A droplet-generating oil, its preparation method and application

Abstract

This invention provides a droplet-generating oil, its preparation method, and its application. The droplet-generating oil comprises a polymer having the structure shown in Formula I. In this invention, by designing the structure of the polymer of Formula I, and further preparing the hydrophobic end of the polymer of Formula I using a low molecular weight perfluoropolyether carboxylic acid, a high-performance droplet-generating oil is obtained, exhibiting good stability and good high-temperature resistance.

Description

Technical Field

[0001] This invention belongs to the field of digital PCR technology, specifically relating to a droplet-generating oil, its preparation method, and its application. Background Technology

[0002] The rapid development and application of digital PCR is particularly important in single-cell sequencing technology. Among them, generating uniform and stable single droplets through microfluidic technology is the most critical step in single-cell sequencing.

[0003] Fluorinated droplet-generating oil, as an oil phase, possesses advantages such as excellent chemical stability and good biocompatibility. Currently, single-cell projects mainly rely on Bio-rad droplet-generating oil, which can meet the high-temperature cycling requirements of digital PCR. However, due to the high molecular weight perfluoropolyether carboxylic acid used in the synthesis of its triblock copolymer, the prepared droplet-generating oil is unstable at room temperature, easily emulsifies and turns white, resulting in the loss of its surfactant function.

[0004] In addition, single-cell sequencing products are constantly being optimized and upgraded, and the requirements for droplet-generated oil are also constantly changing and increasing.

[0005] Therefore, providing a droplet-generating oil with good stability, high temperature resistance, and the ability to meet the changing needs of single-cell sequencing projects has become an urgent technical problem to be solved. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a droplet-generating oil, its preparation method, and its applications. In this invention, by designing the structure of a polymer of formula I, and further preparing the hydrophobic end of the polymer of formula I using a low molecular weight perfluoropolyether carboxylic acid, a high-performance droplet-generating oil is obtained. This droplet-generating oil exhibits good stability and good high-temperature resistance.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a droplet-generating oil, the droplet-generating oil comprising a polymer having the structure shown in Formula I:

[0009]

[0010] Where n represents an integer from 10 to 19;

[0011] x represents an integer from 5 to 10;

[0012] y represents an integer from 10 to 15.

[0013] z represents an integer from 5 to 10.

[0014] In this invention, by designing the structure of polymer of formula I and further controlling the weight-average molecular weight of perfluoropolyether carboxylic acid and polyether diamine, the hydrophobic end of polymer of formula I is prepared by using low molecular weight perfluoropolyether carboxylic acid, and a droplet-generating oil with excellent performance is obtained. This droplet-generating oil has good stability and good high-temperature resistance.

[0015] In this invention, perfluoropolyether carboxylic acid forms the hydrophobic segment of polymer I, and polyether diamine forms the hydrophilic segment of polymer I. By controlling the value of n in the hydrophobic segment of polymer I within a specific range, a droplet-generating oil with excellent performance can be prepared. If the value of n is too large, the stability of the prepared droplet-generating oil is poor; if the value of n is too small, the prepared droplet-generating oil cannot withstand high-temperature PCR reactions.

[0016] In this invention, n can be 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19.

[0017] x can be 5, 6, 7, 8, 9, or 10.

[0018] y can be 10, 11, 12, 13, 14 or 15.

[0019] z can be 5, 6, 7, 8, 9, or 10.

[0020] The following are preferred technical solutions of the present invention, but are not intended to limit the technical solutions provided by the present invention. The purpose and beneficial effects of the present invention can be better achieved and realized through the following preferred technical solutions.

[0021] As a preferred embodiment of the present invention, the raw materials for preparing the polymer of formula I include perfluoropolyether carboxylic acid and polyether diamine.

[0022] Preferably, the molar ratio of the perfluoropolyether carboxylic acid and the polyether diamine is 1:(2-3), for example, it can be 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9 or 1:3, etc.

[0023] As a preferred embodiment of the present invention, the weight-average molecular weight of the perfluoropolyether carboxylic acid is 1800 to 3000, for example, it can be 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900 or 3000.

[0024] Preferably, the weight-average molecular weight of the polyether diamine is 600 to 900, for example, it can be 600, 630, 660, 680, 700, 720, 750, 770, 800, 810, 840, 860, 880 or 900.

