The main technology currently used in diabetic care, monitors blood glucose and involves an invasive “fingerstick” step. However, low patient compliance and non-continuous glucose monitoring imply poor management of diabetes through this technology, which could lead to adverse and potentially life threatening conditions. In this context, non-invasive glucose sensing appears as an alternative that can bring a change in the prevention and management of the diabetic condition, promising to eliminate patient resistance towards more frequent monitoring and, hence, considerably improving diabetic’s control over glycaemia. However, no non-invasive technology has yet succeeded on the market over the long term. The research field is therefore open to innovative and performant non-invasive technologies. This thesis presents the development of a non-invasive biosensor which as a core principle accesses individual, privileged glucose pathways in the skin (such as hair follicles), allowing the extraction of glucose directly from the interstitial fluid, via reverse iontophoresis (RI). The transdermally extracted glucose is then electrochemically detected in a small size sensor with very high sensitivity. A fully developed technology based on this principle will not require fingerpricking and would thus eliminate users’ main barrier to glucose monitoring. The developed sensor is enzymatic (using glucose oxidase), which electrochemically detects the produced H2O2; while the electrode material is graphene produced by Chemical Vapour Deposition, a promising carbon nanomaterial platform for biofunctionalisation and biosensing. The sensor is a miniature one (typically of 9 mm2 area, containing 24 μL of gel encasing the enzyme), with demonstrated performance parameters that are highly competitive (sensitivity of 2.89 μA.mM-1.cm-2 and limit of detection down to 1 μM), with high specificity towards glucose. The combination of this sensor with glucose extraction by reverse iontophoresis was then validated (with proportionality between subdermal and extracted glucose concentrations demonstrated); as well as enhanced extraction through targeting of hair follicles with the miniature device. The electrochemical determination of glucose concentration was further confirmed by 1H quantitative-NMR detection of glucose. Finally, several such sensors were integrated in a multiplex configuration, and independent sensing, with no cross-talk was demonstrated. The steps demonstrated and implemented so far are proof-of-concept of a highly promising non-invasive, transdermal, future technology for diabetic care.

This thesis investigates the potential for monitoring current and historic blood serum concentrations of amino acids via transdermal extraction using phenylalanine as a case study. This work furthers the field of non-invasive monitoring of amino acid disorders which have several advantages over invasive methods such as blood tests. In this thesis we derive models to simulate blood serum concentrations, the formation of the skin reservoir and, finally, transdermal extraction of amino acids under an applied electric field. Chapter 1 concerns itself with the biological background and sets up motivation of the thesis by discussing amino acids, associated amino acid disorders, the overarching clinical problem, skin structure and transdermal extraction methods. Chapter 2 then considers mathematical techniques utilised throughout the thesis. Chapter 3 formulates a model for the distribution of phenylalanine in blood serum. One compartment and two compartment approaches are considered in both a fasting state and a non-fasting state. We consider if these have a noticeable effect on the blood serum concentration of phenylalanine. Having obtained a model for the distribution of phenylalanine in blood serum, chapter 4 models the formation of reservoirs of amino acids in the skin. Prior work has identified the existence of such a reservoir, but its formation has not been addressed. The models developed consider the effect of the removal of outer layers of skin, the stratum disjunctum, and production of amino acids in the skin. Unknown parameters are estimated by comparing the model to in vivo and in vitro data. Chapter 5 and 6 are concerned with transdermal extraction under an applied electric field. Chapter 5 formulates the velocity induced by applying an electric field across a charged interface. Chapter 6 utilises these results for modelling extraction of compounds through the skin under an applied electric field.

It has been shown that prolonged systemic presence of a drug can cause a build up of that drug in the skin. This drug ‘reservoir’, if properly understood, could provide useful and important information about the recent drug-taking history of a patient. In this thesis we create three mathematical models which combine to explore the potential for a drug reservoir to form in the skin and be collected as a method of monitoring compliance. The first model is used to characterise timedependent drug concentrations in plasma and tissue following a customisable drug regimen. Outputs from this model provide boundary conditions for the second, spatio-temporal model of drug build-up and concentration profile in the skin. This then provides initial conditions for the final model which predicts the extraction. These models are then used to identify the scenarios which have the greatest potential for successfully monitoring patient compliance via the skin. We focus in particular on drugs that are highly bound as this will restrict their potential to move freely into the skin but which are lipophilic so that, in the unbound form, they would demonstrate an affinity to the outer layers of the skin (which are built around a lipid matrix). We highlight how this study might be used to inform future experimental design and data collection in order to provide relevant parameter estimates for reservoir formation and its potential to contribute to enhanced drug monitoring techniques.

