CN109342532A - A kind of core-shell nanometer rod and its preparation method and application of negative nickel carburization zone coated ferriferrous oxide - Google Patents
A kind of core-shell nanometer rod and its preparation method and application of negative nickel carburization zone coated ferriferrous oxide Download PDFInfo
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Abstract
The invention belongs to high molecular material and biomedicine fields, and in particular to a kind of core-shell nanometer rod and its preparation method and application of negative nickel carburization zone coated ferriferrous oxide.The core-shell nanometer rod, the negative nickel carburization zone shell including ferriferrous oxide nano stick kernel Yu coated ferroferric oxide nanometer rods.Calcining prepares the core-shell nanometer rod after Fe-NTA complex compound and nickel salt and dopamine chelating.The core-shell nanometer rod has catalytic activity similar with peroxidase, it can be used for preparing peroxidase or peroxidase analogue enztme, also pass through the function served as bridge of biotin-avidin system, DNA signal detection is converted into the electrochemical sensor of electrical signal detection by preparation, selectivity is high, Idiotype is good, testing result is more intuitive, is easier to accurate statistics, and it is more convenient, time saving, precision is high, accuracy is good.
Description
Technical field
The invention belongs to high molecular material and biomedicine fields, and in particular to a kind of negative four oxidation three of nickel carburization zone cladding
Core-shell nanometer rod of iron and its preparation method and application.
Background technique
The sensitive of trace sequence specific DNA, efficient detection are in clinical diagnosis, food analysis, bioterrorism and ring
Significance is all had in the monitoring of border.Common DNA detection technique includes fluorescence colorimetric detection method, molecular imprinting method, polymerase
Chain amplification reacts (PCR) and DNA microarray, however the application of these methods is often limited to complicated experimental procedure, valuableness
Instrument and equipment and limited sensitivity, some methods be difficult to carry out highly sensitive, inexpensive detection to low abundance nucleic acid sample.
In recent years, proposing many methods instead of round pcr to realize DNA Sensitive Detection, such as fluorescent marker, electricity
Chemo-immunity sensor, organic electrochemistry transistor immunosensor, surface plasma body resonant vibration and electrochemical luminescence aptamer pass
Sensor uses enzyme in conjunction with optics, electrochemical techniques, indicator, nano material as signal magnifying tags.These methods
In, electrochemica biological sensor is generally acknowledged quick, cheap, miniaturization detection device.Although these methods are with higher
Sensitivity, but these methods usually require additional probe modification or Conjugation step.Therefore, design it is a kind of more it is simple just
Prompt, low in cost, highly sensitive, high specific DNA detection method is still current research hotspot.
DNA nanoassemble is to hybridize the simple effective method for carrying out signal enhancing by Target-probe.2004
Year, Dirks has been put forward for the first time the DNA detection concept with similar PCR sensitivity with Pierce: hybridization chain reaction (HCR).
HCR is a kind of no enzyme method, and hybrid process is caused by initiator (target dna), and the dsDNA for making oligonucleotides aggregate into long-chain divides
Son.HCR is the reaction that initiator causes, and significantly reduces the interference of background signal.In addition, HCR can be in a mild condition
It carries out, these advantages make HCR become a method preferentially selected in DNA Application in Sensing.
The Fe of the application preparation3O4@C-Ni is a kind of with the active nanometer analogue enztme of class horseradish peroxidase, at it
After upper coating Streptavidin (SA, Streptavidin), closely it connect, urges with the DNA for being modified with biotin (Biotin)
Change tmb substrate liquid, generates electrochemical signals.
Therefore, the application combines HCR with electrochemica biological detection technique, passes through Fe3O4@C-Ni realizes HCR and electrification
The conversion between signal is learned, HCR enhances electrochemical signals, improves detection sensitivity and specificity.
Summary of the invention
The present invention provides a kind of core-shell nanometer rod of negative nickel carburization zone coated ferriferrous oxide, which has class
Peroxidase activity can be catalyzed tmb substrate liquid and redox reaction generation electrochemical signals occur；Core-shell nanometer rod coating
After Streptavidin, in conjunction with the DNA for being modified with biotin, the peroxidase activity based on the core-shell nanometer rod, by DNA
Qualitative and quantitative detection be converted to the qualitative and quantitative detections of electrochemical signals so that the detection of DNA is not necessarily to additional probe
Modification or conjugation, simple and fast, low in cost, high sensitivity, specificity are good.
The present invention also provides a kind of preparation method of the core-shell nanometer rod of negative nickel carburization zone coated ferriferrous oxide, this method
It is simple and easy to operate, it is low in cost, it is suitble to industrialized production.
A kind of core-shell nanometer rod of negative nickel carburization zone coated ferriferrous oxide, including ferriferrous oxide nano stick kernel and packet
The negative nickel carburization zone shell for wrapping up in ferriferrous oxide nano stick, forms rodlike core-shell nano material.
The length of the core-shell nanometer rod of negative nickel carburization zone coated ferriferrous oxide is 1~10 μm, and width is 80~100nm.
A kind of preparation method of the core-shell nanometer rod of negative nickel carburization zone coated ferriferrous oxide, step include:
(1) the alcohol aqueous of PH=8~9 chelating dissolved with Fe-NTA complex compound, nickel salt, dopamine and alkali compounds generates negative
Core-shell nanometer rod (the Fe-NTA@PDA-Ni of nickel poly-dopamine layer cladding Fe-NTA complex compound2+)；
(2) under inert atmosphere, negative nickel is made in the core-shell nanometer rod calcining of negative nickel poly-dopamine layer cladding Fe-NTA complex compound
Core-shell nanometer rod (the Fe of carburization zone coated ferriferrous oxide3O4@C-Ni)。
In the chelating systems of step (1), Fe-NTA complex concentration is 0.8~2mg/mL, preferably 1mg/mL；Fe-NTA
The mass ratio of complex compound and nickel element is 4.5~6:1, preferably 5~5.5:1, more preferably 5.3~5.4:1；Dopamine and nickel
The mass ratio of element is 1~2.5:1, preferably 1.5~2:1, more preferably 1.6:1.
Step (1), stir process while chelating chelate the negative nickel poly-dopamine layer cladding Fe-NTA complex compound of generation
Core-shell nanometer rod also carries out carrying out washing treatment, and concrete operations are that dehydrated alcohol and distilled water alternately wash.
Step (1), alkali compounds include trishydroxymethylaminomethane or phosphate, preferably trihydroxy methyl amino first
Alkane.
Alcohol aqueous and alkali dissolved with nickel salt and dopamine are added into the alcohol aqueous dissolved with Fe-NTA complex compound for step (1)
Property compound or the solution dissolved with alkali compounds, while stir process.Specifically, first to the alcohol water dissolved with Fe-NTA complex compound
Alkali compounds or the solution dissolved with alkali compounds are added in liquid, adds the alcohol aqueous dissolved with nickel salt and dopamine；Or
First into the alcohol aqueous dissolved with Fe-NTA complex compound be added dissolved with nickel salt and dopamine alcohol aqueous, add alkali compounds or
Dissolved with the solution of alkali compounds；Chelating generates the core-shell nanometer rod of negative nickel poly-dopamine layer cladding Fe-NTA complex compound.Dissolved with
The alcohol aqueous of nickel salt and dopamine is slowly added dropwise with alkali compounds or dissolved with the solution selection of alkali compounds.
