WO2019106047A1

WO2019106047A1 - Process for producing bio-functional polymer particles

Info

Publication number: WO2019106047A1
Application number: PCT/EP2018/082896
Authority: WO
Inventors: Jan-Niklas Schönberg; Jürgen RUEHE; Thomas Brandstetter
Original assignee: Albert-Ludwigs-Universität Freiburg
Priority date: 2017-11-28
Filing date: 2018-11-28
Publication date: 2019-06-06
Also published as: DE102017011013A1

Abstract

The present invention relates to a process for producing bio-functional polymer particles, comprising the following steps: (i) production of a solution containing at least one polymer, said polymer being a copolymer with integral cross-linking groups, and at least one bio-molecule; (ii) introduction of the solution produced in step (i) into a carrier fluid to form spatially separate droplets; (iii) solidification of the droplets obtained in step (ii) by the activation of the cross-linking groups using a trigger, and subsequent cross-linking of the copolymers leading to bio-molecular bonding.

Description

Process for the production of biofunctional polymer particles

The specific accumulation of biologically relevant substances from a sample, for example, for later analysis, can i.a. done by biofunctionalized particles. Many different methods are known for the production of such particles.

A problem with known methods, however, is that biofunctionalization must take place in one or more reaction steps after the actual particle preparation.

The synthesis of Janus particles that can be biofunctionalized by click chemistry is described, for example, in B. Li et al., Synthesis of Biofunctional Janus Particles, Macromol. Rapid Commun. 2015, 36, 1200-1204. Therein, styrene is polymerized together with a crosslinker to form polystyrene particles. These are converted into solid polystyrene / polyvinylbenzyl chloride-Janus particles. The Janus particles can then be biofunctionalized in several steps by thiol-click chemistry.

The synthesis of biofunctionalized acrylate-PEG particles is described in KW Bong, "Synthesis of Cell-Adhesive Anisotropy Multifunctional Particles by Stop Flow Lithography and Streptavidin-Biotin Interactions", Langmuir 2015, 31, 13165-13171. In these particles, a covalent bond between the acrylate group of an acrylate PEG-NHS polymer and the NH ₂ group of streptavidin is generated by means of amine-NHS coupling. The resulting streptavidin-PEG-acrylate particles are then mixed with PEG (polyethylene glycol) monomers and briefly treated with UV light (365 nm) to form a PEG network. This can then be functionalized with biotin due to the strong interaction of streptavidin and biotin.

If the particles can only be functionalized with biomolecules after their actual preparation, as described in the above references, this makes the production of particles with different biofunctionality laborious and time-consuming. In addition, the performance of several surface reactions makes it difficult to reproduce the surface density of the biofunctional binding sites. A fast, straightforward, universal and inexpensive production of such biofunctional particles is desirable. An alternative to the subsequent modification (functionalization) of the polymer particles with biomolecules, as described in the above references, is the modification of biomolecules prior to the actual particle production. Thus, WO 2010/138187 A1 discloses a process for the preparation of nucleic acid-modified polymer particles, wherein in the course of the process a reaction mixture of monomers, a part of which has bound an oligonucleotide, is formed, which transforms to a non-aqueous phase with droplet formation and then polymerized to form a network. The monomers which have bound the oligonucleotide are in particular acrydite oligonucleotide.

So far, it is impossible to produce biofunctional solid phases without subsequently modifying the solid phase and / or the function-determining biomolecules in the course of the reaction. Such particle functionalization, as shown above, involves several additional reaction steps. The success of these surface reactions is sometimes not easy to quantify. To make matters worse, typically a specific reaction path has to be developed for each individual biofunctionality. This means that new particles have to be developed and produced for each analytical task. In addition, the production of the polymer particles is typically based on polymerization processes, which often causes impurities due to incomplete reactions and possibly required solvents.

