CN104764782A - Preparation of boron-doped graphene quantum dot electrochemiluminescence sensor for detecting miRNA-20a and application of sensor - Google Patents
Preparation of boron-doped graphene quantum dot electrochemiluminescence sensor for detecting miRNA-20a and application of sensor Download PDFInfo
- Publication number
- CN104764782A CN104764782A CN201510170878.4A CN201510170878A CN104764782A CN 104764782 A CN104764782 A CN 104764782A CN 201510170878 A CN201510170878 A CN 201510170878A CN 104764782 A CN104764782 A CN 104764782A
- Authority
- CN
- China
- Prior art keywords
- electrode
- doped graphene
- quantum dot
- boron doped
- graphene quantum
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the field of biology and relates to preparation of a boron-doped graphene quantum dot electrochemiluminescence sensor for detecting miRNA-20a and application of the sensor. The preparation method comprises the following steps: dropwise adding nanogold and BGQDs-DNA into a platinum electrode, so that the volumes of the nanogold and BGQDs-DNA on the electrode surface are 2-8 mu L/mm<2> and 2-6 mu L/mm<2>; and drying at room temperature, immersing the electrode into a buffer solution containing to-be-detected miRNA-20a of different concentrations, reacting at the temperature of 37 DEG C for 0.5-3 hours, and cleaning the electrode by using PBS buffer solution. The electrochemiluminescence signal of the electrode is detected by using a multi-channel chemiluminescence detection system, the electrolyte refers to 0.1M PBS and contains 0.1M KCl and 0.05M K2S2O8, the voltage is 0.7-1.15V, the scanning rate is 0.1V/s, and the high voltage of a photomultiplier is 600V. Compared with the traditional analysis method, the electrochemiluminescence detection method disclosed by the invention is low in cost, easy to operate and high in practicality.
Description
Technical field
The invention belongs to environmental analysis field, relating to a kind of preparation and application thereof of the boron doped graphene quantum dot electrochemical luminous sensor for detecting miRNA-20a.
Background technology
MiRNAs is the RNA molecule that a class is about the endogenous nonprotein coding of 20-25 nucleotide, can participate in the behavior of regulation and control various biological, as Cell Differentiation, and developmental process, Apoptosis, disease, tumour etc.There are some researches show, the miRNAs of 50% is positioned at the region of tumor-related gene group or the site of fragility, and the expression of dissimilar miRNAs also represents different tumor disease accordingly, the expression of miRNA-20a has been proved to be at least relevant to three kinds of entity tumors.Therefore, the quantitative detection of miRNA-20a has vital effect for the clinical diagnosis of tumour and effect of drugs assessment.
Traditional miRNAs detection method mainly comprises polymerase chain reaction, Hybridized blots method, this several method of micro-array chip.Although the testing result of these methods is relatively more accurate, their operation is all very complicated, and testing cost is higher, consuming time, and sensitivity is low.Electrochemical sensing technology is also used to detect miRNAs in recent years.But these methods are all that magnetic Nano material etc. are as signal amplification technique with enzyme substantially.These materials not only price are higher, and the serviceable life of enzyme is shorter, and require strict to the pH, temperature etc. of testing environment, this is all unfavorable for applying of sensor.Therefore, develop wide, simple to operate, green high-efficient, the high selectivity highly sensitive miRNAs detection method of applicability and there is very important realistic meaning.
Electrochemical luminous sensor, because the feature of itself is more concerned in recent years.This technology equipment needed thereby is simple, detection speed fast, react controlled, and can realize real-time in-situ analysis.Electrochemiluminescence material is the vitals of electrochemical luminous sensor, and the quality of electrochemiluminescence material performance, directly determines the Detection results of electrochemical luminous sensor.Traditional electrochemiluminescence material, ruthenium compound, luminol, all have certain bio-toxicity, if for detecting miRNAs, can have an impact unavoidably to testing result.Graphene quantum dot is also used as electrochemiluminescence material in recent years.But itself is according to the difference of preparation method, stability of photoluminescence is also different.Therefore improve the performance of electrochemiluminescence material, and electrochemiluminescence sensing is used for detecting miRNAs, become problem demanding prompt solution.
The present invention has prepared boron doped graphene quantum dot by electrochemical method, utilize high conductivity, high stability of photoluminescence, the boron doped graphene quantum dot of high bio-compatibility, environmental protection close friend is as electrochemiluminescence material, using the hairpin DNA with miRNA-20a complementation as identifying probe, constructing, to miRNA-20a, there is the quantum dot-based electrochemical luminous sensor of high selectivity highly sensitive boron doped graphene.Solve operated in accordance with conventional methods complexity, loaded down with trivial details consuming time, the shortcoming such as cost is high, poor practicability, application prospect is boundless.
Summary of the invention
The invention solves existing miRNA-20a detection method complicated operation, loaded down with trivial details consuming time, the deficiency such as cost is high, poor practicability, provide the quantitative analysis method of a kind of easy, quick, practical miRNA-20a.
Boric acid is mixed by a certain percentage with graphite oxide, pours in glass tube, under the condition of heating, generate bar-shaped boron doped graphene.With this boron doped graphene rod for working electrode, graphite rod is to electrode, prepares boron doped graphene quantum dot (BGQDs) by electrochemical method.With the hairpin DNA of marking sulfhydryl for probe catches miRNA-20a.By quantum dot-labeled for boron doped graphene one end at hairpin DNA, form electrochemiluminescence probe (being designated as BGQDs-DNA).After platinum electrode is modified by nm of gold (AuNPs), the electrode surface that electrochemiluminescence probe can be fixed on by the combination of sulfydryl and nm of gold, now boron doped graphene quantum dot presses close to electrode surface, can obtain higher electrochemical luminescence signals.When there is target miRNA-20 in system, the foldable structure of hairpin DNA is opened, and forms straight chain, makes boron doped graphene quantum dot away from electrode surface, and electrochemical luminescence signals reduces.The electrochemical luminescence signals of system within the specific limits with the concentration linearly inverse correlation of object miRNA-20, thus provide foundation for the quantitative detection of miRNA-20.
