CN108148204B - Perfluoropolyether-polyethylene glycol block copolymer, preparation method and application thereof - Google Patents
Perfluoropolyether-polyethylene glycol block copolymer, preparation method and application thereof Download PDFInfo
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- CN108148204B CN108148204B CN201711396071.8A CN201711396071A CN108148204B CN 108148204 B CN108148204 B CN 108148204B CN 201711396071 A CN201711396071 A CN 201711396071A CN 108148204 B CN108148204 B CN 108148204B
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- 229920001400 block copolymer Polymers 0.000 title claims abstract description 42
- 229920001223 polyethylene glycol Polymers 0.000 title claims abstract description 42
- 239000002202 Polyethylene glycol Substances 0.000 title claims abstract description 41
- 238000002360 preparation method Methods 0.000 title abstract description 12
- 239000003921 oil Substances 0.000 claims abstract description 47
- 229910052731 fluorine Inorganic materials 0.000 claims abstract description 24
- 239000011737 fluorine Substances 0.000 claims abstract description 24
- 238000000034 method Methods 0.000 claims abstract description 24
- CTSLXHKWHWQRSH-UHFFFAOYSA-N oxalyl chloride Chemical compound ClC(=O)C(Cl)=O CTSLXHKWHWQRSH-UHFFFAOYSA-N 0.000 claims abstract description 24
- YCKRFDGAMUMZLT-UHFFFAOYSA-N Fluorine atom Chemical compound [F] YCKRFDGAMUMZLT-UHFFFAOYSA-N 0.000 claims abstract description 22
- 239000010702 perfluoropolyether Substances 0.000 claims abstract description 17
- 239000004094 surface-active agent Substances 0.000 claims abstract description 14
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- 238000006243 chemical reaction Methods 0.000 claims description 29
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 28
- 239000007788 liquid Substances 0.000 claims description 14
- 238000003752 polymerase chain reaction Methods 0.000 claims description 14
- GETTZEONDQJALK-UHFFFAOYSA-N (trifluoromethyl)benzene Chemical compound FC(F)(F)C1=CC=CC=C1 GETTZEONDQJALK-UHFFFAOYSA-N 0.000 claims description 13
- 239000002904 solvent Substances 0.000 claims description 13
- 239000003054 catalyst Substances 0.000 claims description 10
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- 239000003960 organic solvent Substances 0.000 claims description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 6
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- 150000007530 organic bases Chemical group 0.000 claims description 5
- 125000001153 fluoro group Chemical group F* 0.000 claims description 4
- 230000035484 reaction time Effects 0.000 claims description 4
- IMNIMPAHZVJRPE-UHFFFAOYSA-N triethylenediamine Chemical compound C1CN2CCN1CC2 IMNIMPAHZVJRPE-UHFFFAOYSA-N 0.000 claims description 4
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 3
- DIAIBWNEUYXDNL-UHFFFAOYSA-N n,n-dihexylhexan-1-amine Chemical compound CCCCCCN(CCCCCC)CCCCCC DIAIBWNEUYXDNL-UHFFFAOYSA-N 0.000 claims description 2
- 238000005497 microtitration Methods 0.000 claims 1
- 239000002994 raw material Substances 0.000 abstract description 8
- 238000012408 PCR amplification Methods 0.000 abstract description 6
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- 238000005580 one pot reaction Methods 0.000 abstract 1
- 239000000047 product Substances 0.000 description 24
- 238000000746 purification Methods 0.000 description 9
- WTFAGPBUAGFMQX-UHFFFAOYSA-N 1-[2-[2-(2-aminopropoxy)propoxy]propoxy]propan-2-amine Chemical compound CC(N)COCC(C)OCC(C)OCC(C)N WTFAGPBUAGFMQX-UHFFFAOYSA-N 0.000 description 6
- 239000007810 chemical reaction solvent Substances 0.000 description 6
- 238000001035 drying Methods 0.000 description 6
- 239000011261 inert gas Substances 0.000 description 6
- 238000010586 diagram Methods 0.000 description 5
- 238000002329 infrared spectrum Methods 0.000 description 5
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- 238000003756 stirring Methods 0.000 description 5
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- 230000015572 biosynthetic process Effects 0.000 description 4
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- 150000007523 nucleic acids Chemical class 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 4
- 108020004707 nucleic acids Proteins 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 238000005382 thermal cycling Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- HHBBIOLEJRWIGU-UHFFFAOYSA-N 4-ethoxy-1,1,1,2,2,3,3,4,5,6,6,6-dodecafluoro-5-(trifluoromethyl)hexane Chemical compound CCOC(F)(C(F)(C(F)(F)F)C(F)(F)F)C(F)(F)C(F)(F)C(F)(F)F HHBBIOLEJRWIGU-UHFFFAOYSA-N 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
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- OKIYQFLILPKULA-UHFFFAOYSA-N 1,1,1,2,2,3,3,4,4-nonafluoro-4-methoxybutane Chemical compound COC(F)(F)C(F)(F)C(F)(F)C(F)(F)F OKIYQFLILPKULA-UHFFFAOYSA-N 0.000 description 2
