CN108375612A - A kind of method of composite nano materials Electrochemical Detection alpha-fetoprotein - Google Patents

A kind of method of composite nano materials Electrochemical Detection alpha-fetoprotein Download PDF

Info

Publication number
CN108375612A
CN108375612A CN201810126072.9A CN201810126072A CN108375612A CN 108375612 A CN108375612 A CN 108375612A CN 201810126072 A CN201810126072 A CN 201810126072A CN 108375612 A CN108375612 A CN 108375612A
Authority
CN
China
Prior art keywords
afp
solution
nano
magnetic
added
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201810126072.9A
Other languages
Chinese (zh)
Other versions
CN108375612B (en
Inventor
李桂银
李珊珊
周治德
梁晋涛
陆春婷
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guilin University of Electronic Technology
Original Assignee
Guilin University of Electronic Technology
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Guilin University of Electronic Technology filed Critical Guilin University of Electronic Technology
Priority to CN201810126072.9A priority Critical patent/CN108375612B/en
Publication of CN108375612A publication Critical patent/CN108375612A/en
Application granted granted Critical
Publication of CN108375612B publication Critical patent/CN108375612B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
    • 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
    • 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
    • G01N27/416—Systems
    • G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage

Abstract

A kind of method of composite nano materials Electrochemical Detection alpha-fetoprotein, with Fe3O4@Au are core, and chitosan (CS) is shell, prepares Fe3O4@Au@CS composite nano materials.With alpha-fetoprotein (AFP) for target analytes, people AFP antibody (Ab1) is fixed on Fe as capture probe3O4@Au CS magnetic composite nano material surfaces, mouse anti-human AFP antibody (Ab2) and catalase (HRP) are tagged to a nanometer gold surface as detection probe, utilize the specificity construction interlayer type immunosensor Fe of antigen-antibody3O4@Au@CS‑Ab1@AFP@Ab2‑Au‑HRP；According to H2O2Oxidation reaction can occur under HRP enzyme effects, the size of oxidation current and the concentration of AFP are in a linear relationship, realize that alpha-fetoprotein quantitatively detects.

