CN102914570B - Method for detecting glutathione based on nanogold and thionine signal amplification - Google Patents
Method for detecting glutathione based on nanogold and thionine signal amplification Download PDFInfo
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- CN102914570B CN102914570B CN201210208146.6A CN201210208146A CN102914570B CN 102914570 B CN102914570 B CN 102914570B CN 201210208146 A CN201210208146 A CN 201210208146A CN 102914570 B CN102914570 B CN 102914570B
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- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 title claims abstract description 49
- 238000000034 method Methods 0.000 title claims abstract description 25
- 108010024636 Glutathione Proteins 0.000 title claims abstract description 23
- 229960003180 glutathione Drugs 0.000 title claims abstract description 23
- KUUVQVSHGLHAKZ-UHFFFAOYSA-N thionine Chemical group C=1C=CC=CSC=CC=1 KUUVQVSHGLHAKZ-UHFFFAOYSA-N 0.000 title claims abstract description 21
- 230000003321 amplification Effects 0.000 title claims abstract description 8
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract description 8
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 78
- 239000002608 ionic liquid Substances 0.000 claims abstract description 51
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 44
- 229910021389 graphene Inorganic materials 0.000 claims abstract description 35
- 102100033072 DNA replication ATP-dependent helicase DNA2 Human genes 0.000 claims abstract description 20
- 101000927313 Homo sapiens DNA replication ATP-dependent helicase DNA2 Proteins 0.000 claims abstract description 20
- 229960003067 cystine Drugs 0.000 claims abstract description 16
- 239000000523 sample Substances 0.000 claims abstract description 16
- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 claims abstract description 13
- 238000001514 detection method Methods 0.000 claims abstract description 7
- 239000002105 nanoparticle Substances 0.000 claims abstract description 5
- 239000004158 L-cystine Substances 0.000 claims abstract description 3
- 235000019393 L-cystine Nutrition 0.000 claims abstract description 3
- 239000010931 gold Substances 0.000 claims description 34
- 229910052737 gold Inorganic materials 0.000 claims description 30
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical group [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 27
- -1 quaternary ammonium salt ion Chemical class 0.000 claims description 18
- 238000002360 preparation method Methods 0.000 claims description 13
- 230000000694 effects Effects 0.000 claims description 7
- 239000011324 bead Substances 0.000 claims description 5
- 239000011263 electroactive material Substances 0.000 claims description 5
- 238000004070 electrodeposition Methods 0.000 claims description 5
- GEFGETHETIRZQB-UHFFFAOYSA-N S1C=CC=CC=CC=C1.[Au] Chemical compound S1C=CC=CC=CC=C1.[Au] GEFGETHETIRZQB-UHFFFAOYSA-N 0.000 claims description 4
- 230000008859 change Effects 0.000 claims description 4
- 150000002500 ions Chemical class 0.000 claims description 4
- 230000004048 modification Effects 0.000 claims description 4
- 238000012986 modification Methods 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 3
- 238000007306 functionalization reaction Methods 0.000 claims description 3
- 238000009396 hybridization Methods 0.000 claims description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-O Imidazolium Chemical compound C1=C[NH+]=CN1 RAXXELZNTBOGNW-UHFFFAOYSA-O 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 239000002994 raw material Substances 0.000 claims description 2
- 239000012488 sample solution Substances 0.000 claims description 2
- PNGLEYLFMHGIQO-UHFFFAOYSA-M sodium;3-(n-ethyl-3-methoxyanilino)-2-hydroxypropane-1-sulfonate;dihydrate Chemical compound O.O.[Na+].[O-]S(=O)(=O)CC(O)CN(CC)C1=CC=CC(OC)=C1 PNGLEYLFMHGIQO-UHFFFAOYSA-M 0.000 claims description 2
- 238000005516 engineering process Methods 0.000 abstract description 7
- 238000000835 electrochemical detection Methods 0.000 abstract description 5
- 230000009471 action Effects 0.000 abstract description 3
- 239000004005 microsphere Substances 0.000 abstract 2
- 239000000243 solution Substances 0.000 description 18
- 239000007853 buffer solution Substances 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 239000011325 microbead Substances 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000001903 differential pulse voltammetry Methods 0.000 description 3
- 238000002848 electrochemical method Methods 0.000 description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 208000024893 Acute lymphoblastic leukemia Diseases 0.000 description 2
- 208000014697 Acute lymphocytic leukaemia Diseases 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 208000006664 Precursor Cell Lymphoblastic Leukemia-Lymphoma Diseases 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 238000002484 cyclic voltammetry Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
