201113368 六、發明說明： 【發明所屬之技術領域】 [0001] 本發明有關於一種培養I電極陣列，其包括底層、 黏著層、連接層、隔絕層、導引層所構成，主要用於細 胞的培養、組織切片、電氣切片，可以快速進行細胞組 織之辨識分析，同時也可以對其形態加以量化研究，以 解決耗時主觀等人為分析之缺點，以瞭解細胞組織受損 、死亡等形態變化之機制，並可即時監測所培養細胞的 生長狀況者。 〇 【先前技術】 [0002] 按，習用細胞組織之電氣分析，目前多著重在單電 • 極之生醫電阻頻譜分析，並無電特性分佈影像之分析技 術，顯微鏡之發明，讓我們可以更進一步觀察與分析細 胞與組織之形態變化，顯微鏡影像在很多細胞與組織之 研究上扮演相當重要之角色，自動化顯微鏡醫學影像分 析在細胞等級上之具體研究成果仍然相當有限，過去所 提出之自動化神經細胞組織辨識與分析方法相當有限， 〇 雖然現有市售影像處理與分析軟體相當多，但是對於細 胞組織等分析量化應用，仍然不夠彈性與方便，對於生 醫相關研究人員難以操作與自訂程式，故研發具有合適 簡化人機界面之神經細胞組織影像分析與量化應用軟體 在此益形重要，以往的神經細胞的培養與觀測，無法即 時監測神經細胞之生命狀況與凋零變化，僅能依據分析 圖譜來判斷細胞的變化過程者。 【發明内容】 098133519 表單編號A0101 第3頁/共16頁 0982057384-0 201113368 [0003] 其上述習用的技術，較著重在單電極之生醫電阻頻 譜分析上，並無電特性分佈影像之分析技術，自動化顯 微鏡醫學影像分析在細胞上之具體研究成果仍然相當有 限，在神經細胞組織相關形態學上之量化與辨識分析最 為明顯，無法即時監測神經細胞之生命狀況與凋零變化 ，僅能依據分析圖譜來判斷細胞的變化過程者。 本發明主要目的在於提供一種培養皿電極陣列，可 將電阻抗影像應用在細胞組織的電氣特性分析，配合微 機電製程技術，研發微電阻抗斷層掃描技術，建立電氣 切片，以擷取組織電特性影像並分析，且更進一步整合 細胞組織之電氣特性與形態特性，使得細胞在培養過程 中，便能加以即時監測，建立動態分析者。 為達前揭之目的，本發明培養m電極陣列係在電極 内緣設置導引層^利用導引層的南導電性可將細胞產生 的電氣狀態進行傳遞分析，可即時監測所培養細胞的變 化，該培養瓜電極陣列包含有底層、黏著層、連接層、 隔絕層、導引層所構成；該底層，係以聚醯化合物為載 體，在底層上端設置有黏著層；該黏著層，設置在底層 上方，可供連接層附著；該連接層，設置在黏著層上方 ，可供隔絕層的附著；該隔絕層，設置在連接層上方， 可於通電時隔絕連接層與導引層電子間的置換，避免電 極的使用效能降低；該導引層，設置在隔絕層上，具有 導引電流之作用，可將細胞電氣狀態輸出，依據輸出的 訊號來做細胞的影像重建與細胞變化的即時監測者。 本發明不僅可以快速進行細胞組織之辨識分析，同 時也可以對其形態加以量化研究，以解決耗時主觀等人 098133519 表單編號A0101 第4頁/共16頁 0982057384-0 201113368 為分析之缺點，探討更有意義之參數，以瞭解神經細胞 2又損、死亡等形態變化之_，在細胞組織相關形 悲子上之量化與辨識分析最為明顯者。 【實施方式】 闺 本發明之培養皿電轉列，〔請參㈣-圖〕其包 括底層（1)、黏著層（2)、連接層（3)、隔絕層 (4)、導引層（5)所構成；其中： Ο201113368 VI. Description of the Invention: [Technical Field] [0001] The present invention relates to a cultured I electrode array comprising a bottom layer, an adhesive layer, a connecting layer, an insulating layer and a guiding layer, which are mainly used for cells. Culture, tissue sectioning, and electrical sectioning can quickly identify and analyze cell tissues, and can also quantify its morphology to solve the shortcomings of time-consuming subjective and other human analysis, to understand the morphological changes such as cell damage and death. Mechanism and immediate monitoring of the growth status of the cultured cells. 〇[Prior Art] [0002] According to the electrical analysis of conventional cell tissue, the current focus is on single-electron and ultra-medical resistance spectrum analysis. There is no analysis technique of electrical characteristic distribution image, and the invention of microscope allows us to go further. Observing and analyzing the morphological changes of cells and tissues, microscopic imaging plays a very important role in the research of many cells and tissues. The specific research results of automated microscopy medical image analysis at the cell level are still quite limited. The automated nerve cells proposed in the past The method of tissue identification and analysis is quite limited. Although there are quite a lot of commercially available image processing and analysis software, it is still not flexible and convenient for the analysis and application of cell tissues, etc. It is difficult for biomedical researchers to operate and customize programs. It is important to develop and analyze the neuron tissue image analysis and quantification application software with suitable human-machine interface. The culture and observation of the neural cells in the past can not monitor the life and fade changes of nerve cells in real time, only based on the analysis map. Judgment fine The process of change were. SUMMARY OF THE INVENTION 098133519 Form No. A0101 Page 3 of 16 page 0982057384-0 201113368 [0003] The above-mentioned conventional techniques are more focused on the analysis of the bioelectrical resistance spectrum of a single electrode, and there is no analysis technique of the distribution of electrical characteristics. The specific research results of automated microscopy medical image analysis on cells are still quite limited. The morphological quantification and identification analysis of nerve cell tissue is the most obvious. It is impossible to monitor the life and fade changes of nerve cells in real time. It can only be based on the analysis map. The person who judges the change process of the cell. The main object of the present invention is to provide a petri dish electrode array, which can apply an electrical impedance image to the electrical characteristics analysis of a cell tissue, cooperate with a microelectromechanical process technology, develop a micro-electrical impedance tomography technique, and establish an electrical slice to extract tissue electrical characteristics. Image and analysis, and further integration of the electrical characteristics and morphological characteristics of the cell tissue, so that cells can be monitored in real time during the culture process to establish a dynamic analyst. For the purpose of the prior disclosure, the m electrode array of the present invention is provided with a guiding layer at the inner edge of the electrode. The south conductivity of the guiding layer can be used to transmit and analyze the electrical state generated by the cell, and the change of the cultured cell can be monitored immediately. The cultivating melon electrode array comprises an underlayer, an adhesive layer, a connecting layer, an insulating layer and a guiding layer; the bottom layer is provided with a polyfluorene compound as a carrier, and an adhesive layer is disposed at an upper end of the bottom layer; the adhesive layer is disposed at Above the bottom layer, the connection layer is attached; the connection layer is disposed above the adhesive layer for attachment of the isolation layer; the isolation layer is disposed above the connection layer to isolate the connection layer from the electrons of the guide layer when energized Displacement, to avoid the use of the electrode is reduced; the guiding layer, which is arranged on the insulating layer, has the function of guiding current, can output the electrical state of the cell, and perform image reconstruction and cell change monitoring according to the output signal. By. The invention not only can quickly carry out the identification analysis of the cell tissue, but also can quantitatively study the morphology thereof to solve the time-consuming subjective person 098133519 Form No. A0101 Page 4/16 pages 0982057384-0 201113368 For the shortcomings of the analysis, More meaningful parameters to understand the morphological changes of nerve cells 2 loss, death, etc., the most quantitative and identification analysis of cell structure related sorrows. [Embodiment] The electroplating of the petri dish of the present invention, [see (4) - Figure], includes the bottom layer (1), the adhesive layer (2), the connecting layer (3), the insulating layer (4), and the guiding layer (5). Constituted; where: Ο
