阅读说明：本技术 一种基于癌细胞表面电荷强度的评估肿瘤恶性程度的方法 (Method for evaluating malignancy degree of tumor based on surface charge intensity of cancer cells ) 是由 陈炳地 刘中民 乐文俊 催征 陈景瑶 于 2021-09-01 设计创作，主要内容包括：本发明提供了一种基于癌细胞表面电荷强度的评估肿瘤恶性程度的方法,包括：第一步骤,用于通过建立的电化学器件实现癌细胞表面电荷强度的检测；第二步骤,用于将检测的癌细胞表面电荷强度作为评估肿瘤恶性的指标。(The invention provides a method for evaluating the malignancy degree of a tumor based on the surface charge intensity of cancer cells, which comprises the following steps: a first step for detecting the intensity of the surface charge of the cancer cell by the established electrochemical device; and a second step for using the detected surface charge intensity of the cancer cells as an index for evaluating malignancy of the tumor.)

1. A method for assessing the malignancy of a tumor based on the intensity of surface charge on cancer cells, comprising:

a first step for detecting the intensity of the surface charge of the cancer cell by the established electrochemical device;

and a second step for using the detected surface charge intensity of the cancer cells as an index for evaluating malignancy of the tumor.

2. The method for assessing the malignancy of tumor according to claim 1, wherein the detection method used in the first step is a three-electrode system test method, and the three-electrode system test method uses a three-electrode system comprising a working electrode, a counter electrode and a reference electrode, wherein the working electrode comprises a flexible substrate and a carbon nanotube film arranged in this order, the counter electrode is a platinum wire counter electrode, and the reference electrode is composed of silver coated with silver chloride immersed in a potassium chloride solution, i.e., a silver/silver chloride electrode.

3. The method for assessing malignancy of a tumor according to claim 2, wherein the three-electrode system test comprises:

fixing the pretreated carbon nanotube film on a flexible polydimethylsiloxane substrate to obtain a carbon nanotube film electrode;

connecting the obtained carbon nano tube film electrode to one end of a working electrode of an electrochemical workstation, connecting a platinum wire to one end of a counter electrode of the electrochemical workstation, and connecting a silver/silver chloride electrode to one end of a reference electrode of the electrochemical workstation.

4. The method for assessing the malignancy of a tumor according to claim 3, wherein the detection method used in the first step is a three-electrode system test method comprising: and establishing the relation between the surface charge of the cancer cells and the instant response current, detecting the tumor to be detected in a three-electrode system, and calculating to obtain the intensity level of the surface charge of the cancer cells.

5. The method of claim 1 or 2, wherein establishing the relationship between the surface charge of the cancer cell and the instantaneous response current comprises:

taking phosphate buffer solution as electrolyte, immersing a working electrode, a counter electrode and a reference electrode of a three-electrode system into the electrolyte, and testing a blank curve by a linear sweep voltammetry method of an electrochemical workstation;

and sequentially adding cancer cells with different concentrations or different types into the electrolyte, and testing the change condition of the current under the same voltage by using a linear sweep voltammetry method of an electrochemical workstation.

6. The method for assessing the malignancy of tumors according to claim 1 or 2, wherein the step of detecting the tumor to be detected in a three-electrode system and obtaining the level of the surface charge intensity of the cancer cells by estimation comprises:

and placing the tumor to be detected in phosphate buffer solution as electrolyte, applying an external voltage to the three-electrode system, testing the transfer amount of electrons on the working electrode in the reaction process, calculating the level of the surface charge intensity of the cancer cell according to the established relation between the surface charge of the cancer cell and the instant response current, and realizing the detection of the surface charge intensity of the cancer cell.

7. The method for assessing the malignancy of tumor according to claim 1 or 2, wherein the carbon nanotube film has a thickness of 5 to 20 μm and an area of 0.3 to 0.5cm²。

8. The method for assessing malignancy of tumor according to claim 1 or 2, wherein the flexible polydimethylsiloxane substrate is prepared by: uniformly mixing polydimethylsiloxane and a curing agent according to a predetermined mass ratio, and heating and curing at 70-80 ℃ for 40-60min to obtain the composite material.

