An electrode pad and a cell impedance measuring device including the same

Abstract

The disclosed invention relates to an electrode pad comprising a first electrode and a second electrode spaced apart from and opposite to the first electrode for measuring cell impedance, wherein the first electrode is formed to extend in a manner that depicts a helix with a constant pitch from an outer 1-1 end toward a central 1-2 end, and the second electrode is formed to extend in a manner that depicts a helix with a constant pitch from an outer 2-1 end toward a central 2-2 end, and the first electrode and the second electrode are alternately arranged in the radial direction to form a concentric double helix shape.

Description

Technical Field

[0001] This invention relates to a cell impedance measurement device, which improves the sensitivity and accuracy of cell impedance measurement in cell impedance measurement, a non-destructive, real-time electrocellular analysis. It is also applicable to two-dimensional (2D) cells and three-dimensional (3D) cells, thereby enabling precise analysis of cell electrical signal parameters. It can also be applied to new drug development, animal replacement experiments, etc. Background Technology

[0002] In the processes of new drug development and toxicity evaluation, precise analysis of cell state is essential. Existing cell analysis methods include microscopic cell observation, gene expression analysis, protein expression profiling, immunofluorescence, and flow cytometry. However, these methods struggle to analyze dynamic changes in cells in real time, require additional labeling, and necessitate expensive equipment and lengthy analysis times.

[0003] To overcome these limitations, electrocellular-substrate impedance sensing (ECIS) technology is being researched. However, existing ECIS-based cell chips have simple electrode structures, making them suitable only for 2D cells that are adsorbed, and not for 3D cells that are suspended. Furthermore, the analytical sensitivity is limited because the electrode patterns cannot form a uniform electric field. Therefore, there is a need to develop a cell chip that can achieve accurate real-time impedance measurement in various cellular environments by applying novel electrode structures. Summary of the Invention

[0004] The problem that the invention aims to solve The purpose of this invention is to provide an electrode pad with electrode patterns that improve the sensitivity and accuracy of cell impedance measurement and are also applicable to 2D and 3D cells, and a cell impedance measurement device including the electrode pad.

[0005] 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.

[0006] means for solving problems The present invention relates to an electrode pad comprising a first electrode and a second electrode spaced apart from and opposite to the first electrode for measuring cell impedance, wherein the first electrode is formed to extend in a manner that draws a helix with a constant pitch from an outer 1-1 end toward a central 1-2 end, and the second electrode is formed to extend in a manner that draws a helix with a constant pitch from an outer 2-1 end toward a central 2-2 end, and the first electrode and the second electrode are alternately arranged in the radial direction to form a concentric double helix shape.

[0007] In one embodiment, the first-1 end of the first electrode and the second-1 end of the second electrode may be located on opposite sides of each other relative to the center.

[0008] The distance between the first electrode and the second electrode remains constant along the trajectory of the double helix.

[0009] The spacing between the first electrode and the second electrode may correspond to the width of the first electrode and / or the second electrode.

[0010] The width of the first electrode can be the same as the width of the second electrode.

[0011] In one embodiment, the widths of the first electrode and the second electrode can be in the range of 50 μm to 100 μm.

[0012] In one embodiment, the heights of the first electrode and the second electrode can be in the range of 1.0 ± 0.2 mm.

[0013] In one embodiment, the first electrode further includes a semi-circular first branch electrode extending in a direction opposite to the helix to surround the exterior of the second electrode, and the second electrode further includes a semi-circular second branch electrode extending in a direction opposite to the helix to surround the exterior of the first electrode.

[0014] On the other hand, the present invention provides a cell impedance measurement device, comprising: a plurality of electrode pads arranged longitudinally and / or laterally on a substrate; a plurality of contact pads formed on the substrate, including a first contact pad and a plurality of second contact pads corresponding to the number of electrode pads; and a plurality of transmission lines formed on the substrate, including a first transmission line electrically connecting the first electrode of the plurality of electrode pads to the first contact pad, and a plurality of second transmission lines electrically connecting the second electrode of the plurality of electrode pads to the plurality of second contact pads in a one-to-one correspondence.

