CN101377463B - Method and application for preparing carbon nano tube of histidine label protein function - Google Patents
Method and application for preparing carbon nano tube of histidine label protein function Download PDFInfo
- Publication number
- CN101377463B CN101377463B CN2008101524244A CN200810152424A CN101377463B CN 101377463 B CN101377463 B CN 101377463B CN 2008101524244 A CN2008101524244 A CN 2008101524244A CN 200810152424 A CN200810152424 A CN 200810152424A CN 101377463 B CN101377463 B CN 101377463B
- Authority
- CN
- China
- Prior art keywords
- carbon nano
- tube
- mwcnt
- histidine
- label protein
- 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.)
- Expired - Fee Related
Links
Landscapes
- Carbon And Carbon Compounds (AREA)
Abstract
A preparation method for histidine label protein functional carbon nanotube and an application thereof; the preparation method comprises the following steps: firstly preparing carboxylated carbon nanotube; and then further reacting to obtain activated lactonized carbon nanotube; reacting the histidine label protein expressed by the transgenic colibacillus with the activated nanotube to prepare the histidine label protein functional carbon nanotube. The invention develops a simple, fast, economical method for high-sensitively and high-selectively detecting trace metal elements within complex system by making use of the multifunctional decorative characteristic of the carbon nanotube and the high loading characteristic of the nanometre material. The invention can high-sensitively and high-selectively detect the target element when detecting heavy metals or certain excessive metals within the complex system, can eliminate the influence of other elements.
Description
[technical field]: the present invention relates to the biomolecule functionalized carbon nanotube, the trace metal element that is used for selectivity detection of complex biology and environmental system belongs to the technical field of analysis and detection of metal ingredient in nano material preparation technology and biology, the circumstance complication system.
[background technology]: some specific transitions metals all have toxicity and pathogenic for most of life entity after surpassing finite concentration.Even under very low concentration,, very strong toxicity is arranged all for humans and animals.And it does not possess biodegradability, is easy to pile up in vivo, causes various diseases and imbalance.
Carbon nano-tube be by the single or multiple lift graphite flake around central shaft by certain helix angle reel " microtubule " of seamless, the hollow that forms.According to the difference of formation condition, carbon nano-tube exists multi-walled carbon nano-tubes (MWNTs) and two kinds of forms of Single Walled Carbon Nanotube (SWNTs).Which floor MWNTs generally be made of to the coaxial coiling of tens layers graphite flake, and the interlayer spacing is about 0.34nm, and its typical diameter and length are respectively 2～30nm and 0.1～50 μ m.Carbon nano-tube is because of having characteristics such as size is little, physical strength is high, specific surface is big, conductivity is high, interfacial effect is strong, thereby make it have special machinery, physics, chemical property, in numerous areas such as construction material, catalysis, absorption one separation, energy storage device electrode material, have important application prospects.
Though the preenrichment connection metal of carbon nano-tube before analytic sample done a lot of application, selectivity is not high.Therefore we have developed high sensitivity, high selectivity preenrichment, directly metal ingredient in detection of biological, the circumstance complication system.
Flow injection (FI) on-line adsorption is separated, preenrichment demonstrates great potential, and in the mode that becomes a kind of the most potential automatic sample analysis these years.The online preenrichment of flow injection, separation and NITRATE BY FLAME ATOMIC absorb coupling has become the effective ways that detect trace element by enhancing signal, possesses high sample flux, characteristics such as low consumption.This work promptly adopts this mode to realize the particular characteristic of material property.
[summary of the invention]: the objective of the invention is to solve the enrichment of unmodified carbon nano-tube, separate in the problem of poor selectivity, provide a kind of can high sensitivity, the preparation method of carbon nano-tube metal ingredient, histidine label protein function in high selectivity preenrichment and direct quantitative detection of biological, the circumstance complication system.
