*2.3. Immobilization of GOx/Chitosan Composite on the LIGE*

*2.3. Immobilization of GOx/Chitosan Composite on the LIGE*  The glucose biosensor was prepared by immobilizing the glucose oxidase and chitosan hydrogel homogeneous biocomposite on the LIGE surface. The resulting biocomposite could retain the enzyme bioactivity at considerably extreme conditions [35]. Five milligrams of GOx and three milligrams of chitosan were dissolved in 0.5 mL of deionized water and stirred for 5 min [36]. Subsequently, 5 µL of the mixture was cast onto the sur-The glucose biosensor was prepared by immobilizing the glucose oxidase and chitosan hydrogel homogeneous biocomposite on the LIGE surface. The resulting biocomposite could retain the enzyme bioactivity at considerably extreme conditions [35]. Five milligrams of GOx and three milligrams of chitosan were dissolved in 0.5 mL of deionized water and stirred for 5 min [36]. Subsequently, 5 µL of the mixture was cast onto the surface of the LIGE working electrode. Then, the LIGE sensor was kept in a refrigerator at 4 ◦C for 24 h.

#### face of the LIGE working electrode. Then, the LIGE sensor was kept in a refrigerator at 4 *2.4. Electrochemical Measurements*

°C for 24 h. *2.4. Electrochemical Measurements*  All the electrochemical measurements were carried out using PalmSens 4 potentiostat (PalmSens, Houten, The Netherlands) at room temperature. The electrochemical redox characteristics of the LIGE were measured by Cyclic voltammetry (CV) with different concentrations of potassium ferri (III)cyanide (K3[Fe(CN)6]) in 50 mM of phosphate-buffered solution (PBS). CV measurements were performed at a scan rate of 50 mV/s with a potential range from −0.8 to +0.8 V. Chronoamperometry (CA) experiments for glucose detection with LIGE were performed in 50 mM PBS at the fixed applied voltage of 0.8 V All the electrochemical measurements were carried out using PalmSens 4 potentiostat (PalmSens, Houten, The Netherlands) at room temperature. The electrochemical redox characteristics of the LIGE were measured by Cyclic voltammetry (CV) with different concentrations of potassium ferri (III)cyanide (K3[Fe(CN)6]) in 50 mM of phosphate-buffered solution (PBS). CV measurements were performed at a scan rate of 50 mV/s with a potential range from −0.8 to +0.8 V. Chronoamperometry (CA) experiments for glucose detection with LIGE were performed in 50 mM PBS at the fixed applied voltage of 0.8 V for 60 s. The detection principle of glucose is based on the electron transfer mechanism. GOx reacts with glucose in the presence of O<sup>2</sup> and produces gluconolactone and H2O2. A change in electrical current occurs at the electrode surface during these reactions due to the electron transfer. Additionally, the resulting current response is proportional to the number of glucose molecules present in the sample.

#### for 60 s. The detection principle of glucose is based on the electron transfer mechanism. GOx reacts with glucose in the presence of O2 and produces gluconolactone and H2O2. A *2.5. Optimization of Applied Potential and pH*

change in electrical current occurs at the electrode surface during these reactions due to the electron transfer. Additionally, the resulting current response is proportional to the number of glucose molecules present in the sample. CA measurements were used to determine the optimal applied potential and pH for glucose detection. The CA potential was optimized by varying the potential from 0.3 V to 1.3 V (5 mM Glucose, pH 7), and the resulting CA current was sampled at 60 s. CA

measurements were performed with a LIGE biosensor at 5 mM glucose solution with the applied potential of 0.8 V by varying the pH of the phosphate-buffered solution from a pH of 5 to 9, and the optimal pH was found. glucose detection. The CA potential was optimized by varying the potential from 0.3 V to 1.3 V (5 mM Glucose, pH 7), and the resulting CA current was sampled at 60 s. CA measurements were performed with a LIGE biosensor at 5 mM glucose solution with the applied potential of 0.8 V by varying the pH of the phosphate-buffered solution from a pH

CA measurements were used to determine the optimal applied potential and pH for

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*2.5. Optimization of Applied Potential and pH* 

#### *2.6. Interference Study* of 5 to 9, and the optimal pH was found.

The response of the LIGE biosensor for glucose detection was evaluated in the presence of potential interferences such as 0.1 mM ascorbic acid, 0.1 mM uric acid, and 3 mM urea (pH 7; 5 mM glucose; 0.8 V). *2.6. Interference Study*  The response of the LIGE biosensor for glucose detection was evaluated in the presence of potential interferences such as 0.1 mM ascorbic acid, 0.1 mM uric acid, and 3 mM

