CN104316705A - Preparation method and application of hybridization indicator 5,7-binitro-2-sulfo-acridone - Google Patents
Preparation method and application of hybridization indicator 5,7-binitro-2-sulfo-acridone Download PDFInfo
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Abstract
The invention provides a preparation method and application of a hybridization indicator 5,7-binitro-2-sulfo-acridone and provides a preparation method of an electrochemical biosensor based on a triple signal amplification technology of film modified electrode, exonuclease III auxiliary target sequence circulation and DNA long-range self-assembly by using the 5,7-binitro-2-sulfo-acridone as an indicator. The 5,7-binitro-2-sulfo-acridone is used for detecting extra-high sensitivity and high specificity of a gene relative to a target sequence c-erbB-2 relative to lung cancer. As the linear range of the electrochemical biosensor is 2 aM-50 fM, detection limit reaches 0.5 am, and sequences which are completely complementary and mismatched to each other can be better recognized, so that ultralow content of target sequence in an actual lung cancer serum sample can be clinically detected.
Description
Technical field
The present invention relates to a kind of preparation method and purposes of hybridization indicator 5,7-dinitro-2-sulfo group-acridone, belong to organic synthesis field.
Background technology
Lung cancer is one of major malignant tumor threatening human life in the world, its M & M rises year by year in recent years, occupied first of malignant tumour, wherein non-small cell lung cancer (non-small cell lung cancer, NSCLC) accounts for 80% of all lung cancer.At present, the research for lung cancer therapy obtains greater advance, total but 5 of lung cancer years survival rates still only have 15%, even if I A phase NSCL patient, within postoperative 5 years, survival rate also only has 80%.Therefore, lung cancer Rapid&Early diagnosis for the selection of therapeutic scheme and prognostic evaluation most important.
In recent years, along with the development of the technology such as biological chemistry, molecular biology and immunology, to deepening continuously of lung cancer research, medical workers achieve breakthrough in the screening of lung cancer tumor mark, the marks, particularly tumor markers c-erbB-2 etc. such as the multiple albumen relevant to lung cancer, gene are found at present.Have researchist to find, c-erbB-2 is unconventionality expression in Serum of Patients with Lung Cancer, and stable in properties, become one of novel tumor markers of reliable early diagnosis lung cancer just gradually.Many scholars study the gene marker in close relations with NSCLC by PCR method from gene level.Although PCR can realize the quantitative detection of gene expression, owing to existing, operating process is loaded down with trivial details, cost is high, need the shortcomings such as special technician, limits the application detected in clinical lung cancerous diagnose.Therefore, develop a kind of easy, quick, sensitive, economic c-erbB-2 detection technique and there is extremely wide application prospect, solving this clinical urgent practical problems of NSCLC early diagnosis by being expected to, being extremely important.
Biology sensor utilizes biologic specificity identifying to detect the Sensitive Detector of protein, gene, antibody, toxin etc., is the Measurement for Biotechnique of a new generation.Wherein electrochemical biosensor is as sensitive element by biomaterial (DNA, enzyme, antigen, cell, tissue etc.), electrode is as conversion element, with the sensor that electromotive force or electric current are feature detection signal, the selectivity of its biomolecule identification determines this sensor and has high selectivity.Therefore, scholars devise a lot for detecting the electrochemical biosensor strategy of DNA.Although these sensing strategy can realize the sensitive specific detection of DNA, also there is following defect: 1) complicated and time consumption, lack economy.2) traditional indicator exists expensive, all can have an effect with single double-stranded DNA, and hybridization check specificity is not high.3) less stable.4) some functional group's labeling method, background signal is large, and sensitivity is low.5) false positive and false-negative phenomenon still inevitable.These electric bio-sensing strategies are wanted to replace clinical conventional art in actual sample detects, and still face huge difficulty.Therefore, be necessary to continue a kind of electrochemical biosensor new method of research.
