CN103901020B - Method and the application of DNA transmethylase is detected with electrochemiluminescence biology sensor - Google Patents

Method and the application of DNA transmethylase is detected with electrochemiluminescence biology sensor Download PDF

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CN103901020B
CN103901020B CN201410128514.5A CN201410128514A CN103901020B CN 103901020 B CN103901020 B CN 103901020B CN 201410128514 A CN201410128514 A CN 201410128514A CN 103901020 B CN103901020 B CN 103901020B
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graphene oxide
transmethylase
phenanthroline ruthenium
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CN103901020A (en
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李延
闫曌
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Northwest University
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Abstract

A kind of method detecting dnmt rna with electrochemiluminescence biology sensor that the present invention relates to, contains the hair clip DNA sequence dna self assembly of methylation sites GATC on the surface of gold electrode by S1, composition electrochemiluminescence biology sensor; Modified electrode transmethylase is methylated, again with the digestion with restriction enzyme that methylates, after drip washing, the complementary DNA amino with S2 band is hybridized, again graphene oxide-loaded phenanthroline ruthenium compound is carried out electrochemiluminescence detection by being coupling-connected on electrode of EDC-NHS, realize the mensuration to transmethylase with this.The present invention adopts graphene oxide to provide relatively large specific surface area for load phenanthroline ruthenium, and graphene oxide and phenanthroline ruthenium are Non-covalent binding, can load and be easy to operate and the electronic structure of graphite oxide can be kept effectively, make sensor of the present invention have very high sensitivity to transmethylase as luminescent material.

Description

Method and the application of DNA transmethylase is detected with electrochemiluminescence biology sensor
Technical field
The present invention relates to electrochemiluminescence biology sensor, be specifically related to a kind of method and the application that detect dnmt rna with electrochemiluminescence biology sensor.
Background technology
DNA methylation has important effect in many bioprocess, comprises and transcribing, genomic imprinting, Cell Differentiation, chromatin Structure and embry ogenesis.Research finds that many human diseases methylate relevant with aberrant gene.DNA methylation refers to that biosome is under the catalysis of dnmt rna (DNA methyltransferase, DNA MTase), with S-adenosylmethionine (SAM) for methyl donor, transfers to the process in specific base by methyl.DNA methylation can occur on GATC and CCA/TGG, i.e. the C-5 position of the N-6 position of adenine, the N-4 position of cytimidine, the N-7 position of guanine or cytimidine.The activity of this abnormal dnmt rna is relevant with the pathogenesis of cancer, and this relation provides a potential target at the Diagnosis and Treat of disease.In addition, DNA MTase is the newcomer of the pharmacology family being used for the treatment of cancer.Therefore, a kind of method that is sensitive, that detect DNA methylation and DNA MTase activity fast provides strong means to the early diagnosis of cancer undoubtedly, for the fundamental mechanism studying gene regulation provides opinion, simultaneously for the new cancer therapy drug of research and development provides foundation.Traditional method for the mensuration of DNA methylation transferase active relies on the substrate of labelled with radioisotope, the different technologies of PCR-based, Capillary Electrophoresis and high performance liquid chromatography.These methods time intensives, expend DNA, need the process of effort and special isotope labeling.
Electrochemiluminescence (electrogenerated chemiluminescence), be called for short ECL, that the system containing chemiluminescent substance by electrode pair applies certain voltage or by certain electric current, there is chemical reaction between anodizing reduzate or between anodizing reduzate and other coexisting substances of system and generate the intermediate state material of certain instability, this substance decomposition and the chemiluminescence phenomenon produced.Electrochemiluminescence technology is the detection technique that galvanochemistry combines with chemiluminescence, this technology had both been integrated with advantage that is luminous and electrochemical analysis techniques, have again the two combine produce controllability, selectivity, favorable reproducibility, highly sensitive, detectability is low and the new advantage such as dynamic response wide ranges.
Summary of the invention
The defects such as the prior art sensitivity for mensuration dnmt rna is not high, assay method is complicated, the present invention aims to provide a kind of method and the application that detect dnmt rna with electrochemiluminescence biology sensor.
Applicant proposes the hair clip DNA capture probe of meter containing the specific site that methylates, and utilizes restriction enzyme to the characteristic of recognition site methyl-sensitive, for different transmethylases, sets up the analytical approach of quick methyl transferase activity.Meanwhile, investigate the impact of different cancer therapy drug for methylated transferase activity by the method, establish a kind of new approaches of carrying out drug screening at molecular level.The present invention takes full advantage of character and the function of nucleic acid methylation transferase, restriction enzyme and nucleic acid molecular probe, further develop the application of nucleic acid molecules technology in DNA-protein (enzyme) repercussion study, for research life active procedure provides new measure.
