Fluorine-containing surfactant compound system as well as preparation method and application thereof

Abstract

The invention discloses a fluorine-containing surfactant compound system as well as a preparation method and application thereof, and belongs to the technical field of surfactants. The fluorine-containing surfactant compound system disclosed by the invention comprises two or more than two fluorocarbon surfactants; the fluorocarbon surfactant is formed by covalent coupling of a perfluoropolyether polymer and a hydrophilic compound; the synthetic method of the fluorocarbon surfactant is mild in reaction condition, simple in preparation process and easy to industrially apply; the hydrogel has the advantages of good biocompatibility, temperature resistance, good solubility and the like; the product can be used as an emulsifier applied to a microfluidic droplet technology.

Description

Technical Field

[0001] The invention relates to the technical field of surfactants, and in particular to a fluorine-containing surfactant compound system and a preparation method and application thereof. Background Art

[0002] In recent years, microfluidic droplet technology has been widely used in various fields of biomedical research, including high-throughput drug screening, digital polymerase chain reaction, single-cell sequencing, enzyme immobilization and directed evolution. This method usually uses immiscible two-phase fluids to form a dispersed phase and a continuous phase under the action of shear force. Its typical preparation method is: disperse the working liquid / bioactive substance in an aqueous solution, and use the aqueous solution and the oil phase to interact in a microfluidic chip to form an oil-in-water single emulsion. Based on the above mechanism, the interfacial tension between the two phases when water and oil interact has a great influence on the size and morphology of the actual droplets obtained. In practical applications, the addition of surfactants is often used to reduce the interfacial tension and stabilize the oil-water interface.

[0003] Fluorocarbon oils are both hydrophobic and lipophobic in their structure, and are basically unable to dissolve bioactive molecules. They have a good inhibitory ability on molecular diffusion between droplets. In addition, they have been shown to have high biosafety and gas solubility. Therefore, fluorinated oil-in-water droplets stabilized by fluorinated surfactants are favored in biological research. RANBiotechnologies' fluorinated surfactant (008-FluoroSurfactant) is widely used in the market to stabilize emulsion droplets in fluorinated oil. This is a non-ionic triblock copolymer composed of perfluoropolyether and polyethylene glycol. However, a large number of droplets still merge during thermal cycling (PCR). The poor performance of its surfactant is partly due to its synthetic and structural limitations. Due to the hydrophobicity and thermoresponsiveness of polyethylene glycol molecules, it may destroy the stability of droplets during temperature changes. In addition, due to the polydisperse nature of polyethylene glycol and perfluoropolyether, the exact molar amount or molecular ratio of each molecule cannot be determined during the synthesis of the triblock copolymer. As a result, a mixture of diblock, triblock, unmodified precursor and ion-coupled surfactant molecules is produced, resulting in batch-to-batch variation in the final product. In addition, the market price of this surfactant is around $500 / gram, making the process cost of using this type of surfactant extremely high, limiting its widespread application.

[0004] At present, there are fluorinated surfactants with various hydrophilic groups on the market, including carbohydrate derivatives and polyethylene glycols of various lengths, but these surfactants have poorer interfacial stability than 008-fluorinated surfactants, and it is difficult for a single surfactant to have better properties than 008-fluorinated surfactants. Therefore, it is urgent to develop a low-toxic, stable and inexpensive fluorinated surfactant compound system to expand the application field of microfluidic droplet technology. Summary of the invention

[0005] The purpose of the present invention is to provide a fluorinated surfactant compound system and a preparation method and application thereof. The fluorinated surfactant compound system of the present invention has readily available raw materials, simple preparation, good performance, is suitable for industrial production, and can be used as an emulsifier in microfluidic droplet technology.

[0006] The present invention first provides a fluorocarbon surfactant, which is obtained by covalently coupling a perfluoropolyether polymer and a hydrophilic compound;

[0007] The perfluoropolyether polymer is perfluoropolyether, perfluoropolyether carboxylic acid, perfluoropolyether acyl fluoride, perfluoropolyether diol or perfluoropolyether styrene;

[0008] The hydrophilic compound is polyvinyl alcohol, polyethyleneimine, polyethylene glycol or triglycerol.

