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.
Proteases may serve as valuable diagnostic or prognostic markers for diseases states, and are becoming increasingly important targets for drug discovery and drug development. The long- term goal of this project is to develop a rapid, sensitive, accurate, cost-effective, and field-deployable nanopore sensing method for the multiplex 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.
|Wang, Liang; Han, Yujing; Zhou, Shuo et al. (2014) Real-time label-free measurement of HIV-1 protease activity by nanopore analysis. Biosens Bioelectron 62:158-62|
|Wang, Liang; Han, Yujing; Zhou, Shuo et al. (2014) Nanopore biosensor for label-free and real-time detection of anthrax lethal factor. ACS Appl Mater Interfaces 6:7334-9|