Nano-FET biosensors are an emerging nanoelectronic technology capable of real-time and label-free quantification of soluble biological molecules. This technology promises to enable novel in vitro experimental approaches for investigating complex biological systems. In this study, we first explored osteocyte mechanosensitivity under different mechanical stimuli and found that osteocytes are exquisitely sensitive to different oscillatory fluid flow conditions. We therefore aimed to characterize protein-mediated intercellular communication between mechanically-stimulated osteocytes and other bone cell populations in vitro to elucidate the underlying mechanisms of load-induced bone remodeling. To this end, we devised a novel nano-manipulation based fabrication method for manufacturing nano-FET biosensors with precisely controlled device parameters, and further investigated the effect of these parameters on sensor performance.

Recent biosensors using glucose oxidase enzyme to detect glucose (“blood sugar”) were made with intrinsic conducting polymers such as poly pyrrole (PPY) to mediate the reaction. PPY coated electrodes were difficult to employ via eletropolymerization because PPY is only soluble in solvents potentially damaging to enzymes. Nano crystalline cellulose – poly pyrrole (NCC-PPY) colloid circumvents this by forming natural, enzyme compatible, and hydrophilic films mechanically bound to electrodes using easy-to-disperse colloids. NCC-PPY was studied as mediator to investigate use in biosensor applications. Using NCC-PPY film casted on microfabricated interdigitated electrodes, a glucose biosensor with sensitivity factor of 20 was achieved. NCC-PPY showed enhanced catalysis with no enzyme inactivation and a total current of 2mA. Enhanced sensitivity was attributed to resistance changes of doped PPY, redox mediation, and compatibility of cellulose with enzyme.

Nano-FET biosensors are an emerging nanoelectronic technology capable of real-time and label-free quantification of soluble biological molecules. This technology promises to enable novel in vitro experimental approaches for investigating complex biological systems. In this study, we first explored osteocyte mechanosensitivity under different mechanical stimuli and found that osteocytes are exquisitely sensitive to different oscillatory fluid flow conditions. We therefore aimed to characterize protein-mediated intercellular communication between mechanically-stimulated osteocytes and other bone cell populations in vitro to elucidate the underlying mechanisms of load-induced bone remodeling. To this end, we devised a novel nano-manipulation based fabrication method for manufacturing nano-FET biosensors with precisely controlled device parameters, and further investigated the effect of these parameters on sensor performance.

Recent biosensors using glucose oxidase enzyme to detect glucose (“blood sugar”) were made with intrinsic conducting polymers such as poly pyrrole (PPY) to mediate the reaction. PPY coated electrodes were difficult to employ via eletropolymerization because PPY is only soluble in solvents potentially damaging to enzymes. Nano crystalline cellulose – poly pyrrole (NCC-PPY) colloid circumvents this by forming natural, enzyme compatible, and hydrophilic films mechanically bound to electrodes using easy-to-disperse colloids. NCC-PPY was studied as mediator to investigate use in biosensor applications. Using NCC-PPY film casted on microfabricated interdigitated electrodes, a glucose biosensor with sensitivity factor of 20 was achieved. NCC-PPY showed enhanced catalysis with no enzyme inactivation and a total current of 2mA. Enhanced sensitivity was attributed to resistance changes of doped PPY, redox mediation, and compatibility of cellulose with enzyme.

Methicillin Resistant Staphylococcus aureus (MRSA) is one of the most important threats of nosocomial infections in many regions of the world and Vancomycin Resistant Enterococcus (VRE) is an emerging pathogen that develops full resistance against third-generation glycopeptide antibiotics. Conventional methods for identification of MRSA and VRE generally depend on culturing, which requires incubation of biological samples at least 24-72 hours to get accurate results. These methods are time consuming and necessitate optical devices and experts for evaluation of the results. On the other hand, early diagnosis and initiation of appropriate treatment are necessary to decrease morbidity and mortality rates. Thus, new diagnostic systems are essential for rapid and accurate detection of biological analytes at the point of care. This study presents design, fabrication, and implementation of MEMS based micro electrochemical sensor (&micro / ECS) to detect the methicillin resistance in Staphylococcus aureus and vancomycin resistance in Enterococcus species. To the best of our knowledge, the developed sensor is the first &micro / ECS which utilizes on-chip reference (Ag), working (Au), and counter (Pt) electrodes together with a microchannel to detect MRSA and VRE. The characterization of the designed sensor was achieved analyzing the interactions of the buffer solutions and solvents with the electrodes and Parylene C film layer by using optical and electrochemical methods. Specific parts of genes that are indicators of antimicrobial resistances were used in order to detect the resistances with high selectivity and sensitivity. Thus, synthetic DNA and bacterial PCR product were used as target probes in redox marker based detection and enzyme based detection, respectively. In order to enhance the hybridization, folding structures of the capture probe were investigated by using mfold Web Server. In redox marker based detection, the hybridization of DNA was indirectly detected by using Hoechst 33258 as redox marker with differential pulse voltammetry. The cross reactivity of the tests were performed by using different target probes of femA genes of S. aureus and S. epidermis, which are the major genes detected in methicillin detection assays. Consequently, amplification of signal by using horseradish peroxidase and TMB/H2O2 as substrate was achieved in order to enhance detection sensitivity. The sensor could detect 0.01 nM 23-mer specific part of mecA gene with redox marker based detection and 10 times diluted PCR product with enzyme-based detection in about six hours including the steps of sample preparation from whole blood. This sensor with its compatibility to MEMS fabrication processes and IC technology has a promising potential for a hand-held device for POC through the integration of micropotentiostat.

