A nanobiosensor fluorescence probe, fluorescence sensor and their application in detecting B-type natriuretic peptide

Abstract

The present invention belongs to the field of medicine and biochemistry, and specifically discloses a nano-bioluminescent probe, a fluorescent sensor, and their application in detecting B-type natriuretic peptide. The nano-bioluminescent probe comprises a palladium nanomaterial and a nucleic acid aptamer adsorbed on the palladium nanomaterial, the nucleic acid aptamer being labeled with a fluorescent dye, and the palladium nanomaterial having strong and broad absorption in the range of 600 to 1100 nm; the fluorescent sensor comprises the nano-bioluminescent probe, the nucleic acid aptamer being a nucleic acid aptamer specific for B-type natriuretic peptide, and the emission wavelength of the fluorescent dye-modified nucleic acid aptamer being at 708 nm; when the palladium nanomaterial and the nucleic acid aptamer are sufficiently close to each other, fluorescence resonance energy transfer occurs in the near-infrared region, and the detection of B-type natriuretic peptide, a marker of heart failure, is achieved through the energy transfer process between the fluorescent donor and the acceptor, with the advantages of simple operation, high sensitivity, and stability.

Description

Technical Field

[0001] The present invention relates to the technical field of medicine and biochemistry, and in particular to a nano-biofluorescent probe, a fluorescent sensor and applications thereof in detecting B-type natriuretic peptide. Background Art

[0002] Brain natriuretic peptide (BNP), also known as B-type natriuretic peptide, is a member of a family of structurally related peptide hormones produced by heart cells. When heart function fails, increased cardiac volume or pressure overload causes myocardial stretch or increased ventricular wall pressure, leading to elevated blood BNP concentrations. BNP is the gold standard for heart failure. Currently commercially available BNP detection kits, such as enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatography, rely on the specific recognition of antigens and antibodies. However, antibodies have limited stability and significant batch-to-batch variability. Aptamers are RNA or DNA fragments identified through in vitro screening that specifically bind to targets such as proteins. In contrast, biosensing methods leveraging aptamer recognition are more stable and avoid cross-reactivity. Nanomaterials, such as metal or semiconductor nanoparticles and nanorods, exhibit dimensions similar to those of biomolecules such as proteins (enzymes, antigens, antibodies) or DNA. Nanoparticles possess unique electronic, photonic, and catalytic properties, while biomaterials possess unique recognition, catalytic, and inhibitory properties. Their combination can produce novel hybrid nanobiomaterials with synergistic properties and functions. Therefore, the development of a rapid, simple, highly sensitive, specific, and low-cost BNP detection method would be of great significance for the timely diagnosis of heart failure. Summary of the Invention

[0003] In view of the limitations of the prior art described above, the purpose of the present invention is to provide a nano-biofluorescent probe, a fluorescent sensor and their application in detecting B-type natriuretic peptide, and to provide a simple, sensitive and stable method for the detection of B-type natriuretic peptide.

[0004] To achieve the above-mentioned and other related purposes, the first aspect of the present invention provides a nano-biofluorescent probe, comprising a palladium nanomaterial and a nucleic acid aptamer adsorbed on the palladium nanomaterial, wherein the nucleic acid aptamer is labeled with a fluorescent dye, and the palladium nanomaterial has strong and broad absorption in the range of 600 to 1100 nm.

[0005] Furthermore, the nucleic acid aptamer is a nucleic acid aptamer specific for B-type natriuretic peptide, and the emission wavelength of the fluorescent dye-modified nucleic acid aptamer is at 708 nm.

[0006] Furthermore, the nano-bioluminescent probe is used as a composite probe in a fluorescent sensor for detecting B-type natriuretic peptide, the palladium nanomaterial serves as an energy acceptor, and the nucleic acid aptamer serves as an energy donor. When the distance between the palladium nanomaterial and the nucleic acid aptamer is less than a preset distance threshold, fluorescence resonance energy transfer occurs between the two in the near-infrared region, resulting in fluorescence quenching of the fluorescent dye. When B-type natriuretic peptide is present in the sample, the B-type natriuretic peptide binds to the nucleic acid aptamer through specific binding force to form a complex, so that the nucleic acid aptamer is away from the palladium nanomaterial, allowing the fluorescence of the fluorescent dye to be restored, and the concentration of B-type natriuretic peptide in the sample is positively correlated with the degree of fluorescence recovery.

[0007] Furthermore, the nucleic acid aptamer sequence is: 5'-TTT TTT TAA ACG CTC AAA GGA CAG AGGGTG CGT AGG AAG GGT ATT CGA CAG GAG GCT CAC A-3'.

[0008] Furthermore, the 5' end of the nucleic acid aptamer is labeled with a fluorescent dye.

[0009] Furthermore, the fluorescent dye is selected from cyanine dyes, fluorescein and its derivatives, and rhodamine, preferably cyanine dyes, and more preferably any one of Cy3, Cy3.5, Cy5, Cy5.5, Cy7 and Cy7.5.