#### **3. Results and Discussion** urea (pH 7; 5 mM glucose; 0.8 V).

*3.1. Characterization of LIGE* **3. Results and Discussion** 

#### 3.1.1. Raman Spectra *3.1. Characterization of LIGE*

In this study, a graphene three-electrode system for electrochemical sensing applications was developed by direct laser inscribing on polymer substrate (Polyimide). The prepared LIGE was characterized with Raman spectra, as shown in Figure 2. The Raman spectrum consists of G band at ca. 1592 cm−<sup>1</sup> related to the E2g phonon of the sp<sup>2</sup> carbon atoms, and D band at ca. 1340 cm−<sup>1</sup> corresponds to the disordered grain boundaries [37,38]. Two other bands were observed at 2697 and 2900 cm−<sup>1</sup> . The band at ca. 2700 cm−<sup>1</sup> is known as the 2D band, an indicator of the number of graphene layers. A sharp peak will appear at ca. 2700 cm−<sup>1</sup> for monolayer graphene. Here, the broadened band was observed, which would be attributed to the prepared graphene containing many layers with some defects. The band that appeared at 2900 cm−<sup>1</sup> is called an S3 band, which is a second-order peak derived from the D–G peak combination. The band intensity ratio of S3–2D is proportional to the reduction in defects [38]. This Raman spectra result indicated that the obtained black material on polyimide substrate was carbon-based graphene. 3.1.1. Raman Spectra In this study, a graphene three-electrode system for electrochemical sensing applications was developed by direct laser inscribing on polymer substrate (Polyimide). The prepared LIGE was characterized with Raman spectra, as shown in Figure 2. The Raman spectrum consists of G band at ca. 1592 cm−1 related to the E2g phonon of the sp2 carbon atoms, and D band at ca. 1340 cm−1 corresponds to the disordered grain boundaries [37,38]. Two other bands were observed at 2697 and 2900 cm−1. The band at ca. 2700 cm−1 is known as the 2D band, an indicator of the number of graphene layers. A sharp peak will appear at ca. 2700 cm−1 for monolayer graphene. Here, the broadened band was observed, which would be attributed to the prepared graphene containing many layers with some defects. The band that appeared at 2900 cm−1 is called an S3 band, which is a second-order peak derived from the D–G peak combination. The band intensity ratio of S3–2D is proportional to the reduction in defects [38]. This Raman spectra result indicated that the obtained black material on polyimide substrate was carbon-based graphene.

**Figure 2.** Raman spectra of LIGE. **Figure 2.** Raman spectra of LIGE.

3.1.2. Electrochemical Characterization

Before developing the glucose biosensor with LIGE, validating the LIGE sensor towards electrochemical sensing was necessary. The ferri/ferrocyanide (Fe(CN)<sup>6</sup> <sup>3</sup>−/4−) redox couple is one of the most widely used electron mediators for electrochemical reactions [39]. The performance of an electrochemical sensor towards an electron mediator was considered most relevant to general biochemical sensing applications. Thus, the electrochemical efficacy of the LIGE sensor was evaluated using cyclic voltammetry responses in different concentrations of ferricyanide redox mediator (K3[Fe(CN)6]), as shown in Figure 3a. As seen from Figure 3a, the oxidation peaks' current increased from 35.495 to 65.043 µA

when the ferricyanide concentration ranged from 0.5 to 2.5 mM. The oxidation peak current showed an excellent linear relationship with different ferricyanide concentrations, as shown in Figure 3b. The linear regression equation was *y* = 14.54*x* + 28.69 *R* <sup>2</sup> = 0.998 , where *y* and *x* are the height of oxidation peak current (µA) and (K3[Fe(CN)6]) concentration (mM), respectively. The fabricated LIGE provided a favorable response for varying ferricyanide concentrations, indicating excellent electrocatalytic properties. Moreover, the reproducibility of all CV responses was within 5% RSD (relative standard deviation) (*n* = 4). These results demonstrated the remarkable electrocatalytic response of the fabricated LIGE sensor. when the ferricyanide concentration ranged from 0.5 to 2.5 mM. The oxidation peak current showed an excellent linear relationship with different ferricyanide concentrations, as shown in Figure 3b. The linear regression equation was = 14.54 + 28.69 (<sup>ଶ</sup> = 0.998), where *y* and *x* are the height of oxidation peak current (µA) and (K3[Fe(CN)6]) concentration (mM), respectively. The fabricated LIGE provided a favorable response for varying ferricyanide concentrations, indicating excellent electrocatalytic properties. Moreover, the reproducibility of all CV responses was within 5% RSD (relative standard deviation) (*n* = 4). These results demonstrated the remarkable electrocatalytic response of the fabricated LIGE sensor.

