CC BY
16
Fabrication of Polyvinyl Alcohol Doped CuO Thin Films for Improved Amperometric-Non Enzymatic Hydrogen Peroxide Sensing
Navashree Nagarajan and Parthasarathy Panchatcharam*
Department of Electronics and Communication Engineering, CMR Institute of Technology, Bengaluru 560037, India *e-mail: [email protected]
Abstract. Nanotechnology is an emerging field in science and technology that primarily focus on nanoparticles ranging from 1-100 nm in diameter, which exhibit distinctive properties owing to their small size and large surface area. Among them, copper oxide nanostructures, are major metal oxide nanoparticles, widely used in various fields especially in the development of biosensors due to their unique structural characteristics and biological effects. In this work, copper oxide nanoparticles were synthesized using a simple chemical reduction method and characterized using XRD to study the morphological and structural properties. Those nanoparticles were doped with PVA by sol-gel process and the electrode was fabricated using the spin coating technique on the precleaned glass slide. CV studies showed that the CuO NPs electrode was effective in detecting hydrogen peroxide with high selectivity even in the presence of other substances. A high-level sensitivity of 0.002 mA-mM-1cm-2 and a 0.5 mM to 1.5 mM quick linear response was accomplished due to the large specific surface areas and efficient electron transport in the corresponding reactions, making this electrode a very promising candidate for efficient and accurate non-enzymatic detection of hydrogen peroxide (H2O2). © 2023 Journal of Biomedical Photonics & Engineering.
Keywords: copper oxide nanoparticles; hydrogen peroxide; polyvinyl alcohol; electrochemical sensor; amperometric detection.
Paper #7782 received 24 Feb 2023; revised manuscript received 28 Jun 2023; accepted for publication 8 Jul 2023; published online 23 Aug 2023. doi: 10.18287/JBPE23.09.030309.
1 Introduction
Hydrogen peroxide (H2O2) plays a crucial role as a mediator in biological systems, where it contributes to the regulation of cell growth. It is involved in various cellular processes, including cell signaling, immune activation, and apoptosis. This is primarily attributed to the reactivity of H2O2 with other oxygen species present in the system [1]. It has numerous applications in diverse fields, such as controlling microbes, bleaching textiles and paper, cosmetics, food processing, pharmaceuticals, and environmental practices. In addition, H2O2 has many applications, including treating wastewater, oxidizing cellulose, enhancing rocket fuels, recycling waste paper, detoxifying organic pollutants, and manufacturing
various chemicals and plastics that could potentially contaminate the environment. It is also crucial for various enzymatic reactions in the body, and the H2O2 layer serves as the initial defense mechanism against different pathogens [2]. However, excess H2O2 leakage in biological organisms can be harmful, causing gastrointestinal tract damage, skin and stomach irritation, and protein degradation. Specifically, excessive amounts of H2O2 in the body have been linked to several diseases, including fragmentation of DNA, diabetes, membrane damage, cardiovascular issues, cancer, Parkinson's, Alzheimer's, neurodegeneration, tissue damage, and aging problems [3]. It is also utilized as an oxidant in various fields, including environmental monitoring, food safety, and medical applications. These conditions make
This paper was presented at the International Conference on Nanoscience and Photonics for Medical Applications - ICNPMA, Manipal, India, December 28-30, 2022.
