Electrochemiluminescence immunosensor based on defect porphyrin MOFs as well as preparation method and application of electrochemical luminescence immunosensor

Abstract

The invention belongs to the technical field of electrochemical sensors, and particularly relates to an electrochemical luminescence immunosensor based on defect porphyrin MOFs as well as a preparation method and application of the electrochemical luminescence immunosensor. The preparation method of the electrochemical luminescence immunosensor comprises the following steps: preparation of the defective porphyrin MOFs, preparation of a defective porphyrin MOFs-Ab2 compound and preparation of the electrochemical luminescence immunosensor of the defective porphyrin MOFs. Porphyrin MOFs with different degrees of ligand defects are synthesized by adding a regulator benzoic acid in a gradient manner, the size of the MOFs, the ligand defect degree and the dispersion degree of TCPP are increased along with the increase of the BA content, and an ECL signal presents a change trend of increasing first and then decreasing. The invention opens up a new field of view for developing an ECL luminescent material with excellent performance, also lays a foundation for constructing a high-sensitivity defect porphyrin MOFs electrochemical immunosensor, can realize quantitative detection of A beta1-42, provides a solid foundation for detection and treatment of Alzheimer's disease, and has a wide market application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of electrochemical sensors, and in particular relates to an electrochemiluminescent immunosensor based on defective porphyrin MOFs and a preparation method and application thereof. Background Art

[0002] Alzheimer's disease (AD) is a chronic neurodegenerative disease and the main cause of senile dementia. The disease progresses gradually, from initial memory impairment to severe cognitive loss, which eventually seriously affects the patient's ability to take care of themselves. There is currently a lack of effective treatment. Although the pathogenesis of AD has not been fully elucidated, studies have shown that it is closely related to the accumulation of abnormal proteins in the brain. Among them, β-amyloid protein (Aβ 1-42 Abnormal deposition of Aβ and the formation of amyloid plaques are one of the core pathological features of AD. 1-42 Early diagnosis of AD is of great significance. 1-42 The detection methods include enzyme-linked immunosorbent assay, electrochemical method, fluorescence method, photoelectrochemical method and electrochemiluminescence (ECL), etc. Among them, ECL technology is particularly suitable for the detection and analysis of trace markers in complex biological systems due to its advantages such as high sensitivity, excellent selectivity, low cost and rapid response, providing reliable technical support for the early diagnosis of AD.

[0003] Improving the ECL performance of the luminophore is the key to enhancing the sensitivity of ECL detection. Tetracarboxyphenylporphyrin (TCPP) is regarded as an ideal organic light-emitting material due to its rigid configuration, excellent photoelectric properties and easy functional modification. However, its aggregation-induced quenching (ACQ) effect severely limits the application of ECL. To this end, researchers used TCPP as an organic ligand and constructed a series of TCPP-MOFs materials through metal coordination, which can effectively inhibit the ACQ effect and promote the transport of co-reactants. In recent years, although progress has been made in the research on the ECL performance of TCPP-MOFs, its luminescence efficiency is still difficult to meet actual needs. Therefore, it is of great significance to develop an efficient strategy to improve the ECL efficiency of TCPP-MOFs. In summary, it is urgently needed to develop a porphyrin-based electrochemiluminescence immunosensor that overcomes the problems of the existing technology to achieve higher detection sensitivity and a wider range of applications. Summary of the invention

[0004] The purpose of the present invention is to provide an electrochemiluminescent immunosensor based on defective porphyrin MOFs and a preparation method and application thereof.

[0005] The implementation process of the present invention is as follows:

[0006] A method for preparing an electrochemiluminescent immunosensor based on defective porphyrin MOFs comprises the following steps:

[0007] (1) Preparation of defective porphyrin MOFs

[0008] Tetracarboxyphenyl porphyrin, ZrCl4 and benzoic acid were dispersed in DMF solvent to obtain a dispersion, and then the dispersion was transferred to a high-pressure reaction kettle for hydrothermal reaction. After the reaction, the product was washed and dried to obtain defective porphyrin MOFs;

[0009] (2) Preparation of defective porphyrin MOFs-Ab2 complex

[0010] The defective porphyrin MOFs were dispersed in ultrapure water to obtain a dispersion, and the dispersion was added to a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, and slowly stirred at 4-5 °C in the dark for 5-6 h to fully activate the carboxyl groups of the defective porphyrin MOFs; β-amyloid protein secondary antibody Ab2 was dispersed in the activated defective porphyrin MOFs, and slowly stirred at 4-5 °C in the dark for 10-14 h to obtain a defective porphyrin MOFs-Ab2 complex. The product was centrifuged and washed and then dispersed in ultrapure water to obtain a defective porphyrin MOFs-Ab2 complex dispersion, which was stored at 4-5 °C for subsequent use;

[0011] (3) Preparation of defective porphyrin MOFs electrochemiluminescence immunosensor

[0012] (3.1) The polished and dried glassy carbon electrode GCE was immersed in HAuCl4 solution for electrochemical deposition to obtain an AuNPs-modified electrode DpAu / GCE;

[0013] (3.2) β-amyloid protein primary antibody Ab1 was immobilized on the electrode surface through Au-NH2 bond to obtain an electrode Ab1 / DpAu / GCE;

[0014] (3.3) The blocking agent bovine serum albumin BSA was incubated on the electrode surface to block non-specific binding sites to obtain an electrode BSA / Ab1 / DpAu / GCE;

[0015] (3.4) Based on the specific recognition of antigen and antibody, different concentrations of β-amyloid protein Aβ from 5 fg mL -1 ~1 ng mL -1 were incubated on the surface of the BSA / Ab1 / DpAu / GCE-modified electrode to obtain an electrode Aβ 1-42 / BSA / Ab1 / DpAu / GCE; 1-42 / BSA / Ab1 / DpAu / GCE;

[0016] (3.5) The defective porphyrin MOFs-Ab2 complex dispersion obtained in step (2) was dropped onto Aβ 1-42Incubate on the / BSA / Ab1 / DpAu / GCE modified electrode at room temperature to form defective porphyrin MOFs-Ab2 / Aβ 1-42 The sandwich structure of / BSA / Ab1 / DpAu / GCE, that is, a defective porphyrin MOF-based electrochemiluminescence immunosensor is obtained.

[0017] Furthermore, in step (1), the mass ratio of the tetracarboxyphenylporphyrin, ZrCl4, and benzoic acid is 5:15:(90 - 170).

[0018] Furthermore, in step (1), the temperature of the hydrothermal reaction is 110 - 130 °C, and the reaction time is 22 - 26 h.

[0019] Furthermore, in step (2), the concentration of the dispersion obtained by dispersing the defective porphyrin MOFs in ultrapure water is 0.6 - 1.6 mg mL -1 , and the volume ratio of the dispersion to the mixed solution is 5:1.

[0020] Furthermore, in step (2), the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide in the mixed solution is 40 mmol L -1 , and the concentration of N-hydroxysuccinimide is 10 mmol L -1 .

