CN103901020A - Method for detecting DNA (Deoxyribose Nucleic Acid) transmethylase by using electrochemical luminescence biosensor and application of biosensor - Google Patents

Method for detecting DNA (Deoxyribose Nucleic Acid) transmethylase by using electrochemical luminescence biosensor and application of biosensor Download PDF

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

The invention discloses a method for detecting DNA (Deoxyribose Nucleic Acid) transmethylase by using an electrochemical luminescence biosensor. The method comprises the following steps: self assembling a hairpin DNA sequence of S1 containing a methylated site GATC (Guanine, Ddenine, Thymine and Cytosine) on the surface of a gold electrode to form the electrochemical luminescence biosensor; methylating a modified electrode by using the transmethylase, then carrying out enzyme digestion by using a methylated restriction enzyme, washing, and then hybridizing by using a complementary DNA of S2 containing amino group, connecting graphene oxide loaded phenanthroline ruthenium compound to the electrode through coupling of EDC-NHS (Dichloroethane-N-Hydroxysulfosuccinimide sodium salt) to carry out electrochemical luminescence detection and detect the transmethylase. By adopting the graphene oxide, a relatively large specific surface area is provided for loading the phenanthroline ruthenium; meanwhile, the graphene oxide is in non-covalent bonding with the phenanthroline ruthenium, so that loading can be carried out effectively, the operate is easy and an electronic structure of the graphite oxide can be retained; by adopting the graphene oxide as a luminescence material, the sensor is high in sensitivity to the transmethylase.

Description

Detect method and the application of dnmt rna with electrochemiluminescence biology sensor
Technical field
The present invention relates to electrochemiluminescence biology sensor, be specifically related to a kind of method and application that detects dnmt rna with electrochemiluminescence biology sensor.
Background technology
DNA methylation has important effect in many bioprocess, and comprise and transcribing, genomic imprinting, Cell Differentiation, chromatin Structure and embryo form.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), taking S-adenosylmethionine (SAM) as methyl donor, by Methyl transporters to the process in specific base.DNA methylation can occur in GATC and CCA/TGG is upper, i.e. the N-7 position of the N-4 position of the N-6 position of adenine, cytimidine, guanine or the C-5 position of cytimidine.The activity of this abnormal dnmt rna is relevant with the pathogenesis of cancer, and this relation provides a potential target in diagnosis and the treatment of disease.In addition, DNA MTase is the newcomer who is used for the treatment of the pharmacology family of cancer.Therefore, a kind of method sensitive, that detect fast DNA methylation and DNA MTase activity provides strong means to the early diagnosis of cancer undoubtedly, for the fundamental mechanism of research gene regulation provides opinion, provide foundation for researching and developing new cancer therapy drug simultaneously.The traditional method that is used 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 processing of effort and special isotope labeling.
Electrochemiluminescence (electrogenerated chemiluminescence), be called for short ECL, that the system that contains chemiluminescent substance by electrode pair applies certain voltage or passes through certain electric current, between anodizing reduzate or between anodizing reduzate and other coexisting substances of system, there is chemical reaction and generate certain unsettled intermediate state material, this substance decomposition and the chemiluminescence phenomenon that produces.Electrochemiluminescence technology is the detection technique that galvanochemistry combines with chemiluminescence, the advantage of this technology is both integrated luminous and electrochemical analysis technology, have again the two in conjunction with controllability, selectivity, favorable reproducibility of producing, highly sensitive, detectability is low and the new advantage such as dynamic response wide ranges.
Summary of the invention
For measuring the defects such as the prior art sensitivity of dnmt rna is not high, assay method is complicated, the present invention aims to provide a kind of method and application that detects dnmt rna with electrochemiluminescence biology sensor.
Applicant proposes the hair clip DNA capture probe that meter contains the specific site that methylates, and utilizes restriction enzyme to the recognition site responsive characteristic that methylates, and taking different transmethylases as example, sets up the analytical approach of quick methyl transferase activity.Meanwhile, investigate the impact of different cancer therapy drugs for methylated transferase activity by the method, set up 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 methylated 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, can stable existence in neutral aqueous solution and there is high sensitivity due to its good performance, therefore in electrochemical luminous sensor, obtain research widely.In recent years, nano material becomes the hot fields of people's research, and therefore various nano materials are 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 forms, because there is high surface volume ratio, high dispersibility and the active function groups widely of its surface conjunction in water and in organic solvent, make the material based on graphene oxide have unique attractive force in research and the application of bio-sensing.Graphene assembling Ru (bpy) recently 3 2+compound is in the news, and for example, 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+have better adsorbability, graphene oxide, with also containing a lot of pi-conjugated aromatic groups on negative charge and its basal plane, is therefore easy to by 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 the analytical approach to methylase activity.
