Microfluidic enzyme assays

Abstract

The invention concerns a novel microassay device comprising a plurality of pillars functionalised using antibody specific for the isolation of extracellular vesicles (EVs), typically but not exclusively from human blood or cell culture media, and including electrodes for measuring impedance; wherein said pillars are 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 has an excess of alkene / alkyne functional groups, optionally from about 100:150 to 100:105; a method for the fabrication of said device; use of the device for tissue-selective isolation of EVs, and / or quantifying the amount of said EVs, and / or assaying the enzyme activity of said EVs, such as that of Cytochrome P450 enzymes or UDP-glucuronosyl transferases or, indeed, any other enzyme(s) contained in the cells or EVs; and a kit of parts including said device.

Description

[0001] Microfluidic Enzyme Assays

[0002] Field of the Invention

[0003] The invention concerns a novel microassay device comprising a plurality of pillars functionalised using antibody specific for the isolation of extracellular vesicles (EVs), typically but not exclusively from human blood or cell culture media, and including electrodes for measuring impedance; wherein said pillars are 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 has an excess of alkene / alkyne functional groups, optionally from about 100:150 to 100:105; a method for the fabrication of said device; use of the device for cell type- or tissue-selective isolation of EVs, and / or quantifying the amount of said EVs, and / or assaying the enzyme activity of said EVs, such as that of Cytochrome P450 enzymes or UDP-glucuronosyl transferases or, indeed, any other enzyme(s) contained in the cells or EVs; and a kit of parts including said device.

[0004] Background of the Invention

[0005] Precision medicine is an approach to patient care that allows doctors to select treatments that are most likely to help patients based on personalised information of their disease progression or drug treatment outcomes as well as of the clearance (metabolism) rate of the drugs.

[0006] It has been estimated that the vast majority of drugs - more than 90% - only work in 30 - 50% of the prescribed population. With prescription drugs worth nearly €1 trillion sold annually across the world, this results in a major financial burden to healthcare systems, and it also increases health risks to the individuals (due to adverse drug effects) as well as ecotoxicological hazards posed by human excretions of pharmaceuticals into the environment via sewage systems. This has triggered the development of Precision Medicine to tailor treatment strategies for sub-population groups, in which patients receive individualised specific therapies that work best for them, and no money or time is wasted on ‘trial-and-error’ drug treatments.

[0007] A significant share of sub-optimal drug treatments is related to ‘wrong dosing’ following the currently prevailing ‘one-size-fits-all’ strategy. This problem centres around the inter-individual variation in the Cytochrome P450 (CYP) system, which is a super family of enzymes that catalyses the detoxification (metabolism) of the majority (>70%) of clinically used drugs. CYP enzymes are most abundant in the liver, but also expressed by many extrahepatic tissues. Thus, the majority of drug metabolism reactions also takes place in the liver. The important role of drug metabolism is to protect the body by converting lipophilic, active pharmaceutical drug ingredients into more hydrophilic forms (metabolites), to facilitate their excretion by the kidneys. Without CYP metabolism, the drugs would accumulate in the body, exceeding their therapeutic concentration and becoming toxic; but if CYP conversion is too fast, the drug is eliminated from the body before it is optimally effective. For example, the anticoagulant drug warfarin, widely prescribed to prevent and treat thromboembolism, causes bleeding (too slow metabolism) or results in thromboembolism (too fast metabolism) in, respectively, 16.5% and 25% of patients, due to the inter-individual variation in warfarin elimination kinetics via CYP2C9.

[0008] Thus, the main challenge in determining the optimal drug dose, is the interindividual variation of the drugs’ elimination kinetics via CYPs. This kind of variation can have a genetic origin; many of our critical CYP enzymes are polymorphic. For example, isoenzymes CYP2D6 and CYP2C9 are known to have genetic variants that may present as rapid or slow metabolisers. But a lot of variation is also due to external factors: (i) some drugs may induce the expression of certain CYP isoenzymes that are in charge of elimination of other drugs; (ii) many dietary agents and supplements (e.g., grapefruit juice) also influence CYP expression; (iii) additionally, age, sex, and possible disease state (e.g., inflammation) may alter the expression of functional CYP enzymes and thus, the drug elimination kinetics in vivo. As a result, each patient has a personalized CYP activity profile, which largely impacts one’s drug clearance capacity and determines whether a treatment is safe and efficient. Currently, CYP gene testing services are commercially available, and this has inspired pharmacogenomics-guided precision medicine strategies, with a current market of €12.8 Bn and estimated cost savings in, e.g., depression treatment of nearly US$4000 / patient per year. However, the clinical utility of CYP gene testing is significantly curtailed because these tests are blind to non-genetic variation, and the majority (60-80%) of the inter-individual variation arises from external factors (the phenotype). For example, age, sex, and possible disease state (e.g., inflammation) also influence individual CYP expression in a way that is not detectable by the CYP gene tests. Additionally, many dietary agents and supplements (e.g., grapefruit juice), lifestyle choices (e.g., smoking), as well as medication can alter both the amount and activity of the CYP system in an individual.

[0009] There is therefore a widespread need for commercially available in vitro diagnostics (IVD) for CYP phenotyping that accounts for the impacts of both genetic and external factors on the individual drug elimination speed.

[0010] The current norm for CYP phenotyping (i.e., to measure individual CYP enzyme activities) is to administer to a patient a probe drug (known to be selectively metabolised via the CYP isoform being monitored) and measure its plasma concentration over time, but this is exhausting to the patient and technically laborious, requiring blood sampling at several timepoints. Alternatively, it is possible to determine a patient’s CYP phenotype from a liver biopsy, but this is highly invasive (requires hospitalization, poor patient compliance, risk of complications), and therefore justified only in lifethreatening situations (e.g., cancer or liver diseases). Neither of these methods are suitable for routine diagnostic purposes in the clinic. Recently, others have demonstrated that liver extracellular vesicles (EVs) isolated from human blood samples and incorporating / containing functional CYP enzymes show a correlation with the inter-individual CYP expression and activity levels in the individual’s liver (1 ,2). These EVs (including exosomes, microvesicles, and apoptotic bodies) are released by all cell types into body fluids in both normal and pathological conditions. They carry cellular cargo (e.g., lipids, proteins, coding and non-coding RNAs, mitochondrial DNA), which is representative of the individual’s physiological condition and can thereby support diagnostic needs. This fact has triggered research into the discovery and use of EV-based biomarkers in clinical practice, but the possibility of CYP phenotyping based on liver EVs has only very recently been established. These early experiments were conducted using, e.g., magnetic bead-based immunocapturing protocols for the selective isolation of liver-secreted EVs from human plasma / serum samples, followed by offline characterization of their CYP enzyme expression and activity (phenotyping). These protocols, however, require multi-step sample preparation and characterization by several different techniques, and are therefore complex for routine diagnostic purposes.

[0011] To facilitate activity-based testing of the enzymes incorporated in the EVs, 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 they enable immobilization of vesicles, such as EVs, in the microfluidic assay device, while their scale enables a reduction in the consumption of the patient sample. They can also be automated to enable measurement of enzyme activities, both reproducibly and reliably, and equipped with integrated (thin-film metal) sensors.

[0012] Conventional microfluidic CYP activity assays are performed using micropillars to which, typically, human liver microsomes (HLMs) are tethered whilst a solution, containing the enzyme-specific substrate and cofactor(s) for the enzyme, flows thereby / thereover. The conversion of the substrate to a reaction product (metabolite) is then measured to determine the activity of the enzyme and so provide a measure of the metabolic enzyme activity of the immobilized enzyme source, such as the HLM. Given the ability to assay the effects of multiple parameters simultaneously more than one enzyme activity can be tested, even simultaneously, in this system at more than one substrate concentration. Additionally, the system can be used to determine the metabolic clearance rate of a (novel) therapeutic and whether it has an (inhibitory) effect on the chosen, monitored enzyme activity and if any enzyme inhibition is reversible / irreversible. Thus, the use of microassays in this way represent powerful investigative tools. However, as HLMs are prepared from liver samples (biopsies or donated organs), they are neither practical nor the preferred sample source for measuring the inter-individual variation in CYP activity. In contrast, only minimally invasive blood sampling is required for liver- EV sample collection.

[0013] However, for quantifying the differences in enzyme activity of liver-EV samples between (patient) samples, it is imperative to be able to quantify not only the activity but also the amount of the liver-EVs per blood (plasma) volume.

[0014] Neither immobilization of cell-secreted EVs, isolated e.g., from human liquid biopsies or in vitro cell culture media, on such a device has been described before, nor has assaying of EV enzyme activities under flow-through conditions been described in the prior art.

[0015] Therefore, there is a need for a new microassay device that enables isolation and immobilization of tissue-specific EVs and measurement of their amount (per sample volume) and their enzyme activities under flow-through conditions in an automated manner.

[0016] It is also 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. We have shown, when assaying metabolic enzymes using fluidic devices, biological lipid membranes and the enzymes contained therein, in particular cytochrome P450 (CYP) enzymes, are tethered to micropillars having a thiolene polymer micropillar surface, microsomal CYP enzymes are prone to rapid inactivation if immobilized on a thiol-rich polymer based micropillar reactor.

