CN106248656B - One kind being based on Ni nanoparticle Co2O4The preparation method and application of the electrochemical luminescence immunosensor of@AD bifunctional catalyst - Google Patents
One kind being based on Ni nanoparticle Co2O4The preparation method and application of the electrochemical luminescence immunosensor of@AD bifunctional catalyst Download PDFInfo
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Abstract
The present invention discloses a kind of based on Ni nanoparticle Co2O4The preparation method and application of the electrochemical luminescence immunosensor of@AD bifunctional catalyst, feature are by modifying adamantane to NiCo2O4Nanometer sheet surface and then obtained bifunctional catalyst NiCo2O4@AD, due to the NiCo with bigger serface and excellent electronic conduction ability2O4The electrode of synergistic effect between nanometer sheet and adamantane with good power supply capacity, composite material modification significantly improves S2O8 2−The sensitivity and stability of electrochemiluminescsystem system.Based on NiCo2O4Prostaglandin E1 antibody and prostaglandin E1 are fixed to modified electrode surface, prostaglandin E1 electrochemical luminescence immunosensor are made, for the detection of prostaglandin E1,0.1 fg/ml -1.0 ng/ml of detection range by the good bio-compatibility of@AD.Have many advantages, such as that high specificity, high sensitivity, stability are good.
Description
Technical field
The invention belongs to new function materials and bio-sensing detection technique field, and in particular to one kind is based on nanometer
NiCo2O4The preparation method and application of the electrochemical luminescence immunosensor of@AD bifunctional catalyst.
Background technique
Prostaglandin E1 (PGE1), is widely present in intracorporal bioactive substance, is important in prostaglandin families
Member plays the role of cell function in multiple organ system diversified and powerful.A kind of peanut four as cyclooxygenase
Olefin(e) acid metabolite, PGE1 is related to scorching preceding and anti-inflammatory effect and osteoporosis, and some researchs discovery PGE1 can be reduced spinal cord
It damages caused compression and it has certain beneficial effect to osteoporosis treatment reason.PGE1 has as one kind
The vasodilator of effect has expansion blood vessel, improves peripheral circulation and inhibit platelet aggregation, prevent thrombosis effect,
There is beneficial hemodynamic to severe heart failure patient by increasing cardiac output and reducing filling pressure and pulmonary vascular resistance
Learn effect.PGE1 and vascular diseases and pulmonary disease are closely related, it is monitoring and is quantifying to have in human prostate's element level
Crucial effect, therefore in clinical research, the detection method for developing a kind of quick, easy, sensitive and inexpensive PGE1 has
Huge demand and important meaning.
Electrochemical luminescence immunosensor, using a kind of biosensor of the specific binding between antigen and antibody,
Have many advantages, such as that high sensitivity, selectivity is good, easy to operate, be easy to minimize, continuously can fast and automatically change detection and analysis, has
There is good application prospect.The present invention is prepared for a kind of based on NiCo2O4The electrochemical luminescence of@AD bifunctional catalyst is immune to be passed
Sensor, and realize the highly sensitive detection to prostaglandin E1.Existing document report has the hydrogen-oxygen of the transition metal of a variety of valence states
Compound or oxide, such as Co3O4, NiO, Ni(OH)2 and NiCo2O4, there is certain catalytic action to oxygen evolution reaction process.
Wherein, due to Ni2+/Ni3+And Co2+/Co3+The contribution of oxidation-reduction pair, NiCo2O4With better electronic conduction ability and
Electro-chemical activity.The present invention is by modifying adamantane to NiCo2O4Nanometer sheet surface and then obtained bifunctional catalyst
NiCo2O4@AD, since the compound has bigger serface and good biocompatibility, it is possible to provide more electroactive sites and
Biomolecule fixation site, modified electrode illustrate excellent catalytic performance to oxygen evolution reaction process and can significantly improve S2O8 2 −The sensitivity and stability of electrochemiluminescsystem system.Based on NiCo2O4@AD excellent biocompatibility and high catalytic performance, institute
Immunosensor obtained has the advantages that high specificity, high sensitivity, stability are good, detection limit is low.