Phenanthroline ruthenium (Ru (phen) 3 2+) be conventional electrochemiluminescence material, because its good performance namely can stable existence and have high sensitivity in neutral aqueous solution, therefore obtain in electrochemical luminous sensor and study widely.In recent years, nano material becomes the hot fields of people's research, and therefore various nano material is used to load Ru (phen) 3 2+with ruthenium dipyridine (Ru (bpy) 3 2+).Graphene oxide (GO) is that the graphite oxide of individual layer is formed, because have the active function groups widely of high surface volume ratio, dispersibility high in water and in organic solvent and its surface conjunction, the material based on graphene oxide is made to have unique attractive force at the investigation and application of bio-sensing.Nearest Graphene assembling Ru (bpy) 3 2+compound is in the news, and such as, utilizes perfluorinated sulfonic acid by Ru (bpy) 3 2+good sensitivity and stability are fixed on Graphene for the electrochemiluminescence detection display of TPA.The application of graphene oxide assembling thionine amplifying signal Electrochemical Detection dnmt rna has also had report (Wen Li, Ping Wu, Hui Zhang, and Chenxin Cai.Analytical Chemistry, 2012,84,7583-7590).With electrochemiluminescence reagent Ru (bpy) 3 2+compare, Ru (phen) 3 2+there is better adsorbability, graphene oxide with negative charge and on its basal plane also containing a lot of pi-conjugated aromatic group, therefore easily through electrostatic interaction, pi-pi accumulation effect and Ru (phen) 3 2+strong combination also has outstanding long-time stability (Scott Gilje, Song Han, Minsheng Wang, Kang L.Wang, and Richard B.Kaner, Nano Letters, 2007,7,3394-3398; Yali Yuan, Haijuan Li, Shuang Han, Lianzhe Hu, Saima Parveen, Haoran Cai, and Guobao Xu.Anal.Chim.Acta, 2012,720,38 – 42.).Therefore, based on graphene oxide to phenanthroline ruthenium high load capability, good biocompatibility and stability, we design a kind of based on graphene oxide-loaded electrochemiluminescence active substance to the analytical approach of methylase activity.
The invention provides a kind of method detecting dnmt rna with electrochemiluminescence biology sensor, comprise the steps:
1) S1 is fixed on gold electrode by the self assembly of golden mercapto key; Described S1 is the hair clip DNA containing methylation sites GATC;
2) with transmethylase to be measured (Dam MTase), the S1 that step 1) is fixed on electrode is methylated;
3) cut step 2 with the restriction enzyme that methylates (Dpn I) enzyme) methylate after S1;
4) after cutting with S2 and step 3) enzyme, the S1 be retained on electrode is hybridized; Described S2 is the complementary DNA of band amino;
5) graphene oxide-loaded phenanthroline ruthenium compound (Ru (phen) 3 2+/ GO) by EDC-NHS be coupling-connected to step 4) hybridization after electrode on;
6) electrochemiluminescence detection is carried out to the electrode of step 5), measure methyl transferase activity to be measured thus.
Wherein, the sequence of described S1 is as follows:
5 '-HS-(CH 2) 6-TACTGATTGCGATCGAGAATGCTTTTGCATTCTCGATCGCAAT-3 ' (as shown in SEQ ID No.1).
Wherein, the sequence of described S2 is as follows:
5 '-NH 2-(CH 2) 6-TCGCAATCAGTA-3 ' (as shown in SEQ ID No.2).
Wherein, described in step 1), S1 is fixed on gold electrode by golden mercapto key, concrete steps are: gold electrode to be immersed in the buffer solution of the 500uL of 2 μMs of S1 37 DEG C and to fix 4 hours, self-assembled film is formed in gold electrode surfaces, the S1 of non-specific adsorption is removed with PBS solution drip washing, the 6-sulfydryl hexanol (MCH) of 1mM is used to close, with PBS solution drip washing gold electrode again.
Wherein, gold electrode described in step 1) is pretreated gold electrode as follows:
Chamois leather is used respectively the alumina powder polishing grinding of 0.3 and 0.05 μm to minute surface, again with water, absolute ethyl alcohol supersound washing 2min respectively, to remove the alumina powder that electrode surface adheres to, finally use Milli-Q water wash clean, then electrode is placed in 0.1mol/L H 2sO 4speed of sweeping with 100mV/s in ﹣ 0.2 ~ 1.6V (vs.Ag ∕ AgCl) potential range in solution is scanned up to and obtains stable cyclic voltammogram, uses Milli-Q water wash electrode subsequently, and uses N 2dry up and save backup.
Wherein, described in step 1), the diameter of gold electrode is preferably 2mm.
Wherein, step 2) described in methylate, reaction system used is the Tris-HCl of 500 μ L1 × Dam buffer(50mM pH7.5,10mM NaCl, 10mM EDTA, 5mM mercaptoethanol) and 160mM S-adenosylmethionine (SAM) and transmethylase to be measured.
Wherein, step 2) described in methylate, reaction conditions used is 37 DEG C of insulation 60min.
