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CN109521006A - A kind of preparation method and application of double quenching competitive type Electrochemiluminescsensor sensors based on Au@NiFe MOFs - Google Patents

A kind of preparation method and application of double quenching competitive type Electrochemiluminescsensor sensors based on Au@NiFe MOFs Download PDF

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CN109521006A
CN109521006A CN201811578328.6A CN201811578328A CN109521006A CN 109521006 A CN109521006 A CN 109521006A CN 201811578328 A CN201811578328 A CN 201811578328A CN 109521006 A CN109521006 A CN 109521006A
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赵冠辉
曹伟
王耀光
李小建
东雪
李璇
魏琴
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University of Jinan
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Abstract

本发明涉及一种基于新型纳米材料Au@NiFe MOFs的双重猝灭竞争型型电致化学发光免疫传感器的制备方法及应用，属于电化学发光传感器领域，首次以纳米多孔Au@NiFe MOFs作为双重猝灭标记物标记抗原，利用Ru(bpy)3 2+/Zr‑MOFs作为发光体构建双重猝灭竞争型传感器。鉴于纳米多孔Au@NiFe MOFs材料的双重猝灭效果，传感器的灵敏度将被大大提升。根据不同浓度标准溶液引起的电化学发光信号强度的不同，实现对雌激素己烯雌酚的超灵敏检测。The invention relates to a preparation method and application of a double-quenching competitive electrochemiluminescence immunosensor based on a new nanomaterial Au@NiFe MOFs, which belongs to the field of electrochemiluminescence sensors. For the first time, nanoporous Au@NiFe MOFs are used as a double quenching Antigens were labeled with quenching markers, and Ru(bpy) 3 2+ /Zr‑MOFs were used as luminophores to construct dual quenching competitive sensors. In view of the double quenching effect of the nanoporous Au@NiFe MOFs material, the sensitivity of the sensor will be greatly improved. According to the difference in the intensity of the electrochemiluminescence signal caused by the standard solution with different concentrations, the ultrasensitive detection of the estrogen diethylstilbestrol is realized.

Description

A kind of double quenching competitive type Electrochemiluminescsensor sensors based on Au@NiFe MOFs Preparation method and application
Technical field
The present invention relates to a kind of dual quenching competitive type electrochemiluminescimmunosensor immunosensors based on Au@NiFe MOFs Preparation method and application.More particularly to nano material Ru (bpy)3 2+The system of/Zr-MOFs and double quencher Au@NiFe MOFs The standby and its application in Electrochemiluminescsensor sensor.Utilize Au@NiFe MOFs its excellent bio-compatibility, big ratio Surface area, high porosity increase antigen supported quantity, and it can dual quenching Ru (bpy)3 2+The luminous effect of/Zr-MOFs It answers, and then has achieved the purpose that enhance transducer sensitivity.The invention belongs to electrochemical luminescence detection technique fields.
Background technique
Diethylstilbestrol (DES) is a kind of artificial synthesized non steroidal estrogen substance, can be generated identical as natural estradiol All pharmacology and therapeutic effect.Herbst in 1971 et al. formally proposes DES and youth on " New England Journal of Medicine " Relationship between the rare adenocarcinoma of vagina of women (CCA), therefore DES is to the very harmful of human body.If the parent pregnancy period took DES, female child are easy to miscarry when being developed to adult pregnancy, and being primarily due to DES causes uterine malformation；Male fetus is come It says, the symptom as caused by DES specifically includes that cryptorchidism, carcinoma of testis, cyst of epididymis, infertility etc..DES can promote to move as one kind The estrogen of object protein synthesis is primarily present in the animal foods such as pig Goatfish meat, therefore, People's Republic of China's agricultural The bulletin of portion the 235th all food animals of regulation and all edible in " the animal food herbal medicine highest residual quantity " of revision The highest content of DES is to be not detected in tissue.Therefore important meaning can be had with simple and efficient sensitive DES detection method by finding one kind Justice.Electrochemical luminescence method used in the present invention consume low, easy to control, high sensitivity and detection limit it is low, have electrochemistry and Two methods of the advantage of chemiluminescence is a kind of environmentally friendly method.Therefore the present invention devises a kind of the electroluminescent of competitive type Chemiluminescence immune analysis method is used for the detection of diethylstilbestrol.
In the present invention, using a kind of novel nano-material Ru (bpy)3 2+/ Zr-MOFs is as electrochemical luminescence material. Zr-MOFs because of its porosity there is biggish specific surface area can load more Ru (bpy)3 2+, and then enhance the hair of sensor Luminous intensity.And uses a kind of novel nano porous material Au@NiFe MOFs as quencher for the first time and be introduced into electrochemical luminescence Field.Wherein Au nanoparticle and NiFe MOFs can quench Ru (bpy)3 2+The luminescent effect of/Zr-MOFs reaches dual The effect of quenching.And Au@NiFe MOFs is because there is its porosity biggish specific surface area can load more diethylstilbestrols Antigen.The sensitivity of sensor can be greatly enhanced as described above.The principle of the present invention is based on diethylstilbestrol standard solution Diethylstilbestrol-BSA the coupled antigen of Au@NiFe MOFs label is much stronger than with the binding ability of diethylstilbestrol antibody, with oneself The female phenol concentration of standard solution of alkene become larger and diethylstilbestrol antibody combine Au@NiFe MOFs label coupled antigen it is fewer, Dual quenching decreased effectiveness, luminous intensity enhancing.In addition, the competitive type immunosensor that the present invention designs also is other estrogen The detection of analyte provides a kind of new method.Dual quenching competitive type immunosensor is constructed currently based on Au@NiFe MOFs Method to detect diethylstilbestrol has not been reported.
Summary of the invention
The object of the present invention is to provide a kind of more simple and reliable dual quenching competitive type electricity based on Au@NiFe MOFs The preparation method and application of chemiluminescence immunoassay sensor are caused, are realized to the quick, sensitive, special of estrogen, efficient detection.
