阅读说明：本技术 一种基于金修饰的钴酸镍作为高效共反应加速器构建的无标型ecl传感器的制备及应用 (Preparation and application of standard-free ECL sensor constructed by taking gold-modified nickel cobaltate as efficient coreaction accelerator ) 是由 曹伟 李敏 许晓婷 房靖龙 岳琦 黄心怡 于 2021-10-12 设计创作，主要内容包括：本发明涉及一种基于尖晶石单相二元金属氧化物(NiCo-(2)O-(4))作为高效共反应加速器无标型免疫传感器的制备及其应用,本发明属于生物传感技术领域与新型功能材料的新颖性结合。具体是Au修饰的NiCo-(2)O-(4)与阳极低激发电位氮掺杂酰肼共轭碳点结合作为电致化学发光传感平台,以此构建无标型电致化学发光免疫传感器,用于神经元特异性烯醇化酶的超灵敏检测。NiCo-(2)O-(4)具有丰富的氧化还原化学性质、具有大的比表面积、高电导率、低成本和环境友好等诸多诱人的优点,在许多电极反应中显示出优异的催化活性。这种催化活性归因于Ni～(3+)/Ni～(2+)和Co～(3+)/Co～(2+)提供了丰富的氧化还原反应,使NiCo-(2)O-(4)比相应的单独金属氧化物(NiO和Co-(3)O-(4))具有更好的生物传感效率。通过Au修饰的NiCo-(2)O-(4)不仅能提高材料的电导率和加速电子转移,而且还可以增加抗原抗体的结合数量,增强ECL信号,实现对生物分子的灵敏准确检测。(The invention relates to a spinel-based single-phase binary metal oxide (NiCo) 2 O 4 ) The invention relates to preparation and application of a high-efficiency coreaction accelerator label-free immunosensor, belonging to the novel combination of the technical field of biosensing and novel functional materials. In particular Au modified NiCo 2 O 4 Combining with anode low excitation potential nitrogen-doped hydrazide conjugated carbon points to serve as an electrochemiluminescence sensing platform, so as to construct a standard-free electrochemiluminescence immunosensor for neuron specific enolizationUltrasensitive detection of enzymes. NiCo 2 O 4 The catalyst has the advantages of abundant redox chemical properties, large specific surface area, high conductivity, low cost, environmental friendliness and the like, and shows excellent catalytic activity in a plurality of electrode reactions. This catalytic activity is attributed to Ni 3+ /Ni 2+ And Co 3+ /Co 2+ Provides rich oxidation-reduction reaction to NiCo 2 O 4 Compared with the corresponding metal oxides (NiO and Co) alone 3 O 4 ) Has better biosensing efficiency. NiCo modified by Au 2 O 4 The conductivity of the material can be improved, the electron transfer is accelerated, the combination quantity of antigen and antibody can be increased, ECL signals are enhanced, and sensitive and accurate detection of biomolecules is realized.)

1. NiCo based on gold modification₂O₄The standard-free ECL sensor constructed as an efficient coreaction accelerator comprises the following steps:

(1) polishing a glassy carbon electrode by using polishing powder, cleaning by using deionized water, placing the electrode in a 5 mmol/L potassium ferricyanide solution, and scanning at a potential of-0.2-0.6V to ensure that the difference value of peak potentials is less than 110 mV;

(2) adding 8 mu L of nickel cobaltate @ gold @ nitrogen doped carbon quantum dots (NiCo) with the concentration of 1-3 mg/mL₂O₄@ Au @ NHCDs) nano-composite solution is dripped on an electrode and dried at 4 ℃;

(3) dripping 8 mu L of 1-2 mu g/mL Neuron Specific Enolase (NSE) antibody on an electrode, drying at 4 ℃ in a refrigerator, washing with PBS to remove excessive antibody, and drying at 4 ℃;

(4) dropwise adding 3 mu L of BSA solution with the mass fraction of 1-2% on an electrode to block non-specific binding sites, washing off redundant BSA by using PBS after drying, and drying at 4 ℃;

(5) dropping 8 mu L of NSE antigen standard solution with different concentrations of 0.0001-100 ng/mL on an electrode, drying at 4 ℃, washing with PBS, removing redundant antigen, and drying at 4 ℃.

