Preparation method of a colloidal crystal capillary column based on electric field controllable assembly and application thereof

Abstract

The present application relates to a controllable preparation method of a colloidal crystal capillary column and its application in biomolecule separation, and specifically comprises the following steps: a plunger and an electroosmotic pump are prepared in situ at one end of a capillary; a buffer solution containing monodisperse colloidal particles is filled in an upper reservoir of an electric field assembly device, and a buffer solution is filled in a lower reservoir; the capillary is filled with the buffer solution, and the end of the capillary containing the electroosmotic pump is inserted into the lower reservoir; the dispersion of the colloidal solution is maintained by applying oscillation to the upper reservoir, an electric field is applied between the two reservoirs to make the colloidal particles move to the electroosmotic pump under the action of an electroosmotic flow, and a three-dimensional colloidal crystal is assembled; and the capillary is supplemented with an ultrasonic force to reduce lattice defects. The present application has the advantages of simple preparation process, controlled directional movement of colloidal particles through an electric field, improved assembly efficiency and quality of the three-dimensional colloidal crystal, and controllable assembly of the colloidal particles under the action of an electric field through regulation and control of the length and surface charge of the electroosmotic pump.

Description

Technical Field

[0001] This invention relates to a method for preparing a capillary column, and more particularly to a colloidal crystal capillary column. Specifically, it relates to a method for preparing a colloidal crystal capillary column by controlling the directional movement of colloidal particles within a capillary using an electroosmotic pump and simultaneously relying on oscillation / ultrasonic force, and its application in the efficient separation of biomolecules. Background Technology

[0002] Spatial order at the micrometer and submicrometer scales is crucial for achieving efficient separation in chromatographic materials. Similar to atomic or molecular crystals, monodisperse homogeneous colloidal particles form ordered structures, known as colloidal crystals. While the superior separation performance of colloidal crystals has been proven over the years, assembling high-quality three-dimensional colloidal crystals within capillaries remains a significant challenge. Current methods for assembling three-dimensional colloidal crystals within capillaries primarily rely on external forces to alter the interaction forces between colloidal particles, such as evaporation-induced self-assembly (Anal. Chem., 2014, 86, 1592-1598), pressure-assisted assembly (J. Am. Chem. Soc., 2012, 134, 10780-10782), and gravity deposition (Anal. Chem., 2013, 85, 6820-6825). However, these methods all suffer from poor assembly controllability. To address this problem, this invention develops a method for preparing three-dimensional colloidal crystal capillary columns by directionally controlling the movement and assembly of monodisperse spherical colloidal particles within a capillary using an external electric field. The method achieves ordered assembly of colloidal particles by controlling the total electroosmotic flow during electrophoresis using an electroosmotic pump, while simultaneously relying on oscillation / ultrasonic force to reduce lattice defects within the colloidal crystals, thereby preparing colloidal crystal capillary columns with controllable length and ordered structure. Summary of the Invention

[0003] The purpose of this invention is to propose a controllable preparation method for colloidal crystal capillary columns. This method utilizes an electroosmotic pump to assemble three-dimensional colloidal crystals in a capillary to address the current problem of poor controllability in the preparation of colloidal crystal capillary columns. By adjusting the magnitude of the electroosmotic flow, the assembly speed and length of the colloidal crystals can be controlled. Furthermore, by using oscillation / ultrasonic force-assisted assembly, lattice defects in the colloidal crystals can be reduced, thereby achieving controllable preparation of high-quality colloidal crystal capillary columns.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0005] Electric field assembly device ( Figure 1 The process involves using an electroosmotic pump to directionally move monodisperse colloidal particles in a capillary tube and assemble them into three-dimensional colloidal crystals. The specific steps include:

[0006] (1) A plunger and an electroosmosis pump are prepared in situ at one end of the capillary tube;

[0007] (2) A buffer solution containing monodisperse colloidal particles is loaded into the upper reservoir of the electric field assembly device, and a buffer solution is loaded into the lower reservoir.