Technical scheme is as follows:
To achieve the goals above, The technical solution adopted by the invention is as follows:
1. nano material Ru (bpy)3 2+/ Zr-MOFs labelled antibody, Au@NiFe MOFs mark diethylstilbestrol-BSA coupled antigen The preparation of incubation content solution
(1) Ru (bpy)3 2+The preparation of/Zr-MOFs
The mg 2,2- bipyridyl -5,5- dicarboxylic acids of 30 mg ~ 45 (BPDC) is dissolved in 12 mL N,N-dimethylformamides (DMF) in, the 120 μ L triethylamines of μ L ~ 360 are added, the mL acetic acid of 1 mL ~ 5 is then added, stir the min of 10 min ~ 15 Organosilane precursor liquid solution is obtained, by the mg of 30 mg ~ 50 ZrCl4It is scattered in 16 mL n,N-Dimethylformamide, stirs The min of 10 min ~ 15 obtains metallic solution presoma, and it is anti-that two kinds of precursor solutions are mixed in 50 mL polytetrafluoroethylene (PTFE) high pressures It answers in kettle, 70 DEG C ~ 90 DEG C 24 h of reaction, is centrifugated, washs number respectively with n,N-Dimethylformamide, methanol, ethyl alcohol Secondary, 35 DEG C of 12 h of vacuum drying obtain white solid Zr-MOFs；10 mL second are dispersed by the mg of 18 mg ~ 24 Zr-MOFs In alcoholic solution, after 5 min of stirring and 10 mL contain Ru (bpy)3Cl2·6H2The mg of O(1 mg ~ 3) N, N- dimethyl formyl Amine aqueous solution mixing, 80 DEG C ~ 100 DEG C 12 h of stirring, n,N-Dimethylformamide, ethyl alcohol wash for several times respectively, and 35 DEG C true Dry 12 h of sky, obtain orange/yellow solid Ru (bpy)3 2+/Zr-MOFs；
(2) Ru (bpy)3 2+The preparation of/Zr-MOFs labelled antibody incubation content solution
By the Ru (bpy) of 10 mg3 2+/ Zr-MOFs is scattered in the room temperature concussion reaction h of 4 h ~ 8 in 1 mL EDC/NHS solution, Ultrapure water washes away extra EDC/NHS, is subsequently dispersed in the PBS of 1 pH=7.5 mL, and 4 μ L of μ L ~ 6,6 mg/mL are added Antibody, the oscillation hatching h of 12h ~ 24 in 4 DEG C of constant-temperature shaking incubators, 4 DEG C of centrifuge separations are finally scattered in pH=7.5 PBS buffer solution in, be made the mg/mL of 0.5 mg/mL ~ 5 Ru (bpy)3 2+/ Zr-MOFs marks coupled antibody, is stored in It is spare in 4 DEG C；
(3) preparation of nano-porous materials Au@NiFe MOFs
20 mL are dispersed by the mg of 140 mg ~ 160 Nickel dichloride hexahydrate and the mg two citric acid monohydrate trisodiums of 260 mg ~ 280 5 min of magnetic agitation in ultrapure water equally disperses magnetic force in 20 mL ultrapure waters for the potassium ferricyanide of the mg of 130 mg ~ 150 5 min are stirred, then two kinds of solution are mixed and continue to be stirred at room temperature the h of 8 h ~ 12, are centrifugated suspended matter, using super Pure water and ethyl alcohol wash 2 times respectively, and 35 DEG C of vacuum drying obtain khaki nanoporous NiFe MOFs；
The mg nanoporous NiFe of 30 mg ~ 50 MOFs is dispersed in 50 mL ultrapure waters, the addition mL of 1 mL ~ 3 1% ~ 3% chlorauric acid solution stirs 5 min at room temperature, and the mg of 4 mg ~ 6 polyvinylpyrrolidone (PVP) is added and inhibits Jenner's grain of rice Then reducing agent trisodium citrate (mg of 70 mg ~ 90) is added, sodium borohydride (mg of 0.5 mg ~ 1) room temperature in the reunion of son The lower stirring h of 8 h ~ 12, solution slowly become atropurpureus, are centrifugated suspended matter, and milli-Q water is colourless to supernatant, and 35 DEG C vacuum drying 12 h, obtain atropurpureus solid Au@NiFe MOFs；
(4) preparation of Au@NiFe MOFs labelled antigen incubation content solution
The Au@NiFe MOFs of the mg of 8 mg ~ 12 is distributed in the PBS of 1 pH=7.5 mL, 4 μ L of μ L ~ 6,6 are added 1 mL is added in the antigen of mg/mL, the oscillation hatching h of 12 h ~ 24 in 4 DEG C of constant-temperature shaking incubators, 4 DEG C of centrifuge separations 0.5% ~ 0.9% bovine serum albumin solution, 2 h of oscillation hatching close Au@NiFe MOFs in 4 DEG C of constant-temperature shaking incubators Nonspecific activity site above, 4 DEG C of centrifuge separations, is finally scattered in the PBS buffer solution of pH=7.5, is made 0.5 The Au@NiFe MOFs labelled antigen of the mg/mL of mg/mL ~ 5, is stored in spare in 4 DEG C.
