Dissertations / Theses on the topic 'Biosensiing'

Thesis advisor: Michael J. Naughton
Nanoscale biosensing devices improve and enable detection mechanisms by taking advantage of properties inherent to nanoscale structures. This thesis primarily describes the development, characterization and application of two such nanoscale structures. Namely, these two biosensing devices discussed herein are (1) an extended-core coaxial nanogap electrode array, the ‘ECC’ and (2) a plasmonic resonance optical filter array, the ‘plasmonic halo’. For the former project, I discuss the materials and processing considerations that were involved in the making of the ECC device, including the nanoscale fabrication, experimental apparatuses, and the chemical and biological materials involved. I summarize the ECC sensitivity that was superior to those of conventional detection methods and proof-of-concept bio-functionalization of the sensing device. For the latter project, I discuss the path of designing a biosensing device based on the plasmonic properties observed in the plasmonic halo, including the plasmonic structures, materials, fabrication, experimental equipment, and the biological materials and protocols
Thesis (PhD) — Boston College, 2019
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Physics

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.

Biosensors are often used to detect biochemical species either in the body or from collected samples with high sensitivity and specificity. Those based on piezoelectric sensing methods employ mechanically induced changes to generate an electrical response. Reliable collection and processing of these signals is an important aspect in the design of these systems. To generate the electrical response, specific recognition layers are arranged on piezoelectric substrates in such a way that they interact with target species and so change the properties of the device surface (e.g. the mass or mechanical strain). These changes generate a change in the electrical signal output allowing the device to be used as a biosensor. The characteristics of piezoelectric biosensors are that they are competitively priced, inherently rugged, very sensitive, and intrinsically reliable. In this study, a compound label-free biosensor was developed. This sensor consists of two elements: a Love wave sensor and an electrochemical impedance sensor. The novelty of this device is that it can work in both dry and wet measurement conditions. Whilst the Love wave sensor aspect of the device is sensitive to the mass of adsorbed analytes under both dry and wet conditions with high sensitivity, the sensitivity coefficients in these two conditions may be different due to the different (mechanical) strengths of interaction between the adsorbed analyte and the substrate. The impedance sensor element of the device however is less sensitive to the mechanical strength of the bond between the analyte and the sensing surface and so can be used for in-situ calibration of the number of molecules bound to the sensing surface (with either a strong or weak link): conventional Love wave sensors are not sensitive to material loosely bound to the surface. Thus, a combination of results from these two sensors can provide more information about the analyte and the accuracy of the Love wave sensor measurements in a liquid environment. The device functions with label-free molecules and so special reagents are not needed when carrying out measurements. In addition, the fabrication of the device is not too complicated and it is easy to miniaturise. This may make the system suitable for point-of-care diagnostics and bio-material detection. The substrate used in these sensors is 64°Y–X lithium niobate (LiNbO3) which is a kind of piezoelectric material. On the substrate, there is a pair of interdigital transducers (IDTs) which are composed of 100 Ti/Au split-finger pairs with a periodicity (λ) of 40μm. The acoustic path length, between both IDTs, is 200λ and the aperture between the IDTs is 100λ. On top of the substrate and IDTs, there is a PMMA guiding layer with an optimised thickness ranging from 1000 nm to 1300 nm. In addition, a gold layer with thickness 100 nm is deposited on the guiding layer to act as the electrodes for the electrochemical impedance sensor. The biosensor in this study has been used to measure Protein A, IgG, and GABA molecules. Protein A is often coupled to other molecules such as a fluorescent dye, enzymes, biotin, and colloidal gold or radioactive iodine without affecting the antibody binding site. In addition, the capacity of Protein A to bind antibodies with such high affinity is the driving motivation for its industrial scale use in biologic pharmaceuticals. Therefore, measuring Protein A binding is a useful method with which to verify the function of the biosensor. IgG is the most abundant antibody isotype found in the circulation. By binding many kinds of pathogens including viruses, bacteria, and fungi, IgG protects the body from infection. Also, IgG can bind with Protein A well so the biosensor here could also measure IgG after a Protein A layer is immobilised on the sensing area. GABA is the main inhibitory neurotransmitter in the mammalian central nervous system. It plays an important role in regulating neuronal excitability throughout the nervous system. The conventional method to measure concentrations of GABA under the extracellular conditions is by using liquid chromatography. However, the disadvantages of chromatographic methods are baseline drift and additions of solvent and internal standards. Therefore, it is necessary to develop a simple, rapid and reliable method for direct measurement of GABA, and the sensor here is an attractive choice. When the Love wave sensor works in the liquid media, it can only be used to measure the mass of analytes but does not provide information about the conditions of molecules bound with the sensing surface. In contrast, electrochemical impedance sensing based on the diffusion of redox species to the underlying metal electrode can provide real-time monitoring of the surface coverage of bound macromolecular analytes regardless of the mechanical strength of the analyte-substrate bond: the electrochemical impedance measurement is sensitive to the size and extent of the diffusion pathways around the adsorbed macromolecules used by the redox species probe i.e. it is sensitive to the physical area of the surface covered by the macromolecular analyte and not to the mass of material that is sensed through a mechanical coupling effect (as in a Love wave device). Although electrochemical impedance measurements under the dry state are quite common when studying batteries and their redox/discharge properties, these are quite different sorts of systems to the device in this study. Therefore, integrating these two sensors (Love wave sensor and electrochemical impedance sensor) in a single device is a novel concept and should lead to better analytical performance than when each is used on their own. The new type of biosensor developed here therefore has the potential to measure analytes with greater accuracy, higher sensitivity and a lower limit of detection than found when using either a single Love wave sensor or electrochemical impedance sensor alone.

Analytical chemical information is needed in all areas of human activity including health care, pharmacology, food control and environmental chemistry. Today one of the main challenges in analytical chemistry is the development of methods to perform accurate and sensitive rapid analysis and monitoring of analytes in ‘real’ samples. Electrochemical biosensors are ideally suited for these applications. Despite the wide application of electrochemical biosensors, they have some limitations. Thus, there is a demand on improvement of biosensor performance together with a necessity of simplification required for their mass production. In this thesis the work is focused on the development of electrochemical sensors with improved performance applicable for mass production, e.g. by screen printing. Biosensors using immobilized oxidases as the bio-recognition element are among the most widely used electrochemical devices. Electrical communication between redox enzymes and electrodes can be established by using natural or synthetic electron carriers as mediators. However, sensors based on soluble electronshuttling redox couples have low operational stability due to the leakage of water-soluble mediators to the solution. We have found a new hydrophobic mediator for oxidases – unsubstituted phenothiazine. Phenothiazine and glucose oxidase, lactate oxidase or cholesterol oxidase were successfully co-immobilized in a sol-gel membrane on a screen-printed electrode to construct glucose, lactate and cholesterol biosensors, respectively. All elaborated biosensors with phenothiazine as a mediator exhibited long-term operational stability. A kinetic study of the mediator has shown that phenothiazine is able to function as an efficient mediator in oxidase-based biosensors. To improve sensitivity of the biosensors and simplify their production we have developed a simple approach for production of graphite microelectrode arrays. Arrays of microband electrodes were produced by screen printing followed by scissor cutting, which enabled the realization of microband arrays at the cut edge. The analytical performance of the system is illustrated by the detection of ascorbic acid through direct oxidation and by detection of glucose using a phenothiazine mediated glucose biosensor. Both systems showed enhanced sensitivity due to improved mass transport. Moreover, the developed approach can be adapted to automated electrode recovery. Finally, two enzyme-based electrocatalytic systems with oxidation and reduction responses, respectively, have been combined into a fuel cell generating a current as an analytical output (a so-called self-powered biosensor). This was possible as a result of the development of the phenothiazine mediated enzyme electrodes, which enabled the  construction of a cholesterol biosensor with self-powered configuration. The biosensor generates a current when analyte (cholesterol) is added to the cell. The biosensor has been applied for whole plasma analysis. All developed concepts in the thesis are compatible with a wide range of applications and some of them may even be possible to realize in a fully integrated biosensor unit based on printed electronics.

Thesis advisor: Thomas C. Chiles
Sensitive, real-time detection of biomarkers is of critical importance for rapid and accurate diagnosis of disease for point-of-care (POC) technologies. Current methods, while sensitive, do not adequately allow for POC applications due to several limitations, including complex instrumentation, high reagent consumption, and cost. We have investigated two novel nanoarchitectures, the nanocoax and the nanodendrite, as electrochemical biosensors towards the POC detection of infectious disease biomarkers to overcome these limitations. The nanocoax architecture is composed of vertically-oriented, nanoscale coaxial electrodes, with coax cores and shields serving as integrated working and counter electrodes, respectively. The dendritic structure consists of metallic nanocrystals extending from the working electrode, increasing sensor surface area. Nanocoaxial- and nanodendritic-based electrochemical sensors were fabricated and developed for the detection of bacterial toxins using an electrochemical enzyme-linked immunosorbent assay (ELISA) and differential pulse voltammetry (DPV). Proof-of-concept was demonstrated for the detection of cholera toxin (CT). Both nanoarchitectures exhibited levels of sensitivity that are comparable to the standard optical ELISA used widely in clinical applications. In addition to matching the detection profile of the standard ELISA, these electrochemical nanosensors provide a simple electrochemical readout and a miniaturized platform with multiplexing capabilities toward POC implementation. Further development as suggested in this thesis may lead to increases in sensitivity, enhancing the attractiveness of the architectures for future POC devices
Thesis (PhD) — Boston College, 2016
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology

Microcantilever sensor system as a promising field attracted much attention recently. This system has the potential to be applied for a biosensing technology which is parallel reference, label free, sensitive and real time. In this thesis, polyimide has been selected as a material to fabricate cantilever due to its excellent physical, electrical and mechanical properties, on top of its cost advantage. Importantly, we showed it is feasible to microfabricate large array of microcantilever sensors with high-power UV laser directly. It is low cost and rapid, the parameters for laser direct writing fabrication has been studied. The thesis also shows that it is possible to functionalise the polyimide film first and subsequently cut it to functionalised cantilever sensor array. The unique fabrication and functionalisation process can solve the problem of high-cost microfabrication using silicon and low-efficient functionalisation using capillary tubing all together. In addition, the fabrication process has been further developed to avoid the problem of the cross contamination from receptors on both sides. With this improvement, we developed an internally referenced microcantilever biosensors system for DNA hybridization detection. Different receptors can be coated on each side of the polymer film before fabricating to cantilever biosensors This newly developed capability enables us to coat receptors with similar but slightly different biological properties on each side of the cantilever sensor, a process which is extremely difficult by using conventional capillary tubing methods due to the possibility of thiol exchange on surfaces and hence cross-contamination. A polyimide microcantilever sensor with embedded microfluidic channel has been developed in this thesis. Photoresist material is used to form the precise microfluidic channel within the microcantilever device. The multilayer polymer film device is still soft enough to operate in static mode. The main advantage of the system presented here is that since the device is made entirely of polymer materials, the fabrication process is simple and low-cost. The magnetic beads have been used to amplify the signal of the biosensing processing; the application of polyimide microfluidic microcantilevers to the detection of Cryptosporidium and thrombin is reported in this thesis. Paper based autonomous micocantilever system has also been investigated in this thesis. We build a cantilever system without external pump or force with paper and magnetic field. The limitation of the system is that it takes too much time to pump magnetic beads through the cantilever with capillary. However, we found that it has the potential to develop a long time range timer based on the slowest property. Different methods have been investigated to slow down the speed, when liquid pass through the paper microfluidic. Finally, we demonstrate some timer devices whose ranges are from minutes to month. The devices have the potential to be used as time-based diagnostic assays, food label, etc.

