SECTION 1
SI. No. Content Page No.
0. PRE- INTRODUCTION
a. What is Alzheimer's disease? 1
b. Ten warning signs of Alzheimer's disease 2
c. Treatment for Alzheimer's disease 3
d. Preventive steps for Alzheimer's disease 4
e. Neuropathology of Alzheimer's disease 5
f. Memory defects in Alzheimer's disease and acetylcholine 8
1. INTRODUCTION 9
1.1. Acetylcholine 9
1.1.1. What is ·acetylcholine? 9
1.1.2. Chemistry and electrochemistry of acetylcholine 10
1.1.3. Releasing sites of acetylcholine 12
1.1.4. Pharmacology of acetylcholine 13
1.1.5. Current status of available sensors of acetylcholine 15
1.2. Biosensors 16
1.2.1. What are biosensors? 17
SI. No Content Page No.
1.2.2. Characteristics of biosensors 19
1.2.3. Types and applications of biosensors 20
1.2.4. Metal based electrodes as biosensors 24
1.2.5. Nickel based electrodes as sensors of acetylcholine 25
1.3. The approach of the present work 26
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0. PRE-INTRODUCTION
a. What is Alzheimer's disease?
Alzheimer's disease (AD) was first discovered in 1906 by Alois
Alzheimer [1]. Alzheimer's disease usually affects people above 65 years of
age, although it can appear in people as young as 40, especially in some familial
forms of the disease [2]. Approximately half of men and women over the age of
85 have Alzheimer's disease [1]. Women are more affected by Alzheimer's
disease than men and nearly 1,00,000 people die of complications from
Alzheimer's annually, making it the fourth largest killer of adults in the United
States [1]. Currently there are 5 million Americans with Alzheimer's disease
and it is expected that 13 million will have it by 2050, if no cure is found [3].
Caring for and making decisions on behalf of a loved one with Alzheimer's
disease can be emotionally and physically draining [4]. The cost of Alzheimer's
is high, not only in human suffering but also in economic affairs. Over ninety
billion dollars alone is spent annually for the care of patients with Alzheimer's
disease in United States [ 1]. One cannot care for loved one if they are not well
themselves. It is a healthy and appropriate choice to seek help from others [3].
Alzheimer's disease, the most common form of dementia, is a serious
disease that is usually associated with progressive, degenerative, and
irreversible neurological disease with no cure [1,3,5]. The onset of Alzheimer's
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disease usually begins with periodic forgetfulness that may not be noticeable
[5]. It impairs memory, thinking and behaviour [6]. Alternative names of
Alzheimer's disease is Senile dementia I Alzheimer's type (SDAT) [6]. There
are two types of Alzheimer's disease - early onset and late onset. In early onset
Alzheimer's disease, symptoms first appear before 60 years. Early onset of
Alzheimer's disease is much less common, accounting for only 5 - 10% of
cases. However, it tends to progress rapidly. Late onset Alzheimer's disease, the
most common form of the disease, develops in people aged 60 and above and is
thought to be less likely to occur in families [ 6].
b. Ten warning signs of Alzheimer's disease
. As the disease progresses, a variety of symptoms may become
apparent. Important symptoms of Alzheimer's disease include [2,3,5,6],
(i) Memory loss - the most common early sign.
(ii) Difficulty in performing familiar, everyday tasks.
(iii) Problems with language.
(iv) Disorientation to time and place.
(v) Poor or decreased judgement.
(vi) Problems with abstract thinking.
(vii) Misplacing things.
(viii) Change in mood or behaviour.
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(ix) Change in personality.
(x) Loss of initiative.
These losses are progressive and can last anywhere from 3-20 years [3].
c. Treatment for Alzheimer's disease
Millions of dollars have been spent on Alzheimer's disease research
and yet still no cure has been discovered. There is a correlation between ageing
and contracting Alzheimer's disease, but no way to tum back the clock. Genetic
factors make treating Alzheimer's very hard [5]. The goals in treating
Alzheimer's disease are to,
• Slow the progression of the disease.
• Manage behavioural problems, confusion and agitation.
• Modify the home environment.
• Support family members and other caregivers [6].
Some studies show that exercise, certain drugs, and nutritional
choices may slow the development of Alzheimer's disease and are worth
exploring [5].