2. a kind of preparation method of the dual quenching competitive type Electrochemiluminescsensor sensor of Au@NiFe MOFs, including with Lower step:
(1) glass-carbon electrode of 4 mm of diameter is made at polishing of the aluminum oxide polishing powder of 1.0 mm, 0.3 mm, 0.05 mm respectively Reason, it is clean with ultrapure water；
(2) by the 6 μ L mg/mL of 0.5 mg/mL ~ 5 Ru (bpy)3 2+/ Zr-MOFs marks coupled antibody drop coating to electrode table Face, 4 DEG C are dried；
(3) by 3 μ L volume fractions be 1% bovine serum albumin(BSA) BSA solution in electrode surface, with non-spy on enclosed-electrode surface Specific activities site is rinsed with the PBS buffer solution of pH=7.5, and 4 DEG C are dried；
(4) 6 μ L, certain density determinand standard liquid are added drop-wise to electrode surface, with the PBS buffer solution of pH=7.5 It rinses, 4 DEG C are dried
(5) the Au@NiFe MOFs label determinand antigen hatching solution of 6 μ L, the mg/mL of 0.5 mg/mL ~ 5 are added drop-wise to electricity Pole surface, 4 DEG C are dried, and are rinsed with the PBS buffer solution of pH=7.5, a kind of double quenchings based on Au@NiFe MOFs are made Competitive type electrochemiluminescimmunosensor immunosensor.
3. electrochemical luminescence of the Electrochemiluminescsensor sensor for sample to be tested detects:
(1) it is tested using the three-electrode system of electrochemical workstation, Ag/AgCl electrode is as reference electrode, platinum electrode For to electrode, the glass-carbon electrode of prepared Electrochemiluminescsensor sensor modification is working electrode, by electrochemical workstation and Chemiluminescence detector, which links together, sets 500 V for the high pressure of photomultiplier tube, and cyclic voltammetry scan potential range is 0 ~ 1.4 V, sweep speed are 0.1 V/s；
(2) contain the PBS buffer solution that concentration is the mmol/L tripropyl amine (TPA) of 1 mmol/L ~ 12 in 10 mL, pH 6.0 ~ 8.5 In, by electrochemical luminescence system, it is strong to detect the electrochemical luminescence signals generated to the diethylstilbestrol standard solution of various concentration Degree draws working curve；
(3) testing sample solution is measured instead of standard solution.
Beneficial achievement of the invention
(1) for the first time using nano-porous materials Au@NiFe MOFs as quencher labelled antigen to the luminescent effect of illuminator into The dual quenching of row, and it effectively increases the immobilized of antigen with advantages such as excellent biocompatibility, biggish specific surface areas Amount, to greatly improve the sensitivity of sensor；
(2) with nano material Ru (bpy)3 2+/ Zr-MOFs is that luminescent material is used in the building of Electrochemiluminescsensor sensor, benefit With Ru (bpy)3 2+/ Zr-MOFs high and stable luminous efficiency improve the output of sensor light signal, to obtain higher Sensitivity；
(3) present invention is for the first time by nanoporous Au@NiFe MOFs and Ru (bpy)3 2+/ Zr-MOFs is combined for electroluminescent chemistry The building of luminescence sensor, the sensor based on this building can be applied to the clinical detection of estrogen, have easy to operate, detection Quickly, linearly range wide (ng/mL of 0.1 pg/mL ~ 50) and the advantages of detection limit low (0.039 pg/mL).
Embodiment 1
Nano material Ru (bpy)3 2+/ Zr-MOFs labelled antibody, Au@NiFe MOFs label diethylstilbestrol-BSA coupled antigen are incubated The preparation of compound solution
(1) Ru (bpy)3 2+The preparation of/Zr-MOFs
By 30 mg 2,2- bipyridyl -5,5- dicarboxylic acids (BPDC) is dissolved in 12 mL n,N-Dimethylformamide (DMF), 120 μ L triethylamines are added, the mL acetic acid of 1 mL ~ 5 are then added, 10 min of stirring obtain organosilane precursor liquid solution, by 30 mg ZrCl4It is scattered in 16 mL n,N-Dimethylformamide, 10 min of stirring obtain metallic solution presoma, by body before two kinds Liquid solution is mixed in 50 mL polytetrafluoroethylene (PTFE) autoclaves, 70 DEG C of 24 h of reaction, centrifuge separation, with N, N- dimethyl Formamide, methanol, ethyl alcohol wash for several times respectively, and 35 DEG C of 12 h of vacuum drying obtain white solid Zr-MOFs；By 18 mg Zr-MOFs is scattered in 10 mL ethanol solutions, and after 5 min of stirring and 10 mL contain Ru (bpy)3Cl2·6H2O(1 mg) N, The mixing of dinethylformamide solution, 80 DEG C of 12 h of stirring, n,N-Dimethylformamide, ethyl alcohol wash for several times respectively, and 35 DEG C 12 h are dried in vacuo, orange/yellow solid Ru (bpy) is obtained3 2+/Zr-MOFs；
(2) Ru (bpy)3 2+The preparation of/Zr-MOFs labelled antibody incubation content solution
By the Ru (bpy) of 10 mg3 2+/ Zr-MOFs is scattered in 4 h of room temperature concussion reaction in 1 mL EDC/NHS solution, ultrapure water Extra EDC/NHS is washed away, is subsequently dispersed in the PBS of 1 pH=7.5 mL, the antibody of 4 μ L, 6 mg/mL, 4 DEG C of perseverances are added Oscillation hatching 12h in warm shaken cultivation case, 4 DEG C of centrifuge separations are finally scattered in the PBS buffer solution of pH=7.5, are made The Ru (bpy) of 0.5 mg/mL3 2+/ Zr-MOFs marks coupled antibody, is stored in spare in 4 DEG C；
(3) preparation of nano-porous materials Au@NiFe MOFs
Magnetic agitation 5 in 20 mL ultrapure waters is dispersed by 140 mg Nickel dichloride hexahydrates and 260 mg two citric acid monohydrate trisodiums Min equally disperses 5 min of magnetic agitation in 20 mL ultrapure waters for the potassium ferricyanide of 130 mg, then mixes two kinds of solution Continue that 8 h are stirred at room temperature, be centrifugated suspended matter, is washed respectively 2 times using ultrapure water and ethyl alcohol, 35 DEG C of vacuum drying, Obtain khaki nanoporous NiFe MOFs；
30 mg nanoporous NiFe MOFs are dispersed in 50 mL ultrapure waters, the chlorauric acid solution of 1 mL 1%, room temperature is added 5 min of lower stirring are added the reunion that 4 mg polyvinylpyrrolidones (PVP) inhibit gold nanoparticle, reducing agent lemon are then added Lemon acid trisodium (70 mg), sodium borohydride (0.5 mg) stir 8 h at room temperature, and solution slowly becomes atropurpureus, and centrifuge separation suspends Object, milli-Q water is colourless to supernatant, and 35 DEG C of 12 h of vacuum drying obtain atropurpureus solid Au NiFe MOFs；
(4) preparation of Au@NiFe MOFs labelled antigen incubation content solution
The Au@NiFe MOFs of 8 mg is distributed in the PBS of 1 pH=7.5 mL, the antigen of 4 μ L of addition, 6 mg/mL, 4 12 h of oscillation hatching in DEG C constant-temperature shaking incubator, 4 DEG C of centrifuge separations are added the bovine serum albumin solution of 1 mL 0.5%, and 4 Nonspecific activity site in DEG C constant-temperature shaking incubator above oscillation hatching 2 h closing Au@NiFe MOFs, 4 DEG C of centrifugations Separation, is finally scattered in the PBS buffer solution of pH=7.5, and the Au@NiFe MOFs labelled antigen of 0.5 mg/mL, storage is made It is stored in spare in 4 DEG C.
