WO2017016017A1 - 一种可视化芯片及其制备方法 - Google Patents

一种可视化芯片及其制备方法 Download PDF

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WO2017016017A1
WO2017016017A1 PCT/CN2015/088333 CN2015088333W WO2017016017A1 WO 2017016017 A1 WO2017016017 A1 WO 2017016017A1 CN 2015088333 W CN2015088333 W CN 2015088333W WO 2017016017 A1 WO2017016017 A1 WO 2017016017A1
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chip
biochip
visualization
detected
specific
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PCT/CN2015/088333
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French (fr)
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杨华卫
刘泽涛
古恒森
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江苏猎阵生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

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  • the invention relates to a chip, in particular to a biochip.
  • the invention also relates to a method of preparing the biochip, and a method of visualizing a chip.
  • Chip technology is an emerging industry, mainly divided into gene chip technology, flip chip technology, biochip technology, tissue chip technology, protein chip technology, DNA chip technology, liquid phase chip technology, chip packaging technology and so on.
  • Gene chips are also known as DNA chips or DNA microarrays.
  • the principle is to use a photoconductive in-situ synthesis or micro-printing method to densely and orderly fix a large number of specific sequence probe molecules on a correspondingly processed silicon wafer, glass slide, nitrocellulose membrane, etc., and then add the label.
  • the sample to be tested is subjected to multi-hybridization, and the presence, quantity and sequence of the target molecule are analyzed by the strength and distribution of the hybridization signal, thereby obtaining the genetic information of the sample to be tested (see Gene chip technology and its application).
  • the gene chip can analyze tens of thousands of genes in parallel at the same time, and carry out high-throughput screening and detection analysis, which solves the problems of traditional nucleic acid blot hybridization technology, such as complicated operation, low degree of automation, and small number of molecules for detection (see "Gene Chip Technology in Clinical Practice”. Application and Advantages in Microbial Testing. Tang Guili, China Pharmaceuticals, 5, 2015, 92-94).
  • a protein chip is a high-throughput monitoring system that monitors the interaction between protein molecules by interacting with target molecules and capture molecules.
  • the capture molecules are generally pre-immobilized on the surface of the chip and are widely used as capture molecules due to their high specificity and strong binding properties to antigens.
  • Protein chips can be used for protein expression profiling, protein phosphorylation and protein interactions, immunoassays and enzyme activity assays, antibody screening, proteomics studies, interactions between biomolecules, interactions between proteins and small molecules, drug targets and Research on its mechanism of action, while protein chips are also available. Used in disease diagnosis, food analysis, toxicology and hygiene testing. In clinical applications, protein chips are mainly used in the diagnosis of diseases, disease marker discovery, disease monitoring, evaluation of therapeutic effects (see “Progress in Protein Chip Research". Zhang Aiying, Yin Chengzeng, etc., “Chinese Medical Equipment", 11, 2014, 150-151).
  • Polysaccharide chips are born on the basis of gene chips and protein chips. The basic principles are similar to those of gene chips and protein chips, and they are based on the specific effects between substances. It is a kind of analytical method for testing and analyzing proteins and glycoproteins waiting for samples by covalent or non-covalently immobilizing sugar molecules of different structures on chemically modified substrates (please refer to Sugar Detection and Application of Chips. Huang He, Jia Hongying, et al., Chemical Bulletin, 5, 2009, 401-406). The research of sugar chips is of great significance for the diagnosis and monitoring of epidemics, the identification and identification of endotoxin in pathogenic microorganisms, and the common research on the specific binding of sugars to target proteins.
  • Tissue chip also known as tissue microarrays
  • tissue microarrays is an important branch of biochip technology. It is to arrange many different individual tissue specimens on the same slide in a regular array to carry out the same indicator. Histology research (please refer to "Application and Research Progress of Tissue Chips”. Li Xiaojing, Yang Xiaoli, et al., Journal of Practical Medicine, 21, 2014, 857-859).
  • RNA microarrays use RNA probe hybridization to rapidly and high-throughput expression changes of some RNAs related to specific biological processes or diseases, and provide important guiding significance for related diseases or biological process research.
  • RNA microarray chips are gradually being used in various tumor research and have great application value (please refer to "Application of MicroRNA Microarray Chips in Cancer Research". ⁇ Hong Qing, Zhang Hui et al., Chinese Experimental Surgery Journal, 12, 2011, 2267-2268).
  • Tavazoie et al. used microarray chips to detect the expression of miRNAs in breast cancer MDA-MB-231 cells and subcellular strains that are easily transferred to bone and lung. The low expression of some RNAs in metastatic sub-cells was found, providing a theory for tumor detection and treatment. Based on (see “Endogenous human microRNAs that suppress breast cancer metastasis”. Tavazoie SF, Alarcon C, et al, nature 451, 2008, 147-152).
