CN115166007A - Cell potential non-contact detection device - Google Patents
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- CN115166007A CN115166007A CN202210894479.2A CN202210894479A CN115166007A CN 115166007 A CN115166007 A CN 115166007A CN 202210894479 A CN202210894479 A CN 202210894479A CN 115166007 A CN115166007 A CN 115166007A
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- 238000001514 detection method Methods 0.000 title claims abstract description 19
- 239000000758 substrate Substances 0.000 claims abstract description 35
- 238000012545 processing Methods 0.000 claims abstract description 25
- 238000012546 transfer Methods 0.000 claims abstract description 8
- 238000004113 cell culture Methods 0.000 claims abstract description 7
- 239000004205 dimethyl polysiloxane Substances 0.000 claims description 12
- 235000013870 dimethyl polysiloxane Nutrition 0.000 claims description 12
- 238000001914 filtration Methods 0.000 claims description 12
- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical group C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 claims description 12
- 238000004987 plasma desorption mass spectroscopy Methods 0.000 claims description 12
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 claims description 12
- 238000004528 spin coating Methods 0.000 claims description 12
- 239000011259 mixed solution Substances 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 8
- 238000004140 cleaning Methods 0.000 claims description 6
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 5
- 239000010931 gold Substances 0.000 claims description 5
- 229910052737 gold Inorganic materials 0.000 claims description 5
- 230000008569 process Effects 0.000 claims description 5
- 230000009467 reduction Effects 0.000 claims description 5
- 239000000701 coagulant Substances 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 238000005086 pumping Methods 0.000 claims description 3
- 238000003860 storage Methods 0.000 claims description 3
- 239000000463 material Substances 0.000 abstract description 5
- 230000008878 coupling Effects 0.000 abstract description 4
- 238000010168 coupling process Methods 0.000 abstract description 4
- 238000005859 coupling reaction Methods 0.000 abstract description 4
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 210000004027 cell Anatomy 0.000 description 32
- 230000002107 myocardial effect Effects 0.000 description 6
- 210000002569 neuron Anatomy 0.000 description 5
- 238000010586 diagram Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 229910052751 metal Inorganic materials 0.000 description 3
- 239000002184 metal Substances 0.000 description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 2
- 230000036982 action potential Effects 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910001424 calcium ion Inorganic materials 0.000 description 2
- 210000004413 cardiac myocyte Anatomy 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000001037 epileptic effect Effects 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 229910001425 magnesium ion Inorganic materials 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- NPYPAHLBTDXSSS-UHFFFAOYSA-N Potassium ion Chemical compound [K+] NPYPAHLBTDXSSS-UHFFFAOYSA-N 0.000 description 1
- FKNQFGJONOIPTF-UHFFFAOYSA-N Sodium cation Chemical compound [Na+] FKNQFGJONOIPTF-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000001174 ascending effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000012398 clinical drug development Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 230000028161 membrane depolarization Effects 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 229910001414 potassium ion Inorganic materials 0.000 description 1
- 210000002243 primary neuron Anatomy 0.000 description 1
- 238000011897 real-time detection Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 229910001415 sodium ion Inorganic materials 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
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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
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- Chemical & Material Sciences (AREA)
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- Health & Medical Sciences (AREA)
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- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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Abstract
The invention discloses a cell potential non-contact detection device. The device comprises a non-contact layer, a PCB substrate electrode, a signal transfer port, 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 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 a device for detecting extracellular potential, and more particularly, to a device for detecting extracellular potential without contact.
Technical Field
The cell is the basic unit of organism structure and function, and the measurement of cell electric signal can make us know the survival condition and living environment of the cell, is an important means for researching cell physiological characteristics, and is also an important guide for clinical drug development. The 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 are continuously pursued for higher signal-to-noise ratios and higher integration. On one hand, the biocompatible medium needs to be adhered to the electrode, so that the adhesion between the cell and the electrode is increased; on the other hand, the MEA, signal amplification and signal processing circuits all tend to perform their functions on the same chip. The existing MEA usually cultures cells directly on the surface of an electrode array, the cells grow adherently on the surface of the electrode, and signals enter a detection system through the coupling between a cell membrane and a metal electrode. However, such direct contact detection between cells and electrodes generally requires careful consideration of conditions such as material, size, and spacing of the electrode array, and requires high coupling between cells and electrodes. Generally speaking, 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 and the adhesive force between the cells and the electrodes need to be maintained in a complex process, and the microelectrode array detection mode is not easy to replace due to the integration with a rear-end chip. For this reason, a technique for overcoming the above problems is urgently required.
