Electrochemical biosensor for detection of microRNA associated with acute myocardial infarction

Abstract

The present invention provides an electrochemical biosensor for detecting microRNAs associated with acute myocardial infarction. This method utilizes a tetrahedral DNA scaffold to confine molecular recognition to the nanoscale, reducing molecular diffusion, improving analytical sensitivity, and shortening detection time. This method establishes a sensitive and rapid method for detecting AMI-related microRNAs, providing experimental evidence and technical support for the development of AMI-related microRNA detection technologies.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical detection, and in particular to an electrochemical biosensor for detecting acute myocardial infarction-related microRNA based on the spatial confinement effect of a tetrahedral DNA scaffold and a detection method thereof. Background Art

[0002] Acute myocardial infarction (AMI) is a form of myocardial injury caused by acute, persistent ischemia and hypoxia in the coronary arteries. Sudden onset, delayed diagnosis, and diagnostic uncertainty are the primary causes of high morbidity and mortality in AMI, making AMI a major public health concern. Revascularization therapy for AMI is crucial for repairing ischemic myocardium and significantly reduces mortality in AMI patients. AMI revascularization therapy relies on early, timely, and accurate identification and diagnosis of AMI. Cardiac damage caused by AMI is associated with progressive myocardial cell loss, which induces the time-dependent release of biomarkers such as myoglobin, creatine kinase isoenzyme (CK-MB), and cardiac fatty acid binding protein (H-FABP). However, protein biomarker levels can be affected by non-cardiac diseases, and their low specificity may lead to misdiagnosis. In recent years, numerous biological studies have demonstrated that abnormal expression of several microRNAs is closely associated with the pathogenesis of AMI. Compared with protein biomarkers, AMI-associated microRNAs have higher specificity and timeliness, making them superior biomarkers for the diagnosis of AMI. However, AMI-associated microRNAs are expressed at very low levels in the early stages of the disease, and accurate detection is often interfered with by other homologous microRNAs. Therefore, improving the sensitivity of AMI-associated microRNA detection technology and shortening analysis time are crucial.

[0003] Typically, improving the sensitivity of microRNA detection methods relies on developing signal amplification strategies, such as using nanomaterials or enzymes to amplify biorecognition signals, or employing nucleic acid signal amplification to increase the number of biomarkers, but this often results in prolonged detection times. Consequently, microRNA detection methods face the dual challenges of low sensitivity and long detection times. Electrochemical biosensors, combining the high selectivity of biorecognition with the high sensitivity of electrochemical techniques, have demonstrated potential for sensitive detection of circulating biomarkers. Studies have shown that slow mass transfer of analyte molecules to the sensing interface results in prolonged response times. The timescale of the diffusion process is proportional to the square of the diffusion path length of the diffusing species. Larger sampling volumes are often required to ensure accurate analysis of low-concentration analytes. In recent years, researchers have employed nanostructured electrodes to extend the length scale of the sensing interface or magnetic nanoparticles to reduce the diffusion path length. However, heterogeneous interfaces can lead to reduced hybridization efficiency. Summary of the Invention

[0004] In order to solve the problems in the prior art, the present invention provides an electrochemical biosensor for the detection of acute myocardial infarction-related microRNA. The present invention utilizes a tetrahedral DNA scaffold to confine molecular recognition to the nanoscale, reduce molecular diffusion, improve the sensitivity of the analysis method and shorten the detection time. In this work, four reaction chains are added to the four vertices of the tetrahedral DNA scaffold, wherein the DNA hybridization reaction is confined to the tetrahedral DNA scaffold. Due to the reduction of the diffusion path and the enhancement of the signal, the efficiency of the DNA hybridization reaction and the analytical performance are greatly improved. In addition, the construction process of the sensor is a one-step incubation process, so no additional reagents and transfer of amplification products are required, which greatly reduces the difficulty of operation. This study constructs a sensitive and rapid AMI-related microRNA detection method, providing experimental basis and technical support for the research and development of AMI-related microRNA detection technology.

[0005] An electrochemical biosensor for detecting microRNA associated with acute myocardial infarction, characterized by:

[0006] The TDS-DNAzyme / blocker complex was dropped onto the surface of reduced graphene oxide / Nafion-modified glassy carbon electrode rGO / Nafion / GCE and incubated at room temperature for 2 to 3 hours to prepare an electrochemical biosensor for microRNA-499 detection.