  • the chip substrate may be made of a glass slide, a silicon wafer, a ceramic sheet, a polypropylene film, a nitrocellulose film, and a nylon film.
  • polylysine modification, aldehyde modification, amino modification, thiol modification, agarose coating or acrylamide silanization on the surface of the carrier to make the carrier biospecific. Affinity surface.
  • the prepared probe is fixed to the activated substrate.
  • the most common one on the market is to use the chip spotter to point the probe on the corresponding chip, and then judge the detection result by coloring after hybridization and substrate color development during the detection process;
  • it is necessary to determine the specific type according to the pre-set typesetting from " The clinical significance of human papillomavirus gene chip typing in Shanghai area. Qi Yingxin, Gan Jiemin, et al., Laboratory Medicine, 6, 2015, 595-598) is more complicated and error-prone, causing inconvenience to the interpretation results. Therefore, there is a need in the art for a biochip that is capable of visualizing test results.
  • the present invention provides a visualization biochip characterized in that specific bioprobes are arranged in a pattern on a supporting chip substrate.
  • the method of arranging the biological probes in a graphic form is specifically: in a spotting device (for example, a crystal core PersonalArrayer 16 personal spotting instrument, purchased from Boao Biosystems Co., Ltd.
  • a spotting device for example, a crystal core PersonalArrayer 16 personal spotting instrument, purchased from Boao Biosystems Co., Ltd.
  • Set the matching software on the spotter set from the software to the pattern to be displayed. For example, if you need to display the number “6”, set the print mode to “6” on the software, and then use the spotter.
  • the probe you want to spot can be printed directly on the chip.
  • the corresponding pattern is formed on the spotter by a plurality of points, and finally, when the spotter is spotted, a plurality of points can be combined to form a pattern to be displayed.
  • the visualization biochip utilizes DNA hybridization reaction, DNA and RNA specific hybridization, RNA and cDNA specific hybridization, DNA and cDNA specific hybridization, antigen-antibody specific binding, and DNA-DNA binding protein specificity. Sex binding, RNA-RNA binding protein specific binding, antibody-antibody binding protein (eg G protein-antibody Fc fragment) specific binding, biotin-streptavidin specific binding, or One or more of other specific reactions.
  • the graphic is in the form of a number, a letter, a Chinese character, an iconic symbol or an iconic pattern, or any combination thereof.
  • the letters are preferably English letters.
  • the biochip is one or more of a gene chip, a protein chip, an RNA chip, a polysaccharide chip, a tissue chip, and a neuron chip, preferably a gene chip and/or a protein. chip.
  • the gene chip of the present invention is equivalent to a DNA chip. That is, the gene chip can detect any DNA fragment, not just a gene fragment.
  • the specific bioprobe is one or more of specific DNA, RNA, polysaccharide and protein.
  • the specific biological probe is an oligonucleotide, a cDNA, an RNA, an antibody, an antigen, an antibody binding protein, a fragment of an oligonucleotide, a fragment of a cDNA, and an RNA.
  • a fragment, a fragment of an antibody, a fragment of an antigen, and a fragment of an antibody binding protein is an oligonucleotide, a cDNA, an RNA.
  • the visualization biochip is further provided with a probe as a control, preferably a probe as a positive control and a negative control, and particularly preferably a probe provided as a positive control.
  • the control can be a control for color development and/or a control for PCR amplification.
  • the graphical representation of the graphic is detected by the visualization biochip
  • the key information of the item or the detection result or the key information of the item detected by the visual biochip or the abbreviation of the detection result is preferably one or more of the item name, the age of the detected subject, and the name of the species.
  • the detection result is one or more of a negative, positive, and type name of the detected pathogen or a negative, positive, and a short name of the type name of the detected pathogen.
  • the specific bioprobe is designed to develop color upon binding to its corresponding target product. In this way, the detection result of the biochip can be directly read after the color development process.
  • the color development is chemical color development and/or fluorescent color development. In some more preferred embodiments of the invention, the color development is chemical color development. In still other more preferred embodiments of the invention, the color development is fluorescent color development.
  • the color development is horseradish peroxidase in the chromogenic substrate 3-amino-9-ethylcarbazole (AEC) Under the action, the electrons are lost and the color change and deposition are formed, and the stable red insoluble product is formed to achieve the color development effect.
  • AEC chromogenic substrate 3-amino-9-ethylcarbazole
  • Another object of the present invention is to provide a method of preparing the above-described visualization biochip, comprising the steps of:
  • the method for preparing the above-described visualization biochip comprises the following steps:
  • the specific bioprobe prepared in the step 1) is printed on the supporting chip substrate in the form of a pattern in the step 2) to prepare a visualization chip.
