CN104090012A - Preparation method of carbon nano-horn/ionic liquid composite modified electrode for detecting purine bases in cells - Google Patents
Preparation method of carbon nano-horn/ionic liquid composite modified electrode for detecting purine bases in cells Download PDFInfo
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- CN104090012A CN104090012A CN201410347677.2A CN201410347677A CN104090012A CN 104090012 A CN104090012 A CN 104090012A CN 201410347677 A CN201410347677 A CN 201410347677A CN 104090012 A CN104090012 A CN 104090012A
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Abstract
The invention discloses a preparation method of a carbon nano-horn/ionic liquid composite modified electrode for detecting purine bases in cells, relates to a preparation method of a composite modified electrode for detecting the purine bases in the cells, and aims at solving the technical problems that although a current electrode is good in detection effect on purine standard substances, the detection effect on cytoplasm samples is non-ideal, the detection limit is high and the needed sample amount is large. The method comprises the following steps: (1) acidizing a carbon nano-horn; and (2) preparing a composite electrode. The electrode prepared by adopting the method is very highly sensitive to MCF-7 cells, and the cell amount of 6*10<3>cell/mL can be detected at least. The method is applicable to electrochemical detection on purine components in the cells.
Description
Technical field
The present invention relates to a kind of preparation method who detects the composite modified electrode of purine bases in cell.
Background technology
The diameter of single wall carbon nanohorn (SWNHs) is 2～5nm, and length is 40～50nm, and its form is similar to the Single Walled Carbon Nanotube after brachymemma, but its one end has unique pyramidal structure.Single wall carbon nanohorn is owing to having huge Van der Waals force between pipe, and easily self aggregation together, forms one-level and assembles.Carbon nanohorn is exactly " dahlia " shape aggregation of being assembled the 80～100nm left and right forming by thousands of single wall carbon nanohorns.The specific surface area that single wall carbon nanohorn is large and poriness make it have good compatibility to organism, become the material of potential storage hydrogen and methane simultaneously and are used to delivery system.Dissolubility is the precondition of carbon nanohorn in life science application preferably.Nearest some studies confirm that the single wall carbon nanohorn of functionalization can improve its dissolubility in organic solvent and water.Due to single wall carbon nanohorn by carbon nano-tube structurally polymerization obtain, can believe that it can be applied with the similar field of carbon nano-tube.Similar to Single Walled Carbon Nanotube (SWNTs), can obtain systems such as being conducive to electronics transmission and photovoltaic applications with the chemically derived single wall carbon nanohorn of electroactive material.Raman Chromatogram display single wall carbon nanohorn has more defect than Single Walled Carbon Nanotube.For single wall carbon nanohorn, the D of unordered induction can be with and can be with in intensity similar with the G of graphite mould.In the reaction of carbon nano-tube, amino group can be connected to its end.Can there is similar reaction in single wall carbon nanohorn, even have higher activity.And the existence of defect is also conducive to occur oxidation reaction, generate shuttle base, make follow-up reaction be easy to carry out.
Purine nucleotides can produce different purine bases, such as guanine, adenine, xanthine and hypoxanthine etc. in the metabolic process of cell.In Normocellular metabolic process, purine level is stable, but in the time that abnormal metabolism appears in cell, just there will be the ANOMALOUS VARIATIONS of purine level.Therefore, can reflect the physiological status of cell by the purine level of monitoring cell, and then for the screening active ingredients of medicine.At present, in cell, the detection method of purine mainly contains vapor-phase chromatography, high performance liquid chromatography, capillary electrophoresis and ion pair chromatography etc., but most methods exists the shortcomings such as sample pre-treatments loaded down with trivial details and analysis time is long, thereby be difficult to the real change of reflection purine.Electrochemical analysis as a kind of simply, easily and fast, cheap analytical approach, will greatly shorten the detection time of purine and reduce testing cost.Since bring up Huangsong first etc. the voltammetric signal of proposition intact cell derive from cell guanine fast by the oxidation reaction occurring at electrode surface after cell membrane.Military winter plum teach problem group has further been studied the electrochemical response mechanism of cell, the electrochemical signals (0.7V) of finding intact cell derives from guanine and xanthic oxidation reaction in cell exocrine thing, and xanthic signal (Anal.Biochem. in the highest flight, 2009, 394, 229 and Talanta, 2009, 78, 602), and in 2012, two electrochemical signals (0.7V and 1.1V) of cell are detected, find that it belongs to respectively the oxidation reaction (Analyst of guanine/xanthine and hypoxanthine/adenine, 2012, 137, 3230), find to adopt two signal method, evaluate the physiological status of cell taking purine as biomarker, and set up the electrochemical method of cancer therapy drug and estrogen active with this.But, due to the susceptibility restriction of electrode, sensitive not enough to the detection of cell electric signal.Carbon nanomaterial, as a kind of emerging material, has caused people's interest widely.Carbon nanomaterial is introduced in biology sensor, greatly improved sensitivity and the reappearance of biology sensor, the biology sensor of being combined with carbon nanomaterial has become the study hotspot of electrochemica biological sensor of new generation.Compared with other carbon nanomaterial, single wall carbon nanohorn has advantages of more poriness, the larger specific surface area of height and has a large amount of oxy radicals and is exposed to most advanced and sophisticatedly, becomes more excellent purine and detects electrochemical sensor and prepare material.
