WO2009103985A1

WO2009103985A1 - Method and kit

Info

Publication number: WO2009103985A1
Application number: PCT/GB2009/000465
Authority: WO
Inventors: John Vontas; Janet Hemingway
Original assignee: The Liverpool School Of Tropical Medicine
Priority date: 2008-02-20
Filing date: 2009-02-19
Publication date: 2009-08-27
Also published as: GB0803082D0

Abstract

The present invention relates to a method suitable for but not limited to measuring levels of insecticide compounds such as DDT [(1, 1, 1-trichloro-2, 2-bis (p-chlorophenyl) ethane]. More specifically the present invention relates to a method suitable for measuring the level of xenobiotics, for example DDT in field applications using recombinant glutathione-S transferase enzymes (GST) and either colorimetric detection agents or potentiometric sensors and to a kit for performing same.

Description

METHOD AND KIT

Field of the Invention

The present invention relates to a method suitable for but not limited to measuring levels of insecticide compounds such as DDT [(1 , 1 , 1-trichloro-2, 2-bis (p- chlorophenyl) ethane]. More specifically the present invention relates to a method suitable for measuring the level of xenobiotics, for example DDT in field applications using recombinant glutathione-S transferase enzymes (GST) and either colorimetric detection agents or potentiometric sensors and to a kit for performing same.

Background to the Invention

The use of indoor residual spraying (IRS) is an example of current control measures utilised in the fight against diseases carried by insects, such as malaria.

However, the only way to verify whether or not the control measures are effective and thereby ensure that protection is being provided by such spray interventions is to measure the actual amount of insecticide residues on the surfaces that the insects such as mosquitos are exposed to.

Several testing technologies exist which are employed to measure the amount of insecticides in insecticide treated materials (ITMs) and other such surfaces. These include analytical methods, such as High Performance Liquid Chromatography (HPLC) and Gas Chromatography (GC), enzyme-linked immunosorbent assays (ELISA) based on specific antisera raised against insecticides, taggant/IR technologies and chemical sensors (for example pulse polarography).

Most of the methodologies currently available require technically demanding approaches, and expensive equipment that restricts their use in the required field applications. Less sophisticated assays have also been developed, such as bioassays. These methods whilst simple to use and not requiring multi-step sample clean-up procedures, unfortunately only provide rough estimates of the levels of compounds to be detected. In the ever-increasing fight against diseases such as malaria, rough estimates of the levels of compounds remaining at given location test sites are not sufficient.

The use of biosensor enzymes to measure the amount of certain compounds for example insecticides and xenobiotics is an alternative approach, which allows simple and relatively accurate estimates to be obtained. This technique is based on the recognition of recombinant enzymes for specific xenobiotics and the appropriate methodology for measuring the interaction.

For example, it is possible to detect organophosphate and carbamate insecticides by monitoring colorimetrically the inhibition reaction of recombinant acetylcholinesterase (AChE) that these compounds catalyse (Andreescu et al 2006, Francis et al 2006).

Likewise, in Wong FCM. , et al (2006) there is described an optical biosensor for dichlovos using stacked sol-gel films containing acetylcholinesterase and a lipophilic chromoionophore, Talanta. In contrast to the present invention this method utilises acetylcholinesterase, an enzyme which irreversibly binds organophosphate and carbamate insecticides.

A recombinant glutathione-S transferase (GST) - based assay has also been developed for the quantification of pyrethroid insecticides. This method is based on the inhibition of glutathione-S transferase (GST) - catalysed reactions caused by the insecticide, which is monitored spectrophotometrically, or endpoint determined by iodometric titration (Vontas et al 2000, Enayati et al 2001). In Enayati A. A. et al 2001 , Medical and Veterinary Entomology 15, 58-63) the quantification of pyrethroid insecticides from treated bednets using a mosquito recombinant glutathione S-transferase is described. In contrast to the present invention, the principle of the above quantification method (Enayati et al 2001) is monitoring enzymatic inhibition (non specific), compared to the specific substrate recognition reaction utilised according to the present invention.

However, none of the prior teachings disclose reliable procedures which are both easy and accurate to use and which can be used to detect the levels of specific compounds such as insecticides and xenobiotics for example DDT, [(1 , 1 , 1- trichloro-2, 2-bis (p-chlorophenyl) ethane] in field applications where if is often necessary to take and record numerous measurements easily and quickly and to analyse the data accurately at the test site.

