CN114075506B - Urine exosome extraction reagent tube and manufacturing method thereof - Google Patents

Urine exosome extraction reagent tube and manufacturing method thereof Download PDF

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CN114075506B
CN114075506B CN202010848912.XA CN202010848912A CN114075506B CN 114075506 B CN114075506 B CN 114075506B CN 202010848912 A CN202010848912 A CN 202010848912A CN 114075506 B CN114075506 B CN 114075506B
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nanofiber membrane
chitosan
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plla
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沈宏伟
易路
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Second Xiangya Hospital of Central South University
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Abstract

The invention discloses a urine exosome extraction reagent tube and a manufacturing method thereof. The invention can complete the whole reaction in a short time without waiting for a long time; by utilizing the principle of positive and negative charge attraction, not only is no pollution caused by separating reagent, but also the uncharged lipoprotein with the particle size of 50-200nm in urine can be removed; the pore size of the positive charge porous nanofiber membrane is about 50-200nm, so that high-purity exosomes with particle sizes of 50-200nm can be obtained, and pollution of microvesicles with other particle sizes is prevented; the porous structure on the positively charged porous nanofiber membrane greatly improves the contact area between the membrane and a sample, and increases the extraction efficiency of urine exosomes.

Description

Urine exosome extraction reagent tube and manufacturing method thereof
Technical Field
The invention relates to the technical field of exosome extraction, in particular to a urine exosome extraction reagent tube and a manufacturing method thereof.
Background
Exosomes were first found in 1981, EGTrams et al found vesicles with membrane structures in sheep red blood cell supernatants cultured in vitro and named exosomes. For exosome effects, it was speculated at the time that it was a way for cells to excrete waste. In 1996 GRaposo et al found that immune cells similar to B lymphocytes also secrete antigen presenting exosomes (anti-tumor) which directly stimulate the anti-tumor response of effector CD4+ cells. HValadi et al further found that genetic material can be exchanged between cells by RNA in exosomes. With the increasing research on exosomes, researchers have found that it is widely involved in various biological processes such as immune responses in the body, antigen presentation, cell differentiation, tumor growth invasion, etc.
Almost all types of cells secrete exosomes, while exosomes are also widely present in body fluids, including blood, tears, urine, saliva, milk, ascites fluid, and the like. The substances such as nucleic acid (microRNA, lncRNA, circRNA, mRNA, tRNA and the like), protein, cholesterol and the like carried by the exosomes can be effectively wrapped by the exosomes double-layer membrane, so the exosomes can resist degradation of various factors, the exosomes stably exist in body fluid, along with the deep research of the exosomes in recent years, obvious differences exist between the exosomes in body fluid of tumor patients in early stages of cancer onset and biological substances carried by normal human exosomes in clinic, and the exosomes extraction in body fluid is expected to become a cancer early diagnosis technology.
The extraction of urine exosomes at the present stage mainly comprises a super-high-speed centrifugation method, a magnetic bead antibody capturing method, a hue column size exclusion method, a PEG precipitation column passing method and a positive charge capturing method. Ultra-high speed centrifugation: ultracentrifugation is currently the most commonly used means of exosome purification, whereby vesicles of the same size are precipitated from a sample and purified by high-speed centrifugation. It is necessary to centrifuge the pellet (containing exosomes) in a centrifuge at 100,000-200,000Xg for 120 minutes. Magnetic bead antibody capture method: phosphoserine (PS) specific binding of Tim4 anchored magnetic beads on exosome membrane was then isolated by elution buffer containing EDTA. Hue column size exclusion method: the exosomes are separated from other particle size proteins according to their particle size. PEG precipitation column method: the exosomes are precipitated by PEG, then removed by ion column and finally eluted with high salt solution. Positive charge trapping method: and (3) covering a positively charged coating on the solid phase carrier, and capturing negatively charged exosomes by positive and negative charge attraction.
The existing exosome technology is time-consuming (40 min-10 h is not equal), pollution of extraction reagent with different degrees exists (such as precipitation reagent residue in PEG precipitation method, such as residue of high-salt reagent in column passing method, such as pollution of magnetic bead anchor chain antibody in magnetic bead adsorption method and pollution of eluent EDTA), positive charge capture can not cause pollution of extraction reagent, but micro vesicles with particle size of more than 200nm and exosomes with particle size of 50-200nm can not be distinguished, extraction efficiency of hue column size exclusion method on urine exosomes is low, a large amount of macromolecular substances contained in urine easily block hue column, and are easily polluted by lipoprotein with particle size of about 100 nm.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and provides a urine exosome extraction reagent tube.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention relates to a urine exosome extraction reagent tube which comprises a cap, an outer tube and an inner tube, wherein the cap is sleeved on the top end surface of the outer tube in a threaded manner, the inner tube is sleeved in the outer tube, and a nanofiber membrane is arranged on the inner wall of the inner tube.
As a preferable technical scheme of the invention, the outer walls of the outer tube and the inner tube are provided with capacity scale marks, and the outer tube and the inner tube are made of polycarbonate materials.
The preparation method of the urine exosome extraction reagent tube provided by the invention comprises the following preparation steps:
a: dissolving PLLA with the molecular weight of 9 ten thousand and chitosan in a ratio of 5:1 in trifluoroethanol solution to prepare a mixed solution with the concentration of 8 weight percent, and fully dissolving the PLLA and chitosan mixed solution at the room temperature of 25-30 ℃;
