Quantitative analysis of hydrogen peroxide with special emphasis on biosensors

Abstract

Determination of hydrogen peroxide (H₂O₂) has become essential in pharmaceutical, biological, clinical and environmental studies. The conventional detection methods of H₂O₂ such as colourimetry, titration, chromatography, spectrophotometry, fluorimetry, chemiluminescence have limited success, due to their poor selectivity and sensitivity, long analysis time and lack of long-term reliability and reproducibility. The biosensors overcome these limitations because of their simplicity, rapidity, selectivity and high sensitivity. This review describes the principle, analytic parameters, merits and demerits of various methods of H₂O₂ determination with special emphasis on biosensors. The classification of biosensors based on various materials/nanomaterials and electrodes have been described in detail. The recent advances in vivo sensing and bio-sensing of H₂O₂ by hemoglobin nanoparticles are also presented. The significant challenges and future perspective for highly selective H₂O₂ detection are discussed.

Introduction

Hydrogen peroxide (H₂O₂) is one of the most important substrate in various biological reactions catalyzed by multifunctional oxidases [1]. H₂O₂ (Mr-4.014 g/mol) is a strong oxidant with a skew, chain non-planar structure (Fig. 1). H₂O₂ exhibits several important properties such as oxidizing property, source of energy, gas formation on decomposition, source of free radicals, effects on biological processes and its use in chemical synthesis. Depending on exposure index (> 10%), H₂O₂ is an irritant to skin, eye, gastrointestinal tract, brain and mucous membrane. Accurate determination of H₂O₂ is highly important in the fields of biological, pharmaceutical, clinical and environment sciences, food processing and textile [2]. In industrial and medical areas, it is used for wastewater treatment, bleaching and sterilization. Besides cell cytotoxicity and anti-oxidation, H₂O₂ plays a key role as secondary messenger, modulating vital and complex biological pathways, namely cell growth, cell differentiation, apoptosis, vascular remodeling, immune activation, stomatal movement and root growth [3]. Elevated levels of H₂O₂ have been implicated as an etiological agent in multiple disease conditions including cancer, arthritis, diabetes, neurodegenerative disorders, cardiovascular diseases, asthma, ageing and oxidative stress-related diseases [4, 5]. Table 1 summarizes the various physico-chemical properties of H₂O₂. The detection of H₂O₂ sets the foundation for many analytical techniques and commercial test-kits such as colourimetry, fluorimetry, spectrophotometry, chemiluminescence and electrochemical method [6]. The first four techniques have obvious drawbacks, as these are time-consuming, expensive, require long pre-treatments for preparation of the samples, utilization of expensive reagent and interference [7], Among these methodologies, electro-chemical methods provides a convenient way for H₂O₂ determination, because of its easy fabrication, low cost of instrumentation, intrinsic sensitivity, high selectivity, and suitability for detecting and quantifying analytes in real-time [8]. In electrochemistry, H₂O₂ can be directly reduced/oxidized onto solid electrode surfaces. The detection of H₂O₂ with conventional noble metal electrodes is restricted by high over potential leading to slow electrode kinetics and biofouling, causing interference of other co-existing electroactive species, which downgrade the sensing of real samples [9]. In these studies, conducting research on H₂O₂ detection was focused mainly on electrode modifications to solve the problem of low sensitivity and high over potentials. To fabricate selective H₂O₂ biosensor, platinum nanoparticles, redox polymers, metal oxides, nanofibres, carbon nanotube, a large number of advanced nanomaterials viz carboxylic acid nano-hybrids, fluorophores, graphene capsules, reduced graphene oxide, tetraethyl orthosilicate, polydimethylsiloxane and TTF-modified graphite disc electrodes were used [10]. The present review is aimed to gain an insight into the recent advances in detailed analysis (construction and performance) of the H₂O₂ biosensors over conventional analytical methods for H₂O₂ determination.

Fig. 1

Structure of hydrogen peroxide

Table 1 Physiochemical properties H₂O₂

Conventional methods for determination of H₂O₂

The conventional methods for detection of H₂O₂ can be classified into different categories:

Titrimetry

It is used to calculate unknown amount of H₂O₂ in an identified sample of known concentration. Titrimetric procedures employed iodometry, permanganate and cerium (IV) in acidic media. Klassen et al. [11] estimated 300 µM H₂O₂ using I₃ ⁻ method, after calibrating with permanganate. ε_max was also measured at 351 nm as 25 800 M⁻¹ cm⁻¹ from calibration plot of the I₃ ⁻ method against prepared by titration with potassium dichromate (KMnO₄) [11]. Murty et al. 1981 determined H₂O₂ potentiometrically within a medium containing 8–11 M phosphoric acid [12]. Kieber and Helz used a modified version of the iodometric titration in water matrices, where iodine was liberated as follows: H₂O₂ + 2H⁺ + 2I⁻ = I₂ + 2H₂O. I₂ produced was consumed by adding excess of phenylarsine oxide. To obtain end result, remaining volume of phenylarsine oxide was titrated with I₂ [13].

$${{\text{I}}_2}+\left( 2 \right){{\text{H}}_2}{\text{O}}+{{\text{C}}_6}{{\text{H}}_5}{\text{AsO}}=2{{\text{I}}^ - }+2{{\text{H}}^+}+{{\text{C}}_6}{{\text{H}}_5}{\text{AsO}}{\left( {{\text{OH}}} \right)_2}.$$

The end point was determined, when the intense blue colour of the starch–iodine complex was disappeared. The limit of detection limit (LOD) was 0.02 µM. Another high-throughput two-step absorbance microtitre plate method was developed, titrating an acidified H₂O₂ solution with standard cerium (IV) sulfate. Cerium (IV) sulfate gets converted into cerium (III) sulfate and potassium iodide into iodine [14].

Merits Simple to perform, cost effective and one of the most commonly used method.

Demerits Time consuming, requirement of moderate skill and practice for effective results and proper calibration of the instrument but not accurate at low concentrations.

Spectroscopy

The simplest and most widely available instrumental technique for determination of H₂O_2, generally involves the formation of colored compounds for subsequent absorbance measurements. A method comparing the reaction of methyl blue and toluidine blue with iodine solution was introduced for determination of H₂O₂ based on the following reaction:

$${{\text{H}}_2}{{\text{O}}_2}+2{\text{KI}}+2{\text{HCl}} \to {{\text{I}}_2}\left( {{\text{aq}}} \right)+2{\text{KCl}}+2{{\text{H}}_2}{\text{O}}.$$