Step (1), the preparation method of Fe-NTA complex compound include: that iron ammonium sulfate and nitrilotriacetic acid are dissolved in water
In, hydro-thermal reaction generates Fe-NTA complex compound.
In hydro-thermal reaction system, the concentration of ferro element is 0.15~0.2mol/L, preferably 0.16~0.17mol/L；Iron
The molar ratio of element and nitrilotriacetic acid is 1.5~5:1, preferably 2~2.5:1, more preferably 2:1.
In 150 DEG C~250 DEG C hydro-thermal reaction 5~20 hours.Hydrothermal temperature is preferably 180 DEG C~200 DEG C, and hydro-thermal is anti-
Preferably 10~15 hours, more preferably 12 hours between seasonable.
Step (2), under inert atmosphere, the core-shell nanometer rod of negative nickel poly-dopamine layer cladding Fe-NTA complex compound is in 500 DEG C
~800 DEG C are calcined.Inert atmosphere includes any one in nitrogen, helium, neon or carbon dioxide or combination.
The core-shell nanometer rod of the negative nickel carburization zone coated ferriferrous oxide of the application preparation has similar to peroxidase
Catalytic activity, can be used for preparing peroxidase or peroxidase analogue enztme, be catalyzed peroxide (especially peroxidating
Hydrogen) it is used as the oxygen of electron acceptor substrate, colored indicator (such as 3,3', 5,5'- tetramethyl benzidines, TMB) as electron donor
Change reduction reaction, the ultra-violet absorption spectrum of the constant ratio oxidation product by measuring colored indicator realizes determining for peroxide
Property and/or quantitative detection.The redox reaction of the core-shell nanometer rod catalysis bottom the TMB liquid of negative nickel carburization zone coated ferriferrous oxide
Route is as follows:
The core-shell nanometer rod of negative nickel carburization zone coated ferriferrous oxide is living based on redox similar with peroxidase
Property, it may also be used for the qualitative and/or quantitative detection of the bioactive substance of hydrogen peroxide is generated, bioactive substance includes that gallbladder is solid
Alcohol, glucose, ascorbic acid, glycine or histidine.The hydrogen peroxide that bioactive substance oxidation generates, in negative nickel carburization zone
Under the catalytic action of the core-shell nanometer rod of coated ferriferrous oxide, with colored indicator (3,3', 5,5'- tetramethyl benzidines,
TMB redox reaction) is carried out, by qualitative and/or quantitative determination hydrogen peroxide, and then realizes the qualitative of bioactive substance
And/or quantitative determination.
When detecting hydrogen peroxide, step includes: by sample, negative nickel carburization zone coated ferriferrous oxide core containing hydrogen peroxide
Shell nanometer rods, colored indicator and buffer are uniformly mixed, and 5~30 points are reacted under the conditions of pH=2~10,25 DEG C~65 DEG C
Clock；Negative nickel carburization zone coated ferriferrous oxide core-shell nanometer rod is separated, the absorption spectrum of reaction solution is detected, it is fixed to carry out hydrogen peroxide
Property and/or quantitative determination.
Preferably, pH=2~5；More preferably, pH=3~4, more preferably pH=4；The buffer is acetic acid-acetic acid
Sodium, phosphoric acid-sodium phosphate or phosphoric acid-dibastic sodium phosphate etc., preferably Acetic acid-sodium acetate.
The colored indicator has 3,3', and 5,5'- tetramethyl benzidine (TMB), o-phenylenediamine (OPD), 2,2'- join nitrogen-
Two (3- ethyl-benzothiazole -6- sulfonic acid) di-ammonium salts (ABTS), luminol or fluorescent reagent Amplex Red, preferably 3,3',
5,5'- tetramethyl benzidine.Content of the colored indicator in detection architecture be 0.05~0.2mmol/L, preferably 0.1
~0.2mmol/L, more preferably 0.1mmol/L.
Content of the core-shell nanometer rod of the negative nickel carburization zone coated ferriferrous oxide in detection architecture is 5~100 μ g/
ML, preferably 50~100 μ g/mL, more preferably 100 μ g/mL.
Reaction temperature is preferably 50 DEG C~60 DEG C, more preferably 60 DEG C.
In a preferred embodiment of the invention, using the Acetic acid-sodium acetate buffer of 0.2mol/L pH=4, colour developing refers to
Show that agent is 3,3', 5,5'- tetramethyl benzidines, the Detection wavelength of absorption spectrum is 652nm, in detection architecture, 3,3', 5,5'-
The content of tetramethyl benzidine is 0.1mmol/L, and the content of negative nickel carburization zone coated ferriferrous oxide core-shell nanometer rod is 100 μ
g/mL。
Electrochemica biological sensor is mainly formed by identifying system and with the signal translating system of its close fit, specificity
Determinand in conjunction with identifying system, carry out biochemical reaction, generate or amplification biochemical signals；Biochemical signals are then converted by signal
System is converted into electrically or optically signal that can be qualitative and/or quantitative.
The application electrochemica biological sensor is first based on selective good, the stronger hybridization chain reaction (HCR) of specificity
Selective amplification target dna (target DNA) completes the signal amplification of target dna.It is based on biotin-avidin system again
The function served as bridge of (biotin-avidin system, BAS), the labeled oxide enzyme on the target dna of amplification.Finally it is based on
The redox active of peroxidase, catalysis peroxide (especially hydrogen peroxide) refer to as electron acceptor substrate, colour developing
Show redox reaction of the agent (such as 3,3', 5,5'- tetramethyl benzidines, TMB) as electron donor, forms current signal, it will
Biochemical signals be converted into it is intuitive, can accurate statistics qualitative and/or quantitative electrical signal, complete the qualitative of target dna and/
Or quantitative determination.
The core-shell nanometer rod of negative nickel carburization zone coated ferriferrous oxide is living based on redox similar with peroxidase
Property, as peroxidase or peroxidase analogue enztme, by the function served as bridge of biotin-avidin system, can be used for
Electrochemica biological sensor is prepared, bio signal is converted into electrical signal, the generation of electrical signal is based on negative nickel carburization zone packet
Cover ferroso-ferric oxide core-shell nanometer rod catalysis using peroxide (especially hydrogen peroxide) as electron acceptor substrate, develop the color
Redox reaction of the indicator (such as 3,3', 5,5'- tetramethyl benzidines, TMB) as electron donor.
A kind of electrochemica biological sensor including metal electrode, is attached to the DNA probe (capture of metal electrode
DNA), the amplification target dna and be based on biotin-parent that target dna (target DNA) amplification based on DNA probe capture is formed
The negative nickel carburization zone coated ferriferrous oxide core-shell nanometer rod on amplification target dna is marked on prime system system.DNA probe is modified with
Sulfydryl is fixedly attached to metal (Au) electrode surface by metal (Au)-S key, expands modified biological element on target dna
(Biotin), Streptavidin (Streptavidin, SA) is modified on negative nickel carburization zone coated ferriferrous oxide core-shell nanometer rod.
The metal electrode is gold electrode.
The DNA sequence dna of the DNA probe are as follows: 5 '-CTT TAG GCC AAG AAT TCT GCT ACC-3 ', sulfydryl (SH)
It modifies in 5 ' ends.
