GB2502253A

GB2502253A - Method for detecting a species indicative of microbial growth

Info

Publication number: GB2502253A
Application number: GB201205614A
Authority: GB
Inventors: Jonathan Metters; Dimitrios Kampouris; Craig Banks
Original assignee: Manchester Metropolitan University
Current assignee: Manchester Metropolitan University
Priority date: 2012-03-29
Filing date: 2012-03-29
Publication date: 2013-11-27
Also published as: GB201205614D0

Abstract

A method for detecting a species indicative of microbial growth comprises: contacting a sample of interest with an oxidising agent to generate an oxidised species, subjecting the oxidised species to a varying applied voltage whilst measuring the current response, and analysing said current response to determine whether the oxidised species is indicative of microbial growth. Preferably, the sample contains one or more microbial volatile organic compounds (MVOCs), such as one or more C1-12 alcohol, aldehyde, ketone or ester compounds, and preferably one or more of 1-octen-3-ol, 3-octanone, and 3-octanol. The oxidising agent may be sodium hypochlorite. The method may use cyclic voltammetry, and the voltage may be varied over the range -2 V to +2 V. A handheld device suitable for putting the method into effect is also described. A method for detecting a species derived from a carbonyl compound is also disclosed.

Description

ELECTROCHEMICAL METHOD AND DEVICE

The present invention relates to a method and apparatus for detecting a species indicative of microbial growth. The present invention further provides a method and apparatus for detecting a species derived from a carbonyl compound, such as an alcohol or a ketone.

The unwanted growth of microbes, such as fungi, is not only potentially harmful to the substance upon which the microbes are growing but may also give rise to allergies, irritations, infections and toxic effects in humans exposed to compounds evolved by the growing microbes.

Existing methods for detecting the microbial volatile organic compounds (MVOC)s produced during microbial growth typically require the use of expensive, time-consuming laboratory-based techniques, such as culture methods and/or gas chromatography (GO) in combination with mass spectrometry (MS), to separate and characterise the large number of structurally related organic compounds evolved by the growing microbes. Similarly complicated techniques must also currently be used for the detection and characterisation of carbonyl compounds, such as alcohols, ketones, aldehydes and/or esters, per so across a wide range of different technical fields. Those skilled in the art will appreciate that the direct electrochemical detection of MVOCs is not trivial, hence the current necessity to use the complicated, expensive and time-consuming methods mentioned above.

An object of the present invention is to obviate or mitigate one or more of the problems outlined above in respect of the detection of compounds evolved during microbial growth and/or carbonyl compounds.

According to a first aspect of the present invention there is provided a method for detecting a species indicative of microbial growth comprising: contacting a sample of interest with an oxidising agent to generate an oxidised species; subjecting the oxidised species to a varying applied voltage whilst measuring the current response; and analysing said current response to determine whether the oxidised species is indicative of microbial growth.

According to a second aspect of the present invention there is provided device for detecting a species indicative of microbial growth comprising: a working electrode and a counter electrode operable to apply a varying voltage across said electrodes; a solution containing an oxidising agent in contact with said working electrode; and means to receive a sample of interest such that it contacts said solution.

The present invention further provides a handheld MVOC sensor, or a monitor, for example in the form of a patch or a label, having the features of the device according to the second aspect of the present invention which is suitable to carryout the method according to the first aspect of the present invention.

The present invention provides a method and apparatus, for example in the form of a simple, relatively cheap sensor or a detector, which can be used to monitor the MVOCs indicative of fungal growth without the need for culture methods or specialist equipment (e.g. GC-MS). Such a method and apparatus could be used to protect the health of film archivists who are at risk of exposure to the products of fungal growth and to ensure the safe handling of delicate cinematographic film. Additionally, the method and apparatus of the present invention could be used in any application where substances are at risk of microbial contamination, such as foodstuffs (cereal crops such as wheat maize, barley, grains, peanuts, fruits etc). Furthermore, the present invention provides a means by which carbonyl compounds, such as alcohols, ketones, aldehydes and/or esters, per se can be detected, which could find application across a range of fields unrelated to microbial growth.

According to a third aspect of the present invention there is provided a method for detecting a species derived from a carbonyl compound comprising: contacting a sample of interest with an oxidising agent to generate an oxidised species; subjecting the oxidised species to a varying applied voltage whilst measuring the current response; and analysing said current response to determine whether the oxidised species is derived from a carbonyl compound.

A fourth aspect of the present invention provides a device for detecting a species derived from a carbonyl compound comprising:

I

a working electrode and a counter electrode operable to apply a varying voltage across said electrodes; a solution containing an oxidising agent in contact with said working electrode; and means to receive a sample of interest such that it contacts said solution.