The invention provides a kind of method that detects 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 that contains the site GATC that methylates;
2) S1 with transmethylase to be measured (Dam MTase), step 1) being fixed on electrode methylates;
3) cut step 2 with restriction enzyme (Dpn I) enzyme that methylates) S1 after methylating;
4) be retained in the S1 hybridization on electrode after cutting with S2 and step 3) enzyme; Described S2 is with amino complementary DNA;
5) graphene oxide-loaded phenanthroline ruthenium compound (Ru (phen) 3 2+/ GO) being coupling-connected on the electrode after step 4) hybridization by EDC-NHS;
6) electrode of step 5) is carried out to electrochemiluminescence detection, measure thus methyl transferase activity to be measured.
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 is immersed in the buffer solution of 500uL of 2 μ M S1 to 37 DEG C and fixes 4 hours, form self-assembled film in gold electrode surfaces, remove the S1 of non-specific adsorption with the drip washing of PBS solution, use again 6-sulfydryl hexanol (MCH) sealing of 1mM, with PBS solution drip washing gold electrode.
Wherein, described in step 1), gold electrode is pretreated gold electrode as follows:
On chamois leather, use respectively the alumina powder polishing grinding of 0.3 and 0.05 μ m to minute surface, water, absolute ethyl alcohol supersound washing 2min respectively again, the alumina powder adhering to remove electrode surface, finally uses Milli-Q water wash clean, then electrode is placed in to 0.1mol/L H 2sO 4in solution, in ﹣ 0.2～1.6V (vs.Ag ∕ AgCl) potential range, the speed of sweeping with 100mV/s is scanned up to and obtains stable cyclic voltammogram, uses subsequently Milli-Q water wash electrode, 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, enzyme is cut described in step 3), 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, enzyme is cut described in step 3), and reaction conditions used is 37 DEG C of insulation 2h.
Wherein, hybridize described in step 4), reaction system used be 5 μ L2.0 μ M S2 with amino complementary DNA, 10mM Tris-HCl buffer, pH7.4.
Wherein, graphene oxide-loaded phenanthroline ruthenium compound described in step 5), preparation by the following method: 3mg graphene oxide is joined in the 5 mL aqueous solution that contain 0.5mg phenanthroline ruthenium, ultrasonic 15 minutes, stirred overnight at room temperature, ultrasonic 5 minutes, per minute 8000 leaves the heart and within 10 minutes, removes unnecessary phenanthroline ruthenium, 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, and 59g dag and 2.59g sodium nitrate are mixed with the concentrated sulphuric acid of 115mL, slowly add 15g KMnO in stirring 4keep 2 DEG C of following sustained response l h, transfer them to 35 DEG C of water-baths, reaction 30min progressively adds 250mL deionized water, temperature rises to 98 DEG C and continues after reaction 15min, can obviously observe potpourri and become glassy yellow by sepia, further continuously thin up, and with the H of quality percentage composition 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, in the 5%HCI of volumn concentration solution, wash, to remove metallic ion, in distilled water, cyclic washing is until one-tenth is neutral again, and suction filtration is also put into filter cake 80 DEG C of fully dry graphene oxides that obtain of baking oven.
Wherein, step 5) is being coupling-connected on the electrode after step 4) hybridization by EDC-NHS graphene oxide-loaded phenanthroline ruthenium compound, be specially graphene oxide-loaded phenanthroline ruthenium compound is used before in EDC-NHS mixed solution (5mM EDC, 10mM NHS in Tris-HCl buffer, pH7.4) activation 30min, electrode after step 4) hybridization is immersed in to 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, make on the covalently bound electrode after step 4) hybridization of graphene oxide-loaded phenanthroline ruthenium compound.
Wherein, carry out electrochemiluminescence detection described in step 6), detecting liquid is the 0.1mol/L PBS solution 2.0mL that contains 0.02mol/LTPA.
