Construction and application of bifunctional biosensor based on coordination induced enhancement and interface antifouling

Abstract

The invention discloses construction and application of a bifunctional biosensor based on coordination induced enhancement and interface antifouling, and belongs to the technical fields of light / electrochemistry, biochemical analysis and nano science. According to the invention, tris (4, 4 '-dicarboxylic acid-2, 2'-dipyridyl) ruthenium is used as a ligand, and Zn / Ru-MOF with a coordination induction enhancement effect is prepared and is used as a luminous body. Due to effective aggregation of the ligand and energy transfer from Zn < 2 + > to the ligand, Zn / Ru-MOF has high ECL efficiency. Besides, Zn / Ru-MOF is modified with 3-aminopropyl dimethylamine zwitterions to serve as a sensing substrate, so that interface adsorption of interfering substances is resisted, and integration of self-enhanced luminescence and interface antifouling is realized. Based on pairing of a complementary chain and an aptamer chain, a significant "signal-off" is achieved through resonance energy transfer. The quenching probe is released according to the competitive mechanism of the target, resulting in signal recovery. Based on this, the constructed biosensor shows a low detection limit, and shows a huge application potential in clinical monitoring of alpha fetoprotein in a complex serum medium.

Description

Technical Field

[0001] This invention discloses the construction and application of a bifunctional biosensor based on coordination-induced enhancement and interface antifouling, belonging to the fields of photo / electrochemistry, biochemical analysis, and nanoscience and technology. Background Technology

[0002] As a common serum biomarker, alpha-fetoprotein (AFP) plays a crucial indicative role in the clinical diagnosis of primary liver cancer. Therefore, designing a high-performance analytical platform for trace monitoring of AFP in serum is essential. Among these methods, electrochemiluminescence (ECL) is suitable for sensitive analysis of low-level biological targets in serum due to its high signal-to-noise ratio, wide dynamic range, and good compatibility with automated systems.

[0003] In serum samples, the non-specific adsorption of interfering biomolecules at the sensing interface significantly impairs the detection sensitivity, reliability, and lifespan of biosensors. Developing highly efficient antifouling materials can address these issues. Due to the electroneutrality and hydrophilicity of zwitterionic materials, the charge adsorption and hydrophobic adsorption of interfering substances are effectively suppressed. Based on this, this invention prepares 3-aminopropyldimethylamine (pTAO) as an antifouling zwitterionic material for interface modification.

[0004] The development of highly efficient luminescent materials is crucial for improving the detection sensitivity of ECL biosensors. Polypyridine ruthenium complexes, especially tris(2,2'-bipyridine)ruthenium (Ru(bpy)3), are particularly important. 2+ Ru(bpy)3 is widely chosen as a luminescent material due to its excellent and rapid luminescence properties. In aqueous systems, Ru(bpy)3... 2+ The ECL efficiency of Ru(bpy)3 is limited due to its high solubility. Therefore, achieving the desired ECL efficiency is crucial. 2+ Effective fixation is particularly important. Metal-organic frameworks possess high specific surface area, ordered pores, and tunable internal structures, making them ideal for Ru(bpy)3. 2+ It provides ample loading space and a uniform dispersion environment. Based on this, tris(4,4'-dicarboxylic acid-2,2'-bipyridine)ruthenium (Ru(dcbpy)3) 2+ Ru(bpy)3 was selected as the luminescent ligand for self-assembly, thus enabling the self-assembly of Ru(bpy)3. 2+ Strong anchoring and effective enrichment.

[0005] Based on the above background, this invention constructs a bifunctional biosensor combining coordination-induced enhancement and interfacial antifouling for trace detection of AFP in serum. Firstly, due to Ru(dcbpy)3 2+ Zn aggregation and coordination 2+ To Ru(dcbpy)3 2+Through energy transfer, Zn / Ru-MOF exhibits strong and stable ECL emission. Simultaneously, pTAO zwitterions were designed and attached to Zn / Ru-MOF, achieving the integration of luminescent and antifouling components. The developed Zn / Ru-MOF incorporating pTAO zwitterions was used as a sensing substrate, achieving a strong "signal-on" state and effectively resisting interfacial adsorption of non-specific biomolecules. Due to resonant energy transfer and complementary base pairing, the ECL signal of Zn / Ru-MOF was quenched by Bi2S3@Au NPs. With the introduction of AFP and competition with the complementary chain, the quenching probe detached, and the ECL signal recovered. Based on this, the constructed biosensor exhibited a wide linear range and a low detection limit, indicating its suitability for trace detection of AFP in complex serum environments. Summary of the Invention

