Toward in vivo transdermal pH sensing with a validated microneedle membrane electrode

We present herein the most complete characterization of microneedle (MN) potentiometric sensors for pH transdermal measurements for the time being. Initial in vitro assessment demonstrated suitable analytical performances (e.g., Nernstian slope, linear range of response from 8.5 to 5.0, and fast response time) in both buffer media and artificial interstitial fluid (ISF). Excellent repeatability and reproducibility together with adequate selectivity and resiliency facilitate the appropriateness of the new pH MN sensor for transdermal ISF analysis in healthcare. The ability to resist skin insertions was evaluated in several ex vivo setups using three different animal skins (i.e., chicken, pork, and rat). The developed pH MN sensor was able to withstand from 5 to 10 repetitive insertions in all the skins considered with a minimal change in the calibration graph (<3% variation in both slope and intercept after the insertions). Ex vivo pH measurements were validated by determining the pH with the MN sensor and a commercial pH electrode in chicken skin portions previously conditioned at several pH values, obtaining excellent results with an accuracy of <1% and a precision of <2% in all cases. Finally, pH MN sensors were applied for the very first time to transdermal measurements in rats together with two innovative validation procedures: (i) measuring subcutaneous pH directly with a commercial pH microelectrode and (ii) collecting ISF using hollow MNs and then the pH measurement of the sample with the pH microelectrode. The pH values obtained with pH MN sensors were statistically more similar to subcutaneous measurements, as inferred by a paired sample t-test at 95% of confidence level. Conveniently, the validation approaches could be translated to other analytes that are transdermally measured with MN sensors

Microneedles for Theranostics

Microneedle (MN) arrays were developed to provide a minimally invasive approach to detect biomarkers and deliver drugs into the ISF. Solid, hollow, and dissolvable MNs have been fabricated for various applications and have been evaluated to be very advantageous.These advantages include better patient compliance dur to painless and non-invasive administration, improved permeability and efficiency and provide targeted drug delivery by varying MN dimensions to specific regions in the skin.Techniques to fabricate MNs vary based on the material and potential application requirements. The most common techniques are micro-moulding, wet and dry etching with lithography and laser cutting. Micro-moulding fabrication have been utilized to produce various polymer, hydrogel and dissolvable MNs by filling prepared moulds with a liquid formulation. Alternatively, lithography using wet and dry etching have been used to fabricate MNs. A mask is used as a template for generating the desired pattern on a wafer surface using either a positive or negative photoresist to generate the desired pattern. These wafers are then etched using a strong caustic agent or an etcher.Finally, laser cutting techniques have been used to produce metal MN using a computer aided design to create the desired shape and dimensions.In recent studies, MNs have been created for a wide range of diagnostic and drug delivery applications. A wide range of MNs have been adapted for a variety of disease treatments such as cancer, arthritis and ophthalmic disorders. As diabetes mellitus effects approximately 30 million people and glucose monitoring has advanced from the initial self-monitoring of blood glucose levels to glucose biosensors, a high demand for MNs to be modified for diabetes management has been emphasized.This thesis details the fabrication of MNs using silicon, polyvinylpyrollidone (PVP) and polycarbonate for sensing and drug delivery. Silicon wafers with the combination of photolithography and deep-reactive ion etching (DRIE) techniques are used to create solid and hollow MNs. The MN arrays have sharp tips that provide eased insertion and injectable capabilities.Polycarbonate and PVP MNs were manufactured using micro-moulding techniques to create solid and dissolvable MNs respectively. These MNs were characterized to determine their penetration capabilities through the stratum corneum (SC) to providecontrolled transdermal drug delivery and diagnose biomarkers within the interstitial fluid (ISF).For diagnostic applications, the polycarbonate MNs were modified for glucose sensing using first generation sending strategies wherein oxygen in used as the electron acceptor and the levels of glucose is proportional to the peroxide produced. To determine drug delivery capabilities of MNs, the solid polycarbonate and silicon MNs employed ‘poke and patch’ techniques with the use of a Franz cell to show calcein and FITC-insulin delivery over 24 hours. On the other hand, for drug delivery through the dissolvable PVP MNs, ‘poke and dissolve’ techniques were analysed with the use of Franz cells to show the release of encapsulated calcein and FITC-insulin within the polymeric matrix over 24 hours. The arrays were either left blank for basal drug delivery or metallised with silver for controlled drug delivery using the metals breakdown potential

Non-invasive, transdermal, path-selective and specific glucose monitoring via a graphene-based platform

