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Low-volume multiplexed proteolytic activity assay and inhibitor analysis through a pico-injector array
Ee Xien Ng1, Miles A Miller, Tengyang Jing
1Department of Biomedical Engineering, National University of Singapore, 9 Engineering Drive 1, Singapore 117575, Singapore. biecch@nus.edu.sg.
Lab on a Chip
|January 3, 2015
Summary
A novel pico-injector array enables simultaneous measurement of multiple protease activities and inhibitor effects using minimal sample volumes. This technology advances the characterization of matrix metalloproteinases (MMPs) and ADAMs in complex biological samples.
Area of Science:
- Biochemistry
- Enzymology
- Proteomics
Background:
- Secreted proteases, including matrix metalloproteinases (MMPs) and ADAMs, are crucial in various pathological processes.
- Current methods for analyzing multiple protease activities (proteolytic activity matrix analysis - PrAMA) are limited by sample volume and experimental complexity.
Purpose of the Study:
- To develop a high-throughput platform for simultaneous measurement of multiple specific protease activities and inhibitor effects.
- To overcome sample quantity limitations and simplify the analysis of complex protease activities.
Main Methods:
- Development of a pico-injector array capable of generating 72 distinct reactions in picoliter-volume droplets.
- Utilizing controlled sequential injections for combinational enzyme reactions and inhibitor analyses.
- Simultaneous determination of multiple MMP activities using 9 substrates and 2 inhibitors.
Main Results:
- The pico-injector array platform successfully enabled simultaneous recording of multiple enzyme reactions and inhibitor effects with minimal sample volume (~10 μL).
- Characterization of MMP/ADAM activities in MDA-MB-231 breast cancer cells revealed high activity of MMP-2, MMP-3, and ADAM-10.
- Demonstrated the platform's capability for sensitive analysis through inhibitor effects.
Conclusions:
- The developed pico-injector array platform offers a sensitive and efficient method for analyzing multiple protease activities and their inhibition.
- This technology provides a valuable tool for characterizing protease profiles in various applications, including cancer research.
- The platform's modular design allows customization for diverse applications requiring multiplexed reactions and inhibitor analysis.