[0009] In the above-mentioned fluorocarbon surfactant, the weight average molecular weight of the perfluoropolyether polymer is 1000 to 10000; specifically, it can be 2500, 6000 or 7500;

[0010] The molar ratio of the perfluoropolyether polymer to the hydrophilic compound is 1:0.5-2; specifically, it may be 1:2 or 1:1.5.

[0011] In the above-mentioned fluorocarbon surfactant, the weight average molecular weight of the polyvinyl alcohol is 5000-10000, and specifically may be 10000.

[0012] The present invention also provides a method for preparing the above-mentioned fluorocarbon surfactant, comprising the following steps:

[0013] (1) mixing a perfluoropolyether polymer, oxalyl chloride and fluorocarbon oil to react;

[0014] (2) Adding a hydrophilic compound and triethylamine to the reaction product of step (1) to carry out a reaction to obtain the fluorocarbon surfactant.

[0015] In the above preparation method, the molar ratio of the perfluoropolyether polymer to oxalyl chloride is 1:2 to 8; specifically, it can be 1:4, 2:5 or 1:3;

[0016] The molar ratio of the perfluoropolyether polymer to the hydrophilic compound is 1:0.5-2; specifically, it may be 1:2 or 1:1.5;

[0017] The molar ratio of triethylamine to perfluoropolyether polymer is 1.5 to 3:1; specifically, it can be 1.5:1 or 2:1;

[0018] In step (1), the reaction temperature is room temperature; the reaction time is 8 to 12 hours; the reaction is carried out in an inert atmosphere; specifically, it may be an argon atmosphere;

[0019] In step (2), the reaction temperature is 45-75°C, and the reaction time is 6-48h; specifically, the reaction may be refluxed at 70°C for 48h;

[0020] The reaction in step (2) is carried out in a solvent; specifically, the solvent is fluorocarbon oil.

[0021] In the above-mentioned preparation method, the fluorocarbon oil is at least one of fluorocarbon alkanes, fluorine-substituted ethers, fluorine-substituted esters and fluorine-substituted aromatic hydrocarbons; the number of carbon atoms in the fluorocarbon oil molecule is 3-20;

[0022] Preferably, the fluorocarbon oil is at least one of perfluoropentane, perfluorohexane, perfluoroheptane, perfluorobutyl-methyl ether, perfluorooctane, perfluorononane, perfluorodecane, perfluoroundecane, perfluorododecane, perfluorotridecane, perfluorotetradecane, perfluoropentadecane, perfluorohexadecane, perfluoroheptadecane, perfluorodecalin and Novec HFE series fluorinated liquid.

[0023] Specifically, the fluorocarbon oil is HFE7100 fluorinated oil.

[0024] In the above-mentioned preparation method, after the reaction in step (1) is completed, there is a step of removing volatile by-products by rotary evaporation; specifically, the temperature of the rotary evaporation is 50°C;

[0025] After the reaction in step (2), there are steps of removing organic byproducts by rotary evaporation and purifying the product by liquid-liquid extraction.

[0026] Specifically, the temperature of the rotary evaporation is 50° C.; the solvent of the liquid-liquid extraction is fluorocarbon oil and dichloromethane; and the number of extractions is 1 to 6 times.

[0027] In the above preparation method, in step (1), the perfluoropolyether polymer, oxalyl chloride and fluorocarbon oil are mixed and stirred at room temperature to discharge gaseous products before reacting.

[0028] In the above preparation method, the room temperature is well known to those skilled in the art, and is generally 15-35°C.

[0029] Furthermore, the present invention provides a fluorine-containing surfactant compound system, which includes two or more fluorocarbon surfactants mentioned above.

[0030] The above-mentioned fluorinated surfactant compounding system can be any of the following:

[0031] 1) It is composed of PFPE (2500)-triglycerol and PFPE (6000)-triglycerol; in the fluorinated surfactant compound system, the mass ratio of PFPE (2500)-triglycerol to PFPE (6000)-triglycerol is 1:9;

[0032] 2) It is composed of PFPE (6000)-triglycerol and PFPE (7500)-PVA (10000); in the fluorinated surfactant compound system, the mass ratio of PFPE (6000)-triglycerol to PFPE (7500)-PVA (10000) is 1:9;

