*3.5. Optimization of Applied Potential and Buffer pH*

Chronoamperometry measurements were used to determine the optimal applied potential and pH for glucose detection with the developed enzymatic LIGE biosensor. Figure 6a shows the chronoamperometric response of the LIGE biosensor at 60 s with different applied potential values ranging from 0.3 to 1.3 V. The results showed that the current increased with increasing applied potential from 0.3 to 0.8 V and currents tended to level off when the potential increased beyond 0.8 V. Thus, 0.8 V was selected as the optimized potential for amperometric glucose detection. Figure 6b illustrates the chronoamperometry current response of the biosensor as a function of the pH of PBS containing 2 mM glucose. The current responses at pH 5, pH 6, and pH 7 were almost similar. Considering the pH of a physiological buffer, pH 7 was chosen for the glucose detection experiments. *Polymers* **2021**, *13*, x FOR PEER REVIEW 8 of 11 6a shows the chronoamperometric response of the LIGE biosensor at 60 s with different applied potential values ranging from 0.3 to 1.3 V. The results showed that the current increased with increasing applied potential from 0.3 to 0.8 V and currents tended to level off when the potential increased beyond 0.8 V. Thus, 0.8 V was selected as the optimized potential for amperometric glucose detection. Figure 6b illustrates the chronoamperometry current response of the biosensor as a function of the pH of PBS containing 2 mM glucose. The current responses at pH 5, pH 6, and pH 7 were almost similar. Considering the pH of a physiological buffer, pH 7 was chosen for the glucose detection experiments.

**Figure 6.** (**a**) Chronoamperometry response at 60 s in different applied potential with 5 mM glucose; and (**b**) Chronoamperometry response at 60 s in different buffer pH with 2 mM glucose. **Figure 6.** (**a**) Chronoamperometry response at 60 s in different applied potential with 5 mM glucose; and (**b**) Chronoamperometry response at 60 s in different buffer pH with 2 mM glucose.

#### *3.6. Interference Study 3.6. Interference Study*

than 6%.

The developed LIGE-based enzymatic biosensor was evaluated with possible interferences by comparing the chronoamperometric responses before and after adding some interferents such as ascorbic acid (0.1 mM), uric acid (0.1 mM), and urea (3 mM) in 5 mM glucose. As shown in Figure 7, the chronoamperometric current responses for glucose The developed LIGE-based enzymatic biosensor was evaluated with possible interferences by comparing the chronoamperometric responses before and after adding some interferents such as ascorbic acid (0.1 mM), uric acid (0.1 mM), and urea (3 mM) in 5 mM glucose. As shown in Figure 7, the chronoamperometric current responses for glucose with-

without and with interferents showed practically no interference. The LIGE was modified with GOx, which is the standard enzyme for biosensors and it has relatively higher selec-

The stability of the developed GOx/chitosan-modified LIGE biosensor was evaluated by measuring the amperometric current response in the presence of 5 mM glucose over 25 days stored at 4 °C in a refrigerator. The biosensor exhibited ~90% stability for 10 days, and the response remained approximately 72–85% after 10 days. The reproducibility of the developed biosensor was assessed from the current response of different biosensors prepared independently. In this work, all the measurements were taken from at least three independent sensors (*n* ≥ 3), and the reproducible signals were obtained with the RSD less

*3.7. Stability and Reproducibility of Biosensor* 

**References** 

out and with interferents showed practically no interference. The LIGE was modified with GOx, which is the standard enzyme for biosensors and it has relatively higher selectivity for glucose [48]. Hence, the LIGE biosensor was suggested to possess good selectivity due to the specificity of the GOx enzyme. *Polymers* **2021**, *13*, x FOR PEER REVIEW 9 of 11

**Figure 7.** Chronoamperograms of 5 mM glucose with/without interferences. The inset shows the zoomed part of the result from 45 to 70 s. **Figure 7.** Chronoamperograms of 5 mM glucose with/without interferences. The inset shows the zoomed part of the result from 45 to 70 s.

