GB2265220A - Detecting harmful species in gas or vapour. - Google Patents

Abstract

Harmful chemical species in a gas or vapour are detected and/or measured by associating the gas or vapour with a liquid (by solution or otherwise); contacting the liquid containing the species with suitable biological cells in the presence of an artificial electron acceptor, so that the acceptor is reduced by continuous or transient reaction of the cells to the species being detected; re-oxidising the acceptor; and observing and/or measuring changes in the resulting electric current. Gas is introduced through the line 1 and bubbler 12 into liquid 10 which contains substrates & redox mediators. Pump 7 moves the liquid to electrochemical cell 9 containing a working electrode 4 and reference electrode 6. The working electrode, of platinium or carbon, has biological cells immobilised by a permeable membrane against the electrode. The system may use Escherichia coil as the biological cells and respond to formaldehyde or to dichloromethane. <IMAGE>

Description

MONITORING SYSTEM This invention relates to systems of monitoring or detecting the presence of chemical species using biological cells, by methods in which the effects of the chemical species (for example, but not exclusively, those toxic or otherwise harmful to humans) on the organisms are measured.

It is well known that biological cells can be used for measuring toxic, inhibitory or stimulatory effects of aqueous solutions of chemical species. In particular, microbial toxicology has been studied for many years.

Various organisations, e.g. the Organisation for Economic Cooperation & Development (OECD) and the United States Environmental Protection Agency (EPA), specify a number of microbial tests for evaluating biochemical and environmental effects of chemical substances. See for example OECD Guidelines for the Testing of Chemicals, 201, "Alga growth inhibition test", 7 June 1984; and Walker J. D., A U.S. EPA perspective on ecotoxicity testing using microorganisms", In Dutka BJ and Bitton G (Eds.), Toxicity Testing Using Microorganisms, Volume II, pp.

175-186, CRC Press, Inc., Boca Raton, Florida (1986).

This, however, usually involves the prolonged incubation (many hours or even days) of the microorganisms with the chemical species of interest.

Effects, such as lack of growth or mutation, on the microorganism are measured during or after the incubation.

In recent years, there has been much interest in the development of rapid methods for measuring toxic, inhibitory, stimulatory, mutagenic or other effects using immobilised prokaryotic or eukaryotic cells. In the case of microbial cells, these have frequently been immobilised on a dissolved oxygen sensor which is used to measure the effects of the aqueous solutions on the cell respiration.

Biological cell respiration is known to have been monitored by immobilising microorganisms on an indicator electrode and providing artificial electron acceptors (such as dissolved potassium ferricyanide) to act as mediators which shuttle electrons from the microorganisms to the electrode. The artificial electron acceptor is reduced by the respiring cells, e.g.

This process is measured as a current when the reduced acceptor is re-oxidised at the electrode, e.g.

Inhibition or stimulation of cell respiration by toxicants or stimulants, respectively, is rapidly observed as a perturbation of measured current.

This approach has been developed for the continuous measurement of surface waters which may be used as a source of potable water. In such a case it is important to detect the presence of toxic materials before the water is supplied to the end user.

The present invention provides a method of detecting the transient or continuous presence of chemical species in the gas or vapour phase, by associating the gas or vapour species with a liquid (by solution or otherwise); introducing immobilised biological cells (microbial or other cells), and the liquid containing the gas or vapour species, to each other in the presence of an artificial electron acceptor, whereby the latter becomes reduced by reaction of the organisms to the said species; re-oxidising the acceptor; and observing and/or measuring changes in the resulting electric current.

Broadly, the invention provides: 1. A system for monitoring biological cells perturbed by gases and vapours.

2. A system for making gases and vapours available in the liquid phase for access to biological cells.

3. A system for making gases and vapours, especially those which are harmful to humans, available in the liquid phase for access to biological cells.

4. A system for making gases and vapours, especially those which are harmful to humans but not necessarily harmful to biological cells, available in the liquid phase for access to those biological cells.

5. A system for monitoring gases and vapours.

6. A system for monitoring gases and vapours, especially those which are harmful to humans.

Surprisingly, it has not been known before for immobilised microorganisms to be used in this way for detecting the presence of gases or vapours. This is probably due to the fact that many microorganisms actively respire only when they are in aqueous solution (or at least in a humid atmosphere), so as they are able to take up respiratory substrates from solution.

However, many if not all gases and vapours can be made available in solution, either directly by arranging for the equilibration of gas and liquid phases, or by providing a suitable environment with (for example) micelles, emulsions or miscible solvents. Such gases and vapours, when made available to the biological cells, can exhibit toxic, inhibitory, stimulatory and/or other effects which may be used to detect the presence of the gases or vapours. There are many examples of gases and vapours which are harmful to humans and other higher forms of life which would be expected also to perturb biological cells. In particular, it is not believed to have been known before for transient perturbations of biological cells to be used for detecting gases and vapours.

