DIBLOCK COPOLYMER, A MANUFACTURING METHOD AND SUITED APPLICATIONS

Abstract

It is provided a diblock copolymer consisting of a first block, a second block, and a linker, wherein the second block is covalently bound to the first block by the linker. Thereby, the first block is a glycerol block comprising 1 to 10 glycerol subunits that are optionally substituted, and the second block is a superhydrophobic block comprising a perfluoroether residue having at least 20 carbon atoms.

Claims

1. A diblock copolymer consisting of a first block, a second block, and a linker, wherein the second block is covalently bound to the first block by the linker, wherein the first block is a glycerol block comprising 1 to 10 glycerol subunits that are optionally substituted, and the second block is a superhydrophobic block comprising a perfluoroether residue having at least 20 carbon atoms.

2. The diblock copolymer according to claim 1, wherein the glycerol block comprises an optionally substituted oligoglycerol having 3 to 10 glycerol subunits.

3. The diblock copolymer according to claim 2, wherein the oligoglycerol is a branched oligoglycerol.

4. The diblock copolymer according to any of the preceding claims, wherein the glycerol block corresponds to any of general formulae (I) to (VIII) ##STR00011## ##STR00012## wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8 is independently from each other SH, CH.sub.3, CH.sub.2CH.sub.3, NH.sub.2, OH, OCH.sub.3, OCH.sub.2CH.sub.3, OCH.sub.2OCH.sub.2CH.sub.3, NH.sub.3, OPO.sub.3 (CH.sub.2).sub.2N+(CH.sub.3).sub.3, (CH.sub.3).sub.2N+(CH.sub.2).sub.2OPO.sub.2(OCH.sub.3), (CH.sub.3).sub.2N+(CH.sub.2).sub.2OPO.sub.2(OH), or OSO.sub.3, and X is O or S.

5. The diblock copolymer according to claim 1, wherein the superhydrophobic block comprises two carbon-containing chains that are bound to the same linker but are not covalently crosslinked to each other.

6. The diblock copolymer according to claim 1, wherein the superhydrophobic block comprises a residue according to general formulae (IX) or (X), ##STR00013## wherein n=10 to 50.

7. The diblock copolymer according to claim 1, wherein the linker is chosen from the group consisting of amines, amides, oxygen, and an alkyl residue having 1 to 20 carbon atoms, wherein a hydrocarbon chain of the alkyl residue can be interrupted by one or more oxygen, nitrogen and/or sulfur atoms and/or can be substituted by a group comprising one or more oxygen, nitrogen and/or sulfur atoms.

8. The diblock copolymer according to claim 1, wherein the linker is not a methylene group.

9. A method for manufacturing a diblock copolymer according to claim 1, comprising reacting an oligoglycerol amine comprising protected hydroxyl groups with a perfluorinated polyether acid chloride in an organic solvent, and deprotecting the hydroxyl groups of the obtained diblock copolymer.

10. The method according to claim 9, wherein the oligoglycerol amine comprising protected hydroxyl groups is manufactured by mesylating an oligoglycerol comprising protected hydroxyl groups and at least one unprotected hydroxyl group to introduce a mesyl group into the oligoglycerol, substituting the mesyl group with an azide group and hydrogenating the azide group to obtain the oligoglycerol amine comprising protected hydroxyl groups.

11. The method according to claim 9, wherein the perfluorinated polyether acid chloride is manufactured by reacting a perfluorinated polyether with oxalyl chloride.

12.-13. (canceled)

14. A method for carrying out a reaction in a droplet, wherein the droplet comprises a plurality of diblock copolymers according to claim 1, wherein the reaction is chosen from the group consisting of a PCR reaction and a testing of an effect of a substance on a cell incorporated in the droplet.

15. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Further details of aspects of the solution will be explained with respect to exemplary embodiments and accompanying Figures.