9. The method for assessing the malignancy of a tumor according to claim 1 or 2, wherein the volume of the electrolyte is 10-20mL, and the molar concentration of the phosphate buffer is 0.05-0.2mmol L^-1。

10. The method for assessing the malignancy of a tumor according to claim 1 or 2, wherein the linear sweep voltammetry has a sweep range of 0-3V and a sweep rate of 50-150mV s^-1。

Technical Field

The invention relates to the field of electrochemistry and cell electrophysiology, in particular to a method for evaluating the malignancy degree of a tumor based on the surface charge intensity of a cancer cell, namely a method for detecting the surface charge of the cell and biomedical application thereof, namely detecting the surface charge intensity of the cancer cell of a tumor patient and taking the surface charge intensity as an index for evaluating the malignancy degree of the tumor.

Background

Bioelectricity is an important part of all life activities. However, the electrical properties of animal somatic cells are still not fully understood. Over the past few decades, electrophysiological progress has shown that cell surface charge is an important attribute of cell properties and plays a crucial role in regulating cell function. As early as the 40's of the 20 th century, there were people guessing through some subtle behaviors of cancer cells that the electrical properties of the surface of cancer cells should be different from those of normal cells. Most cancer cells are negatively charged on their surface and produce surface potentials that affect surface activity, the concentration of ions on the cell membrane and thus, important cellular events such as cell adhesion, cellular uptake, intercellular communication, signal transduction, and protein transport.

To measure cell surface charge, a variety of methods have been developed, including electrostatic interaction, isoelectric equilibrium analysis, and electrophoresis. Electrostatic interaction methods are generally based on the electrostatic interaction chromatography (ESIC) technique, which uses charged ion exchange resins/molecules to interact with cells; the affinity of the interaction depends on the charge on the cell surface. Thus, based on the affinity between the cells and the resin, the relative cell surface charge can be assessed. Recently, in terms of measuring the affinity between Nanoprobes (NPs) and cells, Nanoprobes (NPs) having a certain charge have also been used for cell surface charge detection. However, this type of method does not provide direct surface charge measurement and is often time consuming. For the isoelectric equilibrium analysis method, cells are loaded onto a chromatographic column with a linear pH gradient and migrated at the appropriate voltage. The cell surface charge affects the isoelectric point to which the cell migrates after isoelectric equilibration. Although the zeta potential of a cell can be derived from the isoelectric point, this method requires a long time to achieve isoelectric balance. It is not suitable for in situ measurement of surface charge of single cells.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a more sensitive and more accurate universal cell surface charge test method aiming at the defects in the prior art, and provide possibility for the subsequent exploration of the influence of the cancer cell surface charge on the functions of the cancer cell surface charge and even clinical application.

According to the present invention, there is provided a method for assessing malignancy of a tumor based on intensity of surface charge of cancer cells, comprising:

a first step for detecting the intensity of the surface charge of the cancer cell by the established electrochemical device;

and a second step for using the detected surface charge intensity of the cancer cells as an index for evaluating malignancy of the tumor.

Preferably, the detection method adopted in the first step is a three-electrode system test method, and the three-electrode system adopted in the three-electrode system test method comprises a working electrode, a counter electrode and a reference electrode, wherein the working electrode comprises a flexible substrate and a carbon nanotube film which are sequentially arranged, the counter electrode is a platinum wire counter electrode, and the reference electrode is composed of metal silver covered with silver chloride and immersed in a potassium chloride solution, namely a silver/silver chloride electrode.

