200303421 (1) 玖、發明說明 【發明所屬技術領域】 本發明有關分析物分離系統及其應用，尤其有關膠體 電泳系統。 【先前技術】 膠體電泳法係一種用於分離各分析物之混合物之習知 技術。橫越一膠體加上一電場，呈流體樣本形式之混合物 可經由該電場而遷移。在該電場之影響下，每一分析物之 遷移速度可依各種之分析物性質而定，諸如分子量或等電 荷點。其結果是，該分析物沿著該膠體於所加上電場之方 向中分離。 於蛋白質分析物之案例中，典型可藉著等電荷焦集完 成此一分離操作，其中一 pH (酸鹼）値梯度造成蛋白質 依照其等電荷點之分離（即各蛋白質無任何淨電荷之酸鹼 pH値）。根據該“固定不動pH値梯度”（IPG)技術，該 pH値梯度可倂入一膠體中，比方呈黏著至一惰性基材之 膠片形式，該蛋白質之混合物可塗至該膠片。 此“IPG膠片”係早已熟知，且各種技術係可用於其之 製備，比方包含在美國專利第5,993,627號、美國專利第 4，1 3 0,470號、美國專利第5,5 3 4，121號及歐洲專利第 0 3 93 47 8號中所敘述者。一 IPG膠片可能承載於一諸如 Gelbond ™之薄膜背襯上（如於美國專利第5,993,627號 中），在此案例中該背襯層傾向於具有類似該膠片本身之 -6- (2) (2)200303421 尺寸，且稍微大於該膠片。於使用一分離系統時，該膠片 及背襯層係位於諸如一平板或塊材之分離支座上。 典型係能以預先準備及乾燥形式購得IPG膠片，然後 於使用之前以該樣本流體使其再度成爲水化物，以致該樣 本被吸入該膠體。該膠片之處理可涉及許多樣本及/或包 含譬如緩衝及淸洗流體等試劑流體之應用。 於使用一 IPG膠片以進行電泳分離期間，其需要保護 環境免於受汙染及遭受脫水作用。於該分離製程期間典型 係藉著將該膠片浸入矽酮油以達到此目的，或至少以不能 滲透之塑膠薄膜保護層覆蓋該膠片。此保護物造成處理困 難。 於該分離製程期間亦需要冷卻一 IP G膠片，以補償由 S亥施加電場所造成之溫度增加。迫通常亦呈現處理困難性 。典型藉著將該膠片裝入一由導熱材料製成之塊材時需要 進行冷卻，然後藉著習知方法冷卻該塊材之一或多面。然 而既然該電泳分離需要應用一高電場，其必要的是與該膠 片接觸之材料係不導電的，且這限制支撐塊材之可達成之 導熱率。 可藉著進行二連續之分離操作改善一電泳分離之分辨 率。該分析物最初係根據第一種性質分離，且然後將如此 分離之混合物施加至另一膠體及使其遭受一電場以根據第 二、不同性質分離其各成分。首先在 1975年（ O’Farrell，P H[1975]J.Biol.Chem. 250:4007-4021 )報告此 已知爲二次元膠體電泳法之技術。其一般係用於分離生物 -7- (3) (3)200303421 學分析物之混合物，諸如蛋白質。 可使用如上述之IPG膠片進行該“第一次元”分離。然 後典型藉著普通之厚片膠體電泳技術施行該“第二次元”分 離，其中分析物（比方蛋白質）經由所加上電場之流動性 依分子量及電荷程度而定。 在允許該如此分離分析物遷移進入、及經過該第二次 元膠體之前，必須持續進行至完成該第一次元分離這典型 係藉著使已進行第一次元分離之膠體與該第二膠體形成接 觸。再者，已知許多達成該第一至第二次元轉移之方法， 比方如美國專利第 5,993,627號、美國專利第6,013，165 號、歐洲專利第0 3 66 8 97號、及歐洲專利第0 8 7 7 24 5 號、及我們同在申請中之 PCT 專利申請案第 PCT/GB02/0 1 749 號所述。 【發明內容】 根據本發明之第一論點，在此提供一種用於在流體樣 本中分離各分析物之混合物之電泳裝置，該裝置包含： 第一次元分離介質，該分析物可經過該分離介質遷移 ’該分離介質係載送在撓性薄片之一面上，該面之表面積 係大於該分離介質及該薄片間之接觸區域之表面積，其中 言亥第一次元分離介質係位於至少局部藉著該撓性薄片所界 定之第一分離區內； 該裝置尙包含一藉著該撓性薄片由第一次元分離區分 開之流體室，其中該流體可保持與該薄片之一面接觸，該 -8 - (4) (4)200303421 面係與載送該分離介質之面相向； 合適之分離介質爲一水膠體’即傳統用於膠體電泳法 者，諸如聚丙烯醯。當橫越該分析物加上一電場時，其最 好能夠等電荷焦集各分析物。其比方可採取一固定不動 p Η値梯度（IP G )成分之形式’而沿著其各維尺寸之一倂 入一 pH値梯度。 該分離介質最好係呈一修長元件之形式’諸如一膠片 或圓柱體。此種膠體膠片用之典型尺寸係具有於0.1及 1 . 5毫米之間、最好於〇. 4及0.8毫米之間之厚度；於5 0 及5 0 0毫米之間、較好於1 〇 〇及3 5 0毫米之間、更好於 150及320毫米之間、最好大約300毫米之長度（這是在 所使用分析物之移動方向）：及於2及5毫米之間、最好 3.5毫米之寬度。 該分離介質應該支撐在該撓性薄片上，且最好永久固 定至該撓性薄片。其可藉著形成在適當位置或藉著一分開 之黏附處理在製造該介質之後施加至該薄片。一形成在適 當位置之方法係使用一移動式噴嘴以配送一混合膠體成分 至該薄片上。當該噴嘴沿著該分離介質之想要軌道前進時 ，改變該混合成分以給予固定不動pH値之所需梯度。另 一用於形成在適當位置之方法係塗上或配送一鹼基膠體（ 例如聚丙烯醯），然後噴灑固定不動之雙性電離子進入該 膠體，以創造所需之梯度。該分離介質於使用在一分離製 程之前可呈一脫水形式。 該薄片最好係由一增進該分離介質對該薄片之黏附力 -9 - (5) (5)200303421 之材料所製成，或帶有該材料之〜塗層。譬如，該薄片可 爲專有之膠體bond ™型式，其帶有一塗層，一聚丙烯醯 膠體可共價地鍵結至該塗層。 該薄片理想上係可充分撓曲，以便能夠具有下文所述 之密封及冷卻功能。應該要由一惰性及不能滲透流體之材 料、諸如聚酯之適當合成塑膠材料製成該薄片。較佳之薄 片厚度係於20至500微米之範圍中，更佳係於25及200 微米之間，最佳係於5 0及1 5 0微米之間。 相關薄片面之面積較好至少係該分離介質及該薄片間 之接觸區域面積之1 5倍，更好係於2 0及2 0 0倍之間，最 好係於30及1〇〇倍之間。其理想上係充分大，以致其亦 可具有如下文所述用作第二次元分離介質之背襯之作用。 該薄片之合適尺寸係於100乘以40毫米及400乘以600 毫米之間。 在一較大之撓性薄片上支撐諸如IPG膠片之第一次元 分離介質能提供許多優點。其能幫助處理該分離介質及其 在一電泳裝置內之定位。此外，該薄片能如下文所述於橫 越該分離介質所進行之電泳分離期間用作冷卻系統之一部 份。 該薄片之彈性能如下文所述用於可逆地隔絕該電泳裝 置之一或多個含有流體之區域，特別是環繞該第一次元分 離介質之區域，此彈性藉著施加最好相當小之壓力而賦予 該薄片局部變形或位移之能力。 該撓性薄片具有在本發明之裝置內至少局部界定第一 -10- (6) (6)200303421 分離區之作用，該第一分離區包含該第一次元分離介質。 於該第一分離區之區域中最好未藉著任何底板或類似支撐 機構支撐該薄片。 假如本發明之裝置包含用於接近該第一次元分離介質 施壓至該薄片之一或多個區域之機構，以便於該區域或各 區域中變形及/或位移該薄片，這可方便地達成。然後該 變形及/或位移可造成該薄片接觸一在該裝置內之密封元 件’以致該密封元件及薄片在一起至少局部界定一已包圍 之不透水第一分離室，其至少包含及/或接觸該第一次元 分離介質之一部份。該薄片之變形及/或位移最好係可逆 的’以允許該分離室如所想要及當想要時關上或打開。 就此情況而言，該“不透水”一詞涵蓋一具有一或多個 流體入□或出口管道之室，流體可當想要時經由該室引導 至該室或由該室排出。 與該薄片形成接觸之密封元件可爲譬如一墊圈，或該 裝置之一呈不透水接觸之任何其他區域可藉著施加壓力以 迫使該薄片與該區域形成接觸所造成。 如此界定之第一分離室最好係具相當低之容量，例如 在水合作用之後於200微升及2毫升之間，或在該分離介 質之容量之1倍及4倍之間。既然該第一次元分離介質在 該分離製程期間可與所需之流體（譬如樣本流體、試劑流 體 '淸洗流體 '緩衝劑等，亦可能諸如著色劑之成像試劑 )接觸’且理想上浸入所需之流體，其特別適合在此一已 包圍 '低容量室內施行第一次元分離。如此保護該分離介 -11 - (7) (7)200303421 質，而不須油層或其他形式之保護阻擋層。 然而，該第一分離室應具有一尺寸適合允許流體之至 少某種程度地移動環繞著已包圍之第一次元分離介質’以 允許藉著該分離介質吸收流體。 取代之或此外，該薄片之變形及/或位移可用於使該 t 第一次元分離介質本身與該裝置內之一密封元件形成接觸 ‘ ，該分離介質及該密封元件一起至少局部界定上述第一分 離室。 _ 該流體室可具有一控制室之功能，而一已加壓控制流 體可供給至該控制室。最好藉著該薄片至少局部界定該控 制室。可依該裝置之幾何形狀而定，藉著施加一正或一負 壓差至該控制室達成該薄片之想要之變形/位移，藉此改 變該控制流體之壓力。此壓力係施加至該薄片與運送該分 離介質相向之面。 該撓性薄片可另外具有本發明裝置中之一部份溫度控 制系統之功能。該裝置最好包含用於來回傳熱、典型由該 · 薄片之一或多區域傳熱之機構。這最好包含用於供給一溫 度調節流體至該薄片之一或多個區域之機構，較好是供給 至與運送該第一次元分離介質相向之表面，且較好是供給 至該表面接近該分離介質'更好緊接在該分離介質後方之 一區域。因爲該薄片係相當薄，且比該分離介質具有一較 · 大面積，其可用作一有效率之熱傳介質，特別是用作該分 . 離介質及大容量溫度調節流體間之一介面，因此極有助於 進出該分離介質及與其接觸任何流體之熱傳。 -12- (8) (8)200303421 該溫度調節流體典型於一分離製程施加電場期間將用 於冷卻該分離介質及周圍之流體。一適合之冷卻流體爲水 〇 該裝置最好包含一定位置毗連該撓性薄片之適當區域 、且最好與其流體接觸之溫度調節室，該溫度調節流體可 供給至該調節室。 如此該溫度調節室最好至少局部藉著該撓性薄片所界 定。該溫度調節流體及該薄片間之接觸面積應盡可能大， 且應包含緊接圍繞該第一次元分離介質之區域。如此，理 想上，該室係與該薄片之一區域接觸，此區域之面積係一 百及二百倍之間地大於該分離介質及該薄片間之接觸面積 。方便的是，該溫度調節室具有一相當小之深度，例如於 0.2及1 0毫米之間，且該溫度調節流體最好在該撓性薄片 之一大面積上方流經該狹窄之室。 該流體室可適當地具有溫度調節室之功能。最好，該 流體室具有一控制室及一溫度調節室之功能，於此案例中 ’當在一適當壓力下供給時，該溫度調節流體亦可用作該 控制流體。 本發明之裝置可用以施行單一次元之電泳分離，於此 案例中’其只需要包含該第一次元分離介質及相關之機構 ’例如用於橫越該分離介質加上一電場之機構、供給流體 至該分離介質之機構、及於該分離製程期間調節該分離介 質溫度之機構。 然而’根據本發明將該第一次元分離介質定位在一裝 -13- (9) (9)200303421 置中之撓性薄片上亦有助於二次元之分離。該薄片可具有 至少局部界定該裝置內之第二分離區之作用，其中第二分 離介質（典型爲諸如聚丙烯醯膠體之水膠體）可位於該第 二分離區內，以便施行第二次元電泳分離。該第二分離區 可與含有該第一次元分離介質之第一分離區各自獨立或由 其分開（例如使用一可移去之阻擋層，諸如我們同在申請 中之PCT專利申請案第PCT/GB 02/01749號中所述，更好 藉著該薄片本身之局部性變形/位移，如上文所述者）° 又更好的是該第一及第二分離區能彼此可逆地隔絕’ 例如經由該薄片之局部性變形/位移。然後當該第一次元 分離介質係封入第一分離室時（亦即如上文所述於該第一 次元分離介質之區域中施壓至該撓性薄片），可進行第一 次元分離，在此之後可使該第一室與該第二區形成流體接 觸（薄片壓力移除之後），以允許分析物由第一分離介質 遷移至該第二分離區中所包含之第二分離介質。 於此一裝置中，該流體室最好在對應於該第一及第二 分離介質兩者之區域中允許流體與該撓性薄片保持接觸。 於使用中，當達成該第一次元分離時，該第二分離介 質只需要導入該第二分離區。其例如可能以似水液體之形 式導入，而能夠隨後譬如藉著在原位置之聚合反應允許在 該裝置內凝固成一厚片膠體。該第二分離介質典型採取一 於0.5及2毫米厚之厚片膠體之形式，較佳於0.8及1.8 毫米之間，更佳是大約1 . 5毫米厚-因此對該第二分離區 亦有較佳之深度。 -14- 200303421 do) 可引導該第二分離介質，以便接觸、或甚至圍繞該第 一次元分離介質。然而更方便的是’其係以某一方式導入 ，以致於該第一及第二分離介質之間留下一孔腔，該孔腔 可充滿諸如洋菜膠體之適當介質’以允許分析物在想要時 間下移動於該第二分離介質上。於兩案例中，該第二分離 區可有效地倂入該第一分離區、或至少與該第一分離區流 體相通。 在此於該第一及第二分離介質之間允許有此一中介孔 腔，當該分析物前進至該第二區時，其淸楚想要的是應該 保有在該第一分離區中達成之分析物分離。爲此目的，應 該選擇該孔腔之設計及其使用條件，以使該第一區中所達 成之分析物分離扭曲減至最小，這意指使分析物特別是沿 著該第一區施行分離之方向中之移動減至最小。 可容忍之分析物“漂流”數量依所使用分離介質中能達 成之分辨率範圍而定；當該分析物橫過該孔腔時，相關位 元中之分析物移動理想上將橫越比該最佳可達成分辨率較 小之距離。一種目前典型可用之膠體提供一直至大約0.5 毫米之有用分辨率；當使用此膠體時，適當地分析物移動 係少於〇 . 5毫米，理想上少於〇 . 3毫米。 分析物在該中介孔腔內之移動程度能依若干因素而定 ，諸如該孔腔中現有介質或各介質之黏度、該孔腔之長度 (於樣本之移動方向中）、所施加之電場、所施加之壓力 梯度、及該分析物本身之本質且因此其流動性。可藉著諸 如溫度、重力、裝置移動、及甚至於連接裝置中之流體移 -15- (11) (11)200303421 動等外在影響依序施行這些因素、特別是該壓力梯度。 用於該中介孔腔之適合介質係一具相當黏度之流體， 諸如溶化之洋菜（在例如攝氏5 0及7 0度之間之溫度）。 適合之流體黏度可於2及1000微牛頓•秒/平方米（ mPa. s )(在室溫及室壓）之間，較好係於5及5 00 · mPa · s之間，更好係於5及20mPa · s之間，諸如大約 · 1 0 mP a · s。亦可能提供緩衝流體，諸如一般用於膠體電泳 分離者。 · 大致言之，能藉著減少在該中介孔腔內之可能流體移 動程度減少分析物移動。這例如依序能由下列事項所控制 i )以一更具黏度之流體充塡該孔腔，諸如藉著倂入 一膠凝劑，該膠凝劑諸如聚丙烯醯或洋菜； ii )減少該孔腔之長度，最好使該孔腔之長度減至最 小（於分析物移動經過該孔腔之方向中），一適合之長度 可譬如於〇. 5及5毫米之間，較好係於1及3毫米之間， ® 更好係大約2毫米； iii )於該孔腔附近包含流體流量控制閥，以便遍及流 體移動施行控制，該移動可能譬如由於外在影響所造成； 及/或 iv )以一固定支撐方式安裝該裝置，再次於使用期間 - 便於使流體移動減至最小。 · 爲有助於該第二分離介質之導入及假如可對該中介孔 腔應用一介質，一或多個液位感測器可納入本發明之裝置 -16- (12) (12)200303421 。一種方便之感測器形式係一光學液位感測器，例如一種 經過適當之塑形光導將光線導入該相關區域及偵測由該光 導之一內部表面反射回來之光線之感測器，該反射之範圍 及本質係依該區域中所提供流體而定，該區域係該光導所 延伸進入之區域。 該流體室可具有一控制室之功能，能操作該流體室以 與上文該第一分離區所述相同之方式可逆地隔絕該第二分 離區。 亦可以與上文有關該第一次元分離所述相同之方式適 當地控制該第二分離區內之溫度，亦即藉著，在該第二分 離區內接觸該撓性薄片之至少一區域與一溫度調節流體。 該第一及第二分離區兩者可共用該裝置之溫度調節室，理 想上係藉著與該相關薄片面之相當比率（例如百分之90 或更大）面積呈流體接觸。 於本發明之裝置中，如此該撓性薄片最好係具有充分 大之表面積，以便提供一用於諸如厚片膠體等第二次元分 離介質之背襯，然後亦提供一大表面積，而可橫越該表面 積冷卻該第二次元分離介質。 該第一及第二分離區，及理想上亦其相聯之溫度調節 及/或控制室可能方便地提供於二平板之間。該平板可由 在其邊緣密封之玻璃或一類似材料製成，諸如聚二-甲基 丙希酸甲脂或聚碳酸酯。 於根據本發明之一典型裝置中，於使用中該第二分離 介質在分析物移動方向中之長度係於50至500毫米，較 (13) (13)200303421 好係於1 〇 〇及3 5 0毫米之間，更好係於1 5 0及3 2 0毫米之 間，最好大約3 00毫米。該第二分離區在分析物流動之方 向中之長度典型係50至600毫米，較好係於50及400毫 米之間，更好係於6 0及3 5 0毫米之間，最好大約3 0 0毫 米。 · 根據本發明之分離裝置之其他特色可能如同習知一或 - 二次元膠體電泳裝置，例如用於供給流體至該分離區之配 置及橫越他們加上電場。 · 該裝置至少可局部自動地運轉，比方在諸如微處理器 之可程式化控制機構之控制下。此控制機構可特別用於在 適當時機控制該撓性薄片之變形/位移，以允許或防止與 該第一次元分離區流體相通。 根據本發明之一裝置可用於分離各種分析物之混合物 ’諸如蛋白質、縮氨酸、帶電高分子多醣體、合成聚合物 或任何其他能夠於特定蛋白質中電泳分離之化學或生物學 分析物。包含該混合物之樣本應該呈一流體形式，更好係 · 諸如水溶液或懸浮液之液體。於使用該裝置之前，之樣本 製備可爲習知者。 本發明之另一論點提供可施行一項或最好複數電泳分 離之裝置，按照本發明之第一論點，該總成包含至少一個 、較好二或更多個、更好四或六或八或十六或更多電泳裝 · 置。 按照本發明之裝置能允許同時施行複數一次元或二次 元之電泳分離。既然可自動化其每一構成裝置之操作，其 •18- (14) (14)200303421 本身特別適於自動化。該裝置最好包含控制機構，諸如最 好用於個別地操作這些裝置及調節供給至它們之流體、電 力等之微處理器。 現在將只經由範例及參考所附之說明圖面敘述本發明 【實施方式】 下文有相關電泳分離，其中藉著一 IPG膠片施行該第 一次元分離及在一厚片膠體上之第二次元分離（假如可適 用），而橫越該第一及第二分離區加上正交之電場。可使 用本發明實現其他電泳分離技術。 圖1所示電泳裝置可用於進行單一次元或更好二次元 之分離。 該裝置包含一 80微米厚之撓性聚酯薄片1〇1，一膠 體IPG膠片102已形成在該薄片上。該薄片固定在分別標 以1 03,1 04之前後支撐板間之適當位置中。於該薄片1〇】 及該後支撐板1 04之間，一狹窄之後室i 〇 5允許冷卻流體 供給至該薄片之後面，該流體（例如水）係經過入口 i 06 導入及經過出口 1 0 7排出。 該薄片101之另一面具有局部界定一前室108之作用 ，流體可經由該管道109,110導入該前室或由該前室排出 。於該IP G膠片1 02之區域中，一密封墊圈〗〗1係設在該 前板1〇3上。 當該後室1 〇5中之壓力係相當低時，如圖2 A所示， -19- (15) (15)200303421 該IPG膠片不會與該墊圏ill接觸。藉著於該後室ι〇5中 施加一正流體壓力，能迫使該薄片1 0 1與該墊圈1 1 1形成 接觸，如此環繞該IPG膠片（看圖2B )界定一低容量包 圍室。樣本及/或試劑流體（例如包含成像劑，諸如著色 劑）可經由該管道109導入該室，造成該已脫水之IPG膠 ‘ 片鼓脹（圖2C )。可於一受保護及控制之顯微環境中在 · 該IPG膠片上進行一電泳分離。於該分離期間，可經由該 後室1 〇 5輕易地達成該膠片之有效率冷卻。 鲁 爲施行第一次元分離，其需要沿著該IPG膠片之長度 加上一電場。這照慣例係在任一端點使用電極及於它們之 間加上一高電壓所完成。於圖1裝置中，項目1 1 2,1 1 3係 一種電極線，且平行於該IPG膠片之縱軸地延伸橫越該裝 置。管道114，115允許以習知之方式供給緩衝液體至該二 電極，但最好由貯槽（未示出）連續地補充該緩衝液體。 爲了避免受金屬離子之污染，白金線通常係用作該電 極。當施加該電壓時，該水化合膠片之一些成份抵達該電 H 極。爲避免它們干預該膠片之其餘部份，其已知在該電極 及該膠片之間包含一濕氣吸收芯心（通常爲紙片）。達成 相同功能之一方法係顯示在圖3 ，其中與圖1及2所示類 似之零件已給予相同之參考數字。 在對應於該IP G膠片1 02之二端點之位置，圓柱形孔 · 腔120 (典型之橫截面直徑爲2.5毫米）係提供於平板 . 1 03中。於每一該孔腔中倂入一最好用紙製成之多孔塞 1 2 1。在該多孔塞之下係一譬如白金之電極線1 22及用作 -20- (16) (16)200303421 電極緩衝液體之入口及出口之二通口 1 23,1 24。最好藉著 幫浦由一貯槽真空吸入該液體。這有助於防止該膠片被過 多之緩衝液體淹沒。 該液體充滿該孔腔120之其餘部份及滲入該IPG膠片 。於如此做時，其由該電極1 22至該多孔塞1 2 1造成一電 路徑，且亦至與該多孔塞接觸之IPG膠體造成一電路徑。 該緩衝液體不只提供該電接觸，同時也有助於在該膠片之 端點維持 pH値。在該膠片之酸性端點之電極能使用 0.001至0.5莫耳、最好0.005至0.02莫耳之磷酸。在該 鹼性端點之電極能使用一類似莫耳濃度之氫氧化鈉。 當進行電泳時，最好使該緩衝液體慢慢地流動。此流 動有助於移除在該電極所產生之氣泡，及沖走已遷移至該 電極之各種個體。該緩衝液體之流速最好係每分鐘〇. 1至 1 0毫升。 圖4顯示電極配置之另一種形式。再次，與圖1,2 及3類似之零件已標以相同之參考數字。 於圖4配置中，該電極線係與一或多個小金屬管整合 。一金屬管130具有用於緩衝液體入口之作用，且引導其 流動在該多孔塞1 2 1，該第二金屬管1 3 1將過多之液體由 該孔腔1 20排出。該箭頭指示流體於使用中之流動方向。 任一金屬管或該二金屬管可爲金屬及具有一電極之作用。 同理，接合這些金屬管之主體132亦可爲金屬。 假如於該第一次元分離之後隨即進行第二次元分離， 可降低該後室1 〇 5中之壓力，使該薄片1 0 1吸離墊圈1 1 1 -21 - (17) 200303421 (看圖2A)。然後該IPG膠片不再由該前室1〇8之 部份隔絕。造成一聚丙烯醯膠體之試劑能夠以液體之 經由該下入口管道110導入該前室達至一適當之液位 液位可使得其與該IPG膠片接觸或甚至浸入該IPG 。然而，其較佳的是使該第二次元膠體由該IPG膠片 一小量距離，留下一隨後當想要將分析物遷移至該第 離區時可充滿譬如溶化之洋菜之區間孔腔。可經過一 於該室108中且方便地剛好在該IPG膠片102下方之 導入該洋菜，並達至一接觸該IPG膠片或更好浸入該 膠片之液位。 爲有助於該第二分離介質之引導及假如可適用一 該區間孔腔之介質，一或多個液位感測器可納入該裝 一方便之形式係一種光學液位感測器，例如一種經過 之塑形光導將光線導入該相關流體室及偵測由該光導 內部表面反射回來之光線之感測器，該反射之範圍及 係依該區域流體室中所提供流體而定，該區域係該光 延伸進入之區域。 一旦該第二次元液態膠體已凝固，及假如可應用 質、諸如洋菜已導入該區間孔腔及允許其硬化，即可 該第二次元分離，在該IP G膠片上分離之分析物係在 加電場之影響下自由地遷移進入該第二次元膠體。 再次於該第二次元分離期間，能藉著使一冷卻流 過該後室1 〇5而控制該膠體溫度。 於該第二次元分離中之均勻電泳分離要求橫越 其餘 形式 。該 膠片 隔開 二分 提供 入口 IPG 用於 置。 適當 之一 本質 導所 之介 進行 一施 體通 該室 -22- (18) (18)200303421 108中所形成厚片區域之膠體厚度係均勻的。假如未固定 地支撐該薄片1 0 1，則該室1 〇 8知厚度可能有不同變化。 支撐該薄片之一方法係施加一負壓差（相對前室〗〇 8 )， 直至該薄片係堅固地拉抵住該後板1 (H之面。該後板可能 精密地製成平坦狀，然而這將減少該冷卻流體流動於該薄 片區域上方之機會。如此，其可能較佳的是於平板1 〇 4之 內面中提供狹窄之溝槽，及允許該冷卻液體流經它們。該 溝槽係製成具有充分小之間隔，而於薄片1 0 1之各溝槽間 區域及該冷卻液體之間具有足夠之熱耦合作用。 該板104最好係由諸如銘之導熱材料製成。這改善由 該薄片至該冷卻液體之熱流動。該板1 04之導熱率可爲充 分高，以致不需要該液體之冷卻；熱可藉著該面上之散熱 片或其他熱交換裝置之輔助經過該板之厚度散失至在該相 向面上之環境。重要的是該板1 04中之溝槽係狹窄的，以 致該薄片在未被支撐處大致不會變形。溝槽寬度典型可於 〇. 5及3毫米之間。 該薄片101及IPG膠片102典型係用完即丟之項目， 並可單獨地供給至該裝置之其餘部份或與其結合。最好係 當作單一項目供給該薄片及膠片，該單一項目可裝入一包 含如上面所述其餘部份之能再使用之處理卡式匣。 注意該IPG膠片於該第一次元分離期間毋庸必需被包 圍（藉著該薄片101及墊圈111)。其可於放入該系統中 供作電泳分離之前泡入含有樣本之液體中。另一選擇爲在 該裝置中可藉著樣本液體完成該膠片之再成水化物’但不 -23- (19) (19)200303421 須使用一界定密封件11 1。圖5及6中顯示一種適用於此 方法之裝置之一部份。 於此配置中，當控制壓力係施加至該薄片1 0 1時，該 IPG膠片1〇2接觸該前面塊材103之內面。在該接觸區域 內，一溝槽1 4〇係提供於該板1 03之此面中。流體可經由 ， 諸如141，142之一或多個通口進出通過此溝槽。以此方式 . ，樣本液體或試劑可與膠片1 02之至少部份面部形成接觸 ，該面部係泡入該試劑。 φ 既然該膠片典型係可浸透的，該液體可遷移至該膠片 之全部。倘若該膠片之面及該板1 03間之任何間隙爲小的 (例如少於〇 . 3毫米），則該液體可藉著表面張力之作用 保持與該膠片接觸達數小時之時期，而不會有漏損。 於諸如上面所述之裝置中，該板1 03最好係透明的， 以致可觀察到該電泳進展及最後之分離，而不須拆開該裝 置。然而，該膠體中所產生之熱量導致其各面間之一溫差 時可造成一問題；這依序導致電泳分離之差別速率，而於 肇 最後之分離型式中顯示爲各種類之條紋。最好對稱地冷卻 該第二次元膠體以減少此效應。假如該板1 03必須保持透 明，則可加上一冷卻水夾套，如圖7所示裝置，其中一溫 度調節室15〇係提供鄰接該前板103。冷卻液體可引導經 過入口 151及經過出口 152排出。 ， 另一選擇係假如觀看該膠體係可有可無，則該前板 - 1 03可爲設有溝槽之鋁材或類似材料，如上述與該後板 1 04之冷卻有關者。另一新版本係在此使用後一方法，但 -24- (20) (20)200303421 在該冷卻板中包含一小透明窗口，允許觀看一狹窄之膠片 。當沿著一正角於該遷移方向之膠片光學地偵測（例如藉 著附著染料之螢光）各種類之遷移及記錄成分離進程時’ 這可能特別有效用。由此一記錄，其將可能數學地綜合各 種類在一段分離時期之後將如何出現之一合成面積影像。 “ 這可藉著經由超過一膠片之成像而進一步改善及來自該膠 · 片之記錄與時間有相依之關係。 【圖式簡單說明】 圖1係經過根據本發明電泳裝置之縱向剖面圖； 圖2 A52B及2C係經過圖1裝置之各零件之更詳細剖 面圖，其顯示在操作中之不同階段； 圖3係經過根據本發明一裝置零件之一剖面圖，其顯 示另一種電極配置； 圖4係經過根據本發明另一裝置零件之一剖面圖，其 顯示另一種電極配置； Φ 圖5係經過根據本發明另一電泳裝置零件之一剖面圖 圖6係沿著圖5中剖線Vi-vi之一零件剖面圖；及 圖7係經過根據本發明另一裝置零件之一剖面圖。 所有圖式係槪要的。 、 主要元件對照表 101 薄片 -25- (21) 膠片 支撐板 支撐板 後室 入口 出口 前室 管道 管道 墊圏 電極線 電極線 管道 管道 孔腔 多孔塞 電極線 通口 通口 金屬管 金屬管 主體 溝槽 通口 -26- (22) (22)200303421 142 通口 1 50 調節室 15 1 入口 1 52 出口200303421 (1) 发明. Description of the invention [Technical field to which the invention belongs] The present invention relates to an analyte separation system and its application, and particularly to a colloidal electrophoresis system. [Prior art] Colloid electrophoresis is a conventional technique for separating a mixture of analytes. Across a colloid plus an electric field, a mixture in the form of a fluid sample can migrate through the electric field. Under the influence of this electric field, the migration speed of each analyte can depend on various analyte properties, such as molecular weight or isoelectric point. As a result, the analyte is separated along the colloid in the direction of the applied electric field. In the case of protein analytes, this separation operation can typically be accomplished by isocharged coke sets, where a pH (acid-base) 値 gradient causes proteins