CN106442796B - A kind of liquid phase open tubular column of multi-layer nano gold goal and preparation method thereof and application - Google Patents
A kind of liquid phase open tubular column of multi-layer nano gold goal and preparation method thereof and application Download PDFInfo
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- CN106442796B CN106442796B CN201610946040.4A CN201610946040A CN106442796B CN 106442796 B CN106442796 B CN 106442796B CN 201610946040 A CN201610946040 A CN 201610946040A CN 106442796 B CN106442796 B CN 106442796B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/60—Construction of the column
- G01N30/6052—Construction of the column body
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/60—Construction of the column
- G01N30/6095—Micromachined or nanomachined, e.g. micro- or nanosize
Abstract
The invention belongs to field of biotechnology, specially a kind of liquid phase open tubular column of multi-layer nano gold goal and preparation method thereof and application.In the present invention, amino is modified on capillary tube inner wall first；Nano gold sol is placed in gas cylinder, aurosol is pressed into capillary using the gas in steel cylinder, the nano gold spherical in aurosol is in capillary tube inner wall self assembly；It is passed through dimercapto reagent in the capillary for modifying upper first layer nano gold spherical, aurosol is passed through again, independently fills second layer nano gold spherical in the layer gold of capillary tube inner wall；It repeats the above steps, capillary tube inner wall can multi-layer nano gold goal in self assembly；It is finally passed through mercapto reagent into capillary, is fabricated to capillary liquid phase open tubular column.The stationary phase being successful is that the liquid phase open tubular column of multi-layer nano gold goal can be used for the chromatographic isolation of 10-1000 cell protein and peptide.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of liquid phase open tubular column of multi-layer nano gold goal and its production side
Method and application.
Background technique
Nano gold spherical refers to that the miniature spherical particle of gold, diameter have high electron density, dielectric special in 1~100 nm
Property and catalytic action, can be in conjunction with a variety of large biological molecules, and do not influence its bioactivity.It can be with by reduction method by gold chloride
The nano gold spherical of various different-grain diameters is easily prepared, color is taken on a red color according to diameter to purple.Nano gold spherical is because of it
Unique physicochemical properties and by more and more extensive concern.It is mutual that with amino electrostatic can occur for nano gold spherical easily
Effect, generates chemical bond between sulfydryl.Therefore nano gold spherical can be with the conditional autonomous inner surface for being mounted in capillary.
Conventional Si base or capillary glass tube inner surface can carry out interior finishing, such as modification amino or sulfydryl.Work as hair
When being full of aurosol in tubule, the nano gold spherical to suspend in aurosol will be by electrostatic attraction or chemical bond, independently
Mounted in capillary inner surface.As long as the aurosol being passed through is enough, it is possible to form continuous nano gold spherical and independently fill layer.When
When adjacent gold goal is connected using dimercapto reagent, which will can bear ten or more than stronger
The liquid of air pressure washes away.
The metal layer of the nanogold layers of balls of continuous multilayer is the complicated leading step modified as the decorative layer of capillary tube inner wall
Suddenly.Because it not only can transform inner wall as conductor layer, facilitate control of Electric potentials and signal transduction, but also can greatly enrich
The functional molecular type of absorption, this is widely used in fields such as medicine detection, bio-separation, DNA identifications.
Meanwhile the nanogold layers of balls of continuous multilayer is a porous structure, and its Kong Ze from gold goal and gold goal it
Between accumulate the gap of formation.The size in gap is possible to be similar to the hole needed for chromatography retains.Therefore, by further repairing
Decorations, the capillary that inner wall has independently filled multi-layer nano gold goal can become capillary liquid phase open tubular column, carry out separation point to substance
Analysis.The minimum volume of liquid phase open tubular column can be used to separate the trace protein and peptide fragment in a small amount of cell, this is at unicellular point
There is effect in analysis field very much.
Summary of the invention
The purpose of the present invention is to provide liquid phase open tubular columns of a kind of multi-layer nano gold goal and preparation method thereof and application.
