TW201120441A - A dissolved-oxygen electrode array with the integration of cell-patternized culture used for estimation of cellular respiratory activity and its fabrication method - Google Patents

Abstract

The invention is a dissolved-oxygen (DO) electrode array chip integrated with cell-patternized culture, which consists of a sensing chip and a microchannel-levitated slab. The sensing chip includes a substrate containing a cell-cultured area, a counter electrode and multi-DO electrodes and an insulator layer placed on the substrate. Each DO electrode and the counter electrode have a working window. The insulator layer covers the counter electrode and the multi-DO electrodes, and a cell-culture hole, which is used as a cell-adhesion region, is placed on the insulator. The distance between the center of the cell-adhesion region and the working window is smaller than the five times radius of cell-cultured area. The microchannel-levitated layer is combined with the sensing chip. In the invention, the DO electrode array is distributed within the hemispherical diffusion radius of oxygen consumption of cell-cultured area, and it can quantify the respiratory rate of cells.

Description

201120441 VI. Description of the invention: [Technical field to which the invention pertains] The present invention relates to a dissolved oxygen electrode barrier wafer, Yugan β u, and a method for evaluating cellular respiratory activity and patterning cultured cells. Polar array wafer ^ [Prior Art] The cell is the smallest unit of life of the organism, and the cell as a detection unit can represent the direct physiological response of the organism. Since cells need to continuously synthesize many macromolecules (such as proteins) to maintain physiological normal operation, a large number of octopuses must be consumed in this process to overcome the activation energy required for biochemical reactions to promote biochemical synthesis. occur. The synthesis of Ατρ is oxidized by the living body to swallow, digest and absorb the food coming in from the outside, = this physiological need. Please refer to the reaction formula 1. For example, a mulberry grape can produce 38 moles of ATP after glycogenolysis (g|yc〇|ySis) and citric acid cycle (C丨(7)C cycle). This oxidation process also consumes 6 moles of oxygen molecules', so oxygen molecules play an important role in the synthesis of ATP. Therefore, the consumption of oxygen can represent the physiological activity of the living body. When the physiological activity of the cell is higher and a large amount of protein needs to be synthesized, more oxygen will be consumed to synthesize more ATp to assist in protein synthesis. Therefore, direct monitoring of changes in extracellular dissolved oxygen will reveal the cellular respiratory activity. Dissociated. There are many methods for measuring the oxygen consumption of cells (IV), which can be divided into optical methods (such as materials, fluorescent, and twilight, etc.) and electrochemical methods. C6H1206 + 602-> 6C02 + 6h2〇 +3δΑτρ δ〇= _686 kcal/mol

4 [ S 201120441 At present, the electrodes and instruments of the electrochemical method are easy to miniaturize, and no additional calibration is required for dissolved oxygen detection. The electrochemical method has been widely applied to the detection of cellular respiratory activity. A brief introduction of commercially available electrochemical cell respiration assay wafers is as follows: (I) Analyzing system and Metabolic chip from Bionas®, Germany:

The multi-parameter cell physiology analysis system of Bionas® in Germany can treat the extracellular acidification rate (ion-selective field-effect transistor sensor (〇SFET)) The rate (amperometric sensor) and cell attachment (interdigitated electrode conductivity sensor) were integrated for measurement. The main wafer design is to culture the cells in the packaged wafer. The cell culture solution and the test drug are transported by micro-pipes from the upper ends of the wafer. The internal buffer volume is about 1 〇. The characteristics of the sensing chip are It is suitable for high temperature and high pressure during sterilization, and avoids contamination of cells by external cells during culture. It is easy to integrate with microelectronic circuits and has the advantages of rapid detection and low sample reagents. The disadvantage is that the patterned extracellular matrix cannot be surfaced on the surface of the substrate. Modification, causing the cells to be unable to fix the position of the sensing electrode and the cell growth zone when implanted and attached, can only quantify cell viability using relative activity changes. The above-mentioned commercial measurement wafer is liquid-replaced in a closed microchannel, and has a ruthenium as a substrate, and its opacity hinders optical detection of cell type properties. (l!) DOX-96 In 1999, Amano et al. developed a new oxygen electrode sensor.

5 201120441 Combines multi-channel detection with inexpensive disposable electrodes to form a 96_we丨丨 detection disk, each containing a disposable three-pole gold electrode (working, auxiliary, reference electrodes are gold electrodes) 'Oxygen produced in each well is converted to current value by electrode sensing by multipotentiostat, and then output by computer, and the method of analyzing the data is very similar to the traditional 96_we丨丨microplate using optical measurement. It features a disposable electrode that is less cumbersome, easy to use, and provides efficient drug screening and long-term monitoring. It has been applied to detect the response of lung cancer cells to isoflavones, classification of bacteria, and antibacterial sensitivity tests. However, the disadvantage is that it cannot be combined with the fluid system for a single measurement. The random culture may also change due to the change in the distance between the cells and the electrode. (丨丨丨) Broom Electrochemical Microscopy (SCannjng Electrochemical)