[0010] A second aspect of the present invention provides a fluorescence sensor for detecting B-type natriuretic peptide, wherein the fluorescence sensor comprises the nano-biofluorescent probe described in the first aspect.

[0011] Furthermore, in the fluorescence sensor, the concentration of the palladium nanomaterial is 20 to 50 μg / mL, preferably 25 to 35 μg / mL, and more preferably 30 μg / mL.

[0012] Furthermore, the pH of the fluorescence sensor reaction system is 7.0 to 8.5, preferably 7.0 to 8.0, and more preferably 7.4.

[0013] Furthermore, the fluorescence sensor is used to detect samples, and the measurement parameters of the fluorescence intensity of the solution measured on a fluorescence spectrophotometer include: an excitation wavelength of 675 nm, an emission wavelength of 708 nm, and an emission spectrum scanning range of 690 to 750 nm.

[0014] Furthermore, the fluorescence sensor has a good linear relationship when the concentration of B-type natriuretic peptide in the sample is 0.05-10 pg / mL; preferably, the standard curve equation of the fluorescence sensor for quantifying the concentration of B-type natriuretic peptide in the sample solution is: Y=0.98lnX+5.696(r=0.9923).

[0015] The third aspect of the present invention provides a method for detecting B-type natriuretic peptide using the fluorescent sensor described in the second aspect, wherein the method is not for disease or treatment purposes and comprises the following steps:

[0016] The palladium nanomaterial dispersion, buffer solution and nucleic acid aptamer solution labeled with fluorescent dye are mixed and allowed to stand; then the sample solution to be tested is added, mixed and allowed to stand, and the fluorescence intensity of the solution is measured on a fluorescence spectrophotometer.

[0017] Furthermore, the pH of the buffer solution is 7.0 to 8.5, preferably 7.0 to 8.0, and more preferably 7.4.

[0018] Furthermore, the buffer solution is selected from any one of Tris-HCl buffer solution and PBS buffer solution.

[0019] Furthermore, the operation process of the method is carried out at room temperature.

[0020] Furthermore, the palladium nanomaterial dispersion and the nucleic acid aptamer solution labeled with a fluorescent dye are vortex-mixed and then allowed to stand for 5 to 15 minutes before adding the test sample solution.

[0021] Furthermore, after adding the sample solution to be tested and vortex mixing, the solution was allowed to stand for 20 to 40 minutes and then the fluorescence intensity of the solution was measured on a fluorescence spectrophotometer.

[0022] Furthermore, the preparation method of the palladium nanomaterial dispersion includes the following steps: dissolving a palladium precursor salt, citric acid, hexadecyltrimethylammonium bromide, and polyvinylpyrrolidone in an organic solvent, then adding tungsten hexacarbonyl, and reacting by a solvent thermal method; after the reaction is completed, cooling the reaction liquid, centrifuging to obtain a palladium nanomaterial, and then dispersing the palladium nanomaterial in a solvent to obtain a palladium nanomaterial dispersion.

[0023] Furthermore, the preparation method of the palladium nanomaterial dispersion includes the following steps: adding palladium precursor salt, citric acid, hexadecyltrimethylammonium bromide, and polyvinylpyrrolidone to an organic solvent, stirring at a speed of 500 to 800 r / min for 0.5 to 2 hours to dissolve them, thereby obtaining an orange-yellow solution; adding hexacarbonyl tungsten to the orange-yellow solution under a protective gas atmosphere, heating to 75 to 85° C., and reacting by a solvent thermal method for 0.5 to 2 hours to obtain a dark green reaction liquid; after the reaction is completed, cooling the reaction liquid, centrifuging it to obtain a palladium nanomaterial, and then dispersing the palladium nanomaterial in a solvent to obtain a dark blue palladium nanomaterial dispersion.

[0024] Furthermore, in the preparation method of the palladium nanomaterial dispersion, the molar ratio of palladium precursor salt, citric acid, hexadecyltrimethylammonium bromide, polyvinylpyrrolidone and tungsten hexacarbonyl is 1-2:5-20:2-10:0.005-0.03:5-10.

[0025] Furthermore, the palladium precursor salt is a precursor salt of divalent palladium, preferably palladium acetylacetonate.

[0026] Furthermore, the organic solvent is selected from N,N-dimethylformamide.

[0027] Furthermore, the protective gas is selected from argon or helium.

[0028] Furthermore, after the reaction is completed, the reaction solution is cooled to room temperature, acetone is added, mixed, and centrifuged at a speed of 8000-12000 r / min for 10-30 minutes. The supernatant is discarded, and the lower precipitate is resuspended with anhydrous ethanol and then ultrasonically dispersed to obtain a palladium nanomaterial dispersion.

[0029] The fourth aspect of the present invention provides the use of the nanobioluminescent probe according to the first aspect, the fluorescent sensor according to the second aspect, or the method according to the third aspect in detecting B-type natriuretic peptide, wherein the use is for non-disease diagnosis or treatment purposes.