[0007] The preparation steps of the TDS-DNAzyme / blocker complex are as follows: DBCO-DNAzyme / blocker double strands, TDS and horseradish peroxidase (HRP)-conjugated streptavidin are mixed in a fixed buffer (10mM Tris-HCl, 1M NaCl, 1mM EDTA, 10mM TECP, pH 7.4), and the mixture is placed in a gradient PCR instrument and incubated at 20-25°C for 20-30 minutes to obtain the TDS-DNAzyme / blocker complex.

[0008] Preparation of the rGO / Nafion / GCE:

[0009] Graphene oxide suspension and Nafion TMA graphene oxide / Nafion mixture was obtained by mixing a perfluorinated resin solution, and then the graphene oxide / Nafion mixture was dropped onto the pretreated GCE surface. After drying in a vacuum dryer, a graphene oxide / Nafion-modified glassy carbon electrode (GO / Nafion / GCE) was obtained. The GO / Nafion / GCE was placed in phosphate-buffered saline for electrochemical reduction to obtain a reduced graphene oxide / Nafion-modified glassy carbon electrode (rGO / Nafion / GCE).

[0010] The preparation method of the TDS is:

[0011] Four single-stranded DNAs T1, T2, T3 and T4 were mixed in an equimolar ratio in an assembly buffer (20 mM Tris-HCl, 50 mM MgCl2, pH 8.0), then placed in a gradient PCR instrument for annealing, cooled to 3-5°C and maintained for 60-90 minutes.

[0012] Furthermore, the preparation of the above-mentioned TDS-DNAzyme / blocker complex comprises the following steps:

[0013] 1) Preparation of Azide, Pyrene, and Biotinylated TDS: Four single-stranded DNA strands (T1, T2, T3, and T4) were mixed in equimolar proportions in assembly buffer (20 mM Tris-HCl, 50 mM MgCl2, pH 8.0) and annealed in a gradient PCR instrument. The mixture was cooled to 3-5°C and maintained for 60-90 min.

[0014] The DNA sequences of T1, T2, T3, and T4 are as follows:

[0015]

[0016]

[0017] 2) Preparation of DBCO-DNAzyme / blocker: Disperse the DBCO-DNAzyme and blocker chains in hybridization buffer (10 mM Tris-HCl, 20 mM MgCl2, 100 mM NaCl, pH 7.4). Mix equal volumes of blocker and DBCO-DNAzyme, then anneal in a gradient PCR instrument. Cool to 20-25°C at a rate of -1-2°C / min to prepare the DBCO-DNAzyme / blocker.

[0018] 3) Preparation of TDS-DNAzyme / blocker complex: The product prepared in step 1), the DBCO-DNAzyme / blocker double strand prepared in step 2), and horseradish peroxidase (HRP)-conjugated streptavidin were mixed in a fixation buffer (10 mM Tris-HCl, 1 M NaCl, 1 mM EDTA, 10 mM TECP, pH 7.4), and incubated at 20-25°C in a gradient PCR instrument for 20-30 min to obtain the TDS-DNAzyme / blocker complex.

[0019] The preparation steps of the above rGO / Nafion / GCE are as follows:

[0020] Graphene oxide suspension and Nafion TM The perfluorinated resin solution was mixed to obtain a graphene oxide / Nafion mixture, which was then dropped onto the pretreated GCE surface and dried in a vacuum dryer at 20-25°C to obtain a graphene oxide / Nafion-modified glassy carbon electrode (GO / Nafion / GCE). The GO / Nafion / GCE was placed in phosphate buffered saline (10 mM, pH 5.0) for electrochemical reduction, and measured using it ampere with an initial potential of -1.3 V, a sampling interval of 0.1 s, and a standing time of 0 s to obtain a reduced graphene oxide / Nafion-modified glassy carbon electrode (rGO / Nafion / GCE).

[0021] The reduced graphene oxide (rGO) is prepared by dispersing flaky single-layer graphene oxide (GO, 10 mg) in ultrapure water, ultrasonically treating the GO suspension for 30-40 minutes, centrifuging the GO suspension at 8000-9000 rpm / min for 5-10 minutes, and collecting the brown supernatant to obtain the reduced graphene oxide (rGO).

