CN101543755B - Preparation method of encoding microspheres - Google Patents
Preparation method of encoding microspheres Download PDFInfo
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- CN101543755B CN101543755B CN2009100261373A CN200910026137A CN101543755B CN 101543755 B CN101543755 B CN 101543755B CN 2009100261373 A CN2009100261373 A CN 2009100261373A CN 200910026137 A CN200910026137 A CN 200910026137A CN 101543755 B CN101543755 B CN 101543755B
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- 239000004005 microsphere Substances 0.000 title claims abstract description 45
- 238000002360 preparation method Methods 0.000 title claims abstract description 25
- 239000000463 material Substances 0.000 claims abstract description 25
- 238000000034 method Methods 0.000 claims abstract description 14
- 239000000758 substrate Substances 0.000 claims abstract description 13
- 229920002120 photoresistant polymer Polymers 0.000 claims abstract description 10
- 239000007788 liquid Substances 0.000 claims abstract description 9
- 239000004065 semiconductor Substances 0.000 claims abstract description 7
- 239000011248 coating agent Substances 0.000 claims abstract description 5
- 238000000576 coating method Methods 0.000 claims abstract description 5
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- 238000005530 etching Methods 0.000 claims abstract description 4
- 230000008569 process Effects 0.000 claims abstract description 4
- 239000010410 layer Substances 0.000 claims abstract 20
- 239000003795 chemical substances by application Substances 0.000 claims abstract 7
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- 238000005459 micromachining Methods 0.000 claims abstract 2
- 239000002356 single layer Substances 0.000 claims abstract 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical group O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 16
- 229910052581 Si3N4 Inorganic materials 0.000 claims description 8
- 239000000377 silicon dioxide Substances 0.000 claims description 8
- 238000001259 photo etching Methods 0.000 claims description 7
- HQVNEWCFYHHQES-UHFFFAOYSA-N silicon nitride Chemical compound N12[Si]34N5[Si]62N3[Si]51N64 HQVNEWCFYHHQES-UHFFFAOYSA-N 0.000 claims description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 6
- 239000002184 metal Substances 0.000 claims description 4
- 229910052751 metal Inorganic materials 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 229920000642 polymer Polymers 0.000 claims description 3
- 235000012239 silicon dioxide Nutrition 0.000 claims description 3
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- 150000001875 compounds Chemical class 0.000 description 8
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
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- 238000004544 sputter deposition Methods 0.000 description 1
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Abstract
本发明公开了一种编码微球的制备方法，其特征在于包括步骤：在衬底上涂覆牺牲层；进而在其表面生长或沉积制备单层或多层复合的微球材料层；对微球材料层进行光阻涂布、光显影、刻蚀、清洗的半导体微加工制程，生成编码的微图案或阵列；再按制备要求的微球尺寸再次光刻，并清洗去除微球轮廓以外的微球材料层；选取对牺牲层具有溶解性的液剂，将夹有牺牲层的衬底及编码微球浸没于液剂中，最后释放出编码各异的微球悬浮在液剂中。通过微加工技术实现编码微球的制备，提高了微球编码的容量，而且制备工艺过程具有较好的可重复性，便于实施；且可采用常规荧光显微镜来进行检测，有效降低了生物分析的成本。
The invention discloses a method for preparing encoded microspheres, which is characterized by including the steps of: coating a sacrificial layer on a substrate; then growing or depositing a single-layer or multi-layer composite microsphere material layer on its surface; The ball material layer undergoes a semiconductor micro-machining process of photoresist coating, photo development, etching, and cleaning to generate a coded micro pattern or array; it is then photolithographed again according to the microsphere size required for preparation, and cleaned to remove the microsphere outline. Microsphere material layer; select a liquid agent that has solubility for the sacrificial layer, immerse the substrate sandwiched with the sacrificial layer and the coded microspheres in the liquid agent, and finally release microspheres with different codes suspended in the liquid agent. The preparation of encoded microspheres is achieved through micro-processing technology, which increases the encoding capacity of the microspheres, and the preparation process has good repeatability and is easy to implement; and conventional fluorescence microscopes can be used for detection, effectively reducing the cost of biological analysis. cost.
Description
Technical field
The present invention relates to biomedicine field, relate in particular to a kind of method for preparing microsphere of realizing coding by the little pattern of processing.
Background technology
Suspending chip (suspension array), be liquid chip (liquid array) again, its know-why is different from solid phase biological chip technology [annotate 1, annotate 2], the core of suspending chip technology is to adopt multiple differentiable microballoon, can encode to microballoon in other words, when detecting, can realize decoding again.The microsphere chip technology is emerging protein biochip technology, is a kind of new multi-functional liquid phase chip analysis platform that developed recently gets up.This technology has organically been integrated technology such as coloured microballoon technology, laser technology, fluidics, high speed digital signal processor and computer algorithm, has quite high detection specificity and sensitivity.Be generally used for researchs such as immunoassay, Study on Accounting, enzymatic analysis, acceptor and part discriminance analysis.