[0021] Furthermore, in step (3.1), the mass percentage of the HAuCl4 solution is 1%; the potential of the electrochemical deposition is -0.2 V, and the deposition time is 30 s; in step (3.2), the dosage of the β-amyloid primary antibody Ab1 is 10 μL; the fixation time is 12 - 16 h.

[0022] Furthermore, in step (3.3), the mass percentage of the blocking agent bovine serum albumin BSA is 0.25%; the dosage of the blocking agent bovine serum albumin BSA is 10 μL; the incubation time is 25 - 35 min.

[0023] Furthermore, in step (3.4), the dosages of β-amyloid Aβ 1-42 at different concentrations are 10 μL respectively; the incubation time is 1 h; in step (3.5), the dosage of the defective porphyrin MOFs-Ab2 complex dispersion is 10 μL; the incubation time is 2 h.

[0024] The electrochemiluminescence immunosensor based on defective porphyrin MOFs prepared by the above method.

[0025] The above electrochemiluminescence immunosensor based on defective porphyrin MOFs in the detection of β-amyloid Aβ 1-42 in the application.

[0026] Positive effects of the present invention:

[0027] (1) By gradually adding the monocarboxylic acid regulator benzoic acid (BA), the present invention synthesizes porphyrin MOFs with different degrees of ligand defects. The monocarboxylic acid regulator BA competes for coordination with zirconium oxygen clusters, thereby forming ligand defects. While improving the size of MOFs, the degree of ligand defects and the dispersion degree of TCPP in the MOFs framework are also regulated. Under the synergistic action of the three, the ECL performance is significantly improved. With the increase of the BA content, the size of MOFs, the degree of ligand defects and the dispersion degree of TCPP increase accordingly, and the ECL signal shows a trend of first increasing and then decreasing. Furthermore, the relationship between the change of ECL performance and the structure of porphyrin MOFs is studied.

[0028] (2) Based on the excellent ECL performance of defective porphyrin MOFs, the present invention uses them as ECL signal probes and combines a sandwich-type immunosensing strategy to construct a highly sensitive and highly stable defective porphyrin MOFs-based electrochemiluminescence immunosensor for the quantitative detection of Aβ 1-42 . The results show that the ECL signal has a good linear relationship with the logarithm of the Aβ 1-42 concentration (lgc) in the concentration range of 5 fg mL -1 to 1 ng mL -1 , and the detection limit is as low as 3.89 fg mL -1 .

[0029] (3) The present invention opens up a new perspective for the development of excellent ECL luminescent materials, and also lays a foundation for the construction of highly sensitive defective porphyrin MOFs-based electrochemiluminescence immunosensors, and can realize the quantitative detection of Aβ 1-42 , providing a solid foundation for the detection and treatment of AD and having broad market application prospects. Description of the drawings

[0030] Figure 1 is the preparation process diagram of the electrochemiluminescence immunosensor based on defective porphyrin MOFs according to the present invention;

[0031] Figure 2 is the SEM diagram of TCPP and defective porphyrin MOFs prepared in Examples 1-5;

[0032] Figure 3 is the UV-Vis spectrum diagram of defective porphyrin MOFs prepared in Examples 1-5;

[0033] Figure 4 is the PXRD spectrum diagram and FT-IR spectrum diagram of TCPP and defective porphyrin MOFs prepared in Examples 1-5;

[0034] Figure 5TEM images of the defective porphyrin MOFs prepared in Examples 1-5;

[0035] Figure 6 Size distribution plots of the defective porphyrin MOFs prepared in Examples 1-5;

[0036] Figure 7 TEM-EDS images of the defective porphyrin MOFs prepared in Examples 1-5;

[0037] Figure 8 XPS spectra of the defective porphyrin MOF PCN-224-130BA prepared in Example 3, where (A) is the full spectrum, (B) is the Zr 3d spectrum, (C) is the N 1s spectrum, and (D) is the O 1s spectrum;

[0038] Figure 9 TG curves of the defective porphyrin MOFs prepared in Examples 1-5;

[0039] Figure 10 N2 adsorption-desorption isotherms of the defective porphyrin MOFs prepared in Examples 1-5;

[0040] Figure 11 Pore size distribution plots of the defective porphyrin MOFs prepared in Examples 1-5;

[0041] Figure 12 CV curves and ECL curves of TCPP and the defective porphyrin MOFs prepared in Examples 1-5, where (A) is the CV curve and (B) is the ECL curve;

[0042] Figure 13 Influence diagram of the concentration of the defective porphyrin MOF PCN-224-130BA prepared in Example 3 on the ECL intensity;

[0043] Figure 14 For PCN-224-130BA / S2O8 2- ECL intensity-time curve of the system scanned continuously for 500 s;

[0044] Figure 15 Synchronous CV curves of different systems, where (a) is GCE+PBS, (b) is GCE+K2S2O8, and (c) is PCN-224-130BA / GCE+K2S2O8;

[0045] Figure 16 Synchronous ECL curves of different systems, where (a) is GCE+PBS, (b) is GCE+K2S2O8, and (c) is PCN-224-130BA / GCE+K2S2O8;

[0046] Figure 17Influence diagram of the incubation time of the PCN-224-130BA-Ab2 complex on the ECL intensity;

[0047] Figure 18 CV diagram for the preparation process of an electrochemiluminescence immunosensor based on defective porphyrin MOFs, where (a) bare GCE, (b) DpAu / GCE, (c) Ab1 / DpAu / GCE (d) BSA / Ab1 / DpAu / GCE, (e) Aβ 1-42 / BSA / Ab1 / DpAu / GCE, (f) PCN-224-130BA-Ab2 / Aβ 1-42 / BSA / Ab1 / DpAu / GCE;

[0048] Figure 19 EIS curve for the preparation process of an electrochemiluminescence immunosensor, where (a) bare GCE, (b) DpAu / GCE, (c) Ab1 / DpAu / GCE (d) BSA / Ab1 / DpAu / GCE, (e) Aβ 1-42 / BSA / Ab1 / DpAu / GCE, (f) PCN-224-130BA-Ab2 / Aβ 1-42 / BSA / Ab1 / DpAu / GCE;

[0049] Figure 20 ECL response diagram of the electrochemiluminescence immunosensor to different concentrations of Aβ 1-42 where (a) 5 fg mL -1 , (b) 10 fg mL -1 , (c) 100 fg mL -1 , (d) 1 pg mL -1 , (e) 10 pg mL -1 , (f) 100 pg mL -1 , (g) 1 ng mL -1 ;

[0050] Figure 21 Linear regression curve of the electrochemiluminescence immunosensor to different concentrations of Aβ 1-42 where (a) 5 fgmL -1 , (b) 10 fg mL -1 , (c) 100 fg mL -1 , (d) 1 pg mL -1 , (e) 10 pg mL -1 , (f) 100 pg mL -1 , (g) 1 ng mL -1 ;

[0051] Figure 22Performance graph of the electrochemiluminescence immunosensor, where (A) selectivity, (B) stability, (C) storage stability, (D) reproducibility. Detailed implementation mode

[0052] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used can be obtained from commercial channels. The instruments involved in the examples are an electrochemical workstation, a scanning electron microscope, a Fourier transform infrared spectrometer, and an X-ray powder diffractometer. All ECL and EC tests use a three-electrode system. The modified glassy carbon electrode (GCE, Φ = 3 mm) is used as the working electrode, the Ag / AgCl electrode is used as the reference electrode, and the platinum wire is used as the auxiliary electrode.

