Development of non-enzymatic glucose electrode based on Au nanoparticles decorated single-walled carbon nanohorns

Gold nanoparticles (AuNPs)-based composite decorating with single-walled carbon nanohorns (SWCNHs) was synthesized and modified on a gold electrode for non-enzymatic glucose detection. The composite was synthesized by a simple covalent bonding method. The AuNPs with an average particle size of 40 nm are dispersed homogeneously on the surface of the SWCNHs. Therefore, the synergistic effect of the AuNPs and the SWCNHs leads to an excellent glucose sensing performance. The AuNPs/SWCNHs nanocomposites acted as a fast redox probe for non-enzymatic glucose oxidation exhibiting good performance, including a high sensitivity (275.33 and 352.5 μA cm−2 mM−1), a low detection limit of 0.72 μM (S/N = 3). AuNPs/SWCNHs glucose sensor also demonstrates good stability, reproducibility, and selectivity. Moreover, the glucose contents detected by this electrode was in right agreement with real value in blood samples. In summary, this study showed that AuNPs/SWCNHs nanoparticles have the potential to be used in non-enzymatic glucose detection.


Introduction
Diabetes mellitus is a metabolic disorder characterized by elevated levels of blood glucose. The number of people with diabetes mellitus is increasing every year. In 2017, the International Diabetes Federation (IDF) estimated that 1 in 11 adults aged 20-79 years (451 million adults) had diabetes mellitus globally and that patients would increase to 693 million in 2045 [1]. According to the IDF, approximately 5 million deaths worldwide were related to diabetes in the 20-99 years age range in 2017, which accounts for 9.9% of the global mortality for people in this age range [2]. Therefore, the detection and monitoring of the glucose levels is essential for the prevention, the diagnosis, and the treatment of diabetes. Many glucose sensors were developed using enzymes to obtain a high sensitivity and good selectivity [3]. However, the sensitivity, the selectivity, and the stability of enzyme-based glucose sensors largely depend on the activity of the enzymes. The pH, the humidity, and the temperature influence the activity of glucose enzymes [4].
Overall, non-enzymatic glucose sensors have many advantages, such as a high reproducibility, a high stability, and structural simplicity. To date, many noble metals (Pt, Au, and Pd), transition metals (Cu, Ni, and Co), and their oxides or hydroxides have been used in non-enzymatic glucose sensors [9][10][11][12][13][14][15]. Among them, Au is widely used because it produces a high glucose oxidation current in neutral or alkaline conditions [16,17]. Furthermore, Au nanoparticles (AuNPs) are glucose catalysts that have attracted much attention because of their large surface area, their excellent catalytic activity, and a high resistance to toxic Cl - [18]. Cho developed a highly porous gold (Au) electrode for non-enzymatic glucose detection [19]. Zhou synthesized a gold nanotube array for detecting glucose [20]. Au nanomaterials generate larger electrical signals and increase the utilization rate of the noble metal in electrochemical reactions. In addition, the favorable structure of the carrier-catalyst composite is a major advantage for sensors [21][22][23]. The carrier can affect the dispersion of the catalyst, enhance the conductivity and the stability of the material, and make the catalyst work more effectively. Xu reported a novel flexible glucose sensor composed of Au nanoparticles/polyaniline arrays/carbon cloth [24]. Li reported a carbon nanotube supporting Pt nanoparticles in a non-enzymatic glucose sensor [25]. Single-walled carbon nanohorns (SWCNHs) are a nanostructured carbon material with horn-shaped sheaths composed of graphene sheets and has a conical structure with a particularly sharp apical angle [26]. Their excellent electrical conductivity, their high specific surface area, and the vast internal space of the SWCNHs make it an excellent carrier for other particles. Moreover, SWCNHs are produced without using any metal catalyst at a high purity and can be used directly without further purification [27].
