NiO Nanoflowers for Non-Enzymatic Amperometric Detection of Glucose

Summary

The primary objective of this article is to help researchers and scientists understand the process of glucose sensing using electrochemistry and to optimize parameters to maximize the glucose sensing response. The process of fabricating a glucose sensor is described in detail with guidelines for obtaining its best chronoamperometric response.

Abstract

Non-enzymatic glucose sensors employing nanostructured metal oxide materials are amongst the most extensively researched topics currently. Several transition metal oxides (TMOs), such as iron oxides (α-Fe₂O₃, γ-Fe₂O₃, Fe₃O₄, etc.), nickel oxide (NiO), copper oxide (CuO), chromium trioxide (CrO₃), etc., have been utilized as enzyme-mimicking catalysts for glucose oxidation. Along with the use of different TMO materials, considerable efforts have been put into studying the impact of different morphologies of these materials. The literature shows that the flower and wire-shaped catalysts perform better due to their large surface area.

This study presents the non-enzymatic glucose sensing properties of NiO nanoflower decorated glassy carbon electrodes (NiO NF@GCE). The central objective of this work is to shed light on the processes involved in the fabrication and characterization of glucose-sensing NiO nanoflowers on GCE. The amperometric detection of glucose shows a linear increase in current over physiologically relevant glucose concentrations ranging between 0 mM and 15 mM. The electrodes offer an excellent sensitivity of 281.69 µA mM^-1·cm^-2 and a limit of detection of 10 µM. The results show that the linear range of detection increases while the sensitivity decreases with an increase in the loading mass of the nanomaterial after a certain loading concentration. In addition, the electrodes showed high selectivity towards glucose in the presence of other interfering species, such as ascorbic acid, fructose, sucrose, and NaCl. The impact of loading concentration on the selectivity of NiO NF-modified electrodes shows a decrease in selectivity with the increase in loading of the nanomaterial after a certain optimal loading mass.

Introduction

Medical technologies have seen rapid advancement in the last two decades. Biosensing devices enabling real-time disease monitoring represent a major advancement, pioneered by the development of glucose sensors¹. Since the invention of Clark's glucose electrodes², glucose sensors have advanced significantly. They are still the most common in biosensing and hold a magnanimous 80% share of the whole biosensor market³. However, most of the commercial glucose sensors use an enzymatic mode of detection. The enzymatic sensors have high performance dependence on temperature, pH of the analytical solution, storage time and temperature⁴. Due to these drawbacks, non-enzymatic glucose sensors are the new trend in glucose sensing.

The current generation of glucose sensors⁵ uses metal and metal oxide nanoparticles for glucose sensing. These metal oxides are highly stable in ambient conditions and hence are an ideal choice over the enzymes. For a material to be used as a transducer for glucose, it must possess four important characteristics. The first characteristic is the high electrocatalytic activity⁶, making TMOs the ideal choice for this purpose. The availability of multiple stable oxidation states that can be used to oxidize glucose during the electrochemical transition process makes them a suitable candidate. The second one is a high surface area to provide maximum sites for glucose oxidation. Nano-structurization of these metal oxides and manipulation of morphologies give them an edge over their own bulk counterparts. The third one is high electrical conductivity for efficient changes in the electron transfer due to glucose oxidation. Finally, the fourth one is the good redox capability for effective reversibility and antifouling of the electrode. A completely reversible transition in the case of metal oxides is hard to find. However, quasi-reversible processes can be a good alternative. NiO NFs are one of the best options for non-enzymatic glucose sensing as they qualify stupendously in all four criteria. NiO outperforms other TMOs in terms of sensitivity, with only CuO showing comparable performance. Other TMOs, despite possessing multiple oxidative states, have not demonstrated such performance. The highest reported glucose sensitivity in literature⁷ is on a Ni-based electrode with Ni-doped Co₃O₄. Apart from all the above-mentioned advantages, the NiO nanoparticles are easy to synthesize, cost-effective, scalable, and offer high stability in alkaline medium⁸. Considering its alkaline stability, NaOH is the electrolyte of choice for NiO-based non-enzymatic glucose sensing experiments.

The article, therefore, aims to utilize NiO NF as a glucose-sensing material. The base electrode chosen is a glassy carbon electrode (GCE), which is one of the most standard electrodes. However, it is similarly applicable to other substrates, such as metal foam electrodes (Ni and Cu Foam), Indium Tin Oxide (ITO) coated Glass, Fluorine Tin Oxide (FTO) coated Glass, Metal electrodes (Au, Ag, Cu, etc.), and screen-printed electrodes. The GCE can be replaced by other mentioned electrodes, and the processes can be translated equivalently onto them. More importantly, this can also be translated to other analytes such as ascorbic acid, uric acid, etc. A step-by-step guide is provided for experiments related to the chronoamperometric detection of glucose with a focus on the optimization of parameters that maximize the sensor's sensing performance metrics.