[0022] Specifically, an electrochemical biosensor for detecting microRNA associated with acute myocardial infarction is characterized by comprising the following steps:

[0023] (1) Preparation of Tetrahedral DNA Scaffold-DNA Enzyme / Blocker (TDS-DNAzyme / Blocker) Complex

[0024] 1) Preparation of Azide, Pyrene, and Biotinylated TDS: Four single-stranded DNA strands (T1, T2, T3, and T4) were mixed in equimolar proportions in assembly buffer (20 mM Tris-HCl, 50 mM MgCl2, pH 8.0) to a final concentration of 2 μM. The mixture was then annealed at 95°C for 5 min in a gradient PCR instrument, then rapidly cooled to 4°C and maintained for more than 60 min.

[0025] 2) Preparation of dibenzocyclooctyne-DNA enzyme / blocker (DBCO-DNAzyme / blocker): The DBCO-DNAzyme and blocker chains were dispersed in hybridization buffer (10 mM Tris-HCl, 20 mM MgCl2, 100 mM NaCl, pH 7.4); equal volumes of blocker (6 μM) and DBCO-DNAzyme (4 μM) were mixed and annealed at 95°C for 2 min in a gradient PCR instrument. The mixture was then slowly cooled to 25°C at a rate of -1°C / min to prepare DBCO-DNAzyme / blocker (final concentration 2 μM);

[0026] 3) Preparation of TDS-DNAzyme / blocker complex: TDS prepared in step 1) (final concentration of 0.5 μM), DBCO-DNAzyme / blocker double strand prepared in step 2) (final concentration of 0.5 μM) and HRP-conjugated streptavidin (final concentration of 1 ng μL) were added to the mixture. -1 ) were mixed in a fixation buffer (10 mM Tris-HCl, 1 M NaCl, 1 mM EDTA, 10 mM TECP, pH 7.4); incubated in a gradient PCR instrument at 25°C for 20 min to obtain a TDS-DNAzyme / blocker complex with a final concentration of 0.5 μM;

[0027] (2) Preparation of reduced graphene oxide modified glassy carbon electrode:

[0028] 1) Preparation of reduced graphene oxide (rGO): 10 mg of flaky monolayer graphene oxide (GO) was dispersed in 5 mL of ultrapure water and ultrasonically treated for 30 min to obtain a graphene oxide suspension (2 mg mL -1 ); The graphene oxide suspension was then centrifuged at 8000 rpm / min for 5 min, and the graphene oxide brown supernatant was collected;

[0029] 2) Preparation of reduced graphene oxide modified glassy carbon electrode: First, the glassy carbon electrode (GCE) was polished with aluminum oxide powder (300 nm and 50 nm), and then ultrasonically cleaned with ultrapure water for a few seconds; Nafion was washed with 75% ethanol. TM The perfluorinated resin solution was diluted to 2% (wt%). The graphene oxide suspension prepared in step 1) (2 mg mL -1 ) and Nafion TM Equal volumes of a perfluorinated resin solution (2%) were mixed to obtain a graphene oxide / Nafion mixture. 10 μL of the graphene oxide / Nafion mixture was dropped onto the pretreated GCE surface and dried in a vacuum dryer (25°C) to obtain a graphene oxide / Nafion-modified glassy carbon electrode (GO / Nafion / GCE). Finally, the GO / Nafion / GCE was electrochemically reduced in phosphate-buffered saline (10 mM, pH 5.0) and measured using 1 t amperometry with an initial potential of -1.3 V, a sampling interval of 0.1 s, and a rest time of 0 s. The prepared reduced graphene oxide / Nafion-modified glassy carbon electrode (rGO / Nafion / GCE) was stored at room temperature.

[0030] (3) Construction of an electrochemical biosensor for the detection of acute myocardial infarction-related microRNA-499:

[0031] 1) 10 μL of TDS-DNAzyme / blocker complex (0.5 μM) was added dropwise to the rGO / Nafion / GCE surface described in step (2) and incubated at room temperature for 2 h;

[0032] 2) After gently rinsing the electrode obtained in step 1) with assembly buffer, the TDS-DNAzyme / blocker complex-modified electrode (TDS-DNAzyme / blocker / rGO / Nafion / GCE) was stored at 4° C. for subsequent use, thereby obtaining an electrochemical biosensor for microRNA-499 detection.

[0033] In a second aspect, the present invention provides a method for detecting microRNA-499 based on the above electrochemical biosensor.

