CN116698930A - Electrochemical biosensor for acute myocardial infarction related microRNA detection - Google Patents

Electrochemical biosensor for acute myocardial infarction related microRNA detection Download PDF

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CN116698930A
CN116698930A CN202310684164.XA CN202310684164A CN116698930A CN 116698930 A CN116698930 A CN 116698930A CN 202310684164 A CN202310684164 A CN 202310684164A CN 116698930 A CN116698930 A CN 116698930A
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nafion
graphene oxide
dnazyme
gce
buffer
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卿敏
白丽娟
李悦媛
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Chongqing Medical University
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Abstract

The invention provides an electrochemical biosensor for detecting acute myocardial infarction related microRNA. The invention uses tetrahedral DNA bracket to limit molecular recognition in nanometer scale, to reduce molecular diffusion, improve the sensitivity of analysis method and shorten detection time. The invention constructs a sensitive and rapid AMI related microRNA detection method, and provides experimental basis and technical support for the research and development of AMI related microRNA detection technology.

Description

Electrochemical biosensor for acute myocardial infarction related microRNA detection
Technical Field
The invention relates to the technical field of electrochemical detection, in particular to an electrochemical biosensor for detecting acute myocardial infarction related microRNA based on a space limiting effect of a tetrahedral DNA bracket and a detection method thereof.
Background
Acute Myocardial Infarction (AMI) is a type of myocardial injury caused by acute, persistent ischemia and hypoxia of the coronary arteries, and is considered a major public health problem because of sudden onset, delayed diagnosis and uncertain diagnosis, which are major causes of high morbidity and mortality of AMI. The AMI blood circulation reconstruction treatment is crucial to the repair of ischemic cardiac muscle, and can remarkably reduce the death rate of AMI patients. AMI revascularization therapy relies on early, timely, accurate AMI identification and diagnosis. Cardiac injury caused by AMI is associated with progressive loss of cardiomyocytes, and induces time-dependent release of biomarkers such as myoglobin, creatine kinase isozymes (CK-MB), and cardiac fatty acid binding proteins (H-FABP). However, the levels of protein biomarkers may be affected by some non-cardiac diseases, the low specificity of which may lead to misdiagnosis. In recent years, many biological studies have demonstrated that abnormal expression of some micrornas is closely related to the pathogenesis of AMI. Compared with protein biomarkers, the microRNA related to AMI has higher specificity and timeliness, so the microRNA is a more excellent biomarker for diagnosing AMI. However, AMI-related micrornas are extremely low in expression in early disease, and accurate detection is interfered by other homologous micrornas. Therefore, it is important to improve the sensitivity of AMI-related microRNA detection technology and shorten the analysis time.
In general, increasing the sensitivity of microRNA detection methods relies on developing signal amplification strategies, such as amplifying the biological recognition signal using nanomaterials or enzymatic catalysis, or increasing the number of biomarkers using nucleic acid signal amplification, but often increases the detection time. Therefore, the detection method of microRNA faces the double challenges of low sensitivity and long detection time. Electrochemical biosensors combining high selectivity of biological recognition with high sensitivity of electrochemical technology have proven to have the potential to achieve sensitive detection of circulating biomarkers. Studies have shown that the response time is prolonged due to slow mass transport of analyte molecules to the sensing interface. The time scale 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 accuracy in analyzing 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 may result in reduced hybridization efficiency.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides an electrochemical biosensor for detecting acute myocardial infarction related microRNA. The invention uses tetrahedral DNA bracket to limit molecular recognition in nanometer scale, to reduce molecular diffusion, improve the sensitivity of analysis method and shorten detection time. In this work four reaction chains were added to four vertices of a tetrahedral DNA scaffold, wherein DNA hybridization reactions were confined to the tetrahedral DNA scaffold, and DNA hybridization reaction efficiency and analytical performance were greatly improved due to the reduction of diffusion pathways and the enhancement of signals. In addition, the construction process of the sensor is a one-step incubation process, so that no additional reagent or transfer of amplification products is needed, and the operation difficulty is greatly reduced. The research constructs a sensitive and rapid AMI related microRNA detection method, and provides experimental basis and technical support for research and development of AMI related microRNA detection technology.
An electrochemical biosensor for detecting acute myocardial infarction related microRNA, which is characterized in that:
and (3) dripping the TDS-DNAzyme/blocker compound on the surface of a reduced graphene oxide/Nafion modified glassy carbon electrode rGO/Nafion/GCE, and incubating for 2-3 h at room temperature to prepare the electrochemical biosensor for detecting microRNA-499.
The preparation steps of the TDS-DNAzyme/blocking er compound are as follows: the DBCO-DNAzyme/blocker double chain, TDS and horseradish peroxidase HRP marked streptavidin HRP-conjugated streptavidinin are mixed in a fixed buffer solution (10 mM Tris-HCl,1M NaCl,1mM EDTA,10mM TECP,pH 7.4), and placed in a gradient PCR instrument for incubation for 20-30 min at 20-25 ℃ to obtain the TDS-DNAzyme/blocker complex.