Research toward a multiplexed nucleic acid biosensor that uses quantum dots (QDs) as donors in a fluorescence resonance energy transfer (FRET) assay is described. Optical fibers were modified with mixed films composed of different colours of QDs and different oligonucleotide probes that served as scaffolds for the hybridization of the corresponding target nucleic acid sequences. Fluorescent dyes that were suitable as acceptors for each QD donor were associated with hybridization and provided an analytical signal through FRET-sensitized emission. Different detection channels were achieved through the combination of different donors and acceptors: green emitting QDs with Cyanine 3 or Rhodamine Red-X; and red emitting QDs with Alexa Fluor 647. A detection channel that used the direct excitation of Pacific Blue complemented the FRET pairs. One-plex, two-plex, three-plex and four-plex hybridization assays were demonstrated. A sandwich assay format was adopted to avoid target labeling. Detection limits were 1-10 nM (1-12 pmol) and analysis times were 1-4 h. Single nucleotide polymorphisms were discriminated in multiplexed assays, and the potential for reusability was also demonstrated. Non-selective interactions between QDs and oligonucleotides were characterized, and routes toward the optimization of the QD-FRET hybridization assays were identified. A basic model for multiple FRET pathways in a mixed film was also developed. In addition to the advantages of solid-phase assays, the combination of QDs and FRET was advantageous because it permitted multiplexed detection using a single excitation source and a single substrate, in the ensemble, and via ratiometric signals. Spatial registration or sorting methods, imaging or spatial scanning, and single molecule spectroscopy were not required. The research in this thesis is expected to enable new chip-based biosensors in the future, and is an original contribution to both bioanalytical spectroscopy and the bioanalytical applications of nanomaterials.

Fluorescent protein based genetically encoded fluorescent reporters play an improtant role in understanding the cellular physiology by directly monitoring real-time cellular signaling pathways with fluorescent microscope. Quantitative analysis of Ca2+ fluctuations in the endoplasmic/sarcoplasmic reticulum (ER/SR) is essential to defining the mechanisms of Ca2+-dependent signaling under physiological and pathological conditions. Here, we developed a novel class of genetically encoded indicators by designing a Ca2+ binding site in the enhanced green fluorescent protein (EGFP). One of them, CatchER (Calcium sensor for detecting high concentration in the ER), exhibits unprecedented Ca2+ release kinetics with an off-rate estimated at around 700 s-1 and appropriate Ca2+ binding affinity, likely due to local, Ca2+-induced conformational changes around the designed Ca2+ binding site and reduced chemical exchange between two chromophore states. CatchER reported considerable differences in ER Ca2+ dynamics and concentration among epithelial HeLa, kidney HEK 293, and muscle C2C12 cells, enabling us to monitor SR luminal Ca2+ in flexor digitorum brevis (FDB) muscle fibers to determine the mechanism of diminished SR Ca2+ release in aging mice. Moreover, the structure of CatchER has been investigated by nuclear magnetic resonance spectroscope (NMR) and high-resolution X-ray crystal structures to understand the novel mechanism of Ca2+ induced fluorescent enhancement of GFP. It is crucial to investigate the metal selectivity of Ca2+/Mg2+ of these metalloproteins to understand cellular physiology. The major Mg2+ binding sites of proteins have been reviewed and classified based on structural differences, and identified several key factors to determine Mg2+/Ca2+ selectivity with binding constants difference up to 104 in several types of metalloproteins. Thrombin is involved in numerous cellular signaling pathways and plays a crucial role in blood coagulation. I designed a novel class of single EGFP-based thrombin sensors by inserting a thirty-amino acid short peptide with a thrombin cleavage site into the fluorescent sensitive location of EGFP. These designed protease sensors exhibited optimized kcat/Km up to 104 magnitudes higher than that of small peptide based absorption indicator EGR-pNA. The measured Km value is in below 10 mM, in the same magnitude as that of natural thrombin substrate Fibrinogen A.