No description available.

Iontophoresis is a method to enhance and regulate the transdermal drug delivery by application of an electric field to the skin. Application of small electric current (µ A) enhances transport of both charged and neutral molecules across the skin. Reverse of this process enables extraction of analytes across the skin for noninvasive sensing and diagnosis. Hence it is planned to conduct detailed studies on Reverse Iontophoresis. An invitro model is developed to study the extraction of glucose across the skin. Effects of magnitude of electric current, time of application, pH etc. on the extraction of glucose are studied. It is observed that extraction of glucose at the cathode is higher than at the anode. Advantage of invitro model is the possibility of varying parameters to an extent which is impossible invivo. Instrumentation suitable for continuous monitoring of reverse iontophoresis invivo in human subjects is developed. It supplies the required current and acquires the potential profile of the skin during reverse iontophoresis. Potential profiles showed that skin resistance decreases with the application of current. Experimental results revealed that the application of pulsed DC tends to make the reverse iontophoresis more effective by enhancing the flow of analytes which is proved by the fact that skin resistance decreases and stabilizes faster in comparison to the one with direct current reverse iontophoresis. Present work emphasizes the importance of selecting an appropriate duty cycle and frequency for reverse iontophoresis. Duty cycle around 95% and frequency of 250 mHz are good for low frequency reverse iontophoresis. Effect of reverse iontophoresis on the skin recovery is observed by monitoring the potential profiles at the end of the process. In all the reverse iontophoresis experiments, safety of the patient is ensured by fixing a compliance voltage level. Finally, Instrumentation to measure the capacitance of biosensors is developed based on frequency domain technique with a sinusoidal input. Accuracy in capacitance measurements is ±5%. Glucose measurement is demonstrated with the developed instrument using a capacitance type biosensor. The obtained results are in good agreement with the standard UV-Visible spectroscopic measurements based on phenol-sulphuric acid assay method.

Diabetes mellitus is one of the metabolic disorders prevailing all the over world. About 381.8 million people are affected by diabetes mellitus (DM) during 2013 and it is estimated to increase to 80% by 2035. Nephropathy, retinopathy, neuropathy and cardiovascular diseases are common complications arise in the patients suffering from diabetes Type I and Type II. Continuous monitoring of glucose will give greater clinical acumen on glucose metabolism of patients than conventional intensified glucose monitoring. It benefits patients to plan their meals and insulin dosages to prevent hypo-and hyperglycemia. Diabetes is a major cause of chronic kidney disease (CKD). Chronic kidney disease increases the risk of cardiovascular diseases (CVD). CVD and CKD are strongly intertwined. Urea and potassium are the major markers used in the diagnosis of chronic kidney disease and cardiovascular disease, respectively. Continuous monitoring of urea and potassium will help to initiate appropriate medical intervention to decelerate the progression of chronic kidney disease and cardiovascular disease. Conventional invasive blood withdrawal procedure has potential risks like infection, pain and discomfort to the patients. Moreover, invasive techniques deter periodic blood sampling as it requires for frequent vascular puncturing. At present, no medical device is available for continuous monitoring of blood analytes non-invasively. Present investigations aim at developing a non-invasive technique for monitoring blood analytes (glucose, urea and potassium) which have great potential to use as a point of care diagnostics. Interstitial fluid bathes the cells of the body and it is ultrafiltrate of plasma. It contains ions like potassium, sodium, etc., and neutral molecules like glucose, urea, etc. Analytes (glucose, urea and potassium) level in interstitial fluid equilibrates with blood with lag time of 0 – 15 minutes. Reverse iontophoresis is a process in which a small current is applied through the skin to enable the transdermal extraction of interstitial fluid. Reverse iontophoresis is a non-invasive method and it is suitable for developing an integrated system to extract and analyze the extracted analyte. It enables frequent analyte sampling in high risk patients like elderly and paediatric with more comfort than conventional methods. In the present work, investigations are conducted on non-invasive monitoring of blood glucose, urea and potassium using reverse iontophoresis (RI). As part of experimental investigations, in vitro models are developed. In vitro investigations are conducted to optimize the reverse iontophoresis parameters current density and time of extraction. With these optimized parameters, the in vivo investigations are conducted on human subjects. A dedicated instrumentation suitable for extraction of analytes is developed. Screen printed electrochemical glucose sensors suitable for revere iontophoresis applications are developed using mediated carbon ink. Glucose oxidase is immobilized on screen printed sensor using cross linking method. Electrochemical and material characterization studies are conducted on the developed sensors. The obtained results confirm that the suitability of developed sensor can be used for serum glucose measurement as well as for reverse iontophoresis. Screen printed potentiometric urea biosensors are also developed to monitor the blood urea level non-invasively using reverse iontophoresis. The extraction and sensing system consists of a reverse iontophoresis electrodes, a working electrode, and a reference electrode. Unease enzyme is immobilized in the polypyrrole matrix on the working electrode using cyclic voltammetry. The electrochemical and material characterizations are conducted on screen printed sensors. The sensitivity, selectivity and sensing range of sensors show that they have a potential application in reverse iontophoresis applications. The in vitro models are used to evaluate the developed (urea and glucose) sensors. They are further validated by this conducting the clinical investigations on 15 human subjects. A correlation between blood analyte (glucose and urea) level and transdermally extracted analytes (glucose and urea) is established. It is attempted to integrate both the sensors (glucose and urea) and evaluated their performance on human subjects. The effect of potassium present in the stratum corneum of skin during reverse iontophoresis is investigated by conducting in vivo studies on human subjects. Tape stripping technique is used to detect the presence of potassium in stratum corneum. Reverse iontophoresis investigations with and without passive diffusion are also conducted to analyze the effect of potassium in stratum corneum. Skin impedance is measured during reverse iontophoresis in order to study the effect of reverse iontophoreteic current on skin properties. The clinical investigations are conducted on human subjects to validate the performance of the developed sensors (glucose and urea) with the approval of Institute Human Ethical Committee (IHEC), IISc, Bangalore. Non-invasive monitoring of blood analytes (glucose and urea) on human subjects is successfully demonstrated with the indigenously developed sensors through reverse iontophoresis.

