WO2008009046A1

WO2008009046A1 - Detection of enzymes and microorganisms and devices therefor

Info

Publication number: WO2008009046A1
Application number: PCT/AU2007/000991
Authority: WO
Inventors: William Graham Fox Ditcham; Simon Andrew Reid
Original assignee: Environmental Biotechnology Crc Pty Limited
Priority date: 2006-07-17
Filing date: 2007-07-17
Publication date: 2008-01-24
Also published as: WO2008009046A8; TW200817520A

Abstract

The present invention relates to methods for detecting the amount of an enzyme of interest, which may be expressed b a microorganism, in a sample, such methods comprising: (a) incubating a reaction mixture comprising the sample and at least one substrate which can be acted on by the enzyme; (b) contacting an electrochemical sensor with the reaction mixture comprising the sample either: (i) after a selected time of incubation of the reaction mixture or (ii) at the same time as or soon after preparing the reaction mixture, wherein the sensor comprises a working electrode which has immobilised therein or thereto a second enzyme which is capable of acting on a substance selected from one or more of the at least one substrate and a product resulting from action of the enzyme of interest on the at least one substrate; wherein action of the second enzyme on the at least one substrate or product results in a change in electric potential at said working electrode; (c) either: (i) detecting the change in electrical potential at the working electrode after the contacting in step (b)(i); or (ii) determining the period of time required for the electric potential at the working electrode to return to a substantially steady baseline value after the contacting in step (b)(ii); and (d) determining from the change in potential detected at step (c)(i) or period of time determined at step (c)(ii) the amount of enzyme of interest in the sample. The invention also relates to sensors suitable for such methods.

Description

Detection of Enzymes and Microorganisms and Devices Therefor

Technical Field

The present invention relates to sensitive and rapid methods for the detection of microorganisms, such as coliforms in waterways, and enzymes such as may be associated with their presence, and devices for carrying out such methods.

Background Art

Current and established methods for enumerating and identifying microorganisms, including bacteria such as faecal coliforms in potable water, include direct visual detection, such as colony counting, or indirect observation based on colour changes associated with metabolic effects on certain substrates, flow cytometric/ cell sorting techniques, immunogenic detection, detection of specific enzymes associated with particular microorganisms, or detection of specific nucleotide sequences within target microorganisms.

Most of these techniques require amplification of the number of cells of the microorganism, amplification of the amount of target detection material (such as specific nucleic acid sequences), and/or lengthy incubations with substrates if the number of cells of the target microorganism in a given sample is, or is expected to be small. For example, detection of coliform bacteria in water samples requires culturing the microorganism so as to increase the number of cells. Current industry practice is to obtain a small sample of water (-100ml), pass it through a filter to trap bacteria and then culture that filter on an appropriate growth medium to amplify bacterial numbers and/or allow sufficient time to allow a detectable change in the medium, before visually evaluating the growth of specific indicator species for faecal contamination (ie. faecal coliforms). Alternatively, specific nucleotide sequences may be used to detect the presence of specific microorganisms. In view of the limited number of bacterial cells of any specific genus, species or strain that may be present in certain samples (such as water), the preferred technique usually involves PCR to amplify the amount of target nucleic acid before detection by, for example, Southern blot. Because coliform bacteria are a diverse grouping of bacteria, such a technique may require the use of a large number of PCR amplification probes, and detection probes, In addition, poor purity or specificity of amplification probes can lead to incorrect or misleading results.

Thus, current methods used for detecting or enumerating microorganisms, or types of microorganisms in samples are often time consuming and/or require sophisticated laboratory equipment and a reasonable level of technical expertise. The usual time from collection of a sample to recording results is typically at least 24 hours. In addition, there are difficulties in obtaining representative samples from remote sites and the cost per test is typically high.

In addition, the data generated by such techniques can only be used to document past events (i.e. 24 hours ago) rather than enable preventative strategies to be developed/adopted immediately. In addition, the above difficulties preclude the routine implementation of a statistically rigorous testing regime of water supplies.

Object of the Invention

It is an object of the present invention to provide an improved, faster and sensitive method for detecting specific microorganisms, or types of microorganisms, such as coliforms in water supplies/sources, and means/devices for this purpose.

Summary of the Invention

It has now been surprisingly found that specific enzymic activities, such as those associated with specific microorganisms or types of microorganisms, may be detected electrochemically, sensitively and quickly, using uncomplicated techniques and materials.

According to a first embodiment, the present invention provides a method for detecting the amount of an enzyme of interest in a sample, comprising:

(a) incubating a reaction mixture comprising said sample and at least one substrate which can be acted on by said enzyme;

(b) contacting a second enzyme with the reaction mixture, wherein said second enzyme is capable of acting on a substance selected from one or more of said at least one substrate and a product resulting from action of the enzyme of interest on said at least one substrate and wherein action of said second enzyme on said at least one substrate or product results in a change in electric potential at a working electrode; and

(c) observing the electric potential at said working electrode at one or more times after said contacting and determining from the electric potential observation or observations the amount of enzyme of interest in the sample.

According to another embodiment, the present invention provides a method for detecting the amount of an enzyme of interest in a sample, comprising:

(b) contacting a second enzyme with the reaction mixture, wherein said second enzyme is capable of acting on a substance selected from one or more of said at least one substrate and a product resulting from action of the enzyme of interest on said at least one T/AU2007/000991

substrate and wherein action of said second enzyme on said at least one substrate or product results in a change in electric potential at a working electrode; and

(c) detecting the electric potential at said working electrode at one or more times after said contacting and determining from the electric potential or potentials detected the amount of enzyme of interest in the sample.

Said second enzyme may be immobilized in or on said working electrode, and said working electrode may be part of an electrochemical sensor. Although in certain embodiments the second enzyme is physically linked to the working electrode, it is also envisaged that the second enzyme may be unbound but immobilized nonetheless, for example, by retaining the second enzyme in a matrix in or on said working electrode or within a limited volume such that the second enzyme is in sufficient proximity to the working electrode surface such that its action on said at least one substrate or product results in a change in electric potential at the working electrode.

Thus, in another embodiment, the present invention provides a method for detecting the amount of an enzyme of interest in a sample, comprising:

(b) contacting an electrochemical sensor and a second enzyme with the reaction mixture, wherein said sensor comprises a working electrode which has immobilized therein or thereto said second enzyme, wherein said second enzyme is capable of acting on a substance selected from one or more of said at least one substrate and a product resulting from action of the enzyme of interest on said at least one substrate; wherein action of said second enzyme on said at least one substrate or product results in a change in electric potential at said working electrode; and (c) observing the electric potential at said working electrode at one or more times after said contacting and determining from the electric potential observation or observations the amount of enzyme of interest in the sample.

In another embodiment, the present invention provides a method for detecting the amount of an enzyme of interest in a sample, comprising: (a) incubating a reaction mixture comprising said sample and at least one substrate which can be acted on by said enzyme;

(b) contacting an electrochemical sensor and a second enzyme with the reaction mixture, wherein said sensor comprises a working electrode which has immobilized therein or thereto said second enzyme, wherein said second enzyme is capable of acting on a substance selected from one or more of said at least one substrate and a product resulting from action of the enzyme of interest on said at least one substrate; wherein action of said second enzyme on said at least one substrate or product results in a change in electric potential at said working electrode; and (c) detecting the electric potential at said working electrode at one or more times after said contacting and determining from the electric potential or potentials detected the amount of enzyme of interest in the sample.

The electrochemical sensor may, for example, be contacted with the reaction mixture after a selected time of incubation of the reaction mixture, such that an amount of substrate proportionate to the amount of enzyme present in the sample will have been converted to product and, from the change in electric potential observed at the working electrode after said contacting, the amount of enzyme of interest in the sample may be determined.

In another manner of carrying out a method of the invention, the electrochemical sensor may be contacted with said reaction mixture at the same time as, or shortly after forming the reaction mixture. The electric potential at the working electrode may then be monitored frequently, or even continuously, and the period of time required for the potential at said working electrode to return to a substantially steady baseline value after said contacting may be determined and used to determine the amount of the enzyme of interest in the sample. For example, if the second enzyme, bound to the working electrode, acts on the same substrate as the enzyme of interest, more enzyme of interest present in the sample, will result in a shorter period of time for the potential at the working electrode to return to a steady baseline value; if the second enzyme acts on a product of the action of the enzyme of interest on the substrate, more the enzyme of interest present in the sample will result in a longer period of time for the potential at the working electrode to return to a steady baseline value.

Thus, according to another embodiment of the invention, there is provided a method for detecting the amount of an enzyme of interest in a sample, comprising:

(b) contacting an electrochemical sensor and a second enzyme with the reaction mixture comprising the sample either:

(i) after a selected time of incubation of the reaction mixture or (ii) at the same time as or soon after preparing the reaction mixture, wherein said sensor comprises a working electrode which has immobilized therein or thereto said second enzyme, wherein said second enzyme is capable of acting on a substance selected from one or more of said at least one substrate and a product resulting from action of the enzyme of interest on said at least one substrate; wherein action of said second enzyme on said at least one substrate or product results in a change in electric potential at said working electrode;

(c) either:

(i) detecting the change in electric potential at said working electrode after said contacting in step (b)(i); or (ii) determining the period of time required for the electric potential at said working electrode to return to a substantially steady baseline value after said contacting in step (b)(ii); and

(d) determining from the change in electric potential detected in step (c)(i) or period of time determined at step (c)(ii) the amount of the enzyme of interest in the sample.

The working electrode may have the second enzyme immobilized therein or thereon or adsorbed thereto.

The change in electric potential detected at step (c)(i) may be compared to the change in electric potential detected for: a control reaction mixture comprising no enzyme of interest; a reaction mixture comprising a known amount of enzyme of interest; a calibration curve for change in electric potential versus amount of enzyme of interest, or any combination thereof or the period of time determined at step (c)(ii) may be compared with: the period of time taken for the potential at said working electrode to return to a steady baseline value for a reaction mixture comprising a known amount of enzyme of interest; or a calibration curve for periods of time taken for the potential at said working electrode to return to a steady baseline value versus amount of enzyme of interest in reaction mixtures.

The enzyme of interest may be a β-galactosidase or a β-glucuronidase, and the at least one substrate may be a β-galactoside or β-glucuronide derivative respectively. Such substrates may comprise, for example, a 5-bromo-4-chloro-3-indolyl linked compound, such as a 5-bromo-4-chloro-3-indolyl β-linked glycoside or glucuronide, for example 5- bromo-4-chloro-3 -indolyl β-D-galactopyranoside.

The second enzyme may be the same as the enzyme of interest, or may be a related enzyme. For example, the second enzyme may be a β-galactosidase or a β-glucuronidase. Alternatively, the second enzyme may be an enzyme that can act on a product of the T/AU2007/000991

6 action of the enzyme of interest on the substrate. The second enzyme may be bound to the working electrode by means of a high affinity binding pair, such as a streptavidin- biotin binding pair, may be bound directly to the working electrode, or may be bound to the working electrode via a spacer molecule.

The electroconductive material of the electrode may comprise a polymeric material, which may be, for example, polypyrrole or polyaniline or may comprise a non-polymeric material such as, for example, graphite, carbon paste, gold, platinum, or other suitable electroconductive material. The polymeric material may be an electrically conducting polymeric material e.g. electrically conducting polypyrrole (PPy) or polyaniline (PANI). The resistivity of the electrically conducting polymeric material may be in the range of 90 S/cm to 1,500 S/cm or more, or 400 S/cm to 900 S/cm.

The sample may be derived from a water source and the enzyme of interest may be associated with a microorganism or type of microorganism capable of causing a mammalian intestinal disorder, such as coliform bacteria.

Thus, according to another embodiment of the invention, there is provided a method for detecting coliform bacteria in a sample, comprising:

(a) incubating said sample in a reaction mixture with a substrate comprising a β- galactoside or β-glucuronide derivative;

(b) contacting an enzyme with the reaction mixture, wherein said enzyme is capable of acting on said substrate, and wherein action of said enzyme on said substrate results in a change in electric potential at a working electrode; and

(c) observing the electric potential at said working electrode at one or more times after said contacting and determining from the electric potential observation or observations the number of bacteria in the sample.