to be separated according to their isoelectric point (ie, each protein does not have any net charge acid Alkali pH 値). According to the "Fixed pH Gradient" (IPG) technology, the pH Gradient can be incorporated into a gel, such as in the form of a film adhered to an inert substrate, and the protein mixture can be applied to the film. This "IPG film" has long been well known, and various technologies can be used for its preparation, including, for example, US Patent No. 5,993,627, US Patent No. 4,130,470, US Patent No. 5,5 3 4,121, and As described in European Patent No. 0 3 93 47 8. An IPG film may be carried on a film backing such as Gelbond ™ (as in US Pat. No. 5,993,627), in which case the backing layer tends to have a -6- (2) (2) similar to the film itself ) 200303421 size, and slightly larger than the film. When using a separation system, the film and backing layer are located on a separation support such as a flat plate or block. Typically, an IPG film can be purchased in a pre-prepared and dried form, and then rehydrated with the sample fluid before use, so that the sample is sucked into the gel. The processing of the film may involve many samples and / or applications containing reagent fluids such as buffering and washing fluids. During the use of an IPG film for electrophoretic separation, it is necessary to protect the environment from contamination and dehydration. This is typically achieved by dipping the film in silicone oil during the separation process, or at least covering the film with a protective layer of impermeable plastic film. This protection makes handling difficult. It is also necessary to cool an IP G film during the separation process to compensate for the temperature increase caused by the application of electrical fields. Forcing often also presents processing difficulties. Cooling is typically required when the film is loaded into a block made of a thermally conductive material, and then one or more sides of the block are cooled by conventional methods. However, since the electrophoretic separation requires the application of a high electric field, it is necessary that the material in contact with the film is non-conductive, and this limits the achievable thermal conductivity of the support block. The resolution of an electrophoretic separation can be improved by performing two consecutive separation operations. The analyte is initially separated according to the first property, and the mixture thus separated is then applied to another colloid and subjected to an electric field to separate its components according to the second, different property. First in 1975 (O’Farrell, Ph H [1975] J. Biol. Chem. 250: 4007-4021) report this is known as a technique of two-dimensional colloidal electrophoresis. It is generally used to separate mixtures of biological analytes, such as proteins. This "first element" separation can be performed using an IPG film as described above. This “second-dimensional” separation is then typically performed by ordinary thick-film colloidal electrophoresis, where the mobility of the analyte (such as a protein) via the applied electric field depends on the molecular weight and the degree of charge. Before allowing such separated analytes to migrate into and pass through the second colloid, it must be continued until the first meta-separation is completed. This is typically done by bringing the colloid that has undergone the first meta-separation from the second colloid. Make contact. Moreover, many methods are known to achieve this first to second dimension transfer, such as US Patent No. 5,993,627, US Patent No. 6,013,165, European Patent No. 0 3 66 8 97, and European Patent No. 0 8 No. 7 7 24 5 and PCT Patent Application No. PCT / GB02 / 0 1 749 which we are also applying for. [Summary of the Invention] According to a first aspect of the present invention, there is provided an electrophoresis device for separating a mixture of analytes in a fluid sample. The device includes: a primary element separation medium, and the analyte can pass through the separation. Media migration 'The separation medium is carried on one side of a flexible sheet, the surface area of which is greater than the surface area of the contact area between the separation medium and the sheet. A first separation zone defined by the flexible sheet; the device 尙 includes a fluid chamber separated by the first element separation by the flexible sheet, wherein the fluid can remain in contact with one side of the sheet, the -8-(4) (4) 200303421 The surface is opposite to the surface carrying the separation medium; a suitable separation medium is a hydrocolloid, that is, traditionally used in colloid electrophoresis, such as polypropylene. When an electric field is applied across the analyte, it is preferably capable of isoelectrically focusing the analytes. For example, it can take the form of a fixed pΗ 値 gradient (IP G) component 'and insert a pH 値 gradient along one of its dimensions. The separation medium is preferably in the form of an elongated member 'such as a film or a cylinder. The typical size for this colloidal film is less than 0. 1 and 1. 5 mm, preferably 0. 4 and 0. A thickness between 8 mm; a length between 50 and 500 mm, preferably between 1000 and 350 mm, more preferably between 150 and 320 mm, and most preferably about 300 mm ( This is in the direction of movement of the analyte used): and between 2 and 5 mm, preferably 3. 5 mm width. The separation medium should be supported on the flexible sheet and preferably be permanently fixed to the flexible sheet. It can be applied to the sheet by forming it in place or by a separate adhesion process after the medium is manufactured. A method formed in place is to use a mobile nozzle to dispense a mixed colloidal component onto the sheet. As the nozzle advances along the desired orbit of the separation medium, the mixing components are changed to give the desired gradient of fixed pH 値. Another method for forming in place is to coat or dispense a base colloid (such as polypropylene 醯), and then spray immobilized amphoteric ions into the colloid to create the desired gradient. The separation medium may be in a dehydrated form before being used in a separation process. The sheet is preferably made of a material that enhances the adhesion of the separation medium to the sheet -9-(5) (5) 200303421, or a coating with the material. For