Methyl blue reaction yielded a single-peak visible spectrum with higher extinction coefficient, M⁻¹ cm⁻¹ = 49,100 [15]. Matsubara et al. demonstrated the use of mixture of titanium IV and 2,4((5-bromopyridyl)azo)5-(N propyl-N-sulfopropyl amino) phenol disodium for H₂O₂ determination [16]. The molar absorptivity was 5.710⁴ M⁻¹ cm⁻¹ at 539 nm wavelengths. Clapp et al. developed a method to measure H₂O₂ selectively in aqueous solution with titanium (IV) sulphate, yielding a yellow peroxotitanium species at 407 nm [17]. A rapid, reproducible and sensitive method was developed to detect in vitro H₂O₂ activity by 1,10-phenanthroline method [18]. A study reported catalytic decomposition of H₂O₂ by monomeric molybdenum (VI). Hydroquinone, ammonium molybdate, and anilinium sulphate with different H₂O₂ concentrations was mixed and absorbance was determined at 550 nm [19]. Zhang and Wong estimated concentration of H₂O₂ in marine water in the presence of horseradish peroxidase at 592 nm at pH 4.0 by leuco crystal violet oxidation (LCV). LOD was 20 nM with ± 1% precision [20]. A method based on the hydroxylation of phenol was introduced to measure H₂O₂ in water and rainwater samples. The reaction involved presence of iron (II) ions in dilute sulfuric acid medium. The product possessed two bands with maximum absorption at 245 and 300 nm. Linear calibration curve was obtained in 2.0 × 10⁻⁷ to 3.0 × 10⁻⁴ mol/L range with LOD 9.5 × 10⁻⁸ mol L⁻¹ [21]. Huang et al. introduced a rapid and reproducible method for H₂O₂ determination using 4AAP-DEA-βCD-hemin [22]. The LOD obtained through this method was 8.4 × 10⁻⁵ with molar absorption coefficient 1.65 × 10⁴ mol/L/cm. A simple and accurate method for the determination of H₂O₂ in pulp bleaching effluents was introduced in 2013 by Zhang et al. [23]. In this method, H₂O₂ reacted with vanadium pentoxide in H₂SO₄ solution forming a red brown peroxovanadate complex.

Merit These methods exhibit simple and fast analysis.

Demerit They showed analytical interference, slow color development, and low specificity.

Colorimetry

Determination of H₂O₂ by iodide and starch was first reported by Eisenberg in 1943. Color intensities of H₂O₂ sample solutions treated with titanium sulfate reagent was measured. The chemical reaction was as follows: ${\text{T}}{{\text{i}}^{++++}}+{{\text{H}}_2}{{\text{O}}_2}+2{{\text{H}}_2}{\text{O}}={{\text{H}}_2}{\text{Ti}}{{\text{O}}_4}+4{{\text{H}}^+}$.

Formation of pertitanic acid results in formation of yellow color permitting 0.2–3.0 mg/100 ml determination of H₂O₂ concentrations [24]. A sensitive colorimetric method based on iodide oxidation in the presence of (NH4)₂MoO₄ (ammonium molybdate) for direct determination of H₂O₂ in micromolar quantities was reported. Molar absorptivity of the starch–iodine complex was 39.45 mmol⁻¹cm⁻¹L at 570 nm [25]. Enzyme-based colorimetric method was optimized by Fernando and co-workers in 2015 for determination of H₂O₂ scavenging capacity in plant extracts. A significant pink-colored quinoneimine dye was formed when H₂O₂ reacts with phenol, 4-aminoantipyrine, horseradish peroxidase (HRP) in 0.4M phosphate buffer, pH 7.0 [26]. Optimized assay conditions were 30 min as reaction time, pH 7.0, 37 °C, 0.7 mM H₂O₂ concentration and 1 U/ml enzyme concentration. The reported values of limit of quantitation and limit of detection (LOD) were 411 and 136 mM, respectively. A simple and rapid method based on oxidation of phenol red was described for the measurement of H₂O₂ released by cells in tissue culture. A direct linear relationship between concentration of H₂O₂ ranging from 1 to 60 nmol/ml and absorbance at 520 nm was obtained [27]. A direct colorimetric method using 4-nitrophenyl boronic acid for rapid determination of H₂O₂ in aqueous media was developed. Nitrophenylboronic acid reacts with H₂O₂ to produce 4-nitrophenol. An LOD of ~ 1.0 μM was reported [28]. Nitinaivinij et al. proposed a highly sensitive colorimetric method for quantification of H₂O_2, based on chromaticity analysis of silver nanoprisms (AgNPrs). AgNPrs decomposition by H₂O₂ produced yellow color. The protocol recognized H₂O₂ concentration at 1.57 mM with good accuracy and reproducibility [29].

Merit No requirement of complicated apparatus and easy to perform.

Demerit Low sensitivity, false-positive readings, not applicable in turbid samples.

Chromatography

Takahashi reported a method for separating H₂O₂ by HPLC with electrochemical detector and cation-exchange resin gel column of sulphonated styrene divinyl benzene copolymer. The linearity and LOD were 0.9984 and 0.2 pmol, respectively, [30]. H₂O₂ was separated isocratically by HPLC on octadecylsilyl column by Wada et al. in 2003. The LOD was 1.1 µM [31]. A gas-chromatographic method was developed for determination of H₂O₂ in oxidized butyric acid. The absorbance was measured at 517 nm [32]. H₂O₂ was separated by a ligand exchange type column in which a sulfonated polystyrene/divinyl benzene cation-exchange resin was packed [33]. Steinberg determinated H₂O₂ using reverse phase HPLC. The iodovanillic acid thus formed was detected by UV absorption at 280 nm providing ~ 0.1 μM LOD [34]. Gimeno et al. determined H₂O₂ quantitatively in 35 teeth and hair bleaching products by HPLC/UV plus ceric titrimetric method. This method quantified triphenylphosphine oxide (OPPh3) formed by oxidation of triphenylphosphine (PPh3) [35].

Merit Simple, low cost, choice of stationary phase and columns.

Demerit Expensive, require more skilled manpower, time consuming, interferences.

Chemiluminescence methods

Chemiluminescence (CL) is an important method for analysis and quantification of H₂O₂ in different samples. Numerous CL agents such as fluorescein, luminol, dioxetanes, oxalate and its derivatives and acridinium dyes were used as per their specificity. Segawa et al. proposed sensitive H₂O₂ analysis with fluorescein chemiluminescence using HRP as a catalyst. At pH 7.0, the optimum HRP and fluorescein concentrations were 5 × 10⁸ and 6.6 × 10⁴ molar, respectively [36]. A study proposed chemiluminescence detection of H₂O₂ concentrations in various water samples. The method was based on that H₂O₂ induced luminol oxidation in the presence of Co²⁺ ions. The experimental LOD for H₂O₂ was 3.04 × 10⁻⁴ and 6.25 × 10⁻⁵ M, respectively, making the assay suitable for determining micromolar amount of H₂O₂ [37]. An improved chemiluminescence method was developed based on cobalt (II)-catalysed luminol oxidation. This method has enhanced sensitivity towards micromolar determination of H₂O₂ by avoiding quenching effects [38].

Merit Simple to apply, easy to perform, reliable.

Demerit Requirement of complex instrumentation and prone to interferences.

Fluorescence methods

An accurate and simple phosphine-based fluorescent method was developed by Onoda et al. in 2003 to determine H₂O₂ in tissue fractions. Linear calibration curve was attained in the range of 12.5–500 ng H₂O₂ [39]. Paital in 2014 proposed a modified spectrofluorimetric method for determination of H₂O₂ using homovanillic acid oxidation. Biological samples were precipitated with 5% TCA and neutralized by K₂HPO₄ [40]. Chen et al. introduced a new sensitive fluorescent quenching method for H₂O₂ determination in rain water. At pH 3.09, a calibration graph was obtained between 5.0 × 10⁻⁷ to 9.0 × 10⁻⁴ mol/l [41].

Merit Multiple samples analysis, sensitive, more efficient and cost effective.

Demerit Requirement of sophisticated imaging techniques, false positives but not applicable for detection of non-fluorescence compounds.

Chemibioluminescence imaging

Lee et al. 2007 validated the use of peroxalate esters with fluorescent dyes for in vivo H₂O₂ imaging in the peritoneal cavity of mouse. The peroxalate nanoparticles had numerous attractive applications viz nanomolar sensitivity, tunable wavelength emission (460–630 nm), small size, specificity and deep-tissue-imaging capability [42]. An H₂O₂ sensor was developed based on the reaction of phenyl boronic acid with H₂O₂-ACPP (activatable cell-penetrating peptides) to detect H₂O₂ produced endogenously by macrophages, in a model of lung inflammation. The biosensor had an advantage of low micromolar sensitivity [43].