Preferably, the three-electrode system test method comprises:

fixing the pretreated carbon nanotube film on a flexible polydimethylsiloxane substrate to obtain a carbon nanotube film electrode;

connecting the obtained carbon nano tube film electrode to one end of a working electrode of an electrochemical workstation, connecting a platinum wire to one end of a counter electrode of the electrochemical workstation, and connecting a silver/silver chloride electrode to one end of a reference electrode of the electrochemical workstation.

Preferably, the detection method adopted in the first step is a three-electrode system test method, which comprises: and establishing the relation between the surface charge of the cancer cells and the instant response current, detecting the tumor to be detected in a three-electrode system, and calculating to obtain the intensity level of the surface charge of the cancer cells.

Preferably, establishing a relationship between cancer cell surface charge and immediate response current comprises:

taking phosphate buffer solution as electrolyte, immersing a working electrode, a counter electrode and a reference electrode of a three-electrode system into the electrolyte, and testing a blank curve by a linear sweep voltammetry method of an electrochemical workstation;

and sequentially adding cancer cells with different concentrations or different types into the electrolyte, and testing the change condition of the current under the same voltage by using a linear sweep voltammetry method of an electrochemical workstation.

Preferably, the step of detecting the tumor to be detected in the three-electrode system and obtaining the intensity level of the surface charge of the cancer cell by calculation comprises:

and placing the tumor to be detected in phosphate buffer solution as electrolyte, applying an external voltage to the three-electrode system, testing the transfer amount of electrons on the working electrode in the reaction process, calculating the level of the surface charge intensity of the cancer cell according to the established relation between the surface charge of the cancer cell and the instant response current, and realizing the detection of the surface charge intensity of the cancer cell.

Preferably, the carbon nanotube film has a thickness of 5-20 μm and an area of 0.3-0.5cm²。

Preferably, the flexible polydimethylsiloxane substrate is prepared by the following process: uniformly mixing polydimethylsiloxane and a curing agent according to a predetermined mass ratio, and heating and curing at 70-80 ℃ for 40-60min to obtain the composite material.

Preferably, the volume of the electrolyte is 10-20mL, and the molar concentration of the phosphate buffer solution is 0.05-0.2mmol L^-1。

Preferably, the scanning range of the linear sweep voltammetry is 0-3V, and the scanning speed is 50-150mVs^-1。

Compared with the prior art, the beneficial effects and originality of the invention are mainly embodied in the following aspects:

1. the cancer cell surface charge detection device designed by the invention greatly simplifies the structure of the device (most of the existing device structures are three-electrode systems) and completes the direct detection of cancer cells under the condition of not deviating from the physiological condition (most of the devices cannot complete the detection under the PBS condition); moreover, the relation between the surface charge of the cancer cells and the instant response current is established, and the intensity level of the surface charge of the cancer cells can be rapidly calculated. The invention realizes accurate, high-sensitivity and rapid detection of the surface charge of the cancer cell, and has great practical application value.

2. The invention analyzes the breast cancer by adopting a method of combining the surface charge intensity of the cancer cells with pathology, researches the value of the surface charge of the cancer cells in the aspect of evaluating the malignancy degree of the tumor, and better guides clinical treatment and predicts prognosis.

Drawings

A more complete understanding of the present invention, and the attendant advantages and features thereof, will be more readily understood by reference to the following detailed description when considered in conjunction with the accompanying drawings wherein:

fig. 1 schematically shows a general flowchart of a method for assessing malignancy of a tumor based on intensity of surface charge of cancer cells according to a preferred embodiment of the present invention.

Fig. 2 schematically shows a schematic diagram of the principle of a three-electrode system test method employed in the method for assessing malignancy of a tumor based on intensity of surface charge of cancer cells according to a preferred embodiment of the present invention.

