The modification of electrodes is always an important task in electrochemical detection of electroactive and biological molecules. Chemically modified electrodes can offer improved selectivity and sensitivity for the target analyte, which greatly enhance the electrode performance. Various materials such as conducting polymers, metal nanoparticles and carbon nanomaterials have been exploited and widely used for the modification of electrodes. Electrochemical or spontaneous deposition, electrostatic adsorption, layer-by-layer self assembly and covalent binding have also been developed for electrode modification and offer improved performance. Both Prussian blue (PB) and toluidine blue O (TBO) are excellent redox mediators and very popular in electrode modification. PB has shown strong catalytic property for the reduction of hydrogen peroxide, but the application in biosensor fabrication is limited for its instability at neutral pH. Graphene, as a single-atom-thick carbon material, is considered an ideal platform for designing composite nanomaterials for high-performance electrochemical or electrocatalytic devices. The combination of PB with reduced graphene oxide (RGO) and poly(toluidine blue O) (PTBO) will greatly improve the stability of PB. An amperometric biosensor based on glassy carbon (GC) electrode modified with reduced graphene oxide, PB and poly(toluidine blue O) was developed. Experimental results showed that the GC/RGO/PB/PTBO modified electrode offered an excellent electrocatalytic activity toward the reduction of hydrogen peroxide due to the possible synergistic effects of the PB-PTBO composite material. After codeposition of glucose oxidase (GOD) and chitosan (CHIT) coating, the resulting GC/RGO/PB/ PTBO/CHIT-GOD electrode exhibited excellent response to glucose with a sensitivity of 59 mA M1 cm2, a low detection limit of 8.4 μM and a linear range from 0.02 to 1.09 mM at a detection potential of +0.2 V vs. Ag.

Made available in DSpace on 2014-06-11T19:29:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-01-28Bitstream added on 2014-06-13T18:58:37Z : No. of bitstreams: 1 gerola_gp_me_bauru.pdf: 2161665 bytes, checksum: b96d7398df21f2a6ef4bda2cc153bb3a (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / This project has as main objective the development of nanowires for application in biosensors. Nanomaterials have been highlighted by high sensitivity when applied to biological sensors. As they haver virtually the same dimensions as the molecules that should detect, these nanosensors could revolutionize the diagnoses way are made in medical and biological examinations. Therefores, in this research, we prepared and characterized multithreaded nanowires usind different types of metal and polymer. These nanowires were prepared by electrochemical deposition process and characterized by scanning electron microscopy (SEM). The synthesized nanowires have been changed for the attachment of the enzyme glucose oxidase. Several experimental parameters of biosensor was evaluated as the optimization of pH (at 6), as a function of the peak current obtained from glucose; the enzyme concentration of 0,4 mg ml-1 required to prepare the biosensor; as well as the study of electrochemical potential variation, where it was observed that the ideal is to use potential less than 0,4 V. The analyitcal curve for the determination of glucose showed a slope of 7.95 uA mol-1, with a correlation coefficient of 0.997 and detection limit of the biosensor determined was 3.7 x 10-7 mol L-1. The biosensor showed high selectivity only for detection of glucose and maintained a good response to glucose sensing (85%) in 30 days. Thus, the use of nanowires for the preparation of biosensors becomes and alternative to direct detection of glucose; the enzyme concentration of 0.4 mg ml-1 required to prepare the biosensor; as well as the study of electrochemical potential less than 0.4 V. The analytical curve for the determination o glucose showed a slope of 7.95 uA mol-1, with a correlation coefficient of 0.997 and detection limit of the biosensor... / This project has as main objective the development of nanowires for application in bionsensors. Nanomaterials have been highlighted by high sensitivity when applied to biological sensors. As they have virtually the same dimensions as the molecules that should detect, these nanosensors could revolutioneze the diagnoses way are made in medical and biological examinations. Therefore, in this research, we prepared and characterized multithreaded nanowires using different types of metal and polymer. These nanowires were prepared by electrochemical deposition process and characterized by scanning electron microscopy (SEM). The synthesized nanowires have been changed for the attachment of the enzyme glucose oxidase. Several experimental parameters of biosensor was evaluated as the optimization of pH (at 6), as a function of the peak current obtained from glucose; the enzyme concentration of 0.4 mg ml-1 required to prepare the bionsensor; as well as the study of electrochemical potential variation, where it was observed that the ideal is to use potential less than 0.4 V. The analytical curve for the determination of glucose showed a slope of 7.95 uA mol-1, with a correlation coefficient of 0.997 and detection limit of the biosensor determined was 3.7 x 10-7 mol L-1. The biosensor showed high selectivity only for detection of glucose and maintained a good response to glucose sensing (85%) in 30 days. Thus, the use... (Complete abstract click electronic access below) / FAPESP: 12/01402-2