According to another embodiment of the invention, there is provided a method for detecting coliform bacteria in a sample, comprising:

(c) detecting the electric potential at said working electrode at one or more times after said contacting and determining from the electric potential or potentials detected the number of bacteria in the sample. According to another embodiment of the invention, there is provided a method for detecting coliform bacteria in a sample, comprising:

(a) incubating said sample in a reaction mixture with a substrate comprising a β- galactoside or β-glucuronide derivative; (b) contacting a sensor and an enzyme with the reaction mixture, wherein said sensor comprises a working electrode which has immobilized therein or thereto said enzyme, wherein said enzyme is capable of acting on said substrate, and wherein action of said enzyme on said substrate results in a change in electric potential at said working electrode; and (c) observing the electric potential at said working electrode at one or more times after said contacting and determining from the electric potential observation or observations the number of bacteria in the sample.

According to another embodiment of the invention, there is provided a method for detecting coliform bacteria in a sample, comprising: (a) incubating said sample in a reaction mixture with a substrate comprising a β- galactoside or β-glucuronide derivative;

(b) contacting a sensor and an enzyme with the reaction mixture, wherein said sensor comprises a working electrode which has immobilized therein or thereto said enzyme, wherein said enzyme is capable of acting on said substrate, and wherein action of said enzyme on said substrate results in a change in electric potential at said working electrode; and

(c) detecting the electric potential at said working electrode at one or more times after said contacting and determining from the electric potential or potentials detected the number of bacteria in the sample.

According to a particular embodiment of the invention, there is provided a method for detecting coliform bacteria in a sample, comprising:

(b) contacting an electrochemical sensor with the reaction mixture comprising the sample either:

(i) after a selected time of incubation of the reaction mixture or (ii) at the same time as or soon after preparing the reaction mixture, wherein said sensor comprises a working electrode which has immobilized therein or thereto an enzyme which is capable of acting on said substrate; wherein action of said sensor-bound enzyme on said substrate results in a change in electric potential at said working electrode;

(c) either:

(i) detecting the change in electric potential at said working electrode after said contacting in step (b)(i); or

(ii) determining the period of time required for the potential at said working electrode to return to a substantially steady baseline value after said contacting in step (b)(ϋ);

(d) either: (i) comparing the change in electric potential detected at step (c)(i) with: the change in electric potential detected for a control reaction mixture comprising no coliform bacteria; the change in electric potential detected for a known number of coliform bacteria; a calibration curve for change in electric potential versus number of bacteria; or any combination thereof; or (ii) comparing the period of time determined at step (c)(ii) with: the period of time taken for the potential at said working electrode to return to a steady baseline value for a reaction mixture comprising a known number of coliform bacteria; or a calibration curve for periods of time taken for the potential at said working electrode to return to a steady baseline value versus number of bacteria in reaction mixtures; and (e) determining from the change in electric potential observed at step (c) (i) or the period of time observed at step (c)(ii) the amount of coliform bacteria in the sample.

Step (c) (ii) may comprise determining the period of time required for the potential at said working electrode to return to a constant baseline value after said contacting in step (b)(ii).

The invention also relates to sensors for detecting microorganisms using methods of the invention.

Thus, according to another aspect of the invention, there is provided an electrochemical sensor comprising a working electrode which has immobilized therein or thereto an enzyme which is: (i) capable of acting on a substrate, said substrate being one which is capable of being acted on by an enzyme of interest; or (ii) which is capable of acting on a product resulting from action on a substrate by an enzyme of interest; wherein said enzyme of interest is associated with a microorganism or type of microorganism; wherein action of said electrode- immobilised enzyme on said substrate or product results in a change in electric potential at said working electrode. 9

The electrode- immobilised enzyme may be capable of acting on a β-galactoside or β- glucuronide derivative, and may be, for example, a β-galactosidase or a β-glucuronidase.

Thus, according to a particular aspect of the invention, there is provided an electrochemical sensor comprising a working electrode which has immobilized therein or thereto an enzyme which is capable of acting on a β-galactoside or β-glucuronide derivative to result in a change in electric potential at said working electrode, such as a β- galactosidase.

The enzyme may be bound to the electrode by means of a high-affinity binding pair, may be bound to the electrode directly, or may be bound to the electrode via a spacer molecule.

The selected time of incubation may be in the range of 1 minute to 24 hours or more and will depend on the nature of the reaction mixture and the amount of enzyme or number of cells expected to be in the sample. The time of incubation may, for example, be in the range selected from the group consisting of 1 to 24, 2 to 24, 3 to 24, 4 to 24, 5 to 24, 1 to 20, 2 to 20, 3 to 20, 4 to 20, 5 to 20, 1 to 15, 2 to 15, 3 to 15, 4 to 15, 5 to 15, 1 of 10, 2 to 10, 3 to 10, 4 to 10, 5 to 10, 1 to 7, 2 to 7, 3 to 7, 4 to 7, or 5 to 7 hours or from the group consisting of 1 to 60 minutes, such as 5 to 60, 10 to 60, 15 to 60, 20 to 60, 30 to 60, 5 to 50, 10 to 50, 15 to 50, 5 to 45, 10 to 45, 15 to 45, 5 to 30, 10 to 30, 20 to 30, 5 to 20, 10 to 20, 40 to 60, 45 to 60 or 50 to 60 minutes. Where the electrochemical sensor is contacted with the reaction mixture soon after preparing the reaction mixture it may be contacted at any appropriate time, such as a sufficient time which will allow for the reaction catalysed by at least the enzyme in or on the sensor to reach steady-state reaction rate, and which may allow for all enzyme-catalysed reactions to reach steady-state. Thus, for example, the electrochemical sensor may be contacted with the reaction mixture within the range of 1 second to 20 minutes of preparing the reaction mixture, such as within 10 seconds to 10 minutes, 20 seconds to 10 minutes, 30 seconds to 10 minutes, 1 minutes to 10 minutes, 1 minute to 5 minutes, 30 seconds to 5 minutes, 20 seconds to 5 minutes, 10 seconds to 5 minutes, 10 seconds to 3 minutes, 20 seconds to 3 minutes, 30 seconds to 3 minutes, 1 minute to 3 minutes, 1 minute to 2 minutes, 30 seconds to 2 minutes, 20 seconds to 2 minutes, or 10 seconds to 2 minutes, 10 seconds to 1 minute, 20 seconds to 1 minute, or 30 seconds to 1 minute of preparing the reaction mixture.

The invention also relates to devices for detecting microorganisms by methods of the present invention, and which comprise sensors as described above. Brief Description of the Drawings

Figure 1 provides a schematic of the mode of action of an electrode-bound enzyme in a method of the present invention, wherein the enzyme is passively adsorbed to the surface of the electrode. Enzyme of interest to be detected is shown in a box with dashed lines to 5 illustrate the fact that the enzyme may not actually be present in the reaction mixture at the time of contacting the sensor with the reaction mixture.

Figure 2 provides a graph of potential difference between a working electrode of the invention and a reference electrode over time for saturation of the electrode with increasing concentrations of β-galactosidase (2-10μg/mL) immersed in a constant io concentration of 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-GaI - 50μg/mL) in 2x yeast tryptone growth medium.

Figure 3 provides graphs of potential difference between a working electrode of the invention and a reference electrode over time for: Figure 3 A - lOOOng/mL β-galactosidase applied to the electrode and immersed in increasing concentrations of 5-bromo-4-chloro-

I₅ 3-indolyl β-D-galactopyranoside (X-GaI - 1.5-100μg/mL) in 2x Yeast Tryptone growth medium; Figure 3B - lOOOng/mL β-galactosidase applied to the electrode and immersed in 2-fold dilutions of 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-GaI - 50- 0.625μg/mL) in 2x Yeast Tryptone growth medium; Figure 3C - 30U/mL β- galactosidase applied to the electrode and immersed in decreasing concentrations of 5-

20 bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-GaI — 50-0μg/mL) in 2x Yeast Tryptone growth medium; and Figure 3D - 7500ng/mL β-galactosidase applied to the electrode and immersed in decreasing concentrations of 5-bromo-4-chloro-3-indolyl β-D- galactopyranoside (X-GaI - 10-0μg/mL) in 2x Yeast Tryptone growth medium.

Figure 4 shows results for potential difference between a working electrode of the 2₅ invention and a reference electrode wherein the working electrode comprises biotinylated β-galactosidase applied to the electrode by different means or in different amounts, as follows: 7500ng/mL biotinylated β-galactosidase applied to the electrode via streptavidin bound to the electrode surface; 7500ng/mL biotinylated β-galactosidase passively bound to the electrode surface; 7500ng/mL biotinylated β-galactosidase passively bound to the 30 electrode surface; 3250ng/mL biotinylated β-galactosidase passively bound to the electrode surface; 1750ng/mL biotinylated β-galactosidase passively bound to the electrode surface; immersed in 50μg/mL 5-bromo-4-chloro-3-indolyl β-D- galactopyranoside (X-GaI) in 2x Yeast Tryptone growth medium. Figure 4A provides the results in bar chart form and Figure 4B provides the raw data for change in potential over 3₅ time (Streptavidin-bound β-galactosidase results represented by filled triangles, and unfilled symbols representing Oμg/mL X-GaI controls). Figure 5 provides a bar chart illustrating results of different concentrations of non- biotinylated β-galactosidase applied (directly, passively) to working electrodes when immersed in 50μg/mL X-GaI immediately after preparation of the working electrodes or after three days storage of β-galactosidase on dry ice before preparation of working electrodes.

Figure 6 shows a sensor calibration graph, for screen-printed polypyrrole sensors coated with 6U/mL β-galactosidase and exposed to a range of X-GaI concentrations (ranging from lOOμg/mL to Oμg/mL in defined medium). Each bar represents triplicate readings, with error bars. Output expressed as % drop in full scale deflection (FSD).

Figure 7 shows the results of incubating working electrodes with 30U/mL β-galactosidase for increasing amounts of time on the potential difference recorded for the electrode (relative to a reference electrode) on immersion into 50μg/mL X-GaI. Figure 7A is a bar chart showing the potential difference recorded (duplicate readings, with standard error bars) for each electrode at approximately 3 minutes after immersion in 50μg/mL X-GaI, and Figure 7B provides raw data for a first set of readings of potential difference over time after immersion of the working electrodes in 50μg/mL X-GaI from which the maximum readings were taken.

Figure 8 shows results for a repeatability study for 11 sensors according to the invention immersed in 5 Oμg/mL X-GaI in 2x Yeast Tryptone growth medium, the sensors having been prepared by applying a solution comprising 7.5μg/mL biotinylated β-galactosidase to the working electrodes for 1 hour. Figure 8 A shows a bar chart of normalised potential difference (relative to the 0 μg/mL X-GaI control) for each electrode at approximately 3 minutes after immersion in 50μg/mL X-GaI, and Figure 8B provides raw data for potential difference over time after immersion of the working electrodes in 5 Oμg/mL X- Gal.

Figure 9 shows the effect of carry-over of substrate on accuracy of readings obtained for sensors according to the invention. Figure 9A is a bar chart of potential difference (mV) determined for six [decreasing] concentrations of X-GaI (black bars) after which the electrode holder was inverted to then assay the six X-GaI concentrations in reverse order (hatched bars). Figure 9B shows the error introduced by substrate carry over for each pair of sensors.

Figure 10 shows results obtained for sensors of the invention comprising either β- glucuronidase (Figure 10A) or comprising β-glucosidase (Figure 10B). 2007/000991

12

Figure 11 provides a schematic of bacterial detection assays according to the invention. Substrate is shown as filled triangles and bacterial cells as ovals with a lumen.

Figure 12 provides a graph showing bacterial depletion of 5-bromo-4-chloro-3-indolyl β- D-galactopyranoside resulting in lower sensor output: 15 μg/mL 5-bromo-4-chloro-3- indolyl β-D-galactopyranoside was employed throughout with 0-5x10⁶ E.coli cells (ATCC 11775) and a 'no substrate' control.