[0030] As described above, the nano-bioluminescent probe, the fluorescent sensor and the application thereof in detecting B-type natriuretic peptide of the present invention have the following beneficial effects:

[0031] The present invention constructs a nano-bioluminescent probe and a fluorescent sensor, and based on this, constructs a fluorescence method with efficient resonance energy transfer in the near-infrared region. By improving the efficiency of resonance energy transfer, the sensitivity of detecting B-type natriuretic peptide is greatly improved, and the minimum detection limit reaches 45 fg / mL. In addition, the method is stable, can avoid cross-interference, does not require time-consuming and tedious separation of the reaction solution, and does not require magnetic bead separation, which can reduce costs and is suitable for promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Shown is a fluorescence sensing method based on efficient resonance energy transfer in the near-infrared region for detecting BNP in one embodiment of the present application;

[0033] Figure 2 Shown is the UV-visible spectrum of the palladium nanomaterial in one embodiment of the present application;

[0034] Figure 3 Shown is the UV-visible absorption spectrum (black line) of the palladium nanomaterial and the fluorescence emission spectrum (red line) of Cy5.5-aptamer in one embodiment of the present application;

[0035] Figure 4 Shown is a bar graph showing the effect of different concentrations of palladium nanomaterials on the fluorescence quenching of Cy5.5-aptamer and the fluorescence recovery after the addition of BNP in an embodiment of the present application;

[0036] Figure 5 Shown is a bar graph showing the effect of different pH values on the fluorescence quenching of Cy5.5-aptamer and the fluorescence recovery after adding BNP in an embodiment of the present application;

[0037] Figure 6 Shown is a spectrum showing the effect of BNP on the fluorescence recovery of the palladium nanomaterial / Cy5.5-aptamer composite probe in one embodiment of the present application;

[0038] Figure 7 The figure shows a linear relationship fitted by the exponential of fluorescence recovery efficiency relative to BNP concentration in one embodiment of the present application. DETAILED DESCRIPTION

[0039] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0040] An embodiment of the present application provides a nano-bioluminescent probe, comprising a palladium nanomaterial and a nucleic acid aptamer adsorbed on the palladium nanomaterial, wherein the nucleic acid aptamer is labeled with a fluorescent dye, and the palladium nanomaterial has strong and broad absorption in the range of 600 to 1100 nm.

[0041] In a specific embodiment of the present application, the nucleic acid aptamer is a nucleic acid aptamer specific for B-type natriuretic peptide, and the emission wavelength of the fluorescent dye-modified nucleic acid aptamer is at 708 nm; the nano-bioluminescent probe is used as a composite probe in a fluorescent sensor for detecting B-type natriuretic peptide, the palladium nanomaterial serves as an energy acceptor, and the nucleic acid aptamer serves as an energy donor. When the distance between the palladium nanomaterial and the nucleic acid aptamer is less than a preset distance threshold, fluorescence resonance energy transfer occurs between the two in the near-infrared region, resulting in fluorescence quenching of the fluorescent dye; when B-type natriuretic peptide is present in the sample, the B-type natriuretic peptide binds to the nucleic acid aptamer through specific binding force to form a complex, so that the nucleic acid aptamer is away from the palladium nanomaterial, so that the fluorescence of the fluorescent dye is restored, and the concentration of B-type natriuretic peptide in the sample is positively correlated with the degree of fluorescence recovery.

[0042] The palladium nanomaterials used in the embodiments of the present invention exhibit adjustable surface plasmon resonance absorption characteristics in the near-infrared region. In addition to their unique optical properties, they also have a large surface area and have potential application prospects at the biological level. Based on the high energy absorption properties of palladium nanomaterials in the range of 600-1100nm as energy acceptors and fluorescent dye-modified nucleic acid aptamers with an emission wavelength of 708nm as energy donors, when the two are sufficiently close, efficient resonance energy transfer occurs in the near-infrared region. This energy transfer process is linearly positively correlated with BNP concentration. A simple, highly sensitive and stable BNP detection method has been developed. This method does not require magnetic bead separation, which can reduce costs.

[0043] In a specific embodiment of the present application, the preparation method of the palladium nanomaterial comprises the following steps: dissolving a palladium precursor salt, citric acid, hexadecyltrimethylammonium bromide, and polyvinylpyrrolidone in an organic solvent, then adding hexacarbonyl tungsten, and reacting by a solvent thermal method; after the reaction is completed, the reaction liquid is cooled and centrifuged to obtain the palladium nanomaterial. Specifically, the palladium precursor salt, citric acid, hexadecyltrimethylammonium bromide, and polyvinylpyrrolidone are added to an organic solvent, and stirred at a speed of 500 to 800 r / min for 0.5 to 2 hours using a nanosynthesizer to dissolve them to obtain an orange-yellow solution; under a protective gas atmosphere, tungsten hexacarbonyl is added to the orange-yellow solution, heated to 75 to 85°C, and reacted by a solvent thermal method for 0.5 to 2 hours to obtain a dark green reaction liquid; after the reaction is completed, the reaction liquid is cooled and centrifuged to obtain the palladium nanomaterial.

