第9章-微流控分析芯片-制作

第九章 微流控分析芯片Do you know?Biochip生物芯片 Lab-on-a-Chip 芯片实验室Labchip Microfluidic Chip 微流控芯片 Micro Total Analysis System(MicroTAS,TAS)微全分析系统 Research into miniaturization is primarily driven by the need to reduce costs by reducing the consumption of expensive reagents and by increasing throughput and automation.For example,most are aware of the increasing cost of health care,driven in part by the cost of implementing the latest diagnostic assays.These assays,which are usually performed in microtiter plates that consume hundreds of microliters of reagents,would benefit from the use of microfabricated arrays of nanoliter volume vials.By reducing reagent consumption by a factor of 103104,these devices could provide dramatic savings for the repetitive assays often performed in diagnostic laboratories.Why miniaturization？Anal.Chem.2000,72,330A-335AIn the same way that integrated circuits allowed for the miniaturization of computers from the size of a room to the size of a notebook,miniaturization has the potential to shrink a room full of instruments into a compact lab-on-a-chip.Anal.Chem.2000,72,330A-335A尺寸效应（cm100m）尺寸1/100 分子扩散时间1/10,000(1h0.36s)体积1/1,000,000试剂用量1t1g,mlnl 传热速度1500oC/s由此将带来：方法上的变革 理论上的突破 还有巨大的经济和社会效益阵列芯片微流控芯片微流控分析芯片 微流控分析芯片目的是通过化学分析设备的微型化与集成化，最大限度地把分析实验室的功能转移到便携的芯片中。微流控分析芯片通过微机电加工技术把整个实验室的功能，包括采样、稀释、加试剂、反应、分离、检测等集成在几平方厘米的微流控芯片上，且可多次使用，因而极大地减少了样品和分析试剂的用量，降低了分析的成本，加快了分析的速度，具有广泛的适用性。参考文献 D.Figeys,D.Pinto,Lab-on-a-Chip:A Revolution in Biological and Medical Sciences,Analytical Chemistry,2000,72,330A D.R.Reyes,D.Iossifidis,P.A.Auroux,A.Manz,Micro Total Analysis Systems.1.Introduction,Theory,and Technology,Analytical Chemistry,2002,74,2623 P.A.Auroux,D.Iossifidis,D.R.Reyes,A.Manz,Micro Total Analysis Systems.2.Analytical Standard Operations and Applications,Analytical Chemistry,2002,74,2637 T.Vilkner,D.Janasek,A.Manz,Micro Total Analysis Systems.Recent Developments,Analytical Chemistry,2004,76,3373 马立人，蒋中华，生物芯片，化学工业出版社，北京，2002 方肇伦，微流控分析芯片，科学出版社，北京，2003 目 录9-1 发展历史9-2 相关理论9-2-1 基本概念9-2-2 理论描述和模拟9-3 基础技术9-3-1 微制造9-3-2 界面和连接9-3-3 流体控制9-4 单元操作 9-4-1 样品制备 9-4-2 注射 9-4-3 流体和颗粒操作 9-4-4 反应器和混合器 9-4-5 分离 9-4-6 检测9-5 应用9-5-1 细胞培养和操作9-5-2 免疫分析和临床检验9-5-3 蛋白质分析9-5-4 核酸分析目 录9-1 发展历史9-2 相关理论9-2-1 基本概念9-2-2 理论描述和模拟9-3 基础技术9-3-1 微制造9-3-2 界面和连接9-3-3 流体控制9-4 单元操作 9-4-1 样品制备 9-4-2 注射 9-4-3 流体和颗粒操作 9-4-4 反应器和混合器 9-4-5 分离 9-4-6 检测9-5 应用9-5-1 细胞培养和操作9-5-2 免疫分析和临床检验9-5-3 蛋白质分析9-5-4 核酸分析The Early Days:1975-1989 The first analytical miniaturized deviceA gas chromatographic air analyzer fabricated on a silicon wafer Terry,S.C.Ph.D.Thesis,Stanford,Stanford,CA,1975Terry,Stephen C.;et al.IEEE Transactions on Electron Devices,1979,ED-26(12),1880 A miniature gas anal.system based on the principles of gas chromatog.The major components are fabricated in Si using photolithog.and chem.etching techniques,which allows size redns.of nearly 3 orders of magnitude compared to conventional laboratory instruments.consists of a sample injection valve,a 1.5-m-long capillary column.A thermal conductivity detector fabricated