Considering the many merits of AuNPs and SWCNHs, we first developed an Au electrode modified with an AuNPs and SWCNHs composite for the detection of glucose. Covalent bonding is used in the preparation of the Au-SWCNHs composite. The method is simple and has a low cost. Additionally, it avoids the use of toxic reagents and the composite performs very well for the electrochemical detection of glucose. Compared with similar work, Kangkamano's gold nanoparticles decorated multi-walled carbon nanotubes modified electrode for non-enzymatic glucose detection [28], our preparation method is more convenient and our work has also demonstrated a better performance in electrocatalytic glucose, such as higher sensitivity and wider detection range. Because SWCNHs have a large surface area and good electrochemical properties [26], which gives the composite with a stable structure and a high catalytic activity for glucose. There are enough binding sites for the gold nanoparticles on the modified SWCNHs to enable the formation of s-Au bond. Glucose sensing tests confirmed that the fabricated electrode had high sensitivity, good stability, antiinterference, and reproducibility, which is used as the basis for developing the glucose sensor.
Scanning electron microscopy (SEM) images were recorded on a JSM-IT300 (JEOL, Ltd., Japan). We used a Renishaw InVia confocal Raman system (Talos F200S, Thermo Fisher Scientific, United States) to examine the defects in the samples. All the electrochemical experiments were performed on a CS2350 electrochemical workstation (Corrtest Instruments Corp., Ltd., Wuhan, China).

Preparation of the Au-SWCNHs composite
The composite comprised of AuNPs decorating SWCNHs was synthesized by a three-step method. First, oxidized SWCNHs (oxSWCNHs) were prepared via a modified Hummers method [29]. We initially mixed 5 mL H 2 SO 4 (98%) and 15 mL HNO 3 (62%). Next, 30 mg SWCNHs were treated with the mixed acid for 16 h under continuous stirring at 50°C through a hydrothermal reaction. The oxSWCNHs were collected by centrifugation at 12000 rpm for 5 min. Then, the supernatant was removed, and deionized water was added to disperse the oxSWCNHs for further centrifugation. We carried out several rounds of centrifugation and dispersion until the supernatant was neutral. Finally, the collected oxSWCNHs were dried in an oven at 90°C. Then, the oxSWCNHs were dispersed into 30 ml of deionized water, and 50 mg of C 2 H 7 NSÁHCl were added to the dispersion. The mixture was treated at 90°C for 24 h with continuous agitation. The mixture obtained was centrifuged at 12000 rpm and washed with deionized water several times. After centrifugation, the composites were placed into deionized water to form a suspended solution, and 5 mL of an HAuCl 4 solution (0.01 M) were added. The mixture was sonicated for 30 min to promote the interaction between the gold ions and the sulfhydryl (-SH) groups of the oxSWCNHs. The mixture was heated to 90°C and 10 mL of a 4% Na 3 C 6 H 5 O 7 solution were added. The mixed solution was stirred for 30 min until Au ? was reduced to Au nanoparticles. The dispersion obtained was then centrifuged, washed several times with deionized water, and dried at 90°C . The nanocomposite obtained was named Au-SWCNHs. Finally, the Au-SWCNHs black powder was collected and stored at 4°C for further use.