[0062] FIG. 1A shows droplets made from a PFPE-PEG-PFPE triblock copolymer according to U.S. Pat. No. 9,012,390 B2 after performing a PCR at 95 C.

[0063] FIG. 1 B shows droplets made from a PFPE-LPG(OMe)-PFPE triblock copolymer according to Wagner et al. after performing a PCR at 95 C.

[0064] FIG. 1C shows droplets made from an embodiment of a diblock copolymer as described herein after performing a PCR at 95 C.

[0065] FIG. 2A shows a size distribution of the droplets depicted in FIG. 1A.

[0066] FIG. 2B shows a size distribution of the droplets depicted in FIG. 1B.

[0067] FIG. 2C shows a size distribution of the droplets depicted in FIG. 1C.

[0068] FIG. 3A shows different panels depicting differently composed droplets with and without fluorescence dye directly after generating the droplets (0 hours) and after an incubation period of 72 hours.

[0069] FIG. 3B shows droplets stabilized by LG0 with and without fluorescence dye after an incubation period of 72 hours.

[0070] FIG. 3C shows droplets stabilized by MG0 with and without fluorescence dye after an incubation period of 72 hours.

[0071] FIG. 4 shows a plot on the cell viability of a leukemia cell line incorporated within droplets of different composition.

[0072] FIG. 5 shows a reaction scheme of an embodiment of a method for synthesizing a protected oligoglycerol amine.

[0073] FIG. 6 shows a reaction scheme of an embodiment of a method for chlorinating a perfluorinated polyether.

[0074] FIG. 7 shows a reaction scheme of an embodiment of a method for manufacturing a diblock copolymer starting from a protected oligoglycerol amine and a perfluorinated polyether chlorate.

[0075] FIG. 8A shows FT-IR spectra of embodiments of diblock copolymers having three glycerol units.

[0076] FIG. 8B shows FT-IR spectra of embodiments of diblock copolymers having one glycerol unit.

[0077] FIG. 9A shows a reaction scheme for the synthesis of 1,3-bis((2,2-dimethyl-1,3-oxathiolan-5-yl)methoxy)propan-2-ol.

[0078] FIG. 9B shows an 1H-NMR spectrum of the acetal-protected 1,3-bis((2,2-dimethyl-1,3-oxathiolan-5-yl)methoxy)propan-2-ol.

[0079] FIG. 10A shows size distributions of droplets prepared of embodiments of a diblock copolymer before performing a PCR test.

[0080] FIG. 10B shows microscopic pictures of the droplets, the size distributions of which are depicted in FIG. 10A.

[0081] FIG. 10C shows size distributions of droplets of the same diblock copolymers as in FIG. 10A after performing a PCR test.

[0082] FIG. 10D shows microscopic pictures of the droplets, the size distributions of which are depicted in FIG. 10C.

[0083] FIG. 11A shows microgels formed with the help of diblock copolymers serving as surfactants before surfactant removal (lower panels) and after surfactant removal (upper panels).

[0084] FIG. 11B shows the size distribution of microgel particles when MG1 is used as surfactant.

[0085] FIG. 11C shows the size distribution of microgel particles when HG1 is used as surfactant.

[0086] FIG. 11D shows the size distribution of microgel particles when LG1 is used as surfactant.

[0087] FIG. 11E shows precursor macromolecules that were used to prepare the microgel templates and a reaction scheme of the applied chemical reaction.

[0088] FIG. 12A shows a fluorescence image of mixed drop populations of a cross-talking experiment.

[0089] FIG. 12B shows an overlay of bright field and fluorescence of the mixed drop populations of FIG. 12A.

[0090] FIG. 12C shows a fluorescence image of the positive control of the cross-talking experiment.

[0091] FIG. 12D shows an overlay of bright field and fluorescence of the positive control of FIG. 12C.

DETAILED DESCRIPTION

[0092] The Figures will now be explained with respect to exemplary embodiments.