Preparation of rGO/Nafion/GCE:
graphene oxide suspension and Nafion TM And mixing perfluorinated resin solution to obtain graphene oxide/Nafion mixed solution, then dripping the graphene oxide/Nafion mixed solution on the surface of the pretreated GCE, drying in a vacuum dryer to obtain a graphene oxide/Nafion modified glassy carbon electrode (GO/Nafion/GCE), and placing the GO/Nafion/GCE in phosphate buffer saline for electrochemical reduction to obtain a reduced graphene oxide/Nafion modified glassy carbon electrode (rGO/Nafion/GCE).
The preparation method of the TDS comprises the following steps:
equimolar four single-stranded DNA T1, T2, T3 and T4The mixture was mixed in an assembly buffer (20 mM Tris-HCl,50mM MgCl) 2 pH 8.0), then annealing in a gradient PCR instrument, cooling to 3-5 ℃ and maintaining for 60-90 min.
Further, the preparation of the TDS-DNAzyme/blocking er complex comprises the following steps:
1) Preparation of azido, pyrene molecule and biotinylated TDS: the four single stranded DNA T1, T2, T3 and T4 were mixed in equimolar proportions in an assembly buffer (20 mM Tris-HCl,50mM MgCl 2 pH 8.0), then placing in a gradient PCR instrument for annealing, cooling to 3-5 ℃ and then maintaining for 60-90 min;
the DNA sequences of T1, T2, T3, T4 are as follows:
2) Preparation of DBCO-DNAzyme/blocking device: hybridization buffer (10 mM Tris-HCl,20mM MgCl) 2 100mM NaCl,pH 7.4) dispersing DBCO-DNAzyme and a blocker chain, mixing the blocker and the DBCO-DNAzyme in equal volume, then placing the mixture in a gradient PCR instrument for annealing, and cooling to 20-25 ℃ at a cooling rate of-1 to-2 ℃/min to prepare the DBCO-DNAzyme/blocker;
3) Preparation of TDS-DNAzyme/blocking er complex: mixing the product prepared in the step 1), the DBCO-DNAzyme/blocker double chain prepared in the step 2) and horseradish peroxidase HRP-marked streptavidin (HRP-conjugated streptavidin) in a fixed buffer solution (10 mM Tris-HCl,1M NaCl,1mM EDTA,10mM TECP,pH7.4), and placing the mixture in a gradient PCR instrument for incubation at 20-25 ℃ for 20-30 min to obtain the TDS-DNAzyme/blocker complex.
The preparation steps of the rGO/Nafion/GCE are as follows:
mixing graphene oxide suspension with Nafion (TM) perfluorinated resin solution to obtain graphene oxide/Nafion mixed solution, then drying graphene oxide/Nafion mixed solution on the surface of pretreated GCE at 20-25 ℃ in a vacuum dryer to obtain graphene oxide/Nafion modified glassy carbon electrode (GO/Nafion/GCE), placing the GO/Nafion/GCE in phosphate buffered saline (10 mM, pH 5.0) for electrochemical reduction, and measuring by using i-t amperes, wherein the initial potential is-1.3V, the sampling interval is 0.1s, and the standing time is 0s, so as to obtain reduced graphene oxide/Nafion modified glassy carbon electrode (rGO/Nafion/GCE).
The preparation method of the reduced graphene oxide (rGO) comprises the following steps: dispersing flaky single-layer graphene oxide (GO, 10 mg) in ultrapure water, performing ultrasonic treatment for 30-40min to obtain graphene oxide suspension, centrifuging the graphene oxide suspension at 8000-9000rpm/min for 5-10min, and collecting graphene oxide brown supernatant to obtain reduced graphene oxide (rGO).
