Fluorescence-colorimetric double-signal detection method and kit based on spherical nucleic acid probe

Abstract

The invention discloses a fluorescent-colorimetric dual-signal detection method and kit based on a spherical nucleic acid probe, and relates to the technical field of molecular detection. The kit comprises the following components: an aptamer chain AptC, an inhibition chain Inh and gold nanoparticles, the nucleotide sequence of the aptamer chain AptC is as shown in SEQ ID NO. 3; the nucleotide sequence of the inhibition chain Inh is as shown in SEQ ID NO. 1. According to the detection method provided by the invention, rapid, high-sensitivity and high-specificity detection of NT-proBNP can be realized; the kit provided by the invention can be used for detecting NT-proBNP in serum of patients with heart failure, and can meet the clinical requirements of rapid and accurate diagnosis of heart failure.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular detection, and in particular to a fluorescence-colorimetric dual-signal detection method and kit based on spherical nucleic acid probes. Background Art

[0002] N-terminal pro-brain natriuretic peptide (NT-proBNP) has a high concentration and long stability in the blood of heart failure patients and is considered an ideal biomarker for the diagnosis and prognosis of heart failure. Its rapid and accurate detection is of great significance for clinical decision-making.

[0003] Currently, the clinical detection of NT-proBNP mainly relies on enzyme-linked immunosorbent assay (ELISA) and chemiluminescence assay. However, these methods have limitations such as long detection time, dependence on large instruments and professional operators, and high reagent costs, making it difficult to meet the rapid detection needs of emergency or primary medical institutions. Therefore, establishing a simple, sensitive, and low-cost NT-proBNP detection method is of great significance for the early diagnosis and prognosis prediction of heart failure diseases.

[0004] In recent years, biosensing technologies based on nanomaterials have received extensive attention due to their advantages such as high sensitivity and rapid response. Gold nanoparticles (AuNPs) are often used in the construction of colorimetric or fluorescence detection systems due to their unique optical properties and biocompatibility. In terms of colorimetric signal amplification, AuNPs can achieve highly sensitive detection through color changes due to their strong surface plasmon resonance (SPR) effect. At the same time, AuNPs can also be used as fluorescence quenchers to construct "switch-type" fluorescent probes for specific detection of target molecules. However, traditional AuNP functionalization methods mostly rely on chemical modification (such as thiol coupling), which may lead to a decrease in probe activity or impaired stability. In addition, existing detection systems mostly rely on single-signal output and are vulnerable to environmental interference, resulting in risks of false positives or false negatives, limiting the reliability of detection results. Summary of the Invention

[0005] The purpose of the present invention is to provide a fluorescence-colorimetric dual-signal detection method and kit based on spherical nucleic acid probes to solve the problems existing in the above-mentioned prior art. The detection method provided by the present invention can achieve rapid, highly sensitive, and highly specific detection of NT-proBNP; the kit provided by the present invention can be used for the detection of NT-proBNP in the serum of heart failure patients and can meet the clinical needs of rapid and accurate diagnosis of heart failure.

[0006] The present invention designs three DNA probe sequences using NUPACK software: an inhibitory strand (Inh), a control inhibitory strand (n-Inh), and an aptamer strand (AptC). Among them, the AptC strand includes the aptamer sequence of NT-proBNP, and its 3'-end is labeled with a fluorescent group (Cy5) as a reporter group. The Inh strand contains a sequence complementary to the AptC strand. Its 5'-end is designed as a flexible spacer rich in T bases and modified with a thiol group to enhance the adsorption ability to AuNPs. Its 3'-end is designed as a dimer sequence to induce the self-assembly aggregation of AuNPs. The n-Inh strand is used as a control sequence for the Inh strand. Its 3'-end is replaced with a poly-T sequence and cannot induce the aggregation of AuNPs, and is used in a control experiment to verify the key role of the dimer sequence in the aggregation reaction. In the absence of the target NT-proBNP, the Inh strand and the AptC strand form a stable double-stranded structure and are loaded on the surface of AuNPs. The light group is close to the surface of AuNPs, and fluorescence quenching occurs due to the surface plasmon resonance effect. When the target NT-proBNP is present, the AptC strand specifically recognizes the target and thus dissociates from the surface of AuNP, and the fluorescence signal is significantly restored. At the same time, the dimer sequence at the end of the Inh strand is exposed and spontaneously complementary paired, driving the self-assembly aggregation process of AuNPs. In contrast, if the n-Inh strand binds to the AptC strand, no self-assembly process can occur due to the lack of the dimer sequence, and the aggregation of AuNPs cannot be induced.

