CN1940550A - Method for determining cell active oxygen and reduced glutathione simultaneouslly - Google Patents
Method for determining cell active oxygen and reduced glutathione simultaneouslly Download PDFInfo
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- CN1940550A CN1940550A CN 200510047285 CN200510047285A CN1940550A CN 1940550 A CN1940550 A CN 1940550A CN 200510047285 CN200510047285 CN 200510047285 CN 200510047285 A CN200510047285 A CN 200510047285A CN 1940550 A CN1940550 A CN 1940550A
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Abstract
A method for detecting cell active oxygen and reduced glutathion includes utilizing microfluid control chip to carry out detection of cell active oxygen and reduced plutathion simultaneously, setting buff solution concentration in electrophoresis of said chip to be 20-50mM and buffer solution pH value to be 8.0-10.1 as well as separation voltage to be 200-450v / cm and setting distance of detection point to cross point of sample feeding channel and separation channel to be 1.0-3.0cm.
Description
Technical field
The present invention relates to the detection of intracellular reactive oxygen and reduced glutathione, specifically a kind of detection method of measuring cytoactive oxygen and reduced glutathione simultaneously.
Background technology
Active oxygen (ROS) and reduced glutathione (GSH) are two class signal of interest molecules in the intracellular signal transduction process, and be closely related with redox state in the cell.Active oxygen mainly is meant by a class that produces in the aerobic metabolism process in exogenous oxygenant or the cell to have the very oxygen molecule of high bioactivity, as O
2-, NO, H
2O
2, OH etc.When body was in stress situation, cell hypoxia, impaired or Apoptosis, the intracellular reactive oxygen excess generated.Reduced glutathione is the maximum a kind of strong reductant of cell intensive amount, and major function is to remove excessive levels of reactive oxygen species in the body, prevents the albumen sulfhydryl oxidase, and keeps intracellular oxidation-reduction balance.It is research oxygen radical and stress reaction that pair cell active oxygen and reduced glutathione are measured, reach and the important indicator of human diseases correlativity.
The detection method that is used for active oxygen and reduced glutathione at present mainly contains high performance liquid chromatography (HPLC), fluorescence method, zymetology method and flow cytometer etc.Though these methods have his own strong points, in general, required amount of samples is big, and sensitivity is not high, and can not detect simultaneously two kinds of materials.Micro-fluidic chip (claiming chip lab again) is a brand-new analytical technology that grew up in recent years, it can be integrated in the different operating unit in the biochemical reaction process substantially on the chip that has only several square centimeters of sizes and finish, have that analysis speed is fast, amount of samples is little, be easy to characteristics such as integrated, be called as one of of paramount importance cutting edge technology of 21 century.It is applicable to the compartment analysis to nucleic acid, albumen and micromolecule etc. in principle, and might be used for the assay determination of the interior complex component of pair cell such as active oxygen and reduced glutathione.Utilize micro-fluidic chip pair cell active oxygen and reduced glutathione to measure simultaneously, be not reported both at home and abroad at present.
Summary of the invention
The object of the invention is to provide that a kind of amount of samples is few, detection sensitivity is higher, can measures the detection method of cytoactive oxygen and reduced glutathione simultaneously, the present invention has realized on micro-fluidic chip measuring in two kinds of materials, only use 27 seconds analysis time, amount of samples considerably less (3-4uL), and detection sensitivity higher (amol-zmol).
For achieving the above object, the technical solution used in the present invention is:
A kind of detection method of measuring cytoactive oxygen and reduced glutathione simultaneously, adopt micro-fluidic chip pair cell active oxygen and reduced glutathione to measure simultaneously, the buffer concentration that micro-fluid control chip electrophoretic adopts is 20-50mM, the pH of buffer value is 8.0-10.1, separation voltage is 200-450V/cm, and check point is 1.0-3.0cm apart from sample intake passage and split tunnel infall.
The concrete operations content comprises micro-fluidic chip design, intracellular reactive oxygen and glutathione fluorescence labeling and extracting method, micro-fluid control chip electrophoretic separation condition and micro-fluidic chip laser-Induced Fluorescence Detection method.
