CN103439319A - Method for measuring bleomycins by utilizing electrochemical luminescence of carbon nanoparticle modified electrode - Google Patents
Method for measuring bleomycins by utilizing electrochemical luminescence of carbon nanoparticle modified electrode Download PDFInfo
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- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical class N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 title claims abstract description 49
- 108010006654 Bleomycin Proteins 0.000 title claims abstract description 48
- 239000011852 carbon nanoparticle Substances 0.000 title claims abstract description 46
- 238000004020 luminiscence type Methods 0.000 title claims abstract description 17
- 238000000034 method Methods 0.000 title claims abstract description 17
- 239000000523 sample Substances 0.000 claims abstract description 15
- 239000003298 DNA probe Substances 0.000 claims abstract description 7
- 238000009825 accumulation Methods 0.000 claims abstract description 5
- 229960001561 bleomycin Drugs 0.000 claims description 42
- 239000000243 solution Substances 0.000 claims description 31
- HOGDNTQCSIKEEV-UHFFFAOYSA-N n'-hydroxybutanediamide Chemical compound NC(=O)CCC(=O)NO HOGDNTQCSIKEEV-UHFFFAOYSA-N 0.000 claims description 17
- 238000006243 chemical reaction Methods 0.000 claims description 13
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 11
- 229910052737 gold Inorganic materials 0.000 claims description 11
- 239000010931 gold Substances 0.000 claims description 11
- YKWAWOTVGKMRAN-UHFFFAOYSA-L 2-(4-carboxylatopyridin-2-yl)pyridine-4-carboxylate;2-pyridin-2-ylpyridine;ruthenium(2+) Chemical compound [Ru+2].N1=CC=CC=C1C1=CC=CC=N1.N1=CC=CC=C1C1=CC=CC=N1.[O-]C(=O)C1=CC=NC(C=2N=CC=C(C=2)C([O-])=O)=C1 YKWAWOTVGKMRAN-UHFFFAOYSA-L 0.000 claims description 10
- 230000000694 effects Effects 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 9
- 239000004372 Polyvinyl alcohol Substances 0.000 claims description 8
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims description 8
- 229920002451 polyvinyl alcohol Polymers 0.000 claims description 8
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 235000019441 ethanol Nutrition 0.000 claims description 6
- 108020003215 DNA Probes Proteins 0.000 claims description 5
- 230000008859 change Effects 0.000 claims description 5
- 239000008055 phosphate buffer solution Substances 0.000 claims description 5
- 238000003556 assay Methods 0.000 claims description 4
- 229910052697 platinum Inorganic materials 0.000 claims description 4
- -1 succinamide ruthenium Chemical compound 0.000 claims description 4
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims description 3
- 229910021607 Silver chloride Inorganic materials 0.000 claims description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical class [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 3
- 230000004913 activation Effects 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 125000005909 ethyl alcohol group Chemical group 0.000 claims description 3
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- 239000000843 powder Substances 0.000 claims description 3
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- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 claims description 3
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Abstract
The invention relates to a method for detecting the content of bleomycins (BLMs), belonging to the technical fields of electrochemical luminescence and sensors. According to the method, an electrochemical luminescence agent is utilized for marking DNA (deoxyribonucleic acid) to prepare a probe (Ru-DNA), and a carboxylated carbon nanoparticle modified electrode is utilized for constructing a carboxylated carbon nanoparticle modified electrode electrochemical luminescence biosensor through a competitive effect between an electrostatic repulsion force and a pi-pi accumulation action on the basis of non-covalent-bond self-assembly principles of the DNA and the carbon nanoparticle. Target objects, namely the bleomycins, act on the DNA probes to cut the DNA, the DNA probes adsorbing on the carbon nanoparticle modified electrode are reduced, and weak electrochemical luminescence signals are generated, thereby realizing the measurement of the bleomycins. The method has the advantages of simplicity and rapidness.