[0006] One of the technical tasks of this invention is to overcome the shortcomings of the prior art by preparing a Zn / Ru-MOF with pTAO zwitterions that integrates self-enhanced luminescence and interface antifouling functions for ECL analysis. The second technical objective of this invention is to construct a bifunctional biosensor based on coordination-induced enhancement and interfacial antifouling by using the prepared Zn / Ru-MOF with pTAO zwitterionic bonds as the sensing substrate and the Bi2S3@Au NPs-aptamer chain as the quenching probe. The process is simple and the operation is safe. The third technical objective of this invention is to provide the application of a dual-function biosensor based on coordination-induced enhancement and interface antifouling constructed by the aforementioned construction method, namely, for trace detection of AFP in the serum environment, which has certain industrialization prospects.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: 1. Construction of a bifunctional biosensor based on coordination-induced enhancement and interfacial antifouling A glassy carbon electrode with a diameter of 4 mm was polished with Al2O3 powder; 6-10 µL of Zn / Ru-MOF bound with pTAO was dropped onto the treated electrode surface; 8 µL of complementary chain solution of AFP aptamer was added dropwise, and the electrode was incubated at 4 °C for 2 h; 8 μL of Bi2S3@Au NPs-AFP aptamer chain solution was added dropwise, and the electrode was incubated under the same conditions for 2 h; the modified electrode was incubated in AFP solutions of different concentrations for 1 h, thus constructing a bifunctional biosensor based on coordination-induced enhancement and interfacial antifouling. The Zn / Ru-MOF containing pTAO zwitterions is prepared by combining 18 mg of Zn(NO3)2·6H2O and Ru(dcbpy)3 2+40 mg of polyvinylpyrrolidone and 3 mg of pyrazine were dispersed in 64 mL of ultrapure water; the resulting solution was sonicated and then reacted at 80 °C for 24 h; after cooling, the resulting orange-red solution was centrifuged, washed three times with ultrapure water, and vacuum dried at 60 °C for 12 h to obtain Zn / Ru-MOF; 10 g of 3-dimethylaminopropylamine was added to 100 mL of ultrapure water under stirring, and the resulting solution was placed in an oxygen atmosphere and reacted at 60 °C for 6 h, during which 15 mL of H2O2 was added dropwise; the resulting mixed solution was rotary evaporated at 80 °C for 2 h to generate pTAO zwitterions; 1 mL of a mixed solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide was added to 5 mL of the prepared Zn / Ru-MOF solution to activate its carboxyl group; after the reaction was completed, 100 µL of pTAO zwitterions was added, and the reaction was continued for 12 h. h, to obtain Zn / Ru-MOF bound to pTAO zwitterions; The Bi2S3@Au NPs-AFP aptamer chain is prepared by dispersing 1.82 g of Bi(NO3)3·5H2O in 25 mL of ethylene glycol and degassing with nitrogen for 20 min to obtain solution A; dissolving 1.35 g of Na2S in a mixed solution containing 20 mL of ultrapure water and 10 mL of ethylene glycol to obtain solution B; adding solution B to solution A under continuous stirring, followed by adding 20 mL of urea; placing the resulting solution in a polytetrafluoroethylene-lined autoclave and reacting at 180 °C for 12 h; after the reaction, centrifuging the solution, washing the resulting precipitate three times with ethanol, and vacuum drying at 60 °C for 12 h to obtain Bi2S3; dissolving 50 mg of the prepared Bi2S3 in 10 mL of ultrapure water, then adding 5 mg of polyvinylpyrrolidone; stirring for 40 min, adding 4 mL of sodium citrate and 3 mL of HAuCl4, and reacting at 80 °C for 10 min. h; The obtained product was centrifuged, washed, and vacuum dried to obtain Bi2S3@Au NPs; 200 µL of Bi2S3@Au NPs with a concentration of 2 mg / mL was mixed with 40 µL of AFP aptamer chain with a concentration of 100 µM, and then 200 µL of NaCl solution with a concentration of 2 M was added; The obtained product was centrifuged to remove unbound AFP aptamer chain, and washed 3 times with phosphate buffer solution to obtain Bi2S3@Au NPs-AFP aptamer chain; The AFP solutions of different concentrations were prepared by uniformly dispersing AFP antigen at concentrations of 10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, and 100 ng / mL in a phosphate buffer solution with a pH of 7.4.

