CN109682964A - Au@Fe3O4MNPs-Ab2The preparation method of nano enzyme detection probe and the method for detecting multi-component antigen - Google Patents

Au@Fe3O4MNPs-Ab2The preparation method of nano enzyme detection probe and the method for detecting multi-component antigen Download PDF

Info

Publication number
CN109682964A
CN109682964A CN201910091195.8A CN201910091195A CN109682964A CN 109682964 A CN109682964 A CN 109682964A CN 201910091195 A CN201910091195 A CN 201910091195A CN 109682964 A CN109682964 A CN 109682964A
Authority
CN
China
Prior art keywords
mnps
concentration
detection probe
array
nano enzyme
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
CN201910091195.8A
Other languages
Chinese (zh)
Other versions
CN109682964B (en
Inventor
胡锐宣
刘靳一蒙
吴昕玥
杨占军
李娟�
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.)
Yangzhou University
Original Assignee
Yangzhou University
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 Yangzhou University filed Critical Yangzhou University
Priority to CN201910091195.8A priority Critical patent/CN109682964B/en
Publication of CN109682964A publication Critical patent/CN109682964A/en
Application granted granted Critical
Publication of CN109682964B publication Critical patent/CN109682964B/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
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials

Abstract

The present invention relates to a kind of Au@Fe3O4MNPs‑Ab2The preparation method of nano enzyme detection probe and the method for detecting multi-component antigen, the Au@Fe that the present invention synthesizes first3O4Nanoparticle probes are successfully applied to the chemiluminescence array immunization sensor of building nanometer enzyme signal amplification.Secondly, different capture antibody is fixed on the different solid phase interface of immuno-array sensor, antigen samples and Au@Fe are then each led into3O4MNPs‑Ab2Nanowire signal amplifying probe, online incubate forms stable sandwich immunoassay reaction compound, generated optical signals Charged Couple CCD camera is collected after being passed through chemiluminescent substrate, the multi-component antigen chemiluminescence immunoassay detection for realizing nano enzyme catalyzed signal amplification, suitable for being detected while multiple analytes antigen.The analysis method has that testing cost is low, sample consumption is few, expends that the time is short, stability is good, simple operation and other advantages, provides the deep detection platform for having prospect for the clinical detection of poultry disease.

