Advances in Nanoparticles, 2021, 10, 1-25

https://www.scirp.org/journal/anp
ISSN Online: 2169-0529
ISSN Print: 2169-0510

A Brief Review of How to Construct an

Enzyme-Based H2O2 Sensor Involved in
Nanomaterials

Uwitonze Nestor*, Hitimana Frodouard, Muhizi Theoneste

Department of Chemistry, School of Science, College of Science and Technology, University of Rwanda, Kigali, Rwanda

How to cite this paper: Nestor, U., Fro- Abstract

douard, H. and Theoneste, M. (2021) A Brief
Review of How to Construct an Enzyme- This article briefly reviews how to construct an enzyme based hydrogen pe-
Based H2O2 Sensor Involved in Nanomate- roxide sensor involving nanomaterials, which has the advantages of high effi-
rials. Advances in Nanoparticles, 10, 1-25. ciency, good sensitivity and selectivity, fast response time and an extended
https://doi.org/10.4236/anp.2021.101001
range of linearity with lower detection limit. Glucose biosensor is constructed
Received: May 8, 2020 by immobilizing glucose oxidase enzyme on the polycarbonate membrane
Accepted: December 22, 2020 and the protective cover is then filled with a physiological phosphate buffer,
Published: December 25, 2020 pH 7.4. The novel blocking hydrophobic membrane which is only permeable
to hydrogen peroxide is used to eliminate electrochemical interferences. This
Copyright © 2021 by author(s) and
Scientific Research Publishing Inc.
constructed enzyme based H2O2 biosensor is miniaturized by the involvement
This work is licensed under the Creative of nanomaterials like carbon nanotubes, platinum nanoparticles and silver
Commons Attribution International nanoparticles and it can achieve the effective microscopic detection of glu-
License (CC BY 4.0). cose. The introduction of nanomaterials including some pure metals (Ag, Au,
http://creativecommons.org/licenses/by/4.0/
Pd, Ni, Pt, and Cu), metal oxide (ZnO and TiO2), bimetallic (Au/Ag and
Open Access
Au/Pt) and carbon (nanotubes and graphene) nanomaterials in the construc-
tion of the enzyme based H2O2 biosensor improves its sensitivity and perfor-
mance by enhancing the enzymatic activity, and allows the introduction of
many new signal transduction technologies in biosensors. This review article
summarizes the working principles of glucose oxidase based hydrogen pe-
roxide sensor, importance of involving nanomaterials in biosensor manufac-
turing, basic characteristics and components of a biosensor, generations glu-
cose biosensors, procedure of making hydrogen peroxide based biosensor,
synthesis of nanomaterials involved in hydrogen peroxide biosensor, and fi-
nally some examples of nanomaterials which intervene in hydrogen peroxide
biosensor.

Keywords
Biosensor, Glucose Oxidase, Hydrogen Peroxide, Carbon Nanotubes,
Nanoparticles

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U. Nestor et al.

1. Introduction
In past few years, all over the world faced a public health problem of diabetes
mellitus which is a metabolic disorder resulted from the deficiency of insulin
and the unusually high concentration of sugar in blood. The normal range of
concentration of glucose in blood is between 80 - 120 mg/dl or 4.4 - 6.6 mmol/l.
This over concentration disease can be one of the causes of death and disability
in the world. To diagnose and manage the diabetes mellitus, a closed surveil-
lance of levels of blood glucose is required and the higher number of diabetics
tests performed every day has made glucose to be most frequently analyte. This
giant work has stimulating scientists to develop new biosensing concepts and a
glucose biosensor based on enzyme were designed to make ease-to-use blood
sugar testing and expecting their similar importance in continuous glucose moni-
toring [1] [2]. It is in this regard that enzyme based hydrogen peroxide elec-
trodes have been widely developed and used glucose oxidase in the presence of
oxygen for glucose measurements. The glucose probes based on hydrogen perox-
ide are highly generated today and being also used in clinical, industrial, envi-
ronmental, and food analyses [2]. The enzyme based H2O2 sensors have been
commonly used to detect the by-product of oxidases for glucose, alcohol, lactate,
lysine, cholesterol, D-amino acid, urate, oxalate and glutamate respectively and
the hydrogen peroxide is also a substrate for other enzyme like the horse-radish
peroxidase [3] [4]. Before the development of enzyme based glucose/H2O2 sen-
sor, the concentration of glucose/H2O2 is determined by using different analyti-
cal methods such as chromatography, titrimetry, chemiluminscence, spectropho-
tometry, electrochemistry, phosphorescence and fluorescence but, most of them
show their own technical disadvantages including low sensitivity and selectivity,
time losing, vulnerability to electroactive substances and expensive and /or com-
plicated instrumentation. These techniques have been substituted by an en-
zyme based hydrogen peroxide sensors because they are cheapest and have high
selectivity and sensitivity, quick operation, simplicity, rapid response, feasibility,
and with effective commercial applications [5]. Recently, Nanomaterials such as
nanoparticles made from pure metal (Au, Ag, Ni, Pt, Pd and Cu), metal oxide
(ZnO and TiO2); carbon nanotubes and carbon graphene are also involved in
construction of enzyme based hydrogen peroxide biosensor. Particularly, the
nanomaterials have been involved to initiate the development of highest perfor-
mance miniaturized glucose oxidase based hydrogen peroxide biosensors which
have faster response, good stability, higher sensitivity and selectivity with longer
storage time. These resulted biosensors devices are smart, portable and implan-
table [3] [5] [6].
The use of Nanomaterials in electrochemical sensors has shown a potential
positive effect especially in enhancement of sensitivity, optimization of selectivi-
ty, decreasing of detection limit, and extension of storage period [4]. Based on
recent papers published in the field of nanomaterials development, the following
keywords have been used in Google Scholar: NPs, QDs, NTs, NCs, NWs, NRs,

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 U. Nestor et al.

and NFs. Because of the vast progress and revolution in nanotechnology, most
papers were published since 2010 as illustrated in Figure 1(a).
The pie chart in Figure 1(b) indicates the impact factors of nanomaterials
(Top chart) and the variation of citations and articles for nanomaterials (Bottom
chart) since 2014 to 2018. It is obviously clear that the number of citations is
continuously increasing.

