Microscale sample extracellular vesicle enrichment method based on synergistic effect

Abstract

The invention relates to a trace sample extracellular vesicle enrichment method based on a synergistic effect. According to the method, 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine (DSPE) and tetravalent titanium ions (Ti (IV)) are modified in a channel of a micro-fluidic chip, and synergistic enrichment of the extracellular vesicles in a trace sample is realized by utilizing a dual affinity effect. The method is based on a synergistic enrichment principle, can simply, conveniently and efficiently realize high-selectivity, high-purity and high-recovery-rate separation of the extracellular vesicles in the trace sample, has the advantages of few required samples, wide application range, low use cost, high enrichment efficiency and the like, and has important significance on efficient separation of the extracellular vesicles in the trace sample.

Description

Technical Field

[0001] The present invention relates to a method for enriching extracellular vesicles of trace samples based on synergy, which utilizes a microfluidic chip to analyze trace samples and utilizes dual affinity molecules to achieve efficient enrichment of extracellular vesicles. Background Art

[0002] Extracellular vesicles (EVs) are a general term for various membrane-containing vesicle structures released by cells. They carry abundant information about proteins, nucleic acids, lipids and other substances from maternal cells, and participate in physiological and pathological processes such as cell communication, immune response, antigen presentation, cell differentiation, and tumor invasion. They are widely present in a variety of biological fluids such as blood, urine, saliva, tears, milk, and cerebrospinal fluid (R. Kalluri, et al. Science, 2020, 367, 6478). In the past few years, EVs have attracted great interest as biomarkers for diagnosis and treatment. The content of EVs reflects the specific pathophysiological state of patients with different diseases and represents a biomarker for tracking the progression of many diseases (R. Kalluri, et al. Cell, 2023, 186, 8).

[0003] However, due to the heterogeneity of their biogenesis, physical properties and molecular composition, the separation and enrichment of EVs remains extremely challenging. Traditional EV separation methods such as ultracentrifugation, polymer sedimentation, and ultrafiltration have the disadvantages of large sample requirements, low EV yield and purity, and time-consuming and labor-intensive methods, which are not suitable for the analysis of precious clinical biological samples (He Yan, et al. Anal. Chem. 2021 93 11, 4739-4774).

[0004] Affinity separation has developed rapidly in the field of efficient separation of high-purity EVs. Traditional affinity separation methods usually use affinity effects such as antibody-antigen affinity and electrostatic adsorption to separate EVs (Huilin Shao, et al. Chemical Reviews, 2018, 118, 4, 1917-1950). However, due to the high heterogeneity of EVs, this single affinity effect is often only targeted at a certain type of EVs, resulting in a low yield of separated EVs, which seriously limits the separation efficiency.

[0005] Microfluidic chip technology is a versatile technology that is used in a wide range of fields from diagnostics to nanotechnology. Compared with traditional separation technologies, microfluidic chip-based EVs separation technology has attracted attention due to its simplicity, small sample requirements, and high sensitivity, specificity, spatial and temporal resolution, and is increasingly being used for the separation and enrichment of EVs.

[0006] Therefore, based on the advantages of microfluidic chips in processing trace samples and based on dual affinity, efficient enrichment of extracellular vesicles in trace samples is achieved, which has the advantages of small sample requirements, fast separation speed, high separation yield and purity. Summary of the invention

[0007] The purpose of the present invention is to provide a method for enriching extracellular vesicles from trace samples based on synergistic effects, which has the advantages of small sample amount required, fast separation speed, simple operation, high separation purity, and high separation yield, and avoids the defects of the prior art with cumbersome operation steps, low separation purity and yield.

[0008] The present invention provides a method for enriching extracellular vesicles from trace samples based on synergistic effects. The method comprises modifying lipid molecules and tetravalent titanium ions (Ti 2+) having affinity for extracellular vesicles on the surface of a microfluidic chip channel. 4+ ) to achieve efficient enrichment of extracellular vesicles in trace samples.