Microscope, SECM) SECM is a method for scanning and measuring the redox current of electrochemically active analytes in different regions using a probe-type ultramicroelectrode. When the probe electrode φ is scanned close to the surface of the sample, different types The sample will affect the electricity "IL reversal amount', for example, when the sample is an insulating substance, it will block the electroactive substance from diffusing to the electrode surface, resulting in a decrease in the measured current; if the sample is electrically conductive, the regenerative electroactive substance At this time, a large amount of electroactive material is diffused to the probe electrode 丨, so that the detection current is increased, and then the nature of the sample is evaluated according to the stem of the electric stalk. Since SECM can precisely control the distance between electric /, and t, it can accurately assess and analyze changes in cellular respiration rate. However, the cost of the SECM instrument and the micro-f pole production are not easy to limit the potential of SECM to be widely used in the detection of cellular respiratory activity. According to the above background introduction, the current commercially available cell activity sensing 201120441 system still has the following disadvantages: 1. The current cell wafer is in the cell respiratory activity detection, because the cells are randomly cultured, and the electrode is susceptible to cell adsorption. The signal that causes contamination or changes the electrode area, which reduces or interferes with the dissolved oxygen reduction current, results in lower stability and sensitivity of the electrode. The distance between the sensor and the cell also affects the size of the dissolved oxygen current, so the amount of change in cell activity cannot be measured repeatedly. Therefore, if the distance between the fixed electrode and the cell can be patterned, it can be avoided. The above problems; 2, the current cell wafer can not fix the distance between the cell attachment area and the sensing electrode, so the cell activity can only be quantified by the percentage change of activity, and multiple experiments are needed to verify the accuracy; 3. Cellular activity The measurement system uses 矽 as the wafer substrate, which limits the optical observation; 4. The current cell wafer cannot be integrated with the microfluidic control system for the south flux test. SUMMARY OF THE INVENTION The present inventors have arbitrarily cultivated cells due to the existing cell respiratory activity sensing system, which is liable to cause electrode contamination, and the accuracy cannot be improved. Therefore, after a long period of research and continuous testing, this invention was finally invented. Fine = sexual evaluation and combined with patterned cell culture of dissolved oxygen electrode array crystal plate method. SUMMARY OF THE INVENTION The object of the present invention is to provide a ^ ^ ^ ^ cross-supply oxygen electrode array crystal 疋 a combined patterning cell culture 夕 每 every # ^ 胞 cell respiratory activity evaluation. The invention relates to the application of the technology in the present invention to provide a dissolved oxygen electrode array wafer for seed cell culture, which comprises: a sensing wafer, comprising a substrate, a cell attachment region, an auxiliary electrode, a plurality of dissolved oxygen electrodes, an insulating layer, and a plurality of working windows, wherein the cell attachment region, the auxiliary electrode, and the plurality of dissolved oxygen electrodes are spaced apart from the substrate, An insulating layer is coated on the surface of the substrate, and covers at least a portion of the auxiliary electrode and the plurality of dissolved oxygen electrodes, and is provided with a circular cell culture hole to expose the cell attachment region. A working window is respectively disposed on the layer relative to each of the dissolved oxygen electrode and the auxiliary electrode, and a working window working window of each dissolved electrode is adjacent to the cell attaching region, and a working window of each dissolved oxygen electrode is The distance between the center of the cell attachment area is less than five times (5r) of the radius (r) of the cell attachment area, and the working window between the working windows of each dissolved oxygen electrode is less than 〇5r 'the working window of the auxiliary electrode The distance between the center of the cell attachment area is greater than five times (5r) of the radius of the cell attachment area (5r); a micro flow channel pad is attached to the top surface of the sensing chip, including a padded film body, a micro-channel layer and a cell culture tank, wherein the high-film body is located on a top surface of the sensing wafer and a flow-through groove is disposed on a side of the high-film body, and the micro-channel layer is bonded to the high-layer film a top surface of the body, and includes a bottom surface, a micro flow channel and a liquid extraction hole, wherein the micro flow channel is recessed on the bottom surface, and the shape and position of the micro flow channel corresponding to the area of the flow through groove The flow through groove conforms to 'the liquid passage hole is disposed on one side of the micro flow channel layer and communicates with the micro flow channel'. The liquid extraction hole and the flow through groove are located on different sides. The cell culture groove runs through the micro a flow channel layer microchannel layer and the plastic* high film body and communicating with the microchannel and the liquid extraction hole' to exit the cell attachment region and the working window of each dissolved oxygen electric circuit 201120441. The material is glass. The auxiliary electrode and the plurality of dissolved oxygen electrodes are gold layers. The thickness of the body is 50 / / m. The auxiliary electrode and the plurality of dissolved oxygen electrodes and the substrate comprise a layer. wherein 'the cell attachment area has a radius of 200 from m. Where the auxiliary electrode and the The distance between the cell attachment regions is 9.5r. Preferably, the number of the dissolved oxygen electrodes is six. The distance between the working window of each dissolved oxygen electrode and the center of the cell attachment region is 1.25" 1.5 r, 2_25 r, 2.75 Γ, 4.25 r and 4.75 r. wherein the high film system is a layer of poly(dimethyl siloxane) (PDMS) film. The invention also relates to a bonding pattern The method for preparing a dissolved oxygen electrode array wafer for chemical cell culture comprises: providing a cell attachment region on a top surface of a substrate, and depositing a plurality of dissolved oxygen electrodes and an auxiliary electrode at intervals, forming an insulation on a top surface of each electrode; a layer 'and a working window on the insulating layer relative to each electrode to form a working window' to expose the corresponding electrode, and a circular cell culture hole is formed on the insulating layer to display the cell attaching area Form a sense Measuring a wafer; providing a microchannel layer 'a microchannel is recessed from a bottom surface of the microchannel layer, and a liquid hole is bored in the microchannel layer, and the liquid is connected to the microchannel Providing a membrane body, the membrane body is coupled to the lower layer of the microchannel 201120441 to form a microchannel mat upper layer, and a cell culture tank is formed at the upper layer of the microchannel The cell culture tank is in communication with the microchannel and the liquid extraction hole; the microchannel runner layer is assembled on the top surface of the sensing wafer to form the dissolved oxygen electrode array wafer, and the cell culture tank is exposed The cell attachment area and the working window of each dissolved oxygen electrode. Among them, the substrate is glass. Wherein the pad film system is formed by applying the PDMS solution to a top surface of a substrate for curing. Preferably, the high membrane system is combined with the microfluidic layer in an oxygen plasma treatment to form the microchannel mat. Preferably, the thickness of the 塾 high film body is 50 / 7171, the radius of the cell attachment area is 200 // m, and the number of the dissolved oxygen electrodes is six, and each dissolved oxygen electrode and the cell The center of the attachment area is 0 25mm, 0.3mm ' 0.45mm, 0.55mm, 0.85mm and 〇.95mm.

