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CHAPTER 1

INTRODUCTION

Glucose monitoring is a fact of everyday life for diabetic individuals and the
accuracy of such monitoring can literally mean the difference between life and death. To
accommodate a healthy life style and for frequent monitoring of glucose levels, a
number of glucometers are now available which permit the individual to test the glucose
level from blood. A less invasive or a non-invasive method of measuring human glucose
levels is an area of interest in the recent past.
1.1 DIABETES AND ITS IMPACTS:
Glucose (C6H12O6) is a carbohydrate whose most important function is to act as
a source of energy for the human body. The blood glucose concentration is very tightly
regulated. Human body has two hormones released by pancreas that have opposite
effects: insulin and glucagon. Insulin is produced by beta cells of the pancreas while
glucagon is produced by alpha cells. The release of insulin is triggered when high levels
of glucose are found in the bloodstream, and glucagon is released with low levels of
glucose in the blood. Diabetes is a chronic disease characterized by high or low blood
glucose levels, which results from the pancreas not working properly and not producing
enough insulin or when the body cells do not respond to it in the correct way. There are
three types of diabetes:
Type 1 diabetes is also known as juvenile diabetes because it is typically
diagnosed in children and young adults. In this type of diabetes, the body does
not produce insulin. 5% of the population with diabetes has this type of illness.
Type 2 diabetes is the result of the body not producing enough insulin or the cells
not using insulin properly. This is the most common form of diabetes. 90% of the
population with diabetes has this type. Some of the risk factors are physical
inactivity, excess body weight, genetics, age greater than 45 years, and ethnicity.
Gestational diabetes is high blood glucose levels first diagnosed during
pregnancy. This does not mean that the woman will have diabetes after she gives
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birth or that she had it before she conceived, but it is a risk factor for type 2
diabetes in the future.
At this time, there is no cure for diabetes, so lifelong treatment is the only
alternative. These treatments can consist of blood glucose monitoring combined with
insulin injections, keeping to a strict diet to control sugar intake, and exercise. Diabetic
patients must test their blood sugar daily. Some patients have to test themselves up to
eight or more times a day. They prick a finger to draw blood for reading by a handheld
blood glucose meter, and then they inject the necessary amount of insulin determined
by the meter reading. Because of the pain and inconvenience of the testing, many
patients do not monitor their glucose as often as they should[7].
A diabetic person who does not monitor their blood glucose levels runs the risk of
falling into insulin shock and other very serious complications. It is the fifth leading
cause of death in the United States. India is often referred as the diabetes capital of the
world. It is currently experiencing an epidemic of type 2 diabetes mellitus and has the
largest number of diabetic patients. The International Diabetes Federation 2009 report
reveals that the total number of diabetic subjects in India is 50.8 million[7].

Table 1.1.1: Prevalence of diabetes mellitus in rural India(source[7])


Prevalence (%)

Location

Total

Males

Females

Karnataka
Kerala
Tamilnadu
Tamilnadu
Wardha
Maharashtra
Andhra Pradesh
Mysore

16.06
16.5
6.2
10.4
8.8
9.2
14.3
4.58

22.04
13.5
4.4
8.0
8.1
9.9
12.0
2.66

19.78
12.5
5.1
9.2
8.5
9.3
13.2
3.77

National
Kashmir

2.5
3.5

2.5
4.5

2.7
4.0

Fig:1.1.2 Blood glucose levels for non-diabetic and insulin-dependent diabetic


subjects. Even with regular insulin injections, diabetics using current treatment
methods are unable to mimic normal control of glucose levels(source[7]).

1.2 EXISTING INVASIVE METHOD


The most popular method for monitoring blood glucose levels is through the use
of a portable glucose monitor. These devices are made relatively small to maximize
their portability, but still perform their intended function. Portable meters are battery
operated and can analyze a very small sample of blood to a high degree of accuracy.
Over time,these devices have become more user-friendly and more reliable.But the
diabetic patients have to undergo the painful procedure of pricking their finger tip to
place the blood sample in the strip for regular monitoring of glucose level in blood.To
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avoid this, the researchers are working on developing glucosensors for other human
serum(sweat,tear,saliva) and subcutaneous fluids.
1.3 COMPOSITION OF HUMAN PERSPIRATION:
Based on survey we found that there exists a strong correlation between sweat
glucose and blood glucose. A perfusion method was used to rapidly harvest sweat from
forearm sites on human subjects. The sweat samples are analyzed for glucose by highperformance liquid chromatography methods and compared with the results obtained
with a blood glucose meter. Sweat glucose, when properly harvested to prevent
contamination from other sources on the skin's surface, can accurately reflect blood
glucose levels. Water given off by the skin is classified as insensible and sensible
perspiration. Under normal conditions about 600 to 700 c.c. is evaporated from the skin
in twenty four hours. The chief physiological significance of the perspiration is to assist
in regulating the body temperature. The constituents of perspiration are very variable.
The average values calculated from the examination of fourteen male specimens and
ten female specimens are given in Table 1.3.1[1]
Table 1.3.1:Composition of human perspiration (source [1])
Ph

Average
from 10
normal
female
Subjects
Average
from 14
normal
male
subjects

Ammonia

Urea

Amino acid

mgm./100cc

mgm./100c.c mgm./100c.

6.57 6.0

19.2

c
6.5

6.14 4.7

21.44

5.0

1.4 LITERATURE SURVEY


4

Glucose

Chlorides

mgm./100cc

NaCl/1000

20.0

c.c.
3.0

12.6

3.7

B.A.McSwiney (1934) found the constituents of human perspiration for 10 female


and 14 male subjects and described that glucose was an essential constituent of human
perspiration.He also demonstrated that the constituents vary for normal subjects and
rheumatic diseased subjects.He also found that adrenaline has no effect on sweat
glands while pilocarpine excites and atropine paralyses the sweat gland secretion[1].
Herbert.L.Berman.et.al(2003)

investigated

the

non-invasive

glucose

measurement processes for determining blood glucose level in the body.He showed
that after achieving a static level of glucose at a surface of the skin over some period of
time , the glucose may be then measured by a variety of processes.A sample of the
glucose may also first be extracted from the skin and this sample may then be
measured[3].
Moyer.et.al(2012) proved the correlation between sweat glucose level and blood
glucose level by performing liquid chromatography.He also demonstrated that the level
of glucose varies in accordance with the site of sample collection. Fore arm sweat gives
the greater accuracy of glucose level in human body[8].
Namrata Devi(2013) described the basic glucose meter design using Microchips
PIC 8-bit PIC16LF178X XLP device. The glucose meter determines the concentration of
glucose in the solution. The common methods used in the electrochemical
measurement are the colorimetric and the amperometric methods[9].
Matthew Bularzik et.al(2012) changed the glucose strips to colorimetric method
to determine the blood glucose concentrations optically. A separate module was
attached to the USB port for scanning insulin vials and audibly outputting the type of
insulin[5].
Miriam Garcia(2013) showed a basic glucometer design using free scale
products to determine the approximate concentration of glucose. This glucometer was
implemented with K53 microcontroller of the Kinetis family. A free scale USB stack to
show data through graphic user interface in a PC[7].
McAleer et al(2010) described an improved glucose test strip for use in a test
meter of the type which receives a disposable test strip and a sample of blood from a
patient and performs an electrochemical analysis was made using a non conductive
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integrated reagent blood separation layer containing a filler and enzyme effective to
oxidize glucose and a mediator effective to transfer electrons from the enzyme[6].
You Wang.et.al (2008) explained the various electrochemical methods of sensing
human glucose levels. He also showed the third generation enzymeless glucose
detecting methods by using mesoporous platinum electrode. Thus the use of glucose
oxidase and other mediator compounds were eliminated[10].
Jonathon.O.Howell et.al (2011) demonstrated the electroanalytical chemistry
involved in detecting glucose using glucose dehydrogenase and performed CV studies,
choronoamperometric studies using potassium ferricyanide mediator and obtained the
required electron transfer and minimum detection limits for the mediator[4].
Hendrik du toit, Mirella di Lorenzo (2014) successfully implemented a simple and
rapid methodology for the direct immobilization of glucose oxidase into highly porous
gold electrodes. The resulting electrode was tested as glucose sensor with the detection
limit of 25 microM[2].
1.5 LITERATURE SUMMARY
The concentration of glucose in human perspiration was found .It was also stated
that there was strong correlation of sweat glucose with blood glucose level in human
body. The various other non-invasive and surface glucose extraction methods were
found.The construction of a commercial glucometer using two different controller
platforms were studied i.e:using PIC and K53.The methods of glucose detection using
electrochemical and colorimetric methods were discussed. The incubation chemistry
involved in glucose detection was studied. The use of enzymes like glucose
dehydrogenase, glucose oxidase and mediators compounds like ferrocene,PQQ,
potassium ferricyanide and osmium salts were found. The three generations of glucose
biosensors and the enzymeless biosensors were also discussed.The immobilization
techniques involved in immobilization of glucose oxidase on highly porous gold was
studied. The electrochemical analysis techniques using cyclic voltammetry and
chronoamperometry were stated.
1.6 PROBLEM DEFINITION
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The existing portable glucometers use human blood samples to analyse


