CN113533465A - Preparation method of electrochemical immunosensor based on IRMOF-3 internal and external load nitrogen-doped quantum dot composite material - Google Patents

Abstract

The invention relates to a preparation method of an electrochemical immunosensor based on an IRMOF-3 internal and external load nitrogen-doped quantum dot composite material. Compared with graphene quantum dots, the nitrogen-doped quantum dots used in the invention have higher quantum yield and more active sites, and the synthesized IRMOF-3 has the advantages of large specific surface area, good pore regularity, adjustable pore diameter and the like, and can load the nitrogen-doped quantum dots at high quantity. The nitrogen-doped quantum dots are loaded on the inner surface and the outer surface of the IRMOF-3 simultaneously, so that the loading capacity of the luminescent material is greatly improved, a continuous and stable strong electrochemical luminescence signal is provided, and the signal substrate is used. The sandwich burst is constructed by using a gold-modified zinc oxide nanorod combined with a procalcitonin recognition antibody as a secondary antibody marker based on the resonance energy transfer principleThe inactivated electrochemical immunosensor realizes the ultrasensitive detection of procalcitonin, and the detection limit is 12.58 fg mL^‑1。

Description

Preparation method of electrochemical immunosensor based on IRMOF-3 internal and external load nitrogen-doped quantum dot composite material

Technical Field

The invention relates to a preparation method of an electrochemical immunosensor based on an IRMOF-3 internal and external load nitrogen-doped quantum dot composite material, and particularly relates to a sandwich quenching type electrochemical immunosensor for specifically detecting procalcitonin, which is prepared by adopting the IRMOF-3 internal and external load nitrogen-doped quantum dot composite material N-GQDs @ IRMOF-3@ N-GQDs as a substrate luminescent material to be combined with procalcitonin primary antibody to form a primary anti-marker, and adopting gold-modified zinc oxide nanorod ZnO @ Au as a secondary anti-marker to mark a procalcitonin recognition antibody secondary antibody, belonging to the technical field of novel functional materials and biosensing detection.

Background

Sepsis is a systemic inflammatory response caused by infection with bacteria, fungi, etc., and is considered as a global life-threatening disease. Studies have shown that procalcitonin reflects the activity of the systemic inflammatory response and has been explored as a reliable marker for the diagnosis of sepsis. Therefore, sensitive detection of procalcitonin in humans is of great significance for early prevention and diagnosis of sepsis. Electrochemiluminescence is a new product combining electrochemistry and luminescence, has the advantages of low background, wide dynamic range, simple and convenient instrument and equipment, sensitive detection and the like, and is widely concerned in the fields of biological analysis, food safety analysis, environmental pollution monitoring and the like.

The luminescent graphene quantum dot is a zero-dimensional nano material, is composed of graphene, and has the advantages of adjustable size, low toxicity, biocompatibility and the like, but the quantum yield of the bare quantum dot without surface functionalization is low, so that the conductivity and the electrochemical performance of the graphene quantum dot are effectively adjusted by adopting nitrogen doping. The surface of the nitrogen-doped graphene quantum dot has a plurality of oxygen-containing groups, so that the water solubility of the nitrogen-doped graphene quantum dot can be improved, the nitrogen-doped graphene quantum dot is favorable for being combined with other materials, and the nitrogen-doped graphene quantum dot has higher quantum yield and more active sites than luminescent graphene quantum dots. The IRMOF-3 has the advantages of large specific surface area, good pore regularity, adjustable pore diameter and the like, and the internal and external loading of the nitrogen-doped quantum dots is carried out by adopting the IRMOF-3, so that the loading capacity of the luminescent material is greatly improved, and the electrochemical luminescence signal is improved. In addition, the ultraviolet visible absorption spectrum of the zinc oxide and gold modified zinc oxide nano-rod is overlapped with the electrochemical luminescence emission spectrum of the IRMOF-3 carrying the internal and external nitrogen-doped quantum dot composite material, so that the double quenching effect can be realized based on resonance energy transfer, and the sensitivity of the constructed sensor is greatly improved. The electrochemical immunosensor constructed by the invention realizes the ultra-sensitive detection of procalcitonin, and the detection limit is 12.58 fg mL^-1. The electrochemical immunosensor provided by the invention has the advantages of high sensitivity, low detection limit and good stability. Based on the above findings, the inventors have completed the present invention.

Disclosure of Invention

One of the purposes of the invention is to adopt IRMOF-3 to carry out internal and external loading of nitrogen-doped quantum dots as a substrate luminescent material, thereby greatly improving the loading of the nitrogen-doped quantum dots and endowing the nitrogen-doped quantum dots with excellent electrochemical luminescence behavior.

The other purpose of the invention is to use the gold modified zinc oxide nano-rod as a quenching material, because the ultraviolet visible absorption spectra of zinc oxide and gold modified zinc oxide are overlapped with the ECL emission spectra of IRMOF-3 internally and externally loaded with nitrogen-doped quantum dots, the double quenching effect can be realized based on resonance energy transfer, the sensitivity of the constructed sensor is greatly improved, and a new resonance energy transfer donor-acceptor pair is provided for constructing an electrochemical immunosensor.