Figures 13 A and 13B show results obtained for assay of reaction mixtures for X-GaI after incubation of reaction mixtures with (and therefore depletion of X-GaI in the mixture by) decreasing initial inocula of E.coli ATCC 11775, the results being illustrated as % reduction in full scale deflection (relative to 0 bacterial cells control). Figure 13A - 1.2xlO⁷ to 1.2xlO⁵ cells/mL for three hours. Figure 13B - 3.7xlO⁵ to 3.7xlO² cells/mL for five hours. Initial concentration of X-GaI in the reaction mixture was 50 μg/mL in 2x Yeast Tryptone growth medium.

Figures 14A and 14B show results obtained for assay of reaction mixtures for X-GaI (with sensors according to the invention comprising β-galactosidase) after incubation of reaction mixtures comprising (and therefore depletion of X-GaI in the mixture by) increasing initial inocula of E.coli ATCC 11775. Samples were taken hourly after 2 hours until 6 hours after initial inoculation of 2x yeast tryptone growth medium comprising 50 μg/mL X-GaI. The results are provided as % reduction in potential difference observed (relative to 0 bacterial cell control). The data in Figure 14B represent duplicate results for each initial inoculum.

Figures 15A to 15C show calibration curves obtained for determination of initial sample cell concentration (for E. coli ATCC 11775) from % reduction in potential difference observed (relative to 0 bacterial cell control) as determined by sensors according to the invention (comprising β-galactosidase bound to working electrodes) after 4 hours immersion in 50 μg/mL X-GaI.

Figure 16 shows results obtained for assay of reaction mixtures for X-GaI (with sensors

- according to the invention comprising β-galactosidase) after incubation of reaction mixtures with (and therefore depletion of X-GaI in the mixture by) duplicates of decreasing initial inocula of wild type strain 365 after 4 hours incubation in 2x yeast tryptone growth medium comprising 50 μg/mL X-GaI. The results are given as % reduction in potential difference observed (relative to 0 bacterial cell control).

Figure 17 shows show results obtained for assay of reaction mixtures for X-GaI (with sensors according to the invention comprising β-galactosidase) after incubation of reaction mixtures with (and therefore depletion of X-GaI in the mixture by) duplicates of serial dilutions of initial inocula of wild type strain 365 after 5 hours incubation in 2x yeast tryptone growth medium comprising 50μg/mL X-GaI. The results are given as % reduction in potential difference observed (relative to 0 bacterial cell control). Also shown in Figure 16 are the cell numbers in cultures 1 (40,000cfu/mL initial inoculum) and 4 (40cfu/mL initial inoculum) in the cultures over the five hour incubations.

Figure 18 shows the difference in normalised % sensor output drops for a given inoculum of bacteria into 2x yeast tryptone growth medium comprising 5μM IPTG and either 100 or 60μg/mL X-GaI used as substrate.

Figure 19 shows the effect of pH decrease during cell culture on the sensitivity of readings obtained for assay of X-GaI using sensors according to the invention comprising β-galactosidase bound to the working electrodes.

Figure 20 shows the effect of ImM sodium dodecyl sulphate (SDS) on the sensitivity of readings obtained for assay of X-GaI using sensors according to the invention comprising β-galactosidase bound to the working electrodes.

Figure 21 shows the effect of lactose present in the reaction mixture on the sensitivity of readings obtained for assay of X-GaI using sensors according to the invention comprising β-galactosidase bound to the working electrodes.

Figure 22 shows the effect of isopropyl-β-D-thiogalactopyranoside (IPTG) present in the reaction mixture on the sensitivity of readings obtained for assay of X-GaI using sensors according to the invention comprising β-galactosidase bound to the working electrodes.

Figures 23 A and 23B show the use of two types of screen printed carbon paste electrodes to measure X-gal concentration, with β-galactosidase adsorbed to the surface of the sensors at 6U/mL and OU/mL. Outputs read with a millivolt meter.

Figures 24A and 24B show the use of two types of screen printed carbon paste electrodes to measure urea concentration, using urease adsorbed to the surface of the sensors at the three concentrations shown. Outputs read with a millivolt meter.

Definitions

As used herein, the term "comprising" means "including principally, but not necessarily solely". Variations of the word "comprising", such as "comprise" and "comprises", have correspondingly similar meanings. As used herein, the term "detection" includes observation, measurement, and quantification as well as detection, and these terms may be used interchangeably throughout. Variations of the word "detection", such as "detecting", "detect" and "detects" have correspondingly similar scopes.

As used herein, the term "enzyme" refers to any biocatalytic molecule capable of facilitating the conversion of one or more substrates to one or more products and/or of facilitating transport of one or more substrates across a membrane, and may include secreted or intercellular catabolic or anabolic enzymes, membrane transport proteins, catalytic RNA molecules (such as ribozymes).

As used herein, the term "microorganism" refers to any life form of microscopic or submicroscopic size, including bacteria, archaebacteria, protozoa, fungi, viruses and algae.

As used herein, the term "results in a change in electric potential at said working electrode" in the context of enzymes bound to such electrodes means that the action of the enzyme on a substance either directly or indirectly results in a change in electric potential at the electrode. For example, the change in potential at the electrode may arise due to the action of the enzyme on the substance, or due to a subsequent reaction of one or more of the products with one or more components of its environment, the surface of the electrode, or both. The change in potential may comprise a decrease or increase in electrode potential at the working electrode, relative to the electric potential at the working electrode prior to exposure of the electrode-bound enzyme to substrate. The change detected may comprise substantially continuous measurement/detection of the electric potential at the working electrode after exposure to substrate, or may comprise measurement(s) of electric potential at the working electrode made at one or more discrete times after exposure to substrate, or any other suitable means for determining such changes.

Detailed Description

As described above, many, if not most existing standard techniques for detecting and enumerating microbiological entities suffer from the disadvantages of being slow and/or technically demanding, requiring at least 24 hours for a result and/or specialised techniques, apparatus and know-how, especially where numbers of the microbiological entities may be small in a given sample. In many applications, especially environmental monitoring, these disadvantages are significant, as monitoring often needs to be frequent and/or large numbers of samples need to be tested, and results may be required quickly in order to take action, if necessary (for example in the case of sudden contamination of waterways with elevated levels of coliform bacteria).

Detection of Enzymes

The present invention provides a fast and sensitive method for detecting the presence of an enzyme of interest in a sample. A process according to the invention comprises: (a) incubating the sample in a reaction mixture with at least one substrate which can be acted on by the enzyme of interest; followed by (b) contacting an electrochemical sensor with the reaction mixture comprising the sample, wherein the electrochemical sensor comprises a second enzyme capable of acting on at least one of said at least one substrate(s) or on a product, or products of the action of the enzyme of interest on said at least one substrate; (c) determining the change in potential at the working electrode after said contacting; and (d) determining from the change in potential observed at step (c) the amount of enzyme of interest in the sample.

An alternative process according to the invention comprises: contacting the electrochemical sensor with the reaction mixture at the same time as, or soon after preparing the reaction mixture; monitoring the potential at the working electrode and determining the period of time required for the potential at the working electrode to return to a substantially steady baseline value, which may be substantially equivalent to a value previously obtained for the same electrode for a reaction mixture comprising no substrate; and determining from the period of time required for the potential at said working electrode to return to a steady baseline value the amount of enzyme of interest originally present in the sample.

The substantially steady baseline value or the steady baseline value may be a constant baseline value.

Referring to Fig. 1 the sensor comprises a working electrode 101. Second enzyme 102a or 102b may be immobilized or adsorbed to the surface of working electrode 101.

The working electrode 101 may be any appropriate working electrode 101 to which an enzyme 102a or 102b is bound, while retaining its activity, and wherein that enzymic activity results in a changed redox, pH, or ionic state of the product(s) as compared to the substrate(s), with a resulting change in redox state at the working electrode surface. Working electrode 101 may comprise an electroconductive polymeric material 103 derived from suitable monomeric units, such as pyrrole, furan, or thiophene as an electroconductive surface/coating. Working electrode 101 may be screen printed, as described in international patent publication No. WO 03/019171 to Sensor-Tech Limited of Jersey, Great Britain, and as available from Universal Sensors Ltd. (USL), United Kingdom. Alternatively, working electrode 101 may comprise a non-polymeric electroconductive material 103, which may comprise graphite, carbon paste, gold, platinum or other suitable electroconductive material. Suitable screen-printed carbon paste electrodes are available from Gwent Electronic Materials Ltd, of Pontypool, United Kingdom.

Reference electrode 109 is required for a potentiometric detection step, for example a calomel or an Ag/ AgCl reference electrode, can be placed on the same support as the working electrode 101, or an external reference electrode 109 can be employed. Also, the reference electrode 109 may be comprised in, or comprise the receptacle in which steps (b) and (c) are carried out, or may be a common reference electrode 109 for a number of receptacles for carrying out steps (b) and (c), as may be desired if using multi-well plates.

Any lay-outs for carrying out a method of the invention are possible, such as single reaction vessels or cells, dip-stick formats, or multi-well plates, and may contain integrated electrochemical sensors.

A second/sensor-bound enzyme 102a is capable of acting on at least one substrate 104 which is also a substrate for the enzyme of interest 100. Alternatively, second/sensor- bound enzyme 102b is capable of acting on a product 105 of the action of the enzyme of interest 100 on substrate 104. Action of the second enzyme 102a or 102b on the at least one substrate 104 or product 105 respectively produces a change in electric potential at the working electrode 101. This change in potential may then be determined at any appropriate time or times by detector 107, after contacting the sensor 100 with the reaction mixture 108, and can be related to the amount of enzyme of interest in the original sample.

In one method according to the invention, reaction mixture 108 comprising the original sample or a portion thereof and at least one substrate 104 which can be acted on by enzyme of interest 100 is incubated for a desired period of time. In order to avoid interference from the enzyme of interest 100 during contacting of the sensor with reaction mixture 108, the enzyme of interest 100 may be removed from reaction mixture 108 prior to contacting the sensor with reaction mixture 108. Working electrode 101 which has immobilized therein or adsorbed thereto a second enzyme 102a which is capable of acting on substrate 104. Action of said sensor-bound enzyme 102a on said at least one substrate

104 results in a change in electric potential at working electrode 101. The change in potential at working electrode 101, which is electrically connected to detector 107 via electrically conductive line 106a, is determined with respect to reference electrode 109, which is electrically connected to detector 107 via electrically conductive line 106b, after the step of contacting by monitoring the potential as a function of time for a desired period. The presence or absence and/or the amount of the enzyme of interest in the original sample may then be determined from the change in potential as a function of time.

The enzyme 102a bound to the working electrode 101 may be any enzyme capable of acting on the same substrate as any enzyme of interest, or may be any enzyme 102b capable of acting on a product of the action of an enzyme of interest on a substrate, wherein said enzyme 102a or 102b results in a changed redox, pH, or ionic state of the product(s) as compared to the substrate(s), and a resulting change in redox state at the working electrode surface. Such enzymes may include, for example but are not limited to, catabolic enzymes, such as carbohydrate-degrading enzymes, including glycosidases

(such as β-galactosidases, β-glucuronidases), proteolytic or peptidolytic enzymes, ureases, membrane transport proteins and nucleases.

The enzyme of interest may also include, for example, catabolic enzymes, such as carbohydrate-degrading enzymes, including glycosidases (such as coliform-associated β- galactosidases and/or β-glucuronidases, or flu virus-associated neuraminidases, proteolytic or peptidolytic enzymes, ureases (associated with Helicobacter species and some blue-green algae), membrane transport proteins and nucleases, but may also include synthetic enzymes, or disproportionating enzymes/transferases, in which case the one or more substrates are acted upon to produce at least one product 105 upon which an electrode-bound enzyme 102b may act.

The enzyme of interest 100 and an electrode-bound enzyme 102a may be the same enzyme (that is, same enzyme from the same source), be related/same enzymes from different sources, or be entirely different enzymes.

The enzyme of interest 100 and an electrode-bound enzyme 102b may be entirely different enzymes. However, if the reaction catalysed by enzyme of interest 100 and enzyme 102b is reversible, these enzymes may be the same enzyme (that is, same enzyme from the same source), or be related/same enzymes from different sources. However, in such a scenario, substantially all enzyme of interest 100 (or all of enzyme of interest 100) would need to be removed from the reaction mixture 108 prior to contacting the sensor with the reaction mixture 108 in order to obtain meaningful results.