Embodiment 2
Nano material Ru (bpy)3 2+/ Zr-MOFs labelled antibody, Au@NiFe MOFs label diethylstilbestrol-BSA coupled antigen are incubated The preparation of compound solution
(1) Ru (bpy)3 2+The preparation of/Zr-MOFs
37.5 mg 2,2- bipyridyl -5,5- dicarboxylic acids (BPDC) are dissolved in 12 mL N,N-dimethylformamides (DMF) In, 240 μ L triethylamines are added, 3 mL acetic acid are then added, 12.5 min of stirring obtain organosilane precursor liquid solution, by 40 mg ZrCl4It is scattered in 16 mL n,N-Dimethylformamide, 12.5 min of stirring obtain metallic solution presoma, before two kinds Body solution is mixed in 50 mL polytetrafluoroethylene (PTFE) autoclaves, 80 DEG C of 24 h of reaction, centrifuge separation, with N, N- diformazan Base formamide, methanol, ethyl alcohol wash for several times respectively, and 35 DEG C of 12 h of vacuum drying obtain white solid Zr-MOFs；By 21 mg Zr-MOFs is scattered in 10 mL ethanol solutions, and after 5 min of stirring and 10 mL contain Ru (bpy)3Cl2·6H2O(2 mg) N, The mixing of dinethylformamide solution, 90 DEG C of 12 h of stirring, n,N-Dimethylformamide, ethyl alcohol wash for several times respectively, and 35 DEG C 12 h are dried in vacuo, orange/yellow solid Ru (bpy) is obtained3 2+/Zr-MOFs；
(2) Ru (bpy)3 2+The preparation of/Zr-MOFs labelled antibody incubation content solution
By the Ru (bpy) of 10 mg3 2+/ Zr-MOFs is scattered in 6 h of room temperature concussion reaction in 1 mL EDC/NHS solution, ultrapure water Extra EDC/NHS is washed away, is subsequently dispersed in the PBS of 1 pH=7.5 mL, the antibody of 5 μ L, 6 mg/mL, 4 DEG C of perseverances are added 18 h of oscillation hatching in warm shaken cultivation case, 4 DEG C of centrifuge separations are finally scattered in the PBS buffer solution of pH=7.5, make Obtain the Ru (bpy) of 2.5 mg/mL3 2+/ Zr-MOFs marks coupled antibody, is stored in spare in 4 DEG C；
(3) preparation of nano-porous materials Au@NiFe MOFs
Magnetic agitation 5 in 20 mL ultrapure waters is dispersed by 150 mg Nickel dichloride hexahydrates and 170 mg two citric acid monohydrate trisodiums Min equally disperses 5 min of magnetic agitation in 20 mL ultrapure waters for the potassium ferricyanide of 140 mg, then mixes two kinds of solution Continue that 10 h are stirred at room temperature, be centrifugated suspended matter, is washed respectively 2 times using ultrapure water and ethyl alcohol, 35 DEG C of vacuum are dry It is dry, obtain khaki nanoporous NiFe MOFs；
40 mg nanoporous NiFe MOFs are dispersed in 50 mL ultrapure waters, the chlorauric acid solution of 2 mL 2%, room temperature is added 5 min of lower stirring are added the reunion that 5 mg polyvinylpyrrolidones (PVP) inhibit gold nanoparticle, reducing agent lemon are then added Lemon acid trisodium (80 mg), sodium borohydride (0.75 mg) stir 10 h at room temperature, and solution slowly becomes atropurpureus, and centrifuge separation is outstanding Floating object, milli-Q water is colourless to supernatant, and 35 DEG C of 12 h of vacuum drying obtain atropurpureus solid Au NiFe MOFs；
(4) preparation of Au@NiFe MOFs labelled antigen incubation content solution
The Au@NiFe MOFs of 10 mg is distributed in the PBS of 1 pH=7.5 mL, the antigen of 5 μ L of addition, 6 mg/mL, 4 18 h of oscillation hatching in DEG C constant-temperature shaking incubator, 4 DEG C of centrifuge separations are added the bovine serum albumin solution of 1 mL 0.7%, and 4 Nonspecific activity site in DEG C constant-temperature shaking incubator above oscillation hatching 2 h closing Au@NiFe MOFs, 4 DEG C of centrifugations Separation, is finally scattered in the PBS buffer solution of pH=7.5, and the Au@NiFe MOFs labelled antigen of 2.5 mg/mL, storage is made It is stored in spare in 4 DEG C.