Nanoprobes

Jin H et al. 2010 optimized a new approach based on fluorescent SWCNT to understand the stochastic quenching events of H₂O₂ emission from epidermal growth factor (EGF)-stimulated human epidermal carcinoma cells. In response to EGF stimulation, local induction of 2 nM H₂O₂ in 50 min was done by this method [44]. Infrared fluorescent single-walled CNTs were used to investigate single-molecule efflux of H₂O₂ from human umbilical vein endothelial cells (HUVECs). As a result of angiogenic stimulation, a calibration curve of 12.5–400 nM H₂O₂ detection was obtained. The method has impressive sensitivity to nanomolar H₂O₂ detection with 300 nm spatial resolutions [45]. A new luminescent assay using iron oxide magnetic nanoparticles (MNPs) as peroxidase mimetics was designed by Wei and Wang [46]. The developed sensor exhibited strong sensitivity, as shown by its LOD as low as 3 × 10⁻⁶ mol/L with a linear range of 5 × 10⁻⁶ and 1 × 10⁻⁴ mol/L [46].

Emerging detection techniques for H₂O₂

A number of distinctive drawbacks exist in the above conventional H₂O₂ detection methods. Therefore, several efficient and convenient techniques were developed for the rapid detection of H₂O₂ in different samples. In the literature, various methods were reported for the electrochemical estimation of H₂O₂. Table 2 summarizes the reported electrochemical methods revealing the electrode used, immobilization method, linearity, working pH and potential, sensitivity, LOD and their applications.

Table 2 Comparison of different analytic parameters of present H₂O₂ biosensor with those of earlier biosensors

Biosensors

Biosensor is an analytical device that combines biological elements/molecules with microelectronic transducer devices to measure the concentration of analyte. It converts the biological response into electrical signals. An ideal biosensor must be highly specific, selective, accurate, reproducible and independent of interfering components. Biosensors can be classified into enzymatic and nonenzymatic biosensors according to the composition of the bio-recognition element [47]. H₂O₂ biosensors can be classified as follows (Fig. 2):

Fig. 2

Basic principle of biosensor

Potentiometric H₂O₂ biosensors

Potentiometric biosensors measure the electric potential of an electrode at zero current, which is actually the difference in potential between the working and standard electrode. These biosensors are based on the ion-selective electrodes to detect the target-specific ions during the biological reactions. In potentiometric biosensors, enzyme is immobilized onto the surface of electrode through glutaraldehyde crosslinking or adsorption process. The probe of pH meter is surrounded by the membrane, where biological reaction either produces or assimilates hydrogen ions. The variation in hydrogen ions causes a change in pH, which is measure of the concentration of analyte [48]. These potentiometric biosensors could be classified as follows depending on the type of electrode used:

Nafion membrane/Pt electrode

Parrilla et al. [49] used Nafion membrane/Pt electrode for potentiometric detection of H₂O₂. Nafion membrane has advantage of effective permselective barrier, which reduces the response to some redox-active species such as ascorbate. The coupling between redox potential on Pt electrode and Donnan potential plays a role in enhancement of sensitivity to H₂O₂. The current H₂O₂ biosensor showed sensitivity as 125.1 ± 5.9 mV/decade, linear range as 10–1000 µM and LOD as 10 µM [49].

MnO₂ doped/carbon paste electrode (CPE)

Manganese oxide (MnO₂)/carbon paste electrode was fabricated by Zheng [50]. The biosensor exhibited the sensitivity as 21–19.4 mV/decade, linear range as 0.3–363 µM and LOD as 0.12 µM. The analytical parameters of this biosensor were better than the latest potentiometric biosensor based on Nafion membrane/Pt electrode [49] except the sensitivity. It might be due to the use of MnO₂ and CPE-based electrode, as they both enhanced the surface area of the electrode and thus linear range and LOD. However, high sensitivity of Nafion membrane/Pt electrode was due to coupling between redox potential on Pt electrode and Donnan potential [50].

Amperometric H₂O₂ biosensor

Amperometric biosensors are based on generation of a current at a fixed potential applied between two electrodes. Like potentiometric biosensors, amperometric biosensors also exhibited analytical parameters such as response times, linear ranges, selectivity, reproducibility and sensitivities. The conventional amperometric biosensors were based on the Clark O₂ electrode. The electrode contained Pt cathode and Ag/AgCl standard electrode. The reduction of O₂ took place at Pt cathode vs Ag/AgCl as reference electrode. When a fixed potential relative to the reference electrode (Ag/AgCl) is tested on the working electrode (Pt cathode), it allows to generate current, which is directly proportional to the concentration of O₂. Normally both electrodes are kept into the concentrated KCl and kept them away from the bulk solution by employing O₂-permeable membrane onto them. The O₂ reduced at Pt cathode creates O₂ concentration effectively zero. Therefore, the degree of electrochemical reduction depends on the diffusion capacity of O₂ from the bulk solution [51].

Amperometric biosensors are further classified on the basis of generations used:

DO-metric first generation H₂O₂ biosensor

In dissolved oxygen (DO) metric biosensor, hemoglobin (Hb) protein has been immobilized onto the clark electrode surface for detection of H₂O₂. Hb molecule has four electroactive iron heme molecules, which enhance the electron transfer kinetics of heme proteins and help in biosensing application. The principle of biosensor is based on the two factors i.e., electrocatalytic activity of Hb and H₂O₂ reduction during measurement. In the present biosensor, Hb was immobilized onto Teflon membrane via glutaraldehyde crosslinking chemistry [52].

Second-generation H₂O₂ biosensor

An H₂O₂ biosensor, based on modified Au electrode, was fabricated by Kafi et al. 2007. They immobilized Hb onto Au electrode by electrochemical polymerization with o-PD. The fabricated biosensor exhibited good kinetic response, because of the direct immobilization of Hb. It also enhanced the sensitivity and selectivity of biosensor. The amperometric detection of H₂O₂ was carried out at − 300 mV in 0.1 M phosphate buffer solution (PBS) (pH 6.0). The biosensor showed a fast amperometric response towards H₂O₂. The levels of the relative standard deviation (RSDs) (< 3.5%) for the entire analysis reflected a highly reproducible sensor performance. Under the optimized conditions, LOD of the biosensor was 0.1 µM and linear range was from 5 to 125 µM. In addition, the sensor showed long-term stability and good sensitivity [53].

A sensitive amperometric HRP-based biosensor was fabricated via the deposition of AuNPs onto a three-dimensional (3D) porous carbonized chicken eggshell membrane (CESM). Due to the synergistic effects of the unique porous carbon architecture and well-distributed AuNPs, the enzyme-modified electrode showed an excellent electrochemical redox behavior. Compared with bare GC electrode, the cathodic peak current of the enzyme electrode was increased 12.6 times at a formal potential of − 100 mV (vs. SCE) but charge-transfer resistance decreased to 62.8%. Additionally, the AuNPs-CESM electrode exhibited a good biocompatibility, which effectively retains its bioactivity with a surface coverage of HRP 6.39 × 10⁻⁹ mol cm⁻² (752 times higher than the theoretical monolayer coverage of HRP). Furthermore, the HRP-AuNPs-CESM-GC electrode, s had a good accuracy and high sensitivity with the linear range of 10–2700 µM H₂O₂ and the LOD of 3 μM H₂O₂ (S/N = 3) [54].

