier pattern-coded identification. b' is coded as 1 when the coding area is opaque, and c' is coded as 0 when the coding area is transparent. In this way, each point can have two codes, and the number of codes of the point-coded micro-carriers is 2 ⁵ =32. The mask is placed above the flow cell and a 100W high-pressure mercury lamp is irradiated thereon for 50 seconds, then the mercury lamp is turned off and the liquid in the flow cell is poured into a beaker. The microporous-encoded square sheet-shaped microcarriers prepared from the dot-encoded mask template were separated from the solution with a magnet and washed three times with deionized water, and dozens of microcarriers were prepared repeatedly. Then use a similar method to prepare microcarriers with different micropore codes as shown in Figure 3-2 and Figure 3-3. Three types of micropore-coded microcarriers are connected to the sensing material a: 5'-acryloyloxy-TTT TGA TGT AGA TGT TTT ATT AGG GTT GT-3'; b: 5'-acryloyloxy-TAT TGA TGT AGA TGT TTT ATT AGG GTT GT-3'; C: 5'-acryloxy-TCT TGA TGT AGA TGT TTT ATT AGG GTT GT-3'; d: 5'-acryloxy-TGT TGA TGT AGA TGT TTT ATT AGG GTT GT-3' and washed with deionized water to obtain microcarriers capable of detecting nucleic acid sequences a, b, c and d. Three microcarriers from each of the four microcarriers were hybridized with 0.2M sodium chloride and 0.05% Tween-20 containing biotin in Tris-EDTA buffer, and then washed with PBS buffer. The microcarriers were then further mixed with phycoerythrin-linked streptavidin in PBST buffer and incubated at 37°C for 30 minutes. Then wash with PBS and save to obtain the coded microcarrier of the micropore to be tested.

In addition, a circular micropit array with a size of 350 microns, a depth of 60 microns and a pitch of 400 microns is etched on the surface of the silicon wafer by microelectronic technology. Then the basic components of Dow Corning SYLGARD 184 silicone rubber and the curing agent are completely mixed at a weight ratio of 10:1, poured into a container containing a silicon wafer containing a circular micropit array, vacuumed until there are no bubbles, and Curing at 100°C for 1.5h, cooling and demoulding to obtain a PDMS template containing microprotrusions. Then spread 20% polystyrene N,N'-dimethylformamide solution on the PDMS template, dry it and treat it at 200 degrees Celsius for 2 hours, and after cooling, the polystyrene sheet containing the circular micropit array and PDMS separate.

Drop the above-mentioned microcarrier solution to be tested on the micropit array of the polystyrene sheet, and with the help of the gravity of the microcarrier itself and the magnetic force of the magnet on the back of the micropit array, most of the above-mentioned different microporous coded sheets to be tested The micro-carriers are deposited into the micro-pit in the form of one micro-pit and one micro-carrier, and then the excess solution and the micro-carriers outside the micro-pit are gently wiped off with a sponge. Then observe under an inverted fluorescent microscope, and judge whether the measured serum sample contains hepatitis A virus, hepatitis B virus or hepatitis C virus according to the presence or absence of fluorescence on the microcarriers encoded with different microhole patterns.

The above descriptions are only preferred embodiments of the present invention, and the scope of protection of the present invention is not limited to the above embodiments, but all equivalent modifications or changes made by those of ordinary skill in the art according to the disclosure of the present invention should be included within the scope of protection described in the claims.

Claims (5)

1. A detection method for a pattern-coded suspension array chip, characterized in that the method may further comprise the steps: a. Constructing a micropit array capable of fixing pattern-coded microcarriers on the detection substrate; b. Fix the pattern-coded microcarriers to be detected in the micropit array on the detection substrate in such a way that one micropit fixes one pattern-coded microcarrier; c. Imaging the pattern-coded microcarrier array formed in the micropit array on the detection substrate by imaging technology for detection. 2. The detection method of the pattern-coded suspension array chip according to claim 1, wherein the micro-pit array on the detection substrate is a micro-pit array made by etching technology or template replication technology according to the shape of the micro-carrier. 3. according to the detection method of the described pattern coding suspension array chip of claim 1, it is characterized in that, the described pattern coding microcarrier that needs to detect is fixed in the micropit array with a micropit fixing a pattern coding microcarrier mode means The pattern-encoded microcarriers are immobilized into the array of microwells by force. 4. The detection method of the pattern-coded suspension array chip according to claim 1, wherein the imaging technology refers to the use of imaging techniques including optical microscopes, fluorescence microscopes, infrared microscopes, Raman microscopes, scanners, and cameras. The device images an array of pattern-encoded microcarriers. 5. according to the detection method of the described pattern code suspension array chip of claim 1, it is characterized in that, the pattern code microcarrier that needs to detect described, refers to the pattern code microcarrier that is fixed with sensing sensitive material, and this microcarrier completes and comprises Signal extraction is performed in all steps prior to signal extraction including the action of the sample to be tested.

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