[0044] In the preparation method of the nanosheet dispersion, the molar ratio of palladium precursor salt, citric acid, hexadecyltrimethylammonium bromide, polyvinylpyrrolidone and tungsten hexacarbonyl is 1-2:5-20:2-10:0.005-0.03:5-10; the palladium precursor salt is a precursor salt of divalent palladium, preferably palladium acetylacetonate; the organic solvent is selected from N,N-dimethylformamide; the protective gas is selected from argon or helium; after the reaction is completed, the reaction solution is cooled to room temperature, acetone is added, mixed and centrifuged at a speed of 8000-12000 r / min for 10-30 minutes, the supernatant is discarded, and the obtained lower layer precipitate is the palladium nanomaterial.

[0045] In a specific embodiment of the present application, the sequence of the nucleic acid aptamer is: 5'-TTT TTT TAA ACGCTC AAA GGA CAG AGG GTG CGT AGG AAG GGT ATT CGA CAG GAG GCT CAC A-3'; further, the 5' end of the nucleic acid aptamer is labeled with a fluorescent dye, preferably, the fluorescent dye is selected from cyanine dyes, fluorescein and its derivatives, and rhodamine, more preferably cyanine dyes, and most preferably any one of Cy3, Cy3.5, Cy5, Cy5.5, Cy7 and Cy7.5.

[0046] Another embodiment of the present invention provides a fluorescence sensor for detecting B-type natriuretic peptide, wherein the fluorescence sensor includes the nano-bioluminescent probe described in the above embodiment.

[0047] In a specific embodiment of the present application, in the fluorescence sensor, the concentration of the palladium nanomaterial is 20-50 μg / mL, preferably 25-35 μg / mL, and more preferably 30 μg / mL.

[0048] In a specific embodiment of the present application, the pH of the fluorescence sensor reaction system is 7.0 to 8.5, preferably 7.0 to 8.0, and more preferably 7.4.

[0049] In a specific embodiment of the present application, the fluorescence sensor is used to detect samples, and the measurement parameters for measuring the fluorescence intensity of the solution on a fluorescence spectrophotometer include: an excitation wavelength of 675 nm, an emission wavelength of 708 nm, and an emission spectrum scanning range of 690 to 750 nm.

[0050] In a specific embodiment of the present application, the fluorescence sensor has a good linear relationship when the concentration of B-type natriuretic peptide in the sample is 0.05-10 pg / mL; preferably, the standard curve equation of the fluorescence sensor for quantifying the concentration of B-type natriuretic peptide in the sample solution is: Y=0.98lnX+5.696(r=0.9923).

[0051] Another embodiment of the present invention provides a method for detecting B-type natriuretic peptide using the fluorescent sensor described in the above embodiment. The method is not for disease or treatment purposes and comprises the following steps:

[0052] The palladium nanomaterial dispersion, buffer solution and nucleic acid aptamer solution labeled with fluorescent dye are mixed and allowed to stand; then the sample solution to be tested is added, mixed and allowed to stand, and the fluorescence intensity of the solution is measured on a fluorescence spectrophotometer.

[0053] In a specific embodiment of the present application, the pH of the buffer solution is 7.0 to 8.5, preferably 7.0 to 8.0, and more preferably 7.4; the buffer solution is selected from any one of Tris-HCl buffer solution and PBS buffer solution.

[0054] In a specific embodiment of the present application, the operation process of the method is carried out at room temperature.

[0055] In a specific embodiment of the present application, the palladium nanomaterial dispersion and the nucleic acid aptamer solution labeled with a fluorescent dye are vortex-mixed, and then the sample solution to be tested is added after standing for 5 to 15 minutes; after the sample solution to be tested is added and vortex-mixed, the solution is allowed to stand for 20 to 40 minutes before the fluorescence intensity of the solution is measured on a fluorescence spectrophotometer.

[0056] In a specific embodiment of the present application, the method for preparing the palladium nanomaterial dispersion includes the following steps: dissolving a palladium precursor salt, citric acid, hexadecyltrimethylammonium bromide, and polyvinylpyrrolidone in an organic solvent, then adding tungsten hexacarbonyl, and reacting by a solvent thermal method; after the reaction is completed, cooling the reaction solution, centrifuging to obtain a palladium nanomaterial, and then dispersing the palladium nanomaterial in a solvent to prepare a palladium nanomaterial dispersion.

[0057] In another embodiment of the present application, the preparation method of the palladium nanomaterial dispersion includes the following steps: adding palladium precursor salt, citric acid, hexadecyltrimethylammonium bromide, and polyvinylpyrrolidone to an organic solvent, using a nanosynthesizer to stir at a speed of 500-800 r / min for 0.5-2 hours to dissolve them to obtain an orange-yellow solution; under a protective gas atmosphere, adding hexacarbonyl tungsten to the orange-yellow solution, heating to 75-85°C, and reacting by a solvent thermal method for 0.5-2 hours to obtain a dark green reaction liquid; after the reaction is completed, the reaction liquid is cooled and centrifuged to obtain palladium nanomaterial, and then the palladium nanomaterial is dispersed in the solvent to obtain a dark blue palladium nanomaterial dispersion.

[0058] In a specific embodiment of the present application, in the preparation method of the palladium nanomaterial dispersion, the molar ratio of palladium precursor salt, citric acid, hexadecyltrimethylammonium bromide, polyvinylpyrrolidone and hexacarbonyl tungsten is 1-2:5-20:2-10:0.005-0.03:5-10; the palladium precursor salt is a precursor salt of divalent palladium, preferably palladium acetylacetonate; the organic solvent is selected from N,N-dimethylformamide; the protective gas is selected from argon or helium; after the reaction is completed, the reaction solution is cooled to room temperature, acetone is added, mixed and centrifuged at a speed of 8000-12000 r / min for 10-30 min, the supernatant is discarded, the lower precipitate is resuspended with anhydrous ethanol and then ultrasonically dispersed to obtain a palladium nanomaterial dispersion.

[0059] Another embodiment of the present application provides an application of the nano-bioluminescent probe as described in the above embodiment, the fluorescent sensor as described in the above embodiment, or the method as described in the above embodiment in detecting B-type natriuretic peptide, wherein the application is for non-disease diagnosis or treatment purposes. Another embodiment of the present application constructs a nano-bioluminescent probe and a fluorescent sensor by the following method, and verifies its effect in detecting B-type natriuretic peptide through experiments. The specific implementation process is as follows:

[0060] 1. Instruments and Materials

[0061] 1. Instruments

[0062] An RF-5301PC fluorescence spectrophotometer (Shimadzu, Japan) was used to measure the fluorescence intensity of the sample solution; the UV-visible spectrum was measured by a UV-2600 UV-visible spectrophotometer (Shimadzu, Japan); the X-ray diffraction pattern was measured by an X-ray powder diffractometer (Bruker, USA); and the dynamic light scattering (DLS) data were measured by a Zetasizer Nano-ZEN3600 particle size analyzer (Malvem). A BY-R18 high-speed refrigerated centrifuge (Baiyang Medical Equipment, Beijing) was used for centrifugation of FAM-aptamer lyophilized powder and interfering protein samples. A nanosynthesizer was used for the synthesis of nanomaterials.

[0063] 2. Materials

[0064] B-type natriuretic peptide (BNP), amino-terminal pro-brain natriuretic peptide (NT-proBNP), C-reactive protein (CRP), (Hb), cardiac troponin I (cTnI), atrial natriuretic peptide (ANP), pro-brain natriuretic peptide (proBNP), C-type natriuretic peptide (CNP), creatine kinase isoenzyme MB (CK-MB), tumor necrosis factor-α (TNF-α), interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 6 (IL-6), interleukin 18 (IL-18), lactoferrin (LF), bilirubin (BIL), and D-dimer (DD) were purchased from Siemens Medical Diagnostics (USA); palladium nanomaterials (Pd NPs); glutamic acid (Gul), valine (Val), cysteine (Cys), glycine (Lys), human serum albumin (HSA), bovine serum albumin (BSA), arginine (Arg), and peptide (Thr) were purchased from Sigma-Aldrich (USA); buffer (Tris-HCl, 0.1 mol / L) was used to adjust the acidity of the reaction solution; the experimental water was ultrapure water with a resistivity of 18.2 MΩ·cm; acetylacetonate palladium (II) (C 10 H 14 O4Pd-), citric acid (C6H8O7), hexadecyltrimethylammonium bromide (C 19 H42 BrN), polyvinylpyrrolidone (PVP, molecular weight 55000), N, N-dimethylformamide (C3H T NO) and hexacarbonyltungsten (6CO.W) were purchased from Shanghai Aladdin Reagent Company. The oligonucleotide chains used in the experiment were provided by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China). The sequences are as follows:

[0065] BNP-specific aptamer: 5'-Cy5.5-TTT TTT TAA ACG CTC AAA GGA CAG AGGGTG CGT AGG AAG GGT ATT CGA CAG GAG GCT CAC A-3'.

[0066] 2. Detection Principle

[0067] like Figure 1 As shown, palladium nanomaterials have strong and broad absorption in the range of 600-1100nm, and the emission wavelength of the Cy5.5 dye-modified nucleic acid aptamer Cy5.5-aptamer is at 708nm. When the two are close enough in the near-infrared region, fluorescence resonance energy transfer occurs, resulting in efficient quenching of fluorescence; when B-type natriuretic peptide BNP is present in the sample, BNP binds to Cy5.5-aptamer through specific binding force to form a complex, which moves away from the palladium nanomaterial and restores fluorescence. There is a positive correlation between BNP and the degree of fluorescence recovery.

[0068] 3. Preparation and Characterization of Pd Nanomaterials

[0069] 1. The preparation method of Pd nanomaterials comprises the following steps:

[0070] (1) Weigh 32 mg of acetylacetonate palladium (II), 180 mg of citric acid, 120 mg of cetyltrimethylammonium bromide, and 60 mg of PVP (molecular weight 55,000) into a 50 mL round-bottom three-necked flask and dissolve in 20 mL of N,N-dimethylformamide.

[0071] (2) The mixture was stirred at 600 rpm for 1 h using a nanosynthesizer to obtain an orange-red solution (to ensure that the solid was completely dissolved).

[0072] (3) 200 mg of tungsten hexacarbonyl was added under stirring at 600 r / min and argon flow at 200 mL / min. The reaction temperature was raised to 80°C and maintained at this temperature for 1 h. The resulting solution was dark green.

[0073] (4) The reaction solution was cooled to room temperature, 20 mL of acetone was added to the above 20 mL of product, mixed and divided into two 50 mL centrifuge tubes, centrifuged at 10000 r / min for 20 min, the supernatant was discarded, and the lower precipitate of each tube was resuspended with 10 mL of anhydrous ethanol. After ultrasonic dispersion, the solutions in the two tubes were combined together. The final palladium nanomaterial dispersion was an ink-blue solution.

[0074] (5) Store in a refrigerator at 4°C. Ultrasonicate for 1 minute before each use.

[0075] 2. Characterization of Pd nanomaterials

[0076] The synthesized Pd nanomaterials were characterized by UV-visible spectrophotometer, X-ray diffractometer, and Zetasizer Nano-ZEN3600 particle size analyzer, as follows:

[0077] The diluted material was scanned using an ultraviolet-visible spectrophotometer. The synthesized material was diluted with anhydrous ethanol to a light blue solution (300 μg / mL), and its absorption intensity in the range of 200 to 1100 nm was scanned using an ultraviolet-visible spectrophotometer.

[0078] Figure 2 This is the UV-visible spectrum of the Pd nanomaterial prepared in this example. It can be seen that the Pd nanomaterial has a strong and broad absorption in the near-infrared region (600-1100 nm).

[0079] Figure 3 This is the UV-visible absorption spectrum of the Pd nanomaterial prepared in this example (black line) and the fluorescence emission spectrum of Cy5.5-aptamer (red line). It can be seen that the two have good overlap in the near-infrared region, which improves the efficiency of fluorescence resonance energy transfer.

[0080] 4. Solution preparation and detection methods

[0081] 1. Preparation of Cy5.5-aptamer stock solution

[0082] The lyophilized powder of Cy5.5-aptamer was centrifuged at 6000 rpm for 5 min, and 800 μL of ultrapure water was added to prepare a 2 μmol / L stock solution, which was then stored in a refrigerator at 4°C.

[0083] Preparation of Tris-HCl (pH 7.4):

[0084] (1) Preparation of Tris (0.1 M): Weigh 0.485 g of Tris and dissolve it in 40 mL of ultrapure water.

[0085] (2) Preparation of HCl (0.1 M): Use a pipette to take 210 μL of hydrochloric acid (12 M) and drop it into a 24 mL test tube filled with ultrapure water. Add 790 μL of ultrapure water to make the total volume 25 mL.

[0086] (3) Preparation of Tris-HCl (pH 7.4): Place 25 mL of 0.1 M Tris and 21 mL of 0.1 M HCl in a 50 mL test tube and add 4 mL of ultrapure water to make the total volume 5 mL.

[0087] 2. Preparation of BNP standard stock solution

[0088] Use a pipette to add 2 mL of ultrapure water to the BNP freeze-dried powder and equilibrate at room temperature (20-30°C) for 15-20 minutes to completely dissolve the freeze-dried powder. Then gently rotate / invert the reagent until it is uniform. The concentration of the prepared stock solution is 1471 pg / mL. Store in a -20°C refrigerator and dilute before use.

[0089] 3. Determination of the concentration of Pd nanomaterial dispersion

[0090] Prepare different concentrations of raw material acetylacetonate palladium (II), oxidize it to palladium ions using 2 mL of aqua regia, and use a spectrophotometer to measure the ultraviolet absorption at 200-450 nm. Use the concentration as the horizontal axis and the ultraviolet absorption intensity at 275 nm as the vertical axis to establish a standard curve.

[0091] Take 200 μL of the Pd nanomaterial dispersion synthesized by the determination step 1, use 2 mL of aqua regia to oxidize the palladium nanomaterial into palladium ions, wait until the reaction is complete, that is, the solution changes from dark blue to colorless or light yellow, and then dilute with ultrapure water. Use a UV-visible spectrophotometer to measure the ultraviolet absorption at 200-450 nm, substitute the ultraviolet absorption value at 275 nm into the standard curve, and calculate the concentration of the original Pd nanomaterial dispersion.