The DNA sequence dna of the target dna are as follows: 5 '-A TTT GCT CAA CCC ACA TAC CCT GA G GTA GCA
GAA TTC TTG GCC TAA AG-3'；For prostate cancer DNA.
Hairpin structure H in the amplification target dna containing biotin modification1(Biotin-H1) and biotin modification hair
Clamping structure H2(Biotin-H2), the Biotin-H of biotin modification1DNA sequence dna be 5 '-Biotin-TTT TTT TCA GGG
TAT GTG GGT TGA GCA AAT CAA AGT ATT TGC TCA ACC CAC ATA-3 ', biotin modification
Biotin-H2DNA sequence dna be 5 '-A TTT GCT CAA CCC ACA TAC CCT GA TAT GTG GGT TGA GCA
AAT ACT TTG TTT TTT-Biotin-3’。
The electrochemica biological sensor is based on the oxygen using peroxide (especially hydrogen peroxide) as electron acceptor substrate
Change reduction reaction generation for the current signal of detection, such as the bottom TMB containing hydrogen peroxide for electrochemica biological detection
The detection of target dna to be checked, is converted into the detection of current signal by thing liquid, as a result more intuitive, sensitivity is higher, selectivity and spy
It is anisotropic more preferable.
A kind of preparation method of electrochemica biological sensor, step include: that metal electrode is placed in dissolved with sulfydryl modification by (1)
DNA probe and reducing agent PBS solution in, based on the fixed mercapto-modified DNA probe of metal-sulfur element key, preparation modification
The metal electrode of DNA probe；
(2) it by after the metal electrode blocking agent of modifying DNA probe, then is placed in target dna solution, preparation capture
The metal electrode of target dna；
(3) metal electrode for capturing target dna is placed in the hairpin structure H dissolved with biotin modification1And biotin modification
Hairpin structure H2SPSC buffer in, preparation amplification target dna metal electrode；
(4) metal electrode for expanding target dna is placed in the negative nickel carburization zone coated ferriferrous oxide core of Avidin modification
In shell nanometer rods solution, preparation is marked with the electrochemical biosensor of negative nickel carburization zone coated ferriferrous oxide core-shell nanometer rod
Device.
In step (1), metal electrode is gold electrode, and metal electrode is in advance through polishing and cleaning treatment.
In step (1), reducing agent includes sulfur alcohol compound, trialkyl phosphine compound or dithiothreitol (DTT) (DTT), excellent
Select sulfur alcohol compound three (2- carboxyethyl) phosphine (TCEP).
In the PBS solution of step (1), the concentration of PBS solute is 6~10mM, preferably 8.8mM；Mercapto-modified DNA is visited
The concentration of needle is 0.8~10 μM, preferably 1 μM；The molar ratio of mercapto-modified DNA probe and reducing agent is 1:300~800,
Preferably 1:500.
Metal electrode is placed in dissolved in the PBS solution of mercapto-modified DNA probe and reducing agent, in room temperature by step (1)
It stands overnight, prepares the metal electrode of modifying DNA probe.The metal electrode of modifying DNA probe is used in combination with PBS solution repeated flushing
It is dried with nitrogen, the concentration of PBS solution is 10mM.
In step (1), the DNA sequence dna of mercapto-modified DNA probe are as follows: 5 '-SH-CTT TAG GCC AAG AAT TCT
GCT ACC-3’。
In step (2), sealer includes MCH solution, BSA solution, Tween-20 (Tween-20) solution, horse serum solution
Or nonfat milk (No-fat milk) solution, preferred MCH solution.The concentration of MCH solution is 0.8~5mM, preferably 1mM.BSA
The concentration of solution is 5wt%, and the concentration of Tween-20 solution is 0.2wt%, and the concentration of horse serum solution is 10wt%, no rouge ox
The concentration of milk solution is 5wt%.
In step (2), the concentration of target dna solution is 1fM~1nM, preferably 100fM~1nM.
In step (2), the DNA sequence dna of target dna are as follows: 5 '-A TTT GCT CAA CCC ACA TAC CCT GA G
GTA GCA GAA TTC TTG GCC TAA AG-3'；For prostate cancer DNA.
In the SPSC buffer of step (3), the concentration of sodium chloride is 0.5~1.5mM, preferably 0.8~1mM；Phosphoric acid hydrogen
The concentration of disodium is 28~50mM, preferably 40mM；The hairpin structure H of biotin modification1(Biotin-H1) and biotin modification
Hairpin structure H2(Biotin-H2) concentration be 0.05~1 μM, it is preferably to be 0.2 μM.
In step (3), the hairpin structure H of biotin modification1(Biotin-H1) DNA sequence dna be 5 '-Biotin-TTT
TTT TCA GGG TAT GTG GGT TGA GCA AAT CAA AGT ATT TGC TCA ACC CAC ATA-3'；Biotin
The hairpin structure H of modification2(Biotin-H2) DNA sequence dna be 5 '-A TTT GCT CAA CCC ACA TAC CCT GA TAT
GTG GGT TGA GCA AAT ACT TTG TTT TTT-Biotin-3’。
In step (4), the concentration of the negative nickel carburization zone coated ferriferrous oxide core-shell nanometer rod solution of Avidin modification is 5
~50 μ g/mL, preferably 50 μ g/mL.
The preparation method of negative nickel carburization zone coated ferriferrous oxide core-shell nanometer rod of step (4), Avidin modification includes:
It will be uniformly mixed with the PBS solution of negative nickel carburization zone coated ferriferrous oxide core-shell nanometer rod and Avidin, in 30 DEG C~40 DEG C
Reaction prepares the negative nickel of Avidin modification in negative nickel carburization zone coated ferriferrous oxide core-shell nanometer rod pan coating Avidin
Carburization zone coated ferriferrous oxide core-shell nanometer rod.
A kind of electrochemica biological detection device, including reference electrode, to electrode and working electrode and hydrogen peroxide and TMB
Substrate solution or tmb substrate liquid containing hydrogen peroxide make working electrode with electrochemica biological sensor prepared by the application.
Reference electrode is Ag/AgCl or Hg/HgCl, is Pt electrode to electrode.
A method of it is detected using electrochemica biological detection device, step includes: in the TMB containing hydrogen peroxide
The electrochemica biological sensor working electrode for being put into reference electrode in substrate solution, preparing to electrode and the application, is lied prostrate using circulation
Peace method carries out qualitative detection, and when use, m- galvanometry carried out quantitative detection.Reference electrode is Ag/AgCl or Hg/HgCl,
It is Pt electrode to electrode.
The advantages of the application, is: the application preparation negative nickel carburization zone coated ferriferrous oxide core-shell nanometer rod have with
The similar catalytic activity of peroxidase can be used for preparing peroxidase or peroxidase analogue enztme, be catalyzed peroxide
The redox reaction of (especially hydrogen peroxide) as electron acceptor substrate carries out peroxide (especially hydrogen peroxide)
Qualitative and/or quantitative detection.
Function served as bridge based on biotin-avidin system, negative nickel carburization zone coated ferriferrous oxide core-shell nanometer rod with
Target NDA is combined, and bio signal detection is converted into the electricity based on peroxide (especially hydrogen peroxide) redox reaction
Signal detection is learned, can be used for preparing electrochemical sensor and the detection device based on the electrochemical sensor, carry out target dna
Qualitative and/or quantitative detection, and the bio signal amplification of hybridization chain reaction is combined, improve the sensitivity of detection.Pass through
The electrochemical sensor is detected, and selectivity is high, Idiotype is good, and testing result is more intuitive, is easier to accurate statistics, and more square
Just, time saving, precision is high, accuracy is good.