The present invention therefore seeks to provide a novel method of determining the quantity of an insecticide compound, for example DDT, by monitoring the progress of the reaction when the compound, that is the DDT is utilised as a substrate by the glutathione-S transferase enzyme. Detection is based on an ion change, for example the pH change occurring in an appropriately buffered reaction mixture, due to the release of hydrogen ions (H⁺) during the glutathione-S transferase (GST) - DDTase catalysed reaction.

That is the invention relates to a method of assaying recombinant glutathione-S transferase (GST) catalysed reactions of xenobiotics or model substrates which comprises monitoring and measuring the amount of ions generated when a GST enzyme is used to catalyse the reaction wherein the level of detected ions is used to verify the amount of xenobiotic or model substrate in the reaction system. Alternatively the detection procedure measures the amount of chloride ions (Cl^") generated when a GST-DDTase enzyme is used to catalyse the dehydrochlorination of the DDT or a similar test compound. The detection of the change in the ion levels is therefore monitored (a) potentiometrically (pH or Cl electrode), (b) colorimetrically (in the presence of for example a pH marker, or chromogenic Cl detectors), or (c) by commercially available chlorine (Cl) strips.

That is, the method comprises: measuring the amount of chloride ions (Cl^") generated when the GST enzyme catalyses the dehydrochlorination of the xenobiotic or model substrate.

The glutathione-S transferase (GST) enzyme has DDTAse activity and is able to catalyse a dehydrochlorination reaction of the xenobiotic or model substrate.

That is, the glutathione S-transferase (GST) enzyme comprises any glutathione S- transferase (GST) enzyme capable of catalyzing the conjugation of GSH, homoglutathione and other glutathione-like analogs via a sulfhydryl group to hydrophobic and electrophilic compounds and the dehydrochlorination of organochlorine compounds.

The method is performed in a low capacity buffer system and the buffer comprises a concentration of between 0.01 and 100 mM, and more preferably between 0.1 and 50 mM. The ionic strength of the buffer comprises between 1 mM and 2 mM.

The glutathione S-transferase (GST) enzyme is soluble in the buffer system and comprises two subunits. In addition, the glutathione S-transferase (GST) enzyme is present in a concentration of 0.1 to 10 units per ml, most preferably 1 to 5 units per ml and most preferably 3 units per ml. The purity of the glutathione S-transferase (GST) enzyme is between 50 and 100 % and the glutathione S-transferase (GST) enzyme comprises 20 to 50 daltons.

In the method of the present invention the generation of ions is monitored colorimetrically in the presence of a pH marker, or by means of chromogenic detectors for Cl) or by means of a chromatography strip. Alternatively, the generation of ions is monitored potentiometrically using a pH electrode or a Cl electrode or the generation of ions is monitored via a strip - test chromatography means.

Preferably in the method of the present invention the glutathione S-transferase (GST) enzyme is used to catalyse the reaction of one or more of xenobiotics or model substrates selected from the group comprising: a organochlorines compounds selected from the group of comprising DDT [(1 , 1 , 1-trichloro-2, 2-bis (p-chlorophenyl) ethane], endosulphan, dieldrin, aldrin).

The preferred buffer comprises one or more buffer systems selected from: phosphate; tris (1 to 5mM); HEPES (1 to 5 mM) in the presence of absence of sodium chloride (NaCI).

The glutathione S-transferase (GST) enzyme may comprise amino acid sequences longer or shorter than the length of natural GSTs, such as functional hybrid or partial fragments of GSTs, or their analogues.

In addition, the GST enzyme comprises nucleic acid fragments wherein changes in one or more nucleotide bases result in substitution of one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence. The present invention utilises specific glutathione-S transferase enzymes with DDTAse activity, to quantify the parental DDT (insecticidal p.p'DDT [1 ,1 ,1-trichloro- 2,2-bis(p-chlorophenyl) ethane]). In contrast to the aforementioned enzyme systems of the prior art that rely on the inhibition affinity for xenobiotics and which are often subject to interference by non target molecules, the method of the present invention is highly specific as the recombinant aegste2 recognizes the parent insecticide molecule (DDT) as substrate. Quantification of DDT is achieved potentiometrically, by using a pH or Cl electrode and monitoring the pH or Cl ion change resulting from the concomitant release of hydrogen ions (H⁺) or chloride ions (Cl^") during the dehydrochlorination reaction, (b) colorimetrically (in the presence of for example a pH marker, or chromogenic Cl detectors), or (c) by commercially available chlorine (Cl) strips.

