CN105866206B - A kind of method that concentration of hydrogen peroxide in living cells is detected in original position in real time - Google Patents
A kind of method that concentration of hydrogen peroxide in living cells is detected in original position in real time Download PDFInfo
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- CN105866206B CN105866206B CN201610227973.8A CN201610227973A CN105866206B CN 105866206 B CN105866206 B CN 105866206B CN 201610227973 A CN201610227973 A CN 201610227973A CN 105866206 B CN105866206 B CN 105866206B
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- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 title claims abstract description 197
- 238000000034 method Methods 0.000 title claims abstract description 58
- 238000001362 electron spin resonance spectrum Methods 0.000 claims abstract description 29
- 238000001514 detection method Methods 0.000 claims abstract description 21
- 239000012086 standard solution Substances 0.000 claims abstract description 18
- 239000003814 drug Substances 0.000 claims abstract description 16
- 238000011065 in-situ storage Methods 0.000 claims abstract description 15
- 230000007794 irritation Effects 0.000 claims abstract description 14
- 238000012360 testing method Methods 0.000 claims abstract description 12
- 238000000840 electrochemical analysis Methods 0.000 claims abstract description 9
- 238000011897 real-time detection Methods 0.000 claims abstract description 3
- 229960002163 hydrogen peroxide Drugs 0.000 claims description 89
- 238000004435 EPR spectroscopy Methods 0.000 claims description 58
- 239000000243 solution Substances 0.000 claims description 43
- 239000003795 chemical substances by application Substances 0.000 claims description 32
- -1 hydroxyl radical free radical Chemical class 0.000 claims description 30
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims description 22
- 230000004048 modification Effects 0.000 claims description 18
- 238000012986 modification Methods 0.000 claims description 18
- 229910000510 noble metal Inorganic materials 0.000 claims description 16
- 102000004190 Enzymes Human genes 0.000 claims description 15
- 108090000790 Enzymes Proteins 0.000 claims description 15
- 239000011521 glass Substances 0.000 claims description 15
- 239000002082 metal nanoparticle Substances 0.000 claims description 15
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 13
- 229910021389 graphene Inorganic materials 0.000 claims description 12
- GRPQBOKWXNIQMF-UHFFFAOYSA-N indium(3+) oxygen(2-) tin(4+) Chemical compound [Sn+4].[O-2].[In+3] GRPQBOKWXNIQMF-UHFFFAOYSA-N 0.000 claims description 11
- AMGQUBHHOARCQH-UHFFFAOYSA-N indium;oxotin Chemical compound [In].[Sn]=O AMGQUBHHOARCQH-UHFFFAOYSA-N 0.000 claims description 11
- 229910052697 platinum Inorganic materials 0.000 claims description 10
- MRNHPUHPBOKKQT-UHFFFAOYSA-N indium;tin;hydrate Chemical compound O.[In].[Sn] MRNHPUHPBOKKQT-UHFFFAOYSA-N 0.000 claims description 9
- 239000001301 oxygen Substances 0.000 claims description 9
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- 239000010931 gold Substances 0.000 claims description 8
- 239000001257 hydrogen Substances 0.000 claims description 8
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- 238000002360 preparation method Methods 0.000 claims description 7
- 230000001603 reducing effect Effects 0.000 claims description 7
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- QGVLYPPODPLXMB-QXYKVGAMSA-N phorbol Natural products C[C@@H]1[C@@H](O)[C@]2(O)[C@H]([C@H]3C=C(CO)C[C@@]4(O)[C@H](C=C(C)C4=O)[C@@]13O)C2(C)C QGVLYPPODPLXMB-QXYKVGAMSA-N 0.000 claims description 2
- 239000008055 phosphate buffer solution Substances 0.000 claims description 2
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- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims 2
- 235000011330 Armoracia rusticana Nutrition 0.000 claims 1
- 240000003291 Armoracia rusticana Species 0.000 claims 1
- 150000001336 alkenes Chemical class 0.000 claims 1
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- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 6
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- IYMAXBFPHPZYIK-BQBZGAKWSA-N Arg-Gly-Asp Chemical group NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O IYMAXBFPHPZYIK-BQBZGAKWSA-N 0.000 description 4
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- 206010061218 Inflammation Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
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- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
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- 150000002431 hydrogen Chemical class 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 229910003437 indium oxide Inorganic materials 0.000 description 1
- RHZWSUVWRRXEJF-UHFFFAOYSA-N indium tin Chemical compound [In].[Sn] RHZWSUVWRRXEJF-UHFFFAOYSA-N 0.000 description 1
- PJXISJQVUVHSOJ-UHFFFAOYSA-N indium(iii) oxide Chemical compound [O-2].[O-2].[O-2].[In+3].[In+3] PJXISJQVUVHSOJ-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N24/00—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects
- G01N24/10—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects by using electron paramagnetic resonance
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- Molecular Biology (AREA)
- High Energy & Nuclear Physics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention provides a kind of methods of concentration of hydrogen peroxide in detection living cells, the method is to obtain current-time curvel of the living cells after by medicine irritation using electro-chemical test, it is carried out at the same time electron paramagnetic resonance spectrum (EPR) test, ESR spectrogram of the living cells after ultraviolet light in electrochemical cell is obtained, ESR signal strength current relationship curves are established；Electron paramagnetic resonance spectrum (EPR) test equally is carried out to Hydrogen peroxide standard solution, obtain ESR spectrogram of the Hydrogen peroxide standard solution after ultraviolet light, establish ESR signal strength concentration of hydrogen peroxide standard curves, in summary the three kinds of curves established obtain the concentration of the hydrogen peroxide released after the living cells under random time is stimulated, complete the real-time detection of concentration of hydrogen peroxide in living cells.The invention enables the quantitative detection of the concentration of hydrogen peroxide of living cells release is more accurate, can realize the in situ real-time concentration for monitoring the discharged hydrogen peroxide of living cells, be with a wide range of applications.