Wherein, described in step 3), enzyme is cut, and reaction system used is 500 μ L1 × NEB buffer4 (50mM KAc, 20mM Tris-Ac, 10mM Mg (Ac) 2, 1mM DTT, pH7.9) and 80U Dpn I.
Wherein, described in step 3), enzyme is cut, and reaction conditions used is 37 DEG C of insulation 2h.
Wherein, hybridize described in step 4), reaction system used is the complementary DNA of 5 μ L2.0 μMs of S2 band amino, 10mM Tris-HCl buffer, pH7.4.
Wherein, graphene oxide-loaded phenanthroline ruthenium compound described in step 5), prepare by the following method: 3mg graphene oxide is joined in the 5 mL aqueous solution containing 0.5mg phenanthroline ruthenium, ultrasonic 15 minutes, stirred overnight at room temperature, ultrasonic 5 minutes, per minute 8000 leave the heart removes unnecessary phenanthroline ruthenium in 10 minutes, deionized water and absolute ethanol washing, dry, obtain graphene oxide-loaded phenanthroline ruthenium compound.
Wherein, described graphene oxide is standby with Hummers legal system.The standby concrete steps of Hummers legal system are preferably in ice-water bath, 59g dag and 2.59g sodium nitrate are mixed with the concentrated sulphuric acid of 115mL, slowly add 15g KMnO in stirring 4keep less than 2 DEG C sustained response l h, transfer them to 35 DEG C of water-baths, reaction 30min progressively adds 250mL deionized water, after temperature rises to 98 DEG C of continuation reaction 15min, can obviously observe potpourri and become glassy yellow by sepia, further continuously thin up, and with the H of mass percentage 30% 2o 2solution-treated, neutralize unreacted potassium permanganate, by above-mentioned solution while hot suction filtration remove most water and strong acid etc., again filter cake is put into bag filter, wash in the 5%HCI solution of volumn concentration, to remove metallic ion, in distilled water, cyclic washing is until become neutral again, and filter cake is also put into baking oven 80 DEG C of abundant dryings and obtained graphene oxide by suction filtration.
Wherein, step 5) graphene oxide-loaded phenanthroline ruthenium compound by EDC-NHS be coupling-connected to step 4) hybridization after electrode on, to be specially before graphene oxide-loaded phenanthroline ruthenium compound is used (5mM EDC in EDC-NHS mixed solution, 10mM NHS in Tris-HCl buffer, pH7.4) 30min is activated, electrode after step 4) hybridization is immersed in 180min in the graphene oxide-loaded phenanthroline ruthenium complex solution of 500 μ L1mg/mL, the carboxyl reaction of the amino on S2 and graphene oxide activation, on electrode after making graphene oxide-loaded phenanthroline ruthenium compound be covalently attached to step 4) hybridization.
Wherein, carry out electrochemiluminescence detection described in step 6), detection liquid is the 0.1mol/L PBS solution 2.0mL containing 0.02mol/LTPA.
Wherein, described in step 6), carry out electrochemiluminescence detection, at room temperature carry out, electrolytic cell is placed in magazine, connects three-electrode system, in the potential range of 0 ~+1.25V, carry out cyclic voltammetry scan with the speed of sweeping of 100mV/s, record its ECL signal and carry out quantitative test.
Another object of the present invention is to provide the application that described electrochemiluminescence biology sensor detects the method for dnmt rna.
Advantage of the present invention is:
Compared with prior art, the electrochemiluminescence biology sensor that the present invention relates to has following advantage and progress significantly: adopt graphene oxide to provide relative large specific surface area for load phenanthroline ruthenium, and what graphene oxide adopted the load of phenanthroline ruthenium is Non-covalent binding, compared to covalent bond, Non-covalent binding by supermolecule reaction as pi-pi accumulation can load and be easy to operation and can keep the electronic structure of graphite oxide effectively.In addition, experimentally luminous intensity can be found out, graphene oxide-loaded phenanthroline ruthenium compound can as luminescent material, and for when there is no transmethylase or shear enzyme, luminous intensity is well below the situation having transmethylase and shearing enzyme, and this illustrates that this sensor has very high selectivity to transmethylase detection.Therefore, what the present invention designed embodies development prospect well based on graphene oxide electrochemical luminous sensor in structure is to the research method of some toolenzymes.
Accompanying drawing explanation
Fig. 1 is the present invention detects the method for dnmt rna process flow diagram with electrochemiluminescence biology sensor.
Fig. 2 is electrochemical impedance figure (EIS) figure of different modifying electrode.Wherein, a is naked gold electrode; B is S1 modified electrode; C is MCH/S1 modified electrode; D is the electrode after MCH/S1 modified electrode elder generation after Dam and Dpn I process; E is Ru (phen) 3 2+/ GO/S2/MCH/S1 modified electrode.