[0007] Based on the above probes, the present invention has developed a fluorescence-colorimetric dual-signal detection method applicable to NT-proBNP, and the technical principle is as follows:

[0008] (1) Principle of colorimetric signal generation: The color change of the probe solution results from the aggregation reaction of gold nanoparticles (AuNPs). When the target NT-proBNP is present, the aptamer strand (AptC) specifically binds to it, which in turn causes the exposure of the dimer sequence at the end of the inhibitory strand (Inh). The exposed dimer sequence induces the aggregation of gold nanoparticles through base complementary pairing, generating a confinement effect. During this process, the color of the probe solution changes from the dispersed red to the aggregated blue-violet, thus realizing the naked-eye visual qualitative detection of NT-proBNP.

[0009] (2) Principle of fluorescence signal generation: In the absence of the target NT-proBNP, the distance between the fluorescent group (Cy5) at the end of the aptamer chain (AptC) and the surface of the gold nanoparticle (AuNP) is less than 5 nm, triggering fluorescence resonance energy transfer (FRET), resulting in quenching of the fluorescence and an "off" state. When the target NT-proBNP is present, the aptamer chain (AptC) specifically binds to NT-proBNP, causing the AptC chain to detach from the AuNP surface, and the distance between the two increases to more than 10 nm, thereby eliminating the FRET effect and restoring the fluorescence to the "on" state. The change in fluorescence intensity of the fluorophore on the probe is positively correlated with the concentration of the target NT-proBNP. By monitoring the change in fluorescence intensity, the concentration of the target NT-proBNP can be quantitatively detected.

[0010] The above method is a universal detection method. The AptC chain can achieve specific recognition of different targets through its sequence design. The application range of this method can be expanded by simply designing the corresponding aptamer sequence according to the target molecule. Any target that can make the AptC chain detach from the probe surface can trigger changes in fluorescence and colorimetric signals, thereby achieving detection. This makes it suitable for quantitative analysis of a variety of biomarkers and small molecules. Its specific principles are as follows Figure 2 shown.

[0011] Based on this, the present invention provides the following solutions:

[0012] The present invention provides a detection kit for N-terminal B-type natriuretic peptide precursor, comprising the following components:

[0013] Aptamer chain AptC, inhibitory chain Inh and gold nanoparticles;

[0014] The nucleotide sequence of the aptamer chain AptC is shown in SEQ ID NO.3;

[0015] The nucleotide sequence of the inhibitory chain Inh is shown in SEQ ID NO.1.

[0016] Furthermore, the 3' end of the aptamer chain AptC carries a fluorescent group.

[0017] Furthermore, the fluorescent group is a Cy5 fluorescent group.

[0018] Furthermore, the 5' end of the inhibitory chain Inh is modified with a thiol group.

[0019] The present invention also provides a method for preparing a spherical nucleic acid probe for detecting an N-terminal B-type natriuretic peptide precursor, comprising the following steps:

[0020] Annealing the adapter chain AptC and the inhibitory chain Inh to form a DNA double strand;

[0021] Load the DNA duplex on the surface of gold nanoparticles to obtain the spherical nucleic acid probe;

[0022] The nucleotide sequence of the aptamer strand AptC is shown in SEQ ID NO.3, and its 3' end carries a fluorescent group;

[0023] The nucleotide sequence of the inhibitory strand Inh is shown in SEQ ID NO.1.

[0024] Furthermore, the fluorescent group is a Cy5 fluorescent group.

[0025] The present invention also provides a spherical nucleic acid probe for detecting N-terminal pro-brain natriuretic peptide prepared by the above preparation method.

[0026] The present invention also provides a fluorescence-colorimetric dual-signal detection kit for N-terminal pro-brain natriuretic peptide based on the spherical nucleic acid probe, including the above spherical nucleic acid probe.

[0027] The present invention also provides a qualitative detection method for N-terminal pro-brain natriuretic peptide for non-disease diagnosis purposes, including the following steps:

[0028] Add the sample to be tested and the above spherical nucleic acid probe into Tris-HCl buffer for dark incubation, and then observe the color of the solution. If the color of the solution changes from red to blue-violet, it is determined that the sample to be tested contains N-terminal pro-brain natriuretic peptide.