Summary of the invention
An object of the present invention is based on Ni nanoparticle Co2O4@AD material, constructs a kind of unmarked, and stability is good, sensitive
Spend the preparation of high electrochemical luminescence immunosensor.
The second object of the present invention is to the electrochemical luminescence immunosensor is applied to the highly sensitive inspection of prostaglandin E1
It surveys.
To realize that goal of the invention, the present invention adopt the following technical scheme that：
1. one kind is based on NiCo2O4The preparation method of the electrochemical luminescence immunosensor of@AD bifunctional catalyst：
（1）Glass-carbon electrode（GCE）Mechanical grinding polishes first on the chamois leather for be covered with alumina powder, is washed away with secondary water
Remained on surface powder, then move into ultrasonic water bath and clean, until cleaning up, ethyl alcohol is finally sequentially used, diluted acid and water are thoroughly washed
It washs；
（2）The NiCo that 3 μ L concentration are 0.5 mg/mL is added dropwise2O4@AD suspension is red in clean glassy carbon electrode surface
It dries, is cooled to room temperature under outer lamp, NiCo is made2O4@AD modified glassy carbon electrode；
（3）It is the glutaraldehyde solution of 2.0 wt% in NiCo that 30 μ L concentration, which are added dropwise,2O4@AD modified electrode surface active 1
h；
（4）30 μ L prostaglandin E1 antibody are added dropwise（anti-PGE1）Solution is in modified electrode surface and in 4 °C of refrigerators
40 min are incubated for, the anti-PGE1 of physical absorption is washed with deionized water, NiCo is made2O4@AD/ anti-PGE1 modifies glass carbon
Electrode；
（5）It takes 40 μ L concentration to be added drop-wise in the refrigerator of 4 °C of modified electrode surface for the BSA of 1.0 wt.% and is incubated for 1 h, with envelope
Nonspecific activity site on electrode surface is closed, electrode surface is rinsed with deionized water and washes away physical absorption, and be stored in 4 °C of ice
In case；
（6）The prostaglandin E1 standard solution that 30 μ L concentration are 0.1 fg/ml -1.0 ng/ml is taken to drip in NiCo2O4@AD/
Anti-PGE1 modified electrode surface is simultaneously incubated for 40 min in 4 °C of refrigerators, rinses electrode table with the PBS buffer solution of pH7.5
Face is made a kind of and is based on Ni nanoparticle Co2O4The PGE1 electrochemical luminescence immunosensor of@AD dual catalyst.
2. above-mentioned NiCo2O4The preparation of@AD material：1.0mL concentration is the NiCo of 1.0mg/mL2O4It is with 1.0mL concentration
3- aminopropyl triethoxysilane (APTES) the mixing oscillation 12h of 1.0wt %, is collected by centrifugation the NiCo of amino functional2O4
Product is simultaneously dispersed in the deionized water of 2.0 mL again；Then will（1- (3- dimethylamino-propyl) -3- ethyl carbodiimide salt
Hydrochlorate）（EDC）And n-hydroxysuccinimide（NHS）The 1- adamantanecarboxylic acid of activation and the NiCo of amino functional2O4Product is mixed
The NiCo of adamantane modification is made in co oscillation 2h2O4 (NiCo2O4@AD), NiCo is collected by centrifugation2O4@AD product simultaneously disperses again
In the deionized water of 2.0 mL.
