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Synthetic biology is an emerging field which has led to development of many useful applications of engineered biological networks and systems. One of the exciting advancements of the field are living cells which can serve as molecular factories, diagnostics or therapeutics. A widely used chassis in synthetic biology is yeast due to simple and inexpensive culturing conditions and the ability to heterologously express eukaryotic proteins. In this thesis, we present work exploring and expanding biosensing and responding capabilities of engineered lab strain yeast. Chapter 1 gives background information related to synthetic biology, living engineered biosensors, theranostics and more specifically on Saccharomyces cerevisiae general overview and applications in synthetic biology. Chapter 2 describes progress on establishing redox active peptides as a modular electrochemical interfacing language between electronics and engineered yeast. Chapter 3 covers yeast engineering as a heavy metal and metalloid biosensor, as well as the exploration of peptide-containing hydrobeads in conjunction with peptide-responsive yeast as a physical damage biosensor. In Chapter 4, we establish living yeast biosensor for detection of pathogenic fungus Aspergillus fumigatus and expanded biosensing of other Aspergillus species, as well as additional optimization of the biosensing yeast’s signal-to-noise ratio, sensitivity and readout time. Chapter 5 demonstrates the utility of specific peptide proteases in combination with promiscuous GPCRs in living yeast biosensor for detection and differentiation of peptide variants differing in single amino acid. Lastly, in Chapter 6 we implement yeast sense-and-respond community which is activated by pheromone-secreting fungi and as a response secretes a toxin which kills sensed fungi.

Diabetes mellitus is a metabolic disease with abnormally high concentration of glucose in blood in patients. Continuous glucose monitoring, which involves measuring glucose concentration in the patient throughout the day and night, can significantly reduce the risk of diabetes-related complications. Commercially available CGM sensors are not yet suited for long-term applications due to reliability and accuracy issues associated with the irreversible, consumptive nature of the underlying electrochemical reactions. Affinity sensing methods, which are based on reversible affinity binding between glucose and a recognition molecule, hold the potential to address these challenges in CGM applications. These methods do not involve the consumption of glucose and can offer improved stability and accuracy for CGM. When combined with impedance-based transduction methods, affinity sensors can also offer a high level of miniaturization, allow low-cost instrumentation, and are amenable to physical and functional integration. The affinity sensors investigated in this thesis include hydrogel-based affinity microsensors and graphene-based affinity nanosensors. We first present a dielectric affinity microsensor that consists of a pair of coplanar electrodes functionalized in situ with a glucose-responsive hydrogel for dielectrically based affinity measurement of glucose in subcutaneous tissue. We present a study of the effects of the choice of hydrogel compositional parameters on the characteristics of the hydrogel-based microsensor, allowing the identification of the optimal hydrogel composition for the microsensor to sensitively and rapidly respond to changes in glucose concentration. A differential design is then demonstrated, both in vitro and in vivo, to effectively minimize the influence of fluctuations in the environmental conditions, thereby allowing the hydrogel-based microsensor to function appropriately as a subcutaneously implanted device. In addition, we present a preliminary study on affinity nanosensors for non-invasive monitoring of glucose concentrations in physiological media such as tears. The affinity nanosensor is based on a chemically modified graphene field-effect transistor for the electrical measurement of glucose concentrations. The study explores the sensing mechanism of the nanosensors and demonstrates a device with high sensitivity and low limit of detection, which satisfies the requirement for monitoring glucose concentrations in tears. Experimental results demonstrate that these affinity micros and nanosensors are capable of measuring glucose concentrations with a suitable sensitivity and dynamic range for the intended physiological media, with potential applications to minimally invasive or non-invasive continuous glucose monitoring in diabetes care.

In the last two decades, there has been considerable development for biosensor devices as the need to more efficient sensing systems is increasing for monitoring the progress of medicine and help with the early diagnosis of the pathogens and treatment of diseases that would reduce the cost of patient care. DNA sensors, in particular, have attracted attention due to their abundance of practical applications in clinical diagnoses and genetic information which increase the demand for DNA probes. On the other hand, the development of the oxide semiconductors thin film transistors (TFT) devices have been greatly increased, owing to their superior electrical properties, lower cost and large coverage areas. Building a bridge across biological elements and electronic interface using advanced (TFT) platforms are based on materials design and device architecture. Here, a solution-processed multi-layer metal oxide (TFT) is explored as a novel DNA sensor. The device engineering combines the novel hetero-structure metal oxide channel that can sustain a 2-dimensional electron gas (2DEG) which leads to a higher mobility and surface functionalization capacity to create an ultrasensitive, highly stable, and versatile DNA sensor. The prototype solid-state TFT sensor features a sub-10 nm-thick metal oxide heterojunction channel of a In2O3 and a top ZnO layer. The devices developed here rely on a pyrene-based molecule as the receptor unit that is known to intercalate into double stranded DNA with a very high-affinity constant and at very low concentration.

