Exosome separation device, preparation method thereof and exosome separation and purification method

Abstract

The invention discloses an exosome separation device, a preparation method thereof and an exosome separation and purification method in the technical field of biomedical detection. The device comprises a micro-fluidic chip and an electrochemical workstation electrically connected with the micro-fluidic chip; the micro-fluidic chip comprises a substrate defining a fluid channel and a three-electrode assembly embedded in the inner wall of the fluid channel. The substrate is made of polydimethylsiloxane, and electrodes in the three-electrode assembly are carbon fiber electrodes modified with a zirconium-based metal organic framework. The Zr-MOF is modified on the surface of the electrode, the exosome is captured by the affinity of the Zr-MOF, elution and collection are realized by combining an electrochemical method, and the method has the advantages of high efficiency, rapidness, easiness in control and the like; the immunoaffinity of a specific antibody, the phosphoric acid affinity of a Zr-MOF modified electrode, an amphiphilic strategy and an electrochemical method are combined, so that the exosome separation specificity and separation efficiency are remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical detection technology, specifically to the field of microfluidic electrochemical technology, and more specifically to an exosome separation device and a preparation method thereof, as well as an exosome separation and purification method. Background Art

[0002] Exosomes are vesicles secreted by living cells, typically ranging in size from 30 to 200 nm. Exosomes contain a rich array of substances, including nucleic acids, proteins, and lipids, which participate in intercellular molecular transport. They are widely present in cell culture fluids and various body fluids, such as blood and lymph. Exosome research plays a crucial role in biomedical testing. By analyzing the molecular composition and function of exosomes, new biomarkers can be discovered, aiding in the early diagnosis and treatment of diseases.

[0003] However, the difficulty of isolating and extracting exosomes has hindered their development in diagnostic and therapeutic applications. Current methods for isolating exosomes primarily include ultracentrifugation, ultrafiltration, immunoaffinity capture, charge-neutralizing polymer precipitation, size-exclusion chromatography, and microfluidics. Ultracentrifugation, ultrafiltration, and size-exclusion chromatography are time-consuming, yield low purity, and require complex equipment. While immunoaffinity capture offers high specificity, it suffers from low throughput and yield. Microfluidic chip strategies combined with external driving forces can effectively isolate exosomes, but throughput remains low and chip fabrication is complex. Due to the complexity of biological fluid environments, the significant overlap in physicochemical and biochemical properties between exosomes, lipoproteins, viruses, and other extracellular vesicles, and the inherent heterogeneity of exosomes, no single exosome isolation technique is currently considered suitable for all studies. Summary of the Invention

[0004] The purpose of the present invention is to address the above shortcomings and provide an exosome separation device and its preparation method and an exosome separation and purification method. Based on the principle of microfluidic electrochemistry, a dual affinity strategy is adopted to achieve efficient, high-throughput, high-purity and portable separation and capture of exosomes.

[0005] To achieve the above object, the present invention is implemented through the following technical solutions: In a first aspect, the present invention provides an exosome separation device, comprising a microfluidic chip and an electrochemical workstation electrically connected thereto; the microfluidic chip comprises a matrix forming a fluid channel and a three-electrode assembly embedded in the inner wall of the fluid channel; the matrix is made of polydimethylsiloxane (PDMS), and the electrodes in the three-electrode assembly are all carbon fiber electrodes modified with a zirconium-based metal-organic framework (Zr-MOF).

[0006] Furthermore, the three-electrode assembly includes a working electrode, a counter electrode, and a reference electrode; the working electrode and the reference electrode are arranged on the same side of the fluid channel, and the counter electrode is arranged on the side opposite to the working electrode; the surface of the reference electrode is provided with an Ag / AgCl (60 / 40% m / m) coating, and the thickness of the Ag / AgCl coating is 2~3 mm.

[0007] Preferably, the working electrode has a length of 30~75mm, a width of 1~30mm, and a thickness of 0.1~0.3mm; the counter electrode has a length of 35~80mm, a width of 1~30mm, and a thickness of 0.1~0.3mm; the reference electrode has a length of 1~5mm, a width of 1~30mm, and a thickness of 0.1~0.3mm (excluding the Ag / AgCl layer).