The present invention is technology platform with the micro-fluidic chip, is detection means with micro-fluidic chip-laser-induced fluorescence (LIF), and selected laser excitation wavelength is 473nm, detects wavelength 520nm; Adopt probe DHR-123 and NDA to come active oxygen and reduced glutathione in the labeled cell respectively, adopt organic solvent (the fine mixed liquor of perchloric acid and second) to extract intracellular reactive oxygen and reduced glutathione; The weight concentration of described perchloric acid and the fine mixed liquor of second is that 3.3% perchloric acid mixed with acetonitrile in 1: 1 by volume, and wherein the fine volume ratio of perchloric acid and second is 1: 1
The chip material of micro-fluidic chip can adopt glass, quartz or other chip material; Double-T shaped structure is preferably adopted in chip design, and sample feeding adopts folder butt formula to guarantee the accuracy of sample introduction volume; Damping fluid can be phosphate buffer, borate buffer solution etc., is preferably borate buffer solution;
Advantage of the present invention is:
1. detection method amount of samples of the present invention considerably less (3-4uL), on micro-fluidic chip, realized measuring in two kinds of materials (intracellular reactive oxygen and reduced glutathione), only use 27 seconds analysis time, and detection sensitivity higher (amol-zmol), the micro-fluid control chip electrophoretic condition scope of application broad of being optimized (mainly comprises buffer concentration, the pH value, conditions such as electrophoresis sample introduction and separation voltage).
2. the invention provides the universal cytoactive oxygen and the chip detecting method of reduced glutathione, be applicable to the monitoring that the cellular oxidation reducing condition changes when causing the body oxidative stress, and applicable to dissimilar cells such as tumour cell, red blood cell, neuron and embryo's etc. detection, convenient experimental operation is easily gone, and has operability.
Description of drawings
Fig. 1 is adopted the microfluidic chip structure synoptic diagram by the present invention;
Fig. 2 is micro-fluidic chip laser-induced fluorescence (LIF) light path design figure of the present invention;
Fig. 3 is the micro-fluid control chip electrophoretic figure of active oxygen and reduced glutathione in the NB4 cell;
Fig. 4 is the micro-fluid control chip electrophoretic figure of active oxygen and reduced glutathione in the K562 cell, and wherein coordinate is represented relative intensity of fluorescence (mV) and transit time (s) respectively.
Embodiment
Embodiment
1. micro-fluidic chip design:
Micro-fluidic chip that the invention process adopts design is illustrated in fig. 1 shown below, and the glass-chip that adopts is double-T shaped structure, and four circular holes are represented the different solutions pond respectively among the figure, wherein 2 is the damping fluid sample inlet pool, and 4 is the damping fluid waste liquid pool, and 1 is sample cell, 3 is the sample waste liquid pool, and 5 is check point.(from buffering liquid pool to right-angled intersection is 0.5cm to the long 8.5cm of the split tunnel of chip, is 8.0cm from right-angled intersection to separating waste liquid pool), sample intake passage is long to be 1.0cm (being 0.5cm from sample cell to right-angled intersection with from right-angled intersection to sample waste liquid pool), the xsect of passage is approximately half elliptic, going up wide is 50 μ m, and check point is 1.0cm apart from right-angled intersection place.
2. micro-fluidic chip laser induced fluorescence detector:
The selected laser excitation wavelength of micro-fluidic chip-laser induced fluorescence detector is 473nm, and the detection wavelength is 520nm, adopts the burnt detection mode of single-point copolymerization, micro-fluidic chip laser-induced fluorescence (LIF) light path design figure sees Fig. 2, and wherein: 6 is object lens, and 7 is half-reflecting half mirror, 8 is spectroscope, and 9 is first bandpass filter, and 10 is convex lens, 11 is pin hole, 12 is photomultiplier, and 13 is chip, and 14 is second bandpass filter, 15 is laser, and 16 is the inductive coupling device.
3. cell sample preparation:
With people's acute morning of children's grain leukon NB4 cell is example, and cellular incubation is cultivated in 5%CO2 incubator 37 degree in the RPMI1640 nutrient solution that contains 10% NBCS.Collecting cell, in 1000rpm centrifugal 5 minutes, abandon supernatant, cell precipitation is centrifugal with the resuspended back of PBS solution (pH 7.4), and is stand-by after the cell count.
4. intracellular reactive oxygen and reduced glutathione fluorescence labeling and extracting method:
Adopt interior active oxygen of DHR-123 and NDA pair cell and reduced glutathione to carry out fluorescence labeling respectively, at active oxygen, the DHR-123 label concentration is 20-30uM.Because the reaction of NDA and reduced glutathione is very fast, in the experiment NDA is added and reach the dynamically labeled glutathione of final concentration 0.1-0.5mM in the running buffer.Used cell density is 1 * 10 in the experiment
5About/mL, at first with effect in the DHR-123 dyestuff adding cell culture fluid of 20uM 20-30 minute, three times (800 change with the PBS washing with cell suspension then, 6 minutes), abandon supernatant, keep precipitation, add then and the isopyknic fine mixed liquor of perchloric acid/second (3.3%) of precipitation part, through at a high speed (12,000 change) centrifugal 5 minutes.After abandon supernatant, repeat above-mentioned steps twice again, keep cell precipitation, to be checked.