  • the method for preparing the above-described visualization biochip comprises the following steps:
  • step 3 Using the spotter, the specific bioprobe prepared in step 1) is placed on the supporting chip substrate in step 2).
  • the settings are printed in the form of graphics to make a visualization chip.
  • the color development is preferably chemical color development and/or fluorescent color development.
  • the graphic is in the form of a number, a letter, a Chinese character, an iconic symbol or an iconic pattern, or any combination thereof.
  • the biochip is one or more of a gene chip, a protein chip, an RNA chip, a polysaccharide chip, a tissue chip, and a neuron chip.
  • the specific biological probe is one or more of specific DNA, RNA, polysaccharide or protein.
  • the specific biological probe is an oligonucleotide, cDNA, RNA, antibody, antigen, antibody binding protein, or oligonucleotide, cDNA, RNA, antibody, antigen One or more of the fragments of the antibody binding protein.
  • the form of the graphic represents key information or detection result of an item detected by the visualization biochip or key information of an item detected by the visualization biochip or an abbreviation of the detection result.
  • the key information is preferably one or more of the item name, the age of the detected subject, and the name of the species, and the detection result is preferably a negative, positive, and type name of the detected pathogen or negative, positive, and One or more of the abbreviations of the type names of the detected pathogens.
  • the specific bioprobe is designed to develop color upon binding to its corresponding target product.
  • the probe is specifically bound to the target product, and then the substrate is subjected to color development treatment to obtain a visualization effect of genotyping.
  • the invention has the beneficial effects that the key information or the detection result or the abbreviation of the item of the visual biochip detection corresponding to the detection item can be directly displayed on the chip according to a specific hybridization reaction, antigen-antibody binding or other specific reaction.
  • the operation is simple, and the biochip detection result can be quickly obtained, and the observation result is simple and clear; the steps of searching for each detection result according to the preset setting are omitted, the detection result is more convenient to observe and read, and is not easy to be mistaken; and its unique display
  • the effect not only saves the result interpretation time, but also directly views the results at any time, which is convenient and concise.
  • Figure 1 is a schematic diagram showing the arrangement of a specific bioprobe in a biochip on a supporting chip substrate.
  • 16, 18, etc. in the figure are the type names of the HPV detection performed by the gene chip, and the corresponding ones in the figure are in the chip.
  • the spotted probe that is, the probe is directly spotted on the chip, the type aliases 16, 18, etc.; HC, PC is used for process quality control.
  • Both HC and PC are probes for universal templates, where XX is the positive control for color development and XX is the positive control for PCR amplification.
  • Figure 2 is a schematic diagram showing the results of HPV typing. Among them, the corresponding name is directly displayed to indicate that the detection result is positive, such as 16, 33 in the figure, and other blanks indicate that the detection result is negative. HC, PC is always displayed for process quality control.
  • FIG 3 is a schematic diagram of the results of protein chip detection. Among them, “sample 1” to “sample 15” are samples to be tested, and “quality control” is used for process control.
  • Figure 4 is a schematic diagram showing the results of detection of a polysaccharide chip. Among them, A, B, C, and D are respectively the names representing different samples to be detected.
  • Figure 5 is a schematic diagram showing the results of RNA chip detection.
  • the Chinese characters " ⁇ ”, “ ⁇ ”, “ ⁇ ”, “ ⁇ ” all represent the name of the sample to be inspected.
  • the probes are all purchased from Shanghai Biotech, and the spotting instrument is a crystal core PersonalArrayer 16 personal spotting instrument, purchased from Boao Biological Co., Ltd.
  • On-chip probe style setting According to the project requirements, set the corresponding program on the spotter software, that is, set the spotting form of the probe to the number and letter shown in Fig. 1.
  • HPV nucleic acid typing primers and probes including two sets of primers and 30 sets of specific probes, wherein two sets of primers are used for amplification of HPV sequences, one set of probes belongs to universal probes, as long as there is expansion
  • the specific probe can be specifically hybridized to develop color; the other probe is a hybridization reaction quality control probe, as long as the hybridization process is normal, the final display can be displayed; the remaining 28 sets of probes are used for the classification of 28 specific types respectively. , all purchased from Shanghai Biotech.
  • PCR amplification According to the reagents and nucleic acid samples in steps 1, 2, the PCR system was prepared, and a total of 20 ul system, including 19 ul of PCR reaction solution and 1 ul of template. Perform PCR amplification as follows:
  • Chip preparation see embodiment 1;
  • step 4 The amplification product in step 4 is added to the chip, and the hybridization reaction is carried out at a constant temperature of 40 degrees Celsius;
  • a chromogenic substrate is added for color development, and the chromogenic substrate is 3-amino-9-ethylcarbazole;
  • the capture antibody is immobilized on a solid support in the form of a pattern (including numbers and letters) to be displayed, and the solid support is a nitrocellulose membrane.