2. A gold-modified NiCo-based material according to claim 1₂O₄Non-standard ECL sensor constructed as high-efficiency co-reaction accelerator, NiCo thereof₂O₄The preparation method of the @ Au material comprises the following steps:

(1)NiCo₂O₄preparation of

0.75-3 mmol of CoCl is taken₂•4H₂O, 0.375-1.5 mmol of NiCl₂•6H₂Dissolving O and 22.5-90 mmol urea in 20 mL deionized water, continuously stirring at room temperature for 30 min, transferring to a 50 mL high-pressure reaction kettle, reacting at 120 ℃ for 6 h, naturally cooling to room temperature, centrifuging, washing, and drying;

(2)NiCo₂O₄preparation of @ Au

25-100 mg of prepared NiCo₂O₄And 1.5-6 mL of 2% HAuCl₄Adding 10-40% of the mixture in a dispersing wayStirring the deionized water for 5 min, adding 3.5-14 mg of polyvinylpyrrolidone (PVP) into the solution, and stirring for 5 min to prevent the gold nanoparticles from agglomerating; then, 2.5 to 10 mL of 5 mol/L sodium citrate solution and 1 drop of sodium borohydride solution were stirred for 12 hours, and the resultant was washed with deionized water and ethanol until the supernatant was colorless and dried at 50 ℃ overnight.

3. A gold-modified NiCo-based material according to claim 1₂O₄Non-standard ECL sensor constructed as high-efficiency co-reaction accelerator, NiCo thereof₂O₄@[email protected]₁The preparation steps of the material are as follows:

(1) preparation of NHCDs

Dissolving 1.05-4.2 g of citric acid in 5-20 mL of DMF under continuous stirring, then dropwise adding 0.5-2 mL of hydrazine hydrate, transferring to a high-pressure reaction kettle for solvothermal reaction, carrying out heat treatment at 180 ℃ for 12 h, and naturally cooling to room temperature; centrifuging the obtained solution (12000 rpm, 10 min) to remove large particle residues, dialyzing the solution with ionized water (1000da), vacuum drying at 60 deg.C, and storing the obtained black powder in a 4 deg.C closed container;

(2)NiCo₂O₄preparation of @ Au @ NHCDs

Mixing 3-12 mg NiCo₂O₄Dissolving @ Au and 1-4 mg of NHCDs in deionized water, oscillating for 24 hours by using an oscillating machine, and then centrifuging, washing and drying to obtain a target product;

(3)NiCo₂O₄@[email protected]₁preparation of

Weighing 6 mg NiCo₂O₄@ Au @ NHCDs complex, 1mL of a neuron-specific enolase antibody (10. mu.g/mL), 1mL of LPBS (pH =7.38), shaking at-4 ℃ for 12 hours, centrifuging, and adding 1mL of a PBS solution to the resulting precipitate to obtain NiCo₂O₄@[email protected]₁。

4. A gold-modified NiCo-based material according to claim 1₂O₄As an efficient co-reaction accelerationThe detection steps of the built standard-free ECL sensor are as follows:

(1) the method comprises the following steps of (1) testing by using a three-electrode system of an electrochemical workstation, taking an Ag/AgCl electrode as a reference electrode, taking a platinum wire electrode as a counter electrode, taking the prepared electrochemiluminescence immunosensor as a working electrode, connecting the electrochemical workstation and a chemiluminescence detector together, setting the high voltage of a photomultiplier to be 500V, setting the cyclic voltammetry scanning potential range to be 0-0.8V, and setting the scanning rate to be 0.15V/s;

(2) in the presence of 20-80 mM H₂O₂And 10 mL of phosphate buffer solution (PBS, 1/15 mol/L KH)₂PO₄And 1/15 mol/L of Na₂HPO₄) Detecting the intensity of an electrochemiluminescence signal generated by NSE standard solutions with different concentrations by an electrochemiluminescence method, and drawing a working curve;

(3) and (4) replacing the NSE standard solution with the NSE sample solution to be detected for determination.

Technical Field

The invention relates to preparation and application of an Electrochemiluminescence (ECL) -based immunosensor, in particular to gold-modified NiCo₂O₄The invention discloses a standard-free ECL sensor constructed as a high-efficiency coreaction accelerator, belonging to the technical field of novel functional materials and biosensing.

Background

Neuron-specific enolase (NSE) existing in nerve and neuroendocrine tissues is one of enolases participating in glycolysis pathways, the NSE content of normal human serum is 5-15 ng/mL, the NSE content of Small Cell Lung Cancer (SCLC) patients is remarkably increased, and clinical researches show that when the initial level of NSE is lower than 100 ng/mL, the prognosis is good and the survival rate is high; conversely, if the initial level of NSE is higher than the above value, the prognosis is worse and the survival rate is significantly lower; therefore, there is a need to propose a sensitive, broad linear detection strategy to detect NSE in serum.

ECL is a research method combining electrochemical means and chemiluminescence technology, and has attracted much attention in recent years in the fields of immunoassay and the like; the ECL sensor has the advantages of high sensitivity, high selectivity, high specificity, low detection limit and the like, and can quickly and accurately detect the content of an object to be detected.

NiCo₂O₄The nano-composite material has the excellent performances of large surface area, excellent conductivity, good biocompatibility, environmental friendliness and the like; NiCo₂O₄Has excellent electrochemical activity compared with corresponding single-component oxide due to Ni³⁺/Ni²⁺And Co³⁺/Co²⁺Provides more abundant oxidation-reduction reaction, thereby improving catalytic activityProperty, NiCo₂O₄Compared with the corresponding metal oxides (NiO and Co) alone₃O₄) The biosensing efficiency is better; based on the advantages, NiCo₂O₄Becoming an ideal candidate material for constructing an ECL biosensor sensing platform; modification of NiCo by Au₂O₄The conductivity of the material is improved, the electron transfer is accelerated, and the combination quantity of the antigen and the antibody can be increased, so that the luminescent signal is increased, and the sensitivity and the accuracy of detection are improved.

The carbon quantum dots are a new carbon nano material, have very good luminous performance, and are expected to be widely applied to biosensing or imaging due to the outstanding advantages of low cytotoxicity, high stability, good biocompatibility and the like; quantum dots frequently applied in the electrochemiluminescence sensor at present comprise cadmium telluride quantum dots, carbon quantum dots, zinc sulfide quantum dots, molybdenum disulfide quantum dots and the like; the invention uses a novel nitrogen-doped carbon quantum dot with low excitation potential, high stability and good biocompatibility, hydrogen peroxide (H)₂O₂) As a co-reaction active substance, the carbon quantum dots can generate extremely strong ECL emission in the solution; the mechanism is mainly due to NiCo₂O₄Ni in (1)³⁺/Ni²⁺And Co³⁺/Co²⁺Redox couple promotion of H₂O₂Decomposition to produce a large amount of O₂ ^•−Large amount of O produced₂ ^•−And NHCDs^•The reaction produces a stronger ECL signal response.

Disclosure of Invention

One of the objects of the present invention is to prepare a gold-modified NiCo₂O₄The standard-free ECL immunosensor is constructed as an efficient coreaction accelerator.

The second purpose of the invention is to use the sensor for the high-sensitivity, specific and accurate detection of NSE.