Although the appearance of nano material has promoted the development of electrochemical sensor, the structure of Nanoparticle Modified Electrode, has improved the susceptibility of electrode, has significantly reduced the detection lower limit of electro-chemical systems to cell.Especially people by various material construction the modified electrode such as Graphene, carbon nano-tube/ionic liquid, detect the purine content in cell, but although these electrodes detect purine standard items, effect is better, unsatisfactory to the detection of tenuigenin sample, detect limit for height, and need sample size large.
Summary of the invention
Although the object of the invention is in order to solve current electrode detection purine standard items effect better, unsatisfactory to the detection of tenuigenin sample, detect limit for height, and need the technical matters that sample size is large, and a kind of preparation method who detects carbon nanohorn/ionic liquid composite modified electrode of purine bases in cell is provided.
A preparation method who detects carbon nanohorn/ionic liquid composite modified electrode of purine bases in cell, specifically carries out according to the following steps:
One, acidifying carbon nanohorn: carbon nanohorn is evenly mixed with red fuming nitric acid (RFNA), is the 3h～4h that refluxes under the condition of 110 DEG C～130 DEG C in temperature, obtains the carbon nanohorn of acidifying; The quality of the carbon nanohorn described in step 1 and the volume ratio of red fuming nitric acid (RFNA) are 1mg:(3mL～5mL);
Two, prepare combination electrode: the carbon nanohorn of the acidifying that step 1 is obtained mixes with ionic liquid, obtain composite fluid, on glass-carbon electrode after polishing, be evenly coated with one deck composite fluid with knife coating, obtain detecting carbon nanohorn/ionic liquid composite modified electrode of purine bases in cell; Ionic liquid described in step 2 is 1-ethyl-3-methylimidazole hexafluorophosphate, 1-butyl-3-methylimidazole hexafluorophosphate, 1-hexyl-3-methylimidazole hexafluorophosphate, 1-octyl group-3-methylimidazole hexafluorophosphate, O-benzotriazole-N, N, N', N'-tetramethylurea hexafluorophosphate, O-(1H-6-Chloro-Benzotriazole-1-yl)-1,1, the potpourri of one or more in 3,3-tetramethylurea hexafluorophosphate, 1-(chloro-1-pyrrolidinyl methylene) pyrrolidine hexafluorophosphate; The quality of the carbon nanohorn of the acidifying described in step 2 and the volume ratio of ionic liquid are 1mg:(2.5 μ L～10 μ L).
Advantage of the present invention:
Utilize carbon nanohorn/ionic liquid composite modified electrode of purine bases in detection cell prepared by the present invention to detect and mix purine standard solution, in Electrochemical Detection process, can observe two obvious oxidation peak, what wherein occur in 0.7V left and right is guanine and xanthic mixed signal peak, and what occur at 1.1V is the oxidation signal of hypoxanthine and adenine; Guanine and xanthic detectability can reach 2 × 10
-7mol/L, the detectability 5 × 10 of hypoxanthine and adenine
-7mol/L; The prepared electrode of the present invention has very high sensitivity for MCF-7 cell, three signal peaks of cell can be detected, i.e. the uric acid peak of 0.4V left and right, guanine and the xanthine signal peak of 0.7V left and right, and hypoxanthine and the adenine signal peak of about 1.1V, minimumly detect 6 × 10
3the cell concentration of cell/mL.