Compounds that may be monitored and measured in accordance with the present invention, that is compounds for which one or more glutathione S-transferase (GST) enzymes may be used to catalyse the reaction may be one or more compounds selected from the group comprising: a compound selected from the group of organochlorines comprising DDT [(1 , 1 , 1-trichloro-2, 2-bis (p-chlorophenyl) ethane], endosulphan, dieldrin and aldrin.

The method is preferably performed in a low capacity buffer system. Suitable low capacity buffer systems include for example but are not limited to: sodium phosphate buffer, potassium phosphate buffer, TRIS buffer, and HEPES buffer, in the presence or absence of sodium chloride (NaCI). However, it will be appreciated by one skilled in the art that the method of the present invention encompasses a range of buffers, more than the specific exemplary buffer systems used in the examples and Figures presented by the present invention. The term low capacity buffer system is used herein to describe a buffering system that possesses little capacity to preserve its pH when the concentration of hydrogen ions (H⁺) in the reaction mixture is changing.

The buffer preferably has a concentration of between 0.1 mM and 50 mM. Most preferably the buffer concentration is not greater than 10 mM or less than 0.5 mM

The most preferred buffer systems suitable for use in the present invention are selected from the aforementioned, most preferably however phosphate buffers are employed with an ionic strength of between 1 mM and 2 mM ionic strength.

In the method of the present invention it is preferred that the glutathione S- transferase (GST) enzyme is in a soluble form and comprises two subunits.

The term "Glutathione S-Transferase(s)" or "GST(s)" with DDTase activity refers to any GST enzyme capable of catalyzing the dehydrochlorination of organochlorine insecticides such as DDT.

Glutathione S-transferases (GSTs) belong to a large family of multifunctional isoenzymes which are involved in the detoxification of a wide range of xenobiotics, including insecticides. GSTs catalyse the conjugation of electrophilic compounds with the thiol group of reduced glutathione, generally making the resultant products more water soluble then the non-GSH substrate.

The term "GST" also includes amino acid sequences longer or shorter than the length of natural GSTs, such as functional hybrid or partial fragments of GSTs, or their analogues. In addition, the term refers to nucleic acid fragments wherein changes in one or more nucleotide bases result in substitution of one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence. It will therefore be understood by one skilled in the art that the present invention encompasses more than the specific exemplary sequences (and insecticides) listed in the examples.

The glutathione S-transferase (GST) enzyme is preferably present in a concentration of between 0.1 to 10 units per ml, most preferably 1 to 5 units per ml and most preferably 3 units per ml.

,The purity of the glutathione S-transferase (GST) enzyme is preferably between 50 and 100%. The glutathione S-transferase (GST) enzyme preferably comprises 20 to 50 kdaltons.

In the method of the present invention it is possible to monitor the generation of ions colorimetrically. It is also possible to monitor the generation of ions potentiometrically.

Most preferably however the generation of hydrogen ions is monitored using a pH or Cl electrode, or colorimetrically in the presence of the pH marker bromothylmol blue, or chromogenic Cl agents, or using a Chloride Strip.

Accordingly the method of the present invention is most preferably used for monitoring glutathione S-transferase (GST) enzyme catalysed reactions of one or more compounds selected from the group comprising: a compound selected from the group of organochlorines comprising DDT [(1 , 1 , 1-trichloro-2, 2-bis (p-chlorophenyl) ethane], endosulphan, dieldrin and aldrin. The buffer system used in the reaction preferably comprises one or more buffer systems selected from: phosphate; tris (1 to 5mM); HEPES (1 to 5 mM) in the presence of absence of sodium chloride (NaCI 0.1 to 5OmM).

The method of the present invention may also be used to monitor the progress of a reaction wherein the compounds such as insecticides are used as substrates by the enzyme, selected from the group comprising: a compound selected from the group of organochlorines comprising DDT [(1 , 1 , 1-trichloro-2, 2-bis (p-chlorophenyl) ethane], endosulphan, dieldrin and aldrin.

The glutathione S-transferase (GST) enzyme may preferably comprise amino acid sequences longer or shorter than the length of natural GSTs, for example functional hybrids or partial fragments of GSTs, or their analogues.

