BIODEGRADABLE BIOCHEMICAL SENSOR FOR DETERMINING THE PRESENCE AND/OR THE LEVEL OF PESTICIDES OR ENDOCRINE DISRUPTORS: METHOD AND COMPOSITION

Abstract

The present invention is directed to biodegradable biochemical sensor method to perform in a sample multiplex detection and/or quantification of pesticides and/or endocrine disruptors and to provide and logical integrated response to the user. This biochemical sensor is a vesicle encapsulating biochemical networks using enzymes capable of generating, inhibiting or activating specific measurable signal in presence of said target analytes. The biochemical network is able to provide an integrated logical final response to the user. The present invention also relates to a composition or kit comprising said biochemical sensor vesicle.

Claims

1. A method to detect the presence or the absence, and/or to quantify the amount of at least one target analyte in a sample, the method comprising the steps of: from a sample containing or susceptible to contain the target analyte; a) contacting the sample with a composition comprising biochemical elements forming a biochemical network, said biochemical network comprising as biochemical element at least one enzyme having as substrate or as inhibitor or as activator said target analyte which is desired to be detected and/or quantified, and wherein: at least one of said biochemical elements forming a biochemical network is encapsulated in one micro- or nano-vesicle (named vesicle) permeable or not to the target analyte; or at least two of said biochemical elements forming a biochemical network are encapsulated in two distinct vesicles permeable or not to the target analyte, wherein: i) said at least one target analyte which is desired to be detected or quantify in the sample is the glyphosate; ii) the biochemical network is capable of: generating at least one specific readable/measurable output signal only in presence of the target analyte when said target analyte is a substrate of the enzyme of said biochemical network; or inhibiting the specific readable/measurable output signal generated by said biochemical network only in presence of the target analyte when said target analyte is an inhibitor of the enzyme of said biochemical network, and, b) determining the rate and/or level of the specific readable/measurable output signal produced by the biochemical network, the rate and/or level obtained being correlated to the presence or the absence and/or the amount of the target analyte in the sample.

2. The method of claim 1, wherein the sample susceptible to contain the target analyte is selected from the group consisting of fluid or solid material sample, preferably environmental material sample, vegetal material, water, beverage, food products, soil extracts, industrial material, food production, plant extract, physiologic fluid or tissue from living organism.

3. The method of claim 1, wherein the presence and/or absence and/or the amount of the target analyte is detected and/or quantified by the measure of a signal selected from the group consisting of: visible colorimetric measurement, fluorescence, luminescence, spectroscopy (i.e. infra-red, Raman), chemical compound or particle (electron) production.

4. The method of claim 1, to detect the presence or the absence, and/or to quantify the amount of at least a second target analyte in a sample wherein said second analyte is either a substrate, inhibitor or activator of the same at least one biochemical network enzyme wherein at least one of said biochemical elements forming a biochemical network is encapsulated in said vesicle.

5. The method of claim 1, to detect the presence or the absence, and/or to quantify the amount of at least a second target analyte in a sample, wherein: said second analyte is a substrate inhibitor or activator of a second distinct biochemical network enzymes, and one of said biochemical elements forming said second biochemical network is encapsulated in the same or in another distinct vesicle, or set of vesicles; and said two distinct biochemical networks (interconnected or not) generate a different readable/measurable output signal.

6. The method of claim 1, wherein the second target analyte which is desired to detect and/or to quantify is a pesticide or an endocrine disruptor selected from the group consisting of: a) pesticide and/or an endocrine disruptor molecule which is a specific substrate of an enzyme activity, activity which can produce in one step, or more, a specific readable/measurable output signal; b) pesticide and/or an endocrine disruptor molecule which is a specific inhibitor of an enzyme activity, activity which can produce in one step, or more, a specific readable/measurable output signal; and c) pesticide or an endocrine disruptor molecule which is a specific activator of an enzyme activity, activity which can produce in one step, or more, a specific readable/measurable output signal.

7. The method of claim 6, wherein the second target analyte which is desired to detect and/or to quantify is a pesticide or an endocrine disruptor selected from the group consisting of: Chlordecone, Neonicotinoid, Organochlorides, Succinate dehydrogenase inhibitor (SDHI), carbamates, dioxine (PCDD), polychlorobiphenyle (PCB), 17-beta oestradiol, 17-alpha ethylene oestradiol, bisphenol (PBDE), phthalates and heavy metal.

8. The method of claim 1, wherein the at least one biochemical network enzyme which is comprised in the composition in step a), encapsulated in said vesicle or not encapsulated, is selected from the group consisting of: glycine/glyphosate oxidase (EC 1.4.3.19), preferably the native (wild type/WT) glycine/glyphosate oxidase from Bacillus subtilis which can be obtained as recombinant protein, or homolog sequence thereof having at least 70% identity with the WT protein sequence and exhibiting glycine/glyphosate oxidase activity; and 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (EC 2.5.1.19).

