流式细胞仪样品流速及聚焦流测量方法研究
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摘要
为提高流式细胞仪的探测分辨率和数据检测的稳定性,需要精确控制样品流速,并分析样品流速和鞘液流速对样品聚焦流的影响,可通过样品聚焦流直径和样品聚焦流在流动室流道中的相对位置来评价样品的聚焦状况。利用蠕动泵运动特点,设计了一种平均流量称重法测量样品流速的方法,并与微流量传感器测量结果作比较;采用最小二乘法线性拟合蠕动泵的控制电压和样品流速之间的函数关系,并采用显微成像法直接测量和分析样品流速和鞘液流速对样品聚焦流直径、偏离流动室流道中心线的距离的影响。实验结果显示,采用平均流量称重法与微流量传感器测得的样品流速的线性相关系数高达0.982 8;蠕动泵的样品流速与其控制电压的线性相关系数高于0.99,说明利用该线性关系可以实现样品流速的精确控制;采用的显微成像法能快速方便地测得样品聚焦流的直径及位置,为流式细胞仪样品流速、鞘液流速的调控以及液流器件组装精度的测试提供了指导方法。
In order to improve the detection resolution and the stability of data detection,it is usually necessary to precisely control the sample flow rate in the flow system and analyze the effects of the sample flow rate and sheath fluid flow rate on the sample focusing core flow.The focusing state can be evaluated by the sample focusing core flow diameter and the relative position of the sample focusing core flow in the flow channel.First of all,according to the movement characteristics of peristaltic pump,an average flow weighing method was designed to measure the sample flow rate and compared with the measurement result of micro-flow sensor.Then,the relationship between the control voltage of the peristaltic pump and sample flow rate was linearly fitted by the least squares method.Lastly,the microscopic imaging method was used to measure and analyze the impact of the sample flow rate and sheath fluid flow rate on the diameter of the sample focusing core flow and its deviation distance from the flow channel centerline.The experimental data showed that the linear correlation coefficient of sample flow rate measured by the average flow weighing method and the micro-flow sensor was as high as 0.982 8.The correlation coefficient between the sample flow rate of the peristaltic pump and its control voltage was as high as 0.99,which showed that the linear relationship could be used to control the sample flow rate accurately.The diameter and position of sample focusing core flow were quickly and conveniently measured by the microscopic imaging method,which provided a guidance method for the regulation of theflow rate of samples and the sheath fluid in flow cytometer,and the test of the assembly accuracy of the flow device.
In order to improve the detection resolution and the stability of data detection,it is usually necessary to precisely control the sample flow rate in the flow system and analyze the effects of the sample flow rate and sheath fluid flow rate on the sample focusing core flow.The focusing state can be evaluated by the sample focusing core flow diameter and the relative position of the sample focusing core flow in the flow channel.First of all,according to the movement characteristics of peristaltic pump,an average flow weighing method was designed to measure the sample flow rate and compared with the measurement result of micro-flow sensor.Then,the relationship between the control voltage of the peristaltic pump and sample flow rate was linearly fitted by the least squares method.Lastly,the microscopic imaging method was used to measure and analyze the impact of the sample flow rate and sheath fluid flow rate on the diameter of the sample focusing core flow and its deviation distance from the flow channel centerline.The experimental data showed that the linear correlation coefficient of sample flow rate measured by the average flow weighing method and the micro-flow sensor was as high as 0.982 8.The correlation coefficient between the sample flow rate of the peristaltic pump and its control voltage was as high as 0.99,which showed that the linear relationship could be used to control the sample flow rate accurately.The diameter and position of sample focusing core flow were quickly and conveniently measured by the microscopic imaging method,which provided a guidance method for the regulation of theflow rate of samples and the sheath fluid in flow cytometer,and the test of the assembly accuracy of the flow device.