There is further provided a handhold carbonyl compound sensor, or a monitor, for example in the form of a patch or a label, having the features of the device according to the fourth aspect of the present invention which is suitable to carryout the method according to the third aspect of the present invention.

As previously mentioned, the direct electrochemical detection of MVOCs and carbonyl compounds is a complicated problem requiring the use of expensive and time-consuming techniques. The devisors of the present invention have overcome this significant problem by developing a method in which a sample containing MVOCs, or one or more carbonyl compounds, is first contacted with an oxidising agent which then unexpectedly generates a species that is electrochernically active and that exhibits a clear and reproducible signature or finger-print' in terms of current response to a changing applied voltage. This therefore provides an indirect methodology for sensing MVOCs and carbonyl compounds which is both relatively simple and reliable.

The method of the present invention is based upon an electrochemical technique which involves applying a potential (V) and monitoring the current response (A). The most preferred method is cyclic voltammetry, but chronoamperometry, square-wave voltammetry or differential pulse voltammotry may also be used. In the method of the present invention the voltage may be varied over any suitable range depending upon the particular sample under investigation. A preferred voltage range is from -2 V to +2 V more preferably from -1.5 V to +1.5 V. When the preferred method of cyclic voltammetry is employed the results obtained can be used to plot a cyclic voltammograrn which can then be analysed to determine whether the sample under test contained one or more compounds derived from microbial growth. The step of analysing the current response to the applied voltage preferably comprises a comparison of the current generated in the sample of interest to the current generated in a reference sample that does not contain a species that is indicative of microbial growth at a predetermined applied voltage. The identification of an increase in the current generated in the sample of interest as compared to the reference sample at the predetermined voltage confirms the presence of a species indicative of microbial growth in the sample of interest. That is, the feature of the cyclic voltammogram which confirms the presence of oxidised species derived from one or more MVOC or carbonyl compound is a relatively large hump' or peak' around a particular characteristic voltage. While not wishing to be bound by any particular theory, it is currently believed that the peak corresponds to the voltage at which electrochemical reduction of the previously oxidised MVOC species occurs. In a preferred embodiment, the predetermined or characteristic voltage is in the range of around 0 to +1 V, more preferably in the range of around +0.25 V to +0.75 V, and is most preferably at around +0.5 V. The method and apparatus of the present invention preferably employs a working electrode and a counter electrode, and preferably further employs a reference electrode. While a typical, laboratory-type fixed three electrode system may be used, in a preferred embodiment of the apparatus of the present invention screen printed electrodes (SPEs) are used since they can be produced cheaply and can be provided in the form of a disposable, one-shot or single-use sensor.

The sample of interest preferably comprises one or more microbial volatile organic compounds (MVOCs). Such compounds preferably include carbonyl compounds, for example alcohols, aldehydes, ketones and/or esters. Said compounds may be 01-12 alcohols, aldehydes, ketones and esters, more preferably 02-10 and yet more preferably C48 alcohols, aldehydes, ketones or esters.

Initial work on microbial-contaminated cinematographic film utilising headspace solid phase microextraction coupled with GC-MS identified over 150 MVOCs from 16 fungal isolates, with over 40 being common to 2 or more isolates. 1-octen-3-ol was produced from 13 of the isolates analysed, 3-octanone from 10 of the isolates and 3-octanol from 4 isolates. These three MVOCs therefore appear to be key chemical markers indicative of fungal growth on cinematographic film.

Octanol occurs naturally in the form of esters in some essential oils. The primary use of octanol is in the manufacture of various esters (both synthetic and naturally occurring), such as octyl acetate, which are used in perfumery and flavours. Other uses include experimental medical applications utilizing octanol to control Essential Tremor and other types of involuntary neurological tremors. The ascomycetous fungi Pen/cl/I/urn, used in the production of penicillin is known to produce the metabolites 1-Octen-3-ol, 3-Octanone and also 3-Octanol. This information is of importance as Pen/cl/I/urn plays a key role within the natural environment in addition to within food and aforementioned drug production. Thus, in a particularly preferred embodiment of the present invention the sample of interest contains one or more compounds selected from the group consisting of 1 -Octen-3-ol, 3-Octanone and 3-Octanol.

Vacuum headspace analysis of the following substances has indicated the presence of 1-Octen-3-ol: artichoke; beans; beef; beer; bergamot; bread; wheat; peppermint oil; savoury oil summer; and tomato witch hazel leaf oil. Similar analysis has indicated the presence of 3-Octanol in: banana basil flower oil; basil leaf oil; bay leaf oil; Swiss cheese; lavender oil; mushroom; peppermint; strawberry; and truffle.

