Development of ZnO@rGO nanocomposites for the enzyme free electrochemical detection of urea and glucose

Development of ZnO@rGO based nonenzymatic urea and glucose sensors.

dehydration, shock burns, gastrointestinal bleeding, hepatic failure, nephritic syndrome, and cachexia [3,4]. So the monitoring of the urea level has great significance in both the environmental and clinical samples.
Glucose is the primary source of energy in the human body [5]. The acceptable range of blood glucose level in the human body is ~ 3.9-6.2 (empty stomach) or ~ 3.9-7.8 (2h after food) mM [6]. If the human body is unable to control the amount of glucose due to the decreased insulin secretion, the condition results in Diabetics Mellitus [7]. Further, it leads to different health problems like cardiovascular, nervous, ocular, cerebral and peripheral vascular diseases, kidney failure, tissue damage, blindness, etc. [5,8]. Hence, quantitative monitoring of blood glucose level is essential, to avoid the adverse effects of Diabetics [5].
A quantum of research studies has been conducted to develop an efficient and reliable method for urea and glucose sensing. Electrochemical sensing strategy has been considered as the most promising tool for urea and glucose detection [9,10]. Metal oxides such as ZnO, been extensively studied for electrochemical sensors. Among these, ZnO nanoparticles were most widely studied for electrochemical biosensing applications. In electrochemical sensors, nonenzymatic sensors are most popular because they can overcome the disadvantages of enzymatic sensors [11].
Graphene-based nanomaterials are widely used in electrocatalytic sensing applications. rGO is found to be the most preferred sensor platform due to the following reasons: (1) rGO is electrically conductive compared to the non-conductive GO (2) a large number of edges and defects facilitate electron transfer (3) conductivity and surface functional groups could be turned for detection of specific chemical species. Different methods are reported for the preparation of rGO from GO. In 2010, X. Gao et al., investigated that deoxygenation of GO with hydrazine or heat treatment results in rGO [12].
In 2011, P. Cui et al. report a new reducing system i.e., hydriodic acid with trifluroacetic acid, which can chemically convert GO into rGO at subzero temperature (below 0 o C) with a mass production [13]. In 2012, V. H. Pham et al. reported a simple and effective method to reduce the aqueous suspension of GO using nascent hydrogen generated in-situ by the reaction between Al foil and HCl, Al foil and NaOH, and Zn powder and NaOH [14]. In 2012, O. Akhavan et al. reported a single-step green method for the reduction and functionalization of GO by glucose was developed [15]. In 2012, R. S. Dey et al., demonstrated a facile route for the synthesis of rGO sheets by chemical reduction of GO using Zn/acid solution at room temperature [16]. To best of our knowledge, no attempts were made to conduct the reduction of GO using waste materials.
The use of biomaterials as templates has become significant in the green synthesis of nanomaterials with controllable morphology. Biomaterials are cheap, economical, environmentally begin, and renewable. In the present work, ZnO nanoarchitectures with varied morphology were prepared via the biotemplate assisted microwave method. Here dextrose, sucrose, soluble starch, and carboxy methyl cellulose were used as biotemplates. At the same time, rGO was prepared by the chemical reduction of GO using nascent hydrogen.
Zn dust, a waste material obtained from an industry, was used for the generation of nascent hydrogen. It acts as a strong reducing agent. ZnO nanoarchitectures prepared with different biotemplates are attached on rGO via microwave assisted ex-situ technique, which results in ZnO@rGO nanocomposites. Structural and morphological studies were carried out, which shows the formation of homogeneous distribution of ZnO nanoarchitectures on rGO sheets.
The as-prepared ZnO@rGO has been used for the nonenzymatic sensing of urea and glucose.
Since the studies on nonenzymatic ZnO based glucose and urea sensors are rarely explored area, the fabrication and performance of ZnO@rGO based sensors has got much importance.
Similarly, the synergic effect of both ZnO and rGO provides excellent sensing characteristics in the as-prepared nonenzymatic sensor.

Materials
Graphite (99.9%), Nafion solution (5 wt % in lower aliphatic alcohols and water, contains 15-20% water) and Potassium ferri cyanide (C 6 N 6 FeK 3 , 99.9%) were procured from Sigma Zn dust obtained from Binani Zinc Limited was used for the reduction of GO. All the reagents were used as received without any further purification.

