CN106918633A - The detection method of the cytokine TNF α based on fit and golden magnetic nano particle - Google Patents
The detection method of the cytokine TNF α based on fit and golden magnetic nano particle Download PDFInfo
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- CN106918633A CN106918633A CN201710271468.8A CN201710271468A CN106918633A CN 106918633 A CN106918633 A CN 106918633A CN 201710271468 A CN201710271468 A CN 201710271468A CN 106918633 A CN106918633 A CN 106918633A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
Abstract
This case is related to the detection method of the cytokine TNF α based on fit and golden magnetic nano particle, including：Using magnetic glassy carbon electrode as working electrode；Gold is modified in Fe3O4Nano particle, is obtained gold magnetic nano particle；DNA probe 1 is modified in golden magnetic nano particle surface；DNA probe 2 is carried out into hybridization reaction with the DNA probe 1 on golden magnetic nano particle surface；Golden magnetic nano particle is adsorbed in glassy carbon electrode surface by magnetic fields；Reacted during magnetic glassy carbon electrode is inserted into prepare liquid, and gathered corresponding electrochemical response value, according to electrochemical response value and the corresponding relation of cytokine TNF α concentration, obtained the concentration of cytokine TNF α in prepare liquid.The characteristics of this case has sensitivity high, quick, low cost, it is possible to achieve the application of TNF α bio-sensings；By introducing golden magnetic composite nano materials, fit fixation is cannot be only used for, can also realize the recycling of electrode.
Description
Technical field
It is more particularly to a kind of based on fit and golden magnetic nanometer the present invention relates to a kind of detection method of cytokine TNF-α
The detection method of the cytokine TNF-α of particle.
Background technology
TNF (tumor necrosis factor, TNF) is that one kind of appearance in serum can make kinds of tumors
There is the albumen of hemorrhagic necrosis.The main macrophages by activating of TNF, NK cells and T lymphocytes are produced.1985
The TNF of macrophages secrete is named as TNF-α by Shalaby.TNF-α is a kind of small molecular protein, after being combined with specific receptors
Various biological effect, including immune activation, inflammatory reaction, cell growth related gene activation expression, killing or suppression can be played
Tumour cell processed etc..Study carefully and show, TNF-α can substantially increase the damage of liver cell and cause kidney damage, reduce cirrhosis companion
The prognosis of spontaneous peritonitis patient simultaneously significantly rises high mortality.TNF-α antagonism treats the lesion of some diseases of sustainable improvement.
Additionally, there is researcher to find, it is significantly raised that ebv infection can cause body to express TNF-α.Above result of study is proved
The substantial connection that TNF-α reacts with body inflammatory.Existing detection method includes biology detection (using the sensitive targets of TNF
Raji cell assay Raji TNF activity, the relative activity of TNF is drawn according to cell death), immunological detection (ELISA) etc..Biology is examined
Survey method is related to the selection and culture of sensitive specific target cells, complex operation；Immunological detection need to use antibody, and price is more held high
It is expensive.
The content of the invention
For technical problem present in prior art, this case provides a kind of cell based on fit and golden magnetic nano particle
The electrochemical detection method of factor TNF-α.
To achieve the above object, this case is achieved through the following technical solutions：
A kind of detection method of the cytokine TNF-α based on fit and golden magnetic nano particle, it includes：
1) using magnetic glassy carbon electrode as working electrode, magnetic glassy carbon electrode is pre-processed；
2) gold is modified in Fe3O4Nano particle, is obtained gold magnetic nano particle；
3) DNA probe 1 is modified in the golden magnetic nano particle surface；
4) DNA probe 2 is carried out into hybridization reaction with the DNA probe 1 on the golden magnetic nano particle surface, with the golden magnetic
Nano grain surface forms the DNA double chain of complementary pairing；
5) the golden magnetic nano particle is adsorbed in the magnetic glassy carbon electrode surface by magnetic fields；
6) reacted in the magnetic glassy carbon electrode being inserted into prepare liquid, and gathered corresponding electrochemical response value, according to
Electrochemical response value and the corresponding relation of cytokine TNF-α concentration, obtain the concentration of cytokine TNF-α in prepare liquid；
Wherein, the sequence of the DNA probe 1 is 5 '-SH-TTTTTTTTGTCGCCGACTGCGCCATC-3 '；
The sequence of the DNA probe 2 is 5 '-MB-TGGTGGATGGCGCAGTCGGCGACAA-3 '；Wherein, MB is telecommunications
Number molecule methylene blue.