Description
Technical field
The invention belongs to test and analyze technical field, it is related to a kind of method detecting concentration of hydrogen peroxide in living cells, especially
It is related to a kind of method that concentration of hydrogen peroxide in living cells is detected in original position in real time.
Background technology
Active oxygen includes not only some oxygen radicals, and such as superoxide anion, hydroxyl radical free radical etc. further includes having higher work
Some molecules such as hydrogen peroxide and singlet oxygen of property.Compared with other active oxygens, hydrogen peroxide property is stablized, is easy relatively
It is spread between cell and cell peripheral environment.On the one hand, in human body suitable hydrogen peroxide of spontaneous generation be it is considerable,
In signal transduction, neurotransmission, platelet aggregation, immune system control, learning and memory, cell routine growth, important life
It orders in the synthesis of compound and the metabolism of body, all played an important role.However, when hydrogen peroxide generates excessively,
Or the amount of the antioxidant of body itself, when largely reducing, hydrogen peroxide just becomes quite to be harmful to.They pass through oxidation one
A little functional biomolecules so that oxidative damage occurs for cytolipin plasma membrane, proteinaceous tissue, enzyme, carbohydrate and DNA etc.,
Cell damage or death are caused, it is closely related with cancer, inflammation and some the nervous system diseases.In short, in normal physiological ring
In border, a certain amount of hydrogen peroxide is essential for life entity, but excessive active oxygen will result in harm.Therefore,
Accurate measurements cell in real time in situ generates the concentration versus cell signal transduction research of hydrogen peroxide, medical diagnosis on disease has important meaning
Justice.
Currently, most of hydrogen peroxide detection method, such as chromatography, titration, ultravioletvisible spectroscopy, fluorescence method
Deng generally speaking all than relatively time-consuming, being easily disturbed object influence, and be difficult to detection in real time in situ.Electrochemical method is based particularly on
The amperometric sensor of nano material and enzyme modified electrode due to experimentation is simple, high sensitivity, high selectivity and by wide
General concern.In doing the work of quantitative detection to the hydrogen peroxide extracellularly generated by the method for electrochemistry, presently mainly
The standard curve that the concentration of Ampere currents and hydrogen peroxide is first built with modified electrode, is then generated by practical cell system
Ampere currents curve reads on standard curve or calculates the concentration of the hydrogen peroxide of cell generation.The frequent quilt of this method
Means as quantitative analysis, but in living cells system, there are certain defects for this method for making standard curve.One side
Face, if building standard curve after seeding cells into working electrode surface, the part peroxide in the standard solution of addition
Cell interior can be diffused into and be fallen by metabolic breakdowns such as intracellular enzymes, the peroxidating tested in such practical systems by changing hydrogen
Hydrogen concentration is often higher.On the other hand, if building standard curve before seeding cells into working electrode surface,
That is structure standard curve when electrode surface be free from cell, this in terms of electrode active area, electric conductivity with reality
The concentration of hydrogen peroxide for having the working electrode of attached cell inconsistent, therefore testing is grown used in detection architecture
It is inaccurate.
Therefore, it needs to develop a kind of method that can detect concentration of hydrogen peroxide in living cells in real time in situ in this field.
Invention content
In view of the deficiencies of the prior art, the purpose of the present invention is to provide concentration of hydrogen peroxide in a kind of detection living cells
Method is provided in particular in a kind of method that concentration of hydrogen peroxide in living cells is detected in original position in real time.
To reach the invention purpose, the present invention uses following technical scheme：
On the one hand, the present invention provides a kind of method detecting concentration of hydrogen peroxide in living cells, and the method is to utilize electricity
Test chemical obtains current versus time curve of the living cells after by medicine irritation, at the same time carries out electron paramagnetic resonance spectrum (EPR) survey
Examination, obtains ESR spectrogram of the living cells after ultraviolet light in electrochemical cell, establishes ESR signal strengths-current relationship curve；
Electron paramagnetic resonance spectrum (EPR) test equally is carried out to Hydrogen peroxide standard solution, obtains Hydrogen peroxide standard solution in ultraviolet lighting
ESR spectrograms after penetrating establish ESR signal strengths-concentration of hydrogen peroxide standard curve, and the three kinds of curves in summary established obtain
The concentration of the stimulated rear hydrogen peroxide released of living cells under random time, completes concentration of hydrogen peroxide in living cells
Detection in real time.
