An electrical non-destructive real-time cell monitoring device and method

Abstract

An electro-nondestructive real-time cell monitoring device according to an exemplary embodiment of the present invention includes: a first electrode in contact with a cell; a second electrode disposed 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, receiving and processing measurement results. The impedance measuring unit measures the impedance between the first electrode and the second electrode by applying alternating current to the second electrode, and the LFP measuring unit outputs a first digital signal by measuring the local field potential between the first electrode and the second electrode. The impedance measuring unit and the LFP measuring unit operate simultaneously, and the signal processing unit obtains a second digital signal by processing the first digital signal.

Description

Technical Field

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

[0002] The inventors of this invention have conducted research and development on the application of ITO electrodes to an Electric Cell-Substrate Impedance Sensing (ECIS) sensor, and proposed an ECIS system for measuring cell impedance in conjunction with an ECIS sensor. This system, by using a DAQ plate, can achieve more accurate cell response characteristics and lower manufacturing costs. The related technology has been described in Patent Document 1.

[0003] Safety or hazard assessments are essential for substances used in the development of chemicals, cosmetics, medical devices, and pesticides.

[0004] Animal experiments have been used to conduct such assessments, but animal models have limitations in confirming efficacy and effects in humans due to species differences. At the same time, with the increasing ethical issues surrounding animal experiments, research into alternative animal testing methods is ongoing.

[0005] Recently, the U.S. FDA drafted a bill to eliminate the need for animal testing in new drug trials, which has been signed by the U.S. President. It is expected that the requirements for animal alternative testing will increase further in the future.

[0006] On the other hand, non-destructive real-time cell analysis technology can overcome the limitations of existing endpoint cytotoxicity assessment methods based on staining reagents, enabling faster and more accurate cell analysis.

[0007] For non-destructive real-time cell analysis techniques, there are techniques such as electrocellular-substrate impedance measurement and local field potential measurement.

[0008] Electrocellular-substrate impedance sensing (ECIS) is a cell analysis technique developed by Giaver and Keese. By applying an alternating electric field to cells covered with electrodes and simultaneously measuring both in-phase and out-of-phase potentials, frequency-dependent impedance can be obtained. This ECIS method, as a cell analysis technique that allows for long-term measurements without affecting cell function, is suitable for biological research.

[0009] Local field potential (LTP) measurement technology can be used to measure signals of regular beating, such as those of myocardial cells, or to measure various electrical signal changes in nerve cells. It is a cell analysis technology that can monitor changes in cells in response to chemical substances, drugs, anticancer therapeutic agents, etc.

[0010] Impedance measurement and local field potential measurement are suitable techniques for non-destructive real-time cell analysis and monitoring. However, when detecting changes in the electrical properties of cells, simultaneous measurement of impedance and local field potential can lead to mutual interference, making simultaneous measurement impossible. In particular, the reference voltage of the local field potential introduces errors in impedance measurement, while conversely, the AC signal used for impedance measurement can cause unacceptable interference to local field potential measurement.

[0011] Patent Document 1 describes an apparatus and method for parallel recording of impedance spectra and field potentials. It also discusses problems encountered when simultaneously performing impedance and EPR measurements and proposes techniques to address these issues, such as performing impedance and EPR measurements via time division and improving the switching device to reduce noise during method switching. However, Patent Document 1 still fails to resolve the noise problem when performing impedance and LFP measurements at the same time point. Summary of the Invention

[0012] The problem that the invention aims to solve One aspect of the present invention provides an electrically non-destructive real-time cell monitoring technique capable of simultaneously monitoring cell impedance and local field potential.

[0013] The technical problems of this invention are not limited to those mentioned above. Those skilled in the art can clearly understand other technical problems not mentioned through the following description.

[0014] means for solving problems An electro-nondestructive real-time cell monitoring device according to an exemplary embodiment of the present invention includes: a first electrode in contact with a cell; a second electrode disposed 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, receiving and processing measurement results. The impedance measuring unit measures the impedance between the first electrode and the second electrode by applying an alternating current to the second electrode. The LFP measuring unit outputs a first digital signal by measuring the local field potential between the first electrode and the second electrode. The impedance measuring unit and the LFP measuring unit operate simultaneously. The signal processing unit obtains a second digital signal by processing the first digital signal. The second digital signal is a signal obtained by removing noise generated by the impedance measuring unit from the first digital signal.

[0015] At this time, the signal processing unit can convert the first digital signal to the frequency domain, filter the noise, and then convert it to the time domain to obtain the second digital signal.

