CN106872447A - Strengthen the preparation method of the electrochemical luminescence biology sensor of Luminol - Google Patents

Strengthen the preparation method of the electrochemical luminescence biology sensor of Luminol Download PDF

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Publication number
CN106872447A
CN106872447A CN201710026498.2A CN201710026498A CN106872447A CN 106872447 A CN106872447 A CN 106872447A CN 201710026498 A CN201710026498 A CN 201710026498A CN 106872447 A CN106872447 A CN 106872447A
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mirna
dna probe
electrode
luminol
biology sensor
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鲁理平
刘畅
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Beijing University of Technology
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Beijing University of Technology
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage

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Abstract

Strengthen the preparation method of the electrochemical luminescence biology sensor of Luminol, belong to biology sensor preparation field.Step of the present invention：1) determine target miRNA to be detected, and base complementrity principle design dna probe sequence is followed according to sequence；2) by electrochemical cyclic voltammetry, in glassy carbon electrode surface deposited Au nano particle, and the DNA probe (5 ' terminal modified sulfydryl) for designing is connected on gold nano grain by golden sulfide linkage；3) nonspecific binding site on glucose oxidase enclosed-electrode surface is used；4) double-stranded complex will be formed after the electrode reaction after target miRNA and closing, with DNA probe hybridization；5) haemachrome solution is added dropwise on electrode after hybridization, the material is only embedded between miRNA DNA double chains, can effectively be accelerated the decomposition of hydrogen peroxide and be generated superoxide anion, and the enhancing of electrochemical luminescence is substantially realized from mechanism.The present invention solves that Luminol luminous signal is weak, the low problem of biology sensor detection sensitivity.