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- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 229910052755 nonmetal Inorganic materials 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 230000001590 oxidative effect Effects 0.000 description 2
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
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- 239000000126 substance Substances 0.000 description 2
- CWERGRDVMFNCDR-UHFFFAOYSA-N thioglycolic acid Chemical compound OC(=O)CS CWERGRDVMFNCDR-UHFFFAOYSA-N 0.000 description 2
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- YYQRGCZGSFRBAM-UHFFFAOYSA-N Triclofos Chemical compound OP(O)(=O)OCC(Cl)(Cl)Cl YYQRGCZGSFRBAM-UHFFFAOYSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
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- 239000012847 fine chemical Substances 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- FDWREHZXQUYJFJ-UHFFFAOYSA-M gold monochloride Chemical compound [Cl-].[Au+] FDWREHZXQUYJFJ-UHFFFAOYSA-M 0.000 description 1
- 229910002804 graphite Inorganic materials 0.000 description 1
- 239000010439 graphite Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
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- 239000006210 lotion Substances 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- CXKWCBBOMKCUKX-UHFFFAOYSA-M methylene blue Chemical compound [Cl-].C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 CXKWCBBOMKCUKX-UHFFFAOYSA-M 0.000 description 1
- 229960000907 methylthioninium chloride Drugs 0.000 description 1
- HOGDNTQCSIKEEV-UHFFFAOYSA-N n'-hydroxybutanediamide Chemical compound NC(=O)CCC(=O)NO HOGDNTQCSIKEEV-UHFFFAOYSA-N 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- NICDRCVJGXLKSF-UHFFFAOYSA-N nitric acid;trihydrochloride Chemical compound Cl.Cl.Cl.O[N+]([O-])=O NICDRCVJGXLKSF-UHFFFAOYSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
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- 229910052707 ruthenium Inorganic materials 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- DAJSVUQLFFJUSX-UHFFFAOYSA-M sodium;dodecane-1-sulfonate Chemical compound [Na+].CCCCCCCCCCCCS([O-])(=O)=O DAJSVUQLFFJUSX-UHFFFAOYSA-M 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 229960001147 triclofos Drugs 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The embodiment of the invention discloses a method for detecting reduced glutathione (GSH) based on a nanogold and thionine signal amplification technology and an electrochemical detection technology. The method comprises the following steps: modifying a magnetic microsphere by using L-cystine and DNA1, and cutting and releasing the DNA1 on the surface of the magnetic microsphere through the action of cutting an L-cystine disulfide bond by using glutathione; modifying a nanogold/graphene modified ionic liquid carbon paste electrode by using the released DNA1; loading DNA2 on the surfaces of thionine-modified gold nano particles to obtain an electrochemical probe; immersing the DNA1/nanogold/grapheme modified ionic liquid carbon paste electrode in an electrochemical probe solution; and capturing a DNA2/thionine/nanogold signal amplification unit on the electrode surface by the DNA1 to cause the variation of the concentration of the electrochemical probe on the electrode surface so as to carry out the electrochemical detection. By using the nanogold and thionine signal amplification technology and the electrochemical detection technology, the high-sensitivity detection of the glutathione is realized.
Description
Technical field
The invention belongs to technical field of electrochemical detection, particularly a kind of preparation of modified electrode and a kind of development of signal amplification technique, its application relates to the detection of glutathione content.
Background technology
Glutathione there is the abundantest Small molecular sulfur alcohol compound in cell.Reduced glutathione (GSH) (calling glutathione in the following text) is the chief component of non-protein sulfydryl group in cell, has multiple important physiological function in living tissue.It not only has very important physiological action, the direct reflection of the change of its content in cell or some disease and cancer.
Electrochemical method has the advantages such as simple to operate, highly sensitive, instrument and equipment is simple.
Ionic liquid carbon paste electrode is a kind of carbon paste electrode replacing whiteruss to make as bonding agent ionic liquid, because ionic liquid has high electron conduction, good viscosity, wide electrochemical window, therefore the basal electrode of ionic liquid carbon paste electrode Chang Zuowei electrochemical sensor, it has performance better than traditional glass-carbon electrode.
Graphene has the advantages such as thermal conductivity coefficient is high, physical strength good, charge carrier good fluidity, specific surface area are large.
Electrochemical deposition nm of gold has large specific surface area, improves the effect of electron transmission ability.