Fig. 3 is the electrochemiluminescence curve map of electrode after different disposal in different modifying stage.Wherein, a is 1mg/mL Ru (phen) 3 2+/ GO and 0.02M TPA; B is that S1 modified electrode is through Dam, DpnI and S2, Ru (phen) 3 2+/ GO; C is that S1 modified electrode is through DpnI and S2, Ru (phen) 3 2+/ GO; D be S1 modified electrode directly and S2, Ru (phen) 3 2+/ GO; E is that S3 modified electrode is through Dam, DpnI and S2, Ru (phen) 3 2+/ GO.
Fig. 4 is the time-optimized result figure that methylates of hair clip DNA.
Fig. 5 is Ru (phen) 3 2+/ GO concentration optimization result figure.
Fig. 6 is Ru (phen) 3 2+the coupling time optimum results figure of/GO.
Fig. 7 is graphene oxide (GO) and Ru (phen) in embodiment 1 3 2+the phenogram of/GO.Wherein, A is the transmission electron microscope picture of GO, and B is Ru (phen) 3 2+the transmission electron microscope picture of/GO, C is GO and Ru (phen) 3 2+the Raman spectrum of/GO, D is Ru (phen) 3 2+/ GO's can spectrogram.
Fig. 8 is the ECL signal measuring result figure of variable concentrations transmethylase in embodiment 1.Wherein, A is the ECL signal response of variable concentrations transmethylase, and curve a-j is concentration 0,0.05,0.1,0.2,0.5,1.0,5.0,10,20,40U/mL respectively; B is the linear relationship chart of transmethylase concentration and ECL signal.
Fig. 9 is the measurement result figure that in embodiment 2, different gentamicin concentration affects methyl transferase activity.Wherein, A is ECL signal response after different gentamicin concentration process, and B is variable concentrations gentamicin and relative activity curve map, and the concentration of gentamicin is 0,0.1 respectively, 0.3,0.5,0.8,1.0, and 2.0 μMs.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
A kind of method detecting dnmt rna with electrochemiluminescence biology sensor that the present invention relates to, contains the hair clip DNA sequence dna self assembly of methylation sites GATC on the surface of gold electrode by S1, composition electrochemiluminescence biology sensor; Modified electrode transmethylase is methylated, again with the restriction enzyme cutting that methylates, after drip washing, the complementary DNA amino with S2 band is hybridized, again graphene oxide-loaded phenanthroline ruthenium compound is carried out electrochemiluminescence detection by being coupling-connected on electrode of EDC-NHS, realize the mensuration to transmethylase with this.
The present invention is a kind of method detecting dnmt rna with electrochemiluminescence biology sensor, and concrete experimental principle as shown in Figure 1, comprises the following steps:
1) gold electrode (i.e. naked gold electrode) to be invaded in the buffer solution of the 500uL of 2 μMs of S1 37 DEG C and fix 4 hours, self-assembled film is formed in gold electrode surfaces, the S1 of non-specific adsorption is removed with PBS solution drip washing, obtain S1 modified electrode, close with the 6-sulfydryl hexanol of 1mM again, use PBS solution drip washing, obtain MCH/S1 modified electrode.Gold electrode need be handled as follows in advance: on chamois leather, use the alumina powder polishing grinding of 0.3 and 0.05 μm to minute surface respectively, again with water, absolute ethyl alcohol supersound washing 2min respectively, to remove the alumina powder that electrode surface adheres to, finally use Milli-Q water wash clean, then electrode is placed in 0.1mol/L H 2sO 4speed of sweeping with 100mV/s in ﹣ 0.2 ~ 1.6V (vs.Ag ∕ AgCl) potential range in solution is scanned up to and obtains stable cyclic voltammogram, uses Milli-Q water wash subsequently, and uses N 2dry up and save backup.
2) hair clip DNA is methylated, the system that wherein methylates 500 μ L comprises 50mM Tris-HCl pH7.5,10mM NaCl, 10mM EDTA, 5mM mercaptoethanol (2-mercaptohexanol), the transmethylase Dam MTase to be measured of 160mM SAM and variable concentrations, 37 DEG C of insulation 60min.
3) cut by methylated modified electrode restriction enzyme Dpn I enzyme, wherein enzyme is cut system 500 μ L and is comprised 1 × NEB buffer4 (50mM KAc, 20mM Tris-Ac, 10mM Mg (Ac) 2, 1mM DTT, pH7.9) and 80U Dpn I, 37 DEG C of insulation 2h.The electrode obtained is the electrode after MCH/S1 modified electrode elder generation after Dam and Dpn I process.
4) modified electrode that enzyme cuts through is immersed in 5 μ L and comprises 2.0 μMs of S2DNA probes (in10mM Tris-HCl buffer, pH7.4), make to be retained in DNA end on electrode and S2DNA is hybridized.
