1377345 六、發明說明： v 【發明所屬之技術領域】 本發明係有關於一種細胞活性評估晶片，尤其是—種 • 用於檢測多生理參數之細胞活性評估晶片。 【先前技術】 細胞為生物體之最小生命單位，以細胞作為檢測單元 可代表生物體直接的生理反應。當細胞受到外來物理量戋 • 化學性刺激時，細胞會自行調整並改變其營養物質之消耗 量及其代謝物的釋出量；藉由量測細胞攝取或釋出物質的 變化量訊息，可評估細胞當下的生理活性，並且量化外源 v性刺激對細胞生理之影響。而目前於細胞活性狀態之評估 技術大約可分為兩大類，一是量測細胞能量的代謝情形； 其二是檢測細胞特異性變化或接受器（receptor)/配體 (ligand)系統的反應（如膜電位的變化等）。而於量測細胞 能量的代謝情形部份，又以下面三種策略來檢測指標性代 • 謝產物較為常見： 丨·二氧化碳生成量與P Η值的量測 請參照反應式一所示，使用ρΗ值作為新陳代謝指 標的優點之一，是因為細胞在進行醣原酵解和呼吸氧化代 謝反應過程中，會產生許多有機酸並釋出二氧化碳氣體， 當這些有機酸解離或二氧化碳溶解時，皆會產生氫離子而 被排出細胞外，因此可藉由ρΗ值的量測，來得知細胞的 新陳代謝速率，而胞外二氧化碳濃度與ρΗ值之間的關係 s奢參照反應式二所示之Henders丨n Hasse|ba|ch公式描述 1377345 °6Η12〇+6〇2^6002+6Η20+38>ΑΓΡ 反應式一 p// = 6.1 + log^^J [C〇2] 反應式二 Π氧氣消粍的量測技術1377345 VI. Description of the Invention: v Technical Field of the Invention The present invention relates to a cell activity evaluation wafer, and more particularly to a cell activity evaluation wafer for detecting multiple physiological parameters. [Prior Art] A cell is the smallest unit of life of an organism, and a cell as a detection unit can represent a direct physiological reaction of an organism. When the cells are subjected to external physical quantities, chemical stimulation, the cells will adjust and change the consumption of nutrients and the release of their metabolites. By measuring the amount of change in the uptake or release of the cells, the cells can be evaluated. The current physiological activity of the cells, and quantify the effects of exogenous v-stimulation on cell physiology. At present, the evaluation techniques of cell activity state can be divided into two categories, one is to measure the metabolism of cell energy; the other is to detect cell-specific changes or receptor/ligand system responses ( Such as changes in membrane potential, etc.). In the measurement of the metabolic part of cell energy, the following three strategies are used to detect the index generation and the Xie product are more common: 丨·The amount of carbon dioxide produced and the P Η value are measured according to the reaction formula 1, using ρΗ One of the advantages of the value as a metabolic indicator is that the cells produce many organic acids and release carbon dioxide gas during the process of glycogenolysis and respiratory oxidative metabolism. When these organic acids dissociate or dissolve carbon dioxide, they are produced. Hydrogen ions are excreted outside the cell, so the rate of metabolism of the cells can be known by the measurement of the value of ρΗ, and the relationship between the concentration of extracellular carbon dioxide and the value of ρ s is referred to Henders丨n Hasse shown in Equation 2 |ba|ch formula description 1377345 °6Η12〇+6〇2^6002+6Η20+38>ΑΓΡ Reaction formula p// = 6.1 + log^^J [C〇2] Measurement of reactive oxygen dioxide consumption technology
由於細胞需不斷的合成許多大分子（如蛋白質）來維持 生理的正常運作，在這些生化合成的過程中，須消耗大量 的ΑΤΡ，才能克服生化反應所需的活化能以促使生化合 成反應發生。而ΑΤΡ的合成則藉由生命體吞食、消化與 吸收外界進來的食物進行氧化，藉此供生理所需；請再參 照反應式一所示，如一莫耳的葡萄糖分子在經過醣原酵解 (glycolysis)與擰檬酸循環（Cjtnc add cycle)後可生成 38莫耳的ATP，同時在此氧化過程中也消耗6莫耳的氧 分子。因此氧氣分子在ATp的合成過程中扮演著重要的Since cells need to continuously synthesize many macromolecules (such as proteins) to maintain physiological normal function, a large amount of strontium is consumed in these biochemical processes to overcome the activation energy required for biochemical reactions to promote biochemical synthesis. The synthesis of cockroaches is oxidized by the living body to swallow, digest and absorb the food coming in from the outside, thereby providing physiological needs; please refer to the reaction formula 1, such as a molar glucose molecule undergoing glycogenolysis ( Glycolysis and the Cjtnc add cycle produce 38 moles of ATP, while also consuming 6 moles of oxygen molecules during this oxidation. Therefore, oxygen molecules play an important role in the synthesis of ATp.
角色，所以可利用氧氣的消耗量，來代表生命體的生理活 性’當細胞生理活性愈高，需合成大量蛋白質時，將消耗 掉較多的氧氣來合成較多的ATP以協助蛋白質的合成。 目前於市面上商品化之細胞活性檢測晶片系統，其簡 介如下： (a)加拿大 Molecular Devices 公司之 Cytosensor® Microphysiometer ：Role, so the consumption of oxygen can be used to represent the physiological activity of the living body. When the physiological activity of the cell is higher, a large amount of protein needs to be synthesized, and more oxygen is consumed to synthesize more ATP to assist in protein synthesis. The currently commercially available cell activity assay wafer system is as follows: (a) Cytosensor® Microphysiometer from Molecular Devices, Canada:
Cytosensor® Microphysiometer 基本結構是以矽作 為底材，並於上方工作一層氮化矽，藉由光趨電位感測器 5 1377345 ▲ (丨丨ght-addressable potentiometric sensor, LAPS)進行細 胞外酸化率之量測。其主要工作原理是透過光電效應，以 特疋波長的光激發矽基材以產生電子電洞對，此時藉由恆 * 電位儀（potentiostat)於參考電極與工作電極間施與一固定 電位，以趨動電子電洞對產生電流。而電子電洞電流大小 與絕緣層表面電位有關，當細胞代謝產生較多質子而改變 pH值時，同時會改變絕緣層的表面電位，故會改變真正 落在矽基材上趨動電子電洞對移動的電場大小所以可從 • PH量測值的變化量來評估細胞的活性。此系統的特色為 只須少量的樣本液或細胞即可進行量測，並且有良好之靈 敏度及少量的樣本消耗，而藉由雷射光點的照射，可直接 .定位直化待測點細胞之胞外pH值；然而，此系統僅能進 行單一生理指標胞外酸化率之檢測，於實際細胞生理活性 評估上資料不夠廣泛。 (b)德國 Bionas® 公司之 Analyzing system 及 Metabolic chip : 擊德國Bionas®公司的多參數細胞生理分析系統可對胞 外酸化率（離子選擇性場效電晶體感測器（j〇n sensitjve 、fie丨d effect transist〇「，丨SFET))、氧氣消耗率（安培法感測 器）及細胞貼附性（交指狀電極導電度感測器）進行整合性 i測。其主要晶片設計是將細胞培養於封裝製作完成的晶 片中’由晶片上方兩端以微管道的方式輸送細胞培養液及 測試藥物’其内部緩衝液體積約為1 〇 "卜此感測晶片之 特點是能承受滅菌時的高溫高壓，並於培養時細胞不會受 到外界污染’容易與微電子電路整合並具有檢測快速與樣 6 1377345 本試劑量少等優點；但ISFET晶片經反覆實驗測試後之 t 壽命目前仍待驗證。上述商品化量測晶片皆以封閉式微流 道進行液體更換’並以矽為基材，其不透光性阻礙光學法 . 檢測細胞型態的對照性。 