Biosensor based toxicity dissection of tannery and associated environmental samples

Kenyan tannery and associated environmental samples were selected for ecotoxicological assessment. A tool-kit of techniques was developed, including whole-cell biosensor and chemical assays. A luminescence based bacterial biosensor (Escherichia coli HB101 pUCD607) (via a multi-copy plasmid) was used for toxicity assessment. Samples were manipulated prior to biosensor interrogation to identify the nature of the toxic contaminants. Untreated samples (before any manipulations) showed a strong toxic effect at the discharge point in comparison to other sampling points. Sparging was used to identify toxicity associated with volatile organics. The toxicity of contaminants, removed by treatment with activated charcoal was identified for all the sampling points except for those upstream of effluent discharges. Filtration identified toxicity associated with suspended solids. Changes in availability of toxic contaminants due to pH adjustment of most samples from the tannery effluent treatment pits were also associated with the extreme pH values (4.0 and 8.0). The approach used has highlighted the complexicity of toxic pollutants in effluent from the tanning industry and the dissection of toxicity points to possible remediation strategies for effluents from the tanning industry.

Mineralisation of 2,4-dichlorophenol and glucose placed into the same or different hydrological domains as a bacterial inoculant

The spatial location of microorganisms in the soil three-dimensional structure with respect to their substrates plays an important role in the persistence and turnover of natural and xenobiotic organic compounds. To study the effect of spatial location on the mineralisation of ¹⁴C-2,4-dichlorophenol (2,4-DCP, 0.15 or 0.31 μmol g^-1) and ¹⁴C-glucose (2.77 μmol g^-1), columns packed with autoclaved soil aggregates (2-5 mm) were used. Using a chloride tracer of water movement, the existence of 'immobile' water, which was by-passed by preferentially flowing 'mobile' water, was demonstrated. By manipulation of the soil moisture content, the substrates were putatively placed to these conceptual hydrological domains (immobile and mobile water). Leaching studies revealed that approximately 1.7 (glucose) and 3.4 (2.4-DCP) times the amount of substrate placed in mobile water was recovered in the first 4 fractions of leachate when compared to substrate placed in immobile water. The marked difference in the breakthrough curves was taken as evidence of successful substrate placement. The 2,4-DCP degrading bacterium, Burkholderia sp. RASCc2, was inoculated in mobile water (1.8-5.2 × 10⁷ cells g^-1 soil) and parameters (asymptote, time at maximum rate, calculated maximum rate) describing the mineralisation kinetics of 2,4-DCP and glucose previously added to immobile or mobile water domains were compared, For glucose, there was no significant effect (P > 0.1) of substrate placement on any of the mineralisation parameters. However, substrate placement had a significant effect (P < 0.05) on parameters describing 2,4-DCP mineralisation. In particular, 2,4-DCP added in mobile water was mineralised with a greater maximum rate and with a reduced time at maximum rate when compared to 2,4-DCP added to immobile water. The difference in response between the two test substrates may reflect the importance of sorption in controlling the spatial bioavailability of compounds in soil. © 2002 Elsevier Science Ltd. All rights reserved.

Biosensor analysis of dynamics of interleukin 5 receptor subunit beta(c) interaction with IL5:IL5R(alpha) complexes

To gain insight into IL5 receptor subunit recruitment mechanism, and in particular the experimentally elusive pathway for assembly of signaling subunit beta(c), we constructed a soluble beta(c) ectodomain (s(beta)(c)) and developed an optical biosensor assay to measure its binding kinetics. Functionally active s(beta)(c) was anchored via a C-terminal His tag to immobilized anti-His monoclonal antibodies on the sensor surface. Using this surface, we quantitated for the first time direct binding of s(beta)(c) to IL5R(alpha) complexed to either wild-type or single-chain IL5. Binding was much weaker if at all with either R(alpha) or IL5 alone. Kinetic evaluation revealed a moderate affinity (0.2-1 microM) and relatively fast off rate for the s(beta)(c) interaction with IL5:R(alpha) complexes. The data support a model in which beta(c) recruitment occurs with preformed IL5:R(alpha) complex. Dissociation kinetics analysis suggests that the IL5-alpha-beta(c) complex is relatively short-lived. Overall, this study solidifies a model of sequential recruitment of receptor subunits by IL5, provides a novel biosensor binding assay of beta(c) recruitment dynamics, and sets the stage for more advanced characterization of the roles of structural elements within R(alpha), beta(c), and cytokines of the IL5/IL3/GM-CSF family in receptor recruitment and activation.

