CN114075506B - Urine exosome extraction reagent tube and production method - Google Patents

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 production method

Technical field

The present invention relates to the technical field of exosome extraction, and in particular to a urine exosome extraction reagent tube and a production method.

Background technique

Exosomes were first seen in 1981. EG Trams et al. discovered small vesicles with a membrane structure in the supernatant of sheep red blood cells cultured in vitro and named them exosomes. As for the role of exosomes, it was speculated that they were a way for cells to excrete waste. In 1996, GRaposo et al. discovered that immune cells similar to B lymphocytes also secrete antigen-presenting exosomes (antigenpresentingvesicles). The secreted exosomes can directly stimulate the anti-tumor response of effector CD4+ cells. In 2007, HValadi et al. further discovered that cells can exchange genetic material through RNA in exosomes. As more and more studies are conducted on exosomes, researchers have found that they are widely involved in various biological processes such as the body's immune response, antigen presentation, cell differentiation, tumor growth and invasion, etc.

Almost all types of cells can secrete exosomes, and exosomes are also widely found in body fluids, including blood, tears, urine, saliva, breast milk, ascites, etc. The nucleic acids (microRNA, lncRNA, circRNA, mRNA, tRNA, etc.), proteins, cholesterol and other substances carried by exosomes can be effectively wrapped by the double-layer membrane of exosomes, so they can resist degradation by various factors, allowing exosomes to remain in body fluids With the in-depth research on exosomes in recent years, it has been clinically discovered that in the early stages of cancer, there are significant differences in the biological substances carried by exosomes in the body fluids of tumor patients and those carried by normal human exosomes. Therefore, The extraction of exosomes from body fluids is expected to become an early diagnosis technology for cancer. Due to the particularity of the human body structure, exosomes in human urine can be used as new biomarkers for clinical detection of kidney disease, prostate disease, and bladder disease.

At present, the main methods for extracting urinary exosomes include ultra-high-speed centrifugation, magnetic bead antibody capture method, color column size exclusion method, PEG precipitation column method, and positive charge capture method. Ultra-high-speed centrifugation: Ultra-fast centrifugation is currently the most commonly used exosome purification method. Vesicles of the same size are precipitated and purified from the sample through high-speed centrifugation. The pellet (containing exosomes) needs to be centrifuged in a centrifuge at 100,000-200,000xg for 120 minutes. Magnetic bead antibody capture method: Phosphatidylserine (PS) specifically binds Tim4-anchored magnetic beads on the exosome membrane, and then separates through an elution buffer containing EDTA. Color column size exclusion method: Separate exosomes and proteins of other particle sizes based on the particle size of exosomes. PEG precipitation column method: Precipitate exosomes with PEG, remove PEG through an ion column, and finally elute exosomes with high-salt solution. Positive charge capture method: a solid-phase carrier is covered with a positively charged coating, and then negatively charged exosomes are captured through positive and negative charge attraction.

Existing exosome technologies are time-consuming (ranging from 40 minutes to 10 hours), and there are varying degrees of contamination of the extraction reagents (such as residues of precipitation reagents in the PEG precipitation method, residues of high-salt reagents in the column method, such as magnetic In the bead adsorption method, the contamination of the anchor chain antibody of the magnetic beads and the contamination of the eluent EDTA), although positive charge capture does not cause contamination of the extraction reagent, it will affect microvesicles with a particle size greater than 200nm and particles with a particle size of 50-200nm. Exosomes cannot be distinguished. The extraction efficiency of urine exosomes by the color column size exclusion method is low. A large amount of macromolecules contained in urine can easily block the color column and be easily contaminated by lipoproteins with a particle size of about 100 nm. .

Contents of the invention

The technical problem to be solved by the present invention is to overcome the defects of the prior art and provide a urine exosome extraction reagent tube. Another object of the present invention is to provide a method for making the above-mentioned urine exosome extraction reagent tube.

In order to solve the above technical problems, the present invention provides the following technical solutions:

A urine exosome extraction reagent tube of the present invention includes a cap, an outer tube and an inner tube. The top surface of the outer tube is threaded with a cap, and the inside of the outer tube is threaded with an inner tube. The inner wall of the tube is provided with a nanofiber membrane.

As a preferred technical solution of the present invention, the outer walls of the outer tube and the inner tube are both provided with capacity scales, and both the outer tube and the inner tube are made of polycarbonate material.

The invention provides a method for making the above-mentioned urine exosome extraction reagent tube. The making steps are as follows:

A: Dissolve PLLA with a molecular weight of 90,000 and chitosan in a trifluoroethanol solution at a ratio of 5:1 to prepare a mixed solution with a concentration of 8wt%, and fully dissolve PLLA and chitosan at room temperature of 25-30°C. polysaccharide mixture;

B: Use a 27g needle at a high voltage of 8-10kV to advance the syringe at a speed of 0.5-1.0ml/h, and use a static surface to receive the nanofiber membrane;

C: Put the nanofiber membrane into a mixed solvent of ethanol and m-cresol, dissolve it at room temperature of 25-30°C for 60 minutes and then dry it;

D: Soak the membrane in 1.5 mol/L sodium hydroxide methanol aqueous solution for 30 minutes to obtain a porous PLLA/chitosan nanofiber membrane;

E: Add 50 mg of cationic polyacrylamide to 100 ml of deionized water, heat to 50°C and stir, use NaOH to adjust the solution to pH=7.0, immerse the porous PLLA/chitosan nanofiber membrane into the solution, and wait for 25-30 After stirring at room temperature of ℃ for 24 hours, dry;

F: Use UV light for 3 hours to cross-link the cationic polyacrylamide and chitosan to obtain a positively charged porous PLLA/chitosan nanofiber membrane;

G: Adhere the positively charged porous PLLA/chitosan nanofiber membrane to the inner wall of the inner tube, combine the inner tube 3 with the outer tube and screw on the cap.

In the above production method, the ratio of ethanol and m-cresol in step C is 1:1.

In the above production method, the ratio of methanol to water in the sodium hydroxide methanol aqueous solution in step D is 1:1.

Compared with the prior art, the beneficial effects of the present invention are as follows:

The present invention can complete the entire reaction in a short time without waiting for a long time; by utilizing the principle of positive and negative charge attraction, not only is there no pollution from the separation reagent, but it can also remove uncharged lipoproteins with a particle size of 50-200 nm in urine. ; The pore size on the positively charged porous nanofiber membrane is about 50-200nm, and high-purity exosomes with a particle size of 50-200nm can be obtained to prevent contamination of microvesicles with other particle sizes; Positively charged porous nanofiber membrane The porous structure greatly increases the contact area between the membrane and the sample, increasing the extraction efficiency of urine exosomes.

Description of the drawings

The drawings are used to provide a further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention. In the attached picture:

Figure 1 is a schematic diagram of the overall structure of the present invention;

Figure 2 is a cross-sectional view of the structure of Figure 1;

Figure 3 is a production flow chart of the positively charged porous PLLA/chitosan nanofiber membrane of the present invention;

Figure 4 is a flow chart of urine exosome extraction according to the present invention;

In the picture: 1. Cap; 2. Outer tube; 3. Inner tube; 4. Nanofiber membrane.

Detailed ways

The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described here are only used to illustrate and explain the present invention, and are not intended to limit the present invention.

Example 1

As shown in Figures 1-4, the present invention provides a urine exosome extraction reagent tube, which includes a cap 1, an outer tube 2 and an inner tube 3. The top surface of the outer tube 2 is threaded with the cap 1, and the outer tube 2 An inner tube 3 is sleeved inside, and a nanofiber membrane 4 is provided on the inner wall of the inner tube 3 .

The outer walls of the outer tube 2 and the inner tube 3 are both provided with capacity scales, and both the outer tube 2 and the inner tube 3 are made of polycarbonate material.

Urine exosome extraction reagent tube production method, the production steps are as follows:

A: Dissolve PLLA with a molecular weight of 90,000 and chitosan in a trifluoroethanol solution at a ratio of 5:1 to prepare a mixed solution with a concentration of 8wt%, and fully dissolve PLLA and chitosan at room temperature of 25-30°C. polysaccharide mixture;

B: Use a 27g needle at a high voltage of 8-10kV to advance the syringe at a speed of 0.5-1.0ml/h, and use a static surface to receive the nanofiber membrane;

C: Put the nanofiber membrane into a mixed solvent of ethanol and m-cresol, dissolve it at room temperature of 25-30°C for 60 minutes and then dry it;

D: Soak the membrane in 1.5 mol/L sodium hydroxide methanol aqueous solution for 30 minutes to obtain a porous PLLA/chitosan nanofiber membrane;

E: Add 50 mg of cationic polyacrylamide to 100 ml of deionized water, heat to 50°C and stir, use NaOH to adjust the solution to pH=7.0, immerse the porous PLLA/chitosan nanofiber membrane into the solution, and wait for 25-30 After stirring at room temperature of ℃ for 24 hours, dry;

F: Use UV light for 3 hours to cross-link the cationic polyacrylamide and chitosan to obtain a positively charged porous PLLA/chitosan nanofiber membrane;

G: Adhere the positively charged porous PLLA/chitosan nanofiber membrane to the inner wall of the inner tube 3, combine the inner tube 3 and the outer tube 2 and screw on the cap 1.

Further, the ratio of ethanol and m-cresol in step C is 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 positively charged porous nanofiber membrane. When used, the nanofiber membrane 4 is washed once with 5 ml of phosphate buffer, and then 10 ml of urine is sprayed on the nanofiber membrane 4 to cap it. 1. Close the lid and centrifuge for 1 minute at 1000 rpm to remove the waste liquid. Then add 500ul of phosphate buffer to gently wash the membrane once. Aspirate and discard the washing liquid to remove microvesicles larger than 200nm adhered to the membrane. Finally, add 500 ul of phosphate buffer to wash the nanofiber membrane 4 times and five times, centrifuge for 1 minute at 1000 rpm, and collect the membrane washing fluid (exosomes with a diameter of 50-200 nm).

The present invention can complete the entire reaction in a short time without waiting for a long time; by utilizing the principle of positive and negative charge attraction, not only is there no pollution from the separation reagent, but it can also remove uncharged lipoproteins with a particle size of 50-200 nm in urine. ; The pore size on the positively charged porous nanofiber membrane is about 50-200nm, and high-purity exosomes with a particle size of 50-200nm can be obtained to prevent contamination of microvesicles with other particle sizes; Positively charged porous nanofiber membrane The porous structure greatly increases the contact area between the membrane and the sample, increasing the extraction efficiency of urine exosomes.

Finally, it should be noted that the above are only preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, it is still The technical solutions described in the foregoing embodiments may be modified, or some of the technical features may be equivalently replaced. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall 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.