由上述背景簡介得知，目前已商品化之細胞活性感測 系統’仍具有下列缺點： 1、 細胞活性變化為一综合性之生理反應，每次單一 參數的量測無法完全得知細胞對外界刺激的生理響應，但 鲁 進行多參數細胞活性量測時’感測器的量測原理不同大幅 增加了分析儀器之彳賈格，如Bionas®公司之Metabolic chip以ISFET量測胞外酸化率、安培法量測溶氧，阻抗 分析法量測細胞貼附。 2、 目則於細胞活性晶片製作上，皆以梦作為底材以利 半導體製程技術之整合，然而卻限制了以顯微鏡同時觀測 細胞形態的功能。 • 【發明内容】 本發明人有鑑於既有之細胞活性感測系統須要昂貴的 價格及複雜的製程才能進行多參數細胞活性量測，且皆以 發作為底材而限制同時觀測細胞形態的功能，因此經過長 時間的研究以及不斷試驗後，終於發明出此用於檢測多生 理參數之細胞活性評估晶片。 本發明之目的在於提供一 是一種整合晶片式細胞培養、 多生理參數檢測電極陣列之製 種細胞活性評估晶片，尤其 細胞耗氧率與胞外酸化率等 程技術’以進行細胞生理活 7 1377345 性檢測時同步觀測細胞形態，並能簡化不同生理參數量測 電極於玻璃基材之電化學電極陣列之細胞活性評估晶片。 為達上述目的’本發明所運用的技術手段係在於提供 ^ 一種用於檢測多生理參數之細胞活性評估晶片，其係包括 一感測晶片，係包括一基材、一感測平面及一絕緣層 ’該感測平面設於該基材頂面’並包括互相間隔之一辅助 電極、一參考電極及位於該輔助電極該參考電極之間:的二 • 溶氧電極與二ΡΗ電極，該絕緣層係設於該感測平面頂面， 於該絕緣層上相對於各電極的位置穿設一工作窗口，於相 對位於該參考電極與兩pH電極之工作窗口内係設有一氧化 銥層以覆蓋於該參考電極與兩pH電極對應該工作窗口的區 域； 一微流道墊高層，係結合於該感測晶片之頂面，包括 一墊高膜本體、一微流道層及一細胞培養槽，該墊高膜本 體位於該感測晶片之頂面，係包括一流通貫槽，該微流道 # 層結合於該墊高膜本體之頂面，並包括一底面、一微流道 、一抽液孔及一透氣孔’該微流道係凹設於該底面，且該 微流道對應該流通貫槽的區域之形狀與位置係與流通貫槽 相符’該抽液孔及該透氣孔分別穿設於該微流道層的二側 ’且該透氣孔與該流通貫槽係位於同側，該細胞培養槽貫 穿於該微流道層及該墊高膜本體且連通於該微流道、該抽 液孔及及該流通貫槽，以露出兩pH電極、兩溶氧電極之工 作窗口及該輔助電極。 較佳的是，該基材係為玻璃。 8 1377345 f 較佳的是，該感測平面係為金層》 其中’該感測平面與該基材間係包括一鈦層。 較佳的是，該墊高膜本體之厚度為 • 較佳的是，該感測平面厚度為250 nm，該工作窗口面 積為 20 //mx20//m。 其中，該墊高臈本體係為一層聚二甲基矽氧烷 (poly(dimethylsiloxane), PDMS)。 其中’該微流道係為直線型。 • 本發明還關於一種用於檢測多生理參數之細胞活性評 估晶片之製法，係包括： 於一基材頂面形成一辅助電極、一參考電極及位於該 輔助電極該參考電極之間的二溶氧電極與二pH電極，於各 電極上方設置一絕緣層，並於該絕緣層上相對於各電極的 位置分別穿設有一工作窗口，以露出對應之電極，於位在 該參考電極與兩pH電極之工作窗口上沈積一氧化銥層，以 覆蓋於該參考電極與兩pH電極對應該工作窗口的區域； • 提供一微流道層’由該微流道層的底面凹設有一微流 道’該微流道層係穿設該抽液孔及該透氣孔，該抽液孔及 該透氣孔分別形成於該微流道的兩側；提供一藝高膜本體 ’將該墊高膜本體結合於該微流道層的下方而形成一微流 道墊高層，並於該微流道層及該墊高膜本體穿設形成_細 胞培養槽，且該細胞培養槽係與該微流道、該抽液孔及該 透氣孔互相連通； 將該微流道塾咼層组裝於該感測晶片頂面而形成一細 胞活性評估晶片’且該細胞培養槽的位置係相對於該輔 9 1377345 助電極、兩pH電極與兩溶氧電極之工作窗口的區域上 較佳的是，該基材係為玻璃。 其中，該墊高膜本體係將該PDMS溶液塗佈於一基板 頂面固化所形成》其中，該墊高膜本體係以氧氣電漿處理 方式結合於該微流道層的下方而形成該微流道墊高層。 其中’於一基材頂面形成該輔助電極、該參考電極、 兩溶氧電極與兩pH電極前，係以幾屬法將鈦層形成於玆签 • 材頂一面，再沈積金層於該鈦層上形成^威-測^·^，並利用 舉離（lift-off)製程使該感測平面形成該輔助電極、該參考電 極、兩溶氧電極與兩pH電極；再進一步於各電極上方形成 、一絕緣層，並經過曝光、顯影及硬烤而於各電極上分別形 成一工作窗口，於該參考電極與兩pH電極之工作窗口經由 * 電鍍形成一氧化銥層。 較佳的是，該墊高膜本體之厚度為5〇 ，該工作窗 口 面積為 20 //m><20 "m。 籲 其中，將該墊高膜本體結合於該微流道層的下方而形 成一微流道墊高層後，係於該墊高膜本體形成一流通貫槽 ，且該微流道對應該流通貫槽的區域之形狀與位置係與流 通貫槽相符，該流通貫槽係與該透氣孔位於同側，再於該 .微流道層及該墊高膜本體穿設形成該細胞培養槽。 本發明又關於-種用於檢測多生理參數之細胞活性評 估晶片，其係如前述之製法所製成者。 本發明所提供之用於檢測多生理參數之細胞活性評估 晶片，藉由上述技術手段，可以獲得的優點及増進之功效 1377345 至少包括： 1、 本發明將pH電極與溶氧電極共同整合於同一晶片 上’以免標定與非侵入式直接監測細胞活性的變化。 2、 本發明利用該墊高膜本體之設計，於柚出廢液的同 時能控制細胞培養槽内液體之液面高度，且避免抽液時的 流速對細胞的影響而能更精確的評估細胞之活性變化。 3、 本發明之晶片以便宜的透明玻璃材料取代昂貴的石夕 晶圓’作為感測電極微製程加工與細胞培養的基材，除易 於大量製作、降低製作成本與簡化製程程序外，也改良過 去妙基材不利以顯微鏡同時進行光學觀測細胞型態之問題 【實施方式】 為能詳細瞭解本發明的技術特徵及實用功效，並可依 照說明書的内容來實施，詳細說明如後： 請參閱第一、二圖所示，本發明係相關於一種用於檢 測多生理參數之細胞活性評估晶片，其係包括一感測晶片 (10)及一微流道墊高層（20> ; 該感測晶片（1 〇)包括一基材（11 )、一感測平面（1 2)及 —絕緣層（13)，該感測平面（12)位於該基材（11)上，製作 時係可以蒸鍍法或濺鍍法將鉻層或鈦層形成於該基材（11) 頂面作為—黏著層，再沈積金或鉑作為該感測平面（彳2)， 並利用標準的蝕刻（etching)製程或舉離（Mft-0ff)製程完成 電極的圖案化，而形成互相間隔之一輔助電極（122)、一 11 1377345 參考電極（121)及位於該辅助電極（122)和該參考電極 (121)之間的二溶氧電極（123)與二pH電極（124广該絕緣 層（13)係由SU8負型光阻所組成，而塗佈於該感測平面 (12)上，經過曝光、顯影及硬烤（hard bake)以增加該絕緣 層（13)之強度，再於該絕緣層（13)上相對於各電極的尖端 位置分別形成一工作窗口（125)，並於該工作窗口（125)電 沉積一氧化銥層（126); 該微流道墊高層（20)係結合於該感測晶片（1〇)頂面，包 括一墊高膜本體（21)、一微流道層（22)及一細胞培養槽（23) ’該墊高膜本體（21)位於該感測晶片（1〇)之頂面，係包括一 流通貫槽（211)，該微流道層（22)結合於該墊高膜本體（21) 之頂面，並包括一底面、一微流道（221)、一抽液孔（222)及 一透氣孔（223)，該微流道（221)係凹設於該底面，且該微流 道（221)對應該流通貫槽（211)的區域之形狀與位置係與該流 通貫槽（211)相符，該抽液孔（222)及該透氣孔（223)分別穿 設於該微流道層（22)，並分別形成於該微流道（221)的二側 ，且該透氣孔（223)與該流通貫槽（211)係位於同側，該細胞 培養槽（23)貫穿於該微流道層（22)及該墊高膜本體（21)且連 通於該微流道（221)、該抽液孔（222)、該透氣孔（223)及該 流通貫槽（211)，以露出兩pH電極（124)與兩溶氧電極 (123)之工作窗口（125)與該輔助電極（122)。 因此’本發明係可藉由感測晶片（1 〇)之兩pH電極（1 24) 及兩溶氧電極（12 3)同時檢測細胞酸化率與呼吸活性量測， 以評估細胞活性的變化，並且以玻璃為基材（1彳），實現細 12 1377345 ，胞生理活性檢測時同步觀測細胞形態之目的，再者，利用 該墊高膜本體（21)之設計，於抽出廢液的同時能控制細胞 培養槽（23)内液體之液面高度’且避免抽液時的流速對細 . 胞的影響而能更精確的評估細胞之活性變化。 以下實施例係為了具體說明本發明用於檢測多生理參 數之細胞活性評估晶片之製法的技術手段。 實施例 一、製備感測晶片 • (a)基材清潔 請參閱第三（A)圖所示，本實施例所選用之基材（^) 係為玻璃’而為了清除玻璃表面之油污與灰塵，增加金屬 、 濺鍍於玻璃上的附著性，係先將玻璃放入載玻片架内，浸 入異丙醇（isopropano|，丨pA)溶液中，以超音波震盡3〇 m i η，再以二次蒸镏水超音波震盪5 m i η後更換二次水， 重複3~5次清洗乾淨。接著將玻璃吹乾後，浸入食人魚 溶液（piranha solution)中，該食人魚溶液為體積比3 :] _ 之96% H2S〇4與30% H2〇2配製而成，以—8(rc進行隔水 加熱與超音波震盪30 min，取出玻璃後，再以二次蒸館 水超音波震盪重複3〜5次清洗乾淨而完成玻璃清潔❶ (b)電極製作 請參閱第三（B)圖所示，係於清潔後之玻璃上塗佈Az 4620正光阻’經曝光顯影定義出電極的形狀後，進一步 以濺鍍法或蒸鍍法工作一 5〇nm之鈦層作為黏著層，再於 該鈦層上形成厚度250 nm之金層作為該感測平面（12)， 以丨PA或丙酮移除光阻即可於玻璃上形成一辅助電極 13 1377345 ， （122)、一參考電極（121)及位於該辅助電極（122)該參考 電極（121)之間的二溶氧電極（123)與二pH電極（124)。 (c)絕缘層製作 . 將SU8-3025負光阻作為絕緣層（13)並塗佈於各電極 上作第一層塗佈。第一階段係以500rpm旋轉塗佈1 5秒 ’第一階段以3000rpm塗佈40秒，再經於65°C下烘烤 30秒，並分別於75°C與85°C下烘烤2分鐘，再逐步升溫 至95C烘烤10分鐘》再利用旋轉塗佈法將SU8-3025進 _ 行第二層塗佈，其旋佈與軟烤參數同第一層SU8-3025負 光阻之製作過程《再利用設計之光罩透過曝光機於光能 200mJ/cm3下曝光10秒，進行曝後烤加強光交連程度與 結構後，再於65eC下烘烤10分鐘，並分別於75°c與 85 C下烘烤2分鐘’再逐步升溫至i5〇°c硬烤1〇分鐘， 請參閱第三（C)圖所示，最後對晶片顯影即可於各電極上 刀別疋義出一工作窗口（125)，並形成一具有工作窗口之 基材。 # (d)氧化銥pH感測電極製作 請參閱第三（D)圖所示，配製一氧化銥（丨「〇χ)電鍍液， 將該氧化銥電鍍液靜置兩天後，將該具有工作窗口之基材 置入於該氧化銥電錢液中，使用多功能電位儀，連接晶片 之工作電極，並配合外接Ag/AgC丨參考電極及白金絲輔 助電極’利用循環伏安法（CyCljc v〇lta|T1metry, CV)電鑛 丨「〇x ’將電位定於〇V至+〇·6 V之間，電位改變速率為 20 mV s·1 ’循環300次，於該參考電極（彳21)與兩ρΗ電 極（124)之工作窗口（125)工作面積2〇 "mx2〇//m之一氧化銥 1377345 層（126)而形成一氧化銥PH感測電極，請參照附件一（A) 及附件一（B)所示。 請參照第七(A)、（B)圖所示將兩氧化銥pH感測電極以 pH6·00 7 00 8.00的PBS緩衝溶液測試以及評估其靈敏 度，經過線性計算，可得到其靈敏度約為_76mV/pH。 二、製備微流道塾高層 (a)製備微流道層 請參閱第四（A卜（D)圖所示，係先準備一玻璃基板（3〇) 鲁，將該玻璃基板（30)經過清潔後塗佈SU8負型光阻（31)， 經曝光顯影，並移除光阻後即形成母膜（32)。請參閱第五 (E卜（G)圖所示，將聚二曱基矽氧烷 (poly(dimethylsiloxane)，PDMS)主劑與固化劑以 1〇 : 1 (w/w%)授拌混合均勻並除去氣泡後，澆籍於該母模上並 於加熱板上加熱1.5 h後即固化，待其冷卻後自該母膜（32) 取下而形成一微流道層（22)，由該微流道層（22)的底面凹設 有一微流道（221>，其高度約為200 ’再以玻璃毛細管 _ 於該微流道層（22)穿設該抽液孔（222)及該透氣孔（223),該 抽液孔（222)及該透氣孔（223)係分別形成於該微流道（221) 的兩側。 (b>製備墊高膜本體 將PDMS主劑與固化劑以1〇 : 1 (w/w〇/0)的比例調配後 旋轉塗佈於乾淨之一壓克力基板表面，置於加熱板上待其 固化形成一墊高膜本體（21)於壓克力基板上，其厚度約為 50 //m。 (c)製備微流道墊高層 15 1377345 嘩 請參閱第五（Η)圖所示’將該塾高膜本體（21)與該微流 4 道層（22)以功率100W的氧氣電漿處理1〇秒，並使該塾高 膜本體（21)結合於該微流道層（22)的下方，於兩者結合同時 ’該壓克力基板係與該墊高膜本體（21)分離，將該塾高膜 本體（21)的一部分撕除而形成一流通貫槽（211)，且該微流 道（222)對應該流通貫槽（211)的區域之形狀與位置係與流通 貫槽（211)相符，該流通貫槽（211)係與該透氣孔（223)位於 同側’使液體能流至該參考電極（121)以進行量測，並於該 • 微流道墊高層（20)穿設形成細胞培養槽（23)，且該細胞培養 槽（23)係相對位於該微流道（221)的中段位置，且藉由該微 流道（221)連通該抽液孔（222)及該透氣孔（223)，而該細胞 培養槽（23)的底緣與該微流道（221)之間距為50；t/m。 三、 製備細胞活性評估晶片 請參閱第一、六圖所示，將該微流道墊高層（2〇)以壓 克力夾具组裝於該感測晶片（1 0)上方即形成該細胞活性評 估晶片，該細胞培養槽（23)的位置係相對於兩pH電極 鲁 （124)與兩溶氧電極（123)之工作窗口（125)的上方，並與 該微流道（221)、該抽液孔（222)、該透氣孔（223)及該流 通貫槽（211)相連通，且於該絕緣層（13)及該感測平面（12) 上’對應於該細胞培養槽（23)的區域形成一細胞培養平面 (14)〇 四、 晶片表面修飾與細胞培養 請參閱第一、六圖所示，本實施例係以細胞活性評估 晶片對HeLa細胞（50)進行生物活性檢測，該HeLa細胞 (5〇)為人類子宮頸癌上皮細胞株。而由於SU8製成之絕緣 1377345 層（13)為疏水性，但HeLa細胞（5〇)之細胞膜表面帶負電 ，不容易貼附於細胞培養平面（14)上，因此利用細胞外基 質修飾技術’將1〇#丨聚左旋離胺酸（p〇丨丨 • 〇·001% (W/W))滴於細胞培養平面（14)，待其自然揮發後， 再滴入10//I的纖黏連蛋白（fjb「〇nect丨n，1 〇 進行修 飾同樣待其自然揮發後，則可加入細胞培養液於細胞培 養槽（23)内’再加入1〇 #丨且密度為4 71χ1〇5 ce丨丨s/mL之The basic structure of the Cytosensor® Microphysiometer is based on ruthenium as a substrate, and a layer of tantalum nitride is applied above. The amount of extracellular acidification is measured by the 趋 --addressable potentiometric sensor (LAPS). Measurement. The main working principle is to use the photoelectric effect to excite the germanium substrate with a special wavelength of light to generate an electron hole pair. At this time, a fixed potential is applied between the reference electrode and the working electrode by a potentiostat. The current is generated by the pair of electron holes. The current of the electron hole is related to the surface potential of the insulating layer. When the cell metabolizes more protons and changes the pH value, it also changes the surface potential of the insulating layer, so it will change the electronic hole that actually falls on the germanium substrate. For the size of the moving electric field, the activity of the cells can be evaluated from the amount of change in the measured value of PH. The feature of this system is that only a small amount of sample liquid or cells can be measured, and it has good sensitivity and a small amount of sample consumption, and by laser irradiation, it can directly locate and straighten the cells to be measured. Extracellular pH; however, this system can only detect the extracellular acidification rate of a single physiological index, and the data on the evaluation of actual cellular physiological activity is not extensive enough. (b) Analyzing system and Metabolic chip from Bionas®, Germany: The multi-parameter cell physiology analysis system of Germany Bionas® can increase the rate of extracellular acidification (ion-selective field-effect transistor sensor (j〇n sensitjve, fie丨d effect transist〇(,丨SFET)), oxygen consumption rate (amperometric sensor) and cell attachment (interdigital electrode conductivity sensor) for integrated measurement. The main chip design is The cells are cultured in a package-finished wafer. 'The cell culture medium and the test drug are transported by micro-pipes from both ends of the wafer. The internal buffer volume is about 1 〇" The sensor chip is characterized by its ability to withstand sterilization. When the temperature is high and high, and the cells are not polluted by the environment during the cultivation, it is easy to integrate with the microelectronic circuit and has the advantages of rapid detection and the amount of the reagent. However, the life of the ISFET wafer after the repeated experimental test is still To be verified, the above-mentioned commercial measurement wafers are all liquid-replaced in a closed micro-channel and are based on ruthenium, and their opacity hinders the optical method. According to the above background, the commercially available cell activity sensing system still has the following disadvantages: 1. The change of cell activity is a comprehensive physiological reaction, and the measurement of each single parameter cannot be completely completed. Knowing the physiological response of cells to external stimuli, but when measuring the multi-parameter cell activity, the measurement principle of the sensor has greatly increased the Jig of the analytical instrument. For example, the Biobolic® Metabolic chip is measured by ISFET. Extracellular acidification rate, amperometric measurement of dissolved oxygen, impedance analysis method for cell attachment. 2. The purpose of cell production of active wafers, all using dreams as a substrate to facilitate the integration of semiconductor process technology, but limited The function of simultaneously observing the morphology of a cell by a microscope. [Invention] The present inventors have been able to perform multi-parameter cell activity measurement in view of the expensive price and complicated process of the existing cell-activity sensing system, and all of them are based on hair. The material limits the function of simultaneously observing the morphology of the cells, so after a long period of research and continuous testing, this invention was finally invented for testing. The cell activity evaluation wafer of physiological parameters. The object of the present invention is to provide a cell growth activity evaluation wafer for integrating wafer cell culture and multi-physiological parameter detection electrode array, especially the cell oxygen consumption rate and extracellular acidification rate isotactic technique. 