Brief description of the drawings
Fig. 1 is galvanochemistry figure, curve 1 is carbon nanohorn/ionic liquid composite modified electrode of purine bases in the detection cell of test in two galvanochemistry curve in pbs buffer solution, curve 2 be in test three with the glass-carbon electrode after polishing the galvanochemistry curve as working electrode gained in MCF-7 cell pyrolysis liquid, curve 3 is that test detects carbon nanohorn/ionic liquid composite modified electrode of purine bases in cell in three as the galvanochemistry curve of working electrode gained in MCF-7 cell pyrolysis liquid; Signal a is the oxidation peak of uric acid, and signal b is guanine and xanthic mixed oxidization signal peak, and signal c is the mixed oxidization signal peak of hypoxanthine and adenine.
Embodiment
Embodiment one: in present embodiment, a kind of preparation method who detects carbon nanohorn/ionic liquid composite modified electrode of purine bases in cell carries out according to the following steps:
One, acidifying carbon nanohorn: carbon nanohorn is evenly mixed with red fuming nitric acid (RFNA), is the 3h～4h that refluxes under the condition of 110 DEG C～130 DEG C in temperature, obtains the carbon nanohorn of acidifying; The quality of the carbon nanohorn described in step 1 and the volume ratio of red fuming nitric acid (RFNA) are 1mg:(3mL～5mL);
Two, prepare combination electrode: the carbon nanohorn of the acidifying that step 1 is obtained mixes with ionic liquid, obtain composite fluid, on glass-carbon electrode after polishing, be evenly coated with skim composite fluid with knife coating, obtain detecting carbon nanohorn/ionic liquid composite modified electrode of purine bases in cell; Ionic liquid described in step 2 is 1-ethyl-3-methylimidazole hexafluorophosphate, 1-butyl-3-methylimidazole hexafluorophosphate, 1-hexyl-3-methylimidazole hexafluorophosphate, 1-octyl group-3-methylimidazole hexafluorophosphate, O-benzotriazole-N, N, N', N'-tetramethylurea hexafluorophosphate, O-(1H-6-Chloro-Benzotriazole-1-yl)-1,1, the potpourri of one or more in 3,3-tetramethylurea hexafluorophosphate, 1-(chloro-1-pyrrolidinyl methylene) pyrrolidine hexafluorophosphate; The quality of the carbon nanohorn of the acidifying described in step 2 and the volume ratio of ionic liquid are 1mg:(2.5 μ L～10 μ L).
The advantage of present embodiment:
Utilize carbon nanohorn/ionic liquid composite modified electrode of purine bases in detection cell prepared by present embodiment to detect and mix purine standard solution, in Electrochemical Detection process, can observe two obvious oxidation peak, what wherein occur in 0.7V left and right is guanine and xanthic mixed signal peak, and what occur at 1.1V is the oxidation signal of hypoxanthine and adenine; Guanine and xanthic detectability can reach 2 × 10
-7mol/L, the detectability 5 × 10 of hypoxanthine and adenine
-7mol/L; The prepared electrode of present embodiment has very high sensitivity for MCF-7 cell, three signal peaks of cell can be detected, it is the uric acid peak of 0.4V left and right, guanine and the xanthine signal peak of 0.7V left and right, and hypoxanthine and the adenine signal peak of about 1.1V, minimumly detect 6 × 10
3the cell concentration of cell/mL.
Embodiment two: present embodiment is different from embodiment one: the carbon nanohorn described in step 1 is single wall carbon nanohorn.Other are identical with embodiment one.
Embodiment three: present embodiment is different from one of embodiment one or two: step 1 is evenly mixed carbon nanohorn with red fuming nitric acid (RFNA), is the 3h that refluxes under the condition of 120 DEG C in temperature, obtains the carbon nanohorn of acidifying; The quality of the carbon nanohorn described in step 1 and the volume ratio of red fuming nitric acid (RFNA) are 1mg:3.75mL.Other are identical with one of embodiment one or two.
Embodiment four: present embodiment is different from one of embodiment one to three: the massfraction of the red fuming nitric acid (RFNA) described in step 1 is 65%～68%.Other are identical with one of embodiment one to three.
Embodiment five: present embodiment is different from one of embodiment one to four: the quality of the carbon nanohorn of the acidifying described in step 2 and the volume ratio of ionic liquid are 1mg:5 μ L.Other is identical with one of embodiment one to four.
Embodiment six: present embodiment is different from one of embodiment one to five: be 0.1 μ m～1 μ m with the thickness that knife coating is evenly coated with one deck composite fluid on the glass-carbon electrode after polishing described in step 2.Other is identical with one of embodiment one to five.