Kidney failure results in fluid and toxin accumulation within body fluid compartments, contributing to the excess mortality seen in this condition. Such uremic toxins have been measured in plasma, with levels assumed to reflect extraplasmatic concentrations such as in interstitial fluid (ISF). ISF is separated from plasma by nanometre-order microvascular pores; toxins may not circulate “freely” between the two compartments. This work set out to characterise the ISF in uremic subjects, with the hypothesis that there may be differences with plasma. Any such difference may be clinically relevant, owing to the much larger size of the ISF compartment, its proximity to cell metabolic processes, and its expansion in renal impairment.We used a modified microdialysis technique to successfully sample subcorneal ISF of some the uremic toxins (urea, creatinine, urate, phosphate). Reverse iontophoresis (RI) was also used as a non-invasive technique to sample epidermal ISF of urea. Hollow microneedles were developed and their ability to extract ISF tested in CKD patients and controls. The mechanical properties (pressure, volume, permeability) and biochemical composition (proteomic and metabolomic profiles) of the interstitial compartment were also investigated.Microdialysis and RI performed very well as interstitial uremic toxin sampling techniques. Small differences were seen in steady states between ISF and plasma urea, creatinine, phosphate and urate, with slightly lower ISF levels. Dialysis seemed to enhance this difference, with a lag in the clearance of ISF toxins seen in some patients, most remarkable with phosphate. Metabolomic analysis identified several uremic toxins in ISF, whilst proteomics found some significant differences between the two compartments, with toxins like beta-2 microglobulin occurring in ISF only. Microneedle arrays successfully extracted ISF in 68.8% of patients with oedema. Successful extraction of ISF with microneedles occurred mainly in oedematous patients, who were found to have raised interstitial pressures (ISP) and volumes. ISP correlated significantly with body fluid volumes and seemed time-dependent, lower in more chronic oedema. ISP and volumes also correlated with the oedema depitting time (after thumb pressure), a potential novel parameter that probably relates to tissue hydraulic conductivity and hence volume status and fluid mobility within the interstitium.This study demonstrates that interstitial fluid may need to be considered as a separate active compartment in patients with renal dysfunction, with a different “uremic" composition and unique pathophysiological characteristics that cannot be explained by blood compartment based measurements alone. There is a need for more studies, to further characterise this compartment and elucidate its importance.