Highly Efficient Non-Enzymatic Glucose Sensor Based on CuO Modified Vertically-Grown ZnO Nanorods on Electrode

There is a major challenge to attach nanostructures on to the electrode surface while retaining their engineered morphology, high surface area, physiochemical features for promising sensing applications. In this study, we have grown vertically-aligned ZnO nanorods (NRs) on fluorine doped tin oxide (FTO) electrodes and decorated with CuO to achieve high-performance non-enzymatic glucose sensor. This unique CuO-ZnO NRs hybrid provides large surface area and an easy substrate penetrable structure facilitating enhanced electrochemical features towards glucose oxidation. As a result, fabricated electrodes exhibit high sensitivity (2961.7 μA mM−1 cm−2), linear range up to 8.45 mM, low limit of detection (0.40 μM), and short response time (<2 s), along with excellent reproducibility, repeatability, stability, selectivity, and applicability for glucose detection in human serum samples. Circumventing, the outstanding performance originating from CuO modified ZnO NRs acts as an efficient electrocatalyst for glucose detection and as well, provides new prospects to biomolecules detecting device fabrication.

in different morphology with high crystallinity, good optical properties, excellent electrical characterstics 20,21 . In our previous studies, we have shown that vertically-grown ZnO nanostructures on electrode surface have immense capability to hold enzymes because of their nanostructure morphology and high surface area, thereby enhancing overall sensing performance of the enzymatic sensors [22][23][24] . As well, we have also shown that these ZnO nanostructures can provide large surface area for nanostructure modification for the efficient non-enzymatic sensor devices 25 . Interestingly, a few studies on ZnO based hybrid nanostructures have shown enhanced catalytic activity owing to better surface-to-volume ratio of hybrid materials and fast electron transfer ability of ZnO to the supporting electrodes [26][27][28] . On the other hand, CuO nanostructures have been well-studied as an efficient material for the fabrication of non-enzymatic glucose detection owing to the fact that they possess excellent electrochemical and catalytic properties, inexpensive, low temperature, and easy tuning of CuO nanostructures, exhibiting potential outcomes on the sensors sensitivity due to their high surface and volume ratio [29][30][31] .
Keeping in view the various tedious steps in sensors fabrication, mostly previous protocols employed separately synthesized nanostructures that need binders to make slurry and coating for further utilization as an active electrode material for sensor fabrication. This approach further result in reduced electrocatalytic activity by blocking the catalytic active sites with binder, poor reproducibility and low stability of fabricated electrodes due to inhomogeneous and dense film of nanostructures produced via spin-coating/drop-casting. Therefore, fabrication of high performance sensing electrodes requires growth of nanostructures directly on the electrode surface that not only will seamlessly connect nanostructures with electrode but also promote fast electron transfer. To aid in the search for an easy approach to attach nanostructure on electrode surface, herein, we have directly grown ZnO NRs on FTO electrode surface (ZnO NRs/FTO) by low temperature hydrothermal method and functionalized with CuO (CuO-ZnO NRs/FTO electrode) to enhance the electrochemical activity for glucose detection via higher surface area and direct electron transfer. Morphological characterizations of CuO modified directly grown ZnO NRs (CuO-ZnO NRs) confirmed that the ZnO NRs are seamlessly connected to the electrode surface, vertically-oriented, and uniformly CuO-functionalized on the ZnO NRs. Further, the fabrication process was monitored with electrochemical impedance spectroscopy (EIS) measurements in order to get optimized CuO loading on ZnO NRs surface for delivering excellent catalytic properties during glucose detection. The electrochemical analysis of CuO-ZnO NRs electrocatalyst showed excellent electrochemical performance for glucose detection with high reproducibility and repeatability, stability, and selectivity. Additionally, the non-enzymatic sensors were also assessed for glucose detection in serum, illustrating its promising sensing applications in near-real/real time samples.