A simple and low-cost H₂O₂ biosensor was developed using a Yucca filamentosa plant leaf membrane in conjunction with an O₂ sensor. The leaf membrane contained H₂O₂ peroxidase, which decomposed H₂O₂ to produce O₂. The response rate was faster for a Yucca membrane (t ₉₀ response time is ≈ 14 s) than a Yucca membrane with the O₂-permeable membrane (t ₉₀ response time is ≈ 200 s) and the sensitivity was much improved. The biosensor exhibited an excellent linear calibration range from 85 μM to 3750 µM M, H₂O₂ (correlation coefficient r = 0.9999) with a LOD of 15.1 μM and repeatability (1.024%, n = 10). The effects of pH and temperature on the response of the H₂O₂ biosensor were studied in detail. The working life-time of the biosensor was good, as it retained 86.7% of its initial sensitivity at 25 °C even after 2350 determinations of H₂O₂ sample solutions. It was successfully applied for determination of H₂O₂ concentration in some commercial samples [55].

A simple and low-cost H₂O₂ biosensor was fabricated using a beef liver catalase-immobilized eggshell membrane and a dissolved O₂ electrode. Catalase from beef liver extract was covalently immobilized on an eggshell membrane and subsequently covered the surface of an O₂ electrode. The detection scheme was based on the increase in dissolved O₂ content upon exposure to H₂O₂ solution. The increase in O₂ level was then monitored and related to the H₂O₂ concentration. The effects of enzyme loading, dissolved O₂ content, pH, phosphate buffer concentration and temperature on the biosensor were also studied. The response and recovery times of the biosensor (t95) were 1 min, respectively. The detection limit based on 3σb was 3 μM and the relative standard deviation of the response was 1.28% (n = 10) for a solution containing 300 µM of H₂O₂. The H₂O₂ biosensor demonstrated a reasonable long and stable shelf-life. It was successfully applied to the determination of H₂O₂ concentration in some commercial samples [56].

Overall, second generation biosensors exhibited linear range between 5 and 3750 µM and LOD between 0.1 and 15.1 µM. Kaafi et al. 2007 exhibited better LOD as compared to other biosensors due to direct immobilization of Hb onto Au electrode, which enhanced the electrochemical performance of biosensor. However, Yucca membrane-based H₂O₂ biosensor showed wider linear range, due to high catalytical efficiency of Yucca membrane.

Third-generation H₂O₂ biosensor

Nanomaterial electrode-based biosensors are further classified as membrane-based and electrode-based biosensors.

Electrode composition based biosensor

Electrode-based biosensors can be classified on the basis of nanocomposites used in their synthesis:

MWCNTs-nanoNiO/glassy carbon (GCE) A biosensor based on catalase/MWCNTs-nanoNiO composite/GC electrode was employed for detection of H₂O₂. The immobilized catalase displayed excellent electrocatalytic activity towards the reduction of H₂O₂. The developed amperometric biosensor expressed a wider linear range, 200–2.53 mM and LOD of 19.0 µM. The electrochemical impedance measurements revealed that the charge transfer resistance was dropped significantly after enzymatic reaction of nanobiosensor with H₂O₂. The biosensor was highly sensitive to H₂O₂ over a linear range of 19–170 nM with a LOD of 2.4 nM [57].

La _0.6 Sr _0.4 CoO _3−δ (LSC) perovskite oxide/-reduced graphene oxide (RGO) GC electrode La_0.6Sr_0.4CoO_3−δ (LSC) perovskite oxide/-reduced graphene oxide(RGO)-based enzymeless biosensor was designed for detection of H₂O₂. This biosensor overcame the problems of enzyme-based biosensor, as LSC was not sensitive to the pH, temperature, humidity and poisoning chemicals. Moreover, LSC offered high electroactive properties towards the catalytic function of H₂O₂. Additionally, RGO could be added to optimize the analytical performance of biosensor. The working electrode LSC /RGO/GCE exhibited high sensitivity of 500 μA mM⁻¹ cm⁻² for H₂O₂ and LOD of 0.05 μM, respectively (at S/N = 3). The biosensor showed linearity in the concentration range from 0.2 to 3350 μM [58].

Electronic-reduced graphene oxide (ERGO)/GCE A simple, facile and reproducible non-enzymatic H₂O₂ sensor was developed using electrochemically reduced graphene oxide (ERGO)-modified GC electrode. The modified GC electrode was characterized by FTIR, UV–Visible spectra, SEM and atomic force microscopy (AFM). Cyclic voltammetric (CV) analysis revealed that ERGO/GCE exhibited virtuous charge transfer properties for a standard redox systems and showed excellent performance towards electro-reduction of H₂O₂. Amperometric study using ERGO/GCE exhibited high sensitivity (0.3 μA/μM) and faster response upon the addition of H₂O₂ at an applied potential of − 0.25 V vs Ag/AgCl. The LOD was 0.7 μM (S/N = 3) and the time to reach a stable steady state current was < 3 s for a linear range of H₂O₂ concentration (1–16 μM). In addition, the modified GC electrode had good reproducibility and long-term stability [59].

Prussian blue (PB)/ruthenium oxide hexacyanoferrate/GCE A sensor for amperometric detection of H₂O₂ based on Prussian blue (PB)/ruthenium oxide hexacyanoferrate/GC electrode was developed. The electrocatalytic process allowed the determination of H₂O₂ at 0.0 V with a LOD of 1.3 μmol L⁻¹ in a flow-injection analysis (FIA) configuration. Under optimal FIA operational conditions, the linear response of the method was extended up to 500 μmol L⁻¹ H₂O₂ with good stability. The possibility of using the developed sensor in medium-containing sodium ions and the increased operational stability constitute advantages in comparison with PB-based amperometric sensors. The usefulness of the methodology was demonstrated by addition-recovery experiments with rainwater samples and values were in the 98.8–103% range [60].

Cobalt phthalocyanine-modified boron-doped diamond electrode A cobalt phthalocyanine-modified boron-doped diamond (CoPc-BDD) electrode was fabricated. The hydrogen-terminated BDD electrode surface was terminated with the pyridine moieties via photochemical modification with 4-vinylpyridine (4VP), followed by immobilization of CoPc on the surface by immersion of the 4VP-BDD samples in a CoPc solution. X-ray photoelectron spectroscopy studies suggested that coordination of the surface pyridine to the Co atom in CoPc contributes to the surface modification. Electrochemical detection of hydrogen peroxide at the modified electrode was also investigated. Cyclic voltammetry confirmed that the CoPc-BDD electrode exhibits catalytic activity for the electrochemical oxidation of H2O2. Using a flow-injection analysis-electrochemical detection (FIA-EC) system, CoPc-BDD electrode was showed following analytic parameters: Linear range: 10 nM–10 μM, LOD: 10 nM, working potential: − 0.6V [61].

Hb [pluronic P123-nanographene platelet(NGP)] electrode An amperometric H₂O₂ biosensor was fabricated by immobilizing Hb on a pluronic P123-nanographene platelet (NGP) composite. The surface concentration (Γ*) and apparent heterogeneous electron transfer rate constant (k_s) were 1.60 ± 0.17 × 10⁻¹⁰ mol cm⁻² and 48.51 s⁻¹, respectively. In addition, the Hb/Pluronic P123-NGP composite showed excellent bio-electrocatalytic activity toward the reduction of H₂O₂. The biosensor exhibited a linear response to H₂O₂ in the range of 10–150 μM and a LOD of 8.24 μM (S/N = 3) at 0.20 V, pH-7.0 and 25 °C. The apparent Michaelis–Menten constant (K _mapp) was 45.35 μM .The resulting biosensor showed fast amperometric response, with very high sensitivity, reliability and effectiveness [62].

