Construction and application of biosensor for triggering controlled release based on adjacent induced DNA strand displacement

Abstract

The invention discloses construction and application of a biosensor for triggering controlled release based on adjacent induced DNA strand displacement, and belongs to the technical fields of electrochemical luminescence, biological analysis and nano science. According to the invention, by integrating adjacent induced DNA strand displacement and controlled release strategies, the ultra-sensitive self-closing dual-mode biosensor taking the Au-Ag nanocluster as a luminous body is developed, and the ultra-sensitive self-closing dual-mode biosensor is used for trace detection of neuron-specific enolase. Due to the synergistic characteristic between the double metals, the Au-Ag nanocluster has high electrochemical luminescence (ECL) efficiency. Meanwhile, the reduction of S2O8 < 2-> is catalyzed by cyclic conversion between Fe < 2 + > / Mn < 3 + > and Fe < 3 + > / Mn < 4 + >, and a large amount of SO4 <-> is generated for ECL emission. The specific binding between the target antigen and the antibody can drive DNA strand displacement to open a blocked hole, so that the electrochemical probe falls off and the probe is quenched to release, the self-closing of an electrochemical signal and an ECL signal is realized, the detection sensitivity and accuracy of the sensor are effectively improved, and the sensor is suitable for immunoassay of various biomarkers.

Description

Technical Field

[0001] This invention discloses the construction and application of a biosensor based on proximity-induced DNA strand displacement-triggered controlled release, belonging to the fields of electrochemiluminescence, bioanalysis, and nanoscience and technology. Background Technology

[0002] Small cell lung cancer (SCLC) is a highly malignant tumor characterized by high proliferation rate, a significant tendency for early metastasis, and poor prognosis. As a specific biomarker for SCLC, serum levels of neuron-specific enolase (NSE) can serve as an effective assessment standard. To date, researchers have developed several methods incorporating electrochemistry, electrochemiluminescence (ECL), and fluorescence to detect NSE. Among these, ECL analysis combines the controllability of electrochemistry with the high sensitivity of chemiluminescence, offering significant advantages in the trace detection of various serum biomarkers.

[0003] As novel luminescent materials, metal nanoclusters have attracted widespread attention in the field of ECL (Electroluminescent Catalysis) due to their quantum size effect, good structural stability, and biocompatibility. Among them, bimetallic nanoclusters exhibit higher luminescence efficiency due to their specific electronic structure modulation characteristics and synergistic effects. Based on this, this invention develops bimetallic Au-Ag nanoclusters as ECL emitters. In addition to selecting highly efficient luminescent agents, co-reaction promoters can also enhance the ECL signal by catalyzing the decomposition of co-reactants. Specifically, FeMnO4... x With abundant redox active sites, it is an ideal choice as a co-reaction promoter. The combination of the high-performance luminescent material and the co-reaction promoter constructs a dual signal amplification system, effectively improving the sensor's output signal and detection sensitivity.

[0004] Furthermore, introducing efficient analytical methods is crucial for improving sensor detection performance. Controlled release strategies, by releasing functional probes during target recognition to achieve self-on or self-off signal activation, significantly improve detection sensitivity and accuracy. Simultaneously, to achieve better controlled release, a proximity-induced DNA strand substitution strategy was designed. This strategy relies on the high affinity of antigens and antibodies to specifically drive the dynamic process of DNA strand hybridization and dissociation, thereby achieving effective release of the quenching probe. Based on this, the sensor's specificity is also improved. Importantly, the proximity-induced DNA strand substitution strategy provides conditions for combining different signal output modes, thus improving the reliability of detection results through mutual verification of multiple detection modes.

[0005] Based on the above background, this invention proposes a strongly autologous biosensor driven by proximity-induced DNA strand displacement for bimodal analysis of NSE. Specifically, Au-Ag nanoclusters with high luminescence efficiency and stable ECL emission are used as the luminescent agent, along with FeMnO4.x Methylene blue was used as a catalyst and as an electrical signal probe. FeMnO₂ was used as the catalyst. x redox pairs (Fe 2 + / Fe 3+ and Mn 3+ / Mn 4+ It can catalyze S2O8 2- The reduction of SO42- produces more SO42- through a reversible valence state transition. •- This promotes ECL emission, achieving a strong "signal on." Based on the binding of NSE antigen and antibody, and the proximity-induced substitution of S1 and S3, the specificity and sensitivity of target markers are improved. Simultaneously, the S1-first antibody, adsorbed on aminated SiO2 via electrostatic interaction, detaches, opening the "gate" of the controlled-release carrier and releasing ZnS QDs, thus achieving significant self-closing of the ECL signal. Furthermore, the separation of the S2-methylene blue / second antibody from the electrode surface leads to self-closing of the differential pulse voltammetry (DPV) signal. The coexistence of these two detection modes significantly improves detection accuracy. Based on this, the biosensor constructed in this invention exhibits detection limits of 3.23 fg / mL and 31.8 fg / mL in ECL and DPV analyses, respectively, providing a reference for the rapid and accurate clinical diagnosis of small cell lung cancer. Summary of the Invention

[0006] One of the technical objectives of this invention is to overcome the shortcomings of existing technologies by preparing Au-Ag nanoclusters with high ECL efficiency as luminescent materials and FeMnO4 with strong catalytic activity. x As a co-reaction promoter;

[0007] The second technical objective of this invention is to design a controlled release system driven by proximity-induced DNA strand replacement, thereby constructing a biosensor based on proximity-induced DNA strand replacement-triggered controlled release, which is simple in process and safe in operation.

