EP2906337A2 - Production de particules - Google Patents

Production de particules

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Publication number
EP2906337A2
EP2906337A2 EP13798755.8A EP13798755A EP2906337A2 EP 2906337 A2 EP2906337 A2 EP 2906337A2 EP 13798755 A EP13798755 A EP 13798755A EP 2906337 A2 EP2906337 A2 EP 2906337A2
Authority
EP
European Patent Office
Prior art keywords
droplets
liquid phase
particles
enzyme
process according
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP13798755.8A
Other languages
German (de)
English (en)
Inventor
Kevin John LAND
Mesuli Bonani MBANJWA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Council for Scientific and Industrial Research CSIR
Original Assignee
Council for Scientific and Industrial Research CSIR
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Council for Scientific and Industrial Research CSIR filed Critical Council for Scientific and Industrial Research CSIR
Publication of EP2906337A2 publication Critical patent/EP2906337A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2/00—Processes or devices for granulating materials, e.g. fertilisers in general; Rendering particulate materials free flowing in general, e.g. making them hydrophobic
    • B01J2/02—Processes or devices for granulating materials, e.g. fertilisers in general; Rendering particulate materials free flowing in general, e.g. making them hydrophobic by dividing the liquid material into drops, e.g. by spraying, and solidifying the drops
    • B01J2/06—Processes or devices for granulating materials, e.g. fertilisers in general; Rendering particulate materials free flowing in general, e.g. making them hydrophobic by dividing the liquid material into drops, e.g. by spraying, and solidifying the drops in a liquid medium
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2/00—Processes or devices for granulating materials, e.g. fertilisers in general; Rendering particulate materials free flowing in general, e.g. making them hydrophobic
    • B01J2/02—Processes or devices for granulating materials, e.g. fertilisers in general; Rendering particulate materials free flowing in general, e.g. making them hydrophobic by dividing the liquid material into drops, e.g. by spraying, and solidifying the drops
    • B01J2/06—Processes or devices for granulating materials, e.g. fertilisers in general; Rendering particulate materials free flowing in general, e.g. making them hydrophobic by dividing the liquid material into drops, e.g. by spraying, and solidifying the drops in a liquid medium
    • B01J2/08—Gelation of a colloidal solution
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/98—Preparation of granular or free-flowing enzyme compositions