Preferably, the auxiliary electrode and the plurality of dissolved oxygen electrodes are deposited on a substrate, and the titanium layer is first sputtered on the top surface of the substrate, and a gold layer is deposited on the titanium layer. And using the lifting (丨丨ft_off) process to partially dissolve the gold layer to form the auxiliary electrode and the plurality of dissolved oxygen electrodes; further forming an insulating layer on the top surface of each electrode, and forming, exposing, developing and forming on the hard electrode respectively A working window. The present invention is also related to a dissolved oxygen electrode array wafer for cell respiratory activity evaluation and combined cell culture, which is, for example, a producer. The invention provides a dissolved oxygen electrode array wafer provided by the present invention for evaluating cellular respiratory activity and culturing 201120441 cell culture, and a method for preparing the same, and the advantages and enhancements obtained by the above technical means at least include: 1. The present invention By patterning the cultured cells, the amount of change in cell activity can be repeatedly measured, and the oxygen-dissolving electrode array of the present invention is distributed in the diffusion radius of the hemispherical dissolved oxygen consumed by the circular cell pattern region, thereby being quantifiable Analyze the respiratory rate of the cells. 3. The substrate of the present invention can be selected as a translucent glass for observation of cell type. 4. The electrode array wafer of the present invention is integrated with the microfluidic system to increase the convenience of cell culture solution or test agent injection, and to control the environment required for normal cell physiology. 5. The microchannel mat of the present invention can perform large-volume cell culture, so that the cells are detected under normal physiological conditions, and the microchannel can be reduced by lifting the microchannel stilts by the high membrane body. Shear force generated when replacing a liquid. [Embodiment] In order to understand the technical features and practical effects of the present invention in detail, and can be implemented in accordance with the contents of the specification, the detailed description is as follows: Referring to the fourth figure, the positional arrangement of the sensing electrodes according to the present invention is shown. (layout) The electrode sensing position will be designed around the patterned cell attachment area (15) according to the hemispherical diffusion theory. If the cell pattern area is circular and its pattern is 200 m, the effective diffusion radius of the oxygen consumption concentration gradient (, 51) can be half of the pattern area semi-controlled by #, because & Several electrodes were dispersed in this diffusion range, and the dissolved oxygen concentration of 201120441 was detected at different distances from the cell pattern region. The surface oxygen consumption and oxygen consumption rate of the cell (4) region described in the second aspect of the present invention are calculated as the steady-state consumption source, and the surface oxygen concentration, the spherical diffusion, and the oxygen concentration and the concentration around the cell pattern region. The relationship between the distance (7) of the surface of the cell pattern area is as shown in the formula:

ΓΜ - ~ C*X c(r)=z~~T+^~~+c* Equation 1# G is the radius of the cell pattern area; Γ is the position where the electrode is located; c(") is the position where the microelectrode is located Oxygen concentration; 〇Cs is the oxygen concentration on the surface of the test solution and the cell pattern area; C* is 〇2〇9 _ (3rc, 5% c〇2 is broadly determined by C(7) and ~/(/+〇 when plotting The slope of oxygen consumption (F) according to the present invention can be obtained by the formula 2. F = 2nrsD{C* - Cs) Formula 2 D is the oxygen diffusion coefficient, which is 2 ι〇χΐ〇 in the aqueous solution at room temperature. 5cm2/s. Referring to the first figure, the present invention is used for the evaluation of cellular respiratory activity and combined with the patterned cell culture of the dissolved oxygen electrode array wafer and the preparation method thereof, comprising the following steps: 1. Preparing a sensing wafer (a For the cleaning of the substrate, please refer to the first to third figures to remove the oil and dust on the surface of the substrate (H) to increase the adhesion of the metal to the substrate (11). The substrate of the present invention (11) It is made of glass. First put the glass into the slide holder and immerse it in the solution of 201120441 propanol (丨sopr〇pan〇| '丨PA), and oscillate with ultrasonic for 3〇min, then After one person steamed the crane water ultrasonic shock 5, replace the secondary water, repeat the person/mesh clean and then dry the glass, then immerse it in the piranha solution (pj "anha S〇丨Uti〇n", the piranha solution is the volume ratio 3: 1 96% H2S04 and 30 /. Ηζ〇 2 is prepared, 8 〇 it is separated by water heating and ultrasonic shock for 30 min, after taking out the glass, then repeating 3~5 times with double distilled water ultrasonic shock Clean and complete the glass cleaning. (b) Electrode position design clear As shown in the fourth figure, the positional arrangement of the electrode of the present invention (丨ayout) will be around the patterned cell attachment area (15), according to the hemisphere The shape diffusion theory is used to design the electrode sensing position. If the cells in the circular cell attachment area (彳5) are assumed to be oxygen consumption sources, the effective diffusion radius of the dissolved oxygen gradient (15,) is a circular cell attachment area. (15) Five times the radius is calculated. As long as the sensing electrode array is arranged within the effective diffusion radius (1 51), the oxygen consumption at different positions from the cell can be measured. Patterned cell attachment area (1 5) 'The radius is 200 //m, and preset the effective diffusion radius (15" within the range of five times the radius of the preset cell attachment area (15), and preset it to 6 (20//m X 20/ym). An ultramicroband electrode constitutes an electrode array distributed around the cell attachment region (15) and within a predetermined effective diffusion radius (151). The respective preset electrodes are respectively marked with a code (E1). ~E6), and the distance between each preset electrode position from the cell attachment area (15) is 彳·5", 1.25r, 13 201120441 2.25Γ' 4.75Γ' 4.25Γ' 2.75r (?p0.3mm ' 0.25mm '0.45mm, 0_95mm, 0.85mm, 0.55mm), and the auxiliary electrode (121) is a thin film gold electrode with an area of 0.7484 mm2, which is placed at 9.5r (ie 1.9) away from the patterned cell attachment area (15). Mm) Far away, avoid crosstalk with the working electrode. . (c) Electrode fabrication Electrode deposition can be performed by evaporation or sputtering, using chromium or titanium as the adhesion layer, and then depositing gold as the electrochemical sensing layer. The patterning of the electrodes can utilize the standard residual ( Etching) The process or lift-off (uft-off) process is completed to make the preset electrode. Please refer to the first to third figures, apply AZ 4620 positive photoresist on the cleaned glass, define the shape of the electrode after exposure and development, and further deposit a 5 〇nm by sputtering or evaporation. A chrome layer or a titanium layer is used as an adhesive layer, and a gold layer having a thickness of 25 〇 nm is formed on the adhesive layer as a sensing plane (12), and is removed by an etching process or a lift-off technique. The photoresist can form an auxiliary electrode φ (1 21) and a six-dissolved oxygen electrode (1 22) on the glass. (c) Production of insulating layer SU8-3010 negative photoresist was used as an insulating layer (13) and applied to each electrode as a first layer to form an insulating layer (14). The first stage was spin-coated with 5 〇〇 rPm for 15 seconds, the second stage was coated with 3 rpm for 3 rpm, and then red for 6 seconds at 65 ° C, and at 75 t > c and 85, respectively. Bake for 2 minutes under the armpits, then gradually heat up to 1 minute for 1 minute to make the thickness of about 10; then use the designed reticle to expose the light to the light for 3 seconds, and then expose it for 30 seconds. After the degree of cross-linking and the structure of 201120441, it is baked at 65 C for 1 , minutes and at 75 respectively. 〇 Bake with 85 C for 2 minutes' and then gradually heat up to 95. It is baked for 1 minute, and finally the wafer is developed to form a position on the insulating layer (14) relative to each dissolved oxygen electrode (122). A working window 彳 (16), and in the position of the cell attachment region (15) according to the foregoing design, on the glass, a cell attachment region 〇 5) corresponding to the range of the insulating layer (14). Second, the preparation of the micro flow channel pad high-rise (a) Preparation of the micro-channel layer, please refer to the fifth (A) figure (a) ~ (d), first prepare a glass substrate (30) 'the glass base 杈 ( 30) After cleaning, the SU8 negative photoresist (31) is coated and developed by exposure, and the photoresist (31) is removed to form a master mold (32). Please refer to the fifth (B) diagrams (e) to (h) to mix the poly(dimethylsiloxane, PDMS) main agent and curing agent at 10:1 (w/w%). After mixing and removing the bubbles, they are cast on the master mold (32) and solidified on the hot plate for 1.5 h, and then solidified from the master mold (32) after cooling to form a micro-channel layer ( 22) 'A microchannel (222) is recessed from the bottom surface of the microchannel layer (22), the height of which is about 200 // m, and then a glass capillary is used to penetrate the microchannel layer (22). a liquid hole (223) which is formed at a position on the side of the micro flow path (222). (b) Prepare the high-film body. Dispense the PD MS main agent and the curing agent in a ratio of 1 〇·· 1 (w/w%), spin-coat on a clean acrylic surface, and place on a hot plate. It solidifies to form a high film body (21) on the acrylic substrate having a thickness of about 50 Å. (c) Preparation of microchannel mats for high-rises, please refer to the first, second and sixth diagrams. 'The high-film body (21) and 15 201120441. The micro-channel layer (22) with an oxygen plasma of 100 watts. Processing the ι〇 second, and bonding the plastic/South film body (21) to the lower side of the micro flow channel layer (22), and combining the two while the 'acrylic substrate system and the high film body (21) Separating, a part of the height film body (21) is torn off to form a flow through groove (211), and the shape and position of the flow through groove (21 1) and one side of the micro flow channel layer (22) Corresponding 'and the flow through groove (211) is on the same side as the insertion hole (224), so that liquid can flow to the position of the reference electrode for measurement, and a micro flow channel pad high layer (20) is formed. The micro-channel layer (22) is provided with an insertion hole (224), and the insertion hole (224) is opposite to the other side of the micro-channel (222), and then the micro-flow channel is high-rise. (20) forming a cell culture tank (23), and the cell culture tank (23) is located at a middle position of the microchannel (222), and the liquid is connected by the microchannel (221) hole (222) and the insertion hole (224), the cell culture tank (23) and the micro flow channel (222), the liquid inlet hole (223), the insertion hole (224), and the flow through groove (211) Interconnected with each other, and the distance between the bottom edge of the cell culture tank (23) and the microchannel (222) is 50/vrn; (d) preparing a dissolved oxygen electrode array wafer combined with patterned cell culture, see second, Figure 3 and Figure 6 show the upper layer of the microchannel mat (2〇) assembled on the sensing wafer (10) with an acrylic clamp, and the working window of the six dissolved oxygen electrode (1 22) (1 6 And the modified cell attachment region (15) is exposed from the cell culture tank (23) to form a dissolved oxygen electrode array wafer that binds the patterned cell culture. The dissolved oxygen electrode array wafer combined with the patterned cell culture of the present invention comprises a sensing wafer (10) and a microchannel pad high layer (2〇), wherein: the sensing wafer (10) comprises a Substrate (11), a cell attachment area (15), an auxiliary electrode (121), a six dissolved oxygen electrode (122), an adhesive layer, a 201120441 edge layer (14) and a seven working window (16) The substrate (11) is made of glass, and the cell attachment region (15), the auxiliary electrode (121), and the six dissolved oxygen electrode (122) are located on the substrate (11) and are a gold layer, and The six dissolved oxygen electrode (122) is arranged in a strip parallel to each other. The adhesive layer is a layer and is located at the

