Surfactant used in field of microfluidics, and preparation method and application thereof

Abstract

The invention discloses a surfactant used in the field of microfluidics, and a preparation method and application thereof. The preparation method comprises the following steps: adding perfluoropolyether carboxylic acid, dibenzocyclooctyne-polyethylene glycol-amino and a catalyst fluorine-containing silicate compound into a fluorinated solution, stirring, cooling, carrying out suction filtration, adding absolute alcohol into filtrate, recrystallizing to separate out a solid, filtering and drying to obtain the novel surfactant. The surfactant provided by the invention is suitable for micro-fluidic chips of various specifications, the particle size consistency of liquid drops generated under 0.2% of addition amount is stable, and the liquid drops are not fused after 95 DEG C pcr amplification circulation. The liquid drops can be placed at normal temperature for several months without fusion.

Description

Technical Field

[0001] The invention relates to a surfactant, a preparation method and an application, and in particular to a surfactant used in the field of microfluidics, a preparation method and an application. Background Art

[0002] Microfluidic droplet generation technology is widely used in biology, chemistry, medicine, materials and other fields. The droplet volume generated by droplet microfluidics is as small as pL, and the droplet monodispersity is extremely high. It has the advantages of small size, no cross-contamination, and rapid reaction. Each droplet can be used as an independent microreactor. Among them, the surfactant plays a decisive role in the stability of each droplet. Fluorocarbon surfactants are the best of all special surfactants used in the biochemical field. They have the advantages of biological inertness and low interfacial tension. Although the fluorocarbon surfactants currently sold in the market by companies such as EMULSE, RAN BIOTECH and BIO-RAD have excellent performance, they are difficult to purify and expensive. Therefore, it is very meaningful to develop a fluorocarbon surfactant with higher purity, better performance and lower cost, and to widely use it in commercialization.

[0003] Defects and shortcomings of existing technologies: The fluorocarbon surfactant synthesis routes currently sold by companies such as EMULSE, RAN BIOTECH and BIO-RAD require one to three steps of reaction, with complex processes and high purification difficulty. The supply price of the finished product is very high, and general industrial customers cannot afford it, so it is only suitable for scientific research. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to solve the problems of complex synthesis process, high cost, high price and difficult purification of the currently available fluorocarbon surfactants, and to provide a surfactant, preparation method and application for the microfluidics field. The method controls the molecular weight range of the oleophilic end fluorocarbon chain by screening the appropriate hydrophilic end structure and molecular weight. The one-step synthesis is adopted, and the target product is obtained by rotary evaporation, washing and recrystallization.

[0005] The surfactant structure of the present invention is as follows:

[0006]

[0007] Wherein, m=an integer from 4 to 25; n=an integer from 2 to 9.

[0008] The present invention is to screen the molecular weight of perfluoropolyether, the molecular structure and molecular weight of the hydrophilic end respectively. The novel surfactant for droplet generation of the present invention is synthesized in one step. The molecular structure of polyethylene glycol contains hydroxyl groups and exhibits strong water solubility, so it can increase the solubility of poorly soluble drugs in water. Polyethylene glycol can not only have a solubilizing effect, but also increase the stability of the compound, improve physical properties, correct odor and taste, reduce stimulation, improve absorption, and increase pharmacological effects. The introduction of dibenzocyclooctyne structure can increase the biocompatibility and thermal stability of the compound. The perfluoropolyether molecule at the lipophilic end is large, which will affect the yield of the synthesis reaction, and the molecular weight is small, which will affect the stability of the droplet, so a suitable m value should be selected. Preferably, the m value range is 4-25, preferably 20. The molecular weight of the perfluoropolyether at the lipophilic end can be 800, 900, 1000, 1500, 2500, 3500, 4500, 5500. The preferred optimal molecular weight is around 4500.