Detailed description of the invention
Fig. 1 is Fe prepared by embodiment 13O4The SEM of@C-Ni schemes.
Fig. 2 is embodiment 2Fe3O4Catalytic effect diagram of the@C-Ni as peroxidase.
Fig. 3 is Fe under 2 different temperatures of embodiment3O4The catalytic effect diagram of@C-Ni.
Fig. 4 is Fe under 2 difference pH of embodiment3O4The catalytic effect diagram of@C-Ni.
Fig. 5 is 2 difference Fe of embodiment3O4The catalytic effect diagram of@C-Ni dosage.
Fig. 6 is embodiment 3 with H2O2Fe of the concentration as variable3O4@C-Ni dynamic experiment test chart.
Fig. 7 is Fe of the embodiment 3 using TMB concentration as variable3O4@C-Ni dynamic experiment test chart.
Fig. 8 is the naked Au/C-DNA/MCH/T-DNA/H of embodiment 61-H2/Fe3O4The preparation route figure of@C-Ni@SA electrode.
Fig. 9 is the cyclic voltammetry (CV) of the electrochemical sensor of each embodiment self assembly of embodiment 6 to embodiment 10
Test result.
Figure 10 is 11 various concentration Fe of embodiment3O4The Au/C-DNA/MCH/T-DNA/H of@C-Ni@SA self assembly1-H2/
Fe3O4The Electrochemical Detection effect picture of@C-Ni@SA electrochemical sensor.
Figure 11 is 12 various concentration [H of embodiment1/H2] self assembly Au/C-DNA/MCH/T-DNA/H1-H2/Fe3O4@C-
The Electrochemical Detection effect picture of Ni@SA electrochemical sensor.
Figure 12 is that embodiment 13 captures target DNA (Au/C-DNA/MCH/T-DNA/H respectively1-H2/Fe3O4@C-Ni@
SA target DNA (Au/C-DNA/MCH/H is not captured) and1-H2/Fe3O4@C-Ni@SA) self assembly electrochemical sensor
Electrochemical Detection effect picture.
Figure 13 is the Au/C-DNA/MCH/T-DNA/H of 14 various concentration target DNA self assembly of embodiment1-H2/
Fe3O4The Electrochemical Detection effect picture of@C-Ni@SA electrochemical sensor.
Figure 14 is Au/C-DNA/MCH/T- of the embodiment 15 without the HCR various concentration target DNA self assembly expanded
DNA/Fe3O4The Electrochemical Detection effect picture of@C-Ni@SA electrochemical sensor.
Figure 15 is the Au/C-DNA/MCH/T-DNA/H of the different single base mismatch target DNA self assemblies of embodiment 161-
H2/Fe3O4The Electrochemical Detection effect picture of@C-Ni@SA electrochemical sensor.
Specific embodiment
The present invention is described in detail with specific implementation example with reference to the accompanying drawing.
1 Fe of embodiment3O4The synthesis of@C-Ni
(1) preparation of Fe-NTA complex compound
Six ferrous sulfate hydrate ammonium of 2.6g and 0.6g nitrilotriacetic acid are uniformly dissolved in 40mL water, in 180 DEG C of constant temperature
720min is reacted, is cooled to room temperature, filters to take precipitating, alternately for several times, obtaining white precipitate is for washing with ethyl alcohol and distilled water
Fe-NTA。
(2)Fe3O4The preparation of@C-Ni
200mg trishydroxymethylaminomethane is uniformly dissolved in 5mL water, solution A is formed.
15mg dopamine (DA) and 37.6mg Nickel dichloride hexahydrate are uniformly dissolved in the mixing of 2mL ethyl alcohol and 1mL water composition
In solution, B solution is formed.
50mg Fe-NTA is uniformly dissolved in the mixed solution of 30mL dehydrated alcohol and 20mL water composition, forms C solution.
Under stirring condition, solution A and B solution are successively added dropwise into C solution, after being added dropwise, formed mixed solution PH
Value is about 8~9, continues stirring 20 hours, filters to take precipitating, alternately washing for several times, obtains black for precipitating ethyl alcohol and distilled water
Color precipitating is Fe-NTA@PDA-Ni2+。
By Fe-NTA@PDA-Ni2+(500 DEG C) are calcined under nitrogen protection, obtain Fe3O4@C-Ni, SEM figure is such as Fig. 1 institute
Show.
Fe3O4For@C-Ni in rodlike, length is 1~10 μm, and width is 80~100nm；Ferriferrous oxide nano stick is located at
Fe3O4Inside@C-Ni clava, Fe is formed3O4The claval kernel of@C-Ni；Negative nickel carburization zone coated ferroferric oxide nanometer rods
Form Fe3O4The claval shell of@C-Ni.
2 Fe of embodiment3O4The peroxidase activity of@C-Ni
1. peroxidase is active
(1) take respectively the NaAc_HAc buffer solution of the 0.2M pH 4.0 of 285 μ L, 290 μ L, 290 μ L, 295 μ L in
In each centrifuge tube, 6 μ L, 0 μ L, 6 μ L, 0 μ L Fe are successively added into each centrifuge tube3O4@C-Ni aqueous solution (5mg/mL), 6 μ L, 6 μ
L, 0 μ L, 0 μ L aqueous hydrogen peroxide solution (0.01M), 3 μ L, 3 μ L, 3 μ L, 3 μ L3,3', 5,5'- tetramethyl benzidine dehydrated alcohol
Liquid (TMB, 10mM), above-mentioned solution is uniformly mixed；
(2) mixed liquor prepared by step (1) is reacted into 10min at room temperature；
(3) pass through externally-applied magnetic field for Fe3O4@C-Ni is separated with reaction solution；
(4) ultra-violet absorption spectrum of the above-mentioned mixed solution of ultraviolet-ray visible absorbing spectrophotometric determination is used.
As a result as shown in Fig. 2, Fe3O4@C-Ni+TMB+H2O2Ultraviolet absorptivity be about TMB+H2O2Two times, be Fe3O4@
Four times of C-Ni+TMB, individual TMB almost can be ignored, therefore, the synthesized nano material Fe of this experiment3O4@C-Ni
With peroxidase activity.
2. reaction temperature is to Fe3O4The active influence of@C-Ni fermentoid
(1) take the NaAc_HAc buffer solution of 290 μ L 0.2M pH 4.0 in centrifuge tube, successively into centrifuge tube
6 μ L Fe are added3O4@C-Ni aqueous solution (1mg/mL), 3 μ L aqueous hydrogen peroxide solutions (0.1M), 3 μ L 3,3', 5,5'- tetramethyls
Benzidine anhydrous alcohol solution (TMB, 20mM) is uniformly mixed, parallel 9 parts of preparation；
(2) by the mixed liquor of step (1) preparation respectively in 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60
DEG C, react 10min in 65 DEG C of water-baths；
(3) pass through externally-applied magnetic field for Fe3O4@C-Ni is separated with reaction solution；
(4) ultra-violet absorption spectrum of the above-mentioned mixed solution of ultraviolet-ray visible absorbing spectrophotometric determination is used.