Description

Au@Fe3O4MNPs-Ab2The preparation method and detection multicomponent of nano enzyme detection probe The method of antigen
Technical field
The present invention relates to biological molecular chemistry electrochemiluminescent immunoassay to test and analyze technical field, in particular to a kind of Au@ Fe3O4MNPs-Ab2The method of preparation method and its light emitting array the detection multi-component antigen of nano enzyme detection probe.
Background technique
Chemiluminescence immune assay (Chemiluminescence Immunoassay, CLIA) is that have high sensitivity The analysis method that chemiluminescence is combined with the immune response of high specific, is in conventional radioactive immunoassay, enzyme linked immunological One kind for growing up after the immunoassay methods such as analysis, fluoroimmunoassay and time-resolved fluoroimmunoassay is new Detect the novel markings immuno analytical method of trace antigen and antibody.Chemiluminescence imaging immunoassay is that current chemiluminescence is exempted from One of method of greatest concern, combines chemiluminescence immune assay and imaging technique, has both chemiluminescence in epidemic disease analysis detection The advantages of the advantages of immunoassay and imaging analysis flux are high, and energy multicomponent detects simultaneously.Be employed successfully in detection protein, The micro substances such as hormone, cell factor, in the diagnosis of disease, detection body's immunity situation, monitoring therapeuticing effect and monitoring It also plays an important role in the state of an illness.For the sensitivity for improving immunoassay, usually borrow what nano material was amplified as signal Carrier.
It is easy to loss of catalytic activity compared to variation of native enzyme during preservation because of its structure, and its catalysis is lived Property is as the active site of macromolecular peptide is embedded and influences other catalytic activity.And analogue enztme have be synthetically prepared letter The advantages that list, preferable stability are not influenced vulnerable to environment temperature etc., and method of modifying is simple.Therefore research and development stability and Efficient analogue enztme is very necessary.
In the practical application of immunoassay, realize frequently with multiple one pack system analytic approach are measured in parallel in complex sample The measurement of multicomponent content, this method is usually along with the disadvantages of cumbersome, analysis cost is high, time and effort consuming.Currently, more Component immuno analytical method causes people's greatly research interest in immunoassay field, and which overcome conventional analysis modes Deficiency, realize multi-component while detecting in single analysis process, have analysis efficiency is high, reagent consumption less, taken Between short, the advantages such as analysis cost is low.
Summary of the invention
The present invention is for the nanometer material used in chemiluminescence immune assay in the prior art to improve analytical sensitivity The limited problem of the catalytic activity of material amplification carrier, provides a kind of Au Fe3O4MNPs-Ab2The preparation side of nano enzyme detection probe Method, a kind of Au and two Fe is made3O4The nano enzyme detection probe of conjuncted structure, makes Fe3O4Catalytic activity it is unobstructed, mention High detection sensitivity.
Above-mentioned purpose to realize the present invention, Au@Fe of the present invention3O4MNPs-Ab2The preparation method of nano enzyme detection probe, The following steps are included:
Firstly, preparation Au@Fe3O4Nanoparticle: be (1~1.5) by volume ratio: 20 oleic acid is mixed with octadecene, in N2Stream Under be heated to 120 °C of 20 min of heat preservation, in N2Fe (CO) is injected into mixed liquor under layer5, make Fe (CO)5In above-mentioned mixed liquor In concentration be 35~40mg/mL, stir 5 min after, by volume be oleic acid volume half oleyl amine injection reaction after mixing In object, then into mixture, mixed liquor is heated to 300~310 after mixing by the Au nano particle of 10-15 times of quality °C reflux 45 min, after being cooled to room temperature, add isopropanol and separate particle, isolate discrete Au nano particle, will point Obtained dyad Au@Fe after separating out Au nano particle3O4Nanoparticle is distributed in hexane, makes Au@Fe3O4Dispersion it is dense Degree is 5 mg/mL；The Au@Fe of dyad structure is made by this step3O4Nano particle, nanoparticle Au and Fe therein3O4Mutually It does not wrap up, mutual catalytic activity and capture ability are independent of each other.
For convenient for obtained Au@Fe3O4MNPs-Ab2Nano enzyme detection probe continues up step Au@Fe obtained3O4Nanometer The solution of particle is 30:(1~1.5 by volume) addition concentration be 100~200 μ g/mL secondary antibody Ab2, it is slowly stirred 2h, 30 min are centrifuged with 10000 rpm under 4 °C again, to remove excessive Ab2Capture antibody；Will centrifugation liquid precipitate after be scattered in In the phosphate buffer of 0.01M；This step above process is at least repeated twice, and finally obtaining dispersion concentration is 5 mg/mL's Au@Fe3O4MNPs-Ab2Nano enzyme detection probe suspended dispersed liquid.This dispersion liquid can save under 4 °C.