Protocol

1. NiO nanostructured flower synthesis

1. First, prepare a 6 mM solution of NiCl₂·2H₂O in 10 mL of ethanol.
2. In a separate beaker, prepare 10 mL of 3% Polyvinylpyrrolidone (PVP) solution in deionized (DI) water. Ultrasonicate the solution for 5 min at 40 kHz (50 W) and mechanically stir it for an additional 5 min. Repeat the cycle twice to completely dissolve the solid PVP.
   NOTE: If PVP remains, perform another cycle of sonication and stirring.
3. Once the PVP is completely dissolved, add the 10 mL of PVP solution dropwise using a pipette into 10 mL of the Ni precursor solution under vigorous stirring. Continue to stir the mixture for 30 min after PVP addition.
   NOTE: The addition of PVP is a critical step for achieving the desired morphology.
4. Add 18 mM solution of urea (in a 1:1 mixture of DI: ethanol in 10 mL) dropwise to the prepared mixture, bringing the total volume of the solution to 30 mL.
5. Stir the solution for 10 min, transfer it into a polytetrafluoroethylene vessel, and seal it inside a stainless steel autoclave.
6. Once properly sealed, keep the autoclave in an electric oven at 160 °C for 6-8 h. After autoclaving, allow it to cool naturally to room temperature.
7. Centrifuge the resultant product at 26.88 x g and collect the precipitate for further use. The collected, green-colored precipitate Ni(OH)₂ is gelatinous in nature, and the supernatant consists of the clear solvent. Decant the solvent and wash the precipitate using ethanol or DI at least 2-3 times. Dry the washed precipitate at 70 °C for 10 h in an electric oven.
8. Put the dried powder in a box furnace at 450 °C for 4 h to anneal and obtain pure phase NiO NF, which is grey in color.

2. Electrode fabrication

1. Polish GCE with 0.05 µm alumina nanoparticle slurry on a clean electrode polishing pad by repeated motions in the shape of the numeral 8. Rinse immediately using DI water and ultrasonicate using a bath sonicator in IPA and DI for 2 min each at 40 kHz (50 W).
   NOTE: GCE polishing is a simple procedure where the surface of Graphite is cleaned by mechanically rubbing it against fine powder slurry on a cleaning pad. The most common particle size for common cleaning is 0.05 µm.
2. Add a known mass (3-15 mg) of NiO NF powder to 3 mL of DI to prepare a nanoparticle suspension such that the concentration of nanoparticles ranges from 1-5 mg/mL.
3. Put the mixture in a bath ultrasonicator for 10-20 min to obtain a uniform dispersion. Ensure that there are no precipitates or settled particles after sonication.
   NOTE: Avoid excessive sonication if stable suspensions are achieved with shorter ultrasonication times.
4. Cast 10 µL of the aliquot by dropping it on the cleaned surface of the GCE electrodes. Then, depending upon the concentration of the loading solution, name the electrodes NiO NF 1, NiO NF 2, NiO NF 3, NiO NF 4, NiO NF 5, respectively.
   NOTE: For consistent results, carefully put the drop exactly on the surface of the glassy carbon and not on the polymer electrode body. If the 10 µL drop seems excessive, use a smaller volume of the suspension to just cover the GCE surface.
5. Allow the electrodes to dry at room temperature overnight (10-12 h).
6. Finally, use a diluted binder solution (0.5 wt% in ethanol) for compact binding of the nanoparticles onto the surface of the electrode. For this, carefully drop cast 6 µL of the solution on the modified electrodes and allow them to dry in air at room temperature for 30 min.
7. Keep the electrodes in a desiccator until further use.
   NOTE: Avoid long exposure to ambient conditions, as it might change the responses of the electrode due to changes in nafion.

3. Electrochemical detection process

1. For the electroanalytical solution, take 20 mL of 0.1 M NaOH solution in the electrochemical cell.
2. Use a three-electrode setup to analyse the glucose concentration. The Ag/AgCl (3 M KCl) and Pt wire are the reference and counter electrode, respectively.
3. Activate the NiO NF@GCE electrode by stabilizing it with 15 cycles of cyclic voltammetry (CV). The potential window of the CV is 0-0.8 V in a fast scan rate of 200 mV/s. The Ni⁺³/Ni⁺² reduction peak potential is usually around 0.4-0.6 V.
4. After stabilization, run chronoamperometry for real-time detection of glucose using the NiO NF@GCE electrode.
   1. Firstly, establish the baseline by running chronoamperometry at the Ni reduction potential or glucose oxidation potential (as obtained from CV) for 100 s.
      NOTE: The stability time can be chosen to be anything different, keeping in mind that the current becomes constant for at least 10 s till the chosen time for establishing baseline current.
   2. Once the base current is established, pause the chronoamperometric experiment using the Pause button in the software.
5. For the analysis, prepare a 2 M stock solution of glucose.
6. Lift the electrode stand and add 10 µL of the glucose stock solution to the electroanalytical solution, making the glucose concentration of the complete electroanalytical solution 1 mM with negligible change in the total volume. Stir the solution for 30 s to achieve homogeneity.
7. Then, put the electrodes back into their previous state and resume the chronoamperometric measurement after 5 s by pressing the Resume button available in the software.
8. Choose a 30 s analysis window for the chronoamperometric detection of various concentrations of glucose.
9. Repeat the process from step 3.6 to 3.8 to stepwise increase the glucose concentration up to 15 mM and record the chronoamperometric response.