- PVVTWNMXEHROIA-UHFFFAOYSA-N 2-(3-hydroxypropyl)-1h-quinazolin-4-one Chemical group C1=CC=C2NC(CCCO)=NC(=O)C2=C1 PVVTWNMXEHROIA-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 150000001263 acyl chlorides Chemical class 0.000 description 2
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- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 2
- 229910052753 mercury Inorganic materials 0.000 description 2
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- FYSNRJHAOHDILO-UHFFFAOYSA-N thionyl chloride Chemical compound ClS(Cl)=O FYSNRJHAOHDILO-UHFFFAOYSA-N 0.000 description 2
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- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
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- 238000005660 chlorination reaction Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- NBVXSUQYWXRMNV-UHFFFAOYSA-N fluoromethane Chemical compound FC NBVXSUQYWXRMNV-UHFFFAOYSA-N 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
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- 238000010438 heat treatment Methods 0.000 description 1
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- 238000002032 lab-on-a-chip Methods 0.000 description 1
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- 230000003287 optical effect Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
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- -1 polyoxyethylene chain Polymers 0.000 description 1
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- 238000011160 research Methods 0.000 description 1
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- 238000013112 stability test Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
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- C08G81/00—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention relates to a perfluoropolyether-polyethylene glycol block copolymer, a preparation method and application thereof. The structural formula of the perfluoropolyether-polyethylene glycol block copolymer is shown as the following formula I:
the average molecular weight is 5000-16000. It is prepared by one-step reaction of perfluoropolyether, oxalyl chloride and polyamide, and has simple process and low cost. The oil is used as a fluorine surfactant as a solute to be dissolved in fluorine oil to obtain the droplet generation oil used in the micro-droplet processing process of the digital polymerase chain reaction amplification, and has the characteristics of low raw material cost and good performance and stability.
Description
Technical Field
The invention relates to the technical field of macromolecules, in particular to a perfluoropolyether-polyethylene glycol block copolymer, a preparation method and application thereof.
Background
Digital polymerase chain reaction, or Digital PCR (dPCR), is an absolute nucleic acid molecule quantification technique. Compared with qPCR, the digital polymerase chain reaction can directly count the number of DNA molecules, which is an absolute quantification of the starting sample. The digital polymerase chain reaction is also divided into a chip type and a micro-drop type at the present stage, and relatively speaking, the micro-drop type digital polymerase chain reaction system has higher technical content and higher data accuracy. The principle is to divide the reaction system containing nucleic acid molecules into thousands of nanoliter-scale droplets, wherein each droplet contains or does not contain the nucleic acid target molecules to be detected, or contains one to several nucleic acid target molecules to be detected. After amplification by polymerase chain reaction, each droplet is detected one by one and the fluorescence signal is processed using poisson distribution.
The micro-drop digital polymerase chain reaction system has extremely high scientific research content as a technology concerned by the biomedical industry in the 21 st century, and only foreign brands of Bio-Rad and Raindacene have related mature products at the present stage, so that oil required by a digital polymerase chain reaction instrument on the market, including a probe method and a dye method, almost all come from the manufacturers.
The surfactant can avoid the fusion of liquid drops by reducing the tension of an oil-water interface, and can even keep the stability of a liquid drop system under the heating and pressurizing conditions, and the nonionic fluorine surfactants such as perfluoropolyether esterified compounds, perfluoropolyether amidates and the like are reported at present. The stable liquid drop can enable the interior of the liquid drop to be a reaction chamber, and the DNA template can be subdivided into single DNA templates for expansion.