[0009] The specific steps are as follows:

[0010] (1) Chip preparation: The PDMS chip was prepared by soft lithography technology, including an upper straight channel liquid path layer and a lower Y-shaped microcolumn array liquid path layer, with an inlet, an outlet and a microfluidic channel area. The prepolymer was poured onto the SU-8 mold and placed in a constant temperature drying oven at 80°C for curing. The cured chip was separated from the mold, and a handheld puncher was used to punch the inlet, outlet and positioning holes on the chip.

[0011] (2) Chip modification: Use a plasma cleaner to treat the chip surface for 30-60 seconds, immerse the chip in a 5-10% (3-aminopropyl) triethoxysilane (APTES) solution for 30-60 minutes, clean the surface after the reaction with anhydrous ethanol and deionized water, blow dry with clean air or nitrogen, align the positioning hole to close the chip, and place it in a constant temperature drying oven at 130°C for 30-60 minutes. Dissolve 3-phosphopropionic acid, N-hydroxysuccinimide (NHS), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) in a molar ratio of 1:1.5-2:0.8-1 in PBS buffer (pH=7.2-7.4), shake and react for 20-40 minutes, then add lipid molecules with a final concentration of 5-10 mg / mL and ultrasonically dissolve them, pass the above liquid into the chip, and react for 6-12 hours. After the reaction is completed, water is introduced to wash the residual reaction liquid, and then a titanium sulfate aqueous solution with a mass concentration of 0.1-0.5 g / ml is introduced to react for 1-2 hours.

[0012] (3) Extracellular vesicle separation: Use an injection needle to draw up biological fluid, install the injection needle on a syringe pump, and use the syringe pump to pass the test fluid into the microfluidic chip at a flow rate of 5-10 μl / min, and incubate at room temperature for 5 minutes to 2 hours; pump air to remove excess liquid, use PBS buffer to wash away nonspecific adsorption in the channel, and then pump an alkaline solution with a pH of 8-10 to incubate with the chip for 5-10 minutes. Repeat twice to achieve elution of extracellular vesicles and collect extracellular vesicle samples.

[0013] Furthermore, the microfluidic chip is made of polydimethylsiloxane polymer (PDMS), including an upper straight channel liquid path layer and a lower Y-shaped microcolumn array liquid path layer, and is provided with an inlet, an outlet and a microfluidic channel area.

[0014] The lipid molecule having affinity for extracellular vesicles is selected from one or more of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE); tetravalent titanium ion (Ti 4+ ) has a chelating effect on phosphate groups on the surface of vesicles.

[0015] Furthermore, the alkaline solution used includes 100 mM triethylamine aqueous solution and 10% NH3.H2O.

[0016] The sample is a liquid containing extracellular vesicles, including plasma, urine, saliva, tears, milk, cerebrospinal fluid, cell culture fluid, etc.

[0017] The present invention has the following advantages:

[0018] (1) The affinity molecules used in the present invention have common characteristics shared by all extracellular vesicles and can unbiasedly enrich all extracellular vesicles in a sample, which is more advantageous than the current affinity interactions such as specific antibodies and antigens.

[0019] (2) The present invention achieves the capture of extracellular vesicles by bonding dual affinity molecules on the chip. Compared with the currently widely developed single affinity, the capture efficiency is higher and the vesicle yield and purity are higher.

[0020] (3) The efficiency of enriching extracellular vesicles in the present invention is very high, and an enrichment efficiency of 85% can be achieved within 5 minutes, which significantly saves time and energy and facilitates high-throughput processing of clinical samples.

[0021] (4) The present invention introduces samples and reagents through a syringe pump, which is more automated and simplifies manual operations; by setting a Y-shaped microcolumn array in the microfluidic channel, the contact and mixing between the sample and the chip interface is increased, thereby improving the capture effect. The microfluidic chip is used to achieve the enrichment of extracellular vesicles in clinical trace precious samples, providing a solution for clinical diagnosis and treatment based on extracellular vesicles. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the synergistic enrichment of extracellular vesicles based on DSPE and Ti(Ⅳ) dual affinity microfluidic chip.

[0023] Figure 2 This is the actual picture of the DSPE and Ti(Ⅳ) dual-affinity microfluidic chip and the scanning electron microscope image of the surface morphology.

[0024] Figure 3 This is a scanning electron micrograph of extracellular vesicles captured on the surface after plasma separation using a microfluidic chip.

[0025] Figure 4 Transmission electron microscopy images and particle size distribution of extracellular vesicles separated from plasma using microfluidic chips.

[0026] Figure 5 The number of extracellular vesicles enriched after incubation of the microfluidic chip with plasma samples for different time periods.

[0027] Figure 6 Comparison of the purity and yield of extracellular vesicles enriched by microfluidic chip, ultracentrifugation and PEG methods. DETAILED DESCRIPTION

[0028] Example 1 Preparation and modification of microfluidic chip

[0029] The microfluidic chip template was prepared using soft lithography. SU-8 3035 photoresist was spin-coated on silicon wafers at 1400 rpm and 1000 rpm, respectively, and two structural templates with heights of 100 μm and 150 μm were prepared according to the designed pattern. The upper layer of the chip was designed as a straight channel with a height of 100 μm, and the lower layer was a channel containing a Y-shaped micro-pillar array with a height of 150 μm. The microchannel was 22.78 mm long and 4.54 mm wide. The Y-shaped micro-pillars were neatly arranged in the lower channel with a spacing of 260 μm to increase the contact between the sample and the chip and improve the separation efficiency. The PDMS base polymer and the curing agent were evenly mixed in a ratio of 10:1 (w / w) and poured on the SU-8 template, degassed in a desiccator for 30 minutes to remove bubbles, and then cured at 80 °C for 2 hours. The cured chip structure was peeled off from the template, and the positioning holes, sample inlet holes, and outlet holes were drilled.

[0030] After the chip was cleaned with ethanol and deionized water and dried with clean nitrogen, the PDMS surface was plasma treated for 30 seconds using a plasma cleaner (PDC-002, Harrick Plasma). Then the PDMS was immediately immersed in an APTES solution (5% v / v, in ethanol) and reacted at room temperature for 30 minutes to produce primary amine-terminated functional groups on the PDMS surface through a silanol-silanol reaction. The aminated PDMS surface was then cleaned with ethanol and deionized water and dried with clean nitrogen. Align the positioning holes and tightly bond the two PDMS pieces, and keep them in an oven at 130°C for 30 minutes to bond the chip to obtain a closed microchannel. 3-Phosphopropionic acid, NHS, and EDC were dissolved in PBS to a final concentration of 5 mg / mL, 5 mg / mL, and 10 mg / mL, respectively, and shaken at room temperature for 30 minutes to activate the carboxyl group. DSPE-PEG2000-NHS was added to the above solution to a final concentration of 10 mg / mL and ultrasonically dissolved. Subsequently, the solution was injected into the chip and reacted at room temperature for 6 hours. After washing with pure water three times, 25 μl of Ti(SO4)2 aqueous solution (0.2 mg / mL) was injected into the chip and the reaction continued for 1 hour. Finally, the chip was washed with H2O and stored in H2O for subsequent use.

[0031] Example 2 DSPE, Ti (IV) dual affinity microfluidic chip enriches extracellular vesicles in plasma. 20 μl of plasma to be separated is drawn with a sampling needle, and the sampling needle is installed on a syringe pump. The plasma is passed into the microfluidic chip prepared in Example 1 at a flow rate of 10 μl / min by the syringe pump. The extracellular vesicles in the liquid to be tested are captured by the affinity molecules in the chip channel. The nonspecific adsorption in the channel is washed away with 200 μl PBS buffer (pH = 7.4), and this is repeated 3 times. The surface morphology of the chip after enrichment of extracellular vesicles is characterized by scanning electron microscopy (such as Figure 3 As shown in the figure, it was found that vesicles with a particle size of about 100 nm were adsorbed on the chip surface, proving that the chip has a good enrichment effect on extracellular vesicles in plasma.