To overcome these problems, the present invention now proposes a convenient, one-step process for the production of biofunctional polymer particles, which comprises only one chemical reaction step, and avoids contaminating solvents or other by-products. The method according to the invention for producing biofunctional polymer particles according to claim 1 comprises the following substeps:

(i) preparing a solution containing at least one polymer, wherein the polymer is a copolymer having built-in crosslinker groups, and at least one biomolecule,

(ii) introducing the solution prepared in step (i) into a carrier fluid with simultaneous formation of spatially separated droplets, (Iii) solidification of the droplets obtained in step (ii) by activating the crosslinker groups by means of a trigger and subsequent crosslinking of the copolymers with binding of the biomolecules.

Preferred embodiments of the method according to the invention are defined in the subclaims. The invention will be described in detail below.

"Biofunctional polymer particles" are polymer particles to which a biomolecule is bound chemically, in particular covalently. In the context of the present invention, the biofunctional polymer particles are, in particular, the product of the process according to the invention, which will be described in detail below. Thus, if copolymers are used in the process according to the invention for the preparation of the biofunctional polymer particles, the term "biofunctional polymer particles" is synonymous with "biofunctional copolymer particles".

A "biomolecule" in the sense of the invention described herein is a biologically relevant substance, ie a substance which fulfills a biological function in an organism.

In particular, "non-modified biomolecules" are used in the context of the present invention. An "unmodified biomolecule" is a biomolecule that has not been chemically altered in a manner to achieve coupling ability in a polymerization reaction. The biomolecules used in particular in the context of the process according to the invention are therefore not modified (functionalized) in such a way that they are suitable as monomer in a polymerization reaction.

The "copolymer" in the context of the present invention is a polymer which is composed of at least two (chemically) different repeating units. Typically, one of these repeating units contains the crosslinker group, which is thus incorporated into the copolymer in the course of the copolymerization reaction.

The "crosslinker groups" which are incorporated in the copolymer are a structural unit which exists more than once in the copolymer and which can be replaced by an (external) Trigger can be activated to form reactive intermediates such as radicals, nitrene or carbene groups. Activation of the crosslinker groups initiates a reaction that allows insertion into C-H bonds located in the copolymer as well as in the biomolecules. The insertion reaction can take place in a concerted or non-concerted mechanism. Radicals are initially hydrogen abstraction. Recombination leads to the formation of covalent bonds, in particular between adjacent polymer chains and adjacent biomolecules. The crosslinker groups are preferably photoactive crosslinker groups which can be excited by light, in particular by UV light, to form reactive intermediates. The crosslinker groups are typically already present in one repeat unit of the copolymer and are incorporated into the copolymer in the course of the preparation of the copolymer, which is accomplished by polymerizing at least two different repeat units.

The activation of the crosslinker groups under radical formation takes place by means of a "trigger". The trigger is thus an (external) means for activating the crosslinker groups, which leads to a three-dimensional crosslinking of polymer chains and biomolecules in the context of the method according to the invention. Preferably, a hydrogel network is formed in which each biomolecule present in the solution is covalently bound during crosslinking.

In step (i) of the process according to the invention, a solution containing a copolymer with incorporated crosslinker groups and a non-modified biomolecule is prepared. For the preparation of this solution (in short: "copolymer / biomolecule solution"), aqueous solvents as well as organic solvents can be used. All solvents known to those skilled in the art which dissolve the copolymer used and the biomolecule function. Examples of solvents that may be used to prepare the copolymer / unmodified biomolecule solution include, but are not limited to, water, aqueous buffer solutions, mixtures of water and a water-miscible organic solvent such as ethanol, isopropanol, acetone , Acetic acid or ethyl acetate. Of course, the prerequisite for the use of these solvents is that the solvent does not destroy or reduce the activity of the biomolecules. Preferably copolymer and unmodified biomolecule are dissolved in step (i) of the process according to the invention in water or an aqueous buffer solution.