DNA probe 15 ' it is terminal modified have sulfydryl, covalent bonding can occur with gold surface, the sequence of DNA probe 2 contain
The fit sequence of TNF-α to be measured, and it 5 ' terminal modified has methylene blue (MB) this electric signal molecule.This case has synthesized Fe3O4@
Au composite nanometer particles, and by DNA probe 1 modify with its surface, then by the complementary pairing between two segment DNAs, compound
Nano grain surface assembles DNA double chain.
Above-mentioned material is adsorbed in electrode surface using magnetic glassy carbon electrode, electrochemical response is now detected, due to existing
Substantial amounts of methylene blue, it may appear that larger peak current (square wave voltammetry).After adding sample to be tested, TNF-α therein can be with
DNA probe 2 is combined so that duplex structure is destroyed, and DNA probe 2 dissociates in solution, and the methylene blue quantity of electrode surface is big
Width is reduced, so that the peak current for obtaining can be reduced, by analyzing decreasing value, it is possible to achieve the quantitative analysis to TNF-α.
Preferably, described detection method, wherein, step 1) in, carrying out pretreatment to magnetic glassy carbon electrode includes：
Magnetic glassy carbon electrode is soaked in Piranha washing lotion, to remove the impurity of electrode surface absorption；Wherein, with volume
Than being meter, the composition of the Piranha washing lotion is 98%H2SO4: 30%H2O2=3: 1.
Preferably, described detection method, wherein, magnetic glassy carbon electrode is pre-processed also to be included：By magnetic glass
Carbon electrode carborundum paper is polishing in mirror-smooth, then by the magnetic glassy carbon electrode ultrasound in ethanol and distilled water respectively
Cleaning.
Preferably, described detection method, wherein, magnetic glassy carbon electrode is pre-processed also to be included：By magnetic glass
Carbon electrode 0.5M H2SO4Solution is cleaned, and is then used with after with nitrogen drying.
Preferably, described detection method, wherein, the golden magnetic nano particle is obtained by following preparation method：
By Fe3O4Powder is dissolved in 150mL distilled waters, ultrasonic disperse；
Gold chloride is added to Fe3O4In solution, stirred 1 hour at 0 DEG C；
By NaBH4It is slowly added into above-mentioned mixed liquor, is reacted 15 minutes at 0 DEG C；
Magnetic Isolation, ethanol is vacuum dried to obtain golden magnetic nano particle after cleaning 3 times at 50 DEG C.
Preferably, described detection method, wherein, step 3) in, the method for modifying of DNA probe 1 is：By golden magnetic nanometer
Particle is dissolved in distilled water, adds DNA probe 1, is carried out using Ageing solution after placing 16 hours aging；By magnetic point after 1 day
From liquid is removed, cleaned using the 10mM phosphate buffers containing 0.25M NaCl and redissolved；Wherein, the Ageing solution be containing
0.1M KNO310mM phosphate buffers.
The beneficial effects of the invention are as follows：This case is used to detect the characteristics of TNF-α has sensitivity high, quick, low cost, examines
Survey is limited to 1ng mL-1, the combination of SPECIFIC APTAMER and electrochemical techniques by designing, it is possible to achieve TNF-α bio-sensing
Using；By introducing golden magnetic composite nano materials, fit fixation is cannot be only used for, can also realize the recycling of electrode.
