Preparation and application of colloidal crystal capillary column

Abstract

The invention relates to a method for preparing a colloidal crystal capillary column and application of the colloidal crystal capillary column in peptide fragment and protein separation, and the method specifically comprises the following steps: (1) drawing one end of a capillary into a spray needle, inserting the spray needle into a protective sleeve, and tightly connecting the end, far away from the spray needle, of the capillary with a conical outlet in the lower part of a conical liquid storage tank; (2) fixing the conical liquid storage tank above the centrifugal tube, and placing the capillary tube connected with the conical liquid storage tank in the centrifugal tube; and (3) uniformly dispersing the colloidal particles in the solvent, adding the colloidal dispersion liquid into the conical liquid storage tank, arranging a conical liquid storage tank upper cover at the opening end of the upper part of the conical liquid storage tank, applying centrifugal force to the centrifugal tube, and enabling the colloidal particles to enter the capillary tube from the conical liquid storage tank under the centrifugal action to assemble the three-dimensional colloidal crystal capillary column. The preparation flux of the colloidal crystal capillary column is improved, and the colloidal crystal capillary column has a good application prospect in the aspect of separation and characterization of biomolecules.

Description

Technical Field

[0001] This invention relates to a method for preparing colloidal crystal capillary columns by rapidly assembling colloidal particles into three-dimensional colloidal crystals within a capillary, and its application in the efficient separation of biomolecules. Background Technology

[0002] Colloidal crystals are two-dimensional or three-dimensional ordered array structures assembled from monodisperse inorganic or organic particles with diameters in the micrometer or submicrometer range. According to the Van Deemter equation, the small particle size and high orderliness of three-dimensional colloidal crystals can reduce eddy diffusion, thereby improving separation efficiency, making them a highly promising chromatographic separation medium. However, the biggest challenge in using colloidal crystals for separation currently lies in how to assemble highly ordered colloidal crystals within a capillary column. Reported methods include gravity sedimentation and solvent evaporation. Gravity sedimentation involves filling a vertically placed capillary with a dispersion of colloidal particles, allowing them to self-assemble under gravity. While simple to operate, assembly can take several days or even weeks (Anal. Chem., 2013, 85, 6820-6825). Solvent evaporation induces the assembly of colloidal particles into colloidal crystals through solvent evaporation within the capillary, but the slow solvent evaporation rate results in assembly taking several days (Anal. Chem., 2014, 86, 1592-1598). Therefore, there is an urgent need to explore other methods for rapidly assembling ordered three-dimensional colloidal crystals within capillaries. Summary of the Invention

[0003] The purpose of this invention is to propose a method for preparing colloidal crystal capillary columns, which uses centrifugal force to rapidly assemble three-dimensional colloidal crystals in a capillary to solve the problem of low throughput in the current preparation of colloidal crystal capillary columns.

[0004] The present invention provides a method for centrifugally assembling three-dimensional colloidal crystals in a capillary tube, the technical solution of which is as follows:

[0005] Centrifugal assembly device ( Figure 1 The process involves using centrifugal force to rapidly assemble monodisperse colloidal particles into three-dimensional colloidal crystals within a capillary, specifically including the following steps:

[0006] (1) Pull one end of the capillary tube into a nozzle and insert it into the protective sleeve to prevent the nozzle tip from being damaged by environmental factors. Connect the other end of the capillary tube (away from the nozzle end) tightly to the conical outlet at the bottom of the conical storage tank.

[0007] (2) Fix the conical liquid storage tank above the centrifuge tube, and place the capillary tube connected to the conical liquid storage tank inside the centrifuge tube;

[0008] (3) The colloidal particles are uniformly dispersed in the solvent, the colloidal dispersion is added into the conical storage tank and a conical storage tank cover is set at the open end of the conical storage tank. Centrifugal force is applied to the centrifuge tube, and the colloidal particles enter the capillary from the conical storage tank under centrifugal action and assemble into a three-dimensional colloidal crystal capillary column.