Description

A kind of method of composite nano materials Electrochemical Detection alpha-fetoprotein
Technical field
The invention belongs to biomedical engineering fields, and in particular to one kind being based on nanocomposite Electrochemical Detection first tire The method of albumen.
Background technology
Alpha-fetoprotein（Alpha-Fetal Protein, AFP）It is a kind of glycoprotein for belonging to albumin family, more than 590 The single polypeptide chain that a amino acid residue is formed, for molecular weight about in 65-70KD or so, the content in normal adult's body is unusual It is low.The electrochemistry of signal is detected as conversion element, potential or electric current as feature using biomaterial as sensing element, electrode Biosensor, according to the reaction pattern of immunoassay, electrochemical immunosensor mainly has competition law and sandwich method two major classes. In order to ensure that can obtain higher sensitivity, antibody and antigen concentration in competition must have that one is excessive.And sandwich method It is to combine capture probe, determined antigen and label probe according to certain order by the specific binding of antibody-antigene Come, generates " sandwich " type interlayer type compound of " capture probe-antigen-label probe ".Publication No. CN103344634A Patent of invention, be related to a kind of alpha-fetoprotein（AFP）Magnetic microparticle chemiluminescence immunologic detection method, fluorescein isothiocynate （FITC）The AFP monoclonal antibodies and alkaline phosphatase of label（AP）The monoclonal antibody of label and antigen binding form FITC The sandwich immunoassay compound of labelled antibody-antigen-AP labelled antibodies, similar " sandwich " structure.It is then added and is connected with anti-FITC The magnetic particle of antibody makes antigen antibody complex be connected on magnetic particle, the Direct precipitation in externally-applied magnetic field, is not required to centrifugation i.e. The compound that immune response is formed can be detached with unbonded other materials.The patent of invention of Publication No. CN101441218, It is related to a kind of utilization CdTe quantum and Fe3O4The method that-Dextran nano particles detect AFP contents in serum, step packet The preparation of Fe3O4-Dextran- first antibody compounds is included, the preparation of CdTe- secondary antibody compounds, " fluorescence is strong for drafting Degree-AFP concentration " standard working curve measures gained florescent intensity value and obtains the content of hepatocarcinoma patient AFP.
Invention content
Technical problem to be solved by the invention is to provide one kind being based on magnetic Fe3O4@Au@CS composite nano materials electrifications The method for learning detection alpha-fetoprotein realizes high sensitivity, specific good and quickly detection.
In order to solve this technical problem, with Fe3O4@Au are core, using CS as shell, synthesize Fe3O4@Au@CS composite Nano materials Material uses it for structure biosensor, alpha-fetoprotein is detected by electrochemical method.It is as follows：
Step 1: preparing Fe3O4@Au@CS composite nano materials
1, magnetic Fe3O4It is prepared by@Au composite nanoparticles
According to known technique, by FeSO4·7H2O aqueous solutions and the mixing of PEG-20000 aqueous solutions, adjust solution ph, are added H2O2Solution is transferred in autoclave, reacts 5 h in high temperature constant temperature；Successively pure water and absolute ethyl alcohol Magneto separate wash 3 It is secondary, it is then baked to, grinds and obtain Fe3O4Nano-particle.Accurately weigh 10 mg Fe3O4120 mL are added in nano-particle 50 g/L PEG-20000 aqueous solutions are ultrasonically treated 99 min；1 mL, 1% gold chlorides are added, 60 min of sustained response is added 5 ml, 80 mmol/L hydroxylamine hydrochloride solutions sustained responses, 60 min, repeatedly carries out Magneto separate；Then with 50 g/L PEG- 20000 aqueous solutions repeat Magneto separate and clean 3 times, obtain magnetic Fe3O4@Au composite nanoparticles.
2, magnetic Fe3O4The preparation of@Au@CS nano-complex particles
(1) 0.2 g chitosans accurately are weighed, be dissolved in the acetum of 10 ml 1%, and stir.
(2) 1.0 mg Fe accurately are weighed3O4@Au nano-particles are poured into 1 ml secondary waters simultaneously ultrasonic disperse, by it The Fe of a concentration of 1 mg/ml is made3O4@Au nano-particle solutions.
(3) 5 mL Fe are measured3O4@Au solution is added to ultrasound 4 hours in above-mentioned (1) solution, Magneto separate washing, 4 DEG C of guarantors It deposits spare.
3, magnetic Fe3O4The characterization of@Au@CS nanocomposites
(1) infrared detection：Take a little Fe3O4Suitable potassium bromide grinding is added uniformly in mortar in@Au@CS nano-particles, It can be placed in infrared spectrometer and be measured after tabletting.
(2) the step of XRD is detected：Take a little Fe3O4@Au@CS nano-particles are added suitable potassium bromide and grind in mortar Mill is uniform, and tabletting is placed on silicon chip to be placed in x-ray photoelectron spectroscopy instrument and be measured.
