CN106442796A - Liquid phase open tubular column with multiple layers of nano-golden balls and manufacturing method and application thereof - Google Patents
Liquid phase open tubular column with multiple layers of nano-golden balls and manufacturing method and application thereof Download PDFInfo
- Publication number
- CN106442796A CN106442796A CN201610946040.4A CN201610946040A CN106442796A CN 106442796 A CN106442796 A CN 106442796A CN 201610946040 A CN201610946040 A CN 201610946040A CN 106442796 A CN106442796 A CN 106442796A
- Authority
- CN
- China
- Prior art keywords
- capillary tube
- gas
- liquid phase
- nano gold
- tubular column
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000007791 liquid phase Substances 0.000 title claims abstract description 46
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 9
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 77
- 239000010931 gold Substances 0.000 claims abstract description 77
- 229910052737 gold Inorganic materials 0.000 claims abstract description 77
- 229910000831 Steel Inorganic materials 0.000 claims abstract description 25
- 239000010959 steel Substances 0.000 claims abstract description 25
- 238000000034 method Methods 0.000 claims abstract description 18
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 17
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 17
- 239000003774 sulfhydryl reagent Substances 0.000 claims abstract description 11
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 7
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 3
- 239000007789 gas Substances 0.000 claims description 73
- 239000012071 phase Substances 0.000 claims description 34
- 239000010453 quartz Substances 0.000 claims description 30
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 30
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 26
- 239000003153 chemical reaction reagent Substances 0.000 claims description 22
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
- 239000001307 helium Substances 0.000 claims description 21
- 229910052734 helium Inorganic materials 0.000 claims description 21
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 claims description 21
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 18
- 230000002255 enzymatic effect Effects 0.000 claims description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 150000002500 ions Chemical class 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 claims description 10
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 10
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
- -1 amino silicane Chemical compound 0.000 claims description 8
- 239000002904 solvent Substances 0.000 claims description 8
- 239000012798 spherical particle Substances 0.000 claims description 8
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 claims description 6
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 claims description 6
- 229910000077 silane Inorganic materials 0.000 claims description 6
- 210000005239 tubule Anatomy 0.000 claims description 6
- 229910052786 argon Inorganic materials 0.000 claims description 5
- SJECZPVISLOESU-UHFFFAOYSA-N 3-trimethoxysilylpropan-1-amine Chemical group CO[Si](OC)(OC)CCCN SJECZPVISLOESU-UHFFFAOYSA-N 0.000 claims description 4
- 238000010828 elution Methods 0.000 claims description 4
- 238000010438 heat treatment Methods 0.000 claims description 4
- 239000007921 spray Substances 0.000 claims description 4
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 238000005859 coupling reaction Methods 0.000 claims description 3
- 238000005034 decoration Methods 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 claims description 2
- TWWSEEHCVDRRRI-UHFFFAOYSA-N 2,3-Butanedithiol Chemical compound CC(S)C(C)S TWWSEEHCVDRRRI-UHFFFAOYSA-N 0.000 claims description 2
- 108091005804 Peptidases Proteins 0.000 claims description 2
- 239000004365 Protease Substances 0.000 claims description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 2
- 230000008878 coupling Effects 0.000 claims description 2
- 239000007822 coupling agent Substances 0.000 claims description 2
- 238000010168 coupling process Methods 0.000 claims description 2
- CCUOVGPOGBWHAJ-UHFFFAOYSA-N heptane-4,4-dithiol Chemical compound SC(CCC)(CCC)S CCUOVGPOGBWHAJ-UHFFFAOYSA-N 0.000 claims description 2
- ALPIESLRVWNLAX-UHFFFAOYSA-N hexane-1,1-dithiol Chemical compound CCCCCC(S)S ALPIESLRVWNLAX-UHFFFAOYSA-N 0.000 claims description 2
- WKVAXZCSIOTXBT-UHFFFAOYSA-N octane-1,1-dithiol Chemical compound CCCCCCCC(S)S WKVAXZCSIOTXBT-UHFFFAOYSA-N 0.000 claims description 2
- JWUFROLPDIVUOW-UHFFFAOYSA-N pentane-3,3-dithiol Chemical compound CCC(S)(S)CC JWUFROLPDIVUOW-UHFFFAOYSA-N 0.000 claims description 2
- 239000000376 reactant Substances 0.000 claims description 2
- 238000001338 self-assembly Methods 0.000 claims description 2
- BKIMMITUMNQMOS-UHFFFAOYSA-N nonane Chemical compound CCCCCCCCC BKIMMITUMNQMOS-UHFFFAOYSA-N 0.000 claims 2
- 239000003795 chemical substances by application Substances 0.000 claims 1
- WXZKPELXXQHDNS-UHFFFAOYSA-N decane-1,1-dithiol Chemical compound CCCCCCCCCC(S)S WXZKPELXXQHDNS-UHFFFAOYSA-N 0.000 claims 1
- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 claims 1
- HMPSOEYFMTWOFC-UHFFFAOYSA-N propane-2,2-dithiol Chemical group CC(C)(S)S HMPSOEYFMTWOFC-UHFFFAOYSA-N 0.000 claims 1
- 239000000243 solution Substances 0.000 claims 1
- 125000003396 thiol group Chemical group [H]S* 0.000 claims 1
