CN104049010A - Method for detecting apoptosis degree of cells in solution to be tested - Google Patents

Abstract

The invention discloses a method for detecting the degree of cell apoptosis in a solution to be tested, which includes: designing and synthesizing a polypeptide sequence, firmly modifying it on the surface of a working electrode, and making it orderly arranged by occupying molecules, The apoptotic cell is fixed on the surface of the electrode through the property of the polypeptide specifically recognizing the phosphatidylserine on the surface of the apoptotic cell in the solution to be detected. Since the surface of the electrode is covered with a layer of apoptotic cells, the electrochemical response of the working electrode will change accordingly. By comparing the degree of change in the electrochemical signal before and after detection, the degree of apoptosis in the sample to be tested can be detected. This method avoids the specific binding of antigens and antibodies, which can effectively reduce the cost of detection.

Description

A method for detecting the degree of apoptosis in the solution to be tested

technical field

The invention relates to the technical field of electrochemical detection, in particular to an electrochemical detection method of cell apoptosis based on a polypeptide recognition system.

Background technique

Cell death can be divided into necrosis and apoptosis according to the mechanism, inducement, scope of death, morphological characteristics, and biochemical characteristics. Apoptosis is a manifestation of programmed cell death, which induces the pre-existing death program in cells by in vivo and external factors, leading to active cell death, which is different from necrosis in terms of morphology and biochemical characteristics, and the process is controlled by genes. Spontaneous and orderly death will not cause the body's inflammatory response, and can maintain the stability of the internal environment of the human body. It plays a very important role in the normal growth and development of the entire organism, the regulation of the immune system, and the elimination of damaged or aging cells in the body. It has been confirmed that the relevant factors of tumorigenesis, such as the imbalance of cell proliferation and death, abnormal blood vessel formation, and tumor metastasis and spread, are all related to the abnormality of cell apoptosis. The molecular mechanism research and accurate detection of apoptosis can help to clarify the pathogenesis of various diseases and explore new methods for the treatment of these diseases.

A variety of apoptosis detection methods have been developed, which can be divided into morphological, biochemical, immunochemical and molecular biological assays according to methodology. Flow cytometry is currently the most widely used method for cell apoptosis analysis and detection. It has the following advantages: (1) fast and sensitive; (2) good repeatability and accurate results; (3) can analyze living cells, The result is true; (4) The number of apoptotic cells can be quantitatively detected. However, this method relies on large-scale instruments, and the reagents used are very expensive, so it is still necessary to develop some detection methods that are easy to operate and low in cost.

Contents of the invention

The purpose of the present invention is to provide a method for electrochemical detection of cell apoptosis based on a polypeptide recognition system, which fixes apoptotic cells on the surface of the electrode according to the specific sequence polypeptide modified on the working electrode, changes the environment of the working electrode, The changes in the response can be used to qualitatively obtain the degree of apoptosis of the cells in the solution to be tested.

The invention provides a method for detecting the degree of cell apoptosis in the solution to be tested. A working electrode modified with a polypeptide sequence is inserted into the solution to be tested. When there are apoptotic cells in the solution to be tested, the surface of the apoptotic cell The reversed phosphatidylserine reacts with the polypeptide sequence, so that the apoptotic cells are fixed on the surface of the polypeptide sequence on the working electrode, the electrochemical response value of the working electrode is detected, and the apoptotic cells are not fixed but modified with the polypeptide sequence. The electrochemical response values of the working electrodes are compared to determine the degree of cell apoptosis in the solution to be tested; the polypeptide sequence at least includes the artificially synthesized sequence fragment "FNFRLKAGAKIRFG".

Preferably, in the method for detecting the degree of apoptosis in the solution to be tested, the polypeptide sequence is FNFRLKAGAKIRFGRGC.

Preferably, in the method for detecting the degree of apoptosis in the solution to be tested, the polypeptide sequence is synthesized by solid-phase synthesis or liquid-phase synthesis.

Preferably, in the method for detecting the degree of apoptosis in the solution to be tested, the working electrode is modified with a polypeptide sequence and before being inserted into the solution to be tested, a space-occupying molecule is added to assist the effective movement of the polypeptide sequence on the surface of the working electrode. arranged in sequence.

Preferably, in the method for detecting the degree of apoptosis in the solution to be tested, the working electrode is a gold electrode or a carbon electrode.

Preferably, in the method for detecting the degree of cell apoptosis in the solution to be tested, the electrochemical response is measured by cyclic voltammetry or differential pulse voltammetry or AC impedance method.

The beneficial effects of the present invention are: the polypeptide-apoptotic cell recognition system is organically combined with electrochemical technology for the qualitative detection of cell apoptosis, which not only overcomes the dependence on expensive instruments such as flow cytometry, but also avoids the need for antigen-antibody The use of such expensive reagents greatly reduces the cost of detection and contributes to the early diagnosis of related diseases.

Description of drawings

Fig. 1 is a flow chart of the method for detecting the degree of apoptosis in the solution to be tested according to the present invention.

Figure 2 is the characterization data spectrum obtained by the AC impedance method in the electrochemical detection method of the present invention, wherein, (a) bare electrode, (b) polypeptide modified electrode, (c) polypeptide modified electrode after soaking normal cells, (d) ) A polypeptide-modified electrode soaked in apoptotic cells.

Fig. 3 is the cyclic voltammogram in the electrochemical detection method of the present invention, wherein, (a) bare electrode, (b) polypeptide modification electrode, (c) polypeptide modification electrode after soaking normal cells, (d) soaking Peptide-modified electrodes behind apoptotic cells.