[0008] The third technical objective of this invention is to provide the application of a biosensor constructed by the aforementioned method based on proximity-induced DNA strand displacement-triggered controlled release, namely, for sensitive detection of NSE in serum media, 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 proximity-induced DNA strand displacement-triggered controlled release

[0011] The glassy carbon electrode was polished using alumina powder; then, it was sequentially modified with 3 μL of aminated FeMnO4 at a concentration of 3 mg / mL. x6-10 μL of Au-Ag nanoclusters were added; 8 μL of S3-S2-methylene blue / second antibody, 5 μL of 6-mercapto-1-hexanol, and 8 μL of NSE solution were added sequentially, and the mixture was incubated at 4 °C for 2 h; 8 μL of ZnS QDs@SiO2-S1-first antibody was then added, and the mixture was incubated at 4 °C for 2 h to construct a biosensor based on proximity-induced DNA strand displacement-triggered controlled release; S1, S2, and S3 refer to single strands of DNA with different base sequences.

[0012] The aminated FeMnO x The process involves dissolving 0.66 g of terephthalic acid and 8.32 g of NaOH in 200 mL of ultrapure water; adding 50 mL of a mixed solution containing 0.62 g of MnCl₂·4H₂O and 1.18 g of FeCl₂·4H₂O, and reacting for 24 h; centrifuging the resulting solution, washing with ultrapure water, and vacuum drying at 60 °C for 12 h to obtain the MIL-101 precursor; and calcining the obtained MIL-101 precursor in air at 500 °C for 6 h to generate FeMnO₂. x ; 480 mg of FeMnO x 480 μL of 3-aminopropyltriethoxysilane was dissolved in 24 mL of ethanol and refluxed at 80 °C for 12 h; the product was washed with ultrapure water and dried under vacuum at 60 °C to obtain aminated FeMnO. x ;

[0013] The Au-Ag nanoclusters were obtained by adding 17 mg of adenosine 5'-monophosphate disodium salt to 8.4 mL of ultrapure water; then adding 200 μL of 20 mM HAuCl4, 1 mL of 10 mM AgNO3, and 400 μL of 0.5 M citrate buffer solution sequentially, and mixing thoroughly; transferring the mixture to a high-pressure reactor and reacting at 120 °C for 30 min; and finally purifying with acetone to obtain the Au-Ag nanoclusters.

[0014] The S1-first antibody and S3-S2-methylene blue / second antibody are prepared by mixing 60 μL of 6.4 mM N-succinimide-3-maleimide benzoate with 50 μL of 50 μM first antibody solution and activating for 1 h to obtain solution 1; mixing 50 μL of 50 μM thiol-modified S1 and 10 μL of 10 mM tris(2-carboxyethyl)phosphine and reacting for 40 min to obtain solution 2; mixing solution 1 and solution 2 and reacting for 1 h to obtain S1-first antibody; synthesizing S2-methylene blue / second antibody using the same steps except that the second antibody and thiol-modified S2-methylene blue are used instead of the first antibody and thiol-modified S1; adding the prepared S2-methylene blue / second antibody to 50 μL of 50 μM S3 and reacting for 2 h to obtain S3-S2-methylene blue / second antibody;

[0015] The ZnS QDs@SiO2-S1-first antibody was prepared by heating 20 mL of a 15 mM Na2S·9H2O solution to 40 °C, then adding 20 mL of an 8 mM ZnCl2 solution dropwise; centrifuging and washing the resulting mixture, and then vacuum drying at 60 °C for 12 h to obtain a ZnS QDs solution; dissolving 0.5 g of hexadecyltrimethylammonium bromide in 15 mL of ultrapure water, and adding 0.2 g of urea while stirring; sequentially dispersing 1.25 mL of tetraethoxysilane, 12.5 mL of cyclohexane, and 460 μL of isopropanol in the above solution, and stirring at 70 °C for 15 h until a white precipitate appeared; centrifuging the resulting solution, washing three times with ultrapure water and methanol, and then vacuum drying at 60 °C; calcining the resulting powder at 550 °C for 10 h to obtain SiO2; and then calcining 0.2 g of SiO2 and 0.2 g of urea in 0.2 g of urea solution in 0.2 g of urea solution. 3-Aminopropyltriethoxysilane was dispersed in 10 mL of ethanol and refluxed at 80 °C for 12 h. The product was washed with ultrapure water and dried under vacuum at 60 °C to obtain aminated SiO2. 1 mg of aminated SiO2 was dissolved in 1 mL of ZnS QDs solution and shaken for 24 h. 50 μL of S1-first antibody was added and shaken for another 12 h at 4 °C. The product was centrifuged and washed with ultrapure water to obtain ZnS QDs@SiO2-S1-first antibody.

