WO2022095373A1 - Co-reactant self-generating and signal-amplifying electrochemiluminescence system for detecting mirna - Google Patents
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/682—Signal amplification
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6825—Nucleic acid detection involving sensors
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
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Definitions
- the invention belongs to the technical field of miRNA detection, in particular to a self-generated co-reactant signal amplification electrochemiluminescence system for detecting miRNA.
- MicroRNA as a non-coding RNA, plays an important regulatory function in physiological or pathological processes such as embryonic development, apoptosis, gene transcription, protein modification, signal transmission, immunity and disease occurrence. Therefore, the highly sensitive and accurate analysis of miRNA has become the focus and hotspot of research.
- Electrochemiluminescence detection technology has the advantages of low background signal and high stability, and is widely used in the detection of tumor markers such as nucleic acids and proteins.
- the traditional electrochemiluminescence method requires additional co-reactants to be added in the solution, and the performance of sensitivity and other aspects also needs to be improved.
- the purpose of the present invention is to provide a self-generated co-reactant signal amplification electrochemiluminescence system for detecting miRNA, by which the electrochemiluminescence detection of target miRNA can be realized with high sensitivity and high specificity.
- the invention provides a self-generated co-reactant signal amplification electrochemiluminescence system for detecting miRNA, the system comprises a sensor substrate, an immobilized probe and a mercaptoethanol sealing plate connected in sequence; the system further comprises a biotin-modified Probes, streptavidin and glucose oxidase modified gold nanoparticles, heme Hemin and luminol;
- the sensor substrate comprises an FTO electrode electroplated with a layer of Au nanoflowers
- the immobilized probe includes hairpin DNA
- the immobilized probe and the biotin-modified probe can undergo a substitution reaction.
- the immobilized probe comprises hairpin DNA as shown in SEQ ID NO.1.
- the probe modified with biotin includes the probe of the nucleotide sequence shown in SEQ ID NO.2.
- the preparation method of the sensor substrate includes: wrapping the cleaned and dried FTO glass with perforated tape, and immersing it in an electrolyte for electroplating; the electrolyte includes 0.05M phosphate buffer, 0.054M KCl and 5mM HAuCl 4 .
- the cleaning includes cutting the FTO glass into a rectangle with a width and length of 1cm ⁇ 4cm, washing with deionized water, then immersing it in boiling 2-propanol containing 2M KOH for 20min, washing with water and then ultrasonically cleaning for 15min .
- the electroplating is performed by cyclic voltammetry, and the parameters are set as: voltage -0.8-0.3V, 50 cycles, scanning rate of 0.1V/s; heating the solution to 60°C and maintaining a nitrogen atmosphere.
- the preparation method of the streptavidin and glucose oxidase-modified gold nanoparticles comprises: mixing the gold nanoparticles solution with equal volumes of the glucose oxidase solution and the streptavidin solution, and keeping it at 4°C 24h, the precipitate obtained after centrifugation was dispersed in buffer to obtain gold nanoparticles modified with streptavidin and glucose oxidase.
- the volume of the gold nanoparticles solution is 500 ⁇ L
- the particle size of the gold nanoparticles in the solution is 30 nm
- the concentration of the glucose oxidase solution is 15 mg/mL
- the concentration of the streptavidin solution is 1 mg/mL.
- the present invention provides a self-generated co-reactant signal amplification electrochemiluminescence system for detecting miRNA, which utilizes a large surface area nano-sensing substrate and constructs a capture probe with a modified hairpin structure to realize the co-reactant (H 2 O 2 ), and combined with the isothermal signal amplification strategy of strand displacement nucleic acid to amplify the signal, the electrochemiluminescence detection of target miRNA can be realized with high sensitivity and high specificity. Specifically, when the target miRNA appears in the solution, the hairpin structure of the fixed probe is opened, and in the presence of the biotin probe, the opened hairpin DNA undergoes a substitution reaction with the biotin probe, and then the target RNA is replaced.
- RNA is recycled in the presence of biotin probe, and finally a large amount of double-stranded DNA with biotin is generated at the sensing interface, which is combined with gold nanoparticles with avidin and glucose oxidase to catalyze the reaction in solution.
- Fig. 1 is a scanning (SEM) electron microscope image after electrodeposition of gold nanoflowers on the surface of FTO electrode;
- Figure 2 is a schematic diagram of the self-generated coreactant signal amplification type ECL sensing method for miRNA detection
- Figure 3 shows the ECL response curve and working curve
- Figure 5 shows the detection of miRNA in different tumor cell extracts.
- the invention provides a self-generated co-reactant signal amplification electrochemiluminescence system for detecting miRNA, the system includes a sensor substrate, an immobilized probe and a mercaptoethanol sealing plate connected in sequence; the system also includes a biotin modified Probes, streptavidin and glucose oxidase-modified gold nanoparticles, heme Hemin, glucose and luminol;
- the sensor substrate comprises an FTO electrode electroplated with a layer of Au nanoflowers
- the immobilized probe includes hairpin DNA
- the immobilized probe and the biotin-modified probe can undergo a substitution reaction.
- the sensor substrate, the fixed probe and the mercaptoethanol sealing plate constitute the ECL sensor. Then a layer of hairpin DNA was assembled as an immobilized probe, and then mercaptohexanol (MCH) was added to seal the plate.
- the preparation method of the sensor substrate of the present invention preferably includes: wrapping the cleaned and dried FTO glass with perforated tape, and immersing it in an electrolyte for electroplating; the electrolyte includes 0.05M phosphate buffer, 0.054M KCl and 5mM HAuCl 4 .
- the cleaning according to the present invention preferably includes cutting the FTO glass into rectangles with a width and length of 1cm ⁇ 4cm, washing with deionized water to remove tiny fragments, and then immersing it in boiling 2-propanol containing 2M KOH for 20min. After washing with water Ultrasonic cleaning was performed for 15 min.
- the drying temperature in the present invention is preferably 60°C.
- a hole punch is preferably used to punch holes on the adhesive tape, and after wrapping the above-mentioned FTO glass (electrode) with the adhesive tape, a surface with the size of a circular hole is reserved for depositing gold nanoflowers.
- N 2 is preferably charged into the electrolyte to remove oxygen before the electroplating is performed.
- the present invention preferably uses cyclic voltammetry to carry out the electroplating, and the parameters are set as: voltage -0.8-0.3V, 50 cycles, scanning rate of 0.1V/s; heating the solution to 60°C and maintaining a nitrogen atmosphere. With the progress of the reaction, the gold nanoflowers grow on the surface of the FTO, and the colorless glass gradually turns into gold, which indicates that the gold nanoflowers are successfully deposited on the FTO, and the FTO-Au electrode is obtained.
- the preparation method of streptavidin and glucose oxidase-modified gold nanoparticles (GOD-Au-SA) of the present invention preferably includes: mixing the gold nanoparticles solution with equal volumes of glucose solution and streptavidin solution after mixing , kept at 4° C. for 24 h, and the precipitate obtained after centrifugation was dispersed in the buffer to obtain gold nanoparticles modified with streptavidin and glucose oxidase.
- the volume of the gold nanoparticles solution of the present invention is preferably 500 ⁇ L, the particle size of the gold nanoparticles in the solution is preferably 30 nm, the concentration of the glucose oxidase solution is preferably 15 mg/mL, and the concentration of the streptavidin solution is preferably 1 mg/mL.