Electrospun Hb–collagen composite A hemoglobin (Hb)–collagen microbelt-modified electrode with three-dimensional configuration was fabricated via the electrospinning method. Direct electron transfer of the Hb immobilized into the electrospun collagen microbelts was greatly facilitated. The apparent heterogeneous electron transfer rate constant (k_s) was calculated to be 270.6 s⁻¹. The electrospun Hb–collagen microbelt-modified electrode showed an excellent bioelectrocatalytic activity toward the reduction of H₂O₂ at pH 7.0 and 20 °C. The amperometric response of the biosensor varied linearly with the H₂O₂ concentration ranging from 5 × 10⁻⁶ mol L⁻¹ to 30 × 10⁻⁶ mol L⁻¹, with a LOD of 0.37 × 10⁻⁶ mol L⁻¹ (signal-to-noise ratio of 3). The K _mapp was 77.7 μmol L⁻¹. The biosensor exhibited fast amperometric response, high sensitivity, good reproducibility and stability [63].

Hb/Ag sol films/GCE A novel amperometric electrochemical H₂O₂ biosensor was fabricated by immobilizing hemoglobin–silver (Hb–Ag) sol onto GC electrode, which showed a sensitive response to the reduction of H₂O₂ without any electron mediator. Ultraviolet–visible (UV–vis) spectra and reflectance absorption infrared (RAIR) spectra of Hb-Ag/GC suggested that Hb in Hb–Ag sol retained its native secondary structure. Scanning electron microscopy (SEM) demonstrated that the morphology of the Hb film was much different from the Hb–Ag sol film. The Hb–Ag film exhibited a good electrocatalytic activity for the reduction of H₂O₂. Under optimum conditions (at − 0.4V and pH 7.0), the biosensor exhibited linearity for H₂O₂ in the concentration range 1–25 μM and LOD as 0.1 μM at 3σ. The biosensor exhibited high sensibility, good reproducibility, and long-term stability [64].

Gold nanoparticles (AuNPs)/cysteine (L-Cys)/poly(p-aminobenzene sulfonic acid) (ABSA)-based platinum disk electrode An H₂O₂ biosensor was fabricated by self-assembling Hb, nano-Au and L-cysteine (L-cys) on the precursor film formed by electropolymerization of p-aminobenzene sulfonic acid (p-ABSA) on the Pt disk electrode. The EIS and UV–Vis absorption spectroscopy of Hb/AuNPs/L-cys/p-ABSA were carried out to characterize the fabrication process. The biosensor response and factors influencing the biosensor were studied by CV and chronoamperometry. The immobilized Hb showed direct electrochemical behavior toward the reduction of H₂O₂. The biosensor exhibited linear range as 0.21–31 μM and LOD as 0.07 μM (S/N = 3) at 0.1 V, pH: 7.0 and 25 °C. In addition, the biosensor exhibited good accuracy and high sensitivity [65].

Hb–collagen-CNTs An Hb–collagen-CNTs nanofibers modified electrode was constructed by incorporating CNTs into the composite of Hb and collagen, using co-electrospinning technology. The formed Hb–collagen-CNTs composite nanofibers possessed distinct advantage of three-dimensional porous structure, biocompatibility and excellent stability. Hb immobilized in the electrospun nanofibers retained its natural structure .The heterogeneous electron transfer rate constant (k _s) of the direct electron transfer between Hb and electrodes was 5.3 s⁻¹. In addition, the electrospun Hb–collagen-CNT nanofiber-modified electrodes showed good electrocatalytic properties toward H₂O₂ with a linear concentration range, 5–30 μM and LOD of 0.91 μM (S/N = 3) and K _mapp of 32.6 μM at − 0.365 V and pH 7.0 [66].

DNA/Hb/Au A H₂O₂ biosensor was designed by drop-letting of DNA and Hb onto Au electrode surface layer by layer. The sensor based on the direct electron transfer of iron of Hb showed a well electrocatalytic response to the reduction of the H₂O₂. This sensor offered an excellent electrochemical response for H₂O₂ concentration below micromole level with high sensitivity and selectivity and short response time. The levels of the RSD’s (< 5%) for the entire analyses reflected the high reproducibility of the sensor. Using the optimized conditions (potential − 0.75 and pH 5.0), the linearity for H₂O₂ detection was 10–120 μM with LOD of 0.4 μM (S/N = 3) [67].

Platinum nanoparticles (PtNPs)/reduced graphene oxide (RGO)–chitosan (CS)–ferrocene (Fc) carboxylic acid nano-hybrids (Pt NPs/RGO-CS-Fc) A highly sensitive non-enzymatic electrochemical sensor based on platinum nanoparticles/reduced graphene oxide–chitosan–ferrocene carboxylic acid nano-hybrids (Pt NPs/RGO-CS-Fc biosensor) was developed for the measurement of H₂O₂. The RGO-CS-Fc nano-hybrids were characterized by UV–Vis spectrum, FTIR spectroscopy, TEM, Raman spectrometry and EIS. Under optimal experimental conditions, the Pt NPs/RGO-CS-Fc biosensor showed outstanding catalytic activity toward H₂O₂ reduction. The biosensor showed current response at − 0.05 V and 37 °C, a linear relationship with H₂O₂ concentration from 2.0 × 10⁻⁸ to 3.0 × 10⁻⁶ M with a correlation coefficient (R ²) of 0.9968 and with log H₂O₂ conc. from 6.0 × 10⁻⁶M to 1.0 × 10⁻² M with a correlation coefficient (R ²) of 0.9887, the LOD of 20 nM was obtained at the signal/noise (S/N) ratio of 3. Moreover, the Pt NPs/RGO-CS-Fc biosensor exhibited excellent anti-interference capability and reproducibility for the detection of H₂O₂. The biosensor was successfully applied for detection of H₂O₂ from living cells containing normal and cancer cells. All these results proved that the Pt NPs/RGO-CS-Fc biosensor had the potential application in clinical diagnostics to evaluate oxidative stress of different living cells [68].

Dipeptide–AuNP/HRP/GCE A mediate H₂O₂ biosensor was developed based on HRP/dipeptide–AuNP hybrid spheres modified GC electrode. The self-assembled diphenylalanine (FF) or dipeptide(DP)–AuNPs hybrid microspheres with a hollow structure were prepared in aqueous solution by a simple one-step method. The TEM and SEM images of DP-AuNPs hybrid and working electrode showed that formed AuNPs were localized both inside and on the surface of DP spheres. HRP as a model enzyme was further immobilized onto DP–AuNPs hybrid spheres to construct a mediate H₂O₂ amperometric biosensor. UV–Vis spectroscopic study revealed that the immobilized HRP retained its original structure. CV study of HRP/dipeptide–AuNP hybrid spheres/GC electrode showed high electrocatalytic activity to H₂O₂. The biosensor exhibited a wide linear range of 0.05–300 μM with a high sensitivity of 28.3 µA mM⁻¹ and LOD of 0.1 μM (S/N = 3) at 0.05 V and pH 7.0. The biosensor exhibited good reproducibility and long-term stability. Thus, dipeptide-AuNP hybrid sphere was proved to be a promising matrix for application in the fabrication of electrochemical biosensors, due to its excellent biocompatibility and good charge-transfer ability [69].