Electron paramagnetic resonance spectrum (EPR) technology is combined with electrochemical analysis to realize in living cells by the method for the invention
The detection in real time in situ of concentration of hydrogen peroxide so that quantitative detection is more accurate, solves the prior art and cannot achieve living cells
The problem of detection in real time in situ of middle concentration of hydrogen peroxide.
In the present invention, the method for concentration of hydrogen peroxide includes the following steps in the detection living cells：
(1) three electrode work systems are inserted into electrolyte and form three-electrode electrochemical test system, three electrode
It is inoculated with living cells on the working electrode of work system, is when originating to be initially added into medicine irritation living cells to generate hydrogen peroxide
Between, current versus time curve of the living cells after by medicine irritation is obtained using the Amperometric that clocks；
(2) while carrying out step (1), the living cells culture solution in electrochemical cell is taken, hydroxyl radical free radical capture is added
Agent records ESR spectrograms, the current-vs-time that the ESR spectrograms and step (1) obtained using the step is obtained after ultraviolet light
Same time point corresponding ESR signal strengths and size of current establish ESR signal strengths-current relationship curve in curve；
(3) hydrogen peroxide (H is prepared2O2) standard solution, hydroxyl radical free radical capturing agent is added, after ultraviolet light, record
ESR spectrograms establish ESR signal strengths-concentration of hydrogen peroxide standard curve；
(4) time current curve obtained by step (1) obtains the current value under random time, then passes through step
(2) the ESR signal strengths obtained-current relationship curve reads corresponding ESR signal strengths, finally step (3) is utilized to obtain
ESR signal strengths-concentration of hydrogen peroxide standard curve obtains the peroxidating released after living cells under random time is stimulated
The concentration of hydrogen.
The present invention will be by that will have hydrogen peroxide sensitive and accurate response but real-time in situ detects slightly worse electron paramagnetic and is total to
Vibration wave spectral technology is combined with electrochemistry, and standard curve is carried out using electron spin resonance (ESR) assisted electrochemical analytic approach
Structure, this method is not related to the contradiction between electrode and living cells, and the hydrogen peroxide generated outside living cells may be implemented
Accurate quantitative analysis detects, and electrochemical analysis can then generate living cells the process detection in real time in situ of hydrogen peroxide.The present invention carries
The detection method of confession avoids the simple defect detected using electrochemical method and electron paramagnetic resonance so that quantitative detection is more
It is accurate to add, and is particularly suitable for generating living cells system the real-time accurate measurements in original position of concentration of hydrogen peroxide, in disease detection etc.
Biomedical sector has significant application value.
Preferably, step (1) the three electrodes work system is using Ag/AgCl electrodes as reference electrode, using platinized platinum
Or platinum filament is used as to electrode, surface modification is simultaneously inoculated with tin indium oxide (ITO) electrode of living cells as working electrode.
Preferably, tin indium oxide (ITO) electrode of surface modification surface from close to the side of indium-tin oxide electrode
It is successively modified with graphene, noble metal nano particles, hydrogen peroxide catabolic enzyme and cell adherence agent respectively.
Preferably, the preparation method of tin indium oxide (ITO) electrode of the surface modification is as follows：
A, indium oxide tin glass is surface-treated using oxygen plasma；
B, graphene oxide solution is spun to the indium oxide tin glass surface that step A is obtained, is placed in hydrazine steam and carries out
In-situ reducing, obtaining surface modification has the indium oxide tin glass of graphene；
C, noble metal nano particles solution is added into the indium oxide tin glass surface that step B is obtained, it is dry, then by its
Hydrogen peroxide decomposes to be infiltrated in enzyme solutions, obtains tin indium oxide (ITO) electrode for being modified with noble metal nano particles and enzyme；
D, cell adherence agent solution is added into the electrode surface that step C is obtained, is dried to obtain the oxidation of the surface modification
Indium tin electrode.
Preferably, in the preparation of the tin indium oxide of surface modification (ITO) electrode, in utilization oxygen plasma to indium oxide
Before tin glass is surface-treated, indium oxide tin glass is first respectively placed in acetone, ethyl alcohol and ultra-pure water ultrasound 10-30 successively
Minute, such as 10 minutes, 13 minutes, 15 minutes, 18 minutes, 20 minutes, 23 minutes, 25 minutes, 28 minutes or 30 minutes.
In the present invention, the purpose being surface-treated to indium oxide tin glass using oxygen plasma is to increase hydrophily
Be conducive to its follow-up modification with spreadability.
Preferably, a concentration of 0.01-0.1mg/mL of graphene oxide solution described in step B, such as 0.01mg/mL,
0.02mg/mL、0.03mg/mL、0.04mg/mL、0.05mg/mL、0.06mg/mL、0.07mg/mL、0.08mg/mL、0.09mg/
ML or 0.1mg/mL.