Typical coloured microballoon coding techniques is that Luminex company develops, these latex beadses are uniform circular microballoons (about 5.5 microns of diameter) of being made by polystyrene, in its manufacturing process, mix two kinds of fluorescent dyes according to strict ratio, every kind of dyestuff has 10 concentration gradients again, thereby microballoon can be divided into 100 kinds according to its color according to its blend proportion difference.According to the difference that detects thing, each microsphere surface can a kind of nucleic acid of covalent bond or protein detection probe, can add different detection microballoons simultaneously in same reaction system, simultaneously the plurality of target molecule is detected.During detection, can know it is the signal of which kind of detected material according to the detection signal of different fluorescence color microballoons.This method has fast, high flux, highly sensitive, low cost and other advantages, thus more and more be applied to clinical and scientific research in.Present many companies have released reagent corresponding and equipment based on the suspending chip technology of Luminex company.Yet, have only code capacity in trivial 100 obviously can't satisfy the needs of modern biological medicine research, and its detection means is to make of expensive flow cytometer.Along with various novel drugs compositions are developed, the code capacity of the microballoon that is used for the biological medicine analysis is also had higher requirement.
[annotating 1] D.A.A.Vignali, J.Immunol.Methods.2000,243,243-255.
[annotating 2] K.L.Kellar, M.A.Iannone, Exp.Hematol.2002,30,1227-1237.
Summary of the invention
In view of above-mentioned the deficiencies in the prior art, purpose of the present invention aims to provide a kind of preparation method of coding microball, solve the code capacity bottleneck problem of microballoon in preparation process, further expand coding microball carries out multiple bioanalysis, screening at field of medicaments application.
The object of the invention will be achieved through the following technical solutions:
The preparation method of coding microball is characterized in that comprising step:
Step 1: on substrate, apply sacrifice layer, after being used for microballoon preparation and finishing and substrate separation;
Step 2: adopt growth or depositing operation to prepare the compound micro-sphere material layer of single or multiple lift in sacrificial layer surface;
Step 3:, generate the little pattern or the array of coding at the micro-sphere material layer to the semiconductor microactuator procedure for processing that the micro-sphere material layer carries out photoresistance coating, photodevelopment, etching, cleaning;
Step 4:, and clean removal microballoon profile micro-sphere material layer in addition to the microballoon size photoetching once more of the micro-sphere material layer that generates coding by preparation requirements;
Step 5: choose sacrifice layer is had deliquescent liquor, substrate and the coding microball that accompanies sacrifice layer is immersed in the liquor, discharge the different microballoon of coding and be suspended in the liquor.
Further, the component of described micro-sphere material layer comprises the compound of a kind of or these materials in polymer, semi-conducting material, dielectric material, the metal.
Further, the component of described micro-sphere material layer is silica, silicon nitride or the compound of the two.
Further, the little pattern on the microballoon is encoded by these distinctiveness features of position, shape, size, direction or quantity in the described step 3; Perhaps
Further, the array on the microballoon is to encode by the difference of arrangement mode in the described step 3.
Again further, for the situation of photoresist as sacrifice layer, described liquor is elected acetone soln as in the described step 5.
After the preparation method of coding microball of the present invention used, its beneficial effect was:
Realize coding by the little manufacturing process of dot matrix or figure, improved the capacity of microballoon coding, and preparation process has repeatablely preferably, be convenient to implement microballoon; In addition, microspheres prepared can realize functions such as light, magnetic, electricity compound, be convenient to various biomolecule of coupling or little molecule, have the characteristic of harsh conditions such as acid and alkali resistance, heat, the more important thing is and to adopt conventional fluorescence microscope to detect, effectively reduce the cost of bioanalysis.
Description of drawings
Fig. 1 is preparation method's flow chart of coding microball of the present invention;
Fig. 2 is preparation technology's schematic diagram step by step of coding microball of the present invention;
Fig. 3 a～Fig. 3 d is encode little pattern form and a coded system schematic diagram of microballoon of the present invention.
The specific embodiment
Below in conjunction with some specific embodiments of the present invention and accompanying drawing thereof, the preparation method's step and the beneficial effect thereof of coding microball of the present invention is described in further detail:
The present invention prepares the encoding function that the microballoon with little pattern is realized microballoon by micro-processing technology.By processes such as a series of growth, deposition, etchings, on silicon chip or other substrates, prepare microballoon, and by discharging, microballoon is suspended in the solution the most at last.As micro-sphere material preparation method of the present invention is described with the compound of silica and silicon nitride below, shown in Fig. 1 and Fig. 2 preparation method's flow chart and process schematic representation:
At first, on 6 cun silicon chips 1, apply the thick ground floor photoresist 2 of 1 μ m, be used for final step the coding microball of making 6 is spun off from silicon chip 1.