Point-of-care testing (POCT) devices have received increasing attention because of their potential to address the urgent need for quick and accurate diagnostic tools, especially in areas of personal care and clinical medicine. They offer several benefits over current diagnostic systems, including rapid diagnostic results in comparison to microbial cultures, simple interpretation of results, portability, and requiring no specialised laboratory equipment or technical training to operate. These are essential for diagnosing critical illnesses, such as sepsis, in areas of poor healthcare infrastructure. Sepsis, an innate physiological response to infection, is a growing problem worldwide with high associated costs and mortality rates, and affects a wide range of patients including neonates, infants, the elderly, and immunocompromised individuals. A literature review of the biomarkers of sepsis and the currently available diagnostic systems indicates the need for a biosensor capable of meeting the requirements of designing POCT systems and achieving detection of low concentrations of biomarkers. To meet these demands, two significant contributions to developing POCT platforms have been achieved and described in this thesis, including: 1) development of a colorimetric, magnetically separable biosensor that can be easily fabricated and demonstrates an easily identifiable colour response upon analyte detection, as well as the ability to capture and detection target biomarkers at low concentrations from complex solutions; and 2) tuning of the biosensor’s colorimetric response to achieve low detection limits, as well as demonstration of the versatility of the biosensor for sensing different target analytes. The developed biosensor in this work combines colour responsive polydiacetylenes and superparamagnetic iron oxide for the first time to achieve a biosensor capable of meeting these demands. The sensors exhibit identifiable colour responses to biomolecule detection, capture of a target analyte from complex solutions, sensing of different target analytes, a lower detection limit of 0.01 mg/mL, and rapid separation from solution with a common magnet. This work has been a significant demonstration of the capabilities of this biosensor as a new platform for POCT systems to diagnosis sepsis, and potentially other sensing applications.

The Microelectronics Group has been researching sensors useful for detecting and quantifying events in biological molecular chemistry, for example, binding events. Our previous research has been based primarily on quartz resonators. This thesis describes the results of our initial research of Surface Plasmon Resonance (SPR) based technology. This study contains the design and implementation of a fully functional SPR biosensor with detailed disclosure of monolayer construction, digital hardware interfaces and software algorithms for process the SPR sensors output. An antibody monolayer was constructed on the biosensor surface with the goal of setting the strengths, weaknesses and limitation of measuring molecular events with SPR technology. We documented several characteristics of molecular chemistry that directly effect any measurements made using Surface Plasmon Resonance technology including pH, free ions, viscosity and temperature. Furthermore, the component used in our study introduced additional limitations due to wide variations amongst parts, the constraint of a liquid medium and the large surface area used for molecular interrogation. We have identified viable applications for this sensor by either eliminating or compensating for the factors that affect the measured results. This research has been published at the inaugural IEEE sensors conference and to our knowledge is the first time a biosensor has been constructed by attaching a sensor to a PDA and performing all signal processing, waveform analysis and display in the PDAs core processor.

The main subject of this thesis is to design a voltage peak detector for FPW-based IgE measurement systems. Therefore, two different peak detectors are proposed. The first voltage peak detector basically samples the input signal twice (double sampling) to reduce the ripples appearing during the sample and hold modes. This voltage peak detector also resolves the detection error of conventional voltage peak detectors when they are used to detect the output signal of FPW-based biosensors.The fastest signal which this voltage peak detector can detect is 10 MHz. The second voltage peak detector is composed of a coupling capacitor, an unity gain buffer, an 8th order voltage control voltage source(VCVS) low pass filter, and a non-inverting amplifier. The major difference of this design from the previous one is to filter and amplify the input signal. The specification requirements of the operational transconductance amplifier in this voltage peak detector can be relaxed thereafter. The resolution and performance of the sensing system are also improved. By replacing the conventional power MOS by a non-inverting amplifier, the charging time is reduced and over charge hazard is avoided. Besides, the speed of the entire system is enhanced. The fastest signal which this voltage peak detector can detect is 50 MHz and the precision is 0.357 %.