- PCGDBWLKAYKBTN-UHFFFAOYSA-N 1,2-dithiole Chemical compound C1SSC=C1 PCGDBWLKAYKBTN-UHFFFAOYSA-N 0.000 abstract 1
- 230000005526 G1 to G0 transition Effects 0.000 abstract 1
- 238000013375 chromatographic separation Methods 0.000 abstract 1
- RZJRJXONCZWCBN-UHFFFAOYSA-N octadecane Chemical compound CCCCCCCCCCCCCCCCCC RZJRJXONCZWCBN-UHFFFAOYSA-N 0.000 description 10
- VODWYWPXMJISGX-UHFFFAOYSA-N nonane-5,5-dithiol Chemical compound CCCCC(S)(S)CCCC VODWYWPXMJISGX-UHFFFAOYSA-N 0.000 description 9
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 8
- 229940038384 octadecane Drugs 0.000 description 5
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 4
- 235000019253 formic acid Nutrition 0.000 description 4
- 238000007445 Chromatographic isolation Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- OFBQJSOFQDEBGM-UHFFFAOYSA-N n-pentane Natural products CCCCC OFBQJSOFQDEBGM-UHFFFAOYSA-N 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 description 2
- 108010033276 Peptide Fragments Proteins 0.000 description 2
- 102000007079 Peptide Fragments Human genes 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 150000001335 aliphatic alkanes Chemical class 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 229910001873 dinitrogen Inorganic materials 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000001273 butane Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000004020 conductor Substances 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- FDWREHZXQUYJFJ-UHFFFAOYSA-M gold monochloride Chemical compound [Cl-].[Au+] FDWREHZXQUYJFJ-UHFFFAOYSA-M 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- IJDNQMDRQITEOD-UHFFFAOYSA-N n-butane Chemical compound CCCC IJDNQMDRQITEOD-UHFFFAOYSA-N 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 150000003457 sulfones Chemical class 0.000 description 1
Classifications
-
- 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
-
- 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
-
- 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
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention belongs to the technical field of biology and particularly provides a liquid phase open tubular column with multiple layers of nano-golden balls and a manufacturing method and application thereof. The method includes the steps that firstly, amino is modified on the inner wall of a capillary tube; nano gold sol is put in a gas cylinder, the gold sol is pressed into the capillary tube through gas in the steel cylinder, and the nano-golden balls in the gold sol are self-assembled on the inner wall of the capillary tube; a di-thiol reagent s introduced into the capillary tube on which the first layer of nano-golden balls are modified, the gold sol is introduced again, and the second layer of nano-golden balls is self-assembled on the gold layer of the inner wall of the capillary tube; the steps are repeated, and the multiple layers of nano-golden balls can be self-assembled on the inner wall of the capillary tube; finally, a thiol reagent is introduced into the capillary tube, and the capillary tube liquid phase open tubular column is manufactured. The successfully-manufactured stationary phase is the liquid phase open tubular column with the multiple layers of nano-golden balls, and the liquid phase open tubular column can be used for chromatographic separation of 10-1,000 pieces of cell protein and peptide.
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of liquid phase open tubular column of multi-layer nano gold goal and its making side
Method and application.
Background technology
Nano gold spherical refers to the miniature spherical granule of gold, its diameter in 1～100 nm, with high electron density, dielectric spy
Property and catalytic action, can be combined with multiple biomacromolecules, and not affected its biological activity.Permissible by reducing process by gold chloride
The nano gold spherical of various different-grain diameters is easily prepared, and its color is taken on a red color to purple according to diameter.Nano gold spherical is because of which
Unique physicochemical properties and receive more and more extensive concern.Nano gold spherical can occur electrostatic mutual with amino easily
Effect, generates chemical bond between sulfydryl.Therefore nano gold spherical can be with the conditional inner surface for being independently mounted in capillary tube.
Conventional Si base or capillary glass tube inner surface can carry out interior finishing, for example modification amino or sulfydryl.Work as hair
When in tubule full of aurosol, the nano gold spherical for suspending in aurosol will pass through electrostatic attraction or chemical bond, independently
It is 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, the nano gold spherical Iy self-assembled layer will can bear more than ten than stronger
The liquid of air pressure washes away.
The metal level of the nanometer gold layers of balls of continuous multilayer is the complicated leading step that modifies as the decorative layer of capillary tube inner wall
Suddenly.Because which not only can transform inwall as conductor layer, facilitate control of Electric potentials and signal transduction, but also can greatly enrich
The functional molecular species of absorption, this is widely used in fields such as medical science detection, bio-separation, DNA identifications.