[0016] 2. The application of a biosensor based on proximity-induced DNA strand displacement-triggered controlled release constructed by the aforementioned method, characterized in that it is used for the detection of NSE in serum media.

[0017] A three-electrode system was constructed for signal testing, using the prepared biosensor based on proximity-induced DNA strand displacement-triggered controlled release as the working electrode, an Ag / AgCl electrode as the reference electrode, and a platinum electrode as the counter electrode. For ECL testing, a phosphate buffer solution containing 80 mM K2S2O8 with a pH of 7.0–8.0 was used as the detection solution, with a voltage range of 1.6–0 V and a photomultiplier tube voltage of 800 V. For DPV testing, a phosphate buffer solution with a pH of 7.0–8.0 was used as the detection solution, with a voltage range of -0.5–0.1 V, an amplitude of 50 mV, and a pulse width of 0.05 s. Based on the measured signals, working curves were plotted, revealing that the detection limits of the developed biosensor were 3.23 fg / mL and 31.8 fg / mL, respectively. The biosensor exhibited high stability, specificity, and reproducibility, making it suitable for trace detection of NSE in serum media.

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

[0019] 1. This invention prepares Au-Ag nanoclusters with high ECL efficiency as luminescent materials and FeMnO with strong catalytic activity. x As a co-reaction promoter, it significantly enhances the ECL signal of the sensing system, effectively improving the detection sensitivity;

[0020] 2. This invention designs a controlled release system driven by proximity-induced DNA strand displacement, thereby constructing a biosensor based on proximity-induced DNA strand displacement-triggered controlled release; based on the binding of NSE antigen and antibody, and the proximity-induced displacement of S1 and S3, the specificity and sensitivity of target markers are improved; at the same time, the dual-mode analysis of ECL and DPV signals effectively improves the detection accuracy.

[0021] 3. The biosensor constructed in this invention, based on proximity-induced DNA strand displacement-triggered controlled release, exhibits a wide linear range and low detection limit for the target NSE, as well as high stability, selectivity, and reproducibility. It is suitable for trace detection of NSE in serum media and has certain industrialization prospects. Attached Figure Description

[0022] Figure 1 The flowchart for constructing a biosensor based on proximity-induced DNA strand displacement-triggered controlled release is shown below (first antibody: Ab1; second antibody: Ab2; methylene blue: MB; glassy carbon electrode: GCE; Au-Ag nanoclusters: Au-Ag NCs; 6-mercapto-1-hexanol: MCH; resonance energy transfer: RET).

[0023] Figure 2 (A) Transmission electron microscopy (TEM) image and (B) High-resolution transmission electron microscopy (TEM) image of Au-Ag NCs.

[0024] Figure 3 The fluorescence excitation (black) and emission (red) spectra of Au-Ag NCs are shown (fluorescence: FL).

[0025] Figure 4 The X-ray photoelectron spectra of Au-Ag NCs are shown below, where (A) is the X-ray photoelectron spectrum of the whole region; (B) is the high-resolution X-ray photoelectron spectrum of the Ag3d region; (C) is the high-resolution X-ray photoelectron spectrum of the Au4f region; and (D) is the high-resolution X-ray photoelectron spectrum of the N1s region.

[0026] Figure 5 The X-ray diffraction pattern of ZnS QDs is shown.

[0027] Figure 6 (A) Transmission electron microscopy (TEM) image and (B) High-resolution transmission electron microscopy (TEM) image of ZnS QDs.

[0028] Figure 7 FeMnO x X-ray diffraction patterns of Mn3O4, Fe3O4 and Fe2O3.

[0029] Figure 8 FeMnO x The scanning electron microscope image and the corresponding elemental mapping.

[0030] Figure 9 FeMnO x The X-ray photoelectron spectra are shown in the figures, including (A) the full-area X-ray photoelectron spectrum; (B) the high-resolution X-ray photoelectron spectrum of the Fe 2p region; and (C) the high-resolution X-ray photoelectron spectrum of the Mn 2p region.

[0031] Figure 10 This is a transmission electron microscope (TEM) image of SiO2.

[0032] Figure 11 The images show the (A) nitrogen adsorption-desorption isotherm and (B) pore size distribution of SiO2.

[0033] Figure 12 The infrared spectrum of SiO2-NH2 (aminated SiO2: SiO2-NH2).

