Method for separating and enriching exosome by adopting paper-based isoelectric focusing

Abstract

The invention discloses a method for separating and enriching exosomes by using paper-based isoelectric focusing equipment and adopting paper-based isoelectric focusing. The method comprises the following steps: carrying out step-by-step centrifugation and ultrafiltration on an exosome solution to obtain a purified exosome solution, incubating the purified exosome solution and carbon quantum dots at a certain temperature to obtain a fluorescent exosome solution adsorbed with the carbon quantum dots, and respectively taking a glycine-hydrochloric acid solution and a glycine-sodium hydroxide solution as positive and negative electrode solutions of paper-based isoelectric focusing equipment, so as to obtain the fluorescent exosome. And performing isoelectric focusing on the paper-based material fully soaked with the fluorescent exosome solution, and finally recovering different focused exosomes. The invention provides a carrier-free ampholyte focusing platform which is convenient, easy to obtain, environment-friendly, low in cost, small in sample loading amount, low in detection limit, easy for sample enrichment and recovery, and compatible with other subsequent detection methods.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological sample analysis and separation, and particularly relates to a method for separating and enriching exosomes by paper-based isoelectric focusing. Background Art

[0002] Isoelectric focusing is a commonly used electrophoretic separation method that separates amphiphilic substances according to their isoelectric points. It is mostly used to characterize the isoelectric points of peptides and protein drugs. Paper-based analytical equipment provides a lightweight, environmentally friendly, and low-cost platform that is used in pesticide detection, blood analysis, urine testing, food safety, etc. Traditionally, isoelectric focusing is performed using a carrier ampholyte mixture. However, carrier ampholytes are expensive and may interact with samples, resulting in reduced UV detection sensitivity. In addition, they increase ion current during MALDI detection, which is not conducive to subsequent mass spectrometry detection of samples. Paper-based isoelectric focusing of carrier-free ampholyte is characterized by rapidity, simplicity, ease of operation, and compatibility with mass spectrometry. It was first proposed by Xie et al. (Xie SF, Gao H, Niu LL, et al. Carrier ampholyte-free isoelectric focusing on a paper-based analytical device for the fractionation of proteins[J]. Journal of Separation Science, 2018, 41:2085-2091.) and used for the analysis and detection of proteins.

[0003] Exosomes are nano-scale vesicles wrapped in lipid bilayers secreted by cells. They are widely present in body fluids such as blood, urine, and saliva. They participate in a variety of physiological and pathological processes such as immune regulation, angiogenesis, tumorigenesis, and metastasis. Therefore, they show good application prospects in disease diagnosis, treatment, and drug delivery. Carbon quantum dots are a new type of fluorescent material with good optical properties, low biological toxicity, and simple preparation. In recent years, they have received widespread attention and have been applied in many fields. In addition, specific groups (carboxyl, amino, etc.) can be modified to achieve the effect of fluorescent labeling of exosomes.

[0004] In view of this, it is necessary to develop a carrier-free ampholyte paper-based isoelectric focusing method for separating and enriching exosomes. Summary of the invention

[0005] The present invention provides a method for paper-based isoelectric focusing of exosomes after carbon quantum dot labeling, which can realize carbon quantum dot fluorescent labeling and isoelectric focusing enrichment of exosomes.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A method for separating and enriching exosomes by paper-based isoelectric focusing, using a paper-based isoelectric focusing device, including a paper-based material, a positive electrode storage tank, a negative electrode storage tank, and a DC voltage source, characterized in that: the positive electrode storage tank connects one end of the paper-based material with the positive electrode of the DC voltage source, and the negative electrode storage tank connects the other end of the paper-based material with the negative electrode of the DC voltage source.

