CN104049010A - Method for detecting apoptosis degree of cells in solution to be tested - Google Patents
Method for detecting apoptosis degree of cells in solution to be tested Download PDFInfo
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- CN104049010A CN104049010A CN201410300637.2A CN201410300637A CN104049010A CN 104049010 A CN104049010 A CN 104049010A CN 201410300637 A CN201410300637 A CN 201410300637A CN 104049010 A CN104049010 A CN 104049010A
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Abstract
The invention discloses a method for detecting an apoptosis degree of cells in a solution to be tested. The method comprises the steps of designing and synthesizing one section of polypeptide sequence, firmly modifying the polypeptide sequence on the surface of a working electrode, orderly arranging the polypeptide sequence through space occupying molecules, identifying the property of phosphatidylserine on surfaces of apoptotic cells in the solution to be tested through the polypeptide specificity, and fixing the apoptotic cells on the surface of the electrode. Because one layer of apoptotic cells is covered on the surface of the electrode, the electrochemical response of the working electrode is correspondingly changed; through comparing degrees of electrochemical signal changes before and after detection, the apoptosis degree of the cells in a sample to be tested can be detected. The method prevents from using the specific binding of an antigen and an antibody, thus the detection cost can be effectively lowered.
Description
Technical field
The present invention relates to electrochemical measuring technique field, particularly a kind of Apoptosis electrochemical detection method based on polypeptide identification system.
Background technology
Cell death can be divided into necrosis and apoptosis according to mechanism, inducement, dead scope, morphological feature, biochemical character etc.Apoptosis is the form of expression of apoptosis, by the dead program prestoring in the factor inducing cell of inside and outside, thereby cause the death of cell initiative, on morphological and biochemical features, be all different from necrosis, this process is the orderly death autonomous by the cell of gene control, can not cause the inflammatory reaction of body, can maintain the stable of human internal environment, the growth of whole bion normal growth, immune system, body be removed to impaired or senile cell and play very important effect.Confirm that tumorigenic correlative factor is as unbalance, the abnormal vascular of cell proliferation and death form, the transfer of tumour is spread all with apoptotic extremely relevant.Can contribute to the pathogenesis of illustrating various diseases also for new method is explored in these disease treatments to apoptotic Study on Molecular Mechanism and accurate detection.
The detection method of having developed at present apoptosis of many kinds, can be divided into morphology, biological chemistry, immunochemistry and molecular biology determination method according to methodology.Flow cytometry is at present for Apoptosis analysis with detect more method, and it has the following advantages: (1) rapid sensitive; (2) reproducible, result is accurate; (3) can analyze to active somatic cell real result; (4) can quantitatively detect apoptosis cell etc.But the method depends on large-scale instrument, and agents useful for same is very expensive, therefore still needs to develop certain operations convenient, detection method with low cost.
Summary of the invention
The object of the present invention is to provide a kind of Apoptosis electrochemical detection method based on polypeptide identification system, it is according to the particular sequence polypeptide of modifying in working electrode, apoptotic cell is fixed on to electrode surface, change the environment of working electrode, according to the variation of electrochemical response, the qualitative apoptosis degree that draws cell in solution to be measured.
The invention provides a kind of method for detection of Apoptosis degree in solution to be measured, the working electrode that is modified with peptide sequence is inserted in solution to be measured, in the time having apoptotic cell in solution to be measured, the phosphatidyl serine and the peptide sequence that turn up in apoptotic cell surface react, thereby make apoptotic cell be fixed on the peptide sequence surface on working electrode, the electrochemical response value of testing electrode, and be not fixed with apoptotic cell but be modified with the electrochemical response value comparison of the working electrode of peptide sequence, thereby determine the apoptosis degree of cell in solution to be measured; Described peptide sequence at least comprises by the sequence fragment manually synthesizing " FNFRLKAGAKIRFG ".
Preferably, the described method for detection of Apoptosis degree in solution to be measured, described peptide sequence is FNFRLKAGAKIRFGRGC.
Preferably, the described method for detection of Apoptosis degree in solution to be measured, described peptide sequence is synthetic by solid-phase synthesis or liquid phase synthesizing method.
Preferably, the described method for detection of Apoptosis degree in solution to be measured, working electrode adds occupy-place molecule before being modified with after peptide sequence, inserting solution to be measured, to assist the ordered arrangement of peptide sequence at working electrode surface.
Preferably, the described method for detection of Apoptosis degree in solution to be measured, described working electrode is gold electrode or carbon electrode.
Preferably, the described method for detection of Apoptosis degree in solution to be measured, described electrochemical response records by cyclic voltammetry or differential pulse voltammetry or AC impedence method.
The invention has the beneficial effects as follows: polypeptide-apoptotic cell identification system and electrochemical techniques are organically combined, for apoptotic qualitative detection, it has not only overcome the dependence of the expensive instruments such as convection type cell instrument, also avoid the use of the expensive reagent such as antigen-antibody, greatly reduce testing cost, contributed to the early diagnosis of relevant disease.
