A near real-time system for continuously monitoring airborne subtilisin-type enzymes in the industrial atmosphere

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 °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.