In the case of the enzyme of interest and electrode-bound enzyme 102a acting on the same substrate, the presence of the enzyme of interest in the sample being investigated will result in a smaller change in electrical potential at the working electrode 101 at step (c) than that observed where enzyme of interest is not present in the sample, as the enzyme of interest will deplete the amount of substrate 104 available in reaction mixture 108 for action by the electrode-bound enzyme 102a.

In the case of electrode-bound enzyme 102b acting on a product of the action of the enzyme of interest on the at least one substrate 105, the change in electric potential observed at step (c) will be greater if the sample being investigated comprises enzyme of interest.

The at least one substrate 104 may be any compound, or compounds which is/are acted upon by the enzyme of interest and which may either also be acted upon by the electrode- bound enzyme 102 so as to result in a change in potential at the working electrode 101 with respect to reference electrode 109.

Suitable substrates 104 which may be acted upon hydrolytically by at least the electrode- bound enzyme 102a or 102b so as to result in a redox, pH or ionic change (and which would therefore result in such a change in the immediate vicinity of the electrode-bound enzyme 102, thereby resulting in a change in electric potential at the working electrode) are known in the art, and may include, for example, 5-bromo-4-chloro-3-indolyl- or 6- chloro-3-indolyl-linked compounds, fluorescein-linked compounds, methylumbelliferyl derivatives or luciferin-linked compounds.

For example, in one embodiment the at least one substrate 104 may be a glycoside acted upon by both the enzyme of interest and by electrode-bound enzyme 102a so as to result in a change in electric potential at the working electrode 101. According to this embodiment, both enzymes may be glycosidases, such as α- or β-glucosidases, α- or β- galactosidases, α- or β-mannosidases, α- or β-glucuronidases or β-fructosidases (such as invertase), amongst others, and the substrate may be an α- or β-glycopyranoside, such as α- or β-glucopyranosides, α- or β-galactopyranosides, α- or β-mannopyranosides, α- or β- glucuronides, β-fructofuranosides, amongst others.

The enzyme of interest may also be a glycosidase while the electrode-bound enzyme 102b is a dehydrogenase or an oxidase. For example, the enzyme of interest 100 may be a β- galactosidase, the substrate 104 may be a β-galactoside, product 105 may then be galactose and the electrode-bound enzyme 102b may be a β-galactose dehydrogenase. Alternatively, the enzyme of interest 100 may be a β- (or α-) glucosidase, the substrate 104 be a β- (or α-) glucoside, product 105 may be glucose, and electrode-bound enzyme 102b may be a glucose oxidase.

Enzyme 102a or 102b may be bound to the working electrode by any suitable means as are well known in the art. For example, enzyme 102a or 102b may be passively adsorbed to the surface of electrode 101, or may be bound to the electrode 101 by functionalisation of the surface of electrode 101 and, optionally, the enzyme so as to enable direct binding of the enzyme to the electrode. Alternatively, enzyme 102a or 102b may be bound to the electrode 101 by indirect linkage via a spacer molecule, or may be bound to the electrode 101 by a linkage system which may comprise a high affinity binding pair, where each member of such a binding pair is conjugated/bound to either the enzyme or the electrode surface. An example of a suitable high affinity binding pair comprises, for example, a streptavidin- biotin binding pair.

Where a streptavidin-biotin binding pair is used for binding enzyme to the electrode 101, streptavidin may be bound to the electrode surface, and biotin may be conjugated to the enzyme 102a or 102b, for example by recombinant expression of the enzyme with a biotinylated fusion tag. This will allow for highly specific, and strong binding of the enzyme 102a or 102b to the electrode 101. Certain surfaces may not require functionalisation in order to enable binding of enzyme, or streptavidin, and a polypyrrole surface 103 has been found to allow direct binding of streptavidin or for direct binding of β-galactosidase without loss of activity.

The amount of enzyme bound to the working electrode will depend on a number of factors, including the nature of the enzyme itself, the mode of binding to the electrode and the nature of the surface of the electrode. Typically, the amount of enzyme may vary from about 1x10^"6 Units of enzymic activity (μmoles of substrate converted per minute) per electrode to about IxIO^"3 Units per electrode, such as about IxIO^"5 Units per electrode, about 5x10^'5 Units per electrode, about 1x10^"4 Units per electrode, or about 5x10^"4 Units per electrode.

Methods of the invention, and sensors therefor, in which more than one type of enzyme is bound to the working electrode, are also contemplated. For example, one enzyme 102 bound to the surface of working electrode 101 may act on the same substrate 104 as an enzyme of interest, or on a product 105 of the action of the enzyme of interest on the substrate, and a second enzyme bound to the surface of the working electrode may act on a product of the action of enzyme 102, resulting in a further change in potential at the working electrode, potentially resulting in greater sensitivity of the assay. Alternatively, where a sample may contain two or more enzymes of interest, such as may occur with samples comprising microorganisms, two or more enzymes 102 may be bound to the surface of working electrode 101 where each of these may act on a substrate 104 also acted on by an enzyme of interest, or on a product 105 of the action of an enzyme of interest on a substrate. An alternative to detection of multiple enzymic activities of interest may be to carry out methods of the invention using separate sensors comprising working electrodes to which are bound different enzyme types, allowing for separate detection of each enzymic activity in the sample.

Detection of Microorganisms In another embodiment of methods of the invention, the enzyme of interest 100 is associated with a microorganism or type of microorganism, and the method may be a method for detecting the presence of that microorganism, or type of microorganism in a sample, such as a method for detecting coliform bacteria in water samples, or viruses in biological samples.

Specific microorganisms, or types of microorganisms are in many instances associated with particular enzymic activities, at least in a given sample type. For example, β- galactosidase or β-glucuronidase activity in water samples correlates strongly with the number of coliform bacteria in such samples. A number of viruses also have specific enzymes on their external coats. For example the flu virus comprises neuraminidase as an outer coat protein.

Particularly for this purpose, but for the purposes of any method of the invention, the enzyme of interest 100 may be a β-galactosidase or a β-glucuronidase, and the substrate 104 may be a β-galactoside or β-glucuronide derivative respectively. In such methods, the enzyme bound to the electrode may be an enzyme 102a capable of acting on either the β-galactoside or β-glucuronide derivative respectively, or an enzyme 102b capable of acting on a product of hydrolysis or the β-galactoside or β-glucuronide derivative by the β-galactosidase or a β-glucuronidase (such as galactose or glucuronic acid). Where the method is for the detection of coliform bacteria, both the enzyme of interest 100 and an electrode-bound enzyme 102a may be β-galactosidases or β-glucuronidases, and the substrate 104 may be a β-galactoside or a β-glucuronide.

Any suitable β-galactoside or β-glucuronide substrate 104 may be used in such a method, provided it results in a change in electric potential at the working electrode when acted upon by the electrode-bound enzyme 102a or 102b, and is also a suitable substrate 104 for enzymatic action by an enzyme of interest 100 associated with the coliform bacteria (such as β-galactosidase or a β-glucuronidase). Suitable substrates may comprise, for example, 6-chloro-3-indolyl-β-D-galactopyranoside (Red-Gal), 5-bromo-4-chloro-3-indolyl-β-D- galactopyranoside (Blue-gal), fluorescein di(β-D-galactopyranoside), fluorescein mono-β- D-galactopyranoside, 4-methylumbelliferyl-β-D-galactopyranoside, naphthofluorescein di-(β-D-galactopyranoside), 4-trifluoromethylumbelliferyl-β-D-galactopyranoside, and 3,4-Cyclohexenoesculetin β-D-galactopyranoside (S-gal). The substrate 104 may be a 5-bromo-4-chloro-3-indolyl β-linked glycoside or glucuronide. According to a specific embodiment, the substrate 104 is 5-bromo-4-chloro- 3-indolyl β-D-galactopyranoside.

Thus, according to a specific method of the invention, the method is for detecting coliform bacteria in a sample, comprising:

(a) incubating a reaction mixture comprising the sample and a substrate comprising a β-galactoside or β-glucuronide derivative;

(b) contacting an electrochemical sensor with the reaction mixture comprising the sample either: (i) after a selected time of incubation of the reaction mixture or

(ii) at the same time as, or soon after preparing the reaction mixture, wherein the sensor comprises a working electrode which has immobilized therein or adsorbed thereto an enzyme which is capable of acting on the substrate; wherein action of the sensor-bound enzyme on the substrate results in a change in electric potential at the working electrode;

(c) either:

(i) detecting the change in electric potential at the working electrode after said contacting in step (b)(i); or

(d) either:

(i) comparing the change in electric potential detected at step (c) with: the change in electric potential detected for a control reaction mixture comprising no coliform bacteria; the change in electric potential detected for a known number of coliform bacteria; a calibration curve for change in electric potential versus number of bacteria; or any combination thereof; or

(ii) comparing the period of time determined at step (c)(ii) with: the period of time taken for the potential at said working electrode to return to a steady baseline value for a reaction mixture comprising a known number of coliform bacteria; or a calibration curve for periods of time taken for the potential at said working electrode to return to a steady baseline value versus number of bacteria in reaction mixtures; and

(e) relating the change in electric potential observed at step (c)(i) or the period of time observed at step (c)(ii) to the presence or the amount of coliform bacteria in the sample.

Step (c) (ii) may comprise: (ii) the period of time required for the potential at said working electrode to return to a constant baseline value after said contacting in step (b)(ii).

In an embodiment of this aspect, the assay is a depletion assay, in which higher numbers of coliform bacteria are associated with smaller changes in electric potential detected, or shorter periods of time determined at step (c), compared to those detected/determined for control samples comprising no bacteria or no enzyme, as a result of the bacterial- associated enzyme of interest 100 depleting the amount of substrate available for reaction catalysed by an electrode-bound enzyme 102a, and the coliform bacteria are detected quantitatively or semi-quantitatively.

For example, having reference to Figure 10, a depletion assay of the invention may involve assaying a test sample which has been incubated with an unknown sample and substrate, such as X-GaI. If coliform bacteria are present in the reaction mixture, these will deplete the amount of substrate in the reaction mixture, and cell numbers of the coliform bacteria may also increase. The reaction mixture may then be assayed using a sensor according to the invention, such as a sensor comprising a polypyrrole-based working electrode with β-galactosidase adsorbed thereto, optionally being filtered prior to contacting the sensor with it. The potential observed at the working electrode, relative to a reference electrode, may then be compared to that observed for a positive control, comprising a reaction mixture either containing no substrate or substrate and a high inoculum of bacteria with high β-galactosidase activity (such that substantially all or all of the substrate is depleted prior to contacting a sensor of the invention with the reaction mixture), a negative control reaction mixture containing no bacteria (maximum potential difference for the given initial substrate concentration), or a combination thereof. The difference in potential observed for the test and a suitable control may then be equated to an approximate number of coliform cells that would have been present in the initial sample using an appropriate calibration curve previously prepared for the sensor, or batch of sensors.

Coliform bacteria are a problem in a wide range of situations, especially in water sources such as process effluents and water catchment areas/rivers. Thus, according to a specific embodiment, a method of the invention is for detection of coliform bacteria in a sample derived from a water source.

Because of the sometimes very low levels of coliform bacteria in water samples which may still cause a pathologic condition in humans and other animals consuming the water, the sample may be concentrated, for example by filtration/ultrafiltration prior to incubation with substrate. Assay conditions

Generally speaking, in methods of the present invention the substrate may be provided at a concentration which provides the maximum rate of reaction, for best sensitivity, although sub-optimal substrate concentrations may also be employed. For example, if initial slope of potential change is to be used rather than detection of potential change after substantially complete or complete depletion of substrate 104 or product 105, then a substrate concentration approximating the Michaelis constant (K_m) of enzyme 102a or 102b, or slightly higher may be employed so as to ensure more significant differences in rate of potential change detected. The substrate concentration to be used will depend on the reaction kinetics of both the enzyme of interest and the enzyme bound to the electrode, and this can be determined by straight forward procedures, modelling or calculations if or once the basic enzyme parameters are known and/or can be readily determined by those skilled in the art. The cost and/or solubility of substrate may also be a determining factor in deciding on substrate concentrations to be used.

