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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
1
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].
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
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
Glucose
Chlorides
mgm./100cc
NaCl/1000
20.0
c.c.
3.0
12.6
3.7
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
5
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
6
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.
spectroscopy,
Photo
acoustic
laser
spectroscopy,
Polarization
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.
9
processes,
non-invasive
measurement
processes
may
include
spectroscopy
(IR),
Raman
spectroscopy, photoacoustic
spectroscopy,
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].
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
12
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
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].
14
(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).
16
17
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]).
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
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)
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
23
25
The following are the features of the PIC16LF178X device and some of the
peripherals in its integrated measurement engine:
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].
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.
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
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].
30
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
H2O2 2H + O2 +2 e
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.
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
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/).
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
(LB)
,self-assembled
monolayers,
layer-by-layer
electrostatic
43
matrix per se or due to conformational changes in the enzyme molecules induced by the
procedure of immobilization.
solution before and after the immobilization procedure and assuming no enzyme losses
during the process[2].
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
48
one
prefers
to
use
three-electrode
system
for
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.
CHAPTER 5
52
REFERENCES
53
and fuel
cell
applications"
54