it crucial for the determination of hydrogen peroxide [4]. Various techniques have been developed to detect H2O2, including colorimetry, photoelectrochemical, light detection, titration, chromatography, and electrochemical methods. However, chromatography and titration methods are not suitable for in vitro and in vivo detection of H2O2. Similarly, light detection and chemiluminescent techniques are inconvenient for H2O2 detection because of the differences in excitation methods and the interference of H2O2 with chemical compounds during fluorescence measurements. As a result, the electrochemical method has gained popularity in recent years due to its simplicity, accuracy, speed, affordability, and user-friendliness [5]. Extensive research has been conducted on enzyme-based electrochemical biosensors, primarily due to their high sensitivity and excellent selectivity [6]. However, these biosensors suffer from a significant drawback due to a lack of stability and poor reproducibility caused by the inherent fragility of enzymes, which can be easily damaged during biosensor fabrication and testing. Consequently, recent studies have focused on enzyme-free electrochemical biosensors that offer advantages such as high sensitivity, rapid response, affordability, and good stability [7]. Numerous materials have been investigated for enzyme-free electrochemical biosensors, including transition metal nanoparticles, carbon nanotubes, semiconducting metal oxides, graphene, ordered mesoporous carbon, and their composites. These materials possess large specific surface areas and enhanced catalytic activities, enabling sensitive and rapid detection of hydrogen peroxide. Among them, copper oxide stands out as an excellent material due to its remarkable redox properties, non-toxicity, high stability, and conductivity [8]. Conducting polymers have also been employed as modifiers in the fabrication of electrochemical sensors due to their exceptional stability, good conductivity, and ability to maintain stability on the electrode surface [9, 10]. In the present study, we developed enzyme-free H2O2 electrochemical biosensing materials using CuO nanoparticles synthesized through a simple reduction method, which were then doped into polyvinyl alcohol polymer to create a thin film. Our findings demonstrate that the CuO nanostructure enhances the electrochemical performance for H2O2 detection, primarily due to its large specific surface area and efficient electron charge transfer properties.
2 Experimental Section
2.1 Materials
All the chemicals such as Copper (II) sulphate pentahydrate (CuSO4-5H2O), Sodium hydroxide (NaOH), Ascorbic acid, Polyvinyl Alcohol, (PVA, average molecular weight of 85000), and Distilled water were purchased of analytical grade from Kesari Scientific chemicals (Chennai). Nonionic detergent and deionized water were used to clean all glassware and oven-dried before use.
2.2 Copper Oxide Nanoparticle Synthesis
To synthesize the copper oxide nanoparticles, a simple chemical reduction method was employed. The procedure involved the gradual addition of 25 ml of a 10 mM CuSO4^5H2O solution, drop by drop, into 50 ml of a 20 mM NaOH solution with vigorous stirring. Subsequently, 25 ml of a 10 mM ascorbic acid solution was added dropwise while continuously stirring the mixture for 1 hour at room temperature. As a result of the reduction facilitated by sodium hydroxide and ascorbic acid, the solution underwent a color change and formed a yellow precipitate containing copper oxide nanoparticles. The particles were then separated by filtration and subjected to thorough washing with distilled water and acetone. Finally, the washed nanoparticles were left to dry naturally at room temperature for 24 h. Distilled water served as the solvent for the preparation of the solution used in the synthesis process [11].
2.3 PVA Doping with Sol-Gel Technique
To incorporate the copper oxide nanoparticles into the polymer matrix, a sol-gel process was employed. The first step involved preparing a 5% solution of polyvinyl alcohol (PVA) by stirring it for 3 h at 60 °C and subsequently allowing it to cool down to room temperature. Once the PVA solution was prepared, the previously synthesized CuO nanoparticles were added to it. During this addition, a magnetic stirrer was used to ensure rapid and continuous stirring, preventing the nanoparticles from aggregating or clumping together. This process resulted in the formation of a CuO nanoparticle sol that was effectively incorporated within the PVA matrix. Fig. 1 represents the complete flow for the preparation of copper oxide nanoparticle sol.
Fig. 1 Flowchart for Copper oxide nanoparticle doped PVA Sol preparation.
Sodium hydroxide
Ascorbic acid
Polyvinyl alcohol
U-*
I---------------------1 r
Copper sulphate solution
CuO nanoparticles solution
i--------------1
PVA doped
CuO sol
Characterization and electrochemical analysis
Spin coating
Fig. 2 Schematic representation for preparation of CuO NPs/PVA thin film.
Fig. 3 X-ray diffraction pattern for CuO NPs/PVA thin film.
