CN113109243A

CN113109243A - Sensor for detecting bioelectric current between cell and metal and detection method thereof

Info

Publication number: CN113109243A
Application number: CN202110398786.7A
Authority: CN
Inventors: 刘昭; 张继; 李斌; 袁章琴; 王佳媛; 丁玲; 李正逗
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2021-04-14
Filing date: 2021-04-14
Publication date: 2021-07-13
Anticipated expiration: 2041-04-14
Also published as: CN113109243B

Abstract

The invention discloses a sensor for detecting bioelectric current between cells and intermetallics and a detection method thereof, wherein the sensor comprises a generating device and a receiving device, the generating device comprises two identical containers, wherein one container is filled with cell culture solution and cells to be tested, and the other container is only filled with cell culture solution; the bottom of the container is paved with metal plates which are connected with the conduction plate, and the top end of the conduction plate is higher than the highest liquid level of the culture solution; connecting wires are respectively inserted into the cell culture solution of the two containers; the receiving device comprises a data processor, the data processor is connected with the top end of the conducting plate through a conducting wire, and the data processor is connected with an upper computer. The invention utilizes the principle of a primary battery to enable two containers to form a closed loop, and records the potential difference generated by the electrochemical reaction of the cells to be tested on the metal plate through the receiving device, thereby judging the rejection degree of the test cells on the metal and evaluating the condition of the metal material implanted into a human body.

Description

Sensor for detecting bioelectric current between cell and metal and detection method thereof

Technical Field

The invention relates to the technical field of biosensors, in particular to a current sensor for detecting a cell and intermetallic bioelectric current and a detection method thereof.

Background

The sensor is a device which senses detection signals by using a recognition element and converts the detection signals into other usable signals by a conversion element according to a certain rule, and consists of the recognition element, the conversion element and electronic equipment, common sensors comprise a biosensor, a chemical sensor, a gas sensor and a non-gas sensor which are mainly used for analysis and detection, the biosensor takes immobilized enzymes (glucose oxidase membrane, lactate oxidase membrane and the like) and other bioactive substances as biomolecule recognition bases, immobilized oxidase as the recognition element, a hydrogen peroxide base electrode as a transducer, and an analysis tool or a system consisting of a proper physicochemical transducer (such as an oxygen electrode, a photosensitive tube, a field effect tube, a piezoelectric crystal and the like) and a signal amplification device to sense the biological substances and convert the concentration of the biological substances into electric signals for detection, the detecting instrument converts the concentration of the measured substance into digital signals through a proper physicochemical transducer and a signal amplifying device. Patent CN202020774730.8 discloses an immobilized enzyme electrode and an immobilized enzyme sensor, the utility model uses a polycarbonate microporous membrane as a carrier membrane, a glucose oxidase electrode and/or a lactate oxidase electrode as a detection electrode, and a hydrogen peroxide electrode as a basic electrode, and constructs an immobilized enzyme sensor, and the components are measured before the separation of the sample by utilizing the high selectivity of the current enzyme electrode. Patent CN201310531766 discloses a gesture recognition device based on arm muscle current detection and motion sensor, which aims at the defects of unnatural and indirect information input in the traditional human-computer interaction interface, and analyzes the corresponding relationship between arm muscle bioelectric current and gestures by combining with the bioelectronic technology to prepare a novel human-computer interaction interface (HCI) control input device. With the development of science and technology, biomedical materials are more and more popular in application, but limited by technology, some biomedical materials (such as metal stents and artificial joints) are repelled by human bodies after being implanted into human bodies, a series of electrochemical reactions can occur between human body cells and the implanted materials, the implanted materials are damaged due to the biological corrosion phenomenon, and various human body complications are caused. The traditional biological corrosion information is intermittent and accidental, and the sensitivity is low.

Disclosure of Invention

The invention aims to provide a current sensor for detecting bioelectric current between cells and metals and a detection method thereof.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a current sensor for detecting bioelectric current between cells and metal comprises a generating device and a receiving device, wherein the generating device comprises two identical containers, one container is filled with cell culture solution and cells to be tested, and the other container is only filled with cell culture solution; the bottom of the container is paved with metal plates which are connected with the conduction plate, and the top end of the conduction plate is higher than the highest liquid level of the culture solution; two ends of the connecting lead are respectively inserted into the cell culture solution of the two containers; the receiving device comprises a data processor, the data processor is connected with the top end of the conducting plate through a conducting wire, and the data processor is connected with an upper computer.