[0053] Now, the present invention will be further described through specific implementation modes, but it is not limited thereto.

[0054] Example 1 Preparation of defective porphyrin MOFs

[0055] A preparation method of defective porphyrin MOFs includes the following steps:

[0056] Disperse 5 mg of tetracarboxyphenyl porphyrin, 15 mg of ZrCl4, and 90 mg of benzoic acid in 5 mL of DMF solvent, and ultrasonically disperse them evenly to obtain a dispersion. Then transfer the dispersion to a high-pressure reaction kettle and carry out a hydrothermal reaction at 120 °C for 24 h. After the reaction is completed, naturally cool it to room temperature. The obtained product is centrifugally washed three times with DMF to remove unreacted precursors, and then the product is placed in a 60 °C vacuum oven and dried overnight to obtain defective porphyrin MOFs, named PCN-224-90BA.

[0057] Example 2 Preparation of defective porphyrin MOFs

[0058] A preparation method of defective porphyrin MOFs includes the following steps:

[0059] Disperse 5 mg of tetracarboxyphenyl porphyrin, 15 mg of ZrCl4, and 110 mg of benzoic acid in 5 mL of DMF solvent, and ultrasonically disperse them evenly to obtain a dispersion. Then transfer the dispersion to a high-pressure reaction kettle and carry out a hydrothermal reaction at 120 °C for 24 h. After the reaction is completed, naturally cool it to room temperature. The obtained product is centrifugally washed three times with DMF to remove unreacted precursors, and then the product is placed in a 60 °C vacuum oven and dried overnight to obtain defective porphyrin MOFs, named PCN-224-110BA.

[0060] Example 3 Preparation of defective porphyrin MOFs

[0061] A preparation method of defective porphyrin MOFs includes the following steps:

[0062] Disperse 5 mg of tetracarboxyphenyl porphyrin, 15 mg of ZrCl4, and 130 mg of benzoic acid in 5 mL of DMF solvent, and ultrasonically disperse them evenly to obtain a dispersion. Then transfer the dispersion to a high-pressure reaction kettle and carry out a hydrothermal reaction at 120 °C for 24 h. After the reaction is completed, naturally cool it to room temperature. The obtained product is centrifugally washed three times with DMF to remove the unreacted precursors, and then the product is placed in a vacuum oven at 60 °C and dried overnight to obtain defective porphyrin MOFs, named PCN-224-130BA.

[0063] Preparation of Defective Porphyrin MOFs in Example 4

[0064] A preparation method of defective porphyrin MOFs includes the following steps:

[0065] Disperse 5 mg of tetracarboxyphenyl porphyrin, 15 mg of ZrCl4, and 150 mg of benzoic acid in 5 mL of DMF solvent, and ultrasonically disperse them evenly to obtain a dispersion. Then transfer the dispersion to a high-pressure reaction kettle and carry out a hydrothermal reaction at 120 °C for 24 h. After the reaction is completed, naturally cool it to room temperature. The obtained product is centrifugally washed three times with DMF to remove the unreacted precursors, and then the product is placed in a vacuum oven at 60 °C and dried overnight to obtain defective porphyrin MOFs, named PCN-224-150BA.

[0066] Preparation of Defective Porphyrin MOFs in Example 5

[0067] A preparation method of defective porphyrin MOFs includes the following steps:

[0068] Disperse 5 mg of tetracarboxyphenyl porphyrin, 15 mg of ZrCl4, and 170 mg of benzoic acid in 5 mL of DMF solvent, and ultrasonically disperse them evenly to obtain a dispersion. Then transfer the dispersion to a high-pressure reaction kettle and carry out a hydrothermal reaction at 120 °C for 24 h. After the reaction is completed, naturally cool it to room temperature. The obtained product is centrifugally washed three times with DMF to remove the unreacted precursors, and then the product is placed in a vacuum oven at 60 °C and dried overnight to obtain defective porphyrin MOFs, named PCN-224-170BA.

[0069] Example 6 Taking PCN-224-130BA as an example, prepare PCN-224-130BA-Ab2 complex and PCN-224-130BA-based electrochemiluminescence immunosensor

[0070] A preparation method of a PCN-224-130BA-based electrochemiluminescence immunosensor is shown in Figure 1 , including the following steps:

[0071] (1) Preparation of defective porphyrin MOFs. The specific process is as described in Example 3 to obtain defective porphyrin MOFs, named PCN-224-130BA;

[0072] (2) Preparation of PCN-224-130BA-Ab2 complex

[0073] Disperse 1.2 mg of PCN-224-130BA in 1 mL of ultrapure water to obtain a dispersion with a concentration of 1.2 mg mL -1 Add 1 mL of the dispersion to 200 μL of a mixed solution of 40 mmol L -1 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 10 mmol L -1 N-hydroxysuccinimide (NHS). Stir slowly at 4 °C in the dark for 5 h to fully activate the carboxyl groups of PCN-224-130BA. After centrifugation and washing, redisperse it in 1.0 mL of ultrapure water to obtain an activated PCN-224-130BA dispersion. Disperse 150 μL of β-amyloid secondary antibody Ab2 in the activated PCN-224-130BA dispersion and stir slowly at 4 °C in the dark for 12 h to form a PCN-224-130BA-Ab2 complex through amide bond formation. The product is centrifuged and washed, then redispersed in 1.0 mL of ultrapure water to obtain a PCN-224-130BA-Ab2 dispersion, and stored at 4 °C for subsequent use;

[0074] (3) Preparation of defective porphyrin MOFs electrochemical luminescence immunosensor

[0075] (3.1) Polish the glassy carbon electrode GCE with alumina polishing powder, clean it by ultrasonic cleaning, and place the dried GCE in a 1% (mass percentage) HAuCl4 solution for electrochemical deposition at -0.2 V for 30 s to obtain an Au NPs modified electrode DpAu / GCE;

[0076] (3.2) Fix 10 μL of β-amyloid primary antibody Ab1 on the electrode surface through Au-NH2 bond for 14 h to obtain the electrode Ab1 / DpAu / GCE;

[0077] (3.3) Incubate 10 μL of the blocking agent bovine serum albumin BSA (mass percentage 0.25%) on the electrode surface for 30 min to block non-specific binding sites, obtaining the electrode BSA / Ab1 / DpAu / GCE;

[0078] (3.4) Based on the specific recognition of antigen-antibody, respectively add 10 μL of different concentrations (5 fg mL -1 , 10 fg mL -1 , 100 fg mL-1 ,1 pg / mL -1 ,10 pg / mL -1 ,100 pg / mL -1 ,1 ng / mL -1 ) of β-amyloid Aβ 1-42 Incubate on the surface of the BSA / Ab1 / DpAu / GCE modified electrode for 1 h to obtain electrode Aβ 1-42 / BSA / Ab1 / DpAu / GCE;

[0079] (3.5) Drop 10 μL of the PCN-224-130BA-Ab2 complex dispersion obtained in step (2) onto the Aβ 1-42 / BSA / Ab1 / DpAu / GCE modified electrode and incubate at room temperature for 2 h to form a sandwich structure of PCN-224-130BA-Ab2 / Aβ 1-42 / BSA / Ab1 / DpAu / GCE, namely, a defective porphyrin MOFs-based electrochemiluminescence immunosensor is obtained for subsequent ECL testing. It should be noted that each step of modifying the electrode is gently rinsed with ultrapure water to remove non-specific adsorption.