[0034] Figure 13 ECL and (B)CV responses of (a) Au NCs, (b) Ag NCs and (c) Au-Ag NCs in K2S2O8 at a concentration of 80 mM (Inset: Physical image under 365 nm UV irradiation; Cyclic voltammetry: CV).

[0035] Figure 14 for (a) GCE, (b) Au-Ag NCs / GCE and (c) Au-Ag NCs / FeMnO x ECL intensity-voltage curves of / GCE in K2S2O8 at a concentration of 80 mM, and (d) Au-Ag NCs / GCE and (e) Au-Ag NCs / FeMnO x / GCE ECL intensity-voltage curve in phosphate buffer solution.

[0036] Figure 15 This is a diagram showing the overlap between the FL emission spectrum of Au-Ag NCs and the UV absorption spectrum of ZnS QDs.

[0037] Figure 16 This is the electron paramagnetic resonance spectrum of the sensing system.

[0038] Figure 17 Au-Ag NCs / FeMnO x / S2O8 2- ECL mechanism diagram of the system.

[0039] Figure 18 Characterization diagram of the construction process of a biosensor based on proximity-induced DNA strand displacement-triggered controlled release, according to (A) CV and (B) AC impedance tests.

[0040] Figure 19 This is a diagram showing the condition optimization of a biosensor based on proximity-induced DNA strand displacement-triggered controlled release. (A) Solution pH optimization results; (B) K₂S₂O₈ concentration optimization results; (C) FeMnO₂ x -NH2 concentration optimization results diagram.

[0041] Figure 20 The following are calibration curves for (A) ECL response curves (10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL) and (B) corresponding to the biosensor based on proximity-induced DNA strand displacement-triggered controlled release after incubation with different concentrations of NSE; (C) DPV response curves (100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL) and (D) corresponding to the biosensor based on proximity-induced DNA strand displacement-triggered controlled release after incubation with different concentrations of NSE.

[0042] Figure 21The (A, D) selectivity, (B, E) stability, and (D, F) reproducibility of a biosensor based on proximity-induced DNA strand displacement-triggered controlled release (alpha-fetoprotein: AFP; glucose: Glu; prostate-specific antigen: PSA; carcinoembryonic antigen: CEA; alkaline phosphatase: ALP; squamous cell carcinoma antigen: SCCA; bovine serum albumin: BSA; neutrophil gelatinase-associated lipocalin: NGAL; relative standard deviation: RSD). Detailed Implementation

[0043] 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.

[0044] Example 1: Construction of a biosensor based on proximity-induced DNA strand displacement-triggered controlled release

[0045] The glassy carbon electrode was polished using alumina powder; then, it was sequentially modified with 3 μL of aminated FeMnO4 at a concentration of 3 mg / mL. x 6 μL of Au-Ag nanoclusters were added; 8 μL of S3-S2-methylene blue / second antibody, 5 μL of 6-mercapto-1-hexanol, and 8 μL of NSE solution were added sequentially, and the mixture was incubated at 4 °C for 2 h; 8 μL of ZnS QDs@SiO2-S1-first antibody was then added, and the mixture was incubated at 4 °C for 2 h to construct a biosensor based on proximity-induced DNA strand displacement-triggered controlled release; S1, S2, and S3 refer to single strands of DNA with different base sequences.

[0046] The aminated FeMnO x The process involves dissolving 0.66 g of terephthalic acid and 8.32 g of NaOH in 200 mL of ultrapure water; adding 50 mL of a mixed solution containing 0.62 g of MnCl₂·4H₂O and 1.18 g of FeCl₂·4H₂O, and reacting for 24 h; centrifuging the resulting solution, washing with ultrapure water, and vacuum drying at 60 °C for 12 h to obtain the MIL-101 precursor; and calcining the obtained MIL-101 precursor in air at 500 °C for 6 h to generate FeMnO₂. x ; 480 mg of FeMnO x 480 μL of 3-aminopropyltriethoxysilane was dissolved in 24 mL of ethanol and refluxed at 80 °C for 12 h; the product was washed with ultrapure water and dried under vacuum at 60 °C to obtain aminated FeMnO. x ;

[0047] The Au-Ag nanoclusters were obtained by adding 17 mg of adenosine 5'-monophosphate disodium salt to 8.4 mL of ultrapure water; then adding 200 μL of 20 mM HAuCl4, 1 mL of 10 mM AgNO3, and 400 μL of 0.5 M citrate buffer solution sequentially, and mixing thoroughly; transferring the mixture to a high-pressure reactor and reacting at 120 °C for 30 min; and finally purifying with acetone to obtain the Au-Ag nanoclusters.