example, the sheet may be a proprietary colloidal bond ™ type with a coating to which a polypropylene (R) colloid can be covalently bonded. The sheet is ideally flexible enough to have the sealing and cooling functions described below. The sheet should be made of an inert and fluid-impermeable material, such as a suitable synthetic plastic material such as polyester. The preferred sheet thickness is in the range of 20 to 500 microns, more preferably between 25 and 200 microns, and most preferably between 50 and 150 microns. The area of the relevant sheet surface is preferably at least 15 times the area of the contact area between the separation medium and the sheet, more preferably between 20 and 200 times, and most preferably between 30 and 100 times. between. Ideally, it is sufficiently large that it may also function as a backing for a second-dimensional separation medium as described below. Suitable dimensions of the sheet are between 100 by 40 mm and 400 by 600 mm. Supporting primary element separation media such as IPG film on a larger flexible sheet can provide many advantages. It can help handle the separation medium and its positioning within an electrophoresis device. In addition, the sheet can be used as part of a cooling system during an electrophoretic separation performed across the separation medium as described below. The elastic energy of the sheet is used to reversibly isolate one or more fluid-containing regions of the electrophoretic device, as described below, especially the region surrounding the first element separation medium. This elasticity is preferably relatively small by application. The pressure gives the sheet the ability to locally deform or displace. The flexible sheet has the function of at least partially defining a first -10- (6) (6) 200303421 separation zone in the device of the present invention, and the first separation zone contains the first elementary separation medium. It is preferable that the sheet is not supported by any floor or the like in the area of the first separation zone. If the device of the present invention includes a mechanism for applying pressure to one or more regions of the sheet in proximity to the first meta-separation medium to facilitate deformation and / or displacement of the sheet in the region or regions, this may conveniently Reached. The deformation and / or displacement may then cause the sheet to contact a sealing element 'in the device such that the sealing element and the sheet together at least partially define an enclosed, impervious first separation chamber, which at least contains and / or contacts This first element separates a part of the medium. The deformation and / or displacement of the sheet is preferably reversible ' to allow the separation chamber to be closed or opened as desired and when desired. In this context, the term "watertight" encompasses a chamber having one or more fluid inlet or outlet conduits through which fluid can be directed or discharged when desired. The sealing element that comes into contact with the sheet may be, for example, a gasket, or any other area where one of the devices is in impervious contact may be caused by applying pressure to force the sheet into contact with the area. The first separation chamber thus defined preferably has a relatively low capacity, for example between 200 microliters and 2 ml after hydration, or between 1 and 4 times the capacity of the separation medium. Since the first meta-separation medium can be in contact with the required fluid (such as sample fluid, reagent fluid 'wash fluid' buffer, etc., and may also be imaging reagent such as colorant) during the separation process, it is ideally immersed The required fluid, which is particularly suitable for performing the first meta-separation in this enclosed 'low-volume chamber. This protects the separation medium -11-(7) (7) 200303421 without the need for an oil layer or other form of protective barrier. However, the first separation chamber should have a size suitable to allow at least some movement of the fluid around the enclosed first elementary separation medium ' to allow the fluid to be absorbed by the separation medium. Instead of or in addition, the deformation and / or displacement of the sheet can be used to bring the first primary separation medium itself into contact with a sealing element in the device, which together at least partially defines the first A separation room. _ The fluid chamber can function as a control chamber, and a pressurized control fluid can be supplied to the control chamber. Preferably, the control room is at least partially defined by the sheet. Depending on the geometry of the device, the desired deformation / displacement of the sheet can be achieved by applying a positive or negative pressure difference to the control chamber, thereby changing the pressure of the control fluid. This pressure is applied to the side of the sheet facing the conveyance of the separation medium. The flexible sheet may additionally function as a part of the temperature control system in the device of the present invention. The device preferably includes a mechanism for transferring heat back and forth, typically from one or more zones of the sheet. This preferably includes a mechanism for supplying a temperature-regulating fluid to one or more regions of the sheet, preferably to a surface opposite the conveyance of the first element separation medium, and preferably to the surface close to the surface. The separation medium is more preferably located immediately in an area behind the separation medium. Because the sheet is relatively thin and has a larger area than the separation medium, it can be used as an efficient heat transfer medium, especially as the separation. It is an interface between the separation medium and the large-capacity temperature-regulating fluid, so it is very helpful for the heat transfer in and out of the separation medium and any fluids in contact with it. -12- (8) (8) 200303421 The temperature-regulating fluid is typically used to cool the separation medium and the surrounding fluid during the application of an electric field in a separation process. A suitable cooling fluid is water. The device preferably includes a temperature-regulating chamber adjacent to a suitable area of the flexible sheet at a certain position, and preferably in contact with its fluid, and