Electrochemiluminescence biosensor for detecting osteoporosis exosomal miRNAs, its preparation and application

Abstract

This invention belongs to the field of pharmaceutical technology, specifically disclosing an electrochemiluminescence biosensor for the detection of osteoporosis exosomal miRNAs, its preparation, and its application. The electrochemiluminescence biosensor is constructed based on a three-dimensional magnetic walking nanomachine and DNA tetrahedral functionalized Au NPs@g-C3N4 nanomaterials. This invention utilizes a highly efficient 3D DNA walker for signal conversion and amplification, along with superior self-luminescent Au NPs@g-C3N4 nanomaterials, to achieve ultrasensitive detection of target exosomal miRNAs. This demonstrates a reliable and promising strategy for developing ultrasensitive detection systems for osteoporosis exosomal miRNAs and provides a promising tool for the early clinical diagnosis of osteoporosis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to an electrochemiluminescence biosensor for detecting osteoporosis exosome miRNAs, its preparation and application. Background Technology

[0002] Osteoporosis is a systemic bone disease characterized by altered bone quality and decreased bone mass, leading to increased bone fragility and even fractures. Clinically, it is often asymptomatic before the first fracture. Therefore, early diagnosis is crucial for reducing the fracture rate in osteoporosis patients. Currently, routine diagnostic methods for osteoporosis include ultrasound, bone mineral density testing, X-ray examination, and serological testing; however, these methods often suffer from low sensitivity and specificity, hindering early diagnosis. Multiple studies have shown that osteoporosis-related exosomal miRNAs (exomiRNAs) are promising biomarkers for early diagnosis. To date, qRT-PCR, RNA blotting, and microarrays are commonly used for exomiRNA detection. However, the low abundance, high homology, and low expression of exomiRNAs present significant challenges for their clinical detection.

[0003] In recent years, electrochemiluminescence (ECL) technology has received increasing attention in various fields such as biomarker analysis, food detection, and environmental monitoring due to its advantages such as good controllability, high sensitivity, and simple operation. Currently, classic ECL luminescent agents mainly include luminol and ruthenium terpyridine ([Ru(bpy)3]). 2+ Luminol and its derivatives, these luminescent materials possess high electrochemical stability and low excitation potential, and have wide applications. Shen and his colleagues developed an ultrasensitive ECL biosensor based on TCPP-Fe@HMUiO@Au-ABEI nanoluminescent particles for the detection of exomiRNA-155. However, the instability of hydrogen peroxide in the luminol and its derivatives / H2O2 system inevitably affects the luminescence efficiency of ECL in the aqueous phase. Furthermore, [Ru(bpy)3] 2+ Due to their excellent solubility, luminescent materials are difficult to directly fix onto electrodes and require immobilization using nanomaterials [Ru(bpy)3]. 2+ Applications in biosensing. Compared to traditional ECL luminescent materials, graphitic carbon nitride (g-C3N4), as a self-luminescent ECL nanomaterial, possesses advantages such as high luminescence efficiency, ease of immobilization, and good stability. Furthermore, the self-luminescent nanomaterial g-C3N4 utilizes S2O8... 2- As a co-reaction reagent, it can directly generate an ECL signal, achieving highly sensitive detection. Therefore, g-C3N4 / S2O8 is utilized... 2-Building a biosensor for the detection of exomiRNAs could be more efficient and have greater application potential. Summary of the Invention

[0004] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide an electrochemiluminescence biosensor for the detection of osteoporosis exosomal miRNAs, its preparation and application. The electrochemiluminescence biosensor is constructed based on a 3D DNA walker and DNA tetrahedral (TDN) functionalized Au NPs@g-C3N4 nanomaterials. Utilizing the highly efficient 3D DNA walker as a signal transduction and amplification mechanism and the superior self-luminescent Au NPs@g-C3N4 nanomaterials, ultrasensitive detection of target exomiRNAs is achieved. This demonstrates a reliable and promising strategy for developing an ultrasensitive detection system for osteoporosis exosomal miRNAs and provides a promising tool for the early clinical diagnosis of osteoporosis.

[0005] To achieve the above and other related objectives, the first aspect of this invention provides an electrochemiluminescence biosensor for detecting osteoporosis exosomal miRNAs, comprising a three-dimensional magnetic walking nanomachine (3D DNA walker), AuNPs@g-C3N4 nanomaterials and DNA tetrahedrons (TDN), folic acid-modified S4 single-stranded DNA (S4-FA), and a working electrode.

[0006] The three-dimensional magnetic walking nanomachine includes streptavidin-labeled magnetic beads (SMB), biotin-modified S1 / PP double-stranded DNA bound to the magnetic beads and serving as the walking chain, S2 single-stranded DNA bound to the magnetic beads and serving as the DNA orbital, and a restriction endonuclease serving as the driving force; the S1 / PP double-stranded DNA is formed by hybridization of biotin-modified S1 single-stranded DNA and protective probe PP.

[0007] The protective probe PP is used to hybridize with the target exosomal miRNA through site displacement to form a double strand, releasing S1 single-stranded DNA; the S1 single-stranded DNA can hybridize with the S2 single-stranded DNA to form an enzyme cleavage site; the restriction endonuclease is used to specifically recognize the enzyme cleavage site and cleave the short chain S3, while releasing the S1 single-stranded DNA.

