An ECL composite material, a sensor, its preparation, and its application in miRNA-133a detection.

Abstract

This invention discloses an ECL composite material, a sensor, its preparation, and its application in the detection of miRNA-133a. The composite material comprises a nickel-iron Prussian blue analog with peroxidase activity, gold-platinum nanoparticles loaded on the surface of the nickel-iron Prussian blue analog nanoparticles, and an electrochemiluminescent substrate connected to the gold-platinum nanoparticles via gold-nitrogen covalent bonds. The sensor comprises a working electrode and a homogeneous system. The working electrode is modified with the composite material, and a quenching probe, a single-stranded reporter probe PH modified with dopamine, is attached to the composite material. The homogeneous system comprises gold nanoparticles loaded with deoxyribozyme chains and orbital hairpins (TH). The deoxyribozyme chains are double-stranded chains assembled from a long-arm chain (WS) and a blocking chain (BS). This invention utilizes the high ECL output signal of the ECL composite material and the signal amplification capability of the 3D DNAzyme Walker to construct an ECL sensor that achieves high specificity and high sensitivity detection of miRNA-133a.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an ECL composite material, a sensor, its preparation, and its application in the detection of miRNA-133a. Background Technology

[0002] Acute myocardial infarction (AMI) is a serious cardiovascular disease with high morbidity and mortality rates, and in recent years, the affected population has been gradually becoming younger. Early and accurate diagnosis and effective revascularization treatment play a crucial role in reducing the morbidity and mortality of AMI patients. Currently, the gold standard for detecting AMI is the immunological detection of cardiac troponin I (CTnI) due to its high specificity. However, the high cost of immunoassay and the easy inactivation of antibodies limit its widespread application. It has been reported that after the onset of AMI, the concentration of miRNA-133a in plasma increases sharply, and the expression peaks of miRNA-133a and cTnI occur almost simultaneously, which can be used as an auxiliary diagnostic tool for AMI.

[0003] MicroRNAs (miRNAs) are single-stranded, endogenous non-coding regulatory RNAs, approximately 19-23 nucleotides long, that play crucial regulatory roles in early development, cell differentiation, proliferation, and apoptosis. Numerous studies have shown that abnormal miRNA expression is associated with many diseases, particularly tumorigenesis and development. Therefore, analyzing miRNAs in patient serum holds immense potential and value for early disease screening and precision diagnosis. It is worth noting that miRNAs possess many unique characteristics, such as small size, high sequence similarity, easy degradation, and low abundance, posing analytical challenges for accurate detection and quantification. Traditional miRNA analysis methods, including quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and microarray technology, are widely used, but suffer from drawbacks such as low sensitivity, time-consuming methods, high false positive rates, and expensive equipment. Therefore, developing a sensitive, specific, and convenient new detection technique to monitor miRNA-133a levels is of great significance for early screening and diagnosis of acute myeloma (AMI).

[0004] DNA walkers are a type of DNA nanodevice that can precisely control programmed oligonucleotides at the micrometer or nanometer scale, and have broad application potential in fields such as biosensing and drug delivery. Upon entropy-driven strand displacement reactions, enzymes, light, or chemical stimulation, DNA walkers can move along pre-designed trajectories, including one-dimensional (1D) planar trajectories, two-dimensional (2D) DNA origami, or three-dimensional (3D) orbitals. In 3D DNA walkers, all reactive components are concentrated on micrometer or nanometer-scale 3D orbits, resulting in high local effective concentrations and stable signal output. For example, in the field of biosensing, Li and his colleagues designed an enzyme-driven 3D Walker sensor that can achieve isothermal and homogeneous signal amplification for the detection of specific nucleic acids; Fan et al. also programmed a DNA Walker driven by exonuclease III (Exo III) to generate cascade signal amplification for ultrasensitive bioanalysis; Liu and his team developed a 3D DNA Walker for the determination of 8-hydroxy-2'-deoxyguanosine, which reduced the detection limit by three orders of magnitude compared to previously reported methods. Although these methods demonstrate ultra-high sensitivity, they are all enzyme-dependent methods, which suffer from some inherent limitations of enzymes: susceptibility to complex experimental conditions (such as buffers and temperature), high detection costs, and unsuitability for long-term storage. Therefore, developing an enzyme-free 3D DNA Walker is of great significance. Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide an ECL composite material, a sensor, its preparation and application in miRNA-133a detection, which utilizes the high ECL output signal of the composite material and the signal amplification capability of the 3D DNAzymeWalker to achieve high specificity and high sensitivity detection of miRNA-133a.

[0006] To achieve the above and other related objectives, the first aspect of the present invention provides an electrochemiluminescence (ECL) composite material, the composite material comprising a nickel-iron Prussian blue analog (Ni-Fe PBA) with peroxidase activity, gold-platinum nanoparticles (AuPt NPs) loaded on the surface of the nickel-iron Prussian blue analog nanoparticles, and an electrochemiluminescence substrate connected to the gold-platinum nanoparticles via gold-nitrogen covalent bonds (Au-N bonds).

[0007] In some embodiments, the electrochemiluminescent substrate is selected from any one of N-(4-aminobutyl)-N-ethyl isoluminol (ABEI) and luminol, preferably N-(4-aminobutyl)-N-ethyl isoluminol (ABEI).

[0008] In some embodiments, the nickel-iron Prussian blue analogue is nanoscale, with a cube shape and a particle size of 50–60 nm.

[0009] A second aspect of the present invention provides a method for preparing the electrochemiluminescent composite material according to the first aspect, comprising the following steps:

[0010] Nickel-iron Prussian blue analogues were synthesized by a method of standing at room temperature using nickel precursor, trisodium citrate and iron coordination compound as raw materials.

[0011] In situ reduction was performed on the surface of a nickel-iron Prussian blue analogue to load gold-platinum nanoparticles, forming AuPt@Ni-FePBA material.

[0012] The electrochemiluminescent composite material is obtained by connecting the AuPt@Ni-Fe PBA material with the electrochemiluminescent substrate through a gold-nitrogen covalent bond (Au-N bond).