Currently, there is no available needle-free approach for diabetics to monitor glucose levels in the interstitial fluid. Here, we report a path-selective, non-invasive, transdermal glucose monitoring system based on a miniaturized pixel array platform (realized either by graphene-based thin-film technology, or screen-printing). The system samples glucose from the interstitial fluid via electroosmotic extraction through individual, privileged, follicular pathways in the skin, accessible via the pixels of the array. A proof of principle using mammalian skin ex vivo is demonstrated for specific and ‘quantized’ glucose extraction/detection via follicular pathways, and across the hypo- to hyper-glycaemic range in humans. Furthermore, the quantification of follicular and non-follicular glucose extraction fluxes is clearly shown. In vivo continuous monitoring of interstitial fluid-borne glucose with the pixel array was able to track blood sugar in healthy human subjects. This approach paves the way to clinically relevant glucose detection in diabetics without the need for invasive, finger-stick blood sampling

A Functionalised Aptamer Electrochemical Biosensor Platform

The ability to utilise new knowledge of biomarkers from genomic and proteomic data will have a great impact on molecular diagnosis. Biomarker detection could be achieved by utilising a capture molecule that associates specifically with the target biomarker. The work described in this thesis focuses on a platform comprising a lysozyme binding aptamer and an amperometric electrode (an electrochemical aptasensor). To couple the binding reaction to a change in current, the aptamer is modified with a redox group, ferrocene. Two types of signalling aptamer were constructed, one comprised the aptamer self-assembled on gold and hybridised to a short complementary oligonucleotide carrying a ferrocene group. The second incorporated the binding sequence into a molecular beacon, one end of which self-assembled onto the electrode, the other end carried the ferrocene group. Both of these showed a lysozyme dependent change in current on a gold electrode. Further characterisation of the first aptasensor suggested that the nucleic acid formed a multilayer structure on the electrode surface and that lysozyme binding induced conformational change moved ferrocene close to the surface, increasing the current. In contrast, the second aptamer usually showed a decrease in current in the presence of lysozyme suggesting that the binding resulted in the ferrocene moving away from the surface. In order to evaluate the possible use of these aptasensors for continuous in vivo measurement, needle shaped microelectrodes arrays were produced and the beacon aptamer immobilised on the surface. These electrodes had high impedance which resulted in low sensitivity, however lysozyme binding could still be detected using electrochemical impedance spectroscopy with ferrocyanide in solution. These microspike arrays could also be used for glucose sensing following modification with glucose oxidase

Electronic Skin in Robotics and Healthcare: Towards Multimodal Sensing and Intelligent Analysis

Skin-interfaced electronics is gradually transforming robotic and medical fields by enabling noninvasive and continuous monitoring of physiological and biochemical information. Despite their promise, current wearable technologies face challenges in several disciplines: Physical sensors are prone to motion-induced noise and lack the capability for effective disease detection, while existing wearable biochemical sensors suffer from operational instability in biofluids, limiting their practicality. Conventional electronic skin contains only a limited category of sensors that are not sufficient for practical applications, and conventional data processing methods for these wearables necessitate manual intervention to filter noise and decipher health-related information. This thesis presents advances in electronic skin within robotics and healthcare, emphasizing multimodal sensing and data analysis through machine intelligence. Chapter 1 introduces the concept of electronic skin, outlining the emerging sensor technologies and a general machine learning pipeline for data processing. Chapter 2 details the development of multimodal physiological and biochemical sensors that enable long-term continuous monitoring with high sensitivity and stability. Chapter 3 explores the application of integrated electronic skin in robotics, prosthetics, and human machine interactions. Chapter 4 showcases practical implementations of integrated electronic skin with robust sensors for wound monitoring and treatment. Chapter 5 highlights the transformative deployment of artificial intelligence in deconvoluting health profiles on mental health. The last chapter, Chapter 6, delves into the challenges and prospects of artificial intelligence-powered electronic skins, offering predictions for the evolution of smart electronic skins. We envision that multimodal sensing and machine intelligence in electronic skin could significantly advance the field of human machine interactions and personalized healthcare.</p

Development of an Encapsulation Strategy for an Implantable Optical Glucose Sensing Technology Compatible with a Self-Cleaning Membrane