Figure 1. (a) Distribution of publications on one and two-dimensional (1D and 2D) na-
nomaterials in the FET based biosensor literature according to the ISI Web of Science
from 2010 to July 2019; (b) Evolution of Impact Factor, Citations and Publications for
Nanomaterials [7].

2. Working Principle of Enzyme Based Hydrogen Peroxide

Biosensor
A glucose probe operates based to the reaction below which is catalyzed by the
enzyme glucose oxidase and it is oxygen dependent [8].
GLUCOSE OXIDASE
Glucose + O 2  → Gluconic acid + H 2 O 2 (1)

The oxygen consumed during the above oxidative reaction was detected by
applying a negative potential to the cathode electrode made in platinum accord-
ing to the reduction reaction below [8]:
O 2 + 4H + + 4e− → 2H 2 O (2)
The hydrogen peroxide (H2O2) being produced as a by-product of biological
processes is highly sensitive determined by using an enzyme based hydrogen
peroxide biosensor through the anodic monitoring based on the anodic reaction
below that involves the oxidation of H2O2 product [9]
H 2 O 2 → O 2 + 2H + + 2e− (3)

Therefore, the above resulting type of biosensor offered good accuracy and
precision in connection with 100 µL blood samples [9].

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Even though these probes have sufficient stability and precision but in the
presence of electroactive interferences at the platinum electrode, they lack accu-
racy. The interference-free hydrogen peroxide sensors were developed by intro-
ducing an electrochemical interference blocking membrane but which cannot
block the interference from acetaminophen and slightly ascorbic acid drugs
when the probe was left in the measuring solution for a long time. The presence
or the variation of these drugs inhibits the continuous monitoring of glucose by
using this probe.
Another problem of glucose sensors is its limited linearity of (30 mg/dl) and
the required dilution procedures in many practical applications. To overcome
this problem, an interference-free hydrogen peroxide based glucose electrode
with a fast reaction on platinum electrode, good response range or linear range
of 1 - 1200 mg/dl and a detection limit of 0.5 mg/dl glucose is developed [5].

3. Importance of Nanomaterials Involved in Biosensor

Manufacturing
The use of nanomaterials is required today in miniaturization of glucose bio-
sensors that can be synthesized in various shapes and dimension depending on
the desired application. The synthesis method used to make required nanopar-
ticles is widely selected by depending on the desired size, appropriate properties
of surface and type of concerned material for example semiconductors, metals,
ceramics, polymers, etc. The synthesis methods of preparing diverse types of
nanoparticles (NPs) are grouped into three that are physical, chemical and bio-
assisted methods [10]. The involvement of nanomaterials in enzymatic hydrogen
peroxide biosensors has enhanced their working performance like improving the
sensitivity and selectivity, lowering the limit of detection, increasing the re-
sponse time and lengthening the storage period compared to an unmodified
probe which used the direct oxidation systems [11].
The introduction of nanotechnology has had a deep influence in the area of
biosensors in which the highly understood effect is the reduction of devices di-
mensions and the extent increase of sensitivity and selectivity of nanobiosensors.
The novel nanomaterials are used to improve the biological activity of biological
recognition element and the miniaturization of biosensors materials at the na-
noscale level as a result of the nanotechnology development [12]. The introduc-
tion of nanomaterials will increase the loading surface of the enzyme, contact
with more detectors, and improve the speed of electron transfer. The nanomate-
rials, such as Pt nanoparticles, Ag nanoparticles, carbon nanotubes, graphene,
Au nanoparticles, polyaniline and their hybrid nanostructure may enhance ef-
fective performance of the enzyme based biosensors [13].

4. Basic Characteristics and Components of a Biosensor

4.1. Basic Characteristics of a Biosensor
A good enzyme based hydrogen peroxide biosensor should have the following

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discussed characteristics [14]:

 Linearity is the maximum linear value of the sensor calibration curve. Li-
nearity of the sensor must be high for the detection of high substrate concen-
tration;
 Sensitivity is the value of the electrode response per concentration of sub-
strate;
 Selectivity is the way of reducing the interference of chemicals for obtaining
the correct result;
 Response time is the required time for having 95% of the response;
 Stability and Reusability is the ability of the electrode to measure the con-
centration of analyte for at least 4 weeks using the same electrode and same
membrane to produce the good expected results;
 Reproducibility of the biosensor is the ability of the biosensor to generate
the same responses for a duplicated experimental set-up and it is characte-
rized by the precision and accuracy of the transducer and electronics in a
biosensor.
Therefore, a novel enzyme based hydrogen peroxide as well as the glucose
electrodes are useful as analytical device for practical analysis when the examina-
tion of their calibration curves, pH and temperature is done and showing that all
the above discussed characteristics are excellent [14] [15].

4.2. Components of Biosensors

Based on the IUPAC definition, an enzyme based hydrogen peroxide biosensor
is a self-contained analytical device made by a biological recognition element
combined with a chemical or a physical transducer which normally exhibits spe-
cific and powerful interaction to an analyte or to a target molecule including
glucose, alcohol, glutamate, urea, cholesterol, D-amino acid, oxalate etc and
tends to provide the quantitative or semi-quantitative analytical information as
mentioned in Figure 2. It is an electrode that yields a readable signal through its
electronic component that is integrated with a biological component [5] [6].

Figure 2. Schematic presentation of a biosensor [8].