底層⑴，〔請-併參閱第二圖〕係以聚酿化合 物為載體，具有絕緣性、耐熱、耐腐錄及耐電孤性等 性質，在底層（1)上端設置有黏著層（2); 黏著層（2)，設置在底層⑴上方，其黏著層（2 )材質可為環氧樹脂’並可供連接層（3)附著； 連接層（3)’設置在黏著層（2)上方，其連接 層（3)材質可為薄銅膜’可利用壓膜技術，將銅膜壓 合《著層（1 2 )上，其銅膜表面易氧化，使用檸檬 酸做前處理以去除銅表面氧化層，以便隔絕層（4)的 附著： 隔絕層（4)，設置在連接層（3)上方，其隔絕 層（4 )材質可為_合金’可於通電時隔絕連接層（ 3)與導引層（5)電子間的置換，避免電極的使用效 能降低； 導引層（5) ’設置在隔絕層（4)上，具有導引電流 之作用’可將細胞電氣狀態輸出’依據輸出的訊號來做 細胞的影像重建與細胞變化的即時監測，其導引層（5 )材質可為黃金’可提高導電度’亦具有良好生物相容 性者。 098133519 表單編號Α0101 第5頁/共16頁 0982057384-0 201113368 •極2實施騎皿電轉_，〔請參_三圖〕當 I的材質堆疊設置完時，進行光學微影⑹的 "，、先學微影（6)包含有光罩曝光顯影（61) 阻^顯影（62)、關金屬薄膜（63)、移除光 6_占’其光罩曝光顯影（61)係將所需的圖形 :由光罩轉換到光阻下面的電極表面上，讓電極表面產 "光罩轉換的圖形，電極表面經光罩曝光顯影（61 )後進行光阻㈣（62)，將総㈣在電極表面上 ’使經光罩曝光顯影（6 D後的圖形顯現在電極表面 將丄光阻顯#( 6 2 )後的電極進行糊金屬薄膜 (6 3)的動作’將電極表面顯現的圖形以外不需使用 的部份進雜刻動作，其電極經⑽、作將電㈣刻成 32組的陣列電極，其電極具有柔軟性'絕緣性耐腐 純及对電孤性等性質，所以可將電極陣列組彎曲，内 嵌於載玻片上的壓克力圓柱體内側，將經蝕刻後的電極 進行移除光阻（6 4)的動作，將塗佈在電極表面上的 光阻進仃清洗移除，〔請_倂㈣第四圖〕將經光學微 影（6)處裡後的電極設在電路板（7) ±，在電路板 (7 )的-面設置有與電極相㈣數#的連接線路（7 1 )可與導引層（5)相連接’在連接線路（7工）的 -端設置有相對應數量的針腳（7 2)可將電流輸入， 並可將細胞的電氣值輸出，t設置在底層上方的材料堆 疊並钱刻完成時，其底層（^ )可幫曲陣列電極並内嵌 於載玻片上的Μ克力8}柱體内側’其圓柱狀可便於細胞 影像的重建，〔請一倂參閱第五圖〕其導引層（5)可 設置在黏著層（2)上，以減少連接層（3 )與隔絕層 098133519The bottom layer (1), [please - and refer to the second figure] is a polymerized carrier as a carrier, having properties such as insulation, heat resistance, corrosion resistance and electrical resistance, and an adhesive layer (2) is disposed at the upper end of the bottom layer (1); The adhesive layer (2) is disposed above the bottom layer (1), and the adhesive layer (2) is made of epoxy resin and can be attached to the connection layer (3); the connection layer (3) is disposed above the adhesive layer (2). The connecting layer (3) can be made of a thin copper film. The copper film can be pressed onto the layer (1 2 ), and the surface of the copper film is easily oxidized. The citric acid is pretreated to remove the copper surface. An oxide layer for the adhesion of the insulating layer (4): the insulating layer (4) is disposed above the connecting layer (3), and the insulating layer (4) may be made of _alloy, which can isolate the connecting layer (3) when energized The displacement between the electrons of the guiding layer (5) avoids the use efficiency of the electrode; the guiding layer (5) is disposed on the insulating layer (4) and has the function of guiding current to output the electrical state of the cell. The signal is used for real-time monitoring of cell image reconstruction and cell changes, and its guiding layer (5) material can be It is also a good biocompatibility for gold. 098133519 Form number Α0101 Page 5 of 16 page 0982057384-0 201113368 •Pole 2 implementation of riding electric _, [Please refer to _ three pictures] When the material stack of I is set, perform optical lithography (6) ", First learn lithography (6) including reticle exposure development (61) resistance ^ development (62), off metal film (63), remove light 6_ occupies its reticle exposure development (61) will be required Graphics: The reticle is switched to the surface of the electrode under the photoresist, and the surface of the electrode is produced by the reticle conversion pattern. The surface of the electrode is exposed by the reticle (61) and then the photoresist (4) (62) is placed. On the surface of the electrode, the film is exposed and developed (the pattern after 6 D appears on the surface of the electrode, and the electrode after the photoresist is exposed to #(6 2 ) is subjected to the action of the paste metal film (63). The part that is not required to be used for the engraving action, the electrode is (10), and the electric (four) is engraved into 32 sets of array electrodes, and the electrodes have the properties of softness, insulation resistance, corrosion resistance and electrical solicity, so The electrode