on a separate silicon wafer.sepns.of gaseous hydrocarbon mixts.are performed in 10 s.9-1 发展历史 Photograph of a gas chromatograph integrated on a planar silicon wafer fabricated by Terry and co-workers at Stanford University.However,the response of the scientific community to this first silicon chip device was virtually none,presumably because of the lack of technological experience(of the separation scientists)to deal with this kind of device.the research work related to miniaturization on silicon focused on the fabrication of components such as micropumps,microvalves,and chemical sensors.The Renaissance:1990-1993 the reemergence of silicon-based analyzersDesign of an open-tubular column liquid chromatograph using silicon chip technologyManz,A.;et al.Sensors and Actuators,B:Chemical(1990),B1(1-6),249 A novel concept of high pressure liquid chromatog.a silicon chip with an open-tubular column and a conductometric detector.A 55mm chip containing an open-tubular column of 6 m2m15cm was fabricated,which has theor.separation efficiencies of 8000 and 25,000 plates in 1 and 5 min,resp.The total column volume is 1.5 nL and the detection cell volume 1.2 pL.Micrograph of Liquid Chromatograph chip manufactured by Manz and co-workers at Hitachi Ltd.The concept of miniaturized total chemical analysis system or TAS was proposed by Manz et al.the main reason for miniaturization was therefore to enhance the analytical performance of the device rather than to reduce its size.it was also recognized that a small size presented the advantage of a smaller consumption of carrier,reagent,and mobile phase.Growing to Critical Mass:1994-1997 In 1994,the number of published papers related to TAS increased abruptly since more research groups joined the efforts to develop the area.Microfabrication Design Separations Biochemical Reactors Detection 分类与特点 分类：材料：硅、玻璃、石英、聚合物、复合材料功能：分离、采样与前处理、检测、化学合成等 特点：高效、低耗、集成、一致性好、昂贵目 录9-1 发展历史9-2 相关理论9-2-1 基本概念9-2-2 理论描述和模拟9-3 基础技术9-3-1 微制造9-3-2 界面和连接9-3-3 流体控制9-4 单元操作 9-4-1 样品制备 9-4-2 注射 9-4-3 流体和颗粒操作 9-4-4 反应器和混合器 9-4-5 分离 9-4-6 检测9-5 应用9-5-1 细胞培养和操作9-5-2 免疫分析和临床检验9-5-3 蛋白质分析9-5-4 核酸分析9-2 相关理论 9-2-1 基本概念 遵循低雷诺数流动的规律。除了组分间的扩散，两层或者多层流体可以相邻流动而不互相混合，使得样品的混合变得困难。Anal.Chem.2002,74,45-51液体流动的特点液体流动的特点由于比表面积增大，表面张力、摩擦力的影响非常显著。微通道中的液液界面与通道壁平行，因为表面张力和摩擦力大于重力。Anal.Sci.2001,17,89-93液体流动的特点液体的物理性质发生变化，如表观粘度变大纯水通过微通道的时间：理论值2.3ms实验值10msAnal.Chem.2002,74,6170-6176Aq.Org.Aq.Org.Aq.Org.MicrochipMicrochannel多相流的模拟9-2-2 理论描述和模拟扩散的数字模拟Biophysical Journal,2001,80,155160不同尺寸通道的数字模拟Microsystem Engineering of Lab-on-a-Chip Devices,5 Simulations in MicrofluidicsISBN:3-527-30733-8 目 录9-1 发展历史9-2 相关理论9-2-1 基本概念9-2-2 理论描述和模拟9-3 基础技术9-3-1 微制造9-3-2 界面和连接9-3-3 流体控制9-4 单元操作 9-4-1 样品制备 9-4-2 注射 9-4-3 流体和颗粒操作 9-4-4 反应器和混合器 9-4-5 分离 9-4-6 检测9-5 应用9-5-1 细胞培养和操作9-5-2 免疫分析和临床检验9-5-3 蛋白质分析9-5-4 核酸分析微结构的形成1经典的光刻技术Photolithograph procedures for making glass template.(a)Spine coating of photoresist,(b)covered with photo mask,(c)exposure,(d)developing,(e)etching,and(f)removal of photoresist.适合硅、玻璃、石英等材料，与传统的半导体工业的方法一致。