The invention provides a kind of preparation of the boron doped graphene quantum dot electrochemical luminous sensor for detecting miRNA-20a, concrete steps are as follows:
(1) under ice bath state, the concentrated sulphuric acid to be joined in dag and to stir, then potassium permanganate being joined in above-mentioned potpourri, Keep agitation 20-48 hour; Add H
2o static 10-60 minute, add H subsequently
2o and H
2o
2cessation reaction; By potpourri centrifuging, and with concentration be 5% hydrochloric acid and water clean respectively, obtain graphite oxide; The mol ratio of each material wherein used is the concentrated sulphuric acid: dag: potassium permanganate: H
2o:H
2o
2=1:0.3:0.02:10:0.2.
(2) H is added in the graphite oxide obtained to step (1)
3bO
3, potpourri is to 60-100 DEG C of insulation 6-12 hour; Products in water is cleaned, vacuum freeze drying, obtain boron doped Graphene rod.Wherein graphite oxide and H
3bO
3mass ratio be 250-5000.
(3) the boron doped Graphene rod obtained with step (2) is working electrode, with graphite rod for prepare boron doped graphene quantum dot to electrode by electrochemical method, electrolytic solution is the mixed liquor of absolute ethyl alcohol containing 25-50mM NaOH and water, volume ratio=the 99:1-99.5:0.5 of absolute ethyl alcohol and water, current density 180-300mA/cm
2, reaction 1-3 hour, graphite impurities is removed in centrifuging; By the centrifugate dialysis 24-48 hour obtained, dialysis aperture is less than 5000Da, obtains boron doped graphene quantum dot aqueous solution.
(4) NaOH and ClCH is added in the boron doped graphene quantum dot aqueous solution obtained to step (3)
2cOOH, ultrasonic.Add the N-hydroxysuccinimide (NHS) of 150mg/ml and 1-ethyl-3-(3-the dimethyl aminopropyl)-carbodiimides (EDC) of 10mg/ml again, reaction 10-60 minute; And then add the hairpin DNA of 100 μMs of marking sulfhydryls, slowly stir.Finally that potpourri is centrifugal, precipitation is electrochemiluminescence probe BGQDs-DNA.Electrochemiluminescence probe BGQDs-DNA is dispersed in 0.1M Tris-EDTA (TE) pH=8.0 buffer solution, be stored in 4 DEG C for subsequent use.Wherein NaOH and ClCH
2the mass ratio of COOH is 0.25-0.5; The volume ratio 1-3 of NHS and EDC; The volume ratio 120-1200 of the hairpin DNA of boron doped graphene quantum dot aqueous solution and marking sulfhydryl.
(5) AuNPs aqueous solution is dropped in platinum electrode surface, be designated as Pt/Au, AuNPs aqueous solution is at the volume 2-8 μ L/mm of platinum electrode surface
2, drying at room temperature, then the BGQDs-DNA that step (4) is obtained is added drop-wise to Pt/Au surface, BGQDs-DNA is at the volume 2-6 μ L/mm on Pt/Au surface
2, rinse electrode surface with 0.1M phosphate (PBS) pH=7.4 buffer solution after 1-3 hour, obtain boron doped graphene quantum dot electrochemical luminous sensor.
In step (4), the hairpin DNA total bases of marking sulfhydryl is 31-39, its main body section, and corresponding identification miRNA-20a base sequence is 5 '-CTACCTGCACTATAAGCACTTTA-3 '.
In step (5), platinum electrode needs to clean before use, and concrete grammar is as follows:
Platinum electrode is ground in the suspending liquid of the aluminum oxide polishing powder of 0.05-1 μm of particle diameter, uses absolute ethyl alcohol and high purity water ultrasonic cleaning after grinding successively, at N
2protection is lower dry.
Boron doped graphene quantum dot electrochemical luminous sensor is immersed in (concentration range of miRNA-20a is 1-50000pM) in the TE damping fluid of the miRNA-20a contained, hybridize 0.5-3 hour under 37 DEG C of water bath condition, then use 0.1M phosphate (PBS) buffer solution (pH 7.4) to rinse electrode surface.The electrode finally obtained is working electrode, and platinum electrode is to electrode, and silver/silver chloride electrode is contrast electrode, and utilize multi-channel chemiluminescence test macro to detect electrochemical luminescence signals, detection is containing 0.1M KCl and 0.05M K
2s
2o
80.1M PBS buffer solution (pH=7.4) in carry out, voltage range is 0.7 ~ 1.15V, sweep speed 0.1V/s, and photomultiplier high pressure is 600V.
The present invention has following effect:
(1) highly sensitive, detectability can reach 0.05pM (S/N=3).
(2) relative to traditional electrochemiluminescence material, boron doped graphene quanta point biological toxicity is low, stable luminescence, and life cycle is long, can not cause environmental pollution.
(3) this electrochemical luminous sensor is simple to operate, detects fast, does not need the main equipment of complex and expensive, practical.
Accompanying drawing explanation
Fig. 1 is the quantitative detection schematic diagram of miRNA-20a of the present invention.
Fig. 2 is the TEM picture of boron doped graphene quantum dot prepared by this method.