The technical scheme of the invention is as follows:

1. NiCo based on gold modification₂O₄Standard-free ECL (electron cyclotron resonance) sensor constructed as efficient coreaction acceleratorThe preparation of the device is as follows:

(1) polishing a glassy carbon electrode by using polishing powder, cleaning by using deionized water, placing the electrode in a 5 mmol/L potassium ferricyanide solution, and scanning at a potential of-0.2-0.6V to ensure that the difference value of peak potentials is less than 110 mV;

(2) adding 8 mu L of 1-3 mg/mL NiCo₂O₄Dripping the @ Au @ NHCDs nano-composite solution on an electrode, and drying at 4 ℃;

(3) dropwise adding 8 mu L of NSE antibody with the concentration of 1-2 mu g/mL on an electrode, drying at 4 ℃ in a refrigerator, washing with PBS (phosphate buffer solution) to remove excessive antibody, and drying at 4 ℃;

(4) dropwise adding 3 mu L of BSA solution with the mass fraction of 1-2% on an electrode to block non-specific binding sites, washing off redundant BSA by using PBS after drying, and drying at 4 ℃;

(5) dripping 8 mu L of NSE antigen standard solution with different concentrations of 0.0001-100 ng/mL on an electrode, drying at 4 ℃, washing with PBS to remove redundant antigen, and drying at 4 ℃ to obtain NiCo₂O₄The standard-free electrochemiluminescence immunosensor is made of @ Au material.

2. NiCo based on gold modification₂O₄Non-standard ECL sensor constructed as high-efficiency co-reaction accelerator, NiCo thereof₂O₄The preparation method of the @ Au material comprises the following steps:

(1)NiCo₂O₄preparation of

0.75-3 mmol of CoCl is taken₂•4H₂O, 0.375-1.5 mmol of NiCl₂•6H₂Dissolving O and 22.5-90 mmol urea in 20 mL deionized water, continuously stirring at room temperature for 30 min, transferring to a 50 mL high-pressure reaction kettle, reacting at 120 ℃ for 6 h, naturally cooling to room temperature, centrifuging, washing, and drying;

(2)NiCo₂O₄preparation of @ Au

25-100 mg of prepared NiCo₂O₄And 1.5-6 mL of 2% HAuCl₄Dispersing and adding 10-40 mL deionized water, stirring for 5 min, adding 3.5-14 mg polyvinylpyrrolidone (PVP) into the solution, and stirring for 5 min to prevent goldAgglomeration of the nanoparticles; then, 2.5 to 10 mL of 5 mol/L sodium citrate solution and 1 drop of sodium borohydride solution were stirred for 12 hours, and the resultant was washed with deionized water and ethanol until the supernatant was colorless and dried at 50 ℃ overnight.

3. NiCo based on gold modification₂O₄Non-standard ECL sensor constructed as high-efficiency co-reaction accelerator, NiCo thereof₂O₄@[email protected]₁The preparation steps of the material are as follows:

(1) preparation of NHCDs

Dissolving 1.05-4.2 g of citric acid in 5-20 mL of DMF under continuous stirring, then dropwise adding 0.5-2 mL of hydrazine hydrate, and transferring to a high-pressure reaction kettle for solvothermal reaction; carrying out heat treatment for 12 h at 180 ℃, and naturally cooling to room temperature; centrifuging the obtained solution (12000 rpm, 10 min) to remove large particle residues, dialyzing the solution with ionized water (1000da), vacuum drying at 60 deg.C, and storing the obtained black powder in a 4 deg.C closed container;

(2)NiCo₂O₄preparation of @ Au @ NHCDs

Mixing 3-12 mg NiCo₂O₄Dissolving @ Au and 1-4 mg of NHCDs in deionized water, oscillating for 24 hours by using an oscillating machine, and then centrifuging, washing and drying to obtain a target product;

