WO2014057424A2

WO2014057424A2 - Production of particles

Info

Publication number: WO2014057424A2
Application number: PCT/IB2013/059211
Authority: WO
Inventors: Kevin John LAND; Mesuli Bonani MBANJWA
Original assignee: Csir
Priority date: 2012-10-09
Filing date: 2013-10-08
Publication date: 2014-04-17
Also published as: WO2014057424A3; EP2906337A2; ZA201502519B

Abstract

A process for producing product particles includes producing, by means of a droplet-based microfluidic technique, a plurality of primary droplets of a first liquid phase dispersed in a continuous second liquid phase. The first liquid phase is immiscible with the second liquid phase. An emulsion of the primary droplets in the second liquid phase is formed. A liquid adjunct is admixed with the emulsion. The liquid adjunct comprises secondary droplets of a third liquid phase, and/or particles, dispersed in a fourth liquid phase. The third liquid phase and/or the particles and/or the fourth liquid phase include a reagent capable of reacting with a component of the first liquid phase. The secondary droplets and/or the particles (whichever is/are present) are smaller than the primary droplets. The secondary droplets are allowed to coalesce with the primary droplets, and/or the particles are allowed to mix with primary droplets, thereby to form tertiary droplets. In the tertiary droplets, the reagent is allowed to react with the component of the first liquid phase, so that product particles are formed from the tertiary droplets. The product particles are recovered from the second liquid phase.

Description

PRODUCTION OF PARTICLES

THIS INVENTION relates to the production of particles. It relates in particular to a process for producing product particles, to a device for use in the process, and to product particles when produced by the process. The product particles may, in particular, be stabilized hydrocarbon constituent-containing particles, such as stabilized enzyme and/or polymer particles.

Enzymes are proteins, and can be used as biocatalysts to increase the rates of chemical and biological reactions. They have found extensive and growing usage in industries such as food processing, detergent formulations, paper and pulp and textile manufacture. Enzymes are not consumed by the reactions which they catalyse, so recovering these enzymes, which are often costly, is advantageous. In order to recover and recycle these enzymes, they are immobilized . Early enzyme immobilization techniques involved immobilizing the enzymes onto solid carrier supports. However, this introduces a large non-catalytic mass (90-99%), resulting in lower yields and lower productivity. An alternative immobilization technique is to form self immobilized enzyme particles. A number of methods for self- immobilization have been developed, namely cross linked enzyme aggregates (CLEA), cross-linked enzyme crystals (CLEC) and self- supporting enzyme structures such as those described in US 7700335.

Thus, US 7700335 describes a process of manufacturing structured self- immobilized enzymes utilizing batch procedures for emulsion generation . It has been found that immobilized enzymes, retaining much of their activity, can be manufactured utilizing this batch process. However, this process also results in spheres with a non-uniform particle size being manufactured . Typically, visual assessments using scanning electron microscopy have indicated an enzyme particle size range of between 0.5-1 Ομητι in diameter, while a laser light scattering analysis method has indicated that the particles aggregate further to form particles up to 1000 pm. This is not ideal for filter based particle recovery, nor for size based retention in continuous reactors. Moreover, larger particles and aggregates have a lower surface to volume ratio, which results in increased diffusion related kinetic limitations. It is hence an object of this invention to provide a process for producing stabilized enzyme particles which retain the activities found in particles produced utilizing the batch process, while providing a new robust manufacturing technique, with the advantage of a very narrow size distribution of formed particles.

Thus, according to a first aspect of the invention, there is provided a process for producing product particles, which process includes

producing, by means of a droplet-based microfluidic technique, a plurality of primary droplets of a first liquid phase dispersed in a continuous second liquid phase, with the first liquid phase being immiscible with the second liquid phase, and with an emulsion of the primary droplets in the second liquid phase thus being formed;

admixing a liquid adjunct with the emulsion, the liquid adjunct comprising secondary droplets of a third liquid phase, and/or particles, dispersed in a fourth liquid phase, with the third liquid phase and/or the particles and/or the fourth liquid phase including a reagent capable of reacting with a component of the first liquid phase, and the secondary droplets and the particles (whichever is/are present) being smaller than the primary droplets;

allowing the secondary droplets to coalesce with the primary droplets, and/or the particles to mix with the primary droplets, thereby to form tertiary droplets;

allowing, in the tertiary droplets, the reagent to react with the component of the first liquid phase, so that product particles are formed from the tertiary droplets; and

recovering the product particles from the second liquid phase.

"Microfluidic" is a term used to describe the control of very small volumes of liquid inside microchannels, typically 10-200 pm in width. By using microfluidics, i.e. microfluidic techniques, the introduction of reagents into a process, sometimes in a specific sequence, can be precisely controlled . Mixing and reaction times can then also be accurately controlled, allowing for numerous reactions not possible on a macro level . However, the intrinsic laminar flow in continuous microfluidic systems leads to two basic problems: the mixing of reagent streams across the microchannels is slow and dispersion of reagents along the channels is large. These problems are solved with the use of two-phase droplet based microfluidic systems, where droplets are contained in an immiscible continuous phase. Mixing is rapid and precise volumes of reagent are introduced into each droplet and allowed to react.

A number of microfluidic systems or techniques exist for the generation of droplets with very narrow size distributions, with standard deviations typically in the range of 1 -3%. These include co-flowing streams, T-junction and flow- focusing configurations. Each of these methods offers a high level of control over droplet size and uniformity. Droplets act as small reactors, and prevent sample diffusion into the surrounding liquid and also prevent contact with, and subsequent fouling of, channel walls. In addition, droplets can also be precisely controlled in terms of their velocity and a number of other parameters.

The production of the primary droplets in the second liquid phase may thus be effected by using any suitable droplet-based microfluidic technique such as co-flowing streams, T-junction and flow-focussing techniques or configurations that use channel geometry to control droplet formation; however, for stabilized hydrocarbon constituent-containing particle production, especially enzyme and/or polymer particle production, a flow-focussing technique is preferred.

The process may thus be carried out in a flow-focussing microfluidic device comprising a plate, at least one microchannel in the plate and along which the first liquid phase is conveyed to a flow focussing junction; at least one microchannel in the plate and along which the second liquid phase is conveyed to the flow focussing junction; a microchannel in the plate leading from the flow focussing junction and which is in communication with the first and second liquid phase microchannels and which has an inlet portion of reduced width where the droplets of the first liquid phase are formed, with this microchannel leading to a second junction; at least one further microchannel in the plate which leads into the second junction and along which the liquid adjunct is conveyed to the second junction; and a product microchannel leading from the second junction and along which, in use, the secondary droplets and/or particles (i.e. adjunct particles) and the tertiary droplets are conveyed.

The product microchannel may be of such a length that the droplets have a sufficient residence time therein for the reagent to react with the component of the first liquid phase and for the product particles to form. This may be achieved by having a portion of the product channel in convoluted form, e.g. in serpentine form. Due to the nature of flow in the product microchannel, the flow is laminar, resulting in the reagent, e.g. a cross-linking agent as hereinafter described, migrating to the channel centre mostly through diffusion, which is a slow process. This could result in uneven reaction, e.g. uneven cross-linking of a hydrocarbon component as hereinafter described, in the droplets. In order to rectify this, microstructures may be introduced into the microchannels, particularly the product microchannel, to speed up mixing, e.g. of droplets, in the product microchannel. These microstructures provide one or more of the following advantages:

efficient mixing of the secondary emulsion to ensure good contact with the droplets

forcing the reagent to disperse evenly across the microchannel forcing droplets to move away from the microchannel walls

allowing droplets to achieve equal residence times in the microchannel (previously some droplets move to the channel walls where flow is slowest and concentration of reagent highest, both undesirable effects) act to break up any aggregates that form back into a even dispersion, also facilitating easier coalescence or mixing with the primary droplets.