[0009] In the above method, the inner diameter of the capillary column is 20-250 μm, the length of the capillary nozzle column (including the nozzle) is 3-30 cm, the inner diameter of the capillary nozzle opening is 5-20 μm, and the axial length of the nozzle is 0.1-4 cm. In the above method, the colloidal particles can be one or more of monodisperse silica spheres, polystyrene spheres, or organic-inorganic hybrid spheres; the structure of the colloidal particles includes a non-porous structure and / or a core-shell structure; the surface of the colloidal particles has no modifying groups or has modifying groups, the modifying groups including hydrophobic groups such as octadecyl, octyl, hexyl, butyl, phenyl, and biphenyl, or hydrophilic groups such as amide, amino, secondary amine, tertiary amine, ethylene glycol, sugar chain, cyano, and imidazole; the particle size of the colloidal particles can be one of 400-1500 nm, with a coefficient of variation (CV) < 5%.

[0010] In the above method, the volume of the colloidal dispersion can be 10 μL-1 mL, and the concentration of the colloidal dispersion can be 10-200 mg / mL; the homogenizing solvent can be one or a mixture of two or more of the following solvents: n-hexane, methanol, isopropanol, toluene, acetone, chloroform, ethanol, and water. In the above method, the centrifugal force can be 100-5000 × g, the centrifugation time can be 30 s-30 min, and the centrifugation temperature can be 4-25℃.

[0011] After assembly, the prepared colloidal crystal capillary column was placed at room temperature until the solvent inside the capillary completely evaporated, and then compacted and stored for later use.

[0012] This invention enables the rapid assembly of colloidal particles in capillaries through a simple and universal centrifugation technique, thereby increasing the throughput of colloidal crystal capillary column preparation and showing promising application prospects in the separation and characterization of biomolecules.

[0013] The present invention has the following advantages:

[0014] (1) The preparation process of the present invention is simple and has a short cycle. The preparation time of the colloidal crystal capillary column is only 30s-30min, and 36 colloidal crystal capillary columns can be prepared at the same time under one centrifugal force; (2) Compared with conventional packed columns, the colloidal particles are arranged in an orderly manner in the capillary, forming a face-centered cubic lattice structure, which reduces eddy diffusion in the separation process and improves the separation column efficiency. Attached Figure Description

[0015] Figure 1 Schematic diagram of a centrifugal assembly of a colloidal crystal capillary column. In the diagram: 1 is a conical reservoir, 2 is a capillary column, 3 is a centrifuge tube, and 4 is a protective sleeve for the nozzle.

[0016] Figure 2 Electron micrograph of the radial cross-section of the prepared colloidal crystal capillary column

[0017] Figure 3 Colloidal crystal capillary columns are used for efficient separation of HeLa cell enzymatically digested peptides.

[0018] Figure 4 Colloidal crystal capillary columns are used for the efficient separation of peptides from micro-tissue sections via enzymatic digestion.

[0019] Figure 5 Colloidal crystal capillary columns for protein separation Detailed Implementation

[0020] 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.

[0021] Example 1

[0022] High-throughput assembly of 800nm ​​C18 non-porous silica particles in a capillary nozzle column with an inner diameter of 150μm.

[0023] (1) A capillary nozzle column with an inner diameter of 150 μm was drawn by a P-2000 laser microelectrode drawing instrument (Sutter, USA) to produce a nozzle opening inner diameter of 15 μm, a nozzle axial length of 3 mm, and a length (including nozzle length) of 10 cm.

[0024] (2) The nozzle protective sleeve is a hollow cylinder with an open top and a closed bottom. A through hole is opened at the top of the cylindrical sleeve to maintain the internal and external air pressure balance. The height is 2cm. The top open end is equipped with a tube cap. A circular through hole with the same or equivalent outer diameter as the capillary tube is opened in the center of the tube cap. The lower end (needle end) of the capillary nozzle column is inserted into the nozzle protective sleeve through the circular through hole to protect the needle tip from damage.

[0025] (3) The conical storage tank is an open-top, cylindrical container with an inverted conical bottom. A circular through-hole, equal to or the same as the outer diameter of the capillary tube, is provided at the apex of the conical container as the outlet for the colloidal dispersion. The upper end of the capillary nozzle (away from the nozzle end) extends into the conical storage tank from the outlet, ensuring a tight seal between the upper sidewall of the capillary nozzle and the inner wall of the outlet of the conical storage tank, and ensuring that the length of the capillary tube extending into the conical storage tank is 1 mm. A conical storage tank cover is provided at the open end of the upper part of the conical storage tank. The conical storage tank cover is fastened to the upper open end of the conical storage tank to prevent the colloidal dispersion from splashing out, and a through-hole is provided on the conical storage tank cover to maintain the pressure balance inside and outside the protective sleeve.

[0026] (4) Make a circular through hole in the middle of the cap of a 15mL centrifuge tube with an internal height of 12cm to fix the conical liquid storage tank. Insert the lower part of the conical liquid storage tank into the circular through hole. Place the connected capillary nozzle column into the 15mL centrifuge tube. At this time, the outlet of the conical liquid storage tank, the capillary nozzle column and the nozzle protective sleeve are all placed in the centrifuge tube. The lower side wall of the conical liquid storage tank is fixed to the cap of the centrifuge tube, and the capillary is kept vertical (the axis is perpendicular to the horizontal plane) in the centrifuge tube. A through hole is provided on the upper side wall of the centrifuge tube to maintain the internal and external air pressure balance.

[0027] (5) 800nm ​​non-porous silica gel particles (purchased from Nanjing Caina Biotechnology Co., Ltd., China) were modified with octadecyl dimethylchlorosilane and capped with trimethylchlorosilane to obtain C18 non-porous silica gel particles with a particle size of 800nm. The modification amount was 6.0μmol / m 2 The coefficient of variation of particle size is <3%. 80 mg of 800 nm C18 non-porous silica gel particles were dispersed in 600 μL of n-hexane, vortexed and sonicated for 15 min until completely dispersed to obtain a colloidal dispersion.

[0028] (6) Take 50 μL of colloidal dispersion and add it into a conical storage tank. After fastening the top cover of the conical storage tank to the upper end of the tank, a centrifugal assembly colloidal crystal capillary device is obtained, i.e., a centrifugal assembly device.

[0029] Thirty-six of the above-mentioned centrifugal filling devices were symmetrically placed in an ST1RPlus centrifuge (Thermo-Fisher, USA), and centrifuged at a force of 2000 × g for 5 min at a temperature of 20 °C until the colloidal crystals completely filled the capillary column, resulting in 36 colloidal crystal capillary columns. The electron microscope images of their radial cross-sections are shown below. Figure 2 As shown, the silica particles are stacked layer by layer in the capillary, arranged in a tight and orderly manner within and between layers, exhibiting a typical face-centered cubic structure.

[0030] Example 2

[0031] High-throughput assembly of 470nm C18 non-porous silica particles in a 150μm inner diameter capillary nozzle column

[0032] (1) A capillary nozzle column with an inner diameter of 150 μm was drawn by a P-2000 laser microelectrode drawing instrument (Sutter, USA) to produce a nozzle opening inner diameter of 15 μm, a nozzle axial length of 3 mm, and a length (including nozzle length) of 7 cm.

[0033] (2) The nozzle protective sleeve is a hollow cylinder with an open top and a closed bottom. A through hole is opened at the top of the cylindrical sleeve to maintain the internal and external air pressure balance. The height is 2cm. The top open end is equipped with a tube cap. A circular through hole with the same or equivalent outer diameter as the capillary tube is opened in the center of the tube cap. The lower end (needle end) of the capillary nozzle column is inserted into the nozzle protective sleeve through the circular through hole to protect the needle tip from damage.

[0034] (3) The conical storage tank is an open-top, cylindrical container with an inverted conical bottom. A circular through-hole, equal to or the same as the outer diameter of the capillary tube, is provided at the apex of the conical container as the outlet for the colloidal dispersion. The upper end of the capillary nozzle (away from the nozzle end) extends into the conical storage tank from the outlet, ensuring a tight seal between the upper sidewall of the capillary nozzle and the inner wall of the outlet of the conical storage tank, and ensuring that the length of the capillary tube extending into the conical storage tank is 1 mm. A conical storage tank cover is provided at the open end of the upper part of the conical storage tank. The conical storage tank cover is fastened to the upper open end of the conical storage tank to prevent the colloidal dispersion from splashing out, and a through-hole is provided on the conical storage tank cover to maintain the pressure balance inside and outside the protective sleeve.

[0035] (4) Make a circular through hole in the middle of the cap of a 15mL centrifuge tube with an internal height of 12cm to fix the conical liquid storage tank. Insert the lower part of the conical liquid storage tank into the circular through hole. Place the connected capillary nozzle column into the 15mL centrifuge tube. At this time, the outlet of the conical liquid storage tank, the capillary nozzle column and the nozzle protective sleeve are all placed in the centrifuge tube. The lower side wall of the conical liquid storage tank is fixed to the cap of the centrifuge tube, and the capillary is kept vertical (the axis is perpendicular to the horizontal plane) in the centrifuge tube. A through hole is provided on the upper side wall of the centrifuge tube to maintain the internal and external air pressure balance.