9. The method of claim 1, wherein the at least one biochemical network enzyme which is comprised in the composition in step a), encapsulated in said vesicle or not encapsulated, is the glycine oxidase (GO) from the marine bacteria Bacillus licheniformis ((BliGO) which has been cloned and which shows at least 62% similarity to the standard GO from Bacillus subtilis, or homolog BliGO sequence thereof having at least 70% identity with the BliGO WT protein sequence SEQ ID NO:2 and exhibiting GO activity.

10. The method of claim 1, wherein the at least one biochemical network enzyme which is comprised in the composition in step a), encapsulated in said vesicle or not encapsulated, is the mutated glycine oxidase (GO) from the marine bacteria Bacillus licheniformis genetically modified and containing 6 single amino-acids mutation compared to the wild type version BliGO-WT, named BliGO-SCF-4 or the BliGO-Mut having the amino acids sequence SEQ ID NO:4.

11. The method of claim 1, wherein said glycine/glyphosate oxidase comprising a tag which is fused to the glycine/glyphosate oxidase enzyme, preferably a tag selected from the group consisting of maltose-binding protein (MBP), Chitin Binding Protein (CBP), glutathione S-transferase (GST), thioredoxin (TRX), NUS A, ubiquitin (Ub), and SUMO (small ubiquitin-related modifier) tags, preferably SUMO and GST tags.

12. The method of claim 11, wherein said is a SUMO or a GST tag.

13. The method of claim 8, wherein said glycine/glyphosate oxidase comprising a tag is selected from the group of: the GST-BliGO (native/WT) having the DNA sequence SEQ ID NO:5 or the amino acids sequence SEQ ID NOG; the SUMO-BliGO (native/WT) having the DNA sequence SEQ ID NO:9 or the amino acids sequence SEQ ID NO:10; the GST-BliGO-Mut having the DNA sequence SEQ ID NO:7 or the amino acids sequence SEQ ID NO:8 and the SUMO-BliGO-Mut having the DNA sequence SEQ ID NO:11 or the amino acids sequence SEQ ID NO:12 and homolog tagged BliGO sequences thereof as defined above wherein the BliGO sequence exhibits at least 70%.

14. The method of claim 8, wherein the target analyte which is desired to detect and/or to quantify is the glyphosate or derivatives thereof which can be detected or quantified with the same biochemical network enzyme as for glyphosate, and wherein the at least one biochemical network enzyme encapsulated or not in a vesicle is 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (EPSPS) enzyme (EC 2.5.1.19) the composition further comprising 3-phospho-shikimate and phosphoenolpyruvate (PEP).

15. The method of claim 1, wherein the vesicle is selected from the group consisting of unilamellar or multilamellar vesicles, preferred are lipid vesicles, liposomes or self-assembled phospholipids, or vesicles formed from synthetic polymers or copolymers, said vesicles having preferably an average diameter between 0.05 μm to 500 μm, more preferably between 0.1 μm to 100 μm.

16. The method of 1, wherein the vesicles are trapped in a porous polymeric gel, preferably selected from the group consisting of porous polymeric gel, preferably selected from the group consisting of alginate, chitosan, PVP (polyvinylpyrrolidone), PVA (polyvinyl-alcohol), agarose, sephadex, sepharose, sephacryl gel and mixture thereof.

17. A composition to detect the presence or the absence, and/or to quantify the amount of at least one target analyte in a sample said composition comprising biochemical elements forming a biochemical network encapsulated or not in one or in a set of vesicles permeable or not to the target analyte, said biochemical network comprising as biochemical element at least one enzyme selected from the group of: glycine/glyphosate oxidase (EC 1.4.3.19), preferably the native (wild type/WT) glycine/glyphosate oxidase which can be obtained as recombinant protein, or homolog sequence thereof having at least 70% identity with the WT protein sequence and exhibiting glycine/glyphosate oxidase activity; 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (EC 2.5.1.19); the glycine oxidase (GO) from the marine bacteria Bacillus licheniformis ((BliGO) which has been cloned and which shows at least 62% similarity to the standard GO from Bacillus subtilis, or homolog BliGO sequence thereof having at least 70% identity with the BliGO WT protein sequence and exhibiting GO activity; the mutated glycine oxidase (GO) from the marine bacteria Bacillus licheniformis ((BliGO)_SCF4 genetically modified and containing 6 single amino-acids mutation compared to the wild type version BliGO-WT or the BliGO-Mut; a tagged glyphosate oxidase enzyme, preferably with a tag selected from the group consisting of maltose-binding protein (MBP), Chitin Binding Protein (CBP), glutathione S-transferase (GST), thioredoxin (TRX), NUS A, ubiquitin (Ub), and SUMO (small ubiquitin-related modifier) tags, preferably SUMO and GST tags; and the GST-BliGO (native/WT) having the DNA sequence SEQ ID NO:5 or the amino acids sequence SEQ ID NO:6; the SUMO-BliGO (native/WT) having the DNA sequence SEQ ID NO:9 or the amino acids sequence SEQ ID NO:10; the GST-BliGO-Mut having the DNA sequence SEQ ID NO:7 or the amino acids sequence SEQ ID NO:8 and the SUMO-BliGO-Mut having the DNA sequence SEQ ID NO:11 or the amino acids sequence SEQ ID NO:12 and homolog tagged BliGO sequences thereof as defined above wherein the BliGO sequence exhibits at least 70%.

