基于介电电泳的高通量单细胞捕获阵列微流控芯片的研究

  在细胞生物学研究中应当充分考虑细胞异质性，利用传统手段对细胞进行群体分析往往会掩盖个体差异，从而导致实验误差。因此，单细胞研究对现代细胞生物学的发展具有重要意义。微流控芯片是一种具有在极小空间内操作、反应和分析微量液体的新型技术，在芯片上可以利用微结构、流体力学、光学、介电电泳等多种手段实现单细胞的无标签捕获、操控和分析，是一种理想的单细胞分析平台。近年来，越来越多的单细胞分析采用在微流控芯片上构建细胞阵列的方式，这种实验方法相比于传统的单细胞分析效率更高、对照性更强。

  本实验对基于介电电泳的高通量单细胞捕获阵列进行了一定的探索，设计、制造并验证了两种微流控芯片结构。在第一种芯片中，三种尺寸不同的微阱阵列可以在高通量构建单细胞阵列的同时对细胞进行尺寸差异化的自动分选和捕获。通过细胞实验测试表明，该芯片在不同区域捕获的细胞具有尺寸差异性。细胞在不同生命阶段的尺寸具有差异，芯片可以为基于细胞尺寸的单细胞异质性研究提供便利的实验条件。

  对于第二种芯片，实验创新性地提出一种结合双层微阱结构的的三电极排布模式，探索出一种多次上样的细胞实验模式。实验实现了对三种细胞的配对捕获，在芯片上成功构建出三重单细胞阵列，该芯片能为下游细胞异质性分析、细胞间相互作用和细胞融合等多重单细胞研究提供帮助。

[1] TORIELLO N M, DOUGLAS E S, THAITRONG N, et al. Integrated microfluidic bioprocessor for single-cell gene expression analysis[J]. Proceedings of the National Academy of Sciences of the United States of America, 2008, 105(51): 20173-20178.
[2] ALTSCHULER S J, WU L F. Cellular Heterogeneity: Do Differences Make a Difference?[J]. Cell, 2010, 141(4): 559-563.
[3] 林玲. 微/纳流控单细胞分析方法[J]. 生命科学仪器, 2020, 18(04): 19-26+11.
[4] MANZ A, GRABER N, WIDMER H M. Miniaturized Total Chemical-Analysis Systems - a Novel Concept for Chemical Sensing[J]. Sensors and Actuators B-Chemical, 1990, 1(1-6): 244-248.
[5] DUFFY D C, MCDONALD J C, SCHUELLER O J A, et al. Rapid prototyping of microfluidic systems in poly(dimethylsiloxane)[J]. Analytical Chemistry, 1998, 70(23): 4974-4984.
[6] QUAKE S R, SCHERER A. From micro- to nanofabrication with soft materials[J]. Science, 2000, 290(5496): 1536-1540.
[7] UNGER M A, CHOU H P, THORSEN T, et al. Monolithic microfabricated valves and pumps by multilayer soft lithography[J]. Science, 2000, 288(5463): 113-116.
[8] THORSEN T, MAERKL S J, QUAKE S R. Microfluidic large-scale integration[J]. Science, 2002, 298(5593): 580-584.
[9] AU A K, LAI H Y, UTELA B R, et al. Microvalves and Micropumps for BioMEMS[J]. Micromachines, 2011, 2(2): 179-220.
[10] BAO B, WANG Z C, THUSHARA D, et al. Recent Advances in Microfluidics-Based Chromatography-A Mini Review[J]. Separations, 2021, 8(1): 3.
[11] DING Y, HOWES P D, DEMELLO A J. Recent Advances in Droplet Microfluidics[J]. Analytical Chemistry, 2020, 92(1): 132-149.
[12] DALILI A, SAMIEI E, HOORFAR M. A review of sorting, separation and isolation of cells and microbeads for biomedical applications: microfluidic approaches[J]. Analyst, 2019, 144(1): 87-113.
[13] CUI P, WANG S. Application of microfluidic chip technology in pharmaceutical analysis: A review[J]. Journal of Pharmaceutical Analysis, 2019, 9(4): 238-247.
[14] AZIZIPOUR N, AVAZPOUR R, ROSENZWEIG D H, et al. Evolution of Biochip Technology: A Review from Lab-on-a-Chip to Organ-on-a-Chip[J]. Micromachines, 2020, 11(6):599.