The band capture antibody A b as made from the above method2Au@Fe3O4MNPs-Ab2Nano enzyme detection probe, dyad The Au@Fe of structure3O4MNPs magnetic nano-particle has excellent catalytic activity, high stability, improves native enzyme and stablizes Property is poor, and sensitivity is low, is not easy the defects of obtaining.
To further realize the purpose of the present invention, the Au nano particle the preparation method comprises the following steps: by HAuCl4·3H2O is added Into tetrahydronaphthalene, compound concentration is the solution of 2.5~3 mmol/100 mL, and adding volume is tetrahydronaphthalene volume 1/10 Oleyl amine be configured to red solution, the solution is then cooled to room temperature in 0~70 °C of 5-6h h of heating, by ethyl alcohol plus Enter into solution, by being centrifugated gold particle, and with ethanol washing, then in hexane, obtained dispersion concentration is redisperse The Au colloidal state body dispersion of 10mg/mL.
For convenient for the above-mentioned Au@Fe of utilization3O4MNPs-Ab2Nano enzyme detection probe detects the multi-component factor, this hair simultaneously The bright one kind that also provides is based on Au@Fe3O4MNPs-Ab2The chemiluminescence array immunization sensor of nano enzyme detection probe detects multiple groups Divide the method for antigen, comprising the following steps:
B1: using screen printing technique in the reaction array for printing multi-point detection on silanated slides；
B2: chitosan solution is mixed to the capture antibody difference drop coating of different analysans in equal volume in the anti-of B1 step silk-screen printing Answer each point of array；Block buffer closing activity site is used again, and array immunization sensor is made；
B3: the antigen to be detected corresponding with B2 each point of step and Au of 5 μ L is successively added dropwise respectively into each point of immuno-array Fe3O4MNPs-Ab2Nano enzyme detection probe forms after incubation and stablizes sandwich immune complex, is added after buffer solution for cleaning drying Chemiluminescent substrate；
B4: CCD camera point collection luminous signal is used, the luminous intensity of each luminous signal is detected, according to the light of each luminous signal The linear relationship of intensity and anti-source concentration, calculates the concentration of antigen samples.
The present invention successfully constructs a kind of based on Au@Fe3O4Nanoparticle chemistry electrochemiluminescent immunoassay sensor array, and borrow It helps screen printing technique to make high-throughput immune sensing array on silanated slides, by the epoxy group on glass slide and catches It obtains the amino covalence that antibody has to combine, capture antibody is fixed on corresponding binding site.Construct it is a kind of can detect simultaneously it is more Cell factor chemiluminescence imaging immunosensor.After being passed through antigen and enzyme labelled antibody, reacted based on sandwich immunoassay, additionization The chemiluminescence reaction that luminous substrate causes double nano enzymatic is learned, detects different cytokines simultaneously using inductive coupling CCD Chemiluminescence signal, further according to luminous signal intensity and detecting factor concentration linear relationship, determine the dense of detecting factor Degree is, it can be achieved that be used to detect while cytokine profiles, compared to parallel repeatedly one pack system analysis, multi-component immunity analytical side Detection flux can be improved in method, shortens detection time, reduces sample consumption and testing cost；Meanwhile the present invention synthesizes novelly Au@Fe3O4Nanoparticle, will building simulation enzyme signal amplification chemiluminescence array immunization sensor, using chemiluminescence at As immuno analytical method, double-antibody sandwich composite construction is formed by capture antibody, antigen and enzyme labelled antibody, in conjunction with inductive coupling CCD detects the chemiluminescence signal of different cytokines, constructs good chemiluminescence imaging immunoassay system, breaks The detection of the bottom 0.01ng/mL concentration may be implemented in traditional biological detection method, has high-throughput, low cost, consumes less, is easy to operate And there is high sensitivity to the joint-detection of the specific cells factor.
Further, in detection method of the invention, in step B1, the reaction array of silk-screen printing respectively detects the micro- of point Bore dia is 2mm, and the point at edge is 4~6mm away from the spacing at glass slide edge, and each micropore outer rim is green with hydrophobic work Insulating paint.Can make in this way it is hydrophilic in micropore, it is hydrophobic outside micropore, can make micropore carry various reagents, effectively avoid letter Number interference the problems such as.