Preferably, the temperature of in-situ reducing described in step B be 50-70 DEG C, such as 51 DEG C, 52 DEG C, 53 DEG C, 54 DEG C, 56 DEG C,
58 DEG C, 60 DEG C, 62 DEG C, 64 DEG C, 66 DEG C, 68 DEG C or 70 DEG C.
Preferably, the time of in-situ reducing described in step B be 4-12 hours, such as 4 hours, 5 hours, 6 hours, 7 hours,
8 hours, 9 hours, 10 hours, 11 hours or 12 hours.
Preferably, noble metal nano particles described in step C are in silver nano-grain, gold nano grain or Pt nanoparticle
Any one or at least two combination, preferred gold nano grain.
Preferably, the mass percent of noble metal nano particles is in golden noble metal nano particles solution described in step C
0.01-0.05%, for example, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045% or
0.05%.
Preferably, noble metal nano particles solution and the volume ratio of graphene oxide solution described in step B described in step C are
1:2-2:1；Such as 1:2、1:1.8、1:1.5、1:1.3、1:1、1.2:1、1.5:1、1.8:1 or 2:1.
Preferably, hydrogen peroxide catabolic enzyme described in step C is horseradish peroxidase.
Preferably, hydrogen peroxide described in step C decomposes a concentration of 1-5mg/mL of enzyme solutions, such as 1mg/mL, 1.3mg/
mL、1.5mg/mL、1.8mg/mL、2mg/mL、2.5mg/mL、2.8mg/mL、3mg/mL、3.5mg/mL、3.8mg/mL、4mg/
ML, 4.5mg/mL, 4.8mg/mL or 5mg/mL.
Preferably, cell adherence agent described in step D is short for the ring-type being made of arginine-glycine-aspartic acid sequence
Peptide (abbreviation RGD cyclic peptide).
Preferably, a concentration of 1-5mg/mL of the cell adherence agent solution, such as 1mg/mL, 1.3mg/mL, 1.5mg/
mL、1.8mg/mL、2mg/mL、2.5mg/mL、2.8mg/mL、3mg/mL、3.5mg/mL、3.8mg/mL、4mg/mL、4.5mg/
ML, 4.8mg/mL or 5mg/mL.
Preferably, the volume ratio of cell adherence agent solution described in step D and gold nano grain solution described in step C is 1:2-
2:1, such as 1:2、1:1.8、1:1.5、1:1.3、1:1、1.2:1、1.5:1、1.8:1 or 2:1.
Preferably, step (1) described electrolyte is phosphate buffer solution.
Preferably, step (1) described drug is phorbol 12-myristinate-13- acetic acid esters (PMA).
Preferably, step (1) described living cells is the living cells for capableing of adherent growth on the working electrode (s, to living cells kind
There is no limit for class, as long as adherent growth, the specific can be that Hela or other tumour cells.
Preferably, in the detection living cells of the present invention in the method for concentration of hydrogen peroxide, step (2) described hydroxyl free
Base capturing agent is 5,5- dimethyl -1- pyrrolin nitrogen oxides.
Preferably, step (2) the addition hydroxyl radical free radical capturing agent makes its a concentration of 20- in living cells culture solution
80mM, such as 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM or 80mM, it is excellent
Select 40-50mM.
Preferably, the wave-length coverage of step (2) described ultraviolet light be 280-320nm, such as 280nm, 290nm, 300nm,
305nm, 308nm, 310nm, 315nm, 318nm or 320nm.
In the present invention, step (3) ESR signal strengths-concentration of hydrogen peroxide standard curve, is dense with hydrogen peroxide
The logarithm (log values) of degree is abscissa, the relation curve established as ordinate using ESR signal strengths.
In the present invention, a concentration of the 10 of step (3) the Hydrogen peroxide standard solution-9-10-3Mol/L, such as 10- 9mol/L、8×10-8mol/L、5×10-8mol/L、3×10-8mol/L、8×10-7mol/L、5×10-7mol/L、3×10- 7mol/L、5×10-6mol/L、1×10-6mol/L、8×10-5mol/L、4×10-5mol/L、1×10-5mol/L、8×10- 4mol/L、5×10-4Mol/L or 1 × 10-3mol/L。
Preferably, step (3) concentration gradient for preparing selected hydrogen peroxide when Hydrogen peroxide standard solution is 1
×10-6、5×10-6、1×10-5、2×10-5、5×10-5mol/L.It can certainly select the hydrogen peroxide of other concentration gradients
Standard solution, as long as the concentration of each sample is 10 in the standard solution-9-10-3Mol/L.
Preferably, step (3) the hydroxyl radical free radical capturing agent is 5,5- dimethyl -1- pyrrolin nitrogen oxides.
Preferably, step (3) the addition hydroxyl radical free radical capturing agent makes its concentration in Hydrogen peroxide standard solution
Equal to concentration of step (2) the hydroxyl radical free radical capturing agent in living cells culture solution.
Preferably, the wave-length coverage of step (3) described ultraviolet light be 280-320nm, such as 280nm, 290nm, 300nm,
305nm, 308nm, 310nm, 315nm, 318nm or 320nm.