[0008] The Au NPs@g-C3N4 nanomaterials are deposited on the working electrode to obtain an electrochemiluminescence (ECL) response signal and provide active sites. The DNA tetrahedrons can bind to the Au NPs@g-C3N4 nanomaterials through Au-S bonds and be immobilized on the working electrode, serving as a scaffold to construct a bottom-up anchored biological probe. The folic acid-modified S4 single-stranded DNA is used to hybridize with the DNA tetrahedrons on the modified electrode to quench the electrochemiluminescence response signal. The short-chain S3 is used to replace the folic acid-modified S4 single-stranded DNA to restore the quenched electrochemiluminescence response signal.

[0009] Furthermore, the DNA tetrahedron is a DNA tetrahedral nanostructure formed by assembling four single-stranded DNA molecules, and the nucleotide sequences of the four single-stranded DNA molecules are shown in SEQ ID NO. 1 to 4, respectively.

[0010] The nucleotide sequences of the S1 single-stranded DNA, S2 single-stranded DNA, protective probe PP, S3 single-stranded DNA, and S4 single-stranded DNA are shown in SEQ ID NO.5 to 9, respectively.

[0011] Furthermore, the restriction endonuclease specifically recognizes the cleavage site as follows:

[0012] Furthermore, the restriction endonuclease is selected from Nb.BbvCI cleavage enzyme.

[0013] Furthermore, the osteoporosis exosome miRNAs are selected from exomiRNA-214, and their nucleotide sequence is shown in SEQ ID NO. 10.

[0014] Furthermore, the molar ratio of S1 / PP double-stranded DNA to S2 single-stranded DNA is 2-5:5-10, preferably 3:7.

[0015] Furthermore, the S1 / PP double-stranded DNA is formed by hybridization reaction of biotin-modified S1 single-stranded DNA and protective probe PP at 37°C; even further, the hybridization reaction time is not less than 20 min, preferably 20 to 100 min, and more preferably 60 min.

[0016] Furthermore, the streptavidin-labeled magnetic beads (SMB) are combined with the biotin-modified S1 / PP double-stranded DNA and the S2 single-stranded DNA to form modified magnetic beads (S1 / PP-S2-SMB). The method for binding the S1 / PP double-stranded DNA and the S2 single-stranded DNA to the magnetic beads includes the following steps: adding the S1 / PP double-stranded DNA and the S2 single-stranded DNA to the magnetic bead solution and stirring the reaction at 37°C; after the stirring reaction is completed, the modified magnetic beads are obtained by magnetic separation and washing; more further, the stirring reaction time is not less than 20 min, preferably 20-100 min, and more preferably 60 min.

[0017] Furthermore, the modified magnetic beads, the target exosomal miRNA, and the restriction endonuclease are reacted at 37°C; furthermore, the amount of the restriction endonuclease used is 50–500 U / mL. -1 Preferably 100–300 UmL -1 More preferably 200 U / mL -1 The reaction time is not less than 20 min, preferably 20 to 100 min, and more preferably 60 min.

[0018] Furthermore, the Au NPs@g-C3N4 nanomaterial is gold nanoparticles AuNPs immobilized on g-C3N4 nanomaterial. The preparation method of the Au NPs@g-C3N4 nanomaterial includes the following steps: dissolving g-C3N4 nanomaterial in a gold nanoparticle Au NPs solution, and stirring the reaction at room temperature to obtain Au NPs@g-C3N4 nanomaterial; more further, the stirring reaction time is not less than 12 hours, preferably 12 to 48 hours, and more preferably 24 hours.

[0019] Furthermore, the method for synthesizing the gold nanoparticles Au NPs includes the following steps: using tetrachloroauric acid as the gold source, heating and reacting under the action of a reducing agent to obtain gold nanoparticles Au NPs; preferably, the reducing agent is selected from sodium citrate.

[0020] Furthermore, when assembling the DNA tetrahedron, the molar concentrations of the four single-stranded DNA strands are equal.

[0021] Furthermore, the assembly method of the DNA tetrahedron includes the following steps: adding the four single-stranded DNA strands to a buffer solution, heating to 95°C and holding for 5 minutes, and then cooling to 4°C at a rate of 5°C every 18 minutes to obtain the DNA tetrahedron.

[0022] Furthermore, the method for preparing the folic acid-modified S4 single-stranded DNA includes the following steps: reacting folic acid (FA) with S4 single-stranded DNA overnight at 4°C under the action of a coupling agent to obtain the folic acid-modified S4 single-stranded DNA, wherein the coupling agent is preferably 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS); even further, the method for preparing the folic acid-modified S4 single-stranded DNA includes the following steps: adding EDC, NHS and S4 single-stranded DNA to a PBS solution and stirring at 4°C for 2 hours; then adding a folic acid solution and reacting overnight at 4°C to obtain S4-FA.

[0023] Furthermore, the working electrode uses a glassy carbon electrode as the base electrode.

[0024] A second aspect of the present invention provides a method for preparing an electrochemiluminescence biosensor for detecting osteoporosis exosome miRNAs as described in the first aspect, comprising the following steps:

[0025] I. Fabrication of three-dimensional magnetic walking nanomachines:

[0026] The S2 single-stranded DNA and the biotin-modified S1 / PP double-stranded DNA were combined with streptavidin-labeled magnetic beads to obtain modified magnetic beads (S1 / PP-S2-SMB).

[0027] The modified magnetic beads, the target exosomal miRNA, and the restriction endonuclease are mixed and reacted to obtain the short chain S3, including the following process:

[0028] The protective probe PP hybridizes with the target exosomal miRNA through site displacement to form a double strand. The S1 single-stranded DNA hybridizes with the S2 single-stranded DNA to form an enzyme cleavage site. The restriction endonuclease specifically recognizes the enzyme cleavage site and cleaves the short S3 strand, while releasing the S1 single-stranded DNA. The released S1 single-stranded DNA moves autonomously along the DNA track and hybridizes with another S2 single-stranded DNA on the magnetic bead. It is then specifically recognized and cleaved by the restriction endonuclease. After N cycles, a large number of S3 strands are released. The released short S3 strands are collected.