In step B1, silanated slides the preparation method comprises the following steps: disposable glass slide is soaked in Piranha acid solution Activation 10~12 hours makes its surface with amino, after deionized water is rinsed and dried, is then soaked in containing 1% γ-shrink Make its silanization in the toluene solution of glycerol ether oxygen propyl trimethoxy silicane, then is successively rinsed respectively with toluene and ethyl alcohol, nitrogen Silanated slides are made after air-blowing is dry.
Step B2) in, the different capture bodies such as antibody and 0.5 wt% chitosan solution for being 100~200 μ g/mL by concentration It is uniformly dripped after product mixing in the micropore that B1 walks each array point obtained, incubate 10 hours at room temperature or more, then use phosphoric acid It after buffer rinses glass slide, is dried with nitrogen, then confining liquid is added dropwise to each detection site reaction 12h with closing activity site, then It is flushed three times with PBST solution, array immunization sensor is made；The BST solution is that mass content is 0.05% Tween-20 0.01 mol/L pH, 7.4 phosphate buffer.
Further, the confining liquid is the bovine serum albumen solution of mass concentration 1.0~3.0%.
Further, antigen samples to be detected and Au@Fe are added dropwise respectively in the B3 step3O4MNPs-Ab2Nano enzyme detection After probe, the time incubated respectively is 25~30min.
Detection while for convenient for rapidly and accurately carrying out Multi-example, B4 step in, determine luminous signal intensity and anti-source The method of the linear relationship of concentration are as follows: in B3 step, add test sample and Au@Fe to Immuno-sensor arrays3O4MNPs-Ab2 Before nano enzyme detection probe, immunosensor battle array is divided into standard sample detection zone and unknown sample detection zone, the standard Each detection point of sample detection zone is used to be added dropwise the antigen samples and Au Fe of known concentration3O4MNPs-Ab2Nanometer enzyme signal is put Big probe；Then in B4 step, anti-source concentration and light are fitted according to the luminous intensity of the known concentration point of acquisition and known concentration The standard curve of intensity, the standard curve are used to determine concentration value according to the light intensity value of unknown concentration.
In convenient for quick and precisely there is the luminous intensity for measuring respectively to detect point, B4 is walked, each figure in CCD camera is detected As luminous intensity is obtained by software Alpha View SA discriminance analysis.
Detailed description of the invention
Fig. 1 is the spot image array of luminous signal when various concentration ChIL-4 and ChIFN- γ standard sample detect.
Fig. 2 is the concentration of ChIL-4 standard sample and the curve of luminous intensity.
Fig. 3 is the concentration of ChIFN- γ standard sample and the curve of luminous intensity.
Specific embodiment
Embodiment 1
A kind of Au@Fe is made through this embodiment3O4MNPs-Ab2Nano enzyme detection probe, detailed process are as follows:
Au nano particle is prepared first: 1.0g HAuCl43H2O(2.5 mmol), 10 mL oleyl amines (30 mmol) are successively added Enter into 100mL tetrahydronaphthalene, after 65 °C of 5 h of heating, is cooled to room temperature, is collected by centrifugation and obtains Au with ethanol washing and receive Rice grain is dispersed in the dispersion colloid that the Au nano particle that dispersion concentration is 10 mg/ mL is obtained in hexane.
Au@Fe is prepared again3O4Nanoparticle: it will be mixed in 1 mL oleic acid (3 mmol) and 20mL octadecene, in N2Stream Under be heated to 120 °C of 20 min of heat preservation, in N2Under layer, the Fe (CO) that 0.15 mL concentration is 5mg/mL is injected into solution5.It stirs After mixing 5 min, 0.5 mL oleyl amine is injected into reaction mixture, is then injected into the colloid 2 of aforementioned obtained Au nano particle mL.Solution is heated into about 310 °C of 45 min of reflux, after being cooled to room temperature, after addition isopropanol separates particle, takes and isolates Particle is distributed in hexane, obtains the Au@Fe that dispersion concentration is 5mg/mL3O4Nanoparticle.
Finally, preparation Au@Fe3O4MNPs-Ab2Nano enzyme detection probe: Xiang Qianshu Au Fe3O4In nanoparticle dispersion liquid 10 μ L, 100 μ g/mL capture antibody A b is added2, 2 h are slowly stirred, by solution with 10000 rpm centrifugation 30 under 4 °C Min, to remove excessive Ab2；Precipitating is dispersed in again in the PBS phosphate buffer of 0.01 M.It is repeated twice the mistake of this step Journey finally obtains Au@Fe3O4MNPs-Ab2Biological composite is dispersed in suspension in 1.0 mL, 0.01 M PBS, makes Au@ Fe3O4MNPs-Ab dispersion concentration is 5mg/mL.
Band capture antibody A b is made in the above process through this embodiment2Au@Fe3O4MNPs-Ab2Nano enzyme detection is visited Needle, the Au@Fe of dyad structure3O4MNPs magnetic nano-particle has excellent catalytic activity, high stability, improves day Right enzyme stability is poor, and sensitivity is low, is not easy the defects of obtaining.
Embodiment 2
By the Au@Fe obtained of embodiment 1 in the present embodiment3O4MNPs-Ab2The chemiluminescence that nano enzyme detection probe is prepared into The method that array immunization sensor carries out detection multi-component antigen, comprising the following steps:
B1: disposable glass slide is soaked in the H that volume ratio is 7:32SO4With 30% H2O2In Piranha acid solution activation 10~ 12 hours, makes its surface with amino, after deionized water is rinsed and dried, then be soaked in containing 1% GPTMS(γ-glycidol Ether oxygen propyl trimethoxy silicane) toluene solution in make its silanization, then successively rinsed respectively with toluene and ethyl alcohol, nitrogen is blown Silanated slides are made after dry；Then using screen printing technique and what by template by silanization load glass be printed as 4 rows × 12 column formats have the reaction array of 48 detection sites, the bore dia 2 formed in hydrophobicity non-photoactive film therein Mm, 4 mm of pitch-row glass slide edge spacing at edge；
B2: the capture antibody that chitosan solution is mixed to the different chicken cell factors in equal volume takes 5 μ L drop coatings to walk silk screen in B1 respectively Each point of the reaction array of printing；Wherein, 24 point drop coating ChIL-4 of two rows capture antibody, in addition two rows of drop coating ChIFN- γ, the concentration for capturing antibody is 100 μ g/mL, is incubated at room temperature 12 hours, after then rinsing glass slide with phosphate buffer, nitrogen Air-blowing is dry, then the bovine serum albumen solution of mass concentration 2.0% is added dropwise to each detection site reaction 12h as confining liquid to close Active site, then flushed three times with 0.01 mol/L pH, 7.4 PBS of PBST(0.05% Tween-20), array immunization is made Sensor；
B3: immunosensor battle array is divided into standard sample detection zone and unknown sample detection zone, wherein standard sample detection zone is used In the immunoluminescence intensity detection of postorder known concentration sample, it is added dropwise respectively to each detection point of standard sample detection zone by 5 5 μ L are added dropwise to unknown sample detection zone and each point in ChIL-4 the and ChIFN- γ antigen samples of μ L difference known concentration respectively Then ChIL-4 the and ChIFN- γ of different unknown concentrations incubates 25 min at room temperature, then to respectively to standard sample product examine Au Fe made from the embodiment 1 of 5 μ L is added dropwise in each point for surveying area and unknown sample detection zone3O4MNPs-Ab2Nano enzyme detection is visited Needle incubates 30 min at room temperature；In this step, the antigen samples of different known concentrations are 0.01 mol/L by pH 7.4 PBS solution dilutes；Stable folder ChIL-4 and ChIFN- γ heart immune complex is formed after the processing of above-mentioned incubation, is used Chemiluminescent substrate is added after the drying of 0.01 mol/L PBS phosphate buffer nitrogen purge to each detection point；Chemistry used Luminous substrate is luminol, to iodophenol and H2O2Tris-HCl buffer；
B4: generated luminous signal is collected with 10 min of CCD camera dynamic integral, and is shown as the hot spot of varying strength Each hot spot figure is inputted matched software Alpha View SA discriminance analysis image luminescence point by figure, is taken at each micropore center solid The circle for determining diameter calculates the chemiluminescence intensity of each point using Average pixel intensity, wherein standard sample detection zone The image of each antigen samples point is added dropwise as shown in Figure 1, Fig. 2 and Fig. 3 is to be fitted according to the luminous intensity of each light spot image ChIL-4 and ChIFN- γ standard curve, by the standard curve, according to the strong light of each light spot image of unknown sample detection zone Degree can determine the concentration of the sample of each unknown concentration.
Meanwhile to investigate the accuracy and practical application value of immuno-array sensor constructed by the present invention, wherein Standard sample detection zone has respectively selected the ChIL-4 of five kinds of normal concentrations, ChIFN- γ sample, has carried out recovery testu, gained As a result it is compared with Standard entertion amount.Such as table 1, by measurement, to 0.01 ng/mL, 0.02 ng/mL, 0.04 ng/mL, 0.08 ng/mL, 0.10 ng/mL(ChIL-4)；0.01 ng/mL,0.04 ng/mL,0.08 ng/mL,0.12 ng/mL, 0.16 ng/mL(ChIFN- γ) standard sample carry out recovery experiment obtained by the rate of recovery respectively in 96%-104% and 94%- 104.5%, it shows and the good consistency of Standard entertion.It illustrates based on multiple groups constructed by Au@Fe3O4MNPs analogue enztme Divide chemiluminescence imaging immune system that there is acceptable actual sample detectability.
The recovery testu measurement result of 1 ChIL-4 of table, ChIFN- γ
The above is only presently preferred embodiments of the present invention, is not intended to limit the present invention in any form, any ripe Professional and technical personnel is known, without departing from the scope of the present invention, according to the technical essence of the invention, to the above reality Any simple modifications, equivalent substitutions and improvements etc. made by example are applied, it is fallen within the scope of protection of the technical scheme of the present invention It is interior.

