Perfluoropolyether type surfactant with bio-based hydrophilic end as well as preparation method and application of perfluoropolyether type surfactant

Abstract

The invention discloses a perfluoropolyether type surfactant with a bio-based hydrophilic end as well as a preparation method and application of the perfluoropolyether type surfactant with the bio-based hydrophilic end, and the structural formula of the perfluoropolyether type surfactant is as follows: in the formula, R represents any one or more of the formula, or R represents an integer of 5-20, x represents an integer of 1-4, and y represents an integer of 1-5. The perfluoropolyether type surfactant disclosed by the invention can generate liquid drops with good uniformity and thermal stability, and the bio-based hydrophilic end of the perfluoropolyether type surfactant provides excellent biocompatibility. Meanwhile, the synthesis process is simple, convenient and efficient, and a key reagent with high cost performance can be provided for wide application of a ddPCR technology.

Description

Technical Field

[0001] This invention belongs to the field of fluorinated surfactant technology, specifically relating to a novel perfluoropolyether surfactant with a bio-based hydrophilic end for droplet generation and droplet coating. Background Technology

[0002] Droplet digital PCR (polymerase chain reaction) is one of the most accurate and promising techniques for the absolute quantification of tumor genes and pathogenic genes in clinical trials. Its simplicity and reliability have been demonstrated in infectious disease research and genomic analysis. However, a major challenge facing droplet digital PCR (ddPCR) is the high cost of reagents, which limits its widespread commercial application. The surfactants added to the oily mobile phase used in ddPCR microfluidic technology are key reagents.

[0003] One existing commercial surfactant has a structure of fluorocarbon chain linked to polyethylene glycol, as shown in equation a below (Selvaraj, V.; Maheshwari, Y.; Hajeri, S.; Yokomi, R.: Droplet Digital PCR for Absolute Quantification of Plant Pathogens. In Plant Biotechnology: Progress in Genomic Era; Khurana, SMP, Gaur, RK, Eds.; Springer Singapore: Singapore, 2019; pp 583-595.). However, its high-temperature stability is still insufficient for ddPCR applications. Another high-performance surfactant, developed by Harvard University based on perfluoropolyether (PFPE), is shown in formula b below (Chowdhury, MS; Zheng, W.; Kumari, S.; Heyman, J.; Zhang, X.; Dey, P.; Weitz, DA; Haag, R. Dendronized fluorosurfactant for highly stable water-in-fluorinated oil emulsions with minimal inter-droplet transfer of small molecules. Nature Communications 2019, 10, 4546.). However, its complex and costly synthesis of the hydrophilic end hinders its widespread adoption. Therefore, there is an urgent need to develop a surfactant suitable for ddPCR technology that possesses good overall performance, is low in cost, and has a simple manufacturing process.

[0004] Summary of the Invention

[0005] The purpose of this invention is to provide a perfluoropolyether surfactant for the generation of microdroplets in fluorinated oil / water systems. Unlike some existing surfactants, its key hydrophilic end is a bio-based molecule, which has the advantages of simple processing, low cost and excellent biocompatibility.

[0006] The structural formula of the perfluoropolyether surfactant with a bio-based hydrophilic end provided by this invention is shown below:

[0007]

[0008] In the formula, R represents Any one or more of them, or R represents n is an integer from 5 to 20, x and m are integers from 1 to 4, and y is an integer from 1 to 5.

[0009] Furthermore, the perfluoropolyether surfactant of the present invention having a bio-based hydrophilic end is preferably any one of the following formulas I to VI:

[0010]

[0011]

[0012] The preparation method of the perfluoropolyether surfactant with a bio-based hydrophilic end of the present invention includes the following steps:

[0013] Step 1: Dissolve the perfluoropolyether and oxalyl chloride in methoxy-nonafluorobutane, stir the mixture at room temperature until the reaction is complete, and then rotary evaporate to obtain the acyl-chlorinated perfluoropolyether; the reaction equation is as follows:

[0014]

[0015] Step 2: Dissolve acyl chloride perfluoropolyether, any one or more water-soluble sugar molecules of formula A and formula B, or water-soluble glycerol molecules of formula C in tetrahydrofuran, and add triethylamine as a catalyst. Reflux the reaction at 40-60°C for 24-48 hours, separate and purify to obtain a perfluoropolyether surfactant with a bio-based hydrophilic end.

