Polymer nanoparticle-based screening platform for plastic degrading enzymes

Abstract

Disclosed herein is a polymeric nanoparticle comprising a polymeric material that is susceptible to enzymatic degradation and a dye encapsulated within the polymeric material. Also disclosed herein are microfluidic devices and plates of a solid medium (e.g. agar) and methods making use of said nanoparticles.

Description

[0001] POLYMER NANOPARTICLE-BASED SCREENING PLATFORM FOR PLASTIC DEGRADING ENZYMES

[0002] Field of Invention

[0003] The present invention generally relates to screening platforms, and more particularly relates to a polymer nanoparticle-based screening platform for plastic degrading enzymes.

[0004] Background

[0005] The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0006] Over the years various microorganisms capable of degrading certain forms of plastic were identified and the enzymes responsible for the degradation were characterized. These plastics typically include polyethylene (PE), polyethylene terephthalate (PET), polyurethane (PU), polyamide (PA), polystyrene (PS), among others. Incidentally, a discovery made by Yoshida et al. on a novel bacterium, Ideonella sakaiensis, which was isolated from samples obtained around a PET bottle recycling factory in Japan in 2016 has garnered widespread attention. Unlike the previously described enzymes, this one was active at ambient temperatures. This bacterium grows on PET as carbon source and secretes enzyme - PETase and MHETase. PETase enzyme can degrade PET into 2-hydroxyethyl terephthalic acid (MHET) and terephthalic acid (TPA) with some traces of bis(2-hydroxyethyl) terephthalate (BHET) and further MHETase can cleave the MHET into the respective monomer (Yoshida, S., et al., Science 2016, 351(6278), 1196-1199; and Austin, H. P., et a!., P AS 2018, 115(19), E4350- E4357). Ever since, various labs across the world have been working towards engineering this enzyme as the activity of the identified enzyme is rather low. Some improved variants of this enzyme have since been published using rational protein engineering and mutating specific amino acids. The process of identifying an improved mutant is mainly limited by the availability of tools that can screen the enzymes from a library of generated enzymes. The current methods of identifying and screening various plastic degrading enzymes utilize high- performance liquid chromatography (HPLC) analysis which is cumbersome and simply impossible to apply to the vast array of mutants that are generated in mutant libraries.

[0007] The earliest methods used for measuring activity of enzymes include gravimetric method where change is mass of substrate upon treatment is measured and qualitative analysis using SEM imaging. In these quantitative methods, the substrate has to be retrieved after reaction for analysis. The current method of analyzing the activity is by determining the degradation products released using HPLC analysis. HPLC method of analysis provides precise concentration of degradation products but the method requires clean sample for analysis thus a need for prior sample preparation step which makes this method the most labor intensive and discontinuous while requiring several minutes for the analysis of a single sample.

[0008] Some indirect methods have been developed for analysis such as turbidity measurement of PET nanoparticles suspension upon enzymatic reaction. And for a faster detection of enzyme activity, model substrate like p-nitrophenyl acetate is used which provides spectrophotometrically detectable product, p-Nitrophenol (pNP). The dissimilarities between actual PET substrate and model substrate makes for an unreliable comparison between the pNPA activity with actual degradation of PET. There is also a quick saturation in pNPA assay which renders it irrelevant to screen for enzymes with high activity as the difference in activity becomes undistinguishable.

[0009] Another indirect method of analyzing PET degradation includes titration method. As the enzyme-substrate reaction leads to formation of acidic products (MHET and TPA), the amount of base to neutralize the acidic solution provides the estimation of product concentration and thus the enzyme activity, however the method fails to account for BHET as it is not acidic. Recent advancement attained in continuous and faster analysis of activity is by bulk absorbance method in which the degradation products are detected by obtaining a linear absorbance profile of the degradation products using spectrophotometer which cannot be measured using crude enzyme lysates.

[0010] Therefore, there exists an urgent need for the development of platforms that can significantly reduce the time and steps required for screening plastic degrading enzymes.

[0011] Summary of Invention

[0012] Aspects and embodiments of the invention are provided in the following numbered clauses.

[0013] 1. A polymeric nanoparticle comprising: a polymeric material that is susceptible to enzymatic degradation; and a dye encapsulated within the polymeric material. 2. The polymeric nanoparticle according to Clause 1 , wherein at least a portion of the dye is released from the nanoparticle in an environment containing an enzyme suitable for degrading the polymeric material.

[0014] 3. The polymeric nanoparticle according to Clause 1 or Clause 2, wherein the polymeric material is selected from the group consisting of a polypropylene (PP), a polybutylene adipate terephthalate (PBAT), a polyamide (PA), and more particularly, a polyethylene terephthalate (PET), a polybutylene terephthalate (PBT), a polyethylene naphthalate (PEN), a polyethyelene (PE), a polyurethane (PU) and a polystyrene (PS), copolymers thereof and blends thereof.

[0015] 4. The polymeric nanoparticle according to Clause 3, wherein the polymeric material is selected from the group consisting of a polyamide (PA), a polystyrene (PS), a polyester- polyether-polyurethane-copolymer (PU / PCL copolymer); and a polyethylene terepththalate and polybutylene terephthalate blend (PET-PBT blend), and, more particularly, a polystyrene (PS), a polyethylene terephthalate (PET), a polybutylene terephthalate (PBT), a polyethylene naphthalate (PEN), and a polyethyelene (PE).

[0016] 5. The polymeric nanoparticle according to Clause 4, wherein the polymeric material is a PET, a PBT, a PEN, a copolymer thereof, and a blend thereof, optionally wherein the polymeric material is a PET.

[0017] 6. The polymeric nanoparticle according to any one of the preceding clauses, wherein the dye is a fluorescent dye, optionally wherein the dye is selected from one or more of the group consisting of methylene blue, toluidine blue O, acid fuchsin, alcian blue, sulforhodamine, and more particularly rhodamine B, and acridine orange.

[0018] 7. The polymeric nanoparticle according to Clause 6, wherein the dye is selected from one more of the group consisting of methylene blue, toluidine blue O, acid fuchsin, and more particularly, acridine orange.

[0019] 8. The polymeric nanoparticle according to any one of the preceding clauses, wherein the dye is present in an amount of from 0.0001 to 5wt%, such as from 0.01 to 3wt%, such as from 0.1 to 2 wt%.

[0020] 9. The polymeric nanoparticle according to any one of the preceding clauses, wherein the nanoparticle has a crystallinity value (Xc) of from 1 to less than 40%, such as from 5 to 8%, such as about 7.5%. 10. The polymeric nanoparticle according to any one of the preceding clauses, wherein the nanoparticle has a size of from 10 to less than 800 nm, such as from 30 to 600 nm, such as from 100 to 200 nm.

[0021] 11 . The polymeric nanoparticle according to any one of the preceding clauses, wherein:

[0022] (a) the polymeric material is a PET and the dye is acridine orange;

[0023] (b) the polymeric material is a PBT and the dye is acridine orange;

[0024] (c) the polymeric material is a PEN and the dye is acridine orange;

[0025] (d) the polymeric material is a PS and the dye is acridine orange;

[0026] (e) the polymeric material is a PE and the dye is acridine orange;

[0027] (f) the polymeric material is a PA and the dye is acridine orange;

[0028] (g) the polymeric material is a PU / PCL blend and the dye is acridine orange; or

[0029] (h) the polymeric material is a PET-PBT blend and the dye is acridine orange,

[0030] (i) the polymeric material is a PET and the dye is methylene blue;

[0031] (j) the polymeric material is a PBT and the dye is methylene blue;

[0032] (k) the polymeric material is a PEN and the dye is methylene blue;

[0033] (l) the polymeric material is a PS and the dye is methylene blue;

[0034] (m) the polymeric material is a PE and the dye is methylene blue;

[0035] (n) the polymeric material is a PA and the dye is methylene blue;

[0036] (o) the polymeric material is a PU / PCL blend and the dye is methylene blue;

[0037] (p) the polymeric material is a PET-PBT blend and the dye is methylene blue;

[0038] (q) the polymeric material is a PET and the dye is toluidine blue O;

[0039] (r) the polymeric material is a PBT and the dye is toluidine blue O;

[0040] (s) the polymeric material is a PEN and the dye is toluidine blue O;

[0041] (t) the polymeric material is a PS and the dye is toluidine blue O;

[0042] (u) the polymeric material is a PE and the dye is toluidine blue O;

[0043] (v) the polymeric material is a PA and the dye is toluidine blue O;

[0044] (w) the polymeric material is a PU / PCL blend and the dye is toluidine blue O;

[0045] (x) the polymeric material is a PET-PBT blend and the dye is toluidine blue O;

[0046] (y) the polymeric material is a PET and the dye is toluidine blue O;

[0047] (z) the polymeric material is a PBT and the dye is toluidine blue O;

[0048] (s) the polymeric material is a PEN and the dye is toluidine blue O;

[0049] (t) the polymeric material is a PS and the dye is toluidine blue O;

[0050] (u) the polymeric material is a PE and the dye is toluidine blue O;

[0051] (v) the polymeric material is a PA and the dye is toluidine blue O;

[0052] (w) the polymeric material is a PU / PCL blend and the dye is toluidine blue O; (x) the polymeric material is a PET-PBT blend and the dye is toluidine blue O; optionally wherein the polymeric material is selected from the list:

[0053] (i) the polymeric material is a PET and the dye is acridine orange;

[0054] (ii) the polymeric material is a PBT and the dye is acridine orange; or

[0055] (iii) the polymeric material is a PEN and the dye is acridine orange.

[0056] 12. The polymeric nanoparticle according to any one of the preceding clauses, wherein the polymeric material is a PET and the dye is acridine orange.

[0057] 13. A plate suitable for culturing a microbial population, the plate comprising: a solid medium suitable for culturing a microbial population; and a plurality of polymeric nanoparticles according to any one of Claims 1 to 12 homogeneously distributed throughout and / or on a surface layer of the solid medium, optionally wherein the plurality of polymeric nanoparticles are distributed on the surface layer of the solid medium.

[0058] 14. The plate according to Clause 13, wherein the solid medium is agar, optionally wherein the agar is TB (teriffic broth) agar or, more particularly, LB agar.

[0059] 15. A microfluidic device suitable for high throughput identification of one or more of an isolated enzyme, a cell lysate, and a microbe suitable for degrading a polymeric material, the microfluidic device comprising a plurality of polymeric nanoparticles as described in any one of Clauses 1 to 12.