[0008] 2. The application of the bifunctional biosensor based on coordination-induced enhancement and interfacial antifouling constructed by the aforementioned method, characterized in that it is used for AFP detection in complex serum environments. A three-electrode system was constructed for ECL testing: a bifunctional biosensor based on coordination-induced enhancement and interfacial antifouling was used as the working electrode, an Ag / AgCl electrode as the reference electrode, and a platinum electrode as the auxiliary electrode; a phosphate buffer solution containing 50–100 mM K₂S₂O₈ at pH 7.4 was used as the test solution; the applied voltage range was 1.6–0 V, with a high voltage of 500 V; based on the measured signals, a working curve was plotted, revealing that the developed biosensor has a detection range of 10 fg / mL–100 ng / mL and a detection limit of 4.7 fg / mL, exhibiting high stability, selectivity, and reproducibility, making it suitable for trace detection of AFP in complex serum environments.

[0009] The beneficial technical effects of the present invention are as follows: 1. This invention prepares a Zn / Ru-MOF with coordination-induced enhancement properties as the luminescent component and pTAO zwitterion as the antifouling component, and integrates the two as a bifunctional sensing substrate, which effectively improves the sensitivity and accuracy of the biosensor and simplifies its construction process. 2. This invention constructs a bifunctional biosensor based on coordination-induced enhancement and interface antifouling. Through the resonant energy transfer effect and the competition between the complementary chains of AFP antigen and AFP aptamer, the ECL signal of the biosensor decreases linearly with the increase of AFP antigen concentration, thereby achieving sensitive detection of AFP. 3. The bifunctional biosensor based on coordination-induced enhancement and interface antifouling constructed in this invention exhibits a wide linear range and low detection limit for the target AFP, as well as high stability, selectivity and reproducibility. It is suitable for trace detection of AFP in complex serum environments and has certain industrialization prospects. Detailed Implementation

[0010] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the scope of protection of the present invention is not limited thereto. Any changes made to the technical solutions of the present invention by those skilled in the art should fall within the scope of protection of the present invention.

[0011] Example 1: Construction of a bifunctional biosensor based on coordination-induced enhancement and interfacial antifouling. A glassy carbon electrode with a diameter of 4 mm was polished with Al2O3 powder; 6 µL of Zn / Ru-MOF bound with pTAO was dropped onto the treated electrode surface; 8 µL of complementary chain solution of AFP aptamer was added dropwise, and the electrode was incubated at 4 °C for 2 h; 8 μL of Bi2S3@Au NPs-AFP aptamer chain solution was added dropwise, and the electrode was incubated under the same conditions for 2 h; the modified electrode was incubated in AFP solutions of different concentrations for 1 h, thus constructing a bifunctional biosensor based on coordination-induced enhancement and interfacial antifouling. The Zn / Ru-MOF containing pTAO zwitterions is prepared by combining 18 mg of Zn(NO3)2·6H2O and Ru(dcbpy)3 2+ 40 mg of polyvinylpyrrolidone and 3 mg of pyrazine were dispersed in 64 mL of ultrapure water; the resulting solution was sonicated and then reacted at 80 °C for 24 h; after cooling, the resulting orange-red solution was centrifuged, washed three times with ultrapure water, and vacuum dried at 60 °C for 12 h to obtain Zn / Ru-MOF; 10 g of 3-dimethylaminopropylamine was added to 100 mL of ultrapure water under stirring, and the resulting solution was placed in an oxygen atmosphere and reacted at 60 °C for 6 h, during which 15 mL of H2O2 was added dropwise; the resulting mixed solution was rotary evaporated at 80 °C for 2 h to generate pTAO zwitterions; 1 mL of a mixed solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide was added to 5 mL of the prepared Zn / Ru-MOF solution to activate its carboxyl group; after the reaction was completed, 100 µL of pTAO zwitterions was added, and the reaction was continued for 12 h. h, to obtain Zn / Ru-MOF bound to pTAO zwitterions; The Bi2S3@Au NPs-AFP aptamer chain is prepared by dispersing 1.82 g of Bi(NO3)3·5H2O in 25 mL of ethylene glycol and degassing with nitrogen for 20 min to obtain solution A; dissolving 1.35 g of Na2S in a mixed solution containing 20 mL of ultrapure water and 10 mL of ethylene glycol to obtain solution B; adding solution B to solution A under continuous stirring, followed by adding 20 mL of urea; placing the resulting solution in a polytetrafluoroethylene-lined autoclave and reacting at 180 °C for 12 h; after the reaction, centrifuging the solution, washing the resulting precipitate three times with ethanol, and vacuum drying at 60 °C for 12 h to obtain Bi2S3; dissolving 50 mg of the prepared Bi2S3 in 10 mL of ultrapure water, then adding 5 mg of polyvinylpyrrolidone; stirring for 40 min, adding 4 mL of sodium citrate and 3 mL of HAuCl4, and reacting at 80 °C for 10 min. h; The obtained product was centrifuged, washed, and vacuum dried to obtain Bi2S3@Au NPs; 200 µL of Bi2S3@Au NPs with a concentration of 2 mg / mL was mixed with 40 µL of AFP aptamer chain with a concentration of 100 µM, and then 200 µL of NaCl solution with a concentration of 2 M was added; The obtained product was centrifuged to remove unbound AFP aptamer chain, and washed 3 times with phosphate buffer solution to obtain Bi2S3@Au NPs-AFP aptamer chain; The AFP solutions of different concentrations were prepared by uniformly dispersing AFP antigen at concentrations of 10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, and 100 ng / mL in a phosphate buffer solution with a pH of 7.4.