Preparation of GO
GO has been prepared via Hummers method. In a typical procedure 1g graphite and 1g NaNO 3 were dispersed in 50 mL conc. H 2 SO 4 while keeping in an ice bath. 3g of KMnO 4 was added slowly to the reacting mixture and not allowed to increase the temperature to 20 o C. The reaction mixture was then transferred to a water bath kept at a temperature of 35 o C. 50 mL of distilled water was added to the system and the temperature raised to 98 o C. It was followed with the addition of 150 mL of distilled water and 10 mL of 30 % H 2 O 2 . The oxidized graphite was then centrifuged and washed with double distilled water until the pH becomes 7. Finally, on freeze drying graphene oxide with a fluffy nature was obtained [17,18].

Preparation of rGO via Nascent Hydrogen Chemical Reduction
In a typical procedure, 20mg of GO was dispersed in a 200 mL double distilled water using a probe sonicator. It was further treated with conc. HCl (1M) followed by the slow addition of Zn dust (2 g). The whole mixture was stirred overnight using a magnetic stirrer. The gradual change in the colour from orange-brown to black indicates the reduction of GO to rGO. The obtained rGO was then collected by centrifugation. Further, it was washed three times with 0.1 M HCl, followed by washing with double distilled water. After removing all impurities, the rGO nanosheets were freeze-dried. Illustration for the nascent hydrogen chemical reduction is shown in Fig. 1.

Synthesis of ZnO Nanoarchitectures via Biotemplate Method
ZnO nanoarchitectures were prepared via biotemplate assisted microwave technique (Fig. 2 The obtained powder was washed repeatedly with double distilled water to remove the impurities. The final product was dried in the oven at 50 o C. The obtained ZnO samples were denoted as ZD, ZS, ZSs, and ZC, respectively.

Preparation of ZnO@1rGO Nanocomposites
In a typical synthesis of the nanocomposites, 0.

Characterization
The X-ray diffraction patterns of the samples were collected on a X-ray powder diffractometer X' Pert Pro Philips using Cu K radiation  Å) in the diffraction angle (2) ranging between 10 o and 60 o . The crystallite size was calculated using the Debye-Scherrer formula D XRD =k/cos, where D XRD is the average crystallite size in nm, k is the shape factor (0.9),  is the X-ray wavelength in nm,  is the full width at half maximum of (101) peak in radian and  is the Bragg angle (degree) [19].

Fabrication of the Nonenzymatic Electrochemical sensor Electrodes
The nonenzymatic electrochemical sensor electrodes (glassy carbon electrode (GCE)with ZnO or ZnO@1rGO) have been prepared by the following procedure. GCE was first cleaned by polishing with 0.3 µm alumina powder. The polished GCE was further processed by ultrasonication for 5 min each in acetone and double-distilled water, respectively, and dried in ambient air. 1 mg mL -1 of ZnO or ZnO@1rGO was sonicated in nafion for 10 min to get a uniform dispersion. The cleaned GCE was drop cast with 2 µL of ZnO or ZnO@1rGO dispersed in nafion. This thin film of the materials deposited on the surface of the electrodes was left overnight at room temperature for drying the deposited material, and the same was used as a working electrode (nonenzymatic electrochemical sensor electrode) for the detection of urea or glucose.

