Label-Free Nanopore Biosensor for Rapid, Ultrasensitive, and Multiplex Detection

用于快速、超灵敏和多重检测的无标记纳米孔生物传感器

• 批准号: 8689429
• 负责人: Xiyun Guan
• 金额: $ 32.36万
• 依托单位: ILLINOIS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8689429
• 关键词: Acquired Immunodeficiency Syndrome; Alzheimer' s Disease; Amino Acid Sequence; Area; Arginine; Bathing; Biological; Biosensing Techniques; Biosensor; Cleaved cell; Detection; Devices; Diagnostic; Disease; Dose; Drug Industry; Event; Gelatinase A; Generic Drugs; Goals; Grant; HIV-1; Hemolysin; Human; Hydrolysis; Ionic Strengths; Label; Lead; Length; Life; Lysine; Malignant Neoplasms; Measurement; Measures; Methodology; Methods; Modeling; Molecular Profiling; Monitor; Nanotechnology; Peptide Hydrolases; Peptides; Phase; Physiological; Prognostic Marker; Proteins; Research; Series; Societies; Sodium Chloride; Solutions; Staging; Surface; System; Techniques; Time; Trypsin; Work; base; clinical Diagnosis; cost effective; data acquisition; disease diagnosis; drug development; drug discovery; functional group; human disease; improved; instrument; multiplex detection; nanopore; novel; pancreatic secretory trypsin inhibitor I; point of care; point-of-care diagnostics; public health relevance; residence; response; secretase; sensor; serum sodium transport inhibitor; success; voltage

DESCRIPTION (provided by applicant): Measurement of protease activities has broad applications in disease diagnosis and staging, drug discovery and development, and molecular profiling. However, most of the current protease detection methods are laborious and time-consuming, and/or require the use of labels or sophisticated instruments, and hence, improved analytical capability for their more rapid, sensitive, selective, and cost-effective detection remains a high priority. The long-term goal of this project is to develop such a requisite field-deployable sensing system for the multiplexed detection of protease activities for point-of-care diagnostics. Success in this endeavor will have a broad impact on a variety of areas such as clinical diagnosis, pharmaceutical industry, biosensing, and nanotechnology, offering the potential to lead to remarkable improvement in many facets of human life and society. This present proposal describes the first step toward this goal, where we will develop a novel, label-free, real-time nanopore-based sensing methodology for the highly sensitive and selective detection of proteases. The specific aims for the three-year R15 grant period are as follows: 1) Demonstrate the feasibility of utilizing nanopore sensors as an effective generic approach to detect protease activities. For this purpose, during the phase I period of this project, we will us trypsin as a model protease to examine the effects of various factors, including the substrate peptides, experimental conditions, and the inner surface functions of the nanopore, on trypsin activity detection. Further, we will study the sensor selectivity, and investigate trypsin inhibitin; 2) Build on the developed nanopore-based protease detection methodology to explore its applications in measuring protease activities involved in major human diseases. HIV-1 protease, matrix metalloproteinase-2 (MMP-2), and ¿-secretase are valuable diagnostic markers for AIDS, cancer, and Alzheimer's disease, respectively. We will take advantage of highly specific substrate peptides to develop ultrasensitive nanopore sensors for the detection of the activities of these proteases. The selectivities of these protease sensors and protease inhibition will also be studied; 3) Simultaneous detection of HIV-1 protease and MMP-2. To demonstrate the multiplexing capabilities of our proposed nanopore sensor, we will use highly sensitive and selective substrate peptide probes to measure the activities of HIV-1 protease and MMP-2 concurrently.

描述(由申请人提供)：蛋白酶活性的测量在疾病诊断和分期、药物发现和开发以及分子图谱方面有着广泛的应用。然而，目前的大多数蛋白酶检测方法费时费力，并且/或者需要使用标记或复杂的仪器，因此，提高分析能力以进行更快速、灵敏、选择性和成本效益的检测仍然是当务之急。该项目的长期目标是开发这样一种必要的现场可部署的传感系统，用于多路检测用于护理点诊断的蛋白酶活性。这一努力的成功将对临床诊断、制药工业、生物传感和纳米技术等多个领域产生广泛影响，为人类生活和社会的许多方面带来显著改善的潜力。本提案描述了迈向这一目标的第一步，我们将开发一种新的、无标记的、基于纳米孔的实时传感方法，用于高灵敏和选择性的蛋白酶检测。为期三年的R15授权期的具体目标如下：1)证明利用纳米孔传感器作为检测蛋白酶活性的有效通用方法的可行性。为此，在本项目的第一阶段期间，我们将以胰酶为模型酶，考察底物多肽、实验条件以及纳米孔内表面功能等各种因素对胰蛋白酶活性检测的影响。进一步，我们将研究传感器的选择性，并研究胰蛋白酶抑制素；2)建立基于纳米孔的蛋白酶检测方法，探索其在检测涉及人类重大疾病的蛋白酶活性方面的应用。HIV-1蛋白酶、基质金属蛋白酶-2(MMP2)和分泌酶分别是艾滋病、癌症和阿尔茨海默病的有价值的诊断标志物。我们将利用高度特异的底物多肽来开发超灵敏的纳米孔传感器来检测这些蛋白酶的活性。3)同时检测HIV-1蛋白水解酶和基质金属蛋白酶-2。为了展示我们所提出的纳米孔传感器的多路复用能力，我们将使用高灵敏度和选择性的底物多肽探针来同时检测HIV-1蛋白酶和基质金属蛋白酶-2的活性。