Disclosure of Invention
The invention aims to provide a cell potential non-contact detection device aiming at the problems of complex electrode manufacturing process and difficult replacement in the existing cell potential microelectrode array detection device, and the technical scheme is as follows:
a cell potential non-contact detection device is characterized by comprising a non-contact layer, a PCB substrate electrode, a signal transfer port, a cell culture dish, a multi-channel signal processing circuit system board and an upper computer; the non-contact layer is PDMS with the thickness less than 10 μm 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 electrodes are eight round electrodes with the diameter of 50-200 mu m and the center distance of 100-400 mu m, and are made of gold and connected with the multi-channel signal processing circuit system board through signal transfer interfaces; 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; and the upper computer is responsible for displaying, processing and storing data.
Furthermore, the non-contact layer is PDMS with the thickness less than 10 μm, has no biotoxicity, is uniformly coated on the PCB substrate electrode by a spin coater, and has the following manufacturing 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 from PDMS and a PDMS coagulant according to the proportion of 10: 1;
(3) Removing bubbles from the mixed solution by vacuum pumping;
(4) Placing the extracellular potential sensor on a spin coater, and spin-coating the mixed solution onto the surface of the substrate at the rotating speed of 2000 r/s;
(5) And (3) putting the PCB substrate electrode subjected to spin coating into an oven at 80 ℃ for heating and curing for 2h.
Further, the eight electrodes of the PCB substrate electrodes may be arranged in a square, rectangular, circular configuration.
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 muV in the bandwidth of 1Hz-1 kHz.
Furthermore, the upper computer adopts a Qt program to compile, receives signals from the multi-channel signal processing circuit system board through a serial port, adopts high-pass, low-pass and notch filtering algorithms to perform real-time digital noise reduction and filtering processing on the signals, and performs 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 the 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 view 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.
FIG. 4 is a baseline of noise measured for a control group of blank media.
FIG. 5 is a waveform of the action potential of an adventitious neuron detected by using the device under normal culture conditions.
Fig. 6 is a graph of the epileptic discharge signals of neurons detected using the device without induction of calcium and magnesium ions.
Fig. 7 is a typical waveform diagram of a cardiomyocyte.
FIG. 8 is a diagram showing the waveform of extracellular potential of cardiomyocytes detected by the device.
Fig. 9 is a partially enlarged view of fig. 8.
Detailed Description
The invention is described in further detail below with reference to the drawings and specific examples, but the invention is not limited thereto.
The invention discloses a cell potential non-contact detection device, which comprises a non-contact layer, a PCB substrate electrode, a signal transfer port, a cell culture dish, a multi-channel signal processing circuit system board and an upper computer, wherein the non-contact layer is arranged on the PCB substrate electrode; the non-contact layer is PDMS with the thickness less than 10 μm 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 electrodes are eight round electrodes made of gold and are connected with the multi-channel signal processing circuit system board through signal transfer interfaces; 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; and the upper computer is responsible for displaying, processing and storing data.
Furthermore, the non-contact layer has no biotoxicity, is PDMS with the thickness less than 10 μm, is uniformly coated on the PCB substrate electrode through 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 from PDMS and a PDMS coagulant according to the proportion of 10: 1;
(3) Removing bubbles from the mixed solution by vacuum pumping;
(4) Placing the extracellular potential sensor on a spin coater, and spin-coating the mixed solution onto the surface of the substrate at the rotating speed of 2000 r/s;
(5) And (3) putting the PCB substrate electrode subjected to spin coating into an oven at 80 ℃ for heating and curing for 2h.
The PCB substrate electrode layout is shown in FIG. 2, eight round gold electrodes are formed on a metal disc at the center of a PCB by a gold immersion process on a PCB substrate, the diameter of each electrode is 152.4 μm, the center spacing of the electrodes is 381 μm, and the eight electrodes are arranged in a square structure. The electrodes are connected with the pin header adapter interface through metal leads, and the leads of the eight channels are subjected to equal-length wiring treatment by considering the symmetry of the eight channels.
Further, extracellular potential signals detected by the PCB substrate electrodes are transmitted to a multi-channel signal processing circuit system board through a signal transfer interface, buffered through a high input impedance sensor, then input into an AD module, controlled through an MCU (micro control unit), amplified by controllable gain and subjected to digital-to-analog conversion, and 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 in the laboratory, has the input impedance of 150G omega, the input capacitance of 15pF and the noise of 0.9 muV 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. A programmable gain amplifier is built in the chip, and the gain setting of 7 grades in total can be carried out by 1, 2, 3, 4, 6, 8 or 12 times through the control of the MCU module. The MCU is built by using the MSP430 singlechip, is communicated with a computer-side upper computer through a serial port, and can simultaneously control the gain in the ADS1298 chip, the opening and closing of the digital-to-analog conversion module and data transmission.
Furthermore, the upper computer is compiled by adopting a Qt program, receives signals from the multi-channel signal processing circuit system board through a serial port, and carries out real-time digital noise reduction and filtering processing on the signals by adopting 1Hz high-pass, 2kHz low-pass and 50Hz notch filtering algorithms, and carries out real-time display and storage.
Wherein, the cells cultured in the culture dish are GFP mouse pregnant mouse cortex primary neurons and SD newborn mouse myocardial cells, and the device can replace the cells to be tested by replacing the culture dish.
Aiming at the extracellular electric signals of the test cells in the experiment, the original test data are led into matlab, and the test data of the neurons are subjected to noise reduction filtering algorithm processing including 50Hz power frequency notch processing, 2kHz low-pass filtering and 300Hz high-pass filtering, so that typical neuron extracellular potential waveforms recorded by the device can be obtained. Wherein FIG. 4 is a control group noise baseline of eight channels of blank medium, FIG. 5 is a waveform of occasional neurons detected by eight channels under normal culture conditions, the waveform characteristic is represented by rapid decline at a steady position and subsequent rapid rise back, typical action potential characteristics of ascending branch and descending branch caused by sodium ion inflow and potassium ion outflow are presented, the waveform duration is 1-4ms, and the peak-to-peak value is different from dozens of muV to 1 mV. FIG. 6 shows epileptic discharge waveforms due to abnormal discharge of neuronal aggregates tested in a single channel without induction of calcium and magnesium ions in culture.
The testing data of the myocardial cells are subjected to noise reduction filtering algorithm processing including power frequency notch processing of 50Hz, low-pass filtering of 50Hz and high-pass filtering of 1Hz, and the extracellular potential waveform of the myocardial cells recorded by the device can be obtained. FIG. 7 is a typical waveform of a myocardial cell, showing rapid depolarization at plateau and slow decline at negative pole, with waveform duration between 200-300 ms. FIG. 8 is a diagram showing the waveform of extracellular potential of a myocardial cell recorded by using the apparatus, and FIG. 9 is a partially enlarged view of FIG. 8, in which the waveform duration is around 200ms, similar to the typical waveform of a myocardial cell.
The above-described embodiments of the present invention are intended to be illustrative, rather than restrictive, and all such other embodiments as would be known to one skilled in the art to which the present invention pertains are deemed to lie within the scope and spirit of the present invention.
Claims (6)
1. A cell potential non-contact detection device is characterized by comprising a non-contact layer, a PCB substrate electrode, a signal transfer port, a cell culture dish, a multi-channel signal processing circuit system board and an upper computer; the non-contact layer is PDMS with the thickness less than 10 μm 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 a back-off mode; the PCB substrate electrodes are eight round electrodes with the diameter of 50-200 mu m and the center distance of 100-400 mu m, and are made of gold and connected with the multi-channel signal processing circuit system board through signal transfer interfaces; 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; and the upper computer is responsible for displaying, processing and storing data.
2. The device of claim 1, wherein the non-contact layer is PDMS with a thickness less than 10 μm, has no biotoxicity, and is uniformly coated on the PCB substrate electrode by a spin coater, and the method comprises 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 from PDMS and a PDMS coagulant according to the proportion of 10: 1;
(3) Removing bubbles from the mixed solution by vacuum pumping;
(4) Placing the extracellular potential sensor on a spin coater, and spin-coating the mixed solution onto the surface of the substrate at the rotating speed of 2000 r/s;
(5) And (3) putting the PCB substrate electrode subjected to spin coating into an oven at 80 ℃ for heating and curing for 2h.
3. A cell potential non-contact detecting device according to claim 1, wherein the eight electrodes of the PCB substrate electrodes are arranged in a square, rectangular or circular configuration.
4. A cell potential non-contact detection device according to claim 1, wherein one of the eight base electrodes of the PCB serves as a reference electrode.
5. A cell potential non-contact detecting device according to claim 1, wherein the input impedance of the high input impedance sensor is not less than 100G Ω, the input capacitance is not more than 20pF, and the noise is not more than 1 μ V in the bandwidth of 1Hz-1 kHz.
6. The device of claim 1, wherein the upper computer is programmed with a Qt program, receives the signal from the multi-channel signal processing circuit system board through a serial port, performs real-time digital noise reduction and filtering on the signal by using high-pass, low-pass and notch filtering algorithms, 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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| CN115166007B CN115166007B (en) | 2023-07-18 |
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