- the preparation method of the ECL sensor of the present invention preferably includes: drop-coating 10 ⁇ L of 10 ⁇ M immobilized probe on the prepared FTO-Au electrode, overnight at room temperature, and then soaking the electrode in a PBS solution for 2 minutes to remove the unbonded probe Excessive immobilization probe. Subsequently, the remaining reaction sites on the electrode were blocked with 10 ⁇ L of 1 mM mercaptoethanol (MCH), and immersed in PBS solution for 2 min after 1 h.
- MCH mM mercaptoethanol
- miRNA-21 is used as an example to construct the system, wherein the nucleotide sequence of the immobilized probe is shown in SEQ ID NO.1: TTTTTTTCAACATCAGTCTGATAAGCTAGGCGGGTTGGGTAGCTTATCAGACT; the nucleotide of the probe modified with biotin
- the sequence is preferably as shown in SEQ ID NO. 2: Biotin-5'-CTGATAAGCTACCCAACCCGCCTAGCTTATCAGACTGATGTGGGTAGGGCGGGGTTGGG-3'.
- the source of the immobilized probe and the biotin-modified probe is not particularly limited in the present invention, and it is preferably synthesized by Dalian Bao Bio.
- the principle of the system of the present invention is shown in Figure 2.
- the hairpin structure of the fixed probe is opened, and in the presence of the probe modified with biotin, The hairpin structure of the fixed probe is opened to undergo a substitution reaction with the biotin-modified probe, thereby replacing the target miRNA, so that the target miRNA is recycled in the presence of the biotin-modified probe, realizing signal amplification function to improve the detection sensitivity.
- the nucleic acid strand replacement reaction has extremely high sequence accuracy requirements for nucleic acid sequences, which improves the specificity and selectivity of detection.
- the present invention also provides the application of the above system in detecting miRNA.
- the miRNA of the present invention preferably includes miRNA-21 (SEQ ID NO. 3): 5'-UAGCUUAUCAGACUGAUGUUGA-3'.
- a coreactant signal amplification electrochemiluminescence system for detecting miRNA provided by the present invention will be described in detail below with reference to the examples, but they should not be construed as limiting the protection scope of the present invention.
- the instruments used in this method are: micro UV spectrophotometer (Thermo NanoDrop-2000C, USA), transmission electron microscope (JEM-2100, Hitachi), scanning electron microscope (Zeiss Supra 55), CHI660B (Shanghai Chenhua Instrument Co., Ltd., China ) Electrochemical Workstation, MPI-E Multifunctional Electrochemical and Chemiluminescence Analysis System (Xi'an, China).
- a three-electrode system was used in the experiment, consisting of FTO (working electrode), SCE electrode (reference electrode) and platinum wire electrode (counter electrode), and the photomultiplier tube voltage was set to -600V (PMT).
- the experimental reagents used in this method are: KCl, luminol, HAuCl 4 .4H 2 O, MCH purchased from Aladdin-Reagent (Shanghai, China), MgCl 2 .6H 2 O, hydrogen peroxide, hemin (Hemin), tris(2- Carbonyl ethyl) phosphate hydrochloride (TCEP), glucose oxidase (GOD), streptavidin (SA), and glucose (sigma) were purchased from Huji (Shanghai, China), and DNA and RNA were synthesized by Dalian Bao Biotechnology. The sequence is as follows:
- Immobilized probe SEQ ID NO.1, DNA1: SH-5'-TTTTTTTCAACATCAGTCTGATAAGCTAGGCGGGTTGGGTAGCTTATCAGACT-3';
- Biotin-modified probe (SEQ ID NO.2, DNA2): Biotin-5'-CTGATAAGCTACCCAACCCGCCTAGCTTATCAGACTGATGTGGGTAGGGCGGGTTGGG-3';
- miRNA-21 (SEQ ID NO.3): 5'-UAGCUUAUCAGACUGAUGUUGA-3';
- Single base mismatch miRNA-21 (SEQ ID NO.4): 5'-UAGCUUAUCACACUGAUGUUGA-3';
- Three base mismatch miRNA-21 (SEQ ID NO.5): 5'-UAGCUUAUCACUGUGAUGUUGA-3';
- Non-complementary sequence (SEQ ID NO. 6): 5'-GUAUGGUAAGCUGAAUACAACU-3'.
- a self-generated coreactant signal amplification electrochemiluminescence system for detecting miRNA provided by the present invention will be described in detail below with reference to the examples, but they should not be construed as limiting the protection scope of the present invention.
- the FTO glass was cut into 1 cm x 4 cm pieces and washed with deionized water to remove tiny fragments. They were all immersed in boiling 2-propanol containing 2M KOH for 20 min, washed with water and then ultrasonically cleaned for 15 min and dried at 60°C. The tape was punched with a hole punch and then wrapped around the FTO electrode, leaving a surface with the size of a round hole for depositing gold nanoparticles.
- Cyclic voltammetry parameters were set as: voltage -0.8-0.3 V (VS SCE), 50 cycles, and a scan rate of 0.1 V/s.
- the solution was heated to 60 °C and kept under nitrogen atmosphere. As the reaction progressed, the gold nanoflowers grew on the surface of the FTO, and the colorless glass gradually turned into gold, indicating that the gold nanoflowers were successfully deposited on the FTO. As shown in Figure 1.
- Human cervical cancer cells Hela cells and human breast cancer cells MCF-7 cells used in the experiments were provided by KeyGEN Biotech (Nanjing, China). Cells were cultured in a medium consisting of DMEM, 10% fetal bovine serum (FBS) and 1% streptomycin and penicillin in an incubator set at 37°C, 5% CO 2 , 95% air.
- DMEM fetal bovine serum
- FBS fetal bovine serum
- streptomycin and penicillin in an incubator set at 37°C, 5% CO 2 , 95% air.
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Abstract
The present invention provides a co-reactant self-generating and signal-amplifying electrochemiluminescence system for detecting miRNA, and relates to the technical field of miRNA detection. The system provided by the present invention realizes the self-generation of a co-reactant in the presence of a target miRNA by utilizing a nano sensing substrate with a large surface area and by means of the construction of a capture probe with a modified hairpin structure, and amplifies signals in combination with a strand displacement nucleic acid isothermal signal amplification policy, so that high-sensitivity and high-specificity electrochemiluminescence detection of the target miRNA can be realized. When the system provided by the present invention is used for detecting an miRNA, the detection accuracy and sensitivity can be improved, and human interference factors are also eliminated.
Description
本申请要求于2020年11月06日提交中国专利局、申请号为2020112286217、发明名称为“一种检测miRNA的自产生共反应剂信号放大电化学发光体系”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。This application claims the priority of the Chinese patent application filed on November 06, 2020, with the application number of 2020112286217 and the invention titled "A Self-Generating Coreactant Signal Amplification Electrochemiluminescence System for Detecting miRNA", which The entire contents of this application are incorporated by reference.
本发明属于miRNA检测技术领域，具体涉及一种检测miRNA的自产生共反应剂信号放大电化学发光体系。The invention belongs to the technical field of miRNA detection, in particular to a self-generated co-reactant signal amplification electrochemiluminescence system for detecting miRNA.