[0008] Further, the method specifically includes the following steps:

[0009] Step 1: Centrifuge and ultrafilter the sample solution step by step to obtain a purified exosome solution;

[0010] Step 2: incubating the exosome concentrate with carbon quantum dots at a certain temperature to obtain a fluorescent exosome solution adsorbed with carbon quantum dots;

[0011] Step 3: Add glycine-hydrochloric acid solution as the positive electrode liquid into the positive electrode storage tank, and add glycine-sodium hydroxide solution as the negative electrode liquid into the negative electrode storage tank; fix the positive and negative storage tanks, immerse the two electrodes of the DC voltage source into the electrode liquids of the positive and negative storage tanks respectively, and set the voltage of the DC voltage source;

[0012] Step 4: Immerse the paper-based material in the fluorescent exosome solution. After the paper-based material is saturated, take it out and place it between the liquid reservoirs. Both ends of the paper-based material are immersed in the electrode liquid of the positive and negative liquid reservoirs respectively.

[0013] Step 5: Turn on the DC voltage source and start applying pressure to perform isoelectric focusing on the sample. After focusing is completed, turn off the DC voltage source;

[0014] Step 6: Cut the paper-based material, collect the liquid portion on each section of the paper-based material, and perform subsequent characterization and testing.

[0015] Furthermore, the loading solution in step 1 is a complex biological sample containing exosomes such as urine, cell culture medium, fetal bovine serum, etc. after dilution and pH adjustment.

[0016] Furthermore, in step 2, the concentration of carbon quantum dots is 0.001-1 mg / mL.

[0017] In the further step 2, the carbon quantum dots are zero-dimensional carbon nanomaterials with a size less than 10 nm and significant fluorescence properties.

[0018] Furthermore, in step three, the concentration of the glycine-hydrochloric acid solution is 5-100 mmol / L, and the pH is 1-6.

[0019] Furthermore, in step three, the concentration of the glycine-sodium hydroxide solution is 5-100 mmol / L, and the pH is 8-12.

[0020] Furthermore, in step three, 0.2% hydroxyethyl cellulose (HEC) is added to the positive electrode solution.

[0021] Furthermore, in step 2, the reaction temperature is 35-40° C., and the reaction time is 16-24 hours.

[0022] Furthermore, in step three, the DC voltage source is an electrophoresis apparatus, and the voltage of the DC voltage source is set to 0.2-1.5 kV.

[0023] The principle of the present invention is: under a certain voltage, the acid and alkali solutions in the liquid reservoirs at both ends form a pH gradient on the surface of the paper fluid channel. When the sample to be tested migrates to the pH position with the same isoelectric point as its isoelectric point, the net charge carried by the sample is zero and it no longer moves, thus achieving sample focusing; and in a mixed sample, samples with different isoelectric points are respectively focused at the corresponding pH, thus achieving separation of the mixed sample.

[0024] The advantages and beneficial effects of the present invention are: providing an effective method for separation and enrichment of exosomes. Compared with other detection methods, isoelectric focusing can combine the characteristics of the isoelectric point of exosomes to quickly separate and enrich them; compared with other isoelectric focusing methods, paper-based provides a convenient, environmentally friendly, low-cost focusing platform with a small sample load, low detection limit, and easy sample enrichment and recovery; compared with traditional aggregation methods, carrier-free amphoteric electrolyte isoelectric focusing is simpler and compatible with other subsequent detection methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the overall schematic diagram of the isoelectric focusing device for paper-based analysis, which includes the following four parts: paper-based material, positive electrode liquid storage tank, negative electrode liquid storage tank, and DC voltage source.

[0026] Figure 2 This is a graph of the pH gradient formed on the paper-based material.

[0027] Figure 3 This is the result of paper-based isoelectric focusing of exosomes labeled with carbon quantum dots.

[0028] Figure 4 This is the distribution diagram of exosome concentration on the paper substrate surface after focusing. The paper substrate is cut after focusing, and the liquid part on each section of the paper substrate is collected for concentration detection. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Embodiment 1:

[0031] 1. Preparation of purified exosome solution:

[0032] The mid-morning urine was collected, diluted, and adjusted to a pH of about 7.4, and then centrifuged step by step to remove apoptotic cells and cell debris. The urine was then ultrafiltered and washed with PBS to remove contaminating proteins to obtain a purified exosome solution.