[0012] Example 2: Construction of a bifunctional biosensor based on coordination-induced enhancement and interfacial antifouling. A glassy carbon electrode with a diameter of 4 mm was polished with Al2O3 powder; 8 µL of Zn / Ru-MOF bound with pTAO was dropped onto the treated electrode surface; 8 µL of complementary chain solution of AFP aptamer was added dropwise, and the electrode was incubated at 4 °C for 2 h; 8 μL of Bi2S3@Au NPs-AFP aptamer chain solution was added dropwise, and the electrode was incubated under the same conditions for 2 h; the modified electrode was incubated in AFP solutions of different concentrations for 1 h, thus constructing a bifunctional biosensor based on coordination-induced enhancement and interfacial antifouling. The Zn / Ru-MOF containing pTAO zwitterions is prepared by combining 18 mg of Zn(NO3)2·6H2O and Ru(dcbpy)3 2+40 mg of polyvinylpyrrolidone and 3 mg of pyrazine were dispersed in 64 mL of ultrapure water; the resulting solution was sonicated and then reacted at 80 °C for 24 h; after cooling, the resulting orange-red solution was centrifuged, washed three times with ultrapure water, and vacuum dried at 60 °C for 12 h to obtain Zn / Ru-MOF; 10 g of 3-dimethylaminopropylamine was added to 100 mL of ultrapure water under stirring, and the resulting solution was placed in an oxygen atmosphere and reacted at 60 °C for 6 h, during which 15 mL of H2O2 was added dropwise; the resulting mixed solution was rotary evaporated at 80 °C for 2 h to generate pTAO zwitterions; 1 mL of a mixed solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide was added to 5 mL of the prepared Zn / Ru-MOF solution to activate its carboxyl group; after the reaction was completed, 100 µL of pTAO zwitterions was added, and the reaction was continued for 12 h. h, to obtain Zn / Ru-MOF bound to pTAO zwitterions; The Bi2S3@Au NPs-AFP aptamer chain is prepared by dispersing 1.82 g of Bi(NO3)3·5H2O in 25 mL of ethylene glycol and degassing with nitrogen for 20 min to obtain solution A; dissolving 1.35 g of Na2S in a mixed solution containing 20 mL of ultrapure water and 10 mL of ethylene glycol to obtain solution B; adding solution B to solution A under continuous stirring, followed by adding 20 mL of urea; placing the resulting solution in a polytetrafluoroethylene-lined autoclave and reacting at 180 °C for 12 h; after the reaction, centrifuging the solution, washing the resulting precipitate three times with ethanol, and vacuum drying at 60 °C for 12 h to obtain Bi2S3; dissolving 50 mg of the prepared Bi2S3 in 10 mL of ultrapure water, then adding 5 mg of polyvinylpyrrolidone; stirring for 40 min, adding 4 mL of sodium citrate and 3 mL of HAuCl4, and reacting at 80 °C for 10 min. h; The obtained product was centrifuged, washed, and vacuum dried to obtain Bi2S3@Au NPs; 200 µL of Bi2S3@Au NPs with a concentration of 2 mg / mL was mixed with 40 µL of AFP aptamer chain with a concentration of 100 µM, and then 200 µL of NaCl solution with a concentration of 2 M was added; The obtained product was centrifuged to remove unbound AFP aptamer chain, and washed 3 times with phosphate buffer solution to obtain Bi2S3@Au NPs-AFP aptamer chain; The AFP solutions of different concentrations were prepared by uniformly dispersing AFP antigen at concentrations of 10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, and 100 ng / mL in a phosphate buffer solution with a pH of 7.4.