[0036] (5) 470nm non-porous silica gel particles (purchased from Nanjing Caina Biotechnology Co., Ltd., China) were modified with octadecyl dimethylchlorosilane and capped with trimethylchlorosilane to obtain C18 non-porous silica gel particles with a particle size of 470nm. The modification amount was 4.3μmol / m 2 The particle size variation coefficient is <3%. 50 mg of 470 nm C18 non-porous silica gel particles were dispersed in 500 μL of toluene, vortexed, and sonicated for 15 min until completely dispersed to obtain a colloidal dispersion.

[0037] (6) Take 100 μL of colloidal dispersion into a conical storage tank. After fastening the top cover of the conical storage tank to the upper end of the tank, a centrifugal assembly colloidal crystal capillary device is obtained, i.e., a centrifugal assembly device.

[0038] Thirty-six of the above-mentioned centrifugal filling devices were symmetrically placed in an ST1RPlus centrifuge (Thermo-Fisher, USA), and centrifuged for 10 minutes with a centrifugal force of 3000×g. The centrifugation temperature was set to 20°C until the colloidal crystals completely filled the capillary column, resulting in 36 colloidal crystal capillary columns.

[0039] Example 3

[0040] High-throughput assembly of 900nm C18 non-porous silica particles in a 50μm inner diameter capillary nozzle column.

[0041] (1) A capillary nozzle column with an inner diameter of 50 μm was drawn by a P-2000 laser microelectrode drawing instrument (Sutter, USA) to produce a nozzle opening inner diameter of 10 μm, a nozzle axial length of 5 mm, and a length (including nozzle length) of 7 cm.

[0042] (2) The nozzle protective sleeve is a hollow cylinder with an open top and a closed bottom. A through hole is opened at the top of the cylindrical sleeve to maintain the internal and external air pressure balance. The height is 2cm. The top open end is equipped with a tube cap. A circular through hole with the same or equivalent outer diameter as the capillary tube is opened in the center of the tube cap. The lower end (needle end) of the capillary nozzle column is inserted into the nozzle protective sleeve through the circular through hole to protect the needle tip from damage.

[0043] (3) The conical storage tank is an open-top, cylindrical container with an inverted conical bottom. A circular through-hole, equal to or the same as the outer diameter of the capillary tube, is provided at the apex of the conical container as the outlet for the colloidal dispersion. The upper end of the capillary nozzle (away from the nozzle end) extends into the conical storage tank from the outlet, ensuring a tight seal between the upper sidewall of the capillary nozzle and the inner wall of the outlet of the conical storage tank, and ensuring that the length of the capillary tube extending into the conical storage tank is 1 mm. A conical storage tank cover is provided at the open end of the upper part of the conical storage tank. The conical storage tank cover is fastened to the upper open end of the conical storage tank to prevent the colloidal dispersion from splashing out, and a through-hole is provided on the conical storage tank cover to maintain the pressure balance inside and outside the protective sleeve.

[0044] (4) Make a circular through hole in the middle of the cap of a 15mL centrifuge tube with an internal height of 12cm to fix the conical liquid storage tank. Insert the lower part of the conical liquid storage tank into the circular through hole. Place the connected capillary nozzle column into the 15mL centrifuge tube. At this time, the outlet of the conical liquid storage tank, the capillary nozzle column and the nozzle protective sleeve are all placed in the centrifuge tube. The lower side wall of the conical liquid storage tank is fixed to the cap of the centrifuge tube, and the capillary is kept vertical (the axis is perpendicular to the horizontal plane) in the centrifuge tube. A through hole is provided on the upper side wall of the centrifuge tube to maintain the internal and external air pressure balance.

[0045] (5) 900nm non-porous silica gel particles (purchased from Nanjing Caina Biotechnology Co., Ltd., China) were modified with octadecyl dimethylchlorosilane and capped with trimethylchlorosilane to obtain C18 non-porous silica gel particles with a particle size of 900nm. The modification amount was 7.2μmol / m 2 The particle size variation coefficient is <3%. 40 mg of 900 nm C18 non-porous silica gel particles were dispersed in 600 μL of chloroform, vortexed and sonicated for 15 min until completely dispersed to obtain a colloidal dispersion.