Description
Technical field
The invention belongs to analytical chemistry and electrochemical luminous sensor field, be specially method and application that a kind of carbon nano-particles modified electrode electrochemiluminescence is measured bleomycin.
Technical background
For biochemical subject of modern times and biomedical research, the protein sensitive, that Measurement accuracy is relevant with disease is vital aspect a lot, and particularly detection and the accurate prediction for cancer early sign albumen has great importance.Immune detection is a kind of strong analysis determining technology, and it has been widely used in the detection analysis of tumor marker.Because antibody has high specific and strong recognition reaction, thereby be used as recognition element.Yet in its production, the stability of product and the defect on clinical manipulation, people are seeking its substitute due to antibody.
The DNA biology sensor is a kind ofly can, by with the interactional object of DNA, being transformed into the sensing device of the signals such as detectable light, electricity, utilize the interaction of DNA and object can set up many detection techniques for life science.The DNA biology sensor is a kind of strong analysis test method, can substitute in many aspects traditional analytical approach of setting up based on antigen-antibody.The DNA biology sensor is the brand-new biology sensor developed rapidly, molecular biology and galvanochemistry have been opened up, the recent studies on field of the subjects such as spectroscopy, for the research of life science provides new technology, new method, have profound significance to the research of clinical medicine and genetic engineering.
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, chemical reaction occurs between the anodizing reduzate or between anodizing reduzate and other coexisting substances of system and generate certain unsettled intermediate state material, this substance decomposition and the chemiluminescence phenomenon that produces.The 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 the controllability produced, selectivity, favorable reproducibility, highly sensitive, detectability low and the new advantage such as dynamic response wide ranges.
Bleomycin (Bleomycins, BLMs, BLM) is a class glycopeptide antibiotics.It is that Japanese scientist Mei Zebin separated and obtains first in 1996 from the nutrient solution of streptomyces verticillatus.Thin bleomycin at oxygen and redox metallic ion (as Fe
2+deng) existence under can optionally cut off single stranded DNA or double-stranded DNA, thereby cancer cell is killed.Based on this mechanism, the compound of thin bleomycin and metallic ion is widely used in the auxiliary curing of the kinds cancers such as cutaneum carcinoma, lung cancer, the cancer of the esophagus, the cancer of the brain.The assay method of the bleomycin of having reported at present has fluorescence method (Feng Li; Yan Feng; Can Zhao; Peng Li and Bo Tang; A sensitive graphene oxide – DNA based sensing platform for fluorescence " turn-on " detection of bleomycin; Chem. Comm., 2012,48:127; Yoshitsugu Akiyama, Qian Ma, Erin Edgar, Andrei Laikhter and Sidney M Hecht, A novel DNA hairpin substrate for bleomycin, Org. Lett., 2008,10:2127) and (Honglan Qi, Xiaoying Qiu, the Chen Wang such as Electrochemiluminescince, Qiang Gao and Chengxiao Zhang, Anal. Methods, 2013,5:612).The present invention has set up a kind of carboxylated carbon nano-particles modified gold electrode electrochemiluminescence biology sensor, and measures bleomycin with it.A kind of simple and sensitive electrochemiluminescence sensing technology that provides, the mensuration of bleomycin of having utilized the present invention to realize.
Summary of the invention
Demand for the research of the deficiencies in the prior art and this area and application, the purpose of this invention is to provide a kind of simple and sensitive electrochemiluminescence sensing technology, the mensuration of bleomycin of having utilized the present invention to realize; The present invention simultaneously also may be used on other and DNA is had to the protein-based antibiotic mensuration of cutting action, also can expand at an easy rate multi-functional instant pick-up unit.