The preparation method of the carbon nano-tube of histidine label protein function provided by the invention comprises the steps:
(a) in opening wide reaction vessel, add 0.2～2g carbon nano-tube MWCNT, and the concentrated sulphuric acid and red fuming nitric acid (RFNA) are 1: 1～3: 1 acid mixture by volume, the mass volume ratio m/V of carbon nano-tube and acid mixture is 1: 20, the ultrasonic dispersion 60～120min of normal temperature makes homodisperse MWCNT black suspension;
(b) get MWCNT black suspension that 15～25mL (a) step makes in beaker, be heated to 65 ℃ to 75 ℃, 5～15h;
(c) in (b) step, add distilled water diluting, the centrifugal 5min of 7000rpm, supernatant discarded acid solution;
(d) in (c) step, add distilled water again,, use pH=6～7 of distilled water cyclic washing filtrate again to filtered fluid by the polycarbonate membrane filtration of 0.22 μ m; Vacuum drying obtains carbon nano-tube formic acid MWCNT-COOH;
(e) connect pET-22b (+) vector expression by the pMDGLP-D plasmid and produce reorganization pET-22b eight copy expression vectors; The pET-22b eight copy carriers of will recombinating again change e. coli bl21 (DE3) bacterial strain over to and produce e. coli bl21 (DE3)/G8 bacterial strain; In 50mL LB fluid nutrient medium, 37 ℃ of shaken cultivation are spent the night with e. coli bl21 (DE3)/G8 inoculation, and adding derivant IPTG is 0.2～0.6mM to final concentration, abduction delivering; Bacterium liquid under 4 ℃, 8000～12, the centrifugal 5min of 000rpm, cell suspension is handled with ultrasonic disruption, collects soluble protein; Use 6 polyhistidyl label affinity chromatography column separating purifications again, obtain histidine-tagged protein matter;
(f) carbon nano-tube of preparation histidine label protein function adds the recovery product MWCNT-COOH in (d) step in the there-necked flask of 100mL, MWCNT-COOH is activated and be reacted into lactone groups with NHS and EDAC respectively.Suspension separates by the PTFE membrane filtration, and washs with ethanol and distilled water.
(g) go on foot the histidine-tagged protein reaction that obtains with the carbon nano-tube of the activated lactoneization in (f) step and (e), be scattered in the PBS solution, be blended in 2-morpholino b acid (MES) buffer solution (pH=6.4).The mixed solution lucifuge stirs 2～10h, promptly makes the carbon nano-tube of histidine label protein function.
(a) going on foot described acid mixture is that volume ratio is 3: 1 dense H
2SO
4With dense HNO
3
The carbon nano-tube of the histidine label protein function of method for preparing can be used as solid extracting agent, is used for the selective adsorption of transition metal Cu or Ni, and is applied to contain the sample determination of respective element.
Advantage of the present invention and good effect:
But can reach the characteristic of multi-functional modification and trim high capacity amount by the carbon nano-tube of this method preparation, develop a kind of method of trace metal element of detection of complex system of simple, quick, economic, highly sensitive and high selectivity.During some specific transitions metal in the detection of complex system of this method, can high sensitivity, the accurate detection of high selectivity, avoid the interference of other element.
[embodiment]: followingly technical scheme of the present invention is further described by several specific embodiments.
Embodiment 1
(a) in opening wide reaction vessel, adding 1g carbon nano-tube MWCNT and 20mL volume ratio are 3: 1 dense H
2SO
4With dense HNO
3, the ultrasonic dispersion of normal temperature 90min makes homodisperse black suspension;
(b) preparation of MWCNT-COOH: get MWCNT black suspension that 20mL (a) step makes in beaker, be heated to about 70 ℃ 7h;
(c) add distilled water diluting, centrifugal 5 minutes of 7000rpm, supernatant discarded acid solution.3 times repeatedly.
(d) add distilled water again, by the polycarbonate membrane filtration of 0.22 μ m.Be neutral (pH=7) with distilled water cyclic washing filtrate to filtrate again.Vacuum drying obtains carbon nano-tube formic acid MWCNT-COOH.
