基于微流控芯片在线检测全血中内毒素的新方法研究

Endotoxin can mediate sepsis which is major cause of death of critically ill patients. The levels of endotoxin in the blood are closely related to septic condition. This project will build an online, fast, accurate and high-throughput new determination method of whole blood endotoxin content based on a Microfluidic Chip. The endotoxin in blood will be selectively captured by fabricating sandwich-type membrane chip and membrane chip modified by specific capture endotoxin ligands. rFC without fluorescence is choosen as fluorescent reagents of endotoxin. Various sample preconcentration methods are adopted to improve detection sensitivity of microfluidic chip. The analysis processes are observed by using inverted fluorescence microscope. Manual error and contamination induced by offline sample pretreatment will be effectively avoided, and sample-in-answer-out will be achieved. Some parameters, such as adsorption efficiency of endotoxin, endotoxin-ligands interaction sites, the types of interaction forces, will be investigated. Using computer-aided design imitates interaction between ligands and endotoxin. All parameters obtained will provid a theoretical support for new method.The basic application will be investigated by online monitoring endotoxin levels of blood samples of CLP model rats using polymyxin B as a model drug. It is of extremely vital significance for clinical diagnosis and treatment of sepsis according to endotoxin content determined using the fast, ultra-trace detection new method.

内毒素诱发脓毒症是导致危重患者死亡的重要原因。内毒素水平是脓毒症病情诊断的重要指标。本项目拟建立基于微流控芯片的在线快速、准确、高通量测定全血中内毒素含量的新方法。通过含特异性捕捉内毒素配基的内置捕集膜芯片，完成临床血样中内毒素的在线选择性捕集。选择自身无荧光的重组因子C作为内毒素荧光标记物，采用多种富集方法提高检测灵敏度，使用荧光倒置显微镜实时观测分析过程。使用荧光显微镜观查异硫氰酸荧光素标记的内毒素在通道内的吸附情况，评价通道修饰效果。考察内毒素在捕集膜上的吸附效率、作用位点、作用力类型等参数。本项目建立的快速、超微量检测内毒素新方法对脓毒症病情临床诊断、治疗具有重要意义。

内毒素诱发脓毒症是导致危重患者死亡的重要原因。内毒素水平是脓毒症病情诊断的重要指标。本项目建立了基于微流控芯片的在线快速、高通量在线测定全血中内毒素含量的新方法。采用10%的牛血清蛋白修饰PDMS芯片表面，改善亲水性，减少内毒素在PDMS表面的吸附。利用壳聚糖和多粘菌素B制备捕集膜，特异性捕集样品中内毒素分子。采用具有聚集诱导荧光的（AIE）探针分子标记内毒素，有效的减少了探针分子本身对测定的干扰。由于样品中内毒素含量很低，因此发展在线富集技术是关键步骤。基于微流控芯片技术，我们探究了阴离子选择性富集膜对内毒素的在线富集效果，结果较满意。同时，本研究优化了多种在线富集方法，主要有场强放大、胶束解聚、扫集富集及pH连接等多种富集方法连用。本项目建立的多种在线富集方法对生物样本中微量超微量组分分析检测有重要的实用价值，快速、超微量检测内毒素新方法对脓毒症病情临床诊断、治疗具有重要意义。