Before developing the glucose biosensor with LIGE, validating the LIGE sensor towards electrochemical sensing was necessary. The ferri/ferrocyanide (Fe(CN)63−/4−) redox couple is one of the most widely used electron mediators for electrochemical reactions [39]. The performance of an electrochemical sensor towards an electron mediator was considered most relevant to general biochemical sensing applications. Thus, the electrochemical efficacy of the LIGE sensor was evaluated using cyclic voltammetry responses in different concentrations of ferricyanide redox mediator (K3[Fe(CN)6]), as shown in Figure 3a. As seen from Figure 3a, the oxidation peaks' current increased from 35.495 to 65.043 µA

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3.1.2. Electrochemical Characterization

**Figure 3.** (**a**) CV responses of ferricyanide solutions with varying concentrations; and (**b**) oxidation current peaks vs. concentration. Scan rate was 50 mV s<sup>−</sup>1. **Figure 3.** (**a**) CV responses of ferricyanide solutions with varying concentrations; and (**b**) oxidation current peaks vs. concentration. Scan rate was 50 mV s−<sup>1</sup> .

#### *3.2. Characterization of GOx/Chitosan Immobilized LIGE 3.2. Characterization of GOx/Chitosan Immobilized LIGE*

Cyclic voltammetry measurement was performed to confirm the LIGE immobilization with GOx/Chitosan. Figure 4 shows the cyclic voltammograms of potassium ferricyanide at bare LIGE and GOx/chitosan composite-modified LIGE. It can be seen that after the immobilization of GOx/chitosan composite onto the LIGE surface, the peak current decreased to 24.325 from 58.336 µA of the bare LIGE. The electron transfer kinetics of [Fe(CN)6]4−/[Fe(CN)6]3− is significantly hindered after the LIGE surface was modified with GOx/chitosan. This result confirmed that the GOx/chitosan was successfully immobilized on the LIGE surface. Cyclic voltammetry measurement was performed to confirm the LIGE immobilization with GOx/Chitosan. Figure 4 shows the cyclic voltammograms of potassium ferricyanide at bare LIGE and GOx/chitosan composite-modified LIGE. It can be seen that after the immobilization of GOx/chitosan composite onto the LIGE surface, the peak current decreased to 24.325 from 58.336 µA of the bare LIGE. The electron transfer kinetics of [Fe(CN)6]4−/[Fe(CN)6]3<sup>−</sup> is significantly hindered after the LIGE surface was modified with GOx/chitosan. This result confirmed that the GOx/chitosan was successfully immobilized on the LIGE surface. *Polymers* **2021**, *13*, x FOR PEER REVIEW 6 of 11

**Figure 4.** Cyclic voltammograms on bare LIGE and GOx/chitosan-modified LIGE in the presence of 2 mM potassium ferricyanide. **Figure 4.** Cyclic voltammograms on bare LIGE and GOx/chitosan-modified LIGE in the presence of 2 mM potassium ferricyanide.

The chronoamperometry technique was employed to detect glucose using GOx/Chi-

chronoamperometric responses of the LIGE biosensor with glucose concentrations ranging from 0 to 10 mM. The current response increased with increasing glucose concentrations. The steady-state current response at 60 s was chosen for the detection of glucose concentration. The amperometric current response of the LIGE biosensor exhibited a linear relationship with the glucose concentrations ranging from 0 to 8 mM, and the current began to level off at a glucose concentration higher than 8 mM as shown in Figure 5b. The linear regression equation was = 3.05 + 8.54, with a coefficient of determination *R*2 = 0.97 and a sensitivity of 43.15 µA mM−1 cm−2. The limit of detection was calculated according to the 3sa/b criterion, where b was the slope of the calibration curve, and sa was the estimated standard deviation of the y-intercepts of the regression line [3]. The detection limit calculated was 0.431 mM. As seen from Figure 5b, the linear part of the calibration curve includes the normal glucose levels (4.4 to 6.6 mM) in the human blood. Thus, this study could offer a simple approach for the clinical glucose measurement with a disposable LIGE-based biosensor. The performance of the proposed biosensor was compared with other reported glucose biosensors, as shown in Table 1. The developed LIGE-based enzymatic glucose biosensor exhibited good analytical characteristics towards glucose detection such as good linearity and high sensitivity. Moreover, the fabrication and detection procedures of the proposed LIGE-based biosensor were also simple, rapid, and cost-effec-

*3.3. Amperometric Detection of Glucose by the Proposed LIGE* 

tive.