the concentration glucose present in human body.The samples are collected from finger
tips by using a pricking lancet.This traditional method of glucose monitoring is uneasy
and painful for people suffering with Diabetes type 2 .As they are insulin dependent,
they need to examine their glucose levels for atleast three times a day for proper
administration of insulin.The finger tip sites of sample collection are tend to suffer with
infection and swolling due to multiple punctures .The samples collected for other sites of
the body can cause a loss of accuracy which can be lethal at times.The invasive
procedure must be replaced by a continous and non-invasive process using the human
fluids as the sample of analysis.
1.7 OBJECTIVE OF THE PROJECT
To establish a correlation between the sweat glucose and blood glucose levels
and design a calibration graph for the glucometer design purpose.
To design a glucose analysis methodology for sweat samples using an enzyme
and a mediator compound.
To fabricate a working electrode and immobilizing the enzyme on the electrode
using a rapid and cheap immobilization technique.
To construct a glucometer to measure the glucose values from the sweat sample
test strip and display the result on the LED screen provided.
1.8 SCOPE OF THE WORK
The sweat glucometer can reduce the pain and infection problems in blood
glucometers. The accuracy of measuring glucose level in human body can be achieved
in sweat based glucometers by a proper calibration graph. Interpolation techniques
employed and the regression equation used in calibration process can improve the
accuracy and the errors produced in conversion of the current measured to the
concentration present in the sample can be greatly reduced. Various methods of sweat
collection can be employed in harvesting human perspiration. Forearm sweat can be
easily collection due to the presence of large number of sweat glands per cubic
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centimeter of the human forearm. This normal excretion of human body can be obtained
for glucose analysis instead of human blood plasma[8].
The presence of glucose in sweat was practically analyzed using Auto
analyzer and the correlation between blood glucose levels and sweat glucose levels
was found to be in the ratio of 2:1 for 0.5ml of the samples. For patients with diabetes
usually the excess amount of glucose concentration in human blood is made to excrete
through urine using certain drugs.But there is a limitation for excreting glucose through
urine and hence researchers are trying to expel the excess glucose content through
other human serum.
Monitoring glucose levels in sweat can lead to invention of new drugs that can
excrete the excess glucose contents through human perspiration. There are patients
who require monitoring their glucose levels all through the day. Blood glucose monitor
kits require the use of finger pricking lancets which causes pain throughout the
day.These patients undergo this process everytime they need to check their glucose
levels.Since this population is growing by leaps and bounds worldwide, researches are
developing a non-invasive measure, thus can enable the masses of diabetic patients to
monitor their glucose levels without undergoing painful procedure everytime they use.
Atheletes require constant monitoring of glucose, sodium, chloride and potassium
ions in their body when they are in marathon or sprinting tracks to maintain their energy
levels throughout the run. This can be done by monitoring the energy giver (glucose
content in blood) of athletes. Researchers are trying hard to monitor glucose levels
through sweat since athletes generate lot of sweat during their sprints. This proven
correlation between sweat glucose and blood glucose can make researchers to develop
a sweat pad to monitor glucose level constantly. Thus sweat is a potential body fluid that
helps in monitoring the various states of human body.

1.9 ORGANIZATION OF THE REPORT


CHAPTER 1 speaks about the introduction of diabetes and diabetic monitoring
methods. It also contains the literature survey, literature summary, problem
definition, objective and scope of the project work.
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CHAPTER 2 describes the various existing methodologies in non-invasive analytics


like optochemical sensors ,Near infrared spectroscopy ,Raman spectroscopy,
Fluorescent

spectroscopy,

Photo

acoustic

laser

spectroscopy,

Polarization

spectroscopy, Scatter changes and Electrochemical sensors. It also describes the


various methods of glucose extraction methods.
CHAPTER 3 projects the proposed methodology for glucose sensing from sweat
sample using an electrochemical sensor , the fabrication of working electrode using
a porous gold and the immobilization of the glucose oxidase enzyme on the porous
gold electrode.
CHAPTER 4 shows the results of various existing non-invasive methodologies and
the proposed methodology in the form of Cyclic Voltammetry scans of the
electrochemical analysis performed using various electrodes and various cell setup.
CHAPTER 5 conveys the future scope and the conclusion of the present
investigation.It also shows the valid applications of this project and explains the
advantages of the project.

CHAPTER-2
EXISTING METHODOLOGY
Non-invasive glucose measurement and a process for determining blood glucose
levels in the human body upon achieving a static level of glucose at a skin surface over
a period of time is a interest of recent past. Non-invasive processes can ease the pain
involved in obtaining the human blood sample for glucose examination in blood
glucometers.
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2.1 EXISTING MECHANISMS FOR NON-INVASIVE GLUCOSE SENSING


Processes which are able to assess glucose concentrations predictably from a
skin surface may include a step of extracting a sample from the skin and then
measuring that sample from the skin. Such sample extraction processes may include
suction blister extraction, wick extraction, microdialysis extraction, iontophoretic
extraction, sontophoretic extraction, and chemically enhanced extraction.Aside from the
extraction

processes,

non-invasive

measurement

processes

may

include

electrochemical sensors (e.g., glucose electrodes), optochemical sensors (e.g.,


colorimetric strips), near-infrared spectroscopy (NIR), mid-infrared spectroscopy (MIR),
infrared

spectroscopy

(IR),

Raman

spectroscopy, photoacoustic

spectroscopy,

measurement of refractive index or scatter changes, fluorescent spectroscopy, and


polarization spectroscopy[6].
2.1.1 Glucose extraction
Blister suction and wick extraction are some of the most common methods for
sampling subcutaneous interstitial tissue fluid, although blister extraction is less invasive
than the wick extraction technique. Micro dialysis extraction involves calculating the
concentrations of compounds, including skin glucose concentrations, which are in the
extracellular water space. Micro dialysis has been applied to peripheral tissue types,
e.g., skin, muscle, adipose, eye, lung, liver, and blood as well as having micro dialysis
probes implanted subcutaneously and perfused by a portable micro infusion pump.
Finally, iontophoretic extraction involves noninvasive glucose measurement from
subcutaneous tissue[3].
2.1.2 Glucose measurement
The current method of blood glucose self-monitoring is for patients to obtain
finger-prick sample of capillary blood several times daily and to apply this blood to a
reagent strip and portable meter for measurement. Such intermittent testing is
unpopular because of the dislike of multiple sampling with a lancet, and has the
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disadvantage of not being possible during the night or whilst driving a car, and of
missing episodes or hyper- or hypoglycemia which do not occur at the time of sampling.
Some of alternate non-invasive methods are listed below
2.1.2.1 Optochemical Sensors
Optochemical sensors (e.g., colorimetric strips) are based on changes in some
optical parameter due to enzyme reactions or antibody-antigen bonding at a transducer
interface. Such sensors may include enzyme optrodes and optical immunosensors and
may also include different monitoring processes such as densitometric, refractometric or
calorimetric devices[10].

Fig:2.1.2.1 Glucose sensor using optical sources(source:[5])

2.1.2.2 Near-Infrared Spectroscopy (NIR)


Glucose measuring is quite effective on the human skin of the hands and fingers.
The glucose concentration measured correlates very closely with the glucose
concentration determined by a direct determination from a blood sample. This is
surprising in that the IR beam likely passes into the skin, i.e., the stratum corneum, for
only a few microns. It is unlikely in a fingertip that any blood is crossed by that light path.
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The stratum corneum is the outer layer of skin and is substantially unvascularized. The
stratum corneum is the final outer product of epidermal differentiation or keratinization. It
is made up of a number of closely packed layers of flattened polyhedral corneocytes
(also known as squames). These cells overlap and interlock with neighboring cells by
ridges and grooves. In the thin skin of the human body, this layer may be only a few
cells deep, but in thicker skin, such as may be found on the toes and feet, it may be
more than 50 cells deep. The plasma membrane of the corneocyte appears thickened
compared with that of keratinocytes in the lower layers of the skin, but this apparent
deposition of a dense marginal band formed by stabilization of a soluble precursor,
involucrin, just below the stratum corneum[6].
It is sometimes necessary to clean the skin exterior prior before sampling to
remove extraneous glucose from the skin surface. At least when using IR spectra to
measure glucose, it is important to select cleaning materials having IR spectra that do
not interfere with the IR spectra of glucose. Considerations assumed to be suitable for
preparation of the sample skin for the testing are: a.) a glucose solvent, e.g., water or
other highly polar solvent; b.) a solvent for removing the water, e.g., isopropanol, and c.)
a skin softener or pliability enhancer not having significant IR peaks in the noted IR
regions, e.g., mineral oils such as those sold as Nujol. Preferably the b.) and c.)
components are admixed, although they need not be. Certain mixtures of the first two
components may be acceptable, but only if the sampling situation is such that the
solvents evaporate without IR spectrographically significant residue. We have also
found that soap and its residue are sometimes a problem. Consequently, addition of a
weak acid again not having significant IR peaks in the noted IR regions, to the a.)
component, i.e., the solvent for removing glucose, is desirable. The preferred weak acid
is boric acid.
The NIR region of the spectrum extends from 700 to 2500 nm (14,000-4000
cm). In this region, absorption bands are due to overtone vibrations of anharmonic
fundamental absorption bands to combinations of fundamental absorption bands
primarily associated with CH; OH, and NH stretching vibrations. For overtone
vibrations, only the first, second, and third overtones are usually seen with the
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magnitude of the absorption peak diminishing substantially with overtone order. The NIR
region may be attractive for quantitative spectroscopy since NIR instrumentation is
readily available[6].
In measuring aqueous glucose, the NIR region which lies between 2.0 and 2.5
m may be utilized. This region contains a relative minimum in the water absorption
spectrum and has readily identifiable glucose peak information. However, NIR spectra
may generally be sensitive to a host of factors including temperature, pH, and
scattering[3].
2.1.2.3 Raman Spectroscopy
Raman spectra are typically observed when incident light at a frequency v 0=c/0 is
inelastically scattered at frequencies v 0vi. The loss (Stokes shift) or gain (anti-Stokes
shift) of photon energy, and hence frequency, is due to transitions of the rotational and
vibrational energy states within the scattering molecule. Since the Raman spectrum is
independent of excitation frequency, an excitation frequency may be chosen which is
appropriate for a particular sample. However, a drawback may be that scatter and
reabsorption in biological tissues may make detection of Raman shifts due to
physiological

concentrations

difficult.

