[Show abstract][Hide abstract] ABSTRACT: We describe the development and validation of a portable system comprising an air sampler coupled to an automated flow injection analysis device. The system is able to monitor airborne concentrations of subtilisin-type enzymes in the workplace atmosphere on a continuous basis. Sampling is in two stages: using a sampling head that is designed to mimic human respiration at approx. 1 m s(-1) at a sampling rate of 600 l min(-1). In the second stage, the captured particles are deposited by impaction from the air stream onto the inner surface of a cyclone that is continuously washed with a jet of buffer solution. Deposited particles are then washed into a reservoir from which samples are taken every 5-6 min and injected automatically into a continuous flow injection analysis system. Proteolytic enzyme in the sample passes through a bioreactor maintained at about 40 degrees C. This contains a cellulose solid phase matrix on which is covalently immobilised Texas Red-labelled gelatin as substrate. The passing enzyme partially digests the substrate releasing fluorophore that is detected down stream in a flow cell coupled to a fluorimeter. The system is calibrated using enzyme standards and the intensity of the resulting peaks from the ex-air samples is converted to airborne concentrations using a mathematical model programmed into a PC. The system has a limit of detection of 4.8 ng m(-3) and a dynamic range of 5-60 ng m(-3). The within assay precision (RSD) is 6.3-9.6% over this range. The within batch precision is 20.3% at 20 ng m(-3) and the corresponding between batch value is 19.5%. The system has been run for periods up to 8 h in the laboratory and for up to 4 h at a factory site and the values obtained compared with time-averaged values obtained from a conventional Galley sampler and in-house analysis when reasonable agreement of the results was observed. The stability of the system over 21 days of continuous use with standards injected periodically was studied. Linearity was observed for all the standard plots throughout. At the end of 21 days, after a total exposure equivalent to 2395 ng ml(-1) of Savinase, the signal due to the 5.0 ng ml(-1) standard was still easily detectable.
[Show abstract][Hide abstract] ABSTRACT: 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.
The Analyst 01/2001; 125(12):2289-93. DOI:10.1039/b007027k · 4.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An experimental investigation is reported of the flocculation of bentonite and Alcaligenes eutrophus in a batch oscillatory baffled flocculator (OBF), where fluid mixing is achieved by eddies that are generated when fluid passes through a set of equally spaced stationary orifice baffles. Periodically formed vortices can be controlled by a combination of operational and geometrical parameters, such as, oscillation frequency, oscillation amplitude, baffle diameter and baffle spacing. The effect of oscillation frequency and amplitude in the OBF on the percentage of flocculation of both bentonite and Alcaligenes eutrophus was examined, the floc sizes at various operational conditions observed and the strain rates in the OBF measured using a digital particle image velocimetry (DPIV) technique. The results show that the oscillation amplitude is the dominant factor in influencing the percentage of flocculation. The measured strain rate was linked with the percentage of flocculation per ppm polymer dose and the results compared with those obtained in conventional stirred tank flocculators.
Chemical Engineering Research and Design 01/2001; 79(1):33-40. DOI:10.1205/026387601528507 · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A simple assay for some proteolytic enzymes has been developed which can be performed directly on the surface of a cellulose nitrate filter used to capture the analyte during workplace monitoring for health and safety purposes. Following air sampling the analysis is performed on the filter which is retained within the air sampler. This involves two steps: first, a 15 min incubation in which the captured enzyme is dissolved and then digests an alkaline-phosphatase-labelled antibody immobilised as a small dot on the surface of the filter; and second, is a 10 min incubation with substrate solution, which follows an in situ wash under a vacuum. During the incubation colour develops on the spot at the location of the immobilised enzyme antibody conjugate. The intensity of the spot can be assessed visually within the sampler to ascertain the presence or absence of captured enzyme, or alternatively quantitative results can be obtained using an optical scanner. The limit of detection is 5 ng per filter for subtilisin (20 ng for visual discrimination between this standard and the zero). The assay is stable to the effects of ambient air sampling at 31 min(-1) for 18 h.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: We report, for the first time, our direct experimental measurement of velocity vectors and strain-rate distributions in an oscillatory baffled column (OBC) using time-resolved particle image velocimetry (PIV). The technique allowed a time series of spatial velocity maps to be obtained for several phases per oscillation cycle and the results, comprising several thousand such maps, illustrate in detail the variations of velocity and strain rate in a baffled region over a range of oscillation amplitudes and oscillation frequencies and provide a deep insight into the mixing, vortex convection and transport mechanism in such a device. The results show that an OBC not only provides enhanced mixing, but also offers low strain rates, which are lower than those in stirred tank vessels. We have also reported for the first time the quantitative correlation between the strain rate and the power dissipation in an OBC. Using the PIV technique we are able to quantify the strain rates experienced in an OBC.