the temperature-regulating fluid can be supplied to the regulating chamber. Thus, the temperature-regulating chamber is preferably bounded at least in part by the flexible sheet. The contact area between the temperature-regulating fluid and the sheet should be as large as possible, and should include the area immediately surrounding the first element separation medium. Thus, ideally, the chamber is in contact with an area of the sheet, and the area of this area is between one hundred and two hundred times larger than the contact area between the separation medium and the sheet. Conveniently, the temperature-regulating chamber has a relatively small depth, such as at 0. Between 2 and 10 mm, and the temperature-regulating fluid preferably flows through the narrow chamber over a large area of the flexible sheet. The fluid chamber may suitably function as a temperature regulating chamber. Preferably, the fluid chamber has the functions of a control chamber and a temperature regulating chamber. In this case, the 'temperature regulating fluid can also be used as the control fluid when supplied under an appropriate pressure. The device of the present invention can be used to perform a single one-dimensional electrophoretic separation. In this case, 'it only needs to include the first-dimensional separation medium and related mechanisms', such as a mechanism, supply for crossing the separation medium plus an electric field. A mechanism for fluid to the separation medium, and a mechanism for adjusting the temperature of the separation medium during the separation process. However, the positioning of the primary element separation medium according to the present invention on a flexible sheet placed in a container is also helpful for secondary element separation. The sheet may have a function of at least partially delimiting a second separation zone within the device, wherein a second separation medium (typically a hydrocolloid such as a polypropylene gel) may be located in the second separation zone in order to perform a second-dimensional electrophoresis Separation. The second separation zone may be separate from or separated from the first separation zone containing the first meta-separation medium (for example, using a removable barrier layer, such as PCT Patent Application PCT / GB 02/01749, it is better to take advantage of the local deformation / displacement of the sheet itself, as described above) ° It is even better that the first and second separation regions can be reversibly isolated from each other ' For example via local deformation / displacement of the sheet. Then, when the first elementary separation medium is sealed in the first separation chamber (that is, the flexible sheet is pressed in the area of the first elementary separation medium as described above), the first elementary separation can be performed. , After which the first chamber can be brought into fluid contact with the second zone (after the sheet pressure is removed) to allow the analyte to migrate from the first separation medium to the second separation medium contained in the second separation zone . In such a device, the fluid chamber preferably allows fluid to remain in contact with the flexible sheet in a region corresponding to both the first and second separation media. In use, when the first meta-separation is achieved, the second separation medium need only be introduced into the second separation zone. It may, for example, be introduced in the form of a water-like liquid, which can then be allowed to solidify into a thick piece of colloid in the device, for example by polymerisation in place. This second separation medium typically takes less than 0. 5 and 2 mm thick thick film colloidal form, preferably less than 0. 8 and 1. Between 8 mm, more preferably about 1. 5 mm thick-therefore also a better depth to this second separation zone. -14- 200303421 do) the second separation medium can be guided so as to contact, or even surround, the first separation medium. It is more convenient, however, 'that it is introduced in such a way that a cavity is left between the first and second separation media, which cavity can be filled with a suitable medium such as agar colloid' to allow the analyte to pass through Move on the second separation medium in a desired time. In both cases, the second separation zone can effectively penetrate into the first separation zone, or at least communicate with the fluid in the first separation zone. This mesoporous cavity is allowed between the first and second separation media. When the analyte advances to the second zone, it is clear that it should be kept in the first separation zone. Analyte separation. For this purpose, the design of the cavity and its use conditions should be selected to minimize distortion of the analyte separation achieved in the first zone, which means that the separation of analytes, particularly along the first zone, is performed. Movement in the direction is minimized. The number of tolerable "drifts" of the analyte depends on the resolution range that can be achieved in the separation medium used; when the analyte traverses the cavity, the movement of the analyte in the relevant bit will ideally cross the ratio. The best can reach the distance with smaller resolution. A colloid that is currently typically available is provided up to about 0. A useful resolution of 5 mm; when using this colloid, the appropriate analyte movement system is less than 0. 5 mm, ideally less than 0. 