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When developing new techniques to analyze neuro-chemical microenvironments, it is important to realize the incredible variability in the cellular content and the response to stimulation between cells and within a single cell. Conventional analysis techniques yield an average result to describe the content and function of cells. This approach often misses important information since the onset of pathological conditions is always initiated in a small number of cells. New minimally invasive single cell analysis techniques are required for single cell studies in order to gain new insights and understanding of cells' functions. The objective of my Ph.D. study was to fabricate, characterize, and apply submicrometric fluorescence sensors for the analysis of neuron cells. This dissertation will report the fabrication of miniaturized fluorescence sensors for Ca2+, pH and Zn2+ analysis. In the first approach, liposomes (phospholipid vesicles) were used as miniaturized containers for fluorescent sensing reagents. Liposome-based fluorescence sensing technology offers several advantages over commonly used fluorescence sensing techniques including high spatial resolution, protection of the sensing dye from quenchers and high biocompatibility. However, liposome based sensors were found to be unstable in the cellular environment. The second approach was to synthesize submicrometric particle-based fluorescence sensors named lipobeads to replace the fluorescent liposomes in cellular studies. Lipobeads are polystyrene particles that are coated with a phospholipid membrane. One unique advantage of fluorescent sensing lipobeads is the ability to immobilize hydrophobic indicator molecules in the phospholipid membrane. This enables the use of these indicators in aqueous media since the lipobeads are fully water miscible. The lipobeads also proved to be highly biocompatible in cellular studies. This is attributed to their phospholipid bilayer membrane, which is similar in structure to cell membranes. The dissertation will describe the analytical properties of fluorescence sensing lipobeads and their application in studying zinc ion release and pH changes near neuron cells under physiological conditions, conditions of neuronal injury and stress and acidic cortical spreading depression during stroke like conditions.

The objective of my research is to develop new surface optochemical sensors for studying cellular processes by investigating techniques to modify surface properties. The spectral characteristics of the modified surfaces and coatings are designed to show remarkable changes after interaction with analytes from biological fluids and cells. My studies focused on pancreatic cells and addressed the need for improved techniques to measure zinc release from pancreatic cells (chapter 3, 4) and to measure the metastasis potential of cancerous pancreatic cells (chapter 5). Chapter 3 describes the development of zinc sensing glass slides by conjugating a carboxylmodified ZnAF-2 to an amino functionalized glass surface. The sensor was used for the measurement of glucose-stimulated zinc ion release from cultured beta pancreatic cells with impact in diabetes research. In chapter 4 is described conjugation of the carboxyl-modified ZnAF-2 to antibody molecules (A2B5) that specifically recognize pancreatic cells. This enabled for the first time the use of targeted zinc sensors to monitor zinc release events from pancreatic cells. Chapter 5 describes development for the first time of a fluorescence sensor to measure the proteolysis activity of pancreatic cancer cells in microfluidic systems. The sensor was fabricated using a Layer by layer (LbL) deposition of polyelectrolyte. The sensor was based on Fluorescence Resonance Energy Transfer (FRET) between luminescent quantum dots (serve as donors) and rhodamine molecules (serve as acceptors) that are separated by multi-layers of polyelectrolytes. The microfluidic platform enables precise delivery of reactants to assemble the sensor and facilitate unique cellular assays of enzymatic activity and enzymatic expression on pancreatic cancer cells.