Optical biosensors make up a valuable toolkit for label-free biosensing. This thesis presents a detailed study on resonant grating surfaces for biosensing. The focus is on silicon nitride gratings, which exhibit a guided-mode resonance that is highly sensitive to refractive index variations in the vicinity of the grating. A sensitivity of 143 nm/RIU (refractive index units) is measured, leading to a detection limit of 2.4×10−4 RIU. This performance is shown to be sufficient for the detection of biomolecular binding down to ng/mL concentrations. With out-of-plane excitation, these gratings can be used as a sensing surface, enabling a spatially-resolved measurement of variations in refractive index; resonance imaging. The minimum detection distance (sensing depth) is measured to be 183 nm away from the grat- ing, while the spatial resolution of resonance imaging is found to be asymmetric: 2 μm parallel to, or 6 μm perpendicular to the gratings. Using a novel approach of fabricating a resolution test pattern on top of the grating, the relationship between resolution and index contrast is studied - an important question in the context of biosensing - where it is found to decrease with index contrast. All experimental results are supplemented with theoretical and computational models. The resonant gratings are then extensively applied to the study of biofilm development, cellular imaging, and the imaging of cellular secretion. Finally, a miniaturised biosensor is demonstrated, based on a chirped resonant grating. By tuning the resonance wavelength spatially on the chip, the resonance information is directly translated into spatial informa- tion. Instrument read-out requires just a monochromatic light source and a simple CCD camera, resulting in a final device that is inexpensive, compact, robust and can be remotely operated. Performance is proven with successful detection of biomolecular binding.

Inexpensive label-free detection of biomarker panels in serum could revolutionize earlycancer diagnosis and treatment. Such detection capabilities may be possible with dynamicnanochannels in conjunction with electrical impedance measurement. In Dr. Greg Nordin's lab I designed, fabricated and tested several iterations of these sensors with polydimethyl-siloxane microfluidics. The final design yielded a dynamic nanochannel array sensor thatshowed a 140% impedance change when exposed to 14µM bovine serum albumin in phos-phate buffered saline. For the geometry and noise limits of the tested device, simulationsindicated that a minimum detectable concentration of 20pM with specifically bound strep-tavidin should be possible. However, the polydimethylsiloxane approach is also shown to beproblematic in meeting the trade-offs required for a practical device. Consequently, alter-native materials and designs are suggested to reduce the minimum detectable concentrationto the high femtomolar range, which would be attractive for detection of many medicalbiomarkers.

This thesis, Integrated electronics for molecular biosensing, focuses on different approaches to sense the presence and activity of a specific analyte by using integrated electronic platforms. The aim of the first platform is to detect the enzyme telomerase. Telomerase causes the elongation of telomeres, which are part of the chromosomes, and determines the lifespan of cells. Telomerase expression is a marker of malignity in tumoral cells and its evaluation can be exploited for early diagnosis of many types of cancer cells. To detect the telomerase enzyme, a CMOS (complementary metal-oxide semiconductor) biosensor based on CMFET (Charge-Modulated Field Effect Transistor) able to measure kinetics of DNA replication and telomerase reaction was developed. The sensor can be functionalized by immobilizing single strands of DNA that contain the telomeric sequence, used as probes. If telomerase is present, the probes will be elongated by the enzyme and the charge on the sensing area will change, which reflects in a variation of the output current or voltage. The chip includes three different readout schemes (voltage, current- and time-based), each of which has different measuring ranges and operating conditions. The measured data is then digitized, stored, and can be sent off-chip through SPI (Serial Peripheral Interface) protocol. A total of 1024 biosensors have been integrated in a single chip with a size of 10x10 mm2. Each sensor can be independently addressed and functionalized by an electrochemical procedure using an integrated potentiostat, thus requiring no external equipment. Although the sensors have been tailored and optimized to perform telomerase detection, the sensing areas can be functionalized to be used with different analytes. This feature turns the chip into a complete bioassay platform. The second part of this work rises from the idea that bacteria, like Escherichia coli, can detect analytes in solution even at extremely low concentrations and change their movement through a process called chemotaxis, to move towards chemical gradients in the solution. E. coli moves by rotating its flagella either clockwise (for random tumbles) or counterclockwise (for straight runs, when it senses a chemical it is attracted to). Therefore, observing bacteria flagellar rotation can give enough information on the presence of a specific analyte in the solution. To electronically detect bacteria movement, an active surface covered in electrodes has been designed. By measuring the impedance between each pair of electrodes through an integrated LIA (lock-in amplifier), it is possible to know how a single bacterium is moving. By that, the presence or absence of the analyte can be deduced, thus effectively turning bacteria into chemical sensors.

The aim of this work is the study, the design and the nanofabrication of innovative plasmonic nanostructured materials to develop label-free optical biosensors. Noble metalbased nanostructures have gained interest in the last years due to their extraordinary optical properties, which allow to develop optical biosensors able to detect very low concentrations of specific biomolecules, called analyte, down to the picomolar range. Such biosensors rely on the Surface Plasmon Resonance (SPR) excitation which occurs under specific conditions that depend both on the morphology of the nanostructure and on the adjacent dielectric medium. Therefore, the binding of the biomolecules to metal surfaces is revealed as a change in the SPR condition. Four kinds of nanostructures are investigated in this work: ordered and disordered nanohole array (o-NHA, d-NHA), nanoprism array (NPA) and nanodisk array (NDA). The o-NHA and d-NHA consist of a thin metallic film (50 - 100 nm) patterned with, respectively, a hexagonal and a disordered array of circular holes. The NPA consists of a honeycomb lattice of triangle shaped nanoprisms with edges of about 100 - 200 nm and height of 40 - 80 nm. Finally, the NDA consists of a disordered array of non-interacting disks with 100 - 300 nm diameter and 40 - 80 nm height. The first two support the Extended-SPR whereas the last two, due to their three-dimensional confinement, present Localized-SPR property. Two colloidal techniques are employed for the scalable and cost-effective synthesis of wide areas of nanostructures that allow a fine control of the morphology: NanoSphere Lithography (NSL) and Sparse Colloidal Lithography (SCL). Ordered arrays were nanofabricated by NSL (i.e., NPA and o-NHA) whereas disordered nanostructures were synthesized by the SCL (i.e., NDA and d-NHA). Firstly, the nanostructures are simulated by Finite Element Method (FEM) computations and their performances in revealing small variations of the dielectric medium at the interface is evaluated as a function of their geometrical parameters. Simulated local sensitivities range from 3.1 nm/RIU of the o-NHA up to 13.6 nm/RIU of the NPA. Afterwards, the sensing performances are evaluated experimentally with nanofabricated samples and comparable but slightly smaller sensitivities are obtained. Secondly, a proof-of-concept protocol for the detection assay, that relies on the binding of streptavidin protein to the biotinylated gold surfaces, is exploited to test the nanostructures as biosensors. A 4.4 nM limit of detection is reached with the best performing biosensor (NPA) and picomolar ones are expected for NPA and NDA with a suitable improvement of the functionalization protocol. Finally, complementary single stranded RNA molecules were used, respectively, as bioreceptor and analyte. Revealing short sequences of non-coding RNA, called microRNA, is fundamental for the medical research since these oligonucleotides act as biomarkers for specific diseases, like tumors. Signals of about 13 nm are obtained from the binding of bioreceptor to the nanostructure and from the hybridization of the analyte oligonucleotide at saturation concentrations (∼ 1 μM), indicating that for the moment the developed protocol is quite effective down to the 100 nM range. Of course, for reading the nm or even sub-nM range further optimizations are needed.

En aquesta tesi, hem estudiat diferents aspectes relacionats amb transistors orgànics, en particular transistors orgànics electroquímics d’efecte de camp (EGOFETs) i transistors orgànics electroquímics (OECTs). Els dispositius EGOFET es van fabricar dipositant a partir de dissolucions de semiconductors orgànics (OSC) basats en molècules conjugades barrejats amb polímers aïllants, mitjançant la tècnica de Bar-assisted meniscus shearing (BAMS). BAMS és una tècnica ràpida, de baix cost i escalable que permet la formació de pel·lícules cristal·lines i uniformes. Els EGOFET van ser estudiats pel desenvolupament d’un biosensor per a la detecció de la proteïna α-sinucleina, que és un biomarcador per a malalties neurodegeneratives, incloses les malalties de Parkinson. A més, es van utilitzar dispositius OECT també per a la detecció de α-sinucleina, per estudiar el possible ús d’aquests dispositius com a immunosensors, camp molt poc explorat en la literatura. Finalment, es va fabricar un EGOFET completament flexible basat en un nou semiconductor molecular. Per primera vegada, s’ha estudiat la resposta elèctrica sota tensió mecànica per a un EGOFET.
En esta tesis, hemos estudiado diferentes aspectos relacionados con los transistores orgánicos activados por líquido, en particular los transistores de efecto de campo orgánicos activados por electrolitos (EGOFET) y los transistores electroquímicos orgánicos (OECT). Los dispositivos EGOFET se fabricaron depositando a partir de soluciones pequeñas moléculas de semiconductores orgánicos (OSC) mezclados con polímeros aislantes, a través de la técnica de Bar-assisted meniscus shearing (BAMS). BAMS es una técnica rápida, de bajo costo y escalable que permite la formación de películas finas cristalinas y uniformes. Los EGOFET se estudiaron para el desarrollo de un biosensor para la detección de un biomarcador de enfermedades neurodegenerativas, incluidas las enfermedades de Parkinson, es decir, la alpha-sinucleína. Además, se emplearon dispositivos OECT para la biodetección de α-sinucleína, para estudiar el posible uso de estos dispositivos como inmunosensores, campo que aún está menos explorado en la literatura. Finalmente, se fabricó un EGOFET totalmente flexible basado en una pequeña molécula semiconductora mezclada con un polímero aislante y se evaluó su respuesta eléctrica bajo tensión mecánica, por primera vez, hasta donde sabemos, para dispositivos EGOFET.
In this thesis, we have studied different aspects related to liquid-gated organic transistors, in particular electrolyte-gated organic field-effect transistors (EGOFETs) and organic electrochemical transistors (OECTs). EGOFET devices were fabricated by depositing from solution small molecules organic semiconductors (OSC) blended with insulating polymers, through the bar-assisted meniscus-shearing technique (BAMS). BAMS is a rapid, low-cost and scalable technique that allows the formation of crystalline and uniform thin films. The EGOFETs were studied for the development of a biosensor for the detection of a biomarker for neurodegenerative diseases, including Parkinson’s diseases, namely α-synuclein. Further, OECT devices were employed for the biosensing of α-synuclein, to give an insight into the possible use of these devices as immunosensors, field which is still less explored in literature. Finally, an all-flexible EGOFET based on a small molecule OSC blended with an insulating polymer thin film, was fabricated and its electrical response under bending strain was evaluated, for the first time, as far as we know, for liquid-gated OFETs.