Brief description of the drawings
Fig. 1 is Fe3O4The scanning electron microscopy of nano particle and golden magnetic nano particle；Wherein, (a) is Fe3O4Nanometer
The scanning electron microscopy of grain, (b) is the scanning electron microscopy of golden magnetic nano particle.
Fig. 2 is the AC impedance figure in glass-carbon electrode different modifying stage：(a) gold magnetic nano particle, (b) gold magnetic nano particle
With the compound of DNA probe 1, (c) gold magnetic nano particle-DNA double chain, (d) gold magnetic nano particle-DNA double chain and TNF-α generation
After effect.
Fig. 3 is the square wave voltammogram in glass-carbon electrode different modifying stage：(a) gold magnetic nano particle, (b) gold magnetic nano particle
With the compound of DNA probe 1, (c) gold magnetic nano particle-DNA double chain, (d) gold magnetic nano particle-DNA double chain and TNF-α generation
After effect.
(a) is to detect the square wave voltammogram that the TNF-α of various concentrations is finally presented in Fig. 4, and (b) is that TNF-α concentration is relative
The standard curve of the square wave volt-ampere peak point current answered；Error bars represent three relative standard deviations of independent measurement.
Fig. 5 is TNF-α detection limit (100ng mL-1) relative to other interference cell factors (including IL-2, IL-12,
IFN-γ) interference experiment result figure.
Fig. 6 is the result figure for repeated detection TNF-α after glass-carbon electrode regenerates.
Specific embodiment
The present invention is described in further detail below in conjunction with the accompanying drawings, to make those skilled in the art with reference to specification text
Word can be implemented according to this.
The detection method of the cytokine TNF-α of an embodiment is listed in this case, specifically be may include：
1) magnetic glassy carbon electrode (diameter 2mm) is used as working electrode.Electrode is before modification preferably in the water newly matched somebody with somebody
Brave fish solution (98%H2SO4：30%H2O2=3:1) immersion 5 minutes in, remove the material of electrode surface absorption.
2) with after distilled water wash clean, electrode carborundum paper (3000 mesh) is polished to mirror-smooth.Then, by electricity
Pole is cleaned by ultrasonic each 5 minutes in ethanol and distilled water respectively.Then, dried in nitrogen atmosphere using preceding elder generation.
3) treated electrode 0.5M H2SO4Solution carries out electrochemical cleaning.Then, by the drying of electrode nitrogen treating
Further modification.
4) Fe is synthesized using hydro-thermal method3O4Nano particle：The FeCl of 1.35g3·6H2O is dissolved in 40mL ethylene glycol, stirring
Uniformly.After solution is clarified, the sodium acetate of 3.6g is added.The solution continues to stir 0.5 hour, is then heated to 200 DEG C, reaction
9 hours.After being cooled to room temperature, black precipitate is obtained, Fe is separated and collected by magnet3O4Nano particle.3 are cleaned using ethanol
It is secondary, go the removal of impurity.Finally solid is vacuum dried at a temperature of 50 DEG C.
5) by 15mg Fe3O4Powder is dissolved in 150mL distilled waters, ultrasonic disperse.Compound concentration is 6mg mL-1Chlorine gold
Sour 3mL, adds it to above-mentioned Fe3O4In solution, 0 DEG C of temperature is kept, stirred 1 hour.Then compound concentration is 0.2M's
NaBH4Solution 0.9mL, is slowly added into above-mentioned mixed liquor and (adds within 4 minutes).Reacted 15 minutes at a temperature of 0 DEG C.Solution face
Color is changed into dark purple from shallow palm fibre.Equally separated using magnet, cleaned 3 times using ethanol, gone the removal of impurity.Finally in 50 DEG C of temperature
Under be vacuum dried into solid, obtain golden magnetic nano particle (Fe3O4@Au composite nanometer particles).
6) by Fe3O4@Au composite nanometer particles are dissolved in distilled water.Configuration DNA probe 1 solution (10mMTris-HCl, 1mM
EDTA,10mM TCEP,and 0.1M NaCl).Two solution of mixing, make the final concentration of DNA probe 1 reach 5 μM.The mixture
(contain 0.1M KNO using Ageing solution after placing 16 hours310mM phosphate buffers) carry out it is aging.By magnet point after 1 day
From unnecessary reagent is removed, cleaned using the 10mM phosphate buffers containing 0.25M NaCl and redissolved.The material is visited with DNA again
The mixing of pin 2 carries out hybridization reaction, stirring reaction 1 hour, you can realize the complementary pairing of DNA probe 1 and probe 2.Centrifugal purification
Fe is obtained afterwards3O4@Au composite nanometer particles-DNA double chain solution.
7) the electrode immersion concentration that will be pre-processed is 1mg mL-1Fe3O4@Au composite nanometer particles-DNA double chain solution,
Reaction 2 minutes, then prepare a series of TNF-α titer of concentration, by the electrode of modification further immerse above-mentioned standard liquid and
Half an hour is reacted in prepare liquid.Electrochemical Detection is carried out after cleaning.
8) Electrochemical Detection using computer controls CHI 660D electrochemical workstations (CH Instruments, in
State) carry out.Using three-electrode system, wherein, auxiliary electrode is platinum electrode, and saturated calomel electrode makees reference electrode.AC impedance
The buffer solution of spectrum is the 5mM [Fe (CN) containing 1mMKCl6]3-/4-Solution；The electrolyte of square wave voltammetry is to contain 140mMNaCl
With 5mM MgCl220mMTris-HCl (pH 7.4).
From figure 1 it will be seen that Fe3O4The size of nano particle is about 200nm or so, the bright spot on golden magnetic nano particle surface
It is gold grain.
AC impedance in Fig. 2 is used to be characterized in [Fe (CN) in more surface modification6]3-/4-Chemical property, resistance
The half-circle area of anti-spectrum represents the interrupted degree of electric charge transfer.There is not the region in curve a, it was demonstrated that Fe3O4@Au are compound to be received
The electric conductivity of rice grain is good；Curve b occurs in that a less half-circle area, is due to Fe3O4@Au composite nanometer particles
The negative electrical charge carried with the phosphate group of the DNA skeletons in the compound of DNA probe 1 and [Fe (CN)6]3-/4-Between repulsion make
With；Further increase is that electronegativity is further because composite nano materials surface forms DNA double chain for the half-circle area of curve c
Increase.The semicircle area of curve d returns to the degree of curve b, is that can form suitable because a section in determinand and DNA double chain is single-stranded
The compound of body-target protein, so as to replace in double-stranded DNA is single-stranded, reverts to the trim of electrode surface
Fe3O4@Au composite nanometer particles-DNA probe 1.
The trend of the square wave voltammogram in Fig. 3 is consistent with the impedance diagram result of Fig. 2, DNA probe 2 (determinand is fit) subscript
Some methylene blues this electric signal molecules only just occurs obvious current peak in the case of curve c.
SWV peak point currents show the linear relationship with TNF-α concentration in the range of 5 to 100ng/mL in Fig. 4.Return
Equation is y=1.962-0.018x (R2=0.996, number of repetition n=3), wherein y is peak point current (μ A), and x is dense TNF-α
Degree (ng mL-1).Detection is limited to 1ng mL-1。
Fig. 5 verifies the specificity of proposed TNF-α detection method by using the interference cell molecule of some.
TNF-α is determined has significant electrochemical signals difference and control experiment between.Therefore, test result indicate that TNF-α with it is fit
With reference to be it is effective and reliable, and all control molecules reduction current value can be ignored, it was confirmed that proposed method
High selectivity.
Fig. 6 is discharged back into solution by removing electrode-magnetic field, the golden magnetic nano particle for reacting of electrode interface, then
The golden magnetic nano particle of next round detection is adsorbed along with magnetic field, it is found that the new round that it can be used for after regenerating is detected.By reality
Test result to understand, repeatedly the electrode after regeneration still has preferable electrochemical response.