[0033] 3) It is composed of PFPE (2500) -triglycerol, PFPE (7500) -PVA (10000) and fluorocarbon oil; in the fluorinated surfactant compound system, the mass ratio of PFPE (2500) -triglycerol to PFPE (7500) -PVA (10000) is 1:9;

[0034] The PFPE (2500)-triglycerol is obtained by coupling a perfluoropolyether carboxylic acid having a weight average molecular weight of 2500 and triglycerol;

[0035] The PFPE (6000)-triglycerol is obtained by coupling a perfluoropolyether carboxylic acid having a weight average molecular weight of 6000 and triglycerol;

[0036] The PFPE (7500)-PVA (10000) is obtained by coupling a perfluoropolyether carboxylic acid with a weight average molecular weight of 7500 and a polyvinyl alcohol with a molecular weight of 10000.

[0037] The above-mentioned fluorocarbon surfactant or the fluorine-containing surfactant compound system is used as an emulsifier in the preparation of water-in-oil droplets.

[0038] Specifically, the application is to use it as an emulsifier to prepare water-in-oil droplets in microfluidic droplet technology in the biological field.

[0039] The present invention has the following beneficial effects:

[0040] (1) The reaction conditions for preparing the fluorocarbon surfactant provided by the present invention are mild, the preparation process is simple, and it is easy to apply industrially; the fluorocarbon surfactant of the present invention has the advantages of good biocompatibility, temperature resistance and good solubility;

[0041] (2) The fluorinated surfactant compound system provided by the present invention uses two or more fluorocarbon surfactants. The synergistic effect of multiple fluorocarbon surfactants makes it easier to form a uniform oil-in-water single emulsion, which can meet the production of various oil-in-water single emulsions with a wide size distribution;

[0042] (3) At the same time, through the interaction of intramolecular and intermolecular hydrogen bonds, the composite system can form a strong and ordered network structure at the oil-water interface, provide high temperature resistance and high retention of encapsulated small molecules in the emulsion droplets, stabilize the oil-in-water single emulsion in a high temperature environment, and prevent the leakage of substances in the aqueous solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Schematic diagram of the microfluidic chip structure used for performance testing;

[0044] Figure 2 The hydrogen nuclear magnetic resonance spectrum of the fluorocarbon surfactant prepared in Example 1-3;

[0045] Figure 3 The NMR fluorine spectrum of the fluorocarbon surfactant prepared in Example 1-3;

[0046] Figure 4 The infrared spectra of the fluorocarbon surfactants prepared in Examples 1-3;

[0047] Figure 5 The biocompatibility of the fluorocarbon surfactants prepared in Examples 1-3;

[0048] Figure 6 The monodispersity of droplets of different sizes prepared for the fluorocarbon surfactants prepared in Examples 1-3 and the compounded systems of Examples 4-6;

[0049] Figure 7 Temperature stability of droplets prepared from the fluorocarbon surfactants prepared in Examples 1-3 and the compounded systems of Examples 4-6;

[0050] Figure 8 The diffusivity between droplets of the fluorocarbon surfactants prepared in Examples 1-3 and the compounded systems of Examples 4-6 was prepared. DETAILED DESCRIPTION

[0051] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, but not for limiting the scope of the present invention.

[0052] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0053] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0054] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0055] The schematic diagram of the microfluidic chip structure used in the performance test of the following embodiments is as follows: Figure 1 As shown, the microfluidic chip includes a microchannel, a channel inlet 1, a channel inlet 2, a flow focusing junction 3 and a channel outlet 4; wherein the channel inlet 1 serves as a continuous phase inlet, and the channel inlet 2 serves as a dispersed phase inlet; the channel inlet 1 is divided into two branches respectively connected to two microchannels, and the two microchannels connected to the channel inlet 2 intersect at the flow focusing junction 3 to form an oil-in-water single emulsion that flows out from the channel outlet 4; the flow focusing junction 3 is a cross-shaped connection between the microchannels, wherein the microchannel connected to the channel inlet 2 and the microchannel connected to the channel outlet 4 are arranged oppositely. The channel width-to-height ratio of the microfluidic chip is 1:1, and the channels are all on the same horizontal plane; the substrate of the microfluidic chip is an ordinary glass sheet; the material of the microfluidic chip is polydimethylsiloxane; by making corresponding changes to the microchannel width and height of the chip, oil-in-water single emulsions of different sizes are formed. Specifically: The microfluidic channel width of the microfluidic chip used for the oil-in-water single emulsion with a diameter of 25μm is 25μm, and the microfluidic channel height is 25μm. The microfluidic channel width of the microfluidic chip used for the oil-in-water single emulsion with a diameter of 50μm is 50μm, and the microfluidic channel height is 50μm. The microfluidic channel width of the microfluidic chip used for the oil-in-water single emulsion with a diameter of 100μm is 100μm, and the microfluidic channel height is 100μm. The microfluidic channel width of the microfluidic chip used for the oil-in-water single emulsion with a diameter of 150μm is 150μm, and the microfluidic channel height is 150μm.