Alternatively, the glutathione S-transferase (GST) enzyme comprises nucleic acid fragments wherein changes in one or more nucleotide bases result in substitution of one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence.

As indicated above, the method of the present invention finds particular application in measuring the levels of compounds such as DDT in field applications where it is necessary to take numerous readings under most difficult conditions.

Therefore according to a second aspect of the present invention there is provided a kit suitable for use in measuring glutathione S-transferase catalysed reactions of compounds such as xenobiotics comprising: a cartridge or swap filter suitable for allowing rapid removal of insecticide from a small section of for example net: and a vial or strip test with a sensor unit; and an indicator means, for example a 'traffic light' type indicator which will allow visual assessment of the amount if insecticide in the sample.

The indicator in the kit preferably comprises a 'traffic light' type indicator.

In Figure 4 there is illustrated a flow diagram illustrating the steps involved in the kit according to the second aspect of the present invention. A Chloride Strip (such as the Quantab) could replace the pH strip.

In use, the compound to be tested for example insecticide is diluted in a suitable carrier such as an organic solvent for example but not limited to: acetonitrile, ethanol and methanol.

The diluted mixture of compound is then placed in a suitable receptacle such as a plastic vial. The reaction mixture and the biosensor enzyme are then also added to the vial, and depending on the progress of the enzymatic reaction, a colour formation due to pH change, is recorded. The colour of the reaction is then matched for example to a standardised colour chart that relates to the concentration of test compound present. Alternatively, the Chloride Strip indication could match the concentration of test compound present.

In the kit according to the second aspect of the present invention the glutathione S- transferase (GST) enzyme is preferably immobilised onto an electrode or used in solution as a lyophilised enzyme form.

Suitable techniques for immobilising the enzyme onto the electrode include one or more techniques selected from: entrapment within electropolymerised polymers; covalent attachement and cross-linking with glutaraldehye.

Alternatively, it is possible in the kit for the enzyme to be present in the form of an immobilised pH marker on a test strip.

In yet a further alternative embodiment, the enzyme may be present in the kit in the form of an optical biosensor consisting of a chromoionophore sol-gel film (for example immobilised pH marker on a strip test) interfaced with another immobilized enzyme.

According to a third aspect of the present invention there is provided the use of a method according to the first aspect of the present invention or a kit according to a second aspect of the present invention for the assessment of compound quantities, for example but not limited to, the levels of compounds such as insecticides from indoor residual spraying surfaces (IRS).

Detailed Description

The present invention will now be further described by way of the following Figures and examples and with reference to the results wherein:

Figure 1a - illustrates the glutathione S-transferase (GST)-DDTase catalysed 1 ,1 ,1- dichloro-2,2-bis(p-chlorophenyl)ethylene (DDT) dehydrochorination reaction resulting in chloride ion (Cl^") and hydrogen ion (H⁺) release, which can be determined with potentiometric or colorimetric means.

Figure 1 b - illustrates the level of change in pH (as determined by optical density

(OD) in the presense of pH marker) and the associated DDT (1 ,1 ,1-dichloro-2,2- bis(p-chlorophenyl)ethylene) concentrations that can be accurately determined in the presence of the pH marker bromophenol blue using a spectrophotometer. The above experiments were performed using 0.4 units/ml adgste2, 25 to 250 μM DDT

(10% Ethanol), in a 1mM KH₂PO₄/ 1OmM NaCI, pH7 buffer.

Figure 2a - illustrates a chloride electrode determination of DDT concentrations using the GSTe2 Biosensor and is a standard curve of the difference in electrode potential in mV over 1 hour for DDT concentrations between 0 and 100 μg/ml DDT.

Each point is the average of seven independent determinations. Experimental conditions were: 0.1 M sodium phosphate buffer, pH 7.0, temperature 22 ⁰C.

Figure 2b - illustrates DDT determinations using the GSTe2 biosensor and chloride ion strips (Quantab) in place of a chloride electrode.

Figures 3a and 3b respectively - illustrate a comparison of DDT quantification methods when determining the DDT present in swabs from artificially (Potter Tower) sprayed material (cement, wood, rough tile, smooth tile, painted wood, painted tile and shiny tile). Figure 3a illustrates the HPLC vesus GST- DDTase pH assay, and

Figure 3b the HPLC versus GST- DDTase pH Cl electrode assay.