Brief description of the drawings
Fig. 1 is the process flow diagram for detection of the method for Apoptosis degree in solution to be measured of the present invention.
Fig. 2 is AC impedence method gained characterization data collection of illustrative plates in electrochemical detection method of the present invention, wherein, (a) bare electrode, (b) peptide modified electrode, (c) soak the peptide modified electrode after peptide modified electrode, (d) immersion apoptotic cell after normal cell.
Fig. 3 is the cyclic voltammogram in electrochemical detection method of the present invention, wherein, (a) bare electrode, (b) peptide modified electrode, (c) soak the peptide modified electrode after peptide modified electrode, (d) immersion apoptotic cell after normal cell.
Fig. 4 is the differential pulse voltammetry figure that electrochemical method of the present invention detects the hydrogen peroxide cell death inducing of variable concentrations, and concentration is followed successively by 0,25,50,80,100 μ M from top to bottom.
Fig. 5 is the differential pulse voltammetry curve peak current of the electrochemical method of the present invention hydrogen peroxide cell death inducing that detects variable concentrations and the linear relationship chart of hydrogen peroxide concentration.
Embodiment
Below in conjunction with accompanying drawing, the present invention is described in further detail, to make those skilled in the art can implement according to this with reference to instructions word.
The invention provides a kind of method for detection of Apoptosis degree in solution to be measured, comprise the following steps:
Step 1) peptide sequence is modified to working electrode surface, detect its electrochemical response, obtain the first response;
Step 2) working electrode is soaked in solution to be measured, in the time containing apoptotic cell in solution to be measured, utilize the character of the phosphatidyl serine that peptide sequence specific recognition apoptotic cell surface turns up, apoptotic cell is fixed on to the peptide sequence surface on working electrode, and detect the now electrochemical response of working electrode, obtain the second response;
Step 3) determine the apoptosis degree of cell in solution to be measured by relatively the first response and the second response.
Refer to Fig. 1, it has more clearly expressed the general flow chart of this case, and in figure, peptide sequence is
fNFRLKAGAKIRFGrGC, underscore part is " core sequence " fragment, the phosphatidyl serine specific binding that it can turn up with apoptotic cell, " RGC " fragment is " modification sequence ", its effect is by increasing polypeptide length, and " core sequence " and the apoptotic cell that are fixed on electrode surface can not be affected in the time interacting.In " RGC " fragment, the halfcystine of C end provides mercapto groups, can be with working electrode as gold electrode generation covalent reaction, thus be connected to working electrode.Peptide sequence, by manually designing and synthesizing, can synthesize acquisition by solid-phase synthesis or liquid phase synthesizing method.
Above-mentioned steps 2) in working electrode before being modified with after peptide sequence, inserting solution to be measured, should add occupy-place molecule, occupy-place molecule itself has certain length, and it is to assist the ordered arrangement of peptide sequence at working electrode surface by micromolecular space steric effect.Working electrode can be preferably gold electrode or carbon electrode.Electrochemical response can record by cyclic voltammetry or differential pulse voltammetry or AC impedence method.
Embodiment
The occupy-place molecule using in the present embodiment is sulfydryl hexanol, and the cell of use is MCF-7, and the working electrode of use is gold electrode.
1.1, the preparation of peptide modified electrode
A, polishing gold electrode, step comprises that sand paper grinds, and the alundum (Al2O3) slurry of variable grain size grinds, and ethanol/water is ultrasonic, 50% nitric acid dousing, 0.5M sulfuric acid electrochemical cleaning etc.
B, gold electrode are soaked in 0.1mM polypeptide solution, and reaction is spent the night, then washed with de-ionized water electrode.
C, then electrode is soaked to 0.1mM sulfydryl hexanol, reaction 30min, then use deionized water cleaning electrode.
1.2, cultured cell
MCF-7 cell uses DMEM nutrient culture media (10% serum) to cultivate, and temperature is 37 DEG C, and gas concentration lwevel is 5%, when cell is cultivated exponential phase, by cell dissociation, cleans with stand-by.
1.3, electrode surface polypeptide and apoptotic cell interact
Peptide modified electrode is soaked in cell solution to be measured, hatches 1 hour, then clean up, and set it as working electrode.Use platinum electrode as to electrode, saturated calomel electrode, as contrast electrode, builds three-electrode system.
In the high cell solution of apoptosis degree, there are more apoptotic cells to be attached to electrode surface, hinder electronics transmission, by using the electrochemical response of AC impedance, cyclic voltammetric or differential pulse voltammetry testing electrode, thus the apoptosis degree of reflection cell.
Fig. 2 is AC impedence method gained characterization data collection of illustrative plates, wherein, (a) bare electrode, (b) peptide modified electrode, (c) soak the peptide modified electrode after peptide modified electrode, (d) immersion apoptotic cell after normal cell.As seen from the figure, peptide modified electrode causes impedance to increase, and it soaks after normal cell, due to non-specific adsorption, also can cause impedance increase to a certain degree, and peptide modified electrode soaks after apoptotic cell, because polypeptid specificity is caught apoptotic cell, cause impedance sharply to increase.
