1.1 The characteristics and application of biosensors
The biosensors are analytical devices that use immobilized biomolecules to recognize the
chemical and biological substances and convert non-electrical enzymatic responses to the
electric signals, which can be detected by the underneath transducers. They consist of two key
parts, the recognition element and the transducer. The recognition element is basically a
biological substance that could be a protein, enzyme, receptor, antibody, nucleic acid, cell or
even a tissue. The transducer is a hardware instrument component that converts biological,
chemical and physical signals into electric signals [1]. Biosensors exhibit the great usage in
determining serum components, metabolites, pollutants, hormones, drugs, food additives,
pathogens, microbes, toxins, and many more. Biosensors exhibit many advantages over the
conventional analytical methods such as quick response, high sensitivity, instant output, small
sample volume, reliability, ease of use and low cost [2, 3-4].
The applications of biosensors are mainly focused on four categories: (i) Medical
monitoring and clinical diagnosis: this type of biosensors are highly demanded by the clinics
and health care system for the diagnosis and monitoring of the progression diseases and the
therapeutic effect of medicines [5]; (ii) Bioreactor process monitoring: bioreactors are
essential in fermentation industries for mass production of chemicals, biochemicals and
biological substances. To better control the process it is necessary to real-time monitor some
key substances generating or consuming in the this process, such as glucose, ammonia, carbon
dioxide, alcohol, antibiotics and other ingredients [6]; (iii) Environmental monitoring and
protection: biosensors are suitable for monitoring the discharging of pollutants , such as toxic
substances in air or water, from manufacturing factories [7, 8]; (iv) Food and Agricultural
monitoring: for soil and crop pesticide residue detection and drug residues detection in meat
[9, 10].
Biosensors can be divided into two main types based on the recognition element used in
the development. First is thebioaffinity sensors, in which the biological components
recognize target substance by interaction or shape change, which generates signals of mass
change and heats (such as hormones, proteins, antigens or antibodies) [11]. The other is the
biocatalysis sensors, in which the target molecules are recognized by enzymes that catalyze
certain reactions, which can be converted to electric signals [12]. There are three classes of
biocatalysis biosensors based on the signal generated by the catalysis, including
electrochemical biosensors, optical-based biosensors and thermal biosensors. The
electrochemical biosensors detect the electroactive components, the reactants or products, or
ions of the enzymatic reactions in the enzymatic reactions by electrochemical responses or
conductivity [13, 14]. Optical-based biosensors are fabricated to detect the emission of
fluorescence or phosphorescence, ultraviolet or visible light, and the chemiluminescence or
bioluminescence generated during enzymatic reactions [15-17]. Surface plasmon resonance
(SPR) biosensor is another example of optical-based biosensor design, by which the
interaction between molecules can be detected. SPR is the collective oscillation of electrons
on the metal thin film by the incident light photons, which exhibit frequency matching the
oscillation frequency of electrons on the metal surface [18, 19]. In addition to traditional
precious metals, gold nanoparticles can effectively increase the sensitivity of SPR biosensor
and can also enhance SPR for biological measurement of small molecules or ions [20]. The
SPR biosensor has advantages of high sensitivity, low detection limit and label-free [21].
Thermal-detecting biosensors are useful in detecting temperature changes of almost all
chemical reactions, interaction between molecules and acid neutralization [22, 23].
1.2 Hydrogen peroxide and its determination
Hydrogen peroxide (H2O2) is one of the side products of the energy metabolism in the
mitochondria [24] and is also produced by neutrophils and leukocytes to fight microorganisms,
remove damaged tissues and to start the inflammation during the wound-healing process [25].
Killing the invaded microorganisms at the wound sites should buy time for the immune
system to heal [26].
H2O2 may be used as a food additive because of its bactericidal and fungicidal activity.
Interestingly, it also exhibits a bleaching effect. Therefore, H2O2 was popular to be used by
the manufacturers to prevent the darkening of products under the normal storage conditions.
Apparently, the consumers are more willing to buy foods or products with present appearance
or color texture. This consuming behavior prompts manufacturers to use H2O2 to improve the
appearance of food or products. H2O2 is also one of the products of oxidases and is broadly
utilized as a detection target in different areas, such as clinical diagnosis, food analysis,
pharmaceutical and environmental analyses [27]. However, long-term exposure to H2O2 may
cause a variety of lesions, such as gastrointestinal ulceration, mucosal inflammation, due to
the DNA damage and gene mutations in human genome [28]. In addition H2O2 may accelerate
the aging process of the human body and play a role in the development of senile dementia,
especially Alzheimer’s dementia [29].
Determination of H2O2 is important, because it is not only broadly used in many foods
and cosmetic products, but also an essential component in pharmaceutical, clinical, industrial
and environmental analyses [30]. Furthermore, it is one of electroactive substance produced in
the enzymatic reactions of redox enzymes [31]. Conventionally, H2O2 was detected by
peroxidase in the presence of various chromogens, by which the color products or
chemiluminescence could be generated and detected. However, these methods are often
time-consuming, highly prone to interference, and costly.
Electrochemical methods, especially the amperometric biosensor, exhibit advantages,
such as simple, high efficiency and high sensitivity [32] that may overcome the above
drawbacks for determination of H2O2.Under the reductive potential the H2O2 can be reduced
into H2O and generates response currents that allow us to quantitate the amount of H2O2
generated in reactions. The rate of H2O2 generation during the enzymatic reactions may
correlate to the reaction rate of corresponding oxidases [33].