[0080] Example 7

[0081] A preparation method of a defective porphyrin MOFs electrochemiluminescence immunosensor, comprising the following steps:

[0082] (1) Preparation of defective porphyrin MOFs

[0083] Disperse 5 mg of tetracarboxyphenyl porphyrin, 15 mg of ZrCl4 and 160 mg of benzoic acid in 5 mL of DMF solvent, ultrasonically disperse it evenly to obtain a dispersion, then transfer the dispersion to a high-pressure reaction kettle, carry out hydrothermal reaction at 110 °C for 26 h, after the reaction is completed, naturally cool to room temperature, wash the obtained product three times by centrifugation with DMF to remove unreacted precursors, and then place the product in a 50 °C vacuum oven to dry overnight to obtain defective porphyrin MOFs, named PCN-224-160BA.

[0084] (2) Preparation of PCN-224-160BA-Ab2 complex

[0085] Disperse 1.6 mg of PCN-224-160BA in 1 mL of ultrapure water to obtain a dispersion with a concentration of 1.6 mg / mL -1 of the dispersion, add 1 mL of the dispersion to 200 μL of 40 mmol / L -1 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 10 mmol / L - 1In a mixed solution of N-hydroxysuccinimide, the carboxyl groups of PCN-224-160BA were slowly stirred for 6 h at 5 °C in the dark to be fully activated. After centrifugation and washing, it was redispersed in 1.0 mL of ultrapure water to obtain an activated PCN-224-160BA dispersion; 150 μL of β-amyloid secondary antibody Ab2 was dispersed in the activated PCN-224-160BA dispersion, and slowly stirred at 5 °C in the dark for 14 h to form a PCN-224-160BA-Ab2 complex through amide action. The product was centrifuged and washed and then dispersed in 1.0 mL of ultrapure water to obtain a PCN-224-160BA-Ab2 dispersion, which was stored at 5 °C for subsequent use;

[0086] (3) Preparation of defective porphyrin MOFs electrochemiluminescence immunosensor

[0087] (3.1) The glassy carbon electrode GCE was polished clean with alumina polishing powder and ultrasonically cleaned. The dried GCE was placed in a 1% (mass percentage) HAuCl4 solution and electrochemically deposited at -0.2 V for 30 s to obtain an Au NPs modified electrode DpAu / GCE;

[0088] (3.2) 10 μL of β-amyloid primary antibody Ab1 was immobilized on the electrode surface for 12 h through Au-NH2 bond to obtain the electrode Ab1 / DpAu / GCE;

[0089] (3.3) 10 μL of blocking agent bovine serum albumin BSA (mass percentage 0.25%) was incubated on the electrode surface for 25 min to block non-specific binding sites, obtaining the electrode BSA / Ab1 / DpAu / GCE;

[0090] (3.4) Based on the specific recognition of antigen-antibody, 10 μL of β-amyloid Aβ with different concentrations (5 fg mL -1 , 10 fg mL -1 , 100 fg mL -1 , 1 pg mL -1 , 10 pg mL -1 , 100 pg mL -1 , 1 ng mL -1 ) was incubated on the surface of the BSA / Ab1 / DpAu / GCE modified electrode for 1 h to obtain the electrode Aβ 1-42 / BSA / Ab1 / DpAu / GCE; 1-42 / BSA / Ab1 / DpAu / GCE;

[0091] (3.5) 10 μL of the PCN-224-160BA-Ab2 complex dispersion obtained in step (2) was dropped onto Aβ 1-42On the / BSA / Ab1 / DpAu / GCE modified electrode, incubate at room temperature for 2 h to form PCN-224-160BA-Ab2 / Aβ 1-42 The sandwich structure of / BSA / Ab1 / DpAu / GCE, namely, a defective porphyrin MOFs-based electrochemiluminescence immunosensor is obtained.

[0092] Example 8

[0093] A preparation method of a defective porphyrin MOFs electrochemiluminescence immunosensor includes the following steps:

[0094] (1) Preparation of defective porphyrin MOFs

[0095] Disperse 5 mg of tetracarboxyphenyl porphyrin, 15 mg of ZrCl4 and 100 mg of benzoic acid in 5 mL of DMF solvent, and ultrasonically disperse them evenly to obtain a dispersion. Then transfer the dispersion to a high-pressure reaction kettle and carry out hydrothermal reaction at 130 °C for 22 h. After the reaction, naturally cool to room temperature. The obtained product is centrifugally washed three times with DMF to remove unreacted precursors, and then the product is placed in a 60 °C vacuum oven and dried overnight to obtain defective porphyrin MOFs, named PCN-224-100BA.

[0096] (2) Preparation of PCN-224-100BA-Ab2 complex

[0097] Disperse 0.6 mg of PCN-224-100BA in 1 mL of ultrapure water to obtain a dispersion with a concentration of 0.6 mg mL -1 Add 1 mL of the dispersion to 200 μL of a mixed solution of 40 mmol L -1 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 10 mmol L - 1 N-hydroxysuccinimide, and slowly stir at 4 °C in the dark for 6 h to fully activate the carboxyl group of PCN-224-100BA. After centrifugal washing, redisperse it in 1.0 mL of ultrapure water to obtain an activated PCN-224-100BA dispersion; disperse 150 μL of β-amyloid protein secondary antibody Ab2 in the activated PCN-224-100BA dispersion, and slowly stir at 5 °C in the dark for 10 h to obtain a PCN-224-100BA-Ab2 complex formed by amide reaction. The product is centrifugally washed and then dispersed in 1.0 mL of ultrapure water to obtain a PCN-224-100BA-Ab2 dispersion, and store it at 5 °C for subsequent use;

[0098] (3) Preparation of defective porphyrin MOFs electrochemiluminescence immunosensor

[0099] (3.1) The glassy carbon electrode (GCE) was polished clean with alumina polishing powder and ultrasonically cleaned. The dried GCE was placed in a 1% (mass percentage) HAuCl4 solution and subjected to electrochemical deposition at -0.2 V for 30 s to obtain the Au NPs modified electrode DpAu / GCE;

[0100] (3.2) 10 μL of β-amyloid primary antibody Ab1 was immobilized on the electrode surface for 16 h through Au-NH2 bond interaction to obtain the electrode Ab1 / DpAu / GCE;

[0101] (3.3) 10 μL of the blocking agent bovine serum albumin BSA (0.25% mass percentage) was incubated on the electrode surface for 35 min to block non-specific binding sites, obtaining the electrode BSA / Ab1 / DpAu / GCE;

[0102] (3.4) Based on the specific recognition of antigen-antibody, 10 μL of β-amyloid Aβ with different concentrations (5 fg mL -1 , 10 fg mL -1 , 100 fg mL -1 , 1 pg mL -1 , 10 pg mL -1 , 100 pg mL -1 , 1 ng mL -1 ) was incubated on the surface of the BSA / Ab1 / DpAu / GCE modified electrode for 1 h to obtain the electrode Aβ 1-42 / BSA / Ab1 / DpAu / GCE; 1-42 / BSA / Ab1 / DpAu / GCE;

[0103] (3.5) 10 μL of the PCN-224-100BA-Ab2 complex dispersion obtained in step (2) was dropped onto the Aβ 1-42 / BSA / Ab1 / DpAu / GCE modified electrode and incubated at room temperature for 2 h to form a sandwich structure of PCN-224-100BA-Ab2 / Aβ 1-42 / BSA / Ab1 / DpAu / GCE, that is, a defective porphyrin MOFs-based electrochemiluminescence immunosensor was obtained.