4. Optimization of electrochemical process parameters

NOTE: The electrochemical detection process has several parameters, such as potential window determination, determination of the best potential of chronoamperometric detection, time for concentration determination, loading concentration of material, etc. Although there is no fixed method or workflow order for optimizing these parameters, the following steps can help optimize them the best.

1. In a typical initial experiment, start with a low loading concentration of nanomaterial (~1 mg/mL).
   NOTE: In order to optimize the process parameters, two important sensor characteristics are kept in mind. First, the sensitivity or responsivity towards the analyte (in this case, glucose). The primary objective is to maximize the sensitivity or responsivity (change in current response per unit glucose added). Second, but of the same importance, is the linear range of detection. A dynamically wide range of linear behaviour is essential for glucose detection.
2. Optimize the potential window in which the Faradaic reaction occurs.
3. Initially, run a broad scan of -1 V to +1 V using cyclic voltammetry.
4. Now, identify a small potential range where the Faradaic reaction is taking place using the reduction and oxidation peaks of the electrochemical process. For instance, a 0.1-0.8 V window is good enough for NiO NF@GCE based electrodes in 0.1 M NaOH.
5. Collect the cyclic voltammograms for the electrochemical process in that potential window.
6. Add a small amount of glucose (typically 1 mM) to the solution to make redox peaks appear in the cyclic voltammogram (CV) and to resolve multiple peaks by enhancing the peak corresponding to glucose oxidation.
7. Identify the anodic peak potential, which varies with glucose concentration, as the electrochemical potential where glucose is oxidized. Measure chronoamperometric responses slightly above this potential.
8. Once the potential window and the potential of glucose oxidation are identified, find out the optimal loading concentration by changing the concentration of the nanomaterial suspension drop-cast onto the electrode. In the present case, 4 mg/mL is the optimal loading concentration.
   NOTE: Generally, for most NiO-based electrodes described in the literature, the sensitivity increases with an increase in loading suspension concentration up to 3 mg/mL and decreases after that. Usually, the 3 mg/mL suspension shows the highest sensitivity and largest linear range. Thus, the one that consistently shows larger changes in current with each increment in glucose is the best.
9. Fine-tune other parameters, such as the analysis window and analytical potential, by maximizing sensitivity, linear range, and consistency of the obtained results.
   1. Use a 30 s analysis window to obtain repeatable results even without adding glucose.
   2. Observe visual clues in the chronoamperometric curve, ensuring current stabilizes after 30 s.
      NOTE: For tuning parameters, it is essential to focus on improving the consistency of the measurement since maximization has been previously achieved. Look for visual clues in the chronoamperometric curve. The current spike returns to almost the same value when glucose is not added. Additionally, the change of current (ΔI) in the case of simultaneous addition at each increment also remains fairly constant. Avoid longer analysis durations and maintain a balance between consistency and sensitivity.
10. Once all the parameters are optimized, characterize at least ten electrodes at the optimized experimental conditions to plot the calibration curve by linear fitting of the data extracted at different concentrations after 30 s for all ten electrodes.
    NOTE: A smaller set of 5 electrodes has been chosen in this study.

Results

The characterization of NiO nanoflowers was done to affirm their structural and morphological properties. The structural properties were investigated using X-ray diffraction (XRD) and Raman spectroscopic methods. The morphology on the other hand was confirmed using Scanning Electron Microscopy.

The XRD pattern of the NiO nanoflowers is shown in Figure 1A for 20-80° range. The step size was set at 0.02626°/step, and the scan rate for the experiment was 12°/min. The...

Discussion

The amperometric detection of bioanalytes is a common method used for glucose sensing. This process can be exactly transposed with other metal oxides such as ZnO, CuO, Fe₂O₃, CrO₃, MnO_2, etc. The process remains the same even for different analytes such as ascorbic acid, uric acid, etc. However, the process parameters and optimization vary from system to system. The procedure is highly dependent upon two parameters mentioned below.

The optimum nanoma...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ag/AgCl (3M KCl) reference electrodes and	Palmsens	PS111
DI water	N/A	N/A
Electrochemical workstation	Palmsens	Palmsens4
Ethanol(99.9%)	Changshu Song Sheng Fine Chemicals	64-17-5
Glassy Carbon Electrodes	Palmsens	PS103
Micropippete 1 mL	Thermofischer	4642090
Micropippete 20 µL	Thermofischer	4642060
Nafion	Kanopy Techno Pvt. Ltd	KA06
NaOH	Qualigens	1310-73-2
Nickel chloride dihydrate (NiCl2·2H2O)	Sigma Aldrich	69098-15-3
Polyvinyl Pyrrolidinone (PVP)	Thermofischer	227470025
Pt wire Counter Electrode	Palmsens	MW1032
Urea	Qualigens	57-13-6