As a result as shown in figure 3, as can be seen from the figure the absorbance at 652nm as the temperature rises, is first increased and dropped afterwards
It is low, in order to make Fe3O4@C-Ni works under optimum conditions, selects temperature 60 C corresponding to maximum absorbance to react most
Good temperature.
3. reacting pH to Fe3O4The active influence of@C-Ni fermentoid
(1) acetic acid-vinegar of 290 μ L 0.2M pH=2.0,3.0,4.0,5.0,6.0,7.0,8.0,9.0,10.0 is taken respectively
6 μ L Fe are successively added in each centrifuge tube in sour sodium buffer solution into each centrifuge tube3O4@C-Ni aqueous solution (1mg/mL), 3 μ L
Aqueous hydrogen peroxide solution (0.1M), 3 μ L, 3,3', 5,5'- tetramethyl benzidine anhydrous alcohol solution (TMB, 20mM), will be above-mentioned molten
Liquid is uniformly mixed；
(2) mixed liquor prepared by step (1) is reacted into 10min in 60 DEG C of water-baths；
(3) pass through externally-applied magnetic field for Fe3O4@C-Ni is separated with reaction solution；
(4) ultra-violet absorption spectrum of the above-mentioned mixed solution of ultraviolet-ray visible absorbing spectrophotometric determination is used.
As a result as shown in figure 4, as can be seen from the figure with the raising of pH, first increase reduces the absorbance at 652nm afterwards,
In order to make Fe3O4@C-Ni works under optimum conditions, selects pH=4.00 corresponding to maximum absorbance for the best of reaction
pH。
4.Fe3O4@C-Ni concentration is on the active influence of its fermentoid
(1) take the NaAc_HAc buffer solution of 7 part of 290 μ L 0.2M pH=4.00 in each centrifuge tube in parallel, successively
Fe is added into each centrifuge tube3O4@C-Ni aqueous solution forms different final concentrations (0,5,10,15,20,50,100 μ g/mL), 6 μ L mistakes
Aqueous solution of hydrogen (0.1M), 1.5 μ L, 3,3', 5,5'- tetramethyl benzidine (TMB, 10mM) are aoxidized, above-mentioned solution is uniformly mixed；
(2) mixed liquor prepared by step (1) is reacted into 10min in 60 DEG C of water-baths；
(3) pass through externally-applied magnetic field for Fe3O4@C-Ni is separated with reaction solution；
(4) ultra-violet absorption spectrum of the above-mentioned mixed solution of ultraviolet-ray visible absorbing spectrophotometric determination is used.
As a result as shown in figure 5, obviously it can be seen that Fe from figure3O4The concentration and the absorbance at 652nm wavelength of@C-Ni
Linear correlation rule of thumb chooses 20 μ g/mL as Fe3O4The optium concentration of@C-Ni solution.
3 Fe of embodiment3O4The dynamic experiment of@C-Ni
1.H2O2Experiment
[TMB]=0.20mM
(1) take the NaAc_HAc buffer solution of 5 part of 290 μ L 0.2M pH=4.00 in each centrifuge tube in parallel respectively,
6 μ L Fe are successively added into each centrifuge tube3O4@C-Ni aqueous solution (1mg/mL), the aqueous hydrogen peroxide solution of various concentration are (dense eventually
Degree be respectively 50,100,200,500,1000 μM), 3 μ L, 3,3', 5,5'- tetramethyl benzidine anhydrous alcohol solution (TMB,
20mM), above-mentioned solution is uniformly mixed, the concentration of TMB is 0.2mM in each mixed liquor prepared in parallel；
(2) mixed liquor prepared by step (1) is reacted into 10min in 60 DEG C of water-baths；
(3) pass through externally-applied magnetic field for Fe3O4@C-Ni is separated with reaction solution；
(4) ultra-violet absorption spectrum of the above-mentioned mixed solution of ultraviolet-ray visible absorbing spectrophotometric determination is used, with different peroxides
The inverse for changing hydrogen concentration is abscissa, with the inverse of differential responses speed for ordinate, draws the solution curve of Michaelis constant, such as
Shown in Fig. 6.
[TMB]=0.10mM
(1) take the NaAc_HAc buffer solution of 5 part of 290 μ L 0.2M pH=4.00 in each centrifuge tube in parallel respectively,
6 μ L Fe are successively added into each centrifuge tube3O4@C-Ni aqueous solution (1mg/mL), the aqueous hydrogen peroxide solution of various concentration are (dense eventually
Degree be respectively 50,100,200,500,1000 μM), 3 μ L 3,3', 5,5'- tetramethyl benzidine anhydrous alcohol solutions (TMB,
10mM), above-mentioned solution is uniformly mixed, the concentration of TMB is 0.1mM in each mixed liquor prepared in parallel；
(2) mixed liquor prepared by step (1) is reacted into 10min in 60 DEG C of water-baths；
(3) pass through externally-applied magnetic field for Fe3O4@C-Ni is separated with reaction solution；
(4) ultra-violet absorption spectrum of the above-mentioned mixed solution of ultraviolet-ray visible absorbing spectrophotometric determination is used, with different peroxides
The inverse for changing hydrogen concentration is abscissa, with the inverse of differential responses speed for ordinate, draws the solution curve of Michaelis constant, such as
Shown in Fig. 6.
[TMB]=0.05mM
(1) take the NaAc_HAc buffer solution of 5 part of 290 μ L 0.2M pH=4.00 in each centrifuge tube in parallel respectively,
6 μ L Fe are successively added into each centrifuge tube3O4@C-Ni aqueous solution (1mg/mL), the aqueous hydrogen peroxide solution of various concentration are (dense eventually
Degree be respectively 50,100,200,500,1000 μM), 1.5 μ L 3,3', 5,5'- tetramethyl benzidine anhydrous alcohol solutions (TMB,
20mM), above-mentioned solution is uniformly mixed, the concentration of TMB is 0.05mM in each mixed liquor prepared in parallel；
(2) mixed liquor prepared by step (1) is reacted into 10min in 60 DEG C of water-baths；
(3) pass through externally-applied magnetic field for Fe3O4@C-Ni is separated with reaction solution；
(4) ultra-violet absorption spectrum of the above-mentioned mixed solution of ultraviolet-ray visible absorbing spectrophotometric determination is used, with different peroxides
The inverse for changing hydrogen concentration is abscissa, with the inverse of differential responses speed for ordinate, draws the solution curve of Michaelis constant, such as
Shown in Fig. 6.
It is calculated by Fig. 6 it is found that nanometer analogue enztme Fe3O4@C-Ni is to substrate H2O2Michaelis constant Km=0.21, it is maximum anti-
Answer rate Vm=2.64.
2.TMB experiment
[H2O2]=0.10mM
(1) take the NaAc_HAc buffer solution of 5 part of 285 μ L 0.2M pH=4.00 in centrifuge tube in parallel respectively, according to
It is secondary that 6 μ L Fe are added into centrifuge tube3O4@C-Ni aqueous solution (1mg/mL), 3 μ L aqueous hydrogen peroxide solutions (0.01M), various concentration
3,3', 5,5'- tetramethyl benzidine anhydrous alcohol solutions (final concentration is respectively 200,300,400,600,800 μM), will be above-mentioned
Solution is uniformly mixed, H in each mixed liquor prepared in parallel2O2Concentration be 0.1mM；
(2) mixed liquor prepared by step (1) is reacted into 10min in 60 DEG C of water-baths；
(3) pass through externally-applied magnetic field for Fe3O4@C-Ni is separated with reaction solution；
(4) use the above-mentioned mixed solution of ultraviolet-ray visible absorbing spectrophotometric determination ultra-violet absorption spectrum, with difference 3,
3', the inverse of 5,5'- tetramethyl biphenyl amine concentrations are abscissa, with the inverse of differential responses speed for ordinate, draw Michaelis
The solution curve of constant, as shown in Figure 7.
