CN109507174B - Preparation of curcumin composite ZnO nanoparticle based quenching luminol electrochemical luminescence sensor - Google Patents

Abstract

The invention relates to a gold hybridized MoS₂/Bi₂S₃The nanorod-immobilized luminol is used for detecting the electrochemical luminescence sensor of the amyloid beta protein 42. In the present invention, MoS₂/Bi₂S₃The nanorod not only has excellent electrocatalytic performance, but also can be used for immobilizing a large amount of luminol serving as a luminescent material through a gold-sulfur bond, so that the electrochemical luminescence intensity of the luminescent material is enhanced. In order to sensitively detect amyloid beta protein 42, the invention designs a sandwich type quenching electrochemiluminescence immunosensor, which adopts curcumin-ZnO nano material to quench an electrochemiluminescence signal of luminol based on superoxide radical consumption and electrochemiluminescence-resonance energy transfer. Different amounts of the second antibody marker polydopamine @ curcumin-ZnO can be combined according to different concentrations of amyloid beta protein 42, so that the electrochemical luminescence intensity of the sensor is changed differently. The linear range of the detection of amyloid beta protein 42 in the invention is 0.05 pg/mL-10 ng/mL, and the detection limit is 21 fg/mL.

Description

Preparation of curcumin composite ZnO nanoparticle based quenching luminol electrochemical luminescence sensor

Technical Field

The invention relates to a gold hybridized MoS₂/Bi₂S₃The nanorod-immobilized luminol is used for detecting the electrochemical luminescence sensor of the amyloid beta protein 42. In particular to gold hybridized MoS₂/Bi₂S₃The quenching type electrochemical luminescence sensor for detecting amyloid beta protein 42 is prepared by taking nano-rod immobilized luminol as a luminescent material and curcumin composite ZnO nano-particles as a quencher, and belongs to the technical field of electrochemical luminescence detection.

Background

Alzheimer's disease is a degenerative disease of the nervous system that occurs in the elderly and pre-senile stages, and is clinically manifested as memory impairment, aphasia, executive dysfunction, personality and behavioral changes. Therefore, alzheimer's disease seriously threatens the health of human beings and reduces the quality of life of human beings. The deposition of amyloid beta protein has a relationship with the pathogenesis of alzheimer's disease. Amyloid beta protein 42 and amyloid beta protein 40 are two main components of amyloid beta protein, wherein amyloid beta protein 42 is more easily aggregated in the plaque of the alzheimer's disease patient than amyloid beta protein 40. Early detection, early treatment will improve the quality of life of the patient. Therefore, it is of great interest to develop a novel and sensitive immunoassay method using amyloid β protein 42 as a detection target in the present invention.

The electrochemical luminescence (ECL) analysis has high sensitivity and wide linear range; the reaction controllability and the space-time controllability are good; the instrument is simple and the analysis speed is high; the reagent is saved; the analysis has wide application range; can obtain various information simultaneously, is favorable for researching the advantages of rapid luminescence reaction, luminescence reaction mechanism and the like, and has been developed into a branch subject of analytical chemistry. Luminol, as a traditional electrochemical luminescence reagent, has high-efficiency luminescence efficiency, however, how to stably fix luminol on the surface of an electrode to construct a solid-state ECL sensor is very important for solving the stability and sensitivity of the ECL sensor. It is known that label-free sensors achieve detection of targets based on changes in ECL signals caused by the impedance of biomolecules to modified electrodes. Therefore, the discovery that the novel quencher causes the ECL signal of the luminescent material to change through energy transfer has important significance for realizing the trace detection of the target object.

Disclosure of Invention

The invention designs a quenching type electrochemiluminescence immunosensor for detecting amyloid beta protein.