MicroRNA(miRNA)作为一种非编码RNA(non-coding RNA)在胚胎发育、细胞凋亡、基因转录、蛋白修饰、信号传递、机体免疫及疾病发生等生理或病理过程中发挥重要的调控功能。因此，miRNA的高灵敏、准确分析已成为人们研究的重点与热点。MicroRNA (miRNA), as a non-coding RNA, plays an important regulatory function in physiological or pathological processes such as embryonic development, apoptosis, gene transcription, protein modification, signal transmission, immunity and disease occurrence. Therefore, the highly sensitive and accurate analysis of miRNA has become the focus and hotspot of research.
但目前的分析方法仍面临诸多问题，例如探针的稳定性和灵敏度差、操作复杂、噪音大、成本高等，如何实现高灵敏地miRNA的准确分析成为研究的趋势。电化学发光检测技术具有背景信号低，稳定性高等优点，被广泛地应用于核酸、蛋白等肿瘤标志物的检测。传统的电化学发光方法需要在溶液中另外添加共反应剂，且灵敏度等方面的表现也有待提高。However, the current analysis methods still face many problems, such as poor stability and sensitivity of probes, complicated operation, high noise, and high cost. How to achieve accurate analysis of miRNA with high sensitivity has become a research trend. Electrochemiluminescence detection technology has the advantages of low background signal and high stability, and is widely used in the detection of tumor markers such as nucleic acids and proteins. The traditional electrochemiluminescence method requires additional co-reactants to be added in the solution, and the performance of sensitivity and other aspects also needs to be improved.
发明内容SUMMARY OF THE INVENTION
有鉴于此，本发明的目的在于提供一种检测miRNA的自产生共反应剂信号放大电化学发光体系，利用所述体系，可实现目标miRNA的高灵敏、高特异性的电化学发光检测。In view of this, the purpose of the present invention is to provide a self-generated co-reactant signal amplification electrochemiluminescence system for detecting miRNA, by which the electrochemiluminescence detection of target miRNA can be realized with high sensitivity and high specificity.
为了实现上述发明目的，本发明提供以下技术方案：In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:
本发明提供了一种检测miRNA的自产生共反应剂信号放大电化学发光体系，所述体系包括依次连接的传感器基底、固定探针和巯基乙醇封板； 所述体系中还包括修饰有生物素的探针、链霉亲和素和葡萄糖氧化酶修饰的纳米金、血红素Hemin和luminol；The invention provides a self-generated co-reactant signal amplification electrochemiluminescence system for detecting miRNA, the system comprises a sensor substrate, an immobilized probe and a mercaptoethanol sealing plate connected in sequence; the system further comprises a biotin-modified Probes, streptavidin and glucose oxidase modified gold nanoparticles, heme Hemin and luminol;
所述传感器基底包括电镀一层Au纳米花的FTO电极；The sensor substrate comprises an FTO electrode electroplated with a layer of Au nanoflowers;
所述固定探针包括发卡DNA；The immobilized probe includes hairpin DNA;
所述固定探针和修饰有生物素的探针可发生取代反应。The immobilized probe and the biotin-modified probe can undergo a substitution reaction.
优选的，所述固定探针包括如SEQ ID NO.1所示的发卡DNA。Preferably, the immobilized probe comprises hairpin DNA as shown in SEQ ID NO.1.
优选的，修饰有生物素的探针包括如SEQ ID NO.2所示的核苷酸序列的探针。Preferably, the probe modified with biotin includes the probe of the nucleotide sequence shown in SEQ ID NO.2.
优选的，所述传感器基底的制备方法，包括：利用打孔胶带包裹清洗后干燥的FTO玻璃，浸入电解质中进行电镀；所述电解质中包括0.05M磷酸盐缓冲液、0.054M KCl和5mM HAuCl
4。
Preferably, the preparation method of the sensor substrate includes: wrapping the cleaned and dried FTO glass with perforated tape, and immersing it in an electrolyte for electroplating; the electrolyte includes 0.05M phosphate buffer, 0.054M KCl and 5mM HAuCl 4 .
优选的，所述清洗包括将FTO玻璃切成宽和长为1cm×4cm的矩形后，用去离子水洗涤，再浸入含有2M KOH的沸腾2-丙醇中20min，水洗涤后再超声清洗15min。Preferably, the cleaning includes cutting the FTO glass into a rectangle with a width and length of 1cm×4cm, washing with deionized water, then immersing it in boiling 2-propanol containing 2M KOH for 20min, washing with water and then ultrasonically cleaning for 15min .
优选的，利用循环伏安法进行所述电镀，参数设定为：电压-0.8～0.3V、循环50次、扫描速率为0.1V/s；加热溶液至60℃并保持氮气氛围。Preferably, the electroplating is performed by cyclic voltammetry, and the parameters are set as: voltage -0.8-0.3V, 50 cycles, scanning rate of 0.1V/s; heating the solution to 60°C and maintaining a nitrogen atmosphere.
优选的，所述链霉亲和素和葡萄糖氧化酶修饰的纳米金的制备方法，包括：将纳米金溶液与等体积的葡萄糖氧化酶溶液和链霉亲和素溶液混合后，于4℃保持24h，将离心后得到的沉淀分散于缓冲液中，得链霉亲和素和葡萄糖氧化酶修饰的纳米金。Preferably, the preparation method of the streptavidin and glucose oxidase-modified gold nanoparticles comprises: mixing the gold nanoparticles solution with equal volumes of the glucose oxidase solution and the streptavidin solution, and keeping it at 4°C 24h, the precipitate obtained after centrifugation was dispersed in buffer to obtain gold nanoparticles modified with streptavidin and glucose oxidase.
优选的，所述纳米金溶液的体积为500μL，溶液中纳米金的粒径为30nm，葡萄糖氧化酶溶液的浓度为15mg/mL，链霉亲和素溶液的浓度为1mg/mL。Preferably, the volume of the gold nanoparticles solution is 500 μL, the particle size of the gold nanoparticles in the solution is 30 nm, the concentration of the glucose oxidase solution is 15 mg/mL, and the concentration of the streptavidin solution is 1 mg/mL.
本发明提供了一种检测miRNA的自产生共反应剂信号放大电化学发光体系，利用大表面积的纳米传感基底，通过构建修饰发卡结构的捕获探针实现了靶标miRNA存在时共反应剂(H
2O
2)的自产生，并且结合链置换核酸等温信号放大策略对信号进行放大，可实现目标miRNA的高灵敏、高特异性的电化学发光检测。具体的当溶液中出现靶miRNA时，固定探针的发卡结构被打开，在生物素探针的存在下，打开的发卡DNA与生物 素探针发生取代反应，进而替换出靶RNA，由此靶RNA在生物素探针的存在下发生循环利用，最终在传感界面产生大量的带有生物素的双链DNA，并与带有亲和素和葡萄糖氧化酶的纳米金结合，催化溶液中的葡萄糖和氧气反应，产生大量的双氧水，并且打开的发卡DNA末端含有大量的G四联体结构，并与溶液中的血红素Hemin形成Hemin/G四联体结构，该结构具有类过氧化物酶的作用，能够催化双氧水产生过氧自由基，产生的过氧自由基与基底溶液中的Luminol反应产生ECL信号，实现了miRNA的高灵敏检测。利用本发明所述体系进行miRNA的检测时，可提高检测的准确性和灵敏度，同时排除人为干扰因素。
The present invention provides a self-generated co-reactant signal amplification electrochemiluminescence system for detecting miRNA, which utilizes a large surface area nano-sensing substrate and constructs a capture probe with a modified hairpin structure to realize the co-reactant (H 2 O 2 ), and combined with the isothermal signal amplification strategy of strand displacement nucleic acid to amplify the signal, the electrochemiluminescence detection of target miRNA can be realized with high sensitivity and high specificity. Specifically, when the target miRNA appears in the solution, the hairpin structure of the fixed probe is opened, and in the presence of the biotin probe, the opened hairpin DNA undergoes a substitution reaction with the biotin probe, and then the target RNA is replaced. RNA is recycled in the presence of biotin probe, and finally a large amount of double-stranded DNA with biotin is generated at the sensing interface, which is combined with gold nanoparticles with avidin and glucose oxidase to catalyze the reaction in solution. Glucose and oxygen react to generate a large amount of hydrogen peroxide, and the open hairpin DNA end contains a large number of G-quadruplex structures, and forms a Hemin/G-quadruplex structure with the heme Hemin in solution, which has a peroxidase-like structure It can catalyze the generation of peroxy radicals from hydrogen peroxide, and the generated peroxy radicals react with Luminol in the substrate solution to generate ECL signals, realizing highly sensitive detection of miRNAs. When the system of the present invention is used for the detection of miRNA, the accuracy and sensitivity of the detection can be improved, and artificial interference factors can be eliminated at the same time.
