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谱分析中具有广泛的应用。然而，大多数目前的蛋白酶检测方法是费力和耗时的，和/或需要使用标签或复杂的仪器，因此，提高分析能力，使其更快速，灵敏，选择性和成本效益的检测仍然是一个高度优先事项。该项目的长期目标是开发这样一个必要的现场部署的传感系统的多重检测点的护理诊断的蛋白酶活性。这一奋进的成功将对临床诊断、制药工业、生物传感和纳米技术等各个领域产生广泛的影响，并有可能在人类生活和社会的许多方面带来显着的改善。本提案描述了实现这一目标的第一步，我们将开发一种新的，无标记的，实时的基于纳米孔的传感方法，用于蛋白酶的高灵敏度和选择性检测。三年R15资助期的具体目标如下：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，我们将使用胰蛋白酶作为模型蛋白酶来考察各种因素，包括底物肽、实验条件和纳米孔的内表面功能，对胰蛋白酶活性检测的影响。进一步研究传感器的选择性，并探讨胰蛋白酶抑制剂; 2）基于纳米孔的蛋白酶检测方法，探索其在检测人类主要疾病相关蛋白酶活性方面的应用。HIV-1蛋白酶、基质金属蛋白酶-2（MMP-2）和分泌酶分别是AIDS、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性的底物肽来开发超灵敏的纳米孔传感器，用于检测这些蛋白酶的活性。这些蛋白酶传感器的选择性和蛋白酶抑制也将进行研究; 3）同时检测HIV-1蛋白酶和MMP-2。为了证明我们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底物肽探针来同时测量HIV-1蛋白酶和MMP-2的活性。