Although embodiment of the present invention is disclosed as above, it is not restricted to listed in specification and implementation method
With, it can be applied to various suitable the field of the invention completely, for those skilled in the art, can be easily
Other modification is realized, therefore under the universal limited without departing substantially from claim and equivalency range, the present invention is not limited
In specific details and shown here as the legend with description.
The sequence of DNA probe 1 is 5 '-SH-TTTTTTTTGTCGCCGACTGCGCCATC-3 '；
The sequence of DNA probe 2 is 5 '-MB-TGGTGGATGGCGCAGTCGGCGACAA-3 '.
Claims (6)
1. it is a kind of for non-diseases diagnosis the cytokine TNF-α based on fit and golden magnetic nano particle detection method, its
It is characterised by, including：
1) using magnetic glassy carbon electrode as working electrode, magnetic glassy carbon electrode is pre-processed；
2) gold is modified in Fe3O4Nano particle, is obtained gold magnetic nano particle；
3) DNA probe 1 is modified in the golden magnetic nano particle surface；
4) DNA probe 2 is carried out into hybridization reaction with the DNA probe 1 on the golden magnetic nano particle surface, with the golden magnetic nanometer
Particle surface forms the DNA double chain of complementary pairing；
5) the golden magnetic nano particle is adsorbed in the magnetic glassy carbon electrode surface by magnetic fields；
6) reacted in the magnetic glassy carbon electrode being inserted into prepare liquid, and gathered corresponding electrochemical response value, according to electrification
The corresponding relation of response and cytokine TNF-α concentration is learned, the concentration of cytokine TNF-α in prepare liquid is obtained；
Wherein, the sequence of the DNA probe 1 is 5 '-SH-TTTTTTTTGTCGCCGACTGCGCCATC-3 '；
The sequence of the DNA probe 2 is 5 '-MB-TGGTGGATGGCGCAGTCGGCGACAA-3 '；Wherein, MB is electric signal point
Sub- methylene blue.
2. detection method as claimed in claim 1, it is characterised in that step 1) in, magnetic glassy carbon electrode is pre-processed
Including：
Magnetic glassy carbon electrode is soaked in Piranha washing lotion, to remove the impurity of electrode surface absorption；Wherein, it is with volume ratio
Meter, the composition of the Piranha washing lotion is 98%H2SO4: 30%H2O2=3: 1.
3. detection method as claimed in claim 2, it is characterised in that being pre-processed to magnetic glassy carbon electrode also includes：Will
Magnetic glassy carbon electrode carborundum paper is polishing in mirror-smooth, then by magnetic glassy carbon electrode respectively in ethanol and distilled water
Middle ultrasonic cleaning.
4. detection method as claimed in claim 3, it is characterised in that being pre-processed to magnetic glassy carbon electrode also includes：Will
Magnetic glassy carbon electrode 0.5M H2SO4Solution is cleaned, and is then used with after with nitrogen drying.
5. detection method as claimed in claim 1, it is characterised in that the golden magnetic nano particle is obtained by following preparation method
：
By Fe3O4Powder is dissolved in 150mL distilled waters, ultrasonic disperse；
Gold chloride is added to Fe3O4In solution, stirred 1 hour at 0 DEG C；
By NaBH4It is slowly added into above-mentioned mixed liquor, is reacted 15 minutes at 0 DEG C；
Magnetic Isolation, ethanol is vacuum dried to obtain golden magnetic nano particle after cleaning 3 times at 50 DEG C.
6. detection method as claimed in claim 1, it is characterised in that step 3) in, the method for modifying of DNA probe 1 is：By gold
Magnetic nano particle is dissolved in distilled water, adds DNA probe 1, is carried out using Ageing solution after placing 16 hours aging；Pass through after 1 day
Magnetic Isolation removes liquid, is cleaned using the 10mM phosphate buffers containing 0.25M NaCl and redissolved；Wherein, the Ageing solution
It is to contain 0.1M KNO310mM phosphate buffers.
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| CN106918633B (en) | 2019-06-25 |
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