[0016] In addition, the signal processing unit can convert the first digital signal to the frequency domain using mathematical formula 1.

[0017] [Mathematical Expression 1]

[0018] Here, X(k) represents the signal in the frequency domain, x(n) represents the signal in the time domain, k represents the frequency, n represents the time, e represents the natural constant, and i represents the imaginary unit.

[0019] Additionally, the 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.

[0020] Additionally, a main switch may be included, which selectively connects the second electrode to the impedance measuring unit or grounds the second electrode.

[0021] Additionally, it may include: a third electrode disposed at a position spaced apart from the first electrode and the second electrode; and a stimulation signal generation unit that applies an electrical stimulation signal to the third electrode.

[0022] The electrically non-destructive real-time cell monitoring method according to an exemplary embodiment of the present invention is characterized by comprising: step A, outputting the first digital signal by simultaneously measuring impedance and LFP; step B, converting the first digital signal to the frequency domain; step C, filtering the signal converted to the frequency domain by a low-pass filter (LPF) or a notch filter; and step D, generating a second digital signal by converting the signal after step C to the time domain.

[0023] Invention Effects According to one embodiment of the present invention, cellular impedance and local field potential can be monitored simultaneously. As a result, impedance and local field potential can be measured simultaneously on the same experimental subject, thus enabling a more accurate understanding of cellular electrical activity and characteristics by extracting various parameters. For example, impedance measurement can monitor not only cell attachment and viability but also spontaneous contraction. Analysis of the magnitude and cycle of cellular spike potentials based on local field potential measurements allows for the analysis of electrical signals such as changes in cellular ion channels. Furthermore, more diverse cell experiments utilizing microcurrent stimulation signals can be performed. Attached Figure Description

[0024] Figure 1 This is a schematic diagram illustrating an electrically non-destructive real-time cell monitoring device according to an embodiment of the present invention.

[0025] Figure 2 This is a diagram illustrating an electrically non-destructive real-time cell monitoring device according to an embodiment of the present invention.

[0026] Figure 3 This is a diagram illustrating the front end of an electrically non-destructive real-time cell monitoring device according to an embodiment of the present invention.

[0027] Figure 4 This is a flowchart schematically illustrating an electrically non-destructive real-time cell monitoring method according to an embodiment of the present invention.

[0028] Figure 5 This is a diagram illustrating a first digital signal and its converted signal in an electrically non-destructive real-time cell monitoring device according to an embodiment of the present invention.

[0029] Figure 6 This is a diagram illustrating the signal and its conversion signal in an electrically non-destructive real-time cell monitoring device according to an embodiment of the present invention.

[0030] Figure 7 This is a graph used to illustrate the results of impedance measurements on skin keratinocytes.

[0031] Figure 8This is a graph used to illustrate the results of impedance measurements on skin keratinocytes.

[0032] Figure 9 This is a graph used to illustrate the LFP measurement results. Detailed Implementation

[0033] The advantages, features, and methods of implementing the present invention will become apparent from the accompanying drawings and the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below, but can be implemented in various different forms. These embodiments are provided only to complete the disclosure of the invention and to fully inform those skilled in the art of its scope. Throughout this specification, the same reference numerals may denote the same constituent elements.

[0034] The terminology used in this specification is for illustrative purposes and is not intended to limit the invention. In this specification, the singular form includes the plural form unless otherwise specified in the sentence. The terms "comprises" and / or "comprising" as used in this specification refer to the mentioned constituent elements, steps, actions, and / or components, without excluding the presence or addition of more than one other constituent element, step, action, and / or component.

[0035] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms defined in common dictionaries should be interpreted as having the same meaning as in the relevant technical context, and should not be construed as having an idealized or overly formalized meaning unless expressly defined in this application.

[0036] The structure and effects of the present invention will now be described in more detail with reference to the accompanying drawings.

[0037] Figure 1 This is a schematic diagram illustrating an electrically non-destructive real-time cell monitoring device (1000) according to an embodiment of the present invention. Figure 2 This is a diagram illustrating an electrically non-destructive real-time cell monitoring device (1000) according to an embodiment of the present invention. Figure 3 This is a diagram illustrating the front end (210) of an electrically non-destructive real-time cell monitoring device (1000) according to an embodiment of the present invention. Figure 4 This is a flowchart schematically illustrating an electrically non-destructive real-time cell monitoring method according to an embodiment of the present invention. Figure 5 This is a diagram illustrating a first digital signal and its converted signal in an electrically non-destructive real-time cell monitoring device (1000) according to an embodiment of the present invention. Figure 6This is a diagram illustrating the signal and its converted signal in a non-destructive real-time cell monitoring device (1000) according to an embodiment of the present invention. Figure 7 This is a graph used to illustrate the results of impedance measurements on skin keratinocytes. Figure 8 This is a graph used to illustrate the results of impedance measurements on skin keratinocytes. Figure 9 This is a graph used to illustrate the LFP measurement results.