  • the capture antibody binds to a specific protein.
  • a chemically modified sugar molecule is attached to a reactive group, and then immobilized on a solid phase carrier in the form of an image to be displayed (including numbers and letters) to prepare a sugar probe, and the solid phase carrier is Glass slides.
  • the sugar probe on the chip specifically binds to the sample to be tested.
  • Fluorescent dye (Cy5) labeled target sample reacts with the sugar probe, and then rinses the already active chip with the rinse solution. The interference of excess fluorescein is removed and the specific signal emitted by the target bound to the chip is then detected by CCD photography. The name of the test result can be directly read from the captured image without providing an additional comparison table, and the display effect is as shown in FIG. 4.
  • RNA probe was prepared by immobilizing on a solid phase carrier using an Oligo probe in the form of an image to be displayed (including numbers and letters), and the solid phase carrier was a nitrocellulose membrane.
  • Sample processing reverse transcription of the extracted RNA, and amplification of the RNA by in vitro transcription-mediated linear amplification, and reverse transcription and fluorescent labeling of the cDNA amplification product.
  • the RNA probe on the chip specifically binds to the RNA sample to be detected.
  • the color development result is obtained by fluorescence microscopy, and the name of the desired sample can be directly read by the color development result, and the display effect is shown in FIG. 5.

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Abstract

本申请提供了一种可视化生物芯片，特异性的生物探针以图形的形式排布在支持性芯片基片上。本申请还提供了所述生物芯片的制备方法以及使芯片可视化的方法。

Description

可视化芯片及其制备方法和芯片可视化的方法
关联申请的交叉引用
本申请要求于2015年7月30日提交的申请号为201510460246.1的中国专利申请(该申请的发明名称为“可视化芯片及其制备方法和芯片可视化的方法”)的优先权。该申请的全部内容通过引用结合于本文中。
技术领域
本发明涉及一种芯片、尤其涉及一种生物芯片。本发明还涉及所述生物芯片的制备方法，以及一种芯片可视化的方法。
背景技术
芯片技术是一项新兴产业，主要分有基因芯片技术、倒装芯片技术、生物芯片技术、组织芯片技术、蛋白质芯片技术、DNA芯片技术、液相芯片技术、芯片封装技术等等。