The microstructures, when present, may be spaced along at least a portion of the product microchannel, e.g. along the serpentine portion thereof. The microstructures may have any desired shape, and may stand proud of the bottom of the product microchannel and/or be recessed in the microchannel bottom. Thus, for example, a plurality of herringbone-shaped microstructures, a plurality of slanted grooves, or the like can be used.

In a first embodiment of the invention, the liquid adjunct may be a suspension of the secondary droplets of the third liquid phase in the fourth liquid phase. In a second embodiment of the invention, the liquid adjunct may be a solid- liquid suspension of micro- or nanoparticles in the fourth liquid phase, i.e. the micro- and nanoparticles are thus adjunct particles. In a third embodiment of the invention, the liquid adjunct may be a secondary emulsion comprising secondary droplets of the third liquid phase dispersed in the fourth liquid phase. The emulsion with which it is admixed as hereinbefore described is hereinafter referred to as the primary emulsion. The invention will hereinafter be described with reference to the liquid adjunct being in the form of the secondary emulsion; however, it will be appreciated that all aspects hereinafter described will work equally well when the liquid adjunct is in the form of a suspension in accordance with the first and second embodiments. Either the first or the second liquid phase may be a hydrophilic or aqueous phase while the other is then a hydrophobic or oily phase. In particular, the first liquid phase may be a hydrophilic or aqueous phase with the second liquid phase then being a hydrophobic or oily phase. The water immiscible or oily phase, i.e. the hydrophobic phase, may comprise an oil such as mineral, jojoba or avocado oil; a hydrocarbon such as decane, heptane, hexane or isododecane; a fluorocarbon such as Fluorinert FC40 (trademark - 3M), a perfluorocarbon oil and the like; an ether such as dioctyl ether, diphenyl ether, or the like; an ester such as triglyceride, isopropyl palmitate or isopropyl myristate; or the like.

The primary emulsion will normally be a water-in-oil or W/O emulsion; however, instead a oil-in-water or O/W, oil-in-water-in-oil, i.e. O/W/O, or water- in-oil-in-water, i.e. W/O/W, primary emulsion can be formed. The hydrophobic or oily phase may also include, if desired, an emulsifying component or surfactant such as Span 80 (trademark).

The secondary emulsion may, in particular, be a micro-emulsion whose secondary droplets are in a size range of 5-50 nm, a nano-emulsion whose secondary droplets are in a size range of 50-200 nm, or a macro-emulsion whose droplets are in a size range of 1 -1000 pm. Micro-emulsions are thermodynamically stable emulsions that can form spontaneously and do not necessarily require external energy input for their formation; in contrast, other emulsions such a nano-emulsions or macro-emulsions are inherently thermodynamically unstable but can be rendered stable for a finite period, e.g. by means of a surfactant. Thus, the secondary droplets may have sizes (diameters) in the range of 200-1000 nm when the primary droplets have sizes (diameters) in the range of 10-100 pm.

The reagent may be in liquid form, with the secondary emulsion then comprising droplets of the reagent dispersed in a hydrophobic or water- immiscible liquid phase. The water-immiscible phase may comprise an oil as hereinbefore described and, optionally, a surfactant. However, it will be appreciated that in certain cases, the reagent may become of solid particulate form, e.g. due to reaction between its components or constituents or due to dehydration; in such case, the liquid adjunct will be in the form of a solid-liquid suspension as hereinbefore described. The reagent may be a cross-linking agent. The cross-linking agent will typically be selected so that the cross-linking is only effected once a sufficient time period has elapsed, after the primary emulsion formation, e.g. for enzyme orientation at the phase interface to take place when the component with which the reagent reacts is an enzyme as hereinafter described.

The cross-linking agent, when used, is a multifunctional reagent, i.e. a molecule having two or more functional groups or reactive sites which can react with groups on the hydrocarbon constituent to form a cross-linked macromolecule, i.e. a stabilized enzyme and/or polymer structure as hereinafter described. The cross-linking agent may be selected from the following: an isocyanate such as hexamethylene diisocyanate or toluene diisocyanate; an aldehyde such as glutaraldehyde, succinaldehyde and glyoxal; an epoxide; an anhydride; methyl (1 R,2R,6S)-2-hydroxy-9- (hydroxymethyl)-3-oxabicyclo[4.3.0]nona-4,8-diene-5-carboxylate (genipin), or the like. The use of various cross-linking reagents may also allow for modification of the structure's physical and/or chemical properties.

The reagent may comprise a chain extender to enhance immobilization. The chain extender may be ethylene diamine (EDA) when the cross-linking agent is glutaraldehyde. Instead, other double amine or multiple amine compounds can be used for this purpose.

The component with which the reagent reacts may be a hydrocarbon constituent. The aqueous phase will thus comprise at least water and the hydrocarbon constituent. The hydrocarbon constituent may be an enzyme and/or a polymer. In one embodiment of the invention, the hydrocarbon constituent may thus be an enzyme. However, in another embodiment of the invention, the hydrocarbon constituent may be a polymer, such as chitosan and/or polyethyleneimine. For example, the hydrocarbon constituent may then comprise the combination of the cross-linkable polymers chitosan and polyethyleneimine (PEI). The invention will hereinafter be described with reference primarily to enzymes; however, it is to be appreciated that the various features of the invention hereinafter described with reference to enzymes, will apply equally well when the hydrocarbon constituent is a polymer.

The process can hence be used for the production of self-immobilized or stabilized enzyme particles or structures, in particular ones in which enzymes are immobilized with a majority of active sites of the enzymes are oriented either internally or externally; the particles or structures may be of spherical form; the particles may be porous; and may be self-supporting. The invention will thus hereinafter be described further with particular reference to the production of stabilized enzyme particles, structures or spheres. More particularly, the aqueous phase may hence comprise at least water and an enzyme, or a mixture of two or more enzyme classes, i.e. two or more different enzymes, solubilised, suspended or dissolved in the water. Thus, the enzyme constitutes the component of the first liquid phase with which the reagent of the third and/or fourth liquid phase reacts. Enzyme molecules will thus, when the primary droplets are formed, be located at or within interfacial boundaries of the first droplets and the second liquid phase. The reagent will then be a cross-linking agent which reacts with the enzyme molecules, resulting in the formation of the stabilized enzyme particles or structures in which the enzymes are immobilized.

Enzyme molecules often contain both hydrophilic and hydrophobic ends or faces. When such enzymes are used, collection and/or orientation thereof at the interfacial boundaries of the droplets and the second liquid phase, will be enhanced or ensured. Modifications may be made to native enzymes to enhance such properties. Thus, an additive for modifying the hydrophobicity and/or charge of the enzyme may be added to the hydrophilic phase and/or to the hydrophobic phase and/or to the primary emulsion. Examples of additives or modifiers that can be used for this purpose include specific amino acids; amino compounds; proteins; long chain hydrocarbon aldehydes; and other modifiers which bind covalently or otherwise to the enzymes.