[0015] In one embodiment, the plurality of electrode pads, the plurality of contact pads, and the plurality of transmission lines form a conductive pattern, a first protective layer is formed between the substrate and the conductive pattern, and a second protective layer having electrode holes that expose the plurality of electrode pads is formed on the conductive pattern.

[0016] For example, the first protective layer and the second protective layer may each be formed of a polyimide film layer.

[0017] Invention Effects Based on the electrode pad configuration of the present invention as described above, an optimized electrode structure is formed that can improve the sensitivity of electrochemical signals. That is, the concentric double-helix electrode structure forms a uniform electric field throughout the entire region and reduces signal distortion, thereby improving the sensitivity and accuracy of cell impedance measurement.

[0018] Furthermore, the optimized concentric double-helix electrode structure is applicable not only to 2D cells but also to 3D cells. That is, it is suitable for both adsorbed and suspension cell types, and various parameters can be measured simultaneously depending on the culture conditions.

[0019] In addition, when measuring large 3D cells such as cardiomyocytes and other 3D organoids, cell fixation devices are used, and the concentric double helix electrode structure has the characteristic that the electrode contact area of ​​3D cells increases as the contact angle decreases, thereby enhancing the effect and further improving the sensitivity and accuracy of the measurement.

[0020] The effects of this invention are not limited to those mentioned above, but also include other effects that can be clearly understood by those skilled in the art from the entire specification but are not explicitly mentioned. Attached Figure Description

[0021] Figure 1 This is a diagram illustrating an example of a cell impedance measuring device according to an embodiment of the present invention.

[0022] Figure 2 This is a diagram illustrating a cell impedance measuring device according to one embodiment of the present invention.

[0023] Figure 3 It is shown Figure 2 A diagram showing the detailed structure of the electrode pads included in the cell impedance measurement device.

[0024] Figure 4 These are diagrams showing the electric field distribution of the electrode pads of the present invention and the prior art electrode pads, respectively.

[0025] Figure 5 These are diagrams showing the cross-sectional structure of a cell impedance measuring device according to one embodiment of the present invention, as well as the measurement states of 2D cells and 3D cells.

[0026] Figure 6 This is a graph showing the effect of increasing electrode contact area of ​​3D cells as the contact angle decreases. Detailed Implementation

[0027] Preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings. 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 only to complete the disclosure of the invention and to fully inform those skilled in the art of its scope, which is defined only by the scope of the claims. Throughout this specification, the same reference numerals denote the same constituent elements.

[0028] Unless otherwise defined, all terms (including technical and scientific terms) used in this specification are intended to have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. Furthermore, terms defined in common dictionaries are not idealized or over-interpreted unless specifically defined herein. The terminology used in this specification is for illustrative purposes and is not intended to limit the invention. In this specification, singular forms include plural forms unless specifically stated in the sentence.

[0029] The terms “comprises” and / or “comprising” 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.

[0030] Figure 1 This is a diagram illustrating an example of a cell impedance measuring device (20) according to an embodiment of the present invention.

[0031] Reference Figure 1 The cell impedance measuring device (20) of the present invention is combined with a multi-well plate (10), which is formed by a plurality of wells (12) arranged longitudinally and / or laterally to form a receiving portion (14) for culturing or processing cultured cells. In the exemplary drawings, the multi-well plate (10) is composed of a 2×8 16-well plate. The cell impedance measuring device (20) includes a substrate (30) and a conductive pattern (50) formed on the substrate (30).

[0032] The conductive pattern (50) includes a number of electrode pads (100) corresponding to a plurality of wells (12) of the porous plate (10). That is, an electrode pad (100) is assigned to the bottom of the receiving portion (14) of each well (12). Each electrode pad (100) may include a first electrode (110) and a second electrode (120) spaced apart from and opposite to the first electrode (110) to measure cell impedance. The measurement of cell impedance can be used for cell growth, toxicity analysis, etc. When measuring cell impedance, the two electrodes are respectively composed of a working electrode and a counter electrode. As the material of the electrode pad (100), non-toxic materials with excellent conductivity and suitable for cell culture, such as gold (Au), platinum (Pt), and indium tin oxide (ITO), can be used. In order to measure cell impedance, an AC signal in the frequency range of 1 Hz to MHz can be applied to the electrode pad (100).