3. above-mentioned NiCo2O4The preparation of material：The Ni for being 1mmol with 20mL ethyl alcohol and 40mL deionized water compound concentration
(NO3)2·6H2O solution, concentration are the Co (NO of 2 mmol3)2·6H2The hexa-methylene that O solution and concentration are 4.5 mmol
Mixed solution is transferred in 100 mL ptfe autoclave liners, magnetic agitation under 90 oC by the mixed solution of tetramine
Be cooled to room temperature after 24 h, by sediment is collected by centrifugation and with deionized water and ethanol washing for several times, in an oven under 60 oC
12 h are dried, finally, 350 oC calcine the mesoporous NiCo of 2 h acquisition in air2O4Nanometer sheet.
4. one kind of above method preparation of the present invention is based on Ni nanoparticle Co2O4The electrochemical luminescence of@AD bifunctional catalyst is exempted from
Epidemic disease sensor.
5. the detecting step of prostaglandin E1：
（1）It is measured using electrochemical workstation using three-electrode system, nanometer is based on one kind of above-mentioned preparation
NiCo2O4The electrochemical luminescence immunosensor of@AD dual catalyst is working electrode, and Ag/AgCl is reference electrode, platinum electrode
For to electrode, in 0.1mol/ml K2S2O8PH 8.0 PBS buffer solution in tested；
（2）It is detected using prostaglandin E1 standard solution of the cyclic voltammetric voltage to various concentration, scanning voltage model
It encloses for the V of 0.8V--1.8, sweep speed is 100 mV/s, electrochemiluminescence signal is acquired by electrogenerated chemiluminescence equipment,
Photomultiplier tube voltage is 900V, by between resulting electrogenerated chemiluminescence intensity and prostaglandin E1 concentration of standard solution
Relationship draws working curve；
（3）Testing sample solution replaces prostaglandin E1 standard solution to be detected, and the result of detection can pass through work song
Line checks in.
Remarkable advantage of the invention is：
(1) due to Ni2+/Ni3+And Co2+/Co3+The contribution of oxidation-reduction pair, NiCo2O4It is conducted with excellent electronics
Property and electro-chemical activity, while there is the NiCo of bigger serface and meso-hole structure2O4Nanometer sheet can provide more electroactive sites
And biomolecule fixation site.Adamantane has bigger serface and preferable electron supplying capacity.The present invention is by by adamantane
Modify NiCo2O4Bifunctional catalyst NiCo is made in nanometer sheet surface2O4@AD, the compound modified electrode is to oxygen evolution reaction mistake
Journey illustrates excellent catalytic performance and can significantly improve S2O8 2−The sensitivity and stability of electrochemiluminescsystem system, the present invention
The sensitivity with higher of the immunosensor of preparation.
(2)NiCo2O4There is@AD bigger serface and good bio-compatibility to be conducive to antibody consolidating in electrode surface
Fixed, immunosensor prepared by the present invention has preferable stability.
(3) present invention utilizes the immune response of antigen, antibody, improves the specificity of detection method.
Detailed description of the invention
Figure 1A is NiCo2O4The electron emission scanning electron microscope of nanometer sheet（SEM）Figure.
Figure 1B is NiCo2O4The transmission electron microscope of nanometer sheet（TEM）Figure.
Figure 1B illustration is NiCo2O4The high resolution transmission electron microscopy of nanometer sheet（HRTEM）Figure.
Fig. 2 is NiCo2O4The infrared spectroscopy of@AD（IR）Figure.
Fig. 3 is the electrochemical luminescence response signal of immune sensing electrode and the linear pass of prostaglandin E1 concentration of standard solution
System's figure.
Specific embodiment
The present invention is further illustrated by the following examples, but protection scope of the present invention is not limited to following reality
Apply example.
