KR102595673B1 - Apparatus for measuring cell of electrochemical impedance - Google Patents

Abstract

The present invention applies an input signal to a cell measurement device under set frequency scan conditions, determines an input signal for cell state analysis corresponding to cell characteristics by comparing the impedance and phase value for each frequency in response to the input signal, and determines the input signal for cell state analysis corresponding to the determined frequency. Disclosed is an electrochemical impedance device for cells, which is characterized in that the cell state is analyzed by applying an input signal for cell state analysis using voltage.

Description

Electrochemical impedance equipment for cells {APPARATUS FOR MEASURING CELL OF ELECTROCHEMICAL IMPEDANCE}

The present invention relates to electrochemical impedance equipment for cells, and more specifically, to technology for measuring the state of cardiac cells using electrochemical impedance.

Electrochemical Impedance Spectroscopy (EIS) provides a method of scanning from high to low frequencies, sequentially applying sine wave waveforms to the sample, and analyzing the response according to the sine wave coming through the sample. This is a method of analyzing impedance by measuring changes in amplitude and phase.

Electrochemical impedance spectroscopy can extract the equivalent circuit or each parameter of the sample through electrochemical Nyquist analysis (nyquist plot), interpret the impedance graph, and infer changes in other electrochemical reactions. . Electrochemical Nyquist analysis refers to a graph expressed in the form of complex numbers so that one coordinate is drawn at one frequency.

Conventionally, electrochemical phenomena of batteries have been analyzed using electrochemical impedance spectroscopy, but there is a problem in that it is difficult to measure cell conditions such as movement, state, reaction, and abnormality of cells through electrochemical impedance spectroscopy.

More specifically, in the related art, each cell has different unique characteristics and a different unique frequency to which each characteristic responds, and there is a problem in that it is difficult to find changes in the cell's unique impedance and phase only through the frequency scanning method.

Screen printed electrodes are used in the bio field, and are typically used to measure blood sugar, as in patent documents described as prior art. One end of the screen print electrode is connected to the measuring device, and the other end is exposed to contact the sample.

However, conventionally, there is a culture medium for culturing heart cells, and the culture medium may be contaminated or leak on the screen-printed electrode, so there is a problem in that it is difficult to observe the state of heart cells using a screen-printed electrode.

In order to improve this problem and use a screen-printed electrode, the present invention exposes the reaction part where the cells are placed in the screen-printed electrode and injects the culture medium through the holder for reaction exposure, thereby reducing the possibility of contamination or leakage of the culture medium.

Korean Patent No. 10-1352665

In order to solve the above problem, the present invention applies an input signal to a cell measurement device under set frequency scan conditions, compares the impedance and phase value for each frequency in response to the input signal, and generates an input signal for cell state analysis corresponding to cell characteristics. Provides electrochemical impedance equipment for cells that analyzes the state of cells by applying an input signal for cell state analysis with a voltage of a determined frequency.

The electrochemical impedance equipment for cells according to an embodiment of the present invention for the above problem to be solved applies an input signal to the cell measurement device 200 under set frequency scan conditions, and provides impedance for each frequency in response to the input signal. It is characterized by comparing the phase values to determine the input signal for cell state analysis corresponding to the cell characteristics, and analyzing the state of the cell by applying the input signal for cell state analysis with a voltage of the determined frequency.

Electrochemical impedance equipment for cells according to an embodiment of the present invention includes a transceiver 310 that applies an input signal for a cell reaction to a cell measurement device and receives an output signal in response to the cell reaction; A measuring unit 320 that measures the impedance and phase value of the cell using the output signal received from the cell measuring device; An analysis unit 330 analyzes the state of the cell by monitoring changes in measured impedance and phase values, and controls the operation of the transmitting and receiving unit by determining input signals for scanning by frequency under set frequency scan conditions, and scanning by frequency from the measuring unit. It includes a control unit 390 that receives the impedance and phase value generated using the output signal from the input signal, and compares the impedance and phase value for each frequency to determine the input signal for cell state analysis, and the transceiver unit is a control unit. An input signal for cell state analysis may be applied with a voltage of a frequency determined in , and the analysis unit may be characterized in that it analyzes the state of the cell based on an output signal from the input signal for cell state analysis.