Low-molecular-mass small molecules play important roles in biological processes and often serve as disease-related biomarkers for diagnosis. Accurate detection of small molecules remains challenging for conventional sensors due to their limited sensitivities. Two-dimensional (2D) materials, thanks to their atomic level thickness, can be extraordinarily sensitive to external perturbations and therefore well-suited for sensing applications. This dissertation explores the use of 2D materials, including primarily graphene and transition metal dichalcogenides, in the detection of low-molecular-weight and low-charge molecules. This work starts with the study of methods that allow for efficient and clean transfer of graphene grown on Cu using chemical vapor deposition (CVD), which is a critical step for achievement of large-area and high-quality graphene for device fabrication. In addition to the conventional wet-etching transfer method, we have studied on the method of electrochemical delamination, which is more time-efficient and allows for recycling of the Cu foil. Generation of bubbles during the electrochemical reaction is minimized by tuning the experimental parameters, thereby minimizing transfer-induced damages to graphene. We then fabricate the graphene-based field effect transistor (FET) and use the graphene FET as biosensors. First, the sensor is configured as an electrolyte-gated FET. With appropriate biochemical functionalization of the graphene, the FET sensors have been used to detect multiple small-molecule biomarkers including glucose and insulin via their affinity binding with receptors. Then, on a flexible substrate, we demonstrate real-time measurement of tumor necrosis factor alpha, a signal protein that regulates immune cells. We then simplified the sensor structure using a bottom local-gate to replace the external electrode as required in the previous electrolyte gated FET. Using the bottom local-gated FET sensor we have carried out real-time monitoring of the variation of pH in solutions. In addition to the electrical sensors, highly sensitive and multifunctional plasmonic sensors have also been developed by combining the unique optical properties of graphene with engineered metallic metasurfaces. The plasmonic sensors operating in mid-infrared region are configured as either metallic metasurface or hybrid graphene-metallic metasurface. Using a metallic metasurface, we demonstrate simultaneous quantification and fingerprinting of protein molecules. Using a hybrid graphene-metallic metasurface, we demonstrate optical conductivity-based ultrasensitive biosensing. In contrast to refractive-index-based sensors, the sensitivity of the hybrid metasurface sensor is not limited by the molecular masses of analytes. A monolayer of the sub-nanometer chemicals can be readily detected and differentiated on the hybrid metasurface. Reversible detection of glucose is carried out via the affinity binding of glucose with boronic acid immobilized on the graphene of the hybrid metasurface. The lowest detection limit achieved in our work is 36 pg/mL, which is considerably lower than that for the existing optical sensors. Despite the high sensitivity of graphene, the zero band-gap of graphene fundamentally impedes its use in digital electronic devices. In contrast, two-dimensional semiconductors, such as transition metal dichalcogenide (TMDC) with non-zero band gaps, holds great potential for developing practical electronic devices and sensors. Monolayers of TMDC materials are particularly attractive for development of deeply scaled devices, although the contact resistance between metal and the monolayer TMDC has been so large to significantly limit the performance of the devices. We present a high-performance monolayer MoS2 FET with a monolayer graphene as bottom local gate. The graphene gate is found to significantly improve the dielectric strength of the oxide layer compared to the lithographically patterned metal gate. This in turn allows for the use of very thin gate dielectric layer (~5 nm) and application of a strong displacement field to lower the contact resistance. Benefiting from the low contact resistance, the monolayer MoS2 FET offers a high on/off ratio (108) and low subthreshold slope (64 mV/decade). Additionally, thanks to the highly efficient electrostatic coupling through the ultrathin gate dielectric layer, short-channel (50 nm and 14 nm) devices are realized that exhibit excellent switching characteristics. In summary, this dissertation presents significant contributions to 2D material-based electronic and optoelectronic nanosensors, especially for detection of small molecules. Perspectives are made in the end of the thesis, on future studies needed to realize practical applications of these sensors and other 2D material-based products.

This work describes several thiophene-based molecular sensors and various modifications aimed to improve and understand the photophysical and supramolecular properties, such as the association constant (Ka) and selectivity, towards the development of a selective mercury(II) sensor. From the first generation of sensors containing pyridine and thiophene groups, it was determined that thiophene can offer good selectivity for mercury(II) against other transition metal ions, and provide a ratiometric absorption and fluorescent response. The projects following this focused on improving the Ka of the first generation of sensors through several different strategies. Substitution of thiophene for dibenzothiophene was shown to improve the Ka however this resulted in less than ideal photophysics of the dibenzothiophene sensors with absorption and emission in the UV-region. In addition to the effect of the chelating group was examined by incorporating imidazole, and thiazole rings, to compare to the original pyridyl chelating group employed. From this it was determined that pyridine offered the greatest Ka and selectivity for mercury(II). Following this electron-donating groups, including alcohol, octaethyleneglycol monomethyl ether, and amine, were added to a sensor, 2,5-bis(2-pyridyl)thiophene, as an alternative strategy to improving the Ka. Initially these functional groups were placed on the pyridine ring which caused a great increase in affinity for transition metal ions such as iron(II) and copper(II), however this translated to a loss in selectivity. In the final project I functionalized the thiophene ring with the same electron-donating groups which resulted in an increased Ka and maintained good selectivity for the mercury(II) ion, though iron(III) was still a competitive binder. In addition to this one of these thiophene functionalized sensors, 2,5-di(pyridin-2-yl)thiophene-3,4-diol, was shown to have a specific response to copper(II), iron(III), lead(II) and mercury(II) suggesting it could be used as a model for the development of a small-molecule multiplex sensor. Herein I will describe this work in greater detail and focus on the effects the modifications discussed had on the Ka and selectivity for the mercury(II) ion.