Purpose of the present invention is achieved through the following technical solutions: the method for carbon nano-particles modified electrode electrochemiluminescence biosensor assay bleomycin specifically comprises the following steps:
The foundation of carbon nano-particles modified electrode electrochemiluminescence biology sensor is characterized in that specifically comprising the following steps:
Carboxylated carbon nano-particles (cCNP) preparation: 0.1 ~ 1.0 g polyvinyl alcohol (PVA) is dissolved in 10 ml water and makes homogeneous phase solution, then put into micro-wave oven (600 W) and heat 9.5 minutes (min) and change to solution colour.Above-mentioned solution is removed to impurity with centrifugal 30 min of 13000 rpm, obtain carbon nano-particles;
After the nitric acid of 0.1 ~ 2.0 gram carbon nano-particles and 0.5 mL 63% and 0.5 mL dimethyl formamide are mixed under 100 degrees centigrade 8 hours (h) of reaction, obtain carboxylated carbon nano-particles;
Described electrochemiluminescence probe, concrete preparation process is as follows:
Preferably, 200 μ L 2 OD DNA are added to 200 μ L 1.0 * 10
-3mol/L Ru (bpy)
2(dcbpy) NHS(bis-(2,2'-dipyridine) (2,2'-pyridine-4,4'-bis-carbonic acid) succinamide ruthenium).6 ~ 18 h vibrate under room temperature.Subsequently, add 100 μ L 3 mol/L sodium acetates and the cold absolute ethyl alcohols of 2.0 mL in this solution, solution is at-16 ℃ of cooling 24 h.Then centrifugal 30 min under 12000 rotating speeds and-4 ℃.Sediment cleans three times with the cold ethanol of 1 mL, removes unreacted Ru (bpy)
2(dcbpy) NHS.Obtain Ru (bpy)
2(dcbpy) DNA probe of NHS mark (electrochemiluminescence probe) dissolves with 1.0 mL 0.10 mol/L phosphate buffer solutions, is stored in-18 ℃ standby;
Preferably, described aminated dna Sequence is: 5'-CGCTTTAAAAAAAGTG-(CH
2)
6-NH
2-3'(DNA, SEQ ID NO:1).
The modified electrode preparation, concrete preparation process is as follows:
Gold electrode is through 1.0 μ m, 0.3 μ m, α-A1 of 0.05 μ m
2o
3after the burnishing powder polishing, with redistilled water, rinse well, and in ultrasound bath ultrasonic 5 min, with high pure nitrogen, dry up, standby;
The gold electrode of handling well is immersed to 100 μ L(10
-4m) in mercaptoethylmaine, fix 2 ~ 8 h(37 ℃) after, rinse secondary with phosphate buffer solution, get the carboxylated carbon nano-particles solution of 1 mL, add NHS 3.6 mg, EDC 7.2 mg, activation 1 h under 37 ° of C, more above-mentioned electrode is immersed, reaction is spent the night, and makes the carbon nano-particles modified electrode;
Utilize the Competition between electrostatic repulsion and pi-pi accumulation effect, obtain carbon nano-particles modified electrode electrochemiluminescence biology sensor.
The method of carbon nano-particles modified electrode electrochemiluminescence biosensor assay bleomycin is characterized in that specifically comprising the following steps:
[0019] preferred, add the bleomycin sample solution of variable concentrations and the Fe of 0.1 mM in 100 μ L electrochemiluminescence probes
2+, then modified electrode is immersed to reaction a period of time, take out electrode and measure according to a conventional method electrochemical luminescence signals, according to electrochemical luminescence signals, bleomycin is quantitatively detected.
The present invention compared with prior art, has following advantage and beneficial effect:
By the Competition between electrostatic repulsion and pi-pi accumulation effect, utilize DNA and carbon nano-particles non-covalent bond self assembly principle to build carbon nano-particles modified electrode electrochemiluminescence biology sensor;
Utilize the carbon nano-particles modified electrode electrochemiluminescence biology sensor built to set up a kind of new method that detects bleomycin.The method that this design adopts has simply, advantage fast.Therefore, the electrochemical luminous sensor the present invention relates to embodies good development prospect in building the research method that detects object.
The accompanying drawing explanation
Fig. 1 is carbon nano-particles modified electrode electrochemiluminescence biology sensor and measures the bleomycin schematic diagram.