[0034] A method for detecting microRNA-499 based on the above electrochemical biosensor, characterized by comprising the following steps:

[0035] (1) 10 μL of microRNA-499 dispersed in assembly buffer was dropped onto the surface of the above-mentioned electrode (TDS-DNAzyme / blocker / rGO / Nafion / GCE) and incubated at room temperature for 20 min. After rinsing with assembly buffer, the electrode was immersed in HAc-NaAc (0.1 M, pH 4.0) buffer containing a mixture of TMB (0.66 mM) and H2O2 (3.0 mM), and the it curve was measured;

[0036] (2) drawing a working curve based on the linear relationship between the current obtained in step (1) and the logarithmic value of the microRNA-499 concentration;

[0037] (3) Using the electrochemical sensor to detect the sample to be tested, the obtained current value is calculated using the working curve prepared in step (2) to obtain the microRNA-499 concentration of the sample to be tested.

[0038] Compared with the prior art, the preparation method and application of the electrochemical biosensor for detecting microRNA-499 of the present invention have the following outstanding features:

[0039] The present invention explores the confinement effect of tetrahedral DNA scaffolds in interface sensors, improves the sensitivity of AMI-related microRNA-499 detection, and shortens the detection time.

[0040] The present invention reduces the diffusion path length and increases the signal gain, thereby greatly improving the DNA hybridization reaction and analysis performance.

[0041] Furthermore, the detection process is a simple one-step incubation process, which avoids the introduction of additional reagents and the transfer of amplification products. Through the above means, the prepared electrochemical biosensor was successfully used for the sensitive and rapid detection of microRNA-499.

[0042] Compared with traditional microRNA-499 detection methods, the present invention has the advantages of rapid detection, high sensitivity, and simple operation, providing a new analytical method for microRNA-499 detection. The present invention will promote the widespread application of electrochemical biosensors in disease diagnosis.

[0043] The beneficial effects of the present invention are:

[0044] 1) The confinement effect of tetrahedral DNA scaffolds in interfacial sensors can greatly improve the shortcomings of low sensor detection sensitivity and long detection time;

[0045] 2) The tetrahedral DNA scaffold confines the metal-specific catalytic DNA deoxyribozyme (DNAzyme) cleavage reaction to a compact space, reducing the diffusion path length and increasing the signal gain, greatly improving the efficiency of DNA hybridization reactions and analytical performance;

[0046] 3) The materials involved in the present invention can be synthesized under laboratory conditions, are simple to operate, use inexpensive raw materials, are low in toxicity, are environmentally friendly, and are used in very small amounts each time, thus reducing experimental costs;

[0047] 4) The entire detection and analysis method has clear and simple steps, high sensitivity, and fast signal response;

[0048] 5) The electrochemical biosensor prepared by this method can provide a new method for the detection of microRNA-499. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Figure 1 shows the cyclic voltammetry test (A) and electrochemical impedance spectroscopy (EIS) characterization (B) of different modified electrodes in 10 mM PBS (pH 7.0, containing 100 mM NaCl and 5 mM K3[Fe(CN)6] / K4[Fe(CN)6]). The scan rate for the cyclic voltammetry test was set at 0.10 V s -1 The scanning voltage range is -0.1V-0.6V. The electrochemical impedance spectroscopy test was performed with a voltage of 220mV, a boost of 5mV, and a frequency of 1.0×10 -2 Hz-1.0×10 6 Hz.

[0050] Figure 2 The graph shows the IT test of different modified electrodes in HAc-NaAc (0.1M, pH 4.0) buffer containing a mixture of TMB (0.66mM) and H2O2 (3.0mM). The starting voltage was set to 0.1V (versusAg / AgCl), the sampling interval was 0.1s, the test time was 100s, and the sensitivity was 2×10 –5 A / V.

[0051] Figure 3These are the results of detection of microRNA-499 at different concentrations by the sensor of the present invention, wherein Figure A is a response test graph of the sensor to different concentrations of microRNA-499 (0M, 0.5fM, 1fM, 10fM, 50fM, 100fM, 1pM, 10pM) in HAc-NaAc (0.1M, pH 4.0) buffer containing a mixture of TMB (0.66mM) and H2O2 (3.0mM); Figure B is a corresponding relationship curve between the sensor current intensity and different concentrations of microRNA-499 (0.1fM, 0.5fM, 1fM, 10fM, 50fM, 100fM, 1pM, 10pM, 100pM), wherein the inset is a calibration curve of the current intensity and the logarithmic value of different concentrations of microRNA-499 (0.5fM-10pM).