Definitions

  • 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.
  • CLAA cross linked enzyme aggregates
  • CLEC cross-linked enzyme crystals
  • self- supporting enzyme structures such as those described in US 7700335.
  • 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 .
  • visual assessments using scanning electron microscopy have indicated an enzyme particle size range of between 0.5-1 ⁇ 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.
  • 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.
  • 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;
  • Microfluidic is a term used to describe the control of very small volumes of liquid inside microchannels, typically 10-200 pm in width.
  • 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 .
  • 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.
  • 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.
  • a flow-focussing microfluidic device comprising a plate, at least one microchannel in the plate and along which the
  • 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:
  • 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.
  • a plurality of herringbone-shaped microstructures, a plurality of slanted grooves, or the like can be used.
  • the liquid adjunct may be a suspension of the secondary droplets of the third liquid phase in the fourth liquid phase.
  • 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.
  • 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 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • an isocyanate such as hexamethylene diisocyanate or toluene diisocyanate
  • an aldehyde such as glutaraldehyde, succinaldehyde and glyoxal
  • an epoxide an anhydride
  • 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.
  • the hydrocarbon constituent may thus be an enzyme.
  • the hydrocarbon constituent may be a polymer, such as chitosan and/or polyethyleneimine.
  • the hydrocarbon constituent may then comprise the combination of the cross-linkable polymers chitosan and polyethyleneimine (PEI).
  • PEI polyethyleneimine
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • the hydrophilic phase may comprise a buffered solution with pH 7-8.
  • a buffered solution may be phosphate buffered saline (PBS) solution, a Tris-(hydroxymethyl)-aminomethane (TRIS) buffer- containing aqueous solution, or a KH 2 P0 4 NaOH solution.
  • 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.
  • 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.
  • 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.
  • 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 8 shows a series of photographs of the particles of Example 1
  • 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;
  • FIB focussed ion beam
  • 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 ⁇ /min: (A) after collection in oil mixed with cross-linker emulsion, (B) suspended in Triton x100 solution with increased average diameter (109 ⁇ ) due to swelling and (C) SEM with average diameter of 25 ⁇ 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;
  • FIGURE 17 shows, for Example 4, chitosan-PEI microparticles with a shell that wears off with time, leaving the particle core or body exposed.
  • 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 ⁇ (microns or micrometers).
  • the width of the microchannel 18 is 60 ⁇ .
  • microchannel 22 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 ⁇ deep (this is also the depth of all the microchannels hereinafter described).
  • the microchannels 22 are 160 ⁇ wide and narrow down to 60 ⁇ at the junction 20.
  • 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.
  • the microchannel 24 has a narrowed portion 26 which is 35 ⁇ wide and which is the critical flow focussing neck where droplets are formed in use.
  • the remainder of the microchannel 24 is 240 ⁇ 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 microchannel 34 leads from the junction 30 and is aligned with the microchannel 24.
  • the microchannel 34 initially has a width of 240 ⁇ , 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 ⁇ .
  • 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.
  • 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 .
  • the microstructures are spaced apart longitudinally along the microchannel 34, and arranged in herringbone fashion.
  • microstructures 40 are 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.
  • 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.
  • microfluidic device 10 The use of the microfluidic device 10 will be described hereafter in Examples 1 to 4.
  • 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.
  • 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 ⁇ of 0.33 M EDA solution containing 9% (w/v) Triton x-100 was reacted with 100 ⁇ of 25% (w/v) glutaraldehyde solution for 45 minutes. A further 100 ⁇ 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 ⁇ ) and Tris-HCI buffer (100 ⁇ ) were added to the
  • 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.
  • 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.
  • the BSA/lipase solution i.e. hydrophilic phase
  • Mineral oil/surfactant i.e. hydrophobic phase
  • 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.
  • 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 ⁇ /min and 1 ⁇ /min respectively.
  • the aqueous phase flow rate was varied between 0.2 and 2.6 ⁇ /min.
  • 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 ⁇ 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 ⁇ of the resulting reagent mixture was then immediately added to microtitre well containing 10 ⁇ of spherezyme suspension or free enzyme solution. The kinetic reactions were performed in triplicate for all assays.
  • the control assay contained 10 ⁇ 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.
  • Figure 4 and Table 1 show the ability of the microfluidic droplet device 10, 204 to vary droplet sizes by varying flow rates.
  • the oil flow rate remained constant at 4 ⁇ /min, while the aqueous flow rate was varied between 0.2 and 2.6 ⁇ /min.
  • Flow becomes unstable at a flow rate of 2.8 ⁇ /min ( Figure 4h).
  • the droplet diameter of the BSA/Lipase droplets changed between 35 and 61 ⁇ 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 ⁇ /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.
  • Figure 5 shows a graph of droplet diameter and volume fraction as a function of flow rate.
  • FIG. 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.
  • the cross linker was more concentrated at the edges of the channel, particularly when the microstructures 40 were not present.
  • the cross linker was dispersed evenly through the entire width of the channel and was in complete contact with the droplets.
  • the tertiary droplets are collected and allowed to further cross link for up to 12 hours, to produce immobilized enzyme particles.
  • FIG. 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.
  • 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 ⁇ , 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.
  • FIG. 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.
  • 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 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.
  • 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:
  • 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.
  • Micro emulsions which were tested also did not achieve required cross- linking and therefore resulted in non-recovery of immobilized enzyme particles.
  • Example 1 The formulation used in Example 1 was obtained after a number of experiments and optimizations, and meets the four criteria specified above.
  • 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.
  • FIB focused ion beam
  • 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
  • the cross-linking time can also influence the nature of the surface and the internal structure of the particles.
  • bovine serum album (BSA) microspheres In this example, the production of bovine serum album (BSA) microspheres is demonstrated.
  • BSA bovine serum album
  • 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).
  • concentration in the dispersed phase 150, 200, 300mg/ml
  • 250mM tris-HCI buffer solution pH 7.2
  • 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.
  • the cross-linker reagent was prepared by reacting 10 ⁇ of GLA (25% m/v) solution with 120 ⁇ of EDA (0.33M, pH 6) solution containing Triton X-100 (9% m/v) surfactant for 45-minutes. A further 10 ⁇ 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.
  • 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 ( NH 2 ) 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 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 4 ⁇ / ⁇ and ⁇ ⁇ /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 (C 4 ) chain has and thus less diffusional limitations.
  • 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.
  • Psi media 5g/l yeast extract, 20g/l tryptone and 5g/l magnesium sulphate, pH 7.6
  • 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 ⁇ aliquots were stored at -80°C.
  • 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
  • coli competent cells (45 ⁇ ) were mixed with the 5 ⁇ 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. 45 ⁇ of SOC media (20g/l tryptone, 5g/l yeast extract, 0.5g/l sodium chloride, 2.5ml 1 M KCI, 10ml 1 M MgCI 2 , 10ml 1 M MgS0 4 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.
  • SOC media 20g/l tryptone, 5g/l yeast extract, 0.5g/l sodium chloride, 2.5ml 1 M KCI, 10ml 1 M MgCI 2 , 10ml 1 M MgS0 4 and ddH20 per liter
  • 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 dH 2 0.
  • 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.
  • SDS-PAGE SDS-polyacrylamide gel electrophoresis
  • 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), 75 ⁇ 20% (w/v) SDS (use H 2 0 for zymgorame), 6ml Acrylamide/bis-acrylamide (30%/0.8w/v), 75 ⁇ 20% (w/v) ammonium persulphate and 7.5 ⁇ TEMED (Tetramethylethylenediamine).
  • 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.
  • 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.
  • 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.
  • 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 ⁇ . 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.
  • 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.

