Electrically non-destructive real-time cell monitoring apparatus and method

Abstract

An electrically non-destructive real-time cell monitoring device according to an exemplary embodiment of the present invention includes a first electrode into which cells are in contact, a second electrode provided at a position spaced apart from the first electrode, an impedance measuring unit connected to the first electrode and the second electrode, an LFP measuring unit connected to the first electrode and the second electrode, and a signal processing unit connected to the impedance measuring unit and the LFP measuring unit for receiving and processing measurement results, wherein the impedance measuring unit applies alternating current to the second electrode to measure the impedance between the first electrode and the second electrode, the LFP measuring unit measures the local electric field potential between the first electrode and the second electrode and outputs a first digital signal, the impedance measuring unit and the LFP measuring unit operate simultaneously, and the acquired signal processing unit processes the first digital signal to acquire a second digital signal.

Description

Technical Field

[0001] One embodiment of the present invention relates to an electrical non-destructive real-time cell monitoring device.

Background Art

[0002] The inventors of the present invention have conducted intensive research and development to divert an ITO electrode for use in electric cell-substrate impedance sensing (ECIS). As a result, they have devised an ECIS system that measures cell impedance in cooperation with an ECIS sensor and has more accurate response characteristics of cells and can be implemented at a low manufacturing cost by applying a DAQ board. The related technology is introduced in Patent Document 1.

[0003] The evaluation of the safety or harmfulness of substances used in the development of chemicals, cosmetics, medical devices, and agricultural chemicals is essential.

[0004] Animal experiments have been used for such evaluations. However, animal models not only have limitations in confirming the efficacy and effects in the human body due to inter-species differences but also research on alternative methods to animal experiments has continued as ethical issues related to animal experiments have increased.

[0005] In recent years, the FDA has enacted a bill stating that animals do not need to be used for new drug tests, and the US President has signed it. Therefore, it is expected that the demand for alternative methods to animal experiments will further increase in the future.

[0006] On the other hand, non-destructive real-time cell analysis methods overcome the limitations of conventional staining reagent-based endpoint cytotoxicity evaluation methods and enable faster and more accurate cell analysis.

[0007] Examples of non-destructive real-time cell analysis methods include the electric cell-substrate impedance sensing method and the local electric field potential measurement method.

[0008] Electrical cell-substrate impedance sensing (ECIS) is a cell analysis technique developed by Giaver and Keese. It allows for the acquisition of frequency-dependent electrical impedance by measuring in-phase and out-of-phase potentials while applying an alternating electric field to a cell covering electrodes. This type of electrical cell-substrate impedance sensing is a suitable method for biological research because it enables long-term measurements without affecting cell function.

[0009] Local field potential (CAT) measurement is a cell analysis technique that can be used to measure signals with constant pulses, such as those in cardiomyocytes, or to measure changes in various electrical signals in nerve cells, allowing for the monitoring of cellular changes caused by chemicals, drugs, and anticancer agents.

[0010] Impedance measurement and local electric field potential measurement are suitable techniques for non-destructive real-time cell analysis and monitoring. However, when simultaneously measuring impedance and local electric field potential to detect changes in the electrical properties of cells, there is a problem in that mutual interference occurs, making simultaneous measurement impossible. In particular, the reference voltage of the local electric field potential causes errors in impedance measurement, and conversely, the AC signal used for impedance measurement induces interference to an unacceptable degree in the measurement of local electric field potential.

[0011] Patent Document 1 describes an apparatus and method for parallel writing of impedance spectrum and electric field potential. However, Patent Document 1 also discloses problems when impedance measurement and EPR measurement are performed simultaneously. To solve this problem, it proposes a technique that performs impedance measurement and EPR measurement in a time-division manner and improves the switching device to reduce noise generated during the switching process of the measurement method. However, Patent Document 1 was unable to solve the noise problem when impedance measurement and LFP measurement were performed at the same time point in time. [Prior art documents] [Patent Documents]

[0012] [Patent Document 1] Korean Registered Patent No. 1993943 (July 4, 2014) [Overview of the project] [Problems that the invention aims to solve]

[0013] One aspect of the present invention can provide an electrically non-destructive real-time cell monitoring technique that can simultaneously monitor the impedance and local electric field potential of a cell.