18. A composition wherein the target analyte, the biochemical elements, the biochemical network and the vesicles have the characteristic as defined in claim 1.

19. A kit or a device to detect the presence or the absence, and/or to quantify the amount of at least one target analyte in a sample said kit comprising a container containing the composition of claim 17, trapped in a porous polymeric gel, preferably selected from the group consisting of alginate, chitosan, PVP, PVA, agarose, sephadex, sepharose, sephacryl gel and mixture thereof.

Description

FIGURE LEGENDS

[0141] FIG. 1: Schematic representation of the glycine/glyphosate biochemical network #1. The network comprises the GST-BliGO Mut #1 enzyme, the HRP enzyme and the Amplex Red or dianisidine for the colorimetric or fluorescent readout.

[0142] FIGS. 2A-2B: Schematic representation of the EPSP synthase biochemical network #2A and #2B for glyphosate detection.

[0143] The network #2A (FIG. 2A) comprises the EPSP synthase enzyme, the Chorismate synthase enzyme, the Chorimsate lyase enzyme, the lactate dehydrogenase enzyme and the NADH for the absorbance or fluorescence detection.

[0144] The network #2B comprises the EPSP synthase enzyme, the Purine-nucleoside phosphorylase enzyme, the Xanthine oxidase enzyme, the HRP enzyme and the Amplex Red or O-dianisidine for the colorimetric or fluorescent readout.

[0145] FIG. 3: Microfluidic process for vesicle formation (From Courbet et al. Mol. Sys. Biol. 2018, 14(4):e7845. FIG. 4A))

[0146] FIGS. 4A-4B:

[0147] FIG. 4A: Absorbance glyphosate detection by the Glycine/Glyphosate oxidase network #1. Detection of Glyphosate ranging from 0 to 10 mM.

[0148] FIG. 4B: Glycine/Glyphosate oxidase enzyme catalytic activity analysis.

[0149] FIGS. 5A-5B:

[0150] FIG. 5A: Fluorescence Phosphoenol pyruvate (PEP) detection by the Glycine/Glyphosate oxidase network #2B. Detection of PEP ranging from 0 to 100 μM. FIG. 5B: EPSP synthase enzyme catalytic activity analysis.

[0151] FIG. 6: Schematic representation of the Glycine biochemical network #3 for glycine detection. The network comprises the Bacillus Subtilis Glycine oxidase H244K enzyme, the HRP enzyme and the Amplex Red or O-dianisidine for the colorimetric or fluorescent readout.

[0152] FIG. 7: Schematic representation of the Glyphosate OR Glycine biochemical network #4 for glyphosate and glycine detection. The network comprises the GST-BliGO Mut #1 enzyme, the Bacillus Subtilis Glycine oxidase H244K enzyme, the HRP enzyme and the Amplex Red or O-dianisidine for the colorimetric or fluorescent readout.

[0153] FIG. 8A-8B: Fluorescence (upper part) and colorimetric (lower part) glyphosate detection by the Glycine/Glyphosate oxidase network #1. (A-Left) Detection of Glyphosate ranging from 0 to 2 mM in Tris buffer 50 mM pH 7.5. (B-Right) Detection of Glyphosate ranging from 0 to 2 mM in barley seeds extracted in Tris buffer 50 mM pH 7,5.

[0154] FIG. 9A-9B: Fluorescence (upper part) and colorimetric (lower part) glyphosate detection by the Glycine/Glyphosate oxidase network #1 integrated in vesicles. (A-Left) Detection of Glyphosate ranging from 0 to 2 mM in Tris buffer 50 mM pH 7.5. (B-Right) Detection of Glyphosate ranging from 0 to 2 mM in barley seeds extracted in Tris buffer 50 mM pH 7,5.

[0155] FIG. 10: Colorimetric glyphosate detection by the Glycine/Glyphosate oxidase network #1 integrated in alginate beads. (Upper part) Detection of Glyphosate ranging from 0 to 4 mM in Tris buffer 50 mM pH 7,5. (Lower part) Detection of Glyphosate ranging from 0 to 4 mM in barley seeds extracted in Tris buffer 50 mM pH 7,5.