[0008] (3) Fill the capillary with buffer solution, insert one end of the capillary containing the electroosmotic pump into the lower reservoir, and the other end into the upper reservoir.

[0009] (4) The dispersion of the colloidal solution is maintained by oscillation. An electric field is applied between the two reservoirs to make the colloidal particles move towards the electroosmotic pump under the action of electroosmotic flow and assemble into a three-dimensional colloidal crystal. At the same time, ultrasound is used to reduce lattice defects.

[0010] In the above method, the electroosmotic pump is a porous monolithic material with high mechanical strength, surface charge, and a length of 1 cm-1 m; the capillary has an inner diameter of 20-250 μm and a length of 5 cm-2 m (including the electroosmotic pump). In the above method, the colloidal particles can be one or more of monodisperse silica spheres, polymer spheres, and organic-inorganic hybrid spheres; the structure of the colloidal particles includes one or more of a non-porous structure and / or a core-shell structure; the particle size of the colloidal particles can be one or more of 50-1500 nm, with a coefficient of variation (CV) < 5%.

[0011] In the above method, the volume of the colloidal dispersion can be 100 μL-50 mL, and the mass fraction of the colloidal dispersion is 1-30%. The solvent of the colloidal dispersion is one or a mixture of two or more of the following: borate buffer, phosphate buffer, acetate buffer, citrate, succinate, methanol, ethanol, and isopropanol, with a pH value of 3-10. In the above method, the electric field strength can be 30-500 V / cm, and the assembly time can be 10 min-24 h.

[0012] After assembly, the prepared colloidal crystal capillary column was compacted and stored for later use.

[0013] This invention utilizes an electroosmotic pump to achieve the controllable assembly of colloidal particles in a capillary, and applies external oscillation / ultrasonic force to reduce lattice defects in colloidal crystals, which has great application potential in the separation and characterization of intact proteins or peptides.

[0014] This invention prepares a plunger and an electroosmotic pump at the end of a capillary to regulate the total electroosmotic flow during the electrophoresis process, thereby achieving the directional, orderly, and controllable assembly of colloidal particles in the capillary.

[0015] The present invention has the following advantages:

[0016] (1) The preparation process of this invention is simple. The directional movement of colloidal particles is controlled by an electric field, and oscillation / ultrasonic force is used to reduce the internal lattice defects of colloidal crystals, thereby improving the assembly efficiency and quality of three-dimensional colloidal crystals; (2) By adjusting the length and surface charge of the electroosmotic pump, the assembly of colloidal particles under the action of an electric field can be controlled. Attached Figure Description

[0017] Figure 1 A schematic diagram of an electric field driven assembly of a colloidal crystal capillary column. In the diagram, 1 and 11 are electrodes; 2 and 10 are reservoirs; 3 and 9 are the first and second sealing plugs; 4 is an oscillator; 5 is an ultrasonic device; 6 is a high-voltage power supply; 7 is a capillary column; 8 is a plunger electroosmosis pump; and 12 is a fixed support.

[0018] Figure 2 Electron micrograph of the radial cross-section of the prepared BTMSPA-SH-AETMA electroosmotic pump;

[0019] Figure 3 Longitudinal optical microscope image of the prepared colloidal crystal capillary column;

[0020] Figure 4 Electron micrograph of the radial cross-section of the prepared colloidal crystal capillary column;

[0021] Figure 5 Colloidal crystal capillary columns are used for the separation of intact proteins. In the figure: 1 is ribonuclease A, 2 is ubiquitin, 3 is human IGF-1LR3, 4 is myoglobin, and 5 is thioredoxin. Detailed Implementation

[0022] The method provided by the present invention will be described in detail below through examples, but this does not limit the present invention in any way.