[0025] As a preferred embodiment of the present invention, the polymer of formula I is prepared by method A or method B, wherein method A includes the following steps:

[0026] Perfluoropolyether carboxylic acid, polyether diamine, solvent A and reagent A are mixed, reacted, and post-treated to obtain the polymer of formula I.

[0027] Method B includes the following steps:

[0028] (1) Mix perfluoropolyether carboxylic acid, solvent B, oxalyl chloride and catalyst B, and react to obtain an intermediate product;

[0029] (2) The intermediate product, polyether diamine, solvent C and reagent C are mixed and reacted to obtain the polymer of formula I.

[0030] As a preferred embodiment of the present invention, the solvent A is selected from any one or a combination of at least two of HFE 7100 solvent, HFE7500, FC3283 (perfluorotripropylamine) or FC40 fluorinated liquid.

[0031] Preferably, based on 100 parts by volume of the perfluoropolyether carboxylic acid, the volume of solvent A is 100 to 200 parts, for example, 100 parts, 110 parts, 120 parts, 130 parts, 140 parts, 150 parts, 160 parts, 170 parts, 180 parts, 190 parts, or 200 parts.

[0032] Preferably, reagent A is selected from any one or a combination of at least two of potassium hexafluorophosphate, sodium hexafluorophosphate, or lithium hexafluorophosphate.

[0033] Preferably, based on 100 parts by volume of the perfluoropolyether carboxylic acid, the volume of reagent A is 200 to 220 parts, for example, 200 parts, 202 parts, 204 parts, 206 parts, 208 parts, 210 parts, 212 parts, 214 parts, 216 parts, 218 parts, or 220 parts, etc.

[0034] Preferably, the reaction temperature in method A is 30–80°C (e.g., 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C, etc.), and the reaction time is 8–12 h (e.g., 8 h, 8.5 h, 9 h, 9.5 h, 10 h, 10.5 h, 11 h, 11.5 h, or 12 h, etc.).

[0035] Preferably, method A further includes a post-processing step after the reaction, the post-processing step including the following steps:

[0036] A fluorinated solvent is added to the reaction system, and the mixture is filtered. A polar solvent is added to the filtrate to precipitate the polymer of formula I. The mixture is then filtered to remove excess polar solvent, and the polymer of formula I is obtained.

[0037] In this invention, a fluorinated solvent is added to the reaction system to dissolve the crude product obtained from the reaction of perfluorinated polyether carboxylic acid and polyether diamine.

[0038] In this invention, the fluorinated solvent is selected from any one or a combination of at least two of HFE 7100 solvent, HFE7500 or FC40 fluorinated liquid.

[0039] The polar solvent is selected from any one or a combination of at least two of ethanol, ethyl acetate, or difluoromethane.

[0040] As a preferred embodiment of the present invention, the solvent B is selected from any one or a combination of at least two of anhydrous HFE 7100 solvent, HFE7500, FC3283 (perfluorotripropylamine) or FC40 fluorinated liquid.

[0041] Preferably, with 100 parts by volume of the perfluoropolyether carboxylic acid, the volume of solvent B is 100 to 200 parts, for example, 100 parts, 110 parts, 120 parts, 130 parts, 140 parts, 150 parts, 160 parts, 170 parts, 180 parts, 190 parts, or 200 parts.

[0042] Preferably, the catalyst B comprises anhydrous DMF (N,N-dimethylamide).

[0043] Preferably, based on 100 parts of the perfluoropolyether carboxylic acid, the molar amount of the catalyst B is 0.1 to 1 part, for example, it can be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 part, etc.

[0044] Preferably, based on 100 parts of the perfluoropolyether carboxylic acid, the molar amount of the oxaloyl chloride is 100 to 150 parts, for example, it can be 100 parts, 105 parts, 110 parts, 115 parts, 120 parts, 125 parts, 130 parts, 135 parts, 140 parts, 145 parts, or 150 parts, etc.

[0045] Preferably, the reaction temperature in step (1) is 20-30°C (e.g., 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C, etc.), and the reaction time is 20-28h (e.g., 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, or 28h, etc.).

[0046] Preferably, the reaction in step (1) is carried out in a protective atmosphere.

[0047] Preferably, step (1) further includes a pretreatment step before the reaction, wherein the pretreatment step includes dehydrating the raw materials for preparing polymer of formula I.

[0048] Preferably, the reaction in step (1) is followed by a step of removing the solvent.