(3)NiCo₂O₄@[email protected]₁preparation of

Weighing 6 mg NiCo₂O₄@ Au @ NHCDs Complex, 1mL of NSE-Ab was added₁(10. mu.g/mL), 1mLPBS (pH =7.38), centrifuged at-4 ℃ for 12 hours, and a 1mLPBS solution was added to the resulting precipitate to obtain NiCo₂O₄@[email protected]₁。

Assay for NSE, the procedure was as follows:

(1) the method comprises the following steps of (1) testing by using a three-electrode system of an electrochemical workstation, taking an Ag/AgCl electrode as a reference electrode, taking a platinum wire electrode as a counter electrode, taking the prepared electrochemiluminescence immunosensor as a working electrode, connecting the electrochemical workstation and a chemiluminescence detector together, setting the high voltage of a photomultiplier to be 500V, setting the cyclic voltammetry scanning potential range to be 0-0.8V, and setting the scanning rate to be 0.15V/s;

(2) in the presence of 20-80 mM H₂O₂And 10 mL of phosphate buffer solution (PBS, 1/15 mol/L KH)₂PO₄And 1/15 mol/L of Na₂HPO₄) Detecting the intensity of an electrochemiluminescence signal generated by NSE standard solutions with different concentrations by an electrochemiluminescence method, and drawing a working curve;

(3) and (4) replacing the NSE standard solution with the NSE sample solution to be detected for determination.

Advantageous results of the invention

(1) The invention adopts NiCo₂O₄It has large specific surface area, high conductivity, rich redox chemistry and can bear more NHCDs and NiCo₂O₄Ni in (1)³⁺/Ni²⁺And Co³⁺/Co²⁺Provides more abundant oxidation-reduction reaction, thereby improving the catalytic activity, and the Au is used for modifying NiCo₂O₄The conductivity of the material is improved, electron transfer is accelerated, the combination quantity of antigen and antibody can be increased, ECL signals are enhanced, and the sensitivity of detecting NSE is improved.

(2) Adopts nitrogen-doped carbon quantum dots with low excitation potential, high stability and good biocompatibility as a luminous body, and uses H₂O₂As a co-reaction active substance, the carbon quantum dots can generate extremely strong ECL emission in the solution, thereby improving the accuracy and specificity of NSE detection.

(3) The invention adopts NiCo₂O₄The ultrasensitive unlabelled electrochemiluminescence immunosensor constructed by @ Au @ NHCDs can be applied to clinical detection of NSE, and has the advantages of simplicity in operation, rapidness in detection, wide signal linear range (0.0001-100 ng/mL) and low detection limit (0.05 pg/mL).

Detailed Description

Example 1A gold-modified NiCo based on₂O₄Non-standard ECL sensor constructed as high-efficiency co-reaction accelerator, NiCo thereof₂O₄The preparation method of the @ Au material comprises the following stepsThe following:

(1)NiCo₂O₄preparation of

0.75 mmol of CoCl was taken₂•4H₂O, 0.375 mmol of NiCl₂•6H₂Dissolving O and 22.5 mmol urea in 20 mL deionized water, continuously stirring at room temperature for 30 min, transferring to a 50 mL high-pressure reaction kettle, reacting at 120 ℃ for 6 h, naturally cooling to room temperature, centrifuging, washing and drying;

(2)NiCo₂O₄preparation of @ Au

25 mg of prepared NiCo₂O₄And 1.5 mL of 2% HAuCl₄Dispersing and adding 10 mL of deionized water, stirring for 5 min, adding 3.5mg of polyvinylpyrrolidone (PVP), putting into the solution, and stirring for 5 min to prevent the gold nanoparticles from agglomerating; then, 2.5 mL of 5 mol/L sodium citrate solution and 1 drop of sodium borohydride solution were stirred for 12 hours, and the resultant was washed with deionized water and ethanol until the supernatant was colorless and dried overnight at 50 ℃.