5. micro-fluidic chip electrophoresis separation condition:
Before the each run, need water, 0.1M NaOH and damping fluid wash respectively with chip channel.Keep 4 Buffer Pool liquid levels to be in same horizontal level during last sample as far as possible, insert electrode then.See Fig. 1, in the sample introduction process, with sample waste liquid pool 3 ground connection, apply+200V voltage is in sample cell 1, and buffer pool 2 and buffering waste liquid pool 4 apply 150V and 600V voltage, sample injection time 12s respectively.In the detachment process, buffering waste liquid pool 4 ground connection, sample cell 1 and sample waste liquid pool 3 apply 2kV voltage respectively, apply on Buffer Pool 2+3kV voltage.At first conditions such as micro-fluid control chip electrophoretic separation condition such as damping fluid, pH, separation voltage are investigated.Deposition condition through optimized choice is: borate buffer solution 20-50mM, pH of buffer value scope 8.0-10.1, separation voltage 200-450V/cm.
6. application example:
Fig. 3 and Fig. 4 are respectively the micro-fluid control chip electrophoretic figure of active oxygen and reduced glutathione in NB4 cell and the K562 cell, and peak 1 and peak 2 are represented cytoactive oxygen and reduced glutathione respectively in the electrophoretogram.Whole disengaging time is only used 27s, and the actual sample consumption is only used 3-4uL, and detection sensitivity is higher.Table 1 is the main performance index of micro-fluidic chip detection of active oxygen and glutathione. visible its detection sensitivity is higher in the table, can reach pmol and amol level to active oxygen and glutathione detection.
The main performance index of table 1. micro-fluidic chip detection of active oxygen and reduced glutathione (Table 1.Linearity, reproducibility, and detection limits of ROS and GSH)
| Measured object | Relative standard deviation (t/s) RSD | The detectable concentration scope | Equation of linear regression | Regression coefficient (r) | Detectability (S/N=3) |
| ROS | 0.4/3.5 | 2.6×10 -12-1.3×10 -10M | Y＝-0.01188+0.27348X | 0.9956 | 1.9pM(0.2zmol) |
| GSH | 2.2/3.7 | 5×10 -7-5×10 -5M | Y＝-1.67212+0.01469X | 0.9967 | 0.5μM(5.4amol) |
Claims (6)
1. detection method of measuring cytoactive oxygen and reduced glutathione simultaneously, it is characterized in that: adopt micro-fluidic chip pair cell active oxygen and reduced glutathione to measure simultaneously, the buffer concentration that micro-fluid control chip electrophoretic adopts is 20-50mM, the pH of buffer value is 8.0-10.1, separation voltage is 200-450V/cm, and check point is 1.0-3.0cm apart from sample intake passage and split tunnel infall.
2. according to the described detection method of measuring cytoactive oxygen and reduced glutathione simultaneously of claim 1, it is characterized in that: be technology platform with the micro-fluidic chip, with micro-fluidic chip-laser-induced fluorescence (LIF) is detection means, and selected laser excitation wavelength is 473nm, detects wavelength 520nm.
3. according to claim 1 or the 2 described detection methods of measuring cytoactive oxygen and reduced glutathione simultaneously, it is characterized in that: described micro-fluidic chip is double-T shaped structure.
4. according to the described detection method of measuring cytoactive oxygen and reduced glutathione simultaneously of claim 1, it is characterized in that: described damping fluid is a borate buffer solution.
5. according to the described detection method of measuring cytoactive oxygen and reduced glutathione simultaneously of claim 1, it is characterized in that: adopt probe DHR-123 and NDA to come active oxygen and reduced glutathione in the labeled cell respectively, adopt perchloric acid and the fine mixed liquor of second to extract intracellular reactive oxygen and reduced glutathione.
6. according to the described detection method of measuring cytoactive oxygen and reduced glutathione simultaneously of claim 5, it is characterized in that: the weight concentration of described perchloric acid and the fine mixed liquor of second is that 3.3% perchloric acid mixed with acetonitrile in 1: 1 by volume, and wherein the fine volume ratio of perchloric acid and second is 1: 1.