[0013] Example 3: Construction of a bifunctional biosensor based on coordination-induced enhancement and interfacial antifouling. A glassy carbon electrode with a diameter of 4 mm was polished with Al2O3 powder; 10 µL of Zn / Ru-MOF bound with pTAO was dropped onto the treated electrode surface; 8 µL of complementary chain solution of AFP aptamer was added dropwise, and the electrode was incubated at 4 °C for 2 h; 8 μL of Bi2S3@Au NPs-AFP aptamer chain solution was added dropwise, and the electrode was incubated under the same conditions for 2 h; the modified electrode was incubated in AFP solutions of different concentrations for 1 h, thus constructing a bifunctional biosensor based on coordination-induced enhancement and interfacial antifouling. The Zn / Ru-MOF containing pTAO zwitterions is prepared by combining 18 mg of Zn(NO3)2·6H2O and Ru(dcbpy)3 2+ 40 mg of polyvinylpyrrolidone and 3 mg of pyrazine were dispersed in 64 mL of ultrapure water; the resulting solution was sonicated and then reacted at 80 °C for 24 h; after cooling, the resulting orange-red solution was centrifuged, washed three times with ultrapure water, and vacuum dried at 60 °C for 12 h to obtain Zn / Ru-MOF; 10 g of 3-dimethylaminopropylamine was added to 100 mL of ultrapure water under stirring, and the resulting solution was placed in an oxygen atmosphere and reacted at 60 °C for 6 h, during which 15 mL of H2O2 was added dropwise; the resulting mixed solution was rotary evaporated at 80 °C for 2 h to generate pTAO zwitterions; 1 mL of a mixed solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide was added to 5 mL of the prepared Zn / Ru-MOF solution to activate its carboxyl group; after the reaction was completed, 100 µL of pTAO zwitterions was added, and the reaction was continued for 12 h. h, to obtain Zn / Ru-MOF bound to pTAO zwitterions; The Bi2S3@Au NPs-AFP aptamer chain is prepared by dispersing 1.82 g of Bi(NO3)3·5H2O in 25 mL of ethylene glycol and degassing with nitrogen for 20 min to obtain solution A; dissolving 1.35 g of Na2S in a mixed solution containing 20 mL of ultrapure water and 10 mL of ethylene glycol to obtain solution B; adding solution B to solution A under continuous stirring, followed by adding 20 mL of urea; placing the resulting solution in a polytetrafluoroethylene-lined autoclave and reacting at 180 °C for 12 h; after the reaction, centrifuging the solution, washing the resulting precipitate three times with ethanol, and vacuum drying at 60 °C for 12 h to obtain Bi2S3; dissolving 50 mg of the prepared Bi2S3 in 10 mL of ultrapure water, then adding 5 mg of polyvinylpyrrolidone; stirring for 40 min, adding 4 mL of sodium citrate and 3 mL of HAuCl4, and reacting at 80 °C for 10 min. h; The obtained product was centrifuged, washed, and vacuum dried to obtain Bi2S3@Au NPs; 200 µL of Bi2S3@Au NPs with a concentration of 2 mg / mL was mixed with 40 µL of AFP aptamer chain with a concentration of 100 µM, and then 200 µL of NaCl solution with a concentration of 2 M was added; The obtained product was centrifuged to remove unbound AFP aptamer chain, and washed 3 times with phosphate buffer solution to obtain Bi2S3@Au NPs-AFP aptamer chain; The AFP solutions of different concentrations were prepared by uniformly dispersing AFP antigen at concentrations of 10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, and 100 ng / mL in a phosphate buffer solution with a pH of 7.4.

