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ISSN: 2155-6210
Journal of Biosensors & Bioelectronics

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Hydrogen Peroxide Biosensors Based on Horseradish Peroxidase and Hemoglobin

A. J. Saleh Ahammad*

Centre for Advanced Research in Sciences, University of Dhaka, Dhaka-1000, Bangladesh

*Corresponding Author:
A. J. Saleh Ahammad
Centre for Advanced Research in Sciences
University of Dhaka
Dhaka-1000, Bangladesh
Tel: 880-2-966-1920-73
Fax: 880-2-861-5583
E-mail: [email protected]

Received Date: November 11, 2012; Accepted Date: December 08, 2012; Published Date: December 10, 2012

Citation: Saleh Ahammad AJ (2013) Hydrogen Peroxide Biosensors Based on Horseradish Peroxidase and Hemoglobin. J Biosens Bioelectron S9:001. doi: 10.4172/2155-6210.S9-001

Copyright: © 2013 Saleh Ahammad AJ. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Hydrogen peroxide; Biosensors; Electrochemical methods; Horseradish peroxidase; Hemoglobin


Hydrogen peroxide (H2O2) plays a pivotal role in various industrial applications [1]. It is an essential mediator in pharmaceutical, clinical, and environmental research. In addition, it is a byproduct of highly selective oxidases and an important contaminant in several industrial products and wastes [2]. Therefore, a reliable and economical method for the determination of H2O2 is of great significance.

Several methods, such as titrimetry [3], spectrometry [4], chemiluminescence [5], fluorimetry [6], chromatography [7], and electrochemical techniques [8,9], have been reported for this purpose. Among these techniques, the electrochemical techniques are preferable because of their simplicity, low cost, high sensitivity, and selectivity [10-16]. In particular, an amperometric biosensor is an attractive tool for the detection of H2O2. Other electrochemical methods such as cyclic voltammetry, linear sweep voltammetry and differential pulse voltammetry are also used for the development of H2O2 biosensor.

In the electrochemical methods, different types of modified electrodes have been used. Horseradish Peroxidase (HRP) enzyme is one of the mostly used materials for the modification of electrode. Different mediator such as ferrocene, hydroquinone, catechol, methylene blue, methylene green, nile blue, potassium hexacyanoferrate, thionine, toluidine blue etc. have been used with the enzyme modified electrodes. However, mediator-free HRP based biosensor was also reported. In this case, the direct electrochemistry of HRP enzyme plays vital role. Hemoglobin (Hb) protein is also used for the modification of electrodes. In most case, mediator is not necessary for protein modified electrodes. Enzyme free or protein free modified electrodes were also reported for the electrochemical method based H2O2 biosensor. Some other materials such as nanomaterials, conducting polymers, metal oxides, quantum dots, dendrimer, bilayer lipid membrane, kieselguhr membrane, ionic liquid, liquid crystal, etc. have been used with enzyme and protein modified electrodes. The sensitivity and selectivity of the H2O2 biosensor depends on how the electrodes are modified by different materials.

In this review, we have focused on the strategy of the surface modification with HRP and Hb, for the determination of H2O2 based on electrochemical methods.

Detection Mechanism of H2O2 Biosensor

The detection mechanism of H2O2 biosensor depends on how the electrode is modified and whether the mediator is used or not. Direct electron transfer between enzymes and electrodes is not easy and therefore, need to use mediator. The role of the mediator is to shuttle electrons efficiently between electrode and enzyme. In the presence of Mediator (M), the reaction mechanism of the H2O2 biosensor based on the HRP enzyme modified electrodes can be summarized as follows [17]:

H2O2+HRPred → H2O+HRPox



Net reaction: H2O2+2e+2H+→2H2O

First, H2O2 in the solution is reduced by the immobilized HRP. Then the reduced HRP is regenerated with the aid of the mediator, while the mediator itself is oxidized in the enzymatic reaction. Finally, the oxidized mediator is electrochemically reduced on the electrode, leading to an increase in the reduction current, as shown in figure 1.


Figure 1: Schematic diagram of the reaction sequence within the enzyme electrode.

Enzyme (HRP) or protein (Hb), in some modified electrodes, shows direct electrochemistry. In this case, the mediator is not necessary. In the presence of H2O2, the reduction current of voltammogram for the direct electron transfer of enzyme or protein generally increases due to the electrocatalytic behavior of immobilized enzyme or protein to the reduction of H2O2. Furthermore, the reduction peak current increases with increasing H2O2 concentration. The electro catalytic mechanism could be expressed as follows [18,19].