[0046] (6) Take 50 μL of colloidal dispersion and add it into a conical storage tank. After fastening the top cover of the conical storage tank to the upper end of the tank, a centrifugal assembly colloidal crystal capillary device is obtained, i.e., a centrifugal assembly device.

[0047] Thirty-six of the above-mentioned centrifugal filling devices were symmetrically placed in an ST1R Plus centrifuge (Thermo-Fisher, USA), a centrifugal force of 4000×g was applied, and centrifugation was carried out for 5 minutes at a centrifugation temperature of 4°C until the colloidal crystals completely filled the capillary column to obtain 36 colloidal crystal capillary columns.

[0048] Example 4

[0049] High-throughput assembly of 800nm ​​C4 core-shell silica gel particles in a 100μm inner diameter capillary nozzle column.

[0050] (1) A capillary nozzle column with an inner diameter of 100 μm was drawn by a P-2000 laser microelectrode drawing instrument (Sutter, USA) to produce a nozzle opening inner diameter of 15 μm, a nozzle axial length of 6 mm, and a length (including nozzle length) of 10 cm.

[0051] (2) The nozzle protective sleeve is a hollow cylinder with an open top and a closed bottom. A through hole is opened at the top of the cylindrical sleeve to maintain the internal and external air pressure balance. The height is 2cm. The top open end is equipped with a tube cap. A circular through hole with the same or equivalent outer diameter as the capillary tube is opened in the center of the tube cap. The lower end (needle end) of the capillary nozzle column is inserted into the nozzle protective sleeve through the circular through hole to protect the needle tip from damage.

[0052] (3) The conical storage tank is an open-top, cylindrical container with an inverted conical bottom. A circular through-hole, equal to or the same as the outer diameter of the capillary tube, is provided at the apex of the conical container as the outlet for the colloidal dispersion. The upper end of the capillary nozzle (away from the nozzle end) extends into the conical storage tank from the outlet, ensuring a tight seal between the upper sidewall of the capillary nozzle and the inner wall of the outlet of the conical storage tank, and ensuring that the length of the capillary tube extending into the conical storage tank is 1 mm. A conical storage tank cover is provided at the open end of the upper part of the conical storage tank. The conical storage tank cover is fastened to the upper open end of the conical storage tank to prevent the colloidal dispersion from splashing out, and a through-hole is provided on the conical storage tank cover to maintain the pressure balance inside and outside the protective sleeve.

[0053] (4) Make a circular through hole in the middle of the cap of a 15mL centrifuge tube with an internal height of 12cm to fix the conical liquid storage tank. Insert the lower part of the conical liquid storage tank into the circular through hole. Place the connected capillary nozzle column into the 15mL centrifuge tube. At this time, the outlet of the conical liquid storage tank, the capillary nozzle column and the nozzle protective sleeve are all placed in the centrifuge tube. The lower side wall of the conical liquid storage tank is fixed to the cap of the centrifuge tube, and the capillary is kept vertical (the axis is perpendicular to the horizontal plane) in the centrifuge tube. A through hole is provided on the upper side wall of the centrifuge tube to maintain the internal and external air pressure balance.

[0054] (5) 700nm non-porous silica gel particles (purchased from Nanjing Caina Biotechnology Co., Ltd., China) were used to prepare 800nm ​​core-shell silica gel particles by a two-phase method. After modification with butyl dimethylchlorosilane and tailing with trimethylchlorosilane, 800nm ​​C4 core-shell colloidal particles were obtained, with a modification amount of 4.8μmol / m 2 The particle size variation coefficient is <3%. 20 mg of 800 nm C4 core-shell colloidal particles were dispersed in 300 μL of acetone, vortexed and sonicated for 15 min until completely dispersed to obtain a colloidal dispersion.

[0055] (6) Take 20 μL of colloidal dispersion and add it into a conical storage tank. After fastening the top cover of the conical storage tank to the upper end of the tank, a centrifugal assembly colloidal crystal capillary device is obtained, i.e., a centrifugal assembly device.

[0056] Thirty-six of the above-mentioned centrifugal filling devices were symmetrically placed in an ST1RPlus centrifuge (Thermo-Fisher, USA), and centrifuged for 1 minute with a centrifugal force of 2000×g. The centrifugation temperature was set to 10°C until the colloidal crystals completely filled the capillary column, resulting in 36 colloidal crystal capillary columns.

