The Characterization of Screen-Printed Copper Electrodes Modified with Chitosan/Reduced Graphene Oxide for Epinephrine Detection

In this study, the working electrode (WE) from Screen-Printed Copper modified with chitosan/rGO to detect epinephrine has been successfully performed by using electrodeposition method. Samples are conducted by varying the rGO concentration (300, 350, 400, 450, and 500) ppm with chitosan 1:1 (v/v). The obtained working electrodes were tested for FTIR to determine the functional group, the surface area of WE, characterized by SEM, and amperometry method to determine the output voltage optimum. The FTIR analysis confirmed N-H, OH, C-H, NH2, and C-H in all chitosan and chitosan/rGO. The SEM images showed that CS/rGO film morphology was smoother than CS film. It can be assumed the rGO was distributed well onto CS film. The amperometry test results also showed that the working electrode produced the maximum output voltage from CS/rGO 350 ppm at 0.1032 V. It can be concluded that the working electrode from the screen-printed copper electrodes modified with chitosan/reduced graphene oxide can be used to detect the epinephrine.


Introduction
In the medical aspect, epinephrine is a hormone and drug used to treat some emergency health conditions [1].Therefore, epinephrine provides benefits to the nervous, cardiovascular, and metabolic systems that will improve physical performance [2].However, epinephrine has a negative impact because it can cause heart problems, metabolism, blood vessel damage, and mental disorders [3].Meanwhile, in sports, epinephrine is referred to as doping or a prohibited substance athletes use to improve performance in sports competitions.Hence, the World Anti-Doping Agency (WADA) unequivocally announces that the utilization of doping is categorically forbidden due to its harmful impact on the integrity of competition and its potential to pose grave health hazards [4].Therefore, innovation is needed to detect epinephrine that is accurate, fast, and has good sensitivity.
Epinephrine detection methods that are generally used are surface-enhanced Raman scattering (SERS), Liquid Chromatography Mass Spectrometry (LC-MS), High-Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), and capillary electrophoresis [5].However, the method is considered to have many weaknesses due to complicated preparation, long analysis time, high cost, lack of specificity and sensitivity.Electrochemical methods are in great demand with the development of materials science and analytics because of their high selectivity, good response, stability, speed, and relatively low cost [6].Polymer-based electrodes as raw materials are currently being widely highlighted.
Chitosan (CS) is a renewable cationic polysaccharide that can be used as an epinephrine detection material because it has very high chemical reactivity with a tendency to form hydrogen bonds between the nitrogen group in NH2 -of CS and the hydrogen group in epinephrine [7].It is a polymer produced from chitin, a compound found in the shells of marine animals such as crustaceans (shrimp, crabs, and lobsters).CS has unique properties and is useful in a variety of applications.In addition, CS has good film-forming capabilities [8].However, pure CS exhibits high sensitivity to environmental conditions and possesses limited mechanical characteristics.To overcome this problem, adding other ingredients, including reduced graphene oxide (rGO), is necessary.
Reduced Graphene Oxide (rGO) is a nanomaterial that is added to increase the potential of CS as a working electrode material [9].In addition, rGO was chosen because of its properties, namely its expansive surface area, great conductivity, and commendable mechanical resilience.Moreover, it provides faster electron transfer capabilities in redox reactions [10].So, adding rGO expands the surface area, increases the electron transport reaction, and increases the conductivity value of CS as an epinephrine detection material [11].
Apart from the many choices of mixing methods for these two materials, the ionic gelation method is the most widely used because the process is simple, can be controlled easily, the results are more homogeneous, and there is an increase in electron affinity during the process.
The working electrode fabrication process using copper electrodes in Screen-Printed Copper (SPC) modified with CS/rGO was carried out using the electrodeposition method [12].This method has many advantages because it takes little time, is simple, cheap, and utilizes electrochemical phenomena.Subsequently, the obtained working electrode was analyzed using Fourier-transform infrared spectroscopy (FTIR) to determine its chemical composition and scanning electron microscopy (SEM) to examine its morphology.Then, the working electrode characteristics were tested using the amperometry method.

Material and Chemicals
All of the reagents and chemicals were analytical grade and were used precisely as supplied.Phosphate buffer solution prepared from Na2HPO4 and NaH2PO4 was utilized to adjust the electrolyte's pH down to the appropriate level.A PBS solution will be used to dilute epinephrine at pH 7. Chitosan was obtained from Sigma Aldrich, and epinephrine and rGO were obtained from Itnano, Indonesia.

