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

The chronoamperometry technique was employed to detect glucose using GOx/ Chitosan coated LIGE sensor at a constant oxidation potential of +0.8 V. Figure 5a depicts the 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 *y* = 3.05*x* + 8.54, with a coefficient of determination *R* <sup>2</sup> = 0.97 and a sensitivity of 43.15 µA mM−<sup>1</sup> cm−<sup>2</sup> . The limit of detection was calculated according to the 3sa/b criterion, where b was the slope of the calibration curve, and s<sup>a</sup> 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-effective. *Polymers* **2021**, *13*, x FOR PEER REVIEW 7 of 11

**Figure 5.** (**a**) Chronoamperometry response with different concentrations of glucose; (**b**) the relationship between the glucose concentration and the chronoamperometric current response at 60 s. **Figure 5.** (**a**) Chronoamperometry response with different concentrations of glucose; (**b**) the relationship between the glucose concentration and the chronoamperometric current response at 60 s.

**Table 1.** Comparison of the analytical performance of glucose biosensors. **Table 1.** Comparison of the analytical performance of glucose biosensors.


laser-scribed graphene; PBSE—pyrenebutanoic acid–succinimide ester; PtNPs—platinum nanoparticles; MoS2—molybdenum disulfide; PGE—pencil graphite electrode; CPE—carbon paste electrode; SiO2/Lig—silica/lignin; Cys—cysteine; GA—glutaraldehyde; PPy—polypyrrole; SPCE—screen-printed carbon electrodes; GOD/GOx—Glucose oxidase. <sup>a</sup> GC—glassy carbon electrode; MWCNT—multi-walled carbon nanotubes; Fe3O4/PDA—magnetite/polydopamine; LSG—laser-scribed graphene; PBSE—pyrenebutanoic acid–succinimide ester; PtNPs—platinum nanoparticles; MoS2—molybdenum disulfide; PGE—pencil graphite electrode; CPE—carbon paste electrode; SiO2/Lig—silica/lignin; Cys—cysteine; GA—glutaraldehyde; PPy—polypyrrole; SPCE screen-printed carbon electrodes; GOD/GOx—Glucose oxidase.

the enzymatic reaction (௫) and the apparent Michaelis constant (<sup>୫</sup>

complex over triangular silver nanoprisms/platinum biosensor [47].

*3.5. Optimization of Applied Potential and Buffer pH* 

The maximum response current (௫) and the apparent Michaelis–Menten constant

ୟ୮୮) are the corre-

ୟ୮୮ is consistent

ୟ୮୮) were used to analyze the relationship between chronoamperometric signals and

response plateau was observed with the characteristic of Michaelis–Menten kinetic mechanism. From the calibration plot (Figure 5b dotted line), the current response showed hyperbolic dependence on glucose concentration and was in good agreement with Michaelis–Menten kinetics [45]. The kinetic parameters, the maximum current generated during

sponding and parameters of hyperbolic function = /( + ) [46]. The apparent

ics. From the hyperbolic calibration plot (Figure 5b dotted line), the ௫ and Michaelis

Chronoamperometry measurements were used to determine the optimal applied potential and pH for glucose detection with the developed enzymatic LIGE biosensor. Figure

ୟ୮୮ were 40.34 µA and 3.75 mM, respectively. The value of <sup>୫</sup>

ୟ୮୮) is an indication of enzymatic mimics–substrate kinet-

ୟ୮୮ = 3.84 mM) for other GOx immobilized on the chitosan

*3.4. Michaelis–Menten Kinetics* 

Michaelis–Menten constant (<sup>୫</sup>

with the reported value (<sup>୫</sup>

(<sup>୫</sup>

constant <sup>୫</sup>