[0156] FIG. 11: Fluorescence (upper part) and colorimetric (lower part) glycine detection by the Glycine/Glyphosate oxidase network #3. (Left) Detection of glycine ranging from 0 to 1 mM in Tris buffer 50 mM pH 7,5. Note the absence of detection of the glyphosate at 100 μM.

[0157] FIG. 12: Fluorescence glyphosate and glycine detection by the Glycine/Glyphosate oxidase network #4. (Upper part) Kinetic of glyphosate AND/OR glycine degradation by the network. Glyphosate and glycine were present at 1 mM concentration. (Lower part) Glycine/Glyphosate oxidase network #4 logic-gate (OR) response to glycine AND/OR glyphosate presence.

[0158] FIG. 13: Fluorescence glyphosate detection by the EPSP synthase network #2B. Kinetic of glyphosate degradation by the network. Detection of glyphosate ranging from 0 to 1 mM.

[0159] FIG. 14: BliGO_WT (native) Protein: DNA (SEQ ID NO:1) and amino acids (SEQ ID NO:2) sequence

[0160] FIG. 15: BliGO_Mut Protein: DNA (SEQ ID NO:3) and amino acids (SEQ ID NO:4) sequence

[0161] FIG. 16: GST-BliGO_WT (native) Protein: DNA (SEQ ID NO:5) and amino acids (SEQ ID NO:6) sequence

[0162] FIG. 17: GST-BliGO_Mut Protein: DNA (SEQ ID NO:7) and amino acids (SEQ ID NO:8) sequence

[0163] FIG. 18: SUMO-BliGO_WT Protein: DNA (SEQ ID NO:9) and amino acids (SEQ ID NO:10) sequence

[0164] FIG. 19: SUMO-BliGO_Mut Protein: DNA (SEQ ID NO:11) and amino acids (SEQ ID NO:12) sequence.

Example 1: Study Design—Setup of the Biochemical Networks

[0165] Different Biochemical Networks have been designed to detect the presence of different pesticides and/or endocrine disruptors. One originality of our invention resides in the fact that different biochemical networks can be plugged together to allow the detection of different analytes and lead to the delivery of a single output signal if necessary.

[0166] For the specific detection of the glyphosate pesticide we designed two detection systems that can be combined together to improve the specificity of the output signal: [0167] 1. The first network uses the ability of the enzyme Glycine/Glyphosate oxidase to metabolize the Glyphosate (FIG. 1).

[0168] In a first example, this first network comprises:

[0169] a) the enzyme Glycine/Glyphosate oxidase, the enzyme Horseradish Peroxidase and the O-Dianisidine dihydrochloride. In the presence of Glyphosate, 2-amino phosphonate and H.sub.2O.sub.2 are produced by the Glycine/Glyphosate oxidase. Afterward, the H.sub.2O.sub.2 is co-processed with the O-dianisidine by the Horseradish peroxidase to give a colorimetric readout to the reaction with a change in absorbance at 450 nm visible wavelength.

[0170] First, the network has been tested in liquid buffer without vesicle or gel. The 100 μl reaction system comprised 30 mM disodium pyrophosphate (pH 8.5), 0.46 μM (0.0024 units) Glycine/Glyphosate oxidase H244K from Bacillus Subtilis (Biovision #7845), 0.5 mM 0-Dianisidine dihydrochloride, 0.25 units Horseradish peroxidase and Glyphosate at concentrations ranging from 0 to 600 mM. Reaction was followed for 1 hour at 25° C. by registering the absorbance at 450 nm on a spectrophotometer.

[0171] In a second example, this first network can comprise:

[0172] b) the enzyme GST-Bacillus licheniformis Mut #1 or SCF-4 Glycine/Glyphosate oxidase, the enzyme Horseradish Peroxidase and the Amplex red. In the presence of Glyphosate, 2-amino phosphonate and H.sub.2O.sub.2 are produced by the Glycine/Glyphosate oxidase. Afterward, the H.sub.2O.sub.2 is co-processed with the Amplex red by the Horseradish peroxidase to give a colorimetric (red) and fluorescent readout to the reaction.

[0173] First, the network has been tested in liquid buffer without vesicle or gel (FIGS. 8A-8B). The 100 μl reaction system comprised 50 mM Tris (pH 7.5), 0.46 μM (0.0024 units) Glycine/Glyphosate oxidase GST-BliGO Mut #1 from Bacillus Licheniformis genetically modified and derived from the BliGO-SCF-4 containing 6 single amino-acids mutation compared to the wild type version, 0.2 mM Amplex red, 0.25 units Horseradish peroxidase and Glyphosate at concentrations ranging from 0 to 2 mM. The network has also been tested in the presence of barley extracts (FIG. 8B). Reaction was followed for 1 hour at 25° C. by registering the fluorescence (Excitation at 530 nm/Emission at 590 nm) on a spectrophotometer.

[0174] The network has also been tested in vesicle (FIGS. 9A-9B). The vesicles comprised 50 mM Tris (pH 7.5), 0.2 mM Amplex red and 0.25 units Horseradish peroxidase. 0.46 μM (0.0024 units) Glycine/Glyphosate oxidase GST-BliGO Mut #1 from Bacillus licheniformis and Glyphosate at concentrations ranging from 0 to 2 mM were added in the reaction outside of the vesicles. Reaction was followed for 1 hour at 25° C. by registering the fluorescence (Excitation at 530 nm/Emission at 590 nm) on a spectrophotometer.

[0175] The network has also been tested in alginate beads (FIG. 10). The alginate beads comprised 50 mM Tris (pH 7.5), 0.2 mM Amplex red, 0.25 units Horseradish peroxidase. 0.46 μM (0.0024 units), Glycine/Glyphosate oxidase GST-BliGO from Bacillus Licheniformis on. The beads were dipped in 50 mM Tris buffer pH7,5 or barley extracts containing Glyphosate at concentrations ranging from 0 to 4 mM. Reaction (colorimetry of the beads) was followed for 1 hour at 25° C. [0176] 2. The second network combines the activity of 4 enzymes with the first enzyme being the 5-enolpyruvyl Shikimate 3-phosphate-Synthase (EPSP Synthase) (FIGS. 2A-2B).

[0177] This network exploits the ability of the Glyphosate to inhibit the activity of the EPSP Synthase. With this network, the level of inhibition depends on the concentration of glyphosate. The entry of the network is composed of: the EPSP synthase that uses the phospho-enol pyruvate (PEP) and the 3-phospho Shikimate to produce 5-O-(1-caroxyvinyl)-3-phosphoshikimate and inorganic phosphate.

[0178] Then 2 different networks have been tested: [0179] One that converts the inorganic phosphate (Pi): Pi is combined with Inosine in the presence of the purine nucleoside phosphorylase to give Hypoxanthine. The Hypoxantine lead to Xanthine and H.sub.2O.sub.2 in the presence of Xanthine Oxidase. The Horseradish peroxidase uses the H.sub.2O.sub.2 to convert the O-dianisidine or the Amplex Red and give a colorimetric or fluorimetric signal detectable.

[0180] First, the network has been tested in liquid buffer without vesicle or gel. The 100 μl reaction system comprised 50 mM Hepes (pH 7), 50 mM KCl, 0.5 mM Shikimate-3-phosphate, 0.1 unit Xanthine Oxidase, 0.12 μg E. coli EPSP Synthase, 0.2 unit Purine Nucleoside Phosphorylase, 2.25 mM Inosine, 0.5 mM 0-dianisidine dihydrochloride, 0.25 unit Horseradish Peroxidase, Phosphoenol Pyruvate between 0 and 600 μM and Glyphosate at concentrations ranging from 0 to 2 mM. Reaction was followed for 1 hour at 25° C. by registering the absorbance at 450 nm on a spectrophotometer. [0181] One that converts the 5-O-(1-caroxyvinyl)-3-phosphoshikimate produced by the EPSP synthase: 5-O-(1-caroxyvinyl)-3-phosphoshikimate is metabolized by the Chorismate Synthase to give chorismate. This Chorismate is then transformed in pyruvate by the chorismate Lyase. Finaly the pyruvate is used by the Lactate dehydrogenase in the presence of NADH to give lactate and NAD+. The NADH consumption is followed by the change of fluorescence emission at 445 nm (Excitation at 340 nm)—or change of absorbance at 340 nm on a spectrophotometer. [0182] 3. The third network (#3) uses the ability of the Bacillus subtilis H244K (Creative enzyme) enzyme Glycine/Glyphosate oxidase to metabolize the Glycine (FIG. 6).

[0183] The network comprises: the enzyme Bacillus subtilis H244K Glycine/Glyphosate oxidase, the enzyme Horseradish Peroxidase and the Amplex red. In the presence of Glycine but not Glyphosate, glyoxylate and H.sub.2O.sub.2 are produced by the Glycine/Glyphosate oxidase. Afterward, the H.sub.2O.sub.2 is co-processed with the Amplex red by the Horseradish peroxidase to give a colorimetric (red) and fluorescent readout to the reaction.

[0184] First, the network has been tested in liquid buffer without vesicle or gel (FIG. 11). The 100 μl reaction system comprised 50 mM Tris (pH 7.5), 0.46 μM (0.0024 units) H244K Glycine/Glyphosate oxidase from Bacillus subtilis genetically modified containing 1 single amino-acids mutation H244K (see Accession Number 031616 Biovision) compared to the wild type version (Creative enzyme NATE-1674), 0.2 mM Amplex red, 0.25 units Horseradish peroxidase and Glycine at concentrations ranging from 0 to 1 mM. Reaction was followed for 1 hour at 25° C. by registering the fluorescence (Excitation at 530 nm/Emission at 590 nm) on a spectrophotometer. [0185] 4. The fourth network (#4) combines the first and the third networks in order to detect glycine and glyphosate (FIG. 7).

[0186] The network comprises: the enzyme GST-Bacillus licheniformis Mut #1 Glycine/Glyphosate oxidase, the Bacillus subtilis H244K Glycine/Glyphosate oxidase, the enzyme Horseradish Peroxidase and the Amplex red. In the presence of Glycine OR Glyphosate, 2-amino phosphonate, glyoxylate and H.sub.2O.sub.2 are produced by the Glycine/Glyphosate oxidase network #4. Afterward, the H.sub.2O.sub.2 is co-processed with the Amplex red by the Horseradish peroxidase to give a colorimetric (red) and fluorescent readout to the reaction.

[0187] The network has been tested in vesicles (FIG. 12). The vesicles comprised 50 mM Tris (pH 7.5), 0.2 mM Amplex red and 0.25 units Horseradish peroxidase. 0.46 μM (0.0024 units) Glycine/Glyphosate oxidase GST-BliGO Mut #1 from Bacillus Licheniformis, 0.46 μM (0.0024 units) H244K Glycine/Glyphosate oxidase from Bacillus subtilis genetically modified containing 1 single amino-acids mutation H244K compared to the wild type version (Creative enzyme NATE-1674) and Glyphosate at concentrations ranging from 0 to 2 mM were added in the reaction outside of the vesicles. Reaction was followed for 1 hour at 25° C. by registering the fluorescence (Excitation at 530 nm/Emission at 590 nm) on a spectrophotometer.

Example 2: Setup of the Vesicles to Encapsulate the Biochemical Networks (Refer to Courbet et al. Mol. Sys. Biol. 2018)

[0188] We identified a universal and robust macromolecular architecture capable of supporting the modular implementation of in vitro biosensing/biocomputing processes. This architecture is capable of (i) stably encapsulating and protecting arbitrary biochemical circuits irrelevant of their biomolecular composition, (ii) discretizing space through the definition of an insulated interior containing the synthetic circuit, and an exterior consisting of the medium to operate in (e.g. a clinical sample), (iii) allowing signal transduction through selective mass transfer of molecular signals (i.e. biomarker inputs), and (iv) supporting accurate construction through thermodynamically favourable self-assembling mechanisms. The vesicles architecture we propose in this study is made of phospholipid bilayer membranes.

[0189] We relied on the development of a method that would simultaneously support (i) membrane unilamellarity, (ii) encapsulation efficiency and stoichiometry, (iii) monodispersity, and (iv) increased stability/resistance to osmotic stress. For this purpose, we developed a custom microfluidic platform and designed PDMS-based microfluidic chips in order to achieve directed self-assembly of a synthetic phospholipid (DPPC) into calibrated, custom-sized membrane bilayers encapsulating low copy number of biochemical species. Briefly, this strategy relied on flowfocusing droplet generation channel geometries that generate waterin-oil-in-water double-emulsion templates (W-O-W: biochemical circuit in PBS—DPPC in oleic acid—aqueous storage buffer with a low concentration of methanol). After double-emulsion templates formation, DPPC phospholipid membranes are precisely directed to self-assemble during a controlled solvent extraction process of the oil phase by methanol present in buffer (FIG. 3). This microfluidic design also integrates a device known as the staggered herringbone mixer (SHM) (Williams et al, 2008) to enable efficient passive and chaotic mixing of multiple upstream channels under Stokes flow regime. We reasoned that laminar concentration gradients could prevent critical mixing of biochemical parts, precise stoichiometry, and efficient encapsulation. We hypothesized that synthetic biochemical circuits immediately homogenized before assembly could standardize the encapsulation mechanism and reduce its dependency on the nature of insulated materials. Moreover, this design allowed for fine-tuning on stoichiometry via control on the input flow rates, which proved practical to test different parameters for straightforward prototyping of protosensors.

[0190] We used an ultrafast camera to achieve real-time monitoring and visually inspect the fabrication process, which allowed estimating around ˜1,500 Hz the mean frequency of vesicles generation at these flow rates. A strong dependence of vesicles generation yields on flow rates was found, which we kept at 1/0.4/0.4 μl/min (storage buffer/DPPC in oil/biochemical circuit in PBS, respectively) to achieve best assembly efficiency. We then analysed the size dispersion of vesicles using light transmission, confocal, and environmental scanning electron microscopy. Monodispersed vesicles with average size parameter of 10 μm and an apparent inverse Gaussian distribution were observed. Interestingly, biochemical circuit insulation did not appear to influence the size distribution of vesicles, which supports the decoupling of the insulation process from the complexity of the biochemical content. Moreover, no evolution of sizes was recorded after 3 months, which demonstrated the absence of fusion events between vesicles. In order to assess the capability of vesicles to encapsulate protein species without leakage, which is a prerequisite to achieve rational design of biochemical information processing, we assayed encapsulation stability using confocal microscopy. To this end, an irrelevant protein bearing a fluorescent label was encapsulated within vesicles, and the evolution of internal fluorescence was monitored over the course of 3 months. The internal fluorescence was found to remain stable, which demonstrated no measurable protein leakage through the vesicle membrane in our storage conditions. In addition, using phospholipid bilayer specific dye, DiIC18, which undergoes drastic increase in fluorescence quantum yield when specifically incorporated into bilayers (Gullapalli et al, 2008, Phys Chem Chem Phys; 10(24): 3548-3560), the complete extraction of oleic acid from the double emulsion and a well-structured arrangement of the bilayer could be visualized. We next sought to assess the encapsulation of biological enzymatic parts inside vesicles. We found that we could retrieve the molecular signatures of the enzymes in the interior of vesicles. Taken together, these findings show that this setup proved capable of generating stable, modular vesicles with high efficiency, and user-defined finely tunable content.

Example 3: Incorporation of the Vesicles Containing the Biochemical Network of Interest in a Gel Matrix

[0191] Once the vesicles containing the biochemical network of interest are ready, they are incorporated into the final format which is a set of gel matrix based beads. The size of the gel beads can be adjusted depending of the end user needs (i.e. 5 mm diameter). The gel is composed of 10% polyvinyl-alcohol (PVA) and 1% sodium-alginate. The mix containing all the components of the biochemical network in vesicles is incorporated in a liquid solution of 10% polyvinyl-alcohol (PVA), 1% sodium-alginate. The biochemical network/PVA/Alginate mix is then dropped in a 0.8M Boric acid/0.2M CaCl.sub.2 solution under agitation with a stir bar. After 30 minutes, the beads are rinsed 2 times in water and dropped in a 0.5M sodium sulphate buffer for 90 minutes. The beads are rinsed 2 times in cold PBS and conserved in PBS at 4° C.

Example 4: Detection of the Glyphosate/Quantification Results

[0192] 1. Detection of Glyphosate by the Glycine/Glyphosate Oxidase network

[0193] 1.1 By using the 0-Dianisidine dihydrochloride, we followed the Glyphosate oxidation by the Glycine/Glyphosate Oxidase that is dependent of Glyphosate concentration (FIG. 1, 4A, 4B). The color change of the beads was followed by the change of absorbance at 450 nm on a spectrophotometer. It allowed us to determine an affinity (Km) of the Glycine/Glyphosate Oxidase for the glyphosate that is 2.5 mM and a Vmax of 6×10.sup.−9 mol/L/sec.

[0194] 1.2 Detection of Glyphosate in Tris buffer and barley extracts by the Glycine/Glyphosate Oxidase network (#1) in liquid, vesicle or gel)

[0195] 1.2.1 Preparation of Barley extracts for subsequent Glyphosate detection First, Barley grains where grinded and sifted. The powder was ressuspended with Tris 100 mM pH 7,5 and incubated for 30 minutes on a wheel at room temperature. The extract was centrifugated for 10 minutes at 4000 g. The supernatant was filtered with 0.2 micrometer cutoff syringe filter and conserved at 4° C. before analysis.

[0196] 1.2.2 By using the Amplex red, we followed the Glyphosate oxidation by the Glycine/Glyphosate Oxidase that is dependent of Glyphosate concentration (FIG. 8A-8B). The reaction was followed by the change of fluorescence on a fluorimeter (Excitation at 530 nm/Emission at 590 nm). In parallel we followed the color change of the reaction that is dependent on the glyphosate concentration. It allowed us to detect the glyphosate not only in simple buffered medium (FIG. 8A) but also in complex barley extracts (FIG. 8B).

[0197] 1.2.3 Detection of Glyphosate by the Glycine/Glyphosate Oxidase network (#1) in vesicles

[0198] After incorporating a part of the network in the vesicles (HRP, Amplex Red, Tris 50 mM buffer pH 7,5) and outside the vesicles (Glycine/Glyphosate oxidase), we followed the Glyphosate oxidation that is dependent of Glyphosate concentration (FIG. 9A-9B)). The reaction was followed by the change of fluorescence on a fluorimeter (Excitation at 530 nm/Emission at 590 nm). In parallel we followed the color change of the reaction that is dependent on the glyphosate concentration. It allowed us to detect the glyphosate not only in simple buffered medium (FIG. 9A) but also in complex barley extracts (FIG. 9B)

[0199] 1.2.4 Detection of Glyphosate by the Glycine/Glyphosate Oxidase network (#1) in gel beads

[0200] The full Glycine/Glyphosate Oxidase network was incorporated in alginate gel beads.

[0201] We followed the Glyphosate oxidation that is dependent of Glyphosate concentration (FIG. 10). The reaction was followed by the change of beads color (red). Once again, it allowed us to detect the glyphosate not only in simple buffered medium (FIG. 10 (first line)) but also in complex barley extracts (FIG. 10, second line).

[0202] 2. Detection of Glyphosate by the EPSP Synthase network plugged to phosphate detection

[0203] By using the O-Dianisidine dihydrochloride or Amplex red, we followed the Glyphosate inhibition of the EPSP Synthase that is dependent of Glyphosate concentration (FIG. 2B, 5A, 5B, 7). The reaction was followed by the change of fluorescence on a fluorimeter (Excitation at 530 nm/Emission at 590 nm) or by the change of absorbance at 450 nm on a spectrophotometer. It allowed us to determine the activity of the EPSP synthase toward Phosphoenol Pyruvate (PEP). EPSP affinity for PEP is 14 μM and Vmax is at 10.26×10.sup.−9 mol/L/sec. Moreover it allowed us to detect the glyphosate by its inhibitory effect on the EPSP synthase (FIG. 7).

[0204] 3. Detection of Glyphosate by the EPSP Synthase network plugged to 5-O-(1-caroxyvinyl)-3-phospho shikimate detection

[0205] By monitoring the NADH consumption, we followed the Glyphosate inhibition of the EPSP Synthase that is dependent of Glyphosate concentration (FIG. 2A). The NADH consumption was given by the change of fluorescence emission at 445 nm (Excitation at 340 nm)—or change of absorbance at 340 nm on a spectrophotometer.

Example 5: Detection of the Glycine/Quantification Results

[0206] 1. Detection of Glycine in Tris buffer by the Glycine/Glyphosate Oxidase network (#3) in liquid (no vesicle/no gel)

[0207] By using the Amplex red, we followed the Glycine oxidation by the Glycine/Glyphosate Oxidase (Glycine/Glyphosate oxidase H244K Glycine/Glyphosate oxidase from Bacillus subtilis genetically modified containing 1 single amino-acids mutation H244K compared to the wild type version (Creative enzyme NATE-1674)) that is dependent of Glycine concentration (FIG. 11). The reaction was followed by the change of fluorescence on a fluorimeter (Excitation at 530 nm/Emission at 590 nm). It allowed us to detect the glycine.

Example 6: Detection of Glyphosate and Glycine/Quantification Results

[0208] 1. Detection of Glyphosate and glycine in Tris buffer by the Glycine/Glyphosate Oxidase network (#4) in vesicles

[0209] In this example we took advantage of the specificity of the GST-BliGO Mut #1 toward glyphosate compared to glycine and of the specificity of the Glycine/Glyphosate oxidase H244K from Bacillus subtilis toward glycine compared to glyphosate. Indeed, the GST-BliGO Mut #1 is derived from the BliGO SCF4 mutant developed by Zhang et al. (2016). This mutant has an 8-fold increase of affinity (1.58 mM) toward glyphosate and its activity to glycine decreased by 113-fold compared to WT. This mutant was developed to increase plants resistance to glyphosate and we used it as a basis for glyphosate biosensing.

[0210] After incorporating a part of the network in the vesicles (HRP, Amplex Red, Tris 50 mM buffer pH7,5) and outside the vesicles (Glycine/Glyphosate oxidase H244K Glycine/Glyphosate oxidase from Bacillus subtilis genetically modified containing 1 single amino-acids mutation H244K compared to the wild type version (Creative enzyme NATE-1674) and GST-BliGO Mut #1 from Bacillus Licheniformis derived from the BliGO-SCF-4 genetically modified and containing 6 single amino-acids mutation compared to the wild type version, we followed the Glyphosate AND/OR glycine oxidation that is dependent of Glyphosate and Glycine concentration (FIG. 12)). The reaction was followed by the change of fluorescence on a fluorimeter (Excitation at 530 nm/Emission at 590 nm). It allowed us to detect the glyphosate alone, the glycine alone and the glyphosate and glycine combination. (FIG. 12)

CONCLUSION AND DISCUSSION

[0211] This study demonstrated that the method and the composition according to the present invention are highly promising tools to perform detection and quantification of pesticides or endocrine disruptors, potentially multiplexed. We showed that this technology could be successfully applied to solve real environmental problems and demonstrated that the method and the composition of the present invention could overcome several hurdles faced by classical diagnosis tools in this field.