array set is bent and embedded inside the acrylic cylinder on the slide, and the etched The electrode removes the photoresist (6 4), and the photoresist coated on the surface of the electrode is cleaned and removed, [Please _ 倂 (4) 4th] will pass through the optical lithography (6) The electrode is arranged on the circuit board (7) ±, and the connection line (7 1 ) with the electrode phase (four) number # is connected to the surface of the circuit board (7) to be connected with the guiding layer (5) 'on the connection line (7) The end of the work is provided with a corresponding number of pins (7 2) to input current, and the electrical value of the cell can be output, t is set on the material stack above the bottom layer and when the money is completed, the bottom layer (^) can be The Array Electrode is embedded in the inside of the slide on the slide. The cylindrical shape of the cylinder can facilitate the reconstruction of the cell image. [Please refer to Figure 5] The guide layer (5) can be placed in Adhesive layer (2) to reduce the connection layer (3) and the isolation layer 098133519
表單編號删1 第6頁/共16頁 0982057 201113368 (4)的設置工時與成本者。 本發明之細胞影像重建的方式，〔請參閲第四圖〕 將設置好的電極置入培養班（8)内，利用内緣的導引 層（5)與待測物接觸，並傳遞待測物所產生的電氣值 ，利用對邊法與相鄰邊法等兩種方法來做運算，其對邊 法是在電極的對邊兩側加上刺激源，測量阻抗又可分為 兩種：一為旋轉方式，量測刺激源依序旋轉180度所量測 得的阻抗值，連續五次量測，最大值發生處都在微電極 陣列組接合處，且由統計分析發現，隨著時間的過去， 〇 每次量測的平均值、最大值與最小值都隨著遞增，這種 現象，在不同的微電極培養m，也得到相同的結果；也 • 發現培養皿液面的高低與阻抗的平均值有反比的關係， 所以可得之微電極培養皿的阻抗值與液面的蒸發現象有 密切關係，並且培養皿的橫截面面積越小，蒸發與阻抗 值的變化就越明顯，而後旋轉刺激源至下一相鄰電極， 繼續做測量；二為相對方式，刺激源保持不動，測量其 他電極對GND的電壓值，因電流可均勻地流經待測物體， 〇 與待測物有較好的反應，故可得到較佳的測量結果，且 對於形狀的對稱性有良好的評估效果，其對邊法之旋轉 方式與電極位置的關係，其相鄰邊法是在鄰近兩個電極 加上刺激源，測量阻抗與對邊法相同也可分為上述的兩 種方式，因電流密度集中分佈於電極周邊，故可用來檢 測電極之間的相似度，或電極陣列位置的誤差程度，使 用不同刺激方式，將所測量的阻抗值與電極相關位置晝 圖分析，並藉由統計方法分析數據。 其優點在於，利用中空圓柱形的電極，以方便細胞 098133519 表單編號A0101 第7頁/共16頁 0982057384-0 201113368 電氣特性之分析，且可使系統在量測電訊號上提升訊號 雜訊比，將電極蝕刻劃分成3 2組陣列電極可增進量測 的精密度與準確度，使細胞影像重建的精細度增加，不 僅可以快速進行神經細胞組織之辨識分析，同時也可以 對其形態加以量化研究，以解決耗時主觀等人為分析之 缺點，探討更有意義之參數，以瞭解神經細胞組織受損 、死亡等形態變化之機制者。 綜上所述，當知本發明具有新穎性，且本發明未見 之於任何刊物，當符合專利法第2 1、2 2條之規定。 唯以上所述者，僅為本發明之一較佳實施例而已， 當不能以之限定本發明之範圍。即大凡依本發明申請專 利範圍所作之均等變化與修飾，皆應仍屬本發明專利涵 蓋之範圍内。 【圖式簡單說明】 [0005] 第一圖：本發明之立體外觀示意圖 第二圖：本發明之電極剖面示意圖 第三圖：本發明之顯影蝕刻流程示意圖 第四圖：本發明之組合立體外觀示意圖 第五圖：本發明之另一電極剖面示意圖 【主要元件符號說明】 [0006] 1 底層 2 黏著層 3 連接層 4 隔絕層 5 導引層 6 光學微影 6 1 光罩曝光微影 6 2 光阻顯影 6 3 #刻金屬薄膜 6 4 移除光阻 表單編號A0101 第8頁/共16頁 098133519 0982057384-0 201113368 7 電路板 71 連接線路 7 2 針腳 8 培養皿Form number deletion 1 Page 6 of 16 0982057 201113368 (4) Set the working hours and cost. The method for reconstructing the cell image of the present invention, [see the fourth figure], the set electrode is placed in the training class (8), and the guiding layer (5) of the inner edge is used to contact the object to be tested, and is transmitted to The electrical value generated by the object is calculated by two methods: the edge method and the adjacent edge method. The edge method is to add a stimulus source on both sides of the opposite side of the electrode, and the impedance can be divided into two types. : One is the rotation mode, measuring the impedance value measured by the stimulus source rotated by 180 degrees in sequence, and measuring five times in succession, the maximum occurrence occurs at the junction of the microelectrode array group, and it is found by statistical analysis that In the past, the average, maximum and minimum values of each measurement are increasing. This phenomenon, the same result is obtained by culturing m at different microelectrodes; also • the level