## 3 Results and Discussion  
### Characterization of the Electrochemical Aptasensor  
The utilization of graphene in electrochemical biosensors is impactful because no chemical treatment is needed. A uniform distribution of graphene on the electrode surface was observed by depositing only a small volume of GO (\(7\)\(\mu\)L) (Figure 1). A DNA aptamer was coated on the SPCE surface by physical adsorption, but various issues, such as the inefficient orientation of the attached aptamer, the uncontrollability over bonding strength, and strong binding of the aptamer for minimal physical adsorption [22] were challenges. In our experiments, GO was pre-treated with EDC/NHS to activate the carboxyl groups of GO. EDC is a cross-linker that was used to couple carboxyl groups to primary amines by forming an active ester intermediate that was substituted by nucleophilic attack from the primary amine. The intermediate product is prone to be hydrolyzed and reverted to the  
Figure 1: Schematic illustration of the proposed GO-based electrochemical aptasensor for GA protein detection. (**a**) Functionalization of GO; (**b**) drop-coating GO onto SPCEs; (**c**) immobilization of NH\({}_{2}\) aptamer; (**d**) blocking with ethanolamine; (**e**) incubation with GA protein; (**f**) voltametric measurements using \(5\) mM [Fe (CN)\({}_{6}\)]\({}^{+/3-}\) as a redox indicator. original molecule [23]. NHS ester was used to overcome this by forming a steady second intermediate before animation [24]. EDC crosslinking was carried out in a buffer devoid of carboxyl or primary amines. The coupling reaction requires over 20 min for efficient carboxylation [25]. In conventional GO-based electrochemical biosensors, activation of GO was carried out by incubating GO-modified electrodes in EDC/NHS solutions. However, the uniformity of carboxylation is difficult to achieve. In our study, the pre-treatment of GO with EDC/NHS resulted in uniform carboxylation and stable aptamer immobilization. Covalent bonding of carboxyl groups of GO to the amine-modified aptamer was achieved after activation. The covalent binding for the aptamer was more stable than a weak \(\pi\)-\(\pi\) interaction corresponding with physical adsorption [26]. The characterization of the electrochemical aptasensor was confirmed by CV and EIS using 5 mM [Fe (CN)\({}_{6}\)]\({}^{4-/3-}\). Figure 2A represents the cyclic voltammograms of the bare SPCEs and GO-modified SPCEs. After modification of the SPCE surface with GO, both anodic (I\({}_{\text{pa}}\)) and cathodic (I\({}_{\text{pc}}\)) peak currents significantly increased. GO-modified SPCEs exhibited higher peak currents than bare SPCEs. The modification of SPCEs with GO provides a shift in the cathodic (E\({}_{\text{pc}}\)) and anodic (E\({}_{\text{pa}}\)) potentials, thus resulting in smaller peak-to-peak separation (\(\Delta\)E\({}_{\text{p}}\)). The significant increase in the peak current can be attributed to the improved electrochemical activity with a higher charge transfer rate, which in turn leads to the improvement of the total electroactive area of GO/SPCEs. After aptamer immobilization, the redox peak current was decreased because of the large structure of the aptamer and the negative charge on the aptamer phosphate backbone. The peak was further reduced after target incubation due to a change in aptamer conformational structure after specific protein binding. The EIS spectra of the electrochemical sensor are illustrated in Figure 2B. The successful modification of the electrode surface was characterized by EIS data, which revealed the changes in charge transfer resistance (R\({}_{\text{ct}}\)) in each modification step. The simplified Randles circuit, which consists of a series of active electrolyte resistances R\({}_{\text{s}}\) and double layer capacitances C\({}_{\text{dl}}\) that are parallel to the charge transfer resistance R\({}_{\text{ct}}\), was used for EIS fitting. The Nyquist spectrum for the bare SPCEs consists of a semicircle and a linear in high- and low-frequency regions.