[0023] Oscillating / ultrasonic force-assisted electric field assembly device: Includes a fixed support, a sealed reservoir, electrodes, a capillary tube with an electroosmotic pump at one end, and a DC high-voltage power supply. Two sealed reservoirs at different heights are vertically fixed to the fixed support. Each reservoir contains a buffer solution and an electrode immersed in the solution. The high-voltage power supply is connected to the electrodes in both reservoirs. The capillary tube extends downwards from the water surface of a container into the bottom of the container, with its outer wall touching the bottom wall of the container. The capillary tube is sealed and fixed in place. The upper end of the vertically arranged capillary tube is sealed and connected to the interior of the upper storage tank via a first sealing plug, and is immersed in the solution inside. The lower end of the capillary tube is equipped with an electroosmotic pump, and the lower end of the capillary tube equipped with the electroosmotic pump is connected to the lower storage tank via a second sealing plug, and is immersed in the solution inside. At this time, the upper and lower storage tanks are connected through the capillary tube. The upper storage tank is placed in an oscillator, and the capillary tube is placed in a container filled with water after connection. The container is equipped with an ultrasonic transducer (forming an oscillator / ultrasound device), and ultrasonic force is applied to the capillary tube.

[0024] The assembly steps include: (1) preparing a plunger and an electroosmotic pump in situ at one end of the capillary; (2) filling the upper reservoir of the electric field assembly device with a buffer solution containing monodisperse colloidal particles and the lower reservoir with a buffer solution; (3) filling the capillary with a buffer solution, inserting one end of the capillary containing the electroosmotic pump into the lower reservoir and the other end into the upper reservoir; (4) maintaining the dispersion of the colloidal solution by oscillation, applying an electric field between the two reservoirs to make the colloidal particles move towards the electroosmotic pump under the action of electroosmotic flow, assembling into a three-dimensional colloidal crystal, and simultaneously using ultrasonic force to reduce lattice defects.

[0025] Example 1: Preparation of the BTMSPA-SH-AETMA electroosmotic pump

[0026] (1) Clean the inner wall of the capillary column with 1M hydrochloric acid and water in sequence, then dry it with nitrogen. Pour 40% hydrofluoric acid into the capillary column (fill it up), seal it and place it in a 35℃ water bath for 3 hours for etching. Then rinse it with water, 1M sodium hydroxide, water and methanol in sequence, and dry it with nitrogen for later use.

[0027] (2) Weigh 0.024 g of cetyltrimethylammonium bromide (CTAB) into a 1.5 mL centrifuge tube, add 200 μL of methanol, vortex until dissolved, centrifuge, then add 75 μL of (3-mercaptopropyl)trimethoxysilane and 25 μL of bis[3-(trimethoxysilyl)]amine (BTMSPA), vortex, centrifuge; finally add 20 μL of water to the centrifuge tube, vortex to mix, centrifuge to obtain the reaction solution, take a 35 cm long capillary column after treatment, use a syringe to quickly draw the reaction solution into the capillary column 10 cm (near one end of the capillary column), seal it and place it in a 40 °C water bath for 24 h. After the reaction is completed, rinse with methanol to obtain the BTMSPA-SH plunger.

[0028] (3) The solution in the BTMSPA-SH plunger capillary column was replaced with water. 2.7 mg AIBA and 120 mg acryloyloxyethyltrimethylammonium chloride solution (AETMA) were weighed into a 1.5 mL centrifuge tube, 0.9 mL of water was added, and the mixture was vortexed and purged with nitrogen for 1 min. Then, the solution was manually pumped into the BTMSPA-SH plunger capillary column until the water in the capillary column was completely replaced with the reaction solution. The capillary was sealed at both ends and placed in a 65℃ water bath for 24 h. After the reaction, the column was rinsed with water to obtain a BTMSPA-SH-AETMA electroosmotic pump with quaternary ammonium groups on its surface. The radial cross-sectional scanning electron microscope image of the electroosmotic pump is shown below. Figure 2 As shown, the prepared electroosmotic pump has a porous structure and does not detach from the wall, which can intercept colloidal particles.

[0029] Example 2: Preparation of GMA-PEGDA-TEA electroosmotic pump

[0030] (1) Clean the inner wall of the capillary column in sequence with 1M sodium hydroxide, water, 1M hydrochloric acid, water and methanol, and then dry it with nitrogen.

[0031] (2) Take 300 μL of 3-(methacryloyloxy)propyltrimethoxysilane (γ-MAPS) and 300 μL of anhydrous methanol, mix well and centrifuge. Use a manual pump to fill the capillary column with the solution, seal it and place it in a 50℃ oven for 24 h. After the reaction is complete, wash with methanol and dry with nitrogen.

[0032] (3) Weigh 0.0040 g azobisisobutyronitrile (AIBN), 0.1800 g glycidyl methacrylate (GMA), 0.1200 g polyethylene glycol diacrylate (PEGDA), 0.6400 g cyclohexanol, and 0.0600 g n-propanol into a 1.5 mL centrifuge tube, vortex to dissolve, purge with nitrogen for 30 s, and centrifuge. Take a 30 cm long treated capillary column, and use a syringe to draw the supernatant weighed above into the capillary column 10 cm (close to one end of the capillary column), seal both ends, and place in a 55 °C water bath for 24 h. After the reaction, rinse with methanol and seal in water to obtain a capillary column with a GMA-PEGDA plunger at one end.

[0033] (4) Take 300 μL of triethylamine and 300 μL of acetonitrile, vortex mix them, and push them into the GMA-PEGDA plunger capillary column using a manual pump until the solution in the capillary column is completely replaced by the reaction solution. After sealing both ends of the capillary, react at 60°C for 6 hours. After the reaction is completed, wash with methanol to obtain a GMA-PEGDA-TEA electroosmotic pump with quaternary ammonium groups on the surface.

[0034] Scanning electron microscopy of the radial cross-section of the electroosmotic pump reveals that the prepared electroosmotic pump has a porous structure and does not detach from the wall, thus it can intercept colloidal particles.

[0035] Example 3: Assembling 600nm hydrophilic silica particles in a 150μm inner diameter capillary column using an electric field.

[0036] (1) Following the process and conditions of Example 2, a 10cm long GMA-PEGDA-TEA electroosmotic pump was prepared in situ at one end of a capillary tube with a length of 30cm and an inner diameter of 150μm.

[0037] (2) Submicron non-porous silica particles with a particle size of 600 nm were prepared by seed growth method. 0.1 g of hydrophilic silica particles with a particle size of 600 nm were ultrasonically dispersed in 1000 μL of borate buffer (20 mM, pH=9.0). After ultrasonication, the dispersion was added to the upper storage tank of the electric field assembly device. 1000 μL of borate buffer (20 mM, pH=9.0) was ultrasonicated for 15 min and then added to the lower storage tank.

[0038] (3) Fill the capillary completely with borate buffer (20mM, pH=9.0). Pass the capillary from the water surface of the water container downwards through the bottom of the water container. The outer wall of the capillary is sealed and fixed to the bottom wall of the container (the bottom of the water container has a through hole, and the through hole is filled with a sealing rubber plug). Insert the lower end of the capillary equipped with the electroosmotic pump into the lower storage tank through the second sealing plug, and then quickly insert the upper end into the upper storage tank through the first sealing plug.

[0039] (4) Two platinum electrodes were inserted into the upper and lower reservoirs respectively. Oscillating was turned on to maintain the dispersion of the colloidal solution. The platinum electrode in the upper reservoir was grounded, and the platinum electrode in the lower reservoir was connected to positive voltage. The high-voltage power supply was turned on, and the voltage was set to rise to 5kV within 10 seconds. At this time, the silica gel particles moved directionally towards the electroosmotic pump under the action of electroosmotic flow, assembling into a three-dimensional colloidal crystal. Simultaneously, ultrasound was turned on to reduce lattice defects during the assembly process. After 1 hour of continuous assembly, the assembly length of the colloidal crystal in the capillary was 2cm; after 2 hours of continuous assembly, the assembly length was 4cm; and after 5 hours of continuous assembly, the assembly length was 7cm. The results show that by controlling the assembly time, the assembly length can be well controlled, thus achieving the purpose of controllable assembly. When the colloidal crystal column was placed under an optical microscope, obvious Bragg diffraction could be observed. Figure 3 Meanwhile, scanning electron microscopy images of the radial cross-section of the colloidal crystal column show that the silica particles are arranged in a three-dimensional ordered manner within the column. Figure 4 ).

[0040] Example 4: Assembling 800nm ​​hydrophilic silica particles in a 100μm inner diameter capillary column using an electric field.

[0041] (1) Following the process and conditions of Example 1, a 25cm long BTMSPA-SH-AETMA electroosmotic pump was prepared in situ at one end of a capillary tube with a length of 60cm and an inner diameter of 100μm.

[0042] (2) Submicron non-porous silica particles with a particle size of 800 nm were prepared by seed growth method. 0.2 g of hydrophilic silica particles with a particle size of 800 nm were ultrasonically dispersed in 1000 μL of phosphate buffer (20 mM, pH=6.8). After ultrasonication, the dispersion was added to the upper storage tank of the electric field assembly device. 1000 μL of phosphate buffer (20 mM, pH=6.8) was ultrasonicated for 15 min and then added to the lower storage tank.

[0043] (3) Fill the capillary completely with phosphate buffer (20mM, pH=6.8). Pass the capillary from the water surface of the water container downwards through the bottom of the water container. The outer wall of the capillary is sealed and fixed to the bottom wall of the container (the bottom of the water container has a through hole, and the through hole is filled with a sealing rubber plug). Insert the lower end of the capillary equipped with the electroosmotic pump into the lower storage tank through the second sealing plug. Then quickly insert the upper end into the upper storage tank through the first sealing plug.

[0044] (4) Insert two platinum electrodes into the upper and lower reservoirs respectively. Turn on the oscillation to maintain the dispersion of the colloidal solution. Ground the platinum electrode in the upper reservoir and connect the platinum electrode in the lower reservoir to positive voltage. Turn on the high-voltage power supply and set the voltage to rise to 20kV within 10 seconds. At this time, the silica gel particles move directionally towards the electroosmotic pump under the action of electroosmotic flow, assembling into three-dimensional colloidal crystals. Simultaneously, turn on the ultrasound to reduce lattice defects during the assembly process. After 1 hour of continuous assembly, the length of the colloidal crystal assembly in the capillary is 7cm.

[0045] The results showed that obvious Bragg diffraction could be observed under an optical microscope, and the scanning electron microscope image of the radial cross-section of the colloidal crystal column showed that the silica particles were arranged in a three-dimensional ordered manner within the column. Example 5: Assembly of 900nm C1 non-porous silica particles in a 50μm inner diameter capillary column using an electric field.

[0046] (1) Following the process and conditions of Example 2, a 35cm long GMA-PEGDA-TEA electroosmotic pump was prepared in situ at one end of a capillary tube with a length of 80cm and an inner diameter of 50μm.

[0047] (2) Submicron non-porous silica particles with a particle size of 900 nm were prepared by seed growth method. After modification with trimethylchlorosilane, 900 nm non-porous silica particles were obtained as C1 non-porous silica particles with a particle size of 900 nm. The modification amount was 5.3 μmol / m 2 The coefficient of variation for particle size was <3%. 0.1 g of 900 nm C1 non-porous silica gel particles were ultrasonically dispersed in 3000 μL of acetate buffer containing 30% ethanol (30 mM, pH = 5). After ultrasonication, the dispersion was added to the upper reservoir of the electric field assembly device. 3000 μL of acetate buffer containing 30% ethanol (30 mM, pH = 5) was ultrasonicated for 15 min and then added to the lower reservoir.

[0048] (3) Fill the capillary completely with acetate buffer (30mM, pH=5) containing 30% ethanol. Pass the capillary from the water surface of the water container downwards through the bottom of the water container. The outer wall of the capillary is sealed and fixed to the bottom wall of the container (the bottom of the water container has a through hole, and the through hole is filled with a sealing rubber plug). Insert the lower end of the capillary equipped with the electroosmotic pump into the lower reservoir through the second sealing plug, and then quickly insert the upper end into the upper reservoir through the first sealing plug.