[0029] II. Fabrication of a switch-type electrochemiluminescence biosensor:

[0030] The Au NPs@g-C3N4 nanomaterials were deposited on the working electrode to obtain an electrochemiluminescence response signal and provide active sites;

[0031] The DNA tetrahedron was fixed to the working electrode on which the Au NPs@g-C3N4 nanomaterial was deposited via Au-S bonds, serving as a scaffold for constructing a bottom-up anchoring biological probe.

[0032] The folic acid-modified S4 single-stranded DNA was hybridized with the DNA tetrahedron to quench the electrochemiluminescence response signal.

[0033] The folic acid-modified S4 single-stranded DNA was replaced with the short-chain S3 collected in step I to restore the quenched electrochemiluminescence response signal.

[0034] Furthermore, the reaction time of the modified magnetic beads, the target exosome miRNA, and the restriction endonuclease is not less than 20 min, preferably 20 to 100 min, and more preferably 60 min.

[0035] Furthermore, the released short-chain S3 was collected using a magnetic separation method.

[0036] Furthermore, the fabrication process of the three-dimensional magnetic walking nanomachine in step I includes:

[0037] Biotin-modified S1 single-stranded DNA and protective probe PP were incubated at 37°C to obtain S1 / PP double-stranded DNA.

[0038] Take streptavidin-labeled magnetic beads (SMB), wash, and resuspend;

[0039] S2 single-stranded DNA and S1 / PP double-stranded DNA were added to a resuspended magnetic bead solution and stirred at 37°C to bind the biotin-modified S1 / PP double-stranded DNA and S2 single-stranded DNA to the magnetic beads; then magnetic separation and washing were performed; after washing, the modified magnetic beads were resuspended.

[0040] The target exosomal miRNA, resuspended modified magnetic beads, and restriction endonuclease were reacted with stirring at 37°C;

[0041] After the reaction is complete, the enzyme is inactivated by heating; finally, the S3 chains released after the walking process are collected by magnetic separation.

[0042] Furthermore, before depositing the Au NPs@g-C3N4 nanomaterials on the working electrode, the working electrode was pretreated. The pretreatment included polishing, cleaning, and drying the working electrode. More specifically, polishing was performed using aluminum oxide, preferably with a 0.05 μm aluminum oxide slurry, cleaning was performed using deionized water, and drying was performed using nitrogen.

[0043] Furthermore, the method for depositing the Au NPs@g-C3N4 nanomaterial on the working electrode includes: preparing the Au NPs@g-C3N4 nanomaterial into a dispersion, then dropping it onto the surface of the working electrode, and drying it to form a film.

[0044] Furthermore, the method for immobilizing the DNA tetrahedrons on the working electrode deposited with the Au NPs@g-C3N4 nanomaterials via Au-S bonds includes: preparing the DNA tetrahedrons into a solution, then dropping it onto the surface of the working electrode deposited with the Au NPs@g-C3N4 nanomaterials, and incubating it overnight at 4°C to immobilize the DNA tetrahedrons on the working electrode; and further, after immobilizing the DNA tetrahedrons on the working electrode, washing them with PBS solution.

[0045] Furthermore, after the DNA tetrahedron is immobilized on the working electrode, it is washed with PBS solution, then blocked with 6-mercaptohexanol (MCH) at room temperature, and then the folic acid-modified S4 single-stranded DNA is hybridized with the DNA tetrahedron; even further, the blocking time is not less than 20 min, preferably 20 to 100 min, and more preferably 60 min.

[0046] Furthermore, the method for hybridizing the folic acid-modified S4 single-stranded DNA with the DNA tetrahedron includes: adding the folic acid-modified S4 single-stranded DNA to the working electrode and incubating the reaction at 37°C; more further, the reaction time is not less than 20 min, preferably 20 to 100 min, and more preferably 60 min.

[0047] Furthermore, the method of replacing the folic acid-modified S4 single-stranded DNA with the short-chain S3 collected in step I includes: adding the collected short-chain S3 dropwise onto the working electrode and incubating the reaction at 37°C.

[0048] Furthermore, the substitution reaction time for replacing the folic acid-modified S4 single-stranded DNA with the short-chain S3 collected in step I is not less than 20 min, preferably 20 to 100 min, and more preferably 60 min.

[0049] It should be noted that the solvent and buffer used in the embodiments of the present invention are selected from any one of water, Tris-HCl buffer, Cutsmart buffer, PBS buffer, and TM buffer, and the water is preferably deionized water.

[0050] A third aspect of the present invention provides the application of the electrochemiluminescent biosensor prepared according to the first aspect and / or the electrochemiluminescent biosensor prepared according to the method of the second aspect in the preparation of a reagent for detecting osteoporosis exosome miRNAs.

[0051] Furthermore, the concentration of the target exosomal miRNA is measured by detecting the electrochemiluminescence response signal, and the intensity of the electrochemiluminescence response signal is positively correlated with the concentration of the target exosomal miRNA; even further, the intensity of the electrochemiluminescence response signal is positively correlated with the concentration of the target exosomal miRNA within the concentration range of 10.0 fM to 100.0 nM.