[0014] The technical problems of the present invention are not limited to those mentioned above, and other technical problems not mentioned should be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0015] An electrically non-destructive real-time cell monitoring device according to an exemplary embodiment of the present invention includes a first electrode into which cells are in contact, a second electrode provided at a position spaced apart from the first electrode, an impedance measuring unit connected to the first electrode and the second electrode, an LFP measuring unit connected to the first electrode and the second electrode, and a signal processing unit connected to the impedance measuring unit and the LFP measuring unit for receiving and processing measurement results. The impedance measuring unit applies alternating current to the second electrode to measure the impedance between the first electrode and the second electrode, the LFP measuring unit outputs a first digital signal by measuring the local electric field potential between the first electrode and the second electrode, the impedance measuring unit and the LFP measuring unit operate simultaneously, and the acquired signal processing unit obtains a second digital signal by processing the first digital signal, the second digital signal being a signal from which noise generated by the impedance measuring unit has been removed from the first digital signal.

[0016] At this time, the acquired signal processing unit can convert the first digital signal into the frequency domain, filter out noise, and then convert it into the time domain to acquire a second digital signal.

[0017] Furthermore, the acquired signal processing unit can convert the first digital signal into the frequency domain by applying Equation 1.

[0018]

number

[0019] In the formula, X(k) is a signal in the frequency domain, x(n) is a signal in the time domain, k is frequency, n is time, N is any real number greater than 0, e is a natural constant, i is the imaginary unit, and Σ represents the partial sum of a sequence.

[0020] Furthermore, the acquired signal processing unit may include an MCU connected to the impedance measurement unit and the LFP measurement unit, and a computer connected to the MCU.

[0021] The system may also optionally further include a main switch for connecting or grounding the second electrode to the impedance measuring unit.

[0022] The device may further include a third electrode positioned at a distance from the first and second electrodes, and a stimulus signal generating unit that applies an electrical stimulus signal to the third electrode.

[0023] An electrical non-destructive real-time cell monitoring method according to an exemplary embodiment of the present invention includes step A of simultaneously measuring impedance and LFP and outputting the first digital signal, step B of converting the first digital signal into a frequency domain, step C of filtering the signal converted into the frequency domain with a low-pass filter (LPF) or a notch filter, and step D of converting the signal subjected to step C into a time domain to generate a second digital signal.

Advantages of the Invention

[0024] According to an embodiment of the present invention, the impedance and the local electric field potential of a cell can be simultaneously monitored. As a result, since the impedance and the local electric field potential can be simultaneously measured for the same specimen, various parameters can be extracted to more accurately understand the electrical activity and characteristics of the cell. For example, the measurement of impedance can monitor not only the adhesion and viability of the cell but also spontaneous contractions, and the analysis of electrical signals such as changes in the ion channels of the cell can be performed by analyzing the magnitude and period of the spikes of the cell by measuring the local electric field potential. In addition, cell experiments of more diverse events using weak current stimulation signals are also possible.

Brief Description of the Drawings

[0025] [Figure 1] It is a diagram schematically showing an electrical non-destructive real-time cell monitoring device according to an embodiment of the present invention. [Figure 2] It is a diagram for explaining an electrical non-destructive real-time cell monitoring device according to an embodiment of the present invention. [Figure 3] It is a diagram for explaining the front end of an electrical non-destructive real-time cell monitoring device according to an embodiment of the present invention. [Figure 4] It is a flowchart schematically showing an electrical non-destructive real-time cell monitoring method according to an embodiment of the present invention. [Figure 5] This figure illustrates a first digital signal and its converted signal in an electrically non-destructive real-time cell monitoring device according to one embodiment of the present invention. [Figure 6] This figure illustrates a noise-filtered signal and its converted signal in an electrically non-destructive real-time cell monitoring device according to one embodiment of the present invention. [Figure 7] This is a diagram illustrating the measurement results of impedance to keratinocytes. [Figure 8] This is a diagram illustrating the measurement results of impedance to keratinocytes. [Figure 9] This diagram illustrates the measurement results of LFP. [Modes for carrying out the invention]

[0026] The advantages and features of the present invention, as well as methods for achieving them, will become clear with reference to the embodiments detailed below in conjunction with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below and can be embodied in a variety of different forms. These embodiments are provided to complete the disclosure of the present invention and to fully inform those who are ordinary skill in the art to which the invention pertains. The same reference numerals are used throughout the specification for the same components.