Summary of the invention
The suction-operated that the object of the invention is to utilize thionine on golden nanometer particle surface builds signal amplification technique, realizes electrochemical method high sensitivity measuring glutathione with the effect of glutathione cutting disulfide bond.
Method provided by the present invention comprises the following steps:
(1) be that ionic liquid carbon paste electrode prepared by dressing agent with ionic liquid.
Described method, it is quaternary ammonium salt ion, quaternary alkylphosphonium salt ion, imidazolium ion and pyrroles's salt ion that the ionic liquid described in it can be kation; Negative ion is the ionic liquid of halide ion, tetrafluoroborate ion, hexafluorophosphoricacid acid ions composition, as 1-hexyl pyridine hexafluorophosphate etc.
(2) in graphene oxide solution, utilize potentiostatic electrodeposition method at the obtained graphite modified membrane in ionic liquid carbon paste surface, then with HAuCl
4for raw material, use potentiostatic method deposited Au, nm of gold/graphene modified ionic liquid carbon paste electrode can be obtained, obtain working electrode.
Described method, the ionic liquid carbon paste electrode described in it can be ionic liquid carbon paste electrode, metal electrode, non-metal electrode or oxidizing electrode.
(3) with CYSTINE (Cystine) or containing the material of disulfide bond and the DNA1 modified magnetic microballon of functionalization, prepare functional magnetic microballon, magnetic micro-beads contains the material of disulfide bond and the DNA1 of functionalization as carrier loaded.
(4) utilize glutathione to cut the effect of CYSTINE disulfide bond, the DNA1 of the functional magnetic bead surface of preparation in (3) is cut down, released dna 1; Subsequently the DNA1 discharged is measured, realize the mensuration to glutathione accordingly.
(5) utilize the DNA1 discharged in (4) to modify nm of gold/graphene modified ionic liquid carbon paste electrode, forming capture dna at electrode surface, catching electrochemical probe for going in subsequent experimental.
(6) preparation of electrochemical probe (EC probe).Utilize thionine in the suction-operated on nm of gold surface, prepare the golden nanometer particle that thionine is modified, then by DNA2 load on it, preparation DNA2/ thionine/nano-gold signal amplifying unit, i.e. electrochemical probe.
(7) Electrochemical Detection.DNA1/ nm of gold/graphene modified ionic liquid carbon paste working electrode is immersed in electrochemical probe solution, utilize DNA hybridization effect, DNA2/ thionine/nano-gold signal amplifying unit is captured in electrode surface by DNA1, cause the change of electrode surface electroactive material concentration, realize the detection to glutathione accordingly.
Described method, the ionic liquid carbon paste electrode described in it can be ionic liquid carbon paste electrode, metal electrode, non-metal electrode or oxidizing electrode.
Described method, the electroactive material described in it is thionine, bipyridyl ruthenium, methylene blue and the electroactive material containing positive charge.
Accompanying drawing explanation
Fig. 1 is experimental principle schematic diagram of the present invention.
Fig. 2 is that different modifying electrode is at 1.0mmol L
-1[Fe (CN)
6]
3-/4-with 0.5mol L
-1cyclic voltammogram in KCl solution.
Fig. 3 is the differential pulse voltammetry curve of variable concentrations object.From a to f, glutathione concentrations is followed successively by 1.0 × 10
-12, 1.0 × 10
-11, 1.0 × 10
-10, 1.0 × 10
-9, 1.0 × 10
-8, 1.0 × 10
-7mol L
-1.Illustration: the logarithm of concentration is to the relation curve of peak current.
Embodiment
Example below will illustrate method of operating of the present invention, but implementation method of the present invention is not limited thereto.
Example:
The reagent that 1 the present invention is used:
1. 1-hexyl pyridine hexafluorophosphate (HPPF6, > 99%, Chinese Academy of Sciences's Lanzhou Chemistry and Physics Institute Green Chemistry and catalytic center); 2. dag (Shanghai colloidal chemistry factory, granularity < 30 μm); 3. gold chloride (HAuCl
4, Chinese Shanghai reagent one factory); 4. ethyl (3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC, Sigma Co., USA); 5. N-hydroxy-succinamide (NHS, the long biochemical corp of Shanghai Yan); 6. amino magnetic bead (Aminated MB) (happy chromatographic technique development centre is doubly thought in Tianjin); Sodium dodecylsulphonate (SDS, Shanghai Heng Da fine chemicals company limited); 7. the potassium ferricyanide (the special Chemical Company in Rui Jin, Tianjin); 8. CYSTINE (upper sea blue season development in science and technology company limited); 9. glutathione, 10. mercaptoacetic acid (upper sea blue season development in science and technology company limited);
thionine (Chinese Shanghai reagent one factory).