[0016]

[0017] Furthermore, in step 1 above, the molar ratio of the perfluoropolyether to oxaloyl chloride is 1:1 to 10, preferably 1:5 to 6.

[0018] Furthermore, in step 2 above, the molar ratio of the acyl-chlorinated perfluoropolyether to the water-soluble sugar molecule or the water-soluble glycerol molecule is preferably 1:5 to 5:1.

[0019] Furthermore, in step 2 above, the preferred molar ratio of the acyl-chlorinated perfluoropolyether to triethylamine is 1:2 to 4.

[0020] Furthermore, in step 2 above, it is preferable to conduct the reaction in a closed environment at 50°C for 48 hours.

[0021] Furthermore, in step 2 above, the separation and purification method is as follows: repeatedly wash with a mixture of methoxy-nonafluorobutane and dichloromethane in a volume ratio of 1:5 to 5:1 until the upper layer is a clear liquid, remove the upper clear liquid, and rotary evaporate the lower layer solution to obtain a perfluoropolyether surfactant with a purity of over 98% and a bio-based hydrophilic end.

[0022] This invention also provides the use of perfluoropolyether surfactants with bio-based hydrophilic ends in the generation and coating of microdroplets in fluorinated oil / water systems.

[0023] The beneficial effects of this invention are as follows:

[0024] 1. This invention's perfluoropolyether surfactant differs from traditional PEG-type hydrophilic-terminated or complex synthetic hydrophilic-terminated fluorinated surfactants. It utilizes inexpensive, non-toxic (with good biocompatibility), and less environmentally harmful bio-based hydrophilic ends (sugars or glycerol), representing a significant step towards next-generation green and non-toxic emulsifiers. Furthermore, when applied in ddPCR, this surfactant significantly improves the high-temperature stability of droplets after formation, effectively enhancing the quantitative accuracy of droplet contents, thus providing substantial benefits for both medical and basic research.

[0025] 2. The perfluoropolyether surfactant with a bio-based hydrophilic end of the present invention is synthesized in two steps and is simple to purify, which reduces the process complexity and energy consumption required for high-performance emulsifiers. It has broad prospects for mass production and can be further integrated with bio-based refining (e.g., synthetic biology) to form an upstream and downstream value chain, further reducing costs and bringing new growth to cross-industries. Attached Figure Description

[0026] Figure 1 The comparison shows the infrared spectra of the perfluoropolyether surfactant with a monosaccharide hydrophilic end prepared in Example 1 and the perfluoropolyether (PFPE, brand name KrytoxFSH) raw material.

[0027] Figure 2 These are microscopic images (scale bar 100 μm) of 50 μm droplets formed from a perfluoropolyether surfactant with a monosaccharide hydrophilic end prepared in Example 1 after storage at 37°C (a) and 65°C (b) for 3 days.

[0028] Figure 3 This is a microscopic image (scale bar 50 μm) of a 50 μm droplet formed from a perfluoropolyether surfactant with a monosaccharide hydrophilic end prepared in Example 1 after storage at 95°C for 1 day.

[0029] Figure 4 The image shows a comparison of the perfluoropolyether surfactant with a monosaccharide hydrophilic end prepared in Example 1 before and after ddPCR (left) and a microscopic image of the droplet after PCR (scale bar 100 μm).

[0030] Figure 5 This is the infrared spectrum of the perfluoropolyether surfactant with a glycerol hydrophilic end prepared in Example 2.

[0031] Figure 6 These are microscopic images (scale bar 100 μm) of 50 μm droplets formed from a perfluoropolyether surfactant with a hydrophilic glycerol end prepared in Example 2 after storage at room temperature (a), 37°C (b) for 3 days, 65°C for 1 day (c), and 95°C for 8 hours (d). Detailed Implementation

[0032] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. These embodiments are merely illustrative of the invention and not intended to limit it.

[0033] Example 1

[0034]

[0035] Step 1: Add 5g (1eq) of perfluoropolyether (PFPE, CAS: 60164-51-4, brand name Krytox FSH), 0.3mL (6eq) of oxalyl chloride, and 10mL of methoxy-nonafluorobutane (brand name HFE7100) to a three-necked flask, stir at room temperature for 8h, and then remove the methoxy-nonafluorobutane and the remaining oxalyl chloride by rotary evaporation to obtain acyl-chlorinated perfluoropolyether.

[0036] Step 2: 5.18 g (1 eq) of acyl-chlorinated perfluoropolyether, 0.27 g (1.5 eq) of D-(L)-fructose, and 5 mL of tetrahydrofuran were added to a three-necked flask. After stirring until homogeneous, 0.3 mL (3 eq) of triethylamine was added, and the mixture was heated to 50 °C and refluxed for 48 h. After the reaction was complete, the solvent and byproducts were removed by rotary evaporation. A mixture of HFE7100 and dichloromethane (volume ratio 3:1) was added and the mixture was repeatedly washed until a clear liquid was formed in the upper layer. The upper clear liquid was then removed, and the lower layer was rotary evaporated to obtain a perfluoropolyether surfactant with a monosaccharide hydrophilic end. Its infrared spectrum is shown below. Figure 1 1776cm -1 The characteristic peak of -C=OOH disappears at 1688 cm⁻¹. -1The peak at this location represents the ester group that has formed.

[0037] The addition of a perfluoropolyether surfactant with a monosaccharide hydrophilic end, as described in this embodiment, to conduct an emulsification test of cell aqueous solution in fluorinated oil using a microfluidic process, yielded the following results: Figure 2 and Figure 3 As shown. The results indicate that this surfactant can stably and effectively form uniformly sized water-in-fluorine droplets at room temperature, i.e., a stable and homogeneous emulsion system; and the formed droplets can remain stable for at least 3 days at 65°C; and can remain stable for at least 1 day at 95°C, which is the highest temperature during the ddPCR test cycle. This result demonstrates the excellent performance of this surfactant in ddPCR applications. It also performs excellently in actual ddPCR tests, such as... Figure 4 As shown, the microscopic images at the end of the test showed that the droplet clusters were fused relatively little, with only 2% to 3% of the droplets forming clusters.

[0038] Example 2

[0039]

[0040] Step 1: Add 5g (1eq) Krytox FSH, 0.3mL (6eq) oxalyl chloride, and 10mL HFE7100 to a three-necked flask and stir at room temperature for 8 hours. Then, remove methoxy-nonafluorobutane and the remaining oxalyl chloride by rotary evaporation to obtain acyl-chlorinated perfluoropolyether.

[0041] Step 2: 5.18 g (1 eq) of acyl-chlorinated perfluoropolyether, 0.27 g (1.5 eq) of triglycerides, and 7 mL of tetrahydrofuran were added to a three-necked flask. After stirring until homogeneous, 0.3 mL (3 eq) of triethylamine was added, and the mixture was heated to 50 °C and refluxed for 48 h. After the reaction was complete, the solvent and byproducts were removed by rotary evaporation. A mixture of HFE7100 and dichloromethane (volume ratio 3:1) was added and the mixture was repeatedly washed until a clear liquid was formed in the upper layer. The upper clear liquid was then removed, and the lower layer was rotary evaporated to obtain a perfluoropolyether surfactant with a triglyceride hydrophilic end. Its infrared spectrum is shown below. Figure 5 1692cm -1 The peak at 1776 cm⁻¹ represents the newly formed ester group and is not visible. -1 The characteristic peak of the raw material -C=OOH at that location.

[0042] The perfluoropolyether surfactant with a triglyceride hydrophilic end, as described in this embodiment, was added to conduct an emulsification test of cell aqueous solution in fluorinated oil using a microfluidic process. The results are as follows: Figure 6As shown in the figure. The results indicate that the surfactant can form a water-in-fluorine droplet system and maintain uniform and stable droplet size at room temperature. When the temperature rises to 37℃ and 65℃, the droplets exhibit unstable fusion, with a fusion ratio of about 30% to 40%. After storage at a high temperature of 95℃ for 8 hours, the droplet fusion rate is comparable to that at 37℃ and 65℃, remaining at about 30% to 40%.

Claims

1. A perfluoropolyether surfactant with a bio-based hydrophilic end, characterized in that: The structural formula of the surfactant is shown below: In the formula, R represents Any one or more of them, or R represents n is an integer from 5 to 20, x and m are integers from 1 to 4, and y is an integer from 1 to 5.

2. The perfluoropolyether surfactant with a bio-based hydrophilic end according to claim 1, characterized in that: The perfluoropolyether surfactant is selected from any one of the following formulas I to VI:

3. A method for preparing a perfluoropolyether surfactant with a bio-based hydrophilic end as described in claim 1, characterized in that: The preparation method includes the following steps: Step 1: Dissolve perfluoropolyether and oxalyl chloride in methoxy-nonafluorobutane, stir at room temperature until the reaction is complete, and then evaporate by rotary evaporation to obtain acyl chloride perfluoropolyether; Step 2: Dissolve acyl chloride perfluoropolyether, any one or more water-soluble sugar molecules of formula A and formula B, or water-soluble glycerol molecules of formula C in tetrahydrofuran, and add triethylamine as a catalyst. Reflux the reaction at 40-60°C for 24-48 hours, separate and purify to obtain a perfluoropolyether surfactant with a bio-based hydrophilic end.

4. The method for preparing a perfluoropolyether surfactant with a bio-based hydrophilic end according to claim 1, characterized in that: In step 1, the molar ratio of the perfluoropolyether to oxaloyl chloride is 1:1 to 10.

5. The method for preparing a perfluoropolyether surfactant with a bio-based hydrophilic end according to claim 1, characterized in that: In step 1, the molar ratio of the perfluoropolyether to oxaloyl chloride is 1:5 to 6.

6. The method for preparing a perfluoropolyether surfactant with a bio-based hydrophilic end according to claim 1, characterized in that: In step 2, the molar ratio of the acyl-chlorinated perfluoropolyether to the water-soluble sugar molecules or water-soluble glycerol molecules is 1:5 to 5:

1.

7. The method for preparing a perfluoropolyether surfactant with a bio-based hydrophilic end according to claim 1, characterized in that: In step 2, the molar ratio of the acyl-chlorinated perfluoropolyether to triethylamine is 1:2 to 4.

8. The method for preparing a perfluoropolyether surfactant with a bio-based hydrophilic end according to claim 1, characterized in that: In step 2, the reaction is carried out in a closed environment at 50°C for 48 hours.

9. The method for preparing a perfluoropolyether surfactant with a bio-based hydrophilic end according to claim 1, characterized in that: In step 2, the separation and purification method is as follows: repeatedly wash with a mixture of methoxy-nonafluorobutane and dichloromethane in a volume ratio of 1:5 to 5:1 until the upper layer is a clear liquid, remove the upper clear liquid, and rotary evaporate the lower layer solution to obtain a perfluoropolyether surfactant with a purity of over 98% and a bio-based hydrophilic end.

10. The use of the perfluoropolyether surfactant with a bio-based hydrophilic end as described in claim 1 in the generation and coating of microdroplets in fluorinated oil / water systems.

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