# Perspective of Using Vertically Oriented Graphene as an Electrochemical Biosensing Platform
## 2 Materials and Methods
### Characterization Methods  
Raman spectroscopy was carried out using a high-resolution scanning near-field optical microscope (SNOM) equipped with an integrated Raman module (Alpha-SNOM 300S, Witec, Germany) operating at a 532 nm wavelength. The solid-state laser used for the study is diode-pumped and can generate a laser power of up to 145 mW. With a spot size of 1.0, the laser was directed onto the sample using two objectives: 50 x \(\times\) and 100 x.  
The SEM analysis was performed using an FEI NovaTM NanoSEM 630 microscope (FEI Company). We analyze VG in the top view to see the density of the deposited film and the quality of the covering on the substrate, as well as in the cross-section to see the height of the VG film and the grown nanowalls.  
TEM analysis was performed on a Jeol Jem arm 200F microscope equipped with an EDS unit (JEOL USA Inc., Peabody, MA, USA). The sample was prepared by the cross-section method, mechanical thinning to approximately 30 microns, followed by ion thinning in a Gatan Pips machine at an ion beam acceleration voltage of 4 kV and an incidence angle of 7 degrees. A lower voltage of 2 kV was finally used to reduce the amorphous surface layer covering the specimen.  
The topography of the VG was characterized by AFM using a Nano Wizard4 BioScience AFM system (JPK Instruments AG, Berlin, Germany). A 10 \(\upmu\)m \(\times\) 10 \(\upmu\)m area was scanned in the air using intermittent contact mode. Three regions were examined for VG and further processed using Gwyddion 2.49 software. Finally, the surface roughness (Rq) and root mean square (RMS) roughness, representing the surface height standard deviation, were determined.  
For Fourier-transform infrared spectrometry (FTIR), a Bruker Optics Tensor 27 spectrometer was used, equipped with ATR Platinum and a reflection holder. ATR Platinum contains a diamond crystal with a single reflection. All samples were traced after 64 scans, with a resolution of 4 cm\({}^{-1}\), on a spectral domain of 4000-400 cm\({}^{-1}\). VG was analyzed before and after the electrochemical experiments.  
EIS analysis was performed using an Autolab PGSTAT204 equipped with an FRA32M electrochemical impedance spectroscopy module (METROHM AUTOLAB AG., Utrecht, The Netherlands) with a 0.01 V AV amplitude. The initial phase involved analyzing and modeling the collected data using the spectrometer software NOVA 2.1.5. The data were then processed, and the electrochemical impedance characterized the sensor. When a stimulus is applied to the cell, it impacts the cell membrane's permeability. This change in permeability is represented by the impedance, which reflects the conductance of the membrane in the microelectrode array area. The cells were characterized using electrochemical impedance spectroscopy, using a device developed within the project [19]. By applying a small electric voltage between the interdigitated working electrode and the auxiliary electrode, oxidation and reduction reactions occur, resulting in electron release and capture. The solid-liquid interface exhibits capacitor-like behavior, with the capacitance expanding as the interface size increases. This flattens the initial drop and decreases the contact angle.