Low temperature-processed, porous sol-gel glasses represent a new class of materials for the immobilisation of biomolecules. If used to entrap biological recognition elements, these transparent and chemically inert glasses offer a new approach in the development of optical biosensors.

The discovery of monolayer graphene by Manchester group has led to intensive research into a variety of applications across different disciplines. As a monolayer of carbon atoms, graphene presents a high surface to volume ratio and a good electronic conductivity, making it sensitive to its surface bio-chemical environment. This project investigated the fabrication of electronic biosensors using different graphene-based materials. It included the production of graphene, the fabrication of electronic devices, the chemical functionalisation of graphene surface and the specific detection of target bio-molecules. This project first investigated the production of graphene using three different methods, namely mechanical exfoliation, physical vapour deposition and electrochemical reduction of graphene oxide. With respect to the physical vapour deposition method, the production of large area transfer-free graphene from sputtered carbon and metal layers on SiO2 substrate has, for the first time, been achieved. The relationship between growth parameters and the quality of resultant graphene layer has been systematically studied. In addition, a growth model based on the detailed analysis of morphological structures and properties of graphene film was simultaneously proposed. Optical microscopy, Raman spectroscopy and atomic force microscopy were used for the evaluation of the number, the quality and the morphology of resultant graphene layers in each method. To investigate the performance of graphene electronic devices, field effect transistors were fabricated using both exfoliated and chemical vapour deposited graphene. A novel technique for graphene patterning has been developed using deep ultraviolet baking and an improved photolithography method. A new shielding technique for the low damage deposition of Au electrodes on graphene has also been developed in this project. The practical challenges of device fabrication and performance optimisation, such as polymer residue and contact formation, have been studied using Raman spectroscopy and the Keithley 2602A multichannel source meter. For the functionalisation of graphene, a number of chemicals were investigated to provide linking groups that enable binding of bio-probes on the graphene surface. Hydrogen peroxide and potassium permanganate have been demonstrated to have the capability of immobilising oxygen-containing groups onto graphene. The levels of oxidation were estimated by energy dispersive analysis and Fourier transform infrared spectroscopy. In addition, aminopropyltriethoxysilanes and polyallylamine have exhibited good efficiency for immobilising amino groups onto graphene. The resultant graphene was characterised by X-ray photoelectron spectroscopy and cyclic voltammetry measurements. Graphene electrodes modified with electrochemically reduced graphene oxide were developed for the first time which exhibit significantly improved redox currents in electrochemical measurements. Using single stranded DNA immobilised via π-π bonds as probes, these electrodes showed a limit of detection of 1.58 x 10-13 M for the human immunodeficiency virus 1 gene. In parallel, human chorionic gonadotropin sensors were developed by immobilising its antibodies on 1-pyrenebutyric acid N-hydroxysuccinimide ester functionalised graphene field effect transistors. These field effect transistors have been demonstrated to exhibit a quantitative response toward the detection of 0.625 ng/ml antigen. In summary, the fabrications of two types of graphene-based biosensors for the detection of specific DNA sequence and human chorionic gonadotropin have been achieved in this project. Their sensitivity, selectivity, reproducibility and capability of multiple biomarker detection need to be further improved and explored in future work. The outcomes of this project have provided not only ready-made biosensing platforms for the detection beyond these two targets, but also novel techniques applicable to the development of multidisciplinary applications beyond biosensor itself.

Thesis (M.S.)--University of Missouri-Columbia, 2007.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on April 14, 2008) Includes bibliographical references.

Bacterial sensing systems have evolved to detect complex biomolecules, operating near fundamental physical limits for biosensing. No modern engineered biosensor has managed to match the efficiency of bacterial systems, which optimize for each sensing application under constraints on response time and sensitivity. An emerging approach to address this shortfall is to build biosensors that electronically couple microbes and devices to combine the sensing capabilities of bacteria with the communication and data processing capabilities of electronics. This dissertation presents three techniques that advance engineering at the interface between bacteria and electronics, all working towards the integration of living material into hybrid biosensing platforms. In the first technique, we embed current-producing Shewanella oneidensis inside a conductive PEDOT:PSS matrix to electronically interface and structure the bacteria into 3D conductive biocomposite films to our specifications. In the second technique, we observe large numbers of chemotactic bacterial flagellar motor (BFM) behavior to infer environmental conditions, using machine learning to co-opt Escherichia coli's motor response for the front end of a biosensor. In the final technique, we demonstrate progress towards a method to electronically monitor BFM rotation over time for electrochemical biosensing. Together, this body of work contributes to more functional interfaces between silicon- and carbon-based materials for advanced biosensing applications including persistent in situ environmental sensing and microbiorobotics.

The microwave dielectric response of biological solutions and electrolytes has been investigated for a number of decades though applications that utilise the response are few and far between. The dielectric features of many biological fluids are unique across the microwave spectrum and offer a wealth of possibilities for analysis techniques. This thesis documents the development of broadband and resonant microwave techniques that are suitable for applications in biological fluid analysis. Theoretical models concerning the dielectric properties and electromagnetic interaction with polar liquids such as water are examined. The means to conduct experimental observations of the dielectric spectrum of liquids are reviewed and the ability to conduct measurement on small sample volumes discussed. Broadband spectroscopy from 0.2 to 20 GHz has been performed on the simplest constituent of a biological fluid, water, and compared to literature and theoretical models. Other polar liquids such as ethanol, propanol and methanol were also examined. The impact of ions in solution on the high frequency permittivity was studied, in particular the response of alkali metal chlorides, copper sulphate and zinc sulphide. The temperature dependence of the metal chlorides was found to be highly dependent on the effective hydration radius and subsequently a means of calculating the temperature-dependent hydration radius of lithium and sodium was developed. The respective radii at room temperature were found to be 340 ±39 pm and 215± 21 pm. Relaxation processes from ion-association were examined and confirmed to be present in ions with high charge density. Comparative studies between various biological solutes in aqueous environments demonstrated that many proteins possess unique microwave dielectric spectral features based on bound water and protein-water exchange mechanisms. Two techniques for the differentiation of protein solutions are outlined based on the microwave dielectric spectrum and the relaxation processes associated with protein water. Broadband measurements were conducted from 0.5 to 40 GHz to analyse the dielectric response of whole blood and serum from human and murine donors. Based on the dielectric comparison of serum and whole blood a method for the determination of haemoglobin concentration is presented. A 9.4 GHz dielectric resonator was developed with an integrated microfluidic chip for the determination of haemoglobin concentration in samples as small as 2 microlitres. This was subsequently utilised to monitor the progression of haemoglobin levels in APCmin/+ mice with colon cancer. The results demonstrate the first microwave device with proven haematological diagnostic value with an accuracy that is equivalent to or better than existing commercial techniques (comparative standard deviation 0.85 g/dL to Sysmex system - commercial comparison >1.5 g/dL) and is non-destructive.

Photonic crystals have been shown to be a promising technology for improving the performance of light emitting diodes, solar cells and optical communication components. More recently there has been interest in the application of photonic crystals for bio‐chemical sensing since they provide the potential benefits of high sensitivity, label free, real time detection with low limit of detection. Optical sensing mechanisms such as Surface Plasmon Resonance (SPR), and Evanescent Field (EF) sensing methods are currently popular. These are all sensitive to small changes in refractive index (RI) of part of the device. To date SPR methods provide the highest level of sensitivity but have the disadvantage of requiring an expensive gold coating. AROMA Sensor: As a high sensitivity, low cost alternative to conventional SPR methods, this thesis investigates a new concept for bio‐chemical sensing recently developed at Southampton, which uses vertical projection of leaky transmitted modes of a photonic crystal as the sensing method. We call this Angle Resolved Out‐coupled Mode Analysis (AROMA). This method is highly sensitive to small changes in refractive index at the sidewalls of the holes of a photonic crystal resulting in a strong angular shift of an out coupled beam of light. Changes in RI causes a shift in the projected spot position that can be recorded by a CCD/ CMOS camera. Sensor performance is shown to far exceed normal SPR. Simulation and experimental results demonstrate a sensitivity of 10 degree/RIU from a non‐optimised sensor and simulation results indicate an improved sensitivity of 6500 degree/RIU by optimising the sensor operating point. Responsivity of the sensor was investigated by sequentially depositing a series of sub nm ZnO layers, and was found to be highly linear. Photonic crystal coupler and system integration: Apart from the sensor, a new concept for light coupling is developed and optimised. We extend photonic crystal technology to create a combined light coupler/splitter component allowing arbitrary N‐channel light coupling to a simple slab waveguide device. The coupler is combined with multiple sensors to make a fully functional multi‐channel (4‐12 channels) sensor operating at 785nm. This is integrated into a high refractive index (n=1.7) Silicon Oxynitride (SiON) slab waveguide deposited onto a transparent borosilicate glass substrate. The aim for the slab waveguide was to mimic the refractive index of available polymer materials so that the entire system could eventually be fabricated on a flexible polymer substrate by nanoimprint lithography. Design and modelling: This thesis describes the design and optimisation of each component (sensor, coupler and slab waveguide), presenting in depth background physics and rigorous design methods for each component. 3D models were developed based on Rigorous Coupled Wave Analysis (RCWA) and Finite‐Difference Time‐Domain (FDTD) methods. RCWA models allowed accurate prediction and optimisation of light coupling and projection angles for any selected operating wavelength. FDTD methods allowed careful analysis of the interaction between the light field in the slab waveguide and materials placed in the holes. It also predicts the far‐field projected beam pattern for the sensor. Applications: Capability to detect (dry) monolayer coatings was proven for a simple self‐assembled monolayer molecule coating (p‐tolyltrichlorosilane (TTCS)) and also deoxyribonucleic acid (DNA) was successfully detected, close to physiological levels. To achieve this a complex hybridisation process was developed. Sensor response as a function of self‐assemble molecule (SAM) length and distance from the sidewalls was investigated in detail by using reversible chains of long chain charged molecules (lysine, poly‐lysine, bovine serum albumin protein). A detector surface with a layer of poly‐lysine‐g‐PEG was successfully replaced by a poly‐lysine molecule with larger molecule weight. Sequentially additional bovine serum albumin protein binding with the Polylysine was detected. Capability to detect biomolecules in an aqueous environment is intrinsically difficult for most bio‐sensors. By fabricating the device on a transparent glass substrate, and designing the device to project light backwards through the substrate, it became possible to detect small changes in refractive index for liquids placed on the exposed top surface with no detriment to the readout method. The bulk sensitivity of the sensor for liquids was evaluated by measuring a sequence of glucose solutions with increasing concentrations. A highly linear response was again observed.