Some examples of the method of the invention will now be described, by way of example only and with reference to the accompanying drawings, in which: Figure 1 shows diagrammatically an apparatus for detecting the continuous presence of a gas or vapour; Figure 2 is a scrap cross section showing the working electrode of the same apparatus; Figure 3 illustrates detection of transient perturbations resulting from brief exposure of an organism to a vapour; Figure 4 shows the effect on electric current when an organism is exposed to formaldehyde in a steady state; and Figure 5 shows the relationship between change of current and vapour pressure under conditions related to those in Figure 4.

The change between gas or vapour and solution phases in the continuous or steady state, and subsequent detection, can be achieved using, for example, the apparatus shown in Figures 1 and 2. The test gas or vapour is introduced via a gas transfer line 1 and bubbler 12 to a vessel 10, which contains a liquid 11 in which are dissolved suitable substrates and redox mediators. Excess gas is exhausted via a vent 2, whilst a pump 7 is used to move liquid through a fluid transfer line 3 to an electrochemical flow-through cell 9. The electrochemical cell 9 contains a working electrode 4 and a reference electrode 6; applied potentials, and generated currents are controlled via a potentiostat 5 which is connected by wires to each of the electrodes 4 and 6. The solution leaves the flowthrough cell 9 via the pump and a liquid waste pipe 8.

The working electrode 4 (see Figure 2) contains biological cells 17, which are immobilised by using a permeable membrane 18 held by an elastic O-ring 16 against a platinum or carbon electrode 15, which is set into an insulating sheath 13. Electrical connection to the potentiostat 5 is obtained via the electrical conductor 14 of the electrode.

An important aspect of the method is that, using suitable apparatus, it can also be used to detect the transient presence of a gas or vapour. This is illustrated in Figure 3, which shows the transient perturbations resulting from brief exposure of Escherichia coli to dichloromethane. Dichloromethane is a widely used organic solvent which is sparingly soluble in aqueous solution. The vapour is harmful to humans and is a controlled substance. However, at concentrations proscribed by the United Kingdom's Health and Safety Executive there is not necessarily any observable harmful effect on biological cells.

Figure 3, however, shows that clear changes in the observed current appear when immobilised Escherichia coli are briefly exposed to dichloromethane. When the vapour pressure of dichloromethane is low, then the solution concentration is also low and the respiration of the Escherichia coli is apparently little affected by its brief exposure to the dichloromethane.

Analysis of the response from biological cells exposed to gases and vapours is complicated by the fact that there are many biochemical and biophysical routes by which the cells may be perturbed. On the other hand, this makes the use of biological cells a very powerful tool in determining the presence or absence of a wide variety of gases and vapours. The data from each analyte does of course have to be carefully interpreted in order to assess the effect of the analyte on the biological cells. However, the perturbations may be analysed so as to relate the signals to the vapour pressure of the gas or vapour which gave rise to it.

Consequently, these signals may be used to indicate the presence of the vapour even if it is only present very briefly, i.e. in the absence of any apparent lasting toxic, inhibitory or other effect.

The results are particularly useful for monitoring those gases and vapours which are known to be harmful to humans. Formaldehyde, for example, has a vapour which is known to be harmful to humans. The effect on the current from immobilised Escherichia coli when exposed to formaldehyde is shown in Figure 4. The rate of change of current on addition of the formaldehyde can be related to the formaldehyde vapour pressure as shown in Figure 5.

Claims (6)

1. A method of detecting the presence of specific chemical species in a non-liquid fluid phase, including the steps of: (i) associating the said fluid phase with a liquid; (ii) introducing to each other immobilised biological cells and the liquid in association with the said species, in the presence of an artificial electron acceptor, whereby the latter becomes reduced by reaction of the cells to the said species; (iii) re-oxidising the acceptor; and (iv) observing and/or measuring changes in the resulting electric current.

2. A method according to Claim 1, wherein step (i) comprises dissolving the non-liquid phase in the liquid.

3. A method according to Claim 1, wherein step (i) comprises associating the non-liquid phase with the liquid in an environment comprising at least one micelle, emulsion or miscible solvent.

4. A method according to any one of Claims 1 to 3, wherein step (iv) comprises observing and/or measuring transient changes.

5. A method of detecting the transient presence of chemical species in a non-liquid fluid phase, substantially as described in the foregoing description with reference to Figure 3 of the accompanying drawings.

6. A method of detecting the continuous presence of chemical species in a non-liquid fluid phase, substantially as described in the foregoing description with reference to Figures 1, 2, 4 and 5 of the accompanying drawings.