Microfluidic enzyme assays

Abstract

The invention concerns an improved microassay device comprising a plurality of pillars made from, or coated with, a copolymer comprising, as copolymerized units, at least one polythiol monomer ('thiol monomer') and at least one polyalkene / alkyne monomer ('ene' monomer'), wherein the ratio of thiol to alkene / alkyne functional groups in said copolymer is from about 100:100 to 120:100, preferably about 105:100 to 120:100, and more preferably is about 110:100; its method of fabrication; use of the device for assaying enzyme reactions, specifically for use with metabolic enzymes such as Cytochrome P450 enzymes or, indeed, any other enzyme(s) but particularly including those susceptible to inhibition by uncrosslinked thiol monomers; and a kit of parts including said device.

Description

[0001] Microfluidic Enzyme Assays

[0002] Field of the Invention

[0003] The invention concerns an improved microassay device comprising a plurality of pillars made from, or coated with, a copolymer comprising, as copolymerized units, at least one polythiol monomer (‘thiol monomer’) and at least one polyalkene / alkyne monomer (‘ene’ or ‘yne’ monomer, respectively), wherein the ratio of thiol to alkene / alkyne functional groups in said copolymer is from about 100:100 to 120:100, preferably about 105:100 to 120:100, and more preferably is about 110:100; its method of fabrication; use of the device for assaying enzyme reactions, specifically for use with metabolic enzymes such as Cytochrome P450 enzymes or, indeed, any other enzyme(s) but particularly including those enzymes susceptible to inhibition by uncrosslinked thiol monomers; and a kit of parts including said device.

[0004] Background of the Invention

[0005] Adverse reactions to novel therapeutics tend to be characterised by inhibition of metabolic enzymes, such as for example the cytochrome P450 enzymes, and this inhibition can be reversible (competitive or non-competitive) or irreversible. Irreversible inhibition is particularly damaging to the individual as it results in sustained inactivation of the enzyme and even auto-immune reactions. It is therefore a necessary step in drug development to test novel therapeutics for any adverse metabolic effects with a view to eliminating those drugs that inhibit, particularly irreversibly, metabolic enzymes. To facilitate this process, it is highly desirable to use fast, reproducible and reliable in vitro assay techniques. Microfluidic (flow-through) assays (referred to herein as microassays) are particularly favoured because their scale enables reduction in the consumption of bioreagents and they can be automated to enable measurement of transient enzyme activities, both reproducibly and reliably. They are also favoured because they allow multiple interrogations simultaneously on a single sample source, thus enabling the effect of increasing / decreasing concentrations of novel therapeutics to be determined, time course of exposure to be assayed and association with other drugs to be assayed. Additionally, they also enable one to distinguish between reversible and irreversible enzyme inhibitor drugs, in a straightforward manner, as they allow for the test substance to be withdrawn from the feed and the enzyme activity to then be assayed again in the absence of the test substance to determine if enzyme activity has been restored, indicating whether any inhibition is reversible / irreversible. This cannot be done using static conditions where the enzymes are dissolved in a reaction mixture.

[0006] However, we have discovered that certain metabolic enzymes are susceptible to deactivation by the composition of the microassay device or microassay substances. Conventional microfluidic assays are performed using micropillars to which, typically, enzymes to be tested are tethered whilst a solution containing the novel therapeutic to be tested and a substrate and cofactor(s) for the enzyme flows thereby / thereover. The conversion of the substrate to a reaction product is then measured to determine the activity of the enzyme in the presence of the novel therapeutic and so provides a measure of the effect of the therapeutic on metabolic enzyme activity. Given the ability to assay the effects of multiple parameters simultaneously more than one novel therapeutic can be tested in this system and / or more than one enzyme can be tested simultaneously in this system at more than one concentration. Additionally, the system can be used to determine whether any enzyme inhibition is reversible / irreversible. Thus, the use of microassays in this way represent powerful investigative tools.

[0007] It is therefore imperative that the polymer substrate to which the microfluidic assay components are attached is inactive or does not interfere with the activity of the enzyme to be investigated.

[0008] When assaying metabolic enzymes using fluidic devices, biological lipid membranes (microsomes) and membrane-bound proteins contained therein, such as enzymes, in particular cytochrome P450 (CYP) enzymes and UDP glucuronosyl transferase (UGT) enzymes, are tethered to micropillars using biotinylated fusogenic liposomes and a thiol-ene polymer micropillar surface coated with an avidin, such as streptavidin. This is illustrated in Figure 1 . Our work has shown that the CYP enzymes are prone to rapid inactivation when immobilized on such a thiol-ene polymer based micropillar reactor. In fact, we have discovered it is the uncrosslinked thiol monomer(s) that result(s) in CYP inactivation. It is thought that the root cause of the CYP inactivation is the leaching of uncrosslinked thiol monomers from the bulk polymer.

[0009] Notably, not all enzymes are affected in this way, indeed others have shown (Kiiski et al., Eur J Pharm Sci 2021 , 158, 105677 (9 pp)) microfluidic assays for UGTs using pillars comprising, as copolymerised units, pentaerythritol tetrakis (3-mercaptopropionate) (a tetrathiol monomer) and triallyl-1 ,3,5- triazine-2,4,6(1 H,3H,5H)-trione (a triene), in the fabrication of the microreactors, did not result in any kind of UGT enzyme inhibition at a drug testing concentration of up to 1 mM.

[0010] Therefore, there is a need for a new microassay device that overcomes the above-described problems. Our new device avoids or slows down the inactivation of the membrane-bound CYP enzymes during use, via adjustment of the thiol-ene / yne bulk polymer composition (stoichiometric ratio of thiol and ‘ene / yne’ monomers). Additionally, we have made further improvements that increase the reliability of both CYP and UGT assays such as adapting the polymer post-processing protocol and increase the sensitivity by changing the micropillar structure (diamond instead of round shape); these adaptions include optimizing the master (mold) microfabrication process for high quality diamond micropillar arrays, to improve immobilization of membrane-bound enzymes.

[0011] Statements of Invention

[0012] According to a first aspect of the invention there is provided a microassay device comprising a plurality of pillars comprising, as copolymerized units, at least one polythiol monomer (‘thiol’ monomer) and at least one polyalkene / alkyne monomer (‘ene’ or ‘yne’ monomer), wherein the ratio of thiol to alkene / alkyne functional groups in said copolymer is from about 100:100 to 120:100, preferably about 105:100 to 120:100, and more preferably from about 107: 100 to about 115:100, and ideally about 110:100.

[0013] For the avoidance of doubt allyl = ene and refers to a double bond C=C, and the two terms are used herein interchangeably; yne, refers to a triple bond between the carbon atoms.

[0014] As used herein, the term polythiol monomer refers to a compound comprising at least two, preferably at least three, and more preferably at least four thiol (- SH) functional groups.

[0015] In exemplary embodiments, the thiol monomer is a dithiol compound comprising two thiol functional groups. Suitable dithiol compounds include but are not limited to: 1 ,6-hexanedithiol; 2,5-dimercaptomethyl-1 ,4-dithiane; 2,3- dimercapto-1 -propanol; Benzene-1 ,2-dithiol; 1 ,8-octanedithiol; Ethylene glycol bis(3-mercaptopropionate).

[0016] In other exemplary embodiments, the thiol monomer is a trithiol compound comprising three thiol functional groups. Suitable trithiol compounds include but are not limited to: Trimethylolpropane tris(3-mercaptopropionate); Trimethylolpropane tris(3-mercaptoacetate); 2,3-(dimercaptoethylthio)-1 - mercaptopropane.

[0017] In yet other exemplary embodiments, the thiol monomer is a tetrathiol compound comprising four thiol functional groups. Suitable tetrathiol compounds include but are not limited to: pentaerythritol tetrakis)3- mercaptopropionate) (PETMP); Pentaerythritol tetrakis(2-mercaptoacetate).

[0018] As used herein, the term polyalkene / alkyne monomer refers to a compound comprising at least two, and preferably at least three or four unsaturated groups selected from alkene / allyl (C=C) or alkyne groups (C C), or combinations thereof. Most preferably the unsaturated groups are alkene groups.

[0019] In exemplary embodiments, the ‘ene’ monomer is a diene compound comprising two alkene functional groups. Suitable diene compounds include, but are not limited to: Tri(ethylene glycol) divinyl ether; Trimethylolpropane diallyl ether.

[0020] In other exemplary embodiments, the ‘ene’ monomer is a triene compound comprising three alkene functional groups. Suitable triene compounds include, but are not limited to, 1 ,3,5-Triallyl-1 ,3,5-triazine-2,4,6(1 H,3H,5H)-trione (TATATO); Trimethylolpropane-tri(norbom-2-ene-5-carboxylate;

[0021] Pentaerythritol-tri(norborn-2-ene-5-carboxylate).

[0022] In yet other exemplary embodiments, the ‘ene’ monomer is a tetraene compound comprising four alkene functional groups. Suitable tetraene compounds include but are not limited to: Pentaerythritol-tetra(norborn-2-ene- 5-carboxylate); Di(trimethylolpropane)tetra-(norborn-2-ene-5-carboxylate).

[0023] In yet other exemplary embodiments, the ‘ene’ monomer (C=C) is substituted with a polyalkyne compound comprising two or more alkyne (C=C) functional groups. Suitable diyne compounds include but are not limited to: 1 ,6- heptadiyne and 1 ,7-octadiyne.