图1为FTO电极表面电沉积金纳米花后扫描(SEM)电镜图；Fig. 1 is a scanning (SEM) electron microscope image after electrodeposition of gold nanoflowers on the surface of FTO electrode;
图2为自产生共反应剂信号放大型ECL传感方法对miRNA检测的原理图；Figure 2 is a schematic diagram of the self-generated coreactant signal amplification type ECL sensing method for miRNA detection;
图3为ECL响应曲线和工作曲线；Figure 3 shows the ECL response curve and working curve;
图4为检测选择性曲线及稳定性曲线；Fig. 4 is detection selectivity curve and stability curve;
图5为不同肿瘤细胞提取液中miRNA的检测。Figure 5 shows the detection of miRNA in different tumor cell extracts.
下面结合实施例和附图对本发明进一步说明。The present invention will be further described below with reference to the embodiments and accompanying drawings.
本发明提供了一种检测miRNA的自产生共反应剂信号放大电化学发光体系，所述体系包括依次连接的传感器基底、固定探针和巯基乙醇封板；所述体系中还包括修饰有生物素的探针、链霉亲和素和葡萄糖氧化酶修饰的纳米金、血红素Hemin，葡萄糖和luminol；The invention provides a self-generated co-reactant signal amplification electrochemiluminescence system for detecting miRNA, the system includes a sensor substrate, an immobilized probe and a mercaptoethanol sealing plate connected in sequence; the system also includes a biotin modified Probes, streptavidin and glucose oxidase-modified gold nanoparticles, heme Hemin, glucose and luminol;
所述传感器基底包括电镀一层Au纳米花的FTO电极；The sensor substrate comprises an FTO electrode electroplated with a layer of Au nanoflowers;
所述固定探针包括发卡DNA；The immobilized probe includes hairpin DNA;
所述固定探针和修饰有生物素的探针可发生取代反应。The immobilized probe and the biotin-modified probe can undergo a substitution reaction.
本发明所述体系中传感器基底、固定探针和巯基乙醇封板构成ECL传感器，其结构优选为在FTO(掺杂氟的SnO
2导电玻璃)电极表面电镀一层Au纳米花作为传感器的基底，然后组装一层发卡DNA作为固定探针，再加入巯基己醇(MCH)封板。本发明所述传感器基底的制备方法，优选包括：利用打孔胶带包裹清洗后干燥的FTO玻璃，浸入电解质中进行电 镀；所述电解质中包括0.05M磷酸盐缓冲液、0.054M KCl和5mM HAuCl
4。本发明所述清洗优选包括将FTO玻璃切成宽和长为1cm×4cm的矩形后，用去离子水洗涤以除去微小碎片，再浸入含有2M KOH的沸腾2-丙醇中20min，水洗涤后再超声清洗15min。本发明所述干燥的温度优选为60℃。
In the system of the present invention, the sensor substrate, the fixed probe and the mercaptoethanol sealing plate constitute the ECL sensor. Then a layer of hairpin DNA was assembled as an immobilized probe, and then mercaptohexanol (MCH) was added to seal the plate. The preparation method of the sensor substrate of the present invention preferably includes: wrapping the cleaned and dried FTO glass with perforated tape, and immersing it in an electrolyte for electroplating; the electrolyte includes 0.05M phosphate buffer, 0.054M KCl and 5mM HAuCl 4 . The cleaning according to the present invention preferably includes cutting the FTO glass into rectangles with a width and length of 1cm×4cm, washing with deionized water to remove tiny fragments, and then immersing it in boiling 2-propanol containing 2M KOH for 20min. After washing with water Ultrasonic cleaning was performed for 15 min. The drying temperature in the present invention is preferably 60°C.
本发明优选用打孔器在胶带上打孔，用所述胶带包裹上述FTO玻璃(电极)后，留出圆孔大小的面用于沉积纳米金花。本发明在进行所述电镀前优选向所述电解质中充入N
2，以除去氧气。本发明优选利用循环伏安法进行所述电镀，参数设定为：电压-0.8～0.3V、循环50次、扫描速率为0.1V/s；加热溶液至60℃并保持氮气氛围。随着反应的进行的金纳米花在FTO表面上生长，无色玻璃逐渐转变成金色，表明金纳米花成功地沉积在FTO上，得FTO-Au电极。
In the present invention, a hole punch is preferably used to punch holes on the adhesive tape, and after wrapping the above-mentioned FTO glass (electrode) with the adhesive tape, a surface with the size of a circular hole is reserved for depositing gold nanoflowers. In the present invention, N 2 is preferably charged into the electrolyte to remove oxygen before the electroplating is performed. The present invention preferably uses cyclic voltammetry to carry out the electroplating, and the parameters are set as: voltage -0.8-0.3V, 50 cycles, scanning rate of 0.1V/s; heating the solution to 60°C and maintaining a nitrogen atmosphere. With the progress of the reaction, the gold nanoflowers grow on the surface of the FTO, and the colorless glass gradually turns into gold, which indicates that the gold nanoflowers are successfully deposited on the FTO, and the FTO-Au electrode is obtained.
本发明所述链霉亲和素和葡萄糖氧化酶修饰的纳米金(GOD-Au-SA)的制备方法，优选包括：将纳米金溶液与等体积的葡萄糖溶液和链霉亲和素溶液混合后，于4℃保持24h，将离心后得到的沉淀分散于缓冲液中，得链霉亲和素和葡萄糖氧化酶修饰的纳米金。本发明所述纳米金溶液的体积优选为500μL，溶液中纳米金的粒径优选为30nm，葡萄糖氧化酶溶液的浓度优选为15mg/mL，链霉亲和素溶液的浓度优选为1mg/mL。The preparation method of streptavidin and glucose oxidase-modified gold nanoparticles (GOD-Au-SA) of the present invention preferably includes: mixing the gold nanoparticles solution with equal volumes of glucose solution and streptavidin solution after mixing , kept at 4° C. for 24 h, and the precipitate obtained after centrifugation was dispersed in the buffer to obtain gold nanoparticles modified with streptavidin and glucose oxidase. The volume of the gold nanoparticles solution of the present invention is preferably 500 μL, the particle size of the gold nanoparticles in the solution is preferably 30 nm, the concentration of the glucose oxidase solution is preferably 15 mg/mL, and the concentration of the streptavidin solution is preferably 1 mg/mL.