[0027] The terms used herein are for illustrative purposes only and are not intended to limit the invention. In this specification, singular terms include plural terms unless otherwise specified. Components, steps, operations and / or elements described as “comprise” and / or “comprising” in this specification do not preclude the presence or addition of one or more other components, steps, operations and / or elements.

[0028] All terms used herein have the same meaning as generally understood by a person of ordinary skill in the art to which the present invention pertains, unless otherwise defined. Terms defined in commonly used dictionaries should be interpreted in accordance with their meaning in the context of the relevant art and not in an ideal or overly formal sense unless expressly defined herein.

[0029] The configuration and effects of the present invention will be described in more detail below with reference to the attached drawings.

[0030] Figure 1 is a schematic diagram showing an electrically non-destructive real-time cell monitoring device 1000 according to one embodiment of the present invention; Figure 2 is a diagram for explaining the electrically non-destructive real-time cell monitoring device 1000 according to one embodiment of the present invention; Figure 3 is a diagram for explaining the front end 210 of the electrically non-destructive real-time cell monitoring device 1000 according to one embodiment of the present invention; Figure 4 is a flowchart schematicly showing an electrically non-destructive real-time cell monitoring method according to one embodiment of the present invention; Figure 5 is a diagram for explaining the first digital signal and its converted signal in the electrically non-destructive real-time cell monitoring device 1000 according to one embodiment of the present invention; Figure 6 is a diagram for explaining the noise-filtered signal and its converted signal in the electrically non-destructive real-time cell monitoring device 1000 according to one embodiment of the present invention; Figure 7 is a diagram for explaining the measurement results of impedance to keratinocytes; Figure 8 is a diagram for explaining the measurement results of impedance to keratinocytes; and Figure 9 is a diagram for explaining the measurement results of LFP.

[0031] An electrically non-destructive real-time cell monitoring device 1000 according to one embodiment of the present invention includes an impedance measurement unit 200, an LFP measurement unit 300, and an acquired signal processing unit. Furthermore, the electrically non-destructive real-time cell monitoring device 1000 according to one embodiment of the present invention may further include a cell chip 100, a main switch SW1, a stimulus signal generation unit 600, and a computer 800, etc.

[0032] In one embodiment, the first electrode 121 is provided so as to be in contact with the cell, and the second electrode 122 is provided at a position spaced apart from the first electrode 121. In one embodiment, a third electrode 123 can be provided at a position spaced apart from the first electrode 121 and the second electrode 122. In one embodiment, the first electrode 121, the second electrode 122, and the third electrode 123 can be mounted on a cell chip 100, and in particular, on a cell well 110. In one embodiment, the first electrode 121 may be called a measuring electrode or working electrode, the second electrode 122 may be called a reference electrode, and the third electrode 123 may be called a stimulating electrode. In one embodiment, the first terminal T1 and the second terminal T2 connected to the impedance measuring unit 200 and the LFP measuring unit 300 can be connected to the first electrode 121 and the second electrode 122, respectively, and the third terminal T3 connected to the stimulus signal generating unit 600 can be connected to the third electrode 123.

[0033] In one embodiment, the cell chip 100 can be provided with a plurality of cell wells 110. Figure 1 illustrates a case where the impedance measurement unit 200, the LFP measurement unit 300, and the stimulation signal generation unit 600 are connected to the first electrode 121, the second electrode 122, and the third electrode 123 of one cell well 110, but the impedance measurement unit 200, the LFP measurement unit 300, and the stimulation signal generation unit 600 may be connected to other cell wells 110 in a similar manner.