The copolymer used in the process according to the invention comprises a main repeat unit and at least one further repeat unit which is functionalized with the crosslinker group. The main repeating unit is the repeating unit, which represents the percentage of the strongest in the copolymer.

Examples of monomers that can be used in the present invention to form the main repeating unit in the copolymer include, but are not limited to: hydroxyethyl methacrylate (HEMA),

Hydroxyethoxyethyl methacrylate (HEEMA), hydroxyethylethoxyethyl methacrylate (HDEEMA), methoxyethyl methacrylate (MEMA), methoxyethoxyethylmethacrylate (MEEMA), methoxydiethoxyethylmethacrylate (MDEEMA), ethylene glycol dimethacrylate (EGDMA), acrylic acid (AA), N-vinyl-2-pyrrolidone (NVP), N -isopropyl AAm (NIPAAm), vinyl acetate (VAc), N- (2-hydroxypropyl) methacrylamide (HPMA), ethylene glycol (EG), PEG acrylate (PEGA), PEG methacrylate (PEGMA), PEG diacrylate (PEGDA), PEG dimethacrylate (PEGDMA) (where PEG = polyethylene glycol).

As already mentioned above, in the context of the process according to the invention in the crosslinking step (iii), a hydrogel network is preferably formed in which the biomolecules are incorporated. Hydrogels are formed from polymers that can absorb a large amount of water or form a water-swollen polymer network with water. Polymers which form a hydrogel with water are suitable and preferred for use in the present invention. Examples of polymers which form a hydrogel with water include but are not limited to: polyethylene glycols (PEG), polyacrylic acids, polydimethylacrylamides, polyvinyl alcohols, polyhydroxyethyl methacrylates, polyvinylpyrrolidones, alginates. In the context of the present invention, the corresponding copolymers thus derive from the (partly formal) monomers ethylene glycol, acrylic acid, dimethylacrylamide, vinyl alcohol, hydroxyethyl methacrylate, vinylpyrrolidones, and α-L-guluronic acid / β-D-mannuronic acid to form the main repeating unit of the copolymer.

The proportion of the percentage of repeating unit representing the highest percentage in the copolymer is typically 50-99 mol%, based on the molar amount of all in the copolymer repeating units present, with a proportion of 60-98 mol% being preferred, and a proportion of 70-95 mol% being even more preferred.

The crosslinker groups of the copolymer are, for example, benzophenone groups, diazocarbonyl groups such as, for example, diazoester groups and anthraquinone groups, and azide groups, benzophenone groups being preferred in the context of the present invention. Upon excitation by the trigger according to step (iii) of the process according to the invention, the benzophenone and anthraquinone groups react radically, the diazocarbonyl groups react via carbenes, the azides via nitrenes. Common to all crosslinker groups is the crosslinking via C, H insertion reactions.

The crosslinker groups are incorporated in the course of the preparation of the copolymer by preparing the copolymer from at least two different monomers, wherein at least one monomer contains the crosslinker group. For the preparation of such copolymers, those skilled in the art rely on known polymerization processes, for example polycondensation, polyaddition, anionic polymerization, cationic polymerization or free-radical polymerization. The preparation of a copolymer having N, N-dimethylacrylamide as a main monomer component and methacryloyloxybenzophenone as a monomer which introduces the crosslinker group into the copolymer is disclosed in Example 1 of the present invention description. Further examples of monomers with crosslinker group are acrylamide-3-hydroxy-2-anthraquinone (doi 10.1002 / adma.201703469) and diazomalonic acid ester monomer (doi 10.1002 / anie.201704486).

The typical proportion of repeat units with crosslinker group in the copolymer is 1-20 mol%, based on the molar amount of all repeating units present in the copolymer, with a proportion of 2-15 mol% being more preferred and a proportion of 5-10 mol%. % is even more preferred.