Description

Strengthen the preparation method of the electrochemical luminescence biology sensor of Luminol
Technical field
The present invention relates to one kind based on enhancing Luminol electrochemical luminescence signals, build for detecting that miRNA is biological The new method of sensor, belongs to biology sensor preparation field.The method can be applied to miRNA clinical diagnosises and to miRNA Quantitative analysis.
Background technology
MicroRNAs (miRNAs) is the single-stranded microRNA of non-coding that a class contains 19-23 bases, is widely present In the cell of human body and nearly all model organism, these microRNAs are by base pairing and mRNA (mRNA) sequence 3 ' UTR areas or code area combine so as to regulate and control the expression of target gene, development, cell growth differentiation and apoptosis in animals and plants, Important regulating and controlling effect is played during the vital movements such as metabolism.There is result of study to show recently, the unconventionality expression of miRNA with it is many Number cancer is related, such as cancer of pancreas, lung cancer, liver cancer, stomach cancer, breast cancer, colon cancer and oophoroma.The work that these miRNA rise With the function similar to tumor suppressor gene and oncogene.Therefore, the quantitative determination to miRNA in cell or tissue sample is beneficial to People further appreciate that the relation between the expression of miRNA and cancer generation.MiRNA is also expected to be used as valuable mark Thing, cancer diagnosis and prognosis for early stage, this pair is significant with exploration biological evolution and preventing and treating human diseases.
Because miRNA sequence is short, expression in the cell than relatively low, and many same miRNAs (such as let-7 family Race) sequence similarity is high, only differs from 1-2 bases, makes the analysis probe of design low with the efficiency that miRNA hybridizes.So, miRNA Research to analytical chemistry detection sensitivity and selectivity in terms of be proposed new challenge.Currently, the detection side of miRNA Method mainly has Northern traces, micro-array chip, PCR (PCR) amplification and in situ hybridization, but because behaviour Make that cumbersome, time-consuming, these conventional methods of the low shortcoming of sensitivity are above restricted in application.In recent years, in order to improve miRNA The sensitivity of detection, various DNA cloning technologies such as constant temperature rolling circle amplification etc. is applied to miRNA analyses in succession；In order to carry The detection flux of miRNA high, develops various probe labeling techniques and new detecting method in succession.
The detection signal used in this experiment is electrochemical luminescence (ECL) signal.ECL is to apply certain electricity on electrode A kind of light that pressure makes between electrode reaction product or electrode reaction product is electrochemically reacted and produces with certain component in solution Radiation.It includes two processes of electrochemical analysis and chemiluminescence analysis, therefore has both remained chemiluminescence method sensitivity The advantages of height, the range of linearity wide, convenient observation and simple instrument；Incomparable excellent of many chemiluminescence methods is increased simultaneously Point, such as favorable reproducibility, stable reagent, control are easy, so as to attract much attention and are widely used.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of based on enhancing Luminol ECL signals, are used for The biology sensor preparation method of miRNA detections.The method from the reaction mechanism of electrochemical luminescence, by the use of ferroheme as Horseradish peroxidase analog, can the effective decomposition of catalyzing hydrogen peroxide characteristic, Luminol electricity is realized in itself The enhancing of chemiluminescence signal, so that more delicately detection target miRNA.The method sensitivity is high, high specificity, testing cost It is low.
The present invention is adopted the following technical scheme that：
1. the preparation method of the electrochemical luminescence biology sensor of Luminol, its feature are strengthened
It is to comprise the following steps：
1) DNA probe sequence of target miRNA to be detected, design and its base complementrity is determined；
2) pretreatment of glass-carbon electrode：Polishing glass-carbon electrode, is cleaned by ultrasonic, by electrode immersion 5mM chlorine gold after nitrogen drying In acid solution, by cyclic voltammetry, initial voltage and low potential are set to 0V, and high potential is 1V, and interval interior circulation is swept herein Retouch deposited Au nano particle；
3) DNA probe solution is added dropwise in electrode surface, at room temperature the self assembly 16-20h in wet box, with sulfydryl modification Probe gold nano grain surface is fixed to by Au-S keys；
4) glucose oxidase enclosed-electrode surface nonspecific activity site is added dropwise, in cultivating 3h at 4 DEG C；
5) target miRNA is added drop-wise to electrode surface, 30min is hybridized at room temperature, form miRNA-DNA duplex structures；
6) haemachrome solution is added drop-wise to the electrode surface after hybridization, 1h is cultivated at room temperature.
2. further, the DNA probe itself of design will not hybridize.
3. further, the diameter of glass-carbon electrode used is 3mm, and polishing powder is 0.05 μm of Al2O3Powder.
4. further, speed is swept for 50mV/s in electrodeposition process, the scanning number of turns is 5 circles.
5. further, the DNA probe solution of dropwise addition is configured by PBS, and concentration is 25 μM, and volume is 10 μ L；PBS delays Fliud flushing concentration be 5mM, the pH=7.4 of PBS,
6. further, the glucose oxidase concentration of dropwise addition is 0.5mg/mL, and volume is 10 μ L.
7. further, the haemachrome solution concentration of dropwise addition is 10 μM, and volume is 10 μ L.
Preferably, the DNA probe of design itself will not hybridize winding, will not form hair fastener or other structures, related Detection interference reaches minimum.
Preferably, the storing solution of DNA is configured by PBS, and concentration is 5mM, pH=7.4.
Preferably, target miRNA and the DNA probe hybridization time of electrode surface are 30min.
Preferably, the haemachrome solution concentration being added dropwise in electrode surface is 10 μM.
Preferably, the ferroheme insertion miRNA-DNA double-stranded complex reaction time is 1h.
Preferably, when testing electrochemical luminescence signals, concentration of glucose is 1 × 10 in electrolyte-2M。