Figure 4 - illustrates a schematic blueprint for a field applicable kit format for the assay of the present invention.

An example of the kit of the present invention comprises a simple cartridge or swap filter to allow rapid removal of insecticide from for example, a small section of a net and a vial or strip test with a sensor unit and a 'traffic light' type indicator which will allow visual assessment of the amount of a compound to be detected, for example, insecticide in the sample.

EXPERIMENTAL

Materials and Methods.

Reduced glutathione and 1-chloro-2,4-dinitrobenzene were obtained from Sigma-

Aldrich Co (USA). Insecticides with a minimum certified purity of 97.7% were obtained from Merck and Greyhound (UK). Additional reagents and analytical grade chemicals were obtained from Sigma-Aldrich Co (USA).

Cloning, expression and purification of glutathione S-transferase (GST). The cloning of Anopheles gambiae GST epsilon 2 (accession number AAG45164) (aggstE2) into a pET vector and the expression in Esherichia coli BL21 (DE3)plysS was conducted as described by Ranson et al 2001. The recombinant enzyme was purified from bacterial cells as previously described in Ranson et al 2001 , the relevant sections of each are incorporated herein by reference. The eluted enzyme was concentrated using a centriprep-10 centrifugal filter (Amicon, Stonehouse, Gloucestershire, U.K.) following the manufacturer's instructions and made into a 1 : 1 glycerol stock and stored at -80 °C.

Assay of enzyme activity and protein.

Standard glutathione S-transferase (GST) assays were performed by monitoring the formation of a conjugate of 1-chloro-2,4-dinitrobenzene (CDNB) (1 mM) and reduced glutathione (GSH) (1mM) at 340nm at 30 ⁰C. A spectrophotometric assay was performed according to a published method by Habig et al 1974 incorporated herein by reference. The observed reaction velocities were corrected for spontaneous reaction rates when necessary. All of the initial velocities were determined in triplicate in buffers equilibrated at constant temperature. One unit of enzyme is defined as the amount of enzyme that gives 1.0 μmole of product per minute at pH 6.5 at 30 ⁰C. The protein concentration was determined by the method of Bradford (1976) using bovine serum albumin (fraction V) as standard.

1 , 1 , 1 , - dichloro - 2 , 2 - bis ( p - chlorophenyl) ethylene (DDT) dehydrochlorinase activity was assayed as described in Prapanthadara et al. (1998) (the relevant sections of which are incorporated herein by reference). HPLC analysis for the quantification of DDT dehydrochlorination / DDE (4,4'-(2,2-dichloroethene-1 ,1- diyl)bis(chlorobenzene) formation was carried out on reverse phase Acclaim C 18, 12θA 18 250 x 4.6mm, 5μ, Dionex column. The resultant peaks were integrated with an Ultimate 3000 UV-Detector (232 and 245 nm) and analyzed with Dionex Chromeleon™ software. A mobile phase of acetonitrile : water 90:10, was used at a flow rate of 1 ml per minute. Peaks were detected at 232 and 254 nm. The quantities of DDT and DDE were calculated from standard curves established by HPLC analysis of known concentrations of authenticated standards.

Potentiometric/colorimetric assays of the glutathione S-transferase (GST) - catalysed reaction.

The assays of the GST-catalysed reaction were carried out at 20 to 30 ⁰C in a total volume of 1 ml (unless otherwise stated below) in the presence or absence of DDT. The buffer system was adjusted to 1 mM reduced glutathione (GSH), 1 mM potassium phosphate buffer (PPB), 10 mM sodium chloride (NaCI), pH 7.5 and 2.5 mM GSH reduced glutathione. A range of alternative low capacity buffer systems can also be employed in the assay, such as for example but not limited to TRIS (1 to 5mM) and HEPES (1 to 5mM), in the presence or absence of NaCI. The GSH background was completely removed by addition of CuSO₄ 5 mM prior to each determination. The reaction can be measured potentiometrically (single pore glass pH electrode (Hamilton, USA), or Cl^" electrode (for example, halide electrode ISM- 146Cl, Lazar Research Laboratories, Los Angeles, CA, USA) or colorimetrically in the presence of a pH marker, or using a Chloride Strip (Quantab), or using chromogenic substrates for measuring the chloride ion concentration Cl^" (Yokoi 2002). Determination of insecticide residues on treated surfaces