[0033] Figure 2 This is a diagram illustrating a cell impedance measuring device (20) according to one embodiment of the present invention. Figure 2 Image (a) shows a top view of the cell impedance measuring device (20). Figure 2 (b) shows the stacked structure of the cell impedance measuring device (20).

[0034] Reference Figure 2 The cell impedance measuring device (20) includes a substrate (30) and a conductive pattern (50) formed on the substrate (30). In one embodiment, the substrate (30) may be made of FR-4 material, which is widely used in PCB manufacturing. In this case, the cell impedance measuring device (20) may also include a first protective layer (32) and a second protective layer (34), or the first protective layer (32) may be excluded and only the second protective layer (34) may be included.

[0035] Not only has the impact of FR-4 material on organisms not been fully verified, but FR-4 material itself is also fragile to moisture and has the property of absorbing moisture. Therefore, in the high humidity environment of cell culture carried out for a long time on a weekly basis, if the culture material containing moisture comes into contact with the substrate (30), a small leakage current will be generated between the transmission lines (300) on the conductive pattern (50), which may lead to measurement errors. Furthermore, PSR (Photo Solder Resist), which is widely used in the manufacture of ordinary PCBs, is also a material whose impact on organisms has not been fully verified. Therefore, when using existing ordinary PCB manufacturing technology to manufacture the cell impedance measuring device (20), it is impossible to know whether materials such as FR-4 and PSR will cause errors in the cell impedance measurement results. In order to fundamentally eliminate this concern, the cell impedance measuring device (20) may also include a first protective layer (32) and a second protective layer (34). However, it is not limited to this, and the first protective layer (32) may be excluded and only the second protective layer (34) may be included.

[0036] Reference Figure 2 In embodiment (b), a first protective layer (32) is formed between the substrate (30) and the conductive pattern (50), and a second protective layer (34) is formed on the conductive pattern (50) having electrode holes (36) that expose only the electrode pads (100) in the conductive pattern (50). In one embodiment, the first protective layer (32) and the second protective layer (34) may each be formed of a polyimide film layer. Polyimide films have good temperature characteristics and are highly resistant to peeling due to their excellent bonding properties, making them suitable for use in the cell impedance measurement device (20) of the present invention. The polyimide film can be bonded by various techniques such as hot pressing, silicone bonding, or UV curing. However, it is not limited to this; the conductive pattern (50) may be formed on the substrate (30), and the second protective layer (34) may be formed on the conductive pattern (50).

[0037] A conductive pattern (50) is formed on a substrate (30). Specifically, multiple electrode pads (100), multiple contact pads (200), and multiple transmission lines (300) are aggregated to form a conductive pattern (50). The conductive pattern (50) is formed by connecting multiple electrode pads (100) and multiple contact pads (200) through multiple transmission lines (300). Therefore, the constituent elements of the conductive pattern (50) can be formed simultaneously through a series of processes.

[0038] The electrode pad (100) includes a first electrode (110) and a second electrode (120) for measuring cell impedance. (See reference...) Figure 1 and Figure 2In (a), corresponding to a 2×8 16-hole plate, 16 electrode pads (100) are arranged in a 2×8 pattern on the substrate (30). For a detailed description of the electrode pads (100) according to the present invention, refer to... Figure 3 The following will be discussed in conjunction with Figure 7.

[0039] The plurality of contact pads (200) forming terminals for external connection include a first contact pad (210) and a plurality of second contact pads (220) corresponding to the number of electrode pads (100). Figure 2 In one embodiment, the plurality of contact pads (200) consist of one first contact pad (210) and 16 second contact pads (220).

[0040] Furthermore, multiple electrode pads (100) and multiple contact pads (200) are electrically connected via multiple transmission lines (300). Specifically, the first electrode (110) of the multiple electrode pads (100) is electrically connected to the first contact pad (210) via the first transmission line (310). That is, in the illustrated embodiment, a total of 16 first electrodes (110) are electrically connected to 1 first contact pad (210). Furthermore, the second electrodes (120) of the multiple electrode pads (100) are electrically connected to the multiple second contact pads (220) in a one-to-one correspondence via multiple second transmission lines (320). That is, the number of electrode pads (100) is the same as the number of second transmission lines (320), and in the exemplary embodiment, the number of second transmission lines (320) is 16.

