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At least 73 records · Page 4

Phosphate binding protein as the biorecognition element in a biosensor for phosphate

This work explores the potential use of a member of the periplasmic family of binding proteins, the phosphate binding protein (PBP), as the biorecognition element in a sensing scheme for the detection of inorganic phosphate (Pi). The selectivity of this protein originates from its natural role which, in Escherichia coli, is to serve as the initial receptor for the highly specific translocation of Pi to the cytoplasm. The single polypeptide chain of PBP is folded into two similar domains connected by three short peptide linkages that serve as a hinge. The Pi binding site is located deep within the cleft between the two domains. In the presence of the ligand, the two globular domains engulf the former in a hinge-like manner. The resultant conformational change constitutes the basis of the sensor development. A mutant of PBP (MPBP), where an alanine was replaced by a cysteine residue, was prepared by site-directed mutagenesis using the polymerase chain reaction (PCR). The mutant was expressed, from plasmid pSD501, in the periplasmic space of E. coli and purified in a single chromatographic step on a perfusion anion-exchange column. Site-specific labeling was achieved by attaching the fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC), to the protein through the sulfhydryl group of the cysteine moiety. Steady-state fluorescence studies of the MPBP-MDCC conjugate showed a change in the intensity of the signal upon addition of Pi. Calibration curves for Pi were constructed by relating the intensity of the fluorescence signal with the amount of analyte present in the sample. The sensing system was first developed and optimized on a spectrofluorometer using ml volumes of sample. It was then adapted to be used on a microtiter plate arrangement with microliter sample volumes. The system's versatility was finally proven by developing a fiber optic fluorescence-based sensor for monitoring Pi. In all three cases the detection limits for the analyte were in the sub-microMolar range. It was also demonstrated that the sensing system was selective for phosphate over other structurally-similar anions, paving the way for the design and development of a new family of biosensors utilizing the specific binding properties of periplasmic proteins. c2003 Elsevier B.V. All rights reserved.

NASA Program Fundamental Space Biology

Array biosensor for detection of toxins

The array biosensor is capable of detecting multiple targets rapidly and simultaneously on the surface of a single waveguide. Sandwich and competitive fluoroimmunoassays have been developed to detect high and low molecular weight toxins, respectively, in complex samples. Recognition molecules (usually antibodies) were first immobilized in specific locations on the waveguide and the resultant patterned array was used to interrogate up to 12 different samples for the presence of multiple different analytes. Upon binding of a fluorescent analyte or fluorescent immunocomplex, the pattern of fluorescent spots was detected using a CCD camera. Automated image analysis was used to determine a mean fluorescence value for each assay spot and to subtract the local background signal. The location of the spot and its mean fluorescence value were used to determine the toxin identity and concentration. Toxins were measured in clinical fluids, environmental samples and foods, with minimal sample preparation. Results are shown for rapid analyses of staphylococcal enterotoxin B, ricin, cholera toxin, botulinum toxoids, trinitrotoluene, and the mycotoxin fumonisin. Toxins were detected at levels as low as 0.5 ng mL(-1).

Review

Biosensors for DNA sequence detection

DNA biosensors are being developed as alternatives to conventional DNA microarrays. These devices couple signal transduction directly to sequence recognition. Some of the most sensitive and functional technologies use fibre optics or electrochemical sensors in combination with DNA hybridization. In a shift from sequence recognition by hybridization, two emerging single-molecule techniques read sequence composition using zero-mode waveguides or electrical impedance in nanoscale pores.

Review

A comparison of imaging methods for use in an array biosensor

An array biosensor has been developed which uses an actively-cooled, charge-coupled device (CCD) imager. In an effort to save money and space, a complementary metal-oxide semiconductor (CMOS) camera and photodiode were tested as replacements for the cooled CCD imager. Different concentrations of CY5 fluorescent dye in glycerol were imaged using the three different detection systems with the same imaging optics. Signal discrimination above noise was compared for each of the three systems.