[0033] 2. Preparation of fluorescent exosome solution:

[0034] The purified exosome solution was incubated with carbon quantum dots (zero-dimensional carbon nanomaterials with significant fluorescent properties at a concentration of 1 mg / mL and a size of less than 10 nm) at 40°C for 24 h, and then transferred to a centrifugal filter tube after adding a small amount of PBS solution, centrifuged, and washed three times to remove unbound carbon dots to obtain a fluorescent exosome solution.

[0035] 3. Set up the electrophoresis apparatus:

[0036] A mixture of 10 mmol / L glycine-hydrochloric acid solution with a pH value of 3 and 0.2% hydroxyethyl cellulose (HEC) solution was added to the positive electrode storage tank as the positive electrode liquid, and a 10 mmol / L glycine-sodium hydroxide solution with a pH value of 9 was added to the negative electrode storage tank as the negative electrode liquid; the positive and negative storage tanks were fixed, and the two electrodes of the electrophoresis instrument were immersed in the electrode liquids of the positive and negative storage tanks respectively, and the power supply voltage of the electrophoresis instrument was set to 1 kV, and the pressurization time was 5 s.

[0037] 4. Set up the paper base material

[0038] Prepare a paper-based material with a total length of about 45mm and a width of about 3mm, and fold 2.5mm at both ends for immersion in the reservoir solution, that is, the effective length of the paper fluid channel is about 40mm; immerse the paper-based material in the fluorescent exosome solution, take it out after the paper-based material is completely wetted and saturated, and place it between the positive and negative reservoirs. The two ends of the paper-based material are immersed in the electrode liquid of the positive and negative reservoirs respectively to build a complete current loop

[0039] 5. Isoelectric Focusing

[0040] Turn on the power of the electrophoresis instrument and start to apply pressure. Observe the color change of the paper base surface during the isoelectric focusing process. When the sample focusing phenomenon can be clearly observed, stop applying pressure, end the focusing process, and turn off the power. If the sample solution is colorless, record the current change of the power supply. It can be observed that the current first increases rapidly and then decreases slowly. When the current drops below about 60μA and reaches stability, the focusing is considered to be completed.

[0041] 6. Recycling of Focused Exosomes

[0042] The paper-based material was cut and the liquid portion on each section of the paper-based material was collected to obtain different focused exosomes for subsequent characterization and detection.

[0043] Embodiment 2:

[0044] 1. Preparation of purified exosome solution:

[0045] The fetal bovine serum solution was diluted and adjusted to a pH of about 7.4, and then centrifuged step by step to remove apoptotic cells and cell debris, and then ultrafiltration and PBS washing were performed to remove contaminating proteins to obtain a purified exosome solution.

[0046] 2. Preparation of fluorescent exosome solution:

[0047] The purified exosome solution was incubated with carbon quantum dots (zero-dimensional carbon nanomaterials with significant fluorescent properties and a size of less than 10 nm at a concentration of 0.001 mg / mL) at 35°C for 16 h, and then transferred to a centrifugal filter tube after adding a small amount of PBS solution, centrifuged, and washed three times to remove unbound carbon dots to obtain a fluorescent exosome solution.

[0048] 3. Set up the electrophoresis apparatus:

[0049] A 5mmol / L glycine-hydrochloric acid solution with a pH value of 6 was added as the positive electrode liquid into the positive electrode storage tank, and a 5mmol / L glycine-sodium hydroxide solution with a pH value of 8 was added as the negative electrode liquid into the negative electrode storage tank; the positive and negative liquid storage tanks were fixed, and the two electrodes of the electrophoresis instrument were immersed in the electrode liquids of the positive and negative liquid storage tanks respectively, and the power supply voltage of the electrophoresis instrument was set to 0.2kV, and the pressurization time was 5s.