[0017] Our new device enables enzyme activity determination without risk of material triggered inactivation and also incorporates integrated electrodes that can be used to quantify the amount of immobilized EVs.

[0018] Statements of Invention

[0019] 1. According to a first aspect of the invention there is provided 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 / polyalkyne monomer (‘ene / yne’ monomer’), wherein; i) the ratio of thiol to alkene / alkyne functional groups in said copolymer has an excess of alkene / alkyne functional groups; ii) optionally, said device is further provided with at least one pair of electrodes for measuring impedance; and iii) said pillars and / or electrodes are functionalized by coating the alkene / alkyne-rich surfaces of same and / or conducting surfaces of same, respectively, with an antibody specific for lipid vesicles whose amount is to be measured or whose enzyme activity is to be measured.

[0020] Reference herein to the suffix allyl = the suffix ene and refers to a double bond C=C, moreover, the two terms are used herein interchangeably; reference to the suffix yne, refers to a triple bond between the carbon atoms. In a preferred embodiment of the invention the ratio of thiol to alkene / alkyne functional groups in said copolymer is from about 100:150 to 100:105, preferably about 100: 140 to 100: 110, and more preferably about 100: 125.

[0021] Advantageously, we have discovered that adjustment of the thiol-ene / yne bulk polymer composition (off-stoichiometric ratio of thiol and ‘ene’ monomers) in favour of a majority of ene / yne monomers prevents, or minimizes, leaching of uncrosslinked thiol monomers from the bulk polymer. We consider adjustment of this off-stoichiometric ratio, favouring an alkene / alkyne excess avoids, at least metabolic CYP enzyme inactivation, and other metabolic enzyme inactivation, when these metabolic enzymes are immobilized on such a thiolene polymer based micropillar reactor.

[0022] 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.

[0023] 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).

[0024] 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.

[0025] 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).

[0026] 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 (C=C) or alkyne groups (C C), or combinations thereof. Most preferably the unsaturated groups are alkene groups.

[0027] 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.

[0028] 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;

[0029] Pentaerythritol-tri(norborn-2-ene-5-carboxylate).

[0030] 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).

[0031] Preferably, the micropillars are 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. Ideally, these two monomers are in a molar ratio of from about 100:150 to 100:105, preferably about 100: 140 to 100: 110, and more preferably about 100: 125 in terms of the thiol and ene functional groups, yielding a % excess of enes. In yet other exemplary embodiments, the ‘ene’ monomer is substituted with a polyalkyne compound comprising two or more alkyne functional groups. Suitable diyne compounds include but are not limited to: 1 ,6-heptadiyne and 1 ,7-octadiyne.

[0032] 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.

[0033] In a preferred embodiment of the invention, said pillars are provided as an array and more preferably are diamond shape in horizontal cross-section, although conventional, or round shaped pillars may be used.

[0034] Ideally, the microassay device is of a conventional size and nature. This means it includes a microchannel that is typically 30-mm-long and 4-mm-wide, featuring an inlet, an outlet, and an array of micropillars, approx. 14 000 diamond micropillars as shown with exemplary dimensions in Figures 1 & 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 bottom layer.

[0035] In a further preferred embodiment of the invention the microassay device comprises at least a pair of electrodes, ideally two pairs, of electrodes, preferably interdigitated gold electrodes, on the bottom layer inside the sealed micropillar channel, Figure 2 lower panel. Most preferably in the device, the gold electrodes reside in the micropillar-free area matching the ends of the pillar array (Figures 1 and 2). Where the microassay device is to be used to measure enzyme activity and the amount of EVs in a sample both the pillars and electrodes are provided in said device and, further both are functionalized for the capture of said EVs.

[0036] In yet a further preferred embodiment, said pillars and electrodes are functionalized by coating the ene / yne-rich surfaces of same with DTSSP (3,3 - dithiobis(sulfosuccinimidyl propionate) or DSP (dithiobis(succinimidyl propionate)) and protein A / G, including protein A, protein G or recombinant protein A / G. This coating is provided, ideally, on both the pillar array and the gold electrodes.

[0037] The pillars and / or electrodes are further functionalized by attaching an antibody probe, specific for the vesicles to be tethered, ideally the antibodies are attached to the protein A / G. Preferably the antibody is specific for a certain type of cell, in particular extracellular vesicles (EVs) derived therefrom, for example, one antibody to be used in working the invention is HepParl (0CH1 E5) antibody which is a liver cell-specific antibody, thus interacting with and probing specifically for liver cell-secreted EVs. Although other antibodies specific for other types of cells and so their EVs, and known in the art, may be used for binding EVs secreted by other types of cells.

[0038] Reference herein to extracellular vesicles (EVs) is reference to lipid bilayer- delimited particles that are naturally released from almost all types of cells but, unlike a cell, cannot replicate. Notably, EVs include within their definition exosomes, micro vesicles and apoptotic bodies.

[0039] Exemplar antibodies that can be used to work the invention include, but are not limited to, CD235ab (which shows selectivity for erythrocytes and their secreted EVs) and CD172a (which shows selectivity for human pluripotent stem cell differentiated cardiomyocytes and their secreted EVs). Alternatively, exosome-selective antibodies, such CD9, CD37, CD63, CD81 and CD82, may be used to work the invention. It follows that use of the afore antibody / ies enables one to extract and test certain EVs or exosomes from a sample, where the sample can be e.g., cell culture media or human liquid biopsy, such as blood (plasma). In one embodiment of the invention, incubating these samples inside the microassay device, and undertaking a suitable antibody binding reaction, results in, for example, liver cell secreted EVs being selectively isolated from the sample by immobilizing them on a liver-specific antibody. The same applies to the use of other samples and / or other antibodies. Thus, in one embodiment, the technology can be used to isolate selected EVs or exosomes from a sample, typically a biological sample.

[0040] Accordingly, in yet a further preferred embodiment of the invention, said microassay device comprises pillars and / or electrodes that are functionalized with an antibody so as to extract from a sample EVs specific for said antibody.

[0041] Yet more ideally still, this protein A / G functionalized microassay device is provided in a freeze-dried form. Advantageously, we have discovered that the use of the claimed pillars enables us to lyophilize the immobilized protein A / G, inside the microfluidic device. Alternatively, and still yet more ideally, the protein A / G functionalized microassay device can be further functionalized with antibody and provided in a freeze-dried form.

[0042] When ready to use the device, the freeze-dried device can be rehydrated even after a long period of storage, and the immobilized biomaterial can be used without any disadvantages results. Moreover, we have discovered that after freeze-drying the microassay device of the invention, it can be stored at room temperature for long periods (beyond that of the ca. 1 -week period in cold- storage, in wetted state, without freeze-drying) and rehydrated before use. Indeed, our protein A / G coated microassay devices, with or without bound antibody, can be successfully stored, after freeze drying, for several months and then rehydrated without any significant changes in activity / ability to bind lipid membranes, such as EVs in a biological sample. It would therefore appear that our microassay device, as well as preventing / minimizing material triggered enzyme inactivation, also enables long term storage of microfluidic devices coated with the desired protein A / G coating or protein A / G coating and antibody at room temperature.

[0043] According to a further aspect of the invention there is provided a method for the manufacture of a microassay device according to the invention and so including a plurality of pillars 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 has an excess of alkene / alkyne functional groups; ii) optionally, providing in said device at least one pair of electrodes for measuring impedance; iii) coating the ene / yne-rich surfaces of the pillars and / or the conducting surfaces of the electrodes of parts i) and / or ii) with an antibody specific for EVs whose amount is to be measured and / or whose enzyme activity is to be measured.

[0044] In a preferred method of the invention said copolymer comprises, 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 100:150 to 100:105, preferably about 100:140 to 100:110, and more preferably is about 100:125.

[0045] More ideally still the device, after i) making or coating the pillars from / with the co-polymer and before iii) biofunctionalization with antibody, the device is subject to heating to 100°C for 2 hours to help remove any uncross-linked monomers. In a preferred method of the invention, step iii) of the method involves coating the pillars and / or electrodes with DTSSP (3,3'-dithiobis(sulfosuccinimidyl propionate) or DSP (dithiobis(succinimidyl propionate)). Preferably, this is followed by coating the pillars and / or electrodes with protein A / G, including protein A, protein G or recombinant protein A / G, and then attaching the antibody thereto.

[0046] In a preferred method of the invention, the method further comprises providing the device with, ideally two pairs, of electrodes, preferably interdigitated gold electrodes, which yet more preferably reside inside the sealed micropillar channel, ideally, in the micropillar-free area of the device at the ends of the pillar array.

[0047] In a preferred embodiment of the invention said pillars and electrodes are coated with said antibody.

[0048] In yet another embodiment of the invention said pillars are diamond-shaped in cross section.

[0049] In an alternative aspect, or 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, such as a SU-8 master, by spin coating a layer of photoresist 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; and b) exposing the product of step a) to a mask and using UV light for about 30s.