Enzyme/protein (reduced form) +H2O2→Enzyme/protein (oxidized form) +H2O

Enzyme/protein (oxidized form)+2e+2H+→ Enzyme/protein (reduced form) +H2O

Net reaction: H2O2+2e +2H+→2H2O

Enzyme (or protein) is efficiently converted to its oxidized form, which is then reduced at the electrode surface by the direct electron transfer. Therefore, the reductive current increased in the presence of H2O2

HRP based H2O2 Biosensors

Mediated HRP based biosensor

HRP is one of the most extensively studied and commonly used enzymes for the construction of H2O2 biosensors. It contains heme as a prosthetic group, which is the protein active site, along with the hemeiron Fe (III). It can catalyze the oxidation of a wide variety of substrates by H2O2. Moreover, the reduced form of HRP can be chemically reoxidized by H2O2. Generally, direct electron transfer between HRP and an electrode is difficult because the active sites of HRP are deeply buried in a thick protein shell, and because the large distance between the active sites and the electrode surface will slow down the electron transfer. Electron transfer via a mediator, however, is more effective for establishing an electrical connection between the redox centers and the electrode. Lin et al. [20] fabricated a H2O2 biosensor by immobilizing HRP on the methylene blue modified graphite electrode. Here, methylene molecule act as a mediator, which could shuttle electrons between immobilized HRP and graphite substrate. Both ferrocene [21] and carboxylic acid functionalized ferrocene [22], have been used as a mediator with the HRP for the construction of H2O2 biosensor. Wang et al. [23] used potassium hexacyanoferrate with the peroxidase for the fabrication of biosensor, and found a detection limit of 0.5 μM for H2O2. A lower detection limit was obtained when they used tetra thiafulvalene [24], and methylene green [25], as a mediator. Wang et al. [26] utilized catechol mediator with the HRP enzyme for the construction of biosensor. They have used sol-gel/organic hybrid composite material for the immobilization of HRP. For the covalent immobilization of HRP, Gao et al. [27] used thionine modified surface, where thionine itself act as a mediator. A successful entrapment of HRP in a gelatin matrix was proposed by Yao et al. [28], for the development of H2O2 biosensor. The same group also used nano composite of methylene blue and silicon oxide as mediator to construct a biosensor with HRP, co-immobilized in the gelatin matrix and cross-linked with formaldehyde [29].

Nano materials along with mediator have been widely used for the development of HRP based H2O2 biosensor. Among the different nano materials, gold nano particles are the mostly used materials for the immobilization of HRP. Lei et al. [30] used nano gold film modified surface for the efficient and stable immobilization of HRP. The modified electrode exhibited electro catalytical response to the reduction of H2O2. A linear range from 6.1 μM to 1.8 mM, with a detection limit of 6.1 μM was obtained. Lei et al. [31] immobilized HRP labeled nano-gold on a silica sol-gel/alginate hybrid film and found a better performance, compared with the nano gold film modified surface. A more better result was obtained when the immobilizing matrix was replaced with the poly (2,6-pyridinedicarboxylic acid) [32,33], as reported a nano-Au monolayer for the immobilization of HRP. They used thiol functional group-derived Carbon Ceramic Electrode (CCE) and obtained monolayer through covalent linkage between nano-Au and thiol group on the surface of CCE. The fabricated biosensor by immobilizing HRP showed a linear range of 12.2 μM to 1.1 Mm, with a detection limit of 6.1 μM. A lower detection limit was obtained by using PAMAM dendrimer/cyst amine to support the nano-Au monolayer [34]. A Layer-by-layer (LBL) assembly of nano-Au and Toluidine Blue (TB) was also reported for the fabrication of a mediated H2O2 biosensor [35]. Zhu et al. [36] used gold nanoparticles, nafion, Polythionine (PTH), and gelatin as matrixes for the immobilization of HRP and construction of H2O2 biosensor, as well. The biosensor showed the catalytic reduction of H2O2 and the obtained detection limit was 20 μM. Ahammad et al. [16] lowered the detection limit to 1.5 μM by using gold nanoparticleadsorbed conducting PTH modified GCE [37]. The fabrication process of the biosensor is shown in figure 2. PTH nanowires have also been synthesized by electro deposition in porous Anodic Aluminum Oxide (AAO) template, and used to encapsulate HRP and nano- Au by in situ electrochemical copolymerization [38]. The resulting PTHNWs–HRP–nano-Au film modified electrode showed to be excellent amperometric sensors for H2O2. For the fabrication of a H2O2 biosensor, Jia [39] immobilized HRP-Au nanoparticles on a viologenmodified Glassy Carbon Electrode (GCE) by amino cation radical oxidation in basic solution. Viologen acts as a mediator and a covalent linker between GCE and the Au nanoparticles. A nanocomposite of gold nanoparticles–bacterial cellulose nano fibers was also described for the immobilization of HRP [40]. The constructed biosensor showed electro catalytic activities to the reduction of H2O2, in the presence of the mediator hydroquinone (Figure 2).


Figure 2: Schematic diagram of the fabrication of the GCE/PTH/GNP/HRP biosensor electrode [37].