[0060] 16. A method of determining whether one or more of an isolated enzyme, a cell lysate, and a microbe are suitable for degrading a polymeric material, the method comprising the steps of:

[0061] (aa) providing a plurality of polymeric nanoparticles according to Clauses 1 to 12; and

[0062] (ab) contacting the plurality of polymeric nanoparticles with the one or more of the isolated enzyme, the cell lysate, or the microbe in a suitable medium for a period of time and measuring the release of the dye over the period of time to determine whether the enzyme, lysate or microbe are suitable for degrading the polymeric material in the polymeric nanoparticles.

[0063] 17. The method according to Clause 16, wherein the method further comprises providing a control comprising a plurality of polymeric nanoparticles according to Clauses 1 to 12 and a medium so as to provide a background reading over the period of time. 18. The method according to Clause 16 or Clause 17, wherein the dye release is measured using one or both of fluorescence and absorbance (e g. fluorescence).

[0064] 19. A method of determining whether one or more of an isolated enzyme, a cell lysate, and a microbe are suitable for degrading a polymeric material, the method comprising the steps of:

[0065] (I) providing a microfluidic device according to Clause 15; and

[0066] (ii) contacting the plurality of polymeric nanoparticles in the microfluidic device with one or more of the isolated enzyme, the cell lysate or the microbe in a suitable medium for a period of time and measuring the release of the dye over the period of time to determine whether the enzyme or bacteria are suitable for degrading the polymeric material in the polymeric nanoparticles.

[0067] 20. A method of determining whether one or more of an isolated enzyme, a cell lysate, and a microbe are suitable for degrading a polymeric material, the method comprising the steps of:

[0068] (aa) providing a plate suitable for culturing a bacterial population according to Clause 13 or Clause 14; and

[0069] (ab) contacting the plate with a mixture comprising the isolated enzyme, the cell lysate or the bacteria and a suitable medium for a period of time and measuring the release of the dye over the period of time to determine whether the enzyme or bacteria are suitable for degrading the polymeric material in the polymeric nanoparticles.

[0070] 21. The method according to Clause 20, wherein the method further comprises providing a control comprising a plate according to Clause 13 or Clause 14 contacted by a suitable medium identical to that used in Clause 20 so as to provide a background reading over the period of time.

[0071] 22. The method according to Clause 20 or Clause 21, wherein the dye release is measured using one or both of absorbance or fluorescence (e g. fluorescence).

[0072] 23. A method of making a plurality of polymeric nanoparticles according to any one of Clauses 1 to 12, the method comprising the steps of:

[0073] (ai) providing a first mixture comprising a polymeric material that is susceptible to enzymatic degradation, a dye and a first solvent, and second mixture comprising a surfactant and an anti-solvent for the polymeric material; and (aii) adding the first mixture to the second mixture in a dropwise fashion to provide the plurality of polymeric nanoparticles, wherein the dye is soluble in the first solvent and the anti-solvent for the polymeric material.

[0074] 24. The method according to Clause 23, wherein the first solvent is selected from one or more of the group consisting of toluene, dimethyl formamide, xylene, dichloroacetic acid, and, more particularly chloroform, and hexafluoropropan-2-ol, optionally wherein the first solvent is hexafluoropropan-2-ol.

[0075] 25. The method according to Clause 23 or Clause 24, wherein one or more of the following apply:

[0076] (bi) the anti-solvent is water (e.g. ultra-pure water);

[0077] (bii) the surfactant is sodium dodecyl sulfate (SDS) or cetyltrimethylammonium bromide (CTAB), (e.g. SDS);

[0078] (biii) the surfactant is present in an amount of from 1 to 5 wt% relative to the weight of antisolvent, such as from 1.5 to 3 wt%, such as about 2 wt%.

[0079] 26. The method according to any one of Clauses 23 to 25, wherein the polymeric material is selected from the group consisting of a polypropylene (PP), a polybutylene adipate terephthalate (PBAT), a polyamide (PA), and more particularly, a polyethylene terephthalate (PET), a polybutylene terephthalate (PBT), a polyethylene naphthalate (PEN), a polyethyelene (PE), a polyurethane (PU) and a polystyrene (PS), copolymers thereof and blends thereof, optionally wherein the polymeric material is selected from the group consisting of a polyamide (PA), a polystyrene (PS), a polyester-polyether-polyurethane-copolymer (PU / PCL copolymer); and a polyethylene terepththalate and polybutylene terephthalate blend (PET- PBT blend), and, more particularly, a polystyrene (PS), a polyethylene terephthalate (PET), a polybutylene terephthalate (PBT), a polyethylene naphthalate (PEN), and a polyethyelene (PE), yet more optionally wherein the the polymeric material is a PET, a PBT, a PEN, a copolymer thereof, and a blend thereof, optionally wherein the polymeric material is a PET.

[0080] 27. The method according to any one of Clauses 23 to 26, wherein the dye is a fluorescent dye, optionally wherein the dye is selected from one or more of the group consisting of methylene blue, toluidine blue O, acid fuchsin, alcian blue, sulforhodamine, and more particularly rhodamine B, and acridine orange. 28. The method according to any one of Clauses 23 to 27, wherein the dye is present in the first mixture in a concentration of from 0.001 to 10 mg / mL, such as from 0.005 to 1 mg / mL, such as from 0.01 to 0.05 mg / mL. such as about 0.299 mg / mL

[0081] 29. The method according to any one of Clauses 23 to 28, wherein:

[0082] (a) the polymeric material is a PET and the dye is acridine orange;

[0083] (b) the polymeric material is a PBT and the dye is acridine orange;

[0084] (c) the polymeric material is a PEN and the dye is acridine orange;

[0085] (d) the polymeric material is a PS and the dye is acridine orange;

[0086] (e) the polymeric material is a PE and the dye is acridine orange;

[0087] (f) the polymeric material is a PA and the dye is acridine orange;

[0088] (g) the polymeric material is a PU / PCL blend and the dye is acridine orange; or

[0089] (h) the polymeric material is a PET-PBT blend and the dye is acridine orange,

[0090] (i) the polymeric material is a PET and the dye is methylene blue;

[0091] (j) the polymeric material is a PBT and the dye is methylene blue;

[0092] (k) the polymeric material is a PEN and the dye is methylene blue;

[0093] (l) the polymeric material is a PS and the dye is methylene blue;

[0094] (m) the polymeric material is a PE and the dye is methylene blue;

[0095] (n) the polymeric material is a PA and the dye is methylene blue;

[0096] (o) the polymeric material is a PU / PCL blend and the dye is methylene blue;

[0097] (p) the polymeric material is a PET-PBT blend and the dye is methylene blue;

[0098] (q) the polymeric material is a PET and the dye is toluidine blue O;

[0099] (r) the polymeric material is a PBT and the dye is toluidine blue O;

[0100] (s) the polymeric material is a PEN and the dye is toluidine blue O;

[0101] (t) the polymeric material is a PS and the dye is toluidine blue O;

[0102] (u) the polymeric material is a PE and the dye is toluidine blue O;

[0103] (v) the polymeric material is a PA and the dye is toluidine blue O;

[0104] (w) the polymeric material is a PU / PCL blend and the dye is toluidine blue O;

[0105] (x) the polymeric material is a PET-PBT blend and the dye is toluidine blue O;

[0106] (y) the polymeric material is a PET and the dye is toluidine blue O;

[0107] (z) the polymeric material is a PBT and the dye is toluidine blue O;

[0108] (s) the polymeric material is a PEN and the dye is toluidine blue O;

[0109] (t) the polymeric material is a PS and the dye is toluidine blue O;

[0110] (u) the polymeric material is a PE and the dye is toluidine blue O;

[0111] (v) the polymeric material is a PA and the dye is toluidine blue O;

[0112] (w) the polymeric material is a PU / PCL blend and the dye is toluidine blue O;

[0113] (x) the polymeric material is a PET-PBT blend and the dye is toluidine blue O; optionally wherein the polymeric material is selected from the list:

[0114] (i) the polymeric material is a PET and the dye is acridine orange;

[0115] (ii) the polymeric material is a PBT and the dye is acridine orange; or

[0116] (iii) the polymeric material is a PEN and the dye is acridine orange.

[0117] 30. The method according to Clause 29, wherein the polymeric material is a PET and the dye is acridine orange.

[0118] Drawings

[0119] Fig. 1 depicts a schematic of the nanoparticle-based screening platform.

[0120] FIG. 2 depicts differential scanning calorimetry (DSC) curve of nPET.

[0121] FIG. 3 depicts size distribution of nPET as measured using dynamic light scattering (DLS).

[0122] FIG. 4 depicts transmission electron microscopy (TEM) image of nPET.

[0123] FIG. 5 depicts degradation of nPET using WT-PETase enzyme, (a) Purified WT-PETase enzyme at 30 °C and pH 8. (b) Secreted WT-PETase in lysogeny broth (LB) media at pH 7.

[0124] FIG. 6 depicts dye release assay and correlation with degradation of nPET (AO).

[0125] FIG. 7 depicts dye release assay of nPET (AO) at different substrate concentration with purified enzyme and corresponding degradation activity.

[0126] FIG. 8 depicts dye release assay of nPET(AO) at different incubation time with purified enzyme and corresponding degradation activity.

[0127] FIG. 9 depicts dye release assay of nPET(AO) using WT PETase at different concentrations for an incubation of (a) 30 and (b) 60 minutes, with corresponding degradation activity.

[0128] FIG. 10 depicts (a) linear regression of correlation between fluorescence measurement and product released due to degradation measured by HPLC for a reaction period of 30 min, and (b) linear regression of correlation between fluorescence measurement and product released due to degradation measured by HPLC for a reaction period of 60 min. FIG. 11 depicts dye release assay using nPET(AO) with secreted enzyme for (a) 2 hours and (b) 4 hours of incubation.

[0129] FIG. 12 depicts preparation of nPET(AO) incorporated agar plate, (a) LB agar plate without nanoparticles, (b) LB agar plate with nPET(AO).

[0130] FIG. 13 depicts (a) nPET agar plate culture growth of PETase secreting E. coli and (b) image intensity analysis at. / ncubation temperature at 30 °C.

[0131] FIG. 14 depicts (a) nPET agar plate culture growth of PETase secreting E. coli and (b) image intensity analysis at. Incubation temperature at 37 °C.

[0132] FIG. 15 depicts size distribution curve of (a) PBT nanoparticles (nPBT), (b) polyethylene naphthalate nanoparticles (nPEN), (c) PE nanoparticles (nPE), (d) PS nanoparticles (nPS), (e) PU / PCL copolymers nanoparticles (nPU / PCL(Copolymer)), (f) PBT+PET nanoparticles (nBlend(PBT+PET)) and (g) PA nanoparticles (nPA).