[0014] Example 4: An application of a dual-functional biosensor based on coordination-induced enhancement and interfacial antifouling constructed using the methods described in Examples 1, 2, and 3, characterized in that it is used for AFP detection in complex serum environments. A three-electrode system was constructed for ECL testing: a bifunctional biosensor based on coordination-induced enhancement and interfacial antifouling was used as the working electrode, an Ag / AgCl electrode as the reference electrode, and a platinum electrode as the auxiliary electrode; a phosphate buffer solution containing 50 mM K2S2O8 at pH 7.4 was used as the test solution; the applied voltage range was 1.6 ~ 0 V, with a high voltage of 500 V; based on the measured signals, a working curve was plotted, revealing that the developed biosensor has a detection range of 10 fg / mL ~ 100 ng / mL and a detection limit of 4.7 fg / mL, exhibiting high stability, selectivity, and reproducibility, making it suitable for trace detection of AFP in complex serum environments.

[0015] Example 5: An application of a dual-functional biosensor based on coordination-induced enhancement and interfacial antifouling, constructed using the methods described in Examples 1, 2, and 3, characterized by its use in AFP detection in complex serum environments. A three-electrode system was constructed for ECL testing: a bifunctional biosensor based on coordination-induced enhancement and interfacial antifouling was used as the working electrode, an Ag / AgCl electrode as the reference electrode, and a platinum electrode as the auxiliary electrode; a phosphate buffer solution containing 75 mM K2S2O8 at pH 7.4 was used as the test solution; the applied voltage range was 1.6 ~ 0 V, with a high voltage of 500 V; based on the measured signals, a working curve was plotted, revealing that the developed biosensor has a detection range of 10 fg / mL ~ 100 ng / mL and a detection limit of 4.7 fg / mL, exhibiting high stability, selectivity, and reproducibility, making it suitable for trace detection of AFP in complex serum environments.

[0016] Example 6: An application of a dual-functional biosensor based on coordination-induced enhancement and interfacial antifouling constructed using the methods described in Examples 1, 2, and 3, characterized in that it is used for AFP detection in complex serum environments. A three-electrode system was constructed for ECL testing: a bifunctional biosensor based on coordination-induced enhancement and interfacial antifouling was used as the working electrode, an Ag / AgCl electrode as the reference electrode, and a platinum electrode as the auxiliary electrode; a phosphate buffer solution containing 100 mM K2S2O8 at pH 7.4 was used as the test solution; the applied voltage range was 1.6 ~ 0 V, with a high voltage of 500 V; based on the measured signals, a working curve was plotted, revealing that the developed biosensor has a detection range of 10 fg / mL ~ 100 ng / mL and a detection limit of 4.7 fg / mL, exhibiting high stability, selectivity, and reproducibility, making it suitable for trace detection of AFP in complex serum environments. Attached Figure Description

[0017] Figure 1 The construction flowchart of a bifunctional biosensor based on coordination-induced enhancement and interfacial antifouling is shown below (3-dimethylaminopropylamine: DMAPA; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide: EDC; N-hydroxysuccinimide: NHS; glassy carbon electrode: GCE; AFP aptamer complementary chain: S1; AFP aptamer chain: S2; ethylene glycol: EG; coordination-induced enhancement: CIE; resonance energy transfer: RET).

[0018] Figure 2 For (a) Ru(dcbpy)3 2+ X-ray diffraction patterns of (b) Zn / Ru-MOF.

[0019] Figure 3 For (a) Ru(dcbpy)3 2+ (b) Infrared spectra of Zn / Ru-MOF.

[0020] Figure 4 The image shows a scanning electron microscope (SEM) image of Zn / Ru-MOF and its corresponding elemental mapping.

[0021] Figure 5 This is the proton NMR spectrum of pTAO.