5) utilize Hummers method to prepare graphene oxide, then synthesize graphene oxide-loaded phenanthroline ruthenium compound (Ru (phen) further 3 2+/ GO);
Ru (phen) 3 2+before/GO uses in EDC-NHS mixed solution (5mM EDC, 10mM NHS in Tris-HCl buffer, pH7.4)) activation 30min; Electrode after hybridization is immersed in the Ru (phen) of 500 μ L1mg/mL 3 2+180min in/GO solution, obtains Ru (phen) 3 2+/ GO/S2/MCH/S1 modified electrode.
6) electrode modified is carried out electrochemiluminescence measurement, measure system 2.0mL0.10MPBS (pH7.4) and comprise 0.02M tripropyl amine (TPA) (TPA).
Electrode for the above-mentioned different modifying stage draws electrochemical impedance figure, sees Fig. 2.
For testing the electrochemiluminescence behavior of electrode after different disposal in above-mentioned different modifying stage, be provided with 5 process: a gold electrode comprises 1mg/mL Ru (phen) through 0.10M PBS (pH7.4) 3 2+/ GO and 0.02M TPA process; Through 100U/mL Dam, 50U/mL Dpn I process after the electrode that b MCH/S1 modifies is first, S2 hybridization and Ru (phen) 3 2+/ GO compound is coupled; The electrode that c MCH/S1 modifies is directly through 50U/mL Dpn I process, and S2 is hybridized and Ru (phen) 3 2+/ GO compound is coupled; D MCH/S1 modify electrode and S2 is hybridized and Ru (phen) 3 2+/ GO compound is coupled; The electrode that e MCH/S3 modifies is respectively through 100U/mL Dam, 50U/mL Dpn I process, and S2 is hybridized and Ru (phen) 3 2+/ GO compound is coupled.ECL measurement comprises in 0.02M TPA at 0.10M PBS (pH7.4) to be carried out; Sweep velocity: 0.1V/s, sweep limit: 0-+1.25V.
Wherein, S1 is the hair clip DNA sequence dna containing methylation sites GATC: 5 '-HS-(CH 2) 6-TACTGATTGCGATCGAGAATGCTTTTGCATTCTCGA TCGCAAT-3 ' (as shown in SEQ ID No.1)
S2 is the complementary dna sequence of band amino: 5 '-NH 2-(CH 2) 6-TCGCAATCAGTA-3 ' (as shown in SEQ ID No.2)
S3 is the contrast hair clip DNA sequence dna not containing methylation sites GATC: 5 '-HS-(CH 2) 6-TACTGATTGCGACTGAGAATGCTTTTGCATTCTCGA CTG CAAT-3 ' (as shown in SEQ ID No.3)
Concrete outcome is shown in as Fig. 3, adopts S1 to contain the process b electrochemiluminescence behavior of the contrast hair clip DNA of methylation sites GATC obviously, and adopts the process e of the contrast hair clip DNA of S3 containing methylation sites GATC very light current chemiluminescence behavior to be detected.And graphene oxide-loaded phenanthroline ruthenium compound is as luminescent material, and when not having methyl to turn enzyme or shearing enzyme, luminous intensity is far below the situation having transmethylase and shearing enzyme, therefore sensor of the present invention detects transmethylase very high selectivity.
Inventor is also optimized the testing conditions in the present invention, specific as follows:
(1) the methylating the time of hair clip DNA.The process that methylates is arranged to different time span (20U/mL Dam, the 1mg/mL Ru (phen) of 0-120 minute 3 2+/ GO, Ru (phen) 3 2+/ GO and S2 probe coupling 300min, other are with reference to above-mentioned steps 1)-6) carry out), in experimental result as shown in Figure 4,99% of luminous intensity can be reached when methylating time 60min, reach maximum when 120min, in order to save the time of whole test process, we select the time of methylating to be 60min.
(2) Ru (phen) 3 2+/ GO concentration.To Ru (phen) 3 2+/ GO concentration arranges the different concentration gradient of 0-3.0mg/mL, and (20U/mL Dam methylates 60min, Ru (phen) 3 2+/ GO and S2 probe coupling 300min, other are with reference to above-mentioned steps 1)-6) carry out), experimental result as shown in Figure 5, Ru (phen) 3 2+it is very fast that/GO concentration electrochemiluminescence intensity 0 to 0.5mg/mL time increases, and be reach maximal value at 1mg/mL, therefore, we select Ru (phen) 3 2+/ GO concentration is 1mg/mL.
(3) Ru (phen) 3 2+the coupling time of/GO.To Ru (phen) 3 2+/ GO coupling time arranges different length, and (20U/mL Dam methylates 60min, 1mg/mL Ru (phen) 3 2+/ GO, other are with reference to above-mentioned steps 1)-6) carry out), as shown in Figure 6, it is very fast that coupling time electrochemiluminescence intensity 10 to 100min time increases experimental result, and be reach maximum signal level at 180min, therefore, we select coupling time to be 180min.