The invention also aims to realize the construction of the electrochemical immunosensor and sensitively detect procalcitonin, and the detection limit is 12.58 fg mL^-1The application of the electrochemical luminescence sensor in detecting procalcitonin is achieved.

Technical scheme of the invention

1. A preparation method of an electrochemical immunosensor based on an IRMOF-3 internal and external load nitrogen-doped quantum dot composite material is characterized by comprising the following steps:

(1) preparation of nitrogen-doped quantum dot N-GQDs

Dissolving 2-5 mmol of citric acid and 5-15 mmol of urea in 15 mL of ultrapure water, stirring for 5 min, transferring to a 50 mL polytetrafluoroethylene autoclave, reacting at 160 ℃ for 6-12 h, cooling to room temperature after the reaction is finished, washing with absolute ethyl alcohol for three times, and vacuum drying to obtain nitrogen-doped quantum dots N-GQDs;

(2) preparation of N-GQDs @ IRMOF-3 encapsulated nitrogen-doped quantum dots in IRMOF-3

Dissolving 0.2-1 g of PVPK30 and 10-30 mg of nitrogen-doped quantum dots in 18 mL of mixed solution of N, N-dimethylformamide and 12 mL of absolute ethyl alcohol, and simultaneously dissolving 50-80 mg of Zn (NO)₃)₂·6H₂O and 10-30 mg of 2NH₂-BDC is dissolved in 6 mL of N, N-dimethylformamide solution, the two solutions are mixed, stirred for 30 min and then transferred into a 50 mL polytetrafluoroethylene autoclave for reaction at 100 ℃ for 20 h, after the reaction, the product is centrifuged and washed by N, N-dimethylformamide, and after vacuum drying for 12 h, the IRMOF-3 is obtained, and the N-GQDs @ IRMOF-3 of the nitrogen-doped quantum dots is encapsulated in the IRMOF-3;

(3) preparation of IRMOF-3 internal and external load nitrogen doped quantum dot composite material N-GQDs @ IRMOF-3@ N-GQDs

Dissolving 10-30 mg of N-GQDs @ IRMOF-3 in 20 mL of ultrapure water, and then adding 1 mL of 400 mmol of L^-11- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 100 mmol L of^-1The N-hydroxysuccinimide mixed aqueous solution is reacted for 1 hour at the temperature of 4 ℃, then 15 mg of nitrogen-doped quantum dots are added and continuously stirred for 4 hours, the product is washed by absolute ethyl alcohol during centrifugation and dried to obtain an IRMOF-3 internal and external load nitrogen-doped quantum dot composite material N-GQDs @ IRMOF-3@ N-GQDs, and the N-GQDs are dissolved in 2 mg mL of the composite material^-1Storing the chitosan solution at 4 ℃ for later use;

(4) preparation of gold-modified zinc oxide nanorod ZnO @ Au

Mixing 15 mL of the mixture with 0.1-0.5 mol of the mixture^-1Zn (Ac)₂·2H₂O methanol solution, 30 mL and 0.1-1.0 mol L^-1Is mixed with NaOH methanol solution, stirred for 30 min to obtain clear solution, and then transferred to a 50 mL polytetrafluoroethylene autoclaveReacting at 150-200 ℃ for 24 hours to obtain white precipitate, then centrifugally washing with ultrapure water, drying the obtained product in a vacuum oven for 12 hours to obtain a zinc oxide nanorod, then dispersing 50-100 mg of zinc oxide in 50 mL of ultrapure water, stirring for 30 min, then dissolving 0.6-1.2 mmol of CTAB in the solution, heating to 80 ℃, cooling the solution to room temperature until the CTAB is completely dissolved, then adding 5-20 mL of 2% HAuCl₄Continuously stirring for 1 h, and then adding 15 mL of 5-10 mmol NaBH into the solution₄Obviously observing that the yellow solution is changed into purple, and finally centrifugally washing and drying the solution by using ultrapure water to obtain gold modified zinc oxide nano-rod ZnO @ Au;

(5) preparation of PBS buffer solution

11.94 g of disodium hydrogen phosphate dodecahydrate were taken out and dissolved in a 500 mL volumetric flask so as to be prepared into a solution having a concentration of 1/15 mol L^-1As an aqueous solution of (a); 4.54 g of monopotassium phosphate is taken and fixed to a 500 mL volumetric flask, and the volume is configured to be 1/15 mol L^-1As solution b; mixing the solution A and the solution B in proportion to prepare a series of PBS (phosphate buffer solution) with the pH value of 6.0-8.0;

(6) second antibody marker ZnO @ Au-Ab of gold-modified zinc oxide nanorod combined procalcitonin recognition antibody₂Preparation of

20-80 μ L, 10 μ g mL^-1Anti-body Ab of procalcitonin recognition antibody₂Adding the mixture into 5-10 mL and 2 mg mL^-1 Oscillating and incubating ZnO @ Au in PBS (phosphate buffer solution) at 4 ℃ for 12 h, and centrifuging to remove redundant unconjugated substances to obtain a secondary antibody marker ZnO @ Au-Ab of a gold-modified zinc oxide nanorod-conjugated procalcitonin recognition antibody₂Dispersing the mixture in 2 mL of PBS buffer solution, and storing the mixture at 4 ℃ for later use;