[0038] An electrical nondestructive real-time cell monitoring device (1000) according to an embodiment of the present invention includes an impedance measurement unit (200), an LFP measurement unit (300), and a signal collection and processing unit. Further, the electrical nondestructive real-time cell monitoring device (1000) according to an embodiment of the present invention may also include a cell chip (100), a main switch (SW1), a stimulation signal generation unit (600), a computer (800), etc.

[0039] In one embodiment, the first electrode (121) is configured to contact the cell, and the second electrode (122) is configured at a position spaced apart from the first electrode (121). In one embodiment, the third electrode (123) may be configured 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) may be configured on a cell chip (100), particularly in a cell pore (110). In one embodiment, the first electrode (121) may be referred to as the measuring electrode or the working electrode, the second electrode (122) may be referred to as the reference electrode, and the third electrode (123) may be referred to as the stimulation electrode. In one embodiment, the first terminal (T1) and the second terminal (T2) connected to the impedance measurement unit (200) and the LFP measurement unit (300) may be connected to the first electrode (121) and the second electrode (122), respectively, and the third terminal (T3) connected to the stimulation signal generation unit (600) may be connected to the third electrode (123).

[0040] In one embodiment, the cell chip (100) may have multiple cell pores (110). Figure 1 The diagram shows the impedance measurement unit (200), LFP measurement unit (300), stimulation signal generation unit (600) connected to the first electrode (121), second electrode (122), and third electrode (123) of a cell pore (110). However, the impedance measurement unit (200), LFP measurement unit (300), and stimulation signal generation unit (600) can also be connected to other cell pores (110) in a similar manner.

[0041] In one embodiment, the impedance measuring unit (200) is connected to the first electrode (121) and the second electrode (122), and the impedance between the first electrode (121) and the second electrode (122) is measured by applying AC to the second electrode (122).

[0042] In one embodiment, the impedance measurement unit (200) may include a front end (210) and a multiplexer (220). The front end (210) may include a drive power supply (211), a galvanometer (212), switches (213, 214), etc., and the impedance between the first electrode (121) and the second electrode (122) of the cell pore (110) can be measured by switching the switches (213, 214). In one embodiment, the impedance of the cell pore (110) measured by the galvanometer (212) can be converted into a digital signal by an analog-to-digital converter, etc., or it can be amplified or filtered as needed.

[0043] In one embodiment, the LFP measurement unit (300) is connected to a first electrode (121) and a second electrode (122) and measures the local field potential between the first electrode (121) and the second electrode (122). In one embodiment, the LFP measurement unit (300) may include an amplifier (310), a filter (320), and an ADC (330).

[0044] In one embodiment, the signal processing unit can be connected to the impedance measurement unit (200) and the LFP measurement unit (300) and receive the measurement results output by the impedance measurement unit (200) and the LFP measurement unit (300). In one embodiment, the 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 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 receives the first digital signal output by the LFP measurement unit (300), a second digital signal can be obtained.

[0045] When the impedance measurement unit (200) and the LFP measurement unit (300) operate simultaneously and perform measurements, the influence of the AC power applied to the second electrode (122) for impedance measurement is reflected in the LFP measurement result. That is, the first digital signal output by the LFP measurement unit (300) contains noise caused by the AC power supply, and the electrically non-destructive real-time cell monitoring device (1000) according to an embodiment of the present invention can obtain a second digital signal after removing the noise. For this purpose, the signal processing unit can convert the first digital signal to the frequency domain, filter the noise, and convert it to the time domain to obtain the second digital signal. The process of converting the first digital signal to the frequency domain can be performed using the Fourier transform of mathematical formula 1.

[0046] [Mathematical Expression 1]

[0047] Where X(k) represents the signal in the frequency domain, x(n) represents the signal in the time domain, k represents the frequency, n represents the time, e represents the natural constant, i represents the imaginary unit, and ∑ represents the partial sum of the sequence.

[0048] In one embodiment, the signal processing unit can be implemented by the aforementioned MCU (400) and memory, etc.

