This project focused on developing a new strategy for the sensitive and selective measurement of the activity of matrix metalloproteinases (MMPs) and ADAMs (a disintegrin and metalloprotease) for potential point-of-care early cancer detection and diagnosis by combining nanopore stochastic sensing and substrate-based protease assay. 

The current activity-based protease assays have two major issues. One is the use of sophisticated instruments and/or labeled substrates which have to be carefully prepared with complexity and high cost, and may not behave the same as their natural counterparts.  The other is the low specificity of the substrates, which results in their limited diagnostic utility. To address these issues, we developed an innovative nanopore sensing strategy for the detection of proteases by real-time monitoring of the ionic current modulations caused by the protease-substrate peptide interactions in the nanopore and correlating their activities with the event frequency of the substrate breakdown products (Scheme). In addition to the label-free analysis, another significant advantage of our developed nanopore sensor was that we could take advantage of the cleavage site of the substrate to mitigate its specificity issue, thus greatly improving the selectivity of the protease sensor. In our sensing design, we can tolerate substrate degradation by other potential interfering proteases. However, if they cleave the substrate at different positions from that of the target protease, the substrate cleavage site allows the target analyte to be differentiated from others based on their produced quite different substrate breakdown products and their corresponding events with different signatures. To improve turnaround time, reduce sample / reagent volume, and decrease errors between inter-sampling, we developed an innovative non-array based multiplex nanopore sensing system for the simultaneous measurement of the activities of multiple MMPs/ADAMs by using a single nanopore and a single substrate which contained multiple cleavage sites. Although the conventional sensor array, in which each individual sensing probe is highly selective for a different analyte, has advantages of easy operation and simple data analysis, our developed non-array format multiplexing method may further reduce the assay cost. Moreover, in order to obtain the most cancer information with the fewest types of MMPs/ADAMs used, we pioneered an innovative joint-entropy assisted strategy for biomarker panel selection. In addition, to demonstrate the potential clinical diagnosis application of our developed protease detection technology, simulated serum samples were successfully analyzed.  Taken together, our developed nanopore sensing technology may find useful application in the development of stochastic sensors for a wide variety of proteases, and offer the potential as an effective label-free and real-time platform to profile protease activities for point-of-care early disease detection and diagnosis.

The broader impacts of this project were made by establishing a summer scholar research program for high school students and incorporating the new findings from this project in undergraduate and graduate classroom teaching for the benefit of students not directly involved in the research. The latter was demonstrated by developing capstone projects for Analytical Method Development Laboratory and Interprofessional Projects (IPRO) Program students. We also sought for collaboration with industry to initiate a startup company to construct miniature nanopore-based protease sensing devices for point-of-care clinical diagnosis applications and for research and education purpose. During this four-year grant period, six graduate students have worked on various aspects of this program.  Three of them graduated with Ph.D. and currently are doing their postdoctoral research. Two postdoctoral fellows, two undergraduate and one high school students have been trained.

Last Modified: 08/10/2021
Modified by: Xiyun Guan