CN102520171B - Method for detecting pattern code suspended array chip - Google Patents
Method for detecting pattern code suspended array chip Download PDFInfo
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- CN102520171B CN102520171B CN2011104080139A CN201110408013A CN102520171B CN 102520171 B CN102520171 B CN 102520171B CN 2011104080139 A CN2011104080139 A CN 2011104080139A CN 201110408013 A CN201110408013 A CN 201110408013A CN 102520171 B CN102520171 B CN 102520171B
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Abstract
The invention relates to a method for detecting a pattern code suspended array chip. The method provided by the invention is characterized by comprising the following steps of 1, constructing a micro-hole array for fixing pattern code micro-carriers on a detection substrate, 2, fixing pattern code micro-carriers needing to be detected in the micro-hole array of the detection substrate in a way that one pattern code micro-carrier is fixed in one micro-hole, and 3, carrying out imaging of a pattern code micro-carrier array formed in the micro-hole array of the detection substrate by an imaging technology and carrying out detection. The method provided by the invention solves the problem that in a solution, pattern code micro-carriers focus difficultly and produce mutual interference easily, realizes high-throughput, clear and low-cost acquirement of detection information and provides possibility for practical application of a pattern code micro-carrier suspended array chip.
Description
Technical field
The present invention relates to the detection method of pattern code suspended array chip, especially in the situation that pattern code microcarrier and testing sample effect and detection signal can obtain, at first the pattern code microcarrier is fixing in the micro-pit array substrate, then under imaging device, decoding and optical signalling read.When the method not only can solve conventional pattern code microcarrier by Flow cytometry, microcarrier can not detect and detect the limited problem of flux at sheet, and can solve microcarrier be difficult to focus on and obtain the problem of signal due to motion in solution.Therefore be expected to advance the development and application of pattern code suspended array chip.
Background technology
The suspended array chip technology also claims microcarrier, and it is a kind of instrument that carries out the multi-target detection analysis by specificity interaction between sensing responsive material fixing on the coding microparticle and testing sample in fluid.Characteristics that it not only has that common multi-target detection analytical technology possesses contains much information, detection time is short, required detection sample volume is little and the relative traditional detection method of testing cost is low, and no matter be preparation or use it all than another multi-target detection analytical technology-plane micro-array chip technology, many outstanding advantages to be arranged: larger output, detect target arrangement, reaction and higher-quality experimental result faster more flexibly.Therefore, the research and development of suspended array chip just more and more have been subject to showing great attention to of people and have been used widely in fields such as drug screening, health care, food security, anti-terrorisms gradually.
In suspended array chip, the pattern code microcarrier is because pattern is easy to identification and the large extensive concern that obtains people of encoding amount.But current pattern code microcarrier suspension array chip is all detected by flow cytometry, so not only can not detect at sheet (before the detection, reach and detect rear microcarrier all in the detection substrate), and its detection flux is also limited significantly.And why people adopt reason that flow cytometry is detected just to be to be difficult under motion state focus on to obtain signal when microcarrier, and be easy to the phase mutual interference owing to the problem such as blocking in testing process in solution.And by flow cytometry to the pattern code microcarrier detected also moved in the situation that has difficulties of pattern identification, therefore, although the pattern code microcarrier has the large many merits that waits of the encoding amount of comprising, the suspended array chip of current practical application remains take the few optical encoding of group/cording quantity as main.This range of application that had both limited suspended array chip has also affected applying of suspended array chip simultaneously.Therefore the detection technique that exploitation can be carried out effectively identifying to the pattern code microcarrier just becomes a job highly significant.The application proposes constructing the fixing method with by imaging technique, the pattern code microcarrier being detected on micro-pit array the basis that is fixed for the pattern code microcarrier first for this reason.This technology not only can be at the clear coding that reads the pattern code microcarrier of sheet, and cheapness, high flux that imaging technique possesses also make pattern code microcarrier suspension array chip that more advantage has been arranged in the contrast with plane micro-array chip and other code suspended array chip.