[0013] In some embodiments, nickel-iron Prussian blue analogues are synthesized by means of a nickel precursor, trisodium citrate, and potassium ferricyanide, and by a method of standing at room temperature.

[0014] The nickel precursor and trisodium citrate were dissolved in water to form solution A; potassium ferricyanide was dissolved in water to form solution B; solutions A and B were mixed and stirred evenly, and then allowed to stand at room temperature for aging; after the aging was completed, the mixture was centrifuged and the precipitate was washed to obtain the nickel-iron Prussian blue analogue.

[0015] In some embodiments, the molar ratio of nickel, trisodium citrate, and potassium ferricyanide is 10–15:10–20:5–12, preferably 1:1:1, 2:3:2, 4:5:4, 12:15:10, or 12:15:8.

[0016] In some embodiments, the nickel precursor is a nickel-containing soluble salt, which is selected from nickel acetate, nickel chloride, and nickel nitrate. In some embodiments, the static aging time at room temperature is not less than 24 hours, preferably 24-36 hours, for example 24, 26, 28, 30, 32, 34, or 36 hours.

[0017] In some embodiments, in-situ reduction of a nickel-iron Prussian blue analogue onto a surface to load gold-platinum nanoparticles forms an AuPt@Ni-Fe PBA material, comprising:

[0018] A surfactant was added to a nickel-iron Prussian blue analogue solution to form solution C; gold and platinum precursors were dissolved in water to form solution D; solution D was added to solution C, stirred evenly, and then a reducing agent was added and stirred to carry out a reduction reaction; after the reaction was completed, the mixture was centrifuged, the precipitate was washed, and AuPt@Ni-Fe PBA material was obtained.

[0019] In some embodiments, the nickel-iron Prussian blue analog solution is formed by dissolving a nickel-iron Prussian blue analog in a solvent, wherein the solvent used to dissolve the nickel-iron Prussian blue analog is selected from water or ethanol.

[0020] In some embodiments, the surfactant is selected from polyvinylpyrrolidone (PVP).

[0021] In some embodiments, the gold precursor is selected from a soluble salt containing gold, and the soluble salt containing gold is selected from chloroaurate (AuCl4). - ), gold nitrate (Au(NO3)4) - The chloroaurate is selected from any one of potassium dicyanate K[Au(CN)2], wherein the chloroaurate is selected from any one of chloroauric acid, sodium chloroaurate, potassium chloroaurate, and ammonium chloroaurate, and the gold nitrate is selected from gold nitrate.

[0022] In some embodiments, the platinum precursor is selected from platinum-containing soluble salts, wherein the platinum-containing soluble salt is chloroplatinate (PtCl4). - The chloroplatinate is selected from any one of chloroplatinic acid, sodium chloroplatinate, potassium chloroplatinate, and ammonium chloroplatinate.

[0023] In some embodiments, the reducing agent is selected from sodium borohydride. In some embodiments, the molar ratio of gold to platinum is 1:1.

[0024] In some embodiments, the reduction reaction time is not less than 30 min, preferably 30 to 60 min, for example 30, 35, 40, 45, 50, 55, or 60 min.

[0025] In some embodiments, AuPt@Ni-Fe PBA material is linked to an electrochemiluminescent substrate via a gold-nitrogen covalent bond (Au-N bond) to obtain the electrochemiluminescent composite material, comprising:

[0026] AuPt@Ni-Fe PBA material and electrochemiluminescent substrate were added to water and stirred at room temperature to carry out the reaction. After the reaction was completed, excess electrochemiluminescent substrate was removed by washing with water to obtain the electrochemiluminescent composite material.

[0027] In some embodiments, AuPt@Ni-Fe PBA material and electrochemiluminescent substrate are added to water and stirred at room temperature for a reaction time of not less than 30 min, preferably 30 to 60 min, for example 30, 35, 40, 45, 50, 55, or 60 min.

[0028] In some embodiments, the electrochemiluminescent composite material is suspended in a chitosan solution for storage and later use, wherein the concentration of the chitosan solution is 0.05% to 0.2%.

[0029] A third aspect of the present invention provides an electrochemiluminescence sensor, comprising a working electrode and a homogeneous system, wherein the working electrode is modified with an electrochemiluminescence composite material as described in the first aspect, and a quenching probe is attached to the electrochemiluminescence composite material, wherein the quenching probe is a single-chain reporter probe PH modified with dopamine (DA), and the nucleotide sequence of the single-chain reporter probe PH is shown in SEQ ID NO.5;

[0030] The homogeneous system includes gold nanoparticles (Au NPs) loaded with a closed deoxyribozyme (DNAzyme) chain and a cleavable orbital hairpin TH. The deoxyribozyme chain is a double strand assembled from a long arm chain WS and a blocking chain BS. The nucleotide sequences of the long arm chain WS, the blocking chain BS, and the orbital hairpin TH are shown in SEQ ID NO.1, 2, and 4, respectively.

[0031] The detection principle of the electrochemiluminescence sensor of the present invention is as follows: First, the electrochemiluminescence composite material serves as the bottom layer material on the working electrode, providing a high-intensity ECL signal basis; second, a quenching probe, namely a single-stranded reporter probe PH modified with DA, is connected on the bottom layer material. Through the quenching effect of DA on the ECL signal, the signal transition from "on" to "off" is achieved; finally, a 3D DNAzyme Walker is constructed by loading pre-blocked DNAzyme strands (WS+BS) and cleavable orbital hairpins TH onto AuNPs in a homogeneous system, wherein the orbital hairpins TH simultaneously contain sites cleaved by WS and blocked DNAzyme sequences. When the target miRNA-133a is present, the DNAzyme portion of the WS strand is exposed through strand displacement. The specific site of the orbital hairpin TH is cleaved, exposing a new DNAzyme sequence and forming a multi-legged active 3D DNAzyme Walker. When the homogeneous system is dropped onto the surface of the working electrode, the binding arm of the 3D DNAzyme Walker can specifically recognize and cleave the quenching probe on the working electrode. The quenching group DA at the top of the quenching probe is released, thereby generating a measurable ECL signal, enabling the detection of miRNA-133a.