[0024] Preferably, the micropillars were fabricated by mixing tetrathiol (pentaerythritol tetrakis(3-mercaptopropionate), PETMP (Bruno Bock GmbH, Marschacht, Germany) and triene (1 ,3,5-Triallyl-1 ,3,5-triazine- 2, 4, 6(1 H,3H,5H)-trione, TATATO (Sigma-Aldrich, Burlington, MA) monomers in a molar ratio of 110:100 in terms of the thiol and ene functional groups, yielding about 10 mol-% excess of thiols.

[0025] As the skilled reader will readily appreciate, in a preferred embodiment the copolymer is formed via alkene / alkyne hydrothiolation reactions between the thiol functional groups of the thiol monomer(s) and the alkene / alkyne groups of the ‘ene’ / ’yne’ monomer(s). In preferred embodiments, the copolymer is formed by UV initiated free-radical addition.

[0026] We have determined that the use of polymers comprising a thiol: alkene / yne functional group ratio within this range improves enzyme assay sensitivity by approx, a 3-fold increase compared with at least a 25% thiol excess i.e., 125: 100 (as in Kiiski et al. Eur J Pharm Sci 2021 ).

[0027] In a preferred embodiment of the invention, said pillars are provided as an array and more preferably are diamond-shaped in horizontal cross-section, although conventional round shaped pillars may be used. Ideally, the micropillar array is of a conventional size and nature. This means it is typically 30-mm-long and 4 mm wide, it includes a sealed microchannel featuring an array of micropillars approx. 13,800 diamond micropillars as shown with exemplary dimensions in Figure 2. The nominal microchannel (and micropillar) height is 200 pm resulting in an approximate total internal volume of approx. 19 pL (which includes the micropillar-free triangular areas at the ends of the pillar array and the connecting channels to the inlet / outlet, but excluding the space occupied by the micropillars). Preferably, the pillar array is sealed with a planar cover layer.

[0028] We have determined that using this diamond shaped (in cross-section) pillars improves enzyme assay sensitivity by approx, a 1 .7-fold increase.

[0029] In yet a further preferred embodiment, said pillars are functionalized by coating the thiol-rich surfaces of same with an avidin, such as streptavidin. Ideally, this involves coating the micropillar arrays with biotin and then an avidin, such as streptavidin, by using biotin-(PEG)n-alkyne, where n = 4-22, (ideally 0.01 to 1 mM, preferably 0.1 mM in ethylene glycol, ideally with 0.01 % to 5%, preferably 1 %, Igracure TPO-L as a photoinitiator). As acknowledged in the afore, the PEG chain can vary at least between four PEG units (ca. 200 Da) to 22 PEG units (ca. 1 kDa). The avidin, ideally streptavidin, is used at about 0.5 - 5000 pg / mL, preferably 0.5 pg / mL streptavidin in PBS. As shown in Figure 1 , this results in the production of micropillars that can bind biotinylated lipid membranes or vesicles such as liposomes or microsomes (where microsomes are artificial vesicles derived from pieces of endoplasmic reticulum (ER) formed during cell homogenization) or supersomes (recombinantly expressed drug metabolizing enzyme reagents, consisting typically of microsomes prepared from insect cells infected with a virus engineered to express a human CYP isoform).

[0030] Reference herein to microsomes includes, without limitation, reference to human liver microsomes or human intestinal microsomes and supersomes based thereon.

[0031] In yet a further preferred embodiment of the invention, said microassay device comprises pillars having thiol-rich surfaces that are functionalized with an avidin and also having bound thereto biotinylated lipid membranes such as microsomes / supersomes, such as biotinylated commercial human liver or intestinal microsomes. Yet more ideally still, this functionalized and microsome-loaded microassay device is provided in a freeze-dried form. Advantageously, we have discovered that the use of the claimed pillar arrays enables us to lyophilize the immobilized biomaterial, inside the microfluidic device, and then re-solubilization of the immobilized biomaterial after a period of storing (at room temperature) - following the freezing - without any disadvantages results. Indeed, we have discovered that after freezedrying the microassay device of the invention, it can be stored at room temperature for long periods (beyond that of the conventional 2-week period for prior art devices). Indeed, conventional microassay devices have a shelf life of approx. 2 weeks (i.e., in the fridge in wet state). We have been able to successfully store our microassay device, after freeze drying, for at least nine months without a substantial loss of enzyme activity. It would therefore appear that our microassay device, as well as preventing / minimizing enzyme inactivation, also enables long term storage of microfluidic devices loaded with microsomes / supersomes. HLMs as such are commercially available and typically cryopreserved at - 80°C, which enables their long-term storage, for several years at best (according to the supplier). However, when immobilized onto polymer-based microfluidic devices, the HLMs can no longer be cryopreserved, because the crosslinked polymers (the support platforms) tend to lose their viscoelasticity in cryogenic temperatures. This may further compromise their mechanical durability and the bonding strength of the assembled biofunctionalized chips.

[0032] In this study, we evaluated the feasibility of freeze-drying techniques to lyophilization of HLMs readily immobilized inside microfluidic flow reactors, made from off-stoichiometric thiol-ene / yne (OSTE) as described herein.

[0033] Freeze-drying is an established approach for stabilizing proteins, peptides, and organic small molecules. However, extending freeze-drying approach to biological material immobilized on microfluidic devices is much less explored, with only a few previous works focusing on freeze-drying of antibodies, nucleic acids or soluble enzymes. In comparison, lyophilization of membrane-bound enzymes, such as the microsomal CYPs and UGTs, is generally more demanding, requiring careful optimization of the freeze-drying conditions. To our knowledge, there are no previous studies evaluating the feasibility of freeze-drying for preservation of HLMs, neither as such nor immobilized on solid supports.

[0034] From the microfabrication materials’ perspective, freeze-drying enables much shorter exposure times at extreme low temperatures, and thus less material stress, compared with cryopreservation, which by default ensures better stability of the polymer platform in long-term. Moreover, after freeze-drying, the biofunctionalized flow reactors can be stored at room temperature, thus reducing the energy consumption during storing as well as the shipping costs, and thereby overall increasing the technological readiness toward commercialization. In this study, we developed a method specifically for lyophilization of the HLM immobilized onto the OSTE microfluidic chips, to demonstrate the feasibility of freeze-drying for preserving the enzymatic activity of microsomal CYPs and UGTs over the long term. After initial optimizations of the freeze-drying protocol, the long-term stability of the freeze-dried HLM chips stored at room temperature was demonstrated over several months.

[0035] As mentioned above, the OSTE micropillar arrays were fabricated by mixing tetrathiol (pentaerythritol tetrakis(3-mercaptopropionate), PETMP; Bruno Bock GmbH, Marschacht, Germany ) and triene (1 ,3,5-Triallyl-1 ,3,5-triazine- 2, 4, 6(1 H,3H,5H)-trione, TATATO; Sigma-Aldrich, Burlington, MA) monomers in a molar ratio of 110:100 in terms of the thiol and ene functional groups, yielding 10 mol-% excess of thiols.

[0036] This mixture was then degassed, applied on the PDMS molds (the micropillar array and the cover layer) and cured under a UV flood exposure lamp for 5 min (Dymax 5000-EC; Dymax Light Curing Systems, Torrington, CT; nominal intensity 225 mW / cm2). The cured OSTE layers were detached from the mold, preheated on a hot plate at 100 °C for 2 h and laminated against each other. To finalize the bonding, the stack was exposed to UV for 2m in (Dymax 5000- EC).

[0037] According to a further aspect of the invention there is provided a method for the manufacture of a microassay device including a plurality of pillars for measuring the activity of at least one metabolic enzyme comprising: i) making or coating said plurality of pillars from or with a copolymer comprising, as copolymerized units, at least one polythiol monomer and at least one polyalkene / alkyne monomer, wherein the ratio of thiol to alkene / alkyne functional groups in said copolymer is from about 100: 100 to 120: 100, preferably about 105: 100 to 120: 100, and more preferably is about 110:100; and ii) coating the thiol-rich surfaces of the pillars of part i) with an avidin. According to a further aspect of the invention there is provided a method for the manufacture of a microassay device including a plurality of pillars for measuring the activity of at least one metabolic enzyme comprising: i) making or coating said plurality of pillars from or with a copolymer comprising, as copolymerized units, at least one polythiol monomer and at least one polyalkene / alkyne monomer, wherein the ratio of thiol to alkene / alkyne functional groups in said copolymer is from about 100: 100 to 120: 100, preferably about 105: 100 to 120: 100, and more preferably is about 110:100; ii) coating the thiol-rich surfaces of the pillars of part i) with an avidin; and iii) loading the avidin coated pillars of part ii) with biotinylated lipid membranes containing the enzyme to be tested.

[0038] In a preferred method of the invention, part i) involves making or coating said pillars with a copolymer comprising, as copolymerized units, at least one tetrathiol compound and at least one triene compound. In a particularly preferred method of the invention, the tetrathiol compound is PETMP and the triene compound is TATATO.

[0039] In a preferred method of the invention, part ii) of the method involves coating the pillars with biotin-PEGn-alkyne (such as 0.1 mM biotin-PEG4-alkyne in ethylene glycol). Ideally, said biotin-PEGn-alkyne comprises a photoinitiatior such as Igracure TPO-L (ideally, within the range of 0.01 % to 5%, preferably 1 %), then the method involves exposing the biotinylated pillars to an avidin (ideally, 0.5 pg / mL streptavidin in PBS).