Raman

spectroscopy

is

used

for

the

transcutaneous monitoring of glucose concentrations. Unlike many methods of


measuring glucose, with which there are valid questions about whether glucose is being
measured,the strong presence of glucose in the regression vector developed from
Raman measurements provides direct spectral evidence that the measurements result
from the active glucose concentrations[3].
2.1.2.4 Photoacoustic Laser Spectroscopy(PA Laser Spectroscopy)
For a PA signal generation a sample is irradiated with either amplitudemodulated or pulsed light. The molecules of interest present in the sample(glucose in
sweat) absorb radiation of a characteristic part of the spectrum and convert the
incoming optical energy, into a periodic heating of the sample by means of non-radiative
relaxation processes. This leads to a modulated volumetric expansion and to the
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propagation of a thermal and acoustic wave inside the sample. The acoustic wave can
then be detected with a microphone within a PA cell placed on the sample surface or
with a piezoelectric transducer in direct contact with the sample surface.The MIR region
(5-25 m) of the spectrum is particularly attractive for studies of biological samples
since most molecules have a characteristic absorption spectrum in this wavelength
range.
PA spectroscopy is especially interesting for biomedical studies since it is (i) noninvasive (for moderate laser intensities), (ii) much less influenced by scattering effects
than alternative optical techniques, and (iii) readily adaptable to the study of biological
tissues. The MIR region has the big advantage that the glucose spectrum does not
interfere as much with other blood and tissue constituents as it does in the near infrared
and it shows distinct absorption maxima. However water as the main constituent of the
human body tissue absorbs very strongly in the MIR and leads to penetration depths of
less than 100 m depending on the measurement site. These two aspects make the
detection of glucose challenging.
Photoacoustic laser spectroscopy has been utilized for measuring glucose
concentrations of human whole blood samples using pulsed laser photoacoustic
spectroscopy. Such a process may use, e.g., a CO 2 laser operating with J pulse
energy, to measure tiny changes of the absorption coefficient of the sample caused by
the variations of blood glucose concentrations[3].

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Fig:2.1.2.4.1 Schematic of PA setup used for in-vivo measurements(source[3])


2.1.2.5 Refractive Index or Scatter Changes
Measurement of refractive index or scatter changes may be feasible to measure
blood glucose by measuring the scattering coefficient of human skin, e.g., by using
optical sensors attached to the skin. Such techniques may be based on the fact that the
refractive index of sugar solution changes with the concentration of sugar.
2.1.2.6 Fluorescent Spectroscopy
There is an urgent need to develop technology for continuous in vivo glucose
monitoring in subjects with diabetes mellitus. Problems with existing devices based on
electrochemistry have encouraged alternative approaches to glucose sensing in recent
years, and those based on fluorescence intensity and lifetime have special advantages,
including sensitivity and the potential for non-invasive measurement when near infrared
light is used. Several receptors have been employed to detect glucose in fluorescence
sensors, and these include the lectin concanavalin A (Con A), enzymes such as glucose
oxidase, glucose dehydrogenase and hexokinase/glucokinase, bacterial glucosebinding protein, and boronic acid derivatives (which bind the diols of sugars).
Techniques include measuring changes in fluorescence resonance energy transfer
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(FRET) between a fluorescent donor and an acceptor either within a protein which
undergoes glucose-induced changes in conformation or because of competitive
displacement; measurement of glucose-induced changes in intrinsic fluorescence of
enzymes (e.g. due to tryptophan residues in hexokinase) or extrinsic fluorophores (e.g.
using

environmentally

sensitive

fluorophores

to

signal

protein

conformation).

Noninvasive glucose monitoring can be accomplished by measurement of cell auto


fluorescence due to NAD(P)H, and fluorescent markers of mitochondrial metabolism
can signal changes in extracellular glucose concentration[4].
There may be two categories for fluorescent spectroscopy: glucose-oxidase based
sensors and affinity-binding sensors. Sensors in the first category may use the
electroenzymatic oxidation of glucose by glucose-oxidase (GOX) in order to generate
an optically detectable glucose-dependent signal. The oxidation of glucose and oxygen
forms gluconolactone and hydrogen peroxide[10].
Several methods for optically detecting the products of this reaction, and hence the
concentration of glucose driving the reaction, may be utilized. Since oxygen is
consumed in this reaction at a rate dependent on the local concentration of glucose, a
fluorophore which is sensitive to local oxygen concentration can also be used to
quantify glucose concentration.
A method GOX based fluorescent sensor involves the redox mediator
tetrathiafulvalene (TTF) whose oxidized form TTF + reacts with the reduced form of GOX
to reversibly form TTF0. Since TTF+ is absorbed in the 540-580 nm range, a method for
quantifying the presence of TTF + (and hence glucose driving the production of reduced
GOX) is available.
Another method involves the hydrogen peroxide (H 2O2) generated from the GOX
reaction with glucose reacting with bis(2,4,6-trichlorophenyl) oxalate (TCPO) to form a
peroxyoxylate. Here, the peroxyoxylate formed transfers chemiluminescent energy to an
accepting fluorophore which in turn emits photons at a characteristic wavelength. The

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emission by the fluorophore is proportional to the glucose concentration and may be


detected optically[3].
2.1.2.7 Polarization Spectroscopy
Polarimetric quantification of glucose may be based on the principle of optical
rotary dispersion (ORD) where a chiral molecule in an aqueous solution rotates the
plane of linearly polarized light passing through the solution[3]. This rotation is due to a
difference in the indices of refraction n L and nR for left- and right-circularly polarized light
passing through a solution containing the molecule. Because the molecule has a
chirality (or handedness), the angle of rotation depends linearly on the concentration
of the chiral species, the path length through the sample, and a constant for the
molecule called the specific rotation. Glucose in the body is dextrorotatory, i.e., rotates
light in a right-handed direction, and has a specific rotation of +52.6 dm 1 (g/L)1.

Fig 2.1.2.7 Schematic of the turbid polarimeter(source[6]).

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Optical polarimetry is particularly promising in this respect in that its measurable


polarization parameters (e.g., optical rotation) can be directly related to the absolute
glucose levels. Specifically, glucose is an optically active (chiral) molecule that rotates
the plane of linearly polarized light by an amount proportional to its concentration and
the optical pathlength. This proportionality has been verified numerous times in clear
media; in fact, one of earliest application of polarimetry relied on this relationship to
determine sugar concentration in industrial production processes[3] .
2.1.2.8 Electrochemical sensors
Monitoring blood glucose levels is critical for controlling diabetes. The challenge
of providing such tight and reliable glycemic control remains the subject of enormous
amount of research. Blood glucose levels are monitored by a variety of devices in ex
vivo or in vivo configurations. The monitoring devices are part of a larger family of
biosensors[10].
Biosensors are analytical tools for the analysis of bio-material samples to gain an
understanding of their bio-composition, structure and function by converting a biological
response into an electrical signal. The analytical devices composed of a biological
recognition element directly interfaced to a signal transducer which together relate the
concentration of an analyte (i.e.glucose) to a measurable response[10].
Blood glucose biosensors utilize a subset of this family, namely bioelectrodes and
electrochemical biosensors for blood glucose play a leading role in this direction.
Amperometric enzyme electrodes, based on glucose oxidase (GOx) bound to electrode
transducers, have thus been the target of substantial research. Since Clark and Lyons
first proposed the initial concept of glucose enzyme electrodes in 1962 .There has been
significant activity towards the development of reliable devices for self monitoring and
continuous diabetes control. The critical mechanism of the biosensor, and one that has
experienced the most attention, is improved transfer of electrons between the GOx
active site and the electrode surface. The first glucose enzyme electrode relied on a thin
layer of the enzyme GO x (glucose oxidase ) entrapped over an oxygen electrode via a
semipermeable dialysis membrane[6].
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Fig 2.1.2.8.1 Main components of a biosensor, showing (a) the bio reaction, (b)
transducer, (c) processor, (d) amplifier and (e) display(source[10]).

Fig 2.1.2.8.2 Working principles of a biosensor(source[10]).