本发明所述ECL传感器的制备方法，优选包括：在制备的FTO-Au电极上滴涂10μL 10μM的固定探针，室温下过夜，之后将电极浸润在PBS溶液中2min以除去未能键合的过量固定探针。随后用10μL 1mM巯基乙醇封板(MCH)封闭电极上剩余的反应位点，1h后浸入PBS溶液中2min。The preparation method of the ECL sensor of the present invention preferably includes: drop-coating 10 μL of 10 μM immobilized probe on the prepared FTO-Au electrode, overnight at room temperature, and then soaking the electrode in a PBS solution for 2 minutes to remove the unbonded probe Excessive immobilization probe. Subsequently, the remaining reaction sites on the electrode were blocked with 10 μL of 1 mM mercaptoethanol (MCH), and immersed in PBS solution for 2 min after 1 h.
本发明实施例中以miRNA-21为例，构建所述体系，其中所述固定探针的核苷酸序列如SEQ ID NO.1所示：TTTTTTTCAACATCAGTCTGATAAGCTAGGCGGGTTGGGTAGCTTATCAGACT；修饰有生物素的探针的核苷酸序列优选如SEQ ID NO.2所示：Biotin-5’-CTGATAAGCTACCCAACCCGCCTAGCTTATCAGACTGATGTGGGTAGGGCGGGTTGGG-3’。本发明对所述固定探针和修饰有生物素的探针的来源并没有特殊限定，优选由大连宝生物合成。In the examples of the present invention, miRNA-21 is used as an example to construct the system, wherein the nucleotide sequence of the immobilized probe is shown in SEQ ID NO.1: TTTTTTTCAACATCAGTCTGATAAGCTAGGCGGGTTGGGTAGCTTATCAGACT; the nucleotide of the probe modified with biotin The sequence is preferably as shown in SEQ ID NO. 2: Biotin-5'-CTGATAAGCTACCCAACCCGCCTAGCTTATCAGACTGATGTGGGTAGGGCGGGGTTGGG-3'. The source of the immobilized probe and the biotin-modified probe is not particularly limited in the present invention, and it is preferably synthesized by Dalian Bao Bio.
本发明所述体系的原理如图2所示，通过探针DNA的设计，当溶液中出现靶标miRNA时，固定探针的发卡结构被打开，在修饰有生物素的探针存在的情况下，打开固定探针的发卡结构，从而与修饰有生物素的探针发生取代反应，进而替换出靶miRNA，由此靶miRNA在修饰有生物素的探针的存在时被循环利用，实现了信号放大作用，提高了检测的灵敏度。同时利用核酸链替换反应对核酸序列极高的序列准确性要求，提高了检测的特异性和选择性。核酸循环反应之后，最终在传感界面产生大量的带有生物素的双链DNA，与带有亲和素和葡萄糖氧化酶的纳米金结合，金纳米颗粒表面的葡萄糖氧化酶催化溶液中的葡萄糖和氧气反应，产生大量的双氧水，并且打开的发卡DNA末端含有大量的G四联体结构并与溶液中的血红素Hemin形成Hemin/G四联体结构，该结构具有类过氧化物酶的作用，能够催化双氧水产生ECL共反应剂过氧自由基，构建了自产生共反应剂的ECL检测体系，提高了检测的操作便利，同时排除了人为干扰因素。The principle of the system of the present invention is shown in Figure 2. Through the design of the probe DNA, when the target miRNA appears in the solution, the hairpin structure of the fixed probe is opened, and in the presence of the probe modified with biotin, The hairpin structure of the fixed probe is opened to undergo a substitution reaction with the biotin-modified probe, thereby replacing the target miRNA, so that the target miRNA is recycled in the presence of the biotin-modified probe, realizing signal amplification function to improve the detection sensitivity. At the same time, the nucleic acid strand replacement reaction has extremely high sequence accuracy requirements for nucleic acid sequences, which improves the specificity and selectivity of detection. After the nucleic acid recycling reaction, a large amount of double-stranded DNA with biotin is finally generated at the sensing interface, which is combined with gold nanoparticles with avidin and glucose oxidase. The glucose oxidase on the surface of the gold nanoparticles catalyzes the glucose in the solution. Reacts with oxygen to produce a large amount of hydrogen peroxide, and the open hairpin DNA end contains a large number of G-quadruplex structures and forms a Hemin/G-quadruplex structure with the heme Hemin in solution, which has a peroxidase-like effect. , which can catalyze the generation of ECL co-reactant peroxy radicals by hydrogen peroxide, and build an ECL detection system that generates a co-reactant by itself, which improves the convenience of detection and eliminates human interference factors.
本发明还提供了上述体系在检测miRNA中的应用。The present invention also provides the application of the above system in detecting miRNA.
本发明所述miRNA优选包括miRNA-21(SEQ ID NO.3)：5’-UAGCUUAUCAGACUGAUGUUGA-3’。The miRNA of the present invention preferably includes miRNA-21 (SEQ ID NO. 3): 5'-UAGCUUAUCAGACUGAUGUUGA-3'.
本发明在利用上述体系在检测miRNA-21时，优选的取10μL microRNA-21、10μL 10μM生物素修饰的探针滴加在电极表面，37℃孵育2h后，PBS溶液清洗。加入10μL制备的GOD-Au-SA、10μL hemin(10mM)37℃孵育2h后PBS溶液处理2min，最后将组装好的电极浸入在0.01M PBS(0.1mM luminol，20mM葡萄糖)溶液中反应1h后再进行ECL测试。In the present invention, when using the above system to detect miRNA-21, preferably 10 μL of microRNA-21 and 10 μL of 10 μM biotin-modified probe are dropped on the electrode surface, incubated at 37°C for 2 hours, and washed with PBS solution. Add 10 μL of prepared GOD-Au-SA, 10 μL hemin (10 mM) and incubate at 37°C for 2 h and then treat with PBS solution for 2 min. Finally, immerse the assembled electrode in 0.01 M PBS (0.1 mM luminol, 20 mM glucose) solution for 1 h and then react for 1 h. Perform ECL testing.
下面结合实施例对本发明提供的一种检测miRNA的共反应剂信号放大电化学发光体系进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。A coreactant signal amplification electrochemiluminescence system for detecting miRNA provided by the present invention will be described in detail below with reference to the examples, but they should not be construed as limiting the protection scope of the present invention.
本方法所用仪器装置为：微量紫外分光光度计(Thermo NanoDrop-2000C，美国)，透射电子显微镜(JEM-2100，日立)，扫描电子显微镜(Zeiss Supra 55)，CHI660B(上海辰华仪器公司，中国)电化学工作站，MPI-E多功能电化学和化学发光分析系统(中国西安)。The instruments used in this method are: micro UV spectrophotometer (Thermo NanoDrop-2000C, USA), transmission electron microscope (JEM-2100, Hitachi), scanning electron microscope (Zeiss Supra 55), CHI660B (Shanghai Chenhua Instrument Co., Ltd., China ) Electrochemical Workstation, MPI-E Multifunctional Electrochemical and Chemiluminescence Analysis System (Xi'an, China).
实验中采用三电极系统，FTO(工作电极)，SCE电极(参比电极)和铂丝电极(对电极)组成，光电倍增管电压设置为-600V(PMT)。A three-electrode system was used in the experiment, consisting of FTO (working electrode), SCE electrode (reference electrode) and platinum wire electrode (counter electrode), and the photomultiplier tube voltage was set to -600V (PMT).