Example 1: Temperature Stability

[0093] The diblock copolymers described herein have the capability to stabilize emulsions even at high temperatures which can be essential for performing assays in droplets. For example, polymerase chain reaction (PCR) is the most widely used method for DNA amplification which is an indispensable constitute for drop-based biological assays. However, PCR requires as high as 98 C. incubation temperature in amplification cycles. At this high temperature, emulsions with PCR reagents stabilized by PFPE-PEG-PFPE triblock copolymers known from prior art fail to remain stable and coalesce.

[0094] Monodisperse PCR reagent droplets having a diameter of about 75 m stabilized by 2% (w/w) surfactant in HFE 7500 solution were tested. The following surfactants were used for building up the droplets: [0095] a) PFPE-PEG-PFPE triblock copolymer as described in U.S. Pat. No. 9,012,390 B2 (Krytox-PEG-Krytox); [0096] b) PFPE-LPG(OMe)-PFPE triblock copolymer as described in Wagner et al. (Krytox-LPG(OMe)-Krytox); [0097] c) PFPE-PG diblock copolymer according to the following formula (XI) (G1-Krytox; LG1):

##STR00006##

[0098] The used PCR kit was the Phusion kit from Thermo Fisher with detergent-free buffer. Lambda Phage genome fragmented and tagged by Nextera Kit was used as template.

[0099] After performing 35 PCR cycles with 98 C. denaturation temperature (whole cycling conditions: 98 C. for 30 s, then 35 cycles of: 98 C. for 7 s, 60 C. for 30 s, 72 C. for 20 s. After cycling, 72 C. for 10 min), about 25% of emulsions stabilized by PFPE-PEG-PFPE merged into a water layer while only 10% of emulsion stabilized by LG1 merged into water layer.

[0100] The droplets that survived the PCR cycles were examined under microscope. Droplets stabilized by PFPE-PEG-PFPE (FIG. 1A) and PFPE-LPG(OMe)-PFPE (FIG. 1B) were no longer monodisperse. This clearly indicates that they were destabilized due to the temperature treatment. Droplets stabilized by LG1 remained, however, monodisperse (FIG. 1C).

[0101] The size of the droplets stabilized by different surfactants was examined in more detail by measuring the sizes of the droplets depicted in FIGS. 1A to 1C and assigning a size distribution to the corresponding droplets. The results are shown in FIGS. 2A to 2C. PFPE-PEG-PFPE (FIG. 2A) and PFPE-LPG(OMe)-PFPE (FIG. 2B) show a broad size distribution in a diameter range of approximately 25 to 60 m with a highest incidence in both cases at 29 to 33 m. In contrast, the droplets stabilised by LG1 only show a very narrow size distribution in a diameter range of 70 to 82 m with a highest incidence at 74 to 78 m (FIG. 2C).

Example 2: Cross-talking among droplets

[0102] As a superb platform for precisely miniaturizing and compartmentalizing chemical assays, droplet-based microfluidics is of particular interest in a number of biotechnological applications. The technology essentially utilizes water-in-oil emulsions as droplet microreactors, typically at nanoliter or picoliter scale. The extent of application is, however, limited by the so-called cross-talking between droplets. Cross-talking denotes molecular transport between droplets, leading to an inaccuracy of the droplet-based assays. As driven by the thermodynamic disequilibrium of aqueous/oil interfaces, the cross-talking phenomenon is also affected by the type of surfactant used to stabilize the emulsion.

[0103] To test the cross-talking phenomenon, two kinds of aqueous droplets were made and incubated for 72 hours: smaller droplets incorporating fluorescent dye dissolved in phosphate-buffered saline (PBS) (positive droplets) and larger droplets incorporating only PBS without dye (negative droplets). These two types of droplets were prepared with different droplet-stabilizing surfactants using a surfactant concentration of 2% (w/w) in a HFE 7500 solution.

[0104] Images of the droplets were taken immediately after generating the droplets (0 hours) and after an incubation period of 72 hours using a confocal microscope.

