CN114075506A - 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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CN114075506A
CN114075506A CN202010848912.XA CN202010848912A CN114075506A CN 114075506 A CN114075506 A CN 114075506A CN 202010848912 A CN202010848912 A CN 202010848912A CN 114075506 A CN114075506 A CN 114075506A
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nanofiber membrane
chitosan
tube
urine
inner tube
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CN114075506B (en
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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 using the positive and negative charge attraction principle, the method not only has no pollution of a separation reagent, but also can remove uncharged lipoprotein with the particle size of 50-200nm in urine; the pore size on the positive charge porous nanofiber membrane is about 50-200nm, so that exosomes with high purity and particle size of 50-200nm can be obtained, and pollution of microvesicles with other particle sizes is prevented; the porous structure on the positive charge 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, and membrane-structured vesicles were found in supernatants of sheep erythrocytes cultured in vitro by EGTrams et al, and were named exosomes. For the action of exosomes, it was speculated at that time as a way for cells to excrete waste. In 1996, GRaposo et al found that immune cells similar to B lymphocytes also secreted antigen presenting exosomes (antigen presenting exosomes) that directly stimulated the anti-tumor response of effector CD4+ cells. HValadi et al further discovered in 2007 that genetic material could be exchanged between cells via RNA in exosomes. With the increasing research on exosomes, researchers find that it is widely involved in various biological processes such as immune response, antigen presentation, cell differentiation, tumor growth and invasion.
Almost all types of cells secrete exosomes, and exosomes are also widely present in body fluids, including blood, tears, urine, saliva, milk, ascites, and the like. Nucleic acid (microRNA, lncRNA, circRNA, mRNA, tRNA and the like), protein, cholesterol and other substances carried by exosome can be effectively wrapped by an exosome double-layer membrane, so that the exosome can resist the degradation of various factors, and can stably exist in body fluid.
At the present stage, urine exosomes are mainly extracted by an ultra-high speed centrifugation method, a magnetic bead antibody capture method, a hue column size exclusion method, a PEG precipitation column passing method and a positive charge capture method. Ultra-high speed centrifugation: ultracentrifugation is the most common purification method for exosome at present, and vesicles of the same size are precipitated and purified from a sample by high-speed centrifugation. The pellet (containing exosomes) was centrifuged at 100,000-. Magnetic bead antibody capture method: phosphoesterylserine (PS) on the exosome membrane specifically binds to the Tim4 anchored magnetic beads, and is separated by elution buffer containing EDTA. Size exclusion chromatography on chromatography column: exosomes are separated from other particle size proteins according to their particle size. PEG precipitation column-passing method: the exosomes were precipitated by PEG, removed by ion column and finally eluted with high salt solution. Positive charge trapping method: and covering a positively charged coating on the solid phase carrier, and capturing the negatively charged exosomes through positive and negative charge attraction.
The existing exosome technology is time-consuming (40min-10 hours are different), the extraction reagents are polluted to different degrees (such as residual precipitating reagents in a PEG precipitation method, such as residual high-salt reagents in a column passing method, such as pollution of magnetic bead anchor chain antibodies in a magnetic bead adsorption method and pollution of eluent EDTA), positive charge capture does not cause pollution of the extraction reagents, but cannot distinguish microvesicles with the particle size of more than 200nm from exosomes with the particle size of 50-200nm, the extraction efficiency of urine exosomes by a hue column size exclusion method is low, a large amount of macromolecular substances contained in urine easily block a hue column, and the urine is easily polluted by lipoprotein with the particle size of about 100 nm.
Disclosure of Invention
The technical problem to be solved by the present invention is to overcome the defects of the prior art and to provide a urine exosome extraction reagent tube, and another object of the present invention is to provide a manufacturing method for manufacturing the 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 inside 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 pipe and the inner pipe are both provided with capacity scale marks, and the outer pipe and the inner pipe are both made of polycarbonate materials.
The invention provides a preparation method for preparing the urine exosome extraction reagent tube, which comprises the following preparation steps:
a: dissolving PLLA with the molecular weight of 9 ten thousand and chitosan in trifluoroethanol solution according to the proportion of 5:1 to prepare mixed solution with the concentration of 8 wt%, and fully dissolving the mixed solution of the PLLA and the chitosan at the room temperature of 25-30 ℃;
b: pushing the injector with a 27g needle at a speed of 0.5-1.0 ml/h under a high voltage of 8-10kV, and receiving the nanofiber membrane with a standing plane;
c: putting the nanofiber membrane into a mixed solvent of ethanol and m-cresol, dissolving and removing for 60min at the room temperature of 25-30 ℃, and drying;
d: soaking the membrane in 1.5mol/L sodium hydroxide methanol aqueous solution for 30min to obtain porous PLLA/chitosan nanofiber membrane;
e: adding 50mg of cationic polyacrylamide into 100ml of deionized water, heating to 50 ℃, stirring, adjusting the pH value of a solution to be 7.0 by using NaOH, immersing the porous PLLA/chitosan nanofiber membrane into the solution, stirring for 24 hours at the room temperature of 25-30 ℃, and drying;
f: irradiating for 3 hours by using an ultraviolet lamp, and performing crosslinking reaction on the cationic polyacrylamide and chitosan to obtain a porous PLLA/chitosan nanofiber membrane with positive charges;
g: and adhering the positive charge porous PLLA/chitosan nanofiber membrane on the inner wall of the inner tube, combining the inner tube 3 and the outer tube, and screwing the cap on the inner tube.
In the above production method, the ratio of ethanol to m-cresol in step C is 1: 1.
In the above manufacturing method, the ratio of methanol to water in the aqueous solution of sodium hydroxide and methanol in 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 using the positive and negative charge attraction principle, the method not only has no pollution of a separation reagent, but also can remove uncharged lipoprotein with the particle size of 50-200nm in urine; the pore size on the positive charge porous nanofiber membrane is about 50-200nm, so that exosomes with high purity and particle size of 50-200nm can be obtained, and pollution of microvesicles with other particle sizes is prevented; the porous structure on the positive charge 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, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit 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 fabrication 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 in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.
Example 1
As shown in fig. 1 to 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 sleeved on the top end surface of the outer tube 2 in a threaded manner, the inner tube 3 is sleeved inside the outer tube 2, and a nanofiber membrane 4 is arranged on the inner wall of the inner tube 3.
The outer walls of the outer pipe 2 and the inner pipe 3 are both provided with capacity scale marks, and the outer pipe 2 and the inner pipe 3 are both made of polycarbonate materials.
The preparation method of the urine exosome extraction reagent tube comprises the following steps:
a: dissolving PLLA with the molecular weight of 9 ten thousand and chitosan in trifluoroethanol solution according to the proportion of 5:1 to prepare mixed solution with the concentration of 8 wt%, and fully dissolving the mixed solution of the PLLA and the chitosan at the room temperature of 25-30 ℃;
b: pushing the injector with a 27g needle at a speed of 0.5-1.0 ml/h under a high voltage of 8-10kV, and receiving the nanofiber membrane with a standing plane;
c: putting the nanofiber membrane into a mixed solvent of ethanol and m-cresol, dissolving and removing for 60min at the room temperature of 25-30 ℃, and drying;
d: soaking the membrane in 1.5mol/L sodium hydroxide methanol aqueous solution for 30min to obtain porous PLLA/chitosan nanofiber membrane;
e: adding 50mg of cationic polyacrylamide into 100ml of deionized water, heating to 50 ℃, stirring, adjusting the pH value of a solution to be 7.0 by using NaOH, immersing the porous PLLA/chitosan nanofiber membrane into the solution, stirring for 24 hours at the room temperature of 25-30 ℃, and drying;
f: irradiating for 3 hours by using an ultraviolet lamp, and performing crosslinking reaction on the cationic polyacrylamide and chitosan to obtain a porous PLLA/chitosan nanofiber membrane with positive charges;
g: adhering the positive charge porous PLLA/chitosan nanofiber membrane on the inner wall of the inner tube 3, combining the inner tube 3 and the outer tube 2, and screwing the cap 1.
Further, the ratio of ethanol to m-cresol in step C was 1: 1.
In step D, the ratio of methanol to water in the sodium hydroxide methanol aqueous solution is 1: 1.
Specifically, the nanofiber membrane 4 on the inner wall of the inner tube 3 is a positive charge porous nanofiber membrane, when the nanofiber membrane is used, 5ml of phosphate buffer solution is firstly used for washing the nanofiber membrane 4 once, 10ml of urine is sprayed on the nanofiber membrane 4 to cover the cap 1, centrifugation is carried out for 1min at the rotating speed of 1000rpm to remove waste liquid, 500ul of phosphate buffer solution is added for lightly washing the membrane once, washing liquid is sucked and discarded to remove micro-capsule components which are adhered to the membrane and are larger than 200nm, finally 500ul of phosphate buffer solution is added for repeatedly washing the nanofiber membrane for 4 times, centrifugation is carried out for 1min at the rotating speed of 1000rpm, and membrane washing liquid (exosomes 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 using the positive and negative charge attraction principle, the method not only has no pollution of a separation reagent, but also can remove uncharged lipoprotein with the particle size of 50-200nm in urine; the pore size on the positive charge porous nanofiber membrane is about 50-200nm, so that exosomes with high purity and particle size of 50-200nm can be obtained, and pollution of microvesicles with other particle sizes is prevented; the porous structure on the positive charge 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: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (5)

1. The utility model provides a urine exosome draws reagent pipe, includes cap (1), outer tube (2) and inner tube (3), cap (1) has been cup jointed to the top surface screw thread of outer tube (2), inner tube (3) have been cup jointed to the inside of outer tube (2), its characterized in that, the inner wall of inner tube (3) is provided with nanofiber membrane (4).
2. The urine exosome extraction reagent tube and the manufacturing method thereof according to claim 1, characterized in that the outer walls of the outer tube (2) and the inner tube (3) are provided with capacity scale marks, and the outer tube (2) and the inner tube (3) are made of polycarbonate materials.
3. The method for preparing a reagent tube for extracting urine exosomes according to claim 1, which comprises the following steps:
a: dissolving PLLA with the molecular weight of 9 ten thousand and chitosan in trifluoroethanol solution according to the proportion of 5:1 to prepare mixed solution with the concentration of 8 wt%, and fully dissolving the mixed solution of the PLLA and the chitosan at the room temperature of 25-30 ℃;
b: pushing the injector with a 27g needle at a speed of 0.5-1.0 ml/h under a high voltage of 8-10kV, and receiving the nanofiber membrane with a standing plane;
c: putting the nanofiber membrane into a mixed solvent of ethanol and m-cresol, dissolving and removing for 60min at the room temperature of 25-30 ℃, and drying;
d: soaking the membrane in 1.5mol/L sodium hydroxide methanol aqueous solution for 30min to obtain porous PLLA/chitosan nanofiber membrane;
e: adding 50mg of cationic polyacrylamide into 100ml of deionized water, heating to 50 ℃, stirring, adjusting the pH value of a solution to be 7.0 by using NaOH, immersing the porous PLLA/chitosan nanofiber membrane into the solution, stirring for 24 hours at the room temperature of 25-30 ℃, and drying;
f: irradiating for 3 hours by using an ultraviolet lamp, and performing crosslinking reaction on the cationic polyacrylamide and chitosan to obtain a porous PLLA/chitosan nanofiber membrane with positive charges;
g: and (3) adhering the positive charge porous PLLA/chitosan nanofiber membrane to the inner wall of the inner tube (3), combining the inner tube (3) and the outer tube (2), and screwing the cap (1).
4. The method as claimed in claim 3, wherein the ratio of ethanol to m-cresol in step C is 1: 1.
5. The method as claimed in claim 3, wherein the ratio of methanol to water in the aqueous solution of NaOH in methanol in step D is 1: 1.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115786107A (en) * 2022-12-14 2023-03-14 深圳市寰宇生物科技有限公司 Efficient preparation device and method for exosome

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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
CN209485829U (en) * 2018-08-16 2019-10-11 南方医科大学 The device of the workable enrichment urine excretion body of hospital and family

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CN106283396A (en) * 2016-08-22 2017-01-04 王利萍 A kind of preparation method of porous chitosan fibrous membrane
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
CN209485829U (en) * 2018-08-16 2019-10-11 南方医科大学 The device of the workable enrichment urine excretion body of hospital and family

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115786107A (en) * 2022-12-14 2023-03-14 深圳市寰宇生物科技有限公司 Efficient preparation device and method for exosome

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