In particular to an electrochemical biosensor for detecting acute myocardial infarction related microRNA, which is characterized by comprising the following steps:
(1) Preparation of tetrahedral DNA scaffold-DNAzyme/blocking strand (TDS-DNAzyme/blocking er) complexes
1) Preparation of azido, pyrene molecule and biotinylated TDS: the four single stranded DNA (T1, T2, T3, T4) were mixed in equimolar proportions in an assembly buffer (20 mM Tris-HCl,50mM MgCl) 2 pH 8.0), the final concentration is2 mu M, and then the mixture is placed in a gradient PCR instrument for annealing at 95 ℃ for 5min, and the temperature is quickly reduced to 4 ℃ and maintained for more than 60min;
2) Preparation of dibenzocyclooctyne-DNAzyme/blocking Strand (DBCO-DNAzyme/blocking er): hybridization buffer (10 mM Tris-HCl,20mM MgCl) 2 100mM NaCl,pH 7.4) disperse DBCO-DNAzyme and the blocker chains; mixing the same volume of a blocker (6 mu M) and DBCO-DNAzyme (4 mu M), then placing the mixture in a gradient PCR instrument for annealing at 95 ℃ for 2min, and slowly cooling to 25 ℃ at a cooling rate of-1 ℃/min to prepare the DBCO-DNAzyme/blocker (final concentration of 2 mu M);
3) Preparation of TDS-DNAzyme/blocking er complex: the TDS (final concentration of 0.5. Mu.M) prepared in step 1), the DBCO-DNAzyme/blocker duplex (final concentration of 0.5. Mu.M) prepared in step 2) and horseradish peroxidase HRP-labeled streptavidin (HRP-conjugated streptavidinin) (final concentration of 1 ng. Mu.L) were mixed -1 ) Mixing in a fixation buffer (10 mM Tris-HCl,1M NaCl,1mM EDTA,10mM TECP,pH 7.4); incubating for 20min at 25 ℃ in a gradient PCR instrument to obtain a TDS-DNAzyme/blocker complex with the final concentration of 0.5 mu M;
(2) Preparing a reduced graphene oxide modified glassy carbon electrode:
1) Preparation of reduced graphene oxide (rGO): the flaky single-layer graphene oxide (GO, 10 mg) is dispersed in 5mL of ultrapure water, and is subjected to ultrasonic treatment for 30min to obtain graphene oxide suspension (2 mg mL) -1 ) The method comprises the steps of carrying out a first treatment on the surface of the Centrifuging the graphene oxide suspension at 8000rpm/min for 5min, and collecting graphene oxide brown supernatant;
2) Preparing a reduced graphene oxide modified glassy carbon electrode: firstly, respectively polishing Glassy Carbon Electrodes (GCE) by alumina powder (300 nm and 50 nm), and then ultrasonically cleaning by ultrapure water for a few seconds; the NafionTM perfluorinated resin solution was diluted to 2% (wt%) using 75% ethanol. The graphene oxide suspension prepared in step 1) (2 mg mL -1 ) Mixing with Nafion (TM) perfluorinated resin solution (2%) in equal volume to obtain graphene oxide/Nafion mixed solution; drying 10 mu L of graphene oxide/Nafion mixed liquid drops on the surface of the pretreated GCE in a vacuum dryer (25 ℃) to prepare a graphene oxide/Nafion modified glassy carbon electrode (GO/Nafion/GCE); finally, GO/Nafion/GCE was placed in phosphate buffered saline (10 mm, ph 5.0) for electrochemical reduction using an i-t amperometric measurement with an initial potential of-1.3V, a sampling interval of 0.1s, and a resting time of 0s; the prepared reduced graphene oxide/Nafion modified glassy carbon electrode (rGO/Nafion/GCE) is stored at room temperature;
(3) Constructing an electrochemical biosensor for detecting acute myocardial infarction related microRNA-499:
1) Dropwise adding 10 mu L of TDS-DNAzyme/blocker complex (0.5 mu M) on the rGO/Nafion/GCE surface in the step (2), and incubating for 2h at room temperature;
2) After the electrode obtained in the step 1) is lightly rinsed by using an assembly buffer solution, the electrode modified by the TDS-DNAzyme/blocker complex (TDS-DNAzyme/blocker/rGO/Nafion/GCE) is stored at 4 ℃ for subsequent use, and the electrochemical biosensor for detecting microRNA-499 is obtained.
In a second aspect, the invention provides a method for detecting microRNA-499 based on the electrochemical biosensor.
The method for detecting microRNA-499 based on the electrochemical biosensor is characterized by comprising the following steps of:
(1) Dripping 10 μL microRNA-499 dispersed in assembly buffer onto the surface of the above electrode (TDS-DNAzyme/blocker/rGO/Nafion/GCE), incubating at room temperature for 20min, rinsing with assembly buffer, immersing the electrode in a buffer containing TMB (0.66 mM) and H 2 O 2 The i-t curve was measured in HAc-NaAc (0.1M, pH 4.0) buffer of the (3.0 mM) mixture;
(2) Drawing a working curve according to the linear relation between the current obtained in the step (1) and the microRNA-499 concentration logarithmic value;
(3) And (3) detecting a sample to be detected by using the electrochemical sensor, and calculating the obtained current value through the working curve prepared in the step (2) to obtain the microRNA-499 concentration of the sample to be detected.
Compared with the prior art, the preparation method and the application of the electrochemical biosensor for detecting microRNA-499 have the outstanding characteristics that:
the invention explores the finite field effect of the tetrahedral DNA scaffold in the interface sensor, improves the sensitivity of AMI related microRNA-499 detection and shortens the detection time. The invention reduces the diffusion path length and increases the signal gain, and greatly improves the DNA hybridization reaction and analysis performance. In addition, the detection is a simple one-step incubation process, avoiding the introduction of additional reagents and the transfer of amplification products. The prepared electrochemical biosensor is successfully used for sensitive and rapid detection of microRNA-499 by the means. Compared with the traditional microRNA-499 detection method, the method has the advantages of rapid detection, high sensitivity and simple operation, and provides a new analysis method for the microRNA-499 detection. The invention will promote the wide application of electrochemical biosensor in disease diagnosis.