Preferably, in the present invention, ESR spectrograms are recorded using electronic paramagnetic resonance spectrometer.
As optimal technical scheme, the method for concentration of hydrogen peroxide specifically includes following in detection living cells of the present invention
Step：
(1) using Ag/AgCl electrodes as reference electrode, platinized platinum or platinum filament are used as to electrode, and surface modification is simultaneously inoculated with work
The indium-tin oxide electrode of cell forms three electrode work systems as working electrode, inserts it into electrolyte and forms three electrodes
Electrochemical test system generates hydrogen peroxide as initial time, using the Amperometric that clocks to be initially added into medicine irritation living cells
Obtain current versus time curve of the living cells after by medicine irritation；
(2) while carrying out step (1), the living cells culture solution in electrochemical cell is taken, hydroxyl radical free radical capture is added
Agent 5,5- dimethyl -1- pyrrolin nitrogen oxides, after wave-length coverage is the ultraviolet light of 280-320nm, record ESR spectrums
Figure, same time point corresponding ESR letters in the current versus time curve that the ESR spectrograms and step (1) obtained using the step is obtained
Number intensity and size of current establish ESR signal strengths-current relationship curve；
(3) 10 are prepared-4、10-5、10-6、10-7With 10-8The hydrogenperoxide steam generator of mol/L concentration is added hydroxyl radical free radical and catches
Agent 5 is obtained, 5- dimethyl -1- pyrrolin nitrogen oxides, after wave-length coverage is the ultraviolet light of 280-320nm, record ESR is composed
Figure, establishes ESR signal strengths-concentration of hydrogen peroxide standard curve；
(4) time current curve obtained by step (1) obtains the current value under random time, then passes through step
(2) the ESR signal strengths obtained-current relationship curve reads corresponding ESR signal strengths, finally step (3) is utilized to obtain
ESR signal strengths-concentration of hydrogen peroxide standard curve obtains the peroxidating released after living cells under random time is stimulated
The concentration of hydrogen.
Compared with the existing technology, the present invention has the advantages that：
Compared with single electrochemical analysis method, the present invention is by electron paramagnetic resonance spectrum (EPR) technology and electrochemical method knot
It closes, will be using the obtained signal strength of electron paramagnetic resonance spectrum (EPR) technology as an intermediate quantity, the standard curve established
It can more accurately reflect the concentration of hydrogen peroxide in living cells test system；Compared with single electron paramagnetic resonance method, this
Invention is combined that the discharged peroxide of living cells can be monitored in real time in situ with electrochemical method using electron paramagnetic resonance spectrum (EPR) technology
Change the concentration of hydrogen.The present invention is by electron paramagnetic resonance spectrum (EPR) technology and electrochemical method perfect adaptation so that living cells release
The quantitative detection of concentration of hydrogen peroxide is more accurate, solves the original that the prior art cannot achieve concentration of hydrogen peroxide in living cells
It the problem of position detection in real time, is with a wide range of applications.
Description of the drawings
Fig. 1 is the preparation process schematic diagram of working electrode in the present invention.
Fig. 2 is the current-vs-time relation curve that the embodiment of the present invention 1 obtains, and curve 1 is in cell inoculation to working electrode
The lower current-vs-time relation curve generated of addition PMA stimulations afterwards, curve 2 are rear in cell inoculation to working electrode but without PMA
The current-vs-time relation curve generated in the case of stimulation；Curve 3 is only PMA stimulations but non-inoculating cell is to working electrode
In the case of the current-vs-time relation curve that generates.
Fig. 3 is the ESR spectrograms for the living cells culture solution that 1 step of the embodiment of the present invention (3) test obtains
Fig. 4 is the relation curve for ESR signal strengths-electric current that the embodiment of the present invention 1 is established.
Fig. 5 is the ESR spectrums of the Hydrogen peroxide standard solution for the various concentration that 1 step of the embodiment of the present invention (4) test obtains
Figure.
Fig. 6 is ESR signal strengths-concentration of hydrogen peroxide standard curve that the embodiment of the present invention 1 is established.
Specific implementation mode
The technical solution further illustrated the present invention below by specific implementation mode.Those skilled in the art should be bright
, the embodiment, which is only to aid in, understands the present invention, should not be regarded as a specific limitation of the invention.