The liquid phase open tubular column and preparation method thereof of multi-layer nano gold goal provided by the invention, the steps include:
(1) firstly, pre-processing capillary tube inner wall, and it is passed through amino silicane coupling agent reaction, modifies ammonia in capillary tube inner wall
Base；
(2) then, nano gold sol is pressed into inner wall by gas to have modified in the capillary of amino, in capillary tube inner wall
Independently load onto first layer gold goal；
(3) dimercapto reagent is passed through inner wall again to have modified in the capillary of first layer nano gold spherical, in first layer gold goal
Upper modification sulfydryl；
(4) nano gold sol is pressed into inner wall by gas again to have modified in the capillary of the nano gold spherical with sulfydryl, In
Capillary tube inner wall independently loads onto second layer gold goal；
(5) the step of being passed through dimercapto reagent and nano gold sol is repeated, independently loads onto more layers gold in capillary tube inner wall
Ball；
(6) it is finally passed through mercapto reagent into capillary, becomes the capillary of inner wall self assembly multi-layer nano gold goal
Liquid phase open tubular column.
The liquid phase open tubular column that the stationary phase made is multi-layer nano gold goal is used for the protein enzymatic hydrolyzate of 100 cells
Chromatographic isolation utilizes Orbitrap MS on-line checking chromatographic fraction.
The production method of the liquid phase open tubular column of multi-layer nano gold goal provided by the invention, concrete operations process are as follows:
(1) a 200-1000 μm of outer diameter, 5-500 μm of internal diameter, the glass of length 1-1000 cm or quartzy capillary are taken
It manages, at 4-50 DEG C, successively uses 0.1-1 mol/L NaOH, water, 0.1-1 mol/L HCl, water, CH3CN rinses 5-30 respectively
min；
(2) silane coupling agent reaction solution is passed through in capillary obtained by step (1), heating reaction 0.5- at 20-150 DEG C
24 h；Silane coupling reagent include but is not limited to 3- aminopropyl trimethoxysilane, 3- aminopropyl triethoxysilane (1-99%,
V/v) solution, the solvent of solution include but is not limited to methanol, ethyl alcohol, acetonitrile, propyl alcohol, dimethyl imide, dimethyl sulfoxide；Silane
It is cleaned after the completion of coupling reaction with solvent, solvent includes but is not limited to methanol, ethyl alcohol, propyl alcohol, dimethyl imide；
(3) aurosol that nano gold spherical particle diameter is 1-100 nm is placed in bottleneck in the gas cylinder of rubber stopper；Gas
It is connected between bottle and steel cylinder with the quartz capillary that internal diameter is 5-500 μm of internal diameter；Capillary obtained by step (2) is directly inserted in
In aurosol in gas cylinder；Under 4-50 DEG C, 0.1-10 bar pressure, the gas of using gas steel cylinder, gas includes but unlimited
It, will be in 10-1000 times of capillary volume aurosol indentation capillary in nitrogen, argon gas, helium；
(4) dimercapto reagent is placed in bottleneck in the gas cylinder of rubber stopper；Dimercapto reagent includes but is not limited to two mercaptos
Base propane, dimercapto butane, dimercapto pentane, dimercaptohexane, dimercapto heptane, dimercapto octane, dimercapto nonane, two mercaptos
Base decane, concentration 0.1-50%(v/v), solvent includes but is not limited to methanol, ethyl alcohol, acetonitrile, propyl alcohol, dimethyl imide, two
First sulfoxide；It is connected between gas cylinder and steel cylinder with the quartz capillary that internal diameter is 5-500 μm of internal diameter；By capillary obtained by step (3)
It is directly inserted in the dimercapto reagent in gas cylinder；Under 4-50 DEG C, 0.1-10 bar pressure, the gas of using gas steel cylinder, gas
Body includes but is not limited to nitrogen, argon gas, helium, and 10-1000 times of capillary volume dimercapto reagent is pressed into capillary；
(5) aurosol that nano gold spherical particle diameter is 1-100 nm is placed in bottleneck in the gas cylinder of rubber stopper；Gas
It is connected between bottle and steel cylinder with the quartz capillary that internal diameter is 5-500 μm of internal diameter；Capillary obtained by step (4) is directly inserted in
In aurosol in gas cylinder；Under 4-50 DEG C, 0.1-10 bar pressure, the gas of using gas steel cylinder, gas includes but unlimited
It, will be in 10-1000 times of capillary volume aurosol indentation capillary in nitrogen, argon gas, helium；
(6) repetition step (4) and step (5), number of repetition 1 to 20 times；
(7) mercapto reagent is placed in bottleneck in the gas cylinder of rubber stopper；Mercapto reagent include but is not limited to sulfydryl butane,
Mercaptopentane, sulfydryl heptane, sulfydryl octadecane, concentration 0.1-50%(v/v), solvent includes but is not limited to methanol, ethyl alcohol, second
Nitrile, propyl alcohol, dimethyl imide, dimethyl sulfoxide；It is the quartz capillary of 5-500 μm of internal diameter with internal diameter between gas cylinder and steel cylinder
Connection；Capillary obtained by step (6) is directly inserted in the mercapto reagent in gas cylinder；In 4-50 DEG C, 0.1-10 bar pressure
Under, the gas of using gas steel cylinder, gas includes but is not limited to nitrogen, argon gas, helium, by 10-1000 times of capillary volume mercapto
Base reagent is pressed into capillary；Obtain the liquid phase open tubular column that stationary phase is multi-layer nano gold goal.