Preferably, the metal plate is a medical titanium metal plate or a medical stainless steel metal plate with the purity of 99.6%.

Preferably, the thickness of the metal plate is 0.08-0.12 mm.

Preferably, the cell to be tested is a macrophage, an osteoclast or a monocyte.

A method for detecting the bioelectric current between cells and intermetallics, which is implemented by using the current sensor for detecting the bioelectric current between cells and intermetallics, comprises the following steps:

step one, planting cells in a container of the current sensor, placing the current sensor in an incubator, and placing CO in the incubator₂The mass percentage concentration of the gas is 5%, the humidity is 25-65%, the culture solution is replaced every 3-4 days after the culture is carried out for 14-21 days at 37 ℃, and a connecting wire is fixed when the culture solution is replaced;

and secondly, recording the potential value of the culture solution in the container once every 5-8 minutes through a data processor, storing the potential value in an upper computer, recording the period, the strength and the rule of biological corrosion of the cells to be tested and the metal plate in the container, analyzing the type of electrochemical reaction, analyzing the mechanism of biological corrosion of the cells to be tested and the metal plate, judging the rejection degree of the cells to corresponding metal, and further evaluating the condition of the metal material after being implanted into a human body.

The invention utilizes the principle of a galvanic cell to plant cells to be tested in one container, connects cell culture liquids in the two containers through a connecting lead to form a closed loop, records potential difference generated by the cells to be tested in electrochemical reaction on a metal plate at the bottom of the container through a receiving device, further judges the rejection degree of the test cells to metal, and evaluates the condition of a metal material after being implanted into a human body.

Compared with the prior art, the invention can be prepared by adopting conventional experimental materials, has the advantages of low cost and high practicability, and effectively solves the defect that the result can only be seen in the traditional biological corrosion research by continuously recording the electric signals. The use of the data processor and the upper computer greatly improves the measuring efficiency, not only improves the measuring precision, but also reduces the error, saves the manpower, and fundamentally improves the credibility of researching the biological cells to the metal corrosion electric signals.

Drawings

FIG. 1 is a schematic diagram of a current sensor for detecting bioelectric current between cells and metals;

FIG. 2 is 21 days electrorheological experimental data for macrophage bioerosion in example 1;

FIG. 3 is the data of the 24-hour current change experiment for macrophage bioerosion in example 1;

FIG. 4 is a schematic diagram of macrophage corrosion on metal surfaces;

FIG. 5a is a metal surface image before macrophage implantation by white light interferometer detection;

FIG. 5b is a metal surface image of macrophage cells implanted 21 days after detection by white light interferometer.

Detailed Description

The present invention will be described in detail below with reference to the accompanying drawings.

A current sensor for detecting a bioelectric current between a cell and an metal as shown in fig. 1, comprising: the device comprises a generating device and a receiving device, wherein the generating device comprises two identical containers, wherein one container is filled with cell culture solution and cells to be tested, and the other container is filled with only cell culture solution; the bottom of the container is paved with metal plates which are connected with the conduction plate, and the top end of the conduction plate is higher than the highest liquid level of the culture solution; two ends of the connecting lead are respectively inserted into the cell culture solution of the two containers; the receiving device comprises a data processor, the data processor is connected with the top end of the conducting plate through a conducting wire, and the data processor is connected with an upper computer.

The metal plate is a medical titanium metal plate or a medical stainless steel metal plate with the purity of 99.6 percent.

The thickness of the metal plate is 0.08-0.12 mm.

The cells to be tested may be macrophages, osteoclasts or monocytes, the present invention being exemplified by seeded macrophages.

Example 1

step one, THP-1 cells and PMA growth factors are added into a container of the current sensor to culture macrophages, and when the concentration of the macrophages reaches 1 x 10⁴Per cm²Then, the generating device is placed in an incubator, and CO is in the incubator₂The mass percentage concentration of the gas is 5%, the humidity is 25%, the culture solution is replaced every 3-4 days after the culture is carried out for 14 days at 37 ℃, and a connecting wire is fixed when the culture solution is replaced;

macrophages participate in nonspecific defense (innate immunity) and specific defense (cellular immunity) in vertebrates, and can phagocytose and corrode foreign substances invading into human bodies, so that after the macrophages contact the metal plate, the metal plate is corroded, and corresponding electrochemical signals are generated.

And secondly, recording the potential value of the culture solution in the container once every 5 minutes through a data processor, storing the potential value in an upper computer, recording the period, the strength and the rule of the biological corrosion of the macrophages and the metal plate in the container, analyzing the type of the electrochemical reaction, analyzing the mechanism of the biological corrosion of the cells to be tested and the metal plate, judging the rejection degree of the macrophages to corresponding metal, and further evaluating the condition of the metal material implanted into a human body.

Comparative example 1

THP-1 cells were added to a vessel of a current sensor for detecting bioelectric current between cells and an intermetallic compound, and the cells were cultured in the same manner as in the first step of example 1, and the potential value of the culture solution was recorded in the same manner as in the second step.

Comparative example 2

And (4) not planting cells in a container of a current sensor for detecting the bioelectric current between the cells and the metal, and recording the potential value of the culture solution according to the method of the step two.

The electrical signals generated when the cells and the metal plate are subjected to the biological corrosion are respectively tested by the experimental method, each embodiment/comparative example is repeated three times and averaged, and the change curve of the signals generated by the biological corrosion is drawn, so that the current change trend of the cells to be tested within 21 days (shown in figure 2) and 24 hours (shown in figure 3) after the cells to be tested are corroded with the metal in the sensor disclosed by the invention can be obtained.

According to the image curve, the macrophage with rejection function can generate biological corrosion effect with metal, when the macrophage contacts with the metal, the macrophage can release hydrogen ions on the contact surface and combine with chloride ions to form an acid environment, so that the metal surface is corroded (as shown in figure 4), and according to the obtained curve, the electrochemical reaction between cells and metal becomes stronger along with the increase of the cells, and a certain corrosion mark is left on the metal surface.

To further verify the corrosion effect, the roughness of the surface of the macrophage-seeded metal material was tested by a white light interferometer, which verifies the occurrence of corrosion signs (as shown in fig. 5). Fig. 5a is the metal surface before macrophage seeding, and fig. 5b is the metal surface 21 days after macrophage seeding.

The detection result of the white light interferometer is consistent with the analysis result of the sensor, so that the biological corrosion phenomenon of the metal can be evaluated within a certain time after the metal is implanted into a human body due to the rejection of immune cells such as macrophages, the hidden danger that the surface material of the metal is damaged exists, and the risk that the structure of the implanted material is damaged and harmful metal nano particles are generated exists.

Claims

1. A current sensor for detecting a bioelectric current between a cell and an metal, comprising: the device comprises a generating device and a receiving device, wherein the generating device comprises two identical containers, wherein one container is filled with cell culture solution and cells to be tested, and the other container is filled with only cell culture solution; the bottom of the container is paved with metal plates which are connected with the conduction plate, and the top end of the conduction plate is higher than the highest liquid level of the culture solution; two ends of the connecting lead are respectively inserted into the cell culture solution of the two containers; the receiving device comprises a data processor, the data processor is connected with the top end of the conducting plate through a conducting wire, and the data processor is connected with an upper computer.

2. The current sensor for detecting a bioelectric current between a cell and an metal according to claim 1, wherein: the metal plate is a medical titanium metal plate or a medical stainless steel metal plate with the purity of 99.6 percent.

3. The current sensor for detecting a bioelectric current between a cell and an metal according to claim 1 or 2, wherein: the thickness of the metal plate is 0.08-0.12 mm.

4. The current sensor for detecting a bioelectric current between a cell and an metal according to claim 3, wherein: the cells to be tested are macrophages, osteoclasts or monocytes.

5. A method for detecting a cell-to-metal bioelectric current, which is performed by using the current sensor for detecting a cell-to-metal bioelectric current according to any one of claims 1 to 4, characterized in that: the method comprises the following steps:

step one, planting cells in a container of the current sensor, and placing the current sensor in an incubator in which CO is arranged₂The mass percentage concentration of the gas is 5%, the humidity is 25-65%, the culture solution is replaced every 3-4 days after the culture is carried out for 14-21 days at 37 ℃, and a connecting wire is fixed when the culture solution is replaced;