[0052] Figure 4 This is a selectivity evaluation graph for the sensor, where the interfering agents are other AMI-related microRNAs (including microRNA-208, microRNA-1, and microRNA-133a) and base mismatches. The concentration of microRNA-499 is 1 pM, and the interfering agent concentration is 50 pM.

[0053] Figure 5 The figure shows the sensor stability test results. The rGO / nafion / GCE modified electrode was subjected to continuous cyclic voltammetry in 10 mM PBS (pH 7.0, containing 100 mM NaCl and 5 mM K3[Fe(CN)6] / K4[Fe(CN)6]). The cyclic voltammetry test was performed at a scan rate of 0.10 V s. -1 , the scanning voltage range is -0.1V-0.6V.

[0054] Figure 6 A certain concentration of microRNA-499 (10pM-1fM) was added to 10% healthy human serum samples and buffer solutions to simulate real samples, and the electrochemical biosensor was tested in HAc-NaAc (0.1M, pH 4.0) buffer containing a mixture of TMB (0.66mM) and H2O2 (3.0mM) to obtain the detection results. DETAILED DESCRIPTION

[0055] The present invention will be further described below with reference to the embodiments, but the present invention is not limited thereto.

[0056] The main chemical reagents used in the embodiments of the present invention are as follows:

[0057] TrackIt TMUltra-low range DNA ladder (10–300 bp) was purchased from Thermo Fisher Scientific (Shanghai, China); ammonium persulfate (APS), tetramethylethylenediamine (TEMED), BeyoRed DNA fixation buffer (6×), Gel-Red (10000×) fluorescent indicator, and tris-borate-EDTA premix (5×, 445 mM tris-borate, 10 mM EDTA, pH 7.0) were purchased from 8.3) was purchased from Biyuntian Biotechnology Co., Ltd. (Shanghai, China); horseradish peroxidase HRP-labeled streptavidin (0.4 mg / mL, Catalog No. D111054), 40% acrylamide / bisacrylamide solution (9:1), DEPC-treated water (DNase-free, RNase-free), and tris(2-carboxyethyl)phosphate hydrochloride (TCEP) were purchased from Sangon Co., Ltd. (Shanghai, China); 3,3',5,5'-tetramethylbenzidine (TMB), hydrogen peroxide (H2O2, 30.0%, w / w), and Nafion TM Perfluorinated resin solution (D520CS, 5.0–5.4% wt) was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Monolayer graphite oxide (GO, 0.5–5 μm in diameter and 0.8–1.2 nm in thickness, Catalog No. XF002-1) was provided by Xianfeng Nanotechnology Co., Ltd. (Nanjing, China). Ultrapure water used in all experiments was obtained using a water purification system from Aoside Instrument (Chongqing, China).

[0058] The oligonucleotides involved were synthesized and purified by Shanghai Sangon Co., Ltd. The specific sequences are as follows:

[0059]

[0060] Note: Italic letters represent substrate sequences, underlined letters represent Mg(II)-DNAzyme sequences; bold letters represent mismatched sites.

[0061] Equipment used and technical parameters:

[0062] Instruments: The electron binding energy of the materials was determined by X-ray photoelectron spectroscopy (XPS, Thermo Scientific K-Alpha, USA). Raman spectroscopy was performed using a Raman spectrometer from Horiba LabRAM HR Evolution, Japan, with an excitation wavelength of 455 nm. Fourier transform infrared (FT-IR) spectroscopy was performed using an infrared spectrometer (Thermo Scientific Nicolet iS20, USA). The stained gels were imaged using an imaging system (Sinsage ChampChemi TM610, China); ImageJ software (Softonic) was used to calculate the grayscale intensity of different gel bands; electrochemical tests were performed with an electrochemical workstation (Chenhua CHI 660E, China) using a traditional three-electrode system, with a modified glassy carbon electrode (GCE, 3.0 mm diameter) as the working electrode, a platinum wire as the counter electrode, and an Ag / AgCl electrode (saturated with KCl) as the reference electrode.

[0063] Example 1

[0064] Prepare the tetrahedral DNA scaffold-DNA enzyme / blocker (TDS-DNAzyme / blocker) complex as follows:

[0065] 1) Preparation of Azide, Pyrene, and Biotinylated TDS: Four single-stranded DNA strands (T1, T2, T3, and T4) were mixed in equimolar proportions in assembly buffer (20 mM Tris-HCl, 50 mM MgCl2, pH 8.0) to a final concentration of 2 μM. The mixture was then annealed at 95°C for 5 minutes in a gradient PCR instrument, followed by rapid cooling to 4°C and holding for over 60 minutes.