'Simultaneous observation of cell morphology during cell physiological activity 7 1377345, and simplification of cell activity evaluation wafers for measuring electrochemical electrodes of electrodes on glass substrates with different physiological parameters. For the above purposes, the present invention is utilized. The technical means is to provide a cell activity evaluation wafer for detecting a plurality of physiological parameters, comprising a sensing chip, comprising a substrate, a sensing plane and an insulating layer, wherein the sensing plane is disposed on the base The top surface of the material includes a plurality of auxiliary electrodes, a reference electrode, and a second dissolved oxygen electrode and a second electrode disposed between the auxiliary electrodes, and the insulating layer is disposed on the top surface of the sensing plane a working window is disposed on the insulating layer relative to each electrode, and is located opposite to the reference electrode and the two pH electrodes a working layer is provided with a layer of ruthenium oxide to cover the region where the reference electrode and the two pH electrodes correspond to the working window; a microchannel mat is attached to the top surface of the sensing wafer, including a high film body a micro flow channel layer and a cell culture tank, wherein the height film body is located on a top surface of the sensing wafer, and includes a flow through groove, and the micro flow channel layer is bonded to a top surface of the height film body. And including a bottom surface, a micro flow channel, a liquid extraction hole and a venting hole. The micro flow channel is recessed on the bottom surface, and the shape and position of the micro flow channel corresponding to the region of the flow through groove are continuous The groove matching 'the liquid venting hole and the venting hole are respectively disposed on two sides of the micro flow channel layer' and the venting hole is located on the same side as the circulation through groove system, and the cell culture groove penetrates through the micro flow channel layer and The high film body is connected to the micro flow channel, the liquid extraction hole and the flow through groove to expose the working windows of the two pH electrodes, the two dissolved oxygen electrodes, and the auxiliary electrode. Preferably, the substrate is glass. 8 1377345 f Preferably, the sensing plane is a gold layer, wherein the sensing plane and the substrate comprise a titanium layer. Preferably, the thickness of the height film body is: • Preferably, the sensing plane has a thickness of 250 nm and the working window area is 20 //mx20//m. Among them, the sorghum system is a layer of poly(dimethylsiloxane) (PDMS). Wherein the microchannel is linear. The invention also relates to a method for preparing a cell activity evaluation wafer for detecting a plurality of physiological parameters, comprising: forming an auxiliary electrode on a top surface of a substrate, a reference electrode, and a dissolving solution between the reference electrode of the auxiliary electrode An oxygen electrode and a two-pH electrode are disposed with an insulating layer above each electrode, and a working window is respectively disposed on the insulating layer relative to each electrode to expose a corresponding electrode at the reference electrode and the two pHs Depositing a ruthenium oxide layer on the working window of the electrode to cover the area where the reference electrode and the two pH electrodes correspond to the working window; • providing a micro flow channel layer 'a micro flow channel is recessed from the bottom surface of the micro flow channel layer The microchannel layer penetrates the liquid guiding hole and the venting hole, and the liquid venting hole and the venting hole are respectively formed on both sides of the micro flow channel; and providing a high film body to the high film body Forming a microchannel mat upper layer under the microchannel layer, and forming a cell culture tank in the microchannel layer and the pad membrane body, and the cell culture tank system and the microchannel , the liquid hole and The venting holes are connected to each other; the microchannel layer is assembled on the top surface of the sensing wafer to form a cell activity evaluation wafer' and the cell culture tank is positioned relative to the auxiliary 9 1377345 auxiliary electrode, two pH Preferably, the substrate is glass in the region of the working window of the electrode and the two dissolved oxygen electrodes. Wherein, the high-film system applies the PDMS solution to a top surface of a substrate to be formed, and the high-film system is combined with the micro-flow layer to form the micro-oxygen plasma treatment. The runner pad is high-rise. Wherein, before the auxiliary electrode, the reference electrode, the two dissolved oxygen electrodes and the two pH electrodes are formed on the top surface of the substrate, the titanium layer is formed on the top side of the material by a few methods, and the gold layer is deposited thereon. Forming a