1.3 Surface treatment of screen-printed carbon electrode (SPCE)
Screen-printing carbon electrode (SPCE) was broadly adopted by manufacturers for the
production of the test strips of glucose meter due to its simplicity, inexpensive and easy mass
production [34]. With these characteristics a variety of the amperometric biosensors were
developed for a wide variety of applications by using SPCE in the enzyme electrode
preparation [35, 36].
The SPCE is generated by screen-printing the carbon ink that contains ultrafine graphite
powder, organic oil, pasting binder and other minor reagents, onto a solid support. However,
the surface of the SPCE is often covered by the organic oil, paste binder and some pollutants,
which may block the access of reactants to the graphite particles or attenuate the electron
transfer on the surface. Some simple electrochemical surface treatments have been proposed
to remove the blocking substances on SPCE, such as pre-anodization [37, 38],
electrochemical cycling [39] and oxygen plasma treatment [40]. Surface treatment of the
electrode with electrochemical methods can not only remove surface organic contaminants
and impurities [37-40], but can possibly adding some oxygenated functionalities, such as CO,
O–C–O and O–C O [40]. Electrochemical surface treatment has been demonstrated to be
successfully enhanced electrocatalytic activity of electrochemical biosensors [37-41]. The
presence of surface oxygenated functionalities may provide adequate micro-space for the
electrode surface at the nano level and may contribute to the increase in electrochemical
activity of the electrode [40].
1.4 Diazotization and electro-addressing
Diazotization and coupling reactions are widely applied in chemical industry for organic
synthesis, such as in the synthesis of dyes and pigment [42]. It is because diazonium salt
exhibits a very lively chemical property and can occur in a variety of reactions and react with
a variety of materials. Diazotization reaction is exothermic with a fast reaction rate and, hence,
can be carried out under ambient condition. Once the diazonium salt solution is prepared the
next experimental procedure should be conducted as soon as possible [43].
Diazonium organic salt has been extensively used for the surface modification. It is not
only because of its broad selectivity to wide range of metal and semiconductor materials but
also because of its simplicity, efficiency, and speed [44]. Depends on the functional groups it
carried, the diazonium organic salt-modified electrode can be functionalized with phenolic,
imidazole, or amino group, achieving different types of surface derivatization [45]. The
chemistry involved in the diazonium-based surface modification including (i) the diazotation
reaction of an aniline derivative to form an aryl diazonium; (ii) generating an aryl radical of
this latter species to form a covalent bond to the surface of electrode by the electrochemical
reaction. [46]; (iii) the surface of the electrode can be functionalized by functional groups
(such as -SH) associated with the diazonium salt [47].
In this thesis, the surface SPCE was first functionalized with carboxylic groups by a
layer of 4-aminophenylacetic acid (CMA) through electrochemical deposition. The carboxylic
groups on the surface of SPCE were then activated by EDC/NHS. Immediately, gold
nanoparticles (AuNPs) or the cysteamine-modified AuNPs were coated on top of the CMA
layer by either physical absorption or covalent bonding. The electrochemical properties of
CMA-modified, AuNPs-coated SPCE were characterized and analyzed [48].
1.5 Gold nanoparticles (AuNPs) and its applications
Nanotechnology has been widely adopted and integrated with the biosensing technology
recently for the detection biomolecules [49]. Nanomaterials, materials with size below 100
nm, have recently attracted eyes of scientists due to their unique optical, electrical and
magnetic properties. Silver (Ag) and gold (Au) nanoparticles are two types of metal
nanomaterials that have been used in the construction of biosensors due to their unique
chemical and physical properties and the surface plasmon resonance (SPR) activity. Gold
nanoparticle (AuNP) exhibits its own unique surface plasmon resonance absorption peak [50].
The size of synthesized AuNPs can be controlled in a range of 1-100 nm by the salt
concentration and temperature [51, 52]. AuNP has been widely used in numerous areas, such
as acting as electron microscopy contrast reagents, mediator of chemical and biochemical
sensors, electrical conduction medium, dyes and as catalysts. AuNPs are good bridging
substance to link biomolecules, such as enzymes, proteins, and nucleic acids via their surface
charges or via surface functionalization. For example, the negatively charged substances can
associated with AuNPs directly via their surface positive charged. The AuNPs can be easily
functionalized through the sulfur-containing linkers. The sulfur can be readily bound to the
AuNP surface by a sulfur-Au bond. AuNPs have been used for construction of biosensors
because of their excellent ability to immobilize biomolecules to retain the biocatalytic
activities of those biomolecules and to enhance the electrochemical redox responses of the
biosensors [53]. AuNPs have some fascinating properties, such as favorable micro
environment, good biocompatibility and high electron transfer ability (Figure 1), that make
them suitable for protein immobilization in biosensors fabrication [54]. In this study the
electrocatalytic activity of SPCE after modified by AuNPs with difference configurations was
investigated. In the first arrangement the SPCE was first modified with a thin layer of
cysteamine-coated AuNPs (CNH2AuNPs), followed by immobilizing the glucose oxidase
(GOx) on top of AuNPs, forming a sandwich-like structure. In the second arrangement the
SPCE was modified with the mixture of CNH2AuNPs and GOx. The electrochemical
responses of H2O2 and glucose on the above enzyme electrodes were then studied [55]. The
effect of gold nanoparticles size or surface modification on the electrochemical responses to
H2O2 was also investigated [56].