[0029] The present invention also provides a quantitative detection method for N-terminal pro-brain natriuretic peptide for non-disease diagnosis purposes, including the following steps:

[0030] Add the sample to be tested and the above spherical nucleic acid probe into Tris-HCl buffer for dark incubation, and then detect the fluorescence intensity of the reaction system;

[0031] Substitute the fluorescence intensity into the standard curve to obtain the content of N-terminal pro-brain natriuretic peptide in the sample to be tested.

[0032] The present invention discloses the following technical effects:

[0033] The present invention has developed a fluorescence-colorimetric dual-signal detection method based on spherical nucleic acid probes (SNAs) for highly sensitive and highly specific detection of N-terminal pro-brain natriuretic peptide (NT-proBNP). The spherical nucleic acid probe uses 13-nm gold nanoparticles (AuNPs) as the core, and DNA is efficiently loaded on its surface by the freeze-thaw method without chemical modification, ensuring the activity and stability of the probe. The present invention utilizes the binding of a specific aptamer strand (AptC) to NT-proBNP and the dimer sequence at the 3'-end of the inhibitory strand (Inh) to induce the self-assembly aggregation of AuNPs, realizing the restoration of the fluorescence signal and the change of the solution color, and achieving highly sensitive and highly specific detection of NT-proBNP through the synergistic output of dual signals. Experimental results show that the fluorescence recovery efficiency of the present invention has a good linear relationship with the NT-proBNP concentration in the range of 0.1-5 pg / mL (R 2 = 0.996), and the detection limit of the fluorescence signal is as low as 0.043 pg / mL. This method can complete the detection within 20 minutes and has the characteristics of rapidity, high sensitivity, and high specificity. The present invention provides an innovative detection method for NT-proBNP and provides an efficient and reliable solution for the early diagnosis of heart failure. The present invention specifically has the following advantages:

[0034] (1) Ultra-high sensitivity: Based on the highly specific recognition ability of the aptamer (AptC) and combined with the fluorescence quenching / restoration mechanism, ultra-sensitive detection of NT-proBNP is achieved. The detection limit (LOD) of the present invention in the fluorescence mode is as low as 0.043 pg / mL, and the linear range covers 0.1-5 pg / mL. This ultra-low detection limit can meet the detection requirements of ultra-low concentrations in early heart failure or occult cases.

[0035] (2) Significantly improved anti-interference ability: The present invention introduces the aptamer strand (AptC) of NT-proBNP, and realizes signal readout through its specific binding to the target. There is no significant response to coexisting interferents (such as BNP, cTnI, IL-6), showing excellent specificity. At the same time, the present invention adopts a fluorescence-colorimetric dual-signal output mode. Through the cross-validation of the two signals, the incidence of false positives and false negatives is significantly reduced. The dual-mode complementary mechanism ensures the reliability of the detection results in complex biological samples and provides a strong guarantee for high-precision biomarker detection.

[0036] (3) Fast and low cost: The detection time of the present invention is only 20 min, which can meet the rapid diagnosis requirements of the emergency department for acute heart failure. In addition, the probe synthesis and detection process of the present invention support naked-eye colorimetric interpretation and quantitative analysis with a portable fluorometer, without relying on professional large-scale equipment, and has the characteristics of low cost and easy operation, and is applicable to a variety of detection scenarios.

[0037] (4) Wide generality: The present invention adopts a modular probe design and has the potential for multiplex biomarker detection. By directionally replacing the aptamer sequences, it is possible to simultaneously detect heart failure biomarkers such as NT-proBNP, cTnT, and D-dimer in the same detection system. Combining with multi-wavelength fluorescence labeling (such as FAM, Cy5), it can achieve high-throughput discrimination of multi-target signals, avoid cross-interference, and significantly improve the detection throughput and accuracy. Further, this technology can be extended to non-clinical fields. By targeted reprogramming of the aptamer sequences, a detection system for environmental pollutants (such as heavy metal ions, organic toxins) or foodborne hazards (such as pathogens, mycotoxins) can be quickly constructed, forming an interdisciplinary platform technology applicable to diversified scenarios such as environmental monitoring and food safety. Description of the Drawings

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0039] Figure 1 Flow chart of DNA loading by freeze-thaw method;

[0040] Figure 2 Schematic diagram of the principle of SNAs for detecting the target NT-proBNP; where A is the schematic diagram of the principle of the target NT-proBNP triggering the fluorescence-colorimetric dual-signal output of SNAs; B is the flow chart of the self-assembly of gold nanoparticles;

[0041] Figure 3 Comparison chart of the triggering signals of n-SNAs and SNAs;