Meanwhile, the nanometer gold layers of balls of continuous multilayer is a loose structure, and its Kong Ze come from gold goal and gold goal it
Between pile up the space of formation.The size in space is possible to retain required hole similar to chromatograph.Therefore, through further repairing
Decorations, inwall has independently filled the capillary tube of multi-layer nano gold goal can become capillary tube liquid phase open tubular column, material is carried out separating and is divided
Analysis.The minimum volume of liquid phase open tubular column can be used to the trace protein in a small amount of cell of separation and peptide fragment, and this is at unicellular point
There is effect in analysis field very much.
Content of the invention
It is an object of the invention to provide a kind of liquid phase open tubular column of multi-layer nano gold goal and preparation method thereof and application.
Liquid phase open tubular column of multi-layer nano gold goal that the present invention is provided and preparation method thereof, its step is：
（1）First, pretreatment capillary tube inner wall, and amino silicane coupling agent reaction is passed through, amino is modified in capillary tube inner wall；
（2）Then, nano gold sol is pressed into inwall by gas to have modified in the capillary tube of amino, autonomous in capillary tube inner wall
Load onto ground floor gold goal；
（3）Again dimercapto reagent is passed through in the capillary tube that inwall has modified ground floor nano gold spherical, repaiies on ground floor gold goal
Decorations sulfydryl；
（4）Again nano gold sol is pressed into inwall by gas to have modified in the capillary tube with the nano gold spherical of sulfydryl, in capillary
Inside pipe wall independently loads onto second layer gold goal；
（5）Repeating the step of being passed through dimercapto reagent and nano gold sol, more layers gold goal is independently loaded onto in capillary tube inner wall；
（6）Sulfhydryl reagent is passed through in most backward capillary tube, makes the capillary tube of inwall self assembly multi-layer nano gold goal become liquid phase
Open tubular column.
By the fixing phase that makes for multi-layer nano gold goal liquid phase open tubular column for the protein enzymatic hydrolyzate of 100 cells
Chromatographic isolation, using Orbitrap MS on-line checking chromatographic fraction.
The manufacture method of the liquid phase open tubular column of the multi-layer nano gold goal that the present invention is provided, concrete operations flow process is as follows：
（1）Take a 200-1000 μm of external diameter, 5-500 μm of internal diameter, the glass of length 1-1000 cm or quartz capillary, 4-
At 50 DEG C, 0.1-1 mol/L NaOH, water, 0.1-1 mol/L HCl, water, CH is used successively3CN rinses 5-30 min respectively；
（2）Silane coupler reactant liquor is passed through step（1）In gained capillary tube, reacting by heating 0.5-24 h at 20-150 DEG C；
Silane coupling reagent includes but is not limited to 3- aminopropyl trimethoxysilane, 3- aminopropyl triethoxysilane（1-99%, v/v）
Solution, the solvent of solution includes but is not limited to methanol, ethanol, acetonitrile, propanol, dimethyl imide, dimethyl sulfoxide；Silane coupled
Cleaned with solvent after the completion of reaction, solvent includes but is not limited to methanol, ethanol, propanol, dimethyl imide；
（3）Nano gold spherical particle diameter is placed in bottleneck for the aurosol of 1-100 nm have in the gas cylinder of rubber stopper；Gas cylinder and
With the quartz capillary connection that internal diameter is 5-500 μm of internal diameter between steel cylinder；By step（2）Gained capillary tube is directly inserted in gas cylinder
In interior aurosol；Under 4-50 DEG C, 0.1-10 bar pressure, using the gas of gas bomb, gas includes but is not limited to nitrogen
Gas, argon, helium, 10-1000 times of capillary tube volume aurosol is pressed in capillary tube；
（4）Dimercapto reagent is placed in bottleneck have in the gas cylinder of rubber stopper；Dimercapto reagent includes but is not limited to dimercapto third
Alkane, dimercapto butane, dimercapto pentane, dimercaptohexane, dimercapto heptane, dimercapto octane, dimercapto nonane, the dimercapto last of the ten Heavenly stems
Alkane, concentration is 0.1-50%（v/v）, solvent including but not limited to methanol, ethanol, acetonitrile, propanol, dimethyl imide, diformazan Asia