Summary of the invention
Technical matters:The detection method that the purpose of this invention is to provide a kind of applicable pattern code microcarrier suspension array chip, particularly in the situation that fixing with micro-pit array, will need the pattern code microcarrier that detects fixing on micro-pit array for the pattern code microcarrier detecting in substrate to construct, then by imaging technique to the pattern code microcarrier array image-forming of needs detection to be detected.
Technical scheme:For solving the problems of the technologies described above, the invention provides a kind of detection method of pattern code suspended array chip, the method comprises the steps:
A, detect in substrate construct can fixed pattern coding microcarrier micro-pit array;
The mode that b, the pattern code microcarrier that will need to detect are fixed a pattern code microcarrier with a micro-hole is fixed on and detects in suprabasil micro-pit array;
C, by imaging technique to the pattern code microcarrier array image-forming that forms in micro-pit array on detecting substrate to be detected.
Preferably, detecting suprabasil micro-pit array is the micro-pit array by lithographic technique or template duplicating fabrication techniques according to the microcarrier shape.
Preferably, describedly will need the pattern code microcarrier that detects to fix a pattern code microcarrier mode with a micro-hole to be fixed in micro-pit array and to refer to by acting force the pattern code microcarrier is fixed in micro-pit array.
Preferably, described imaging technique refers to adopt and comprises that the imaging device of optical microscope, fluorescent microscope, infrared microscope, Raman microscope, scanner, camera carries out imaging to pattern code microcarrier array.
Preferably, the described pattern code microcarrier need detected refer to the pattern code microcarrier that is fixed with the sensing responsive material complete comprise with the acting on of testing sample in signal extraction before carry out in steps the pattern code microcarrier of signal extraction.
Beneficial effect:The present invention first is fixed to the pattern code microcarrier first and detects in substrate in micro-pit array then the detection of carrying out the pattern code microcarrier by imaging technique and not only solved the pattern code microcarrier be difficult to the problem that focuses on, is easy to interfere with each other in solution, and can be at sheet high flux, clear and obtain at an easy rate detection information, therefore for the practical application of pattern code microcarrier suspension array chip provides may.
The accompanying drawing explanation
Fig. 1 is pattern code suspended array chip detection method schematic diagram;
Wherein a is containing the detection substrate of micro-pit array; B pattern code microcarrier; C fluorescence labeling compound;
Fig. 2 is the micro-pit array vertical view;
Fig. 3 a is a kind of some coding templet;
Fig. 3 b is another some coding templet;
Fig. 3 c is the third coding templet; Wherein
3-1 the first coding; 3-2 the second coding; 3-3 the 3rd coding;
The direction identifier of a ' direction cog region; B ' code area is encoded to 1; C ' code area is encoded to 0.
Embodiment
The present invention will be described below with reference to accompanying drawings.
The detection method of pattern code suspended array chip of the present invention adopts following step to carry out:
A, detect in substrate construct can fixed pattern coding microcarrier micro-pit array;
The mode that b, the pattern code microcarrier that will need to detect are fixed a pattern code microcarrier with a micro-hole is fixed on and detects in suprabasil micro-pit array;
C, by imaging technique to the pattern code microcarrier array image-forming that forms in micro-pit array on detecting substrate to be detected.
Detecting suprabasil micro-pit array is the micro-pit array by lithographic technique or template duplicating fabrication techniques according to the microcarrier shape.
Describedly will need the pattern code microcarrier that detects to fix a pattern code microcarrier mode with a micro-hole to be fixed in micro-pit array and to refer to by acting force the pattern code microcarrier is fixed in micro-pit array.
Described imaging technique refers to adopt and comprises that the imaging device of optical microscope, fluorescent microscope, infrared microscope, Raman microscope, scanner, camera carries out imaging to pattern code microcarrier array.
The described pattern code microcarrier need detected refer to the pattern code microcarrier that is fixed with the sensing responsive material complete comprise with the acting on of testing sample in signal extraction before carry out in steps the pattern code microcarrier of signal extraction.