[0049] In another embodiment, the process of processing the first digital signal to obtain a second digital signal can also be performed in a computer (800) connected to the MCU (400). In this case, the signal processing unit may include the MCU (400) and the computer (800). At this time, the computer (800) and the MCU (400) can be connected via various data cables such as a USB cable. In addition, the MCU (400), power supply unit (700), impedance measurement unit (200), LFP measurement unit (300), stimulation signal generation unit (600), main switch (SW1), etc., may be included and referred to as the measuring device body (MB). The measuring device body (MB) can be separately packaged in a separate housing (not shown), can be connected to the computer (800) via a USB cable, etc., and the measuring device body can be connected to various cell chips (100).

[0050] In one embodiment, the measuring instrument body (MB) can be controlled or data received from the measuring instrument body (MB) can be processed via a separate driver program running on a computer (800). In one embodiment, the data collected by the measuring instrument body (MB) can be transmitted to the computer (800) via a USB cable and processed to display to the user various indicators such as numerical values ​​and graphs. For example, Figure 7 and Figure 8The results show the impedance measurements at a frequency of 100 kHz while varying the injected concentration of keratinocytes. Figure 9 The results of electrical signal detection after administration of various drugs to human cardiac stem cells are shown, and the analysis... Figure 9 The charts and graphs shown can help determine whether an arrhythmia has occurred.

[0051] In one embodiment, a main switch (SW1) can also be provided to selectively ground the second terminal (T2). For example, in cases where LFP measurement is required but impedance measurement is not required, LFP measurement can be performed more easily by grounding the second electrode (122) using the main switch (SW1). That is, with the second electrode (122) grounded, it can operate in the same manner as existing LFP measurement devices.

[0052] In one embodiment, the main switch (SW1) can connect the second electrode (122) to an AC power source. That is, when performing impedance measurement and LFP measurement simultaneously, the main switch (SW1) can apply AC power to the second electrode (122). Alternatively, the selective connection of the main switch (SW1) can be determined according to control instructions from the MCU (400) or the computer (800).

[0053] In one embodiment, when LFP measurement is performed alone, the second electrode (122) can be grounded. In another embodiment, when LFP measurement and impedance measurement are performed simultaneously, an AC signal with a frequency higher than the local field potential signal of the target cell can be applied to the second electrode (122). This minimizes interference with impedance measurement caused by LFP measurement. However, in the case of simultaneous measurement, noise is generated when measuring the local field potential due to the influence of the AC signal used for impedance measurement, making it difficult to perform index analysis based solely on the raw data. According to an embodiment of the present invention, the problem of LFP measurement caused by the AC signal used for impedance measurement can be solved, and index analysis can be performed.

[0054] In one embodiment, the stimulation signal generation unit (600) can perform the function of applying an electrical stimulation signal to the third electrode (123) and may include a DAC (610) and a constant current circuit (620).

[0055] In one embodiment, the user can set the characteristics of the stimulus signal using a computer (800) or the like.

[0056] In one embodiment, the preset stimulation signal can be a digital signal. After receiving the digital signal, the stimulation signal generation unit (600) can convert the digital signal into an analog signal through a DAC (610), stabilize it through a constant current circuit (620), and then provide it to the third electrode (123). Generally, a balanced two-phase pulse waveform is used as the stimulation signal, and the timing and amplitude of the two-phase pulse can be controlled by software and built-in algorithms.

[0057] An electrically non-destructive real-time cell monitoring method according to an embodiment of the present invention may include the following steps: outputting a first digital signal by simultaneously measuring impedance and LFP; converting the first digital signal to the frequency domain; filtering the signal converted to the frequency domain by a low-pass filter (LPF) or a notch filter; and generating a second digital signal by converting to the time domain.

[0058] Reference Figure 4 First, impedance measurement and LFP measurement are performed simultaneously (S110).

[0059] Next, the measurement results are output. At this time, the LFP measurement results are output as the first digital signal (S120).

[0060] Next, the first digital signal is converted to the frequency domain (S130). Figure 5 (a) shows the first digital signal. Figure 5 (b) shows the result of converting the first digital signal to the frequency domain. Figure 5 (b) indicates the presence of noise (NS). This noise is caused by the influence of the AC power supply used during the impedance measurement.

[0061] Next, the noise filtering process is performed (S140). In one embodiment, the noise filtering process can be performed by using a low-pass filter (LPF) or a notch filter to filter the signal converted to the frequency domain.

