Christopher R. Lloyd

Reagentless detection of microorganisms by intrinsic fluorescence

Quick and accurate detection of microbial contamination is accomplished by a unique combination of leading-edge technologies described in this and the accompanying paper. In this contribution, a hand-held prototype instrument is described which is capable of statistically sampling the environment for microbial contamination and determining cell viability. The technology is sensitive enough to detect very low levels ( approximately 20 cells/cm(2) or cm(3)) of microbes in seconds.

Taxonomic identification of microorganisms by capture and intrinsic fluorescence detection.

Quick and accurate detection of microbial contamination is accomplished by a unique combination of leading edge technologies described in this and the accompanying article. Microbe capture chips, used with a prototype fluorescence detector, are capable of statistically sampling the environment for pathogens (including spores), identifying the specific pathogens/exotoxins, and determining cell viability where appropriate.

Ferrate(VI) oxidation of aniline

A detailed kinetic and thermodynamic study of ferrate(VI) oxidation of aniline (aminobenzene) has been carried out in isotopic solvents, H2O and D2O, as a function of reductant concentration, solution pH, temperature and pressure by means of conventional stopped-flow and high-pressure stopped-flow spectrophotometric methods. Under pseudo first-order conditions with reductant in at least 10-fold excess over ferrate, these redox processes give rise to a simple exponential change of optical density. The temperature profile reveals relatively low activation enthalpies, and the activation entropies found for these processes are very negative. In addition, the significant negative activation volumes estimated from the pressure dependence of the rate constants indicate a substantial decrease in partial molar volume during the formation of the transition state, suggesting that highly structured transition states are formed in these reactions. An EPR result indicates a free radical reaction mechanism. The kinetic isotopic results for aniline systems measured in H2O and in D2O solvent indicate that the amino hydrogen/deuterium plays a role in the formation of the transition states.

Is what you eat and drink safe? Detection and identification of microbial contamination in foods and water

Current technologies for assessing the microbial load in food and water require cellular outgrowth to increase the cell count to a detectable level. This step therefore makes real-time assessment of microbial loads impossible. To circumvent this problem, we have developed a high-sensitivity, multiwavelength fluorescence detector and signal- collection and processing software. This detection system enables one to distinguish between abiotic matter, sporulated bacteria, vegetative bacteria, dead bacteria, and other biomaterials containing aromatic amino acids. Our detection limit is in the 10-100 cell/cm/sup 2/ range (or per cm/sup 3/ if bulk aqueous samples are used), and there is no sample contact. To supplement this detection system, we have developed a variety of ligands, including hemin derivatives, peptides, glycoconjugates, and siderophores, for cell capture to circumvent problems associated with antibodies. These materials are tethered to disposable surfaces (glass, plastics) in arrays for use in capturing biomaterials (e.g., live bacterial cells, dead cells, spores, and toxins); identification is based on which sector of an array it binds to. The detection system can serve as a reader for these coated materials, and variants have been tested for use in scanning food surfaces and food wash water.

Algorithm for recognition of optical spectra in an environment containing interferants

An algorithm for quickly determining the presence of bacteria based on their intrinsic fluorescence signals has been developed. Applications of this algorithm are discussed.

Real-time In-situ detection of microbes

Currently, no methods exist for the real-time detection and quantification of microbes in the environment or for the detection and identification of pathogenic organisms in clinical specimens. We have developed technologies which overcome these limitations and provide detection limits as low as a ten microbial cells per cm2 on abiotic surfaces, and per mL in fluids. The detection and quantification of microbes [total microbial load] is based on the intrinsic fluorescence of microbial metabolites and protein cofactors, and provides an estimate of the total microbial load as well as the relative distribution of live cells, dead cells, and endospores. Unlike existing methods, no additional reagents or sample contact is needed. This technology has been applied to the in-situ measurements of two sub-glacial microbial communities at sites in the Svalbard Archipelago, Norway, and to the efficacy of disinfection of contact lenses. In the rapid spread of a life-threatening infection, early diagnosis is of great importance. In such situations, pathogen counts will be very low, which also presents a significant challenge to diagnostic methods. We have developed a point of care disposable diagnostic based on the en masse capture of blood-borne microbes from 1 mL of fresh whole blood with surface-tethered, small molecule ligands. Quantification is based on the intrinsic fluorescence of captured cells.

Molecular engineering of sensors for biological materials

Molecular engineering methods have been used to develop sensors for the capture of DNA, viruses, and bacterial cells, spores, and toxins. The capture technology exploits the molecular basis of pathogenesis and capture events are detected using the intrinsic fluorescence of the captured microbial components. Capabilities include statistically sampling the environment in minutes and sensitivity of /spl sim/100 cells/cm/sup 3/.

Real-time in situ detection and quantification of bacteria in the Arctic environment

At present, there are no methods that determine the total microbial load on an abiotic substrate in real time. The utility of such a capability ranges from sterilization and medical diagnostics to the search for new microorganisms in the environment and study of their ecological niches. We report the development of a hand-held, fluorescence detection device and demonstrate its applicability to the field detection of Arctic bacteria. This technology is based on the early pioneering work of Britton Chance which elucidated the intrinsic fluorescence of a number of metabolites and protein cofactors in cells, including reduced pyridine nucleotides, cytochromes and flavins. A PDA controls the device (fluorescence excitation and data collection) and processes the multiwavelength signals to yield bacterial cell counts, including estimates of live cells, dead cells and endospores. Unlike existing methods for cell counting, this method requires no sample contact or addition of reagents. The use of this technology i...

Interactions between sol-gel encapsulated myoglobin and carbon dioxide in supercritical CO2

The interaction between biomolecules and CO{sub 2} is of crucial importance because CO{sub 2} is produced during mammalian metabolism and must be removed in order for cells to function properly. The ideal solvent for investigations into reactivity of proteins and enzymes towards CO{sub 2} is CO{sub 2} itself since this choice of solvent eliminates any other interactions that would complicate data collection and analysis. The development of an isolated reaction vessel suitable for use with supercritical CO{sub 2} is an important step towards conducting valid spectroscopic experiments because contamination from the high-pressure vessel is avoided. This is crucial when biomolecules are the subject of investigations due to the ease with which contaminants perturb their reactivity. The combination of sol-gel encapsulation of biologically active molecules with supercritical fluid spectroscopy provides an experimental method for studying CO{sub 2} interactions previously considered inaccessible. The molecules retain their biological activity even under conditions required to maintain the CO{sub 2} in the supercritical state.