Sulfone；With the quartz capillary connection that internal diameter is 5-500 μm of internal diameter between gas cylinder and steel cylinder；By step（3）Gained capillary tube is direct
It is inserted in the dimercapto reagent in gas cylinder；Under 4-50 DEG C, 0.1-10 bar pressure, using the gas of gas bomb, gas bag
Include but nitrogen, argon, helium is not limited to, 10-1000 times of capillary tube volume dimercapto reagent is pressed in capillary tube；
（5）Nano gold spherical particle diameter is placed in bottleneck for the aurosol of 1-100 nm have in the gas cylinder of rubber stopper；Gas cylinder and
With the quartz capillary connection that internal diameter is 5-500 μm of internal diameter between steel cylinder；By step（4）Gained capillary tube is directly inserted in gas cylinder
In interior aurosol；Under 4-50 DEG C, 0.1-10 bar pressure, using the gas of gas bomb, gas includes but is not limited to nitrogen
Gas, argon, helium, 10-1000 times of capillary tube volume aurosol is pressed in capillary tube；
（6）Repeat step（4）And step（5）, number of repetition 1 to 20 times；
（7）Sulfhydryl reagent is placed in bottleneck have in the gas cylinder of rubber stopper；Sulfhydryl reagent includes but is not limited to sulfydryl butane, sulfydryl
Pentane, sulfydryl heptane, sulfydryl octadecane, concentration is 0.1-50%（v/v）, solvent include but is not limited to methanol, ethanol, acetonitrile, third
Alcohol, dimethyl imide, dimethyl sulfoxide；With the quartz capillary connection that internal diameter is 5-500 μm of internal diameter between gas cylinder and steel cylinder；
By step（6）Gained capillary tube is directly inserted in the sulfhydryl reagent in gas cylinder；Under 4-50 DEG C, 0.1-10 bar pressure, use
The gas of gas bomb, gas includes but is not limited to nitrogen, argon, helium, by 10-1000 times of capillary tube volume sulfhydryl reagent pressure
Enter in capillary tube；Fixing phase liquid phase open tubular column for multi-layer nano gold goal is obtained.
The liquid phase open tubular column of the multi-layer nano gold goal prepared by the present invention, can be used to separate 10-1000 cell protein
And peptide.Concretely comprise the following steps：
（1）The liquid phase open tubular column of multi-layer nano gold goal is loaded chromatographic system；
（2）Using liquid phase open tubular column sample introduction of the chromatographic system to multi-layer nano gold goal, sample is the protease of 10-1000 cell
Solution liquid；
（3）Using chromatographic system elution step（2）The protein enzymatic hydrolyzate of 10-1000 cell in gained liquid phase open tubular column, stream
Dynamic is mutually A phase water, 0.1% formic acid, B phase acetonitrile, 0.1% formic acid；
（4）Using Orbitrap MS to step（3）Gained chromatographic fraction carries out on-line checking, and ion source is electric spray ion source.
It is characteristic of the invention that the porous layer fixing phase of open tubular column is built easily and fast using nano gold spherical, can be very
Operate in the capillary tube of wide inside diameter ranges.The liquid phase open tubular column column volume of gained is minimum, when separating a small amount of cell, peptide fragment and albumen
The diluting effect of matter is little, beneficial to detection.
Description of the drawings
Fig. 1 is the scanning electron microscope (SEM) photograph of liquid phase open tubular column cross section that 20 μm of internal diameter fixing phases of gained are four layers of nano gold spherical.
Fig. 2 is opened by the liquid phase that 20 μm of internal diameter fixing phases that Orbitrap MS is detected are four layers of nano gold spherical for gained
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 of liquid phase open tubular column cross section that 40 μm of internal diameter fixing phases of gained are six layers of nano gold spherical.
Fig. 4 is opened by the liquid phase that 40 μm of internal diameter fixing phases that Orbitrap MS is detected are six layers of nano gold spherical for gained
The total ion current figure of the protein enzymatic hydrolyzate fraction of 500 HeLa cells of tubing string.
Specific embodiment
The following examples are that the present invention is further illustrated, rather than limit the scope of the present invention.