[0133] FIG. 16 depicts size distribution curve of (a) nPET(AO), (b) nPET(methylene blue (MB)), (c) nPET (toluidine blue O (TBO)) and (d) nPET (acid fuchsin (AF)).

[0134] FIG. 17 depicts stability of nanoparticles loaded with (a) AO, (b) MB, (c) TBO and (d) AF.

[0135] FIG. 18 depicts dye release assay using purified WT PEtase with nPET loaded with AO, MB, TBO and AF.

[0136] Description

[0137] It has been surprisingly found that a combination of a dye and a polymeric material in the form of nanoparticles (the polymeric material encapsulating the dye) can be used to rapidly screen for microbes, cell lysates, or isolated enzymes that are capable of breaking down said polymeric material. In certain embodiments, specific combinations of a dye and a polymeric material may show particular utility in this regard.

[0138] Thus, in a first aspect of the invention, there is disclosed a polymeric nanoparticle comprising: a polymeric material that is susceptible to enzymatic degradation; and a dye encapsulated within the polymeric material. For the avoidance of doubt, it will be appreciated that the polymeric material is provided in the form of nanoparticles that encapsulate the dye.

[0139] In embodiments herein, the word “comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components / features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of” or “consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of’ or the phrase “consists essentially of’ or synonyms thereof and vice versa.

[0140] The phrase, “consists essentially of’ and its pseudonyms may be interpreted herein to refer to a material where minor impurities may be present. For example, the material may be greater than or equal to 90% pure, such as greater than 95% pure, such as greater than 97% pure, such as greater than 99% pure, such as greater than 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure.

[0141] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “a first solvent” includes mixtures of two or more solvent materials, reference to “the catalyst” includes mixtures of two or more such catalysts, and the like.

[0142] The polymeric material in the form of nanoparticles encapsulates a (e.g. fluorescent) dye. These particles or compartments may be made of various polymeric materials depending on the type of plastic (or polymeric) degrading enzyme that needs to be screened for. During the synthesis of these particles, the dye is encapsulated throughout the polymer. When these dye- encapsulated nanoparticles are subjected to treatment with the respective degrading enzymes, the encapsulated fluorescent dye is released from the particles which serves as a read out for the activity the degrading enzyme. This significantly reduces the time and processing required to analyze the activity of the degrading enzymes, thus enabling a rapid screening process for identification of novel enzymes and variants.

[0143] As will be appreciated therefore, the polymeric nanoparticles disclosed herein may be used for the rapid identification of novel enzymes and variants (or their producing microbes), and their respective activities, thereby expanding the pool of such enzymes / microbes and enabling new strategies for dealing with plastic / polymeric waste materials.

[0144] Given the above, it will be appreciated that the polymeric nanoparticles disclosed herein will release at least a portion of the dye from the nanoparticle in an environment containing an enzyme suitable for degrading the polymeric material. This enzyme may be in the form of an isolated enzyme (where a particular enzyme is known or suspected to have suitable degradation properties), a cell lysate or in the form of a microbe (which are suspected of producing such an enzyme). When used herein the term “at least a portion of the dye” is intended to refer to the release of a sufficient amount of the dye from the polymeric material that can be measured by a suitable means or apparatus (this may be, for example, the eye (e.g. with a suitable light source to enable fluorescence to occur), a fluorescence spectrometer or an absorbance spectrometer). These methods may allow for a qualitative and / or a quantitative reading to be taken for the particular enzyme / microbe expressing an enzyme to be taken.

[0145] The polymeric material used in the current invention is not particularly limited, though it should be a material that contains chemical bonds that can be cleaved by an enzyme. It may be, for example, selected from one or more of the group consisting of a polypropylene (PP), a polybutylene adipate terephthalate (PBAT), a polyamide (PA), and more particularly, a polyethylene terephthalate (PET), a polybutylene terephthalate (PBT), a polyethylene naphthalate (PEN), a polyethyelene (PE), a polyurethane (PU) and a polystyrene (PS), copolymers thereof and blends thereof. In more particular embodiment, the polymeric material may be selected from the group consisting of a polyamide (PA), a polystyrene (PS), a polyester-polyether-polyurethane-copolymer (PU / PCL copolymer); and a polyethylene terepththalate and polybutylene terephthalate blend (PET-PBT blend), and, more particularly, a polystyrene (PS), a polyethylene terephthalate (PET), a polybutylene terephthalate (PBT), a polyethylene naphthalate (PEN), and a polyethyelene (PE). For example, the polymeric material may be a PET, a PBT, a PEN, a copolymer thereof, and a blend thereof, optionally wherein the polymeric material may be a PET.

[0146] As noted herein in the examples section, the polymeric material may be in the form of copolymers or blends of polymeric materials and these combinations are explicitly intended to be covered by the embodiments discussed in the current application.

[0147] While technically any suitable dye may be used, it is particularly preferred that the dye be a fluorescent dye. The combination of the dye and the polymeric material may require tuning to ensure that the dye does not have particularly high binding affinity to the polymeric material it is to be paired with. This may be experimentally determined through the combination of the polymeric material and dye and subjecting the combined material to degradation with an enzyme of known activity for said polymeric material.

[0148] Particular fluorescent dyes that may be mentioned herein include, but are not limited to, methylene blue, toluidine blue O, acid fuchsin, alcian blue, sulforhodamine, rhodamine B, acridine orange, and combinations thereof. In yet more particular embodiments of the invention that may be mentioned herein, the dye may be selected from methylene blue, toluidine blue O, acid fuchsin, and acridine orange. In particular embodiments of the invention that may be mentioned herein, the dye may be acridine orange.

[0149] The amount of the dye within the nanoparticles may be any sufficient amount to provide a release amount that can be detected by the apparatus being used for that purpose. For example, the dye may be present in an amount of from 0.0001 to 5 wt%, such as from 0.01 to 3 wt%, such as from 0.1 to 2 wt%. This weight percent may be relative to the weight of the dye versus the total weight of the polymeric nanoparticle(s). Note however that the amount of dye is not particularly limited and may depend on the specific dye and polymer(s) to be tested and can be adjusted accordingly by the skilled person following the teachings provided in the current application.

[0150] The polymeric nanoparticles disclosed herein may be partly crystalline. For example, the nanoparticle may have a crystallinity value (Xc) of from 1 to less than 40%, such as less than 10% such as from 5 to 8%, such as about 7.5%. The crystallinity value may be measured as described in the examples section below.

[0151] The polymeric nanoparticles may have a spherical morphology, though other shapes may be suitable too.

[0152] The polymeric nanoparticles may have a size of from 10 to less than 800 nm, such as from 30 to 600 nm, such as from 100 to 200 nm.

[0153] Specific combinations of polymeric materials and dyes that may be mentioned herein include but are not limited to the following list:

[0154] (a) the polymeric material is a PET and the dye is acridine orange;

[0155] (b) the polymeric material is a PBT and the dye is acridine orange;

[0156] (c) the polymeric material is a PEN and the dye is acridine orange; (d) the polymeric material is a PS and the dye is acridine orange;

[0157] (e) the polymeric material is a PE and the dye is acridine orange;

[0158] (f) the polymeric material is a PA and the dye is acridine orange;

[0159] (g) the polymeric material is a PET and the dye is methylene blue;

[0160] (h) the polymeric material is a PBT and the dye is methylene blue;

[0161] (I) the polymeric material is a PEN and the dye is methylene blue;

[0162] (j) the polymeric material is a PS and the dye is methylene blue;

[0163] (k) the polymeric material is a PE and the dye is methylene blue;

[0164] (l) the polymeric material is a PA and the dye is methylene blue;

[0165] (m) the polymeric material is a PET and the dye is toluidine blue O;

[0166] (n) the polymeric material is a PBT and the dye is toluidine blue O;

[0167] (o) the polymeric material is a PEN and the dye is toluidine blue O;

[0168] (p) the polymeric material is a PS and the dye is toluidine blue O;

[0169] (q) the polymeric material is a PE and the dye is toluidine blue O;

[0170] (r) the polymeric material is a PA and the dye is toluidine blue O;

[0171] (s) the polymeric material is a PET and the dye is acid fuchsin;

[0172] (t) the polymeric material is a PBT and the dye is acid fuchsin;

[0173] (u) the polymeric material is a PEN and the dye is acid fuchsin;

[0174] (v) the polymeric material is a PS and the dye is acid fuchsin;

[0175] (w) the polymeric material is a PE and the dye is acid fuchsin;

[0176] (x) the polymeric material is a PA and the dye is acid fuchsin;

[0177] (y) the polymeric material is a PU / PCL copolymer and the dye is acridine orange;

[0178] (z) the polymeric material is a PET-PBT blend and the dye is acridine orange;

[0179] (aa) the polymeric material is a PU / PCL copolymer, and the dye is methylene blue;

[0180] (ab) the polymeric material is a PET-PBT blend and the dye is methylene blue;

[0181] (ac) the polymeric material is a PU / PCL copolymer and the dye is toluidine blue O;

[0182] (ad) the polymeric material is a PET-PBT blend and the dye is toluidine blue O;

[0183] (ae) the polymeric material is a PU / PCL copolymer and the dye is acid fuchsin; and

[0184] (af) the polymeric material is a PET-PBT blend and the dye is acid fuchsin.

[0185] In more particular embodiments, the combination of polymeric materials and dyes that may be mentioned herein include but are not limited to the following list:

[0186] (i) the polymeric material is a PET and the dye is acridine orange;

[0187] (ii) the polymeric material is a PBT and the dye is acridine orange; and

[0188] (iii) the polymeric material is a PEN and the dye is acridine orange. In yet more particular embodiments, the polymeric material may be a PET and the dye may be acridine orange.

[0189] The nanoparticles disclosed herein may be amendable to handling in such a way that they can be integrated into different products, thereby allowing the formation of useful tools for the detection of enzymes / microbes thatcan degrade polymeric materials. Thus, in a further aspect of the current invention, there is provided a plate suitable for culturing a microbial population, the plate comprising: a solid medium suitable for culturing a microbial population; and a plurality of polymeric nanoparticles as described herein homogeneously distributed throughout and / or on a surface layer of the solid medium, optionally wherein the plurality of polymeric nanoparticles are distributed on the surface layer of the solid medium.

[0190] The terms “microbial” and “microbe” when used herein may refer to any suitable microbial species, such as a bacteria or a yeast or any suitable combination thereof (e g. two or more bacteria, two or more yeasts, a yeast and a bacteria, two yeasts and a bacteria etc...).

