CN115166007B - Cell potential non-contact detection device - Google Patents

Abstract

The invention discloses a cell potential non-contact detection device. The device includes a non-contact layer, a PCB base electrode, a signal transfer interface, a cell culture dish, a multi-channel signal processing circuit system board and an upper computer. The non-contact layer is added on the basis of the existing extracellular potential sensor, and the non-contact detection of the extracellular potential can be realized by reasonably selecting the material and thickness of the non-contact layer. The device improves the coupling between cells and electrodes, increases the flexibility of electrode size and material selection, simplifies electrode production, is easy to replace, and is convenient for detection.

Description

A non-contact detection device for cell potential

technical field

The invention relates to an extracellular potential detection device, in particular to a non-contact detection device for cell potential.

technical background

Cells are the basic unit of the structure and function of organisms. The measurement of cell electrical signals can enable us to understand the survival and living environment of cells. It is an important means of studying the physiological characteristics of cells and an important guide for clinical drug development. Extracellular detection is usually measured using a microelectrode array (MEA). This method has many advantages, such as less damage to cells, and the detection results of multiple cell potentials can be output in parallel. The MEA-based cell potential measurement device has been pursuing higher signal-to-noise ratio and higher integration. On the one hand, a biocompatible medium needs to be attached to the electrodes to increase the adhesion between cells and electrodes; on the other hand, MEA, signal amplification and signal processing circuits all tend to realize their functions on the same chip. In the existing MEA, the cells are usually cultured directly on the surface of the electrode array, the cells grow adherently on the electrode surface, and the signal enters the detection system through the coupling between the cell membrane and the metal electrode. However, this kind of direct contact detection between cells and electrodes usually requires careful consideration of the material, size, spacing and other conditions of the electrode array, and requires high coupling between cells and electrodes. In general, the microelectrode array detection method, the direct contact between the cells and the electrodes, has high requirements on the electrodes, and requires complex processes to maintain the biocompatibility of the electrodes and the adhesion between the cells and the electrodes, and because of the integration with the back-end chip Not easy to replace. For this reason, there is an urgent need for a technology to overcome the above problems.

Contents of the invention

The purpose of the present invention is to propose a non-contact detection device for cell potential in view of the problems that the electrode manufacturing process is complicated and difficult to replace in the existing cell potential microelectrode array detection device, and its technical scheme is as follows:

A non-contact detection device for cell potential, characterized in that it includes a non-contact layer, a PCB base electrode, a signal transfer interface, a cell culture dish, a multi-channel signal processing circuit system board, and an upper computer; the non-contact layer has a thickness less than The 10 μm PDMS is processed on the PCB base electrode by spin coating; the PCB base electrode is connected to the cell culture dish through an undercut; the PCB base electrode is eight 50-200 μm in diameter, The center distance is 100-400 μm, and the material is a circular electrode made of gold, which is connected to the multi-channel signal processing circuit system board through the signal transfer interface; the multi-channel signal processing circuit system board is composed of a high input impedance sensor, an AD module and an MCU, and The data is transmitted to the host computer through the serial port of the MCU; the host computer is responsible for the display, processing and storage of the data.

Further, the non-contact layer is PDMS with a thickness of less than 10 μm, which is non-biologically toxic, and is evenly coated on the PCB base electrode through a glue spreader. The manufacturing steps are as follows:

(1) Use the PCB base electrode plate as the spin-coating base, and use the standard cleaning process to clean the surface;

(2) PDMS and PDMS coagulant are configured as a mixed solution in a ratio of 10:1;

(3) the mixed solution is removed through vacuum pumping;

(4) Put the extracellular potential sensor on the homogenizer, and spin-coat the mixed solution onto the surface of the substrate at a speed of 2000r/s;

(5) Put the spin-coated PCB base electrode into an oven at 80° C. for 2 hours for heating and curing.

Further, the eight electrodes of the PCB base electrode can be arranged in a square, rectangular or circular structure.

Further, one of the eight base electrodes of the PCB can be used as a reference electrode.

Further, the input impedance of the high input impedance sensor is no less than 100GΩ, the input capacitance is no more than 20pF, and the noise within the bandwidth of 1Hz-1kHz is no more than 1μV.

Further, the host computer is written with Qt program, receives the signal from the multi-channel signal processing circuit system board through the serial port, and uses high-pass, low-pass, notch filter algorithm to perform real-time digital noise reduction and filtering processing on the signal, and performs real-time display and storage.