Claims (10)

1. a kind of Au@Fe3O4MNPs-Ab2The preparation method of nano enzyme detection probe, comprising the following steps:
A1: preparation Au@Fe3O4Nanoparticle: be (1~1.5) by volume ratio: 20 oleic acid is mixed with octadecene, in N2It flows down 120 °C of 20 min of heat preservation are heated to, in N2Fe (CO) is injected into mixed liquor under layer5, make Fe (CO)5In above-mentioned mixed liquor Concentration is 35~40mg/mL, after stirring 5 min, in the mixture after the oleyl amine injection that volume is oleic acid volume half is reacted, Then into mixture, mixed liquor is heated to 300~310 °C and returned by the Au nano particle of 10-15 times of quality after mixing 45 min are flowed, after being cooled to room temperature, isopropanol is added and separates particle, discrete Au nano particle is isolated, will isolate Obtained dyad Au@Fe after Au nano particle3O4Nanoparticle is distributed in hexane, makes Au@Fe3O4Dispersion concentration be 5 mg/mL；
A2: preparation Au@Fe3O4MNPs-Ab2Nano enzyme detection probe: to Au@Fe made from step A13O4The solution of nano particle By volume be 30:(1~1.5) addition concentration be 100~200 μ g/mL secondary antibody Ab2, it is slowly stirred 2h, then under 4 °C 30 min are centrifuged with 10000 rpm, to remove excessive Ab2Capture antibody；The phosphorus being scattered in after liquid precipitate in 0.01M will be centrifuged In phthalate buffer；This step above process is at least repeated twice, and finally obtains the Au@that dispersion concentration is 5 mg/mL Fe3O4MNPs-Ab2Nano enzyme detection probe suspended dispersed liquid.
2. Au@Fe according to claim 13O4MNPs-Ab2The preparation method of nano enzyme detection probe, which is characterized in that institute State Au colloidal dispersion the preparation method comprises the following steps: by HAuCl4·3H2It is 2.5~3 that O, which is added to compound concentration in tetrahydronaphthalene, The solution of mmol/100 mL adds the oleyl amine that volume is tetrahydronaphthalene volume 1/10 and is configured to red solution, then should Solution is cooled to room temperature, ethyl alcohol is added in solution in 0~70 °C of 5-6h h of heating, by being centrifugated gold particle, And with ethanol washing, then in hexane, the Au colloidal state body dispersion that dispersion concentration is 10mg/mL is made in redisperse.
3. one kind is based on Au@Fe3O4MNPs-Ab2The chemiluminescence array immunization sensor of nano enzyme detection probe detects multicomponent The method of antigen, which comprises the following steps:
B1: using screen printing technique in the reaction array of silanated slides printing multi-point detection；
B2: chitosan solution is mixed to the capture antibody drop coating of different analysans in equal volume in the reaction battle array of B1 step silk-screen printing Each point of column；Block buffer closing activity site is used again, and array immunization sensor is made；
B3: the antigen to be detected corresponding with B2 each point of step and Au of 5 μ L is successively added dropwise respectively into each point of immuno-array Fe3O4MNPs-Ab2Nano enzyme detection probe forms after incubation and stablizes sandwich immune complex, is added after buffer solution for cleaning drying Chemiluminescent substrate；
B4: CCD camera point collection luminous signal is used, the luminous intensity of each luminous signal is detected, according to the light of each luminous signal The linear relationship of intensity and anti-source concentration calculates the concentration of Proantigen sample.
4. according to claim 3 be based on Au@Fe3O4MNPs-Ab2The chemiluminescence array immunization of nano enzyme detection probe The method of sensor detection multi-component antigen, which is characterized in that in step B1, the reaction array of silk-screen printing respectively detects point Micro-pore diameter is 2mm, and the point at edge is 4~6mm away from the spacing at glass slide edge, and micropore outer rim is green with hydrophobic work Insulating paint.
5. according to claim 3 be based on Au@Fe3O4MNPs-Ab2The chemiluminescence array immunization of nano enzyme detection probe Sensor detection multi-component antigen method, which is characterized in that in step B1, silanated slides the preparation method comprises the following steps: can Throwing formula glass slide is soaked in Piranha acid solution and activates 10~12 hours, makes its surface with amino, and deionized water is rinsed simultaneously After drying, being then soaked in the toluene solution containing 1% γ-glycidyl ether oxygen propyl trimethoxy silicane makes its silane Change, then successively rinsed respectively with toluene and ethyl alcohol, silanated slides are made after being dried with nitrogen.
6. according to claim 3 be based on Au@Fe3O4MNPs-Ab2The chemiluminescence array immunization of nano enzyme detection probe The method of sensor detection multi-component antigen, which is characterized in that step B2) in, the difference that concentration is 100~200 μ g/mL is caught It obtains and is uniformly dripped after antibody mixes in equal volume with 0.5 wt% chitosan solution in the micropore that B1 walks each array point obtained, room It the lower incubation of temperature 10 hours or more, after then rinsing glass slide with phosphate buffer, is dried with nitrogen, then confining liquid is added dropwise to each detection Site reaction 12h is flushed three times with closing activity site, then with PBST solution, and array immunization sensor is made；The BST solution For 0.01 mol/L pH, 7.4 phosphate buffer that mass content is 0.05% Tween-20.
7. according to claim 6 be based on Au@Fe3O4MNPs-Ab2The chemiluminescence array immunization of nano enzyme detection probe The method of sensor detection multi-component antigen, which is characterized in that the confining liquid is the cow's serum of mass concentration 1.0~3.0% Protein solution.
8. according to claim 3 be based on Au@Fe3O4MNPs-Ab2The chemiluminescence array immunization of nano enzyme detection probe The method of sensor detection multi-component antigen, which is characterized in that antigen samples to be detected and Au@are added dropwise respectively in the B3 step Fe3O4MNPs-Ab2After nano enzyme detection probe, the time incubated respectively is 25~30min.
9. according to claim 6 be based on Au@Fe3O4MNPs-Ab2The chemiluminescence array immunization of nano enzyme detection probe The method of sensor detection multi-component antigen, which is characterized in that in B4 step, determine the intensity and anti-source concentration of luminous signal The method of linear relationship are as follows: in B3 step, add test sample and Au@Fe to Immuno-sensor arrays3O4MNPs-Ab2Nano enzyme Before detection probe, immunosensor battle array is divided into standard sample detection zone and unknown sample detection zone, the standard sample product examine Each detection point for surveying area is used to be added dropwise the antigen samples and Au Fe of known concentration3O4MNPs-Ab2Nanometer enzyme signal amplification is visited Needle；Then in B4 step, anti-source concentration and luminous intensity are fitted according to the luminous intensity of the known concentration point of acquisition and known concentration Standard curve, which is used to according to the light intensity value of unknown concentration determine concentration value.
10. according to claim 6 be based on Au@Fe3O4MNPs-Ab2The chemiluminescence array immunization of nano enzyme detection probe The method of sensor detection multi-component antigen, which is characterized in that in B4 step, each image luminescence intensity detected in CCD camera is logical It crosses software Alpha View SA discriminance analysis and obtains.
CN201910091195.8A 2019-01-30 2019-01-30 Au@Fe3O4MNPs-Ab2Preparation method of nano enzyme detection probe and method for detecting multi-component antigen Active CN109682964B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910091195.8A CN109682964B (en) 2019-01-30 2019-01-30 Au@Fe3O4MNPs-Ab2Preparation method of nano enzyme detection probe and method for detecting multi-component antigen