(e) connect pET-22b (+) vector expression by the pMDGLP-D plasmid and produce reorganization pET-22b eight copy expression vectors, the pET-22b eight copy expression vectors of will recombinating again change e. coli bl21 (DE3) bacterial strain over to and produce e. coli bl21 (DE3)/G8 bacterial strain; In 50ml LB fluid nutrient medium, 37 ℃ of shaken cultivation are spent the night with e. coli bl21 (DE3)/G8 inoculation, and adding derivant IPTG is 0.4mM to final concentration, abduction delivering.Bacterium liquid under 4 ℃, 12, the centrifugal 5min of 000rpm, cell suspension is handled with ultrasonic disruption, collects soluble protein.Use 6 polyhistidyl label affinity chromatography column separating purifications again, obtain histidine-tagged protein matter.
(f) the recovery product MWCNT-COOH in adding (d) step in the there-necked flask of 100mL can activate and be reacted into lactone groups with NHS and EDAC respectively with MWCNT-COOH.Suspension separates by the PTFE membrane filtration, and washs with ethanol and distilled water.
(g) with the carbon nano-tube of the activated lactoneization in (f) step, it is scattered in the PBS solution and adds histidine-tagged protein matter and is blended in 2-morpholino b acid (MES) buffer solution (pH=6.4).The mixed solution lucifuge stirs 10h., promptly makes the carbon nano-tube of histidine label protein function.
Embodiment 2
(a) in opening wide reaction vessel, adding 1.5g carbon nano-tube MWCNT and 30mL volume ratio are 1: 1 dense H
2SO
4With dense HNO
3, the ultrasonic dispersion of normal temperature 120min makes homodisperse MWCNT black suspension;
(b) preparation of MWCNT-COOH: get MWCNT black suspension that 20mL (a) step makes in beaker, be heated to about 65 ℃ 14h;
(c) add distilled water diluting, the centrifugal 5min of 7000rpm, supernatant discarded acid solution.3 times repeatedly.
(d) add distilled water again, by the polycarbonate membrane filtration of 0.22 μ m.Be neutral with distilled water cyclic washing filtrate to filtrate again.Vacuum drying obtains MWCNT-COOH.
(e) connect pET-22b (+) vector expression by the pMDGLP-D plasmid and produce reorganization pET-22b eight copy expression vectors; The pET-22b eight copy carriers of will recombinating again change e. coli bl21 (DE3) bacterial strain over to and produce e. coli bl21 (DE3)/G8 bacterial strain; With e. coli bl21 (DE3)/G8 inoculation in 50mL LB fluid nutrient medium, 37 ℃ of shaken cultivation 6h, adding derivant IPTG is 0.3mM to final concentration, abduction delivering.Bacterium liquid under 4 ℃, 8, the centrifugal 5min of 000rpm, cell suspension is handled with ultrasonic disruption, collects soluble protein.Obtain histidine-tagged protein matter with 6 polyhistidyl label affinity chromatography column separating purifications again.
(f) the recovery product in adding (d) step in the there-necked flask of 100mL can activate and be reacted into lactone groups with NHS and EDAC respectively with MWCNT-COOH.Suspension separates by the PTFE membrane filtration, and washs with ethanol and distilled water.
(g) with the carbon nano-tube of the activated lactoneization in (f) step, it is scattered in the PBS solution and adds histidine-tagged protein matter and is blended in 2-morpholino b acid (MES) buffer solution (pH=6.4).The mixed solution lucifuge stirs 2h., promptly makes the carbon nano-tube of histidine label protein function.