The beneficial effects of the invention are as follows:
1) The defects of low detection sensitivity and long detection time of the sensor can be greatly improved by utilizing the limiting effect of the tetrahedral DNA bracket in the interface sensor;
2) The tetrahedral DNA bracket limits the metal specificity catalysis DNA deoxyribozyme (DNAzyme) shearing reaction in a compact space, reduces the diffusion path length, increases the signal gain, and greatly improves the DNA hybridization reaction efficiency and the analysis performance;
3) The materials related by the invention can be synthesized under laboratory conditions, and the method has the advantages of simple operation, low raw material price, low toxicity, environmental friendliness, extremely small usage amount each time and reduced experiment cost;
4) The whole detection analysis method has clear and simple steps, high sensitivity and quick signal response;
5) The electrochemical biosensor prepared by the method can provide a new method for detecting microRNA-499.
Drawings
FIG. 1 is a schematic of different modified electrodes in 10mM PBS (pH 7.0, containing 100mM NaCl and 5 mMK) 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ]) Cyclic voltammetry (a) and electrochemical impedance characterization (B) were performed in solution. The sweep rate is set to be 0.10V s in the cyclic voltammetry test -1 The scanning voltage ranges from-0.1V to 0.6V. The electrochemical impedance test set voltage is 220mV, the boost voltage is 5mV, and the frequency is 1.0X10 -2 Hz-1.0×10 6 Hz。
FIG. 2 is a schematic diagram of a different modified electrode containing TMB (0.66 mM) and H 2 O 2 I-t test pattern in HAc-NaAc (0.1M, pH 4.0) buffer of (3.0 mM) mixture. Setting an initial voltage of 0.1V (versus Ag/AgCl), sampling interval of 0.1s, test time of 100s and sensitivity of 2×10 –5 A/V。
FIG. 3 shows the results of detection of different concentrations of microRNA-499 by the sensor of the present invention, wherein FIG. A shows a sample containing TMB (0.66 mM) and H 2 O 2 Response i-t test patterns of sensors in HAc-NaAc (0.1M, pH 4.0) buffer of (3.0 mM) mixture to different concentrations of microRNA-499 (0M, 0.5fM,1fM,10fM,50fM,100fM,1pM,10 pM), respectively; FIG. B is a plot of sensor amperometric vs. microRNA-499 at various concentrations (0.1 fM,0.5fM,1fM,10fM,50fM,100fM,1pM,10 pM)100 pM) wherein the inset is a calibration curve of amperometric vs. microRNA-499 (0.5 fM-10 pM) vs. different concentrations.
FIG. 4 is a graph of a sensor's selectivity evaluation, wherein interferents are other AMI-related microRNAs (including microRNA-208, microRNA-1, and microRNA-133 a) and base mismatches. microRNA-499 was at a concentration of 1pM and the interferent at a concentration of 50pM.
Fig. 5 is a sensor stability test result. rGO/nafion/GCE modified electrode in 10mM PBS (pH 7.0, containing 100mM NaCl and 5mM K) 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ]) Continuous cyclic voltammetry was performed in solution. The cyclic voltammetry test sets the sweep rate to 0.10Vs -1 The scanning voltage ranges from-0.1V to 0.6V.
FIG. 6 is a sample of healthy human serum and buffer solution with a concentration of microRNA-499 (10 pM-1 fM) added to simulate a real sample, respectively, an electrochemical biosensor in a sample containing TMB (0.66 mM) and H 2 O 2 Detection results of i-t test in HAc-NaAc (0.1M, pH 4.0) buffer of (3.0 mM) mixture.
Detailed Description
The invention is further illustrated, but is not limited, by the following examples.
The main chemical reagents used in the examples of the present invention are as follows:
TrackIt TM ultra low range DNA ladder (10-300 bp) was purchased from Thermo Fisher Scientific (Shanghai, china); ammonium Persulfate (APS), tetramethyl ethylenediamine (TEMED), beyoRed DNA immobilization buffer (6×), gel-Red (10000×) fluorescent indicator, and triboronic acid-EDTA premix powder (5×,445mM tris-borate,10mM EDTA, ph 8.3) were purchased from the yunskia biology company (Shanghai, china); horseradish peroxidase HRP-labeled streptavidin (0.4 mg/mL, cat No. D111054), 40% acrylamide/bisacrylamide solution (9:1), DEPC treated water (DNase-free, RNase-free) and tris (2-carboxyethyl) phosphate (TCEP) were purchased from the manufacturer (Shanghai, china); 3,3', 5' -Tetramethylbenzidine (TMB), hydrogen peroxide (H) 2 O 2 30.0%, w/w) and Nafion TM Perfluorinated resin solutions (D520 CS,5.0-5.4% wt.)Purchased from aladine biochemical technologies, inc (Shanghai, china); single-layer graphite oxide (GO, diameter 0.5-5 μm, thickness 0.8-1.2nm, product number XF 002-1) is provided by Nanfeng nanometer Co., ltd. (Nanjing, china); the ultra-pure water used in all experiments was obtained by a water purification system of Aoside Instrument company (Chongqing in China);
the oligonucleotides involved were synthesized and purified by Shanghai Biotechnology, inc., and the specific sequences are as follows:
note that: italics indicates substrate sequence, underlined indicates sequence of Mg (II) -DNAzyme; bold letters indicate mismatched sites.