In the present invention, gold hybridized MoS is used₂/Bi₂S₃The nano-rod carries luminol which is a luminescent material, so that the ECL intensity of the luminol is greatly enhanced. MoS₂/Bi₂S₃The nano-rod not only has a certain catalytic action on the decomposition of hydrogen peroxide, but also can be combined with more gold nano-particles through Au-S bonds, thereby leading the gold-NH bonds to pass through₂The bond immobilized more luminol and amyloid beta 42 antibody. The invention uses MoS₂/Bi₂S₃Nanorods as substrate material using MoS₂And Bi₂S₃The heterojunction formed between the two improves the electrochemical characteristics of the single MoS₂And Bi₂S₃Compared with the nanometer material, the composite nanometer material immobilized luminol has stable and strong electrochemical luminescence signals, and the stability and the sensitivity of the sensor are improved. In order to sensitively detect the amyloid beta protein 42, the curcumin composite ZnO nano particle is used as a quencher, and the electrochemical luminescence intensity of luminol is reduced. The antioxidant curcumin can react with superoxide radical (O)₂ ^•−) Reaction, and the electrochemiluminescence intensity of luminol and O₂ ^•−Is proportional because of O₂ ^•−Is one of the indispensable substances for generating luminol free radical and excited 3-amino phthalate. In addition, the ultraviolet-visible absorption peak of curcumin and the fluorescence emission peak of luminol have certain spectral overlap, namely certain energy transfer exists between the ultraviolet-visible absorption peak and the fluorescence emission peak of luminol, and the electrochemical luminescence intensity of luminol is further quenched. The hydrophobicity of the curcumin limits the application of the curcumin in the field of biological analysis, and the curcumin is fixed on the nano material, so that the dispersibility of the curcumin in water is improved, and the curcumin in organisms is widenedApplication in the field of sensors. In the invention, curcumin and ZnO nanoparticles are compounded, polydopamine is self-polymerized on the surface of the composite material for stably and simply combining secondary antibodies, and a benzoquinone functional group on the polydopamine can form a covalent bond with an amino group on a biomolecule through a Michael reaction. Different concentrations of amyloid beta 42 can bind different amounts of the second antibody polydopamine @ curcumin-ZnO, resulting in luminol-gold hybridized MoS₂/Bi₂S₃The change of the nanorod luminescence intensity realizes the detection of amyloid beta protein 42, and the preparation process of the sensor is shown in fig. 1.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

1. gold-hybridized MoS₂/Bi₂S₃The electrochemical luminescence sensor for detecting amyloid beta protein 42 by using the nanorod immobilized luminol comprises the following preparation steps:

(1) pretreating a glassy carbon electrode with the diameter of 4 mm by using polishing powder, and washing the glassy carbon electrode with ultrapure water;

(2) 7 mu L10 mg/mL luminol-gold hybridized MoS₂/Bi₂S₃Dripping the nanorod solution on the surface of the electrode, and storing at room temperature until the nanorod solution is dried;

(3) 6 μ L of 500 μ g/mL primary anti-Ab was drop-coated₁Placing the solution on the surface of a glassy carbon electrode, preserving the solution in a refrigerator at 4 ℃ until the solution is dried, and cleaning the solution with ultrapure water;

(4) dripping 3 μ L bovine serum albumin BSA with mass fraction of 1%, blocking nonspecific active sites, storing in a refrigerator at 4 deg.C until dried, and cleaning with ultrapure water;

(5) 6 mu L of amyloid beta protein 42 with different concentrations is dripped on the surface of an electrode, stored in a refrigerator at 4 ℃ until being dried, and cleaned by ultrapure water;

(6) 6 mu L1-7 mg/mL Ab₂And (3) dropwise coating the polydopamine @ curcumin-ZnO solution on the surface of an electrode, storing in a refrigerator at 4 ℃ until the electrode is dried, and cleaning with ultrapure water to obtain the electrochemical luminescence biosensor for detecting the amyloid beta protein 42.