[0092] 4. Detection method:

[0093] Blank group 1: 100 μL Tris-HCl buffer (pH 7.4) and 10 μL Cy5.5-aptamer (100 nmol / L) were added to a 1.5 mL centrifuge tube in sequence, and ultrapure water was added to a total volume of 400 μL and vortexed to mix. The fluorescence intensity of the solution was measured on a fluorescence spectrophotometer (excitation wavelength: 675 nm; emission wavelength: 708 nm; emission spectrum scanning range: 690-750 nm; scanning speed: Very Fast; slit width: ex: 5 nm, em: 5 nm). The measured fluorescence intensity was recorded as F0.

[0094] Blank group 2: 100 μL Tris-HCl buffer (pH 7.4), 10 μL Cy5.5-aptamer solution (100 nmol / L) and 15 μL Pd nanomaterial dispersion (800 μg / mL) were added to a 1.5 mL centrifuge tube in sequence, and ultrapure water was added to make up the total volume to 400 μL. The mixture was vortexed and allowed to stand at room temperature for 10 min. The fluorescence intensity of the solution was measured on a fluorescence spectrophotometer (excitation wavelength: 675 nm; emission wavelength: 708 nm, emission spectrum scanning range: 690-750 nm; scanning speed: Very Fast; slit width: ex: 5 nm, em: 5 nm). The measured fluorescence intensity was recorded as F.

[0095] Experimental group: 100 μL of Tris-HCl buffer (pH 7.4), 15 μL of Pd nanomaterial dispersion (800 μg / mL), and 10 μL of Cy5.5-aptamer (100 nmol / L) were added to a 1.5 mL centrifuge tube in sequence, vortexed and mixed, and allowed to stand for 10 min. Then, a certain concentration of BNP sample solution was added, and ultrapure water was added to a total volume of 400 μL. The mixture was vortexed and allowed to stand at room temperature for 30 min. The fluorescence intensity of the solution was measured on a fluorescence spectrophotometer (excitation wavelength: 675 nm; emission wavelength: 708 nm, emission spectrum scanning range: 690-750 nm; scanning speed: Very Fast; slit width: ex: 5 nm, em: 5 nm). The measured fluorescence intensity was recorded as F1.

[0096] V. Investigation of influencing factors

[0097] Referring to the above method, the effects of Pd nanomaterial concentration and system pH on the test results were studied as follows:

[0098] 1. Influence of Pd nanomaterial concentration:

[0099] 100 μL of Tris-HCl buffer (pH 7.4) was mixed with 10 μL of Cy5.5-aptamer (100 nmol / L), and the volume was adjusted to 400 μL with ultrapure water. The fluorescence intensity was measured and recorded as F0; Pd nanomaterial dispersions of different concentrations were added to the mixture of 100 μL of Tris-HCl buffer (pH 7.4) and 10 μL of Cy5.5-aptamer (100 nmol / L) in sequence, the volume was adjusted to 400 μL, and the fluorescence intensity was measured after standing for 10 minutes, which was recorded as F; BNP sample solution of the same concentration was added to the above mixture, the volume was adjusted to 400 μL with ultrapure water, vortex mixed, and stood at room temperature for 30 minutes. The fluorescence parameters set in the detection method in step 3 were measured, and the measured fluorescence intensity was recorded as F1.

[0100] When the concentration of Pd nanomaterials is 0-70 μg / mL, the fluorescence emission intensity of Cy5.5-aptamer is as follows: Figure 3 As shown. Figure 6 It can be seen that with the increase of Pd nanomaterial concentration, the fluorescence intensity of Cy5.5-aptamer gradually decreases. When the Pd nanomaterial concentration is 30 μg / mL, the fluorescence recovery efficiency (F1 / F) is the highest. Therefore, a Pd nanomaterial concentration of 30 μg / mL was selected as the optimal condition for subsequent experiments.

[0101] 2. Influence of system pH:

[0102] PBS buffer solution at pH 6.5 and Tris-HCl buffer solutions at pH 7.0, 7.4, 8.0, and 8.5 were prepared, mixed with 10 μL of Cy5.5-aptamer (100 nmol / L), and then diluted to 400 μL with ultrapure water. The fluorescence intensity was measured, denoted as F0. Pd nanomaterial dispersions of varying concentrations were then added to the mixture of 100 μL of the various pH buffers and 10 μL of Cy5.5-aptamer (100 nmol / L), diluted to 400 μL, and allowed to stand for 10 minutes. The fluorescence intensity was measured, denoted as F. BNP sample solutions of the same concentration were added to the mixture, diluted to 400 μL with ultrapure water, vortexed, and allowed to stand at room temperature for 30 minutes. The fluorescence parameters set in step 3 were used for the detection method, and the measured fluorescence intensity was recorded as F1. Each experiment was repeated three times.

[0103] The results of the test are as follows Figure 7 As shown. Figure 5 It can be seen that the quenching efficiency of the probe fluorescence by a fixed concentration of Pd nanomaterial remains almost unchanged at different pH values, while the fluorescence recovery efficiency increases with increasing pH. Taking all factors into consideration, the physiological buffer value of pH 7.4 was selected as the subsequent detection condition.