[0034] In one embodiment, the impedance measuring unit 200 is connected to the first electrode 121 and the second electrode 122, and an alternating current is applied to the second electrode 122 to measure the impedance between the first electrode 121 and the second electrode 122.

[0035] In one embodiment, the impedance measurement unit 200 may include a front end 210 and a multiplexer 220. In this case, the front end 210 may include a drive power supply 211, an ammeter 212, and switches 213, 214, etc., and the impedance between the first electrode 121 and the second electrode 122 of the cell well 110 can be measured by switching the switches 213, 214. In one embodiment, the impedance of the cell well 110 measured by the ammeter 212 may be converted into a digital signal by an analog / digital converter, etc., and may undergo signal amplification and filtering processes as needed.

[0036] In one embodiment, the LFP measuring unit 300 is connected to a first electrode 121 and a second electrode 122, and measures the local electric field potential between the first electrode 121 and the second electrode 122. In one embodiment, the LFP measuring unit 300 may also include an amplifier 310, a filter 320, and an ADC 330.

[0037] In one embodiment, the acquired signal processing unit is connected to the impedance measurement unit 200 and the LFP measurement unit 300, and can receive the measurement results output from the impedance measurement unit 200 and the LFP measurement unit 300. In one embodiment, the acquired signal processing unit may include an MCU 400, which can be connected to the impedance measurement unit 200, the LFP measurement unit 300, the electrical stimulation generation unit 600, the main switch SW1, etc. In one embodiment, the acquired signal processing unit can process the measurement results received from the impedance measurement unit 200 and the LFP measurement unit 300. In particular, after the signal processing unit that acquires the first digital signal output by the LFP measurement unit 300 receives it, a second digital signal can be acquired.

[0038] When the impedance measurement unit 200 and the LFP measurement unit 300 operate simultaneously during measurement, the influence of the AC power supply applied to the second electrode 122 for impedance measurement is reflected in the LFP measurement result. In other words, this means that the first digital signal output by the LFP measurement unit 300 contains noise from the AC power supply, and the electrically non-destructive real-time cell monitoring device 1000 according to one embodiment of the present invention can acquire a second digital signal from which the noise has been removed. For this purpose, the acquired signal processing unit can obtain the second digital signal by converting the first digital signal to the frequency domain, filtering out the noise, and then converting it to the time domain. The process of converting the first digital signal to the frequency domain can be performed by a Fourier transform using Equation 1.

[0039]

number

[0040] Here, X(k) is a signal in the frequency domain, x(n) is a signal in the time domain, k is the frequency, n is the time, N is any real number greater than 0, e is a natural constant, i is the imaginary unit, and Σ represents the partial sum of the sequence.

[0041] In one embodiment, the acquired signal processing can be implemented using the aforementioned MCU400 and memory.

[0042] In another embodiment, the process of processing the first digital signal and acquiring a second digital signal can be performed in a computer 800 connected to the MCU 400 described above. In this case, the acquired signal processing may include the MCU 400 and the computer 800. In this case, the computer 800 and the MCU 400 can be connected by various cables such as a USB cable. On the other hand, the MCU 400, power supply unit 700, impedance measurement unit 200, LFP measurement unit 300, stimulus signal generation unit 600, main switch SW1, etc., can be called the measuring instrument body MB. This measuring instrument body MB may be individually packaged in a separate housing (not shown), or it may be connected to the computer 800 by a USB cable or the like, and various cell chips 100 can be connected to the measuring instrument body.

[0043] In one embodiment, the measuring instrument body MB can be controlled by a separate drive program driven by the computer 800, or data received from the measuring instrument body MB can be processed. In one embodiment, the data collected by the measuring instrument body MB can be supplied to the computer 800 via a USB cable and processed, and presented to the user in various indicators such as numerical values ​​and graphs. For example, Figures 7 and 8 show the results of measuring impedance at a frequency of 100 kHz while varying the injection concentration of keratinocytes, and Figure 9 exemplifies the results of detecting electrical signals after administering various drugs to human-derived cardiomyocyte stem cells. By analyzing the graphs shown in Figure 9, it is possible to determine whether or not an arrhythmia has occurred.