[0049] As a preferred embodiment of the present invention, the solvent C is selected from anhydrous HFE 7100 solvent and / or anhydrous tetrahydrofuran (THF).

[0050] Preferably, based on 100 parts by volume of the perfluoropolyether carboxylic acid, the volume of the solvent C is 200 to 220 parts, for example, 200 parts, 202 parts, 204 parts, 206 parts, 208 parts, 210 parts, 212 parts, 214 parts, 216 parts, 218 parts, or 220 parts, etc.

[0051] Preferably, reagent C is selected from any one or a combination of at least two of triethylamine, 4-dimethylpyridine (DMAP) or 1,8-diazabicyclo(5,4,0)-7-undecene (commonly known as bicyclic amidine, DBU).

[0052] Preferably, with 100 parts per fluoropolyether carboxylic acid, the molar amount of reagent C is 100 to 150 parts, for example, it can be 100 parts, 105 parts, 110 parts, 115 parts, 120 parts, 125 parts, 130 parts, 135 parts, 140 parts, 145 parts, or 150 parts, etc.

[0053] Preferably, the reaction temperature in step (2) is 20-30°C (e.g., 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C, etc.), and the reaction time is 10-15h (e.g., 10h, 11h, 12h, 13h, 14h, or 15h, etc.).

[0054] Preferably, step (2) further includes a post-processing step after the reaction, and the post-processing method includes the following steps: adding a polar solvent to the reaction system, filtering, precipitating the polymer of formula I, and obtaining the polymer of formula I.

[0055] The polar solvent is selected from any one or a combination of at least two of ethanol, ethyl acetate, or difluoromethane.

[0056] As a preferred embodiment of the present invention, the polymer of formula I is prepared by method A or method B, wherein method A specifically includes the following steps:

[0057] Perfluorinated polyether carboxylic acid, polyether diamine, solvent A and reagent A are mixed and reacted at 30-80°C for 8-12 hours. A fluorinated solvent is added to the reaction system, and the mixture is filtered. A polar solvent is added to the filtrate to precipitate the polymer of formula I. The mixture is then filtered to obtain the polymer of formula I.

[0058] Wherein, based on 100 parts of perfluoropolyether carboxylic acid, the molar amount of the polyether diamine is 200-300 parts, and the molar amount of reagent A is 200-220 parts;

[0059] Based on 100 parts by volume of the perfluoropolyether carboxylic acid, the volume of solvent A is 100 to 200 parts.

[0060] The preparation process of polymer I in method A is shown below:

[0061]

[0062] Method B specifically includes the following steps:

[0063] (1) The dehydrated perfluoropolyether carboxylic acid, solvent B, oxalyl chloride and catalyst B are mixed and reacted at 20-30°C for 20-28 hours in a protective atmosphere to obtain an intermediate.

[0064] Of which, based on 100 parts of perfluoropolyether carboxylic acid, the molar amount of catalyst B is 0.1 to 1 part, and the molar amount of oxaloyl chloride is 100 to 150 parts.

[0065] Based on 100 parts by volume of the perfluoropolyether carboxylic acid, the volume of solvent B is 100 to 200 parts.

[0066] (2) Mix the intermediate, polyether diamine, solvent C and reagent C, and react at 20-30°C for 10-15 h. Add a polar solvent to the reaction system, and polymer of formula I precipitates. Filter to obtain polymer of formula I.

[0067] Wherein, based on 100 parts of perfluoropolyether carboxylic acid, the molar amount of the polyether diamine is 200-300 parts, and the molar amount of reagent C is 100-150 parts;

[0068] Based on 100 parts by volume of the perfluoropolyether carboxylic acid, the solvent C comprises 100 to 200 parts by volume.

[0069] The preparation process of polymer I in method B is shown below:

[0070]

[0071] As a preferred embodiment of the present invention, the droplet-generating oil further includes a solvent.

[0072] Preferably, the mass percentage of the polymer of formula I in the droplet generating oil is 2-6%, for example, it can be 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5% or 6%, etc.

[0073] Preferably, the solvent is selected from any one or a combination of at least two of anhydrous HFE 7100 solvent, HFE7500 or FC40 fluorinated liquid.

[0074] In this invention, a high-performance droplet-generating oil was prepared by controlling the amount of solvent within a specific range. If the content of Formula I polymer in the droplet-generating oil is too low or too high, the generated droplets will be unable to withstand high-temperature PCR.

