基于纳应力传感和微接触印刷技术的肝癌标志物组合高灵敏SERS检测研究

Developing a simple, highly specific, and highly sensitive multiplex detection method is critical for the early diagnosis of liver cancer. The current state-of-the-art hepatocellular carcinoma (HCC) diagnosis methods for early-stage liver cancer detection however still have great limitations. This project is focused on combining SERS-based nano-stress sensing with micro-contact printing to achieve multiple biomarker detection with high sensitivity, which has rarely been reported until now. Compared with the traditional SERS method which relies solely on sensing via the enhanced Raman spectrum of the molecule or protein of interest, the SERS nano-stress technique utilizes instead the sensitive response of a Raman reporter to changes in its chemical environment due to (for instance) the binding of a target molecule/protein. This new SERS-based biosensor has the potential to decrease the influence of nonspecific protein adsorption and thus increase detection accuracy. It also allows for easier Raman signal acquisition by use of a standard Raman reporter, overcoming the disadvantage of relying too heavily on material performance. Integration of the micro-contact printing technique and self-assembly methods could build different functional domains for the efficient immobilization of multiple antibodies on a substrate surface and subsequent recognition of their corresponding antigens (biomarkers), thus realizing highly sensitive, multiplex detection. By analyzing the shifts in the Raman spectrum of the reporter before and after introducing antibody and antigen, respectively, and combining quantum mechanics as well as DFT theoretical modeling, we aim to reveal more about the mechanisms of nano-stress regulation in the chemical bonds, and to provide experimental and theoretical guidance for designing and preparing new biosensors with higher sensitivity.

早期肝癌检出率低的现状表明目前肝癌诊断方法还有很大的局限性。研究并建立简便、高特异性和高灵敏度的多种标志物同时检出的方法对肝癌早诊具有重要意义。本项目提出将纳应力(nano-stress)传感与微接触印刷技术相结合对肝癌标志物组合进行高灵敏SERS检测，目前未见相关报道。该方法的优势在于（1）克服了传统SERS检测灵敏度过分依赖材料性能的缺点，（2）避免了传统检测过程中较长免疫反应链带来的较强非特异性干扰，提高检测的准确性。本项目利用微接触印刷技术在材料表面构筑多功能微区，实现不同微区固定不同抗体及对相应抗原的特异性识别，利用拉曼光谱对化合物结构变化的高灵敏响应实现对肝癌标志物的联合检测。对比分析引入抗体及识别抗原前后信号分子拉曼峰的位移变化，探讨抗体和抗原对信号分子的作用区别，结合理论计算，揭示纳应力对信号分子的微观作用机制，为进一步设计超高灵敏的纳米生物传感器提供实验基础和理论指导。

研究并建立简便、高特异性和高灵敏度的多种标志物同时检出的方法对肝癌早诊具有重要意义。申请人提出将纳应力(nano-stress)传感与微接触印刷技术相结合对肝癌标志物组合进行高灵敏SERS检测。通过理论模拟和计算，揭示纳应力对信号分子化学键的微观作用机制，为进一步设计超高灵敏的纳米生物传感器提供理论指导。在本项目执行期间内，我们利用纳应力传感技术重点围绕肝癌蛋白标志物和基因标志物的多组分、高灵敏联合检测展开了研究，取得了一些重要的结果，主要研究结果和数据如下：（1）对多种类肝癌标志物进行了高灵敏、联合检测，实现血清中microRNA 飞摩尔(fM)量级的检测限，克服了传统方法不能测得循环肿瘤microRNA（miRNA）在血清中绝对含量的缺点；通过联合检测microRNA与AFP，实现对肝癌易混类型 (HCC与ICC)的精准诊断；（2）设计了枝状Y-DNA探针实现信号放大，进一步提高纳应力传感技术对microRNA的检测灵敏度，达到10 aM；（3）基于纳应力传感技术和酶促循环放大对肺癌标志物循环肿瘤DNA的超灵敏检测，检测限达到0.1 fM，并且能从10000个正常DNA 分子中，识别出1个基因突变的ctDNA；（4）基于MOF 材料构建人工嗅觉系统进行肺癌呼出标志物的高灵敏、联合检测；（5）构建了一种免标记、灵敏度高和特异性强的Ag-HBA传感器，利用拉曼频移检测手段检测痕量锌离子，检出限低至10 fM。在此基础上，我们将纳应力传感方法拓展应用到纳米药物与蛋白的相互作用研究中，探讨了口服纳米药物与关键消化蛋白酶的相互作用。以上这些工作具有重要的学术价值，有助于解决肝癌早期诊断困难，灵敏度差等挑战性难题，在纳应力传感和微接触印刷技术相结合的基础上，实现对肝癌标志物组合的高灵敏检测，同时也希望为其他肿瘤的早期诊疗开拓新思路。