1、生物芯片技术生物芯片技术何为生物芯片何为生物芯片?生物芯片主要指通过平面微细加工技术在固体芯片生物芯片主要指通过平面微细加工技术在固体芯片表面构建的微流体分析单元和系统，以实现对细胞表面构建的微流体分析单元和系统，以实现对细胞、蛋白质、核酸以及其他生物组分的准确、快速、蛋白质、核酸以及其他生物组分的准确、快速、大信息量的检测。大信息量的检测。他是继大规模集成电路之后的又一次具有深远意义他是继大规模集成电路之后的又一次具有深远意义的科学技术革命。的科学技术革命。生物芯片分类生物芯片分类D N A C h ip s P ro te in C h ip sL a b C h ip sB io c h
2、 ip s蛋白芯片蛋白芯片(Protein Chips)a microarray-based high-throughput protein assay methodChemiluminescence or Fluorescence based detection methods can be used to visualize bound antibodies.蛋白芯片应用蛋白芯片应用 Diagnostic immunoassay that allows the simultaneous high-throughput analysis of known autoantigens.In or
3、der to quantify antibodies in the sera of patients with autoimmune diseases,Recombinant antigens and control proteins were immobilized on slides with reactive aldehyde groups as replicas in serial dilutions of the various antigens thereby allowing accurate determination of autoantibody titer using m
4、inimal amounts of serum.Miniaturized and highly paralleled immunoassays like these will reduce cost by decreasing reagent consumption and improve performance by greatly increasing the number of assays that can be performed with a single serum sample.Protein-Protein Interaction.DNA-Protein Interactio
5、n 基因芯片(Gene chip)技术是指通过微阵列(Microarray)技术将高密度DNA片段阵列通过高速机器人或原位合成方式以一定的顺序或排列方式使其附着在如玻璃片等固相表面，以荧光标记的DNA探针，借助碱基互补杂交原理，进行大量的基因表达及监测等方面研究的最新革命性技术基因芯片基因芯片（Gene chip）Types of DNA ChipsExpression ChipsGenomic ChipsSequencing ChipsDNA ChipsComparison of DNA Chip Technologies Sensitivity of DNA chip based assa
6、ys is a function of:Probe and target DNA/RNA(Complexity)Chip surface(autofluorescence&non-spec.bkg)Attachment chemistry/methodology(hyb.efficiency&crosshyb.)Hybridization efficiency(lots of factors)Detection technology(signal type,efficiency,noise)Why Genomic Biochips?Screen specimens to determine g
7、ene copy number changes Establish correlations between gene copy number changes and disease biology Determine the interaction of multiple genes on the initiation and progression of disease Accelerate development of products for genomic disease management to guide therapeutic intervention Combined wi
8、th expression chips,gives full understanding of disease processDNA Chip Technology Solid support(glass,plastic,metal,silicon)Miniaturized array of DNA(genetic material)Work on the biochemical principle of DNA/DNA hybridization Hybridized probes(DNA molecules)are fluorescently labeledAssay Formats Ar
9、eas of Biochip Applications Academic research of genetic diseasesCancerPrenatal geneticsGeneral genetic diseases Infectious diseases Drug discovery Animal farming/veterinary Industrial(fermentation,corrosion)Environmental应用之一 基因表达谱基因表达谱（gene expression pattern）Research Use.Clinical Diagnostic Use.Re
10、search UseFrom Sequence to function 计算计算Ratio 值值(=Cy3/Cy5)在在 0.5-2.0 之外的定义为在两样本中有之外的定义为在两样本中有明显差异表达。进而获取初步功能信息。明显差异表达。进而获取初步功能信息。ClusteringResearch UseFrom Sequence to function 庞大的数据库庞大的数据库 数学模型数学模型 可以大大推进包括人类（后）基因组计划在内可以大大推进包括人类（后）基因组计划在内的各类基因组研究，通过比较不同个体或物的各类基因组研究，通过比较不同个体或物种之间以及同一个体在不同生长发育阶段、种之间以