3 mm. The degree of movement of the analyte in the mesopore can depend on several factors, such as the viscosity of the existing medium or each medium in the cavity, the length of the cavity (in the direction of sample movement), the applied electric field, The pressure gradient applied, and the nature of the analyte itself and therefore its fluidity. These factors, in particular the pressure gradient, can be implemented in sequence by external influences such as temperature, gravity, device movement, and even fluid movement in the connected device -15- (11) (11) 200303421. A suitable medium for the mesoporous cavity is a fluid of considerable viscosity, such as melted agar (at a temperature of, for example, between 50 and 70 degrees Celsius). Suitable fluid viscosities are between 2 and 1000 micronewtons / second / square meter (mPa. s) (at room temperature and room pressure), preferably between 5 and 500 · mPa · s, and more preferably between 5 and 20 mPa · s, such as approximately · 10 mP a · s. Buffer fluids may also be provided, such as those commonly used in colloidal electrophoretic separators. · Roughly speaking, analyte movement can be reduced by reducing the degree of possible fluid movement within the mesoporous cavity. This can, for example, be controlled in sequence by i) filling the cavity with a more viscous fluid, such as by pouring in a gelling agent such as polypropylene tincture or agar; ii) reducing The length of the cavity is preferably to minimize the length of the cavity (in the direction in which the analyte moves through the cavity), a suitable length may be, for example, 0. Between 5 and 5 mm, preferably between 1 and 3 mm, and more preferably about 2 mm; iii) a fluid flow control valve is included near the cavity to control the movement of the fluid, which may be, for example, Due to external influences; and / or iv) the device is installed with a fixed support, again during use-to minimize fluid movement. In order to facilitate the introduction of the second separation medium and if a medium can be applied to the mesoporous cavity, one or more liquid level sensors can be incorporated into the device of the present invention -16- (12) (12) 200303421. A convenient sensor form is an optical liquid level sensor, such as a sensor that guides light into the relevant area through an appropriate shaped light guide and detects light reflected from an internal surface of the light guide. The extent and nature of the reflection depends on the fluid provided in the area, which is the area into which the light guide extends. The fluid chamber may have the function of a control chamber capable of operating the fluid chamber to reversibly isolate the second separation zone in the same manner as described above for the first separation zone. It is also possible to appropriately control the temperature in the second separation region in the same manner as described above in relation to the first element separation, that is, by contacting at least one region of the flexible sheet in the second separation region With a temperature-regulating fluid. Both the first and second separation zones can share the temperature-regulating chamber of the device, and ideally, they come into fluid contact by a ratio (e.g., 90% or more) of area relative to the relevant sheet surface. In the device of the present invention, the flexible sheet preferably has a sufficiently large surface area so as to provide a backing for a second-dimensional separation medium such as a thick piece of colloid, and then also provides a large surface area, which can The second-dimensional separation medium is cooled across the surface area. The first and second separation zones, and ideally their associated temperature regulation and / or control room, may conveniently be provided between the two plates. The plate may be made of glass or a similar material sealed at its edges, such as polydimethyl-methylpropanoate or polycarbonate. In a typical device according to the present invention, in use, the length of the second separation medium in the moving direction of the analyte is 50 to 500 mm, which is better than (13) (13) 200303421 than 100 and 35. Between 0 mm, more preferably between 150 and 32 mm, and most preferably about 300 mm. The length of the second separation zone in the direction of analyte flow is typically 50 to 600 millimeters, preferably between 50 and 400 millimeters, more preferably between 60 and 350 millimeters, and most preferably about 3 millimeters. 0 0 mm. · Other features of the separation device according to the present invention may be similar to conventional one- or two-dimensional colloidal electrophoresis devices, such as a configuration for supplying fluid to the separation zone and applying an electric field across them. · The device can operate at least partially automatically, for example under the control of a programmable control mechanism such as a microprocessor. This control mechanism may be particularly used to control the deformation / displacement of the flexible sheet at an appropriate timing to allow or prevent fluid communication with the first element separation region. An apparatus according to the present invention can be used to separate mixtures of various analytes, such as proteins, peptides, charged polymer polysaccharides, synthetic polymers, or any other chemical or biological analyte capable of being separated by electrophoresis in a specific protein. The sample containing the mixture should be in the form of a fluid, preferably a liquid such as an aqueous solution or suspension. Before using the device, the sample preparation can be known. Another aspect of the present invention provides a device capable of performing one or preferably plural electrophoretic separations. According to the first aspect of the present invention, the assembly contains at least one, preferably two or more, better four or six or eight Or sixteen or more electric swimsuits. The device according to the present invention allows simultaneous electrophoretic separation of plural primary or secondary elements. Since the operation of each of its constituent devices can be automated, its 18- (14) (14) 200303421 itself is particularly suitable for automation. The device preferably includes control mechanisms, such as a microprocessor, which is best used to individually operate these devices and to regulate the fluid, power, etc. supplied to them. The present invention will now be described only by way of example and with reference to the accompanying drawings. [Embodiment] The following is related to electrophoretic separation, in which the first element separation and the second dimension on a thick gel are performed by an IPG film Separate (if applicable), and apply orthogonal electric fields across the first and second separation regions. The present invention can be used to implement other electrophoretic separation techniques. The electrophoretic device shown in Fig. 1 can be used to perform single or better separation. The device contains an 80 micron thick flexible polyester sheet 101, on which a colloidal IPG film 102 has been formed. The sheet is fixed in place between the support plates marked before and after 03, 104 respectively. Between the thin sheet 10] and the rear support plate 