The concerning rate of diabetes mellitus prevalence and its associated chronic complications accentuates the urgency for continuous glucose monitoring. Optical techniques, especially fluorescence-affinity assays, offer a strategy that transcends current transcutaneous sensors by enabling subcutaneous implantation and interrogation. Biosensors can initiate an immune response that ultimately leads to a dense fibrous capsule surrounding the sensor. This biological interference, termed “biofouling,” severely limits implantable sensor lifetimes by slowing analyte diffusion and decreasing optical signal propagation. In an effort to control biofouling, a thermoresponsive, “self-cleaning,” hydrogel membrane based on poly(N-isopropylacrylamide) (PNIPAAm) has been proposed. Further, a continuous glucose monitoring system based on a competitive binding assay using Concanavalin A (ConA) as a component in a Förster resonance energy transfer (FRET) approach is being developed for encapsulation within the thermoresponsive hydrogel. In this research, a double network nanocomposite PNIPAAm (DNNC) hydrogel’s glucose diffusion, thermosensitivity, cytocompatibility, in vitro cellular release, and in vivo compatibility and efficacy were thoroughly investigated. Further, an encapsulation strategy was developed for retaining the glucose assay within the hydrogel and yet allowing glucose diffusion. The methods and systems for obtaining the in vitro and in vivo results of the hydrogel are presented along with the glucose encapsulation strategies. In general, the research showed that the hydrogel could adequately diffuse glucose at temperatures associated with subcutaneous implantation, maintain a stable thermal cycling profile, promote cellular release without toxic effects in vitro, and decrease the extent of biofouling in vivo. Furthermore, the hydrogel revealed its feasibility to be embedded with layer-by-layer (LbL) microsphere assemblies, which exhibited the ability to encapsulate the components of a competitive binding glucose assay. Overall, the results of this research demonstrate that the hydrogel combined with the encapsulated glucose assay is a promising approach for an implantable continuous glucose monitoring biosensor

Integrated Microsystems for Wireless Sensing Applications

Personal health monitoring is being considered the future of a sustainable health care system. Biosensing platforms are a very important component of this system. Real-time and accurate sensing is essential for the success of personal health care model. Currently, there are many efforts going on to make these sensors practical and more useful for such measurements. Implantable sensors are considered the most widely applicable and most reliable sensors for such accurate health monitoring applications. However, macroscopic (cm scale) size has proved to be a limiting factor for successful use of these systems for long time and in large numbers. This work is focused to resolve the issues related with miniaturizing these devices to a microscopic (mm scale) size scale which can minimize many practical difficulties associated with their larger counterparts currently. To accomplish this goal of miniaturization while retaining or even improving on the necessary capabilities for such sensing platforms, an integrated approach is presented which focuses on system-level miniaturization using standard fabrication procedures. First, it is shown that a completely integrated and wireless system is the best solution to achieve desired miniaturization without sacrificing the functionality of the system. Hence, design and implementation of the different components comprising the complete system needs to be done according to the requirements of the overall integrated system. This leads to the need of on-chip functional sensors, integrated wireless power supply, integrated wireless communication and integrated control system for realization of such system. In this work, different options for implementation of each of these subsystems are compared and an optimal solution is presented for each subsystem. For such complex systems, it is imperative to use a standard fabrication process which can provide the required functionality for all subsystems at smallest possible size scale. Complementary Metal Oxide Semiconductor (CMOS) process is the most appropriate of the technologies in this regard and has enabled incredible miniaturization of the computing industry. It also provides options for designing different subsystems on the same platform in a monolithic process with very high yield. This choice then leads to actual designs of subsystems in the CMOS technology using different possible methods. Careful comparison of these subsystems provides insights into different design adjustments that are made until the desired functions are achieved at the desired size scale. Integration of all these compatible subsystems in the same platform is shown to provide the smallest possible sensing platform to date. The completely wireless system can measure a host of different important analyte and can transmit the data to an external device which can use it for appropriate purpose. Results on measurements in phosphate buffer solution, blood serum and whole blood along with wireless communication in real biological tissues are provided. Specific examples of glucose and DNA sensors are presented and the use for many other relevant applications is also proposed. Finally, insights into animal model studies and future directions of the research are discussed. </p

Evaluation of safety of transdermal drug delivery using electroporation by In vitro and In vivo studies