Insecticide sprayed surfaces (cement, wood, rough tile, smooth tile, painted wood, painted tile, shiny tile) in the range of World Health Organisation (WHO) recommended concentrations (that is, 100 to 200 μg/cm² for DDT, and 2 to 3 μg/cm² for lambda-cyhalothrin were extracted from tiles (total sprayed area 54 cm²) by either swabbing or using adhesive tape. For the swabbing method, glass filter surface swabs of 5 cm diameter (SKC, Eighty Four, PA, USA) for bioaerosols and xenobiotic contaminations were used. The tile surfaces were swabbed by a dry fiberglass filter that was subsequently transferred to a glass tube, and insecticide residue was extracted by adding 2 ml of acetone (3 times). Extracts from each method were combined, evaporated to dryness under N₂, and re-dissolved in 2 ml of methanol. Aliquots of these samples were used either for HPLC analysis or the biosensor enzyme assay.

RESULTS AND DISCUSSION

1. Quantification of insecticides which are recognised by GSTs as substrates , such as DDT by GST / DDTase).

The determination of insecticides that are recognised by GSTs as substrates is possible in a direct way, for example when hydrogen ions (H⁺) or chloride ions Cl^" are released by a metabolic reaction (that is dehydrochlorination of DDT, Figure 1a). The reaction can be measured potentiometrically (pH or Cl^' electrode for Cl^" released reactions) or colorimetrically in the presence of a pH marker, or using a Chloride Strip, or using chromogenic substrates for measuring the chloride ion concentration Cl^" (Yokoi 2002) and the initial amount of DDT determined. The assay is highly specific, as only the parental DDT (insecticidal p.p'DDT [1 ,1 ,1-trichloro-2,2-bis(p- chlorophenyl) ethane]) is being metabolised by the GSTe2. Figure 1 b shows the calibration curve obtained for DDT, using 0.4 units/ml aggstE2, 0 to 100 μg/ml DDT (10% Ethanol), in a 1 mM KH₂PO₄ / 1OmM NaCI buffer, at 25 ⁰C. A number of alternative low capacity buffer systems, such as TRIS (1 to 5mM) and HEPES (1 to 5mM) may be also employed in the assay, as mentioned above such that the present invention is understood to encompass more than the specific buffer system used in the specific examples. The pH change was determined spectrophotometrically at 433nm (formation of yellow colour) and was linear for this range of concentrations. Figure 2a shows the determination of DDT concentrations using the GSTe2 Biosensor and a chloride electrode (standard curve of difference in electrode potential in mV over 1 hour for DDT concentrations between 0 and 100 ug/ml DDT. Each point is the average of seven independent determinations). Figure 2b shows respective DDT determinations using the GSTe2 and chloride ion strips (Quantab). The AgCI peak on Quantab strips for different concentration of DDT is measured. The quantab reading ranged from 0.6 to 1.2, for DDT concentrations 0 to 300 mictog per ml.

Figures 3a and 3b illustrate the correlation between biosensor DDT quantification methods of Potter Tower sprayed material swabs (Figure 3a, HPLC versus GST- DDTase Cl^" electrode assay, Figure 3b, HPLC versus GST- DDTase pH assay).

Recombinant biosensor Glutathione S-Transferase.

In the context of this disclosure, the term "Glutathione S-Transferase(s)" or "GST(s)" refers to any glutathione S-transferase (GST) enzyme capable of catalyzing the conjugation of GSH, homoglutathione and other glutathione-like analogs via a sulfhydryl group to hydrophobic and electrophilic compounds. GST-DDTase refers to any glutathione S-transferase (GST) enzyme capable of catalyzing the dehydrochlorination of DDT. The term "GST" includes amino acid sequences longer or shorter than the length of natural GSTs, such as functional hybrid or partial fragments of GSTs, or their analogues. In addition, the term refers to nucleic acid fragments wherein changes in one or more nucleotide bases result in substitution of one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence. It is therefore understood that the invention encompasses more than the specific exemplary sequences (and insecticides).

Possible adaptation of the assays into a kit format.

The potentiometric or colorimetric assays described above offers clear and distinct advantages over standard analytical methods (for example, HPLC) for the direct monitoring of insecticides in the field, such as low cost, real-time detection with minimum sample preparation and handling. In addition, the potentiometric assay is rapid because potential measurements could be made as soon as a measurable change in potential was achieved at the surface of the indicating electrode. The use of several potentiometric biosensor formats based on the immobilized enzyme is possible. The enzyme could be immobilized onto electrodes using different procedures, such as entrapment within electropolymerized polymers, covalent attachment and cross-linking with glutaraldehyde (Vidal et al 2006). An optical biosensor consisting of a chromoionophore sol-gel film (for example, immobilised pH marker on a strip test) interfaced with another immobilized enzyme can be also employed.

The present invention finds particular application for use in the assessment of insecticide quantities, as a quality control tool or a procurement tool, in a wide range of applications, such as but not limited to indoor residual spraying (IRS). Therefore in summary, the present invention provides a novel method of determining the quantity of insecticide by monitoring, either the inhibition of GST catalysed reactions, or the progress of the reaction when insecticides are used as substrates by the enzyme. Detection is performed with simple colorimetric, potentiometric or strip chromatography means, by measuring pH change or Cl ion concentration in the reaction mixture

References:

1. Andreescu S, Marty JL (2006) Twenty years research in cholinesterase biosensors: From basic research to practical applications Biomolecular Engineering 23, 1-15.

2. Bradford, M. (1976) A rapid and sensitive method for the detection of microgram quantities of proteins. Anal. Biochem. 72, 248-254.

3. Enayati AA, Vontas JG, Small GJ, et al. (2001) Quantification of pyrethroid insecticides from treated bednets using a mosquito recombinant glutathione S- transferase Med. Vet. Entomol. 15, 58-63.

4. Francis CM. Wong, Musa Ahmad, Lee Yook Heng, Lim Boon Peng (2006) An optical biosensor for dichlovos using stacked sol-gel films containing acetylcholinesterase and a lipophilic chromoionophore Talanta 69 888-893

5. Habig, W.H., Pabst, M. J., Fleischner, G., Gatmaitan, Z., Arias, I M. and Jakoby, W. B. (1974) The identity of glutathione S-transferase B with ligandin, a major binding protein of liver. Proc. Natl. Acad. Sci. U S A 71 , 3879-3882

6. Labrou, N. E., MeIIo, LV. and Clonis, Y.D. (2001) The conserved Asn49 of maize glutathione S-transferase I modulates substrate binding, catalysis and intersubunit communication. Eur. J. Biochem. 268, 3950-3957.

7. Mulchandani A, Chen W, Mulchandani P, Wang J, Rogers KR. (2001) Biosensors for direct determination of organophosphate pesticides. Biosens Bioelectron. 16, 225-30.

8. Prapanthadara LA, Ranson H, Somboon P, et al. (1998) Cloning, expression and characterization of an insect class I glutathione S-transferase from Anopheles dirus species Insect Biochem MoI Biol 28, 321-329.

9. Ranson H, Rossiter L, Ortelli F., Jensen B, Wang X., Roth C. W., Collins F. H. and Hemingway J (2001 ) Identification of a novel class of insect glutathione S- transferases involved in resistance to DDT in the malaria vector Anopheles gambiae. Biochem. J. 359, 295±304 (Printed in Great Britain) 295 10. Vidal JC, Esteban S, Gil J, Castillo JR (2006) A comparative study of immobilization methods of a tyrosinase enzyme on electrodes and their application to the detection of dichlorvos organophosphorus insecticide (2006) Talanta 68791-799

11. Vontas JG, Enayati AA, Small GJ, et al. (2000) A simple biochemical assay for glutathione S-transferase activity and its possible field application for screening glutathione S-transferase-based insecticide resistance, Pest Biochem Physiol 68, 184-192.

12. Wong F. C. M., Ahmad M., Heng L.Y, Peng L. B. (2006) An optical biosensor for dichlovos using stacked sol-gel films containing acetylcholinesterase and a lipophilic chromoionophore, Talanta 69, 888-893.

13. Yokoi K (2002) Colorimetric determination of chloride in biological samples by using mercuric nitrate and diphenylcarbazone, Biological Trace Emement Research 85, 87-94.

Claims

CLAIMS.

1. A method of assaying recombinant glutathione-S transferase (GST) catalysed reactions of xenobiotics or model substrates which comprises monitoring and measuring the amount of ions generated when a GST enzyme is used to catalyse the reaction wherein the level of detected ions is used to verify the amount of xenobiotic or model substrate in the reaction system.

2. A method according to claim 1 wherein the method comprises: measuring the amount of chloride ions (Cl^") generated when the GST enzyme catalyses the dehydrochlorination of the xenobiotic or model substrate.

3. A method according to claim 2 wherein the glutathione-S transferase (GST) enzyme has DDTAse activity and is able to catalyse a dehydrochlorination reaction of the xenobiotic or model substrate.

4. A method according to any of claims 1 to 3 wherein the reaction is performed in a low capacity buffer system.

5. A method according to claim 4 wherein the buffer comprises a concentration of between 0.1 and 50 mM.

6. A method according to any of claims 1 to 5 wherein the ionic strength of the buffer comprises between 1 mM and 2 mM.

7. A method according to any of the preceding claims wherein the glutathione S- transferase (GST) enzyme comprises any glutathione S-transferase (GST) enzyme capable of catalyzing the conjugation of GSH, homoglutathione and other glutathione-like analogs via a sulfhydryl group to hydrophobic and electrophilic compounds and the dehydrochlorination of organochlorine compounds.

8. A method according to claim 7 wherein the glutathione S-transferase (GST) enzyme is soluble in the buffer system and comprises two subunits.

9. A method according to any of claims 1 to 8 wherein the glutathione S- transferase (GST) enzyme is present in a concentration of 0.1 to 10 units per ml, most preferably 1 to 5 units per ml and most preferably 3 units per ml.

10. A method according to any of the preceding claims wherein the purity of the glutathione S-transferase (GST) enzyme is between 50 and 100 %.

11. A method according to any of the preceding claims wherein the glutathione S- transferase (GST) enzyme comprises 20 to 50 daltons.

12. A method according to any of preceding claims 1 to 11 wherein the generation of ions is monitored colorimetrically in the presence of a pH marker, or by means of chromogenic detectors for Cl) or by means of a chromatography strip.

13. A method according to any of preceding claims 1 to 12 wherein the generation of ions is monitored potentiometrically using a pH electrode or a Cl electrode.

14. A method according to any of preceding claims 1 to 12 wherein the generation of ions is monitored via a strip - test chromatography means.

15. A method according to any of the preceding claims wherein the glutathione S- transferase (GST) enzyme is used to catalyse the reaction of one or more of xenobiotics or model substrates selected from the group comprising: a organochlorines compounds selected from the group of comprising DDT [(1 , 1 , 1-trichloro-2, 2-bis (p-chlorophenyl) ethane], endosulphan, dieldrin, aldrin);

16. A method according to any of the preceding claims wherein the buffer comprises one or more buffer systems selected from: phosphate; tris (1 to 5mM); HEPES (1 to 5 mM) in the presence of absence of sodium chloride (NaCI).

17. A method according to any of the preceding claims wherein the glutathione S- transferase (GST) enzyme comprises amino acid sequences longer or shorter than the length of natural GSTs, such as functional hybrid or partial fragments of GSTs, or their analogues.

18. A method according to any of claims 1 to 17 wherein the GST enzyme comprises nucleic acid fragments wherein changes in one or more nucleotide bases result in substitution of one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence.

19. A kit suitable for measuring glutathione S-transferase catalysed reactions of compounds such as xenobiotics as claimed in any of claims 1 to 18 comprising: a cartridge or swap filter suitable for allowing rapid removal of the test compound from a test sight: and a vial or strip test with a sensor unit; and an indicator means for visual assessment of the amount of compound in the sample.

20. A kit according to claim 19 wherein the indicator means comprises a 'traffic light' type indicator.

21. A kit according to claim 19 or 20 wherein the enzyme is immobilised onto an electrode.

22. A kit according to claim 21 wherein the enzyme is immobilised onto the electrode by means of one or more techniques selected from: entrapment within electropolymerised polymers; covalent attachement and cross-linking with glutaraldehye.

23. A kit according to any of claims 19 to 22 wherein the enzyme is present in the form of a co- immobilised enzyme - pH marker on a test strip, or lyophilised enzyme in solution

24. A kit according to any of claims 19 to 22 wherein the enzyme is present in the form of an optical biosensor consisting of a chromoionophore sol-gel film, for example, an immobilised pH marker on a strip test, interfaced with another immobilized enzyme, or lyophilised enzyme in solution.

25. Use of a method according to any of claims 1 to 18 or a kit according to any of claims 19 to 24 for the assessment of compounds such as insecticides on indoor residual spraying surfaces (IRS).