2.4 Electrode Fabrication
The CuO NPs thin films were deposited onto glass substrates via a sol-gel/spin coating process. The glass slides were pre-treated with alumina slurry and washed with deionized water and acetone. The CuO NPs sol was deposited onto the glass slides using a spin coater at 2500 rpm for 30 s, followed by drying and annealing at different temperatures (100-400 °C) for 5 min. Fig. 2 represents the complete flow for the preparation of thin film CuO NPs working electrode.
3 Result and Discussion
The structural properties of the coated films were determined using a Phillips X'Pert Pro X-ray
diffractometer with Cu Kal radiation (1.5406). The electrochemical measurements, specifically the cyclic voltammetry (CV) curve and the CV VS H2O2 concentration, were performed in a three-electrode cell system at room temperature. The platinum was used as the counter electrode, Ag/AgCl saturated with potassium chloride was used as the reference electrode, and the newly developed thin film CuO NPs/PVA slide acted as the working electrode, with a diameter of 3mm. The Wireless potentiostat was used for the electrochemical measurements.
3.1 Materials Characterization
The crystal structure was identified with the aid of XRD analysis, which allowed for the detection of diffraction peaks in the copper oxide. The diffraction peaks at 20 = 36.36°, 42.18°, and 50.91° (JCPDS 45-0937) were attributed to the (002), (111), and (202) crystallographic planes of the CuO monoclinic crystal structure, as shown in Fig. 3. These diffraction peaks were found to be responsible for the characteristic pattern of the copper oxide, confirming the crystalline nature of the copper nanostructures that were present in PVP.
The mean size of the nanoparticles was determined using the Scherrer formula based on the high-resolution X-ray diffraction peaks. The Scherrer formula is a mathematical equation that relates the size of crystalline domains to the width of X-ray diffraction peaks. It can be expressed as follows in Eq. (1):
t =
K\ pcose
(1)
In Eq. (1), т represents the mean size of the ordered (crystalline) domains, K is a dimensionless shape factor (approximately unity), X is the X-ray wavelength, p is the line broadening at half the maximum intensity (full width at half maximum, FWHM, in radians) represented as 20, and 0 is the Bragg angle. By performing the calculations using the Scherrer formula, the average size of the nanoparticles was determined to be approximately 19.72 nm.
3.2 Electrochemical Analysis of H2O2
electroreduction generates free electrons, corresponding to the concentration of H2O2.
When hydrogen peroxide (H2O2) molecules come into contact with the electrode surface, they undergo a reaction where they release 2H+ + 2e- ions. This process is accompanied by the production of an electric current when a potential is applied to the electrode. Fig. 5 depicts a schematic representation of this electrochemical process. By measuring the resulting current value, it is possible to estimate the quantity or concentration of H2O2 present in the sample.
3.2.1 Mechanism of Interaction
Despite the distinct morphological properties of copper oxide (CuO), it is capable of forming a compound with the polymer polyvinyl alcohol (PVA). Fig. 4 illustrates that the hydroxyl group present in PVA interacts with the oxygen atoms of CuO nanoparticles, leading to the formation of CuO-doped PVA through hydrogen bonding. This interaction between the polymer and nanoparticles allows for their integration at a molecular level. However, when a reactive species such as hydrogen peroxide (H2O2) comes into contact with the surface of CuO, it serves as a redox carrier and facilitates a chemical reduction process. This interaction with H2O2 can lead to changes in the properties or behavior of the CuO nanoparticles, potentially impacting their functionality or reactivity in certain applications. This
PVA
Solution casting 60oC
о
H H
ОН
PVA- CuO nanocomposite
,H ,H
Binding mechanism depicted through dotted lines (Hydrogen bonding)
„
Fig. 4 Reaction mechanism between CuO and PVA [12].
Fig. 5 Schematic representation of the working mechanism of developed CuO NPs/PVA thin film.
Fig. 6 Cyclic Voltammogram of H2O2 in CuO NPs/PVA thin film electrode at different concentrations with 500 mV/sec scan rate.
This mechanism forms the basis for the development of electrochemical sensors specifically designed for the detection and quantification of H2O2. These sensors utilize the electrochemical response generated by the interaction between H2O2 and the electrode surface to provide a means of detecting and measuring H2O2 levels in various applications.