Electrochemical Studies
The

Characterization of rGO Prepared via Zn Mediated Nascent Hydrogen Reduction
Zn dust obtained as a waste by-product from industry was used for the generation of nascent hydrogen for the chemical reduction of GO. The detailed characterization studies of freezedried GO prepared via the Hummers method had been reported in our earlier work [18].The XRD pattern of the Zn dust is given in Fig. S1. no. 00-004-0831) [21] confirms the presence of pure Zn metal in the waste by-product. The XRD analysis of rGO sheets prepared using nascent hydrogen reduction with Zn dust is given in Fig. 3A. The XRD pattern of GO is given in the inset of Fig. 3A. The main intensity peak of GO is at 10.8 o corresponding to the (001) reflection plane. After reduction with Zn dust, this peak was completely disappeared, and a new broad diffraction peak centred at ~ 24.5 o was appeared indicating the complete conversion of GO to rGO. This peak corresponds to the (002) plane of rGO nanosheets [22]. The appearance of a low-intensity peak exhibited by rGO nanosheets at ~ 44 o corresponds to the (100) reflection plane of rGO [23].
The UV-vis absorption spectrum of rGO, studied by dispersing rGO nanosheets in DMF is given in Fig. 3B. The optical property of GO is also given in the inset of The chemical reduction of GO involves the removal of the above mentioned functional groups. It involves the deoxygenation of epoxide groups through ring-opening mechanism and decarboxylation of carboxylic acid groups [16]. Both reactions led to the restoration of  conjugation [14] and resulted in olefins. Similarly, carbonyl groups are reduced to the corresponding alcohols, which further give olefins. The conversion of GO to rGO is again clear from the colour change of the reactant mixture from dark brown to black [24].
The BET surface area of GO and rGO were measured using N 2 adsorption desorption isotherm and is presented in the supporting information electrolyte solution is in Fig. S3. Extremely low value of the charge transfer resistance (250 mΩ) of rGO clearly indicates that the material is having high electrical conductivity.  No other impurity peaks arising due to carbohydrate/cellulose as well as no remarkable shift in all diffraction peaks were detected in the XRD patterns of as-prepared samples, which confirm the phase purity of the products. The lattice parameters of the as-prepared samples    The optical property of ZnO nanoarchitectures synthesized with different carbohydrates/cellulose is studied using UV-vis absorption spectroscopy and is given in Fig.   5B.  using soluble starch as a structure-directing agent were found to be spherical in shape with smaller particle size. But some agglomeration was also observed in this case. Carboxy methyl cellulose leads to the formation of ZnO nanoarchitectures (ZC) with agglomerates of spherical particles of relatively larger size compared to ZS and ZSs nanoarchitectures.

Characterization of ZnO Nanoparticles Prepared via Biotemplate Assisted Method
Similar observations are obtained in the TEM imaging also. The synthesized ZD nanoarchitectures having a rod shape with~800.8 nm length and ~140.5 nm diameter (Fig.   7A). The spherical shape of the ZS nanoarchitectures is more clearly seen in the TEM image nm (Fig. 7D).    Table   2.   nanocomposite is confirmed by the EDS spectrum of ZSs@1rGO. Additional peaks of C and O are also seen in the composite. The elemental mapping of ZSs@1rGO is shown in Fig. S5.

Characterizations of ZnO@1rGO Nanocomposites
The presents of elements such as C, O, and Zn are well mapped in the images. addition of urea into the electrolyte. In the case of ZSs@1rGO modified GCE, the oxidation current increases from 0.1 mA to 0.36 mA (Fig. 13C). The enhancement in the oxidation current was more than double with ZSs@1rGO nanocomposite modified GCE compared to electrochemical activity of ZSs@1rGO is due to the smaller crystallite size or particle size, as well as, the higher BET surface area of ZSs@1rGO compared to other nanocomposites.  resistance (R ct ), which reflects the conductivity and the electron transfer process [26]. A large semicircle arc with high R ct indicates that the system has a higher resistance to the flow of electrons. In Fig. 15A and B the ZnO modified GCE showed a higher R ct with a larger semicircle diameter (R ct =169 Ω), indicating that ZnO nanoarchitectures were successfully immobilized on the GCE surface, which hindered the electron transfer of the electrochemical probe. This hindrance was decreased (R ct =74 Ω) after the incorporation of rGO in nanocomposites. The small semicircular diameter of the ZSs@1rGO modified GCE implies it has low resistance towards the electron transfer process. These results showed the efficiency of ZSs@1rGO modified GCE compared to ZnO modified GCE [27,9].

Electrochemical Detection of Urea
The calibration study was conducted using CV by the addition of 0.02  to 32  urea to the electrolyte solution (Fig. 16A). From the plot of I P versus concentration of urea given in the Fig. 16B, the linear range was calculated and is between 0.02x10 -3 to7.2x10 -3 mM. The effect of scan rate for 50, 100, 150, 200, 300, 400, and 500 mVs -1 towards the electrochemical oxidation of urea using cyclic voltammetry as determining mode is shown in the Fig. 16C. The oxidation/reduction peak current increases with increasing scan rate. It is evident that the oxidation peak current has a linear dependence upon the square root of scan rate (Fig. 16D) compared to I P versus scan rate. It means that the electrochemical oxidation process is purely diffusion-controlled on the surface of ZSs@1rGO modified GCE surface.
The lowest detection limit calculated was 0.012M, and the sensitivity was found to be 682.8 A mM -1 cm -2 . The proposed sensing mechanism for the fabricated urea sensor is described as CO(NH 2 ) 2 + 8OH -6 H 2 O + CO 3 2-+N 2 + e -2

Electrochemical Detection of Glucose
The calibration study was conducted using CV by the addition of 0.02 M to 160  glucose (Fig. 17A). The I P was plotted against concentration (Fig. 17B), and the linear range was calculated (0.02x10 -3 to 18x10 -3 mM). The effect of scan rate on the electrochemical behavior of ZSs@1rGO modified electrode towards the oxidation of glucose has been conducted using cyclic voltammetry and is shown in Fig. 17C. From the graph it is clear that the oxidation/reduction peak current increases with an increase in the scan rate from 50-500 mV s -1 . The linearity of oxidation peak current (I p ) with the square root of scan rates was observed within the scan rate of 50-500 mV s -1 (Fig. 17D). This indicates that the electrochemical oxidation process is purely diffusion controlled. The lowest detection limit calculated was 0.008 M, and the sensitivity was found to be 481 A mM -1 cm -2 .

Fig. 17(A)
Calibration study of ZSs@1rGO modified electrode towards 4 mM glucose(B) Plot of I P versus concentration (C) Scan rate study using ZSs@1rGO modified electrode with different scan rates (D) Plot of I P versus square root of scan rate.
The proposed sensing mechanism for the fabricated glucose sensor is described as Glucose + Oglucono--lactone +2e -5 glucono--lacone gluconic acid 6 The present study confirms that the ZnO nanoarchitectures act an efficient electron mediator for the fabrication of efficient nonenzymatic sensor.
The results obtained in the present study are compared with the literature data on similar types of nonenzymatic sensors for detecting urea or glucose. The comparison shown in Table 3 reveals that the fabricated urea and glucose sensor based on ZSs@1rGO modified GCE is highly effective in the accurate detection of urea and glucose. The developed ZSs@1rGO modified GCE has showed lowest detection (0.008 M for glucose and 0.012 M for urea), excellent sensitivity (481 mA mM -1 cm -2 for glucose and 682.8 mA mM -1 cm -2 for urea) in the concentration range of 0.02x10 -3 -18x10 -3 M for glucose and of 0.02x10 -3 -7.2x10 -3 M for urea.

Conclusions
In the present work, the as-prepared GO was reduced with a nascent hydrogen reduction mechanism using metallic zinc obtained as a waste by-product from an industry. The rGO formed then analyzed with XRD, UV-vis, SEM, and TEM. The peak shift from 10.8 to 24.5 o in the XRD pattern and the redshift in the UV-vis absorption spectra from 230 to 267 nm confirms the complete reduction of GO to rGO. In addition to this, ZnO nanoarchitectures with various morphologies were prepared using carbohydrate/cellulose as a bio-template. The as-prepared ZnO nanoarchitectures were used to decorate rGO sheets, which results in the ZnO@rGO nanocomposite. Due to the synergistic effect of ZnO and rGO, the prepared ZnO@rGO nanocomposite was found to be an excellent probe for the nonenzymatic electrochemical sensing of urea and glucose. The electrochemical studies revealed that the fabricated electrode was sensitive to urea in the concentration range of 0.02x10 -3 -7.2x10 -3 mM with a detection limit of 0.012 M. The developed nonenzymatic sensor electrode was sensitive to glucose in the concentration range of 0.02x10 -3 -18x10 -3 mM with a detection limit of 0.008 M. The developed sensor exhibited ultra-high sensitivity of 682.8 A mM -1 cm -2 towards urea and 481 A mM -1 cm -2 towards glucose.

Conflicts of interest
There are no conflicts to declare.