Evaluation Studies

Improved Ion-Channel Biosensors

An effort is underway to develop improved biosensors of a type based on ion channels in biomimetic membranes. These sensors are microfabricated from silicon and other materials compatible with silicon. As described, these sensors offer a number of advantages over prior sensors of this type.

Nadeau, Jay

Miniature Biosensor with Health Risk Assessment Feedback

Heart rate (HR) monitoring is a medical requirement during exercise on the International Space Station (ISS), fitness tests, and extravehicular activity (EVA); however, NASA does not currently have the technology to consistently and accurately monitor HR and other physiological data during these activities. Performance of currently available HR monitor technologies is dependent on uninterrupted contact with the torso and are prone to data drop-out and motion artifact. Here, we seek an alternative to the chest strap and electrode based sensors currently in use on ISS today. This project aims to develop a high performance, robust earbud based biosensor with focused efforts on improved HR data quality during exercise or EVA. A health risk assessment algorithm will further advance the goals of autonomous crew health care for exploration missions.

Hanson, Andrea

Biosentinel: Improving Desiccation Tolerance of Yeast Biosensors for Deep-Space Missions

BioSentinel is one of 13 secondary payloads to be deployed on Exploration Mission 1 (EM-1) in 2019. We will use the budding yeast Saccharomyces cerevisiae as a biosensor to determine how deep-space radiation affects living organisms and to potentially quantify radiation levels through radiation damage analysis. Radiation can damage DNA through double strand breaks (DSBs), which can normally be repaired by homologous recombination. Two yeast strains will be air-dried and stored in microfluidic cards within the payload: a wild-type control strain and a radiation sensitive rad51 mutant that is deficient in DSB repairs. Throughout the mission, the microfluidic cards will be rehydrated with growth medium and an indicator dye. Growth rates of each strain will be measured through LED detection of the reduction of the indicator dye, which correlates with DNA repair and the amount of radiation damage accumulated. Results from BioSentinel will be compared to analog experiments on the ISS and on Earth. It is well known that desiccation can damage yeast cells and decrease viability over time. We performed a screen for desiccation-tolerant rad51 strains. We selected 20 re-isolates of rad51 and ran a weekly screen for desiccation-tolerant mutants for five weeks. Our data shows that viability decreases over time, confirming previous research findings. Isolates L2, L5 and L14 indicate desiccation tolerance and are candidates for whole-genome sequencing. More time is needed to determine whether a specific strain is truly desiccation tolerant. Furthermore, we conducted an intracellular trehalose assay to test how intracellular trehalose concentrations affect or protect the mutant strains against desiccation stress. S. cerevisiae cell and reagent concentrations from a previously established intracellular trehalose protocol did not yield significant absorbance measurements, so we tested varying cell and reagent concentrations and determined proper concentrations for successful protocol use.

BioSentinel

BioSentinel: Mission Development of a Radiation Biosensor to Gauge DNA Damage and Repair Beyond Low Earth Orbit on a 6U Nanosatellite.