3.2.2 Different Concentration
Cyclic Voltammetric measurements were performed on a CuO-doped PVA thin film working electrode in 10 mM PBS (pH 7.4) with 0.5 mM to 1.5 mM H2O2. The newly generated CuO NPs doped PVA thin film electrode displayed a significant response in reduction current. Prior to this, the CV results were obtained for the PBS solution without H2O2 for comparison. The cathodic peak observed between -0.4 to 0.2 V confirmed the electrocatalytic behavior of the thin film in H2O2 electroreduction. Fig. 6 displays the CVs of a CuO thin film electrode for varying concentrations of H2O2 in 10 mM PBS (pH 7.4) with a 500 mV/sec scan rate. The results reveal that the catalytic reduction current increased with increasing H2O2 concentrations (0.5-1.5 mM), indicating that the sensor is suitable for quantitative analysis.
3.2.3 Different Scan Rate
Fig. 7 illustrates the cyclic voltammograms (CVs) obtained from a CuO-doped PVA thin film electrode in 10 mM phosphate-buffered saline (PBS) with a pH of 7.4 and 1.0 mM hydrogen peroxide (H2O2) at 500 mV/sec scan rate. The cathodic reduction current of H2O2 exhibits a linear correlation with the square root of the scan rate. This suggests that the electron transfer reaction at the thin film electrode surface is diffusion-controlled. In other words, the rate of the electron transfer reaction is limited by the diffusion of H2O2 to the electrode surface. As the scan rate increases, the diffusion layer thickness decreases, allowing more H2O2 molecules to access the electrode surface, leading to a higher reduction current. This linear relationship between the reduction current
and the square root of the scan rate agrees with the Randles-Sevcik equation, which describes the behavior of electrochemical systems under diffusion-controlled conditions. The Randles-Sevcik equation is an empirical relationship used in electrochemistry to describe the behavior of a reversible redox reaction at an electrode surface. It relates the peak current of a cyclic voltammogram to the concentration of the electroactive species, the scan rate, and the electrochemical properties of the system.
The Eq. (2) is expressed as:
= (2.69 x 105)n2AC(Dv)1
(2)
where ip is the peak current (in A), n is the number of electrons involved in the redox reaction, A is the electrode surface area (in cm2), D is the diffusion coefficient of the electroactive species (in cm2/s), C is the concentration of the electroactive species (in mol/cm3), and v is the scan rate (in V/s).
0.0005 -
^ 0.0000 -«A
¡■5 -0.0005 -
-
3
O -0.0010-
-----100mV
-----200mV
-----300mV
-----400mV
-----500mV
r**''
1000 500 0 -500 -1000
Potential (mV)
Fig. 7 Cyclic Voltammogram of H2O2 at different scan rates with 1.0 mM concentration.
3.2.4 Effect of Annealing Temperature
To investigate the impact of annealing temperature on the performance of the CuO-doped PVA thin film electrode, a series of thin films were prepared and subjected to annealing at different temperatures ranging from 100 °C to 400 °C. The cyclic voltammograms of these films were recorded and subjected to analysis. The outcomes, as shown in Fig. 8, indicate that an increase in annealing temperature resulted in a corresponding increase in current within the current-voltage response curve. This observation can be attributed to the heightened reactivity between CuO nanoparticles and H2O2 molecules, facilitated by the greater surface area of the CuO agglomerates formed at higher annealing temperatures. The enhanced reactivity leads to increased current generation during the electrochemical reaction. These findings are consistent with prior research [13-15], which has demonstrated the influence of annealing on the structural and morphological properties of thin films,
p
subsequently affecting their electrochemical behavior. By optimizing the annealing temperature, it becomes possible to customize the performance of the thin film electrode to meet specific application requirements. These results have significant implications for the advancement of electrochemical sensors that aim to achieve sensitive and selective detection of H2O2.
Fig. 8 Cyclic Voltammogram of H2O2 with CuO NPs/PVA thin films annealed at various temperatures.