Graphene capsules (GRCAPS)/horseradish peroxidase (HRP)/indium titanium oxide (ITO) To solve the problems of enzymatic loss and inactivation of the biosensor, HRP was initially encapsulated in graphene capsules (GRCAPS) using porous CaCO₃ as sacrificial templates to mimic the existence form of bio-enzymes in the organisms, and then GRCAPS and graphene-poly (sodium 4-styrenesulfonate) were assembled onto the substrate of indium tin oxide (ITO) for constructing multilayer films of the biosensor. TEM and field-emission SEM analyses revealed that GRCAPS and multilayer films were prepared. The resulting biosensor showed a wide linear range of 0.01–12 μM and, LOD of 3.3 μM (S/N = 3), excellent anti-interference ability, and long-term stability under optimum condition at − 0.45V, pH 7.0 and 37 °C [70].

HRP/poly (aniline-co-N-methylthionine) (PAN-PNMThH) A H₂O₂ biosensor was fabricated using electrochemical doping to immobilize HRP in a new conducting polymer, poly(aniline-co-N-methylthionine) (PAN-PNMThH). Amperometric detection of H₂O₂ was evaluated by holding the PAN-PNMThH HRP electrode at − 0.25 V [versus saturated calomel electrode (SCE)]. PAN-PNMThH showed excellent redox activity and high porosity and acted as an electron transfer mediator. The biosensor showed a wide linear range from 5.0 μM to 60.0 mM H₂O₂ with a sensitivity of 35 mA M⁻¹ cm⁻², a LOD of 3.2 μM (S/N ratio of 3) and a K_mapp of 2.79 mM at pH 6.0. The biosensor had good analytical performance and storage stability [71].

3,3′,5,5′-teramethyl bencidine (TMB)/polydimethylsiloxane (PDMS)/tetraethyl orthosilicate (TEOS)/silicon oxide nanoparticles (SiO ₂ NPs) A H₂O₂ biosensor was fabricated based on the co-immobilization of the reagent 3,3′,5,5′-teramethylbenzidine (TMB) and HRP in a PDMS-TEOS-SiO₂NPs support to enhance the performance of colorimetric biosensors. The HRP, in presence of H₂O₂, catalyzes the oxidation of TMB, producing a blue color. The generated biosensor, doped with the substrate (TMB) and HRP (entrapped or adsorbed), was used to determine H₂O₂ in real samples. Firstly, the immobilization of TMB and HRP in the composite was studied to find the best suitable configuration. The kinetic parameters i.e., V _max and K _mapp of the different assayed systems were n determined and compared. Second, the analytical properties of the H₂O₂ method were studied. The biosensor was simple, inexpensive, highly sensitive and selective for determination of H₂O₂, with LOD of 1.3 μM and a good linearity in the range 4.2–72 μM at pH 7.0. The LOD could be improved to 0.4 μM by acidifying the solution with sulphuric acid. The relative standard deviation (RSD) was < 10%. The biosensor had a reagent-release support, which significantly simplified the analytical measurements, as it avoided the need to prepare derivatization reagents and sample handling. It allowed in situ measurements [72].

Turnip tissue paper (TTP)/screen-printed carbon electrode (SPCE) A novel inexpensive turnip tissue paper-based mediated amperometric H₂O₂ biosensor was developed based on screen-printed carbon electrodes (SPCEs). The use of cellulose paper proved to be an “ideal” and simple biocompatible immobilization matrix for raw turnip peroxidase as it was successfully embedded within the fiber matrix of paper via physical adsorption. The mediator potassium hexacyanoferrate (II) was also embedded onto the paper matrix together with the raw enzyme. The biosensor allowed a minute amount (0.5 μL) of sample solution for analysis. The biosensor had a linear range as 20–500 μM, LOD as 4.1 μM, at − 0.1 V, pH, 7.0, correlation coefficient (R ²) as 0.999, retention capacity as 70% of initial activity and storage stability—25 days at 4 °C. The biosensor was employed for analysis and the results matched with the classical titration method [73].

Graphene-supported platinum nanostructure (GPtNPs) A facile and highly sensitive platform was developed for synthesizing branched Pt nanostructures on graphene. Graphene support was employed to increase the performance of platinum nanostructures (PtNs) in electrochemical sensing/biosensing. The graphene-supported PtNs (GPtNs)-modified electrode efficiently oxidises the H₂O₂ at a lower potential in neutral phosphate buffer without employing the enzymes or redox mediators. The GPtNs electrode exhibited high sensitivity of 811.26 μAmM⁻¹ cm and linear range, 0–0.32 mM, with a very low LOD (1 nM) for H₂O₂ at potential 0.45 V and pH 7.0. The sensor showed excellent reproducibility, long-time storage and operational stability. It measured H₂O₂ concentration in the real samples (rainwater) [74].

Platinium–ruthenium (PtRu)/three-dimensional graphene flakes (3DGF) An amperometric H₂O₂ biosensor was developed exploiting the large surface, excellent dispersion and high degree of sensitivity of bimetallic nanocatalysts. To achieve it, graphene foam (GF), a three-dimensional (3D) porous architecture consisting of extremely large surface and high-conductive pathways was incorporated into platinum–ruthenium (PtRu) bimetallic nanoparticles, as an electrochemical nanocatalyst for detection of H₂O₂. PtRu/3D GF nanocatalyst brought electrochemical oxidation of H₂O₂ without any additional mediator showing a high sensitivity (1023.1 µA mM⁻¹cm⁻²) and a LOD of 0.04 µM for H₂O₂ at 0.2 V and pH 7.4. The amperometric results revealed that GF provided a promising platform for the development of electrochemical sensors in biosensing and PtRu/3D GF nanocatalyst possessed the excellent catalytic activity toward the H₂O₂ detection. A small particle size and a high degree of the dispersion in obtaining of large active surface area were important for the nanocatalyst for the best H₂O₂ detection in biosensing. Ascorbic acid and uric acid had practically no effect [75].

Cytochrome C (Cytc)/nickel oxide nanoparticles (NiONPs)/multiwalled carboxylated carbon nanotubes (c-MWCNT)/polyaniline/Au An amperometric H₂O₂ biosensor was constructed based on covalent immobilization of Cytochrome c onto nickel oxide nanoparticles/carboxylated multiwalled carbon nanotubes/polyaniline composite (NiO-NPs/cMWCNT/PANI) electrodeposited onto Au electrode. The modified Au electrode was characterized by CV, EIS, SEM and FTIR spectroscopy. CV studies of the electrode at different stages demonstrated that the modified Au electrode had enhanced electrochemical oxidation of H₂O₂, which offered a number of attractive features to develop an amperometric biosensor based on split of H₂O₂. There was a good linear relationship between the current (mA) and H₂O₂ concentration in the range 3–700 μM at − 0.28 V, pH 6.5 and 30 °C. The sensor had a LOD of 0.2 μM (S/N = 3) with a high sensitivity of 3.3 mA μM⁻¹ cm⁻² .It measured accurately the level of H₂O₂ in different fruit juices [76].

