Abstract
We employed the wavelength-interrogated surface plasmon resonance (SPR) method to characterize the real-time kinetics of urea-urease hydrolysis reaction in response to a CO2-free N2 environment and CO2-enriched ambient reaction medium. We established that a simple label-free SPR probe could accurately extract kinetic parameters from the nature of the sharp jump of the SPR wavelength shift in the reaction profile. The kinetic analysis showed that CO2 production increases with increasing reaction time irrespective of CO2-free N2 or CO2-enriched reaction environment. We also explored the essential insights into the isotopic fractionations of 12CO2, 13CO2, 12C18O16O in the reaction medium utilizing integrated cavity output spectroscopy. The plasmonic system measured the reaction rate in the order of 10-7 M/s for urea species in the presence of the urease enzyme. This study deepens our understanding of plasmonic-based enzymatic urea hydrolysis in real time and opens a new way to quantify chemical reaction kinetics for various other systems.
Graphical abstract
This is the first detailed experimental investigation of the real-time kinetics of urea-urease hydrolysis reaction exploiting wavelength-interrogated surface plasmon resonance method in response to produced CO2 in the CO2-free N2 environment and CO2-enriched ambient reaction medium utilizing integrated cavity output spectroscopy.

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Acknowledgements
J. Banerjee would like to acknowledge the Science & Engineering Research Board, Government of India, for funding the project (PDF/2020/001422).
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Jayeta Banerjee: Conceptualization, Methodology, Validation, Formal Analysis, Investigation, Writing – original draft, Manik Pradhan: Supervision, Writing - Review & Editing, Funding Acquisition.
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Banerjee, J., Pradhan, M. A combined surface plasmonic and isotope-selective spectroscopic study toward a deeper understanding of real-time enzymatic urea hydrolysis. J Chem Sci 135, 53 (2023). https://doi.org/10.1007/s12039-023-02175-0
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DOI: https://doi.org/10.1007/s12039-023-02175-0