The equipment and technical parameters used are as follows:
instrument: electron binding energy of the material was measured by X-ray photoelectron spectroscopy (XPS, thermo Scientific K-Alpha, usa); the raman spectrum experiment adopts a raman spectrometer of the company Horiba LabRAM HR Evolution in japan, and the excitation wavelength is 455 nm; fourier transform infrared (FT-IR) spectroscopy employs an infrared spectrometer (Thermo Scientific Nicolet iS, USA); the stained gel was imaged by an imaging system (Sinsage ChampChemiTM610, china); gray scale intensities of the different gel bands were calculated using ImageJ software (Softonic); the electrochemical test adopts an electrochemical workstation (Chenhua CHI 660E, china), a traditional three-electrode system, a modified glassy carbon electrode (GCE, diameter 3.0 mm) is used as a working electrode, a platinum wire is used as a counter electrode, and an Ag/AgCl electrode (saturated KCl) is used as a reference electrode.
Example 1
Preparation of tetrahedral DNA scaffold-DNAzyme/blocker complexes the procedure was as follows:
1) Preparation of azido, pyrene molecule and biotinylated TDS: four single stranded DNAs (T1)T2, T3, T4) in equimolar proportions in an assembly buffer (20 mM Tris-HCl,50mM MgCl 2 pH 8.0) was 2. Mu.M. Then placing the mixture in a gradient PCR instrument for annealing at 95 ℃ for 5min, rapidly cooling to 4 ℃ and maintaining for more than 60min;
2) Preparation of dibenzocyclooctyne-DNAzyme/blocking Strand (DBCO-DNAzyme/blocking er): hybridization buffer (10 mM Tris-HCl,20mM MgCl) 2 100mM NaCl,pH 7.4) disperse the DBCO-DNAzym and the blocker chains. The blocker (6. Mu.M) and DBCO-DNAzyme (4. Mu.M) were mixed in equal volumes and then placed in a gradient PCR apparatus for annealing at 95℃for 2min, and slowly cooled to 25℃at a cooling rate of-1℃per min. DBCO-DNAzyme/blocking (final concentration 2. Mu.M) was prepared.
3) Preparation of TDS-DNAzyme/blocking er complex: TDS (final concentration of 0.5. Mu.M), DBCO-DNAzyme/blocker duplex (final concentration of 0.5. Mu.M) and horseradish peroxidase HRP-labeled streptavidin (HRP-conjugated streptavidinin) (final concentration of 1 ng. Mu.L) -1 ) Mix in a fixed buffer (10 mM Tris-HCl,1M NaCl,1mM EDTA,10mM TECP,pH 7.4). After incubation for 30min at 25℃in a gradient PCR instrument, the final concentration of TDS-DNAzyme/blocker complex was obtained at 0.5. Mu.M.
Example 2
Preparing a reduced graphene oxide modified glassy carbon electrode, and operating according to the following steps:
1) The flaky single-layer graphene oxide (GO, 10 mg) is dispersed in 5mL of ultrapure water, and is subjected to ultrasonic treatment for 30min to obtain graphene oxide suspension (2 mg mL) -1 ). Centrifuging the graphene oxide suspension at 8000rpm/min for 5min, and collecting graphene oxide brown supernatant;
2) The Glassy Carbon Electrodes (GCE) were first polished with alumina powder (300 nm and 50 nm), respectively, and then ultrasonically cleaned with ultrapure water for several seconds.
3) The NafionTM perfluorinated resin solution was diluted to 2% (wt%) using 75% ethanol. The graphene oxide suspension prepared in step 1) (2 mg mL -1 ) And mixing with Nafion (TM) perfluorinated resin solution (2%) in an equal volume to obtain graphene oxide/Nafion mixed solution.
4) And (3) placing the 10 mu L graphene oxide/Nafion mixed liquid drop obtained in the step (3) on the surface of the GCE obtained in the step (2) in a vacuum drier (25 ℃) for drying to obtain the graphene oxide/Nafion modified glassy carbon electrode (GO/Nafion/GCE).