(7) preparation of electrochemical immunosensor

1) Polishing a glassy carbon electrode with the diameter of 4 mm by using aluminum oxide polishing powder, cleaning by using ultrapure water, and then polishing 6 muL and 1.0-3.0 mg mL^-1Dropwise adding the N-GQDs @ IRMOF-3@ N-GQDs solution to the surface of the electrode, and airing at room temperature;

2) then, 6. mu.L of 10. mu.g mL of the solution was added dropwise^-1Procalcitonin-anti Ab of₁After a period of time, washing with PBS buffer solution, and drying at room temperature;

3) continuously dropwise adding bovine serum albumin solution with the mass fraction of 0.1% and 3 muL to the surface of the electrode, washing with PBS buffer solution after a period of time, and airing at room temperature;

4) continuously dropwise adding 6 muL and 0.0001-100 ng mL^-1A series of procalcitonin antigens with different concentrations are applied to the surface of the electrode, incubated for 2 hours, washed by PBS buffer solution and dried at room temperature;

5) finally dropwise adding 6 mu L of second antibody marker ZnO @ Au-Ab of gold-modified zinc oxide nano-rod combined procalcitonin recognition antibody₂And (3) washing the solution with a PBS (phosphate buffer solution) after a period of time, and airing at room temperature to prepare the electrochemical immunosensor for detecting procalcitonin.

2. The detection method of the electrochemical immunosensor comprises the following steps:

(1) Ag/AgCl is used as a reference electrode, a platinum wire is used as a counter electrode, the prepared electrochemical luminescence sensor is used as a working electrode and is correctly connected in a cassette of a chemiluminescence detector, an electrochemical workstation is connected with the chemiluminescence detector, the high voltage of a photomultiplier is set to be 500-800V, and the electrochemical luminescence sensor contains 20-100 mmoL L^-1The test was performed in potassium persulfate in PBS buffer;

(2) detecting the procalcitonin standard solution by an electrochemical luminescence method, wherein the voltage test range is-1.6-0V;

(3) observing the electrochemical luminescence intensity of the sensor before and after the procalcitonin is added, then recording the linear relation between the electrochemical luminescence intensity value and the procalcitonin concentration, and drawing a working curve;

(4) and replacing the procalcitonin antigen sample solution to be detected with the procalcitonin antigen standard solution for detection.

The linear range of the sensor for detecting procalcitonin antigen is 0.0001-100 ng mL^-1The detection limit is 12.58 fg mL^-1。

The chemicals required for the synthesis were all purchased at the local reagent store and were not reprocessed.

Advantageous results of the invention

(1) According to the invention, the IRMOF-3 is adopted to carry out internal and external loading on the nitrogen-doped quantum dot composite material as the substrate luminescent material, so that the loading capacity of the nitrogen-doped quantum dot is greatly improved, the excellent electrochemical luminescence behavior is endowed, and the method has important significance for the extended application of the nitrogen-doped quantum dot.

(2) The invention provides a novel donor-acceptor pair for resonance energy transfer, wherein IRMOF-3 loaded with nitrogen-doped quantum dots inside and outside is used as a substrate luminescent material, and a gold-modified zinc oxide nanorod is used as a quenching material, so that a sandwich quenching type electrochemical immunosensor is constructed.

(3) The electrochemical immunosensor prepared by the invention sensitively detects procalcitonin, and the detection range is 0.0001-100 ng mL^-1The detection limit is 12.58 fg mL^-1And the detection with simplicity, convenience, rapidness, high sensitivity, high specificity and high stability can be realized.

Detailed Description

EXAMPLE 1 preparation of electrochemical immunosensor

(1) Preparation of nitrogen-doped quantum dot N-GQDs

Dissolving 2 mmol of citric acid and 5 mmol of urea in 15 mL of ultrapure water, stirring for 5 min, transferring to a 50 mL polytetrafluoroethylene autoclave, reacting at 160 ℃ for 6-12 h, cooling to room temperature after the reaction is finished, washing with absolute ethyl alcohol for three times, and vacuum drying to obtain nitrogen-doped quantum dots N-GQDs;

(2) preparation of N-GQDs @ IRMOF-3 encapsulated nitrogen-doped quantum dots in IRMOF-3

0.2 g of PVPK30 and 10 mg of nitrogen-doped quantum dots were dissolved in 18 mL of a mixed solution of N, N-dimethylformamide and 12 mL of anhydrous ethanol, while 50 mg of Zn (NO) was added₃)₂·6H₂O and 10 mg of 2NH₂-BDC is dissolved in 6 mL of N, N-dimethylformamide solution, the two solutions are mixed, stirred for 30 min and then transferred into a 50 mL polytetrafluoroethylene autoclave for reaction at 100 ℃ for 20 h, after the reaction, the product is centrifuged and washed with N, N-dimethylformamide, and after vacuum drying for 12 h, N-GQDs @ IRMOF-3 is obtained;

(3) preparation of IRMOF-3 internal and external load nitrogen doped quantum dot composite material N-GQDs @ IRMOF-3@ N-GQDs

