1249031 九、發明說明： 【發明所屬之技術領域】 本發明係有關於一種建立快速分析蛋白質的整合型微流體 晶片系統及其分析方法，前述系統係整合固相萃取機制或免疫分 析機制與電泳分離模式於系統中，於免疫分析機制中，利用抗原 與抗體有特殊專一性之結合’發展出一次抗體與一次抗體對於欲 分析蛋白質之研究。 【先前技術】 毛細管電泳（Capillary electrophoresis，CE )自發展以來，以其快 速的分離時間、微量的樣品注射體積、高靈敏度以及儀器操作的便利 性專優越條件’已廣泛的應用在各種分析領域[R· Kuhn，S. Hof. Kuhn， Capillary Electrophoresis ·· Principles and Practice， 1993, Springer-Verlag，BerlinHeidellerggN.Y.(U.S.A·)]，尤其近幾年來生物 科技的快速發展，毛細管電泳所發展出來的分析技術，更是大量應用 在以去氧核糖核酸（deoxyribonucleic acid，DNA)分析為基礎的研究 [Roche, M.E.; Oda, R.P.; Landers, J.P. BIOTECHNOLOGY PROGRESS, 1997, 13, 659-668]。隨著生化科技及半導體製程技術的進步，1992年 Manz將毛細管電泳微小化應用於微晶片上進行樣品分離之實驗 [Manz, A.; Harrison, D.J.; Verpoorte, Fettinger, J.C.; Paulus, A.;1249031 IX. Description of the Invention: [Technical Field] The present invention relates to an integrated microfluidic wafer system for rapidly analyzing proteins and an analysis method thereof, the system is integrated with a solid phase extraction mechanism or an immunoassay mechanism and electrophoresis separation In the system, in the immunoassay mechanism, the combination of antigen and antibody has special specificity to develop a primary antibody and a primary antibody for the study of proteins to be analyzed. [Prior Art] Capillary electrophoresis (CE) has been widely used in various analytical fields since its development due to its rapid separation time, small sample injection volume, high sensitivity, and convenience of instrument operation. R· Kuhn, S. Hof. Kuhn, Capillary Electrophoresis ·· Principles and Practice, 1993, Springer-Verlag, BerlinHeidellerggN.Y. (USA·)], especially the rapid development of biotechnology in recent years, developed by capillary electrophoresis Analytical techniques are used in a large number of studies based on deoxyribonucleic acid (DNA) analysis [Roche, ME; Oda, RP; Landers, JP BIOTECHNOLOGY PROGRESS, 1997, 13, 659-668]. With advances in biotechnology and semiconductor process technology, Manz applied capillary electrophoresis miniaturization to microchips for sample separation experiments in 1992 [Manz, A.; Harrison, D.J.; Verpoorte, Fettinger, J.C.; Paulus, A.;
Ludi，H.; Widmer，H.M.J· Chromatogr· 1992,593,253-258]，因此將傳統 之毛細管電泳技術推進到另一層更高科技之領域-晶片式電泳。 晶片式電泳對於微量物種的分析具有高效率的分析方法[Κ·Ludi, H.; Widmer, H.M.J. Chromatogr. 1992, 593, 253-258], thus advancing conventional capillary electrophoresis technology to another layer of higher technology - wafer electrophoresis. Wafer-based electrophoresis has a highly efficient analytical method for the analysis of trace species [Κ·
Seiler，D.Jed Harrison，A· Manz. Analytical Chemistry 65(1993)， 1481]’例如·將純化過的樣品（如：經過聚合酶鍊反應(polymerase chain 1249031 reaction，PCR)放大後的去氧核糖核酸(DNA)產物、酵素和受質、抗體 和抗原）置於晶片樣品槽内進行分析專相關之報導[Ν·-Η· Chiem，D.J. Harrison. Electrophoresis 19 (1998), 3040]; [N.-H. Chiem5 DJ. Harrison.Seiler, D. Jed Harrison, A. Manz. Analytical Chemistry 65 (1993), 1481] 'For example, a purified sample (eg, polymerase chain 1249031 reaction, PCR) amplified deoxyribose Nucleic acid (DNA) products, enzymes and substrates, antibodies and antigens are placed in wafer sample chambers for analysis of specific correlations [Ν·-Η·Chiem, DJ Harrison. Electrophoresis 19 (1998), 3040]; [N. -H. Chiem5 DJ. Harrison.