[0057] Example 5

[0058] High-throughput assembly of 900nm hydrophilic silica particles in a 150μm inner diameter capillary nozzle column.

[0059] (1) A capillary nozzle column with an inner diameter of 150 μm was drawn by a P-2000 laser microelectrode drawing instrument (Sutter, USA) to produce a nozzle opening inner diameter of 15 μm, a nozzle axial length of 2 mm, and a length (including nozzle length) of 7 cm.

[0060] (2) The nozzle protective sleeve is a hollow cylinder with an open top and a closed bottom. A through hole is opened at the top of the cylindrical sleeve to maintain the internal and external air pressure balance. The height is 2cm. The top open end is equipped with a tube cap. A circular through hole with the same or equivalent outer diameter as the capillary tube is opened in the center of the tube cap. The lower end (needle end) of the capillary nozzle column is inserted into the nozzle protective sleeve through the circular through hole to protect the needle tip from damage.

[0061] (3) The conical storage tank is an open-top, cylindrical container with an inverted conical bottom. A circular through-hole, equal to or the same as the outer diameter of the capillary tube, is provided at the apex of the conical container as the outlet for the colloidal dispersion. The upper end of the capillary nozzle (away from the nozzle end) extends into the conical storage tank from the outlet, ensuring a tight seal between the upper sidewall of the capillary nozzle and the inner wall of the outlet of the conical storage tank, and ensuring that the length of the capillary tube extending into the conical storage tank is 1 mm. A conical storage tank cover is provided at the open end of the upper part of the conical storage tank. The conical storage tank cover is fastened to the upper open end of the conical storage tank to prevent the colloidal dispersion from splashing out, and a through-hole is provided on the conical storage tank cover to maintain the pressure balance inside and outside the protective sleeve.

[0062] (4) Make a circular through hole in the middle of the cap of a 15mL centrifuge tube with an internal height of 12cm to fix the conical liquid storage tank. Insert the lower part of the conical liquid storage tank into the circular through hole. Place the connected capillary nozzle column into the 15mL centrifuge tube. At this time, the outlet of the conical liquid storage tank, the capillary nozzle column and the nozzle protective sleeve are all placed in the centrifuge tube. The lower side wall of the conical liquid storage tank is fixed to the cap of the centrifuge tube, and the capillary is kept vertical (the axis is perpendicular to the horizontal plane) in the centrifuge tube. A through hole is provided on the upper side wall of the centrifuge tube to maintain the internal and external air pressure balance.

[0063] (5) 900nm non-porous silica gel particles (purchased from Nanjing Caina Biotechnology Co., Ltd., China) were modified with amide groups to obtain 900nm non-porous colloidal particles after being polymerized with acrylamide and 3-methacryloyloxypropyltrimethoxysilane. The modification amount was 7.1μmol / m 2 The particle size variation coefficient is <3%. 70 mg of amide-modified non-porous colloidal particles with a particle size of 900 nm were dispersed in 600 μL of ethanol, vortexed and sonicated for 15 min until completely dispersed to obtain a colloidal dispersion.

[0064] (6) Take 50 μL of colloidal dispersion and add it into a conical storage tank. After fastening the top cover of the conical storage tank to the upper end of the tank, a centrifugal assembly colloidal crystal capillary device is obtained, i.e., a centrifugal assembly device.

[0065] Thirty-six of the above-mentioned centrifugal filling devices were symmetrically placed in an ST1RPlus centrifuge (Thermo-Fisher, USA), a centrifugal force of 500×g was applied, and centrifugation was carried out for 5 minutes at a centrifugation temperature of 20°C until the colloidal crystals completely filled the capillary column, resulting in 36 colloidal crystal capillary columns.

[0066] Example 6

[0067] The colloidal crystal capillary column prepared in Example 1 was used for the efficient separation and high-throughput identification of HeLa cell enzymatically digested peptides.