A potentiometric biosensor for rapid on-site disease diagnostics

Quantitative point-of-care (POC) devices are the next generation for serological disease diagnosis. Whilst pathogen serology is typically performed by centralized laboratories using Enzyme-Linked ImmunoSorbent Assay (ELISA), faster on-site diagnosis would infer improved disease management and treatment decisions. Using the model pathogen Bovine Herpes Virus-1 (BHV-1) this study employs an extended-gate field-effect transistor (FET) for direct potentiometric serological diagnosis. BHV-1 is a major viral pathogen of Bovine Respiratory Disease (BRD), the leading cause of economic loss ($2 billion annually in the US only) to the cattle and dairy industry. To demonstrate the sensor capabilities as a diagnostic tool, BHV-1 viral protein gE was expressed and immobilized on the sensor surface to serve as a capture antigen for a BHV-1-specific antibody (anti-gE), produced in cattle in response to viral infection. The gE-coated immunosensor was shown to be highly sensitive and selective to anti-gE present in commercially available anti-BHV-1 antiserum and in real serum samples from cattle with results being in excellent agreement with Surface Plasmon Resonance (SPR) and ELISA. The FET sensor is significantly faster than ELISA (<10 min), a crucial factor for successful disease intervention. This sensor technology is versatile, amenable to multiplexing, easily integrated to POC devices, and has the potential to impact a wide range of human and animal diseases.

Hydrogel-Forming Microneedle Arrays Allow Detection of Drugs and Glucose In Vivo: Potential for Use in Diagnosis and Therapeutic Drug Monitoring

We describe, for the first time the use of hydrogel-forming microneedle (MN) arrays for minimally-invasive extraction and quantification of drug substances and glucose from skin in vitro and in vivo. MN prepared from aqueous blends of hydrolysed poly(methyl-vinylether-co-maleic anhydride) (11.1% w/w) and poly(ethyleneglycol) 10,000 daltons (5.6% w/w) and crosslinked by esterification swelled upon skin insertion by uptake of fluid. Post-removal, theophylline and caffeine were extracted from MN and determined using HPLC, with glucose quantified using a proprietary kit. In vitro studies using excised neonatal porcine skin bathed on the underside by physiologically-relevant analyte concentrations showed rapid (5 min) analyte uptake. For example, mean concentrations of 0.16 μg/mL and 0.85 μg/mL, respectively, were detected for the lowest (5 μg/mL) and highest (35 μg/mL) Franz cell concentrations of theophylline after 5 min insertion. A mean concentration of 0.10 μg/mL was obtained by extraction of MN inserted for 5 min into skin bathed with 5 μg/mL caffeine, while the mean concentration obtained by extraction of MN inserted into skin bathed with 15 μg/mL caffeine was 0.33 μg/mL. The mean detected glucose concentration after 5 min insertion into skin bathed with 4 mmol/L was 19.46 nmol/L. The highest theophylline concentration detected following extraction from a hydrogel-forming MN inserted for 1 h into the skin of a rat dosed orally with 10 mg/kg was of 0.363 μg/mL, whilst a maximum concentration of 0.063 μg/mL was detected following extraction from a MN inserted for 1 h into the skin of a rat dosed with 5 mg/kg theophylline. In human volunteers, the highest mean concentration of caffeine detected using MN was 91.31 μg/mL over the period from 1 to 2 h post-consumption of 100 mg Proplus® tablets. The highest mean blood glucose level was 7.89 nmol/L detected 1 h following ingestion of 75 g of glucose, while the highest mean glucose concentration extracted from MN was 4.29 nmol/L, detected after 3 hours skin insertion in human volunteers. Whilst not directly correlated, concentrations extracted from MN were clearly indicative of trends in blood in both rats and human volunteers. This work strongly illustrates the potential of hydrogel-forming MN in minimally-invasive patient monitoring and diagnosis. Further studies are now ongoing to reduce clinical insertion times and develop mathematical algorithms enabling determination of blood levels directly from MN measurements.