993 resultados para Chemical sensor.


Relevância:

30.00% 30.00%

Publicador:

Resumo:

The two-component system DcuSR of Escherichia coli regulates gene expression of anaerobic fumarate respiration and aerobic C4-dicarboxylate uptake. C4-dicarboxylates and citrate are perceived by the periplasmic domain of the membrane-integral sensor histidine kinase DcuS. The signal is transduced across the membrane by phosphorylation of DcuS and of the response regulator DcuR, resulting in activation of DcuR and transcription of the target genes.rnIn this work, the oligomerisation of full-length DcuS was studied in vivo and in vitro. DcuS was genetically fused to derivatives of the green fluorescent protein (GFP), enabling fluorescence resonance energy transfer (FRET) measurements to detect protein-protein interactions in vivo. FRET measurements were also performed with purified His6-DcuS after labelling with fluorescent dyes and reconstitution into liposomes to study oligomerisation of DcuS in vitro. In vitro and in vivo fluorescence resonance energy transfer showed the presence of oligomeric DcuS in the membrane, which was independent of the presence of effector. Chemical crosslinking experiments allowed clear-cut evaluation of the oligomeric state of DcuS. The results showed that detergent-solubilised His6-DcuS was mainly monomeric and demonstrated the presence of tetrameric DcuS in proteoliposomes and in bacterial membranes.rnThe sensor histidine kinase CitA is part of the two-component system CitAB of E. coli, which is structurally related to DcuSR. CitAB regulates gene expression of citrate fermentation in response to external citrate. The sensor kinases DcuS and CitA were fused with an enhanced variant of the yellow fluorescent protein (YFP) and expressed in E. coli under the control of an arabinose-inducible promoter. The subcellular localisation of DcuS-YFP and CitA-YFP within the cell membrane was studied by means of confocal laser fluorescence microscopy. Both fusion proteins were found to accumulate at the cell poles. The polar accumulation was slightly increased in the presence of the stimulus fumarate or citrate, respectively, but independent of the expression level of the fusion proteins. Cell fractionation demonstrated that polar accumulation was not related to inclusion bodies formation. The degree of polar localisation of DcuS-YFP was similar to that of the well-characterised methyl-accepting chemotaxis proteins (MCPs), but independent of their presence. To enable further investigations on the function of the polar localisation of DcuS under physiological conditions, the sensor kinase was genetically fused to the flavin-based fluorescent protein Bs2 which shows fluorescence under aerobic and anaerobic conditions. The resulting dcuS-bs2 gene fusion was inserted into the chromosome of various E. coli strains.rnFurthermore, a protein-protein interaction between the related sensor histidine kinases DcuS and CitA, regulating common metabolic pathways, was detected via expression studies under anaerobic conditions in the presence of citrate and by in vivo FRET measurements.

Relevância:

30.00% 30.00%

Publicador:

Resumo:

With the increasing importance that nanotechnologies have in everyday life, it is not difficult to realize that also a single molecule, if properly designed, can be a device able to perform useful functions: such a chemical species is called chemosensor, that is a molecule of abiotic origin that signals the presence of matter or energy. Signal transduction is the mechanism by which an interaction of a sensor with an analyte yields a measurable form of energy. When dealing with the design of a chemosensor, we need to take into account a “communication requirement” between its three component: the receptor unit, responsible for the selective analyte binding, the spacer, which controls the geometry of the system and modulates the electronic interaction between the receptor and the signalling unit, whose physico-chemical properties change upon complexation. A luminescent chemosensor communicates a variation of the physico-chemical properties of the receptor unit with a luminescence output signal. This thesis work consists in the characterization of new molecular and nanoparticle-based system which can be used as sensitive materials for the construction of new optical transduction devices able to provide information about the concentration of analytes in solution. In particular two direction were taken. The first is to continue in the development of new chemosensors, that is the first step for the construction of reliable and efficient devices, and in particular the work will be focused on chemosensors for metal ions for biomedical and environmental applications. The second is to study more efficient and complex organized systems, such as derivatized silica nanoparticles. These system can potentially have higher sensitivity than molecular systems, and present many advantages, like the possibility to be ratiometric, higher Stokes shifts and lower signal-to-noise ratio.

Relevância:

30.00% 30.00%

Publicador:

Resumo:

This work presents an innovative integration of sensing and nano-scaled fluidic actuation in the combination of pH sensitive optical dye immobilization with the electro-osmotic phenomena in polar solvents like water for flow-through pH measurements. These flow-through measurements are performed in a flow-through sensing device (FTSD) configuration that is designed and fabricated at MTU. A relatively novel and interesting material, through-wafer mesoporous silica substrates with pore diameters of 20 -200 nm and pore depths of 500 µm are fabricated and implemented for electro-osmotic pumping and flow-through fluorescence sensing for the first time. Performance characteristics of macroporous silicon (> 500 µm) implemented for electro-osmotic pumping include, a very large flow effciency of 19.8 µLmin-1V-1 cm-2 and maximum pressure effciency of 86.6 Pa/V in comparison to mesoporous silica membranes with 2.8 µLmin-1V-1cm-2 flow effciency and a 92 Pa/V pressure effciency. The electrical current (I) of the EOP system for 60 V applied voltage utilizing macroporous silicon membranes is 1.02 x 10-6A with a power consumption of 61.74 x 10-6 watts. Optical measurements on mesoporous silica are performed spectroscopically from 300 nm to 1000 nm using ellipsometry, which includes, angularly resolved transmission and angularly resolved reflection measurements that extend into the infrared regime. Refractive index (n) values for oxidized and un-oxidized mesoporous silicon sample at 1000 nm are found to be 1.36 and 1.66. Fluorescence results and characterization confirm the successful pH measurement from ratiometric techniques. The sensitivity measured for fluorescein in buffer solution is 0.51 a.u./pH compared to sensitivity of ~ 0.2 a.u./pH in the case of fluorescein in porous silica template. Porous silica membranes are efficient templates for immobilization of optical dyes and represent a promising method to increase sensitivity for small variations in chemical properties. The FTSD represents a device topology suitable for application to long term monitoring of lakes and reservoirs. Unique and important contributions from this work include fabrication of a through-wafer mesoporous silica membrane that has been thoroughly characterized optically using ellipsometry. Mesoporous silica membranes are tested as a porous media in an electro-osmotic pump for generating high pressure capacities due to the nanometer pore sizes of the porous media. Further, dye immobilized mesoporous silica membranes along with macroporous silicon substrates are implemented for continuous pH measurements using fluorescence changes in a flow-through sensing device configuration. This novel integration and demonstration is completely based on silicon and implemented for the first time and can lead to miniaturized flow-through sensing systems based on MEMS technologies.