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910091195.8A CN109682964B (en) 2019-01-30 2019-01-30 Au@Fe3O4MNPs-Ab2Preparation method of nano enzyme detection probe and method for detecting multi-component antigen

Publications (2)

Publication Number Publication Date
CN109682964A true CN109682964A (en) 2019-04-26
CN109682964B CN109682964B (en) 2022-04-29

Family

ID=66195080

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910091195.8A Active CN109682964B (en) 2019-01-30 2019-01-30 Au@Fe3O4MNPs-Ab2Preparation method of nano enzyme detection probe and method for detecting multi-component antigen

Country Status (1)

Country Link
CN (1) CN109682964B (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112730338A (en) * 2020-12-22 2021-04-30 扬州大学 Ag @ Au-based porous structure dual-signal nano amplification probe and SPR immunodetection method thereof

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102707049A (en) * 2012-05-14 2012-10-03 宁波大学 Preparation method and application of magnetic sandwich nano immunosensor
CN102914648A (en) * 2012-10-09 2013-02-06 重庆医科大学 Electrochemical immunosensor for detecting toxoplasma gondii IgM antibody and preparation method thereof
CN103823061A (en) * 2014-02-17 2014-05-28 广西壮族自治区兽医研究所 Au/Fe3O4 immunity chromatography test paper, preparation method and applications thereof
CN105301030A (en) * 2015-10-26 2016-02-03 上海师范大学 Detection reagent and method for Listeria monocytogenes
CN105866105A (en) * 2016-04-06 2016-08-17 扬州大学 Preparation and analysis method for chemiluminiscence imaging immunosensor for detecting multiple chicken cytokines
CN106018806A (en) * 2016-05-09 2016-10-12 山东理工大学 Preparing method and application of sandwich type immunosensor marked based on Au-Fe3O4@Pd-ZIF-8
CN107328928A (en) * 2017-06-09 2017-11-07 扬州大学 Based on Hemin@Fe3O4The method that the chemiluminescence immunoassay of MPs analogue enztmes detects the chicken cell factor

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102707049A (en) * 2012-05-14 2012-10-03 宁波大学 Preparation method and application of magnetic sandwich nano immunosensor
CN102914648A (en) * 2012-10-09 2013-02-06 重庆医科大学 Electrochemical immunosensor for detecting toxoplasma gondii IgM antibody and preparation method thereof
CN103823061A (en) * 2014-02-17 2014-05-28 广西壮族自治区兽医研究所 Au/Fe3O4 immunity chromatography test paper, preparation method and applications thereof
CN105301030A (en) * 2015-10-26 2016-02-03 上海师范大学 Detection reagent and method for Listeria monocytogenes
CN105866105A (en) * 2016-04-06 2016-08-17 扬州大学 Preparation and analysis method for chemiluminiscence imaging immunosensor for detecting multiple chicken cytokines
CN106018806A (en) * 2016-05-09 2016-10-12 山东理工大学 Preparing method and application of sandwich type immunosensor marked based on Au-Fe3O4@Pd-ZIF-8
CN107328928A (en) * 2017-06-09 2017-11-07 扬州大学 Based on Hemin@Fe3O4The method that the chemiluminescence immunoassay of MPs analogue enztmes detects the chicken cell factor