Results
Synthesis and structural properties. Direct nanostructure synthesis and electrode fabrication strategy is briefly illustrated in Fig. 1 along with the details presented in method section. Hydrothermally grown ZnO NRs on electrode surface were modified with CuO through dip-coating and annealing. Figure 2a shows the XRD patterns of the as-synthesized ZnO NRs before and after CuO modification. For bare ZnO NRs spectrum, all diffraction peaks were well-indexed as hexagonal wurtzite structure of bulk ZnO (JCPDS 36-1451). After modification with CuO, the CuO-ZnO spectrum displays all the ZnO NRs diffraction peaks along the additional peaks corresponding to CuO modification and is indexed to the monoclinic CuO (JCPDS 48-1548). Next, morphologies of the as-synthesized ZnO NRs before and after CuO modification were examined by FESEM, as shown in Fig. 2b-f. From the images, the ZnO NRs were found to be uniformly and vertically grown on electrode surface in large scale. Compared to the smooth surface of bare ZnO NRs (Figure 2b-c), rough morphology was observed for CuO-ZnO NRs due to surface decoration with CuO ( Fig. 2d-f). The cross-sectional image in Fig. 2f shows the length and diameter of ZnO NR as ~1.2 µm and ~80-90 nm, respectively.  [32][33][34] . Compared to the pristine ZnO NRs, CuO-ZnO NRs showed a little shift to the higher binding energy which indicates the ZnO surface modification with CuO 12 . Further, both high resolution Zn 2p spectra of ZnO NRs and CuO modified ZnO NRs both showed two symmetric peaks (Fig. 3c). The ZnO NRs show peaks at around 1021.6 eV and around 1044.2 eV which corresponds to the Zn 2p 3/2 and Zn 2p 1/2 , respectively. After CuO modification, both peaks of as-synthesized ZnO NRs were shifted to the higher binding energy, confirming the different chemical environment of as-synthesized CuO-ZnO NRs. In addition, to find out the presence of Cu 2+ on ZnO NRs, the high resolution Cu 2p spectra of CuO-ZnO NRs were presented in the Fig. 3d. In the figure, there are two peaks located at approximately 934.6 eV and 954.3 eV, corresponds to the Cu 2p 3/2 and Cu 2p 1/2 , respectively, that confirms the Cu 2+ presence on ZnO NRs surface 35,36 . Additionally, two shake-up satellite peaks for Cu 2p 3/2 and Cu 2p 1/2 were observed at higher binding energy at around 943.2 eV and 962.8 eV, attributed to the partially filled d-block (3d9) of Cu 2+ and hence further confirms the formation of CuO over ZnO NRs surface 36 .
Electrochemical properties.    suggests that the controlled CuO-modification over ZnO NRs/FTO electrode is important, as less (curve c) and more (curve e) modification gives high electron transfer resistance. The low R et reflects that the diffusion-limited process occurs between surface of electrode and the solution. Therefore, 20 s CuO-ZnO NRs/FTO electrodes were selected for further electrochemical characterizations and non-enzymatic glucose detection.
The electrochemical tests for different electrodes (working electrodes) were performed in a three-electrode cell system with Pt wire as the counter electrode and Ag/AgCl as reference electrode within the potential range of 0 to +0.8 V. Fig. 5 presents the CV profile of electrochemical responses in 10 mL of 0.1 M NaOH solution with/ without glucose. In the blank NaOH solution, no oxidation peak was observed (Fig. 5a). The addition of 0.1 mM glucose causes a negligible oxidation current for FTO and ZnO NRs/FTO electrodes (Fig. 5b) which confirms that these electrodes have no specific catalytic property like CuO modified electrodes. In contrast, CuO-ZnO NRs/ FTO electrodes showed well-defined oxidation peak at +0.62 V in the presence of glucose. The obtained result clearly suggests that the oxidation peak corresponds to the electro-oxidation of glucose at the CuO modified electrode. The possible mechanism of non-enzymatic glucose detection on CuO modified electrode is presented in Fig. 1. The electrocatalytic oxidation of glucose can be ascribed to the conversion of Cu(II) to Cu(III) in NaOH solution, as suggested by Marioli and Kuwana 37 . In brief, during electrocatalytic oxidation of glucose, Cu(II) is electrochemically oxidized to Cu(III) which acts as an electron delivery system, and the glucose oxidized to gluconolactone is further oxidized to gluconic acid. In addition, CV response of CuO-ZnO NRs/FTO electrode was measured in 0.1 M NaOH solution at different scan rates from 20 to 200 mV s −1 in the presence of 0.1 mM glucose, as depicted in Fig. 5c. The corresponding calibration plot of peak current versus scan rate presented in Fig. 5d showed a linear change in current response with an increasing scan rate which indicates the surface-controlled electrochemical process over CuO-ZnO NRs/FTO electrode 38 .
Analytical performance of non-enzymatic glucose sensor. In order to demonstrate the analytical parameters (i.e. sensitivity, linear range, detection limit and response time), amperometric response of CuO-ZnO NRs/FTO electrode was performed at a fixed voltage of +0.62 V (versus Ag/AgCl) in 0.1 M NaOH solution by stepwise addition of glucose at different concentration. From the Fig. 6a, a well-defined and fast amperometric response for CuO-ZnO NRs/FTO electrode was noticed. Inset of Fig. 6a shows the current response at lower glucose concentration (0.001-3.45 mM). Upon addition of glucose, the current response quickly reached a steady-state and attains ~ 96% of response within 2 s. The response current was lineally increased with increasing glucose concentration, as shown in the calibration plot (current response verses glucose concentration) of amperometric response in Fig. 6b. The calibration plot was found to be linear in the concentration range of 0.001 to 8.45 mM with a correlation coefficient (R 2 ) of 0.9997. Further, sensitivity was calculated as 2961.7 μA mM −1 cm −2 by dividing the slope of the linear portion of calibration curve with electrode surface area. The lower detection limit (LOD) was observed to be as low as 0.40 μM at the signal-to-noise ratio (S/N) of 3. A comparative analytical performance of our sensing electrode with previously published non-enzymatic glucose sensor is also presented in Table 1. As shown in Table, CuO-ZnO NRs/FTO electrode showed high sensitivity (2961.7 μA mM −1 cm −2 ) in wide linear range (0.001 to 8.45 mM), which is superior to previously reported values for CuO based sensor electrodes 12,13,16,[39][40][41][42] . Other parameters like response time and detection limits were also satisfactory. While comparing only sensitivity, our fabricated sensor showed less response than sensors fabricated using electrospinning and electrochemical anodization methods 12,39,42 . It is worthwhile to notice that the linear range of those sensors were ~4-16 folds less than our sensing electrodes. Overall, the enhanced sensing performance of non-enzymatic glucose sensor is ascribed to the direct growth of ZnO NRs on FTO electrodes which offers high surface area for CuO modification, resulting in fast electron transfer during electrochemical process of glucose oxidation occurring between the electrolyte and electrode. Importantly, we have used hydrothermal approach to fabricate non-enzymatic glucose sensing electrodes which bestow controllable nanostructures with good reproducibility and cost-effective fabrication process for stable glucose sensing devices.
Anti-interference ability, reproducibility, reusability, and stability tests. Anti-interference ability of non-enzymatic based glucose sensing devices is a major challenge, which could affect the electrode's sensing performance. Herein, to verify the selectivity of CuO-ZnO NRs/FTO electrode in the presence of interfering Reproducibility, reusability, and stability are other vital parameters for measuring the efficiency of sensing devices. The reproducibility of CuO-ZnO NRs/FTO electrode was investigated by employing 10 freshly prepared non-enzymatic glucose sensors. Their CV response was recorded in 0.1 M NaOH solution containing 0.1 mM glucose at the scan rate of 100 mVs −1 (Supplementary Figure S2a). The peak current of CV response is presented in Supplementary Figure S2b, which showed relative standard deviation (RSD) of 4.6%. Similarly, the reusability CuO-ZnO NRs/FTO electrode was measured in 10 samples, each containing 0.1 mM of glucose at 100 mVs −1 scan rate and the obtained data is shown in Supplementary Figure S2c and S2b. After 10 times usage, CuO-ZnO NRs/FTO glucose sensing electrode retains around 98% of its original response, suggesting excellent reproducibility and reusability of our sensing electrode. In addition, the long-term stability of the CuO-ZnO NRs/FTO electrode was explored by measuring CV response in the presence of 0.1 mM glucose in 0.1 M NaOH solution at the scan rate of 100 mV s −1 (Supplementary Figure S2e). The CV response was evaluated once every three days and after completion of measurement, the electrodes were stored at room temperature. As shown in the histogram (Supplementary Figure S2f), after 30 days of usage the current response was found to be 93.2% of its original response (measured after fabricating the electrode). The excellent stability of electrode was due to the directly grown nanorods on electrode surface, which provided robust mechanical stability to the sensing device.