Adopt following verification experimental verification effect of the present invention:
Test one: a kind of preparation method who detects carbon nanohorn/ionic liquid composite modified electrode of purine bases in cell of this test carries out according to the following steps:
One, acidifying carbon nanohorn: carbon nanohorn is evenly mixed with red fuming nitric acid (RFNA), is the 3h that refluxes under the condition of 120 DEG C in temperature, obtains the carbon nanohorn of acidifying; The quality of the carbon nanohorn described in step 1 and the volume ratio of red fuming nitric acid (RFNA) are 1mg:3.75mL; Carbon nanohorn described in step 1 is single wall carbon nanohorn; The massfraction of the red fuming nitric acid (RFNA) described in step 1 is 67%;
Two, prepare combination electrode: the carbon nanohorn of the acidifying that step 1 is obtained mixes with ionic liquid, obtain composite fluid, on glass-carbon electrode after polishing, be evenly coated with skim composite fluid with knife coating, obtain detecting carbon nanohorn/ionic liquid composite modified electrode of purine bases in cell; Ionic liquid described in step 2 is 1-butyl-3-methylimidazole hexafluorophosphate; The quality of the carbon nanohorn of the acidifying described in step 2 and the volume ratio of ionic liquid are 1mg:5 μ L; On the glass-carbon electrode after polishing described in step 2, be 0.5 μ m with the thickness that knife coating is evenly coated with one deck composite fluid.
Test two: by the Na of 17.9g
2hPO
412H
2the KH of O and 6.8g
2pO
4be dissolved in the distilled water of 250mL, obtain pbs buffer solution; In the test one detection cell obtaining, carbon nanohorn/ionic liquid composite modified electrode of purine bases is as working electrode, and effectively material area is 0.7cm
2, silver/silver chloride is as contrast electrode, and platinum filament, as to electrode, adopts traditional three-electrode system to test by cyclic voltammetric method, and voltage is increased to 1.20V from 0V, sweep speed 50mV/s, thus obtain the current-responsive of this modified electrode to pbs buffer solution.
Test three: 2.4 × 10
3in individual MCF-7 cell, add the pbs buffer solution in the test two of 0.4mL, constant temperature 30min in the water-bath of 50 DEG C, naturally cool at normal temperatures room temperature, obtain MCF-7 cell pyrolysis liquid, glass-carbon electrode in the test one detection cell obtaining after carbon nanohorn/ionic liquid composite modified electrode and the polishing of purine bases is respectively as working electrode, and effectively material area is 0.7cm
2, silver/silver chloride is as contrast electrode, and platinum filament, as to electrode, adopts traditional three-electrode system to test by cyclic voltammetric method, and voltage is increased to 1.20V from 0V, sweep speed 50mV/s, thus obtain respectively the current-responsive of electrode pair MCF-7 cell pyrolysis liquid.
Fig. 1 is galvanochemistry figure, curve 1 is the test two galvanochemistry curves that obtain, curve 2 be test three in the glass-carbon electrode after polishing the galvanochemistry curve as working electrode gained, curve 3 is in test three, to detect carbon nanohorn/ionic liquid composite modified electrode of purine bases in cell as the galvanochemistry curve of working electrode gained, signal a is the oxidation peak of uric acid, signal b is guanine and xanthic mixed oxidization signal peak, and signal c is the mixed oxidization signal peak of hypoxanthine and adenine.From figure, 3 contrasts of curve 1 and curve can find out that the carbon nanohorn/ionic liquid composite modified electrode that detects purine bases in cell in test three is all to be come by the MCF-7 cell in test three as the signal of the galvanochemistry curve of working electrode gained, in 3 contrasts of curve 2 and curve can be found out the detection cell of test three, to detect the electric signal of cell stronger for the glass-carbon electrode of carbon nanohorn/ionic liquid composite modified electrode of purine bases than the polishing of unmodified, in the detection cell of test three, carbon nanohorn/ionic liquid composite modified electrode of purine bases has very high sensitivity for MCF-7 cell, can detect 6 × 10
3the cell concentration of cell/mL.