Compared with the existing devices, the present invention proposes to add an additional non-contact layer on the basis of the multi-electrode array detection device to realize non-contact detection. By rationally selecting the material and thickness of the non-contact layer, the real-time detection of extracellular cell electrical signals can be realized. detection. The device improves the coupling between the cells and the electrodes, has more flexible selection of the size and material of the electrodes, simplifies the production of the electrodes, is easy to replace, and is convenient for detection.

Description of drawings

Fig. 1 is a schematic diagram of the non-contact detection device for cell potential of the present invention.

Figure 2 is the PCB substrate electrode layout.

Fig. 3 is a realization block diagram of the multi-channel signal processing circuit system board.

Figure 4 is the measurement noise baseline of the blank medium control group.

Fig. 5 is a waveform diagram of sporadic neuron action potentials detected by the device under normal culture conditions.

Fig. 6 is a signal diagram of neuron epileptic discharge detected by the device without induction of calcium and magnesium ions.

Figure 7 is a typical waveform diagram of cardiomyocytes.

Fig. 8 is a waveform diagram of the extracellular potential of cardiomyocytes detected by the device.

FIG. 9 is a partially enlarged view of FIG. 8 .

Detailed ways

The present invention will be described in further detail below in conjunction with the accompanying drawings and specific examples, but the present invention is not limited.

The invention discloses a cell potential non-contact detection device, as shown in Figure 1, comprising a non-contact layer, a PCB base electrode, a signal transfer interface, a cell culture dish, a multi-channel signal processing circuit system board and a host computer; The non-contact layer is PDMS with a thickness less than 10 μm, which is processed on the PCB base electrode by spin coating; the PCB base electrode is connected to the cell culture dish by an undercut; the PCB base electrode is Eight circular electrodes made of gold are connected to the multi-channel signal processing circuit system board through the signal transfer interface; The data is transmitted to the upper computer; the upper computer is responsible for the display, processing and storage of data.

Further, the non-contact layer is non-biologically toxic and is PDMS with a thickness of less than 10 μm, which is uniformly coated on the PCB base electrode through a glue spreader, and its manufacturing steps are as follows:

(1) Use the PCB base electrode plate as the spin-coating base, and use the standard cleaning process to clean the surface;

(2) PDMS and PDMS coagulant are configured as a mixed solution in a ratio of 10:1;

(3) the mixed solution is removed through vacuum pumping;

(4) Put the extracellular potential sensor on the homogenizer, and spin-coat the mixed solution onto the surface of the substrate at a speed of 2000r/s;

(5) Put the spin-coated PCB base electrode into an oven at 80° C. for 2 hours for heating and curing.

Among them, the PCB base electrode layout is shown in Figure 2. On the PCB base board, eight circular gold electrodes are formed on the metal disc in the center of the PCB through the immersion gold process. The diameter of a single electrode is 152.4 μm, and the center-to-center spacing of the electrodes is 381 μm. , eight electrodes are arranged in a square structure. The electrodes are connected to the pin header transfer interface through metal leads. Considering the symmetry of the eight channels, the leads of the eight channels are routed with equal lengths.

Further, the extracellular potential signal detected by the PCB base electrode is transmitted to the multi-channel signal processing circuit system board through the signal transfer interface, buffered by the high input impedance sensor, and then input into the AD module, and controlled by the MCU. The signal is amplified with controllable gain and digital-to-analog conversion, and the processed cell signal is transmitted to the host computer on the computer side through the serial port.

Among them, the high input impedance sensor is a chip independently developed by the laboratory, with an input impedance of 150GΩ, an input capacitance of 15pF, and a noise of 0.9μV within a bandwidth of 1Hz-1kHz, which is used to efficiently collect and buffer the extracellular potential. The AD module is built with the ADS1298 chip, which can realize 8-channel high-precision 4000Hz synchronous sampling. The chip has a built-in programmable gain amplifier, which can be set to 1, 2, 3, 4, 6, 8 or 12 times with a total of 7 gain settings by using the MCU module for control. The MCU is built with MSP430 single-chip microcomputer, communicates with the host computer on the computer side through the serial port, and can simultaneously control the gain inside the ADS1298 chip, the opening and closing of the digital-to-analog conversion module, and data transmission.

Further, the host computer is programmed with Qt program, receives the signal from the multi-channel signal processing circuit system board through the serial port, and uses 1Hz high-pass, 2kHz low-pass, 50Hz notch filter algorithm to perform real-time digital noise reduction and filter processing on the signal, And real-time display and storage.