[0041] Based on this connection structure of electrode pads (100), contact pads (200), and transmission lines (300), in the cell impedance measuring device (20) of the present invention, the counter electrode is composed of one common electrode, and the working electrodes are composed of 16 electrodes. That is, the first contact pad (210) is used as the common electrode, and the second contact pad (220) corresponds to the working electrode of each electrode pad (100).

[0042] Here, regarding the lengths of the multiple second transmission lines (320), for a pair of electrode pads (100) facing each other in the left-right direction (longitudinal direction) of the substrate (30), they can be manufactured in the same way because they are symmetrical structures. However, for the electrode pads (100) forming a row in the transverse direction, their lengths will necessarily be different due to the difference in distance from the second contact pad (220). If the lengths of the transmission lines (300) are different, slight deviations will occur in the signal output due to the resistance difference. To prevent this, it is necessary to reduce the resistance deviation of the second transmission lines (320) with different lengths to a negligible level. The resistance of a wire is directly proportional to its length and inversely proportional to its width. Taking advantage of this physical property, for the electrode pads (100) forming a row in the transverse direction, the resistance deviation can be significantly reduced by designing the dimensionless number of each second transmission line (320) calculated from "length / width" to be the same or similar. To allow for greater design flexibility, each second transmission line (320) can be designed with a different width, and a second transmission line (320) can also be designed with different widths for several segments.

[0043] Figure 3 This is a diagram showing the detailed structure of the electrode pads (100) included in the cell impedance measuring device (20). Figure 2 In order to clearly understand the present invention, some complex structures of the electrode pad (100) are not shown. Therefore, it should be understood that... Figure 3 The structure of the electrode pad (100) is also applicable to Figure 2 Each of the 16 electrode pads (100) illustrated in the cell impedance measuring device (20).

[0044] exist Figure 3 In order to understand the present invention, the first electrode (110) and the second electrode (120) are shown in colors (shading) that can distinguish them from each other. The first electrode (110) is formed in a spiral with a constant pitch (the distance between the spirals) extending from the outer first-1 end (110-1) toward the central (representing the central region of the electrode pad, hereinafter used in the same sense) first-2 end (110-2). The second electrode (120) is formed almost identically to the first electrode (110) in a spiral with a constant pitch extending from the outer second-1 end (120-1) toward the central second-2 end (120-2). Moreover, the first electrode (110) and the second electrode (120) are arranged alternately in the radial direction to form a concentric double helix shape.

[0045] In this specification, the following shape is referred to as a concentric double-spiral electrode: [e.g.] Figure 3As shown, two spiral-shaped electrodes place their respective ends (end 1-2, end 2-2) at the center of the electrode pad (100) and extend outward in the same direction (counterclockwise based on the figure) without touching or crossing each other. In one embodiment, end 1-1 (110-1) of the first electrode (110) and end 2-1 (120-1) of the second electrode (120) may be located on opposite sides of each other relative to the center (representing the center of the electrode pad).

[0046] In the concentric double-helix electrode structure, the distance between the first electrode (110) and the second electrode (120) remains constant along the double helix trajectory. That is, the distance and width of the first electrode (110) and the second electrode (120) are constant respectively. In this respect, the helical structure of the first electrode (110) and the second electrode (120) is mathematically equivalent to the Archimedean spiral.

[0047] In addition, please refer to the detailed information. Figure 3 The first electrode (110) and the second electrode (120) also include additional electrodes extending in a direction opposite to the helical direction. Specifically, the first electrode (110) further includes a semi-circular first branch electrode (112) extending in a direction opposite to the helical direction (clockwise) to surround the exterior of the second electrode. Furthermore, the second electrode (120) also includes a semi-circular second branch electrode (122) extending in a direction opposite to the helical direction to surround the exterior of the first electrode (110). The first branch electrode (112) and the second branch electrode (122) increase the overall area of ​​the electrode pad (100), thereby expanding the region where the electric field is formed. However, in various embodiments of the concentric double helix electrode structure of the present invention, the first branch electrode (112) of the first electrode (110) and the second branch electrode (122) of the second electrode (120) are structures that can additionally obtain the effect of expanding the region forming the electric field. The first electrode (110) and the second electrode (120) may also be constructed without the first branch electrode (112) and the second branch electrode (122), respectively.

[0048] In one embodiment, the spacing between the first electrode (110) and the second electrode (120) may correspond to the width of the first electrode (110) and / or the second electrode (120). In the illustrated exemplary embodiment, the width of the first electrode (110) is the same as the width of the second electrode (120). Thus, the spacing between the first electrode (110) and the second electrode (120) is also the same as the width of the first electrode (110) and the second electrode (120).

[0049] Figure 4These are diagrams showing the electric field distribution of the electrode pad (100) of the present invention and the electrode pad of the prior art, respectively. Figure 4 (b) shows the electric field of an electrode pad according to the prior art (refer to Patent Document 001). The prior art electrode pad is equivalent to an interdigitated wave electrode (IWE) structure and exhibits a strong edge effect because the size and direction of the radius of curvature change drastically with the wave. This increases the non-uniformity of the electric field distribution, resulting in concerns about unpredictable effects in cell impedance measurements. In contrast, reference is made to the electrode pad (100) of the present invention, showing the electric field... Figure 4 In (a), the concentric double-helix electrode structure exhibits only a small edge effect at the two ends of the first electrode (110) and the second electrode (120), forming a uniform electric field distribution over the entire region. Therefore, the sensitivity, accuracy, and reliability of cell impedance measurement are improved by the electrode pad (100) according to the present invention.

[0050] Figure 5 These are diagrams showing the cross-sectional structure of the cell impedance measuring device (20) according to one embodiment of the present invention, as well as the measurement states of 2D cells and 3D cells. According to the present invention, when designing the width and height of the first electrode (110) and the second electrode (120), it is preferable to design it to be able to simultaneously handle the impedance measurement of both adsorbed 2D cells and suspended 3D cells.

[0051] Figure 5 (a) in the figure schematically illustrates the impedance measurement of adsorbed 2D cells. Figure 5 (b) schematically illustrates the impedance measurement of suspended 3D cells. Comparing approximate cell sizes, 2D cells are 10 μm to 20 μm in size, while 3D cells (3D organoids or spheroids) are approximately 100 μm to 2 mm in size. As mentioned above, the size of 2D cells is significantly smaller than that of 3D cells. Considering this size difference, preferably, the widths of the first electrode (110) and the second electrode (120) can be designed in the range of 50 μm to 100 μm, and the heights of the first electrode (110) and the second electrode (120) can be designed in the range of 1.0 ± 0.2 mm.

[0052] In the case of small 2D cells, if the space between the first electrode (110) and the second electrode (120) is formed as a space for the 2D cells to adhere, it is advantageous to obtain cell impedance measurement as the 2D cells are located between the electrodes. Conversely, in the case of large 3D cells, the first electrode (110) and the second electrode (120) serve to fix the position of the suspended 3D cells, which is beneficial for impedance measurement. Taking into account these measurement advantages, it is preferable to design the width of the first electrode (110) and the second electrode (120) in the range of 50 μm to 100 μm, and the height of the first electrode (110) and the second electrode (120) in the range of 1.0 ± 0.2 mm, thereby fixing the large 3D cells on the electrodes and placing the small 2D cells between the electrodes.

[0053] Figure 6 This is a graph showing the effect of increasing electrode contact area of ​​3D cells as the contact angle decreases. To stably maintain the position of the suspended 3D cells, the cell impedance measurement device (20) may include a cell fixation device (16). The cell fixation device (16) may be made of a flexible material that does not damage the 3D cells. The cell fixation device (16) applies slight pressure to the 3D cells and presses them down, thereby fixing the position of the 3D cells in the culture medium and inducing good contact with the electrode pad (100).