For bacterial glycosidases, for example, a wide range of Michaelis constants (K_ms) are known, from the micromolar range (such as about 25 μM) to the millimolar range (such as about 25mM). To achieve maximal reaction rate, the substrate concentration should be at least about 3-5 times the Michaelis constant (K_m), and ideally higher, such as about 10 times the K_m of a given enzyme for the particular substrate or higher. Thus, for endpoint potential change determinations, if the highest Michaelis constant for one of the enzyme of interest 100 or the electrode-bound enzyme 102a or 102b is approximately ImM, suitable substrate concentrations may be as high as about 10OmM or higher, although cost of substrate may require lower concentrations, such as, for example, about 75mM, about 5OmM, about 4OmM, about 3OmM, about 2OmM, about 15mM, about 1OmM, about 7.5mM, about 5mM, about 4mM, about 3mM or even less. If substrate concentrations nearing the Michaelis constant, or lower are employed, care should be taken to ensure consistency of the substrate concentration between assays, as small differences in substrate concentration may result in large difference in reaction rates. Alternatively, suitable standards should be tested between substrate batches.

Where change in potential at the working electrode is determined at set times after contacting the sensor with the reaction mixture (see below), for best sensitivity the amount of substrate 104 to be used in such assays should be such that amount of substrate 104 available for electrode-bound enzyme 102a or of product 105 for an enzyme 102b to act upon (and therefore amount of substrate not converted by the enzyme of interest, or amount of product 105 released by action of the enzyme of interest) is in the range of about 0 to about 2-3 times the Michaelis constant (K_m) of the electrode-bound enzyme for this substrate. If higher substrate concentrations are employed, accuracy may be compromised, and this may also occur where very low substrate concentrations, which may be difficult to accurately reproduce, are employed and/or where the substrate saturation curve for the electrode-bound enzyme is very steep (that is, the enzyme is saturated, and therefore the rate of reaction is approximately maximal) at very low substrate concentrations.

So as to increase the chance of detection of an enzymic activity of interest, the sample may be concentrated, for example by filtration/ultrafiltration prior to incubation with substrate 104.

So as to eliminate any interference from microorganisms after incubation with the substrate, the reaction mixture 108 may be filtered after step (a) to remove microorganisms from the mixture to be applied to the sensor.

Generally speaking, the temperature to be employed for steps (a) or (b) in a method of the invention is not important, although temperatures closer to the optimal temperature for activity of the enzyme of interest 100 and the electrode-bound enzyme 102a or 102b may provide for greater sensitivity or speed of the assay. If meaningful comparison with other assay runs are to be made, the temperature should ideally be the same between different assay runs, or suitable standards and/or controls should be run at different temperatures. The temperature at which steps (a) and (b) are carried out may be readily optimised by those skilled in the art by straight forward procedures and modeling once the basic enzyme and substrate parameters are known.

For the reaction catalysed by β-galactosidase from E. coli, the optimum temperature is reported as being about 37⁰C, however, temperatures from about 20°C to about 50°C, such as about 2O⁰C, 22⁰C, 24°C, 26⁰C, 28°C, 30⁰C, 32⁰C, 34°C, 36°C, 38⁰C, 40^°C, 42⁰C, 44°C, 46°C, 48°C, or 50°C, or even outside this region may be employed, although β- galactosidase from E. coli is known to be unstable at temperatures above 37^°C.

The optimum temperatures for the enzyme of interest 100 and the sensor-bound enzyme 102 will not necessarily be the same, and therefore steps (a) and (b) may need to be carried out at different temperatures.

The reaction environment should have a pH which is within about 1 to 2 pH units of the pH optimum for the enzyme of interest 100, the sensor-bound enzyme 102, or both. However where the enzyme has a relatively flat pH activity profile, broader pH ranges may be applicable. Reaction pH may also affect the reactant concentrations, and this may be a determining factor, and the enzyme's pH optimum may not necessarily provide the optimum reaction conditions. Once the basic enzyme and reactant parameters are known, 2007/000991

25 pH optimisation may be readily performed by those of skill in the art by straight forward procedures and modeling.

The pH optimum for the enzyme of interest 100 and the sensor-bound enzyme 102 will not necessarily be the same, and therefore there may be a need to adjust the pH of the reaction mixture after step (a).

β-galactosidase from E. coli has a pH optimum of 6.6-8.0, depending on the substrate and the buffer used and, while being stable at pH 6.0 is unstable at pH 5.0, and is unstable at pH values greater than pH 9.0. Accordingly, where β-galactosidase from coliforms is present in the reaction mixture, and β-galactosidase from E. coli is bound to the working electrode the reaction environment pH may be from about pH 5.5 to about pH 9.0, such as from about pH 6.0 to about pH 8.0, such as about pH 6.0, about pH 6.2, about pH 6.4, about pH 6.6, about pH 6.8, about pH 7.0, about pH 7.2, about pH 7.4, about pH 7.6, about pH 7.8 or about pH 8.0.

Thus, although not necessary, the reaction mixture may comprise a suitable buffer to maintain the pH within a desired range. Suitable buffers, and concentrations thereof, are well known in the art and can be determined by those skilled in the art for any given set of desired reaction conditions by no more than routine experimentation.

Where the method involves detection of an enzymic activity of interest associated with the presence of a microorganism in a sample, and step (a) comprises incubating a reaction mixture comprising the sample and a substrate, unless the incubation period is very short or the amount of microorganism present in the sample is very low, acidification of the reaction mixture may be expected to occur. This may reduce the pH to a level significantly below the pH optimum for the enzyme, or at which the working electrode surface is not as responsive, potentially reducing the sensitivity of the sensor. This effect may be avoided or limited by appropriate buffering of the reaction mixture, avoidance of excessive cell numbers in the sample(s), suitable incubation timing, or a combination thereof.

Methods of the invention may also require inclusion of one or more co-factors or co- substrates in the incubation medium, for the activity of the enzyme of interest, or in the assay medium, for the activity of the second/sensor-bound enzyme, depending on the enzyme and the reaction being catalyzed.

The potential effect of inhibitors on the activity of the enzyme of interest or of the second/sensor-bound enzyme, or factors which may otherwise affect the sensitivity of sensors of the invention should also be considered. For example, where the enzyme of interest, the second/sensor-bound enzyme, or both are β-galactosidases, inclusion of lactose or isopropyl-β-D-thiogalactopyranoside (IPTG) may reduce the sensitivity of detection of X-GaI remaining in the reaction mixture as both of these compounds compete with X-GaI for the action of β-galactosidase. This may be a problem where the method of the invention comprises detection of coliform bacteria by detection of β-galactosidase activity, as lactose or IPTG are often used to induce expression of β-galactosidase. For better sensitivity of assays using methods according to the invention for the detection of microorganisms it may therefore be preferable to not induce β-galactosidase using lactose or IPTG. Alternatively, low levels of such β- galactosidase-inducing agents may be used at low, substantially non-interfering concentrations, and this may allow for more reliable results.

Also, in the course of these studies it has been found that the anionic detergent SDS (which may be used to permeabilise microbial cells to release, or increase extracellular levels of the enzyme of interest) interferes with the sensitivity of assays for X-GaI using polypyrrole-based sensors as sourced from Universal Sensors Ltd. (USL), United Kingdom with β-galactosidase bound to it. Part of the reason for this may be due to the charge because SDS is also a dopant in the preparation of the working electrodes of these sensors, or the charge provided by the SDS may be an interfering factor. It is expected that other ionic detergents may also interfere with the sensitivity of such sensors and therefore use of nonionic detergents may be preferable for permeabilisation of microbial cells if polypyrrole sensors are to be used.

If assay sensitivity is not of the utmost importance, appropriate controls could be run to account for the effect of any inhibitors or interfering substances.

Determination of the amount of enzyme or microorganisms Methods of the invention may involve semi-quantitative determination of the enzyme of interest 100 as well as quantitative determination. In the former case, accurate substrate concentrations and temperatures may not be necessary, and such methods may be more suitable for methods carried out in the field, rather than a laboratory, using small, possibly portable devices (even microfluidics devices), which may lack temperature control, but which may optionally be incubated in a shirt or jacket pocket if necessary.

If the method is to be semi-quantitative, there may also be no need for numerical determination of the electric potential at the working electrode 101 and/or comparison with changes in electric potentials determined for standards or controls, a threshold change in electric potential, or slope of change in electric potential being sufficient to provide a positive or negative result, if this is desired. Alternatively, where the method involves quantitative determination of the enzyme of interest in a sample, the change in electric potential observed at step (c) may be compared to: the change in electric potential observed for a control reaction mixture 108 comprising no enzyme of interest; the change in electric potential observed for a reaction mixture 108 comprising a known amount of enzyme of interest 100; a calibration curve for change in electric potential versus amount of enzyme of interest 100, or any combination thereof.

The change in potential at the working electrode may be determined at any appropriate time or times. Detection of each molecule of substrate 104 or product 105 upon which the electrode-bound enzyme 102a or 102b may act, respectively, may require an extended amount of time (as the amount of substrate for the electrode-bound enzyme is depleted, the rate of reaction slows, providing an asymptotic depletion curve). Accordingly, the potential at the working electrode 101 may be determined at one or more set times, and the actual value, or the slope in change in potential may be used to relate back to amount of enzyme of interest 100 in the sample. Such a process may allow significantly accelerated assay times due to the need to only determine an initial rate of reaction, rather than allow the reaction to proceed substantially to equilibrium or to equilibrium.

Alternatively, the potential at a working electrode may be monitored continuously or periodically after contacting an electrochemical sensor of the invention with a reaction mixture (at the same time as, or soon after preparing the reaction mixture) and determining the period of time required for the potential observed at the working electrode to return to a steady baseline value, which may be a value substantially equivalent or equivalent to a value previously observed for the same, or similar electrode when contacted with a reaction mixture comprising no substrate. The period of time required for the potential at the working electrode may then be compared to a suitable control or standard so as to determine the amount of enzyme of interest or microorganisms present in a sample. For example, the period of time observed may be compared to the period of time taken for the potential at said working electrode to return to a steady baseline value for a reaction mixture comprising a known number of coliform bacteria. Alternatively, the period of time observed may be compared to a calibration curve for periods of time taken for the potential at a working electrode to return to a steady baseline value versus number of bacteria in reaction mixtures. Such methods may also be automated, as software for detecting asymptotic endpoints is readily available.

In methods of the invention where results obtained for a sample are to be compared to a suitable control, the sample, a positive control (a control with no substrate, or which has a known amount of microorganism) and/or a negative control (a control with substrate but no microorganism) may be tested using separate electrodes. However, to allow for possible inconsistencies between electrodes, the same electrode may be used to assay the sample and a positive control, a negative control or both. In such a method, so as to reduce inaccuracies due to carry over of substrates or products, the electrode may be washed with an appropriate medium between readings, or the readings may be carried out in the order: positive control; sample; negative control.

Specific Advantages of methods of the invention

The methods of the present invention lend themselves to automation, due to the simplicity of the methods, and the robustness of the materials involved. Thus, according to an embodiment, at least steps (b) and (c) of a method of the invention are automated. According to another embodiment, all of the steps of a method of the invention are automated.

The invention may thus provide a system to automatically obtain and present samples, such as a sample of water to an assay that detects and quantifies the amount of enzymic activity of interest and/or microorganisms present using a biosensor. Thus, methods of the present invention may allow automatic deployment of new assays and biosensors as required, allowing a self contained environmental monitoring system to run unattended.

Also, as sensors for use in methods of the present invention can be screen-printed, and are thus capable of being employed in small assay vessels/cells, at least steps (b) and (c) may be performed in multi-well plates, such as a 96-well plate.

Screen printed sensors may also be employed in sensor arrays for detection of multiple enzymic activities. This may allow for detection of patterns of enzyme expression by microorganisms, possibly allowing for detection of conditions under which a microorganism has been cultured, or allowing for detection of microorganisms with specific enzyme expression patterns.