分为湿法和干法两种，干法的分辨率较湿法高，相应的制造成本也高。Analyst,2004,129,3053089-3 基础技术 9-3-1 微制造微结构的形成2模版浇注法（模塑法）Process overview for mass manufacturing of plastic microfluidic systems 适合聚合物材料。大批量生产时成本低。Anal.Chem.,2002,74,78A-86A微结构的形成3模版热压法Schematic representation of the fabrication method involving hotembossing of thermoplastic polymer pellets and thermal bonding.适合热塑性聚合物。Applied Physics Letters,2002,80,3614-3616微结构的形成4激光刻蚀法用激光直接在聚合物或玻璃上加热形成微结构.Anal.Chem.,1997,69,2035-2042 芯片的封装1热键合对玻璃和石英材质刻蚀的微结构一般使用热键合对玻璃和石英材质刻蚀的微结构一般使用热键合方法，将加工好的基片和相同材质的盖片洗净烘干对方法，将加工好的基片和相同材质的盖片洗净烘干对齐紧贴后平放在高温炉中，在基片和盖片上下方各放齐紧贴后平放在高温炉中，在基片和盖片上下方各放一块抛光过的石墨板，在上面的石墨板上再压一块重一块抛光过的石墨板，在上面的石墨板上再压一块重0.5kg的不锈钢块，在高温炉中加热键合。玻璃芯片键的不锈钢块，在高温炉中加热键合。玻璃芯片键合时，高温炉升温速度为合时，高温炉升温速度为10oC/分，在分，在620oC时保温时保温3.5小时，再以小时，再以10oC/分的速率降温。石英芯片键合温度高分的速率降温。石英芯片键合温度高达达1000oC以上。此方法对操作技术要求较高。以上。此方法对操作技术要求较高。现代科学仪器，2001，4，8-12芯片的封装2阳极键合 在玻璃、石英与硅片的封接中已广泛采用阳极键合的方法。即在键合过程中，施加电场，使键合温度低于软化点温度。在500-760伏电场下，升温到500oC时，可使两块玻璃片键合。在两块玻璃板尚未键合时，板间空气间隙承担了大部分电压降，玻璃板可视为平行板电容器，板间吸引力与电场强度的平方成正比，因此，键合从两块玻璃中那些最接近的点开始，下板中可移动的正电荷（主要是Na+）与上板中的负电荷中和，生成一层氧化物（正是这层过渡层，使两块玻璃板封接），该点完成键合后，周围的空气间隙相应变薄，电场力增大，从而键合扩散开来，直至整块密合。现代科学仪器，2001，4，8-12芯片的封装3室温键合Anal.Chem.2004,76,5597-5602芯片的封装4贴合 将聚合物薄片直接覆将聚合物薄片直接覆盖在玻璃或石英板上。盖在玻璃或石英板上。5压合Schematic illustration of sealing and connection method.The top and bottom plates are pressed by a screw and holders.Anal.Chem.2002,74,1724-1728表面修饰1作为色谱固定相 coating of channel walls with something,e.g.octadecylsilanes,to create stationary phasesAnal.Chem.2002,74,784-789Anal.Chem.2003,75,64A-69A表面修饰2固定化酶 Schematic diagram of streptavidin-conjugated alkaline phosphatase bound to a supported lipid bilayer containing biotinylated lipids.Anal.Chem.2002,74,379-385表面修饰3固定化生物膜Langmuir 2003,19,1624-1631表面修饰4亲水或疏水修饰Analyst 2004,129,284-287与外部溶液连接Anal.Chem.2000,72,1711-17149-3-2 界面和连接与质谱仪连接Basic features of an electrospray interface.(A)Diagram of the layout of an electrospray-chip incorporating needle sources(electrospray emitters).(B)Scanning electron micrograph of an actual chip.Lab on a Chip,2001,1,7N12N外加压力驱动Anal.Chem.2000,72,1711-17149-3-3 流体控制重力驱动Anal.Chem.2005,77,1330-1337离心力驱动Centrifugal Microfluidics PlatformAnal.Chem.2002,74,5569-5575毛细作用驱动Microfilter device design and detail:(a)top view of generic device design with narrow and expanded channels,(b)filter detail area showing filter pores and expanded channel layout,(c)microfilter cross section.LabChip,2005,5,922-929利用电渗流驱动与控制Generation and control of electroosmotic flow onmicrofabricated devices.Anal.Chem.2000,72,330A-335A利用表面性质控制Anal.Chem.2003,75,5097-5102单向阀 Cylinder diameters are approximately 100 and 25 m.Anal.Chem.2002,74,4913-4918目 录9-1 发展历史9-2 相关理论9-2-1 基本概念9-2-2 理论描述和模拟9-3 基础技术9-3-1 微制造9-3-2 界面和连接9-3-3 流体控制9-4 单元操作 9-4-1 样品制备 9-4-2 注射 9-4-3 流体和颗粒操作 9-4-4 反应器和混合器 9-4-5 分离 9-4-6 检测9-5 应用9-5-1 细胞培养和操作9-5-2 免疫分析和临床检验9-5-3 蛋白质分析9-5-4 核酸分析9-4 单元操作 9-4-1 样品制备Anal.Chem.2002,74,1565-1571SonicationSonication A Minisonicator To Rapidly Disrupt Bacterial Spores for DNA Analysis Anal.Chem.,1999,71,4232-4236 The minisonicator and plastic spore lysis cartridge.Samples