Construction and application of a biosensor based on efficient antifouling interface and double signal amplification

Abstract

The application discloses a kind of construction and application of biosensor based on high-efficiency antifouling interface and double signal amplification, belong to photo / electro analytical chemistry, biosensing, material science and technology field.In the present application, by being modified polytannin acid doped polyaniline as antifouling film on electrode interface, resist the non-specific adsorption of interfering biomolecules in complex medium;Prepared EuTb-MOF as high-efficiency luminophore, wherein, Tb 3+ The energy of itself and ligand can be transferred to Eu 3+ , realize effective signal self-enhancement;Utilize the valence reversible conversion of Co 2+ / Co 3+ And Fe 2+ / Fe 3+ , and the synergistic catalysis of Ag NPs, promote the reduction of S2O8 2‑ , to further improve the electrochemiluminescence signal of EuTb-MOF.Based on the above strategy, the biosensor constructed has high detection sensitivity and precision, and long service life, linear range is 10fM~100nM, and detection limit is as low as 3.62fM.

Description

Technical Field

[0001] This invention discloses the construction and application of a biosensor based on a highly efficient antifouling interface and dual signal amplification, belonging to the fields of photo / electroanalytical chemistry, biosensing, and materials science and technology. Background Technology

[0002] Thrombin (TB), an important protease in molecular biology, provides crucial information for the diagnosis and monitoring of various blood diseases. Therefore, it is necessary to develop an efficient sensing platform for rapid and sensitive detection of TB in human serum. Electrochemiluminescence (ECL) analysis, with its low background interference and good controllability, is considered an ideal choice. However, the detection performance of biosensors is affected by the non-specific adsorption of many interfering substances in complex serum media. Developing efficient antifouling materials for interface modification can effectively solve these problems.

[0003] The development of high-performance luminescent materials is crucial for improving the detection sensitivity of biosensors. Previous reports have indicated that lanthanide metal-organic frameworks possess tunable luminescence properties, long decay rates, and high energy transfer efficiency, making them suitable for optical sensing analysis. Typically, lanthanide metals exhibit stable luminescence properties, but their efficiency is relatively low. To improve luminescence efficiency, conjugated ligands are introduced to induce an antenna effect for energy transfer. However, triggering the antenna effect requires a specific bandgap. Therefore, under specific conditions, additional metal ions or second ligands can be introduced as intermediate bridges to achieve efficient internal energy transfer.

[0004] In addition to developing self-enhanced luminescent materials with high ECL efficiency, effective amplification strategies can be designed to further improve the detection sensitivity of biosensors. Traditional signal amplification methods rely on the concentrations of the luminescent material and co-reactants, resulting in low amplification efficiency. To avoid these limitations, co-reaction promoters can be introduced to catalyze the redox reaction of the co-reactants, thereby enhancing ECL emission. Among these, multivalent metals can promote the redox reaction of co-reactants through reversible valence state transitions, generating a large number of free radicals. Furthermore, noble metal nanoparticles can also achieve highly efficient catalysis of co-reactants.

[0005] In this invention, an ultrasensitive ECL antifouling biosensor with a dual signal amplification strategy was constructed for trace detection of TB in serum. First, polytannic acid with multiple hydroxyl groups was modified onto the electrode surface as an antifouling component, effectively preventing interfacial adsorption of interfering substances and extending the lifespan of the biosensor. Furthermore, EuTb-MOF with a strong and stable luminescence signal was prepared as the luminescent agent. Based on the effect of terephthalic acid on Tb... 3+ and Tb 3+ For Eu 3+ Dual sensitization, Tb 3+It can act as an energy transfer bridge to achieve the conversion of terephthalic acid and Eu. 3+ The antenna effect between them; third, Ag / CoFe-LDH@GO was designed as a co-reaction promoter to further improve the ECL emission of EuTb-MOF. With Co 2+ / Co 3+ and Fe 2+ / Fe 3+ The reversible conversion and the synergistic catalysis of Ag NPs generated a large amount of SO4. ·- Furthermore, the introduction of GO allows for the loading of more catalysts and accelerates electron transfer. Therefore, the constructed biosensor enables trace detection of TB with a detection limit as low as 3.62 fM. Summary of the Invention

[0006] One of the technical objectives of this invention is to overcome the shortcomings of the prior art, based on the effect of terephthalic acid on Tb 3+ and Tb 3 + For Eu 3+ By leveraging the dual sensitization properties, a signal-self-enhancing EuTb-MOF luminescent material was prepared.

[0007] The second technical objective of this invention is to construct an ECL biosensor based on a highly efficient antifouling interface and dual signal amplification by combining co-reactant catalysis and an interface antifouling strategy. The raw materials used are low-cost, the process is simple, and the operation is safe.

[0008] The third technical objective of this invention is to provide an application for a biosensor constructed using the aforementioned method, based on a highly efficient antifouling interface and dual signal amplification, specifically for trace monitoring of TB in serum, which has certain industrialization prospects.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] 1. Construction of a biosensor based on a highly efficient antifouling interface and dual signal amplification

[0011] The glassy carbon electrode was polished to a mirror finish using Al2O3 powder; polytannic acid-doped polyaniline was electropolymerized on the electrode surface as an antifouling film; AuNPs were electroplated on the polytannic acid-doped polyaniline-modified electrode to improve the interfacial electron transfer capability; 10 μL of Ag / CoFe-LDH@GO was added to the electrode; 6–10 μL of EuTb-MOF bound to the complementary chain of the TB aptamer was added to further modify the electrode; 10 μL of ferrocene bound to the TB aptamer was added to the electrode, and the electrode was incubated at 4 °C for 2 h to construct a biosensor based on a highly efficient antifouling interface and dual signal amplification.

[0012] The polytannic acid-doped polyaniline was obtained by immersing an electrode in a 5 mL mixed solution containing 0.3 M aniline, 2 M HClO4, and 0.53 μM polystyrene sulfonate using IT technology, and electropolymerizing at a constant potential of 0.76 V for 60 s to obtain a polyaniline film; then immersing the polyaniline-modified electrode in 10 mL of a 15 mg / mL tannic acid solution, and electropolymerizing at a voltage range of -0.4 to 1.2 V and a scan rate of 50 mV / s using CV technology for 600 s to obtain the polytannic acid-doped polyaniline.

[0013] The Au NPs were obtained by immersing a polytannic acid-doped polyaniline-modified electrode in 5 mL of a 1% (w / w) HAuCl4 solution using IT technology and running it at a constant potential of -0.2 V for 30 s. The Ag / CoFe-LDH@GO was obtained by dispersing 3.49 g of Co(NO3)2·6H2O, 0.48 g of FeCl3, and 20 mL of a 1 mg / mL GO solution in 40 mL of ultrapure water; adding 5 mL of NH3·H2O and stirring for 5 min to obtain CoFe-LDH@GO; dissolving 40 mg of CoFe-LDH@GO in 30 mL of ultrapure water under dark conditions using ultrasonic treatment; adding 10 mL of a 6 mg / mL AgNO3 solution under magnetic stirring; centrifuging the mixture; and drying the resulting precipitate at 60 °C for 12 h to obtain Ag / CoFe-LDH@GO.

[0014] The EuTb-MOF with complementary TB aptamer strands is prepared by dissolving 82 mg of TbNO3 and 29 mg of EuCl3·6H2O in ultrapure water and stirring for 30 min to obtain solution A; dissolving 78 mg of terephthalic acid in a mixed solution containing 15 mL of N,N-dimethylformamide and 15 mL of ethanol under ultrasonic treatment to obtain solution B; thoroughly mixing solutions A and B under stirring and letting stand for 3 h, then reacting the mixed solution at 80 °C for 24 h. The resulting solution was centrifuged and washed, then vacuum dried at 60 °C for 12 h to obtain EuTb-MOF; 10 μL of a mixed solution containing 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.1 M N-hydroxysuccinimide was added to 1 mL of Ag / CoFe-LDH@GO solution with a concentration of 1 mg / mL; 100 μL of an aminated TB aptamer complementary chain was added, and the reaction was carried out for 2 h to obtain EuTb-MOF bound with the TB aptamer complementary chain;

[0015] The TB aptamer-bound ferrocene is obtained by adding 10 μL of a mixed solution containing 0.4 M of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.1 M of N-hydroxysuccinimide to 1 mL of a carboxylated ferrocene solution with a concentration of 1 mg / mL; adding 100 μL of an aminated TB aptamer; and reacting for 2 h to obtain TB aptamer-bound ferrocene.

[0016] 2. The application of the biosensor constructed using the described method, based on a highly efficient antifouling interface and dual signal amplification, is for the detection of trace amounts of TB in serum.

[0017] A three-electrode system was constructed using an Ag / AgCl electrode as the reference electrode and a platinum electrode as the auxiliary electrode. This system, based on a highly efficient antifouling interface and dual signal amplification, served as the working electrode. The three electrodes were immersed in TB at different concentrations for ECL testing. A phosphate buffer solution containing 20–80 mM K₂S₂O₈ at pH 7.5 was used as the detection solution. The applied scanning voltage range was 1.8–0 V, with a high voltage of 800 V. Based on the measured signals, a working curve was plotted, revealing that the ECL biosensor has a detection range of 10 fM–100 nM, a detection limit as low as 3.62 fM, and exhibits high stability, specificity, and reproducibility, making it suitable for trace monitoring of TB in serum.

[0018] The beneficial technical effects of the present invention are as follows:

[0019] 1. This invention prepares a signal-enhancing EuTb-MOF as a light emitter. Wherein, Tb 3+ It can act as an energy transfer bridge to trigger the reaction of terephthalic acid and Eu. 3+ The antenna effect between them transfers energy from themselves and their ligands to Eu. 3+ This enables effective signal amplification.

[0020] 2. This invention constructs a biosensor based on a highly efficient antifouling interface and dual signal amplification. Ag / CoFe-LDH@GO is developed as a co-reaction promoter, utilizing Co... 2+ / Co 3+ and Fe 2+ / Fe 3+ The reversible conversion and synergistic catalysis of Ag NPs significantly improved the ECL emission of EuTb-MOF; polyhydroxy polytannic acid, as an antifouling component, effectively prevented the interfacial adsorption of interfering substances and extended the service life of the biosensor.

[0021] 3. The biosensor constructed in this invention, based on a highly efficient antifouling interface and dual signal amplification, exhibits a wide linear range and low detection limit for the target TB, as well as high stability, specificity, and reproducibility. It is suitable for trace monitoring of TB in serum and has certain industrialization prospects. Detailed Implementation

[0022] 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.

[0023] Example 1: Construction of a biosensor based on a highly efficient antifouling interface and dual signal amplification

[0024] The glassy carbon electrode was polished to a mirror finish using Al2O3 powder; polytannic acid-doped polyaniline was electropolymerized on the electrode surface as an antifouling film; AuNPs were electroplated on the polytannic acid-doped polyaniline-modified electrode to improve the interfacial electron transfer capability; 10 μL of Ag / CoFe-LDH@GO was added to the electrode; 6 μL of EuTb-MOF bound to the complementary chain of the TB aptamer was added to further modify the electrode; 10 μL of ferrocene bound to the TB aptamer was added to the electrode, and the electrode was incubated at 4 °C for 2 h to construct a biosensor based on a highly efficient antifouling interface and dual signal amplification.

[0025] The polytannic acid-doped polyaniline was obtained by immersing an electrode in a 5 mL mixed solution containing 0.3 M aniline, 2 M HClO4, and 0.53 μM polystyrene sulfonate using IT technology, and electropolymerizing at a constant potential of 0.76 V for 60 s to obtain a polyaniline film; then immersing the polyaniline-modified electrode in 10 mL of a 15 mg / mL tannic acid solution, and electropolymerizing at a voltage range of -0.4 to 1.2 V and a scan rate of 50 mV / s using CV technology for 600 s to obtain the polytannic acid-doped polyaniline.

[0026] The Au NPs were obtained by immersing a polytannic acid-doped polyaniline-modified electrode in 5 mL of a 1% (w / w) HAuCl4 solution using IT technology and running it at a constant potential of -0.2 V for 30 s. The Ag / CoFe-LDH@GO was obtained by dispersing 3.49 g of Co(NO3)2·6H2O, 0.48 g of FeCl3, and 20 mL of a 1 mg / mL GO solution in 40 mL of ultrapure water; adding 5 mL of NH3·H2O and stirring for 5 min to obtain CoFe-LDH@GO; dissolving 40 mg of CoFe-LDH@GO in 30 mL of ultrapure water under dark conditions using ultrasonic treatment; adding 10 mL of a 6 mg / mL AgNO3 solution under magnetic stirring; centrifuging the mixture; and drying the resulting precipitate at 60 °C for 12 h to obtain Ag / CoFe-LDH@GO.