Embodiment 1
(1) (i.e. surface modification has graphene, gold nano grain, horseradish peroxidase and cell adherence agent to working electrode
The ITO electrode of RGD cyclic peptide simultaneously has living cells in its surface seeding) preparation, specifically include following steps (its preparation process as scheme
Shown in 1)：
A, ito glass is placed in acetone, ethyl alcohol, ultra-pure water ultrasound 20 minutes successively, then uses oxygen plasma pair
Its surface is handled, and is conducive to subsequently modify to increase hydrophily and spreadability；
B, the graphene oxide solution of a concentration of 0.06mg/mL is spun to above-mentioned ito glass surface, is subsequently placed in hydrazine steaming
In 60 DEG C of in-situ reducings 10 hours in vapour；
C, 10 microlitres of gold nano grain solution are added drop-wise to the above-mentioned surfaces ITO for being modified with graphene, it is naturally dry at room temperature
It is dry, then under the conditions of 4 DEG C, above-mentioned electrode is infiltrated by least 24 hours in horseradish peroxidase solution, phosphoric acid is then used
Salt buffer solution rinses surface three times, obtains tin indium oxide (ITO) electrode for being modified with gold nano grain and enzyme；
D, 10 microlitres of RGD cyclic peptide solution are added drop-wise to the electrode surface that step C is obtained, and are spontaneously dried at 4 DEG C, obtain the table
The indium-tin oxide electrode of face modification, then the indium-tin oxide electrode surface of Hela cell inoculations to surface modification, obtains work electricity
Pole.
(2) foundation of current-vs-time relation curve
Using Ag/AgCl electrodes as reference electrode, it is used as to electrode using platinized platinum or platinum filament, utilizes surface obtained above
Modify and be inoculated with living cells indium-tin oxide electrode as working electrode, above-mentioned three electrode is inserted into the phosphorus of 0.01mol/L
In hydrochlorate buffer solution, a three-electrode electrochemical test system is formed, was generated with being initially added into PMA medicine irritation living cells
Hydrogen oxide is initial time (0min), and electric current-of the living cells after by medicine irritation in 0 to 60 minutes is obtained using the Amperometric that clocks
Time graph, the results are shown in Figure 2.
(3) foundation of ESR signal strengths-current relationship curve
While carrying out step (2), every 5 or 10 minutes, i.e., when 0,10,20,25,30,35,40,50min
The living cells culture solution in electrochemical cell is taken with capillary, hydroxyl radical free radical capturing agent 5,5- dimethyl -1- pyrrolin nitrogen is added
Oxide makes its a concentration of 50mM in living cells culture solution make after the ultraviolet light 30min of 280-320nm wave bands
Hydrogen peroxide in living cells culture solution is fully decomposed into hydroxyl radical free radical, using X-band electronic paramagnetic resonance spectrometers
(FA200, Japanese JEOL companies) records ESR spectrograms, and the results are shown in Figure 3.
The current versus time curve obtained to obtained ESR spectrograms and step (2) is analyzed, and same time point is taken
(current value at certain time point cuts starting for ESR signal strengths (average value of two signal peak strengths among spectrogram) and current value
The current value of time), using current value as abscissa, using ESR signal strengths as ordinate, fitting is obtained such as the pass in Fig. 4
It is curve, equation of linear regression y=60.3+731.5x, ESR signal strength is in good linear relationship with current value, related
Coefficient is 0.995.
(4) foundation of ESR signal strengths-concentration of hydrogen peroxide standard curve
Compound concentration is respectively 1 × 10-6、5×10-6、1×10-5、2×10-5、5×10-5The Hydrogen peroxide standard of mol/L
Hydroxyl radical free radical capturing agent 5 is added in solution, and 5- dimethyl -1- pyrrolin nitrogen oxides makes it in Hydrogen peroxide standard solution
A concentration of 50mM make the peroxidating in Hydrogen peroxide standard solution after the ultraviolet light 30min of 280-320nm wave bands
Hydrogen is fully decomposed into hydroxyl radical free radical, records ESR spectrograms using X-band electronic paramagnetic resonance spectrometers, the results are shown in Figure 5.
Same time point corresponding ESR signals in the current versus time curve that the ESR spectrograms and step (1) obtained using the step is obtained
Intensity and size of current, using ESR signal strengths as ordinate, are obtained using the logarithm of concentration of hydrogen peroxide as abscissa
ESR signal strengths-concentration of hydrogen peroxide standard curve as indicated with 6, equation of linear regression y=1995.3+273.0x, linearly
Related coefficient is 0.997.
(5) according to the curve as above established, the current value at arbitrary time point after medicine irritation is added can be read from Fig. 2；
Current value is converted into ESR signal strengths according to Fig. 4；Finally, the peroxidating corresponding to this ESR signal strength is read from Fig. 6
Thus the concentration value of hydrogen completes the real-time detection to concentration of hydrogen peroxide in living cells.
Applicant states that the present invention illustrates the process of the present invention, but the present invention not office by above-described embodiment
It is limited to above-mentioned processing step, that is, does not mean that the present invention has to rely on above-mentioned processing step and could implement.Technical field
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to raw material selected by the present invention
Addition, the selection etc. of concrete mode, all fall within protection scope of the present invention and the open scope.
Claims (32)
1. a kind of method of concentration of hydrogen peroxide in detection living cells, which is characterized in that the method is to utilize electro-chemical test
Current versus time curve of the living cells after by medicine irritation is obtained, electron paramagnetic resonance spectrum (EPR) test is at the same time carried out, obtains electricity
Electron paramagnetic resonance spectrum figure of the living cells after ultraviolet light in chemical bath, establishes electron paramagnetic resonance signal strength-electric current
Relation curve；Electron paramagnetic resonance spectrum (EPR) test equally is carried out to Hydrogen peroxide standard solution, obtains Hydrogen peroxide standard solution
It is bent to establish electron paramagnetic resonance signal strength-concentration of hydrogen peroxide standard for electron paramagnetic resonance spectrum figure after ultraviolet light
Line, the three kinds of curves in summary established obtain the dense of the hydrogen peroxide released after the living cells under random time is stimulated
Degree completes the real-time detection of concentration of hydrogen peroxide in living cells.