Embodiment 3
Nano material Ru (bpy)3 2+/ Zr-MOFs labelled antibody, Au@NiFe MOFs label diethylstilbestrol-BSA coupled antigen are incubated The preparation of compound solution
(1) Ru (bpy)3 2+The preparation of/Zr-MOFs
By 45 mg 2,2- bipyridyl -5,5- dicarboxylic acids (BPDC) is dissolved in 12 mL n,N-Dimethylformamide (DMF), 360 μ L triethylamines are added, 5 mL acetic acid are then added, 15 min of stirring obtain organosilane precursor liquid solution, by 50 mg ZrCl4 It is scattered in 16 mL n,N-Dimethylformamide, 15 min of stirring obtain metallic solution presoma, by two kinds of precursor solutions Be mixed in 50 mL polytetrafluoroethylene (PTFE) autoclaves, 90 DEG C of 24 h of reaction, be centrifugated, with n,N-Dimethylformamide, Methanol, ethyl alcohol wash for several times respectively, and 35 DEG C of 12 h of vacuum drying obtain white solid Zr-MOFs；By 24 mg Zr-MOFs points It dissipates in 10 mL ethanol solutions, after 5 min of stirring and 10 mL contain (Ru (bpy)3 2+) Cl2·6H2O(3 mg) N, N- bis- The mixing of methylformamide solution, 100 DEG C of 12 h of stirring, n,N-Dimethylformamide, ethyl alcohol wash for several times respectively, and 35 DEG C true Dry 12 h of sky, obtain orange/yellow solid Ru (bpy)3 2+/Zr-MOFs；
(2) Ru (bpy)3 2+The preparation of/Zr-MOFs labelled antibody incubation content solution
By the Ru (bpy) of 10 mg3 2+/ Zr-MOFs is scattered in 8 h of room temperature concussion reaction in 1 mL EDC/NHS solution, ultrapure water Extra EDC/NHS is washed away, is subsequently dispersed in the PBS of 1 pH=7.5 mL, the antibody of 6 μ L, 6 mg/mL, 4 DEG C of perseverances are added 24 h of oscillation hatching in warm shaken cultivation case, 4 DEG C of centrifuge separations are finally scattered in the PBS buffer solution of pH=7.5, make Obtain the Ru (bpy) of 5 mg/mL3 2+/ Zr-MOFs marks coupled antibody, is stored in spare in 4 DEG C；
(3) preparation of nano-porous materials Au@NiFe MOFs
Magnetic agitation 5 in 20 mL ultrapure waters is dispersed by 160 mg Nickel dichloride hexahydrates and 280 mg two citric acid monohydrate trisodiums Min equally disperses 5 min of magnetic agitation in 20 mL ultrapure waters for the potassium ferricyanide of 150 mg, then mixes two kinds of solution Continue that 12 h are stirred at room temperature, be centrifugated suspended matter, is washed respectively 2 times using ultrapure water and ethyl alcohol, 35 DEG C of vacuum are dry It is dry, obtain khaki nanoporous NiFe MOFs；
50 mg nanoporous NiFe MOFs are dispersed in 50 mL ultrapure waters, the chlorauric acid solution of 3 mL 3%, room temperature is added 5 min of lower stirring are added the reunion that 6 mg polyvinylpyrrolidones (PVP) inhibit gold nanoparticle, reducing agent lemon are then added Lemon acid trisodium (90 mg), sodium borohydride (1 mg) stir 12 h at room temperature, and solution slowly becomes atropurpureus, and centrifuge separation suspends Object, milli-Q water is colourless to supernatant, and 35 DEG C of 12 h of vacuum drying obtain atropurpureus solid Au NiFe MOFs；
(4) preparation of Au@NiFe MOFs labelled antigen incubation content solution
The Au@NiFe MOFs of 12 mg is distributed in the PBS of 1 pH=7.5 mL, the antigen of 6 μ L of addition, 6 mg/mL, 4 24 h of oscillation hatching in DEG C constant-temperature shaking incubator, 4 DEG C of centrifuge separations are added the bovine serum albumin solution of 1 mL 0.9%, and 4 Nonspecific activity site in DEG C constant-temperature shaking incubator above oscillation hatching 2 h closing Au@NiFe MOFs, 4 DEG C of centrifugations Separation, is finally scattered in the PBS buffer solution of pH=7.5, and the labelled antigen of 5 mg/mL is made, is stored in standby in 4 DEG C With.
Embodiment 4
A kind of preparation method of double quenching competitive type Electrochemiluminescsensor sensors based on Au@NiFe MOFs, including following step It is rapid:
(1) glass-carbon electrode of 4 mm of diameter is made at polishing of the aluminum oxide polishing powder of 1.0 mm, 0.3 mm, 0.05 mm respectively Reason, it is clean with ultrapure water；
(2) by 6 μ L, 0.5 mg/mL Ru (bpy)3 2+/ Zr-MOFs marks coupled antibody drop coating to electrode surface, and 4 DEG C are dried；
(3) by 3 μ L volume fractions be 1% bovine serum albumin(BSA) BSA solution in electrode surface, with non-spy on enclosed-electrode surface Specific activities site is rinsed with the PBS buffer solution of pH=7.5, and 4 DEG C are dried；
(4) 6 μ L, certain density determinand standard liquid are added drop-wise to electrode surface, with the PBS buffer solution of pH=7.5 It rinses, 4 DEG C are dried
(5) the Au@NiFe MOFs of 6 μ L, 0.5 mg/mL label determinand antigen hatching solution is added drop-wise to electrode surface, 4 It DEG C dries, is rinsed with the PBS buffer solution of pH=7.5, it is electroluminescent that a kind of double quenching competitive types based on Au@NiFe MOFs are made Chemiluminescence immunoassay sensor.
