CN115368576B - Perfluoropolyether-adipic acid dihydrazide block copolymer and preparation method and application thereof - Google Patents
Perfluoropolyether-adipic acid dihydrazide block copolymer and preparation method and application thereof Download PDFInfo
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- CN115368576B CN115368576B CN202211008338.2A CN202211008338A CN115368576B CN 115368576 B CN115368576 B CN 115368576B CN 202211008338 A CN202211008338 A CN 202211008338A CN 115368576 B CN115368576 B CN 115368576B
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Abstract
The invention discloses a perfluoropolyether-adipic dihydrazide block copolymer, and also discloses a preparation method and application of the block copolymer. The prepared perfluoropolyether-adipic dihydrazide segmented copolymer can be used as a surfactant of fluorine oil and has excellent surface performance; when the fluorine oil is used as an oil phase for generating PCR microdroplets, the fluorine oil can be applied to liquid drop type digital PCR, and the microdroplets have certain thermal stability. In the preparation process, triethylamine is used as a catalyst, so that the reaction process can be accelerated, the reaction efficiency is improved, and the product yield is improved; and finally, purifying by using a mode of alternately washing with sodium hydroxide solution and ethanol, protecting amino groups by using sodium hydroxide, and improving the droplet quantity, the droplet morphology and the fluorescence intensity of the fluorine oil.
Description
Technical Field
The invention relates to the technical field of microfluidics, in particular to a perfluoropolyether-adipic acid dihydrazide block copolymer and a preparation method and application thereof.
Background
Microfluidic droplet-based technology (DMF) is continually being developed in the biomedical engineering and analytical chemistry fields for monitoring a variety of chemical and biological analytes, such as viruses, bacteria, nucleic acids and proteins. This technique provides various advantages such as high throughput, short reaction times, higher efficiency and cost effectiveness. For single molecule analysis using DMF, the droplets must remain stable in various reactions. Including thermal cycling and long-term incubation. The oils and surfactants used for encapsulation must therefore be highly biocompatible and provide stability in the droplets to avoid breakage and cross-talk between droplets.
In recent years, various surfactants and oils have been explored in DMF for sample distribution and various applications. The most successful commercial application of these is probably digital micro-drop PCR (ddPCR). Microdroplet Digital PCR (ddPCR) is a single molecule basedThe nucleic acid quantifying method for counting by the PCR method mainly adopts a microfluidic technology to carry out microdroplet treatment on a sample to form tens of thousands of nanodroplets, and each droplet contains no nucleic acid target molecules or one or a plurality of nucleic acid target molecules. After PCR amplification, the droplets are counted and detected one by one, and finally the copy number and concentration of the nucleic acid can be obtained according to the Poisson distribution principle and the number of positive droplets. In order to maintain the original shape of the droplets after PCR thermal cycling, a product oil having high thermal stability is required as an oil phase of the droplets. The more commonly used oil phase is a fluorooil, for example: the surfactants used in combination with HFE-7500, HFE-7100 and FC-40 are PFPE-PEG-PFPE and ABIL TM EM90, 1, 2-tetrahydroperfluorodecanol, etc., but the existing surfactants for fluorooils are often only applicable to normal temperature environments and cannot maintain their surface properties after PCR thermal cycling.
Chinese patent document CN110042151a discloses an oil phase composition for preparing droplets in droplet-type digital PCR, which uses mineral oil as an oil phase, and uses a macromolecular nonionic surfactant with a low hydrophilic-lipophilic balance value and polydimethylsiloxane as a skeleton, a polyethylene glycol small molecular nonionic surfactant with a high hydrophilic-lipophilic balance value, and an anti-evaporation agent as stabilizers in the oil phase. The composition of the patent is simple to synthesize and low in cost, but an additional anti-evaporation agent is needed when the surfactant is used, which indicates that the surfactant is not high in thermal stability and complex in formula composition.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is that the existing surfactant for the fluorine oil has low thermal stability and complex formula, so as to provide the perfluoropolyether-adipic dihydrazide block copolymer and the preparation method and the application thereof.
Therefore, the invention adopts the following technical scheme:
the invention provides a perfluoropolyether-adipic dihydrazide segmented copolymer, the structure of which is shown as formula I:
wherein n is 20-80.