Fig. 2 is modified electrode at bleomycin and probe, Fe
2+in solution after effect, soak time is to electrochemical luminescence signals (Δ I
eCL) affect graph of a relation.
The canonical plotting that Fig. 3 is electrochemiluminescence intensity and bleomycin concentration.
Embodiment
Be below the specific embodiment the present invention relates to, technical scheme of the present invention is done to further the description, but protection scope of the present invention is not limited to these embodiment.Every do not deviate from the change of the present invention design or be equal to substitute include within protection scope of the present invention.
Embodiment
1. experimental section
1.1 instrument and reagent
1-(3-dimethylamino aldehyde)-3-ethyl diimmonium salt hydrochlorate (EDC), N-hydroxy-succinamide (NHS), be purchased from Tianjin BASF chemical industry company limited; Ru (bpy)
2(dcbpy) NHS(bis-(2,2'-dipyridine) (2,2'-pyridine-4,4'-bis-carbonic acid) succinamide ruthenium, Ru), HS-(CH
2)
6-NH
2be purchased from Sigma-Aldrich.
DNA artificial sequence synthetic used (match Parkson, Beijing bioengineering company limited buys) is as follows:
5'-CGCTTTAAAAAAAGTG-(CH
2)
6-NH
2-3'（DNA，SEQ?ID?NO：1）。
CHI660B electrochemical workstation (Shanghai Chen Hua instrument company); Anke-TGL-16C flies father-in-law's board supercentrifuge (Shanghai City An Ting scientific instrument factory); Experiment adopts three-electrode system: gold electrode and modified gold electrode are working electrode, the saturated KCl of Ag/AgC1() electrode is contrast electrode, platinum electrode is to electrode.
1.2 experimental procedure
1.2.1 the preparation of electrochemiluminescence probe
200 μ L 2 OD DNA add 200 μ L 1.0 * 10
-3mol/L Ru (bpy)
2(dcbpy) NHS.6 ~ 18 h vibrate under room temperature.Subsequently, add 100 μ L 3 mol/L sodium acetates and the cold absolute ethyl alcohols of 2.0 mL in this solution, solution is at-16 ℃ of cooling 24 h.Then centrifugal 30 min under 12000 rotating speeds and-4 ℃.Sediment cleans three times with the cold ethanol of 1 mL, removes unreacted Ru (bpy)
2(dcbpy) NHS.Obtain Ru (bpy)
2(dcbpy) DNA probe of NHS mark (electrochemiluminescence probe) dissolves with 1.0 mL 0.10 mol/L phosphate buffer solutions, is stored in-18 ℃ standby.
1.2.2 the preparation of carboxylated carbon nano-particles
(1) preparation of carbon nano-particles: 0.5 g polyvinyl alcohol (PVA) is dissolved in 10 ml water and makes homogeneous phase solution, then put into micro-wave oven (600 W) and heat 9.5 min and change to solution colour.Above-mentioned solution is removed to impurity with centrifugal 30 min of 13000 rpm, then use dialysis membrane (molecular cut off 2000) in pure water to its dialysis, continue 4 days to remove remaining polyvinyl alcohol (PVA), obtain carbon nano-particles.
(2) utilize carbon nano-particles to prepare carboxylated carbon nano-particles: after the nitric acid of 1 gram carbon nano-particles and 0.5 mL 63% and 0.5 mL dimethyl formamide are mixed under 100 degrees centigrade reaction 8 h.Then, product utilization 0.22 μ M ultra filtration membrane is filtered, by under the centrifugal 10 min(14000 rmp conditions of filtrate), and with second distillation water washing three times, obtain carboxylated carbon nano-particles, then being dispersed in redistilled water and being prepared into concentration is 2.0 mg/mL solution.
