CN115558097A - Surfactant for PCR detection reagent and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a surfactant for a PCR detection reagent, a preparation method and application thereof, wherein two hydroxyl groups are symmetrically distributed on the upper side and the lower side of the perfluoropolyether fluorocarbon glycol surfactant, and the perfluoropolyether fluorocarbon glycol surfactant can be used as the surfactant to prepare an oil phase in a PCR detection method, particularly a micro-drop digital PCR method, so as to form a water-in-oil micro-drop, and realize the application of the surfactant in the micro-fluidic drop treatment process of PCR amplification. The molecules of the adjacent perfluoropolyether type fluorocarbon glycol surfactants are mutually connected through hydrogen bonds between two mutually parallel molecules, so that a stable layered film is formed on the surface layer close to the droplets, the stability of the droplets can be effectively enhanced by the film in a PCR amplification circulation experiment, the small molecule transfer among the droplets is inhibited, the droplets subjected to the PCR amplification are stabilized by changing the surface tension of an oil-water interface, the droplets are prevented from being fused and broken, and the accuracy of a PCR detection result is improved.
Description
Technical Field
The invention relates to the technical field of PCR detection, and particularly relates to a surfactant for a PCR detection reagent, and a preparation method and application thereof.
Background
The Digital PCR technology (Digital PCR, dPCR) is an absolute nucleic acid molecule quantification technology based on a single molecule PCR method, and compared with a fluorescence quantification PCR technology, i.e., a qPCR technology, the Digital PCR technology can directly realize the absolute quantification of an initial DNA template by a direct counting method, and usually a microfluidic or microdroplet method is adopted, a large amount of diluted nucleic acid solution is dispersed into micro droplets or micro reaction cavities of a chip, i.e., the whole reaction system is divided into a plurality of tiny independent reaction systems, the nucleic acid templates are fully diluted in the separation process, the number of the nucleic acid templates in each reaction system is less than or equal to 1, after PCR cycles, the fluorescence of all droplets is identified and counted one by one, and an absolute quantification result of the DNA template can be obtained without constructing a standard curve, the reaction system with one nucleic acid molecule template can give a fluorescence signal, the reaction system without the template has no fluorescence signal, and the nucleic acid concentration of the original solution can be calculated according to the relative proportion and the volume of the reaction system. However, because the surface tension of the droplets is high, the droplets are easy to aggregate and tend to fuse, and the detection result is influenced, so that the improvement of the stability of the micro-droplets as a reaction chamber in the digital PCR is very critical to accurately realize the absolute quantification of the initial DNA template. In the field of digital PCR technology, a reaction system needs to be subjected to micro-droplet treatment before being subjected to PCR amplification treatment, wherein fluorinated oil is widely used as a continuous corresponding liquid drop micro-fluidic system due to good biological inertia and oxygen dissolving capacity.
The key to the digital PCR in microdroplet is to maintain the stability of the water-in-oil droplets. According to the water-in-oil principle, oil-water two-phase liquid is mixed under specific conditions, a reaction system containing nucleic acid molecules is divided into a plurality of water-in-oil micro-droplets with uniform sizes, and from the thermodynamic point of view, the water-in-oil droplets are naturally an unstable system, the droplets are only relative and temporarily stable systems, and the instability of the droplets is mainly reflected in the crushing, aggregation and fusion of the droplets. Particularly, PCR amplification and high-low temperature circulation are realized, and liquid drops are more easily broken and fused. Therefore, in order to ensure the stability of the water-in-oil micro-droplets, it is necessary to add a surfactant to the fluorinated oil to adjust the magnitude of the surface tension of the oil-water interface to stabilize the droplets.
At present, the most common surfactant for PCR detection is polyethylene glycol (PEG), and the PEG and perfluoropolyether resin (PFPE) are synthesized into the fluorine-containing surfactant PEG-PFPE 2 The surfactant can obviously improve the stability of liquid drops generated at normal temperature. But PEG-PFPE 2 The PEG molecule in the probe has certain hydrophobicity and is sensitive to temperature change, the generated liquid drop is easy to crack in the process of carrying out PCR thermal cycle, small molecule transfer is easy to occur among the liquid drops, and the final detection result is influenced, so that the surfactant for PCR detection with more excellent performance needs to be developed, the generated micro liquid drop is more stable, and the problem of fusion and fragmentation is avoided.
Disclosure of Invention
In order to solve the technical problems that in the prior art, in the technical field of digital PCR, in the detection process of realizing absolute quantification of an initial DNA template by a direct counting method, a sample is subjected to micro-titration by a micro-fluidic or micro-titration method before being subjected to PCR amplification treatment, the sample containing nucleic acid molecules is difficult to be divided into thousands of water-in-oil micro-droplets with uniform size, good stability and moderate surface tension, the micro-droplets tend to be converged and tend to fuse, the micro-droplets are easy to break in the process of PCR thermal cycle, and small molecule transfer is easy to occur among the droplets, so that the final detection result is influenced, and therefore, the absolute quantification of the DNA template is difficult to be accurately realized, the invention discloses a perfluoropolyether type fluorocarbon glycol surfactant with the structural characteristics shown in formula I on the first aspect, wherein n is a positive integer and n =1-43.
In some embodiments of the present invention, the method for preparing the perfluoropolyether fluorocarbon glycol surfactant comprises the steps of:
the method comprises the following steps: performing acyl chlorination on the perfluoropolyether of the formula II to obtain acylated perfluoropolyether of the formula III, wherein the average molecular weight of the perfluoropolyether is 2500-7000, n is a positive integer, and n =1-43;
step two: the preparation method comprises the following steps of (1) reacting acylated perfluoropolyether with (2- (1,3-dihydroxypropyl-2-amino) -2-hydroxymethyl propane-1,3-diol) to prepare the perfluoropolyether type fluorocarbon diol surfactant in the formula I;
in some embodiments of the present invention, the step of subjecting the perfluoropolyether of formula II to acylchlorination reaction specifically comprises:
adding fluorine oil into perfluoropolyether of a formula II for dissolving, then adding thionyl chloride, and carrying out acyl chlorination reaction for 10-15h at the temperature of 30-80 ℃;
in some embodiments of the present invention, the acyl chlorination reaction is followed by the steps of: cooling to room temperature, sequentially carrying out reduced pressure distillation and vacuum pumping treatment to remove the solvent and thionyl chloride, and then carrying out column chromatography extraction to prepare acylated perfluoropolyether;
in some embodiments of the present invention, the specific steps of step two include:
adding a reaction solvent into acylated perfluoropolyether, then adding (2- (1,3-dihydroxypropyl-2-amino) -2-hydroxymethyl propane-1,3-diol) for reaction, stirring and refluxing for reaction for 13-18h at 55-65 ℃, sequentially carrying out vacuum pumping treatment and liquid separation treatment to remove unreacted (2- (1,3-dihydroxypropyl-2-amino) -2-hydroxymethyl propane-1,3-diol), removing the solvent by reduced pressure evaporation, and then carrying out column chromatography extraction to prepare the perfluoropolyether type fluorocarbon glycol surfactant;
in some embodiments of the invention, the molar ratio of perfluoropolyether to thionyl chloride is in the range of from 0.04 to 0.1:1;
in some embodiments of the invention, the mass ratio of the acylated perfluoropolyether to (2- (1,3-dihydroxypropyl-2-amino) -2-hydroxymethylpropane-1,3-diol) is 0.01 to 0.05;
in some embodiments of the invention, the reaction solvent is a combination of 2- (trifluoromethyl) -3-ethoxydodecafluorohexane and dimethyl sulfoxide, and the volume ratio of 2- (trifluoromethyl) -3-ethoxydodecafluorohexane to dimethyl sulfoxide is 2.5-5:1;
the invention discloses fluorinated oil which comprises the perfluoropolyether type fluorocarbon glycol surfactant with the mass percentage concentration of 1-5%.
In some embodiments of the present invention, the perfluoropolyether fluorocarbon diol surfactants in the fluorinated oil are hydrogen bonded together to form a chain structure of formula IV:
in some embodiments of the invention, the structural formula of (2- (1,3-dihydroxypropyl-2-amino) -2-hydroxymethylpropane-1,3-diol) is shown in structure V:
the third aspect of the invention discloses an application of perfluoropolyether fluorocarbon glycol surfactant in microfluidic droplet generation.
Compared with the prior art, the method has the following beneficial effects:
the invention makes perfluoropolyether react with (2- (1,3-dihydroxypropyl-2-amino) -2-hydroxymethyl propane-1,3-diol), wherein (2- (1,3-dihydroxypropyl-2-amino) -2-hydroxymethyl propane-1,3-diol) is in a tree structure, two hydroxyl groups are distributed on the upper side and the lower side of the tree structure, the perfluoropolyether fluorocarbon diol surfactant with the structural characteristics shown in formula I is prepared after the reaction with the perfluoropolyether, two hydroxyl groups are distributed on the upper side and the lower side of the prepared perfluoropolyether fluorocarbon diol surfactant symmetrically, the perfluoropolyether fluorocarbon diol surfactant is added into fluorinated oil with the mass percentage concentration of 1-5 percent and is used as a surfactant in a PCR method for preparing oil phase, before the PCR amplification treatment is carried out on a reaction system, adding fluorinated oil drops containing 1-5% of fluoropolyether type fluorocarbon surfactant in mass percentage concentration into a reaction system, carrying out micro-titration treatment on the fluorinated oil drops, mixing oil-water two-phase liquid under specific conditions, dividing the reaction system containing nucleic acid molecules into a plurality of water-in-oil micro-droplets with uniform nano-scale size, wherein on the surfaces of the generated micro-droplets, fluorine-containing groups of the perfluoropolyether type fluorocarbon glycol surfactant are positioned in an oil phase, and polar group carbonyl groups are positioned in a water phase, in an aqueous solution, because two hydroxyl groups are symmetrically distributed on the left side and the right side of the perfluoropolyether type fluorocarbon glycol surfactant, the two hydroxyl groups are arranged in parallel on the inner surface of the droplets, and the adjacent perfluoropolyether type fluorocarbon glycol surfactant molecules are mutually connected through hydrogen bonds between two molecules which are mutually parallel, and then a stable 'layered film' is formed on the surface layer close to the liquid drops, the stable 'layered film' can effectively enhance the stability of the micro liquid drops and inhibit the small molecule transfer among the micro liquid drops, the micro liquid drops are stabilized by changing the surface tension of an oil-water interface, the fusion and the breakage of the micro liquid drops are avoided, the accuracy of a detection result is improved, and in a PCR amplification circulation experiment, the perfluoropolyether type fluorocarbon glycol surfactant prepared by the method is uniform in size after 50 thermal cycles, the stability of the micro liquid drops is excellent, and the problem of fusion and breakage does not occur.
Drawings
In order to more clearly explain the technical solution of the present application, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious to those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of a perfluoropolyether fluorocarbon glycol surfactant obtained in the first example;
FIG. 2 is a schematic diagram of generating water-in-oil droplets by a cross-convergent microfluidic chip according to the third embodiment;
FIG. 3 is a microscopic view (at 20X magnification) of a droplet generated in example III using the fluorinated oil prepared in example II after thermal cycling for PCR amplification;
FIG. 4 shows the third embodiment of the present invention in which PEG-PFPE with a concentration of 3% by mass is used 2 Microscopic observation of surfactant-containing oil-fluorinated droplets after thermal cycling of the PCR amplification (20-fold magnification);
FIG. 5 is a schematic representation of the addition of perfluoropolyether fluorocarbon glycol surfactants of the present application and the prior art fluorosurfactant PEG-PFPE 2 The result of the PCR amplification experiment of the generated water-in-oil droplets is shown.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present application more comprehensible, the present application is described in further detail with reference to the following embodiments.
Example one
A perfluoropolyether type fluorocarbon diol surfactant is synthesized by the following steps:
the preparation method of the perfluoropolyether fluorocarbon glycol surfactant comprises the following steps:
the method comprises the following steps: weighing 0.583g of perfluoropolyether (PFPE-COOH (obtained from Kemu, krytox157FSH, the average molecular weight is 6500, n is a positive integer, and n = 1-43) in a three-neck flask, dissolving the perfluoropolyether in 40mL of fluorine oil (HFE-7500, obtained from 3M company), dropwise adding 0.128mL of thionyl chloride, stirring and refluxing for 12 hours at the temperature of 60 ℃ under the protection of nitrogen, carrying out acyl chlorination on perfluoropolyether carboxylic acid, cooling to room temperature, carrying out distillation and concentration by using a rotary evaporator to obtain solid acylated perfluoropolyether, removing excessive thionyl chloride and solvent fluorine oil through vacuum pumping treatment, and then carrying out purification treatment on the solid acylated perfluoropolyether through column chromatography and extraction;
step two: dissolving the solid acylated perfluoropolyether subjected to column chromatography extraction in 60ml of a solvent with the volume ratio of 3:1 from 2- (trifluoromethyl) -3-ethoxydodecafluorohexane: adding 23.134g of 2- (1,3-dihydroxypropyl-2-amino) -2-hydroxymethyl propane-1,3-diol into a reaction solvent consisting of dimethyl sulfoxide for reaction, stirring and refluxing for 15 hours at 60 ℃, vacuumizing to remove a byproduct HCL, performing liquid separation treatment by using a separating funnel to remove unreacted 2- (1,3-dihydroxypropyl-2-amino) -2-hydroxymethyl propane-1,3-diol, performing rotary evaporation by using a rotary evaporator to remove the solvent, and performing column chromatography extraction to prepare the perfluoropolyether type fluorocarbon glycol surfactant.