The invention also provides a preparation method of the perfluoropolyether-adipic acid dihydrazide segmented copolymer, which comprises the following steps:
s1: reacting the perfluoropolyether shown in the formula II with oxalyl chloride to generate the perfluoropolyether acyl chloride shown in the formula III;
s2: reacting the perfluoropolyether acyl chloride shown in the formula III with adipic acid dihydrazide and triethylamine to generate the perfluoropolyether-adipic acid dihydrazide segmented copolymer shown in the formula I;
s3: alternately washing the obtained perfluoropolyether-adipic acid dihydrazide block copolymer by using a sodium hydroxide solution and an ethanol solution, and drying;
wherein formula II is
Formula III is
Further, the average molecular weight of the perfluoropolyether is 2500-7000;
preferably, the perfluoropolyether of model 157FSL from Krytox is used.
In the step S1, the reaction condition is carried out under the condition of nitrogen, the reaction temperature is 60-80 ℃, the reaction time is 12-24h, and the mass ratio of the perfluoropolyether to the oxalyl chloride is 0.1-0.5:1.
step S2 is that perfluoropolyether acyl chloride reacts with adipic acid dihydrazide and triethylamine dissolved in methylene dichloride to generate the perfluoropolyether-adipic acid dihydrazide segmented copolymer.
Preferably, the molar ratio of perfluoropolyether acyl chloride to adipic acid dihydrazide is 1:0.5-1;
the mass ratio of the methylene dichloride to the adipic dihydrazide is 20:0.1-0.174;
the mass ratio of the triethylamine to the dichloromethane is 1:4-10..
In the step S3, the concentration of the sodium hydroxide solution is 0.1M-1M, the ethanol solution is absolute ethanol, the washing times are 1-3 times, and the vacuum drying is carried out after the washing.
In both steps S1 and S2, the reaction is carried out in a fluorooil.
The fluorine oil used in the step S1 is HFE-7100, and the fluorine oil used in the step S2 is HFE-7500.
The invention also provides application of the perfluoropolyether-adipic acid dihydrazide segmented copolymer, which is used as a surfactant in fluorine oil.
The mass percentage of the perfluoropolyether-adipic acid dihydrazide block copolymer in the fluorine oil is 0.5-2 wt%.
The technical scheme of the invention has the following advantages:
(1) The prepared perfluoropolyether-adipic dihydrazide segmented copolymer can be used as a surfactant of fluorine oil and has excellent surface performance; when the fluorine oil is used as an oil phase for generating PCR microdroplets, the fluorine oil can be applied to liquid drop type digital PCR, and the microdroplets have certain thermal stability.
(2) In the preparation process of the invention, triethylamine is used as a catalyst, so that the reaction process can be accelerated, the reaction efficiency is improved, and the product yield is improved.
(3) In the preparation process of the invention, the hydrolysis of oxalyl chloride can be avoided by using HFE-7100, and the final product can be dissolved in the HFE-7500, so that the later purification step is convenient.
(4) The invention uses sodium hydroxide solution and ethanol to alternatively wash and purify, uses sodium hydroxide to protect amino, and can improve the droplet quantity and the morphology of the fluorine oil and the fluorescence intensity.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the Fourier infrared signature of the perfluoropolyether-adipic acid dihydrazide block copolymer obtained in example 1 of the present invention;
FIG. 2 is a microscopic image of the droplets produced in test example 1 of the present invention before PCR thermocycling amplification;
FIG. 3 is a microscopic image of the droplets produced in test example 1 of the present invention after PCR thermal cycling amplification;
FIG. 4 is a graph showing the number of droplets produced in test example 1 of the present invention after PCR thermal cycle amplification;
FIG. 5 is a graph showing amplification efficiency of the microdroplet produced in test example 1 of the present invention after PCR thermal cycle amplification;
FIG. 6 is a microscopic image of the droplets produced in test example 2 of the present invention after PCR thermal cycling amplification;
FIG. 7 is a graph showing the number of droplets produced in test example 2 of the present invention after PCR thermal cycle amplification;
FIG. 8 is a graph showing amplification efficiency of the microdroplet produced in test example 2 of the present invention after PCR thermal cycle amplification.
Detailed Description
The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.
The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field.
Perfluoropolyethers (PFPE) available from Krytox under the model 157FSL
HFE-7500 and HFE-7100 were purchased from 3M company.
Other reagents were all analytically pure grade, purchased from Sigma Aldrich.
Example 1
The embodiment provides a perfluoropolyether-adipic dihydrazide segmented copolymer, the structure of which is shown as a formula I:
where n=20.
The synthetic route is as follows:
the preparation method comprises the following steps:
(1) 2.5g of PFPE of formula II was taken and added to 20g of HFE-7100 and sonicated for 3min. Then 10mL of oxalyl chloride is added, and reflux is carried out for 24 hours at 65 ℃ under the nitrogen atmosphere, so as to obtain the perfluoropolyether acyl chloride shown in the formula III.
(2) The above solution was spin-distilled at 40 ℃ for 10min, followed by heating to 70 ℃ for 10min, and then cooled to room temperature.
(3) 0.087g adipic acid dihydrazide was added to 20g methylene chloride, followed by 2mL triethylamine and ultrasonic dissolution for 3min.
(4) 20g of HFE-7500 was added to dissolve the product of step (2), then the mixed liquid of step (3) was added, and the mixture was sonicated for 3min.
(5) And (3) reacting the mixed liquid obtained in the step (4) for 24 hours at room temperature under the protection of nitrogen to obtain the perfluoropolyether-adipic dihydrazide block copolymer shown in the formula I.
(6) After the reaction was completed, excess HFE-7500 was removed by rotary evaporation at 90 ℃.
(7) The washing was performed 3 times with 0.1M sodium hydroxide and absolute ethanol solution, respectively.
(8) Vacuum drying at room temperature for 24h.
And carrying out Fourier infrared characterization on the obtained product, wherein a specific characterization map is shown in figure 1.
In the infrared spectrum, the characteristic seal at 3432cm-1 corresponds to-OH mainly from hydroxyl remained after alcohol washing, at 1712cm -1 The characteristic peak of the position corresponds to the amide of the product-c=o double bond at 2889cm -1 、1227cm -1 And 1183cm -1 The characteristic peak of the position corresponds to the-CH of the product 2 -C-F and-C-N bonds.
Comparative example
This comparative example provides a perfluoropolyether-adipic acid dihydrazide block copolymer, differing from example 1 in that triethylamine is not added at the time of step (3).
Test example 1
The perfluoropolyether-adipic acid dihydrazide block copolymer obtained in example 1 was added as a fluorosurfactant to HFE-7500 containing 0.1wt% of 1h,2 h-perfluoro-1-octanol, the perfluoropolyether-adipic acid dihydrazide block copolymer being 3wt%, to obtain a droplet-forming oil.
The above-mentioned droplet-generating oil was used as a PCR droplet-generating oil phase. The preparation method of the aqueous phase for PCR microdroplet generation comprises mixing 60. Mu.l of 2 Xbuffer (Supermix, available from Biorad Co.), 10. Mu.l of forward primer Prime-F (10. Mu.M), 10. Mu.l of reverse primer Prime-R (10. Mu.M), 10. Mu.l of lambda DNA (10 ng/mL), 10ul of probe (10. Mu.M) uniformly, and adding 60. Mu.l of ultrapure water dropwise to mix uniformly.
The oil phase and the water phase with the volume ratio of 4:1 are taken, and water-in-oil droplets (20-120 μm) with good uniformity are generated in the microfluidic chip, as shown in figure 2.
PCR thermocycling amplification experiments were then performed. The morphology of the droplets observed under the microscope before PCR thermocycling amplification is shown in FIG. 2, and the morphology of the droplets observed under the microscope after PCR thermocycling amplification is shown in FIG. 3. As shown by observation under a microscope, after 30-50 circles of thermal cycles of PCR thermal cycles, the droplets generated by the surface activity prepared in the example 1 have no demulsification and fusion phenomenon, most of droplets still keep good uniformity, regular honeycomb arrangement is still shown, and CV values (variation coefficients) of demulsified and fused droplets are lower than 5%. And droplets generated by surface activities in other proportions have most of demulsification phenomena after PCR thermal cycling.
The PCR thermocycling amplified droplets were subjected to on-machine detection (machine manufactured by Biorad Co., model: QX 200), the number of droplets after PCR thermocycling amplification was as shown in FIG. 4, and the number of droplets after PCR thermocycling was kept at 17719, indicating that most of the droplets were not broken. As shown in FIG. 5, it can be seen that after PCR thermal amplification, there are two very distinct bands, the negative and positive bands, respectively. The 20 μl copy number calculated by Quanta soft software is 10100, which indicates that the interior of the microdroplet was successfully amplified, the surfactant did not affect the amplification efficiency, and the positive and negative bands were very concentrated, indicating that the morphology of the microdroplet was still very uniform after PCR thermal cycling.
Test example 2
The procedure of test example 1 was repeated using the perfluoropolyether-adipic acid dihydrazide block copolymer obtained in comparative example 1 as a fluorosurfactant.
As shown in FIG. 6, which is a morphology graph of water-in-oil droplets after PCR thermal cycling, it can be seen that droplets generated by surface activity without catalyst are fused after thermal cycling, indicating that the purity of the generated surfactant is not high enough.
As shown in FIG. 7, the number of droplets after PCR thermal cycle amplification was significantly smaller than that of FIG. 4 in test example 1.
As shown in FIG. 8, it can be seen that the distribution of the whole positive band after PCR thermal amplification is relatively dispersed, indicating that the morphology of the droplets is not uniform enough and the fluorescence value is low.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (9)
1. The perfluoropolyether-adipic acid dihydrazide block copolymer is characterized in that the structure is shown as a formula I:
wherein n is 20-80;
the preparation method of the perfluoropolyether-adipic acid dihydrazide block copolymer comprises the following steps:
s1: reacting the perfluoropolyether shown in the formula II with oxalyl chloride to generate the perfluoropolyether acyl chloride shown in the formula III;
s2: reacting the perfluoropolyether acyl chloride shown in the formula III with adipic acid dihydrazide and triethylamine to generate the perfluoropolyether-adipic acid dihydrazide segmented copolymer shown in the formula I;
s3: alternately washing the obtained perfluoropolyether-adipic acid dihydrazide block copolymer by using sodium hydroxide solution and absolute ethyl alcohol, and then drying;
wherein formula II is
；
Formula III is
。
2. The method for preparing the perfluoropolyether-adipic acid dihydrazide block copolymer according to claim 1, which is characterized by comprising the following steps:
s1: reacting the perfluoropolyether shown in the formula II with oxalyl chloride to generate the perfluoropolyether acyl chloride shown in the formula III;
s2: reacting the perfluoropolyether acyl chloride shown in the formula III with adipic acid dihydrazide and triethylamine to generate the perfluoropolyether-adipic acid dihydrazide segmented copolymer shown in the formula I;
s3: alternately washing the obtained perfluoropolyether-adipic acid dihydrazide block copolymer by using sodium hydroxide solution and absolute ethyl alcohol, and then drying;
wherein formula II is
；
Formula III is
。
3. The preparation method according to claim 2, wherein in the step S1, the reaction conditions are performed under nitrogen, the reaction temperature is 60-80 ℃, the reaction time is 12-24 hours, and the mass ratio of the perfluoropolyether to oxalyl chloride is 0.1-0.5:1.
4. the process of claim 3, wherein step S2 is the reaction of the perfluoropolyether acid chloride with adipic acid dihydrazide and triethylamine dissolved in methylene chloride to produce the perfluoropolyether-adipic acid dihydrazide block copolymer.
5. The method according to claim 4, wherein,
the molar ratio of the perfluoropolyether acyl chloride to the adipic dihydrazide is 1:0.5-1;
the mass ratio of the methylene dichloride to the adipic dihydrazide is 20:0.1-0.174;
the mass ratio of the triethylamine to the dichloromethane is 1:4-10.
6. The method according to claim 5, wherein in step S3, the concentration of the sodium hydroxide solution is 0.1M to 1M and the number of washing is 1 to 3.
7. The method according to claim 6, wherein in steps S1 and S2, the reaction is carried out in a fluorine oil.
8. The method according to claim 7, wherein the fluorine oil used in the step S1 is HFE-7100 and the fluorine oil used in the step S2 is HFE-7500.
9. Use of the perfluoropolyether-adipic dihydrazide block copolymer according to claim 1, as surfactant in a fluorooil.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101367932A (en) * | 2008-10-10 | 2009-02-18 | 张永明 | Fluorine-containing block polymer and synthesis thereof |
| CN108148204A (en) * | 2017-12-21 | 2018-06-12 | 广东顺德墨赛生物科技有限公司 | Perfluoropolyether-polyethylene glycol blocked copolymer, preparation method and applications |
| CN108285537A (en) * | 2018-01-30 | 2018-07-17 | 肇庆市华师大光电产业研究院 | A kind of fluorine-containing nonionic surfactant of amide bond and its preparation method and application |
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| WO2010009365A1 (en) * | 2008-07-18 | 2010-01-21 | Raindance Technologies, Inc. | Droplet libraries |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101367932A (en) * | 2008-10-10 | 2009-02-18 | 张永明 | Fluorine-containing block polymer and synthesis thereof |
| CN108148204A (en) * | 2017-12-21 | 2018-06-12 | 广东顺德墨赛生物科技有限公司 | Perfluoropolyether-polyethylene glycol blocked copolymer, preparation method and applications |
| CN108285537A (en) * | 2018-01-30 | 2018-07-17 | 肇庆市华师大光电产业研究院 | A kind of fluorine-containing nonionic surfactant of amide bond and its preparation method and application |
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