基于CMOS兼容硅纳米线FET的超灵敏肺癌标志物-miRNA传感器

Among all cancers, lung cancer is the most life threatening and prevalent worldwide. In most cases, lung cancer is diagnosed at an advanced stage when treatment outcomes are unfavorable. Biomarker detection is the preferred approach for early stage screening of lung cancer because of their unique association with genomic changes. Recent studies demonstrated that miRNAs play important roles in gene regulation and are considered as useful diagnostic and prognostic biomarkers for early dignosis of lung cancer. Due to the short length, very similar nucleotide sequences, and quite low expression levels, it is challenging to develop rapid and sensitive methods for miRNA analysis.In this proposal, we develop a semiconducting silicon nanowire (SiNW)-based field-effect transistor (FET) biosensor array for ultrasensitive, label-free and real-time detection of miRNAs, which are up-regulated in lung cancer. SiNWs with high surface-to-volume-ratios are top-down fabricated with complementary metal oxide semiconductor (CMOS) compatible anisotropic etching technique. To functionalize the surfaces of the SiNWs, SiNW chips were immersed in a solution of 3-aminopropyltriethoxysilane (APTES). Peptide nucleic acids (PNAs), which serve as capture probes to recognize miRNAs are covalently modified onto the surfaces of the SiNWs. MiR-21 and miR-205 are choosed as the target miRNAs associated with lung cancer. Conductivity (or current) change will be measured after hybridization correlated to a series of concentrations of target miRNA, and the calibration curve is obtained for the miRNA assay. In order to further test the selectivity of this nanosensor, one-base mismatched miRNA sequences are analyzed. To demonstrate the performance of the biosensor in the detection of clinically relevant samples, total RNA samples isolated from human lung cancer are analyzed by SiNW-based nanosensors.

寻找新的分子标志物对于肺癌的早期诊断及治疗具有重要的意义。已有的研究表明，作为肺癌早期诊断的一种新型生物标志分子，miRNA在基因调控中起着关键的角色。但是miRNA 存在长度短、相似度高及表达量低等问题，实现对其超灵敏、无标记、快速检测具有很大的挑战性。本项目旨在建立一种新型的硅纳米线FET生物传感器，通过自上而下的方法、各向异性自停止湿法腐蚀技术制作出CMOS兼容的硅纳米线；对硅纳米线表面硅烷化处理，将新型探针分子PNA通过化学作用自组装在纳米线的表面，选择肺癌中上调表达的miR-21和miR-205作为目标分析对象；对目标物进行定量分析，建立相关的工作曲线；进一步对相应的碱基错配序列进行考察，分析传感器的选择性；最终实现对肺癌实际样本total RNA的超灵敏、无标记、快速检测，为肺癌的早期诊断提供指导。

(1)项目背景：.发展简单快速灵敏的miRNA传感器对于肿瘤早期诊断和治疗等具有重要意义。但目前硅纳米线miRNA的检测仍存在一些问题：大部分传感器硅纳米线采用自下而上方法制得，往往存在器件制作困难、难以实现批量生产以及与硅集成工艺相兼容问题；硅纳米线传感器对其检测存在本底噪音大，信噪比相对低，以及测试易受外界因素干扰等问题。.(2)主要研究内容：.本项目通过建立CMOS兼容硅纳米线FET生物传感器，实现了对肺癌相关miRNA超灵敏、无标记、实时检测，为肺癌早期诊断和早期治疗提供了指导。研究内容包括：1)自上而下硅纳米线批量制造优化。进一步发展各向异性湿法腐蚀技术，解决均一性良好硅纳米线批量制造问题。2)微流控一体化硅纳米线芯片设计制备。通过设计合理PDMS模块和微流控通道，解决密闭反应环境和样品自动流入流出问题。3)实验体系选择和目标物miRNA分析检测。选择肺癌中上调表达明显的miR-21和miR-205作为目标分析对象；优化实验条件下，分析miRNA传感器的灵敏度和选择性。4)在前面实验基础上，最后对肺癌实际样本进行检测分析。.(3)重要结果、关键数据及科学意义.1)针对硅纳米线高精度和批量加工制造要求，通过机理研究以及各向异性腐蚀和自限制氧化，开展了硅纳米线制造一致性方法研究，实现了硅纳米线尺寸精确可控和批量加工制造。申请专利3项，授权专利1项。.2)针对硅纳米线芯片一体化结构设计和功能设计要求，结合微流控技术，通过MEMS加工工艺和表面修饰等，构建了PDMS微流控输送集成硅纳米线生物芯片。授权专利1项。.3)基于硅纳米线器件以及生物探针分子精确组装，发展了硅纳米线传感器对两种肺癌标志物miRNA分子无标记、高灵敏检测，最低检测量可至1 zmol，并实现了对肺癌真实样品的初步检测。文章发表在2014年Small。.4)基于CMOS兼容硅纳米线芯片和抗原/抗体免疫反应，发展了一种对蛋白质分子无标记电学检测新策略，该方法响应快速，灵敏度高，选择性好。文章发表在2014年ACS Appl. Mater. & Inter.。.5) 通过MEMS微加工工艺，构建了PDMS双通道微流控集成硅纳米线FET阵列传感器，用于两种不同肿瘤蛋白标志物的无标记、超灵敏、特异性电检测，溶液体系及人血清样品最低分别检测至1和10 fg/mL。文章发表在2015年Anal. Chem.。