To improve the water-solubility of the copolymer, in the course of the preparation of the copolymer, it is optionally also possible to introduce further hydrophilic groups by copolymerization with monomers which contain corresponding hydrophilic groups, for example hydroxyl, sulfonic or carboxylic acid groups. The copolymer prepared in Example 1 of the present invention description contains besides N, N-dimethylacrylamide (main component) and methacryloyloxybenzophenone (crosslinking group component), also Na-4-styrenesulfonate, which serves to improve the water solubility of the copolymer.

The proportion of repeating units having a further hydrophilic group in the copolymer is up to 10 mol%, based on the molar amount of all repeating units present in the copolymer, a proportion of up to 5 mol% is preferred. When present in the copolymer, a minimum proportion of the repeating unit having another hydrophilic group is at least 0.5 mol%, preferably at least 1 mol%, even more preferably at least 2 mol%.

Typical molecular weights of the copolymer used in the process according to the invention are in the range of 100,000 g / mol to 1,000,000 g / mol, molecular weights of between 200,000 and 500,000 g / mol being preferred.

As already mentioned above, the solvent for preparing the solution in step (i) of the process according to the invention is preferably water or an aqueous buffer solution. The aqueous buffer solution serves to set a certain pH at which the biomolecule goes into solution. The pH ranges which can be used within the scope of the present invention are thus ultimately determined by the biomolecule itself. As long as it does not denature at a certain pH, the pH at which the biomolecule goes into solution is also usable. Typically, the pH of the solution is in the range of from 2 to 12, more preferably between 3 and 1 1. As buffer, buffer systems known to those skilled in the art may be used, for example sodium acetate, HEPES, sodium citrate, sodium succinate, Na-K-Phoshpat, TRIS, TRIS maleate, imidazole maleate, bistrispropane, CAPSO, CHAPS, MES, imidazole, DI-H20, PBS, PBS-T, NaPi-Tween (0.1), TNE or RiPa. The buffer phase can definitely participate in the crosslinking reaction, but the process according to the invention can still be carried out.

Any biomolecule is suitable for use in the method of the invention because each biomolecule has CH bonds that can be used to form the covalent bond with the solid phase. Examples of biomolecules that can be used in the process of the invention include, but are not limited to, oligo- and polynucleotides, synthetic nucleotides, DNA (Deoxyribonucleic acid), RNA (ribonucleic acid), including mRNA and microRNA (miRNA), oligo- and polypeptides, proteins, antibodies, enzymes and vitamins such as biotin.

The droplets in step (ii) of the method according to the invention are produced by means of two-phase microfluidics. For this purpose, the solution containing the unmodified biomolecule and the copolymer in dissolved form is introduced into a carrier fluid.

The carrier fluid is generally a liquid or gas which is not miscible with the solution prepared in step (i) of the process of the invention, which means that a two-phase system is obtained. Thus, if the solution prepared in step (i) is an aqueous solution (which can be prepared by dissolving the copolymer and the biomolecule in water or an aqueous buffer solution), the carrier fluid is, for example, a hydrophobic liquid, in particular to oil, may be the oil is fluorinated oil such as FC43 (perfluorotributylamine, C _I2 F ₂₇ N)), or silicone oils. Air can also be used as a carrier fluid, since a two-phase system is also obtained with air.

By entry of the copolymer / biomolecule solution prepared in step (i) of the process according to the invention, for example an aqueous copolymer / biomolecule solution, into a carrier fluid, for example into a hydrophobic liquid, in particular into an oil, a two-phase system is formed. With the entry spatially separated droplets are generated at the same time. This can be done, for example, by combining the solution containing the copolymer and the biomolecule in a microfluidic channel system at a T-junction with the carrier fluid, the T-junction resulting in a train of droplets of the copolymer / biomolecule solution in the carrier fluid leads.