Fig. 4 is a differential pulse voltammogram of cell apoptosis induced by different concentrations of hydrogen peroxide detected by the electrochemical method of the present invention, the concentrations from top to bottom are 0, 25, 50, 80, 100 μM.

Fig. 5 is a graph showing the linear relationship between the peak current of the differential pulse voltammetry curve and the concentration of hydrogen peroxide for detecting cell apoptosis induced by different concentrations of hydrogen peroxide by the electrochemical method of the present invention.

Detailed ways

The present invention will be further described in detail below in conjunction with the accompanying drawings, so that those skilled in the art can implement it with reference to the description.

The invention provides a method for detecting the degree of apoptosis in the solution to be tested, comprising the following steps:

Step 1) modifying the polypeptide sequence on the surface of the working electrode, detecting its electrochemical response, and obtaining the first response value;

Step 2) Soak the working electrode in the solution to be tested. When the solution to be tested contains apoptotic cells, the apoptotic cells are fixed on the the surface of the polypeptide sequence on the working electrode, and detect the electrochemical response of the working electrode at this time to obtain the second response value;

Step 3) Determine the degree of apoptosis of the cells in the solution to be tested by comparing the first response value with the second response value.

Please refer to Figure 1, which shows the overall flowchart of this case more clearly. The polypeptide sequence in the figure is FNFRLKAGAKIRFG RGC, and the underlined part is the "core sequence" fragment, which can specifically bind to the phosphatidylserine in apoptotic cells. In combination, the "RGC" fragment is a "modified sequence", and its function is to increase the length of the polypeptide so that the "core sequence" fixed on the surface of the electrode and the apoptotic cell will not be affected when interacting. The cysteine at the C-terminus of the "RGC" fragment provides a sulfhydryl group that can covalently react with a working electrode, such as a gold electrode, thereby connecting to the working electrode. The polypeptide sequence is artificially designed and synthesized, and can be obtained by solid-phase synthesis or liquid-phase synthesis.

The working electrode in the above step 2) should be added with a space-occupying molecule after being modified with a polypeptide sequence and before being inserted into the solution to be tested. The space-occupying molecule itself has a certain length. Orderly arrangement of the working electrode surface. The working electrode may preferably be a gold electrode or a carbon electrode. The electrochemical response can be measured by cyclic voltammetry or differential pulse voltammetry or AC impedance.

Example

The space-occupying molecule used in this example is mercaptohexanol, the cell used is MCF-7, and the working electrode used is a gold electrode.

1.1. Preparation of peptide-modified electrodes

a. Grinding the gold electrode, the steps include grinding with sandpaper, grinding with aluminum oxide slurry of different particle sizes, ultrasonication with ethanol/water, soaking in 50% nitric acid, electrochemical cleaning with 0.5M sulfuric acid, etc.

b. Soak the gold electrode in 0.1mM peptide solution, react overnight, and then wash the electrode with deionized water.

c. Then soak the electrode in 0.1mM mercaptohexanol, react for 30min, and then clean the electrode with deionized water.

1.2. Cell culture

MCF-7 cells were cultured in DMEM medium (10% serum) at a temperature of 37°C and a carbon dioxide concentration of 5%. When the cells were cultured to the exponential phase, the cells were digested and washed for later use.

1.3. Interaction between electrode surface peptides and apoptotic cells

Soak the polypeptide-modified electrode into the cell solution to be tested, incubate for 1 hour, then clean it, and use it as the working electrode. A platinum wire electrode was used as the counter electrode, and a saturated calomel electrode was used as the reference electrode to construct a three-electrode system.

In the cell solution with a high degree of apoptosis, more apoptotic cells are bound to the electrode surface, hindering electron transfer, and the electrochemical response of the working electrode is detected by using AC impedance, cyclic voltammetry or differential pulse voltammetry to reflect the apoptosis of the cells. degree of death.

Figure 2 is the characterization data spectrum obtained by AC impedance method, in which, (a) bare electrode, (b) polypeptide-modified electrode, (c) polypeptide-modified electrode soaked in normal cells, (d) polypeptide-modified electrode soaked in apoptotic cells . It can be seen from the figure that the polypeptide-modified electrode leads to an increase in impedance. After soaking normal cells, due to non-specific adsorption, the impedance will also increase to a certain extent. After soaking apoptotic cells, the polypeptide-modified electrode will specifically capture apoptotic cells. cells, leading to a sharp increase in impedance.

Fig. 3 is a cyclic voltammogram, wherein, (a) a bare electrode, (b) a polypeptide-modified electrode, (c) a polypeptide-modified electrode soaked in normal cells, (d) a polypeptide-modified electrode soaked in apoptotic cells. The larger the impedance, the smaller the peak current in the cyclic voltammogram. The cyclic voltammogram results in Figure 3 are consistent with the AC impedance spectra in Figure 2.

Fig. 4 is a differential pulse voltammogram of cell apoptosis induced by different concentrations of hydrogen peroxide, and the concentrations are 0, 25, 50, 80, and 100 μM from top to bottom. It can be seen from the figure that as the concentration of hydrogen peroxide increases, the degree of cell apoptosis increases, and the peak current value detected by the electrochemical system also decreases accordingly.

Fig. 5 is a graph showing the linear relationship between the peak current of the differential pulse voltammetry curve and the concentration of hydrogen peroxide induced by different concentrations of hydrogen peroxide. It can be seen from the figure that the concentration of hydrogen peroxide is positively correlated with the peak current drop.

Although the embodiment of the present invention has been disclosed as above, it is not limited to the use listed in the specification and implementation, it can be applied to various fields suitable for the present invention, and it can be easily understood by those skilled in the art Therefore, the invention is not limited to the specific details and examples shown and described herein without departing from the general concept defined by the claims and their equivalents.

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.