[0056] Before use, the inner surface of the microchannel of the microfluidic chip is subjected to hydrophobic and sterilization treatment; the specific steps are as follows:

[0057] (1) Use a syringe to draw in Aquapel hydrophobic agent (Pittsburgh Glass Works LLC. 47100), then connect a needle and connect the polytetrafluoroethylene tube;

[0058] (2) Fill the microchannels with a syringe to inject the hydrophobic agent. When all microchannels are filled with the hydrophobic agent, cover the microfluidic chip with tape to prevent evaporation.

[0059] (3) placing the microfluidic chip at room temperature for 5 minutes;

[0060] (4) Finally, fluorinated oil (3M TM Novec TMIntroduction 7100 Engineered Fluid) replaces the hydrophobic agent in the flow channel; then anhydrous ethanol is introduced and sterilized under ultraviolet light for 15 minutes.

[0061] Example 1: Fluorocarbon surfactant PFPE (2500)-triglycerol

[0062] 0.001 mol of perfluoropolyether carboxylic acid (Typda New Materials Co., Ltd., CAS: 51798-33-5) with a weight average molecular weight of 2500 was mixed with 20 mL of HFE7100 fluorinated oil (3M TM Novec TM Introduction 7100 Engineered Fluid) was dissolved and then 0.004 mol of oxalyl chloride (McLean reagent, O815154) was added to react. After being fully stirred at room temperature for 30 minutes, the gaseous product was discharged and stirred for 8 hours under argon atmosphere at room temperature. After removing volatile by-products by rotary evaporation at 50℃ for 6 hours, the reaction system was dissolved again with 20mL HFE7100 and 0.002 mol of triglycerol and 0.002 mol of triethylamine were added in sequence. After the reaction system was fully mixed, it was placed in a 70℃ oil bath for reflux reaction for 48 hours. After the reaction was completed, the temperature was cooled to room temperature and then the organic by-products were removed by rotary evaporation at 50℃. The reaction system was re-dissolved in 8mL HFE7100, and 2mL of dichloromethane was added and stirred. After standing and layering, the upper organic solvent was removed. This process was repeated 4 times to obtain the lower emulsifier solution. Finally, the emulsifier product, i.e., the fluorocarbon surfactant PFPE (2500)-triglycerol, was obtained after being fully evaporated at 70℃.

[0063] Example 2: Fluorocarbon surfactant PFPE (6000)-triglycerol

[0064] 0.002 mol of the perfluoropolyether carboxylic acid raw material with a weight average molecular weight of 6000 was dissolved in 50 mL of HFE7100 fluorinated oil, and then 0.005 mol of oxalyl chloride was added for reaction. After being fully stirred at room temperature for 30 minutes, the gaseous product was released, and then the stirring was continued for 12 hours in an argon environment at room temperature; after being rotary evaporated at 50 ° C for 6 hours to remove volatile by-products, the reaction system was dissolved again in 50 mL of HFE7100 and 0.004 mol of triglycerol and 0.004 mol of triethylamine were added in sequence. After the reaction system was fully mixed, it was placed in a 70 ° C oil bath for reflux reaction for 48 hours. After the reaction was completed, the temperature was cooled to room temperature, and then the organic by-products were removed by rotary evaporation at 50 ° C. The reaction system was re-dissolved in 8 mL of HFE7100, and 2 mL of dichloromethane was added and stirred thoroughly, and then the layers were allowed to stand, and the upper organic solvent was removed. This process was repeated 3 times to obtain the lower emulsifier solution. Finally, the emulsifier product, i.e., the fluorocarbon surfactant PFPE (6000)-triglycerol, was obtained after sufficient evaporation at 70°C.