[0066] 2) Preparation of Dibenzocyclooctyne-DNAzyme / Blocker: Disperse the DBCO-DNAzyme and blocker chains in hybridization buffer (10 mM Tris-HCl, 20 mM MgCl2, 100 mM NaCl, pH 7.4). Mix equal volumes of blocker (6 μM) and DBCO-DNAzyme (4 μM), then anneal at 95°C for 2 minutes in a gradient PCR instrument. Slowly cool to 25°C at a rate of -1°C / min. This yields DBCO-DNAzyme / blocker (final concentration 2 μM).

[0067] 3) Preparation of TDS-DNAzyme / blocker complex: TDS (final concentration of 0.5 μM), DBCO-DNAzyme / blocker duplexes (final concentration of 0.5 μM) and HRP-conjugated streptavidin (final concentration of 1 ng μL -1 ) were mixed in immobilization buffer (10 mM Tris-HCl, 1 M NaCl, 1 mM EDTA, 10 mM TECP, pH 7.4) and incubated at 25°C for 30 minutes in a gradient PCR instrument to obtain a TDS-DNAzyme / blocker complex with a final concentration of 0.5 μM.

[0068] Example 2

[0069] Prepare reduced graphene oxide modified glassy carbon electrode by following the steps below:

[0070] 1) 10 mg of GO was dispersed in 5 mL of ultrapure water and ultrasonically treated for 30 min to obtain a GO suspension (2 mg mL -1 ). The graphene oxide suspension was then centrifuged at 8000 rpm / min for 5 min, and the graphene oxide brown supernatant was collected;

[0071] 2) Glassy carbon electrodes (GCEs) were first polished with alumina powder (300 nm and 50 nm), respectively, and then ultrasonically cleaned with ultrapure water for a few seconds.

[0072] 3) Use 75% ethanol to remove Nafion TM The perfluorinated resin solution was diluted to 2% (wt%). The graphene oxide suspension prepared in step 1) (2 mg mL -1 ) and Nafion TM Perfluorinated resin solution (2%) was mixed in equal volumes to obtain a graphene oxide / Nafion mixed solution.

[0073] 4) 10 μL of the graphene oxide / Nafion mixed solution described in step 3) was dropped onto the surface of the GCE described in step 2), and the mixture was dried in a vacuum dryer (25° C.) to obtain a graphene oxide / Nafion modified glassy carbon electrode (GO / Nafion / GCE).

[0074] 5) The GO / Nafion / GCE described in step 4) was electrochemically reduced in phosphate buffered saline (10 mM, pH 5.0) using 1 t amperometric measurements with an initial potential of -1.3 V, a sampling interval of 0.1 s, and a rest time of 0 s. The prepared reduced graphene oxide / Nafion-modified glassy carbon electrode (rGO / Nafion / GCE) was stored at room temperature for further experiments.

[0075] Example 3

[0076] To construct an electrochemical biosensor for the detection of acute myocardial infarction-related microRNA-499, follow the steps below:

[0077] 1) 10 μL of the TDS-DNAzyme / blocker complex (0.5 μM) described in Example 1 was added dropwise to the rGO / Nafion / GCE surface described in Example 2 and incubated at room temperature for 2 h;

[0078] 2) After gently rinsing the modified electrode obtained in step 1) with assembly buffer, the TDS-DNAzyme / blocker complex-modified electrode (TDS-DNAzyme / blocker / rGO / Nafion / GCE) was stored at 4° C. for subsequent use.

[0079] Example 4

[0080] The electrochemical biosensor constructed in Example 3 was used to detect microRNA-499, and the following steps were followed:

[0081] 1. Draw a working curve

[0082] 1) The modified electrodes prepared in step 2), step 4) and step 5) of Example 2 were placed in 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution and subjected to cyclic voltammetry (CV) characterization. The results are shown in FIG. Figure 1 As shown in A, rGO / Nafion / GCE has the best electron transport properties.

[0083] 2) The modified electrodes of step 5) of Example 2 and step 2) of Example 3 were placed in 5mM K3[Fe(CN)6] / K4[Fe(CN)6] solution for electrochemical impedance spectroscopy (EIS) characterization. 10μL microRNA-499 (1pM) dispersed in assembly buffer was added dropwise to the electrode surface described in Example 3 and incubated at room temperature for 20min. After rinsing with assembly buffer, the modified electrode was placed in 5mM K3[Fe(CN)6] / K4[Fe(CN)6] solution for electrochemical impedance spectroscopy (EIS) characterization. The results are shown in FIG. Figure 1 As shown in B, it proves that the electrochemical sensor was successfully prepared.