surface on the titanium layer, and using a lift-off process to form the auxiliary electrode, the reference electrode, the two dissolved oxygen electrodes and the two pH electrodes; further to each electrode An insulating layer is formed on the upper surface, and a working window is formed on each of the electrodes by exposure, development and hard baking, and a ruthenium oxide layer is formed by electroplating on the working electrode of the reference electrode and the two pH electrodes. Preferably, the height of the height film body is 5 〇, and the working window area is 20 // m >< 20 " m. Wherein, after the high-film body is bonded under the micro-channel layer to form a micro-flow channel upper layer, a high-flow film body forms a flow-through groove, and the micro-flow channel corresponds to the flow. The shape and position of the groove region are consistent with the flow through groove, and the flow through groove is located on the same side of the gas permeable hole, and the cell culture tank is formed by piercing the micro flow channel layer and the high film body. The present invention is further directed to a cell activity evaluation wafer for detecting a plurality of physiological parameters, which is produced by the above-described method. The cell activity evaluation wafer for detecting multiple physiological parameters provided by the present invention, the advantages and the effect of the advancement obtained by the above technical means 1377345 include at least: 1. The present invention integrates the pH electrode and the dissolved oxygen electrode in the same The wafer was 'directly calibrated and non-invasively monitored for changes in cell viability. 2. The invention utilizes the design of the high-membrane body to control the liquid level of the liquid in the cell culture tank while the waste liquid is discharged, and to avoid the influence of the flow rate on the cells during the liquid extraction, and can more accurately evaluate the cells. The activity changes. 3. The wafer of the present invention replaces the expensive Shishi wafer with an inexpensive transparent glass material as a substrate for the microelectrode processing and cell culture of the sensing electrode, and is improved in addition to being easy to mass-produce, reducing the manufacturing cost and simplifying the process procedure. In the past, the problem of the substrate is unfavorable for optical observation of the cell type by the microscope. [Embodiment] In order to understand the technical features and practical effects of the present invention in detail, it can be implemented in accordance with the contents of the specification, and the detailed description is as follows: As shown in FIGS. 1 and 2, the present invention relates to a cell activity evaluation wafer for detecting a plurality of physiological parameters, which comprises a sensing wafer (10) and a microchannel pad high layer (20); the sensing wafer (1 〇) includes a substrate (11), a sensing plane (12), and an insulating layer (13). The sensing plane (12) is located on the substrate (11) and can be evaporated during fabrication. A chromium or titanium layer is formed on the top surface of the substrate (11) as an adhesion layer by sputtering or depositing, and gold or platinum is deposited as the sensing plane (彳2), and a standard etching process is utilized. Or lift off (Mft-0ff) The patterning of the electrodes is completed, and one auxiliary electrode (122), an 11 1377345 reference electrode (121), and a two dissolved oxygen electrode between the auxiliary electrode (122) and the reference electrode (121) are formed. 123) and two pH electrodes (124) the insulating layer (13) is composed of SU8 negative photoresist, and is coated on the sensing plane (12), exposed, developed and hard bake Increasing the strength of the insulating layer (13), forming a working window (125) on the insulating layer (13) with respect to the tip positions of the electrodes, and electrodepositing a ruthenium oxide layer in the working window (125) ( 126); the microchannel mat upper layer (20) is coupled to the top surface of the sensing wafer (1), including a padded film body (21), a microchannel layer (22), and a cell culture tank ( 23) 'The padded film body (21) is located on the top surface of the sensing wafer (1), and includes a flow through groove (211), and the micro flow channel layer (22) is coupled to the high film body ( 21) a top surface, including a bottom surface, a micro flow channel (221), a liquid extraction hole (222), and a venting hole (223), the micro flow channel (221) is recessed The bottom surface, and the shape and position of the micro flow channel (221) corresponding to the region of the flow through groove (211) coincide with the flow through groove (211), the liquid suction hole (222) and the gas permeable hole (223) The microchannel layer (22) is respectively disposed on the microchannel layer (22), and is formed on the two sides of the microchannel (221), and the venting hole (223) is located on the same side as the circulation channel (211). a culture tank (23) penetrating the microchannel layer (22) and the high membrane body (21) and communicating with the microchannel (221), the liquid extraction hole (222), the gas permeable hole (223), and The flow through groove (211) exposes the working window (125) of the two pH electrodes (124) and the two dissolved oxygen electrodes (123) and the auxiliary electrode (122). Therefore, the present invention can simultaneously measure cell acidification rate and respiratory activity measurement by sensing two pH electrodes (1 24) of the wafer (1 )) and two dissolved oxygen electrodes (12 3 ) to evaluate changes in cell activity, And using glass as the substrate (1彳), the purpose of synchronizing the cell morphology during the detection of the physiological activity of the fine 12 1377345 is achieved, and further, the design of the high membrane body (21) can be used to extract the waste liquid. By controlling the liquid level of the liquid in the cell culture tank (23) and avoiding the influence of the flow rate at the time of the pumping on the cells, the activity of the cells can be more accurately evaluated. The following examples are intended to specifically illustrate the technical means for the preparation of the cell activity evaluation wafer for detecting multiple physiological parameters of the present invention. Example 1: Preparation of Sensing Wafer • (a) Cleaning of Substrate Please refer to the third (A) diagram, the substrate (^) selected in this embodiment is made of glass 'in order to remove oil and dust on the surface of the glass. To increase the adhesion of metal and sputtered on glass, first put the glass into the slide holder, immerse it in isopropano|(丨pA) solution, and shock it by 3〇mi η, then After the second steamed water ultrasonic shock 5 mi η, replace the secondary water, repeat 3 to 5 times to clean. The glass is then blown dry and immersed in a piranha solution prepared by volumetric ratio of 3:] _ 96% H2S 〇 4 and 30% H 2 〇 2, with -8 (rc) After the water is heated and the ultrasonic wave is oscillated for 30 min, the glass is taken out, and then the glass is cleaned by repeated ultrasonic vibration for 3 to 5 times in the secondary steaming water. (b) Electrode production Please refer to the third (B) diagram. After the surface of the cleaned glass is coated with Az 4620 positive photoresist, the shape of the electrode is defined by exposure and development, and then a titanium layer of 5 nm is used as an adhesive layer by sputtering or evaporation, and then A gold layer having a thickness of 250 nm is formed on the titanium layer as the sensing plane (12), and an auxiliary electrode 13 1377345, (122) and a reference electrode (121) are formed on the glass by removing the photoresist with 丨PA or acetone. And a two-dissolved oxygen electrode (123) and a two-pH electrode (124) located between the auxiliary electrode (122) and the reference electrode (121). (c) Insulation layer fabrication. Using SU8-3025 negative photoresist as an insulating layer ( 13) and applied to each electrode for the first layer coating. The first stage is spin-coated at 500 rpm for 15 seconds. The stage was coated at 3000 rpm for 40 seconds, then baked at 65 ° C for 30 seconds, and baked at 75 ° C and 85 ° C for 2 minutes, and then gradually heated to 95 ° for 10 minutes. The fabric is applied to the second layer of SU8-3025. The rotation and soft-bake parameters are the same as the first layer of SU8-3025 negative photoresist. The re-use design of the reticle through the exposure machine at 200mJ/ Exposure for 10 seconds under cm3, after exposure and post-bake to enhance the degree of light cross-linking and structure, then bake at 65eC for 10 minutes, and bake at 75 °c and 85 C for 2 minutes respectively, then gradually increase the temperature to i5 〇 °c Hard baking for 1 minute, please refer to the third (C) figure. Finally, the wafer is developed to form a working window (125) on each electrode and form a substrate with a working window. (d) Preparation of yttrium oxide pH sensing electrode Please refer to the third (D) diagram to prepare cerium oxide (丨"〇χ) plating solution. After the cerium oxide plating solution is allowed to stand for two days, it will work. The substrate of the window is placed in the cerium oxide liquid solution, and the multi-function potentiometer is used to connect the working electrode of the wafer with the external Ag/AgC reference. Pole and platinum wire auxiliary electrode 'Using cyclic voltammetry (CyCljc v〇lta|T1metry, CV) electric ore "丨x ' set the potential between 〇V to +〇·6 V, the potential change rate is 20 mV s·1 'cycle 300 times, the working window (125) of the reference electrode (彳21) and the two ρΗ electrode (124) has a working area of 2〇"mx2〇//m one of the yttrium oxide layers 1377345 (126) To form a ruthenium oxide PH sensing electrode, please refer to Annex 1 (A) and Annex 1 (B). Please refer to the seventh (A) and (B) diagrams to test the pH of the cerium oxide pH sensing electrode in PBS buffer solution of pH 6.00 00 8.00 and evaluate its sensitivity. After linear calculation, the sensitivity is about _ 76mV/pH. Second, the preparation of the micro-channel 塾 high-rise (a) preparation of the micro-channel layer, please refer to the fourth (A (D) diagram, first prepare a glass substrate (3 〇) Lu, the glass substrate (30) After cleaning, apply SU8 negative photoresist (31), develop through exposure, and remove the photoresist to form the mother film (32). Please refer to the fifth (E (G) diagram, the polydifluorenyl