[0032] In some embodiments, the homogeneous system further includes Mn 2+ When the target is present, the exposed WS can be in Mn 2+ TH is cut with the assistance of [unclear].

[0033] In some embodiments, the single-stranded reporter probe PH, orbital hairpin TH, deoxyribonuclease chain (WS+BS), and Mn 2+ The molar ratio is 10:10:1:5000 to 15000, preferably 10:10:1:10000 to 12000, for example 10:10:1:10000, 10:10:1:11000, 10:10:1:12000.

[0034] In some embodiments, the method for preparing the deoxyribonuclease chain (WS+BS) includes the following steps:

[0035] The long-arm chain WS and the blocking chain BS are annealed and denatured to assemble into a double chain, thus obtaining the deoxyribozyme chain.

[0036] In some embodiments, the molar ratio of the long arm chain WS to the blocking chain BS is 1:1.1 to 1.3, for example 1:1.1, 1:1.2, or 1:1.3.

[0037] In some embodiments, the nucleotide sequence of the target TA of the long-arm chain WS that can be exposed by chain substitution is shown in SEQ ID NO.4.

[0038] In some embodiments, the concentration of dopamine in the moving average system is 1.0–1.20 mM.

[0039] In some embodiments, the working electrode is a glassy carbon electrode.

[0040] In some embodiments, the method of modifying the working electrode with the electrochemiluminescence composite material includes the following steps:

[0041] The electrochemiluminescent composite material is suspended in a chitosan solution, then dropped onto the working electrode and dried to form the underlying material.

[0042] In some embodiments, the concentration of the chitosan solution is 0.05% to 0.2%.

[0043] In some embodiments, the electrochemiluminescent composite material is suspended in a chitosan solution, then dropped onto the working electrode and dried to form a material layer on the working electrode.

[0044] In some embodiments, the drying temperature is 37°C.

[0045] In some embodiments, the drying time is not less than 20 minutes, preferably 20 to 30 minutes, for example 20, 25, or 30 minutes.

[0046] In some embodiments, the method of attaching a quenching probe to the electrochemiluminescent composite material includes the following steps:

[0047] A single-chain reporter probe PH modified with dopamine was dropped onto the working electrode modified with the electrochemiluminescence composite material, and the reaction was allowed to proceed.

[0048] In some embodiments, the static reaction temperature is 0–8°C, preferably 4°C.

[0049] In some embodiments, the static reaction time is not less than 12 hours, preferably 12 to 16 hours, for example 12, 14, or 16 hours.

[0050] In some embodiments, the method for preparing the quenching probe includes the following steps:

[0051] Carboxyl and thiol groups were modified at the 5' and 3' ends of the single-chain reporter probe PH, respectively. After activating the carboxyl group, it was mixed with dopamine solution to react and form a single-chain reporter probe PH modified with dopamine, namely the quenching probe.

[0052] In some embodiments, the carboxyl group is activated using a reaction system containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

[0053] In some embodiments, the concentration of the dopamine solution is 1.0 to 1.20 mM.

[0054] In some embodiments, the reaction time with the dopamine solution is not less than 8 hours, preferably 8 to 12 hours, for example 8, 10, or 12 hours.

[0055] The fourth aspect of the present invention provides the use of the electrochemiluminescent composite material according to the first aspect and the electrochemiluminescent sensor according to the third aspect in the preparation of a miRNA-133a detection kit.

[0056] The fifth aspect of this invention provides a method for detecting miRNA-133a, which uses the electrochemiluminescence sensor described in the third aspect to detect the presence of miRNA-133a in a sample, and includes the following steps:

[0057] The sample to be tested is added to the homogeneous system, mixed, and then subjected to the first cycle reaction. After the reaction is completed, it is dropped onto the working electrode and subjected to the second cycle reaction. After the reaction is completed, the electrochemiluminescence (ECL) signal is measured, and the presence of miRNA-133a in the sample is determined based on the measured electrochemiluminescence signal.

[0058] In some embodiments, the reaction temperature of the first and second cycle reactions is 35–40°C, preferably 37°C, and the reaction time is 1–3 h, preferably 2 h.

[0059] As described above, the ECL composite material, sensor, and their preparation and application in miRNA-133a detection of the present invention have the following beneficial effects:

[0060] This invention utilizes the high ECL output signal of ECL composite materials and the signal amplification capability of 3D DNAzyme Walker to construct an ECL sensor, achieving high specificity and high sensitivity detection of miRNA-133a. The sensitive, rapid, and simple ECL analysis technique established based on this technology is expected to enable early diagnosis and timely treatment of acute myocardial infarction (AMI) in clinical practice.

[0061] The ECL composite material ABEI@AuPt@Ni-Fe PBA provided by this invention has strong peroxidase activity, while the AuPt NPs on the surface are sufficiently small (2-3 nm) and uniformly dispersed, which can load a large amount of ABEI and significantly improve the ECL signal.

[0062] This invention utilizes the cleavage effect of DNAzymes to construct a dual DNA Walker cascade amplification system, which is expected to achieve higher detection sensitivity. The enzyme-free design reduces the requirements for the experimental environment. At the same time, this sensor has a certain degree of versatility and is expected to be used for the detection of other biomarkers and research in related medical fields, providing new ideas and technical guidance for the detection of various other nucleic acid biomarkers. Attached Figure Description

[0063] Figure 1 The diagram shows the sensor construction process and detection principle in an embodiment of the present invention.

[0064] Figure 2 The image shown is a scanning electron microscope (SEM) characterization image of the Ni-Fe PBA material in Example 1 of this invention.

[0065] Figure 3 The image shown is a transmission electron microscope (TEM) characterization image of the Ni-Fe PBA material in Example 1 of this invention.