[0105] Panels a and b of FIG. 3A show droplets stabilized by PFPE-PEG-PFPE. At 0 hours, the positive droplets 1 show a strong green fluorescence (light grey in FIG. 3A), whereas the negative droplets 2 do not show any green fluorescence at all (dark grey in FIG. 3A). After an incubation period of 72 hours, the fluorescence of the previously positive droplets 1 diminished, whereas the fluorescence of the previously negative droplets 2 increased. Rather, an almost equal distribution of the fluorescence between previously positive droplets 1 and previously negative droplets 2 could be observed.

[0106] Panels c and d of FIG. 3A show droplets stabilized by LG1. At 0 hours, the positive droplets 3 show a strong green fluorescence (light grey in FIG. 3A), whereas the negative droplets 4 do not show any green fluorescence at all (dark grey in FIG. 3A). After an incubation period of 72 hours, the fluorescence of the positive droplets 3 almost remained unchanged, whereas the negative droplets 4 did still not show a significant fluorescence. This clearly indicates that LG1-stabilized droplets decrease the extent of cross-talking between individual droplets. Thus, if a substance is incorporated into an LG1-stabilized droplet, it remains within this droplet and is not transported to another droplet to a significant extent.

[0107] Panels e and f of FIG. 3A show droplets stabilized by MG1. MG1 has a similar structure like LG1 but a higher molecular weight than LG1. It corresponds to formula (XII):

##STR00007##

[0108] At 0 hours, the positive droplets 5 show a strong green fluorescence (light grey in FIG. 3A), whereas the negative droplets 6 do not show any green fluorescence at all (dark grey in FIG. 3A). After an incubation period of 72 hours, the fluorescence of the positive droplets 5 almost remained as strong as at 0 hours, whereas the negative droplets 6 did still not show any fluorescence. This clearly indicates that also MG1-stabilized droplets do not show cross-talking between individual droplets. Thus, if a substance is incorporated into an MG1-stabilized droplet, it remains within this droplet and is not transported to another droplet.

[0109] Panels g and h of FIG. 3A show droplets stabilized by HG1. HG1 has a similar structure like LG1 and MG1 but an even higher molecular weight than MG1. It corresponds to formula (XIII):

##STR00008##

[0110] At 0 hours, the positive droplets 7 show a strong green fluorescence (light grey in FIG. 3A), whereas the negative droplets 8 do not show any green fluorescence at all (dark grey in FIG. 3A). After an incubation period of 72 hours, the fluorescence of the positive droplets 7 almost remained as strong as at 0 hours, whereas the negative droplets 8 did still not show any fluorescence. This clearly indicates that also HG1-stabilized droplets do not show cross-talking between individual droplets. Thus, if a substance is incorporated into an HG1-stabilized droplet, it remains within this droplet and is not transported to another droplet.

[0111] FIG. 3B shows droplets stabilized by LG0. LG0 has a similar molecular structure like LG1, but only a single glycerol unit in the glycerol block. It corresponds to formula (XIV):

##STR00009##

[0112] After an incubation period of 72 hours, positive droplets 9 showed a strong green fluorescence (light grey in FIG. 3B), whereas the negative droplets 10 did not show any significant green fluorescence (dark grey in FIG. 3B), Thereby, the fluorescence of the positive droplets 9 was almost as strong as directly after generating the droplets. This clearly indicates that also LG0-stabilized droplets did not show cross-talking between individual droplets. Thus, if a substance is incorporated into an LG0-stabilized droplet, it remains within this droplet and is not transported to another droplet.

[0113] FIG. 3C shows droplets stabilized by MG0. MG0 has a similar structure like LG0 but a higher molecular weight than LG0. It corresponds to formula (XV):

##STR00010##

[0114] After an incubation period of 72 hours, positive droplets 11 showed a strong green fluorescence (light grey in FIG. 3C), whereas the negative droplets 12 did not show any significant green fluorescence (dark grey in FIG. 3C), Thereby, the fluorescence of the positive droplets 11 was almost as strong as directly after generating the droplets. This clearly indicates that also MG0-stabilized droplets did not show cross-talking between individual droplets. Thus, if a substance is incorporated into an MG0-stabilized droplet, it remains within this droplet and is not transported to another droplet.