Fabrication of Chitosan/Reduced Graphene Oxide Electrodes
The modified material was manufactured using the electrodeposition method.The cathode and anode are connected according to their poles.The screen-printed electrode (SPC), which has previously been cleaned using several processes to remove dirt on its surface, is clamped with a clamp connected to an electric current.SPC was immersed in a solution of CS/rGO modified material with several variations in rGO concentration, namely 300, 350, 400, 450, and 500 ppm.Then, the deposited material is dried in an oven at a certain temperature so that the film formation process occurs on the copper electrode.Next, the optimum CS/rGO concentration was determined using the amperometry method to obtain the output stress of the modified material.The chitosan/rGO electrode was placed on top of the testing chamber, and the anode and cathode were adjusted, then 0.01 mg/L epinephrine was dripped onto the CS/rGOmodified copper electrode [13].

Fourier-Transform Infrared Spectroscopy (FTIR) of CS and CS/rGO Electrodes
FTIR analysis was conducted to evaluate changes in functional groups of chitosan and the chitosan/rGO bonding process.The FTIR instrument analysis used the Shimadzu IR Prestige-21 was carried out for the 500-4000 cm -1 wavenumber range.The FTIR spectra of the CS and CS/rGO are displayed in Figure 1.
Figure 1(a) shows the FT-IR spectrum of chitosan, where there is a widening of the N-H group that overlaps with the hydroxyl group (OH -) at 3236.99 cm -1 .The band at 3200-3425 cm -1 showed the hydroxyl group (OH -).In addition, the absorption spectrum at 2877.04 cm -1 demonstrated the C-Hgroup, which shows the stretching vibration of the -CH2 group.The band at 1540.78 cm -1 showed bending vibration on the primary amine group (NH2).Also, the bending vibration of the C-H group was found at 1379.00 cm -1 [8].
Meanwhile, in Figure 1(b), The FTIR spectrum of chitosan modified with rGO revealed the presence of an OH-group at a wave number of 3207.77cm -1 and the band at 2853.30 cm -1 , which showed the CH stretching vibration.Next, the FTIR spectrum of CS/rGO also showed the interaction between chitosan and rGO resulted in a decrease in the C-O vibration of chitosan at 1019.39 cm -1 .Additionally, asymmetric stretching of the C-O-C at 1062.01 cm -1 indicated the presence of rGO in chitosan.The FTIR spectrum analysis indicated that CS/rGO exhibits a nearly identical FT-IR spectrum to pure chitosan, as the presence of practically perfect rGO does not produce an obvious peak.

Scanning Electron Microscopy (SEM) of CS/rGO Electrodes
The SEM analysis results indicate that the particle morphology of the samples can be classified as chitosan films and optimal chitosan/rGO films.The morphology SEM results can be displayed in Figure 2.
Figure 2(a) demonstrates that the surface of chitosan exhibits a morphology characterized by a rough layer and tiny perforations, which are attributed to the structural bands resulting from the inherent tendency of chitosan to self-aggregate in solution [14].On the other hand, the chitosan/rGO film has a generally homogeneous surface.The rGO is evenly distributed inside the CS matrix.The polymer material can be impregnated, resulting in the formation of a stratified structure.The alignment of the rGO sheet is a result of the flow generated by the filtration process and the presence of rGO filler [15].This morphology demonstrates that the chitosan envelops the rGO sheets and fills in the spaces to produce a compact morphology.
In this study, the chitosan/rGO film exhibits a significantly smoother surface compared to the chitosan film, which indicates that the distribution of rGO inside the chitosan matrix is uniform.Therefore, the most effective electrode made of chitosan/rGO copper can be used to detect epinephrine concentrations.

Data Analysis on the Effect of rGO Addition on Chitosan Modified Screen Printed Copper
Data analysis on the effect of rGO addition on chitosan-modified screen-printed copper electrodes in detecting epinephrine concentrations of 0.01 mg/L at a ratio of 1:1 shown in Table 1.
These results show the optimal rGO concentration offered to the chitosan copper electrode was 350 mg/L.Figure 3 displays the curve representing the addition of rGO.

Conclusion
In conclusion, the working electrode from Screen-Printed Copper modified with chitosan/rGO to detect epinephrine has been successfully carried out using the electrodeposition method.Based on the results of the FTIR test, it confirmed that N-H, OH, C−H, NH2, and C-H in all chitosan and chitosan/rGO without showed the chemical interaction.Correspondingly, the SEM images showed that CS/rGO film morphology was smoother than CS film because the rGO was successfully

Figure 3 .
Figure 3.Effect of adding rGO on the output voltage of chitosan electrodes.

Table 1 .
Effect of rGO Concentration in Detecting Epinephrine Concentration 0.01 mg/L Against Output Voltage.