A method for improving the encapsulation efficiency of phospholipid vesicle-encapsulated polymers

Abstract

The application relates to a method for improving the encapsulation rate of a phospholipid vesicle-encapsulated polymer, and belongs to the fields of biotechnology and biochemistry. The application aims to solve the problem of low encapsulation rate of a polymer with relatively large molecular weight in the existing method for preparing a phospholipid vesicle. The method comprises the following steps: one, preparation of a PEG-Dextran two-water-phase solution; two, preparation of a phospholipid vesicle-encapsulated PEG-Dextran two-water-phase solution. The application is used for improving the encapsulation rate of a phospholipid vesicle-encapsulated polymer.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology and biochemistry. BACKGROUND

[0002] Cell is the basic structure and function unit of organism. All life phenomena are embodied in the basic properties of cells. The understanding of cell structure, function and behavior has very important significance in exploring the mystery of life, the mechanism of disease, diagnosis and treatment, etc. Although great progress has been made in the study of cell biology, there are still many problems to be solved due to the inherent complexity and fragility of cells, for example, biological cells are easy to lose activity or die in vitro. In order to effectively overcome these problems, an artificial synthetic life-like cell which is more easily controlled and stable than biological cells is constructed. As a bionic system, life-like cell is conducive to understanding the properties of biological cells, studying the dynamic changes of biological cells after reducing the interference of biological cell complexity, helping to establish the connection between life system and non-life system, and providing corresponding theoretical basis for the study of life origin.

[0003] The ideal life-like cell should not only have a biological membrane structure similar to that of cells, but also simulate the biochemical reaction environment in cells. Cytoplasm is the main place for cell metabolism, which provides important guarantee for the orderly progress of life. The earliest stage of cell is assumed to be formed in a dilute solution, and as the evolution of cell, the concentration of encapsulated components can play a key role in cell growth rate and vitality. The non-covalent interaction between biological macromolecules such as proteins and nucleic acids improves the stability and activity of macromolecules, making the cytoplasm more crowded. Even in the earliest stage of living cells, internal simple organization parts, such as phase separation, may be the key to the aggregation of organic molecules. Special structures promote the production and retention of polymers and improve the reaction speed of precursor molecules.

[0004] At present, one or more polymers with relatively large molecular weight are mostly used to simulate the "macromolecular crowding" effect in real cytoplasm. However, the current method for preparing phospholipid vesicles has the defect of low encapsulation rate for polymers with relatively large molecular weight, because the larger polymer molecules are difficult to embed in the lipid layer during the hydration of phospholipid. Therefore, in order to better construct life-like cells, it is an urgent problem to be solved to improve the encapsulation rate of phospholipid vesicles for high polymer. SUMMARY

[0005] The present application solves the problem of low encapsulation rate of polymers with relatively large molecular weight in the current method for preparing phospholipid vesicles, and further provides a method for improving the encapsulation rate of phospholipid vesicles for polymers.

[0006] A method for improving the encapsulation rate of phospholipid vesicles for polymers, which is carried out according to the following steps:

[0007] I. Preparation of PEG-Dextran aqueous two-phase solution:

[0008] Polyethylene glycol and dextran were added to a container, then distilled water was added, heated to completely dissolve the solid powder, and a homogeneous phase solution was obtained.

[0009] II. Preparation of PEG-Dextran aqueous two-phase solution encapsulated by phospholipid vesicles:

[0010] Two pieces of ITO glass electrodes were coated with phospholipid solution, and after the solvent was volatilized, ITO glass electrodes with phospholipid film on the surface were obtained. A polytetrafluoroethylene rectangular pool was placed between the two pieces of ITO glass electrodes with phospholipid film on the surface, and PEG-Dextran aqueous two-phase solution was added to the inside of the polytetrafluoroethylene rectangular pool. The signal generator was connected to the two pieces of ITO glass electrodes with phospholipid film on the surface, and the alternating current was turned on. Under the conditions of sinusoidal wave, electric field of 3V-15V, frequency of 60Hz-140Hz, and temperature of 40℃-60℃, the power was turned on for 2h-3h, and a solution containing giant phospholipid vesicles was obtained, and the PEG-Dextran aqueous two-phase solution was encapsulated in the giant phospholipid vesicles.

[0011] The beneficial effects of the present application are:

[0012] The present application constructs a phase separation system with real cytoplasmic properties to simulate cytoplasmic environment, and encapsulates synthetic cytoplasm in phospholipid vesicles. By changing the parameters such as waveform, frequency and voltage in the electric formation method, the problem of low encapsulation rate of phospholipid vesicles encapsulating high polymer is solved, and the encapsulation rate can reach more than 70%, improving the efficiency of encapsulating large relative molecular mass polymer in phospholipid vesicles. Thus, it provides a reference and research basis for early preparation of the smallest cell with similar complexity and functionality to living cells, and provides a new idea and concept for life science research. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 Figure 1 is a control diagram of PEG-Dextran aqueous two-phase solution before and after phase separation at 37℃ in step one of the embodiment, a is before phase separation, and b is after phase separation;

[0014] Figure 2 Figure 2 is a physical diagram of PEG-Dextran aqueous two-phase solution at 37℃, a is comparative experiment one, b is comparative experiment two, c is comparative experiment three, and d is the embodiment one;

[0015] Figure 3 Figure 3 is a structural schematic diagram of PEG-Dextran aqueous two-phase solution encapsulated in giant phospholipid vesicles in the embodiment one;

[0016] Figure 4 Figure 1 is a micrograph of giant phospholipid vesicles prepared in Example 1, a is giant phospholipid vesicles prepared in Example 1, b is dextran in giant phospholipid vesicles prepared in Example 1;

[0017] Figure 5 Figure 2 is a micrograph of giant phospholipid vesicles with different phospholipid compositions, a is Comparative Experiment 4, b is Comparative Experiment 5, c is Example 1;

[0018] Figure 6 Figure 3 is a column chart of the encapsulation efficiency of PEG (20kDa)-Dextran (500kDa) in giant phospholipid vesicles prepared in Comparative Experiments 6-10 and Example 1 under different waveforms, voltages and frequencies, a is Comparative Experiment 6, b is Comparative Experiment 7, c is Comparative Experiment 8, d is Comparative Experiment 9, e is Example 1, and f is Comparative Experiment 10. DETAILED DESCRIPTION

[0019] Specific embodiment 1: The present embodiment is a method for improving the encapsulation efficiency of polymer-encapsulated phospholipid vesicles, which is carried out according to the following steps:

[0020] I. Preparation of PEG-Dextran aqueous two-phase solution:

[0021] Add polyethylene glycol and dextran to a container, then add distilled water, heat until the solid powder is completely dissolved, and obtain a homogeneous phase solution, then place the homogeneous phase solution at room temperature to obtain a PEG-Dextran aqueous two-phase solution;

[0022] II. Preparation of phospholipid vesicles encapsulating PEG-Dextran aqueous two-phase solution:

[0023] Coat phospholipid solution on two pieces of ITO glass electrodes, and after the solvent is volatilized, obtain ITO glass electrodes coated with phospholipid membranes, place a polytetrafluoroethylene rectangular cell between the two pieces of ITO glass electrodes coated with phospholipid membranes, and add PEG-Dextran aqueous two-phase solution inside the polytetrafluoroethylene rectangular cell, connect a signal generator to the two pieces of ITO glass electrodes coated with phospholipid membranes and turn on the alternating current, under the conditions of sinusoidal wave, electric field of 3V-15V, frequency of 60Hz-140Hz, and temperature of 40℃-60℃, and power on for 2h-3h, to obtain a solution containing giant phospholipid vesicles, and the giant phospholipid vesicles encapsulate the PEG-Dextran aqueous two-phase solution.

[0024] Main steps of the present embodiment:

[0025] (1) Construct a polymer environment to simulate the cytoplasmic environment:

[0026] The reason for selecting the polyethylene glycol / dextran system to simulate the cytoplasm is that the system composed of polyethylene glycol, dextran and water has similar environmental characteristics to the actual cytoplasm, such as being in a multi-phase and phase separation state, and the polyethylene glycol / dextran aqueous two-phase system has the advantages of high water content, high biocompatibility, and the like, and the active substances such as proteins are not easy to deform, and the viscosity is small, and the like, so in this embodiment, the polyethylene glycol / dextran aqueous two-phase system is used to simulate the artificial cytoplasmic environment, which lays a foundation for subsequent biochemical reactions in the artificial cytoplasm. After mixing polyethylene glycol and dextran, they can be in a homogeneous solution state above a certain temperature. As the temperature of the solution decreases, the polyethylene glycol / dextran aqueous two-phase system is gradually formed.

[0027] The polyethylene glycol / dextran solution has the properties of the real cytoplasm, such as about 80% water content, cytoplasm viscosity of 5cp-30cp, high concentration of macromolecular crowding state, phase separation solution in the cytoplasm under the condition of human body temperature of 37℃, and the like, and is used to simulate the cytoplasmic environment.

[0028] (2) Giant phospholipid vesicles for simulating cytoplasmic environment of aqueous two-phase system:

[0029] There are many methods for preparing giant phospholipid vesicles in ordinary aqueous solution environment, and the effect is also ideal, but the efficiency of phospholipid vesicles for wrapping high polymers is low. In this embodiment, the quality and effect of the phospholipid vesicles for wrapping polyethylene glycol / dextran are improved by changing the phospholipid composition and process, and the phospholipid vesicles prepared by the electric formation method are uniform in size, complete in structure, and contain PEG-Dextran aqueous two-phase solution.

[0030] The electric formation of giant phospholipid vesicles can be generally divided into three processes: ① phospholipid molecules spontaneously form a phospholipid bilayer membrane structure; ② under the repulsion (hydration and electrostatic interaction) between the external field and the phospholipid membrane, the phospholipid membrane is raised to form small-sized vesicles; ③ the vesicles gradually fuse and the size increases. The above three processes usually proceed simultaneously.

[0031] The beneficial effects of this embodiment are:

[0032] This embodiment constructs a phase separation system with the properties of real cytoplasm to simulate the cytoplasmic environment, and wraps the synthetic cytoplasm in the phospholipid vesicles. By changing the parameters such as waveform, frequency and voltage in the electric formation method, the problem of low encapsulation efficiency of phospholipid vesicles for wrapping high polymers is solved, and the encapsulation efficiency can reach more than 70%, thereby improving the efficiency of encapsulating polymers with relatively large molecular weight in phospholipid vesicles. Thus, it provides a reference and research basis for early preparation of the smallest cells with similar complexity and functionality to living cells, and provides a new idea and concept for life science research.

[0033] Specific embodiment two: the difference between this embodiment and specific embodiment one is that the relative molecular weight of the polyethylene glycol in step one is 8kDa-20kDa; the relative molecular weight of the dextran in step one is 200kDa-500kDa. The others are the same as specific embodiment one.

[0034] Specific embodiment three: the difference between this embodiment and one of specific embodiment one or two is that the mass ratio of the polyethylene glycol to the dextran in step one is 1:(0.12-6.71); the mass of the polyethylene glycol in step one to the volume of distilled water is 1g:(10.37-57.64)mL. The others are the same as specific embodiment one or two.

[0035] Specific embodiment four: the difference between this embodiment and one of specific embodiment one to three is that in step one, the solid powder is heated to completely dissolve at a temperature of 50℃-70℃ to obtain a homogeneous phase solution. The others are the same as specific embodiment one to three.

[0036] Specific embodiment five: the difference between this embodiment and one of specific embodiment one to four is that the ITO glass electrode in step two is specifically immersed in a sodium hydroxide aqueous solution with a concentration of 8mol / L-12mol / L, ultrasonic cleaning for 8min-12min, taking out the ITO glass electrode and rinsing with flowing distilled water, then immersing in an ethanol solution, ultrasonic cleaning again for 8min-12min, placing the ITO glass electrode in distilled water for ultrasonic cleaning for 8min-12min, and finally blowing the ITO glass electrode dry with flowing nitrogen for standby. The others are the same as specific embodiment one to four.

[0037] Specific embodiment six: the difference between this embodiment and one of specific embodiment one to five is that the phospholipid solution in step two is specifically dissolving dipalmitoyl phosphatidylcholine in chloroform to obtain a phospholipid solution with a concentration of 7mg / mL-10mg / mL. The others are the same as specific embodiment one to five.

[0038] Specific embodiment seven: the difference between this embodiment and one of specific embodiment one to six is that in step two, 3 microliters-5 microliters of phospholipid solution are added to the surface of every 4cm 2 ITO glass electrode. The others are the same as specific embodiment one to six.

[0039] Specific embodiment eight: the difference between this embodiment and one of specific embodiment one to seven is that the thickness of the phospholipid film in step two is 15nm-25nm. The others are the same as specific embodiment one to seven.

[0040] Specific embodiment nine: different from one of the specific embodiments one to eight is that: the distance between the two ITO glass electrodes in step two is 1mm-3mm; the area of the side of the polytetrafluoroethylene rectangular pool in contact with the ITO glass electrode in step two is 4cm 2 ~6cm 2 . The others are the same as specific embodiments one to eight.

[0041] Specific embodiment ten: different from one of the specific embodiments one to nine is that: the particle size of the giant phospholipid vesicle prepared in step two is 2μm-5μm. The others are the same as specific embodiments one to nine.

[0042] The beneficial effects of the present application are verified by the following examples:

[0043] Example one:

[0044] A method for improving the encapsulation efficiency of a polymer wrapped by a phospholipid vesicle, which is carried out according to the following steps:

[0045] I. Preparation of PEG-Dextran two-aqueous phase solution:

[0046] 0.1928g of polyethylene glycol (PEG) and 0.0238g of dextran are added to a container, then 2mL of distilled water is added, and the container is heated to 70℃ until the solid powder is completely dissolved to obtain a homogeneous phase solution. The homogeneous phase solution is placed at room temperature to obtain a PEG-Dextran two-aqueous phase solution;

[0047] II. Preparation of PEG-Dextran two-aqueous phase solution wrapped by a phospholipid vesicle:

[0048] Phospholipid solution is coated on two ITO glass electrodes, and after the solvent is volatilized, ITO glass electrodes with phospholipid membranes on the surface are obtained. A polytetrafluoroethylene rectangular pool is placed between the two ITO glass electrodes with phospholipid membranes on the surface, and PEG-Dextran two-aqueous phase solution is added to the inside of the polytetrafluoroethylene rectangular pool. A signal generator is connected to the two ITO glass electrodes with phospholipid membranes on the surface and an alternating current is turned on. Under the conditions of a sine wave, an electric field of 8V, a frequency of 80Hz, and a temperature of 50℃, the current is turned on for 2.5h to obtain a solution containing giant phospholipid vesicles, and the PEG-Dextran two-aqueous phase solution is wrapped in the giant phospholipid vesicles.

[0049] The relative molecular mass of the polyethylene glycol in step one is 20kDa; the relative molecular mass of the dextran in step one is 500kDa.

[0050] The ITO glass electrode in step two is specifically prepared by immersing the ITO glass electrode in a 10 mol / L sodium hydroxide aqueous solution, ultrasonic cleaning for 10 min, taking out the ITO glass electrode, rinsing with flowing distilled water, then immersing in an ethanol solution, ultrasonic cleaning again for 10 min, placing the ITO glass electrode in distilled water for ultrasonic cleaning for 10 min, and finally blowing the ITO glass electrode dry with flowing nitrogen for standby.

[0051] The phospholipid solution in step two is specifically prepared by dissolving dipalmitoyl phosphatidylcholine (DPPC) in chloroform to obtain a phospholipid solution with a concentration of 8 mg / mL.

[0052] In step two, 4 microliters of the phospholipid solution is added dropwise to the surface of each 4 cm 2 ITO glass electrode.

[0053] The thickness of the phospholipid film in step two is 15 nm to 25 nm.

[0054] The distance between the two ITO glass electrodes in step two is 1 mm.

[0055] In step two, the area of the side of the polytetrafluoroethylene rectangular pool in contact with the ITO glass electrode is 4 cm 2 .

[0056] Comparative Test One: The comparative test is different from Example One in that the relative molecular mass of the polyethylene glycol in step one is 2 kDa; and the relative molecular mass of the dextran in step one is 10 kDa. The others are the same as Example One.

[0057] Comparative Test Two: The comparative test is different from Example One in that the relative molecular mass of the polyethylene glycol in step one is 2 kDa; and the relative molecular mass of the dextran in step one is 500 kDa. The others are the same as Example One.

[0058] Comparative Test Three: The comparative test is different from Example One in that the relative molecular mass of the polyethylene glycol in step one is 20 kDa; and the relative molecular mass of the dextran in step one is 10 kDa. The others are the same as Example One.

[0059] Comparative Test Four: The comparative test is different from Example One in that the phospholipid solution in step two is specifically prepared by dissolving dioleoyl lecithin DOPC in chloroform to obtain a phospholipid solution with a concentration of 8 mg / mL. The others are the same as Example One.

[0060] Comparative Test Five: The comparative test is different from Example One in that the phospholipid solution in step two is specifically prepared by dissolving dimyristoyl phosphatidylcholine DMPC in chloroform to obtain a phospholipid solution with a concentration of 8 mg / mL. The others are the same as Example One.

[0061] Comparative Test Six: The difference between this comparative test and Example One is that in Step One, the triangular wave, electric field is 3V, frequency is 50Hz and temperature is 50℃, and the power is on for 2.5h. The others are the same as Example One.

[0062] Comparative Test Seven: The difference between this comparative test and Example One is that in Step One, the square wave, electric field is 8V, frequency is 80Hz and temperature is 50℃, and the power is on for 2.5h. The others are the same as Example One.

[0063] Comparative Test Eight: The difference between this comparative test and Example One is that in Step One, the square wave, electric field is 15V, frequency is 150Hz and temperature is 50℃, and the power is on for 2.5h. The others are the same as Example One.

[0064] Comparative Test Nine: The difference between this comparative test and Example One is that in Step One, the sine wave, electric field is 8V, frequency is 50Hz and temperature is 50℃, and the power is on for 2.5h. The others are the same as Example One.

[0065] Comparative Test Ten: The difference between this comparative test and Example One is that in Step One, the sine wave, electric field is 15V, frequency is 150Hz and temperature is 50℃, and the power is on for 2.5h. The others are the same as Example One.

[0066] Figure 1 The control diagram of PEG-Dextran aqueous two-phase solution in Example One before and after phase separation at 37℃, a is before phase separation, b is after phase separation; as can be seen from the figure, after mixing polyethylene glycol and dextran into a homogeneous phase in aqueous solution, polyethylene glycol and dextran components respectively gather and appear phase separation phenomenon, i.e. form aqueous two-phase solution.

[0067] The feasibility test of PEG-Dextran aqueous two-phase solution prepared under the same concentration and mixing ratio in Comparative Test One to Three, Figure 2 The actual picture of PEG-Dextran aqueous two-phase solution at 37℃, a is Comparative Test One, b is Comparative Test Two, c is Comparative Test Three, d is Example One; as can be seen from the figure, Comparative Test One to Three do not appear phase separation phenomenon, while PEG(20KDa)-Dextran(500KDa) combination in Example One can appear phase separation state, i.e. form aqueous two-phase solution.

[0068] Figure 3 The structural schematic diagram of PEG-Dextran aqueous two-phase solution wrapped in giant phospholipid vesicle in Example One; Figure 4The images are microscopic images. A shows the giant phospholipid vesicles prepared in Example 1, and B shows the dextran in the giant phospholipid vesicles prepared in Example 1. As can be seen from the figures, under the conditions of sinusoidal wave, 8V voltage, and 80Hz frequency, phospholipid vesicles encapsulating an aqueous two-phase solution can be successfully prepared. At the same time, obvious phase separation phenomenon is observed in the aqueous two-phase solution within the vesicles. Through fluorescent labeling and tracking experiments of the dextran component, it was found that the central part of the vesicle interior space is dextran, and the outer layer is polyethylene glycol. This is because hydrogen bonds are formed between the -OH groups in the polyethylene glycol molecules and the phospholipid molecules, causing the polyethylene glycol to tend to be distributed in the outer layer of the vesicle, while the dextran is aggregated in the core of the vesicle.

[0069] Figure 5 The figures show giant phospholipid vesicles with different phospholipid components. a is comparative experiment four, b is comparative experiment five, and c is example one. As can be seen from the figures, DPPC phospholipids have the best effect in preparing PEG-Dextran aqueous two-phase solutions, which can form relatively rational vesicles with a size of 2μm to 5μm, while other phospholipid components cannot form vesicles.

[0070] The prepared solution containing giant phospholipid vesicles was filtered using a 1 μm pore size filter membrane, and the amount of PEG (20 kDa)-Dextran (500 kDa) in the filtrate, i.e., C, was measured. f Subsequently, the surfactant Triton X-100 was added to the giant phospholipid vesicles and vortexed to break the phospholipid membrane, thereby completely releasing the internal PEG (20kDa)-Dextran (500kDa). The polymer content within the vesicles was measured, and C was added. f That is, the sum C t The percentage of cytoplasm encapsulation within giant phospholipid vesicles was calculated using formula (1), and bar charts of the experimental results calculated under different experimental conditions were plotted.

[0071] EN% = (1 - C f / C t )×100% (1)

[0072] In the formula C f The amount of free cytoplasm; C t It represents the total amount of cytoplasm in a solution containing giant phospholipid vesicles.

[0073] Figure 6Figure 6 shows the encapsulation efficiency of PEG(20kDa)-Dextran(500kDa) in the giant phospholipid vesicles prepared in Comparative Experiment 6 to 10 and Example 1 under different waveforms, voltages and frequencies, wherein a is Comparative Experiment 6, b is Comparative Experiment 7, c is Comparative Experiment 8, d is Comparative Experiment 9, e is Example 1, and f is Comparative Experiment 10. As shown in the figure, the encapsulation efficiency of PEG(20kDa)-Dextran(500kDa) in the DPPC phospholipid vesicles is up to 73.4% under the conditions of sinusoidal wave, voltage 8V and frequency 80Hz.

Claims

1. A method of increasing the encapsulation efficiency of a polymer encapsulated by a phospholipid vesicle, characterized by It is carried out according to the following steps: I. Preparation of PEG-Dextran aqueous two-phase solution: Polyethylene glycol and dextran are added to a container, then distilled water is added, heated to completely dissolve the solid powder, and a homogeneous phase solution is obtained, which is placed at room temperature to obtain a PEG-Dextran aqueous two-phase solution; The mass ratio of polyethylene glycol to dextran is 1:(0.12-6.71); the mass of polyethylene glycol to the volume of distilled water is 1g:10.37mL; The relative molecular mass of the polyethylene glycol is 8kDa-20kDa; the relative molecular mass of the dextran is 200kDa-500kDa; II. Preparation of PEG-Dextran aqueous two-phase solution encapsulated by phospholipid vesicles: Two pieces of ITO glass electrodes are coated with phospholipid solution, and after the solvent is volatilized, ITO glass electrodes with phospholipid film on the surface are obtained. A polytetrafluoroethylene rectangular pool is placed between the two pieces of ITO glass electrodes with phospholipid film on the surface, and PEG-Dextran aqueous two-phase solution is added to the inside of the polytetrafluoroethylene rectangular pool. Connect the signal generator to the two pieces of ITO glass electrodes with phospholipid film on the surface and turn on the alternating current. Under the conditions of sinusoidal wave, electric field of 8V, frequency of 60Hz-80Hz, and temperature of 40℃-60℃, power on for 2.5h-3h, obtain a solution containing giant phospholipid vesicles with a particle size of 2μm-5μm, and the PEG-Dextran aqueous two-phase solution is encapsulated in the giant phospholipid vesicles; The phospholipid solution is specifically prepared by dissolving dipalmitoyl phosphatidylcholine in chloroform to obtain a phospholipid solution with a concentration of 7mg / mL-10mg / mL. Every 4 cm 2 3-5 μl of phospholipid solution was dropped on the surface of ITO glass electrode; the thickness of the phospholipid film was 15-25 nm; The distance between the two ITO glass electrodes is 1 mm to 3 mm; the area of the side of the polytetrafluoroethylene rectangular pool in contact with the ITO glass electrode is 4 cm 2 ~6 cm 2 .

2. The method of claim 1, wherein the phospholipid vesicle is a liposome. In step one, heat to completely dissolve the solid powder at a temperature of 50℃-70℃ to obtain a homogeneous phase solution.

3. The method of claim 1, wherein the phospholipid vesicle is a liposome. In step two, the ITO glass electrode is specifically prepared by immersing the ITO glass electrode in a 8mol / L-12mol / L sodium hydroxide aqueous solution, ultrasonic cleaning for 8min-12min, taking out the ITO glass electrode and rinsing with flowing distilled water, then immersing in ethanol solution, ultrasonic cleaning again for 8min-12min, then placing the ITO glass electrode in distilled water for ultrasonic cleaning for 8min-12min, and finally blowing the ITO glass electrode dry with flowing nitrogen for standby.

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