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Abstract

L'invention concerne un procédé de production de particules de produit qui comprend la production, au moyen d'une technique micro-fluidique à base de gouttelettes, d'une pluralité de gouttelettes primaires d'une première phase liquide dispersée dans une deuxième phase liquide continue. La première phase liquide et la deuxième phase liquide sont immiscibles. Une émulsion des gouttelettes primaires dans la deuxième phase liquide est formée. Un additif liquide est mélangé avec l'émulsion. L'additif liquide comprend des gouttelettes secondaires d'une troisième phase liquide et/ou des particules, dispersées dans une quatrième phase liquide. La troisième phase liquide et/ou les particules et/ou la quatrième phase liquide comprennent un réactif apte à réagir avec un constituant de la première phase liquide. Les gouttelettes secondaires et/ou les particules (celles qui sont présentes) sont plus petites que les gouttelettes primaires. Les gouttelettes secondaires sont amenées à coalescer avec les gouttelettes primaires et/ou les particules sont amenées à se mélanger avec les gouttelettes primaires, permettant de former ainsi des gouttelettes tertiaires. Dans les gouttelettes tertiaires, le réactif est amené à réagir avec le constituant de la première phase liquide, de sorte que les particules de produit sont formées à partir des gouttelettes tertiaires. Les particules de produit sont récupérées à partir de la deuxième phase liquide.
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EP2962751B1 (fr) * 2014-07-03 2017-04-05 Helmholtz-Zentrum Geesthacht Zentrum für Material- und Küstenforschung GmbH Procédé de formation de gouttelettes dans un système à phases multiples
CN105056820B (zh) * 2015-07-10 2017-04-05 清华大学 一种串联放大的微结构装置
EP3391958B1 (fr) * 2017-04-19 2020-08-12 The Procter & Gamble Company Procédé de fabrication de particules polymères absorbant l'eau à surface enduite dans un dispositif microfluidique
EP3917500A2 (fr) 2019-01-31 2021-12-08 Elektrofi, Inc. Formation et morphologie de particules
WO2021050953A1 (fr) 2019-09-13 2021-03-18 Elektrofi, Inc. Compositions et procédés pour l'administration de produits biologiques thérapeutiques pour le traitement d'une maladie
WO2021168271A1 (fr) * 2020-02-19 2021-08-26 Elektrofi, Inc. Formation de gouttelettes et morphologie de particules
US20230158502A1 (en) 2020-04-17 2023-05-25 Sphere Fluidics Limited Droplet spacing
CN113791016B (zh) * 2021-09-16 2023-08-11 中国石油大学(华东) 乳状液生成及微观渗流监测一体化实验装置、监测方法

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