DÜZENLEME KURULU / ORGANIZING COMMITTEES

Recently, electrochemical methods are of growing interest. Electrochemical methods used in the development of sensors have many advantages such as high sensitivity, short response time, and low cost. Screen-printed electrodes (SPEs) are one of the electrochemical devices utilized as sensors that are used in a wide range of applications from health to food. In this study, the surface functionalization of commercially available screen-printed carbon electrodes (SPCE) were carried out, and the layers formed on SPCE surface were examined by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) method. Textural and micro-structural properties of SPCE were investigated by scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) spectroscopy. Bonding of a self-assembling monolayer (SAM), an antibody, and an antigen on SPCE were also successfully achieved. After each layer formation, EIS tests were performed using the redox probe potassium ferricyanide (K 3 Fe(CN) 6 ) at the both optimum concentration and parameters. The impedance data were presented in the Nyquist curves. The Nyquist plot provides charge transfer resistance (R ct ) when employed along with the equivalent circuit model. The results showed that the R ct values increased with the formation of additional layers. The increasing resistance showed a successful achievement of the immobilization process. modeled by using a constant-phase element (CPE) with α c and Q c . The modeling results showed that the charge-transfer resistance increased after each of the immobilization steps. The increase in R ct demonstrated the formation of layers on the SPCE.


Introduction
Screen-printed electrodes (SPEs) are widely used in electrochemical sensor systems owing to the advantageous features including low-cost, ease of use, high reproducibility, and high sensitivity. The presented advantages of SPEs enable these electrodes use in different type of applications such as analytical chemistry, biochemistry, medicine, environmental protection, food safety, and pharmacy. Compared to the conventional electrodes, the SPEs exhibit rapid analysis and reliable results 1 . The SPEs also provide in situ analyses 2 . Therefore, techniques in electrochemical processes. EIS is a non-destructive measurement that can provide beneficial insights into the identification and quantification of electrode characteristics 3 . EIS measurements allow discriminating time constants related to the processes occurring at the electrolyte/electrode interfaces 4 . Cyclic voltammetry analyses provide information for the electrochemical reaction mechanisms between the electrolyte and SPEs. Compared to the conventional CV method, EIS measurements provide more detailed it is of great significance to characterize the features of the SPEs. To determine the characteristic properties of the SPEs, numerous methods have been applied. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) are the most widely used information about the electrochemical processes 5 .
In this study, the immobilization of SPCEs was carried out to prepare the SPE-based biosensor. EIS analysis was performed by using potassium ferricyanide to present the steps of stable immobilization onto the SPCEs surface. The changes on the surface of SPCEs were characterized by SEM and EDX.

Materials and Method
Immobilization and electrochemical experiments were performed on commercial screen printed carbon electrode. Cyclic voltammetry and impedance spectroscopy were used to monitor changes associated with immobilization steps.

Materials
Commercial 110 AT SPCEs with the electrode surface are area of 0.1256 cm 2 were purchased from Metrohm DropSense. EIS and CV measurements were performed using a Gamry 3000 Potentiostat/Galvanostat/ZRA connected to a desktop computer. The experimental setup for the electrochemical measurements is presented in Figure 1. The external morphology of SPCEs was characterized by a Hitachi Regulus 8230 scanning electron microscope (SEM) at 20 kV. The elemental composition of SPCEs surface was also investigated by using combined SEM-EDX instrument mentioned above.

Methods
The surface of SPCE is first coated with a self-assembled monolayer (SAM) using 11-mercaptoundecanoic acid (11-MUA), followed by immobilization of streptavidin (SPV) by covalent bonding with 11-MUA through the carbodiimide reaction in an immobilization procedure. The SPCE was rinsed by anhydrous ethanol and 11-mercaptoundodecanoicacid (MUA, 10 mM prepared in anhydrous ethanol, Sigma). After one-hour incubation of MUA, it is rinsed with ethanol and Dulbecco's phosphate-buffered saline to remove unbounded thiols. Then desired layers were formed on the surface of SPCEs. Then, antibodies were attached to SPV via strong interaction between SPV and biotin. The electrochemical characterization of SPCEs was carried out based on the change of electron transfer kinetics of redox probe potassium ferricyanide (5 mM K3Fe(CN)6). CV measurements were performed at a potential range of 0-1.6 V and 100 mV/s scan rate. Impedance measurements were performed at the open circuit potential with the 10 kHz-100 mHz frequency range.

Result and Discussion
The immobilization steps are presented in Figure 2. The surface was activated through the formation of carboxylic groups on the SPCE. The activation of surface was achieved by the CV with the phosphate-buffered saline solution. The cyclic voltammogram is presented in Figure 3. In the second step (Figure 2.b), the surface of SPCE was functionalized by immobilizing a self-assembling monolayer (SAM) from spacers containing thiol groups on one end and carboxyl groups on the other terminal. SAM formation enables the surface to be more stable for the subsequent interactions. After the functionalization step, antibody was attached to the antigen via strong interaction. In the end, the SPCE can attach the toxin onto its surface. The impedance response for the immobilization steps including SAM, antibody, and toxin attachment is presented in Figure 4. The impedance data were modeled to capture the physically meaningful circuit parameters. An equivalent circuit was used to reflect the impedance behavior of the immobilization steps. The equivalent circuit consists of three elements. Re, CPE, and Rct parameters represent the ohmic resistance, capacitive behavior, and charge-transfer resistance, respectively. The impedance data could not be described simply by a Randles circuit design since the Nyquist plot is not a perfect semi-circle. The high frequency response was modeled by using a constant-phase element (CPE) with αc and Qc. The modeling results showed that the charge-transfer resistance increased after each of the immobilization steps. The increase in Rct demonstrated the formation of layers on the SPCE.

Fig. 4 Electrochemical impedance spectroscopy analysis for the immobilization steps: (A) Nyquist representation of the impedance responses and the equivalent circuit model used to fit the impedance data; (B) charge transfer resistance values during the immobilization steps
SEM images of the surface of SPCE are presented in Figure 5. The surface after the immobilization was smoother while the bare electrode surface was rough. Compared with the surface of immobilized SPCE, the surface of bare electrode was more uniform. The SEM images indicated that the additional layer formed on the bare electrode surface after the immobilization. The thicker surface caused the increase in Rct. EDX analyses showed that the working surface of bare electrode consisted of carbon (96 wt.%), oxygen (3 wt.%) and chlorine (1 wt.%). After the immobilization processes, the elemental composition of the surface was changed. The contents of carbon, nitrogen, oxygen, and chlorine were 70 wt.%, 12 wt.%, 14 wt.%, and 2 wt.%, respectively.

Conclusions
This work provides a detailed characterization of the screen-printed carbon electrodes. The applied of cyclic voltammetry in 0-1.6 V potential range formed additional layers on the surface of SPCE. Electrochemical impedance responses were modeled by the developed equivalent circuit model. The charge-transfer resistance values obtained by the equivalent circuit modeling and the subsequent analyses including SEM and EDX demonstrated formation of layers on the surface of SPCE after the immobilization steps. This work provides guidance for the characterization and optimum use of commercially available screen printed electrodes.