基因芯片技术是同时将大量的探针分子固定到固相支持物上,借助核酸分子杂交配对的特性对DNA样品的序列信息进行高效的解读和分析。基因芯片又称DNA芯片(DNA chip)或DNA微阵列(DNA microarray)。其原理是采用光导原位合成或显微印刷等方法将大量特定序列的探针分子密集、有序地固定于经过相应处理的硅片、玻片、硝酸纤维素膜等载体上,然后加入标记的待测样品,进行多元杂交,通过杂交信号的强弱及分布,来分析目的分子的有无、数量及序列,从而获得受检样品的遗传信息(请参见《基因芯片技术及其应用》。嵇常宇，《黑龙江生态工程职业学院学报》，2，2014，23-24)。基因芯片能够同时平行分析数万个基因,进行高通量筛选与检测分析，解决了传统核酸印迹杂交技术操作复杂、自动化程度低、检测目的分子数量少等不足(请参见《基因芯片技术在临床微生物检验中的应用和优势》。汤桂丽，《中国药业》，5，2015，92-94)。
蛋白质芯片是一种高通量监测系统，通过靶分子和捕捉分子相互作用来监测蛋白分子之间的相互作用。捕获分子一般都预固定在芯片表面，由于抗体的高度特异性和与抗原强结合特性所以被广泛的用做捕获分子。蛋白芯片可用于蛋白表达谱、蛋白质磷酸化及蛋白质相互作用，免疫检测及酶活性测定，抗体筛选，蛋白质组研究，生物分子间的相互作用研究，蛋白质和小分子间的相互作用，药物靶标及其作用机理的研究，同时蛋白芯片也可 应用于疾病诊断，食品分析，毒理学中及卫生检验。在临床应用上，蛋白芯片主要表现在对疾病的诊断、疾病标志物发现、病程监控、治疗效果的评价等方面(请参见《蛋白芯片研究进展》。张爱英，尹成增等，《中国医学装备》，11，2014，150-151)。
多糖芯片是在基因芯片和蛋白芯片的基础上诞生的，其基本原理与基因芯片和蛋白芯片相似，都是基于物质之间的特异性作用。它是将多个不同结构的糖分子通过共价或非共价作用固定于经化学修饰的基质上，进而对蛋白质、糖蛋白等待测样品进行测试、分析的一类分析方法(请参见《糖芯片的检测及应用》。黄河，贾红英等，《化学通报》，5，2009，401-406)。糖芯片的研究，对于流行病的诊断监测、病原微生物内毒素的识别与鉴定以及常见的糖与靶蛋白特异性结合的研究具有重要的意义。
组织芯片(tissue chip)，也称组织微阵列(tissue microarrays)，是生物芯片技术的一个重要分支，是将许多不同个体组织标本以规则阵列方式排布于同一载玻片上，进行同一指标的原位组织学研究(请参见《组织芯片的应用及研究进展》。李小京，杨晓莉等，《实用医学杂志》，21，2014，857-859)。
RNA芯片是利用RNA探针杂交，能够快速高通量的获得与特定生物学过程或者疾病相关的一些RNA的表达变化，为相关疾病或生物学过程研究提供重要指导意义。RNA微阵列芯片逐渐被应用于各种肿瘤研究中，并呈现出巨大的应用价值(请参见《微小RNA微阵列芯片在肿瘤研究中的应用》。禚洪庆，张辉等，《中华实验外科杂志》，12，2011，2267-2268)。Tavazoie等使用微阵列芯片检测了乳腺癌MDA-MB-231细胞及容易转移到骨和肺的亚细胞株miRNA表达，发现了转移亚细胞总的一些RNA的低表达，为肿瘤检测及治疗提供理论依据(请参见《Endogenous human microRNAs that suppress breast cancer metastasis》.Tavazoie SF,Alarcon C,et al,nature 451,2008,147-152)。
芯片基片可用材料有玻片、硅片、瓷片、聚丙烯膜、硝酸纤维素膜和尼龙膜。为保证探针稳定固定于载体表面,需要对载体表面进行多聚赖氨酸修饰、醛基修饰、氨基修饰、巯基修饰、琼脂糖包被或丙烯酰胺硅烷化,使载体形成具有生物特异性的亲和表面。最后将制备好的探针固定到活化基片上。目前市面上最常见的是利用芯片点样仪将探针点在对应的芯片上，然后在检测的过程中通过杂交及底物显色后通过是否显色来判断检测结果；当同时检测的型别过多时，如hpv基因分型检测，可能同时涉及到近30种型别，当在芯片上杂交显色后，还需对照事先设定好的排版来确定具体每一个型别(来源于《上海地区人乳头瘤病毒基因芯片法分型检测的临床意义》。缪应新，甘洁民等，《检验医学》，6，2015，595-598)，比较复杂且容易出错，给判读结果带来不便。因此，本领域亟需一种能够将检测结果可视化的生物芯片。
发明内容
为了解决上述现有技术中存在的问题，本发明提供了一种可视化生物芯片，其特征在于，特异性的生物探针以图形的形式排布在支持性芯片基片上。
在本发明的一些优选的实施方式中，将生物探针以图形的形式排布的方法具体为：在点样仪(例如为晶芯PersonalArrayer 16个人点样仪，购自博奥生物有限公司生物)的点样仪配套软件上设置：从软件上设置成想要显示的图样形式，比如需要显示数字“6”，则在软件上设置打印模式为“6”的样式，然后通过点样仪将想要点样的探针直接打印在芯片上即可。又或者：在点样仪上以多个点的排列形式组成相应的图样，最终在点样仪点样时即可以多个点共同组成需要显示的图样形式。