FIG. 3 shows the cell surface charge amount of a human osteosarcoma cell line with a cell number of 80W; (degree of malignancy: U2OS < ZOS < SJSA-1)

FIG. 4 shows the cell surface charge amount at a lung cancer cell count of 80W; (degree of malignancy: A549> H460)

FIG. 5 cell surface charge at 80W for colorectal cancer cell count; (degree of malignancy: HCT116> HT29)

FIG. 6 shows the cell surface charge amount when the number of hepatoma cells is 80W; (degree of malignancy: HCCLM3> Hep-3B)

FIG. 7 cell surface charge at 80W of bladder cancer cells; (malignancy from top to bottom, from low to high)

It is to be noted, however, that the appended drawings illustrate rather than limit the invention. It is noted that the drawings representing structures may not be drawn to scale. Also, in the drawings, the same or similar elements are denoted by the same or similar reference numerals.

Detailed Description

In order that the present disclosure may be more clearly and readily understood, reference will now be made in detail to the present disclosure as illustrated in the accompanying drawings.

In the invention, the detection of the surface charge intensity of the cancer cells is realized through the established electrochemical device, and the detected surface charge intensity of the cancer cells is used as an index for evaluating the malignancy of the tumor.

< first embodiment >

Fig. 1 schematically shows a general flowchart of a method for assessing malignancy of a tumor based on intensity of surface charge of cancer cells according to a preferred embodiment of the present invention.

As shown in fig. 1, the method for evaluating malignancy of a tumor based on intensity of surface charge of cancer cells according to a preferred embodiment of the present invention includes:

first step S1: the detection of the surface charge intensity of the cancer cells is realized through the established electrochemical device;

second step S2: the detected surface charge intensity of the cancer cells is used as an index for evaluating the malignancy of the tumor.

The surface charge of the cancer cell can evaluate the energy metabolism level of the cancer cell (glycolysis process is the main reason of negative charge on the surface of the cancer cell), indirectly reflects the malignancy degree of the tumor (the glycolysis degree of the malignancy is enhanced), and can be used as an index for noninvasive evaluation of tumor biology. The invention relates to a method for retrospectively analyzing cancers such as breast cancer by combining the surface charge intensity of cancer cells and pathology, aiming at researching the value of the surface charge of the cancer cells in the aspect of evaluating the malignancy degree of tumors so as to better guide clinical treatment and predict prognosis.

< specific examples >

The detection method adopted in the first step is a three-electrode system test method. Specifically, fig. 2 schematically shows a schematic diagram of the principle of a three-electrode system test method employed in the method for assessing malignancy of a tumor based on intensity of surface charge of cancer cells according to a preferred embodiment of the present invention. As shown in fig. 2, the three-electrode system used in the three-electrode system test method includes a working electrode, a counter electrode, and a reference electrode, wherein the working electrode includes a flexible substrate and a carbon nanotube film sequentially disposed,the counter electrode was a platinum wire counter electrode and the reference electrode consisted of metallic silver covered with silver chloride immersed in a potassium chloride solution. Wherein the short arrow shows the electron e^-In the direction of flow.

For example, in specific operation, the relationship between the surface charge of cancer cells and the instant response current can be first established, and the tumor to be detected is detected in a three-electrode system and the intensity level of the surface charge of cancer cells is obtained by calculation. Specifically, the tumor to be detected can be placed in phosphate buffer solution as electrolyte, an external voltage is applied to the three-electrode system, the transfer amount of electrons on the working electrode in the reaction process is tested, the intensity level of the surface charge of the cancer cell is calculated according to the established relationship between the surface charge of the cancer cell and the instant response current, and the detection of the surface charge intensity of the cancer cell is realized.

Compared with the prior art, the invention can detect the surface charges of different types of cancer cells through a three-electrode system, and further evaluate the malignancy degree of different tumors.

Preferably, the flexible substrate of the working electrode is polydimethylsiloxane and has a thickness of 0.8-1.5mm, preferably 1.0 mm. Preferably, the thickness of the carbon nanotube film is 5-20 μm, preferably 10 μm, and the area of the carbon nanotube film is 0.3-0.5cm²Preferably 0.4cm²。

Specifically, for example, the three-electrode system test method includes:

(a) fixing the pretreated carbon nanotube film with a certain size on a flexible polydimethylsiloxane substrate to obtain a carbon nanotube film electrode;

(b) connecting the carbon nano tube film electrode obtained in the step (a) to one end of a working electrode of an electrochemical workstation, connecting a platinum wire to one end of a counter electrode of the electrochemical workstation, and connecting a silver/silver chloride electrode to one end of a reference electrode of the electrochemical workstation;

(c) immersing the three electrodes of step (b) in a volume of Phosphate Buffered Saline (PBS) as an electrolyte, and testing the blank curve by Linear Sweep Voltammetry (LSV) of an electrochemical workstation;

(d) and sequentially adding cancer cells with different concentrations or different types into the electrolyte, and testing the change condition of the current under the same voltage through an LSV curve of an electrochemical workstation.

Specifically, for example, in step (a), the pretreatment of the carbon nanotube film is specifically: making the carbon nano tube film at the molar concentration of 8-12mol L^-1Soaking in nitric acid for 8-12h, washing with deionized water, soaking in deionized water for 8-12h, washing with deionized water, and adsorbing water on the surface of the carbon nanotube film with dust-free paper.

Specifically, for example, in the step (a), the carbon nanotube film has a thickness of 5 to 20 μm and an area of 0.3 to 0.5cm²。

Specifically, for example, in step (a), the flexible polydimethylsiloxane substrate is prepared by the following process: uniformly mixing polydimethylsiloxane and a curing agent according to the mass ratio of (5-10) to 1, and heating and curing at 70-80 ℃ for 40-60min to obtain the composite material.

Specifically, for example, in step (c), the volume of the electrolyte is 10-20mL, and the molar concentration of PBS is 0.05-0.2mmol L^-1。

Specifically, for example, in steps (c) and (d), the LSV is scanned in the range of 0-3V and at a scanning speed of 50-150mV s^-1。

Specifically, for example, in step (d), the types of cancer cells include: 10 kinds of cancer cells, such as breast cancer cell, lung cancer cell, liver cancer cell, etc. The concentration of the cancer cells is 20W/mL.

Specifically, for example, in step (d), primary extracted cancer cells of tumor tissues of different breast cancer patients are collected as samples; the concentration of cancer cells was 20W/mL.

Wherein, the volume of PBS is preferably 10-20mL, preferably 15mL, and the molar concentration of PBS is 0.05-0.2 mmols L^-1Preferably 0.067mmo L^-1. Preferably, the sweep range of the linear sweep voltammetry is 0-3V, preferably 0-1.6V, and the sweep rate is 50-150mV s^-1Preferably 100mV s^-1。

The method detects the surface charge of the cells by a three-electrode test method, takes the carbon nano tube film as a working electrode, a platinum wire electrode as a counter electrode, a silver/silver chloride electrode as a reference electrode, PBS (phosphate buffer solution) as electrolyte and LSV (localized surface plasmon resonance) of an electrochemical workstation as a test method, and detects the change of response current after the electrolyte is added into different cancer cells. The invention drives the cancer cells with charges on the surface to migrate by applying an electric field, so as to cause the charge accumulation on the surface of the electrode, and evaluates the malignancy degree of the tumor according to the change of response current after different cancer cells are added.

The device can be used for detecting the charge carrying quantity of macromolecules, bacteria, cells and the like, particularly, after cancer cells are primarily extracted from tumor tissues of a breast cancer patient, the cancer cells are added into a detection chamber of the device (PBS buffer solution is added in advance indoors to ensure the physiological activity in the cancer cell detection process), after an external voltage is applied, the detection of the surface charge strength of the cancer cells is realized by testing the electron transfer quantity in the reaction process on a working electrode, and experimental results show that the two-electrode system electrochemical device can realize accurate and rapid detection of the surface charge of the cancer cells.

Example 1

The detection device is prepared by the following steps:

(1) the pretreatment of the carbon nanotube film comprises the following specific steps: making the carbon nano tube film at the molar concentration of 8-12mol L^-1Soaking in nitric acid for 8-12h, washing with deionized water, soaking in deionized water for 8-12h, washing with deionized water, and adsorbing water on the surface of the carbon nanotube film with dust-free paper.

(2) The carbon nanotube film has a thickness of 5-20 μm and an area of 0.3-0.5cm²。

(3) The preparation process of the flexible polydimethylsiloxane substrate comprises the following steps: uniformly mixing polydimethylsiloxane and a curing agent according to the mass ratio of (5-10) to 1, preferably 10:1, and heating and curing at 70-80 ℃ for 40-60min to obtain the polydimethylsiloxane-containing curing agent. The polydimethylsiloxane and the curing agent are both purchased from Dow Corning company and used in combination, and the type of the curing agent is 184.

(4) The volume of the electrolyte is 10-20mL, and the molar concentration of the PBS is 0.05-0.2mmol L-1.

(5) Setting LSV scanning range to 0-3V and scanning speed to 50-150mV s^-1。

(6) And (3) sequentially adding samples to be detected with the concentration of 20W/mL, namely different types of cancer cells such as MDA-MB-231, K562, A549 and the like into the electrolyte, and testing the current change condition through the LSV curve test of the electrochemical workstation under the same scanning range and scanning speed as those in the step (5).

The experimental results prove that the three-electrode system cancer cell surface charge detection device designed and prepared by the invention successfully realizes accurate and rapid detection of cancer cells and has great advantages.

Example 2

The detection device is prepared by the following steps:

(1) the pretreatment of the carbon nanotube film comprises the following specific steps: making the carbon nano tube film at the molar concentration of 8-12mol L^-1Soaking in nitric acid for 8-12h, washing with deionized water, soaking in deionized water for 8-12h, washing with deionized water, and adsorbing water on the surface of the carbon nanotube film with dust-free paper.

(2) The carbon nanotube film has a thickness of 5-20 μm and an area of 0.3-0.5cm²。

(3) The preparation process of the flexible polydimethylsiloxane substrate comprises the following steps: uniformly mixing polydimethylsiloxane and a curing agent according to the mass ratio of (5-10) to 1, preferably 10:1, and heating and curing at 70-80 ℃ for 40-60min to obtain the polydimethylsiloxane-containing curing agent. The polydimethylsiloxane and the curing agent are both purchased from Dow Corning company and used in combination, and the type of the curing agent is 184.

(4) The volume of the electrolyte is 10-20mL, and the molar concentration of the PBS is 0.05-0.2mmol L-1.

(5) Setting LSV scanning range to 0-3V and scanning speed to 50-150mV s^-1。

(6) And (3) adding the breast cancer patient A with the concentration of 20W/mL and the primary extracted cancer cells of the tumor tissue into the electrolyte in sequence, and testing the current change condition through the LSV curve test of the electrochemical workstation under the same scanning range and scanning speed as those in the step (5).

(7) Pathological analysis was performed on the same breast cancer patient sample as in step (6), the tumor stroma ratio was evaluated, and the lesions were divided into two groups, i.e., a stroma-rich group (tumor stroma ratio ≦ 50%) and a stroma-poor group (> 50%) according to the tumor stroma ratio. Grading the lesions according to Scarff-Bloom-Richardson grading, wherein 8-9 are graded as 3, poor differentiation and high malignancy; grade 2, moderate differentiation and moderate malignancy are divided into 6-7; grade 1 in 3-5, good differentiation, low grade malignancy; scores of 3 or less were considered as grade-unknown and could not be evaluated.

(8) And (4) statistically processing the data obtained in the step (7), and analyzing the data by using SPSS 19.0 statistical software. The measurement data conforming to normal distribution is expressed by mean plus or minus standard deviation (x plus or minus s), and two samples t test is adopted for comparison among 2 groups; the abnormally distributed measures were expressed as median M (P25, P75) and the Mann-Whitney U test was used for the 2-group comparisons. The count data are expressed in units (%) and the 2-group comparison was performed by the X2 test. Spearman correlation was used to analyze the correlation of tumor maximum diameter, tumor-to-stroma ratio, and tumor grade to the intensity of the surface charge on cancer cells. And analyzing the relation between the tumor charge intensity value and the tumor characteristics by adopting multiple linear regression and stepwise regression. P < 0.05 is statistically significant.

Example 3

The detection device is prepared by the following steps:

(1) the pretreatment of the carbon nanotube film comprises the following specific steps: making the carbon nano tube film at the molar concentration of 8-12mol L^-1Soaking in nitric acid for 8-12h, washing with deionized water, soaking in deionized water for 8-12h, washing with deionized water, and adsorbing water on the surface of the carbon nanotube film with dust-free paper.

(2) The carbon nanotube film has a thickness of 5-20 μm and an area of 0.3-0.5cm²。

(3) The preparation process of the flexible polydimethylsiloxane substrate comprises the following steps: uniformly mixing polydimethylsiloxane and a curing agent according to the mass ratio of (5-10) to 1, preferably 10:1, and heating and curing at 70-80 ℃ for 40-60min to obtain the polydimethylsiloxane-containing curing agent. The polydimethylsiloxane and the curing agent are both purchased from Dow Corning company and used in combination, and the type of the curing agent is 184.

(4) The volume of the electrolyte is 10-20mL, and the molar concentration of the PBS is 0.05-0.2mmol L-1.

(5) Setting LSV scanning range to 0-3V and scanning speed to 50-150mV s^-1。

(6) And (3) adding the breast cancer patient B with the concentration of 20W/mL and the primary extracted cancer cells of the tumor tissue into the electrolyte in sequence, and testing the current change condition through the LSV curve test of the electrochemical workstation under the same scanning range and scanning speed as those in the step (5).

(7) Pathological analysis was performed on the same breast cancer patient sample as in step (6), the tumor stroma ratio was evaluated, and the lesions were divided into two groups, i.e., a stroma-rich group (tumor stroma ratio ≦ 50%) and a stroma-poor group (> 50%) according to the tumor stroma ratio. Grading the lesions according to Scarff-Bloom-Richardson grading, wherein 8-9 are graded as 3, poor differentiation and high malignancy; grade 2, moderate differentiation and moderate malignancy are divided into 6-7; grade 1 in 3-5, good differentiation, low grade malignancy; scores of 3 or less were considered as grade-unknown and could not be evaluated.

(8) And (4) statistically processing the data obtained in the step (7), and analyzing the data by using SPSS 19.0 statistical software. The measurement data conforming to normal distribution is expressed by mean plus or minus standard deviation (x plus or minus s), and two samples are adopted for comparison among 2 groups for t test; the abnormally distributed measures were expressed as median M (P25, P75) and the Mann-Whitney U test was used for the 2-group comparisons. The count data are expressed in units (%) and the 2-group comparison was performed by the X2 test. Spearman correlation was used to analyze the correlation of tumor maximum diameter, tumor-to-stroma ratio, and tumor grade to the intensity of the surface charge on cancer cells. And analyzing the relation between the tumor charge intensity value and the tumor characteristics by adopting multiple linear regression and stepwise regression. P < 0.05 is statistically significant.

Example 4

The detection device is prepared by the following steps:

(1) the pretreatment of the carbon nanotube film comprises the following specific steps: forming a carbon nanotube film onThe molar concentration is 8-12mol L^-1Soaking in nitric acid for 8-12h, washing with deionized water, soaking in deionized water for 8-12h, washing with deionized water, and adsorbing water on the surface of the carbon nanotube film with dust-free paper.

