血小板粘附的SPR传感检测方法研究

发布时间：2017-12-26 18:41

本文关键词：血小板粘附的SPR传感检测方法研究　出处：《重庆大学》2016年硕士论文　论文类型：学位论文

【摘要】：随着我国经济发展和生活水平的提高,心血管系统疾病越来越成为严重威胁人民健康的一个重要因素。其中,血栓性疾病发病率的上升趋势尤为明显。这类疾病的发病机制是与病理性血栓形成有关。目前预防血栓形成是这类疾病的一种重要防治手段。抗血小板药物的药理机制就是抑制血小板的粘附和聚集。在抗血小板新药研发中,药物对不同患者的生物效应并不一致,使得监测个体患者的抗血小板治疗药物最佳治疗剂量,最大限度地降低血栓和减少出血变得尤为重要。因此,各类体外检测血小板功能的方法和仪器被广泛应用于抗血小板治疗的监测中。但是,传统的血小板功能检测装置及方法普遍存在操作复杂、用时长、需标记、精度不高、价格昂贵等缺点,限制了其推广运用。为解决这一问题,本文选用了表面等离体激元共振传感技术用于血小板功能的研究。该分析技术具有高灵敏度、无需标记、操作简单、高通量等优势,通过测定血小板的粘附值来评估血小板功能的改变,从而实现抗血小板治疗中药物的监测。本文首先利用COMSOL仿真软件分析了几组具有不同宽度及高度的微通道。经过仿真研究通道底部剪切率的分布情况,确定出了一组剪切率分布最为均匀的通道并完成了芯片的加工制作。在气相及液相中分别测试了血小板在胶原上的粘附情况。实验结果表明,血液流经通道后,金膜表面折射率有所增加,SPR角增大,通过计算SPR角的增量就可算得血小板粘附的具体数值。这证明了SPR传感器用于研究以粘附值为参考标准的血小板功能检测的可行性。同时,该技术能实现通道任意位置的精确检测,定量分析血小板在通道中的分布情况,而且实验重复性高,分析区域被划分成的多个小分析点差异性小。采用多点分析的方法可以在一次实验中就实现传统分析需要多次检测的效果,大大提高了测试分析的效率。通过比较分析不同测试条件下的血小板粘附检测方法,发现液相中的血小板粘附检测效果更好。在液相条件下,检测了不同因素对血小板粘附的影响,分别从剪切率和不同基质蛋白方面展开研究。结果表明,在一定范围内,血小板粘附值随剪切率的增加而增加,在500s-1时,粘附值达到2.958ng/mm2;此后,继续增加剪切率,粘附值逐渐下降。在不同浓度的多种基质蛋白实验中发现,血小板的粘附依赖于胶原蛋白溶液浓度,浓度越大,粘附就越多;而纤维蛋白原分子的结构决定了血小板的粘附值;血清蛋白的惰性使得血小板几乎不与其发生结合反应。随后,设计了一种齿状通道作为实验模型,用于分析体内血管狭窄时血小板粘附的分布状况。实验中发现,在低剪切率下,在齿状通道的任何位置血小板的粘附值都要高于中高剪切率,且随着剪切率增加,通道中游区血小板数量逐渐低于上下游。最后,将不同浓度的阿司匹林作用于正常人的血液,结果发现阿司匹林用量越多,血小板粘附量就越低,说明药物的用量是与抑制程度成正比的。用二磷酸腺苷(ADP)试剂激活血小板来模拟血栓性疾病患者血小板的活化状态。施加不同浓度的阿司匹林抑制剂后,低剂量的阿司匹林并不能完全抑制血小板的粘附,粘附值仍高于正常水平,药物药效不明显;加大用量后,粘附值逐渐降低,趋于正常。但在高剂量下,粘附明显较低,说明血小板的凝血功能在减弱,在体内会发生出血事件。
[Abstract]:With the development of our country's economy and the improvement of the living standard, the disease of cardiovascular system has become an important factor that seriously threatens the people's health. Among them, the trend of the incidence of thrombotic diseases is particularly obvious. The pathogenesis of these diseases is related to the formation of pathological thrombus. At present, the prevention of thrombosis is an important means of prevention and treatment of these diseases. The pharmacological mechanism of antiplatelet drugs is to inhibit the adhesion and aggregation of platelets. In the development of antiplatelet drugs, the biological effects of drugs on different patients are not consistent, which makes monitoring individual patients' optimal treatment dose of antiplatelet drugs, minimizing thrombosis and reducing bleeding. Therefore, various methods and instruments for the detection of platelet function in vitro are widely used in the monitoring of antiplatelet therapy. However, the traditional platelet function testing devices and methods generally have many shortcomings, such as complex operation, long time use, marking, low accuracy and high price, which limits their popularization and application. In order to solve this problem, this paper uses a surface plasmon resonance sensing technique to study the function of platelets. The analysis technology has the advantages of high sensitivity, no labeling, simple operation, high throughput and so on. By measuring the platelet adhesion value, we can evaluate the change of platelet function, so as to achieve the monitoring of drugs in antiplatelet therapy. In this paper, the COMSOL simulation software is used to analyze several groups of microchannels with different width and height. Through the simulation of the distribution of the shear rate at the bottom of the channel, a group of channels with the most uniform distribution of shear rate are determined and the fabrication of the