Carbon nanotubes are also used for the development of H2O2 biosensor. Tripathi et al. [41] developed an amperometric H2O2 biosensor by entrapping HRP in an ormosil composite, doped with ferrocene monocarboxylic acid–bovine serum albumin conjugate and Multiwall Carbon Nanotubes (MWCNTs). The proposed H2O2 biosensor exhibited a linear range of 0.02–4.0 Mm, with a detection limit of 5.0 μM. The detection limit is lowered to 0.5 μM, when the fabrication of the biosensor was based on the co-immobilization of HRP, Methylene Green (MG), and MWCNTs, within ormosils [42]. It is still better compared with the Layer-by-layer (LBL) assembly of the nanocomposite of Methylene Blue-Multiwalled Carbon Nanotubes (MB-MWNTs) and HRP [43]. However, a higher sensitive H2O2 biosensor can be constructed by using the nanocomposite of brilliant cresyl blue-MWCNTs [44], nile blue-MWCNTs [45] and toluidine blue-MWCNTs [46], methylene blue-MWCNTs [47]. Moreover, a very high sensitive H2O2 biosensor can be constructed by wrapping the nanocomposite of MB-Sodium Dodecylsulfate (SDS) with the MWCNTs [48].

Chitosan (CHIT) is extensively used for the development of mediated HRP based H2O2 biosensor. Wang et al. [49] described the fabrication of a H2O2 biosensor via an easy and effective enzyme immobilization method with the “sandwich” configuration: ferrocene- CHIT: HRP: CHIT-glyoxal. The biosensor surface was cross-linked with glyoxal, in order to prevent the loss of immobilized HRP. Ferrocene was selected and immobilized on the GCE surface as a mediator. Wang and Zhang [50] prepared a sol-gel organic-inorganic hybrid material from natural CHIT and tetrakis(2-hydroxyethyl) orthosilicates. An amperometric H2O2 biosensor was developed by immobilizing HRP onto the hybrid gel matrix. The linear range for the determination of H2O2 was found to be from 1.0 μM to 0.3 Mm, with a detection limit of 0.4 μM. Another organic–inorganic hybrid was prepared by Chen et al. [51] by dispersing colloidal carbon microspheres in CHIT solution. Then, they constructed a H2O2 biosensor by entrapping HRP in hybrid material. Also, organic-inorganic hybrid material composed of zirconia-CHIT sol–gel and Au nanoparticles have been used for entrapping HRP and fabricating H2O2 biosensor, as shown in figure 3 [52]. A little lower detection limit was obtained than the previous one. Fe3O4/CHIT modified GCE can be used for the immobilization of HRP and fabrication of H2O2 biosensor, as well [53]. However, the sensitivity of the biosensor is not as good as previous one. The sensitivity of the biosensor increased when magnetic carbon-coated iron nanoparticles were used to immobilize HRP on the surface of a PTH modified GCE, in combination with CHIT and cross-linking of glutaraldehyde. A very high sensitive biosensor was reported by Liu et al. [54], based on HRP and γ-Al2O3/CHIT composite film at a GCE. The sensitivity of the biosensor again decreased when HRP and CHITwrapped NiFe2O4 nanoparticles [55], or MgO nanoparticles-CHIT composite matrix [56], were used. Kafi et al. [57] developed a H2O2 biosensor based on the co-immobilization of HRP and CHIT, onto Aumodified TiO2 nanotube arrays. Electrochemical measurements show that the Au-modified TiO2 nanotube arrays provide excellent matrices for the immobilization of HRP, and that the optimized electrochemical biosensor exhibits long linearity, a low detection limit, high stability, and very good reproducibility for the detection of H2O2 (Figure 3).


Figure 3: Illustration of the fabrication process of the biosensor (redrawn from reference [52], [80]).

Various polymer films have been reported for the development of mediated HRP based H2O2 biosensor. Qu et al. [58] proposed a H2O2 biosensor by encapsulating HRP in situ, in poly (neutral red) nanowires (PNRNWs) by electrochemical copolymerization. The PNRNWs showed excellent efficiency of electron transfer between the HRP and the GCE for the reduction of H2O2, and the PNRNWs–HRP modified GC electrode showed to be excellent amperometric sensors for H2O2 at -0.1V, with a linear response range of 1 μM to 8 mM. For high enzyme loading density and long-term retention of bioactivity, Zeng et al. [59] synthesized a looped HRP-poly amidoamine nanohybrid for the construction of a H2O2 biosensor. Polythiolated-β-cyclodextrin polymer also synthesized for immobilizing adamantane-modified HRP via supramolecular associations [60]. Then the enzyme-containing electrode was used as an amperometric H2O2 biosensor. Another amperometric H2O2 biosensor was fabricated by immobilizing HRP on a GCE by poly (glycidyl methacrylate-co-vinylferrocene) (poly(GMAco- VFc)) film [61].

A polymeric electron transfer mediator containing copolymers of Gycidyl Methacrylate (GMA) and Vinylferrocene (VFc), with different molar ratios, was prepared by free-radical copolymerization. The mediated H2O2 biosensor showed a fast response of less than 4 s, with a linear range 2.0-30.0 mM. Some other meterials, such as β-cyclodextrin-branched carboxymethylcellulose residues [62], manganese oxide [63], zinc oxide [64], magnetic dextran microsphere [65], bilayer lipid membrane [66], and ionic liquid [67], are reported for the immobilization of HRP and development of mediated H2O2 biosensor. Table 1 summarizes characteristics of the mediated HRP based H2O2 biosensors.

