Raman spectroelectrochemistry was employed for the detection of aldehyde dehydrogenase (ALDH) in aqueous solution. Electrochemical SERS (EC-SERS) protocol requires two steps in a single experiment: the electrochemical activation of SERS features of Ag SPEs, and then the spectroscopic detection of the sample.

Electrochemical activation consists of scanning the potential from +0.50 V to produce the initial oxidation of the silver surface, followed by its reduction up to -0.60 V to generate nanostructures with the SERS effect. The spectroelectrochemical experiment is performed in presence of the analyte to be detected (in this case ALDH), but chloride ions are also present in solution to favor the generation of silver SERS substrate [1].

In order to evaluate the evolution of Raman bands during the entire experiment, and therefore to optimize the optical detection, SPELEC RAMAN 638 performs measurements in operando mode. This way, spectra are recorded continuously during the whole experiment and not only at certain potentials.

Figure 2a displays the cyclic voltammogram of ALDH, and the characteristic Raman spectrum of ALDH is shown in Figure 2b. The results were obtained for 1 mg/mL ALDH in 0.1 mol/L KCl aqueous solution. Although the acquisition of spectra was continuously performed, the Raman spectrum in Figure 2b was recorded at -0.50 V as this potential provides the highest Raman intensity.

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The combination of the proposed EC-SERS procedure for the easy activation of Ag SPEs with a Raman spectroelectrochemical instrument (638 nm laser) offers a fast and interesting alternative for the characterization of ALDH in solution not previously reported in the literature.

Conventional electrodes were also used for the EC-SERS detection of different enzymes such as cytochrome c. The protocol for this type of electrode follows the same steps as for SPEs. An initial oxidation of the surface is required followed by the subsequent reduction to generate silver nanostructures with SERS effect. The potential window was adjusted according to the electrodes used in this experiment. The best results were obtained by scanning the potential of 0.1 mg/mL cytochrome c in 0.1 mol/L KCl aqueous solution from +0.80 V to -0.80 V. The Raman spectrum with the highest intensity (Figure 3) was recorded at -0.70 V.

Assignments of vibrational modes of each Raman band of cytochrome c are listed in Table 2.

Considering that cytochrome c exists as interconvertible reduced and oxidized forms, the oxidation state of the Fe ion can be determined according to the position of their characteristic Raman bands. Reduced cytochrome c shows one Raman band centered at 1604 cm^-1, while the oxidized form displays an upshifted band centered at 1636 cm^-1. According to the Raman spectrum (Figure 3) and the vibrational assignments (Table 2), the reduced form of the analyte is detected during the EC-SERS experiment. This experiment demonstrates the possibility to detect cytochrome c as well as characterize its oxidation state.

Raman spectroelectrochemistry is a multi-response technique that provides outstanding results when studying a wide variety of systems. In the particular case of the 638 nm laser, the energy provided at this wavelength is suitable for biological applications.

In this work, the proposed electrochemical protocols for the activation of SERS properties of different silver electrodes (screen-printed as well as conventional) offers an easy and fast procedure to enhance the Raman intensity. This enhancement of Raman intensity allows the detection of different molecules present in solution. Fingerprint Raman bands of ALDH, not previously reported, are defined in this work. In the study of cytochrome c, the characteristic Raman bands are defined in addition to characterization of the redox state of the Fe ion.