(3) SEM is characterized：Conducting resinl or Double-face gummed paper are first cut into different shapes, it, will such as triangle or square It is bonded on specimen holder, and then equably powder to be measured is sprinkling upon above again, finally blows away the powder not sticked, then plate one Layer conductive film, you can be scanned Electronic Speculum observation.
Step 2: the preparation of nano-Au solution
1, the chlorauric acid solution for measuring 100 mL a concentration of 0.01% is added in round-bottomed flask, and heating under agitation is boiled；
2, the sodium citrate solution for measuring 2 mL 1% is added drop-wise in the chlorauric acid solution of boiling, and observation solution is slowly become by colourless Change to claret, static cooling, acquired solution is nano-Au solution, and grain size is about 13 nm, is finally stored with brown bottle molten Liquid refrigerates spare in 4 DEG C of refrigerators.
Step 3: structure is with Fe3O4@Au@CS composite nano materials are the electrochemica biological sensor of carrier
1, magnetic Fe3O4@Au@CS mark Ab1 antibody
(1) first with pure water by magnetic Fe3O4@Au@CS composite nanoparticle Magneto separates wash 3 times, then will with pH7.4 PBS Fe3O4@Au@CS composite nanoparticle Magneto separates wash 3 times, its concentration is then adjusted to 1 mg/mL and ultrasound point with pure water It dissipates uniform；
(2) 5 mL magnetic Fes are taken3O4@Au@CS composite nanoparticle solution, adds 150 μ g Ab1 antibody, and antibody is dripped by several times It adds to and constantly shakes in composite nanoparticle solution, place 2 h under shaking table shake state after being added dropwise to complete, BSA is then added It closes extra nonspecific activity site and adjusts a concentration of 1%, continue 1 h of placing response under shake；
(3) it after the completion of reacting, carries out Magneto separate with the PBS solution of the pH7.4 containing 1% BSA and washs, finally use containing 1% BSA The concentration for the magnetic Fe_3O_4@Au@CS nano-particles for being marked with Ab1 antibody is adjusted to 1 mg/mL by the PBS solution of pH7.4, note For Fe3O4@Au@CS-Ab1。
2, nano gold mark HRP enzymes and Ab2 antibody
(1) 0.1M Na are first used2CO3It is 9 that solution nano-Au solution, which is adjusted to pH, and the HRP of the Ab2 and 100 μ g of 30 μ g is added Enzyme is stirred to react 30 min, adds 1%BSA and continues to stir 1h, closes nonspecific activity position that may be present in nanogold Point；
(2) solution is moved in 1mL centrifuge tubes, centrifuges 30 min with 4800 r/min, after centrifugation, sucks supernatant, Precipitation is scattered in the PBS solution of the pH7.4 containing 1% BSA again, Ab2-Au-HRP is made, be stored in 4 DEG C it is spare.
3, immunoreaction process
100 μ L magnetic Fes are drawn with liquid-transfering gun3O4@Au@CS-Ab1 nano-particle solutions are in the circular hole of polystyrene micropore plate In, be added the 10 appropriate concentration of μ L AFP antigens and 10 μ L, 30 μ g/ml Ab2-Au-HRP after mixing, in 37 DEG C 30 min of lower incubation.It is combined through the specific immunity of antigen and antibody, both obtains Fe3O4@Au@CS-Ab1@AFP@Ab2-Au-HRP。
Step 4: the quantitative detection of AFP AFP
1, the processing of electrode
Electrochemical Detection uses traditional three-electrode system：Platinum electrode is to electrode, and saturated calomel electrode is reference electrode, magnetic Glass-carbon electrode is working electrode.Before detection, by magnetic glassy carbon electrode respectively with 0.3 μm, 0.05 μm of alundum (Al2O3) powder After polishing and polishing, washed 3 times with distilled water, absolute ethyl alcohol respectively；The electrode of wash clean is placed in 0.5 mol/L H2SO4In Cyclic voltammetry scan activation is carried out, it is totally spare with pure water rinsing after the completion of scanning.
2, the detection of alpha-fetoprotein
With liquid-transfering gun carefully by Fe3O4@Au@CS-Ab1@AFP@Ab2-Au-HRP liquid is drawn from micropore to be come out, and by several times will It is added drop-wise on processed magnetic glassy carbon electrode, sucks supernatant；Three-electrode system is assembled, 5mL is containing 2mmol/L to benzene two The solution of the pH7.4 PBS of phenol is as test bottom liquid；It is measured using i-t methods, the current potential of measurement is 0.1V-0.2V, scanning Rate is 50 MV/s, and under shake state, the H of 100 μ L, 0.1 mol/L is added into test bottom liquid2O2, after electric current stabilization, According to the amount and addition H that AFP is added2O2Former and later two current-responsive values quantify AFP.
Step 5: the detection of practical serum sample
0.2 mL serum samples are taken, 1.0 mL are diluted to pH7.4 PBS.The 10 diluted serum of μ L are then taken to replace step 3 AFP antigens, build Fe3O4@Au@CS-Ab1@AFP@Ab2-Au-HRP immunosensors.It is measured according to step 4.According to The amount of AFP and addition H in the serum of addition2O2Former and later two current-responsive values realize the quantitative analysis to AFP in serum.
The present invention has the following advantages that compared with prior art：