2. The invention relates to a gold-hybridized MoS₂/Bi₂S₃Nanorod immobilized luminal riceElectrochemiluminescence sensor for detecting amyloid beta 42, luminol-gold hybridized MoS₂/Bi₂S₃The preparation method of the nanorod solution comprises the following steps:

（1）MoS₂/Bi₂S₃preparation of nanorods

0.242 g of Na₂MoO₄·2H₂O and 0.765 g Bi (NO)₃Preparation of nanorods

0.242 g of Na₂MoO₄·2H₂O and 0.765 g Bi (NO)₃)₃·5H₂O was dispersed in 60 mL of ultrapure water, 0.76 g of thiourea was added, and the mixture was magnetically stirred for 1 hour. Transferring the solution into a 100 mL reaction kettle, reacting for 24 h at 220 ℃, centrifuging, washing and drying to obtain MoS₂/Bi₂S₃A nanorod;

(2) luminol-gold hybridized MoS₂/Bi₂S₃Preparation of nanorods

The prepared MoS₂/Bi₂S₃The nanorods were dispersed in 50 mL of ultrapure water and sonicated for 1 h. Then, 2 mL of 1% HAuCl₄And 5 mg PVP was added to the above solution, and after stirring for 6 h, 2 mL of 50 mmol/L sodium citrate solution and a small amount of NaBH were added dropwise₄Solution reduction of HAuCl₄. After stirring for 6 h, centrifuging to remove unbound gold nanoparticles to obtain gold-hybridized MoS₂/Bi₂S₃And (4) nanorods. Then dispersing the nano-rods in 5 mL of ultrapure water, adding 1-5 mL of 5mmol/L luminol, stirring overnight, and passing through gold-NH₂Bonding luminol on the surface of the nanorod material, and centrifugally separating to remove unbound luminol to obtain luminol-gold hybridized MoS₂/Bi₂S₃And (4) nanorods.

3. The invention relates to a gold-hybridized MoS₂/Bi₂S₃Electrochemical luminescence sensor for detecting amyloid beta protein 42 by using nanorod immobilized luminol, and Ab₂-polydopamine @ curcumin-ZnO, the preparation steps being as follows:

(1) preparation of curcumin-ZnO

5 mg of curcumin was dispersed in 50 mL of ultrapure water and refluxed at 90 ℃ until curcumin was completely dissolved. 50 mL of a 0.1 mol/L zinc nitrate solution was added, and the mixture was refluxed at 90 ℃ for 1 hour. When the solution is cooled to room temperature, adding 5 mL of 0.2 mol/L KOH into an ice-water bath, stirring for 1 h to form an orange-yellow colloidal suspension, washing with ultrapure water and acetone to remove unbound curcumin, and drying in vacuum to obtain curcumin-ZnO;

（2）Ab₂-polydopamine @ curcumin-ZnO

Dispersing 20 mg of prepared curcumin-ZnO and 1-5 mg of dopamine in 30 mL of ultrapure water, stirring for 6 h, and centrifuging to remove unbound dopamine. The mixture was dispersed in 10 mL 10 mmol/L Tris-HCl (pH = 8.5) solution, stirred for 6 h, and centrifuged to remove unbound polydopamine. Then, 1-10 mg polydopamine @ curcumin-ZnO was dispersed in 5 mL phosphate buffer solution (pH = 7.4), 500 μ L500 μ g/mL secondary Ab was added₂Stirring for 6 h at 4 ℃. Then, 100. mu.L of 1% bovine serum albumin was added to block the nonspecific sites, and Ab was obtained by centrifugation₂Polydopamine @ curcumin-ZnO, dispersed in 1 mL of phosphate buffer solution (pH = 7.4), stored in a refrigerator at 4 ℃ until use.

4. The MoS prepared by the preparation method based on luminol-gold hybridization₂/Bi₂S₃The method for constructing the electrochemical luminescence sensor by the nanorods for detecting the amyloid beta protein 42 comprises the following steps:

(1) connecting a reference electrode-Ag/AgCl electrode, a counter electrode-platinum wire electrode and the prepared electrochemical luminescence sensor as a working electrode in a cassette of a chemiluminescence detector, connecting an electrochemical workstation and the chemiluminescence detector together, setting the high voltage of a photomultiplier to be 600V, and setting the scanning voltage to be-0.2-0.6V;

(2) detecting the intensity of an electrochemiluminescence signal generated by amyloid beta protein 42 with different concentrations by an electrochemiluminescence method by using a PBS (phosphate buffer solution) containing 1 mmol/L-8 mmol/L hydrogen peroxide;

the PBS buffer solution has pH = 6.5-8.5 and is prepared from 1/15 mol/L Na₂HPO₄And 1/15 mol/L KH₂PO₄Preparing;

(3) and drawing a working curve according to the linear relation between the obtained electrochemiluminescence intensity value and the logarithm of the amyloid beta protein 42 concentration.