[0062] Next, a second digital signal is generated by converting the noise-removed signal back to the time domain (S150).

[0063] As a result, it can solve the existing problem of difficulty in simultaneously measuring impedance and local field potential due to mutual interference when detecting changes in the electrical properties of cells. Furthermore, it can simultaneously measure impedance and local field potential for the same experimental subject, thus allowing for a more accurate understanding of the electrical activity and properties of cells by extracting multiple parameters.

[0064] The foregoing has described in detail typical embodiments of the present invention. However, those skilled in the art will understand that various modifications can be made to the above embodiments without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the described embodiments, but should be defined according to the following claims and their equivalents.

[0065] Explanation of reference numerals in the attached figures 1000: Electrically non-destructive real-time cell monitoring device; 100: Cell chip; 110: Cell pores; 121: First electrode; 122: Second electrode; 123: Third electrode; 200: Impedance measurement section; 210: Front-end; 211: Drive power supply; 212: Ammeter; 220: Multiplexer; 300: LFP Measurement Section; 310: Amplifier; 320: Filter; 330: ADC; T1: First terminal; T2: Second terminal; T3: Third terminal; 400: MCU; 500: Main switch; 600: Stimulus signal generation unit; 610: DAC; 620: Constant current circuit; 700: Power Supply Section; 800: Computer; MB: Measuring instrument body.

Claims

1. A non-destructive real-time cell monitoring device for monitoring cell impedance and local field potential (LFP), characterized in that, The electrically non-destructive real-time cell monitoring device includes: The first electrode is in contact with the cell; The second electrode is positioned at a distance from the first electrode; An impedance measuring unit is connected to the first electrode and the second electrode, and an AC power supply is applied to it. The LFP measuring unit is connected to the first electrode and the second electrode; and The signal processing unit is connected to the impedance measurement unit and the LFP measurement unit to receive and process the measurement results. The impedance measuring unit measures the impedance between the first electrode and the second electrode by applying alternating current to the second electrode. The LFP measurement unit outputs a first digital signal by measuring the local field potential between the first electrode and the second electrode. The impedance measuring unit and the LFP measuring unit operate simultaneously, measuring the impedance and LFP at physically identical time points. The signal processing unit obtains the second digital signal by processing the first digital signal. The second digital signal is the signal obtained by removing the noise generated by the impedance measurement unit from the first digital signal.

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

3. The electrically non-destructive real-time cell monitoring device according to claim 2, characterized in that, The signal processing unit converts the first digital signal to the frequency domain using mathematical formula 1. [Mathematical Expression 1] X(k) represents the signal in the frequency domain, x(n) represents the signal in the time domain, k represents the frequency, n represents the time, N represents any real number greater than 0, e represents the natural constant, and i represents the imaginary unit.

4. The electrically non-destructive real-time cell monitoring device according to claim 1, characterized in that, The signal processing unit includes: The MCU is connected to the impedance measurement unit and the LFP measurement unit; and A computer connected to the MCU.

5. The electrically non-destructive real-time cell monitoring device according to claim 1, characterized in that, The electro-nondestructive real-time cell monitoring device also includes a main switch that selectively connects the second electrode to the impedance measurement unit or grounds the second electrode.

6. The electrically non-destructive real-time cell monitoring device according to claim 1, characterized in that, The electrically non-destructive real-time cell monitoring device also includes: The third electrode is disposed at a position spaced apart from the first electrode and the second electrode; and The stimulation signal generation unit applies an electrical stimulation signal to the third electrode. The stimulation signal generation unit includes: a DAC, which converts the received digital signal into an analog signal; and a constant current circuit, which provides the stabilized analog signal to the third electrode.

7. A non-destructive real-time cell monitoring method, utilizing the device according to claim 1, characterized in that, The electrically non-destructive real-time cell monitoring method includes: Step A: Output the first digital signal by simultaneously measuring the impedance and LFP; Step B: Convert the first digital signal to the frequency domain; Step C involves filtering the signal converted to the frequency domain using a low-pass filter (LPF) or a notch filter; and Step D involves generating a second digital signal by converting the signal obtained after step C to the time domain.

8. The electrically non-destructive real-time cell monitoring method according to claim 7, characterized in that, In step B, the first digital signal is converted to the frequency domain using mathematical formula 1. [Mathematical Expression 1] X(k) represents the signal in the frequency domain, x(n) represents the signal in the time domain, k represents the frequency, n represents the time, N represents any real number greater than 0, e represents the natural constant, and i represents the imaginary unit.

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