1, with Fe3O4It is shell for core Au, magnetic Fe is prepared by hydroxylamine hydrochloride reduction method3O4@Au nano materials, by chitosan It is re-used as shell, obtains magnetic Fe3O4@Au@CS composite nano materials have good magnetic effect and stability.
2, the electrochemical immunosensor Fe for detecting alpha-fetoprotein is prepared for by the way of sandwich immunoassay reaction3O4@ Au@CS-Ab1@AFP@Ab2-Au-HRP, for detecting alpha-fetoprotein, the results showed that the AFP concentration within the scope of 0.01-8 μ g/mL It is in more good linear relationship, equation of linear regression with its current-responsive value：y=0.16679x+0.17813（Y is sensor Current-responsive value, x are AFP concentration）, related coefficient 0.99715.
3, sandwich immunoassay reaction is a kind of reaction that standard is homogeneous, can improve the reaction efficiency of AFP antigens and antibody.
Description of the drawings
Fig. 1 magnetic Fes3O4@Au@CS nano materials are used for the schematic diagram of Electrochemical Detection alpha-fetoprotein
Fig. 2 magnetic Fes3O4The infared spectrum of@Au@CS nanocomposites
Fig. 3 magnetic Fes3O4The XRD diagram of@Au@CS nanocomposites
Fig. 4 magnetic Fes3O4The SEM of@Au@CS nanocomposites schemes
The current-responsive curve of Fig. 5 immunosensors
The working curve of Fig. 6 alpha-fetoproteins.
Specific implementation mode
Below in conjunction with the accompanying drawings, further this patent is illustrated.
Fig. 1 is magnetic Fe3O4@Au@CS nano materials are used for the schematic diagram of Electrochemical Detection alpha-fetoprotein.
Specific implementation step is as follows：
1. preparing Fe3O4@Au@CS composite nano materials
（1）Magnetic Fe3O4Preparation
1. weighing 2.50g Fe3O4·7H2O is first added in 30 mL pure water and makes it dissolve, add 10 mL's in beaker After 50 g/L PEG-20000 aqueous solutions, stirred under 30 DEG C of constant temperatures；
3. the H of 0.27 mL 30% of accurate measuring2O2Solution is at the uniform velocity added under conditions of being stirred continuously in (2) solution, at this time Solution colour blackening, Fe (OH)2Fe in precipitation2+Ion is oxidized to Fe3+Ion, and continue to be stirred to react 20 min；
4. will be stirred to react complete（3）Solution is transferred in autoclave, 5 h of isothermal reaction at 160 DEG C；
5. after the reaction was complete it is cooling after, solution is poured out from autoclave, is taken up in order of priority with pure water and absolute ethyl alcohol magnetic point It 3 times from washed product, is transferred in beaker, is placed under 70 DEG C of constant temperature and dries；
6. after drying, reaction product is ground up to magnetic Fe with agate mortar3O4Nano particle, by finished product under room temperature It is sealed.
（2）Magnetic Fe3O4The preparation of@Au nano-complex particles
1. accurately weighing 10 mg Fe3O4, the PEG-20000 aqueous solutions of 120 mL, 50 g/L are added in supersonic cell powder Broken machine（Power 300W, work 2 s, 3 s of gap）99 min of middle supersound process；
2. by the Fe after ultrasound3O4Dispersion liquid pours into 500 mL conical flasks, with 160 r/min of rotating speed that circles round, 25 DEG C of temperature Condition is circled round concussion in gas bath isothermal vibration device, and 1 mL1% gold chlorides, 60 min of sustained response is added dropwise；
3. be then added in the hydroxylamine hydrochloride solution to conical flask of 5 ml 80 mmol/L, 60 min of sustained response, after will Solution imports small beaker and is performed in multiple times Magneto separate；
4. then repeating Magneto separate with the PEG-20000 aqueous solutions of 50 g/L to clean three times to get magnetic Fe3O4@Au are compound Nano-particle.
（3）Magnetic Fe3O4The preparation of@Au@CS nano-complex particles
1. accurately weighing 0.2 g chitosans, it is dissolved in the acetum of 10 ml 1%, and stir.
2. accurately weighing 1.0 mg Fe3O4@Au nano-particles are poured into 1 ml secondary waters simultaneously ultrasonic disperse, are made At the Fe of a concentration of 1 mg/mL3O4@Au nano-particle solutions.
3. measuring 5 mL Fe3O4@Au solution is added to ultrasound 4 hours in above-mentioned (1) solution, Magneto separate washing, 4 DEG C of preservations It is spare.
（4）Magnetic Fe3O4The characterization of@Au@CS nanocomposites
1. infrared detection：Take a little Fe3O4In mortar suitable potassium bromide grinding is added uniformly, pressure in@Au@CS nano-particles It can be placed in infrared spectrometer and be measured after piece.Collection of illustrative plates is shown in attached drawing 2.
2. XRD is detected：Take a little Fe3O4Suitable potassium bromide grinding is added uniformly in mortar in@Au@CS nano-particles, Tabletting is placed on silicon chip to be placed in x-ray photoelectron spectroscopy instrument and be measured.Collection of illustrative plates is shown in attached drawing 3.
3. SEM is characterized：Conducting resinl or Double-face gummed paper are first cut into different shapes, such as triangle, are bonded in sample On seat, then equably powder to be measured is sprinkling upon above again, finally blows away the powder not sticked, then plates last layer conductive film, i.e., Electronic Speculum observation can be scanned.Scanning electron microscope collection of illustrative plates is shown in attached drawing 4.