There are medications currently available that have been approved
through the U. S. Food and Drug Administration that can slow the progression
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of Alzheimer's disease. These medications do not cure or improve the disease,
but slow the progression of memory loss, and allow people to enjoy a htgher
quality of life for a longer time. There are also medications available which help
to reduce some of the behavioural disturbances that can coincide with
Alzheimer's disease, including depression, sleeplessness and agitation [3].
Alternative treatments for Alzheimer's disease that have limited research
backing them include; dynamic psychotherapy, aromatherapy, music therapy,
usage of Vitamin E and ginkgo biloba extract [3]. Ginkgo biloba is a herb
widely used in Europe for treating dementia. It improves blood flow in the brain
and contains flavanoids (plant substances) that acts as antioxidants [ 6].
d. Preventive steps for Alzheimer's disease
Although there is no proven way to prevent Alzheimer's disease,
there are some practices that may be worth incorporating into the daily routine.
• Consume a low - fat diet.
• Eat cold - water fish (like tuna, salmon and mackerel) rich in
omega - 3 fatty acids, at least 2 to 3 times per week.
•
•
•
Maintain a normal blood pressure .
Stay mentally and socially active throughout your life .
Reduce intake of linoleic acid found in margarine, butter and
diary products.
Introduction 5
• Increase antioxidants like carotenoids, Vitamin E and Vitamin C
by eating plenty of darkly coloured fruits and vegetables [ 6]. ·
e. Neuropathology of Alzheimer's disease
The risk factors for Alzheimer's disease include
• Age and family history.
• Long-standing high blood pressure.
• History of head trauma.
• High levels of homocysteine ( a body chemical that contributes to
chronic illness such as heart disease, depression and possibly
Alzheimer's disease).
• Female gender - because women usually live longer than men,
they are more likely to develop Alzheimer's disease [6].
Alzheimer's disease is a degenerative disease of nerve cells in the
cerebral cortex that leads to atrophy of the brain and senile dementia. The
disease is characterized by abnormal accumulation of plaques and by
neurofibrillary tangles (malformed nerve cells). The plaques result from the
release and accumulation of excessive amounts of beta - amy loid proteins [2, 7].
The neurofibrillary tangles prevent transportation of synthesized products with
in the cell body to organells and target sites. The plaques and neurofibrillary
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tangles prevent proper transmission of electrochemical signals necessary for
information, processing and retrieval. The plaques also suffocate neuron's by
inhibiting proper blood supplies from reaching them [2]. Although these
changes occur to some extent in all brains with age, there are many more of
them in the brains of people with Alzheimer's disease [6].
The pathogenesis of Alzheimer's disease has been linked to a
deficiency in the brain neurotransmitter acetylcholine [8]. The correct balance
of neurotransmitter is critical to the brain [6]. This was based on the observation
that corrected cholinergic system abnormalities with intellectual impairment
[8,9]. The memory loss in Alzheimer's disease is due to decline in neurons in
the cortex. Figure 0.1. shows a healthy normally aged neuron and one in the
later stages of Alzheimer's disease.
Introduction------------------------ 7
(a) (b)
Figure 0.1. (a) A healthy normally aged neuron and (b) neuron in the later stages of
Alzheimer's disease.
Figure 0.2. shows a healthy normally aged brain and one in the later
stages of degeneration from Alzheimer 's disease. Extensive spaces in the
fissures and sulci of the brain reflects the loss of brain tissue caused by dead and
dying neurons. The only sure way to confirm Alzheimer's disease remains to
examine brain tissue under a microscope, which is usually done upon an
autopsy [1].
(a) (b)Figure 0.2. (a) A healthy normally aged brain and (b) brain in the later stages of
Alzheimer's disease.
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f. Memory defects in Alzheimer's disease and acetylcholine
Over the years, both evidence for and challenges to the relationship
between acetylcholine dysfunction and Alzheimer's disease have been put
forward [8]. Acetylcholine is critical for an adequately functioning memory, and
it is major target of scientists who are working on treatments for memory
deficit, like those found in Alzheimer's disease. Acetylcholine is also used in
the brain, where it tends to cause excitatory actions. The glands that receive
impulses from the parasympathetic part of the autonomic nervous system are
also stimulated in the same way [10]. Attempts at correcting acetylcholine
deficiency in the brain of affected individuals produced the first licensed
medication for the symptomatic treatment of Alzheimer's disease in the form of
acetylcholinesterase inhibitors (AChEls) [8]. One of the characteristic changes
that occur in Alzheimer's disease is the loss of memory and the loss of
acetylcholinesterase (AChE) from both cholinergic and non-cholinergic neurons
of the brain. However, AChE activity is increased around amyloid plaques
[1,11]. This increase in AChE may be of significance for therapeutic strategies
using AChE inhibitors.
