A Glucose-Responsive Enzymatic Electrode on Carbon Nanodots for Glucose Biosensor and Glucose/Air Biofuel Cell

In this study, an enzymatic electrode for glucose biosensing and bioanode of glucose/air biofuel cell has been fabricated by immobilizing poly (methylene green) (polyMG) for electrocatalytic NADH oxidation and NAD-dependent glucose dehydrogenase (GDH) for oxidizing glucose on carbon nanodots (CNDs). The polyMG-CNDscomposites obtained by electro-polymerization of dye MG molecules adsorbed on CNDs display excellent electrocatalytic activity toward NADH electro-oxidation at a low overpotential of ca. −0.10 V (vs. Ag/AgCl) and the integrated enzymatic electrode shows fast response to glucose electrooxidation. Using the fabricated GDH-based enzymatic electrode, a glucose biosensor was constructed and exhibits a wide linear dynamic range from 0 to 8 mM, a low detection limit of 0.02 μM (S/N = 3), and fast response time (ca. 4 s) under the optimized conditions. The developed glucose biosensor was used to detect glucose content in human blood with satisfactory results. The fabricated GDH-based enzymatic electrode was also employed as bioanode to assembly a glucose/air biofuel cell with the laccase-CNDs/GC as the biocathode. The maximum power density delivered by the assembled glucose/air biofuel cell reaches 3.1 μW∙cm at a cell voltage of 0.22 V in real sample fruit juice. The present study demonstrates that potential applications of GDH-based CNDs electrode in analytical and biomedical measurements.


Introduction
It is the essential groundwork to fabricate enzymatic electrodes for constructing enzyme-based electrochemical biosensors and enzymatic biofuel cells (BFCs) [1] [2] [3] [4] [5]. Glucose-responsive enzymatic electrodes, which are responsible for electrochemical glucose biosensors or bioanodes of glucose-based BFCs with glucose as fuels, have attracted much attention [6] [7] [8] [9] [10]. Two types of commercially available enzymes, glucose oxidase (GOx) and glucose dehydrogenases (GDH), are generally employed to construct glucose-responsive enzymatic electrodes [1]- [10]. The GDH family is classified into three different types on the basis of its cofactors, nicotinamide adenine dinucleotide-dependent GDH (NAD-GDH), flavin adenine dinucleotide-dependent GDH (FAD-GDH), and pyrroloquinoline quinone-dependent GDH (PQQ-GDH) [11]- [16]. Among these enzymes, NAD-GDH is widely favored to be chosen to construct enzymatic electrode because it is insensitive to oxygen and highly specific to glucose [11]- [16]. As a result, the NAD-GDH based enzymatic electrode is allowed to improve the accuracy and selectivity of electrochemical biosensor of glucose and reduce the cross-talk of glucose/O 2 BFC. In order to expand the bioelectrochemical applications of NAD-GDH enzymatic electrode, extensive studies are focused on recycling the cofactor by regenerating NADH from the enzymatically produced NAD + cofactor through electrochemistry methods [11]- [20]. However, direct electrochemical oxidation of NADH generally requires an applied potential as high as Ca. 0.75 V (versus Ag/AgCl) although the theoretical thermodynamic potential for NADH/NAD+ couple is as low as −0.32 V (versus NHE) [ [26].
In this study, a NAD-GDH based enzymatic electrode was fabricated, on which NAD-GDH was immobilized on carbon nanodots (CNDs) with in-situ electrochemical polymerized methylene green (polyMG) on CNDs as electro-oxidation electrocatalyst for NADH, to construct glucose electrochemical biosensor and the bioanode for glucose oxidation in glucose/air BFC. For the assembly of glucose/air BFC, the GDH-polyMG-CNDs/GC and laccase-CNDs/GC electrode were used as bioande for glucose oxidation and biocathode for oxygen reduction, respectively. Fruit juice containing glucose was used as the fuel solution to characterize the performances of the as-prepared BFC at ambient air atmosphere. The real sample fruit juice was adjusted to pH 7.0 with 0.1 M PBS and the ratio for buffer solution and soft drinks was about 3:2 (v/v).

Typical SEM and TEM Images of Carbon Nanodots
CNDs were prepared according to previous reports [18] [25] [26]. Figure 1 shows the low magnification SEM (Figure 1(A)) and high magnification TEM (Figure 1(B)) images of the obtained CND. From the figures, we can see that the obtained products consist of a large amount of nanodots. The images reveal that the diameters of the nanodots are in the range of 40 -60 nm. The uniform nanostructures provide a significant increase in effective electrode surface for loading enzymes and accelerating electron transfer.  are presented in Figure 2(B). As shown in this figure, the potential for NADH oxidation at the polyMG-CNDs/GC electrode was observed at −0.10 V, which has a good agreement with the value reported previously [27]. These results indicate that the presence of polyMG-CNDs resulted in a substantial decrease of the overpotential to about 650 mV for NADH oxidation at conventional bare GC electrode. From Figure 2(B), we also can clearly see that the peak current increases significantly with the addition of an increasing amount of NADH.

Preparation and Catalytic Performances of Different Electrode
These results demonstrate that the polyMG-CNDs composites show excellent electrocatalytic activity to NADH oxidation and could be further fabricated NAD-dependent dehydrogenase based enzymatic electrodes.
In order to construct a biosensor of glucose and a bioanode of glucose/O 2 BFC, GDH was further immobilized on polyMG-CNDs composites to obtain GDH-polyMG-CNDs/GC electrode.

Real Applications of the Fabricated GDH-PolyMG-CNDs/GC Electrode
Based on the catalytic currents dependent on the glucose concentrations, a glucose biosensor was fabricated. As shown in Figure 3 Table 1. From this Table, it can be seen that the determination results are good agreement with the results provided by the hospital and the RSD values are smaller than 2.97%, indicating that the proposed glucose biosensor displays good accuracy and repeatability in complex samples and therefore shows potentially practical applications. It has been attracted much attention to develop NAD + -dependent dehydrogenase based bioanodes for constructing BFCs because more than 300 dehydrogenases have been known today and therefore different substrates of dehydrogenases can be employed for biofuels [17] [18] [19]. Glucose, as a most active fuel of enzymatic BFCs, is also a common ambient fuel which exists widely in human, animals, plants, various foods. Many kinds of soft drinks, which are rich in glucose, are suitable fuels for BFCs owing to the advantages of green fuel, cheapness and availability. In this study, by using GDH-polyMG-CNDs composites as the bioelectrocatalysts for the catalytic electrooxidation of glucose at the bioanode and laccase-CNDs composites as the direct bioelectrocatalyst for oxygen reduction at the biocathode, a membrane-less glucose/air BFC was successfully constructed. The investigations on the direct electron transfer (DET) of blue-copper oxidases including bilirubin oxidase and laccase on CNDs have been studied in our previous studies [18] [26]. These studies have demonstrated that CNDs can efficiently facilitate the DET behaviours of bilirubin oxidase and laccase, and also retain their bioactivity for bio-catalyzing oxygen reduction. Based on previous studies, a laccase-CNDs enzymatic electrode was prepared as before and used as the biocathode for oxygen reduction [18] [26]. In this study, the performances of the constructed glucose/air BFC was further investigated in glucose-containing fruit juice in order to demonstrate its suitability for implantable applications. Figure 3(B) shows the polarization curve and power curve of the assembled glucose/air BFC in the quiescent fruit juice containing 20 mM NAD + under ambient air atmosphere in 0.1 M, pH 7.0 phosphate buffer solution. The open-circuit voltage (OCV) of the BFC is ca. 0.48 V and the power density reaches 0.31 µW/cm 2 at 0.22 V. This result is quite comparable to that of glucose/oxygen BFC reported recently [28]. Therefore, the assembled miniature BFC can directly generate energy from soft drinks. When the cell operated continuously with an external loading resistance of 1 MΩ in a quiescent 0.1 M, pH 7.0 buffer solution containing 20 mM NAD + under ambient air, it lost ca. 9.8% of its original power in the first day and the power output remained ca. 73.4% of its original power after a week continuous work. The performance of the assembled CNDs-based glucose/air BFC is dominated by the current density of the laccase-CNDs/GC biocathode. The decreased OCV and power density may be explained by the deactivation of enzymes by some compounds within fruit juice, and low glucose and oxygen concentration in fruit juice under air atmosphere.

Conclusion
In this work, we have synthesized and characterized a carbon-based material, carbon nanodots. Using carbon nanodots as supporting matrixes, GDH-polyMG-CNDs and laccase-CNDs were prepared and used for glucose oxidation and oxygen reduction. A glucose biosensor was fabricated with the GDH-polyMG-CNDs/GC electrode and also used for blood glucose determination in serum samples. Moreover, the GDH-polyMG-CNDs/GC electrode as a promising bioanode and DET-type laccase-CNDs /GC electrode for as biocathode four-electron reduction of oxygen were used to construct glucose/air BFC. The bioelectrocatalytic performances of the prepared enzyme electrodes using carbon nanodots as supporting matrixes were studied systematically and therefore a novel glucose/air BFC is assembled by using GDH-polyMG-CNDs/ GC electrode as bioanode and laccase-CNDs/GC as biocathode. In all, the present studies indicate that CNDs can be employed as promising immobilizing materials and electrochemical transducer in bioelectrochemistry area.