[0052] As described above, the electrochemiluminescence biosensor for detecting osteoporosis exosome miRNAs of the present invention, its preparation and application, have the following beneficial effects:

[0053] 1. This invention utilizes a three-dimensional (3D) DNA walker and self-luminescent nanomaterial (g-C3N4) to construct an ultrasensitive ECL biosensor for the detection of osteoporosis exosomal miRNAs, particularly exomiRNA-214. The ECL biosensor established in this invention has a limit of detection of 3.66 fM and a linear range from 10.0 fM to 100.0 nM. This invention provides a reliable strategy for the effective detection of osteoporosis exosomal miRNAs, especially exomiRNA-214, and offers a promising tool for the early clinical diagnosis of osteoporosis.

[0054] 2. In this invention, the 3D DNA walker can effectively convert the target exomiRNA-214 into an amplified biosensing signal, thereby improving the sensitivity of the constructed biosensor.

[0055] 3. This invention utilizes AuNPs functionalized with self-luminescent nanomaterials (g-C3N4) and tetrahedral DNA nanostructures (TDN) to construct a self-luminescent biosensing platform.

[0056] 4. This invention utilizes a three-dimensional tetrahedral DNA nanostructure with superior orientation and distance control as a scaffold to construct a "bottom-up" anchored biological probe, thereby improving the conversion efficiency of biosensors.

[0057] In summary, this invention successfully constructed an ECL biosensor for detecting osteoporotic exosomal miRNAs. Furthermore, the ECL biosensor exhibits high sensitivity, strong stability, and good reproducibility in the measurement of exomiRNA-214. Compared with existing technologies, the ECL biosensor of this invention has higher sensitivity and faster response speed. Therefore, the ECL biosensor provided by this invention holds promise for application in clinical specimen analysis, developing into a sensor with clinical value and providing a promising tool for the early clinical diagnosis of osteoporosis. Attached Figure Description

[0058] Figure 1The diagram shows the construction process and detection principle of the ECL biosensor in an embodiment of the present invention.

[0059] Figure 2 The figure shown is a result obtained by characterizing the constructed ECL biosensor in Example 2 of the present invention.

[0060] Figure 3 The figure shown is a graph of the results of the optimization of experimental parameters obtained in Example 3 of the present invention.

[0061] Figure 4 The figure shown is a result of evaluating the analytical performance of the constructed ECL biosensor in Example 4 of this invention.

[0062] Figure 5 The image shown is a result obtained by using the constructed ECL biosensor to detect an actual sample in Example 5 of this invention. Detailed Implementation

[0063] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0064] exomiRNA-214 is one of the exomiRNA biomarkers for early osteoporosis. Given the extremely low abundance of exomiRNA biomarkers in early osteoporosis, various signal amplification strategies are typically employed in the analytical field. Among various amplification techniques, DNA walkers are specialized molecular nanomachines programmed using the Watson-Crick pairing principle. Precisely designed DNA walkers can be driven by endonucleases, DNases, or strand displacement to move autonomously along specific trajectories, effectively converting small amounts of target signals into large amounts of output signals. Compared to one-dimensional and two-dimensional DNA walkers, three-dimensional (3D) DNA walkers typically walk on particulate nanomaterials such as gold nanoparticles (Au NPs) and magnetic beads, which have larger specific surface areas and higher DNA loading densities, resulting in better signal amplification performance and higher walking efficiency. Therefore, this invention designs a 3D DNA walker to construct an efficient analytical platform for target conversion and signal amplification, and combines it with DNA tetrahedral (TDN) functionalized Au NPs@g-C3N4 nanomaterials to construct a self-luminescent ECL biosensor for ultrasensitive detection of exomiRNA-214. This self-luminescent ECL biosensor utilizes a highly efficient 3D DNA walker for signal conversion and amplification, along with superior Au NPs@g-C3N4 nanomaterials, demonstrating a promising strategy for developing ultrasensitive detection systems.

[0065] Please refer to Figure 1 This invention provides a self-luminescent ECL biosensor, which is constructed based on Au NPs@g-C3N4 nanomaterials functionalized with three-dimensional magnetic walking nanomachines (3D DNA walkers) and DNA tetrahedrons (TDN). Its construction process and detection principle are as follows:

[0066] like Figure 1 As shown in Figure A, to convert the target exomiRNA-214 into an amplified biosensing signal (S3 strand), the 3D DNA walker designed in this embodiment of the invention consists of three main parts: ① biotin-labeled S1 / PP double-stranded DNA modified on magnetic beads SMB as the walking strand; ② the S2 strand as the DNA track; and ③ Nb.BbvCI cleavage enzyme as a driving force. In the presence of exomiRNA-214, the protective probe (PP) forms exomiRNA-214 / PP through foothold replacement, releasing the S1 strand. Subsequently, the S1 strand hybridizes with the S2 strand to form cleavage sites. This site is specifically recognized by the Nb BbvCI cleaving enzyme, which cleaves the short S3 strand and releases the S1 strand. The released S1 strand moves autonomously along a pre-designed DNA track, hybridizes with another S2 strand on the magnetic bead, and is subsequently specifically recognized and cleaved by the Nb BbvCI cleaving enzyme. After N cycles, a large number of S3 strands are released.

[0067] like Figure 1 As shown in Figure B, in the fabrication of the ECL biosensor, pre-prepared Au NPs were first immobilized on the surface of g-C3N4 to form Au NPs@g-C3N4 self-luminescent nanomaterials, which were then deposited on the electrode to obtain an ECL signal and provide a large number of active sites (the first "signal-on" state). Subsequently, pre-assembled TDN was immobilized on the modified electrode via Au-S bonds, and the folic acid-modified S4 chain (S4-FA) was hybridized with the TDN to quench the initial ECL response (the "signal-off" state). Finally, the S3 chain from the 3D DNA walker replaced the S4-FA chain, restoring the quenched ECL response (the second "signal-on" state).