The measuring unit may measure impedance according to frequency using a lock-in amplifier algorithm.

The control unit corrects the impedance and phase value for each frequency using a smooth algorithm, and differentiates the corrected impedance and phase value using a differentiation algorithm, and the change value of the differentiated impedance and phase value for each frequency is the maximum. It may be characterized in that the input signal for cell state analysis is determined by selecting the frequency.

The present invention determines the optimal frequency corresponding to the unique characteristics of each cell, and by applying a voltage of the optimal frequency corresponding to the unique characteristics of each cell, the state of each cell can be monitored using electrochemical impedance spectroscopy, and the impedance and phase The accuracy or sensitivity of value measurement can be improved.

Figure 1 shows an electrochemical impedance-type cell measurement system according to an embodiment of the present invention.
Figure 2 is an example showing a screen printed electrode.
Figure 3 is an example showing an assembled cytometry device.
Figure 4 is an example showing a base holder.
Figure 5 is an example showing a holder for electrodes.
Figure 6 is an example showing a substrate.
Figure 7 is an example showing a fog pin.
Figure 8 shows an example of coupling the electrode holder to the base holder using the first locking screw.
Figure 9 is an example showing the state of partial coupling and complete coupling of the base holder and the electrode holder.
Figure 10 is an example showing the state after combination of the base holder and the reaction exposure holder.
Figure 11 is an example showing a holder for reaction exposure.
Figure 12 is an example illustrating a conventional electrochemical impedance analysis concept.
Figure 13 is an example of measuring the impedance and phase of a cell through conventional electrochemical impedance analysis.
Figure 14 is a block diagram showing an electrochemical impedance device for cells according to an embodiment of the present invention.
Figure 15 is an example showing a method of measuring impedance according to frequency.
Figure 16 is a graph showing impedance and phase values at 50Hz, 150Hz, and 2KHz.
Figure 17 is a graph showing impedance and phase values to which the smooth algorithm is applied.
Figure 18 is an example showing a method of measuring impedance and phase value using a differential algorithm.
Figure 19 is a graph showing the impedance and phase values to which the differentiation algorithm is applied.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings and the contents described in the accompanying drawings, but the present invention is not limited or limited by the embodiments.

Figure 1 shows an electrochemical impedance-type cell measurement system according to an embodiment of the present invention. The cell measurement system 10 includes a screen print electrode 100, a cell measurement device 200, and an electrochemical impedance device 300. ) includes.

Cells are placed on the screen print electrode 100, and the cell measurement device 200 fixes the screen print electrode 100. The electrochemical impedance device 300 applies a signal to the cell measurement device 200 and receives a response from the cell measurement device to measure the state of the cell.

Figure 2 is an example of a screen print electrode, and the screen print electrode 100 includes a hand part 110, a reaction part 120, and an electrode part 130. The hand portion 110 is in contact with a person's hand or a tool such as tongs or tweezers. For example, the hand part 110 is picked up by hand.

The reaction unit 120 is where cells are placed, and the electrode unit 130 is for connection to the electrode fog pin 211, which will be described later. The reaction unit 120 and the electrode unit 130 are connected by a printed electrode. Electrodes are classified into Working Electrode (WE), Counter Electrode (CE), and Reference Electrode (RE).

The working electrode is an electrode for viewing actual cell reactions, the counter electrode is an electrode that sends a current to cause a reaction to occur on the surface of the working electrode, and the reference electrode is an electrode that sets the standard for the potential applied to the working electrode, providing precise control and Used for measurement.

Figure 3 is an example showing an assembled cell measurement device, the cell measurement device 200 includes a holder (230, 240, 250), a fog pin for electrodes (211), a fog pin for equipment connection (212), and a substrate (220). ) and locking screws (213, 214).