The nonionic type product in the existing fluorine surfactant mainly takes a polyoxyethylene chain as a hydrophilic group, the preparation process of the surfactant is very complicated, the general preparation steps are more than two steps, and the intermediate product is not sold in the market, so that the consumable material required by the generation of the liquid drop is high in price, and the wider market cannot be met.
The C.Holtz et al of Harvard university, mentioned in the Lab on a chip journal, can be prepared by reacting fluoropolyether carboxylic acid with thionyl chloride to produce acyl chloride, and then dissolving and adding self-made PEG-Block under the protection of inert gas to react to produce fluoropolyether carboxylic acid amide. However, the intermediate product acyl chloride is very easy to react with water in the reaction process, which causes poor effect of the subsequent steps and even product scrapping, and the difficulty in preparing PEG-Block is very high. In addition, the purification method using centrifugation results in low yield of reactants, which is not suitable for commercial production.
Disclosure of Invention
In view of the above, there is a need to provide a method for preparing a perfluoropolyether-polyethylene glycol block copolymer, which has the advantages of simple preparation, excellent performance and high yield, and can be used as a fluorosurfactant for preparing a droplet formation oil for use in a microdroplet treatment of digital pcr amplification.
A perfluoropolyether-polyethylene glycol block copolymer, the structural formula of which is shown in formula I below:
wherein n is more than or equal to 20 and less than or equal to 80, o + q is more than or equal to 3 and less than or equal to 7, and p is more than or equal to 9 and less than or equal to 14.
In one embodiment, the average molecular weight of the perfluoropolyether-polyethylene glycol block copolymer is 5000-16000.
The invention also provides a preparation method of the perfluoropolyether-polyethylene glycol block copolymer, which comprises the following steps of reacting perfluoropolyether, oxalyl chloride and polyamide in a solvent to obtain the perfluoropolyether-polyethylene glycol block copolymer, wherein the solvent is fluorine oil, the structural formula of the perfluoropolyether is shown as the following formula II, the structural formula of the oxalyl chloride is shown as the following formula III, and the structural formula of the polyamide is shown as the following formula IV:
wherein n is more than or equal to 20 and less than or equal to 80, o + q is more than or equal to 3 and less than or equal to 7, and p is more than or equal to 9 and less than or equal to 14.
The invention uses the commercial polyamide (purchased from Hensman, model number JEFFAMINE series) with the structural formula shown as formula IV as the raw material to replace the PEG-amine which is a compound with complex preparation and difficult purification, so that the preparation method is simpler and is suitable for commercial production.
The specific reaction equation is as follows:
in the reaction process, oxalyl chloride shown in formula III plays a role in acyl chlorination, COOH of raw material perfluoropolyether (PFPE-COOH) is changed into COCl, and then the COCl reacts with commercial polyamide shown in formula IV to obtain a perfluoropolyether-polyethylene glycol block copolymer product shown in formula I. The by-product in the reaction is hydrochloric acid, which is dissolved in the fluorine oil 7100 in the later cleaning process, and other impurities can be easily separated out and cleaned.
In one embodiment, the reaction temperature is 60-120 ℃ and the reaction time is 36-72 h. The control reaction is carried out according to the limited conditions, so that the occurrence of side reactions can be reduced, and the product yield can be improved. Preferably, the reaction temperature is 80-110 ℃ and the reaction time is 48-62 h. The preferred reasons are: the temperature range and the reaction time range can ensure that the reaction between the raw materials is more complete.
In one embodiment, the solvent is a fluoro oil. The fluorine oil can avoid the hydrolysis of oxalyl chloride, and can dissolve each reaction raw material to react to obtain a final product.
In one embodiment, the fluoro oil is methoxy-nonafluorobutane. The methoxy-nonafluorobutane is used as a solvent during the reaction, and has the advantages of good matching with a polar organic solvent and high controllability.