chip is completed. The adhesion of platelets on collagen was tested in the gas phase and the liquid phase. The experimental results showed that after the passage of blood, the refractive index of the gold film increased and the SPR angle increased. By calculating the increment of SPR angle, the specific value of platelet adhesion could be calculated. This proves that the SPR sensor is used to study the feasibility of platelet function detection with the reference standard of adhesion. At the same time, the technology can detect accurately any position of the channel, quantitatively analyze the distribution of platelets in the channel, and the experimental repeatability is high. The analysis area is divided into multiple small analysis points with little difference. The method of multipoint analysis can be used in an experiment to realize the effect of multiple detection in the traditional analysis, which greatly improves the efficiency of the test analysis. By comparing and analyzing the method of platelet adhesion detection under different test conditions, it is found that the effect of platelet adhesion detection in the liquid phase is better. In liquid phase, the effect of different factors on platelet adhesion was detected, and the shear rate and different matrix protein were studied. The results showed that within a certain range, the platelet adherence value increased with the increase of shear rate. When 500s-1, the adhesion value reached 2.958ng/mm2. Thereafter, the shear rate continued to increase, and the adhesion value gradually decreased. Found in a variety of experiments with different concentrations of matrix protein, platelet adhesion on collagen solution concentration, the greater the concentration, the more structure and adhesion; fibrinogen molecules determines the platelet adhesion value; serum protein and its inertness makes almost no platelet binding reaction. Subsequently, a dentate channel was designed as an experimental model to analyze the distribution of platelet adhesion during vascular stenosis in the body. In experiment, it was found that at low shear rate, the adhesion value of platelets at any location of dentate channel was higher than that of mid high shear rate, and with the increase of shear rate, the number of platelets in the middle passage of the channel was gradually lower than that of the upstream and downstream. Finally, different concentrations of aspirin were applied to the blood of normal people. It was found that the more the aspirin dosage, the lower the platelet adherence, which indicated that the dosage of drugs was directly proportional to the degree of inhibition. Platelet activation by adenosine two (ADP) reagents is used to simulate the activation of platelet in patients with thrombotic disease. After applying different concentration of aspirin inhibitor, low dose aspirin did not completely inhibit platelet adhesion. The adhesion value was still higher than the normal level. The drug efficacy was not obvious. After increasing the dosage, the adhesion value gradually decreased and tended to be normal. But at high dose, the adhesion is obviously lower, indicating that the blood coagulation function of the platelets is weakened, and the bleeding event will occur in the body.
【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R54

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