[H2O2]=0.075mM
(1) take the NaAc_HAc buffer solution of 5 part of 285 μ L 0.2M pH=4.00 in each centrifuge tube in parallel respectively,
6 μ L Fe are successively added into each centrifuge tube3O4@C-Ni aqueous solution (1mg/mL), 3 μ L aqueous hydrogen peroxide solutions (0.01M), difference
The 3,3' of concentration, 5,5'- tetramethyl benzidine anhydrous alcohol solutions (final concentration is respectively 200,300,400,600,800 μM) will
Above-mentioned solution is uniformly mixed, H in each mixed liquor prepared in parallel2O2Concentration be 0.075mM；
(2) mixed liquor prepared by step (1) is reacted into 10min in 60 DEG C of water-baths；
(3) pass through externally-applied magnetic field for Fe3O4@C-Ni is separated with reaction solution；
(4) use the above-mentioned mixed solution of ultraviolet-ray visible absorbing spectrophotometric determination ultra-violet absorption spectrum, with difference 3,
3', the inverse of 5,5'- tetramethyl biphenyl amine concentrations are abscissa, with the inverse of differential responses speed for ordinate, draw Michaelis
The solution curve of constant, as shown in Figure 7.
[H2O2]=0.05mM
(1) take the NaAc_HAc buffer solution of 5 part of 285 μ L 0.2M pH=4.00 in each centrifuge tube in parallel respectively,
6 μ L Fe are successively added into each centrifuge tube3O4@C-Ni aqueous solution (1mg/mL), 1.5 μ L aqueous hydrogen peroxide solutions (0.01M), no
With the 3,3' of concentration, 5,5'- tetramethyl benzidines (final concentration is respectively 200,300,400,600,800 μM), by above-mentioned solution
It is uniformly mixed, H in each mixed liquor prepared in parallel2O2Concentration be 0.05mM；
(2) mixed liquor prepared by step (1) is reacted into 10min in 60 DEG C of water-baths；
(3) pass through externally-applied magnetic field for Fe3O4@C-Ni is separated with reaction solution；
(4) use the above-mentioned mixed solution of ultraviolet-ray visible absorbing spectrophotometric determination ultra-violet absorption spectrum, with difference 3,
3', the inverse of 5,5'- tetramethyl biphenyl amine concentrations are abscissa, with the inverse of differential responses speed for ordinate, draw Michaelis
The solution curve of constant, as shown in Figure 7.
It is calculated by Fig. 7 it is found that nanometer analogue enztme Fe3O4Michaelis constant Km=0.35 of the@C-Ni to substrate TMB, maximum reaction
Rate Vm=1.13.
The pre-treatment of 4 Au electrode of embodiment
(1) electrode is polished
Suitable 0.05 μm of alumina powder on polishing flannelet, uniformly, vertical Au electrode is in alumina powder for paving
" 8 " word is uniformly drawn, rinses electrode with deionized water (Q water)；Au is placed in ultrasound 5min in Q water, and Q water rinses, and surpasses in ethanol solution
Sound 5min, Q water rinses, again the ultrasound 5min in Q water, and Q water rinses, and is dried with nitrogen stand-by.
(2) electrode clean
It by occasion China electrochemical workstation, is detected using three-electrode system, in 0.5M H2SO4Middle cleaning electrode.
1. sweeping (check gold electrode cleannes) slowly
Potential range is -0.25-1.5V
Sweep speed is 0.1V/S
Scanning Section is 2
2. sweeping (removal electrode surface impurity, activated electrode) fastly
Potential range is -0.25-1.5V
Sweep speed is 4V/S
Scanning Section is 60
Au electrode after polishing is placed in H2SO4Electrolytic cell in, start sweep slowly, if 1V~1.5V appearance three companies
Continuous oxidation peak then proves that electrode surface is clean.If there are three continuous oxidation peaks, needs to be swept fastly, repeatedly follow
Ring is swept fastly-is swept slowly, until there are continuous three oxidation peaks.Au electrode, nitrogen (N are rinsed with Q water2) dry up electrode and electrode
Cap covers electrode cap, electrode is vertically put on foamed plastics, for use.
5 Fe of embodiment3O4@C-Ni is coated with SA (Streptavidin)
By the Fe of 20 μ g/mL of 1mL3O4@C-Ni aqueous solution and the SA-PBS solution of 25 μ L 1mg/mL are placed in centrifuge tube,
In 37 DEG C of reaction overnights, in Fe3O4@C-Ni pan coating SA (Fe3O4@C-Ni@SA), it is centrifuged 10min in 10000rpm, is collected
Supernatant is rinsed repeatedly with 1 × PBS (0.01M), disperses gains in 1 × PBS of 1mL, and ultrasound is uniformly, spare.
Embodiment 6 prepares naked Au/C-DNA/MCH/T-DNA/H1-H2/Fe3O4@C-Ni@SA electrode
It is performed the following operation according to preparation route shown in Fig. 8:
(1) Au electrode modification capture DNA
10 μ L 5mM TCEP aqueous solutions, the mercapto-modified capture aqueous dna of 50 μM of 2 μ L are added in centrifuge tube
(SH-capture DNA), 88 μ 1 × PBS of L react at room temperature 30min, obtain mixed liquor；1 μM of 6 above-mentioned mixed liquor of μ L is added dropwise in
On the Au electrode set, electrode cap is covered, at room temperature overnight, SH-capture DNA is modified on Au electrode based on Au-S key.With 1
× PBS repeated flushing electrode, is dried with nitrogen.
(2) Target DNA is captured
10 μ L 1mM MCH aqueous solutions (6- sulfydryl hex- 1- alcohol) are added dropwise on the Au electrode of SH-capture DNA modification,
React at room temperature 1h；With 1 × PBS (0.01M) repeated flushing electrode, it is dried with nitrogen；The target DNA that 6 μ L 1nM are added dropwise again is water-soluble
Liquid reacts 2h on corresponding electrode at room temperature.With 1 × PBS (0.01M) repeated flushing electrode, it is dried with nitrogen.
(3) Target DNA is expanded
80 1 × SPSC of μ L (1mM NaCl, 50mM Na is added in centrifuge tube2HPO4) buffer, 10 μ L, 1 μM of Biotin-
H1Aqueous solution, 10 μ L, 1 μM of Biotin-H2Aqueous solution is uniformly mixed；The 6 above-mentioned mixed liquors of μ L are added drop-wise to capture Target DNA
Au electrode surface, react at room temperature 2h.With 1 × PBS repeated flushing electrode, it is dried with nitrogen.
(4) Fe is modified3O4@C-Ni@SA
6 μ L, 20 μ g/mL Fe is added dropwise3O4@C-Ni@SA aqueous solution is reacted at room temperature on the Au electrode of amplification Target DNA
1h rinses electrode with 1 × PBS, Au/C-DNA/MCH/T-DNA/H is made1-H2/Fe3O4@C-Ni@SA electrode, using " embodiment
17 electrochemical detection methods " carry out Electrochemical Detection, as a result as shown in Figure 9.