Example 2A gold-modified NiCo₂O₄Non-standard ECL sensor constructed as high-efficiency co-reaction accelerator, NiCo thereof₂O₄The preparation method of the @ Au material comprises the following steps:

(1)NiCo₂O₄preparation of

1.5 mmol of CoCl was taken₂•4H₂O, 0.75 mmol of NiCl₂•6H₂Dissolving O and 45 mmol urea in 20 mL deionized water, continuously stirring at room temperature for 30 min, transferring to a 50 mL high-pressure reaction kettle, reacting at 120 ℃ for 6 h, naturally cooling to room temperature, centrifuging, washing and drying;

(2)NiCo₂O₄preparation of @ Au

50 mg of prepared NiCo₂O₄And 3 mL of 2% HAuCl₄Dispersing and adding 20 mL of deionized water, stirring for 5 min, adding 7 mg of polyvinylpyrrolidone (PVP), putting into the solution, and stirring for 5 min to prevent the gold nanoparticles from agglomerating; then, 5mL of 5 mol/L sodium citrate solution and 1 drop of sodium borohydride solution are stirred for 12 hours, and the resultant is washed with deionized water and ethanolThe precipitate was precipitated until the supernatant was colorless and dried at 50 ℃ overnight.

Example 3A gold-modified NiCo₂O₄Non-standard ECL sensor constructed as high-efficiency co-reaction accelerator, NiCo thereof₂O₄The preparation method of the @ Au material comprises the following steps:

(1)NiCo₂O₄preparation of

3 mmol of CoCl was taken₂•4H₂O, 1.5 mmol of NiCl₂•6H₂Dissolving O and 90 mmol urea in 20 mL deionized water, continuously stirring at room temperature for 30 min, transferring to a 50 mL high-pressure reaction kettle, reacting at 120 ℃ for 6 h, naturally cooling to room temperature, centrifuging, washing, and drying;

(2)NiCo₂O₄preparation of @ Au

100 mg of prepared NiCo₂O₄And 6 mL of 2% HAuCl₄Dispersing and adding 40 mL of deionized water, stirring for 5 min, adding 14 mg of polyvinylpyrrolidone (PVP), putting into the solution, and stirring for 5 min to prevent the gold nanoparticles from agglomerating; then, 10 mL of 5 mol/L sodium citrate solution and 1 drop of sodium borohydride solution were stirred for 12 hours, and the resultant was washed with deionized water and ethanol until the supernatant was colorless and dried at 50 ℃ overnight.

Example 4A gold-modified NiCo₂O₄Non-standard ECL sensor constructed as high-efficiency co-reaction accelerator, NiCo thereof₂O₄@[email protected]₁The preparation steps of the material are as follows:

(1) preparation of NHCDs

Dissolving 1.05 g of citric acid in 5mL of DMF under continuous stirring, then dropwise adding 0.5 mL of hydrazine hydrate, and transferring to a high-pressure reaction kettle for solvothermal reaction; carrying out heat treatment for 12 h at 180 ℃, and naturally cooling to room temperature; centrifuging the obtained solution (12000 rpm, 10 min) to remove large particle residues, dialyzing the solution with ionized water (1000da), vacuum drying at 60 deg.C, and storing the obtained black powder in a 4 deg.C closed container;

(2)NiCo₂O₄preparation of @ Au @ NHCDs

3 mg of NiCo₂O₄Dissolving @ Au and 1 mg NHCDs in deionized water, vibrating for 24 h by using a vibrator, and then centrifuging, washing and drying to obtain a target product;

(3)NiCo₂O₄@[email protected]₁preparation of

Weighing 6 mg NiCo₂O₄@ Au @ NHCDs Complex, 1mL of NSE-Ab was added₁(10. mu.g/mL), 1mLPBS (pH =7.38), centrifuged at-4 ℃ for 12 hours, and a 1mLPBS solution was added to the resulting precipitate to obtain NiCo₂O₄@[email protected]₁。

Example 5A gold-modified NiCo based on₂O₄Non-standard ECL sensor constructed as high-efficiency co-reaction accelerator, NiCo thereof₂O₄@[email protected]₁The preparation steps of the material are as follows:

(1) preparation of NHCDs

Dissolving 2.1 g of citric acid in 10 mL of DMF under continuous stirring, then dropwise adding 1.0 mL of hydrazine hydrate, and transferring to a high-pressure reaction kettle for solvothermal reaction; carrying out heat treatment for 12 h at 180 ℃, and naturally cooling to room temperature; centrifuging the obtained solution (12000 rpm, 10 min) to remove large particle residues, dialyzing the solution with ionized water (1000da), vacuum drying at 60 deg.C, and storing the obtained black powder in a 4 deg.C closed container;

(2)NiCo₂O₄preparation of @ Au @ NHCDs

6 mg of NiCo₂O₄Dissolving @ Au and 2 mg NHCDs in deionized water, vibrating for 24 h by using a vibrator, and then centrifuging, washing and drying to obtain a target product;

(3)NiCo₂O₄@[email protected]₁preparation of

Weighing 6 mg NiCo₂O₄@ Au @ NHCDs Complex, 1mL of NSE-Ab was added₁(10. mu.g/mL), 1mLPBS (pH =7.38), centrifuged at-4 ℃ for 12 hours, and a 1mLPBS solution was added to the resulting precipitate to obtain NiCo₂O₄@[email protected]₁。

Example 6A method for preparing a catalyst based onGold-modified NiCo₂O₄Non-standard ECL sensor constructed as high-efficiency co-reaction accelerator, NiCo thereof₂O₄@[email protected]₁The preparation steps of the material are as follows:

(1) preparation of NHCDs

Dissolving 4.2 g of citric acid in 20 mL of DMF under continuous stirring, then dropwise adding 2.0 mL of hydrazine hydrate, and transferring to a high-pressure reaction kettle for solvothermal reaction; carrying out heat treatment for 12 h at 180 ℃, and naturally cooling to room temperature; centrifuging the obtained solution (12000 rpm, 10 min) to remove large particle residues, dialyzing the solution with ionized water (1000da), vacuum drying at 60 deg.C, and storing the obtained black powder in a 4 deg.C closed container;

(2)NiCo₂O₄preparation of @ Au @ NHCDs

12 mg of NiCo₂O₄Dissolving @ Au and 4 mg NHCDs in deionized water, vibrating for 24 h by using a vibrator, and then centrifuging, washing and drying to obtain a target product;

(3)NiCo₂O₄@[email protected]₁preparation of

Weighing 6 mg NiCo₂O₄@ Au @ NHCDs Complex, 1mL of NSE-Ab was added₁(10. mu.g/mL), 1mLPBS (pH =7.38), centrifuged at-4 ℃ for 12 hours, and a 1mLPBS solution was added to the resulting precipitate to obtain NiCo₂O₄@[email protected]₁。

Example 7A gold-modified NiCo based on₂O₄The standard-free ECL sensor constructed as an efficient coreaction accelerator comprises the following steps:

(1) polishing a glassy carbon electrode by using polishing powder, cleaning by using deionized water, placing the electrode in a 5 mmol/L potassium ferricyanide solution, and scanning at a potential of-0.2-0.6V to ensure that the difference value of peak potentials is less than 110 mV;

(2) mu.L of 2 mg/mL NiCo₂O₄Dripping the @ Au @ NHCDs nano-composite solution on an electrode, and drying at 4 ℃;

(3) mu.L of 1. mu.g/mL NSE antibody was added dropwise to the electrode, dried at 4 ℃ in a refrigerator, washed with PBS to remove excess antibody, and dried at 4 ℃;

(4) dropwise adding 3 mu L of BSA solution with the mass fraction of 1% on an electrode to block non-specific binding sites, washing off redundant BSA by using PBS after drying, and drying at 4 ℃;

(5) dripping 8 mu L of NSE antigen standard solution with different concentrations of 0.0001-100 ng/mL on an electrode, drying at 4 ℃, washing with PBS to remove redundant antigen, and drying at 4 ℃ to obtain NiCo₂O₄@ Au material is a standard-free electrochemiluminescence immunosensor.