Wherein, carry out electrochemiluminescence detection described in step 6), at room temperature carry out, electrolytic cell is placed in to magazine, connects three-electrode system, in the potential range of 0～+ 1.25V, the speed of sweeping with 100mV/s is carried out cyclic voltammetry scan, records its ECL signal and carries out quantitative test.
Another object of the present invention is to provide the application of the method for described electrochemiluminescence biology sensor detection dnmt rna.
Advantage of the present invention is:
Compared with prior art, the electrochemiluminescence biology sensor the present invention relates to has following advantage with progressive 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 bond, than covalent bond, non-covalent combination is reacted as pi-pi accumulation load and easy operating and can keep the electronic structure of graphite oxide effectively by supermolecule.In addition, can find out according to experiment luminous intensity, graphene oxide-loaded phenanthroline ruthenium compound can be used as luminescent material, and for there is no transmethylase or shearing in the situation of enzyme, luminous intensity is well below the situation that has transmethylase and shearing enzyme, and this illustrates that this sensor detects and has very high selectivity transmethylase.What therefore, the present invention designed embodies development prospect well based on graphene oxide electrochemical luminous sensor in building the research method of some toolenzymes.
Brief description of the drawings
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 through Dam and Dpn I electrode after treatment after MCH/S1 modified electrode elder generation; E is Ru (phen) 3 2+/ GO/S2/MCH/S1 modified electrode.
Fig. 3 is the electrode in the different modifying stage electrochemiluminescence curve map after different disposal.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 of methylating 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 in embodiment 1 (GO) and Ru (phen) 3 2+the phenogram of/GO.Wherein, the transmission electron microscope picture that A is GO, 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 respectively concentration 0,0.05,0.1,0.2,0.5,1.0,5.0,10,20,40U/mL; B is the linear relationship chart of Methyl transporters enzyme concentration and ECL signal.
Fig. 9 is the measurement result figure that in embodiment 2, different gentamicin concentrations affect methyl transferase activity.Wherein, A is ECL signal response after different gentamicin concentrations are processed, and B is variable concentrations gentamicin and relative activity curve map, and the concentration of gentamicin is respectively 0,0.1,0.3,0.5,0.8,1.0,2.0 μ M.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.
A kind of method that detects dnmt rna with electrochemiluminescence biology sensor the present invention relates to, the hair clip DNA sequence dna self assembly that S1 is contained to the site GATC that methylates, on the surface of gold electrode, forms electrochemiluminescence biology sensor; Modified electrode is methylated with transmethylase, again with the restriction enzyme cutting that methylates, after drip washing, hybridize with amino complementary DNA with S2, again graphene oxide-loaded phenanthroline ruthenium compound is carried out to 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 that detects dnmt rna with electrochemiluminescence biology sensor, and concrete experimental principle as shown in Figure 1, comprises the following steps:
1) gold electrode (being naked gold electrode) is invaded in the buffer solution of 500uL of 2 μ M S1 to 37 DEG C and fix 4 hours, form self-assembled film in gold electrode surfaces, remove the S1 of non-specific adsorption with the drip washing of PBS solution, obtain S1 modified electrode, seal with the 6-sulfydryl hexanol of 1mM again, with the drip washing of PBS solution, obtain MCH/S1 modified electrode.Gold electrode need be handled as follows in advance: on chamois leather, use respectively the alumina powder polishing grinding of 0.3 and 0.05 μ m to minute surface, water, absolute ethyl alcohol supersound washing 2min respectively again, the alumina powder adhering to remove electrode surface, finally use Milli-Q water wash clean, then electrode is placed in to 0.1mol/L H 2sO 4in solution, in ﹣ 0.2～1.6V (vs.Ag ∕ AgCl) potential range, the speed of sweeping with 100mV/s is scanned up to and obtains stable cyclic voltammogram, uses subsequently Milli-Q water wash, and uses N 2dry up and save backup.
2) hair clip DNA is methylated, the system that wherein methylates 500 μ L comprise 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) methylated modified electrode is cut with 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 obtaining is through Dam and Dpn I electrode after treatment after MCH/S1 modified electrode elder generation.
4) modified electrode of enzyme being cut is immersed in 5 μ L and comprises 2.0 μ M S2DNA probes (in10mM Tris-HCl buffer, pH7.4), makes to be retained in DNA end and S2DNA hybridization on electrode.