#### **4. Conclusions**  *3.7. Stability and Reproducibility of Biosensor*

We developed a simple laser-induced graphene-based enzymatic biosensor for glucose detection. The proposed detection strategy could offer an easy and low-cost route to mass-produce sensitive biosensing electrodes. The chronoamperometric measurements successfully detected the glucose over a linear range from 0 to 8 mM with a detection limit of 0.431 mM. The biosensor response was not affected by interfering compounds (ascorbic acid, uric acid and urea) and demonstrated the high specificity and selectivity of this LIGE biosensor in glucose detection. The proposed LIGE biosensor holds excellent promise in point-of-care diagnosis. Our future study aims to validate the biosensor response in hu-The stability of the developed GOx/chitosan-modified LIGE biosensor was evaluated by measuring the amperometric current response in the presence of 5 mM glucose over 25 days stored at 4 ◦C in a refrigerator. The biosensor exhibited ~90% stability for 10 days, and the response remained approximately 72–85% after 10 days. The reproducibility of the developed biosensor was assessed from the current response of different biosensors prepared independently. In this work, all the measurements were taken from at least three independent sensors (*n* ≥ 3), and the reproducible signals were obtained with the RSD less than 6%.

#### man blood samples for real-life applications. **4. Conclusions**

Grown ZnO Nanorods. *Sci. Rep.* **2018**, *8*, 13722, doi:10.1038/s41598-018-32127-5.

biosensing of glucose. *Biosensors* **2019**, *9*, 46.

**Author Contributions:** K.S.: conceptualization, supervision, validation, writing—review and editing; P.-T.C.: methodology, validation, data curation, writing—original draft; Y.-M.H.: validation, data curation. All authors have read and agreed to the published version of the manuscript. **Funding:** This research was supported under Grants MOST 110-2222-E-305-001 and MOST 107- 2221-E-305-012-MY3 by the Ministry of Science and Technology, Taiwan, and Grant 109- NTPU\_ORDA-F-008 by National Taipei University, Taiwan. **Institutional Review Board Statement:** Not applicable. **Informed Consent Statement:** Not applicable. We developed a simple laser-induced graphene-based enzymatic biosensor for glucose detection. The proposed detection strategy could offer an easy and low-cost route to mass-produce sensitive biosensing electrodes. The chronoamperometric measurements successfully detected the glucose over a linear range from 0 to 8 mM with a detection limit of 0.431 mM. The biosensor response was not affected by interfering compounds (ascorbic acid, uric acid and urea) and demonstrated the high specificity and selectivity of this LIGE biosensor in glucose detection. The proposed LIGE biosensor holds excellent promise in point-of-care diagnosis. Our future study aims to validate the biosensor response in human blood samples for real-life applications.

**Conflicts of Interest:** The authors declare no conflict of interest. **Author Contributions:** K.S.: conceptualization, supervision, validation, writing—review and editing; P.-T.C.: methodology, validation, data curation, writing—original draft; Y.-M.H.: validation, data curation. All authors have read and agreed to the published version of the manuscript.

1. Ramanavicius, S.; Ramanavicius, A. Charge Transfer and Biocompatibility Aspects in Conducting Polymer-Based Enzymatic Biosensors and Biofuel Cells. *Nanomaterials* **2021**, *11*, 371, doi:10.3390/nano11020371. 2. Ramanavicius, S.; Ramanavicius, A. Conducting Polymers in the Design of Biosensors and Biofuel Cells. *Polymers* **2021**, *13*, 49, doi:10.3390/polym13010049. **Funding:** This research was supported under Grants MOST 110-2222-E-305-001 and MOST 107-2221- E-305-012-MY3 by the Ministry of Science and Technology, Taiwan, and Grant 109-NTPU\_ORDA-F-008 by National Taipei University, Taiwan.

3. Ridhuan, N.S.; Razak, K.A.; Lockman, Z. Fabrication and Characterization of Glucose Biosensors by Using Hydrothermally

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.