The liquid phase open tubular column of multi-layer nano gold goal prepared by the present invention, can be used for separating 10-1000 cell protein
And peptide.Specific steps are as follows:
(1) the liquid phase open tubular column of multi-layer nano gold goal is packed into chromatographic system；
(2) using chromatographic system to the liquid phase open tubular column sample introduction of multi-layer nano gold goal, sample is the egg of 10-1000 cell
White enzymolysis liquid；
(3) using the proteolysis of 10-1000 cell in liquid phase open tubular column obtained by chromatographic system elution step (2)
Liquid, mobile phase are A phase water, 0.1% formic acid, B phase acetonitrile, 0.1% formic acid；
(4) on-line checking carried out to chromatographic fraction obtained by step (3) using Orbitrap MS, ion source be electron spray from
Component.
It is characteristic of the invention that construct the porous layer stationary phase of open tubular column easily and fast using nano gold spherical, it can be very
It is operated in the capillary of wide inside diameter ranges.Resulting liquid phase open tubular column column volume is minimum, when separating a small amount of cell, peptide fragment and albumen
The diluting effect of matter is small, is conducive to detection.
Detailed description of the invention
Fig. 1 is the scanning electron microscope (SEM) photograph for the liquid phase open tubular column cross section that 20 μm of internal diameter stationary phases of gained are four layers of nano gold spherical.
Fig. 2 is that gained is opened by the liquid phase that 20 μm of internal diameter stationary phases that Orbitrap MS is detected are four layers of nano gold spherical
The total ion current figure of the protein enzymatic hydrolyzate fraction of 100 Hep3B cells of tubing string.
Fig. 3 is the scanning electron microscope (SEM) photograph for the liquid phase open tubular column cross section that 40 μm of internal diameter stationary phases of gained are six layers of nano gold spherical.
Fig. 4 is that gained is opened by the liquid phase that 40 μm of internal diameter stationary phases that Orbitrap MS is detected are six layers of nano gold spherical
The total ion current figure of the protein enzymatic hydrolyzate fraction of 500 HeLa cells of tubing string.
Specific embodiment
The following examples are not intended to limit the scope of the invention to further explanation of the invention.
1:20 μm of internal diameter stationary phase of embodiment is the production of the liquid phase open tubular column of four layers of nano gold spherical:
(1) 2 m long, 365 μm of outer diameters, the quartz capillary of 20 μm of internal diameters, at room temperature, successively with 0.2 mol/ are taken
L NaOH rinses 10 min, is rinsed with water 5 min, rinses 5 min with 0.1 mol/L HCl, is rinsed with water 5 min, uses CH3CN
Rinse 5 min；
(2) 100 μ L 3- aminopropyl trimethoxysilanes are dissolved in 400 μ L acetonitriles, acquired solution is passed through step
(1) in gained quartz capillary, 14 h of heating reaction at 60 DEG C；
(3) aurosol that nano gold spherical particle diameter is 60 nm is placed in bottleneck in the gas cylinder of rubber stopper；Gas cylinder and
With the quartz capillary connection that internal diameter is 100 μm of internal diameters between steel cylinder；Quartz capillary obtained by step (2) is directly inserted in gas cylinder
In interior aurosol；Under 30 DEG C, 2 bar pressure, using the helium of helium steel cylinder, by 500 times of capillary volume aurosol pressures
Enter in capillary；
(4) dimethyl sulfoxide solution of dimercapto nonane is placed in bottleneck has in the gas cylinder of rubber stopper, concentration 20%(v/