Thus the present invention allows rapid (-4-6 hours) enumeration of low numbers of microorganisms, such as bacteria in water, automatically. This short assay time distinguishes the methods of the present invention from current, industry standard, culture-based methods of detection, which require an overnight incubation step. The ability to process samples automatically distinguishes the invention from other biosensor based systems, which require a human operator to manipulate the assay at various stages through the process. In addition, the central assay employed is electrochemical, utilising robust, cheap and disposable electrodes suited to field conditions and unattended operation, essential in the area of environmental monitoring. This assay is also generic in nature and could be adapted to detect a large number of enzymic activities of interest, microbial pathogens or indicator micro-organisms, both in the field of environmental monitoring and in other areas such as aquaculture or clinical diagnostics.

The greater speed of the methods of the present invention, especially where detection of microorganisms (such as pathogens in the environment e.g. E. coli and other coliforms) is concerned, allows results to be obtained more rapidly, and for decisions based on those results to be more effective. Additionally, the low cost of the assay, and the automation of the sampling procedure will allow many more samples to be taken in a given period of time. In the field of water monitoring this will enable a better predictive model of the effects of environmental events on the contamination of water sources to be built up or, for instance, in the field of aquaculture early indications of the presence of disease causing organisms that will enable preventive interventions. Monitoring of other pathogens in water, waste water, aquaculture, biosolids etc can also be achieved using the method of the invention. For example, Enterococci which are a major problem for the water industry and Legionella which is an important pathogen disseminated in water cooling systems may be monitored.

Devices for detection of microorganisms or types of microorganisms comprising one or more electrochemical sensors of the invention are also contemplated by the present invention. In such devices, the one or more sensors may be in contact with, or be contactable with a potentiometer. The potentiometer may be in communication with at least a data collecting module, either directly or through remote communication means. In addition, the device may comprise, or be contactable with a data processing module for relating a change in electric potential detected by the one or more sensors to the amount of microorganism or type of microorganism in a sample, either quantitatively or semi- quantitatively. For quantitative determination of amount of microorganism or type of microorganism, the processing module may also comprise data for: change in electric potential relating to the absence of microorganism in a sample; change in electric potential relating to presence of a known number of microorganism cells in a sample; a calibration curve relating change in electric potential to number of microorganism cells; or any combination thereof; and said module is capable of comparing a change in electric potential detected by the sensor with said data to quantitatively or semi-quantitatively determine the amount of said microorganism or type of microorganism in said sample.

Devices contemplated by the present invention include automated devices for remote monitoring of an environment (such as a waterway), or part automated or fully manual devices for laboratory use, as well as pocket-sized devices for environmental monitoring, which may comprise at least one or more sensors, a potentiometer and either data- acquiring or data transmitting means. Preferred forms of the present invention will now be described, by way of example only, with reference to the following examples, and which are not to be taken to be limiting to the scope or spirit of the invention in any way.

Examples

Example 1 - Preparing the sensors

Screen-printed electrochemical sensors with polypyrrole-coated working electrodes are sourced from Universal Sensors Ltd. (USL) (Unit 2 Suite 2, Abbey Barns, Duxford Road, Ickleton, Cambridge CB 10 ISX United Kingdom).

The sensors are removed from their protective wrapping, and heated to 15O⁰C for 2 minutes.

All subsequent steps are performed in the wells of a 96 well polypropylene microplate.

Direct, passive adsorption of enzyme to the working electrode

The sensors are immersed in an appropriate volume of solution comprising a desired concentration of enzyme for a sufficient period of time to allow for binding of the desired amount of enzyme to the working electrode. For example, for preparation of sensors suitable for detection of X-GaI in a reaction mixture, the sensors may be immersed in 160μl of 6U/ml β-galactosidase in 0.1M phosphate buffer (pH7.3) for a time in the range 1 minute to 1 hour or more (e.g. 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.5, 4, 4.5 , 5 hours or more). The sensors may be immersed in 160μl of 6U/ml β-galactosidase in 0.1M phosphate buffer (pH7.3) for about 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15,, 16,

17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55 or

58 minutes for example.

The sensors are then washed to remove unbound enzyme. In the specific example given above, the wash may comprise immersing the sensors in three aliquots of 160 μl phosphate buffer.

Sensors are now ready for immediate use, or covering with a protective coating (such as Arrayguard™, or Postcoating buffer, available from TropBio, Townsville, Queensland, Australia) for storage. Binding of enzyme to the working electrode via a streptavidin-biotin linkage

The sensors are immersed in an appropriate volume of solution comprising streptavidin (Sigma; Cat No. S0677) at 80 μg/ml in sucrose buffer. 7μl of streptavidin solution are required for each lmm² working electrode.

The streptavidin solution is then dried onto the electrode (37°C/3hr minimum) and the sensor then washed to remove unbound streptavidin.

Biotin-conjugated β-galactosidase (Sigma; Cat. No. G5025) is dissolved in a proprietary enzyme stabilising solution, (Stabilzym, Surmodics Inc.) at 1 mg/ml. A further dilution of this stock is made in PBS (pH 7.0) to give the desired enzyme concentration, and 7 μl of this is pipetted onto the washed streptavidin-coated electrode. The biotinylated β- galactosidase is specifically oriented and bound by the interaction of the biotin molecule with the specific receptors for this molecule on the streptavidin on the polypyrrole electrode. The sensors are then washed to remove unbound enzyme. The captured enzyme (β-galactosidase, or any other diagnostic bacterial enzyme) is then dried on to the sensor (3 hrs 37°C), and a protective layer of microarray coating (such as Array guard™, or Postcoating buffer, available from TropBio, Townsville, Queensland, Australia) optionally applied and dried. Sensors are then stored at 4⁰C until use.

Binding of enzyme to the working electrode via antibody attachment

Antibody could also be applied to the surface of sensors by pipetting sufficient volume of antibody solution (concentration determined empirically) in a suitable buffer, such as phosphate buffered saline pH 7.4 (for example, 30μL for carbon electrodes and 3μL for polypyrrole electrodes).

Sensors could then be dried at 37°C until almost dry (such as for about 15 min), then 30μL or 3μL of protective solution (for example, TropBio post coating buffer) could be applied, optionally, and the sensor fully dried, and stored at 4°C, dehydrated.

Immediately prior to use, if a protective layer has been applied sensors could then be washed with buffer to remove the protective layer.

Enzyme solution may then be either pipetted onto the working electrode, or the electrode immersed in enzyme solution. After a period of incubation to allow capture of the enzyme by the antibody, the sensor could be rinsed to remove unbound antibody.

Sensors could then be immediately used to assay for a substrate either as described above, or by immersion in the substrate solution, with data collected by, for example, a USL reader in the case of USL polypyrrole sensors or using a millivolt meter in the case of other electrodes, such as carbon electrodes.

If the antibody is to be attached to sensors by specific linkage, such as streptavidin/biotin, the streptavidin could be applied to the electrode by any suitable method known in the art. For example, for linkage of streptavidin to USL polypyrrole sensors, this may be carried out as described in the USL technical manual.

A solution of streptavidin (for example, 80μg/mL in a 10% sucrose solution in 0.005M phosphate buffer pH 8.0) may be placed on the working electrode surface (for example, 30μL for carbon electrodes, 3μL for USL polypyrrole electrodes) and dried at 37°C for 4 hours. The sensors can then be stored desiccated at 4⁰C.

Prepared sensors could then be washed in PBS, and a solution of biotinylated antibody conjugated to the enzyme of interest applied to the working electrode (for example, 30μL for carbon electrodes or 3μL for USL polypyrrole electrodes as before), or the sensor immersed in biotinylated antibody-enzyme solution, and incubated for a period to allow interaction of the streptavidin with the biotin tag on the antibody.

After a rinse to remove unbound antibody, the sensor would be used in the assay as above.

Antibody can be biotinylated using any proprietary kit or method as known in the art.

Example 2 - Development of sensors and methods for detection of substrates and, thereby, enzymic activities

Sensors were prepared as described in Example 1, with E. coli β-galactosidase bound to the working electrode either directly (adsorbed passively) or via a streptavidin-biotin linkage. The concentration of β-galactosidase applied to the sensors was varied as desired.

Sensors were held in individual channels of a device that measures the electrical potential of individual sensors (produced by USL of Unit 2 Suite 2, Abbey Barns, Duxford Road, Ickleton, Cambridge CB 10 ISX United Kingdom). A total of 12 sensors can be held in this device, which enables 6 sets of control and test readings. Each sensor is located in a well of a 96 well polypropylene plate. The electrical output from each sensor is measured and displayed using proprietary software provided by USL.

For the purposes of assessing the performance of sensors prepared by methods as described in Example 1, each was immersed in X-GaI (5-bromo-4-chloro-3-indolyl-β-D- galactoside) in 2 x yeast tryptone growth medium at the desired concentration (50mg/mL X-GaI stock solution diluted in 2 x yeast tryptone as required). The concentration of X- GaI in the growth medium was varied as desired.

In a first series of experiments, the effect of different concentrations of β-galactosidase applied to sensors during their preparation was investigated, using β-galactosidase concentrations of from 2μg/mL to lOμg/mL. The change in electric potential observed at the working electrode of each β-galactosidase treatment was monitored after immersion of the sensors in 50μg/mL X-GaI. The results are shown in Figure 3.

In another series of experiments, the sensitivity of sensors prepared using different β- galactosidase concentrations, at different dilutions of X-GaI was investigated. The results are illustrated in Figures 3A to 3D, showing good sensitivity of all of the sensors to a wide range of X-GaI concentrations.

In another series of experiments, the effect of binding β-galactosidase to the sensors by direct/passive adsorption, as compared to binding of the β-galactosidase to the sensor via a streptavidin-biotin linkage was investigated. The results are illustrated in Figures 4A and 4B: Figure 4 A provides a bar chart of electrical potential at each electrode (duplicates for each β-galactosidase binding method/treatment) after 3 minutes immersion in 2x yeast tryptone growth medium comprising 50μg/mL X-GaI. Figure 4B shows the raw potential traces obtained for the data represented in Figure 4A; filled triangles in Figure 4B represent the sole streptavidin-biotin sample, and hollow symbols represent Oμg/mL X-GaI controls; remaining [filled] symbols represent directly bound/passively adsorbed samples. The results show that β-galactosidase can be bound to polypyrrole- coated sensors directly by passive adsorption, without significant loss in activity as compared to binding of the β-galactosidase to the sensor by a streptavidin-biotin linkage.

The results provided in Figures 4A and 4B also show that the electrodes are almost saturated with β-galactosidase when a solution comprising 1.75μg/mL β-galactosidase is applied to the sensors during their preparation.

In a following series of experiments, saturation of sensors with β-galactosidase was further investigated, β-galactosidase in the solution applied to the sensors during their preparation being varied from 0-100 Units/mL. Figure 5 shows that, with a new batch of β-galactosidase (non-biotinylated) only 6U/mL in the coating buffer was required to give maximum output. This figure also shows that the β-galactosidase is substantially unaffected by storage on dry ice for three days. Electrodes prepared using 6U/mL β-galactosidase had an activity of approximately 8x10 ,-5 units/electrode (μmoles substrate converted per minute/electrode; working electrode surface area = ~1.3mm).

In a following series of experiments, a calibration curve was obtained for 6U/mL β- galactosidase sensors for a range of X-GaI concentrations (0-lOOμg/mL) in defined medium (EZRich medium - F. C. Neidhardt, P. L. Bloch, and D. F. Smith (1974),

"Culture medium for enterobacterial J Bacteriol. 119(3): 736-747; http : //www, genome . wise . edu/resources/protocols/TekNo va.htm) . The results are shown in Figure 6 (data shown as % of full scale deflection achieved with lOOμg/mL X-GaI), which shows good sensitivity of these electrodes in the range of from about 2.5μg/mL to about 85μg/mL X-GaI under these conditions.