The holders 230, 240, and 250 are composed of a plurality of holders and are assembled together. Once assembly is completed, the screen print electrode 100 is fixed and the reaction portion 120 on which the cells are placed is exposed in the screen print electrode 100. . The cell measurement device allows the injection of culture medium into the reaction section where the cells are placed by assembling the holders (230, 240, 250), provides an environment for measuring the state of the cultured cells, and provides an environment for measuring the state of the cultured cells. Use to prevent leakage and contamination of culture medium.

Figure 4 is an example of a base holder, and the holders 230, 240, and 250 include a base holder 230, an electrode holder 240, and a reaction exposure holder 250. The base holder 230 is combined with the electrode holder 240 and surrounds the substrate 220 and the electrode fog pin 211.

The base holder 230 is formed with a plurality of holes for coupling the electrode holder 240 and the reaction exposure holder 250. The base holder 230 may include a seating portion for a substrate 231, a seating portion for an electrode 232, a grip portion 233, and a retractable space portion 234.

The substrate seating portion 231 provides seating of the substrate 220. The electrode seating portion 232 is formed on one side of the substrate seating portion 231 and provides seating for the screen print electrode 100. The grip portion 233 consists of a pair and is formed on one side of the electrode seating portion 232, and provides a grip function. The retractable space 234 is formed between the grip portions 233 and provides a space for inserting or withdrawing the screen print electrode 100.

The user can move or fix the cell measurement device 200 by holding the grip portion 233 with one hand, and hold the hand portion 110 with the other hand to attach the screen print electrode 100 to the electrode seating portion 232. ) can be inserted by sliding, and when inserting the screen print electrode 100 into the electrode seating part 232, it can be inserted through the free space of the withdrawal/inlet space 234.

The present invention can stably replace the screen print electrode 100 through the retractable space 234. In addition, the present invention allows the screen print electrode 100 to be stably inserted into the electrode seating portion 232 due to the structural characteristics of the base holder 230, and the screen print electrode 100 cannot be damaged during the insertion process. Existing risk factors can be removed, and with the risk factors removed, accurate and reliable measurement data can be obtained.

Figure 5 is an example showing an electrode holder, and Figure 6 is an example showing a substrate, and the electrode holder 240 is used to fix the substrate. The substrate 220 provides electrical connection between the fog pin 211 for electrodes and the fog pin 212 for equipment connection. The substrate 220 may be a printed circuit board (PCB) to provide electrical connections.

The substrate 220 is formed with a plurality of holes for coupling the base holder 230 and the electrode holder 240 and the fog pins 211 and 212. The fog pin 212 for equipment connection may have a predetermined protrusion length in order to protrude from the electrode holder 240 and connect to the cable. The fog pins 211 and 212 can prevent the risk of damage to the electrode and provide stable contact or height adjustment.

Figure 7 is an example showing a fog pin, where the fog pin 211 for electrode provides a connection with the electrode part 130 of the screen print electrode 100, and the fog pin 212 for equipment connection is an electrochemical impedance equipment. Provides connection to (300). The fog pin 211 for electrodes or the fog pin 212 for equipment connection may be fixed to the board 220 through soldering or interference fitting.

Figure 8 shows an example of coupling the electrode holder to the base holder using the first locking screw, and the locking screws 213 and 214 include the first locking screw 213 and the second locking screw 214. do. The first locking screw 213 is used to fix the electrode holder 240 to the base holder 230, and the second locking screw 214 is used to fix the reaction exposure holder 250 to the base holder 230. It is used for. The first locking screw 213 and the second locking screw 214 may have the same material and shape.

Figure 9 is an example showing a state of partial coupling and complete coupling of the base holder and the electrode holder, and the cell measurement device 200 may further include an elastic means 260. The elastic means 260 is formed between the electrode holder 240 and the base holder 230, and elasticity changes occur by tightening and loosening the first locking screw 213. The elastic means 260 may typically be a spring.

The first locking screw 213 is partially tightened to secure the electrode holder 240 to the base holder 230, and when the screen print electrode 100 is inserted in a sliding manner and connected to the electrode fog pin 211, Fully tightened.

One end of the equipment connection fog pin 212 is coupled to the substrate 220, and the other end protrudes outward through a hole formed in the electrode holder 240. Conventionally, alligator-claw cables were used to directly electrically connect a device that measures impedance with a sample such as a battery, but it may be difficult to directly connect an alligator-claw cable to a sample such as cells due to contamination issues.