We are designing and developing a "6U" (10 x 22 x 34 cm; 14 kg) nanosatellite as a secondary payload to fly aboard NASA's Space Launch System (SLS) Exploration Mission (EM) 1, scheduled for launch in late 2017. For the first time in over forty years, direct experimental data from biological studies beyond low Earth orbit (LEO) will be obtained during BioSentinel's 12- to 18- month mission. BioSentinel will measure the damage and repair of DNA in a biological organism and allow us to compare that to information from onboard physical radiation sensors. In order to understand the relative contributions of the space environment's two dominant biological perturbations, reduced gravity and ionizing radiation, results from deep space will be directly compared to data obtained in LEO (on ISS) and on Earth. These data points will be available for validation of existing biological radiation damage and repair models, and for extrapolation to humans, to assist in mitigating risks during future long-term exploration missions beyond LEO. The BioSentinel Payload occupies 4U of the spacecraft and will utilize the monocellular eukaryotic organism Saccharomyces cerevisiae (yeast) to report DNA double-strand-break (DSB) events that result from ambient space radiation. DSB repair exhibits striking conservation of repair proteins from yeast to humans. Yeast was selected because of 1) its similarity to cells in higher organisms, 2) the well-established history of strains engineered to measure DSB repair, 3) its spaceflight heritage, and 4) the wealth of available ground and flight reference data. The S. cerevisiae flight strain will include engineered genetic defects to prevent growth and division until a radiation-induced DSB activates the yeast's DNA repair mechanisms. The triggered culture growth and metabolic activity directly indicate a DSB and its successful repair. The yeast will be carried in the dry state within the 1-atm P/L container in 18 separate fluidics cards with each card having 16 independent culture microwells, with integral microchannels and filters to supply nutrients and reagents, confine the yeast to the wells, and enable optical measurement. The measurement subsystem will monitor each subgroup of culture wells continuously for several weeks, optically tracking DSBtriggered cell growth and metabolism. BioSentinel will also include physical radiation sensors based on the TimePix sensor, as implemented by JSC's RadWorks group, which record individual radiation events including estimates of their linear-energytransfer (LET) values. Radiation-dose and LET data will be compared directly to the rate of DSB-and-repair events measured by the S. cerevisiae biosentinels. The spacecraft bus will operate in a deep space environment with functions that include command and data handling, communications, power generation (via deployable solar panels) and storage, and attitude determination-and-control system with micropropulsion. Development of the BioSentinel spacecraft will mature and prove multiple nanosatellite advances in order to function well beyond LEO: Communications from distances of ≥ 500,000 km; Autonomous attitude control, momentum management, and safe mode of nanosatellites in deep space; Shielding-, hardening-, design-, and software-derived radiation tolerance for electronics; Reliable functionality for 12 - 18 months of key subsystems for biofluidics, memory, communications, power, etc.; Close integration of living biological radiation event monitors with miniature physical radiation spectrometers; Biological measurement of solar particle events beyond Earth orbit In addition to providing the first biological results from beyond LEO in over 4 decades, BioSentinel will provide an adaptable small-satellite instrument platform to perform a range of human-exploration-relevant measurements that characterize the biological consequences of multiple outer space environments. BioSentinel is being developed under NASA's Advanced Exploration Systems program.

DNA damage

BioSentinel: Mission Development of a Radiation Biosensor to Gauge DNA Damage and Repair Beyond Low Earth Orbit on a 6U Nanosatellite

We are designing and developing a 6U (10 x 22 x 34 cm; 14 kg) nanosatellite as a secondary payload to fly aboard NASAs Space Launch System (SLS) Exploration Mission (EM) 1, scheduled for launch in late 2017. For the first time in over forty years, direct experimental data from biological studies beyond low Earth orbit (LEO) will be obtained during BioSentinels 12- to 18-month mission. BioSentinel will measure the damage and repair of DNA in a biological organism and allow us to compare that to information from onboard physical radiation sensors. In order to understand the relative contributions of the space environments two dominant biological perturbations, reduced gravity and ionizing radiation, results from deep space will be directly compared to data obtained in LEO (on ISS) and on Earth. These data points will be available for validation of existing biological radiation damage and repair models, and for extrapolation to humans, to assist in mitigating risks during future long-term exploration missions beyond LEO. The BioSentinel Payload occupies 4U of the spacecraft and will utilize the monocellular eukaryotic organism Saccharomyces cerevisiae (yeast) to report DNA double-strand-break (DSB) events that result from ambient space radiation. DSB repair exhibits striking conservation of repair proteins from yeast to humans. Yeast was selected because of 1) its similarity to cells in higher organisms, 2) the well-established history of strains engineered to measure DSB repair, 3) its spaceflight heritage, and 4) the wealth of available ground and flight reference data. The S. cerevisiae flight strain will include engineered genetic defects to prevent growth and division until a radiation-induced DSB activates the yeasts DNA repair mechanisms. The triggered culture growth and metabolic activity directly indicate a DSB and its successful repair. The yeast will be carried in the dry state within the 1-atm PL container in 18 separate fluidics cards with each card having 16 independent culture microwells, with integral microchannels and filters to supply nutrients and reagents, confine the yeast to the wells, and enable optical measurement. The measurement subsystem will monitor each subgroup of culture wells continuously for several weeks, optically tracking DSB-triggered cell growth and metabolism. BioSentinel will also include physical radiation sensors based on the TimePix sensor, as implemented by JSCs RadWorks group, which record individual radiation events including estimates of their linear-energy-transfer (LET) values. Radiation-dose and LET data will be compared directly to the rate of DSB-and-repair events measured by the S. cerevisiae biosentinels.