104, a narrow rear chamber i05 allows cooling fluid to be supplied to the rear face of the thin sheet. The fluid (for example, water) is introduced through the inlet i06 and passed through the outlet 10 7 evacuated. The other side of the sheet 101 has the function of partially defining a front chamber 108, and fluid can be introduced into the front chamber through the pipes 109, 110 or discharged from the front chamber. In the area of the IP G film 102, a sealing gasket 1 is provided on the front plate 103. When the pressure in the back chamber 105 is relatively low, as shown in FIG. 2A, the IPG film does not come into contact with the pad ill as shown in FIG. 2A. By applying a positive fluid pressure in the back chamber 05, the sheet 101 can be forced into contact with the gasket 1 11, thus defining a low-capacity enclosure around the IPG film (see FIG. 2B). Samples and / or reagent fluids (eg containing imaging agents, such as colorants) can be introduced into the chamber via the tube 109, causing the dehydrated IPG gel's to swell (Figure 2C). An electrophoretic separation can be performed on the IPG film in a protected and controlled microscopic environment. During the separation, efficient cooling of the film can be easily achieved through the back chamber 105. To perform the first meta-separation, Lu needs to add an electric field along the length of the IPG film. This is conventionally done using electrodes at either end and applying a high voltage between them. In the device of Fig. 1, item 1 1 2, 1 1 3 is an electrode wire that extends parallel to the device parallel to the longitudinal axis of the IPG film. The pipes 114, 115 allow the buffer liquid to be supplied to the two electrodes in a conventional manner, but it is preferred that the buffer liquid be continuously replenished from a storage tank (not shown). To avoid contamination by metal ions, platinum wire is usually used as the electrode. When the voltage is applied, some components of the hydrated film reach the H electrode. To prevent them from interfering with the rest of the film, it is known to include a moisture-absorbing core (usually paper) between the electrode and the film. One way to achieve the same function is shown in Figure 3, where parts similar to those shown in Figures 1 and 2 have been given the same reference numerals. At a position corresponding to the end point of the IP G film 102 02, a cylindrical hole · cavity 120 (typical cross-sectional diameter of 2. 5 mm) is provided on the tablet. 1 in 03. A porous plug 1 2 1 preferably made of paper is inserted into each of the holes. Below the porous plug are, for example, platinum electrode wires 1 22 and two ports 1 23, 1 24 which are used as inlets and outlets of -20- (16) (16) 200303421 electrode buffer liquid. The liquid is preferably sucked in by a pump from a storage tank under vacuum. This helps prevent the film from being flooded with too much buffer liquid. The liquid fills the rest of the cavity 120 and penetrates the IPG film. In doing so, it creates an electrical path from the electrode 122 to the porous plug 121, and also to an IPG colloid in contact with the porous plug creates an electrical path. The buffer liquid not only provides the electrical contact, but also helps maintain pH at the end of the film. The electrode at the acidic end of the film can use 0. 001 to 0. 5 moles, preferably 0. 005 to 0. 02 Mor's Phosphoric Acid. The electrode at the basic end point can use a sodium hydroxide with a similar molarity. When performing electrophoresis, it is preferable to allow the buffer liquid to flow slowly. This flow helps to remove air bubbles generated at the electrode and flushes away various individuals that have migrated to the electrode. The flow rate of the buffer liquid is preferably 0. 1 to 10 ml. Figure 4 shows another form of electrode arrangement. Again, parts similar to those in Figures 1, 2, and 3 have been given the same reference numerals. In the configuration of FIG. 4, the electrode line is integrated with one or more small metal tubes. A metal tube 130 has a function of buffering the liquid inlet, and guides it to flow through the porous plug 1 2 1, and the second metal tube 1 3 1 discharges excess liquid from the cavity 120. This arrow indicates the direction of fluid flow in use. Any metal tube or the two metal tubes can be metal and have the function of an electrode. Similarly, the main body 132 joining these metal pipes may be metal. If the second-dimensional separation is performed immediately after the first-dimensional separation, the pressure in the back chamber 105 can be reduced, and the sheet 10 can be sucked away from the gasket 1 1 1 -21-(17) 200303421 (see the figure) 2A). The IPG film is then no longer blocked by a portion of the front room 108. The reagent that creates a polypropylene gel can be introduced into the front chamber through the lower inlet pipe 110 as a liquid to a proper level. The liquid level can make it come into contact with the IPG film or even immerse in the IPG. However, it is preferable to make the second-dimensional colloid a small distance from the IPG film, leaving an interval cavity that can then be filled with, for example, dissolved amaranth, when the analyte is to be migrated to the first separation zone. . The agar can be introduced through the chamber 108 and conveniently just below the IPG film 102, and reach a liquid level that contacts the IPG film or better immerses the film. In order to facilitate the guidance of the second separation medium and if a medium of the interval cavity is applicable, one or more liquid level sensors may be incorporated into the device in a convenient form such as an optical liquid level sensor, such as A shaped light guide that passes light into the relevant fluid chamber and a sensor that detects light reflected back from the inner surface of the light guide. The range and extent of the reflection depends on the fluid provided in the fluid chamber in the area. It is the area into which the light extends. Once the second-dimensional liquid colloid has solidified, and if applicable substances such as agar have been introduced into the cavity of the interval and allowed to harden, the second-dimensional separation can be performed, and the analyte separated on the IP G film is in Freely migrate into the second-dimensional colloid under the influence of the