Fig. 3 is the standard working curve of the quantitative detection miRNA-20a that method of the present invention obtains.
Embodiment
Below in conjunction with accompanying drawing and technical scheme, further illustrate the specific embodiment of the present invention.
Embodiment 1
The preparation of electrochemiluminescence probe BGQDs-DNA, comprises the steps:
(1) under ice bath state, the 23ml concentrated sulphuric acid to be joined in 1g high purity graphite powder and to stir, then 3g potassium permanganate being joined in said mixture several times, Keep agitation 24 hours.Add 50ml water and static 15 minutes, add 150ml water and 10ml H subsequently
2o
2cessation reaction.By potpourri centrifuging, and be hydrochloric acid and the water respectively washing and precipitating of 5% by concentration, finally obtain graphite oxide.
(2) 0.002g H is added in the graphite oxide obtained to step (1)
3bO
3, within ultrasonic 10 minutes, make it mix.Then potpourri is transferred in glass tube, in air dry oven, is heated to 80 DEG C of insulations 12 hours.By products in water cleaning several times, then vacuum freeze drying, finally obtains boron doped Graphene rod.
(3) the boron doped Graphene rod obtained with step (2) is working electrode, with graphite rod for prepare boron doped graphene quantum dot to electrode by electrochemical method.Electrolytic solution is the mixed liquor (volume ratio=99.5:0.5) of 10ml absolute ethyl alcohol containing 0.01g NaOH and water, current density range 200-250mA/cm
2.React after 2 hours, remove graphite impurities by centrifuging.The centrifugate obtained is put into bag filter (1000Da), dialyse 24 hours with pure water, finally obtain boron doped graphene quantum dot aqueous solution.
(4) 0.015gNaOH and 0.025g ClCH is added in the boron doped graphene quantum dot aqueous solution obtained to 10ml step (3)
2cOOH, ultrasonic 3 hours (frequency: 40KHZ, power: 200W).400 μ L N-hydroxysuccinimide (NHS, 150mg/ml) and 250 μ L 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides (EDC, 10mg/ml) are joined in said mixture and react 30 minutes.Then the hairpin DNA (100 μMs) of 50 μ L marking sulfhydryls is joined in said mixture, and slowly stirring is spent the night.Finally that potpourri is centrifugal, precipitation is electrochemiluminescence probe BGQDs-DNA.Electrochemiluminescence probe is dispersed in 0.1M Tris-EDTA (TE) buffer solution (pH 8.0) be stored in 4 DEG C for subsequent use.
Embodiment 2
The preparation of electrochemiluminescence probe BGQDs-DNA, comprises the steps:
(1) under ice bath state, the 30ml concentrated sulphuric acid to be joined in 1.2g high purity graphite powder and to stir, then 1.8g potassium permanganate being joined in said mixture several times, Keep agitation 24 hours.Add 100ml water and static 15 minutes, add 180ml water and 15ml H subsequently
2o
2cessation reaction.By potpourri centrifuging, and be hydrochloric acid and the water respectively washing and precipitating of 5% by concentration, finally obtain graphite oxide.
(2) 0.0025g H is added in the graphite oxide obtained to step (1)
3bO
3, within ultrasonic 10 minutes, make it mix.Then potpourri is transferred in glass tube, in air dry oven, is heated to 80 DEG C of insulations 12 hours.By products in water cleaning several times, then vacuum freeze drying, finally obtains boron doped Graphene rod.
(3) the boron doped Graphene rod obtained with step (2) is working electrode, with graphite rod for prepare boron doped graphene quantum dot to electrode by electrochemical method.Electrolytic solution is the mixed liquor (volume ratio=99.5:0.5) of 10ml absolute ethyl alcohol containing 0.015g NaOH and water, current density range 200-230mA/cm
2.React after 2 hours, remove graphite impurities by centrifuging.The centrifugate obtained is put into bag filter (1000Da), dialyse 24 hours with pure water, finally obtain boron doped graphene quantum dot aqueous solution.
(4) 0.018gNaOH and 0.028g ClCH is added in the boron doped graphene quantum dot aqueous solution obtained to 10ml step (3)
2cOOH, ultrasonic 3 hours (frequency: 40KHZ, power: 200W).500 μ L N-hydroxysuccinimide (NHS, 150mg/ml) and 300 μ L 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides (EDC, 10mg/ml) are joined in said mixture and react 30 minutes.Then the hairpin DNA (100 μMs) of 80 μ L marking sulfhydryls is joined in said mixture, and slowly stirring is spent the night.Finally that potpourri is centrifugal, precipitation is electrochemiluminescence probe BGQDs-DNA.Electrochemiluminescence probe is dispersed in 0.1M Tris-EDTA (TE) buffer solution (pH 8.0) be stored in 4 DEG C for subsequent use.
Embodiment 3
The mensuration of miRNA-20a content in configuration sample:
(1) under ice bath state, the 25ml concentrated sulphuric acid to be joined in 1.2g high purity graphite powder and to stir, then 1.5g potassium permanganate being joined in said mixture several times, Keep agitation 24 hours.Add 45ml water and static 15 minutes, add 200ml water and 15ml H subsequently
2o
2cessation reaction.By potpourri centrifuging, and be hydrochloric acid and the water respectively washing and precipitating of 5% by concentration, finally obtain graphite oxide.
(2) 0.0045g H is added in the graphite oxide obtained to step (1)
3bO
3, within ultrasonic 10 minutes, make it mix.Then potpourri is transferred in glass tube, in air dry oven, is heated to 80 DEG C of insulations 12 hours.By products in water cleaning several times, then vacuum freeze drying, finally obtains boron doped Graphene rod.