[0040] In a further preferred method of the invention, part i) of the method is followed by heating the device, ideally to 70-110°C for 1 -3h, preferably to 100°C for approx. 2h. This post-processing by heat was additionally added as part of the process to further remove uncrosslinked thiol monomers before bonding the pillars with the biotinylated lipid membranes. We have determined that heating the device to 100°C for approx. 2h improves enzyme activity by approx, a 9-fold increase.

[0041] In part iii) the biotinylated lipid membranes containing the enzyme to be tested are prepared by exposing microsomes or supersomes to biotin-containing fusogenic liposomes (b-FL), these b-FL are prepared from commercially available liposomes e.g., via Avanti Polar Lipids. Preferably, to prepare unilamellar vesicles for merging the biotin tag to the microsomes or supersomes and attaching them to the avidin coated pillars, the multilamellar liposome mixture is passed through a polycarbonate membrane (pore size 100 nm) 51 times (suggested supplier protocol) using a benchtop extruder (Avanti Polar Lipids). More preferably still, to prepare biotinylated lipid membranes such as microsomes or supersomes, equal volumes of the b-FL dispersion (2 mg / mL total lipid) and the lipid membrane stock, such as human liver microsomes (HLM, 20 mg / mL total protein) or human intestinal microsomes (HIM, 10 mg / mL total protein), are mixed and incubated at 37°C for 15 min to transfer the biotin tag to the microsomes via spontaneous fusion. These biotinylated lipid microsomes containing the enzyme to be tested are then incubated in the device and the device is stored in the fridge until used or freeze-dried for longer storage at room temperature. Alternatively, it is possible to use the ability of liposomes and lipids membranes to spontaneously fuse in a different way: by producing the b-FL as afore and then incubating the b-FL product with the avidin coated pillars and then exposing these now pillar tethered b-FLs to the lipid membranes, such as microsomes, containing the enzymes to be tested. This device can then be stored as afore: stored in the fridge until used or freeze-dried for longer storage at room temperature.

[0042] In an alternative aspect, or a yet a further preferred, method of the invention, particularly where diamond shaped pillars are to be used, the above method may be preceded by the manufacture of the microassay device, and this comprises: a) creating a photolithographic master cast, such as a SU-8 master cast, by spin coating a first layer of photoresist on an untreated wafer, preferably a silicon wafer, and then soft baking, preferably at about 65°C for about 10 min, then at about 95°C for about 40 min to yield ca. 100-pm-thick layer, b) repeating step a); and c) exposed the product of step b) to curing via UV light for about 30s.

[0043] Reference herein to UV light is ideally to UV collimated light.

[0044] In the preferred preceded method, step a) is repeated and comprises step b) thus building the coating up in two steps, whereby two nominally ca. 100 pm layers are created (instead of one-step spin coating of 200-pm-thick layer). In this method, the soft bake is performed separately for the first layer before spin coating of the next layer, and for the second layer the soft bake is performed before UV lithographic patterning (of both layers simultaneously). As a result, the apparent layer thickness uniformity is improved from 6.7% RSD to 0.9% RSD (n=12 data points around the master wafer, as illustrated in Figures 3b and c).

[0045] In an alternative preferred preceded method, step a) comprises building a single 200-pm-thick layer which is then exposed to a separate edge bead removal (EBR) step by spraying acetone to the edge of the silicon substrate (UD-3b dispenser, Laurell Technologies; 800 rpm / 15 s) after spin coating of the SU-8 layer.

[0046] The afore method creates the master cast (step i of Table 1 ), casting of the polydimethylsiloxane (PDMS) molds (step ii of Table 1 ) is performed by mixing the base elastomer and a curing agent in a ratio of about 10: 1 (w / w), degassing in vacuum for about 30 min, and pouring the mixture onto the photolithographic master cast, such as a SU-8 master cast, prior then curing by heat (using standard protocol of about 70°C for at least 8 hours), as per the standard protocol. In yet a further aspect of the invention there is provided a method for measuring the activity of a metabolic enzyme comprising use of the device according to the invention.

[0047] In yet a further aspect of the invention there is provided a method for measuring the activity of a metabolic enzyme comprising: i) exposing a microassay device according to the invention and so comprising a plurality of pillars having thiol-rich surfaces, made from a copolymer comprising, as copolymerized units, at least one polythiol monomer and at least one polyalkene / alkyne monomer, wherein the ratio of thiol to alkene / alkyne functional groups in said copolymer is from about 100:100 to 120:100, preferably about 105: 100 to 120: 100, and more preferably is about 110:100, that are functionalized with avidin and also have bound thereto biotinylated lipid membranes or microsomes / supersomes containing the enzyme to be assayed, to a sample comprising a therapeutic to be tested and a substrate for the enzyme; ii) measuring the activity of said enzyme; iii) comparing the measured activity in part ii) with that of the enzyme when not exposed to said therapeutic and, where the activity is reduced, concluding the therapeutic has an inhibitory effect on the activity of said enzyme.

[0048] In yet a further aspect of the invention there is provided a method for measuring the activity of a metabolic enzyme comprising: i) exposing a microassay device according to the invention and so comprising a plurality of pillars having thiol-rich surfaces, made from a copolymer comprising, as copolymerized units, at least one polythiol monomer and at least one polyalkene / alkyne monomer, wherein the ratio of thiol to alkene / alkyne functional groups in said copolymer is from about 100:100 to 120:100, preferably about 105:100 to 120:100, and more preferably is about 110: 100, that are functionalized with avidin and, optionally, biotinylated lipid membranes, or biotinylated microsomes / supersomes containing the enzyme to be assayed, to produce a loaded microassay device; ii) exposing the loaded microassay device of part i) to a sample comprising a therapeutic to be tested and a substrate for the enzyme and, optionally, biotinylated lipid membranes or microsomes / supersomes containing the enzyme to be assayed; iii) measuring the activity of said enzyme either before or after and / or during step ii); iv) comparing the measured activity in part iii) of the enzyme when exposed to said therapeutic and when not exposed to said therapeutic and, where the activity is reduced when exposed to said therapeutic, concluding the therapeutic has an inhibitory effect on the activity of said enzyme.

[0049] In either method for measuring the activity of a metabolic enzyme the said pillars are round or diamond-shaped in cross-section but diamond-shaped in cross-section is preferred. Additionally, or alternatively, the microassay device is provided in a freeze-dried form.

[0050] Reference herein to metabolic enzymes includes reference to metabolic enzymes such as Cytochrome P450 enzymes, UGT enzymes or, indeed, any other enzyme(s) / membrane-bound enzyme(s) involved in a metabolic reaction for the metabolism of drugs or susceptible to inhibition by therapeutics, but particularly including those susceptible to deactivation by uncrosslinked thiol monomers.

[0051] The most severe drug-drug interactions arise from irreversible binding of the pharmaceutical or its metabolites to the critical CYP isoforms. However, it is still difficult to identify irreversible CYP inhibitors using static in vitro assays. Currently, reversible and irreversible CYP inhibitors can only be distinguished based on time- and resource-intensive dialysis assays. Instead, flow-through microreactors functionalized with human liver microsomes (HLM) or human intestinal microsomes (HIM) are an enabling technique for studying human drug metabolism and related drug-drug interactions in vitro.

[0052] For example, HLMs are heterogeneous vesicle-like artifacts re-formed from the endoplasmic reticulum of liver cells via centrifugation, and they readily embed membrane-bound drug metabolizing enzymes such as the CYPs and UGTs. Compared to static enzyme activity assays, flow-through assay setups exploiting immobilized HLMs offer significant advantages by enabling the establishment of time-dependent concentration gradients of the test substance over time (e.g., for determining half-maximal inhibitory concentrations, IC50) and even elimination of the test substance from the feed to easily distinguish between reversible and irreversible enzyme inhibition. Moreover, the HLM chips, same as other immobilized enzyme microreactors, are also well suitable for producing drug metabolites for subsequent flow-through assays, such as microfluidic organ-on-a-chip systems, to facilitate examination of also the metabolites’ effects in a streamlined manner.

[0053] Similarly, HIMs are heterogeneous vesicle-like artifacts re-formed from the endoplasmic reticulum of intestinal cells via centrifugation, and they too readily embed membrane-bound drug metabolizing enzymes such as the CYPs and UGTs.

[0054] According to a yet further aspect of the invention there is provided a kit of parts comprising: i) a microassay device comprising a plurality of pillars made from or coated with a copolymer comprising, as copolymerized units, at least one polythiol monomer and at least one polyalkene / alkyne monomer, wherein the ratio of thiol to alkene / alkyne functional groups in said copolymer is from about 100:100 to 120:100, preferably about 105:100 to about 120:100, and more preferably is about 110:100, and are functionalized by the attachment of avidin thereto; and ii) biotinylated lipid membranes containing the enzyme to be tested; and / or iii) microsomes / supersomes containing the enzyme to be assayed; and / or; iv) biotinylated microsomes / supersomes containing the enzyme to be assayed.

[0055] In a preferred kit of parts said ii), iii) or iv) are freeze dried. Additionally, or alternatively, said pillars are diamond-shaped in cross-section.

[0056] Preferred features of each aspect of the invention may be as described in connection with any of the other aspects.

[0057] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of the words, for example “comprising” and “comprises”, mean “including but not limited to” and do not exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0058] All references, including any patent or patent application, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. Further, no admission is made that any of the prior art constitutes part of the common general knowledge in the art.