[0084] 3) A non-HRP-labeled TDS-DNAzyme / blocker complex was prepared as described in Example 1, and a non-HRP-labeled TDS-DNAzyme / blocker / rGO / Nafion / GCE modified electrode was prepared as described in Example 3. After rinsing with assembly buffer, the modified electrode was placed in a HAc-NaAc (0.1 M, pH 4.0) buffer containing a mixture of TMB (0.66 mM) and H2O2 (3.0 mM), and the it curve was measured. A TDS-DNAzyme / blocker / rGO / Nafion / GCE modified electrode was prepared as described in Example 3. After rinsing with assembly buffer, the modified electrode was placed in a HAc-NaAc (0.1 M, pH 4.0) buffer containing a mixture of TMB (0.66 mM) and H2O2 (3.0 mM), and the it curve was measured. 10 μL of microRNA-499 (10 fM) dispersed in assembly buffer was dropwise applied to the electrode surface described in Example 3 and incubated at room temperature for 20 min. After rinsing with assembly buffer, the modified electrode was placed in HAc-NaAc (0.1 M, pH 4.0) buffer containing a mixture of TMB (0.66 mM) and H2O2 (3.0 mM), and the it curve was measured. Figure 2 As shown, (a) TDS-DNAzyme / blocker / rGO / Nafion / GCE without HRP labeling, (b) TDS-DNAzyme / blocker / rGO / Nafion / GCE labeled with HRP, and (c) TDS-DNAzyme / blocker / rGO / Nafion / GCE labeled with HRP detected 10 fM microRNA-499. This demonstrates that the electrochemical sensor can be used for microRNA-499 detection.

[0085] 4) 10 μL of microRNA-499 at different concentrations dispersed in assembly buffer was dripped onto the electrode surface described in Example 3 and incubated at room temperature for 20 min. After rinsing with assembly buffer, the electrode was immersed in HAc-NaAc (0.1 M, pH 4.0) buffer containing a mixture of TMB (0.66 mM) and H2O2 (3.0 mM) and the it curve was measured. Figure 3 As shown in A, the concentration gradient of microRNA-499 was set to 0M, 0.5fM, 1fM, 10fM, 50fM, 100fM, 1pM, and 10pM.

[0086] 5) Based on the linear relationship between the obtained current value and the logarithmic value of microRNA-499 concentration, a standard curve (such as Figure 3The results showed that the current response value and the logarithm of microRNA-499 concentration showed a good linear relationship in the range of 0.5 fM-10 pM, with a linear correlation coefficient of 0.9963. The detection limit was calculated to be 0.16 fM using 3σ / s.

[0087] 2. Sensor specificity test: To investigate the specificity of the proposed sensor, the sensor was investigated to distinguish microRNA-499 from other AMI-related microRNAs (including microRNA-208, microRNA-1, and microRNA-133a) and base mismatches. The current signal difference between the blank sample and the interfering substances was negligible, while the electrochemical biosensor showed a significant current change in response to microRNA-499 (e.g., Figure 4 The results showed that the proposed electrochemical biosensor for microRNA-499 detection had good specificity.

[0088] 3. Sensor stability test: The sensor prepared in Example 2 was placed in a 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution and subjected to cyclic voltammetry (CV) testing. After 50 consecutive scans, the relative standard deviations (RSDs) of the current and voltage values ​​were less than 5% (e.g. Figure 5 This shows that the sensor has good stability.

[0089] 4. Actual Sample Analysis Application

[0090] To further verify the practical application of the proposed method, a certain concentration of microRNA-499 (10 pM to 1 fM) was added to 10% healthy human serum samples and buffer solution to simulate real samples, and then the prepared electrochemical biosensor was used for detection. Figure 6 As shown, the current difference between 10% healthy human serum and buffered blood is negligible, indicating that the electrochemical biosensor has application potential for detecting microRNA-499 in clinical samples.