Preferably, the initial potential and low potential set by deposited Au nano particle process are 0V, and high potential is 1V, the scanning number of turns is 5 circles, sweeps speed for 50mV/s.
The present invention compared with prior art, with advantages below and good result：
1) H can be catalyzed using ferroheme2O2The characteristic of generation superoxide anion is decomposed, with reference to Particles in Electrochemiluminescofce ofce Luminol machine Reason, inherently realizes the enhancing of the ECL signals of luminol, and for the detection of miRNA.
2) high specific：Because combining form is embeddeding action between ferroheme and miRNA-DNA, for other miRNA, its Hybridization efficiency will be far smaller than target miRNA, therefore the ferroheme quantity for combining is substantially reduced, and cause finally to measure ECL enhancings Effect is significantly different.
3) high sensitivity：Luminol itself illumination effect is relatively low, with reference to the humidification of ferroheme after, for various concentrations Target miRNA sensitive can be responded.
4) it is pollution-free：Whole detection process need not use toxic reagent.
Brief description of the drawings
Fig. 1 is schematic diagram prepared by biology sensor of the present invention based on enhancing Luminol electrochemical luminescence signals.
Fig. 2 is that the ECL signals measured by three kinds of different modifying state electrodes compare figure.
Fig. 3 is that the biology sensor of present invention design realizes high specific detection figure for target miRNA.
Specific embodiment
In order that present disclosure, object, technical solution and advantage become more apparent, below in conjunction with Figure of description And embodiment, the present invention will be described in further detail.It should be appreciated that specific embodiment described herein is only used to explain The present invention, is not intended to limit the present invention.General replacement known to those skilled in the art is also covered by of the invention In protection domain.
Prepared by the biology sensor based on enhancing Luminol electrochemical luminescence signals, comprise the following steps：
1) target miRNA to be detected is determined, it then follows base complementrity principle, the DNA probe sequence that design is complementary to.
2) pretreatment of glass-carbon electrode：Polishing glass-carbon electrode, is cleaned by ultrasonic, nitrogen drying.Electrode is immersed into 5mM gold chlorides In solution, by cyclic voltammetry, initial voltage and low potential are set to 0V, and high potential is 1V, herein interval interior electro-deposition Gold nano grain.
3) 10 μ L DNA probes are added dropwise in electrode surface, at room temperature the self assembly 16-20h in wet box, 5 ' terminal modified mercaptos The probe of base by Au-S keys so that be fixed to gold nano grain surface.
4) 10 μ L glucoses oxidizing ferment are added dropwise is used for the nonspecific activity site on enclosed-electrode surface, in cultivation at 4 DEG C 3h。
5) target miRNA is added drop-wise to electrode surface, is hybridized at room temperature, form miRNA-DNA duplex structures.
6) 10 μ L haemachrome solutions are added drop-wise to the electrode surface after hybridization, to be embedded in duplex structure.
7) after electrode is cleaned, electrochemical luminescence is measured in PBS solutions of the 5mL containing glucose and luminol.
Embodiment one
The design of 1.DNA probes
The target miRNA of selection is let-7d, its base sequence：
5’-AGAGGUAGUAGGUUGCAUAGUU-3’
DNA probe sequence is：
5’-HS-CCACCACGAACTATGCAACCTACTACCTCT-3’
, wherein it is desired to explanation, underscore part is and target miRNA complementary series in the DNA probe sequence of design. DNA probe itself will not be intertwined, and will not form hair fastener or other structures, and structure minimum free energy is zero, with phase When stabilization single-stranded structure and with the genome that has found in human body keep minimum similarity degree, coherent detection interference reaches minimum.
2. experimental section
1) the DNA probe dry powder that will be ordered is centrifuged 5min, and rotating speed is set to 6000 revs/min, adds 5mM PBSs (pH=7.4) 25 μM of concentration, is made into, 5min is full and uniform to solution for concussion.
2) the target miRNA dry powder that will be ordered is centrifuged 5min, and rotating speed is set to 10000 revs/min, adds DEPC treatment Water, concussion 5min to fully dissolving.
3) with 0.05 μm of Al2O3Powder polishing glass-carbon electrode, is cleaned by ultrasonic with secondary water, ethanol, secondary water successively, Nitrogen is dried up.In immersion 5mM chlorauric acid solutions, by cyclic voltammetry electro-deposition nm of gold, initial voltage and low-voltage set 0V is set to, high voltage is set to 1V, and sweep speed is 50mV/s, the deposition number of turns is 5 circles.
4) the DNA probe solution of 10 μ L25 μM is added dropwise, at room temperature the self assembly 16-20h in wet box, with sulfydryl modification DNA probe can be scheduled on gold nano grain surface by Au-S keyings.Fully cleaned with 5mM PBS, after nitrogen drying, 10 μ L are added dropwise The glucose oxidase enclosed-electrode surface residual avtive spot of 0.5mg/mL, 3h is cultivated at 4 DEG C.
5) target miRNA is added drop-wise to the electrode surface after closing, 30min is hybridized at room temperature.Cleaning electrode, nitrogen blows After dry, the ferroheme of 10 μ L10 μM is added dropwise, 1h is cultivated at room temperature.
6) it is electrode washing is clean, electrochemical luminescence detection is carried out, bottom liquid is 0.1M PBS (pH=7.4；Containing 1 × 10-4M Luminol and 2 × 10-2M glucose), electric potential scanning scope is 0.2V~0.8V, sweeps speed for 100mV/s, Photomultiplier tube voltage It is set to 800V.
7) feasibility tactful in order to verify enhancing, tests the electrode ECL signal (accompanying drawings under three kinds of different modifying states 2)；In order to investigate the specificity of biology sensor, selection is made with other three kinds of nucleotide sequences that target miRNA is homologous family It is control (accompanying drawing 3).
Although the present invention is disclosed as above with preferred embodiment, it is not restricted to institute in specification and implementation method Row are used, and the embodiment for those skilled in the art, is not departing from essence of the invention only for the purposes of explanation Some changes and retouching can be made on the premise of god and scope, the protection domain that the present invention is advocated should be with described in claims It is defined.