2. the preparation of nano-Au solution
（1）The chlorauric acid solution for measuring 100 mL a concentration of 0.01% is added in round-bottomed flask, and heating under agitation is boiled；
（2）The sodium citrate solution for measuring 2 mL 1% is added drop-wise in the chlorauric acid solution of boiling, observation solution by it is colourless slowly It is changed to claret, static cooling, acquired solution is nano-Au solution, and grain size is about 13 nm, is finally stored with brown bottle Solution refrigerates spare in 4 DEG C of refrigerators.
3. the preparation of the sensor for detecting AFP
（1）Use magnetic Fe3O4@Au@CS mark Ab1 antibody
1. first with pure water by magnetic Fe3O4@Au@CS composite nanoparticle Magneto separates wash 3 times, then with pH7.4 PBS by Fe3O4@ Au@CS composite nanoparticle Magneto separates wash 3 times, its concentration is then adjusted to 1 mg/mL with pure water and ultrasonic disperse is equal It is even；
2. taking 5 mL magnetic Fes3O4@Au@CS composite nanoparticle solution, adds 150 μ g Ab1 antibody, and antibody is added dropwise by several times It is shaken in composite nanoparticle solution to continuous, 2 h is placed under shaking table shake state after being added dropwise to complete, BSA envelopes are then added It closes extra nonspecific activity site and adjusts a concentration of 1%, continue 1 h of placing response under shake；
3. after the completion of reaction, carrying out Magneto separate with the PBS solution of the pH7.4 containing 1% BSA and washing, finally use containing 1% BSA The PBS solution of pH7.4 will be marked with the magnetic Fe of Ab1 antibody3O4The concentration of@Au@CS nano-particles is adjusted to 1 mg/mL, note For Fe3O4@Au@CS-Ab1。
（2）Nano gold mark HRP enzymes and Ab2 antibody
1. with 0.1mol/L Na2CO3It is 9 that solution nano-Au solution, which is adjusted to pH, and the HRP of the Ab2 and 100 μ g of 30 μ g is added Enzyme is stirred to react 30 min, adds 1%BSA and continues to stir 1h, closes nonspecific activity position that may be present in nanogold Point；
2. solution is moved in 1mL centrifuge tubes, 30 min are centrifuged with 4800 r/min, after centrifugation, suck supernatant, it will Precipitation is scattered in the PBS solution of the pH7.4 containing 1% BSA again, and Ab2-Au-HRP is made, be stored in 4 DEG C it is spare.
（3）Immunoreaction process
100 μ L magnetic Fes are drawn with liquid-transfering gun3O4@Au@CS-Ab1 nano-particle solutions are in the circular hole of polystyrene micropore plate In, be added the 10 appropriate concentration of μ L AFP antigens and 10 μ L, 30 μ g/ml Ab2-Au-HRP after mixing, in 37 DEG C 30 min of lower incubation.It is combined through the specific immunity of antigen and antibody, both obtains Fe3O4@Au@CS-Ab1@AFP@Ab2-Au-HRP。
4. the quantitative detection of AFP AFP
（1）The processing of electrode
Electrochemical Detection uses traditional three-electrode system：Platinum electrode is to electrode, and saturated calomel electrode is reference electrode, magnetic Glass-carbon electrode is working electrode.Before detection, by magnetic glassy carbon electrode respectively with 0.3 μm, 0.05 μm of alundum (Al2O3) powder After polishing and polishing, washed 3 times with distilled water, absolute ethyl alcohol respectively；The electrode of wash clean is placed in 0.5 mol/L H2SO4In Cyclic voltammetry scan activation is carried out, it is totally spare with pure water rinsing after the completion of scanning.
（2）The detection of alpha-fetoprotein
With liquid-transfering gun carefully by Fe3O4@Au@CS-Ab1@AFP@Ab2-Au-HRP liquid is drawn from micropore to be come out, and by several times will It is added drop-wise on processed magnetic glassy carbon electrode, sucks supernatant；Three-electrode system is assembled, 5mL is containing 2mmol/L to benzene two The pH7.4 PBS of phenol are as test bottom liquid；It is measured using i-t methods, the current potential of measurement is 0.1V-0.2V, and sweep speed is The H of 100 μ L, 0.1 mol/L is added in 50 mV/s under shake state into test bottom liquid2O2, after electric current stabilization, according to adding Enter the amount and addition H of AFP2O2Former and later two current-responsive values quantify AFP.The current-responsive curve of immunosensor See that attached drawing 5, the AFP working curves of gained are shown in attached drawing 6.The sensor AFP concentration and its electric current within the scope of 0.01 ~ 8 μ g/mL Response is in more good linear relationship（Y=0.17813+0.16679 X, R2=0.99715）, it can be achieved that alpha-fetoprotein is quantitatively examined It surveys.
5. the detection of practical serum sample
0.2 mL serum samples are taken, 1.0 mL are diluted to pH7.4 PBS.The 10 diluted serum of μ L are then taken to replace step 3 AFP antigens, build Fe3O4@Au@CS-Ab1@AFP@Ab2-Au-HRP immunosensors.It is measured, obtains according to step 4 Current-responsive difference.The content of AFP in serum is calculated according to AFP working curves, and is compareed with clinical effectiveness, and recycling is calculated Rate the results are shown in Table 1.The rate of recovery is 90.42-109.67%, which can be used for the detection of practical serum sample.
The testing result of 1 practical serum sample of table