本方法所用实验试剂为：KCl、luminol、HAuCl
4.4H
2O、MCH购于Aladdin-Reagent(中国上海)，MgCl
2.6H
2O、双氧水、氯高铁血红素(Hemin)、三(2-羰基乙基)磷盐酸盐(TCEP)、葡萄糖氧化酶(GOD)、链霉亲和素(SA)、葡萄糖(sigma)购于沪剂(中国上海)，DNA和RNA由大连宝生物合成，序列如下：
The experimental reagents used in this method are: KCl, luminol, HAuCl 4 .4H 2 O, MCH purchased from Aladdin-Reagent (Shanghai, China), MgCl 2 .6H 2 O, hydrogen peroxide, hemin (Hemin), tris(2- Carbonyl ethyl) phosphate hydrochloride (TCEP), glucose oxidase (GOD), streptavidin (SA), and glucose (sigma) were purchased from Huji (Shanghai, China), and DNA and RNA were synthesized by Dalian Bao Biotechnology. The sequence is as follows:
固定探针(SEQ ID NO.1，DNA1)：SH-5’-TTTTTTTCAACATCAGTCTGATAAGCTAGGCGGGTTGGGTAGCTTATCAGACT-3’；Immobilized probe (SEQ ID NO.1, DNA1): SH-5'-TTTTTTTCAACATCAGTCTGATAAGCTAGGCGGGTTGGGTAGCTTATCAGACT-3';
修饰有生物素的探针(SEQ ID NO.2，DNA2)：Biotin-5’-CTGATAAGCTACCCAACCCGCCTAGCTTATCAGACTGATGTGGGTAGGGCGGGTTGGG-3’；Biotin-modified probe (SEQ ID NO.2, DNA2): Biotin-5'-CTGATAAGCTACCCAACCCGCCTAGCTTATCAGACTGATGTGGGTAGGGCGGGTTGGG-3';
miRNA-21(SEQ ID NO.3)：5’-UAGCUUAUCAGACUGAUGUUGA-3’；miRNA-21 (SEQ ID NO.3): 5'-UAGCUUAUCAGACUGAUGUUGA-3';
单碱基错配miRNA-21(SEQ ID NO.4)：5’-UAGCUUAUCACACUGAUGUUGA-3’；Single base mismatch miRNA-21 (SEQ ID NO.4): 5'-UAGCUUAUCACACUGAUGUUGA-3';
三碱基错配miRNA-21(SEQ ID NO.5)：5’-UAGCUUAUCACUGUGAUGUUGA-3’；Three base mismatch miRNA-21 (SEQ ID NO.5): 5'-UAGCUUAUCACUGUGAUGUUGA-3';
非互补序列(SEQ ID NO.6)：5’-GUAUGGUAAGCUGAAUACAACU-3’。Non-complementary sequence (SEQ ID NO. 6): 5'-GUAUGGUAAGCUGAAUACAACU-3'.
下面结合实施例对本发明提供的一种检测miRNA的自产生共反应剂信号放大电化学发光体系进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。A self-generated coreactant signal amplification electrochemiluminescence system for detecting miRNA provided by the present invention will be described in detail below with reference to the examples, but they should not be construed as limiting the protection scope of the present invention.
实施例1Example 1
制备FTO-Au电极Preparation of FTO-Au electrodes
将FTO玻璃切成1cm×4cm片大小，然后用去离子水洗涤以除去微小碎片。将它们全部浸入含有2M KOH沸腾的2-丙醇中保持20min，水洗涤后超声清洗15分钟并在60℃下干燥。将胶带用打孔器打孔后包裹FTO电极，留出圆孔大小的面用于沉积纳米金。将FTO玻璃片浸入5.0mL由0.05 M磷酸盐缓冲液(pH＝2)，0.054M KCl和5mM HAuCl
4组成电解质中。电镀前向溶液中通入N
2以除去氧气。循环伏安法参数设定为：电压-0.8-0.3V(VS SCE)、循环50次、扫描速率为0.1V/s。加热溶液至60℃并保持在氮气氛围下，随着反应的进行的金纳米花在FTO表面上生长，无色玻璃逐渐转变成金色，表明金纳米花成功地沉积在FTO上，其扫描电镜图如图1所示。
The FTO glass was cut into 1 cm x 4 cm pieces and washed with deionized water to remove tiny fragments. They were all immersed in boiling 2-propanol containing 2M KOH for 20 min, washed with water and then ultrasonically cleaned for 15 min and dried at 60°C. The tape was punched with a hole punch and then wrapped around the FTO electrode, leaving a surface with the size of a round hole for depositing gold nanoparticles. FTO glass slides were immersed in 5.0 mL of an electrolyte consisting of 0.05 M phosphate buffer (pH=2), 0.054 M KCl and 5 mM HAuCl 4 . N2 was bubbled through the solution to remove oxygen prior to electroplating. Cyclic voltammetry parameters were set as: voltage -0.8-0.3 V (VS SCE), 50 cycles, and a scan rate of 0.1 V/s. The solution was heated to 60 °C and kept under nitrogen atmosphere. As the reaction progressed, the gold nanoflowers grew on the surface of the FTO, and the colorless glass gradually turned into gold, indicating that the gold nanoflowers were successfully deposited on the FTO. As shown in Figure 1.
实施例2Example 2
ECL传感器的构建Construction of the ECL sensor
在500μL 30nM纳米金溶液中加入等体积的葡萄糖氧化酶(15mg/mL)与链霉亲和素(1mg/mL)4℃下保持24h后离心清洗分离，将得到的沉淀重新分散于缓冲液中得到链霉亲和素与葡萄糖氧化酶修饰的纳米金(GOD-Au-SA)，4℃保存备用。Add equal volume of glucose oxidase (15mg/mL) and streptavidin (1mg/mL) to 500μL of 30nM nano-gold solution at 4°C for 24h, then centrifuge and wash and separate, and redisperse the obtained precipitate in the buffer solution Streptavidin and glucose oxidase-modified gold nanoparticles (GOD-Au-SA) were obtained and stored at 4°C for later use.
在制备的FTO-Au电极上滴涂10μL 10μM DNA1室温下过夜，之后将电极浸润在PBS溶液中2min以除去未能键合的过量DNA1。随后用10μL 1mM MCH封闭电极上剩余的反应位点，1h后浸入PBS溶液中2min。On the prepared FTO-Au electrode, 10 μL of 10 μM DNA1 was dropped overnight at room temperature, and then the electrode was soaked in PBS solution for 2 min to remove excess DNA1 that failed to bond. The remaining reaction sites on the electrode were then blocked with 10 μL of 1 mM MCH, and immersed in PBS solution for 2 min after 1 h.