Related molecules with varying carbon-chain length include the following, which have been identified in the substance listed: 1-Hepten-3-ol: banana; beer; patchouli oil china; green pepper; and red pepper; 3-Heptanol: banana; brandy; butter; coffee; and spearmint; 3-Hexanol: Clary sage oil; Propane-i,3-diol: No natural occurrence; Propanoic Acid: bread wheat; blue cheese; cheddar cheese; lavender; and whiskey; and Ethanol: alcoholic products.

From the above it will be appreciated that the ability to detect the presence of carbonyl compounds, such as MVOCs, would be of interest across a wide range of

fields.

It is particularly preferred that the carbonyl compounds in the sample of interest are oxidisable, that is, that they are susceptible to oxidation by a suitable oxidising agent, such as sodium hypochlorite (NaOCI) or another oxidising agent of similar oxidising strength, i.e. which has the potential to oxidise similar compounds to NaCCI.

Preferred carbonyl compounds include alcohols that can be oxidised to their corresponding aldehyde or ketone, and then subsequently oxides through to their corresponding carboxylic acid or derivative carboxylic acids. Forming carboxylic acids is particularly advantageous since they are usually more soluble in aqueous media than their corresponding aldehyde, ketone or alcohol. Oxidation of the compounds preferably generates an electrochemically active substance which can itself be reduced under suitable electrochemical conditions. Said reduction typically involves the transfer of electrons to the oxidised species.

Advantages of the apparatus of the present invention include the fact that it can be embodied in a small hand-held electrochemical sensor that could fit into a user's pocket or jacket, which is clearly a significant advantage over existing techniques such as GO-MS. A further advantage of both the method and apparatus is that the result would be available very quickly. A typical GO-MS analysis can take many hours or days, the present invention could produce a result in minutes, or less. It will also be appreciated that the apparatus of the present invention could be set-up and used rapidly without any sample preparation or calibration. Furthermore, the method and apparatus of the present invention would only require relatively small sample sizes as compared to existing techniques, such as GC-MS.

The present invention thus provides an electrochemical sensor, which could be placed into areas to monitor MVOOs indicating microbial contamination which are detrimental to health. By way of example, it is envisaged that an MVOC sensor according to the present invention could be placed into a Oinefilm can' or a foodstuff container for grains, peanuts or the like during transportation onboard container ships where conditions are such that microbial contamination can readily occur which could damage the Cinefilm' or spoil the foodstuff, and potentially pose a health risk.

Preferred embodiments of the various aspects of the present invention will now be described with reference to the following Experimental information, non-limiting Examples and Figures, in which: Figure 1 is a composite cyclic voltammogram of two systems: a first system in which the sample is just a pHil buffered solution; and a second system using the same pHi 1 buffered solution containing an exemplary MVOO (20 mM 3-Octanol) following exposure to the oxidising agent, sodium hypochlorite (NaOCl); Figure 2 is a calibration plot showing the maximum current observed at the characteristic applied voltage (Peak Height') as the volume of the oxidising agent (NaCCI) was increased; Figure 3 is a calibration plot showing the maximum current observed at the characteristic applied voltage (Peak Height') as the concentration of a particular exemplary MVOO (3-Octanol) was increased; Figure 4 is a calibration plot showing the maximum current observed at the characteristic applied voltage (Peak Height') as the concentration of a particular exemplary MVOC (i-Octen-3-ol) was increased; Figure 5 is a calibration plot showing the maximum current observed at the characteristic applied voltage (Peak Height') as the concentration of a particular exemplary MVOO (i-Heptan-3-ol) was increased; Figure 6 is a calibration plot showing the maximum current observed at the characteristic applied voltage (Peak Height') as the concentration of a particular exemplary MVOC (3-Heptanol) was increased; Figure 7 is a calibration plot showing the maximum current observed at the characteristic applied voltage (Peak Height') as the concentration of a particular exemplary MVOO (3-Hexanol) was increased; Figure 8 is a calibration plot showing the maximum current observed at the characteristic applied voltage (Peak Height') as the concentration of a particular exemplary MVOO (Propanoic acid) was increased; Figure 9 is a calibration plot showing the maximum current observed at the characteristic applied voltage (Peak Height') as the concentration of a particular exemplary MVOC (Propane-i,3-diol) was increased; and Figure 10 is a calibration plot showing the maximum current observed at the characteristic applied voltage (Peak Height') as the concentration of a particular exemplary MVOC (Ethanol) was increased.