Embodiment 3
(a) in opening wide reaction vessel, adding 0.5g carbon nano-tube MWCNT and 10mL volume ratio are 2: 1 dense H
2SO
4With dense HNO
3, the ultrasonic dispersion of normal temperature 120min makes homodisperse MWCNT black suspension;
(b) preparation of MWCNT-COOH: get MWCNT black suspension that 15mL (a) step makes in beaker, be heated to about 65 ℃ 9h;
(c) add distilled water diluting, the centrifugal 5min of 7000rpm, supernatant discarded acid solution.3 times repeatedly.
(d) add distilled water again, by the polycarbonate membrane filtration of 0.22 μ m.Be neutral with distilled water cyclic washing filtrate to filtrate again.Vacuum drying obtains MWCNT-COOH.
(e) connect pET-22b (+) vector expression by the pMDGLP-D plasmid and produce reorganization pET-22b eight copy expression vectors; The pET-22b eight copy carriers of will recombinating again change e. coli bl21 (DE3) bacterial strain over to and produce e. coli bl21 (DE3)/G8 bacterial strain; With e. coli bl21 (DE3)/G8 inoculation in 50mL LB fluid nutrient medium, 37 ℃ of shaken cultivation 6h, adding derivant IPTG is 0.6mM to final concentration, abduction delivering.Bacterium liquid under 4 ℃, 8, the centrifugal 5min of 000rpm, cell suspension is handled with ultrasonic disruption, collects soluble protein.Obtain histidine-tagged protein matter with 6 polyhistidyl label affinity chromatography column separating purifications again.
(f) the recovery product in adding (d) step in the there-necked flask of 100mL can activate and be reacted into lactone groups with NHS and EDAC respectively with MWCNT-COOH.Suspension separates by the PTFE membrane filtration, and washs with ethanol and distilled water.
(g) with the carbon nano-tube of the activated lactoneization in (f) step, it is scattered in the PBS solution and adds histidine-tagged protein matter and is blended in 2-morpholino b acid (MES) buffer solution (pH=6.4).The mixed solution lucifuge stirs 2h., promptly makes the carbon nano-tube of histidine label protein function.
Embodiment 4
(a) the histidine protein functionalized carbon nanotube 15mg of microtrabeculae filling.
(b) online NITRATE BY FLAME ATOMIC absorbs coupling mensuration Cu
2+In, with respect to the carbon nano-tube of unmodified, aspect other metallic ions coexistence anti-interference, 1700 lifting is being arranged at most.
(c) sample enrichment on 60s, flow velocity are 5.0mL min
-1Process in, Adsorption of Cu
2+The enrichment multiple is 28 times.
(d) the sample flux is 40h
-1The detection limit of measuring (3 σ) is 0.31 μ gL
-1Repeat 11 times continuously and measure 10 μ gL
-1The precision of Cu (RSD) is 2.4%.
Embodiment 5
(a) the histidine protein functionalized carbon nanotube 15mg of microtrabeculae filling.
(b) online NITRATE BY FLAME ATOMIC absorbs coupling mensuration Ni
2+In, with respect to the carbon nano-tube of unmodified, aspect other metallic ions coexistence anti-interference, 20000 times lifting is being arranged at most.
(c) sample enrichment on 60s, flow velocity are 5.0mL min
-1Process in, absorption Ni
2+The enrichment multiple be 29 times.
(d) the sample flux is 40h
-1The detection limit of measuring (3 σ) is respectively 0.32 μ g L
-1Repeat 11 times continuously and measure 15 μ gL
-1The precision of Ni (RSD) is 2.5%.