Introduction: Transdermal electroporation involves the application of high voltage electrical pulses for microsecond to millisecond duration to produce reversible increase in permeability of skin. It can provide an alternative route to intravenous injection for the fast delivery of macromolecules molecules such as proteins and peptide drugs in clinically effective amounts for the patient. However, the mechanism of electroporation and its safety are unclear. Hence, electrical parameters for delivery of individual drugs have to be chosen empirically or by careful optimization. -- Objectives: To carry out optimization of electrical parameters using fuzzy rat skin tissue in vitro for delivery of terazosin hydrochloride (TRZ), followed by use of these parameters in live fuzzy rats (in vivo), to study their safety and effectiveness. To design in vitro and in vivo tests to predict the safety of this technique. -- Materials: Side-by-side diffusion cells were used for in vitro and in vivo studies. Ag/AgCI electrodes of different areas were used to deliver the exponentially decaying electroporation pulses from a Gene Pulser® (BioRad Laboratories, USA). Fuzzy rats and freshly excised full thickness skin from fuzzy rats were used for in vivo and in vitro studies respectively. -- Methods: Pulse length and rate of pulsing were evaluated with respect to their ability to reverse the increased permeability caused by electroporation. The correlation between TRZ concentration and increase in electroporative delivery was studied. Based on above studies optimal parameters were chosen to deliver TRZ in vivo. Their safety and effectiveness were compared to delivery without electroporation (control). Pharmacokinetic parameters were estimated by giving drug intravenously and subcutaneously. In vivo impedance recovery of skin after electroporation to pre-electroporated state was studied to predict safety. Similarly, uptake of glucose by skin with or without electroporation was studied to predict change in viability (damage) in vitro. Finally, electrodes of different area were characterized in vitro with respect to the electrical parameters. These parameters in conjunction with the in vitro and in vivo drug delivery and safety studies would throw some light on the mechanism of electroporation and the effect of electrode area on drug delivery and safety by electroporation. -- Results and discussion: If electroporation is completely reversible then the rate of transport of drug through the skin after stopping electroporation pulses should be the same as that through non-pulsed skin. Using this method an applied voltage (Uelectrode.0) of 400V, a pulse length of 20 millisecond and a rate of pulsing of 10 pulse per minute (ppm) were found to be relatively safe and delivered significantly higher drug compared to passive drug delivery. Increased donor concentration gave higher delivery and may help in reducing exposure to higher electrical conditions to produce same amount of drug delivery. In vivo studies showed that TRZ can be delivered safely and effectively with electroporation. However, the effect of electrode area needs to be studied further. Pharmacokinetic studies indicated depot formation within skin after electroporation and this could be due to limited blood flow to the skin. In vitro biochemical studies showed a lag time in lactate production when a very high voltage electroporation pulse was used and there was a general stimulation of lactate production (as a result of glucose utilization) after electroporation compared to non-electroporated skin. The lag time may be used to predict damage due to electroporation. In vitro electrode characterization studies gave considerable insight into the observed drug delivery profiles and the differences in safety profiles between the different electrodes. In vivo impedance studies showed that complete recovery of skin impedance after electroporation might take hours to days. Recovery was faster with shorter pulse lengths and lower number of pulses. -- Conclusions: An applied voltage of 400 V, 20 pulses of 20 millisecond at the rate of 10 pulses per minute with 10% TRZ in contact with skin and using a small area electrode was found to be relatively safe and effective for delivery of TRZ in vitro and in vivo, with higher delivery for higher electrode area as predicted. The in vitro electrode characterization experiments could explain some of the observed differences between the drug delivery by different electrodes and in vitro viability studies could predict damaging electrical conditions. In vivo impedance studies showed that the parameters which can cause appreciable recovery of impedance after electroporation, are those that have not shown to deliver appreciable amount of drug in vitro, at least by electroporation alone. New electrode designs or new methods will have to be devised to increase safety of electroporation before electroporation can be considered useful or tested on humans

MICRONEEDLE-ASSISTED TRANSDERMAL DELIVERY OF NALTREXONE SPECIES: \u3cem\u3eIN VITRO\u3c/em\u3e PERMEATION AND \u3cem\u3eIN VIVO\u3c/em\u3e PHARMACOKINETIC STUDIES

Naltrexone (NTX) is a drug used primarily in the management of alcohol dependence and opioid dependence. Based on several drawbacks associated with the oral and injectable intramuscular dosage forms of naltrexone currently available on the market, there is substantial interest in delivering naltrexone transdermally. Although naltrexone does not permeate skin at the rate sufficient to reach therapeutic plasma concentrations in humans, novel flux enhancement methods such as microneedles help address this challenge. Earlier work in humans has demonstrated that the use of microneedles achieves plasma concentrations in the lower end of expected therapeutic values. Further flux enhancement is desired to decrease the patch area while increasing drug transport rates. In the present work, several strategies aiming at in vitro flux maximization were employed including: formulation optimization, naltrexone salt screening, and naltrexone prodrug design. While naltrexone prodrugs did not reveal any improved permeation characteristics formulation optimization through decrease in vehicle microviscosity allowed a 5-fold increase in the percutaneous transport rates, and naltrexone glycolate salt selection provided an additional 1.5-fold enhancement in flux. One of the key observations was a good correlation (R2 = 0.99) between vehicle microviscosity and drug transport rates across the microchannel pathway. This finding alone allowed for formulation optimization and, at the same time, provided a potential explanation for the low permeation of high-concentration naltrexone salts and prodrugs. In vivo studies were carried out in Yucatan minipigs using a “poke and patch” microneedle method to deliver NTX•HCl. These studies demonstrated that initial plasma concentrations spiked to 2.5 ng/ml but rapidly dropped to a plateau of below 1 ng/ml. This pharmacokinetic profile could be explained by the use of a mathematical model which identified the importance of microchannel closure kinetics on drug transport. Also, an estimate of diffusional resistance of the viable tissue associated with percutaneous NTX•HCl delivery through microchannels was obtained. Its relatively large value suggests that the effect of diffusional resistance of the dermis in vivo should not be ignored and must be accounted for in order to obtain a good in vitro-in vivo correlation