[0104] 6. Drawing of the standard curve

[0105] In a 1.5 mL centrifuge tube, 100 μL of Tris-HCl buffer (pH 7.4), 15 μL of Pd nanomaterial dispersion (800 μg / mL) and 10 μL of Cy5.5-aptamer (100 nmol / L) were added in sequence and vortexed to mix. The mixture was allowed to stand for 10 minutes. Then, sample solutions of BNP with different concentrations were added and the total volume was supplemented with ultrapure water to 400 μL. The mixture was vortexed to mix and allowed to stand at room temperature for 30 minutes. The fluorescence intensity of the solution was measured on a fluorescence spectrophotometer (excitation wavelength of 675 nm; emission wavelength of 708 nm, emission spectrum scanning range: 690-750 nm; scanning speed: Very Fast; slit width: ex: 5 nm, em: 5 nm). The measured fluorescence intensity was recorded as F1. The experiment was repeated three times.

[0106] Depend on Figure 6 It can be seen that the fluorescence intensity of Cy5.5-aptamer gradually recovered with the increase of BNP concentration. Figure 7 As can be seen, a linear fit, with the fluorescence recovery efficiency F1 / F as the ordinate and the logarithm of the BNP concentration as the abscissa, shows a good linear relationship within the concentration range of 0.05 to 10 pg / mL. The standard curve equation is: Y = 0.98lnX + 5.696 (r = 0.9923), and the minimum detection limit reaches 45 fg / mL. Therefore, the method provided by the present invention is simple, rapid, and highly sensitive, and can quantify BNP in a sample solution according to a linear equation.

[0107] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention.

Claims

1. A nano-bioluminescent probe, characterized in that: The invention comprises a palladium nanomaterial and a nucleic acid aptamer adsorbed on the palladium nanomaterial, wherein the nucleic acid aptamer is specific for B-type natriuretic peptide and is labeled with a fluorescent dye. The nucleic acid aptamer sequence is: 5'-Cy5.5-TTT TTT TAA ACG CTC AAA GGA CAG AGGGTG CGT AGG AAG GGT ATT CGA CAG GAG GCT CAC A-3'. The emission wavelength of the fluorescent dye-modified nucleic acid aptamer is 708 nm, and the palladium nanomaterial has strong and broad absorption in the range of 600-1100 nm.

2. A fluorescent sensor for detecting B-type natriuretic peptide, characterized in that: The fluorescent sensor comprises the nano-bioluminescent probe according to claim 1, and the concentration of the palladium nanomaterial is 20-50 μg / mL.

3. The fluorescent sensor for detecting B-type natriuretic peptide according to claim 2, characterized in that: The pH of the fluorescence sensor reaction system is 7.0-8.

5.

4. The fluorescent sensor for detecting B-type natriuretic peptide according to claim 2, characterized in that: The fluorescence sensor is used to detect samples, and the measurement parameters of the fluorescence intensity of the solution measured on a fluorescence spectrophotometer include: an excitation wavelength of 675 nm, an emission wavelength of 708 nm, and an emission spectrum scanning range of 690-750 nm.

5. The fluorescent sensor for detecting B-type natriuretic peptide according to claim 2, characterized in that: The fluorescent sensor has a good linear relationship when the concentration of B-type natriuretic peptide in the sample is 0.05-10 pg / mL.

6. A method for detecting B-type natriuretic peptide using the fluorescent sensor according to any one of claims 2 to 5, characterized in that: The method is not for the purpose of disease diagnosis or treatment, and comprises the following steps: mixing a palladium nanomaterial dispersion, a buffer solution, and a nucleic acid aptamer solution labeled with a fluorescent dye, and allowing the mixture to stand; then adding a sample solution to be tested, mixing the mixture, allowing the mixture to stand, and measuring the fluorescence intensity of the solution on a fluorescence spectrophotometer.

7. The method according to claim 6, characterized in that: The pH of the buffer solution is 7.0-8.

5.

8. The method according to claim 6, wherein: The buffer solution is selected from any one of Tris-HCl buffer solution and PBS buffer solution.

9. The method according to claim 6, wherein: The method is carried out at room temperature.

10. The method according to claim 6, wherein: After the palladium nanomaterial dispersion and the fluorescent dye-labeled nucleic acid aptamer solution are vortexed and mixed, they are allowed to stand for 5-15 minutes before adding the test sample solution; And / or, after adding the sample solution to be tested and vortex mixing, let it stand for 20 to 40 minutes and then measure the fluorescence intensity of the solution on a fluorescence spectrophotometer.

11. Use of the nanobioluminescent probe according to claim 1, the fluorescent sensor according to any one of claims 2 to 5, or the method according to any one of claims 6 to 10 in detecting B-type natriuretic peptide, wherein the use is for non-disease diagnosis or treatment purposes.

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