11、及同一个体在不同生长发育阶段、正常和疾病状态下基因及其表达的差异，寻正常和疾病状态下基因及其表达的差异，寻找和发现新的基因，研究基因的功能以及生找和发现新的基因，研究基因的功能以及生物体在进化、发育、遗传等过程中的规律。物体在进化、发育、遗传等过程中的规律。基因芯片可为研究不同层次多基因协基因芯片可为研究不同层次多基因协同作用的生命过程提供手段。同作用的生命过程提供手段。将在研究人类重大疾病如癌症、心血将在研究人类重大疾病如癌症、心血管疾病等相关基因及作用机理方面发管疾病等相关基因及作用机理方面发挥巨大的作用。挥巨大的作用。Clinical Diagnostic Use Academic re
12、search of genetic diseases Cancer Prenatal genetics General genetic diseases Infectious diseases HAV、HBV、HCV.HIV One Disease=One Expression Pattern 生物芯片能为现代医学科学及医学诊断学生物芯片能为现代医学科学及医学诊断学的发展提供强有力的手段，促进医学从的发展提供强有力的手段，促进医学从“系统、血管、组织和细胞层次系统、血管、组织和细胞层次”(通常称通常称之为之为“第二阶段医学第二阶段医学”)向向“DNA、RNA、蛋白质及其相互作用层次蛋白质及其相
13、互作用层次”(第三阶段医学第三阶段医学)过 渡，使 之 快 速 进 入 实 际 应 用。过 渡，使 之 快 速 进 入 实 际 应 用。Clinical Diagnostic UsePrototype AmpliOnc I Biochip 12345678910111213141516171819202122XYPDGFBEGFR1PDGFRAMETFGFR2WNT1MYBHER2YES1HRAS1CND1RAF1GLIMYCMDM220q13RELMYCL1FGRFESABLINT2PIK3CANMYCAKT2FGFR1JUNBAKT1KRAS2CDK4ARRDA Protocol RNA
14、extraction and cDNA preparation from archived tissue specimens(tester and driver)Generation of amplified cDNA fragments(amplicons)Subtractive hybridization of amplicons Enrichment of cDNA fragments from differentially expressed genes Microarray 用于验证用于验证RDA Shotgun subcloning of RDA fragments Picking
15、 transformed libraries for long-term propagation Amplification of RDA inserts in 96-well plate format for arraying Hybridization of cDNA amplicons to microarrays References aboutCoupling of RDA&Microarray Schena,M.et al.(2019)Science,270,467 Lockhart,et al.(2019).Nature Biotechnology,14,1675 DeRisi,
16、et al.(2019).Nature Genet.,14,457.Microarray 还可用于验证还可用于验证 SSH Differential Display PCR应用之三应用之三SNPs&STRs analysis Single Nucleotide Polymorphisms Short Tandem Repeats Polymerase LigaseSNPs Typing by LigaseSNPs Typing by Polymerase（1）SNPs Typing by Polymerase（2）STRs Typing（1）STRs Typing（2）STRs Typing（
17、3）应用之四应用之四 LCM俘获细胞的基因表达分析俘获细胞的基因表达分析LCM:Laser Capture MicrodissectionGene expression profiles of laser-captured adjacent neuronal subtypesLarge-and Small-sized Dorsal Root Ganglion(DRG)Rat Neurons,Nissl stainedLarge DRG Neurons:myelinated fast-conductingtransmit mechanosensory informationSmall DRG N
18、eurons:unmyelinatedslow-conductingtransmit nociceptive informationGene expression profiles of laser-captured adjacent neuronal subtypesDorsal Root Ganglion(DRG)Rat Neurons,Nissl stainedcDNA microarray expression patterns of small(S)and large(L)neuronsmRNA enriched in large DRG neuronsmRNA enriched i
19、n small DRG neurons放射性原位杂交验证结果放射性原位杂交验证结果应用之五应用之五Development of therapeutic Drugs drug target discovery evaluation of animal models of human disease test for drug efficacy test for drug specificity test for drug toxicity 在预防医学方面，可以使人们尽早地认识自在预防医学方面，可以使人们尽早地认识自身潜在的疾病，并实施有效的防治措施身潜在的疾病，并实施有效的防治措施 法医学中，进
20、行基因指纹快速识别，以及亲法医学中，进行基因指纹快速识别，以及亲子鉴定子鉴定 监测流行病和传染病扩散监测流行病和传染病扩散 监测有害微生物的发生和传播监测有害微生物的发生和传播 农、林、牧、鱼等产业的品种改良和农、林、牧、鱼等产业的品种改良和病虫防治病虫防治生物芯片制作方法分类生物芯片制作方法分类 片片基基 显显色色及及检检测测方方式式 原原位位合合成成 (i in n s si it tu u s sy yn nt th he es si is s)钢钢性性片片基基如如玻玻片片、半半导导体体硅硅片片等等 荧荧光光，激激光光共共聚聚焦焦扫扫描描、定定量量分分析析；生生物物传传感感器器等等 预预