Chemical Engineering Science 08/2000; 55(16-55):3195-3208. DOI:10.1016/S0009-2509(99)00577-1 · 2.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A simple and relatively rapid enzyme-linked immunosorbant assay method for the anti-emetic drug ondansetron has been developed for its quantitation in solution. This has been optimised for use with samples that have been obtained following extraction of filters after the drug's capture from air samples in the workplace. The assay has the sample throughput (40 duplicate samples in 3 h), specificity, sensitivity (LOD of 10.5 ng drug ml-1) and precision (RSD < 11%) necessary for its use in determining airborne concentrations of ondansetron in such samples as part of an occupational health and hygiene monitoring programme.
The Analyst 05/1998; 123(5):1121-6. DOI:10.1039/A708204E · 4.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A biosensor for collagenase detection was developed which detected the change in impedance caused by proteolytic digestion of gelatin coated interdigitated gold electrodes. The concentration of gelatin in the layer did not greatly affect the impedance measurements. Enzyme degradation of the layer produced a rapid rise in impedance when a critical thickness was reached. The change in impedance with protease digestion was correlated with solubilisation of the gelatin layer as measured by weight loss of the gelatin layer from the sensor surface. The response time of the device was greatly reduced by stirring the fluid around the biosensor. The ability to detect the gelatin coating on the sensor was severely impaired by the presence of electrolyte. The implications of these findings for further biosensor development are discussed.
[Show abstract][Hide abstract] ABSTRACT: We report, for the first time, the preliminary results of flocculation of bentonite particles in a batch oscillatory baffled column. The effects of floe settling time, mixing intensity, polymer dose, and polymer injecting mode on percentage of flocculation were investigated. The optical density of bentonite suspension prior to and after polymer injection was measured as the means to quantify the percentage of flocculation. The results show clearly that the intensity of oscillation is an important factor to the flocculation rate, and the two-stage mixing mode produced a higher percentage of flocculation than the single-stage mode for low polymer dosages up to 10 ppm. The advantages of the incremental polymer injection method over the slug method are also discussed.
[Show abstract][Hide abstract] ABSTRACT: A simple competitive enzyme-linked immunoassay for the antibiotic ceftazidime and structurally similar beta-lactam antibiotics has been developed which can be performed directly on the surface of a cellulose nitrate filter used to capture the airborne drug during workplace monitoring for health and safety purposes. Post sampling analysis is performed on the filter retained within the air sampler. It involves two steps; the first a 10 min incubation in which the captured drug is dissolved and competes with drug immobilised within a protein conjugate on the surface of the filter for an enzyme-labelled antibody reagent, and the second, following washing under vacuum in situ, a 5 min incubation of substrate solution when colour develops on the spot at the location of the immobilised drug-protein conjugate. The intensity of the spot can be assessed visually within the sampler to ascertain the presence or absence of captured drug, or quantitative results can be obtained using an optical scanner. The intensity of the spots in linear from 10 ng to 1 microgram (r2 = 0.9996, n = 3) and the limit of detection is 1.9 ng of captured drug (10 ng for visual discrimination between this standard and the zero). The assay is precise with between-assay RSD values of < 4% over the linear range of the assay.
The Analyst 12/1997; 122(12):1505-8. DOI:10.1039/a704728b · 4.11 Impact Factor
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.