Le dépôt de couche atomique (ALD) est devenu une technique essentielle de dépôt en phase vapeur de couches minces pour de nombreuses applications. La demande croissante de composants électroniques et de matériaux nanostructurés a fait de gls{ald} l'un des processus de fabrication clés du marché des nanotechnologies.Dans ce travail, nous présentons de nouveaux matériaux nanostructurés pouvant être utilisés comme transducteurs dans des dispositifs à biocapteurs. Ces matériaux ont été préparés en combinant gls{ald} avec des techniques "top-down" et "bottom-up" telles que la lithographie par nanosphère (gls{nsl}), le dépôt physique en phase vapeur (gls{pvd}), la gravure chimie assistée par des métaux (gls{mace}) et électrodéposition.En tant que premier candidat prometteur, des nanofils de silicium (gls{sinws}) recouverts de ZnO par gls{ald} ont été fabriqués. Ces structures 3D sont très attractives pour les applications de biocapteurs optiques en raison de leur activité intense de photoluminescence (gls{pl}) à température ambiante. Dans une première approche, ces nanostructures coe ur/coquille ont été entièrement caractérisées et testées en tant que capteurs possibles pour la détection du peroxyde d’hydrogène, qui est un produit de réaction courant de plusieurs oxydoréductases.De plus, des nanostructures creuses en ZnO semblables à des oursins recouvertes de Au ont été préparées avec une taille contrôlée en combinant NSL, gls{ald}, électrodéposition et évaporation par faisceau d'électrons (gls{ebeam}). L’influence de l’épaisseur du film Au sur les capacités de diffusion Raman (gls{sers}) améliorées en surface des substrats a été étudiée. Les structures optimisées ont été utilisées pour détecter des molécules de thiophénol avec une limite de détection (gls{lod}) de SI{e-8}{Molar}. De plus, l'adénine peut être détectée avec une concentration aussi basse que SI{e-6}{Molar}. L'excellente uniformité et la répétabilité lot par lot des substrats en font d'excellents candidats pour une détection et une biocapture SERS fiables.En outre, un groupe diversifié de nouveaux matériaux présentant des caractéristiques attrayantes qui peuvent être facilement appliqués à la détection, à la catalyse et à la plasmonique est présenté. Des nanoparticules bimétalliques de Pd/Au supportées sur gls{sinws} avec gls{ald} et un remplacement galvanique ont été fabriquées. De plus, des structures ZnO creuses de type urchin avec ZIF-8 par électrodéposition ont été fabriquées pour de possibles applications SERS
Atomic layer deposition (gls{ald}) has emerged as an essential vapor deposition technique of thin films for countless applications. The rising demand for electronic components and nanostructured materials has established gls{ald} as one of the key fabrication processes in the nanotechnology market.In this work, novel nanostructured materials that can be used as transducers in biosensor devices are presented. These materials have been prepared by a combination of gls{ald} with top-down and bottom-up techniques such as nanosphere lithography (gls{nsl}), physical vapor deposition (gls{pvd}), metal-assisted chemical etching (gls{mace}), and electrodeposition.As a first promising candidate, silicon nanowires (gls{sinws}) covered with ZnO by gls{ald} were fabricated. These 3D structures are quite attractive for optical biosensing applications thanks to their intense photoluminescence (gls{pl}) activity at room temperature. As a first approach, these core/shell nanostructures were fully characterized and tested as possible sensors for the detection of hydrogen peroxide, which is a common reaction product of several oxidoreductases.In addition, Au-covered hollow urchin-like ZnO nanostructures were prepared with controlled size by combining NSL, gls{ald}, electrodeposition, and electron beam (gls{ebeam}) evaporation. The influence of the Au film thickness on the surface-enhanced Raman scattering (gls{sers}) capabilities of the substrates was investigated. The optimized structures were used to detect thiophenol molecules with a limit of detection (gls{lod}) of SI{e-8}{Molar}. Additionally, adenine can be detected with a concentration as low as SI{e-6}{Molar}. The excellent uniformity and batch-to-batch repeatability of the substrates makes them excellent candidates for reliable SERS sensing and biosensing.Moreover, a miscellaneous group of novel materials with enticing features that can be readily applied in sensing, catalysis, and plasmonics is presented. Bimetallic Pd/Au nanoparticles supported on gls{sinws} with gls{ald} and galvanic replacement were fabricated. Furthermore, hollow urchin-like ZnO structures with ZIF-8 via electrodeposition were fabricated for possible SERS applications

Les nanoparticules dopées au lanthanide (LnNPs) sont devenues une classe importante de fluorochromes pour la biodétection et la bioimagerie avancées. Cependant, le développement de LnNP brillants, stables et bioconjugués reste un défi important pour leur traduction en applications bioanalytiques fonctionnelles. Ici, nous présentons la détection in situ de la liaison ligand-récepteur sur les cellules a été accompli avec des conjugués TbNP-anticorps (Matuzumab) qui pourraient se lier spécifiquement aux récepteurs transmembranaires du facteur de croissance épidermique (EGFR). Une spécificité et une sensibilité élevées ont été démontrées par l'imagerie temporelle de l'EGFR sur des lignées cellulaires exprimant fortement (A431) et faiblement (HeLa et Cos7) l'EGFR, alors que les lignées cellulaires non exprimantes (NIH3T3) et les cellules A431 passives de l'EGFR n'ont montré aucun signal. Malgré la taille relativement importante des conjugués TbNP-anticorps, ils ont pu être internalisés par les cellules A431 lors de leur liaison à l'EGFR extracellulaire, qui a montré leur potentiel en tant que marqueurs de luminescence brillants et stables pour la signalisation Des tests de stabilité des TbNP sur une gamme de valeurs de pH, de tampons différents, dans le temps, et d'effet de sonication sur ces NPs ont été effectués en utilisant la DLS et la ZP
Lanthanide-doped nanoparticles (LnNPs) have become an important class of fluorophores for advanced biosensing and bioimaging. However, developing bright, stable, and bioconjugated LnNPs remains an important challenge for their translation into functional bioanalytical applications. Here, we present in-situ detection of ligand-receptor binding on cells, which was accomplished with TbNP-antibody (Matuzumab) conjugates that could specifically bind to transmembrane epidermal growth factor receptors (EGFR). High specificity and sensitivity were shown by time-gated imaging of EGFR on both strongly (A431) and weakly (HeLa and Cos7) EGFR-expressing cell lines, whereas non-expressing cells did not show any signals. Despite the relatively large size of TbNP-antibody conjugates, they could be internalized by A431 cells upon binding to extracellular EGFR, which showed their potential as bright and stable luminescence markers for intracellular signaling. Stability tests of TbNPs over a range of pH values, different buffers, over time, and sonication effect on these NPs were performed by using dynamic light scattering (DLS) and zeta potential (ZP) measurements

Plasmonics could bridge the gap between photonics and electronics at the nanoscale, by allowing the realization of surface-plasmon-based circuits and plasmonic chips in the future. To build up such devices, elementary components are required, such as a passive plasmonic lens to focus free-space light to nanometre area and an active plasmonic modulator or switch to control an optical response with an external signal (optical, thermal or electrical). This thesis partially focuses on designing novel passive and active plasmonic devices, with a specific emphasis on the understanding of the physical principles lying behind these nanoscale optical phenomena. Three passive plasmonic devices, designed by conformal transformation optics, are numerically studied, including nanocrescents, kissing and overlapping nanowire dimers. Contrary to conventional metal nanoparticles with just a few resonances, these devices with structural singularities are able to harvest light over a broadband spectrum and focus it into well-defined positions, with potential applications in high efficiency solar cells and nanowire-based photodetectors and nanolasers. Moreover, thermo-optical and electrooptical modulation of plasmon resonances are realized in metallic nanostructures integrated with either a temperature-controlled phase transition material (vanadium dioxide, VO2), or ferroelectric thin films. Taking advantage of the high sensitivity of particle plasmon resonances to the change of its surrounding environment, we develop a plasmon resonance nanospectroscopy technique to study the effects of sizes and defects in the metal-insulator phase transition of VO2 at the single-particle level, and even single-domain level. Finally, we propose and examine the use of two-dimensional metallic nanohole arrays as a refractive index sensing platform for future label-free biosensors with good surface sensitivity and high-throughput detection ability. The designed plasmonic devices have great potential implications for constructing nextgeneration optical computers and chip-scale biosensors. The developed plasmon resonance nanospectroscopy has the potential to probe the interfacial or domain boundary scattering in polycrystalline and epitaxial thin films.

This thesis describes a novel approach to the fabrication and characterisation of metallic nanopores and their application for the detection of single DNA molecules. Metallic nanopores with apparent diameters below 20 nm are produced using electrochemical deposition and real-time ionic current feedback. Beginning with large nanopores (diameter 100-200 nm) milled into gold silicon nitride membranes using a focused ion beam, platinum metal is electrodeposited onto the gold surface, thus reducing the effective pore diameter. By simultaneously observing the ion current feedback, the shrinking of the nanopore can be monitored and terminated at any pre-defined value of the pore conductance in a precisely controlled and reproducible way. The ion transport properties of the metallic nanopore system are investigated by characterising the pore conductance at varying potentials across the nanopore and concentrations of electrolyte. The results are compared to conventional bare silicon nitride nanopore systems. Chemical modification at the nanopore surface is also studied using thiolisation to reduce the capacitive charging effects observed with metallic nanopores. Further to this, impedance measurements are carried out to study the resistive behaviour exhibited in these systems. An equivalent circuit model is proposed to validate the results obtained from the experimental studies. To evaluate the suitability of these nanopores for applications in single-molecule biosensing, translocation experiments using λ-DNA are performed. DNA molecules are electrokinetically driven through the nanopore under an applied electric field, hence as the DNA translocates through the pore, current blockade events are detected. Each event is the result of a single molecular interaction of DNA with the nanopore and is characterised by its dwell time and amplitude. Characterisation studies and noise analysis towards the applicability of metallic nanopores as single molecule detectors are also studied and compared to current bare silicon nitride pore systems.