[0009] Technical solution: On the one hand, the present invention provides a method for preparing the surfactant used in the microfluidics field, wherein perfluoropolyether carboxylic acid, dibenzocyclooctyne-polyethylene glycol-amino and a catalyst fluorine-containing silicate compound are added to a fluorinated liquid, stirred, cooled and filtered, anhydrous alcohol is added to the filtrate, solids are precipitated by recrystallization, filtered and dried to obtain a new surfactant.

[0010] Further, stirring at 50-70°C for 8-12h, cooling and filtering, adding a certain amount of anhydrous alcohol to the filtrate, recrystallizing to precipitate solid at 2-10°C, filtering and drying to obtain a new surfactant.

[0011] Furthermore, the catalyst is a fluorine-containing silicate compound; the catalyst of the present invention is lithium perfluorosilicate;

[0012] Furthermore, in the synthesis route of the novel surfactant described in the present invention, the mass ratio of perfluoropolyether carboxylic acid and dibenzocyclooctyne-polyethylene glycol-amino substance is 1:2-2.5, preferably 1:2, 1:2.05, 1:2.1, 1:2.15, 1:2.2, 1:2.25, 1:2.3, 1:2.35, 1:2.4, 1:2.45, 1:2.5.

[0013] Furthermore, the mass ratio of the perfluoropolyether carboxylic acid to the catalyst substance in the present invention is 1:0.01-0.05, preferably 1:0.01, 1:0.02, 1:0.03, 1:0.04, 1:0.05.

[0014] Furthermore, the fluorinated liquid described in the present invention is not limited to a mixture of one or more fluorinated liquids including HFE7100, HFE7500, FC-40, FC-3283, FC-43, and Flou-Oil135.

[0015] The molecular formula of dibenzocyclooctyne-polyethylene glycol-amino is as follows:

[0016]

[0017] Furthermore, the n value in the dibenzocyclooctyne-polyethylene glycol-amino structure is 2-9, and a more preferred n value is 4. The preferred molecular weight is 600, 800, 1000, 1300, 1700, 2000, 2500, and a more preferred molecular weight is 1000.

[0018] Another object of the present invention is to provide the surfactant.

[0019] The third object of the present invention is to provide a novel structured surfactant for use in digital PCR droplet gene sequencing and single cell sequencing droplet generation, wherein the generated droplets undergo multiple thermal cycles at 95°C without fusion; the surfactant is added in an amount of 0.05%-1.0% by mass fraction, preferably 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1.0%. More preferably, the added amount is 0.2% or 0.5%.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The product prepared by the invention is a novel fluorocarbon surfactant, which is synthesized by a one-step method, has high product purity, low synthesis cost and selling price, and is suitable for industrial production.

[0022] 1. The novel surfactant of the present invention is widely used in the field of microfluidics. It can be applied not only to the field of digital PCR gene sequencing, but also to the field of single-cell sequencing.

[0023] 2. The novel surfactant described in the present invention is suitable for microfluidic chips of various specifications. The droplet size consistency is stable when the addition amount is 0.2%. After PCR amplification cycle at 95°C, the droplets do not fuse. The droplets can be placed at room temperature for several months without fusion.

[0024] 3. The surfactant of the present invention has low synthesis cost, simple process and high product purity. A 0.1%-0.5% addition amount can generate stable water-in-oil droplets, and the droplets do not fuse after PCR amplification at 95°C thermal cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the H-NMR spectrum of a new surfactant;

[0026] Figure 2 The figure is a comparison result. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be further described below.

[0028] Embodiment 1:

[0029] The perfluoropolyether carboxylic acid with m value = 20 and molecular weight of 4500, dibenzocyclooctyne-polyethylene glycol-amino with n value = 4 and molecular weight of 1000 and catalyst lithium perfluorosilicate were added into a reactor containing fluorinated liquid HFE7500 in a mass ratio of 1:2.05:0.03, stirred at 50°C for 11 hours, cooled to 16°C and filtered, 40g of anhydrous alcohol was added to the filtrate, the solid was recrystallized at 2°C, filtered and dried to obtain a new surfactant.

[0030] Embodiment 2:

[0031] The perfluoropolyether carboxylic acid with m value = 20 and molecular weight of 4500, dibenzocyclooctyne-polyethylene glycol-amino with n value = 4 and molecular weight of 1000 and catalyst lithium perfluorosilicate were added into a reactor containing fluorinated liquid HFE7500 in a mass ratio of 1:2.05:0.03, stirred at 60°C for 11 hours, cooled to 16°C and filtered, 40g of anhydrous alcohol was added to the filtrate, the solid was recrystallized at 2°C, filtered and dried to obtain a new surfactant.

[0032] Embodiment three:

[0033] The perfluoropolyether carboxylic acid with m value = 20 and molecular weight of 4500, dibenzocyclooctyne-polyethylene glycol-amino with n value = 4 and molecular weight of 1000 and catalyst lithium perfluorosilicate were added into a reactor containing fluorinated liquid HFE7500 in a mass ratio of 1:2.05:0.03, stirred at 70°C for 11 hours, cooled to 16°C and filtered, 40g of anhydrous alcohol was added to the filtrate, the solid was recrystallized at 2°C, filtered and dried to obtain a new surfactant.

[0034] Embodiment 4:

[0035] Perfluoropolyether carboxylic acid m=20, dibenzocyclooctyne-polyethylene glycol-amino n=2, molecular weight 600 and catalyst lithium perfluorosilicate were added into a reactor containing fluorinated liquid HFE7500 in a molar ratio of 1:2.05:0.03, stirred at 50°C for 11 hours, cooled to 16°C and filtered, 40g of anhydrous alcohol was added to the filtrate, recrystallized at 2°C to precipitate a solid, filtered and dried to obtain a new surfactant.

[0036] Embodiment five:

[0037] Perfluoropolyether carboxylic acid m=20, dibenzocyclooctyne-polyethylene glycol-amino n=3, molecular weight 800 and catalyst lithium perfluorosilicate are added into a reactor containing fluorinated liquid HFE7500 in a molar ratio of 1:2.05:0.03, stirred at 50°C for 11 hours, cooled to 16°C and filtered, 40g of anhydrous alcohol is added to the filtrate, solids are recrystallized at 2°C, filtered and dried to obtain a new surfactant.

[0038] Embodiment six:

[0039] Perfluoropolyether carboxylic acid m=20, dibenzocyclooctyne-polyethylene glycol-amino n=5 molecular weight 1300 and catalyst lithium perfluorosilicate are added into a reactor containing fluorinated liquid HFE7500 in a molar ratio of 1:2.05:0.03, stirred at 50°C for 11 hours, cooled to 16°C and filtered, 40g of anhydrous alcohol is added to the filtrate, recrystallized at 2°C to precipitate a solid, filtered and dried to obtain a new surfactant.

[0040] Embodiment seven:

[0041] Perfluoropolyether carboxylic acid m=20, dibenzocyclooctyne-polyethylene glycol-amino n=4, molecular weight 1000 and catalyst lithium perfluorosilicate are added in sequence to a reactor containing fluorinated liquid HFE7500 in a molar ratio of 1:2:0.03, stirred at 50°C for 11 hours, cooled to 16°C and filtered, 40g of anhydrous alcohol is added to the filtrate, solids are recrystallized at 2°C, filtered and dried to obtain a new surfactant.

[0042] Embodiment eight:

[0043] Perfluoropolyether carboxylic acid m=20, dibenzocyclooctyne-polyethylene glycol-amino n=2, molecular weight 600 and catalyst lithium perfluorosilicate were added into a reactor containing fluorinated liquid HFE7500 in a molar ratio of 1:2.1:0.03, stirred at 50°C for 11 hours, cooled to 16°C and filtered, 40g of anhydrous alcohol was added to the filtrate, recrystallized at 2°C to precipitate a solid, filtered and dried to obtain a new surfactant.