Preparation of the Au-SWCNHs modified Au electrode
Before its modification, the Au electrode was polished, washed several times with deionized water and anhydrous ethanol under sonication, and dried in a nitrogen stream. First, we added 10 mg of the Au-SWCNHs powder into 5 mL of anhydrous ethanol and sonicated the mixture for 20 min to obtain an Au-SWCNHs ethanol suspension at 2 mg mL -1 . Then, we dropped 5 lL of the Au-SWCNHs ethanol suspension on the Au electrode and let it dry naturally to obtain the Au-SWCNHs/Au electrode (Au-SWCNHs/Au). The electrode was stored at 4°C for further use. Figure 1 illustrates the preparation of the Au-SWCNHs composite. First, the SWCNHs were treated with mixed acid (H 2 SO 4 /HNO 3 ). Upon oxidation, oxygen-containing groups like carboxyl groups (-COOH) were generated on the surface of the SWCNHs. The subsequently added C 2 H 7 NSÁHCl was ionized in water to produce positively charged sulfur-containing ions while the oxygen-containing groups on the surface of the oxSWCNHs were deprotonated and became negatively charged. Both positively and negatively charged ions were then combined by electrostatic forces. After the addition of HAuCl 4 , it was dispersed by sonication and fully mixed with the SWCNHs, H ? of -SH was replaced by Au 3? . Finally, AuNPs were formed when Au 3? was reduced by Na 3 C 6 H 5 O 7 . Figure S1 in the Supplementary information shows the Raman spectra of the SWCNHs and oxSWCNHs samples. The D band (1334 cm -1 ) and the G band (1564 cm -1 ) of both samples were detected. The intensity ratio of D and G peaks (I D /I G ) is an indicator of the level of defects [30]. The oxSWCNHs (1.24) have a higher I D /I G ratio than the original SWCNH (0.53). This indicates the structural deformation of the functional SWCNHs by combing functional groups. The unmodified surface of the SWCNH is inert and does not promote the chemical synthesis of surface materials. Therefore, a surface modification is required to introduce functional groups to provide a large number of chemically active binding sites for the modification by other materials. The functional groups on the surface of the SWCNHs provided binding sites for C 2 H 7 NSÁHCl and enabled further modification with the AuNPs. Figure 2 shows the SEM images of the SWCNHs and Au-SWCNHs samples. Figure 2a and b compares the Au nanoparticles dispersed on the surface of the SWCNHs. Figure 2c shows that several isolated Au nanoparticles were uniformly adhered on the surface of SWCNHs surface and the diameter of most gold nanoparticles is 40-60 nm. This structure has a high surface area than bulk gold and provides more active sites. Glucose can better contact composite nanoparticles in the electrochemical reaction. Figure 3a and b respectively shows the elemental composition of oxSWCNHs and Au-SWCNHs determined by energy dispersive spectroscopy (EDS). Table 1 shows the elemental composition data of each EDS spectrum. The modification of the Au nanoparticles clearly leads to the reduction of the oxygen atoms, which is consistent with other results in the literature [31]. When comparing with the different samples, the N content was higher in the samples with a higher Au content. This is because the AuNPs modification occurs spontaneously with the formation of an Au-S bond, which binds to the oxSWCNHs via the N-containing C 2 H 7 NSÁHCl. The AuNPs were successfully synthesized by chemical bonding to the SWCNHs and are expected to have good surface properties in electrochemical sensors.

Electrochemical characterization of the Au-SWCNHs/Au electrode
A three-electrode cell was used for the electrochemical measurements. It contained a platinum counter electrode, an Ag/AgCl reference electrode, and the modified electrodes as the working electrode. The electrochemical behavior of the Au-SWCNHs/Au electrode and the bare Au electrode was first studied by cyclic voltammetry (CV). As shown in Fig. 4a Fig. 4a shows that both redox peaks had the same direction as the red line. The value of the peak current of the Au-SWCNHs/Au electrode was significantly larger than that for the bare Au electrode, because of the synergistic effect of SWCNHs and AuNPs. In the  electrochemical reaction, SWCNHs can accelerate the formation of active sites on the surface of gold nanoparticles due to their rapid electron transfer rate, and the special structure increases the loading of nanoparticles on the surface [26,27]. Compared with the bulk gold electrode, gold nanoparticles and its special