引文
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[14]于虎,祝连庆,郭阳宽,等.流式细胞仪流动室流场特性仿真[J].计算机仿真,2015,32(8):230-234.YU Hu,ZHU Lian-qing,GUO Yang-kuan,et al.Simulation of fluid field characteristic of flow cytometry flow chamber[J].Computer Simulation,2015,32(8):230-234.
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[16]MATHIAS Busek,MARTIN N9tzel,CHRISTOPH Polk,et al.Peristaltic pneumatic pump-characterisation viaμPIV,non-invasive pressure measurement and simulation[J].Journal of Sensors and Sensor Systems,2013,2(2):165-169.
[2]HOWARD M Shapiro.Practical flow cytometry[M].New Jersey:Wiley-Liss,2003:1-2.
[3]BENDALL S C,SIMONDS E F,QIU P,et al.Singlecell mass cytometry of differential immune and drug responses across a human hematopoietic continuum[J].Science,2011,332(6030):687-696.
[4]BJORNSON Z B,NOLAN G P,FANTL W J.Singlecell mass cytometry for analysis of immune system functional states[J].Current Opinion in Immunology,2013,25(4):484-494.
[5]RUBEN Props,KERCKHOF Frederiek-Maarten,PETER Rubbens,et al.Absolute quantification of microbial taxon abundances[J].The ISME Journal,2017,11(2):584-587.
[6]WENG X,NEETHIRAJAN S.Ensuring food safety:quality monitoring using microfluidics[J].Trends in Food Science&Technology,2017,65:10-22.
[7]NEVEL S V,KOETZSCH S,PROCTOR C R,et al.Flow cytometric bacterial cell counts challenge conventional heterotrophic plate counts for routine microbiological drinking water monitoring[J].Water Research,2017,113:191-206.
[8]MENAKE E Piyasena,STEVEN W Graves.The intersection of flow cytometry with microfluidics and microfabrication[J].Lab Chip,2014,14(6):1044-1059.
[9]NORASYIKIN Selamat,EHSAN A A.Particles trajectories simulation of hydrodynamic focusing in circular and rectangular polymer microflow cytometer[J].International Journal of Applied Engineering Research,2017,12(7):1311-1315.
[10]SHIVHARE P K,BHADRA A,SAJEESH P,et al.Hydrodynamic focusing and interdistance control of particle-laden flow for microflow cytometry[J].Microfluidics&Nanofluidics,2016,20(6):1-14.
[11]OLSON R J,SHALAPYONOK A,KALB D J,et al.Imaging FlowCytobot modified for high throughput by in-line acoustic focusing of sample particles[J].Limnology&Oceanography Methods,2017,15(10):867-874.
[12]AHMAD Ahsan Nawaz,ZHANG Xiang-jun,MAO Xiao-le,et al.Sub-micrometer-precision,three-dimensional(3D)hydrodynamic focusing via“microfluidic drifting”[J].Lab on a Chip,2014,14(2):415-423.
[13]马玉婷,严心涛,陈忠祥,等.流式细胞仪液流聚焦系统仿真分析与设计[J].分析仪器,2014(1):17-22.MA Yu-ting,YAN Xin-tao,CHEN Zhong-xiang,et al.Simulated analysis and design of flow cytometer focusing system[J].Analytical Instrumentation,2014(1):17-22.
[14]于虎,祝连庆,郭阳宽,等.流式细胞仪流动室流场特性仿真[J].计算机仿真,2015,32(8):230-234.YU Hu,ZHU Lian-qing,GUO Yang-kuan,et al.Simulation of fluid field characteristic of flow cytometry flow chamber[J].Computer Simulation,2015,32(8):230-234.
[15]OLA Jakobsson,CARL Grenvall,MARIA Nordin,et al.Acoustic actuated fluorescence activated sorting of microparticles[J].Lab on a Chip,2014,14(11):1943-1950.
[16]MATHIAS Busek,MARTIN N9tzel,CHRISTOPH Polk,et al.Peristaltic pneumatic pump-characterisation viaμPIV,non-invasive pressure measurement and simulation[J].Journal of Sensors and Sensor Systems,2013,2(2):165-169.