group The main agent of poly(dimethylsiloxane) (PDMS) and the curing agent are mixed uniformly at 1〇: 1 (w/w%) and the bubbles are removed, and then poured onto the master mold and heated on a heating plate. After h, it is solidified, and after it is cooled, it is removed from the mother film (32) to form a microchannel layer (22), and a microchannel (221) is recessed from the bottom surface of the microchannel layer (22). The height is about 200 ′ and the glass capillary tube _ is inserted into the microchannel layer (22) through the liquid extraction hole (222) and the vent hole (223), the liquid extraction hole (222) and the vent hole (223) ) are formed on both sides of the microchannel (221). (b> Preparation of the padded film body. The PDMS main agent and the curing agent are formulated in a ratio of 1〇: 1 (w/w〇/0) and then spin coated. Deployed on the surface of one of the acrylic substrates The film is cured on the heating plate to form a high-film body (21) on the acrylic substrate, and the thickness thereof is about 50 //m. (c) Preparation of the micro-flow pad high-rise 15 1377345 哗 See the fifth (Η As shown in the figure, 'the high film body (21) and the microfluidic layer (22) are treated with oxygen gas of 100 W for 1 second, and the high film body (21) is bonded to the micro Below the flow channel layer (22), when the two are combined, the acrylic substrate is separated from the high film body (21), and a part of the high film body (21) is peeled off to form a flow through. a groove (211), wherein the shape and position of the region of the microchannel (222) corresponding to the flow through groove (211) coincide with the flow through groove (211), and the flow through groove (211) is connected to the gas permeable hole ( 223) on the same side 'to enable liquid to flow to the reference electrode (121) for measurement, and to form a cell culture tank (23) on the upper layer (20) of the microfluidic pad, and the cell culture tank ( 23) is relatively located in the middle of the microchannel (221), and the microchannel (221) is connected to the liquid inlet (222) and the vent (223), and the cell culture The distance between the bottom edge of the culture tank (23) and the microchannel (221) is 50; t/m. 3. Preparation of Cell Activity Evaluation Wafer Please refer to the first and sixth figures, and the microchannel mat is high-rise ( 2)) The cell activity evaluation wafer is formed by assembling an acrylic clamp over the sensing wafer (10), and the cell culture tank (23) is positioned relative to the two pH electrodes Lu (124) and the two solutions. Above the working window (125) of the oxygen electrode (123), and communicating with the micro flow channel (221), the liquid discharging hole (222), the gas permeable hole (223), and the circulation through groove (211), and Forming a cell culture plane (14) on the insulating layer (13) and the sensing plane (12) corresponding to the cell culture tank (23), wafer surface modification and cell culture, see first, As shown in the six figures, in this example, the activity of HeLa cells (50) was measured by a cell activity evaluation wafer, and the HeLa cells (5〇) were human cervical cancer epithelial cell lines. Since the insulating layer 1377345 (13) made of SU8 is hydrophobic, the surface of the cell membrane of HeLa cells (5〇) is negatively charged and is not easily attached to the cell culture plane (14), so the technique of extracellular matrix modification is utilized. 1〇#丨poly-L-amino acid (p〇丨丨•〇·001% (W/W)) was dropped on the cell culture plane (14), and after it naturally evaporated, it was dropped into 10//I fiber. Adhesin (fjb "〇nect丨n, 1 〇 is also modified after it is naturally volatilized, then you can add cell culture solution in the cell culture tank (23)' and add 1〇#丨 and the density is 4 71χ1〇5 Ce丨丨s/mL
HeLa細胞懸字液，而後靜置15分鐘讓HeLa細胞（50)貼 φ 附於表面上，再利用微注射泵（40)插入該抽液孔（222)，並 將培養液抽乾’同時觀察抽液時對HeLa細胞貼附之影響 〇 . 由於本發明係採用非密閉式培養的方式，且該細胞培 養槽（23)係以50 //m的高度以該微流道（22彳）連通該抽液孔 (222) ’如此再抽出廢液的同時能控制細胞培養槽内液體之 液面高度’且纟田胞培養平面（14)與該微流道（221)相距有5〇 ’因此抽液時的流速快慢產生的剪切力對細胞的影響不 鲁大’可改善HeLa細胞因受到流體之剪切力而被沖走之問 題’使HeLa細胞可以穩定的貼附於細胞培養平面（14)上， 而能更精確的評估細胞之活性變化。五、以細胞活性評估 晶片對HeLa細胞進行量測HeLa cells were suspended, and then allowed to stand for 15 minutes to attach HeLa cells (50) to the surface, and then inserted into the wells (222) by a microinjection pump (40), and the culture solution was drained while observing The effect of the solution on the adhesion of HeLa cells 〇. Since the present invention adopts a non-closed culture method, and the cell culture tank (23) is connected at a height of 50 // m in the microchannel (22 彳). The liquid suction hole (222) 'can control the liquid level of the liquid in the cell culture tank while withdrawing the waste liquid, and the field cell culture plane (14) is 5 〇 from the micro flow channel (221). The effect of the shear force generated by the flow rate during pumping on the cells is not large, which can improve the problem that HeLa cells are washed away by the shearing force of the fluid', so that HeLa cells can be stably attached to the cell culture plane ( 14), and can more accurately assess changes in cell activity. V. Evaluation of cell activity Wafer measurement of HeLa cells
