Nanoelectrode Array Based Electronic Biosensors for Rapid Profiling of Cancerous

描述（由申请人提供）：各种酶如激酶、磷酸酶和蛋白酶的过度表达已被认为是导致癌症的原因。酶活性分析可用于癌症诊断、治疗监测和药物发现。在这里，我们提出了一种无标记的电子方法，用于快速，超灵敏和高度可靠的癌性蛋白酶活性分析。我们假设，连接到独立定位电极阵列的肽库可以通过绘制单个电极上的电化学信号来分析癌症相关蛋白酶的混合物的活性。电活性标签（即二茂铁，Fc）可以附着在每个肽的远端，它产生一个特征氧化还原信号，其振幅通过交流伏安法测量。这种信号是可靠的，不像其他电子技术那样容易受到非特定因素的干扰。在特定的蛋白酶如豆蔻蛋白酶和基质酶的水解作用下，特定电极位点上的肽将被切割，附着的Fc片段将离开电极表面。结果，氧化还原信号将随时间衰减，衰减速率与特定蛋白酶的活性成反比。一种新的纳米技术，即嵌入式纳米电极阵列（NEAs），将用于提高检测速度和灵敏度。NEAs是基于分离良好的垂直排列的碳纳米纤维（VACNFs）生长在单独定位的微电极垫上，然后用SiO2包裹。顶部表面抛光或等离子蚀刻，以暴露直径约100纳米的碳纳米纤维（CNFs）的末端。肽底物被共价功能化到CNFs的末端。NEA允许对非常少量的肽分子进行选择性功能化，使用高频（~3 kHz）交流伏安法技术促进极其敏感和快速的电子检测。从长远来看，NEA可以制造成单独寻址的多路芯片，使用5-10微升的样品使用许多（多达100个）肽底物同时检测多种蛋白酶。这将显著加快蛋白酶谱分析在癌症诊断、分期、预后预测和治疗选择方面的应用。为了实现我们的长期目标，在本领域的应用范围内设计了三个特定目标：特定目标1:CNF NEA的制造和特性表征，特定目标2：用豆类和基质酶检测肽蛋白水解的方法开发，特定目标3：各种乳腺癌细胞系的蛋白酶谱。通过三位物理科学和癌症生物学的研究人员的合作，这个AREA应用程序将促进纳米生物传感器研究向潜在的临床癌症诊断和治疗监测的过渡，通过快速分析癌症相关蛋白酶。它将激励堪萨斯州立大学的研究生和本科生通过在这个癌症检测项目中的研究经验，在健康科学和相关领域进一步发展。

DESCRIPTION (provided by applicant): The overexpression of various enzymes such as kinases, phosphatases, and proteases has been recognized to lead to cancers. The enzyme activity profiling can be used for cancer diagnosis, therapeutic monitoring, and drug discovery. Here we propose a label-free electronic method for rapid, ultrasensitive, and highly reliable profiling of the activities of cancerous proteases. We hypothesize that a library of peptides attached to an independently addressed electrode array can be used to profile the activities of a mixture of cancer-related proteases through mapping the electrochemical signals at individual electrodes. An electroactive tag (i.e. a ferrocene, Fc) can be attached to the distal end of each peptide, which produces a characteristic redox signal with amplified amplitude measured by an alternate current (AC) voltammetry technique. This signal is reliable and not as easily interfered by nonspecific factors as other electronic techniques. Upon proteolysis by specific proteases such as legumain and matriptase, the peptides at a specific electrode site will be cleaved and the attached Fc moiety will leave the electrode surface. As a result, the redox signal will decay vs. time with the decay rate inversely proportional to the activity of that particular protease. A novel nanotechnology, i.e. embedded nanoelectrode arrays (NEAs), will be used to enhance the detection speed and sensitivity. The NEAs are based on well- separated vertically aligned carbon nanofibers (VACNFs) grown on individually addressed microelectrode pads and then encapsulated with SiO2. The top surface is polished or plasma etched to expose the very end of the carbon nanofibers (CNFs) of ~100 nm in diameter. Peptide substrates are covalently functionalized to the end of the CNFs. The NEA allows selective functionalization of very small amount of peptide molecules, facilitating extremely sensitive and fast electronic detection using high-frequency (~3 kHz) AC voltammetry techniques. In long term, the NEA can be fabricated into individually addressed multiplex chip, enabling the simultaneous detection of multiple proteases using many (up to 100) peptide substrates using only 5-10 microliter samples. This will significantly expedite protease profiling for cancer diagnosis, staging, outcome prediction, prognosis, and treatment selection. Three specific aims are designed within the scope of this AREA application toward our long-term goals: Specific Aim 1: CNF NEA fabrication and property characterization, Specific Aim 2: Method development for detecting peptide proteolysis with legumain and matriptase, Specific Aim 3: Protease profiling of various breast cancer cell lines. This AREA application will facilitate the transition of nanobiosensors research to potential clinical cancer diagnosis and therapeutic monitoring by rapid profiling cancer-related proteases through the collaboration of three researchers in physical sciences and cancer biology. It will stimulate graduate and undergraduate students at Kansas State University for further career development in health sciences and related fields through research experience in this cancer detection project. PUBLIC HEALTH RELEVANCE: Reliable, highly specific, ultrasensitive, and low-cost electronic technology is important for rapid screening of cancer diseases and monitoring cancer treatments. This proposed work is to develop a nanotechnology based electronic method for cancer detection through rapid profiling of the activities of cancerous proteases. This will significantly expedite cancer diagnosis, staging, outcome prediction, prognosis, and treatment selection.