A variety of approaches have been explored in the operation of glucose enzyme
electrodes (i.e., bioelectrodes). In addition to diabetes control, such devices offer great
promise for other important applications, ranging from food analysis to bioprocess
monitoring. Additionally, the great importance of glucose has generated an enormous
number of publications, the flow of which shows no sign of diminishing. Yet, despite of
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impressive advances in glucose biosensors, there are still many challenges related to
the achievement of clinically accurate tight glycemic monitoring. This biosensor offered
good accuracy and precision but required a blood sample size of 100 L. Following this
initial work, a wide range of amperometric enzyme electrodes differing in electrode
design or material, immobilization approach, or membrane composition were
developed[6].
Electrochemical biosensors for glucose play a leading role in this direction.
Amperometric enzyme electrodes, based on glucose oxidase (GO x) bound to electrode
transducers, are used extensively for home monitoring and have been the target of
substantial research and development. Most electrochemical glucose biosensors rely on
electron transfer from glucose to the electrode via the active site of the enzyme (GOx).
However, enzymeless thin film biosensors have also been developed[4].
Electrochemical biosensors may be constructed on the amperometric
principle which is based on the oxidation or reduction of electrochemically active
substances. Such sensor may also be constructed to measure the changes in local pH
due to the gluconic acid produced at a potentiometric sensor, usually a coated wire pHselective electrode or an ion selective field effect transistor (ISFET). Also, electrical
resistance changes during the overall process may be used as a basis for
conductometric biosensors.
Conductometric sensors are based on the measurement of electrolyte
conductivity, which varies when the cell is exposed to different environments. The
sensing effect is based on the change of the number of mobile charge carriers in the
electrolyte. If the electrodes are prevented from polarizing, the electrolyte shows ohmic
behavior. Conductivity measurements are generally performed with AC supply. The
conductivity is a linear function of the ion concentration; therefore, it can be used for
sensor applications. However, it is nonspecific for a given ion type. On the other hand,
both the polarization and the limiting current operation mode must be avoided. Thus,
small amplitude alternating bias is used for the measurements with frequencies where
the capacitive coupling is still not determining the impedance measurement.
20

Coulometry is an electrochemical technique, related to amperometry, where


the amount of charge (coulombs) passing between two electrodes is measured. The
amount of charge passing between the electrodes is directly proportional to oxidation or
reduction of an electroactive substance at one of the electrodes. The number of
coulombs transferred in this process is related to the absolute amount of electroactive
substance by Faradays Law[10].
Moreover, potentiometric glucose sensors (e.g., coated wire sensors) may
potentially be utilized for implantable use. Coated wire sensors are general easy to
fabricate and are suitable for miniaturization to diameters of 50-200 m. They may also
be used in combination with a standard cardiographic (EKG) reference electrode[10].
An electrochemical sensor including a glucose permeable membrane comprising
an oxygen permeable, hydroxyl endblocked diorganopolysilox- ane, preferably
dimethylpolysiloxane, having an average molecular weight of at least 5,000, the
membrane 35 covering the glucose oxidase immobilized anode, thereby retaining said
glucose oxidase between said membrane and said anode.
The electrode of the sensor may be, a Clark electrode, having a platinum anode
and a silver cathode, which preferably are electrically insulated from each other by
epoxy. A layer of material capable of catalyzing the reaction of glucose with oxygen,
suitably an enzyme, such as glucose oxidase or glucose dehydrogenase is immobilized
adjacent to, on, or integrally bonded to the electrode. The catalyst can be immobilized to
the anode only or can be adhered to the entire electrode, without loss of accuracy[6].
2.1.2.9 Generation of glucose sensors
The following list summarizes the advancement of blood glucose biosensors, and
Figure 2.1.2.9.1 summarizes three generations based on different mechanisms of
electron transfer, including the use of natural secondary substrates, artificial redox
mediators, or direct electron transfer:

21

First generation: First generation glucose biosensors relied on the use of the natural
oxygen cosubstrate, generation and detection of hydrogen peroxide. Electrons are
transferred from glucose to the electrode via the active site of the enzyme.The
biocatalytic reaction involves reduction of the flavin group (FAD) in the enzyme by
reaction with glucose to give the reduced form of the enzyme (FADH 2). Followed by
reoxidation of the flavin by molecular oxygen to regenerate the oxidized form of the
enzyme GOx(FAD)

GOx(FAD) + glucose GOx(FADH2) + gluconolactone


GOx(FADH2) + O2 GOx(FAD) + H2O2

Measurements of peroxide formation are best made using miniaturized devices which
are commonly carried out on a Pt electrode at a moderate anodic potential of around +
0.6 V (vs Ag/AgCl reference). A YSI probe is most often used, which involves the
entrapment of GOx between an inner anti interference cellulose acetate membrane and
an outer diffusion limiting/biocompatible one.
Second generation: Further improvements were achieved by replacing the oxygen with
a nonphysiological (synthetic) electron acceptor capable of shuttling electrons from the
redox center of the enzyme to the surface of the electrode. This improved transfer of
electrons between the GOx active site and the electrode surface, which the limiting
factor in the operation of first generation amperometric glucose biosensors. o Enzyme
wiring with a redox polymer offered additional improvements in the electrical contact
between the redox center of GOx and electrode surfaces, as shown in Figures 2 and 3.
An elegant nondiffusional route for establishing a communication link between GOx and
electrodes was accomplished by wiring the enzyme to the surface with a long flexible
hydrophilic polymer backbone [poly(vinylpyridine) or poly(vinylimidazole)] having a
dense array of covalently linked osmium-complex electron relays.
22

Third generation: It is desirable to eliminate the mediator and develop a reagentless


glucose biosensor with a low operating potential, close to that of the redox potential of
the enzyme. In this case, the electron would be transferred directly from glucose to the
electrode via the active [10]

Fig 3.1.2.9.1 Three generations of amperometric enzyme electrodes for glucose


based on the use of natural oxygen cofactor (A), artificial redox mediators (B), or
direct electron transfer between GOx and the electrode (C)(source[10]).

23

Fig 3.1.2.9.2 The principle schematic of:


(a) first generation amperometric biosensors,
(b) second generation amperometric biosensors(source[10]).
Nanomaterials have become an extremely popular theme in recent
electrochemical sensing research, due to their electrical conductivity, unique structural
and catalytic properties, high loading of biocatalysts, good stability and excellent
penetrability.Carbon nanotubes (CNTs) can be used as electrode materials with useful
properties for various potential applications including miniature biological devices. The
subject of electrochemical sensing utilizing CNTs has been extensively studied and
reviewed by various authors, including CNTs paste electrodes . These sensors achieved
higher response current, low work potential and low interference. A soluble carbon
nanofiber was used to modify a glucose sensor, which performed the electroreduction of
dissolved oxygen at a low operating potential, breaking through the limit of the
insolubility of CNTs in their application in designing sensors[2].
The other metal nanomaterials applied in fabrication of glucose sensor such as
copper (Cu), which showed good selectivity and sensitivity and iridium, which could
detect H2O2 released from the enzymatic reaction at a relatively low applied potential
with a favorable signal-to-noise ratio. The silica nanoparticles and the non-doped
nanocrystalline diamond also have been reported[2].
An amperometric sensor based on polypyrrole nanotube array deposited on a Pt
plated nano-porous alumina substrate was described. The use of nano-porous template
electrodes lead to an efficient enzyme loading and provided an increased surface area
for sensing the reaction. Another nanoelectrode sensor, polypyrrole nanofibers
containing entrapped GOx, was fabricated via a two-step process. These sensors
demonstrated good biocatalytic activity to glucose[6].
24

2.2 GENERAL BLOCK DIAGRAM OF GLUCOMETER


This section details the hardware design and the software development of the
glucose meter using the Amperometric method. Fig 2.2.1 illustrates the block diagram of
a typical glucose meter. The glucose meter can be implemented using the
PIC16LF178X device.

Fig:3.2.1 Block diagram of glucometer(source[4])


Glucose regression equation
Y = mX + C
where,
Y = Glucose concentration in mg/dl
m = Slope
X = Average ADC reading of op amp output voltage
C= Constant

25

The following are the features of the PIC16LF178X device and some of the
peripherals in its integrated measurement engine:

Extreme low-power (XLP) operation


Two op amps
2x8-bit DAC
12-bit Successive Approximation (SAR) ADC,
up to 11 channels
Internal EEPROM
Inter-Integrated Circuit (I2C)
16-bit Timer1
When the solution sample is placed on the test strip, glucose undergoes a

chemical reaction and electrons are produced. The flow of electrons (i.e., the current
flowing through the working electrode) can be measured. This current will change
according to the glucose concentration. The current is measured with the help of the
current-to-voltage conversion using the internal op amp of the PIC16LF178X device and
the filtering of high-frequency signals. The filtered signal is fed to the 12-bit ADC module
of the device.
The PIC16LF178X device starts capturing the voltage at the ADC channel after
about 1.5 seconds of placing the solution sample. An average of about 2048 ADC
readings is taken. This average value is substituted into the regression equation.The
glucose concentration is determined using this regression equation and the value is
displayed on the LCD in units of mg/dl or mmol/l. Up to 32 glucose readings can be
stored in the internal EEPROM and can be viewed later on the LCD. The power to the
Glucose Meter can be supplied from theon-board lithium battery (3V 225 mAH
CR2032).
The time to start capturing the ADC values (i.e., one second to 1.5 seconds) and
the number of ADC readings taken should be modified to match the type and
characteristics of the test strip to be used[4].

2.3 TEST STRIP WORKING PRINCIPLE IN AMPEROMETRIC METHOD

26

The majority of glucose meters are electrochemical and use the Amperometric method.
Figure 2.3.1 illustrates the glucose meter test strip working principle.

Fig 2.3.1 Glucose meter test working strip principle(source[4])


The test strip forms the main biochemical sensor where the sample of solution is
placed. The test strip has the following electrodes:
27

Working electrode: Electrons are produced here during the chemical reaction.
This electrode is connected to the current-to-voltage amplifier.
Reference electrode: Held at a constant voltage with respect to the working
electrode to push the desired chemical reactions.
Counter electrode: Supplies current to the working electrode.
Most of the glucose meter designs use only two electrodes, reference electrode
and working electrode. A precise reference voltage (VREF) is applied to the reference
electrode and a precise bias voltage (VBIAS) is applied to the op amp. This way the
precise potential difference is maintained across the working electrode and the
reference electrode. This voltage is the stimulus which drives the test strips output
current. The magnitude of the output current is then used to calculate the number of
electrons produced. The solution sample is placed on the test strip and the reaction of
the glucose with the enzyme takes place. Electrons are generated during the chemical
reaction. Flow of electrons will correspond to the flow of current through the working and
the reference electrode. This current will change according to the glucose
concentration.
The current is measured using a trans impedance amplifier (current-to-voltage
converter) for the measurement with an Analog-to-Digital Converter (ADC).
The output of the trans impedance amplifier will be seen as a variation in the voltage
with varying glucose concentrations in the solution[4].