(3) the boron doped Graphene rod obtained with step (2) is working electrode, with graphite rod for prepare boron doped graphene quantum dot to electrode by electrochemical method.Electrolytic solution is the mixed liquor (volume ratio=99:1) of 10ml absolute ethyl alcohol containing 0.015g NaOH and water, current density range 180-220mA/cm
2.React after 2 hours, remove graphite impurities by centrifuging.The centrifugate obtained is put into bag filter (1000Da), dialyse 24 hours with pure water, finally obtain boron doped graphene quantum dot aqueous solution.
(4) 0.02gNaOH and 0.035g ClCH is added in the boron doped graphene quantum dot aqueous solution obtained to 10ml step (3)
2cOOH, ultrasonic 3 hours (frequency: 40KHZ, power: 200W).450 μ L N-hydroxysuccinimide (NHS, 150mg/ml) and 300 μ L 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides (EDC, 10mg/ml) are joined in said mixture and react 30 minutes.Then the hairpin DNA (100 μMs) of 150 μ L marking sulfhydryls is joined in said mixture, and slowly stirring is spent the night.Finally that potpourri is centrifugal, precipitation is electrochemiluminescence probe BGQDs-DNA.Electrochemiluminescence probe is dispersed in 0.1M Tris-EDTA (TE) buffer solution (pH 8.0) be stored in 4 DEG C for subsequent use.
(5) the AuNPs aqueous solution of 3 μ L is dropped in platinum electrode surface (being designated as Pt/Au), after natural drying at room temperature, the BGQDs-DNA dripping 2 μ L steps (4) more obtained is added drop-wise to Pt/Au surface, rinse electrode surface with 0.1M phosphate (PBS) buffer solution (pH 7.4) after 2 hours, obtain boron doped graphene quantum dot electrochemical luminous sensor.
(6) the boron doped graphene quantum dot electrochemical luminous sensor that step (5) obtains is immersed in the TE damping fluid of the miRNA-20a containing variable concentrations, hybridize 1 hour under 37 DEG C of water bath condition, then use 0.1M phosphate (PBS) buffer solution (pH 7.4) to rinse electrode surface.With the electrode finally obtained for working electrode, platinum electrode is to electrode, and silver/silver chloride electrode is contrast electrode, and utilize multi-channel chemiluminescence test macro to detect electrochemical luminescence signals, detection is containing 0.1M KCl and 0.05M K
2s
2o
80.1M PBS buffer solution (pH=7.4) in carry out, voltage range is 0.7 ~ 1.15V, sweep speed 0.1V/s, and photomultiplier high pressure is 600V.
(7) in step (6) along with the increase of miRNA-20a concentration in sample, the electrochemical luminescence signals of system constantly declines, within the scope of 5 ~ 10000pM, electrochemiluminescence and miRNA-20a concentration have good linear relationship, linearly dependent coefficient R=0.982.
(8) mensuration of miRNA-20a concentration in sample is configured:
With work damping fluid, (0.1M PBS contains 0.1M KCl and 0.05M K
2s
2o
8, pH 7.4) and configure the miRNA-20a testing sample that activity is 7pM, detect by the method for step (6), the standard working curve that testing result and step (7) obtain contrasts, and calculates the concentration value of miRNA-20a.The concentration that experimental result measures miRNA-20a is 7.5pM, and the recovery is 107.1%, relative standard deviation RSD is 2.34% (n=3).
The miRNA-20a sequence of embodiment 3 and DNA sequence dna as shown in table 1.
Table 1miRNA-20a sequence and DNA sequence dna
Embodiment 4
The mensuration of miRNA-20a content in configuration sample:
(1) under ice bath state, the 11.5ml concentrated sulphuric acid to be joined in 0.5g high purity graphite powder and to stir, then 1.5g potassium permanganate being joined in said mixture several times, Keep agitation 24 hours.Add 23ml water and static 15 minutes, add 70ml water and 5ml H subsequently
2o
2cessation reaction.By potpourri centrifuging, and be hydrochloric acid and the water respectively washing and precipitating of 5% by concentration, finally obtain graphite oxide.
(2) 0.002g H is added in the graphite oxide obtained to step (1)
3bO
3, within ultrasonic 10 minutes, make it mix.Then potpourri is transferred in glass tube, in air dry oven, is heated to 80 DEG C of insulations 12 hours.By products in water cleaning several times, then vacuum freeze drying, finally obtains boron doped Graphene rod.
(3) the boron doped Graphene rod obtained with step (2) is working electrode, with graphite rod for prepare boron doped graphene quantum dot to electrode by electrochemical method.Electrolytic solution is the mixed liquor (volume ratio=99:1) of 10ml absolute ethyl alcohol containing 0.01g NaOH and water, current density range 200-250mA/cm
2.React after 2 hours, remove graphite impurities by centrifuging.The centrifugate obtained is put into bag filter (1000Da), dialyse 24 hours with pure water, finally obtain boron doped graphene quantum dot aqueous solution.
(4) 0.02gNaOH and 0.03g ClCH is added in the boron doped graphene quantum dot aqueous solution obtained to 15ml step (3)
2cOOH, ultrasonic 3 hours (frequency: 40KHZ, power: 200W).400 μ L N-hydroxysuccinimide (NHS, 150mg/ml) and 250 μ L 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides (EDC, 10mg/ml) are joined in said mixture and react 30 minutes.Then the hairpin DNA (100 μMs) of 75 μ L marking sulfhydryls is joined in said mixture, and slowly stirring is spent the night.Finally that potpourri is centrifugal, precipitation is electrochemiluminescence probe BGQDs-DNA.Electrochemiluminescence probe is dispersed in 0.1M Tris-EDTA (TE) buffer solution (pH 8.0) be stored in 4 DEG C for subsequent use.