Dissolving 10 mg of N-GQDs @ IRMOF-3 in 20 mL of ultrapure water, and adding 1 mL of 400 mmol L^-11- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 100 mmol L of^-1The N-hydroxysuccinimide mixed aqueous solution is reacted for 1 hour at the temperature of 4 ℃, then 15 mg of nitrogen-doped quantum dots are added and continuously stirred for 4 hours, the product is washed by absolute ethyl alcohol during centrifugation and dried to obtain an IRMOF-3 internal and external load nitrogen-doped quantum dot composite material N-GQDs @ IRMOF-3@ N-GQDs, and the N-GQDs are dissolved in 2 mg mL of the composite material^-1Storing the chitosan solution at 4 ℃ for later use;

(4) preparation of gold-modified zinc oxide nanorod ZnO @ Au

Mixing 15 mL of the solution with 0.1 mol of L^-1Zn (Ac)₂·2H₂O methanol solution and 30 mL, 0.1 mol L^-1Mixing the obtained NaOH methanol solution, stirring for 30 min to obtain a clear solution, transferring the clear solution into a 50 mL polytetrafluoroethylene autoclave, reacting at 150 ℃ for 24 h to obtain a white precipitate, then centrifugally washing with ultrapure water, drying the obtained product in a vacuum oven for 12 h to obtain a zinc oxide nano rod, then dispersing 50 mg of zinc oxide in 50 mL of ultrapure water, stirring for 30 min, then dissolving 0.6 mmol of CTAB in the solution, heating to 80 ℃, cooling the solution to room temperature until the CTAB is completely dissolved, and then adding 5 mL of 2% HAuCl₄Stirring for 1 h, and adding 15 mL of 5 mmol NaBH into the solution₄Obviously observing that the yellow solution is changed into purple, and finally centrifugally washing and drying the solution by using ultrapure water to obtain gold modified zinc oxide nano-rod ZnO @ Au;

(5) preparation of PBS buffer solution

11.94 g of disodium hydrogen phosphate dodecahydrate were taken out and dissolved in a 500 mL volumetric flask so as to be prepared into a solution having a concentration of 1/15 mol L^-1As an aqueous solution of (a); 4.54 g of monopotassium phosphate is taken and fixed to a 500 mL volumetric flask, and the volume is configured to be 1/15 mol L^-1As solution b; mixing solution A and solution B at a certain proportion to obtain PBS buffer solution with pH of 6.0；

(6) Second antibody marker ZnO @ Au-Ab of gold-modified zinc oxide nanorod combined procalcitonin recognition antibody₂Preparation of

20. mu.L of 10. mu.g mL^-1Anti-body Ab of procalcitonin recognition antibody₂Added to 5 mL, 2 mg mL^-1Oscillating and incubating ZnO @ Au in PBS (phosphate buffer solution) at 4 ℃ for 12 h, and centrifuging to remove redundant unconjugated substances to obtain a secondary antibody marker ZnO @ Au-Ab of a gold-modified zinc oxide nanorod-conjugated procalcitonin recognition antibody₂Dispersing the mixture in 2 mL of PBS buffer solution, and storing the mixture at 4 ℃ for later use;

(7) preparation of electrochemical immunosensor

1) Polishing a glassy carbon electrode with the diameter of 4 mm by using aluminum oxide polishing powder, cleaning by using ultrapure water, and polishing by using 6 mu L and 1.0 mg mL^-1Dropwise adding the N-GQDs @ IRMOF-3@ N-GQDs solution to the surface of the electrode, and airing at room temperature;

2) then, 6. mu.L of 10. mu.g mL of the solution was added dropwise^-1Procalcitonin-anti Ab of₁After a period of time, washing with PBS buffer solution, and drying at room temperature;

3) continuously dropwise adding bovine serum albumin solution with the mass fraction of 0.1% and 3 muL to the surface of the electrode, washing with PBS buffer solution after a period of time, and airing at room temperature;

4) continuously dropwise adding 6 muL and 0.0001-100 ng mL^-1A series of procalcitonin antigens with different concentrations are applied to the surface of the electrode, incubated for 2 hours, washed by PBS buffer solution and dried at room temperature;

5) finally dropwise adding 6 mu L of second antibody marker ZnO @ Au-Ab of gold-modified zinc oxide nano-rod combined procalcitonin recognition antibody₂And (3) washing the solution with a PBS (phosphate buffer solution) after a period of time, and airing at room temperature to prepare the electrochemical immunosensor for detecting procalcitonin.

EXAMPLE 2 preparation of electrochemical immunosensor

(1) Preparation of nitrogen-doped quantum dot N-GQDs

Dissolving 3 mmol of citric acid and 10 mmol of urea in 15 mL of ultrapure water, stirring for 5 min, transferring to a 50 mL polytetrafluoroethylene autoclave, reacting at 160 ℃ for 10 h, cooling to room temperature after the reaction is finished, washing with absolute ethyl alcohol for three times, and vacuum drying to obtain nitrogen-doped quantum dots N-GQDs;

(2) preparation of N-GQDs @ IRMOF-3 encapsulated nitrogen-doped quantum dots in IRMOF-3

0.5 g of PVPK30 and 20 mg of nitrogen-doped quantum dots were dissolved in 18 mL of a mixed solution of N, N-dimethylformamide and 12 mL of anhydrous ethanol, while 60 mg of Zn (NO) was added₃)₂·6H₂O and 20 mg of 2NH₂-BDC is dissolved in 6 mL of N, N-dimethylformamide solution, the two solutions are mixed, stirred for 30 min and then transferred into a 50 mL polytetrafluoroethylene autoclave for reaction at 100 ℃ for 20 h, after the reaction, the product is centrifuged and washed by N, N-dimethylformamide, and after vacuum drying for 12 h, the IRMOF-3 is obtained, and the N-GQDs @ IRMOF-3 of the nitrogen-doped quantum dots is encapsulated in the IRMOF-3;

(3) preparation of IRMOF-3 internal and external load nitrogen doped quantum dot composite material N-GQDs @ IRMOF-3@ N-GQDs

Dissolving 20 mg of N-GQDs @ IRMOF-3 in 20 mL of ultrapure water, and adding 1 mL of 400 mmol of L^-11- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 100 mmol L of^-1The N-hydroxysuccinimide mixed aqueous solution is reacted for 1 hour at the temperature of 4 ℃, then 15 mg of nitrogen-doped quantum dots are added and continuously stirred for 4 hours, the product is washed by absolute ethyl alcohol during centrifugation and dried to obtain an IRMOF-3 internal and external load nitrogen-doped quantum dot composite material N-GQDs @ IRMOF-3@ N-GQDs, and the N-GQDs are dissolved in 2 mg mL of the composite material^-1Storing the chitosan solution at 4 ℃ for later use;

(4) preparation of gold-modified zinc oxide nanorod ZnO @ Au

Mixing 15 mL of the solution with 0.3 mol of the solution^-1Zn (Ac)₂·2H₂O methanol solution and 30 mL, 0.5 mol L^-1Mixing with methanol solution of NaOH, stirring for 30 min to obtain clear solution, transferring into 50 mL polytetrafluoroethylene autoclave, reacting at 180 deg.C for 24 h to obtain white precipitate, centrifuging with ultrapure water, drying in vacuum oven for 12 h to obtain zinc oxide nanorod, dispersing 80 mg zinc oxide in 50 mL ultrapure water, stirring for 30 min, dissolving 0.8 mmol CTAB in the solution, adding CTAB, stirring, and dryingHeated to 80 ℃ until CTAB is completely dissolved, the solution is cooled to room temperature, then 10 mL of 2% HAuCl is added₄Stirring for 1 h, and adding 15 mL of 8 mmol NaBH into the solution₄Obviously observing that the yellow solution is changed into purple, and finally centrifugally washing and drying the solution by using ultrapure water to obtain gold modified zinc oxide nano-rod ZnO @ Au;

(5) preparation of PBS buffer solution

11.94 g of disodium hydrogen phosphate dodecahydrate were taken out and dissolved in a 500 mL volumetric flask so as to be prepared into a solution having a concentration of 1/15 mol L^-1As an aqueous solution of (a); 4.54 g of monopotassium phosphate is taken and fixed to a 500 mL volumetric flask, and the volume is configured to be 1/15 mol L^-1As solution b; mixing the solution A and the solution B in proportion to prepare PBS buffer solution with pH of 7.4;

(6) second antibody marker ZnO @ Au-Ab of gold-modified zinc oxide nanorod combined procalcitonin recognition antibody₂Preparation of

50. mu.L of 10. mu.g mL^-1The anti-second Ab of the procalcitonin-recognizing antibody of (1)₂Added to 8 mL, 2 mg mL^-1Oscillating and incubating ZnO @ Au in PBS (phosphate buffer solution) at 4 ℃ for 12 h, and centrifuging to remove redundant unconjugated substances to obtain a secondary antibody marker ZnO @ Au-Ab of a gold-modified zinc oxide nanorod-conjugated procalcitonin recognition antibody₂Dispersing the mixture in 2 mL of PBS buffer solution, and storing the mixture at 4 ℃ for later use;

(7) preparation of electrochemical immunosensor

1) Polishing a glassy carbon electrode with the diameter of 4 mm by using aluminum oxide polishing powder, cleaning by using ultrapure water, and polishing by using 6 mu L and 2.0 mg mL^-1Dropwise adding the N-GQDs @ IRMOF-3@ N-GQDs solution to the surface of the electrode, and airing at room temperature;

2) then, 6. mu.L of 10. mu.g mL of the solution was added dropwise^-1Procalcitonin-anti Ab of₁After a period of time, washing with PBS buffer solution, and drying at room temperature;

3) continuously dropwise adding bovine serum albumin solution with the mass fraction of 0.1% and 3 muL to the surface of the electrode, washing with PBS buffer solution after a period of time, and airing at room temperature;

4) continuously dropwise adding 6 mu L,0.0001 ~ 100 ng mL^-1A series of procalcitonin antigens with different concentrations are applied to the surface of the electrode, incubated for 2 hours, washed by PBS buffer solution and dried at room temperature;

5) finally dropwise adding 6 mu L of second antibody marker ZnO @ Au-Ab of gold-modified zinc oxide nano-rod combined procalcitonin recognition antibody₂And (3) washing the solution with a PBS (phosphate buffer solution) after a period of time, and airing at room temperature to prepare the electrochemical immunosensor for detecting procalcitonin.

EXAMPLE 3 preparation of electrochemical immunosensor

(1) Preparation of nitrogen-doped quantum dot N-GQDs

Dissolving 5 mmol of citric acid and 15 mmol of urea in 15 mL of ultrapure water, stirring for 5 min, transferring to a 50 mL polytetrafluoroethylene autoclave, reacting at 160 ℃ for 12 h, cooling to room temperature after the reaction is finished, washing with absolute ethyl alcohol for three times, and vacuum drying to obtain nitrogen-doped quantum dots N-GQDs;

(2) preparation of N-GQDs @ IRMOF-3 encapsulated nitrogen-doped quantum dots in IRMOF-3

1 g of PVPK30 and 30 mg of nitrogen-doped quantum dots were dissolved in 18 mL of a mixed solution of N, N-dimethylformamide and 12 mL of anhydrous ethanol, while 80 mg of Zn (NO) was added₃)₂·6H₂O and 30 mg of 2NH₂-BDC is dissolved in 6 mL of N, N-dimethylformamide solution, the two solutions are mixed, stirred for 30 min and then transferred into a 50 mL polytetrafluoroethylene autoclave for reaction at 100 ℃ for 20 h, after the reaction, the product is centrifuged and washed by N, N-dimethylformamide, and after vacuum drying for 12 h, the IRMOF-3 is obtained, and the N-GQDs @ IRMOF-3 of the nitrogen-doped quantum dots is encapsulated in the IRMOF-3;

(3) preparation of IRMOF-3 internal and external load nitrogen doped quantum dot composite material N-GQDs @ IRMOF-3@ N-GQDs

30 mg of N-GQDs @ IRMOF-3 is dissolved in 20 mL of ultrapure water, and 1 mL of 400 mmol L of N-GQDs @ IRMOF-3 is added^-11- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 100 mmol L of^-1Reacting the N-hydroxysuccinimide mixed aqueous solution for 1 h at the temperature of 4 ℃, adding 15 mg of nitrogen-doped quantum dots, continuously stirring for 4 h, washing the product with absolute ethyl alcohol during centrifugation, and drying to obtain IRMOF-3 with internal and external negative polaritiesDissolving the N-GQDs @ IRMOF-3@ N-GQDs in 2 mg mL^-1Storing the chitosan solution at 4 ℃ for later use;

(4) preparation of gold-modified zinc oxide nanorod ZnO @ Au

Mixing 15 mL of the solution with 0.5 mol of the solution^-1Zn (Ac)₂·2H₂O methanol solution and 30 mL, 1.0 mol L^-1Mixing the obtained NaOH methanol solution, stirring for 30 min to obtain a clear solution, transferring the clear solution into a 50 mL polytetrafluoroethylene autoclave, reacting at 200 ℃ for 24 h to obtain a white precipitate, then centrifugally washing with ultrapure water, drying the obtained product in a vacuum oven for 12 h to obtain a zinc oxide nano rod, then dispersing 100 mg of zinc oxide in 50 mL of ultrapure water, stirring for 30 min, then dissolving 1.2 mmol of CTAB in the solution, heating to 80 ℃, cooling the solution to room temperature until the CTAB is completely dissolved, then adding 20 mL of 2% HAuCl₄Stirring for 1 h, and adding 15 mL of 10 mmol NaBH into the solution₄Obviously observing that the yellow solution is changed into purple, and finally centrifugally washing and drying the solution by using ultrapure water to obtain gold modified zinc oxide nano-rod ZnO @ Au;

(5) preparation of PBS buffer solution

11.94 g of disodium hydrogen phosphate dodecahydrate were taken out and dissolved in a 500 mL volumetric flask so as to be prepared into a solution having a concentration of 1/15 mol L^-1As an aqueous solution of (a); 4.54 g of monopotassium phosphate is taken and fixed to a 500 mL volumetric flask, and the volume is configured to be 1/15 mol L^-1As solution b; mixing the solution A and the solution B in proportion to prepare PBS buffer solution with the pH value of 8.0;

(6) second antibody marker ZnO @ Au-Ab of gold-modified zinc oxide nanorod combined procalcitonin recognition antibody₂Preparation of

80. mu.L, 10. mu.g mL^-1Anti-body Ab of procalcitonin recognition antibody₂Added to 10 mL, 2 mg mL^-1Oscillating and incubating ZnO @ Au in PBS (phosphate buffer solution) at 4 ℃ for 12 h, and centrifuging to remove redundant unconjugated substances to obtain a secondary antibody marker ZnO @ Au-Ab of a gold-modified zinc oxide nanorod-conjugated procalcitonin recognition antibody₂Divide it intoDispersing in 2 mL PBS buffer solution, and storing at 4 ℃ for later use;

(7) preparation of electrochemical immunosensor

1) Polishing a glassy carbon electrode with the diameter of 4 mm by using aluminum oxide polishing powder, cleaning by using ultrapure water, and polishing by using 6 mu L and 3.0 mg mL^-1Dropwise adding the N-GQDs @ IRMOF-3@ N-GQDs solution to the surface of the electrode, and airing at room temperature;

2) then, 6. mu.L of 10. mu.g mL of the solution was added dropwise^-1Procalcitonin-anti Ab of₁After a period of time, washing with PBS buffer solution, and drying at room temperature;