[0059] Other features of the present invention will become apparent from the following examples. Generally speaking, the invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including the accompanying claims and drawings). Thus, features, integers, characteristics, compounds or chemical moieties described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein, unless incompatible therewith.

[0060] Moreover, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.

[0061] The Invention will now be described by way of example only with reference to the Examples below and to the following Figures wherein:

[0062] Figure 1. Upper: Shows a schematic illustration of the biotinylation of microsomes / supersomes with the help of biotinylated fusogenic liposomes (b- FL) to yield biotinylated microsomes / supersomes that can be immobilized on avidin-coated surfaces. Middle: Schematic illustration of the functionalization workflow to obtain avidin-coated micropillars. Lower: Schematic illustration of the microfluidic (flow-through) pillar array (device) and the setup used in enzyme activity determination of enzymes incorporated in microsomes / supersomes, such as cytochromes P450 (CYP) and UDP- glucuronosyl transferases (UGT).

[0063] Figure 2. Upper: Shows the layout of the micropillar reactor and the exemplary critical dimensions (all in mm). Middle: Shows the layout of each type of pillar array with dimensions (all in mm): round (initial), small diamonds, and wide diamonds. Lower: Shows exemplary scanning electron micrographs of each type of pillar array.

[0064] Figure 3. Shows visualizations of (a) the edge bead resulting from spincoating of thick SU-8 layers and (b) its impact on the layer thickness (non)uniformity. Impact of the application of (c) the two-step spincoating protocol and (d) the EBR step on the layer thickness uniformity. Comparison of the feature resolution of (e) the initial spincoating process resulting in rounding of the comers of the diamond-shaped micropillars and (f) the new process employing EBR step resulting in sharp corners. Figure 4. Shows impact of postprocessing conditions on the cumulative (1 h) CYP enzyme activity of immobilized HLMs in flow-through conditions at a flow rate of 5 pL / min. Two temperature (70°C and 100°C) and two heating times (2h vs. 4h) were compared. Temperatures >130°C resulted in mechanical damage to the bulk polymer. Times >4h did not improve the performance.

[0065] Figure 5. A Shows Impact of bulk polymer composition (three different) on the cumulative (1 h) CYP enzyme activity of immobilized HLMs in flow-through conditions at a flow rate of 5 pL / min. B Shows impact of bulk polymer composition (five different) on the cumulative (1 h) CYP enzyme activity of immobilized HLMs in flow-through conditions at a flow rate of 5 pL / min. In B, the cumulative activities obtained with each bulk composition are normalized to the activity of the thiol-allyl 110:100 composition that gives the highest activity in A. Thiol-allyl ratio refers to the stoichiometric ratio of thiol and ‘ene’ functional groups in the bulk polymer.

[0066] Figure 6. Shows impact of the pillar array design on the cumulative (1 h) CYP enzyme activity of immobilized HLMs in flow-through conditions at a flow rate of 5 pL / min.

[0067] Figure 7*. Shows the cumulative impact of the optimizations in Figures 4 - 6 on the cumulative (1 h) CYP enzyme activity of immobilized HLMs in flow- through conditions at a flow rate of 5 pL / min, when using biotin-PEG4-alkyne or biotin-PEGI kDa alkyne, for functionalization of the thiol-rich pillar arrays prior to avidin coating. Conventional refers to a copolymer composition ratio thiol : alkene 125:100, without post-processing by heating and using round shaped pillars. Optimized refers to a copolymer composition ratio thiol : alkene 110:100, post-processing heating 100°C / 2h, diamond pillars (wide). The difference between the two bars is only the biotin-PEGn-alkyne linker type.

[0068] *For the data in Figures 4 - 7 (as well as Figures 9-10) the metabolite amount of each collected fraction (in arbitrary units) is determined by substracting the background signal (feed solution) from the sample (effluent) signal and summing the so obtained metabolite amounts from six subsequent fractions (a 10 min) in each case (microfluidic device). When initial number of samples of a given set is four or higher, results deviating by >1.25 (standard deviations) from mean are excluded as outliers and not included in the final calculated mean + / - standard deviation.

[0069] Figure 8 A. Shows the transient marker metabolite concentrations in the effluents of the microfluidic devices, implemented using the optimized protocol (n=4 devices per each enzyme) and functionalized with biotinylated human liver microsomes (HLM), for selected CYP (upper panel) and UGT (lower panel) marker reactions in flow-through assays performed as described in Table 4, demonstrating good stability over time.

[0070] Figure 8 B. Shows the transient marker metabolite (resorufin) concentration in the effluent of the microfluidic devices implemented using the optimized protocol (n=3 devices) and functionalized with biotinylated human intestinal microsomes (HIM), for a nonselective CYP marker reaction (benzoxyresorufin dealkylation) in flow-through assays performed as described in Table 4, demonstrating good stability over time. Not all CYP isoforms are similarly expressed in the human intestine, thus a nonselective marker is used for assaying intestinal CYP activity, but CYP3A4 is known to be the most abundant intestinal isoform.

[0071] Figure 9 A. Shows the effect of storing time on the cumulative (1 h) CYP (upper) and on the cumulative (2h) UGT (lower) activity, when the microfluidic devices incorporating the immobilized human liver microsomes (HLM) are stored in the fridge in wetted stage and enzyme activities measured underflow- through conditions at a flow rate of 5 pL / min.

[0072] Figure 9 B. Shows the effect of storing time on the cumulative (1 h) CYP (benzoxyresorufin) activity, when the microfluidic devices incorporating the immobilized human intestinal microsomes (HIM) are stored in the fridge in wetted stage and enzyme activities measured under flow-through conditions at a flow rate of 2.5 pL / min.

[0073] Figure 10. Shows recovered CYP (upper) and UGT (lower) activities after freeze-drying and storing of the microfluidic devices incorporating the immobilized human liver microsomes (HIMs) in room temperature in dry state. The enzyme activities were measured under flow-through conditions at a flow rate of 5 pL / min, after resolubilization. The control activity represents the average enzyme activity of microfluidic devices, prefilled with sucrose (similar to freezedried devices before freeze-drying), but stored in fridge in wetted stage for 5-7 days.

[0074] Figure 11. Shows comparison of the relative CYP2C9 enzyme activity over time in static vs. microreactor experiments (with optimum bulk composition and post-processing conditions; at flow rate of 5 pL / min). The CYP2C9 enzyme activities in static conditions were determined in duplicate in a total volume of 100 pL with 200 pM Luciferin-H (model substrate), 2 mM NADPH (cosubstrate), and 0.2 mg / mL human liver microsomes.

[0075] Figure 12*. Shows the liver microsomal CYP2C9 (upper panel) and CYP2D6 (lower panel) activities in a flow-through inhibition assay (n=3 devices each), before, during and after introduction of the (known) enzyme-specific inhibitors: fluconazole and sulfaphenazole examples of reversible inhibitors of CYP2C9, and quinidine and paroxetine examples of respectively a reversible and an irreversible inhibitor of CYP2D6.

[0076] *Two examples of inhibition assays are shown with CYP enzymes that are known to have significant inter-individual variation (polymorphism) in their catalytic activity, which can result in a clinically significant variation in their therapeutic plasma concentration as such and especially together with enzyme inhibition by other concomitantly administered therapeutics:

[0077] Upper the first one illustrates inhibition of CYP2C9 (diclofenac 4-hydroxylation as the marker reaction) by sulfaphenazole and fluconazole, both of which are reversible inhibitors, but have quite different dissociation constants (sulfaphenazole dissociates from the enzyme much faster than fluconazole ). Lower illustrates inhibition of CYP2D6 (bufuralol 1 -hydroxylation as the marker reaction) by quinidine (reversible inhibitor) and paroxetine (irreversible inhibitor), which exemplifies how reversible and irreversible inhibitors can be distinguished by the microassay.

[0078] MATERIALS & METHODS

[0079] 2.1.1 Microreactor design

[0080] The conventional microreactor design comprises of a 30-mm-long and 4 mm wide, sealed microchannel featuring an array of ca. 14 400 round-shaped micropillars and ca. 360 semicircular micropillar structures on the channel walls (Figure 2). The nominal microchannel (and micropillar) height is 200 pm resulting in approximate total internal volume of ca. 25 pL, which includes the micropillar-free triangular areas at the ends of the pillar array and the connecting channels to the inlet / outlet, and excluding the space occupied by the micropillars. The pillar array is sealed with a planar cover layer.

[0081] Alternatively, new microreactor designs comprise of micropillar arrays of the same size and about same total micropillar amount (precisely 13 840 and 346 half-pillars on the walls) as the initial design, but diamond-shaped instead of round micropillars. The impact of micropillar shape on the CYP enzyme activity level is compared between the initial round shape (0 50 pm) and two different diamond shapes including ‘small diamonds’ with equal perimeter to that of the round pillars (Figure 2), and ‘wide diamonds’ with ca. 43% increase in perimeter (and thus, ca. 33% in the total surface-over-volume ratio) compared with that of the round pillars (Figure 2). The approximate internal volumes of the microchannels incorporating ‘small diamond’ and ‘wide diamond’ pillar arrays are ca. 22 pL and 19 pL, respectively.

[0082] 2.1.2 Microfabrication protocol

[0083] Microfabrication of the thiol-ene based micropillar reactors comprises of four steps protocol including: (i) UV-lithography based fabrication of the initial SU- 8 masters and (ii) casting of the PDMS molds (soft lithography) for the micropillar and cover layers, (iii) UV replica-molding and (iv) bonding of the thiol-ene based micropillar and cover layers by lamination. The comparison of the initial and re-designed microfabrication protocols is described in Table 1.