Advantageous results of the invention

(1) Prepared gold-hybridized MoS₂/Bi₂S₃The nano rod not only enables luminol to stably exist on the surface of the electrode, but also can pass through gold nano particles and MoS₂/Bi₂S₃The nanorod has catalytic activity to hydrogen peroxide, so that the electrochemical luminescence intensity of the luminescent material is increased;

(2) prepared MoS₂/Bi₂S₃The heterojunction formed by the nanorods can improve the electrochemical characteristics of the composite material, and can fixedly carry a large amount of gold nanoparticles through gold-sulfur bonds, so that more luminol and primary antibody can be combined, and the sensitivity of the sensor is improved;

(3) the curcumin is compounded with zinc oxide, so that the bioavailability of curcumin is improved, and luminol/H is improved₂O₂The electrochemiluminescence of the system has a high-efficiency quenching effect, polydopamine is polymerized on the surface of curcumin-ZnO, and is combined with secondary antibodies through a Michael reaction;

(4) the quenching type electrochemical luminescence sensor prepared by the invention is used for detecting amyloid beta protein 42, has the advantages of small using amount of luminescent substances and short response time, and can realize simple, quick and high-sensitivity detection. The linear range of the detection of amyloid beta protein 42 in the invention is 0.05 pg/mL-10 ng/mL, and the detection limit is 21 fg/mL.

Drawings

FIG. 1 is a flow chart of a sensor designed according to the present invention.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1 preparation of luminol-gold hybridized MoS₂/Bi₂S₃Nanorod solution

（1）MoS₂/Bi₂S₃Preparation of nanorods

0.242 g of Na₂MoO₄·2H₂O and 0.765 g Bi (NO)₃)₃·5H₂O was dispersed in 60 mL of ultrapure water, 0.76 g of thiourea was added, and the mixture was magnetically stirred for 1 hour. Transferring the solution into a 100 mL reaction kettle, reacting for 24 h at 220 ℃, centrifuging, washing and drying to obtain MoS₂/Bi₂S₃A nanorod;

(2) luminol-gold hybridized MoS₂/Bi₂S₃Preparation of nanorods

The prepared MoS₂/Bi₂S₃The nanorods were dispersed in 50 mL of ultrapure water and sonicated for 1 h. 2 mL of 1% HAuCl₄And 5 mg PVP was added to the above solution, and after stirring for 6 h, 2 mL of 50 mmol/L sodium citrate solution and a small amount of NaBH were added dropwise₄Solution reduction of HAuCl₄. After stirring for 6 h, centrifuging to remove unbound gold nanoparticles to obtain gold-hybridized MoS₂/Bi₂S₃And (4) nanorods. The nanorods were then dispersed in 5 mL of ultrapure water, added 5 mL of 5mmol/L luminol and stirred overnight, over gold-NH₂Bonding luminol on the surface of the nanorod material, and centrifugally separating to remove unbound luminol to obtain luminol-gold hybridized MoS₂/Bi₂S₃And (4) nanorods.