在本发明中，所述可视化生物芯片利用了DNA杂交反应、DNA和RNA特异性杂交、RNA和cDNA特异性杂交、DNA和cDNA特异性杂交、抗原-抗体特异性结合、DNA-DNA结合蛋白特异性结合、RNA-RNA结合蛋白特异性结合、抗体-抗体结合蛋白(例如G蛋白-抗体Fc片段)特异性结合、生物素-链霉亲和素特异性结合中的一种或多种，或者其他特异性反应中的一种或多种。
在本发明的一个优选的实施方式中，所述图形的形式为数字、字母、汉字、标志性符号或标志性图案，或者其任意组合的形式。所述字母优选为英语字母。
在本发明的一个优选的实施方式中，所述生物芯片为基因芯片、蛋白质芯片、RNA芯片、多糖芯片、组织芯片和神经元芯片中的一种或多种，优选为基因芯片和/或蛋白质芯片。
需要注意的是，本发明所述的基因芯片与DNA芯片等价。即所述基因芯片能检测任何DNA片段，而不仅仅是基因片段。
在本发明的一个优选的实施方式中，所述特异性的生物探针为特异性的DNA、RNA、多糖和蛋白质中的一种或多种。
在本发明的一个优选的实施方式中，所述特异性的生物探针为寡核苷酸、cDNA、RNA、抗体、抗原、抗体结合蛋白、寡核苷酸的片段、cDNA的片段、RNA的片段、抗体的片段、抗原的片段和抗体结合蛋白的片段中的一种或多种。
在本发明的一个优选地实施方式中，所述可视化生物芯片中还设置有作为对照的探针，优选设置有作为阳性对照和阴性对照的探针，尤其优选设置有作为阳性对照的探针。所述对照可以为对于显色的对照和/或对于PCR扩增的对照。
在本发明的一个优选的实施方式中，所述图形的形式表示被所述可视化生物芯片检测 的项目的关键信息或者检测结果或者被所述可视化生物芯片检测的项目的关键信息或者检测结果的简称，所述关键信息优选为项目名称、被检测主体的年龄和物种名称中的一种或多种，所述检测结果优选为阴性、阳性和被检测的病原体的型别名称或阴性、阳性和被检测的病原体的型别名称的简称中的一种或多种。
在本发明的一个优选的实施方式中，所述特异性的生物探针被设计为和与其对应的目标产物结合后显色。这样，在显色处理后可直接读取该生物芯片的检测结果。
在本发明的一些优选的实施方式中，所述显色为化学显色和/或荧光显色。在本发明的一些更加优选的实施方式中，所述显色为化学显色。在本发明的另一些更加优选的实施方式中，所述显色为荧光显色。
在本发明的一些更加优选的实施方式中，所述显色为辣根过氧化物酶在显色底物3-氨基-9-乙基咔唑(3-amino－9－ethylcarbazole；AEC)的作用下，失去电子而呈现出颜色变化和沉积，形成稳定性红色不溶性产物而达到显色效果。
本发明的另一个目的在于，提供制备上述可视化生物芯片的方法，其包括如下步骤：
1)制备与待检测样品相应的特异性的生物探针；
2)利用点样仪来将步骤1)制得的特异性的生物探针以图形的形式打印在支持性芯片基片上，制得可视化芯片。
在本发明的一个优选的实施方式中，制备上述可视化生物芯片的方法包括如下步骤：
1)制备与待检测样品相应的特异性的生物探针；
2)在与点样仪相连接的终端系统(例如电脑系统、手机系统等)上对点样仪设置点样的图形形式，
3)用点样仪将步骤1)制得的特异性的生物探针在支持性芯片基片上按步骤2)中的设置打印成图形的形式，制得可视化芯片。
在本发明的另一个优选的实施方式中，制备上述可视化生物芯片的方法包括如下步骤：
1)制备与待检测样品相应的特异性的生物探针；
2)在点样仪上设置点样的图形形式；
3)用点样仪将步骤1)制得的特异性的生物探针在支持性芯片基片上按步骤2)中
的设置打印成图形的形式，制得可视化芯片。
本发明的再一个目的在于，提供一种生物芯片可视化的方法，包括如下步骤：
1)根据上述方法制备可视化生物芯片，其中，所述图形的形式表示被所述可视化生物芯片检测的项目的关键信息或者检测结果或者被所述可视化生物芯片检测的项目的关 键信息或者检测结果的简称；
2)将待检测样品与可视化生物芯片上的探针结合并显色，在芯片上直接读取被所述可视化生物芯片检测的项目的关键信息或者检测结果或者被所述可视化生物芯片检测的项目的关键信息或者检测结果的简称，其中，所述显色优选为化学显色和/或荧光显色。
在本发明的一个优选的实施方式中，所述图形的形式为数字、字母、汉字、标志性符号或标志性图案，或者其任意组合的形式。在本发明的一个优选的实施方式中，所述生物芯片为基因芯片、蛋白质芯片、RNA芯片、多糖芯片、组织芯片和神经元芯片中的一种或多种。
在本发明的一个优选的实施方式中，所述特异性的生物探针为特异性的DNA、RNA、多糖或蛋白质中的一种或多种。