[0065] Example 3, Fluorocarbon surfactant PFPE (7500) -PVA (10000)

[0066] 0.002 mol of the perfluoropolyether carboxylic acid raw material with a weight average molecular weight of 7500 was dissolved in 50 mL of HFE7100 fluorinated oil, and then 0.006 mol of oxalyl chloride was added for reaction. After being fully stirred at room temperature for 30 minutes, the gaseous product was released and stirred for 12 hours under argon gas at room temperature. After 6 hours of rotary evaporation at 50 ° C to remove volatile by-products, the reaction system was dissolved again in 50 mL of HFE7100 and 0.003 mol of polyvinyl alcohol with a molecular weight of 10000 and 0.003 mol of triethylamine were added in sequence. After the reaction system was fully mixed, it was placed in a 70 ° C oil bath for reflux reaction for 48 hours. After the reaction was completed, the temperature was cooled to room temperature, and then the organic by-products were removed by rotary evaporation at 50 ° C. The reaction system was redissolved in 8 mL of HFE7100, and 2 mL of dichloromethane was added and stirred thoroughly, and then the layers were allowed to stand and the upper organic solvent was removed. This process was repeated 4 times to obtain the lower emulsifier solution. Finally, after sufficient evaporation at 70°C, the emulsifier product, namely the fluorocarbon surfactant PFPE (7500)-PVA (10000), was obtained.

[0067] Example 4 PFPE (2500) -triglycerol / PFPE (6000) -triglycerol compound system

[0068] 0.1 g PFPE(2500)-triglycerol and 0.9 g PFPE(6000)-triglycerol were mixed to obtain a PFPE(2500)-triglycerol / PFPE(6000)-triglycerol composite system.

[0069] Example 5: PFPE (6000) -triglycerol / PFPE (7500) -PVA (10000) compound system

[0070] The preparation method of the composite system of this embodiment is the same as that of Example 4, except that PFPE(2500)-triglycerol and PFPE(6000)-triglycerol are replaced by PFPE(6000)-triglycerol and PFPE(7500)-PVA(10000).

[0071] Example 6: PFPE (2500) -triglycerol / PFPE (7500) -PVA (10000) compound system

[0072] The preparation method of the composite system of this embodiment is the same as that of Example 4, except that the PFPE(6000)-triglycerol in Example 4 is replaced by PFPE(7500)-PVA(10000).

[0073] Comparative example, RAN-008FS

[0074] Neat (un-dissolved) 008-FluoroSurfactant (cat#: 008-FluoroSurfactant-1g) was purchased from RAN biotechnologies for performance comparison testing.

[0075] Embodiment 7, performance test

[0076] (1) Nuclear magnetic resonance spectroscopy test: The hydrogen spectrum and fluorine spectrum were measured using a German Bruker-400M to identify the chemical structure of the surfactant. The fluorocarbon surfactants prepared in Examples 1, 2, and 3 were tested. The results are shown in Figure 2 and Figure 3 ;Depend on Figure 2 As shown in the hydrogen spectrum, the characteristic peak around 2.5ppm belongs to the CH characteristic peak, which is Figure 3 As shown in the fluorine spectrum, Examples 1-3 all showed CF characteristic peaks, and the hydrogen spectrum and fluorine spectrum jointly confirmed the successful synthesis of the fluorocarbon surfactant.

[0077] (2) Infrared spectroscopy test: The spectra were measured using a Thermo Fisher Nicolet IS5 to identify the structure and composition of the surfactant to determine whether it was successfully prepared. This test was performed on the fluorocarbon surfactants prepared in Examples 1, 2, and 3. The results are shown in Table 1. Figure 4 ;Depend on Figure 4 The spectrum shows a range of 2980-2990 cm -1The stretching peak in the range is the CH absorption peak, which can also confirm the successful preparation of fluorocarbon surfactant.