Claims (6)
1. detect a preparation method for carbon nanohorn/ionic liquid composite modified electrode of purine bases in cell, it is characterized in that the preparation method who detects carbon nanohorn/ionic liquid composite modified electrode of purine bases in cell carries out according to the following steps:
One, acidifying carbon nanohorn: carbon nanohorn is evenly mixed with red fuming nitric acid (RFNA), is the 3h～4h that refluxes under the condition of 110 DEG C～130 DEG C in temperature, obtains the carbon nanohorn of acidifying; The quality of the carbon nanohorn described in step 1 and the volume ratio of red fuming nitric acid (RFNA) are 1mg:(3mL～5mL);
Two, prepare combination electrode: the carbon nanohorn of the acidifying that step 1 is obtained mixes with ionic liquid, obtain composite fluid, on glass-carbon electrode after polishing, be evenly coated with one deck composite fluid with knife coating, obtain detecting carbon nanohorn/ionic liquid composite modified electrode of purine bases in cell; Ionic liquid described in step 2 is 1-ethyl-3-methylimidazole hexafluorophosphate, 1-butyl-3-methylimidazole hexafluorophosphate, 1-hexyl-3-methylimidazole hexafluorophosphate, 1-octyl group-3-methylimidazole hexafluorophosphate, O-benzotriazole-N, N, N', N'-tetramethylurea hexafluorophosphate, O-(1H-6-Chloro-Benzotriazole-1-yl)-1,1, the potpourri of one or more in 3,3-tetramethylurea hexafluorophosphate, 1-(chloro-1-pyrrolidinyl methylene) pyrrolidine hexafluorophosphate; The quality of the carbon nanohorn of the acidifying described in step 2 and the volume ratio of ionic liquid are 1mg:(2.5 μ L～10 μ L).
2. a kind of preparation method who detects carbon nanohorn/ionic liquid composite modified electrode of purine bases in cell according to claim 1, is characterized in that the carbon nanohorn described in step 1 is single wall carbon nanohorn.
3. a kind of preparation method who detects carbon nanohorn/ionic liquid composite modified electrode of purine bases in cell according to claim 1, it is characterized in that step 1 evenly mixes carbon nanohorn with red fuming nitric acid (RFNA), be the 3h that refluxes under the condition of 120 DEG C in temperature, obtain the carbon nanohorn of acidifying; The quality of the carbon nanohorn described in step 1 and the volume ratio of red fuming nitric acid (RFNA) are 1mg:3.75mL.
4. a kind of preparation method who detects carbon nanohorn/ionic liquid composite modified electrode of purine bases in cell according to claim 1, the massfraction that it is characterized in that the red fuming nitric acid (RFNA) described in step 1 is 65%～68%.
5. a kind of preparation method who detects carbon nanohorn/ionic liquid composite modified electrode of purine bases in cell according to claim 1, is characterized in that the quality of carbon nanohorn and the volume ratio of ionic liquid of the acidifying described in step 2 is 1mg:5 μ L.
6. a kind of preparation method who detects carbon nanohorn/ionic liquid composite modified electrode of purine bases in cell according to claim 1, the thickness that it is characterized in that on the glass-carbon electrode after polishing described in step 2 being evenly coated with knife coating one deck composite fluid is 0.1 μ m～1 μ m.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104765962A (en) * | 2015-04-08 | 2015-07-08 | 河海大学 | Temperature variation considering type state estimation method of power system |
| CN105954338A (en) * | 2016-04-21 | 2016-09-21 | 福建师范大学 | Electrochemical sensing method for food-borne pollutants based on carbon nanohorn and triethoxysilane |
| CN108828033A (en) * | 2017-12-19 | 2018-11-16 | 浙江大学 | A kind of solid ionic electrodes selective and preparation method based on single corner electrode |
-
2014
- 2014-07-21 CN CN201410347677.2A patent/CN104090012A/en active Pending
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| FEI XIAO, ET AL.: "Voltammetric Determination of Xanthine with a Single-Walled Carbon Nanotube-Ionic Liquid Paste Modified Glassy Carbon Electrode", 《ELECTROANALYSIS》 * |
| JINLIAN LI,ET AL.: "Two-signal electrochemical method for evaluation suppression and proliferation of MCF-7 cells based on intracellular purine", 《ANALYTICAL BIOCHEMISTRY》 * |
| QUANPING YAN,ET AL.: "Voltammetric Determination of Uric Acid with a Glassy Carbon Electrode Coated by Paste of Multiwalled Carbon Nanotubes and Ionic Liquid", 《ELECTROANALYSIS》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104765962A (en) * | 2015-04-08 | 2015-07-08 | 河海大学 | Temperature variation considering type state estimation method of power system |
| CN105954338A (en) * | 2016-04-21 | 2016-09-21 | 福建师范大学 | Electrochemical sensing method for food-borne pollutants based on carbon nanohorn and triethoxysilane |
| CN108828033A (en) * | 2017-12-19 | 2018-11-16 | 浙江大学 | A kind of solid ionic electrodes selective and preparation method based on single corner electrode |
| WO2019120314A1 (en) * | 2017-12-19 | 2019-06-27 | 浙江大学 | Solid ion-selective electrode based on single-wall carbon nanohorn electrode, and preparation method |
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Application publication date: 20141008 |