F.J. Rowell

Detection of protease activity in the wetted surface of gelatin-coated electrodes in air by AC impedance spectroscopy

As a step towards developing a biosensor which can detect airborne protease droplets, a biosensor which had previously been developed to detect protease in solution is shown to be capable of detecting different concentrations of protease in liquid films on the sensor surface in air. The biosensor measured impedance change due to proteolytic digestion of its gelatin coating. In saturated air there was a rise in impedance, with a loss in weight of the gelatin, in proportion to collagenase concentration. The addition of glycerol to the gelatin caused a lower impedance response and smaller loss in weight. A critical thickness of the gelatin layer prior to a more rapid change in the rate of impedance was noted, with and without the addition of glycerol. In low air humidity (40%), with gelatin, all collagenase concentrations produced a very similar rapid increase in impedance. However, with glycerol-enhanced gelatin, there was a clear distinction between the extent of impedance change with different collagenase concentrations. The application of these findings for use in the field of bioaerosol sampling is discussed.

Rapid Determination of Warfarin by Sequential Injection Analysis with Cyclodextrin-Enhanced Fluorescence Detection

Abstract A procedure for rapid and reproducible measurement of warfarin is described. This is based on a computer-controlled sequential injection analysis (SIA) system and utilises the phenomenon of enhancement of fluorescence seen when the drug forms inclusion complexes with β-cyclodextrin in aqueous solution. Cyclodextrin and warfarin solutions are sequentially injected and mixed within seconds using the automated system and the fluorescence is then monitored immediately. The calibration curve is linear over the measured concentration range of 0.1 to 1 μg/ml (r2 0.998, n=5) with a limit of detection of 0.02 μg/ml. The measurement is fast with response time of 20 seconds, and is also reproducible with coefficient of variation at 1.5% (n=10) at a concentration of 0.5 μg/ml. Water samples spiked with warfarin were successfully measured using the method.

Environmental analysis in the workplace; development of a rapid, sensitive ELISA for monitoring airborne alcalase

Abstract Alcalase, in common with other proteases is a respiratory sensitiser and long term and short term occupational exposure limits of 60 ng m−3 for periods of 8 h and 15 min respectively have been set for workers who may inhale these proteases in the work-place. Polyclonal antibodies have been raised in rabbits that specifically recognise alcalase. These have been purified using immunoaffinity chromatography and the resulting anti-alcalase antibodies used with alkaline-phosphatase labelled anti-rabbit antibodies to generate a single reagent for use in a competitive ELISA for alcalase that can be used in a rapid semi-quantitative mode to screen samples from conventional filters following monitoring of worker exposure. Presence of alcalase in excess of the occupational exposure standard (OES) results in a lack of colour at the endpoint of the assay while samples with levels less than the OES produce a yellow colour. The ELISA can process 45 samples in duplicate in 60 min. The limit of detection of the assay is 1 pg ml−1 in a 50 μl sample volume. The assay has good precision with coefficients of variation less than 10%. and is better suited than current colorimetric enzyme assays for use on site by semi-skilled operatives immediately following the monitoring period when exposure of workers above the OES can be rapidly ascertained and action taken.

Evaluation of a model for the detection of aerosol-containing protease in real-time using chromogenic substrates in the sampling fluid of a cyclone and a bubbler

Abstract A model previously developed for predicting the colour change in a liquid sampler containing a chromogenic substrate was tested for the detection of aerosols of the protease enzyme, alcalase. Aerosols were generated in a bioaerosol test chamber at 20°C and relative humidity of 40%. The detection system responded to changes in alcalase concentration in real time. The performance of a high flow rate Aerojet cyclone was contrasted with that of a low flow rate bubbler. The model was tested with both a constant aerosol concentration and also with the sudden release of an aerosol for a short time. It was found that the model needed correcting for fluid loss from both samplers. The model predicted the performance of the bubbler better than the cyclone. Possible reasons are discussed.

