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A high sensitive assay platform based on surface-enhanced Raman scattering for quantification of protease activity
Nazife Nur Yazgan1, Ismail Hakki Boyaci, Erhan Temur
1Department of Food Engineering, Faculty of Engineering, Hacettepe University, Ankara, Turkey.
Talanta
|July 7, 2010
Summary
A new surface-enhanced Raman scattering (SERS) assay using gold nanoparticles rapidly and sensitively measures proteolytic enzyme activity. This SERS method offers a flexible platform for analyzing enzyme preparations and substrate specificity.
Area of Science:
- Analytical Chemistry
- Biochemistry
- Nanotechnology
Background:
- Proteolytic enzymes play crucial roles in biological processes and industrial applications.
- Accurate and sensitive measurement of enzyme activity is essential for research and quality control.
- Existing methods for enzyme activity measurement can be time-consuming or lack sensitivity.
Purpose of the Study:
- To develop a novel, sensitive, and rapid assay for quantifying proteolytic enzyme activity.
- To utilize surface-enhanced Raman scattering (SERS) probes for enhanced detection.
- To explore the potential of gold nanoparticles in developing advanced biosensing platforms.
Main Methods:
- Synthesis of gold nanospheres and nanorods as surface-enhanced Raman scattering (SERS) probes.
- Coating nanoparticles with 5,5-Dithiobis (2-Nitrobenzoic acid) (DTNB) as a SERS label.
- Development of two SERS-based platforms: gold-coated glass slides and polystyrene microtiter plates.
- Optimization of nanoparticle shape and assay platform for protease detection.
Main Results:
- A linear correlation (R(2)=0.979) was established between SERS signal intensity and protease concentration (0.1-2 mU/mL).
- The assay demonstrated high sensitivity with a limit of detection (LOD) of 0.43 mU/mL and a limit of quantification (LOQ) of 1.30 mU/mL.
- Excellent precision was achieved, with intra-day and inter-day relative standard deviations (RSD) of 2.5% and 3.6%, respectively.
- The method was successfully applied to analyze commercial enzyme preparations and investigate protease substrate specificity.
Conclusions:
- A robust and sensitive SERS-based assay for quantitative protease activity measurement was successfully developed.
- The optimized assay using rod-shaped SERS probes on gold-coated glass slides offers superior performance.
- The developed SERS method provides a flexible and adaptable platform for diverse enzyme activity analyses and substrate specificity studies.