EXAMPLE 2 preparation of Ab₂-polydopamine @ curcumin-ZnO

(1) Preparation of curcumin-ZnO

5 mg of curcumin was dispersed in 50 mL of ultrapure water and refluxed at 90 ℃ until curcumin was completely dissolved. 50 mL of a 0.1 mol/L zinc nitrate solution was added, and the mixture was refluxed at 90 ℃ for 1 hour. When the solution is cooled to room temperature, adding 5 mL of 0.2 mol/L KOH into an ice-water bath, stirring for 1 h to form an orange-yellow colloidal suspension, washing with ultrapure water and acetone to remove unbound curcumin, and drying in vacuum to obtain curcumin-ZnO;

（2）Ab₂-polydopamine @ curcumin-ZnO

Dispersing 20 mg of prepared curcumin-ZnO and 1 mg of dopamine in 30 mL of ultrapure water, stirring for 6 h, and centrifuging to remove unbound substancesThe dopamine of (1). The mixture was dispersed in 10 mL 10 mmol/L Tris-HCl (pH = 8.5) solution, stirred for 6 h, and centrifuged to remove unbound polydopamine. Then, 1 mg polydopamine @ curcumin-ZnO was dispersed in 5 mL phosphate buffer solution (pH = 7.4) and 500 μ L of 500 μ g/mL secondary Ab was added₂Stirring for 6 h at 4 ℃. Then, 100. mu.L of 1% bovine serum albumin was added to block the nonspecific sites, and Ab was obtained by centrifugation₂Polydopamine @ curcumin-ZnO, dispersed in 1 mL of phosphate buffer solution (pH = 7.4), stored in a refrigerator at 4 ℃ until use.

EXAMPLE 3 Ab preparation₂-polydopamine @ curcumin-ZnO

(1) Preparation of curcumin-ZnO

5 mg of curcumin was dispersed in 50 mL of ultrapure water and refluxed at 90 ℃ until curcumin was completely dissolved. Then, 50 mL of a 0.1 mol/L zinc nitrate solution was added, and the mixture was refluxed at 90 ℃ for 1 hour. When the solution is cooled to room temperature, adding 5 mL of 0.2 mol/L KOH into an ice-water bath, stirring for 1 h to form an orange-yellow colloidal suspension, washing with ultrapure water and acetone to remove unbound curcumin, and drying in vacuum to obtain curcumin-ZnO;

（2）Ab₂-polydopamine @ curcumin-ZnO

20 mg of the prepared curcumin-ZnO and 5 mg of dopamine were dispersed in 30 mL of ultrapure water, stirred for 6 hours, and centrifuged to remove unbound dopamine. The mixture was dispersed in 10 mL 10 mmol/L Tris-HCl (pH = 8.5) solution, stirred for 6 h, and centrifuged to remove unbound polydopamine. Then, 5 mg polydopamine @ curcumin-ZnO was dispersed in 5 mL phosphate buffer solution (pH = 7.4) and 500 μ L of 500 μ g/mL secondary Ab was added₂Stirring for 6 h at 4 ℃. Then, 100. mu.L of 1% bovine serum albumin was added to block the nonspecific sites, and Ab was obtained by centrifugation₂Polydopamine @ curcumin-ZnO, dispersed in 1 mL of phosphate buffer solution (pH = 7.4), stored in a refrigerator at 4 ℃ until use.

EXAMPLE 4 preparation of Ab₂-polydopamine @ curcumin-ZnO

(1) Preparation of curcumin-ZnO

5 mg of curcumin was dispersed in 50 mL of ultrapure water and refluxed at 90 ℃ until curcumin was completely dissolved. Then, 50 mL of a 0.1 mol/L zinc nitrate solution was added, and the mixture was refluxed at 90 ℃ for 1 hour. When the solution is cooled to room temperature, adding 5 mL of 0.2 mol/L KOH into an ice-water bath, stirring for 1 h to form an orange-yellow colloidal suspension, washing with ultrapure water and acetone to remove unbound curcumin, and drying in vacuum to obtain curcumin-ZnO;

（2）Ab₂-polydopamine @ curcumin-ZnO

20 mg of the prepared curcumin-ZnO and 10 mg of dopamine were dispersed in 30 mL of ultrapure water, stirred for 6 hours, and centrifuged to remove unbound dopamine. The mixture was dispersed in 10 mL 10 mmol/L Tris-HCl (pH = 8.5) solution, stirred for 6 h, and centrifuged to remove unbound polydopamine. Then, 10 mg polydopamine @ curcumin-ZnO was dispersed in 5 mL phosphate buffer solution (pH = 7.4) and 500 μ L of 500 μ g/mL secondary Ab was added₂Stirring for 6 h at 4 ℃. Then, 100. mu.L of 1% bovine serum albumin was added to block the nonspecific sites, and Ab was obtained by centrifugation₂Polydopamine @ curcumin-ZnO, dispersed in 1 mL of phosphate buffer solution (pH = 7.4), stored in a refrigerator at 4 ℃ until use.