[0068] As described above, the self-luminescent ECL biosensor provided in this embodiment of the invention utilizes a highly efficient 3D DNA walker for signal conversion and amplification and excellent Au NPs@g-C3N4 nanomaterials to achieve ultrasensitive detection of exomiRNA-214, providing a promising strategy for the development of early clinical diagnostic reagents for osteoporosis and the early clinical diagnosis of osteoporosis.

[0069] The implementation process of the present invention will be further described in detail below through specific embodiments.

[0070] Example 1

[0071] Preparation of an ECL biosensor for detecting osteoporosis exomiRNA-214

[0072] 1.1 Reagents and Materials

[0073] Streptavidin-labeled magnetic beads (SMB) were purchased from Selvinth Biotechnology Co., Ltd. (Chengdu, China). N-hydroxysuccinimide (NHS), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), chloroauric acid hexahydrate (HAuCl4·6H2O), folic acid (FA), tris(2-carbonylethyl)phosphohydrochloride (TCEP), and 6-mercaptohexanol (MCH) were purchased from Sigma-Aldrich Chemical Company (St. Louis, Missouri, USA). g-C3N4 was purchased from Frontier Nanomaterials Technology Co., Ltd. (Nanjing, China). Nb.BbvCI cleavage enzyme was purchased from New England Biolabs (Ipswich, Massachusetts, UK). Other reagents were supplied by Chemical Reagent Co., Ltd. (Chongqing, China). DNA oligonucleotides (Table 1) were synthesized and purified by Sangon Biotech Co., Ltd. (Shanghai, China).

[0074] 1.2 Instruments

[0075] Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were performed using a CHI660D electrochemical workstation (Shanghai Chenhua Instruments, China). All ECL measurements were performed on an MPI-E multifunction analyzer (Xi'an Ruimai Electronic Technology Co., Ltd., China). PCR detection was performed using a Bio-Rad analyzer (Bio-Rad, USA).

[0076] 1.3 Detection Principle

[0077] Figure 1 This study demonstrates the detection principle of a self-luminescent ECL biosensor for the ultrasensitive detection of osteoporosis exosomal miRNA-214 (exomiRNA-214), constructed from Au NPs@g-C3N4 nanomaterials functionalized with 3D DNA walkers and TDN. In the presence of exomiRNA-214, the protective probe (PP) undergoes site displacement to form exomiRNA-214 / PP, releasing the S1 strand. Subsequently, the S1 strand hybridizes with the S2 strand to form an enzyme cleavage site. This site is specifically recognized by the Nb BbvCI cleaving enzyme, which cleaves the short S3 strand and releases the S1 strand. The released S1 strand moves autonomously along a pre-designed DNA track, hybridizes with another S2 strand on the magnetic bead, and is subsequently specifically recognized and cleaved by the Nb BbvCI cleaving enzyme. After N cycles, a large number of S3 strands are released. Figure 1 A). Fabrication of ECL biosensors as follows Figure 1As shown in Figure B: First, pre-prepared Au NPs were immobilized on the surface of g-C3N4 to form Au NPs@g-C3N4 nanomaterials. Subsequently, Au NPs@g-C3N4 were deposited on the surface of a pre-treated glassy carbon electrode (GCE) to obtain an ECL signal and provide a large number of active sites (first "ON" state). Pre-assembled DNA tetrahedra (TDN) were immobilized on the modified electrode via Au-S bonds, serving as a scaffold to construct a "bottom-up" anchored biological probe to improve the conversion efficiency of the biosensor. Then, a folic acid-modified S4 strand (S4-FA) was hybridized with TDN to quench the initial ECL response ("OFF" state). Finally, an S3 strand from a 3D DNA walker replaced the S4-FA strand, restoring the quenched ECL response (second "ON" state). Based on this, the self-luminescent ECL biosensor utilizes the efficient signal conversion and amplification strategy of the 3D DNA walker and the superior self-luminescent Au NPs@g-C3N4 nanomaterial to achieve ultrasensitive detection of osteoporosis exomiRNA-214.

[0078] 1.4 Preparation and Detection Methods

[0079] 1.4.1 Fabrication of Three-Dimensional Magnetic Walking Nanomachines

[0080] Biotin-modified S1 strand (10 μL, 10 μM) and protective probe PP (10 μL, 10 μM) were added to 80 μL of 20 mM Tris-HCl buffer (1 mM MgCl2, 1 mM CaCl2, 5 mM KCl, 20 mM Tris, 140 mM NaCl, pH 7.4) and reacted at 37 °C for 1 h to obtain S1 / PP double-stranded DNA. Then, 10 μL of streptavidin-labeled magnetic beads (SMB) were washed three times with magnetic bead washing buffer 1 (10 mM Tris, 1 mM EDTA, 1 M NaCl, pH 7.5). After the last wash, the magnetic beads were resuspended in 10 μL of 20 mM Tris-HCl buffer. Subsequently, 70 μL of 1 μM S2 single-chain and 30 μL of 1 μM S1 / PP double-chain were added to 10 μL of resuspended magnetic bead solution, and the mixture was stirred at 37 °C for 1 h. The biotin-modified S1 / PP double-chain and S2 single-chain could bind to the magnetic beads. Then, after magnetic separation, the magnetic beads were washed three times with magnetic bead washing solution 2 (10 mM Tris, 1 mM EDTA, 1 M NaCl, 0.1% Tween-20, pH 7.5) and magnetic bead washing solution 1, respectively. After washing, the modified magnetic beads were resuspended in 40 μL of Cutsmart buffer (1×). Finally, 10 μL of Cutsmart buffer (10×) (20 mM Tris, 10 mM Mg(Ac)2, 500 mM KAc, 100 μg / mL) was added to the solution. -1 BSA (pH 7.9), 10 μL of target exomiRNA-214 at different concentrations, 10 μL of resuspended modified magnetic beads, 68 μL of DEPC water, and 2 μL of 10 U μL of [unclear - possibly a specific ingredient or solution] -1 The Nb.BbvCI cleavage enzyme was stirred at 37°C for 1 h. Subsequently, the mixture was inactivated at 80°C for 20 min. Finally, the S3 chains released after the walking process were collected by magnetic separation.

