Oxidase-Coupled Amperometric Glucose and Lactate Sensors with Integrated Electrochemical Actuation System

Unpredictable baseline drift and sensitivity degradation during continuous use are two of the most significant problems of biosensors including the amperometric glucose and lactate sensors. Therefore, the capability of on-demand in situ calibration/diagnosis of biochemical sensors is indispensable for reliable long-term monitoring with minimum attendance. Another limitation of oxidase enzyme-based biosensors is the dependence of enzyme activity on the background oxygen concentration in sample solution. In order to address these issues, the electrolytic generation of oxygen and hydrogen bubbles were utilized 1) to overcome the background oxygen dependence of glucose and lactate sensors and 2) to demonstrate the feasibility of in situ self-calibration of the proposed glucose and lactate sensors. Experimental data assure that the proposed techniques effectively establish the zero calibration value and significantly improve the measurement sensitivity and dynamic range in both glucose and lactate sensors

Automated Oxidase-Coupled Amperometric Microsensor with Integrated Electrochemical Actuation System for Continuous Sensing of Saccharoids

Recent developments for biosensors have been mainly focused on miniaturization and exploratory use of new materials. It should be emphasized that the absence of a novel in-situ self-calibration/diagnosis technique that is not connected to an external apparatus is a key obstacle to the realization of a biosensor for continuous use with minimum attendance. To address this deficiency, a novel needle-type biosensor system with fully automated operations is being developed, in which a novel oxidase-coupled amperometric sensor with oxygen depleting/generating actuator is interfaced with an electrochemical instrument and a perfusion system. Labview virtual instrument has been also developed to oversee the automatic control of the prototype sensor. Using the proposed system, a large amount of data can be rapidly collected for more effective sensor characterization and more advanced sensor designs. Autonomous and continuous sensing and self-calibration with minimal human intervention is also envisioned

Carbon Nanotube-Enzyme Conjugates for the Fabrication of Diagnostic Biosensors

The fabrication of multi-analyte biotransducers continues to be a major technical challenge when the length scales of the individual transducer elements are on the order of microns Generation-3 (Gen-3) biosensors and advanced enzyme biofuel cells will benefit from direct electron transfer to oxidoreductases facilitated by single-walled carbon nanotubes (SWNTs). Direct electron transfer helps to mitigate errors from the instability in oxygen tension, eliminate use of a mediator and produce a device with low operating potential close to the redox potential of the enzymes. Supramolecular conjugates of SWNT-glucose oxidase (GOx-SWNT) may be produced via ultrasonic processing. Using a Plackett-Burman experimental design to investigate the process of tip ultrasonication, conjugate formation was investigated as a function of ultrasonication times and functionalized SWNTs of various tube lengths. Supramolecular conjugates formed from shorter, -OH functionalized SWNTs using longer sonication times gave the most favored combination for forming bioactive conjugates

Nanomaterials for Healthcare Biosensing Applications

In recent years, an increasing number of nanomaterials have been explored for their applications in biomedical diagnostics, making their applications in healthcare biosensing a rapidly evolving field. Nanomaterials introduce versatility to the sensing platforms and may even allow mobility between different detection mechanisms. The prospect of a combination of different nanomaterials allows an exploitation of their synergistic additive and novel properties for sensor development. This paper covers more than 290 research works since 2015, elaborating the diverse roles played by various nanomaterials in the biosensing field. Hence, we provide a comprehensive review of the healthcare sensing applications of nanomaterials, covering carbon allotrope-based, inorganic, and organic nanomaterials. These sensing systems are able to detect a wide variety of clinically relevant molecules, like nucleic acids, viruses, bacteria, cancer antigens, pharmaceuticals and narcotic drugs, toxins, contaminants, as well as entire cells in various sensing media, ranging from buffers to more complex environments such as urine, blood or sputum. Thus, the latest advancements reviewed in this paper hold tremendous potential for the application of nanomaterials in the early screening of diseases and point-of-care testing

Real-time monitoring of metabolic function in liver-on-chip microdevices tracks the dynamics of mitochondrial dysfunction