The enzyme can be selected from enzyme classes such as Lipases, Esterases, Proteases, Nitrilases, Nitrile hydratases, Oxynitrilases, Epoxide hydrolases, Halohydrin dehalogenases, Polyphenoloxidases (e.g. laccase), Penicillin amidases, Amino acylases, Ureases, Uricases, Lysozymes Asparaginases, Elastases; however, it may, in particular, be a lipase.

The lipase can be chosen from microbial, animal, or plant sources, including any one of the following: Pseudomonas cepacia lipase, Pseudomonas fluoresceins lipase, Pseudomonas alcaligenes lipase Candida rugosa lipase, Candida antarctica lipase A, Candida antarctica lipase B, Candida utilis lipase, Thermomyces lanuginosus lipase, Fthizomucor miehei lipase, Aspergillus niger lipase, Aspergillus oryzae lipase, Penicillium sp lipase, Mucor javanicus lipase, Mucor miehei lipase, Fthizopus arrhizus lipase, Rhizopus delemer lipase, Rhizopus japonicus lipase, Rhizopus niveus lipase, and Porcine Pancreatic lipase.

The hydrophilic phase may also include a filler material which aids in cross- linking of lysine groups of the enzyme. The filler material may be bovine serum albumin (BSA). Instead, any other amine rich molecules can be used in this process, for example poly-L-lysine, polyethyleneimine (PEI), and/or chitosan. Protection of the active sites of an enzyme from being occupied by, or reacting with, the cross-linking agent may be achieved by the addition of a temporary protectant that can occupy the active sites during cross-linking. In the case of lipase, this temporary enzyme active site protectant may, for example, be tributyrin, or other triglyceride or ester compound, or menthol . Specific enzymes (even within specific classes) require different protectants to minimise or prevent activity loss during cross-linking.

While the hydrophilic phase in which the enzymes are dissolved or solubilized may comprise pure water, it is believed that improved results may be achieved if it includes a suitable buffer. The buffer should be selected to facilitate the cross-linking of the enzyme molecules, while ensuring enzyme stability. Thus, for example, the hydrophilic phase may comprise a buffered solution with pH 7-8. Such a buffered solution may be phosphate buffered saline (PBS) solution, a Tris-(hydroxymethyl)-aminomethane (TRIS) buffer- containing aqueous solution, or a KH₂P0₄ NaOH solution.

Thus, for example, when the enzyme is lipase, the primary emulsion is preferably a water-in-oil emulsion to ensure that most of the lipase active sites, which are hydrophobic, are oriented outwardly, thus increasing the total effective activity of the structures.

According to a second aspect of the invention, there is provided a process for producing stabilized enzyme particles, which process includes

providing, by means of droplet-based microfluidics, a plurality of droplets of a first liquid phase dispersed in a second liquid phase, with the first liquid phase being immiscible in the second liquid phase, and with enzyme molecules being located at or within interfacial boundaries of the droplets and the second liquid phase; and

cross-linking the enzyme molecules of the respective droplets so that individual enzyme structures, which are stable and in which the enzymes are immobilized with a majority of active sites of the enzymes being orientated either internally or externally, are formed from individual droplets.

According to a third aspect of the invention, there is provided an enzyme particle, which has a particle body comprising cross-linked enzyme molecules, with the particle body being stable and porous, and with the particle body having an outer surface which is also porous.

The enzyme particle may be as hereinbefore described with reference to the first aspect of the invention. Thus, for example, the particle may be spherical and/or it may be self-supporting, etc.

The enzyme particle may thus be that produced by the process of the first aspect of the invention.

The invention extends also to a flow-focussing or droplet-based microfluidic device as hereinbefore described.

The invention extends further to particles when produced by the process of the first or second aspect of the invention.

The invention will now be described in more detail with reference to the accompanying non-limiting drawings and specific examples. In the drawings,

FIGURE 1 shows, schematically, a three-dimensional view of a microfluidic device for use in producing stabilized enzyme particles according to the invention, with portions thereof shown on an enlarged scale; FIGURE 2 shows three-dimensional views of different microstructures that can be used in the microchannel 34 of the microfluidic device 10 of Figure 1 , instead of the microstructures 40 shown in Figure 1 ;

FIGURE 3 is a schematic representation of the enzyme immobilization process used in the Example 1 , i.e. in accordance with the invention;

FIGURE 4 shows, for Example 1 , a series of photographs of a portion of the device of Figure 1 , showing (when taken with Table 1 ) the dependence of droplet diameter, volume fraction and droplet frequency on flow rate of the aqueous or hydrophilic phase;

FIGURE 5 is a graph of droplet diameter and volume fraction as a function of flow rate, based on Table 1 ;

FIGURE 6 shows two photographs of (a) a primary emulsion formed at the flow focussing junction 20, and (b) a secondary emulsion introduced into the microfluidic circuit of the device of Figure 1 , in Example 1 ;

FIGURE 7 shows two series of photographs of the microchannel 34 of

Figure 1 in Example 1 , immediately downstream of the junction 30, for the case where there are no microstructures 40 in the microchannel 34 (photographs (a)-(e) and for the case where there are microstructures 40 present in the microchannel 34 (photographs (f)-(j));

FIGURE 8 shows a series of photographs of the particles of Example 1

(in an embodiment in which there were no microstructures 40 in the microchannel 34), imaged on a microscope slide, the top half of the picture being the originally captured image, while the bottom half shows the image after editing and particle identification;

FIGURE 9 shows, similarly to Figure 8, a series of photographs of the particles of Example 1 (in an embodiment in which there were microstructures 40 in the microchannel 34), imaged on a microscope slide;

FIGURE 10 shows, for Example 1 , a comparison of corrected and uncorrected relative enzymatic activities of the particles;

FIGURE 1 1 shows, for Example 1 , the activities of the particles after washing with different surfactants;

FIGURE 12 shows, for Example 1 , a SEM micrograph of the enzyme microparticle at (a) 1818x, (b) 5000x, and (c) 20000x magnifications showing the cross-linked porous outer surface structure of the microparticles; FIGURE 13 shows, for Example 1 , a SEM micrograph of the enzyme microparticle at 3000x magnification showing its porous body or interior; this exposure of the internal cross-section was achieved by using focussed ion beam (FIB) to meticulously ablate the immobized enzyme particle;

FIGURE 14 shows, for Example 2, manipulation of droplet size by variation of the flow rates of the dispersed phase and the continuous phase;

FIGURE 15 shows, for Example 2, micrographs of monodispersed BSA-lipase particles manufactured at a dispersed phase flow rate of 1 μΙ/min: (A) after collection in oil mixed with cross-linker emulsion, (B) suspended in Triton x100 solution with increased average diameter (109μιη) due to swelling and (C) SEM with average diameter of 25μηπ due to shrinking during drying;

FIGURE 16 shows, for Example 3, cross-linked FAE1 -BSA microspheres after 6 hours at 25°C (a) before washing and (b) after washing; and

FIGURE 17 shows, for Example 4, chitosan-PEI microparticles with a shell that wears off with time, leaving the particle core or body exposed.

Referring to Figure 1 , reference numeral 10 generally indicates a microfluidic device for use in producing stabilized enzyme particles according to the invention, and which was used in the specific example (Example) described hereunder.

