Easy detection of enzymes with the electrochemical-SERS effect

Significance of the Topic

Raman spectroscopy offers label-free molecular fingerprinting but its sensitivity is often insufficient for detecting low-concentration biomolecules. Combining electrochemical activation with surface-enhanced Raman scattering (EC-SERS) produces plasmonic nanostructures on silver electrodes, boosting Raman signals and enabling rapid, in situ enzyme analysis.

Objectives and Study Overview

This work demonstrates simple EC-SERS protocols using a 638 nm Raman spectroelectrochemical instrument to detect two model enzymes, aldehyde dehydrogenase (ALDH) and cytochrome c. The aim is to validate activation routines on screen-printed and conventional silver electrodes and to characterize enzyme Raman fingerprints and redox states.

Methodology

The analytical workflow comprises two steps in a single experiment: (1) electrochemical activation of silver surfaces via an oxidation–reduction potential sweep to form SERS-active nanostructures; (2) operando Raman measurements recorded continuously during potential scanning. For ALDH detection, potentials were cycled between +0.50 V and –0.60 V in 0.1 mol/L KCl with 1 mg/mL enzyme on Ag screen-printed electrodes. For cytochrome c, the potential range was +0.80 V to –0.80 V with 0.1 mg/mL analyte on a conventional silver disk electrode.

Instrumentation Used

• SPELEC RAMAN 638 spectroelectrochemical system (638 nm laser, Class 3B)
• Raman probe matched to 638 nm excitation
• Raman spectroelectrochemical cells: RAMANCELL for screen-printed electrodes, RAMANCELL-C for conventional electrodes
• Ag screen-printed electrodes (C013) and conventional Ag working electrode (6.09395.044)
• Steel counter electrode (6.0343.110) and Ag/AgCl reference electrode (6.0728.120)
• Connection cables (CAST for SPEs, CABSTAT for conventional cells)
• DropView SPELEC software for synchronized optical and electrochemical control and data processing

Main Results and Discussion

• ALDH detection: After activation, the highest Raman intensity was observed at –0.50 V. Characteristic ALDH bands were recorded, marking the first report of its SERS fingerprint in solution.
• Cytochrome c detection: Optimal enhancement occurred at –0.70 V. Key SERS bands at 713, 969, 1123, 1220, 1358, 1426, 1528, 1578, and 1604 cm⁻¹ were assigned to heme vibrations. The position of the 1604 cm⁻¹ band confirmed detection of the reduced Fe(II) form; an oxidized Fe(III) form would shift the signature to 1636 cm⁻¹.

Benefits and Practical Applications

The EC-SERS approach enables rapid, label-free detection and redox characterization of enzymes at physiological laser energy, minimizing fluorescence and sample damage. Its simplicity and speed suit real-time monitoring in biochemical assays, quality control, and clinical diagnostics.

Future Trends and Potential Applications

Advances may include multiplexed EC-SERS assays for simultaneous detection of multiple biomolecules, integration with microfluidic platforms for automation, portable spectroelectrochemical devices for field use, adaptation to other excitation wavelengths for broader molecular coverage, and exploration of dynamic redox kinetics in living cells.

Conclusion

The combination of electrochemical activation and 638 nm Raman spectroscopy on silver electrodes provides a versatile, high-sensitivity platform for enzyme analysis. The protocols yield clear SERS fingerprints for ALDH and cytochrome c, demonstrate redox-state discrimination, and open avenues for rapid, in situ biomolecular sensing.

References

1. Martin-Yerga D., Pérez-Junquera A., González-Garcia M. B. et al. Quantitative Raman Spectroelectrochemistry Using Silver Screen-Printed Electrodes. Electrochimica Acta 2018, 264, 183–190.
2. Brazhe N. A., Evlyukhin A. B., Goodilin E. A. et al. Probing Cytochrome c in Living Mitochondria with Surface-Enhanced Raman Spectroscopy. Scientific Reports 2015, 5, 13793.
3. Hu S., Morris I. K., Singh J. P. Complete Assignment of Cytochrome c Resonance Raman Spectra via Enzymic Reconstitution with Isotopically Labeled Hemes. Journal of the American Chemical Society 1993, 115(26), 12446–12458.