[0022] Figure 6 The X-ray photoelectron spectra of pTAO-Zn / Ru-MOF are shown below. (A) Full-region X-ray photoelectron spectrum; (B) High-resolution X-ray photoelectron spectrum of Zn 2p region; (C) High-resolution X-ray photoelectron spectrum of Ru 3p region; (D) High-resolution X-ray photoelectron spectrum of C 1s region; (E) High-resolution X-ray photoelectron spectrum of N 1s region; (F) High-resolution X-ray photoelectron spectrum of O 1s region.

[0023] Figure 7 The image shows the X-ray diffraction pattern of Bi2S3.

[0024] Figure 8 This is a scanning electron microscope image of Bi2S3.

[0025] Figure 9 The X-ray photoelectron spectrum of Bi2S3 is shown below. (A) X-ray photoelectron spectrum of the whole region; (B) High-resolution X-ray photoelectron spectrum of the Bi4f region; (C) High-resolution X-ray photoelectron spectrum of the S2p region; (D) High-resolution X-ray photoelectron spectrum of the S2s region.

[0026] Figure 10 The image shows the scanning electron microscope (SEM) image and corresponding elemental mapping of Bi2S3@Au NPs.

[0027] Figure 11 Contact angle diagrams for (a) GCE and (b) pTAO-Zn / Ru-MOF / GCE.

[0028] Figure 12 Fluorescence imaging of ITO and pTAO-Zn / Ru-MOF / ITO after incubation in fluorescein-labeled bovine serum albumin (Indium Tin Oxide: ITO).

[0029] Figure 13ECL intensity plots of (a) Zn / Ru-MOF / GCE and (b) pTAO-Zn / Ru-MOF / GCE incubated in different concentrations of FBS, and corresponding signal attenuation rate line plots (B); ECL intensity plots of (a) Zn / Ru-MOF / GCE and (b) pTAO-Zn / Ru-MOF / GCE incubated in different concentrations of human serum, and corresponding signal attenuation rate line plots (D) (fetal bovine serum: FBS).

[0030] Figure 14 For (a) Ru(dcbpy)3 2+ ECL intensity-time curves for (b) Zn / Ru-MOF.

[0031] Figure 15 For (a) Ru(dcbpy)3 2+ (b) FL spectrum of Zn / Ru-MOF (fluorescence: FL).

[0032] Figure 16 The image shows the overlap region between the UV-vis absorption spectrum of Bi2S3@Au NPs and the FL emission spectrum of Zn / Ru-MOF (UV-Vis: UV-vis).

[0033] Figure 17 For (a) pTAO-Zn / Ru-MOF / GCE, (b) Bi2S3@Au NPs-S2 / S1 / pTAO-Zn / Ru-MOF / GCE, (c) AFP / Bi2S3@Au NPs-S2 / S1 / pTAO-Zn / Ru-MOF / GCE in S2O8 2- ECL intensity-time curve in solution.

[0034] Figure 18 Zn / Ru-MOF / S2O8 2- ECL mechanism diagram of the system.

[0035] Figure 19 Characterization diagram of the construction process of a bifunctional biosensor based on coordination-induced enhancement and interfacial antifouling, according to (A) cyclic voltammetry and (B) AC impedance testing.

[0036] Figure 20 The diagram shows the condition optimization results of a bifunctional biosensor based on coordination-induced enhancement and interfacial antifouling. (A) Optimization results of K2S2O8 concentration; (B) Optimization results of solution pH; (C) Optimization results of Zn / Ru-MOF concentration.

[0037] Figure 21The following are the ECL response curves (A) (10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 10 ng / mL, 100 ng / mL) and the corresponding calibration curves (B) of the bifunctional biosensor based on coordination-induced enhancement and interfacial antifouling after incubation with different concentrations of AFP.

[0038] Figure 22 For a bifunctional biosensor based on coordination-induced enhancement and interfacial antifouling, consider its (A) selectivity, (B) signal stability, (C) long-term stability, and (D) reproducibility (carcinoembryonic antigen: CEA; bovine serum albumin: BSA; adenosine triphosphate: ATP; glucose: Glu; prostate-specific antigen: PSA; neuron-specific enolase: NSE; squamous cell carcinoma antigen: SCCA).

Claims

1. Construction of a bifunctional biosensor based on coordination-induced enhancement and interfacial antifouling, characterized in that, A glassy carbon electrode with a diameter of 4 mm was polished with Al2O3 powder. 6–10 µL of Zn / Ru-MOF containing zwitterions of 3-aminopropyldimethylamine were added dropwise to the treated electrode surface. 8 µL of a complementary chain solution of alpha-fetoprotein aptamer was then added dropwise, and the mixture was incubated at 4 °C for 2 h. 8 μL of a Bi2S3@Au NPs-alpha-fetoprotein aptamer chain solution was added dropwise, and the mixture was incubated under the same conditions for 2 h. The modified electrode was then incubated for 1 h in alpha-fetoprotein solutions of different concentrations, thus constructing a bifunctional biosensor based on coordination-induced enhancement and interfacial antifouling.

2. The construction of a bifunctional biosensor based on coordination-induced enhancement and interface antifouling as described in claim 1, characterized in that, The Zn / Ru-MOF conjugated with 3-aminopropyl dimethylamine zwitterions was prepared by dispersing 18 mg of Zn(NO3)2·6H2O, tris(4,4'-dicarboxylic acid-2,2'-bipyridine)ruthenium, 40 mg of polyvinylpyrrolidone, and 3 mg of pyrazine in 64 mL of ultrapure water; the resulting solution was sonicated and then reacted at 80 °C for 24 h; after cooling, the resulting orange-red solution was centrifuged, washed three times with ultrapure water, and vacuum dried at 60 °C for 12 h to obtain Zn / Ru-MOF; 10 g of 3-dimethylaminopropylamine was added to 100 mL of ultrapure water with stirring, and the resulting solution was placed in an oxygen atmosphere and reacted at 60 °C for 6 h, during which 15 mL of H2O2 was added dropwise; the resulting mixed solution was rotary evaporated at 80 °C for 2 h to generate 3-aminopropyl dimethylamine zwitterions; 1 A mixed solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide was added to 5 mL of the prepared Zn / Ru-MOF solution to activate its carboxyl group. After the reaction was complete, 100 µL of 3-aminopropyldimethylamine zwitterion was added, and the reaction was continued for 12 h to obtain Zn / Ru-MOF bound with 3-aminopropyldimethylamine zwitterion.

3. The construction of a bifunctional biosensor based on coordination-induced enhancement and interfacial antifouling as described in claim 1, characterized in that, The Bi2S3@Au NPs-alpha-fetoprotein aptamer chain is prepared by dispersing 1.82 g of Bi(NO3)3·5H2O in 25 mL of ethylene glycol and degassing with nitrogen for 20 min to obtain solution A; and dissolving 1.35 g of Na2S in a mixed solution containing 20 mL of ultrapure water and 10 mL of ethylene glycol to obtain solution B. Solution B was added to solution A with continuous stirring, followed by 20 mL of urea. The obtained solution was placed in a polytetrafluoroethylene-lined autoclave and reacted at 180 °C for 12 h. After the reaction, the solution was centrifuged, the precipitate was washed three times with ethanol, and then vacuum dried at 60 °C for 12 h to obtain Bi2S3. 50 mg of the prepared Bi2S3 was dissolved in 10 mL of ultrapure water, and then 5 mg of polyvinylpyrrolidone was added. After stirring for 40 min, 4 mL of sodium citrate and 3 mL of HAuCl4 were added, and the reaction was carried out at 80 °C for 10 h. The obtained product was centrifuged, washed, and vacuum dried to obtain Bi2S3@Au NPs. 200 µL of Bi2S3@Au NPs with a concentration of 2 mg / mL was mixed with 40 µL of alpha-fetoprotein aptamer chain with a concentration of 100 µM, and then 200 µL of NaCl solution with a concentration of 2 M was added. The obtained product was centrifuged to remove unbound alpha-fetoprotein aptamer chains, and washed three times with phosphate buffer solution to obtain Bi2S3@Au NPs-alpha-fetoprotein aptamer chains.

4. The construction of a bifunctional biosensor based on coordination-induced enhancement and interfacial antifouling as described in claim 1, characterized in that, The different concentrations of alpha-fetoprotein solutions were obtained by uniformly dispersing alpha-fetoprotein antigen at concentrations of 10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, and 100 ng / mL in a phosphate buffer solution with a pH of 7.

4.

5. The use of a bifunctional biosensor based on coordination-induced enhancement and interfacial antifouling constructed by the method described in claim 1, characterized in that, Applications for alpha-fetoprotein (AFP) detection.

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