[0042] Figure 4 Characterization result chart of AuNPs and SNAs; where (A) is the TEM image of AuNPs (a) and SNAs (b); (B) is the ultraviolet-visible spectrum of AuNPs and SNAs; (C) is the particle size distribution and polydispersity index of AuNPs and SNAs;

[0043] Figure 5Results graph for verifying the feasibility of spherical nucleic acid probe aggregation; among them, (A) is the gel electrophoresis characterization graph of SNAs, and "+" and "-" respectively indicate the presence and absence of the corresponding components; (B) is the TEM graph of the different responses of n-SNAs (a) and SNAs (b) to NT-proBNP; (C) is the ultraviolet-visible absorption spectrum of SNAs and n-SNAs under the condition of NT-proBNP (the inset is the color change under different conditions); (D) is the particle size distribution graph of SNAs and n-SNAs under the condition of NT-proBNP.

[0044] Figure 6 Is the fluorescence spectrum graph of SNAs and n-SNAs under the condition of NT-proBNP;

[0045] Figure 7 Results graph for detecting the sensitivity of spherical nucleic acid probes: among them, (A) is the fluorescence spectrum graph of SNAs under the condition of different concentrations of NT-proBNP; (B) is the fluorescence response intensity graph of SNAs and n-SNAs to different concentrations of NT-proBNP, and the inset is the linear correlation curve of SNAs in the concentration range of 0.1 - 5 pg / mL (LOD = 0.043 pg / mL);

[0046] Figure 8 Results graph for verifying the time-dependence of spherical nucleic acid probe for detecting NT-proBNP: among them, (A) is the fluorescence kinetics graph of SNAs and n-SNAs under the condition of NT-proBNP; (B) is the bar graph of the fluorescence change of SNAs and n-SNAs under the condition of NT-proBNP at a fixed time interval of 20 min;

[0047] Figure 9 Results graph for verifying the specificity of spherical nucleic acid probe for detecting NT-proBNP: among them, (A) is the fluorescence spectrum graph of SNAs added with NT-proBNP and different interfering substances; (B) is the fluorescence response heat map of SNAs added with NT-proBNP and different interfering substances. Detailed implementation mode

[0048] Now, various exemplary implementation modes of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0049] It should be understood that the terms used in this invention are only for describing specific embodiments and are not intended to limit the invention. Additionally, for the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0050] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0051] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the specification of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the specification of this invention are obvious to those skilled in the art. The specification and examples of this invention are merely exemplary.

[0052] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0053] Example 1 Synthesis of Gold Nanoparticle (AuNPs) Material

[0054] Using chloroauric acid (HAuCl4) as the raw material, 13nm AuNPs were prepared by the sodium citrate reduction method. To ensure uniform particle size of the synthesized AuNPs, all glassware was soaked in aqua regia for 30 min before synthesis, then rinsed thoroughly with ultrapure water and dried in an oven. The specific steps are as follows:

[0055] S1: Place 100 mL of 1 mmol / L HAuCl4 solution in a 250 mL conical flask and heat to boiling under vigorous stirring.

[0056] S2: After the solution boils, quickly add 10 mL of 38.8 mmol / L sodium citrate solution to the center of the vortex in the conical flask.

[0057] S3: After the solution color stabilizes to wine red, continue boiling for 15 min, then remove the heat source and keep stirring until the solution cools to room temperature to obtain the AuNPs solution.

[0058] S4: Store the synthesized AuNPs solution in the dark at 4 °C for future use.

[0059] The uniformity of the AuNPs synthesized in this invention was verified by transmission electron microscopy (TEM), dynamic light scattering (DLS), and ultraviolet-visible spectroscopy (UV-Vis). The results are shown in Figure 4 .

[0060] Example 2 Synthesis of SNAs and n-SNAs nucleic acid probes

[0061] The nucleotide sequences of the nucleic acids involved in this invention are shown in Table 1.

[0062] Table 1 Nucleotide sequence information

[0063] Name Nucleotide sequence (5’-3’) Sequence number Inh SH-TTTTTTTTTGGCCACACTCAGCTGCAGCTG SEQ ID NO.1 n-Inh SH-TTTTTTTTTGGCCACACTCAGCTGTTTTTT SEQ ID NO.2 AptC CAGGGGGACGGGTCGGGTTACAGCTGAGTGTGGCC-Cy5 SEQ ID NO.3

[0064] As Figure 1 shown, spherical nucleic acid probes were synthesized by modifying DNA on the surface of gold nanoparticles using the freeze-thaw method. The specific steps are as follows:

[0065] Synthesis of SNAs spherical nucleic acid probes:

[0066] S1: Dissolve DNA in enzyme-free water to a concentration of 100 μM, aliquot, and store at -20 °C for future use.