[0084] Table 1. Comparison of the critical steps of initial (starting point) and redesigned (new) microfabrication protocols.

[0085] With reference to Table 1 , for fabrication of the photolithographic master cast according to the invention, the photolithographic polymer is spin coated on an untreated silicon wafer. The spin coating involves either

[0086] Spincoating two layers each: at 500 rpm / 40s, then 2500 rpm / 30s; 100 pm per layer thick, and baking at 65°C / 10 min, then 95°C / 40 min

[0087] OR Spincoating one layer: 500 rpm / 40s, then 1150 rpm / 30s +800 rpm / 15s; 200 pm layer thick (and undertaking edge bead removal with acetone) and baking 65°C / 25 min, then 95°C / 110 min.

[0088] Either method produces a ca. 200-pm-thick layer, which is then exposed through the plastic photomask under collimated UV light for 30 s (OAI LS 30 / 5, OAI Instrument; nominal intensity of 40 mW / cm2). After UV exposure, the master is post exposure baked (for example 95°C / 40min).

[0089] The exposed microstructures are then developed in propylene glycol methyl ether acetate for 20 min with stirring, rinsed with isopropanol, and dried with nitrogen gas, followed by a conditioning bake (95 °C / 30 min).

[0090] When fabricating the masters of the diamond-shaped micropillar arrays, the initial (standard) spincoating protocol yields insufficient feature resolution, which necessitates customization of the early parts (spincoating and softbake) of the master fabrication process, whereas the later parts (UV exposure, post exposure bake and conditioning bake) follow the standard procedure. The impact of the spincoating protocol on the quality of the diamond-shaped micropillar arrays is described in section 2.2.1.

[0091] Casting of the PDMS molds (step ii) is performed by mixing the base elastomer and the curing agent in an approximate ratio of 10:1 (w / w), degassing in vacuum for about 30 min, and pouring the mixture onto the SU-8 master(s) prior to curing according to standard protocol (e.g., 70°C, overnight). No changes applied to the standard protocol.

[0092] UV replica molding of the thiol-ene polymer layers (step iii) is prepared by mixing the PETMP (tetrathiol) and TATATO (triene) monomers in the given molar ratio (with respect to free thiol and ‘ene’ functional groups) and the monomer mixture is poured onto the PDMS mold, degassed in vacuum, and cured under UV for 5 min (Dymax 5000-EC Series UV flood exposure lamp, Dymax Corporation; nominal power 225 mW cm-2). In the initial protocol, the molar ratio of thiol over ‘ene’ functional groups was 125:100. In the new protocol, the molar ratio is optimized to obtain thiol-rich bulk composition with minimal leaching of the tetrathiol monomer (see Section 2.2.3). A postprocessing by heat was additionally adapted as part of the process to further remove uncrosslinked tetrathiol monomers before bonding. Both micropillar and cover layers (with inlet / outlet holes) are prepared in the same manner. The impacts of monomer ratio (bulk composition) and post-processing conditions on the CYP activity and stability are elaborated in section 2.2.2.

[0093] The bonding (step iv) is performed according to the prior art protocol, or standard protocol, by preheating the micropillar and cover layers at 70-100 °C before lamination and UV exposure (Table 1 ).

[0094] 2.1.3 Microreactor functionalization with human liver microsomes

[0095] The microreactor functionalization comprises of two steps as previously described (Figure 1 ): (i) biotinylation of commercial human liver microsomes (Corning® Gentest 20-Donor Pool, BD Biosciences) with the help of biotinylated fusogenic liposomes, and (ii) sequential coating of the micropillar arrays with biotin-PEG4-alkyne, streptavidin, and biotinylated HLM. In the alternative protocol, biotin-PEG4-alkyne can be replaced by biotin-PEGI kDa- alkyne (Figure 7). In yet another alternative protocol, HLMs are replaced by human intestinal microsomes (Figure 8A & 8B).

[0096] Preparation of the biotinylated fusogenic liposomes preparation and fusion with lipid membranes (step i): The lipids used for preparation of biotin-containing fusogenic liposomes (b-FL) are from Avanti Polar Lipids and include 1 ,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP), 1 ,2-dioleoyl-sn-glycero-3-phophoethanolamine (DOPE), 1 ,2-dioleoyl-sn- glycero-3-phophoethanolamine-N-(Cap biotinyl) (sodium salt) (biotin-cap- DOPE), and 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (Lissamine Rhodamine B-DOPE). The b-FLs are prepared by mixing stock solutions (each in chloroform) of DOPE (10 mg / mL), DOTAP (10 mg / mL), biotin-cap-DOPE (10 mg / mL), and Lissamine Rhodamine B-DOPE (1 mg / mL) in a lipid mass ratio of 1 :1 :0.1 :0.05, respectively. After mixing, the bulk solvent is evaporated under a stream of nitrogen. To remove residual solvent, the lipid mixture is kept in vacuum for 2 h. Next, the dry lipid film is solvated in PBS to yield a total lipid concentration of 2 mg / mL and vortexed for 1 h at room temperature. To prepare unilamellar vesicles, the multilamellar liposome mixture is passed through a polycarbonate membrane (pore size 100 nm) 51 times using a benchtop extruder (Avanti Polar Lipids).

[0097] For HLM biotinylation, equal volumes of the b-FL dispersion (2 mg / mL total lipid) and the HLM stock solution (20 mg / mL total protein) are mixed and incubated at 37°C for 15 min to transfer the biotin tag to the HLMs via spontaneous fusion.

[0098] Alternatively, the biotinylated liposomes are exposed to streptavidin functionalized pillars where they are retained and so available for spontaneous fusion with microsomes / supersomes.

[0099] Alternatively, human intestinal microsomes (10 mg / mL total protein) can be / are used instead of HLMs (Figure 8A & 8B).

[0100] Functionalization of the micropillar reactors (step ii) is performed by coating the thiol-rich surfaces first with biotin-PEG4-alkyne (0.1 mM in ethylene glycol with 1 % Igracure TPO-L as the photoinitiator) using photopolymerization under UV-LED (A = 365 nm) for 1 min. Next, the microreactor is sequentially rinsed with methanol and deionized water (ca. 2 mL each), filled with Alexa Fluor® labeled Streptavidin (0.5 pg / mL in PBS) and incubated at room temperature for 40 min. Next, the micropillar channel is rinsed with ca. 2 mL of PBS and filled with the b-HLM suspension (10 mg / mL total protein) and incubated at 4°C overnight (or until use, but no longer than two weeks at maximum unless the device is freeze-dried).

[0101] Alternatively, biotin-PEGI kDa-alkyne is used instead of biotin-PEG4-alkyne.

[0102] 2.1.4 Determination of the CYP activity in flow-through conditions

[0103] Before use, the microreactors featuring immobilized biotinylated microsomes were equilibrated to room temperature and rinsed with ca. 2-5 mL of fresh PBS (to remove the excess of unimmobilized microsomes). Next, the microreactor is assembled with a syringe pump with the help of nanoport fluidic connectors and capillaries and placed on a PID-controlled heating block at 37°C. The CYP activities of the b-HLMs immobilized were assessed by infusing the feed solution into the microreactor at a constant flow rate (here, 5 pL / min) and collecting fractions of the effluent (a 50 pL) every 10 min with the help of CMA 470 refrigerated fraction collector (Harvard Apparatus). The feed solution contains a CYP2C9-spesific, prelum inescent model substrate (Luciferin-H, 200 pM) and the CYP cosubstrate, NADPH (1 mM), in PBS (pH 7.4). After the experiment, the fractions collected from the microreactor are mixed with 50 pL of the detection reagent (P450-Glo Assay, Promega), incubated for 20 min, and analysed offline for luminescence (arising from the Luciferin metabolite produced in CYP2C9 reaction) using Varioskan LUX well plate reader (Thermo Scientific). The enzyme activity (in arbitrary units) is determined by subtracting the background (feed solution) luminescence from the collected (effluent) fractions to quantitate the concentration of the reaction product (Luciferin metabolite) of the marker molecule (Luciferin-H). The enzyme kinetics (moles per time) can be calculated by multiplying the metabolite concentration with the flow rate in order to proportion the metabolite yield to the reaction (residence) time.

[0104] The different experimental conditions used to assay different metabolic enzymes besides CYP2C9 described here, are shown in Table 4.

[0105] 2.1.5 Determination of the UGT activity in flow-through conditions

[0106] To assess the UGT activity, the microreactors were prepared as described in section 2.1.4, but the feed solution contained the non-specific UGT model substrate (8-hydroxyquinoline, 50 pM) as well as the UGT co-substrate, UDPGA (1 mM), in 0.1 M Tris buffer with 5 mM MgCI2 (pH 7.5). The fractions (100 pL) were collected every 20 min and mixed with 10 pL of 4 M HCIO4 before analysis by fluorescence (ex. 245 nm, em. 475 nm) using Varioskan LUX well plate reader (Thermo Scientific).

[0107] 2.2 Results 2.2.1 Impact of master fabrication protocol on the quality of the micropillar structures

[0108] Microfabrication of thick (here, nominally 200 pm) and high aspect ratio microstructures (here, ca. 4:1 for the micropillar arrays) is nonstandard methodology. The spincoating and soft bake steps critically affect the layer thickness uniformity (i.e., the micropillar height uniformity), because of the so- called edge bead effect. Edge bead refers to a buildup of the coated photoresist (Sll-8) at the edge of the (silicon) substrate, as illustrated in Figure 3a. During the softbake, the edge bead transforms into an uneven Sll-8 layer thickness around the silicon wafer (Figure 3b), which further impairs the lithographic patterning resolution. In the new microfabrication protocol, the edge bead effect is minimized by spincoating the Sll-8 100 photoresist onto the silicon wafer in two steps, each nominally ca. 100 pm layer (instead of one- step spincoating of 200-pm-thick layer). In this case, the soft bake is performed separately for the first layer before spincoating of the next, and for the second layer before UV lithographic patterning (of both layers simultaneously). As a result, the apparent layer thickness uniformity is improved from 6.7% RSD to 0.9% RSD (n=12 data points around the master wafer, as illustrated in Figures 3b and c). Alternatively, a separate edge bead removal (EBR) step can be adapted as part of the master fabrication protocol by spraying acetone to the edge of the silicon substrate (UD-3b dispenser, Laurell Technologies; 800 rpm / 15 s) after spincoating of the Sll-8 layer. In our process, application of the EBR step to the one-step spincoating protocol improves the apparent layer thickness uniformity from the initial 6.7% RSD to 4.7% RSD (n=12 data points around the master wafer, as illustrated in Figures 3b and d. The impact of layer thickness uniformity on the feature resolution is additionally illustrated in Figures 3e (poorer resolution, initial process) and 3f (better resolution, here with the EBR process).

[0109] On the basis of these results, the master fabrication protocol was customized by adapting the two-step or two-layer spincoating protocol (Table 1 ) to ensure sufficiently high feature resolution in diamond-shaped micropillar arrays. 2.2.2 Impact of bulk polymer post-processing on CYP activity in flow- through experiments

[0110] Functionalization of the micropillar reactors with microsomes (e.g., human liver or intestine) is based on specific, multi-step protocol built on thiol-ene polymer. In the first step, a biotin-PEGn-alkyne is covalently bound to the free surface thiols of a thiol-rich (initially, +25% molar excess over ‘enes’) thiol-ene polymer. At the same time, however, the use of off-stoichiometric (thiol-rich) bulk composition results in substantial leaching of the uncrosslinked tetrathiol (PETMP) monomers into the microchannel, which are hypothesized to inactivate the immobilized CYP enzymes (residing on the cytosolic side of the membrane) in an irreversible manner.

[0111] Here, an additional polymer post-processing protocol is adapted as part of the microfabrication of thiol-rich thiol-ene microreactors (Table 1 ). The postprocessing is performed between the thiol-ene replica-molding and bonding steps for functionalizing the thiol-rich pillar surfaces, by heat-treating the cured thiol-ene replicas at different temperatures (70 or 100 °C) for 2 or 4h (last step of Table 1 ) prior to functionalization and enzyme immobilization.

[0112] The impact of polymer post-processing temperature (70 or 100°C) on the model CYP enzyme activity is illustrated in Figure 4, suggesting that the higher the post-processing temperature, the more effectively the uncrosslinked thiols are vaporized during post-processing prior to functionalization, which eventually yields greater initial enzyme activity level and slows down monomer-induced inactivation. This results in ca. 9-fold increase in the cumulative (1 h) CYP enzyme activity.

[0113] The impact of heating time (2h vs. 4h) on the model CYP enzyme activity is illustrated in Figure 4. This data suggests that extending the heat-treatment time beyond 2h is unnecessary and does not result in substantial improvement in the performance of the microreactor. On the basis of these results, it can be concluded that heat treatment of the replica-molded thiol-ene parts at 100°C for 2h is the overall best protocol for removing the uncrosslinked thiols from the bulk thiol-ene polymer considering both the CYP enzyme activity (improved by about 9-fold compared with initial protocol) and material stability (affecting bonding).

[0114] By applying this post-processing, at a flow rate typically used in the flow- through enzyme activity experiments (5 pL / min), the concentration of the leaching thiols in the microreactor effluent is between 1.5 and 2.5 pM, when using a bulk composition that has +25 mol-% excess of thiol over alkene functional groups.

[0115] 2.2.3 Impact of bulk polymer composition on the CYP activity in flow- through experiments

[0116] On the basis of the previous results, it can be concluded that even the optimum post-processing protocol (100°C / 2h) is not sufficient in removing all uncrosslinked thiol monomers from the bulk polymer, if the initial PETMP:TATATO ratio of 125:100 is used. This warrants for further optimization of the bulk composition to reduce the residual uncrosslinked thiol amount, while still maintaining the thiol-rich surface (necessary for further functionalization).

[0117] The impact of the bulk polymer composition on the model CYP enzyme activity is illustrated in Figures 5A & B. Figure 5A suggests that reducing the excess of thiol functional groups in the bulk from initial +25 mol-% to about +10 mol-% increases the cumulative (1 h) CYP activity about 3-fold. Figure 5B further shows that reducing the excess of thiol functional groups in the bulk from initial +25 mol-% to a range including +5 mol-% to +20 mol-% favorably increases the cumulative (1 h) CYP activity with no significant differences within this range. However, where the thiol excess is +5 mol-% yields lower initial enzyme activity, and thus PETMP:TATATO ratio of 110:100 is concluded optimum for replica molding (step iii) of thiol-ene micropillar reactors, because it provides best mechanical durability compared with 115:100 and 120:100 compositions (increasing excess of thiol functional groups decreases the crosslinking degree). However, 2h heat treatment at 100°C is necessary to remove the residual uncrosslinked thiols from the bulk polymer to be able to reach the aforementioned enzyme activity levels (Figure 5).

[0118] With the optimized bulk composition (PETMP:TATATO 110:100) and the optimized post-processing (100°C / 2h), the concentration of leached thiols in the microreactor effluent is negligible, even at very low flow rates. The relative enzyme activity (decay) is also identical to that of the inherent CYP decay also observed in static enzyme incubations (Figure 11 ) and features a somewhat linear decline over time with half-life of 70-100 min, instead of exponential decline with half-life of ca. 40 min as is the case of the initial protocol.

[0119] In Figure 11 , the relative enzyme activities in static and flow-through experiments are calculated as follows:

[0120] The average reaction rate as a function of time are first determined separately for static and flow-through setups using following equations:

[0121] _ k

[0122] ^t, static where vT, static is the average reaction rate (a.u. / min) for an incubation period t and h is the average luminescence intensity (of the sample) at timepoint t, for the static setup, and where V^IMER is the average reaction rate (a.u. / min) for an experiment period of t and h is the average luminescence intensity (of sample) at timepoint t (elapsed time), for the flow-through setup. The relative reaction rate is then obtained by dividing the average reaction rate at given timepoint with the highest reaction rate (t=20 min in the flow-through experiments).

[0123] The term "IMER" is used to refer to the flow-through microfluidic device.

[0124] 2.2.4 Impact of micropillar shape on the CYP activity in flow-through experiments Maximizing the surface-to-volume ratio of the micropillar arrays provides additional means to further increase the apparent ‘concentration’ of the immobilized microsomes, and thus, the initial CYP enzyme activity level. From the microfabrication perspective, the density of the micropillar array (ca. 120 pillars / mm2) cannot be much increased without compromising the replicamolding process (qualitative finding). However, the total surface area can be impacted by altering the micropillar shape.

[0125] The impact of the micropillar shape on the model CYP enzyme activity is illustrated in Figure 6, indicating that changing the micropillars from the initial round shape to the diamond shape, both featuring the same pillar perimeter (identical surface area), already increases the cumulative (1 h) CYP activity by about 40% (small diamonds, Figure 6). Such increase is likely associated with the different fluid dynamics in each type of pillar array, as the diamond shape effectively eliminates the ‘silent (no flow) zone’ behind the micropillars (in flow direction), which is characteristic for the round shape pillars.

[0126] Increasing the perimeter (surface area) of the (wide / elongated) diamond arrays by about 43% further increases the cumulative (1 h) CYP activity by about 70% (wide diamonds, Figure 6) compared with round pillars.

[0127] When using the optimum bulk polymer composition (thiol : alkene 110:100 (PETMP:TATATO), Figure 5) and post-processing conditions (100°C / 2h, Figure 4), and the wide diamond pillar shape (Figure 6), the combined increase in the cumulative (1 h) CYP activity compared with the initial protocol (ca. 15*103a.u.) is ca. 30-fold (Figure 7, ca. 450*103a.u.) for the preferred functionalization protocol using biotin-PEG4-alkyne, streptavidin, and biotinylated human liver microsomes (HLMs). When using the alternative protocol with biotin-PEGI kDa, streptavidin, and biotinylated HLMs, the increase compared with the initial / conventional protocol is ca. 20-fold.

[0128] Finally, the validity of the optimized protocol for measuring different liver microsomal CYP activities (using isoenzyme-selective marker substrates, Table 4) as well as the UGT activity (using a nonselective marker substrate, Table 4) was demonstrated under flow-through conditions, indicating good stability of the enzymatic marker activities over time (Figure 8). The absolute isoenzyme activity levels depend on the marker substrate’s clearance rate (varies between substrates and enzymes). The validity of the optimized protocol for measuring intestinal CYP activities (using a nonselective marker substrate, Table 4) was also demonstrated under flow-through conditions with immobilized, biotinylated human intestinal microsomes instead of biotinylated liver microsomes (Figure 8B).

[0129] Testing of the inhibitory effects of therapeutics using the microassay device

[0130] The feed solution contains a CYP marker substrate (isoenzyme-specific) and a cofactor (NADPH), and the possible inhibitor (the test pharmaceutical). To measure the inhibitory impacts of the test pharmaceutical, its concentration in the feed is gradually increased, during the course of the assay, and the enzyme activity measured based on the conversion of the marker substrate into its specific (marker) metabolite (Figure 12). To measure the inhibition mechanism, i.e. , whether the inhibitory impact was reversible or irreversible, the pharmaceutical is eventually excluded from the feed and the recovery of the inhibited enzyme activity is measured (based on the marker substrate conversion) (Figure 12). The enzymatic reaction time in the flow-through system is controlled by the flow rate (and the internal volume of the microfluidic device), which defines the residence time of the feed solution inside the microfluidic device. In the inhibition assays, the flow rate is kept constant over time and adjusted so that the enzyme kinetics of the marker reaction follows first order kinetics (i.e., marker metabolite concentration in the effluent is linearly proportional to the residence time).

[0131] As the invention facilitates the measurement of the enzyme activities under flow-through conditions, the system most importantly enables examination of not only the inhibitory concentrations of pharmaceuticals on the given enzyme activities (particularly the CYPs are prone to inhibition by pharmaceuticals) but also the inhibition mechanisms (whether reversible or irreversible) in a manner that is not feasible for current state-of-the-art static in vitro assays.

[0132] Furthermore, by excluding the marker substrate and the co-factor from the feed, which are necessary for monitoring the enzyme activities, but including the therapeutic / pharmaceutical, it is also possible to use the same microfluidic device for assessment of the nonspecific binding of the given therapeutic / pharmaceutical to the immobilized microsomes in order to determine so called free ‘unbound fraction’ (fu). Besides the inhibitory constants (IC50) and inhibition mechanism, the fu is another critical factor to correct for the in vitro metabolic clearance rate (CLint) of pharmaceuticals, in the absence of cofactor triggered metabolism. The current equivalent state-of- the-art static assay for determining the fu is a static assay, called rapid equilibrium dialysis.

[0133] 3. CONCLUSIONS

[0134] Overall, we report on adjustments to the microfabrication protocol, including customization of the master fabrication (for the production of diamond-shaped micropillars), optimization of the thiol-ene / yne ratio of the polymer composition and post-processing of the thiol-ene / yne replicas by heating (Figure 7). The model CYP2C9 activity can be increased from ca. 15,000 (initial protocol, Figure 4) to ca. 450,000 arbitrary units for wide diamond-based micropillar arrays fabricated using PETMP:TATATO 110:100 polymer and postprocessed by heat at 100°C for 2h (Figure 7).

[0135] Preparing the functionalized microfluidic devices for freeze-drying

[0136] After incubating the microsome-coated devices in fridge (2-6°C) for 5 or 7 days, the microfluidic devices are rinsed and filled with 10% (w / V) sucrose solution (a cryoprotection reagent) and stored overnight before loading into the freezedryer (FTS LyoStar II freeze dryer (SP Industries, Inc., Stone Ridge, NY).

[0137] The freeze-drying protocol The freeze-drying protocol comprises four steps (Table 3). The optimized conditions used in each step are given in Table 3. After freeze-drying, the microreactors are kept at room temperature, protected from light and moisture, until use.

[0138] Further adjustment and optimization of these conditions may have an impact on the overall performance (recovered enzyme activity), but the proof-of- concept data presented herein provides the evidence of the feasibility of freeze-drying to lyophilization of immobilized microsomes in order to extend the shelf life (storing stability) of the microfluidic devices compared with storing in wetted state in fridge.

[0139] Table 3. The freeze-drying protocol used in cryopreservation experiments enabling long-term storage.

[0140] After manufacture and functionalization, the microfluidic devices are stored in a ‘wet’ state in a fridge, i.e., after immobilization of the pre-biotinylated microsomes and until use (enzyme activity testing). In this manner, the CYP and UGT activities are at their highest about 7-9 days after storage, after which the activities start to decline and eventually fade within ca. 3 weeks (Figure 9). Similar trend is observed with both immobilized HLMs (Figure 9A) and immobilized HIMs (Figure 9B).

[0141] The impact of storing time on the enzyme activities of the HLM-functionalized devices were evaluated using Luciferin-H (CYP2C9-specific marker, section 2.1.4) and 8-hydroxyquinoline (nonselective UGT marker, Table 4), and of the HIM-functionalized devices using benzoxyresorufin (nonselective CYP marker, Table 4) as the marker substrates representative of the CYP and UGT activities.

[0142] By using the freeze-drying protocol presented above, the microfluidic devices can be stored at room temperature in dry state until use, and the storing time can be extended to several months. The impact of freeze-drying on the recovered enzyme activities was assessed with three similarly prepared batches of microfluidic devices, the first one of which had been stored for >12 months, the second for >9 months, and the third for approx. 6 months at this point. At best, recovered enzyme activities similar to control activity can be achieved even after 9 months of storing in room temperature after freeze- drying (Figure 10). However, we have recently obtained data (not shown) confirming these recovered enzyme activities can be achieved even after 16 months of storing in room temperature after freeze-drying. The impact of storing time on the recovered enzyme activities was also evaluated using Luciferin-H (CYP2C9) and 8-hydroquinoline (UGT) as marker substrates representative of the CYP and UGT activities.

[0143] Our data suggests that freeze-drying is not detrimental to immobilized microsomes nor their membrane bound enzymes and so is a feasible method for the long-term preservation of microsomal enzyme activities in microsomes on chips.

[0144] DETERMINATION OF THE STABILITY OF CYP AND UGT ACTIVITIES DURING USE

[0145] The human liver microsomes (HLMs) incorporate several different CYP (cytosolic) and UGT (luminal) enzyme isoforms. Here, we evaluated the activity of selected CYP isoforms, known to be most important to elimination (metabolism) of active pharmaceutical ingredients, as a function of time (assay duration) using enzyme specific marker substrates (Table 4). In addition, the liver microsomal UGT enzyme activity was evaluated using a nonselective UGT marker substrate, 8-hydroxyquinoline, known to metabolize via several UGT isoforms (Table 4). Furthermore, the intestinal CYP enzyme activity was evaluated using a nonselective CYP marker substrate, benzoxyresorufin (Table 4), known to metabolize via several CYP isoforms, most notably by CYPs 1A1 , 1 B1 and 3A4 / 5.

[0146] Before use, the microfluidic devices incorporating the immobilized microsomes were equilibrated to room temperature and rinsed with approx. 2-5 mL of fresh run buffer. Next, the microfluidic device was coupled to a pressure-induced pumping system with the help of nanoport fluidic connectors and capillaries and placed on a heating block or a heated chip housing at 37°C. The CYP and UGT activities of the immobilized microsomes were assessed by infusing a feed solution containing the enzyme-specific marker substrate and the cosubstrate in the run buffer, at a constant flow rate (Table 4). The enzyme activities were quantified by analyzing the effluent of the microfluidic devices for the metabolites produced from the marker enzyme substrates by the microsomal enzymes. The effluent was collected and appropriately fractionated using a CMA 470 refrigerated fraction collector (Harvard Apparatus), and the samples were prepared for analysis, as indicated in Table 4.

[0147] Using the assay protocols described herein, enzyme stability for a range of CYP / UGT enzymes was demonstrated (Figures 8 & 11 ).

[0148] Table 4. The experimental conditions used to determined the CYP and UGT enzyme activities (Figures 8-12).

[0149] Experiments undertaken using the new microassay device to determine enzyme inhibition activity both reversible and irreversible

[0150] 5 To assess the inhibitory effects of therapeutic drugs on CYP activities in the immobilized microsomes, the microassays were undertaken using the described protocol (Table 4). The transient CYP enzyme activities were assessed similar to enzyme stability during use, except for the fact that the therapeutic was introduced to the microassay in a step-wise increasing 10 concentration (0.01 to 100 pmol / L). Typically, three fractions were collected before introducing the therapeutic, and thereafter three fractions at each concentration level (Figure 12). The first fraction always represents the intermediate state, whereas the next two represent the equilibrium of the new condition. After collecting three fractions at the highest concentration, the test 15 substance was withdrawn from the feed solution to assess if the inhibited enzyme activity recovers (reversible inhibition) or persists (irreversible inhibition). In the exemplary data (Figure 12 upper), the validity of this protocol for monitoring the recovery phase is shown by using two known reversible inhibitors of CYP2C9 (diclofenac 4-hydroxylation as the marker reaction), 0 namely fluconazole and sulphaphenazole. In addition, the validity of the protocol for distinguishing irreversible (paroxetine) and reversible (quinidine) CYP2D6 inhibitors is shown using bufuralol 1 -hydroxylation as the marker reaction (Figure 12 lower). The activity of the marker reactions before, during, and after exposure to the test substance (inhibitor) was assessed as described in Table 4. The test substance concentration in the feed was adjusted by the pressure-induced pumping system.

Claims

CLAIMS1. A microassay device comprising a plurality of pillars made from or coated with a copolymer comprising, as copolymerized units, at least one polythiol monomer (‘thiol monomer’) and at least one polyalkene / alkyne monomer (‘ene / yne’ monomer’), wherein the ratio of thiol to alkene / alkyne functional groups in said copolymer is from about 100:100 to 120:100, preferably about 105:100 to 120:100, and more preferably is about 110:100.

2. The microassay device according to claim 1 wherein the thiol monomer is a dithiol, trithiol or a tetrathiol compound comprising two, three or four thiol functional groups.

3. The microassay device according to claim 1 wherein the polythiol compound is selected from the group comprising: 1 ,6-hexanedithiol;2.5-dimercaptomethyl-1 ,4-dithiane; 2,3-dimercapto-1 -propanol;Benzene-1 ,2-dithiol; 1 ,8-octanedithiol; Ethylene glycol bis(3- mercaptopropionate);Trimethylolpropane tris(3-mercaptopropionate); Trimethylolpropane tris(3-mercaptoacetate); 2,3-(dimercaptoethylthio)- 1 -mercaptopropane; pentaerythritol tetrakis)3-mercaptopropionate) (PETMP); and Pentaerythritol tetrakis(2-mercaptoacetate) .

4. The microassay device according to any one of claims 1 - 3 wherein the polyalkene / alkyne monomer comprises a compound comprising at least two, and preferably at least three or four alkene / allyl (C=C) or alkyne (C C) functional groups.

5. The microassay device according to claim 4 wherein the polyalkene or polyalkyne compound is selected from the group comprising: 1 ,3,5- Triallyl-1 ,3,5-triazine-2,4,6(1 H,3H,5H)-trione, TATATO. Tri(ethylene glycol) divinyl ether; Trimethylolpropane diallyl ether; 1 ,3,5-Triallyl-1 .3.5-triazine-2,4,6(1 H,3H,5H)-trione (TATATO); Trimethylolpropane- tri(norborn-2-ene-5-carboxylate; Pentaerythritol-tri(norbom-2-ene-5- carboxylate);Pentaerythritol-tetra(norborn-2-ene-5-carboxylate); andDi(trimethylolpropane)tetra-(norbom-2-ene-5-carboxylate); 1 ,6- heptadiyne; 1 ,7-octadiyne.

6. The microassay device according to any one of claims 1 - 5 wherein said pillars are provided as an array and are diamond shape in crosssection.

7. The microassay device according to claim 6 comprising approx. 14,000 diamond-shaped in cross-section micropillars.

8. The microassay device according to any one of claims 1 - 7 wherein said pillars are functionalized by coating the thiol-rich surfaces of same with an avidin.

9. The microassay device according to claim 8 wherein the pillars are coated with biotin-PEGn-alkyne and then an avidin.

10. The microassay device according to any one of claims 1 - 9 wherein the pillars are also coated with biotinylated lipid membranes containing the enzyme to be assayed.11 . The microassay according to claim 10 wherein the lipid membranes are microsomes or supersomes.

12. The microassay according to claim 10 or claim 11 wherein the lipid membranes are derived from liver or intestinal tissue.

13. The microassay device according to any one of claims 1 - 12 wherein the device has been freeze-dried.

14. A method for the manufacture of a microassay device including a plurality of pillars for measuring the activity of at least one metabolic enzyme comprising: i) making or coating said plurality of pillars from or with a copolymer comprising, as copolymerized units, at least one polythiol monomer and at least one polyalkene / alkyne monomer, wherein the ratio of thiol to alkene / alkyne functional groups in said copolymer is from about 100:100 to 120:100, preferably about 105:100 to about 120:100, and more preferably is about 110:100; and ii) coating the thiol-rich surfaces of the pillars of part i) with an avidin.

15. The method according to claim 14 wherein step i) involves making said pillars from or coating said pillars with the tetrathiol compound PETMP and the triene compound TATATO.

16. The method according to anyone of claims 14 - 15 wherein step ii) of the method also involves coating the pillars with a biotin-PEGn-alkyne.

17. The method according to claims 16 wherein said biotin-PEGn-alkyne also comprises a photoinitiatior, including, Igracure TPO-L.

18. The method according to anyone of claims 14 - 17 wherein step i) is followed by: heating the device to 100°C for approx. 2h before step ii) is performed.

19. The method according to any one of claims 14 - 18 wherein step ii) or iii) is followed by: iii) loading the avidin coated pillars with biotinylated lipid membranes or biotinylated lipid membranes containing the enzyme to be tested.

20. The method according to claim 19 wherein the biotinylated lipid membranes containing the enzyme to be tested are prepared by exposing said lipid membranes, including microsomes or supersomes, to biotincontaining fusogenic liposomes (b-FL).

21. The method according to any one of claims 14 - 20 wherein the biotinylated lipid membranes containing the enzyme to be tested are microsomes derived from liver or intestinal tissue or supersomes.

22. The method according to any one of claims 14 - 21 wherein the device has been freeze-dried.

23. The method according to any one of claims 14 - 22 wherein the method is preceded by the manufacture of the microassay device, and this comprises: a) creating a photolithographic master cast by spin coating a first photoresist layer on an untreated wafer, preferably a silicon wafer, and then soft baking, preferably at about 65°C for about 10 min, then at about 95°C for about 40 min to yield ca. 100-pm-thick layer, b) repeating step a); and c) exposed the product of step b) to curing via UV light for about 30s.

24. The method according to any one of claims 14 - 23 wherein the method is preceded by the manufacture of the microassay device, and this comprises:a) creating a photolithographic master cast by spin coating a layer on an untreated wafer, preferably a silicon wafer, and then soft baking, preferably at about 65°C for about 25 min, then at about 95°C for about 110 min to yield ca. 200-pm-thick layer, b) exposing said layer of part a) to a separate edge bead removal (EBR) step by spraying acetone to the edge of the silicon substrate; c) exposed the product of step b) to curing via UV light for about 30s.

25. A method for measuring the activity of a metabolic enzyme comprising: i) exposing a microassay device comprising a plurality of pillars having thiol-rich surfaces, made from a copolymer comprising, as copolymerized units, at least one polythiol monomer and at least one polyalkene / alkyne monomer, wherein the ratio of thiol to alkene / alkyne functional groups in said copolymer is from about 100: 100 to 120: 100, preferably about 105: 100 to 120: 100, and more preferably is about 110:100, that are functionalized with an avidin and also have bound thereto biotinylated lipid membranes, such as microsomes / supersomes, containing the enzyme to be assayed, to a sample comprising a therapeutic to be tested and a substrate for the enzyme; ii) measuring the activity of said enzyme during step i); iii) comparing the measured activity in part ii) with that of the enzyme when not exposed to said therapeutic and, where the activity is reduced, concluding the therapeutic has an inhibitory effect on the activity of said enzyme.

26. A method for measuring the activity of a metabolic enzyme comprising: i) exposing a microassay device comprising a plurality of pillars having thiol-rich surfaces, made from a copolymer comprising, as copolymerized units, at least one polythiol monomer and at least one polyalkene / alkyne monomer, wherein the ratio of thiol to alkene / alkyne functional groups in said copolymer is from about 100:100 to 120: 100, preferably about 105:100 to 120:100, and preferably is about 110:100, that are functionalized with an avidin and, optionally, biotinylated lipid membranes, or biotinylatedmicrosomes / supersomes containing the enzyme to be assayed, to produce a loaded microassay device; ii) exposing the loaded microassay device of part i) to a sample comprising a therapeutic to be tested and a substrate for the enzyme and, optionally, lipid membranrs, such as microsomes / supersomes, containing the enzyme to be assayed; iii) measuring the activity of said enzyme either before or after and / or during step ii); iv) comparing the measured activity in part iii) of the enzyme when exposed to said therapeutic and when not exposed to said therapeutic and, where the activity is reduced when exposed to said therapeutic, concluding the therapeutic has an inhibitory effect on the activity of said enzyme.

27. The method according o claim 25 or claim 26 wherein measuring the activity of said enzyme occurs before, during and after exposure to said therapeutic whereby the nature of any inhibition can be determined to be either reversible or irreversible.

28. The method according to any one of claims 25 - 27 wherein said sample comprising a therapeutic to be tested also includes an enzyme substrate co-factor.

29. The method according to any one of claims 25 - 28 wherein said pillars are diamond-shaped in cross-section.

30. The method according to any one of claims 25 - 29 wherein said device has been freeze-dried.

31. The method according to anyone of claims 25 - 30 wherein part i) or part ii) comprises exposing said enzyme to be tested to a sample comprising the therapeutic to be tested, in the absence of a substrate and / or its co-factor, to obtain a measure of nonspecific binding of said therapeutic to the lipid membranes in order to determine ‘unbound fraction’ (fu).

32. The method according to anyone of claims 25 - 31 wherein said metabolic enzyme is an enzyme that metabolizes a drug, including aCytochrome P450 enzyme, or a UGT enzyme or an enzyme that is susceptible to deactivation by uncrosslinked thiol monomers.

33. The method according to anyone of claims 25 - 32 wherein said microsomes / supersomes are human liver microsomes (HLM) and / or human intestinal microsomes (HIM),34. A kit of parts comprising: i) a microassay device comprising a plurality of pillars made from or coated with a copolymer comprising, as copolymerized units, at least one polythiol monomer and at least one polyalkene / alkyne monomer, wherein the ratio of thiol to alkene / alkyne functional groups in said copolymer is from about 100:100 to 120:100, preferably about 105:100 to 120:100, and more preferably is about 110:100, and are functionalized by the attachment of an avidin thereto; and ii) biotinylated lipid membranes containing the enzyme to be tested; and / or iii) microsomes / supersomes containing the enzyme to be tested; and / or; iv) biotinylated microsomes / supersomes containing the enzyme to be tested and / or.

Images