[0075] In a second aspect, the present invention provides a method for preparing droplet-generating oil as described in the first aspect, the method comprising the following steps:

[0076] The polymer and solvent shown in Formula I are mixed evenly to obtain the droplet-generating oil.

[0077] Thirdly, the present invention provides an application of the droplet-generating oil as described in the first aspect, wherein the droplet-generating oil is used in the field of microfluidics technology for digital PCR.

[0078] Compared with the prior art, the present invention has the following beneficial effects:

[0079] In this invention, by designing the structure of polymer of formula I and further controlling the weight-average molecular weight of perfluoropolyether carboxylic acid and polyether diamine, the hydrophobic end of polymer of formula I is prepared by using low molecular weight perfluoropolyether carboxylic acid, and a droplet-generating oil with excellent performance is obtained. This droplet-generating oil has good stability and good high-temperature resistance. Attached Figure Description

[0080] Figure 1 The proton NMR spectrum of polymer 1 provided in Preparation Example 1;

[0081] Figure 2 The stability test results of the droplet-generating oil provided in Example 1 are shown in the figure.

[0082] Figure 3 Figure showing the stability test results of the droplet-generating oil provided in Comparative Example 1;

[0083] Figure 4 A microscope image of the droplet morphology generated by the droplet-generating oil provided in Example 1 via a microfluidic chip;

[0084] Figure 5 Microscopic photographs of the morphology of droplets generated by the microfluidic chip from the droplet-generating oil provided in Example 1 after three days of storage.

[0085] Figure 6 A microscope image of droplets generated by a microfluidic chip from the droplet-generating oil provided in Example 1 before digital PCR testing;

[0086] Figure 7 Microscopic images of droplets generated by a microfluidic chip from the droplet-generating oil provided in Example 1 during digital PCR testing. Detailed Implementation

[0087] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be considered as specific limitations thereof.

[0088] The sources of some components in the following examples and comparative examples are as follows:

[0089] Perfluorinated polyether carboxylic acid 1 (n=15): Taipuda New Materials;

[0090] Perfluoropolyether carboxylic acid 2 (n=20): Taipuda New Materials;

[0091] Perfluoropolyether carboxylic acid 3 (n=8): Taipuda New Materials;

[0092] Polyether diamine (y = 9, x + z = 4, x = 1~3, z = 1~3): Merck, brand name: 14526;

[0093] HFE 7100 solvent: 3M Company, grade: 7100003767;

[0094] Polyethylene glycol diamine ( m represents 12-13):; Pengshuo Biotechnology, brand name: PS2-N-600.

[0095] Preparation Example 1

[0096] This preparation example provides a polymer 1 and a method for preparing the same. The method for preparing polymer 1 is as follows:

[0097] Perfluoropolyether carboxylic acid 1 (1 mmol), polyether diamine (2 mmol), HFE 7100 solvent (10 mL) and potassium hexafluorophosphate (2.1 mmol) were mixed and reacted at 50 °C for 10 h. Then, HFE 7100 solvent (10 mL) was added to the reaction system, and the mixture was filtered. Ethanol (50 mL) was added to the filtrate to precipitate the polymer of formula I. The mixture was then filtered to obtain the polymer of formula I.

[0098] The 1H NMR spectrum of the polymer of Formula I provided in this preparation example is shown below. Figure 1 As shown.

[0099] Preparation Example 2

[0100] This preparation example provides a polymer 2 and a method for preparing the same. The method for preparing polymer 2 is as follows:

[0101] (1) Add 1 mmol of perfluoropolyether carboxylic acid 1 to a 50 mL three-necked flask, and remove the water by depressurizing the oil pump for 1 h in an oil bath at 120 °C. After returning to room temperature, add 10 mL of anhydrous HFE 7100 solvent and stir well. Then add 0.1 mmol of anhydrous DMF and 1.2 mmol of oxaloyl chloride. React at 25 °C for 24 h in a nitrogen atmosphere. Remove excess solvent by depressurizing the oil pump and in an oil bath at 100 °C to obtain the intermediate.

[0102] (2) Stir the intermediate and 10 mL of anhydrous HFE 7100 solvent evenly, then add 1.2 mmol of anhydrous triethylamine and stir evenly. Dissolve 2.5 mmol of polyether diamine in 10 mL of anhydrous THF and add it to the above reaction system. React at 25°C for 12 h. Add polar solvent ethanol to the reaction system. Polymer of Formula I precipitates out to obtain polymer of Formula I.