Microfluidic organ-on-a-chip technology aims to replace animal toxicity testing, but thus far has demonstrated few advantages over traditional methods. Mitochondrial dysfunction plays a critical role in the development of chemical and pharmaceutical toxicity, as well as pluripotency and disease processes. However, current methods to evaluate mitochondrial activity still rely on end-point assays, resulting in limited kinetic and prognostic information. Here, we present a liver-on-chip device capable of maintaining human tissue for over a month in vitro under physiological conditions. Mitochondrial respiration was monitored in real time using two-frequency phase modulation of tissue-embedded phosphorescent microprobes. A computer-controlled microfluidic switchboard allowed contiguous electrochemical measurements of glucose and lactate, providing real-time analysis of minute shifts from oxidative phosphorylation to anaerobic glycolysis, an early indication of mitochondrial stress. We quantify the dynamics of cellular adaptation to mitochondrial damage and the resulting redistribution of ATP production during rotenone-induced mitochondrial dysfunction and troglitazone (Rezulin)-induced mitochondrial stress. We show troglitazone shifts metabolic fluxes at concentrations previously regarded as safe, suggesting a mechanism for its observed idiosyncratic effect. Our microfluidic platform reveals the dynamics and strategies of cellular adaptation to mitochondrial damage, a unique advantage of organ-on-chip technology

Novel optofluidic sensor systems for quantitative chemical imaging and on-chip sensor calibration

The design, fabrication and characterization of optofluidic biosensor systems for quantitative oxygen imaging with a color charge-coupled device (CCD) camera as well as on-chip self-calibration of sensors utilizing gas bubbles was investigated. This dissertation was prepared in publication format. The first and second papers demonstrate that color imaging devices can be used in quantitative chemical analysis. The final paper explores the feasibility of using electrolytically generated bubbles for a novel functionality of reagentless, on-chip, in situ calibration of optical biosensors. Work in the first paper includes the use of a color CCD camera for fluorescence intensity imaging. This involves extracting the red color element to determine the dissolved oxygen content from the color image of a sample. The linearity and sensitivity of oxygen detection based on the red intensity analysis was improved to those of spectrometric measurement and total color intensity analysis. In the second paper, the color extraction technique used in the dissolved oxygen sensor was extended to gaseous oxygen detection to eliminate the need of optical filters and replace the blue light emitting diode (LED) excitation source with a general broad-band white LED. This new method has potential applications in multi-analyte monitoring and simultaneous structural/functional imaging of biological samples with a single broad-band light source. In the final paper, a double-layered optofluidic system was developed to demonstrate on-chip, self-calibration of dissolved oxygen sensor. A multilayers of dry film resist was used for preparing a 3-D fluidic structure. A thin black polydimethylsiloxane membrane was used for oxygen diffusion and optical isolation. The sensor calibration result with the on-chip bubble was shown to be in good agreement with that of standard calibrants --Abstract, page iv

Microfluidic biosensors for intelligent metabolite monitoring

Baseline (zero-value) drift and sensitivity degradation are two common problems related with biosensors. In order to overcome these problems, there is a great need for integrating an on-demand, in situ self-diagnosis and self-calibration unit along with the sensor. Utilizing the microfluidic technology, it is possible to explore the feasibility of implementing this function without any externally coupled bulky apparatus. A microsystem including a microfluidic channel and calibration electrodes are prepared by microfabrication techniques --Abstract, page iv

Implantable Multi-panel Platform for Continuous Monitoring of Exogenous and Endogenous Metabolites for Applications in Personalized Medicine

Nowadays, scientific advances are leading to the discovery of newer, better, more targeted treatments that will improve the human health. However, despite the promising results and the major advantages in treatments offered to patients, these personalized medical treatments are limited to few cases. Translational medicine research with animals is needed to find innovative, safe and life-saving solutions for patients, especially in drug development. Although technological improvements may lead one day to the end of animal testing, today those strategies are not sufficient, due to the complexity of living organisms. The living conditions of these animals are of primary importance because high stress levels can affect the experimental results. In this respect, the monitoring of the animals in a small living space by means of a fully implantable device, can contribute to minimize the human intervention, increasing the comfort for the animals. The objective of this thesis is the design and characterization of a fully implantable biosensor array for the real-time detection of endogenous and exogenous metabolites, for the monitoring of small caged animals in drug development, and for future applications in personalized medicine. The fully implantable device consists of: a passive sensing platform consisting of an array of four independent electrochemical biosensors, together with a pH sensor and a temperature sensor for the optimization of the sensing performances in different physiological conditions; integrated circuits capable of performing multiple electrochemical measurements; a coil for remote powering of the integrated circuit and the short-range data transmission to an external device; a membrane packaging ensuring measurements with high signal-to-noise ratio, biocompatibility and selectivity against possible interfering molecules in biological fluids. ⢠In vitro monitoring of four anti-cancer drugs and an anti-inflammatory drug within the pharmacological ranges in undiluted human serum; ⢠Demonstration of the in vitro functionality of the complete system, showing that the external powering system correctly operate the device, and receive the data from the sensors; ⢠In vivo biocompatibility tests of the packaging, showing after 30 days a significant reduction of the inflammatory response in time, suggesting normal host recovery; ⢠In vivo continuous monitoring of an anti-inflammatory drug, demonstrating the proof of-concept of the system for future personalized medicine applications