Radiation Biosensor

New Enzymes for Plastics Recycling from Biosensor-Guided Evolution and Machine Learning: Cooperative Research and Development Final Report, CRADA Number CRD-23-24335

The Agile BioFoundry (ABF) is a consortium of national laboratories dedicated to accelerating biomanufacturing and enabling the bioeconomy. The ABF operates a flexible biotechnology platform that can adjust to the needs of numerous government, academic, and industrial partners, thus enabling them to rapidly develop and optimize the production of a wide range of bioproducts. In this project, the ABF partnered with Birch Biosciences, Inc. to use ABF technology to assist in the development of high-performance enzymes that enable circular, sustainable recycling of poly(ethylene terephthalate) (PET) plastics. A primary goal of this project is to enable cost-effective, high yield, sustainable recycling of PET plastic packaging products.

09 BIOMASS FUELS

A biosensor for cadmium based on bioconvective patterns

An 'in vitro' method for monitoring cadmium, one of the most lethal bivalent heavy metals, can detect biologically active levels. The effects of cadmium tend to concentrate in protozoa far above natural levels and therein begin transferring through freshwater food chains to animals and humans. In a small sample volume (approximately 5 ml) the method uses the toxic response to the protozoa, Tetrahymena pyriformis, to cadmium. The assay relies on macroscopic bioconvective patterns to measure the toxic response, giving a sensitivity better than 1 micro-g/1 and a toxicity threshold to 7 micro-g/1 for Cd(2+). Cadmium hinders pattern formation in a dose-dependent manner. Arrested organism growth arises from slowed division and mutation to non-dividing classes. Unlike previous efforts, this method can be performed in a shallow flow device and does not require electronic or chemical analyses to monitor toxicity.

Noever, David A.

A chemical sensor and biosensor based totally automated water quality monitor for extended space flight: Step 1

The result of a literature search to consider what technologies should be represented in a totally automated water quality monitor for extended space flight is presented. It is the result of the first summer in a three year JOVE project. The next step will be to build a test platform at the Authors' school, St. John Fisher College. This will involve undergraduates in NASA related research. The test flow injection analysis system will be used to test the detection limit of sensors and the performance of sensors in groups. Sensor companies and research groups will be encouraged to produce sensors which are not currently available and are needed for this project.

Smith, Robert S.

Evaluation of Flow Biosensor Technology in a Chronically-Instrumented Non-Human Primate Model

The Physiology Research Branch of Brooks AFB conducts both human and non-human primate experiments to determine the effects of microgravity and hypergravity on the cardiovascular system and to indentify the particular mechanisms that invoke these responses. Primary investigative research efforts in a non-human primate model require the calculation of total peripheral resistance (TPR), systemic arterial compliance (SAC), and pressure-volume loop characteristics. These calculations require beat-to-beat measurement of aortic flow. We have evaluated commercially available electromagnetic (EMF) and transit-time flow measurement techniques. In vivo and in vitro experiments demonstrated that the average error of these techniques is less than 25 percent for EMF and less than 10 percent for transit-time.

SC Koenig