Concentration (mM)
Fig. 9 Peak current density VS H2O2 concentration.
Table 1 Parameters for intercept and R-Square calculation.
Equation y = a + b x x
Plot B
Weight No Weighting
Intercept 6.01167E-4 ± 8.48109E-6
Slope 6.16E-5 ± 7.85196E-6
Residual Sum of 3.08267E-11
Squares
Pearson's r 0.99197
R-Square (COD) 0.98401
3.2.5 Sensitivity and Detection Limit
To assess the analytical effectiveness of a CuO-doped PVA thin film, a series of amperometric tests were carried out using known concentrations of H2O2 in a 10 mM PBS (pH 7.4) solution. The scan rate of 500 mV/sec was utilized, and standard solutions of H2O2 were added in successive amounts to generate a calibration curve. The linear range of the calibration curve was found to be 0.5 mM to 1.5 mM, and the corresponding peak current was determined. The resulting peak current for each concentration of H2O2 was plotted in Fig. 9. The relative sensitivity of the proposed sensor was found to be approximately 0.002 mA-mM-lcm-2. The limit of detection (LOD) was calculated using the 3S/k approach [16] and was determined to be 0.78 mM. Overall, the results demonstrate that the CuO NPs/PVA thin film provides great performance in detecting H2O2 and acts as a prominent sensor for this application.
3.2.6 Stability and Reproducibility
Another requirement that a sensor must meet is long-term storage stability, which has been examined for theCuO-doped PVA thin film produced and tested in mM H2O2. The modified electrode was stored at room temperature when not in use. Results showed that after two weeks of storage, the electrode retained 96% of its initial reaction, as previously reported, and after a month, it could still sustain 98% of its original current. No reaction was detected over a longer period, indicating the high durability and stability of the newly developed electrode.
The reproducibility of the electrode was evaluated by measuring responses to 1.0 mM H2O2 solution for 20 successive measurements, which demonstrated peak currents at ±0.067 mA, as shown in Fig. 10. These results confirm the electrode's long-term stability, durability, and reproducibility.
A Peak current
0.000674 -
0.000672 - ▲
0.000670 - ▲ ▲
0.000668 - ▲
-g 0.000666 - A
A
s- ^ 0.000664 - A
s
0.000662 - 4 ▲
0.000660 - ▲ ▲
0.000658 - ▲ A
0.000656 -
1 0.0 1 1 1 1 1 ■ 1 0.5 1.0 1.5 2.0
Concentration (mM)
Fig. 10 Peak current VS 1.0 mM H2O2 concentration for 20 successive measurements.
4 Conclusion
In summary, a CuO nanostructure-based thin film was successfully synthesized using a reduction method without any additives, and it was incorporated into PVA for the detection of H2O2. The electrochemical properties of this material were thoroughly investigated. The analysis revealed that the morphology of the CuO nanostructures primarily depended on the initial amount of CuSO4. The resulting CuO nanoparticle-doped PVA thin film demonstrated effective non-enzymatic detection of H2O2. The modified glass electrode with cubic CuO nanoparticles displayed outstanding catalytic performance towards H2O2, exhibiting high sensitivity
(0.002 mA^mM-1cm-2), rapid response, relatively low detection limit, good stability, and favorable selectivity. Consequently, this electrode holds great promise for sensing applications due to its advantageous features and ease of production.
Acknowledgment
This work is supported by DST-SERB, New Delhi, Project File number: EEQ/2021/000467EEQ.
Disclosures
The authors declare no conflict of interest.
References
1. M. Shafa, I. Ahmad, S. Hussain, M. Asif, Yi Pan, R. Zairov, A. A. Alothman, M. Ouladsmane, Z. Ullah, N. Ullah, C. Lai, and U. Jabeen, "Ag-Cu nanoalloys: An electrochemical sensor for H2O2 detection," Surfaces and Interfaces 36, 102616 (2023).
2. N. Amini, B. Rashidzadeh, N. Amanollahi, A. Maleki, J. K. Yang, and S. M. Lee, "Application of an electrochemical sensor using copper oxide nanoparticles/polyalizarin yellow R nanocomposite for hydrogen peroxide," Environmental Science and Pollution Research 28, 38809-38816 (2021).