of the liquid level of the culture dish is found. It is inversely proportional to the average value of the impedance, so the impedance value of the microelectrode culture dish is closely related to the evaporation phenomenon of the liquid surface, and the smaller the cross-sectional area of the culture dish, the more obvious the change of evaporation and impedance value. And then rotate the stimulus to the next The adjacent electrode continues to measure; the second is the relative mode, the stimulus source remains stationary, and the voltage value of the other electrode to GND is measured. Since the current can flow uniformly through the object to be tested, the 〇 and the object to be tested have a good reaction, so Better measurement results are obtained, and the shape symmetry is well evaluated. The relationship between the rotation method of the edge method and the position of the electrode is adjacent to the two electrodes plus the stimulus source, and the impedance is measured. The same as the opposite side method can also be divided into the above two ways, because the current density is concentrated around the periphery of the electrode, it can be used to detect the similarity between the electrodes, or the degree of error in the position of the electrode array, using different stimulation methods, The measured impedance values are compared with the electrode-related position map analysis, and the data is analyzed by statistical methods. The advantage is that the hollow cylindrical electrode is used to facilitate the analysis of the electrical characteristics of the cell 098133519 Form No. A0101, page 7 / 16 page 0982057384-0 201113368, and the system can improve the signal noise ratio on the measuring signal. Dividing electrode etching into 32 sets of array electrodes can improve the precision and accuracy of measurement, and increase the fineness of cell image reconstruction. It can not only quickly identify and analyze nerve cell tissue, but also quantify its morphology. In order to solve the shortcomings of time-consuming subjective and other human analysis, explore more meaningful parameters to understand the mechanism of morphological changes such as nerve cell tissue damage and death. In summary, it is known that the present invention is novel, and the present invention is not found in any publication, and is in compliance with the provisions of Articles 21 and 22 of the Patent Law. The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto. That is, the equivalent changes and modifications made by the applicant in accordance with the scope of the patent application of the present invention are still within the scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a perspective view of a stereoscopic appearance of the present invention. FIG. 2 is a schematic cross-sectional view of an electrode according to the present invention. FIG. 3 is a schematic view showing a development etching process of the present invention. Figure 5: Schematic diagram of another electrode of the present invention [Description of main components] [0006] 1 Underlayer 2 Adhesive layer 3 Connection layer 4 Insulation layer 5 Guide layer 6 Optical lithography 6 1 Mask exposure lithography 6 2 Photoresist development 6 3 #刻金属膜6 4 Remove photoresist form No. A0101 Page 8 of 16 098133519 0982057384-0 201113368 7 Circuit board 71 Connection line 7 2 Pin 8 Petri dish
098133519 表單編號A0101 第9頁/共16頁 0982057384-0098133519 Form No. A0101 Page 9 of 16 0982057384-0