Between the substrate (11) and the sensing plane (12), the insulating layer (14) is composed of a SU8 negative photoresist (31) which is coated on the sensing plane (12) and the base The surface of the material (11) is provided with a circular cell culture hole (141) on the insulating layer (14), and the cell culture hole (141) is disposed at the application position of the six dissolved oxygen electrode (122). Exposing the cell attachment region (15) on the substrate, and respectively inserting a position on the insulating layer (14) with respect to each of the dissolved oxygen electrode (122) and the auxiliary electrode (121) Working window (16), please refer to the third and fourth figures, the working window (16) of each dissolved oxygen electrode (122) is adjacent to the cell attaching area (15), and each dissolved oxygen electrode (122) The distance between the working window (16) and the center of the cell attachment region (15) is less than five times (5r) of the radius (r) of the cell attachment region (彳5), and each dissolved oxygen electrode (122) The distance between the working window σ (彳6) is less than 0.5 "' the working window (16) of the auxiliary electrode (121) is greater than the center of the cell attaching area (15) by more than (5"). In this embodiment, Each dissolved oxygen electrode (122) The distance between the window (16) and the center of the cell attachment area (15) is 1.25 and 4,75 1.5 r, 2.25 r, 2.75 r' 4.25", and the auxiliary electrode (121) is used as a window (16). The distance from the center of the cell attachment region (15) is 9.5r; 凊 Referring to the first and second 'six diagrams, the microchannel mat high layer is bonded to the top surface of the sensing wafer (10) to include a high film. a body (21), a first-order layer (22) and a cell culture tank (23), the high-film body (21) is located on the top surface of the sensing wafer (1), and includes a flow-through groove (21) The microflow 17 201120441 layer (22) is bonded to the top surface of the high film body (21), and includes a bottom surface (221), a micro flow channel (222), a liquid extraction hole (223), and a plug. a hole (224) is defined, the micro flow channel (222) is recessed on the bottom surface (221), and the micro flow channel (222) corresponds to the shape and position of the region through which the groove (211) flows. (211) conforming, the liquid suction hole (223) is disposed on one side of the micro flow channel layer (22) and communicates with the micro flow channel (222), and the insertion hole (224) is disposed in the micro hole The other side of the runner layer (22) is 'connected to The microchannel (222) and the insertion hole (224) are located on the same side as the flow channel (211), the cell culture tank (23) and the microchannel (222), the liquid extraction hole (223), the insertion hole (224) and the flow through groove (211) are in communication with each other, and a distance between the bottom edge of the cell culture tank (23) and the micro flow channel (222) is 50/ym, and is exposed. The cell attachment region (15) and the working window (16) of the six dissolved oxygen electrode (122). The embodiment is used in the detection of the present invention, and is attached to the upper layer of the microchannel mat (2〇). Before the combination of the far sensing wafer (1 〇), the micromolding technique can be used to locally modify the extracellular matrix. The sensing wafer (彳〇) on the glass allows the cells to be selectively attached to the surface of the sensing wafer (10), as shown in the seventh figure, the size of the first fabrication and cell attachment region (彳5) One of the same master molds, and then through the PDMS mold to form the PDMS layer (60) 'and a hole (61) is applied to the PDMS layer (60) by the laser processing machine, and the hole of the pDMS layer (6〇> (61) Aligning the cell attachment region (15) of the sensing wafer (10), first injecting 0.001% poly-L-amino acid (70) (p〇|y_L_|ysjne) to statically modify π min, and then injecting 10 / /g mL·1 fibronectin (fjbronectjn) (7l) After modification for 1 h, the PDMS layer (60) was separated, washed with double distilled water, and finally injected with 18 201120441 0.3 mg mL-1 bovine serum albumin (bovine serum) Albumin, BSA) (72) modified insulating layer (14) SU8 surface 1 h; due to poly-L-amino acid (70) is easier to adsorb on the hydrophilic glass surface, and poly left After modification with amino acid (70), the surface of the substrate (11) of the glass is provided with an amino group, so that fibronectin (71) is easily adsorbed, and bovine serum albumin (72) is more easily adsorbed on the hydrophobic SU8. Therefore, the bovine serum albumin (72) can be modified in the region of the insulating layer (14) to which the fibronectin (71) is not modified. After the modification, the phosphate buffer saline (PBS) is used for washing. The pretreatment step of cell pattern refining. After the pretreatment is completed, please refer to the eighth figure, and cover the cell suspension of human hepatocarcinoma cell line (HepG2) with 30#L density of lxi〇-6ce丨丨/mL. Above the cell attachment area (15) of fibronectin (71), the cells were sedimented for about 15-20 min, washed with PBS, and then added to the culture medium for about 2 days in the cell culture incubator. Depending on the type of cells, the patterned cells are completed on the sensing wafer (1 〇). Please refer to the figure 6 in Fig. 6 to complete the sensing wafer (1 0) and the edge micro flow channel of the patterned cells. After the high-rise (2〇) is combined, the extrapolated commercial® Ag/AgCI is used as the reference electrode (4〇) and inserted In the insertion hole (224), a thin film gold electrode is used as an auxiliary electrode (121), and a syringe pump (50) is inserted into the liquid extraction hole (223) and communicates with the micro flow path (222). To facilitate fluid exchange, 1 mM mM cell culture fluid (HBS) solution was used to perform the respiratory activity of the cells in the absence of nutrient sources. . Referring to the fourth and ninth (a) figure, add 3 细胞 cell culture medium (HBS) with hydroxyethyl acetate (hepes) as buffer molecule, and measure the oxygen consumption rate for 10 minutes; Measure 1〇 separately