[0049] (4) Insert two platinum electrodes into the upper and lower reservoirs respectively. Turn on the oscillation to maintain the dispersion of the colloidal solution. Ground the platinum electrode in the upper reservoir and connect the platinum electrode in the lower reservoir to positive voltage. Turn on the high-voltage power supply and set the voltage to rise to 15kV within 20 seconds. At this time, the silica gel particles move directionally towards the electroosmotic pump under the action of electroosmotic flow, assembling into three-dimensional colloidal crystals. Simultaneously, turn on the ultrasound to reduce lattice defects during the assembly process. After 7 hours of continuous assembly, the length of the colloidal crystal assembly in the capillary is 8cm.

[0050] The results showed that obvious Bragg diffraction could be observed under an optical microscope, and the scanning electron microscope image of the radial cross-section of the colloidal crystal column showed that the silica particles were arranged in a three-dimensional ordered manner within the column. Example 6: Assembly of 800nm ​​C1 non-porous silica particles in a 150μm inner diameter capillary column using an electric field.

[0051] (1) Following the process and conditions of Example 1, a 15cm long BTMSPA-SH-AETMA electroosmotic pump was prepared in situ at one end of a capillary tube with a length of 35cm and an inner diameter of 150μm.

[0052] (2) Submicron non-porous silica particles with a particle size of 800 nm were prepared by seed growth method. After modification with trimethylchlorosilane, 800 nm non-porous silica particles were obtained as C1 non-porous silica particles with a particle size of 800 nm. The modification amount was 6.4 μmol / m 2 The coefficient of variation for particle size was <3%. 0.2 g of 800 nm C1 non-porous silica gel particles were ultrasonically dispersed in 5000 μL of borate buffer (20 mM, pH = 4) containing 30% ethanol. After ultrasonication, the dispersion was added to the upper reservoir of the electric field assembly device. 5000 μL of borate buffer (20 mM, pH = 4) containing 30% ethanol was ultrasonicated for 15 min and then added to the lower reservoir.

[0053] (3) Fill the capillary completely with borate buffer (20mM, pH=4) containing 30% ethanol. Pass the capillary from the surface of the water in the container downwards through the bottom of the container. The outer wall of the capillary is sealed and fixed to the bottom wall of the container (the bottom of the container has a through hole, and the through hole is plugged with a sealing rubber stopper). Insert the lower end of the capillary with the electroosmotic pump into the lower reservoir through the second sealing stopper. Then quickly insert the upper end into the upper reservoir through the first sealing stopper. (4) Insert the two platinum electrodes into the upper and lower reservoirs respectively. Turn on the oscillation to maintain the dispersion of the colloidal solution. The platinum electrode in the upper reservoir is grounded, and the platinum electrode in the lower reservoir is connected to positive voltage. Turn on the high voltage power supply and set the voltage to rise to 10kV within 5s. At this time, the silica particles move directionally towards the electroosmotic pump under the action of electroosmotic flow and assemble into a three-dimensional colloidal crystal. At the same time, turn on the ultrasound to reduce lattice defects during the assembly process. After 5 hours of continuous assembly, the length of the colloidal crystal assembly in the capillary was 6 cm.

[0054] The results show that obvious Bragg diffraction can be observed under an optical microscope, and the scanning electron microscope image of the radial cross-section of the colloidal crystal column shows that the silica particles are arranged in a three-dimensional order within the column.

[0055] Example 7: Assembling 1000nm C4 core-shell silica gel particles in a 100μm inner diameter capillary column using an electric field.

[0056] (1) Following the process and conditions of Example 1, a 25cm long BTMSPA-SH-AETMA electroosmotic pump was prepared in situ at one end of a capillary tube with a length of 50cm and an inner diameter of 100μm.

[0057] (2) Submicron non-porous silica particles with a particle size of 900 nm were prepared by seed growth method, and 1000 nm core-shell silica spheres were further prepared by two-phase method. 0.3 g of 1000 nm C4 core-shell silica particles were ultrasonically dispersed in 10 mL of phosphate buffer (35 mM, pH=5.4) containing 30% isopropanol. After ultrasonication, the dispersion was added to the upper reservoir of the electric field assembly device. 10 mL of phosphate buffer (35 mM, pH=5.4) containing 30% isopropanol was ultrasonicated for 15 min and then added to the lower reservoir.