Embodiment 5
A kind of preparation method of double quenching competitive type Electrochemiluminescsensor sensors based on Au@NiFe MOFs, including following step It is rapid:
(1) glass-carbon electrode of 4 mm of diameter is made at polishing of the aluminum oxide polishing powder of 1.0 mm, 0.3 mm, 0.05 mm respectively Reason, it is clean with ultrapure water；
(2) by 6 μ L, 2.5 mg/mL Ru (bpy)3 2+/ Zr-MOFs marks coupled antibody drop coating to electrode surface, and 4 DEG C are dried；
(3) by 3 μ L volume fractions be 1% bovine serum albumin(BSA) BSA solution in electrode surface, with non-spy on enclosed-electrode surface Specific activities site is rinsed with the PBS buffer solution of pH=7.5, and 4 DEG C are dried；
(4) 6 μ L, certain density determinand standard liquid are added drop-wise to electrode surface, with the PBS buffer solution of pH=7.5 It rinses, 4 DEG C are dried
(5) the Au@NiFe MOFs of 6 μ L, 2.5 mg/mL label determinand antigen hatching solution is added drop-wise to electrode surface, 4 It DEG C dries, is rinsed with the PBS buffer solution of pH=7.5, it is electroluminescent that a kind of double quenching competitive types based on Au@NiFe MOFs are made Chemiluminescence immunoassay sensor.
Embodiment 6
A kind of preparation method of double quenching competitive type Electrochemiluminescsensor sensors based on Au@NiFe MOFs, including following step It is rapid:
(1) glass-carbon electrode of 4 mm of diameter is made at polishing of the aluminum oxide polishing powder of 1.0 mm, 0.3 mm, 0.05 mm respectively Reason, it is clean with ultrapure water；
(2) by 6 μ L, 5 mg/mL Ru (bpy)3 2+/ Zr-MOFs marks coupled antibody drop coating to electrode surface, and 4 DEG C are dried；
(3) by 3 μ L volume fractions be 1% bovine serum albumin(BSA) BSA solution in electrode surface, with non-spy on enclosed-electrode surface Specific activities site is rinsed with the PBS buffer solution of pH=7.5, and 4 DEG C are dried；
(4) 6 μ L, certain density determinand standard liquid are added drop-wise to electrode surface, with the PBS buffer solution of pH=7.5 It rinses, 4 DEG C are dried
(5) the Au@NiFe MOFs of 6 μ L, 5 mg/mL label determinand antigen hatching solution is added drop-wise to electrode surface, 4 DEG C It dries, is rinsed with the PBS buffer solution of pH=7.5, a kind of double quenching competitive type electroluminescentization based on Au@NiFe MOFs are made Learn electrochemiluminescent immunoassay sensor.
Embodiment 7
Electrochemical luminescence of the Electrochemiluminescsensor sensor for diethylstilbestrol detects:
(1) it is tested using the three-electrode system of electrochemical workstation, Ag/AgCl electrode is as reference electrode, platinum electrode For to electrode, the glass-carbon electrode of prepared Electrochemiluminescsensor sensor modification is working electrode, by electrochemical workstation and Chemiluminescence detector, which links together, sets 500 V for the high pressure of photomultiplier tube, and cyclic voltammetry scan potential range is 0 ~ 1.4 V, sweep speed are 0.1 V/s；
(2) in the PBS buffer solution for being 10 mmol/L tripropyl amine (TPA)s containing concentration of 10 mL, pH 7.5, pass through electrochemical luminescence System detects the electrochemical luminescence signals intensity generated to the diethylstilbestrol antigen of various concentration, draws working curve；
(3) testing sample solution is measured instead of standard solution.
Embodiment 8
The detection of estrogen diethylstilbestrol in flesh of fish extracting solution
The diethylstilbestrol standard solution of various concentration is added, using Standard Addition Method for Determination sample in 1 mL of flesh of fish extracting solution thereto The average recovery rate of diethylstilbestrol, the results are shown in Table 1. in product
The testing result of diethylstilbestrol in 1 sample of table
1 testing result of table can be seen that the rate of recovery of diethylstilbestrol detection result in flesh of fish extracting solution in 95.0 ~ 105% ranges It is interior, show that the present invention can be used for the detection of practical biological sample, the precision of method is high, as a result accurately and reliably.

