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材料和设备方面取得的进展的基础上，包括纳米级场效应管(FET)、微流体和生物传感器。越来越清楚的是，开发技术平台对于癌症的早期发现、预后评估和治疗监测至关重要，能够以经济高效的方式对多个血清蛋白生物标记物以及多个遗传和表观遗传病变进行定量分析。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，用于一步检测血清蛋白标记物、DNA突变、多态和位点特异性甲基化。这项应用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，基于In_2O_3、NW和SWNT的FET可以作为有效的生物传感器，具有实现高特异性和高灵敏度的潜力。我们建议开发和测试NW/NT FET阵列在模型系统以及临床血清样本中用于多路传感的适用性。各种捕获配体的独特表面功能化将通过我们成熟的微流控传递系统实现，并将采用半导体工艺来创建建议的生物传感器。在具体的目标1到3中，我们将使用新型的NW/SWNT传感器来检测1)癌症相关蛋白质生物标记物(包括PSA、CEA、CA-125等标记物作为模型)；2)与癌症相关的DNA核酸序列变化(以K-ras突变为模型)；3)位置特异性DNA甲基化(表观遗传)癌症相关变化(以p16基因为模型)。在具体的目标4和5中，我们将集成一种新型的微流控传递和测试系统，并开发能够同时询问多个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，这是一种功能强大的设计，可实现分析物结合信号的互补，最大限度地减少复杂混合物的假阴性或阳性信号。将对这些传感器阵列的灵敏度、选择性、稳定性和重复性进行研究。在特定的目标6中，我们将检测癌症患者的血清，这些血清的相关生物标志物水平已知。这里涉及的应用将产生传感器阵列平台，这些平台极有可能产生用于癌症分子诊断和监测治疗效果的无标记、低成本、多重分析，并且可以很容易地扩展到其他疾病的应用。 项目描述/相关性：越来越清楚的是，为了实现使用基于血清的生物标记物(包括蛋白质、DNA突变和DNA的其他变化)对癌症进行早期发现和治疗监测的目标，关键是开发能够同时以经济高效的方式对多个生物标记物进行定量分析的技术平台。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，用于一步检测基于血清的蛋白质和DNA生物标志物。这一应用领域的研究极有可能产生新的设备，允许检测多个癌症相关生物标记物，能够用于癌症的早期检测和治疗监测。这种设备也很可能很容易扩展到其他疾病的应用中。

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.