生物芯片技术,生物芯片技术,生物芯片原理和应用,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 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、 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 的科学技术革命。,生物芯片分类,蛋白芯片(Protein Chips),a microarray-based high-throughput protein assay method,Chemiluminescence or Fluorescence based detection methods can be used to visualize bound antibodies.,蛋白芯片应用,Diagnostic immunoassay that allows the simultaneous high-throughput analysis of known autoantigens. In order 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 minimal 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 Interaction,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（Gene chip）,基因芯片发展历史,Southern color composite of red, blue and green image,This biochip contains all genomic regions that have been reported to be amplified in cancers.,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,X,Y,Oncogene Targets On the AmpliOnc I Biochip,PDGFB,EGFR1,PDGFRA,MET,FGFR2,WNT1,MYB,HER2,YES1,HRAS1,CND1,RAF1,GLI,MYC,MDM2,20q13,REL,MYCL1,FGR,FES,ABL,INT2,PIK3CA,NMYC,AKT2,FGFR1,JUNB,AKT1,KRAS2,CDK4,AR,RDA Protocol,RNA 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RDA,Shotgun subcloning of RDA fragments Picking 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. (1995)Science, 270, 467 Lockhart,et al.(1996).Nature Biotechnology, 14, 1675 DeRisi, et al. (1996). Nature Genet., 14, 457.,Microarray 还可用于验证,SSH Differential Display PCR,应用之三SNPs & STRs analysis,Single Nucleotide Polymorphisms Short Tandem Repeats Polymerase Ligase,SNPs Typing by Ligase,SNPs Typing by Polymerase（1）,SNPs Typing by Polymerase（2）,STRs Typing（1）,STRs Typing（2）,STRs Typing（3）,应用之四 LCM俘获细胞的基因表达分析,LCM: Laser Capture Microdissection,原理,Gene expression profiles of laser-captured adjacent neuronal subtypes,Differential Gene Expression between Large- and Small-sized Dorsal Root Ganglion (DRG) Rat Neurons, Nissl stained Large DRG Neurons: myelinated fast-conducting transmit mechanosensory information Small DRG Neurons: unmyelinated slow-conducting transmit nociceptive information,Gene expression profiles of laser-captured adjacent neuronal subtypes,Dorsal Root Ganglion (DRG) Rat Neurons, Nissl stained,cDNA microarray expression patterns of small (S) and large (L) neurons,mRNA enriched in large DRG neurons,mRNA enriched in small DRG neurons,放射性原位杂交验证结果,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身潜在的疾病，并实施有效的防治措施 法医学中，进行基因指纹快速识别，以及亲子鉴定,其它应用,监测流行病和传染病扩散 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病虫防治,其它应用,生物芯片制作技术 及相关产品介绍,生物芯片制作方法分类,原位合成(In Situ Synthesis),Light directed oligonucleotide synthesis. A solid support is derivatized with a covalent linker molecule terminated with a photolabile protecting group. Light is directed through a mask to deprotect and activate selected sites, and protected nucleotides couple to the activated sites. The process is repeated, activating different sets of sites and coupling different bases allowing arbitrary DNA probes to be constructed at each site.,预先合成后点样(off-chip synthesis),接触式点样 非接触式点样,接触式点样,Best!,接触式点样：Chipmaker Pin （Telechem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 volume 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：nQUAD Technology,Characteristics of nQUAD Technology,Wide dispense range Low nanoliter to high microliter Excellent linearity Precise and accurate CVs 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,多针头自动同时取样，取样板为96孔或384孔板 单针取样量为250到500nL，每次点样量为10