If the alligator clip cable is directly connected to the electrode portion 130 of the screen print electrode 100, damage to the electrode portion 130 may occur, so it is not recommended.

In order to solve the problem of contamination of the sample and damage to the electrode unit 130, the present invention can accurately measure the impedance of a sample such as a cell by indirectly connecting an alligator clip cable to the fog pin 212 for connecting equipment.

The elastic means 260 provides a sliding space when the screen print electrode 100 is inserted, and prevents damage to the electrode fog pin 211 and the electrode portion 130 of the screen print electrode 100 when they contact each other. It can be prevented.

Figure 10 is an example showing the state after the combination of the base holder and the reaction exposure holder, and Figure 11 is an example showing the reaction exposure holder, where the reaction exposure holder 250 is on one side of the electrode holder 240. It is formed in, is coupled to the base holder 230, and provides exposure of the reaction portion 120 of the screen print electrode 100. The second locking screw 214 is used to secure the reaction exposure holder 250 to the base holder 230.

The reaction exposure holder 250 has a brighter color than the base holder 230 for visual identification of the cells, and may be combined with an O-ring 251 to prevent the culture medium from leaking into the internal space.

The base holder 230, the electrode holder 240, and the reaction exposure holder 250 may be manufactured from materials with high sterility and contamination resistance, and may be typically manufactured using polycarbonate materials. The reason why the colors of the base holder 230 and the electrode holder 240 and the color of the reaction exposure holder 250 are different or the reaction exposure holder 250 is manufactured in a transparent color is to measure the impedance of the cell. This is because the user can determine the contamination status of the cells only by changing the color of the cells.

Cells require a culture medium to survive, and in the present invention, the culture medium can be formed only in the reaction part 120 through sealing using the O-ring 251, and leakage of the culture medium can be prevented.

In the cell management and impedance measurement method, the cell measurement device 200 into which the screen printed electrode 110 is inserted is sterilized and then placed in an incubator to culture or manage the cells. For cell culture, cells are attached to the reaction unit 120, and the culture medium is injected into the inner space of the reaction exposure holder 250. When measuring impedance, the cell measuring device 200 with the screen printed electrode 110 inserted is taken out from the incubator and the impedance of the cells is measured using the cell electrochemical impedance device 300.

The present invention can provide an environment for measuring electrical signals of cells, such as their movement, status, reaction, and presence of abnormalities, through a cell measuring device 200, and can be measured using a screen-printed electrode 100 and electrochemical impedance spectroscopy. Changes in heart cells can be measured.

Figure 12 is an example showing the concept of conventional electrochemical impedance analysis. Electrochemical Impedance Spectroscopy (EIS) provides a method of scanning from high frequency to low frequency, and sequentially applies sine waves to the sample. ) This is a method of analyzing impedance by applying a waveform and measuring changes in amplitude and phase according to the sine wave of the response coming through the sample.

Electrochemical impedance spectroscopy can extract the equivalent circuit or each parameter of the sample through electrochemical Nyquist analysis (nyquist plot), interpret the impedance graph, and infer changes in other electrochemical reactions. . Electrochemical Nyquist analysis refers to a graph expressed in the form of complex numbers so that one coordinate is drawn at one frequency.

Figure 13 is an example of measuring the impedance and phase of cells through conventional electrochemical impedance analysis. Conventionally, electrochemical phenomena of batteries were analyzed using electrochemical impedance spectroscopy, but through electrochemical impedance spectroscopy, the movement of cells, There is a problem that it is difficult to measure the state of cells, such as condition, reaction, and presence of abnormalities.

More specifically, if a conventional electrochemical impedance analysis method is used as shown in FIG. 13, it may be difficult to measure the impedance and phase values of cardiac cells, such as their motility, state, or response.

Figure 14 is a block diagram showing an electrochemical impedance device for cells according to an embodiment of the present invention. The electrochemical impedance device 300 for cells applies an input signal to the cell measurement device 200 under set frequency scan conditions. , the input signal for cell state analysis corresponding to the cell characteristics is determined by comparing the impedance and phase value for each frequency in response to the input signal, and the state of the cell is analyzed by applying the input signal for cell state analysis with a voltage of the determined frequency. .