Embodiment 1 electrochemiluminescence biology sensor detects transmethylase concentration
1. experimental section
1.1 instruments and reagent
MPI – E type Electrochemiluminescprocess process system (Xi'an Rui Mai Analytical Instrument Co., Ltd); CHI660D type electrochemical workstation (Shanghai Chen Hua Instrument Ltd.); TGL – 16G type hydro-extractor (Anting Scientific Instrument Factory, Shanghai); KH3200B type ultrasonic cleaner (Kunshan He Chuan ultrasonic instrument company limited); Milli-Q type ultrapure water instrument (Millipore company of the U.S.); Experiment adopts three-electrode system: the gold electrode working electrode being modified with hair clip DNA, the saturated KCl of Ag/AgCl() be contrast electrode, platinum electrode is to electrode.
Dag (99.998%), three-(2-carboxyethyl) phosphate hydrochlorate (TCEP, 98%), N-hydroxysuccinimide (NHS), ethyl { 3-(dimethylamino) Bing Ji ﹜ carbodiimide hydrochloride (EDC), tripropyl amine (TPA) (TPA), 6-sulfydryl hexanol (MCH), phenanthroline ruthenium (Ru (phen) 3 2+), above reagent all purchased from Sigma-Aldrich (Sigma-Aldrich) company limited, without the need to secondarily purified during use.S-adenosylmethionine (SAM), E.coli transmethylase Dam, E.coli restriction enzyme Dpn I buys in Beijing Niu Yinglun Bioisystech Co., Ltd.Oligonucleotide chain is purchased from Shanghai bioengineering company limited, and base sequence is as follows:
S1(contains the hair clip DNA of methylation sites GATC):
5 '-HS-(CH 2) 6-TACTGATTGCGATCGAGAATGCTTTTGCATTCTC GATCGCAAT-3 ' (as shown in SEQ ID No.1)
The complementary DNA that S2(band is amino):
5 '-NH 2-(CH 2) 6-TCGCAATCAGTA-3 ' (as shown in SEQ ID No.2)
The contrast hair clip DNA of S3(not containing methylation sites GATC):
5 '-HS-(CH 2) 6-TACTGATTGCGACTGAGAATGCTTTTGCATTCTC GACTG CAAT-3 ' (as shown in SEQ ID No.3)
1.2 experimental procedure
1.2.1 graphene oxide and Ru (phen) 3 2+the synthesis of/GO compound
In ice-water bath, 59g dag and 2.59g sodium nitrate are mixed with the concentrated sulphuric acid of 115mL, in stirring, slowly adds 15g KMnO 4keep less than 2 DEG C sustained response l h, transfer them to 35 DEG C of water-baths, reaction 30min progressively adds 250mL deionized water, after temperature rises to 98 DEG C of continuation reaction 15min, can obviously observe potpourri and become glassy yellow by sepia, further continuously thin up, and with the H of mass percentage 30% 2o 2solution-treated, neutralize unreacted potassium permanganate, by above-mentioned solution while hot suction filtration remove most water and strong acid etc., again filter cake is put into bag filter, wash in the 5%HCI solution of volumn concentration, to remove metallic ion, in distilled water, cyclic washing is until become neutral again, and filter cake is also put into baking oven 80 DEG C of abundant dryings and obtained graphene oxide by suction filtration.
3mg graphene oxide is joined in the aqueous solution of the 5mL of the phenanthroline ruthenium containing 0.5mg, to mixed solution after ultrasonic 15 minutes, at room temperature stir and spend the night.To mixed solution ultrasonic 5 minutes afterwards, leave the heart with per minute 8000 and remove unnecessary phenanthroline ruthenium in 10 minutes, then wash with deionized water and absolute ethyl alcohol, finally drying in vacuum drying chamber, obtained Ru (phen) 3 2+/ GO.
Obtained graphene oxide (GO) and Ru (phen) 3 2+the phenogram of/GO compound is shown in Fig. 7.
Fig. 7 A is the transmission electron microscope picture (TEM) of GO, and B is Ru (phen) 3 2+the transmission electron microscope picture (TEM) of/GO, the graphene oxide synthesized in figure and the compound number of plies thereof seldom, are translucent and have some folds.Fig. 7 C is GO and Ru (phen) 3 2+the Raman spectrum of/GO, as can be seen from the figure D and the G peak of sample, I d/ I gratio often can be used as show graphite carbon sample on chemical modification level.GO and Ru (phen) 3 2+the I of/GO d/ I gratio is 0.78 and 0.81 respectively, this ratio increased reflects the increase of unordered degree, because the donor atom on graphene oxide causes.In addition, Fig. 7 D is Ru (phen) 3 2+the energy spectrogram of/GO, this compound is made up of three kinds of elements as can be seen from Figure, and Ru (phen) is described 3 2+the synthesis success of/GO compound.