2. according to the method described in claim 1, it is characterized in that, the described method comprises the following steps：
(1) three electrode work systems are inserted into electrolyte and form three-electrode electrochemical test system, the three electrodes work
Be inoculated with living cells on the working electrode of system, using be initially added into medicine irritation living cells generate hydrogen peroxide as initial time,
Current versus time curve of the living cells after by medicine irritation is obtained using the Amperometric that clocks；
(2) while carrying out step (1), the living cells culture solution in electrochemical cell is taken, hydroxyl radical free radical capturing agent, warp is added
After ultraviolet light, electron paramagnetic resonance spectrum figure is recorded, the electron paramagnetic resonance spectrum figure obtained using the step and step (1) are obtained
To current versus time curve in same time point corresponding electron paramagnetic resonance signal strength and size of current establish electron paramagnetic
Resonance signal intensity-current relationship curve；
(3) Hydrogen peroxide standard solution is prepared, hydroxyl radical free radical capturing agent is added, after ultraviolet light, records electron paramagnetic
Resonate spectrogram, establishes electron paramagnetic resonance signal strength-concentration of hydrogen peroxide standard curve；
(4) time current curve obtained by step (1) obtains the current value under random time, is then obtained by step (2)
The electron paramagnetic resonance signal strength arrived-current relationship curve reads corresponding electron paramagnetic resonance signal strength, finally utilizes
Electron paramagnetic resonance signal strength-concentration of hydrogen peroxide standard curve that step (3) obtains obtain under random time living cells by
The concentration of the hydrogen peroxide released after stimulation.
3. according to the method described in claim 2, it is characterized in that, step (1) the three electrodes work system is with Ag/AgCl
Electrode is used as reference electrode, platinized platinum or platinum filament to electrode, surface modification and the indium-tin oxide electrode work for being inoculated with living cells
For working electrode.
4. according to the method described in claim 3, it is characterized in that, the surface modification and being inoculated with the tin indium oxide of living cells
The indium-tin oxide electrode of surface modification surface layer-by-layer from close to the side of indium-tin oxide electrode is modified with graphite respectively in electrode
Alkene, noble metal nano particles, hydrogen peroxide catabolic enzyme and cell adherence agent.
5. according to the method described in claim 4, it is characterized in that, the preparation method of the indium-tin oxide electrode of the surface modification
It is as follows：
A, indium oxide tin glass is surface-treated using oxygen plasma；
B, graphene oxide solution is spun to the indium oxide tin glass surface that step A is obtained, is placed in hydrazine steam and carries out original position
Reduction, obtaining surface modification has the indium oxide tin glass of graphene；
C, noble metal nano particles solution is added into the indium oxide tin glass surface that step B is obtained, it is dry, then by it in peroxide
Change and infiltrated in hydrogen decomposition enzyme solutions, obtains the indium-tin oxide electrode for being modified with noble metal nano particles and enzyme；
D, cell adherence agent solution is added into the electrode surface that step C is obtained, is dried to obtain the tin indium oxide of the surface modification
Electrode.
6. according to the method described in claim 5, it is characterized in that, using oxygen plasma to tin indium oxide described in step A
Before glass is surface-treated, indium oxide tin glass is first respectively placed in acetone, ethyl alcohol and ultra-pure water ultrasound 10-30 points successively
Clock.
7. according to the method described in claim 5, it is characterized in that, a concentration of 0.01- of graphene oxide solution described in step B
0.1mg/mL。
8. according to the method described in claim 5, it is characterized in that, the temperature of in-situ reducing described in step B is 50-70 DEG C.
9. according to the method described in claim 5, it is characterized in that, the time of in-situ reducing described in step B is 4-12 hours.
10. according to the method described in claim 5, it is characterized in that, noble metal nano particles described in step C are silver nanoparticle
In grain, gold nano grain or Pt nanoparticle any one or at least two combination.
11. according to the method described in claim 10, it is characterized in that, the noble metal nano particles are gold nano grain.
12. according to the method described in claim 5, it is characterized in that, noble metal in noble metal nano particles solution described in step C
The mass percent of nano particle is 0.01-0.05%.
13. according to the method described in claim 5, it is characterized in that, noble metal nano particles solution described in step C and step B
The volume ratio of the graphene oxide solution is 1:2-2:1.
14. according to the method described in claim 5, it is characterized in that, hydrogen peroxide catabolic enzyme described in step C is horseradish peroxidating
Object enzyme.