Frequency scan conditions are conditions regarding time, frequency, and voltage. More specifically, frequency scan conditions may include frequency range, increase frequency, and voltage and time conditions. Increased frequency refers to a frequency that increases step by step in the frequency range.

For example, in the present invention, the frequency range is set from 100Hz to 1000HZ, the increase frequency is set to 100Hz, the applied voltage is set to 10mV, and the time is set to 10 seconds. In the present invention, once the setting is completed, an input signal of 10 mV at 100 Hz is applied for 10 seconds to obtain the impedance and phase value for 100 Hz, and then an input signal of 10 mV at 200 Hz is applied for 10 seconds to obtain the impedance and phase value for 200 Hz. Acquire the value and perform a frequency scan operation in the same way until the frequency range is reached.

The electrochemical impedance equipment for cells 300 includes a transmitter/receiver 310, a measurement unit 320, an analysis unit 330, and a control unit 390. The transceiver unit 310 applies an input signal for a cell response to a cell measurement device and receives an output signal in response to the cell response. The transmitting and receiving unit 310 is connected to the fog pin 212 for equipment connection using an alligator clip cable.

The measurement unit 320 measures the impedance and phase value of the cell using the output signal received from the cell measurement device 200, and the analysis unit 330 monitors changes in the measured impedance and phase value to determine the state of the cell. Analyze.

The control unit 390 controls the operation of the transmitter and receiver by determining the input signal for scanning by frequency under the set frequency scan conditions, and receives the impedance and phase value generated using the output signal by the input signal for scanning by frequency from the measuring unit. The input signal for cell state analysis is determined by comparing the impedance and phase values for each frequency.

The transmitting and receiving unit 310 applies an input signal for cell state analysis with a voltage of the frequency determined by the control unit 390, and the analysis unit 330 analyzes the state of the cell based on the output signal from the input signal for cell state analysis. do.

Figure 15 is an example of a method for measuring impedance according to frequency, and the measurement unit 320 can measure impedance according to frequency using a lock-in amplifier algorithm.

The lock-in amplification algorithm is an algorithm that provides amplification and signal detection functions and is used to detect and amplify signals of a specific frequency.

The lock-in amplification algorithm is an algorithm that, when an input signal and a reference signal are input, operates only on the signal for which the reference signal is to be detected and searches for amplitude information of the signal to be searched. The reference signal is a signal of the same frequency as the input signal.

The measuring unit 320 sets a reference signal for the input signal to use the reference signal to extract only the signal to be searched among the input signals, and even if noise is buried in the signal to be measured (output signal), the signal related to the reference signal is generated. The amplitude and phase can be detected.

Figure 16 is a graph showing impedance and phase values at 50 Hz, 150 Hz, and 2 KHz, and the control unit 390 compares the impedance and phase values for each frequency to determine the input signal for cell state analysis. In Figure 16, among 50Hz, 150Hz, and 2KHz, the frequency of 150Hz shows the best sensitivity, and almost no response occurs at 50Hz and 2KHz.

If the graph values of FIG. 16 are applied, the control unit 390 can determine a frequency of 150 Hz as an input signal for cell state analysis. High sensitivity means that the range of change in impedance and phase values over time is large, as shown in the 150Hz graph in FIG. 16.

As there are environmental factors such as cell size, type, and electrode area, it is very important to find the optimal frequency in order to accurately measure the state of the cell. The present invention can automatically find the optimal frequency using the frequency index condition and the smooth algorithm and differentiation algorithm described later.

Figure 17 is a graph showing the impedance and phase values to which the smooth algorithm is applied, and the control unit corrects the impedance and phase values for each frequency using the smooth algorithm.

The smooth algorithm generates an intermediate value between two values, and the concept of interpolation is applied. The smooth algorithm can reduce the influence of noise values that are not related to the cell state and reduce errors in measurement values.

