Biosensors Development for Hydrogen Peroxide Detection


30 Jan 2018

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An approach towards clinical diagnosis.

1. Introduction:

The molecular oxygen is the basic source of energy for aerobic systems. Its consumption by the living systems produces the radical such as superoxide anion (O2-). This is a highly reactive toxic radical and is implicated in numerous pathologies. The toxicity of the triplet oxygen is prevented by the involvement of enzymes. A real paradox is that hydrogen peroxide and superoxide ion have to be present in the living systems but their concentrations have to be controlled precisely so that they persist only for a short period in cells.

Hydrogen peroxide belongs to a class of non-radicals reactive oxygen species [1, 2]. It is an important intermediate species in many biological and environmental processes. Moreover, H2O2 which is known as a cell killer due to its oxidizing power is required as a substrate for many enzymes. It has been shown to be present in the atmospheric and hydrospheric environments [3, 4]. H2O2 is a major reactive oxygen species in living organisms, better known for its cytotoxic effects and it also plays an important role as a second messenger in cellular signal transduction. Oxidative damages resulting from the cellular imbalance of H2O2 and other reactive oxygen species are related to aging and severe human diseases such as cancers and cardiovascular disorders. [5, 6] Furthermore, H2O2 is one of the products of reactions mediated by almost all oxidases. [7] H2O2 is generated in response to various stimuli, including cytokines and growth factors and is also involved in regulating diverse biological processes from immune cell activation and vascular remodeling in mammals [8] to stomatal closure and root growth in plants[9]. In unicellular organisms an important response to the increased levels of H2O2 is the increased production of antioxidants and repair proteins to allow adaptation to these oxidative conditions[10]. Most biological sources of H2O2 involve in the spontaneous or catalytic breakdown of superoxide anions, produced by the partial reduction of oxygen during aerobic respiration and following the exposure of cells to a variety of physical, chemical and biological agents. As for example, activation of NADPH oxidase complexes generate superoxide and hence the H2O2.

2. A brief outlook of the hydrogen peroxide assays

Unlike other reactive oxygen species H2O2 (a mild reducing and oxidizing agent) needs an initiator for the activation by the transitional metal or enzymes. This robust chemical characteristic of H2O2 made the assay rather difficult in the quantification compared to its other reactive oxygen species such as superoxide, hydroxyl radical, singlet oxygen, peroxyl radical, and such others. Methods useful for its assay include ultra-violet, infrared, Raman scattering, ESR, electro analytical techniques, metal-H2O2 complexes, enzyme mediated reactions, nanotechnogy, flow injection analysis, and biosensors.

2.1 Analytical methods based on the physical properties

Numerous methods have been reported in the literature for the quantification of H2O2 based on its physical properties. These include electrochemical, optical thermal, ultrasonic, chromatographic methods and mass spectra [11]. The analytical methods based on its physical properties are rather restricted due to its relatively poor robustness and sensitivity for biomedical application. Methods assessible for the enzyme assay techniques include

  • Synthetic labeled substrates including fluorogenic and chromogenic substrates
  • Isotopically labeled substrates
  • Fluorescence Resonance Energy Transfer (FRET) substrates
  • Substrates with fluorescent labels with indirect detection
  • Electrochemical assays
  • Chemiluminiscence assays
  • Bioluminescent assays
  • Mass spectroscopy
  • Nanotechnology
  • Enzyme immobilization techniques

For the development of analytical techniques and methods for the hydrogen peroxide assays, analytical chemists play a crucial role as they are mainly devoted to the development of methodologies or have been too much concerned with the analysis of isolated targeted material.

2.2 Quantification based on the electrochemical biosensor

The modern concept of biosensors is a rapidly expanding field of instruments to determine the concentration of substances and other parameters of biological interest since the invention by Clark and lyons in 1962. Electrochemical biosensors are the analytical devices that detect biochemical and physiological changes. Early techniques of biosensors in the analysis of chemical and biological species involved reactions that took place in a solution in addition to catalysts and samples. In recent years, however the biosensor techniques have provided alternative systems that allowed the reactions without reagents to take place at a surface of an electrode. The immobilization techniques include physical adsorption, cross-linking, entrapment, covalent-bond or sometimes combination of all the techniques.

  1. International Status

As it has been mentioned in the introduction of the proposal, a tremendous burst in research activities in the field of hydrogen peroxide measurement has increased over understanding about its role. Over the last few years, studies have suggested that oxidative stress plays a role in the regulation of hematopoietic cell homeostasis.[12] The generation of H2O2 is increased in response to various stresses, in which previous exposure to one stress can induce tolerance of subsequent exposure to the same or different stresses. [13] Oxidative stress is an important cause of cell damage associated with the initiation of many diseases.[14] It is also investigated that tissue injury due to free radical liberated by H2O2 during oxidative stress is the heart of periodontal diseases. [15]

Many research papers describes that high levels of H2O2 is cytotoxic to a wide range of animal, plant and bacterial cells. Hydrogen peroxide has the ability to penetrate the cell membrane and form the hydroxyl radical OH. which is capable of causing high levels of DNA damage. [16] Evidences show that increase in the cellular levels of H2O2 play a major role directly or indirectly in sensitizing cancer cells to H2O2-induced cell death. Indeed, there is a growing literature showing that H2O2 can be used as an inter- and intra-cellular signalling molecule. [17]

  1. National status:

A tremendous growth is taking place in developing hydrogen peroxide biosensor all over the world and also in India. As per the Indian scenario, different national research institutes and private companies have been working in this field. NPL, CEERI Pilani, IISC, IIT, Bengal Engineering and Science University and many more national institutes are working on this. Many of the private sectors like Biosensor Interventional technologies (India) Pvt. Ltd,

Clearly a major obstacles in studying the roles of hydrogen peroxide has been the lack of widely available specific tools and methodologies


The whole idea of the project is to develop a new ultrasensitive reagent, versatile, non-carcinogenic easily available so that there are no earlier reports.

The proposal of new reagents for enzyme peroxidase based hydrogen peroxide assay.

Principal investigator is interested to have an extensive catch over the kinetic assay by developing new kinetic equations by controlling different parameters such as pH, effect of co-substrate concentration etc.,

(iv) Methodology:

The simple oxidative reaction of the H2O2 in the presence of enzyme can be explored by converting the co-substrates into optically detectable product. This includes a variety of oxidizing reagents based on the oxidative property of metals such as Co(II), Fe(II), Cu(II), and other metal ion catalysts. The assay based on simple oxidation comprises the optical methods such as spectrophotometry, spectroflurimetry, and chemiluminiscence. To overcome the relatively poor sensitivity and selectivity of the optical methods described below, peroxidase or metal porphyrins can be introduced to enhance the sensitivity of specific H2O2 detection. It is based on the specific H2O2 reaction with hydrogen donors on the catalysis of peroxidase or metal porphyrins, instead of oxidizing reagents. These optical methods of analysis are sensitive to the extent of micromolar and nanomolar H2O2 concentrations.

As one of the most sensitive optical methods, chemiluminiscence is based on the reaction of luminol with H2O2 in basic solution in the presence of metal ions such as Fe(II), Cu(II), Co(II) and other metal ion catalysts [7]. Further chemiluminiscent methods were developed by the use of different oxidizing agents such as KIO4-K2CO3 [8]. Also attempts were made for the enhancement of chemuminiscent reaction by the use of p-iodopenol [9], gold nanoparticles [10], chitosan [11], resin [12] and DNAzyme [13].

Alternatively, fluorescent quantification has been applied to H2O2 based on the oxidation [14]. The generation of oxidized form can be measured by the fluorescent probes such as p-(hydroxyphenyl)propionic acid [15], β- Cyclodextrin (CD)–hemin [16], N,N’-dicyanomethyl-o-phenylenediamine- hemin [17], Rhodamine B hydrazide-iron(III)-tetrasulfonatophthalocyanine [18], Fluorescein hydrazide [19], Haemin-L-tyrosine [20], Fluorescin [21], and ninhydrin [22]. Another approach was mainly based on the ROS fluorogenic reaction, which generally involves formation of oxidized forms which are highly fluorescent products [23-26].

There are also many spectrophotometric methods for the assay of H2O2 which are based on the oxidation and formation of the colored product. The spectrophotometry involves the methods based on guaiacol [27], 4-amino-5-(p-aminophenyl)-1-methyl-2-phenyl-pyrazol-3-one (DAP) N-ethyl-N-sulpho- propylaniline sodium salt (ALPS) [28], Phenol-AAP [29], Photofenton reaction-metavanadate [30], Fenton reaction [31], Pyrocatecol-aniline [32], H2O2-molybdate [33], Naphthalene-Agrocybe aegerita peroxidase [34], and phenol red-HRP [35]. The sensitivity of these optical methods can be further enhanced by the involvement of sequential flow injection analysis system. Principal investigator is interested to carry out modest research for the development of newer analytical procedures for the enzyme based substrate assay.

Implementation of the project proposal involves of developing new reagents for the assay of peroxidase involving the use of amine, phenol related co-substrate assay for peroxidase. The main proposal of the research work will be dealt with alternative reagents to guaiacol, benzidine which are having it own disadvantages such as solubility in water, carcinogenic, economic viability.


[1] C.L. Murrant, M.B. Reid, Detection of reactive oxygen and reactive nitrogen species in skeletal muscle, Microscopy Research and Technique, 55 (2001) 236-248.

[2] M.P. Fink, Role of reactive oxygen and nitrogen species in acute respiratory distress syndrome, Current Opinion in Critical Care, 8 (2002) 6-11.

[3] D. Price, P.J. Worsfold, R.F. C. Mantoura, Hydrogen peroxide in the marine environment: cycling and methods of analysis, TrAC Trends in Analytical Chemistry, 11 (1992) 379-384.

[4] J.M. Anglada, P. Aplincourt, J.M. Bofill, D. Cremer, Atmospheric Formation of OH Radicals and H2O2 from Alkene Ozonolysis under Humid Conditions, ChemPhysChem, 3 (2002) 215-221.

[5] M.C.Y. Chang, A. Pralle, E.Y. Isacoff, C.J. Chang, A Selective, Cell-Permeable Optical Probe for Hydrogen Peroxide in Living Cells, Journal of the American Chemical Society, 126 (2004) 15392-15393.

[6] E.W. Miller, A.E. Albers, A. Pralle, E.Y. Isacoff, C.J. Chang, Boronate-Based Fluorescent Probes for Imaging Cellular Hydrogen Peroxide, Journal of the American Chemical Society, 127 (2005) 16652-16659.

[7] D.A. Abramowicz, C.R. Keese, Enzyme-catalyzed reactions involving diphenyl carbonate, in, Google Patents, 1990.

[8] M. Geiszt, T.L. Leto, The Nox Family of NAD(P)H Oxidases: Host Defense and Beyond, Journal of Biological Chemistry, 279 (2004) 51715-51718.

[9] C. Laloi, K. Apel, A. Danon, Reactive oxygen signalling: the latest news, Current Opinion in Plant Biology, 7 (2004) 323-328.

[10] D.J. Jamieson, Oxidative stress responses of the yeast Saccharomyces cerevisiae, Yeast, 14 (1998) 1511-1527.

[11] D. Harms, H. Luftmann, F.K. Muller, B. Krebs, U. Karst, Selective determination of hydrogen peroxide by adduct formation with a dinuclear iron(iii) complex and flow injection analysis/tandem mass spectrometry, Analyst, 127 (2002) 1410-1412.

[12] A. Nogueira-Pedro, T.A.M. Cesario, C. Dias, C.S.T. Origassa, L.P.M. Eca, E. Paredes-Gamero, A. Ferreira, Hydrogen peroxide (H2O2) induces leukemic but not normal hematopoietic cell death in a dose-dependent manner, Cancer Cell International, 13 (2013) 123.

[13] B. Halliwell, M.V. Clement, L.H. Long, Hydrogen peroxide in the human body, FEBS Letters, 486 (2000) 10-13.

[14] E.A. Veal, A.M. Day, B.A. Morgan, Hydrogen Peroxide Sensing and Signaling, Molecular Cell, 26 (2007) 1-14.

[15] A. Mendi, B. Aslım, Antioxidant Lactobacilli Could Protect Gingival Fibroblasts Against Hydrogen Peroxide: A Preliminary In Vitro Study, Probiotics & Antimicro. Prot., (2014) 1-8.

[16] B. Halliwell, J.M.C. Gutteridge, Oxygen free radicals and iron in relation to biology and medicine: Some problems and concepts, Archives of Biochemistry and Biophysics, 246 (1986) 501-514.

[17] S. Neill, R. Desikan, J. Hancock, Hydrogen peroxide signalling, Current Opinion in Plant Biology, 5 (2002) 388-395.


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