[0032] Example 3 Characterization of extracellular vesicles enriched after incubation of microfluidic chip with plasma

[0033] 20 μl of plasma to be separated was drawn by a sampling needle, and the sampling needle was installed on a syringe pump. The plasma was passed into the microfluidic chip prepared in Example 1 at a flow rate of 10 μl / min by the syringe pump and incubated for 1 hour. The extracellular vesicles in the liquid to be tested were captured by the affinity molecules in the chip channel. Nonspecific adsorption in the channel was washed away by 200 μl PBS buffer (pH=7.4), and the process was repeated 3 times. 50 μl triethylamine aqueous solution (100 mM) was pumped into the chip and incubated for 5 minutes, and the process was repeated 2 times to achieve elution of the extracellular vesicles, and the extracellular vesicle sample was collected. The size distribution of the extracellular vesicles was determined by a nanoparticle size tracking instrument (NTA), and the specific operation was as follows: 5 μl of the collected extracellular vesicle sample was taken, diluted to 1 ml with PBS buffer (pH=7.4), and the sample was injected into the sample pool. The laser wavelength was 488 nm, and the high-resolution sCMOS camera was used to shoot, and the measurement was repeated 3 times. The extracellular vesicle samples were negatively stained using a transmission electron microscope to characterize the structure. The specific operation was as follows: 10 μl of the collected extracellular vesicle sample was dripped onto a copper grid, 10 μl of 2% (v / v) tungsten phosphate was added to the copper grid, and after drying at room temperature for 5 minutes, electron microscopy was performed at 80-120 kV for imaging. The results are shown in Figure 2. Figure 4 As shown, the size of the enriched extracellular vesicles is distributed between 30-350 nm, and the electron microscopy structure shows a typical double-layer membrane structure, proving the successful enrichment of extracellular vesicles.

[0034] Example 4 The number of extracellular vesicles enriched after incubation of the microfluidic chip with plasma samples for different time periods

[0035] 20 μl of plasma to be separated was drawn with an injection needle, and the injection needle was installed on a syringe pump. The plasma was passed into the microfluidic chip prepared in Example 1 at a flow rate of 10 μl / min using the syringe pump. The plasma was incubated for 5 minutes, 10 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes respectively. After the incubation was completed, 200 μl of PBS buffer (pH=7.4) was used to wash away the nonspecific adsorption in the channel, and the process was repeated 3 times. 50 μl of triethylamine aqueous solution (100 mM) was pumped in and incubated with the chip for 5 minutes, and the process was repeated 2 times to achieve the elution of the extracellular vesicles, and the extracellular vesicle sample was collected. The number of extracellular vesicles enriched was determined using a nanoparticle size tracker (NTA). The specific operation was as follows: 5 μl of the collected extracellular vesicle sample was taken, diluted to 1 ml with PBS buffer (pH=7.4), and the sample was injected into the sample pool. The laser wavelength was 488 nm, and the high-resolution sCMOS camera was used to shoot the sample, and the measurement was repeated 3 times. The results are shown in FIG. Figure 5 As shown, as the incubation time increased, the number of vesicles increased and reached a plateau at 1 hour.

[0036] Example 5 Comparison of Purity and Yield of Extracellular Vesicles Enriched by Microfluidic Chip, Ultracentrifugation Method, and PEG Method

[0037] Microfluidic chip method: 20 μl of plasma to be separated was drawn with an injection needle, and the injection needle was installed on a syringe pump. The plasma was passed into the microfluidic chip prepared in Example 1 at a flow rate of 10 μl / min using a syringe pump. After incubation for 1 hour, nonspecific adsorption in the channel was washed away with 200 μl PBS buffer, and the process was repeated 3 times. 50 μl of triethylamine aqueous solution (100 mM) was pumped in and incubated with the chip for 5 minutes, and the process was repeated 2 times to achieve elution of the extracellular vesicles and collect the extracellular vesicle samples.

[0038] Ultracentrifugation method: 20 μl of plasma was centrifuged at 100,000 g for 70 min using a Beckman centrifuge, the supernatant was removed, the precipitate was resuspended in 50 μl of PBS buffer, and centrifuged again at 100,000 g for 70 min, and resuspended in PBS buffer to obtain an extracellular vesicle sample.

[0039] Polyethylene glycol (PEG) method: Take 20 μl of plasma sample, dilute with 80 μl PBS buffer, add 25 μl of 50% PEG aqueous solution and mix evenly, precipitate at 4°C overnight, centrifuge at 4000g for 20 minutes, discard the supernatant, and re-dissolve the precipitate with 50 μl PBS buffer to obtain the extracellular vesicle sample.

[0040] The number of extracellular vesicles was determined using a nanoparticle tracking instrument (NTA). The specific operation was as follows: 5 μl of the extracellular vesicle samples obtained by the above three methods were taken, diluted to 1 ml with PBS buffer, and the sample was injected into the sample pool. The laser wavelength was 488 nm, and the high-resolution sCMOS camera was used to shoot. The measurement was repeated 3 times. The protein concentration of the extracellular vesicle sample was tested using the BCA kit. The ratio of the total number of extracellular vesicle particles measured by NTA to the total protein amount measured by BCA is the purity of the extracellular vesicles. The results are shown in Figure 6 As shown in the figure, the extracellular vesicles enriched by the microfluidic chip method are superior to the current classical ultracentrifugation method in terms of yield and purity. Since the purity of the vesicles enriched by the PEG method is very low, although the yield is high, many of them are impurities, so the microfluidic chip method also has advantages in comparison.

Claims

1. A method for enriching extracellular vesicles from trace samples based on synergy, characterized in that: The surface of the microfluidic chip channel is modified with lipid molecules and tetravalent titanium ions (Ti) that have an affinity for extracellular vesicles. 4+ ), achieving efficient enrichment of extracellular vesicles in trace samples.

2. The enrichment method according to claim 1, characterized in that: The lipid molecule having affinity for extracellular vesicles is selected from one or more of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE); tetravalent titanium ion (Ti 4+ ) has a chelating effect on phosphate groups on the surface of vesicles.

3. The enrichment method according to claim 1, characterized in that: The microfluidic chip is made of polydimethylsiloxane polymer (PDMS), comprises an upper straight channel liquid path layer and a lower Y-shaped microcolumn array liquid path layer, and is provided with an inlet, an outlet and a microfluidic channel area.

4. The method according to claim 1, characterized in that: The microfluidic chip channel surface modification process includes the following steps: (1) Pretreatment of the microfluidic chip: place the microfluidic PDMS chip in a plasma cleaning machine to treat the surface for 30-60 seconds, then immerse the chip in a 5-10% by volume (3-aminopropyl) triethoxysilane (APTES) solution for 30-60 minutes; wash with ethanol and deionized water, and blow dry with nitrogen; (2) Modification of lipid molecules: 3-phosphopropionic acid, N-hydroxysuccinimide (NHS), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were dissolved in PBS buffer (pH = 7.2-7.4) and oscillated for 20-40 minutes, and then lipid molecules were added for ultrasonic dissolution. The above liquid was passed into the pretreated chip and reacted for 6-12 hours; (3) Tetravalent titanium ion (Ti 4+ ) modification, passing 20-30 μl of a 0.1-0.5 g / ml titanium sulfate aqueous solution into the PDMS chip after the reaction in step (2), reacting for 1-2 hours to obtain a modified PDMS chip.

5. The enrichment method according to claim 1, characterized in that: In step (2), the molar ratio of 3-phosphopropionic acid, EDC and NHS is 1:1.5-2:0.8-1, and the final concentration of lipid molecules is 5-10 mg / ml.

6. The enrichment method according to claim 1, characterized in that: The sample to be tested was pumped into the chip at 5-10 μl / min using a syringe pump and incubated at room temperature for 5 minutes to 2 hours to capture the extracellular vesicles in the biological sample on the chip surface; PBS buffer (pH = 7.2-7.4) was passed to remove non-specific adsorption, and an alkaline solution with a pH between 8 and 10 was passed and incubated with the chip for 5-10 minutes to achieve extracellular vesicle elution.

7. The enrichment method according to claim 1, characterized in that: The basic solution includes 50-100 mM triethylamine aqueous solution, 5%-10% NH3.H2O.

8. The enrichment method according to claim 7, characterized in that: The sample is a liquid containing extracellular vesicles, including plasma, urine, saliva, tears, milk, cerebrospinal fluid, cell culture fluid, etc.

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