Claims (3)
1. the preparation method of the carbon nano-tube of histidine label protein function is characterized in that this method comprises the steps:
(a) in opening wide reaction vessel, add 0.2～2g carbon nano-tube MWCNT, and the concentrated sulphuric acid and red fuming nitric acid (RFNA) are 1: 1～3: 1 acid mixture by volume, the mass volume ratio m/V of carbon nano-tube and acid mixture is 1: 20, the ultrasonic dispersion 60～120min of normal temperature makes homodisperse MWCNT black suspension;
(b) get MWCNT black suspension that 15～25mL (a) step makes in beaker, be heated to 65 ℃ to 75 ℃, 5～15h;
(c) add distilled water diluting, the centrifugal 5min of 7000rpm, supernatant discarded acid solution in the black suspension after the heating of (b) step;
(d) add distilled water in the final product after (c) step supernatant discarded acid solution again,, use pH=6～7 of distilled water cyclic washing filtrate again to filtered fluid by the polycarbonate membrane filtration of 0.22 μ m; Vacuum drying obtains carbon nano-tube formic acid MWCNT-COOH;
(e) connect pET-22b (+) vector expression by the pMDGLP-D plasmid and produce reorganization pET-22b eight copy expression vectors; The pET-22b eight copy carriers of will recombinating again change e. coli bl21 (DE3) bacterial strain over to and produce e. coli bl21 (DE3)/G8 bacterial strain; In 50mL LB fluid nutrient medium, 37 ℃ of shaken cultivation are spent the night with e. coli bl21 (DE3)/G8 inoculation, and adding derivant IPTG is 0.2～0.6mM to final concentration, abduction delivering; Bacterium liquid under 4 ℃, 8000～12, the centrifugal 5min of 000rpm, cell suspension is handled with ultrasonic disruption, collects soluble protein; Use 6 polyhistidyl label affinity chromatography column separating purifications again, obtain histidine-tagged protein matter;
(f) carbon nano-tube of preparation histidine label protein function, add the recovery product MWCNT-COOH in (d) step in the there-necked flask of 100mL, MWCNT-COOH is activated and is reacted into lactone groups with NHS and EDAC respectively, suspension separates by the PTFE membrane filtration, and washs with ethanol and distilled water;
(g) go on foot the histidine-tagged protein reaction that obtains with the carbon nano-tube of the activated lactoneization in (f) step and (e), be scattered in the PBS solution, be blended in the 2-morpholino b acid MES buffer solution pH=6.4; The mixed solution lucifuge stirs 2～10h., promptly makes the carbon nano-tube of histidine label protein function.
2. method according to claim 1 is characterized in that described acid mixture of (a) step is that volume ratio is 3: 1 dense H
2SO
4With dense HNO
3
3. the application of the carbon nano-tube of the histidine label protein function of the described method preparation of claim 1, with the carbon nano-tube of histidine label protein function as solid extracting agent, be used for the selective adsorption of transition metal Cu or Ni, and be applied to contain the sample determination of respective element.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101524244A CN101377463B (en) | 2008-10-22 | 2008-10-22 | Method and application for preparing carbon nano tube of histidine label protein function |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101524244A CN101377463B (en) | 2008-10-22 | 2008-10-22 | Method and application for preparing carbon nano tube of histidine label protein function |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101377463A CN101377463A (en) | 2009-03-04 |
| CN101377463B true CN101377463B (en) | 2011-11-23 |
Family
ID=40421109
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2008101524244A Expired - Fee Related CN101377463B (en) | 2008-10-22 | 2008-10-22 | Method and application for preparing carbon nano tube of histidine label protein function |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101377463B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101325282B1 (en) * | 2011-08-18 | 2013-11-01 | 연세대학교 산학협력단 | Bioactive carbon nanotube functionalized by β-sheet block copolypeptide and preparing method the same |
| CN102642825B (en) * | 2012-04-16 | 2013-12-25 | 天津师范大学 | Histidine-functionalized carbon nanotube and preparation method and application thereof |
| CN113735935A (en) * | 2021-07-27 | 2021-12-03 | 罗义 | Detection method of new coronavirus capable of enriching free nucleic acid by selectively adsorbing nucleoprotein |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1860370A (en) * | 2003-10-29 | 2006-11-08 | 英特尔公司 | Methods and device for analyte characterization |