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102175679A (en) * | 2011-01-24 | 2011-09-07 | 苏洋 | Quantitative measurement of concentration of glutathione |
| CN102175678A (en) * | 2011-01-24 | 2011-09-07 | 苏洋 | Quantitative measuring method for concentration of glutathione (GSII) |
| CN102183504A (en) * | 2011-01-25 | 2011-09-14 | 山东师范大学 | Microfluidic unicellular active oxygen automatic analyzer |
| CN101855538B (en) * | 2007-11-13 | 2012-08-08 | 蒂埃里·帕特里斯 | Method for measuring the ability of a sample to withstand reactive oxygen species (ROS) |
| RU2506579C1 (en) * | 2012-09-18 | 2014-02-10 | Федеральное государственное бюджетное образовательное учреждение высшего профессионального образования "Национальный исследовательский Томский политехнический университет" | Method of determining glutathione in standard test aqueous solutions via cyclic voltammetry on graphite electrode, modified with colloidal gold particles |
| CN104730051A (en) * | 2015-03-06 | 2015-06-24 | 山东大学 | Method for detecting content of glutathione (GSH) in each cell by using microfluidic chip based laser induced fluorescence system |
| CN105241942A (en) * | 2015-09-06 | 2016-01-13 | 常州大学 | Method of quickly detecting concentration of glutathione on the basis of capillary electrophoresis |
| CN107478618A (en) * | 2016-06-07 | 2017-12-15 | 国家纳米科学中心 | A kind of method of the intracellular ROS detections based on fluorescence gold nanoclusters |
| CN107490614A (en) * | 2017-09-15 | 2017-12-19 | 福建中医药大学 | A kind of quality determining method of Kangfuxin Liquid |
| CN110669937A (en) * | 2019-11-07 | 2020-01-10 | 华东理工大学 | Method for improving metal biological leaching rate in waste lithium battery by using glutathione |
| CN110987891A (en) * | 2019-12-20 | 2020-04-10 | 吉林大学 | Method for testing oxidative stress biomarkers in blood plasma with low coefficient of variation |
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2005
- 2005-09-28 CN CN 200510047285 patent/CN1940550A/en active Pending
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101855538B (en) * | 2007-11-13 | 2012-08-08 | 蒂埃里·帕特里斯 | Method for measuring the ability of a sample to withstand reactive oxygen species (ROS) |
| CN102175678A (en) * | 2011-01-24 | 2011-09-07 | 苏洋 | Quantitative measuring method for concentration of glutathione (GSII) |
| CN102175679B (en) * | 2011-01-24 | 2012-03-28 | 宁波市鄞州奥胜生物科技有限公司 | Quantitative measurement of concentration of glutathione |
| CN102175678B (en) * | 2011-01-24 | 2012-03-28 | 宁波市鄞州奥胜生物科技有限公司 | Quantitative measuring method for concentration of glutathione (GSII) |
| CN102175679A (en) * | 2011-01-24 | 2011-09-07 | 苏洋 | Quantitative measurement of concentration of glutathione |
| CN102183504A (en) * | 2011-01-25 | 2011-09-14 | 山东师范大学 | Microfluidic unicellular active oxygen automatic analyzer |
| RU2506579C1 (en) * | 2012-09-18 | 2014-02-10 | Федеральное государственное бюджетное образовательное учреждение высшего профессионального образования "Национальный исследовательский Томский политехнический университет" | Method of determining glutathione in standard test aqueous solutions via cyclic voltammetry on graphite electrode, modified with colloidal gold particles |
| CN104730051B (en) * | 2015-03-06 | 2017-10-24 | 山东大学 | The method that micro-fluidic laser-induced fluorescing system detects individual cells Glutathione peptide content |
| CN104730051A (en) * | 2015-03-06 | 2015-06-24 | 山东大学 | Method for detecting content of glutathione (GSH) in each cell by using microfluidic chip based laser induced fluorescence system |
| CN105241942A (en) * | 2015-09-06 | 2016-01-13 | 常州大学 | Method of quickly detecting concentration of glutathione on the basis of capillary electrophoresis |
| CN105241942B (en) * | 2015-09-06 | 2018-01-02 | 常州大学 | A kind of method based on Capillary Electrophoresis quick detection glutathione concentrations |
| CN107478618A (en) * | 2016-06-07 | 2017-12-15 | 国家纳米科学中心 | A kind of method of the intracellular ROS detections based on fluorescence gold nanoclusters |
| CN107490614A (en) * | 2017-09-15 | 2017-12-19 | 福建中医药大学 | A kind of quality determining method of Kangfuxin Liquid |
| CN107490614B (en) * | 2017-09-15 | 2019-11-26 | 福建中医药大学 | A kind of quality determining method of Kangfuxin Liquid |
| CN110669937A (en) * | 2019-11-07 | 2020-01-10 | 华东理工大学 | Method for improving metal biological leaching rate in waste lithium battery by using glutathione |
| CN110987891A (en) * | 2019-12-20 | 2020-04-10 | 吉林大学 | Method for testing oxidative stress biomarkers in blood plasma with low coefficient of variation |
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