of spores were loaded into the chamber through a channel in the cartridge wall and subjected to sonication for 30 s.A microfluidic cartridge to prepare spores for PCR analysis The spore disruption cartridge system.Components included the fluidic control unit,the cartridge,and the minisonicator.Biosensors&Bioelectronics,2000,14,849852Extraction Extraction Side view schematic of miniaturized affinity dialysis and concentration system Anal.Chem.,2001,73,2048-2053 Concept of sequential ion-sensing system using single microchip Anal.Chem.2001,73,5551 Schematic illustration of experimental setup and ion pair extraction model Anal.Chem.2001,73,1382-1386 determination of Co(II)as 2-nitroso-1-naphthol chelates by solvent extraction and thermal lens microscopyLab Chip 2001,1,72-75 PreconcentrationPreconcentration Microfabricated Porous Membrane Structure for Sample Concentration and Electrophoretic AnalysisAnal.Chem.1999,71,1815-1819 Electrokinetic Preconcentration of Proteins on thin PDMS MembranesmicrTAS,2003,Vol.2,1171-1174 Schematic diagrams of CE procedures with pinched injection(top panels),floating injection(middle),and(C)simplest injection mode(bottom).Anal.Chem.,2001.73,2656-26629-4-2 注射 Schematic of microchip used for gated valving.The double lines represent microfabricated channels,and the single lines represent electrical connections.The high-voltage relay is in the dispense(open)position.Anal.Chem.,1999,71(15),3273-3276,White light image of(a)m i c r o c h i p v a l v e a n d fluorescence images of(b)sample loading,(c)dispensing,and(d)analysis modes.The injection time was 0.4 s.In the sample loading mode,550,550,and 0 V were applied to the buffer,sample,and waste reservoirs,respectively.In the dispensing mode,550 and 0 V were applied to the sample and waste reservoirs,respectively.(a)S c h e m a t i c o f t h e c a p i l l a r y electrophoresis microdevice equipped with external voltage electrodes:1,injector waste port;2,separation port inlet;3,sample inlet port/injector port;4,external voltage electrode;5,separation waste port;6,external voltage electrode.Total size of the structure:2.547.62cm.(b)Micrograph of the injection(150pL)zone within the separation channel.The sample is injected from reservoir 3 to reservoir 1 and separated from reservoir 2 to reservoir 5 using applied voltage fields.(c)Micrograph of external voltage electrodes on the capillary electrophoresis microdevice.Electrodes are placed 50 m away from the surface of the channel(30m wide 10m deep).Anal.Chem.,2000,72(5),1088-1092(a)(b)(c)9-4-3 流体和颗粒操作 Magnetic Field-Controlled Microfluidic Transport(A)Gravity-driven flow in the absence of a magnetic field(B=0).(B)MHD flow at B=1.0 T.J.Am.Chem.Soc.2002,124,462-467 Microfabrication InsideCapillaries Using MultiphaseLaminar Flow PatterningScience 1999,285,83Mechanistic Investigation of Nanoparticle Motionin Pulsed Voltage Miniaturized Electrical FieldFlow Fractionation Device by in Situ FluorescenceImagingAnal.Chem.2004,76,2719-2724Microchip Flow Cytometry Using Electrokinetic Focusing Anal.Chem.1999,71,4173 A Picoliter-Volume Mixer for Microfluidic Analytical Systems Anal.Chem.2001,73,1942-1947 9-4-4 反应器和混合器Rapid Microfluidic MixingAnal.Chem.2002,74,45-51 Ultrasonic Mixing in Microfluidic Channels Using Integrated Transducers Anal.Chem.,2004;76;3694-3698 Chemical ReactorsChemical ReactorsA New Synthetic Method for Controlled Polymerization Using a Microfluidic System J.AM.CHEM.SOC.2004,126,9880-9881General idea of polymer membrane formation under organic/aqueous two-phase flow in an X-shaped microchannel Anal.Chem.2003,75,350-354Photothermal Temperature Control of a Chemical Reaction on a Microchip Using an Infrared Diode Laser Anal.Chem.2001;73(16);4037-4044 Microfluidic Devices for Energy Conversion:Planar Integration and Performance of a Passive,Fully Immersed H2-O2 Fuel CellLangmuir 2004,20,6974-6976Enzymatic ReactorsEnzymatic ReactorsMicrofabricated Electrophoresis Chips for Simultaneous Bioassays of Glucose,Uric Acid,Ascorbic Acid,and Acetaminophen Anal.Chem.2000,72,2514-2518 Measurement of Enzyme Kinetics Using aContinuous-Flow Microfluidic SystemAnal.Chem.2003,75,3161-3167Substrate permeation and subsequent enzyme reaction experiment Anal.Chem.2003,75,350-354Immunoassay Reactors.Immunoassay Reactors.Schematic illustrations of microchip-based immunosorbent assay.Anal.Chem.2001,73,1213-1218Glass microchip for immunosorbent assay:PostcolumnPostcolumn Labeling LabelingSchematic of microchip used for protein separation with postcolumn labeling Anal.Chem.;2000;72,4608-4613 9-4-5 分离Top view of the HDC separation of fluorescent nanoparticles and a marker Anal.Chem.;2003;75(24);6761-6768 ChromatographyElectrophoresis Surface Modification of the Channels of Poly(dimethylsiloxane)Microfluidic Chips with Polyacrylamide for Fast Electrophoretic Separations of ProteinsAnal.Chem.2004,76,2055-2061Distillation Continuous laminar evaporation:micron-scale distillationChem.Commun.,2004,266 267MicrofilterSensors and Actuators B 67 2000 203208 Characterization of micromachined silicon membranes forimmunoisolation and bioseparation applicationsSchematic of assembled biocapsule consisting of two micromachined membranes bonded together to form a cell-containing cavity bounded by membranes.Journal of Membrane Science 159(1999)221-2319-4-6 检测Anal.Chem.;1999;71;5309-5314 FluorescenceChemiluminescenceChemiluminescenceA Two-Channel Microfluidic Sensor That Uses Anodic Electrogenerated Chemiluminescence as a Photonic Reporter of Cathodic Redox ReactionsAnal.Chem.2003,75,313-318The thermal lens microscope.The thermal lens microscope.ElectrochemistryElectrochemistry Characteristic Electrochemical Responses of Polymer Microchannel-Microelectrode ChipsAnal.Chem.2003,75,2086-2091MSMS Chip-Based P450 Drug Metabolism Coupled to Electrospray Ionization-Mass Spectrometry DetectionAnal.Chem.2003,75,6430-6436AmplificationAmplificationDetection of Viable Cryptosporidium parvumUsing DNA-Modified Liposomes in a Microfluidic Chip目 录9-1 发展历史9-2 相关理论9-2-1 基本概念9-2-2 理论描述和模拟9-3 基础技术9-3-1 微制造9-3-2 界面和连接9-3-3 流体控制9-4 单元操作 9-4-1 样品制备 9-4-2 注射 9-4-3 流体和颗粒操作 9-4-4 反应器和混合器 9-4-5 分离 9-4-6 检测9-5 应用9-5-1 细胞培养和操作9-5-2 免疫分析和临床检验9-5-3 蛋白质分析9-5-4 核酸分析Development of a Microchip-Based Bioassay System Using Cultured Cells Concept of the microchip-based bioassay system Anal.Chem.,2005.77(7),2125-21319-5 应用9-5-1 细胞培养和操作(A)Illustration and(B)photo showing the arrangement of Peltier elements.(C)Photo of the temperature control device(A)Illustration and(B)photograph of the bioassay microchip.(C)An enlarged illustration of the microchamber for cell culture Complete system for the microchip-based bioassay(A)Photo of the temperature control device with the microchip.(B)Thermograph of the microchip surface Micrographs of(A)mouse peritoneal macrophages and(B,C)J774.1 cells in the microchip.The dam successfully kept cells in the microchamber.Micrographs of J774.1 cells cultured in the microchip for 2 days(A)under continuous medium flow conditions and(B)static conditions.The cells stained in blue were dead.Chemical processes carried out in the microchip for bioassay of macrophage-stimulating agent Calibration curves of NO dissolved in the medium(A)determined on the bulk scale and(B)in a microchip.Typical results of NO released from J774.1 cells.Design of a Compact Disk-like Microfluidic Platform for Enzyme-Linked Immunosorbent Assay Schematics of(a)a CD-ELISA design with 24 sets of assays,(b)a single assay,(1,waste;2,detection;3,first antibody;4,6,8,10,washing;5,blocking protein;7,antigen/sample;9,second antibody;and 11,substrate),and(c)photo of a single assay.Anal.Chem.,2004,76(7),1832-1837 9-5-2 免疫分析和临床检验(a)Schematic of five-step flow sequencing CD(1,waste;2,detection;7,antigen/sample;8,9,washing;10,second antibody;and 11,substrate)and(b)a CNC-machined CD.Anal.Chem.,2004.76(3),742-748Schematic and image of 2-D protein separation platform using plastic microfluidics.9-5-3 蛋白质分析 Integration of Isoelectric Focusing with Parallel Sodium Dodecyl Sulfate Gel Electrophoresis for Multidimensional Protein Separations in a Plastic Microfludic NetworkFluorescent images of on-chip 2-D separation of four model proteins using single separation medium.(A)Non-native IEF with focusing order of(i)actin,(ii)bovine serum albumin and trypsin inhibitor,and(iii)parvalbumin from left to right;(B)electrokinetic transfer of focused proteins;(C)SDS gel electrophoresis.Fluorescent images of on-chip 2-D separation of five model proteins using multiple separation media.(A)Non-native IEF with focusing order of(i)actin,(ii)bovine serum albumin,ovalbumin,and trypsin inhibitor,and(iii)parvalbumin from left to right;(B)electrokinetic transfer of focused proteins;(C)SDS gel electrophoresis.Anal.Chem.2004,76,1824-18319-5-4 核酸分析 Self-Contained,Fully Integrated Biochip for Sample Preparation,Polymerase Chain Reaction Amplification,and DNA Microarray DetectionMicrofabricated Device for DNA and RNA Amplification by Continuous-Flow Polymerase Chain Reaction and Reverse Transcription-Polymerase Chain Reaction with Cycle Number Selection Anal.Chem.2003,75,288-295