[0048] The S1-first antibody and S3-S2-methylene blue / second antibody are prepared by mixing 60 μL of 6.4 mM N-succinimide-3-maleimide benzoate with 50 μL of 50 μM first antibody solution and activating for 1 h to obtain solution 1; mixing 50 μL of 50 μM thiol-modified S1 and 10 μL of 10 mM tris(2-carboxyethyl)phosphine and reacting for 40 min to obtain solution 2; mixing solution 1 and solution 2 and reacting for 1 h to obtain S1-first antibody; synthesizing S2-methylene blue / second antibody using the same steps except that the second antibody and thiol-modified S2-methylene blue are used instead of the first antibody and thiol-modified S1; adding the prepared S2-methylene blue / second antibody to 50 μL of 50 μM S3 and reacting for 2 h to obtain S3-S2-methylene blue / second antibody;

[0049] The ZnS QDs@SiO2-S1-first antibody was prepared by heating 20 mL of a 15 mM Na2S·9H2O solution to 40 °C, then adding 20 mL of an 8 mM ZnCl2 solution dropwise; centrifuging and washing the resulting mixture, and then vacuum drying at 60 °C for 12 h to obtain a ZnS QDs solution; dissolving 0.5 g of hexadecyltrimethylammonium bromide in 15 mL of ultrapure water, and adding 0.2 g of urea while stirring; sequentially dispersing 1.25 mL of tetraethoxysilane, 12.5 mL of cyclohexane, and 460 μL of isopropanol in the above solution, and stirring at 70 °C for 15 h until a white precipitate appeared; centrifuging the resulting solution, washing three times with ultrapure water and methanol, and then vacuum drying at 60 °C; calcining the resulting powder at 550 °C for 10 h to obtain SiO2; and then calcining 0.2 g of SiO2 and 0.2 g of urea in 0.2 g of urea solution in 0.2 g of urea solution. 3-Aminopropyltriethoxysilane was dispersed in 10 mL of ethanol and refluxed at 80 °C for 12 h. The product was washed with ultrapure water and dried under vacuum at 60 °C to obtain aminated SiO2. 1 mg of aminated SiO2 was dissolved in 1 mL of ZnS QDs solution and shaken for 24 h. 50 μL of S1-first antibody was added and shaken for another 12 h at 4 °C. The product was centrifuged and washed with ultrapure water to obtain ZnS QDs@SiO2-S1-first antibody.

[0050] Example 2: Construction of a biosensor based on proximity-induced DNA strand displacement-triggered controlled release

[0051] The glassy carbon electrode was polished using alumina powder; then, it was sequentially modified with 3 μL of aminated FeMnO4 at a concentration of 3 mg / mL. x 8 μL of Au-Ag nanoclusters were added; 8 μL of S3-S2-methylene blue / second antibody, 5 μL of 6-mercapto-1-hexanol, and 8 μL of NSE solution were added sequentially, and the mixture was incubated at 4 °C for 2 h; 8 μL of ZnS QDs@SiO2-S1-first antibody was then added, and the mixture was incubated at 4 °C for 2 h to construct a biosensor based on proximity-induced DNA strand displacement-triggered controlled release; S1, S2, and S3 refer to single strands of DNA with different base sequences.

[0052] The aminated FeMnO xThe process involves dissolving 0.66 g of terephthalic acid and 8.32 g of NaOH in 200 mL of ultrapure water; adding 50 mL of a mixed solution containing 0.62 g of MnCl₂·4H₂O and 1.18 g of FeCl₂·4H₂O, and reacting for 24 h; centrifuging the resulting solution, washing with ultrapure water, and vacuum drying at 60 °C for 12 h to obtain the MIL-101 precursor; and calcining the obtained MIL-101 precursor in air at 500 °C for 6 h to generate FeMnO₂. x ; 480 mg of FeMnO x 480 μL of 3-aminopropyltriethoxysilane was dissolved in 24 mL of ethanol and refluxed at 80 °C for 12 h; the product was washed with ultrapure water and dried under vacuum at 60 °C to obtain aminated FeMnO. x ;

[0053] The Au-Ag nanoclusters were obtained by adding 17 mg of adenosine 5'-monophosphate disodium salt to 8.4 mL of ultrapure water; then adding 200 μL of 20 mM HAuCl4, 1 mL of 10 mM AgNO3, and 400 μL of 0.5 M citrate buffer solution sequentially, and mixing thoroughly; transferring the mixture to a high-pressure reactor and reacting at 120 °C for 30 min; and finally purifying with acetone to obtain the Au-Ag nanoclusters.

[0054] The S1-first antibody and S3-S2-methylene blue / second antibody are prepared by mixing 60 μL of 6.4 mM N-succinimide-3-maleimide benzoate with 50 μL of 50 μM first antibody solution and activating for 1 h to obtain solution 1; mixing 50 μL of 50 μM thiol-modified S1 and 10 μL of 10 mM tris(2-carboxyethyl)phosphine and reacting for 40 min to obtain solution 2; mixing solution 1 and solution 2 and reacting for 1 h to obtain S1-first antibody; synthesizing S2-methylene blue / second antibody using the same steps except that the second antibody and thiol-modified S2-methylene blue are used instead of the first antibody and thiol-modified S1; adding the prepared S2-methylene blue / second antibody to 50 μL of 50 μM S3 and reacting for 2 h to obtain S3-S2-methylene blue / second antibody;

