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

Illinois Institute Of Technology, Chicago IL

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Abstract

Summary MMPs and ADAMs are currently under intensive investigation as novel biomarkers and potential therapeutic targets for the early detection and treatment of human cancers. However, most of the current techniques to detect MMPs/ADAMs are laborious and time-consuming, require the use of labels or sophisticated instruments, and/or have low specificity, which results in their limited diagnostic utility. Hence, improved analytical capability for their more rapid, sensitive, selective, and cost-effective detection remains a high priority. Our research effort of this 3-year R15 project is mainly focused on addressing such important aspects of the current substrate-based MMP/ADAM assay by demonstrating a novel, label-free, real-time, and multiplexed nanopore sensing system for the simultaneous quantification of the activities of multiple MMPs/ADAMs. This is the first but a crucial step toward our long-term project goal, i.e., to develop a cost-effective, portable device for profiling MMPs/ADAMs in serum samples for potential point-of-care early cancer detection and diagnosis. Aim 1: Demonstrate the feasibility of utilizing our proposed real-time label-free nanopore sensing strategy as an effective generic approach for the sensitive and accurate quantitative determination of the activities of MMPs/ADAMs. For this purpose, during the phase I period of this project, we will use ADAM-9 (a biomarker for pancreatic cancer) and ?-hemolysin protein nanopore as a model system to systematically examine the effects of various factors, including the substrate peptides, experimental conditions, and the inner surface functions of the nanopore, on ADAM-9 activity measurement. Further, we will study the sensor selectivity, investigate ADAM-9 inhibition, determine ADAM-9 kinetics, construct dose-response curve, etc.; Aim 2: Build on the nanopore-based ADAM-9 detection methodology developed in aim #1 to develop multiplexed nanopore sensors for the simultaneous quantification of the activities of multiple MMPs/ADAMs. Such a sensing system which uses a panel of MMPs/ADAMs instead of a single protease as cancer biomarkers can improve diagnosis accuracy and minimize false positives. Two types of multiplex systems will be constructed and evaluated: one involves the use of an array of nanopores and multiple substrates for different MMPs/ADAMs, while the other employs a single nanopore and a single substrate which contains multiple cleavage sites for different MMPs/ADAMs. Aim 3: Analyze mock serum samples. To proof-of-concept demonstrate the feasibility of utilizing our developed nanopore sensing platform for cancer detection and diagnosis, the multiplex nanopore sensor developed in aim #2 will be used to analyze 5 simulated clinical serum samples spiked with MMP-7, MMP-14, ADAM-9, and ADAM17 at concentrations / activities similar to those found in healthy people, pancreatic cancer, and ovarian cancer patients at various stages.