Thesis (Ph.D.)--Boston University
The large electromagnetic field enhancement provided by nanostructured noble metal surfaces forms the foundation for a series of enabling optical analytical techniques, such as surface enhanced Raman spectroscopy (SERS), surface enhanced IR absorption spectroscopy (SEIRA), surface enhanced fluorescent microscopy (SEF), to name only a few. Critical sensing applications have, however, other substrate requirements than mere peak signal enhancement. The substrate needs to be reliable, provide reproducible signal enhancements, and be amenable to a combination with microfluidic chips or other integrated sensor platforms. These needs motivate the development of engineerable SERS substrate "chips" with defined near- and far-field responses. In this dissertation, two types of rationally designed SERS substrates - nanoparticle cluster arrays (NCAs) and SERS stamp - will be introduced and characterized. NCAs were fabricated through a newly developed template guided self-assembly fabrication approach, in which chemically synthesized nanoparticles are integrated into predefined patterns using a hybrid top-down/bottom-up approach. Since this method relies on chemically defined building blocks, it can overcome the resolution limit of conventional lithographical methods and facilitates higher structural complexity. NCAs sustain near-field interactions within individual clusters as well as between entire neighboring clusters and create a multi-scale cascaded E-field enhancement throughout the entire array. SERS stamps were generated using an oblique angle metal deposition on a lithographically defined piston. When mounted on a nanopositioning stage, the SERS stamps were enabled to contact biological surfaces with pristine nanostructured metal surfaces for a label-free spectroscopic characterization. The developed engineered substrates were applied and tested in critical sensing applications, including the ultratrace detection of explosive vapors, the rapid discrimination of bacterial pathogens, and the label-free monitoring of the enzymatic degradation of pericellular matrices of cancer cells.

In this thesis, a novel electrochemical array is reported. The array consists of two planar halves, each having four carbon screen-printed band electrodes (SPEs), orthogonally facing each other and separated by a spacer to yield 16 two-electrode electrochemical cells with 1 mm² working electrode areas. The 16 counter electrodes were converted to Ag/AgCl by electrodeposition and anodization. These electrodes were stable for at least 30 days with potentials under the current densities used in our experiments. The 16 working electrodes were modified by Au electrodeposition, and were examined by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS).

Immobilization strategies for biomolecules are of paramount importance for successful fabrication of biosensors. This thesis reports a new immobilization method that is based on patterned deposition of alkyl thiosulfates (Bunte salts). Monolayers were formed through electrochemical oxidation of Bunte salts at Au-modified electrodes. Single-component and mixed monolayers were investigated, where the mixed monolayers involved one component with a terminal carboxylic acid functional group to allow immobilization of biomolecules.

Applications of the newly developed immobilization method to an enzyme-based biosensor and an immunosensor were investigated. Glucose and biotin were chosen as model analytes, respectively. Glucose oxidase (GOx) and avidin were covalently immobilized onto the mixed-monolayer-modified electrodes through the carboxylic acid groups. Under the optimized conditions for the fabrication and operation of the biosensors, the new electrochemical array showed linearity up to 10 mM glucose with a sensitivity of 4. 7 nA mM^-1 and a detection limit of 0. 8 mM (S/N=3), and linearity up to 12. 8 µM biotin with a detection limit of 0. 08 µM (S/N=3).

Conducting polymers have emerged as a promising material for optoelectronics and chemical sensing application. Polyaniline (PANI) is a conductive polymer which can be easily functionalized to be specific for various biomolecules and has ideal sensor characteristics. The protonation and deprotonation of the polyaniline’s backbone by derivatization can result in color and conductive change responses. This makes it ideal for the construction of a real time, naked eye sensor. Derivatized polyaniline has previously been reported as a colorimetric sensor in solution. We plan to create a more practical sensor by synthesizing hydroxyl functionalized polyaniline thin films. In this study, we designed a process to functionalize polyaniline and deposit it as a thin film on quartz or silicon substrate via a dip coating process. To demonstrate the use of derivatized PANI in biosensing applications, derivatized and underivatized PANI thin films were treated with solutions of L-aspartic (Asp) acid at concentrations ranging from 10-8 mM to 103 mM and monitored utilizing UV-Vis spectroscopy. We found that the derivatized thin films change from deep blue to green color upon addition of Asp solution and showed a decrease in the characteristic quinoid ring peak at 600nm and the appearance of a new polaron peak at 425nm. The underivatized PANI films showed no colorimetric response indicating the hydroxyl functionalized PANI films are a more ideal material for a biosensing and naked eye detection. The polyaniline derivative was characterized using FT-IR spectroscopy, 1H NMR spectroscopy, UV-VIS spectroscopy, and Scanning Electron Microscopy. Additionally, conductivity studies were utilized to explore the material’s effectiveness as an electronic sensor using a 4-point probe to measure resistance.

The term "Lab-on-a-Chip" (LoC) describes highly miniaturized systems in which the functionalities of entire laboratories are scaled down to the size of transportable microchips. Particularly in the field of chemical and bio-analysis, such platforms are desired for a fast and highly sensitive sample analysis at the point of care. This work focuses on silicon nanowire (SiNW) based sensors. Innovative device fabrication concepts are developed from various directions, for a facile and reliable assembly of LoC analysis systems. Firstly, a multifunctional microfluidic set-up is developed which allows for a facile reversible sealing of channel structures on virtually any kind of substrate while maintaining the possibility of a rapid prototyping of versatile channel designs and the applicability of high working pressures of up to 600 kPa. Secondly, a 3-(triethoxysilyl)propylsuccinic anhydride (TESPSA) based surface modification strategy for the attachment of specific receptor molecules without additional binding site passivation is explored. Thirdly, bottom-up grown SiNWs are utilized for producing parallel arrays of Schottky barrier field-effect transistors (FETs) via contact printing. Using the initially developed microfluidic set-up, the concept of the TESPSA-based receptor immobilization is proved via fluorescence microscopy and by applying the SiNW FETs as biosensors. Using a receptor-analyte system based on a set of antibodies and a peptide from human influenza hemagglutinin, it is shown that antibodies immobilized with the developed method maintain the specificity for their antigens. The fourth major research field in this work is the microfluidics-based alignment of one-dimensional nanostructures and their deposition at predetermined trapping sites for reliably fabricating single NW-based FETs. Such devices are expected to provide superior sensitivity over sensors based on parallel arrays of FETs. Consequently, within this work, innovative LoC devices fabrication approaches over a broad range of length scales, from micrometer scale down to the molecular level, are investigated. The presented methods are considered a highly versatile and beneficial tool set not only for SiNW-based biosensors, but also for any other LoC application
Unter dem Begriff „Lab-on-a-Chip“ (LoC) fasst man stark miniaturisierte Systeme zusammen, die die Fähigkeiten eines ganzen Labors auf einen transportablem Mikrochip übertragen. Insbesondere im Bereich der Analyse chemischer und biologischer Proben werden solche Plattformen bevorzugt eingesetzt, da sie direkt am Ort der Probenentnahme schnelle, hoch sensible Messungen ermöglichen. Im Mittelpunkt dieser Doktorarbeit stehen Sensoren auf Basis von Siliziumnanodrähten (SiNWs). Auf verschiedenen Gebieten werden innovative Konzepte zur einfachen und zuverlässigen Herstellung von LoC Systemen entwickelt. Zu Beginn wird ein multifunktionaler Mikrofluidik-Aufbau vorgestellt, der ein einfaches reversibles Verschließen von Mikrofluidik-Kanälen auf nahezu allen möglichen Substraten erlaubt. Der Aufbau ermöglicht das schnelle Anfertigen und Testen verschiedener Kanalstrukturen sowie das Betreiben von Fluidik-Experimenten mit hohen Arbeitsdrücken von bis zu 600 kPa. Der zweite Schwerpunkt der Arbeit ist die Entwicklung einer Methode zur Funktionalisierung von Sensor-Oberflächen mittels 3-(Triethoxysilyl) Propyl Bernsteinsäure Anhydrid (TESPSA) für die Immobilisierung spezifischer Rezeptormoleküle. Bei dieser Methode entfällt die Notwendigkeit einer zusätzlichen Passivierung ungenutzter Anbindungsstellen. Des Weiteren erfolgt die Herstellung von Parallelschaltungen von Schottky-Barrieren-Feld-Effekt-Transistoren (SB-FETs) aus „bottom-up“ gewachsenen SiNWs durch mechanisches Abreiben der SiNWs vom Wachstumssubstrat auf ein Empfängersubstrat. Unter Verwendung des eingangs entwickelten Mikrofluidik-Aufbaus wird die prinzipielle Anwendbarkeit der TESPSA-basierten Rezeptor-Immobilisierung nachgewiesen, sowohl anhand von Fluoreszenzmikroskopie-Untersuchungen als auch mit Hilfe der SiNW FETs als Biosensoren. Mittels eines Rezeptor-Analyt-Systems, bestehend aus verschiedenen Antikörpern und einem Peptid des Influenzavirus A, wird gezeigt, dass Antikörper, die über TESPSA an Oberflächen gebunden werden, ihre Spezifizität für ihre Antigene beibehalten. Der vierte große Forschungsabschnitt dieser Arbeit widmet sich der mikrofluidischen Ausrichtung eindimensionaler Nanomaterialien und deren Ablage an vorgegebenen Fangstellen, wodurch eine zuverlässige Herstellung von FETs aus Einzelnanodrähten erreicht wird. Es wird davon ausgegangen, dass Einzelnanodraht-FETs gegenüber Parallelschaltungen von Nanodraht-FETs verbesserte Sensoreigenschaften aufweisen. Folglich beinhaltet diese Arbeit viele zukunftsweisende Ansätze für die Herstellung von LoC Systemen. Untersuchungen über eine Bandbreite von Längenskalen, von Mikrometer großen Strukturen bis hinab zur molekularen Ebene, werden präsentiert. Es wird davon ausgegangen, dass die vorgestellten Methoden als eine vielfältige Sammlung von Werkzeugen nicht nur bei der Herstellung von Biosensoren auf SiNW-Basis Einsatz finden, sondern ganz allgemein den Aufbau verschiedenster LoC Systeme vorantreiben

The object of this thesis, is to demonstrate the potential capabilities of injection moulded chiral plasmonic nanostructures for enhanced sensing in biological systems. The key phenomenon employed throughout this thesis is the generation of electromagnetic fields, that produce a greater chiral asymmetry than that of circularly polarised light, termed ‘superchiral’ fields. These superchiral fields will be demonstrated as being an incisive probe into the structure, conformation, and orientation of proteins immobilised on the nanostructure surface of these injection moulded substrates. Initially, it will be shown how this phenomenon is sensitive to higher order changes in protein structure induced upon ligand binding, using an asymmetry parameter extracted from the optical rotatory dispersion (ORD) spectra. Where these changes would not be routinely detected by conventional chiroptical spectroscopy techniques, such as circular dichroism (CD). Further to this, as these nanostructures display the plasmonic analogue of the interference effect, electromagnetically induced transparency (EIT), a narrow transparency window is created within a broad reflectance spectrum. Where the spectra can be modelled using a simple coupled oscillator model, and the retardation phase effects extracted. This allows two new asymmetry parameters to be introduced for characterising any changes induced by the biological samples, the experimental separation parameter ∆∆S, and the modelled retardation phase asymmetries. These will be used to characterise the orientation of three structurally similar protein fragments, called Affimers, with the modelled phase asymmetries being shown as a particularly incisive probe into the surface immobilised orientation. Furthermore, conformational changes in the cancer relevant protein, HSP90, will be characterised upon the addition of increasing concentrations of the inhibitor molecule 17-AAG. With the orientation of the immobilised HSP90 protein being shown to influence the sensitivity observed for any protein-ligand interactions that occur. Finally, this phenomenon will be used to quantitatively detect elevated protein levels in a complex solution. Elevated levels of IgG will be measured in human blood serum solutions, utilising the isoelectric point of the proteins in solution to enhance the level of IgG adsorbed in the protein corona. This will demonstrate for the first time, the use of superchiral fields generated around injection moulded chiral nanostructures, to detect protein changes in complex real life solutions, such as human blood serum. Representing the first step in creating a high-throughput ultrasensitive system for a range of diagnostic applications.