21、先先合合成成后后点点样样 (o of ff f-c ch hi ip p s sy yn nt th he es si is s)玻玻片片、薄薄膜膜片片基基如如 N NC C、N Ny yl lo on n 膜膜等等 荧荧光光，激激光光共共聚聚焦焦扫扫描描、定定量量分分析析；生生物物传传感感器器等等 原位合成(In Situ Synthesis)Light directed oligonucleotide synthesis.A s o l i d s u p p o r t i s derivatized with a covalent linker molecule terminated
22、w i t h a p h o t o l a b i l e protecting group.Light is directed through a mask to deprotect and activate s e l e c t e d s i t e s,a n d protected nucleotides couple to the activated sites.The process is repeated,activating different sets of sites and coupling different bases allowing arbitrary D
23、NA probes to be constructed at each site.预先合成后点样(off-chip synthesis)接触式点样接触式点样 非接触式点样非接触式点样接触式点样接触式点样：接触式点样：Chipmaker Pin（Telechem专利）专利）ArrayIt ChipMaker Pins Developed in conjunction with Stanford University Single dip in sample multiple spots Fine(EDM)slot in tip of stainless steel pin Take-up vol
24、ume 250 nL(saves sample)Spot volume of 100 to 500 pL 100 m to 250 m spot size(high density arrays)Requires 10 to 30 pre-prints to“prime”pin非接触式点样：非接触式点样：nQUAD TechnologyCharacteristics of nQUAD Technology Wide dispense range Low nanoliter to high microliter Excellent linearity Precise and accurate C
25、Vs typically less than 10%Precision less than 5%Non-contact dispense mechanism Easier mechanical alignment(384,1536,)“On-the-fly”printing possible Capable of dispensing onto membranes or slides Multiple liquid handling modes Aspirate/Dispense Continuous Reagent Dispensing生物芯片点样系统必备性能生物芯片点样系统必备性能 多针头
26、自动同时取样，取样板为多针头自动同时取样，取样板为96孔或孔或384孔板孔板 单针取样量为单针取样量为250到到500nL，每次点样量为，每次点样量为100到到150pL 点样直径为点样直径为100到到150um，系统点阵分辨率为，系统点阵分辨率为10um 可程控进行连续点样操作可程控进行连续点样操作 固相介质如玻璃片须通过适当机制进行位置固定固相介质如玻璃片须通过适当机制进行位置固定 点样针尖必须具备单次取样连续点样的性能点样针尖必须具备单次取样连续点样的性能Cartesian-PixSys Series合成后点样板合成后点样板-Cartesian Tech.PixSys Series 特点
27、：特点：Combines with TWO dispensing technologies Contact and Non-contact printing mode ChipMaker quill pins and nQUAD dispensers Pin array Spot Size:75 to 200um nQUAD quantity:10nL 10 m positioning resolution 50 slide capacityMethods in Microarray Detection CCD Confocal Scanning MicroscopyGeneral Scann
28、ing Inc.ScanArray 5000/4000/3000SCANARRAY3000/4000/5000特点：特点：全自动检测及分析系统全自动检测及分析系统 适用于适用于DNA微阵列生物芯片应用微阵列生物芯片应用 共聚焦激光扫描探测系统共聚焦激光扫描探测系统 灵敏度最高：灵敏度最高：0.5分子分子/u2 极高分辨率：极高分辨率：5um像素点像素点 多种激发及检测通道多种激发及检测通道 适用于多种生物标记荧光探针适用于多种生物标记荧光探针GENERAL SCANNINGSummary(2)：生物芯片应用优势：生物芯片应用优势 Experiments can be performed at
29、a faster rate Users can simultaneously screen hundreds to thousands of targets(104-106)in a single experiment Analysis of biochips can be automated through hardware/software Less reagents are needed for experiments Low cost per target with biochipsSummary(3)：生物芯片现有技术及发展趋势生物芯片现有技术及发展趋势 Photolithography(Affymetrix)Drop-on-demand(Cartesian)Surface contact(TeleChem,Cartesian)Greater density Accelerated automation Cost reduction