Claims (1)

1. a kind of method of composite nano materials Electrochemical Detection alpha-fetoprotein, including magnetic Fe3O4@Au composite nanoparticle systems It is standby, it is characterised in that：Further include following steps：
1) magnetic Fe3O4The preparation of@Au@CS nano-complex particles
Chitosan is weighed, acetum is dissolved in, is stirred evenly；
Weigh Fe3O4@Au nano-particles, are made Fe3O4@Au solution；
Take Fe3O4@Au solution is added to ultrasonic reaction in chitosan solution, and Fe is made3O4@Au@CS solution, saves backup；
2）The preparation of nano-Au solution
Chlorauric acid solution is measured, agitating and heating is boiled；Sodium citrate solution is measured, is added drop-wise in the chlorauric acid solution of boiling anti- It answers, obtains the nano-Au solution that grain size is 13 nm, refrigeration is spare；
3）Fe3O4The structure of@Au@CS-Ab1@AFP@Ab2-Au-HRP electrochemical immunosensors
Magnetic Fe3O4@Au@CS mark Ab1 antibody
Take magnetic Fe3O4Ab1 antibody is added to the Fe constantly shaken by@Au@CS composite nanoparticle solution by several times3O4@Au@CS are multiple It closes in nano-particle solution, is placed certain time under shaking table shake state, BSA is added and closes extra nonspecific activity position It puts and adjusts concentration；After the completion of reaction, carries out Magneto separate with the PBS solution containing BSA and wash, mark is adjusted with the PBS solution containing BSA Note has the magnetic Fe of Ab1 antibody3O4The concentration of@Au@CS nano-particle solutions, is denoted as Fe3O4@Au@CS-Ab1；
Nano gold mark HRP enzymes and Ab2 antibody
First use Na2CO3Solution adjusts the pH value of nano-Au solution, Ab2 and HRP enzymes are added, and after being stirred to react a period of time, is added BSA solution ＆ stirs；Solution is moved in centrifuge tube and is centrifuged, supernatant is sucked after centrifugation, precipitation is scattered in again and is contained The PBS solution of BSA is made Ab2-Au-HRP, saves backup；
Immunoreaction process
Draw magnetic Fe3O4@Au@CS-Ab1 nano-particle solutions in the circular hole of polystyrene micropore plate, be added AFP antigens and Ab2-Au-HRP, which is uniformly mixed, to be incubated；Specific immune response through antigen and antibody obtains Fe3O4@Au@CS-Ab1@AFP@ Ab2-Au-HRP electrochemical immunosensors；
4）The quantitative detection of AFP AFP
The processing of electrode
Electrochemical Detection uses traditional three-electrode system：Platinum electrode is to electrode, and saturated calomel electrode is reference electrode, magnetic Glass-carbon electrode（MGCE）For working electrode；Before detection, by magnetic glassy carbon electrode（MGCE）It is pre-processed；It will be pretreated Electrode is placed in H2SO4Middle progress cyclic voltammetry scan activation, it is totally spare with pure water rinsing after the completion of scanning；
The quantitative detection of alpha-fetoprotein
By Fe3O4@Au@CS-Ab1@AFP@Ab2-Au-HRP liquid is drawn from micropore to be come out, and is added drop-wise to by several times processed On magnetic glassy carbon electrode, supernatant is sucked；Three-electrode system is assembled, uses the PBS solution containing hydroquinone as test bottom liquid, adopts It is measured with I-T methods；H is added into test bottom liquid under shake state2O2, after electric current stabilization, record current value；According to The amount and addition H of AFP is added2O2Former and later two current-responsive values quantify AFP；
5）The detection of practical serum sample
After the PBS of the serum sample of acquisition pH value 7.4 is diluted 5 times, 1 part of diluted serum is taken to replace step 3）AFP it is anti- Original builds Fe3O4@Au@CS-Ab1@AFP@Ab2-Au-HRP immunosensors；
According to step 4）It is analyzed, according to the amount of AFP in the serum of addition and addition H2O2Former and later two current-responsive values are realized Quantitative analysis to AFP in serum.
CN201810126072.9A 2018-02-08 2018-02-08 A kind of method of composite nano materials Electrochemical Detection alpha-fetoprotein Active CN108375612B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810126072.9A CN108375612B (en) 2018-02-08 2018-02-08 A kind of method of composite nano materials Electrochemical Detection alpha-fetoprotein