In one embodiment, the solvent for the reaction may further include an organic solvent having polarity. The organic solvent is at least one of trifluorotoluene, dimethylformamide, tetrahydrofuran and dimethyl sulfoxide, and is preferably trifluorotoluene. The organic solvent with polarity and the fluorine oil are simultaneously used as reaction solvents, so that the purity of the perfluoropolyether-polyethylene glycol block copolymer can be improved.
In one embodiment, the volume ratio of the fluorine oil to the organic solvent is (0.5-2.0): 1.
In one embodiment, a catalyst may also be used during the reaction. The catalyst is an organic base. The organic base may be 4-dimethylaminopyridine, trihexylamine, or triethylenediamine. Preferably, the organic base is 4-dimethylaminopyridine. The preferred reasons are: 4-dimethylamino pyridine is used as a catalyst, has higher catalytic capability, can realize acylation on reactants, and has extremely obvious effect on improving the yield of products.
In one embodiment, the reaction further comprises a purification and/or centrifugation step after completion of the reaction. And in the purification step, adding the obtained product into the fluorine oil again for dissolving, then dropwise adding an organic solvent to separate out the perfluoropolyether-polyethylene glycol block copolymer shown in the formula I in the product, and drying. The purity of the product can be greatly improved through the purification step. After purification, a centrifugation step can be carried out, or the mixture obtained by the reaction can be directly centrifuged, namely, after centrifugation, the product is dried to obtain the product.
The invention also provides application of the perfluoropolyether-polyethylene glycol block copolymer. The PFPE-PEG block copolymers are useful as fluorosurfactants.
The invention also provides a droplet generating oil. The liquid drop generating oil comprises a fluorine surfactant and fluorine oil, wherein the fluorine surfactant is perfluoropolyether-polyethylene glycol block copolymer. The liquid drop generated oil can ensure the stability of the liquid drops and has better effect.
In one embodiment, the mass percent of the fluorinated surfactant in the fluorinated oil solution is 2% -6%.
In one embodiment, the fluorine oil used in the droplet generating oil is HFE-7500.
In one embodiment, the droplet forming oil is used in a process of microdroplet processing for digital polymerase chain reaction amplification. The fluorine surfactant is applied to the droplet-type digital polymerase chain reaction, so that the surface tension of droplets can be reduced, and the droplet stability is effectively improved.
The perfluoropolyether-polyethylene glycol block copolymer can be used as a fluorine surfactant and can be widely applied to the field of microfluidics as a reagent, such as single cell packaging or microorganism detection. The perfluoropolyether-polyethylene glycol block copolymer is applied to a droplet type digital polymerase chain reaction, the stability of the droplets can be ensured, and in addition, the perfluoropolyether-polyethylene glycol block copolymer used as a fluorine surfactant has excellent surface performance and biocompatibility.
Compared with the prior art, the invention has the following beneficial effects:
the perfluoropolyether-polyethylene glycol block copolymer is used as a fluorosurfactant and can be used as an oil phase of a polymerase chain reaction probe method, so that the application on the liquid drop type digital polymerase chain reaction is realized, the stability of liquid drops can be ensured, and the perfluoropolyether-polyethylene glycol block copolymer has the biocompatibility characteristic and does not hinder the amplification reaction of a biological water phase reagent.
The preparation method of the perfluoropolyether-polyethylene glycol block copolymer is a one-step preparation process, has the advantages of simple process and low cost, and is suitable for commercial application.
The perfluoropolyether-polyethylene glycol block copolymer is used as the fluorosurfactant to prepare the droplet generating oil, and has the characteristics of low raw material cost and good performance and stability.
Drawings
FIG. 1 is a comparison of the IR spectra of the reaction feed and product of example 1, wherein: a represents the infrared spectrum of the product perfluoropolyether-polyethylene glycol block copolymer, b represents the infrared spectrum of the raw material perfluoropolyether, and c represents the infrared spectrum of the raw material polyamide.
FIG. 2 is the fluorine spectrum of the perfluoropolyether-polyethylene glycol block copolymer of example 1.
FIG. 3 is a microscopic observation image of a sample taken immediately after the formation of a droplet in example 6, wherein the length of the image is 200. mu.m.
FIG. 4 is a drawing showing an observation under a sampling microscope of example 6 in which the length of the drawing is 200 μm after the droplet formation is allowed to stand for 30 days.