[15] LEVATO R, JUNGST T, SCHEURING R G, et al. From Shape to Function: The Next Step in Bioprinting[J]. Advanced Materials, 2020, 32(12): 1906423.
[16] CHIRCOV C, GRUMEZESCU A M. Microelectromechanical Systems (MEMS) for Biomedical Applications[J]. Micromachines, 2022, 13(2): 164.
[17] TANIGUCHI Y, CHOI P J, LI G W, et al. Quantifying E-coli Proteome and Transcriptome with Single-Molecule Sensitivity in Single Cells[J]. Science, 2010, 329(5991): 533-538.
[18] 董盛华, 张晶, 葛胜祥. 微流控芯片细胞捕获分离方法概述[J]. 生物化学与生物物理进展, 2016, 43(11): 1102-1110.
[19] ZHU L, LIN H B, WAN S, et al. Efficient Isolation and Phenotypic Profiling of Circulating Hepatocellular Carcinoma Cells via a Combinatorial-Antibody-Functionalized Microfluidic Synergetic-Chip[J]. Analytical Chemistry, 2020, 92(22): 15229-15235.
[20] MISHRA A, DUBASH T D, EDD J F, et al. Ultrahigh-throughput magnetic sorting of large blood volumes for epitope-agnostic isolation of circulating tumor cells[J]. Proceedings of the National Academy of Sciences of the United States of America, 2020, 117(29): 16839-16847.
[21] XI H D, ZHENG H, GUO W, et al. Active droplet sorting in microfluidics: a review[J]. Lab on a Chip, 2017, 17(5): 751-771.
[22] 程丹彤. 集成单细胞捕获、阵列化、释放及快速染色的微流控装置的研究[D]. 上海：上海交通大学, 2018.
[23] ZHENG S Y, LIN H K, LU B, et al. 3D microfilter device for viable circulating tumor cell (CTC) enrichment from blood[J]. Biomedical Microdevices, 2011, 13(1): 203-213.
[24] DI CARLO D, AGHDAM N, LEE L P. Single-cell enzyme concentrations, kinetics, and inhibition analysis using high-density hydrodynamic cell isolation arrays[J]. Analytical Chemistry, 2006, 78(14): 4925-4930.
[25] TAN W H, TAKEUCHI S. A trap-and-release integrated microfluidic system for dynamic microarray applications[J]. Proceedings of the National Academy of Sciences of the United States of America, 2007, 104(4): 1146-1151.
[26] JIN D, DENG B, LI J X, et al. A microfluidic device enabling high-efficiency single cell trapping[J]. Biomicrofluidics, 2015, 9(1): 014101.
[27] 孙东, 罗涛. 基于微流控的细胞操纵方法与应用[J]. 科技导报, 2018, 36(16): 29-38.
[28] YAMADA M, NAKASHIMA M, SEKI M. Pinched flow fractionation: Continuous size separation of particles utilizing a laminar flow profile in a pinched microchannel[J]. Analytical Chemistry, 2004, 76(18): 5465-5471.
[29] TAKAGI J, YAMADA M, YASUDA M, et al. Continuous particle separation in a microchannel having asymmetrically arranged multiple branches[J]. Lab on a Chip, 2005, 5(7): 778-784.
[30] VIG A L, KRISTENSEN A. Separation enhancement in pinched flow fractionation[J]. Applied Physics Letters, 2008, 93(20):203507.
[31] KUNTAEGOWDANAHALLI S S, BHAGAT A A S, KUMAR G, et al. Inertial microfluidics for continuous particle separation in spiral microchannels[J]. Lab on a Chip, 2009, 9(20): 2973-2980.
[32] SHEN S F, TIAN C, LI T B, et al. Spiral microchannel with ordered micro-obstacles for continuous and highly-efficient particle separation[J]. Lab on a Chip, 2017, 17(21): 3578-3591.
[33] SONG H J, ROSANO J M, WANG Y, et al. Continuous-flow sorting of stem cells and differentiation products based on dielectrophoresis[J]. Lab on a Chip, 2015, 15(5): 1320-1328.
[34] HUANG L R, COX E C, AUSTIN R H, et al. Continuous particle separation through deterministic lateral displacement[J]. Science, 2004, 304(5673): 987-990.