5) utilize Hummers method to prepare graphene oxide, more further synthetic graphene oxide-loaded phenanthroline ruthenium compound (Ru (phen) 3 2+/ GO);
Ru (phen) 3 2+/ GO uses front in EDC-NHS mixed solution (5mM EDC, 10mM NHS in Tris-HCl buffer, pH7.4)) activation 30min; Electrode after hybridization is immersed in to 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 of having modified is carried out to 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 is drawn electrochemical impedance figure, sees Fig. 2.
For the electrode of testing the above-mentioned different modifying stage electrochemiluminescence behavior after different disposal, be provided with 5 processing: a gold electrode comprises 1mg/mL Ru (phen) through 0.10M PBS (pH7.4) 3 2+/ GO and 0.02M TPA process; After the electrode elder generation that b MCH/S1 modifies, through 100U/mL Dam, 50U/mL Dpn I processes, S2 hybridization and Ru (phen) 3 2+the coupling of/GO compound; The electrode that c MCH/S1 modifies is directly processed through 50U/mL Dpn I, S2 hybridization and Ru (phen) 3 2+the coupling of/GO compound; The electrode that d MCH/S1 modifies and S2 hybridization and Ru (phen) 3 2+the coupling of/GO compound; The electrode that e MCH/S3 modifies is respectively through 100U/mL Dam, and 50U/mL Dpn I processes, S2 hybridization and Ru (phen) 3 2+the coupling of/GO compound.ECL measures and carries out in 0.10M PBS (pH7.4) comprises 0.02M TPA; Sweep velocity: 0.1V/s, sweep limit: 0-+1.25V.
Wherein, S1 is the hair clip DNA sequence dna that contains the site GATC that methylates: 5 '-HS-(CH 2) 6-TACTGATTGCGATCGAGAATGCTTTTGCATTCTCGA TCGCAAT-3 ' (as shown in SEQ ID No.1)
S2 is with amino complementary dna sequence: 5 '-NH 2-(CH 2) 6-TCGCAATCAGTA-3 ' (as shown in SEQ ID No.2)
S3 is the contrast hair clip DNA sequence dna that does not contain the site GATC that methylates: 5 '-HS-(CH 2) 6-TACTGATTGCGACTGAGAATGCTTTTGCATTCTCGA CTG CAAT-3 ' (as shown in SEQ ID No.3)
Concrete outcome is shown in as Fig. 3, adopt the processing b electrochemiluminescence behavior of the contrast hair clip DNA that S1 contains the site GATC that methylates obvious, and the processing e that adopts S3 not contain the contrast hair clip DNA of the site GATC that methylates detects very light current chemiluminescence behavior.And in the situation that not having methyl turn enzyme or shear enzyme, luminous intensity is far below the situation that has transmethylase and shearing enzyme as luminescent material for graphene oxide-loaded phenanthroline ruthenium compound, and therefore sensor of the present invention detects and has very high selectivity transmethylase.
Inventor is also optimized the testing conditions in the present invention, specific as follows:
(1) methylating the time of hair clip DNA.Process the different time spans (20U/mL Dam, the 1mg/mL Ru (phen) that arrange 0-120 minute to methylating 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, in the time methylating time 60min, can reach 99% of luminous intensity, in the time of 120min, reach maximum, in order to save the time of whole test process, we select the time of methylating is 60min.
(2) Ru (phen) 3 2+/ GO concentration.To Ru (phen) 3 2+/ GO concentration arranges different concentration gradients (the 20U/mL Dam 60min that methylates, the Ru (phen) of 0-3.0mg/mL 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+/ GO concentration 0 when the 0.5mg/mL electrochemiluminescence strength increase very fast, be to 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 (the 20U/mL Dam 60min that methylates, 1mg/mL Ru (phen) 3 2+/ GO, other are with reference to above-mentioned steps 1)-6) carry out), as shown in Figure 6, coupling time electrochemiluminescence strength increase very fast 10 to 100min time, is to reach maximum signal level at 180min to experimental result, therefore, we select coupling time is 180min.