2 different types of buffer solution are as follows: 1. 1 × TEA buffer solution (40.0mmol/L Tris, 1.0mmol/L EDTA, 40.0mmol/L acetic acid, pH=8.0), 2. 50.0mmol/L Tris-HCl buffer solution (pH=7.0), 50.0mmol/LTris-EDTA (TE, pH=8.0); 3. experimental water is redistilled water.
3 graphene oxides are synthesized by the Hummer method improved (Hummers, W.S., Offeman, R.E., 1958.J.Am.Chem.Soc.80,1339).
4 1. DNA1,2. DNA2 sequence buied by Shanghai Sheng Gong Bioisystech Co., Ltd, sequence is as follows:
4.1 DNA1：3’-NH
2-CCA GCA AGT TGG AGT CTG-5’；
4.2 DNA2：3’-SH-CAG ACT CCA ACT-5’；
The series of 4.3 DNA1 and DNA2 is preferably: the 3 ' end of DNA1 is for amino; Its functional group and the carboxyl of the CYSTINE that magnetic bead is connected realize the fixing of DNA1.3 ' the end of DNA2 is sulfydryl; The gold of its functional group and electrode surface forms S-Au key and realizes the fixing of DNA2.5 ' the end of DNA1 is complementary with the 3 ' end portion of DNA2, realizes hybridization.
5 electrodeposition processes prepare nm of gold/graphene modified ionic liquid carbon paste electrode:
5.1 ionic liquid carbon paste electrode preparations.3.2g carbon dust, 1.6g 1-hexyl pyridine hexafluorophosphate and 500 μ L whiterusss are mixed, will insert in glass tube in right amount after 80 DEG C of grindings evenly, connect copper wire as wire, polish to minute surface on pan paper during use.
5.2 graphene modified ionic liquid carbon paste electrode preparations:
5.2.1 freshly prepd ionic liquid carbon paste electrode is immersed in the graphene oxide solution of 1.0mg/mL, slowly stirs and pass into nitrogen;
5.2.2 at-1.3V (vs.SCE) electroreduction 600s, after taking-up redistilled water fully rinses, N is used
2dry up, can form the stable electrochemical reduction graphene modified film of one deck on ionic liquid carbon paste electrode surface, this modified electrode is expressed as graphene modified ionic liquid carbon paste electrode.
5.3 nm of gold/graphene modified ionic liquid carbon paste electrode preparation: by freshly prepd graphene modified ionic liquid carbon paste electrode at 5.0mmol/L HAuCl
4with 0.5mol/L KNO
3in-0.4V (vs.SCE) potential range, use potentiostatic method electro-deposition in mixed solution, nm of gold/graphene modified ionic liquid carbon paste electrode can be obtained.Rinse this electrode with intermediate water, use N
2dry up rear for subsequent use.
5.4 under room temperature (about 25 DEG C) containing 10.0mmol/L [Fe (CN)
6]
3-/4-with in 0.5mol/L KCl solution, electrode is characterized.
The preparation of 6 collaurums:
Preparing, storing gold size solution glass container (volumetric flask, brown, wide-mouth bottle, round-bottomed flask etc.) used, with chloroazotic acid, (acid of hydrochloric acid and nitric acid ratio is 1: 3, takes care in 6.1 elder generations! ) foam washing 30 minutes;
Then 6.2 rinse well with intermediate water, dry for standby;
6.3 add 500mL in 1000mL round-bottomed flask, the HAuCl of 1mM
4, be heated with stirring to boiling;
6.4 add 50mL fast, the Na of 38.8mM
3c
6h
5o
7, then heat 10 minutes, stir 15 minutes, be then cooled to the nylon membrane metre filter with 0.8 μm after room temperature, transfer in brown bottle and preserve in cool place place;
6.5 is 20-25nm with the particle diameter of scanning electron microscopic observation collaurum.