Modified Electrode Mediator Linear range DetectionLimit (µM) Reference
GE/MB/HRP methylene blue 10–560 µM 3.0 [20]
SGCE/PPy/HRP ferrocene carboxylic acid 0.9 µM–0.2 mM 50.0 [22]
GCE/SG/HG/HRP potassium hexacyanoferrate 0.1 µM–3.4 mM 0.5 [23]
SGOIH/HRP Tetrathiafulvalene up to 1.3 mM 0.3 [24]
SPCE/ TH/HRP thionine 5–65  µM 0.5 [27]
GCE/GNP/HRP Hydroquinone 6.1 µM–1.8 mM 6.1 [30]
GCE/SSG/AH/GNP/HRP Hydroquinone 12.2 µM–1.5 mM 0.6 [31]
PPDA/GNP/HRP Hydroquinone 0.3 µM–2.0 mM 0.1 [32]
CSG/GNP/HRP Hydroquinone 12.2 µM–1.1 mM 6.1 [33]
Cys/PAMAM/GNP/HRP Hydroquinone 10 µM–2.5 mM 2.0 [34]
TB/GNP/HRP toluidine blue 0.2 µM–8.6 mM 70 nM [35]
GL/PTH/GNP/HRP Polythionine 50 µM–30.2 mM 20.0 [36]
GCE/PTH/GNP/HRP Hydroquinone 5.0 µM–0.2 mM 1.5 [37]
PTHNW/GNP/HRP Polythionine 0.5 µM–13.0 mM 0.3 [38]
GCE/BAPV/GNP/HRP BAPV 0.2 µM–2.0 mM 0.1 [39]
BCN/GNP/HRP Hydroquinone 0.3 µM–1.0 mM 0.1 [40]
FMC/BSA/MWNTs/OC/HRP ferrocene carboxylic acid 20 µM–1.0 mM 5.0 [41]
MG/MWCNTs/HRP methylene green 0.5 µM–20.0  µM 0.5 [42]
MB/MWCNTs/HRP methylene blue 4.0 µM–3.8 mM 1.0 [43]
BCB/MWCNTs/HRP brilliantcresyl blue 0.3 µM–10.0 mM 0.1 [44]
NB/MWCNTs/HRP nile blue 0.2 µM–38.0 mM 0.1 [45]
TB/MWCNTs/HRP toluidine blue up to 40 mM 1.7 [46]
MB/SDS/MWCNTs/HRP methylene blue 0.2 µM–1.4 mM 5 nM [48]
GCE/FC-CHIT/HRP/CHIT-GLY Ferrocene 35.0 µM–1.1 mM 8.0 [49]
GCE/CMS/CHIT/HRP Hydroquinone 0.1–1.6  mM 0.93 [51]
ZrO2/CHIT/GNP/HRP 2,6-pyridinediamine -copper 0.8 µM–3.7 mM 0.3 [52]
GCE/ Fe3O4/CHIT/HRP methylene blue 0.2–12.0 mM 100 [53]
GCE/ γ-Al2O3/CHIT/HRP ferrocene carboxylic acid 0.5 µM–0.7 mM 70 nM [54]
GCE/ NiFe2O4/CHIT/HRP ferrocene carboxylic acid 10.0 µM–2.0 mM 2.0 [55]
Nano-MgO/CHIT/HRP Hydroquinone 0.1 µM–1.3 mM 50 nM [56]
TiO2 nanotube/CHIT/HRP methylene blue 5.0 µM–40.0 mM 2.0 [57]
GCE/ PNRNWs/HRP neutral red 1.0 µM–8.0 mM 1.0 [58]
GCE/PAMAM/HRP Hydroquinone 3.1 µM–2.0 mM 0.8 [59]
GDE/PTHCD/HRP Hydroquinone 28.0 µM–5.5 mM 7.0 [60]
PGMAVF/HRP Ferrocene 2.0–30.0 mM 2.6 [61]
ZnO/CHIT/HRP Hydroquinone 0.5 µM–70 mM 0.3 [64]
GCE/MDMS/HRP Hydroquinone 0.2 µM–0.7 mM 78 nM [65]
PLM/PDA/HRP Hydroquinone 0.3 µM–3.1 mM 0.1 [66]
GCE/PTBA/RTIL/HRP Hydroquinone 5.0 µM–17.5 mM 0.5 [67]

Table 1: Characteristics of the mediated HRP based H2O2 biosensors.

Mediator-free HRP based biosensor

The direct electrochemical behavior of the enzyme or protein at the electrode surface provides a foundation for the fabrication of the mediator-free biosensors. It simplifies the preparation processes of sensing devices and avoids the toxicity of the mediator, in comparison with the mediator based biosensors. It features the advantages of high sensitivity and selectivity, and therefore, attracts considerable attention.

Depending on the immobilizing matrix, HRP exhibited direct electrochemical behavior towards the reduction of H2O2. For example, a quasi-reversible electron transfer was observed in the absence of mediator, when HRP is incorporated in a salt bridge-supported Bilayer Lipid Membrane (sb-BLM), modified with Lauric Acid (LA). Zhang et al. [68] utilize this quasi-reversible electron transfer to construct a H2O2 biosensor.

Different nano materials are widely used for the development of mediator-free HRP enzyme based biosensor. Liu and Ju [69] proposed a renewable reagentless H2O2 biosensor based on the direct electron transfer of HRP, which was immobilized on a gold nano particlemodified carbon paste electrode. Electrochemical methods were used to investigate the direct electrochemistry of HRP. The biosensor displayed an excellent electrocatalytic response to the reduction of H2O2, without the aid of an electron mediator. Gold nanoparticlemodified ITO electrode was also used for the immobilization of HRP and investigation of the direct electrochemistry of HRP [70,71]. However, the performance of the mediator-free biosensor increased when gold nanoparticle and HRP embedded simultaneously on sol–gel network [72]. Yin et al. [73] reported the preparation of HRP–Gold Nanoparticles (GNPs)–Silk Fibroin (SF) modified GCE by one step procedure, and investigated the direct electrochemistry of HRP at the modified electrode. The fabricated mediator-free biosensor showed an excellent and quick electrocatalytic response to the reduction of H2O2. A very sensitive mediator-free biosensor was prepared based on the layer-by-layer assembly of Mercapto Propionic Acid (MPA), Cystinebased polymethylene-bridged cyclic bisureas (CBU)/GNPs and HRP on gold electrode [74]. A lower detection limit of 50 nM for H2O2 was obtained. Gold electrode was further modified with one-dimensional gold nanowires (Au NWs) and TiO2 nanoparticles (nano-TiO2) [75]. However, the sensitivity of the biosensor using the modified electrode was not as high as the previous one. A better performance of the biosensor was obtained when a layered Calcium carbonate–Gold Nanoparticles (CaCO3–GNPs) inorganic hybrid composite was used for the immobilization of HRP [76].

Carbon nanotubes are widely used for the development of mediator-free HRP based H2O2 biosensor. Qian and Yang [77] developed an amperometric H2O2 biosensor, based on cross-linking HRP by glutaraldehyde with multiwall carbon nanotubes/CHIT (MWNTs/CHIT) composite film coated on a GCE. The linear range of detection towards H2O2 was from 16.7 μM to 0.7 mM, with correction coefficient of 0.998. A nanocomposite film of Tetrathiafulvalene– Tetracyanoquinodimethane (TTF–TCNQ)/MWCNTs modified gold electrode was prepared for the immobilization of HRP [78]. The prepared enzyme electrode was used for the bioelectrocatalytic reduction of H2O2, without using any mediator. A remarkable detection limit of 1.5 nM was achieved when HRP was covalently attached to layered nonoriented MWNTs modified electrode [79]. The composites of MWCNTs and core–shell [email protected] CHIT crosslinked nanospheres as an immobilization matrix for the construction of an amperometric H2O2 biosensor was described by Chen et al. [80]. HRP was immobilized onto the composite film through electrostatic interaction between oppositely charged [email protected] CHIT nanospheres and HRP, as shown in figure 4. The direct electron transfer of HRP was achieved at HRP /[email protected]/MWNTs/ GCE, which exhibited excellent electrocatalytic activity for the reduction of H2O2. Huang and Tsai [81] found direct electrochemistry and bioelectrocatalytic ability of HRP towards the reduction of H2O2 at the Multiwalled Carbon Nanotube/Alumina-coated Silica (MWCNT/ ACS) nanocomposite modified GCE. The HRP/MWCNT/ACS nanobiocomposite modified GCE was prepared by casting HRP onto the surface of GCE. Direct electrochemistry of HRP was also achieved when HRP was cross-linked with MWCNTs, by using glutaraldehyde and bovine serum albumin. The sensitivity was found a little higher without using alumina-coated silica with MWCNTs. Besides CNTs, some other nanomaterials have been used for the development of HRP based mediator-free H2O2 biosensor, such as graphene [82-84], quantum dots [85], silver nanoparticles [86,87], silver nanowires [88], dendritic silver/silicon dioxide nanocomposites [89], graphene nano platelet [90], etc. (Figure 4).


Figure 4: Illustration of the preparation process of the biosensor (redrawn from reference [80]).

Conducting polymer has been widely used for the construction of HRP based mediator-free H2O2 biosenosr. Kong et al. [91] investigated the direct electron transfer process of immobilized HRP on a conducting poly (5, 2’: 5’, 2’’-terthiophene-3’-carboxylic acid). The HRP was immobilized by covalent bonding between amino group of the HRP and carboxylic acid group of the polymer. Amine group containing conducting polymer also can be used for the immobilization of HRP, by using cross-linking agent. For example, a H2O2 biosensor is constructed by cross-linking between HRP and Polyaniline (PANI), using glutaraldehyde as a cross-linking agent on F-doped Tin Oxide (FTO) electrode [92].The immobilization of HRP onto ordered mesoporous polymaniline was reported by Xua et al. [93], to construct a mediator-free H2O2 biosensor. The mesoporous polyaniline film was fabricated by electrodepositing from the hexagonal lyotropic liquid crystalline (LLC). The proposed biosensor combined the advantages of the good conductivity of polyaniline and the higher surface area of the ordered mesoporous film. The PANI nanofibers also reported for the immobilization of HRP [94]. Pt Nanoparticle (PtNP) was electrochemically deposited on the PANI nanofibers, which was electropolymerized on a gold electrode surface. Then, the hybrid film of GNPs, CHIT, and HRP was cast onto the modified electrode to form a stable biofunctional film. A higher sensitive H2O2 biosensor can be fabricated by using the hybrid film of chitosan and HRP onto the nanocomposite film of Au-Pt alloy Nanoparticles (NPs) and polyaniline nanotube [95].

Some other conducting polymers, copolymers and materials are reported for the construction of HRP based mediator-free H2O2 biosenosr such as polypyrrole [96,97], poly-(vinylferrocenium perchlorate) [98], poly 2,6-pyridinediamine [99], poly(3,4- ethylenedioxythiophene) [100], poly (N-isopropylacyamide-co-3- methacryloxy-propyltrimethoxysilane) [101], ionic liquids [102-104], 4-Carboxyphenyl [105], zirconia enhanced grafted collagen tri-helix scaffold [106], DNA films [107], Nafion–Sonogel–Carbon composite [108], silica–hydroxyapatite hybrid film [109], porous structure of screen-printed electrode [110], kieselguhr membrane [111], lipid membrane [112,113] etc. Table 2 summarizes characteristics of the mediator-free HRP based H2O2 biosensor.

Modified Electrode Linear range Detection Limit (µM) Reference
CPE/GNP/HRP 0.5–50 µM 0.2 [69]
ITO/APTMS /GNP /HRP 20.0 µM–8.0 mM 8.0 [70]
ITO/GNP/HRP 8.0 µM–3.0 mM 2.0 [71]
GDE/SSG/GNP/HRP 1.6 µM–3.2 mM 0.5 [72]
GCE/SF/GNP/HRP 10.0 µM–1.8 mM 5 [73]
GDE/MPA/CBU/GNP/HRP 0.4–0.9  µM 50 nM [74]
GDE/nano-TiO2/AuNWs/HRP 2.3 µM–2.4 mM 0.7 [75]
GDE/ATP/CaCO3–GNPs/HRP 0.5 µM–5.2 mM 0.1 [76]
GCE/MWCNTs/CHIT/HRP 16.7 µM–0.7 mM 10.3 [77]
GDE/MWCNTs/TTF–TCNQ/HRP 5.0 µM–1.1 mM 0.5 [78]
PDDA/MWNTs/HRP Up to 120 nM 1.5 nM [79]
MWNTs/[email protected]/HRP 0.7 µM–2.8 mM 0.3 [80]
GCE/ACS/MWCNTs/HRP 1.0 µM–0.5 mM 0.6 [81]
GCE/CHIT/GP/HRP 5.0 µM–5.3 mM 1.7 [82]
ITO/CHIT/GP-Fe3O4/HRP 5.0 µM–3.8 mM 0.6 [83]
GP/GNP/CdTe–CdS/GNP/HRP 0.1–12.0 nM 0.03 nM [84]
GCE/QDs/HRP 5.0 µM–0.1 mM 0.3 [85]
GDE/SNP/HRP 3.3 µM–9.4 mM 0.8 [86]
GDE/DNA/SNP/HRP 1.5 µM–2.0 mM 0.5 [87]
GDE/SNW/HRP 4.8 nM–0.31 µM 1.2 nM [88]
GCE/DSSD/HRP 0.7 µM–0.1 mM 50 nM [89]
TPA/SLGnP/HRP 0.6–16.8 µM 0.1 [90]
FTO/PANI/HRP up to 20 mM --- [92]
GCE/PANI/HRP 1.0 µM–2.0 mM 0.6 [93]
PANI/PNP/CHIT/GNP/HRP 7.0 µM–14.0 mM 2.8 [94]
Nano-PANI/GNP-PNP/CHIT/HRP 1.0 µM–2.2 mM 0.5 [95]
PPy/PNP/GNP/HRP 4.9 µM–4.8 mM 1.3 [97]
Pt/PPA/GNP/HRP 0.4 µM–1.5 mM 0.1 [99]
Au/PEDOT-PSS/HRP 0.2 µM–38.0 mM 0.1 [100]
GCE/PNM/HRP 0.2–1.4 µM 47.5 nM [101]
GDE/DNA/ BMITB/HRP 10.0 µM–7.4 mM 3.5 [102]
β-CD//ILs/ BMIMBF4/HRP 4.0–84.0 µM 2.7 [103]
GDE/4-CP/HRP 20.0 µM–20.0 mM 5.0 [105]
SGE/HRP 4.0 µM–0.1 mM 1.6 [108]
GCE/SHAP/HRP 1.0 µM–0.1 mM 0.4 [109]
PSPCE/HRP 5.9–35.4 µM 0.5 [110]

Table 2: Characteristics of the mediator-free HRP based H2O2 biosensor.

Hb based H2O2 biosensor

It is well known that HRP is the most commonly used enzyme for the fabrication of H2O2 electrochemical biosensors. Owing to its expensiveness and unstableness, finding alternatives to reduce the cost and to improve the performance of H2O2 biosensors is scientifically interesting and important practically. Hb, a protein, can be utilized as the HRP substitute in the detection of H2O2, in virtue of the low cost and the stable property of in solution. Although Hb does not play a role as an electron transfer carrier in biological systems, it has been shown to possess enzyme-like catalytic activity. Its bioelectrocatalytic activity to reduce H2O2 is also well documented.

Carbon nanotubes and GNPs are widely used with Hb for the construction of biosensor. Qi et al. [114] designed a mediator-free H2O2 biosensor by immobilizing Hb on MWCNTs modified with GCE. The direct electron transfer of the Hb was observed. A linear in the concentration range from 6.0 μM to 6.0 mM, with a detection limit of 1.2 μM was obtained. The direct electron transfer of Hb also observed onto MWCNTs enhanced grafted collagen matrix [115]. However, the fabricated biosensor by this way showed lower sensitivity. A high sensitive Hb based mediator-free biosensor can be constructed by using the nanocomposite of MWNTs, CeO2 nanoparticles, and CHIT as an immobilizing matrix [116]. The composite matrix combined the advantages of MWNTs, CeO2 nanoparticles, and CHIT, with good electron-transfer ability, attractive biocompatibility, and fine filmforming ability, which could increase Hb attachment quantity and H2O2 detection sensitivity. The nanocomposite of MWCNTs and GNPs also used for the fabrication of high sensitive Hb based mediator-free biosensor [117].

Zhang and Oyama [118] prepared a modified ITO electrode surface, with spherical and rod-shaped GNPs surfactant-assisted seeding growth approach, which provided a biocompatible matrix for the immobilization of Hb. The Hb immobilized GNPs-modified ITO (Hb/Au/ITO) electrode exhibited an effective catalytic response to the reduction of H2O2. The linear relationship existed between the catalytic current and the H2O2 concentration, in the range of 10 μM to 7 mM. The detection limit (S/N=3) was 4.5 μM. The nano molar detection limit can be achieved by using gold electrode, instead of ITO [119]. In this case 1,6-hexanedithiol (HDT) need to be used as a molecule bridge. The layer-by-layer assemble of the composite of C at SiO2 with GNPs shown in figure 5, was also reported to achieve the lower detection limit [120] (Figure 5).


Figure 5: Procedure for preparation of Hb–GNPs–[email protected]2(redrawn from reference [120]).

Tang et al. [121] described a facile strategy of a mediator-free H2O2 biosensor based on the direct electrocatalysis of Hb immobilized on GNPs/1,6-diaminohexane (DAH) modified GCE. A uniform monolayer film of DAH was initially covalently bound on a GCE surface, by virtue of the electrooxidation of one amino group of DAH, and another amino group was modified with GNPs and Hb, successively. In another way, a biocompatibile nanocomposite of colloidal gold and Hydroxyapatite nanotubes (Hap) was first prepared, then the Hb was immobilized on the nanocomposite [122]. The immobilized Hb showed fast direct electron transfer and excellent electrocatalytic behavior toward reduction of H2O2. Another composite film of gold colloid (nano-Au)/l-cysteine (l-cys)/nano-Au/nanoparticles Pt (nano- Pt)/CHIT was also reported for the fabrication of high sensitive Hb based mediator-free H2O2 biosensor [123].

One-dimensional (1D) GNPs were prepared and used for the immobilization of Hb, to construct an amperometric biosensor for H2O2 [124]. The linear range for the determination of H2O2 was 0.6 to 12.4 μM, with detection limit of 24 nM was obtained. Threedimensionally ordered macroporous gold-nanoparticle-doped titanium dioxide (3DOM GTD) film by Wei et al. [125] to fabricate a mediator-free H2O2 biosensor. However, the detection limit was not as low as one-dimensional (1D) GNPs. Nevertheless, three-dimensional nanoporous Au networks modified Ti substrate can lower the detection limit to 20 nM [126]. Some other nanomaterials have been reported for the construction of Hb based mediator-free H2O2 biosensor, such as zirconium dioxide nanoparticles [127-129], iron nanoparticles [130-132], Fe3O4 nanoparticles [133], Ag nanoparticles [134], Cadmium telluride (CdTe) nanoparticles [135], titania nanotubes [136], nanographene [137], activated carbon nanoparticles [138], carbon nanofiber [139], etc.

Polymer and copolymer have been used for the development of Hb based mediator-free H2O2 biosensor. Jia et al. [140] prepared an unmediated H2O2 biosensor by co-immobilizing Hb, with platinum nanoparticles enhanced poly(chloromethyl thiirane) cross-linked CHIT (CCCS-PNs) hybrid film. CCCS could provide a biocompatible microenvironment for Hb, and PNs could accelerate the electron transfer between Hb and the electrode. A linear range for H2O2 from 0.4 to 44 μM, with a limit of detection of 28 nM was obtained. A film of triblock copolymer EO20PO70EO20 (P123) was used to incorporate Hb onto the surface of Pyrolytic Graphite (PG) electrode [141]. The amphiphilic, biomembrane like structure of the film provided a favorable microenvironment around Hb to retain its biological activity and native structure. Immobilized Hb in the film displayed good electrocatalytic response to H2O2. Another film of copolymer poly(acrylonitrile-co-acrylic acid) (PAN-co-PAA) was also used for the immobilization of Hb [142]. The Hb in PAN-co-PAA matrix acted as a biologic catalyst to catalyze reduction of H2O2. Some other materials have been used for the fabrication of mediator-free Hb based H2O2 biosensor, such as ionic liquid [143-145], liquid crystal [146], sol–gel film [147-149], kieselgubr film [150], DNA membrane [151], triton X-100 [152], niobate [153], collagen [154], magnetite–graphene [155], etc. Table 3 summarizes characteristics of the Hb based H2O2 biosensors.

Modified Electrode Linear range DetectionLimit (µM) Reference
GCE/MWCNTs/Hb 6.0- µM–6.0 mM 1.2 [114]
grafted collagen-MWNTs/Hb 0.6–30.0 µM 0.1 [115]
CHIT/MWCNTs/CeO2/Hb 5.0 µM–0.5 mM 0.6 [116]
GNP/MWCNTs/Hb 0.2 µM–3.0 mM 80 nM [117]
ITO/GNP/Hb 10.0 µM–7.0 mM 4.5 [118]
HDT/GNP/Hb 50.0 nM–1.0 µM 10 nM [119]
[email protected]/GNP/Hb 5.0–80.0  µM 80 nM [120]
HAP/GNP/Hb 0.5–25.0  µM 0.2 [122]
GNP/PNP/CHIT/Hb 0.1 µM–6.6 mM 45 nM [123]
ODGNP/Hb 0.6–12.4  µM 24 nM [124]
ITO/TiO2/GNP/Hb 5.0 µM–1.0 mM 0.6 [125]
Ti/TDN-Au/Hb 50.0 nM–0.2 µM 20 nM [126]
PG/ZrO2/Hb 1.5–30.2 µM 0.1 [127]
ZrO2/Hb 0.8 µM–0.1 mM 0.1 [128]
SA/HZMS/Hb 1.8 µM–4.9 mM 0.6 [129]
CIN/Hb 3.1 µM–4.0 mM 1.2 [130]
CFN/CHIT/Hb 50 nM–1.0 mM 10 nM [131]
GCE/MCMS/CHIT/Hb 69.0 µM–0.3 mM 21.0 [132]
Fe3O4/CHIT/Hb 50.0 µM–1.8 mM 4.0 [133]
GCE/AgNPs/Hb 1.0 µM–0.1 mM 0.3 [134]
CdTe/CHIT/Hb 7.4 µM–0.7 mM 2.2 [135]
TNT/Hb 1.0 µM–0.1 mM 0.9 [136]
PP123-NGP/Hb 10.0 µM–0.1 mM 8.2 [137]
GCE/CAN/NF/Hb 0.9–17.0  mM 0.4 [138]
PG/P123/Hb 1.0 µM–0.5 mM 0.5 [142]
GCE/PAN-co-PAA/Hb 9.2 µM–2.0 mM 4.5 [143]
GCE/BMIM·PF6–MSFs/Hb 0.2–28.0 µM 80 nM [144]
BPPF6/MWCNTs/Hb 50.0 nM – 0.7 µM 3.8 nM [145]
GCE/LCCP/Hb 7.0 µM–0.2 mM 3.1 [146]
Fe3O4-GP/Hb 1.5 µM–0.6 mM 0.5 [155]

Table 3: Characteristics of the Hb based H2O2 biosensors.


The goal of this review paper was to provide an updated summary of the HRP and Hb based electrochemical H2O2 biosensor. Modification of the electrode is an important task for the construction of biosensor. Nanomaterials modified electrode showed better performance, in terms of sensitivity and detection limit. However, less attention has been given to the fundamental research into understanding the mechanisms by which nano materilas give such excellent electrochemical performance. Enzyme based biosensor is still dominating, although they are costly. Moreover, the activity of enzymes is affected by the temperature, pH, humidity, and toxic chemicals. Much attention should be given on the simple fabrication, and cost should be considered during the construction of the biosensor. This would be helpful for the commercial production of the H2O2 biosensor.


I am grateful to my colleagues and collaborators for their contributions to our electrochemical biosensor research. This work was supported by the Centre for Advanced Research in Sciences (CARS), University of Dhaka.


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