HRP/toluidine/graphite powder/rigid ceramic HRP and water-soluble mediator toluidine blue were immobilized covalently onto 3-aminopropyl trimethoxy silane precursor through glutaraldehyde crosslinker. A rigid ceramic composite electrode was constructed from this modified silane along with graphite powder, which caused electrochemical oxidation of H₂O_2, as proved by CV studies in the potential range, 0.2V to -0.4V vs SCE. The biosensor showed a stable voltammogram with cathodic peak at − 0.234 V and anodic peak at − 0.172 V, with a formal potential of − 0.203 V. The biosensor was optimized. The biosensor exhibited linearity in the range, 0.429 μM–0.455 mM with a LOD of 0.171 μM at − 0.25 V, pH 7.0 and 30 °C. The biosensor was robust for long-term usage besides showing the high sensitivity, rapid response and an advantage of surface renewability by simple mechanical polishing [77].

Hb/dipeptide graphene nanostructure/GCE Hb was immobilized onto self-assembled nanowires of diphenylalanine (FF) and graphene (G) nanocomposite-modified GC electrode to fabricate an amperometric H₂O₂ biosensor. The immobilized Hb, retained its original structure and bioactivity, as revealed by UV spectroscopy. The Hb/FF-G/GC electrode exhibited high electrocatalytic activity to H₂O₂, with wider linearity in the range, 5.0 × 10⁻⁷ to 5.0 × 10⁻⁴ mol L⁻¹, with a LODf 1.0 × 10⁻⁷ mol L⁻¹ at pH 7.0. These results indicated the excellent biocompatibility and good charge-transfer ability of nanowires of FF-G nanocomposites [78].

Catalase/bilirubin oxidase/multi-copper enzyme/carbon nanotube-modified electrodes (catalase/box/multicopper enzyme/GCE) A highly sensitive and selective amperometric H₂O₂ biosensor was developed based on adsorption of catalase (Cat) and either laccase (Lac) or bilirubin oxidase (BOX) onto MWCNT-modified GC electrode. The stability and durability of the electrode was improved by glutaraldehyde crosslinking of enzymes. CV and chrono-amperometry proved the synergy of the laccase and catalase co-adsorbed on MWCNT. Catalase catalyzed the transformation of H₂O₂ into H₂O and O₂, which was further changed into water by multi-copper enzymes (MCO), either laccase or bilirubin oxidase. The unique property of such a bienzymatic sensing layer was able to detect O₂ originating both from catalase activity and the self-decomposition of H₂O₂. This helped in evaluating the initial concentration of H₂O₂ in the analyzed sample [79].

Platinum nanoflower and graphene modified indium titanium electrode (PtNF/N-graphene/ITO) The nitrogen-doped graphene (N-graphene) was synthesized by annealing graphene oxide with urea at 900 °C. A non-enzymatic H₂O₂ biosensor was fabricated by a simple layer-by-layer electrophoretic and electrochemical sequential deposition of nitrogen-doped graphene (N-graphene) and Pt nanoflower (Pt NF) with different N-graphene loadings, onto ITO-coated glass plate. The structure and morphology of PtNF/N-graphene/ITO electrode were studied by XRD field emission electron microscopy (FEEM), TEM, Raman and X-ray photoelectron spectra. The Pt NF-N-graphene-modified ITO electrodes with different N-graphene loadings were used as a non-enzymatic electrode for detection of H₂O₂. The Pt NF-N-graphene-modified ITO electrode with a 0.05 mg ml⁻¹ N-graphene loading showed the linearity in the range, 1–1000 μM with LOD as 340 μM at pH 7.2 and − 0.4 V. It also exhibited excellent stability and reproducibility for non-enzymatic H₂O₂ detection because of the synergistic effect between the electrocatalytic activity of Pt NF and the high conductivity and large surface area of N-graphene [80].

Gold nanoparticle-decorated silver-bipyridine nanobelts (AuNPs/bipyridine nanobelts/GCE) AuNPs modified with 4-mercaptopyridine and 6-mercapto-1-hexanol were employed as coordination agents to prepare a novel hybrid nanomaterial with Ag:4,4′-bipyridine nanobelts. This nanohybrid was electrodeposited onto GC electrodes. A mediatorless amperometric biosensor for H₂O₂ was constructed by immobilizing HRP onto this modified GC electrode. The electrode showed a rapid response within 4 s at − 0.1 V and pH 7.0 and a linearity in the range, 90 pM–6.5 nM with a LOD of 45 nM (S/N = 3.0) for H₂O₂. The biosensor had a high sensitivity of 283 A/M cm². The enzyme electrode retained 96 and 78% of its initial activity after 15 and 30 days of storage at 4 °C, respectively [81].

Metal-organic framework-based electrode A new Co-metal-organic framework [Co(pbda)(4,4-bpy)·2H₂O]n Co-MOF] [bpy = 4,4-Bipyridine; H₂pbda = 3-(pyridine-3-yloxy)benzene-1,2-dicarboxylic acid] or CoMOF was synthesized under hydrothermal conditions and then electrodeposited onto GC electrode to construct a high performance amperometric H₂O₂ sensor. Single-crystal X-ray analysis exhibited a three-dimensional supramolecular architecture with a uninodal 4-connected net with the gis topology. The CV of the Co-MOF-modified GC electrode showed a pair of reduction peaks at ca. − 0.40 V in 0.1 M NaOH solution corresponding to CoIII-MOF/CoII-MOF couple. The highly electrocatalytic activity of the Co-MOF-modified GC electrode on H₂O₂ reduction showed a wide linear range from 5 μM to 9.0 mM, a low detection limit of 3.76 μM and a high sensitivity of 83.10 μA/mM cm² at an applied potential of − 0.40 V. Further, the sensor had favorable selectivity and long-term stability. The Co-MOF possessed highly efficient intrinsic peroxidase-like activity to produce hydroxyl radical from H₂O_2, which react with oxidized peroxidase substrate (terephthalic acid) to produce color [82].

Porphyrinic iron-based metal-organic framework/ordered mesoporous carbon (pFeMOF/OMC) A novel hybrid of porphyrinic iron metal-organic framework (pFeMOF) and ordered mesoporous carbon (OMC) was synthesized via a simple one-step hydrothermal method. This hybrid was electrodeposited onto GC electrode for non-enzymic amperometric determination of H₂O_2, released from viable cells. The pFeMOF/OMC hybrid materials were synthesized Fe (III) ion could coordinate with carboxylates of porphyrin groups strongly, leading to more stable MOFs. As pFeMOF mimic peroxidase property, the electrode gave amplified electrochemical signal. The carbon skeleton of OMC revealed a function of restriction for the growth of pFeMOF crystallites, resulting in more active sites to reduce H₂O₂. The increased amount of mesoporous brings faster diffusion. Moreover, the electrical conductivity and stability was improved due to the introduction of OMC. The electrocatalytic reduction of H₂O₂ displayed two segments linearity range from 0.5 to 70.5 μM and 70.5 to 1830.5 μM, with high sensitivity of 67.54 μAmM⁻¹ in low concentration range and 22.29 μAmM⁻¹ of high concentration. The LOD was as low as 0.45 μM. The pFeMOF/OMC/GC electrode showed outstanding property to resist interference, long-term stability and repeatability. Due to these excellent analytical performances, the sensor was able to detect H₂O₂ satisfactorily released from living cells [83].

Carbon paste electrode modified with nano-composite of reduced graphene oxide and CuFe ₂ O ₄ nanoparticles A highly sensitive voltammetric H₂O₂ sensor (RGO/CuFe₂O₄/CPE) was developed by modifying a carbon paste electrode(CPE) with reduced graphene oxide (RGO) and CuFe₂O₄ nanoparticle CuFe₂O₄ nanoparticles were synthesized by co-precipitation method and characterized by SEM, TEM, XRD, and FTIR. The electrocatalytic reduction of H₂O₂ was measured by CV chronoamperometry, amperometry and differential pulse voltammetry (DPV). Under the optimum conditions (pH 5.0), the modified CP electrode showed a fast amperometric response within < 2 s, a good linear range of 2–200 μM, LOD of 0.52 μM for H₂O₂. The current obtained through DPV, was increased linearly with increase in H₂O₂ concentration in the ranges, 2–10 μM and 10–1000 μM with LOD as 0.064 μM The sensor measured H₂O₂ level in biological and pharmaceutical samples such as milk, green tea, and hair dye cream and mouthwash solution [84].