Described belowly design and synthesize a kind of novel acridone derivatives-" 5 had compared with highly-water-soluble and electrochemical activity for the present inventor, 7-dinitro-2-sulfo group-acridone ", and as hybridization indicator, have developed a kind of electrochemical sensor measuring c-erbB-2 related gene---based on " film modified electrode ", the electrochemica biological sensor of " exonuclease III auxiliary target sequence loops " and " DNA is long apart from formula self assembly " triple signal amplification technique: 1B is modified at glassy carbon electrode surface, by covalent modification method, hair fastener DNA probe is fixed on the terminal carboxyl group of polylysine.By the hybridization of DNA probe and target DNA, cut after circulation through exonuclease III again, flexible short oligonucleotide sequence is left on modified electrode surface, it can be hybridized with assist probes Gene A P1 and AP2 and forms super sandwich structure, select 5,7-dinitro-2-sulfo group-acridone is as hybridization indicator, and it can be embedded in the double-spiral structure of DNA, successfully realizes the development of the electrochemica biological sensor of triple signal amplification technique.Because polylysine contains carboxyl, be conducive to fixing at electrode surface of DNA, " electric conductivity that polylysine is good ", " exonuclease III auxiliary target sequence loops " and " DNA is long apart from formula self assembly " all significantly enhance the detection sensitivity of sensor in addition.Thus establish the c-erbB-2 related gene testing method of high sensitivity, high specific.From now on, we will be successfully applied in this new technology on the basis of lung cancer early diagnosis, and be applied to the early diagnosis of other types tumour and screen in anti-cancer agent work, thus the present invention has huge potential using value and profound significance.
Summary of the invention
The object of the present invention is to provide a kind of hybridization indicator 5, the preparation method of 7-dinitro-2-sulfo group-acridone and purposes, a kind of preparation method of the electrochemica biological sensor based on " film modified electrode ", " exonuclease III auxiliary target sequence loops " and " DNA is long apart from formula self assembly " triple signal amplification technique is provided, and uses it for lung cancer and to be correlated with the ultra-high sensitive of target sequence c-erbB-2 related gene, high specific detection.
For achieving the above object, the present invention adopts following technical scheme:
A kind of hybridization indicator 5,7-dinitro-2-sulfo group-acridone, described 5,7-dinitro-2-sulfo group-acridone structural formulas are
.
A kind of preparation method of hybridization indicator 5,7-dinitro-2-sulfo group-acridone, described step is: take 0.6 g acridone, adds the dense H of 5 mL
2sO
4, after stirring and dissolving, be heated to 100 DEG C of reaction 30 min, poured into by solution in frozen water (0 ~ 4 DEG C) while hot, solid is separated out, and filtration drying obtains light green solid 2-sulfo group-acridone; Take 0.6 g 2-sulfo group-acridone, add 3 mL 36 % acetic acid, add the red fuming nitric acid (RFNA) of 0.36 mL and the glacial acetic acid of 0.8 mL more successively, at 56 ~ 60 DEG C, stirring reaction 2 h, pours in frozen water by solution while hot, solid is separated out, filter, with 10 ~ 20 mL water flushings, and with the further recrystallization purifying of absolute ethyl alcohol, obtain yellow needles powder 5,7-dinitro-2-sulfo group-acridone.
The application of a kind of hybridization indicator 5,7-dinitro-2-sulfo group-acridone on electrochemical sensor.
Described electrochemical sensor preparation method comprises: the 1) pre-service of electrode
By the Al of glass-carbon electrode at 0.05 μm
2o
3powder is polished, and priority absolute ethyl alcohol and distilled water ultrasonic vibration 5 min, to remove attached impurity at the electrode surface, electrode surface is characterized with the 10 mM potassium ferricyanides, until front and back peak position voltage difference is less than 80 V, distilled water is cleaned and is dried rear taking-up, and nitrogen dries up;
2) preparation of probe modification electrode
Glass-carbon electrode after process is placed in pH 8.0, the 0.2 M PBS damping fluid containing 0.01 M/L lysine, close with cyclic voltammetry electricity and modify polylysine film, use distilled water cleaning down electrode again and at room temperature drying, obtain Mercapto-group modification electrode, be inverted by Mercapto-group modification electrode, surface drips 20 μ L coupling activator, room temperature nature evaporate to dryness, then clean 10 s with ultrapure water, remove the coupling activator not being combined in modified electrode surface, nitrogen dries up stand-by; Then the probe gene P solution of 10 μ L, 1 μM of hairpin structure is dripped at electrode surface, room temperature nature evaporate to dryness, removes with ultrapure water cleaning the probe gene P not being combined in electrode surface, then uses 10 mM pH7.0 PBS buffer solution for cleaning, drying at room temperature, obtained hair fastener probe modification electrode;
3) enzyme auxiliary target sequence loops
Hair fastener probe modification electrode is placed in target sequence c-erbB-2 related gene (1 aM ~ 100 fM) solution of a series of concentration, after hybridizing 1 h under room temperature, cleaning dries up, electrode after hybridization is placed in the damping fluid of 10 U exonuclease III, after reacting 30 min at 37 DEG C, cleaning dries up stand-by;
4) DNA is long apart from formula self assembly and Electrochemical Detection
Assist probes AP1 and the AP2 solution (being synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd) droplet of getting 10 μ L 1 μM are respectively coated in above-mentioned electrode surface, and carry out the self assembly of DNA long distance formula under room temperature, after 2 h, cleaning dries up; Containing 5 mM K respectively
3[Fe (CN)
6]/K
4[Fe (CN)
6] 100 mM pH 7.0 PBS+0.1 M KCl buffer solution of (1:1) measure the CV curve of different modifying electrodes, sweep speed is 0.05 V/s, and sampling interval is 0.001 V, and time of repose is 2 s; Be placed in the pH 7.0 PBS buffer solution containing 100 μM of 5,7-dinitro-2-sulfo group-acridone DSA by long for the DNA electrode after formula self assembly, after 20 min, cleaning dries up; Then electrode is placed in pH 5.0 phosphate PB buffer solution and scans CV and square wave voltammetry (SWVs) curve.
The condition of described cyclic voltammetry is: voltage-1.0 ~+2.4 V, and sweep speed is 100 mVs-1, and characterizing the number of turns is 14 circles.
Described coupling activator is pH 7.4 phosphate buffer containing the EDC of 5 mM and the NHS of 8 mM.
The sweep speed of described CV method is 0.50 V/s, and sampling interval is 0.001 V, and time of repose is 2 s; The scanning potential range of square wave voltammetry is-0.2 V ~+0.15 V, and current potential increment is 0.005 V, and amplitude is 0.025 V.
Described probe P sequence is 5'-NH2-AAA AAT TTA TTT GAT AGG CGA ACT ATT TGT TTT TAA TAT CAA ATA ATG GTT-3'; Described probe AP1 sequence is 5'-CAA AAT ATA T GA TAG GCG AA-3'; Probe AP2 sequence is 5'-ATA TAT TTT GTT CGC CTA TC-3';
The invention has the advantages that:
1, synthesized a kind of novel electrochemical hybridization indicator acridone derivatives-" 5,7-dinitro-2-sulfo group-acridone " (DSA), it has higher water-soluble and electrochemical activity, and stronger to the selectivity of single double-strand.
2, take DSA as hybridization indicator, have developed a kind of electrochemica biological sensor based on " film modified electrode ", " exonuclease III auxiliary target sequence loops " and " DNA is long apart from formula self assembly " triple signal amplification technique, lung cancer can be used it for and to be correlated with the ultra-high sensitive of target sequence c-erbB-2 related gene, high specific detection.
3, the range of linearity of this sensor is 2 aM ~ 50 fM, and detection limit reaches 0.5 aM, and can identify complete complementary and mismatch preferably, can realize the detection of super low loading target sequence in clinical practice Sera of Lung Cancer sample.
Accompanying drawing explanation
Fig. 1 is the mass spectrogram of electrochemical hybridization indicator 5,7-dinitro-2-sulfo group-acridone.
Fig. 2 is the [Fe (CN) of different modifying electrode of the present invention
6]
3-/4-phenogram.[Fe (CN) in figure on different modifying electrode
6]
3-/4-characterize: (c) and rear (b), DNA long distance formula self assembly (d) before film modified electrode (a), exonuclease III auxiliary target sequence loops
Fig. 3 is electrochemical signals detection figure of the present invention.Electrochemical signals A, B, C, D, E, F, G of variable concentrations target dna compare.