[0104] (I) Characterization of the defective porphyrin MOFs prepared in Examples 1-5

[0105] In the synthesis process of Zr-based MOFs, the regulator BA and the ligand compete with the coordination of zirconium oxygen clusters, slowing down the crystallization process to improve the crystallinity; MOFs synthesized without the addition of the regulator BA have the disadvantages of low crystallinity and easy agglomeration. Therefore, in the synthesis process of defective porphyrin MOFs (PCN-224-90BA ~ PCN-224-170BA), the introduced monocarboxylic acid regulator BA competes with TCPP for the coordination of zirconium oxygen clusters to slow down the nucleation rate and improve the crystallinity. In general, the acid regulator plays the following roles: guiding the framework to assemble into a pure phase MOFs structure with high crystallinity; controlling the size; and adjusting the defect level.

[0106] The morphologies of TCPP and PCN-224-90BA to PCN-224-170BA were characterized by SEM. Figure 2 As shown in the figure, TCPP presents an irregular shape, while PCN-224-90BA to PCN-224-170BA are spherical, indicating that the unique framework structure of MOFs breaks the aggregation state of TCPP. It can be further observed that the amount of regulator has a great influence on the size of MOFs crystals. With the increase of BA dosage, the particle size of defective porphyrin MOFs gradually increases. This is because BA is used as a regulator to participate in the competitive coordination of MOFs, which can be explained by (Equation 1) and (Equation 2), where K is the equilibrium constant. During the reaction, as the amount of BA increases, its coordination with the zirconium cluster causes small molecules to occupy the position of TCPP and generate additional ligand defects, which slows down the nucleation rate of defective porphyrin MOFs, and the number of nuclei gradually decreases, resulting in a gradual increase in the particle size of defective porphyrin MOFs, confirming the regulatory effect of BA on the size of MOFs during the crystallization process.

[0107]

[0108] UV-Vis spectra of TCPP and defective porphyrin MOFs Figure 3 As shown, the defective porphyrin MOFs show one Soret and four Q absorption bands of porphyrin, which are characteristic absorption bands of porphyrin MOFs, once again indicating the successful preparation of defective porphyrin MOFs. With the increase of particle size and scattering, the absorption peak around 420nm shows a slight red shift. This phenomenon can be explained by Mie theory, that is, when the size of the particle increases, the scattering of the incident light by the particle becomes more significant. Larger particles will cause the peak wavelength of the scattering spectrum to move toward the long-wave direction, and the degree of red shift is proportional to the particle size. Figure 4The PXRD of A observed the crystal structure of defective porphyrin MOFs. The diffraction peaks at 6.4°, 7.9°, 9.1°, 11.2° and 13.7° correspond to the characteristic diffraction crystal planes (022), (222), (004), (224) and (006) of defective porphyrin MOFs, respectively, which are consistent with the reported crystal structure of porphyrin MOFs, indicating that a series of defective porphyrin MOFs materials were successfully prepared. In addition, it can be seen that the position of the diffraction peak of the defective porphyrin MOFs remains basically unchanged with the change of the amount of BA added, indicating that although the exchange reaction between BA and TCPP can adjust the coordination balance to form coordination defects, it does not destroy the structure of the defective porphyrin MOFs, which is very beneficial for the subsequent study of the dispersion degree of TCPP molecules in the regular framework of MOFs. FT-IR of TCPP molecules before and after synthesis of MOFs Figure 4 As shown in B, compared with the TCPP ligand, the C-OH (1257 cm -1 ) and C=O(1656cm -1 ) group's asymmetric vibration absorption intensity is greatly weakened, and 663cm -1 The characteristic vibration peak of Zr-O bond appears at 4+ The coordination effect with the -COOH group in the TCPP ligand further verified the successful preparation of the defective porphyrin MOFs series of materials. The morphology and size distribution of the luminescent materials were further characterized by TEM. The five defective porphyrin MOFs prepared were all spherical ( Figure 5 ), and the particle size analysis statistics ( Figure 6 ), with sizes of 35.0±3.6nm, 56.0±8.5nm, 88.5±6.4nm, 211.0±25.4nm and 290.5±43.3nm, respectively. Figure 7 The elemental composition of PCN-224-130BA was analyzed by TEM-EDS. The TEM-EDS image showed that C, N, O, and Zr elements coexisted and were evenly distributed in the defective porphyrin MOFs. The TEM-EDS image of PCN-224-130BA was consistent with its XPS full spectrum.

[0109] Taking PCN-224-130BA with the best ECL performance as an example, the surface composition and bonding structure were characterized by XPS spectroscopy, and the binding energy data were calibrated using the C1s peak (284.8 eV). Figure 8 B) has two characteristic peaks at 185.2eV and 182.8eV, corresponding to Zr 4+ 3d 3 / 2 and Zr 4+ 3d 5 / 2, the characteristic peaks of N 1s are located at 400.1 eV and 397.6 eV, corresponding to C-N-C and C=N-C( Figure 8 C), the O 1s spectrum shows absorption peaks at 533.6 eV, 531.9 eV and 530.3 eV, which are attributed to O-H, C=O and Zr-O bonds respectively( Figure 8 D), from which Zr 4+ and the carboxyl group of TCPP coordinate and bond to form PCN-224-130BA in the form of Zr-O bonds. The above analysis results jointly verify the successful preparation of defective porphyrin MOFs.

[0110] BET and TGA characterizations were used together to study the ligand defect degree in defective porphyrin MOFs. When BA replaces TCPP, additional porphine ring defects will occur in the MOFs, resulting in higher pore size, pore volume and specific surface area. Adding more regulators during the synthesis makes the N2 adsorption capacity( Figure 10 ) and pore size( Figure 11 ) of defective porphyrin MOFs gradually increase. And with the increase of the amount of BA, the BET specific surface area of defective porphyrin MOFs increases from 837.06 m 2 g -1 to 1204.78 m 2 g -1 gradually, and its pore volume increases from 1.06 cm 3 g -1 to 1.54 cm 3 g -1 , indicating that the ligand defect degree in defective porphyrin MOFs increases in turn (Table 1).