[0103] Comparative Preparation Example 1

[0104] This comparative preparation example provides a polymer 3 and its preparation method. The only difference from preparation example 1 is that perfluoropolyether carboxylic acid 1 in preparation example 1 is replaced with perfluoropolyether carboxylic acid 2, while other conditions are the same as in preparation example 1.

[0105] Comparative Preparation Example 2

[0106] This comparative preparation example provides a polymer 3 and its preparation method. The only difference from preparation example 1 is that perfluoropolyether carboxylic acid 1 in preparation example 1 is replaced with perfluoropolyether carboxylic acid 3, while other conditions are the same as in preparation example 1.

[0107] Comparative preparation example 3

[0108] This comparative preparation example provides a polymer 3 and its preparation method. The only difference from preparation example 1 is that the polyether diamine in preparation example 1 is replaced with polyethylene glycol diamine, while the other conditions are the same as in preparation example 1.

[0109] Examples 1-9 and Comparative Examples 1-3

[0110] Examples 1-9 and Comparative Examples 1-3 respectively provide a droplet-generating oil and its preparation method, wherein the droplet-generating oil is composed of a polymer of Formula I and an HFE 7100 solvent, wherein:

[0111] In Example 1: the polymer of Formula I was provided by Preparation Example 1, and the mass percentage of the polymer of Formula I in the droplet-generating oil was 4%;

[0112] In Example 2: the polymer of Formula I was provided by Preparation Example 2, and the mass percentage of the polymer of Formula I in the droplet-generating oil was 3%;

[0113] In Example 3: the polymer of Formula I was provided by Preparation Example 2, and the mass percentage of the polymer of Formula I in the droplet-generating oil was 5%;

[0114] In Example 4: the polymer of Formula I was provided by Preparation Example 1, and the mass percentage of the polymer of Formula I in the droplet-generating oil was 2%;

[0115] In Example 5: the polymer of Formula I was provided by Preparation Example 1, and the mass percentage of the polymer of Formula I in the droplet-generating oil was 6%;

[0116] In Example 6: the polymer of Formula I was provided by Preparation Example 1, and the mass percentage of the polymer of Formula I in the droplet-generating oil was 1%;

[0117] In Example 7: the polymer of Formula I was provided by Preparation Example 1, and the mass percentage of the polymer of Formula I in the droplet-generating oil was 8%;

[0118] In Comparative Example 1: the polymer of Formula I was provided by Comparative Preparation Example 1, and the mass percentage of the polymer of Formula I in the droplet-generating oil was 4%;

[0119] In Comparative Example 2: the polymer of Formula I was provided by Comparative Preparation Example 2, and the mass percentage of the polymer of Formula I in the droplet-generating oil was 4%;

[0120] In Comparative Example 3: the polymer of Formula I was provided by Comparative Preparation Example 3, and the mass percentage of the polymer of Formula I in the droplet-generating oil was 4%;

[0121] The method for preparing the droplet-generating oil is as follows:

[0122] The polymer of formula I and HFE 7100 solvent are mixed evenly to obtain the droplet-generating oil.

[0123] Comparative Example 4

[0124] This comparative example provides a droplet generating oil, which is Droplet Generation Oil for Probes, catalog number 1863005.

[0125] The performance of the droplet-generating oils provided in the above embodiments and comparative examples was tested, and the specific test methods are as follows:

[0126] Stability: The droplet-generating oils provided in the above examples and comparative examples were left to stand for 3 months, 6 months and 9 months at 25°C, and their appearance was observed.

[0127] The morphology of droplets after standing for 3 months, 6 months, and 9 months was observed using a microscope. After being placed at room temperature for 3 days, the morphology of the droplets after 3 days was observed again using a microscope, and the morphology of the droplets before and after 3 days was compared.

[0128] High temperature resistance: The droplets generated by the droplet-generating oil provided in the above examples and comparative examples through the microfluidic chip were characterized by digital PCR test using a microscope. Then, the droplets were subjected to digital PCR test respectively, and the morphology of the droplets after the PCR test was observed.