1.2.2 the structure of electrochemiluminescence biology sensor
1. the process of gold electrode.Gold electrode (Φ=2mm) uses the careful polishing grinding of the alumina powder of 0.3 and 0.05 μm to minute surface respectively on chamois leather, again with water, absolute ethyl alcohol supersound washing 2min respectively, to remove the alumina powder that electrode surface adheres to, finally use Milli-Q water wash clean.Then electrode is placed in 0.1mol/L H 2sO 4speed of sweeping with 100mV/s in ﹣ 0.2 ~ 1.6V (vs.Ag/AgCl) potential range in solution is scanned up to and obtains stable cyclic voltammogram, uses Milli-Q water wash subsequently, and uses N 2dry up and save backup.
2. the preparation of electrochemiluminescence biology sensor.The gold electrode processed to be invaded in the buffer solution of the 500uL of 2 μMs of S1 37 DEG C and fix 4 hours, self-assembled film is formed in gold electrode surfaces, remove the S1 of non-specific adsorption with PBS solution drip washing, then close, with PBS solution drip washing gold electrode with the 6-sulfydryl hexanol of 1mM.Thus obtained electrochemiluminescence biology sensor.
1.2.3 electrochemiluminescence detects dnmt rna
Methylate to hair clip DNA, the system that wherein methylates 500 μ L comprises 50mM Tris-HCl pH7.5,10mM NaCl, 10mM EDTA, the Dam MTase of 5mM2-mercaptohexanol, 160mM SAM and variable concentrations, 37 DEG C of insulation 60min; Cut by methylated modified electrode restriction enzyme Dpn I enzyme, wherein enzyme is cut system 500 μ L and is comprised 1 × NEB buffer4 (50mM KAc, 20mM Tris-Ac, 10 mM Mg (Ac) 2,1mM DTT, pH7.9) and 80U Dpn I, 37 DEG C of insulation 2h; The modified electrode cut through by enzyme is immersed in 5 μ L and comprises 2.0 μMs of S2DNA probes (in10mM Tris-HCl buffer, pH7.4), makes to be retained in DNA end on electrode and S2DNA is hybridized.Electrode after hybridization is immersed in the Ru (phen) of 500 μ L1mg/mL 3 2+180min in/GO solution; The electrode modified is carried out electrochemiluminescence measurement, measurement system 2.0mL0.10M PBS (pH7.4) comprises 0.02M TPA, electrolytic cell is placed in magazine, connect three electrodes, in the potential range of 0-+1.25, carry out its ECL signal of cyclic voltammetry scan record with the speed of sweeping of 0.1V/s and carry out quantitative test, the results are shown in Figure 8.
1.3 results and discussion
The present invention, under preferred best experiment condition, have studied the transmethylase of variable concentrations and the relation of electrochemiluminescence intensity, obtains the typical curve of transmethylase, the range of linearity and linear equation.
Under the experiment condition of the best, when the concentration of transmethylase is in 0.05U/mL to 40U/mL scope, along with the increase of transmethylase Dam concentration, electrochemical luminescence signals strengthens gradually, its linear equation is I=912.32+545.78lgC (the unit U/mL of C), r=0.9976.
The Measures compare for detecting MTase activity with reporting, the results are shown in Table 1: the method range of linearity of the present invention is wider, detection limit lower (0.02U/mL, S/N=3).The relative standard deviation of the transmethylase of 0.5U/mL is 4.65% (n=5).Show that the method has good sensitivity and reappearance.
Table 1 difference detects the detection limit of transmethylase method
[1]Zheng,X.J.,Qiu,J.D.,Zhang,L.,Wang,Z.X.,Liang,R.P.,2013.Chemical Communications49,3546-3548.
[2]Wang,G.L.,Zhou,L.Y.,Luo,H.Q.,Li,N.B.,2013.Analytica Chimica Acta768,76–81.
[3]He,X.X.,Su,J.,Wang,Y.H.,Wang,K.M.,Ni,X.Q.,Chen,Z.F.,2011.Biosensors and Bioelectronics28,298–303.
[4]Wang,Y.H.,He,X.X.,Wang,K.M.,Su,J.,Chen,Z.F.,Yan,G.P.,Du,Y.D.,2013.Biosensors and Bioelectronics41,238-243.
[5]Ouyang,X.Y.,Liu,J.H.,Li,J.S.,Yang,R.H.,2012.Chemical Communications48,88–90.
Embodiment 2 electrochemiluminescence biology sensor assessment inhibitor affects methyl transferase activity
Be model with gentamicin, having investigated this medicine affects Dam methyl transferase activity: arranging transmethylase concentration is 40U/mL, and add variable concentrations gentamicin wherein, other steps are with embodiment 1.
The results are shown in Figure 9.Dam methyl transferase activity reduces along with the increase of gentamicin drug concentration, and inhibiting effect and drug concentration positive correlation, when 0.3uM gentamicin joins 40U/mL transmethylase system, chemiluminescence intensity have dropped 16%.This electrochemiluminescence biology sensor that this result indicates invention may be used for the screening to cancer therapy drug.