Example 3: Superior Biocompatibility

[0115] The biocompatibility of LG1, MG1, and HG1 was assessed by means of a cell viability test. In the test, individual cells of the leukemia cell line K562 were encapsulated in the droplets (100 m). Fluorinated oil was used as the continuous phase and cell culture medium (DMEM) supplemented with 10% FBS was used as the aqueous phase. After 3 days of incubation, the droplets were destabilized by perfluorooctanol (PFO) and the cell viability was evaluated using a live/dead staining kit. As shown in FIG. 4, the cell viability in droplets stabilized by PFPE-PEG-PFPE (comparative example; negative control) was found to be 54%, whereas the cell viability in LG1-, MG1-, and HG1-stabilized droplets was above 90% for all three types of droplets stabilized by a diblock copolymer as described herein. The cell viability in LG1-, MG1-, and HG1-stabilized droplets almost achieved a viability rate of 97% observed for cells incubated for 3 days in cell culture medium (DMEM) supplemented with 10% FBS (comparative example; positive control). No droplets were formed in this positive control.

Example 4: Synthesis of an Embodiment of a Diblock Copolymer

[0116] First, a protected oligoglycerol amine was manufactured according to the reaction scheme depicted in FIG. 5. The starting material was an acetal protected [G1] polyglycerol dendron hydroxy that was dried in high vacuum at 60 C. overnight. Thereafter, G1 dendron-N.sub.3 was prepared in a two-step process. First, the mesylation of the hydroxyl groups was carried out using 1.5 equivalent of methane sulfonyl chloride (MsCl) and two equivalents of triethyl amine in dichloromethane (DCM) at 0 C. to room temperature (RT). The mesylated compound was extracted in DCM and then dried under high vacuum (HV). Afterwards, the dried compound was dissolved in dimethylformamide (DMF) under argon, and NaN.sub.3 (3 eq.) was added to the above reaction mixture. Substitution of the mesyl group with azide was carried out overnight at 70 C. The final compound was obtained in 95% yield after extraction in DCM followed by drying in HV.

[0117] Finally, G1 dendron-N.sub.3 was dissolved in methanol and hydrogenation of azide (N.sub.3) functional groups was conducted in presence of palladium catalyst (10% Pd/Charcoal) in a reactor with vigorous stirring for two days. The reduced compound was filtered off using a bed of Cellite545. Subsequently, the solvent was removed and the residue was purified by column chromatography on silica gel using a hexane:isopropanol mixture as eluent. The thus purified product was obtained with 75% yield after drying in HV.

[0118] Separate from this, a perfluorinated polyether (PFPE) was activated to acid chloride overnight by reacting it with oxalyl chloride according to the reaction scheme depicted in FIG. 6. The protected oligoglycerol amine synthesized according to the reaction scheme depicted in FIG. 5 and the PFPE-COCl synthesized according to the reaction scheme depicted in FIG. 6 were then reacted according to the reaction scheme in FIG. 7 so that per-fluorinated diblock-copolymer based surfactants were synthesized in a three-step process. An amide bond is formed in the final step, wherein the amine group acts as linker between the PFPE block and the oligoglycerol block.

[0119] Firstly, a catalytic amount of DMF and oxalyl chloride (10 eq.) was added under argon atmosphere to a solution of perfluorinated polyethers (PFPE) in HFE-7100 to convert the terminal carboxylic group of PFPE to acid chloride. In the meantime, the argon filled balloon was removed and the outlet of a Schleck flask was connected to a Schleck line to let the gases escape during the reaction. After several minutes, the outlet valve was closed and an argon balloon was introduced again. The reaction mixture was stirred overnight at RT. Next, HFE-7100, DMF, and all volatile gases were removed under HV using an addition cold trap. The PFPE species Krytox 157 FSH, Krytox 157 FSM, and Krytox 157 FSL were used as high, medium, and low molecular weight block, respectively, and all of them were activated to acid chloride in a similar fashion.