[0129] The specific process for digital PCR testing is as follows:

[0130]

[0131] After conducting stability tests (6 months) on the droplet-generating oils provided in Example 1 and Comparative Example 1, the test results are as follows: Figure 2-3 As shown ( Figure 2 The graph shows the stability test results of the droplet-generating oil provided in Example 1. Figure 3 (Figure showing the stability test results of the droplet-generating oil provided in Comparative Example 1). Figure 2 It can be seen that the droplet-generating oil provided by this invention showed no significant changes after standing at room temperature for 6 months, indicating that it has good stability; Figure 3 It can be seen that the droplet-generating oil provided in Comparative Example 1 showed obvious emulsification and whitening after standing at room temperature for 6 months, indicating that its stability was poor.

[0132] The droplet-generating oil provided in Example 1 of this invention was subjected to microfluidic droplet generation. In a microfluidic chip, the droplet-generating oil was used as the oil phase and single cells as the aqueous phase. Droplets were generated under stable pressure (16 kPa). The formed droplets were observed using a microscope, and the droplet morphology is as follows. Figure 4As shown, after the droplet was placed at room temperature for 3 days, the droplet was observed again using a microscope. The morphology of the droplet is as follows. Figure 5 As shown. By Figure 4-5 It can be seen that there was no significant difference in the morphology of the droplets before and after placement under a microscope, and the droplet size was uniform, indicating that the droplets generated by the oil have excellent stability when left to stand at room temperature.

[0133] The droplet-generating oil provided in Example 1 was subjected to digital PCR testing. The PCR amplification cycle involved a 98°C high-temperature cycle. The droplet morphology before PCR amplification was observed under a microscope (e.g., ...). Figure 6 As shown), after 10 heating cycles, the droplet was taken and its morphology was observed under a microscope (as shown). Figure 7 (As shown). By Figure 6-7 As can be seen, after digital PCR testing, the droplet morphology remained intact and the size was uniform. No droplet fusion was observed, indicating that the droplet-generating oil provided by this invention can withstand high-temperature PCR cycling heating.

[0134] The performance test results are detailed in Table 1 below.

[0135] Table 1

[0136]

[0137]

[0138] Note: When conducting high temperature resistance tests, if the droplet size remains uniform after 9 months of standing, it indicates that the droplet oil is within its shelf life. The droplet size will also be uniform after 3 months and 6 months of standing. The high temperature resistance test results only record the test results of the sample after 9 months of standing, and also record the droplet situation after 3 / 6 months.

[0139] As can be seen from the above, in this invention, by designing the structure of polymer of formula I, and further by controlling the weight-average molecular weight of perfluoropolyether carboxylic acid and polyether diamine, the hydrophobic end of polymer of formula I is prepared by using low molecular weight perfluoropolyether carboxylic acid, thus obtaining a droplet-generating oil with excellent performance. This droplet-generating oil has good stability and good high-temperature resistance. After the droplet-generating oil is left to stand for 9 months, its appearance remains clear and transparent. The droplet morphology is uniform before PCR, and there is no demulsification of the droplets after PCR. The high-temperature resistance is 98℃.

[0140] As can be seen from Examples 1-7, by controlling the content of Formula I polymer in the droplet generating oil within a specific range, a droplet generating oil with excellent performance can be prepared in this invention.

[0141] As can be seen from Examples 1-5 and Comparative Examples 1-4, in this invention, by designing the structure of polymer of formula I and further preparing the hydrophobic end of polymer of formula I using low molecular weight perfluoropolyether carboxylic acid, a droplet-generating oil with excellent performance was obtained.

[0142] In summary, this invention, by designing the structure of the polymer of formula I, prepared a droplet-generating oil with excellent performance, which exhibits good stability and high-temperature resistance.

[0143] The applicant declares that the detailed process flow of this invention is illustrated by the above embodiments, but this invention is not limited to the above detailed process flow, that is, it does not mean that this invention must rely on the above detailed process flow to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product of this invention, addition of auxiliary components, and selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

Claims

1. A droplet-generating oil, characterized in that, The droplet-generating oil comprises a polymer having the structure shown in Formula I: Where n represents an integer from 10 to 19; x represents an integer from 5 to 10; y represents an integer from 10 to 15. z represents an integer from 5 to 10.

2. The droplet-generating oil according to claim 1, characterized in that, The raw materials for preparing the polymer of formula I include perfluoropolyether carboxylic acid and polyether diamine; Preferably, the molar ratio of the perfluoropolyether carboxylic acid and the polyether diamine is 1:(2-3).