V)；With the quartz capillary connection that internal diameter is 100 μm of internal diameters between gas cylinder and steel cylinder；Quartz capillary obtained by step (3) is direct
It is inserted in the dimercapto nonane reagent in gas cylinder；Under 30 DEG C, 2 bar pressure, using the helium of helium steel cylinder, by 100 times of hairs
Tubule volume dimercapto nonane reagent is pressed into capillary；
(5) aurosol that nano gold spherical particle diameter is 60 nm is placed in bottleneck in the gas cylinder of rubber stopper；Gas cylinder and
With the quartz capillary connection that internal diameter is 100 μm of internal diameters between steel cylinder；Quartz capillary obtained by step (4) is directly inserted in gas cylinder
In interior aurosol；Under 30 DEG C, 2 bar pressure, using the helium of helium steel cylinder, by 500 times of capillary volume aurosol pressures
Enter in capillary；
(6) step (4) and (5) are repeated, number of repetition 2 times；
(7) propanol solution of sulfydryl octadecane is placed in bottleneck has in the gas cylinder of rubber stopper, concentration 30%(v/v)；Gas
With the quartz capillary connection that internal diameter is 100 μm of internal diameters between bottle and steel cylinder；Quartz capillary obtained by step (6) is directly inserted in
In sulfydryl octadecane reagent in gas cylinder；Under 30 DEG C, 2 bar pressure, using the helium of helium steel cylinder, by 300 times of capillaries
Volume dimercapto nonane reagent is pressed into capillary.
Fig. 1 is the scanning electron microscope (SEM) photograph for the liquid phase open tubular column cross section that 20 μm of internal diameter stationary phases of gained are four layers of nano gold spherical.
Embodiment 2: the liquid phase open tubular column that 1 20 μm of internal diameter stationary phases of gained of embodiment are four layers of nano gold spherical is used for 100
The chromatographic isolation and Orbitrap MS on-line checking of the protein enzymatic hydrolyzate of a cell:
(1) 1 gained liquid phase open tubular column of example is packed into nanoliter chromatographic system；
(2) using the automatic sample handling system of nanoliter chromatographic system to 1 gained liquid phase open tubular column sample introduction of embodiment, sample is
The protein enzymatic hydrolyzate of 100 Hep3B cells, sample volume are 20 fmol；
(3) using the protease of 100 Hep3B cells in nanoliter chromatographic system elution 1 gained liquid phase open tubular column of example
Solve liquid, 100 nL/min of flow velocity, gradient elution, from 99% mobile phase A phase (water, 0.1% formic acid), 1% Mobile phase B phase (acetonitrile,
0.1% formic acid) to 60% mobile phase A phase, 40% Mobile phase B phase；
(4) on-line checking carried out to chromatographic fraction obtained by step (10) using Orbitrap MS, ion source be electron spray from
Component.
Fig. 2 is that gained is opened by the liquid phase that 20 μm of internal diameter stationary phases that Orbitrap MS is detected are four layers of nano gold spherical
The total ion current figure of the protein enzymatic hydrolyzate fraction of 100 Hep3B cells of tubing string.
3:40 μm of internal diameter stationary phase of embodiment is the production of the liquid phase open tubular column of six layers of nano gold spherical:
(1) 3 m long, 365 μm of outer diameters, the quartz capillary of 40 μm of internal diameters, at room temperature, successively with 0.1 mol/ are taken
L NaOH rinses 5 min, is rinsed with water 5 min, rinses 15 min with 0.2 mol/L HCl, is rinsed with water 35 min, uses
CH3CN rinses 3 min；
(2) 100 μ L 3- aminopropyl trimethoxysilanes are dissolved in 500 μ L acetonitriles, acquired solution is passed through step
(1) in gained quartz capillary, 12 h of heating reaction at 80 DEG C；