The microfluidic device 10 comprises a flat rectangular (when seen in plan view) plate or chip 12. The chip 12 has end portions 14 and 16 which are thus spaced from each other.

A centrally located microchannel 18 is located centrally in the end portion 14 of the chip 12, and terminates in a flow focussing junction which is generally indicated by reference numeral 20 and which is also shown on an enlarged scale. The depth of the microchannel 18 is 68 μηη (microns or micrometers). The width of the microchannel 18 is 60 μιτι.

On either side of the microchannel 18 is provided a microchannel 22 which is L-shaped when seen in plan view and which each also terminate at the flow focussing junction 20. The channels 22 are also 68 μιη deep (this is also the depth of all the microchannels hereinafter described). The microchannels 22 are 160 μηη wide and narrow down to 60 μηη at the junction 20.

The microchannels 22, at and adjacent the flow focussing junction 20, are aligned with each other, and extend perpendicularly to the microchannel 18 and the microchannel 24 which are in turn aligned with each other, as seen in Figure 1 .

A microchannel 24 leads from the flow junction 20. At the flow junction 20, the microchannel 24 has a narrowed portion 26 which is 35 μιτι wide and which is the critical flow focussing neck where droplets are formed in use. The remainder of the microchannel 24 is 240 μηη wide with the narrowed portion or section 26 thus flaring into the wider portion of the channel 24. The microchannel 24 leads to a junction 30, which is also shown on an enlarged scale.

A pair of microchannels 32 lead into the junction 30 and are located at an oblique angle a, where a=45° to the microchannel 24. A microchannel 34 leads from the junction 30 and is aligned with the microchannel 24. The microchannel 34 initially has a width of 240 μιτι, and then flares into a wider serpentine portion, generally indicated by reference numeral 36. The width of the microchannel 34, in the serpentine portion 36, is 400 μιτι. The serpentine portion 36 thus comprises a plurality of parallel runs of the microchannel 34 which eventually terminates in the end portion 16 of the chip 12.

If desired, a plurality of spaced microstructures 40 may be provided in the microchannel 34, in particular in the serpentine portion 36 thereof, to speed up contact and mixing of a micro emulsion (secondary emulsion) introduced along the microchannels 32, with primary droplets of a primary emulsion flowing along the microchannels 24, 34. These microstructures 40 are in the form of longitudinally spaced herringbone-shaped grooves in the base of the microchannel 34. Each herringbone-shaped groove has a long limb 42 and a short limb 44 arranged at an obtuse angle to the long limb 42 so that each groove has a vertex 46. The vertices of the grooves point in an operationally upstream direction. The grooves are arranged in groups 48 of five, with the grooves of one group being of opposite hand to those of an adjacent group.

Figure 2 shows different microstructures 150, 160, 170 and 180 that can be used instead of the microstructures 40, in the microchannel 34 of the device of Figure 1 . In each case the microstructures are spaced apart longitudinally along the microchannel 34, and arranged in herringbone fashion.

With reference also to Figure 1 , it will be appreciated that instead of the microstructures 40 being recessed into the base of the microchannel 34, i.e. being in the form of grooves, they can stand proud of the microchannel base, i.e. be in the form of ridges. Thus, the microstructures 150, 160, 170 and 180 described hereunder and which are in the form of ridges, can equally well be in the form of grooves in the microchannel base, with the grooves being the same shape as the ridges.

The microstructures 150 are in fact similar to the grooves 40 in shape, each having a long limb 152 which forms an obtuse angled vertex 156 with the shorter limb 154. The microstructures 150 are arranged in groups 150 of five, with the ridges of one group being of opposite hand to those of an adjacent group.

The microstructures 160 are similar to the microstructures 150 and are also arranged in groups 158 of five. However, four of the groups 158 form a larger group 162. The vertices 156 of the microstructures 160 of one group 162 face downstream, i.e. in an opposite direction to those of the microstructures 160 of an adjacent group 162.

The microstructures 170 each comprise limbs 172, 174 of equal length and defining at their vertices 176 an obtuse angle. The vertices are directed upstream, and the microstructures are not arranged in groups as are the microstructures 150 and 160.

The microstructures 180 are similar to the microstructures 170, except that they are arranged in groups 182 of 23 microstructures 180. The vertices of the microstructures 180 of one group 182 face in an opposite direction to those of the microstructures 180 of an adjacent group 182.

The use of the microfluidic device 10 will be described hereafter in Examples 1 to 4.

Thus, the microfluidic device 10 was used in Example 1 : EXAMPLE 1

Materials

Pseudomonas Fluorencens lipase (Lipase AK "Amano") was purchased from Amano Enzyme Europe Limited (Oxfordshore, UK) and partially purified before use. Glutaraldehyde cross linker, ethylene diamine (EDA) chain extender, Span 80 surfactant, p -nitrophenyl palmitate (PNPP), p -nitrophenyl butyrate (PNPB) and Triton x-100 were all purchased from Sigma Aldrich. Caltex Pharma 15 mineral oil and de-ionized water were used in the continuous (hydrophobic) and dispersed (hydrophilic) phases respectively. Bovine Serum Albumin (BSA), was purchased from Sigma Aldrich and used as received.

Preparation The powdered lipase preparation was purified as follows:

A suspension of crude Amano lipase (500% m/v) in deionized water was centrifuged at 10000 rpm. 60% m/v polyethylene glycol 6000 (PEG 6000) was added and dissolved in the supernatant at 4°C. The mixture was centrifuged in conditions similar to previously stated and diluted by 1000% voume of deionized water and was washed in ultrafiltration unit (Amicon) fitted with membrane of 10 kDa molecule weight cut off (MWCO). The retentate was then frozen at ultra-low temperature (-80°C) and lyophilized. The powdered lipase was kept refrigerated at 4°C until use.

Process feedstock liquids were prepared as follows: secondary emulsion: 120 μΙ of 0.33 M EDA solution containing 9% (w/v) Triton x-100 was reacted with 100 μΙ of 25% (w/v) glutaraldehyde solution for 45 minutes. A further 100 μΙ of glutaraldehyde solution was added into the reaction mixture. The final mixture was then emulsified in 1 .2 ml of mineral oil containing 5% (w/w) Span 80 by magnetically stirring in a beaker for 15 minutes;

hydrophilic phase (for formation of primary droplets): BSA (60 mg) and lipase (240 mg) were weighed to obtain the correct ratio (20%/80%) by mass, and mixed together. While continuously stirring, de-ionized water (900 μΙ) and Tris-HCI buffer (100 μΙ) were added to the

BSA/lipase mixture. Stirring was continued for 5 minutes until no further solids were observed;

hydrophobic phase (for primary emulsion): the continuous oil phase consisted of mineral oil and Span-80 surfactant (3% w/w).

BSA was used as a proteic feeder. It facilitates the cross-linking process where the lipase concentration is low or where the enzyme activity is affected by a high concentration of glutaraldehyde required to obtain cross linking.

Experimentally, all solutions are introduced into the microfluidic device by means of syringe pumps (not shown).