[0111] Table 1 BET specific surface area and pore volume of defective porphyrin MOFs

[0112]

[0113] To further study the defects of the defective porphyrin MOF framework, thermogravimetric analysis (TGA) was carried out on the prepared defective porphyrin MOFs, as Figure 9As shown, the trends of these five curves are similar, and there are two obvious weight loss steps (the final weight is normalized to 100%). The first weight loss below 160 °C corresponds to the evaporation of water, DMF, and acetone. In the temperature range of 160 - 430 °C, the weight loss is not obvious. The temperature for the destruction of organic bonds is above 430 °C. With the loss of ligands, the framework structure begins to collapse, indicating that the defective porphyrin MOFs have good thermal stability and can be used as signal probes for immunosensors. The second weight loss step can measure the degree of defect of the defective porphyrin MOFs. The increase in the content of BA in the framework is inversely proportional to the mass loss of the MOFs and the content of TCPP in the framework, and is directly proportional to the degree of ligand defect. The change trend of the TGA curve is consistent with the specific surface area, indicating that with the increase in the addition amount of BA, the specific surface area, the degree of ligand defect, and the dispersion degree of TCPP in the framework increase accordingly.

[0114] The ECL performance of TCPP and defective porphyrin MOFs was investigated by CV and ECL-time curves. Compared with the reduction peak of TCPP (black line) at about -1.0 V, the peak position of the defective porphyrin MOFs shifted positively from -1.0 V to about -0.84 V, indicating an enhanced electrocatalytic effect on the reduction of the coreactant S2O8 2- ( Figure 12 A), that is, S2O8 2- is more easily electrochemically reduced to form the active intermediate SO4 ·- , accelerating the electron transfer rate and thus enhancing the ECL intensity. The ECL intensity of the best-performing PCN-224-130BA is 5.34 times that of TCPP Figure 12 (

[0115]

[0116] B), indicating that the synthesis of defective porphyrin MOFs with TCPP as the ligand overcomes the ACQ effect. The ECL efficiency of the defective porphyrin MOFs system was further calculated. The ECL efficiency is defined as the number of photons per electron transfer. I and i represent the ECL intensity and current value respectively. The calculation formula for the ECL efficiency is as follows: 2- The relative ECL efficiencies of the defective porphyrin MOFs are 4.23, 5.59, 6.23, 4.45, and 3.19 times that of TCPP respectively. In summary, PCN-224-130BA with an appropriate degree of ligand defect exhibits the best ECL performance because the increased specific surface area and active sites in the defective porphyrin MOFs enhance the interaction between TCPP and S2O8 2-Diffusion in the framework is slow, and the content of the luminescent ligand TCPP in the framework continuously decreases, and the ECL signal shows a decreasing trend. Therefore, as the content of BA increases, the ECL intensity and efficiency show a trend of first increasing and then decreasing, which is the result of the synergistic effect of particle size, ligand defects, and TCPP dispersion degree.

[0117] (II) Electrochemiluminescence / Electrochemical Performance Test

[0118] Experimental procedure: The quantitative detection of Aβ was achieved by using the ECL analysis method. 1-42 The ECL test parameters were as follows: the potential range was -1.3 to 0 V, the scanning rate was 0.1 V s -1 , and the voltage of the photomultiplier tube (PMT) was 650 V. The ECL test was carried out in a 4 mL PBS (0.1 M, pH 7.4) reaction bottom solution containing 50 mmol L - 1 K2S2O8. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to characterize the step-by-step assembly process of the defective porphyrin MOFs electrochemiluminescence immunosensor. The CV characterization parameters for the immunosensor assembly process were as follows: the scanning rate was 0.1 V s -1 , and the potential range was -0.2 to 0.6 V; the EIS characterization parameters were as follows: it was carried out in the frequency range of 1 Hz to 100 KHz, and the alternating current voltage was 5 mV.

[0119] (1) Optimization and Stability Test of Materials

[0120] The ECL intensity and stability of the luminescent body are crucial for the construction of the sensor. In order to achieve the best detection performance, the concentration of the luminescent body was optimized. As Figure 13 shown, when the concentration of PCN-224-130BA was in the range of 0.6 - 1.2 mg mL -1 , the ECL intensity gradually increased with the increase of the concentration. When the concentration was greater than 1.2 mg mL -1 , the ECL intensity gradually decreased with the increase of the concentration. This is because an excessive amount of the luminescent body will hinder the electron transfer on the electrode surface, resulting in a decrease in the ECL intensity. In addition to the ECL intensity, its stability is also important for the preparation of the immunosensor. Therefore, under the optimal luminescent body concentration conditions, the ECL stability of the PCN-224-130BA modified electrode was studied. As Figure 14 shown, after continuous scanning for 500 s in the co-reactant bottom solution, the ECL signal remained stable, and the relative standard deviation (RSD) was 0.96%, indicating that the system has good ECL stability and reproducibility.

[0121] (2) ECL Response Mechanism

[0122] To explore the possible ECL mechanism of the PCN-224-130BA / S2O8 2- system, the CV and ECL curves were synchronously measured in the potential range of -1.3 to -0 V. In PBS, there were no obvious redox peaks on the bare electrode in PBS ( Figure 15 , curve a), and an obvious reduction peak at -0.90 V appeared in the 50 mM K2S2O8 solution, which was related to the reduction of S2O8 2- on the electrode surface ( Figure 15 , curve b), and the bare electrode had almost no ECL signal in PBS without K2S2O8 ( Figure 16 , curve a) and in PBS containing K2S2O8 ( Figure 16 , curve b). When testing the PCN-224-130BA-modified electrode in the PBS buffer solution containing K2S2O8, the reduction peak of the CV curve shifted positively to -0.84 V. Compared with the reduction peak potential of -0.90 V of K2S2O8 in PBS, a positive shift of 0.06 V occurred, promoting the reduction of S2O8 2- , and the peak current also increased significantly ( Figure 15 , curve c), corresponding to a strong ECL signal ( Figure 16 , curve c). The test results of CV and ECL showed that PCN-224-130BA could be used as a co-reaction promoter to promote the generation of more SO4 ·- in the system, and the increase in peak current indicated that PCN-224-130BA improved the electron transfer rate in the reaction system.

[0123] Through the above tests and referring to relevant literature, the ECL mechanism of the PCN-224-130BA / S2O8 2- system is summarized as follows:

[0124] PCN-224-130BA + e - → PCN-224-130BA ·- (Equation 4)

[0125] PCN-224-130BA ·- + S2O8 2- → PCN-224-130BA + SO4 2- + SO4 ·- (Equation 5)

[0126] S2O8 2- + e - → SO4 2- + SO4 ·- (Equation 6)

[0127] PCN-224-130BA·- +SO4 ·- →PCN-224-130BA * +SO4 2- (Formula 7)

[0128] PCN-224-130BA * →PCN-224-130BA+hv (Formula 8)

[0129] (3) Optimization of experimental conditions

[0130] PCN-224-130BA with the best ECL performance was selected as the luminophore to construct a defective porphyrin MOFs electrochemiluminescence immunosensor. In order to achieve the best detection performance, the incubation time of the PCN-224-130BA-Ab2 complex was optimized. As Figure 17 shown, with the increase of the incubation time of the complex, the ECL intensity first increased and then decreased with the increase of the incubation time. When the incubation time was 120 min, the ECL signal reached the highest value. This may be because too much protein was loaded on the electrode surface, hindering electron transfer. Therefore, the optimal incubation time of the PCN-224-130BA-Ab2 complex was 120 min.

[0131] (4) Characterization of the construction process of the defective porphyrin MOFs electrochemiluminescence immunosensor

[0132] To verify the successful construction of this immunosensor, the assembly process of the defective porphyrin MOFs electrochemiluminescence immunosensor was characterized step by step. As Figure 18 shown, a pair of obvious redox peaks were observed on the CV curve of the bare electrode (curve a). Due to the good conductivity of Au NPs, which can effectively promote electron transfer, the peak current of DpAu / GCE increased significantly (curve b). When Ab1 was incubated, the peak current value decreased significantly (curve c), because the antibody hindered electron transfer. Subsequently, the incubation of BSA blocked the remaining active sites, further hindering electron transfer and the peak current decreased further (curve d). After incubating Aβ 1-42 , due to the steric hindrance effect of the antibody-antigen complex, which hindered electron transfer, the peak current continued to decrease (curve e). After incubating PCN-224-130BA-Ab2, due to its steric hindrance effect, the peak current decreased further (curve f), which preliminarily proved the successful construction of this defective porphyrin MOFs electrochemiluminescence immunosensor.

[0133] EIS is another characterization technique for characterizing the changes in the electrode interface characteristics. The semicircle diameter of this curve is related to the electron transfer resistance (R et ). As Figure 19As shown, compared with the electron transfer resistance of the smaller bare GCE (curve a), due to the good conductivity of AuNPs, the EIS of DepAu / GCE is almost a straight line (curve b). This is because AuNPs have excellent conductivity, which promotes the 3- electron transfer of the [Fe(CN)6] 4- / [Fe(CN)6] 1-42 redox electron pair. After gradually modifying Ab1 (curve c), BSA (curve d), Aβ et (curve e) and PCN-224-130BA-Ab2 complex (curve f), due to the influence of steric hindrance, the

[0134] R value gradually increases because the resistance of non-conductive substances slows down the electron transfer rate of the redox electron pair on the electrode surface. Therefore, the EIS results further demonstrate the successful preparation of this immunosensor. 1-42 Analysis performance of the defective porphyrin MOFs electrochemiluminescence immunosensor for Aβ

[0135] Under the optimal experimental conditions, a series of quantitative detections of Aβ 1-42 concentrations were carried out to evaluate the analysis performance of the immunosensor for the target. As Figure 20 shown, as the concentration of Aβ 1-42 increases, the ECL intensity gradually increases. When the target concentration increases from 5 fg mL -1 to 1 ng mL -1 (curves a - g), the ECL intensity shows a good positive correlation with the logarithm (lg c) of the Aβ 1-42 concentration ( Figure 21 ), and the regression equation is I ECL = 695.321 lg c + 4693.70, with a correlation coefficient of 0.9993. The detection limit calculated according to the 3σ / k rule is 3.89 fg mL -1 . In addition, Table 2 compares this sensor with other sensors in the literature, and the results show that this sensor has a lower detection limit for the detection of Aβ 1-42 , which can be attributed to the excellent ECL performance of PCN-224-130BA.

[0136] Table 2 Comparison of different Aβ 1-42 detection methods

[0137]

[0138] Reference 1: Sheng M., Yu L., Peng Y., et al. Combination of ternary electrochemiluminescence system of BNQDs / AgMOG-K2S2O8 and electrochemiluminescence resonance energy transfer strategy for ultrasensitive immunoassay of amyloid-β Protein[J]. Analytical Chemistry, 2023, 96(1): 41-48.

[0139] Reference 2: Yang J., Qin D., Wang N., et al. Aggregation-induced electrochemiluminescence based on a zinc-based metal-organic framework and a double quencher Au@UiO-66-NH2 for the sensitive detection of amyloid β42 via resonance energy transfer[J]. Analytical Chemistry, 2023, 95(17): 7045-7052.

[0140] Reference 3: Wang Y., Yang M., Wang X., et al. Construction of built-in correction photoelectrochemical sensing platform for diagnosis of Alzheimer's disease[J]. Biosensors and Bioelectronics, 2024, 249: 116020.

[0141] Reference 4: Huang Z., Li M., Zhang L., et al. Electrochemical immunosensor based on superwettable microdroplet array for detecting multiple Alzheimer’s disease biomarkers[J]. Frontiers in Bioengineering and Biotechnology, 2022, 10: 1029428.

[0142] Reference 5: Abbasi H.Y., Tehrani Z., Devadoss A., et al. Graphene based electrochemical immunosensor for the ultra-sensitive label free detection of Alzheimer's beta amyloid peptides Aβ(1-42)[J]. Nanoscale Advances, 2021, 3(8): 2295-2304.

[0143] (6) Selectivity, stability, and reproducibility of the defective porphyrin MOFs electrochemiluminescence immunosensor

[0144] To evaluate the selectivity of the defective porphyrin MOFs electrochemiluminescence immunosensor, human serum albumin (HSA), BSA, immunoglobulin (IgG), and procalcitonin (PCT) with a concentration of 100 pg mL -1 were selected as interferents for detection. As Figure 22 the results showed that the ECL responses of the interferents were similar to those of the blank samples, while the mixed solution (including 100 pg mL -1 of the above interferents and 10 pg mL -1 of Aβ 1-42 ) showed an ECL response value similar to that of the target Aβ 1-42 (10 pg mL -1 ), indicating that the influence of the interferents on the detection of Aβ 1-42 could be ignored. The above results indicated that the prepared immunosensor had good selectivity for Aβ 1-42It has excellent selectivity. The stability of the immunosensor was evaluated, and the results showed that the ECL signal remained stable after continuous scanning for 500 s, with an RSD of 0.50%, indicating that the sensor has excellent stability. Subsequently, a batch of prepared sensors was stored long-term at 4 °C, and the above electrodes were taken out every 2 - 4 days to test and record the ECL signal. The results showed that after 15 days, the ECL signal only decreased by 10.3% without obvious fluctuations, indicating that the sensor has excellent storage stability. In addition, the reproducibility of the immunosensor is also important for the quantitative detection of the target. The RSDs of intra-assays and inter-assays were 0.42% and 0.70% respectively, indicating that the sensor has good reproducibility. In summary, the immunosensor constructed in this work has excellent selectivity, stability, and reproducibility in the detection of Aβ 1-42 with excellent selectivity, stability, and reproducibility.

[0145] (7) Analysis of actual samples

[0146] To verify the applicability of the prepared immunosensor in actual samples, different concentrations of Aβ 1-42 (1.00, 10.0, and 100 pg mL -1 ) were added to human serum samples diluted 50-fold (obtained from the University Hospital of Northwest University) and ECL tests were carried out. As shown in Table 3, the spiked recoveries were 94.8% - 103% and the RSDs were 3.48% - 4.97%. The results showed that the sensor is suitable for the quantitative detection of Aβ 1-42 in biological samples. Table 3 Results of the spiked recovery experiment of the defective porphyrin MOFs electrochemiluminescence immunosensor in human serum samples

[0147]

[0148] The present invention adopts a regulator-assisted strategy to introduce different degrees of ligand defects into porphyrin MOFs to construct porphyrin MOFs with defective structures, and explores the relationship between different defect degrees and the material structure and ECL performance. The successful introduction of different ligand defect degrees was jointly demonstrated by BET and TGA characterizations. The results showed that with the increase in the amount of BA added, the size, specific surface area, ligand defect degree, and the dispersion degree of TCPP in the MOF framework of the defective porphyrin MOFs increased, and the ECL signal of the defective porphyrin MOFs showed a trend of first increasing and then decreasing. This is because with the increase in the ligand defect degree, the increased active sites and specific surface area enhanced the interaction between TCPP and S2O8 2- , accelerated the electron transfer rate, and the ECL intensity increased accordingly; with the addition of excessive BA, the too large crystal size would delay S2O8 2-The free time in the framework and the decrease in the content of the luminescent ligand TCPP in the framework jointly lead to the decrease in the ECL intensity of the defective porphyrin MOFs. The ECL intensity and ECL efficiency of the PCN-224-130BA with the best performance are 5.34 times and 6.23 times that of TCPP, respectively. An immunosensor constructed with it as a signal probe was used for the quantitative detection of Aβ 1-42 with a detection limit as low as 3.89 fg mL -1 . This work provides a new idea for the application of regulating ligand defects in the field of ECL sensing and expands the application scope of defective MOFs in the sensing field.

[0149] After considering the specification and the content disclosed herein, those skilled in the art will readily conceive of other embodiments of the present application. The present application is intended to cover any variations, uses, or adaptations of the present application, which follow the general principles of the present application and include the common general knowledge or conventional technical means in the technical field not disclosed in the present application. The specification and examples are only regarded as exemplary, and the present application is not limited to the precise structures described above and shown in the drawings, and various modifications and changes can be made without departing from its scope.

Claims

1. A preparation method of an electrochemiluminescence immunosensor based on defective porphyrin MOFs, characterized in that, It includes the following steps: (1) Preparation of defective porphyrin MOFs Tetracarboxyphenyl porphyrin, ZrCl4 and benzoic acid are dispersed in DMF solvent to obtain a dispersion liquid, and then the dispersion liquid is transferred to a high-pressure reactor for hydrothermal reaction. After the reaction ends, the product is washed and dried to obtain defective porphyrin MOFs; (2) Preparation of defective porphyrin MOFs-Ab2 complex The defective porphyrin MOFs are dispersed in ultrapure water to obtain a dispersion liquid. The dispersion liquid is added to a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, and slowly stirred at 4-5 °C in the dark for 5-6 h to fully activate the carboxyl groups of the defective porphyrin MOFs. β-amyloid protein secondary antibody Ab2 is dispersed in the activated defective porphyrin MOFs, and slowly stirred at 4-5 °C in the dark for 10-14 h to obtain a defective porphyrin MOFs-Ab2 complex. The product is centrifuged and washed and then dispersed in ultrapure water to obtain a defective porphyrin MOFs-Ab2 complex dispersion liquid, which is stored at 4-5 °C for subsequent use; (3) Preparation of an electrochemiluminescence immunosensor based on defective porphyrin MOFs (3.1) The polished and dried glassy carbon electrode GCE is immersed in HAuCl4 solution for electrochemical deposition to obtain an AuNPs-modified electrode DpAu / GCE; (3.2) β-amyloid protein primary antibody Ab1 is immobilized on the electrode surface through Au-NH2 bond to obtain an electrode Ab1 / DpAu / GCE; (3.3) The blocking agent bovine serum albumin BSA is incubated on the electrode surface to block non-specific binding sites to obtain an electrode BSA / Ab1 / DpAu / GCE; (3.4) Incubate β-amyloid Aβ with different concentrations of 5 fg mL -1 ~1 ng mL -1 onto the surface of the BSA / Ab1 / DpAu / GCE modified electrode to obtain the electrode Aβ 1-42 / BSA / Ab1 / DpAu / GCE; 1-42 ​ (3.5) Drop the defective porphyrin MOFs-Ab2 complex dispersion obtained in step (2) onto the Aβ 1-42 / BSA / Ab1 / DpAu / GCE modified electrode, incubate at room temperature to form a sandwich structure of defective porphyrin MOFs-Ab2 / Aβ 1-42 / BSA / Ab1 / DpAu / GCE, that is, a defective porphyrin MOFs-based electrochemiluminescence immunosensor is obtained.

2. The preparation method of the electrochemiluminescence immunosensor based on defective porphyrin MOFs according to claim 1, characterized in that: In step (1), the mass ratio of the tetracarboxyphenyl porphyrin, ZrCl4, and benzoic acid is 5:15:(90-170).

3. The preparation method of the electrochemiluminescence immunosensor based on defective porphyrin MOFs according to claim 1, characterized in that: In step (1), the temperature of the hydrothermal reaction is 110-130 °C, and the reaction time is 22-26 h.

4. The preparation method of the electrochemiluminescence immunosensor based on defective porphyrin MOFs according to claim 1, characterized in that: In step (2), the concentration of the dispersion obtained by dispersing the defective porphyrin MOFs in ultrapure water is 0.6 - 1.6 mg mL -1 , and the volume ratio of the dispersion to the mixed solution is 5:

1.

5. The preparation method of the electrochemiluminescence immunosensor based on defective porphyrin MOFs according to claim 1, characterized in that: In step (2), the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide in the mixed solution is 40 mmol / L -1 , and the concentration of N-hydroxysuccinimide is 10 mmol / L -1 .

6. The preparation method of the electrochemiluminescence immunosensor based on defective porphyrin MOFs according to claim 1, wherein: In step (3.1), the mass percentage of the HAuCl4 solution is 1%; the potential of the electrochemical deposition is -0.2 V, and the deposition time is 30 s; in step (3.2), the dosage of the β-amyloid protein primary antibody Ab1 is 10 μL; the immobilization time is 12-16 h.

7. The preparation method of the electrochemiluminescence immunosensor based on defective porphyrin MOFs according to claim 1, characterized in that: In step (3.3), the mass percentage of the blocking agent bovine serum albumin BSA is 0.25%; the dosage of the blocking agent bovine serum albumin BSA is 10 μL; the incubation time is 25-35 min.

8. The preparation method of the electrochemiluminescence immunosensor based on defective porphyrin MOFs according to claim 1, characterized in that: In step (3.4), the dosages of amyloid-β Aβ at different concentrations are 10 μL respectively; the incubation time is 1 h; in step (3.5), the dosage of the defective porphyrin MOFs-Ab2 complex dispersion is 10 μL; the incubation time is 2 h. 1-42 ​ 9. An electrochemiluminescence immunosensor based on defective porphyrin MOFs prepared by the method according to claim 1.

10. Use of the electrochemical luminescence immunosensor based on defective porphyrin MOFs according to claim 9 in the detection of β-amyloid Aβ 1-42 in.

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