CN115166007B - Cell potential non-contact detection device - Google Patents
Cell potential non-contact detection device Download PDFInfo
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- CN115166007B CN115166007B CN202210894479.2A CN202210894479A CN115166007B CN 115166007 B CN115166007 B CN 115166007B CN 202210894479 A CN202210894479 A CN 202210894479A CN 115166007 B CN115166007 B CN 115166007B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/301—Reference electrodes
Abstract
The invention discloses a cell potential non-contact detection device. The device comprises 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 added on the basis of the existing extracellular potential sensor, and the non-contact detection of extracellular potential can be realized by reasonably selecting the material and the thickness of the non-contact layer. The device improves the coupling condition between cells and electrodes, increases the flexibility of electrode size and material selection, simplifies electrode manufacture, is easy to replace and is convenient to detect.
Description
Technical Field
The present invention relates to an extracellular potential detection device, and more particularly, to a cell potential non-contact detection device.
Technical Field
The cell is a basic unit of organism structure and function, and the measurement of the cell electric signal can enable people to know the survival condition and living environment of the cell, so that the cell is an important means for researching the physiological characteristics of the cell and is also an important guide for clinical drug development. Extracellular detection is usually performed by using a microelectrode array (MEA), and the method has the advantages of small damage to cells, capability of outputting detection results of a plurality of cell potentials in parallel, and the like. MEA-based cell potential measurement devices have been pursued for higher signal-to-noise ratios and higher integration levels. On the one hand, it is necessary to adhere biocompatible medium to the electrode, increasing the adhesion between the cells and the electrode; on the other hand, MEA, signal amplification and signal processing circuits all tend to perform their functions on the same chip. The existing MEA is usually to directly culture cells on the surface of an electrode array, the cells grow on the surface of the electrode, and signals enter a detection system through coupling between a cell membrane and a metal electrode. However, such detection of direct contact between the cells and the electrodes generally requires careful consideration of the conditions such as the material, size, and pitch of the electrode array, and requires high coupling requirements between the cells and the electrodes. In general, in the microelectrode array detection mode, cells are in direct contact with electrodes, the requirements on the electrodes are high, the biocompatibility of the electrodes, the adhesion between the cells and the electrodes are required to be maintained by a complicated process, and the microelectrode array detection mode is not easy to replace due to integration with a back-end chip. For this reason, a technology is urgently needed to overcome the above-mentioned problems.
Disclosure of Invention
The invention aims to solve the problems of complex electrode manufacturing process and difficult replacement in the existing cell potential microelectrode array detection device, and provides a cell potential non-contact detection device, which has the following technical scheme:
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.
Further, the non-contact layer is PDMS with the thickness smaller than 10 μm, the non-contact layer is non-biological toxicity, and is uniformly coated on the PCB substrate electrode by a spin coater, and the preparation steps are as follows:
(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.
Further, eight electrodes of the PCB substrate electrode may be arranged in a square, rectangular, circular structure.
Further, one of the eight base electrodes of the PCB may serve as a reference electrode.
Further, the input impedance of the high input impedance sensor is not less than 100G omega, the input capacitance is not more than 20pF, and the noise is not more than 1 mu V within the bandwidth of 1Hz-1 kHz.
Further, the upper computer adopts a Qt program to write, receives signals from the multichannel signal processing circuit system board through a serial port, adopts a high-pass, low-pass and notch filtering algorithm to carry out real-time digital noise reduction and filtering processing on the signals, and carries out real-time display and storage.
Compared with the existing device, the invention provides that a non-contact layer is additionally added on the basis of the multi-electrode array detection device to realize non-contact detection, and the real-time detection of extracellular cell electric signals can be realized by reasonably selecting the material and thickness of the non-contact layer. The device improves the coupling condition between cells and electrodes, has more flexible selection of the size and the material of the electrodes, simplifies the manufacture of the electrodes, is easy to replace and is convenient to detect.
Drawings
FIG. 1 is a schematic diagram of a cell potential non-contact detection device according to the present invention.
Fig. 2 is a PCB substrate electrode layout.
Fig. 3 is a block diagram of an implementation of a multi-channel signal processing circuitry board.
Figure 4 is a baseline of noise measured for the blank media control.
FIG. 5 is a waveform of action potentials of sporadic neurons detected under normal culture conditions using the device.
FIG. 6 is a graph of epileptic discharge signals of neurons detected using the device without the induction of calcium and magnesium ions.
FIG. 7 is a typical waveform of cardiomyocytes.
FIG. 8 is a waveform of extracellular potential of myocardial cells detected using the present device.
Fig. 9 is a partial enlarged view of fig. 8.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and specific examples, but the present invention is not limited thereto.
The invention discloses a cell potential non-contact detection device, which is shown in figure 1 and comprises a non-contact layer, a PCB (printed circuit board) 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 round electrodes made of gold and is 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.
Further, the non-contact layer is non-bio-toxic, is PDMS with the thickness less than 10 μm, is uniformly coated on the PCB substrate electrode by a spin coater, and is prepared by the following steps:
(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.
The PCB substrate electrode layout is shown in figure 2, eight round gold electrodes are formed on a metal disc in the center of the PCB on the PCB substrate board through a gold deposition process, the diameter of each electrode is 152.4 mu m, the center distance of each electrode is 381 mu m, and the eight electrodes are arranged in a square structure. The electrode is connected with the pin header through a metal lead, and the symmetry of eight channels is considered to perform equal-length wiring treatment on the leads of the eight channels.
Further, the extracellular electric signals detected by the PCB substrate electrode are transmitted to a multichannel signal processing circuit system board through a signal conversion interface, buffered through a high input impedance sensor, then input into an AD module, controlled through an MCU, amplified and digital-to-analog converted into controllable gains, and the processed cell signals are transmitted to an upper computer at a computer end through a serial port.
The high input impedance sensor is a chip independently developed by the laboratory, the input impedance is 150G omega, the input capacitance is 15pF, and the noise is 0.9 mu V in the bandwidth of 1Hz-1kHz, and is used for efficiently collecting and buffering extracellular potential. The AD module is built by using an ADS1298 chip, and 8-channel high-precision 4000Hz synchronous sampling can be realized. The programmable gain amplifier is arranged in the chip, and the control is carried out by using the MCU module, so that the gain setting of 7 gears of 1, 2, 3, 4, 6, 8 or 12 times can be carried out. The MCU is built by using the MSP430 singlechip, and is communicated with the upper computer at the computer end through the serial port, so that the gain inside the ADS1298 chip, the opening and closing of the digital-to-analog conversion module and the data transmission can be controlled simultaneously.
Further, the upper computer adopts a Qt program to write, receives signals from the multichannel signal processing circuit system board through a serial port, adopts a 1Hz high-pass, 2kHz low-pass and 50Hz notch filtering algorithm to carry out real-time digital noise reduction and filtering processing on the signals, and carries out real-time display and storage.
Wherein, the cells cultured in the culture dish are GFP mouse pregnant mouse cortex primary neuron and SD neonatal mouse myocardial cell, and the device can replace the tested cells by replacing the culture dish.
For the extracellular electric signals of the test cells in the experiment, the tested original data are imported into matlab, the test data of the neurons are processed by a noise reduction filtering algorithm comprising power frequency notch processing of 50Hz, low-pass filtering of 2kHz and high-pass filtering of 300Hz, and typical extracellular electric waveforms of the neurons recorded by the device can be obtained. Wherein FIG. 4 is a noise baseline of a control group of eight channels of a blank medium, FIG. 5 is a waveform of sporadic neurons detected by eight channels under normal culture conditions, the waveform characteristic is shown to be rapidly declining at a plateau and then rapidly rising, the characteristic of typical action potentials of rising branches and falling branches caused by sodium ion inflow and potassium ion outflow is shown, the waveform duration is 1-4ms, and the peak-to-peak value is different from tens of μV to 1 mV. FIG. 6 is a waveform of epileptic discharge induced by abnormal discharge of neurons in a single pass under the induction of a culture solution without calcium and magnesium ions.
The test data of the myocardial cells are processed by a noise reduction filtering algorithm comprising power frequency notch processing of 50Hz, low-pass filtering of 50Hz and high-pass filtering of 1Hz, so that the extracellular potential waveforms of the myocardial cells recorded by the device can be obtained. FIG. 7 is a typical waveform of a cardiomyocyte showing rapid depolarization at a plateau, slow descent at a negative electrode, and a waveform duration of between 200-300 ms. FIG. 8 is a graph of the extracellular potential of a cardiomyocyte recorded using the present apparatus, and FIG. 9 is an enlarged partial view of FIG. 8, with a waveform duration clock of about 200ms, similar to a typical waveform of a cardiomyocyte.
The above examples of the invention are intended to be illustrative, and not limiting, of the invention, and any other embodiments which may be obtained by those skilled in the art in light of the present teachings are deemed to be within the scope of the invention without departing from the principles thereof.
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.
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| CN202210894479.2A CN115166007B (en) | 2022-07-27 | 2022-07-27 | Cell potential non-contact detection device |
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| CN202210894479.2A CN115166007B (en) | 2022-07-27 | 2022-07-27 | Cell potential non-contact detection device |
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| CN115166007B true CN115166007B (en) | 2023-07-18 |
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