[0055] The ZnS QDs@SiO2-S1-first antibody was prepared by heating 20 mL of a 15 mM Na2S·9H2O solution to 40 °C, then adding 20 mL of an 8 mM ZnCl2 solution dropwise; centrifuging and washing the resulting mixture, and then vacuum drying at 60 °C for 12 h to obtain a ZnS QDs solution; dissolving 0.5 g of hexadecyltrimethylammonium bromide in 15 mL of ultrapure water, and adding 0.2 g of urea while stirring; sequentially dispersing 1.25 mL of tetraethoxysilane, 12.5 mL of cyclohexane, and 460 μL of isopropanol in the above solution, and stirring at 70 °C for 15 h until a white precipitate appeared; centrifuging the resulting solution, washing three times with ultrapure water and methanol, and then vacuum drying at 60 °C; calcining the resulting powder at 550 °C for 10 h to obtain SiO2; and then calcining 0.2 g of SiO2 and 0.2 g of urea in 0.2 g of urea solution in 0.2 g of urea solution. 3-Aminopropyltriethoxysilane was dispersed in 10 mL of ethanol and refluxed at 80 °C for 12 h. The product was washed with ultrapure water and dried under vacuum at 60 °C to obtain aminated SiO2. 1 mg of aminated SiO2 was dissolved in 1 mL of ZnS QDs solution and shaken for 24 h. 50 μL of S1-first antibody was added and shaken for another 12 h at 4 °C. The product was centrifuged and washed with ultrapure water to obtain ZnS QDs@SiO2-S1-first antibody.

[0056] Example 3: Construction of a biosensor based on proximity-induced DNA strand displacement-triggered controlled release

[0057] The glassy carbon electrode was polished using alumina powder; then, it was sequentially modified with 3 μL of aminated FeMnO4 at a concentration of 3 mg / mL. x 10 μL of Au-Ag nanoclusters were added; 8 μL of S3-S2-methylene blue / second antibody, 5 μL of 6-mercapto-1-hexanol, and 8 μL of NSE solution were added sequentially, and the mixture was incubated at 4 °C for 2 h; 8 μL of ZnS QDs@SiO2-S1-first antibody was then added, and the mixture was incubated at 4 °C for 2 h to construct a biosensor based on proximity-induced DNA strand displacement-triggered controlled release; S1, S2, and S3 refer to single strands of DNA with different base sequences.

[0058] The aminated FeMnO xThe process involves dissolving 0.66 g of terephthalic acid and 8.32 g of NaOH in 200 mL of ultrapure water; adding 50 mL of a mixed solution containing 0.62 g of MnCl₂·4H₂O and 1.18 g of FeCl₂·4H₂O, and reacting for 24 h; centrifuging the resulting solution, washing with ultrapure water, and vacuum drying at 60 °C for 12 h to obtain the MIL-101 precursor; and calcining the obtained MIL-101 precursor in air at 500 °C for 6 h to generate FeMnO₂. x ; 480 mg of FeMnO x 480 μL of 3-aminopropyltriethoxysilane was dissolved in 24 mL of ethanol and refluxed at 80 °C for 12 h; the product was washed with ultrapure water and dried under vacuum at 60 °C to obtain aminated FeMnO. x ;

[0059] The Au-Ag nanoclusters were obtained by adding 17 mg of adenosine 5'-monophosphate disodium salt to 8.4 mL of ultrapure water; then adding 200 μL of 20 mM HAuCl4, 1 mL of 10 mM AgNO3, and 400 μL of 0.5 M citrate buffer solution sequentially, and mixing thoroughly; transferring the mixture to a high-pressure reactor and reacting at 120 °C for 30 min; and finally purifying with acetone to obtain the Au-Ag nanoclusters.

[0060] The S1-first antibody and S3-S2-methylene blue / second antibody are prepared by mixing 60 μL of 6.4 mM N-succinimide-3-maleimide benzoate with 50 μL of 50 μM first antibody solution and activating for 1 h to obtain solution 1; mixing 50 μL of 50 μM thiol-modified S1 and 10 μL of 10 mM tris(2-carboxyethyl)phosphine and reacting for 40 min to obtain solution 2; mixing solution 1 and solution 2 and reacting for 1 h to obtain S1-first antibody; synthesizing S2-methylene blue / second antibody using the same steps except that the second antibody and thiol-modified S2-methylene blue are used instead of the first antibody and thiol-modified S1; adding the prepared S2-methylene blue / second antibody to 50 μL of 50 μM S3 and reacting for 2 h to obtain S3-S2-methylene blue / second antibody;