EXAMPLE 5 preparation of an electrochemiluminescence sensor for detecting amyloid beta 42

(1) Pretreating a glassy carbon electrode with the diameter of 4 mm by using polishing powder, and washing the glassy carbon electrode with ultrapure water;

(2) 7 mu L10 mg/mL luminol-gold hybridized MoS₂/Bi₂S₃Dripping the nanorod solution on the surface of the electrode, and storing at room temperature until the nanorod solution is dried;

(3) 6 μ L of 500 μ g/mL primary anti-Ab was drop-coated₁Placing the solution on the surface of a glassy carbon electrode, preserving the solution in a refrigerator at 4 ℃ until the solution is dried, and cleaning the solution with ultrapure water;

(4) dripping 3 μ L of bovine serum albumin with mass fraction of 1%, sealing nonspecific active sites, storing in a refrigerator at 4 deg.C until dried, and cleaning with ultrapure water;

(5) 6 mu L of amyloid beta protein 42 with different concentrations is dripped on the surface of an electrode, stored in a refrigerator at 4 ℃ until being dried, and cleaned by ultrapure water;

(6) 6 mu L3 mg/mL Ab₂And (3) dropwise coating the polydopamine @ curcumin-ZnO solution on the surface of an electrode, storing in a refrigerator at 4 ℃ until the electrode is dried, and cleaning with ultrapure water to obtain the electrochemical luminescence biosensor for detecting the amyloid beta protein 42.

EXAMPLE 6 preparation of an electrochemiluminescence sensor for detecting amyloid beta 42

(1) Pretreating a glassy carbon electrode with the diameter of 4 mm by using polishing powder, and washing the glassy carbon electrode with ultrapure water;

(2) 7 mu L10 mg/mL luminol-gold hybridized MoS₂/Bi₂S₃Dripping the nanorod solution on the surface of the electrode, and storing at room temperature until the nanorod solution is dried;

(3) 6 μ L of 500 μ g/mL primary anti-Ab was drop-coated₁Placing the solution on the surface of a glassy carbon electrode, preserving the solution in a refrigerator at 4 ℃ until the solution is dried, and cleaning the solution with ultrapure water;

(4) dripping 3 μ L of bovine serum albumin with mass fraction of 1%, sealing nonspecific active sites, storing in a refrigerator at 4 deg.C until dried, and cleaning with ultrapure water;

(5) 6 mu L of amyloid beta protein 42 with different concentrations is dripped on the surface of an electrode, stored in a refrigerator at 4 ℃ until being dried, and cleaned by ultrapure water;

(6) 6 μ L7 mg/mL Ab₂And (3) dropwise coating the polydopamine @ curcumin-ZnO solution on the surface of an electrode, storing in a refrigerator at 4 ℃ until the electrode is dried, and cleaning with ultrapure water to obtain the electrochemical luminescence biosensor for detecting the amyloid beta protein 42.

Example 7 detection method of amyloid beta protein 42

(1) Connecting a reference electrode-Ag/AgCl electrode, a counter electrode-platinum wire electrode and the prepared electrochemical luminescence sensor as a working electrode in a cassette of a chemiluminescence detector, connecting an electrochemical workstation and the chemiluminescence detector together, setting the high voltage of a photomultiplier to be 600V, and setting the scanning voltage to be-0.2-0.6V;

(2) detecting the intensity of an electrochemiluminescence signal generated by amyloid beta protein 42 with different concentrations by an electrochemiluminescence method by using PBS buffer solution containing 3 mmol/L hydrogen peroxide;

the PBS buffer solution, pH =6.5,is prepared from 1/15 mol/L Na₂HPO₄And 1/15 mol/L KH₂PO₄Preparing;

(3) and drawing a working curve according to the linear relation between the obtained electrochemiluminescence intensity value and the logarithm of the amyloid beta protein 42 concentration.