[0120] Next, the viscous PFPE acid chloride was diluted adding small amount of HFE-7100 oil. Then a freshly prepared solution of G1 dendron-NH.sub.2 (dried under HV at 50 C.) (1.07 eq. to PFPE acid chloride) in dry DCM was mixed with HFE-7100 oil in a ratio of 1:0.75. Later, dropwise addition of G1 dendron-NH.sub.2 solution into the activated Krytox compound was performed under inert condition using a dropping funnel over a period of 1 h. The reaction mixture was then refluxed overnight at 45-50 C. The crude product was transferred into a one neck round bottom flask. DCM was evaporated under reduced pressure followed by removal of HFE-7100 in HV using an additional cold trap. Lastly, the dried viscous compound was dissolved in a mixture of HFE-7100 and methanol solution and deprotection acetal groups was carried out overnight at 50 C. adding 0.5-1 mL of 1M HCl solution to the above reaction mixture. The thus prepared high, medium, and low MW diblock-copolymer surfactants (having 15, 27 or 43 repeating units) were obtained with a yield of 75-85% after drying in HV.

Example 5: Characterization of the Dendronized Fluorosurfactants

[0121] The successfully synthesized surfactants with different generation oligoglycerols, e.g. monoglycerol (G0) and triglycerol (G1), and Krytox blocks were characterized by Fourier transform infrared (FT-IR) spectroscopy. FIG. 8A shows the FT-IR spectra of HG1, MG1, and LG1. FIG. 8B shows the FT-IR spectra of MG0 and LG0.

[0122] Typically, the terminal carbonyl stretch (CO) of the starting perfluorinated block (PFPE) shows, irrespective of its molecular weight, an absorbance peak at 1775 cm.sup.1. Therefore, a representative spectrum of the PFPE polymer is presented by a dashed line. On the other hand, the absorption peaks at about 1675 cm.sup.1 and 1725 cm.sup.1 indicate the amide stretch vibration (CONH) in the synthesized surfactants when G0 and G1 dendrons are incorporated into the PFPE block.

Example 6: Synthesis of a Protected Dithiolated G1-OH Dendron

[0123] A protected dithiolated G1 dendron [1,3-bis((2,2-dimethyl-1,3-oxathiolan-5-yl)methoxy)propan-2-ol] was synthesized using epichlorohydrin and (2,2-dimethyl-1,3-oxathiolan-5-yl)methanol according to the reaction scheme depicted in FIG. 9A. This synthetic procedure includes a reaction of one mole equivalent of epichlorohydrin with four mole equivalents of (2,2-dimethyl-1,3-oxathiolan-5-yl)methanol and a base e.g. sodium hydroxide (NaOH). The 1H-NMR spectrum depicted in FIG. 9B confirms the successful synthesis of the acetal-protected dithiolated G1-OH dendron. The secondary hydroxyl (OH) group can itself be a functional group and be modified to any other functional groups, e.g. amine, azide, halogen etc. Moreover, after acetal deprotectionapart from the possibility to modify the hydroxyl groups to other functional groupsthe dithiols can be protected with any suitable thiol protecting groups, e.g. with 2,2-dipyridyldisulfide.

[0124] Finally, both monothiolated G0 dendron ((2,2-dimethyl-1,3-oxathiolan-5-yl)methanol) and dithiolated G1 dendron can be used to prepare a variety of dendronized diblock copolymer fluorosurfactants. The novel protected dithiolated G1-OH dendron can also be utilized to formulate other nanostructures. In particular, micelles, vesicles, liposomes, and oil-in-water (O/W) emulsions can be also obtained using monothiolated and dithiolated G1 dendron as polar head groups along with any fluorinated block e.g. fluorinated alkyl chain, PFPE, and classical alkyl chain derivatives.