1.2.3 the preparation of carbon nano-particles modified electrode
(1) pre-service of gold electrode
Gold electrode is through 1.0 μ m, 0.3 μ m, α-A1 of 0.05 μ m
2o
3after the burnishing powder polishing, with redistilled water, rinse well, and in ultrasound bath ultrasonic 5 min, with high pure nitrogen, dry up.The employing three-electrode system detects, and working electrode is gold electrode, to electrode, is platinum electrode, and contrast electrode is the Ag/AgCl electrode, at K
3[Fe (CN)
6] in solution, it is 0～0.8 V that voltage is set, and gold electrode is carried out to cyclic voltammetric (CV) scanning.If the redox current peak in 0～100 mV, has illustrated that electrode surface is processed good.Otherwise again process, until meet the requirements.After having surveyed, with intermediate water, rinse electrode, dry up electrode surface, standby.
(2) preparation of modified electrode
The above-mentioned gold electrode of handling well is immersed to 100 μ L(10
-4m) in mercaptoethylmaine, fix 4 h(37 ℃) after, rinse secondary with phosphate buffer solution, get the carboxylated carbon nano-particles solution of 1 mL, add NHS 3.6 mg, EDC 7.2 mg, activation 1 h under 37 ° of C, above-mentioned electrode is immersed, reaction is spent the night again, and makes the carbon nano-particles modified electrode.
1.2.4 the detection of bleomycin
The bleomycin solution and the 0.1 mM Fe that add variable concentrations in 100 μ L electrochemiluminescence probes
2+, react 30 min.Then the carbon nano-particles modified electrode is immersed to reaction 7 min under 37 ° of C.Rinse electrode three times with the phosphate buffer solution (pH 7.4) of 10 mM.Using this electrode as working electrode, is platinum electrode to electrode, and contrast electrode is the Ag/AgCl electrode, take electrochemical luminescence signals as analytic signal, carries out the mensuration of bleomycin.Parameter is set to: high pressure 800 V, and number of stages of amplification is 3, controlling current potential is 0-1.30 V.
1.3 result and discussion
Experimental principle
As shown in Figure 1, in this invention, utilize the cutting action after molecular recognition, thereby produce signal intensity, set up accordingly the electrochemiluminescence biology sensor of measuring bleomycin.Utilize single stranded DNA for the molecular recognition element, take bleomycin as analyzing determination object, this invention is verified.
Designed DNA partial sequence (5'-CGCTTTAAAAAAAGTG-(CH
2)
6-NH
2-3'), by a Ru for end (bpy)
2(dcbpy) NHS carries out mark as the electrochemical luminescence signals probe.When bleomycin exists, Ru-DNA is cut, forms the sheet segment DNA, and it reduces at carbon nano-particles modified electrode surface excess, a little less than electrochemical luminescence signals.When bleomycin does not exist, Ru-DNA in solution is not cut, and due to electrostatic repulsion and the pi-pi accumulation effect of carbon nano-particles and DNA, Ru-DNA is attracted to carbon nano-particles modified electrode surface, electrochemical luminescence signals is strong, realizes accordingly the mensuration to bleomycin.
As shown in Figure 2, the carbon nano-particles modified electrode is immersed in DNA probe, Fe
2+, after the bleomycin sample effect solution time to electrochemical luminescence signals (Δ I
eCL) there is impact.In 1～5 min scope, the increase of electrochemical luminescence signals intensity soak time and strengthening, after 5 min, electrochemical luminescence signals intensity slowly increases, until reach maximal value during 7 min, be greater than after 7 min change little.Therefore determine that the carbon nano-particles modified electrode is immersed in DNA probe, Fe
2+, the Best Times of solution is 7 min after the bleomycin sample effect.
Studied the impact of metallic ion on the reaction of bleomycin cutting DNA in the present embodiment.Add respectively Fe in reaction system
2+, Co
2+, Zn
2+, Ba
2+, Mg
2+, Ca
2+, Mn
2+during metallic ion, experiment finds to only have Fe
2+obvious effect is arranged, and other metallic ion is inoperative to the bleomycin cutting DNA.This oxidation cutting action that shows bleomycin and DNA needs Fe
2+thereby ion forms compound as accessory factor and makes the fracture of DNA chain.