CHAPTER 3
PROPOSED METHODOLOGY
28

The sensor used for detecting glucose in sweat is a electrochemical


amperometric enzyme based sensor. The electrochemical reaction involves redox
reactions of glucose which is initiated by glucose oxidase(GOX).This electrochemical
reaction in turn produces an equivalent flow of electrons(current)at the working
electrode. To facilitate the transfer of electrons from the glucose molecule to the
electrode ,we use mediators like ferrocene,benzoquinone ,Au-nanoparticles etc.The
redox reactions of

glucose is initiated by either glucose oxidase(GOX) or glucose

dehydrogenase(GDH).
3.1 GLUCOSE DEHYDROGENASE
The chemistry is relatively complex and the sample chamber contains many
constituents (e.g., stabilizers, processing aids, etc.). However, for this discussion it will
be treated more simply as an enzyme and mediator. An enzyme overcomes many of
the problems associated with a variable biological sample matrix. Because glucose
and enzymes do not readily exchange electrons directly with an electrode, an
electrochemical measurement requires a mediator to facilitate (or mediate) the electron
transfer. The chemistry is summarized as:
Glucose first reacts with the enzyme glucose dehyrogenase. Glucose is
oxidized to gluconic acid and the enzyme is temporarily reduced by two
electrons transferred from glucose to the enzyme.
The reduced enzyme next reacts with the mediator (Mox), transferring a
single electron to each of two mediator ions. The enzyme is returned to its
original state and the two Mox are reduced to Mred.
At the electrode surface, Mred is oxidized back to Mox and the measured
current is used to determine the concentration of glucose in the sample.The
enzyme (a protein catalyst) glucose dehydrogenase was chosen because it
is highly specific for, and accelerates the oxidation of, glucose to gluconic
acid. It is also less susceptible than glucose oxidase to common
interferences[4].
Its specificity enables it to selectively react with glucose in the presence of the
thousands of compounds that could potentially interfere within the complex sample fluid,
blood. This specificity is critical because glucose levels vary widely over time in a single
29

healthy patient along with many other factors such as hematocrit, oxygen levels,
metabolic byproducts, etc.
The well-known mediator, potassium ferricyanide is used in this process. The redox
couple ferricyanide/ferrocyanide is capable of rapidly transferring electrons with and
electrode (electrochemically reversible on the timescale of the experiment). It also has a
relatively low oxidation potential[4].
This allows for a lower applied potential at the working electrode, thereby
minimizing the amount of oxidation of extraneous compounds in the sample. The end
result is that electrons may thus be transferred between glucose and the electrode via
enzyme and mediator[6].

Fig3.1.1 Incubation chemistry and detection mechanism(source[4]).

3.2 GLUCOSE OXIDASE

30

Glucose oxidase is an enzyme extracted from the growth medium of Aspergillus


niger.Glucose oxidase catalyse the oxidation of Beta D-glucose present in the plasma to
D-glucono-1,5-lactone with the formation of hydrogen peroxide; the lactone is then
slowly hydrolysed to D-gluconic acid.The hydrogen peroxie produced is then broken
down to oxygen and water by a peroxidase enzyme.. In cells, it aids in breaking the
sugar down into its metabolites.
Glucose oxidase is widely used for the determination of free glucose in body fluids
(diagnostics), in vegetal raw material, and in the food industry.

Fig 3.2.1 PDB model of glucose oxidase(source:Wikipedia.org/)


GOx is a dimeric protein, the 3D structure of which has been elucidated. The
active site where glucose binds is in a deep pocket. The enzyme, like many proteins
that act outside of cells, is covered with carbohydrate chains. At pH 7, glucose exists in
solution in cyclic hemiacetal form as 63.6% -D-glucopyranose and 36.4% -Dglucopyranose, the proportion of linear and furanose form being negligible. The glucose
oxidase binds specifically to -D-glucopyranose and does not act on -D-glucose. It is
able to oxidise all of the glucose in solution because the equilibrium between the and
anomers is driven towards the side as it is consumed in the reaction.
31

Glucose oxidase catalyzes the oxidation of -D-glucose into D-glucono-1,5lactone, which then hydrolyzes to gluconic acid. In order to work as a catalyst, GOx
requires a cofactor, flavin adenine dinucleotide (FAD). FAD is a common component in
biological oxidation-reduction (redox reactions). Redox reactions involve a gain or loss
of electrons from a molecule. In the GOx-catalyzed redox reaction, FAD works as the
initial electron acceptor and is reduced to FADH 2. Then FADH2 is oxidized by the final
electron acceptor, molecular oxygen(O2), which can do so because it has a higher
reduction potential. O2 is then reduced to hydrogen peroxide (H2O2).
Glucose oxidase is widely used coupled to peroxidase reaction that visualizes
colorimetrically the formed H2O2, for the determination of free glucose in sera or blood
plasma for diagnostics, using spectrometric assays manually or with automated
procedures, and even point of use rapid assays. Similar assays allows to monitor
glucose levels in fermentation, bioreactors, and to control glucose in vegetal raw
material and food products. In the glucose oxidase assay, the glucose is first oxidized
by glucose oxidase to produce gluconate and hydrogen peroxide. The hydrogen
peroxide is then oxidatively coupled with a chromogen to produce a colored compound
which may be measured spectroscopically. For example, hydrogen peroxide together
with 4 amino-antipyrene (4-AAP) and phenol in the presence of peroxidase yield a red
quinoeimine dye that can be measured at 505nm. The absorbance at 505 nm is
proportional to concentration of glucose in the sample.
Enzymatic glucose biosensors use an electrode instead of O2 to take up the
electrons needed to oxidize glucose and produce an electronic current in proportion to
glucose concentration. This is the technology behind the disposable glucose sensor
strips used by diabetics to monitor serum glucose levels. In manufacturing, GOx is used
as an additive thanks to its oxidizing effects: it prompts for stronger dough in bakery,
replacing oxidants such as bromate. It also helps remove oxygen from food packaging,
or D-glucose from egg white to prevent browning[source:Wikipedia.org/].
Glucose oxidase is found in honey and acts as a natural preservative. GOx at the
surface of the honey reduces atmospheric O2 to hydrogen peroxide (H2O2), which acts
as an antimicrobial barrier. GOx similarly acts as a bactericide in many cells
32

(fungi, immune cells)[10].


D-glucose + O2 + H2O

D-gluconic acid + H2O2

H2O2 2H + O2 +2 e

3.3 SENSOR AND ITS MEMBRANES


The integrated reagent/blood separation layer comprises reagents for the
electrochemical detection of the analyte and to exclude blood cells from the surface of
the first conductive element (working electrode)While permitting access to the first
conductive element by soluble electroactive species[6].
A glucose test strip is formed with an integrated reagent/blood separation layer
comprising a filter which has both hydrophobic and hydrophilic surface regions, an
enzyme effective to oxidize glucose, e.g., glucose oxidase, and a mediator effective to
transfer electrons from the enzyme to the conductive element. Glucose electrochemical
sensors are essentially made up of two major components: an electrode and a user
replaceable, disposable membrane assembly including a primary membrane and a
secondary membrane. The electrode is based on The Clark electrode which includes a
platinum anode and a silver cathode. A voltage of .7 volts is applied to the electrode and
current between the cathode and anode is measured. The difference between this
measured potential and that produced upon glucose reaction is proportional to the
glucose concentration in the sweat sample assayed. The membrane assembly serves
three distinct functions[6].
The primary membrane serves to selectively allow the passage of glucose
therethrough while keeping out interferants. The primary membrane,
typically a plasma etched polycarbonate, is designed to have a pore size
of 300 A.
An enzyme disposed between the primary and secondary membranes
provides a second function of catalyzing the reaction between glucose
33

and oxygen passing through the primary membrane to produce hydrogen


peroxide.
The secondary membrane is a cellulose acetate layer or can be a silicone
rubber layer which is not glucose-permeable and is a relatively
nonporous, extremely thin membrane The overall function of the
membrane assembly is to protect the electrode from contaminants[6].

Fig 3.3.1 Stack of membranes coated on working electrode(source[6])

3.4 FABRICATION OF ELECTRODE FOR SWEAT SAMPLE


Single use electrode strips are mass produced by the rapid and simple thick film
(screen printing) microfabrication or vapor deposition process. Screen printing
technology involves printing patterns of conductors and insulators onto the surface of
planar solid (plastic or ceramic) substrates based on pressing the corresponding inks
through a patterned mask. Each strip contains printed working and reference electrodes
,with the working one coated with the necessary reagents (i.e., enzyme, mediator,
stabilizer, surfactant, linking, and binding agents) and membranes. The reagents are
34

commonly dispensed by ink jet printing technology and deposited in the dry form. A
counter electrode and an additional (baseline) working electrode may also be included.
Various membranes (mesh, filter) are often incorporated into the test strips and along
with surfactants are used to provide a uniform sample coverage and separate the blood
cells. Such single-use devices eliminate problems of carry over, cross contamination, or
drift. Overall, despite their low cost and mass production such sensor strips are based
on a high degree of sophistication essential for ensuring high clinical accuracy[10]. As
screen printing technology is currently unavailable in India, the working electrode for
sweat sample was fabricated and the electrochemical studies was conducted using
cyclic voltammetry.

