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A high sensitive assay platform based on surface-enhanced Raman scattering for quantification of protease activity.
Talanta. 2010 Jul 15; 82(2):631-9.T

Abstract

In this study, a new, sensitive, and rapid assay was developed to quantitatively measure the proteolytic enzyme activity using the surface-enhanced Raman scattering (SERS) probe. Two different shapes of gold nanoparticles, gold nanosphere and nanorod particles were produced. SERS label, comprising self-assembled monolayers (SAMs) of Raman reporter molecule (5,5-Dithiobis (2-Nitrobenzoic acid), DTNB), was coated on the surface of the nanoparticles. Two different SERS-based analysis platforms were designed using gold-coated glass slide and polystyrene microtiter plate. The calibration curves were obtained by plotting the intensity of the SERS signal of symmetric NO(2) stretching of DTNB at 1326 cm(-1) vs. the protease concentration. The effects of nanoparticle geometry and assay platform on the protease assay were investigated and the best working combination of the parameters was selected as rod shaped SERS probe and gold-coated glass slide. The correlation between the protease activity and SERS signal was found to be linear within the range of 0.1-2 mU/mL (R(2)=0.979). The limit of detection (LOD) and limit of quantification (LOQ) values of the validated method were found as 0.43 and 1.30 mU/mL, respectively. The intra-day and inter-day precisions of the method, as relative standard deviation (RSD), were determined as 2.5% and 3.6%, respectively. The developed method was successfully applied for quantitative analysis of the commercial enzyme preparate that is used in cheese making process. It was also used for investigation of substrate specificity of protease enzyme towards the casein and bovine serum albumin. The proposed method has a flexibility to try different substrates for the detection of various enzyme activities.

Authors+Show Affiliations

Department of Food Engineering, Faculty of Engineering, Hacettepe University, Ankara, Turkey.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

20602947

Citation

Yazgan, Nazife Nur, et al. "A High Sensitive Assay Platform Based On Surface-enhanced Raman Scattering for Quantification of Protease Activity." Talanta, vol. 82, no. 2, 2010, pp. 631-9.
Yazgan NN, Boyaci IH, Temur E, et al. A high sensitive assay platform based on surface-enhanced Raman scattering for quantification of protease activity. Talanta. 2010;82(2):631-9.
Yazgan, N. N., Boyaci, I. H., Temur, E., Tamer, U., & Topcu, A. (2010). A high sensitive assay platform based on surface-enhanced Raman scattering for quantification of protease activity. Talanta, 82(2), 631-9. https://doi.org/10.1016/j.talanta.2010.05.023
Yazgan NN, et al. A High Sensitive Assay Platform Based On Surface-enhanced Raman Scattering for Quantification of Protease Activity. Talanta. 2010 Jul 15;82(2):631-9. PubMed PMID: 20602947.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A high sensitive assay platform based on surface-enhanced Raman scattering for quantification of protease activity. AU - Yazgan,Nazife Nur, AU - Boyaci,Ismail Hakki, AU - Temur,Erhan, AU - Tamer,Uğur, AU - Topcu,Ali, Y1 - 2010/05/16/ PY - 2010/02/20/received PY - 2010/05/05/revised PY - 2010/05/10/accepted PY - 2010/7/7/entrez PY - 2010/7/7/pubmed PY - 2010/11/17/medline SP - 631 EP - 9 JF - Talanta JO - Talanta VL - 82 IS - 2 N2 - In this study, a new, sensitive, and rapid assay was developed to quantitatively measure the proteolytic enzyme activity using the surface-enhanced Raman scattering (SERS) probe. Two different shapes of gold nanoparticles, gold nanosphere and nanorod particles were produced. SERS label, comprising self-assembled monolayers (SAMs) of Raman reporter molecule (5,5-Dithiobis (2-Nitrobenzoic acid), DTNB), was coated on the surface of the nanoparticles. Two different SERS-based analysis platforms were designed using gold-coated glass slide and polystyrene microtiter plate. The calibration curves were obtained by plotting the intensity of the SERS signal of symmetric NO(2) stretching of DTNB at 1326 cm(-1) vs. the protease concentration. The effects of nanoparticle geometry and assay platform on the protease assay were investigated and the best working combination of the parameters was selected as rod shaped SERS probe and gold-coated glass slide. The correlation between the protease activity and SERS signal was found to be linear within the range of 0.1-2 mU/mL (R(2)=0.979). The limit of detection (LOD) and limit of quantification (LOQ) values of the validated method were found as 0.43 and 1.30 mU/mL, respectively. The intra-day and inter-day precisions of the method, as relative standard deviation (RSD), were determined as 2.5% and 3.6%, respectively. The developed method was successfully applied for quantitative analysis of the commercial enzyme preparate that is used in cheese making process. It was also used for investigation of substrate specificity of protease enzyme towards the casein and bovine serum albumin. The proposed method has a flexibility to try different substrates for the detection of various enzyme activities. SN - 1873-3573 UR - https://www.unboundmedicine.com/medline/citation/20602947/A_high_sensitive_assay_platform_based_on_surface_enhanced_Raman_scattering_for_quantification_of_protease_activity_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0039-9140(10)00363-2 DB - PRIME DP - Unbound Medicine ER -