The magnetic micro-beads preparation process that 7L-cystine/DNA1 modifies is as shown in schematic diagram, and detailed process is as follows:
7.1 get 2mL 10
-3the CYSTINE solution of M, adds 1mL EDC+NHS mixed solution (5.0mM EDC, NHS and 10.0mM Tris-HCl buffer solution, pH=7.4), to activate-the COOH of CYSTINE, reacts 30 minutes;
7.2 add 50 μ L amino magnetic beads, continue reaction under 37 DEG C of constant temperature;
7.3 reacted material PBS buffer solution wash three times, obtain the magnetic micro-beads that CYSTINE is modified;
7.4 add 200 μ L 10 in the magnetic micro-beads solution of CYSTINE modification
-6the aminated dna 1 of M, reacts 12h at constant temperature 37 DEG C;
7.5 reacted material PBS buffer solution wash three times, obtain the magnetic micro-beads that CYSTINE/DNA1 modifies.
The preparation of 8 load thionine gold nanoparticle probe:
Add acetate buffer solution (pH=5.2) and 10 μ L trichloroethyl phosphate in 8.1 to sulfydryl DNA2 solution, reaction 1h, in order to activated thiol groups;
8.2 get fresh colloidal gold solution just synthetic in 1mL above-mentioned 8.1, add thionine solution, and reaction 0.5h, makes thionine fully be adsorbed on collaurum;
8.3 collaurums having adsorbed thionine join in the sulfydryl DNA2 activated, and concussion reaction 16 hours, makes sulfydryl DNA2 and collaurum be coupled together by S-Au key;
8.4 potpourris to be placed in the hydro-extractor of 15000 turns centrifugal 30 minutes, obtain red precipitate;
8.5 red precipitate PBS buffer solution washings obtained are also centrifugal, in triplicate, with PBS buffer solution (pH=8.0) dispersion, namely obtain DNA2/ thionine gold nanoparticle probe, keep in Dark Place at being placed on 4 DEG C.
The detection of 9 glutathione contents:
The 9.1 sample solution 50 μ L got containing glutathione join in the magnetic micro-beads solution of DNA1/L-cystine modification, reaction 12h, and magnetic resolution, obtains the DNA supernatant containing-SH;
Supernatant drop in 5.0 μ L above-mentioned 10.1 is coated in nm of gold/graphene modified ionic liquid carbon paste electrode surface by 9.2, after making it at room temperature hybridize 20 minutes, successively with 0.5% SDS solution and intermediate water washing, namely obtain DNA1/ nm of gold/graphene modified ionic liquid carbon paste electrode;
DNA1/ nm of gold obtained above/graphene modified ionic liquid carbon paste electrode immerses in DNA2/ thionine gold nanoparticle probe solution by 9.3, after making it at room temperature hybridize 20 minutes, successively with 0.5% SDS solution and intermediate water wash;
9.4 electrochemical gaging.To state 9.3 the electrode obtaineds for working electrode, utilize the sweep speed of differential pulse voltammetry with 100mV/s in PBS buffer solution (pH=8.0) to scan, electric potential scanning scope is-0.7-0.1V.
10 results and discussion:
The electrochemical Characterization of 10.1 nm of gold/graphene modified ionic liquid carbon paste electrode:
10.1.1 Fig. 2 is ionic liquid carbon paste electrode, graphene modified ionic liquid carbon paste electrode, and nm of gold/graphene modified ionic liquid carbon paste electrode is at 1.0mmol L
-1[Fe (CN)
6]
3-/4-with 0.5mol L
-1cyclic voltammogram in KCl solution;
10.1.2 curve a can see the redox peak of a pair symmetry, spike potential difference is 98mV, and this illustrates that the ionic liquid in ionic liquid carbon paste electrode serves good electric action;
10.1.3 on graphene modified ionic liquid carbon paste electrode (curve b), peak current obviously increases, potential difference (PD) is obviously reduced to 74mV, and this is because the graphene modified film of the surperficial high conductivity of ionic liquid carbon paste electrode significantly increases electron transmission ability; Can there is π-pi-conjugated effect with the aromatic rings of ionic liquid in Graphene, be therefore the electronics passage that swift electron transmits from graphene film to ionic liquid carbon paste electrode surface construction;
10.1.4 response current continues to increase on nm of gold/graphene modified ionic liquid carbon paste electrode (curve c), and simultaneously potential difference (PD) is reduced to 62mV, and this is the collaborative enlarge-effect of nm of gold due to electrode surface and graphene layer.Electrochemical reduction Graphene not only can increase the electric conductivity of modified electrode, can also provide larger surface area, so nm of gold/Graphene compound ulcer can reduce [Fe (CN) for the deposition of nm of gold simultaneously
6]
3-/4-to the resistance of electrode surface metastatic electron;
The drafting of 10.2 typical curves and the collection of sample measure.