In another series of experiments, the effect of β-galactosidase coating/binding time on the resulting sensitivity of the resulting sensors was investigated. Non-biotinylated β- galactosidase, and direct/passive adsorption was used for binding β-galactosidase to the sensor. As can be seen from Figures 7 A and7, 10 minutes coating time is sufficient to reliably saturate the electrode surface with β-galactosidase.

In another series of experiments, the repeatability of methods for coating β-galactosidase onto sensors was investigated (β-galactosidase bound to the sensors by streptavidin-biotin linkage). 7.5μg/mL biotinylated β-galactosidase was applied to 11 sensors as described in Example 1 and then immersed in 2x yeast tryptone growth medium comprising 50μg/mL X-GaI. The results, shown in Figures 8A and 8B, show very good repeatability of the β- galactosidase coating procedure (streptavidin-biotin linkage method), as particularly illustrated by the small standard deviation bar on the mean/average result for all 11 sensors, and the raw data traces for change in electrical potential for each of the sensors (Figure 8B).

In another series of experiments the effects of carryover of substrate when sensors are used to read samples sequentially was investigated. As can be seen by the results shown in Figures 9A and 9B, carry over of substrate can introduce significant errors into readings obtained, especially when a sensor is moved from a sample with a high substrate concentration to one with a low substrate concentration.

In the course of these studies: the saturating concentration of β-galactosidase on the sensor was determined - the concentration of β-galactosidase in the solution applied to the working electrode can be cut down to 6U/mL while retaining full scale change in potential; the minimum detectable concentration of X-GaI over no substrate control, using β- galactosidase-saturated sensors was determined, concentrations of X-GaI below 0.5μg/mL being detectable; different methods of coating β-galactosidase onto the electrodes were examined, passive adsorption and linkage via a streptavidin-biotin binding pair providing substantially similar results with polypyrrole-coated working electrodes; methods of stabilising the enzyme were also evaluated, enzyme can be dried to the sensor surface and stored under a protective layer (such as Postcoating buffer, available from TropBio, Townsville, Queensland, Australia) for at least several days at 4⁰C. Also β-galactosidase was shown to be resistant to storage on dry ice for three days; some problems were encountered with aberrant output from individual sensors, possibly due to inconsistent coating of sensor surface or electrochemical deposition of polypyrrole, which resulted in an increase in variability of sensor output, reducing sensitivity and repeatability.

Example 3 - Sensors with β-glucuronidase or β-glucosidase bound thereto

Sensors were prepared as described in Example 1, with E. coli β-glucuronidase or β- glucosidase from almonds bound/adsorbed to the working electrode directly. The concentration of enzyme applied to the sensors was varied as desired. Sensors were held in individual channels of a device that measures the electrical potential of individual sensors (produced by USL of Unit 2 Suite 2, Abbey Barns, Duxford Road, Ickleton, Cambridge CB 10 ISX United Kingdom). A total of 12 sensors can be held in this device, which enables 6 sets of control and test readings. Each sensor is located in a well of a 96 well polypropylene plate. The electrical output from each sensor is measured and displayed using proprietary software provided by USL.

For the purposes of assessing the performance of sensors prepared by methods as described in Example 1, β-glucuronidase sensors were immersed in X-GIcA (5-bromo-4- chloro-3-indolyl-β-D-glucuronide) dissolved in 2 x yeast tryptone growth medium at the desired concentration and β-glucosidase sensors were immersed in X-GIc (5-bromo-4- chloro-3-indolyl-β-D-glucoside) dissolved in 2 x yeast tryptone growth medium at the desired concentration.

The results shown in Figure 1OA show preliminary results from the depletion of X-GIcA by β-glucuronidase. A decrease in signal is observed as the concentration of enzyme bound onto the sensor decreases, but output/sensitivity is not as high as with β- galactosidase. Orientation of the enzyme onto the sensor with a specific interaction should improve this.

The results illustrated in Figure 1OB provide an indication that the sensors will also work with β-glucosidase bound to them, as a difference in signal could be seen as the concentration of β-glucosidase decreased. The sensitivity in these preliminary experiments was not ideal, but further work on enzyme concentration, orientation and assay conditions should improve the results.

Example 4 - Electrochemical detection of enzymic activity in a sample

A schematic of the principle behind methods for detecting enzymic activities according to the invention (exemplified by secreted or cell-surface bacterial enzymes) is provided in Figure 11.

For a control measurement, a sensor is immersed in 400μL of solution comprising no substrate (control) and, after three minutes, the output of the sensor is read for 90s, with an initial 10s OmV potentiostat clamp. The reading at 90s is the "Baseline" reading.

A "Test" reading is obtained as follows:

A reaction mixture comprising an initial substrate concentration "[Sj]" and sample containing enzyme of interest (but otherwise of the same composition as that for the "Baseline" reading) is incubated for a set amount of time. A sensor is then immersed in 400μL of the reaction mixture, which has been optionally filtered or otherwise treated to remove or deactivate the enzyme and, after three minutes, the output of the sensor is read for 90s, with an initial 10s 0 mV potentiostat clamp. The reading at 90s is the "Test" reading.

Output is expressed as:

"Test" - "Baseline" = "Difference"

To quantify the amount of substrate depleted by the enzyme in the 'Test' sample, and represented by this difference, the difference may be compared to a standard curve prepared using one or more 'standard' determinations as described below, where the 'Baseline' readings are subtracted from each of the 'Standard' readings.

Suitable 'standards' may be prepared by dissolving known amount(s) of substrate in a solution of the same composition as that used for the 'Baseline' determination or by incubating one or more solutions comprising known amount(s) of enzymic activity and an initial substrate concentration [Sj] for the same set time period(s) as used for the 'Test' sample. A sensor is then immersed in the 'standard' reaction mixture, which has been optionally filtered or otherwise treated to remove or deactivate the enzyme and, after three minutes, the output of the sensor is read for 90s, with an initial 10s OmV potentiostat clamp. The reading at 90s is the "Standard" reading.

Where a single known enzyme activity is used as a 'standard', the enzymic activity in the test sample may be calculated as:

"Test" - "Baseline" x amount of enzymic activity in the standard sample "Standard"-"Baseline"

Where a single known concentration of substrate has been used as a standard, the amount of substrate remaining in the sample may be calculated as: "Test" - "Baseline" x Amount or Concentration of substrate in the standard "Standard"-"Baseline" sample

The difference between that amount/concentration and the amount/concentration originally present in the reaction mixture may then be equated to the number of moles of substrate depleted, and then equated to the amount of enzymic activity (as, for example, moles/minute, or more typically μmoles/minute) by dividing the number of moles of substrate depleted by the length of the original reaction mixture incubation time. A similar procedure applies where a calibration curve of substrate concentration against "Standard"-"Baseline" values is prepared, and the amount of substrate remaining in the test sample is read off against this curve.

Example 5 - Electrochemical detection of bacteria, such as coliform bacteria in a sample

For detection of the presence of bacteria in test samples, the samples were incubated for between 2 and 7 hours in 2 x yeast tryptone growth medium comprising 50μg/mL X-GaI (5-bromo-4-chloro-3-indolyl-β-D-galactoside) at 37°C. This was carried out in 5mL polypropylene vials with shaking at 225 r.p.m. to aerate. An aliquot was then centrifuged in a 1.5mL Eppendorf tube to remove bacterial cells (12,00Og, 2 min) and the supernatant tested for the presence of residual or non-depleted X-GaI using a sensor of the invention as follows: - Immerse sensor in 400μL of 2x yeast tryptone bacterial growth medium (Positive control, representing total depletion of X-GaI) Wait 3 minutes.

Read for 90s, with an initial 10s OmV potentiostat clamp. Reading at 90s is 'Baseline" reading.

- Move sensor to 400μL of filtered depleted substrate (2YT + 50μg/mL starting concentration of X-GaI) Wait 3 min.

Read for 90s, with initial 10s OmV clamp. Reading at 90s is "Depleted" reading.

- Move sensor to negative control (2YT + 50μg/mL X-GaI is currently used, lower concentration should improve sensitivity) Wait 3 minutes. Read as section 3 and 7. Final reading is "Maximum." Output is expressed as:

(Maximum-Baseline)-(DepIeted-Baseline) = 'Difference'

Difference/(Maximum-BaseIine) x 100 = Drop in output as % of Full Scale

Deflection (FSD).

Results may also be represented by electric potential - raw or adjusted (compared to an appropriate control).

The results may also be read against results obtained for known concentrations of bacteria under the same assay conditions in order to estimate the initial inoculum of bacterial cells present in the sample before incubation in the growth medium.

The results shown in Figures 12 to 17 show results of experiments carried out as described above for different initial inocula, and incubation times using E. coli strain ATCC 11775 or wild-type strain 365 and illustrate also the effect of initial inoculum size and cell multiplication on the sensitivity of bacterial detection by the sensors. The data illustrated in Figure 12 were obtained using a sensor on which β-galactosidase was bound via a streptavidin-biotin linkage, the remaining data were obtained using sensors to which the β-galactosidase was passively adsorbed.

High negative control readings were observed in some experiments, emphasising the need to carry out appropriate controls if quantitative assays are desired.

The highest sensitivity achieved was between 500 and 1000 log phase E. coli in 500μL of broth, although detection of as low as about 100 cells has been achieved after incubation of the reaction mixture for 5 hours.

Further work has shown that, at least for USL polypyrrole electrodes to which a solution of β-galactosidase (6U/mL) has been applied, sensitivity is greatest in the range of about 2.5μg/mL to about 60-85μg/mL (see, for example, Figure 6). Accordingly, where anticipated concentrations of target bacteria are high, a concentration of X-GaI higher than 50μg/mL, such as lOOμg/mL X-GaI may be used. Where anticipated concentrations of target bacteria are low, 50μg/mL X-GaI may be a more appropriate starting concentration.

Further work has also shown that more uniform results are achieved for detection of coliform bacteria if the sample comprising such bacteria is pre-treated with IPTG, which induces expression of β-galactosidase. Thus, 5μM IPTG may be included in the sample medium and negative control medium, to induce β-galactosidase activity without significant depression of signal from the sensor (see Example 6, below).

Figure 18 shows the difference in normalised % drops for a given inoculum of bacteria into 2x yeast tryptone growth medium comprising 5μM IPTG and either 100 or 60μg/mL X-GaI. Consistent with Figure 6, the non-linear fitted line gives a larger % drop at lower initial inocula of bacteria inoculated at 60μg/mL X-GaI than in lOOμg/mL X-gal, but as initial bacterial inocula increase the fitted line gets steeper for the lOOμg/mL X-GaI line.

Example 6 - Interfering factors

In the experiments described herein, sensors comprising β-galactosidase passively bound/adsorbed to the working electrodes were prepared as described in Example 1, and measurements taken as described in Example 2. All reaction mixtures comprised 2 x yeast tryptone growth medium comprising 50μg/mL X-GaI, and additional substances as described below. Figure 19 shows the effect of pH changes on the sensitivity of the sensors. If cultures are left to grow for more than 4 hours or so, the pH of the medium can drop by up to 1 pH unit or more, this has a small effect on the sensor output, as can be seen by the reduced signal obtained from a totally depleted sample spiked back up to 50 μg/ml X-gal after culture (as compared to the response achieved for spiked samples that had no bacterial cells in them). Better buffering of the growth/assay medium, or a defined medium, or culture times of less than 4 hours may remove this problem.

Figure 20 shows the results of an experiment examining the effect of SDS, a potential permeabiliser of the bacteria (to increase apparent β-galactosidase activity/cell) but also a dopant in the polypyrrole that alters the potential of the electrode and decreases the overall signal from the sensor. While ImM SDS in the growth/assay medium did reduce sensitivity, changes in electric potential observed (as compared to the 1 mM SDS positive control) were still proportional to initial inoculum size.

Figures 21 and 22 show the effect of the presence of lactose and IPTG (respectively) in the reaction mixture on detection of X-GaI. The data show that lactose and IPTG affect the sensor output in a concentration-dependent manner. This is not unexpected as lactose and IPTG are substrates for β-galactosidase and would therefore be expected to compete with X-GaI for the active site of the enzyme. The presence of lactose or IPTG may be an issue in detecting coliform bacteria, as these compounds can be used to induce expression of β-galactosidase in coliform bacteria to enhance their detection in dilute samples.

