MEDIATORS FOR BIOELECTROCHEMICAL CELLS

Abstract: l,4-Benzoquinone can act as a mediator of electron transfer in bioelectrochemical cells for measuring microbiological activity in a sample. In combination with ferricyanide it can provide a surprisingly high level of mediator activity with many microorganisms, and a more even response when the sample may contain a mixture of microorganisms.

Description: Field of the Invention This invention relates to bioelectrochemical cells, for use in measuring microbiological activity in a sample. Background In bioelectrochemical cells, especially for measuring bacterial activity, ferricyanide has been proposed as a mediator for electron transfer between the bacteria and the electrodes. l,4-Benzoquinone is also known as a mediator of electron transfer. Different bacteria present different levels of response, and for example Pseudomonas species (e.g. P . aeruginosa ) normally have a rather poor response compared with, say, E . coli using ferricyanide as mediator. Large differences in response from different microorganisms makes it difficult to relate the signal to the amount of microbiological activity, unless one is expecting a particular microorganism to predominate. Summary of the Invention It has now been found that a ferricyanide and a l,4-benzoquinone can exhibit an unexpectedly high level of mediator activity compared with ferricyanide alone, or even compared with l,4-benzoquinone alone, whereas in some cases the response is not significantly higher, and can even be lower, than using the mediators separately. The effect is such that a more even response can normally be expected from general or non-specific microbiological activity using the cocktail of mediators; while a particularly enhanced response can be expected when testing for many individual microorganisms using the mediator cocktail. Description of Preferrred Embodiments The invention will be further illustrated by reference to the following examples. EXAMPLE l Use of l,4-benzoquinone and ferricyanide cocktail in a two electrode bioelectrochemical cell (BEC) The BEC was set up at pH 7.0 in a sodium phosphate buffer. Benzoquinone was prepared at 25mM concentration and ferricyanide at 250mM concentration. Initially the E . coli (l50µl) BEC response using 500µl ferricyanide was compared with that of 500µl ferricyanide + 500µl benzoquinone added together as a cocktail. The results are shown in Table l. From the foregoing it will be seen that the cocktail gave about a 7.5-fold enhancement over the ferricyanide alone for E . coli . This experiment was repeated using P . aeruginosa . In this case the cocktail gave about a 750-fold enhancement over the ferricyanide alone. EXAMPLE 2 Further investigation of the cocktail of Example 1 was made. The response of the benzoquinone cocktail was compared with that of the two individual components. The results are shown in Table 2. Comparison of the BEC response of the benzoquinone cocktail with that of the two individual components 150µl P. aeruginosa 1.2 x 10¹⁰ org/ml Mediator BEC response µA/min benzoquinone (500µl) 0.64 benzoquinone (1000µl) 1.1 benzoquinone (500µl) + ferricyanide (500µl) 2.9 ferricyanide (500µl) 0.06 These results showed that the rate of change of current with time obtained with the mediator cocktail was more than the sum of the slopes of the individual components. In this case the cocktail gave a 48-fold enhancement over ferricyanide alone. EXAMPLE 3 It was noted that the BEC response using 1ml of benzoquinone was greater than that using 0.5ml; therefore the concentration of benzoquinone relative to ferricyanide was increased in an attempt to further enhance the BEC response. The results are shown in Table 3. Effect of varying benzoquinone concentration in the cocktail on the BEC response 150µl P. aeruginosa 1.2 x 10¹⁰ org/ml Mediator BEC response µA/min benzoquinone (1000µl) 0.88 benzoquinone (1000µl) + ferricyanide (500µl) 1.22 benzoquinone (500µl) + ferricyanide (500µl) 1.37 ferricyanide (500µl) 0.09 It will be seen that increasing the relative amount of benzoquinone in the mediator cocktail did not further enhance the BEC response. 1ml Benzoquinone + 500µl ferricyanide produced a 13.6-fold enhancement of the response whereas 500µl benzoquinone + 500µl ferricyanide resulted in a l5.2-fold enhancement. EXAMPLE 4 The effect of the mediator cocktail with a variety of bacteria The bacteria to be examined were grown up overnight, and then tested in a BEC using the following composition: l50µl Glucose (25mM) 500µl Bacterial suspension Mediator: (i) 250µl Ferricyanide (250mM), or (ii) 250µl Benzoquinone (25mM), or (iii) 250µl Ferricyanide (250mM) + 250µl Benzoquinone (25mM). Sodium phosphate buffer (50mM, pH 7.0) containing KCl (to a final concentration of l00mM in the BEC) to a final BEC volume of l0ml. The work was carried out at 30°C using a platinum working electrode and a silver/silver chloride reference electrode. The results were obtained as mean values of a minimum of four runs. The standard BEC solution was prepared and the response was observed with each of the three mediator combinations given above. The results are shown in Table 4. Measured Rates ORGANISM FERRICYANIDE (%) (a) BENZOQUINONE (%) (a) COCKTAIL (%) (b) Acinetobacter 8.3 44.4 189.8 Agrobacterium tumefaciens 208.5 36.9 32.4 Alcaligenes faecalis 80.4 47.5 78.1 Bacillus licheniformis 176.7 65.1 41.3 Bacillus megaterium 205.7 67.9 36.6 Escherichia coli 6.2 41.1 211.2 Micrococcus lysodeikticus 2.4 85.6 113.6 Pseudomonas aeruginosa 0.6 61.4 161.4 Pseudomonas fluorescens 4.5 122.6 78.7 Staphylococcus aureus 18.3 70.6 112.5 Streptococcus faecalis 4.7 79.8 118.2 (a): rate as % of cocktail response (b): cocktail rate as % of response of both mediators combined Table 4 was compiled from measured data, examples of which are shown in Table 5. Organism at 1 x 10⁹/ml BEC RESPONSE (µA/min) Cocktail Ferricyanide Escherichia coli 8.57 0.53 Bacillus licheniformis 0.75 1.33 Pseudomonas aeruginosa 1.39 0.007 Total variation in relative responses 11-fold 200-fold The Table 5 data include E . coli , which is the best responder with the cocktail (out of those tested), P . aeruginosa , which is the poorest responder with ferricyanide alone, and B . licheniformis , a strain whose response is decreased with the cocktail relative to ferricyanide alone. It will be seen that the cocktail has greatly evened out the response of a mixed culture at a given concentration. Tables 6 and 7 illustrate the effect of varying the relative concentrations of the two mediator components. BENZOQUINONE CONCENTRATION (µM) BEC RESPONSE (µA/min) 250 3.95 100 1.05 25 0.74 2.5 0.58 Ferricyanide concentration: 2.5mM Sample: 5 x 10⁶ org/ml E . coli COCKTAIL COMPOSITION BEC RESPONSE (µA/min) FERRICYANIDE (mM) BENZOQUINONE (mM) 12.5 1.25 10.70 12.5 0.25 10.62 6.25 0.625 11.70 2.5 1.875 10.67 1.0 1.25 9.98 Sample: P . aeruginosa 5 x 10⁸ org/ml In Table 6, with E . coli , the ferricyanide:benzoquinone ratio varies from 1000:1 to 10:1 with only a seven-fold change in signal. In Table 7, with P . aeruginosa , that ratio varies from 50:l to 0.8:l with virtually no change in signal. The ratios therefore do not appear to be critical. Likewise the overall concentrations of the mediators does not seem to be critical, and can be lower than those exemplified, especially with the more sensitive microorganisms. We have further demonstrated above, the use of the mediator cocktail with examples of gram positive and gram negative bacteria, motile and non-motile organisms, rods and cocci.