Embodiment 1 electrochemiluminescence biology sensor detects Methyl transporters enzyme 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: be modified with the gold electrode working electrode of 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-maloyl imines (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 is all purchased from Sigma's aldrich (Sigma-Aldrich) company limited, when use without secondarily purified.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:
The hair clip DNA that S1(contains the site GATC that methylates):
5 '-HS-(CH 2) 6-TACTGATTGCGATCGAGAATGCTTTTGCATTCTC GATCGCAAT-3 ' (as shown in SEQ ID No.1)
S2(is with amino complementary DNA):
5 '-NH 2-(CH 2) 6-TCGCAATCAGTA-3 ' (as shown in SEQ ID No.2)
S3(does not contain the contrast hair clip DNA of the site GATC that methylates):
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+synthesizing 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 add 15g KMnO 4keep 2 DEG C of following sustained response l h, transfer them to 35 DEG C of water-baths, reaction 30min progressively adds 250mL deionized water, temperature rises to 98 DEG C and continues after reaction 15min, can obviously observe potpourri and become glassy yellow by sepia, further continuously thin up, and with the H of quality percentage composition 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, in the 5%HCI of volumn concentration solution, wash, to remove metallic ion, in distilled water, cyclic washing is until one-tenth is neutral again, and suction filtration is also put into filter cake 80 DEG C of fully dry graphene oxides that obtain of baking oven.
3mg graphene oxide is joined in the aqueous solution of 5mL of the phenanthroline ruthenium that contains 0.5mg, mixed solution, after ultrasonic 15 minutes, is at room temperature stirred and spent the night.To mixed solution ultrasonic 5 minutes afterwards, leave the heart with per minute 8000 and within 10 minutes, remove unnecessary phenanthroline ruthenium, then wash with deionized water and absolute ethyl alcohol, finally in vacuum drying chamber, be dried, obtain Ru (phen) 3 2+/ GO.
The graphene oxide (GO) making 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, graphene oxide and the compound number of plies thereof synthetic in figure are little, are translucent and have some folds.Fig. 7 C is GO and Ru (phen) 3 2+the Raman spectrum of/GO, the as can be seen from the figure D of sample and G peak, I d/ I gratio often can be as the chemical modification level of showing on the carbon sample of graphite.GO and Ru (phen) 3 2+the I of/GO d/ I gratio is respectively 0.78 and 0.81, the ratio of this increase has reflected 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 synthetic success of/GO compound.
1.2.2 the structure of electrochemiluminescence biology sensor
1. the processing of gold electrode.Gold electrode (Φ=2mm) uses respectively the careful polishing grinding of alumina powder of 0.3 and 0.05 μ m to minute surface on chamois leather, water, absolute ethyl alcohol supersound washing 2min respectively again, the alumina powder adhering to remove electrode surface, finally uses Milli-Q water wash clean.Then electrode is placed in to 0.1mol/L H 2sO 4in solution, in ﹣ 0.2～1.6V (vs.Ag/AgCl) potential range, the speed of sweeping with 100mV/s is scanned up to and obtains stable cyclic voltammogram, uses subsequently Milli-Q water wash, and uses N 2dry up and save backup.
2. the preparation of electrochemiluminescence biology sensor.The gold electrode of processing is invaded in the buffer solution of 500uL of 2 μ MS1 to 37 DEG C and fix 4 hours, form self-assembled film in gold electrode surfaces, remove the S1 of non-specific adsorption with the drip washing of PBS solution, then with the 6-sulfydryl hexanol sealing of 1mM, use PBS solution drip washing gold electrode.Thereby make electrochemiluminescence biology sensor.
1.2.3 electrochemiluminescence detects dnmt rna
To hair clip, DNA methylates, and the system that wherein methylates 500 μ L comprise 50mM Tris-HCl pH7.5,10mM NaCl, 10mM EDTA, 5mM2-mercaptohexanol, the Dam MTase of 160mM SAM and variable concentrations, 37 DEG C of insulation 60min; Methylated modified electrode is cut with restriction enzyme Dpn I enzyme, and 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 that enzyme was cut is immersed in 5 μ L and comprises 2.0 μ M S2DNA probes (in10mM Tris-HCl buffer, pH7.4), makes to be retained in DNA end and S2DNA hybridization on electrode.Electrode after hybridization is immersed in to the Ru (phen) of 500 μ L1mg/mL 3 2+180min in/GO solution; The electrode of having modified is carried out to electrochemiluminescence measurement, measurement system 2.0mL0.10M PBS (pH7.4) comprises 0.02M TPA, electrolytic cell is placed in to magazine, connect three electrodes, in the potential range of 0-+1.25, the speed of sweeping with 0.1V/s is carried out cyclic voltammetry scan and is recorded its ECL signal 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, has studied the transmethylase of variable concentrations and the relation of electrochemiluminescence intensity, has obtained typical curve, the range of linearity and the linear equation of transmethylase.