Embodiment
followingly to describe the present invention in conjunction with the accompanying drawings and embodiments:
embodiment 1
The characterization data of DSA is as follows:
The ultimate analysis of DSA: C, 42.83 %; H, 1.99 %; N, 11.52 %. (calculated value: C, 42.75 %; H, 1.93 %; N, 11.50 %); The infrared analysis IR (KBr) ν: 3410 (υ NH) of DSA, 1592 (υ C-C), 1642 (υ C=C), 1389 (υ C-NO2), 1190 (υ SO2OH).
The mass spectrophotometry FAB-MS:m/z 366 ([M+1]+) of DSA.
Hydrogen analysis of spectrum 1H NMR (CDCl3, δ) of DSA: 9.04 (s, ArH), 8.76 (s, ArH), 8.13 (s, ArH), 7.85 (d, ArH), 6.87 (d, ArH), 4.21 (s, NH).
embodiment 2
Electrochemical sensor of the present invention comprises glass-carbon electrode GCE, and surface is coated with sensitive membrane, ExoIII enzyme, auxiliary sequencel AP1 and AP2.Sensitive membrane is made up of polylysine PLLy and fixing hair fastener probe gene P (5'-NH2-AAA AAT TTA TTT GAT AGG CGA ACT ATT TGT TTT TAA TAT CAA ATA ATG GTT-3'), the glass-carbon electrode that will secure DNA probe immerses containing the PBS(phosphate pH 7.0 of certain density complementary DNA (T1)) in buffer solution, carry out hybridization reaction.Electrode after hybridization is placed in the damping fluid of 10 U ExoIII enzymes, shear for 37 DEG C and eliminate, due to ExoIII can hold level with both hands from 3 ' progressively to degrade probe gene P, be completely degraded until hybridize with T1 the part forming double-strand, the portion gene being left not hybridize with T1 residues in electrode surface.Meanwhile, T1 is discharged by complete and can continue again to hybridize with another P.The cyclic process of hybridizing, degrading, being hybridized like this, a T1 just can make many digested degradeds of P.After above-mentioned circulation completes, probe gene P can be transformed into flexible short linear structures from hairpin structure.Assist probes Gene A P1(5'-CAAAATATATGATAGGCG AA-3' is dripped at electrode surface) and AP2(5'-ATATATTTTGTTCGCCTATC-3') solution, it is long apart from formula self assembly to carry out DNA under room temperature.Long electrode after formula self assembly is placed in the pH 7.0 PBS buffer solution containing 5,7-dinitro-2-sulfo group-acridone, makes it be embedded in the double-spiral structure of DNA.Namely Electrochemical Detection is can be used for after above-mentioned electrode clean dries up.
The preparation method of the sensor
1) preparation of electrochemical hybridization indicator 5,7-dinitro-2-sulfo group-acridone: take 0.6 g acridone, adds the dense H of 5 mL
2sO
4, after stirring and dissolving, be heated to 100 DEG C of reaction 30 min, pour in frozen water by solution while hot, solid is separated out, and filtration drying obtains light green solid 2-sulfo group-acridone; Take 0.6 g 2-sulfo group-acridone, add 3 mL 36 % acetic acid, then add the red fuming nitric acid (RFNA) of 0.36 mL and the glacial acetic acid of 0.8 mL successively, stirring reaction 2 h at 56 ~ 60 DEG C.While hot solution is poured into (0 ~ 4 DEG C) in frozen water, solid is separated out.Filter, rinse with 10 ~ 20 mL water, and with the further recrystallization purifying of absolute ethyl alcohol, obtain yellow needles powder 5,7-dinitro-2-sulfo group-acridone.
2) preparation of Mercapto-group modification glass-carbon electrode: the glass-carbon electrode after process is placed in 0.2 M PBS (pH 8.0) damping fluid containing 0.01 M 1B, close with cyclic voltammetry electricity and modify polylysine film, condition is: voltage-1.0 ~+2.4 V, sweep speed is 100 mVs-1, characterizing the number of turns is 14 circles, use distilled water cleaning down electrode again and at room temperature drying, obtain Mercapto-group modification electrode.