Fig. 3 is cyclic voltammogram, and wherein, peptide modified electrode, (d) that (a) bare electrode, (b) peptide modified electrode, (c) soak after normal cell soak the peptide modified electrode after apoptotic cell.Impedance is larger, and the peak current of cyclic voltammogram is less.Cyclic voltammogram result in Fig. 3 is consistent with the ac impedance spectroscopy in Fig. 2.
Fig. 4 is the differential pulse voltammetry figure of the hydrogen peroxide cell death inducing of variable concentrations, and concentration is followed successively by 0,25,50,80,100 μ M from top to bottom.As seen from the figure, along with the increase of hydrogen peroxide concentration, Apoptosis degree increases, and the peak point current that electro-chemical systems detects is corresponding decline also.
Fig. 5 is the differential pulse voltammetry curve peak current of hydrogen peroxide cell death inducing of variable concentrations and the linear relationship chart of hydrogen peroxide concentration.As seen from the figure, the concentration of hydrogen peroxide and peak current drop-out value are proportionate.
Although embodiment of the present invention are open as above, but it is not restricted to listed utilization in instructions and embodiment, it can be applied to various applicable the field of the invention completely, for those skilled in the art, can easily realize other amendment, therefore do not deviating under the universal that claim and equivalency range limit, the present invention is not limited to specific details and illustrates here and the legend of describing.
Claims (6)
1. the method for detection of Apoptosis degree in solution to be measured, it is characterized in that, the working electrode that is modified with peptide sequence is inserted in solution to be measured, in the time having apoptotic cell in solution to be measured, the phosphatidyl serine and the peptide sequence that turn up in apoptotic cell surface react, thereby make apoptotic cell be fixed on the peptide sequence surface on working electrode, the electrochemical response value of testing electrode, and be not fixed with apoptotic cell but be modified with the electrochemical response value comparison of the working electrode of peptide sequence, thereby determine the apoptosis degree of cell in solution to be measured, described peptide sequence at least comprises by the sequence fragment manually synthesizing " FNFRLKAGAKIRFG ".
2. the method for detection of Apoptosis degree in solution to be measured according to claim 1, is characterized in that, described peptide sequence is FNFRLKAGAKIRFGRGC.
3. the method for detection of Apoptosis degree in solution to be measured according to claim 1, is characterized in that, described peptide sequence is synthetic by solid-phase synthesis or liquid phase synthesizing method.
4. the method for detection of Apoptosis degree in solution to be measured according to claim 1, it is characterized in that, working electrode adds occupy-place molecule before being modified with after peptide sequence, inserting solution to be measured, to assist the ordered arrangement of peptide sequence at working electrode surface.
5. the method for detection of Apoptosis degree in solution to be measured according to claim 1, is characterized in that, described working electrode is gold electrode or carbon electrode.
6. the method for detection of Apoptosis degree in solution to be measured according to claim 1, is characterized in that, described electrochemical response records by cyclic voltammetry or differential pulse voltammetry or AC impedence method.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105445248A (en) * | 2015-12-14 | 2016-03-30 | 上海应用技术学院 | Method for rapid determination of cyanobacteria cell apoptosis rate |
| CN106680337A (en) * | 2016-12-20 | 2017-05-17 | 中国科学院苏州生物医学工程技术研究所 | Quantitative detection method of heparin |
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| CN101750435A (en) * | 2009-12-31 | 2010-06-23 | 首都医科大学 | Method for detecting early apoptosis with electrorotation technology |
| KR100973042B1 (en) * | 2008-03-18 | 2010-07-29 | 연세대학교 산학협력단 | Cell sensor and Method of monitoring cell growth and apoptosis in real time |
| US20140141453A1 (en) * | 2011-06-30 | 2014-05-22 | Ge Healthcare Bio-Sciences Ab | Cell binding assay |
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2014
- 2014-06-27 CN CN201410300637.2A patent/CN104049010A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| KR100973042B1 (en) * | 2008-03-18 | 2010-07-29 | 연세대학교 산학협력단 | Cell sensor and Method of monitoring cell growth and apoptosis in real time |
| CN101750435A (en) * | 2009-12-31 | 2010-06-23 | 首都医科大学 | Method for detecting early apoptosis with electrorotation technology |
| US20140141453A1 (en) * | 2011-06-30 | 2014-05-22 | Ge Healthcare Bio-Sciences Ab | Cell binding assay |
Non-Patent Citations (1)
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| PENG MIAO 等: "Peptide-based electrochemical approach for apoptosis evaluation", 《BIOSENSORS AND BIOELECTRONICS》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105445248A (en) * | 2015-12-14 | 2016-03-30 | 上海应用技术学院 | Method for rapid determination of cyanobacteria cell apoptosis rate |
| CN106680337A (en) * | 2016-12-20 | 2017-05-17 | 中国科学院苏州生物医学工程技术研究所 | Quantitative detection method of heparin |
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Application publication date: 20140917 |