Embodiment mono-:
At first by microelectronics photoengraving technique on silicon chip respectively preparation size be that 350 micron pitch are 500 micrometer depth be 10 microns character A, B, and the convex formwork of C.Then the solvent of DOW CORNING SYLGARD 184 silicon rubber is mixed by the 10:1 weight ratio fully with hardening agent, be poured into containing in the container of convex character silicon chip template, be evacuated to without bubble, and solidify 1.5h under 100 ℃ of conditions, cooling, the demoulding obtains the PDMS template containing matrix retrography character A, B and C array.Then preparation contains 2% silicon dioxide, 1% polyvinyl alcohol (PVA) 1750,10% acrylamides and 5% N, and the aqueous solution of N-methylene-bisacrylamide is at the solution with front adding 1% ammonium persulfate (APS), standby after mixing.Get the above-mentioned solution of 0.1 microlitre by its point sample on the PDMS template that retrography character A, B and C array are arranged respectively with point sample instrument.Then in the situation that being greater than the 80%RH polymerized at room temperature, nitrogen protection and humidity within 8 hours, obtains the roly-poly shape microparticle containing convex character pattern A, B, C coding.The tumbler microparticle of convex character A, B and C pattern code is connected respectively to the polyacrylamide tumbler microcarrier that HAAb, hepatitis B antibody, antibody to hepatitis C (these antibody are the sensing responsive material) obtain convex character pattern coding by glutaraldehyde.Get respectively 7-8 and connected the convex character pattern coding polyacrylamide tumbler microcarrier of anti-HAV, hbv antibody and c-hepatitis antibody and people's serum sample after 37 degrees centigrade of hybrid reaction 2h, use the PBS buffer solution for cleaning, then obtain convex character code tumbler microcarrier to be measured with the fluorescently-labeled anti-HAV of CY3, hbv antibody, c-hepatitis antibody solution reaction 1h cleaning.
Etch and be of a size of 400 microns at silicon chip surface by microelectronic technique in addition, the degree of depth is the 200 micron pitch micro-pit array that is 500 microns.Above-mentioned microcarrier solution to be detected dripped on the micro-pit array of silicon chip and make most of above-mentioned tumbler microcarrier to be measured be deposited in micro-hole by the Action of Gravity Field of microcarrier self, then with sponge, wiping gently the tumbler microcarrier outside unnecessary solution and micro-hole away.Then at metallographic fluorescence microscopy Microscopic observation, can judge in surveyed blood serum sample whether contain hepatitis A virus, hepatitis B or hepatitis C virus according to having or not of fluorescence on kinds of characters coding tumbler microcarrier.
Embodiment bis-:
It is circle, the square and triangular array that 500 micron-scales are 400 microns that the range of size that is first 20wt% by Epson R230 ink-jet printer by concentration prints spacing between the crosslinked polymethylmethacrylaparticles microballoon that is of a size of the tri-iron tetroxide of 10 nanometers containing 5% superparamagnetism of 100 nanometer to 250 nanometers respectively containing the aqueous solution of 40% ethylene glycol on transparency base (contact angle of solution is 80 degree).After drying at room temperature, under 150 degrees centigrade, process 1 hour, the circle then by deionized water, crosslinked polymethylmethacrylaparticles built, square and triangle sheet microparticle rinse also cleaning-drying from the transparency base.After obtained circle, square and triangle sheet crosslinked polymethylmethacrylaparticles microparticle are first processed to 1 minute by ammonia plasma, be placed on 1% glutaraldehyde PB damping fluid room temperature reaction 4 hours, within 10 minutes, clean three times with the PBS damping fluid subsequently.Then process circular microparticles 12 hours with 4 degrees centigrade, the PBS buffer solution of the anti-HAV of 0.2mg/ml respectively, process square microparticles 12 hours with 4 degrees centigrade, the PBS buffer solution of the hbv antibody of 0.2mg/ml, with 4 degrees centigrade, the PBS buffer solution of the c-hepatitis antibody of 0.2mg/ml, process the triangle microparticles 12 hours.Unreacted glutaraldehyde is blocked with the PBS buffer solution reaction of 1% BSA in 2 hours.After the PBS buffer solution for cleaning, with the concentration of 3-4/10 microlitres, be stored in the PBS damping fluid of 4 degrees centigrade, obtain detecting the circular pattern coding sheet microcarrier of hepatitis A, detect the square pattern coding sheet microcarrier of hepatitis B and detect the triangle pattern coding sheet microcarrier of the third liver.Respectively get before detection that the storage liquid of the storage liquid of the circle codification microcarrier storage liquid of 20 microlitres, square coding microcarrier and triangle coding microcarrier is mixed to be incorporated in 37 degrees centigrade and to hatch 2 hours with 180rpm speed in oscillator with the test serum sample, then clean three times with PBST solution.Add CY3 mark anti-HAV, hbv antibody and the c-hepatitis antibody PBS buffer solution of 4 ug/ml, hatch 1 hour 37 degrees centigrade of speed with 60RPM, clean and obtain different pattern coding microcarrier to be measured for three times with PBS subsequently.