Figure 3 shows schematically (generally indicated by reference numeral 100) the process of the invention for immobilized enzyme particle synthesis. Lipase 102 is added to the aqueous phase 104 (Figure 3a). Droplets of this aqueous solution are formed in the microfluidic device 10, resulting in an excess of mineral oil (106), and forming a water-in-oil emulsion (Figure 3b). Lipase migrates to the phase boundary and orientates its hydrophobic active site to this boundary. The chemical protein cross linker 108 is then added making this structure permanent (Figure 3c, d). The solvent is removed (Figure 3e), thereby obtaining immobilized enzyme particles 1 10.

Referring now to Figure 1 , the BSA/lipase solution, i.e. hydrophilic phase, is introduced along microchannel 18. Mineral oil/surfactant, i.e. hydrophobic phase, is introduced along the microchannels 22, and as the continuous phase at the junction 20, with droplets of the hydrophilic phase forming. Droplets sizes are strongly dependent on the flow rate of these two phases and the geometry of the junction 20. The pre-emulsified glutaraldehyde/EDA micro emulsion is introduced along the microchannels 32, at the junction 30. The serpentine portion of the microchannel 34 allows sufficient residence time for coalescence of the micro emulsion with the larger droplets of BSA/lipase. The flow is largely laminar, so that the micro emulsion is not immediately in contact with the droplets. This also allows time for the lipase to migrate to the surfaces of the droplets.

The microstructures 40, when present in the microchannel 34, assist in speeding up mixing of the microemulsion with the emulsion of BSA/lipase droplets in the continuous mineral oil/surfactant phase, thereby ensuring good contact thereof with the larger droplets of BSA/lipase.

The continuous oil phase flow rate and the micro-emulsion flow rate were kept constant at 4 μΙ/min and 1 μΙ/min respectively. The aqueous phase flow rate was varied between 0.2 and 2.6 μΙ/min.

Determination of enzyme hydrolytic activity

The activity of the resultant immobilized enzyme particles was measured by the hydrolysis of p-nitrophenyl esters (p-nitrophenyl palmitate and p-nitrophenyl butyrate) to p-nitrophenol and an aliphatic carbocylic acid. The release of p-nitrophenol yielded a yellow colour which was spectrophotometrically measured. A substrate stock solution was made by dissolving 13.3 μΙ of PNPB or 24 mg PNPP was dissolved in 8 ml isopropanol at 35°C from which 1 ml was used per assay. An assay reagent was prepared by dissolving 40 mg of sodium deoxycholate and 10 mg of gum arabic (Acacia) in 9 ml of tris-HCI buffer (50 mM, pH 8.0). The assay reagent was also prepared at 35°C. The latter two preparations were used within 10 minutes of preparation. All assays were performed using PowerWave HT Microplate Spectrophotometer (BioTek) with a temperature-controlled microtire plate reader. The spherezyme water suspension was first sonicated using an ultranosonic probe at 10% power for 5 seconds to ensure even and good dispersion of suspended particles before the amount required for the assay was pipetted out into microtitre wells. The microtritre plate reader (PowerWave HT spectrophotometer) was pre-heated to 35°C with the microtitre plate inside before the kinetic experiments. 1 ml of the substrate stock solution was added to 9 ml of the assay reagent solution and stirred at 35°C for 20 seconds. 240 μΙ of the resulting reagent mixture was then immediately added to microtitre well containing 10 μΙ of spherezyme suspension or free enzyme solution. The kinetic reactions were performed in triplicate for all assays. The control assay contained 10 μΙ tris-HCI buffer (50 mM, pH 8.0). The reactions occurred at a temperature of 35°C and the kinetic absorbance was read at a wavelength of 410 nm. A unit of enzyme activity was determined from the product of kinetic absorbance and a constant that is inversely proportional to light path of microtitre well and molar extinction coefficient of p-nitrophenol at 410 nm. The molar extinction co-efficient of p- nitrophenol at pH 8.0 was calculated as 15.1 M^~1.cm^"1. One unit of enzyme activity (U) is the amount of enzyme necessary to produce 1 pmol of p- nitrophenol per minute. Particle size measurements

Particle size measurements were done utilizing automated microscopic techniques. Data given is based on the measurement of the diameters of a minimum of 200 individual particles.

Droplet and particle sizes

Figure 4 and Table 1 show the ability of the microfluidic droplet device 10, 204 to vary droplet sizes by varying flow rates. In Figure 4, the oil flow rate remained constant at 4 μΙ/min, while the aqueous flow rate was varied between 0.2 and 2.6 μΙ/min. Flow becomes unstable at a flow rate of 2.8 μΙ/min (Figure 4h). The droplet diameter of the BSA/Lipase droplets changed between 35 and 61 μηη at these flow rates, with the droplet frequency increasing from approximately 150 droplets/s to 360 droplets/s. The volume fraction, defined as the volume fraction of BSA/Lipase to oil, increased from 5% to 65%. Droplet size and frequency could also be controlled by varying the oil flow rate. This study utilized aqueous flow rates of 1 μΙ/min, due to the high stability in this region and the production of 50 pm diameter droplets, which is desired. The volume fraction and droplet frequency, although not in the higher ranges, were acceptable.

Table 1

Figure 5 shows a graph of droplet diameter and volume fraction as a function of flow rate.

Flow introduced into a microfluidic channel is typically laminar and is characterized by low Reynold's numbers. This is clearly shown in Figures 6a and 6b. Figure 6a shows flow and droplet formation at the flow focussing junction 20. Figure 6b shows the junction 30 where the pre-emulsified glutaraldehyde/EDA was introduced shortly after primary droplet formation. At this point, there was no contact between the cross linker and the primary droplets. However, along the serpentine channel section 36, mixing by mainly diffusive processes had taken place, although the cross linker was more concentrated at the edges of the channel, particularly when the microstructures 40 were not present. By the time the droplets (now tertiary droplets) reached the outlet of the serpentine channel, the cross linker was dispersed evenly through the entire width of the channel and was in complete contact with the droplets. Upon leaving the microfluidic device, the tertiary droplets are collected and allowed to further cross link for up to 12 hours, to produce immobilized enzyme particles.

These particles were then centrifuged and resuspended in deionized water. The particles were sufficiently robust to enable recovery by centrifugation, and no coalescence of the particles was observed.

Flow in the microchannel 34, with and without microstructures 40 being present therein, was investigated. The results are shown in Figure 7. Figure 7 shows the advantage of using the microstructures 40. From Figures 7(a)-(e) it can be seen that 104 mm downstream of the junction 30 and where no microstructures are present, the micro-emulsion (cross-linker) is still not entirely mixed with the primary emulsion. However, when microstructures are present, then there is already emulsion mixing of the micro-emulsion with the primary emulsion 10.8 mm downstream of the junction 30. In other words, with the microstructures 40 mixing is at least 10 times more rapid than without the microstructures.

Visualisation

Particle sizes and size distributions were characterized by measuring optically obtained microscope images with in-house particle sizing software. Figure 8 shows an image of the particles immediately after being manufactured, in an embodiment where no microstructures 40 were present in the microchannel 34. The software automatically identified the particles and provided an average particle diameter and standard deviation. Some manual editing was required where the software incorrectly identified particles or clearly calculated the incorrect diameter, generally in cases such as when particles overlapped.

The experiment produced particles with an average particle diameter of 49.7 μιτι, with a coefficient of variation (CV) of less than 3%. 412 particles were counted in Figure 8. Figure 9 is similar to Figure 8, and thus also shows an image of particles immediately after being manufactured, but for an embodiment where microstructures 40 were present in the microchannel 34. From Figure 9, a marked improvement in particle quality, as compared to Figure 8, can be seen.