b: advancing the syringe with a 27g needle at a high voltage of 8-10kV at a speed of 0.5-1.0ml/h, and receiving the nanofiber membrane with a resting plane;
c: putting the nanofiber membrane into a mixed solvent of ethanol and m-cresol, dissolving at room temperature of 25-30 ℃ for 60min, and drying;
d: soaking the membrane in 1.5mol/L sodium hydroxide methanol water solution for 30min to obtain a porous PLLA/chitosan nanofiber membrane;
e: adding 50mg of cationic polyacrylamide into 100ml of deionized water, heating to 50 ℃ and stirring, adjusting the pH value to 7.0 by NaOH, immersing a porous PLLA/chitosan nanofiber membrane into the solution, stirring at the room temperature of 25-30 ℃ for 24 hours, and drying;
f: irradiating for 3 hours by an ultraviolet lamp, and performing a crosslinking reaction on the cationic polyacrylamide and chitosan to obtain a positively charged porous PLLA/chitosan nanofiber membrane;
g: and (3) adhering the positively charged porous PLLA/chitosan nanofiber membrane on the inner wall of the inner tube, combining the inner tube 3 with the outer tube, and screwing a cap.
In the preparation method, the ratio of ethanol to m-cresol in the step C is 1:1.
In the preparation method, the ratio of methanol to water of the sodium hydroxide methanol aqueous solution in the step D is 1:1.
Compared with the prior art, the invention has the following beneficial effects:
the invention can complete the whole reaction in a short time without waiting for a long time; by utilizing the principle of positive and negative charge attraction, not only is no pollution caused by separating reagent, but also the uncharged lipoprotein with the particle size of 50-200nm in urine can be removed; the pore size of the positive charge porous nanofiber membrane is about 50-200nm, so that high-purity exosomes with particle sizes of 50-200nm can be obtained, and pollution of microvesicles with other particle sizes is prevented; the porous structure on the positively charged porous nanofiber membrane greatly improves the contact area between the membrane and a sample, and increases the extraction efficiency of urine exosomes.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:
FIG. 1 is a schematic view of the overall structure of the present invention;
FIG. 2 is a cross-sectional view of the structure of FIG. 1;
FIG. 3 is a flow chart of the positively charged porous PLLA/chitosan nanofiber membrane of the present invention;
FIG. 4 is a flow chart of urine exosome extraction of the present invention;
in the figure: 1. capping; 2. an outer tube; 3. an inner tube; 4. a nanofiber membrane.
Detailed Description
The preferred embodiments of the present invention will be described below with reference to the accompanying drawings, it being understood that the preferred embodiments described herein are for illustration and explanation of the present invention only, and are not intended to limit the present invention.
Example 1
As shown in fig. 1-4, the invention provides a urine exosome extraction reagent tube, which comprises a cap 1, an outer tube 2 and an inner tube 3, wherein the cap 1 is in threaded socket connection with the top surface of the outer tube 2, the inner tube 3 is in socket connection with the inner tube 2, and a nanofiber membrane 4 is arranged on the inner wall of the inner tube 3.
The outer walls of the outer tube 2 and the inner tube 3 are provided with capacity graduation marks, and the outer tube 2 and the inner tube 3 are made of polycarbonate materials.
The preparation method of the urine exosome extraction reagent tube comprises the following preparation steps:
a: dissolving PLLA with the molecular weight of 9 ten thousand and chitosan in a ratio of 5:1 in trifluoroethanol solution to prepare a mixed solution with the concentration of 8 weight percent, and fully dissolving the PLLA and chitosan mixed solution at the room temperature of 25-30 ℃;
b: advancing the syringe with a 27g needle at a high voltage of 8-10kV at a speed of 0.5-1.0ml/h, and receiving the nanofiber membrane with a resting plane;
c: putting the nanofiber membrane into a mixed solvent of ethanol and m-cresol, dissolving at room temperature of 25-30 ℃ for 60min, and drying;
d: soaking the membrane in 1.5mol/L sodium hydroxide methanol water solution for 30min to obtain a porous PLLA/chitosan nanofiber membrane;
e: adding 50mg of cationic polyacrylamide into 100ml of deionized water, heating to 50 ℃ and stirring, adjusting the pH value to 7.0 by NaOH, immersing a porous PLLA/chitosan nanofiber membrane into the solution, stirring at the room temperature of 25-30 ℃ for 24 hours, and drying;
f: irradiating for 3 hours by an ultraviolet lamp, and performing a crosslinking reaction on the cationic polyacrylamide and chitosan to obtain a positively charged porous PLLA/chitosan nanofiber membrane;
g: the positive charge porous PLLA/chitosan nanofiber membrane is adhered to the inner wall of the inner tube 3, and the cap 1 is screwed after the inner tube 3 and the outer tube 2 are combined.
Further, the ratio of ethanol to m-cresol in step C is 1:1.
In the step D, the ratio of methanol to water of the sodium hydroxide methanol aqueous solution is 1:1.
Specifically, when in use, the nanofiber membrane 4 on the inner wall of the inner tube 3 is a positively charged porous nanofiber membrane, firstly, 5ml of phosphate buffer solution is used for washing the nanofiber membrane 4 once, then 10ml of urine is sprayed on the nanofiber membrane 4 to cover the cover cap 1, the centrifugation is carried out for 1min to remove waste liquid, then 500ul of phosphate buffer solution is added to lightly wash the membrane once, the washing solution is sucked and removed to remove microvesicle components adhered on the membrane, finally 500ul of phosphate buffer solution is added to repeatedly wash the nanofiber membrane for 4 five times, the centrifugation is carried out for 1min at 1000rpm, and the membrane washing solution (the exosome with the diameter of 50-200 nm) is collected.
The invention can complete the whole reaction in a short time without waiting for a long time; by utilizing the principle of positive and negative charge attraction, not only is no pollution caused by separating reagent, but also the uncharged lipoprotein with the particle size of 50-200nm in urine can be removed; the pore size of the positive charge porous nanofiber membrane is about 50-200nm, so that high-purity exosomes with particle sizes of 50-200nm can be obtained, and pollution of microvesicles with other particle sizes is prevented; the porous structure on the positively charged porous nanofiber membrane greatly improves the contact area between the membrane and a sample, and increases the extraction efficiency of urine exosomes.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (4)