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This thesis describes work carried out on the research, development and implementation of new processes for the fabrication of surface plasmon waveguide biosensors. Fabrication of surface plasmon resonance (SPR) based waveguides embedded in a thick CYTOP cladding with the incorporation of fluidic channels was achieved with improved quality and operability compared to previous attempts. The fabrication flow was modified in key areas including lithography for feature definition, gold evaporation and the upper cladding deposition procedure. The combined result yielded devices with sharper resolution of waveguides, gold surfaces with minimal aberrations, reduced surface roughness and minimization of waveguide deformation due to reduction of solvent diffusion into the lower cladding. The fabricated waveguides consisted of a thin, 35 nm, patterned gold film, embedded in a thick, 18 µm, CYTOP fluoroploymer cladding. The gold devices were exposed by O2 plasma etching through the upper cladding to form fluidic channels for the facilitation of flow of an index matched sensing medium. Optical and physical characterization of devices revealed structures of significantly improved quality over previous attempts, rendering the platform competitive for biosensing applications.

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Guanosine tetraphosphate (ppGpp) is a bacterial signaling molecule involved in activating the stringent response, a cellular reaction to environmental stress that downregulates cell division and metabolism processes to conserve nutrients. The stringent response is implicated in some instances of antibiotic resistance, so broadening the current understanding of ppGpp signaling is useful. This experiment seeks to generate a ppGpp biosensor that will bind ppGpp and emit fluorescent light in its presence which will allow for improved research into the pathways and functions of the signaling molecule. To generate a novel ppGpp biosensor, I converted a biosensor previously used to detect cyclic di-GMP (a different signaling molecule) to contain a binding site transformed to now bind specifically with ppGpp. The genetic sequence for the cyclic di-GMP binding site was replaced with the ppGpp hydrolase domain which has a specific affinity for ppGpp; however, hydrolase activity would provide unwanted breakdown of the ppGpp, so it is mutated further to neutralize hydrolase activity. The desired outcome of this experiment results in a biosensor with an active site that has a specific and sufficient binding affinity for the ppGpp molecule. This sensor is designed to have the active site (ppGpp binding site) flanked by two fluorescent proteins that will interact more closely and transfer fluorescent light. When the first fluorescent protein is activated and the second fluorescent protein is in close proximity, emitted light can be transferred to the second. Binding of ppGpp will cause a conformational shift in the biosensor’s structure, causing the two fluorescent proteins to move further apart. This results in them losing the ability to transfer the fluorescent energy between fluorescent proteins. Using a fluorescent microscope or fluorescent plate reader, I will be able to determine the level of transferred fluorescent energy, and in turn measure the amount of ppGpp in the sample. Having generated the biosensor, I must now determine the ppGpp detection level, intensity of change in fluorescent transfer upon ppGpp binding, and the binding affinity for other nucleotides that might give me an incorrect signal. Using this, we can determine how ppGpp levels are regulated in bacteria under conditions of stress.

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