Embodiment 1
1. one kind is based on Ni nanoparticle Co2O4The preparation of the electrochemical luminescence immunosensor of@AD bifunctional catalyst：
（1）Glass-carbon electrode（GCE）Mechanical grinding polishes first on the chamois leather for be covered with alumina powder, is washed away with secondary water
Remained on surface powder, then move into ultrasonic water bath and clean, until cleaning up, ethyl alcohol is finally sequentially used, diluted acid and water are thoroughly washed
It washs；
（2）The NiCo that 3 μ L concentration are 0.5 mg/mL is added dropwise2O4@AD suspension is red in clean glassy carbon electrode surface
It dries, is cooled to room temperature under outer lamp, NiCo is made2O4@AD modified glassy carbon electrode；
（3）It is the glutaraldehyde solution of 2.0wt % in NiCo that 30 μ L concentration, which are added dropwise,2O4@AD modified electrode surface active 1
h；
（4）30 μ L prostaglandin E1 antibody are added dropwise（anti-PGE1）Solution is in modified electrode surface and in 4 °C of refrigerators
40 min are incubated for, the anti-PGE1 of physical absorption is washed with deionized water, NiCo is made2O4@AD/ anti-PGE1 modifies glass carbon
Electrode；
（5）It takes 40 μ L concentration to be added drop-wise in the refrigerator of 4 °C of modified electrode surface for the BSA of 1.0 wt.% and is incubated for 1 h, with envelope
Nonspecific activity site on electrode surface is closed, electrode surface is rinsed with deionized water and washes away physical absorption, and be stored in 4 °C of ice
In case；
（6）The PGE1 standard solution that 30 μ L concentration are 0.1 fg/ml -1.0 ng/ml is taken to drip in NiCo2O4@AD/ anti-
PGE1 modified electrode surface is simultaneously incubated for 40 min in 4 °C of refrigerators, rinses electrode surface, system with the PBS buffer solution of pH7.5
It obtains a kind of based on Ni nanoparticle Co2O4The PGE1 electrochemical luminescence immunosensor of@AD dual catalyst.
Embodiment 2
NiCo2O4The preparation of material：
Ni (the NO for being 1 mmol with 20 mL ethyl alcohol and 40 mL deionized water compound concentrations3)2·6H2O solution is dense
Degree is the Co (NO of 2 mmol3)2·6H2The mixed solution for the hexa that O solution and concentration are 4.5 mmol, will
Mixed solution is transferred in 100 mL ptfe autoclave liners, is cooled to room temperature after 24 h of magnetic agitation under 90 oC,
By sediment is collected by centrifugation and with deionized water and ethanol washing for several times, dry 12 h under 60 oC in an oven, finally,
350 oC calcine 2 h and obtain mesoporous NiCo in air2O4Nanometer sheet.NiCo2O4The electron emission scanning electron microscopy of nanometer sheet
Mirror（SEM）Figure and transmission electron microscope（TEM）Figure, as illustrated in figures 1A and ib, shows NiCo2O4Nanometer sheet is by NiCo2O4
Nanoparticle unit is interconnected to constitute, and forms a large amount of intergranular pore, and such porous structure will greatly increase electroactive
Site.High resolution transmission electron microscopy（HRTEM）Figure, as shown in illustration in Figure 1B, it is seen that the clearly lattice of about 0.25 nm
Striped, with spinelle NiCo2O4（311）Crystal face is consistent.
Embodiment 3
NiCo2O4The preparation of@AD material：
1.0 mL concentration are the NiCo of 1.0 mg/mL2O4Three ethoxy of 3- aminopropyl for being 1.0 wt% with 1.0 mL concentration
Base silane (APTES) mixing oscillation 12h, is collected by centrifugation the NiCo of amino functional2O4Product is simultaneously dispersed in 2.0 mL's again
In deionized water；Then will（1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride）（EDC）And N- hydroxysuccinimidyl acyl
Imines（NHS）The 1- adamantanecarboxylic acid of activation and the NiCo of amino functional2O4Product mixing oscillation 2h, is made adamantane modification
NiCo2O4 (NiCo2O4@AD), NiCo is collected by centrifugation2O4@AD product is simultaneously dispersed in the deionized water of 2.0 mL again.
NiCo2O4The infrared spectroscopy of@AD（IR）Figure, as shown in Fig. 2, in 1666 cm-1With 3185 cm-1There is absorption peak appearance at place, this is
The stretching vibration absworption peak of C=O and N-H group in typical amide group, shows NiCo2O4The successful preparation of@AD.