3. The droplet-generating oil according to claim 2, characterized in that, The weight-average molecular weight of the perfluoropolyether carboxylic acid is 1800–3000. Preferably, the polyether diamine has a weight-average molecular weight of 600 to 900.

4. The droplet-generating oil according to any one of claims 1-3, characterized in that, The polymer of Formula I is prepared by method A or method B, wherein method A includes the following steps: Perfluoropolyether carboxylic acid, polyether diamine, solvent A and reagent A are mixed and reacted to obtain the polymer of formula I. Method B includes the following steps: (1) Mix perfluoropolyether carboxylic acid, solvent B, oxalyl chloride and catalyst B, and react to obtain an intermediate; (2) The intermediate, polyether diamine, solvent C and reagent C are mixed and reacted to obtain the polymer of formula I.

5. The droplet-generating oil according to claim 4, characterized in that, Solvent A is selected from any one or a combination of at least two of HFE 7100 solvent, HFE7500, FC3283 or FC40 fluorinated liquid; Preferably, based on 100 parts by volume of the perfluoropolyether carboxylic acid, the volume of solvent A is 100 to 200 parts; Preferably, reagent A is selected from any one or a combination of at least two of potassium hexafluorophosphate, sodium hexafluorophosphate, or lithium hexafluorophosphate; Preferably, based on 100 parts by volume of the perfluoropolyether carboxylic acid, the volume of reagent A is 200 to 220 parts; Preferably, the reaction temperature in method A is 30–80°C, and the reaction time is 8–12 h; Preferably, method A further includes a post-processing step after the reaction, the post-processing step including the following steps: adding a fluorinated solvent to the reaction system, filtering, adding a polar solvent to the filtrate, precipitating the polymer of formula I, filtering, and obtaining the polymer of formula I.

6. The droplet-generating oil according to claim 4 or 5, characterized in that, The solvent B is selected from any one or a combination of at least two of the following: anhydrous HFE 7100 solvent, HFE7500, FC3283 or FC40 fluorinated liquid; Preferably, based on 100 parts by volume of the perfluoropolyether carboxylic acid, the volume of solvent B is 100 to 200 parts; Preferably, the catalyst B comprises anhydrous DMF; Preferably, based on 100 parts of the perfluoropolyether carboxylic acid, the molar amount of catalyst B is 0.1 to 1 part; Preferably, based on 100 parts of the perfluoropolyether carboxylic acid, the molar amount of the oxaloyl chloride is 100 to 150 parts; Preferably, the reaction temperature in step (1) is 20–30°C, and the reaction time is 20–28 h; Preferably, the reaction in step (1) is carried out in a protective atmosphere; Preferably, step (1) further includes a pretreatment step before the reaction, wherein the pretreatment step includes dehydrating the raw materials for preparing polymer of formula I; Preferably, the reaction in step (1) is followed by a step of removing the solvent.

7. The droplet-generating oil according to any one of claims 4-6, characterized in that, The solvent C is selected from anhydrous HFE7100 solvent and / or anhydrous tetrahydrofuran; Preferably, based on 100 parts by volume of the perfluoropolyether carboxylic acid, the solvent C comprises 100 to 200 parts by volume; Preferably, reagent C is selected from any one or a combination of at least two of triethylamine, 4-dimethylpyridine, or 1,8-diazabicyclo(5,4,0)-7-undecene; Preferably, based on 100 parts of the perfluoropolyether carboxylic acid, the molar amount of reagent C is 100 to 150 parts; Preferably, the reaction temperature in step (2) is 20–30°C, and the reaction time is 10–15 h; Preferably, step (2) further includes a post-processing step after the reaction, and the post-processing method includes the following steps: A polar solvent was added to the reaction system, and the mixture was filtered to obtain the polymer of formula I.

8. The droplet-generating oil according to any one of claims 1-7, characterized in that, The droplet-generating oil also includes a solvent; Preferably, the mass percentage of the polymer of formula I in the droplet-generating oil is 2-6%; Preferably, the solvent is selected from any one or a combination of at least two of anhydrous HFE 7100 solvent, HFE7500 or FC40 fluorinated liquid.

9. A method for preparing droplet-generating oil as described in any one of claims 1-8, characterized in that, The preparation method includes the following steps: The polymer and solvent shown in Formula I are mixed evenly to obtain the droplet-generating oil.

10. An application of the droplet-generating oil as described in any one of claims 1-8, characterized in that, The droplet-generating oil is used in the field of microfluidics for digital PCR.

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