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810126072.9A CN108375612B (en) 2018-02-08 2018-02-08 A kind of method of composite nano materials Electrochemical Detection alpha-fetoprotein

Publications (2)

Publication Number Publication Date
CN108375612A true CN108375612A (en) 2018-08-07
CN108375612B CN108375612B (en) 2019-07-30

Family

ID=63017352

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810126072.9A Active CN108375612B (en) 2018-02-08 2018-02-08 A kind of method of composite nano materials Electrochemical Detection alpha-fetoprotein

Country Status (1)

Country Link
CN (1) CN108375612B (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110146581A (en) * 2019-06-03 2019-08-20 桂林电子科技大学 A method of alpha-fetoprotein is detected based on RGO-CS-Fc/Au NPs nanocomposite combination aptamers
CN111781351A (en) * 2020-07-15 2020-10-16 桂林电子科技大学 Electrochemical immunochromatographic test strip for quantitative detection of novel coronavirus antigen and preparation method thereof
CN113804633A (en) * 2021-09-15 2021-12-17 中国石油大学(华东) Based on Au-Fe3O4Preparation method and application of nano-material salmonella identification immune probe

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004093406A (en) * 2002-08-30 2004-03-25 Fujirebio Inc Solution measuring micro electrode, electrochemical measuring apparatus, electrode manufacturing method, and solution measuring method
WO2011109214A2 (en) * 2010-03-01 2011-09-09 University Of Florida Research Foundation, Inc. Near-ir indocyanine green doped multimodal silica nanoparticles and methods for making the same
CN102608189A (en) * 2012-03-30 2012-07-25 山东理工大学 Method for manufacturing nanometer magnetic ferroferric oxide modified immunosensor
CN104133067A (en) * 2014-08-05 2014-11-05 桂林电子科技大学 Method for electrochemical detection of human immune globulin E (hIgE)on the basis of magnetic Fe3O4 @ Au nano composite material
WO2017167633A1 (en) * 2016-03-31 2017-10-05 Sony Corporation Sensor for the detection of biomolecules

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004093406A (en) * 2002-08-30 2004-03-25 Fujirebio Inc Solution measuring micro electrode, electrochemical measuring apparatus, electrode manufacturing method, and solution measuring method
WO2011109214A2 (en) * 2010-03-01 2011-09-09 University Of Florida Research Foundation, Inc. Near-ir indocyanine green doped multimodal silica nanoparticles and methods for making the same
CN102608189A (en) * 2012-03-30 2012-07-25 山东理工大学 Method for manufacturing nanometer magnetic ferroferric oxide modified immunosensor
CN104133067A (en) * 2014-08-05 2014-11-05 桂林电子科技大学 Method for electrochemical detection of human immune globulin E (hIgE)on the basis of magnetic Fe3O4 @ Au nano composite material
WO2017167633A1 (en) * 2016-03-31 2017-10-05 Sony Corporation Sensor for the detection of biomolecules