And (3) verifying the structure of the perfluoropolyether fluorocarbon glycol surfactant.
The structure of the perfluoropolyether fluorocarbon glycol surfactant is verified by nuclear magnetic resonance hydrogen spectrum, and H in figure 1 1 The results of NMR spectroscopy are shown in Table 1, and are represented by H in FIG. 1 1 NMR spectrum analysis shows that: the product structure is not only perfluoropolyether type fluorocarbon glycol surfactant.
Table 1: perfluoropolyether fluorocarbon glycol surfactant nuclear magnetic resonance hydrogen spectrum proton peak
Example two
A fluorinated oil was prepared by dissolving the perfluoropolyether fluorocarbon glycol surfactant prepared in example one in 3% by weight in a fluorinated oil (HFE-7500 from 3M).
EXAMPLE III
Use of the fluorinated oil prepared in example two in microfluidic droplet processing for PCR amplification:
PCR oil phase: with the fluorinated oil of example two as the PCR oil phase, the PCR water phase comprises: 4 xreactional mix (made by manufacturer) 21.3 μ L; VIC (dye) 4.3. Mu.L; 59.5 μ L of water.
The PCR oil phase and the PCR water phase were mixed according to 7:3 volume ratio, generating water-in-oil droplets (80-100 μm) with good uniformity by a cross-converging microfluidic chip, taking a small amount of generated micro-droplet samples to place on a slide glass made of PMMA, and observing the droplet shapes as shown in figure 2, wherein the droplet surfaces are uniform and are not damaged.
Setting a control experiment: PEG-PFPE with 3 percent of same mass concentration of two phases of the embodiment is prepared 2 Fluorinated oil of surfactant as PCR oil phase (wherein polyethylene glycol (PEG) and perfluoropolyether resin (PFPE) are adopted to synthesize fluorine-containing surfactant PEG-PFPE 2 ) And the PCR aqueous phase comprises: 4 xreactional mix (made by manufacturer) 21.3 μ L; VIC (dye) 4.3. Mu.L; water 59.5 μ L, PCR oil phase and PCR water phase were mixed according to 7:3 volume ratio, and generating water-in-oil droplets by a cross convergent microfluidic chip.
Then, a PCR amplification thermal cycle experiment is carried out, the PCR amplification step is shown in Table 2, the micro-droplet morphology under a microscope after the water-in-oil micro-droplets (80 μm-100 μm) with good generation uniformity generated by the method are subjected to PCR amplification thermal cycle is shown in FIG. 3, and the micro-droplets are observed under the microscope, the uniformity state of the micro-droplets is still good after 50 cycles of thermal cycle, the micro-droplets still have regular honeycomb morphology, and the existence rate (CV value) of damaged and fused droplets is far lower than 5%.
TABLE 2 PCR amplification thermal cycling Experimental procedure
The shape of the microdroplet generated in the control experiment under a microscope after thermal cycling through PCR amplification is shown in FIG. 4, and the microdroplet is observed under the microscope, after 50 cycles of thermal cycling, the homogeneity state of the microdroplet is poor, the shape of a microdroplet honeycomb is damaged, and the existence rate (CV value) of the damaged and fused microdroplet is far more than 15%.
Therefore, in example 2 of the present application, the fluorinated oil containing 3% by mass of fluoropolyether type fluorocarbon surfactant was used as the PCR oil phase, and the ratio of the fluorinated oil to the PCR water phase was determined according to 7:3, after being uniformly mixed in a volume ratio, a cross-shaped convergent microfluidic chip can generate water-in-oil microdroplets (80-100 microns) with good uniformity, fluorine-containing groups of perfluoropolyether fluorocarbon glycol surfactants are located in an oil phase on the surfaces of the generated microdroplets, polar group carbonyl groups are located in a water phase, as shown in formula I, two hydroxyl groups are symmetrically distributed on the upper side and the lower side of the perfluoropolyether fluorocarbon glycol surfactants and are arranged in parallel on the inner surfaces of the microdroplets, two parallel intermolecular hydrogen bonds between the adjacent perfluoropolyether fluorocarbon glycol surfactants are mutually connected to form a chain structure in formula IV, and a stable layered film is further formed on the surface layer close to the microdroplets, so that the stability of the microdroplets can be effectively enhanced, small molecule transfer among the microdroplets is inhibited, the microdroplets are stabilized in size by changing the surface tension of the oil-water interface, the microdroplets are prevented from being subjected to micro-fused and crushed, the accuracy of detection results is improved, and in a PCR amplification cycle experiment, the perfluoropolyether fluorocarbon glycol surfactants prepared by the application have uniform microdroplets and excellent stability and have no problem of micro-fused drops after 50 thermal cycles.
Fluorinated surfactant PEG-PFPE in the prior art 2 The polyethylene glycol (PEG) and perfluoropolyether resin (PFPE) are synthesized, the structural formula is VI, wherein in the structural formula VI, n is more than or equal to 20 and less than or equal to 80,3 and less than or equal to o + q is more than or equal to 7,9 and less than or equal to 14, and the average molecular weight of the structural formula VI is 13000.
The perfluoropolyether type fluorocarbon glycol surfactant in the application and the fluorine-containing surfactant PEG-PFPE in the prior art are mixed 2 Added to fluorinated oil according to the procedure in example threeGenerating water-in-oil droplets, and respectively adding the perfluoropolyether fluorocarbon glycol surfactant in the application and the fluorine-containing surfactant PEG-PFPE in the prior art without performing PCR amplification thermal cycle experiment 2 The generated micro-droplet system generates water-in-oil droplets according to the steps in the third embodiment, the water-in-oil droplet system is placed outdoors for 7 hours, and the result is shown in figure 5, and the result shows that the fluorine-containing surfactant PEG-PFPE is added before a PCR amplification thermal cycle experiment is not carried out and along with the prolonging of the placing time 2 The more serious the fusion of the droplets formed in the oil phase system (a); the droplets generated by the oil phase system added with the surfactant prepared in the first embodiment of the present application are stable as time goes on, and no large-area droplet fusion occurs after the fluorine oil volatilizes, so that the water-in-oil micro-droplets generated by the surfactant prepared in the present application can play a key role in stabilizing the droplet morphology.
Secondly, the perfluoropolyether type fluorocarbon glycol surfactant in the application and the fluorine-containing surfactant PEG-PFPE in the prior art are respectively mixed 2 Adding the mixture into fluorinated oil, generating water-in-oil droplets according to the third step in the example, then carrying out a PCR amplification thermal cycle experiment according to the second step, and respectively adding the perfluoropolyether type fluorocarbon glycol surfactant in the application and the fluorine-containing surfactant PEG-PFPE in the prior art 2 After the generated micro-droplet system was subjected to PCR amplification thermal cycling experiments according to the third step of the example, the micro-droplet system was left outdoors for 7 hours, and the results are shown in FIG. 5.
The results show that as the standing time is prolonged and as the fluorine oil is volatilized, the fluorine-containing surfactant PEG-PFPE is added 2 The generated droplets of the oil phase system prepared by adding the surfactant prepared by the embodiment of the application are very stable along with the prolonging of the time, and the droplets generated by the oil phase system are not fused into large-area droplets after the fluorine oil is volatilized, so that the water-in-oil micro-droplets generated by adding the surfactant prepared by the application can play a key role in stabilizing the droplet form after the PCR amplification thermal cycle experiment stage.
The present application has been described in detail with reference to particular embodiments and illustrative examples, but the description is not intended to be construed as limiting the application. Those skilled in the art will appreciate that various equivalent substitutions, modifications or improvements may be made to the presently disclosed embodiments and implementations thereof without departing from the spirit and scope of the present disclosure, and these fall within the scope of the present disclosure. The protection scope of this application is subject to the appended claims.
Claims (10)
1. The perfluoropolyether fluorocarbon glycol surfactant has the structural characteristics shown in the formula I, and the structure is as follows:
n is a positive integer, n =1-43.
2. A process for preparing a perfluoropolyether fluorocarbon glycol surfactant of claim 1 comprising the steps of:
the method comprises the following steps: performing acyl chlorination on the perfluoropolyether of the formula II to obtain acylated perfluoropolyether of the formula III, wherein the average molecular weight of the perfluoropolyether is 2500-7000, n is a positive integer, and n =1-43;
step two: and (2- (1,3-dihydroxypropyl-2-amino) -2-hydroxymethylpropane-1,3-diol) reacts with the acylated perfluoropolyether to prepare the perfluoropolyether type fluorocarbon diol surfactant in the formula I.
3. The method for preparing perfluoropolyether fluorocarbon diol surfactant according to claim 2, wherein the step one of acylchlorination of perfluoropolyether of formula II comprises the following steps:
and (2) adding the perfluoropolyether in the formula II into fluorine oil for dissolving, then adding thionyl chloride, and carrying out acyl chlorination reaction for 10-15h at the temperature of 30-80 ℃.
4. The method of claim 3, further comprising the following steps after said acyl chlorination reaction: cooling to room temperature, removing the solvent and thionyl chloride through reduced pressure distillation and vacuum pumping treatment in sequence, and then performing column chromatography extraction to prepare the acylated perfluoropolyether.
5. The method for preparing a perfluoropolyether type fluorocarbon glycol surfactant as claimed in claim 2, wherein said step two comprises the specific steps of:
adding a reaction solvent into the acylated perfluoropolyether, then adding (2- (1,3-dihydroxypropyl-2-amino) -2-hydroxymethyl propane-1,3-diol) for reaction, stirring and refluxing for reaction for 13-18h at 55-65 ℃, sequentially carrying out vacuum pumping treatment and liquid separation treatment to remove unreacted (2- (1,3-dihydroxypropyl-2-amino) -2-hydroxymethyl propane-1,3-diol), removing the solvent by reduced pressure evaporation, and then carrying out column chromatography extraction to prepare the perfluoropolyether fluorocarbon glycol surfactant.
6. A process for preparing a perfluoropolyether fluorocarbon glycol surfactant according to claim 3, wherein the molar ratio of perfluoropolyether to sulfoxide chloride is 0.04-0.1:1.
7. the method of claim 5, wherein the mass ratio of said acylated perfluoropolyether to (2- (1,3-dihydroxypropyl-2-amino) -2-hydroxymethylpropane-1,3-diol) is 0.01-0.05.
8. The method of claim 5, wherein the reaction solvent is a mixture of 2- (trifluoromethyl) -3-ethoxydodecafluorohexane and dimethyl sulfoxide, and the volume ratio of 2- (trifluoromethyl) -3-ethoxydodecafluorohexane to dimethyl sulfoxide is 2.5-5:1.
9. a fluorinated oil comprising from 1% to 5% by weight of the perfluoropolyether fluorocarbon diol surfactant of claim 1.
10. Use of the perfluoropolyether fluorocarbon glycol surfactant of claim 1 in microfluidic droplet processing for PCR amplification.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020052465A1 (en) * | 1999-03-12 | 2002-05-02 | George Fleet | Bis(1,3-dihydroxy-prop-2-yl) amine and derivatives thereof in the manufacture of polymers |
| CN106823990A (en) * | 2017-01-20 | 2017-06-13 | 广东顺德工业设计研究院(广东顺德创新设计研究院) | perfluoropolyether fluorocarbon surfactant and its preparation method and application |
| CN111233664A (en) * | 2020-01-17 | 2020-06-05 | 中国科学院上海高等研究院 | Perfluoropolyether ester-based derivative and preparation method and application thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020052465A1 (en) * | 1999-03-12 | 2002-05-02 | George Fleet | Bis(1,3-dihydroxy-prop-2-yl) amine and derivatives thereof in the manufacture of polymers |
| CN106823990A (en) * | 2017-01-20 | 2017-06-13 | 广东顺德工业设计研究院(广东顺德创新设计研究院) | perfluoropolyether fluorocarbon surfactant and its preparation method and application |
| CN111233664A (en) * | 2020-01-17 | 2020-06-05 | 中国科学院上海高等研究院 | Perfluoropolyether ester-based derivative and preparation method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| MOHAMMAD SUMAN CHOWDHURY，等: "Dendronized fluorosurfactant for highly stable water-in-fluorinated oil emulsions with minimal inter-droplet transfer of small molecules" * |
| MOHAMMAD SUMAN CHOWDHURY，等: "Dendronized fluorosurfactant for highly stable water-in-fluorinated oil emulsions with minimal inter-droplet transfer of small molecules", 《NATURE COMMUNICATIONS》 * |
| YA-LING CHIU,等: "Synthesis of Fluorosurfactants for Emulsion-Based Biological Applications", 《ACS NANO》 * |
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