10.2.1 the drafting of typical curve.The glutathione standard solution of preparation variable concentrations, measure its electrochemical signals, oxidation peak current becomes good linear relationship with the logarithm value of glutathione concentrations, and the range of linearity is 1.0 × 10
-12mol L
-1~ 1.0 × 10
-7mol L
-1, detect and be limited to 4.1 × 10
-13mol L
-1(as Fig. 3).
10.2.2 the collection of sample measures.
10.2.3 cultivate chmice acute lymphocytic leukemia cell system p388, when adherent growth reaches more than 80%, carry out cell liquid extraction:
1, adhere-wall culture cell 2mL trypsinization 2-3 minute, outwells pancreatin, adds about 5mL nutrient culture media, is dispelled by cell and cell is suspended, centrifugal collecting cell;
2, in cell, the PBS (pH7.2,0.01M) of 3mL 4 DEG C of precoolings is added, washed cell, centrifugal, then discard washing lotion, repeat above operation twice, wash cell three times altogether to wash away nutrient solution;
3, cell Eddy diffusion is in 3mL PBS-EDTA buffer solution (PBSE, pH7.4,0.10M), adds 3% perchloric acid with the protein substance in sedimentation cell after homogenizing, and whole operating in is carried out on ice;
4, at 4 DEG C 14,000rpm centrifugal 5 minutes;
5, collect supernatant to be used for, detect;
6, the sweep speed of differential pulse voltammetry with 100mV/s in PBS buffer solution (pH=8.0) scans, and electric potential scanning scope is-0.7-0.1V;
7, under optimum experimental condition, detect the chmice acute lymphocytic leukemia cell system p388 supernatant extracted (every part of 400 μ L), measuring content is 1.62 × 10
-8mol/L.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; the change made under other any deviates from Spirit Essence of the present invention and principle, modification, replacement, combination, simplification; comparatively the substitute mode of equivalence, is included within protection scope of the present invention.
Claims (4)
1., based on a method for nm of gold and thionine signal amplification detection glutathione, it is characterized in that it comprises the following steps:
A () take ionic liquid as dressing agent, prepare ionic liquid carbon paste electrode; In graphene oxide solution, utilize potentiostatic electrodeposition method at the obtained graphene modified film in ionic liquid carbon paste surface, then with HAuCl
4for raw material, use potentiostatic method deposited Au, prepare nm of gold/graphene modified ionic liquid carbon paste electrode, i.e. working electrode;
B (), with the DNA1 modified magnetic microballon of CYSTINE and functionalization, prepares functional magnetic microballon; Described DNA1 sequence is: 3 '-NH
2-CCA GCA AGT TGG AGT CTG-5 ';
C the preparation of () electrochemical probe: utilize thionine in the suction-operated on nm of gold surface, prepares the golden nanometer particle that thionine is modified, then by DNA2 load on it, and preparation DNA2/ thionine/nano-gold signal amplifying unit, i.e. electrochemical probe; Described DNA2 sequence is: 3 '-SH-CAG ACT CCA ACT-5 ';
D sample solution that () gets containing glutathione joins in the functional magnetic microballon of DNA1/L-cystine modification, glutathione is utilized to cut the effect of CYSTINE disulfide bond, cut down by functional magnetic bead surface DNA1, release obtains the DNA1 containing-SH;
E () modifies nm of gold/graphene modified ionic liquid carbon paste electrode electrode with the DNA1 containing-SH discharged, obtain DNA1/ nm of gold/graphene modified ionic liquid carbon paste electrode;
F DNA1/ nm of gold obtained above/graphene modified ionic liquid carbon paste electrode immerses in DNA2/ thionine gold nanoparticle probe solution by (), utilize DNA hybridization effect, DNA2/ thionine/nano-gold signal amplifying unit is captured in carbon paste electrode surface by DNA1, cause the change of electrode surface electroactive material concentration, realize the detection to glutathione accordingly.
2. method according to claim 1, is characterized in that: ionic liquid described in step (a) is kation is quaternary ammonium salt ion, quaternary alkylphosphonium salt ion, imidazolium ion or pyrroles's salt ion; Negative ion is the ionic liquid of halide ion, tetrafluoroborate ion, hexafluorophosphoricacid acid ions composition.
3. method according to claim 2, is characterized in that: described ionic liquid is 1-hexyl pyridine hexafluorophosphate.
4. method according to claim 1, is characterized in that: the electroactive material described in step (f) is thionine.
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