在本发明的一个优选的实施方式中，所述特异性的生物探针为寡核苷酸、cDNA、RNA、抗体、抗原、抗体结合蛋白，或者寡核苷酸、cDNA、RNA、抗体、抗原、抗体结合蛋白的片段中的一种或多种。
在本发明的一个优选的实施方式中，所述图形的形式表示被所述可视化生物芯片检测的项目的关键信息或者检测结果或者被所述可视化生物芯片检测的项目的关键信息或者检测结果的简称，所述关键信息优选为项目名称、被检测主体的年龄、物种名称中的一种或多种，所述检测结果优选为阴性、阳性和被检测的病原体的型别名称或阴性、阳性和被检测的病原体的型别名称的简称中的一种或多种。
在本发明的一个优选的实施方式中，所述特异性的生物探针被设计为与其对应的目标产物结合后显色。
在本发明的一个优选的实施方式中，探针与目的产物的特异性结合，再通过底物显色处理而得到基因分型的可视化效果。
本发明的有益效果在于：能够根据特定的杂交反应、抗原-抗体结合或者其他特异性反应在芯片上直接显示出与检测项目相对应的可视化生物芯片检测的项目的关键信息或者检测结果或者其简称，操作简单，能够快速得到生物芯片检测结果，结果观察简单明了；省去了根据预先设置来查找各检测结果的步骤，检测结果更方便观察读取，且不容易出错；还有其独特的显示效果不仅节省结果判读时间，还能随时直接查看结果，方便简洁。
附图说明
图1为可视化生物芯片中特异性的生物探针在支持性芯片基片上的排布示意图。其中，图中的16、18等为该基因芯片进行的HPV检测的型别名称，本图中对应的是在芯片 上点样的探针，即把探针在芯片上直接点样成分型的型别名16、18等；HC、PC用于过程质控。HC和PC均为针对通用模板的探针，其中，XX是显色的阳性对照，XX是PCR扩增的阳性对照。
图2为HPV分型检测结果示意图。其中，直接显示出对应的名称表示检测结果为阳性，如图中的16，33等，其他空白处即表示检测结果阴性。HC、PC始终显示，用于过程质控。
图3为蛋白质芯片检测结果示意图。其中“样1”至“样15”分别为待测样品，“质控”用于过程控制。
图4为多糖芯片检测结果示意图。其中，A、B、C、D分别为代表待检测的不同的样品的名称。
图5为RNA芯片检测结果示意图。其中汉字“一”、“二”、“三”、“四”均代表待检的样本名称。
具体实施方式
以下结合非限制性的具体实施例来对本发明进行进一步的说明。
实施例1基因芯片的制备
1.探针及设备的采购：探针均采购上海生工生物，点样仪为晶芯PersonalArrayer 16个人点样仪，购自博奥生物有限公司生物。
2.芯片上探针样式的设置：根据项目需求，在点样仪软件上设置相应的程序，即把要点样的探针的点样形式设置为图1中所示的数字和字母。
3.点样：把需要点样的膜放置在点样仪的膜放置处，根据设定进行点样，如图1，并进行烘干处理。即获取了对应的芯片。
探针的点样：利用点样仪设置程序将探针溶液以对应检测项名称点样在芯片上如图1，并烘干固定。
实施例2人乳头瘤病毒(HPV)核酸分型(28型)的检测
1.HPV核酸分型引物及探针的购买：包括两组引物和30组特异性探针，其中两组引物用于HPV序列的扩增，其中一组探针属于通用探针，只要存在扩增即可特异性杂交而显色；另一组探针为杂交反应质控探针，只要杂交过程正常，最终即可显示；其余28组探针分别用于28个特异性型别的分型，均采购自上海生工生物。
2.HPV核酸样本的提取，所述HPV核酸样本是从江苏省某医院HPV患者的宫颈脱落细胞标本提取的。
3.PCR扩增：根据步骤1，2中的试剂及核酸样本进行PCR体系配制，共20ul体系，包括19ul的PCR反应液和1ul的模板。按照如下程序进行PCR扩增：
50℃2min,94℃4min；
94℃30sec,60℃45sec,72℃20sec,35cycles；
4℃hold.
扩增完成后保存待用。
4.芯片的准备：见实施例1；
5.将步骤4中的扩增产物加入到芯片中，在40摄氏度恒温环境下进行杂交反应；
6.杂交反应结束后，加入显色底物进行显色处理，显色底物为3－氨基－9－乙基咔唑；
7.显色完成后显示效果如图2，可直接读取检测结果，无需再提供另外的对照表。
实施例3蛋白质芯片的制备及检测
1.蛋白质芯片的制备：将捕获抗体以需要显示的图样形式(包括数字和字母)固定在固相载体上，固相载体为硝酸纤维素膜。
2.样品的处理：对样品进行蛋白生物素标记，将活化的生物素与蛋白的氨基共价偶联。
3.生物素标记后的样品加入到芯片上一起孵育反应后，捕获抗体可结合特异性的蛋白。
4.加入HRP-链霉亲和素与化学发光底物、或者荧光染色剂一链霉亲和素结合作为信号检测，反应10分钟后即可在芯片上观察对应的实验结果。显色效果如图3所示，可直接读取检测结果，无需再提供另外的对照表。
实施例4多糖芯片的制备及检测
1.糖芯片的制备：将用化学修饰过的糖分子连上活性基团，然后以需要显示的图像形式(包括数字和字母)固定在固相载体上制成糖探针，固相载体为玻璃载片。