[0067] S2: Mix the Inh strand and the AptC strand to obtain Solution 1. Anneal Solution 1 at 95 °C for 5 min and let it cool to room temperature naturally to obtain a double-stranded solution.

[0068] S3: Add the formed double-stranded solution to a 10 nM AuNPs solution (DNA:AuNPs = 300:1), vortex to mix well, and then freeze in a -20 °C refrigerator for 2 h.

[0069] S4: Thaw in the dark at room temperature and centrifuge the mixture 3 times (13000 rpm, 10 min) to remove the supernatant.

[0070] S5: Redisperse the product in Tris-HCl buffer to obtain SNAs spherical nucleic acid probes and store at 4 °C for future use.

[0071] Synthesis of n-SNAs control nucleic acid probes:

[0072] S1: Dissolve DNA in enzyme-free water to a concentration of 100 μM, aliquot, and store at -20 °C for future use.

[0073] S2: Mix the n-Inh strand and the AptC strand to obtain Solution 2. Anneal Solution 2 at 95 °C for 5 min and let it cool to room temperature naturally to obtain a double-stranded solution.

[0074] S3: Add the formed double-stranded solution to a 10 nM AuNPs solution (DNA:AuNPs = 300:1). After vortexing and mixing, place it in a refrigerator at -20 °C and freeze for 2 h.

[0075] S4: Thaw in the dark at room temperature. Centrifuge the mixture (13000 rpm, 10 min) three times and remove the supernatant.

[0076] S5: Redisperse the product in Tris-HCl buffer to obtain the n-SNAs control nucleic acid probe, and store it at 4 °C for later use.

[0077] In the above method, the Tris-HCl buffer has a concentration of 10 mM, a pH of 7.2, and contains 50 mM NaCl, 10 mM MgCl2, and 10 mM KCl.

[0078] As Figure 4 shown in (A) below, a DNA halo is clearly visible around the synthesized spherical probe, and the probes are evenly dispersed. Figure 4 The ultraviolet absorption spectrum in (B) below shows that the absorption peak of the synthesized spherical probe is slightly red-shifted to 524 nm. Figure 4 The particle size distribution diagram in (C) below shows that the particle size of the probe after loading DNA increases slightly. In summary, these results indicate that DNA is successfully modified on the surface of AuNPs.

[0079] Example 3 verifies the feasibility of the aggregation reaction when detecting the target NT-proBNP based on spherical nucleic acid probes

[0080] Figure 2 is the schematic diagram of SNAs for detecting the target NT-proBNP; Figure 3 is the comparison diagram of the triggering signals of n-SNAs and SNAs.

[0081] Add 50 pg / mL of NT-proBNP to 100 μL of SNAs and the control probe n-SNAs respectively. After mixing, place them in Tris-HCl buffer (10 mM, pH 7.2, containing 50 mM NaCl, 10 mM MgCl2, and 10 mM KCl), and incubate in the dark at room temperature for 20 min. Then observe the color change of the solution, and analyze each solution using a dynamic light scattering instrument (DLS) and an ultraviolet-visible spectrometer (UV-Vis).

[0082] The results are as Figure 5 shown. As Figure 5 shown in the transmission electron microscope (TEM) image in (B) below, when the target NT-proBNP is added to the control probe (n-SNAs) system, no aggregation occurs in this system, and the nanoparticles have good dispersibility.Figure 5 The ultraviolet spectrum analysis results shown in (C) are consistent with this, and the characteristic absorption peak hardly undergoes red shift, and the solution color also has no obvious change. On the contrary, when the target NT-proBNP is added to the experimental group probe (SNAs) system, significant aggregation occurs in the system. As Figure 5 shown in (C), after adding the target, the solution color of the SNAs system changes from red to blue-violet, and the characteristic absorption peak red-shifts to 565 nm. In addition, as Figure 5 shown in (D), the dynamic light scattering (DLS) detection results show that only the experimental group probe (SNAs) aggregates after adding the target, and the particle size increases to 285 nm.

[0083] These results fully prove that the dimer sequence at the end of the Inh chain can induce the aggregation of gold nanoparticles (AuNPs) when responding to the target NT-proBNP, significantly enhancing the confinement effect of the SNAs system and realizing the detection of NT-proBNP by observing the change of the solution color with the naked eye.