[0078] (3) Biocompatibility test: Monodisperse droplets with a diameter of about 150 μm were prepared in HFE7500 fluorinated oil using a microfluidic chip device. The continuous phase was HFE7500 fluorinated oil containing 1.0% w / v fluorocarbon surfactant at a flow rate of 1000 μL / h, and the dispersed phase was a cell culture medium solution containing mesenchymal stem cells (Starfish Biotechnology, ADHX-G101-500) (concentration of 2.5×10 6 / mL), the flow rate is 100μL / h, the prepared droplets are collected in a 96-well microtiter plate, and cultured at 37°C and 5% CO2 for 24h. Then, HFE7500 fluorinated oil containing 20% ​​(v / v) perfluorooctanol is added for demulsification, and the Calcein / PI cell activity and cytotoxicity detection kit is used to determine the ratio of live / dead cells. The detection principle of this kit is that the two probes can detect intracellular esterase activity and cell membrane integrity, respectively, thereby reflecting cell activity and cytotoxicity. Among them, Calcein AM stains living cells and exhibits green fluorescence; while propidium iodide (PI) stains dead cells and exhibits red fluorescence. The surfactants of Examples 1, 2, 3 and the comparative example were subjected to this test, and the results are shown in Table 1. Figure 5 ;Depend on Figure 5 It can be seen that the surfactant-stabilized microdroplets prepared using Examples 1-3 exhibit good biocompatibility with mesenchymal stem cells, and the cell viability of all microdroplets is higher than 87.54%, thus demonstrating that these surfactants are suitable for cell-based experiments.

[0079] (4) Monodispersity test of droplets: Monodisperse droplets with diameters of approximately 25 μm, 50 μm, and 100 μm were prepared using a microfluidic chip device. The continuous phase reagents were prepared by dissolving the surfactants in Example 1 (PFPE (2500) -triglycerol), Example 2 (PFPE (6000) -triglycerol), Example 3 (PFPE (7500) -PVA (10000)), Example 4 (PFPE (2500) -triglycerol / PFPE (6000) -triglycerol compound system), Example 5 (PFPE (6000) -triglycerol / PFPE (7500) -PVA (10000) compound system), Example 6 (PFPE (2500) -triglycerol / PFPE (7500) -PVA (10000) compound system) and Comparative Example (Neat (un-dissolved) 008 - FluoroSurfactant) into HFE7500 fluorinated oil at 1.0% w / v. The dispersed phase aqueous solution for preparing droplets was 1×PBS (pH=7.4). The continuous phase flow rate of 25μm and 50μm droplets is 500μL / h, and the dispersed phase flow rate is 100μL / h; the continuous phase flow rate of 100μm droplets is 1000μL / h, and the dispersed phase flow rate is 150μL / h. The photos were taken under a microscope and the dispersion was calculated by Image j software. The results are shown in Figure 6 .Depend on Figure 6 It can be seen that the dispersibility index of droplets of all sizes is less than 0.01. Among them, the comparative example, Example 1 and Example 2 are more uniform when forming droplets with a diameter of 100 μm. And Example 3 forms the highest uniformity for all three droplet sizes. For the compounded systems of Examples 4-6, it was found that the monodispersity of 50 μm and 100 μm droplets was improved after compounding.

[0080] (5) Temperature stability test of droplets: A microfluidic chip device was used, and the continuous phase reagents were Example 1 (PFPE (2500) -triglycerol), Example 2 (PFPE (6000) -triglycerol), Example 3 (PFPE (7500) -PVA (10000)), Example 4 (PFPE (2500) -triglycerol / PFPE (6000) -triglycerol composite system), Example 5 (PFPE (6000) -triglycerol / PFPE (7500) -PVA (10000)), Example 6 (PFPE (2500) -triglycerol / PFPE (6000) -triglycerol composite system), Example 7 (PFPE (6000) -triglycerol / PFPE (7500) -PVA (10000)), Example 8 (PFPE (7500) -PVA (10000)), Example 9 (PFPE (2500) -triglycerol / PFPE (6000) -triglycerol composite system), Example 10 (PFPE (6000) -triglycerol / PFPE (7500) -PVA (10000)), Example 11 (PFPE (7500) -PVA (10000)), Example 12 (PFPE (7500) -PVA (10000)), Example 13 (PFPE (7500) -PVA (10000)), Example 14 (PFPE (2500) -triglycerol / PFPE (6000) -triglycerol composite system), Example 15 (PFPE (6000) -triglycerol / PFPE (7500) -PVA (10000)), Example 16 (PFPE (7500) -PVA (10000)), Example 17 (PFPE (7500) -PVA (10000)), Example 18 (PFPE (7 The surfactant in Example 6 (PFPE (2500) -triglycerol / PFPE (7500) -PVA (10000) compound system), Example 6 (PFPE (2500) -triglycerol / PFPE (7500) -PVA (10000) compound system) or the comparative example (Neat (un-dissolved) 008-FluoroSurfactant) was dissolved in HFE7500 fluorinated oil at a concentration of 0.5% w / v, 1.0% w / v or 2.0% w / v) to prepare monodisperse droplets with a diameter of about 50 μm. The aqueous solution used to prepare the droplets was 1×PBS (pH = 7.4). Among them, the flow rate of the continuous phase was 1000 μL / h, and the flow rate of the dispersed phase was 500 μL / h. 40 μL of droplets were collected in a PCR tube, and 100 μL of liquid paraffin was added to prevent the liquid from evaporating. Then the PCR program was performed, the PCR program was 95℃ for 30 seconds; then 95℃ for 7 seconds, 60℃ for 30 seconds and 72℃ for 20 seconds as one cycle, and the cycle was repeated 30 times; finally, 72℃ for 10 minutes. Use a pipette to measure the volume of the unfused droplets and calculate the fusion rate. Fusion rate calculation formula:

[0081]

[0082] The experimental results are shown in Figure 7 (surfactant concentration is 1.0% w / v), by Figure 7 The analysis showed that the ability of surfactants to stabilize droplets was ranked in the following order: PFPE(2500)-triglycerol / PFPE(7500)-PVA(10000) complex system > PFPE(6000)-triglycerol / PFPE(7500)-PVA(10000) complex system > PFPE(2500)-triglycerol / PFPE(6000)-triglycerol complex system > PFPE(7500)-PVA(10000) > PFPE(6000)-triglycerol > Neat(un-dissolved)008-FluoroSurfactant > PFPE(2500)-triglycerol.

[0083] (6) Diffusion test between droplets: Two groups of droplets with a diameter of about 100 μm were prepared simultaneously using a microfluidic chip device. The continuous phase was HFE7500 fluorinated oil containing 1.0% w / v surfactant, with a flow rate of 1000 μL / h, and the dispersed phase was 1×PBS (pH=7.4) or 1×PBS (pH=7.4) containing 4 μM fluorescein isothiocyanate. The two groups of droplets were collected simultaneously into the same 1.5 mL centrifuge tube at a flow rate of 150 μL / h. One droplet population contained PBS+fluorescein isothiocyanate dye, and the other droplet population contained only PBS. The mixed droplet population was then incubated at 37°C and imaged by microscopy on days 0, 1, 2, and 3. The green fluorescence intensity of the PBS droplets alone was quantified using Imageview software, and the results are shown in Figure 2. Figure 8 .Depend on Figure 8 It can be seen that the quantification of fluorescence intensity in the seven groups of PBS droplets from day 0 to day 3 showed no significant increase. This indicates that the fluorosurfactant is able to prevent the transfer of the dye within the droplets and highly retain the aqueous solution in the droplets.

[0084] The surfactant concentration % w / v in the continuous phase mentioned above refers to g / mL.

[0085] In summary, the fluorocarbon surfactant of the present invention has readily available raw materials, simple synthesis, good performance, can reduce oil-water interfacial tension, has high biocompatibility, and is suitable for industrial production. Using it to prepare a fluorosurfactant compound system can not only stabilize droplets in a high temperature environment, but also encapsulate the aqueous solution in the droplets without leakage. This makes the compound system have great potential to become a powerful and economical droplet stabilizer. In addition, combined with a microfluidic device, the synthesis of highly stable droplets provides a basis for genetic analysis, high-throughput single-cell research and drug screening under thermal cycling conditions.

Claims

1. A fluorocarbon surfactant obtained by covalently coupling a perfluoropolyether polymer and a hydrophilic compound; The perfluoropolyether polymer is perfluoropolyether, perfluoropolyether carboxylic acid, perfluoropolyether acyl fluoride, perfluoropolyether diol or perfluoropolyether styrene; The hydrophilic compound is polyvinyl alcohol, polyethyleneimine, polyethylene glycol or triglycerol.