[0050] Reference herein to UV light is ideally to collimated UV light. In a further preferred method of the invention said master is post exposure- heated at 65 °C for 10 min, then at 95 °C for 40 min. More ideally still, the master is then developed in propylene glycol methyl ether acetate for about 30 min. The master is then baked at 95 °C for 30 min.

[0051] The afore method creates the master for casting of the polydimethylsiloxane (PDMS) molds, which 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, such as a Sll-8 master, then curing by heat (using standard protocol of about 70°C for at least 8 hours).

[0052] In yet an alternative aspect of the invention there is provided a method for measuring the amount of EVs in a sample comprising: i) exposing a microassay device comprising at least one pair of electrodes for measuring impedance and a plurality of pillars having ene / yne-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 has an excess of alkene / alkyne functional groups and further wherein at least said electrodes are functionalized with an antibody specific for extracellular vesicles (EVs); to said extracellular vesicles (EVs) in the sample to produce a loaded microassay device; ii) measuring the impedance of the microassay electrodes of part i) before and after loading with said EVs; iii) using the change in impedance as an indicator of the amount of EVs in said sample.

[0053] In a preferred method of the invention the said pillars are also functionalized with an antibody specific for extracellular vesicles (EVs). In a preferred method for measuring the amount of EVs in a sample, part ii) involves removing unbound EVs by use of a washing step, such as washing with buffer, before measuring impedance as an indicator of the amount of EVs in said sample.

[0054] In a preferred method the EVs whose amount is to be measured in said sample are provided in a biological sample.

[0055] According to a further aspect of the invention there is provided a method for measuring the activity of an enzyme in an extra cellular vesicle (EV) comprising: i) exposing a microassay device comprising pillars 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 has an excess of alkene / alkyne functional groups; wherein said device is further provided with at least one pair of electrodes for measuring impedance; and at least said pillars are functionalized with an antibody specific for said extracellular vesicles (EVs) containing the enzyme whose activity is to be measured; ii) exposing said pillars to a sample containing EVs containing the enzyme whose activity is to be measured; and iii) measuring the enzyme activity of the enzymes within said EVs.

[0056] In a preferred method of the invention said electrodes are also functionalized with an antibody specific for said extracellular vesicles (EVs) containing the enzyme whose activity is to be measured.

[0057] In a preferred method of this aspect of the invention said ratio of thiol to alkene / alkyne functional groups in said copolymer is from about 100:150 to 100:105, preferably about 100:140 to 100:110, and more preferably is about 100:125.

[0058] Preferably the enzyme is a metabolic enzyme.

[0059] Reference herein to a metabolic enzyme is reference to an enzyme that catalyzes the metabolism of drugs and / or other xenobiotics, ideally the enzyme is also polymorphic or exhibits genetic variation.

[0060] In a preferred embodiment of the invention said metabolic enzyme comprises one of the following enzymes: cytochrome P450 (CYP) enzymes such as, but not limited to, CYP1A1 , CYP1A2, CYP1 B1 , CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 , CYP3A4, CYP3A5, CYP79B2, CYP79B3, CYP71A12, CYP71A13, CYP71 B15.

[0061] In a preferred embodiment of the invention said metabolic enzyme comprises one of the following enzymes: Uridine 5'-diphospho-glucuronosyltransferase enzymes (UDP-glucuronosyltransferase, UGT), including, but not limited to B3GAT1 , B3GAT2, B3GAT3. UGT1A1 , UGT1A3, UGT1A4, UGT1A5, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2A1 , UGT2A2, UGT2A3, UGT2B4, UGT2B7, UGT2B10, UGT2B11 , UGT2B15 or UGT2B17.

[0062] In a preferred embodiment of the invention, the enzyme activity of the immobilized EVs is measured by monitoring the concentration of the metabolite produced from the catalysis of the enzyme-specific substrate (included in the feed solution) from the effluent collected out of the microassay device.

[0063] In a preferred method for measuring the enzyme activity of the EVs in a sample, part ii) involves removing unbound EVs by use of a washing step, before measuring enzyme activity in part iii). Advantageously, our technology enables one to measure different enzyme activities by using a single patient (blood) sample or a single sample of cell culture media.

[0064] In a preferred method the EVs containing the enzyme to be tested are provided in a biological sample.

[0065] In yet a further aspect of the invention there is provided a method for isolating EVs and / or quantifying the amount of said extracellular vesicles in a sample and / or measuring the activity of a metabolic enzyme contained in said EVs comprising: use of the device according to the invention. Ideally, but not exclusively, the method is undertaken to perform metabolic phenotyping that accounts for the impact of both genetic and external factors on an individual’s metabolic enzyme activity.

[0066] In a preferred method for measuring the activity of the metabolic enzyme, the method further comprises measuring the amount of EVs interacting with the said antibody in the sample, or tethered to said device / pillars / electrodes via said antibody, ideally via measuring the impedance of a suitable electrical conductor such as at least one pair of electrodes, ideally interdigitated gold electrodes, ideally inside the sealed micropillar channel, to which said EVs are attached.

[0067] In a preferred method of the invention the method comprises measuring the activity of an enzyme in the immobilized EVs and normalizing it to the measured amount of EVs in the sample.

[0068] In a yet preferred method for measuring the activity of a metabolic enzyme, the method further comprises comparing the measured activity in part iii) with that of the enzyme when exposed to a therapeutic and, where the activity is reduced in the presence of said therapeutic, concluding the therapeutic has an inhibitory effect on the activity of said enzyme. In any of the afore methods the said pillars are round or diamond-shaped in cross-section but diamond-shaped in cross-section is preferred.

[0069] 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 (‘thiol monomer’) and at least one polyalkene / polyalkyne monomer (‘ene’ monomer’), wherein the ratio of thiol to alkene / alkyne functional groups in said copolymer has an excess of alkene / alkyne functional groups; and further wherein said device is provided with at least one pair of electrodes for measuring impedance; ii) at least one antibody specific for EVs whose amount in a sample is to be measured or specific for EVs containing an enzyme whose activity is to be measured and, optionally protein A / G; and / or iii) at least one enzyme substrate and, optionally, at least one cofactor for the enzyme whose activity is to be measured.

[0070] In a preferred kit of the invention the ratio of thiol to alkene / alkyne functional groups in said copolymer is from about 100:150 to 100:105, preferably about 100: 140 to about 100: 110, and more preferably is about 100: 125.

[0071] In yet a further preferred kit of the invention, the antibody of part ii) is omitted and said pillars and / or electrodes are functionalized by coating the alkene / alkyne-rich surfaces of same or the conducting surface of same, respectively, with an antibody specific for EVs whose amount in a sample is to be measured and / or whose enzyme activity is to be measured.

[0072] In a preferred kit of parts one or more of said i) or ii) or iii) are freeze dried. Additionally, in a preferred kit of parts said pillars are diamond-shaped in crosssection.

[0073] Preferred features of each aspect of the invention may be as described in connection with any of the other aspects or embodiments of the invention.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] Moreover, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.

[0078] The Invention will now be described by way of example only with reference to the Examples below and to the following Figures wherein: Figure 1. Upper: Shows a schematic illustration of the immunocaptured EVs immobilized on antibody-coated surfaces and a photograph of a four-in-parallel microassay device test configuration. Middle: Schematic illustration of the architecture of the device and a scanning electron micrograph of the pillar array. Lower: Photograph of the computer-controlled impedance meter coupled with the integrated gold electrodes and schematic illustration of the microfluidic (flow-through) pillar array (device) and the setup used in enzyme activity determination of enzymes incorporated in EVs, such as cytochromes P450 (CYP) and UDP-glucuronosyl transferases (UGT).

[0079] Figure 2. Upper: Shows the layout of the top part, the micropillar array and the exemplary critical dimensions (all in mm). Lower: Shows the layout of the bottom part, the interdigitated gold electrodes and the exemplary critical dimensions (all in mm).

[0080] Figure 3. Shows the selectivity of the microassay device for isolation and impedance-based quantitation of the desired extracellular vesicles (EV) type by immunocapturing with the desired antibody.

[0081] Figure 4. Exemplary graph showing the linear relationship between concentration of Evs isolated from in vitro culture media of human 3D hepatoma cell spheroids (Huh7) measured by nanoparticle tracking analysis (NTA) and the impedance signal (AR / Ra) measured by the HepParl -coated gold electrodes, which provides the evidence of the feasibility of the microassay device for quantitation of the EV concentration in the samples introduced into the device with approximate detection limit of ca. 1 x1 O10particles / mL. Assumptions: All Evs in the Huh7 culture medium represent hepatic phenotype and interact with HepParl (+).

[0082] Figure 5. Shows the impact of the surface area (5A), which is a combination of the impacts of finger number and wire width (5B), of the interdigitated gold electrodes (functionalized with HepParl antibody) on the impedance signal (AR / Ra) arising from the immobilized HepPar1 (+) extracellular vesicles (EVs) isolated by the microassay device from an in vitro culture media of human 3D hepatoma cell spheroids (Huh7-EV) and from human plasma (plasma-EV). (The same in vitro culture media sample or the same human plasma sample is used with all the different electrode designs.)