HBS+25 _glucose, 1 mM HBS+25 mM 201120441 glucose + 1. /Insulin was compared and finally the cells were removed with trypsin. Comparing the effects of g|uc〇se or insulin molecules on the extracellular respiration activity in the culture medium, it was found that the electrode (E2) 1 to 25 r from the center of the cell pattern region contained 25 mM g|UC0se and 25 mM glucose +1. In the cell culture medium of % insu|jn, the oxygen consumption rate was increased to 0.4222 /zM/min and 0.3835 /zM/min, respectively, compared with 10 mM HEPES as the buffer cell culture medium; The oxygen consumption rate of the electrode (E6) at 2.75 r in the center of the zone is 0.2355 " M/min and 0.3834 μ M/min respectively. Therefore, the distance between the cell pattern region and the dissolved oxygen electrode is correlated, and the electrode is closer to the cell pattern region. The measured oxygen consumption rate is higher. The change in cellular respiratory activity can be quantitatively compared by the oxygen consumption rate or oxygen consumption. The ninth (b) graph uses the oxygen concentration measured by the different electrodes in the ninth graph. The value, corresponding to the distance from the center of the cell pattern region, is fitted by the hemispherical diffusion model according to the least square method, and can be obtained after the measurement is stabilized for 5 min, and after the application of the drug for about 5 min (adding g|UC0Se) and 1〇min (added • insulin) concentration and distance diagram, the results are It shows that there is a high correlation coefficient (r2 > 〇·9404) in HBS solution, glucose addition or insuMfl, indicating that the diffusion behavior caused by this cell can be explained by hemispherical mode. The slope obtained by the fitting curve of the ninth (b) graph is the difference between the oxygen concentration of the host solution (bu丨k so丨ution) and the surface of the cell pattern region (Δ C), and then the oxygen consumption is calculated by the second formula. The rate (F) is about 5 49χ1〇-14 丨/s when the measurement is stable for 5 min and about 5 times after the application of the drug (adding the lang sentence and 彳〇(7) called adding insulin). , 9 1〇m〇l/s, and 9·67χ1〇-'_, the results verify that the cells in the environment with gIUC〇Se nutrient source, the respiratory rate is no nutrient source

20 [S 201120441 1_09 times' When adding 1% insulin solution, because the sputum called sputum can help glucose easily enter the cell, it can increase the activity of the cell, so the oxygen consumption rate increases to 1.14 times in HBS solution. . Repeat the test of the respiratory activity test of the cell patterned culture, and compare the single point oxygen consumption rate at different distances in the center of the cell pattern region, and the electrode fitting according to the least square method fitting half-sphere expansion model W oxygen consumption rate for comparison, please refer to ^ diffusion

HBS Array fitting (,· mole/s) yr N el 祀 祀 罕 罕 single single single point E2 (β M/min) E6 (β M/min) 92.9+20.4 — glucose 117_3±26·5 2.72±4.22 1· 97±2·76 insulin 141.5 + 59.0 0.76 soil 0.81 0.91+0.65 ~~π compensated for the analysis of the oxygen consumption rate reproducibility, it can be seen that the single-point electrode analysis can not calculate the oxygen consumption rate in the HBS solution, and reproducibility Poor... 2 and E6 electrodes are 154.78% and ·0% in glucose, respectively, and ins_t is 1〇6 9_7U6% respectively; and the electricity (4) is used to reproduce oxygen consumption rate in HBS, glucose and In 丨nsulin, the 219% 24 4% disk was 22.9%, and the reproducibility was higher than that of the single-point electrode. From the above, it can be seen that the method of calculating the dissolved oxygen electrode array by the hemispherical diffusion model can obtain an accurate oxygen consumption rate compared with 21 • 201120441. Therefore, the present invention uses glass as a substrate, which is more convenient when optically observing cells, and since the oxygen-dissolving electrode is made of a metal thin film, which is suitable for steaming or deplating on a glass substrate, the sensing electrode fabrication procedure can be simplified. Furthermore, by designing the cell patterning technique and the electrode array, the distance between the cell and the sensing electrode in each experiment can be fixed, and the data analysis of the cell oxygen consumption rate can be explained by the hemispherical diffusion model to improve The reproducibility is measured, and the electrode array is located in the oxygen-consuming diffusion layer of the patterned cells, which can quantify the respiration rate of the cells. In addition, the wafer is easily integrated with the microfluidic control system, and can perform high-throughput of drugs and poisons. The microfluidic pad of the present invention can perform large-volume cell culture, and the cells are detected under normal physiological conditions, and the microchannel can be reduced by lifting the microchannel body to lift the microchannel. Shear force generated when replacing a liquid. However, the above is only the preferred embodiment of the present invention, and should not be construed as limiting the scope of the present invention, the simple equivalent changes and modifications made in accordance with the scope of the invention and the contents of the specification should be It is still within the scope of this patent. BRIEF DESCRIPTION OF THE DRAWINGS The first drawing is a perspective view of a dissolved oxygen electrode array wafer in accordance with a preferred embodiment of the present invention. The second drawing is a perspective exploded view of a dissolved oxygen electrode array wafer in accordance with a preferred embodiment of the present invention. 22.201120441 The third drawing is a top plan view of a dissolved oxygen electrode array wafer in accordance with a preferred embodiment of the present invention. The fourth figure is a schematic diagram of the design of the sensing electrode array of the preferred embodiment of the present invention. FIG. 0 is a schematic view showing the manufacturing process of the high-flow channel pad high-level embodiment of the preferred embodiment of the present invention. The fifth (B) diagrams (e) to (h) are schematic diagrams showing the manufacturing process of the microchannel mat high-layer of the preferred embodiment of the present invention. Figure 6 is a schematic diagram of a dissolved oxygen electrical system in accordance with a preferred embodiment of the present invention.