[0044] In one embodiment, a main switch SW1 for selectively grounding the second terminal T2 may be further provided. For example, if it is necessary to measure LFP but impedance measurement is not required, LFP measurement can be made simpler by grounding the second electrode 122 using the main switch SW1. In other words, when the second electrode 122 is grounded, it can operate in the same manner as a conventional LFP measuring instrument.

[0045] In one embodiment, the main switch SW1 may connect the second electrode 122 to an AC power supply. That is, when impedance measurement and LFP measurement are performed simultaneously, the main switch SW1 can be made to apply an AC power supply to the second electrode 122. On the other hand, the selective connection of the main switch SW1 can be determined according to a control command from the MCU 400 or computer 800.

[0046] In one embodiment, when LFP measurement is performed alone, the second electrode 122 may be grounded. In one embodiment, when LFP measurement and impedance measurement are performed simultaneously, an AC signal with a frequency higher than the frequency of the local electric field potential signal of the target cell can be applied to the second electrode 122. This minimizes interference to impedance measurement caused by LFP measurement. However, when simultaneous measurement is performed, noise is generated in the measurement of the local electric field potential due to the influence of the AC signal used for impedance measurement, making it difficult to analyze the index using raw data. According to one embodiment of the present invention, the problem of LFP measurement caused by the AC signal used for impedance measurement is resolved, and the index can be analyzed.

[0047] In one embodiment, the stimulus signal generation unit 600 can perform the function of applying an electrical stimulus signal to the third electrode 123, and includes a DAC 610 and a constant current circuit 620.

[0048] In one embodiment, the user can set the characteristics of the stimulus signal using a computer 800 or the like.

[0049] In one embodiment, the preset stimulus signal may be a digital signal. The stimulus signal generation unit 600 receives the digital signal, converts it to an analog signal using the DAC 610, stabilizes it using the constant current circuit 620, and then provides it to the third electrode 123. Typically, a balanced two-phase pulse waveform is used as the stimulus signal, and the timing and amplitude of the two-phase pulse can be controlled by software and an internal algorithm.

[0050] An electrically non-destructive real-time cell monitoring method according to one embodiment of the present invention may include the steps of: simultaneously measuring impedance and LFP and outputting the first digital signal; converting the first digital signal to the frequency domain; filtering the signal converted to the frequency domain with a low-pass filter (LPF) or notch filter; and converting it to the time domain to generate a second digital signal.

[0051] Referring to Figure 4, first, impedance measurement and LFP measurement are performed simultaneously (S110).

[0052] Next, the measured results are output, and at this time, the LFP measurement results are output as a first digital signal (S120).

[0053] Next, the first digital signal is converted to the frequency domain (S130). Figure 5a shows the first digital signal, and Figure 5b shows the result of converting the first digital signal to the frequency domain. Noise NS can be seen in Figure 5b. This noise is caused by the influence of the AC power supply used in the impedance measurement process.

[0054] Next, a noise filtering process S140 is performed. In one embodiment, the noise filtering process can be performed by filtering the signal converted to the frequency domain with a low-pass filter (LPF) or a notch filter.

[0055] Next, a second digital signal is generated by converting the noise-removed signal back into the time domain (S150).

[0056] As a result, this method solves the conventional problem where simultaneous measurement of impedance and local electric field potential was difficult due to mutual interference when detecting changes in the electrical properties of cells. Furthermore, it allows simultaneous measurement of impedance and local electric field potential for the same experimental specimen, enabling the extraction of various parameters and a more accurate understanding of the characteristics of the electrical activity of cells.