[0050] 4. Set up the paper base material

[0051] Prepare a paper-based material with a total length of about 45mm and a width of about 3mm, and fold 2.5mm at both ends for immersion in the reservoir solution, that is, the effective length of the paper fluid channel is about 40mm; immerse the paper-based material in the fluorescent exosome solution, take it out after the paper-based material is completely wetted and saturated, and place it between the positive and negative reservoirs. The two ends of the paper-based material are immersed in the electrode liquid of the positive and negative reservoirs respectively to build a complete current loop

[0052] 5. Isoelectric Focusing

[0053] Turn on the power of the electrophoresis instrument and start to apply pressure. Observe the color change of the paper base surface during the isoelectric focusing process. When the sample focusing phenomenon can be clearly observed, stop applying pressure, end the focusing process, and turn off the power. If the sample solution is colorless, record the current change of the power supply. It can be observed that the current first increases rapidly and then decreases slowly. When the current drops below about 60μA and reaches stability, the focusing is considered to be completed.

[0054] 6. Recycling of Focused Exosomes

[0055] The paper-based material was cut and the liquid portion on each section of the paper-based material was collected to obtain different focused exosomes for subsequent characterization and detection.

[0056] Embodiment 3:

[0057] 1. Preparation of purified exosome solution:

[0058] The cell culture medium was diluted and the pH value was adjusted to about 7.4, and then centrifuged step by step to remove apoptotic cells and cell debris, and then ultrafiltration and PBS washing were performed to remove contaminating proteins to obtain a purified exosome solution.

[0059] 2. Preparation of fluorescent exosome solution:

[0060] The purified exosome solution was incubated with carbon quantum dots (zero-dimensional carbon nanomaterials with significant fluorescent properties and a size of less than 10 nm at a concentration of 0.1 mg / mL) at 40°C for 16 h, and then transferred to a centrifugal filter tube after adding a small amount of PBS solution, centrifuged, and washed three times to remove unbound carbon dots to obtain a fluorescent exosome solution.

[0061] 3. Set up the electrophoresis apparatus:

[0062] A 50mmol / L glycine-hydrochloric acid solution with a pH value of 2 and a 0.2% hydroxyethyl cellulose (HEC) solution were mixed and added to the positive electrode storage tank as the positive electrode liquid, and a 100mmol / L glycine-sodium hydroxide solution with a pH value of 12 was added to the negative electrode storage tank as the negative electrode liquid; the positive and negative storage tanks were fixed, and the two electrodes of the electrophoresis instrument were immersed in the electrode liquids of the positive and negative storage tanks respectively, and the power supply voltage of the electrophoresis instrument was set to 0.8kV, and the pressurization time was 5s.

[0063] 4. Set up the paper base material

[0064] Prepare a paper-based material with a total length of about 45mm and a width of about 3mm, and fold 2.5mm at both ends for immersion in the reservoir solution, that is, the effective length of the paper fluid channel is about 40mm; immerse the paper-based material in the fluorescent exosome solution, take it out after the paper-based material is completely wetted and saturated, and place it between the positive and negative reservoirs. The two ends of the paper-based material are immersed in the electrode liquid of the positive and negative reservoirs respectively to build a complete current loop

[0065] 5. Isoelectric Focusing

[0066] Turn on the power of the electrophoresis instrument and start to apply pressure. Observe the color change of the paper base surface during the isoelectric focusing process. When the sample focusing phenomenon can be clearly observed, stop applying pressure, end the focusing process, and turn off the power. If the sample solution is colorless, record the current change of the power supply. It can be observed that the current first increases rapidly and then decreases slowly. When the current drops below about 60μA and reaches stability, the focusing is considered to be completed.

[0067] 6. Recycling of Focused Exosomes

[0068] The paper-based material was cut and the liquid portion on each section of the paper-based material was collected to obtain different focused exosomes for subsequent characterization and detection.

[0069] Embodiment 4:

[0070] 1. Preparation of purified exosome solution:

[0071] The mid-morning urine was collected, diluted, and adjusted to a pH of about 7.4, and then centrifuged step by step to remove apoptotic cells and cell debris. The urine was then ultrafiltered and washed with PBS to remove contaminating proteins to obtain a purified exosome solution.

[0072] 2. Preparation of fluorescent exosome solution:

[0073] The purified exosome solution was incubated with carbon quantum dots (zero-dimensional carbon nanomaterials with significant fluorescent properties and a size of less than 10 nm at a concentration of 0.5 mg / mL) at 38°C for 20 h, and then transferred to a centrifugal filter tube after adding a small amount of PBS solution, centrifuged, and washed three times to remove unbound carbon dots to obtain a fluorescent exosome solution.