As shown in Figure 9, Au/C-DNA/MCH/T-DNA/H1-H2/Fe3O4The redox peaks of@C-Ni@SA electrode are obviously high
In embodiment 7 to the other kinds of electrochemical sensor of embodiment 10, which create significant redox signals.
Embodiment 7. prepares naked Au/C-DNA/MCH/H1-H2/Fe3O4@C-Ni@SA electrode
This preparation method is the same as " embodiment 6. prepares naked Au/C-DNA/MCH/T-DNA/H1-H2/Fe3O4@C-Ni@SA electricity
Pole ", the difference is that: after step (1) Au electrode modification capture DNA, without step (2) capture Target DNA behaviour
Make, directly amplification capture DNA, and carries out step (4) modification Fe3O4@C-Ni@SA.Expand the operation of capture DNA are as follows:
80 1 × SPSC of μ L, 10 μ L, 1 μM of Biotin-H are added in centrifuge tube1、10μL 1μM Biotin-H2, it is uniformly mixed；By 6 μ L
Above-mentioned mixed liquor is added drop-wise to the Au electrode surface of modification capture DNA, reacts at room temperature 2h.With 1 × PBS repeated flushing electrode,
It is dried with nitrogen, prepares Au/C-DNA/MCH/H1-H2/Fe3O4@C-Ni@SA electrode, using " 17 electrochemical detection method of embodiment "
Electrochemical Detection is carried out, as a result as shown in Figure 9.
Embodiment 8. prepares naked Au/C-DNA/MCH/T-DNA/H1-H2Electrode
This preparation method is the same as " embodiment 6. prepares naked Au/C-DNA/MCH/T-DNA/H1-H2/Fe3O4@C-Ni@SA electricity
Pole ", the difference is that: Fe is modified without step (4)3O4The operation of@C-Ni@SA prepares Au/C-DNA/MCH/T-DNA/
H1-H2Electrode carries out Electrochemical Detection using " 17 electrochemical detection method of embodiment ", as a result as shown in Figure 9.
As shown in Figure 9, the signal that Au/C-DNA/MCH/T-DNA/H1-H2 is generated almost can be ignored.
Embodiment 9. prepares naked Au/C-DNA/MCH/T-DNA electrode
This preparation method is the same as " embodiment 6. prepares naked Au/C-DNA/MCH/T-DNA/H1-H2/Fe3O4@C-Ni@SA electricity
Pole ", the difference is that: without (2) capture Target DNA, step (3) amplification Target DNA and step (4) modification
Fe3O4The operation of@C-Ni@SA, prepare Au/C-DNA/MCH/T-DNA electrode, using " 17 electrochemical detection method of embodiment " into
Row Electrochemical Detection, as a result as shown in Figure 9.
Embodiment 10. prepares naked Au/C-DNA/MCH electrode
This preparation method is the same as " embodiment 6. prepares naked Au/C-DNA/MCH/T-DNA/H1-H2/Fe3O4@C-Ni@SA electricity
Pole ", the difference is that: Target DNA is expanded without step (3) and step (4) modifies Fe3O4The operation of@C-Ni@SA.
10 μ L 1mM MCH need to only be added dropwise on the Au electrode of SH-capture DNA modification, react at room temperature 1h；With 1 × PBS (0.01M)
Repeated flushing electrode, is dried with nitrogen, and prepares Au/C-DNA/MCH electrode, carries out electricity using " 17 electrochemical detection method of embodiment "
Chemical detection, as a result as shown in Figure 9.
As shown in Figure 9, signal can be enhanced in HCR process, is essential.Fe3O4@C-Ni@SA can be catalyzed the bottom TMB liquid,
Generate electrochemical signals.
11. self assembly various concentration Fe of embodiment3O4@C-Ni@SA electrochemical sensor
This preparation method is the same as " embodiment 6. prepares naked Au/C-DNA/MCH/T-DNA/H1-H2/Fe3O4@C-Ni@SA electricity
Pole ", the difference is that: the Fe of step (4) introducing various concentration3O4@C-Ni@SA constructs various concentration Fe3O4@C-Ni@SA
Electrochemical sensor, using " 17 electrochemical detection method of embodiment " carry out Electrochemical Detection, the results are shown in Figure 10.
As shown in Figure 10, with Fe3O4The increase of@C-Ni@SA concentration, self assembly electrochemical sensor electric current detected
Signal gradually increases, but works as Fe3O4When the concentration of@C-Ni@SA reaches 50 μ g/mL, current signal reduces, therefore with 20 μ g/mL
As Fe3O4The preferred concentration of@C-Ni@SA.
12. self assembly various concentration [H of embodiment1/H2] electrochemical sensor
This preparation method is the same as " embodiment 6. prepares naked Au/C-DNA/MCH/T-DNA/H1-H2/Fe3O4@C-Ni@SA electricity
Pole ", the difference is that: [the H of step (3) introducing various concentration1/H2] (0.05,0.1,0.2 μM), construct various concentration [H1/
H2] electrochemical sensor, using " 17 electrochemical detection method of embodiment " carry out Electrochemical Detection, as a result as shown in figure 11.
As shown in Figure 11, with H1/H2Concentration increases, and the current signal that self assembly electrochemical sensor detects gradually increases
Greatly, and in [H1/H2]=0.2 μM reaches maximum value, therefore chooses [H1/H2]=0.2 μM is used as preferred concentration.
The electrochemical sensor of 13 self assemblies of embodiment ± target DNA
This preparation method is the same as " embodiment 6. prepares naked Au/C-DNA/MCH/T-DNA/H1-H2/Fe3O4@C-Ni@SA electricity
Pole ", the difference is that: step (2) selects capture target DNA respectively and does not capture target DNA, component composition
The electrochemical sensor of target DNA and unassembled target DNA carry out electricity using " 17 electrochemical detection method of embodiment "
Chemical detection, as a result as shown in figure 12.
As shown in Figure 12, the electrochemical sensor that 1nM target DNA is self-assembly of, which is added, can be detected 2000nA's
Current signal, and the current signal that the electrochemical sensor that target DNA is self-assembly of only detects 90nA or so is not added,
Therefore, the electrochemical sensor of the application building can realize the highly sensitive detection of DNA.
The electrochemical sensor of 14 self assembly various concentration target DNA of embodiment
This preparation method is the same as " embodiment 6. prepares naked Au/C-DNA/MCH/T-DNA/H1-H2/Fe3O4@C-Ni@SA electricity
Pole ", the difference is that: step (2) introduces the target DNA of various concentration, constructs the electrification of various concentration target DNA
Sensor is learned, Electrochemical Detection is carried out using " 17 electrochemical detection method of embodiment ", as a result as shown in figure 13.
As shown in Figure 13, the electrochemical sensor that 1fM target DNA is self-assembly of is added can detecte 200nA
The current signal of left and right, one times high when than target DNA is not added, detection sensitivity is high.And with target DNA concentration liter
Height, detection sensitivity are substantially increased.