3.4.1 Materials and reagents


Glucose oxidase (Aspergillus niger) of analytical grade and purchased from SigmaAldrich. All aqueous solutions were prepared with reverse osmosis purified water. Gold
electrode was obtained commercially.This gold electrode was polished using Tegramin25(250 mm discs)[2].
All analytical experiments were performed in phosphate buffered saline (PBS).
Phosphate buffered saline (abbreviated as PBS) is a buffer solution commonly used in
biological research. It is a salty solution containing sodium chloride, sodium phosphate,
and (in some formulations) potassium chloride and potassium phosphate. The buffer
helps to maintain a constant pH. The osmolarity and ion concentrations of the solution
usually match those of the human body (isotonic).PBS has many uses because it is
isotonic and non-toxic to cells. It can be used to dilute substances. It is used to rinse
containers containing cells. PBS can be used as a diluent in methods to dry
biomolecules, as water molecules within it will be structured around the substance
(protein, for example) to be dried and immobilized to a solid surface. The thin film of
water that binds to the substance prevents denaturation or other conformational
changes. Carbonate buffers may be used for the same purpose but with less
effectiveness. PBS can be used to take a reference spectrum when measuring the
protein adsorption in ellipsometry.

35

Additives can be used to add function. For example, PBS with EDTA is also used
to disengage attached and clumped cells. Divalent metals such as zinc, however,
cannot be added as this will result in precipitation. For these types of applications,
Goods buffers are recommended.
There are many different ways to prepare PBS. Some formulations do not contain
potassium, while others contain calcium or magnesium[2].
One of the most common preparations is described below.

137 mM NaCl
2.7 mM KCl
10 mM Na2HPO4
2 mM KH2PO4
After complete mixing, top up final solution to 10 L. The pH of the 10X stock is will

be approximately 6.8, but when diluted to 1x PBS it should change to 7.4.When making
buffer solutions, it is good practice to always measure the pH directly using a pH meter.
If necessary, pH can be adjusted using hydrochloric acid or sodium hydroxide.
Table :3.4.1.1 The most common composition of PBS (1X)(source:Wikipedia.org/)
Salt

Concentration (mmol/L)

Concentration (g/L)

NaCl

137

8.0

KCl

2.7

0.2

Na2HPO4

10

1.44

KH2PO4

1.8

0.27

3.4.2 Etching of gold electrode

36

The etching of gold is a key enabling technology in the fabrication of many micro
devices and is widely used in the electronic, optoelectronic and micro electro
mechanical systems (MEMS) industries.Nano-porous gold (nPG) electrodes (porous
gold electrodes witha pore size distribution limited to the nanometre range) are
considered a very promising alternative for the development of new generation
bioelectrochemical devices with implantable capability. These non-toxic electrodes have
remarkable properties, such as high conductivity, large surface area, three-dimensional
open porosity, and biocompatibility.An even more promising material for the production
of high sensitivity biosensors is highly porous gold (hPG)[2].
While retaining the morphology observed with nPG , hPG electrodes present large
micro-pores that are lined with nano-pores themselves. As a consequence, hPG
electrodes have a very wide pore size distribution, leading to extremely large surface
areas and hence larger current densities in comparison to conventional nPG .A new and
rapid method of producing hPG electrodes by direct electro deposition of porous gold
films onto gold electrodes was recently reported .These electrodes were characterised
by a3D foam-like structure, with a wide pore size distribution (ranging from 10 nm to 30
nm), and a roughness factor (calculated interms of electrochemically effective surface
area) approximately 103 times higher than polished gold. The hPG electrodes showed
excellent glucose electro oxidation activity with a detection limit as low as
5microM.However, the high specificity required for some applications, such as
implantable biofuel cell devices where the fuel (e.g. glucose) and the oxidant (e.g.
oxygen) are fed to the system as a mixture, demands the implementation of enzymatic
electrodes.The large surface area of hPG electrodes and their complex morphology
make them an ideal support for enzyme immobilisation at high loadings. This hypothesis
is encouraged by the successful production of GOx-immobilised nPG electrodes
recently reported .In these cases the GOx immobilisation protocols involved the nPG
functionalization with thiol-linker molecules or with conductive polymers, such as
poly(3,4-ethylenedioxythiophene), to enhance the electron transfer process .An efficient,
simple, cost-effective, and rapid method for the functional immobilisation of GOx onto
hPG surfaces was used to obtained better accuracy and sensitivity. The immobilisation

37

protocol does not require any electrode pre-treatments with linker molecules orpolymers
and it is simple to reproduce. In particular, GOx is immobilised onto the hPG surface via
a one-step electrochemical adsorption process in a phosphate buffer with no additional
chemicals. The use of the resulting GOx-immobilised hPG electrode is tested for
glucose sensing[2].

Gold has a very high density of 19.3 g/cm 3, its crystal structure is cubic face
centred. With a standard potential of 1.5, gold belongs to the noble metals. The
electron configuration [Xe]4f145d106s1 strongly prevents the oxidation of gold: The
completely occupied 5d orbital extends beyond the single valence electron which
hereby is well shielded against any reaction partners. Wet chemical etching of gold
therefore requires a strong oxidizer for the separation of the unpaired valence
electron, as well as a complexing agent which suppresses the reassembly of oxidized
gold atoms back into the crystal.
The gold was polished before etching to remove the surface contaminants.The
polished gold (2*1*1mm) was then treated with aqua regia. It was etched for 23 seconds
and a highly porous gold was obtained.Mixtures of nitric acid and hydrochloric acid (in a
mixing ration of 1 : 3 also called aqua regia) are able to etch gold at room temperature.
The very strong oxidative effect of this mixture stems from the formation of nitrosyl
chloride (NOCl) via
HNO3+ 3 HCl

NOCl + 2 Cl + 2 H2O,

while free Cl radicals formed in the solution keep the noble metal dissolved as Clcomplex(tetrachloro gold-(III)-acid = HAuCl4). HNO3/HCl mixtures are not stable and
decompose accompanied by the formation of nitrogen oxides and Cl2.
The etch rate of aqua regia for gold is approx. 10 m/min (at room temperature)
and can be increased to several 10 m/min at elevated temperatures.Palladium,
aluminium, copper and molybdenum are also etched by aqua regia. For etching
platinum or rhodium, the etching solution has to be heated to attain a reasonable etch
rate. Etching of iridium requires strongly heated (boiling) aqua regia.
38

Silver is not attacked by aqua regia due to the formation of a silver chloride
passivation film. Chromium, titanium, tantalum, zirconium, hafnium and niobium also
form a very stable passivation film (in many cases the metal oxide) protecting the metal
against the attack of aqua regia at least at room temperature. For same reason,
tungsten reveals a very slow etch rate in aqua regia(source:Wikipedia.org/).

3.5 IMMOBILIZATION OF ENZYME ON POROUS GOLD ELECTRODE


Enzymes are protein molecules which serve to accelerate the chemical reactions
of living cells (often by several orders of magnitude).

Without enzymes, most

biochemical reactions would be too slow to even carry out life processes. Enzymes
display great specificity and are not permanently modified by their participation in
reactions. Since they are not changed during the reactions, it is cost-effective to use
them more than once. However, if the enzymes are in solution with the reactants and/or
products it is difficult to separate them. Therefore, if they can be attached to the reactor
in some way, they can be used again after the products have been removed. The term
"immobilized" means unable to move or stationary.
Immobilized enzyme is an enzyme that is physically attached to a solid support
over which a substrate is passed and converted to product. Enzyme immobilization may
be defined as confining the enzyme molecules to a distinct phase from the one in which
the substrates and the products are present; this may be achieved by fixing the enzyme
molecules to or within some suitable material. It is critical that the substrates and the
products move freely in and out of the phase to which the enzyme molecules are
confined. Immobilization of enzyme molecules does not necessarily render them
immobile; in some methods of immobilization, e.g. entrapment and membrane
confinement, the enzyme molecules move freely within their phase, while in cases of
adsorption and covalent bonding they are, in fact, immobile(source:Wikipedia.org/).
The achievement of efficient electron transfer between the enzyme active centre
and the electrode is critical. Usually a mediated electron transfer (MET) mechanism is
39

required. This might involve the use of small redox active particles and polymers as
electron carriers (mediators), such as organic dyes, ferrocene and its derivatives,
modified vitamin complexes,and conducting salts If the mediator is in solution their diffusion to the electrode surface allows for a more rapid electron transfer compared to the
direct transfer from the enzyme itself. Alternatively, the mediators can be polymerised
directly onto the electrode surface or co-immobilized with the reacting enzymes to
further enhance the rate of electron transfer .However, the use of redox active electron
carriers can have several drawbacks, such as short lifespans, poor biocompatibility,risk
of leaching away from the electrode surface, potential toxicity. Consequently, the
achievement of a direct electron transfer (DET)process is preferred[2].
In the case of glucose oxidase (GOx), DET is more difficult to achieve, due to the
fact that the GOx redox centre is buried inside the enzyme structure, and is far from any
feasible electrode binding sites. To achieve efficient electron transfer, the use of GOx
has been often combined with mediator compounds, of which ferrocene is the most
common. Most of the GOx immobilzation protocols reported, while effective, are usually
very expensive, due to the reagents required. These protocols are often very laborious,
involving multi-steps in the immobilization procedure that can be sources of
experimental errors. Moreover the resulting bioelectrodes can be unstable and
inefficient with limited opportunity for practical implementations, due to the leaching of
the mediator implemented and the dependence of the electrontransfer process on the
capability

of

the

mediator

to

be

rapidly

oxidised

and

reduced[2].