[0068] The prepared colloidal crystal capillary column was coupled to a Dionex Ultimate 3000 (Thermo-Fisher, San Jose, CA) liquid chromatography system and an Orbitrap Fusion Lumos (Thermo-Fisher, San Jose, CA) mass spectrometer for efficient separation and high-throughput identification of HeLa cell enzymatically digested peptides (i-FASP sample pretreatment; trypsin digestion at 37°C for 12 h). The chromatographic conditions were as follows: colloidal crystal capillary column: 150 μm id × 6 cm; sample concentration: 100 ng / μL; loading volume: 1 μL; flow rate: 600 nL / min; mobile phase A (V / V) was 0.1% formic acid + 99.9% water, mobile phase B (V / V) was 80% acetonitrile + 19.9% ​​water + 0.1% formic acid; gradient: 0-5-5.1-10.1 min, 14%-50%-95%-95% B (V / V);

[0069] like Figure 3 As shown, the obtained colloidal crystal capillary column can achieve reversed-phase chromatographic separation of HeLa cell enzymatically digested peptides with a short gradient of 5 min. For randomly extracted different precursor ions, the average half-peak width is 0.014 min. For three mass spectrometry repetitions, the average number of peptides and proteins identified per injection at a gradient time of 5 min were 2830±37 (n=3) and 860±19 (n=3), respectively. This indicates that the colloidal crystal capillary column can achieve efficient separation of peptides, and even with a short gradient time of 5 min, it can achieve high-throughput identification of HeLa cell enzymatically digested peptides.

[0070] Example 7

[0071] The colloidal crystal capillary column prepared in Example 3 was used for the efficient separation and high-sensitivity identification of enzymatically digested peptides in micro-tissue sections.

[0072] The prepared colloidal crystal capillary column was coupled to an Eatonano 1200 (Thermo-Fisher, San Jose, CA) liquid chromatography system and an Orbitrap Fusion Lumos (Thermo-Fisher, San Jose, CA) mass spectrometer for efficient separation and high-sensitivity identification of enzymatically digested peptides (one-pot digestion, trypsin digestion at 37°C for 12 h) from 300×300 μm tissue sections. The chromatographic conditions were as follows: colloidal crystal capillary column: 50 μm id × 6 cm; flow rate: 500 nL / min; mobile phase A (V / V): 2% acetonitrile + 97.9% water + 0.1% formic acid; mobile phase B (V / V): 98% acetonitrile + 1.9% water + 0.1% formic acid; gradient: 0-15-15.1-17.5-17.51-20-25 min, 10%-30%-80%-80%-0%-0%-10% B (V / V);

[0073] like Figure 4 As shown, the obtained colloidal crystal capillary column can achieve efficient separation and high-sensitivity identification of enzymatically digested peptides in micro-tissue sections with short gradients. The peaks are sharp. For tissue sections with a size of 300μm×300μm, the number of peptides identified within a 15-minute gradient time is 9461, corresponding to 1470 proteins. The results indicate that the colloidal crystal capillary column exhibits good separation and identification performance in high-throughput, in-depth visualization of the proteomic landscape of tissue regions.

[0074] Example 8

[0075] The colloidal crystal capillary column prepared in Example 4 was used for protein separation.

[0076] The prepared colloidal crystal capillary column was coupled to a Dionex Ultimate 3000 (Thermo-Fisher, San Jose, CA) liquid chromatography system and an LTQ Orbitrap Velos (Thermo-Fisher, San Jose, CA) mass spectrometer for the efficient separation of aprotinin, ribonuclease A, ubiquitin, human IGF-1LR3, myoglobin, and thioredoxin. The chromatographic conditions were as follows: colloidal crystal capillary column: 100 μm·d. × 6 cm; flow rate: 300 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, 2%-2%-95%-95%-2%-2% B (V / V);

[0077] like Figure 5As shown, the obtained colloidal crystal capillary column can achieve good separation of proteins. The chromatographic peaks of the proteins are sharp and symmetrical. Under a 40-minute gradient, the average half-width of the six proteins is 0.21 min. The results indicate that the colloidal crystal capillary column has a narrow half-width and high peak capacity in the separation of intact proteins, showing good development potential.

Claims

1. A method for preparing a colloidal crystal capillary column, characterized in that, A centrifugal assembly device is used to rapidly assemble monodisperse colloidal particles into three-dimensional colloidal crystals in a capillary tube by centrifugal force. The centrifugal assembly device includes a nozzle protective sleeve, a centrifuge tube, and a conical storage tank. The steps include: (1) pulling one end of the capillary tube into a nozzle and inserting it into the protective sleeve to prevent the nozzle tip from being damaged by environmental factors, and tightly connecting the other end of the capillary tube (away from the nozzle end) to the conical outlet at the bottom of the conical storage tank; (2) fixing the conical storage tank above the centrifuge tube, and placing the capillary tube connected to the conical storage tank inside the centrifuge tube; (3) uniformly dispersing the colloidal particles in a solvent, adding the colloidal dispersion into the conical storage tank, and setting a conical storage tank cover at the open end of the upper part of the conical storage tank, applying centrifugal force to the centrifuge tube, and the colloidal particles enter the capillary tube from the conical storage tank under centrifugal action and assemble into a three-dimensional colloidal crystal capillary column.