As illustrated from Figure 3, a good working biosensor consists of three major

components discussed below [8]:

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 Bioreceptors that bind the specific form to the sample and they are con-
nected to an electrochemical interface where specific biological processes
occur by giving rise to a signal;
 A transducer that converts the specific biochemical reaction in an electrical
signal;
 A signal processor that converts the electronic signal into a meaningful
physical parameter and finally a proper interface displays the results to the
operator.

Figure 3. Components of a typical Biosensors [8].

5. Generations Glucose Biosensors

5.1. First-Generation Glucose Biosensors
First-generation glucose biosensors employ the molecular oxygen as natural

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coenzyme to produce and detect hydrogen peroxide (Equations (1) and (3)
above). The hydrogen peroxide can also be generated by the biocatalytic reaction
involving the reduction of the enzymatic flavin group (FAD) by reacting with
glucose into a reduced form of the enzyme (FADH2) and gluconolactone [14].
GOx ( FAD ) + glucose → GOx ( FADH 2 ) + gluconolactone (4)

The obtained reduced enzymatic Flavin group is reoxidized by natural mole-

cular oxygen to reproduce its oxidized form and hydrogen peroxide.
GOx ( FADH 2 ) + O 2 → GOx ( FAD ) + H 2 O 2 (5)

The detections of hydrogen peroxide formation have the advantage of being

simpler, especially when miniaturized devices are used. Such detections are
commonly carried out on a platinum electrode at a moderate anodic potential of
around +0.6 V (vs Ag/AgCl). The selective detection of the GOx-generated hy-
drogen peroxide at low-potential is possible also by coupling with another en-
zyme such as horseradish peroxidase (HRP) that catalyzes the oxidation of pe-
roxide (Refer to Figure 4). The coupling of carbon nanotubes (CNTs) with pla-
tinum nanoparticles has been shown to be extremely useful for improving the
sensitivity and speed of GOx-based glucose biosensors (down to 0.5 µM within 3
s) [14].

Figure 4. Amperometric enzyme electrodes for glucose based on the use of natural oxy-
gen cofactor [14].

5.2. Second-Generation Glucose Biosensors

The second generation of glucose biosensor did not use oxygen as an electron
acceptor but it is replaced by a nonphysiological electron acceptor known as a
mediator. Electrons are not directly transferred by Glucose oxidase to conven-
tional electrodes due to the presence of a thick protein layer that covers its flavin
adenine dinucleotide (FAD) redox center and introducing an intrinsic barrier to
direct electron transfer. The reaction scheme below demonstrates how electrons
are carried by synthetic mediator between the FAD redox center and the surface
of the electrode [14] [16].

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Glucose + GOx ( ox ) → gluconic acid + GOx ( red ) (6)

GOx ( red ) + 2M ( ox ) → GOx ( ox ) + 2M ( red ) + 2H + (7)

2M ( red ) → 2M ( ox ) + 2e − (8)

where M(ox) and M(red) are the oxidized and reduced forms of the mediator.
The reoxidation of the reduced form of the mediator at the electrode surface
produces a current signal which is proportional to the glucose concentration.
Such mediation cycle is displayed in Figure 5 [16].

Figure 5. Sequence of events that occur in “second-generation” (mediator-based) glucose

biosensors-mediated system [16].

The mediator should be insoluble, nontoxic and chemically stable in its both
reduced and oxidized forms. If the rate of electron transfer through the mediator
is higher than the rate of the reaction of enzyme with oxygen, the competition of
oxygen will be reduced (Refer to Figure 6) [17].

Figure 6. Amperometric enzyme electrodes for glucose based on the use of artificial re-
dox mediators [17].

5.3. Third-Generation Glucose Biosensor

The third generation of glucose biosensor would like to remove the mediator and
develop a non-reagent glucose biosensor that operates at low potential closed to
the redox potential of the enzyme (Refer to Figure 7). The main advantage of
the third generation glucose biosensor is the absence of mediators that leads to
their very high selectivity at very low operating potential. This will also allow a di-
rect transfer of electron from glucose to the electrode through the active enzyme
site. Some critical challenges should be overcome to make the realization of this
direct electron transfer successful. One route for constructing third-generation

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amperometric glucose biosensors is to employ conducting organic salt electrodes

based on charge-transfer complexes such as tetrathiafulvalene-tetracyanoqui-
nodimethane (TTF-TCNQ). Some authors claimed that this type of biosensor
cannot allow a direct electron transfer because of the direct oxidation of the en-
zyme at the surface of crystal and the selectivity of glycose measurements at 0.1
V against Ag/AgCl electrode. Later they proposed that the electron transfer of
GOx at TTF-TCNQ electrodes is mediated and involves corrosion of the TTF-
TCNQ to produce dissolved components of these organic salts that mediate the
electron transfer of the enzyme. Thereafter, an oxidized boron-doped diamond
mediatorless electrodes based on direct electron transfer was developed for glu-
cose detection [14] [18].

Figure 7. Amperometric enzyme electrodes based on direct electron transfer between

GOx and the electrode [18].

6. Procedure of Making Hydrogen Peroxide Based Sensor

An electrochemical HPLC detector of a Tacussel PRG-GLUC is used to perform
the studies of constant anodic potential at +0.65 V. Gould medium gain pream-
plifier, model 13-4615-10 and Gould differentiator amplifier model 13-4615-71
are both used for measuring the rate. In an alternative way, the Universal Sen-
sors Amperometric Biosensor Detector (ABD, cat. no. 3001, Universal Sensors
Inc. Metairie, LA) is used for both constant potential studies and rate measure-
ments. A dual pen Houston Microscribe strip chart recorder is used to record
the steady-state current and rate measurements. The combined working elec-
trode (Pt) and reference electrode (silver/silver chloride) in a Universal Sensors
Hydrogen Peroxide Electrode (Cat. no. 4106) contained in the same electrode
jacket made the base sensor. A protective cover glass wall beaker is thermostated
by an MS-20 LAUDA Heating Circulator from Brinkmann Instruments to study
temperatures [14] [19].
The enzyme based hydrogen peroxide sensor is constructed by protecting a
1cm2 piece of the blocking membrane made in Polycarbonate on the extreme
end of a reversed electrode protective cover with an O-ring. Glucose oxidase en-
zyme is immobilized on the polycarbonate membrane and the protective cover is
then filled with a physiological phosphate buffer, pH 7.4. Inside the protecting
cover, the combined working/reference electrode is introduced and screwed

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down until the end of the platinum electrode is pressed against the blocking
membrane in polycarbonate. Then, a thin layer of electrolytic solution is formed
between the working electrode and the inner membrane while the reference
electrode is exposed to the body of the filling solution. The very thin enzyme
membranes with a thickness of 5 - 10 µm approximatively is prepared by mixing
20 U of glucose oxidase with 10 µL of phosphate buffer of pH 7.5 that contains
2.5% glutaraldehyde on the blocking polycarbonate membrane. There is a good
conduction between the working and the reference electrodes because they are
on the same side of the hydrophobic membrane. The sensor is then dunked in 10
mL of the physiological buffer for balancing and a recording of the current and
rate of current change is done after injection of aliquots of hydrogen peroxide
[14].
The construction procedure of the enzyme based hydrogen peroxide sensor is
summarized in the following membrane configuration [2]
INNER MIDDLE
Hdrogen peroxide membrane Enzyme membrane
OUTER
Polycarbonate blocking membrane
Hence, the enzyme based hydrogen peroxide sensor is assembled as follow:
Pt/Hydrogen Peroxide membrane/Glucose Oxidase enzyme/Polycarbonate mem-
brane [15].
The glucose oxidase enzyme is highly used in the construction of the enzyme
based glucose sensor since 950 s. During the construction of this enzyme based
sensing electrode, the electroactive species such as uric and ascorbic acid from
samples of human blood or body fluids are excluded. The sensing performance
of enzyme based glucose biosensor is determined by enzyme immobilization and
dependent to nanomaterials morphology [20].

7. Synthesis of Nanomaterials Involved in Hydrogen

Peroxide Biosensor
The construction and measurement of enzyme based hydrogen peroxide bio-
sensor use platinum as working electrode and silver/silver chloride as standard
reference electrode [4] [5]. Therefore, the Pt NPs, Ag NPs and carbon nanotubes
(CNTs) nanomaterials can be synthetized to miniaturize the hydrogen peroxide
electrode. The chemical methods involving microemulsion technique is used to
synthetize platinum and silver nanoparticles. The reduction strategy is used in
microemulsion technique for easy preparation of metal NPs. The effective syn-
thesis of metal NPs is controlled by some key parameters such as water/surfactant
molar ratio (u), type of continuous phase, metal ion concentration, type and
concentration of the reducing agent, structure and amount of the surfactant em-
ployed. The production of Pt cubic NPs is performed by reducing H2PtCl6 pre-
cursor with sodium borohydride and various amount of hydrochloric acid were
introduced in the H2PtCl6/NaBH4 microemulsion system to control the Pt NPs
shape/surface. The results of the studies of the Ag NPs synthesis and investiga-

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tion of the effect of water/surfactants ratio on the particle size and distribution
revealed that the size of particles decreases continuously from 5 to 1.5 nm while
the water/surfactants ratio reduced from 15 to 2.5. Therefore, during the synthe-
sis of metal NPs, the particle size is reduced by increasing the concentration of
surfactants such as sodium bis(2-ethylhexyl) sulfosuccinate (AOT) concentra-
tion on the Ag NPs synthesis and an appropriate concentration of a reducing
agent is used because both less and excess amount of reducing agent inhibits the
synthesis process by affecting the colloids stability [1] [21]. Carbon nanotubes
(CNTs) have therefore been employed as electrical connectors between the elec-
trode surface and the redox center of the enzyme. For instance, glucose oxidase
can be reconstituted on a 1.4 nm gold nanocrystal functionalized with the FAD
cofactor. The dithiol connector allows gold nanoparticle to be immobilized onto
the gold electrode surface and acts as an “electrical nanoplug” for the electrical
wiring of its redox-active center [14]. An example of the whole process of nano-
particles synthesis involved in hydrogen peroxide biosensor is presented in Fig-
ure 8 and Figure 9.
The other metals nanomaterials such as Pd, Rd, Au, Ni, Cu and Ir NPs can be
synthetized by using the reverse micelle microemulsion strategy and employing
Pd(NH2)4Cl2, RhCl2, NiCl2 and IrCl3, as the precursors with the reducing agents
which are the active hydrogen and hydrazine. These nanomaterials are synthe-
tized and used as matrix to immobilize the heme enzymes for improving the
sensing performances of hydrogen peroxide (Refer to Figure 10). The synthesis
of the above metal nanomaterials requires different reagent such as hydrazine in
synthesis of Cu NPs which displayed the growing of water and oxide content
during the control of NPs size and structure respectively; cationic water to oil
ratio of water/CTAB (cetyl trimethylammonium bromide)/n-hexanol system
to reduce nickel chloride by using hydrazine to synthetize Ni NPs; the alkaline 2,
7-dihydroxynaphthalene (DNP) in CTAB micellar media under 30 min conti-
nuous UV-irradiation is used to reduce the Au(III) ions for synthetizing the size
and shape-selective Au NPs. The size and shape of Au NPs is dependent to the
Au(III): CTAB ratio. The Rh NPs are synthetized in the similar way with Au NPs
by using DNP as reducing agent and CTAB as surfactant with 6hours of conti-
nuous UV-irradiation [4] [5].

(a) (b)

Figure 8. (a) Synthesis process of PAMAM-Au nanocomposite, (b) Preparation of the

enzyme modified electrode [5].