Embodiment 1：20 μm of internal diameter fixing phases are the making of the liquid phase open tubular column of four layers of nano gold spherical：
（1）Take 2 m length, 365 μm of external diameters, the quartz capillary of 20 μm of internal diameters, under room temperature, use 0.2 mol/L successively
NaOH rinses 10 min, rinses 5 min with water, rinses 5 min with 0.1 mol/L HCl, rinses 5 min with water, uses CH3CN is rushed
Wash 5 min；
（2）100 μ L 3- aminopropyl trimethoxysilane are dissolved in 400 μ L acetonitriles, resulting solution is passed through step（1）Institute
Obtain in quartz capillary, 14 h of reacting by heating at 60 DEG C；
（3）The aurosol that nano gold spherical particle diameter is 60 nm is placed in bottleneck have in the gas cylinder of rubber stopper；Gas cylinder and steel cylinder
Between with the connection of quartz capillary that internal diameter is 100 μm of internal diameters；Step（2）Gained quartz capillary is directly inserted in gas cylinder
In aurosol；At 30 DEG C, under 2 bar pressure, using the helium of helium steel cylinder, 500 times of capillary tube volume aurosols are pressed into hair
In tubule；
（4）The dimethyl sulfoxide solution of dimercapto nonane is placed in bottleneck have in the gas cylinder of rubber stopper, concentration is 20%（v/v）；Gas
With the quartz capillary connection that internal diameter is 100 μm of internal diameters between bottle and steel cylinder；Step（3）Gained quartz capillary is directly inserted in
In dimercapto nonane reagent in gas cylinder；At 30 DEG C, under 2 bar pressure, using the helium of helium steel cylinder, by 100 times of capillary tubies
In volume dimercapto nonane reagent press-in capillary tube；
（5）The aurosol that nano gold spherical particle diameter is 60 nm is placed in bottleneck have in the gas cylinder of rubber stopper；Gas cylinder and steel cylinder
Between with the connection of quartz capillary that internal diameter is 100 μm of internal diameters；Step（4）Gained quartz capillary is directly inserted in gas cylinder
In aurosol；At 30 DEG C, under 2 bar pressure, using the helium of helium steel cylinder, 500 times of capillary tube volume aurosols are pressed into hair
In tubule；
（6）Repeat step（4）With（5）, number of repetition 2 times；
（7）The propanol solution of sulfydryl octadecane is placed in bottleneck have in the gas cylinder of rubber stopper, concentration is 30%（v/v）；Gas cylinder and
With the quartz capillary connection that internal diameter is 100 μm of internal diameters between steel cylinder；Step（6）Gained quartz capillary is directly inserted in gas cylinder
In interior sulfydryl octadecane reagent；At 30 DEG C, under 2 bar pressure, using the helium of helium steel cylinder, by 300 times of capillary tube volumes
In dimercapto nonane reagent press-in capillary tube.
Fig. 1 is the scanning electron microscope (SEM) photograph of liquid phase open tubular column cross section that 20 μm of internal diameter fixing phases of gained are four layers of nano gold spherical.
Embodiment 2：By liquid phase open tubular column that embodiment 1 gained, 20 μm of internal diameter fixing phases are four layers of nano gold spherical for 100
The chromatographic isolation of the protein enzymatic hydrolyzate of individual cell and Orbitrap MS on-line checking：
（1）1 gained liquid phase open tubular column of example is loaded nanoliter chromatographic system；
（2）Using nanoliter chromatographic system automatic sample handling system to 1 gained liquid phase open tubular column sample introduction of embodiment, sample be
The protein enzymatic hydrolyzate of Hep3B cell, sample size is 20 fmol；
（3）Using the protein enzymatic hydrolyzate of 100 Hep3B cells in 1 gained liquid phase open tubular column of nanoliter chromatographic system eluting example,
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% first
Acid) to 60% mobile phase A phase, 40% Mobile phase B phase；
（4）Using Orbitrap MS to step（10）Gained chromatographic fraction carries out on-line checking, and ion source is electron spray ion
Source.
Fig. 2 is opened by the liquid phase that 20 μm of internal diameter fixing phases that Orbitrap MS is detected are four layers of nano gold spherical for gained
The total ion current figure of the protein enzymatic hydrolyzate fraction of 100 Hep3B cells of tubing string.
Embodiment 3：40 μm of internal diameter fixing phases are the making of the liquid phase open tubular column of six layers of nano gold spherical：
（1）Take 3 m length, 365 μm of external diameters, the quartz capillary of 40 μm of internal diameters, under room temperature, use 0.1 mol/L successively
NaOH rinses 5 min, rinses 5 min with water, rinses 15 min with 0.2 mol/L HCl, rinses 35 min with water, uses CH3CN
Rinse 3 min；
（2）100 μ L 3- aminopropyl trimethoxysilane are dissolved in 500 μ L acetonitriles, resulting solution is passed through step（1）Institute
Obtain in quartz capillary, 12 h of reacting by heating at 80 DEG C；
（3）The aurosol that nano gold spherical particle diameter is 30 nm is placed in bottleneck have in the gas cylinder of rubber stopper；Gas cylinder and steel cylinder
Between with the connection of quartz capillary that internal diameter is 100 μm of internal diameters；Step（2）Gained quartz capillary is directly inserted in gas cylinder
In aurosol；At 25 DEG C, under 1 bar pressure, using the helium of helium steel cylinder, 400 times of capillary tube volume aurosols are pressed into hair
In tubule；
（4）The methanol solution of dimercapto nonane is placed in bottleneck have in the gas cylinder of rubber stopper, concentration is 15%（v/v）；Gas cylinder and
With the quartz capillary connection that internal diameter is 100 μm of internal diameters between steel cylinder；Step（3）Gained quartz capillary is directly inserted in gas cylinder
In interior dimercapto nonane reagent；At 25 DEG C, under 1 bar pressure, using the helium of helium steel cylinder, by 70 times of capillary tube volumes
In dimercapto nonane reagent press-in capillary tube；
（5）The aurosol that nano gold spherical particle diameter is 30 nm is placed in bottleneck have in the gas cylinder of rubber stopper；Gas cylinder and steel cylinder
Between with the connection of quartz capillary that internal diameter is 100 μm of internal diameters；Step（4）Gained quartz capillary is directly inserted in gas cylinder
In aurosol；At 25 DEG C, under 1 bar pressure, using the helium of helium steel cylinder, 400 times of capillary tube volume aurosols are pressed into hair
In tubule；
（6）Repeat step（4）With（5）, number of repetition 4 times；
（7）The propanol solution of sulfydryl octadecane is placed in bottleneck have in the gas cylinder of rubber stopper, concentration is 40%（v/v）；Gas cylinder and
With the quartz capillary connection that internal diameter is 100 μm of internal diameters between steel cylinder；Step（6）Gained quartz capillary is directly inserted in gas cylinder
In interior sulfydryl octadecane reagent；At 25 DEG C, under 1 bar pressure, using the helium of helium steel cylinder, by 400 times of capillary tube volumes
In dimercapto nonane reagent press-in capillary tube.