[0191] As noted above, the polymeric nanoparticles can be homogenously distributed throughout the solid medium. Alternatively, they may be attached to the surface of the solid medium (i.e. the surface that is to be exposed to a microbe / enzyme. Yet further alternatively, they may be both homogeneously distributed throughout the solid medium and attached to the surface thereof too. As will be appreciated, it may be preferred to ensure that the nanoparticles are either attached to the surface of the solid medium or are distributed on the surface layer and within a portion of the solid medium adjacent thereto. This is because it is likely that only the nanoparticles on the surface (or close thereto) will be functionally susceptible to the enzyme / microbe and so distributing the nanoparticles throughout the solid medium may result in nanoparticles that cannot be affected by the enzyme / microbe. Therefore, from a cost and waste perspective, such an arrangement may be preferred.

[0192] Any suitable solid medium compatible with biological systems may be used. For example, the solid medium may be an agar. For example, the agar may be Terrific broth (TB) agar or, more particularly, LB agar. For the avoidance of doubt, LB agar refers to Luria-Bertani agar.

[0193] Microfluidics are a powerful tool that enables screening of enzyme variants in the millions range. The versatility of the nanoparticles disclosed herein enables it to be coupled with microfluidics, drastically increasing the potential throughput of the system. Thus, in a further aspect of the invention, there is provided a microfluidic device suitable for high throughput identification of one or more of an isolated enzyme, a cell lysate or a microbe suitable for degrading a polymeric material, the microfluidic device comprising a plurality of polymeric nanoparticles as described herein. Such a microfluidic device can be designed and operated using the knowledge of the skilled person and the details of the current invention.

[0194] Such a microfluidics system and process may entail the production of droplets containing nanoparticles of the current invention and droplets containing enzymes / microbes, or the droplets produced may contain both. The droplets can then be subjected to sorting and the best performing enzymes / microbes can be isolated (it will be appreciated that the cell lysate’s most important component will be the enzyme of interest).

[0195] As intimated hereinbefore, the polymeric nanoparticles mentioned above may be particularly useful for the identification of enzymes / microbes that have the ability to degrade the polymeric material(s) used in the polymeric nanoparticles. Such methods may also be capable of quantifying the activity of the isolated enzymes, cell lysates or microbes, thereby helping to identify the best-performing enzyme / microbe for use in the degradation of a particular polymeric material.

[0196] Thus, in a further aspect of the invention, there is provided a method of determining whether an isolated enzyme, a cell lysate or a microbe are suitable for degrading a polymeric material, the method comprising the steps of:

[0197] (aa) providing a plurality of polymeric nanoparticles as described hereinbefore; and

[0198] (ab) contacting the plurality of polymeric nanoparticles with the isolated enzyme, the cell lysate or the microbe in a suitable medium for a period of time and measuring the release of the dye over the period of time to determine whether the enzyme or microbe are suitable for degrading the polymeric material in the polymeric nanoparticles.

[0199] In certain embodiments of this method, a control may be used. That is, the method may further comprise providing a control comprising a plurality of polymeric nanoparticles as described herein and a medium so as to provide a background reading over the period of time.

[0200] Liquid phase format: In this method, cell lysates or cells secreting the enzymes can be cultured in multi-well plates containing the dye encapsulated nanoparticles. Upon degradation of the polymer, the fluorescent dye gets released into the liquid phase which can be readily measured using a plate reader allowing us to identify cells that contain a better degrading enzyme with respect to the WT control. An added advantage with this method is the ability to screen for enzymes at different conditions such as pH and temperature by using cell lysates. It will be appreciated that any suitable medium (e.g. a liquid medium) that can maintain the biological activity of the enzyme and / or microbe may be used herein and is not particularly limited. Examples of suitable media are provided, but not limited to, those in the examples.

[0201] It will be appreciated that the dye release may be measured by any suitable means. This may be completely qualitative (e.g. by eye detecting fluorescence) or it may be measured in a way that may be somewhat more quantitative (e.g. one or both of fluorescence and absorbance (e.g. fluorescence) as determined with a suitable spectrometer). It will be appreciated that these detection means may be used in the other methods discussed hereinbelow too.

[0202] In a further aspect of the invention, there is provided a method of determining whether an isolated enzyme, a cell lysate or a microbe are suitable for degrading a polymeric material, the method comprising the steps of:

[0203] (i) providing a microfluidic device as discussed herein; and

[0204] (ii) contacting the plurality of polymeric nanoparticles in the microfluidic device with the isolated enzyme, a cell lysate or the microbe in a suitable medium for a period of time and measuring the release of the dye over the period of time to determine whether the enzyme or bacteria are suitable for degrading the polymeric material in the polymeric nanoparticles.

[0205] As noted above, the nanoparticles can be provided as plates, which are suitable use in a detection method also. Thus, in a further aspect of the invention, there is provided a method of determining whether one or more of an isolated enzyme, a cell lysate, and a microbe are suitable for degrading a polymeric material, the method comprising the steps of:

[0206] (aa) providing a plate suitable for culturing a bacterial population as described hereinbefore; and

[0207] (ab) contacting the plate with a mixture comprising the isolated enzyme, the cell lysate or the bacteria and a suitable medium for a period of time and measuring the release of the dye over the period of time to determine whether the enzyme or bacteria are suitable for degrading the polymeric material in the polymeric nanoparticles.

[0208] In such embodiments, the method may further comprise providing a control comprising a plate as described hereinbefore contacted by a suitable medium identical to that used in the aspect above so as to provide a background reading over the period of time.

[0209] It will be appreciated that the isolated enzyme(s), cell lysate, or microbe(s) will be kept in a suitable medium to retain their activity and provide any desired nourishment they require before being applied to the plates mentioned herein. For example, this may be LB or TB. As noted hereinbefore, the solid medium suitable for culturing a microbial population of the plates may be formed from a material that has properties enabling this to occur (e.g. LB or TB agar).

[0210] As will be appreciated, the dye released from the nanoparticles may be taken up by any cells (or parts of cells) in the mixture applied to the plates. In this case, the dyes may intercalate into the cells or the cell parts, thereby amplifying the detectible signal (e.g. fluorescence). Given this, it may be useful to have a background reading from a blank plate contacted by the same medium used to contact the isolated enzyme(s), cell lysate, or microbe(s) with the detection plate. It will be appreciated that the same plate may be used for both detection and as a background by having defined and delineated regions for this purpose.

[0211] Solid phase format: In this screening modality, the nanoparticles are embedded directly into the agar plate and colonies secreting the degrading enzyme are grown and induced on these plates. The enzymes degrade the nanoparticles, and the dye is thereby released into the agar which gets readily taken up by the cells in the colony. These dyes intercalate to the nucleic acids in the cells, amplifying the fluorescence greatly with respect to the background. These plates can be imaged in a blue light imager and simple image processing tools can be deployed for measuring the intensity of the fluorescence thereby giving a fluorescence intensity values which let us pick the colonies with high fluorescence.

[0212] In a further aspect of the invention there is provided a method of making a plurality of polymeric nanoparticles as described hereinbefore, the method comprising the steps of:

[0213] (ai) providing a first mixture comprising a polymeric material that is susceptible to enzymatic degradation, a dye and a first solvent, and second mixture comprising a surfactant and an anti-solvent for the polymeric material; and

[0214] (aii) adding the first mixture to the second mixture in a dropwise fashion to provide the plurality of polymeric nanoparticles, wherein the dye is soluble in the first solvent and the anti-solvent for the polymeric material.

[0215] As will be appreciated, the first solvent may be a single solvent or it may be a mixture of different solvents to achieve the desired formation of the nanoparticles. To that end, it may be desired that the solvent and anti-solvent are immiscible together, or are miscible together. The selection of the solvents may ultimately depend on the polymer(s) being tested and these can be selected accordingly. It will be appreciated that the first solvent should be able to solubilise the polymeric material and the dye. In certain embodiments that may be mentioned herein, the first solvent may be selected from one or more of the group consisting of toluene, dimethyl formamide, xylene, dichloroacetic acid, and, more particularly chloroform, and hexafluoropropan-2-ol, optionally wherein the first solvent is hexafluoropropan-2-ol. As an example of a first solvent mixture, the first solvent may be formed from chloroform and hexafluoropropan-2-ol in any suitable ratio.

[0216] The anti-solvent used in the method of manufacture may be any suitable material that causes precipitation of the polymer from solution. For example, the anti-solvent may be water (e.g. ultra-pure water).

[0217] Any suitable surfactant may be used herein. For example, the surfactant may be sodium dodecyl sulfate (SDS) or cetyltrimethylammonium bromide (CTAB), (e.g. SDS). The surfactant may be used in any suitable amount to effect the formation of the desired polymeric nanoparticles. For example, the surfactant may be present in an amount of from 1 to 5 wt% relative to the weight of anti-solvent, such as from 1 .5 to 3 wt%, such as about 2 wt%.

[0218] The polymeric material used herein is not particularly limited. As will be appreciated, the list of polymeric materials discussed above for the polymeric nanoparticles perse may be used. For the sake of brevity, this list is not repeated here.

[0219] The dye used in the method may be chosen from the dyes mentioned hereinbefore in relation to the polymeric nanoparticles perse. Again, this list is not repeated here for the sake of brevity.

[0220] The dye may be present in any suitable amount in the first mixture. For example, the dye may be present in the first mixture in a concentration of from 0.001 to 10 mg / mL, such as from 0.005 to 1 mg / mL, such as from 0.01 to 0.05 mg / mL, such as about 0.299 mg / mL. In certain embodiments, it is believed that the loading efficiency of the dye may be from 0.01% to 5%.

[0221] In certain embodiments of the method, that may be mentioned herein:

[0222] (a) the polymeric material is a PET and the dye is acridine orange;

[0223] (b) the polymeric material is a PBT and the dye is acridine orange;

[0224] (c) the polymeric material is a PEN and the dye is acridine orange;

[0225] (d) the polymeric material is a PS and the dye is acridine orange;

[0226] (e) the polymeric material is a PE and the dye is acridine orange;

[0227] (f) the polymeric material is a PA and the dye is acridine orange;

[0228] (g) the polymeric material is a PET and the dye is methylene blue;

[0229] (h) the polymeric material is a PBT and the dye is methylene blue;

[0230] (I) the polymeric material is a PEN and the dye is methylene blue; (j) the polymeric material is a PS and the dye is methylene blue;

[0231] (k) the polymeric material is a PE and the dye is methylene blue;

[0232] (l) the polymeric material is a PA and the dye is methylene blue;

[0233] (m) the polymeric material is a PET and the dye is toluidine blue O;

[0234] (n) the polymeric material is a PBT and the dye is toluidine blue O;

[0235] (o) the polymeric material is a PEN and the dye is toluidine blue O;

[0236] (p) the polymeric material is a PS and the dye is toluidine blue O;

[0237] (q) the polymeric material is a PE and the dye is toluidine blue O;

[0238] (r) the polymeric material is a PA and the dye is toluidine blue O;

[0239] (s) the polymeric material is a PET and the dye is acid fuchsin;

[0240] (t) the polymeric material is a PBT and the dye is acid fuchsin;

[0241] (u) the polymeric material is a PEN and the dye is acid fuchsin;

[0242] (v) the polymeric material is a PS and the dye is acid fuchsin;

[0243] (w) the polymeric material is a PE and the dye is acid fuchsin;

[0244] (x) the polymeric material is a PA and the dye is acid fuchsin;

[0245] (y) the polymeric material is a PU / PCL copolymer and the dye is acridine orange;

[0246] (z) the polymeric material is a PET-PBT blend and the dye is acridine orange;

[0247] (aa) the polymeric material is a PU / PCL copolymer, and the dye is methylene blue;

[0248] (ab) the polymeric material is a PET-PBT blend and the dye is methylene blue;

[0249] (ac) the polymeric material is a PU / PCL copolymer and the dye is toluidine blue O;

[0250] (ad) the polymeric material is a PET-PBT blend and the dye is toluidine blue O;

[0251] (ae) the polymeric material is a PU / PCL copolymer and the dye is acid fuchsin; and

[0252] (af) the polymeric material is a PET-PBT blend and the dye is acid fuchsin.

[0253] For example:

[0254] (i) the polymeric material is a PET and the dye is acridine orange;

[0255] (ii) the polymeric material is a PBT and the dye is acridine orange; or

[0256] (iii) the polymeric material is a PEN and the dye is acridine orange.

[0257] In yet more particular embodiments the polymeric material may be a PET and the dye may be acridine orange.

[0258] Advantages associated with the currently disclosed invention may include one or more of the following. • Our method describes the screening of plastic degrading enzymes using polymer nanoparticles for the identification of enzymes with enhanced activity.

[0259] • Unlike the traditional agar plate screening methods that use zone of clearance as a readout, our method uses fluorescence signals which give an amplified readout that can be easily measured. Traditional methods use zone of clearance as read out.

[0260] • Our method is versatile and can be adapted to cytosolic and secretory expression of plastic degrading enzymes while also allowing them to be used for screening in both solid and liquid phase screening methods.

[0261] • This method eliminates the use culturing and purification of enzymes. Cell lysates or enzymes secreted from cells can be used directly in the assay drastically reducing the number of steps required for screening.

[0262] • Unlike existing methods that use proxy substrates such as pNPA , our platform uses real plastic substrate in the form of nanoparticles which helps identify the enzymes that are able to degrade the polymer structure in real case scenarios.

[0263] • In our method, enzymes can be directly secreted on the agar plate without the need for any further culturing making it a one step process for mutant identification.

[0264] The current methods are extremely labor intensive and require sample preparation and analysis steps that require several minutes to analyze a single sample. The disclosed products and methods are very efficient as allow for measurements in less than a minute for up to 96 samples, or even more. Conventional prior art fluorescent methods require co-hydrolysis of the substrate as well as the fluorescent moiety where parameters such as pH and temperature can affect the co-hydrolysis resulting in unreliable readouts. The currently disclosed method can be easily adapted for various scenarios, such as pH, temperature, time of analysis and can be performed in solid or liquid phase and can employ cytosolic or secretory expression of PET hydrolases (or similar enzymes for other polymeric materials). Moreover, this platform is not limited to the identification of just PET hydrolases but can be employed to other plastic polymers such as PE, PS and PU among others, as mentioned hereinbefore (and as exemplified in the examples).

[0265] While some reported methods use molecules that are similar to a PET polymer (e g., containing ester bonds) referred to as a proxy substrate, they fail to show that the activity results are translatable to a real PET substrate. That is, if high activity is detected on proxy substrate, it does not necessarily mean that it will have high activity on the real PET substrate. In fact, we have observed the reverse. The disclosed method is as easy as adding the dye-loaded polymer (e g. mPET) into the reaction mixture and let the reaction run for a pre-determined time. At the end of the reaction time, the reaction mixture is spun to remove any debris and the released dye will be measured using a spectrophotometer. In contrast, some of the current methods will require an additional reagent (e.g., dye) as a reporter / indicator.

[0266] The disclosed nanoparticles provide a screening platform that is a powerful tool for the identification of improved and efficient plastic degrading enzymes. It may find application as an assay following random mutagenesis or directed evolution strategies that result in vast sets of plastic degrading enzyme mutants. Thus the disclosed platform may aid in the rapid identification of mutants with superior performance which is currently only limited by availability of screening tools.

[0267] With the discovery of enzymes such as PETase that have the ability to degrade PET plastics, the focus is quickly shifting towards development of processes for biocatalysts of plastic wastes as an alternate route to recycling. Platforms such as the ones disclosed herein are key to attaining these goals where the first step is the identification of best performing enzymes. These platforms are may be important for establishing commercial scale development of plastic waste recycling strategies.

[0268] Certain aspects of the invention that may be mentioned herein relate to the following numbered statements.

[0269] Further aspects and embodiments of the invention will now be discussed by reference to the following non-limiting examples.

[0270] Examples

[0271] Materials

[0272] The chemicals used were purchased from Sigma Aldrich unless mentioned otherwise. The solvents used in the synthesis of nanoparticles are 1 ,1 ,1,3,3,3-hexafluoro-2-propanol, dimethyl formamide, toluene anhydrous, dichloroacetic Acid, The polymers used for the synthesis of nanoparticles are polybutylene terephthalate (sheet, thickness 9.5 mm, size 150 x 150 mm), polyethylene naphthalate (film, thickness 0.125 mm, L 0.5 m), polyethylene (powder, mean particle size 150micron), polystyrene (granule, 3.5 mm nominal granule size), polyamide Nylon 6 (powder, max. particle size 50micron), and poly[4,4 ' - methylenebis(phenyl isocyanate)-alt-1 ,4-butanediol / di(propylene glycol) / polycaprolactone] (pellets, MDI-polyester / polyether polyurethane). The dyes used for the synthesis of nanoparticles are Acridine Orange hemi(zinc chloride) salt, Methylene Blue, Acid Fuchsin and Toluidine Blue O (Pure).

[0273] Example 1. Nanoparticle-based screening platform

[0274] To address the challenge of screening libraries of plastic degrading enzymes, we envisioned the development of nano compartments that encapsulate fluorescent dye. These compartments or particles may be made of various plastic polymers depending on the type of plastic degrading enzyme that needs to be screened for. During the synthesis of these particles, fluorescent dyes are encapsulated throughout the polymer. When these dye encapsulated particles are subjected to treatment with the respective degrading enzymes, the encapsulated fluorescent dye is released from the particles which serves as a read out for the activity the degrading enzyme. This significantly reduces the time and processing required to analyze the activity of the degrading enzymes, thus enabling a rapid screening process for identification of novel enzymes and variants.

[0275] The screening platform 100 disclosed herein can be used in two different formats (FIG. 1):

[0276] (i) solid phase format 110: In this screening modality, synthesized PET nanoparticles acridine orange (nPET (AO)) 130 are embedded directly into the agar plate 111 and colonies secreting the degrading enzyme are grown and induced on these plates 112. The enzymes degrade the nanoparticles, and the dye is thereby released into the agar which gets readily taken up by the cells in the colony. These dyes intercalate to the nucleic acids in the cells, amplifying the fluorescence greatly with respect to the background. These plates 113 can be imaged in a blue light imager and simple image processing tools can be deployed for measuring the intensity of the fluorescence thereby giving a fluorescence intensity values which let us pick the colonies with high fluorescence; and

[0277] (ii) liquid phase format 120: In this method, synthesized nPET (AO) 130 are suspended directly into reaction buffer in multi-well plates 121. Cell lysates 123 or cells secreting the enzymes 124 can be cultured in multi-well plates containing the dye encapsulated nanoparticles. Upon degradation of the polymer 125, the fluorescent dye gets released into the liquid phase which can be readily measured using a plate reader 126 allowing us to identify cells that contain a better degrading enzyme with respect to the WT control. An added advantage with this method is the ability to screen for enzymes at different conditions such as pH and temperature by using cell lysates.

[0278] Example 2. Synthesis and characterization of PET nanoparticles (nPET) encapsulated with AO nPET can be synthesized by dissolution of 100 mg PET film and 10 mg AO in 10 ml 1,1 ,1 ,3,3,3-hexafluoro-2-propanol (HFIP) followed by precipitation by slow release at a rate of 5ml / hr in 100 ml of 2% SDS in ultra-pure water. The suspension was centrifuged at 12,000 g for 10 minutes and the pellet was resuspended in ultra-pure water. This wash was repeated twice and the particles were suspended in 10 ml ultra-pure water. The suspension was sonicated for 10 minutes in water bath to ensure uniform dispersion.

[0279] DSC

[0280] The percentage crystallinity of nPET was measured by using differential scanning calorimeter. The freeze dried nPET particles were weighed on aluminum pan and crimped with a cover lid. Similarly, an empty pan was crimped with lid which will be used as reference. The samples were subjected to heating at the rate of 10 °C per min from 25 °C to 300 "C followed by 5 min hold at 300 C and the cycle was completed at the same rate with a cooling sequence from 300 °C to 25 °C. The enthalpy of fusion (AHf) and enthalpy of cold crystallization(AHcc) was analyzed from the thermogram of first heating cycle. The percentage of crystallinity was calculated using the equation below.

[0281] AHOf is the enthalpy of fusion of 100% crystalline PET which is 140.1 J / g.

[0282] Characterization

[0283] The synthesized nPET has a spherical morphology, lower (<10%) crystallinity, and shows faster degradability by WT PETase than PET powder or film used for studying degradation.

[0284] The growth of E. coli expressing secretory PETase in LB media containing nPET also shows degradation of the particles upon secretion of WT PETase as described in Example 4 below.

[0285] Example 3. Characterization of nPET(AO)

[0286] Size distribution The size distribution was measured by using the Malvern zetasizer Nano-ZS (Malvern panalytical).

[0287] TEM

[0288] The morphology was observed from micrograph obtained using Transmission electron microscope (JE-1400Flash Electron Microscope).

[0289] Degradability

[0290] Purified VVT PETase enzyme was used to observe the degradation of nPET. The enzymesubstrate reaction was carried out with 10 pM WT PETase and 4 mg / mL concentration of substrate in 0.1 M phosphate buffer, pH 8. The reaction mixture was incubated in thermoshaker at 30 °C, and the product release was analyzed after 24 hours (day 1) and 48 hours (day 2). The sample (50 pL) was centrifuged at 15000 g for 10 min. The supernatant was added with an equal volume of dimethyl sulphoxide (DMSO). The prepared samples were then analyzed for degraded products by HPLC method (Agilent 1260 HPLC). The samples were run using solvent A -1% formic acid in DI water and solvent B - acetonitrile as mobile phase at a flow rate of 1.0 ml / min over a course of 13 mins sequence, as follows - 15% to 35% of acetonitrile for 8 mins, 35% to 50% of acetonitrile for 0.1min, 50% acetonitrile for 1 min, 50% to 15% of acetonitrile for 0.1 mins 15% acetonitrile for 4 mins. The separated components were detected at 260 nm wavelength. The number of monomers in the sample was calculated from the standard curve prepared with TPA, MHET, and BHET. The percentage degradation was calculated from equation. g L

[0291] 1000

[0292] As will be appreciated, variations of this equation may be used for other polymers.

[0293] Results and discussion

[0294] DSC measurements of nPET (FIG. 2) show the percentage crystallinity of prepared nPET to be 7.5%. The size distribution of nPET was determined to be around 105 nm (FIG. 3). The morphology of the nPET (FIG. 4) was observed from micrograph obtained using TEM. FIG. 4 shows spherical PET nanoparticles.

[0295] The degradability of nPET using WT-PETase was measured. The degradability of synthesized nPET using purified enzyme is shown in FIG. 5a. FIG. 5b shows the degradation of nPET by secreted WT-PETase in media. Over 50% degradation is observed within 24 hours using purified enzyme while 15% degradation is obtained using secreted WT-PETase during the same time.

[0296] Example 4. Dye release assay and correlation with degradation of nPET(AO) - liquid phase screening platform 120 (FIG. 1) nPET(AO) was prepared by following the protocol in Example 2.

[0297] Dye release assay

[0298] Reaction was set up with nPET(AO) -10mg (10 mg of AO / 10 mg of PET) and nPET(AO) -1mg (1 mg of AO / 100 mg of PET) as substrate, suspended in 0.1 M Phosphate buffer, pH 8 at 4 mg / mL concentration. To the substrate, 10 pM PETase was added and incubated in a thermoshaker at 30 °C. After 1 hour of incubation, the sample were centrifuged at 15000 g for 10 min, and the supernatant was transferred to a 96 well microplate. Immediately, the fluorescence of supernatant was measured using Spectromax M5 microplate reader at excitation wavelength of 487 nm and emission wavelength at 523 nm. These sample was taken and prepared for HPLC analysis.

[0299] Results and discussion nPET with 10 mg of AO per 100 mg of PET gives 5-fold higher fluorescence than nPET with 1 mg of AO per 100 mg of PET (FIG. 6). 60% degradation was observed in 1 hour and the corresponding fluorescence reading was 1000. The nanoparticles worked well in the liquid phase format of the assay.

[0300] Example 5. Development and characterization of liquid phase screening method

[0301] The liquid phase screening method is as described in Example 4 except different substrate and enzyme concentrations and incubation periods were used.

[0302] Effect of substrate concentration nPET(AO) at different concentration of 0.5, 1 and 1.5 mg / mL was suspended in 200 pL 0.1M phosphate buffer, pH 8 in a microwell plate. To the substrate, purified WT PETase was added at 200nM concentration and incubated for 1 hour. The reaction mixture was centrifuged at 15000 g for 5 mins and fluorescence of the supernatant was measured at excitation wavelength of 487 nm and emission wavelength at 523 nm. These sample was further prepared for HPLC analysis to determine the amount of product released. Effect of incubation time nPET(AO) at 1 mg / mLwas suspended in 200 pL of 0.1 M phosphate buffer, pH 8 in a microwell plate. To the substrate, purified WT PETase was added at 200 nM concentration and incubated at different time point of 15, 30, 60 and 90 mins. The reaction mixture was centrifuged at 15000 g for 5 mins and fluorescence of the supernatant was measured at excitation wavelength of 487 nm and emission wavelength at 523 nm. These sample was further prepared for HPLC analysis to determine the amount of product released.

[0303] Enzyme dose response nPET(AO) at concentration of 1 mg / mL suspended in 200 pL of 0.1 M phosphate buffer, pH 8 were incubated were incubated with purified WT PETase at different concentrations of 5 nM, 20 nM, 50 nM and 100 nM for 30 minutes and 60 minutes incubation period. The reaction mixture was centrifuged at 15000 g for 5 minutes and fluorescence of the supernatant was measured at excitation wavelength of 487 nm and emission wavelength at 523 nm. These sample was further prepared for HPLC analysis to determine the amount of product released.

[0304] Dye release assay using secretory enzyme

[0305] The E. coli SHuffle cells expressing secretory PETase genes (WT, V3 and FAST PETase) were inoculated in TB media (1 mL) with ampicillin (100 pg / mL) in a deep well plate and incubated at 30 °C for 6 hours and induced at 16 °C for 16 hours. As a control, E. coli Shuffle (with empty vector) was inoculated in TB media. The culture was centrifuged at 4,500 g for 10 min, and the cell pellet and media supernatant were separated. The synthesized nPET(AO) was suspended in 100 pL of 0.2 M Phosphate buffer, pH 8. The collected media supernatant (100 pL) was transferred to a 96 well plate containing the nPET suspension and incubated for 2 hours and 4 hours. After incubation, the sample were centrifuged at 15000 g for 10 minutes, and the supernatant was transferred to a 96 well microplate followed by measurement of fluorescence at excitation wavelength of 487 nm and emission wavelength at 523 nm. After measurement, the sample was added with equal volumes of DMSO and filtered using a 0.2 pm syringe filter. The prepared sample was analysed by HPLC to measure the amount of PET monomer released.

[0306] Results and discussion

[0307] Effect of substrate concentration

[0308] The dye release assay was performed using different substrate concentration at 0.5 mg / mL, 1.0 mg / mL and 1.5 mg / mL using purified WT PETase at 200 nM concentration. The fluorescence measured after 30 minutes of incubation shows that with increase in substrate concentration, fluorescence reading also increases (bar graph). This trend is also observed in amount of products released in the reaction as measured using HPLC (scatter plot) shown in FIG. 7.

[0309] Effect of incubation time

[0310] Enzyme substrate reaction was carried out at 200 nM WT PETase and 1 mg / mL of nPET(AO). The fluorescence recorded at 15 min, 30 min, 60 min and 90 min shows a trend of increase in fluorescence, validating the progress of reaction catalyzed by PETase (FIG. 8).

[0311] Enzyme dose response

[0312] The enzyme dose response, as measured using fluorescence output, carried out using WT PETase at series of concentrations (5 nM, 20 nM, 50 nM and 100 nM) for 30 minutes and 60 minutes of incubation time is as shown in FIG. 9. The samples were analyzed to determine the product released by HPLC method which shows an increase in product release from 0.05 mg to 0.09 mg at 30 minutes of incubation and from 0.06 mg to 0.102 mg at 60 minutes with increase in concentration of enzyme from 5 nM to 100 nM. The linear regression also shows a strong correlation with fluorescence due to dye released and amount of product measured by HPLC within the range of enzyme concentration - 5 nM to 100 nM (FIG. 10). This establishes a tight correlation of our assay with traditional means of measurement of degradation using HPLC analysis.

[0313] Dye release assay using secretory enzyme

[0314] The assay disclosed herein may be applicable to wider formats of PET degradation analysis where there is no need to purify the enzyme, and the measurements can be obtained directly from the supernatants of enzyme secreting cells. Dye release assay performed with secreted PETase from E. coli culture is as observed with mutants of varied degradation ability, as shown in FIG. 11. In a deep well plate, E. coli SHuffle cells expressing secretory WT PETase, V3 PETase and FASTPETase were grown and induced in TB media (1 mL). 100 pL of supernatant from the culture (separated by centrifugation) was incubated with nPET substrate suspension in 96 well plate at 45 °C for 2 hours and 4 hours. Secreted V3 exhibits higher activity compared to secreted FASTPETase which is higher than WT. This trend observed using our fluorescence assay is in agreement with the product release measured by HPLC.

[0315] Example 6. Solid phase screening platform 110 (FIG. 1)

[0316] Agar plate-based method using PET nanoparticles with dye

[0317] Table 1. Colony growth condition.

[0318] Table 2. E.coli shuffle with empty vector.

[0319] The intensities of fluorescence emission were measured using Imaged, an image analysis software.

[0320] Solid phase screening platform

[0321] For the preparation of nPET(AO) agar plate, 35 mm diameter and 15 mm height petri dishes were used. LB agar was prepared with 90 % of total volume (45 mL). The nPET(AO) particles were suspended in sterile DI water (5 mL) and added to the LB agar at 1 mg / mL concentration of nPET(AO) then ampicillin (100 pg / mL) was added which was poured into petri dish to solidify. The nPET AO incorporated agar plates were spread with 0.5 mM IPTG and stored in 4°C overnight. On the nPET AO agar plate, one microlitre of overnight culture of E. coli SHuffle with WT PETase and E. coli SHuffle with STAR PETase expression was placed on top of the agar as drop. As a control, E. coli SHuffle with empty pET-22b vector was used. Each of these plates were incubated at 30 °C and 37 °C for 24 hours. After incubation the plates were observed under UV illumination using iBright imaging system to check for fluorescence.

[0322] Results and discussion

[0323] For the solid phase screening platform, the nPET loaded with AO was incorporated into LB agar media which is used in preparing agar plate with uniform distribution of nPET (AO) as shown in FIG. 12. An opaque distribution of the nanoparticles was observed in the plate.

[0324] The plate inoculated with 1 pl of overnight culture of E. coli SHuffle cells as control, E. coli SHuffle cells secreting WT and V3 PETase was incubated for 24 hours for growth of the cells. The agar plate cultivation observed under blue light illumination showed fluorescence emission around E. coli secreting WT-PETase and V3 PETase. From the image of cells cultivated on nPET(AO), the intensity of the fluorescence ring was analyzed. As shown in FIGS. 13 and 14, due to increased activity of secreted V3 PETase, the intensity is higher compared to WT and control. The nanoparticles worked well in the solid phase format of the assay. Example 7. Microfluidics based high throughput screening using nPET(AO)

[0325] Microfluidics is a powerful tool that enables screening of enzyme variants in the millions range. The versatility of our platforms enables it to be coupled with microfluidics drastically exemplifying the scale. This process entails production of droplets containing nPET encapsulating reporters (e.g., fluorescent dyes) and bacteria producing PETase mutants (secreting or otherwise). The droplets will be subjected to sorting and the best performing mutants under specified parameters to be determined in the future will be isolated. This can be extrapolated likewise to other polymers and enzymes that degrade them.

[0326] Example 8. Extension of the platform to other polymers

[0327] Synthesis ofnPBT, nPEN, nPE, nPS, nPUPCL(Copolymer), nBlend(PBT+PET), and nPA The synthesis of fluorescent nanoparticles was extended to other polymers for applications in the liquid and solid screening platforms described in the Examples 4 and 6, which includes PBT, PEN, PE, PS, PA, PU / PCL copolymer and a blend of PET and PBT. The synthesis of nPBT, nPA, and nPU / PCL(Copolymer) were carried out by following the protocol for synthesizing nPET as described in Example 2.

[0328] For the synthesis of nPE, PE was dissolved in a solvent mixture of HFIP and toluene at 1 :10 ratio by volume, and heated at 110 °C overnight for complete dissolution.

[0329] Similarly, PS was dissolved in dimethyl formamide and heated at 60 °C, followed by releasing in 2% sodium dodecyl sulphate (SDS) solution at the rate of 5 ml / hr. The nPS formed were centrifuged at 15000 g for 15 minutes and resuspended in deionized (DI) water.

[0330] The synthesis of nPEN is similar to the synthesis of nPET and nPBT but to dissolve PEN, along with 1 ,1 ,1 ,3,3,3-hexafluoro-2-propanol, dichloroacetic acid was used in the ratio of 1 :1 , followed by heating at 60 °C for 30 min.

[0331] The synthesis of nPET+PBT blend was carried out by following the protocol for synthesizing nPET as described in Example 2 except equal amounts of PBT and PET were taken for dissolution in 1 ,1 ,1,3,3,3-hexafluoro-2-propanol.

[0332] Synthesis of nPBT, nPEN, nPE, nPS, nPUPCL(Copolymer), nBlend(PBT+PET), and nPA, encapsulating different dyes Nanoparticles encapsulating different dyes were synthesized by following the protocol described in Example 2. In addition to AO, the dyes used for encapsulation include methylene blue (MB), toluidine blue O (TBO) and acid fuchsin (AF) at 1 mg / mL concentration.

[0333] Results and discussion

[0334] The size distribution of these particles is presented in the DLS data in FIGS. 15 and 16.

[0335] Example 9. Stability of nanoparticles loaded with dye

[0336] The stability of the nanoparticles prepared in Examples 2 and 8 were investigated.

[0337] Stability of nanoparticles

[0338] The nanoparticles were incubated in 0.1 M phosphate buffer pH 8 for a time period of 30, 60 and 120 minutes at different temperatures of 30, 40 and 50 °C.

[0339] Results and discussion

[0340] The supernatant of the reaction in each case gave a fluorescence reading (FIG. 17), which was stable over the time of incubation at the respective temperature for each dye.

[0341] Example 10. Dye loading efficiency and dye release assay of nPET with different dyes

[0342] To test the applicability of a wider range of dyes in our synthesized nanoparticles of various polymers, the synthesized nanoparticles were tested with a variety of dyes to assess their effectiveness as a reporter fluorescent dye. As such, nPET loaded with various dyes, namely AO, MB, TBO and AF, were tested. The loading was performed as described in Example 8.

[0343] Loading efficiency

[0344] The loading efficiency was calculated by measuring the amount of dye retained in the particles as a percentage of the total dye added during synthesis.

[0345] Loading capacity

[0346] The loading capacity was calculated by measuring the mass of dye encapsulated in the partcles as a percentage of the total mass of the particles with the dye

[0347] Results and discussion

[0348] The loading efficiencies and the loading capacities of the various dyes in nPET are summarized in Table 3. The release of these dyes in the presence of enzymes was measured by subjecting the dye loaded-nPET to 2 different concentrations of WT PETase at 20 nM and 200 nM (FIG. 18). The difference in dye release in these conditions while having no enzyme control was measured by fluorescence. All the tested dyes exhibited elevated release when a higher concentration of the enzyme was used. The difference in dye release in these conditions was pronounced in the cases of AO, MB and TBO, while the difference with AF was not very high. All the dyes may be employed for application with varied efficiencies.

[0349] Table 3. Encapsulation efficiency and loading capacity of the various dyes loaded in nPET.

[0350] Comparative Example 1

[0351] Table 4. Comparison of liquid and solid screening platforms with recently reported screening methods.

[0352] Comparison of liquid and solid screening platforms with recently reported screening methods

[0353] Parameters Fe2+reagent-based BHET-OH based FP co hydrolysis Liquid phase screening Solid phase screening screening screening conjugated screening method using method using

[0354] (H. S. Zurier, J. M. (L. Shi et al. 2023) (K. Liu et al. 2023) nanoparticles nanoparticles

[0355] Goddard 2023) (The present disclosure) (The present disclosure)

[0356] Substrate used PET microfibre BHET (Proxy PET microfibre PET nanoparticles (low PET substrate) embedded on well crystalline) nanoparticles plate (low crystalline)

[0357] Mechanism Fenton reaction Fenton reaction FDL co hydrolysis - Dye release upon Dye release upon C c conjugated conjugated (TPA-OH) Fluorescein detection degradation degradation

[0358] (fluorescent byproduct detection)

[0359] Detected Fluorescence of Fluorescence of TPA- Fluorescein Dye released - acridine Dye released - acridine species TPA-OH OH orange orange

[0360] Number of 4 3 4 2 1 steps pH and No No No Yes No temperature control

[0361] Comparative Example 2

[0362] Table 5. Comparison between conventional method HPLC and PET nanoparticles of the present disclosure.

[0363] Parameters Conventional method HPLC PET nanoparticles

[0364] Solid phase Liquid phase

[0365] Time for 10,000 mutants 666 days 7 days 42 days considering 1 person (5 agar plates per screening cycle) (5 plates per screening cycle) working full time on one (100 samples per week) equipment

[0366] Time of analysis per ~ 7 day per screening 24 hours per screening 2 days per screening screening (For production, purification, and characterization of 50 mutants)

[0367] Number of steps involved 5 1 2

[0368] Involves cell lysis Yes No No pH and temp control for Yes No Yes assay

[0369] Crystallinity of the Any substrate can be used 7.5% 7.5% substrate

[0370] Remarks Not suitable for screening Advantages Advantages

[0371] • Simple, one step method • Can be used for second

[0372] • Can be used for environmental round screening after agar isolates plates

[0373] Disadvantages • pH and temperature of the

[0374] • pH and temperature of the assay can be altered assay cannot be altered

[0375] Steps involved in the assay 1. Production and harvest 1. Transformation of mutant library 1. Transformation and obtaining

[0376] 2. Purification of PETase on agar plate and overnight colonies on plate overnight.

[0377] 3. PETase - substrate incubation on nPET containing 2. Inoculation from agar plate to reaction agar plate to obtain colonies the 96-well plate containing nPET.

[0378] 4. HPLC analysis of following day which can be readily 3. Incubate for 1 hour and obtain product released imaged. fluorescence reading

[0379] Conclusion

[0380] Temperature and pH of the assay

[0381] PET degrading enzymes may be functional at different temperatures ranging from 30 °C to 70 ° C and at pH ranging from 7 to 9. In the solid phase method which uses the agar plate, it may not be possible to incubate the plate at over 37 °C and the pH was near neutral at 7.

[0382] Thus, solid phase (agar plate) based method may be used between 30 °C and 37 °C and pH 7.

[0383] Liquid phase method, however, may be carried out at higher temperatures as the final incubation step may be carried out at required temperatures and there is a certain level of flexibility with the pH as well.

Claims

Claims1. A polymeric nanoparticle comprising: a polymeric material that is susceptible to enzymatic degradation; and a dye encapsulated within the polymeric material.

2. The polymeric nanoparticle according to Claim 1 , wherein at least a portion of the dye is released from the nanoparticle in an environment containing an enzyme suitable for degrading the polymeric material.

3. The polymeric nanoparticle according to Claim 1 or Claim 2, wherein the polymeric material is selected from the group consisting of a polypropylene (PP), a polybutylene adipate terephthalate (PBAT), a polyamide (PA), and more particularly, a polyethylene terephthalate (PET), a polybutylene terephthalate (PBT), a polyethylene naphthalate (PEN), a polyethyelene (PE), a polyurethane (PU) and a polystyrene (PS), copolymers thereof and blends thereof.

4. The polymeric nanoparticle according to Claim 3, wherein the polymeric material is selected from the group consisting of a polyamide (PA), a polystyrene (PS), a polyester- polyether-polyurethane-copolymer (PU / PCL copolymer); and a polyethylene terepththalate and polybutylene terephthalate blend (PET-PBT blend), and, more particularly, a polystyrene (PS), a polyethylene terephthalate (PET), a polybutylene terephthalate (PBT), a polyethylene naphthalate (PEN), and a polyethyelene (PE).

5. The polymeric nanoparticle according to Claim 4, wherein the polymeric material is a PET, a PBT, a PEN, a copolymer thereof, and a blend thereof, optionally wherein the polymeric material is a PET.

6. The polymeric nanoparticle according to any one of the preceding claims, wherein the dye is a fluorescent dye, optionally wherein the dye is selected from one or more of the group consisting of methylene blue, toluidine blue O, acid fuchsin, alcian blue, sulforhodamine, and more particularly rhodamine B, and acridine orange.

7. The polymeric nanoparticle according to Claim 6, wherein the dye is selected from one more of the group consisting of methylene blue, toluidine blue O, acid fuchsin, and more particularly, acridine orange.

8. The polymeric nanoparticle according to any one of the preceding claims, wherein the dye is present in an amount of from 0.0001 to 5wt%, such as from 0.01 to 3wt%, such as from 0.1 to 2 wt%.

9. The polymeric nanoparticle according to any one of the preceding claims, wherein the nanoparticle has a crystallinity value (Xc) of from 1 to less than 40%, such as from 5 to 8%, such as about 7.5%.

10. The polymeric nanoparticle according to any one of the preceding claims, wherein the nanoparticle has a size of from 10 to less than 800 nm, such as from 30 to 600 nm, such as from 100 to 200 nm.11 . The polymeric nanoparticle according to any one of the preceding claims, wherein:(a) the polymeric material is a PET and the dye is acridine orange;(b) the polymeric material is a PBT and the dye is acridine orange;(c) the polymeric material is a PEN and the dye is acridine orange;(d) the polymeric material is a PS and the dye is acridine orange;(e) the polymeric material is a PE and the dye is acridine orange;(f) the polymeric material is a PA and the dye is acridine orange;(g) the polymeric material is a PU / PCL blend and the dye is acridine orange; or(h) the polymeric material is a PET-PBT blend and the dye is acridine orange,(i) the polymeric material is a PET and the dye is methylene blue;(j) the polymeric material is a PBT and the dye is methylene blue;(k) the polymeric material is a PEN and the dye is methylene blue;(l) the polymeric material is a PS and the dye is methylene blue;(m) the polymeric material is a PE and the dye is methylene blue;(n) the polymeric material is a PA and the dye is methylene blue;(o) the polymeric material is a PU / PCL blend and the dye is methylene blue;(p) the polymeric material is a PET-PBT blend and the dye is methylene blue;(q) the polymeric material is a PET and the dye is toluidine blue O;(r) the polymeric material is a PBT and the dye is toluidine blue O;(s) the polymeric material is a PEN and the dye is toluidine blue O;(t) the polymeric material is a PS and the dye is toluidine blue O;(u) the polymeric material is a PE and the dye is toluidine blue O;(v) the polymeric material is a PA and the dye is toluidine blue O;(w) the polymeric material is a PU / PCL blend and the dye is toluidine blue O;(x) the polymeric material is a PET-PBT blend and the dye is toluidine blue O;(y) the polymeric material is a PET and the dye is toluidine blue O;(z) the polymeric material is a PBT and the dye is toluidine blue O;(s) the polymeric material is a PEN and the dye is toluidine blue O;(t) the polymeric material is a PS and the dye is toluidine blue O;(u) the polymeric material is a PE and the dye is toluidine blue O;(v) the polymeric material is a PA and the dye is toluidine blue O;(w) the polymeric material is a PU / PCL blend and the dye is toluidine blue O;(x) the polymeric material is a PET-PBT blend and the dye is toluidine blue O; optionally wherein the polymeric material is selected from the list:(i) the polymeric material is a PET and the dye is acridine orange;(ii) the polymeric material is a PBT and the dye is acridine orange; or(iii) the polymeric material is a PEN and the dye is acridine orange.

12. The polymeric nanoparticle according to any one of the preceding claims, wherein the polymeric material is a PET and the dye is acridine orange.

13. A plate suitable for culturing a microbial population, the plate comprising: a solid medium suitable for culturing a microbial population; and a plurality of polymeric nanoparticles according to any one of Claims 1 to 12 homogeneously distributed throughout and / or on a surface layer of the solid medium, optionally wherein the plurality of polymeric nanoparticles are distributed on the surface layer of the solid medium.

14. The plate according to Claim 13, wherein the solid medium is agar, optionally wherein the agar is TB (teriffic broth) agar or, more particularly, LB agar.

15. A microfluidic device suitable for high throughput identification of one or more of an isolated enzyme, a cell lysate, and a microbe suitable for degrading a polymeric material, the microfluidic device comprising a plurality of polymeric nanoparticles as described in any one of Claims 1 to 12.

16. A method of determining whether one or more of an isolated enzyme, a cell lysate, and a microbe are suitable for degrading a polymeric material, the method comprising the steps of:(aa) providing a plurality of polymeric nanoparticles according to Claims 1 to 12; and(ab) contacting the plurality of polymeric nanoparticles with the one or more of the isolated enzyme, the cell lysate, or the microbe in a suitable medium for a period of time and measuring the release of the dye over the period of time to determine whether the enzyme, lysate or microbe are suitable for degrading the polymeric material in the polymeric nanoparticles.

17. The method according to Claim 16, wherein the method further comprises providing a control comprising a plurality of polymeric nanoparticles according to Claims 1 to 12 and a medium so as to provide a background reading over the period of time.

18. The method according to Claim 16 or Claim 17, wherein the dye release is measured using one or both of fluorescence and absorbance (e.g. fluorescence).

19. A method of determining whether one or more of an isolated enzyme, a cell lysate, and a microbe are suitable for degrading a polymeric material, the method comprising the steps of:(i) providing a microfluidic device according to Claim 15; and(ii) contacting the plurality of polymeric nanoparticles in the microfluidic device with one or more of the isolated enzyme, the cell lysate or the microbe in a suitable medium for a period of time and measuring the release of the dye over the period of time to determine whether the enzyme or bacteria are suitable for degrading the polymeric material in the polymeric nanoparticles.

20. A method of determining whether one or more of an isolated enzyme, a cell lysate, and a microbe are suitable for degrading a polymeric material, the method comprising the steps of:(aa) providing a plate suitable for culturing a bacterial population according to Claim 13 or Claim 14; and(ab) contacting the plate with a mixture comprising the isolated enzyme, the cell lysate or the bacteria and a suitable medium for a period of time and measuring the release of the dye over the period of time to determine whether the enzyme or bacteria are suitable for degrading the polymeric material in the polymeric nanoparticles.21 . The method according to Claim 20, wherein the method further comprises providing a control comprising a plate according to Claim 13 or Claim 14 contacted by a suitable medium identical to that used in Claim 20 so as to provide a background reading over the period of time.

22. The method according to Claim 20 or Claim 21 , wherein the dye release is measured using one or both of absorbance or fluorescence (e.g. fluorescence).

23. A method of making a plurality of polymeric nanoparticles according to any one of Claims 1 to 12, the method comprising the steps of:(ai) providing a first mixture comprising a polymeric material that is susceptible to enzymatic degradation, a dye and a first solvent, and second mixture comprising a surfactant and an anti-solvent for the polymeric material; and(aii) adding the first mixture to the second mixture in a dropwise fashion to provide the plurality of polymeric nanoparticles, wherein the dye is soluble in the first solvent and the anti-solvent for the polymeric material.

24. The method according to Claim 23, wherein the first solvent is selected from one or more of the group consisting of toluene, dimethyl formamide, xylene, dichloroacetic acid, and, more particularly chloroform, and hexafluoropropan-2-ol, optionally wherein the first solvent is hexafluoropropan-2-ol.

25. The method according to Claim 23 or Claim 24, wherein one or more of the following apply:(bi) the anti-solvent is water (e.g. ultra-pure water);(bii) the surfactant is sodium dodecyl sulfate (SDS) or cetyltrimethylammonium bromide (CTAB), (e g. SDS);(biii) the surfactant is present in an amount of from 1 to 5 wt% relative to the weight of antisolvent, such as from 1.5 to 3 wt%, such as about 2 wt%.

26. The method according to any one of Claims 23 to 25, wherein the polymeric material is selected from the group consisting of a polypropylene (PP), a polybutylene adipate terephthalate (PBAT), a polyamide (PA), and more particularly, a polyethylene terephthalate (PET), a polybutylene terephthalate (PBT), a polyethylene naphthalate (PEN), a polyethyelene (PE), a polyurethane (PU) and a polystyrene (PS), copolymers thereof and blends thereof, optionally wherein the polymeric material is selected from the group consisting of a polyamide (PA), a polystyrene (PS), a polyester-polyether-polyurethane-copolymer (PU / PCL copolymer); and a polyethylene terepththalate and polybutylene terephthalate blend (PET- PBT blend), and, more particularly, a polystyrene (PS), a polyethylene terephthalate (PET), a polybutylene terephthalate (PBT), a polyethylene naphthalate (PEN), and a polyethyelene (PE),yet more optionally wherein the the polymeric material is a PET, a PBT, a PEN, a copolymer thereof, and a blend thereof, optionally wherein the polymeric material is a PET.

27. The method according to any one of Claims 23 to 26, wherein the dye is a fluorescent dye, optionally wherein the dye is selected from one or more of the group consisting of methylene blue, toluidine blue O, acid fuchsin, alcian blue, sulforhodamine, and more particularly rhodamine B, and acridine orange.

28. The method according to any one of Claims 23 to 27, wherein the dye is present in the first mixture in a concentration of from 0.001 to 10 mg / mL, such as from 0.005 to 1 mg / mL, such as from 0.01 to 0.05 mg / mL. such as about 0.299 mg / mL29. The method according to any one of Claims 23 to 28, wherein:(a) the polymeric material is a PET and the dye is acridine orange;(b) the polymeric material is a PBT and the dye is acridine orange;(c) the polymeric material is a PEN and the dye is acridine orange;(d) the polymeric material is a PS and the dye is acridine orange;(e) the polymeric material is a PE and the dye is acridine orange;(f) the polymeric material is a PA and the dye is acridine orange;(g) the polymeric material is a PU / PCL blend and the dye is acridine orange; or(h) the polymeric material is a PET-PBT blend and the dye is acridine orange,(i) the polymeric material is a PET and the dye is methylene blue;(j) the polymeric material is a PBT and the dye is methylene blue;(k) the polymeric material is a PEN and the dye is methylene blue;(l) the polymeric material is a PS and the dye is methylene blue;(m) the polymeric material is a PE and the dye is methylene blue;(n) the polymeric material is a PA and the dye is methylene blue;(o) the polymeric material is a PU / PCL blend and the dye is methylene blue;(p) the polymeric material is a PET-PBT blend and the dye is methylene blue;(q) the polymeric material is a PET and the dye is toluidine blue O;(r) the polymeric material is a PBT and the dye is toluidine blue O;(s) the polymeric material is a PEN and the dye is toluidine blue O;(t) the polymeric material is a PS and the dye is toluidine blue O;(u) the polymeric material is a PE and the dye is toluidine blue O;(v) the polymeric material is a PA and the dye is toluidine blue O;(w) the polymeric material is a PU / PCL blend and the dye is toluidine blue O;(x) the polymeric material is a PET-PBT blend and the dye is toluidine blue O;(y) the polymeric material is a PET and the dye is toluidine blue O;(z) the polymeric material is a PBT and the dye is toluidine blue O;(s) the polymeric material is a PEN and the dye is toluidine blue O;(t) the polymeric material is a PS and the dye is toluidine blue O;(u) the polymeric material is a PE and the dye is toluidine blue O;(v) the polymeric material is a PA and the dye is toluidine blue O;(w) the polymeric material is a PU / PCL blend and the dye is toluidine blue O;(x) the polymeric material is a PET-PBT blend and the dye is toluidine blue O; optionally wherein the polymeric material is selected from the list:(i) the polymeric material is a PET and the dye is acridine orange;(ii) the polymeric material is a PBT and the dye is acridine orange; or(iii) the polymeric material is a PEN and the dye is acridine orange.

30. The method according to Claim 29, wherein the polymeric material is a PET and the dye is acridine orange.

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