3) designing probe of hair fastener probe P, c-erbB-2 related gene, AP1 and AP2: hair fastener probe P:5'-NH2-AAA AAT TTA TTT GAT AGG CGA ACT ATT TGT TTT TAA TAT CAA ATA ATG GTT-3'(is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd); C-erbB-2 related gene: 5'-AAC CAT TAT TTG ATA TTA AAA CAA ATA GGC TTG-3' is specific sequence DNA (the c-erbB-2 related gene also synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd); AP1:5'-CAA AAT ATA T GA TAG GCG AA-3'(is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd); AP2:5'-ATA TAT TTT GTT CGC CTA TC-3'(is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd); Probe P is dissolved in the probe solution (prepared by 10 mMTris, 1.0 mM EDTA and 0.10 mM NaCl, and be adjusted to pH 7.4 with 10 mmol/L HCl) in TE damping fluid and make 100 μMs; Target dna is dissolved in pH7.0 PBS damping fluid the target dna solution making 100 μMs.
4) hair fastener probe gene fixing on modified electrode: the Mercapto-group modification glass-carbon electrode lysine that fully washing removing is unnecessary in ethanol, intermediate water.20 μ L coupling activator (phosphate buffer containing the pH 7.4 of the EDC of 5 mM and the NHS of 8 mM) are dripped again at electrode surface, room temperature nature evaporate to dryness, then clean 10 s with ultrapure water, remove the coupling activator not being combined in modified electrode surface, nitrogen dries up.Then the probe gene P solution of 10 μ L1 μM hairpin structures is dripped at electrode surface, the probe gene P covalency of end modified amino is fixed on the carboxyl of polylysine, room temperature nature evaporate to dryness, the P not being combined in electrode surface is removed with ultrapure water cleaning, use pH 7.0 PBS buffer solution for cleaning again, drying at room temperature, obtained hair fastener probe modification electrode.
5) enzyme auxiliary target sequence loops: target sequence c-erbB-2 related gene solution hair fastener probe modification electrode being placed in a series of concentration, after hybridizing 1 h under room temperature, cleaning dries up.Electrode after hybridization is placed in the damping fluid of 10 U exonuclease III, after reacting 30 min at 37 DEG C, cleaning dries up stand-by.
6) DNA is long apart from formula self assembly: get the assist probes AP1 of 10 μ L 1 μM and AP2 solution respectively and drip and be coated in above-mentioned electrode surface, and it is long apart from formula self assembly to carry out DNA under room temperature, and after 2 h, cleaning dries up.The cyclic voltammetry curve of different modifying electrode is measured respectively at 100 mM pH 7.0 PBS+0.1 M KCl buffer solution containing 5 mM K3 [Fe (CN) 6]/K4 [Fe (CN) 6] (1:1), sweep speed is 0.05 V/s, sampling interval is 0.001 V, and time of repose is 2 s.From experimental result known (see accompanying drawing 2), the DNA content of electrode surface is more, and peak point current (Ip) is less; The DNA content of electrode surface is fewer, and peak point current (Ip) is larger.This is because DNA phosphate backbones is electronegative, hinders [the Fe (CN) of band like charges
6]
3-/4-to electrode surface generation redox reaction, cause its electron transmission to be obstructed, peak current reduces.
Required sensor can be obtained by above method.
Embodiment 3
Above-mentioned electrochemical sensor is for detecting c-erbB-2 related gene, and concrete detection method is: immerse the glass-carbon electrode securing hair fastener DNA probe containing certain density complementary DNA solution, carry out hybridization reaction.Electrode after hybridization is placed in the damping fluid of 10 U exonuclease III, shear for 37 DEG C and eliminate, probe P is transformed into flexible short linear structures from hairpin structure.Drip assist probes AP1 and AP2 solution at electrode surface, under room temperature, carry out the self assembly of DNA long distance formula.Long electrode after formula self assembly is placed in the pH 7.0 PBS buffer solution containing 5,7-dinitro-2-sulfo group-acridone, allows it be embedded in the double-spiral structure of DNA.PB buffer solution square wave voltammetry can be placed in after above-mentioned electrode clean dries up detect.