applied electric field. During the second dimensional separation again, the colloid temperature can be controlled by passing a cooling through the back chamber 105. Uniform electrophoretic separation in this second-dimensional separation requires crossing the remaining forms. The film is divided in two to provide an entrance IPG for placement. Appropriately, one of the essence guides is to perform one application. The thickness of the colloidal region formed in the chamber -22- (18) (18) 200303421 108 is uniform. If the sheet 101 is not fixedly supported, the chamber 108 may have different thicknesses. One method of supporting the sheet is to apply a negative pressure difference (relative to the front chamber) 08 until the sheet is firmly pulled against the face of the back plate 1 (H. The back plate may be precisely flat, However this will reduce the chance that the cooling fluid will flow over the lamella area. As such, it may be preferable to provide narrow grooves in the inner surface of the plate 104 and allow the cooling liquid to flow through them. The groove The grooves are made with sufficiently small intervals, and there is sufficient thermal coupling between the regions between the grooves of the sheet 101 and the cooling liquid. The plate 104 is preferably made of a thermally conductive material such as Ming. This improves the heat flow from the sheet to the cooling liquid. The thermal conductivity of the plate 104 can be sufficiently high that cooling of the liquid is not required; heat can be assisted by heat sinks on the surface or other heat exchange devices Passes through the thickness of the plate to the environment on the opposite side. It is important that the grooves in the plate 104 are narrow so that the sheet is not substantially deformed when unsupported. The width of the groove is typically less than . 5 and 3 mm. The sheet 101 and the IPG film 102 are typically disposable items, and can be supplied separately to the rest of the device or combined with it. Preferably, the sheets and films are supplied as a single item which can be loaded into a reusable process cartridge containing the remainder as described above. Note that the IPG film need not be enclosed during the first meta-separation (by the sheet 101 and the gasket 111). It can be bubbled into a sample-containing liquid before being placed in the system for electrophoretic separation. Another option is that the film can be rehydrated by the sample liquid in the device 'but not -23- (19) (19) 200303421 A definitive seal 11 1 must be used. Figures 5 and 6 show part of a device suitable for this method. In this configuration, when a control pressure is applied to the sheet 101, the IPG film 102 contacts the inner surface of the front block 103. In the contact area, a groove 1440 is provided in this side of the board 103. Fluid can enter and exit through this channel through one or more ports such as 141,142. In this way. The sample liquid or reagent can come into contact with at least part of the face of the film 102, and the face is soaked with the reagent. φ Since the film is typically permeable, the liquid can migrate to all of the film. If any gap between the face of the film and the plate 103 is small (e.g. less than 0. 3 mm), the liquid can be kept in contact with the film for a period of several hours by surface tension without leakage. In devices such as those described above, the plate 103 is preferably transparent so that the progress and final separation of the electrophoresis can be observed without disassembling the device. However, the heat generated in the colloid can cause a problem when it causes a temperature difference between its sides; this in turn results in a different rate of electrophoretic separation, which appears as various types of stripes in the last separation pattern. It is best to cool the second-dimensional colloid symmetrically to reduce this effect. If the plate 103 must be kept transparent, a cooling water jacket can be added, as shown in Fig. 7, where a temperature adjustment chamber 15 is provided adjacent to the front plate 103. The cooling liquid can be discharged through the inlet 151 and through the outlet 152. Another option is that if the glue system is optional, the front panel-03 may be an aluminum or similar material with grooves, such as those mentioned above related to the cooling of the rear panel. Another new version uses the latter method here, but -24- (20) (20) 200303421 includes a small transparent window in the cooling plate to allow viewing of a narrow film. This can be particularly useful when the various types of migration are optically detected (e.g., by fluorescent light attached to the dye) and recorded as a separation process along the film at a positive angle in the direction of migration '. From this record, it will be possible to mathematically synthesize a composite area image of how each category will appear after a period of separation. "This can be further improved by imaging through more than one film and the recording from the film has a time-dependent relationship. [Brief Description of the Drawings] Figure 1 is a longitudinal sectional view of an electrophoresis device according to the present invention; 2 A52B and 2C are more detailed cross-sectional views of various parts of the device of FIG. 1, which are shown at different stages in operation; FIG. 3 is a cross-sectional view of one of the device parts according to the present invention, which shows another electrode configuration; 4 is a cross-sectional view of another part of the device according to the present invention, which shows another electrode configuration; Φ FIG. 5 is a cross-sectional view of a part of another electrophoretic device according to the present invention; FIG. 6 is a section line Vi along FIG. 5. -vi is a cross-sectional view of a part; and Fig. 7 is a cross-sectional view passing through one of the parts of another device according to the present invention. All the diagrams are essential. Table of main components 101 Sheet-25- (21) Film support plate Support plate rear chamber entrance and exit front chamber pipe piping pad electrode wire electrode wire pipe pipe cavity porous plug electrode wire port opening metal pipe metal pipe body groove opening -26- (22) (22) 200303 421 142 Port 1 50 Room 15 1 Entrance 1 52 Exit
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