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Figure 9. Carbon nanotube (CNT) connectors with long-range electrically contacted to

glucose oxidase electrode [22].

Figure 10. SEM (a) and TEM ((b), (c)) images of Au/Pt bimetallic nanoparticles sup-
ported on coaxial nanocables. (d) Procedure to design diverse metal nanoparticles/coaxial
nanocable composite nanostructures [24].

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Bimetallic nanoparticles can be also synthetized and used in hydrogen perox-

ide sensing since they show a highest catalytic selectivity and activity and a best
resistance to deactivation compare to their corresponding monometal nanopar-
ticles. These properties are due to the addition of the second metal which brings
indispensable variations in particle shape, size, and surface morphology. They
are usually in forms of alloys resulted to the mixture of monometallic nanopar-
ticles. Recently, the most synthetized bimetallic nanoparticles are Au/Ag and
Au/Pt NPs that are used to catalyze the detection of hydrogen peroxide. Mean-
while, the construction of third generation H2O2 biosensor will be easy when
using the Au/Pt bimetallic nanoparticles [20] [23]. The synthesis of Gold-silver
bimetallic NPs with variable mole fractions is performed under water-in-oil mi-
croemulsions of water/aerosol OT/isooctane system by using hydrazine to re-
duce HAuCl4 and AgNO3 [20]. The Gold-Platinum bimetallic nanoparticles were
synthetized by preparing a series of room-temperature ionic liquids (RTILs)
containing various functional groups that were used as substrate for electrode-
deposition to deposit the Au/Pt nanostructures. In addition, the other bimetallic
nanoparticles including Pt/Pd, Pt/Ir, Pt/Cu, Pd/Cu, Rh/Pd and Ru/Rh were uti-
lized to construct an enzyme based H2O2 biosensor but also used for construc-
tion the non-enzymatic electrochemical biosensor for sensing and determining
glucose and H2O2 [20] [23].
Another novel biosensor used to detect H2O2 was reported by Zhao et al. [25]
as an electrode with the surface of 0.3 cm2 which is constructed by directly in-
creasing mesoporous ZnO NFs based on chitosan enzyme. The SEM images of
ZnO NFs and 3D porous ZnO framework for high and low magnification re-
spectively are shown by Figure 11(a) and Figure 11(b). All the above discussed
nanomaterials contributed to the higher performance of enzyme based H2O2
biosensors [4].

Figure 11. (a) SEM images of ZnO NF and (b) 3D porous ZnO framework and Inset SEM
image of nature connection between ZnO NFs [4].

8. Examples of Nanomaterials Involved in Enzyme Based

Hydrogen Peroxide Biosensor
The carbon nanotubes have been shown to be useful for development of glucose

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nanobiosensors based on conductivity due to the unique electrical properties of

unidimensional nanomaterials. In this case, a glucose sensing devices were made
by connecting two carbon band microelectrodes with polyaniline(PANI) film
covered with a glucose oxidase enzyme/poly(1, 2-diaminobenzene) layer. A typ-
ically small pocket sized, lighter and battery operated is manufactured based on
its precision and it is used for home blood glucose testing (Figure 12). Such de-
vices offer significant affirmation for obtaining the desired clinical information
in a simpler, faster, and cheaper manner compared to traditional attempts. These
blood glucose personal testing electrochemical biosensors are fully suited and
have played a crucial role in the move to simple one-step blood sugar testing. Since
blood glucose home testing devices are used daily to diagnose potentially life-
threatening events they must be of extremely high quality. The majority of home
blood glucose monitors rely on disposable screen-printed enzyme electrode test
strips. Such single-use electrode strips are mass produced by the rapid and sim-
ple thick-film microfabrication or vapor deposition process. In these devices
each strip contains both printed working and reference electrodes in which the
working one is coated with the needed reagents such as enzyme, mediator, stabi-
lizer, surfactant, linking, and binding agents and membranes as shown in figure
below [1].

Figure 12. Cross section of a commercial strip for self-testing of blood glucose: (a) Elec-
trode system; (b) Hydrophobic layer (drawing the blood) [1] [14].

Most of these devices count on a ferricyanide mediator, except for the Abbott
devices that employ a ferrocene derivative or an osmium-based redox polymer.
In all cases, the diabetic patient pricks the finger, places the small blood droplet
on the sensor strip, and obtains the blood glucose concentration within 5 - 30 s.
Some of the modern meters allow a collection of submicroliter amples of blood
from the forearm and this minimizes the pain and discomfort associated with
puncturing the skin. For instance, the FreeStyle monitor of Abbott counts on
coulometric strips with a 50 µm gap thin-layer cell for attempts of 300 nL blood
samples.

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Gold nanoparticles have been widely employed for biomaterials immobiliza-

tion for the efficiency retaining of their activity and their electron transfer im-
provement for the application in the arrangement of biosensors. Au nanopar-
ticles are biocompatible materials that can interact strongly with a certain num-
ber of proteins by promoting greatly the direct electron transfer in the redox
reactions of proteins. The enzyme based hydrogen peroxide sensors involving
Au nanomaterials can work with or without mediators. Table 1 and Table 2
summarize the sensing performance parameters of some examples for enzyme
based H2O2 sensors involving Au nanomaterials with or without mediators [26].
The percentage was obtained by comparing the response current after a period
of storage with the original response current of the modified electrode.

Table 1. Examples of enzymatic nano-Au based mediator H2O2 biosensor [4].

Immobilization mode Linear range Detection limit

Sensing materials Stability References
of mediators (µM) (µM)
HRP-labeled Au colloids In test solution 0.39 - 330 0.15 90% (2 weeks) [27]
Au/SPAN-HRP-CS In test solution 10 - 2000 1.6 85% (1 week) [28]
HRP-nano-Au In test solution 12 - 1100 6.1 75% (5 weeks) [29]
HRP/nano-Au/Thi/p-ABSA Adsorption 2.6 - 8800 0.64 70% (1 month) [30]
HRP-[1]n Adsorption 0.15 - 8600 0.07 85% (30 days) [31]
CSHMs/HRP-GNPs-Fe(CN)6 3−/4
-CSHMs Entrapment 3.5 - 1400 0.8 78% (1 month) [32]
HRP-GNPs/Thi/GNPs/MWCNTs-Chits Electropolymerization 0.5 - 1500 0.0375 97% (2 weeks) [33]
HRP/GNPs-Thi/Chit Covalent linking 0.1 - 100 0.05 92% (20 days) [34]
Hb/CMCS-GNPs/TiO2-PTATB Covalent linking 1.4 - 1600 0.37 94% (20 days) [35]

MWCNTs, multi-walled carbon nanotubes, SPAN, self-doped polyaniline nanofibers, CS or Chit chitosan, HRP, horse-radish peroxidase, ABSA, amino-
benzene sulfonic acid, CSHMs, chitosan/silica sol-gel hybrid membranes, GNPs or AuNPs gold nanoparticles.

Table 1 shows that the mediator based hydrogen peroxide biosensors could
hold a larger linear range and the very low detection limits of about 0.1 - 0.01
µM.

Table 2. Examples of enzymatic nano-Au based mediatorless H2O2 biosensor [4].

Linear Detection
Sensing materials range limit Stability
(µM) (µM)

Hb-GNACs 1 - 140 0.93 93.6% (1 month)

HRP/AuNPs/poly(St-co-AA)
8 - 7000 4 97.8% (60 days)
nanospheres

HRP/AuNPs 8 - 3000 2 83% (12 weeks)

HRP/{Au/Cys}4/Nf 1.6 - 2400 0.5 86% (2 weeks)
HRP/AuNPs/sol-gel 5 - 10000 2 No change 120 days
cyt. c/nanoporous Au/ITO 10 - 12000 6.3 Constant for at least 1 month
HRP/3D-PAMAM-Au NC 18 - 20800 6.72 82% (4 weeks)

Hb/Au nanoflowers/CNTs/GCE 1 - 600 7.3 -

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U. Nestor et al.

Continued
HRP/GNPs-TNTs 5 - 1000 2.1 90% (3 days)
HRP/CaCO3-AuNPs/ATP/Au 0.5 - 5200 0.1 96.4% (30 days)
HRP/AuNPs/CdTe-CdS/G-AuNP 0.0001 - 0.012 0.000032 97.2% (2 weeks)
Au/TiO2 0.1 - 12000 0.45 No changes half a month

Cat/AuNPs/graphene-NH2 0.3 - 600 0.05 95% (15 days)

Hb/GNPs/Hb/MWNT 0.21 - 3000 0.08 85% (1 month)

Hb/Au@Fe3O4 3.4 - 4000 0.67 92% (1 week)

Hb/C@Au 50 - 13500 1.67 93.6% (1 month)

GNACs, gold nanoparticle-assembled capsules, St-co-AA, styrene-co-acrylic acid, AuNPs or GNPs: Au na-
noparticles, 3D-PAMAM-Au NC, three-dimensional polyamidoamine Au nanocomposite, Hb, hemoglobin,
MWNTs, multi-wall nanotubes, G, graphene.

Table 2 shows that the mediatorless based hydrogen peroxide biosensors

could hold a greater extent linear range and the very high detection limits than
the ones with mediators.
The sensing parameters of this electrode were improved by the introduction
of metal oxides nanomaterials and they are listed and compared in Table 3. The
functionalized glucose oxidase, GOx, enzyme based biosensor involving Au NPs,
chitosan, conducting polymers, CNTs, graphene and other metal nanomaterials
shown a high sensing performance. The noble metals with a good resistance to
oxidation like Au and Pt are better to be functionalized on electrode while for
other metal it is better to use the metal oxide nanomaterials because the sensing
performance parameters of nonoxide metal nanomaterials based electrodes were
destroyed and the respective layer of insulating native oxide is obtained at its
sensing interface. The detection range GOx operated enzyme glucose sensors
based on Au NPs was extended from 10 µM to 52 mM with a very low detection
limit of less than 0.0986 µM after incorporation of some other nanomaterials like
for example molybdenum sulphide, carbon nanotubes and conducted polymers.
Nevertheless, the results from the recent experimental studies show that the com-
mercial applications are obstructed by expensiveness of enzymes, critical condi-
tions of operation, complicated immobilization procedure and lower stability.
The stability of an enzyme based electrode sensor depends on environmental
storage conditions such as pH, humidity, temperature, ionic detergents and oth-
er toxic chemicals that affect significantly the activity of immobilized glucose oxi-
dase enzyme [14].

Table 3. Performance parameters of glucose oxidase functioned enzyme glucose sensor

based on nanomaterials [3].

Retention
Linear Detection Sensitivity Response
Materials period References
range (µM) limit (µM) (µA/mM) time (s)
(days)

70
Au NPs-Pb NWs 5 - 2200 2 135.5 <5 [36]
(80.7%)

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 U. Nestor et al.

