基于荧光化学传感阵列的氨基酸类癌症标志物检测研究

Cancer has become a major disease that seriously jeopardizes human life and health, so new methods and technologies for early detection of cancer are important for the early screening and treatment of cancer patients and improvement of survival rate. However, current detection methods are cumbersome, has single cancer markers detection, low sensitivity and specificity, which may result in “false positive” results..To obtain fluoresce bioprobes with specific selectivity, this project will focus on the molecular design of smart fluoresce bioprobes for the detection of precancerous small molecule markers amino acids from human metabolite. Based on intelligent fusion of a number of fluorescent probes, we study on the design of fluorescence chemical sensor for multi-marker detections. The pH value difference of precancerous population metabolites and the specificity detection of amino acid as cancer marker are considered by taking advantage of the flowing three important factors into account. (1) The fluorescence probe optical signal being controlled by metabolites pH; (2) the specific recognition of amino acid small molecule; (3) multi-marker detections by intelligent fusion of a number of fluorescent probes..This project intends to study the model construction of fluorescent probes, microfluidic chip technology and surface molecular modification techniques. Meanwhile, through the detailed investigation of spectral signals, electronic transport and other information, we plan to establish and refine fluorescent chemical sensor array models with amino acid molecular detection functions. And we develop multi-marker co-detection technology of precancerous population metabolites for early detection of cancer with the improvement of the detection selectivity and sensitivity to obtain the new cancer early detection model.

癌症是威胁人类生命健康的一类重要疾病，研究早期检测的新方法和新技术对于癌症人群的早期筛查和治疗、提高生存率具有重要的意义。但现存检测方法过程繁琐、标志物检测单一、敏感性和特异性低，可能会带来“假阳性”结果。本项目围绕开发特异性强、可从人体代谢物中高选择性检出癌前人群癌症标志物的荧光探针，并进行多探针的智能融合，构建多标志物联合检测的荧光化学传感器。利用癌前人群代谢物pH差异及癌症标志物氨基酸检测的特异性，从以下三方面入手：1）荧光信号强度受检测物pH差异；2）探针满足氨基酸小分子特异性识别；3）多探针融合实现多标志物联合检测。研究荧光探针的模型构建、微流控芯片技术和表面分子修饰机理，在此基础上协同光谱信号和电子输运等信息，筛选和完善具有氨基酸小分子检测功能的荧光化学传感阵列模型。发展癌前人群代谢物的多标志物联合检测技术，提高对癌症标志物检测的特异选择性及灵敏度，获得癌症早期诊断的新模型。

癌症的早期检测对于癌症人群的早期筛查和治疗、提高生存率具有重要的意义。但现存检测方法具有过程繁琐、标志物检测单一、敏感性和特异性低的不足，可能会带来“假阳性”结果。本项目针对这些问题做了如下研究：（1）首先从探针材料设计方面，设计并合成了3个对生物硫醇类小分子选择性检测的荧光探针，以及1个对pH检测的荧光探针。以NBD-Cl为识别位点，构建对生物硫醇类的氨基酸具有选择性的荧光探针Rhodol-NBD、DPM-NBD和MPM-NBD。对探针分子的结构进行表征并对光谱性能做了分析。Rhodol-NBD、DPM-NBD对半胱氨酸的检测限分别达到3.4×10-9 M、3.3×10-8 M，探针MPM-NBD对L-Cys/Hcy/GSH的检测限分别为1.63×10-9 M，1.69×10-9 M和1.18×10-9 M，线性关系分别为R2 = 0.996，0.997，0.999。检测限低于同类型已报道的探针检测限，说明探针MPM-NBD对L-Cys/Hcy/GSH有较高的检测灵敏度。通过一步合成方法，设计合成了1个对pH高灵敏、高选择性响应的荧光探针PH-2Cy。在pH值1.0-10.0范围内探针PH-2Cy紫外光谱的pKa = 6.2（R2= 0.997），激发波长520 nm的荧光光谱中探针的pKa = 4.95（R2 = 0.999），这意味着探针在pH值附近非常敏感，实现了我们将探针PH-2Cy用于pH条件的预期目标。（2）荧光传感阵列的设计方面，采用物理包埋法制备海绵芯片负载荧光探针，用于小分子的检测。该PDMS海绵结构芯片，其核心为吸附染料的PDMS孔洞芯片，由于PDMS海绵结构具有良好的吸附性，并且具有优良柔韧性，易加工及成本低等特性，通过将荧光染料吸附在PDMS海绵结构中，避免了化学键连及物理旋涂的不足。因需要设计流道结构，优化了荧光传感阵列模型，设计了翻转式芯片结构，制备的阵列式储液池可以实现多种目标物同时检测及不同浓度梯度检测。（3）最后设计温度模块，对加热模块进行了仿真处理，初步进行升温曲线的测试，确定双排蛇形结构作为温控模块加热电极。进一步需要对荧光化学传感阵列模型优化，并利用该模型实现对氨基酸的检测。通过本项目的开展，期望用于氨基酸类癌症标志物的特异性及灵敏度检测，为癌症的早期筛查提供相关科学依据。