2. The preparation method according to claim 1, characterized in that, The protective sleeve includes a hollow cylindrical sleeve and a cap that is fastened to the open end of the cylindrical sleeve. A circular through hole with the same or equivalent outer diameter as the capillary tube is provided in the middle of the capillary tube. A through hole is provided on the upper part of the cylindrical sleeve or on the capillary tube to maintain the air pressure balance inside and outside the protective sleeve. The lower end (needle end) of the capillary nozzle column passes through the circular through hole and is sleeved inside the nozzle protective sleeve. The conical storage tank is an open-top, cylindrical container with an inverted conical bottom. A circular through-hole, equal to or the same as the outer diameter of the capillary tube, is located at the apex of the conical shape as the outlet for the colloidal dispersion. The upper end of the capillary nozzle (away from the nozzle tip) extends into or is inserted into the conical storage tank from the outlet. The side wall of the upper end of the capillary nozzle is tightly sealed to the inner wall of the outlet of the conical storage tank, ensuring that the length of the capillary tube inserted into the conical storage tank is less than or equal to 3 mm. A conical storage tank cover is located at the open end of the conical storage tank, fastening to the upper open end to prevent the colloidal dispersion from splashing out. A through-hole is located on the conical storage tank cover to maintain the pressure balance inside and outside the protective sleeve. Make a circular through hole in the middle of the centrifuge tube cap, insert the lower part of the conical liquid storage tank into the through hole, and fix the lower part of the conical liquid storage tank to the centrifuge tube cap. The outlet of the conical liquid storage tank, the capillary nozzle column and the nozzle protective sleeve are all placed inside the centrifuge tube, and the capillary is in a vertical position inside the centrifuge tube. A through hole is provided at the top of the cap or centrifuge tube to maintain the balance of internal and external air pressure.

3. The preparation method according to claim 1, characterized in that, The centrifugal force is 100-5000×g, the centrifugation time is 30s-30min, and the centrifugation temperature is 4-25℃.

4. The preparation method according to claim 1, characterized in that, The colloidal particles are one or more of monodisperse silica spheres, polystyrene spheres, and organic-inorganic hybrid spheres; the structure of the colloidal particles includes a non-porous structure and / or a core-shell structure; the surface of the colloidal particles has no modifying groups or has modifying groups, the modifying groups including hydrophobic groups such as octadecyl, octyl, hexyl, butyl, phenyl, biphenyl, etc., or hydrophilic groups such as amide, amino, secondary amine, tertiary amine, ethylene glycol, sugar chain, cyano, imidazole, etc., the modification amount is 1-20 μmol / m 2 One of them; The colloidal particles have a particle size of 400-1500 nm and a particle size variation coefficient (CV) of <5%.

5. The preparation method according to claim 1, characterized in that, The solvent for dispersing colloidal particles is one or a mixture of two or more of the following: hexane, methanol, isopropanol, toluene, acetone, chloroform, ethanol, and water.

6. The preparation method according to claim 1, 4, or 5, characterized in that, The volume of colloidal dispersion added to the conical storage tank is 10 μL-1 mL, and the concentration of colloidal dispersion is 10-200 mg / mL.

7. The preparation method according to claim 1, wherein the tip of the capillary nozzle is a hollow inverted cone, the axial length of the nozzle is 0.1-4 cm, and the inner diameter of the nozzle opening is 5-20 μm; The inner diameter of the capillary column is 20-250 μm, and the length of the capillary nozzle column (including the nozzle) is 3-30 cm.

8. The preparation method according to any one of claims 1 to 7, characterized in that, At least two or more centrifugal loading devices can be placed in a centrifuge at the same time for the batch and rapid assembly of three-dimensional colloidal crystal capillary columns. It can simultaneously load two or more colloidal crystal capillary columns, which is a high-throughput preparation method with short centrifugation time and fast loading speed.

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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