In the medical world the precise identification of a disease can take longer than it is safe to wait to start treatment so there is a need for faster and more precise biosensors. Bond Rupture is a new sensor technique that maybe able to improve disease detection. It does this by inducing bonds to rupture from the surface, and also measuring the point at which this rupture occurs this enables the forces to be measured on the surface. Specifically, this project has focused on the application of Bond Rupture to detecting antigens when bound to a surface using their specific antibodies, and the idea that the rupture force of these antigens can also be measured. The sensor that this project is based around is the Quartz Crystal Microbalance (QCM), which oscillates horizontally when a voltage is applied, and can also be used to measure mass change on its surface via change in resonant frequency. The aim of this project was to investigate possible Bond Rupture detection methods and techniques and has involved the development of a high speed digital electronics system, for the purposes of inducing and detecting Bond Rupture. This has involved the development of a FPGA based high speed transceiver board which is controlled by a Digital Signal Processor (DSP), as well as the development of various graphical user interfaces for end user interaction. Bond rupture testing was carried out by rupturing beads from the surface of a QCM in an experiment taking as little as 20 seconds. The Bond Rupture effect has been observed via the high accuracy measurement of the frequency change while inducing Bond Rupture on the sensor, proving that the Bond Rupture effect indeed exists. The research performed is believed to be a world first in terms of the method used and accuracy acquired.

The use of a conductive polypyrrole urea biosensor for the detection of urea in blood plasma is investigated.Urease was incorporated into a polypyrrole film by galvanostatic polymerisation. The presence of urea was verified, and the activity of the enzyme in the polypyrrole film was confirmed. The inherent electroactive properties of the polypyrrole-urease film has enabled the production of a flow injection amperometric biosensor for the reliable determination of urea. Greater sensitivity and stability was achieved when a pulsed amperometric detection system was implemented. The analysis of urea in human blood plasma by potentiometry, amperometry and pulsed amperometry was achieved with the assistance of an anion exchange separation prior to the electrochemical detection of urea. A polypyrrole-sulfite oxidase film was developed for use in an amperometric biosensor for sulfite determination. The response of the biosensor to sulfate was linear from 0 to 80 mg/L and the minimum detectable amount was 5 mg/L. Useful interferants in sulfite determination such as ascorbic acid, sodium nitrate and sodium sulfate did not respond to the biosensor. The excellent reproducibility of the sulfite response provides the basis for the construction of a disposable or renewable biosensor for sulfite determination. The analysis of sulfite in wine and beer was accomplished and no pretreatment was required. / Doctor of Philosophy (PhD)

Thesis (Ph. D.)--State University of New York at Binghamton, Dept. of Chemistry, 2006. / Includes bibliographical references.

The objective of this research was to develop the initial constituents of a highly scalable and label-free electronic biosensing platform. Current immunoassays are becoming increasingly incapable of taking advantage of the latest advances in disease biomarker identification, hindering their utility in the potential early-stage diagnosis and treatment of many diseases. This is due primarily to their inability to simultaneously detect large numbers of biomarkers. The platform presented here - termed the electronic microplate - embodies a number of qualities necessary for clinical and laboratory relevance as a next-generation biosensing tool. Silicon nanowire (SiNW) sensors were fabricated using a purely top-down process based on those used for non-planar integrated circuits on silicon-on-insulator wafers and characterized in both dry and in biologically relevant ambients. Canonical pH measurements validated the sensing capabilities of the initial SiNW test devices. A low density SiNW array with fluidic wells constituting isolated sensing sites was fabricated using this process and used to differentiate between both cancerous and healthy cells and to capture superparamagnetic particles from solution. Through-silicon vias were then incorporated to create a high density sensor array, which was also characterized in both dry and phosphate buffered saline ambients. The result is the foundation for a platform incorporating versatile label-free detection, high sensor densities, and a separation of the sensing and electronics layers. The electronic microplate described in this work is envisioned as the heart of a next-generation biosensing platform compatible with conventional clinical and laboratory workflows and one capable of fostering the realization of personalized medicine.