(5) the AuNPs aqueous solution of 4 μ L is dropped in platinum electrode surface (being designated as Pt/Au), after natural drying at room temperature, the BGQDs-DNA dripping 4 μ L steps (4) more obtained is added drop-wise to Pt/Au surface, rinse electrode surface with 0.1M phosphate (PBS) buffer solution (pH 7.4) after 2 hours, obtain boron doped graphene quantum dot electrochemical luminous sensor.
(6) the boron doped graphene quantum dot electrochemical luminous sensor that step (5) obtains is immersed in the TE damping fluid of the miRNA-20a containing variable concentrations, hybridize 1.5 hours under 37 DEG C of water bath condition, then use 0.1M phosphate (PBS) buffer solution (pH 7.4) to rinse electrode surface.With the electrode finally obtained for working electrode, platinum electrode is to electrode, and silver/silver chloride electrode is contrast electrode, and utilize multi-channel chemiluminescence test macro to detect electrochemical luminescence signals, detection is containing 0.1M KCl and 0.05M K
2s
2o
80.1M PBS buffer solution (pH=7.4) in carry out, voltage range is 0.7 ~ 1.15V, sweep speed 0.1V/s, and photomultiplier high pressure is 600V.
(7) in step (6) along with the increase of miRNA-20a concentration in sample, the electrochemical luminescence signals of system constantly declines, within the scope of 10 ~ 10000pM, electrochemiluminescence and miRNA-20a concentration have good linear relationship, linearly dependent coefficient R=0.987.
(8) mensuration of miRNA-20a concentration in sample is configured:
With work damping fluid, (0.1M PBS contains 0.1M KCl and 0.05M K
2s
2o
8, pH 7.4) and configure the miRNA-20a testing sample that activity is 20pM, detect by the method for step (6), the standard working curve that testing result and step (7) obtain contrasts, and calculates the concentration value of miRNA-20a.The concentration that experimental result measures miRNA-20a is 19.7pM, and the recovery is 98.5%, relative standard deviation RSD is 5.49% (n=3).
The miRNA-20a sequence of embodiment 4 and DNA sequence dna as shown in table 2.
Table 2miRNA-20a sequence and DNA sequence dna
Embodiment 5
The mensuration of miRNA-20a content in configuration sample:
(1) under ice bath state, the 60ml concentrated sulphuric acid to be joined in 2g high purity graphite powder and to stir, then 6g potassium permanganate being joined in said mixture several times, Keep agitation 24 hours.Add 92ml water and static 15 minutes, add 280ml water and 20ml H subsequently
2o
2cessation reaction.By potpourri centrifuging, and be hydrochloric acid and the water respectively washing and precipitating of 5% by concentration, finally obtain graphite oxide.
(2) 0.008g H is added in the graphite oxide obtained to step (1)
3bO
3, within ultrasonic 10 minutes, make it mix.Then potpourri is transferred in glass tube, in air dry oven, is heated to 80 DEG C of insulations 12 hours.By products in water cleaning several times, then vacuum freeze drying, finally obtains boron doped Graphene rod.
(3) the boron doped Graphene rod obtained with step (2) is working electrode, with graphite rod for prepare boron doped graphene quantum dot to electrode by electrochemical method.Electrolytic solution is the mixed liquor (volume ratio=99.5:0.5) of 20ml absolute ethyl alcohol containing 0.01g NaOH and water, current density range 260-300mA/cm
2.React after 1.5 hours, remove graphite impurities by centrifuging.The centrifugate obtained is put into bag filter (1000Da), dialyse 48 hours with pure water, finally obtain boron doped graphene quantum dot aqueous solution.
(4) 0.025gNaOH and 0.035g ClCH is added in the boron doped graphene quantum dot aqueous solution obtained to 20ml step (3)
2cOOH, ultrasonic 3 hours (frequency: 40KHZ, power: 200W).500 μ L N-hydroxysuccinimide (NHS, 150mg/ml) and 350 μ L 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides (EDC, 10mg/ml) are joined in said mixture and react 30 minutes.Then the hairpin DNA (100 μMs) of 100 μ L marking sulfhydryls is joined in said mixture, and slowly stirring is spent the night.Finally that potpourri is centrifugal, precipitation is electrochemiluminescence probe BGQDs-DNA.Electrochemiluminescence probe is dispersed in 0.1M Tris-EDTA (TE) buffer solution (pH 8.0) be stored in 4 DEG C for subsequent use.
(5) the AuNPs aqueous solution of 6 μ L is dropped in platinum electrode surface (being designated as Pt/Au), after natural drying at room temperature, the BGQDs-DNA dripping 6 μ L steps (4) more obtained is added drop-wise to Pt/Au surface, rinse electrode surface with 0.1M phosphate (PBS) buffer solution (pH 7.4) after 2 hours, obtain boron doped graphene quantum dot electrochemical luminous sensor.
(6) the boron doped graphene quantum dot electrochemical luminous sensor that step (5) obtains is immersed in the TE damping fluid of the miRNA-20a containing variable concentrations, hybridize 2 hours under 37 DEG C of water bath condition, then use 0.1M phosphate (PBS) buffer solution (pH 7.4) to rinse electrode surface.With the electrode finally obtained for working electrode, platinum electrode is to electrode, and silver/silver chloride electrode is contrast electrode, and utilize multi-channel chemiluminescence test macro to detect electrochemical luminescence signals, detection is containing 0.1M KCl and 0.05M K
2s
2o
80.1M PBS buffer solution (pH=7.4) in carry out, voltage range is 0.7 ~ 1.15V, sweep speed 0.1V/s, and photomultiplier high pressure is 600V.