实施例3Example 3
对不同浓度的样品进行检测以及标准曲线的绘制Detection of samples with different concentrations and drawing of standard curve
取10μL不同浓度的microRNA-21(0,1fM,5fM,10fM,50fM,0.1pM,1pM,10pM)、10μL 10μM DNA2滴加在电极表面37℃孵育2h后，PBS溶液清洗。加入10μL制备的GOD-Au-SA、10μL hemin(10mM)37℃孵育2h后PBS溶液处理2min，最后将组装好的电极浸入在0.01M PBS(0.1mM luminol，20mM葡萄糖)溶液中反应1h后再进行ECL测试，测试结果如图3中A所示，随着待测液中microRNA-21浓度的增加，检测的电化学发光响应信号也出现增强趋势，根据电化学发光响应信号与目标物浓度的关系作图得标准曲线(如图3中B所示)，并根据标准曲线计算线性回归方程为y＝720.855x+11937.582，其中y为鲁米诺的电化学发光强度(图3中B的纵坐标)，x为目标物microRNA-21的浓度的对数(图3中B的横坐标)。Take 10 μL of different concentrations of microRNA-21 (0, 1fM, 5fM, 10fM, 50fM, 0.1pM, 1pM, 10pM) and 10 μL of 10μM DNA2 dropwise on the electrode surface and incubated at 37°C for 2h, then washed with PBS solution. Add 10 μL of prepared GOD-Au-SA, 10 μL hemin (10 mM) and incubate at 37°C for 2 h and then treat with PBS solution for 2 min. Finally, immerse the assembled electrode in 0.01 M PBS (0.1 mM luminol, 20 mM glucose) solution for 1 h and then react for 1 h. The ECL test was carried out, and the test results are shown in Figure 3A. With the increase of the concentration of microRNA-21 in the test solution, the detected electrochemiluminescence response signal also showed an increasing trend. According to the difference between the electrochemiluminescence response signal and the target concentration. The relationship is plotted to obtain a standard curve (as shown in B in Figure 3), and the linear regression equation is calculated according to the standard curve as y=720.855x+11937.582, where y is the electrochemiluminescence intensity of luminol (the vertical of B in Figure 3). coordinates), x is the logarithm of the concentration of target microRNA-21 (abscissa of B in Figure 3).
实施例4Example 4
对传感器的选择性测定及稳定性测定Selectivity determination and stability determination of sensors
(1)选择性实验：使用目标miRNA-21(SEQ ID NO.3)、单碱基错配miRNA-21(SEQ ID NO.4)、三碱基错配miRNA-21(SEQ ID NO.5)、非互补序列(SEQ ID NO.6)以及空白组(不存在目标miRNA-21)来验证该传感器的选择性。(1) Selective experiment: using target miRNA-21 (SEQ ID NO.3), single base mismatch miRNA-21 (SEQ ID NO.4), three base mismatch miRNA-21 (SEQ ID NO.5 ), non-complementary sequence (SEQ ID NO.6), and blank group (no target miRNA-21 present) to verify the selectivity of the sensor.
结果如图4所示，当存在过量的单碱基错配miRNA-21(10pM)、三碱基错配miRNA-21(10pM)及非互补序列(10pM)时，与空白试验相比，ECL信号响应非常微小。当目标miRNA-21存在时，ECL信号强度显著增强。表明由于核酸链置换反应的高度序列依赖性，使得该传感器对于miRNA-21的检测方法具有良好的选择性；The results are shown in Figure 4. When there is an excess of single-base mismatch miRNA-21 (10 pM), three-base mismatch miRNA-21 (10 pM) and non-complementary sequence (10 pM), compared with the blank test, ECL The signal response is very tiny. When the target miRNA-21 was present, the ECL signal intensity was significantly enhanced. It shows that due to the high sequence dependence of the nucleic acid strand displacement reaction, the sensor has good selectivity for the detection method of miRNA-21;
(2)稳定性实验：使用浓度为1pM的miRNA-21样品反复检测多次，发现ECL信号响应变化非常稳定，证明检测体系具有比较好的稳定性。(2) Stability experiment: The miRNA-21 sample with a concentration of 1 pM was used for repeated detection for many times, and it was found that the ECL signal response change was very stable, which proved that the detection system had relatively good stability.
实施例5Example 5
针对不同肿瘤细胞提取液中miRNA的检测Detection of miRNA in different tumor cell extracts
实验中所使用的人宫颈癌细胞Hela细胞以及人乳腺癌细胞MCF-7细胞由KeyGEN Biotech(中国南京)提供。细胞在由DMEM，10％胎牛血清(FBS)和1％链霉素和青霉素组成的培养液中培养，恒温孵育箱设定为37℃、5％CO
2、95％空气。
Human cervical cancer cells Hela cells and human breast cancer cells MCF-7 cells used in the experiments were provided by KeyGEN Biotech (Nanjing, China). Cells were cultured in a medium consisting of DMEM, 10% fetal bovine serum (FBS) and 1% streptomycin and penicillin in an incubator set at 37°C, 5% CO 2 , 95% air.
实验时使用指数生长期的细胞，使用前用冰冷的无菌PBS洗涤两次。RNA通过试剂盒(Invitrogen Biotechnology)提取得到。结果如图5所示，两种细胞中提取液中miRNA的含量有所不同，相同细胞浓度下MCF-7细胞提取液的miRNA-21含量要高于Hela细胞，且随着细胞浓度的增加，两种细胞提取液中miRNA-21的含量也随之增加。Cells in exponential growth phase were used for the experiments and were washed twice with ice-cold sterile PBS before use. RNA was extracted by a kit (Invitrogen Biotechnology). The results are shown in Figure 5. The content of miRNA in the extracts of the two cells was different. The content of miRNA-21 in the extracts of MCF-7 cells was higher than that of Hela cells at the same cell concentration. The content of miRNA-21 in the two cell extracts also increased.
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.
Claims (11)
- 一种检测miRNA的自产生共反应剂信号放大电化学发光体系，所述体系包括依次连接的传感器基底、固定探针和巯基乙醇封板；所述体系中还包括修饰有生物素的探针、链霉亲和素和葡萄糖氧化酶修饰的纳米金、血红素Hemin和luminol；A self-generated co-reactant signal amplification electrochemiluminescence system for detecting miRNA, the system comprises a sensor substrate, an immobilized probe and a mercaptoethanol sealing plate connected in sequence; the system further comprises a biotin-modified probe, Streptavidin and glucose oxidase modified gold nanoparticles, heme Hemin and luminol;所述传感器基底包括电镀一层Au纳米花的FTO电极；The sensor substrate comprises an FTO electrode electroplated with a layer of Au nanoflowers;所述固定探针包括发卡DNA；The immobilized probe includes hairpin DNA;所述固定探针和修饰有生物素的探针能够发生取代反应。The immobilized probe and the biotin-modified probe can undergo a substitution reaction.
- 根据权利要求1所述体系，其特征在于，所述固定探针包括如SEQ ID NO.1所示的发卡DNA。The system according to claim 1, wherein the immobilized probe comprises a hairpin DNA as shown in SEQ ID NO.1.
- 根据权利要求1所述体系，其特征在于，修饰有生物素的探针的核苷酸序列包括如SEQ ID NO.2所示的核苷酸序列的探针。The system according to claim 1, wherein the nucleotide sequence of the probe modified with biotin comprises the probe of the nucleotide sequence shown in SEQ ID NO.2.
- 根据权利要求1所述体系，其特征在于，所述传感器基底的制备方法，包括：利用打孔胶带包裹清洗后干燥的FTO玻璃，浸入电解质中进行电镀；所述电解质中包括0.05M磷酸盐缓冲液、0.054M KCl和5mM HAuCl 4。 The system according to claim 1, wherein the method for preparing the sensor substrate comprises: wrapping the cleaned and dried FTO glass with perforated tape, and immersing it in an electrolyte for electroplating; the electrolyte includes 0.05M phosphate buffer solution, 0.054M KCl and 5mM HAuCl4.