Monitoring proteolytic enzymes for health and safety in the manufacturing environment. A review

Abstract The widespread application of proteolytic enzymes in many industrial sectors brings considerable benefits to modern industrial manufacturing and to the quality of life for human beings. However, as the enzymes are known potent respiratory sensitising agents, there are potential risks to workers on site and to the general public off-site, if these compounds are released into the atmosphere during their manufacture and processing. To ensure adequate containment within the factory, to protect factory workers and the general public, and to comply with health and safety legislations, it is necessary to monitor airborne concentrations of the enzymes in the workplace atmosphere. At present, however, workplace monitoring of industrial enzymes can only provide exposure data for retrospective use. There is an urgent need to develop rapid and sensitive monitoring methods which provide continuous data on a near real-time basis and detect sudden release of the sensitising material. This paper reviews the existing monitoring methods for proteolytic enzymes in the industrial atmosphere and some recent developments in this important field. Possible strategies for developing integrated sampling and detecting systems for near real-time monitoring of industrial proteolytic enzymes in the manufacturing environment are discussed.

Monitoring of hazardous biochemicals in the work-place atmosphere

Abstract Regulatory bodies are increasingly expressing an interest in the monitoring of the work-place air for hazardous biochemicals but examples in the literature of this form of monitoring are not common. In contrast, there are many examples of sensitive assays being developed for the quantitation of very small quantities of biochemicals in fluids which use a multi-step process. An aerosol sensing system has two components: an aerosol collection device and the biochemical detector. The coupling of the two has rarely been investigated. The requirements for such a sensing system are reviewed, together with an appraisal of the types of detector devices that might be suitable. It is suggested that there is a need to develop one-step sensitive sensors for real-time aerosol monitoring.

Rapid immunological assay methods for ceftazidime and alcalase in the workplace atmosphere

Abstract Rapid ELISAs are reported for the protease enzyme, alcalase, and the cephalosporin, ceftazidime. Both assays have the sensitivity, specificity, precision and speed necessary for rapid analysis (25–45 min for 40 samples) of samples eluted from filters following sampling of airborne aerosols in the workplace. Recovery of the analytes from spiked filters showed near quantitative recovery for alcalase with glass fibre filters and for ceftazidime with PTFE filters. Under controlled release conditions for aerosols at a concentration corresponding to the Occupational Exposure Standard for ceftazidime, use of glass fibre, PTFE and polycarbonate filters all gave poor recovery ranging from 34.6 to 59.2% when analysed by ELISA and HPLC. Evidence is provided that ceftazidime undergoes hydrolysis on exposure to glass-fibre filters.

A Rapid Homogeneous Fluorescence Assay for Subtilisin

Abstract A rapid and sensitive homogeneous assay method has been developed for the determination of subtilisin. The method employs a protein substrate labelled with two fluorescent dyes with fluorescence energy transfer (FET) characteristics. The doubly-labelled substrate was prepared by chemically coupling bovine serum albumin with lucifer yellow and rhodamine dyes. The fluorescence emission from the lucifer labels was initially quenched due to the FET to the adjacent rhodamine labels. However, upon the addition of subtilisin into the labelled substrate solution, increased fluorescence was observed as the enzyme hydrolyzed the substrate and reduced the FET effect. The rate of increase in fluorescence due to substrate hydrolysis was used to calibrate the subtilisin assay. It was linear over the range 0–150 ng of the enzyme (n=8, r2=0.985). The assay was fast with a time of 30 sec to exceed the limit of detection (LOD) signal for 60 ng of subtilisin in 600 μl. In this volume, the LOD for the enzyme was 4.2...

Mathematical analysis for impedance change with proteolytic digestion of gelatin

A mathematical analysis is proposed to demonstrate an inter-relationship between the proteolytic digestion of gelatin on the surface of an interdigitated gold electrode and the resulting rate of impedance change, at different collagenase concentrations, in a biosensor used to detect protease in solution. The impedance change due to digestion of the gelatin layer by collagenase for the overall digestion process was expressed in two different stages: an initial exponential period where the rate of impedance change with enzymic digestion was slow, leading to a critical thickness; after which there was a greater change in impedance associated with subsequent dissolution of the layer and partial or complete uncoating of the digits on the electrode surface. An inter-relationship between the rate of impedance change and collagenase concentration within the range 0.2-0.6 mg ml-1 was predicted for the early stages of the digestion process. A kinetic theory for the rapid rate of impedance change with collagenase concentrations could not be developed owing to the rate remaining almost constant for all concentrations of collagenase, after the critical thickness had been reached. An inter-relationship between the rate of impedance change and stirrer speed was also demonstrated.