15 self assembly various concentration target DNA of embodiment and the electrochemical sensor expanded without HCR
This preparation method is the same as " embodiment 6. prepares naked Au/C-DNA/MCH/T-DNA/H1-H2/Fe3O4@C-Ni@SA electricity
Pole ", the difference is that: step (2) introduces the target DNA of various concentration, and expands Target DNA without step (3), from
Various concentration target DNA and the electrochemical sensor without HCR amplification are assembled, using " 17 electrochemical detection method of embodiment "
Electrochemical Detection is carried out, as a result as shown in figure 14.
As shown in Figure 14, with the increase of target DNA concentration, the current signal that electrochemical sensor detects is gradually
Enhancing, sensitivity gradually rise.14 Figure 13 is it is found that due to lacking HCR amplification in conjunction with the embodiments, with concentration target DNA,
The current signal that the present embodiment detects is well below the embodiment 14 for carrying out HCR amplification.
The electrochemical sensor of the target DNA of 16 self assembly single base mismatch (SNP) of embodiment
This preparation method is the same as " embodiment 6. prepares naked Au/C-DNA/MCH/T-DNA/H1-H2/Fe3O4@C-Ni@SA electricity
Pole ", the difference is that: step (2) introduces the target DNA (1MM) an of base mispairing, two base mispairings respectively
The target DNA (3MM) of target DNA (2MM), three base mispairings, respectively correspond to form one base mistake of self assembly degree
Target DNA that matches, the target DNA (2MM) of two base mispairings, three base mispairings target DNA (3MM)
Electrochemical sensor carries out Electrochemical Detection using " 17 electrochemical detection method of embodiment ", as a result as shown in figure 15.
As shown in Figure 15, the electrochemical sensor of the application building is high to the selectivity of target DNA.
17. electrochemical detection method of embodiment
Different types of electrochemical sensor prepared by embodiment 6 to embodiment 16, according to following Electrochemical Detection sides
Method is detected.
1. being detected by occasion China electrochemical workstation using three-electrode system, Ag/AgCL electrode is reference electrode,
Platinum electrode is to electrode, and the Au electrode of self assembly is working electrode, and electricity is carried out in the bottom TMB liquid of the 1.5mL containing hydrogen peroxide
Chemical detection.
2. carrying out qualitative detection to prostate cancer DNA with cyclic voltammetry, parameter area is as follows:
Potential range is 0-0.8V
Sweep speed is 0.1V/S
Used time m- electric current (I-t) carries out quantitative detection to prostate cancer DNA, and parameter area is as follows:
Time range is 0-100s
Sweep speed is 0.1V/S
3. it is bent to make electric current-concentration according to the concentration of electric current I and the target DNA in gained time current curve
Line.
Utilize current -voltage curve such as Fig. 9 of the electrochemical workstation detection different types of electrochemical sensor of self assembly
It is shown, only Au/C-DNA/MCH/T-DNA/H1-H2/Fe3O4@C-Ni@SA produces significant redox signal.Au/C-
DNA/MCH/T-DNA/Fe3O4The signal that@C-Ni@SA is generated is smaller, it was demonstrated that signal can be enhanced in HCR process, is essential
's.The signal that Au/C-DNA/MCH/T-DNA/H1-H2 is generated almost can be ignored, and illustrate Fe3O4@C-Ni@SA can be urged
Change the bottom the TMB liquid containing hydrogen peroxide, generates electrochemical signals.
The above-mentioned description to embodiment is for ease of ordinary skill in the art to understand and use the invention.
Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general
Principle is applied in other embodiments without having to go through creative labor.Therefore, the present invention is not limited to above-mentioned embodiment,
Those skilled in the art's announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be in this hairs
Within bright protection scope.
Claims (10)
1. a kind of core-shell nanometer rod of negative nickel carburization zone coated ferriferrous oxide, which is characterized in that including ferriferrous oxide nano
The negative nickel carburization zone shell of stick kernel and coated ferroferric oxide nanometer rods.
2. the preparation method of core-shell nanometer rod described in claim 1, which is characterized in that step includes:
(1) negative nickel is generated dissolved with the alcohol aqueous chelating of PH=8~9 of Fe-NTA complex compound, nickel salt, dopamine and alkali compounds
The core-shell nanometer rod of poly-dopamine layer cladding Fe-NTA complex compound；
(2) under inert atmosphere, negative nickel carbonization is made in the core-shell nanometer rod calcining of negative nickel poly-dopamine layer cladding Fe-NTA complex compound
The core-shell nanometer rod of layer coated ferriferrous oxide.
3. the core-shell nanometer rod of negative nickel carburization zone coated ferriferrous oxide described in claim 1 be used to prepare peroxidase or
Mimics of peroxidase.
4. the core-shell nanometer rod of negative nickel carburization zone coated ferriferrous oxide described in claim 1 is used for qualitative and/or quantitative detection
Hydrogen peroxide or the bioactive substance that can produce hydrogen peroxide.
5. a kind of method for detecting hydrogen peroxide, which is characterized in that sample, negative nickel carburization zone containing hydrogen peroxide are coated four oxygen
The core-shell nanometer rod, colored indicator and buffer for changing three-iron are uniformly mixed, anti-under the conditions of pH=2~10,25 DEG C~60 DEG C
It answers 5~30 minutes；The core-shell nanometer rod for separating negative nickel carburization zone coated ferriferrous oxide, detects the absorption spectrum of reaction solution, into
Row hydrogen peroxide is qualitative and/or quantitative determines.
6. the core-shell nanometer rod of negative nickel carburization zone coated ferriferrous oxide described in claim 1 is used to prepare electrochemical biosensor
Device.
7. a kind of electrochemica biological sensor, which is characterized in that including metal electrode, the DNA probe for being attached to metal electrode, base
Amplification is marked in the amplification target dna of the target DNA amplification formation of DNA probe capture and based on biotin-avidin system
The core-shell nanometer rod of negative nickel carburization zone coated ferriferrous oxide on target dna.
8. a kind of preparation method of electrochemica biological sensor, which is characterized in that step include: (1) metal electrode is placed in it is molten
In the PBS solution for having mercapto-modified DNA probe and reducing agent, visited based on the fixed mercapto-modified DNA of metal-sulfur element key
Needle prepares the metal electrode of modifying DNA probe；
(2) it after the metal electrode blocking agent of modifying DNA probe, then is placed in DNA solution, preparation capture target dna
Metal electrode；
(3) metal electrode for capturing target dna is placed in the hairpin structure H dissolved with biotin modification1With the hair clip of biotin modification
Structure H2SPSC buffer in, preparation amplification target dna metal electrode；
(4) the negative nickel carburization zone coated ferriferrous oxide nucleocapsid that the metal electrode for expanding target dna is placed in Avidin modification is received
In rice stick solution, preparation is marked with the electrochemica biological sensor of negative nickel carburization zone coated ferriferrous oxide core-shell nanometer rod.
9. a kind of electrochemica biological detection device, including reference electrode, to electrode and working electrode, which is characterized in that with right
It is required that 7 electrochemica biological sensors make working electrode, it further include hydrogen peroxide with tmb substrate liquid or containing hydrogen peroxide
Tmb substrate liquid.
10. a kind of electrochemica biological detection method for detecting target dna, which is characterized in that step includes: to contain hydrogen peroxide
Tmb substrate liquid in be put into reference electrode, to electrochemica biological sensor working electrode described in electrode and claim 7, use
Cyclic voltammetry carries out qualitative detection, and when use, m- galvanometry carried out quantitative detection.
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