This thesis describes the realisation of devices and techniques based on evanescent field sensing using planar optical waveguides for mid-infrared (MIR) absorption spectroscopy, to provide bio-chemical sensing capabilities for medical diagnostics. The fundamental vibrations of bio-chemical molecules occur in the MIR region, where their absorption is orders of magnitude stronger than their overtone bands in the near-infrared making it suitable for highly sensitive and specific absorption spectroscopy. Realisation of waveguides is an essential step towards mass-producible and low-cost integrated lab-on-chip devices. Two chalcogenide compositions were used to make waveguides, germanium telluride (GeTe4) as waveguide core and zinc selenide (ZnSe) as waveguide lower cladding. Two approaches were followed for waveguide fabrication: GeTe4 waveguides on bulk ZnSe and GeTe4 waveguides on thin films ZnSe deposited on Si. High contrast (Δn ~ 0.9) GeTe4 channel waveguides on ZnSe were fabricated using photolithography and lift-off. Waveguiding was demonstrated for the wavelength range between 2.5 and 9.5 μm for GeTe4 channel waveguides on bulk ZnSe substrates. GeTe4 waveguides fabricated on Si with ZnSe isolation layers were characterised for waveguiding and propagation losses in the wavelength range between 2.5 and 3.7 μm. ZnSe rib waveguides were also fabricated on oxidised Si by photolithography and dry etching and were characterised for propagation losses in the wavelength region of 2.5-3.7 μm. Absorption spectroscopy of liquid mixtures absorbing in the MIR was performed on the surface of the waveguide and the results were compared with a theoretical model.

Monolithically integrating multiple sensing technologies shows a great potential to perform quantitative measurements for multiple biomarkers of diseases and also provide more insight towards one single biochemical event. The localised surface plasmon resonance spectroscopy measures the change in the refractive index arising from the molecular adsorption on the metallic nanostructures. Acoustic sensors, such as surface acoustic wave sensor and quartz crystal microbalance, measure the variation of its mechanical oscillation caused by the sum of the deposited molecules and the solvent coupled to the adsorbed molecules. Both techniques are known independently as having applications in in-situ, label-free sensing and analysis of biological binding reactions. Due to their complementary properties, the integration of both can prove to be a valuable tool for studying biomolecules on sensing surface. This thesis reports on the development of a hybrid biosensing device that integrates localised surface plasmonic sensing and acoustic sensing technologies. Gold nanodisk arrays as localised surface plasmon resonance sensing device was studied in visible region using three substrates: borosilicate glass, lithium niobate and quartz. The design, simulation, fabrication and characterisation of the gold nanodisk arrays, and the sensing performance optimisation were investigated using glass substrate. Lithium niobate, as a piezoelectric material has surface acoustic wave compatibility and this study can pave the way towards the development of hybrid sensing devices. The study on lithium niobate demonstrated the feasibility of a localised surface plasmon resonance device utilising a high refractive index, birefringent and piezoelectric substrate. Using quartz as the substrate, the design and fabrication of a hybrid sensor were performed, which integrated localised surface plasmonic resonance into a quartz crystal microbalance for studying biochemical surface binding reactions. The coupling of localised plasmon resonance nanostructures and a quartz crystal microbalance allows optical spectra and quartz crystal microbalance resonant frequency shifts to be recorded simultaneously, and analysed in real time for a given surface adsorption process. This integration has the potential to be miniaturised for application in point-of-care diagnostics.

This dissertation presents the synthesis and application of nanodiamond based materials for electrochemical biosensors. In this research work, nanodiamond particles have been used to prepare doped and undoped nanocrystalline diamond films, and conducting polymer composites for enhanced biosensing. The performance of the synthetized materials towards sensing applications was evaluated against glucose amperometric biosensing. Besides, cholesterol biosensing was attempted to prove the capabilities of the platform as a generic biosensing substrate. Biosensors have been proved to provide reliable detection and quantification of biological compounds. The detection of biological markers plays a key factor in the diagnosis of many diseases and, even more importantly, represents a major aspect in the survival rate for many patients. Among all of the biosensors types, electrochemical biosensors have demonstrated the best reliability to cost ratio. Amperometric biosensors, for example, have been used for decades as point of care sensing method to monitor different conditions such as glucose. Despite the amount the research presented, the sensitivity, selectivity, stability, low cost and robustness are always driving forces to develop new platforms for biosensor devices. In the first phase of this dissertation, we synthesized undoped and nitrogen doped nanocrystalline diamond films. The synthetic material was thoroughly studied using different material characterization techniques and taken through a chemical functionalization process. The functionalization process produced a hydrogen rich surface suitable for enzymatic attachment. Glucose oxidase was covalently attached to the functionalized surface to form the biosensing structure. The response of the biosensor was finally recorded following voltammetry and amperometric techniques under steady state and dynamic conditions. The experimental results demonstrated that conductivity induced by the doping process enhanced the sensitivity of the sensing structure with respect to the undoped substrate. Also, the functionalization procedure showed strong bonding to avoid enzyme leaching during the measurements. Later, in the second phase of this dissertation, the nanodiamond particles were used as filler for conducting polymer composites. The objective for developing these composite materials was to overcome the high resistivity observed for nanocrystalline films. The experimental results demonstrated that the inclusion of nanodiamond particles increased the sensitivity of the overall structure towards the quantification of glucose with respect to the nanocrystalline films and the bare polymer. Besides, the experiment showed a noticeable enhancement in the signal-to-noise ratio and the mechanical stability of the sensing platform due to the nanodiamond addition. The best structures from the previous experiments were further grafted with iron oxide nanoparticles to attempt signal amplification. Initial experiments with nanodiamond based composited showed similar current for low glucose concentrations for two different active electrochemical sensing areas. This observation indicates that more area is still available to transport signal and to enhance even further the sensing action. Oxidation of iron oxide nanoparticles after initial enzymatic decomposition of glucose has been proved to provide higher current for the same glucose concentration; thus, creating amplification effect for the signal. Finally, the toxicity of the nanomaterial synthesized during this dissertation was evaluated in mammalian cells. The advances in biosensing techniques indicate the potential application of amperometric platform for continuous implantable devices; hence, the toxicity of the materials becomes a key aspect of the platform design.

Molecular transport characterization is an active part of the research field in nanotechnology. In this interesting branch the self-assembly approach is highly exploited; it consists in spontaneous formation of highly ordered monolayers on various substrate surfaces. Self-assembled monolayers (SAMs) have found their applications in various areas, such as nanoelectronics, surface engineering, biosensing, etc. An important area in biosensing is the electrochemical detection, that enables sensing of dierent biomarkers with an important role, for many dierent applications in biomedical diagnostics or in monitoring of biological systems. Various test structures have been developed in order to carry out characterizations of self-assembled molecules, and numerous reports have been published in the past several years on the transport characteristics. This thesis' purpose is the single protein biomolecular sensing, that could become the starting point for monitoring drugs, developing clean energy systems, realizing bio-opto-electronic transistors... The possibility to cover so many fields is related to the kind of proteins, molecules, bioelements that will be inserted inside sensors. Biomolecular sensing has to be thought in order to reach a result with the better compromise between instrumentation versatility and measurements precision. The main underlying idea is to use single molecules as active elements in nano-devices. As a consequence, the proper realization of a molecule-electrode contact is a crucial issue. What is needed by author is something versatile, precize, cheap, at single molecule level and able to record measurements in few time in order to do statistical characterizations. The final goal of this work is a platform system adapt for both industry and research field. Electrical nanogap devices are the main character of this work. They have proven good performances as element for detecting small quantities of biomolecules, allowing direct transduction of biomolecular signals into useful electrical ones such as resistance/impedance, capacitance/dielectric, or field effect. Nanogaps are now one of the most busy area of research in the nanotechnology world. Moreover, these structures do not require feedback to maintain the mutual arrangement (comparing with conducting tip AFM) and are less stochastic with respect to electrochemical cells. Several techniques can be applied to nanogap fabrication: mechanically broken or positioned junctions, nano-scale lithography by Synchrotron radiation sources, electrochemical deposition and etching, and electromigration. None of these techniques is presently able to give precise control as to thefinal gap size. In this thesis the electromigration approach has been choosen, because of several useful characteristics. It is cost eective, because of the relatively low complexity of the required equipment. It can be embedded into a lab-on-chip system, thus exploiting the possibility to tailor the gap formation process by means of a digital loop control system. To this end, it just requires a conventional microchip fabrication process. It allows the parallelization with a smart packaging through which it is possible to produce more probes at the same time and perform many measurements in contemporary. The employment of nanogaps, as an instrumentation for the molecular charac- terization, has also some issues that have to be considered in order to obtain useful measurements. To characterize molecules the leakedge must be not higher than some pA to avoid the noise overcome the signal. Nanogap platform is perfect for molecular electronics. The experiments have been developed in dry way, as a consequence the solutions were evaporated before the measurement starting. This brought several problems cause biochemical analysis requires liquid solution in order to avoid an untimely death of the bio-elements tha has to be characterized. Considering a future developement, an improvement is necessary in terms of a system able to work with salty solutions without damaging the microchip's probes. Therefore it is a necessary a set-up allowing the anchorage of a microfluidic part. At the same time it is necessary to keep in mind that the presence of a new system has to not overcome the molecule signal, maintaining the leakedge under some tens of pA.