Embodiment 4
The detecting step of prostaglandin E1：
（1）It is measured using electrochemical workstation using three-electrode system, nanometer is based on prepared one kind
NiCo2O4The electrochemical luminescence immunosensor of@AD dual catalyst is working electrode, and Ag/AgCl is reference electrode, platinum electrode
For to electrode, in 0.1mol/ml K2S2O8PH 8.0 PBS buffer solution in tested；
（2）It is detected using prostaglandin E1 standard solution of the cyclic voltammetric voltage to various concentration, scanning voltage model
It encloses for the V of 0.8V--1.8, sweep speed is 100 mV/s, electrochemiluminescence signal is acquired by electrogenerated chemiluminescence equipment,
Photomultiplier tube voltage is 900V, by between resulting electrogenerated chemiluminescence intensity and prostaglandin E1 concentration of standard solution
Relationship, draws working curve, and Fig. 3 is dense for the electrochemical luminescence response signal and prostaglandin E1 standard solution of immune sensing electrode
The linear relationship chart of degree；
（3）Testing sample solution replaces prostaglandin E1 standard solution to be detected, and the result of detection can pass through work song
Line checks in.
Claims (4)
1. a kind of Ni nanoparticle Co based on adamantane modification2O4The preparation of the electrochemical luminescence immunosensor of bifunctional catalyst
Method, which is characterized in that include the following steps：
（1）Glass-carbon electrode mechanical grinding first on the chamois leather for be covered with alumina powder polishes, and washes away remained on surface with secondary water
Powder, then move into ultrasonic water bath and clean, until cleaning up, ethyl alcohol is finally sequentially used, diluted acid and water thoroughly wash；
（2）The Ni nanoparticle Co that the adamantane that 3 μ L concentration are 0.5 mg/mL is modified is added dropwise2O4Suspension is in clean glass carbon electricity
Pole surface is dried under infrared lamp, is cooled to room temperature；
（3）It is the glutaraldehyde solution of 2.0 wt% in step that 30 μ L concentration, which are added dropwise,（2）Modified electrode surface active 1 obtained
h；
（4）30 μ L prostaglandin E1 antibody-solutions are added dropwise in step（3）It is simultaneously incubated in 4 °C of refrigerators on modified electrode surface obtained
40 min are educated, the prostaglandin E1 antibody of physical absorption is washed with deionized water；
（5）Taking 40 μ L concentration is that the BSA of 1.0 wt.% is added drop-wise to step（4）It is incubated in 4 °C of refrigerators in modified electrode surface obtained
1 h is educated, with nonspecific activity site on enclosed-electrode surface, electrode surface is rinsed with deionized water and washes away physical absorption, and
It is stored in 4 °C of refrigerators；
（6）The prostaglandin E1 standard solution that 30 μ L concentration are 0.1 fg/mL -1.0 ng/mL is taken to drip in step（5）It is obtained
Modified electrode surface is simultaneously incubated for 40 min in 4 °C of refrigerators, rinses electrode surface with the PBS buffer solution of pH7.5, is made one
The Ni nanoparticle Co that kind is modified based on adamantane2O4The electrochemical luminescence immunosensor of the prostaglandin E1 of bifunctional catalyst；
The Ni nanoparticle Co of the adamantane modification2O4What suspension was prepared by the following method：1.0 mL concentration are 1.0 mg/mL
Ni nanoparticle Co2O4Oscillation 12h is mixed with the 3- aminopropyl triethoxysilane that 1.0 mL concentration are 1.0 wt%, is collected by centrifugation
The Ni nanoparticle Co of amino functional2O4Product is simultaneously dispersed in the deionized water of 2.0 mL again；Then by 1- (3- dimethylamino
Propyl) -3- ethyl-carbodiimide hydrochloride and n-hydroxysuccinimide activation 1- adamantanecarboxylic acid and amino functional
Ni nanoparticle Co2O4Product mixing oscillation 2h, is made the Ni nanoparticle Co of adamantane modification2O4, receiving for adamantane modification is collected by centrifugation
Rice NiCo2O4Product is simultaneously dispersed in the deionized water of 2.0 mL again.
