Evaluation of the Ellman’s Reagent Protocol for Free Sulfhydryls Under Protein Denaturing Conditions
Early detection of cancer can dramatically improve long-term prognosis and survivability in a variety of different cancer types. However, for many cancer types, the ability to effectively detect early-developing tumors is challenging, especially in physiological locations with limited visibility or...
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MDPI AG
2025-05-01
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| Series: | Analytica |
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| author | Sophia R. Ginet Frank Gonzalez Maxine L. Marano Megha D. Salecha Joseph E. Reiner Gregory A. Caputo |
| author_facet | Sophia R. Ginet Frank Gonzalez Maxine L. Marano Megha D. Salecha Joseph E. Reiner Gregory A. Caputo |
| author_sort | Sophia R. Ginet |
| collection | DOAJ |
| description | Early detection of cancer can dramatically improve long-term prognosis and survivability in a variety of different cancer types. However, for many cancer types, the ability to effectively detect early-developing tumors is challenging, especially in physiological locations with limited visibility or access. Previously, we reported a sensing platform and methodology to detect biomarker peptides found in urine from ovarian cancer patients. This sensing platform relies on peptide interactions with gold nanoclusters through thiol-mediated linkages; thus, the sensitivity of the biomarker assay is directly related to appropriate redox states of the biomarkers in question. Here, we report on an expansion of the traditional thiol-reactivity assay originally developed by Ellman to include and evaluate a variety of solution modifications that may be used in conjunction with the biomarker-sensing platform. Because biomarker peptides may be isolated from a variety of biological tissues or fluids, depending on the target condition or disease, we screened numerous solution conditions that may be directly used in sample preparation and peptide extraction. The data demonstrate that the assay maintains linearity under these various conditions. The assay was then applied to a variety of models and biomarker peptides and exhibits the expected linear response. These results demonstrate the applicability of the thiol-reactivity assay to biologically derived samples, and the flexibility to ensure sample preparation and treatment will retain the appropriate sample redox conditions to ensure optimal interactions with the biosensor platform. It also facilitates the ability to perform quality control on clinically derived biological samples to ensure appropriate preparations, and concentrations are available for application to the nanopore biosensor platform. |
| format | Article |
| id | doaj-art-be7777dde9b0492f9b24eca30a61b5d8 |
| institution | Kabale University |
| issn | 2673-4532 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | MDPI AG |
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| series | Analytica |
| spelling | doaj-art-be7777dde9b0492f9b24eca30a61b5d82025-08-20T03:24:29ZengMDPI AGAnalytica2673-45322025-05-01621810.3390/analytica6020018Evaluation of the Ellman’s Reagent Protocol for Free Sulfhydryls Under Protein Denaturing ConditionsSophia R. Ginet0Frank Gonzalez1Maxine L. Marano2Megha D. Salecha3Joseph E. Reiner4Gregory A. Caputo5Department of Chemistry and Biochemistry, Rowan University, Glassboro, NJ 08028, USADepartment of Chemistry and Biochemistry, Rowan University, Glassboro, NJ 08028, USADepartment of Chemistry and Biochemistry, Rowan University, Glassboro, NJ 08028, USADepartment of Chemistry and Biochemistry, Rowan University, Glassboro, NJ 08028, USADepartment of Physics, Virginia Commonwealth University, Richmond, VA 23284, USADepartment of Chemistry and Biochemistry, Rowan University, Glassboro, NJ 08028, USAEarly detection of cancer can dramatically improve long-term prognosis and survivability in a variety of different cancer types. However, for many cancer types, the ability to effectively detect early-developing tumors is challenging, especially in physiological locations with limited visibility or access. Previously, we reported a sensing platform and methodology to detect biomarker peptides found in urine from ovarian cancer patients. This sensing platform relies on peptide interactions with gold nanoclusters through thiol-mediated linkages; thus, the sensitivity of the biomarker assay is directly related to appropriate redox states of the biomarkers in question. Here, we report on an expansion of the traditional thiol-reactivity assay originally developed by Ellman to include and evaluate a variety of solution modifications that may be used in conjunction with the biomarker-sensing platform. Because biomarker peptides may be isolated from a variety of biological tissues or fluids, depending on the target condition or disease, we screened numerous solution conditions that may be directly used in sample preparation and peptide extraction. The data demonstrate that the assay maintains linearity under these various conditions. The assay was then applied to a variety of models and biomarker peptides and exhibits the expected linear response. These results demonstrate the applicability of the thiol-reactivity assay to biologically derived samples, and the flexibility to ensure sample preparation and treatment will retain the appropriate sample redox conditions to ensure optimal interactions with the biosensor platform. It also facilitates the ability to perform quality control on clinically derived biological samples to ensure appropriate preparations, and concentrations are available for application to the nanopore biosensor platform.https://www.mdpi.com/2673-4532/6/2/18peptide biomarkerscysteine reactivitysulfhydryl reactivity |
| spellingShingle | Sophia R. Ginet Frank Gonzalez Maxine L. Marano Megha D. Salecha Joseph E. Reiner Gregory A. Caputo Evaluation of the Ellman’s Reagent Protocol for Free Sulfhydryls Under Protein Denaturing Conditions Analytica peptide biomarkers cysteine reactivity sulfhydryl reactivity |
| title | Evaluation of the Ellman’s Reagent Protocol for Free Sulfhydryls Under Protein Denaturing Conditions |
| title_full | Evaluation of the Ellman’s Reagent Protocol for Free Sulfhydryls Under Protein Denaturing Conditions |
| title_fullStr | Evaluation of the Ellman’s Reagent Protocol for Free Sulfhydryls Under Protein Denaturing Conditions |
| title_full_unstemmed | Evaluation of the Ellman’s Reagent Protocol for Free Sulfhydryls Under Protein Denaturing Conditions |
| title_short | Evaluation of the Ellman’s Reagent Protocol for Free Sulfhydryls Under Protein Denaturing Conditions |
| title_sort | evaluation of the ellman s reagent protocol for free sulfhydryls under protein denaturing conditions |
| topic | peptide biomarkers cysteine reactivity sulfhydryl reactivity |
| url | https://www.mdpi.com/2673-4532/6/2/18 |
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