Singlemolecule experiments have been attracting interest since they can pave the way towards the realization of new molecular devices and biosensors. Molecular electronics could be an alternative to classical electronics to overcome the technologic dimensional limit of CMOS technology. On the other hand, biosensor downscaling can open to new detection techniques that are impossible at the conventional dimensional scale. Nowadays, single molecule experiments are mainly based on scanning probe techniques to manipulate and characterize molecules at the nanoscale. Although these techniques have revealed effective tools, real applications are restricted by problems of miniaturization, cost, integration and portability. Nanogap electrodes are an emerging new probing tool for single molecule experiments that can serve a function equivalent to classical probing systems, but guaranteing the integration and portability required in real applications. For these reasons, nanogap electrodes are the object of this thesis, from the fabrication to their employment in molecular electronics and biosensing.

2009/2010
Le nanotecnologie sono descrivibili come lo studio della manipolazione della materia con precisione atomica e molecolare, generalmente circoscrivibile a strutture di dimensione compresa tra 1 e 100 nanometri. Questo settore è molto vario e spazia dall’estensione della fisica convenzionale applicata all’ideazione di nuovi approcci basati sull’auto-assemblamento di molecole. Per questo motivo le nanotecnologie sono in continuo ampliamento e possono vantare applicazioni nei più svariati settori, come la medicina, lo sviluppo di nuovi biomateriali e l’elettronica. In questo lavoro di tesi riporterò degli esempi di come le nanotecnologie siano state impiegate nella progettazione di sistemi innovativi per la rilevazione di biomolecole o per il rilascio controllato di farmaci. La principale parte del lavoro svolto concerne la realizzazione di un biosensore basato sull’impiego di nanotubi di carbonio, per il rilevamento della palitossina. La nascita del progetto deriva dalla diffusione di un particolare tipo di microalga nel Mediterraneo e nelle coste italiane, comprese quelle del Friuli-Venezia-Giulia, produttrice di palitossina duranti i periodi di fioritura. Questa tossina marina è caratterizzata da una forte tossicità e ha infatti registrato numerosi casi di ricovero ospedaliero in bagnanti esposti all’aerosol contaminato durante attività ricreative balneari. Dal momento che il biosensore è concepito per raggiungere la massima sensibilità possibile, immunochimica ed elettrochemiluminescenza sono state combinate in un sistema ibrido che soddisfacesse questo requisito: la capacità unica degli anticorpi di legare specificamente il loro antigene, insieme all’eccellente sensibilità ottenibile dai trasduttori basati sul rilevamento della luminescenza, rappresenta il punto chiave per poter rilevare quantità di analita nel range del picogrammo. Al fine di ottimizzare riconoscimento tra anticorpo e antigene e segnale di risposta del biosensore, occorre avere a disposizione un elemento che predisponga al meglio la comunicazione tra elementi biologici e componenti elettrochimiche del sistema. I nanotubi di carbonio sono ottimi candidati per questo scopo, in virtù delle loro peculiari caratteristiche, come l’alto rapporto area superficiale-peso e la versatilità nella funzionalizzazione, che li rendono particolarmente adatti per il legame con bio-macromolecole, come gli anticorpi. I nanotubi di carbonio sono stati funzionalizzati per predisporre al meglio il legame con l’anticorpo anti-palitossina e successivamente sono stati legati covalentemente a un elettrodo di ITO. Un immuno-sandwich è stato costruito sull’elettrodo aggiungendo la tossina, seguita da un anticorpo secondario legato a un’etichetta fosforescente. Il fluoroforo è stato eccitato indirettamente tramite l’applicazione di uno specifico potenziale all’elettrodo al fine di ottenere l’emissione di luce. Dal momento che la luminescenza ottenuta è proporzionale alla quantità di tossina riconosciuta dall’anticorpo, la rilevazione quantitativa della palitossina è possibile tramite la costruzione di una retta di calibrazione. La seconda parte del lavoro è riportata nell’ultima sezione della tesi e riguarda la realizzazione di matrici di silicio poroso (PSi) per il rilascio controllato di farmaci. L’idea è quella si sfruttare le proprietà peculiari di questo materiale, come la vasta area superficiale, la biocompatibilità e la possibilità di essere monitorato in-vivo, per il trasporto di farmaci all’interno del corpo umano. Inoltre, il PSi presenta una particolare cinetica dissolutiva in condizioni fisiologiche simulate, proporzionale alla basicità della soluzione tampone. Questa caratteristica aggiuntiva è di grande interesse per il trasporto di quei farmaci che, facilmente solubili a pH gastrico, risultano poco assorbiti a livello intestinale. Campioni di silicio poroso con diverse porosità sono stati fabbricati attraverso un processo elettrochimico, funzionalizzati e dissolti in diversi tamponi fisiologici, al fine di identificare il candidato migliore per le prove di caricamento del farmaco. Il caricamento del principio attivo è avvenuto attraverso l’impiego della CO2 supercritica e le matrici sono state infine caratterizzate tramite calorimetria differenziale a scansione. Entrambi i nano-sistemi investigati hanno prodotto risultati interessanti, specialmente dal punto di vista della riproducibilità e dell’attendibilità dei dati.
Nanotechnology is the study of manipulating matter on an atomic and molecular scale, generally dealing with structures sized between 1 to 100 nanometre. Nanotechnology is very diverse, ranging from extensions of conventional device physics to completely new approaches based upon molecular self-assembly. It represent a fast-growing research field, due to the potential applications in a wide range of domains, such as in medicine, biomaterials and electronics. In this thesis I will give you some examples of how nanotechnologies have been exploited to the development of novel systems for biosensing and drug delivery. The main part of the thesis work is focused on the realization of a carbon nanotubes (CNTs)-based biosensor for palytoxin detection. The necessity to develop the sensing device arises from the diffusion of particular microseaweeds in the Italian coasts, Friuli-Venezia-Giulia included, producing palytoxins during bloom events. This marine toxin present remarkable toxicity and has already recorded several cases of hospitalization cases from patients exposed to the marine aerosol. Since the biosensor is conceived in order to be as sensitive as possible, we have combined immunochemistry and electrochemiluminescence in a hybrid system. The unique capacity of antibodies to bind specifically the analyte of interest, and the excellent sensitivity afforded by luminescence-based transducers, were coupled together in order to detect analyte quantities in the range of the picogram. The crucial point in reaching this aim is arranging biological elements with an electrochemical component, in order to optimize the immuno recognition between antibody and antigene and at the same time the response signal from the biosensor. To this aim, carbon nanotubes are excellent candidates due to the high surface area-to-weight-ratio and to the versatility in functionalization, making them suitable for attachment of biomolecules such as antibodies. Carbon nanotubes functionalized with specific antibodies anti-palytoxin are covalently attached to the electrode. An immuno sandwich is build on the electrode by adding the toxin, followed by a secondary antibody labeled with a fluorophore. The fluorophore is excited at certain voltages, in order to produce an emission of light. Since luminescence produced by the label is proportional to the amount of toxin recognized by the antibody, quantitative detection of palytoxin is achievable by constructing a calibration line. The second part of the work presented in the last section of the thesis concerns the realization of porous silicon (PSi) matrix for drug delivery. The idea was to exploit the very peculiar properties exhibited by this material, such as wide superficial area, biocompatibility and in-vivo monitoring, to carry drugs inside the human body. Furthermore, PSi showed a particular dissolution behaviour in simulated physiological conditions, proportional to the basicity of the buffer solution. This additional peculiarity is of great interest for the delivery of those drugs that cannot be absorbed through the intestine since they dissolve at the acid pH of the stomach. PSi samples with different grade of porosity were fabricated through an electrochemical procedure, functionalized and dissolved in different physiological buffers, in order to identify the most suitable one for drug loading experiments. Loading with the drug was performed through supercritical CO2 and the silicon carriers characterized by differential scanning calorimetry. Both of the nano systems investigated produced very interesting results, especially concerning the reproducibility of the devices and the reliability of the results obtained.
XXIII Ciclo
1983

A health condition called “Oxidative Stress” (OS), resulting from an excessive level of Reactive Oxygen Species (ROS) is a “state harmful to the body, which arises when oxidative reactions exceed antioxidant reactions because the balance between them has been lost”[1] OS appears to be associated with and might be a cause of, many serious diseases such as cardio-vascular accidents, cancer, Parkinson’s and Alzheimer’s[2]. This is not surprising as ROS are free oxygen radicals that can attack lipids, proteins, cellular membranes, enzymes and even modify DNA. Extensive correlation studies have shown that the complex impedance spectrum of blood samples from patients diagnosed with an OS syndrome differs significantly from the spectra obtained from the blood of healthy people, which is quite normal as the presence of an excessive amount of ROS should affect the physico-chemical properties of a blood sample. Measuring the complex impedance spectrum of a blood sample can be done quickly by means of low-cost electronic devices, making possible and affordable the early detection of OS among a large population. In order to quantitatively evaluate the OS, the impedance spectra being insufficient, the concentration of oxidative stress markers such as hydrogen peroxyde, malondialdehyde or F2 isoprostanes needs to be measured. Such measurements can, for instance, be used for monitoring the severity of a disease during a treatment. These concentration measurements are traditionally based upon analytical techniques but recently biosensors acting as transducers transforming directly a specific biochemical reaction into a measurable signal have been developed. They are essentially obtained by modifying the surface of metal or carbon electrodes using biomaterials such as enzymes antibodies or DNA that allow bindings or catalytic reactions with other specific biomaterials to occur on the surface of the electrodes. The resulting modifications of the electrical properties of the medium separating the electrodes can be analyzed through ad-hoc electronic and signal processing systems to yield the desired concentration. Biosensors have the advantages of rapid analysis, low-ost and high-precision. They are widely used in various fields, such as medical care, disease diagnosis and food analysis [3]. Hydrogen peroxide (H2O2) generated by cellular processes directly via two-electron reduction of molecular oxygen or indirectly via dismutation of superoxide, is the most widely studied ROS and its overproduction results in OS. Therefore, an ability to quantify the level of hydrogen peroxide and by ricochet the assessment of oxidative stress can be useful in order to assess certain health conditions occurring inside the body and as a result, an integrated electrochemical biosensor coupled with the hydrogen peroxide quantification can become a practical solution as a point of care device at home[4] Most of the time, H2O2 biosensors are based on HRP (Horseradish peroxidase) which is the most commonly used enzyme in the design of biosensors that can supervise the activity of oxidases and determine in terms of concentration, oxidase substrate such as lactate oxidase, cholesterol oxidase, or glucose oxidase, which all induce the production of hydrogen peroxide (HRP’s substrate). In the first part of this research, we explore the development of low-cost and compact measurement systems aiming to determining the impedance of biological samples as they grant access to information from electrical cellular characteristics. It is indeed possible to measure capacitance or conductance that are dependent on the health state of cells. The development of such measurement systems allowing the portability of biological essays requires sensitive electronics. Afterward, in the second part of our work, we explore the design of an electrochemical biosensor by immobilizing an enzyme (HRP) onto the surface of golden electrodes in order to detect and assess the analyte, hydrogen peroxide (H2O2). We also discuss the design of a potentiostat readout circuit to measure and convert the biosensor’s current. The combined results of the two parts of this work can be considered as a first prototype of a low cost and robust instrument easy to use in the field, away from a biological laboratory, with the goal of reaching the so called “point of care diagnostic” [5] The present thesis is organized as follows: Chapter I, introduces the present thesis. In Chapter II, we provide an overview in the field of biosensing technology. Chapter III deals with the design of a portable EIS measurement system to investigate reactive oxygen species in blood. Chapter IV presents an improved version of the previously designed instrument. Moreover, it points out the significance of EIS-based blood analysis through relevant medical diagnosis parameters such as hematocrit and erythrocyte sedimentation rate, extracted from the measured impedance spectra. In Chapter V we discuss on one hand the design of the H2O2 biosensor, and on the other hand the realization of the front-end circuit of the amperometric sensor. Finally, in Chapter VI, a conclusion is drawn..