請參閱第八圖所示，其係以細胞活性評估晶片之溶氧 電極對HeLa細胞進行呼吸活性量測。先將密度為2〇〇〇 cells mm·2之HeLa細胞培養於細胞培養平面上，並加入 30 "L之細胞培養液，該細胞培養液係以1 〇 mM ( N-2-经 乙基派嗪-N-2-乙磺酸（HEPES)為緩衝分子，且含有25 mM 17 137.7345 之甘露醇（mannitol)’再進行耗氧率量測i〇分鐘；同前述 步驟再分別量測10 mM HEPES + 25 mM葡萄糖（g|UC0se) 及 10 mM HEPES+25 mM glucose + 1 % insulin，最後以 1 * /〇 ΤΓΚ〇η X_1〇〇將細胞移除。比較培養液中含有g|ucose 或胰島素（insu丨in)分子時對胞外呼吸活性的影響，其結果發 現在含有 25 mM glucose 與 25 mM glucose +1% insulin 的細胞培養液中，耗氧率較含有25 mM mannito丨時的耗氧 率分別增加了 1·23及1.54倍。Referring to Figure 8, the respiratory activity of HeLa cells was measured by a dissolved oxygen electrode of the cell activity evaluation wafer. First, HeLa cells with a density of 2〇〇〇cells mm·2 were cultured on the cell culture plane, and 30 "L cell culture solution was added, and the cell culture solution was 1 〇 mM (N-2-ethyl group). Piperazine-N-2-ethanesulfonic acid (HEPES) is a buffer molecule and contains 25 mM 17 137.7345 mannitol's and then the oxygen consumption rate is measured for 1 〇 minutes; and 10 mM is measured separately with the previous steps. HEPES + 25 mM glucose (g|UC0se) and 10 mM HEPES + 25 mM glucose + 1 % insulin, and finally remove the cells with 1 * /〇ΤΓΚ〇η X_1〇〇. Compare culture medium containing g|ucose or insulin (insu丨in) The effect of molecular on extracellular respiration activity, and it was found that in cell culture medium containing 25 mM glucose and 25 mM glucose +1% insulin, the oxygen consumption rate was lower than that of oxygen containing 25 mM mannito丨. The rates increased by 1.23 and 1.54 times respectively.
• 請再參閱第九圖所示，其係以細胞活性評估晶片之氧 化銥pH感測電極對HeLa細胞進行胞外酸化率的量測。先 將密度為1016 cells mm·2之HeLa細胞培養於晶片中，並 加入1〇 "L以1 mM HEPES為緩衝分子之細胞培養液，並 進行酸化率量測10分鐘；同前述步驟分別量測1 mM 參 HEPES + 25 mM glucose 、1 mM HEPES+25 mM glucose + 1 % insulin 進行比較，最後以 1 〇/。Triton X-l〇〇 將細胞移除。比較培養液中含有g|UCOSe或jnsu|jn分子時 # 對胞外酸化率的影響’其結果發現在含有25 mM glucose 與25 mM glucose +1% insulin的細胞培養液中，酸化率較 含有25 mM mannitol時的酸化率分別增加了 2.0及3.0倍 〇 請再參閱第十圖所示，利用本發明之細胞活性評估晶 片以OCP法（氧化銥pH感測電極）與安培法（溶氧電極）同時檢 測溶氧與pH的變化。在大約240秒處注入30//L的1 Μ亞硫酸 納（Na2S03，pH 10.20，[〇2] = 〇.〇9 mg/丨)於培養槽内，進行 18 1377345 氧氣的去除與pH的鹼化，其與pH相關的氧化銥表面電位與 溶氧電流的變化分別-146 mV與-3.747 nA，之後在420 s處 重新換液回原本的1 mM HEPES溶液，其氧化銥表面電位 與溶氧電流又可回復。 因此’本發明能同時檢測細胞酸化率與呼吸活性之細 胞/舌性評估晶片，以評估細胞活性的變化’並且以玻璃為 基材，能實現細胞生理活性檢測時同步觀測細胞形態之目 的’再者’本發明係由含有離底部一定高度之微流道與細 胞培養槽進行大體積的細胞培養，其液體的更換可藉由微 流道與幫浦系統共同完成，且由於流道開口離底部約5〇 "m’可降低流體剪切力對細胞與代謝產物濃度梯度的影 響’大幅簡化了長時間培養與固定換液的程序。 惟以上所述者，僅為本發明之較佳實施例而已，當不 能以此限定本發明實施之範圍，凡依本發明申請專利範圍 及說明書内容所作之簡單的等效變化與修飾，皆應仍屬本 發明專利涵蓋之範圍内。 【圖式簡單說明】 第一圖係本發明較佳實施例之細胞活性評估晶片立體 示意圖。 第二圖係本發明較佳實施例之細胞活性評估晶片立體 分解示意圖。 第三（A)~(D)圖係本發明較佳實施例之感測晶片製作流• Please refer to the ninth figure, which is a measure of the extracellular acidification rate of HeLa cells by the osmium pH sensing electrode of the cell activity evaluation wafer. HeLa cells with a density of 1016 cells mm·2 were first cultured on a wafer, and a cell culture medium containing 1 mM HEPES as a buffer molecule was added, and the acidification rate was measured for 10 minutes; 1 mM ginseng HEPES + 25 mM glucose, 1 mM HEPES + 25 mM glucose + 1 % insulin was compared and finally 1 〇 /. Triton X-l〇〇 removes the cells. Comparing the effect of g|UCOSe or jnsu|jn on the extracellular acidification rate in the culture medium, the results showed that the acidification rate was 25 in the cell culture medium containing 25 mM glucose and 25 mM glucose +1% insulin. The acidification rate of mM mannitol increased by 2.0 and 3.0 times, respectively. Please refer to the tenth figure, and use the cell activity evaluation wafer of the present invention to use OCP method (yttrium oxide pH sensing electrode) and amperometric method (dissolved oxygen electrode). Simultaneously detect changes in dissolved oxygen and pH. Inject about 30//L of 1 Μ sulfite (Na2S03, pH 10.20, [〇2] = 〇.〇9 mg/丨) in a culture tank at about 240 seconds, and perform 18 1377345 oxygen removal and pH alkali. The pH-dependent yttrium oxide surface potential and dissolved oxygen current change were -146 mV and -3.77 nA, respectively, and then re-transformed back to the original 1 mM HEPES solution at 420 s, and its surface potential and dissolved oxygen The current can be recovered again. Therefore, the present invention can simultaneously detect cell acidification rate and respiratory activity of cell/lingual evaluation wafers to evaluate changes in cell activity' and use glass as a substrate to achieve the purpose of simultaneously observing cell morphology when detecting physiological activity of cells. The present invention consists of a large volume of cell culture containing a microchannel at a certain height from the bottom and a cell culture tank, and the replacement of the liquid can be accomplished by the microchannel and the pump system, and the flow channel opening is off the bottom. Approximately 5 〇"m' reduces the effect of fluid shear on cell and metabolite concentration gradients' greatly simplifies the long-term culture and fixed exchange procedures. 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 the invention patent. BRIEF DESCRIPTION OF THE DRAWINGS The first drawing is a schematic view of a cell activity evaluation wafer of a preferred embodiment of the present invention. The second drawing is a schematic exploded perspective view of a cell activity evaluation wafer of a preferred embodiment of the present invention. Third (A) to (D) drawings are sensing wafer fabrication streams in accordance with a preferred embodiment of the present invention