(3) aurosol that nano gold spherical particle diameter is 30 nm is placed in bottleneck in the gas cylinder of rubber stopper；Gas cylinder and
With the quartz capillary connection that internal diameter is 100 μm of internal diameters between steel cylinder；Quartz capillary obtained by step (2) is directly inserted in gas cylinder
In interior aurosol；Under 25 DEG C, 1 bar pressure, using the helium of helium steel cylinder, by 400 times of capillary volume aurosol pressures
Enter in capillary；
(4) methanol solution of dimercapto nonane is placed in bottleneck has in the gas cylinder of rubber stopper, concentration 15%(v/v)；Gas
With the quartz capillary connection that internal diameter is 100 μm of internal diameters between bottle and steel cylinder；Quartz capillary obtained by step (3) is directly inserted in
In dimercapto nonane reagent in gas cylinder；Under 25 DEG C, 1 bar pressure, using the helium of helium steel cylinder, by 70 times of capillaries
Volume dimercapto nonane reagent is pressed into capillary；
(5) aurosol that nano gold spherical particle diameter is 30 nm is placed in bottleneck in the gas cylinder of rubber stopper；Gas cylinder and
With the quartz capillary connection that internal diameter is 100 μm of internal diameters between steel cylinder；Quartz capillary obtained by step (4) is directly inserted in gas cylinder
In interior aurosol；Under 25 DEG C, 1 bar pressure, using the helium of helium steel cylinder, by 400 times of capillary volume aurosol pressures
Enter in capillary；
(6) step (4) and (5) are repeated, number of repetition 4 times；
(7) propanol solution of sulfydryl octadecane is placed in bottleneck has in the gas cylinder of rubber stopper, concentration 40%(v/v)；Gas
With the quartz capillary connection that internal diameter is 100 μm of internal diameters between bottle and steel cylinder；Quartz capillary obtained by step (6) is directly inserted in
In sulfydryl octadecane reagent in gas cylinder；Under 25 DEG C, 1 bar pressure, using the helium of helium steel cylinder, by 400 times of capillaries
Volume dimercapto nonane reagent is pressed into capillary.
Fig. 3 is the scanning electron microscope (SEM) photograph for the liquid phase open tubular column cross section that 40 μm of internal diameter stationary phases of gained are six layers of nano gold spherical.
Embodiment 4: the liquid phase open tubular column that 1 40 μm of internal diameter stationary phases of gained of embodiment are six layers of nano gold spherical is used for 500
The chromatographic isolation and Orbitrap MS on-line checking of the protein enzymatic hydrolyzate of a cell:
(1) 1 gained liquid phase open tubular column of example is packed into nanoliter chromatographic system；
(2) using the automatic sample handling system of nanoliter chromatographic system to 1 gained liquid phase open tubular column sample introduction of embodiment, sample is
The protein enzymatic hydrolyzate of 500 HeLa cells, sample volume are 100 fmol；
(3) using the proteolysis of 500 HeLa cells in nanoliter chromatographic system elution 1 gained liquid phase open tubular column of example
Liquid, 300 nL/min of flow velocity, gradient elution, from 99% mobile phase A phase (water, 0.1% formic acid), 1% Mobile phase B phase (acetonitrile,
0.1% formic acid) to 60% mobile phase A phase, 40% Mobile phase B phase；
(4) on-line checking carried out to chromatographic fraction obtained by step (10) using Orbitrap MS, ion source be electron spray from
Component.
Fig. 4 is that gained is opened by the liquid phase that 40 μm of internal diameter stationary phases that Orbitrap MS is detected are six layers of nano gold spherical
The total ion current figure of the protein enzymatic hydrolyzate fraction of 500 HeLa cells of tubing string.