The development of wearable devices to monitor biosignals and collect real-time data from biological systems at all scales from cellular to organ level has played a significant role in the field of medical engineering. The current coronavirus disease 2019 (COVID-19) pandemic has further increased the demand for remote monitoring and smart healthcare where patient data can be also be accessed from a remote distance. Recent efforts to integrate wearable devices with artificial intelligence and machine learning have transformed conventional healthcare into smart healthcare, which requires reliable and robust recording data. The biomedical devices that are mechanically stretchable and compliant have provided the capability to form a seamless interface with the curvilinear, soft surface of tissues and body, enabling accurate, continuous acquisition of physical and electrophysiological signals. This dissertation presents a comprehensive set of functional materials, design principles, and fabrication strategies to develop mechanically stretchable and compliant biomedical devices tailored for various applications, including (1) a stretchable sensor patch enabling the continuous monitoring of swallowing function from the submental/facial area for the telerehabilitation of patients with dysphagia, (2) a human hand-like sensory glove for advanced control of prosthetic hands, (3) a mechanically compliant manipulator for the non-invasive handling of delicate biomaterials and bioelectronics, and (4) a stretchable sensors embedded inside a tissue scaffold enabling the continuous monitoring of cellular electrophysiological behavior with high spatiotemporal resolution.<br>

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<p>Microbes play key roles in processes that shape the world around us having direct impact in crop  production,  food  safety,  digestion,  and  overall  health.  Developing  tools  to  monitor  their activity in-situ is the key towards better understanding the true impact of microbial activity in these processes and, eventually, harnessing their potential. Many conventional techniques for microbial activity assessment require sample collection, expensive benchtop equipment, skilled technicians, and   destructive   sample   processing   which   makes   their   adaptation   for   in-situ   monitoring cumbersome. The need for technologies for in-situ monitoring has led to the development of many sensordesigns,  capable  of  detecting  single  strains  of  bacteria  to  low  limits  of  detection  (LOD). These designs, however, are limited to their complex manufacturing procedures, cost, and delicacy which makes them difficult to implement outside of a laboratory setting into harsh environments.</p> <p>In  the  last  25  years,  impedimetric  sensing  methods  have  been used  as  powerful  analytical tools  to  characterize  the  degradation  and  dissolution  of  polymers.  Known  for  their  robustness, these techniqueswere mainly used for characterizing polymer’s properties as corrosion-protective layers on metals. At the time, someresearchers pondered onthe potential use of this technique for biosensing  applications.In  this  thesis,  the  ability  of  monitoring  microbial  activity  in-situ  was explored by  integratingdifferent  bioresponsive  polymers  with  low-cost electronic  impedimetricplatformsand assessing their degradation kinetics in response to microbes</p> <p>This  novel  use  of  impedimetric  sensing  methods  and  approach  towards  microbial  activity sensing was systematically studied in different  areas including  agriculture, food packaging, and healthcare.  Microbes,  the  good,  the  bad,  and  the  ugly,  were  studied  within  their  ecosystems  to demonstrate  the  ability  of  using  the  described  systems  in  in-situ  monitoring.  In  agriculture, polymer  degradation  was  successfully  correlated  to  the  concentration  of  decomposing  bacteria directly in soil. In food packaging, spoilage of chicken samples was successfully detected within their package through a non-reversible system. In healthcare, a wireless and electronic-free wound monitoring  system  capable of  detecting  early  onset  of  infection  while  delivering  therapeutics without the need of external actuation was achieved. Further developments of this technology will present the key towards monitoring microbial activity in-situ in a large scale, providing solutions to  humanity’s  toughest  upcoming  challenges  including  food  production,  food  safety,  and healthcare.</p>

Early detection of illness is essential in preventing symptoms from escalating and infectious diseases from spreading. Electrochemical biosensors are a promis- ing tool in healthcare detection. Previously, the collaboration between the Arold and Inal labs has led to the design of organic electrochemical transistors (OECT) capable of rapidly detecting coronavirus in saliva by using nanobody constructs as biorecognition units. In this project, I aimed to prove the versatility of nanobody- functionalized OECT biosensors in detecting other relevant viruses, specifically, Zika and Dengue. Both viruses pose a risk to multiple populations around the world, including the Kingdom of Saudi Arabia. I designed and produced nanobod- ies that are reported to bind to the NS1 glycoprotein, which is released by Zika and Dengue into the blood of the patient. Then, I confirmed the binding of the nanobodies to their associated targets. I also developed a robotic liquid handling script to automate the biosensing operations. Ultimately, this project aims to support the design of a multiplex OECT biosensor for blood-borne pathogens.

Our ability to sense small molecules with high specificity, over a broad range of concentrations, is limited and difficult to accomplish in a way that is inexpensive and continuous. The most commercially successful biosensor is the enzyme-based blood glucose electrochemical biosensor, yet for nearly all other biomolecules, detection and monitoring require specialized equipment, trained personnel, and long lead times, and are not amenable to continuous monitoring. Industries in need of enzyme-based small-molecule biosensors, including medical diagnostics, industrial production, environmental monitoring, food safety analysis, and international security, would benefit greatly from the development of new devices capable of measuring biomolecules of interest. Environmental microbes have been gaining attention because of the vast array of biomolecules that they are capable of sensing and degrading. These microbes do so, in part, through redox enzymes with diverse substrate specificities that represent an immense resource for developing electrochemical biosensors. However, the development of new enzyme biosensors has largely been limited by the lack of a general high-throughput method to identify these redox enzymes, making discovery slow, laborious, and ad hoc. To address this need, a high-throughput functional screening approach has been developed to isolate microbial oxidase enzymes from complex metagenomic DNA libraries based solely on the enzyme-mediated degradation of any target analyte. The approach can be applied to DNA isolated from any complex microbial sample, including unidentified or unculturable bacteria. In this research, I first describe the development of a general assay to capture the activity of oxidase enzymes expressed in E. coli cells. I then demonstrate how the assay can be used to screen for the nicotine degrading oxidase NicA2 from a genomic DNA library generated from the microbe P. putida. Lastly, I describe the use of this screen to identify a new hydrocortisone-responsive oxidase from a pooled genomic DNA library of eight microbes, representing over 43 Mb of DNA sequence space. This hydrocortisone oxidase represents the first of many new enzymes that can be discovered leveraging our screening platform, which is poised to revolutionize the electrochemical biosensing field and substantially broaden the number of molecules these electrochemical biosensors can detect continuously and quantitatively. / 2023-02-17T00:00:00Z

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