[0058] (3) Fill the capillary completely with phosphate buffer (35mM, pH=5.4) containing 30% isopropanol. Pass the capillary from the water surface of the water container downwards through the bottom of the water container. The outer wall of the capillary is sealed and fixed to the bottom wall of the container (the bottom of the water container has a through hole, and the through hole is plugged with a sealing rubber stopper). Insert the lower end of the capillary with the electroosmotic pump into the lower reservoir through the second sealing stopper. Then quickly insert the upper end into the upper reservoir through the first sealing stopper. (4) Insert the two platinum electrodes into the upper and lower reservoirs respectively. Turn on the oscillation to maintain the dispersion of the colloidal solution. The platinum electrode in the upper reservoir is grounded, and the platinum electrode in the lower reservoir is connected to positive voltage. Turn on the high voltage power supply and set the voltage to rise to 15kV within 10s. At this time, the silica particles move directionally towards the electroosmotic pump under the action of electroosmotic flow and assemble into a three-dimensional colloidal crystal. At the same time, turn on the ultrasound to reduce lattice defects during the assembly process. After 10 hours of continuous assembly, the length of the colloidal crystal assembly in the capillary was 10 cm.

[0059] The results show that obvious Bragg diffraction can be observed under an optical microscope, and the scanning electron microscope image of the radial cross-section of the colloidal crystal column shows that the silica particles are arranged in a three-dimensional order within the column.

[0060] The colloidal crystal capillary columns prepared in Examples 8 and 6 were used to separate intact proteins.

[0061] The colloidal crystal capillary column prepared in Example 6 was drawn into a capillary nozzle column with an inner diameter of 10 μm at the nozzle opening and a length (including the nozzle length) of 6 cm at the end of the electroosmotic pump plunger (far from the opening end) using a P-2000 laser microelectrode drawing instrument (Sutter, USA). The prepared capillary nozzle column was coupled to a Dionex Ultimate 3000 (Thermo-Fisher, San Jose, CA) liquid chromatography system and an LTQ OrbitrapVelos (Thermo-Fisher, San Jose, CA) mass spectrometer for the efficient separation of ribonuclease A (0.01 mg / mL), ubiquitin (0.01 mg / mL), human IGF-1LR3 (0.01 mg / mL), myoglobin (0.01 mg / mL), and thioredoxin (0.01 mg / mL). The chromatographic conditions were as follows: colloidal crystal capillary column: 150 μm·d. × 6 cm; flow rate: 500 nL / min; mobile phase A (V / V): 98% water + 2% formic acid; mobile phase B (V / V): 80% acetonitrile + 18% water + 2% formic acid; gradient: 0-10-50-60-60.1-70 min, 5%-5%-95%-95%-5%-5%B (V / V), with a linear gradient within each time period; Figure 5As shown, the colloidal crystal column assembled by the electric field has high resolution when separating intact proteins, with an average half-width of 0.085 min for the five proteins. These results indicate that the colloidal crystal column prepared by the electric field can significantly improve the column efficiency of chromatographic separation and has significant advantages in the separation of biomolecules.

