Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

Improved detection of 2,3-diaminophenazine for horseradish peroxidase quantification using surface-enhanced Raman spectroscopy

Evgeniy G. Evtushenko
* ORCID logo ,
Elizaveta S. Gavrilina
,
Alexandra D. Vasilyeva
ORCID logo ,
Lyubov V. Yurina
ORCID logo ,
Ilya N. Kurochkin
Version 1 : Received: 12 December 2023 / Approved: 12 December 2023 / Online: 12 December 2023 (21:10:11 CET)

A peer-reviewed article of this Preprint also exists.

Evtushenko, E.G.; Gavrilina, E.S.; Vasilyeva, A.D.; Yurina, L.V.; Kurochkin, I.N. Highly Sensitive Measurement of Horseradish Peroxidase Using Surface-Enhanced Raman Scattering of 2,3-Diaminophenazine. Molecules 2024, 29, 793. Evtushenko, E.G.; Gavrilina, E.S.; Vasilyeva, A.D.; Yurina, L.V.; Kurochkin, I.N. Highly Sensitive Measurement of Horseradish Peroxidase Using Surface-Enhanced Raman Scattering of 2,3-Diaminophenazine. Molecules 2024, 29, 793.

Abstract

The enzyme-linked immunosorbent assay (ELISA) coupled with surface-enhanced Raman spectroscopy (SERS) detection is a growing area of analytical chemistry emerged as a solution to a problem of sensitivity improvement. SERS-based ELISA with horseradish peroxidase as an enzymatic label, o-phenylenediamine (oPD) substrate, and 2,3-diaminophenazine (DAP) enzymatic product was one of the first examples of such systems. Surprisingly, the full potential of this long-known approach has not been revealed yet. The current study addresses a previously neglected problem of SERS detection stage performance. Using silver nanoparticles and model mixtures of oPD and DAP, the effects of pH, concentration of aggregating agent, and particle surface chloride stabilizer were extensively evaluated. At optimal mildly acidic pH of 3, 0.93 to 1 M citrate buffer, and AgNPs stabilized with 20 mM chloride, at least two orders of magnitude advantages in the detection limits of SERS over colorimetry both for DAP and HRP were demonstrated. We suppose that this improved detection system could become a useful tool for the development of SERS-based ELISA protocols.

Keywords

Surface-enhanced Raman spectroscopy; SERS; horseradish peroxidase; HRP; ELISA; 2,3-diaminophenazine; DAP; o-phenylenediamine; 1,2-diaminobenzene; oPD

Subject

Chemistry and Materials Science, Analytical Chemistry

Copyright: This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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