[0083] Figure 6. Upper panel: Shows the cumulative count (per mL) of HepParl (+)- reactive EVs isolated from the in vitro culture media of 3D human primary hepatocyte spheroids on different days of culture and the cumulative total EV count (per mL) measured by nanoparticle tracking analysis. Lower panel: Shows in the increase in the HepParl (+)-EV count and in the total EV count in the in vitro culture media of 3D human primary hepatocytes as a function of culture time.

[0084] Figure 7. Shows the HepParl (+)-EV concentration in human plasma collected from ten (randomised) blood donors.

[0085] Figure 8. Shows the stability of the CYP3A4 activity (assessed with Luciferin- IPA marker) of the HepParl (+)-EVs collected from the 3D primary human hepatocyte (PHH) cell culture media on different days of culture and isolated / immobilized by the microassay devices, in four subsequent fractions of the effluent (a 50 pL) collected from the microassay devices.

[0086] Figure 9. Left panel: Shows the cellular CYP3A4 and CYP2C9 activities of primary human hepatocytes (PHH) cultured in 3D (average of n=3 macrowells a ca. 1200 spheroids equivalent to initially seeded 0.06M cells). Right panel: Shows the corresponding CYP3A4 and CYP2C9 activities of the HepParl (+)- EVs collected from the matching PHH cell culture media and isolated / immobilized by the microassay devices and assayed for enzyme activity under flow-through conditions (summed activity of EVs isolated from n=3 macrowells, total of ca. 3x1200 spheroids equivalent to initially seeded 3x0.06M cells). Figure 10. Shows the normalized CYP3A4 activity of the HepPar1 (+)-EVs collected from the PHH cell culture media and isolated / immobilized by the microassay devices, assayed for enzyme activity under flow-through conditions, and quantitated for HepPar1 (+)-EV concentration by impedance. The bars represent the summed activity of EVs isolated from 3 macrowells (total of ca. 3x1200 spheroids) and normalized to the amount of immobilized HepParl (+)-EVs (in 19 pL volume applied to the chip).

[0087] Figure 11. Shows the normalized enzyme activities of the HepParl (+)-EVs isolated / immobilized from ten different human plasma samples, assayed for enzyme activity under flow-through conditions, and quantitated for HepParl (+)-EV concentration by impedance. The normalization is done with respect to the amount of immobilized HepParl (+)-EVs (in 19 pL volume applied to the chip). Top panel: Activities of CYP2D6 and CYP3A4 (n.d.=not detected). Lower panel: Activities of CYP1A2, CYP2C9, and UGT (nonselective qualifier).

[0088] Figure 12. Shows the normalized enzyme activities of the HepParl (+)-Evs isolated / immobilized from ten different human plasma samples, assayed for enzyme activity under flow-through conditions, and quantitated for HepParl (+)-EV concentration by impedance. The normalization is done with respect to the amount of immobilized HepParl (+)-EVs (in 19 pL volume applied to the chip) as well as the original individual plasma volume. Top panel: Activities of CYP2D6 and CYP3A4 (n.d.=not detected). Lower panel: Activities of CYP1A2, CYP2C9, and UGT (nonselective qualifier).

[0089] Figure 13. Shows the impact of the protein A / G incubation time over 1 day (overnight), 5 days, and 10 days in the fridge, on the CYP3A4 activity of HepParl (+)-EVs isolated from the in vitro culture medium of 3D primary human hepatocytes (four different samples collected on culture days 2, 4, 8 and 10; same samples tested at all storing times). The data indicates no differences in the measured enzyme activities if the microassay device is stored in the fridge (upon protein A / G incubation) for 1 -5 days but storing the chip for 10 days impairs the performance of the protein A / G coating.

[0090] Figure 14. Investigates the impact of freeze-drying, and subsequent storing of the biofunctionalized microassay devices at room temperature (protected from light and moisture) for up to 3 months following freeze-drying. The figure shows the impedance-based quantitation of the amounts EVs in the sample (upper panel) and the enzyme activity, measured based on Luciferin-IPA (CYP3A4 / 5) activity (lower panel) of EVs collected from 3D primary human hepatocyte (PHH) culture media and isolated / immobilized on rehydrated chips after storing. The control data represents the chip-to-chip (n=4) variation of non- freeze-dried chips rinsed with PBS or non-freeze-dried chips exposed to 10%, w / V, sucrose and then rinsed with PBS prior to application of the EV sample and subsequent impedance and enzyme activity measurement. The data demonstrates that the sucrose exposure prior to freeze-drying has no significant impact on the impedance reading or enzyme activity. The data for the freeze-dried chips represents the chip-to-chip (n=4) variation of microassay devices freeze-dried after the protein A / G coating (l.h.s. series 1 ) or after the antibody (here, HepParl ) coating (r.h.s. series 2). The same PHH-EV sample was used in all assays and was a prepurified (ExoEasy) culture medium collected from 3D PHH cultures on Day 9.

[0091] MATERIALS & METHODS

[0092] Microreactor design

[0093] The microreactor design comprises of a 30-mm-long and 4-mm-wide, sealed microchannel featuring an array of ca. 14 000 diamond-shaped micropillars and ca. 360 semicircular micropillar structures on the channel walls. The nominal microchannel (and micropillar) height is 200 pm resulting in approximate total internal volume of ca. 19 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. Advantageously, the microreactor design comprises at least one pair of gold interdigitated electrodes. In fact, two pairs of interdigitated gold electrodes, typically but not exclusively, each of which have 19 fingers, each 20 pm wide with 20 pm spacing (gap) and cover a total area of 0.562 mm2(of which 0.296 mm2is occupied by the gold coating, Figure 2 lower panel). In the final device, the bottom part is sealed with the top part, so that the gold electrodes reside in the micropillar-free triangular area at the ends of the pillar array (Figure 1 ).

[0094] Design of the microfluidic device

[0095] The microfluidic chip design comprises of two polymer-based parts, top and bottom, in this embodiment, both made of a tetrathiol monomer PETMP and a triene monomer TATATO, using a thiol : ene ratio 100:125, which yields +25 mol-% excess of ene functional groups on the surface and bulk.

[0096] The top part incorporates the microchannel and the bottom part incorporates two pairs of interdigitated gold electrodes. In the final device, the bottom part is sealed with the top part, so that the gold electrodes reside in the micropillar- free areas, at the ends of the pillar array and near the connecting channels for the inlet / outlet, but excluding the space occupied by the micropillars.

[0097] After fabrication, the ene / yne-rich microfluidic device is functionalized sequentially with DTSSP (3,3'-dithiobis(sulfosuccinimidyl propionate) and protein A / G, this functionalization includes both the pillar array and the gold electrodes. After functionalization, the device is stored in wet state in the fridge until use (by sealing the inlet and outlet). These functionalized chips can be stored in the fridge for at least 5 days.

[0098] Microfabrication protocol

[0099] Fabrication of the thiol-ene based micropillar chip

[0100] The chip comprises of two layers, a planar bottom part and the micropillar part, both fabricated based on UV-replica molding of off-stoichiometric thiol- enes / ynes (OSTE). Two sets of gold-coated electrodes are additionally patterned to the bottom layer to serve as electrodes for electrical impedance spectroscopy (EIS), the patterning of the electrodes occurs before bonding the micropillar layers together: the top on the bottom part.

[0101] This fabrication process comprises eight steps:

[0102] 1. Fabrication of two SU-8 masters, one for the bottom and one for the top (micropillar) part

[0103] SU-8 100 is spin coated on an untreated silicon wafer using a 3-step protocol: first spinning at 500 rpm for 40 s (spreading step), then 1150 rpm for 30 s (coating step), and finally 700 rpm for 25 s (edge bead removal with acetone). This yields ca. 200-p.m -thick SU-8 layer. After spin coating, the SU-8 is soft- baked on a hot plate first at 65°C for 25 min and then at 95°C for 110 min, and then exposed for 30 s to a collimated UV light (OAI LS 30 / 5, OAI Instrument; nominal intensity of 40 mW / cm2) through a plastic mask featuring the aforementioned micropillars, an inlet, an outlet, and the chip edges (top part) or the chip edges (bottom part) .

[0104] Finally, the SU-8 master is post exposure baked first at 65 °C for 10 min, then at 95 °C for 40 min, and developed in propylene glycol methyl ether acetate for 30 min with mixing. The final master is rinsed with isopropanol, dryed with nitrogen stream, and heated at 95 °C for 30 min.

[0105] 2. Casting of poly(dimethyl siloxane) (PDMS) molds using the SU-8 masters

[0106] Casting of the PDMS molds 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, 8 hours).

[0107] 3. UV-replicamolding of the thiol-ene parts using the PDMS molds

[0108] UV replica molding of the thiol-ene polymer layers 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 / cm2). In the preferred protocol, the molar ratio of thiol over ‘ene’ functional groups is 100:125. Both micropillar and cover layers (with inlet / outlet holes) are prepared in the same manner.