Clinical Chemistry 44(3)591] o 免疫分析法(immunoassay)對於臨床檢驗、藥物分析的運用越來越 廣泛，它可用來檢驗小分子藥物、何爾蒙、大分子的腫瘤標誌蛋白， 血清蛋白等。此種檢測方法，通常是將欲檢測的項目當抗原 (antigen)，而將產生的抗體（antibody)當反應物，利用抗原與抗 體的專一性來檢測。 利用抗體-抗原結合原理來發展免疫分析技術時，最重要係能分 開結合之抗體或抗原（bound antibody or antigen)及游離之抗體或抗 原（freeantibodyorantigen)。此種分離抗原-抗體複合物和游離型抗 原（或抗體），可利用（1)溶解度；（2)顆粒性凝集性；（3)活 性碳吸附小分子抗原；以及（4)固相結合（s〇lidphase)來達成。而 固相結合更是現今最常用的免疫分析法，其特徵係在於抗體抗原反應 後’藉由簡單之沖洗動作即能將結合之抗體（抗原）及游離之抗體（抗 原）分開。欲達此目的，設計免疫試劑時，將抗體（抗原）附著在一 固相（solid phase，例如：96 well之塑膠盤或agarose bead)上，將抗 原（抗體）與固定化在固相上之抗體（抗原）進行免疫反應，產生抗 原-抗體複合物結合於固相上，之後，加入第二抗體（此抗體上可結 合染料或酵素）即可與固相上之聚合體反應，透過偵啦統侧前述 染料或酵素標記，即可達収性或定量分析。這種方法所用之第二抗 體對抗原並不具專一性，純粹只是用於偵測訊號之放大，因此false positive的機率並不小。 第與弟一抗體辨識特定蛋白質（例如：碟酸化蛋白質）的 124903i 貫驗方式與 免疫泳：跡、丄〃 ip，Clinical Chemistry 44(3)591] o Immunoassay is widely used in clinical testing and drug analysis. It can be used to test small molecule drugs, hormones, macromolecular tumor marker proteins, serum proteins, etc. . Such a detection method usually uses an antigen to be detected as an antigen, and an antibody produced as a reactant, which is detected by the specificity of the antigen and the antibody. When using the antibody-antigen binding principle to develop immunoassay techniques, the most important is the ability to separate bound antibodies or antigens and free antibodies or anti-antigens. Such an isolated antigen-antibody complex and an episomal antigen (or antibody) can utilize (1) solubility; (2) particulate agglutination; (3) activated carbon adsorption of small molecule antigen; and (4) solid phase binding ( S〇lidphase) to achieve. Solid phase binding is the most commonly used immunoassay today, characterized by the separation of bound antibodies (antigens) and free antibodies (antigens) by simple washing action after antibody antigen reaction. To achieve this goal, when designing an immunological reagent, attach the antibody (antigen) to a solid phase (for example, a 96 well plastic plate or agarose bead), and immobilize the antigen (antibody) on the solid phase. The antibody (antigen) is subjected to an immune reaction to produce an antigen-antibody complex bound to the solid phase, and then a second antibody (which can bind a dye or an enzyme) can be reacted with the polymer on the solid phase. The aforementioned dye or enzyme label can be used for quantitative or quantitative analysis. The second antibody used in this method is not specific to the antigen and is purely used to detect amplification of the signal, so the probability of false positive is not small. The first and second antibody identification methods for specific proteins (eg, acidified proteins) are observed in the 124903i method and immunobloom: trace, ip ip,