Example 8A gold-modified NiCo₂O₄The standard-free ECL sensor constructed as an efficient coreaction accelerator comprises the following steps:

(1) polishing a glassy carbon electrode by using polishing powder, cleaning by using deionized water, placing the electrode in a 5 mmol/L potassium ferricyanide solution, and scanning at a potential of-0.2-0.6V to ensure that the difference value of peak potentials is less than 110 mV;

(2) mu.L of 1 mg/mL NiCo₂O₄Dripping the @ Au @ NHCDs nano-composite solution on an electrode, and drying at 4 ℃;

(3) mu.L of 1.5. mu.g/mL NSE antibody was dropped onto the electrode, dried at 4 ℃ in a refrigerator, washed with PBS to remove excess antibody, and dried at 4 ℃;

(4) dropwise adding 3 mu L of BSA solution with the mass fraction of 1.5% on an electrode to block non-specific binding sites, washing off redundant BSA by using PBS after drying, and drying at 4 ℃;

(5) dripping a series of NSE antigen standard solutions with different concentrations of 0.0001-100 ng/mL on an electrode, drying at 4 ℃, washing with PBS to remove redundant antigens, and drying at 4 ℃ to obtain NiCo₂O₄@ Au material is a standard-free electrochemiluminescence immunosensor.

Example 9A gold-modified NiCo based on₂O₄The standard-free ECL sensor constructed as an efficient coreaction accelerator comprises the following steps:

(1) polishing a glassy carbon electrode by using polishing powder, cleaning by using deionized water, placing the electrode in a 5 mmol/L potassium ferricyanide solution, and scanning at a potential of-0.2-0.6V to ensure that the difference value of peak potentials is less than 110 mV;

(2) mu.L of 3 mg/mL NiCo₂O₄Dripping the @ Au @ NHCDs nano-composite solution on an electrode, and drying at 4 ℃;

(3) mu.L of 2. mu.g/mL NSE antibody was added dropwise to the electrode, dried at 4 ℃ in a refrigerator, washed with PBS to remove excess antibody, and dried at 4 ℃;

(4) dropwise adding 3 mu L of BSA solution with the mass fraction of 2% on an electrode to block non-specific binding sites, washing off redundant BSA by using PBS after drying, and drying at 4 ℃;

(5) dripping a series of NSE antigen standard solutions with different concentrations of 0.0001-100 ng/mL on an electrode, drying at 4 ℃, washing with PBS to remove redundant antigens, and drying at 4 ℃ to obtain NiCo₂O₄@ Au material is a standard-free electrochemiluminescence immunosensor.

Example 10 assay for NSE, the procedure was as follows:

(1) the method comprises the following steps of (1) testing by using a three-electrode system of an electrochemical workstation, taking an Ag/AgCl electrode as a reference electrode, taking a platinum wire electrode as a counter electrode, taking the prepared electrochemiluminescence immunosensor as a working electrode, connecting the electrochemical workstation and a chemiluminescence detector together, setting the high voltage of a photomultiplier to be 500V, setting the cyclic voltammetry scanning potential range to be 0-0.8V, and setting the scanning rate to be 0.15V/s;

(2) in the presence of 20-80 mM H₂O₂And 10 mL of phosphate buffer solution (PBS, 1/15 mol/L KH)₂PO₄And 1/15 mol/L of Na₂HPO₄) Detecting the intensity of an electrochemiluminescence signal generated by NSE standard solutions with different concentrations by an electrochemiluminescence method, and drawing a working curve;

(3) substituting the NSE sample solution to be detected for the NSE standard solution to perform determination;

(4) the linear range of NSE detection is 0.0001-100 ng/mL, and the detection limit is 0.05 pg/mL.