[0109] 4. Gold coating on the surface of the bottom part

[0110] For coating the gold layer on the bottom part of the device, a sputter coater is used for 210 s (three steps of 70 s) with 18 mA current in Argon gas condition to create the gold-coated chip. Next, AZ photoresist is spin-coated on top of the gold layer at 4000 rpm for 30 s and prebaked for 2 min at 100 °C. Next, the interdigitated electrodes are patterned onto the AZ layer by photolithography using collimated UV exposure for 15 s, after which uncrosslinked AZ is developed in AZ 351 B developer (diluted 1 :4 with deionized water) for 30 s and rinsed with water. Finally, the gold layer is etched by immersing the substrate in gold etchant (Sigma, CAS 7681 -11 -0) for 10-20 s and the chip is cleaned with acetone.

[0111] 5. Post-processing by heat

[0112] Before bonding, the top layer is post-processed by heat (100°C for 2 h) to remove any possible uncrosslinked monomers by vaporization. The bottom part may also be heated similarly before or after gold coating.

[0113] 6. Bonding of the top and bottom parts two layers, preheated on a hot plate, by lamination, followed by 2 min UV exposure

[0114] The bonding 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.

[0115] 7. Functionalization of the thiol-ene / yne and gold surfaces

[0116] To functionalize the thiol:ene / yne surfaces, a mixture of 2 mM DTSSP (3,3 - dithiobis(sulfosuccinimidyl propionate)), 20 mM DTT (dithiotreitol), and 0.1 % v / v Lucirin TPO-L in PBS is introduced to the channel and the chip is exposed to UV-LED 365 nm for 2 min to functionalize the bare thiol-ene surfaces via thiol-ene reaction. Next, the channel is washed rigorously with plain PBS, and a fresh 2 mM solution of DTSSP in MQ water is incubated in the channel for 1 h (room temperature) to further functionalize the gold electrodes via thiol-gold interaction.

[0117] The thiol moieties of DTSSP will react with both (i) the surface enes on the thiol-ene pillars and (ii) the gold (electrodes), but the reaction chemistry is different. i. In the first step, DTSSP is incubated inside the chip with DTT, to reduce the thiol bond of DTSSP so that DTSSP can react with the surface enes via thiol-ene reaction chemistry. ii. In the second step, DTSSP+DTT is washed away and DTSSP (nonreduced) alone is incubated inside the chip and left to react with gold surfaces forming thiol-gold bonds.

[0118] As a result, we have thiol residues (arising from DTSSP) on both the pillars and the gold electrodes. Both of these steps i. and ii. are performed in a closed channel, so both pillars and gold surface experience exactly the same treatment.

[0119] 8. Functionalization of the thiol-ene / yne and DTSSP surfaces

[0120] The DTSSP coated pillar and electrode surfaces are functionalized with protein A / G. To do so, the channel is rinsed with PBS and 0.5 mg / mL protein A / G solution is introduced and incubated in the channel at 4°C for at least overnight or maximum of 5 days, before the antibody is added.

[0121] Preparation of microfluidic device for use

[0122] After protein A / G incubation (or after freeze-drying), the chip is rinsed with PBS, and then incubated with 1 M ethanolamine hydrochloride solution for 1 h to block the residual acting sites of protein A / G. Next, the chip is rinsed with PBS, filled with the antibody (in the exemplary experiments 10 pg / mL HepParl in PBS), and left to incubate at room temperature for about 30 min. Then, the chip is again rinsed with PBS and the impedance of both electrode arrays is measured. Alternatively, the chip may be freeze-dried after antibody coating, and the impedance measured after rehydration of the freeze-dried chip with PBS.

[0123] Using the microassay device for EV characterization

[0124] Finally, the EV-containing sample, such as pre-purified (e.g., ExoEasy) human plasma or in vitro cell culture media, is placed in the chip, and incubated in still conditions for about 1 h, to let the antibody-responsive EVs immobilize onto the antibody-coated surfaces. Then, the chip is rinsed with PBS and the impedance of both electrode arrays is again measured to quantify the amount of immobilized Evs based on AR / Ra as explained in a later section. Following impedance measurement, the enzyme activities of the immobilized, antibody- reactive Evs are measured under flow-through conditions as explained in another later section.

[0125] EV sample collection and primary EV purification

[0126] For technical validation of the invention reported herein, two different kinds of samples were used.

[0127] Cell culture media collected from 3D cultures of human hepatocyte spheroids (both human hepatoma Huh7 cell line and primary human hepatocytes (PHH) cultured in 3D using EV-depleted medium so that the medium includes only Evs secreted by the cells during the culture period). The 3D spheroids were cultured on commercial AggreWell™ microwell plates (Stemcell Technologies), which have 24 macrowells, each of which contain ca. 1200 microwells (diameter 400 pm) per macrowell. By treating the wells with AntiAdherence Rinsing Solution (Stemcell Technologies) before use, the cells cannot adhere to the surface of the wells but aggregate with each other forming uniform cell spheroids in each microwell (i.e., ca. 1200 spheroids per macrowell). In this system, the size of the spheroids is easily controllable by adjusting the density of the seeded cell suspension. For the exemplary experiments, the cells have been seeded on the AggreWell™ plates using density of 25 000 cells (Huh7) or 60 000 cells (PHH) per culture volume (per macrowell), which results in the initial cell count of ca. 21 cells (Huh7) or 50 cells (PHH) per each spheroid formed in the 1200 microwells.

[0128] The Huh7 spheroids were cultured in 1 .5 mL volume of DMEM - EV depleted FBS (10%) - Penicillin-streptomycin (1 %) - Non-Essential Amino acids (1 %) medium for 4 days, before collecting and pooling the culture media from a total of 7 macrowells, for later EV isolation. The PHH spheroids were cultured in initial volume of 1 .0 mL 3D-HPM medium supplemented with 5% EV depleted FBS for total of 10 days, by adding 0.5 mL more media on days 4 and 8.

[0129] The culture media was collected and pooled from 3 macrowells, for later EV isolation, on Days 2, 3, 4, 6, 8, 9, and 10.

[0130] Additionally, the CYP3A4 activity of the PHH spheroids was measured on Days 0, 2, 4, 6, 8, and 10 using Luciferin-IPA (Promega P450-GloTM) as the ‘marker’ substrate, and the CYP2C9 activity on Days 0, 3, and 9 using Luciferin-H (Promega P450-GloTM) as the ‘marker’ substrate, according to the supplier’s protocol.

[0131] Besides in vitro cell culture media, human plasma was collected from ten randomized blood donors (containing EVs secreted by all / any organs). For this purpose, raw blood samples were pretreated with citrate (anticoagulant) and the plasma were separated by centrifugation (standard procedure).

[0132] For the exemplary experiments, the samples were filtered (0.8 pm) and prepurified with a commercial ExoEasy kit (Qiagen), according to manufacturer’s instructions. Briefly, 1.25x volume of buffer XBP was added to 1x volume of the sample (culture media or human plasma) and mixed well by gently inverting the tubes 5 times. The initial sample volume may vary between 1 -5 mL. Next, the sample / XBP (1 :1 .25, v / v) mixture was applied to the ExoEasy spin column and centrifuged at 400x g for 2 min. The eluate was discarded, and the column was placed back into the same collection tube. Next, 10 ml of buffer XWP was added to the tube, and it was centrifuged at 3500x g for 5 min to remove residual buffer from the column. The eluate together with the collection tube were discarded, the spin column was transferred to a fresh collection tube and 1 ml of buffer XE (prefiltered with 0.1 pm filter) was added to the ExoEasy spincolumn membrane and incubated for 2 min. The tube was centrifuged at 400x g for 2 min to collect the eluate and then the eluate was re-applied to the ExoEasy spin column membrane and incubated for another 2 min to get ready for the last ultracentrifugation step to wash the EVs away from the membrane. Finally, the tube was centrifuged at 3500x g for 5 min to collect the eluate and this final, pretreated sample was transferred to a low-protein binding tube until further testing by the microfluidic device. The enrichment factor was defined as the ratio of the initial sample volume per the volume of the eluate.

[0133] Pre-characterization of the EV samples

[0134] After EV purification (ExoEasy), all samples were characterized for the amount of HepParl (+) responsive EVs by using the on-chip impedance electrodes and for selected CYP and UGT enzyme activities (of the immobilized PHH-EVs or plasma EVs) using the on-chip flow-through assays, as explained in the following sections.

[0135] Additionally, all EV samples were characterized by standard techniques to produce data for calibration and / or normalization purposes: for total particle (EV) count using ZetaView® nanoparticle tracking analyzer (NTA) and for total EV protein using Pierce BCA Protein Assay Kit (Thermo scientific).