Etch and be of a size of 450 microns at silicon chip surface by microelectronic technique in addition, the degree of depth is the 150 micron pitch circular micro-pit array that is 500 microns.Above-mentioned microcarrier solution to be detected dripped on the micro-pit array of silicon chip and make most of above-mentioned circle to be measured, square and triangle sheet microcarrier be deposited in micro-hole by the magneticaction of the magnet at the Action of Gravity Field of microcarrier self and the micro-pit array back side, then with sponge, wiping gently the microcarrier outside unnecessary solution and micro-hole away.Then observe under inverted fluorescence microscope, can judge in surveyed blood serum sample whether contain hepatitis A virus, hepatitis B or hepatitis C virus according to having or not of fluorescence on difformity coding microcarrier.
Embodiment tri-:
At first under the lucifuge condition, preparation is about the 2M acrylamide of colloid of iron oxide of 10 nanometers and the 2-hydroxy-2-methyl propiophenone aqueous solution of 2mM methylene diacrylamide and 1.5%v/v containing the size of 5wt% superparamagnetic.This solution is placed in to the flow cell that a thickness is about the square ultraviolet light that 50 microns length of sides are 500 microns and keeps motionless.Then taking-up has as Fig. 3-1 mask plate that the rectangle size dimension is about the some encoding array of 150 microns of 300 microns *.Wherein a ' is reference position and the direction of coding orientation distinguishing mark to provide the microcarrier pattern code to identify.For code area, it is encoded to 1 to b ' when light tight, and it is encoded to 0 to c ' during for the code area printing opacity.Each point can have two codings like this, and the group/cording quantity of this some coding microcarrier is just 2
5=32.This mask plate is placed on to the top of flow cell and, in the above by the high voltage mercury lamp radiation of 100W 50 seconds, closes immediately mercury lamp and the liquid in flow cell is poured in beaker.Micropore that will be prepared by the mask plate of a coding with the magnet square sheet microcarrier of encoding separates and uses washed with de-ionized water three times from solution, repeats to prepare the dozens of microcarrier.And then the microcarrier that adopts the similarity method preparation to encode as the different micropores in Fig. 3-2 and Fig. 3-3.The microcarrier of getting three kinds of micropore codings connects respectively sensing responsive material a:5 '-acryloxy-TTT TGA TGT AGA TGT TTT ATT AGG GTT GT-3 '; B:5 '-acryloxy-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 ' also uses washed with de-ionized water, and acquisition can detect the microcarrier of nucleotide sequence a, b, c and d.Respectively get the sodium chloride of three microcarriers and 0.2M and is connected testing sample hybridization containing the biotin in the Tris-EDTA damping fluid of 0.05% Tween-20 from four kinds of microcarriers and use afterwards the PBS buffer solution for cleaning.Subsequently microcarrier is further mixed and hatches 30 minutes at 37 degrees centigrade with the PBST damping fluid of the streptavidin that is connected with phycoerythrin.Clean and preserve with PBS subsequently and obtain micropore to be measured coding microcarrier.