[0084] The products of the SNAs system were characterized by gel electrophoresis. Prepare the reaction solution: the concentrations of Inh and AptC are both 400 nM in Tris-HCl buffer, and the concentration of NT-proBNP is 50 pg / mL; the reaction is incubated in the dark at room temperature for 1 h. After mixing the reaction solution with loading buffer and GelRed, add it to a 9% acrylamide gel. Set the voltage of the electrophoresis apparatus to 100 V, take out the gel after 2 h, and finally make the DNA visible under ultraviolet light through a chemiluminescence imaging system.

[0085] The electrophoresis results are as Figure 5 shown in (A). After adding the target NT-proBNP to the SNAs system, the inhibitory strand (Inh) undergoes self-assembly, forming high-molecular-weight products and aggregating at the top of the gel. This result is consistent with the red shift of the characteristic absorption peak observed in the ultraviolet spectrum experiment, further confirming the occurrence of the Inh strand self-assembly and AuNPs aggregation reaction induced by NT-proBNP.

[0086] Example 4 verifies the fluorescence feasibility of detecting the target NT-proBNP based on spherical nucleic acid probes

[0087] 50 pg / mL of NT-proBNP was added to 100 μL of SNAs and the control probe n-SNAs respectively. After mixing, it was placed in Tris-HCl buffer (10 mM, pH 7.2, containing 50 mM NaCl, 10 mM MgCl2 and 10 mM KCl), and incubated at room temperature in the dark for 20 min. The fluorescence intensity of the system was measured using a fluorescence spectrometer (excitation wavelength 642 nm, excitation slit 3 nm, emission wavelength 666 nm, emission slit 3 nm, wavelength scanning range 650 - 800 nm), and the fluorescence intensity at the maximum emission wavelength of 666 nm was recorded.

[0088] As Figure 6 shown, after adding the target NT-proBNP to the system, due to the high affinity between AptC and NT-proBNP, the fluorescence signal of the system was significantly restored after their binding, indicating that it is feasible to detect NT-proBNP using spherical nucleic acid probes.

[0089] Example 5 Sensitivity Detection of Spherical Nucleic Acid Probes

[0090] The target NT-proBNP with gradient dilution (from low to high: 0.1, 0.4, 2, 3, 5, 15, 30, 50, 100 pg / mL) was added to 100 μL of SNAs or the control probe n-SNAs respectively. After mixing, it was placed in Tris-HCl buffer (10 mM, pH 7.2, containing 50 mM NaCl, 10 mM MgCl2 and 10 mM KCl), and incubated at room temperature in the dark for 20 min. The fluorescence intensity of the system was measured using a fluorescence spectrometer (excitation wavelength 642 nm, excitation slit 3 nm, emission wavelength 666 nm, emission slit 3 nm, wavelength scanning range 650 - 800 nm), and the fluorescence intensity at the maximum emission wavelength of 666 nm was recorded.

[0091] As Figure 7 shown in (A) below, when different concentrations of the target NT-proBNP were added to the system, the change value of the fluorescence intensity of the SNAs system was positively correlated with the target concentration, indicating that this system can be used for quantitative detection of NT-proBNP. As Figure 7 shown in (B) below, with the increase of NT-proBNP concentration, the fluorescence intensities (λ = 666 nm) of both the n-SNAs and SNAs systems gradually increased. Under the same concentration conditions, the fluorescence intensity recovery of the experimental group probe (SNAs) was significantly higher than that of the control probe (n-SNAs). The inset shows that SNAs exhibited a good linear relationship in the concentration range of 0.1 - 5 pg / mL (ΔF = 3093.6C + 8773.0, R 2(= 0.996), the calculated detection limit was 0.043 pg / mL, while that of n-SNAs was 0.073 pg / mL. This result indicates that due to its unique confinement effect, the SNAs system can achieve highly sensitive detection of NT-proBNP.

[0092] Example 6 Time-dependent Verification of Spherical Nucleic Acid Probe for Detecting NT-proBNP

[0093] 50 pg / mL of NT-proBNP was added to 100 μL of SNAs and n-SNAs respectively. After thorough mixing, it was placed in Tris-HCl buffer (10 mM, pH 7.2, containing 50 mM NaCl, 10 mM MgCl2 and 10 mM KCl). Immediately, the fluorescence intensity of the system was measured every 2 minutes using a fluorescence spectrometer (excitation wavelength 642 nm, excitation slit 3 nm, emission wavelength 666 nm, emission slit 3 nm, wavelength scanning range 650 - 800 nm), and the fluorescence intensity at the maximum emission wavelength of 666 nm was recorded.

[0094] As Figure 8 shown, when the target NT-proBNP was not added, the aptamer strand (AptC) was complementary paired with the inhibitor strand (Inh) and stably bound to the surface of AuNPs. In this state, with the extension of time, only minor changes in fluorescence occurred, indicating that the system was in a stable "off" state. In contrast, when the target NT-proBNP was present, the AptC strand specifically recognized and bound to the target, causing it to detach from the surface of AuNPs, and the fluorescence was significantly restored to the "on" state. Further comparison found that compared with the control probe (n-SNAs), the fluorescence intensity recovery of the experimental group probe (SNAs) was more significant, and the reaction time was shorter, only 20 min. This result is consistent with the sensitivity detection, indicating that due to the confinement effect of the SNAs system, it has higher sensitivity and faster response speed in detecting NT-proBNP.

[0095] Example 7 Specificity and Anti-interference Test of Spherical Nucleic Acid Probe

[0096] To demonstrate the high-efficiency selectivity of the present invention for NT-proBNP detection, 2 μL of various potential interferent standards (including IL-18, IL-6, IL-1, pro-BNP, CNP, TNF-α, BIL, CRP, CK-MB, LF, Hb, BSA, ANP, DD, HSA, BNP, and NT-proBNP) were separately added to 100 μL of SNAs solution. After mixing, the solution was placed in Tris-HCl buffer (10 mM, pH 7.2, containing 50 mM NaCl, 10 mM MgCl2, and 10 mM KCl), incubated in the dark at room temperature for 20 min, and the fluorescence intensity of each system was measured using a fluorescence spectrometer (excitation wavelength 642 nm, excitation slit 3 nm, emission wavelength 666 nm, emission slit 3 nm, wavelength scanning range 650 - 800 nm). The fluorescence intensity at the maximum emission wavelength at 666 nm was recorded.

[0097] As Figure 9 shown, only when interacting with the target NT-proBNP, the fluorescence intensity of the system increased significantly. In contrast, there were significant differences in the fluorescence changes caused by coexisting interferents such as BNP, HSA, DD, ANP, and BSA compared to those caused by NT-proBNP, while the fluorescence changes caused by other potential interferents (such as IL-18, IL-6, IL-1, pro-BNP, CNP, TNF-α, BIL, CRP, CK-MB, LF, Hb) were extremely small and almost negligible. The above results indicate that the method of the present invention has excellent selective recognition ability for NT-proBNP, can effectively distinguish the target from other potential interferents, and ensure the accuracy and reliability of the detection results.

[0098] The term descriptions of this example are as follows:

[0099] IL-18: Interleukin-18;

[0100] IL-6: Interleukin-6;

[0101] IL-1: Interleukin-1;

[0102] pro-BNP: Pro-brain natriuretic peptide (or B-type natriuretic peptide precursor);

[0103] CNP: C-type natriuretic peptide;

[0104] TNF-α: Tumor necrosis factor-α;

[0105] BIL: Bilirubin;

[0106] CRP: C-reactive protein;

[0107] CK-MB: Creatine kinase isoenzyme MB;

[0108] LF: Lactoferrin;

[0109] Hb: Hemoglobin;

[0110] BSA: Bovine Serum Albumin;

[0111] ANP: Atrial Natriuretic Peptide;

[0112] DD: D-Dimer;

[0113] HSA: Human Serum Albumin;

[0114] BNP: B-Type Natriuretic Peptide.

[0115] Example 8 Spiking Recovery Experiment

[0116] To evaluate the detection accuracy of the present invention for actual samples, a spiking recovery experiment was conducted. Serum samples containing different spiking concentrations of NT-proBNP at low (0.5 pg / mL), medium (1 pg / mL), and high (3 pg / mL) levels were set up for the experiment, with three parallels in each group. The serum samples were respectively added to 100 μL of SNAs solution, mixed and then placed in Tris-HCl buffer (10 mM, pH 7.2, containing 50 mM NaCl, 10 mM MgCl2, and 10 mM KCl), incubated in the dark at room temperature for 20 min, and the fluorescence intensity of each system was measured using a fluorescence spectrometer (excitation wavelength 642 nm, excitation slit 3 nm, emission wavelength 666 nm, emission slit 3 nm, wavelength scanning range 650 - 800 nm). The fluorescence intensity at the maximum emission wavelength at 666 nm was recorded, and the recovery rate was calculated according to the following formula:

[0117] Spiking recovery rate = (measured value of spiked sample - measured value of sample) / spiked amount × 100%.