到特定的蛋白質。 IP，immunoprecipitation )加上點潰法 ‘續似，對於免疫沈澱法（IP)而言，此法先利用第一 用的蛋白質（抗原）抓下來並將溶液中少量的蛋白質做 再利用點潰法（Blotting)表現出來，並觀察是否有抓 貝。對於免疫沈澱法與點潰法而言，對於蛋白質的專一 ϋ (speeifie)並不南，因為在免疫沈澱法時只要是與抗體會作用的蛋 白質均會被抓下來，且蛋白f經免疫沈驗之魏f會失去活性，因 此，習知技術所述之免疫分析法之步驟相當繁雜、耗時且無法連續性 分析。 【發明内容】 有鑑於習知技術對於蛋白質分析上之缺陷及弊端，本發明係 提供一種整合型微流體晶片系統，用於整合免疫分析或固相萃取 機制與電泳分離模式於系統中以分析蛋白質。 本發明之整合型微流體晶片系統，至少包含：一微流體晶 片，係用於分離樣品之用；及一卡匣，係作為免疫分析及/或樣品 濃縮之用。To specific proteins. IP, immunoprecipitation) plus point collapse method 'continuation, for immunoprecipitation (IP), this method first uses the first protein (antigen) to grab down and use a small amount of protein in the solution for re-use point collapse (Blotting) showed up and observed if there was a grab. For the immunoprecipitation method and the point-expansion method, the specific peek of the protein is not south, because in the immunoprecipitation method, as long as the protein that interacts with the antibody will be captured, and the protein f is immunostained. The Wei f loses activity, so the steps of the immunoassay described in the prior art are quite complicated, time consuming, and incapable of continuous analysis. SUMMARY OF THE INVENTION In view of the defects and drawbacks of the prior art for protein analysis, the present invention provides an integrated microfluidic wafer system for integrating immunoassay or solid phase extraction mechanism and electrophoretic separation mode in a system for analyzing proteins. . The integrated microfluidic wafer system of the present invention comprises at least: a microfluidic wafer for separating samples; and a cassette for use in immunoassays and/or sample concentration.
之用。 南分子塑膠材料或 前述晶片材質玎為石英、玻璃、矽晶片、 其他具有相同功效之材質。 例如··碳十八小珠或蛋 前述卡匣主要係利用一填充有固相， 白質（例# :第—抗冑），並於晶片上之微管道中加人特定之蛋 白質（例如：第二抗體），使樣品經由前述卡£後再沖堤至晶片 上進行反應後之即時伯測。 本發明之另一目的係提供一 種利用微流體晶片系統分析蛋 1249031 白質之方法，至少包含下列步驟：將樣品導入微流體晶片系統中 之卡匣；讓樣品與卡匣内填充之固相反應；加入特定之蛋白質溶 液於微流體晶片上之微管道中；及將卡ϋ内之樣品沖堤至晶片上 之微管道中，使樣品與前述特定之蛋白質進行反應。 本發明之整合型微流體晶片系統之第一種實施態樣為整合 固相萃取及電泳分離模式，前述系統主要係將微流體晶片連接一 卡匣，此卡匣填充液相層析之碳十八小珠，可進行固相萃取，使 蛋白質濃縮、純化，濃縮後之蛋白質連續進樣至微流體晶片中進 行即時偵測。 本發明之整合型微流體晶片系統之第二種實施態樣為整合 免疫分析機制及電泳分離模式，前述系統主要係將微流體晶片連 接一卡匣，此卡匣填充固定化之第一抗體，並於微流體晶片上之 微管道中加入第二抗體，使蛋白質樣品進行連續之免疫分析反應 並進行線上即時偵測。 前述第一抗體例如：anti-BSA，其係於卡匣内填充anti-BSA 之小珠為固相。 前述第二抗體例如：anti-P，可用於辨識特定蛋白質（磷酸化 蛋白質）。 【實施方式】 本發明係有關一種整合型微流體晶片系統100及其分析方法，其 諸多優與特徵將從下述詳細說明並配合圖式得到進一步的瞭解。 首先參考圖一，係顯示本發明之整合型微流體晶片系統1〇〇之裝 置示意圖，至少包含：一微流體晶片3，係用於分離樣品之用；及一 卡匣2，係作為免疫分析及/或樣品濃縮之用。本系統100之操作流程 如下：首先將樣品由自動進樣裝置1注入卡匣2中，進行免疫分析或 1249031 樣品預濃縮反應’之後再針E 2巾之樣品沖堤至微趙^ 3之微 管道3i中，並藉由f源供應n 4之㈣將樣品進行電齡離模式，最 後經由偵測及訊號處理單元5將晶片3上之分析結果輪出。 南分子塑膠材 前述晶片2之材質可為石英、玻璃、石夕晶片 料或其他具有相同功效之材質。 前述卡Ε 2主要係利用-填充有_，例如：碳十八小珠或蛋白 質（例如抗體）’並於晶片3上之微管道31中加人特定之蛋 白質（例如：第二抗體），使樣品經由前述卡£ 2後再沖堤至晶片3 上進行反應後做即時偵測。 圖二係顯示利用本發明之微流體晶片系統1〇〇分析蛋白質之方法 之流程圖’至少包含下列步驟：將樣品導人微流體晶片系統_中之 卡Ef讓樣品與卡Ε 2㈣充之_反應，前述_例如：填充固 定有前述樣品（蛋白質）之第一抗體之固相；於微流體晶片3上之微 管道31中加人特定之蛋白f溶液，例如：前述蛋白質樣品之第二抗 體；及將卡ϋ 2内之樣品沖堤至晶片3上之微管道中3卜使樣品與前 述特定之蛋白質進行反應。前述蛋白之分析方法係主要係作為免疫分 析方法之用。 本發明之整合型微流體晶片系統100，將透過下述實施例做更進 一步說明。 !难例一：具变里萃取與電泳分離之整合型微流體晶片系統 本發明之整合型微流體晶片系統100之第一種實施態樣為整合了 固相萃取及電泳分離模式。前述系統1〇〇如圖一所示，其中系統1〇〇中之卡 匣2係填充液相層析之碳十八小珠（C18 bead ，Particle size=5pm，Vydac， 218TPB5) ’可進行固相萃取，使蛋白質濃縮、純化，濃縮後之蛋白質連續 進樣至微流體晶片中進行即時偵測。 1249031 兔十八小珠為十八個碳鍊長之疏水性固相，當蛋白質由幫浦 (syringe pump)推入碳十八卡匣中，蛋白質之疏水性區域會與碳十 八之石夕烧基作用，使蛋白質吸附於碳十八小珠上，利用低比例之有機 溶劑進行螢光蛋白質之清洗（wash)，將不純物（impurity)移除， 接著提高有機溶劑比例，進行沖堤(elution)使蛋白質由碳十八小珠脫 附，在這清洗與沖堤動作之間，蛋白質在碳十八卡匣中有固相萃取 (solid phase extraction)的效應，具有濃縮（concentrati〇n)及純化 (purification )的功能。 為了證明連接碳十八卡匣有濃縮效果，因此實驗分成兩部分進 行：（A)微流體晶片連接碳十八卡匣，（B)微流體晶片未接碳十 八卡匣；樣品為未純化之Cy5-BSA (2·8*10-6Μ)溶液25μ1 (未經 desalting column，Microcon ΥΜ-30 純化），比照（A)、（B)兩者 結果之差異。貫驗結果如圖三所示，A波峰之螢光強度明顯較B波峰 高，由此可知，Cy5-BSA經由碳十八卡匣（A波峰）後具有濃縮（約 濃縮10倍左右）及純化的效果。 實施例二：昇有立羞分臭電泳分離之整合型檄流體晶片系統 本發明之整合型微流體晶片系統之第二種實施態樣為整合免疫分 析機制及電泳分離模式。前述系統100，如圖一所示，其中卡匣2係填 充固定化之第一抗體，並於微流體晶片3上之微管道31中加入第二抗體， 使蛋白質樣品進行連續之免疫分析反應並進行線上即時偵測。 石粦酸化之蛋白貝在汛息傳遞上、基因表現之調控、蛋白質合成等 扮演相當重要的角色，但如果磷酸化蛋白質過渡累積會造成過渡表現 而產生疾病。纟實施例係_本發明之整合型微流體晶片熟有免疫 反應卡匣之系統，以Cy5-P-BSA (抗原，BSA conjugated 1249031 phosphotyrosine，填酸化蛋白質）與第一抗體anti-BSA和弟一抗體 anti-phosphotyrosine (anti-P)來研究碟酸化蛋白質之辨識與鑑定。 Cy5-P-BSA 經 anti-BSA agarose bead (第一抗體）後’與第二抗—體· (anti_P)反應之實驗 將Cy5-P-BSA樣品注入卡匣中與第一抗體（固定化anti-BSA) 反應後，將沖堤出之抗原（Cy5-P-BSA)直接導入微流體晶片中與晶 片上之第二抗體（anti-P)進行反應，形成抗原/抗體複合物，其結果 如圖四所示，其中A波峰··僅只Cy5-P-BSA，B波峰··開始於微流體 晶片中加入第二抗體（anti-P) 9·5*1(Τ8Μ，C波峰··加入第二抗體 (anti-P) 1·9*10·7Μ，D 波峰··加入第二抗體（anti-P) 4·8*1(Τ7Μ，通 常親和性作用力的產生可以由電泳圖上波峰形狀及其遷移時間的改 變得知，圖四中加上抗體後波峰明顯變寬且其遷移時間縮短。由圖四 結果可知，當anti-P含量增加時，抗原/抗體複合物的波峰會漸漸增 加，且波峰會位移，可知Cy5-P-BSA經第一抗體反應，經清洗及沖堤 步驟，仍與anti-P有特異性之作用。 有上述結果顯示，經第一抗體濃縮之磷酸化蛋白質（Cy5-p_BSA) 離開第一抗體後仍具有活性，因此嶙酸化蛋白質（Cy5_p_BSA)可被 專-性之第二抗體（anti.P)賴，比起傳統免疫分析之免疫沈殿法 (IP ’ imimmoprecipitation)及點潰法（m〇tting)所有反應步驟、試 劑用量和反應時間均大幅下降’而且於整合型晶片中可達連續進樣， 即時制之功能，這是傳統免疫分析所達不到的。除外，蛋白質於此 研究裝置中並不會失去活性’因此’藉由本發明之整合型微流體晶片 系統可應用於研究酵素活性反應。 對照實驗（Negative control) 此對照實驗係觀察Cy5_BSA與第一抗體（anti_BSA吻職 Ϊ24903i bead)、第二抗體（anti_P)之間的反應。以5•職與-_ρ兩者之 作用並不具特異性，由圖五所示，A波峰：僅只Cy5_BSA，b波峰： 於微流體晶片中開始加入第二抗體（anti_p) i 9*ι〇·7μ，c波峰：力口 · 入第二抗體（anti-P) 4洲-7Μ Μ貞測之結果顯示，當ip含量增 ，時’並無抗原/抗體複合物的波峰出現。@此藉^此對照實驗可知 W挪A與固定化anti_BSA反應後，ami p可辨識ο”撼而 Cy5-BSA則與感P不具特異性作用由上述結果可證實 ，經由本發 明之整合型微流體晶片系統確實可以辨識特殊蛋白質，而且經此裝置 中疋出來之蛋白貝亚不會失去活性，因此絲可將此裝置用於研究親 和力鍵結（binding-affinity)上。 · 當然’本發明之實施m不脫離本發明之要旨，可進行種種 之變更，其保護範圍由町之巾請專利範圍所界定。 本發明係關於-種快速分析蛋白質的整合型微流體晶片系統及 其分析方法’ μ线健合_萃取機制或免疫分析_與電泳分 離模式於系統中’藉由整合固相萃取機制可於線上蛋白質濃縮、純 濃縮後之蛋自質連續進樣至微流體晶片中進行即時偵測。另外， 稭由本發明’可整合m相免疫分析法，對蛋白質進行連雜之免疫分 析’相較於傳統技術，具有節省分析時間、降低成本、蛋白質不失去鲁 活性…等優點；且本發明係為—微流體晶片系統，容易整合至其他分 析#置’例如·貝邊儀（LC_MS)，因此可廣泛利用於醫療债測，快 速樣品分析等各種分析領域以增進人齡會之福祉。 12 1249031 【圖式簡單說明】 圖一係本發明之整合型微流體晶片系統之裝置圖示意圖。 圖二係本發明之微流體晶片系統分析蛋白質之方法流程示意圖。 圖二係顯示利用本發明之第一種實施態樣，整合碳十八固相萃取及 電泳分離模式之;^流體晶片系統之樣品（Cy5_BSA)預濃縮電泳圖。 圖四係顯示利用本發明之第二種實施態樣，整合免疫分析及電泳分 離杈式之;ft流體晶片系統之樣品（Cy5_BSA)與固定化anti_BSA (第一抗 體）、anti"*P (第二抗體）線上（On-line)免疫反應之電泳圖。 圖五係顯示本發明之第二種實施態樣之對照組實驗（Negative control)之 Cy5-BSA 經第一抗體（&ηΑΒ§[Α agarose bead)後再與第 二抗體（anti-P)反應之電泳圖。 [主要元件符號對照說明] 【主要元件符號說明】 1 自動進樣裝置 2 …卡匣 3 —晶片 4 電源供應器 5 --偵測及訊號處理單元 31…微管道 100—-整合型微流體晶片系統Use. The southern molecular plastic material or the aforementioned wafer material is quartz, glass, germanium wafer, and other materials having the same function. For example, carbon eight beads or eggs are mainly made up of a solid phase, white matter (example #: first-anti-caries), and a specific protein is added to the microchannels on the wafer (for example: The second antibody) allows the sample to pass through the aforementioned card and then rushed to the wafer for immediate measurement after the reaction. Another object of the present invention is to provide a method for analyzing egg 1249031 white matter using a microfluidic wafer system, comprising at least the steps of: introducing a sample into a cassette in a microfluidic wafer system; allowing the sample to react with a solid phase filled in the cassette; A specific protein solution is added