EXPERIMENTAL

Work was conducted to quantify microbial contamination and growth on cinematographic film utilising headspace solid phase microextraction coupled with GO-MS. MVOCs were produced only when mould was actively growing on the cinematographic film. Over 150 volatile compounds were detected from 16 fungal isolates, with over 40 being common to 2 or more isolates. i-octen-3-ol was produced from 13 of the isolates analysed, 3-octanone from 10 of the isolates and 3-octanol from 4 isolates. These three MVOCs are therefore key chemical markers indicative of fungal growth on cinematographic film.

EXAMPLES

To afford a more convenient method of detecting MVOCs the devisors of the present invention developed an indirect sensing methodology which involves first contacting the MVOCs with an oxidising agent and then subjecting the resulting species to a varying applied voltage whilst measuring the current response by cyclic voltammetry using screen printed electrodes.

Figure 1 is a typical cyclic voltammogram showing the response of a sample containing 20 mM 3-Octanol following exposure to the relatively strong oxidising agent, sodium hypochlorite (NaOCI). The feature of the cyclic voltammogram which confirms the presence of oxidised species derived from the 3-Octanol is the large hump' or peak' at around +0.5 V, corresponding to the electrochemical reduction of the previously oxidised species.

A series of experiments were conducted to determine the optimum volume of the oxidising agent, sodium hypochlorite, for the greatest achievable sensitivity of the electrochemical system. As can be observed in Figure 2, the optimum volume of sodium hypochlorite within each of the solutions was found to be 100 pL. This volume of sodium hypochlorite was used in the subsequent analyses of the following MVOCs of interest: 3-Octanol; 1-Octen-3-ol; 1-Hepten-3-ol; 3-Heptanol; 3-Hexanol; Propane-i,3-diol; and Ethanol. Each of these compounds was observed to generate a similar characteristic peak on a cyclic voltammogram when tested in the same was as described above. The cyclic voltammetry testing was repeated in respect of each compound over a range of concentrations of the compound under test. The results of these analyses are shown in Figures 3 to 10 respectively. In each case the results were obtained using a screen printed electrode system. The results shown in Figures 3 to 8 are the average results from carrying out each test three times. The results shown in Figure 9 and 10 are the results of carrying out the test on each compound once.

Claims

CLAIMS1. A method for detecting a species indicative of microbial growth comprising: contacting a sample of interest with an oxidising agent to generate an oxidised species; subjecting the oxidised species to a varying applied voltage whilst measuring the current response; and analysing said current response to determine whether the oxidised species is indicative of microbial growth.
2. A method according to claim 1, wherein the sample contains one or more microbial volatile organic compounds.
3. A method according to claim 1, wherein the sample contains one or more Ci 12 alcohol, aldehyde, ketone or ester compounds.
4. A method according to claim 1, wherein the sample contains one or more compounds selected from the group consisting of 1-Octen-3-ol, 3-Octanone and 3-Octanol.
5. A method according to any preceding claim, wherein the oxidising agent is sodium hypochorite or an oxidising agent of similar oxidising strength.
6. A method according to any preceding claim, wherein the voltage is varied over a range of from -2 V to +2 V.
7. A method according to any one of claims 1 to 5, wherein the voltage is varied over a range of from -1.5 V to +1.5 V.
8. A method according to any preceding claim, wherein said analysis comprises a comparison of the current generated in the sample of interest to the current generated in a reference sample that does not contain a species that is indicative of microbial growth at a predetermined applied voltage.
9. A method according to claim 8, wherein the identification of an increase in the current generated in the sample of interest as compared to the reference sample at the predetermined voltage confirms the presence of a species indicative of microbial growth in the sample of interest.
10. A method according to claim 8 or 9, wherein the predetermined voltage is in the range of around 0 to +1 V.
11. A method according to claim 8 or 9, wherein the predetermined voltage is around +0.5 V.
12. A device for detecting a species indicative of microbial growth comprising: a working electrode and a counter electrode operable to apply a varying voltage across said electrodes; a solution containing an oxidising agent in contact with said working electrode; and means to receive a sample of interest such that it contacts said solution.
13. A device according to claim 12, wherein the device further comprises a reference electrode.
14. A device according to claim 12 or 13, wherein the device comprises screen printed electrodes.
15. A method for detecting a species derived from a carbonyl compound comprising: contacting a sample of interest with an oxidising agent to generate an oxidised species; subjecting the oxidised species to a varying applied voltage whilst measuring the current response; and analysing said current response to determine whether the oxidised species is derived from a carbonyl compound.
16. A device for detecting a species derived from a carbonyl compound comprising: a working electrode and a counter electrode operable to apply a varying voltage across said electrodes; a solution containing an oxidising agent in contact with said working electrode; and means to receive a sample of interest such that it contacts said solution.