Claims (2)

1.一种基于Au@NiFe MOFs的双重猝灭型电致化学发光免疫传感器的制备方法及应用，其特征在于，步骤如下：1. A preparation method and application of a double quenching electrochemiluminescence immunosensor based on Au@NiFe MOFs, characterized in that the steps are as follows: （1）Ru(bpy)3 2+/Zr-MOFs的制备(1) Preparation of Ru(bpy) 3 2+ /Zr-MOFs 将30 mg ~ 45 mg 2, 2-联吡啶-5, 5-二羧酸溶于12 mL N, N-二甲基甲酰胺中，加入120 μL ~ 360 μL三乙胺，然后加入1 mL ~ 5 mL醋酸，搅拌10 min ~ 15 min得到有机前驱体溶液，将30 mg ~ 50 mg ZrCl4分散于16 mL N, N-二甲基甲酰胺中，搅拌10 min ~ 15min得到金属溶液前驱体，将两种前躯体溶液混合于50 mL聚四氟乙烯高压反应釜中，70 ℃~ 90 ℃反应24 h，离心分离，用N, N-二甲基甲酰胺、甲醇、乙醇分别洗涤数次，35 ℃真空干燥12 h，得到白色固体Zr-MOFs；将18 mg ~ 24 mg Zr-MOFs分散于10 mL乙醇溶液中，搅拌5 min后和10 mL含有Ru(bpy)3Cl2·6H2O（1 mg ~ 3 mg）的N, N-二甲基甲酰胺溶液混合，80 ℃ ~ 100 ℃搅拌12 h，N, N-二甲基甲酰胺、乙醇分别洗涤数次，35 ℃真空干燥12 h，得到橙黄色固体Ru(bpy)3 2+/Zr-MOFs；Dissolve 30 mg ~ 45 mg 2, 2-bipyridine-5, 5-dicarboxylic acid in 12 mL N, N-dimethylformamide, add 120 μL ~ 360 μL triethylamine, then add 1 mL ~ 5 mL of acetic acid, stirred for 10 min to 15 min to obtain an organic precursor solution, dispersed 30 mg to 50 mg of ZrCl in 16 mL of N, N-dimethylformamide, and stirred for 10 min to 15 min to obtain a metal solution precursor, The two precursor solutions were mixed in a 50 mL polytetrafluoroethylene autoclave, reacted at 70 ℃ to 90 ℃ for 24 h, centrifuged, and washed several times with N, N-dimethylformamide, methanol, and ethanol, respectively. Vacuum drying at 35 ℃ for 12 h gave white solid Zr-MOFs; 18 mg ~ 24 mg Zr-MOFs were dispersed in 10 mL ethanol solution, stirred for 5 min and 10 mL containing Ru(bpy) 3 Cl 2 ·6H 2 O (1 mg ~ 3 mg) of N, N-dimethylformamide solution, stirred at 80 ℃ ~ 100 ℃ for 12 h, washed with N, N-dimethylformamide and ethanol several times, and dried in vacuum at 35 ℃ for 12 h. h, the orange-yellow solid Ru(bpy) 3 2+ /Zr-MOFs was obtained; （2）Ru(bpy)3 2+/Zr-MOFs标记抗体孵化物溶液的制备(2) Preparation of Ru(bpy) 3 2+ /Zr-MOFs labeled antibody incubation solution 将10 mg的Ru(bpy)3 2+/Zr-MOFs分散于1 mL EDC/NHS溶液中室温震荡反应4 h ~ 8 h，超纯水洗去多余EDC/NHS，然后分散到1 mL pH = 7.5的PBS中，加入4 μL ~ 6 μL、6 mg/mL的抗体，4 ℃恒温振荡培养箱中振荡孵化12h ~ 24 h，4 ℃离心分离，最后分散于pH = 7.5的PBS缓冲溶液中，制得0.5 mg/mL ~ 5 mg/mL的Ru(bpy)3 2+/Zr-MOFs标记偶联抗体，储存于4 ℃中备用；Disperse 10 mg of Ru(bpy) 3 2+ /Zr-MOFs in 1 mL EDC/NHS solution and shake it at room temperature for 4 h ~ 8 h, wash with ultrapure water to remove excess EDC/NHS, and then disperse to 1 mL pH = 7.5 Add 4 μL ~ 6 μL, 6 mg/mL antibody, shake and incubate in a constant temperature shaking incubator at 4 °C for 12 h ~ 24 h, centrifuge at 4 °C, and finally disperse in PBS buffer solution with pH = 7.5 to prepare Get 0.5 mg/mL ~ 5 mg/mL Ru(bpy) 3 2+ /Zr-MOFs-labeled conjugated antibody, store at 4 ℃ for use; （3）纳米多孔材料Au@NiFe MOFs的制备(3) Preparation of nanoporous Au@NiFe MOFs 将140 mg ~ 160 m六水合氯化镍和260 mg ~ 280 mg二水合柠檬酸三钠分散于20 mL超纯水中磁力搅拌5 min，同样将130 mg ~ 150 mg的铁氰化钾分散于20 mL超纯水中磁力搅拌5 min，然后将两种溶液混合继续在室温下搅拌8 h ~ 12 h，离心分离悬浮物，使用超纯水和乙醇分别洗涤2次，35 ℃真空干燥，得到土黄色纳米多孔NiFe MOFs；Disperse 140 mg ~ 160 mg of nickel chloride hexahydrate and 260 mg ~ 280 mg of trisodium citrate dihydrate in 20 mL of ultrapure water with magnetic stirring for 5 min, and similarly disperse 130 mg ~ 150 mg of potassium ferricyanide in 20 mL of ultrapure water was magnetically stirred for 5 min, then the two solutions were mixed and stirred at room temperature for 8 h to 12 h, the suspended solids were separated by centrifugation, washed twice with ultrapure water and ethanol, and dried in vacuum at 35 °C to obtain Earthy yellow nanoporous NiFe MOFs; 将30 mg ~ 50 mg纳米多孔NiFe MOFs分散在50 mL超纯水中，加入1 mL ~ 3 mL 1% ~3%的氯金酸溶液，室温下搅拌5 min，加入4 mg ~ 6 mg聚乙烯吡咯烷酮抑制金纳米粒子的团聚，然后加入70 mg ~ 90 mg还原剂柠檬酸三钠，0.5 mg ~ 1 mg硼氢化钠，室温下搅拌8h ~ 12 h，溶液慢慢变成紫黑色，离心分离悬浮物，超纯水洗涤至上清液无色，35 ℃真空干燥12 h，得到紫黑色固体Au@NiFe MOFs；Disperse 30 mg ~ 50 mg nanoporous NiFe MOFs in 50 mL ultrapure water, add 1 mL ~ 3 mL 1% ~ 3% chloroauric acid solution, stir at room temperature for 5 min, add 4 mg ~ 6 mg polyethylene Pyrrolidone inhibits the agglomeration of gold nanoparticles, then add 70 mg ~ 90 mg reducing agent trisodium citrate, 0.5 mg ~ 1 mg sodium borohydride, stir at room temperature for 8h ~ 12 h, the solution slowly turns purple black, centrifuge and suspend The product was washed