Place the electrochemical cell prepared with the gap mode SERS substrate on the stage of an inverted optical microscope. Secure the substrate to the microscope stage by taping the edges to prevent movement during spectro electrochemical measurements due to the tension of the wires connecting the cell to the potentiostat. Place the silver-silver chloride reference electrode into the home-built stand and fix its position by tightening the screw on the electrode holder stand.
Attach the reference electrode to the potentiostat's reference electrode alligator clip. Then attach the platinum wire counter electrode to potentiostat's counter electrode alligator clip. Finally, clip the copper wire attached to the silver film to the potentiostat's working electrode alligator clip.
Insert the platinum wire and alligator clip into the electrode holder and tighten the screw to secure its position. Place the electrode holder over the electrochemical cell to insert the electrodes. Then turn on the 642-nanometer laser and adjust the power to 500 microwatts.
Next, add a drop of immersion oil to the objective lens. Then, move the focus knob to raise the objective until the oil contacts the bottom of the substrate. Focus the laser onto the surface of the gap mode SERS substrate.
After removing one of the eye pieces from the microscope, insert the adapter in its place. Change the mode to video on the camera application and zoom in as much as possible. Scan the gap mode SERS substrate by moving the microscope stage to search for an isolated donut-shaped SERS emission pattern.
Once the donut-shaped emission pattern is located, move the light diverter lever of the microscope to direct the admitted light to the spectrometer. Set the grading position to 1000 wave numbers to detect Stokes shifted ramen scattering from 400 to 1600 wave number region. Keeping the laser light focused on the donut-shaped emission pattern, add 3 milliliters of a 0.1-molar phosphate buffer of pH 5 into the electrochemical cell.
In the potentiostat's software prepare a cyclic voltammogram experiment with at least three cycles from 0 to minus 0.6 volts versus silver-silver chloride and a scan rate of 50 millivolts per second. Then, run the simultaneous cyclic voltammetry and SERS experiments. Finally, move the light diverter lever, so that the light is directed to the phone camera and start recording a video while running the cyclic voltammetry experiment.
Individual silver nanoparticles on the silver thin film can be unambiguously identified by a donut-shaped emission pattern in contrast to a solid emission pattern produced by nanoparticle dimers, trimers, or multimers. The SERS cyclic voltammograms were measured for single nanoparticles, and the Nile blue molecules in and around the gap between the silver nanoparticle and the silver film were electrochemically reduced. Spectroelectrochemical measurements were done with the same applied potential range.
Electrochemical modulation of the Nile blue SERS spectrum by stepping the potential causes the peak intensity at the 592 wave number region to decrease with time due to the reduction of the Nile blue molecules. The magnitude of the electric bias altered the reduction kinetics, as evidenced by the decay of the area under the 592 wave number region peak.
