KR102131217B1 - Daq board based cell impedance measurement sysetm - Google Patents

Abstract

The present invention, the cell is attached to the electrical cell-based impedance sensor (ECIS) for measuring the electrical properties of the cell in the impedance measurement system of the cell to detect the electrical properties based on an analog, the signal generated from the sinusoidal wave generator A DAC channel unit for applying the input signal to the electrode of the ECIS to which cells are attached by converting it into an analog input signal, and an analog response signal generated from the electrode of the ECIS as the input signal of the DAC channel unit is applied. A DAQ board having an ADC channel unit for receiving and converting it into a digital signal; And a processor unit that receives a digitally converted response signal from the ADC channel unit and analyzes amplitude and phase characteristics to measure cell impedance, and measures the impedance of the cell in an analog region through the DAQ board. do.

Description

DAQ board-based cell impedance measurement system{DAQ BOARD BASED CELL IMPEDANCE MEASUREMENT SYSETM}

The present invention relates to a cell impedance measurement system, and in particular, by using a DAQ board, the present invention relates to a cell impedance measurement system having excellent dispersibility at a low manufacturing cost.

Analysis of cells using cell-substrate electric cell-substrate impedance sensing (ECIS) technology, rather than fluorescent label staining, is emerging as an alternative or adjunct to traditional biochemical methods. The ECIS sensor refers to an electrode sensor for measuring electrical cell-substrate impedance, and enables the mobility and mortality of cells attached to the microelectrode of ECIS to be detected in real time without staining the fluorescent label. ECIS is mainly used for drug testing and cell monitoring at the cellular level, and is recognized as a powerful technology for cell research, according to non-destructive and real-time diagnostic properties without fluorescent labeling. ECIS can quantify the properties of a cell-cell or cell-substrate junction based on data of electrical properties that change with cell adhesion, proliferation, micromotor, toxicity, necrosis, viral infection or differentiation.

In general, the ECIS system consists of a biocompatible microelectrode-based cell chip and a system for measuring bioelectrical impedance. As the material of the electrode, a material of Au, Pt, or ITO (Indium Tin Oxide) having biocompatibility and stable electrochemical properties is used. The ECIS system must protect the cells from electrical damage by appropriately applying the amount of current flowing through the electrodes, while at the same time being set so that the noise signal does not increase. The impedance spectrum of the cells measured via the ECIS system can be verified by well-known theoretical or mathematical models.

Cells adsorbed to the electrodes of the ECIS cause morphological and physiological changes in the cells depending on the adsorption power, and phenomena such as proliferation, necrosis, or cell death cause changes in model parameters of the electrical equivalent circuit. Accordingly, although the development of digital signal processing (DSP) technology has been pursued to implement high performance of ECIS, it has a great disadvantage in terms of portability and cost for application to point-of-care applications.

Accordingly, there is a need for a device and method for measuring the impedance of cells in consideration of portability of an ECIS system as a measuring device to which a small micro-controller and a cost-effective DSP are applied.

1 relates to a conventional electrical cell-substrate impedance sensor (ECIS), and shows a representative view of U.S. Patent Publication No. 2016-0178605 (hereinafter referred to as a prior art document). Referring to FIG. 1, the electrical cell-substrate impedance sensor comprises a substrate 101, a catalyst layer 103 formed on the substrate 101, and a nanowire electrode array 104 grown on a plurality of catalyst layers 103. The nanowire electrode 104 measures the electrical response of biological cells. The prior document discloses a biosensor that monitors the proliferation of biological cells, and provides an electrical cell-substrate impedance sensor for detecting the cancer characteristics of cells by monitoring the proliferation of cells on nanoelectrodes.

The present applicant, as a system for measuring the impedance of cells in connection with the above ECIS, devised an ECIS system capable of realizing more accurate response characteristics of cells and low manufacturing cost by applying a DAQ board.

Meanwhile, a DAQ (Data Acquisition) board means hardware that converts a voltage value output through a sensor or a signal conditioning module into a digital signal that can be recognized by a computer. The DAQ board is equipped with an ADC (Analog to Digital Convertor) chip, and converts a continuous analog signal of voltage into a discrete digital signal of 0 or 1.

The data acquisition performed by the DAQ board can target electrical or physical phenomena such as voltage, current, temperature, pressure or noise. The DAQ board is usually associated with a sensor that measures physical phenomena such as voltage and current, and consists of a processor with hardware and programming software that converts the signal.

U.S. Patent Publication No. 2016-0178605

The present invention is to provide an impedance measurement system for a cell that can measure the impedance of a cell in an analog region by connecting a DAQ board to an electrical cell-substrate impedance sensor (ECIS), and can be supplied at a low cost.

In order to achieve the above object, the present invention, in connection with an electrical cell-substrate impedance sensor (ECIS) to which cells are attached to measure the electrical properties of cells, is an impedance measurement system of cells for analog-based detection of electrical properties, sinusoidal wave The DAC channel unit that receives the signal generated from the generator and converts it into an analog input signal to apply the input signal to the electrode of the ECIS to which cells are attached, and the electrode of the ECIS as the input signal of the DAC channel unit is applied. A DAQ board having an ADC channel for receiving an analog response signal and converting it into a digital signal; And a processor unit that receives a digitally converted response signal from the ADC channel unit and analyzes amplitude and phase characteristics to measure cell impedance, and measures the impedance of the cell in an analog region through the DAQ board. do.

Preferably, the processor unit includes a lock-in amplifier having a phase-locked loop, a mixer, and a low-pass filter, and has an in-phase and an out-of-phase potential. It can be configured with software to extract and calculate the impedance spectrum.

Preferably, the DAC channel unit outputs an input signal in the range of 1 Vrms ±0.5 Vrms, and the input signal passes a resistance set to 0 to 1 ㏁ or less to limit the maximum amplitude, and a current of 0 to 1 ㎂ or less flows. Can be done.

According to the present invention, the DAQ board is connected to an electrical cell-substrate impedance sensor (ECIS) to measure the impedance of cells in the analog region. The present invention analyzes the impedance spectrum with the analog response characteristics of cells, and through experiments, the resistance, frequency, and input voltage ranges have been optimized for analog analysis. Accordingly, the present invention can reduce the manufacturing cost of the existing expensive ECIS system to tens of millions of won by up to 1/10, and has an advantage suitable for portability and cost for application.

1 relates to a conventional electrical cell-substrate impedance sensor (ECIS), and shows a representative view of US Patent Publication No. 2016-0178605.
2 is a block diagram of a cell impedance measurement system according to an embodiment of the present invention.
3 is a schematic diagram showing a circuit of a cell impedance measurement system according to an embodiment of the present invention.
4 is an experimental example of the present invention, showing the configuration of the cell impedance measurement system by implementing a sinusoidal wave generator and a processor as a lab view and hardware of the fabricated cell impedance measurement system.
5 shows an ECIS chip used as an experimental example of the present invention.
Figure 6 shows a photographed image of the cultured cells according to this experimental example.
7 shows impedance data measured in a frequency range of 10 Hz to 100 kHz according to the size of a resistor (100 Ω, 1 ㏀, 10 ㏀, 100 ㏀, 1 ㏁) using a DAQ board.
FIG. 8 shows the impedance magnitude (a) and phase (b) of C2C12 cells, and shows the measurement results of this experiment with fitting lines applying an electrical equivalent circuit model designed during cell culture.
9 shows standardized impedance measurement (a) data of C2C12 cells and impedance measurement (b) data according to cell culture time at a frequency of 10 kHz.
Figure 10 shows the H2O2 cytotoxic effect test data of HEK293 cells evaluated by impedance analysis.

Hereinafter, the present invention will be described in detail with reference to the contents described in the accompanying drawings. However, the present invention is not limited or limited by the exemplary embodiments. The same reference numerals in each drawing denote members that perform substantially the same function.

The objects and effects of the present invention may be naturally understood or more apparent by the following description, and the objects and effects of the present invention are not limited only by the following description. In addition, in the description of the present invention, when it is determined that the detailed description of the known technology related to the present invention may unnecessarily obscure the subject matter of the present invention, the detailed description will be omitted.

2 shows a configuration diagram of a cell impedance measurement system 1 according to an embodiment of the present invention. FIG. 3 schematically shows the circuit of the impedance measurement system 1 for cells according to an embodiment of the present invention.

2 and 3, the cell impedance measurement system 1 may include a DAQ board 30, a processor unit 50, a sine wave generator 20, and an ECIS 10. .

In this embodiment, the ECIS 10 used in the cell impedance measurement system 1 may be an ECIS developed by Giaever and Keese. ECIS 10 is a biosensor capable of attaching and culturing cells 3 to a microelectrode and detecting the mobility and mortality of attached cells in real time without fluorescent labeling, even if conventional ECIS is used.

The sinusoidal wave generator 20 generates an input signal of the ECIS 10, and the generated signal is applied to the DAQ board 30. The sinusoidal wave generator 20 may be configured together on the DAQ board 30, or may be provided as a separate power device and connected to the DAQ board 30. The processor unit 50 includes software for analyzing a response signal of the DAQ board 30. The processor unit 50 may be implemented on the DAQ board 30, or may be mounted on a separate portable device. Hereinafter, the DAQ board 30, which is a characteristic configuration of the present invention, will be described.

The DAQ board 30 refers to hardware including a DAC channel portion 301 and an ADC channel portion 303. DAQ hardware acts as a device that digitizes the incoming analog signal and allows the computer to decode it. Although the DAQ board 30 is not shown, it can be used by inserting it as a card to a main board such as a computer or a separate portable device, including a computer bus, and interfacing an interface to various devices. The DAQ board 30 is connected to an electrical cell-substrate impedance sensor (ECIS) 10 to which the cells 3 are attached to measure the electrical properties of the cells 3, so that electrical properties can be detected based on analog.

The DAC channel unit 301 receives a signal generated from the sinusoidal wave generator 20 and converts it into an analog input signal to apply an input signal to the electrode of the ECIS 10 to which the cells 3 are attached.

The DAC channel unit 301 outputs an input signal in the range of 1 Vrms ±0.5 Vrms, and the input signal passes a resistance set to 0 to 1 ㏁ or less to limit the maximum amplitude so that a current of 0 to 1 ㎂ or less flows. can do. The range of the input signal and the range of the resistance of the DAC channel unit 301 are numerically selected experimentally, and will be described later through experimental examples.

The ADC channel unit 303 may receive an analog response signal generated from the electrode of the ECIS 10 as the input signal of the DAC channel unit 301 is applied, and convert it into a digital signal.

The processor unit 50 may measure the impedance of the cell by receiving the digitally converted response signal from the ADC channel unit 303 and analyzing the amplitude and phase characteristics.

The processor unit 50 includes a lock-in amplifier having a phase-locked loop, a mixer, and a low-pass filter, and is capable of in-phase and out-of-phase potential. It can be configured with software to extract and calculate the impedance spectrum.

Hereinafter, an example of an experiment performed to verify the validity of a cell impedance measurement system according to an embodiment of the present invention will be described.

This experimental example was performed by measuring C2C12 cell growth and analyzing DAPI, limiting HEK293 cells exposed to H ₂ O ₂ , and analyzing viability of MTT cells.

4 is an experimental example of the present invention, showing the configuration of the cell impedance measurement system by implementing a sinusoidal wave generator and a processor as a lab view and hardware of the fabricated cell impedance measurement system. 5 shows an ECIS chip used as an experimental example of the present invention.

In this experimental example, the AC input signal for the ECIS chip used is applied to the cells 3 of the electrode through the DAC channel 301 of the DAQ board 30 and a resistance element that limits the current. The response signal was recorded on the ADC channel 303 of the DAQ board 30, and the amplitude and phase were analyzed by software including a lock-in amplifier, mixer, and low-pass filter. Impedance measurement according to C2C12 cell growth on the ITO electrode and mast cell viability analysis of HEK293 cells exposed to H2O2 were tested, and the validity of the system (1) was evaluated by DAPI analysis.

In the ECIS chip according to this experimental example, an ITO electrode was used. The ECIS chip has a 2 μm thick ITO electrode on a 75 mm x 25 mm substrate. The bared working electrode was set to a circle with a radius of 250 μm, and the polystyrene chamber was attached to the electrode chip using silicone adhesive.

C2C12 cells were cultured on the ECIS chip to monitor cell growth. 500 C2C12 cells were inoculated with 500 μl culture medium (DMEM, 10% fetal calf serum, 1% penicillin) on an ITO microelectrode and cultured at 5% CO ₂ . The temperature was maintained at 37°C during cell growth, and the impedance spectrum of C2C12 cells was measured in a frequency range of 10 Hz to 10 kHz. Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature and washed with PBS. Thereafter, the cells were cooled with PBS containing 0.27% Triron X-100, incubated with DAPI staining, and washed with PBS. Figure 6 shows a photographed image of the cultured cells according to this experimental example. Figure 6a shows a micrograph of the culture time of the C2C12 cells cultured in the ITO electrode, Figure 6b shows the nuclear stained C2C12 cells photographed through a microscope.

In the cytotoxicity test, H ₂ O ₂ was prepared by deionized water and applied to HEK293 cells cultured on an ITO electrode for 1 day. The viability analysis of the cells was tested by comparing the size of the cells exposed to H ₂ O ₂ at an impedance of 10 kHz and conventional MTT, and measuring absorbance at a wavelength of 450 nm.