The materials used for immobilization of enzymes, called carrier matrices, are
usually inert polymers or inorganic materials. The ideal carrier matrix has the following
properties:
(i) low cost
(ii) inertness
(iii) physical strength
(iv) stability
(v) regenerability after the useful lifetime of the immobilized enzyme
(vi) enhancement of enzyme specificity
(vii) reduction in product inhibition
40

(viii) a shift in the pH optimum for enzyme action to the desired value for the process
(ix) reduction in microbial contamination and non-specific adsorption.
Clearly, most matrices possess only some of the above features. Therefore, carrier
matrix for the immobilization of an enzyme must be chosen with care keeping in view
the properties and limitations of various matrices.
3.5.1Methods of Immobilization
The various methods used for immobilization of enzymes may be grouped into the
following four types:
(i) Adsorption
(ii) Covalent Bonding
(iii)Entrapment and
(iv) Membrane Confinement
(i) Adsorption:
In case of adsorption, the enzyme molecules adhere to the surface of carrier matrix
due to a combination of hydrophobic effects and the formation of several salt links per
enzyme molecule. The binding of enzyme molecules to the carrier matrix is usually very
strong, but it may be weakened during use by many factors e.g. addition of substrate,
pH or ionic strength.
(ii) Covalent Binding:
In this system the enzyme molecules are attached to the carrier matrix by formation
of covalent bonds. As a result the strength formation occurs with the side chains of
amino acids of the enzyme, their degree of reactivity being dependent on their charged
status.

Roughly

the

following

relation

-S > -SH > -0 > -NH2 >- COO > OH >> -NH3+
(iii) Entrapment:

41

is

observed

in

reactivity.

In this approach, enzyme molecules are held or entrapped within suitable gels or
fibers and there may or may not be covalent bond formation between the enzyme
molecules and the matrix. A non-covalent entrapment may be viewed as putting the
enzyme molecule in a molecular cage just as a caged bird / animal. When covalent
binding is also to be generated, the enzyme molecules are usually treated with a
suitable reagent.
(iv) Membrane Confinement:
Enzyme molecules, usually in an aqueous solution, may be confined within a
semipermeable membrane which, ideally, allows a. free movement in either direction to
the substrates and products but does not permit the enzyme molecules to escape.
Most of the techniques described above have been used for the immobilization of
biocatalyst for biosensor applications. The choice of the support and the technique for
the preparation of membranes has been dictated by the low diffusional resistance of the
membrane coupled with its ability to incorporate optimal amount of enzyme per unit
area. In this respect, stable membranes have been prepared by binding glucose
oxidase to cheese cloth in the fabrication of a glucose biosensor. Enzymes entrapped
inside the reversed micelle have also shown promise in the fabrication of biosensors.
Cross-linked enzyme crystals (CLCs) described above provide their own supports and
so achieve enzyme concentration close to the theoretical packing limit in excess of even
highly concentrated enzyme solutions. In view of this, CLCs are particularly attractive in
biosensor applications where the largest possible signal per unit volume is often
critical(source:Wikipedia.org/).
Sensors based on small transducer or thinner enzyme immobilized membranes
(miniature biosensors) are also emerging. The development of molecular devices
incorporating a sophisticated and highly organized biological information processing
function, is a long-term goal of bioelectronics. For this purpose, it is necessary in the
future to develop suitable methods for micro immobilizing the proteins/enzymes into an
organized array/pattern, as well as designing molecular structures capable of
42

performing the required function. A typical example is the micro immobilization of


proteins into organized patterns on a silicone wafer based on a specific binding reaction
between strepatavidin and biotin combined with photolithography techniques.
Immobilized enzymes have also been used for various other analytical purposes[6].
There are many kinds of assembling techniques for protein immobilization, such as
Langmuir-Blodgett

(LB)

,self-assembled

monolayers,

layer-by-layer

electrostatic

adsorption of alternate multilayers and so on. Glutaraldehyde is a traditional material for


cross-linking enzymes, but some new chemicals, such as chitosan, are used and good
effects have been obtained ;electrically conductive ultrananocrystalline diamond thin
films which realized covalent immobilization of GOx via the tethered aminophenyl
functional groups have served as a robust platform for a new class of bioinorganic
interfaces and electrochemical sensors, two types of sol-gel precursor mixture of 3glycidoxypropyltrimethoxysilane with methyltrimethoxysilane or tetraethoxysilane , ionic
liquidmethylimidazolium
hexafluophosphate providing a unique microenvironment for the immobilization of GOx ,
hexagonal mesoporous silica adsorbing GOx retained its bioactivity and stability[10].
LB films had been employed for the immobilization of GOx, since the very thin
nature in nanoscale might produce a highly sensitive sensor with ultrafast response
time, and catalysts were involved to improve the response current.Conducting polymers
have been receiving great and broad interests in clinical diagnosis and environmental
monitoring. There are many advantages in preparing sensors with conducting
polymers, such as efficient transfer of electric charge, and considerable flexibility in
available chemical structure.
3.5.2 Effects of Immobilization on Enzyme
Often kinetic behavior of an immobilized enzyme may differ significantly from that
of its free molecules. Different enzymes respond differently to the same immobilization
protocol. Therefore, a suitable immobilization protocol has to be worked out for a given
enzyme. The effects on enzyme kinetics (i.e. activity) may be due to the influence of

43

matrix per se or due to conformational changes in the enzyme molecules induced by the
procedure of immobilization.

3.5.3 Advantage of Immobilization


Enzymes are costly items, and can be used repeatedly only if they can be
recovered from the reaction mixtures. Immobilization permits their repeated use since
such enzyme preparations can be easily separated from the reaction system.
1. Immobilized enzymes can be used in non-aqueous systems as well, which may be
highly desirable in some cases.
2. Continuous production systems can be used, which is not possible with free
enzymes.
3. Thermo stability of some enzymes may be increased. For example, glucose
isomerase denatures at 45C in solution, but is stable for about 1 yr even at 65C when
suitably immobilized.
4. Recovery of enzyme may also reduce effluent handling problems.
5. Enzymes can be used at much higher concentrations than free enzyme.
3.5.4 Immobilization of GOx onto hPG electrodes
GOx was electrochemically adsorbed onto the prepared hPG disk electrodes by
conducting a total of 6 CV scans between 0.42 V and 0.60 V (vs. SCE) at a scan rate of
1 mV s1, in a PBS solution containing 0.45 mg ml1GOx (approximately 8 U ml1as
per activity rating of manufacturer).As a term of comparison of performance, GOx was
also immobilized by absorption. In this case, the hPG electrodes were incubated with
the GOx solution in PBS (0.45 mg ml1) for 1 hour at room temperature, without
conducting any CV scans. In both cases ,the GOx-hPG electrodes were then thoroughly
rinsed three times with PBS to remove any weakly bonded enzyme, and stored in PBS
at 4C until used. The amount of GOx immobilized onto the hPG electrodes, was
estimated by performing a kinetic assay (provided by MegazymeLtd.) of the enzyme
44

solution before and after the immobilization procedure and assuming no enzyme losses
during the process[2].

3.6 ELECTROCHEMICAL STUDIES USING CYCLIC VOLTAMMETRY


Cyclic voltammetry or CV is a type of potentiodynamic electrochemical
measurement. In a cyclic voltammetry experiment, the working electrode potential is
ramped linearly versus time. Unlike in linear sweep voltammetry, after the set potential
is reached in a CV experiment, the working electrode's potential is ramped in the
opposite direction to return to the initial potential. These cycles of ramps in potential
may be repeated as many times as desired. The current at the working electrode is
plotted versus the applied voltage (i.e., the working electrode's potential) to give the
cyclic voltammogram trace. Cyclic voltammetry is generally used to study the
electrochemical properties of an analyte in solution(source:Wikipedia.org/).

Fig3.6.1 Typical cyclic voltammogram where ipc and ipa show the peak cathodic
and anodic current for reversible reaction(source:Wikipedia.org/)
In cyclic voltammetry, the rate of voltage change over time during each of these
phases is known as the experiment's scan rate (V/s). The potential is applied between
45

the working electrode and the reference electrode while the current is measured
between the working electrode and the counter electrode. These data are plotted as
current (i) vs. applied potential (E, often referred to as just 'potential'). In Figure 2, during
the initial forward scan (from t0 to t1) an increasingly reducing potential is applied; thus
the cathodic current will, at least initially, increase over this time period assuming that
there are reducible analytes in the system. At some point after the reduction potential of
the analyte is reached, the cathodic current will decrease as the concentration of
reducible analyte is depleted. If the redox couple is reversible then during the reverse
scan, the reduced analyte will start to be re-oxidized, giving rise to a current of reverse
polarity (anodic current) to before. The more reversible the redox couple is, the more
similar the oxidation peak will be in shape to the reduction peak. Hence, CV data can
provide information about redox potentials and electrochemical reaction rates.
For instance, if the electron transfer at the working electrode surface is fast and
the current is limited by the diffusion of analyte species to the electrode surface, then
the peak current will be proportional to the square root of the scan rate. This relationship
is described by the Cottrell equation. In this situation, the CV experiment only samples a
small portion of the solution, i.e., the diffusion layer at the electrode surface.
3.6.1 Experimental setup of cyclic voltammetry
A standard CV experiment uses a reference electrode, working electrode, and
counter electrode. This combination is sometimes referred to as a three-electrode
setup. An electrolyte is usually added to the sample solution to ensure sufficient
conductivity. The solvent, electrolyte, and material composition of the working electrode
will determine the potential range that can be accessed during the experiment.
The electrodes are immobile and sit in unstirred solutions during cyclic
voltammetry. This "still" solution method gives rise to cyclic voltammetry's characteristic
diffusion-controlled peaks. This method also allows a portion of the analyte to remain
after reduction or oxidation so that it may display further redox activity. Stirring the
solution between cyclic voltammetry traces is important in order to supply the electrode
46