15. according to the method described in claim 5, it is characterized in that, hydrogen peroxide described in step C decomposes a concentration of of enzyme solutions
1-5mg/mL。
16. according to the method described in claim 5, it is characterized in that, cell adherence agent described in step D is by the sweet ammonia of arginine-
The cyclic annular small peptide of acid-aspartic acid sequence composition.
17. according to the method described in claim 5, it is characterized in that, a concentration of 1-5mg/mL of the cell adherence agent solution.
18. according to the method described in claim 5, it is characterized in that, described in cell adherence agent solution described in step D and step C
The volume ratio of noble metal nano particles solution is 1:2-2:1.
19. according to the method described in claim 2, it is characterized in that, step (1) described electrolyte is phosphate buffer solution.
20. according to the method described in claim 2, it is characterized in that, step (1) described drug is phorbol 12-myristic acid
Ester -13- acetic acid esters.
21. according to the method described in claim 2, it is characterized in that, step (1) described living cells is can be on the working electrode (s
The living cells of adherent growth.
22. according to the method described in claim 2, it is characterized in that, step (2) the hydroxyl radical free radical capturing agent is 5,5- bis-
Methyl-1-pyrrolin nitrogen oxides.
23. according to the method described in claim 2, it is characterized in that, step (2) the addition hydroxyl radical free radical capturing agent makes it
A concentration of 20-80mM in living cells culture solution.
24. according to the method described in claim 2, it is characterized in that, step (2) the addition hydroxyl radical free radical capturing agent makes it
A concentration of 40-50mM in living cells culture solution.
25. according to the method described in claim 2, it is characterized in that, the wave-length coverage of step (2) described ultraviolet light is 280-
320nm。
26. according to the method described in claim 2, it is characterized in that, step (3) the Hydrogen peroxide standard solution it is a concentration of
10-9-10-3mol/L。
27. according to the method described in claim 2, it is characterized in that, step (3) the preparation Hydrogen peroxide standard solution when institute
The concentration gradient of selected hydrogen peroxide is 1 × 10-6、5×10-6、1×10-5、2×10-5、5×10-5mol/L。
28. according to the method described in claim 2, it is characterized in that, step (3) the hydroxyl radical free radical capturing agent is 5,5- bis-
Methyl-1-pyrrolin nitrogen oxides.
29. according to the method described in claim 2, it is characterized in that, step (3) the addition hydroxyl radical free radical capturing agent makes it
It is dense in living cells culture solution that concentration in Hydrogen peroxide standard solution is equal to step (2) the hydroxyl radical free radical capturing agent
Degree.
30. according to the method described in claim 2, it is characterized in that, the wave-length coverage of step (3) described ultraviolet light is 280-
320nm。
31. method according to claim 1 or 2, which is characterized in that suitable using electronic paramagnetic resonance spectrometer record electronics
Magnetic resonance spectrogram.
32. according to the method described in claim 1, it is characterized in that, the method for detecting concentration of hydrogen peroxide in living cells
Include the following steps：
(1) using Ag/AgCl electrodes as reference electrode, platinized platinum or platinum filament are used as to electrode, and surface modification is simultaneously inoculated with living cells
Indium-tin oxide electrode as working electrode form three electrode work systems, insert it into electrolyte and form three electrode electrochemicals
Test system is learned, generates hydrogen peroxide as initial time to be initially added into medicine irritation living cells, Amperometric obtains using clocking
Current versus time curve of the living cells after by medicine irritation；
(2) while carrying out step (1), the living cells culture solution in electrochemical cell is taken, hydroxyl radical free radical capturing agent 5 is added,
5- dimethyl -1- pyrrolin nitrogen oxides, after wave-length coverage is the ultraviolet light of 280-320nm, record electron paramagnetic is total
Shake spectrogram, the same time in the current versus time curve that the electron paramagnetic resonance spectrum figure and step (1) obtained using the step is obtained
The corresponding electron paramagnetic resonance signal strength of point and size of current establish electron paramagnetic resonance signal strength-current relationship curve；
(3) 10 are prepared-4、10-5、10-6、10-7With 10-8The hydrogenperoxide steam generator of mol/L concentration gradients is added hydroxyl radical free radical and catches
Agent 5 is obtained, 5- dimethyl -1- pyrrolin nitrogen oxides records electronics after wave-length coverage is the ultraviolet light of 280-320nm
Paramagnetic resonance spectrogram establishes electron paramagnetic resonance signal strength-concentration of hydrogen peroxide standard curve；
(4) time current curve obtained by step (1) obtains the current value under random time, is then obtained by step (2)
The electron paramagnetic resonance signal strength arrived-current relationship curve reads corresponding electron paramagnetic resonance signal strength, finally utilizes
Electron paramagnetic resonance signal strength-concentration of hydrogen peroxide standard curve that step (3) obtains obtain under random time living cells by
The concentration of the hydrogen peroxide released after stimulation.
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| KR101041462B1 (en) * | 2009-04-08 | 2011-06-16 | 전남대학교산학협력단 | Cobalt porphyrin nanowire, the synthesis of cobalt porphyrin nanowire and the dertermination of hydrogen peroxide by cobalt porphyrin nanowire |
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