Polar Array Wafer Scrape FIG. 7 is a schematic diagram of a patterned surface modification process in accordance with a preferred embodiment of the present invention. Figure 8 is a graph showing the results of HepG2 cells of the preferred embodiment of the present invention. The ninth (4) drawing is a dissolved oxygen of the preferred embodiment of the present invention. 2 Cell Patterns for Oxygen Concentration Measured by Glucose or 姨 姨 第九 第九 第九 较佳 较佳 较佳 较佳 较佳 He He He He He He He He He He He He He He He He He He He He He He He He He He He He He H degree corresponding to W [Main component symbol description] (10) Sensing wafer (11) Substrate (12) Sensing plane

23 201120441 (121) Auxiliary electrode (122) Dissolved oxygen electrode (14) Insulation layer (141) Cell culture hole (1 5) Cell attachment area (151) Effective diffusion radius (16) Working window (20) Micro flow channel pad High-rise (21) high-film body (211) flow through groove (22) micro-channel layer (221) bottom surface (222) micro-flow channel (223) liquid extraction hole (224) insertion hole (23) cell culture tank ( 30) Glass substrate (31) Photoresist (40) Reference electrode (50) Microinjection pump (60) PDMS layer (61) Hole (70) Poly-L-amino acid (71) Fibronectin (72) Bovine serum white protein

Claims (1)