[0118] As shown in Table 2, the spiking recovery rate of this method for NT-proBNP in serum was 93.7% - 105.6%, and the relative standard deviation obtained from three parallel experiments was 2.22% - 6.37% (n = 3). The above results indicate that the present invention has good accuracy and repeatability in the detection of NT-proBNP in actual serum samples.

[0119] Table 2 Spiking Recovery Rate of SNAs System for NT-proBNP in Actual Serum Samples (n = 3)

[0120]

[0121] Note: RSD represents the coefficient of variation; Recovery represents the recovery rate.

[0122] Example 9

[0123] An NT-proBNP detection kit, comprising the following components: aptamer strand AptC, inhibitor strand Inh, and AuNPs.

[0124] Example 10

[0125] A fluorescence-colorimetric dual-signal detection kit for NT-proBNP based on spherical nucleic acid probes, comprising spherical nucleic acid probes.

[0126] Among them, the preparation method of the spherical nucleic acid probe is as follows:

[0127] S1: Dissolve DNA in enzyme-free water to a concentration of 100 μM, dispense and store at -20 °C for subsequent use.

[0128] S2: Mix the Inh strand and the AptC strand to obtain a mixed solution, anneal the mixed solution at 95 °C for 5 min, and naturally cool to room temperature to obtain a double-stranded solution.

[0129] S3: Add the formed double-stranded solution to a 10 nM AuNPs solution (DNA:AuNPs = 300:1), vortex and mix well, then place in a -20 °C refrigerator and freeze for 2 h.

[0130] S4: Thaw in the dark at room temperature, centrifuge the mixture (13000 rpm, 10 min) 3 times, and remove the supernatant.

[0131] S5: Redisperse the product in Tris-HCl buffer to obtain spherical nucleic acid probes, and store at 4 °C for standby.

[0132] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A detection kit for N-terminal pro-B-type natriuretic peptide, characterized in that, It comprises the following components: Aptamer strand AptC, inhibitor strand Inh, and gold nanoparticles; The nucleotide sequence of the aptamer strand AptC is shown as SEQ ID NO.3; The nucleotide sequence of the inhibitor strand Inh is shown as SEQ ID NO.

1.

2. The detection kit according to claim 1, wherein The 3'-end of the aptamer strand AptC carries a fluorophore.

3. The detection kit according to claim 2, wherein, The fluorophore is a Cy5 fluorophore.

4. The detection kit according to claim 1, wherein The 5'-end of the inhibitor strand Inh has a thiol modification.

5. A method for preparing a spherical nucleic acid probe for detecting N-terminal pro-B-type natriuretic peptide, characterized in that, It comprises the following steps: Anneal the aptamer strand AptC and the inhibitor strand Inh to form a DNA double strand; Load the DNA double strand onto the surface of gold nanoparticles to obtain the spherical nucleic acid probe. The nucleotide sequence of the aptamer strand AptC is shown as SEQ ID NO.3, and its 3'-end carries a fluorophore; The nucleotide sequence of the inhibitor strand Inh is shown as SEQ ID NO.

1.

6. The preparation method according to claim 5, wherein The fluorophore is a Cy5 fluorophore.

7. A spherical nucleic acid probe for detecting N-terminal pro-brain natriuretic peptide, prepared by the preparation method according to claim 5 or 6.

8. A fluorescence-colorimetric dual-signal detection kit for N-terminal pro-B-type natriuretic peptide based on spherical nucleic acid probes, characterized in that, It comprises the spherical nucleic acid probe according to claim 7.

9. A qualitative detection method for N-terminal pro-B-type natriuretic peptide for non-diagnostic purposes, characterized in that, It comprises the following steps: Add the sample to be detected and the spherical nucleic acid probe according to claim 7 into Tris-HCl buffer for incubation in the dark, and then observe the color of the solution. If the color of the solution changes from red to blue-violet, it is determined that the sample to be detected contains N-terminal pro-brain natriuretic peptide.

10. A method for quantitatively detecting N-terminal pro-B-type natriuretic peptide for non-diagnostic purposes, characterized in that, It comprises the following steps: Add the sample to be detected and the spherical nucleic acid probe according to claim 7 into Tris-HCl buffer for incubation in the dark, and then detect the fluorescence intensity of the reaction system; Substitute the fluorescence intensity into the standard curve to obtain the content of N-terminal pro-brain natriuretic peptide in the sample to be detected.

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