The present invention is the electrochemiluminescence biology sensor of a kind of highly sensitive detection transmethylase developed based on the amplification technique of graphene oxide.The sensor of development has following advantage: graphene oxide provides relatively large specific surface area for load phenanthroline ruthenium; What graphene oxide adopted the assembling of phenanthroline ruthenium is Non-covalent binding, compared to covalent bond Non-covalent binding by supermolecule reaction as pi-pi accumulation can load and be easy to operation and can keep the electronic structure of graphite oxide effectively.Therefore the sensor operations designed is simple, highly sensitive, embodies development prospect well in the research method of some toolenzymes.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Claims (10)

1. detect a method for dnmt rna with electrochemiluminescence biology sensor, it is characterized in that, comprise the steps:
1) S1 is fixed on gold electrode by the self assembly of golden mercapto key; Described S1 is the hair clip DNA sequence dna containing methylation sites GATC;
2) with transmethylase to be measured to step 1) S1 be fixed on electrode methylates;
3) by the digestion with restriction enzyme step 2 that methylates) methylate after S1;
4) with S2 and step 3) enzyme be retained in after cutting on electrode S1 hybridization; Described S2 is the complementary DNA of band amino;
5) graphene oxide-loaded phenanthroline ruthenium compound is coupling-connected to step 4 by EDC-NHS) on electrode after hybridization;
6) to step 5) electrode carry out electrochemiluminescence detection, measure transmethylase to be measured thus.
2. method according to claim 1, is characterized in that, the sequence of described S1 is as follows:
5’-HS-(CH 2) 6-TACTGATTGCGATCGAGAATGCTTTTGCATTCTCGATCGCAAT-3’；
The sequence of described S2 is as follows:
5’-NH 2-(CH 2) 6-TCGCAATCAGTA-3’。
3. method according to claim 1, it is characterized in that, step 1) described S1 is fixed on gold electrode by the self assembly of golden mercapto key, concrete steps are: gold electrode to be immersed in the buffer solution of the 500uL of 2 μMs of S1 37 DEG C and to fix 4 hours, self-assembled film is formed in gold electrode surfaces, remove the S1 of non-specific adsorption with PBS solution drip washing, then close, with PBS solution drip washing gold electrode with the 6-sulfydryl hexanol of 1mM.
4. method according to claim 1, is characterized in that, step 2) described in methylate, reaction system used is 500 μ L 1 × Dam buffer and 160mM S-adenosylmethionine and transmethylase to be measured; Described Dam buffer comprises the Tris-HCl of 50mM pH 7.5,10mM NaCl, 10mM EDTA, 5mM mercaptoethanol.
5. method according to claim 1, is characterized in that, step 2) described in methylate, reaction conditions used be 37 DEG C insulation 60min.
6. method according to claim 1, is characterized in that, step 3) described enzyme cuts, and reaction system used is 500 μ L 1 × NEB buffer 4 and 80U Dpn I; Described NEB buffer 4 comprises 50mM KAc, 20mM Tris-Ac, 10mM Mg (Ac) 2, 1mMDTT, pH 7.9.
7. method according to claim 1, is characterized in that, step 4) described hybridization, reaction system used is the complementary DNA of 5 μ L, 2.0 μMs of S2 band amino, 10mMTris-HCl buffer, pH 7.4.
8. method according to claim 1, it is characterized in that, step 5) described graphene oxide-loaded phenanthroline ruthenium compound, prepare by the following method: 3mg graphene oxide is joined in the 5mL aqueous solution containing 0.5mg phenanthroline ruthenium, ultrasonic 15 minutes, stirred overnight at room temperature, ultrasonic 5 minutes, per minute 8000 leave the heart removed unnecessary phenanthroline ruthenium, deionized water and absolute ethanol washing in 10 minutes, drying, obtains graphene oxide-loaded phenanthroline ruthenium compound.
9. method according to claim 1, it is characterized in that, step 5) graphene oxide-loaded phenanthroline ruthenium compound is coupling-connected to step 4 by EDC-NHS) on electrode after hybridization, be specially before graphene oxide-loaded phenanthroline ruthenium compound is used and activate 30min in EDC-NHS mixed solution, step 4) hybridization after electrode be immersed in 180min in the graphene oxide-loaded phenanthroline ruthenium complex solution of 500 μ L 1mg/mL, the carboxyl reaction of the amino on S2 and graphene oxide activation, make graphene oxide-loaded phenanthroline ruthenium compound be covalently attached to step 4) hybridization after electrode on, described EDC-NHS mixed solution refer to EDC concentration be 5mM, NHS concentration be 10mM and pH value be 7.4 Tris-HCl damping fluid.
10. electrochemiluminescence biology sensor described in any one of claim 1-9 detects the application of the method for dnmt rna.
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