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YULIN YUAN 等: "A Fe3O4@Au-basedpseudo-homogeneous electrochemical immunosensor for AFP measurement using AFP antibody-GNPs-HRP as detection probe", 《ANALYTICAL BIOCHEMISTRY》 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110146581A (en) * 2019-06-03 2019-08-20 桂林电子科技大学 A method of alpha-fetoprotein is detected based on RGO-CS-Fc/Au NPs nanocomposite combination aptamers
CN111781351A (en) * 2020-07-15 2020-10-16 桂林电子科技大学 Electrochemical immunochromatographic test strip for quantitative detection of novel coronavirus antigen and preparation method thereof
CN111781351B (en) * 2020-07-15 2023-03-28 桂林电子科技大学 Electrochemical immunochromatographic test strip for quantitative detection of novel coronavirus antigen and preparation method thereof
CN113804633A (en) * 2021-09-15 2021-12-17 中国石油大学(华东) Based on Au-Fe3O4Preparation method and application of nano-material salmonella identification immune probe
CN113804633B (en) * 2021-09-15 2023-07-11 中国石油大学(华东) Based on Au-Fe 3 O 4 Preparation method and application of salmonella recognition immune probe of nano material

Also Published As

Publication number Publication date
CN108375612B (en) 2019-07-30

Similar Documents

Publication Publication Date Title
CN106442994B (en) A kind of preparation method and application of the electrochemical immunosensor based on Ag@Au nano composite materials
CN102818893B (en) Preparation and application of Au-Pd core-shell material constructed lung cancer tumor marker immunosensor
CN108593743B (en) Preparation method and application of platinum-palladium composite molybdenum diselenide marked sandwich type immunosensor
CN108051491B (en) It is a kind of for detecting the electrochemical immunosensor of LAG-3 albumen
CN101216429A (en) SERS biological probe and method for making same
CN106596969A (en) Production method, product, detection method and application of electrochemiluminescence immunosensor
CN108375612B (en) A kind of method of composite nano materials Electrochemical Detection alpha-fetoprotein
CN110687182A (en) Preparation method of electrochemical immunosensor for detecting prostate specific antigen
CN106568973A (en) Preparation method and application of MoS2/Au-Pd compound-based procalcitonin electrochemical immunosensor
CN105954339B (en) A kind of preparation method and application of the interlayer type immunosensor based on CeO2@Cu2O/Au@Pt
CN108344783A (en) A kind of electro-chemical cells sensor and its preparation method and application
CN109613244B (en) Preparation method and application of Ag @ Pt-CuS labeled immunosensor
CN108918853B (en) Pd @ Ag @ CeO2Preparation method and application of labeled immunosensor
CN110133252A (en) For detecting kit and detection method and its application of carcinomebryonic antigen
CN108896638B (en) Preparation method and application of immunosensor based on titanium dioxide doped graphene loaded sea cucumber-like gold-palladium core-shell nanoparticles
CN108469461B (en) Preparation method and application of sandwich type lung cancer marker electrochemical sensor
CN108663424A (en) A kind of preparation method and application of the immunosensor based on the hud typed gold@palladium nanospheres of sea urchin shape
CN106093396A (en) A kind of preparation method and application of immunosensor based on Au GQD@PtPd
CN108956743A (en) A kind of preparation method and its detection method of the field effect transistor biosensor that can be enhanced with gold nanoparticle
CN106770530B (en) A kind of preparation method and application of squamous cell carcinoma marker interlayer type immunosensor
Zheng et al. A Label-Free Immunosensor for CEA based on Pd–Ir bimetallic nanoparticles
Ma et al. The preparation of C-reactive protein immunosensor based on nano-mimetic enzyme Co3O4
CN106802314B (en) A kind of preparation method and application of the immunosensor of the graphene based on load TaC and gold cladding decahedron Nano silver grain
CN110031447A (en) A kind of detection method that substrate being immunized using magnetic surface enhancing Raman
CN106248657B (en) One kind is based on dendroid Fe2O3The preparation method and application of@Au Electrochemiluminescsensor sensor

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant