An electrochemically reduced copper/reduced graphene oxide film modified electrode for sensitive non-enzymatic glucose detection in human serum

Numerous studies suggest that modification with functional nanomaterials can enhance the electrode electrocatalytic activity, sensitivity, and selectivity of the electrochemical sensors. Here, a highly sensitive and cost-effective disposable non-enzymatic glucose sensor based on copper(II)/reduced graphene oxide modified screen-printed carbon electrode is demonstrated. Facile fabrication of the developed sensing electrodes is carried out by the adsorption of copper(II) onto graphene oxide modified electrode, then following the electrochemical reduction. The proposed sensor illustrates good electrocatalytic activity toward glucose oxidation with a wide linear detection range from 0.10 mM to 12.5 mM, low detection limit of 65 µM, and high sensitivity of 172 μA mM –1 cm –2 along with satisfactory anti-interference ability, reproducibility, stability, and the acceptable recoveries for the detection of glucose in a human serum sample (95.6–106.4%). The copper(II)/reduced graphene oxide based sensor with the superior performances is a great potential for the quantitation of glucose in real samples. ,

of GO to adsorb Cu(II) ion and the electrochemical reduction of Cu(II)/GO have been proposed to synthesize the Cu(II)/rGO and surface-reduced Cu(0)/rGO complexes.
In this study, a facile preparation of Cu(II)/rGO complexes with respect to electrocatalytic oxidation of glucose is demonstrated. The GO modified SPCE is used as catalysts support for Cu(II) adsorption. After the complexation of Cu(II)/GO was formed on the surface of GO, the Cu(II)/GO modified SPCE was subsequently reduced by electrochemical technique to give the Cu(II)/rGO nanocomplexes that employed as a glucose sensing platform.
A schematic diagram of the device fabrication is shown in Figure 1. The proposed sensor showed good electrocatalytic activity towards glucose. A fast electron transfer between the electrode and the electroactive species would enhance due to the high electrical property of rGO. Additionally, a wide linear range, a good sensitivity, and a low limit of detection (LOD) are observed, and the proposed sensor was applied for the determination of glucose in a biological sample.

Results
Optimal conditions for the fabrication of Cu(II)/GO modified electrodes. Under the adsorption period of 60 min, the concentration of Cu(NO3)2 (0.00−100 mM) in an adsorption solution was optimized to acquire the sensing platform with high electrocatalytic performance.
To study the effect of Cu(II) concentration on the electrochemical property, cyclic voltammestry (CV) of each Cu(II)/GO modified electrode was performed over the potential range between −0.80 to 1.0 V in 0.20 M acetate buffer (pH 4.5) (supporting information, Figure S1(a)). The redox peak currents of Cu(II) increase accordingly and the peaks potential gradually shift negatively and positively with the increment of Cu(NO3)2 concentration from 0.25 to 100 mM. This behavior resulted from the presence of a large amount of electroactive species at the electrode surface. At higher concentrations of Cu(NO3)2, the amount of Cu(II) adsorbed on GO modified SPCE was also high. When the Cu(II) complexes are formed, more electrons are transferred led to the anodic and cathodic potential shifts their position to a more positive direction. In this work, the anodic peak current was considered for the optimization of Cu concentration. The correlation graph between anodic current response of Cu(II) and concentration of Cu(NO3)2 is shown in supporting information, Figure S1 The result indicates that the GO modified SPCE can be used to adsorb the Cu(II) onto the electrode surface. Moreover, the surface coverage (Γ) of the Cu(II)/GO modified electrode was calculated by integration of the anodic peak area using the following equation 26 .
where n is the number of electrons transferred in the electrode (n = 2), F is the Faraday constant (96,487 C mol -1 ), A is the surface area of the electrode (7.07 × 10 -2 cm 2 ), and v is the scan rate (V s -1 ). The estimated surface coverage of the modified electrode with the maximum uptake of Cu(II) is 5.20 × 10 -10 mol cm -2 , and the amount of Cu(II) in the GO-modified SPCE is of 4.90 µmol g -1 GO (Cu(II) = 3.68 × 10 -11 mol and GO = 7.5 μg). Furthermore, the relationship between the Cu(NO3)2 concentration and the peak current of glucose oxidation at each Cu(II)/GO modified SPCE prepared from different Cu(NO3)2 concentrations is displayed in supporting information, Figure S1(b) curve(b). The peak current significantly increases when the amount of Cu(II) catalyst increase with adsorption concentration from 0.25 to 2.50 mM while the current response with insignificant change occurs at higher concentration (5.00−100 mM). This result suggests that an increase in the amount of Cu(II) provides more active sites, facilitating better electrooxidation of glucose at the electrode surface 5 . However, the electrocatalysis for glucose oxidation is not enhanced by a larger number of Cu(II) on the electrode surface. It is caused by the limitation of the electrode, which would involve the accessibility of glucose into catalytic Cu(II) centers inside the Cu(II)/GO nanostructure of the electrode 27 . The Cu(II)/GO-modified SPCE prepared using 2.50 mM Cu(NO3)2 (a Cu(II) uptake = 1.44 µmol g -1 GO (Cu(II) = 1.08 × 10 -11 mol and GO = 7.5 μg) and surface coverage = 1.52 × 10 -10 mol cm -2 ) exhibits sufficient electrocatalytic activity for the oxidation of glucose. Therefore, this concentration is considered to be the optimal concentration, and is further employed for the construction of the sensing platform in this work. In addition, Cu(II) sitting on the surface of GO is also depended on the adsorption time, which influences the electrocatalytic performance of the modified electrode (supporting information, Figure S2(a) and (b)). The adsorption time of 60 min is the optimal adsorption period in term of ability to catalyze glucose oxidation. The amount of GO could also affect the electrochemical property of the modified electrode (supporting information, Figure S3). In this work, a 3.0 mg mL -1 GO dispersion solution for the fabrication of Cu(II)/GO complex exhibits the highest current response of glucose oxidation, which is considered to be the optimal GO concentration.
Characterization of morphological surface of modified electrodes. The field emission scanning electron microscope (FE-SEM) was employed to characterize the surface morphology of the modified SPCEs as shown in Figure 2(a-e). The surface morphology of the bare electrode ( Figure 2(a)) significantly changes after modification with the GO. As shown in Figure 2(b), the entire electrode surface is covered by the GO nanosheet, and the typical crumpled and wrinkled nanosheet structure is clearly observed. The large surface area of GO could facilitate the adsorption of Cu(II), which can increase the active sites or catalytic centers on the electrode surface. Although, the Cu(II)/GO-modified SPCE surface has no obvious change from the GOmodified SPCE as shown in Figure 2(c), the existance of Cu element in the nanocomposite can be proved by the energy dispersive X-ray spectroscopic technique (EDS) and the EDS spectrum is showed in supporting information, Figure S4(c). The rGO-modified SPCE (Figure 2 manifestly exhibits a pattern of wrinkled nanostructure and becomes more rough compared to the GO-modified SPCE. Also, more stacking and aggregation of rGO is observed which is the characteristic structure of rGO 28 . The electrochemical reduction can eliminate the oxygenated functional group on GO surface, resulting in the formation of rGO due to the increased π-π interaction between the rGO layer 29 . Likewise, the morphology of Cu(II)/rGO nanocomplex has no change as compared with that of the rGO sheet as shown in Figure 2(e) and the remaining of Cu on the Cu(II)/rGO-modified SPCE is cleary observed in the EDS spectrum (supporting information, Figure S4 and Cu contents can prove the successful adsorption of Cu(II) onto the GO/rGO surface. The peak intensities in the two spectra were different, especially the C 1s spectrum. As seen in and O=C−O, significantly decrease, suggesting that most of oxygen functional groups on the GO surface were removed during the electrochemical reduction process. Furthermore, the peak intensities of sp 2 hybridization of C atoms distinctly increase, which it can be proved that the GO was successfully reduced to be rGO under the optimal condition. Additionally, synchrotron-based X-ray absorption spectroscopy (XAS) technique was employed to determine the valence state of Cu in the prepared Cu(II)/GO-and Cu(II)/rGO-modified electrodes. The Cu K-edge X-ray absorption near-edge structure (XANES) spectra were recorded in transmission modes using a 4-element Si drift detector 31,32 , as the result shown in probably remained from the physisorption of Cu 2+ precursors on the graphenic carbon site due to metal interactions with π-electrons in the graphene layer 33,34 . These adsorptive properties of GO contributed to having an abundance of Cu(II) species located in the nanocomposite.
Furthermore, some Cu 2+ in the nanocomplex can be reduced during the electrochemical reduction to form Cu 0 or Cu nanoparticles 35 ; therefore, the electrocatalysis activity at the modified electrode could be involved by two forms of active species including Cu(0) and Cu(II), which are located in the proposed sensing platform. (equations (4)-(5)). Then, glucose is catalytically oxidized by Cu(III) species to produce gluconolactone and hydrolyzation to gluconic acid (equation (6) and (7)

Electrochemical
Although various forms of Cu can be observed on our platform confirming by the XPS spectrum, the formation of Cu(III) species is essential for the electrocatalytic glucose oxidation.
The different forms of Cu including Cu(0) or Cu(II) in the nanocomposite can be electrochemically oxidized into Cu(II) and eventually into Cu(III) species (equation 2-5).
Therefore, the electrocatalytic glucose oxidation on Cu-based modified electrode could be generated under the similar mechanism as represent in equation (6). The Cu(II)/rGO-modified SPCE was further employed as a sensing platform for the detection of glucose. The anodic peak current for sensing platform increases with increasing in the glucose concentration as shown in supporting information, Figure S9. This mechanism promoted by Cu(III) species; therefore, more Cu (III) would be consumed at high concentration, leading to the large oxidation peak current 45  proposed sensors were stored at room temperature for 20 days, and the change in current response toward 2.5 mM glucose solution was measured as shown in Figure 5(b). It was found that the 87% current response of the initial response was obtained after storage for 20 days.
The result indicates good stability of the developed sensor, which can be applied for glucose detection in the real samples. To prolong the electrode shelf life, the electrodes might require the storage in a proper atmosphere such as inert atmosphere, low temperature, low humidity atmosphere, and low pressure atmosphere or vacuum.
In order to study the practical feasibility of the developed sensor, the Cu(II)/rGO nanocomplex-modified electrode was subsequently employed for the determination of glucose in human serum sample. The human serum sample was diluted with 0.10 M NaOH for 50 times and then glucose stock solutions (0.05, 1.00, 2.50, 4.00, and 5.00 mM) were spiked into the diluted serum solution. The chronoamperometry was carried out to investigate the glucose oxidation at a Cu(II)/rGO modified electrode and each measurement was performed for three times (n = 3). As seen in Table 2, the percent recovery values for glucose determination by our electrode are in the range from 95.6% to 106.4% and the R.S.D. values are found to be less than 5%. This suggests that the proposed sensing platform offers a good selectivity and repeatability for determination of glucose in real sample matrix (human serum). Hence, it demonstrated that the proposed modified electrode can be applied for the assay of glucose in real sample.