[0008] Furthermore, the fluid channel is also connected with an input tube and an output tube for injecting sample solution, elution solution, etc.; the input tube is arranged at one end close to the reference electrode, and the output tube is arranged at one end away from the reference electrode.

[0009] In a second aspect, the present invention provides a method for preparing the exosome separation device according to the first aspect, comprising: Preparation of carbon fiber electrodes modified with zirconium-based metal-organic frameworks; constructing a microfluidic chip embedded with a three-electrode assembly; Connect each electrode in the microfluidic chip to an electrochemical workstation.

[0010] Furthermore, the preparation of the carbon fiber electrode modified with a zirconium-based metal-organic framework comprises: a. ZrCl4 is dissolved in a mixed solvent of formic acid and ethanol to form a metal solution; b. The NH2-BDC was dissolved in a mixed solution containing formic acid and pure water, and a homogeneous ligand solution was obtained by ultrasonic treatment; c. The carbon cloth was immersed in a metal solution and then in a ligand solution. After rinsing with ethanol, a carbon fiber electrode modified with a zirconium-based metal-organic framework (UiO / CF) was obtained.

[0011] Preferably, in step a, the volume ratio of formic acid to ethanol is 1:(2-4), and the concentration of ZrCl4 in the metal solution is 1-10 mM; in step b, the volume ratio of formic acid to pure water is 1:(1-2), and the concentration of NH2-BDC in the homogeneous ligand solution is 1-3 mM.

[0012] Preferably, the carbon cloth is immersed in the metal solution for 1 to 2 hours, immersed in the ligand solution for 4 to 5 hours, and ultrasonically treated for 1 to 2 hours.

[0013] Furthermore, the fluid channel in the microfluidic chip is prepared by a molding method, comprising: Three electrodes are pasted into two half-channel molds and a needle with a flexible tube is installed; the reference electrode is coated with an Ag / AgCl layer; The polydimethylsiloxane and the curing agent are stirred evenly and then vacuum treated to prepare a matrix mixed solution; The two half-channel molds with electrodes attached are merged, and the matrix mixed solution is poured into the assembled molds and solidified.

[0014] Preferably, the half-channel mold is washed with ethanol before use and then dried in an oven at 40° C.; the reference electrode is coated with an Ag / AgCl layer before pasting and then dried in an oven at 80° C.

[0015] Preferably, the mass ratio of the polydimethylsiloxane to the curing agent is (8-15):1, and the curing agent can be a commonly used curing agent prepared by microfluidic chips in the prior art; the curing treatment temperature is 70°C-90°C, and the time is 1-2 hours.

[0016] In a third aspect, the present invention further provides a method for separating and purifying exosomes, based on the exosome separation device described in the first aspect, the method comprising: Injecting the specific antibody solution into the fluid channel of the exosome isolation device and pushing it back and forth; The sample solution containing exosomes is injected into the fluid channel of the exosome separation device and pushed back and forth; Remove the remaining solution in the fluid channel and inject PBS buffer into the fluid channel; An electric field is applied to the working electrode by an electrochemical workstation, and the PBS buffer is pushed back and forth during the action time of the electric field; The electrochemical workstation then applies an electric field to the counter electrode, pushing the PBS buffer back and forth during the electric field action time.

[0017] Preferably, the intensity of the electric field is -0.5V to -3V; and the action time of the electric field is 15s to 180s.

[0018] Preferably, the specific antibody solution is a CD9 antibody solution or a CD63 antibody solution.

[0019] In the present invention, pushing back and forth in the fluid channel means injecting the reagent from the input tube at one end, allowing it to flow into the fluid channel, and then being sucked out from the output tube at the other end, which is considered one push.

[0020] Preferably, the concentration of the specific antibody solution is 20-25 μg / mL, and the sample is pushed back and forth through the fluid channel 5 times every 10 minutes for 1 hour.