(7) in step (6) along with the increase of miRNA-20a concentration in sample, the electrochemical luminescence signals of system constantly declines, within the scope of 20 ~ 1000pM, electrochemiluminescence and miRNA-20a concentration have good linear relationship, linearly dependent coefficient R=0.991.
(8) mensuration of miRNA-20a concentration in sample is configured:
With work damping fluid, (0.1M PBS contains 0.1M KCl and 0.05M K
2s
2o
8, pH 7.4) and configure the miRNA-20a testing sample that activity is 100pM, detect by the method for step (6), the standard working curve that testing result and step (7) obtain contrasts, and calculates the concentration value of miRNA-20a.The concentration that experimental result measures miRNA-20a is 112.7pM, and the recovery is 112.7%, relative standard deviation RSD is 2.34% (n=3).
The miRNA-20a sequence of embodiment 5 and DNA sequence dna as shown in table 3.
Table 3miRNA-20a sequence and DNA sequence dna
Claims (5)
1., for detecting a preparation method for the boron doped graphene quantum dot electrochemical luminous sensor of miRNA-20a, it is characterized in that, step is as follows:
(1) under ice bath state, the concentrated sulphuric acid to be joined in dag and to stir, then potassium permanganate being joined in above-mentioned potpourri, Keep agitation 20-48 hour; Add H
2o static 10-60 minute, add H subsequently
2o and H
2o
2cessation reaction; By potpourri centrifuging, and with concentration be 5% hydrochloric acid and water clean respectively, obtain graphite oxide; The mol ratio of each material wherein used is the concentrated sulphuric acid: dag: potassium permanganate: H
2o:H
2o
2=1:0.3:0.02:10:0.2;
(2) H is added in the graphite oxide obtained to step (1)
3bO
3, potpourri is to 60-100 DEG C of insulation 6-12 hour; Products in water is cleaned, vacuum freeze drying, obtain boron doped Graphene rod; Wherein graphite oxide and H
3bO
3mass ratio be 250-5000;
(3) the boron doped Graphene rod obtained with step (2) is working electrode, with graphite rod for prepare boron doped graphene quantum dot to electrode by electrochemical method, electrolytic solution is the mixed liquor of absolute ethyl alcohol containing 25-50mM NaOH and water, volume ratio=the 99:1-99.5:0.5 of absolute ethyl alcohol and water, current density 180-300mA/cm
2, reaction 1-3 hour, graphite impurities is removed in centrifuging; By the centrifugate dialysis 24-48 hour obtained, dialysis aperture is less than 5000Da, obtains boron doped graphene quantum dot aqueous solution;
(4) NaOH and ClCH is added in the boron doped graphene quantum dot aqueous solution obtained to step (3)
2cOOH, ultrasonic; Add the N-hydroxysuccinimide of 150mg/ml and 1-ethyl-3-(3-the dimethyl aminopropyl)-carbodiimides of 10mg/ml again, reaction 10-60 minute; And then add the hairpin DNA of 100 μMs of marking sulfhydryls, slowly stir; Finally that potpourri is centrifugal, precipitation is electrochemiluminescence probe BGQDs-DNA; Electrochemiluminescence probe BGQDs-DNA is dispersed in 0.1MTris-EDTApH=8.0 buffer solution, be stored in 4 DEG C for subsequent use; Wherein NaOH and ClCH
2the mass ratio of COOH is the volume ratio 1-3 of 0.25-0.5, NHS and EDC, the volume ratio 120-1200 of the hairpin DNA of boron doped graphene quantum dot aqueous solution and marking sulfhydryl;
(5) AuNPs aqueous solution is dropped in platinum electrode surface, be designated as Pt/Au, AuNPs aqueous solution is at the volume 2-8 μ L/mm of platinum electrode surface
2, drying at room temperature, then the BGQDs-DNA that step (4) is obtained is added drop-wise to Pt/Au surface, BGQDs-DNA is at the volume 2-6 μ L/mm on Pt/Au surface
2, rinse electrode surface with 0.1M phosphate pH=7.4 buffer solution after 1-3 hour, obtain boron doped graphene quantum dot electrochemical luminous sensor.
2. preparation method according to claim 1, it is characterized in that, in step (4), the hairpin DNA total bases of marking sulfhydryl is 31-39, its main body section, and corresponding identification miRNA-20a base sequence is 5 '-CTACCTGCACTATAAGCACTTTA-3 '.
3. preparation method according to claim 1 and 2, it is characterized in that, clean method is as follows before use for platinum electrode in step (5): ground in the suspending liquid of the aluminum oxide polishing powder of 0.05-1 μm of particle diameter by platinum electrode, absolute ethyl alcohol and high purity water ultrasonic cleaning is used successively, at N after grinding
2protection is lower dry.
4. the application of the boron doped graphene quantum dot electrochemical luminous sensor that the preparation method described in claim 1 or 2 obtains, it is characterized in that, boron doped graphene quantum dot electrochemical luminous sensor is immersed in the TE damping fluid of the miRNA-20a contained, the concentration of miRNA-20a is 1-50000pM, 0.5-3 hour is hybridized under 37 DEG C of water bath condition, then electrode surface is rinsed with 0.1M phosphate pH=7.4 buffer solution, the electrode obtained is working electrode, platinum electrode is to electrode, silver/silver chloride electrode is contrast electrode, multi-channel chemiluminescence test macro is utilized to detect electrochemical luminescence signals, detection is containing 0.1M KCl and 0.05MK
2s
2o
80.1M PBS pH=7.4 buffer solution in, voltage range is 0.7 ~ 1.15V, sweep speed 0.1V/s, and photomultiplier high pressure is 600V.