[0081] 1.4.2 Preparation of Au NPs@g-C3N4 nanomaterials

[0082] The synthesis method of gold nanoparticles (Au NPs) is as follows: 85.2 mL of deionized water and 3 mL of tetrachloroauric acid solution (29.4 mM) were added to a round-bottom flask, and the flask was placed in an oil bath mixer at 120 °C. After the solution boiled, 10.1 mL of sodium citrate (34.0 mM) was quickly poured in. The solution color changed from light yellow to dark red within minutes. Stirring was continued for 10 min to obtain Au NPs. Subsequently, 4.0 mg of g-C3N4 was completely dissolved in 4 mL of the Au NPs solution, and the mixture was stirred continuously at room temperature for 24 h to obtain Au NPs@g-C3N4 nanomaterials. Finally, the obtained Au NPs@g-C3N4 nanomaterials were washed three times with deionized water and stored at 4 °C until use.

[0083] 1.4.3 Assembly of DNA tetrahedra

[0084] DNA tetrahedral nanostructures were assembled using four single-stranded DNAs (TDN-AQ, TDN-BS, TDN-CS, and TDN-DS) in TM buffer (10 mM Tris, 50 mM MgCl2, 1 mM EDTA, pH 8.0). The specific process was as follows: four single-stranded DNAs of equal concentration were added to the buffer, and the mixture was heated to 95°C and held for 5 min. The mixture was then cooled to 4°C at a rate of 5°C every 18 minutes to obtain DNA tetrahedra, which were then stored at 4°C until use.

[0085] 1.4.4 Preparation of S4-FA

[0086] 40 μL of 100 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 10 μL of 100 mM N-hydroxysuccinimide (NHS), and 10 μL of S4 chain were added to 140 μL of PBS solution (0.1 M, pH 7.4), and stirred at 4 °C for 2 h. Finally, 5.0 mM folic acid (FA) solution (folic acid powder dissolved in 0.1 M, pH 7.4 PBS solution) was added to the above mixture, and the reaction was carried out overnight at 4 °C to obtain S4-FA.

[0087] 1.4.5 Isolation of exosomes and exosomal miRNAs

[0088] The exosome isolation method was as follows: First, serum samples from multiple osteoporosis patients and healthy individuals were centrifuged at 2000×g for 30 min. After centrifugation, the supernatant was filtered through a 0.22 μm filter. Then, the obtained filtrate was ultracentrifuged at 110,000×g and 4℃ for 70 min. Subsequently, the supernatant was discarded, and the precipitate was resuspended in PBS buffer (0.1M, pH 7.4). To remove protein impurities, the resuspended solution was ultracentrifuged again at 110,000×g and 4℃ for 70 min. Finally, an appropriate amount of supernatant was discarded, and the obtained nanoscale vesicles were mixed and stored in the remaining 300 μL solution, and stored at -80℃ before use. Throughout the process, all sample processing and centrifugation were performed at 4℃. Exosomal miRNA was extracted using a universal miRNA extraction and purification kit according to the manufacturer's instructions.

[0089] 1.4.6 Fabrication of a Switch-Type ECL Biosensor

[0090] First, a bare glassy carbon electrode (GCE) was polished with 0.05 μm alumina slurry, washed with deionized water, and dried under nitrogen. Next, 10 μL of Au NPs@g-C3N4 nanomaterials was added to the prepared electrode surface and dried to form a film. Then, 10 μL of 1 μM DNA tetrahedra was dropped onto the modified GCE surface and incubated overnight at 4°C. The DNA tetrahedra can bind to the Au NPs@g-C3N4 nanomaterials via Au-S bonds. Subsequently, the modified GCE was washed with PBS solution and blocked with 1 mM 6-mercaptohexanol (MCH) at room temperature for 1 h. Then, 10 μL of S4-FA strands was added to the modified GCE and reacted at 37°C for 1 h. Finally, 10 μL of collected S3 strands was dropped onto the modified GCE for hybridization and reacted at 37°C for 1 h. Throughout the reaction, the patient was washed with PBS solution (0.01 M, pH 7.4) at each step, and the ECL signal was recorded. The increase in ECL signal was closely related to the concentration of exomiRNA-214.

[0091] Table 1. Nucleotide sequence list

[0092]

[0093]

[0094] T 50 : indicates 50 T bases; T 10 : indicates 10 T bases.

[0095] Example 2

[0096] Characterization of the constructed ECL biosensor

[0097] The ECL biosensor constructed in Example 1 was characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV), respectively, in a solution containing 5 mM [Fe(CN)6]. 3- / 4- It is carried out in 0.1M potassium chloride. For example... Figure 2 As shown in Figure A, the bare GCE (curve a) exhibits a pair of distinct redox peaks due to the free electron transfer process. After immobilizing TDN on the modified electrode, the peak current decreases due to weaker electron transfer at the electrode surface (curve b). For the same reason, the peak current significantly decreases after hybridization with S4-FA (curve c). When S3 chains are added, the peak current increases significantly (curve d), indicating that S4-FA is replaced by S3 chains released from the three-dimensional magnetic walking nanomachine. The EIS results are consistent with the CV results. Figure 2 B), where R et The values ​​varied with immobilization, hybridization, and substitution. These results indicate that the ECL biosensor has been successfully established.

[0098] Example 3

[0099] Optimization of key experimental conditions for ECL biosensors

[0100] To construct an ultrasensitive exomiRNA-214 detection system, the key experimental conditions of the ECL biosensor constructed in Example 1 were optimized, and the analytical performance of the biosensor was established.

[0101] First, the ECL intensity was measured at different Nb.BbvCI cleavage enzyme treatment times (20, 40, 60, 80, 100 min), and the results are as follows: Figure 3 As shown in Figure A, the ECL intensity gradually increased with the extension of the Nb.BbvCI cleavage enzyme reaction time, reaching a plateau at 60 min, and then slightly decreased. Therefore, 60 min was selected as the optimal reaction time for the Nb.BbvCI cleavage enzyme.

[0102] Subsequently, the ECL intensity was measured at different substitution reaction times (20, 40, 60, 80, 100 min) for the S4-FA probe, and the results are as follows: Figure 3 As shown in Figure B, the ECL intensity gradually increases with increasing substitution reaction time, reaching a plateau at 60 min. Therefore, the optimal substitution reaction time for S4-FA is selected as 60 min.

[0103] Example 4

[0104] Analytical performance of the constructed ECL biosensor

[0105] 4.1 Sensitivity of the constructed ECL biosensor

[0106] Under the optimal conditions obtained in Example 3, the ECL biosensor constructed in Example 1 was used to quantitatively measure different concentrations of exomiRNA-214.

[0107] Figure 4 A showed that the ECL intensity increased with increasing exomiRNA-214 concentration from 10.0 fM to 100.0 nM. The ECL intensity showed a good correlation with exomiRNA-214 concentration, with a linear equation of I = 855.08lg c + 928.39(R²). 2 =0.9969), where I is the ECL intensity and c is the concentration of exomiRNA-214. The limit of detection (LOD) was calculated to be 3.66 fM by adding three standard deviations (95% confidence interval) to the mean of negative samples. Compared with previous miRNA detection strategies, the ECL biosensor constructed in this embodiment of the invention shows a lower LOD (Table 2), which is due to the efficient signal conversion and amplification of the superior Au NPs@g-C3N4 self-luminescent nanomaterials and the three-dimensional magnetic walking nanomachines.

[0108] Table 2. Comparison with existing miRNA detection methods

[0109] Fluorescence From 100.0pM to 25.0nM 77.40pM Xu et al., 2022(1) DPV From 0.1 nM to 10 μM 29pM Kong et al., 2022(2) ECL From 0.6pM to 4000pM 0.26pM Du et al., 2022(3) ECL From 30fM to 20nM 10fM Guo et al., 2023(4) ECL From 10.0fM to 100.0nM 3.66fM This work

[0110] DPV: Differential pulse voltammetry.

[0111] Note:

[0112] (1)Xu,Y.,Li,XM,Niu,CC,Wu,HP,Yong,YT,Qi,CH,Gong,W.,Bai,HJ,Chen,YR,Ding,SJ,Liao,P.,2022.Biosens.Bioelectron.212,114405.

[0113] (2)Kong,L.,Lv,S.,Qiao,Z.,Yan,Y.,Zhang,J.,Bi,S,2022.Biosens.Bioelectron.207,114188.

[0114] (3)Du,JF,Chen,JS,Liu,XP,Mao,CJ,Jin,BK,2022.Microchim.Acta189,264.

[0115] (4)Guo, Y., Nie, Y., Wang, P., Li, Z., Ma, Q., 2023. Talanta 259, 124559.

[0116] 4.2 Stability and specificity of the constructed ECL biosensor

[0117] To evaluate the stability of the constructed ECL biosensor, three concentrations (100.0 fM, 100.0 pM, and 10.0 nM) of exomiRNA-214 were used, and the relative standard deviation (RSD) of the ECL intensity was investigated. Figure 4 B shows that the RSDs are 4.17%, 4.16%, and 4.29% (n=3), indicating that the developed ECL strategy has good stability.

[0118] To evaluate the specificity of the constructed ECL biosensor, four different exomiRNAs (exomiRNA-214, exomiRNA-133, exomiRNA-155, and exomiRNA-373) were used in the study. Figure 4 As shown in Figure C, the ECL intensity containing exomiRNA-214 showed a significant change, while other exomiRNAs (exomiRNA-133, exomiRNA-155, and exomiRNA-373) did not show strong signal changes, and the difference was statistically significant (P<0.001). These results indicate that only exomiRNA-214 can promote the signal amplification and conversion effect based on the three-dimensional magnetic walking nanomachine. Therefore, the constructed ECL biosensor has good specificity.

[0119] Example 5

[0120] Actual sample analysis

[0121] To evaluate whether the biosensor constructed in Example 1 can be used to measure exomiRNA-214 in real samples, this example conducted comparative and recovery experiments, measuring the exomiRNA-214 in osteoporosis patients ( Figure 5 c, d, e, f, g) and healthy people ( Figure 5 exomiRNA-214 in serum samples a) and b) from [the sample]. Figure 5 As shown in A and 5B, the ECL intensity in the osteoporosis group was significantly higher than that in the healthy group, which is consistent with the results of qRT-PCR detection, and the difference was statistically significant (P<0.001).

[0122] Furthermore, different concentrations of exomiRNA-214 (10.0 pM, 100.0 pM, and 1.0 nM) were added to the extracted miRNAs for recovery experiments. The results showed that the average recoveries of the constructed ECL biosensor in detecting exomiRNA-214 in serum samples were 109.21%, 106.54%, and 97.80%, respectively, with acceptable RSDs ranging from 3.88% to 7.17%. This indicates that the two methods can clearly distinguish between healthy individuals and osteoporosis patients, thus demonstrating good analytical value in clinical applications.

[0123] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. An electrochemiluminescence biosensor for detection of osteoporosis exosome miRNAs, characterized in that, This includes a three-dimensional magnetic walking nanomachine, Au NPs@g-C3N4 nanomaterials and DNA tetrahedra, folic acid-modified S4 single-stranded DNA, and a working electrode. The three-dimensional magnetic walking nanomachine includes streptavidin-labeled magnetic beads, biotin-modified S1 / PP double-stranded DNA bound to the magnetic beads and serving as the walking chain, S2 single-stranded DNA bound to the magnetic beads and serving as the DNA orbital, and a restriction endonuclease serving as the driving force; the S1 / PP double-stranded DNA is formed by hybridization of biotin-modified S1 single-stranded DNA and a protective probe PP. The protective probe PP is used to hybridize with the target exosomal miRNA through site displacement to form a double strand, releasing S1 single-stranded DNA; the S1 single-stranded DNA can hybridize with the S2 single-stranded DNA to form an enzyme cleavage site; the restriction endonuclease is used to specifically recognize the enzyme cleavage site and cleave the short chain S3, while releasing the S1 single-stranded DNA. The Au NPs@g-C3N4 nanomaterials are deposited on the working electrode to obtain an electrochemiluminescence response signal and provide active sites. The DNA tetrahedrons can bind to the Au NPs@g-C3N4 nanomaterials through Au-S bonds and be immobilized on the working electrode, serving as a scaffold to construct a bottom-up anchored biological probe. The folic acid-modified S4 single-stranded DNA is used to hybridize with the DNA tetrahedrons on the working electrode to quench the electrochemiluminescence response signal. The short-chain S3 is used to replace the folic acid-modified S4 single-stranded DNA to restore the quenched electrochemiluminescence response signal. The DNA tetrahedron is a DNA tetrahedral nanostructure formed by assembling four single-stranded DNA molecules, and the nucleotide sequences of the four single-stranded DNA molecules are shown in SEQ ID NO. 1~4, respectively. The nucleotide sequences of the S1 single-stranded DNA, S2 single-stranded DNA, protective probe PP, and short-stranded S3 and S4 single-stranded DNA are shown in SEQ ID NO. 5~9, respectively.

2. The electrochemiluminescence biosensor according to claim 1, characterized in that: The restriction endonuclease specifically recognizes the cleavage site as follows: .

3. The electrochemiluminescence biosensor according to claim 1, characterized in that: The restriction endonuclease is Nb.BbvCI cleavage enzyme.

4. The electrochemiluminescence biosensor according to claim 1, characterized in that: The osteoporosis exosomal miRNAs are exomiRNA-214, and their nucleotide sequence is shown in SEQ ID NO.

10.

5. The method for preparing the electrochemiluminescence biosensor according to any one of claims 1 to 4, characterized in that, Includes the following steps: I. Fabrication of three-dimensional magnetic walking nanomachines: The S2 single-stranded DNA and the biotin-modified S1 / PP double-stranded DNA were combined with streptavidin-labeled magnetic beads to obtain modified magnetic beads. The modified magnetic beads, the target exosomal miRNA, and the restriction endonuclease are mixed and reacted to obtain the short chain S3, including the following process: The protective probe PP hybridizes with the target exosomal miRNA through site displacement to form a double strand. The S1 single-stranded DNA hybridizes with the S2 single-stranded DNA to form an enzyme cleavage site. The restriction endonuclease specifically recognizes the enzyme cleavage site and cleaves the short S3 strand, simultaneously releasing the S1 single-stranded DNA. The released S1 single-stranded DNA moves autonomously along the DNA track and hybridizes with another S2 single-stranded DNA on the magnetic bead. It is then specifically recognized and cleaved by the restriction endonuclease. After N cycles, a large number of S3 strands are released. The released short S3 strands are collected by magnetic separation. II. Fabrication of a switch-type electrochemiluminescence biosensor: The Au NPs@g-C3N4 nanomaterials were deposited on the working electrode to obtain an electrochemiluminescence response signal and provide active sites; The DNA tetrahedron was fixed to the working electrode on which the Au NPs@g-C3N4 nanomaterial was deposited via Au-S bonds, serving as a scaffold for constructing a bottom-up anchoring biological probe. The folic acid-modified S4 single-stranded DNA was hybridized with the DNA tetrahedron to quench the electrochemiluminescence response signal; The folic acid-modified S4 single-stranded DNA was replaced with the short S3 collected in step I to restore the quenched electrochemiluminescence response signal.

6. The preparation method according to claim 5, characterized in that: The reaction time for the modified magnetic beads, the target exosomal miRNA, and the restriction endonuclease is no less than 20 min.

7. The preparation method according to claim 5, characterized in that: The substitution reaction time for replacing the folic acid-modified S4 single-stranded DNA with the short S3 collected in step I shall not be less than 20 min.

8. The application of the electrochemiluminescent biosensor according to any one of claims 1 to 4 or the electrochemiluminescent biosensor prepared by the method according to any one of claims 5 to 7 in the preparation of osteoporosis exosome miRNA detection reagents.

9. The application according to claim 8, characterized in that: The concentration of target exosomal miRNA is measured by detecting the electrochemiluminescence response signal, and the intensity of the electrochemiluminescence response signal is positively correlated with the concentration of target exosomal miRNA.

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