[0057] Although representative embodiments of the present invention have been described in detail above, those with ordinary skill in the art to which the present invention pertains will understand that various modifications can be made to the embodiments described above without departing from the scope of the present invention. Therefore, the scope of the rights of the present invention should not be limited to the disclosed embodiments, but should be defined not only by the claims described below, but also by the claims and their equivalents. [Explanation of symbols]

[0058] 1000 Electrical Non-destructive Real-Time Cell Monitoring Device 100 cell chip 110 cell wells 121 First electrode 122 Second electrode 123 Third electrode 200 Impedance Measurement Section 210 Front End 211 Power supply 212 Ammeter 220 Multiplexer 300 LFP measurement section 310 Amplifier 320 filters 330 ADC T1 First terminal T2 Second terminal T3 Third terminal 400 MCU 500 Main Switch 600 Stimulus signal generation section 610 DAC 620 constant current circuit 700 Power supply section 800 Computers MB measuring instrument body

Claims

1. In an electrically non-destructive real-time cell monitoring device that monitors cell impedance and local field potential (LFP), The first electrode into which the cells are in contact, A second electrode is provided at a position spaced apart from the first electrode, An impedance measuring unit connected to the first electrode and the second electrode, to which an AC power supply is applied, An LFP measuring unit connected to the first electrode and the second electrode, A signal processing unit connected to the impedance measurement unit and the LFP measurement unit receives and processes the measurement results, Includes, The impedance measuring unit applies alternating current to the second electrode to measure the impedance between the first electrode and the second electrode. The LFP measuring unit measures the local electric field potential between the first electrode and the second electrode and outputs a first digital signal. The impedance measurement unit and the LFP measurement unit operate simultaneously and measure the impedance and LFP at the same time point in time. The acquired signal processing unit processes the first digital signal to acquire a second digital signal. An electrically non-destructive real-time cell monitoring device, characterized in that the second digital signal is a signal obtained by removing noise generated by the impedance measuring unit from the first digital signal.

2. The electrically non-destructive real-time cell monitoring apparatus according to claim 1, characterized in that the acquired signal processing unit converts the first digital signal to the frequency domain, filters out noise, converts it to the time domain, and acquires a second digital signal.

3. The electrical non-destructive real-time cell monitoring apparatus according to claim 2, characterized in that the acquired signal processing unit converts the first digital signal into the frequency domain by applying formula 1: [Math 1] In the formula, X(k) is a signal in the frequency domain, x(n) is a signal in the time domain, k is frequency, n is time, N is any real number greater than 0, e is a natural constant, i is the imaginary unit, and Σ represents the partial sum of a sequence.

4. The aforementioned acquired signal processing unit is: The impedance measurement unit and the LFP measurement unit are connected to the MCU, The electrically non-destructive real-time cell monitoring device according to claim 1, characterized by comprising a computer connected to the MCU.

5. The electrically non-destructive real-time cell monitoring apparatus according to claim 1, further characterized by selectively including a main switch for connecting or grounding the second electrode to the impedance measuring unit.

6. A third electrode is provided at a position separated from the first electrode and the second electrode, A stimulation signal generation unit that applies an electrical stimulation signal to the third electrode, It further includes, The stimulus signal generation unit is, A DAC that converts the received digital signal into an analog signal, The electrically non-destructive real-time cell monitoring apparatus according to claim 1, comprising a constant current circuit for supplying the stabilized analog signal to the third electrode.

7. In an electrically non-destructive real-time cell monitoring method using the apparatus described in claim 1, Step A involves simultaneously measuring impedance and LFP and outputting the first digital signal, Step B, which converts the first digital signal into the frequency domain, Step C involves filtering the signal converted to the frequency domain with a low-pass filter (LPF) or a notch filter, Step D involves converting the signal from step C into a time domain and generating a second digital signal. A method for electrically non-destructive real-time cell monitoring, characterized by including the following:

8. The method for electrically non-destructive real-time cell monitoring according to claim 7, characterized in that step B involves applying formula 1 to convert the first digital signal into a frequency domain: [Math 2] In the formula, X(k) is a signal in the frequency domain, x(n) is a signal in the time domain, k is frequency, n is time, N is any real number greater than 0, e is a natural constant, i is the imaginary unit, and Σ represents the partial sum of a sequence.

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