Non-enzymatic glucose detection in real human blood. The excellent sensing performance of CuO-ZnO NRs/FTO electrode suggests the suitability for glucose detection in real samples for future practical applications. In order to justify the above statement, we measured amperometric response of CuO-ZnO NRs/FTO electrode in 9.5 mL of 0.1 M NaOH solution after injecting 0.5 mL blood serum and freshly drawn whole human blood at an applied potential of +0.62 V (versus Ag/AgCl), shown in Fig. 6d. The blood samples used in the experiments were obtained from same donor, which was also analyzed using blood chemistry analyzer VetScan VS2 (Abaxis, Inc., Union City, CA 94587) and the obtained data (5.2 mM glucose concentration) was further compared (inset of Fig. 6d). As presented in histogram, the measured glucose concentration in the serum sample was in good agreement with the analytically measured value. However, whole human blood sample showed ~4% less concentration of glucose due to the presence of different types of molecules (such as cells, protein fragments, etc.) 43 . Therefore, as-prepared CuO-ZnO NRs/FTO electrode may hold potential practical application for glucose detection in real samples.

Discussion
We have successfully presented the fabrication of highly-efficient non-enzymatic glucose sensor electrode by directly growing ZnO NRs on FTO electrode followed by CuO modification. The uniqueness of CuO-ZnO NRs/ FTO electrode is that, the directly-grown ZnO NRs on electrode surface provides easy substrate penetrable structure, and large surface area for CuO modification which in turn enhances electrochemical activity for glucose detection. The sensing electrode exhibited remarkable high performance in terms of sensitivity, wide response range, response time, selectivity, reproducibility, repeatability, and stability. Additionally, the glucose detection in real human blood shows the electrodes suitability for practical or real-time applications. This improved sensing performance is mainly attributed to the directly grown nanostructures that provide an excellent contact between the nanostructure and electrode with high surface area for catalytic sites, facilitating suitable path for electron transport during electrochemical activity 16,40,42,[44][45][46][47][48][49][50][51][52][53][54][55] . Overall, the fabricated electrodes can be envisioned as promising design for practical application of non-enzymatic glucose measurement in real clinical samples which may garner considerable benefits for different biomolecule detection. Characterization and measurements. The surface morphology of the as-synthesized ZnO NRs before and after CuO modification was inspected using field emission scanning electron microscopy (FESEM, Hitachi S4700, and SUPRA 40VP), transmission electron microscopy (TEM), and high-resolution transmission electron microscopy (HRTEM) equipped with digital charge-coupled device (JEOL-JEM-2010 equipped with CCD camera). The elemental chemical composition was determined by TEM-EDX-line scan. The crystalline structure of ZnO NRs before and after CuO modification was analyzed using X-ray diffractometer (XRD, Rigaku) with Cu-Kα radiation (λ = 1.54178 Ǻ) in the range of 30-70° with 8°/min scanning speed. The chemical states were analyzed by X-ray photoelectron spectroscopy (M/s. AXIS-NOVA, Kratos Inc.) using X-ray source of monochromatic Al K (1486.6 eV) 150 W. All the electrochemical measurements were conducted at room temperature using an electrochemical measurement station (Ivium CompactStat.e; Ivium Technologies) connected to computer with a conventional three-electrode cell system: a working electrode (Nafion/CuO-ZnO NRs/FTO), platinum (Pt) wire as counter electrode, and Ag/AgCl as reference electrode. During amperometric measurements, solution was continuously stirred at 150 rpm. The electrochemical impedance spectroscopy (EIS) measurements were performed in a mixture of 5 mM [Fe(CN) 6 ] 3-/4and 0.1 M KCl solutions. The EIS measurements were taken within a frequency range of 0.01 Hz-100 MHz with applied amplitude of ± 5 mV.