[0061] The ZnS QDs@SiO2-S1-first antibody was prepared by heating 20 mL of a 15 mM Na2S·9H2O solution to 40 °C, then adding 20 mL of an 8 mM ZnCl2 solution dropwise; centrifuging and washing the resulting mixture, and then vacuum drying at 60 °C for 12 h to obtain a ZnS QDs solution; dissolving 0.5 g of hexadecyltrimethylammonium bromide in 15 mL of ultrapure water, and adding 0.2 g of urea while stirring; sequentially dispersing 1.25 mL of tetraethoxysilane, 12.5 mL of cyclohexane, and 460 μL of isopropanol in the above solution, and stirring at 70 °C for 15 h until a white precipitate appeared; centrifuging the resulting solution, washing three times with ultrapure water and methanol, and then vacuum drying at 60 °C; calcining the resulting powder at 550 °C for 10 h to obtain SiO2; and then calcining 0.2 g of SiO2 and 0.2 g of urea in 0.2 g of urea solution in 0.2 g of urea solution. 3-Aminopropyltriethoxysilane was dispersed in 10 mL of ethanol and refluxed at 80 °C for 12 h. The product was washed with ultrapure water and dried under vacuum at 60 °C to obtain aminated SiO2. 1 mg of aminated SiO2 was dissolved in 1 mL of ZnS QDs solution and shaken for 24 h. 50 μL of S1-first antibody was added and shaken for another 12 h at 4 °C. The product was centrifuged and washed with ultrapure water to obtain ZnS QDs@SiO2-S1-first antibody.

[0062] Example 4: An application of a biosensor based on proximity-induced DNA strand displacement-triggered controlled release, constructed using the methods described in Examples 1, 2, and 3, characterized in that it is used for the detection of NSE in serum media.

[0063] A three-electrode system was constructed for signal testing, using the prepared biosensor based on proximity-induced DNA strand displacement-triggered controlled release as the working electrode, an Ag / AgCl electrode as the reference electrode, and a platinum electrode as the counter electrode. For ECL testing, a phosphate buffer solution containing 80 mM K2S2O8 at pH 7.0 was used as the detection solution, with a voltage range of 1.6–0 V and a photomultiplier tube voltage of 800 V. For DPV testing, a phosphate buffer solution at pH 7.0 was used as the detection solution, with a voltage range of -0.5–0.1 V, an amplitude of 50 mV, and a pulse width of 0.05 s. Based on the measured signals, working curves were plotted, revealing that the detection limits of the developed biosensor were 3.23 fg / mL and 31.8 fg / mL, respectively. The biosensor exhibited high stability, specificity, and reproducibility, making it suitable for trace detection of NSE in serum media.

[0064] Example 5: An application of a biosensor based on proximity-induced DNA strand displacement-triggered controlled release, constructed using the methods described in Examples 1, 2, and 3, characterized in that it is used for the detection of NSE in serum media.

[0065] A three-electrode system was constructed for signal testing, using the prepared biosensor based on proximity-induced DNA strand displacement-triggered controlled release as the working electrode, an Ag / AgCl electrode as the reference electrode, and a platinum electrode as the counter electrode. For ECL testing, a phosphate buffer solution containing 80 mM K2S2O8 at pH 7.4 was used as the detection solution, with a voltage range of 1.6–0 V and a photomultiplier tube voltage of 800 V. For DPV testing, a phosphate buffer solution at pH 7.4 was used as the detection solution, with a voltage range of -0.5–0.1 V, an amplitude of 50 mV, and a pulse width of 0.05 s. Based on the measured signals, working curves were plotted, revealing that the detection limits of the developed biosensor were 3.23 fg / mL and 31.8 fg / mL, respectively. The biosensor exhibited high stability, specificity, and reproducibility, making it suitable for trace detection of NSE in serum media.

[0066] Example 6: An application of a biosensor based on proximity-induced DNA strand displacement-triggered controlled release, constructed using the methods described in Examples 1, 2, and 3, characterized in that it is used for the detection of NSE in serum media.

[0067] A three-electrode system was constructed for signal testing, using the prepared biosensor based on proximity-induced DNA strand displacement-triggered controlled release as the working electrode, an Ag / AgCl electrode as the reference electrode, and a platinum electrode as the counter electrode. For ECL testing, a phosphate buffer solution containing 80 mM K2S2O8 at pH 8.0 was used as the detection solution, with a voltage range of 1.6–0 V and a photomultiplier tube voltage of 800 V. For DPV testing, a phosphate buffer solution at pH 8.0 was used as the detection solution, with a voltage range of -0.5–0.1 V, an amplitude of 50 mV, and a pulse width of 0.05 s. Based on the measured signals, working curves were plotted, revealing that the detection limits of the developed biosensor were 3.23 fg / mL and 31.8 fg / mL, respectively. The biosensor exhibited high stability, specificity, and reproducibility, making it suitable for trace detection of NSE in serum media.

Claims

1. Construction of a biosensor based on proximity-induced DNA strand displacement-triggered controlled release, characterized in that, The glassy carbon electrode was polished using alumina powder; then, it was sequentially modified with 3 μL of aminated FeMnO4 at a concentration of 3 mg / mL. x 6–10 μL of Au-Ag nanoclusters were added; 8 μL of S3-S2-methylene blue / second antibody, 5 μL of 6-mercapto-1-hexanol, and 8 μL of neuron-specific enolase solution were added sequentially, and the mixture was incubated at 4 °C for 2 h; 8 μL of ZnSQDs@SiO2-S1-first antibody was then added and incubated at 4 °C for 2 h to construct a biosensor based on proximity-induced DNA strand displacement-triggered controlled release; S1, S2, and S3 refer to single strands of DNA with different base sequences.

2. The construction of a biosensor based on proximity-induced DNA strand displacement-triggered controlled release as described in claim 1, characterized in that, The aminated FeMnO x The process involves dissolving 0.66 g of terephthalic acid and 8.32 g of NaOH in 200 mL of ultrapure water; adding 50 mL of a mixed solution containing 0.62 g of MnCl₂·4H₂O and 1.18 g of FeCl₂·4H₂O, and reacting for 24 h; centrifuging the resulting solution, washing with ultrapure water, and vacuum drying at 60 °C for 12 h to obtain the MIL-101 precursor; and calcining the obtained MIL-101 precursor in air at 500 °C for 6 h to generate FeMnO₂. x ; 480 mg of FeMnO x 480 μL of 3-aminopropyltriethoxysilane was dissolved in 24 mL of ethanol and refluxed at 80 °C for 12 h; the product was washed with ultrapure water and dried under vacuum at 60 °C to obtain aminated FeMnO. x .

3. The construction of a biosensor based on proximity-induced DNA strand displacement-triggered controlled release as described in claim 1, characterized in that, The Au-Ag nanoclusters were obtained by adding 17 mg of adenosine 5'-monophosphate disodium salt to 8.4 mL of ultrapure water; then adding 200 μL of 20 mM HAuCl4, 1 mL of 10 mM AgNO3, and 400 μL of 0.5 M citrate buffer sequentially, and mixing thoroughly; transferring the mixture to a high-pressure reactor and reacting at 120 °C for 30 min; and finally purifying with acetone to obtain the Au-Ag nanoclusters.

4. The construction of a biosensor based on proximity-induced DNA strand displacement-triggered controlled release as described in claim 1, characterized in that, The S1-first antibody and S3-S2-methylene blue / second antibody are prepared by mixing 60 μL of 6.4 mM N-succinimide-3-maleimide benzoate with 50 μL of 50 μM first antibody solution and activating for 1 h to obtain solution 1; mixing 50 μL of 50 μM thiol-modified S1 and 10 μL of 10 mM tris(2-carboxyethyl)phosphine and reacting for 40 min to obtain solution 2; and mixing solution 1 and solution 2 and reacting for 1 h to obtain S1-first antibody. Except that the first antibody and the thiol-modified S1 were replaced with the second antibody and the thiol-modified S2-methylene blue, the same steps were used to synthesize the S2-methylene blue / second antibody; the prepared S2-methylene blue / second antibody was added to 50 μL of S3 with a concentration of 50 μM and reacted for 2 h to obtain S3-S2-methylene blue / second antibody.

5. The construction of a biosensor based on proximity-induced DNA strand displacement-triggered controlled release as described in claim 1, characterized in that, The ZnS QDs@SiO2-S1-first antibody was prepared by heating 20 mL of a 15 mM Na2S·9H2O solution to 40 °C, then adding 20 mL of an 8 mM ZnCl2 solution dropwise; centrifuging and washing the resulting mixture, and then vacuum drying at 60 °C for 12 h to obtain a ZnS QDs solution; dissolving 0.5 g of hexadecyltrimethylammonium bromide in 15 mL of ultrapure water, and adding 0.2 g of urea while stirring; sequentially dispersing 1.25 mL of tetraethoxysilane, 12.5 mL of cyclohexane, and 460 μL of isopropanol in the above solution, and stirring at 70 °C for 15 h until a white precipitate appeared; centrifuging the resulting solution, washing three times with ultrapure water and methanol, and then vacuum drying at 60 °C; calcining the resulting powder at 550 °C for 10 h to obtain SiO2; and then calcining 0.2 g of SiO2 and 0.2 g of urea in the solution. 3-Aminopropyltriethoxysilane was dispersed in 10 mL of ethanol and refluxed at 80 °C for 12 h. The product was washed with ultrapure water and dried under vacuum at 60 °C to obtain aminated SiO2. 1 mg of aminated SiO2 was dissolved in 1 mL of ZnS QDs solution and shaken for 24 h. 50 μL of S1-first antibody was added and shaken for another 12 h at 4 °C. The product was centrifuged and washed with ultrapure water to obtain ZnS QDs@SiO2-S1-first antibody.

6. The use of a biosensor based on proximity-induced DNA strand displacement-triggered controlled release constructed by the method described in claim 1, characterized in that, Applications for detecting neuron-specific enolases.

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