5. the application of boron doped graphene quantum dot electrochemical luminous sensor that obtains of preparation method according to claim 3, it is characterized in that, boron doped graphene quantum dot electrochemical luminous sensor is immersed in the TE damping fluid of the miRNA-20a contained, the concentration of miRNA-20a is 1-50000pM, 0.5-3 hour is hybridized under 37 DEG C of water bath condition, then electrode surface is rinsed with 0.1M phosphate pH=7.4 buffer solution, the electrode obtained is working electrode, platinum electrode is to electrode, silver/silver chloride electrode is contrast electrode, multi-channel chemiluminescence test macro is utilized to detect electrochemical luminescence signals, detection is containing 0.1M KCl and 0.05MK
2s
2o
80.1M PBS pH=7.4 buffer solution in, voltage range is 0.7 ~ 1.15V, sweep speed 0.1V/s, and photomultiplier high pressure is 600V.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510170878.4A CN104764782B (en) | 2015-04-10 | 2015-04-10 | A kind of preparation and its application for detecting the boron doped graphene quantum dot electrochemical luminescence sensor of miRNA 20a |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510170878.4A CN104764782B (en) | 2015-04-10 | 2015-04-10 | A kind of preparation and its application for detecting the boron doped graphene quantum dot electrochemical luminescence sensor of miRNA 20a |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104764782A true CN104764782A (en) | 2015-07-08 |
| CN104764782B CN104764782B (en) | 2017-06-13 |
Family
ID=53646746
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510170878.4A Active CN104764782B (en) | 2015-04-10 | 2015-04-10 | A kind of preparation and its application for detecting the boron doped graphene quantum dot electrochemical luminescence sensor of miRNA 20a |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104764782B (en) |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105223189A (en) * | 2015-09-24 | 2016-01-06 | 福建医科大学 | A kind of Electrochemiluminescsensor sensor based on tungsten oxide quanta point material |
| CN105862057A (en) * | 2016-04-15 | 2016-08-17 | 北京科技大学 | Phosphorus-doped graphene quantum dot and electrochemistry preparing method thereof |
| CN106323951A (en) * | 2016-08-24 | 2017-01-11 | 济南大学 | Preparing method for electrogenerated chemiluminescence cell sensor flexible detection of cancer cells |
| CN106770143A (en) * | 2017-02-28 | 2017-05-31 | 济南大学 | A kind of biology sensor of detection MiRNA and preparation method thereof |
| CN107121454A (en) * | 2017-04-17 | 2017-09-01 | 云南大学 | A kind of different element doping graphene quantum dots are respectively with molecular engram compound gas sensitive and preparation method and application |
| 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 |
| CN108559497A (en) * | 2018-04-28 | 2018-09-21 | 华南师范大学 | Generate the doped graphene quantum dot and its preparation method and application of second harmonic |
| CN109856214A (en) * | 2019-03-07 | 2019-06-07 | 广西师范学院 | The electrode modification method and its application of electrochemical signals can be amplified |
| CN109856215A (en) * | 2019-03-07 | 2019-06-07 | 广西师范学院 | The method of carboxylated graphene oxide modified electrode detection miRNA-21 |
| CN111122551A (en) * | 2019-12-11 | 2020-05-08 | 南京大学 | Three-channel electrochemical luminescence sensor and application thereof in cell detection |
| CN112986361A (en) * | 2021-04-27 | 2021-06-18 | 上海执诚生物科技有限公司 | Application of electrochemical biosensor based on gold-graphene quantum dots in detection of ctDNA in cells |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703594A (en) * | 2012-06-12 | 2012-10-03 | 华南师范大学 | Method for detecting miRNA (micro ribonucleic acid) based on graphene/nucleic acid dye platform |
| CN103922319B (en) * | 2013-01-15 | 2016-03-23 | 海洋王照明科技股份有限公司 | Boron doped graphene nano belt and preparation method thereof |
-
2015
- 2015-04-10 CN CN201510170878.4A patent/CN104764782B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| HUANSHUN YIN,ET AL.: "Electrochemical determination of microRNA-21 based on graphene, LNA integrated molecular beacon, AuNPs and biotin multifunctional bio bar codes and enzymatic assay system", 《BIOSENSORS AND BIOELECTRONICS》 * |
| TINGTING ZHANG,ET AL.: "Electrolytic exfoliation synthesis of boron doped graphene quantum dots: a new luminescent material for electrochemiluminescence detection of oncogene microRNA-20a", 《ELECTROCHIMICA ACTA》 * |
| ZETAN FAN,ET AL.: "Surrounding media sensitive photoluminescence of boron-doped graphene quantum dots for highly fluorescent dyed crystals, chemical sensing and bioimaging", 《CARBON》 * |
Cited By (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105223189B (en) * | 2015-09-24 | 2019-02-01 | 福建医科大学 | A kind of Electrochemiluminescsensor sensor based on tungsten oxide quanta point material |
| CN105223189A (en) * | 2015-09-24 | 2016-01-06 | 福建医科大学 | A kind of Electrochemiluminescsensor sensor based on tungsten oxide quanta point material |
| CN105862057A (en) * | 2016-04-15 | 2016-08-17 | 北京科技大学 | Phosphorus-doped graphene quantum dot and electrochemistry preparing method thereof |
| CN105862057B (en) * | 2016-04-15 | 2018-07-31 | 北京科技大学 | A kind of p-doped graphene quantum dot and its electrochemical preparation method |
| CN106323951A (en) * | 2016-08-24 | 2017-01-11 | 济南大学 | Preparing method for electrogenerated chemiluminescence cell sensor flexible detection of cancer cells |