Figure 22 illustrates results for two separate experiments, one testing the effect of IPTG at concentrations of 1OmM, 5mM, 2.5mM, 1.25mM and 0.625mM on detection of X-GaI (50μg/mL), and another testing the effect of IPTG at concentrations of ImM, 0.5mM, 0.25mM, 0.125mM and 0.062mM on detection of X-GaI (50μg/mL).

Example 7 - Testing of water samples for the presence of coliform bacteria

A representative water sample is obtained, optionally sub-sampled, and concentrated if, and as required and then mixed with an equal volume of double strength (2x concentration) bacterial growth medium comprising the desired concentration of substrate to form a reaction mixture. This can be a crude medium, a selective medium or a defined medium, to engender rapid growth and metabolism of the substrate (currently X-gal {5- Bromo-4-chloro-3-indolyl β-D-galactopyranoside} but any substrate that gives an electrochemical signal upon metabolism by β-galactosidase can be used).

The reaction mixture is incubated at 37⁰C for a period of time (such as -1.5 hours) that may be adjusted depending on the requirements of the use (i.e. the sensitivity of the assay will increase with increased duration of culture). A negative control containing no bacteria (or a control containing a known number of bacteria) or both are incubated under identical conditions.

Both test and control cultures are filtered to remove the bacteria and the number of coliform bacteria in the reaction mixture (at the begimiing of the incubation) may then be determined by a method as described in Example 4 or Example 5.

Detection of other enzymes or microorganisms of interest

The β-galactosidase/X-Gal: enzyme/substrate system used to generate the signal from the electrochemical sensor in the proposed assay for coliforms in water could be adapted by using different substrates for the β-galactosidase that may generate a larger signal for a given concentration. The enzyme itself can be replaced by other species specific enzymes, i.e. β-glucuronidase, specific for E. coli or any other enzyme diagnostic of a pathogen or indicator species that generates an electrical signal on exposure to its substrate, which could be expressed in a recombinant system to biotinylate it allowing specific immobilisation on the streptavidin-coated sensor.

Example 8 - Enzymes bound to carbon-based electrodes/sensors

Carbon paste electrodes (types C10903P1 and C2000802P2) were sourced from Gwent Electronic Materials Ltd, Monmouth House, Mamhilad Park, Pontypool, NP4 OHZ, United Kingdom

β-Galactosidase Sensors were coated with either 6U/mL β-galactosidase in 0. IM Phosphate buffer pH 7.3, or phosphate buffer pH 7.3 alone (control). 30μL of either solution was applied directly to the working electrode, and incubated for 1 hour at room temperature. Sensors were then washed with 200 μL of phosphate buffer, by pipetting the buffer on and then removing it immediately.

The sensors were then tested for response with 2x yeast tryptone (2YT) broth comprising increasing concentrations of X-GaI applied to the electrode sequentially by pipetting 200 μL of each test solution onto the electrode, ensuring coverage of the reference electrode. Readings were taken with a millivolt meter 60 seconds after applying the sample.

Sensors were then rinsed with 2YT broth comprising the next [higher] X-GaI concentration, then 200μL of that 2YT X-GaI sample applied to the sensor and measured as before. Figures 23 A and 23B show the response achieved for each carbon paste electrode type for the given X-GaI concentrations (0-lOOμg/mL).

Urease

Sensors coated by direct application of 30μL of 295U/mL urease in 0.2M Phosphate 5 buffer pH 7.1 , 29.5U/mL urease in 0.2M Phosphate buffer pH 7.1 , or phosphate buffer pH 7.1 alone (control) to the working electrode, and incubated for 1 hour at RT. Sensors were then washed with 200μL of phosphate buffer, by pipetting the buffer on and then removing it immediately.

The sensors were then tested for response with samples of buffer comprising increasing ic concentrations of urea (0-lOmg/mL urea) applied to the electrode sequentially by pipetting 200 μL of each test solution onto the electrode, ensuring coverage of the reference electrode. Readings taken with a millivolt meter 60 seconds after applying the sample.

Sensors were then rinsed with the next [higher urea concentration] sample, then 200μL of is that sample applied to the sensor and measured as before.

Figures 24A and 24B show the response achieved for each carbon paste electrode type, comprising the three different urease concentrations, for the given urea concentrations (0- lOmg/mL).

It will be appreciated that, although a specific embodiment of the invention has been 20 described herein for the purpose of illustration, various modifications may be made without deviating from the spirit and scope of the invention as defined in the following claims.

Claims

Claims:

1. A method for detecting the amount of an enzyme of interest in a sample, comprising:

(a) incubating a reaction mixture comprising said sample and at least one s substrate which can be acted on by said enzyme;

(i) after a selected time of incubation of the reaction mixture or (ii) at the same time as or soon after preparing the reaction mixture,o wherein said sensor comprises a working electrode which has immobilized therein or thereto a second enzyme which is capable of acting on a substance selected from one or more of said at least one substrate and a product resulting from action of the enzyme of interest on said at least one substrate; wherein action of said second enzyme on said at least one substrate or product results in a change in electric potential at said workings electrode;

(c) either:

(ii) determining the period of time required for the electric potential at said0 working electrode to return to a substantially steady baseline value after said contacting in step (b)(ii); and

(d) determining from the change in electric potential detected at step (c)(i) or period of time determined at step (c)(ii) the amount of enzyme of interest in the sample.

2. A method according to claim 1, wherein the enzyme of interest and the₅ second enzyme are both capable of acting on the same substrate, and wherein the presence of the enzyme of interest in the reaction mixture results in a smaller change in electric potential or smaller period of time observed at step (c) than that observed in the absence of said enzyme.

3. A method according to claim 1 or claim 2, wherein said enzyme of interest is0 associated with a microorganism or type of microorganism.

4. A method according to claim 3, wherein said microorganism or type of microorganism comprises coliform bacteria.

5. A method according to any one of claims 1 to 4, wherein the enzyme of interest is a β-galactosidase or a β-glucuronidase, and the substrate is a β-galactoside or β-₅ glucuronide derivative respectively.

6. A method according to claim 5, wherein the enzyme of interest is a β- galactosidase.

7. A method according to claim 5 or claim 6, wherein the second enzyme is a β- galactosidase.

5 8. A method according to any one of claims 1 to 7, wherein said at least one substrate is a 5-bromo-4-chloro-3-indolyl or a 5-bromo-6-chloro-3-indolyl linked compound.

9. A method according to claim 8, wherein said substrate is a 5-bromo-4-chloro- 3-indolyl β-linked glycoside or glucuronide. o

10. A method according to claim 8, wherein said substrate is 5-bromo-4-chloro-3- indolyl β-D-galactopyranoside.

11. A method according to any one of claims 1 to 10 wherein the sample is derived from a water source and the enzyme of interest is associated with a microorganism or type of microorganism. s

12. A method according to any one of claims 1 to 11, wherein the reaction mixture is filtered after step (a) to remove enzyme of interest or bacteria or enzyme of interest and bacteria from the mixture to be applied to the sensor.

13. A method for detecting coliform bacteria in a sample, comprising:

(a) incubating a reaction mixture comprising said sample and a substrate0 comprising a β-galactoside or β-glucuronide derivative;

(i) after a selected time of incubation of the reaction mixture or (ii) at the same time as or soon after preparing the reaction mixture,s wherein said sensor comprises a working electrode which has immobilized therein or thereto an enzyme which is capable of acting on said substrate; wherein action of said sensor-bound enzyme on said substrate results in a change in electric potential at said working electrode;

(c) either: 0 (i) detecting the change in electric potential at said working electrode after said contacting in step (b)(i); or

(ii) determining the period of time required for the electric potential at said working electrode to return to a substantially steady baseline value after said contacting in step (b)(ii); 5 (d) either: (i) comparing the change in electric potential detected at step (c)(i) with: the change in electric potential detected for a control reaction mixture comprising no coliform bacteria; the change in electric potential detected for a known number of coliform bacteria; a calibration curve for change in electric potential versus number of

5 bacteria; or any combination thereof; or

(ii) comparing the period of time determined at step (c)(ii) with: the period of time taken for the electric potential at said working electrode to return to a steady baseline value for a reaction mixture comprising a known number of coliform bacteria; or a calibration curve for periods of time taken for the potential at said working electrode toQ return to a steady baseline value versus number of bacteria in reaction mixtures; and

(e) determining from the change in electric potential observed at step (c)(i) or the period of time observed at step (c)(ii) the amount of coliform bacteria in the sample.

14. A method according to claim 13, wherein said enzyme is a β-galactosidase or a β-glucuronidase. s

15. A method according to claim 13, wherein said substrate is a 5-bromo-4- chloro-3-indolyl β-linked glycoside or glucuronide.

16. A method according to claim 13, wherein said substrate is 5-bromo-4-chloro- 3-indolyl β-D-galactopyranoside.

17. An electrochemical sensor comprising a working electrode which has0 immobilized therein or thereto an enzyme which is: (i) capable of acting on a substrate, said substrate being one which is capable of being acted on by an enzyme of interest; or (ii) which is capable of acting on a product resulting from action on a substrate by an enzyme of interest; wherein the enzyme of interest is associated with a microorganism or type of microorganism; wherein action of said electrode-immobilised enzyme on said5 substrate or product results in a change in electric potential at said working electrode.

18. A sensor according to claim 17, wherein the enzyme of interest and the electrode-immobilised enzyme are both capable of acting on said substrate.

19. A sensor according to claim 18, wherein the said electrode- immobilised enzyme is capable of acting on a β-galactoside or β-glucuronide derivative. o

20. A sensor according to claim 19, wherein said electrode- immobilised enzyme is a β-galactosidase or a β-glucuronidase.

21. An electrochemical sensor comprising a working electrode which has immobilized therein or thereto an enzyme which is capable of acting on a β-galactoside or β-glucuronide derivative to produce a change in electric potential at said working5 electrode.

22. A sensor according to claim 21, wherein said enzyme is a β-galactosidase or a β-glucuronidase.

23. A sensor according to claim 22 which comprises polypyrrole or a polypyrrole coating as electroconductive material.

24. A sensor according to claim 23 which comprises a β-galactosidase adsorbed directly to said polypyrrole.

25. A device for detecting the amount of a microorganism or a type of microorganism in a sample, said device comprising at least one electrochemical sensor according to any one of claims 17 to 22 in contact with, or contactable with a potentiometer.

26. A method for detecting the amount of an enzyme of interest in a sample, comprising:

(a) incubating a reaction mixture comprising said sample and at least one substrate which can be acted on by said enzyme; (b) contacting a second enzyme with the reaction mixture, wherein said second enzyme is capable of acting on a substance selected from one or more of said at least one substrate and a product resulting from action of the enzyme of interest on said at least one substrate and wherein action of said second enzyme on said at least one substrate or product results in a change in electric potential at a working electrode; and (c) detecting the electric potential at said working electrode at one or more times after said contacting and determining from the electric potential or potentials detected the amount of enzyme of interest in the sample.

27. A method for detecting the amount of an enzyme of interest in a sample, comprising: (a) incubating a reaction mixture comprising said sample and at least one substrate which can be acted on by said enzyme;

28. A method for detecting coliform bacteria in a sample, comprising:

5 (a) incubating said sample in a reaction mixture with a substrate comprising a β- galactoside or β-glucuronide derivative;

(b) contacting an enzyme with the reaction mixture, wherein said enzyme is capable of acting on said substrate, and wherein action of said enzyme on said substrate results in a change in electric potential at a working electrode; and io (c) detecting the electric potential at said working electrode at one or more times after said contacting and determining from the electric potential or potentials detected the number of bacteria in the sample.

29. A method for detecting coliform bacteria in a sample, comprising:

(a) incubating said sample in a reaction mixture with a substrate comprising a β- I₅ galactoside or β-glucuronide derivative;

(b) contacting a sensor and an enzyme with the reaction mixture, wherein said sensor comprises a working electrode which has immobilized therein or thereto said enzyme, wherein said enzyme is capable of acting on said substrate, and wherein action of said enzyme on said substrate results in a change in electric potential at said working

20 electrode; and