2. the method according to claim 1, wherein the Ni nanoparticle Co2O4Material is prepared by the following method
's：Ni (the NO for being 1 mmol with 20 mL ethyl alcohol and 40 mL deionized water compound concentrations3)2·6H2O solution, concentration 2
Co (the NO of mmol3)2·6H2The mixed solution for the hexa that O solution and concentration are 4.5 mmol, by mixed solution
It is transferred in 100 mL ptfe autoclave liners, is cooled to room temperature after 24 h of magnetic agitation under 90 oC, passes through centrifugation
Collect sediment and with deionized water and ethanol washing for several times, 12 h are dried under 60 oC in an oven, finally, in air 350
OC calcines 2 h and obtains mesoporous NiCo2O4Nanometer sheet.
3. a kind of Ni nanoparticle Co based on adamantane modification of any method preparation of claim 1-22O4Double-function catalyzing
The electrochemical luminescence immunosensor of agent.
4. a kind of Ni nanoparticle Co based on adamantane modification as claimed in claim 32O4The electrochemical luminescence of bifunctional catalyst is exempted from
Epidemic disease sensor, which is characterized in that for the detection of prostaglandin E1, detecting step is as follows：
（1）It is measured using electrochemical workstation using three-electrode system, with as claimed in claim 3 a kind of based on adamantane
The Ni nanoparticle Co of modification2O4The electrochemical luminescence immunosensor of bifunctional catalyst is working electrode, and Ag/AgCl is reference electricity
Pole, platinum electrode are to electrode, in the K of 0.1mol/mL2S2O8PH 8.0 PBS buffer solution in tested；
（2）It is detected using prostaglandin E1 standard solution of the cyclic voltammetric voltage to various concentration, scanning voltage range is
The V of 0.8V--1.8, sweep speed are 100 mV/s, acquire electrochemiluminescence signal, photoelectricity by electrogenerated chemiluminescence equipment
Multiplier voltage is 900V, passes through the pass between resulting electrogenerated chemiluminescence intensity and prostaglandin E1 concentration of standard solution
Working curve is drawn by system；
（3）Testing sample solution replaces prostaglandin E1 standard solution to be detected, and the result of detection can be looked by working curve
?.
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| CN107256972A (en) * | 2017-05-27 | 2017-10-17 | 武汉理工大学 | A kind of method based on the sour nickel nano film of the porous cobalt of hollow carbon sphere template growth |
| CN108318477B (en) * | 2018-02-05 | 2020-06-26 | 福建省妇幼保健院 | Based on TiO2Electrochemiluminescence probe prepared by metal organic framework and competitive immunosensing method of electrochemiluminescence probe for vomitoxin |
| CN108445062B (en) * | 2018-03-22 | 2020-05-19 | 福建医科大学附属第一医院 | Based on NiCo2O4Construction of nano sheet material and trypsin electrochemical sensing platform with peptide chain cleavage effect |
| CN109799273B (en) * | 2019-03-04 | 2021-05-11 | 福建师范大学 | Based on nanometer Co3O4Zearalenone impedance sensor for simulating signal double amplification of enzyme catalysis |
| CN110031529B (en) * | 2019-05-18 | 2021-07-13 | 福建师范大学 | Based on NiCo2O4Nano sheet thermal sensitization type thyroglobulin electrogenerated chemiluminescence immunosensor |
| CN111766288B (en) * | 2020-06-22 | 2022-10-28 | 济南大学 | Based on oxygen boosting vacancy NiCo 2 O 4 Preparation method of electrochemiluminescence sensor |
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