Fig. 3 is the scanning electron microscope (SEM) photograph of liquid phase open tubular column cross section that 40 μm of internal diameter fixing phases of gained are six layers of nano gold spherical.
Embodiment 4：By liquid phase open tubular column that embodiment 1 gained, 40 μm of internal diameter fixing phases are six layers of nano gold spherical for 500
The chromatographic isolation of the protein enzymatic hydrolyzate of individual cell and Orbitrap MS on-line checking：
（1）1 gained liquid phase open tubular column of example is loaded nanoliter chromatographic system；
（2）Using nanoliter chromatographic system automatic sample handling system to 1 gained liquid phase open tubular column sample introduction of embodiment, sample be
The protein enzymatic hydrolyzate of HeLa cell, sample size is 100 fmol；
（3）Using the protein enzymatic hydrolyzate of 500 HeLa cells in 1 gained liquid phase open tubular column of nanoliter chromatographic system eluting example,
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% first
Acid) to 60% mobile phase A phase, 40% Mobile phase B phase；
（4）Using Orbitrap MS to step（10）Gained chromatographic fraction carries out on-line checking, and ion source is electron spray ion
Source.
Fig. 4 is opened by the liquid phase that 40 μm of internal diameter fixing phases that Orbitrap MS is detected are six layers of nano gold spherical for gained
The total ion current figure of the protein enzymatic hydrolyzate fraction of 500 HeLa cells of tubing string.
Claims (7)
1. a kind of manufacture method of the liquid phase open tubular column of multi-layer nano gold goal, it is characterised in that concretely comprise the following steps：
（1）First, pretreatment capillary tube inner wall, and amino silicane coupling agent reaction is passed through, amino is modified in capillary tube inner wall；
（2）Then, nano gold sol is pressed into inwall by gas to have modified in the capillary tube of amino, autonomous in capillary tube inner wall
Load onto ground floor gold goal；
（3）Again dimercapto reagent is passed through in the capillary tube that inwall has modified ground floor nano gold spherical, repaiies on ground floor gold goal
Decorations sulfydryl；
（4）Again nano gold sol is pressed into inwall by gas to have modified in the capillary tube with the nano gold spherical of sulfydryl, in capillary
Inside pipe wall independently loads onto second layer gold goal；
（5）Repeating the step of being passed through dimercapto reagent and nano gold sol, more layers gold goal is independently loaded onto in capillary tube inner wall；
（6）Sulfhydryl reagent is passed through in most backward capillary tube, makes the capillary tube of inwall self assembly multi-layer nano gold goal become liquid phase
Open tubular column.
2. manufacture method according to claim 1, it is characterised in that concrete operations flow process is as follows：
（1）Take a 200-1000 μm of external diameter, 5-500 μm of internal diameter, the glass of length 1-1000 cm or quartz capillary, 4-
At 50 DEG C, 0.1-1 mol/L NaOH, water, 0.1-1 mol/L HCl, water, CH is used successively3CN rinses 5-30 min respectively；
（2）Silane coupler reactant liquor is passed through step（1）In gained capillary tube, reacting by heating 0.5-24 h at 20-150 DEG C；
Cleaned with solvent after the completion of silane coupled reaction；
（3）Nano gold spherical particle diameter is placed in bottleneck for the aurosol of 1-100 nm have in the gas cylinder of rubber stopper；Gas cylinder and
With the quartz capillary connection that internal diameter is 5-500 μm of internal diameter between steel cylinder；By step（2）Gained capillary tube is directly inserted in gas cylinder
In interior aurosol；Under 4-50 DEG C, 0.1-10 bar pressure, using the gas of gas bomb, by 10-1000 times of capillary tube
In volume aurosol press-in capillary tube；
（4）Dimercapto reagent is placed in bottleneck have in the gas cylinder of rubber stopper；It it is 5-500 μm with internal diameter between gas cylinder and steel cylinder
The quartz capillary connection of internal diameter；By step（3）Gained capillary tube is directly inserted in the dimercapto reagent in gas cylinder；In 4-50
DEG C, under 0.1-10 bar pressure, using the gas of gas bomb, 10-1000 times of capillary tube volume dimercapto reagent is pressed into hair
In tubule；
（5）Nano gold spherical particle diameter is placed in bottleneck for the aurosol of 1-100 nm have in the gas cylinder of rubber stopper；Gas cylinder and
With the quartz capillary connection that internal diameter is 5-500 μm of internal diameter between steel cylinder；By step（4）Gained capillary tube is directly inserted in gas cylinder
In interior aurosol；Under 4-50 DEG C, 0.1-10 bar pressure, using the gas of gas bomb, by 10-1000 times of capillary tube
In volume aurosol press-in capillary tube；
（6）Repeat flow process（4）And flow process（5）, number of repetition 1 to 20 times；
（7）Sulfhydryl reagent is placed in bottleneck have in the gas cylinder of rubber stopper；Between gas cylinder and steel cylinder with internal diameter it is in 5-500 μm
The quartz capillary connection in footpath；By flow process（6）Gained capillary tube is directly inserted in the sulfhydryl reagent in gas cylinder；4-50 DEG C,
Under 0.1-10 bar pressure, using the gas of gas bomb, 10-1000 times of capillary tube volume sulfhydryl reagent is pressed into capillary tube
In；Fixing phase liquid phase open tubular column for multi-layer nano gold goal is obtained.