Activity Studies Tests were completed where EDA was utilized together with glutaraldehyde as a cross linker. It is believed that the increase in cross linker chain length provided by the EDA results in improved access of the substrate to the enzyme. Initial experiments utilizing palmitate as the substrate, resulted in retained activities of 4-6%. This low activity was not unexpected due to the fact that PNPP has 15 carbon atoms in its acyl chain, in contrast to PNPB which has only 4. The catalysis in the hydrolysis of the bigger molecules is less effective. The results discussed hereon are only those involving PNPB substrate. However, utilizing PNPB as the substrate resulted in activities increasing 10 fold. In Figure 10, a series of activity results has been plotted as compared against fluctuating activity values (uncorrected) of the free enzyme as well as against a mean value (corrected). The uncorrected values take account of variations in free enzyme activity which was the benchmark on which the activity of the the manufactured particles was compared against. The average specific activity of the free lipase was 0.12 U/mg lipase with coefficient of variation (CV) of around 25%.

This shows an improvement when compared to the results obtained previously where no active site protectant had been used. It was observed that during the process of the recovering the manufactured particles from oil and also during conducting of assays, a considerable amount of particles were lost due to their sticking on the walls of the plastic tubes and pipette tips. These losses resulted in incorrect results of the activity when the particle samples were manufactured in smaller quantities. However in larger amounts the losses were negligible. A water-soluble surfactant was therefore used in the water used for washing in order to minimise the losses which were difficult to quantify. Figure 1 1 shows the activity results from particles which were washed with deionised water containing 1 % surfactant. 100% relative activity is taken as the activity of the samples washed with de- ionised water. The surfactants tested were Tween 80, sodium dodecyl sulphate (SDS) and Triton x-100. The particles was with deionised water was also include in the test as a control.

The particles washed with 1 % Tween 80 surfactant were not adversely affected but those that were washed with SDS and Triton x-100 showed lower activity compared to particle washed with deionised water. The activity of the particles washed with Tween 80 was higher than that of the particles washed with deionised water. In could be concluded from the results that Tween 80 surfactant was suitable additive to deionised water for washing the particles during recovery in order to reduce the adhering of particle on the walls.

Glutaraldehyde (GLUT) can be used as an active ingredient in the covalent bonding (or cross-linking reaction) of enzyme molecules in a process of making immobilized enzyme structures such as that of US 7700335. It is also known that ethylene diamine (EDA) can be added to glutaraldehyde prior to enzyme immobilization to improve the process. The two components, usually in aqueous solutions, are usually mixed in specific amounts which can be tailored for a specific process. However, hitherto, it has not been known to emulsify GLUT-EDA solutions. In the process of the invention, the secondary emulsions of GLUT-EDA are formulated as water-in-oil emulsions, where a solution of GLUT-EDA is emulsified in oil. Mineral oil is the oil of choice but other hydrocarbon oils, fluorocarbon oils or organic solvents could also be utilized to achieve similar emulsions. Various types of commercially available surfactants which are compatible with and soluble in hydrocarbon oils, fluorocarbon oils or solvents can be used, together with water-soluble surfactants, in the formulation of the secondary emulsions. The secondary emulsions can be prepared in volumes between 1 -10 ml in a beaker using magnetic stirring. Other forms of industrial-type agitation or droplet break-up could also be utilized for obtaining smaller sizes and for producing large quantities of secondary emulsion.

The "ideal" GLUT-EDA emulsion would be one that would (i) remain stable for duration of production, (ii) homogenous in its composition, (iii) with good flow properties that are compatible with microchannel flow, and (iv) enable cross- linking of the proteins/enzymes droplets formed in the microchannel. Different emulsion formations were attempted but problems were encountered and had to be overcome - these can be summarized in the following manner:

- Emulsions with poor stability that resulted in GLUT-EDA droplets in the emulsion merging and heterogeneous cross-linking. The enzymatic activity of the immobilized enzyme structures produced from these emulsions was generally low.

Formulations stabilized with very high concentrations of surfactants resulted in progressively viscous fluids. High surfactant concentrations can lead to highly viscous fluids that cause flow difficulties in microchannels.

Too stable emulsions, under certain flow conditions, prohibited optimum coalescence between the enzyme droplets and GLUT-EDA droplets leading to insufficient cross-linking and poor or no recovery of immobilized enzyme particles.

Micro emulsions which were tested also did not achieve required cross- linking and therefore resulted in non-recovery of immobilized enzyme particles.

The formulation used in Example 1 was obtained after a number of experiments and optimizations, and meets the four criteria specified above.

Analysis of surface and internal structure using scanning electron microscospy (SEM)

The prepared particles were washed 3 times in a centrifuge using a 0.1 % Triton x-100 solution and then dried on a copper sheet at room temperature for 48 hours. The particles were then imaged using SEM. Separate samples were sputter-coated with gold; a single particle was then ablated using focused ion beam (FIB) and then imaged using an electron microscope.

The SEM results show that the particles produced by the process of the invention have a porous structure on the surface (Figure 12) and in their interiors or bodies/cores (Figure 13). This physical characteristic of the particles is unique and novel for self-immobilised enzyme particles.

The porosity of the structure could be varied by manipulation of starting concentration(s) of the material(s) in the droplet phase or/and the cross-linker solution. The cross-linking time (i.e. contact time between the droplet phase and the cross-linker) can also influence the nature of the surface and the internal structure of the particles. EXAMPLE 2

Preparation of BSA-immobilised enzyme micorspheres

In this example, the production of bovine serum album (BSA) microspheres is demonstrated.

Particles from different concentrations of BSA were prepared by varying the concentration in the dispersed phase (150, 200, 300mg/ml) in 250mM tris-HCI buffer solution (pH 7.2). For preparation of enzyme particles, a solution of BSA (200mg/ml) and partially purified Lipase AK "Amano" (30 mg/ml ) was prepared in the buffer solution mention above. The Pseudomonas Fluorencens lipase was purchased from Amano Enzyme Europe Limited in crude form and partially purified before use. Briefly, a suspension of crude Amano lipase (500% m/v) in deionized water was centrifuged at 10 000 rpm. 60% m/v polyethylene glycol 6000 (PEG 6000) was added and dissolved in the supernatant at 4°C. The mixture was centrifuged in conditions to similar to previously stated and diluted by 1000% volume of deionized water and was washed in ultrafiltration (UF) unit fitted with a 10 kDa molecular weight cut off (MWCO) membrane. The retentate was frozen at ultra-low temperature (-80°C), lyophilized and kept refrigerated at 4°C until use. The continuous phase was prepared by dissolving 3% (m/m) Span 80 in mineral oil. The cross-linker reagent was prepared by reacting 10ΟμΙ of GLA (25% m/v) solution with 120μΙ of EDA (0.33M, pH 6) solution containing Triton X-100 (9% m/v) surfactant for 45-minutes. A further 10ΟμΙ GLA solution was added into the mixture. The reacted mixture, exhibiting a yellowish colour, was then emulsified by magnetic stirring in 1 .2ml of mineral oil containing 5% m/m Span 80 for 15 minutes. Particles with BSA only could be prepared with or without the inclusion of the EDA in the cross-linker phase.

All fluids were freshly prepared before use in microfluidic experiments. Syringe pumps (Fusion, Chemyx) were used for delivering the fluids to the microfluidic device via PTFE tubing. After manufacturing the microparticles were separated from oil by centrifuging at 4000rpm after washing twice with aqueous solution of Triton X100 (1 %).

The activity of the resultant immobilised enzyme particles was determined in the hydrolysis of p-nitrophenyl esters, p-nitrophenyl palmitate (PNPP) and p- nitrophenyl butyrate (PNPB) to p-nitrophenol and an aliphatic carboxylic acid. The calorimetric assays were performed using a PowerWave HT Microplate Spectrophotometer (BioTek Instruments) with a temperature-controlled microtitre plate reader. The kinetic measurements were conducted at wavelength of 410 nm and temperature of 35^°C. Results

The droplets of aqueous polymer solution are generated in the continuous stream of mineral oil, and the GLA emulsion is introduced in the second junction (junction 30 in Figure 1 ). The protein droplets and the emulsion mix in the 120 mm-long serpentine microchannel section (serpentine portion 30 in Figure 1 ) where the cross-linker reacts with the proteins via Schiff-base and Michael-type reactions. The primary amines (=NH₂) of the protein and the aldehydes (-CHO) of the cross-linker, react in condensation reaction where a water molecule is a released and a covalent bond is formed. This reaction leads to denaturing and solidification of the protein. Albumins have an abundance of lysine amino groups in their structures which contain two primary amines, making them ideal proteins to cross-link with a di-functional aldehyde molecule. The use of herringbone microstructures (microstructures 40 in Figure 1 ) has been previously demonstrated to improve the mixing of protein droplets and the cross-linker emulsion. The flow rate of the dispersed phase could be varied to manipulate the droplet size (Figure 14) while the flow rates of the continuous phase and the cross-linker emulsion were kept constant at 4μΙ/ιηίη and Ι μΙ/min, respectively (see also Figure 15). Control of droplet size by varying dispersed phase was preferable since changing the continuous phase varies the overall residence time of the particles in the microfluidic circuit.

The cross-linker emulsion prepared showed good homogeneity and kinetic stability and could be used in experiment up to 12 hours without showing signs of phase separation or settling. The starting solution had an average specific activity of 577 U/g protein on PNBB and 2507 U/g on PNPP. The immobilized lipase was able to retain on average 50.5% of its initial enzymatic activity on PNPB and only 1 % on PNPP. The low activity of the microspheres on hydrolysis of PNPP was not unexpected due to the fact that PNPP has a long acyl (Ci6) chain, which exhibits diffusional limitations on immobilized enzymes. This is in contrast to the hydrolytic activity of the microspheres on PNPB, which has a shorter acyl (C₄) chain has and thus less diffusional limitations.

EXAMPLE 3

Immobilization of ferulic acid esterase The aim in this example was to demonstrate immobilization of ferulic acid esterase (FAE1 ). Preparation and culturing of competent bacterial cells

FAE1 used herein was isolated from the hindgut of a termite found in the central region of South Africa. Top10 E. coli competent cells (Invitrogen)s were prepared using rubidium chloride method. A single colony was used to inoculate 100 ml Psi media (5g/l yeast extract, 20g/l tryptone and 5g/l magnesium sulphate, pH 7.6) followed by incubation at 37°C until optical density at 550nm is 0.48. The cells were cooled on ice for 15 min before centrifugation (5000 x g for 5 min) and then washed with 40ml Tfb 1 buffer (potassium acetate 30mM, rubidium chloride 100mM, calcium chloride 10 mM, manganese chloride 50mM and glycerol 15% v/v, pH 5.8). The pelleted cells were re-suspended in 4ml Tfb II buffer (MOPS 10mM, rubidium chloride 10mM, calcium chloride 75mM, manganese chloride 50mM and glycerol 15% v/v, pH 6.5), and 50 μΙ aliquots were stored at -80°C. The E. coli competent cells (45μΙ) were mixed with the 5μΙ of the FAE 1 plasmid then incubated on ice for 20 min. The transformation mixture was heat-shocked at 42°C for 45 sec, followed by cold shock on ice for 5 min. 45μΙ of SOC media (20g/l tryptone, 5g/l yeast extract, 0.5g/l sodium chloride, 2.5ml 1 M KCI, 10ml 1 M MgCI₂, 10ml 1 M MgS0₄ and ddH20 per liter) was immediately added and the mixture was incubated at 37°C for 1 hour with shaking incubator. The transformants were selected on LB agar medium and plated on ampicillin LB agar overnight. Single colonies were cultured inoculated in three 50ml LB broth conical flasks with ampicillin (50μΙ). The flasks were incubated in a 3 shaking incubators at 25°C, 30°C and 37°C overnight. The cultures were recovered by centrifuging (JA-14 rotor, 14.000rpm for 30 minutes) in a Beckman centrifuge and the pellets (cells) were recovered. A 5ml B-per (Sigma Aldrich) was added in the cells followed by vigorously vortexing to lyse the cells and release the protein from the cells. The above centrifuging method was applied but the supernatant was recovered instead of the pellet. The supernatant was stored in a cold room prior to processing. To visualize the size and protein production SDS-PAGE gel following an existing protocol was run by adding 10μΙ of sample and 10μΙ of loading dye. Enzyme recovery

The following percentage sample of ammonium sulfate was prepared 0%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, 60%, and 65% using ammonium sulfate table. The sample was incubated overnight in a cold room (-4°C). The samples were centrifuged at 14.000 rpm for 30 minutes in a Beckman centrifuge and the pellets were recovered. The pellets were re-suspended in 50ml dH₂0. The sample was filtered through a filter paper and washed 3 times through a 10 kDa NMWL ultrafiltration membrane with 500ml of 20mM Tris-HCI to remove ammonium sulfate. The final wash sample was stored at - 80°C.

12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and zymogram were prepared using the following protocol: for separation gel, 5.1 ml of deionised water, 3.7ml of 1 .5M Tis-HCI (Ph 8.8), 75μΙ 20% (w/v) SDS (use H₂0 for zymgorame), 6ml Acrylamide/bis-acrylamide (30%/0.8w/v), 75μΙ 20% (w/v) ammonium persulphate and 7.5μΙ TEMED (Tetramethylethylenediamine). for stalking gel 3.07ml of dH₂0, 1 .25ml Tis-HCI (PH 6.8), 25μΙ 20% (w/v) SDS, 670μΙ acrylamide/bis-acrylamide (30%/0.8w/v), 25μΙ 20% (w/v) ammonium persulphate and 5μΙ TEMED (tetramethylethylenediamine). Molecular weight standard (M) (Thermo scientific, United states) consisting of protein markers of the following size 10, 15, 25, 35, 40, 55, 70, 100, 130 and 170 kDa was used to determine the size of the FAE1 . Bradford assay was performed following the Quick Start™ Bradford manual's instruction using microtitre plate reader (BioTek PowerWave). FAE1 activity assay was performed on p-nitrophenyl butyrate substrate using spectrophotometric assay.

Immobilization using microfluidic process The immobilization process used was similar to that of Example 1 . The concentrations of the FAE1 and BSA in aqueous phase were 0.77mg/ml and 200mg/ml, respectively, making a total of 200.77mg/ml. The activity retention of the FEA 1 -BSA particles was measured by following the enzyme kinetics (Figure 16) assay similar to one used for lipase immobilized enzymes. The FAE 1 immobilized enzymes retain 3% activity. EXAMPLE 4

Preparation of particles from chitosan and polyethyleneimine (PEI)

The aim in this example was to demonstrate utilization of the process of the invention in the preparation of particles from cross-linkable polymers, viz chitosan and PEI. The work also demonstrated that a blend of the polymers could be used.

Preparation

The continuous phase and the cross-linker phase were as described in Example 1 . The microfluidic systems and condition were as described in Example 1 . The aqueous phase was prepared by mixing 2.5% w/v chitosan solution in 5% acetic acid with 2.5% PEI solution. Particles from only chitosan or only PEI could also be prepared by running only the individual polymer solution in the aqueous phase.

Results Figure 17 shows a microscopic image of the particles formed from chitosan- PEI blend. The particles have a shell that wears off after a certain time. This unique feature has a possibility for use in multiple enzyme immobilisation and drug delivery systems. Self-immobilized enzymes have advantages over carrier bound immobilized enzymes as hereinbefore described. In addition to known self-immobilized enzymes, such as CLEAs and CLECs, US 7700335 teaches production of such enzymes using batch processing techniques. However, these batch techniques do not easily lend themselves to monodisperse particle size formation. Microfluidic techniques, however, have the advantage of producing particles with very narrow size distributions, as shown above.

Droplet-based microfluidic systems many advantages in the fields of biology and chemistry over conventional methods. They allow for the compartmentalisation of reactions into droplets containing extremely small volumes, typically less than nanolitres. This in turn allows for the precise temporal control of the mixing of reagents. It also allows one to control surface properties and the transport of the droplets on the microfluidic circuit. The present study shows a simple, robust method of manufacturing Spherezymes, which are a novel self-immobilized enzyme, overcoming a number of problems experienced utilizing the conventional batch process method of manufacture. It has thus been shown that microfluidic methods can indeed be utilized for the manufacture of immobilized enzyme structures, and that retained activity is relatively high while producing a narrow distribution of particle sizes. These particles can be recovered by centrifuging. The particles produced had a mean diameter of 49.7 μιτι. It has also been shown that the microfluidic technique lends itself ideally to the control of particle size, so that additional studies relating to activity as a function of particle volume and surface area are achievable.

It has thus been shown that microfluidic platform techniques can be utilized to successfully manufacture immobilized enzyme particles. These particles retain activity and can easily be recovered utilizing standard bench top laboratory centrifuges. Microfluidic techniques allow for optimum control over droplets and the introduction of reagents, and this should allow for further improvement of the activity and robustness of the immobilized enzyme particles. In addition, the control offered by microfluidic systems could be beneficial for the development of more advanced biocatalysts.

Claims

CLAIMS:

1 . A process for producing product particles, which process includes

admixing a liquid adjunct with the emulsion, the liquid adjunct comprising secondary droplets of a third liquid phase, and/or particles, dispersed in a fourth liquid phase, with the third liquid phase and/or the particles and/or the fourth liquid phase including a reagent capable of reacting with a component of the first liquid phase, and the secondary droplets and/or the particles (whichever is/are present) being smaller than the primary droplets;

allowing the secondary droplets to coalesce with the primary droplets, and/or the particles to mix with primary droplets, thereby to form tertiary droplets;

recovering the product particles from the second liquid phase.

2. The process according to claim 1 , wherein the liquid adjunct is a secondary emulsion, with the emulsion with which is admixed thus being a primary emulsion.

3. The process according to claim 2, wherein the microfluidic technique is a flow-focussing technique.

4. The process according to claim 3, which is carried out in a flow- focussing microfluidic device comprising a plate, at least one microchannel in the plate and along which the first liquid phase is conveyed to a flow focussing junction; at least one microchannel in the plate and along which the second liquid phase is conveyed to the flow focussing junction; a microchannel in the plate leading from the flow focussing junction and which is in communication with the first and second liquid phase microchannels and which has an inlet portion of reduced width where the droplets of the first liquid phase are formed, with this microchannel leading to a second junction; at least one further microchannel in the plate which leads into the second junction and along which the liquid adjunct is conveyed to the second junction; and a product microchannel leading from the second junction and along which, in use, the secondary droplets and/or particles and the tertiary droplets are conveyed.

5. The process according to claim 4, wherein the product microchannel is of convoluted form so that the droplets have a sufficient residence time therein for the reagent to react with the component of the first liquid phase and for the product particles to form.

6. The process according to claim 4 or 5, wherein microstructures are provided in at least the product microchannel to speed up mixing of droplets in the product microchannel.

7. The process according to any one of claims 2 to 6 inclusive, wherein the first liquid phase is an aqueous phase with the second liquid phase being an oily phase.

8. The process according to claim 7, wherein the component with which the reagent reacts, is a hydrocarbon constituent, with the aqueous phase thus comprising at least water and the hydrocarbon constituent.

9. The process according to claim 8, wherein the oily phase comprises an oil, a hydrocarbon, a fluorocarbon, an ether, and/or an ester.

10. The process according to claim 8 or 9, wherein the primary emulsion is a water-in-oil (or W/O) emulsion.

1 1 . The process according to any one of claims 8 to 10 inclusive, wherein the oily phase also includes an emulsifying component or surfactant,

12. The process according to any one of claims 8 to 1 1 inclusive, wherein the secondary emulsion is a micro-emulsion whose secondary droplets are in a size range of 5-50 nm, a nano-emulsion whose secondary droplets are in a size range of 50-200 nm, or a macro-emulsion whose droplets are in a size range of 1 -1000 pm.

13. The process according to claim 12, wherein the secondary droplets have sizes in the range of 200-1000 nm when the primary droplets have sizes in the range of 10-100 μιτι.

14. The process according to any one of claims 8 to 13 inclusive, wherein the reagent is in liquid form with the secondary emulsion comprising droplets of the reagent dispersed in a hydrophobic or water-immiscible liquid phase.

15. The process according to claim 14, wherein the reagent comprises a cross-linking agent.

16. The process according to claim 15, wherein the cross-linking agent is an isocyanate, an aldehyde and/or an anhydride.

17. The process according to any one of claims 8 to 16 inclusive, wherein the hydrocarbon constituent is an enzyme and/or a polymer.

18. The process according to claim 17, wherein the hydrocarbon constituent is an enzyme, with the particles thus being stabilized enzyme particles in which enzymes are immobilized with a majority of active sites of the enzymes being oriented either internally or externally, and with the particles being porous and self-supporting.

19. The process according to claim 18, wherein the enzyme comprises enzyme molecules having both hydrophilic and hydrophobic ends or faces, with the process including adding to the aqueous phase and/or to the oily phase and/or to the primary emulsion an additive for modifying the hydrophobicity and/or the charge of the enzyme molecules.

20. The process according to claim 18 or claim 19, wherein the enzyme is a lipase, with the reagent being a cross-linging agent which reacts with molecules of the lipase, resulting in the formation of the stabilized lipase particles or structures in which the lipase is immobilized.

21 . The process according to Claim 17, wherein the hydrocarbon constituent is a polymer, and is chitosan and/or polyethyleneimine.

22. A process for producing stabilized enzyme particles, which process includes

23. An enzyme particle, which has a particle body comprising cross- linked enzyme molecules, with the particle body being stable and porous, and with the particle body having an outer surface which is also porous.