The present invention uses the square wave voltammetry electrochemical measuring technique of prior art to observe the redox signal intensity of indicator.Experimental result is shown in accompanying drawing 3, as seen from the figure, in certain limit, along with target DNA concentration increases, double stranded DNA (dsDNA) amount that hybridization is formed increases, and the flexible short data records that enzyme cuts generation is more, thus it is more to form the long amount apart from formula self assembly of DNA, indicator embedded quantity is increased, and peak current signal strengthens.The spike potential of indicator 5,7-dinitro-2-sulfo group-acridone is-0.028V.The condition measured: measuring medium is PB buffer solution, pH=5.0.Square wave voltammetry location parameter: current potential increment is 0.005 V, amplitude is 0.025 V.Its range of linearity is: 2 aM ~ 50 fM.Regression equation is I (μ A)=0.5955 Log [C/ (aM)]+0.5814, linearly dependent coefficient r is 0.9947, and the lowest detection lower limit of the method to specific sequence DNA is 0.5 aM.
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
SEQUENCE LISTING
<110> Medical University Of Fujian
The preparation method of <120> hybridization indicator 5,7-dinitro-2-sulfo group-acridone and purposes
<130> 4
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 51
<212> DNA
<213> probe P
<400> 1
aaaaatttat ttgataggcg aactatttgt ttttaatatc aaataatggt t 51
<210> 2
<211> 20
<212> DNA
<213> probe AP1
<400> 2
caaaatatat gataggcgaa 20
<210> 3
<211> 20
<212> DNA
<213> probe AP2
<400> 3
atatattttg ttcgcctatc 20
<210> 4
<211> 33
<212> DNA
<213> c-erbB-2 related gene
<400> 4
aaccattatt tgatattaaa acaaataggc ttg 33
Claims (8)
1. hybridization indicator 5,7-dinitro-2-sulfo group-acridone, is characterized in that: described 5,7-dinitro-2-sulfo group-acridone structural formulas are
.
2. the preparation method of a kind of hybridization indicator 5,7-dinitro-2-sulfo group-acridone as claimed in claim 1, is characterized in that: described step is: take 0.6 g acridone, adds the dense H of 5 mL
2sO
4, after stirring and dissolving, be heated to 100 DEG C of reaction 30 min, poured into by solution while hot in 0 ~ 4 DEG C of frozen water, solid is separated out, and filtration drying obtains light green solid 2-sulfo group-acridone; Take 0.6 g 2-sulfo group-acridone, add 3 mL 36 % acetic acid, add the red fuming nitric acid (RFNA) of 0.36 mL and the glacial acetic acid of 0.8 mL more successively, at 56 ~ 60 DEG C, stirring reaction 2 h, pours in frozen water by solution while hot, solid is separated out, filter, with 10 ~ 20 mL water flushings, and with the further recrystallization purifying of absolute ethyl alcohol, obtain yellow needles powder 5,7-dinitro-2-sulfo group-acridone.
3. the application of a kind of hybridization indicator 5,7-dinitro-2-sulfo group-acridone as claimed in claim 1 on electrochemical sensor.