surface with fresh analyte for each new experiment. The solubility of an analyte can
change drastically with its overall charge; as such it is common for reduced or oxidized
analyte species to precipitate out onto the electrode. This layering of analyte can
insulate the electrode surface, display its own redox activity in subsequent scans, or
otherwise alter the electrode surface in a way that affects the CV measurements. For
this reason it is often necessary to clean the electrodes between scans.
Common materials for the working electrode include glassy carbon, platinum, and
gold. These electrodes are generally encased in a rod of inert insulator with a disk
exposed at one end. A regular working electrode has a radius within an order of
magnitude of 1 mm. Having a controlled surface area with a well-defined shape is
necessary for being able to interpret cyclic voltammetry results. To run cyclic
voltammetry experiments at very high scan rates a regular working electrode is
insufficient. High scan rates create peaks with large currents and increased resistances,
which result in distortions. Ultra microelectrodes can be used to minimize the current
and resistance. The counter electrode, also known as the auxiliary or second electrode,
can be any material which conducts current easily and will not react with the bulk
solution. Reactions occurring at the counter electrode surface are unimportant as long
as it continues to conduct current well. To maintain the observed current the counter
electrode will often oxidize or reduce the solvent or bulk electrolyte[2].
3.6.2 Application of cyclic voltammetry
Cyclic voltammetry (CV) has become an important and widely used electro
analytical technique in many areas of chemistry. It is often used to study a variety of
redox processes, to determine the stability of reaction products, the presence of
intermediates in redox reactions, reaction and electron transfer kinetics, and the
reversibility of a reaction. CV can also be used to determine the electron stoichiometry
of a system, the diffusion coefficient of an analyte, and the formal reduction potential of
an analyte, which can be used as an identification tool. In addition, because
concentration is proportional to current in a reversible, Nernstian system, the

47

concentration of an unknown solution can be determined by generating a calibration


curve of current vs. concentration[2].
3.6.3 Biofuel cell setup of cyclic voltammetry
The immobilisation mechanism achieved during the CV scans in the presence of
GOx is not merely absorption, but an electrochemically driven physical adsorption.
Since GOx is anionic at pH 7.0, and since a relatively high positive scan range is used,
the CV scans are likely to promote an electrostatic attraction between the gold surface
and the free enzyme. In contrast, when the electrodes are simply placed in con-tact with
the enzymatic solution, absorption occurs into the pores of the electrode. Consequently,
the resulting electrode is less stable as GOx can easily leach out[2].

Fig3.6.3.1 Electrochemical cell for cyclic voltammetry studies

48

Fig3.6.3.2 Electrochemical cell setup


Normally,

one

prefers

to

use

three-electrode

system

for

voltammetry/amperometric measurements because it offers good control of the


interfacial potential (the driving force of the reaction) at the working electrode.
Conventional wisdom in the electrochemical arts suggests biosensors of the following
types:
1) a three electrode system, wherein a working electrode is referenced against a
reference electrode (such as silver/silver chloride) and a counter electrode provides a
means for current flow;
2) a two electrode system, wherein the working and counter electrodes are made of
different electrically conducting materials;
3) a two electrode system, wherein the working and counter electrodes are made of the
same electrically conducting materials, but the counter electrode is larger than the
working electrode

49

CHAPTER 4
RESULTS AND DISCUSSION
4.1CV SCANS OF hPG ELECTRODE Vs SCE
The CV scans might actively draw the enzyme to the surface of the electrode, thus
significantly increasing the loading. For the case of electrochemical adsorption in fact an
average of 31.7% reduction of activity in solution after the immobilisation process was
observed. On the other hand, the amount of enzyme immobilised by absorption was so
low that no significant changes were observed with the activity tests in solution prior and
after the incubation with the hPG electrode.

Fig 4.1.1 Electrochemical response to glucose in PBS of the hPG electrodes. A:


CV scans of the GOxads-hPG electrode in the absence and presence of 10 mM of
glucose.B: CVscans of the GOxabs-hPG electrode in the absence and presence
of 10 mM of glucose. The control was performed with the hPG electrode (no GOx)
in the presence of 10 mM of glucose. The scan rate for each test was 1 mV
s1.Extract from ref [2]
The amperometric response of the two GOx-hPG electrodes to glucose (10 mM)
was tested and compared at a potential of 0.52 Vvs SCE (the potential at which the
50

electrodes showed the highest sensitivity to glucose).This potential is within the


0.4 - 0.6 V range of values previously reported for the oxidation peak of H2O2on
polished gold .H2O2 is formed through the oxidation of glucose by GOx and, since its
formation is related to the amount of glucose in solution , many glucose sensors rely on
the resultant oxidation of H2O2 on gold to determine the concentration of glucose in a
sample.
4.2 CV SCANS OF POROUS GOLD ELECTRODE Vs Ag/AgCl2
Cyclic voltammetry (CV) test was performed in a three-electrode electrochemical
set-up with a Ag/AgCl2 reference electrode and a platinum rod counter electrode. CV
scans were performed at a scan rate of 1 mV s1 in the potential range of 0.42 - 0.60 V
(vs. Ag/AgCl2) in order to determine the optimum potential for the amperometric
analysis(this range incorporates the normal oxidation potential of H2O2 on polished
gold).

Fig 4.2.1 Cyclic voltammograms of GOx immobilized gold electrode Vs Ag/AgCl2


at different scan rates with a PBS electrolyte of pH 7.0
51

The immobilization mechanism achieved during the CV scans in the presence of


GOx is not merely absorption, but an electrochemically driven physical adsorption.
Since GOx is anionic at pH 7.0, and since a relatively high positive scan range is used,
the CV scans are likely to promote an electrostatic attraction between the gold surface
and the free enzyme.
In contrast, when the electrodes are simply placed in contact with the enzymatic
solution, absorption occurs into the pores of the electrode. Consequently, the resulting
electrode is less stable as GOx can easily leach out. The CV scans might actively draw
the enzyme to the surface of the electrode, thus significantly increasing the loading. For
the case of electrochemical adsorption in fact an average of 31.7% reduction of activity
in solution after the immobilization process was observed.

Fig 4.2.2 Cyclic voltammogram of GOx immobilised gold electrode Vs Ag/AgCl2


reference electrode for glucose concentration of 0.1mM at 1m/V

CHAPTER 5
52

CONCLUSION AND FUTURE SCOPE


The actual amount of oxidized form of the redox mediator needed in the
reagent is governed by the concentration range of the analyte sought to be measured.
When the counter electrode is smaller than the working electrode, the amount of
oxidized form of the redox mediator used for the cv scans must be increased. For
example, it has been shown that when the counter electrode is about half the size of the
working electrode, a mixture of about 2700 nanomoles (nmol) of ferricyanide and about
900 nmol of ferrocyanide (dissolved in 20 u] of water) satisfied the requirements stated
above. The buffer having a higher oxidation potential than the reduced form of the redox
mediator and being of sufficient type(i.e phosphate buffered saline) and in sufficient
amount to provide and maintain a pH(i.e pH 7.0) at which the Gox catalyzes the
reaction is required to complete the process of detecting the concentration of the
analyte(i.e beta-D-glucose).
On the other hand, the amount of enzyme immobilized by absorption was so low
that no significant changes were observed with the activity tests in solution prior and
after the incubation with the hPG electrode. The amount of enzyme included in the
reagent may vary depending upon the time period desired for completion of the reaction
involving analyte, enzyme, and oxidized form of the redox mediator. The more enzyme
added, the shorter the time period for completion of the reaction.
Thus the minimum concentration of glucose found in human perspiration was
measured by conducting CV scans using the fabricated porous Au GOX electrode. The
future work is focused on removal of electrolyte interferences in human perspiration to
improve the current obtained in the working electrode. The primary focus is on resins
which can filter out the interferences (urea, sodium and other ions) to avoid electrode
corrosion. The interferences if removed could yield better results in detecting of glucose
levels from human perspiration.

REFERENCES
53

[1] B.A.McSwiney.et.al, "The composition of human perspiration",1934,Royal Soceity


of medicine proceedings.
[2] Hendrik du toit, Mirella di lorenzo "Glucose oxidase directly immobilized onto
highly porous gold

electrodes for sensing

and fuel

cell

applications"

2014,Electrochimica Acta, Elsevier Publications.


[3] Herbert .L.Berman.et.al "Glucose measurement utilizing non-invasive assesment
methods",2003,US patent application.
[4] Jonathon.O.Howell et.al, Bioanalytical systems Inc "Glucose test strips and
electroanalytical chemistry,2011,US patent application,US7807031 B2,Page nos.:
1-12.
[5] Matthew Bularzik et.al "Accessible blood glucose monitor", University of
Connecticut,2012,Page nos.:3-30.
[6] McAleer et al Disposable test strip with integrated blood separation layer
2010, US 7807031B2 ,Page nos.:2-8
[7] Miriam Garcia "Glucose meter fundamentals and design",2013,Freescale
..semiconductors,AN4364,Page nos.:2-20
[8] Moyer.et.al ,"Correlation between sweat glucose and blood glucose in subjects
..with diabetes",2012 ,PubMed journals.
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54