nanoparticle morphology caused electrolyte can contact the active sites on the surface of the composite nanoparticle more easily, which can increase the reaction efficiency and generate a larger reaction current [24,25,28]. Then, we compared the electrochemical glucose sensing with the Au-SWCNHs/Au electrode to that of the bare Au electrode by cyclic voltammetry in a 0.1 M NaOH solution without and with glucose at a scan rate of 20 mV s -1 . Figure 4b shows the CV response of the Au-SWCNHs/Au electrode. In the absence of glucose (black curve), we observed an anodic oxidation peak from the oxidization of Au to Au 2 O 3 and a cathodic peak from the reduction of Au 2 O 3 [33] on the Au-SWCNHs/Au electrode. In the positive scan, the first peak related to the adsorption of OHon the electrode surface, and the Au nanoparticles were being oxidized to AuOH site. With increasing potential, AuOH site formed Au 2 O 3 on the electrode surface at the second peak. In the negative scan, the oxide of Au was reduced to AuOH site at ? 0.18V. And AuOH site was further reduced, and OHwas desorbed at the same time at -0.2V [34][35][36][37]. After the addition of the glucose, the CV result of Au-SWCNHs/Au electrode was significantly altered and a series of complex electrochemical processes took place. During the forward scanning, two new oxidation peaks were observed at -0.3 V (peak 1) and at ? 0.52 V (peak 2). In the positive scan, Glucose was adsorbed on the surface of the electrode at peak 1. At the same time, the OHions in the NaOH solution were adsorbed onto the surface of the gold at a certain potential to form an AuOH site and glucose was oxidized to form gluconolactone. We find that the current changes little under different glucose concentrations, because of the limited amount of AuOH sites and the oxidation of glucose is incomplete at the low potential of peak 1. When the potential increases, the amount of AuOH sites increases and gluconolactone is further oxidized at peak 2. The AuOH site is considered as a key factor in the catalytic oxidation of glucose and the oxidation of glucose on Au depends largely on the number of AuOH sites available [38]. The peak 2 current changed little in the different concentrations of glucose because the oxidation of gluconolactone blocked the adsorption of OHand AuOH sites could not continually form on the surface of the electrode. Then, an excessively high potential ([? 0.52 V) leads to the formation of Au 2 O 3 and the oxidation of the AuOH active site, which terminates the oxidation of gluconolactone. In the negative scan, the AuOH, which did not participated in the catalytic reaction, and Au 2 O 3 were reduced and desorbed from the surface of the electrode. A significant anodic peak (peak 3) appears at ? 0.2 V. We can observe a large current change at peak 3, when the concentration of glucose changed. In the electrolyte with a high glucose concentration, the amount of AuOH participating in the electrocatalytic reaction was large, so a stronger current was generated during the reduction [39][40][41]. All the peak currents increase with the increase of concentrations of the glucose, which indicated the Au-SWCNHs/Au electrode is sensitive to the glucose. Figure 4c shows the CV response of the bare Au electrode. Though the curves of the bare electrode are similar with those of Au-SWCNHs/Au electrode, the peak currents of the bare electrode were lower than those of Au-SWCNHs/Au electrode. It indicated that the modification of the Au-SWCNHs composite material significantly improves its catalytic activity for the oxidation of glucose. Figure 4d shows the CV response of the bare Au electrode and the Au-SWCNHs/Au electrode in a 0.1 M NaOH solution with 8 mM glucose. The current at the Au-SWCNHs/Au electrode was generally higher and a significant peak at ? 0.52 V appeared. This indicated that the Au-SWCNHs can significantly improve the sensitivity to glucose and further improved the electrochemical performance of the non-enzymatic glucose sensing approach used. Overall, the synergistic effect of the AuNPs and the SWCNHs improves the performance of the detection of glucose.
To determine the voltage at which the continuous injection of glucose must be conducted, the amperometric reaction of the Au-SWCNHs/Au electrode to the addition of 4 mM glucose at different voltages was studied in a 0.1 M NaOH solution (Fig. S2). The electrode had the maximum response at ? 0.3 V, which was therefore selected for the subsequent experiments.