Wherein, the cells cultured in the culture dish are the primary neurons of the cortex of the GFP mouse pregnant mouse and the cardiomyocytes of the SD newborn mouse, and the device can replace the tested cells by changing the culture dish.

For the extracellular electrical signals of the test cells in this experiment, the original test data were imported into matlab, and the test data of the neurons were processed including 50Hz power frequency notch processing, 2kHz low-pass filtering and 300Hz high-pass filtering. The typical neuron extracellular potential waveform recorded by this device can be obtained by processing the noise reduction filtering algorithm. Accompanying drawing 4 is the noise baseline of the control group in the eight channels of the blank culture medium, and accompanying drawing 5 is the occasional neuron waveform detected in the eight channels under normal culture conditions. , showing typical action potential characteristics of ascending branch and descending branch caused by sodium ion inflow and potassium ion outflow, the waveform duration is 1-4ms, and the peak-to-peak value ranges from tens of μV to 1mV. Accompanying drawing 6 is the waveform of epileptic discharge caused by collective abnormal discharge of neurons tested in a single channel under the induction of calcium and magnesium ion-free culture solution.

The test data of cardiomyocytes is processed by noise reduction filtering algorithm including 50Hz power frequency notch processing, 50Hz low-pass filtering and 1Hz high-pass filtering, and the extracellular potential waveform of cardiomyocytes recorded by this device can be obtained. Figure 7 is a typical waveform diagram of cardiomyocytes. The waveform shows a rapid depolarization at a stable position and a slow decrease at a negative pole. The duration of the waveform is between 200-300 ms. Accompanying drawing 8 is the waveform diagram of the extracellular potential of cardiomyocytes recorded by the device, and accompanying drawing 9 is a partial enlarged view of accompanying drawing 8, and its waveform duration clock is about 200 ms, which is similar to the typical waveform of cardiomyocytes.

The above embodiments of the present invention are illustrative, rather than limiting the present invention. Without departing from the principles of the present invention, any other implementations obtained by those skilled in the art under the inspiration of the present invention are considered within the protection of the present invention.

Claims (6)

1. The cell potential non-contact detection device is characterized by comprising a non-contact layer, a PCB substrate electrode, a signal conversion interface, a cell culture dish, a multichannel signal processing circuit system board and an upper computer; the non-contact layer is PDMS with the thickness smaller than 10 mu m, and is processed on the PCB substrate electrode in a spin coating mode; the PCB substrate electrode is connected with the cell culture dish in an inverted manner; the PCB substrate electrode is eight circular electrodes with the diameter of 50-200 mu m, the center distance of 100-400 mu m and gold, and the circular electrodes are connected with the multichannel signal processing circuit system board through the signal conversion interface; the multichannel signal processing circuit system board consists of a high input impedance sensor, an AD module and an MCU, and transmits data to an upper computer through a serial port of the MCU; the upper computer is responsible for displaying, processing and storing data.

2. The device according to claim 1, wherein the noncontact layer is PDMS with a thickness of less than 10 μm, is non-bio-toxic, and is uniformly coated on the PCB substrate electrode by a spin coater, and comprises the steps of:

(1) Taking a PCB substrate electrode plate as a spin-coating substrate, and cleaning the surface by adopting a standard cleaning process;

(2) Preparing a mixed solution of PDMS and a PDMS coagulant according to the proportion of 10:1;

(3) Removing bubbles from the mixed solution through vacuum pumping;

(4) Placing an extracellular potential sensor on a spin coater, and spin-coating the mixed solution onto the surface of a substrate at a rotating speed of 2000 r/s;

(5) And (5) placing the spin-coated PCB substrate electrode into an oven at 80 ℃ for heating and curing for 2 hours.

3. The device of claim 1, wherein the eight electrodes of the PCB substrate electrode are arranged in a square, rectangular, circular configuration.

4. The device of claim 1, wherein one of the eight substrate electrodes of the PCB is used as a reference electrode.

5. The device of claim 1, wherein the high input impedance sensor has an input impedance of not less than 100gΩ, an input capacitance of not more than 20pF, and a noise of not more than 1 μv within a bandwidth of 1Hz-1 kHz.

6. The device of claim 1, wherein the host computer is programmed with a Qt program, receives signals from the multi-channel signal processing circuit system board through the serial port, and performs real-time digital noise reduction and filtering processing on the signals by using a high-pass, low-pass and notch filtering algorithm, and performs real-time display and storage.