Etch and be of a size of 350 microns at silicon chip surface by microelectronic technique in addition, the degree of depth is the circular micro-pit array that 60 micron pitch are 400 microns.Then the solvent of DOW CORNING SYLGARD 184 silicon rubber is mixed by the 10:1 weight ratio fully with hardening agent, be poured in the container of having placed the silicon chip that contains circular micro-pit array, be evacuated to without bubble, and solidify 1.5h under 100 ℃ of conditions, cooling, the demoulding obtains the PDMS template containing micro-protuberance.Sprawl 20% polystyrene N on the PDMS template, N '-dimethyl formamide solution, process 2 hours at 200 degrees centigrade after drying again, and the polystyrene sheet material that will contain circular micro-pit array after cooling separates with PDMS.
Above-mentioned microcarrier solution to be detected dripped on the micro-pit array of polystyrene sheet material and make most of above-mentioned different micropores coding sheet microcarriers to be measured be deposited in micro-hole with the form of the microcarrier in a micro-hole by the magneticaction of the magnet at the Action of Gravity Field of microcarrier self and the micro-pit array back side, then with sponge, wiping gently the microcarrier outside unnecessary solution and micro-hole away.Then observe under inverted fluorescence microscope, can judge in surveyed blood serum sample whether contain hepatitis A virus, hepatitis B or hepatitis C virus according to having or not of fluorescence on different micropore pattern coding microcarriers.
The foregoing is only better embodiment of the present invention; protection scope of the present invention is not limited with above-mentioned embodiment; in every case the equivalence that those of ordinary skills do according to disclosed content is modified or is changed, and all should include in the protection domain of putting down in writing in claims.
Claims (5)
1. the detection method of a pattern code suspended array chip, is characterized in that, the method comprises the steps:
A, detect in substrate construct can fixed pattern coding microcarrier micro-pit array;
The mode that b, the pattern code microcarrier that will need to detect are fixed a pattern code microcarrier with a micro-hole is fixed on and detects in suprabasil micro-pit array;
C, by imaging technique to the pattern code microcarrier array image-forming that forms in micro-pit array on detecting substrate to be detected.
2. the detection method of pattern code suspended array chip according to claim 1, is characterized in that, detecting suprabasil micro-pit array is the micro-pit array by lithographic technique or template duplicating fabrication techniques according to the microcarrier shape.
3. the detection method of pattern code suspended array chip according to claim 1, it is characterized in that, describedly will need the pattern code microcarrier that detects to fix a pattern code microcarrier mode with a micro-hole to be fixed in micro-pit array and to refer to by acting force the pattern code microcarrier is fixed in micro-pit array.
4. the detection method of pattern code suspended array chip according to claim 1, it is characterized in that, described imaging technique refers to adopt and comprises that the imaging device of optical microscope, fluorescent microscope, infrared microscope, Raman microscope, scanner, camera carries out imaging to pattern code microcarrier array.
5. the detection method of pattern code suspended array chip according to claim 1, it is characterized in that, the described pattern code microcarrier detected that needs, refer to the pattern code microcarrier that is fixed with the sensing responsive material, this microcarrier complete comprise with the acting on of testing sample in signal extraction before institute carry out in steps signal extraction.
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| CN102788779B (en) * | 2012-09-07 | 2015-04-01 | 中国科学院苏州纳米技术与纳米仿生研究所 | Coding suspension microchip and preparation method and application thereof |
| CN109745934B (en) * | 2019-03-18 | 2023-11-21 | 中国人民解放军军事科学院军事医学研究院 | Array type synthesizer and inkjet synthesizer |
| CN110068528A (en) * | 2019-04-23 | 2019-07-30 | 中国石油大学(华东) | Particle detection technique in detection device and suspension |
| CN114414546B (en) * | 2022-01-28 | 2023-07-28 | 福州大学 | High-flux liquid-phase biomolecule detection method and device |
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