Example 7: Temperature Stability (PCR Reaction in Droplets)

[0125] This example was performed using the same reaction conditions as in Example 1.

[0126] Different surfactants based on G1 dendrons along with PFPE-PEG-PFPE as a control were used to perform the PCR reaction. Droplets having a diameter of approximately 100 m were prepared with a coefficient of variation (CV) of around 2% in all cases. The corresponding size distributions before performing a PCR are depicted in FIG. 10A. FIG. 10B shows microscopic pictures of these droplets.

[0127] After PCR test, HG1, MG1, and LG1 surfactants demonstrated excellent single drop resolution stability with CVs of about 19%, 10%, and 11%, respectively. The corresponding size distributions after performing the PCR test are depicted in FIG. 10C. On the other hand, the control group showed instability due to the PCR reaction at elevated temperature. To be more specific, it exhibited a 2.5 times higher CV (25%) as compared to MG1 and LG1. FIG. 10D shows microscopic pictures of the droplets after the PCR test.

Example 8: Synthesis of Microgel Template

[0128] Triglycerol polar head group based surfactants were used to prepare microgel particles. A strain-promoted azide alkyne cycloaddition (SPAAC) reaction was performed to form a three-dimensional cross-linked network, i.e. the microgels. Different dye labelled cross linkers were used to identify different microparticles prepared incorporating HG1, MG1, and LG1 surfactants. However, the formation of microgel particles is not limited to the macromolecules used in this work. Ideally, any polymeric material suitable for microparticle formation in an aqueous environment can be used to make templates of different sizes ranging from one micrometer to several hundred micrometers using the diblock copolymer surfactants as the stabilizer of the aqueous phase. Here, microparticles with a diameter of about 50 m were prepared with a CV of about 2% with all three types of fluorosurfactants (HG1, MG1, and LG1). FIG. 11A shows templated microgels before surfactant removal (lower panels) and after surfactant removal (upper panels). FIGS. 11B to 11D show the corresponding size distributions, namely of MG1 (FIG. 11B), HG1 (FIG. 11C), and of LG 1 (FIG. 11D). FIG. 11E shows precursor macromolecules that were used to prepare the microgel templates and a reaction scheme of the applied chemical reaction.

Example 9: Drug Screening Mimetic Biological Assay (Cross Talking)

[0129] To determine cross-talking phenomena among droplets under biological environment Human Embryonic Kidney cell 293 (HEK 293) was genetically modified using Piggybac transposition gene editing tool. Two plasmid vectors, namely hyPBase (vector was obtained from the Wellcome Trust Sanger Institute, UK) and XLone-GFP (vector was obtained from Xiaojun Lian Lab (Pennsylvania State University, USA) through Addgene) were incorporated to insert a gene of interest (GOI) in the host cell's genome. The genetically modified stable HEK293 cells can express a fluorescent protein, e.g. green fluorescence protein (GFP), only in presence of a suitable drug, e.g. doxycycline (Dox). Herein, as positive control, HEK293 cells were dispersed in doxycycline-containing cell culture medium. Afterwards, drops having a diameter of about 80 m were prepared.

[0130] To investigate the cross-talking phenomena, two different sets of drops were co-mixed, wherein one set of drops contained the Dox-inducible HEK293 cells and the other set of drops contained doxycycline solution. On day 1, images of drops from positive control and mixed drop populations were taken using fluorescence microscope.

[0131] FIG. 12A shows a fluorescence image and FIG. 12B an overlay of bright field and fluorescence of mixed drop populations. FIG. 12C shows a fluorescence image and FIG. 12D an overlay of bright field and fluorescence of the positive control. The arrows in the images are used only to guide the eyes to facilitate distinguishing GFP+ from GFP cells.