2. The fluorocarbon surfactant according to claim 1, characterized in that: The weight average molecular weight of the perfluoropolyether polymer is 1000 to 10000; The molar ratio of the perfluoropolyether polymer to the hydrophilic compound is 1:0.5-2.

3. The method for preparing the fluorocarbon surfactant according to claim 1 or 2, comprising the following steps: (1) mixing a perfluoropolyether polymer, oxalyl chloride and fluorocarbon oil to react; (2) Adding a hydrophilic compound and triethylamine to the reaction product of step (1) to carry out a reaction to obtain the fluorocarbon surfactant.

4. The preparation method according to claim 3, characterized in that: The molar ratio of the perfluoropolyether polymer to oxalyl chloride is 1:2 to 8; The molar ratio of the perfluoropolyether polymer to the hydrophilic compound is 1:0.5-2; The molar ratio of triethylamine to perfluoropolyether polymer is 1.5 to 3:1; In step (1), the reaction temperature is room temperature; the reaction time is 8 to 12 hours; In step (1), the reaction is carried out in an inert atmosphere; In step (2), the reaction temperature is 45 to 75° C., and the reaction time is 6 to 48 hours; The reaction in step (2) is carried out in a solvent; specifically, the solvent is fluorocarbon oil.

5. The preparation method according to claim 3 or 4, characterized in that: After the reaction in step (1) is completed, there is a step of removing volatile by-products by rotary evaporation; After the reaction in step (2), there are steps of removing organic byproducts by rotary evaporation and purifying the product by liquid-liquid extraction.

6. The preparation method according to any one of claims 3 to 5, characterized in that: The fluorocarbon oil is at least one of fluorocarbon alkanes, fluorine-substituted ethers, fluorine-substituted esters and fluorine-substituted aromatic hydrocarbons; the number of carbon atoms in the fluorocarbon oil molecule is 3-20; Preferably, the fluorocarbon oil is at least one of perfluoropentane, perfluorohexane, perfluoroheptane, perfluorobutyl-methyl ether, perfluorooctane, perfluorononane, perfluorodecane, perfluoroundecane, perfluorododecane, perfluorotridecane, perfluorotetradecane, perfluoropentadecane, perfluorohexadecane, perfluoroheptadecane, perfluorodecalin and Novec HFE series fluorinated liquid.

7. A fluorine-containing surfactant compound system, comprising two or more fluorocarbon surfactants as described in claim 1 or 2.

8. The fluorinated surfactant compound system according to claim 7, characterized in that: The fluorinated surfactant compounding system is any of the following: 1) It is composed of PFPE (2500)-triglycerol and PFPE (6000)-triglycerol; in the fluorinated surfactant compound system, the mass ratio of PFPE (2500)-triglycerol to PFPE (6000)-triglycerol is 1:9; 2) It is composed of PFPE (6000)-triglycerol and PFPE (7500)-PVA (10000); in the fluorinated surfactant compound system, the mass ratio of PFPE (6000)-triglycerol to PFPE (7500)-PVA (10000) is 1:9; 3) It is composed of PFPE (2500) -triglycerol, PFPE (7500) -PVA (10000) and fluorocarbon oil; in the fluorinated surfactant compound system, the mass ratio of PFPE (2500) -triglycerol to PFPE (7500) -PVA (10000) is 1:9; The PFPE (2500)-triglycerol is obtained by coupling a perfluoropolyether carboxylic acid having a weight average molecular weight of 2500 and triglycerol; The PFPE (6000)-triglycerol is obtained by coupling a perfluoropolyether carboxylic acid having a weight average molecular weight of 6000 and triglycerol; The PFPE (7500)-PVA (10000) is obtained by coupling a perfluoropolyether carboxylic acid with a weight average molecular weight of 7500 and a polyvinyl alcohol with a molecular weight of 10000.

9. Use of the fluorocarbon surfactant according to claim 1 or 2 or the fluorinated surfactant complex system according to claim 7 or 8 as an emulsifier in the preparation of water-in-oil droplets.

10. The use according to claim 9, characterized in that: The application is to use the emulsifier as an emulsifier to prepare water-in-oil droplets in microfluidic droplet technology in the biological field.

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