2.样品的处理：对样品进行荧光染料Cy5荧光素标记。
3.将待检测样品加入到芯片上一起孵育反应后，芯片上的糖探针与待检样品特异性结合。
4.荧光染料(Cy5)标记的靶样与糖探针反应，再用冲洗液冲洗已经发生作用的芯片, 除去多余荧光素的干扰,然后用CCD照相技术对结合到芯片上的靶样所发出的特定信号进行检测。从拍出的图像中即可直接读取检测结果的名称，无需提供额外的对照表，显示效果如图4所示。
实施例5RNA芯片的制备及检测
1.RNA芯片的制备：使用Oligo探针以需要显示的图像形式(包括数字和字母)固定在固相载体上制成RNA探针，固相载体为硝酸纤维素膜。
2.样品的处理：对提取的RNA进行逆转录，再通过进行体外转录介导的线性扩增法对RNA进行扩增，进行对cDNA扩增产物进行反转录和荧光标记。
3.将待检测样品加入到芯片上一起孵育反应后，芯片上的RNA探针与待检的RNA样品特异性结合。
4.荧光标记的样本与RNA探针杂交反应后，通过荧光显微技术获取显色结果，通过显色结果可直接读取希望检测样本的名称，显示效果如图5所示。
应当注意的是，以上所述的实施例仅用于解释本发明，并不构成对本发明的任何限制。通过参照典型实施例对本发明进行了描述，但应当理解为其中所用的词语为描述性和解释性词汇，而不是限定性词汇。可以按规定在本发明权利要求的范围内对本发明作出修改，以及在不背离本发明的范围和精神内对本发明进行修订。尽管其中描述的本发明涉及特定的方法、材料和实施例，但是并不意味着本发明限于其中公开的特定例，相反，本发明可扩展至其他所有具有相同功能的方法和应用。

Claims (10)

  1. 一种可视化生物芯片，其特征在于，特异性的生物探针以图形的形式排布在支持性芯片基片上。
  2. 根据权利要求1所述的可视化生物芯片，其特征在于，所述图形的形式为数字、字母、汉字、标志性符号或标志性图案，或者其任意组合的形式。
  3. 根据权利要求1或2所述的可视化生物芯片，其特征在于，所述生物芯片为基因芯片、蛋白质芯片、RNA芯片、多糖芯片、组织芯片和神经元芯片中的一种或多种，优选为基因芯片和/或蛋白质芯片。
  4. 根据权利要求1-3中任意一项所述的可视化生物芯片，其特征在于，所述特异性的生物探针为特异性的DNA、RNA、多糖和蛋白质中的一种或多种。
  5. 根据权利要求4所述的可视化生物芯片，其特征在于，所述特异性的生物探针选自寡核苷酸、cDNA、RNA、抗体、抗原、抗体结合蛋白、寡核苷酸的片段、cDNA的片段、RNA的片段、抗体的片段、抗原的片段和抗体结合蛋白的片段中的一种或多种。
  6. 根据权利要求1-5中任意一项所述的可视化生物芯片，其特征在于，所述图形的形式表示被所述可视化生物芯片检测的项目的关键信息或者检测结果或者被所述可视化生物芯片检测的项目的关键信息或者检测结果的简称，所述关键信息优选为项目名称、被检测主体的年龄和物种名称中的一种或多种，所述检测结果优选为阴性、阳性和被检测的病原体的型别名称以及阴性、阳性和被检测的病原体的型别名称的简称中的一种或多种。
  7. 根据权利要求1-6中任意一项所述的可视化生物芯片，其特征在于，所述特异性的生物探针被设计为与其对应的目标产物结合后显色，所述显色优选为化学显色和/或荧光显色。
  8. 制备根据权利要求1-7中任意一项所述的可视化生物芯片的方法，包括如下步骤：
    1)制备与待检测样品相应的特异性的生物探针；
    2)利用点样仪来将步骤1)制得的特异性的生物探针以图形的形式打印在支持性芯片基片上，制得可视化芯片。
  9. 一种生物芯片可视化的方法，包括如下步骤：
    1)根据权利要求8所述的方法制备可视化生物芯片，其中，所述图形的形式表示被所述可视化生物芯片检测的项目的关键信息或者检测结果或者被所述可视化生物芯片检测的项目的关键信息或者检测结果的简称；
    2)将待检测样品与可视化生物芯片上的探针结合并显色，在芯片上直接读取被所述可视化生物芯片检测的项目的关键信息或者检测结果或者被所述可视化生物芯片检测的 项目的关键信息或者检测结果的简称，其中，所述显色优选为化学显色和/或荧光显色。
  10. 根据权利要求9所述的方法，其特征在于，所述图形的形式为数字、字母、汉字、标志性符号或标志性图案，或者其任意组合的形式，所述生物芯片为基因芯片、蛋白质芯片、RNA芯片、多糖芯片、组织芯片和神经元芯片中的一种或多种。
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WO2017096561A1 (zh) * 2015-12-09 2017-06-15 杨华卫 用于生物芯片的印刷方法及其应用
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