So far, polymers could only be crosslinked in the dry / virtually solvent-free and therefore highly concentrated state. It is obvious that the use of dry polymers in a fluidic system would not be possible. Surprisingly, it has been found that a certain window exists for the concentration of the copolymer used in the process according to the invention for solution in corresponding buffers / solvents, within which the copolymer solution is sufficient is low in viscosity in order to achieve the desired compartmentation effects in the microfluidic system and, at the same time, sufficient polymer chains per volume are still present to allow intermolecular crosslinks to predominate. Typical concentrations for the copolymer in the solution produced in step (i) of the method of the invention are 10-1000 mg / mL, more preferably 40-500 mg / mL, even more preferably 70-300 mg / mL. Typical concentrations for the biomolecule in the solution produced in step (i) of the method of the invention are 0.2-2000 mM, more preferably 0.5-1500 mM, even more preferably 1-1000 mM.

In step (iii) of the process according to the invention, the droplets are hardened to form a solid phase. This is done by activating the crosslinker groups by means of an external trigger, such as light. This initiates a reaction that causes the cleavage of C-H bonds in the copolymer and biomolecule, thereby resulting in the rejoining of covalent bonds for three-dimensional cross-linking of copolymer and biomolecules in solution. The biomolecules are thus covalently incorporated into a solid polymeric network, which prevents the migration of biomolecules from the particle. Preferably, a hydrogel network is built, in which the biomolecules are incorporated.

The trigger is, for example, light, in particular UV light having a wavelength in the range of 200 to 400 nm, more preferably 250 to 385 nm, even more preferably 300 to 370 nm. The radiation passes through those contained in the copolymer Crosslinker groups in the case of benzophone groups an h-tt ^* transition into a biradical triplet state, which abstracts almost every hydrogen atom from adjacent CH groups in close proximity. Thus, two radicals are formed which can recombine to form a covalent bond linking the two chains, resulting in a (formal) C, H insertion-crosslinking reaction (CHic). Similar C, H insertion reactions can also be achieved via nitrene or carbene intermediates. As basically every biomolecule possesses CH groups, each biomolecule present in the solution is covalently bound to the network during crosslinking. If, for example, DNA, RNA, polynucleotides, oligonucleotides or synthetic nucleotides are used as biomolecules, crosslinking takes place via the base thymine. When proteins are used as biomolecules, Crosslinking takes place via the amino acids tyrosine and tryptophan, since these building blocks have CH groups with crosslinking properties.

Alternatively, the crosslinking reaction can be initiated by solidification of the particles by increasing the temperature, wherein the height of the temperature is in turn determined by the temperature stability of the biomolecule. Unsuitable are temperatures at which the biomolecule denatures. A typical temperature window for initiating the crosslinking reaction is 60-120 ° C under the previous premise. However, the preferred trigger is the irradiation of the droplets with UV light.

The solid biofunctional particles resulting from the solidification reaction typically have a diameter of 50 nm to 5 mm. The size of the particles is determined by the size of the channels in the microfluidic system, the flow rates in the system, and the structure of the microfluidic system in the area where the two phases meet. The particle size can be measured optically by light microscopy or scanning electron microscopy.

The carrier fluid is washed out and recycled after the solidification reaction. Since the carrier fluid is immiscible, a clean phase separation forms and the particles produced have no residues.

In a particular embodiment, the biofunctional polymer particles of the invention are substantially transparent and / or translucent.

In a further embodiment, the biomolecules are contained or embedded or incorporated in the interior of the biofunctional polymer particles according to the invention. It may also be on the surface of the polymer particles biomolecules, which are connected to the polymer.

The biofunctional polymer particles according to the invention are preferably hydrogel particles.

The advantages of the method according to the invention can therefore be summarized as follows. The inventive method allows the flexible production of biofunctional polymer particles directly in one stage (ie in a chemical Step) of copolymer and biomolecule, without pre- and post-processing of biomolecules or biofunctional particles is necessary. Since all biomolecules have CH bonds, the method can be used for a variety of biomolecules. Since the biomolecules are also covalently bound, migration of the biomolecules is prevented. The method according to the invention makes it possible for the user to dispense with technically demanding, time-intensive, expensive and multistage modifications to the biofunctionalization and purification steps. The biomolecules are enriched by the production of biofunctional polymer particles according to the method of the invention and can be easily analyzed or detected in this way. This is important for the detection of clinically relevant analytes.

