Au nanoparticles modified CuO nanowire electrode based non-enzymatic glucose detection with improved linearity

This paper explores gold nanoparticle (GNP) modified copper oxide nanowires(CuO NWs)based electrode grown on copper foil for non-enzymatic glucose detection in a wide linear ranging up to 31.06 mM, and 44.36 mM at 0.5 M NaOH and 1 M NaOH concentrations. The proposed electrode can be used to detect a very low glucose concentration of 0.3 µM with a high linearity range of 44.36mM and sensitivity of 1591.44 µA mM−1 cm−2. The electrode is fabricated by first synthesizing Cu (OH)2 NWs on a copper foil by chemical etching method and then heat treatment is performed to convert Cu (OH)2 NWs into CuO NWs. The GNPs are deposited on CuO NWs to enhance the effective surface-to-volume ratio of the electrode with improved catalytic activity. The surface morphology has been investigated by XRD, XPS, FE-SEM and HR-TEM analysis. The proposed sensor is expected to detect low-level of glucose in urine, and saliva. At the same time, it can also be used to measure extremely high sugar levels (i.e. hyperglycemia) of ~ 806.5454 mg/dl. The proposed sensor is also capable of detecting glucose after multiple bending of the GNP modified CuO NWs electrode. The proposed device is also used to detect the blood sugar level in human being and it is found that this sensor’s result is highly accurate and reliable.

www.nature.com/scientificreports/ It is reported that performance improvement of such non-enzymatic glucose sensors can be achieved using electrodes with high conductivity, large specific surface area, high stability, good selectivity and good reproducibility with high capability of effective electron transfer from the electro-catalyst to the conductive surface of the electrode 27 . Several noble metals such as Pd, Au and Pt as well as their composites like Pt-Pd, Pt-Au, Au-Pd etc. have explored for glucose sensing [28][29][30] . However, the cost of these metals have encouraged to explore the possibility of metal oxides such as CuO, NiO, ZnO, Cu 2 O, MnO 2 , Fe 2 O 3 , SnO 2 and Ag 2 O for developing low-cost non-enzymatic glucose 31 . Among them, CuO has been widely used for developing low-cost glucose sensors due to its good electrochemical and electro-catalytic properties, and abundant availability 11,16,25,27,32,33 . The intrinsically p-type semiconductor CuO with a band-gap of ~ 1.2 eV has also been extensively used in electrochemical sensors, photoelectric devices, gas sensors and lithium-ion batteries due to its interesting electrical and optical properties 15,[34][35][36] . Various CuO nanostructures (e.g. nanowires, nanorods, nano flower, etc.) grown on a copper foil using easy and simple synthesis techniques have been reported for low-cost glucose sensors with a fast response, high sensitivity, and stable detection due to their enhanced catalytic property over the other metal oxide nanostructures 11 .
Li et al. 27 have synthesized CuO nanowires (NWs) on 3-D copper foam by anodization process for glucose detection. They 27 have achieved high sensitivity and wider linear range up to 18.8 mM at a concentration of 1 M NaOH. The high catalytic activity of the metal oxide and high conductivity of the noble metals have also encouraged to use nanocomposites of ≥ 95% metal oxide and ≤ 5% noble metal for glucose-sensing applications. Li et al. 37 have tried Au/CuO (metal and metal oxide)nano-composite (nano-cauliflower)to sense glucose for the first time.
They 37 have achieved a detection limit of 0.3 µM, the sensitivity of 708.7 µA mM −1 cm −2 and linear range from 0.0001 mM to 30 mM at a concentration of 1 M of NaOH. Xio et al. 38 have used Au/CuO Nanohybrids to get a sensitivity of 374 µA mM −1 cm −2 with linearity up to 12 mM at a concentration of 0.1 M NaOH (pH = 13) while Wang et al 19 have got the sensitivity of 709.52µA mM −1 cm −2 and linearity up to 8 mM at a concentration of 0.1 M NaOH. The nano-composite of CuO (metal oxide) nanostructure and Au (metal) nanostructure shows the enhancement of conductivity to accelerate the rate of electron transfer, selectivity and sensitivity of glucose sensing 37 . In such glucose sensors, the direct electron transfer property of CuO NWs acts as catalysts whereas the large surface-to-volume ratio of Au nanoparticles can act as co-catalyst for enhancing the linearity and sensitivity in a drastic manner of the glucose sensors.