[0066] Figure 4 The image shown is a scanning electron microscope (SEM) image of the material AuPt@Ni-Fe PBA in Example 1 of this invention.

[0067] Figure 5 The image shows the X-ray diffraction (XRD) results of the materials Ni-Fe PBA and AuPt@Ni-Fe PBA in Example 1 of this invention.

[0068] Figure 6 The image shown is a TMB colorimetric result diagram of the materials Ni-Fe PBA and AuPt@Ni-Fe PBA in Example 1 of this invention.

[0069] Figure 7 The figure shows the effect of Ni-Fe PBA and AuPt@Ni-Fe PBA on the ECL signal of ABEI in Example 1 of this invention.

[0070] Figure 8 The image shown is an ECL signal result diagram of the final material ABEI@AuPt@Ni-Fe PBA in Example 1 of this invention.

[0071] Figure 9 The image shown is a polyacrylamide gel electrophoresis (PAGE) result used in Example 2 of this invention to verify the feasibility of the dual-cycle system.

[0072] Figure 10 The image shown is a fluorescence signal result diagram used in Example 3 of this invention to verify the feasibility of the dual-cycle system.

[0073] Figure 11The image shown is a graph illustrating the quenching effect of the dopamine (DA)-modified quenching probe PH on the ECL signal in Example 5 of this invention.

[0074] Figure 12 The figure shown is a result of verifying the feasibility of the ECL sensor in detecting the target miRNA-133a in Example 6 of the present invention. Detailed Implementation

[0075] 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.

[0076] Electrochemiluminescence (ECL) is a chemiluminescence phenomenon triggered by electrochemical methods. Compared to chemiluminescence, ECL does not require any additional light source. Instead, it cleverly combines the ultrasensitivity of chemiluminescence with the high controllability of electrochemistry, giving it unique advantages such as simple operation, rapid response, high sensitivity, strong controllability, and low background signal. ECL biosensors have been widely used in biomedicine, food safety, environmental monitoring, and other fields. The electrochemiluminescence substrate N-(4-aminobutyl)-N-ethylisoluminol (ABEI) is a novel derivative of luminol, which is more reactive than the original and reacts more readily with co-reactants, thus yielding a stronger ECL signal.

[0077] Prussian blue (PB) and its analogues (PBA) exhibit diversity in composition and structure, and display unique magnetic, optical, host-guest, redox, and adsorption properties. First, PB(A)s possess a variety of redox properties, including Fenton catalysis, oxygen evolution reaction (OER), and hydrogen evolution reaction (HER) catalysis. Furthermore, they exhibit significant absorption in the near-infrared (NIR) region, possess relatively high photothermal conversion coefficients, and radiation resistance, making them suitable as photothermal agents (PTAs) for photothermal therapy (PTT) in cancer or bacterial eradication. Finally, chemically modified PB(A)s can be used as nanoprobes for magnetic resonance imaging (MRI), scintillation scanning, photoacoustic, or computed tomography (CT) imaging. The applications of PB(A)s offer the following advantages: (1) significant chemical stability even under low pH conditions; (2) several types of porosity allow for the capture of monovalent cations, small molecules, and complexes; (3) the presence of unsaturated metal centers allows for surface functionalization; and (4) the tunability of their chemical composition allows for the introduction of different transition metal ions or lanthanides through substitution or doping without structural changes. These multiple properties and advantages make PB(A)s widely applicable in biomedical, gas capture and storage, battery, catalysis, sensing, and decontamination technologies.

[0078] Deoxyribozymes (DNAzymes) are single-stranded DNA fragments with catalytic functions synthesized using in vitro molecular evolution techniques. They possess highly efficient catalytic activity and structure recognition capabilities. Firstly, they have a relatively small molecular weight, stable structure, high catalytic efficiency, and are easy to synthesize and modify. Secondly, they only require the assistance of metal ions to achieve high enzymatic activity, without the need for other auxiliary proteins. Furthermore, they can precisely recognize and cleave targets through complementary binding arms at both ends, exhibiting high specificity. Based on these advantages, they have found wide application in the biomedical field.

[0079] Based on the above aspects, this invention mainly focuses on improving the sensitivity and specificity of detection. It integrates multiple disciplines and technologies such as molecular biology, bioanalytical chemistry, and nanomaterials science to construct a highly sensitive electrochemiluminescence (ECL) sensor to achieve low abundance and high specificity detection of miRNA.

[0080] The construction process and detection principle of the ECL sensor of this invention are as follows: Figure 1As shown, firstly, a nickel-iron Prussian blue analogue (Ni-Fe PBA) with peroxidase activity was synthesized, and gold-platinum nanoparticles (AuPt NPs) were reduced in situ on its surface to achieve a combined effect of high loading of the electrochemiluminescent substrate N-(4-aminobutyl)-N-ethyl isoluminol (ABEI) and promoting the ECL signal. The synthesized final material ABEI@AuPt@Ni-Fe PBA served as the bottom layer material on the electrode, providing a basis for a high-intensity ECL signal. Secondly, a single-chain reporter probe PH modified with dopamine (DA) was attached to the bottom layer material. The quenching effect of DA on the ECL signal enabled the signal to switch from "on" to "off". Finally, a 3D deoxyribozyme walker (3D DNAzyme) was constructed by loading a pre-blocked deoxyribozyme (DNAzyme) chain (a double strand assembled from a long-arm chain WS and a blocking chain BS) and a cleavable orbital hairpin TH onto the gold nanoparticles (AuNPs) in a homogeneous system. The DNAzyme Walker consists of a hairpin (TH) containing both a cleavage site (WS) and a blocked DNAzyme sequence. When the target miRNA-133a is present, the DNAzyme portion of the long-arm WS is exposed through strand displacement. The specific site of the hairpin (TH) is cleaved, exposing a new DNAzyme sequence, forming a multi-legged, active 3D DNAzyme Walker. When this homogeneous system is dropped onto the working electrode surface, the binding arms of the 3D DNAzyme Walker specifically recognize and cleave the quenching probe on the electrode. The quenching group (DA) at the tip of the quenching probe is released, thereby generating a measurable ECL signal, enabling the detection of miRNA-133a.

[0081] Specifically, the construction process of the ECL composite material and sensor, and the detection method of the target miRNA-133a of the present invention are mainly as follows:

[0082] 1. Construction of ECL composite material ABEI@AuPt@Ni-Fe PBA

[0083] First, Ni-Fe PBA with peroxidase activity was synthesized using a simple method of room temperature incubation. Second, AuPt NPs were uniformly loaded onto the material surface through in-situ reduction to form AuPt@Ni-Fe PBA. The AuPt NPs serve two purposes: (1) they possess peroxidase activity, catalyzing the reaction substrate H2O2 and promoting the ECL signal of ABEI; (2) they act as a linking intermediate, connecting ABEI and Ni-Fe PBA. Finally, by linking ABEI, a composite material with a good initial ECL signal was obtained.

[0084] 2. Nucleic acid sequence design and construction of 3D DNAzyme Walker

[0085] The main components of the 3D DNAzyme Walker are the nucleic acid chains that form the Walker body and Au NPs that act as 3D orbitals. First, a suitable nucleic acid sequence was designed to enable target-driven cascade cycling, and its feasibility was verified using polyacrylamide gel electrophoresis (PAGE) and a fluorescence platform. Second, uniformly sized and well-dispersed Au NPs were synthesized to serve as the carrier portion of the 3D Walker. Finally, the nucleic acid chains of the Walker component were linked to the Au NPs via Au-S bonds using a salt aging method, ultimately yielding a homogeneous multi-armed 3D DNAzyme Walker.

[0086] 3. Integration of the overall ECL sensor for the detection of the target miRNA-133a.

[0087] The target was added to the homogeneous system, and the first cycle was started. The reaction was carried out at 37°C for 1 hour. The resulting active 3D DNAzymeWalker was dropped onto the solid phase platform of the ECL electrode for the second cycle, which was carried out at 37°C for 1 hour. The quenching probe was cleaved, and the recovery of the ECL signal was detected. Based on the final ECL signal, the content of miRNA-133a could be quantitatively detected.

[0088] The technology provided by this invention is simple, fast, and sensitive, and has excellent signal amplification performance. It will be developed for ultrasensitive detection of low-abundance target miRNAs.

[0089] In addition, to improve detection efficiency and realize practical clinical application, based on the new biosensor detection strategy established by the above-mentioned ECL platform, this invention intends to collect blood samples from clinical AMI patients, apply the constructed sensor to perform rapid and ultrasensitive detection of the target miRNA-133a, evaluate the methodology in terms of accuracy, precision, and linear range, and carry out clinical detection applications.

[0090] The following specific examples illustrate the present invention in detail. It should also be understood that the following examples are only for specific illustrative purposes and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0091] Example 1

[0092] Synthesis and characterization of composite material ABEI@AuPt@Ni-Fe PBA

[0093] 1. Synthesis of Ni-Fe PBA

[0094] Ni-Fe PBA was synthesized from nickel acetate, trisodium citrate dihydrate, and potassium ferricyanide via a static incubation method at room temperature. This method is simple and quick, requiring no additional heating or stirring. All chemical reagents were purchased from Shanghai Sangon Biotech. The specific steps are as follows:

[0095] 1.1. Dissolve 0.30 g of nickel acetate(II) tetrahydrate [Ni(CH3COO)2·4H2O] and 0.441 g of trisodium citrate dihydrate [Na3C6H5O7·2H2O] in 40 mL of deionized water to form solution A;

[0096] 1.2. Dissolve 0.264 g of potassium hexacyanoferrate(III) (potassium ferricyanide) [K3Fe(CN)6] in 60 mL of deionized water to form solution B;

[0097] 1.3. Add solution B to solution A under magnetic stirring, stir continuously for 1 min, and then let the resulting mixed solution stand at room temperature for 24 h to age.

[0098] 1.4. The product solution was aliquoted and centrifuged in 100 mL portions (9000 rpm, 5 min) to obtain a dark brown precipitate. This precipitate was washed three times with deionized water to remove unreacted reagents, and finally reconstituted into 20 mL of Ni-Fe PBA aqueous solution, which was brownish-yellow in color. A portion was temporarily stored at 4°C for subsequent experiments, while the remaining portion was dried overnight at 65°C, stored as a powder, and subjected to X-ray diffraction (XRD) characterization.

[0099] 2. Synthesis of AuPt@Ni-Fe PBA

[0100] In a deionized water system containing the surfactant polyvinyl pyrrolidone (PVP), gold-platinum was reduced in situ using HAuCl4 and H2PtCl6 as raw materials, relying on the reducing agent NaBH4, to obtain AuPt@Ni-Fe PBA. All steps were carried out at room temperature. The specific steps are as follows:

[0101] 2.1. Take 2 mL of Ni-Fe PBA aqueous solution, centrifuge to remove the supernatant, redissolve in anhydrous ethanol to obtain 8 mL of system, add 0.01 g PVP, and stir for 30 min;

[0102] 2.2. Dissolve 0.144 mL of H2PtCl6 (20 mM) / 0.098 mL of HAuCl4 (29.4 mM) in 9.6 mL of deionized water, add the entire solution to the above system at once, and continue stirring for 10 min.

[0103] 2.3. Prepare NaBH4 solution by dissolving 0.005 g of NaBH4 in 10 mL of deionized water (to minimize the generation of bubbles, the EP tube can be placed in an ice bath);

[0104] 2.4. Add 0.5 mL of NaBH4 solution to the above system and stir for 30 min;

[0105] 2.5. The above reaction system was centrifuged (9000 rpm, 5 min) to obtain the precipitate, washed three times with deionized water, and finally reconstituted into a 4 mL AuPt@Ni-Fe PBA aqueous solution (diluted 2 times), and stored at 4℃ for later use.

[0106] 3. Synthesis of ABEI@AuPt@Ni-Fe PBA

[0107] The luminescent substrate ABEI was directly stirred in deionized water to obtain the final material ABEI@AuPt@Ni-Fe PBA via Au-N bonds. After modification onto a glassy carbon electrode, it generated a high ECL signal in an alkaline PBS system. The specific steps are as follows:

[0108] 3.1. Dilute 2 mL of AuPt@Ni-Fe PBA aqueous solution with deionized water to a total volume of 20 mL, add 500 μL of 10 mL MABEI, and stir at room temperature for 30 min;

[0109] 3.2. The precipitate was obtained by centrifugation and washed three times with deionized water to remove excess ABEI, yielding the final material ABEI@AuPt@Ni-Fe PBA. Finally, it was suspended in 2 mL of 0.1% chitosan solution and stored at 4 °C for later use.

[0110] 4. Characterization of the composite material ABEI@AuPt@Ni-Fe PBA

[0111] The materials were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and X-ray diffraction (XRD). Figure 2 and Figure 3 The images are SEM and TEM images of Ni-Fe PBA, respectively. It can be seen that the material is a cube with a very uniform size, with a particle size between 50-60 nm and good dispersibility. Figure 4The TEM image of AuPt@Ni-Fe PBA shows that the surface of the cube becomes relatively rough, indicating the loading of AuPt NPs. At the same time, the loading does not change the morphology of Ni-Fe PBA. Figure 5 The XRD results for the two intermediate materials are consistent with the characteristic diffraction peaks of standard Ni-Fe PBA. Furthermore, the loading of AuPt NPs did not alter their internal crystal structure, indicating the successful synthesis of the materials.

[0112] 5. Material performance verification

[0113] 5.1. TMB colorimetric experiment

[0114] The peroxidase activity of Ni-Fe PBA and AuPt@Ni-Fe PBA materials was preliminarily verified by TMB colorimetric assay. The colorimetric system consisted of 100 μL TMB solution + 2 μL 10M H₂O₂ solution + 1 μL of the material. Results are as follows: Figure 6 As shown, due to the loading of AuPt NPs, the color development of AuPt@Ni-Fe PBA is significantly higher than that of Ni-Fe PBA.

[0115] 5.2. ECL Detection Experiment

[0116] 5.2.1. The promoting effect of the material (Ni-Fe PBA / AuPt@Ni-Fe PBA) and ABEI was verified by dropwise addition of the material to the solution. Reaction system: 2 mL 0.1 M PBS (pH 8.0) + 2 μL 10 M H₂O₂ solution + 1 μL 10 M ABEI + 2 μL of the material. Experimental parameters: Potential range: 0-0.5 V; Scan rate: 0.15 W / s; Photomultiplier tube high voltage: 800 V. Results are as follows... Figure 7 As shown, both materials significantly enhance the ECL signal of ABEI. Due to the loading of AuPt NPs, the color development of AuPt@Ni-Fe PBA is significantly higher than that of Ni-Fe PBA.

[0117] 5.2.2. After mixing the chitosan solution of the final material ABEI@AuPt@Ni-Fe PBA, 10 μL of the mixture was applied to glassy carbon electrodes. The electrodes were dried in a 37°C oven for approximately 30 min until a smooth material layer was formed. The signal was then detected using an ECL instrument. The reaction system was as follows: 2 mL 0.1 M PBS (pH 8.0) + 2 μL 10 M H₂O₂ solution. Experimental parameters: potential range: 0-0.5 V; scan rate: 0.15 V / s; photomultiplier tube high voltage: 800 V. The results are as follows... Figure 8 As shown, the material generates a high ECL signal, and the signal does not change significantly in multiple consecutive scanning cycles, indicating good stability.

[0118] Example 2

[0119] The feasibility of the dual-cycle system was verified by polyacrylamide gel electrophoresis (PAGE).

[0120] The feasibility of the dual-cycle system of the present invention was verified by PAGE experiments. The nucleotide sequences involved in the system are shown in Table 1.

[0121] Table 1 Nucleotide sequence list

[0122]

[0123] Note: The underlined part represents the DNAzyme active sequence; / rA / represents ribonucleotide.

[0124] 1. Assembly of double chains and hairpins

[0125] All nucleotide chains were melted using Tris-HCl Buffer (170 mM NaCl, 20 mM Tris, 5.0 mM KCl, 1.0 mM MgCl2, pH 7.4) to a final concentration of 100 μM. The nucleotides were aliquoted and stored, with 10 μL of each of the five components diluted to 20 μL (10 μM) for later use. The WS and BS single strands were annealed in Tris-HCl Buffer at a molar ratio of 1:1.1. The annealing process involved denaturation at 95°C for 5 min, followed by slow cooling to room temperature, and finally storage at 4°C to prepare a *WS+BS double strand with a final concentration of 10 μM. The hairpin TH was diluted to 10 μM and annealed in the same manner to form a stable hairpin structure *TH.

[0126] 2. PAGE system reaction and electrophoresis

[0127] A 12% PAGE gel was prepared for electrophoresis. Experimental parameters: final concentration of each nucleotide fraction was 1 μM; Mn 2+ The concentration was 10 mM; the reaction temperature was 37℃; the reaction time was 1 h; the total reaction volume was 10 μL; the sample loading volume in each lane was 6 μL; the voltage was 110 V; and the electrophoresis time was 50 min. The systems for each lane are shown in Table 2. The sample loading volume for each component was 1 μL, and any system with a volume less than 10 μL was made up with deionized water.

[0128] Table 2 Electrophoresis System

[0129] Components / lanes 1 2 3 4 5 6 7 8 9 10 11 12 13 WS + + BS + TA + + + + *TH + + + + + + PH + + + + + *WS+BS + + + + + + <![CDATA[Mn 2+ ]]> + + + + + +

[0130] Electrophoresis results as follows Figure 9As shown, lane M represents a 20bp DNA marker; lanes 1, 2, 3, 4, and 5 correspond to WS, BS, TA, TH, and PH, respectively; lane 6 shows that WS and BS successfully assembled into a double strand; lane 7 shows that the target TA strand replaced the blocking strand BS to form a double-stranded structure, exposing the WS single strand; lane 8 shows that when TA is absent, the WS in the double strand cannot cleave the orbital hairpin *TH, while in lane 9, when TA is present, the exposed WS in lane Mn 2+ With the assistance of [unclear], the TH chain was cleaved; lanes 10 and 11 are the positive and negative groups of the complete dual-cycle system. It can be seen that in the absence of TA, the hairpin TH and probe PH are rarely cleaved, while in the presence of TA, TH and PH are almost completely cleaved; in lane 12, it can be seen that WS alone cannot cleave PH, indicating that the DNAzyme in the first cycle cannot cleave the probe in the second cycle; in lane 13, it can be seen that before the hairpin *TH is cleaved, the DNAzyme sequence within it hardly cleaves the probe PH, thus demonstrating the sequential operation of the dual-cycle system. In summary, the feasibility of the dual-cycle system is proven.

[0131] Example 3

[0132] The feasibility of the dual-cycle system was verified using fluorescence.

[0133] The probe PH was modified with a fluorescent group FAM and a quencher group BHQ1 at both ends, named PHf. The feasibility of the dual-cycle system was further verified by fluorescence detection. Experimental parameters: total volume 100 μL; final concentration of PHf 800 nM, final concentration of other nucleotide components 400 nM; Mn 2+ The final concentration was 10 mM; the reaction temperature was 37℃; the reaction time was 1 h; the reaction system is shown in Table 3.

[0134] The above reactants were added to a quartz cuvette, and the measurements were taken using a fluorescence spectrophotometer. The specific steps included:

[0135] 1. Cleaning of fluorescent cuvettes: Soak the fluorescent cuvettes in alcohol and then clean them with ddH2O;

[0136] 2. Parameter settings: Set the excitation wavelength to 490nm, the emission wavelength range to 500nm, and the voltage to 600V;

[0137] 3. Zeroing: Add ddH2O to the fluorescent cuvette to zero it;

[0138] 4. Perform the detection: Add the reaction solution to the fluorescent cuvette, click the detection button, and you can obtain the fluorescence signal.

[0139] Fluorescence results as follows Figure 10 As shown, the fluorescence signal was significantly recovered when the target TA was present, thus verifying the feasibility of the dual-cycle system.

[0140] Table 3. Verification system for fluorescence feasibility

[0141] Components / samples Negative group (a) Positive group (b) *WS+BS + + TA + *TH + + PHf + + <![CDATA[Mn 2+ ]]> + +

[0142] Example 4

[0143] Construction of 3D DNAzyme Walker

[0144] 1. Synthesis of Au NPs with a particle size of 13 nm:

[0145] Add 85.2 mL of deionized water and 3 mL of HAuCl4 (29.4 mM) to a beaker equipped with a magnetic stir bead, place it in an oil bath on a magnetic stirrer, and heat at 130°C until boiling.

[0146] To prepare a trisodium citrate trihydrate solution, dissolve 0.1g of powder in 10.1mL of deionized water, then quickly add the entire solution to the boiling solution above, and continue heating and boiling for 20 minutes. The solution color changes from golden yellow to black, and finally to wine red.

[0147] Stop heating in the oil bath, continue stirring, slowly cool to room temperature, then transfer to a light-proof bottle and store in a refrigerator at 4°C.

[0148] 2. Construction of 3D DNAzyme Walker using salt aging method:

[0149] 2.1. The thiol-modified long-arm chain SH-WS and the blocking chain BS are annealed at a ratio of 1:1.1 to form a double chain (SH-WS+BS);

[0150] 2.2. Mix 4.5 μL of SH-WS+BS (50 μM), 44.5 μL of thiol-modified orbital hairpin SH-TH (50 μM), 5 μL of sodium acetate (500 mM, pH 5.2) and 0.15 μL of tris(2-carboxyethyl)phosphine (TCEP) (100 mM) at room temperature and let stand for 1 h to activate the thiol groups;

[0151] 2.3. Add 1.6 mL of the synthesized Au NPs to the above system and let it stand at room temperature in the dark for 16 h;

[0152] 2.4. Add 16 μL of Tris-acetic acid (500 mM, pH 8.2) and 40 μL of NaCl (1 M) to the above system. Then add 40 μL of NaCl dropwise every 4 h for a total of 4 drops, for a total of 160 μL. After the last addition, let the mixture stand at room temperature in the dark overnight (about 10 h).

[0153] 2.5. Centrifuge the above system (12,000 rpm, 10 min) to remove excess reagents. The resulting precipitate is reconstituted with 1.6 mL PBS (0.01 M, pH 7.4) and stored at 4 °C in the dark for later use.

[0154] Example 5

[0155] Verify the quenching effect of the quenching probe PH on the ECL signal.

[0156] 1. The 5' and 3' ends of the probe pH were modified with carboxyl and thiol groups, respectively, and the carboxyl groups were activated by treatment in EDC-NHS solution. The system was: 145 μL PBS (0.01 M, pH 7.4) + 5 μL pH chain (100 μM) + 40 μL EDC (0.1 M) + 10 μL NHS (0.1 M), and incubated at 4 °C for 2 h.

[0157] 2. Prepare 1 mL of 0.1 M dopamine (DA) solution. Specifically, weigh 0.019 g of DA powder and dissolve it in 1 mL of PBS (0.01 M, pH 7.4). Use this as the original DA solution, and then dilute it 100 times and 120 times with PBS respectively, and set aside for later use.

[0158] 3. Divide 200 μL of the PH chain after activating the carboxyl group in step 1 into two portions, and mix them with 100 μL of 100-fold and 120-fold DA solutions respectively. In a refrigerator at 4°C, shake the mixture overnight with a rotary mixer for at least 8 hours to form quenching probes modified with two concentrations of DA (100-fold DA chain and 120-fold DA chain), which can be used directly.

[0159] 4. Clean the glassy carbon electrode, modify it with 10 μL of the composite material ABEI@AuPt@Ni-Fe PBA, dry it in an oven at 37℃, then add 10 μL of Tris-HCl Buffer, 100 times the amount of DA chain and 120 times the amount of DA chain respectively, let it stand in a refrigerator at 4℃ for 12 h, rinse the electrode with PBS (0.01M, pH 7.4) to remove unbound chains, and then measure the ECL signal.

[0160] 5. Results are as follows Figure 11 As shown, the composite material exhibits a high ECL signal, but the signal is significantly reduced after modification with DA chains. When the DA concentration is reduced (from 100-fold dilution to 120-fold dilution), the quenching effect decreases slightly, allowing the signal to rise, thus proving the quenching effect of DA on the ECL signal.

[0161] Example 6

[0162] Integration of ECL sensor with detection of target miRNA-133a

[0163] 1. The homogeneous reaction was carried out in a 30 μL system. The positive group contained 10 μL of the synthesized 3D DNAzyme Walker (see Example 4 for specific operation), 3 μL of 100 mM MnCl2, 5 μL of Tris-HCl Buffer (170 mM NaCl, 20 mM Tris, 5.0 mM KCl, 1.0 mM MgCl2, pH 7.4), 2 μL of deionized water and 10 μL of target miRNA-133a. In the negative group, deionized water was used instead of the target. The reaction was carried out at 37°C for 1 h.

[0164] 2. The electrode modification was carried out the night before. After the glassy carbon electrode was thoroughly cleaned, the surface was dried with nitrogen. 10 μL of the composite material ABEI@AuPt@Ni-Fe PBA was modified, dried at 37°C, and 120 times the amount of DA chain was added (see Example 5 for specific operation). The electrode was then left to stand overnight in a refrigerator at 4°C.

[0165] 3. The homogeneous reaction system was added dropwise to three modified electrodes (10 μL / electrode), and reacted at 37°C for 1 h. The electrodes were then rinsed with PBS (0.01 M, pH 7.4), and the ECL signal was measured. Reaction system: 2 mL 0.1 M PBS (pH 8.0) + 2 μL 10 M H₂O₂ solution. Experimental parameters: Potential range: 0-0.5 V; Scan rate: 0.15 V / s; Photomultiplier tube high voltage: 800 V.

[0166] 4. Results are as follows Figure 12 As shown, when the target is absent, the ECL signal remains in a quenched state (curve a) with a low signal level, while when the target is present, the signal is significantly recovered (curve b). This indicates that the recovery of the ECL signal is related to the target miRNA-133a, thus proving the feasibility of the ECL sensor.

[0167] 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 sensor, characterized in that: The invention includes a working electrode and a homogeneous system. The working electrode is modified with an electrochemiluminescence composite material, and a quenching probe is attached to the electrochemiluminescence composite material. The quenching probe is a single-chain reporter probe PH modified with dopamine, and the nucleotide sequence of the single-chain reporter probe PH is shown in SEQ ID NO.

5. The homogeneous system includes gold nanoparticles Au NPs, on which are loaded closed deoxyribozyme chains and cleavable orbital hairpins TH. The deoxyribozyme chains are double strands assembled from long arm chains WS and blocking chains BS. The nucleotide sequences of the long arm chains WS, blocking chains BS and orbital hairpins TH are shown in SEQ ID NO.1, 2 and 4, respectively. The electrochemiluminescent composite material includes a nickel-iron Prussian blue analog Ni-FePBA with peroxidase activity, gold-platinum nanoparticles AuPt NPs loaded on the surface of the nickel-iron Prussian blue analog nanoparticles, and an electrochemiluminescent substrate connected to the gold-platinum nanoparticles via gold-nitrogen covalent bonds.

2. The electrochemiluminescence sensor according to claim 1, characterized in that: The electrochemiluminescent substrate is selected from either N-(4-aminobutyl)-N-ethylisoluminol or luminol.

3. The electrochemiluminescence sensor according to any one of claims 1 to 2, characterized in that, The preparation method of the electrochemiluminescence composite material includes the following steps: Nickel-iron Prussian blue analogues were synthesized by a method of standing at room temperature using nickel precursor, trisodium citrate and iron coordination compound as raw materials. In situ reduction was performed on the surface of a nickel-iron Prussian blue analogue to load gold-platinum nanoparticles, forming AuPt@Ni-Fe PBA material. The electrochemiluminescent composite material is obtained by linking AuPt@Ni-Fe PBA material with an electrochemiluminescent substrate through a gold-nitrogen covalent bond.

4. The electrochemiluminescence sensor according to claim 1, characterized in that: The homogeneous system also includes Mn 2+ .

5. The electrochemiluminescence sensor according to claim 1, characterized in that: The method for attaching a quenching probe to the electrochemiluminescent composite material includes the following steps: adding a single-chain reporter probe PH modified with dopamine to the working electrode modified with the electrochemiluminescent composite material, and allowing the reaction to proceed statically.

6. The electrochemiluminescence sensor according to claim 1, characterized in that: The preparation method of the quenching probe includes the following steps: modifying the 5' end and 3' end of the single-chain reporter probe PH with carboxyl and thiol groups respectively, activating the carboxyl groups, and then mixing with dopamine solution to react and form a single-chain reporter probe PH modified with dopamine, i.e., the quenching probe.

7. The use of the electrochemiluminescence sensor according to any one of claims 1 or 4 to 6 in the preparation of a miRNA-133a detection kit.

8. A method for detecting miRNA-133a, wherein the detection method is for non-disease detection or treatment purposes, characterized in that: The detection of the presence of miRNA-133a in a sample using the electrochemiluminescence sensor according to any one of claims 1 or 4-5 includes the following steps: The sample to be tested is added to the homogeneous system, mixed, and then subjected to the first cycle reaction. After the reaction is completed, it is dropped onto the working electrode and subjected to the second cycle reaction. After the reaction is completed, the electrochemiluminescence signal is measured, and the presence of miRNA-133a in the sample is determined based on the measured electrochemiluminescence signal.

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