Nanoelectrode Array Based Electronic Biosensors for Rapid Profiling of Cancerous

描述（由申请人提供）：已经认识到各种酶如激酶、磷酸酶和蛋白酶的过表达会导致癌症。酶活性谱可用于癌症诊断、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，高度可靠的分析癌性蛋白酶的活性。我们假设，一个图书馆的肽连接到一个独立寻址的电极阵列，可用于通过映射在各个电极的电化学信号的癌症相关的蛋白酶的混合物的活动。电活性标签（即二茂铁，Fc）可以连接到每个肽的远端，其产生具有通过交流（AC）伏安法技术测量的放大幅度的特征性氧化还原信号。这种信号是可靠的，不像其他电子技术那样容易受到非特异性因素的干扰。在通过特异性蛋白酶如豆荚蛋白酶和间质蛋白酶进行蛋白水解时，特异性电极位点处的肽将被切割，并且所连接的Fc部分将离开电极表面。结果，氧化还原信号将随时间衰减，衰减速率与该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。NEA基于在单独寻址的微电极垫上生长的良好分离的垂直排列的碳纳米纤维（VACNF），然后用SiO2封装。将顶表面抛光或等离子体蚀刻以暴露直径约100 nm的碳纳米纤维（CNF）的最末端。肽底物共价官能化至CNF的末端。NEA允许非常少量的肽分子的选择性功能化，促进使用高频（~3 kHz）AC伏安法技术的极其灵敏和快速的电子检测。从长远来看，NEA可以被制造成单独寻址的多路芯片，使得仅使用5-10微升样品就可以使用许多（多达100种）肽底物同时检测多种蛋白酶。这将显著加快蛋白酶谱用于癌症诊断、分期、结果预测、预后和治疗选择。在本AREA应用范围内，针对我们的长期目标设计了三个具体目标：具体目标1：CNF NEA制造和特性表征，具体目标2：用豆荚蛋白和间质蛋白酶检测肽蛋白水解的方法开发，具体目标3：各种乳腺癌细胞系的蛋白酶谱分析。这一领域的应用将促进纳米生物传感器研究过渡到潜在的临床癌症诊断和治疗监测，通过三名物理科学和癌症生物学研究人员的合作，快速分析癌症相关蛋白酶。它将刺激研究生和本科生在堪萨斯州立大学的进一步职业发展，在健康科学和相关领域通过研究经验，在这个癌症检测项目。 公共卫生相关性：可靠、高特异性、超灵敏和低成本的电子技术对于快速筛查癌症疾病和监测癌症治疗非常重要。这项拟议的工作是开发一种基于纳米技术的电子方法，通过快速分析癌性蛋白酶的活性来检测癌症。这将显著加快癌症诊断、分期、结果预测、预后和治疗选择。

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.