The range of linearity and the detection limit of the present embodiment are as follows: preferred, under optimal conditions, the effects the relation between variable concentrations bleomycin and electrochemiluminescence intensity, obtained typical curve, the range of linearity and the linear equation of detection bleomycin.When the concentration of bleomycin 1.0 * 10
-10～3.0 * 10
-7in the time of between M, the electrochemiluminescence intensity of system increases (as shown in Figure 3) along with the increase of bleomycin concentration.Nonlinear regression equation is Δ I
eCL=-5618.44*exp (C/79.05)-1156.30*exp (C/3.67)+6933.49(Δ I
eCLelectrochemiluminescence intensity for system; The concentration that C is bleomycin, 10
-9m; N=7, R=0.9980).The range of linearity of bleomycin is 1.0 * 10
-10～3.0 * 10
-8m, equation of linear regression is Δ I
eCL=198.75C+120.72.The method detects and is limited to 4.0 * 10
-11m (3 σ).To concentration 5.0 * 10
-9the RSD that the bleomycin of M carries out 7 parallel replications is 3.5%, shows that the present invention has reappearance preferably.
Claims (5)
1. carbon nano-particles modified electrode electrochemiluminescence is measured the method for bleomycin, and its feature comprises the following steps:
A is dissolved in 0.1 ~ 1.0 g polyvinyl alcohol (PVA) in 10 ml water and makes homogeneous phase solution, then puts into micro-wave oven (600 W) and heats 15 min(minute) to the solution colour change, above-mentioned solution is removed to impurity with centrifugal 30 min of 13000 rpm, obtain carbon nano-particles; After the nitric acid of 0.1 ~ 2.0 gram carbon nano-particles and 0.5 mL 63% and 0.5 mL dimethyl formamide are mixed under 100 degrees centigrade 8 hours (h) of reaction;
B adds 200 μ L 1.0 * 10 by 200 μ L 2 OD DNA
-3mol/L Ru (bpy)
2(dcbpy) NHS(bis-(2, the 2'-dipyridine) (2,2'-pyridine-4,4'-bis-carbonic acid) the succinamide ruthenium), 6 ~ 18 h vibrate under room temperature, subsequently, add 100 μ L 3 mol/L sodium acetates and the cold absolute ethyl alcohols of 2.0 mL in this solution, solution is at-16 ℃ of cooling 24 h, then centrifugal 30 min under 12000 rotating speeds and-4 ℃, sediment cleans three times with the cold ethanol of 1 mL, removes unreacted Ru (bpy)
2(dcbpy) NHS, obtain Ru (bpy)
2(dcbpy) DNA probe of NHS mark (electrochemiluminescence probe) dissolves with 1.0 mL 0.10 mol/L phosphate buffer solutions;
The c gold electrode is through 1.0 μ m, 0.3 μ m, α-A1 of 0.05 μ m
2o
3after the burnishing powder polishing, with redistilled water, rinse well, and in ultrasound bath ultrasonic 5 min, with high pure nitrogen, dry up, standby, the gold electrode of handling well is immersed to 100 μ L(10
-4m) in mercaptoethylmaine, fix 2 ~ 8 h(37 ℃) after, rinse secondary with phosphate buffer solution, get the carboxylated carbon nano-particles solution of 1 mL, add N-hydroxy-succinamide (NHS) 3.6 mg, 1-(3-dimethylamino aldehyde)-3-ethyl diimmonium salt hydrochlorate (EDC) 7.2 mg, activation 1 h under 37 ° of C, again above-mentioned electrode is immersed, reaction is spent the night, make the carbon nano-particles modified electrode, utilize electrostatic repulsion between DNA and probe and carbon nano-particles modified electrode and the Competition between the pi-pi accumulation effect, obtain carbon nano-particles modified electrode electrochemiluminescence biology sensor.