with ultrapure water until the supernatant was colorless, and dried in vacuum at 35 °C for 12 h to obtain Au@NiFe MOFs as a purple-black solid; （4）Au@NiFe MOFs标记抗原孵化物溶液的制备(4) Preparation of Au@NiFe MOFs labeled antigen incubation solution 将8 mg ~ 12 mg的Au@NiFe MOFs分散到1 mL pH = 7.5的PBS中，加入4 μL ~ 6 μL、6mg/mL的抗原，4 ℃恒温振荡培养箱中振荡孵化12 h ~ 24 h，4 ℃离心分离，加入1 mL0.5% ~ 0.9%的牛血清白蛋白溶液，4 ℃恒温振荡培养箱中振荡孵化2 h封闭Au@NiFe MOFs上面的非特异性活性位点，4 ℃离心分离，最后分散于pH = 7.5的PBS缓冲溶液中，制得0.5mg/mL ~ 5 mg/mL的Au@NiFe MOFs标记抗原，储存于4 ℃中备用；Disperse 8 mg to 12 mg of Au@NiFe MOFs into 1 mL of PBS with pH = 7.5, add 4 μL to 6 μL, 6 mg/mL of antigen, and incubate in a constant temperature shaking incubator at 4 °C for 12 h to 24 h. Centrifuge at 4 °C, add 1 mL of 0.5% to 0.9% bovine serum albumin solution, incubate in a constant temperature shaking incubator at 4 °C for 2 h to block the non-specific active sites on Au@NiFe MOFs, and separate by centrifugation at 4 °C. Finally, disperse in PBS buffer solution with pH = 7.5 to prepare 0.5 mg/mL ~ 5 mg/mL Au@NiFe MOFs labeled antigen, and store it at 4 ℃ for later use; （5）分别用1.0 mm、0.3 mm、0.05 mm的氧化铝抛光粉对直径4 mm的玻碳电极做抛光处理，用超纯水冲洗干净；(5) Polish the glassy carbon electrode with a diameter of 4 mm with 1.0 mm, 0.3 mm, and 0.05 mm of alumina polishing powder, and rinse it with ultrapure water; （6）将6 µL 0.5 mg/mL ~ 5 mg/mL Ru(bpy)3 2+/Zr-MOFs标记偶联抗体滴涂至电极表面，4 ℃晾干；(6) Apply 6 µL of 0.5 mg/mL ~ 5 mg/mL Ru(bpy) 3 2+ /Zr-MOFs-labeled conjugated antibody onto the surface of the electrode, and dry at 4 °C; （7）将3 μL体积分数为1%的牛血清白蛋白BSA溶液于电极表面，以封闭电极表面上非特异性活性位点，用pH = 7.5的PBS缓冲溶液冲洗，4 ℃晾干；(7) Put 3 μL of bovine serum albumin BSA solution with a volume fraction of 1% on the surface of the electrode to block the non-specific active sites on the surface of the electrode, rinse with PBS buffer solution with pH = 7.5, and dry at 4 °C; （8）将6 μL、一定浓度的待测物标准溶液滴加到电极表面，用pH = 7.5的PBS缓冲溶液冲洗，4 ℃晾干(8) Add 6 μL of the standard solution of the analyte at a certain concentration to the surface of the electrode dropwise, rinse with PBS buffer solution with pH = 7.5, and dry at 4 °C （9）将6 μL、0.5 mg/mL ~ 5 mg/mL的Au@NiFe MOFs标记待测物抗原孵化溶液滴加到电极表面，4 ℃晾干，用pH = 7.5的PBS缓冲溶液冲洗，制得一种基于Au@NiFe MOFs的双猝灭竞争型电致化学发光免疫传感器。(9) Add 6 μL, 0.5 mg/mL ~ 5 mg/mL Au@NiFe MOFs-labeled analyte antigen incubation solution dropwise to the surface of the electrode, dry it at 4 °C, and rinse with PBS buffer solution with pH = 7.5 to prepare A double-quenching competitive electrochemiluminescence immunosensor based on Au@NiFe MOFs was obtained. 2.如权利要求1所述制备方法制得的一种基于Au@NiFe MOFs的双猝灭竞争型电致化学发光免疫传感器用于己烯雌酚的检测，其特征在于，检测步骤如下：2. A double-quenching competitive electrochemiluminescence immunosensor based on Au@NiFe MOFs prepared by the preparation method according to claim 1 is used for the detection of diethylstilbestrol, wherein the detection steps are as follows: （1）使用电化学工作站的三电极体系进行测试，Ag/AgCl电极作为参比电极，铂丝电极为对电极，所制备的电致化学发光免疫传感器修饰的玻碳电极为工作电极，将电化学工作站和化学发光检测仪连接在一起将光电倍增管的高压设置为500 V，循环伏安扫描电位范围为0 ~ 1.4 V，扫描速率为0.1 V/s；(1) The three-electrode system of the electrochemical workstation was used for testing, the Ag/AgCl electrode was used as the reference electrode, the platinum wire electrode was used as the counter electrode, and the glassy carbon electrode modified by the prepared electrochemiluminescence immunosensor was used as the working electrode. The chemical workstation and the chemiluminescence detector are connected together, the high voltage of the photomultiplier tube is set to 500 V, the scanning potential range of cyclic voltammetry is 0 ~ 1.4 V, and the scanning rate is 0.1 V/s; （2）在10 mL、pH 6.0 ~ 8.5的含浓度为1 mmol/L ~ 12 mmol/L三丙胺的PBS缓冲溶液中，通过电化学发光方法，检测对不同浓度的己烯雌酚标准溶液产生的电化学发光信号强度，绘制工作曲线；(2) In 10 mL, pH 6.0 ~ 8.5 PBS buffer solution containing 1 mmol/L ~ 12 mmol/L tripropylamine, by electrochemiluminescence method, the electrochemical reaction to different concentrations of diethylstilbestrol standard solution was detected. Luminescent signal intensity, draw the working curve; （3）将待测己烯雌酚样品溶液代替己烯雌酚标准溶液进行测定。(3) The diethylstilbestrol sample solution to be tested was replaced by the diethylstilbestrol standard solution for determination.
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