[0136] Quantitation of the immobilized EVs by electrical impedance spectroscopy

[0137] To quantitate the amount of EVs in the sample immobilized into the micropillar chip, the EIS spectra were measured before and after EV sample application, using the integrated gold electrodes at both ends of the micropillar array (i.e. , two replicate measurements) and a PalmSens4 impedance analyzer (AC 0.01 V; 1 nA-10 mA; 5-100 000 Hz; 44 logarithmic steps). The spectra (Nyquist plots) obtained in each measurement was analysed using EIS Spectrum Analyser program, using the equivalent electrical circuit with an unweighted function and the Nelder-Mead (NM Simp) algorithm, to obtain AR / RAntibody. Briefly, the average charge transfer impedance values (R2) were analyzed, and the difference in R2 before and after sample application (AR = REV - RAntibody) was measured and the relative change percentage (AR / RAntibody) was calculated. With this method, both impedance values and relative estimated errors of the calculated parameters can be obtained. Elsewhere in the text, RAntibody is referred by Ra.

[0138] Characterization of the EVs’ enzyme activities on chip

[0139] For characterization of the EVs’ enzyme activities in the EVs isolated (by using the microassay device) from pretreated cell culture media or plasma samples, the micropillar arrays (4 in parallel) were coupled to a 4-syringe pump (CMA 4004 Syringe Pump, Harvard Apparatus) with the help of nanoport fluidic connectors and capillaries and placed on an aluminum heating block to ensure physiological temperature during enzyme activity testing (Figure 1 ).

[0140] Using this setup, the (EVs’) enzyme activities can be measured for selected enzymes, such as CYP and UGT enzymes under flow-through conditions by incorporating appropriate enzyme specific ‘marker’ substrate as well as enzyme co-substrate (1 mM NADPH for CYPs or 1 mM UDPGA for UGTs) in the feed solution, and analyzing the concentrations of the respective marker metabolites in effluents of the microfluidic devices. The different marker substrate concentrations and the information of other experimental variables between assays as well as the preparation of the microreactor effluent for analysis of the respective marker metabolite concentrations are given in Table 1.

[0141] To determine the respective enzyme activities, four subsequent 50 pL aliquots of chip effluent were collected, and their metabolite concentrations analysed using Varioskan wellplate reader (ThermoFisher). Before enzyme activity determination, the flow rate was optimized for each marker reaction separately, as it defines the residence time of the marker substrate inside the micropillar array. The flow rate of the feed solution is adjusted so that the assay operates in the regime where the marker metabolite concentration increases linearly along with the residence (i.e., reaction) time of the sample solution inside the chip (so as to enable measurement of the first order reaction kinetics of the enzyme in question).

[0142] Table 1. Experimental conditions used in flow-through enzyme activity assays.

[0143] In the exemplary experiments, the enzyme activities (pmol metabolite per minute) of the EVs isolated from the culture media of primary human hepatocyte (PHH) spheroids on different days of culture (Figures 8-10) as well as EVs isolated from human plasma (10 samples from different donors; Figures 11 and 12) were determined with respect to selected CYP isoforms and UGTs. In each case, negative controls were run without the co-substrate to ensure that no metabolite is formed out of the marker compound through nonenzymatic routes.

[0144] In Figures 8-12 (and also Figures 13 and 14), the enzyme activities of the immobilized EVs in flow-through experiments are calculated as follows:

[0145] The concentrations of the marker metabolite (nmol / L) in the collected chip effluents (in each fraction / = 1 -n) are first multiplied by the flow rate (pL / min) to acquire the activity of the marker reaction (representative of the enzyme activity of the given marker) in each fraction (like in Figure 8) and then averaged, using the following equations, to obtain the enzyme activity (pmol / h) representative of the EV containing sample applied into the microassay device:

[0146] Activitymarker reaction i— cmarkermetabolite, i x Flow rate

[0147] Finally, the validity of the optimized protocol for measuring different liver exosomal CYP activities (using isoenzyme-selective marker substrates, Table 1 ) as well as the UGT activity (using a nonselective marker substrate, Table 1 ) was demonstrated under flow-through conditions.

[0148] The absolute isoenzyme activity levels depend on the marker substrate’s clearance rate, which varies between substrates and enzymes.

[0149] Preparing the functionalized microfluidic devices for freeze-drying

[0150] After incubating the protein A / G-coated microassay devices in a fridge (2-6°C) overnight or for a maximum of 5 days, the microfluidic devices are first rinsed with PBS and then filled with 10% (wA / ) sucrose solution (a cryoprotection reagent) before loading into the freezedryer (FTS LyoStar II freeze dryer (SP Industries, Inc., Stone Ridge, NY). Alternatively, the microassay devices may be functionalized with the chosen antibody, then rinsed with PBS and filled with 10% (w / V) sucrose solution in the same way, and finally freeze-dried with the antibody readily coated onto the micropillar and electrode surfaces.

[0151] The freeze-drying protocol

[0152] The freeze-drying protocol comprises four steps (Table 2). After freeze-drying, the microreactors are kept at room temperature, protected from light and moisture, until use.

[0153] Table 2. The freeze-drying protocol used in cryopreservation experiments enabling long-term storage.

[0154] The impact of freeze-drying on the performance of the protein A / G coated chip was assessed by coating the freezedried chips (after rehydration) with HepParl antibody and loading with the PHH-EV sample followed by analysis of the EV amount and enzyme activities, as described above. The impact of freeze-drying on the performance of the antibody (here, HepParl ) coated chip was assessed by loading the freezedried chips (after rehydration) with PHH- EV sample followed by analysis of the EV amount and enzyme activities, as described above.

[0155] Results

[0156] Impact of bulk polymer post-processing on CYP activity in flow-through experiments

[0157] Functionalization of the micropillar reactors with EVs (e.g., human hepatocyte, erythrocyte, cardiomyocyte or plasma) is based on specific, multi-step protocol built on thiol-ene / yne polymer. In the first step, DTSSP (3,3'- dithiobis(sulfosuccinimidyl propionate) is covalently bound to the free surface of a ene / yne-rich (initially, +25% molar ene / yne excess over ‘thiols’) polymer. We know, however, the use of off-stoichiometric bulk composition (especially thiol-rich) can result in substantial leaching of uncrosslinked monomers into the microchannel, which can inactivate the CYP enzymes (incorporated in the extracellular vesicles) in an irreversible manner. Here, as well as adjusting the thiol:ene / yne ratio to be ene / yne-rich we use an additional polymer post-processing protocol as part of the microfabrication of ene / yne-rich thiol-ene / yne microreactors. The processing is performed between the thiol-ene / yne replica-molding and sealing of the micropillar (top) with the planar (bottom) surfaces, by heat-treating the cured thiol-ene replicas. The heat treatment can be done at different temperatures (70 or 100 °C) for 2 or 4h prior to functionalization and EV loading, ideally at 100°C / 2h.

[0158] With the optimized bulk composition range 100:150 to 100:105, preferably about 100:140 to 100:110, and more preferably a composition ratio of about 100:125 in terms of free thiol and ene / yne functional groups. Using PETMP:TATATO monomers (thiol:ene / yne 100:125) and post-processing (100°C / 2h), the concentration of leached monomers in the microreactor effluent is negligible, even at very low flow rates. There is therefore no substantial inactivation of the EV enzyme activity over time, not at least within the first 3h, in the flow-through assays performed by using the microassay device (Figure 8).

[0159] Quantitation of the antibody-reactive EVs with the microassay device

[0160] By using this microassay device, it is possible to coat the protein A / G functionalized surfaces with the desired antibody and further immobilize EVs that bind to the antibody used in the device. The selectivity of the microassay device for isolation and immobilization of EVs secreted by only the desired tissue / cell type is thus dependent on the selectivity of the antibody used. Experimentally, the selectivity of the antibody coating used can be confirmed by measuring the impedance signal (AR / Ra) of the integrated gold electrodes before and after application of the EV sample, as the impedance signal responds to the amount of the biological material attached onto the electrodes. In the exemplary experiments, this is confirmed by coating the protein A / G functionalized devices with the liver-specific HepParl antibody (as described above) or alternatively, with the red blood cell-specific CD235ab antibody, and incubating both kinds of devices separately with EVs isolated from either the in vitro culture medium of 3D human hepatocytes (Huh7) or human plasma.

[0161] The Huh7 cells are an immortalized human hepatocarcinoma cell line, and thus the EVs secreted by these cells, by default, represent a hepatic phenotype only. Conversely, the plasma samples, by default, contain EVs secreted by all cell types of the human body, including hepatocytes as well as red blood cells. As illustrated in Figure 3, the EVs isolated from human plasma react with both liver-specific antibody HepParl and red blood cell-specific antibody CD235ab, whereas the EVs isolated from in vitro culture media of Huh7 spheroids only react with the liver-specific HepParl antibody, but not CD235ab antibody.

[0162] Furthermore, the impedance signal (AR / Ra) can also be used for quantitation of the amount of immobilized EVs. This is illustrated in Figure 4, which shows the correlation of the total particle concentration (measured by standard NTA technique) of EVs isolated from Huh7 cultures (representing only HepParl (+)- EVs of hepatic phenotype) with the impedance signal of HepParl coated microassay devices when incubated with the same EV samples (Huh7). On this basis, it can be concluded that the amount of immobilized EVs (and the impedance signal) is directly proportional to the concentration of the antibody- reactive EVs in the sample solution applied into the microassay device, with an approximate lower limit of detection of ca. 1 x1 O10particles (EVs) per mL (Figure 4).

[0163] However, if the microassay device is to be used for quantitation of the antibody binding EVs, it is imperative that the impedance signal (AR / Ra) is as sensitive as possible, but also that it correlates linearly with the electrode area. Figure 5 shows that the default electrode design (with gold-coated area of 0.296 mm2and / or 19 interdigitated fingers) gives the maximal sensitivity (highest AR / Ra signal), when the finger (electrode) width is between 20 and 30 pm, while still in the linearly increasing range of electrode area. In Figure 5, this has been confirmed by testing the sensitivity of different sized, HepParl -coated electrodes with the same EV samples, including a human plasma sample and a sample from in vitro cultures of Huh7 spheroids. With this design, the absolute impedance signal is thus dependent on the amount of antibody- reactive EVs in the sample applied into the microassay devices, as already shown in Figures 3 and 4.

[0164] In further exemplary experiments, the microassay device was challenged by incubating HepParl coated devices with EV samples isolated from in vitro culture medium of 3D primary human hepatocytes (PHH) on different days of culture. It is well known that in the in vitro cultures, the primary hepatocytes dedifferentiate very rapidly and may therefore start secreting apoptotic vesicles or EVs that represent not only hepatic but also dedifferentiated phenotypes. However, it is also known that culturing PHHs in 3D (as spheroids) helps revive their differentiated (hepatic) phenotype. Figure 6 shows the comparison of the total particle (EV) concentration, measured by NTA, and the concentration of HepParl -reactive EVs in the culture media collected from 3D PHH cultures on different days of culture, calculated based on the on-chip impedance signal and the correlation established with Huh7-EVs in Figure 4. This data illustrates that on Day 2, the total EV concentration (NTA) is somewhat identical to the concentration of HepParl (+)-EVs (impedance). Instead, from Day 2 onward and until at least Day 4, the concentration of HepParl (+)-EVs does not much increase (likely because of dedifferentiation), in the same way as the total EV concentration (NTA). However, after about 6 days of 3D culture, the spheroids have likely achieved a more mature state, which triggers the release of more HepParl (+)-reactive EVs (Figure 6), as can be expected based on the commonly knowledge of PHH biology. Thus, it may be concluded that the microassay device enables selective isolation / immobilization and reliable quantitation of the antibody-reactive EVs.

[0165] These results provide the grounds for selective isolation / immobilization and quantitation of liver-secreted EVs from human plasma samples with the help of the microassay device. Figure 7 shows the impedance-based quantitation of the liver-EVs in human plasma collected from ten randomised, volunteer blood donors and analysed by using the HepParl -coated microassay device, based on the correlation established in Figure 4 and accounting for the enrichment factors of the pre-purification step (ExoEasy kit).

[0166] Quantitation of the enzyme activities of immobilized, antibody-reactive EVs with the microassay device

[0167] In the exemplary experiments, the possibility to measure the enzyme activities of the EVs immobilized onto the microassay device under flow-through conditions was first confirmed by using EVs isolated from the in vitro culture medium of metabolically active primary human hepatocytes (PHH) on different days of the 3D culture (Figure 8). It is well known that upon dedifferentiation, the primary hepatocytes also lose their inherently high metabolic activity, but this too can be revived by culturing the PHHs in 3D, which can be seen as an increased activity level over culture time (Figure 8). In the exemplary experiments, the EVs isolated from the culture media collected on different days of the 3D PHH culture were further assessed, in comparison to matching cell cultures, for their CYP3A4 and CYP2C9 activity, as detailed in Table 1 , by using HepParl -coated microassay devices. The corresponding enzyme activities of the matching PHH spheroids were assessed after collection of the culture media (the ‘EV sample’). Figure 9 shows the comparison of the cellular (spheroids) and the EV enzyme activities. This data illustrates how the PHHs rapidly lose their initially (Day 0, immediately after thawing) high metabolic activity (both CYP3A4 and CYP2C9). However, toward the end of the culture period, the cellular CYP3A4 activity (known to be inducible by 3D culture) recovers back to the original level, whereas the cellular CYP2C9 activity remains very low even after 9 days of 3D culture (Figure 9, left panel). As illustrated in Figure 9 (right panel), the CYP3A4 and CYP2C9 activities of HepParl (+)-EVs isolated from the in vitro culture media and immobilized by the microassay device follow exactly the same trend (qualitative correlation). It should be noted however that the cellular enzyme activities (pmol / h; average activity of ca. 1200 spheroids grown in one macrowell) are many orders of magniture higher than the enzyme activities of HepPar1 (+)-EVs (fmol / h; summed activity of the EVs isolated from 3 macrowells, ca. 3x1200 spheroids). Nevertheless, by normalizing the enzyme activity to the amount of HepParl (+)- EVs applied to the chip (19 pL) and quantitated by on-chip impedance (Figure 10), it can be concluded that the increasing CYP3A4 activity of HepParl (+)- EVs observed toward the end of the 3D culture period is not only because of higher number of HepParl (+)-EVs secreted, but also because of their higher metabolic activity, like in their originator cells (the PHH spheroids). This provided the evidence of the feasibility of the microassay device for measuring inter-individual variation in the liver metabolic activity, based on isolation / immobilization of the liver cell-secreted EVs from human plasma, followed by quantitation of their metabolic activity, and normalizing it to the amount of the HepParl (+)-reactive EVs.

[0168] Quantitation of the inter-individual variation in the enzyme activities of liver-secreted EVs in human plasma with the microassay device

[0169] In the next set of exemplary experiments, the microassay device was used for selective isolation of the HepParl (+)-EVs from ten (randomized) human plasma samples. The amount and enzyme activities of the immobilized HepParl (+)-EVs in each sample were quantitated similar to that of PHH-EVs using experimental parameters given in Table 1 . In terms of enzyme activities, our data shows that the liver-EVs isolated from human plasma contain not only functional CYP3A4 / 5 and CYP2D6, but also functional CYP1 A2, CYP2C9, and UGT enzymes (Figure 11 ). As shown, only 3 donors contained measurable amounts of CYP2C9 activity (the rest have very low activity which was close to baseline). Normalization of these enzyme activities to the amount of HepParl (+)-EVs immobilized on chip, as illustrated in Figure 11 , shows that the liver-EVs isolated from human plasma have inter-individual variation in their metabolic activity, as expected. Further normalization of the measured enzyme activities to the collected plasma volume reveals the actual donor-to- donor variation (Figure 12), which likely represents the variation of the liver metabolic activity of these enzymes between individuals. Impact of freeze-drying on the shelf-life of the new microassay device

[0170] By using the freeze-drying protocol presented above, the protein A / G coated or antibody coated microassay devices can be stored at room temperature in a dry state until use, and the storing time can be extended from ca. 5 days (in fridge, Figure 13) to several months (at room temperature after freeze-drying, Figure 14).

[0171] Experiments undertaken using the new microassay device to quantitate the amount and / or determine the enzyme activities of other EV types

[0172] Besides characterization of hepatic EVs (as described above), the microassay device can be used with another antibody showing selectivity toward other EV types. In our exemplary experiments, we have shown that impedance signal (AR / Ra) correlates linearly with the concentration of, e.g., cardiomyocyte- secreted EVs (isolated from in vitro cultures of cardiomyocytes differentiated from human induced pluripotent stem cells), when a cardiomyocyte-selective antibody CD172a is used, as well as erythrocyte-secreted EVs, when the red blood cell-specific antibody CD235ab is used. For calibration purposes, the selectivity and the linear relationship between the on-chip impedance signal and the total particle (EV) concentration measured by NTA was established in the same way as with HepParl antibody and liver-EVs in Figures 3 and 4.

[0173] Experiments undertaken using the new microassay device to determine enzyme inhibition by therapeutics

[0174] To assess the inhibitory effects of therapeutic drugs on CYP activities in the immobilized EVs, the microassays can be undertaken using the protocols described in Table 1 . The transient CYP enzyme activities are assessed similar to enzyme stability, except for the fact that a therapeutic is introduced to the microassay in a step-wise increasing concentration (typically from 0.01 to 100 pmol / L). Two to three fractions are collected before introducing the therapeutic, and thereafter two to three fractions at each concentration level. The first fraction always represents the intermediate state, whereas the next fractions represent the equilibrium of the new condition and gives information of the (inhibitory) impact of the therapeutic, at given concentration, toward the monitored enzyme activity. After collecting the fractions at the highest concentration, the therapeutic can be withdrawn from the feed solution to assess if the inhibited enzyme activity recovers (reversible inhibition) or persists (irreversible inhibition).

[0175] 3. CONCLUSIONS

[0176] We have shown herein that a microassay device can be developed to isolate EVs from a sample, via an immunobinding technique, and the captured EVs can be quantitated and used to measure the activity of enzymes incorporated / contained therein.

[0177] We expect there to be a (quantitative) correlation between the EVs and liver enzyme activities, which will allow us to identify the biological scaling factors between organ and EV content, rendering yet more useful the nature of the technology described herein.

[0178] References

[0179] 1. Achour, B., et al. (2021 ). Clin. Pharmacol. Then, 109(1 ), 222-232. DOI & Rowland, A., et al. (2019). British J Clin. Pharmacol., 85(1 ), 216-226. DOI

[0180] 2. Rodrigues, A. B., et al. (2021 ). Clinical Pharmacology & Therapeutics, 110(1 ), 248-258. DOI

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 / polyalkyne monomer (‘ene / yne’ monomer’), wherein; i) the ratio of thiol to alkene / alkyne functional groups in said copolymer has an excess of alkene / alkyne functional groups; ii) optionally, said device is further provided with at least one pair of electrodes for measuring impedance; and iii) said pillars and / or electrodes are functionalized by coating the alkene / alkyne-rich surfaces of same and / or conducting surfaces of same, respectively, with antibody specific for EVs to be isolated or whose amount is to be measured or whose enzyme activity is to be measured.

2. The microassay device according to claim 1 wherein said ratio of thiol to alkene / alkyne is from about 100:150 to 100:105, preferably about 100: 140 to 100: 110, and more preferably is about 100: 125.

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

4. The microassay device according to any one of claims 1 - 3 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) .

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

6. The microassay device according to claim 5 wherein the polyalkene / 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; T rimethylolpropane-tri(norborn-2-ene-5-carboxylate;Pentaerythritol-tri(norborn-2-ene-5-carboxylate);Pentaerythritol- tetra(norborn-2-ene-5-carboxylate); and Di(trimethylolpropane)tetra- (norborn-2-ene-5-carboxylate); 1 ,6-heptadiyne; 1 ,7-octadiyne.

7. The microassay device according to any one of claims 1 - 6 wherein said pillars are provided as an array and are round or diamond shape in cross-section.

8. The microassay device according to any one of claims 1 - 7 wherein the antibody is bound to said pillars and / or electrodes using DTSSP (3,3 - dithiobis(sulfosuccinimidyl propionate) or DSP (dithiobis(succinimidyl propionate)) and / or protein A / G.

9. The microassay device according to any one of claims 1 - 8 wherein the pillars and / or electrodes are also coated with said lipid vesicles.

10. The microassay according to any one of claims 1 - 9 wherein the lipid vesicles are extracellular vesicles (EVs).

11. The microassay according to claim 9 or claim 10 wherein the lipid vesicles or EVs are derived from a human liquid biopsy, such as plasma, or from in vitro cell culture medium, comprising one or more of the following phenotypes: liver, intestinal, or cardiac tissue.

12. The microassay device according to any one of claims 1 - 11 wherein the device has been freeze-dried.

13. The microassay device according to any one of claims 1 - 12 wherein the device comprises at least one or two pairs of electrodes.

14. The microassay device according to any one of claims 1 - 13 wherein, said electrodes are interdigitated gold electrodes.

15. The microassay device according to any one of claims 1 - 14 wherein the electrodes are located in a micropillar-free area of the device.

16. The microassay device according to any one of claims 1 - 15 wherein both the pillars and electrodes are coated with said antibody.

17. The microassay device according to any one of claims 1 - 16 wherein the width of the gold-coated electrodes is from about 5 to 50 pm, preferably about 10 to 30 pm, and ideally 20 pm.

18. The microassay device according to any one of claims 1 - 17 wherein the electrodes have a surface area of at least 0.296 mm2.

19. A method for the manufacture of a microassay device according to any one of claims 1 - 18 including a plurality of pillars 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 has an excess of alkene / alkyne functional groups; ii) optionally, providing in said device at least one pair of electrodes for measuring impedance; iii) coating the ene / yne-rich surfaces of the pillars and / or the conducting surfaces of the electrodes of parts i) and / or ii) with an antibody specific for EVs to be investigated.

20. The method according to claim 19 wherein the ratio of thiol to alkene / alkyne functional groups is from about 100:150 to 100:105, preferably about 100:140 to about 100:110, and more preferably is about 100:125.

21. The method according to claim 19 or claim 20 wherein step i) involves making said pillars from or coating said pillars with the tetrathiol compound PETMP and the triene compound TATATO.

22. The method according to anyone of claims 19 - 21 wherein step iii) of the method involves coating the pillars and / or electrodes with DTSSP (3,3'-dithiobis(sulfosuccinimidyl propionate) or DSP(dithiobis(succinim idyl propionate)) and / or protein A / G before attaching the antibody.

23. The method according to anyone of claims 19 - 22 wherein step i) or ii) is followed by: heating the device to 100°C for approx. 2h before step iii) is performed.

24. The method according to any one of claims 19 - 23 wherein the method further comprises freeze-drying the device.

25. The method according to any one of claims 19 - 24 wherein the device is provided with at least one or two pairs of electrodes.

26. The method according to any one of claims 19 - 25 wherein, said electrodes are interdigitated gold electrodes.

27. The method according to any one of claims 19 - 26 wherein the electrodes are located in a micropillar-free area of the device.

28. The method according to any one of claims 19 - 27 wherein said both said pillars and electrodes are coated with said antibody.

29. A method for measuring the amount of EVs in a sample using the microassay device according to any one of claims 1 - 18 comprising: i) exposing a microassay device according to any one of claims 1- 18 comprising at least electrodes functionalized with an antibody specific for extracellular vesicles (EVs); to said extracellular vesicles (EVs) to produce a loaded microassay device; ii) measuring the impedance of the microassay device of part i) when loaded with said EVs and when not loaded with said EVs; iii) using the change in impedance as an indicator of the amount of EVs in said sample.

30. The method according to claim 29 wherein part ii) involves removing unbound EVs by use of a washing step before measuring impedance.

31. A method for measuring the activity of a metabolic enzyme using the microassay device according to any one of claims 1 - 18 comprising: i) exposing the microassay device according to any one of claims 1 - 18 comprising at least pillars functionalized with an antibodyspecific for extracellular vesicles (EVs) containing the enzyme whose activity is to be measured, to said extracellular vesicles (EVs); and ii) measuring the activity of said enzyme.

32. The method according to any one of claims 31 wherein part i) or part ii) comprises exposing said enzyme whose activity is to be measured to a sample comprising the enzyme’s substrate and, optionally its co-factor, and measuring the catalysis of said substrate as an indicator of enzyme activity.

33. The method according to claim 32 wherein catalysis is determined by measuring the enzyme product.

34. The method according to anyone of claims 31 - 33 wherein the method further comprises comparing the measured enzyme activity in part ii) with that of the enzyme when exposed to a therapeutic and, where the activity is reduced in the presence of said therapeutic, concluding the therapeutic has an inhibitory effect on the activity of said enzyme.

35. The method according to anyone of claims 30-34 wherein part i) or part ii) comprises exposing said enzyme whose activity is to be measured to a sample comprising a therapeutic to be tested, in the absence of its substrate and its co-factor, to obtain a measure of nonspecific binding of said therapeutic to the EVs in order to determine ‘unbound fraction’ (fu).

36. The method according to any one of claims 30-35 wherein said enzyme is selected from the list comprising: a cytochrome P450 (CYP) enzyme, CYP1A1 , CYP1A2, CYP1 B1 , CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 , CYP3A4, CYP3A5, CYP79B2, CYP79B3, CYP71A12, CYP71A13, and CYP71 B15; or a Uridine 5'-diphospho- glucuronosyltransferase enzyme (UDP-glucuronosyltransferase, UGT) B3GAT1 , B3GAT2, B3GAT3. UGT1A1 , UGT1A3, UGT1A4, UGT1A5, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2A1 , UGT2A2, UGT2A3, and UGT2B4, UGT2B7, UGT2B10, UGT2B11 , UGT2B15 and UGT2B17.

37. A method for isolating EVs from a sample comprising the use of the device according to any one or more of claims 1 - 18.

38. A kit of parts comprising: i) a microassay device according to any one of claims 1 - 18; ii) at least one antibody specific for EVs whose amount in a sample is to be measured or specific for EVs containing an enzyme whose activity is to be measured and, optionally protein AG; and / or iii) at least one enzyme substrate and, optionally at least one cofactor, for the enzyme whose activity is to be measured.

39. The kit of parts according to claim 38 wherein the antibody of part ii) is omitted and the pillars and / or electrodes of the microassay device are functionalized by coating the alkene / alkyne-rich surfaces of same and / or the conducting surface of same, respectively, with an antibody specific for EVs whose amount in a sample is to be measured and / or whose enzyme activity is to be measured.

40. The kit of parts according to claim 38 or claim 39 wherein one or more of said i) or ii) or iii) is / are freeze dried.41 . The kit of parts according to any one of claims 38-40 wherein the pillars of said microassay device are round or diamond-shaped in crosssection.

42. A method for isolating EVs in a sample using the microassay device according to any one of claims 1 - 18 comprising: exposing a microassay device according to any one of claims 1 - 18 comprising at least electrodes functionalized with an antibody specific for extracellular vesicles (EVs); to said sample whereby extracellular vesicles (EVs) in said sample bind said antibody to be isolated from said sample.

43. The method according to claim 42 further comprising removing unbound EVs by use of a washing step.

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