4. the application of a kind of hybridization indicator 5,7-dinitro-2-sulfo group-acridone according to claim 3 on electrochemical sensor, is characterized in that: described electrochemical sensor preparation method comprises:
1) pre-service of electrode
By the Al of glass-carbon electrode at 0.05 μm
2o
3powder is polished, and priority absolute ethyl alcohol and distilled water ultrasonic vibration 5 min, to remove attached impurity at the electrode surface, electrode surface is characterized with the 10 mM potassium ferricyanides, until front and back peak position voltage difference is less than 80 V, distilled water is cleaned and is dried rear taking-up, and nitrogen dries up;
2) preparation of probe modification electrode
Glass-carbon electrode after process is placed in pH 8.0, the 0.2 M PBS damping fluid containing 0.01 M/L lysine, close with cyclic voltammetry electricity and modify polylysine film, use distilled water cleaning down electrode again and at room temperature drying, obtain Mercapto-group modification electrode, be inverted by Mercapto-group modification electrode, surface drips 20 μ L coupling activator, room temperature nature evaporate to dryness, then clean 10 s with ultrapure water, remove the coupling activator not being combined in modified electrode surface, nitrogen dries up stand-by; Then the probe gene P solution of 10 μ L, 1 μM of hairpin structure is dripped at electrode surface, room temperature nature evaporate to dryness, removes with ultrapure water cleaning the probe gene P not being combined in electrode surface, then uses 10 mM pH7.0 PBS buffer solution for cleaning, drying at room temperature, obtained hair fastener probe modification electrode;
3) enzyme auxiliary target sequence loops
Hair fastener probe modification electrode is placed in the target sequence c-erbB-2 related gene solution of a series of concentration, after hybridizing 1 h under room temperature, cleaning dries up, electrode after hybridization is placed in the damping fluid of 10 U exonuclease III, after reacting 30 min at 37 DEG C, cleaning dries up stand-by;
4) DNA is long apart from formula self assembly and Electrochemical Detection
Get the assist probes Gene A P1 of 10 μ L 1 μM and AP2 solution respectively to drip and be coated in above-mentioned electrode surface, it is long apart from formula self assembly to carry out DNA under room temperature, and after 2 h, cleaning dries up; Respectively at the 5 mM K containing mass ratio being 1:1
3[Fe (CN)
6]/K
4[Fe (CN)
6] 100 mM pH 7.0 PBS+0.1 M KCl buffer solution measure the CV curve of different modifying electrodes, sweep speed is 0.05 V/s, and sampling interval is 0.001 V, and time of repose is 2 s; Be placed in the 10 mM pH 7.0 PBS buffer solution containing 100 μM of 5,7-dinitro-2-sulfo group-acridone by long for the DNA electrode after formula self assembly, after 20 min, cleaning dries up; Then electrode is placed in 100 mM pH 5.0 phosphate PB buffer solution and scans CV and square wave voltammetry curve.
5. a kind of hybridization indicator 5 according to claim 4, the application of 7-dinitro-2-sulfo group-acridone on electrochemical sensor, it is characterized in that: the condition of described cyclic voltammetry is: voltage-1.0 ~+2.4 V, sweep speed is 100 mVs-1, and characterizing the number of turns is 14 circles.
6. the application of a kind of hybridization indicator 5,7-dinitro-2-sulfo group-acridone according to claim 4 on electrochemical sensor, is characterized in that: described coupling activator is pH 7.4 phosphate buffer containing the EDC of 5 mM and the NHS of 8 mM.
7. the application of a kind of hybridization indicator 5,7-dinitro-2-sulfo group-acridone according to claim 4 on electrochemical sensor, is characterized in that: the sweep speed of described CV method is 0.50 V/s, and sampling interval is 0.001 V, and time of repose is 2 s; The scanning potential range of square wave voltammetry is-0.2 V ~+0.15 V, and current potential increment is 0.005 V, and amplitude is 0.025 V.
8. a kind of hybridization indicator 5 according to claim 4, the application of 7-dinitro-2-sulfo group-acridone on electrochemical sensor, is characterized in that: described probe P sequence is 5'-NH2-AAA AAT TTA TTT GAT AGG CGA ACT ATT TGT TTT TAA TAT CAA ATA ATG GTT-3'; Described probe AP1 sequence is 5'-CAA AAT ATA T GA TAG GCG AA-3'; Probe AP2 sequence is 5'-ATA TAT TTT GTT CGC CTA TC-3'.
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| CN113552191A (en) * | 2021-07-28 | 2021-10-26 | 江苏师范大学 | Construction method of proportional electrochemical sensor for detecting methylated DNA based on multilayer DNA amplification loop |
| CN113552191B (en) * | 2021-07-28 | 2023-11-21 | 江苏师范大学 | Construction method of proportional electrochemical sensor for detecting methylated DNA based on multilayer DNA amplification loop |
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