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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。
Objective To develop a simple microfluidic chip technology for the analysis of platelet adhesion and aggregation under physiological fluid conditions. Methods The basic structure of the microfluidic chip was composed of straight microchannels and sample cells and exits on both sides. The theoretical analysis and finite element numerical software ANSYS were used to analyze the microchannel hydrodynamic behavior. The microchannels were modified with type Ⅰ collagen, Blood was flowed through the microchannels at a physiological shear rate of 200 s -1 and a physiological shear rate of 1000 s -1, respectively, and fluorescent markers of blood were dynamically recorded by fluorescence microscopy Adhesion and aggregation images of platelets and collagen type Ⅰ surface. Results The theoretical and numerical results show that the microchannels with the size of 700μm × 70μm (width to depth ratio of 10:1) have the best size distribution of fluid shear rate. Compared with the low shear rate of 200 s -1, (-1) shear rate, the initial adhesion time, the aggregation rate and the maximum surface area of platelets were significantly decreased (P <0.05). At the shear rate of 1000 s -1, the anticoagulant drug tirofiban Class treatment in a concentration-dependent manner to reduce platelet surface aggregation rate, IC50 value of 23.8 nmol / L. Conclusion The microfluidic chip technology can dynamically analyze platelet adhesion and aggregation under physiologically relevant fluid shear rate conditions with few blood samples. The method is simple and easy to use and can be used for the clinical detection of platelet function, anticoagulant efficacy and coagulation in small animal models analysis.