to the microchannel on the microfluidic wafer; and the sample in the cassette is flushed into the microchannel on the wafer to react the sample with the specific protein described above. The first embodiment of the integrated microfluidic wafer system of the present invention is an integrated solid phase extraction and electrophoretic separation mode. The system is mainly for connecting a microfluidic wafer to a cassette, and the cassette is filled with carbon chromatography. Eight beads can be solid phase extracted to concentrate and purify the protein. The concentrated protein is continuously injected into the microfluidic wafer for immediate detection. A second embodiment of the integrated microfluidic wafer system of the present invention is an integrated immunoassay mechanism and an electrophoretic separation mode. The system mainly connects a microfluidic wafer to a cassette, and the cassette is filled with the immobilized first antibody. A second antibody is added to the microchannel on the microfluidic wafer to allow the protein sample to undergo a continuous immunoassay and to perform an on-line detection. The aforementioned first antibody is, for example, anti-BSA, which is a solid phase in which the beads filled with anti-BSA in the cassette are solid. The aforementioned second antibody, for example, anti-P, can be used to recognize a specific protein (phosphorylated protein). [Embodiment] The present invention relates to an integrated microfluidic wafer system 100 and an analysis method thereof, and various advantages and features will be further understood from the following detailed description and drawings. Referring first to Figure 1, there is shown a schematic view of an integrated microfluidic wafer system of the present invention, comprising at least: a microfluidic wafer 3 for separating samples; and a cassette 2 for immunoassay And / or sample concentration. The operation flow of the system 100 is as follows: first, the sample is injected into the cassette 2 from the automatic sample introduction device 1 for immunoassay or pre-concentration reaction of the 1249031 sample, and then the sample of the E 2 towel is rushed to the micro-Zhao ^ 3 micro In the pipe 3i, the sample is subjected to the electric age separation mode by the f source supply n 4 (4), and finally the analysis result on the wafer 3 is rotated by the detection and signal processing unit 5. South Molecular Plastic Material The material of the above wafer 2 may be quartz, glass, stone wafer or other materials having the same effect. The aforementioned cassette 2 is mainly made by using -filled with _, for example, carbon eighteen beads or protein (for example, antibody)' and adding a specific protein (for example, a second antibody) to the microchannel 31 on the wafer 3, The sample is rushed to the wafer 3 via the aforementioned card and then reacted for immediate detection. Figure 2 is a flow chart showing a method for analyzing proteins using the microfluidic wafer system of the present invention. The method includes at least the following steps: introducing a sample into a microfluidic wafer system _ a card Ef to charge a sample and a cassette 2 (4) Reaction, for example, filling a solid phase of a first antibody immobilized with the aforementioned sample (protein); adding a specific protein f solution to the microchannel 31 on the microfluidic wafer 3, for example, a second antibody of the aforementioned protein sample And the sample in the cassette 2 is flushed into the microchannel on the wafer 3 to react the sample with the specific protein described above. The aforementioned methods for analyzing proteins are mainly used as immunoassay methods. The integrated microfluidic wafer system 100 of the present invention will be further described by way of the following examples. Difficult Example 1: Integrated Microfluidic Wafer System with Variable Extraction and Electrophoretic Separation The first embodiment of the integrated microfluidic wafer system 100 of the present invention integrates a solid phase extraction and electrophoretic separation mode. The foregoing system 1 is shown in FIG. 1 , wherein the cassette 2 in the system 1 is filled with carbon 18 beads of liquid chromatography (C18 bead, Particle size=5pm, Vydac, 218TPB5) The phase extraction allows the protein to be concentrated and purified, and the concentrated protein is continuously injected into the microfluidic wafer for immediate detection. 1249031 Rabbit 18 beads are the hydrophobic solid phase of 18 carbon chains. When the protein is pushed into the carbon eighteen card by the syringe pump, the hydrophobic region of the protein will be the same as the carbon eighteen The base effect is to adsorb the protein on the carbon eighteen beads, use a low proportion of the organic solvent to wash the fluorescent protein, remove the impurity, and then increase the ratio of the organic solvent to perform the embankment. The protein is desorbed from the carbon eighteen beads. Between this cleaning and the action of the dyke, the protein has a solid phase extraction effect in the carbonic acid, which has a concentration and concentration. The function of purification. In order to prove that there is a concentration effect on the carbon eight-clamp, the experiment was divided into two parts: (A) the microfluidic wafer was connected to the carbon eight-calendar, and (B) the microfluidic wafer was not connected with the carbon eight-calendar; the sample was not purified. Cy5-BSA (2·8*10-6Μ) solution 25μ1 (without desalting column, Microcon®-30 purification), the difference between the results of (A) and (B). The results of the test are shown in Figure 3. The fluorescence intensity of the A peak is significantly higher than that of the B peak. It can be seen that Cy5-BSA is concentrated (about 10 times concentrated) and purified by carbon eighteen calves (A peak). Effect. Example 2: Integrated enthalpy fluid wafer system with immersive odor separation. The second embodiment of the integrated microfluidic wafer system of the present invention is an integrated immunoassay mechanism and an electrophoretic separation mode. The system 100, as shown in FIG. 1, wherein the cassette 2 is filled with the immobilized first antibody, and a second antibody is added to the microchannel 31 on the microfluidic wafer 3 to perform a continuous immunoassay reaction on the protein sample. Perform online instant detection. The protein of the ascorbate plays a very important role in the transmission of asphyxiation, the regulation of gene expression, and the synthesis of proteins, but if the phosphorylated protein is accumulated, it will cause a transitional performance and cause disease.纟Examples _ The integrated microfluidic wafer of the present invention is cooked with an immunoreactive cassette system, Cy5-P-BSA (antigen, BSA conjugated 1249031 phosphotyrosine, acid-filled protein) and the first antibody anti-BSA and brother The antibody anti-phosphotyrosine (anti-P) was used to study the identification and identification of acidified proteins. Cy5-P-BSA After the anti-BSA agarose bead (first antibody) and the second anti-body (anti_P) reaction, the Cy5-P-BSA sample was injected into the cassette with the first antibody (immobilized anti -BSA) After the reaction, the antigen (Cy5-P-BSA) from the embankment is directly introduced into the microfluidic wafer to react with the second antibody (anti-P) on the wafer to form an antigen/antibody complex. The result is as follows. As shown in Figure 4, the A peak is only Cy5-P-BSA, and the B wave peak begins with the addition of a second antibody (anti-P) 9·5*1 to the microfluidic wafer (Τ8Μ, C wave··· Two antibodies (anti-P) 1·9*10·7Μ, D crest··Adding a second antibody (anti-P) 4·8*1 (Τ7Μ, usually the affinity force can be generated by the peak shape on the electropherogram And the change of migration time, it is known that the peak of the antibody is significantly broadened and the migration time is shortened after adding the antibody in Fig. 4. From the results of Fig. 4, when the anti-P content increases, the peak of the antigen/antibody complex gradually increases. And the peak shifts, it is known that Cy5-P-BSA reacts with the first antibody, and after washing and rushing the bank, it still has a specific effect with anti-P. It is shown that the phosphorylated protein (Cy5-p_BSA) concentrated by the first antibody remains active after leaving the first antibody, so the citrated protein (Cy5_p_BSA) can be mediated by a specific secondary antibody (anti.P). All the reaction steps, reagent dosage and reaction time of IP 'imimmoprecipitation and dot 法 method are greatly reduced from the traditional immunoassay', and continuous injection can be achieved in integrated wafers. Function, which is not achieved by traditional immunoassay. Except that the protein does not lose activity in this research device. Therefore, the integrated microfluidic wafer system of the present invention can be applied to study the enzyme active reaction. Negative control) This control experiment observed the reaction between Cy5_BSA and the first antibody (anti_BSA kisser 24903i bead) and the second antibody (anti_P). The role of both 5 and -_ρ is not specific. As shown in Figure 5, A wave peak: only Cy5_BSA, b wave peak: Start adding secondary antibody (anti_p) in microfluidic wafer i 9*ι〇·7μ, c wave peak: force port · into second antibody anti-P) 4 continent-7Μ The results of the test show that when the ip content increases, there is no peak of the antigen/antibody complex. @本借^ This control experiment shows that W move A reacts with immobilized anti_BSA , ami p can recognize ο”撼 and Cy5-BSA has no specific effect with sensible P. It can be confirmed from the above results that the integrated microfluidic wafer system of the present invention can surely identify a specific protein and is extracted by the device. Protein Beyer does not lose activity, so silk can be used to study binding-affinity. Of course, the implementation of the present invention can be variously modified without departing from the gist of the present invention, and the scope of protection is defined by the scope of the patent of the Toshio Towel. The present invention relates to an integrated microfluidic wafer system for rapid analysis of proteins and an analytical method thereof. [μ line fitness_extraction mechanism or immunoassay_with electrophoresis separation mode in a system" can be performed online by integrating solid phase extraction mechanism Protein concentrate, purely concentrated eggs are continuously injected into the microfluidic wafer for immediate detection. In addition, the straw can be integrated with the m-phase immunoassay method of the present invention, and the immunoassay of the protein is compared with the conventional technology, which has the advantages of saving analysis time, reducing cost, and not losing the activity of the protein. It is a microfluidic wafer system that can be easily integrated into other analytical methods, such as the Bayesian Instrument (LC_MS), so it can be widely used in various analytical fields such as medical debt testing and rapid sample analysis to enhance the well-being of the elderly. 12 1249031 BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic view of a device of an integrated microfluidic wafer system of the present invention. 2 is a schematic flow chart of a method for analyzing proteins by the microfluidic wafer system of the present invention. Figure 2 is a diagram showing the preconcentration electrophoresis pattern of the sample (Cy5_BSA) of the fluid wafer system by using the first embodiment of the present invention to integrate the carbon eight-phase solid phase extraction and electrophoresis separation mode. Figure 4 shows a second embodiment of the present invention, an integrated immunoassay and electrophoretic separation method; a sample of the ft fluid wafer system (Cy5_BSA) and an immobilized anti_BSA (first antibody), anti"*P (p. Two antibodies) Electropherograms of the on-line immune response. Figure 5 is a diagram showing the Cy5-BSA of the second control of the second embodiment of the present invention after the first antibody (&ηΑΒ§[Α agarose bead) followed by the second antibody (anti-P) Electropherogram of the reaction. [Main component symbol comparison description] [Main component symbol description] 1 Automatic sample introduction device 2 ... cassette 3 - wafer 4 power supply 5 - detection and signal processing unit 31 ... micro-pipe 100 - integrated microfluidic chip system