FIG. 5 is a microscopic observation image of a sample taken immediately after the formation of a droplet in example 6, wherein the length of the image is 100. mu.m.
FIG. 6 is a drawing showing the observation under a sampling microscope of example 6 after allowing the droplets to stand for 30 days, wherein the length of the drawing is 100. mu.m.
FIG. 7 is a schematic diagram of the generation of uniform water-in-oil droplets using a microfluidic chip in example 6.
FIG. 8 is a microscopic image of PCR amplification of example 6 before thermal cycling, wherein the length of the image is 200 μm.
FIG. 9 is a microscopic image of the PCR amplification of example 6 after thermal cycling, wherein the length of the image is 200 μm.
FIG. 10 is an enlarged view of the black line frame of FIG. 9, in which the length of the drawing is 100 μm.
FIG. 11 is a schematic diagram of the fluorescence of the droplets of FIG. 10 after exposure adjustment in a mercury lamp environment.
FIG. 12 is a schematic diagram of the fluorescence detection of the liquid drop entering the microfluidic chip in example 6.
FIG. 13 is a waveform diagram of fluorescent signal acquisition performed by the droplet in example 6.
FIG. 14 is a data diagram of FIG. 13 after processing by a software algorithm.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The starting materials used in the following examples are all commercially available.
Example 1:
the reaction formula of the perfluoropolyether-polyethylene glycol block copolymer is as follows:
wherein n is more than or equal to 20 and less than or equal to 80, o + q is more than or equal to 3 and less than or equal to 7, and p is more than or equal to 9 and less than or equal to 14.
Specifically, under the protection of inert gas, the perfluoropolyether (from DuPont, type number is FSH157, the average molecular weight of the perfluoropolyether is 5000-7500), the oxalyl chloride of the formula III, the polyamide of the formula IV (from Henscman, type number is JEFFAMINE series) and the catalyst 4-Dimethylaminopyridine (DMAP) are dissolved in the mixed reaction solvent of the fluorine oil (from DuPont, type number is HFE-7100) and the benzotrifluoride. Wherein the volume ratio of the HFE-7100 to the benzotrifluoride is 1: 1. Stirring and reacting for 50h at the temperature of 100 ℃, and performing suction filtration and spin-drying on the solvent to obtain the product.
And (3) purification: and adding the product obtained by the reaction into HFE-7100 again for dissolving, dropwise adding dichloromethane for cleaning, and precipitating and carrying out rotary evaporation on the final product. Then vacuum drying is carried out to obtain a product of white transparent oily matter, namely the perfluoropolyether-polyethylene glycol block copolymer of the formula I. The average molecular weight of the resulting perfluoropolyether-polyethylene glycol block copolymer is 5000-16000.
The perfluoropolyether-polyethylene glycol block copolymer obtained is subjected to infrared spectrum and nuclear magnetic spectrum (fluorine spectrum) tests, and the test results are shown in fig. 1 and fig. 2.
As can be seen from fig. 1, the characteristic peak at position 1716cm-1 corresponds to the amide C ═ O double bond in the product, and the characteristic peak at position 1635cm-1 corresponds to the amine group in the product.
As can be seen from FIG. 2, a series of formants with chemical shifts in the range of-83 to-74 correspond to perfluoropolyether.
From the results of both FIG. 1 and FIG. 2, it was confirmed that the reaction yielded a perfluoropolyether-polyethylene glycol block copolymer of formula I.
Example 2:
the reaction procedure and purification procedure of example 2 were the same as in example 1. The method comprises the following specific steps:
under the protection of inert gas, perfluoropolyether (from DuPont, type FSH157, the average molecular weight of which is 5000-7500), oxalyl chloride of formula III, polyamide (from Henschel, type JEFFAMINE series) of formula IV and catalyst 4-Dimethylaminopyridine (DMAP) are dissolved in a mixed reaction solvent of fluoro-oil (from DuPont, type HFE-7100) and benzotrifluoride. Wherein the volume ratio of HFE-7100 to benzotrifluoride is 1.5: 1. Stirring and reacting for 42h at the temperature of 110 ℃, and performing suction filtration and spin-drying on the solvent to obtain the product.