[0044] Embodiment nine:

[0045] Perfluoropolyether carboxylic acid m=20, dibenzocyclooctyne-polyethylene glycol-amino n=2, molecular weight 600 and catalyst lithium perfluorosilicate were added into a reactor containing fluorinated liquid HFE7500 in a molar ratio of 1:2.15:0.03, stirred at 50°C for 11 hours, cooled to 16°C and filtered, 40g of anhydrous alcohol was added to the filtrate, recrystallized at 2°C to precipitate a solid, filtered and dried to obtain a new surfactant.

[0046] Embodiment ten:

[0047] Perfluoropolyether carboxylic acid m=20, dibenzocyclooctyne-polyethylene glycol-amino n=2, molecular weight 600 and catalyst lithium perfluorosilicate were added into a reactor containing fluorinated liquid HFE7500 in a molar ratio of 1:2.05:0.01, stirred at 50°C for 11 hours, cooled to 16°C and filtered, 40g of anhydrous alcohol was added to the filtrate, recrystallized at 2°C to precipitate a solid, filtered and dried to obtain a new surfactant.

[0048] Embodiment eleven:

[0049] Perfluoropolyether carboxylic acid m=20, dibenzocyclooctyne-polyethylene glycol-amino n=2, molecular weight 600 and catalyst lithium perfluorosilicate were added into a reactor containing fluorinated liquid HFE7500 in a molar ratio of 1:2.05:0.02, stirred at 50°C for 11 hours, cooled to 16°C and filtered, 40g of anhydrous alcohol was added to the filtrate, recrystallized at 2°C to precipitate a solid, filtered and dried to obtain a new surfactant.

[0050] Embodiment 12:

[0051] Perfluoropolyether carboxylic acid m=20, dibenzocyclooctyne-polyethylene glycol-amino n=2, molecular weight 600 and catalyst lithium perfluorosilicate were added into a reactor containing fluorinated liquid HFE7500 in a molar ratio of 1:2.05:0.04, stirred at 50°C for 11 hours, cooled to 16°C and filtered, 40g of anhydrous alcohol was added to the filtrate, recrystallized at 2°C to precipitate a solid, filtered and dried to obtain a new surfactant.

[0052] Stability Implementation Case 1

[0053] Take the optimal conditions involved in the above implementation cases 1 to 12 for stability experiments, the specific plan is as follows:

[0054] Perfluoropolyether carboxylic acid m=20, dibenzocyclooctyne-polyethylene glycol-amino n=4, molecular weight 1000 and catalyst lithium perfluorosilicate are added into a reactor containing fluorinated liquid HFE7500 in a molar ratio of 1:2.05:0.03, stirred at 50°C for 11 hours, cooled to 16°C and filtered, 40g of anhydrous alcohol is added to the filtrate, solids are recrystallized at 2°C, filtered and dried to obtain a new surfactant.

[0055] Application Example 1:

[0056] The new surfactant was added to the fluorinated oil Flou-Oil135 at a mass fraction of 0.2%, mixed with the aqueous phase premix, and droplets were generated on the self-developed microfluidic platform, with an oil phase speed of 25ul / min and an aqueous phase speed of 15ul / min. After the generated droplets were placed in the PCR for thermal cycling, the droplets did not fuse, and the thermal cycling parameters are shown in the following table.

[0057] Table 1 PCR amplification parameters

[0058]

[0059] Application Example 2:

[0060] The new surfactant was added to the fluorinated oil Flou-Oil135 at a mass fraction of 0.5%, mixed with the aqueous phase premix, and droplets were generated on the self-developed microfluidic platform, with an oil phase speed of 30ul / min and an aqueous phase speed of 12ul / min. After the generated droplets were placed in the PCR for thermal cycling, the droplets did not fuse, and the thermal cycling parameters are shown in Application Case Table 1. It can be seen from Application Case 1 and Application Case 2 that as the amount of the new surfactant added to the fluorinated oil Flou-Oil135 increases, the droplet generation rate has been affected.

[0061] Comparative application case 1:

[0062] By comparing the droplet generation oil product number (S0100010101) produced by Guangzhou Yongnuo with the droplet generation oil doped with a new surfactant, it can be seen that the comparison conditions are room temperature 23°C, and the state of the droplets before and after amplification is observed under a microscope after 3 days, 7 days, 15 days, and 30 days. Figure 2 It can be seen that the droplets generated by the droplet generation oil prepared with the new surfactant were placed at 23°C for the first day, the third day, the seventh day, the fifteenth day, and the 30th day. After amplification, the droplets were stable and did not fuse. The Yongnuo droplet generation oil was placed at 23°C for the first day, the third day, the seventh day, the fifteenth day, and the 30th day. After amplification, the droplets began to fuse from the seventh day.

[0063] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any technician in the relevant technical field, without departing from the scope of the technical solution of the present invention, makes any form of equivalent replacement or modification to the technical solution and technical content disclosed in the present invention, which does not depart from the content of the technical solution of the present invention and still falls within the protection scope of the present invention.

Claims

1. A method for preparing a surfactant for use in the field of microfluidics, characterized in that: Perfluoropolyether carboxylic acid, dibenzocyclooctyne-polyethylene glycol-amino and catalyst fluorine-containing silicate compounds are added to the fluoridation liquid, stirred, cooled and filtered, anhydrous alcohol is added to the filtrate, solids are precipitated by recrystallization, filtered and dried to obtain a new surfactant.

2. The method for preparing a surfactant for microfluidics according to claim 1, characterized in that: The mass ratio of the perfluoropolyether carboxylic acid and dibenzocyclooctyne-polyethylene glycol-amino substance is 1:2-2.5, preferably 1:2, 1:2.05, 1:2.1, 1:2.15, 1:2.2, 1:2.25, 1:2.3, 1:2.35, 1:2.4, 1:2.45, 1:2.

5.

3. The method for preparing a surfactant for microfluidics according to claim 1, characterized in that: The mass ratio of the perfluoropolyether carboxylic acid to the catalyst substance is 1:0.01-0.05, preferably 1:0.01, 1:0.02, 1:0.03, 1:0.04 or 1:0.

05.

4. The method for preparing a surfactant for microfluidics according to claim 1, characterized in that: The m value of the perfluoropolyether carboxylic acid is in the range of 4-25, preferably 20, and the molecular weight is 800, 900, 1000, 1500, 2500, 3500, 4500, 5500, preferably 4500; the n value of the dibenzocyclooctyne-polyethylene glycol-amino is 2-9, preferably 4, and the molecular weight of n=4 is 600, 800, 1000, 1300, 1700, 2000 or 2500, preferably 1000.

5. The method for preparing a surfactant for microfluidics according to claim 1, characterized in that: The stirring temperature is 50-70°C for 8-12h; recrystallization is carried out at 2-10°C.

6. The method for preparing a surfactant for microfluidics according to claim 1, characterized in that: The fluorinated liquid includes one or more of HFE7100, HFE7500, FC-40, FC-3283, FC-43 or Flou-Oil135.

7. The method for preparing a surfactant for microfluidics according to claim 1, characterized in that: The fluorine-containing silicate compound is lithium perfluorosilicate.

8. The surfactant prepared by the method according to any one of claims 1 to 7 has the following structure: in, m=an integer of 4-25, n=an integer of 2-9.

9. Use of the surfactant according to claim 8 in digital PCR droplet gene sequencing and single-cell sequencing droplet generation.

10. The use according to claim 9, characterized in that The surfactant is added in an amount of 0.05%-1.0% by mass, preferably 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1.0%, more preferably 0.2% or 0.5%.

Images