Under best experiment condition, 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 (U/mL of unit of C), r=0.9976.
The method comparison for detection of 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.
The different detection limits that detect Methyl transporters enzyme method of table 1
Figure BDA0000485149480000131
Figure BDA0000485149480000141
[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 affect methyl transferase activity
Taking gentamicin as model, having investigated this medicine affects Dam methyl transferase activity: it is 40U/mL that Methyl transporters enzyme concentration is set, and adds therein variable concentrations gentamicin, and 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, and in the time that 0.3uM gentamicin joins 40U/mL Methyl transporters enzyme system, chemiluminescence intensity has declined 16%.This result has shown that this electrochemiluminescence biology sensor of invention can be for the screening of antagonism cancer drug.
The present invention is the electrochemiluminescence biology sensor of a kind of highly sensitive detection transmethylase of the amplification technique development based on 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 bond, reacts as pi-pi accumulation load and easy operating and can keep the electronic structure of graphite oxide effectively by supermolecule than the non-covalent combination of covalent bond.Therefore the sensor of design is simple to operate, highly sensitive, to embodying 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, do not departing under the prerequisite of 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.
Figure IDA0000485149560000011

Claims (10)

1. a method that detects dnmt rna with electrochemiluminescence biology sensor, is characterized in that, comprises 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 that contains the site GATC that methylates;
2) S1 with transmethylase to be measured, step 1) being fixed on electrode methylates;
3) by the digestion with restriction enzyme step 2 that methylates) S1 after methylating;
4) be retained in the S1 hybridization on electrode after cutting with S2 and step 3) enzyme; Described S2 is with amino complementary DNA;
5) being coupling-connected on the electrode after step 4) hybridization by EDC-NHS graphene oxide-loaded phenanthroline ruthenium compound;
6) electrode of step 5) is carried out to electrochemiluminescence detection, measure thus transmethylase to be measured.
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 ' (as shown in SEQ ID No.1);
The sequence of described S2 is as follows:
5 '-NH 2-(CH 2) 6-TCGCAATCAGTA-3 ' (as shown in SEQ ID No.2).
3. method according to claim 1, it is characterized in that, described in step 1), S1 is fixed on gold electrode by the self assembly of golden mercapto key, concrete steps are: gold electrode is immersed in the buffer solution of 500uL of 2 μ M S1 to 37 DEG C and fixes 4 hours, form self-assembled film in gold electrode surfaces, remove the S1 of non-specific adsorption with the drip washing of PBS solution, then with the 6-sulfydryl hexanol sealing of 1mM, use PBS solution drip washing gold electrode.
4. method according to claim 1, it is characterized in that, 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 160mMS-adenosylmethionine and transmethylase to be measured.
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, enzyme is cut described in step 3), 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.
7. method according to claim 1, is characterized in that, hybridize described in step 4), reaction system used be 5 μ L2.0 μ M S2 with amino complementary DNA, 10mM Tris-HCl buffer, pH7.4.
8. method according to claim 1, it is characterized in that, graphene oxide-loaded phenanthroline ruthenium compound described in step 5), by the following method preparation: 3mg graphene oxide is joined in the 5mL aqueous solution that contains 0.5mg phenanthroline ruthenium to ultrasonic 15 minutes, stirred overnight at room temperature, ultrasonic 5 minutes, per minute 8000 left the heart and within 10 minutes, removes unnecessary phenanthroline ruthenium, deionized water and absolute ethanol washing, dry, obtain graphene oxide-loaded phenanthroline ruthenium compound.
9. method according to claim 1, it is characterized in that, step 5) is being coupling-connected on the electrode after step 4) hybridization by EDC-NHS graphene oxide-loaded phenanthroline ruthenium compound, be specially graphene oxide-loaded phenanthroline ruthenium compound is used before in EDC-NHS mixed solution (5mM EDC, 10mM NHS in Tris-HCl buffer, pH7.4) activation 30min, electrode after step 4) hybridization is immersed in to 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, make on the covalently bound electrode after step 4) hybridization of graphene oxide-loaded phenanthroline ruthenium compound.
10. the application of the method for electrochemiluminescence biology sensor detection dnmt rna described in claim 1-9 any one.
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CN109580597A (en) * 2019-01-28 2019-04-05 青岛科技大学 It is a kind of based on the electrochemical luminescence biosensor and its preparation method of DNA nanotube and application
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