Continued

120
Au NPs/graphene 43.6 - 261.6 8.9 64 - [37]
(80%)

27
Au NPs/MoS2 10 - 300 2.8 - - [38]
(89%)

30
Au NPs/CNTs 500 - 8000 - 16.6 <10 [39]
(94.6%)

60
Au NPs/GR-MWNTs 10 - 52,000 4.1 - 950 0.695 - 0.238 - [40]
(94.37%)

Au NPs/DTP-Ph-NH2 100 - 25,000 50 - - [41]

Au NPs/DTP-
50 - 1000 0.0986 - -
Ph-Ph-NH2

10
Au nanodendrite 28 - 8400 7 47.76 -3
(84%)

ZnO NF 250 - 19,000 1 70.2 <4 120 [43]

40
ZnO/chitosan NFs 200 - 12,000 - - 0.016 3 [25]
(85%)
21
Porous ZnO film 200 - 5600 10 50.58 <3 [43]
(94%)
ZnO NRs/prickly 15
1000 - 15,000 40 0.097 <6 [44]
ZnO-Cu (95%)
45
ZnO NRs 10 - 23,000 0.1 110.76 <1 [45]
(94%)
10
CP NFs 125 - 11,250 - 1900 30 - 35 [46]
(85%)

GOx, glucose oxidase, NPs, nanoparticles, NWs, nanowires, NFs, nanofibers, NRs, nanorods, CP, conduct-
ing polymers, CNT, carbon nanotubes, MWNTs, muti-wall carbon nanotubes, NBs, nanobelts, G, graphene,
GO, graphene oxide, IL, ionic liquid, MFs, microfibers, NTs, nanotubes, MWs, microwires, NCs, nanocubes.

Hydrogen peroxide is the very important byproduct of glucose and its deter-
mined concentration is helpful in determining the glucose content in the blood.
Various nanomaterials from metals and metal oxides are involved to improve
sensing parameters of the biosensors used in the detection of hydrogen perox-
ide/glucose based on the glucose oxidase enzyme or on the horse-radish perox-
idase enzyme. These improved parameters are mainly sensitivity, linear range,
detection limit, and response time and retention period. Table 4 enumerated
and compared the distinctive sensing performance parameters of the enzyme
based H2O2 sensors involving nanomaterials are shown in Table 3 [4].

Table 4. Performance parameters of H2O2 sensor based on nanomaterials.

Linear Detection Retention

Sensitivity Response
Materials range limit period References
(µA/mM) time (s)
(µM) (µM) (days)

Au/TiO2 NTs 5 - 40,000 2 ≈170 <5 21 (95%) [47]

Au NPs/BC NFs 1 - 500 <1 ≈60 <10 - [48]

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U. Nestor et al.

Continued

G/Cu2O NCs 300 - 7800 20.8 - <7 - [49]

ZnO NFs/Chitosan 10 - 1560 1 573 <3 28 (90%) [25]

G/peptide NFs-Ag NWs 50 - 5000 10.4 ≈10 - 15 (87%) [50]

Pt NPs/ OMCs 0.1 - 3200 0.08 24.43 4-5 14 (92.6) [51]

Pt/Ni nanofilm 10 - 60 2.2 200 <5 60 (>90%) [52]

Pt NPs/CNTs 100 - 75,000 0.61 ≈0.07 - 15 (95.5%) [53]

WO3/TiC/carbon NFs 10 - 500 0.003 386 <15 30 (90%) [54]

Co3O4 NPs 0.4 - 200 0.24 389.7 <4 60 (−100%) [55]

NTs, nanotubes, BC, bacteria cellulose, NFs, nanofibers, G, graphene, NCs, nanocubes, NWs, nanowires,
NPs, nanoparticles, OMCs, ordered mesoporous carbons, CNTs, carbon nanotubes.

The oxidation of H2O2 is also catalyzed by the heme enzyme horse-radish pe-
roxidase (HRP) during the recycling of hydroquinone and benzoquinone in
which the reduction current is increased significantly as shown in Figure 13 of
the mechanism of phosphate buffer solution enzyme-based hydrogen peroxide
biosensor [4].

Figure 13. Mechanism of PBS enzyme-based H2O2 sensor [4].

As proposed by Kafi and his co-workers [47], a novel biosensor used to de-
termine the concentration of hydrogen peroxide as well as of glucose is the one
based on Gold-modified TiO2 NTs which resulted to their modification with
HRP and chitosan. It is highly advantageous due to its high quality detection
performance and unique properties of TiO2 NTs including the effective bio-
compatibility, cheap, environmental friendly, and chemical/thermal stability. Its
sensing performance parameters are shown in first row of Table 3 and it must
be kept in 0.1M PBS with a pH = 7 at 4˚C to maintain its bioactivity. Even if the
enzyme based biosensors show the high performance activity in detecting and
determining the concentration of H2O2 and glucose but the high cost, tempera-
ture susceptibility, pH and electrochemical interfering species as well as their
natural instability will hinder their commercial applications. Therefore, the syn-
thetic enzyme based biosensors are explored and carried out in the future works
as the best feasible solution [4].
Table 5 indicates the analytical performance of Ag nanoparticles based non-
enzymic H2O2 sensors. As observed from Table 5, they also exhibited favourable
catalysis effect with wide linear range and achieved the detection of H2O2 at low
potential. Furthermore, the non-enzymicH2O2 sensors obtained relatively higher

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 U. Nestor et al.

stability than enzyme biosensors (as shown in Table 5). However, the non-enzy-
mic H2O2 sensors generally hold the detection limits of 10−5 - 10−7 M, which is
not as low as 10−7 - 10−8 M for the enzyme H2O2 sensors [5].

Table 5. Examples of Ag nanoparticles based non-enzymic H2O2 sensors [5].

Sensing Detection
L. R. (M) D. L. (M) Stability
material potential (V)

Ag-DNA/GCE −0.45 2.0 × 10−6 - 2.5 × 10−3 6.0 × 10−7

89.92%
AgPs-SWCNT electrode −0.3 1.6 × 10−5 - 1.8 × 10−2 2.76 × 10−6
(30 days)

Ag NPs/ZnO/GCE −0.25 2.0 × 10−6 - 5.5 × 10−3 4.2 × 10−7

Ag NPs/CHIT-GO/ 98.8%
−0.4 6.0 × 10−6 - 1.8 × 10−2 7.0 × 10−7
cysteamine/Au (30 days)

AgNPs/PoPD/ 98.2%
−0.5 6.0 × 10−6 - 6.73 × 10−2 1.5 × 10−6
GCE (15 days)

no change
Ag NPs/ATP/GCE −0.3 1.0 × 10−5 - 2.15 × 10−2 2.4 × 10−6
(2 weeks)

95%
Ag/GC electrode −0.44 4.0 × 10−5 - 9.0 × 10−1 1.0 × 10−5
(100 days)

PQ11-AgNPs/GCE −0.30 1.0 × 10−4 - 1.8 × 10−1 3.39 × 10−5

Ag/GN-R/GCE −0.40 1.0 × 10−4 - 4.0 × 10−2 2.8 × 10−5

Ag@PMPD-Ag/GCE −0.30 1.0 × 10−4 - 1.7 × 10−1 2.5 × 10−6

SWCNT/AgNPs/CCE −0.20 1.0 × 10−5 - 8.0 × 10−3 2 × 10−7

92%
Ag-3D/graphite electrode 0.6 5.0 × 10−5 - 2.5 × 10−3 1.0 × 10−6
(30 days)

Polydopamine-Ag 90%
−0.30 9.2 × 10−5 - 2.0 × 10−2 1.97 × 10−6
hollow/GCE (10 days)

Ag/graphene/GCE −0.3 1.0 × 10−4 - 1.0 × 10−1 5.0 × 10−7

AgPs or Ag NPs silver particles, SWCNT, single-walled carbon nanotube, CHIT, chitosan, GO, Graphene
oxide, PoPD, poly(ophenylenediamine), PEDOT, poly[3,4-ethylenedioxythiophene], ATP, attapulgite, GN,
graphene nanosheet, PMPD, poly(m-phenylenediamine), MWCNT or MWNTs: multiwalled carbon nano-
tubes, CCE, carbon-ceramic electrode, DA, dopamine, AA, ascorbic acid, UA, uric acid, AC, acetaminophen.

In the recent study conducted by Tavakkoli and his team [56], they have pre-
pared a nanobiocomposite from multiwalled carbon nanotubes and zein nano-
particles. It was dispersed in water/ethanol and drop cast onto a glassy carbon
electrode. The modified electrode can be used for electroreduction of H2O2 (typ-
ically at a working potential of −0.71 V vs. Ag/AgCl). The electrochemical prop-
erties of the electrode were performed by cyclic voltammetry Figure 14(a), li-
near sweep voltammetry Figure 14(b), chronoamperometry Figure 14(c), and
electrochemical impedance spectroscopy Figure 14(d) Response to H2O2 was li-
near in the 0.049 to 22 μM concentration range, and the detection limit was 35
nM at pH 7.0. The sensor was successfully utilized for the measurement of H2O2
in a synthetic urine sample, and for monitoring the release of H2O2 from human
dermal fibroblasts and human hepatocellular carcinoma cells.

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U. Nestor et al.

Figure 14. (a) The CVs of GC/ZNBs/fMWCNTs in the absence and presence of 5.0
μMH2O2 in the range of 1.0 to −1.0 V vs. Ag/AgCl, at 100 mV∙s−1; (b) The CVs of bare
GCE and GC/ZNBs/ fMWCNTs in the absence ((a) and (b)) and presence ((c) and (d)) of
5.0 μM H2O2, respectively, at 100 mV∙s−1; (d) Chronoamperometry of (a) GC/ZNBs, (b)
bare GCE, (c) GC/ MWCNTs and GC/ZNBs/fMWCNTs in 5.0 μM H2O2 at −0.75 V. (d)
Amperometry of 1.0 - 10.0 μM H2O2 at (a) bare GCE and (b) GC/ZNBs/fMWCNTs. All
experiments were performed in Ar-saturated phosphate buffer (pH 7.0) [56].

9. Conclusions
The combination of the platinum, hydrogen peroxide membrane, glucose oxidase
enzyme and 0.015 µm polycarbonate membrane makes the electrode which is
very useful for glucose sensing in many practical applications such clinical anal-
ysis, industrial and environmental processes.
A novel enzyme based hydrogen peroxide probe was developed and widely
studied. This constructed electrode has showed a great performance in terms of
its basic characteristics such stability reproducibility, sensitivity, response time,
reusability, large linear range, and high selectivity without electrochemical inter-
ferences resulting from the protection of the platinum electrode surface by new
blocking membrane.
The use of carbon nanotubes as nanomaterials has led to the fabrication of
home blood glucose testing sensor used by many diabetic patients because it is
easy-to-use device.
Even if the enzyme based biosensors show the high performance activity in
detecting and determining the concentration of H2O2 and glucose but the high
cost, temperature susceptibility, pH and electrochemical interfering species as
well as their natural instability will hinder their commercial applications. There-
fore, the synthetic enzyme based biosensors are explored and carried out in the
future works as the best feasible solution. Metals and Metal oxides nanoparticles
have been widely employed for biomaterials immobilization for the efficiency
retaining of their activity and their electron transfer improvement for the appli-
cation in the arrangement of biosensors and they can work with or without me-

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 U. Nestor et al.

diators. Besides, the use of metals and metal oxides nanomaterials, the bimetallic
nanoparticles including Au/Ag, Au/Pt, Pt/Pd, Pt/Ir, Pt/Cu, Pd/Cu, Rh/Pd and
Ru/Rh were also utilized to construct an enzyme based H2O2 biosensor for sens-
ing and determining glucose and H2O2. Therefore, the combination of nanoma-
terials and the miniaturized sensing electrodes may offer a high performance
and adequate platform for H2O2 detection.

Acknowledgements
Authors are grateful to the University of Rwanda, School of Science and De-
partment of Chemistry for providing the facilities.

Conflicts of Interest
The authors declare no conflicts of interest regarding the publication of this pa-
per.

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