Claims

1. An electrochemical biosensor for detecting microRNA associated with acute myocardial infarction, characterized by: The TDS-DNAzyme / blocker complex was added dropwise onto the surface of reduced graphene oxide / Nafion-modified glassy carbon electrode (rGO / Nafion / GCE) and incubated at room temperature for 2-3 hours to prepare an electrochemical biosensor for microRNA-499 detection. The preparation of the TDS-DNAzyme / blocker complex comprises the following steps: 1) Preparation of Azide, Pyrene, and Biotinylated TDS: Four single-stranded DNA strands, T1, T2, T3, and T4, were mixed in an equimolar ratio in assembly buffer containing 20 mM Tris-HCl and 50 mM MgCl2, pH 8.0, and then annealed in a gradient PCR instrument, cooled to 3-5°C, and maintained for 60-90 minutes. 2) Preparation of DBCO-DNAzyme / blocker: The DBCO-DNAzyme and blocker chains were dispersed in hybridization buffer, and equal volumes of blocker and DBCO-DNAzyme were mixed. The mixture was then annealed in a gradient PCR instrument and cooled to 20-25°C at a rate of -1-2°C / min to prepare the DBCO-DNAzyme / blocker. The hybridization buffer contained 10 mM Tris-HCl, 20 mM MgCl2, and 100 mM NaCl, and the pH of the hybridization buffer was 7.

4. 3) Preparation of TDS-DNAzyme / blocker complex: The product prepared in step 1), the DBCO-DNAzyme / blocker double strand prepared in step 2), and horseradish peroxidase (HRP)-labeled streptavidin were mixed in a fixation buffer, and the mixture was incubated at 20-25°C for 20-30 minutes in a gradient PCR instrument to obtain the TDS-DNAzyme / blocker complex; the fixation buffer contained 10 mM Tris-HCl, 1 M NaCl, 1 mM EDTA, and 10 mM TECP; the pH of the fixation buffer was 7.

4. Preparation of rGO / Nafion / GCE: graphene oxide suspension and Nafion TM A graphene oxide / Nafion mixture was obtained by mixing a perfluorinated resin solution, and then the graphene oxide / Nafion mixture was dropped onto the pretreated GCE surface. After drying in a vacuum dryer, a graphene oxide / Nafion-modified glassy carbon electrode (GO / Nafion / GCE) was prepared. The GO / Nafion / GCE was placed in phosphate buffered saline for electrochemical reduction to obtain a reduced graphene oxide / Nafion-modified glassy carbon electrode (rGO / Nafion / GCE). The TDS preparation method is as follows: four single-stranded DNA strands T1, T2, T3, and T4 are mixed in an equimolar ratio in an assembly buffer, and then annealed in a gradient PCR instrument, cooled to 3-5°C, and maintained for 60-90 minutes; the assembly buffer contains 20 mM Tris-HCl and 50 mM MgCl2, and the pH of the assembly buffer is 8.0; the DNA sequences of T1, T2, T3, and T4 are as follows:

2. The electrochemical biosensor according to claim 1, wherein The preparation steps of rGO / Nafion / GCE are as follows: graphene oxide suspension and Nafion TM A perfluorinated resin solution was mixed to obtain a graphene oxide / Nafion mixture, which was then dropped onto the pretreated GCE surface and dried in a vacuum dryer at 20-25°C to obtain a graphene oxide / Nafion-modified glassy carbon electrode GO / Nafion / GCE. The GO / Nafion / GCE was placed in 10 mM pH 5.0 phosphate buffered saline for electrochemical reduction, and measured using it ampere with an initial potential of -1.3 V, a sampling interval of 0.1 s, and a standing time of 0 s to obtain a reduced graphene oxide / Nafion-modified glassy carbon electrode rGO / Nafion / GCE.

3. A method for detecting microRNA-499 for non-diagnostic and / or therapeutic purposes based on the electrochemical biosensor according to claim 1 or 2, characterized in that: The steps include: (1) 10 μL of microRNA-499 dispersed in assembly buffer was dropped onto the surface of TDS-DNAzyme / blocker / rGO / Nafion / GCE and incubated at room temperature for 20 min. After rinsing with assembly buffer, the electrode was immersed in 0.1 M HAc-NaAc buffer (pH 4.0) containing a mixture of 0.66 mM TMB and 3.0 mM H2O2, and the it curve was measured. (2) drawing a working curve based on the linear relationship between the current obtained in step (1) and the logarithmic value of the microRNA-499 concentration; (3) Using the electrochemical sensor to detect the sample to be tested, the obtained current value is calculated using the working curve prepared in step (2) to obtain the microRNA-499 concentration of the sample to be tested.

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