Claims: A bioelectrochemical cell for use in measuring microbiological activity in a sample, characterised by the incorporation of a ferricyanide and a l,4-benzoquinone as a mediator of electron transfer. A method of testing for microbiological activity in a sample which comprises measuring an electrical signal from a bioelectrochemical cell containing the sample, characterised in that the cell incorporates a ferricyanide and a l,4-benzoquinone as a mediator of electron transfer. A method according to claim 2 wherein the microorganisms comprise gram positive bacteria. A method according to claim 2 wherein the microorganisms comprise gram negative bacteria. A method according to claim 2 wherein the microorganisms comprise E . coli . A method according to claim 2 wherein the microorganisms comprise Pseudomonas aeruginosa . A method according to claim 2 wherein the microorganisms comprise Micrococcus lysodeikticus . A method according to claim 2 wherein the microorganisms comprise Staphylococcus aureus . A method according to claim 2 wherein the microorganisms comprise Streptococcus faecalis . A method according to claim 2 wherein the microorganisms comprise Acinetobacter . A method according to any one of claims 2 to l0 wherein the sample contains a mixture of different species of genera or microorganism.

Inventors: CARDOSI, MARCO, RAMSAY, GRAHAM, SCHNEIDER, BERNARD HENRY, SWAIN, ANN, TURNER, ANTHONY PETER FRANCIS
Applicants: PAUL DE LA PENA LIMITED
Application year: 1987
Main classification: C12Q1
IPC codes: C12Q1