3. manufacture method according to claim 2, it is characterised in that flow process（1）In, the silane coupling reagent is selected from 3-
Aminopropyl trimethoxysilane or 3- aminopropyl triethoxysilane solution, the solvent selected from methanol of solution, ethanol, acetonitrile, third
Alcohol, dimethyl imide or dimethyl sulfoxide；Solution concentration is 1-99%（v/v）.
4. manufacture method according to claim 2, it is characterised in that flow process（4）And flow process（7）In, the dimercapto examination
Agent is selected from dimercaptopropane, dimercapto butane, dimercapto pentane, dimercaptohexane, dimercapto heptane, dimercapto octane, two mercaptos
Base nonane, dimercapto decane, concentration is 0.1-50%（v/v）, solvent selected from methanol, ethanol, acetonitrile, propanol, dimethyl Asia acyl
Amine, dimethyl sulfoxide.
5. manufacture method according to claim 2, it is characterised in that the cyclinder gas is nitrogen, argon or helium.
6. the liquid phase open tubular column of the multi-layer nano gold goal that a kind of manufacture method described in one of claim 1-5 is obtained.
7. the liquid phase open tubular column of multi-layer nano gold goal as claimed in claim 6, in 10-1000 cell is separated protein with
The application of peptide, it is characterised in that concretely comprise the following steps：
（1）The liquid phase open tubular column of multi-layer nano gold goal is loaded chromatographic system；
（2）Using liquid phase open tubular column sample introduction of the chromatographic system to multi-layer nano gold goal, sample is the protease of 10-1000 cell
Solution liquid；
（3）Using chromatographic system elution step（2）The protein enzymatic hydrolyzate of 10-1000 cell in gained liquid phase open tubular column；
（4）Using Orbitrap MS to step（3）Gained chromatographic fraction carries out on-line checking, and ion source is electric spray ion source.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610946040.4A CN106442796B (en) | 2016-10-26 | 2016-10-26 | A kind of liquid phase open tubular column of multi-layer nano gold goal and preparation method thereof and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610946040.4A CN106442796B (en) | 2016-10-26 | 2016-10-26 | A kind of liquid phase open tubular column of multi-layer nano gold goal and preparation method thereof and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106442796A true CN106442796A (en) | 2017-02-22 |
| CN106442796B CN106442796B (en) | 2019-12-03 |
Family
ID=58177703
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610946040.4A Expired - Fee Related CN106442796B (en) | 2016-10-26 | 2016-10-26 | A kind of liquid phase open tubular column of multi-layer nano gold goal and preparation method thereof and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106442796B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115356425A (en) * | 2022-07-27 | 2022-11-18 | 上海奥浦迈生物科技股份有限公司 | NanoHPLC-Titer system applied to culture medium supernatant albumin quantification |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103336081A (en) * | 2013-07-04 | 2013-10-02 | 福州大学 | Preparation method of gold nanoparticle modification based open tubular column |
| CN103983769A (en) * | 2014-03-25 | 2014-08-13 | 中国海洋大学 | Preparation method for nano-gold immunity chromatography capillary |
| WO2014121781A1 (en) * | 2013-02-11 | 2014-08-14 | RENNEBERG, Max | Method for visually ruling out acute diseases using colour-marked proteins |
| CN104606919A (en) * | 2014-12-31 | 2015-05-13 | 华南师范大学 | Preparation method of novel nucleic acid aptamer/nano-gold modified solid phase microextraction quartz capillary tube |
| CN104624167A (en) * | 2013-11-08 | 2015-05-20 | 中国科学院大连化学物理研究所 | Preparation of nucleic acid aptamer immobilized integral material, and applications of nucleic acid aptamer immobilized integral material in protein online detection |
| TW201607542A (en) * | 2014-08-19 | 2016-03-01 | 行政院原子能委員會核能研究所 | Method of bonding nano-gold and doxorubicin |
-
2016
- 2016-10-26 CN CN201610946040.4A patent/CN106442796B/en not_active Expired - Fee Related
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014121781A1 (en) * | 2013-02-11 | 2014-08-14 | RENNEBERG, Max | Method for visually ruling out acute diseases using colour-marked proteins |