2. utilize the method for the prepared carbon nano-particles modified electrode electrochemiluminescence biosensor assay bleomycin of claim 1, its feature comprises the following steps:
The bleomycin solution and the 0.1 mM Fe that add variable concentrations in 100 μ L electrochemiluminescence probes
2+then modified electrode is immersed to reaction 7 min, with the phosphate buffer solution (pH 7.4) of 10 mM, rinses electrode three times, then carry out electrochemiluminescence mensuration, according to effect rear electrode surface electrochemistry electrochemical luminescence signals that luminescence probe produces, bleomycin is quantitatively detected.
3. electrochemiluminescence according to claim 2 is measured and is specially: adopt three-electrode system to detect, working electrode is gold electrode, to electrode, is platinum electrode, contrast electrode is the Ag/AgCl electrode, parameter is set to: high pressure 800 V, and number of stages of amplification is 3, controlling current potential is 0-1.30 V.
4. it is characterized in that according to claim 1: the DNA partial sequence is 5'-CGCTTTAAAAAAAGTG-(CH
2)
6-NH
2-3'.
5. it is characterized in that according to claim 1: the DNA partial sequence is that the 3' end is amido modified.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104007152A (en) * | 2014-06-18 | 2014-08-27 | 青岛科技大学 | DNA determining electrochemical sensor and method based on platinum nano particle catalysis electrochemistry circulation signal amplification technology |
| CN105385770A (en) * | 2015-12-18 | 2016-03-09 | 山东大学 | Dual-ring hairpin probe mediate label-free strand displacement amplification method for detecting bleomycin |
| CN105950757A (en) * | 2016-06-17 | 2016-09-21 | 山东大学 | Method for detecting bleomycin based on double-ring hairpin probe and enzyme-mediated cascade amplification strategy |
| CN110426440A (en) * | 2019-07-16 | 2019-11-08 | 曲阜师范大学 | A kind of Photoelectrochemistrbiosensor biosensor and its detection method to BLM |
| CN114487066A (en) * | 2022-01-27 | 2022-05-13 | 上海市肿瘤研究所 | Ultrasensitive DNA-biomacromolecule sensor, construction and application thereof |
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- 2013-09-03 CN CN201310396240.3A patent/CN103439319B/en not_active Expired - Fee Related
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104007152A (en) * | 2014-06-18 | 2014-08-27 | 青岛科技大学 | DNA determining electrochemical sensor and method based on platinum nano particle catalysis electrochemistry circulation signal amplification technology |
| CN104007152B (en) * | 2014-06-18 | 2016-05-25 | 青岛科技大学 | Measure the method for electrochemical sensor and the mensuration DNA of DNA based on nano platinum particle catalytic electrochemical cycle signal amplifying technique |
| CN105385770A (en) * | 2015-12-18 | 2016-03-09 | 山东大学 | Dual-ring hairpin probe mediate label-free strand displacement amplification method for detecting bleomycin |
| CN105385770B (en) * | 2015-12-18 | 2018-09-28 | 山东大学 | The Dual-ring hairpin probe for detecting bleomycin mediates label-free strand displacement amplification method |
| CN105950757A (en) * | 2016-06-17 | 2016-09-21 | 山东大学 | Method for detecting bleomycin based on double-ring hairpin probe and enzyme-mediated cascade amplification strategy |
| CN105950757B (en) * | 2016-06-17 | 2019-11-22 | 山东大学 | Method based on the cascade amplification strategy detection bleomycin that Dual-ring hairpin probe and enzyme mediate |
| CN110426440A (en) * | 2019-07-16 | 2019-11-08 | 曲阜师范大学 | A kind of Photoelectrochemistrbiosensor biosensor and its detection method to BLM |
| CN110426440B (en) * | 2019-07-16 | 2022-02-01 | 曲阜师范大学 | Photoelectrochemistry biosensor and detection method of photoelectrochemistry biosensor for BLM |
| CN114487066A (en) * | 2022-01-27 | 2022-05-13 | 上海市肿瘤研究所 | Ultrasensitive DNA-biomacromolecule sensor, construction and application thereof |
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