There are still challenges to develop glucose sensors with a wide range of linearity and high sensitivity for detecting the glucose levels in moderate to severe diabetic patients. Recently, Mishra et al. 39 have observed a significant increase in sensitivity, linearity, and selectivity in gold nanoparticles (GNPs) modified CuO NWs based glucose sensors at low-concentration of 0.1 M of NaOH solvent. In this article, we have demonstrated the improvement of linearity and sensitivity of the GNPs modified CuO NWs electrode based glucose sensor using the higher levels of electrolyte (NaOH) concentrations at 0.5 M and 1 M.

Results and discussions
Characterizations of electrodes. Synthesis and electorde fabrication is briefly illustrated in Fig. 1 HRTEM image analysis gives us better investigations of the surface morphology of CuO NWs with GNP as shown in Supplementary Fig. S1. The free-standing of TEM images of CuO NWs with GNP is obtained after scrapping from the Cu substrate shown in Supplementary Fig. S1(a). The image of Supplementary Fig. S1(b) clearly shows the dark spot of well-distributed gold nano-particles on CuO NWs. In Supplementary Fig. S1 [40][41][42] . The XPS of Cu 2p core level is expressed in Fig. 3(c) where two peaks of energy 934 eV and 954 eV are shown in Fig. 3(c) which corresponds to Cu 2p 3/2 and Cu 2p 1/2 respectively which confirms the existence of the Cu +2 . Two more peaks present at 941.8 eV and 961.9 eV are the satellite peaks of Cu2p 3/2 and Cu2p 1/2 respectively. They show the presence of the unfilled shell of 3d which again proves the presences of Cu +2 in the sample. Gold (Au) 4f core of the Au/CuO nanostructure (shown in Fig. 3(d) has been filled with two peaks i.e. Au 4f 7/2 and Au 4f 5/2 with binding energy 84.0 eV and 87.7eVwhich prove the presences of gold on CuO nanowires. For the selection of the better electrode the different copper oxide has been chosen like Cu (OH) 2 NWs, CuO NWs and CuO NWs with GNP. The current which is observed in the case of Cu(OH) 2 NWs is very low, moderate for CuO NWs and extremely high for CuO NWs with GNP in the solution of 0.5 M NaOH and 1 mM glucose concentrations.
Due to the very high current observed in CuO NWs with GNP, we can use it as a working electrode for this research work. The drastic improvement with CuO NWs with GNP is due to intensive increments in surface to volume ratio for CuO NWs with GNP electrode. The C-V graph of the different working electrodes has been shown in Fig. 4(a).
CuO NWs with GNP electrode are dipped in the solution of 0.5 M NaOH with different concentrations of glucose at 0 mM, 1 mM, 2 mM, 3 mM 4 mM, and 5 mM. It is observed that the current increases with an increase in glucose concentration and linearity is found to be maintained as elucidated in Fig. 4(b). Figure 4(c) again shows the C-V plot same as Fig. 4(b) which covers the complete range of glucose for the working electrode at a solution concentration of 0.5 M NaOH up to till 31.05 mM which linearity is maintained. The final relation between current and concentration is shown in Fig. 4(d). It shows the sensitivity of the sensors 1429.43 µA cm −2 with R 2 = 0.9927 and linearity shows a maximum value of 31.05 mM.
The current-voltage graphs for the GNPs modified CuO NWs based electrodes with different scan rates are shown in Fig. 5(a). This figure confirms the redox reaction model. The linearity graph of the cathodic peak current and anodic peak current with different scan rates (5 mV s −1 to 500 mV s −1 ) is shown in Fig. 5(b). In view of the above discussion, it can be easily observed that the redox reaction model is surface-confined process 43,44 and the glucose molecule directly oxidizes the composite surface of CuO NWs with GNP and the electrons were directly transferred without any mediators.
The studied electronics properties over the surface are corroborated from the measurements of the electrochemical impedance spectra (EIS). EIS measurements are clearly shown in Fig. 5(c) depicts the gradual enhancement of conductivity from Cu (OH) 2 NWs electrode to CuO NWs with the GNP electrode. Figure 5 Uric acid (UA), ascorbic acid (AA), dopamine (DA), sucrose, lactose, and maltose are the various interferences components along with glucose present in human blood serum. The concentration glucose in human blood serum is approximately 30 times more than the components of the above interference 45,46 . Due to the aforesaid reasons, the selectivity of CuO NWs with GNP electrode-based glucose sensor under study was examined at 1 mM glucose with these interfering elements of 0.2 mM each of lactose, sucrose, and maltose, UA, AA, and DA. Due to increased oxidation of glucose in comparison to other interferences components, the change in current was significantly high for glucose incomparison to other interferences species 47 which is as shown in Fig. 5(d). At the concentration of 1 M NaOH solution, working electrode CuO NWs with GNP has been used for measuring the current with an increase in glucose concentration. At the fixed voltage of 0.55 V, the increment in current was directly proportional to the amount of glucose added in the solution.