Relevância:

30.00% 30.00%

Publicador:

Resumo:

Gas sensors have been used widely in different important area including industrial control, environmental monitoring, counter-terrorism and chemical production. Micro-fabrication offers a promising way to achieve sensitive and inexpensive gas sensors. Over the years, various MEMS gas sensors have been investigated and fabricated. One significant type of MEMS gas sensors is based on mass change detection and the integration with specific polymer. This dissertation aims to make contributions to the design and fabrication of MEMS resonant mass sensors with capacitance actuation and sensing that lead to improved sensitivity. To accomplish this goal, the research has several objectives: (1) Define an effective measure for evaluating the sensitivity of resonant mass devices; (2) Model the effects of air damping on microcantilevers and validate models using laser measurement system (3) Develop design guidelines for improving sensitivity in the presence of air damping; (4) Characterize the degree of uncertainty in performance arising from fabrication variation for one or more process sequences, and establish design guidelines for improved robustness. Work has been completed toward these objectives. An evaluation measure has been developed and compared to an RMS based measure. Analytic models of air damping for parallel plate that include holes are compared with a COMSOL model. The models have been used to identify cantilever design parameters that maximize sensitivity. Additional designs have been modeled with COMSOL and the development of an analytical model for Fixed-free cantilever geometries with holes has been developed. Two process flows have been implemented and compared. A number of cantilever designs have been fabricated and the uncertainty in process has been investigated. Variability from processing have been evaluated and characterized.

Relevância:

30.00% 30.00%

Publicador:

Resumo:

Bacteriorhodopsin (bR), an optoelectric protein found in Halobacterium salinarum, has the potential for use in protein hybrid sensing systems. Bacteriorhodopsin has no intrinsic sensing properties, however molecular and chemical tools permit production of bR protein hybrids with transducing and sensing properties. As a proof of concept, a maltose binding protein-bacteriorhodopsin ([MBP]-bR) hybrid was developed. It was proposed that the energy associated with target molecule binding, maltose, to the hybrid sensor protein would provide a means to directly modulate the electrical output from the MBP-bR bio-nanosensor platform. The bR protein hybrid is produced by linkage between bR (principal component of purified purple membrane [PM]) and MBP, which was produced by use of a plasmid expression vector system in Escherichia coli and purified utilizing an amylose affinity column. These proteins were chemically linked using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), which facilitates formation of an amide bond between a primary carboxylic acid and a primary amine. The presence of novel protein hybrids after chemical linkage was analyzed by SDSPAGE. Soluble proteins (MBP-only derivatives and unlinked MBP) were separated from insoluble proteins (PM derivatives and unlinked PM) using size exclusion chromatography. The putatively identified MBP-bR protein hybrid, in addition to unlinked bR, was collected. This sample was normalized for bR concentration to native PM and both were deposited onto indium tin oxide (ITO) coated glass slides by electrophoretic sedimentation. The photoresponse of both samples, activated using 100 Watt tungsten lamp at 10 cm distance, were equal at 175 mV. Testing of deposited PM with 1 mM sucrose or 1 mM maltose showed no change in the photoresponse of the xiv material, however addition of 1 mM maltose to the deposited MBP-bR linked hybrid material elicited a 57% decrease in photoresponse indicating a positive response for targeting of maltose. This chemically linked MBP-bR hybrid protein, with bacteriorhodopsin, as a photoresponsive transducing substrate, shows promise for creation of a universal sensing array by attachment of other pertinent sensing materials, in lieu of the maltose binding protein utilized. This strategy would allow significant reduction in sensor size, while increasing responsiveness and sensitivity at nano and picomolar levels.

Relevância:

30.00% 30.00%

Publicador:

Resumo:

The Holocene Twin Slides form the most recent of recurrent mass wasting events along the NE portion of Gela Basin within the Sicily Channel, central Mediterranean Sea. Here, we present new evidence on the morphological evolution and stratigraphic context of this coeval slide complex based on deepdrilled sediment sequences providing a >100 ka paleo-oceanographic record. Both Northern (NTS) and Southern Twin Slide (STS) involve two failure stages, a debris avalanche and a translational slide, but are strongly affected by distinct preconditioning factors linked to the older and buried Father Slide. Core-acoustic correlations suggest that sliding occurred along sub-horizontal weak layers reflecting abrupt physical changes in lithology or mechanical properties. Our results show further that headwall failure predominantly took place along sub-vertical normal faults, partly through reactivation of buried Father Slide headscarps.