Example 3:
the reaction procedure and purification procedure of example 3 were the same as in example 1. The method comprises the following specific steps:
under the protection of inert gas, the perfluoropolyether (from DuPont, type number is FSH157, the average molecular weight of which is 5000-7500), the oxalyl chloride of formula III, the polyamide of formula IV (from Henscman, type number is JEFFAMINE series) and the catalyst 4-Dimethylaminopyridine (DMAP) are dissolved in the mixed reaction solvent of the fluoro-oil (from DuPont, type number is HFE-7100) and the dimethylformamide. Wherein the volume ratio of the HFE-7100 to the benzotrifluoride is 0.5: 1. Stirring and reacting at the temperature of 80 ℃ for 65 hours, and performing suction filtration and spin-drying on the solvent to obtain the product.
Example 4:
the reaction procedure of example 4 was the same as in example 1, after which the product was centrifuged and the bottom solution was dried to give a perfluoropolyether-polyethylene glycol block copolymer of formula I.
The specific process is as follows: under the protection of inert gas, the perfluoropolyether (from DuPont, type number is FSH157, the average molecular weight of which is 5000-7500), the oxalyl chloride of formula III, the polyamide of formula IV (from Henscman, type number is JEFFAMINE series) and the catalyst 4-Dimethylaminopyridine (DMAP) are dissolved in the mixed reaction solvent of the fluoro-oil (from DuPont, type number is HFE-7100) and tetrahydrofuran. Wherein the volume ratio of the HFE-7100 to the benzotrifluoride is 0.8: 1. Stirring and reacting for 72h at the temperature of 60 ℃, and performing suction filtration and spin-drying on the solvent to obtain a product.
Example 5:
the reaction procedure of example 5 was the same as in example 1, and after the reaction, the product was purified and centrifuged, respectively, to obtain a perfluoropolyether-polyethylene glycol block copolymer of formula I.
The specific process is as follows: under the protection of inert gas, dissolving perfluoropolyether (purchased from DuPont, the model is FSH157, the average molecular weight of the perfluoropolyether is 5000-7500), oxalyl chloride of formula III, polyamide (purchased from Hensman, the model is JEFFAMINE series) of formula IV and catalyst 4-Dimethylaminopyridine (DMAP) in a mixed reaction solvent of fluoro-oil (purchased from DuPont, the model is HFE-7100) and dimethyl sulfoxide, wherein the volume ratio of the HFE-7100 to the trifluorotoluene is 2:1, stirring and reacting for 36h at 120 ℃, and performing suction filtration and spin drying on the solvent to obtain the product.
Example 6:
the perfluoropolyether-polyethylene glycol block copolymer having the average molecular weight of 5000-16000 prepared in example 1 was used as a fluorosurfactant, and dissolved in HFE-7500 fluorocarbon oil (available from DuPont, model No. HFE-7500) at a mass percentage concentration of 3% to obtain a droplet-forming oil.
Droplet generation experiments were performed using the droplet generation oil of example 6:
first, standing stability test
The droplet standing stability of the droplet-forming oil of example 6 was tested, and the droplet sampled immediately after the generation was compared with the droplet after 30 days of standing, as shown in fig. 3 to 6, it was observed by a microscope that there was little difference between the two, and the droplets exhibited a honeycomb-like uniform arrangement, indicating that the droplets produced from the oil phase had excellent standing stability.
Two, drop digital polymerase chain reaction test
The droplet-wise digital polymerase chain reaction test was performed on the droplet-forming oil of example 6, specifically: the Hotstart master mix is used as a water phase (as shown in FIG. 7) to generate uniform water-in-oil droplets in the microfluidic chip for PCR amplification thermal cycling.
As shown in fig. 8 and 9, under the observation of a microscope, after 40 to 50 cycles of thermal cycling of the pcr, the uniformity of the droplets remained good, regular honeycomb morphology was still observed, and the existence rate (CV value) of damaged and fused droplets was much lower than 5%.
The partial view of fig. 10 is obtained by enlarging a part of the black line box in fig. 9. The exposure rate was adjusted in the mercury lamp environment, and it was clearly observed that the fluorescence intensity of the amplified positive droplets was brighter than that of the other negative droplets, as shown in fig. 11, which is the same view in fig. 10.