3. G. He, F. Gao, W. Li, P. Li, X. Zhang, H. Yin, B. Yang, Y. Liu, and S. Zhang, "Electrochemical sensing of H2O2 released from living cells based on AuPd alloy-modified PDA nanotubes," Analytical Methods 11(12), 1651-1656 (2019).
4. J. S. Kumar, S. Ghosh, N. C. Murmu, N. Mandal, and T. Kuila, "Electrochemical detection of H2O2 using copper oxide-reduced graphene oxide heterostructure," Journal of Nanoscience and Nanotechnology 19(8), 5295-5302
(2019).
5. P. Parthasarathy, "Synthesis and UV detection characteristics of TiO2 thin film prepared through sol gel route," IOP Conference Series: Materials Science and Engineering 360, 012056 (2018).
6. P. Parthasarathy, S. Vivekanandan, "Investigation on uric acid biosensor model for enzyme layer thickness for the application of arthritis disease diagnosis," Health Information Science and Systems 6, 5 (2018).
7. P. Panchatcharam, "Synthesis and Characterization of CoO-ZnO-Based Nanocomposites for Gas-Sensing Applications," Chapter 5 in Multilayer Thin Films - Versatile Applications for Materials Engineering, S. Basu (Ed.), IntechOpen, London, UK (2020). ISBN: 978-1-83968-463-0.
8. A. A. Mathew, P. Parthasarathy, and S. Vivekanandan, "Development of Copper Nanoparticles from E-waste for Biomedical Applications," in Advances in Automation, Signal Processing, Instrumentation, and Control Select Proceedings of i-CASIC 2020. Lecture Notes in Electrical Engineering, V. L. N. Komanapalli, N Sivakumaran, and S Hampannavar (Eds.), Springer, Singapore, 703-715 (2021).
9. P. Gao, D. Liu, "Facile synthesis of copper oxide nanostructures and their application in non-enzymatic hydrogen peroxide sensing," Sensors and Actuators B: Chemical 208, 346-354 (2015).
10. P. Parthasarathy, S. Vivekanandan, "A comprehensive review on thin film-based nano-biosensor for uric acid determination: arthritis diagnosis," World Review of Science, Technology and Sustainable Development 14, 52-71 (2018).
11. M. A. Bhosale, S. C. Karekar, and B. M. Bhanage, "Room temperature synthesis of copper oxide nanoparticles: morphological evaluation and their catalytic applications for degradation of dyes and C-N bond formation reaction," ChemistrySelect 1(19), 6297-6307 (2016).
12. K. K. Dey, P. Kumar, R. R. Yadav, A. Dhar, and A. K. Srivastava, "CuO nanoellipsoids for superior physicochemical response of biodegradable PVA," RSC Advances 4, 10123-10132 (2014).
13. A. Muthumariappan, K. Sakthivel, S. M. Chen, T.-W. Chen, M. Govindasamy, and B.-S. Lou, "Effects of annealing temperature on crystal structure and glucose sensing properties of cuprous oxide," Sensors and Actuators B: Chemical 266, 655-663 (2018).
14. P. Parthasarathy, S. Vivekanandan, "Biocompatible TiO2-CeO2 nano-composite synthesis, characterization and analysis on electrochemical performance for uric acid determination," Ain Shams Engineering Journal 11, 777-785
(2020).
15. P. Parthasarathy, S. Vivekanandan, "Structural, optical and electrochemical response studies of TiO2-ZrO2 nanocomposite for uric acid detection," in 2019 Innovations in Power and Advanced Computing Technologies, Vellore, India, 1-6 (2019).
16. E. Gricar, K. Kalcher, B. Genorio, and M. Kolar, "Highly Sensitive Amperometric Detection of Hydrogen Peroxide in Saliva Based on N-Doped Graphene Nanoribbons and MnO2 Modified Carbon Paste Electrodes," Sensors 21(24), 8301 (2021).