(2) The carbon nanotube film has a thickness of 5-20 μm and an area of 0.3-0.5cm²。

(3) The preparation process of the flexible polydimethylsiloxane substrate comprises the following steps: uniformly mixing polydimethylsiloxane and a curing agent according to the mass ratio of (5-10) to 1, preferably 10:1, and heating and curing at 70-80 ℃ for 40-60min to obtain the polydimethylsiloxane-containing curing agent. The polydimethylsiloxane and the curing agent are both purchased from Dow Corning company and used in combination, and the type of the curing agent is 184.

(4) The volume of the electrolyte is 10-20mL, and the molar concentration of the PBS is 0.05-0.2mmol L-1.

(5) Setting LSV scanning range to 0-3V and scanning speed to 50-150mV s^-1。

(6) And (3) adding the breast cancer patient C with the concentration of 20W/mL and the primary extracted cancer cells of the tumor tissue into the electrolyte in sequence, and testing the current change condition through the LSV curve test of the electrochemical workstation under the same scanning range and scanning speed as those in the step (5).

(7) Pathological analysis was performed on the same breast cancer patient sample as in step (6), the tumor stroma ratio was evaluated, and the lesions were divided into two groups, i.e., a stroma-rich group (tumor stroma ratio ≦ 50%) and a stroma-poor group (> 50%) according to the tumor stroma ratio. Grading the lesions according to Scarff-Bloom-Richardson grading, wherein 8-9 are graded as 3, poor differentiation and high malignancy; grade 2, moderate differentiation and moderate malignancy are divided into 6-7; grade 1 in 3-5, good differentiation, low grade malignancy; scores of 3 or less were considered as grade-unknown and could not be evaluated.

(8) And (4) statistically processing the data obtained in the step (7), and analyzing the data by using SPSS 19.0 statistical software. The measurement data conforming to normal distribution is expressed by mean plus or minus standard deviation (x plus or minus s), and two samples are adopted for comparison among 2 groups for t test; the abnormally distributed measures were expressed as median M (P25, P75) and the Mann-Whitney U test was used for the 2-group comparisons. The count data are expressed in units (%) and the 2-group comparison was performed by the X2 test. Spearman correlation was used to analyze the correlation of tumor maximum diameter, tumor-to-stroma ratio, and tumor grade to the intensity of the surface charge on cancer cells. And analyzing the relation between the tumor charge intensity value and the tumor characteristics by adopting multiple linear regression and stepwise regression. P < 0.05 is statistically significant.

Compared with the prior art, the beneficial effects and originality of the invention are mainly embodied in the following aspects:

1. the cancer cell surface charge detection device designed by the invention greatly simplifies the structure of the device (most of the existing device structures are three-electrode systems) and completes the direct detection of cancer cells under the condition of not deviating from the physiological condition (most of the devices cannot complete the detection under the PBS condition); moreover, the relation between the surface charge of the cancer cells and the instant response current is established, and the intensity level of the surface charge of the cancer cells can be rapidly calculated. The invention realizes accurate, high-sensitivity and rapid detection of the surface charge of the cancer cell, and has great practical application value.

2. The invention analyzes the breast cancer by adopting a method of combining the surface charge intensity of the cancer cells with pathology, researches the value of the surface charge of the cancer cells in the aspect of evaluating the malignancy degree of the tumor, and better guides clinical treatment and predicts prognosis.

It should be noted that the terms "first", "second", "third", and the like in the description are used for distinguishing various components, elements, steps, and the like in the description, and are not used for indicating a logical relationship or a sequential relationship between the various components, elements, steps, and the like, unless otherwise specified.

It is to be understood that while the present invention has been described in conjunction with the preferred embodiments thereof, it is not intended to limit the invention to those embodiments. It will be apparent to those skilled in the art from this disclosure that many changes and modifications can be made, or equivalents modified, in the embodiments of the invention without departing from the scope of the invention. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention are still within the scope of the protection of the technical solution of the present invention, unless the contents of the technical solution of the present invention are departed.