CHAPTER 1INTRODUCTION 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 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 handheldblood 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 leadingcause 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]) Location Prevalence (%) Total

MalesFemales

Karnataka16.0622.0419.78

Kerala16.513.512.5

Tamilnadu6.24.45.1

Tamilnadu10.48.09.2

Wardha8.88.18.5

Maharashtra9.29.99.3

Andhra Pradesh14.312.013.2

Mysore4.582.663.77

National2.52.52.7

Kashmir3.54.54.0

Fig:1.1.2 Blood glucose levels for non-diabetic and insulin-dependent diabeticsubjects. 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 METHODThe 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 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 high-performance 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])PhAmmoniamgm./100ccUreamgm./100c.cAmino acidmgm./100c.cGlucosemgm./100ccChloridesNaCl/1000c.c.

Average from 10 normal femaleSubjects6.576.019.26.520.03.0

Average from 14 normal male subjects6.14

4.7

21.44

5.0

12.6

3.7

1.4 LITERATURE SURVEY

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 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 DEFINITIONThe 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 WORKThe 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 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.

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-2EXISTING 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.

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 measurementThe 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 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 below2.1.2.1 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. 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 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 v0=c/0 is inelastically scattered at frequencies v0vi. 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 amplitude-modulated 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 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) non-invasive (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 CO2 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].

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 infraredlight 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 glucose-binding protein, and boronic acid derivatives (which bind the diols of sugars). Techniques include measuring changes in fluorescence resonance energy transfer(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 (H2O2) 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 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 nL 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 dm1 (g/L)1.

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

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 clearmedia; in fact, one of earliest application of polarimetry relied on this relationship todetermine 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 GOx (glucose oxidase ) entrapped over an oxygen electrode via a semipermeable dialysis membrane[6].

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 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 (GOx) 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 pH-selective 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. 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: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 (FADH2). 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. 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]).

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].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

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 METHODThe 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: 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 glucoseconcentration. 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 3PROPOSED METHODOLOGY 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 manyconstituents (e.g., stabilizers, processing aids, etc.). However, for this discussion it willbe treated more simply as an enzyme and mediator. An enzyme overcomes many ofthe problems associated with a variable biological sample matrix. Because glucoseand enzymes do not readily exchange electrons directly with an electrode, anelectrochemical measurement requires a mediator to facilitate (or mediate) the electrontransfer. 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 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 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 itsmetabolites. 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 adimericprotein, 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 carbohydratechains. At pH 7, glucose exists in solution in cyclic hemiacetal form as 63.6% -D-glucopyranose and 36.4% -D-glucopyranose, the proportion of linear andfuranoseform 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. Glucose oxidasecatalyzesthe oxidation of -D-glucose intoD-glucono-1,5-lactone, which thenhydrolyzestogluconic acid. In order to work as a catalyst, GOx requires acofactor,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 FADH2. Then FADH2is oxidized by the final electron acceptor, molecularoxygen(O2), which can do so because it has a higher reduction potential. O2is then reduced tohydrogen peroxide(H2O2). Glucose oxidase is widely used coupled to peroxidasereaction that visualizes colorimetrically the formed H2O2, for the determination of free glucose inseraor blood plasmafor 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 achromogento produce a colored compound which may be measured spectroscopically. For example, hydrogen peroxide together with4 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 glucosebiosensorsuse an electrodeinstead of O2to 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 diabeticsto monitor serum glucose levels. In manufacturing, GOx is used as an additive thanks to its oxidizing effects: it prompts for stronger dough inbakery, 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 O2tohydrogen peroxide(H2O2), which acts as anantimicrobialbarrier. GOx similarly acts as a bactericide in many cells (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 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 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 Sigma-Aldrich. All aqueous solutions were prepared with reverse osmosis purified water. Gold electrode was obtained commercially.This gold electrode was polished using Tegramin-25(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. 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/)SaltConcentration (mmol/L)Concentration (g/L)

NaCl1378.0

KCl2.70.2

Na2HPO4101.44

KH2PO41.80.27

3.4.2 Etching of gold electrode 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 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/cm3, 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 Cl-complex(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. 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 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 dif-fusion 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 ofthe 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 (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 is observed in reactivity. -S > -SH > -0 > -NH2 >- COO > OH >> -NH3+ (iii) Entrapment: 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 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 3-glycidoxypropyltrimethoxysilane with methyltrimethoxysilane or tetraethoxysilane , ionic liquidmethylimidazoliumhexafluophosphate 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 conductingpolymers, 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 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 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 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 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 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

Fig3.6.3.2 Electrochemical cell setup Normally, one prefers to use a 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

CHAPTER 4RESULTS 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 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/AgCl2Cyclic 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 7.0The 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 5CONCLUSION 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

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