5) The GO/Nafion/GCE from step 4) was subjected to electrochemical reduction in phosphate buffered saline (10 mM, pH 5.0), measured using i-t amperes, with an initial potential of-1.3V, a sampling interval of 0.1s, and a resting time of 0s. The prepared reduced graphene oxide/Nafion modified glassy carbon electrode (rGO/Nafion/GCE) was stored at room temperature for further experiments.
Example 3
An electrochemical biosensor for detecting acute myocardial infarction related microRNA-499 is constructed, and the method is operated according to the following steps:
1) mu.L of TDS-DNAzyme/blocker complex (0.5. Mu.M) as described in example 1 was added dropwise to the rGO/Nafion/GCE surface as described in example 2 and incubated for 2h at room temperature;
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℃for subsequent use.
Example 4
microRNA-499 was detected using the electrochemical biosensor constructed in example 3, and the procedure was as follows:
1. drawing a working curve
1) The modified electrodes of example 2, step 2), step 4) and step 5) were placed in 5mM K, respectively 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ]Cyclic Voltammetry (CV) characterization was performed in solution. The results are shown in FIG. 1A, where rGO/Nafion/GCE has the best electron transport properties.
2) Placing the modified electrodes of example 2, step 5) and example 3, step 2) at 5mM K, respectively 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ]Electrochemical Impedance (EIS) characterization was performed in solution. 10. Mu.L of microRNA-499 (1 pM) dispersed in the 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 at 5mM K 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ]Electrochemical Impedance (EIS) characterization was performed in solution. The results are shown in fig. 1B, which demonstrate the success of the electrochemical sensor fabrication.
3) An HRP-free TDS-DNAzyme/blocker complex was prepared as described in example 1 and an HRP-free 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 buffer containing TMB (0.66 mM) and H 2 O 2 The i-t curve was measured in HAc-NaAc (0.1M, pH 4.0) buffer of the (3.0 mM) mixture. TDS-DNAzyme/blocker/rGO/Nafion/GCE modified electrodes were prepared as described in example 3. After rinsing with assembly buffer, the modified electrode was placed in a buffer containing TMB (0.66 mM) and H 2 O 2 The i-t curve was measured in HAc-NaAc (0.1M, pH 4.0) buffer of the (3.0 mM) mixture. 10. Mu.L microRNA-499 (10 fM) 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 a buffer containing TMB (0.66 mM) and H 2 O 2 The i-t curve was measured in HAc-NaAc (0.1M, pH 4.0) buffer of the (3.0 mM) mixture. The results are shown in FIG. 2, (a) TDS-DNAzyme/blocker/rGO/Nafion/GCE without HRP label, (b) TDS-DNAzyme/blocker/rGO/Nafion/GCE with HRP label, and (c) TDS-DNAzyme/blocker/rGO/Nafion/GCE with HRP label for detection of 10fMmicroRNA-499. The electrochemical sensor can be used for microRNA-499 detection.
4) 10. Mu.L of microRNA-499 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 electrode was immersed in a buffer containing TMB (0.66 mM) and H 2 O 2 The i-t curve was measured in HAc-NaAc (0.1M, pH 4.0) buffer of the (3.0 mM) mixture. As shown in FIG. 3A, the microRNA-499 concentration gradient was set to 0M,0.5fM,1fM,10fM,50fM,100fM,1pM,10pM.
5) A standard curve (shown in FIG. 3B) was drawn from the linear relationship between the current values obtained and microRNA-499 concentration vs. value. The measurement result shows that the current response value and microRNA-499 concentration logarithmic value have good linear relation within the range of 0.5fM-10pM, the linear correlation coefficient is 0.9963, and the detection limit is 0.16fM calculated by 3 sigma/s.
2. Sensor specificity test: in order to study the specificity of the proposed sensor, the sensor was studied to distinguish microRNAs-499 from other AMI-related microRNAs (including microRNA-208, microRNA-1, and microRNA-133 a) and base mismatches. The difference in current signal between the blank sample and the interfering substance was negligible, while the electrochemical biosensor response to microRNA-499 showed a significant current change (as shown in FIG. 4). The result shows that the proposed electrochemical biosensor for detecting microRNA-499 has good specificity.
3. Sensor stability test: the sensor prepared in example 2 was placed at 5mM K 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ]The Cyclic Voltammetry (CV) test was performed in the solution, and after 50 consecutive scans, the current and voltage values were less than 5% Relative Standard Deviation (RSD) (as shown in FIG. 5), indicating good stability of the sensor.
4. Practical sample analysis application
To further verify the practical application of the proposed method, a concentration of microRNA-499 (10 pM to 1 fM) was added to 10% healthy human serum sample and buffer solution, respectively, to mimic a real sample, and then detected with the prepared electrochemical biosensor. The detection result is shown in fig. 6, the current difference between the serum of 10% healthy people and the buffer blood is negligible, and the electrochemical biosensor has the application potential of detecting microRNA-499 in clinical samples.

Claims (6)

1. An electrochemical biosensor for detecting acute myocardial infarction related microRNA, which is characterized in that: dropwise adding the TDS-DNAzyme/blocker compound on the surface of a reduced graphene oxide/Nafion modified glassy carbon electrode rGO/Nafion/GCE, and incubating for 2-3 h at room temperature to prepare an electrochemical biosensor for detecting microRNA-499;
the preparation steps of the TDS-DNAzyme/blocking er compound are as follows: uniformly mixing DBCO-DNAzyme/blocker double chains, TDS and horseradish peroxidase HRP-marked streptavidin HRP-conjugated streptavidinin in a fixed buffer solution, and placing the mixture in a gradient PCR instrument for incubation at 20-25 ℃ for 20-30 min to obtain a TDS-DNAzyme/blocker compound; the fixing buffer contains 10mM Tris-HCl,1M NaCl,1mM EDTA and 10mM TECP, and the pH of the fixing buffer is 7.4;
preparation of rGO/Nafion/GCE: graphene oxide suspension and Nafion TM Mixing perfluorinated resin solution to obtain graphene oxide/Nafion mixed solution, then dripping the graphene oxide/Nafion mixed solution on the surface of the pretreated GCE, drying in a vacuum dryer to obtain a graphene oxide/Nafion modified glassy carbon electrode (GO/Nafion/GCE), and placing the GO/Nafion/GCE in phosphate buffer saline for electrochemical reduction to obtain a reduced graphene oxide/Nafion modified glassy carbon electrode rGO/Nafion/GCE;
the preparation method of the TDS comprises the following steps: mixing four single-stranded DNA T1, T2, T3 and T4 in an assembly buffer solution according to an equimolar ratio, then placing the mixture in a gradient PCR instrument for annealing, cooling to 3-5 ℃ and maintaining for 60-90 min; the assembly buffer contains 20mM Tris-HCl and 50mM MgCl 2 The pH of the assembly buffer was 8.0; the DNA sequences of T1, T2, T3, T4 are as follows:
2. the electrochemical biosensor of claim 1, wherein the preparation of the TDS-DNAzyme/blocker complex comprises the steps of:
1) Preparation of azido, pyrene molecule and biotinylated TDS: mixing four single-stranded DNA T1, T2, T3 and T4 in an assembly buffer solution according to an equimolar ratio, then placing the mixture in a gradient PCR instrument for annealing, cooling to 3-5 ℃ and maintaining for 60-90 min; the assembly buffer contains 20mM Tris-HCl and 50mM MgCl 2 The pH of the assembly buffer was 8.0;
2) Preparation of DBCO-DNAzyme/blocking device: dispersing DBCO-DNAzyme and a blocker chain by using a hybridization buffer solution, mixing the blocker and the DBCO-DNAzyme in equal volume, then placing the mixture in a gradient PCR instrument for annealing, cooling to 20-25 ℃ with the cooling rate of-1 to-2 ℃/min, and preparing the DBCO-DNAzyme/blocker; the hybridization buffer contains 10mM Tris-HCl,20mM MgCl 2 And 100mM NaCl, pH7.4 of hybridization buffer;
3) Preparation of TDS-DNAzyme/blocking er complex: mixing the product prepared in the step 1), the DBCO-DNAzyme/blocker double chain prepared in the step 2) and the streptavidin marked by horseradish peroxidase HRP in a fixed buffer solution, and placing the mixture in a gradient PCR instrument for incubation for 20-30 min at 20-25 ℃ to obtain a TDS-DNAzyme/blocker compound; the immobilization buffer contained 10mM Tris-HCl,1M NaCl,1mM EDTA and 10mM TECP, pH7.4 of the immobilization buffer.
3. The electrochemical biosensor of claim 1, wherein the steps of preparing rGO/Nafion/GCE are: graphene oxide suspension and Nafion TM And mixing perfluorinated resin solution to obtain graphene oxide/Nafion mixed solution, then dripping the graphene oxide/Nafion mixed solution on the surface of the pretreated GCE, drying the mixture in a vacuum dryer at 20-25 ℃ to obtain graphene oxide/Nafion modified glassy carbon electrode GO/Nafion/GCE, placing the GO/Nafion/GCE in 10mM phosphate buffer saline with pH of 5.0 for electrochemical reduction, and measuring by using i-t amperes, wherein the initial potential is-1.3V, the sampling interval is 0.1s, and the standing time is 0s to obtain reduced graphene oxide/Nafion modified glassy carbon electrode rGO/Nafion/GCE.
4. The electrochemical biosensor of claim 1, wherein the reduced graphene oxide is prepared by the steps of: dispersing flaky single-layer graphene oxide in ultrapure water, performing ultrasonic treatment for 30-40min to obtain graphene oxide suspension, centrifuging the graphene oxide suspension at 8000-9000rpm/min for 5-10min, and collecting graphene oxide brown supernatant to obtain reduced graphene oxide rGO.
5. The electrochemical biosensor of claim 1, comprising the steps of:
(1) Preparation of tetrahedral DNA scaffold-DNAzyme/blocking strand TDS-DNAzyme/blocking er complexes
1) Preparation of azido, pyrene molecule and biotinylated TDS: mixing four single-stranded DNA T1, T2, T3 and T4 in an assembly buffer solution according to an equimolar ratio, wherein the final concentration is2 mu M, and then placing the mixture in a gradient PCR instrument for annealing at 95 ℃ for 5min, rapidly cooling to 4 ℃ and maintaining for more than 60min; the assembly buffer contains 20mM Tris-HCl and 50mM MgCl 2 The pH of the assembly buffer was 8.0;
2) Preparation of dibenzocyclooctyne-DNAzyme/blocker: dispersing DBCO-DNAzyme and a blocker chain using a hybridization buffer; mixing 6 mu M of a blocker with 4 mu M of DBCO-DNAzyme in an equal volume, then placing the mixture in a gradient PCR instrument for annealing at 95 ℃ for 2min, slowly cooling to 25 ℃ at a cooling rate of-1 ℃/min, and preparing the DBCO-DNAzyme/blocker with a final concentration of 2 mu M; the hybridization buffer contains 10mM Tris-HCl,20mM MgCl 2 And 100mM NaCl, pH7.4 of hybridization buffer;
3) Preparation of TDS-DNAzyme/blocking er complex: mixing the TDS prepared in the step 1), the DBCO-DNAzyme/blocker double chain prepared in the step 2) and the horseradish peroxidase HRP-marked streptavidin in a fixed buffer solution; incubating for 20min at 25 ℃ in a gradient PCR instrument to obtain a TDS-DNAzyme/blocker complex with the final concentration of 0.5 mu M; the fixing buffer contains 10mM Tris-HCl,1M NaCl,1mM EDTA and 10mM TECP, and the pH of the fixing buffer is 7.4;
(2) Preparing a reduced graphene oxide modified glassy carbon electrode:
1) Preparation of reduced graphene oxide: dispersing flaky single-layer graphene oxide in ultrapure water, and performing ultrasonic treatment for 30min to obtain graphene oxide suspension; centrifuging the graphene oxide suspension at 8000rpm/min for 5min, and collecting graphene oxide brown supernatant;
2) Preparing a reduced graphene oxide modified glassy carbon electrode: firstly, respectively polishing a glassy carbon electrode GCE by alumina powder, and then ultrasonically cleaning the glassy carbon electrode GCE by ultrapure water for a few seconds; nafion using 75% ethanol TM Diluting the perfluorinated resin solution to 2wt%, and mixing the graphene oxide suspension prepared in the step 1) with Nafion TM Mixing perfluorinated resin solution in equal volume to obtain graphene oxide/Nafion mixed solution; drop graphene oxide/Nafion mixture in pretreatmentDrying the GCE surface in a vacuum dryer at 25 ℃ to obtain a graphene oxide/Nafion modified glassy carbon electrode GO/Nafion/GCE; finally, GO/Nafion/GCE was placed in 10mm pH5.0 phosphate buffered saline for electrochemical reduction, measured using i-t amps, with an initial potential of-1.3V, sampling interval of 0.1s, and rest time of 0s; the prepared reduced graphene oxide/Nafion modified glassy carbon electrode rGO/Nafion/GCE is stored at room temperature;
(3) Constructing an electrochemical biosensor for detecting acute myocardial infarction related microRNA-499:
1) Dropwise adding 0.5 mu M TDS-DNAzyme/blocker complex on the rGO/Nafion/GCE surface in the step (2), and incubating for 2h at room temperature;
2) And (3) rinsing the electrode obtained in the step (1) with an assembly buffer solution to obtain the electrochemical biosensor for detecting microRNA-499.
6. A method for detecting microRNA-499 based on the electrochemical biosensor of any one of claims 1-5, comprising the steps of:
(1) Dripping 10L of microRNA-499 dispersed in an assembly buffer onto the surface of TDS-DNAzyme/blocker/rGO/Nafion/GCE, incubating at room temperature for 20min, rinsing with the assembly buffer, immersing the electrode in a buffer containing 0.66mM TMB and 3.0mM H 2 O 2 The i-t curve was measured in HAc-NaAc buffer at pH4.0 at 0.1M of the mixture;
(2) Drawing a working curve according to the linear relation between the current obtained in the step (1) and the microRNA-499 concentration logarithmic value;
(3) And (3) detecting a sample to be detected by using the electrochemical sensor, and calculating the obtained current value through the working curve prepared in the step (2) to obtain the microRNA-499 concentration of the sample to be detected.
CN202310684164.XA 2023-06-11 2023-06-11 Electrochemical biosensor for acute myocardial infarction related microRNA detection Pending CN116698930A (en)

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