[0074] 3. Set up the electrophoresis apparatus:

[0075] A 100 mmol / L glycine-hydrochloric acid solution with a pH of 1 was added as the positive electrode liquid into the positive electrode storage tank, and a 50 mmol / L glycine-sodium hydroxide solution with a pH of 10 was added as the negative electrode liquid into the negative electrode storage tank; the positive and negative liquid storage tanks were fixed, and the two electrodes of the electrophoresis instrument were immersed in the electrode liquids of the positive and negative liquid storage tanks respectively, and the power supply voltage of the electrophoresis instrument was set to 0.5 kV, and the pressurization time was 5 s.

[0076] 4. Set up the paper base material

[0077] Prepare a paper-based material with a total length of about 45mm and a width of about 3mm, and fold 2.5mm at both ends for immersion in the reservoir solution, that is, the effective length of the paper fluid channel is about 40mm; immerse the paper-based material in the fluorescent exosome solution, take it out after the paper-based material is completely wetted and saturated, and place it between the positive and negative reservoirs. The two ends of the paper-based material are immersed in the electrode liquid of the positive and negative reservoirs respectively to build a complete current loop

[0078] 5. Isoelectric Focusing

[0079] Turn on the power of the electrophoresis instrument and start to apply pressure. Observe the color change of the paper base surface during the isoelectric focusing process. When the sample focusing phenomenon can be clearly observed, stop applying pressure, end the focusing process, and turn off the power. If the sample solution is colorless, record the current change of the power supply. It can be observed that the current first increases rapidly and then decreases slowly. When the current drops below about 60μA and reaches stability, the focusing is considered to be completed.

[0080] 6. Recycling of Focused Exosomes

[0081] The paper-based material was cut and the liquid portion on each section of the paper-based material was collected to obtain different focused exosomes for subsequent characterization and detection.

[0082] Embodiment 5:

[0083] 1. Preparation of purified exosome solution:

[0084] The mid-morning urine was collected, diluted, and adjusted to a pH of about 7.4, and then centrifuged step by step to remove apoptotic cells and cell debris. The urine was then ultrafiltered and washed with PBS to remove contaminating proteins to obtain a purified exosome solution.

[0085] 2. Preparation of fluorescent exosome solution:

[0086] The purified exosome solution was incubated with carbon quantum dots (zero-dimensional carbon nanomaterials with significant fluorescent properties and a size of less than 10 nm at a concentration of 0.05 mg / mL) at 40°C for 18 h, and then transferred to a centrifugal filter tube after adding a small amount of PBS solution, centrifuged, and washed three times to remove unbound carbon dots to obtain a fluorescent exosome solution.

[0087] 3. Set up the electrophoresis apparatus:

[0088] Add 80mmol / L glycine-hydrochloric acid solution with pH=3 as the positive electrode liquid into the positive electrode storage tank, and add 30mmol / L glycine-sodium hydroxide solution with pH=10 as the negative electrode liquid into the negative electrode storage tank; fix the positive and negative liquid storage tanks, immerse the two electrodes of the electrophoresis instrument into the electrode liquids of the positive and negative liquid storage tanks respectively, set the power supply voltage of the electrophoresis instrument to 0.2kV, and the pressurization time to 5s.

[0089] 4. Set up the paper base material

[0090] Prepare a paper-based material with a total length of about 45mm and a width of about 3mm, and fold 2.5mm at both ends for immersion in the reservoir solution, that is, the effective length of the paper fluid channel is about 40mm; immerse the paper-based material in the fluorescent exosome solution, take it out after the paper-based material is completely wetted and saturated, and place it between the positive and negative reservoirs. The two ends of the paper-based material are immersed in the electrode liquid of the positive and negative reservoirs respectively to build a complete current loop

[0091] 5. Isoelectric Focusing

[0092] Turn on the power of the electrophoresis instrument and start to apply pressure. Observe the color change of the paper base surface during the isoelectric focusing process. When the sample focusing phenomenon can be clearly observed, stop applying pressure, end the focusing process, and turn off the power. If the sample solution is colorless, record the current change of the power supply. It can be observed that the current first increases rapidly and then decreases slowly. When the current drops below about 60μA and reaches stability, the focusing is considered to be completed.