1. The manufacturing method of the urine exosome extraction reagent tube is characterized by comprising a cap (1), an outer tube (2) and an inner tube (3), wherein the cap (1) is sleeved on the top end surface of the outer tube (2) in a threaded manner, the inner tube (3) is sleeved in the outer tube (2), and a nanofiber membrane (4) is arranged on the inner wall of the inner tube (3), and the manufacturing steps are as follows:
a: dissolving PLLA with the molecular weight of 9 ten thousand and chitosan in a ratio of 5:1 in trifluoroethanol solution to prepare a mixed solution with the concentration of 8 weight percent, and fully dissolving the PLLA and chitosan mixed solution at the room temperature of 25-30 ℃;
b: advancing the syringe with a 27g needle at a high voltage of 8-10kV at a speed of 0.5-1.0ml/h, and receiving the nanofiber membrane with a resting plane;
c: putting the nanofiber membrane into a mixed solvent of ethanol and m-cresol, dissolving at room temperature of 25-30 ℃ for 60min, and drying;
d: soaking the membrane in 1.5mol/L sodium hydroxide methanol water solution for 30min to obtain a porous PLLA/chitosan nanofiber membrane;
e: adding 50mg of cationic polyacrylamide into 100ml of deionized water, heating to 50 ℃ and stirring, adjusting the pH value to 7.0 by NaOH, immersing a porous PLLA/chitosan nanofiber membrane into the solution, stirring at the room temperature of 25-30 ℃ for 24 hours, and drying;
f: irradiating for 3 hours by an ultraviolet lamp, and performing a crosslinking reaction on the cationic polyacrylamide and chitosan to obtain a positively charged porous PLLA/chitosan nanofiber membrane;
g: the positive charge porous PLLA/chitosan nanofiber membrane is adhered to the inner wall of the inner tube (3), and the inner tube (3) and the outer tube (2) are combined and then the cap (1) is screwed on.
2. The method for manufacturing the urine exosome extraction reagent tube according to claim 1, wherein capacity scale marks are arranged on the outer walls of the outer tube (2) and the inner tube (3), and the outer tube (2) and the inner tube (3) are made of polycarbonate materials.
3. The method of claim 1, wherein the ratio of ethanol to m-cresol in step C is 1:1.
4. The method of claim 1, wherein the ratio of methanol to water in the aqueous solution of sodium hydroxide and methanol in step D is 1:1.
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CN107254430A (en) * 2017-08-11 2017-10-17 上海浦美生物医药科技有限公司 A kind of method based on positive charge adsorbing separation excretion body
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