To determine the dependence of the electrochemical signals on the glucose concentration, the amperometric reaction of the Au-SWCNH/Au electrode to the continuous injection of glucose was studied at a potential of ? 0.3 V in 0.1 M NaOH. Figure 5a shows the corresponding current-time (i-t) curve. The intensity of the current at the electrode increased with the addition of glucose. Figure 5b shows the calibration curve, and both linear ranges with slopes of 413 lA mM -1 cm -2 and 528.75 lA mM -1 cm -2 , respectively. The sensitivity in the first and second linear range was 275.33 lA mM -1 cm -2 and 352.5 lA mM -1 cm -2 , respectively. The limit of detection (LOD) was determined experimentally at 0.72 lM with a signal-to-noise ratio (S/N) of 3. Table 2 compares the electrochemical performances of the Au-SWCNHs/Au electrode with other nonenzymatic glucose sensors using AuNPs. The developed electrode has a wider linear concentration range and a higher sensitivity than those of in most of cited reports. The unique structure of Au-SWCNHs creating a large surface area and abundant active sites makes glucose easily migrate to the electrode surface for a high sensitivity.

Selectivity, stability, and reproducibility of the Au-SWCNHs/Au electrodes
To evaluate the selectivity of the Au-SWCNHs/Au electrode, we examined the effects of interfering substances like ascorbic acid (AA), uric acid (UA), dopamine (DA), galactose, lactose, sucrose, which typically coexist with glucose in human serum [42].
Therefore, we tested the Au-SWCNHs/Au electrode in a solution where the molar ratio of glucose to each interfering substance was 10:1. Figure 6 shows that the change in the current was insignificant in the presence of interfering substances while the current was significant with the increment of the glucose. This indicated that Au-SWCNHs/Au electrode has excellent selectivity toward glucose. For the stability study, we determined that the current response remained at 90.4% and 78.3% of the maximum value for 600 s and 2000 s, respectively (Fig. S3). The Au-SWCNHs/Au electrodes were stored in an airtight container at room temperature for 19 days and the current response to 4 mM glucose was checked every other day (Fig. S4). After 15 days of storage, the current response only decreased by 8.6%. To study the influence of environmental variables such as humidity, the fabricated electrode was exposed to air for 3 days and tested under similar conditions. The current response remained around 89%. To evaluate the reproducibility, we prepared five electrodes by the same method and used them to detect 4 mM glucose. The relative standard deviation (RSDs) of the glucose response was less than 8.7%. Overall, our results demonstrated that the Au-SWCNHs/Au electrode had an excellent selectivity toward glucose, good long-term stability, and good reproducibility.

Human blood serum sample analysis
The feasibility of the Au-SWCNHs/Au electrode in the practical application was performed by testing  Table 3. Glucose was added to human serum with and without glucose continuously, and the detection recovery was 93.50-97.67%. This indicated that Au-SWCNHs/Au electrode here reported can be potentially utilized for the real sample analysis.

Conclusion
AuNPs with an average particle size of 40 nm were synthesized on SWCNHs using an effective method to produce a stable chemical bond. The nanocomposite-modified electrode had a high sensitivity, a wide linear response, good selectivity, a high stability, and a high reproducibility. The AuNPs and the unique structure of the SWCNHs significantly enhanced the catalytic activity of the electrode for glucose. The stable chemical bond between them greatly improved the performance and the storage of the glucose electrode. The large surface area of the microstructure of the SWCNHs provides a good basis to bind a large number of AuNPs, thereby effectively preventing aggregation and deactivation. Moreover, the excellent conductivity of the SWCNHs results in a faster electron transfer between the Au-SWCNHs composite and the Au electrode. Meanwhile, due to the significant catalytic activity of the Au-SWCNHs/ Au electrode in the low concentration range of glucose, the electrode has great potential for the detection of glucose in sweat. We believe that our work opens a new route for the preparation of Au-SWCNHs composites and sets an example for the production of high-quality nanomaterials for sensing applications.