[0093] 6. Recycling of Focused Exosomes

[0094] The paper-based material was cut and the liquid portion on each section of the paper-based material was collected to obtain different focused exosomes for subsequent characterization and detection.

Claims

1. A method for separating and enriching exosomes by paper-based isoelectric focusing, using a paper-based isoelectric focusing device, including a paper-based material, a positive electrode liquid storage tank, a negative electrode liquid storage tank, and a DC voltage source, characterized in that: The positive electrode liquid storage tank is connected to one end of the paper-based material and the positive electrode of the DC voltage source, and the negative electrode liquid storage tank is connected to the other end of the paper-based material and the negative electrode of the DC voltage source.

2. The method for separating and enriching exosomes by paper-based isoelectric focusing according to claim 1, characterized in that: The following steps are involved: Step 1: Centrifuge and ultrafilter the sample solution step by step to obtain a purified exosome solution; Step 2: incubating the exosome concentrate with carbon quantum dots at a certain temperature to obtain a fluorescent exosome solution adsorbed with carbon quantum dots; Step 3: Add glycine-hydrochloric acid solution as the positive electrode liquid into the positive electrode storage tank, and add glycine-sodium hydroxide solution as the negative electrode liquid into the negative electrode storage tank; fix the positive and negative storage tanks, immerse the two electrodes of the electrophoresis instrument into the electrode liquids of the positive and negative storage tanks respectively, and set the voltage of the DC voltage source; Step 4: Immerse the paper-based material in the fluorescent exosome solution. After the paper-based material is saturated, take it out and place it between the liquid reservoirs. Both ends of the paper-based material are immersed in the electrode liquid of the positive and negative liquid reservoirs respectively. Step 5: Turn on the DC voltage source and start applying pressure to perform isoelectric focusing on the sample. After focusing is completed, turn off the DC voltage source; Step 6: Cut the paper-based material, collect the liquid portion on each section of the paper-based material, and perform subsequent characterization and testing.

3. The method for separating and enriching exosomes by paper-based isoelectric focusing according to claim 2, characterized in that: The loading solution in step 1 is a complex biological sample containing exosomes such as urine, cell culture medium, fetal bovine serum, etc. after dilution and pH adjustment.

4. The method for separating and enriching exosomes by paper-based isoelectric focusing according to claim 3, characterized in that: In step 2, the concentration of carbon quantum dots is 0.001-1 mg / mL.

5. The method for separating and enriching exosomes by paper-based isoelectric focusing according to claim 4, characterized in that: In step 2, the carbon quantum dots are zero-dimensional carbon nanomaterials with a size less than 10 nm and significant fluorescence properties.

6. The method for separating and enriching exosomes by paper-based isoelectric focusing according to claim 3 or 5, characterized in that: In step 3, the concentration of the glycine-hydrochloric acid solution is 5-100 mmol / L, and the pH is 1-6.

7. The method for separating and enriching exosomes by paper-based isoelectric focusing according to claim 6, characterized in that: In step 3, the concentration of the glycine-sodium hydroxide solution is 5-100 mmol / L, and the pH is 8-12.

8. The method for separating and enriching exosomes by paper-based isoelectric focusing according to claim 7, characterized in that: In step three, 0.2% hydroxyethyl cellulose (HEC) is also added to the positive electrode solution.

9. The method for separating and enriching exosomes by paper-based isoelectric focusing according to claim 8, characterized in that: In step 2, the reaction temperature is 35-40° C. and the reaction time is 16-24 hours.

10. The method for separating and enriching exosomes by paper-based isoelectric focusing according to claim 8, characterized in that: In step three, the DC voltage source is an electrophoresis apparatus, and the voltage of the DC voltage source is set to 0.2-1.5 kV.

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