FIG. 18 is an example showing a method of measuring impedance and phase values to which a differential algorithm is applied, and FIG. 19 is an example of a graph showing impedance and phase values to which a differential algorithm is applied. The control unit 390 is configured to measure The impedance and phase values are differentiated, and the input signal for cell state analysis can be determined by selecting the frequency at which the change in the differentiated impedance and phase values for each frequency is maximum.

Differentiation algorithms can be used to generate measured values after differentiation that have a larger variation than the measured values before differentiation. [Equation 1], which represents the Taylor series, can be applied to the differentiation algorithm. [Equation 2] is an expanded equation using Taylor series.

[Formula 1]

[Formula 2]

Here, f ⁽ⁿ⁾ means differentiating the function f(x) n times.

The present invention determines the optimal frequency corresponding to the unique characteristics of each cell, and by applying a voltage of the optimal frequency corresponding to the unique characteristics of each cell, the state of each cell can be monitored using electrochemical impedance spectroscopy, and the impedance and phase The accuracy or sensitivity of value measurement can be improved.

The present invention can automatically find a reliable optimal frequency by applying a lock-in amplification algorithm, smooth algorithm, and differentiation algorithm. In the present invention, the optimal frequency of heart cells was automatically found at 150Hz, but the optimal frequency may vary depending on the unique characteristics of the sample.

The electrochemical impedance device 300 for cells may further include an input unit 340 and a display unit 350. The input unit 340 can receive user conditions, and the display unit 350 can output information about the cell state in the form of a graph.

10: Cell measurement system 100: Screen print electrode
110: hand part 120: reaction part
130: electrode unit 200: cell measurement device
211: Fog pin for electrode 212: Fog pin for equipment connection
213: first locking screw 214: second locking screw
220: substrate 230: base holder
231: Seating part for substrate 232: Seating part for electrode
233: Grip part 234: In/out space part
240: Holder for electrode 250: Holder for reaction exposure
251: O-ring 260: Elastic means
300: Impedance equipment 310: Transmitter and receiver
320: measurement unit 330: analysis unit
340: input unit 350: display unit
390: Control unit

Claims (4)

An input signal is applied to the cell measurement device 200 under set frequency scan conditions, and the impedance and phase value for each frequency responding to the input signal are compared to determine an input signal for cell state analysis corresponding to the cell characteristics, and determine the input signal of the determined frequency. Analyzes the state of cells by applying an input signal for cell state analysis using voltage.
A transceiver unit 310 that applies an input signal for a cell response to a cytometry device and receives an output signal in response to the cell response;
A measuring unit 320 that measures the impedance and phase value of the cell using the output signal received from the cell measuring device;
An analysis unit 330 that analyzes the state of the cell by monitoring changes in the measured impedance and phase value, and
Controls the operation of the transmitter and receiver by determining the input signal for scanning by frequency based on the set frequency scan conditions, and receives the impedance and phase value generated using the output signal by the input signal for scanning by frequency from the measurement unit, and impedance by frequency. It includes a control unit 390 that compares the phase value and determines an input signal for cell state analysis,
The transmitting and receiving unit applies an input signal for cell state analysis with a voltage of a frequency determined by the control unit, and the analysis unit analyzes the state of the cell based on an output signal from the input signal for cell state analysis,
The measuring unit measures impedance according to frequency using a lock-in amplifier algorithm,
The control unit corrects the impedance and phase value for each frequency using a smooth algorithm, and differentiates the corrected impedance and phase value using a differentiation algorithm, and the change value of the differentiated impedance and phase value for each frequency is the maximum. Select the frequency to determine the input signal for cell state analysis,
The lock-in amplification algorithm detects the amplitude and phase of the signal related to the reference signal to be searched even if noise is buried in the signal of the measurement target, the smooth algorithm generates an intermediate value between the two values, and the differentiation algorithm detects the amplitude and phase of the signal before differentiation. It is used to generate measured values after differentiation with a change greater than the measured value,
Electrochemical impedance equipment for cells, characterized in that the frequency corresponding to the unique characteristics of each cell is determined by applying the lock-in amplification algorithm, smooth algorithm, and differentiation algorithm. delete delete delete