Claims (6)
1. a kind of production method of the liquid phase open tubular column of multi-layer nano gold goal, which is characterized in that specific steps are as follows:
(1) firstly, pre-processing capillary tube inner wall, and it is passed through amino silicane coupling agent reaction, modifies amino in capillary tube inner wall；
(2) then, nano gold sol inner wall is pressed by gas to have modified in the capillary of amino, it is autonomous in capillary tube inner wall
Load onto first layer gold goal；
(3) dimercapto reagent is passed through inner wall again to have modified in the capillary of first layer nano gold spherical, is repaired on first layer gold goal
Adorn sulfydryl；
(4) nano gold sol is pressed into inner wall by gas again to have modified in the capillary of the nano gold spherical with sulfydryl, in capillary
Inside pipe wall independently loads onto second layer gold goal；
(5) the step of being passed through dimercapto reagent and nano gold sol is repeated, independently loads onto more layers gold goal in capillary tube inner wall；
(6) it is finally passed through mercapto reagent into capillary, the capillary of inner wall self assembly multi-layer nano gold goal is made to become liquid phase
Open tubular column；
Concrete operations process is as follows:
(1) glass or quartz capillary of a 200-1000 μm of outer diameter, 5-500 μm of internal diameter, length 1-1000 cm, 4- are taken
At 50 DEG C, 0.1-1 mol/L NaOH, water, 0.1-1 mol/L HCl, water, CH are successively used3CN rinses 5-30 min respectively；
(2) amino silicane coupling agent reaction solution is passed through in capillary obtained by process (1), heating reaction 0.5- at 20-150 DEG C
24 h；It is silane coupled to be cleaned after the reaction was completed with solvent；
(3) aurosol that nano gold spherical particle diameter is 1-100 nm is placed in bottleneck in the gas cylinder of rubber stopper；Gas cylinder and
It is connected between steel cylinder with the quartz capillary that internal diameter is 5-500 μm of internal diameter；Capillary obtained by process (2) is directly inserted in gas cylinder
In interior aurosol；Under 4-50 DEG C, 0.1-10 bar pressure, the gas of using gas steel cylinder, by 10-1000 times of capillary
Volume aurosol is pressed into capillary；
(4) dimercapto reagent is placed in bottleneck in the gas cylinder of rubber stopper；It with internal diameter is 5-500 μm between gas cylinder and steel cylinder
The quartz capillary of internal diameter connects；Capillary obtained by process (3) is directly inserted in the dimercapto reagent in gas cylinder；In 4-50
DEG C, under 0.1-10 bar pressure, 10-1000 times of capillary volume dimercapto reagent is pressed into hair by the gas of using gas steel cylinder
In tubule；
(5) aurosol that nano gold spherical particle diameter is 1-100 nm is placed in bottleneck in the gas cylinder of rubber stopper；Gas cylinder and
It is connected between steel cylinder with the quartz capillary that internal diameter is 5-500 μm of internal diameter；Capillary obtained by process (4) is directly inserted in gas cylinder
In interior aurosol；Under 4-50 DEG C, 0.1-10 bar pressure, the gas of using gas steel cylinder, by 10-1000 times of capillary
Volume aurosol is pressed into capillary；
(6) repetition process (4) and process (5), number of repetition 1 to 20 times；
(7) mercapto reagent is placed in bottleneck in the gas cylinder of rubber stopper；It with internal diameter is in 5-500 μm between gas cylinder and steel cylinder
The quartz capillary of diameter connects；Capillary obtained by process (6) is directly inserted in the mercapto reagent in gas cylinder；4-50 DEG C,
Under 0.1-10 bar pressure, 10-1000 times of capillary volume mercapto reagent is pressed into capillary by the gas of using gas steel cylinder
In；Obtain the liquid phase open tubular column that stationary phase is multi-layer nano gold goal.
2. manufacturing method according to claim 1, which is characterized in that in process (2), the amino silicane coupling agent is selected from
3- aminopropyl trimethoxysilane or 3- aminopropyl triethoxysilane solution, the solvent of solution be selected from methanol, ethyl alcohol, acetonitrile,
Propyl alcohol, dimethyl imide or dimethyl sulfoxide；Solution concentration is 1-99%(v/v).
3. manufacturing method according to claim 1, which is characterized in that in process (4), the dimercapto reagent is selected from two mercaptos
Base propane, dimercapto butane, dimercapto pentane, dimercaptohexane, dimercapto heptane, dimercapto octane, dimercapto nonane, two mercaptos
Base decane, concentration 0.1-50%(v/v), solvent is selected from methanol, ethyl alcohol, acetonitrile, propyl alcohol, dimethyl imide, dimethyl sulfoxide.
4. manufacturing method according to claim 1, which is characterized in that the cyclinder gas is nitrogen, argon gas or helium.
5. a kind of liquid phase open tubular column for the multi-layer nano gold goal that the production method as described in one of claim 1-4 obtains.
6. the liquid phase open tubular column of multi-layer nano gold goal as claimed in claim 5, in separating 10-1000 cell protein with
The application of peptide, which is characterized in that specific steps are as follows:
(a) the liquid phase open tubular column of multi-layer nano gold goal is packed into chromatographic system；
(b) using chromatographic system to the liquid phase open tubular column sample introduction of multi-layer nano gold goal, sample is the protease of 10-1000 cell
Solve liquid；
(c) using the protein enzymatic hydrolyzate of 10-1000 cell in liquid phase open tubular column obtained by chromatographic system elution step (b)；
(d) on-line checking is carried out to chromatographic fraction obtained by step (c) using Orbitrap MS, ion source is electric spray ion source.
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