The DAQ board 30 according to this experimental example was impedance measured with 24-bit resolution and 102 kS/s sampling. The DAQ board 30 was set to generate a signal of 1 Vrms in the DAC channel 301, and through a current limiting resistor of 1 ㏁ to limit the maximum amplitude, the current flowing through the electrode was set to be 1 ㎂ or less. To measure the response potential, after connecting to the ADC channel 303 of the DAQ board 30, the in-phase and reverse-phase potentials of the signal passing through the lock-in amplifier were extracted by software of the processor unit 50.

As a result of the experimental example, FIG. 7 shows impedance data measured in a frequency range of 10 Hz to 100 kHz according to the size of the resistance (100, 1 ㏀, 10 ㏀, 100 ㏀, 1 ㏁) using the DAQ board 30 .

Referring to FIG. 7, the cell impedance measurement system 1 according to the present embodiment exhibited an accuracy of 95% or more in a resistance condition of 100 Hz to 100 Hz in a frequency range of 10 Hz to 10 kHz. As shown in FIG. 7, in the impedance measurement system of the DAQ board 30 according to this experimental example, it was confirmed that the accuracy of impedance measurement at a resistance of 1 kHz is limited in the frequency range.

Figure 8 shows the impedance magnitude (a) and phase (b) of C2C12 cells, and shows the measurement results of this experiment with fitting lines applied with an electrical equivalent circuit model designed during cell culture.

Referring to FIG. 8, it can be seen that the magnitude and phase of the measured impedance are in agreement with the fitted curve Fit using an equivalent circuit model. In this experimental example, the impedance change due to cell growth was not observed at a low frequency of 100 Hz or less, but as the frequency range increased to 100 Hz or more, an increase in phase and impedance due to cell growth was confirmed. In this experimental example, it can be confirmed that the DAQ board 30-based ECIS system operates precisely and can be described as a theoretical circuit model by matching the measured impedance spectrum with the fitting result.

9 shows standardized impedance measurement (a) data of C2C12 cells and impedance measurement (b) data according to cell culture time at a frequency of 10 kHz. Referring to FIG. 9, experimentally significant inflection was observed when the frequency was 10 kHz. The measured impedance showed an S-shaped curve of typical cell growth when the frequency was 10 kHz. In the limited region of the electrode after cell saturation, the value did not increase with cell growth.

Figure 10 shows the H ₂ O ₂ cytotoxic effect viability test data of HEK293 cells evaluated by impedance analysis. FIG. 10 shows measurement data of MTT(a) when n=4 and impedance (b) when n=3 when the concentrations of H ₂ O ₂ are 0, 0.5, 1, 1.5, and 2 mM.

Referring to FIG. 10, it can be seen that in both the MTT and the impedance measurement data, as the concentration of H ₂ O ₂ increases, the viability of the cells decreases.

As the experiment in the above, the cell impedance measurement system (1) according to this embodiment is verified by the theory of the existing impedance evaluation model, while the DAQ board (30) is used, low manufacturing cost and excellent dispersibility are possible. It was confirmed that the system can be implemented.

Although the present invention has been described in detail through exemplary embodiments above, those skilled in the art to which the present invention pertains understand that various modifications are possible within the limits of the embodiments described above without departing from the scope of the present invention. will be. Therefore, the scope of rights of the present invention should not be limited to the described embodiments, but should be determined by all modified or modified forms derived from the claims and equivalent concepts as well as the claims described below.

1: Cell impedance measurement system
3: cell
10: ECIS
20: sine wave generator
30: DAQ board
50: processor unit

Claims (3)

In the cell attached to the electrical cell-based impedance sensor (ECIS) for measuring the electrical properties of the cell, the impedance measurement system of the cell to detect the electrical properties based on an analog,
A DAC channel unit that receives a signal generated from a sinusoidal wave generator, converts it into an analog input signal, and applies the input signal to an electrode of the ECIS with cells attached thereto;
A DAQ board having an ADC channel portion that receives an analog response signal generated from an electrode of the ECIS and converts it into a digital signal as the input signal of the DAC channel portion is applied; And
And a processor unit that receives a digitally converted response signal from the ADC channel unit and analyzes amplitude and phase characteristics to measure cell impedance.
The processor unit,
Includes a phase-lock loop, a mixer, and a lock-in amplifier with a low-pass filter, and extracts the potential of in-phase and out-of-phase to calculate the impedance spectrum. It consists of software
The DAC channel unit,
It is characterized in that it outputs an input signal in the range of 1 Vrms ±0.5 Vrms, and the input signal passes through a resistance set to 0 to 1 ㏁ or less to limit the maximum amplitude so that a current of 0 to 1 ㎂ or less flows. A cell impedance measurement system characterized by measuring the impedance of cells in an analog region through a DAQ board.
delete delete

Images