Another advantage is that the polymer particles have a high capacity for loading with biomolecules, especially since the biomolecules are also incorporated in the interior of the polymer particles. If the polymer particles are essentially transparent, a fluorescence signal emitted by particles according to the invention can also be better detected by a detector than a signal from conventional particles which have biomolecules only on their surface.

Description of the pictures:

Figure 1: Schematic representation of the production of biofunctionalized polymer particles by the method according to the invention. The syringe pumps deliver the carrier fluid (oil) and the copolymer solution with the biomolecules. Both liquids are combined at a T-junction, resulting in a train of copolymer solution droplets within the carrier fluid. The copolymer within the droplets contains photoactive moieties which covalently crosslink with adjacent polymer chains and the biomolecules upon exposure in the downstream UV chamber. The solidified particles are collected in an Eppendorf tube and used in the respective experiments.

Figure 2: Light intensity of fluorescence uptake of polymer particles functionalized with either a) biotin or b) DNA probes. The inserts show typical Fluorescence micrographs of the respective polymer particles. The graphs are normalized to the respective intensity of the positive controls.

The following embodiments are intended to illustrate and further illustrate the process of the invention without limiting the subject matter of the invention.

Example 1: Preparation of an N, N-dimethylacrylamide-based copolymer with incorporated benzophenone groups

A random copolymer consisting of three molecular building blocks was synthesized using a radical polymerization method. In a typical run, N, N-dimethylacrylamide (DMAA, 92.5 mol%),

Methacryloyloxybenzophenone (MaBP, 5 mol%), and Na-4-styrenesulfonate (SSNa, 2.5 mol%) dissolved under nitrogen in methanol at a concentration of 2 mol / 1 and 0.1 mol% o, g'-azoisobutyronitrile (AIBN) added. After five cycles of freezing and thawing, the solution was placed in a preheated water bath and held at 60 ° C for 20 hours. After completion of the polymerization reaction, the polymer was precipitated by dropwise addition of the solution to a large excess of diethyl ether, filtered off and purified by reprecipitation from methanol. After freeze-drying, the copolymer was obtained as a white powder with a typical yield of 50% and Mw of about 300,000 g / mol.

The major component of the copolymer is N, N-dimethylacrylamide, which serves as a hydrophilic matrix to prevent nonspecific interactions with the biological environment (e.g., protein adsorption). 4-methacryloxybenzophenone (MABP, 5 mol%) serves as a second building block and provides photoreactive groups to the polymer. To increase the water solubility of the final copolymer, the sodium salt of 4-styrenesulfonate (SSNa, 2.5 mol%) is used as the third component, so that the resulting copolymer has a solubility of more than 300 g / L.

Example 2: Preparation of biofunctional polymer particles

The system chosen for the embodiment is based on the photocrosslinking of an aqueous solution of the copolymer prepared in Example 1. As biomolecules i) biotin or ii) DNA (HPV oligo 19, 36mer) are used. 200 mg copolymer (100 mg / ml_) and 0.2 mg biomolecule (0.1 mg / ml_) are dissolved in 2 ml_ in aqueous phase. The aqueous phase is a PBS (0.25x) buffer system for the biotin example, and a NaPi (100 mM) buffer system for the DNA example.

Using microfluidics, 20 μl of the aqueous solutions thus obtained are sheared off in individual droplets at a T junction in FC 43. The photocrosslinking of the droplets is carried out by UV irradiation at 365 nm in a redesigned UV chamber at a dose of 8 J / cm ² . This particle generation process with integrated biofunctionalization is shown in Figure 1.

Example 3 Analysis of the Biofunctional Prepared by the Above Method

polymer particles

The biofunctional polymer particles obtained by the method described above and unmodified, i. non-biofunctionalized, polymer particles (prepared from the polymers of Example 1, the particles were obtained analogously to the above procedure according to Example 2 without biomolecule) were incubated with a corresponding fluorescently labeled counterpart. For the protein assay, the fluorescently-labeled counterpart was cyanine 5-labeled streptavidin and HPV-19 antisense labeled with cyanine 5 for the DNA assay. The background-corrected light intensities of the respective particles, which were measured by fluorescence microscopy, as well as a schematic representation of the respective binding principle are shown in Figures 2 a) and 2 b).

The respective modified particles act as positive controls and the unmodified particles as negative controls.

The data show a successful immobilization and homogeneous staining of biomolecules. A significantly higher positive signal compared to the signal of the unmodified polymer particles from the negative control indicates the low unspecific adsorption of the polymer used. The coefficient of variation of the fluorescence signal from the functionalized particles is 5% for the biotin-functionalized particles and 3% for the DNA-functionalized particles. These values can compete well with the reported values in the literature in which biofunctionality has been brought about by several upstream or downstream steps. The detection limit of the labeled protein target streptavidin-Cy5 of the biotinylated hydrogel particles was investigated by gradually reducing the concentration of the labeled protein target streptavidin-Cy5 in the incubation solution. This gives a detection limit of 9 pM for the presented assay. In addition, the fluorescence signal shows the expected linear dependence on the concentration, as shown in Figure 2c.

Claims

claims

A method of producing biofunctional polymer particles, comprising the following steps:

(ii) introducing the solution prepared in step (i) into a carrier fluid with simultaneous formation of spatially separated droplets,

(Iii) solidification of the droplets obtained in step (ii) by activating the crosslinker groups by means of a trigger and subsequent crosslinking of the copolymers with binding of the biomolecules.

2. Process according to claim 1, wherein the crosslinker groups incorporated in the copolymer are benzophenone groups, diazocarbonyl groups, azide groups or anthraquinone groups, preferably benzophenone groups.

3. The method according to any one of the preceding claims, wherein it is the solution prepared in step (i) is an aqueous solution or an aqueous buffer solution.

4. The method according to any one of the preceding claims, wherein the biomolecule is oligo- and polynucleotides, synthetic nucleotides, DNA, RNA, oligo- and polypeptides, protein, antibodies, enzymes or vitamins.

5. The method according to any one of the preceding claims, wherein the generation of the droplets in step (ii) takes place in that the solution prepared in step (i) (copolymer / biomolecule solution) in a channel system at a T-junction merged with the carrier fluid with the T-junction resulting in a train of droplets of the copolymer / biomolecule solution in the carrier fluid.

A method according to any one of the preceding claims, wherein the carrier fluid is oil or air.

7. The method according to any one of the preceding claims, wherein in step (iii) forms a hydrogel network, in which the biomolecules are covalently bound.

8. The method according to any one of the preceding claims, wherein it is the trigger used in step (iii) is UV light, preferably UV light in a wavelength range of 200-400 nm.

9. Biofunctional polymer particles obtainable by the process according to any one of claims 1-8.

10. Biofunctional polymer particles according to claim 9, characterized in that they are substantially transparent.

1 1. Biofunctional polymer particles according to claim 9 or 10, characterized in that they are translucent.

12. Biofunctional polymer particles according to any one of claims 9 to 1 1, characterized in that biomolecules are contained or embedded in the interior of the polymer particles.

13. Biofunctional polymer particles according to claim 12, characterized in that there are also on the surface of the polymer particles biomolecules which are connected to the polymer.

14. Biofunctional polymer particles according to any one of claims 9 to 13, characterized in that they are hydrogel particles.

15. Use of biofunctional polymer particles according to any one of claims 9 to 14 for the analysis of biomolecules.