Example 8 detection of amyloid beta protein 42

(1) Connecting a reference electrode-Ag/AgCl electrode, a counter electrode-platinum wire electrode and the prepared electrochemical luminescence sensor as a working electrode in a cassette of a chemiluminescence detector, connecting an electrochemical workstation and the chemiluminescence detector together, setting the high voltage of a photomultiplier to be 600V, and setting the scanning voltage to be-0.2-0.6V;

(2) detecting the intensity of an electrochemiluminescence signal generated by amyloid beta protein 42 with different concentrations by an electrochemiluminescence method by using PBS buffer solution containing 5mmol/L hydrogen peroxide;

the PBS buffer solution, pH =8.0, was treated with 1/15 mol/L Na₂HPO₄And 1/15 mol/L KH₂PO₄Preparing;

(3) and drawing a working curve according to the linear relation between the obtained electrochemiluminescence intensity value and the logarithm of the amyloid beta protein 42 concentration.

Example 9 detection of Artificial cerebrospinal fluid amyloid beta 42

(1) Different concentrations of amyloid beta protein 42 were added to the diluted artificial cerebrospinal fluid and the average recovery of amyloid beta protein 42 in the samples was determined using standard addition methods and the results are shown in table 1.

TABLE 1 detection of amyloid beta 42 in samples

The detection results in table 1 show that the recovery rate of the detection result of amyloid beta protein 42 in the sample is 94-112%, which indicates that the method can be applied to the detection of practical biological samples and the result is accurate and reliable.

Claims (4)

1. Gold-hybridized MoS₂/Bi₂S₃The preparation method of the electrochemical luminescence sensor for detecting amyloid beta protein 42 by using the nanorod immobilized luminol comprises the following preparation steps:

(1) pretreating a glassy carbon electrode with the diameter of 4 mm by using polishing powder, and washing the glassy carbon electrode with ultrapure water;

(2) 7 mu L10 mg/mL luminol-gold hybridized MoS₂/Bi₂S₃Dripping the nanorod solution on the surface of the electrode, and storing at room temperature until the nanorod solution is dried;

(3) dripping 6 mu L of 500 mu g/mL primary antibody solution on the surface of the glassy carbon electrode, storing in a refrigerator at 4 ℃ until the solution is dried, and cleaning with ultrapure water;

(4) dripping 3 μ L of bovine serum albumin with mass fraction of 1%, sealing nonspecific active sites, storing in a refrigerator at 4 deg.C until dried, and cleaning with ultrapure water;

(5) 6 mu L of amyloid beta protein 42 with different concentrations is dripped on the surface of an electrode, stored in a refrigerator at 4 ℃ until being dried, and cleaned by ultrapure water;

(6) dripping 6 mu L of 1-7 mg/mL secondary antibody-polydopamine @ curcumin-ZnO solution on the surface of an electrode, storing in a refrigerator at 4 ℃ until the solution is dried, and cleaning with ultrapure water to obtain the electrochemical luminescence biosensor for detecting amyloid beta protein 42.

2. A gold-hybridized MoS as claimed in claim 1₂/Bi₂S₃Preparation method of electrochemical luminescence sensor for detecting amyloid beta protein 42 by using nanorod immobilized luminol, and luminol-gold hybridized MoS₂/Bi₂S₃The preparation method of the nanorod solution comprises the following steps:

（1）MoS₂/Bi₂S₃preparation of nanorods

0.242 g of Na₂MoO₄·2H₂O and 0.765 g Bi (NO)₃)₃·5H₂Dispersing O in 60 mL of ultrapure water, adding 0.76 g of thiourea, magnetically stirring for 1 h, transferring the solution to a 100 mL reaction kettle, reacting at 220 ℃ for 24 h, centrifuging, washing and drying to obtain MoS₂/Bi₂S₃A nanorod;