- 根据权利要求4所述体系，其特征在于，所述清洗包括将FTO玻璃切成宽和长为1cm×4cm的矩形后，用去离子水洗涤，再浸入含有2M KOH的沸腾2-丙醇中20min，水洗涤后再超声清洗15min。The system according to claim 4, wherein the cleaning comprises cutting the FTO glass into rectangles with a width and length of 1cm×4cm, washing with deionized water, and then immersing in boiling 2-propanol containing 2M KOH 20min, washed with water and then ultrasonically cleaned for 15min.
- 根据权利要求4所述体系，其特征在于，所述电镀利用循环伏安法进行，参数设定为：电压-0.8～0.3V、循环50次、扫描速率为0.1V/s；加热溶液至60℃并保持氮气氛围。The system according to claim 4, wherein the electroplating is performed by cyclic voltammetry, and the parameters are set as: voltage -0.8-0.3V, 50 cycles, and a scan rate of 0.1V/s; heating the solution to 60 °C and maintain a nitrogen atmosphere.
- 根据权利要求1所述体系，其特征在于，所述链霉亲和素和葡萄糖氧化酶修饰的纳米金的制备方法，包括：将纳米金溶液与等体积的葡萄糖氧化酶溶液和链霉亲和素溶液的混合液混合后，于4℃保持24h后离心，将离心得到的沉淀分散于缓冲液中，得链霉亲和素和葡萄糖氧化酶修饰的纳米金。The system according to claim 1, wherein the preparation method of the streptavidin and glucose oxidase-modified nano-gold comprises: mixing the nano-gold solution with an equal volume of glucose oxidase solution and streptavidin After mixing the mixed solution of the protein solution, the mixture was kept at 4° C. for 24 hours and then centrifuged, and the precipitate obtained by centrifugation was dispersed in the buffer to obtain the nano-gold modified with streptavidin and glucose oxidase.
- 根据权利要求7所述体系，其特征在于，所述纳米金溶液的体积 为500μL，溶液中纳米金的粒径为30nm；所述混合液中葡萄糖氧化酶的浓度为15mg/mL，链霉亲和素的浓度为1mg/mL。The system according to claim 7, wherein the volume of the gold nanoparticles solution is 500 μL, the particle size of the gold nanoparticles in the solution is 30 nm; the concentration of glucose oxidase in the mixed solution is 15 mg/mL, the streptavidin And the concentration of 1 mg/mL.
- 权利要求1～8任一项所述体系在检测miRNA中的应用。Application of the system according to any one of claims 1 to 8 in detecting miRNA.
- 根据权利要求9所述应用，其特征在于，所述miRNA包括miRNA-21，所述miRNA-21的核苷酸序列如SEQ ID NO.3所示。The application according to claim 9, wherein the miRNA comprises miRNA-21, and the nucleotide sequence of the miRNA-21 is shown in SEQ ID NO.3.
- 一种利用权利要求1～8任一项所述体系检测miRNA-21的方法，包括以下步骤：取10μL待测样品、10μL 10μM修饰有生物素的探针滴加在FTO电极表面，37℃孵育2h后，PBS溶液清洗；A method for detecting miRNA-21 using the system described in any one of claims 1 to 8, comprising the steps of: taking 10 μL of the sample to be tested and 10 μL of 10 μM biotin-modified probes and dropping them on the surface of an FTO electrode, incubating at 37°C After 2h, wash with PBS solution;加入10μL链霉亲和素和葡萄糖氧化酶修饰的纳米金、10μL血红素Hemin，37℃孵育2h后PBS溶液处理2min，所述血红素Hemin的浓度为10mM，得到电镀一层Au纳米花的FTO电极；Add 10 μL of streptavidin and glucose oxidase-modified gold nanoparticles, 10 μL of heme Hemin, incubate at 37°C for 2 h, and then treat with PBS solution for 2 min. The concentration of heme Hemin is 10 mM to obtain FTO electroplated with a layer of Au nanoflowers. electrode;最后将电镀一层Au纳米花的FTO电极浸入在0.01M PBS溶液中反应1h后再进行ECL测试；所述0.01M PBS溶液中还包括0.1mM luminol和20mM葡萄糖；Finally, the FTO electrode electroplated with a layer of Au nanoflowers was immersed in a 0.01M PBS solution and reacted for 1 hour before the ECL test; the 0.01M PBS solution also included 0.1mM luminol and 20mM glucose;进行所述ECL测试采用ECL传感器进行，所述ECL传感器的制备方法，包括：在制备的电镀一层Au纳米花的FTO电极上滴涂10μL 10μM的固定探针，室温下过夜，之后将电极浸润在PBS溶液中2min以除去未能键合的过量固定探针；随后用10μL 1mM巯基乙醇封板封闭电极上剩余的反应位点，1h后浸入PBS溶液中2min。The ECL test is carried out using an ECL sensor. The preparation method of the ECL sensor includes: drip-coating 10 μL of 10 μM fixed probe on the prepared FTO electrode electroplated with a layer of Au nanoflowers, overnight at room temperature, and then soaking the electrode In PBS solution for 2 min to remove excess immobilized probes that failed to bond; then seal the remaining reaction sites on the electrode with 10 μL of 1mM mercaptoethanol, and immerse in PBS solution for 2 min after 1 h.
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| CN114113267A (en) * | 2021-12-28 | 2022-03-01 | 郑州大学 | Construction method and application of aptamer sensor based on TdT and G4/hemin mimic enzyme amplification technology |
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| CN114113264A (en) * | 2021-12-11 | 2022-03-01 | 郑州大学 | Tobramycin double-amplification detection method based on EXO III auxiliary chain circulation and CHA reaction and application |
| CN114113267A (en) * | 2021-12-28 | 2022-03-01 | 郑州大学 | Construction method and application of aptamer sensor based on TdT and G4/hemin mimic enzyme amplification technology |
| CN115184428A (en) * | 2022-06-27 | 2022-10-14 | 东南大学 | Electrochemical luminescence sensor for detecting mutant BRAF gene and preparation method and application thereof |
| CN115184428B (en) * | 2022-06-27 | 2024-01-26 | 东南大学 | Electrochemical luminescence sensor for detecting mutant BRAF gene, and preparation method and application thereof |
| CN115032258A (en) * | 2022-06-28 | 2022-09-09 | 南京邮电大学 | miRNA tumor marker detection kit |
| CN115032258B (en) * | 2022-06-28 | 2024-01-19 | 南京邮电大学 | miRNA tumor marker detection kit |
| CN116024313A (en) * | 2022-09-20 | 2023-04-28 | 华南农业大学 | Programmable nucleic acid molecule detection method and platform |
| CN115728254A (en) * | 2022-11-08 | 2023-03-03 | 中国海洋大学 | Electrochemical colorimetric dual-signal sensor, and preparation method and application thereof |
| CN116297769A (en) * | 2023-05-18 | 2023-06-23 | 山东大学 | Group selective functionalized potential resolution type electrochemiluminescence nucleic acid detection method |
| CN116297769B (en) * | 2023-05-18 | 2023-08-08 | 山东大学 | Group selective functionalized potential resolution type electrochemiluminescence nucleic acid detection method |
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| Publication number | Publication date |
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| CN112226492B (en) | 2022-08-19 |
| CN112226492A (en) | 2021-01-15 |
| ZA202104691B (en) | 2021-07-28 |
| LU500321B1 (en) | 2022-05-10 |
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