And then quantitatively analyzing the fluorescence intensity of the liquid drop, collecting the fluorescence signal of each liquid drop passing through the detection port of the microfluidic chip by using an optical machine module, and performing software algorithm processing, as shown in fig. 12 to 14. The peak intensity of a plurality of positive peaks is about 3 times of that of a plurality of negative peaks, which can be clearly seen in a random section of collected fragments, and the result accords with the observation result of a microscope. After the data is processed by a software algorithm, two obvious yin and yang strips on a data graph can be clearly seen, and the expected result of an experiment is met.
Example 7:
the droplet-forming oil of example 7 differs from example 6 only in that the fluorosurfactant is present at a concentration of 2% by mass.
Example 8:
the droplet-forming oil of example 8 differs from example 6 only in that the fluorosurfactant concentration is 4% by mass.
Example 9:
the droplet-forming oil of example 9 differs from example 6 only in that the fluorosurfactant is present at a concentration of 5% by mass.
Example 10:
the droplet-forming oil of example 10 differs from example 6 only in that the fluorosurfactant concentration is 6% by mass.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A perfluoropolyether-polyethylene glycol block copolymer is characterized in that the structural formula of the perfluoropolyether-polyethylene glycol block copolymer is shown as the following formula I:
wherein n is more than or equal to 20 and less than or equal to 80, o + q is more than or equal to 3 and less than or equal to 7, and p is more than or equal to 9 and less than or equal to 14.
2. The perfluoropolyether-polyethylene glycol block copolymer according to claim 1, wherein the average molecular weight of the perfluoropolyether-polyethylene glycol block copolymer is 5000 to 16000.
3. The method for preparing the perfluoropolyether-polyethylene glycol block copolymer as claimed in claim 1 or 2, wherein perfluoropolyether, oxalyl chloride and polyamide are reacted in a solvent to obtain the perfluoropolyether-polyethylene glycol block copolymer, wherein the solvent is fluorine oil, the perfluoropolyether has the formula shown in formula II, the oxalyl chloride has the formula shown in formula III, and the polyamide has the formula shown in formula IV:
wherein n is more than or equal to 20 and less than or equal to 80, o + q is more than or equal to 3 and less than or equal to 7, and p is more than or equal to 9 and less than or equal to 14.
4. The method for preparing the perfluoropolyether-polyethylene glycol block copolymer according to claim 3, wherein the reaction temperature is 60-120 ℃ and the reaction time is 36-72 h.
5. The method of claim 3, wherein a catalyst is added in the reaction, and the catalyst is an organic base, and the organic base is at least one of 4-dimethylaminopyridine, trihexylamine and triethylenediamine.
6. The method of claim 3, wherein the solvent further comprises an organic solvent, and the organic solvent is at least one of benzotrifluoride, dimethylformamide, tetrahydrofuran, and dimethylsulfoxide.
7. Use of a perfluoropolyether-polyethylene glycol block copolymer according to claim 1 or 2 as fluorosurfactant.
8. The liquid drop generating oil is characterized by comprising a fluorine surfactant and fluorine oil, wherein the fluorine surfactant is a perfluoropolyether-polyethylene glycol block copolymer.
9. The droplet forming oil according to claim 8, wherein the mass percentage of the fluorinated surfactant in the droplet forming oil is 2 to 6%.
10. The droplet generating oil of claim 8 or 9, wherein the droplet generating oil is used in a process of micro-titration for polymerase chain reaction.
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| EP4351786A1 (en) * | 2021-06-11 | 2024-04-17 | Samplix ApS | Fluorosurfactants for stabilizing single- and double-emulsion droplets |
| CN116200440A (en) * | 2021-11-30 | 2023-06-02 | 万华化学集团股份有限公司 | Rhamnolipid fermentation process |
| CN114539536B (en) * | 2022-02-18 | 2023-04-28 | 华南师范大学 | Preparation method and application of perfluoro polyether surfactant containing amide bond |
| CN115322798B (en) * | 2022-08-22 | 2023-09-05 | 苏州中科医疗器械产业发展有限公司 | Fluorine oil composition and preparation method thereof |
| CN115368576B (en) * | 2022-08-22 | 2023-06-16 | 苏州中科医疗器械产业发展有限公司 | Perfluoropolyether-adipic acid dihydrazide block copolymer and preparation method and application thereof |
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