(2) luminol-gold hybridized MoS₂/Bi₂S₃Preparation of nanorods

The prepared MoS₂/Bi₂S₃Dispersing the nano rods in 50 mL of ultrapure water, and performing ultrasonic treatment for 1 h; then, 2 mL of 1% HAuCl₄And 5 mg PVP was added to the above solution, and after stirring for 6 h, 2 mL of 50 mmol/L sodium citrate solution and a small amount of NaBH were added dropwise₄Solution reduction of HAuCl₄(ii) a After stirring for 6 h, centrifuging to remove unbound gold nanoparticles to obtain gold-hybridized MoS₂/Bi₂S₃A nanorod; then dispersing the nano-rods in 5 mL of ultrapure water, adding 1-5 mL of 5mmol/L luminol, stirring overnight, and passing through gold-NH₂Bonding luminol on the surface of the nanorod material, and centrifugally separating to remove unbound luminol to obtain luminol-gold hybridized MoS₂/Bi₂S₃And (4) nanorods.

3. A gold-hybridized MoS as claimed in claim 1₂/Bi₂S₃The preparation method of the electrochemical luminescence sensor for detecting amyloid beta protein 42 by using the nanorod immobilized luminol comprises the following steps:

(1) preparation of curcumin-ZnO

Dispersing 5 mg of curcumin in 50 mL of ultrapure water, and refluxing at 90 ℃ until the curcumin is completely dissolved; then, adding 50 mL of 0.1 mol/L zinc nitrate solution, and refluxing for 1 h at 90 ℃; when the solution is cooled to room temperature, adding 5 mL of 0.2 mol/L KOH into an ice-water bath, stirring for 1 h to form an orange-yellow colloidal suspension, washing with ultrapure water and acetone to remove unbound curcumin, and drying in vacuum to obtain curcumin-ZnO;

(2) secondary antibody-polydopamine @ curcumin-ZnO

Dispersing 20 mg of prepared curcumin-ZnO and 1-5 mg of dopamine in 30 mL of ultrapure water, stirring for 6 h, and centrifuging to remove unbound dopamine; dispersing the mixture in 10 mL 10 mmol/L Tris-HCl solution with pH =8.5, stirring for 6 h, and centrifuging to remove unbound polydopamine; then, dispersing 1-10 mg of polydopamine @ curcumin-ZnO into 5 mL of phosphate buffer solution with pH =7.4, adding 500 mu L of 500 mu g/mL secondary antibody, and stirring for 6 h at 4 ℃; then, 100 μ L of 1% bovine serum albumin was added, the non-specific site was blocked, and centrifuged to obtain a secondary antibody, polydopamine @ curcumin-ZnO, which was dispersed in 1 mL of pH =7.4 phosphate buffer solution and stored in a refrigerator at 4 ℃ for use.

4. An electrochemiluminescence sensor prepared by the preparation method of claim 1 is used for detecting amyloid beta protein 42, and the method comprises the following steps:

(1) connecting a reference electrode-Ag/AgCl electrode, a counter electrode-platinum wire electrode and the prepared electrochemical luminescence sensor as a working electrode in a cassette of a chemiluminescence detector, connecting an electrochemical workstation and the chemiluminescence detector together, setting the high voltage of a photomultiplier to be 600V, and setting the scanning voltage to be-0.2-0.6V;

(2) detecting the intensity of an electrochemiluminescence signal generated by amyloid beta protein 42 with different concentrations by an electrochemiluminescence method by using a PBS (phosphate buffer solution) containing 1 mmol/L-8 mmol/L hydrogen peroxide;

the PBS buffer solution has pH = 6.5-8.5 and is prepared from 1/15 mol/L Na₂HPO₄And 1/15 mol/L KH₂PO₄Preparing;

(3) and drawing a working curve according to the linear relation between the obtained electrochemiluminescence intensity value and the logarithm of the amyloid beta protein 42 concentration.

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