| CN106323951B (en) * | 2016-08-24 | 2019-03-29 | 济南大学 | The preparation method of the electrogenerated chemiluminescence cell sensor of super sensitivity detection cancer cell |
| CN106770143A (en) * | 2017-02-28 | 2017-05-31 | 济南大学 | A kind of biology sensor of detection MiRNA and preparation method thereof |
| CN106770143B (en) * | 2017-02-28 | 2020-03-13 | 济南大学 | Biosensor for detecting MiRNA and preparation method thereof |
| CN107121454A (en) * | 2017-04-17 | 2017-09-01 | 云南大学 | A kind of different element doping graphene quantum dots are respectively with molecular engram compound gas sensitive and preparation method and application |
| CN107121454B (en) * | 2017-04-17 | 2019-08-30 | 云南大学 | A kind of difference element doping graphene quantum dot is respectively with molecular engram compound gas sensitive and the preparation method and application thereof |
| 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 |
| CN108559497A (en) * | 2018-04-28 | 2018-09-21 | 华南师范大学 | Generate the doped graphene quantum dot and its preparation method and application of second harmonic |
| CN108559497B (en) * | 2018-04-28 | 2021-03-30 | 华南师范大学 | Doped graphene quantum dot for generating second harmonic and preparation method and application thereof |
| CN109856214A (en) * | 2019-03-07 | 2019-06-07 | 广西师范学院 | The electrode modification method and its application of electrochemical signals can be amplified |
| CN109856215A (en) * | 2019-03-07 | 2019-06-07 | 广西师范学院 | The method of carboxylated graphene oxide modified electrode detection miRNA-21 |
| CN109856215B (en) * | 2019-03-07 | 2021-09-17 | 宁波远志立方能源科技有限公司 | Method for detecting miRNA-21 by using carboxylated graphene oxide modified electrode |
| CN111122551A (en) * | 2019-12-11 | 2020-05-08 | 南京大学 | Three-channel electrochemical luminescence sensor and application thereof in cell detection |
| CN112986361A (en) * | 2021-04-27 | 2021-06-18 | 上海执诚生物科技有限公司 | Application of electrochemical biosensor based on gold-graphene quantum dots in detection of ctDNA in cells |
| CN112986361B (en) * | 2021-04-27 | 2022-02-01 | 上海执诚生物科技有限公司 | Application of electrochemical biosensor based on gold-graphene quantum dots in detection of ctDNA in cells |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104764782B (en) | 2017-06-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104764782A (en) | Preparation of boron-doped graphene quantum dot electrochemiluminescence sensor for detecting miRNA-20a and application of sensor | |
| CN106706607B (en) | High quantum production rate electrogenerated chemiluminescence gold nano cluster probe and preparation method thereof | |
| CN103913443B (en) | A kind of purposes based on DNA-silver nanoclusters (DNA-Ag NCs) aptamer sensor and detection method | |
| CN103063715B (en) | Method for detecting surviving gene based on graphene-gold composite material electrochemical DNA (Deoxyribose Nucleic Acid) biosensor | |
| CN104155445B (en) | Preparation and application of unmarked electrochemiluminescent tumor marker immunosensor | |
| CN107543852B (en) | A kind of Electrochemiluminescsensor sensor based on functional metal organic framework materials | |
| CN103364353B (en) | A kind of aptamer nanogold Resonance Rayleigh Scattering Spectra method measuring lysozyme | |
| CN104237344A (en) | Electrochemical aptamer electrode for kanamycin detection and preparation method of electrochemical aptamer electrode | |
| CN103217406B (en) | Based on halfcystine and the Cu of Au/Ag core/shell quantum dot 2+the method for making of fluorescence probe | |
| JP7039481B2 (en) | Branched-chain amines in electrochemical luminescence detection | |
| CN102818893A (en) | Preparation and application of Au@Pd core-shell material constructed lung cancer tumor marker immunosensor | |
| CN102854235B (en) | Preparation method and application of gynecological tumor marker immune sensor constructed with ordered mesoporous carbon | |
| CN104655616A (en) | Preparation method and application of electrochemiluminescence aptamer sensor for detecting tumor marker MUC1 | |
| CN106645107A (en) | CdSe/ZnS quantum dot nano-cluster based electrochemiluminescence biological sensor, as well as manufacturing method and application thereof | |
| CN107044978B (en) | Glutathione electrogenerated chemiluminescence measuring method based on gold nano cluster probe | |
| CN106066324A (en) | A kind of preparation method and application of electroluminescent chemiluminescence biosensor label | |
| CN104049087B (en) | A kind of preparation method and application thereof detecting the biology sensor of L-Histidine | |
| CN104330453A (en) | Chiral sensor based on chitosan and derivatives of chitosan and preparation method of chiral sensor | |
| CN103439319B (en) | Carbon nano-particles modified electrode electrochemiluminescence measures the method for bleomycin | |
| CN105044194A (en) | Method used for detecting acrylamide concentration of solutions | |
| CN103616357A (en) | Visual biosensor device and preparation method thereof | |
| CN105886596A (en) | Cervical cancer cell detection kit | |
| CN103487573B (en) | Electrogenerated chemiluminescence immunosensor for detecting human interleukin 6 as well as preparation method and detection method of immunosensor | |
| CN103616420B (en) | The Optical Electro-Chemistry assay method of glucose in serum | |
| CN106198503B (en) | A kind of electrochemiluminescence sandwich biosensor and preparation and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| EXSB | Decision made by sipo to initiate substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |