1355418 九、發明說明： 【發明所屬之技術領域】 本發明係關於一種細胞數量及細胞蛋白質量 測裝置與方法，尤指一種可對貼附或懸浮型細胞之 數量及特異蛋白質作非破壞性連續監測的裝置與 方法。 【先前技術】 φ 細胞行為的連續偵測： 有鑒於許多針對細胞的實驗及方法都將對細 胞造成傷害，因而無法對單一樣品做長時間的監 測，所以非破壞性、可以連續偵測細胞狀態的感測 方式或裝置非常有其必要，但卻不多見且仍有限 制。 1984年Giaever和Keese共同提出了一套稱為 細胞阻抗分析（Electrical Cell Impedance Spectroscopy, ECIS)的方法，利用電學的方式來測 量貼附型細胞之行為，並連續記錄細胞貼附電極介 ® 面所產生的電阻與電容抗。他們發現在使用金電極 材質或施加電場之下都對此種細胞沒有影響，細胞 無論在貼附、伸展、生長上都一直表現正常。此外， 在連續的量測紀錄中，總電阻值確實會隨貼附生長 的細胞而增加。 此類的測量方法上，阻抗分析必須以交流訊號 來做阻抗的量測，這是因為細胞膜如同電容器一般 有隔離直流耦合交流的特性，因此必須使用交流訊 號來對細胞進行量測。在許多傳統的細胞阻抗分析 中，多使用小於1 μ A的電流對細胞做量測，再藉 5 由量測待測電極兩端的微小電壓來做進一步分 析。因為其測量電路中的電流小於ΙμΑ，且所需的 測量時間極為短暫’在許多文獻中也表示此種測量 方式對細胞正常的生理並無影響，所以可視為一種 非破壞性的量測方式。 在研究的細胞種類中，細胞阻抗分析已經實際 應用在許多不同貼附型細胞數量的感測上，例如： 纖維母細胞（fibroblast)、上皮細胞（end〇thelial cell)、星狀細胞（astrocyte)、腎細胞（kidney cell)、 肝癌細胞、表皮性癌細胞等，這些細胞包括細胞株 (Cell line)及初級培養(Primary culture)細胞，但目 前為止仍無法對懸浮型細胞做數目的量測。 在電極設計上，傳統的細胞阻抗分析裝置多將 金電極設置在塑膠培養盤上，其中又分為一個微小 的偵測電極和一個較大的計數電極。研究發現如果 將此二電極改為兩個等大的電極，將會無法測量到 ^胞的變化。但另有學者使用鉑製作兩個等面積之 J ^電，(IDES)，成功地連續偵測細胞貼附生長的 。這表示在細胞阻抗量測分析時，電極的形式 。對可測虽性有很大的影響。而近年來在類似 ，雖然有許多的研究嘗試利用不同的電極形 二=計來做細胞阻抗的量測，但從未有人提出對懸 g ^細胞數量偵測與細胞蛋白質偵測相關的解^ 大類現行測量細胞蛋白質方式約略可分為下列兩 1 ·破壞細胞型態的測量方法： 一般是將細胞溶解後做蛋白質的分離純化及 /里，或疋做蛋白質晶片的檢測，甚至藉由萃 取核糖^酸(RNA)來做分子生物的分析。常見的蛋 白質測量方法方法很多，例如做蛋白質膠片電泳 (SDS-PAGE)後以西方墨潰法（Western b〇〗t)比較蛋 白的表現量；又如高效液相層析（HPLC)後做分子 質譜(MS)分析；又如毛細管電泳(CE)後做免疫染色 (Immunostain)等等。這些方法需要破壞細胞，如果 需要做時間上的觀察，就需要準備許多不同時間點 的樣本’因此無法對樣本作連續非破壞的監測。 2.保留細胞型態的測量方法 一般以細胞免疫螢光染色 (Immunocytochemistry)為主，其是以帶有螢光物質 的抗體標示特定蛋白質。透過細胞免疫螢光染色， 觀祭者可以在螢光顯微鏡（Fluorescence microscope)下觀察蛋白質在細胞的位置，甚至以雷 射共輛焦顯微鏡(Confocal microscopy)觀察更清晰 的立體成像及動態變化。細胞免疫螢光染色雖然保 留了完整的型態，但細胞仍需移出培養箱，所以並 無法在顯微鏡下做長時間的觀察。此外，細胞免疫 螢光染色更具有操作時需要避光、螢光物質有其半 衰期、光學儀器昂貴等等的缺點。 蛋白質生物感測晶片之概況： 目前以電學方法進行量測蛋白質的感測晶片 主要為免疫晶片。關於免疫晶片，其主要是利用抗 原抗體特異性結合的原理而對蛋白質做量測，其方 法是將抗原蛋白塗佈在半導體表面，以測量其電容 特性的改變。研究結果發現當抗原抗體結合時，電 容的測量值將會下降。此外；也有在導電性高分子 表面接上抗體’以測量抗原抗體結合後導電性的變 化’研究也發現隨著待測抗原濃度的增加，導電度 的變化也越大。而這些檢測晶片的樣本皆需取自於 1355418 ϊΐ質fif液丄而樣本的準備如傳統的檢測方法 瓜，都品先將實驗細胞破壞，所以仍難以 樣本做連續性的測量。 早 職是之故，申請人鑑於習知技術中所產生缺 失，乃經悉心試驗與研究，並一本 ί置=本工r數量及細胞蛋白= 裝置與方法」，以下為本案之簡要說明。 【發明内容】 白質ϊίίΐΐίί提供一種細胞數量及細胞蛋 t Λ 藉由在目標細胞上通過一 =測，入帶導電金屬=== 裝置下對細胞特異蛋白質作非破壞性: ^ t ί i ^ 5^ 雷栖卜剎田々队x、<驟l括.（a)於一氧化銦錫 胞之-細胞蛋白質弟;該細 以性，-第以ϊΐ;ί= 過-：ί Ht子,:(c)於該氧化銦錫電極上通 i或ί ί該氧化銦錫電極之電阻抗 值或電容抗值換為—賴細胞蛋白質^見電量阻抗 雷托戶f述之方法’其中步驟（a)於該氧化銦锡 電極上同時培養—種以上之細胞。乳化銦錫 如上所述之方法，其中步驟(b)之該培養液包 8 1355418 a . 含複數種第一級抗體，該複數種第一級抗體分別可 與不同之細胞蛋白質特異性結合。 ' 如上所述之方法，其中步驟（b)加入分別可與 * 該複數種第一級抗體特異性結合的複數種第二級 •- 抗體，該複數種第二級抗體上均帶有金屬粒子。 . 如上所述之方法，其中步驟（c)之該電流每經 過一期間後通過該氧化銦錫電極。 為達上述目的，本案提供一種連續測量細胞蛋 白質表現的裝置，該裝置包含：一細胞；一細胞培 • 養液，用以提供該細胞生長，其中該培養液更包 含：一結合物，用以與該細胞之一細胞蛋白質特異 性結合，其中該結合物上帶有一金屬粒子，該金屬 粒子可於該結合物與該細胞蛋白質特異性結合 後，改變細胞的交流阻抗特性；一電極，與該培養 液電連接，用以提供該裝置之可感測性；及一交流 阻抗偵測裝置，與該電極電連接，用以偵測該電極 通過一電流時之一交流阻抗變化。 如上所述之裝置，其中該電極為由二線狀電極 所形成的一雙電極，該二線狀電極之個別線寬為 _ 0.4mm 〇 如上所述之裝置，其中該二線狀電極相互平 行，該二線狀電極之間距為4mm。 如上所述之裝置，其中該電極為一氧化銦錫電 極。 如上所述之裝置，其中該氧化銦錫電極位於一 絕緣基板上，其中該絕緣基板之材質為玻璃、石英 及塑膠其中之一或其組合。 如上所述之裝置，其中該結合物為一第一級抗 9 該第一級抗體特異性結合之-第二級抗 體之組合，該第二級抗體上帶有—金屬粒子。 子。如上所述之方法，其中該金屬粒子為一金粒 如上所述之裝置，其中該電極為一多樣本陣 賴触不同㈣料之樣本或同 日寸取付冋一樣本之複數組數據。 通過述之裝置’其中該電流每經過一期間後 細胞如上所述之方法’其中該細胞包含一種以上之 合物+胃培養液包含複數種結 生、，·σδ，且忒複數種結合物上均帶有金屬粒 -·^ί ί 之農置’其中該阻抗偵測裝置更包含 λ唬放大早凡，用以放大該阻抗變化。 方法為ίίϊ 本案提供-種連續細胞數目的 一細胞液;.⑷於一氧化銦錫電極上利用 試算表將該連續ί 值換為一連績細胞數目改變量。 mi ^ 時，其血為一懸浮型細胞 1355418 % . 如上所述之方法，其中步驟（b)之該電流每經 過一期間後通過該氧化銦錫電極。 ' 為達上述目的，本案提供一種連續測量細胞蛋 白質表現的裝置，該裝置包含：一細胞；一細胞培 •- 養液，用以提供該細胞生長；一電極，與該培養液 電連接，用以提供該裝置之可感測性；及一交流阻 ' 抗偵測裝置，與該電極電連接，用以偵測該電極通 過一電流時之一交流阻抗變化。 如上所述之裝置，其中該電極為由二線狀電極 φ 所形成的一雙電極，該二線狀電極之個別線寬為 0.4mm 〇 如上所述之裝置，其中該二線狀電極相互平 行，該二線狀電極之間距為4mm。 如上所述之裝置，其中該電極為一氧化銦錫電 極。 如上所述之裝置，其中該氧化銦錫電極位於一 絕緣基板上，其中該絕緣基板之材質為玻璃、石英 及塑膠其中之一或其組合。 φ 如上所述之裝置，其中該電極為一多樣本陣 歹|J，用以同時偵測複數個不同的實驗條件之樣本或 同時取得同一樣本之複數組數據。 如上所述之裝置，其中該電流每經過一期間後 通過該電極。 如上所述之裝置，其中該阻抗偵測裝置更包含 一訊號放大單元，用以放大該阻抗變化。 本發明經由上述構想的解說，即能看出所運用 之檢測裝置及其方法，確能對懸浮型或貼附型細胞 之數量及細胞蛋白質作非破壞性連續的測量。而為 11 1355418 了易於說明，本發明可藉由下述較佳實施例及圖示 以更加瞭解之。 【實施方式】 請參閱第一圖，其為本發明之一種不影響細胞 正常生長之氧化銦錫電極晶片。該氧化銦錫（ITO) 電極晶# 01包含氧化銦錫電極001、細胞培養區 002、一細胞培養間格003、一絕緣基板004及一 線路接觸片005。 此氧化銦錫電極晶片01，係在於絕緣基板004 上，製作出特殊規格的氧化銦錫電極001後，再透 過細胞培養間格003將氧化姻錫電極001間隔出細 胞培養區0 0 2。欲進行細胞或蛋白質檢測前’先將 細胞培養在細胞培養間格003中，而使細胞及其偵 測培養液105成為此感測電路的一部份。當細胞數 目或是細胞阻抗特性有變化時，則氧化銦錫電極 001之阻抗值亦可從線路接觸片005的輸出觀察出 變化。 因細胞本身為電的不良導體，而一般的細胞培 養液主要成分為離子溶液，故為良導體。在貼附型 細胞偵測時，細胞成為橋接兩端電極的電路，因此 細胞的變化即為電路的變化，並可在半導體氧化銦 錫電極裝置中可產生一阻抗特性，故而經由偵測整 體電路的阻抗特性可以測得細胞的行為。而在懸浮 型細胞培養環境中，細胞未與電極接觸，因此電流 會經由培養溶液連接電極兩端形成迴路而略過細 胞本身，因此這樣的操作無法偵測懸浮型細胞。因 此必須改變以純血清作為培養溶液，由於血清的電 阻較高，因此可驅使電流通過懸浮型細胞。藉由對 12 1355418 . 不同型態細胞處理以不同的培養溶液，使得懸浮型 或貼附型細胞在數目有變化時，可以從電阻抗值或 • 電容抗值中作連續觀察。 在細胞蛋白質的感測中，利用抗原抗體之原 •- 理，將會與標的蛋白質特異性結合之第一級抗體及 帶有導電金屬粒子且會與前述第一級抗體特異性 ' 結合之第二級抗體加入細胞培養液中共同培養。藉 由導電能力的差別（電阻係數：導電金屬粒子与 ΙΟ'6 Ωοιη ； ΙΤΟ = 2χ10"4 Qcin ； Cell = 140 Ωοιη) > 導電金屬粒子的導電性優於氧化銦錫電極及細 # 胞，藉由氧化銦錫電極、導電金屬粒子與細胞三者 之間導電性的差異，可以達成其偵測蛋白質變化的 高靈敏度，並可對所欲偵測的蛋白質做連續的監 測。 請參閱第二圖，其為透過第一圖的氧化銦錫電 極001，藉以對細胞特異蛋白質作非破壞性連續測 量之示意圖。該示意圖包含一氧化銦錫電極001、 一絕緣基板004、細胞101、細胞蛋白質102、會 與該細胞蛋白質102特異性結合之第一級抗體103 以及會舆第一級抗體103特異性結合且帶有導電 • 金屬粒子之第二級抗體104。 當欲利用氧化銦錫電極001進行細胞101上之 細胞蛋白質102的檢測時，需先使細胞101於該氧 化銦錫電極001上穩定生長，並通過一電流以得到 一初始電阻阻抗值。隨後，於細胞偵測培養液105 中加入第一級抗體103與細胞蛋白質102特異性結 合接著，而加入之第二級抗體104會與第一級抗體 103特異性結合，且第二級抗體104上帶有導電金 屬粒子，所以會使得細胞101之交流阻抗特性改 變。隨著細胞101上的細胞蛋白質102表現量之變 13 1355418 化，藉由於不同時間點在氧化銦錫電極001上通過 同樣電流大小之該電流，以獲得不同時間點之電阻 阻抗值，並可因之換算出細胞蛋白質102的表現 量0 •- 請參閱第三圖，其為本發明細胞數量及細胞蛋 白質的檢測裝置示意圖，該裝置03包含氧化銦錫 (ITO)電極晶片01、交流訊號源201、訊號放大單 元202、訊號擷取單元203、記錄及控制介面204 以及電腦205。其中氧化銦錫電極晶片01、201、 訊號放大單元202、訊號擷取單元203、記錄及控 # 制介面204以及電腦205之間均電連接。 當欲利用氧化姻錫電極晶片01進行細胞蛋白 質的檢測時，需先使得一待測細胞（圖中未顯示）於 氧化姻錫電極晶片01生長穩定’並於培養該待測 細胞之培養液中，添加帶有導電金屬之一結合物 (圖中未顯示），其中該結合物會與該待測細胞之一 目標蛋白（圖中未顯示）特異性結合。利用交流訊號 源201於不同但連續之時間點供以不傷害該待測 細胞之微小交流訊號，並量測氧化銦錫電極001 兩端的電位差，所產生的電位訊號經訊號放大單元 • 202放大，並經訊號擷取單元203擷取後，藉由記 錄及控制介面204以及電腦205，即可換算出該連 續阻抗變化值，並得知細胞數量之變化或目標蛋白 的表現量變化。在一較佳的實施例中，氧化銦錫電 極晶片01可於細胞培養箱中連續培養無須取出， 並可藉由記錄及控制介面204以及電腦205，使用 者即可得到長時間且非壞性的監測記錄。 關於懸浮型細胞之生長數目監測，請參閱第四 圖。其細胞為HL-60細胞株(BCRCNo.60027)，本 實驗使用胎牛血清為細胞培養液，並以三種不同的 14 13554181355418 IX. Description of the invention: [Technical field of the invention] The present invention relates to a device and method for measuring cell number and cell protein, in particular to a non-destructive continuous quantity of specific or specific proteins of attached or suspended cells. Devices and methods of monitoring. [Prior Art] Continuous detection of φ cell behavior: In view of the fact that many cell-based experiments and methods will cause damage to cells, it is impossible to monitor a single sample for a long time, so non-destructive, continuous detection of cell status The sensing method or device is very necessary, but it is rare and still limited. In 1984, Giaever and Keese jointly proposed a method called Electro-Electrical Cell Impedance Spectroscopy (ECIS), which uses electrical methods to measure the behavior of adherent cells and continuously records the cell attachment electrode surface. The resulting resistance and capacitance are resistant. They found that there was no effect on the cells under the use of gold electrode materials or the application of an electric field. Cells remained normal regardless of attachment, stretching, and growth. In addition, in continuous measurement records, the total resistance value does increase with the cells attached to the growth. In this type of measurement method, the impedance analysis must be measured by the AC signal. Because the cell membrane is like a capacitor, it has the characteristics of isolated DC-coupled AC. Therefore, the AC signal must be used to measure the cells. In many traditional cell impedance analyses, cells are measured using a current of less than 1 μA, and further analysis is performed by measuring the small voltage across the electrode to be tested. Because the current in the measurement circuit is less than ΙμΑ, and the required measurement time is extremely short'. In many literatures, this measurement method also has no effect on the normal physiology of cells, so it can be regarded as a non-destructive measurement method. Among the cell types studied, cell impedance analysis has been applied to the sensing of many different adherent cell numbers, such as: fibroblast, end〇thelial cell, astrocyte , kidney cells, liver cancer cells, epidermal cancer cells, etc. These cells include cell lines and primary culture cells, but the number of suspension cells has not been measured so far. In the electrode design, the conventional cell impedance analysis device mostly places the gold electrode on the plastic culture plate, which is further divided into a tiny detection electrode and a large counting electrode. The study found that if the two electrodes were changed to two equal-sized electrodes, the change in the cell could not be measured. However, another scholar used platinum to make two equal-area J ^ electricity, (IDES), and successfully detected cell attachment growth continuously. This represents the form of the electrode during cell impedance measurement analysis. It has a great influence on the measurability. In recent years, similarly, although many studies have attempted to measure cell impedance using different electrode shapes, there has never been a solution related to the detection of suspended g ^ cells and cell protein detection. The current methods for measuring cellular proteins in a large class can be roughly divided into the following two types: Measurement methods for destroying cell type: Generally, the cells are lysed and separated, and the protein is separated and purified, or the protein wafer is detected, or even by extracting ribose. Acid (RNA) to do molecular biology analysis. There are many common methods for measuring proteins, such as protein film electrophoresis (SDS-PAGE), and Western blotting (Western b〇 t) compares the amount of protein expression; and, after high performance liquid chromatography (HPLC), Mass spectrometry (MS) analysis; as well as immunostaining (Immunostain) after capillary electrophoresis (CE). These methods require disruption of the cells, and if time-consuming observations are required, samples of many different time points need to be prepared' so that continuous non-destructive monitoring of the samples is not possible. 2. Measurement method for retaining cell type Generally, immunocytotochemistry is used, which is a specific protein labeled with an antibody having a fluorescent substance. Through cellular immunofluorescence staining, the spectator can observe the position of the protein in the cell under a fluorescence microscope (Fluorescence microscope), and even observe clear stereoscopic imaging and dynamic changes with a Confocal microscopy microscope. Cellular immunofluorescence staining retains the intact form, but the cells still need to be removed from the incubator, so they cannot be observed under the microscope for a long time. In addition, cellular immunofluorescence staining has the disadvantage of requiring light protection during operation, half-life of the fluorescent material, and expensive optical instruments. Overview of Protein Biosensing Wafers: Sensing wafers that currently measure proteins electronically are primarily immunological wafers. Regarding immunochips, proteins are mainly measured by the principle of specific binding of anti-antigen antibodies by coating antigenic proteins on a semiconductor surface to measure changes in their capacitance characteristics. The study found that when antigen-antibody is combined, the measured capacitance will decrease. Further, there is also a case where the antibody is attached to the surface of the conductive polymer to measure the change in conductivity after antigen-antibody binding. The study also found that as the concentration of the antigen to be tested increases, the degree of change in conductivity increases. The samples of these test wafers need to be taken from the 1355418 enamel fif liquid helium and the sample preparation is like the traditional detection method. The product is destroyed first, so it is still difficult to measure the continuity of the sample. In the early years, the applicants were carefully tested and studied in view of the defects in the prior art, and a copy of the quantity and the cellular protein = device and method. The following is a brief description of the case. SUMMARY OF THE INVENTION White matter ϊίίίίί provides a cell number and cell egg t Λ by using a = test on the target cell, the conductive metal === device is non-destructive to the cell-specific protein: ^ t ί i ^ 5 ^ 雷栖卜刹田々队x, <<>> (a) in the indium tin oxide cell-cell protein brother; the fine sex, - the first to ϊΐ; ί = over -: ί Ht sub,: (c) on the indium tin oxide electrode, i or ί ί, the inductive value or capacitance value of the indium tin oxide electrode is replaced by the lysed cell protein ^ see the electric quantity impedance of the method of the Reto households, wherein the step (a And cultivating more than one type of cells on the indium tin oxide electrode. Emulsified Indium Tin The method described above, wherein the culture solution of the step (b) comprises a plurality of first-level antibodies, and the plurality of first-level antibodies can specifically bind to different cellular proteins, respectively. The method as described above, wherein the step (b) incorporates a plurality of second-stage antibodies, each of which specifically binds to the plurality of first-order antibodies, each of which has a metal particle . The method as described above, wherein the current of the step (c) passes through the indium tin oxide electrode after a period of time. In order to achieve the above object, the present invention provides a device for continuously measuring the expression of a cellular protein, the device comprising: a cell; a cell culture solution to provide the cell growth, wherein the culture solution further comprises: a combination for Binding to a cell protein of one of the cells, wherein the conjugate carries a metal particle, and the metal particle can change the AC impedance characteristic of the cell after the conjugate specifically binds to the cell protein; The electrolysis fluid is electrically connected to provide sensibility of the device; and an AC impedance detecting device is electrically connected to the electrode for detecting an alternating current impedance change of the electrode when passing a current. The device as described above, wherein the electrode is a double electrode formed by a two-line electrode having an individual line width of _ 0.4 mm 〇 as described above, wherein the two linear electrodes are parallel to each other The distance between the two linear electrodes is 4 mm. The device as described above, wherein the electrode is an indium tin oxide electrode. The device as described above, wherein the indium tin oxide electrode is located on an insulating substrate, wherein the insulating substrate is made of one of glass, quartz and plastic or a combination thereof. The device as described above, wherein the conjugate is a combination of a first-stage antibody, a second-stage antibody specifically bound to the first-stage antibody, and a metal particle on the second-stage antibody. child. The method as described above, wherein the metal particle is a gold particle device as described above, wherein the electrode is a multi-sample array of samples of different (four) materials or the same array data of the same day. By the device described herein, wherein the cell has a method as described above after each period of time, wherein the cell comprises more than one compound + the gastric culture solution comprises a plurality of knots, σδ, and a plurality of conjugates Each has a metal particle -·^ί ί of the farm set 'The impedance detection device further includes λ 唬 amplification to reduce the impedance change. The method provides a cell fluid with a continuous number of cells in the case of ίίϊ; (4) using a trial calculation table on the indium tin oxide electrode to change the continuous value to a change in cell number. At mi ^ , the blood is a suspended cell of 1355418%. As described above, the current of step (b) passes through the indium tin oxide electrode after a period of time. For the above purposes, the present invention provides a device for continuously measuring the expression of cellular proteins, the device comprising: a cell; a cell culture solution to provide growth of the cell; and an electrode electrically connected to the culture solution. To provide sensibility of the device; and an AC resistance anti-detection device electrically connected to the electrode for detecting an AC impedance change of the electrode when passing a current. The device as described above, wherein the electrode is a double electrode formed by a two-line electrode φ having an individual line width of 0.4 mm 〇 as described above, wherein the two-line electrodes are parallel to each other The distance between the two linear electrodes is 4 mm. The device as described above, wherein the electrode is an indium tin oxide electrode. The device as described above, wherein the indium tin oxide electrode is located on an insulating substrate, wherein the insulating substrate is made of one of glass, quartz and plastic or a combination thereof. φ The device as described above, wherein the electrode is a multi-sample array J|J for simultaneously detecting a plurality of samples of different experimental conditions or simultaneously obtaining complex array data of the same sample. The device as described above, wherein the current passes through the electrode after a period of time has elapsed. The device as described above, wherein the impedance detecting device further comprises a signal amplifying unit for amplifying the impedance change. The present invention, as explained above, shows that the detection device and method therefor can indeed perform non-destructive continuous measurement of the number of suspended or attached cells and cellular proteins. For ease of description, the present invention can be better understood by the following preferred embodiments and illustrations. [Embodiment] Please refer to the first figure, which is an indium tin oxide electrode wafer of the present invention which does not affect the normal growth of cells. The indium tin oxide (ITO) electrode crystal # 01 comprises an indium tin oxide electrode 001, a cell culture zone 002, a cell culture compartment 003, an insulating substrate 004, and a line contact sheet 005. The indium tin oxide electrode wafer 01 is formed on an insulating substrate 004, and a special specification of indium tin oxide electrode 001 is formed, and then the oxidized samarium electrode 001 is separated from the cell culture region 0 0 2 through the cell culture compartment 003. Prior to cell or protein detection, the cells are first cultured in cell culture compartment 003, and the cells and their detection broth 105 are part of the sensing circuit. When the number of cells or the impedance characteristics of the cells change, the impedance value of the indium tin oxide electrode 001 can also be observed from the output of the line contact piece 005. Since the cell itself is a poor conductor of electricity, and the general cell culture solution is mainly composed of an ionic solution, it is a good conductor. In the case of attached cell detection, the cell becomes a circuit that bridges the electrodes at both ends, so the change of the cell is a change of the circuit, and an impedance characteristic can be generated in the semiconductor indium tin oxide electrode device, so that the whole circuit is detected. The impedance characteristics measure the behavior of the cell. In the suspension cell culture environment, the cells are not in contact with the electrodes, so the current bypasses the cells by connecting the ends of the electrodes through the culture solution, so that the operation cannot detect the suspended cells. Therefore, it is necessary to change the pure serum as a culture solution, and since the resistance of the serum is high, the current can be driven through the suspension type cells. By treating different types of cells with different culture solutions, 12 1355418 can be continuously observed from the electrical impedance value or the capacitance resistance value when the number of suspended or attached cells changes. In the sensing of cellular proteins, the original antibody that specifically binds to the target protein and the first antibody with conductive metal particles and which are specifically associated with the aforementioned first-order antibody are utilized. Secondary antibodies are added to the cell culture medium for co-cultivation. By the difference in electrical conductivity (resistance coefficient: conductive metal particles and ΙΟ'6 Ωοιη; ΙΤΟ = 2χ10"4 Qcin ; Cell = 140 Ωοιη) > conductive metal particles are superior to indium tin oxide electrodes and fine cells, By the difference in conductivity between the indium tin oxide electrode, the conductive metal particles and the cells, the high sensitivity for detecting protein changes can be achieved, and the proteins to be detected can be continuously monitored. Please refer to the second figure, which is a schematic diagram of non-destructive continuous measurement of cell-specific proteins by passing through the indium tin oxide electrode 001 of the first figure. The schematic diagram includes an indium tin oxide electrode 001, an insulating substrate 004, a cell 101, a cellular protein 102, a first-order antibody 103 that specifically binds to the cellular protein 102, and a specific binding of the first-order antibody 103. A second-order antibody 104 having a conductive metal particle. When the detection of the cell protein 102 on the cell 101 is to be carried out by using the indium tin oxide electrode 001, the cell 101 is first stably grown on the indium tin oxide electrode 001, and a current is passed to obtain an initial resistance resistance value. Subsequently, the first-stage antibody 103 is specifically added to the cell-detecting culture medium 105 to specifically bind to the cellular protein 102, and the second-stage antibody 104 to be added specifically binds to the first-stage antibody 103, and the second-stage antibody 104 is With conductive metal particles on it, the AC impedance characteristics of the cell 101 are changed. As the amount of expression of the cellular protein 102 on the cell 101 is changed, the resistance of the same current is obtained on the indium tin oxide electrode 001 at different time points to obtain resistance values at different time points, and The amount of expression of the cellular protein 102 is converted to 0. - Please refer to the third figure, which is a schematic diagram of the apparatus for detecting the number of cells and the cellular protein of the present invention. The device 03 comprises an indium tin oxide (ITO) electrode wafer 01 and an alternating current signal source 201. The signal amplifying unit 202, the signal capturing unit 203, the recording and control interface 204, and the computer 205. The indium tin oxide electrode wafers 01 and 201, the signal amplifying unit 202, the signal capturing unit 203, the recording and control interface 204, and the computer 205 are electrically connected. When the cell protein is to be detected by using the oxidized samarium electrode wafer 01, a cell to be tested (not shown) is stably grown on the oxidized samarium electrode wafer 01 and cultured in the culture medium of the cell to be tested. A conjugate having a conductive metal (not shown) is added, wherein the conjugate specifically binds to a target protein (not shown) of one of the cells to be tested. The alternating current signal source 201 is used to supply a small alternating current signal that does not damage the cell to be tested at different but continuous time points, and the potential difference between the indium tin oxide electrodes 001 is measured, and the generated potential signal is amplified by the signal amplifying unit • 202. After being captured by the signal acquisition unit 203, the continuous impedance change value can be converted by the recording and control interface 204 and the computer 205, and the change in the number of cells or the change in the expression amount of the target protein can be known. In a preferred embodiment, the indium tin oxide electrode wafer 01 can be continuously cultured in a cell culture incubator without taking out, and the user can obtain long-term and non-defective functions by recording and controlling the interface 204 and the computer 205. Monitoring record. For the monitoring of the number of growth of suspended cells, please refer to the fourth figure. The cell is an HL-60 cell line (BCRC No. 60027). This experiment uses fetal bovine serum as a cell culture medium and uses three different 14 1355418 cells.
縱座標為細胞生長所產生 的交流阻抗變化（ΔΩ)。 cells/mL)，將 HL-60 备電極001上，並置於 箱中繼續培養。橫座標 分鐘自動偵測一次，為 所偵測得到的交流阻抗變化(ΔΩ)，為對實驗組 (有接種細胞於氧化銦錫電極〇〇1上）通過一恭a德 ，得之交流阻抗值減去對照組（沒#接種=於 氧化銦錫電極001上）通過該電流後所測得之交流 阻抗值。 透過第四圖可以清楚觀察到，越高密度的懸浮 細胞有越高的交流阻抗變化量；且隨著細胞的i長 數目增加’交流阻抗值也會隨之增高。 貼附型細胞之數目量測’請參閱第五圖。其第 五A圖為細胞之不同生長密度以及交流阻抗變化 ΐ關係圖。其中該細胞是MG-63細胞株（BCRC No.60279) ’所使用的細胞培養液為含有1〇%胎牛 血清與1 %抗生素的high glucose DMEM培養基， 並置於細胞培養箱中培養。在本發明氧化銦錫電極 001上’於24小時等待細胞完成貼附後，所測得 交流阻抗變化(ΑΩ)之關係圖，橫座標為接種細胞密 度（cells/mL)，縱座標則為交流阻抗變化（δω)，該 交流阻抗變化的計算方式為： 於時間T1對實驗組（有接種細胞於氧化銦錫 電極001上）通過一電流後所測得之電阻抗值減去 於時間T1對照組（沒有接種細胞於氧化銦錫電極 001上）通過該電流後所測得之電阻抗值。其中時 間T1為細胞貼附後之任一適當時間。 15 1355418 &透過第五A圖可以清楚發現，當接種細胞數 量愈多，即生長密度愈高時，其測得之交流阻抗變 化也愈高。The ordinate is the change in AC impedance (ΔΩ) produced by cell growth. Cells/mL), place HL-60 on electrode 001 and place in a box to continue the culture. The abscissa minute is automatically detected once, and the detected AC impedance change (ΔΩ) is obtained for the experimental group (the inoculated cells are on the indium tin oxide electrode 〇〇1), and the AC impedance value is obtained. The AC impedance value measured after passing this current was subtracted from the control group (no #inoculation = on indium tin oxide electrode 001). It can be clearly seen from the fourth graph that the higher density of suspended cells has a higher amount of AC impedance change; and as the number of cells i increases, the AC impedance value increases. Measurement of the number of attached cells' Please refer to the fifth figure. Figure 5A shows the relationship between the different growth densities of cells and the change in AC impedance. The cell culture medium used in the MG-63 cell line (BCRC No. 60279) was a high glucose DMEM medium containing 1% fetal bovine serum and 1% antibiotic, and cultured in a cell culture incubator. On the indium tin oxide electrode 001 of the present invention, the relationship between the measured AC impedance changes (ΑΩ) after waiting for the cells to be attached for 24 hours, the abscissa is the inoculated cell density (cells/mL), and the ordinate is the alternating current. Impedance change (δω), the AC impedance change is calculated as follows: The resistance value measured after passing a current to the experimental group (with inoculated cells on the indium tin oxide electrode 001) is subtracted from the time T1 at time T1. The electrical impedance value measured after passing this current (no inoculated cells on the indium tin oxide electrode 001). The time T1 is any suitable time after the cells are attached. 15 1355418 & It can be clearly seen from Figure 5A that the higher the number of cells inoculated, ie the higher the growth density, the higher the measured AC impedance.
請參閱第五B圖’其為利用MTTPlease refer to Figure 5B' for the use of MTT
(3，[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazd ium bromide )法測量接種細胞MG-63貼附（於本實 驗是指接種細胞MG-63經過24小時後）於本發明 氧化銦錫電極〇〇1上於時間T1所測得之細胞活性 與接種細胞MG-63濃度之關係圖。其中第五B圖 之橫座標為接種細胞MG-63密度（ceiis/mL)，縱座 標則為測定波長570nm、參考波長690nm之吸光 值（O.D.)。透過圖五B可以清楚發現，當接種細胞 MG-63數量愈多，即生長密度愈高時，利用Μττ 法於時間T1所測得之細胞MG-63活性也愈高。(3,[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazd ium bromide) method was used to measure the attachment of MG-63 inoculated cells (in this experiment, after inoculation of cells MG-63 after 24 hours) A graph showing the relationship between the cell activity measured at time T1 and the concentration of inoculated cells MG-63 on the indium tin oxide electrode 本1 of the present invention. The abscissa of the fifth B diagram is the density of the inoculated cells MG-63 (ceiis/mL), and the ordinate is the absorbance (O.D.) of the measurement wavelength of 570 nm and the reference wavelength of 690 nm. It can be clearly seen from Fig. 5B that the higher the number of inoculated cells MG-63, that is, the higher the growth density, the higher the activity of the cell MG-63 measured by the Μττ method at time T1.
上▲請參閱第五C圖，其為第五A圖所測得之阻 才几變化(ΔΩ)與第五B圖所測得代表細胞活性之吸 光^(O^D.)之線性迴歸分析圖，其中第五c圖之橫 座標為第五A圖所測得之交流阻抗變化，縱座標 =為測定波長570nm、參考波長的如瓜之吸光值 (O.D.)。透過圖五c可以清楚發現，結果顯示代表 ，胞活性之吸光值與同樣細胞MG_63接種濃度下 父仙·阻杬變化，具有高度的線性相關（R2=〇.9883 ’ Ρ<0·005) ’並可知其交流阻抗變化正比於貼附於晶 上的細胞數量，說明本發明氧化銦錫電極〇〇1 上可用於連續觀測細胞的生長情形，未來更可以應 用在細胞毒性實驗、快速藥物開發等相關領域。〜 以下關於細胞蛋白質之感測。請參閱第六圖， "為利用本發明之一種連續測量細胞蛋白質 ”,置03測量Integrin此細胞蛋白質造成細胞阻 抗受化之曲線圖’其橫座標為細胞培養時間 16 1355418 • (Hours)，縱座標則為交流阻抗變化（ΔΩ);另外， Integrin蛋白則是已知會隨著細胞增生而表現之一 • 種細胞膜蛋白質。首先，將細胞MG-63以四種不 • 同的密度·· 5xl05cells/mL、2.5xl〇5cells/mL、1.2x 105 cells/mL、5xl04 cells/mL分別接種至本發明之 實驗組與對照組之氧化銦錫電極，其中所使用的細 • 胞培養液為含有10%胎牛血清與1%抗生素的 high glucose DMEM 培養基’並置於 37°C、5% 的 C02細胞培養箱中培養。待培養細胞MG-63經過 24小時後’於貫驗組之培養液中加入mouse 肇 anti-Integrin βΐ IgG之第一級抗體及帶金粒子的 goat anti-Mouse IgG-Au之第二級抗體，於對照組 之培養液中則加入PBS及帶金粒子的g〇at anti-Mouse IgG-Au ’並且將包含實驗組及對照組之 氧化銦錫電極晶片置回細胞培養箱中，以前述所揭 露之連續測量細胞蛋白質表現的裝置連續量測24 小時。 請續參閱第六圖’在前述所揭露之連續測量細 胞蛋白質表現的裝置連續量測24小時後，將實驗 組之交流阻抗值減去對照組之交流阻抗值，以得到 # 此圖之交流阻抗變化（ΔΩ)，並藉以分別得到不同接 種細胞濃度之交流阻抗變化的連續曲線。其中曲線 A代表細胞接種密度為5xl〇5 ceus/mL之交流阻抗 變5化連續曲線、曲線B代表細胞接種密度為2.5x 10 cells/mL之父流阻抗變化連續曲線、曲線c代 表細胞接種密度為1.2xl〇5 cells/mL之交流阻抗變 化連續曲線、曲線D代表細胞接種密度為5χ1〇4 cells/mL之交流阻抗變化連續曲線。 請續參閱第六圖’曲線A於第48小時之交流 阻抗變化量為37Ω、曲線B於第48小時之交流阻 17 1355418 • 抗變化量為20 Ω、曲線C於第48小時之交流阻抗 變化量為14 Ω、曲線D於第48小時之交流阻抗變 • 化量為11 Ω。因此，透過第六圖可以知道，當細 胞MG-63接種密度愈向時，Integrin蛋白的表現量 . 愈高，因而使得細胞所產生的交流阻抗變化也愈 高。另外，同樣透過第六圖的數據，也可以清楚觀 - 察到’細胞隨時間增生後，阻抗變化量亦會隨著▲Please refer to the fifth C diagram, which is the linear regression analysis of the resistance change (ΔΩ) measured in Figure 5A and the absorbance of the representative cell activity measured in Figure 5B (O^D.) In the figure, the abscissa of the fifth c diagram is the AC impedance change measured in the fifth A diagram, and the ordinate = the absorption value (OD) of the reference wavelength of 570 nm and the reference wavelength. It can be clearly seen from Fig. 5c that the results show that the absorbance of the cell activity is highly linearly correlated with the change of the father's sputum in the same cell MG_63 concentration (R2=〇.9883 'Ρ<0·005)' It can be seen that the change of the AC impedance is proportional to the number of cells attached to the crystal, indicating that the indium tin oxide electrode 本1 of the present invention can be used for continuous observation of cell growth, and can be applied to cytotoxicity experiments, rapid drug development, etc. in the future. Related field. ~ The following is about the sensing of cellular proteins. Please refer to the sixth figure, "To utilize a continuous measurement of cellular protein of the present invention, and measure the curve of cell impedance induced by Integrin as a cell protein, and its abscissa is cell culture time 16 1355418 • (Hours), The ordinate is the change in AC impedance (ΔΩ); in addition, the Integrin protein is known to be one of the cell membrane proteins as the cell proliferates. First, the cell MG-63 has four different densities. · 5xl05cells /mL, 2.5xl〇5cells/mL, 1.2x 105 cells/mL, 5xl04 cells/mL were inoculated to the indium tin oxide electrode of the experimental group and the control group of the present invention, respectively, wherein the fine cell culture solution used contained 10 % fetal bovine serum and 1% antibiotic high glucose DMEM medium' were cultured in a 5% C02 cell culture incubator at 37 ° C. After 24 hours of cultured cells MG-63 was added to the culture medium of the test group. Mouse 肇anti-Integrin βΐ IgG first-level antibody and goat anti-Mouse IgG-Au second-stage antibody with gold particles. In the culture medium of the control group, PBS and g〇at anti-with gold particles were added. Mouse IgG-Au ' And the indium tin oxide electrode wafer containing the experimental group and the control group was placed back into the cell culture incubator, and the apparatus for continuously measuring the cell protein expression disclosed above was continuously measured for 24 hours. Please refer to the sixth figure 'in the foregoing After continuously measuring the apparatus for continuously measuring the protein expression of the cells for 24 hours, the AC impedance value of the experimental group was subtracted from the AC impedance value of the control group to obtain the AC impedance change (ΔΩ) of the graph, and the difference was respectively obtained. A continuous curve of the change in AC impedance of the inoculated cell concentration, wherein curve A represents a continuous impedance curve with a cell seeding density of 5 x 10 ceus/mL, and curve B represents a parent cell with a cell seeding density of 2.5 x 10 cells/mL. Continuous curve of impedance change, curve c represents a continuous curve of AC impedance change with a cell seeding density of 1.2×1〇5 cells/mL, and curve D represents a continuous curve of AC impedance change with a cell seeding density of 5χ1〇4 cells/mL. In the six graphs, the change in the AC impedance of curve A at the 48th hour is 37Ω, and the impedance of curve B at the 48th hour is 17 1355418. 20 Ω, curve C has an AC impedance change of 14 Ω at 48 hours, and curve D has an AC impedance of 11 Ω at 48 hours. Therefore, it can be seen from the sixth figure that when cell MG-63 is inoculated The higher the density, the higher the amount of Integrin protein expression, and thus the higher the AC impedance generated by the cells. In addition, through the data in the sixth figure, it is also clear that the amount of impedance change will follow along with the proliferation of cells over time.
Integrin蛋白的表現量而增高。 請參閱第七圖，其為在接種MG-63細胞第48 小時’做Integrin蛋白免疫螢光染色後之螢光密度 • 柱狀圖。其利用96孔盤培養MG-63細胞，以相同 於第六圖所揭露之細胞接種密度（即5 X 105 cells/mL、2,5xl05 cells/mL、1.2xl05 cells/mL、5x 104 cells/mL)以及相同的培養與實驗條件。其橫座 標為細胞接種密度(cells/mL)，縱座標則為榮光密 度（FITC intensity)。在第48小時，細胞接種密度 為5xl05cells/mL組的螢光值為229.65±4.52、細胞 接種密度為 2.5><105 cells/mL 營光值為 224.39±3.93、細胞接種密度為 l.2xl〇5 celis/mL 螢 光值為221·64±4.33、細胞接種密度為5χ104 φ cells/mL螢光值為217.51±4.20。因此，透過第七 圖及上述數值，可以知道接種的細胞MG-63愈 多，Integrin蛋白質的表現量也愈高。 請參閱第八圖，其為.將第七圖所測得之不同細 胞MG-63接種濃度之第48小時的交流阻抗變化 (△Ώ)與同時間所測的得Integrin蛋白螢光讀值之線 性迴歸分析圖，發現兩者間有良好的線性相關 (R2=0.9178 ’ p<0.〇〇5)，亦即愈高地 Integrin 蛋白 質表現量，交流阻抗變化有愈高的趨勢存在。第八 圖的結果5兒明本氧化姻錫電極晶片在適當的抗體 18 1355418 4 . 加入後，可以藉由非破壞的連續阻抗量測，來對活 細胞之蛋白質表現，作連續的觀察，亦可為研究細 • 胞蛋白質的新方法。 請參閱第九圖，其為利用本發明所述之細胞數 .. 量檢測方法時，可以用以培養所欲檢測其數量之懸 浮細胞（圖中未顯示）之培養皿04示意圖。培養皿 ' 04包含一對電極41、一培養皿本體42、一培養液 43及一導線44,其中對電極41分別嵌入培養孤本 體42兩側、且對電極41與培養皿本體42中之培 養液43電連接，而導線44則與對電極41電連接， • 培養皿本體42則為一絕緣材質所製成。另外，當 利用培養皿04以及本發明所述之細胞數量檢測方 法檢測懸浮細胞之數量及生長情況時，如本說明書 前述所揭露的，培養液43可為一血清培養液。 當培養皿04取代第三圖中之氧化銦錫電極晶 片01、並透過導線44與第三圖所述之細胞數量及 細胞蛋白質的檢測裝置03電連接後，即可藉由本 說明書前述所揭露的原理及方法，對所欲檢測之懸 浮細胞數量及生長情況進行連續且非破壞性之監 測。當然，培養皿04亦可以用來進行本發明所述 ® 之針對特定細胞蛋白質的檢測。 培養m 04為本領域所十分常見之培養皿形式 稍加修飾後便可得之；換句話說，採用本發明所述 之細胞數量及細胞蛋白質的檢測裝置及方法，其實 驗成本或裝置製備成本均十分低廉，卻可大幅提昇 細胞數量或細胞蛋白質長時間測量之便利性。是 故，從培養皿04此一實施例中，再次說明了本發 明顯著之進步性以及產業可利用性。 綜上所述，藉由該等實施例當可說明本發明之 細胞數量及細胞蛋白質的檢測裝置與方法，確可達 19 1355418 連續自動的量測;且該連續 法，=法，不同於免疫染色之定量方 成:有 勢，而與習知之姑介八局邊差的優 外，該荨介技f相較具有顯著之進步性；另 s:較: ?上述之各項具體實施而A發;ί;=ίϊ; 思而為諸般修飾’然不脫如附申、;範 【圖式簡單說明】 第一圖係為本發明之一種可# 之氧化銦錫電極晶片；^供、·，田胞正吊生長 ΪΪ係為透過本發明之氧化銦錫電極〇〇1 對越特異蛋白質作非破壞性連續測量之示意圖； 白質Siiiii，-種細胞數量及細胞蛋 生』===，不同生長密度與其 第五A圖係為細胞MG-63之不同生長穷产盥 交流阻抗變化之關係圖；第五The amount of Integrin protein is increased. Please refer to the seventh panel, which is the fluorescence density after the 48th hour of inoculation of MG-63 cells by immunofluorescence staining of Integrin protein. The MG-63 cells were cultured in 96-well plates to the same cell seeding density as disclosed in Figure 6 (ie, 5 X 105 cells/mL, 2, 5 x 105 cells/mL, 1.2 x 105 cells/mL, 5 x 104 cells/mL). ) and the same culture and experimental conditions. The abscissa is the cell seeding density (cells/mL) and the ordinate is the FITC intensity. At the 48th hour, the fluorescence density of the cell seeding density of 5xl05cells/mL was 229.65±4.52, and the cell seeding density was 2.5><105 cells/mL camp light value was 224.39±3.93, and the cell seeding density was l.2xl. 〇5 celis/mL fluorescence value was 221.64±4.33, cell seeding density was 5χ104 φ cells/mL, and the fluorescence value was 217.51±4.20. Therefore, the seventh graph and the above values show that the more cells MG-63 are inoculated, the higher the expression of Integrin protein. Please refer to the eighth figure, which is the 48th hour AC impedance change (ΔΏ) of the different cell MG-63 inoculation concentration measured in the seventh figure and the simultaneous detection of the Integrin protein fluorescence reading. Linear regression analysis showed that there was a good linear correlation between the two (R2=0.9178 'p<0.〇〇5), that is, the higher the Integrin protein expression, the higher the AC impedance change. The results of the eighth figure show that after the addition of the appropriate antibody 18 1355418 4 , the non-destructive continuous impedance measurement can be used to observe the protein performance of living cells. It can be a new method for studying fine cell proteins. Please refer to the ninth figure, which is a schematic diagram of a culture dish 04 which can be used to culture the suspension cells (not shown) of the number to be detected by using the cell number detection method of the present invention. The culture dish '04 includes a pair of electrodes 41, a petri dish body 42, a culture solution 43, and a wire 44, wherein the counter electrode 41 is respectively embedded in the cultured solitary body 42 and the counter electrode 41 and the culture dish body 42 are cultured. The liquid 43 is electrically connected, and the wire 44 is electrically connected to the counter electrode 41. • The culture vessel body 42 is made of an insulating material. Further, when the number and growth of suspended cells are detected using the culture dish 04 and the cell number detecting method of the present invention, the culture solution 43 may be a serum culture solution as disclosed in the foregoing specification. When the petri dish 04 is substituted for the indium tin oxide electrode wafer 01 in the third figure and electrically connected to the cell number detecting device 03 described in the third figure through the wire 44, the foregoing disclosure of the present specification can be utilized. Principles and methods for continuous and non-destructive monitoring of the number and growth of suspended cells to be tested. Of course, Petri dish 04 can also be used to perform specific cell protein-based assays of the present invention. The culture m 04 can be obtained by slightly modifying the culture dish form which is very common in the field; in other words, the experiment cost and device preparation cost using the cell number and cell protein detection device and method according to the present invention. They are very inexpensive, but can greatly increase the number of cells or the convenience of long-term measurement of cellular proteins. Therefore, from the embodiment of the petri dish 04, the remarkable progress and industrial applicability of the present invention are again illustrated. In summary, the apparatus and method for detecting the number of cells and the cellular protein of the present invention can be used for the continuous automatic measurement of 19 1355418 by the embodiments; and the continuous method, the method is different from the immunity. The quantification of the dyeing is as follows: there is a potential, and compared with the well-known advantages of the eight-game margin, the 荨 荨 f 相 相 相 相 相 相 相 相 ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ;; ϊ; ϊ 诸 诸 诸 诸 诸 诸 诸 诸 诸 诸 诸 诸 诸 诸 诸 诸 诸 诸 诸 ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; The field cell growth lanthanide is a schematic diagram for non-destructive continuous measurement of the more specific protein by the indium tin oxide electrode 本1 of the present invention; white matter Siiii, - cell number and cell egg production ===, different The relationship between the growth density and the fifth A-picture is the change of the AC impedance of the different growth and poor production of the cell MG-63;
濃度與其細胞活性之關係圖I ;、ίί迴歸irr㈣胞活性之吸光值㈣ 第/、圖係為利用本發明之一種連續測量細胞 20 ·.〆 1355418 . 蛋白質表現的裝置測量Integrin蛋白造成細胞交流 阻抗值變化之曲線圖； ' 第七圖係為利用96孔盤以相同於第六圖所揭 露之細胞接種濃度以及培養與實驗條件同時培養 - MG-63細胞後，在接種細胞第48小時後做免疫螢 光染色後之螢光密度柱狀圖；及 第八圖係為將第七圖所測得之不同細胞接種 濃度之第48小時的交流阻抗變化(ΔΩ)與同時間所 測的得Integrin蛋白質螢光讀值之線性迴歸分析 • 圖。 第九圖係為本發明所述之細胞數量及細胞蛋 白質檢測方法所可採用的一種電極形式暨培養皿 示意圖。 【主要元件符號說明】 01氧化姻锡電極晶片 001氧化銦錫電極 002細胞培養區 • 003細胞培養間格 004絕緣基板 005線路接觸片 101細胞 102細胞蛋白質 103可與細胞蛋白質102特異性結合之第一 級抗體 104可與第一級抗體103特異性結合且帶有 導電金屬粒子之第二級抗體 201交流訊號源 21 13:55418 / • . 202訊號放大單元 203訊號擷取單元 • 204記錄及控制介面以及 ' 205電腦 … 03連續測量阻抗變化之檢測裝置 • 04培養皿 41對電極 42培養m本體 43培養液 籲 44導線 22Relationship between concentration and cell viability Figure I; ίί regression irr (IV) absorbance of cell activity (IV) / / Figure is a continuous measurement of cells using the present invention 20 ·. 〆 1355418. Protein expression device to measure the cell impedance of Integrin protein The graph of the change in value; 'The seventh figure is the same as the cell inoculation concentration as disclosed in the sixth figure and the culture and experimental conditions simultaneously - the MG-63 cells are used after the 48th hour of seeding the cells. Fluorescence density histogram after immunofluorescence staining; and the eighth graph is the 48th hour AC impedance change (ΔΩ) of the different cell inoculation concentrations measured in the seventh graph and the Integrin measured at the same time. Linear regression analysis of protein fluorescence readings • Figure. The ninth diagram is a schematic diagram of an electrode form and a culture dish which can be used for the method of detecting the number of cells and the method for detecting cellular proteins. [Main component symbol description] 01 oxidation of tin electrode wafer 001 indium tin oxide electrode 002 cell culture zone • 003 cell culture compartment 004 insulation substrate 005 line contact piece 101 cell 102 cell protein 103 can specifically bind to cell protein 102 The primary antibody 104 can specifically bind to the first-level antibody 103 and has a second-level antibody 201 with conductive metal particles. The AC signal source 21 13:55418 / • . 202 signal amplification unit 203 signal extraction unit • 204 recording and control Interface and '205 computer... 03 Continuous measurement of impedance change detection device • 04 Petri dish 41 for electrode 42 culture m body 43 culture solution 44 wire 22