Biomedical subjects

Mona Wells

Publications and source records attributed to Mona Wells.

6 recordsLinked to original sources

PubMed2006

Bioreporters: gfp versus lux revisited and single-cell response.

Genetically engineered organisms expressing spectroscopically active reporter molecules in response to chemical effectors display great potential as living transducers in sensing applications. Green fluorescent protein (gfp gene) bioreporters have distinct advantages over luminescent couterparts (lux gene), including applicability at the single-cell level, but are typically less sensitive. Here we describe a gfp-bearing bioreporter that is sensitive to naphthalene (a poorly water soluble pollutant behaving like a large class of hydrophobic compounds), is suitable for use in chemical assays and bioavailability studies, and has detection limits comparable to lux-bearing bioreporters for higher efficiency detection strategies. Simultaneously, we find that the exploitation of population response data from single-cell analysis is not an algorithmic conduit to enhanced signal detection and hence lower effector detection limits, as normally assumed. The assay reported functions to equal effect with or without biocide.

Biosensing Techniques↗

PubMed2006

Advances in optical detection strategies for reporter signal measurements.

Many recent advances in bioreporter technology focus on challenges related to bioengineering, yet in many applications implementation of optical signal measurement is equally susceptible to improvement. For bioluminescent bioreporters, one area of effort lies in the development of semiconductor chip-based detector modules; this holds great promise for ultra-compact and field-deployable instrumentation, but has not yet had a palpable impact on improved detection limits. Regarding lower detection limits, single-molecule detection techniques have seen their first application to bioreporters, and preliminary results serve as an indication of future promise. Another technique applicable to fluorescent bioreporters is fluorescence flow cytometry, which is rapid, suitable for high-throughput screening, and lends itself to increased analytical specificity through simple algorithmic approaches to data treatment.

Biosensing Techniques↗

PubMed2006

A new green fluorescent protein-based bacterial biosensor for analysing phenanthrene fluxes.

The polycyclic aromatic hydrocarbon (PAH)-degrading strain Burkholderia sp. RP007 served as host strain for the design of a bacterial biosensor for the detection of phenanthrene. RP007 was transformed with a reporter plasmid containing a transcriptional fusion between the phnS putative promoter/operator region and the gene encoding the enhanced green fluorescent protein (GFP). The resulting bacterial biosensor--Burkholderia sp. strain RP037--produced significant amounts of GFP after batch incubation in the presence of phenanthrene crystals. Co-incubation with acetate did not disturb the phenanthrene-specific response but resulted in a homogenously responding population of cells. Active metabolism was required for induction with phenanthrene. The magnitude of GFP induction was influenced by physical parameters affecting the phenanthrene flux to the cells, such as the contact surface area between solid phenanthrene and the aqueous phase, addition of surfactant, and slow phenanthrene release from Model Polymer Release System beads or from a water-immiscible oil. These results strongly suggest that the bacterial biosensor can sense different phenanthrene fluxes while maintaining phenanthrene metabolism, thus acting as a genuine sensor for phenanthrene bioavailability. A relationship between GFP production and phenanthrene mass transfer is proposed.

Biodegradation, Environmental↗

PubMed2005

Ultrasensitive reporter protein detection in genetically engineered bacteria.

We demonstrate the use of laser-induced fluorescence confocal spectroscopy to measure analyte-stimulated enhanced green fluorescent protein (egfp) synthesis by genetically modified Escherichia coli bioreporter cells. Induction is measured in cell lysates and, since the spectroscopic focal volume is approximately the size of one bioreporter cell, also in individual live bacteria. This is, to our knowledge, the first ever proof-of-concept work utilizing instrumentation with single-molecule detection capability to monitor bioreporter response. Although we use arsenic inducible bioreporters here, the method is extensible to gfp/egfp bioreporters that are responsive to other substances.

Arsenites↗

PubMed2005

Model polymer release system study of PAH bioaccessibility: the relationship between "rapid" release and bioaccessibility.

This paper examines bacterial uptake of polycyclic aromatic hydrocarbons (PAHs) entrained within model polymer release systems (MPRSs) whose release kinetics, particularly for operationally defined "slow" release, are similarto PAH release kinetics from sediments and soils. We find that biodegradation is not restricted to the fraction "rapidly" released, f1, as quantified by an empirical biphasic exponential fitting parameter. Though our results indicate that f1 does not predict bioaccessibility (defined by a recent paper calling for a standard definition of same), we analyze the causes of the reported limitation of biodegradation to rapidly released PAHs and we find that, for the MPRSs, there are very strong correlations between an ad hoc bioaccessibility and a wide range of fitting parameters from various kinetic expressions used to phenomenologically characterize release. These results indicate that fitting parameters may be used to predict ad hoc bioaccessibility; however, it is not clear if this is actually a particularly useful quantity. We also report experimental results which indicate that bacteria may influence their environment and cause biological uptake to exceed that expected from abiotic release data obtained under quasi-infinite sink conditions. When this occurs, fitting parameters from simple empirical expressions are even inadequate to predict ad hoc bioaccessibility.

Biodegradation, Environmental↗

PubMedDate not supplied

Parallel dual-color fluorescence cross-correlation spectroscopy using diffractive optical elements.

Dual-color cross-correlation spectroscopy allows the detection and quantification of labeled biomolecules at ultra-low concentrations, whereby the sensitivity of the assay correlates with the measurement time. We now describe a parallel multifocal dual-color spectroscopic configuration employing multiple avalanche photodiodes and hardware correlators. Cross-correlation curves are obtained from several dual-color excitation foci simultaneously. Multifocal dual-color excitation is achieved by splitting each of two laser beams (488 and 633 nm) into four sub-beams with the help of two 2x2 fan-out diffractive optical elements (DOEs), and subsequent superposition of the two sets of four foci. The fluorescence emission from double-labeled biomolecules is detected by two 2x2 fiber arrays.

Equipment Design↗