Reusability, reproducibility, and stability tests. The important factors for measuring the efficiency of the sensing devices are reusability, reproducibility, and stability. For the reusability, CuO NWs with the GNP electrode was dipped into a freshly prepared sample containing 1 mM of glucose in 0.5 M NaOH solutions at 50 mVs −1 scan rate and the resultant data were shown in Supplementary Fig. S3(a) and (b). The electrode was dipped in the solution for at least 10 times in the same solution or 10 different solutions. It is observed that CuO NWs GNP glucose sensing electrode retains more than 99% of its original response which shows its reusability. To test reproducibility, 10 freshly preparedAu/CuONWs with GNP electrode have been used in 1 mM of glucose solution of 0.5 M NaOH with a scan rate of 50 mV s −1 . The peak current of each electrode is mention in the C-V graph as shown in Fig. Supplementary Fig. S3(c) and (d). The response of these electrodes is found to have a relative standard deviation of 5%. It can be concluded from the above discussion that the sensors based on CuO NWs with GNP electrode are good enough to reproduce approximately the same results which show the sensor reproducibility. To add more functionality to the sensor, stability test was evaluated by the C-V response of the CuONWs with the GNP electrode at an interval of 3 days for a month as shown in Supplementary Fig. S3 (e). The electrode was stored at room temperature upto the completion of the measurement. After the completion www.nature.com/scientificreports/ of 30 days, the response of the proposed CuO NWs with the GNP electrode was compared with the response of the electrode on the first day. The result is shown in Supplementary Fig. S3(f) as a histogram plot which conveys the information that the proposed sensor is very much stable which is able to retain 95% (measured on day 30) of its originals response (measured on day 0). The excellent stability response of the electrode was due to the stable grown of CuO NWs with GNP on the surface of the electrode which provides strong mechanical stability to the sensing device. In all the above discussion the C-V measurement is done at 0.5 M NaOH. To test the sensor feasibility, we have also measured the C-V characteristics at 1 M NaOH solution as shown in Fig. 6(a) along with different concentrations of glucose. At a fixed potential of 0.55 V and the scan rate of 50 mV s −1 , it is observed that the increment in current density is directly proportional to the amount of glucose added in the solutions. The linearity between current density and glucose concentration is shown in Fig. 6(b). The sensitivity of 1591.44 mA M −1 cm -2 with a wide linear range up to 44.36 mM is observed for the proposed sensor with 1 M NaOH. Due to this significant improvement in linearity with 1 M NaOH, the sensor is capable enough to detect the glucose level in highly diabetic patients. Figure 6(c) shows the C-V characteristics with different scan rates. The significance of this plot is to show that the proposed sensor is also showing linearity with varying scan rates. Figure 6(d) shows C-V characteristics with varying scan rates keeping the potential (voltage) constant. Here also we can observe that the linearity is still maintained by varying scan rates. Sensitivity and linearity of the different Au-nanoparticles decorated CuO NWs (working electrode) have been given in Table 1. From the Table 1 it can be easily conclude that at 0.5 M and 1M NaOH solutions, this sensors are works for an extremely serious diabetic patients(linearity up to 31.05 mM and up to 44.36 mM) with good sensitivity.
To see the practical application of proposed device, real blood samples have been taken from our research group for testing in a private pathology laboratory named Dr. Lal Pathology Lab just located outside of our institute. The real time blood reports have been compared with the results obtained from our proposed sensor in Table 2. Approximately 100 μL blood is added into 9.90 ml of 0.5 M NaOH solutions, and the current densityconcentrations responses are measured at Voltage = 0.55 V. The blood glucose concentration in the human blood serum is measured through the calibrated curve shown in Fig. 4(d). Each sample has been used 3 times and the average value has been taken for the comparison. It has been observed that our proposed sensor data are closely matched with the data provided by the Dr. Lal Pathology Lab. The maximum difference between our proposed www.nature.com/scientificreports/ sensor data and the data provided by the Lal Pathology Lab is ~ 3.6%. Thus, we safely conclude that our proposed sensor can be used for commercial glucose sensing applications.
In the summary, a facile step is involved in the fabrication of GNPs modified CuO NWs electrode-based nonenzymatic glucose sensors. GNPs coated CuO NWs enhance the effective surface-to-volume ratio of the electrode with respect only CuO, NWs based electrodes. This improves the catalytic property of the electrodes which, in turn, enhances the oxidation and reduction properties of the GNPs coated CuO NWs electrode. These enhanced properties in the proposed sensor gives a sensitivity of 1591.44 µA mM −1 cm −2 and 1,440.63 µA mM −1 cm −2 at a concentration of 1 M and 0.5 M NaOH, respectively. The linearity ranges of the glucose sensor are 44.36 mM and 31.05 mM at 1M and 0.5 M NaOH solvent concentrations respectively. The low detection limit of the sensor at different concentrations shows that the sensors can be used to detect extremely low glucose levels in salvia and urine. The results reported here are highly accurate and stable.

Experimental section
The fabrication and characterization methods of the electrode are nearly similar to our previous works reported elsewhere 38 . However, we have briefly discussed the fabrication and characterization of the electrode here for the clarity of the readers. We have also included some new characterization results for the better understanding of the readers.

Materials
Alfa Aesar, Thermo Fisher Scientific (India) has provided the highly pure copper foil (99.9%) used as substrate for CuO NWs. Sodium hydroxide (NaOH), hydrochloric acid (HCl), ammonium persulfate [(NH 4 ) 2 S 2 O 8 ], acetone, isopropanol, and Malt extract powder have been bought from Merck Life Science Private Limited (India). Sisco Research Laboratories Private Limited (India) has provided glucose, sucrose, and uric acid. All the chemicals used are ultra-pure and of analytical grade, hence there is no need for further cleaning. DI water of high resistivity (18MΩ-cm) obtained from Merck Millipore system is used for cleaning purposes. 2 NWs electrode. Small pieces of (5 mm × 5 mm) of copper foil have been prepared from the bulk copper sheet. These copper foils are first cleaned ultrasonically in DI water and HCl. Again these copper foils have been cleaned by acetone and isopropanol sequentially. After the Formation of CuO NWs electrode. Deep blue colors of Cu(OH) 2 on Cu foil were kept in alumina boat inside the furnace till half an hour in presences of Ar gas. The flow of Ar gas has been stopped after 30 min and foils are heated at 120 °C for three hours. For better crystallization, the foils were heated further at temapture180°C for two hours. The blue film of copper foils converted into black one and this is CuO NWs in the Cu foils 33 .

Electrodes preparation. Formation of Cu (OH)
Formation of gold NPs decorated CuO NWs electrode. CuO NWs were dipped in the solution of 3 ml DI water containing 8 mg tetra chloro auric Acid (HAuCl 4 ) for 10 min. After that rinse these foil in DI water. Again these foils were dipped in a solution containing 9 mg sodium borohydride (NaBH 4 ) dissolve in 3 ml methanol. Finally, the foil is again rinsed with DI water for 2 min, to get gold nanoparticle decorated CuO NWs electrode on copper foil. The following reaction takes place during the formation of a gold nanoparticle on the CuO NWs electrode 39 .
Electrode characterizations and electrochemical set-up. The copper foils, Cu(OH) 2 NWs, CuO NWs, and GNP modified CuO NWs are characterized using X-ray diffraction (XRD) (RIGAKU-SmartXDMAX, PC-20, 18-kW Cu rotating anode, Rigaku, Tokyo). The surface morphology of GNP modified CuO NWs is investigated by (3) NaBH 4 + 4CH 3 OH ⇔ NaB(OCH 3 ) 4 + 4H 2 (4) 4HAuCl 4 + 3NaBH 4 → 4Au + 6H 2 + 3NaCl + 3BCl 3 + 4HCl The linearity graph between current density and potential with different scan rates. www.nature.com/scientificreports/ FE-SEM (Model-Nova Nano SEM FEI Company of USA (S.E.A.) PTE, LTD) and (HR-TEM) (Model-Tecnai G2 20 TWIN FEI Company of USA (S.E.A.) PTE, LTD).The electrochemical measurements were performed in a 3-electrode electrochemical cell configuration with CuO based electrodes as working electrodes, a platinum wire as counter electrode, and Ag/AgCl as a reference electrode with an electrochemical workstation (ModelCS: 350, S/N: 1,609,178, Corr test Instrument, China). The electrode preparation processes have been shown in Fig. 1(a) in which the steps involved in the formation of CuO NWs with GNP have been shown. Different concentration of NaOH (0.5 M and 1 M NaOH) solution has been used an electrolyte for measurement of cyclic voltammetry (C-V) at room temperature. In Fig. 1(b), bendable clean strip of copper foil is shown which is used in Fig. 1(a).