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HENG YU 等: "Dumbbell-like bifunctional Au-Fe3O4 nanoparticles", 《NANO LETTERS》 *
侯建国等: "可再生使用的磁性纳米修饰C反应蛋白电流型免疫传感器", 《传感技术学报》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112730338A (en) * 2020-12-22 2021-04-30 扬州大学 Ag @ Au-based porous structure dual-signal nano amplification probe and SPR immunodetection method thereof
CN112730338B (en) * 2020-12-22 2023-03-24 扬州大学 Ag @ Au-based porous structure dual-signal nano amplification probe and SPR immunodetection method thereof

Also Published As

Publication number Publication date
CN109682964B (en) 2022-04-29

Similar Documents

Publication Publication Date Title
CN108802133A (en) A kind of preparation method and application of detection stomach neoplasms tumor markers interlayer type immunosensor
CN103837675A (en) Homogeneous luminescence immunoassay method for quantitatively analyzing multiple components simultaneously and kit used for method
CN103575896B (en) Highly sensitive disposable multicomponent chemical luminescence imaging immunosensor
CN111693571B (en) Method for detecting GPC3 based on light addressing potential sensor
CN105866105A (en) Preparation and analysis method for chemiluminiscence imaging immunosensor for detecting multiple chicken cytokines
CN110361370A (en) A kind of single embryo's secretory protein quantitative detecting method based on Microfluidic droplet
CN109613244B (en) Preparation method and application of Ag @ Pt-CuS labeled immunosensor
CN105954339B (en) A kind of preparation method and application of the interlayer type immunosensor based on CeO2@Cu2O/Au@Pt
CN107677806B (en) The preparation and detection method of the highly sensitive visualization joint inspection immuno-chromatographic test paper strip of fluorescent quantitation based on magnetic enrichment
CN108918853B (en) Pd @ Ag @ CeO2Preparation method and application of labeled immunosensor
CN110133255A (en) A kind of quickly SERS- immunochromatography detection method with highly sensitive detection mycoplasma pneumoniae infection
CN105115961A (en) Method for preparing electrochemical luminescence sensor made of nano-composites
Yin et al. A microfluidic chip-based MRS immunosensor for biomarker detection via enzyme-mediated nanoparticle assembly
CN110220889A (en) A kind of pair of quenching electrochemical luminescence strategy is applied to transducer production method and the application of the detection of procalcitonin
CN110441295A (en) One kind is based on ferritin encapsulation Ir (ppy)3Biosensor preparation method
Zhong et al. Multiplex immunoassay of chicken cytokines via highly-sensitive chemiluminescent imaging array
CN113933500A (en) Portable high-sensitivity immunoassay kit for ractopamine and application thereof
CN105842460A (en) Preparation method of electro-chemiluminescence immunosensor based on silver-hybridized bismuth sulfide
CN109444240A (en) A kind of electrochemistry immuno-sensing method established based on Prussian blue electrochemical immunosensor and based on the sensor and application
CN110441293A (en) A kind of electrochemical luminescence sensor preparation method and application based on protein active protection
CN107328928A (en) Based on Hemin@Fe3O4The method that the chemiluminescence immunoassay of MPs analogue enztmes detects the chicken cell factor
CN109682964A (en) Au@Fe3O4MNPs-Ab2The preparation method of nano enzyme detection probe and the method for detecting multi-component antigen
CN110530949B (en) Preparation method of immunosensor constructed based on copper nanocluster-resonance energy transfer system
CN109991298B (en) Preparation method and application of Pt @ MOF-GO labeled electrochemical sensor
CN111426667A (en) Fluorescence method for β -lactoglobulin detection based on quantum dot-aptamer-graphene oxide

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
CB03 Change of inventor or designer information

Inventor after: Yang Zhanjun

Inventor after: Hu Ruixuan

Inventor after: Liu Jinyimeng

Inventor after: Wu Xinyue

Inventor after: Li Juan

Inventor before: Hu Ruixuan

Inventor before: Liu Jinyimeng

Inventor before: Wu Xinyue

Inventor before: Yang Zhanjun

Inventor before: Li Juan

CB03 Change of inventor or designer information
GR01 Patent grant
GR01 Patent grant