Claims

1. A method for preparing a colloidal crystal capillary column based on controllable assembly of an electric field, characterized in that, A device employing oscillation / ultrasonic force-assisted electric field assembly is described, with a plunger and electroosmotic pump fabricated at the end of a capillary. During application, the total electroosmotic flow is controlled to achieve directional, ordered, and controllable assembly of colloidal particles within the capillary. The specific structure includes an oscillator / ultrasonic device, a fixed support, reservoirs, electrodes, a capillary with an electroosmotic pump at one end, and a DC high-voltage power supply. Two sealed reservoirs at different heights are fixed to the fixed support. Each reservoir contains a buffer solution and an electrode immersed in the solution within it. The DC high-voltage power supply is connected to the electrodes in both reservoirs. The capillary passes downwards from the surface of a water-filled container into a water-filled container. At the bottom of the container, the outer wall of the capillary is sealed and fixed to the bottom wall of the container. The upper end of the capillary extends from the bottom of the upper storage tank into the interior of the upper storage tank, with its open end immersed in the solution inside. The outer wall of the capillary is sealed to the bottom wall of the upper storage tank. An electroosmotic pump is installed at the lower end of the capillary, and the lower end of the capillary with the electroosmotic pump extends into the solution in the lower storage tank. The upper storage tank is placed inside the shaker, and the capillary between the two storage tanks is placed in a water-filled container after connection (preferably, part of the electroosmotic pump area is placed inside the water-filled container). An ultrasonic transducer or ultrasonic oscillator is installed inside or on the outer wall of the water-filled container, forming an oscillator / ultrasound. The assembly steps include: (1) preparing a plunger and an electroosmotic pump in situ at one end of the capillary; (2) filling the upper reservoir of the electric field assembly device with a buffer solution containing monodisperse colloidal particles and the lower reservoir with a buffer solution; (3) filling the capillary with a buffer solution, inserting one end of the capillary containing the electroosmotic pump into the lower reservoir and the other end into the upper reservoir; (4) maintaining the dispersion of the colloidal solution by applying oscillation to the upper reservoir, applying an electric field between the two reservoirs to make the colloidal particles move towards the electroosmotic pump under the action of electroosmotic flow, assembling into a three-dimensional colloidal crystal, and simultaneously applying ultrasonic force to the capillary to reduce lattice defects.

2. The preparation method according to claim 1, characterized in that: The plunger and electroosmotic pump used are a section of porous monolithic material with adjustable surface charge prepared in situ at one end of the capillary tube; the porous structure of the monolithic material is adjusted to enable it to intercept colloidal particles; the type and ratio of polymeric monomers or organic bridged siloxanes are adjusted to control the surface charge properties of the monolithic material, thereby adjusting the magnitude and direction of the total electroosmotic flow; the length of the monolithic material is 1cm-1m.

3. The preparation method according to claim 1, characterized in that, The colloidal particles include one or more of monodisperse silica spheres, polymer spheres, and organic-inorganic hybrid spheres; the structure of the colloidal particles includes one or more of non-porous structures and / or core-shell structures; the particle size of the colloidal particles is one or more of 50-1500 nm, and the coefficient of variation (CV) of the particle size value is <5%.

4. The preparation method according to claim 1, characterized in that, The solvent for dispersing colloidal particles in the reservoir and capillary is one or a mixture of two or more of the following: borate buffer, phosphate buffer, acetate buffer, citrate, succinate, methanol, ethanol, and isopropanol, with a pH value of 3-10.

5. The preparation method according to claim 1, 3, or 4, characterized in that, The volume of the colloidal dispersion in the storage tank is 100μL-50mL, and the mass fraction of the colloidal dispersion is 1-30%.

6. The preparation method according to claim 1, characterized in that, The inner diameter of the capillary is 20-250 μm, and the length of the capillary (including the electroosmotic pump) is 5 cm-2 m.

7. The preparation method according to claim 1, characterized in that, The electric field strength is 30-500V / cm, and the assembly time is 10min-24h.

8. The preparation method according to any one of claims 1 to 7, characterized in that, The total electroosmotic flow during electrophoresis can be controlled by changing the length, surface charge, and electric field strength of the electroosmotic pump, thereby enabling the controllable assembly of colloidal particles in the capillary. Colloidal crystal capillary columns prepared by electric field assembly have colloidal particles arranged in a three-dimensional order inside the capillary column and exhibit Bragg diffraction.

9. A colloidal crystal capillary column, characterized in that, The colloidal crystal capillary column is prepared by the preparation method according to any one of claims 1 to 8.

10. An application of the colloidal crystal capillary column according to claim 9, characterized in that, The colloidal crystal capillary column described above can be used for the efficient separation and characterization of biomolecules such as peptides or proteins.

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