Claims (7)

1. the preparation method of the electrochemical luminescence biology sensor of Luminol is strengthened, it is characterised in that comprised the following steps：
1) DNA probe sequence of target miRNA to be detected, design and its base complementrity is determined；
2) pretreatment of glass-carbon electrode：Polishing glass-carbon electrode, is cleaned by ultrasonic, after nitrogen drying that electrode immersion 5mM gold chlorides is molten In liquid, by cyclic voltammetry, initial voltage and low potential are set to 0V, and high potential is 1V, herein interval interior circulation scanning electricity Deposition gold nano grain；
3) DNA probe solution is added dropwise in electrode surface, at room temperature the self assembly 16-20h in wet box, the spy with sulfydryl modification Pin is fixed to gold nano grain surface by Au-S keys；
4) glucose oxidase enclosed-electrode surface nonspecific activity site is added dropwise, in cultivating 3h at 4 DEG C；
5) target miRNA is added drop-wise to electrode surface, 30min is hybridized at room temperature, form miRNA-DNA duplex structures；
6) haemachrome solution is added drop-wise to the electrode surface after hybridization, 1h is cultivated at room temperature.
2. according to the method for claim 1, it is characterised in that the DNA probe of design will not hybridize itself.
3., according to the method for claim 1, it is characterised in that the diameter of glass-carbon electrode used is 3mm, polishing powder is 0.05 μm Al2O3Powder.
4. according to the method for claim 1, it is characterised in that sweep speed for 50mV/s in electrodeposition process, the scanning number of turns is 5 Circle.
5., according to the method for claim 1, it is characterised in that the DNA probe solution of dropwise addition is configured by PBS, concentration is 25 μM, volume is 10 μ L；PBS concentration be 5mM, the pH=7.4 of PBS,
6., according to the method for claim 1, it is characterised in that the glucose oxidase concentration of dropwise addition is 0.5mg/mL, volume is 10μL。
7., according to the method for claim 1, it is characterised in that the haemachrome solution concentration of dropwise addition is 10 μM, volume is 10 μ L.
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CN111220666A (en) * 2020-01-09 2020-06-02 济南大学 Efficient miRNA detection based on hemin-induced biocatalysis photoelectric sensitive interface
CN113358727A (en) * 2021-05-20 2021-09-07 东南大学 Electrochemical luminescence sensor for detecting microRNA, and preparation method and application thereof
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Cited By (11)

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Publication number Priority date Publication date Assignee Title
CN108152274A (en) * 2017-12-25 2018-06-12 汕头大学医学院 A kind of method for carrying out quantitative detection to serum miRNA using RNase ONE nucleases and chemiluminescence
CN108690870A (en) * 2018-05-15 2018-10-23 北京工业大学 The preparation method of biosensor is quenched in tris (bipyridine) ruthenium electrochemical luminescence
CN109632903A (en) * 2018-12-05 2019-04-16 泰山学院 A method of in glassy carbon electrode surface fabricated in situ single layer uniform gold nanoparticle
CN110687172A (en) * 2019-10-17 2020-01-14 山东师范大学 Electrochemical luminescence biosensor, preparation method and application thereof in detection of base excision repair enzyme
CN110687172B (en) * 2019-10-17 2021-12-03 山东师范大学 Electrochemical luminescence biosensor, preparation method and application thereof in detection of base excision repair enzyme
CN111220666A (en) * 2020-01-09 2020-06-02 济南大学 Efficient miRNA detection based on hemin-induced biocatalysis photoelectric sensitive interface
CN113358727A (en) * 2021-05-20 2021-09-07 东南大学 Electrochemical luminescence sensor for detecting microRNA, and preparation method and application thereof
CN113358727B (en) * 2021-05-20 2023-07-21 东南大学 Electrochemical luminescence sensor for detecting microRNA and preparation method and application thereof
CN114410749A (en) * 2022-01-25 2022-04-29 河南中医药大学 Electrochemiluminescence detection kit and method based on click chemistry and ARGET-ATRP amplification strategy
CN114410749B (en) * 2022-01-25 2023-10-24 河南中医药大学 Electrochemiluminescence detection kit and method based on click chemistry and ARGET-ATRP amplification strategy
CN115058489A (en) * 2022-06-18 2022-09-16 福州大学 Construction and application of electrochemical sensor based on tandem catalysis hairpin assembly technology and adopting double-target nucleic acid sequence response

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