| CN1922106A (en) * | 2004-02-16 | 2007-02-28 | 独立行政法人科学技术振兴机构 | Carbon nanotube structure-selective separation and surface fixation |
| CN101194162A (en) * | 2005-06-28 | 2008-06-04 | 韩国化学研究院 | Carbon nanotube transistor biosensors with aptamers as molecular recognition elements and method for sensing a target material using the same |
-
2008
- 2008-10-22 CN CN2008101524244A patent/CN101377463B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1860370A (en) * | 2003-10-29 | 2006-11-08 | 英特尔公司 | Methods and device for analyte characterization |
| CN1922106A (en) * | 2004-02-16 | 2007-02-28 | 独立行政法人科学技术振兴机构 | Carbon nanotube structure-selective separation and surface fixation |
| CN101194162A (en) * | 2005-06-28 | 2008-06-04 | 韩国化学研究院 | Carbon nanotube transistor biosensors with aptamers as molecular recognition elements and method for sensing a target material using the same |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101377463A (en) | 2009-03-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Bezzon et al. | Carbon nanostructure‐based sensors: a brief review on recent advances | |
| Yilmaz et al. | Functionalized nanomaterials for sample preparation methods | |
| Wang et al. | A SiO2@ MIP electrochemical sensor based on MWCNTs and AuNPs for highly sensitive and selective recognition and detection of dibutyl phthalate | |
| Liu et al. | Nanomaterials for analysis and monitoring of emerging chemical pollutants | |
| Scida et al. | Recent applications of carbon-based nanomaterials in analytical chemistry: critical review | |
| Singh et al. | Recent advancement of carbon nanomaterials engrained molecular imprinted polymer for environmental matrix | |
| Liu et al. | A novel electrochemical sensing platform of JUC-62 metal-organic framework/platelet ordered mesoporous carbon for high selective detection of nitro-aromatic compounds | |
| Hun et al. | Aptamer biosensor for highly sensitive and selective detection of dopamine using ubiquitous personal glucose meters | |
| Li et al. | Recent advances in nanomaterials for analysis of trace heavy metals | |
| Gong et al. | Application of nanotechnology in analysis and removal of heavy metals in food and water resources | |
| Mavumengwana-Khanyile et al. | Recent advances in sorbents applications and techniques used for solid-phase extraction of atrazine and its metabolites deisopropylatrazine and deethylatrazine: a review | |
| CN101377463B (en) | Method and application for preparing carbon nano tube of histidine label protein function | |
| CN101806766B (en) | Hydroxypropyl /carbon nanotube decoration electrochemical sensor and preparation method and application thereof | |
| Wei et al. | A review of current status of ratiometric molecularly imprinted electrochemical sensors: From design to applications | |
| Yu et al. | Nanomaterials-based ion-imprinted electrochemical sensors for heavy metal ions detection: a review | |
| Azzouz et al. | Advancements in nanomaterial-based aptasensors for the detection of emerging organic pollutants in environmental and biological samples | |
| F Samanidou et al. | Carbon nanotubes in sample preparation | |
| CN102642825B (en) | Histidine-functionalized carbon nanotube and preparation method and application thereof | |
| Shoala | Carbon nanostructures: Detection, controlling plant diseases and mycotoxins | |
| CN102313768A (en) | Modified glassy carbon electrode (GCE) capable of simultaneously determining dopamine and uric acid in presence of ascorbic acid as well as preparation method and application thereof | |
| Jinadasa et al. | Smart materials for mercury and arsenic determination in food and beverages | |
| Huang et al. | Recent progresses on biosensors for Escherichia coli detection | |
| Xue et al. | Research progress in detection technology of polycyclic aromatic hydrocarbons | |
| Anthemidis et al. | Unmodified multi-walled carbon nanotubes as sorbent material in flow injection on-line sorbent extraction preconcentration system for cadmium determination by flame atomic absorption spectrometry | |
| Çalışır et al. | Microwave-assisted cross-coupling synthesis of aryl functionalized MWCNTs and investigation of hydrogen storage properties |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20111123 Termination date: 20121022 |