[0027] The EuTb-MOF with complementary TB aptamer strands is prepared by dissolving 82 mg of TbNO3 and 29 mg of EuCl3·6H2O in ultrapure water and stirring for 30 min to obtain solution A; dissolving 78 mg of terephthalic acid in a mixed solution containing 15 mL of N,N-dimethylformamide and 15 mL of ethanol under ultrasonic treatment to obtain solution B; thoroughly mixing solutions A and B under stirring and letting stand for 3 h, then reacting the mixed solution at 80 °C for 24 h. The resulting solution was centrifuged and washed, then vacuum dried at 60 °C for 12 h to obtain EuTb-MOF; 10 μL of a mixed solution containing 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.1 M N-hydroxysuccinimide was added to 1 mL of Ag / CoFe-LDH@GO solution with a concentration of 1 mg / mL; 100 μL of an aminated TB aptamer complementary chain was added, and the reaction was carried out for 2 h to obtain EuTb-MOF bound with the TB aptamer complementary chain;

[0028] The TB aptamer-bound ferrocene is obtained by adding 10 μL of a mixed solution containing 0.4 M of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.1 M of N-hydroxysuccinimide to 1 mL of a carboxylated ferrocene solution with a concentration of 1 mg / mL; adding 100 μL of an aminated TB aptamer; and reacting for 2 h to obtain TB aptamer-bound ferrocene.

[0029] Example 2: Construction of a biosensor based on a highly efficient antifouling interface and dual signal amplification. A glassy carbon electrode was polished to a mirror finish using Al2O3 powder; polytannic acid-doped polyaniline was electropolymerized on the electrode surface as an antifouling film; AuNPs were electroplated onto the polytannic acid-doped polyaniline-modified electrode to improve interfacial electron transfer capability; 10 μL of Ag / CoFe-LDH@GO was added to the electrode; 8 μL of EuTb-MOF bound to the complementary chain of the TB aptamer was further added; 10 μL of ferrocene bound to the TB aptamer was added to the electrode, and the mixture was incubated at 4°C for 2 h to construct a biosensor based on a highly efficient antifouling interface and dual signal amplification.

[0030] The polytannic acid-doped polyaniline was obtained by immersing an electrode in a 5 mL mixed solution containing 0.3 M aniline, 2 M HClO4, and 0.53 μM polystyrene sulfonate using IT technology, and electropolymerizing at a constant potential of 0.76 V for 60 s to obtain a polyaniline film; then immersing the polyaniline-modified electrode in 10 mL of a 15 mg / mL tannic acid solution, and electropolymerizing at a voltage range of -0.4 to 1.2 V and a scan rate of 50 mV / s using CV technology for 600 s to obtain the polytannic acid-doped polyaniline.

[0031] The Au NPs were obtained by immersing a polytannic acid-doped polyaniline-modified electrode in 5 mL of a 1% (w / w) HAuCl4 solution using IT technology and running it at a constant potential of -0.2 V for 30 s. The Ag / CoFe-LDH@GO was obtained by dispersing 3.49 g of Co(NO3)2·6H2O, 0.48 g of FeCl3, and 20 mL of a 1 mg / mL GO solution in 40 mL of ultrapure water; adding 5 mL of NH3·H2O and stirring for 5 min to obtain CoFe-LDH@GO; dissolving 40 mg of CoFe-LDH@GO in 30 mL of ultrapure water under dark conditions using ultrasonic treatment; adding 10 mL of a 6 mg / mL AgNO3 solution under magnetic stirring; centrifuging the mixture; and drying the resulting precipitate at 60 °C for 12 h to obtain Ag / CoFe-LDH@GO.

[0032] The EuTb-MOF with complementary TB aptamer strands is prepared by dissolving 82 mg of TbNO3 and 29 mg of EuCl3·6H2O in ultrapure water and stirring for 30 min to obtain solution A; dissolving 78 mg of terephthalic acid in a mixed solution containing 15 mL of N,N-dimethylformamide and 15 mL of ethanol under ultrasonic treatment to obtain solution B; thoroughly mixing solutions A and B under stirring and letting stand for 3 h, then reacting the mixed solution at 80 °C for 24 h. The resulting solution was centrifuged and washed, then vacuum dried at 60 °C for 12 h to obtain EuTb-MOF; 10 μL of a mixed solution containing 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.1 M N-hydroxysuccinimide was added to 1 mL of Ag / CoFe-LDH@GO solution with a concentration of 1 mg / mL; 100 μL of an aminated TB aptamer complementary chain was added, and the reaction was carried out for 2 h to obtain EuTb-MOF bound with the TB aptamer complementary chain;

[0033] The TB aptamer-bound ferrocene is obtained by adding 10 μL of a mixed solution containing 0.4 M of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.1 M of N-hydroxysuccinimide to 1 mL of a carboxylated ferrocene solution with a concentration of 1 mg / mL; adding 100 μL of an aminated TB aptamer; and reacting for 2 h to obtain TB aptamer-bound ferrocene.

[0034] Example 3: Construction of a biosensor based on a highly efficient antifouling interface and dual signal amplification. A glassy carbon electrode was polished to a mirror finish using Al2O3 powder; polytannic acid-doped polyaniline was electropolymerized on the electrode surface as an antifouling film; AuNPs were electroplated onto the polytannic acid-doped polyaniline-modified electrode to improve interfacial electron transfer capability; 10 μL of Ag / CoFe-LDH@GO was added to the electrode; 10 μL of EuTb-MOF bound to the complementary chain of the TB aptamer was further added; 10 μL of ferrocene bound to the TB aptamer was added to the electrode, and the mixture was incubated at 4°C for 2 h to construct a biosensor based on a highly efficient antifouling interface and dual signal amplification.

[0035] The polytannic acid-doped polyaniline was obtained by immersing an electrode in a 5 mL mixed solution containing 0.3 M aniline, 2 M HClO4, and 0.53 μM polystyrene sulfonate using IT technology, and electropolymerizing at a constant potential of 0.76 V for 60 s to obtain a polyaniline film; then immersing the polyaniline-modified electrode in 10 mL of a 15 mg / mL tannic acid solution, and electropolymerizing at a voltage range of -0.4 to 1.2 V and a scan rate of 50 mV / s using CV technology for 600 s to obtain the polytannic acid-doped polyaniline.

[0036] The Au NPs were obtained by immersing a polytannic acid-doped polyaniline-modified electrode in 5 mL of a 1% (w / w) HAuCl4 solution using IT technology and running it at a constant potential of -0.2 V for 30 s. The Ag / CoFe-LDH@GO was obtained by dispersing 3.49 g of Co(NO3)2·6H2O, 0.48 g of FeCl3, and 20 mL of a 1 mg / mL GO solution in 40 mL of ultrapure water; adding 5 mL of NH3·H2O and stirring for 5 min to obtain CoFe-LDH@GO; dissolving 40 mg of CoFe-LDH@GO in 30 mL of ultrapure water under dark conditions using ultrasonic treatment; adding 10 mL of a 6 mg / mL AgNO3 solution under magnetic stirring; centrifuging the mixture; and drying the resulting precipitate at 60 °C for 12 h to obtain Ag / CoFe-LDH@GO.

[0037] The EuTb-MOF with complementary TB aptamer strands is prepared by dissolving 82 mg of TbNO3 and 29 mg of EuCl3·6H2O in ultrapure water and stirring for 30 min to obtain solution A; dissolving 78 mg of terephthalic acid in a mixed solution containing 15 mL of N,N-dimethylformamide and 15 mL of ethanol under ultrasonic treatment to obtain solution B; thoroughly mixing solutions A and B under stirring and letting stand for 3 h, then reacting the mixed solution at 80 °C for 24 h. The resulting solution was centrifuged and washed, then vacuum dried at 60 °C for 12 h to obtain EuTb-MOF; 10 μL of a mixed solution containing 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.1 M N-hydroxysuccinimide was added to 1 mL of Ag / CoFe-LDH@GO solution with a concentration of 1 mg / mL; 100 μL of an aminated TB aptamer complementary chain was added, and the reaction was carried out for 2 h to obtain EuTb-MOF bound with the TB aptamer complementary chain;

[0038] The TB aptamer-bound ferrocene is obtained by adding 10 μL of a mixed solution containing 0.4 M of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.1 M of N-hydroxysuccinimide to 1 mL of a carboxylated ferrocene solution with a concentration of 1 mg / mL; adding 100 μL of an aminated TB aptamer; and reacting for 2 h to obtain TB aptamer-bound ferrocene.

[0039] Example 4 describes the application of a biosensor based on a highly efficient antifouling interface and dual signal amplification constructed using the methods described in Examples 1, 2, and 3, for the trace detection of TB in serum. An Ag / AgCl electrode is used as the reference electrode, a platinum electrode as the auxiliary electrode, and the constructed biosensor based on a highly efficient antifouling interface and dual signal amplification is used as the working electrode, forming a three-electrode system. The three electrodes are immersed in TB of different concentrations for ECL testing. A phosphate buffer solution containing 20 mM K₂S₂O₈ at pH 7.5 is used as the detection solution. The applied scanning voltage range is 1.8–0 V, with a high voltage of 800 V. Based on the measured signals, a working curve is plotted, showing that the detection range of the ECL biosensor is 10 fM–100 nM, with a detection limit as low as 3.62 fM, and it exhibits high stability, specificity, and reproducibility, making it suitable for trace monitoring of TB in serum.

[0040] Example 5 describes the application of a biosensor based on a highly efficient antifouling interface and dual signal amplification, constructed using the methods described in Examples 1, 2, and 3, for the trace detection of TB in serum. An Ag / AgCl electrode is used as the reference electrode, a platinum electrode as the auxiliary electrode, and the constructed biosensor based on a highly efficient antifouling interface and dual signal amplification is used as the working electrode, forming a three-electrode system. The three electrodes are immersed in TB of different concentrations for ECL testing. A phosphate buffer solution containing 50 mM K₂S₂O₈ at pH 7.5 is used as the detection solution. The applied scanning voltage range is 1.8–0 V, with a high voltage of 800 V. Based on the measured signals, a working curve is plotted, showing that the detection range of the ECL biosensor is 10 fM–100 nM, with a detection limit as low as 3.62 fM, and it exhibits high stability, specificity, and reproducibility, making it suitable for trace monitoring of TB in serum.

[0041] Example 6 describes the application of a biosensor based on a highly efficient antifouling interface and dual signal amplification constructed using the methods described in Examples 1, 2, and 3, for the trace detection of TB in serum. An Ag / AgCl electrode is used as the reference electrode, a platinum electrode as the auxiliary electrode, and the constructed biosensor based on a highly efficient antifouling interface and dual signal amplification is used as the working electrode, forming a three-electrode system. The three electrodes are immersed in TB of different concentrations for ECL testing. A phosphate buffer solution containing 80 mM K₂S₂O₈ at pH 7.5 is used as the detection solution. The applied scanning voltage range is 1.8–0 V, with a high voltage of 800 V. Based on the measured signals, a working curve is plotted, showing that the detection range of the ECL biosensor is 10 fM–100 nM, with a detection limit as low as 3.62 fM, and it exhibits high stability, specificity, and reproducibility, making it suitable for trace monitoring of TB in serum. Attached Figure Description

[0042] Figure 1 This is a schematic diagram of the construction process of a biosensor based on a highly efficient antifouling interface and dual signal amplification (polytannic acid: PTA; polyaniline: PANI; complementary strand of TB aptamer: cDNA; ferrocene: Fc; energy transfer: ET; terephthalic acid: H2BDC).

[0043] Figure 2 The image shows the X-ray diffraction pattern of EuTb-MOF.

[0044] Figure 3 Infrared spectra of H2BDC and EuTb-MOF.

[0045] Figure 4 This is a scanning electron microscope image of EuTb-MOF.

[0046] Figure 5 The images show the X-ray photoelectron spectra of EuTb-MOF, where (A) is the full-area X-ray photoelectron spectrum; (B) is the high-resolution X-ray photoelectron spectrum of the Eu 3d region; (C) is the high-resolution X-ray photoelectron spectrum of the Tb 3d region; (D) is the high-resolution X-ray photoelectron spectrum of the C 1s region; and (E) is the high-resolution X-ray photoelectron spectrum of the O 1s region.

[0047] Figure 6 The X-ray diffraction pattern of Ag / CoFe-LDH@GO is shown.

[0048] Figure 7 This is a scanning electron microscope image of Ag / CoFe-LDH@GO.

[0049] Figure 8 The X-ray photoelectron spectra of Ag / CoFe-LDH@GO are shown below. (A) X-ray photoelectron spectrum of the whole region; (B) High-resolution X-ray photoelectron spectrum of Ag 3d region; (C) High-resolution X-ray photoelectron spectrum of Co 2p region; (D) High-resolution X-ray photoelectron spectrum of Fe 2p region.

[0050] Figure 9 Contact angle diagrams for (a) GCE, (b) PTA-PANI / GCE and (c) Ag / CoFe-LDH@GO / Au NPs / PTA-PANI / GCE (glassy carbon electrode: GCE).

[0051] Figure 10 Differential pulse voltammetry plots of (A) GCE and (B) PTA-PANI / GCE incubated in different concentrations of bovine serum albumin, and (C) corresponding signal attenuation plot.

[0052] Figure 11Fluorescence imaging of ITO and PTA-PANI / ITO after incubation in fluorescein-labeled bovine serum albumin (Indium Tin Oxide: ITO).

[0053] Figure 12 The fluorescence spectra of (A) Tb-MOF, (B) Eu-MOF, and (C) EuTb-MOF are shown.

[0054] Figure 13 H2BDC to Tb in EuTb-MOF 3+ and Tb 3+ To Eu 3+ A model diagram of the energy transfer process (intersystem crossing: ISC).

[0055] Figure 14 For (a) GCE, (b) EuTb-MOF / GCE, (c) Ag / CoFe-LDH@GO / EuTb-MOF / GCE and (d) Ag / CoFe-LDH@GO / GCE in S2O8 containing 50 mM 2- ECL intensity-voltage curves in phosphate buffered saline solution; and ECL intensity-voltage curves of (e)Ag / CoFe-LDH@GO / GCE and (f)Ag / CoFe-LDH@GO / EuTb-MOF / GCE in phosphate buffered saline solution.

[0056] Figure 15 For (a) EuTb-MOF / GCE, (b) CoFe-LDH@GO / EuTb-MOF / GCE and (c) Ag / CoFe-LDH@GO / EuTb-MOF / GCE in S2O8 containing 50 mM 2- (A) ECL intensity-voltage curve and (B) cyclic voltammetry curve in phosphate buffer solution.

[0057] Figure 16 The electron paramagnetic resonance spectra of the reaction systems containing (pink curve) and without (blue curve) Ag / CoFe-LDH@GO (5,5-dimethyl-1-pyrrolline-N-oxide: DMPO).

[0058] Figure 17 Characterization diagram of the construction process of a biosensor based on an efficient antifouling interface and dual signal amplification, based on (A) cyclic voltammetry and (B) AC impedance testing.

[0059] Figure 18The diagram shows the optimization results of a biosensor based on a highly efficient antifouling interface and dual signal amplification. Among them, (A) is the optimization result of K2S2O8 concentration; (B) is the optimization result of solution pH; (C) is the optimization result of EuTb-MOF concentration; and (D) is the optimization result of Ag / CoFe-LDH@GO concentration.

[0060] Figure 19 The following are the ECL response curves (A) (10fM, 100fM, 1pM, 10pM, 100pM, 1nM, 10nM and 100nM) and the corresponding calibration curves (B) of the biosensor based on a high-efficiency antifouling interface and dual signal amplification after incubation with different concentrations of TB.

[0061] Figure 20 The (A) selectivity, (B) signal stability, (C) reproducibility, and (D) storage stability of a biosensor based on an efficient antifouling interface and dual signal amplification are evaluated.

Claims

1. A method for constructing a biosensor based on a highly efficient antifouling interface and dual signal amplification, characterized in that, The glassy carbon electrode was polished to a mirror finish using Al2O3 powder; polytannic acid-doped polyaniline was electropolymerized on the electrode surface as an antifouling film; Au NPs were electroplated on the polytannic acid-doped polyaniline-modified electrode to improve the interfacial electron transfer capability; 10 μL of Ag / CoFe-LDH@GO was added to the electrode; 6-10 μL of EuTb-MOF bound to the thrombin aptamer complementary chain was added to further modify the electrode; 10 μL of ferrocene bound to the thrombin aptamer was added to the electrode, and the electrode was incubated at 4 °C for 2 h to construct a biosensor based on a highly efficient antifouling interface and dual signal amplification.

2. The method for constructing a biosensor based on a highly efficient antifouling interface and dual signal amplification as described in claim 1, characterized in that, The polytannic acid-doped polyaniline was obtained by immersing an electrode in a 5 mL mixed solution containing 0.3 M aniline, 2 M HClO4, and 0.53 μM polystyrene sulfonate using IT technology, and electropolymerizing at a constant potential of 0.76 V for 60 s to obtain a polyaniline film. The polyaniline-modified electrode was then immersed in 10 mL of a tannic acid solution with a concentration of 15 mg / mL, and electropolymerized at a voltage range of -0.4 to 1.2 V and a scan rate of 50 mV / s using CV technology for 600 s to obtain polytannic acid-doped polyaniline.

3. The method for constructing a biosensor based on a highly efficient antifouling interface and dual signal amplification as described in claim 1, characterized in that, The Au NPs are obtained by immersing a polytannic acid-doped polyaniline-modified electrode in 5 mL of a 1% (w / w) HAuCl4 solution using IT technology and running it at a constant potential of -0.2 V for 30 s.

4. The method for constructing a biosensor based on a highly efficient antifouling interface and dual signal amplification as described in claim 1, characterized in that, The Ag / CoFe-LDH@GO is prepared by dispersing 3.49 g of Co(NO3)2·6H2O, 0.48 g of FeCl3, and 20 mL of GO solution with a concentration of 1 mg / mL in 40 mL of ultrapure water; adding 5 mL of NH3·H2O and stirring for 5 min to obtain CoFe-LDH@GO; under dark conditions, dissolving 40 mg of CoFe-LDH@GO in 30 mL of ultrapure water by ultrasonic treatment; adding 10 mL of AgNO3 solution with a concentration of 6 mg / mL under magnetic stirring; centrifuging the mixed solution and drying the resulting precipitate at 60 °C for 12 h to obtain Ag / CoFe-LDH@GO.

5. The method for constructing a biosensor based on a highly efficient antifouling interface and dual signal amplification as described in claim 1, characterized in that, The EuTb-MOF bound to the thrombin aptamer complementary chain was prepared by dissolving 82 mg of TbNO3 and 29 mg of EuCl3·6H2O in ultrapure water and stirring for 30 min to obtain solution A; dissolving 78 mg of terephthalic acid in a mixed solution containing 15 mL of N,N-dimethylformamide and 15 mL of ethanol under ultrasonic treatment to obtain solution B; thoroughly mixing solutions A and B under stirring and allowing to stand for 3 h, then reacting the mixture at 80 °C for 24 h; centrifuging and washing the resulting solution, then vacuum drying at 60 °C for 12 h to obtain EuTb-MOF; adding 10 μL of a mixed solution containing 0.4 M of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.1 M of N-hydroxysuccinimide to 1 mL of a 1 mg / mL EuTb-MOF solution; adding 100 μL of an aminoated thrombin aptamer complementary chain, and reacting for 2... h, EuTb-MOF bound to the thrombin aptamer complementary chain was obtained.

6. The method for constructing a biosensor based on a highly efficient anti-fouling interface and dual signal amplification as described in claim 1, characterized in that, The thrombin aptamer-bound ferrocene is prepared by adding 10 μL of a mixed solution containing 0.4 M of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.1 M of N-hydroxysuccinimide to 1 mL of a carboxylated ferrocene solution with a concentration of 1 mg / mL; then adding 100 μL of an aminolated thrombin aptamer and reacting for 2 h to obtain the thrombin aptamer-bound ferrocene.

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