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1. INTRODUCTION
1.lAcetylcholine
Acetylcholine was first identified in 1914 by Henry Hallett Dale for
its actions on heart tissue. It was confirmed as a neurotransmitter by Otto 1:oewi
who initially gave it the name Vagustoff because it was released from the vagus
nerve. Both received the 1936 Nobel Prize in Physiology or Medicine for their
work [10].
1.1.1. What is Acetylcholine?
Acetylcholine often abbreviated as ACh, was the first chemical
compound identified as neurotransmitter [ 1 O]. This neurotransmitter can be
found in brain, neuro-muscular junctions, spinal cord and both in the post
ganglionic terminal buttons of the parasympathetic division of the autonomic
nervous system and the ganglia of the autonomic nervous system [ 1]. It is
released by stimulation of vagus nerve that alters heart muscle contractions.
Acetylcholine binds to acetylcholine receptors on striated muscle fibres, opened
channels in the membrane. Sodium ions then enter the muscle cells stimulating
muscle contraction [10]. It is important when it comes to the movement of other
muscles as well. Acetylcholine induces movement by the locomotion of an
impulse across a nerve that causes it to release neurotransmitter molecules onto
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the surface of the next cell. This causes stimulation on that cell. After this
process, the acetylcholine is broken into acetate and choline. These travel back
to the first cell to be recycled into acetylcholine to start the process again.
1.1.2. Chemistry and electrochemistry of acetylcholine
Acetylcholine is an ester of acetic acid and choline with chemical
formula CH3COOCH2CH2W(CH3)3 . This structure is reflected in the systematic
name, 2 - (acetyloxy) - N, N, N - trimethyl ethanaminium ion [10]. The
skeletal structure of acetylcholine is,
Acetylcholine is synthesized in certain neurons by the enzyme
choline acetyltransferase from the compounds choline and acetyl - coenzyme A.
The chemical reactions in brain for the production of acetylcholine(ACh) is,
Introduction-----------�-----------11
CH3
I+ II H C-C-S-CoA
3 + H
3C-N -C8i-CH
2-0H
I CH
3
Acetyl-CoA Choline
CH3
0
I+ II H C-N -Cl-l-CH -0-C-CH +HS-CoA
3 �"'2 2 3
I CH
3
Acetylcholine Coenzyme A
Cholineacetyl
transferase
The inhibition of choline acetyltransferase may lead to acetylcholine
deficiency and can have consequences on motor function. The enzyme
acetylcholinesterase converts acetyl choline into the inactive metabolites
choline and acetate [12].
+ CH3COO(CH2)2 N(CH3)3 +H20
Acetylchol inesterase
The inhibition of acetylcholinesterase leads to accumulation of
acetylcholine, which results in continuous stimulation of the muscles, glands
and central nervous system [ 1 O]. The chemistry of acetylcholine can be
abbreviated in table 1.1.
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Table 1.1. Chemistry of acetylcholine
Acety lcho line
Precursors Choline and acetyl- CoA
Synthesizing enzyme Choline acetyltransferase
Metabolizing enzyme acety lcho linesterase
Metabolites Choline and acetate
1.1.3. Releasing sites of acetylcholine
Acetylcholine is released by,
a) Neurons of central nervous system (CNS), arising from 3 key areas.
• Dorsolateral pons, which have broad targets through the brain, and
are involved in REM sleep.
• Basal forebrain, the major source of cholinergic innervations
throughout the cortex, implicated in the facilitation of learning.
• Medical septum, which projects largely to the limbic system, and
may induce rhythmic firing in the hippocampus critical to learning.
Many intemeurons of the basal ganglia are also cholinergic.
b) Some neurons of parasympathetic nervous system (PNS), including
• Motor neurons ( of somatic nervous system), causing muscle
Introduction-----------------------13
contraction of striated muscle.
• The neurons of autonomic nervous system:
o Pre and post - ganglionic parasympathetic neurons
o Preganglionic sympathetic neurons ( and also post ganglionic
sympathetic neurons, i.e., the ones that control sweating)
1.1.4. Pharmacology of acetylcholine
Like other transmembrane receptors, acetylcholine receptors (AChR)
are classified according to their pharmacology or according to their relative
affinities and sensitivities to different molecules.
• Nicotinic acetylcholine receptors (nAChR) also known as "ionotropic"
acetylcholine receptors are particularly responsive to nicotine.
• Muscarinic acetylcholine receptors (mAChR) also known as
metabotropic acetylcholine receptors are particularly responsive to
muscarine.
Nicotinic acetylcholine receptors are ionotropic receptors permeable
to sodium, potassium and chloride ions. They are stimulated by nicotine and
blocked by curare. All peripheral AChRs are nicotinic, such as those on the
heart or at the neuromuscular junction. They are also found in wide distribution
through the brain, but in relatively low numbers [10].
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Muscarinic acetylcholine receptors are metabotropic and affect
neurons over a longer time frame. They are stimulated by muscarine and
blocked by atropine, which is the poison found in the belladonna plant. Extracts
from the plant included this compound and its action on muscarinic AChRs that
increased pupil size was used for attractiveness in many European cultures in
the past. Now acetylcholine is sometimes used during cataract surgery to
produce rapid constriction of the pupil. It must be administered intraocularly
because corneal cholinesterase metabolizes topically administered ACh before it
can diffuse into the eye. It is sold by the trade name Miochol-E (CIBA vision).
Similar drugs are used to induce mydriasis (dilation of the pupil) in
cardiopulmonary resuscitation and many other situations [10].
The disease Myasthenia gravis, characterized by muscle weakness
and fatigue, occurs when the body inappropriately produces antibodies against
acetylcholine receptors, and thus inhibits proper acetylcholine signal
transmission. Drugs that competitively inhibit acetylcholinesterase (eg.,
neostigmine or physostigmine) are effective in treating this disorder [10].
Blocking, hindering or mimicking the action of acetylcholine has
many uses in medicine. Cholinesterase inhibitors increase the action of
acetylcholine by delaying its degradation. Some have been used as nerve agents
or pesticides. Clinically they are used to reverse the action of muscle relaxants,
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to treat Myasthenia gravis and in Alzheimer's disease (rivastigmine increases
cholinergic activity in the brain) [10].
1.1.5. Current status of available sensors of acetylcholine
The enzyme acetylcholinesterase (AChE) promotes the hydrolysis of
the natural neurotransmitter acetylcholine (ACh) and its inhibition (by the
blocking of active sites of the enzyme) terminates the propagation of the nerve
impulse [13, 14]. Hence, it is very important to develop a sensitive method for
the detection of cholinesterase inhibitors and acetylcholine in clinical diagnosis.
Methods for analysing acetylcholine include HPLC using an immobilized
enzyme column [15,16] and Radioimmuno assay to detect acetylcholine in very
low concentration in blood and plasma [ 15, 17]. Potentiometric measurements
employ ion selective field emission transmitter (ISFET) as sensing device and
use acetylcholinesterase to modify the sensing interface [15, 18-20] and signal
transducer antimony pH electrode with immobilization of acetylcholinesterase
(AChE) [13].
In the amperometric measurement, the acetylcholinesterase (AChE)
and cholineoxidase (ChO) are also immobilized on electrode surface [15, 21
26]. Then choline is oxidized by ChO and produces H202 detected by Pt
electrode. Multilayer enzyme networks assembled by a stepwise synthesis onto
Au electrodes are also used for sensing acetylcholine. Nickel electrodes were
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used as acetylcholine sensors in several studies [15, 27 - 29]. Other enzymatic
methods include the immobilization of various enzymes on carbon fibre '[30]
and conducting polymer [31 - 33]. Sol - gel and screen-printed electrodes used
as acetylcholine sensors were also reported [22, 34]. More recently ceramic -
based microelectrodes were also used for the detection of acetylcholine [35, 36].
1.2. Biosensors
Professor Leland C. Clark Jnr. was known as the father of the
biosensor concept. He published his work on oxygen electrode in 1956 [37, 38].
Biosensors represent a rapidly expanding field, at the present time, with an
estimated 60% annual growth rate; the major impetus coming from the health
care industry ( e.g., 6% of the western world are diabetic and would benefit from
the availability of a rapid, accurate and simple biosensor for glucose) but with
some pressure from other areas, such as food quality appraisal and
environmental monitoring. Research and development in this field is wide and
multidisciplinary, spanning over biochemistry, bioreactor science, physical
chemistry, electrochemistry, electronics and software engineering. Most of this
current endeavour concerns potentiometric and amperometric biosensors and
colourimetric paper enzyme strips. However, all the main transducer types are
likely to be thoroughly examine, for use in biosensors, over the next few years
[39].
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1.2.1. What are biosensors?
A biosensor can be defined as any substance which detect a bio
active component or any sensor that uses a biological component, such as an
antibody, enzyme or even a microorganism, to bind specifically an analyte of
interest and provide a physical signal ( e.g., · optical, amperometric, impedance)
that is in proportion to the amount of analyte [ 40]. A biosensor should convert a
biological response into an electrical signal. The schematic diagram showing the
main components of a biosensor is given in figure 1.1.
Reference
Figure 1.1. The schematic diagram showing the main components of a biosensor (a) biocatalyst (b) transducer (c) amplifier (d) processor (e) displayer (s) substrate and (p) product.
The biocatalyst (a) converts the substrate to product. This reaction is
determined by the transducer (b) which converts it into an electrical signal. The
output from the transducer is amplified ( c) processed ( d) and displayed ( e ).
The key part of the biosensor is the transducer (b ), which makes use
of a physical change accompanying the reaction. This may be,
1. The heat output ( or absorbed) by the reaction ( calorimetric biosensors)
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2. Changes in the distribution of charges causing an electrical potential to be
produced (potentiometric biosensors)
3. Movement of electrons produced in a redox reaction ( amperometric
biosensors)
4. Light output during the reaction or a light absorbance difference between
the reactants and products ( optical biosensors ), or
5. Effects due to the mass of the reactants or products (piezo-electric
biosensors) [39].
There are three so-called 'generations' of biosensors; First generation
biosensors where the normal product of the reaction diffuses to the transducer
and causes the electrical response, second generation biosensors which involve
specific 'mediators' between the reaction and the transducer in order to generate
improved response, and third generation biosensors where the reaction itself
causes the response and no product or mediator diffusion is directly involved [39].
The electrical signal from the transducer is often low and
superimposed upon a relatively high and noisy (i.e., containing a high frequency
signal component of an apparently random nature, due to electrical interference
or generated within the electronic components of the transducer) baseline. The
signal processing normally involves subtracting a 'reference' baseline signal,
derived from a similar transducer without any biocatalytic membrane, from the
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sample signal, amplifying the resultant signal difference and electronically
filtering (smoothing) out the unwanted signal noise. The relatively slow nature
of the biosensor response considerably eases the problem of electrical noise
filtration. The analogue signal produced at this stage may be output directly but
is usually converted to a digital signal and· passed to a microprocessor stage
where the data is processed, converted to concentration units and output to a
display device or data store [39].
1.2.2. Characteristics of biosensors
A successful biosensor must possess at least some of the following
beneficial features.
1. The biocatalyst must be highly specific for the purpose of the analyses
(i.e. selectivity), be stable under normal storage conditions except in the
case of colourimetric enzyme strips and dipsticks show good stability
over a large number of assays (i.e. much greater than 100).
ii. The reaction should be independent of such physical parameters as
stirring, pH and temperature as is manageable. This would allow the
analysis of samples with minimal pre-treatment. If the reaction involves
cofactors or coenzymes, these should, preferably, be co-immobilized
with the enzyme.
m. The response should be accurate, precise, reproducible and linear over
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the useful analytical range, without dilution or concentration. It should
also be free from electrical noise.
iv. If the biosensor is to be used for invasive monitoring m chemical
situations, the probe must be tiny and biocompatible, having no toxic or
antigenic effects. If it is to be used in fermenters, it should be sterilisable.
This is preferably performed by autoclaving but no biosensor enzyme can
presently withstand such drastic wet-heat treatment. In either case, the
biosensor should not be prone to fouling or proteolysis.
v. The complete biosensor should be cheap, small, portable and capable of
being used by semi-skilled operators.
vi. There should be a market for the biosensor. There is clearly little purpose
in developing a biosensor if other factors ( e.g., government subsidies, the
continued employment of skilled analysts, or poor customer perception)
encourage the use of traditional methods and discourage the
decentralization of laboratory testing [3 9].
1.2.3. Types and applications of biosensors
a) Types ofbiosensors
The major types ofbiosensors are,
1. Piezoelectric biosensors
11. Optical biosensors
Introduction 21
111. Electrochemical biosensors
1v. Thermometric biosensors
v. Magnetic biosensors
Both piezoelectric and optical biosensors based on the phenomenon
of surface plasmon resonance and evanescent wave techniques [ 41]. This
utilizes a property shown of gold and other materials; specifically that a thin
layer of gold on a high refractive index glass surface can absorb laser light,
producing electron waves (surface plasmons) on the gold surface. This occurs
only at a specific angle and wavelength of incident light and is highly dependent
on the surface of the gold, such that binding of a target analyte to a receptor on
the gold surface produces a measurable signal.
Other optical biosensors are mainly based on changes in absorbance
or fluorescence of an appropriate indicator compound.
Piezoelectric sensors utilize crystals which undergo a phase
transformation when an electrical current is applied to them. An alternating
current (A.C.) produces a standing wave in the crystal at a characteristic
:frequency. This frequency is highly dependent on the surface properties of the
crystal, such that if a crystal is coated with a biological recognition element the
binding of a (large) target analyte to a receptor will produce a change in the
Introduction-----------------------22
resonant frequency, which gives a binding signal.
Electrochemical biosensors are normally based on enzymatic
catalysis of a reaction that produces ions. The sensor contains three electrodes, a
reference electrode, an active electrode and a sink electrode. A counter electrode
may also be present as an ion source. The target analyte is involved in the
reaction that takes place on the active electrode surface, and the ions produced
cre�te a potential which is subtracted from that of the reference electrode to give
a signal.
Thermometric and magnetic based biosensors are rare. In magnetic
based biosensors magnetic permeability is measured for biosensing applications
[42, 43]. The first blood p02 electrode was introduced by Clark et al., in 1953
[42, 44] and the first biosensor applying an enzyme membrane on to the
electrode was constructed in 1962 by Clark and Lyons [42, 45].
b) Applications of biosensors
When attempting to design a new biosensor, the first question to
answer is "What parameter is the sensor to be used to detect?" There are many
potential applications of biosensors of various types [ 41]. The main
requirements for a biosensor approach to be valuable in terms of research and
commercial applications are the identification of a target molecule, availability
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of a suitable biological recognition element, and the potential for disposable,
portable detection systems to be preferred to sensitive laboratory - based
techniques in some situations. Some examples are given below:
•!• Glucose monitoring in diabetes patients
•!• Other medical health related targets
•!• Environmental applications, e.g., the detection of pesticides and river
water contaminants
•!• Remot� sensing of air-born bacteria e.g., in counter bioterrorist activities
•!• Detection of pathogens
•!• Determining levels of toxic substances before and after bio-remediation
•!• Detection and determining of organophosphate [ 41]
Biosensors can also meet the need for continuous, real-time in vivo
monitoring to replace the intermittent analytical techniques used in industrial
and clinical chemistry [ 42, 46]. It is even possible to measure the concentrations
of neurotransmitter molecules by means of a neuronal biosensor [42, 47, 48].
Antigens and antibodies have been measured using imrnuno-sensors [ 42, 49].
Most recently, the development ofbiosensors for the detection of DNA damage,
mutation [ 42, 50,. 51] , the identification of DNA sequences and hybridization
[42, 52] offers considerable promise in several medical fields.
Introduction--------------------------- 24
1.2.4. Metal based electrodes as biosensors
New developments in biosensor design are appearing a high rate as
these devices play increasingly important roles in daily life. In recent years,
biosensors have been increasingly used for continuous monitoring of biological
and synthetic processes and to aid our understanding of these processes [42].
Many parameters have been suggested to characterize a biosensor. Some are
commonly used to evaluate the functional properties and quality of the sensor,
such as sensitivity, selectivity, stability and response time; while other
parameters are related to the application rather than to sensor function, for
example the biocompatibility of sensors for clinical monitoring.
Electrochemical electrodes have been used for pH monitoring for
over 100 years [ 42, 53] and the principle established in this technique provides
the basis for the most widely used electrochemical biosensors. The
electrochemical principle is now well established and both chemical and
mathematical models have been developed, including both two and three
dimensions [42, 54]. In the simplest applications, the electrochemical reactions
occurred directly on the electrode surface or in the space between the electrodes,
by the restoration of redox balance between the target molecule, or ion, and the
electrolyte [42, 44].
Metals and carbon are commonly used to prepare solid electrode
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'
systems and supporting electrolyte. Metals such as platinum, gold, silver and
stainless steel have long been used for electrochemical electrodes due to their
excellent electrical and mechanical properties. Carbon-based materials such as
graphite, carbon black and carbon fibre are also used to construct the conductive
phase. These materials have a high chemical inertness and provide a wide range
of anode working potentials with low electrical resistivity'. They also have a
very pure crystal structure that provides low residual currents and a high signal
to-noise ratio [42, 55]. Carbon fibres could be valuable in sensor construction
and it showed how a parallel array consisting of a large number of carbon fibres,
separated by insulators can be prepared to obtain a very high signal-to-noise
ratio [ 42, 56].
1.2.5. Nickel based electrodes as sensors of acetylcholine
The selection of material and fabrication techniques of the electrode
is crucial for adequate sensor function and the performance of a biosensor
depends upon these factors [ 42]. Many metals were used as acetylcholine
sensors by the immobilization of enzymes. But the enzymatic method gave a
slow response time and it is very difficult to preserve the enzyme. The nickel
electrodes can be applied to develop novel electrochemical sensors to alcohol at
a lower potential, because the anodic oxidation of alcohol is catalyzed by nickel
in an aqueous alkaline solution in which nickel perhydroxide is formed on the
Introduction------------------------26
nickel electrode [15, 29, 57 - 60]. Nickel electrodes have been applied to
electrochromic devices [57, 61], alkaline batteries [57, 6,2] an� as an
electrocatalyst [57, 63, 64]. Most of the applications are on the basis of the
redox pair, Ni(OH)2/NiOOH [57, 65, 66]. Chemically modified nickel
electrodes were used to detect aliphatic alcohols [57, 67]. Nickel electrodes
exhibited a very good linear relationship between the oxidation current and
acetylcholine concentration, a major criteria for its application as biosensor for
acetylcholine [15, 28].
1.3. The approach of the present work
Acetylcholine is an important neurotransmitter in the sympathetic
nervous system. There is clinical evidence indicating that some neuropsychiatric
disorders such as Parkinson's disease, Alzheimer's disease and Myasthenia
gravis are correlated with dysfunctional acety lcholine regulation [ 15, 68, 69].
Early detection of these diseases would help in giving proper treatment at the
early stage. As discussed in section 1.3.5., nickel electrodes found very much
applications in sensing acetylcholine. But the background current of pure nickel
electrode is unstable, and there is low sensitivity in the electrochemical sensing
system [29]. According to the steady state limiting current equation [29, 57, 58,
70], ilim = nFAD0Cc/8 where, 'ilim' is the steady-state limiting current, 'n' is the
electron transfer number, 'F' is the Faraday constant, 'A' is the catalytic area,
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'D0' is the diffusion coefficient, 'C0 ' is the bulk concentration and 'o' is the
thickness of the boundary layer. The sensitivity is defined as the slope of
nF ADc/8. The slope is dependent on the catalytic area A, which can be
increased by using electrochemical deposition of nickel on the substrate [29].
The instability of the background current is also avoided by electrodeposition of
nickel.
A significant improvement m nickel-coated electrode can be
achieved if the factors like cost, ease of fabrication, process modification,
compositional modification and structural modification are considered. In the
present study, nickel electroplating was made on graphite substrate. The good
electrical conductivity, low cost and ease of fabrication makes graphite a
suitable substrate material for development of electrodes [57, 71-78]. The
process modification of the electrodes by co-electrodepostion of nickel,
compositional modification by alloying of nickel with other metals and
structural modification by incorporation of nano nickeloxide was carried out.
All the modified electrodes were characterized by different techniques like
scanning electron microscopy, cyclic voltammetry and chronoamperometry.
The complete discussion of the present study is given in following sections.