| CN103336081A (en) * | 2013-07-04 | 2013-10-02 | 福州大学 | Preparation method of gold nanoparticle modification based open tubular column |
| CN104624167A (en) * | 2013-11-08 | 2015-05-20 | 中国科学院大连化学物理研究所 | Preparation of nucleic acid aptamer immobilized integral material, and applications of nucleic acid aptamer immobilized integral material in protein online detection |
| CN103983769A (en) * | 2014-03-25 | 2014-08-13 | 中国海洋大学 | Preparation method for nano-gold immunity chromatography capillary |
| TW201607542A (en) * | 2014-08-19 | 2016-03-01 | 行政院原子能委員會核能研究所 | Method of bonding nano-gold and doxorubicin |
| CN104606919A (en) * | 2014-12-31 | 2015-05-13 | 华南师范大学 | Preparation method of novel nucleic acid aptamer/nano-gold modified solid phase microextraction quartz capillary tube |
Non-Patent Citations (2)
| Title |
|---|
| SINÉAD CURRIVAN等: "Production of polymer monolithic capillary columns with integrated gold nano-particle modified segments for on-capillary extraction", 《MICROCHEMICAL JOURNAL》 * |
| 王梦兰等: "石英毛细管内壁镀金工艺的研究", 《表面技术》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115356425A (en) * | 2022-07-27 | 2022-11-18 | 上海奥浦迈生物科技股份有限公司 | NanoHPLC-Titer system applied to culture medium supernatant albumin quantification |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106442796B (en) | 2019-12-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Slovakova et al. | Use of self assembled magnetic beads for on-chip protein digestion | |
| Wilson et al. | Nano liquid chromatography columns | |
| US6387257B1 (en) | Apparatus for screening compound libraries | |
| Feng et al. | Advances in coupling microfluidic chips to mass spectrometry | |
| Gibson et al. | Nanoelectrospray emitters: trends and perspective | |
| US6656739B2 (en) | Methods for screening compound libraries | |
| Liu et al. | Capillary electrophoresis-based immobilized enzyme reactor using particle-packing technique | |
| Wang et al. | Plasma-assisted alignment in the fabrication of microchannel-array-based in-tube solid-phase microextraction microchips packed with TiO2 nanoparticles for phosphopeptide analysis | |
| Barceló‐Barrachina et al. | State‐of‐the‐art of the hyphenation of capillary electrochromatography with mass spectrometry | |
| US20060214099A1 (en) | Polymer entrapped particles | |
| Bagheri et al. | An unbreakable on-line approach towards sol–gel capillary microextraction | |
| JP7099965B2 (en) | Systems and methods for analyzing analytes extracted from samples using adsorbent materials | |
| Li et al. | High-efficiency nano/micro-reactors for protein analysis | |
| CN105126793B (en) | A kind of preparation method based on single stranded DNA nucleic acid aptamers modification hybrid quartz capillary integral post | |
| Huang et al. | Facile synthesis of bifunctional polymer monolithic column for tunable and specific capture of glycoproteins and phosphoproteins | |
| Lin et al. | Preparation of iminodiacetic acid functionalized silica capillary trap column for on-column selective enrichment of N-linked glycopeptides | |
| CN106442796B (en) | A kind of liquid phase open tubular column of multi-layer nano gold goal and preparation method thereof and application | |
| CN101406828B (en) | Method for producing open tubular electrophoresis column coated with nano titanic oxide and uses thereof | |
| JP2010044066A (en) | Lab-on-chip having micro fluid circuit and electrospray nozzle on the same plane | |
| CN109865315A (en) | A kind of organic-inorganic hybrid mesoporous material coating preparation method as chromatographic stationary phases | |
| Safdar et al. | Microscale enzyme reactors comprising gold nanoparticles with immobilized trypsin for efficient protein digestion. | |
| CN103990298B (en) | A kind of preparation method of surface and hydrophilic outer macropore organic-inorganic hybridization monolithic column | |
| Behrmann et al. | A microfluidic porous solid phase suitable for mass production | |
| CN1982890A (en) | Liquid-phase separating system and mass-spectrum connecting interface, its production and use | |
| Nissilä et al. | Fully polymeric integrated microreactor/electrospray ionization chip for on-chip digestion and mass spectrometric analysis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20191203 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |