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Use of the Protease Fluorescent Detection Kit to Determine Protease Activity
Published on: August 4, 2009
Quantitative and label-free technique for measuring protease activity and inhibition using a microfluidic cantilever
Digvijay A Raorane1, Mark D Lim, Fanqing Frank Chen
1Department of Mechanical Engineering, University of California, Berkeley, California 94720, USA.
Nano Letters
|August 30, 2008
Summary
This study demonstrates a novel microcantilever sensor for precisely measuring protease activity and inhibition. The silicon nitride-based sensor system offers a sensitive platform for enzyme kinetics and drug discovery.
Area of Science:
- Biotechnology
- Nanotechnology
- Biochemistry
Background:
- Protease activity is crucial in biological processes and disease.
- Developing sensitive and quantitative methods for protease analysis is essential.
- Microcantilever technology offers a label-free, high-sensitivity platform for biomolecular detection.
Purpose of the Study:
- To develop and validate a silicon nitride-based microcantilever array for quantitative protease activity measurement.
- To assess the inhibition of immobilized trypsin using a model inhibitor.
- To establish a sensitive platform for enzyme kinetics and drug screening.
Main Methods:
- Immobilization of trypsin onto a gold-coated silicon nitride microcantilever array.
- Surface passivation using silanated polyethylene glycol.
- Quantitative measurement of enzyme activity via optical laser-deflection detection of cantilever motion.
- Assay of trypsin inhibition using soybean trypsin inhibitor (SBTI).
Main Results:
- Microcantilever deflections directly correlated with trypsin turnover of a synthetic fibronectin substrate.
- The Michaelis constant (K M) for trypsin was determined to be 0.58 x 10^-6 M.
- Successful quantitative observation of trypsin inhibition by SBTI.
Conclusions:
- The developed gold-coated silicon nitride microcantilever array is a viable tool for quantitative protease activity and inhibition measurements.
- This sensor platform provides a sensitive method for studying enzyme kinetics.
- The system holds potential for high-throughput screening in drug discovery and diagnostics.