[0054] like Figure 6 As shown, by pressing with the cell immobilization device (16), the contact angle of the 3D cell relative to the electrode pad (100) decreases. That is, the 3D cell is pressed into a slightly flattened shape, thereby reducing the contact angle relative to the electrode pad (100) and increasing the contact area. In particular, since the electrode pad (100) of the present invention has a concentric double helix electrode structure, an increase in the contact area in all 360° directions can be expected when the contact angle of the 3D cell decreases. That is, compared with the prior art (e.g., Patent Document 001), the effect of increasing the contact area due to the reduction of the 3D cell contact angle in the electrode pad (100) of the present invention is more significant, and this characteristic also plays a positive role in improving the sensitivity and accuracy of cell impedance measurement.

[0055] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, those skilled in the art will understand that the present invention can be implemented in other specific ways without changing its technical concept or essential features. Therefore, it should be understood that the above embodiments are exemplary in all respects and not restrictive.

[0056] Explanation of reference numerals in the attached figures 10: Perforated plate; 12: Hole; 14: Reception area; 16: Cell fixation device; 20: Impedance measuring device; 30: substrate; 32: First protective layer; 34: Second protective layer; 36: Electrode hole; 50: Conductive pattern; 100: Electrode pad; 110: First electrode; 110-1: End portion 1-1; 110-2: End portion 1-2; 112: First branch electrode; 120: Second electrode; 120-1: End portion 2-1; 120-2: End portion 2-2; 122: Second branch electrode; 200: Contact pad; 210: First contact pad; 220: Second contact pad; 300: Transmission line; 310: First transmission line; 320: Second transmission line.

Claims

1. An electrode pad, wherein, The electrode pad includes a first electrode and a second electrode spaced apart from and opposite to the first electrode, for measuring cell impedance. The first electrode is formed by extending in a helix with a constant pitch from the outermost 1-1 end toward the central 1-2 end. The second electrode is formed by extending in a helix with a constant pitch from the outer 2-1 end toward the central 2-2 end. The first electrode and the second electrode are arranged alternately along the radial direction to form a concentric double helix shape.

2. The electrode pad according to claim 1, wherein, The first electrode's first-1 end and the second electrode's second-1 end are located on opposite sides of each other relative to the center.

3. The electrode pad according to claim 1, wherein, The distance between the first electrode and the second electrode remains constant along the trajectory of the double helix.

4. The electrode pad according to claim 3, wherein, The spacing between the first electrode and the second electrode corresponds to the width of the first electrode and / or the second electrode.

5. The electrode pad according to claim 4, wherein, The width of the first electrode is the same as the width of the second electrode.

6. The electrode pad according to claim 5, wherein, The widths of the first electrode and the second electrode are in the range of 50 μm to 100 μm.

7. The electrode pad according to claim 5, wherein, The heights of the first electrode and the second electrode are within the range of 1.0 ± 0.2 mm.

8. The electrode pad according to claim 1, wherein, The first electrode further includes a semi-circular first branch electrode that extends in the opposite direction to the helix to surround the exterior of the second electrode. The second electrode also includes a semi-circular second branch electrode that extends in the opposite direction to the spiral to surround the exterior of the first electrode.

9. A cell impedance measuring device, wherein, The cell impedance measurement device includes: substrate; A plurality of electrode pads, wherein the plurality of electrode pads are electrode pads according to any one of claims 1 to 7, and the plurality of electrode pads are arranged longitudinally and / or laterally on the substrate; A plurality of contact pads are formed on the substrate, including a first contact pad and a plurality of second contact pads corresponding to the number of electrode pads; and A plurality of transmission lines are formed on the substrate, including a first transmission line electrically connecting the first electrode of the plurality of electrode pads to the first contact pad, and a plurality of second transmission lines electrically connecting the second electrode of the plurality of electrode pads to the plurality of second contact pads in a one-to-one correspondence.

10. The cell impedance measuring device according to claim 9, wherein, The plurality of electrode pads, the plurality of contact pads, and the plurality of transmission lines form a conductive pattern. A first protective layer is formed between the substrate and the conductive pattern. A second protective layer with electrode holes that expose the plurality of electrode pads is formed on the conductive pattern.

11. The cell impedance measuring device according to claim 10, wherein, The first protective layer and the second protective layer are each formed of a polyimide film layer.

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