The promoter region of the katG gene of Escherichia coli has been fused to two reporter genes GFPuv, encoding green fluorescent protein derived from Aequorea victoria and luxCDABE, encoding bacterial luciferase, from Photorhadbus luminescens to compare the qualities of these two reporters in microbial biosensor applications. In Escherichia coli both reporter systems produce stable signals. The lux construct was more sensitive at lower concentrations of hydrogen peroxide and the response time was shorter when compared with GFPuv. The latter, however, was better able to sense oxidative stress at concentrations that impaired signal output in the E. coli lux system. Low level non-induced bioluminescence was observed using the P. luminescens reporter system and this was utilised to measure EC50. As many compounds produce an increase in luminescence when incubated with this system, there is no means of specifically identifying any oxidative pollutants in the unknown sample. The system is limited to compounds that produce oxidative stress. Here we describe a system to add specificity to the stress-response whole-cell biosensor using glucose oxidase, which produces from glucose, hydrogen peroxide and gluconate. On incubation of these two adjuncts, glucose and glucose oxidase, with the pkatGlux whole cell biosensor, we found that the system was specific for glucose and had a range of sensitivity from 2 to 12 mM glucose. We propose that by adding glucose oxidase to the oxidative stress whole cell biosensor the specificity of the oxidative stress response can be increased, and by adding other oxidase enzymes the range of compounds that can be detected is expanded. There are enzymes of which the products of metabolism include glucose, beta-galactosidase converts lactose into glucose and galactose. The enzymes, beta-amylase and beta-amlyglucosidase digest starch to produce glucose and cellulases that act on cellulose to liberate glucose. Glucose oxidase then converts glucose to hydrogen peroxide and gluconate, the latter of which induces an increase in luminescence from the E. coli lux system. Thus it is possible to further develop the theme of adding in specificity to the stress response whole cell biosensor in the use of dual enzyme systems, where the first enzyme acts on the first substrate to yield glucose on to which glucose oxidase can metabolise, to yield hydrogen peroxide. If pkatGlux is incubated with a dual enzyme system then the number of compounds that can be biosensed can be increased and a greater specificity introduced. Samples may originate from lake, river or soil samples. These will not be 'clean'; they may contain organic debris, dirt and other bacteria that could interfere with the biosensing process. To this end lake and soil samples were spiked with substrates to see if direct sensing is possible, without the need for sample preparation. It was indicated that biosensing could take place in samples that originated from an aqueous environment. Where there were high levels of soil present, luminescence signal was quenched, which was restored on extraction of the substrate with appropriate solvent.

As a self-assembled mimetic structure of biological membranes, polydiacetylene liposomes have been studied for the development of platforms for various applications including nano-containers, nano-transporters, and nano-delivery systems for biological-, life- and materials-science applications. Liposomes incorporating amphiphilic polymer poly(10, 12 pentacosadiynoic acid) (PDA) was used as a building block for investigations mimicking cellular reaction and processes at the membrane cell. Changes in local membrane micro-organization and packing as a result of biomolecular and bioparticle reactions and processes at the liposomal membrane were investigated through the use of colorimetric and emission responses of PDA liposomes in solution phase. My dissertation comprises of six chapters. I provide brief overview of each chapter in the following paragraphs: Chapter 1: Introduction. In this chapter, an introduction is given on structure and function of lipid bilayer and multilayer of liposomes from a perspective of shared features with biological membranes. Amphiphilic molecules along with natural lipids at (or higher) critical micelle concentration self-assemble in aqueous medium, thereby, forming a lipid bilayer or multilayer to reduce the free energy of the system. When one of the components of the lipid bilayer is a polymerizable monomer, micelles/liposomes with enhanced mechanical and chemical stability are achieved. The lipid bilayer of liposomes is a boundary that includes at least three different regions: inside aqueous cavity, hydrophobic membrane zone, and membrane-aqueous interfaces. The membrane surface is available for further functionalization. In general, all three regions of the liposomes are utilized for both fundamental and applied studies. For example, the PDA liposomes have been employed for biosensing, drug/protein/nucleic acid transport and delivery and target release, and various probing cellular-like reactions and processes at the membranes. Here, in this chapter, literature on PDA was reviewed for a time period of 2008-2015. Furthermore, emphasis was given to application of PDA liposomes as (bio) sensing elements utilizing colorimetric, fluorescence, and FRET mechanisms. Chapter 2. Polydiacetylene (PDA) liposomes have been accepted as attractive colorimetric bionanosensors. The molecular recognition elements, either embedded within the liposomal membrane or covalent bound at the membrane surface, are available for interaction with biological and chemical analytes. Usually, PDA liposomes perform transduction activity through perturbation of the conjugated polymer backbone, which provides a colorimetric change in solution or solid-state phase. Here, we report that trapping self-quenched fluorescent specie within inner cavity of the liposomes is a simple and effective analytical tool for evaluating biomolecular binding events at the membrane surface. The release of fluorophores in response to the membrane binding event led to amplified emission signal which was utilized for probing reactions at the membrane surface that mimics reactions occurring at the cellular membrane surface. Specifically, a covalent binding on enzyme-substrate reaction resulted in a change of membrane fluidity, thereby releasing inner fluorophore content of the PDA liposomes. Fluorescent markers were loaded at or higher self-quenched concentration in the cavity of the liposome. Amplification of the fluorescence intensity was positively correlated with the concentration of protein added in the solution. The bilayer fluidity alteration also appears to depend on the molecular weight of the protein bound at the membrane. Overall, binding of protein with membrane promoted changes in the local PDA membrane organization and packing that enhanced the membrane permeability. The encapsulated content therefore leaked through “transient pores” formed in the membrane yielding substantial emission amplification. Chapter 3. Inspired by stability of the PDA liposomes, surface functionalization with a variety of molecules and loading within bilayer and inner cavity of the liposomes, we utilized liposomes as biocatalytical nanoreactors. Removable template molecules were embedded in the lipid bilayer and active protein encapsulated in the internal cavity was used for studying the transport properties of liposomes through substrate-enzyme reactions. Detergent Triton X-100 was used to remove a small portion of lipid and template molecules embedded in the membrane. The removal of lipid/template molecules not only affected the membrane fluidity but also provided transient pores in the membrane, allowing transport of substrate for enzymatic oxidation of glucose and 2-deoxy-glucose. Three important biological-relevant properties of cellular membrane: transport, bioavailability, and bio-reactivity of enzyme and substrate were studied. We found that enzyme molecules retained their reactivity when encapsulated within the aqueous inner cavity of the PDA liposomes, and that their activity was comparable to that in the bulk solution. Chapter 4. This chapter introduces studies on (at least partially) answering important questions how and if anchored enzyme activity at the liposome surface is affected through limited diffusion and spatial constraints. A further crucial question was investigated what effect of protein binding at the surface of the liposomes to enzymatic activity was. These relevant questions were important for increasing our fundamental knowledge related to reactions, interactions, and transport processes in biological cellular systems. A functionalized liposome system containing enzyme (Trypsin) covalently attached at the PDA liposome surface was synthesized. Using PDA liposomes as an immobilization scaffold, we evaluated and compared the cleavage behaviors of Trypsin in either immobilized at the membrane surface or in the free form. The covalent binding interaction and tryptic cleavage at the membrane-water interface was monitored by UV-vis and fluorescent spectroscopy, fluorescent anisotropy and spectro-micro-imaging. Trypsin binding at the membrane appeared to be significantly affected the enzymatic activity of the bound enzyme as seen from colorimetric response of the PDA liposomes. Chapter 5. Hierarchical structures support structures with new functionalities, therefore, advances in fabrication and characterization of biomimetic systems based on biological building blocks may present substantial potential rewards in material science. We take advantage of non-covalent forces known in biology for creating spatial organization by assembly tobacco mosaic virus-liposome polymeric hierarchical systems through biotin-streptavidin linkages. The advantage of using the biological thin rods such as TMV is that it can span the whole liposomal membrane allowing us to create microscopic hinge structures that connected liposomes. Our findings through electron and fluorescence microscopy confirmed that SA-TMV motif was able to stay inserted within the lipid bilayer of liposomes which yielded hierarchical structures after binding with Bt-liposomes. These hierarchical structures may find potential applications in targeted load (drug/protein/DNA) delivery, investigations involving virus-cell interactions, and sensing of virus particles. Chapter 6. Conclusions and Future work The present work in this dissertation utilized exploitation of biological self-assembly of small lipid molecules and larger biological-like motifs for enhancing our understanding of reactions and processes occurring at the cellular membrane surface. Overall the following four major studies were accomplished; 1. Sensing through amplified delivery, 2. Triggering an encapsulated bioreactor system at nanometric size, 3. Holding active biological elements when liposomes perform an attachment matrix, 4. Formation of hierarchical structures promoted by self-assembling of biological motifs with mimickers of cell membrane From our findings by mimicking the lipid bilayer of cell structures through liposomal membrane future work holds different ways to contribute in enhancing fundamental understanding of biological behavior. Active transport is an important function of all natural cells, playing important roles in intercellular communication. Liposomes composed of natural and polymerizable lipids may allow investigation involving exocytosis, formation of filopodia, vesicle fusion, budding and reproduction of neural synapses. Our liposome system may also mediate a broader range of highly selective and sensitive detection and sensing of cellular reactions and processes in physiological condition. I hope that this work in collaboration with multiple PIs will contribute to the fields at the interface of biology and material science.