Integration of biomolecular logic principles with electronic transducers on a chip

Boolean operations applied in biology and integrated with electronic transducers allow the development of a new class of digital biosensors for the detection of multiple input signals simultaneously and in real-time. With the help of Boolean functions (AND, OR, etc.), an electrical output signal will be directly delivered, representing a ”1” or “0” binary notation, corresponding to a “true” or “false” statement, respectively. Such digital biosensors have the future potential to create medical devices and systems for intelligent or smart diagnostics. The present thesis describes the realization of different enzyme-based biomolecular logic gates combined with electronic transducers for the possible application in medicine or food industry. In a first concept, a so called BioLogicChip is developed combining a “sense-act-treat” function integrated on one chip. The present system exemplarily mimics an “artificial pancreas” designed as a closed-loop drug-release system. A glucose sensor is constructed as enzyme-based AND logic gate, a temperature-depending hydrogel imitates the actuator function switching ON and OFF with its shrinking or swelling property, and an additional insulin sensor is developed to monitor and control the release of the drug (here: insulin) from the actuator. In this study, the results of the individual components such as the amperometric glucose sensor, the temperature-dependent hydrogel and the amperometric insulin sensor are presented, which are necessary to create such BioLogicChip. Moreover, a digital adrenaline biosensor is developed to proof the catheter position during adrenal vein sampling. The sensor consists of an oxygen electrode modified by a bi-enzyme system with the enzymes laccase and pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) to realize substrate-recycling principle to detect low adrenaline concentrations (in the nanomolar concentration range). The sensor`s behavior at different pH values and at different temperatures is studied. Measurements in Ringer`s solution are performed. In addition, the sensitivity of the biosensor to other catecholamines such as noradrenaline, dopamine and dobutamine is investigated. Furthermore, the adrenaline biosensor is successfully examined in human blood plasma. Finally, “proof-of-principle” experiments have been performed by combining the adrenaline biosensor with Boolean operations to get a rapid qualitative statement of the presence or absence of adrenaline, thus validating the correct position of the catheter in a YES/NO form. This adrenaline biosensor is further miniaturized as a thin-film platinum adrenaline biosensor. Here, the bioelectrocatalytical measurement principle is applied by immobilization of the enzyme PQQ-GDH to detect adrenaline in the nanomolar concentration range, too. The measurement conditions such as pH value, glucose concentration in the analyte solution and temperature are optimized with regard to a high sensitivity and low detection limit. Also, this sensor has been verified towards other catecholamines (noradrenaline, dopamine and dobutamine). The platinum thin-film adrenaline biosensor is successfully applied in blood plasma for the detection of different spiked adrenaline concentrations. Furthermore, the developed adrenalin biosensor is able to detect the concentration difference between adrenal blood and peripheral blood. In contrast to the above-mentioned amperometric biosensor examples for biomolecular gates, also a field-effect-based platform is given attention in this thesis. The field-effect electrolyte-insulator-semiconductor (EIS) sensor consists of a layer structure of Al/p-Si/SiO2/Ta2O5 and is used to create an acetoin biosensor for the first time to control different fermentation processes. The sensor chip is modified by the enzyme acetoin reductase from B. clausii DSM 8716T for the catalytical reaction of (R)-acetoin to (R,R)-butanediol and meso-butanediol, respectively, in the presence of NADH. The linear measurement range, the optimal immobilization strategy (cross-linking by using glutaraldehyde and adsorptive binding) as well as the optimal working pH value and long-term stability are investigated by means of constant-capacitance measurements. Finally, the acetoin sensor was successfully applied in wine probes to detect different spiked acetoin concentrations. The sensor shows opportunities to be further developed as digital acetoin biosensor