[0021] Preferably, the exosome-containing sample solution is injected into the fluid channel at a rate of 1 mL / s and pushed back and forth 10 times. The exosome-containing sample solution is a pre-treated exosome-containing solution sample, such as lung cancer cell culture fluid, wherein the pre-treatment includes removing impurities such as cell debris.

[0022] Preferably, the PBS buffer is injected into the fluid channel at a rate of 0.3 mL / s.

[0023] Furthermore, when using the exosome isolation device, the microfluidic chip is placed between two custom acrylic plates. Screws are inserted through holes in the acrylic plates to secure the chip between the plates. The screws and nuts are tightened evenly to ensure uniform clamping of the chip while avoiding overtightening that could damage the chip or clog the microchannels. After securing, the microchannels are inspected for patency and to ensure no blockage. The entire device is then tested for leaks to ensure experimental accuracy.

[0024] Preferably, the three electrodes can be connected to an electrochemical workstation using three steel needles and alligator clips; after the connection, cyclic voltammetry can be used for quality detection, that is, the first five cycles of cyclic voltammetry are performed between -0.1 V and +0.1 V. If a current of μA level is generated, the stability of the system is confirmed.

[0025] Compared with the prior art, the present invention has the following beneficial effects: The exosome separation device provided by the present invention modifies the electrode surface with Zr-MOF, utilizes its affinity to capture exosomes, and combines it with electrochemical methods to achieve elution and collection, with the advantages of high efficiency, rapidity, and easy control. The device integrates Zr-MOF-modified electrodes and an electrochemical control system to achieve automated and high-throughput separation and purification of exosomes, simplifying the operation process, improving separation efficiency, and reducing human error. The device is small in size, has low energy consumption, and can be used in field conditions, with important clinical significance and application value. The exosome separation and purification method described in the present invention combines the immunoaffinity of specific antibodies with the phosphate affinity of Zr-MOF modified electrodes, and the dual affinity strategy is combined with electrochemical methods to significantly improve the specificity and separation efficiency of exosome separation. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 A schematic structural diagram of a microfluidic chip in an exosome separation device provided in an embodiment of the present invention; Figure 2 This is a comparison diagram of the scanning electron microscope (SEM) images of the electrodes described in the examples; Figure 3 This is a specification diagram of the acrylic plate fixture described in the embodiment; Figure 4This is a working principle diagram of the exosome separation and purification method described in the examples; Figure 5 The cyclic voltammetry curve for monitoring the changes in the electrode surface as described in the embodiment; Figure 6 Nanoparticle tracking analysis (NTA) results of exosomes obtained using the exosome separation and purification method provided in the examples. (a) shows a control CF electrode without Zr-MOF modification, (b) shows a modified 2-UiO / CF electrode prepared in Example 2, and (c) shows a modified 5-UiO / CF electrode prepared in Example 3. Figure 7 This is a photo of the assembled system described in the embodiment.

[0027] In the figure: 1. PDMS matrix; 2. fluid channel; 3. working electrode; 4. counter electrode; 5. reference electrode; 6. input tube; 7. output tube. DETAILED DESCRIPTION

[0028] The preferred embodiments of the present invention will be described in more detail below with reference to the accompanying drawings and specific examples.

[0029] Example 1: This embodiment provides an exosome separation device, comprising a microfluidic chip and an electrochemical workstation electrically connected thereto.

[0030] like Figure 1 As shown, the microfluidic chip comprises a PDMS matrix 1 that encloses a fluid channel 2 and a three-electrode assembly embedded within the inner wall of the fluid channel 2. The three-electrode assembly includes a working electrode, a counter electrode, and a reference electrode. The working and reference electrodes are positioned on the same side of the fluid channel, while the counter electrode is positioned on the opposite side. Each of the working, counter, and reference electrodes is a carbon fiber electrode modified with a zirconium-based metal-organic framework (Zr-MOF). The fluid channel also has inlet and outlet tubes for injecting sample solutions, eluents, and the like. The inlet tube is positioned near the reference electrode, while the outlet tube is positioned away from the reference electrode.

[0031] In this embodiment, the working electrode has a length of 53 mm, a width of 8 mm, and a thickness of 0.15 mm; the counter electrode has a length of 58 mm, a width of 8 mm, and a thickness of 0.15 mm; the reference electrode has a length of 3 mm, a width of 8 mm, and a thickness of 0.15 mm. The surface of the reference electrode is further provided with an Ag / AgCl coating, and the thickness of the Ag / AgCl coating is 2 mm.

[0032] Example 2: This embodiment provides a method for preparing the exosome separation device described in Example 1, which specifically includes the following steps: (1) Preparation of UiO / CF electrode ZrCl4 was dissolved in a mixed solvent of 7 mL formic acid and 20 mL ethanol to form a metal solution with a ZrCl4 concentration of 2 mM; then NH2-BDC was dissolved in a mixed solvent of 7 mL formic acid and 8 mL pure water and ultrasonically treated for 1 h to obtain a homogeneous ligand solution with an NH2-BDC concentration of 2 mM.

[0033] The carbon cloth was immersed in the metal solution for 1 h, then taken out and further immersed in the homogeneous ligand solution for 4 h; after that, the surface of the carbon cloth was rinsed with ethanol to remove the unreacted NH2-BDC to obtain the UiO / CF electrode, which was then immersed in deionized water until use.

[0034] (2) Construction of a microfluidic chip embedded with three-electrode components Prepare two half-channel molds and cut out three electrodes according to the fluid channel size designed in the mold. Apply a layer of Ag / AgCl paste (60 / 40% m / m, 901773-50G, Sigma-Aldrich) on the reference electrode and place it in a normal pressure drying oven and heat it to 80°C for later use.

[0035] Wash the two half-channel molds with alcohol, assemble them after drying, use tape to stick the three electrodes to the corresponding positions, install the needle with a hose (input tube, output tube), stick glass sheets on all four sides of the mold, and wrap it with tin foil to prevent leakage.

[0036] Put 25g of PDMS and 2.5g of curing agent into a beaker, stir evenly, and then put it into a vacuum pump for treatment. Then pour it into the assembled mold, and then put the whole thing into an oven to shape the mixed solution of PDMS and curing agent.

[0037] The cured PDMS was cut and the tape was removed, ultimately forming a microfluidic chip with a fluid channel size of 200 μm high, 58 mm long, and 8 mm wide, and embedded with three electrodes.

[0038] (3) Connect each electrode in the microfluidic chip to the electrochemical workstation In this embodiment, three steel needles are inserted into and connected to three electrodes, which are then clamped with alligator clips and connected to an electrochemical workstation.

[0039] Example 3: This embodiment provides a method for preparing an exosome separation device, wherein the concentration of ZrCl4 in the metal solution is 5 mM, and the other steps are the same as those in Example 2.

[0040] Electron microscope scanning was performed on the electrode without Zr-MOF modification and the UiO / CF electrode prepared in Example 2 and Example 3, and the images obtained were as follows: Figure 2 As shown in the figure, 0 is an electrode without Zr-MOF modification, 2 is a UiO / CF electrode prepared in Example 2, and 5 is a UiO / CF electrode prepared in Example 3. It can be clearly seen from the figure that the surface morphology of the electrode before and after Zr-MOF modification.

[0041] Example 4: This embodiment provides a method for separating and purifying exosomes from a lung cancer cell culture medium. The separation and purification method uses the exosome separation device prepared in Example 2 or Example 3, and specifically includes the following steps: (1) Assemble the system and test The microfluidic chip was placed between two custom acrylic plates (the specifications of the two acrylic plates used in this example are as follows Figure 3 As shown in the figure), screws are passed through the holes in the acrylic plate and the screws and nuts are tightened evenly to achieve uniform clamping of the microfluidic chip. At the same time, avoid over-tightening that may cause damage to the microfluidic chip or blockage of the fluid channel. The actual picture of the assembled system is shown in the figure. Figure 7 After fixation, check the microchannel for unobstructed flow to ensure there is no blockage. Check the entire device for leaks to ensure experimental accuracy.

[0042] The three electrodes were connected to an electrochemical workstation and quality tested: cyclic voltammetry was performed between -0.1 V and +0.1 V for the first five cycles. If a current in the μA range was generated, the stability of the system was confirmed.

[0043] (2) Modified antibodies 300 μL of a 21.3 μg / mL CD9 antibody solution was directly injected into the fluid channel, and the sample was pushed back and forth through the fluid channel 5 times every 10 minutes for one hour. In some other embodiments, a CD63 antibody solution may also be used.

[0044] (3) Injecting samples to capture exosomes 3 mL of pretreated lung cancer cell culture fluid was injected into the fluid channel at a rate of 1 mL / s and pushed back and forth 10 times, and then the remaining solution was removed from the device.

[0045] (4) Elution and collection of exosomes 20 mL of PBS buffer was injected into the fluid channel at a rate of 0.3 mL / s to remove contaminants and capture exosomes.

[0046] A potential of -1.5 V was applied to the working electrode by an electrochemical workstation for 60 seconds, during which the PBS solution was pushed back and forth in the fluid channel; the instrument needles connected to the working electrode and the counter electrode were interchanged, and the process was repeated once.

[0047] like Figure 4 As shown, the working principle of the separation and purification method described in this embodiment is as follows: Because the electrodes on the fluid channel surface are modified with Zr-MOF, when CD9 antibodies flow through the channel, they bind to the Zr-MOF, forming a relatively stable structure with the CD9 antibody as a ligand. When exosomes in lung cancer cell culture fluid flow through the channel, they bind to the antibody-bound Zr-MOF due to immunoaffinity, capturing the exosomes in the lung cancer cell culture fluid. By applying a negative potential to the three-electrode system, charge repulsion and changes in electrode surface pH are used to achieve controlled release of the exosomes.

[0048] Electrostatic repulsion between negatively charged exosomes and the negative working electrode: When the potential is below -1.23 V, water molecules undergo electrolysis, producing H2 and O2 on the working and counter electrode surfaces, respectively. These gases repel the exosomes and prevent reassociation. Local pH changes on the electrode surface cause conformational changes in the antibody, weakening its affinity for exosomes.

[0049] Example 5: This example is based on the separation method provided in Example 4. Cyclic voltammetry is used to scan in three stages: step 1 (UiO / CF electrode), step 2 (after modification of specific antibodies), and step 3 (after capture of exosomes). The scanning range is -0.1~0.1V (scanning 10 times at a speed of 0.2V / s). The cyclic voltammetry curve is plotted as shown in Figure 4. Figure 5 As shown. Figure 5 As can be seen, compared to the pure electrode, the current gradually increases after antibody modification and exosome capture. This result may be attributed to the introduction of CD9 antibody molecules and the negative charge of exosomes, which leads to a change in the charge density on the electrode surface. In this way, the efficiency of electrode modification and the success of exosome capture and release can be evaluated, providing a basis for precise control of the experimental process.

[0050] Example 6: In this example, NTA was used to analyze the size distribution concentration of the exosomes purified by the method described in Example 4, wherein the devices used were a control device containing a CF electrode with unmodified Zr-MOF, and the exosome separation devices prepared in Examples 2 and 3. The analysis results are shown in FIG. Figure 6 As shown, from Figure 6 It can be seen that all exosome populations separated by the device prepared in the embodiment of the present invention showed a typical size distribution between 30-300 nm. The exosome concentration obtained by the control CF electrode without Zr-MOF modification was 5.36×10 7 ±1.01×10 7The exosome concentration obtained by the modified 2-UiO / CF electrode prepared in Example 2 was 7.11×10 7 ±2.43×10 7 The exosome concentration obtained by the modified 5-UiO / CF electrode prepared in Example 3 was 7.08×10 7 ±3.04×10 7 / ml, the exosome concentration obtained by the modified Zr-MOF electrode was higher, among which the concentration obtained by the 2-UiO / CF electrode was slightly higher than that of the 5-UiO / CF electrode. Therefore, the optimal amount of ZrCl4 added when preparing the UiO / CF electrode was 2mM.

[0051] While various embodiments of the present invention have been described above, the foregoing description is intended to be illustrative and non-exhaustive, and is not intended to be limiting of the disclosed embodiments. Numerous modifications and variations will be apparent to those skilled in the art without departing from the scope and technical principles of the described embodiments, and such modifications and variations should be considered within the scope of the present invention.

Claims

1. An exosome separation device, characterized in that: The invention comprises a microfluidic chip and an electrochemical workstation electrically connected thereto; the microfluidic chip comprises a matrix forming a fluid channel and a three-electrode assembly embedded in the inner wall of the fluid channel; the matrix is made of polydimethylsiloxane, and the electrodes in the three-electrode assembly are all carbon fiber electrodes modified with a zirconium-based metal-organic framework.

2. The exosome separation device according to claim 1, characterized in that The three-electrode assembly includes a working electrode, a counter electrode, and a reference electrode; the working electrode and the reference electrode are arranged on the same side of the fluid channel, and the counter electrode is arranged on the side opposite to the working electrode; the surface of the reference electrode is provided with an Ag / AgCl coating, and the thickness of the Ag / AgCl coating is 2-3 mm.

3. The exosome separation device according to claim 2, wherein The fluid channel is further connected with an input tube and an output tube; the input tube is arranged at one end close to the reference electrode, and the output tube is arranged at one end away from the reference electrode.

4. A method for preparing the exosome separation device according to any one of claims 1 to 3, characterized in that: include: Preparation of carbon fiber electrodes modified with zirconium-based metal-organic frameworks; constructing a microfluidic chip embedded with a three-electrode assembly; Connect each electrode in the microfluidic chip to an electrochemical workstation.

5. The method for preparing the exosome separation device according to claim 4, wherein: The method for preparing a carbon fiber electrode modified with a zirconium-based metal-organic framework comprises: a. ZrCl4 is dissolved in a mixed solvent of formic acid and ethanol to form a metal solution; b. The NH2-BDC was dissolved in a mixed solution containing formic acid and water, and a homogeneous ligand solution was obtained by ultrasonic treatment; c. The carbon cloth was immersed in a metal solution, then immersed in a ligand solution, and rinsed with ethanol to obtain a carbon fiber electrode modified with a zirconium-based metal-organic framework.

6. The method for preparing the exosome separation device according to claim 5, characterized in that: In step a, the volume ratio of formic acid to ethanol is 1:(2-4), and the concentration of ZrCl4 in the metal solution is 1-10 mM; And / or, in step b, the volume ratio of formic acid to water is 1:(1-2), and the concentration of NH2-BDC in the homogeneous ligand solution is 1-3 mM.

7. The method for preparing the exosome separation device according to claim 4, wherein: The fluid channel in the microfluidic chip is prepared by a molding method, comprising: Three electrodes are pasted into two half-channel molds and a needle with a flexible tube is installed; the reference electrode is coated with an Ag / AgCl layer; The polydimethylsiloxane and the curing agent are stirred evenly and then vacuum treated to prepare a matrix mixed solution; The two half-channel molds with electrodes attached are merged, and the matrix mixed solution is poured into the assembled molds and solidified.

8. A method for separating and purifying exosomes, characterized in that: Based on the exosome separation device according to any one of claims 1 to 3, the method comprises: The specific antibody solution is injected into the fluid channel of the exosome isolation device and pushed back and forth; The sample solution containing exosomes is injected into the fluid channel of the exosome separation device and pushed back and forth; Remove the remaining solution in the fluid channel and inject PBS buffer into the fluid channel; An electric field is applied to the working electrode by an electrochemical workstation, and the PBS buffer is pushed back and forth during the action time of the electric field; The electric field action electrode was replaced with the counter electrode, and the electric field was applied again, pushing the PBS buffer back and forth during the electric field action time.

9. The method for separating and purifying exosomes according to claim 8, wherein: The intensity of the electric field is -0.5V to -3V; and the action time of the electric field is 15s to 180s.

10. The method for separating and purifying exosomes according to claim 8, wherein: The specific antibody solution is a CD9 antibody solution or a CD63 antibody solution.

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