Then, be about the silica 3 of 500nm at the ground floor photoresist 2 surface magnetic control sputterings layer thickness of growing, and on these silicon dioxide layer 3 surfaces again the magnetron sputtering layer thickness of growing be about the silicon nitride 4 of 500nm.Here magnetron sputtering is a kind of of growing method, not as limit.
Then, further apply the second layer photoresist 5 that is used to generate little pattern on these silicon nitride layer 4 surfaces, and this MULTILAYER SUBSTRATE carried out photoetching, on this MULTILAYER SUBSTRATE, form single or multiple square, circular, triangles or little pattern of other geometric arrangements.
Then, adopt the silicon nitride layer 4 of ion beam dry etching (IBE) 500nm, and remove this second layer photoresist with oxygen gas plasma; Have in this generation again and apply the 3rd layer photoetching glue that is used for photoetching microballoon profile on the MULTILAYER SUBSTRATE of little pattern, it is carried out after the photoetching silicon dioxide layer 3 with this 500nm of BOE solvent corrosion, on silicon chip 1 and ground floor photoresist 2, form separately, and have the microballoon of little pattern.
At last, the silicon chip substrate 1 that above step is made is immersed in the acetone soln dissolves ground floor photoresist 2 fully, so just can discharge the microballoon of realizing coding and be suspended among the solution.
From these preparation method's steps as seen, preparation process of the present invention has repeatable preferably, is convenient to implement.More than generate in the step of little pattern, the used mask plate of photoetching has variation (rule that is the microballoon coding is that the distinctiveness features such as position, shape, size, direction and quantity by pattern are determined, and more can determine by the arrangement mode of pattern).
In above-mentioned embodiment by silica and epontic silicon nitride compound as the micro-sphere material, use silica separately or use silicon nitride to prepare coding microball separately also for feasible.In addition, the compound of a kind of or these materials in each base polymer, semi-conducting material, dielectric material, the metal also provides alternative widely for micro-sphere material.
Shown in Fig. 3 a～Fig. 3 d, be encode little pattern form and coded system schematic diagram of microballoon of the present invention.As seen from the figure: what Fig. 3 a reflected is a kind of coded system of arranging according to rectangular patterns, realizes distinguishing the coding of microballoon type from the arrangement difference of two microsphere surface patterns.This mode of arranging has diversity, is not limited to diagram and arranges.And the arrangement mode of this pattern is equally applicable to the array coded system; What Fig. 3 b reflected is a kind of mode of encoding according to pattern form; What Fig. 3 c reflected is a kind of mode of encoding according to pattern magnitude; What Fig. 3 d reflected is a kind of mode of encoding according to number of patterns.
From diagram and other feasible little pattern correlative coding modes as seen, the microballoon that the present invention is prepared from has the code capacity of super prior art far away.And according to the difference of microballoon selection, the various microballoons of making by polymer, semi-conducting material, dielectric material, metal etc. or its compound can realize functions such as light, magnetic, electricity compound, be convenient to various biomolecule of coupling or little molecule, have the characteristic of harsh conditions such as acid and alkali resistance, heat, and can adopt conventional fluorescence microscope to detect, need not to purchase again flow cytometer or other special equipments.
To sum up, the preparation method of coding microball of the present invention can be widely used in the field such as detection, drug screening, combinatorial chemistry of DNA, RNA, protein, glycan molecule etc., has outstanding superiority.
Claims (6)
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
| CN103645308B (en) * | 2013-12-09 | 2015-06-03 | 东南大学 | Two-dimensional coding method of micro-carrier |
| CN103675258A (en) * | 2013-12-09 | 2014-03-26 | 东南大学 | Microcarrier preparation system |
| US10620197B2 (en) * | 2016-04-14 | 2020-04-14 | Suzhou Institute Of Nano-Tech And Nano-Bionics (Sinano), Chinese Academy Of Sciences | Magnetic microchip having graph code as well as preparation method and application thereof |
| CN110878147B (en) * | 2019-11-05 | 2021-08-17 | 嘉兴学院 | A kind of multi-segment bar code nanorod and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1355883A (en) * | 1999-04-16 | 2002-06-26 | 泰博特克有限公司 | Encoding of microcarriers |
| CN1434284A (en) * | 2003-02-26 | 2003-08-06 | 华中科技大学 | Spectrum identifying resin microball capable of self-coding |
| CN101216415A (en) * | 2008-01-08 | 2008-07-09 | 东南大学 | A photonic crystal composite coded microsphere and its preparation method |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1355883A (en) * | 1999-04-16 | 2002-06-26 | 泰博特克有限公司 | Encoding of microcarriers |
| CN1434284A (en) * | 2003-02-26 | 2003-08-06 | 华中科技大学 | Spectrum identifying resin microball capable of self-coding |
| CN101216415A (en) * | 2008-01-08 | 2008-07-09 | 东南大学 | A photonic crystal composite coded microsphere and its preparation method |
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