Carbon-supported Cu@Pt core-shell nanoparticles A nanocomposite of carbon-supported Cu@Pt/C core-shell nanoparticles was synthesized via two-step reduction method. The electrochemical sensor based on Cu@Pt/C showed better electrocatalytic activity for the reduction of H₂O₂ than that based on Pt/C. The Cu@Pt/C sensor had a wide linear range between 0.50 μM and 32.56 mM, with a high sensitivity of 351.3 μA mM⁻¹ cm⁻², and LOD of 0.15 μM (S/N = 3). The sensor exhibited excellent long-time stability, good reproducibility and acceptable selectivity [85].

Ratiometric fluorescent-based H₂O₂ biosensor

The ratiometric fluorescent biosensing of H₂O₂ is based on the facility of dLys-AgNCs ratiometric probe, assigned to the ability of lysozyme (dLys) to work as a stabilizing agent as well as fluorescence signal unit. In the presence of Fenton reagents, dLys-AgNCs emission at 640 nm was quenched by·OH, whereas the emission at 450 was improved as a result of·OH-induced oxidation of tyrosine in the lysozyme. This probe was applied for extreme sensitive determination of H₂O₂. The fluorescence changes of F450/F640 had wider linearity for H₂O₂ in the range, 0.8–200 μmol/L (R ² = 0.9993), with a LOD as 0.2 μmol/L. The dLys-AgNCs also had a sensitive response to variation of ·OH levels in living cells, which showed its promising application for studying ·OH-induced oxidative damage to proteins [86] (Fig. 3).

Fig. 3

Schematic representation of ratiometric fluorescent biosensing of hydrogen peroxide

Chemiluminescence-based H₂O₂ biosensor

The chemiluminescence-based H₂O₂ biosensor was fabricated using poly(ethylene-co-polyvinyl alcohol) (PVA-co-PE) membrane. The PVA-co-PE nanofibers (Av. diameter in the range from 200 to 500 nm) were modified by cyanuric chloride and 1,3-propanediamine and subsequently biotinylated, then HRP was immobilized onto this membrane. The HRP-immobilized PVA-co-PE nanofiber membrane showed high activity, efficiency, sensitivity and reusability, when used for the luminol-H₂O₂ reaction. The relative light unit (RLU) could run up to 5 × 10¹¹ µM, when the concentration of H₂O₂ was as low to 10⁻⁹ µM. In the biosensor, the HRP-immobilized onto the PVA-co-PE nanofiber membrane, which acted as potential chemiluminescence transducer [87].

Supercapacitor-based H₂O₂ biosensor

Tellurium nanoparticles (Te Nps)-based H₂O₂ biosensor exhibited specific capacitance (586 F/g) at 2 mA/cm² and 100 F/g at 30 mA/cm² with an excellent cycle life (100% after 1000 cycles). The biosensor exhibited better analytical performances such as, linear range of 0.67–8.04 μM for H₂O₂, high sensitivity (0.83 mA mM⁻¹ cm⁻²) with good correlation coefficient i.e., 0.995. The response time was < 5 s [88].

Nanoneedle-based H₂O₂ biosensor

A biosensor based on KNbO₃ nanoneedles (KNs) was constructed to catalyze H₂O_2, directly. The mechanism of detection was based on molecular orbital principles, with the development of σ-bonding within e _g orbital of surface niobium ions and surface-adsorbed oxygen-related intermediate species. It allows the direct electron transfer between HRP and electrode surface. The designed biosensor revealed high sensitivity of 750 μA mM⁻¹ cm⁻² and rapid response i.e. 1–2 s towards H₂O₂ [89].

Protein nanoparticle-based H₂O₂ biosensor

A new approach of construction of a nanomaterial based- but simple amperometric H₂O₂ biosensor was designed by Narwal et al. 2017. The biosensor was developed by covalent immobilization of haemoglobin nanoparticles (HbNPs) onto polycrystalline Au electrode. The working electrode exhibited optimum response at pH 6.5 and 30 °C. A linear range of 1–1200 μM with a LOD of 1.0 μM, 2 V against Ag/AgCl with rapid response time of 2.5 s. The HbNPs/AuE offered Vmax of 5.161 ± 0.1 μA cm⁻² with Michaelis–Menten constant Km of 0.1 ± 0.01 mM and sensitivity of 129 ± 0.25 μA cm⁻² mM⁻¹. The measured analytical recovery of biosensor was 98.01%. Within and between batch coefficients of variation (CV) were 3.16 and 3.36%, respectively. The biosensor showed a good correlation coefficient (r = 0.99) between standard method and present method. The HbNPs/Au electrode lost 10% of its initial activity after 90 days of its regular uses, when stored dry at 4 °C. The biosensor-measured H₂O₂ in blood of diabetic patients [90].

A comparison of various electrochemical H₂O₂ biosensors is summarized in the Table 2.

Technical challenges in detection of hydrogen peroxide

Lack of reliability, false-positive results, cost, portability, lower detection limit, interferences, and careful calibration are potential methodological challenges of non-electrochemical methods. This would likely to limit the use of these methods in electrochemical analyte detection. Its detection by voltammetry on carbon electrodes has proved to be more complex due to irreversible slow electron transfer kinetics. Commercial production/availability of simple, easy-to-use measurement devices as portable or pocket-sized analyzers is still challenging sensor technology. Traditional approaches to measure H₂O₂ in vivo suffers with multiple problems: (1) nonspecific probes that react with other reactive oxygen and nitrogen species (2) sensitivity issues related with production of H₂O₂ at low concentration and short half-life (3) rapid diffusion of H₂O₂ across membranes (4) detection of H₂O₂ levels requires deep tissue imaging techniques with high resolution.

Conclusion and future perspectives

This review article addresses the updated summary of electrochemical H₂O₂ biosensor. In conclusion, biosensing methods are better than conventional methods (colorimetry, titration, chromatography, spectrophotometry, fluorimetry, chemiluminescence) for determination of H₂O₂ as they are comparatively simple, sensitive, specific, and rapid and even can work with complete automation. The nanomaterial-based biosensor exhibited better analytical performances in terms of working potential (− 0.6–0.45), detection limit (0.00045–340 µM) and sensitivity (0.3 μAmp/mM/cm²) which was owing to the large surface area, high electrical and optical properties of nanomaterials. Among the electrochemical H₂O₂ biosensor, enzymes and proteins (HRP, catalase and Hb-based biosensors are dominating. The HRP/Hb based H₂O₂ biosensors was upgraded further with the use of nanoparticles of enzymes/proteins. Beside increased surface area protein nanomolecules also exhibit exceptional optical, electronic, mechanical and thermal properties [91]. Hence the future research could be focus on application of protein nanoparticles in construction of highly sensitive H₂O₂ biosensors. Attempt can also be made by designing electronic chip for the laboratory model of H₂O₂ biosensor, which could be used outside the laboratory. The development, optimization and characterization of microsensors can be done to ensure a successful transfer of the technology from in vitro proof of concept to in vivo analysis.