在生理流动条件下分析血小板黏附聚集的简易微流控芯片技术

摘要

Objective To develop a simple microfluidic chip technology for analyzing platelet adhesion and aggregation under the condition of physiological flow .Methods The basic structure of the proposed microfluidic chip was a straight microchannel , where a sample pool and an outlet were located on each side respectively .The fluid dynamic behavior ,of the fluid in the microchannel was analyzed by theoretical calculation and finite element numerical analysis software ANSYS . The microchannel was first coated with type Ⅰ collagen protein.Then,blood flowed through the microchannel at 200 s-1 venous physiological shear rate and 1000 s-1 arterial physiological shear rate respectively .The behavior of the fluorescence labeled platelet adhesion and aggregation was recorded by fluorescence microscopy .Results Theoretical calculation and finite element numerical analysis showed that the microchannel with a width of 700 μm and a height of 70 μm ( aspect ration 10:1 ) had a uniform fluidic shear rate distribution .Compared with those at the 200 s-1 shear rate, the initial adhesion time, aggregation rate and the maximum surface cover rate of the platelet were significantly reduced at the 1000 s-1 shear rate ( P<0.05 ) .At the 1000 s-1 shear rate, tirofiban, an anticoagulant drug , significantly reduced the platelet aggregation in a concentration-dependent manner and the IC 50 was 23.8 nmol/L.Conclusion The technology developed in this paper can dynamically assay platelet adhesion and aggregation under the condition of physiological flow . The proposed microfluidic methods have the advantage of simple implementation and low sample cousumpation , and can be used for point-of-care detection of human platelet function , assay of the efficacy of anticoagulant drugs and for inspection of the thrombin behaviors of small animal models .%目的 发展一种在生理流动条件下分析血小板黏附聚集的简易微流控芯片技术.方法 微流控分析芯片基本结构由直微通道及两侧的样品池和出口组成;利用理论计算和有限元数值软件ANSYS对微通道流体动力学行为进行分析;微通道用Ⅰ型胶原蛋白修饰,分别在200 s-1静脉生理性剪切率和1000 s-1动脉生理性剪切率条件下使血液流过微通道,同时通过荧光显微摄像动态记录血液中荧光标记血小板与Ⅰ型胶原蛋白表面的黏附聚集图像.结果 理论和数值分析显示,700μm×70μm(宽深比10:1)的微通道具有最优的流体剪切率大小分布;相比200 s-1低剪切率,在1000 s-1剪切率条件下血小板初始黏附时间、聚集速率和最大表面覆盖面积均显著降低(P<0.05);在1000 s-1剪切率条件下,抗凝药物替罗非班处理呈浓度依赖性地降低血小板表面聚集率,IC50值为23.8 nmol/L.结论 该微流控芯片技术可在生理相关性流体剪切率环境下动态分析血小板黏附聚集,血样少,方法简单易行,未来可用于血小板功能临床检测、抗凝药物药效和小型动物模型凝血分析.