Multiplexed Nanosensing for DNA and Proteins

描述（由申请人提供）：本申请建立在南加州大学研究人员在纳米级材料和器件（包括纳米级场效应晶体管（FET）、微流体和生物传感器）方面取得的进展的基础上。越来越清楚的是，关键是要开发技术平台，允许以具有成本效益的方式定量分析多种血清蛋白质生物标志物以及多种遗传和表观遗传病变，用于癌症的早期检测、预后评估和治疗监测。为了解决这一关键的公共卫生问题，该项目的总体目标是开发和验证新型纳米线（NW）和单壁碳纳米管（SWNT）传感器阵列，用于血清蛋白标志物，DNA突变，多态性和位点特异性甲基化的单步检测。该申请的重点是开发可用于临床环境的多路复用纳米传感器阵列。我们已经证明了In 2 O3 NW和SWNT基FET可以用作有效的生物传感器，具有实现高特异性和灵敏度的潜力。我们建议开发和测试的适用性的NW/NT FET阵列的多路传感模型系统以及在临床血清样本。独特的表面功能化的各种捕获配体将通过我们证明微流体输送系统实现，和半导体加工将被用来创建拟议的生物传感器。在特定目标1至3中，我们将使用新型NW/SWNT传感器来检测1）癌症相关蛋白质生物标志物（包括PSA、CEA、CA-125和其他标志物作为模型）; 2）与癌症相关的DNA核酸序列变化（使用K-ras突变作为模型）;和3）位点特异性DNA甲基化（表观遗传）癌症相关变化（使用p16基因作为模型）。在具体目标4和5中，我们将整合一种新的微流体输送和测试系统，并开发能够同时询问多种生物标志物的纳米传感器阵列。基于NW和SWNT的设备将并排集成到同一阵列中，这是一种强大的设计，将允许分析物结合的信号互补，最大限度地减少复杂混合物的假阴性或阳性信号。这些传感器阵列的灵敏度，选择性，稳定性和重现性将进行研究。在具体目标6中，我们将测试来自具有已知水平的相关生物标志物的癌症患者的血清。本文所述的应用将导致传感器阵列平台，其极有可能产生用于癌症的分子诊断和监测治疗功效的无标记、低成本、多重测定，并且其也可以容易地扩展到其他疾病的应用。 项目叙述/相关性：越来越清楚的是，为了实现使用基于血清的生物标志物（包括蛋白质、DNA突变和DNA中的其他变化）对癌症进行早期检测和治疗监测的目标，关键是开发允许同时定量分析多种生物标志物并且具有成本效益的技术平台。为了解决这一关键的公共卫生问题，该项目的总体目标是开发和验证新型纳米线（NW）和单壁碳纳米管（SWNT）传感器阵列，用于基于血清的蛋白质和DNA生物标志物的单步检测。本申请中的研究极有可能产生新型装置，其将允许检测能够用于癌症的早期检测和治疗监测的多种癌症相关生物标志物。这种设备也很可能很容易扩展到其他疾病的应用。

DESCRIPTION (provided by applicant): This application builds on the advances that the USC investigators have made in nanoscale materials and devices, including nanoscale field effect transistors (FETs), microfluidics and biosensors. It has become increasingly clear that it is critical to develop technology platforms that permit quantitative analysis of multiple serum protein biomarkers as well as multiple genetic and epigenetic lesions in a cost-effective manner for early detection, prognostic evaluation and therapeutic monitoring of cancer. To address this critical public health issue, the overall goal of this project is to develop and validate novel nanowire (NW) and single-walled carbon nanotube (SWNT) sensor arrays for single-step detection of serum protein markers, and of DNA mutation, polymorphism and site-specific methylation. This application is focused on the development of multiplexed nanosensor arrays that can be used in a clinical setting. We have demonstrated that In2O3 NW and SWNT- based FETs can be used as effective biosensors with a potential to achieve both high specificity and sensitivity. We propose to develop and test the applicability of the NW/NT FET arrays for multiplexed sensing in model systems as well as in clinical serum samples. Unique surface functionalization for various capture ligands will be achieved via our proven microfluidic delivery system, and semiconductor processing will be employed to create the proposed biosensors. In Specific Aims 1 to 3, we will used novel NW/SWNT sensors to detect 1) cancer related protein biomarkers (including PSA, CEA, CA-125 and other markers as the model); 2) DNA nucleic acid sequence changes related to cancer (using K-ras mutations as the model); and 3) site- specific DNA methylation (epigenetic) cancer related changes (using p16 gene as the model). In Specific Aims 4 and 5, we will integrate a novel microfluidic delivery and test system and develop nanosensors arrays capable of interrogating multiple biomarkers simultaneously. Both NW and SWNT-based devices will be integrated side-by-side into the same array, a powerful design which will allow complementarily of signals for analyte binding, minimizing false negative or positive signals for complex mixtures. The sensitivity, selectivity, stability and reproducibility of these sensor arrays will be investigated. In Specific Aim 6, we will test serum from cancer patients with known levels of relevant biomarkers. The applications addressed here will result in sensor array platforms that are highly likely to yield label-free, low-cost, multiplexed assays for molecular diagnosis and monitoring therapeutic efficacy for cancer, and which can easily be extended to applications to other diseases as well. Project Narrative/Relevance: It is becoming increasingly clear that in order to achieve the goal of early detection and therapeutic monitoring of cancer using serum based biomarkers, including proteins, DNA mutations, and other changes in DNA, it is critical to develop technology platforms that permit quantitative analysis of multiple biomarkers simultaneously and a cost effective manner. In order to address this critical public health issue, the overall goal of this project is to develop and validate novel nanowire (NW) and single-walled carbon nanotube (SWNT) sensor arrays for single step detection of serum based protein and DNA biomarkers. The research in this application is highly likely to result in novel devices that will allow for the detection of multiple cancer associated biomarkers capable of use in early detection and therapeutic monitoring of cancer. It is also likely that this device will be easily extended to applications in other diseases as well.