201120441 VII. Patent application scope: 1. A dissolved oxygen electrode array wafer combined with patterned cell culture, comprising: 'sensing aa piece, comprising a substrate, a cell attachment area, an auxiliary electrode, a plurality of dissolved oxygen An electrode, an insulating layer and a plurality of working windows, the cell attaching region, the It auxiliary electrode and the plurality of dissolved oxygen electrodes are spaced apart from the substrate, the insulating layer is coated on the surface of the substrate, and Covering at least a portion of the auxiliary electrode and the plurality of dissolved oxygen electrodes, and penetrating a circular cell culture hole to expose the cell attachment region, wherein the insulating layer is opposite to each of the dissolved oxygen electrode and the auxiliary electrode The positions of the respective dissolved oxygen electrodes are adjacent to the cell attachment area, and the distance between the working window of each dissolved oxygen electrode and the center of the cell attachment area is smaller than the cell attachment area. Five times the radius (r) (5"), the distance between the working windows of each dissolved oxygen electrode is less than 0.5", and the distance between the working window of the auxiliary electrode and the center of the cell attachment region is greater than the radius of the cell attachment region (r) ) Five times (5"); _ a micro-channel 塾 high-level, is attached to the top surface of the sensing wafer, and includes a sputum film body, a micro-channel layer and a cell culture tank, wherein the 塾-high film body is located Detecting the top surface of the wafer and providing a flow through groove on the side of the high film body, the micro flow channel layer is coupled to the top surface of the high film body, and includes a bottom surface, a micro flow channel and a pumping a liquid hole, the micro flow channel is recessed on the bottom surface, and a shape and a position of the micro flow channel corresponding to a region through which the groove is formed is consistent with the flow through groove, and the liquid suction hole is bored in the micro flow channel layer One side of the microfluidic channel and the flow channel are located on the opposite side of the flow channel, the cell culture channel is inserted through the microchannel layer and the high membrane body and communicates with the microchannel The liquid venting electrode array wafer, wherein the substrate is 3. As in the application of the patent scope, the dissolved oxygen electrode array wafer, , , , The auxiliary electrode and the plurality of dissolved oxygen electrodes are gold layers. 4. The dissolved oxygen electrode array wafer according to claim 2, wherein the auxiliary electrode and the plurality of dissolved oxygen electrodes are gold layers 5. The dissolved oxygen electric array wafer according to any one of the items of the present invention, wherein the thickness of the high-film body is 50 ′ as described in any one of the claims. a dissolved oxygen electrode array wafer, wherein the auxiliary electrode substrate comprises n U plurality of electrodes and the dissolved oxygen electrode array wafer according to claim 5, wherein the auxiliary electrode and the auxiliary electrode a plurality of dissolved oxygen electrodes and the substrate-titanium layer. 8. The dissolved oxygen electrode array crystal #' S + according to any one of claims 1 to 4, wherein the cell attachment region has a radius of 2 〇〇 (4). 9. The dissolved oxygen electric conversion wafer according to claim 5, wherein the cell attachment area has a radius of 2 〇〇 from m. 1) The dissolved oxygen electrode array wafer as described in claim 7 wherein the cell attachment region has a radius of 2 〇〇 from m. 11. The oxygen-dissolving electrode array wafer according to any one of the items (4), wherein the high-film system is a layer of polydimethyl oxaxane (poly(dimethylsj|〇xane). ), PDMS) film. Soil 12, as described in claim 9 of the dissolved oxygen electrode array wafer 26 201120441, the mat is still a layer of polydimethyl methoxyoxane (p〇ly (dimethy|Siloxane), PDMS) film. 13. The dissolved oxygen electrode array wafer according to any one of claims 1 to 4, wherein the auxiliary electrode has a distance of 9.5 r from the cell attachment region. 14, of which 15' Wherein, wherein, the dissolved oxygen electrode array wafer according to claim 5 of the patent application has a distance of 9.5r from the cell attachment region. The dissolved oxygen electrode array wafer according to claim 12 of the patent application has a distance of 9.5 r from the cell attachment region. The number of dissolved oxygen electrodes described in the dissolved oxygen electrode array wafer of claim 12 is six. 17 ', wherein the number of dissolved oxygen electrodes described in the dissolved oxygen electrode array wafer according to claim 13 is six. 1 8. The dissolved oxygen electrode array wafer according to the 14th box tearing, +-々々# & Gu Di 14 of the patent application scope, wherein the number of the dissolved oxygen electrodes is six. 19, wherein, the distance is 4.75 r. - .>4 I dry yjj 工作 working window of each dissolved oxygen electrode and center of the cell attachment area from 1.25", 15", 2·25", 2.75", 4.25 r, -呗 described electrode array wafer Wherein, the distance between the working window of each of the four electrodes of each lyochlor and the 丫I curry is 1 25 r, 1 5 Γ, 〇1 3 Γ, 厶25 γ, 2 75 4·25 r and 4.75 r. Electricity 21, such as any of the pole array wafers of the scope of the patent application, wherein the dissolved oxygen port of the working window of each dissolved oxygen electrode is attached to the cell center 27 201120441 The distances are 1.25 I·, 1.5 r, 2.25 “, 2.75 “' 4.25 r, and 4.75 r. 22. A method for fabricating a dissolved oxygen electrode array wafer combined with patterned cell culture, comprising: providing a top surface of a substrate a cell attachment region, and a plurality of dissolved oxygen electrodes and an auxiliary electrode are deposited at intervals, an insulating layer is formed on the top surface of each electrode, and a working window is formed on the insulating layer relative to each electrode to expose a corresponding Electrode and a circular shape on the insulating layer a cell culture well is formed to display the cell attachment region to form a sensing wafer; a microchannel layer is provided, a microchannel is recessed from the bottom surface of the microchannel layer, and is passed through the microchannel layer a liquid extraction hole is provided, the liquid extraction hole is connected to the micro flow channel; a high film body is provided, and the high film body is coupled under the micro flow channel layer to form a micro flow channel pad upper layer, and The microchannel layer and the high membrane body are bored to form a cell culture tank, and the cell culture tank system is in communication with the microchannel and the liquid extraction hole; Forming the dissolved oxygen electrode array wafer on the top surface of the sensing wafer, and the cell culture tank exposes a working window of the cell attaching region and each dissolved oxygen electrode. 23. Dissolving as described in claim 22 The method for producing an oxygen electrode array wafer, wherein the substrate is a glass. The method for preparing a dissolved oxygen electrode array wafer according to claim 22, wherein the high film system coats the PDMS solution. Formed on the top surface of a substrate. The method for preparing an oxygen-dissolving electrode array wafer 28 201120441 according to claim 23, wherein the padding 膣* 膜 膜 本 本 将该 将该 PD PD PD PD PD PD PD PD PD 、 、 、 、 、 、 、 、 、 、囹坌 囹坌 ^ ^ ^ ^ ^ The oxygen-dissolving electrode array wafer described in item 25, -, the 尚 尚 尚 膜 膜 本 以 以 以 氧气 氧气 氧气 氧气 氧气 氧气 氧气 氧气 氧气 氧气 氧气 氧气 氧气 氧气 氧气 氧气 氧气 氧气 氧气 氧气 氧气 氧气 氧气 氧气 氧气 氧气塾 塾 。 ... ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ 专利 专利 专利 专利 专利 专利 专利 专利 专利 专利 专利 专利 ' ' ' ' ' ' ' ' ' ' ' ' 〇〇_, the number of dissolved oxygen electrodes is six 'each dissolved oxygen electrode and the center of the cell attachment area are 〇.25mm, 〇.3mm, 0.45mm' 〇.55mm, 〇85mm to / 0.95 Mm » p 28, as in the solution of the dissolved oxygen electrode array wafer described in the patent application 帛27, before depositing the auxiliary electrode and the plurality of dissolved oxygen electrodes on the substrate, the titanium layer is first sputtered on the substrate On the top surface of the material, a gold layer is deposited on the seed layer and the partial gold layer is dissolved by the lift (|ift_Gff) process. Forming the auxiliary electrode and the plurality of dissolved oxygen electrodes; further forming on the top surface of each electrode, in the edge layer, and forming a working window on each electrode after exposure, development and hard baking, respectively, 29 for one cell The respiratory activity is evaluated and combined with a patterned cell culture of a cardiooxidic electrode array wafer, which is produced by the method described in any one of claims 22 to 28. Schematic · (such as the next page) 29