3) continuously dropwise adding bovine serum albumin solution with the mass fraction of 0.1% and 3 muL to the surface of the electrode, washing with PBS buffer solution after a period of time, and airing at room temperature;

4) continuously dropwise adding 6 muL and 0.0001-100 ng mL^-1A series of procalcitonin antigens with different concentrations are applied to the surface of the electrode, incubated for 2 hours, washed by PBS buffer solution and dried at room temperature;

5) finally dropwise adding 6 mu L of second antibody marker ZnO @ Au-Ab of gold-modified zinc oxide nano-rod combined procalcitonin recognition antibody₂And (3) washing the solution with a PBS (phosphate buffer solution) after a period of time, and airing at room temperature to prepare the electrochemical immunosensor for detecting procalcitonin.

Example 4 detection of Procalcitonin

(1) Ag/AgCl is used as a reference electrode, a platinum wire is used as a counter electrode, the prepared electrochemical luminescence sensor is used as a working electrode and is correctly connected in a cassette of a chemiluminescence detector, an electrochemical workstation and the chemiluminescence detector are connected together, the high voltage of a photomultiplier is set to be 500V, and the electrochemical luminescence sensor contains 20 mmoL L^-1The test was performed in potassium persulfate in PBS buffer;

(2) detecting the procalcitonin standard solution by an electrochemical luminescence method, wherein the voltage test range is-1.6-0V;

(3) and observing the electrochemical luminescence intensity of the sensor before and after the procalcitonin is added, recording the linear relation between the electrochemical luminescence intensity value and the procalcitonin concentration, and drawing a working curve.

Example 5 detection of Procalcitonin

(1) Ag/AgCl is used as a reference electrode, a platinum wire is used as a counter electrode, the prepared electrochemical luminescence sensor is used as a working electrode and is correctly connected in a cassette of a chemiluminescence detector, an electrochemical workstation and the chemiluminescence detector are connected together, the high voltage of a photomultiplier is set to be 600V, and the electrochemical luminescence sensor contains 50 mmoL L^-1The test was performed in potassium persulfate in PBS buffer;

(2) detecting the procalcitonin standard solution by an electrochemical luminescence method, wherein the voltage test range is-1.5-0V;

(3) and observing the electrochemical luminescence intensity of the sensor before and after the procalcitonin is added, recording the linear relation between the electrochemical luminescence intensity value and the procalcitonin concentration, and drawing a working curve.

Example 6 detection of Procalcitonin

(1) Ag/AgCl is used as a reference electrode, a platinum wire is used as a counter electrode, the prepared electrochemical luminescence sensor is used as a working electrode and is correctly connected in a cassette of a chemiluminescence detector, an electrochemical workstation and the chemiluminescence detector are connected together, the high voltage of a photomultiplier is set to be 700V, and the electrochemical luminescence sensor contains 80 mmoL L^-1The test was performed in potassium persulfate in PBS buffer;

(2) detecting the procalcitonin standard solution by an electrochemical luminescence method, wherein the voltage test range is-1.4-0V;

(3) and observing the electrochemical luminescence intensity of the sensor before and after the procalcitonin is added, recording the linear relation between the electrochemical luminescence intensity value and the procalcitonin concentration, and drawing a working curve.

Example 7 detection of Procalcitonin

(1) Ag/AgCl is used as a reference electrode, a platinum wire is used as a counter electrode, the prepared electrochemical luminescence sensor is used as a working electrode and is correctly connected in a cassette of a chemiluminescence detector, an electrochemical workstation and the chemiluminescence detector are connected together, the high voltage of a photomultiplier is set to be 800V, and the electrochemical luminescence sensor contains 100 mmoL L^-1The test was performed in potassium persulfate in PBS buffer;

(2) detecting the procalcitonin standard solution by an electrochemical luminescence method, wherein the voltage test range is-1.3-0V;

(3) and observing the electrochemical luminescence intensity of the sensor before and after the procalcitonin is added, recording the linear relation between the electrochemical luminescence intensity value and the procalcitonin concentration, and drawing a working curve.

Claims (1)

1. A preparation method of an electrochemical immunosensor based on an IRMOF-3 internal and external load nitrogen-doped quantum dot composite material is characterized by comprising the following steps:

(1) preparation of nitrogen-doped quantum dot N-GQDs

Dissolving 2-5 mmol of citric acid and 5-15 mmol of urea in 15 mL of ultrapure water, stirring for 5 min, transferring to a 50 mL polytetrafluoroethylene autoclave, reacting at 160 ℃ for 6-12 h, cooling to room temperature after the reaction is finished, washing with absolute ethyl alcohol for three times, and vacuum drying to obtain nitrogen-doped quantum dots N-GQDs;

(2) preparation of N-GQDs @ IRMOF-3 encapsulated nitrogen-doped quantum dots in IRMOF-3