[35] ASHKIN A, DZIEDZIC J M, BJORKHOLM J E, et al. Observation of a Single-Beam Gradient Force Optical Trap for Dielectric Particles[J]. Optics Letters, 1986, 11(5): 288-290.
[36] ASHKIN A, DZIEDZIC J M, YAMANE T. Optical trapping and manipulation of single cells using infrared laser beams[J]. Nature, 1987, 330(6150): 769-771.
[37] RODRIGO P, ERIKSEN R, DARIA V, et al. Interactive light-driven and parallel manipulation of inhomogeneous particles[J]. Optics Express, 2002, 10(26): 1550-1556.
[38] 赵磊. 基于微流控的多重单细胞阵列构建及应用[D]. 咸阳：西北农林科技大学, 2016.
[39] WANG X L, CHEN S X, KONG M, et al. Enhanced cell sorting and manipulation with combined optical tweezer and microfluidic chip technologies[J]. Lab on a Chip, 2011, 11(21): 3656-3662.
[40] WU M, OZCELIK A, RUFO J, et al. Acoustofluidic separation of cells and particles[J]. Microsystems & Nanoengineering, 2019, 5(1):32.
[41] PETERSSON F, NILSSON A, HOLM C, et al. Separation of lipids from blood utilizing ultrasonic standing waves in microfluidic channels[J]. Analyst, 2004, 129(10): 938-943.
[42] 吴春卉, 姜有为, 程鑫. 微流控芯片在单细胞捕获中的应用[J]. 科技导报, 2018, 36(16): 39-45.
[43] DING X Y, LIN S C S, KIRALY B, et al. On-chip manipulation of single microparticles, cells, and organisms using surface acoustic waves[J]. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(28): 11105-11109.
[44] POHL H A. The Motion and Precipitation of Suspensoids in Divergent Electric Fields[J]. Journal of Applied Physics, 1951, 22(7): 869-871.
[45] 徐溢, 郝敦玲, 曾雪, et al. 生化样本的芯片介电电泳富集和分离研究进展[J]. 化学通报, 2009, 72(01): 4-9.
[46] LUO T, FAN L, ZENG Y X, et al. A simplified sheathless cell separation approach using combined gravitational-sedimentation-based prefocusing and dielectrophoretic separation[J]. Lab on a Chip, 2018, 18(11): 1521-1532.
[47] 陈礼, 郑小林, 胡宁, et al. 基于介电电泳的微流控细胞分离芯片的研究进展[J]. 分析化学, 2015, 43(02): 300-309.
[48] MODARRES P, TABRIZIAN M. Alternating current dielectrophoresis of biomacromolecules: The interplay of electrokinetic effects[J]. Sensors and Actuators B-Chemical, 2017, 252: 391-408.
[49] DOH I, CHO Y H. A continuous cell separation chip using hydrodynamic dielectrophoresis (DEP) process[J]. Sensors and Actuators a-Physical, 2005, 121(1): 59-65.
[50] THOMAS R S, MORGAN H, GREEN N G. Negative DEP traps for single cell immobilisation[J]. Lab on a Chip, 2009, 9(11): 1534-1540.
[51] MAZUTIS L, GILBERT J, UNG W L, et al. Single-cell analysis and sorting using droplet-based microfluidics[J]. Nature Protocols, 2013, 8(5): 870-891.
[52] HOSOKAWA M, HAYATA T, FUKUDA Y, et al. Size-Selective Microcavity Array for Rapid and Efficient Detection of Circulating Tumor Cells[J]. Analytical Chemistry, 2010, 82(15): 6629-6635.
[53] ZHAO L, MA C, SHEN S, et al. Pneumatic microfluidics-based multiplex single-cell array[J]. Biosensors & Bioelectronics, 2016, 78: 423-430.
[54] WU C H, CHEN R F, LIU Y, et al. A planar dielectrophoresis-based chip for high-throughput cell pairing[J]. Lab on a Chip, 2017, 17(23): 4008-4014.
[55] 潘洋. 基于微液滴阵列的生物大分子检测技术研究[D]. 合肥：中国科学技术大学, 2020.
[56] 段玉. 用于单微球阵列捕获与单细胞力学分析的微流体芯片研究[D]. 深圳：深圳大学, 2019.