Dissolving 0.2-1 g of PVPK30 and 10-30 mg of nitrogen-doped quantum dots in 18 mL of mixed solution of N, N-dimethylformamide and 12 mL of absolute ethyl alcohol, and simultaneously dissolving 50-80 mg of Zn (NO)₃)₂·6H₂O and 10-30 mg of 2NH₂-BDC is dissolved in 6 mL of N, N-dimethylformamide solution, the two solutions are mixed, stirred for 30 min and then transferred into a 50 mL polytetrafluoroethylene autoclave for reaction at 100 ℃ for 20 h, after the reaction, the product is centrifuged and washed by N, N-dimethylformamide, and after vacuum drying for 12 h, the IRMOF-3 is obtained, and the N-GQDs @ IRMOF-3 of the nitrogen-doped quantum dots is encapsulated in the IRMOF-3;

(3) preparation of IRMOF-3 internal and external load nitrogen doped quantum dot composite material N-GQDs @ IRMOF-3@ N-GQDs

Dissolving 10-30 mg of N-GQDs @ IRMOF-3 in 20 mL of ultrapure water, and then adding 1 mL of 400 mmol of L^-11- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 100 mmol L of^-1Reacting the N-hydroxysuccinimide mixed aqueous solution for 1 h at the temperature of 4 ℃, adding 15 mg of nitrogen-doped quantum dots, continuously stirring for 4 h, and centrifuging the product by using anhydrousWashing with ethanol, drying to obtain IRMOF-3 internal and external load nitrogen doped quantum dot composite material N-GQDs @ IRMOF-3@ N-GQDs, and dissolving the IRMOF-3 internal and external load nitrogen doped quantum dot composite material N-GQDs in 2 mg mL^-1Storing the chitosan solution at 4 ℃ for later use;

(4) preparation of gold-modified zinc oxide nanorod ZnO @ Au

Mixing 15 mL of the mixture with 0.1-0.5 mol of the mixture^-1Zn (Ac)₂·2H₂O methanol solution, 30 mL and 0.1-1.0 mol L^-1Mixing the obtained NaOH methanol solution, stirring for 30 min to obtain a clear solution, transferring the clear solution into a 50 mL polytetrafluoroethylene autoclave, reacting at 150-200 ℃ for 24 h to obtain a white precipitate, centrifugally washing with ultrapure water, drying the obtained product in a vacuum oven for 12 h to obtain a zinc oxide nano rod, dispersing 50-100 mg of zinc oxide in 50 mL of ultrapure water, stirring for 30 min, dissolving 0.6-1.2 mmol of CTAB in the solution, heating to 80 ℃, cooling the solution to room temperature until the CTAB is completely dissolved, adding 5-20 mL of 2% HAuCl₄Continuously stirring for 1 h, and then adding 15 mL of 5-10 mmol NaBH into the solution₄Obviously observing that the yellow solution is changed into purple, and finally centrifugally washing and drying the solution by using ultrapure water to obtain gold modified zinc oxide nano-rod ZnO @ Au;

(5) preparation of PBS buffer solution

11.94 g of disodium hydrogen phosphate dodecahydrate were taken out and dissolved in a 500 mL volumetric flask so as to be prepared into a solution having a concentration of 1/15 mol L^-1As an aqueous solution of (a); 4.54 g of monopotassium phosphate is taken and fixed to a 500 mL volumetric flask, and the volume is configured to be 1/15 mol L^-1As solution b; mixing the solution A and the solution B in proportion to prepare a series of PBS (phosphate buffer solution) with the pH value of 6.0-8.0;

(6) second antibody marker ZnO @ Au-Ab of gold-modified zinc oxide nanorod combined procalcitonin recognition antibody₂Preparation of

20-80 μ L, 10 μ g mL^-1Anti-body Ab of procalcitonin recognition antibody₂Adding the mixture into 5-10 mL and 2 mg mL^-1 ZnO @ Au in PBS, incubated at 4 ℃ with shaking for 12 h, and centrifuged to remove excess unbound materialObtaining a secondary antibody marker ZnO @ Au-Ab of the gold modified zinc oxide nanorod combined procalcitonin recognition antibody₂Dispersing the mixture in 2 mL of PBS buffer solution, and storing the mixture at 4 ℃ for later use;

(7) preparation of electrochemical immunosensor

1) Polishing a glassy carbon electrode with the diameter of 4 mm by using aluminum oxide polishing powder, cleaning by using ultrapure water, and then polishing 6 muL and 1.0-3.0 mg mL^-1Dropwise adding the N-GQDs @ IRMOF-3@ N-GQDs solution to the surface of the electrode, and airing at room temperature;

2) then, 6. mu.L of 10. mu.g mL of the solution was added dropwise^-1Procalcitonin-anti Ab of₁After a period of time, washing with PBS buffer solution, and drying at room temperature;

3) continuously dropwise adding bovine serum albumin solution with the mass fraction of 0.1% and 3 muL to the surface of the electrode, washing with PBS buffer solution after a period of time, and airing at room temperature;

4) continuously dropwise adding 6 muL and 0.0001-100 ng mL^-1A series of procalcitonin antigens with different concentrations are applied to the surface of the electrode, incubated for 2 hours, washed by PBS buffer solution and dried at room temperature;

5) finally dropwise adding 6 mu L of second antibody marker ZnO @ Au-Ab of gold-modified zinc oxide nano-rod combined procalcitonin recognition antibody₂And (3) washing the solution with a PBS (phosphate buffer solution) after a period of time, and airing at room temperature to prepare the electrochemical immunosensor for detecting procalcitonin.