200526960 九、發明說明： 【發明所屬之技術領域】 本發明係關於一種使用均一且 層合物之方味^^ &里的試劑浸染膜或膜 層口物之方法，在特殊態樣中，本 'h Θ係關於製備会p、、君 刮的臈層合物，其中該等試劑受限於該層合物之單： 的個別層’且本發明係關於此 ^ 包含其之分析裳置。 …之胰及層合物以及 【先前技術】 由於便利性、速度及減少了 、 史用者對试劑進行操作的需 求，所以普遍使用運用/基# 士久 絲格式之診斷分析，其中使用診 斷试劑來浸染諸如纖維條或膜之 夕孔材科。在製造此等裝 置時，或劑之均一且可複現之塗霜 λΑ , ^ 兄l 土復很重要以確保測試結果 的一致性及準確性。 好的控制’因而可減少分散至層合物中下面層的風險。然 而，藉由此等方法所產生的塗層亦趨向於不均一，從而產 生在塗覆區域之中央具有較多試劑的濃度梯度。 如EIM 329 724 A2及DE 102 04 606中所述，另一種將試 已運用了多種方法以將試劑塗覆於此等測試條。舉例而 言，可僅藉由使膜接觸一試劑溶液即可使多孔膜浸染滿該 武劑。為達成均-浸染，通f使用飽和量的溶液。然而， 此方法通常會產生試劑之不均—分佈。具體而纟，對於層 合式膜而言是不滿意的，因為其通常不會限制試劑分散至 單個層，特別是若該等層具有相似材料時。將試劑喷塗或 注射塗佈至膜或層合物表面可提供對所塗覆之試劑的量更 95372.doc 200526960 劑塗覆於-層合物中之不同層的方法為獨立地塗佈或浸染 該等層，緊接著對其進行層厂堅。上述文獻揭示了包含一試 劑^及-HDL-反應膜之高密度脂蛋白⑽l)分析裝置。可 错由以-定分配率將包含硫酸葡聚糖之水溶液分配於不對 稱膜上而產生該試劑墊。使 私…九咖 至使用相同的程序，使用含水調配 勿π木HDL反應膜。為完成上述分析 接來單獨地或同時附著兩個經浸染的膜。3… 轨：二缺點疋此超音波層合物或熱層合物可能不適用於 :::導另外，層之間使用黏合劑可減少對加熱之需 卢亦;=析化學的可能污染。保持多孔膜之孔隙 度亦很重要，且任-此等技術在此態樣中皆有宝。 膜==技術問題係提供一種用以均-塗覆試劑 膜之方法且具體而言係分離一層合物中的膜，而 之：產生交又污染’且不會將試劑曝露於過熱下。理：地： 可：一地將受控用量之試劑塗覆於此層合物的-側 戍兩側。本發明之另-問題係提供一種包括均 的分析袈置以判定可靠的測試結果。 【發明内容】 在-態樣中，本發明提供了 一種將一試劑 :-層合式膜及第二層合式膜的膜層合物中之」：： 法。該方法包括·· Μ的方 2供—具有第—膜及第：膜之層合物，其具有 *、表面、一層合式第一臈表面、一 及-外部第二膜表面； 曰口式弟〜膜表面 95372.doc 200526960 (b)藉由使該外邱楚 之表面的支撐件，將；:表面接觸一具有—包含-圖案 面，及 又工用置的第一試劑溶液塗覆於該表 (C)乾燥该層合物中的第一膜， 產生-包含浸染有第_試劑之第—膜的 第：試劑不接觸該層合物内之第二膜。 … 較佳:也4方法藉由一包括填充有該溶液的凹槽之圖 7 2 1由匕括塗覆該溶液的突出物的圖案來塗覆該溶 液:另外’該切件較佳包括—平面或圓柱形狀。 車乂佳地，該方法進—步包括使用第二試#j來㈣b 2此藉由以y步驟達成：⑷將受控用量之第二試劑溶液 土後於5亥外部第二膜表面，此藉由使該表面接觸一包含填 2該溶液的凹槽的固體支撐件而達成，及⑷乾燥該層合 中的苐二膜以產生—包含被第：試劑浸 合物其中該第二試劑不接觸層合物中的第一膜广曰 通常’固體支撐件為一旋轉凹版印刷式圓柱體，其具有 形成於表面中之複數個凹槽或單元，如下文所論述了較佳 地’該塗覆同時發生於第一膜外部表面之整個寬度上，以 在該膜寬度上產生均一的塗覆。 關於個別膜的吸收能力，”受控用量”的溶液通常僅為〆 飽和量的溶液，且通常為不完全飽和量，如下文所界定。 在某些實施例中，一給定(第一或第二)試劑不接觸所完成之 層合物内的個別膜的層合表面；意即’其僅部分地渗透穿 過個別膜的深度。在其它實施例中，—給定(第一或第二） 95372.doc 200526960 試!1接觸所完成之層合物内的個別膜的層合表面；音即， 其次染個別膜的全部厚度，但 在士 -不接觸層合物中的其它膜。 在列中，至少第-膜為-不對稱膜，音即，且有 一小的有孔表面及一大的有孔 ^ 〃、有 稱膜，在所選之實施例中，外㈣可為-不對 的有孔表面。在另一，J…膜表面為第-膜之大 夕丨_ a财，外部第二膜表面為第二膜 ^有孔表面。圖1說明了此實施例，該圖展示了 I㈣ ㈣層合物10’其具有外部第-膜表面 _膜表面22^5表面18、層合式第項表面2G及外部第 开 T:: 式第一膜表面及層合式第二膜表面結合 形成一層合物界面。 至少-種試劑可為熱感應試劑，使得將包含該試劑之膜 :合至另一膜將在試劑中產生有害變化。在-實施例中，、 劑包括能有效用以自流過第一膜的企液流體樣品選 ^生地f除非HDL脂蛋白的試劑，且第二試劑包括能有效 生與弟—膜内之HDL相關膽固醇之量成比例的視覺上可 该測到的訊號的試劑。 才關:樣中，本發明提供了一種將一試劑塗覆至膜的 藉由將叉控用1之該試劑溶液塗覆至膜的第一表 面，藉由使該表面接觸一具有一包含填充有該溶液的凹槽 ^圖案的表面之平面或圓柱體支撐件來達成。並且，該支 擇件通吊為一旋轉凹版印刷式圓柱體。另外，較佳使用描 述用於將β式劑塗覆至一膜層合物内之膜的技術及設備。 工里的5式劑可為不完全飽和量。在一實施例中，在塗 95372.doc 200526960 後了 且較佳地乾燥之後n刺不接觸該膜的相反表 -即，其僅部分地滲透穿過膜的深度。試劑之塗 ，同時發生於膜的第—表面之整個寬度。因此，較佳地，乂 試劑濃度在該膜的長度及寬度上係均-的。 膜可為一不對稱膜，其具有一小的有孔表面及一大的有 孔表面；實施例中，塗覆有試劑的第—表面為大的有 二在其它實施例中，第一表面為小的有孔表面。在 法較佳貫施例中，試劑包括能有效用以自流過該膜的血液 流體樣品中選擇性地移除非祖脂蛋白的試劑；或者，試 劑包括能有效用以選擇性地沈«品中的非hdl(ldl: VLDL)脂蛋白微粒的試劑，且藉由過濾來防止經沈澱之 L士DL及几沉微粒流過膜。在此實施例中，當膜為不對稱膜 /將„式劑塗覆至大的有孔表面。在另一較佳實施例中， 试劑包括能有效用以產生與膜内之HDL相關膽固醇之量成 比例的視覺上可偵测到的訊號的試劑。在此實施例中，當 膜為不對稱膜時，將試劑塗覆至小的有孔表面。 在另心樣中，本發明提供了一種用於診斷分析之膜層 合物’其包括在一層合物界面處結合的第一層合式膜及第 -層合式膜，其中第一試劑浸染該層合物内的第一膜，第 -试劑浸染第：膜’且至少—個財的試劑不存在於與該 層合物界面相鄰膜之層合區域中，使得可由至少該層合區 域來物理分離兩個膜内的試劑。 幸乂佳地，# 5式劑或試劑系統之濃度在個別膜之長度及 寬度上是均一的。 95372.doc 200526960 在所選實施例中’至少-種該試劑為熱感應試劑，使得 將-包含該試劑之膜層合至另一膜將在試劑中產生有害變 化。 較佳地，至少第一膜為不對稱膜，其具有一小的有孔表 面及-大的有孔表面。在所選實施例中，該小的有孔表面 面對該層合物界面。第二膜亦可為不對稱膜；在所選實施 例中’其大的有孔表面面對該層合物界面。 較佳實施例中，第一試劑包括能有效用以自流過該 第一膜的血液流體樣品中選擇性地移除非hdl脂蛋白的試 劑；或者’第-試劑包括能有效用以選擇性地沈殺樣品中 的非HDL(LDL及VLDL)脂蛋白微粒的試劑，且藉由過濾來 防止經沈澱之LDL及VLDL微粒流過層合物界面。在此等實 施例中’第二試劑包括能有效用以產生與第二膜内之 相關膽固醇之量成比例的視覺上可偵測到的訊號的試劑。 較佳地，在此層合物中，第二膜内之試劑不存在於與層合 界面相鄰膜之層合區域中。 在一相關態樣中’本發明提供了一種用以分析血液或血 清樣品中之HDL的分析裝置，其包括： (a)—基板，其具有一用於接收樣品之等分試樣的樣品接 收井， 、（b)膜層&物，其包括在層合物界面處結合的第一層合 式膜及第二層合式膜，其中 (!)使用能有效用以選擇性地沈澱樣品中2Ldl&vldl 脂蛋白微粒的言式劑來浸染第一m ’且藉由過渡來防止經過 95372.doc -10- 200526960 濾之LDL及VLDL·微粒流過層合物界面； ⑻使用能有效產生與第二膜内之hdl相關膽固醇之量 成比例的視覺上可偵測到的訊號的試劑來浸染第二膜；且 (111)至夕個膜(且車乂佳為第二膜)内的試劑不存在於與 層合物界面相鄰膜的層合區域中，使得可由至少該層合區 域來物理分離兩個膜内的試劑。 曰σ 如上所述’在層合物中， 曰口奶甲，較佳地，每一試劑之濃度在膜 之長度及寬度上是均一的。該裝置亦包括⑷一位於樣品接 收井與層合物之第一膜之間的過濾器，其用於當樣品自樣 品接收井遷移至層合物時移除該樣品中之紅血細胞，其中 井、過濾、器及層合物係以或可以彼此進行流體交流的 放置。 當結合隨附圖式來閱讀本發明之如下詳細描述時，本發 明之此等及其它目的與特徵將變得更加顯而易見。 【實施方式】 I·试劑塗覆方法 本么月提供了使用至少一種試劑來浸染或塗覆一具有至 少兩個膜的層合物的方法，使得每—試劑均被限定在其個 別膜中。較佳地，該層合物包括至少兩個膜，#中每一膜 包含不同的試劑或試劑系統。 _此層合物之一實例為一用於一裝置中的膜總成，該裝置 又1·以用於里測血液樣品（其亦包含Ldi^几沉微粒）中 之職相關膽固醇之濃度。舉例而[可參考仂⑽等人之 美國申請公開案第細則66291號及美國申請案序列號第 95372.doc 200526960 1 0/410,671號，其以引用的方式倂人本文 '然而，本發明適 用於運用如上所述之層合物的任一應用，其中在至少一個 膜層且較佳為兩個膜層中浸染試劑，#中每—試劑必須被 限定在其個別層中。 、如下文進一步所述之例不性HDL分析層合物包括hdl測 試膜(其中量測祖濃度)及—包含結合及/或沈澱試劑的試 d膜，使知在樣品接觸測試膜之前，該膜能有效地自流體 樣品中選擇性地移除非HDL脂蛋白。 較佳地’層合物中的每一膜為多孔不對稱膜；意即，為 -沿在其厚度具有孔徑大小梯度的膜。包括兩個不對稱膜 12與14的例示性二膜層1〇展示於圖丨的橫截面圖中，其展示 了較佳定向’且在膜12中之24處具有較大的孔而在％處具 有較小的孔。 為了使HDL分析能準確地發揮作用，HDU賴試劑必須 受限在其個別層中4HDL試劑存在於試劑層巾，則分析 :應中所產生的顏色除了分析物、咖相關膽固醇之外通 常將代表某一部分非HDL膽固醇。 申請人已發現一凹版印刷式印刷程序，其中自具有平面 或任意形狀之固體板或通常旋轉圓柱體之凹槽將：體塗覆 在基板上、允許將不同試劑之受控用量沈積至層合式膜之 相對表面上。藉由控制沈㈣量，每—試财被限制在層 合物中其個別層内。 藉由活版印刷技術將試 為此’支撐件具有平面 根據本發明之另一較佳實施例 劑塗覆至分離的膜或膜層合物上 95372.doc 200526960 或 < 〜適且之構型’較佳為圓柱體構型。該支撐件包 =將忒劑塗覆至膜的突出物之圖案。較佳_，所提供的該 等试劑具有黏稠性可暫時地黏附在該等突出物 以受控方式塗覆。 /、此 在凹版印刷式塗佈中，通常將為緊m隔單^的凹以 之網耗刻於金屬圓柱體表面上。該等單元負载有流體处 且該旋轉圓柱體將該流體轉移至基板。該等單元具有一 _ 界定之體積，因而限制了可被轉移至基板的液體量、。通常坐 圓柱體的體積約為每平方英吋圓柱體表面3〇_5〇 ^(約4.5_8 W /cm2)。（就此而言，”圓柱體表面，，假定為一平滑表面，其 對應於圓柱體所接觸的基板之表面面積。其不包括凹槽自' 身所提供的額外表面面積。）亦可使用其它體積容量。該等 凹槽通常具有均一尺寸且緊密間隔，使得液滴一經轉移至 膜，其便聚合進入均一液體層。 圖2中之30處展示了凹版印刷式印刷系統，其可用於執行 本文所描述的程序。（如本文所使用，”塗佈”一膜包括塗覆 一試劑，使得其滲透超出表面且可浸染膜之整個厚度广f 自捲筒34饋入層合式膜或”網膜”32。凹版印刷式圓柱體 36通常為銅或鍍銅鋼或铭；通常塗覆一細薄層鉻以達成耐 用性。如上所述，圓柱體表面蝕刻有能有效用以固持一界 定量的液體之複數個小單元。選擇具有若干井之圖案的圓 柱體，該圓柱體可藉由給定塗佈系統來有效地塗覆均一塗 層。該圓柱體可部分地浸沒於待塗覆之流體浴槽或流體盤 内。或者’作為本方法之車义佳貫例’試劑腔室3 8可用於妙 95372.doc -13 - 200526960 由吕C 40將減劑〉谷液傳遞至圓柱體。該腔室可減少試劑曝 露至空氣。 … 。當圓柱體轉動時，藉由在試劑腔室或盤與膜”之間接觸 圓柱體的可撓性刮漿刀41將過量溶液自圓柱體擦除。接著 將殘留細曹式單元内的溶液轉移至所要的膜表面。 在貝施例中，藉由張緊圓柱體42使膜32保持與凹版印 刷式圓柱體切向接觸（意即，接觸圓柱體圓周的一些角度）， 該程序稱為，，親吻”凹版印刷。或I，網膜可在凹版印刷式 圓柱體36與壓印或”備份”圓柱體之間通過，如42,處所示。 在此程序中，通常不存在張緊圓柱體42。在一平凹版印刷 程序（未圖示）中，將流體自凹版印刷式圓柱體轉移至經橡膠 塗佈的轉移捲筒，接著該捲筒將溶液塗覆至基板。 接著使基板通過乾燥器44，較佳為IR乾燥吹風器，且使 其在張緊捲筒46上張緊。接著以相似方式塗佈層合物的另 一表面。 以直接模式可執行凹版印刷式塗佈程序，其中圓柱體以 與膜饋入方向相同的方向旋轉。或者，在反向凹版印刷中， 如圖2中所說明，圓柱體之旋轉方向與膜之行進方向相反。 此配置導致當溶液被轉移至基板時會產生施加給該溶液的 剪切力，此導致塗層更光滑。 如上所述，當膜接觸凹版印刷捲筒時，塗覆至該膜的試 劑溶液之用量受到圓柱體表面上之單元體積的限制。實際 自單元轉移至膜的量視諸如圓柱體速度、捲筒壓力、網膜 速度及溶液組份之額外因數而定。因此，轉移至膜的量可 95372.doc -14- 200526960 進步藉由適當選擇系統構型、膜與圓柱體之間的接觸時 間（由相對於凹版印刷旋轉速度及方向的網膜速度及方向 判疋）及界面活性劑含量來控制。包含了界面活性劑以有助 於將命液轉移至膜表面，否則可排斥水溶液，此視膜材料 之疏水性而定。 對於不對稱膜而言，試劑滲透進人膜亦視孔徑大小及分 佈而定。例如，參看,，與塗覆至表面22(小孔隙）的溶液 相比塗覆至表面16(大孔隙）的溶液可藉由毛細作用被牵引 至膜的程度顯著更大。 因此’將党控用量的試劑溶液塗覆至位於層合物外部表 之膜的^，接著乾燥，可產生層合物，其中試劑不 ::超：該膜。受控量可為不完全飽和量；意即，低於若 :後至選定膜便會藉由毛細流動而滲透整個膜的溶液用 里。在一些狀況中，可使用飽和或接近飽和的用量。 根據本發明，將-定量試劑溶液塗覆至膜 r度達所要的量。藉由在職度上同時塗覆受控量t 产f塗覆方法亦可在膜長度及寬度上提供試劑之均-漠 又。均一”意指在膜長度及寬声上、力古— 變化及冤度上-有顯者的濃度梯度或 之不V 性可有效地為樣品給出出現在層合物表面上 1不同點的-致分析讀數。較佳地，繼塗覆試劑及 後’出現於層合物中之膜夺 /、之 面的終不同點處或平行於此表 ^層合表面上之不同點處的試劑量變化僅為約5%。 下：：:：適用於熱感應試劑，其中在正常熱層合條件 射包含試劑之膜進行声人脾户 仃層口將在層合物上所執行的分析 95372.doc 200526960 :對心]及/或其功能產生有害改變。此改變可包括試劑形 態或分佈的改變以及試劑之化學改變。 該塗覆方法亦可有利地用於以均—方式將一受控量的試 d土後至早個膜’此藉由使膜表面與具有_包含填充有溶 液的凹槽之圖案的表面之平面或圓柱形支撐件接觸而達 成。並且，如上所述，支撑件通常為旋轉凹版印刷式圓柱 體。在層合物塗佈時，藉由此方法來塗覆試劑較佳在膜第 面的整個寬度上同時發生；因而，試劑濃度在膜長度 及见度上大體上是均一的。 試劑的受控量可為不完全飽和量。在—實施例中，繼塗 覆試劑及較佳地乾燥之後，試劑不接觸膜的相反表面；意 即，其僅部分地滲透穿過膜深度。 4方法甚至在塗覆了飽和量時亦有利，因為其與諸如浸 沒在飽和量的溶液或喷塗之先前技術之方法相比產生了盆 中試劑被浸染得更均一的膜。 /' 、本發明亦提供了根據本方法浸染有試劑的膜。尤為使人 感興趣的是用於診斷分析的膜。 膜可為不對稱臈，其具有一小的有孔表面及一大的有孔 表面’可將试劑塗覆至任一表面。在一較佳實施例中，試 劑包括能有效地用以自流過膜的血液流體樣品中選擇性地 移除非咖脂蛋白的試劑；或者，試劑包括能有效地用以 選擇性地賴樣品中的非舰(既及vldl)脂蛋白微粒的 試劑’且藉由過濾、來防止經沈殿之咖及凡沉微粒流過 膜。在此等實施例中’當膜為不對稱膜時，較佳將試劑塗 95372.doc 200526960 覆至大的有孔表面。在另-較佳實施例中，試劑包括能有 效地用以產生與膜内之脈相關膽固醇的量成比例的視覺 上可偵測道的訊號的試劑。在此實施射，t膜為不對稱 膜日守’較佳將遠試劑塗覆至小的有孔表面。 II.膜層合物 在另一態樣中，本發明提供具有浸染於至少一個較佳為 兩個膜層中的試劑的層合式膜，其中每一試劑(或試劑系統) 被限制至其個別層。通常，T同層包含不同的試劑系統。 較佳地，根據上述方法將試劑塗覆至層合式臈。尤為使人 感興趣的是用於診斷分析的膜層合物。 此層合物包括分別浸染有第一試劑及第二試劑的第一層 合式膜及第二層合式膜，其在層合物界面處結合。第一試 劑及第二試劑中的至少_個試劑不存在於與層合物界面相 鄰的個別膜的層合區域中，使得藉由至少該層合區域來物 理分離兩個膜内的試劑。 較佳地，每一該試劑之濃度在包含該試劑的個別膜之長 度及寬度上是均一的。此均一性可有效地為樣品給出出現 在層合物表面上之不同點的一致分析讀數。較佳地，出現 於層合物中之膜表面上之不同點處或平行於此表面的給定 層合表面上之不同點處的試劑量變化僅為約5%。 在一實Μ例中，層合物為一設計用於判定流體樣品中 HDL相關膽固醇的含量。舉例而言，可參考上述j〇nes等人 之美國申請公開案第20030166291號及美國申請案序列號 第10/410,671號。在此情形中，第一膜包括能有效地用於自 95372.doc 200526960 通過膜的血液流體樣品中選擇性地移除非概脂蛋白的試 劑。舉例而言，可藉由選擇性地結合至包含或岐於膜内 的試劑來移除非祖脂蛋白。或者或此外，藉由由試劑來 進行選擇性的沈爾除此等組份，且藉由過濾來防止此 等組份通過層合物界面而進入第二膜 此等試劑包括與諸如Mg“、Mn2+、*〜+之族陽離子 結合的多陰離子化合物’諸如磺化多糖、肝素或磷鶴酸鹽。 較佳試劑為與乙酸鎮或氯化鎂結合的具有分子量為 KDa的續化m諸如硫酸葡聚糖’可對其進行選擇性地 緩衝以維持中性pH。亦可將試劑固定至膜。 如此項技術中所熟知，HDL測試臈包含用於量化hdl相 關膽固醇的試劑。例如，可參見共同擁有的美國專利第 :，213,964號、第 5,213,965號、第 5,316，196號與第 5,451，37〇 號，其中每一專利皆以引用的方式倂入本文中。通常，分 析試劑包括用於自HDL釋放自由膽固醇的膽固醇酯酶、= 於藉由與自由膽固醇反應而產生Hah的膽固醇氧化酶、過 氧化物酶及一經耦合之染色系統，該染色系統可在過氧化 物酶及H2〇2之存在下被轉換成經不同著色之訊號反應產 物。已知在此項技術中分析試劑用於量化其它血液組份， 例如，總膽㈣、甘油三酷或葡萄糖，且其經常包括類似 之酶/經耦合之染色系統。 較佳地，至少第一膜為不對稱膜，其具有一小的有孔表 面及一大的有孔表面。該小的有孔表面較佳面對層合物界 面，使得大的有孔表面位於層合物的外部表面上。此定向 95372.doc -18- 200526960 允許樣品通過較大的孔隙自由進入膜内，且能防止任一經 沈㈣其它㈣材料通過較小孔隙。第二膜亦可為不對^ 膜，較佳地使其大的有孔表面面對層合物界面，且使小的 有孔表面位於層合物之另一表面上^此定向提供了更均一 的膜表面以用於光學掃描及量化分析結果。 舉例而言，不對稱膜的製備描述於：美國專利第4,629,563 號、第 5，171，445 號、第 5,886,059 號、第 5,536,408 號、第 5,562,826 號、及第 4,774，192 號中；ACS 座談會 269 : 1-21(1985)之 D· R· Lloyd的”Materials Science 〇f Synthetic200526960 IX. Description of the invention: [Technical field to which the invention belongs] The present invention relates to a method for dipping a membrane or a membrane layer using a uniform and laminated recipe ^^ & in a special aspect, The 'h Θ is related to the preparation of the plutonium and plutonium laminates, in which these reagents are limited to the single layer of the laminate: the individual layers' and the present invention is related to this . … Of pancreas and laminates and [prior art] Due to the convenience, speed, and reduction, the user needs to operate the reagents, so the diagnostic analysis using the / basic # Shijisi format is commonly used, which uses diagnostics Reagents to impregnate such materials as fiber strips or membranes. In the manufacture of such devices, the uniform and reproducible cream λΑ, ^ ^ Du Fu is important to ensure the consistency and accuracy of the test results. Good control 'thus reduces the risk of dispersion to the lower layers in the laminate. However, the coatings produced by these methods also tend to be non-uniform, resulting in a concentration gradient with more reagents in the center of the coating area. As described in EIM 329 724 A2 and DE 102 04 606, another method has been used to apply reagents to these test strips. For example, the porous membrane can be impregnated with the military agent simply by contacting the membrane with a reagent solution. To achieve homo-dip, a saturated amount of solution is used. However, this method often produces a non-uniform-distribution of reagents. Specifically, it is unsatisfactory for laminated membranes because it usually does not restrict the dispersion of reagents to individual layers, especially if the layers have similar materials. Spraying or injecting reagents onto the surface of a film or laminate can provide more amount of reagents to be applied. 95372.doc 200526960 Agents are applied to different layers of the laminate by independent coating or These layers are impregnated and then layer-hardened. The above document discloses a high-density lipoprotein ⑽1) analysis device comprising a reagent and a -HDL-reactive membrane. This reagent pad can be produced by dispensing an aqueous solution containing dextran sulfate on an asymmetric membrane at a fixed distribution ratio. Use private ... Nine coffees to use the same procedure, use water to prepare the HDL reaction membrane. To complete the above analysis, two impregnated membranes were attached individually or simultaneously. 3 ... Rail: Two disadvantages: This ultrasonic or thermal laminate may not be suitable for ::: guide. In addition, the use of adhesives between layers can reduce the need for heating. Lu Yi; = possible pollution of analytical chemistry. It is also important to maintain the porosity of the porous membrane, and any of these techniques are valuable in this way. Membrane == Technical problem is to provide a method for uniformly coating a reagent membrane and specifically to separate the membrane from a layer of composition, and that: cross-contamination is generated 'and the reagent is not exposed to overheating. Physics: Ground: You can: Apply a controlled amount of reagent to the -side and the side of the laminate in one place. Another aspect of the present invention is to provide a homogeneous analysis set to determine a reliable test result. [Summary of the Invention] In the aspect, the present invention provides a method of combining a reagent:-a laminated film and a laminated film of a second laminated film. The method includes: · The formula 2 of the M—a laminate having a first film and a first film, which has a surface, a first-layer composite surface, and a second external film surface; ~ Membrane surface 95372.doc 200526960 (b) By making the outer surface of the surface of the support member, the: surface contact with a-including-pattern surface, and the first reagent solution applied to the Table (C) The first film in the laminate was dried to produce the first film containing the first reagent-impregnated film: the reagent did not contact the second film in the laminate. … Better: Also 4 methods apply the solution by a pattern including protrusions that fill the solution with the solution 7 2 1 coating the solution with a pattern of protrusions: In addition, 'the cut piece preferably includes- Flat or cylindrical shape. In a good way, the method further includes using a second test #j to b 2 This is achieved by step y: ⑷ a controlled amount of the second reagent solution is placed on the outer surface of the second membrane, and Achieved by contacting the surface with a solid support containing a groove filled with the solution, and drying the second membrane in the laminate to produce—including the first reagent reagent in which the second reagent does not The first film in the contact laminate is generally called 'the solid support is a rotary gravure printing cylinder having a plurality of grooves or cells formed in the surface, as discussed below preferably' the coating Covering occurs simultaneously over the entire width of the outer surface of the first film to produce a uniform coating over the width of the film. With regard to the absorptive capacity of individual membranes, a "controlled amount" solution is usually only a saturated amount solution, and is usually an incompletely saturated amount, as defined below. In certain embodiments, a given (first or second) agent does not contact the laminated surfaces of individual membranes within the finished laminate; that is, ' it only partially penetrates through the depth of the individual membranes. In other embodiments, given a (first or second) 95372.doc 200526960 test! 1 contact the laminated surface of the individual film within the completed laminate; that is, the second is to dye the entire thickness of the individual film, But the other films in the laminate are not contacted. In the column, at least the -membrane is -asymmetric membrane, that is, there is a small perforated surface and a large perforated ^ 〃, a nominal membrane. In the selected embodiment, the outer diaphragm may be- Incorrect perforated surface. On the other hand, J ... the film surface is the first film, and the outer second film surface is the second film. FIG. 1 illustrates this embodiment, which shows an I㈣ ㈣ laminate 10 ′ having an outer first film surface_a film surface 22 ^ 5 surface 18, a laminated surface 2G, and an external surface T :: A film surface and a laminated second film surface are combined to form a single-layer interface. At least one of the reagents can be a heat-sensing reagent, such that combining a film containing the reagent with another film will produce a deleterious change in the reagent. In the embodiment, the agent includes a reagent that is effective for selecting a liquid sample of the enterprise fluid flowing through the first membrane except HDL lipoprotein, and the second reagent includes a reagent that is effective to associate with the HDL in the membrane. The amount of cholesterol is proportional to the visually detectable signal of the reagent. Key: In the sample, the present invention provides a method for applying a reagent to a film by applying the reagent solution of fork control 1 to a first surface of the film, and by contacting the surface with a This solution is achieved by a flat surface or a cylindrical support member with a surface of the groove pattern of the solution. Also, the option is suspended as a rotary gravure printing cylinder. In addition, it is preferred to use techniques and equipment that describe a method for applying a beta agent to a film in a film laminate. The type 5 agent in the process may be incompletely saturated. In one embodiment, after the application of 95372.doc 200526960 and preferably drying, the n-stab does not contact the opposite surface of the film-that is, it penetrates only partially through the depth of the film. The coating of the reagent occurs simultaneously over the entire width of the first-surface of the membrane. Therefore, preferably, the concentration of the rhenium reagent is uniform over the length and width of the membrane. The membrane may be an asymmetric membrane having a small perforated surface and a large perforated surface; in an embodiment, the first-surface coated with the reagent is large and there are two. In other embodiments, the first surface For small perforated surfaces. In a preferred embodiment, the reagent includes a reagent effective for selectively removing non-proprolipids from a blood fluid sample flowing through the membrane; or, the reagent includes a reagent effective for selectively depositing a product It is a reagent for non-hdl (ldl: VLDL) lipoprotein particles in the medium, and the precipitated L + DL and a few sediment particles are prevented from flowing through the membrane by filtration. In this embodiment, when the membrane is an asymmetric membrane / coating agent is applied to a large perforated surface. In another preferred embodiment, the reagent includes an agent effective to produce HDL-related cholesterol in the membrane. The amount of reagent is proportional to the visually detectable signal. In this embodiment, when the membrane is an asymmetric membrane, the reagent is applied to a small perforated surface. In another aspect, the present invention provides A film laminate for diagnostic analysis includes a first laminated film and a first laminated film combined at a layer interface, wherein a first reagent impregnates a first film in the laminate, and -Reagent-impregnated: the membrane 'and at least one reagent is not present in the laminated area of the membrane adjacent to the laminate interface, so that the reagents in the two membranes can be physically separated by at least the laminated area. Fortunately, the concentration of # 5 type agent or reagent system is uniform in the length and width of individual membranes. 95372.doc 200526960 In the selected embodiment 'at least one of the reagents is a heat sensitive reagent, so that- Lamination of a film containing the reagent to another film will cause harmful effects in the reagent Preferably, at least the first membrane is an asymmetric membrane having a small perforated surface and a large perforated surface. In selected embodiments, the small perforated surface faces the laminate Interface. The second membrane may also be an asymmetric membrane; in the selected embodiment, its large perforated surface faces the laminate interface. In a preferred embodiment, the first reagent includes a material that can effectively flow through the interface. A reagent that selectively removes non-hdl lipoproteins from the blood fluid sample of the first membrane; or the "first-agent" includes reagents effective to selectively kill non-HDL (LDL and VLDL) lipoprotein particles in the sample And to prevent the precipitated LDL and VLDL particles from flowing through the laminate interface by filtration. In these embodiments, the 'second reagent includes a component that is effective to produce an amount proportional to the amount of related cholesterol in the second membrane. Visually detectable signal reagent. Preferably, in this laminate, the reagent in the second film does not exist in the lamination area of the film adjacent to the lamination interface. In a related aspect 'The invention provides a method for analyzing HDL in blood or serum samples. A device comprising: (a) a substrate having a sample receiving well for receiving an aliquot of a sample, (b) a membrane layer & including a first bonded at a laminate interface Laminated membrane and second laminated membrane, in which (!) Is used to effectively precipitate the 2Ldl & vldl lipoprotein particles in the sample to impregnate the first m 'and prevent the passage of 95372 by transition. doc -10- 200526960 The filtered LDL and VLDL · particles flow through the laminate interface; 浸 dipping with a reagent that can effectively produce a visually detectable signal proportional to the amount of HDL-related cholesterol in the second membrane The second film; and the reagents in (111) to the first film (and the second film is Chelajia) are not present in the lamination area of the film adjacent to the laminate interface, so that at least the lamination area can be used Physical separation of reagents in both membranes. Σ is as described above 'In the laminate, it is said that the concentration of each reagent is uniform over the length and width of the membrane. The device also includes a filter located between the sample receiving well and the first membrane of the laminate for removing red blood cells in the sample when the sample migrates from the sample receiving well to the laminate, where the well , Filters, filters, and laminates are placed or can be in fluid communication with each other. These and other objects and features of the invention will become more apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings. [Embodiment] I. Reagent coating method This month provides a method of using at least one reagent to impregnate or coat a laminate with at least two membranes, so that each reagent is limited to its individual membrane. . Preferably, the laminate comprises at least two membranes, each of which comprises a different reagent or reagent system. An example of this laminate is a membrane assembly for use in a device that is used to measure the concentration of job-related cholesterol in a blood sample (which also contains Ldi ^ sink particles). For example, [see U.S. Published Application Rule No. 66291 and U.S. Application Serial No. 95372.doc 200526960 1 0 / 410,671, which are incorporated herein by reference. 'However, the present invention is applicable to Any application using the laminate as described above, wherein the reagent is impregnated in at least one film layer, and preferably two film layers, each of the # reagents must be confined to its individual layers. An example of an insulative HDL analysis laminate as described further below includes an HDL test film (where the ancestral concentration is measured) and a test d film containing binding and / or precipitation reagents, so that before the sample contacts the test film, the The membrane is effective in selectively removing non-HDL lipoproteins from a fluid sample. Preferably each of the membranes in the laminate is a porous asymmetric membrane; that is, a membrane having a gradient of pore size along its thickness. An exemplary two-layer layer 10 including two asymmetric membranes 12 and 14 is shown in the cross-sectional view of FIG. 丨, which shows a better orientation, and has larger holes at 24 in the membrane 12 and at% There are smaller holes everywhere. In order for HDL analysis to work accurately, HDU reagents must be limited to their individual layers. 4HDL reagents are present in the reagent layer. The analysis: the color produced in the application will usually represent Some portion of non-HDL cholesterol. The applicant has discovered a gravure printing process in which a solid plate having a flat or arbitrary shape or a groove of a generally rotating cylinder is coated with a body on a substrate, allowing a controlled amount of different reagents to be deposited in a laminated type On the opposite surface of the film. By controlling the amount of sinking, each test is restricted to its individual layers in the laminate. This type of 'support member has a flat surface according to another preferred embodiment of the present invention is applied to a separate film or film laminate by letterpress printing technology 95372.doc 200526960 or < appropriate configuration 'Preferably a cylindrical configuration. The support package = a pattern of projections that apply tincture to the film. Preferably, the provided reagents are viscous and can temporarily adhere to the protrusions and are applied in a controlled manner. / 、 This In gravure coating, usually a concave mesh with a thickness of m is used to engrav on the surface of a metal cylinder. The units are loaded with fluid and the rotating cylinder transfers the fluid to the substrate. These units have a defined volume, thus limiting the amount of liquid that can be transferred to the substrate. Generally, the volume of a sitting cylinder is about 30-50 square meters per square inch of the cylinder surface (about 4.5-8 W / cm2). (For this purpose, "the surface of a cylinder is assumed to be a smooth surface that corresponds to the surface area of the substrate that the cylinder is in contact with. It does not include the additional surface area provided by the groove itself.) Others can also be used Volume capacity. The grooves are usually of uniform size and closely spaced so that once the droplets are transferred to the membrane, they converge into a uniform liquid layer. The gravure printing system is shown at 30 in Figure 2 and can be used to perform this article The procedure described. (As used herein, "coating" a film includes coating a reagent such that it penetrates beyond the surface and is diptable over the entire thickness of the film. F Feeds a roll 34 into a laminated film or "reticulum" 32. Gravure printing cylinder 36 is usually copper or copper-plated steel or inscription; usually a thin layer of chromium is applied to achieve durability. As mentioned above, the surface of the cylinder is etched to effectively hold a defined amount of liquid A plurality of small units. A cylinder having a pattern of several wells is selected, and the cylinder can be effectively coated with a uniform coating by a given coating system. The cylinder can be partially immersed in the coating to be coated In a fluid bath or a fluid pan. Or the reagent chamber 38, which is the "car yi example" of this method, can be used for Miao 95372.doc -13-200526960 Lu C 40 transfers the reducing agent> valley to the cylinder. This chamber reduces the exposure of reagents to the air .... When the cylinder rotates, the excess solution is wiped from the cylinder by a flexible doctor blade 41 that contacts the cylinder between the reagent chamber or disc and the membrane ". The solution in the residual fine Cao unit is then transferred to the desired film surface. In the example, the film 32 is held in tangential contact with the gravure printing cylinder by tensioning the cylinder 42 (meaning, contacting the cylinder Some angles of the body circumference), the procedure is called, kissing "gravure printing." Or I, the omentum can be passed between the gravure printing cylinder 36 and the embossed or "backup" cylinder, as shown at 42 ,. In this procedure, there is usually no tensioning cylinder 42. In a flat gravure printing process (not shown), the fluid is transferred from the gravure cylinder to a rubber-coated transfer roll, and the roll will then The solution is applied to the substrate. An over-dryer 44, preferably an IR drying blower, is tensioned on a tension roll 46. The other surface of the laminate is then coated in a similar manner. Gravure coating can be performed in a direct mode Procedure in which the cylinder rotates in the same direction as the film feed direction. Or, in reverse gravure printing, as illustrated in Figure 2, the rotation direction of the cylinder is opposite to the direction of travel of the film. This configuration results in the When transferred to the substrate, a shearing force is applied to the solution, which results in a smoother coating. As mentioned above, when the film contacts the gravure roll, the amount of reagent solution applied to the film is affected by the surface of the cylinder Limit of unit volume. The actual amount transferred from the unit to the membrane depends on additional factors such as cylinder speed, roll pressure, omentum speed, and solution components. Therefore, the amount transferred to the membrane can be 95372.doc -14- 200526960 Progress is controlled by proper selection of system configuration, contact time between film and cylinder (determined by screen speed and direction relative to the rotation speed and direction of gravure printing) and surfactant content system. A surfactant is included to help transfer life fluid to the membrane surface, otherwise it can repel aqueous solutions, depending on the hydrophobicity of the membrane material. For asymmetric membranes, the penetration of reagents into human membranes also depends on the pore size and distribution. For example, see, the solution applied to surface 16 (large pores) can be drawn to the membrane by capillary action to a significantly greater extent than the solution applied to surface 22 (small pores). Therefore, a party-controlled amount of the reagent solution is applied to the film on the outer surface of the laminate, followed by drying, to produce a laminate, where the reagent is not :: ultra: the film. The controlled amount can be an incompletely saturated amount; that is, less than: if the selected membrane will penetrate the entire membrane by capillary flow, the solution will be used. In some cases, saturated or near-saturated amounts may be used. According to the present invention, the quantitative reagent solution is applied to the membrane at a desired degree. The coating method can also provide uniformity of the reagents in the length and width of the membrane by applying a controlled amount of coatings simultaneously. “Homogeneous” means that in terms of film length and broad sound, Ligu—variation and inconsistency—concentration gradient or non-V of the obvious can effectively give the sample a different point appearing on the surface of the laminate. -Analytical readings. Preferably, after applying the reagents and after the reagents appearing in the laminate at different final points on the surface or parallel to the surface at different points on the lamination surface The amount of change is only about 5%. Bottom :::: Applicable to heat-sensing reagents, in which a film containing the reagent is shot under normal thermal lamination conditions to perform an analysis of the spleen spleen layer on the laminate 95372 .doc 200526960: Detrimental changes to the heart] and / or its function. This change may include changes in the form or distribution of the reagents as well as chemical changes in the reagents. The coating method can also be advantageously used to uniformly control a controlled After measuring the amount of soil to the previous film, this is achieved by bringing the film surface into contact with a planar or cylindrical support having a surface containing a pattern of grooves filled with a solution. And, as described above, the support Usually a rotary gravure cylinder. When laminating is applied The coating of reagents by this method preferably occurs simultaneously over the entire width of the first surface of the membrane; therefore, the concentration of the reagents is generally uniform over the length and visibility of the membranes. The controlled amount of reagents can be incompletely saturated In the examples, following the application of the reagent and preferably drying, the reagent does not contact the opposite surface of the membrane; that is, it penetrates only partially through the depth of the membrane. 4 The method even when a saturated amount is applied It is advantageous because it results in a more uniform film of the reagent in the pot being impregnated compared to prior art methods such as immersing in a saturated amount of solution or spraying. Membrane. Of particular interest are membranes for diagnostic analysis. Membrane can be asymmetrical, having a small perforated surface and a large perforated surface, which can apply reagents to any surface. In a preferred embodiment, the reagent includes a reagent that is effective for selectively removing non-caproprotein from a blood fluid sample flowing through the membrane; or, the reagent includes a reagent that is effective for selectively relying on the sample Non-ship (both vl dl) Reagent for lipoprotein particles 'and to prevent filtration through Shen Dianzhi and Fan Shen particles through the membrane. In these examples,' when the membrane is asymmetric membrane, the reagent is preferably coated with 95372.doc 200526960 covers a large perforated surface. In another preferred embodiment, the reagent includes a reagent that is effective to produce a visually detectable signal in proportion to the amount of vein-associated cholesterol in the membrane. According to this implementation, the t-film is an asymmetric membrane. It is preferable to apply a remote reagent to a small porous surface. II. Film Lamination In another aspect, the present invention provides a method for impregnating at least one A laminated film that is a reagent in two layers, where each reagent (or reagent system) is limited to its individual layer. Generally, the same layer of T contains different reagent systems. Preferably, the reagents are coated according to the method described above Cover to laminated 臈. Of particular interest are membrane laminates for diagnostic analysis. This laminate includes a first laminated film and a second laminated film which are impregnated with a first reagent and a second reagent, respectively, which are bound at the laminate interface. At least one of the first reagent and the second reagent does not exist in the lamination region of the individual membrane adjacent to the laminate interface, so that the reagents in the two membranes are physically separated by at least the lamination region. Preferably, the concentration of each of the reagents is uniform over the length and width of the individual membranes containing the reagents. This uniformity effectively gives the sample consistent analytical readings at different points on the surface of the laminate. Preferably, the amount of reagent present at different points on the film surface in the laminate or at different points on a given laminated surface parallel to this surface is only about 5%. In one example, the laminate was designed to determine the amount of HDL-related cholesterol in a fluid sample. For example, reference may be made to the aforementioned US Patent Application Publication No. 20030166291 and US Application Serial No. 10 / 410,671. In this case, the first membrane includes a reagent effective for selectively removing non-proteoprotein from a blood fluid sample passing through the membrane at 95372.doc 200526960. For example, non-prolipid proteins can be removed by selectively binding to a reagent that is contained or disproportionate in the membrane. Alternatively or in addition, these components are selectively removed by reagents, and filtered to prevent these components from entering the second membrane through the laminate interface. These reagents include compounds such as Mg, Mn2 +, * ~ + cation-bonded polyanionic compounds' such as sulfonated polysaccharides, heparin, or phosphatidsate. Preferred reagents are conjugated m with molecular weight KDa, such as dextran sulfate, combined with acetate or magnesium chloride. 'It can be selectively buffered to maintain a neutral pH. Reagents can also be immobilized to membranes. As is well known in the art, HDL tests include reagents to quantify HDL-related cholesterol. For example, see commonly owned U.S. Patent Nos .: 213,964, 5,213,965, 5,316,196, and 5,451,370, each of which is incorporated herein by reference. In general, analytical reagents are included for free release from HDL Cholesterol esterase of cholesterol, = cholesterol oxidase, peroxidase that generates Hah by reacting with free cholesterol, and a coupled staining system that can In the presence of enzymes and H2O2, they are converted into signal products of different coloration. Analytical reagents are known in the art to quantify other blood components, such as total biliary tract, triglyceride or glucose, and their Similar enzyme / coupled staining systems are often included. Preferably, at least the first membrane is an asymmetric membrane having a small perforated surface and a large perforated surface. The small perforated surface is preferably For the laminate interface, the large porous surface is located on the outer surface of the laminate. This orientation 95372.doc -18- 200526960 allows samples to freely enter the membrane through larger pores, and can prevent any one from sinking into the other The material passes through smaller pores. The second film may also be a misaligned film, preferably with its large perforated surface facing the laminate interface, and the small perforated surface on the other surface of the laminate ^ This orientation provides a more uniform film surface for optical scanning and quantitative analysis results. For example, the preparation of asymmetric films is described in: US Patent Nos. 4,629,563, 5,171,445, 5,886,059, No. 5,536,408, Nos. 5,562,826 and 4,774,192; ACS Symposium 269: 1-21 (1985) by D. R. Lloyd, "Materials Science 〇f Synthetic
Membranes”中。其可以多種孔徑大小及孔徑大小比之形式 而購得。製造材料包括聚砜、聚醚砜、聚醯胺、聚醚醯胺、 聚氨酯、醋酸纖維素、聚乙烯吡咯烷酮、聚苯乙烯及經改 貝之聚苯乙烯以及摻合物、共聚物及層合複合物。對於較 佳膜類型之進一步描述而言，可參考（例如）上述美國申請公 開案第20030166291號與美國申請案序列號第1〇/41〇,671 號。例示性不對稱膜為聚砜或聚醚颯膜，諸如由pall公司 (San Diego，CA)所提供的 BTS 83膜。 在例不性HDL分析層合物中，每一膜通常具有約1〇〇_15Q^m 之厚度及約80 μΐ/sq in(約12·4 y/cm2)之吸收能力。最小孔 I大小通4自〇·〇1變化至丨〇 ，最大/最小孔徑大小比高達 i〇〇或更大。 在一實施例中，層合物包括至少一種熱感應試劑，使得 將包含試劑的膜層合至另一膜在試劑中可產生有害變化。 该有害變化可包括試劑形態、分佈或實際的化學結構中的 95372.doc 200526960 任-變化或所有變化。藉由本文所描述的試劑塗覆方法可 有利地製備此等層合物。 III.分析裝置 為便利使用，-般將用於診斷分析之諸如本文所描述的 祖分析層合物的膜層合物倂入—分析襄置中。因此，本 發明提供了包含如本文所述之膜層合物的分析裝置。例 如，一種用於分析血液或血清樣品中的HDL之分析裝置包 括： ⑷基板，其具有—用於接收血液流體樣品之等分試樣 的樣品接收井， ⑻-膜層合物，其包括結合於層合物界面處的第一層合 式膜及第二層合式膜，及 ⑷-位於樣品接收井與層合物之第—膜之間的過遽器， 其用於當樣品自樣品接收井遷移至層合物時移除樣品中的 紅血細胞。⑻之膜層合物為如上所述之層合物，其中使第 -膜浸染滿能有效地選擇性沈澱該樣品中非舰(弧及 几叫脂蛋白微粒的試劑，且藉由過遽來防止所沈殿之咖 及v微粒流過層合物界面；使第二膜浸染滿能有效地產 生與該第二膜内之H D L相關膽固醇量成比例之光學上可偵 測訊號的試劑’·且此等臈内至少一個膜中的試劑不存在於 ，層合物界面相鄰膜的層合區域中，使得兩膜内的試劑可 糟由至少該層合區域以物理方式分離。尤其較佳的是，Hdl 分析試劑不存在於與層合物界面相鄰之第二膜的層合區域 95372.doc -20- 200526960 之如上料，每一試劑之濃度在包含該試劑的膜 、又見度上I均一#。膜較佳為如上述定向之不對稱 膜，其中試劑膜的外部表面為大的有孔（黯淡）表面，且 膜的外部表面為小的有孔（明亮）表面。 ⑷-⑷之樣品井、過滤器及層合物係以或可以彼此進行流 體交流的方式放置。例如，此等元件可位於共同基板上， 或其可位於-個或多個分離的基板上且在分析期間開始進 行流體流通。此分析裝置之不同實施例包括上述ws等人 ，吴國中睛公開案第期66291號與美时請案序列號 第10/410,671號中所描述的該等實施例。 圖3中展示了一實施例。設備5〇包括可界定樣品井54的主 體或支撐件52。该井與篩塾6〇成流體接觸，該筛塾⑼可被 帶入一形成於支擇件之上部邊緣中的缺口區域58内。流體 接觸可為直接的接觸，或如在圖3所示之裝置中，由形成於 井基底處之板内的毛細管管道56提供該流體接觸。支撐件 較佳為一塑料板，且藉由標準模製或機械加工方法而形成 井、缺口區域及/或毛細管管道。 士圖所不田樣品以底部至頂部的方向遷移通過襯墊矩 陣時’帶入區域58中之篩墊6〇起部分地移除大微粒物質（包 、、田肊）的作用。襯墊6〇較佳由材料之玻璃纖維矩陣形 成’其經設計以藉由表面濕潤來牵引水流，且當血液樣品 被牵引通過矩陣時能延遲血細胞的移動。一例示性襯墊為 破呙、截維過濾杰，诸如由Whatmai^供應的或 過攄器。使襯墊成尺寸以吸收樣品流體之一卩定用量，例 95372.doc 200526960 如約15_25 μΐ。篩墊60可額外地包含諸如外原凝集素、特定 用於紅血細胞表面膜蛋白質的抗體、纖維蛋白酶、或離子 交換劑之紅血細胞捕獲試劑。 篩墊60又接觸沿板52之上部邊緣延伸的延長 八 配矩陣62。亦可藉由泡沫墊層或其它支撐件支撐此條。矩 陣62用於將來自與襯墊6〇產生流體接觸的中央樣品塗覆區 域64之樣品流體分佈至矩陣内的樣品收集區域，諸如μ、 68。該矩陣較佳由玻璃纖維形成。矩陣之充填密度及厚度 可使得能將供應給條之樣品塗覆區域的樣品流體之用量 (例如，10-25 μΐ)吸收且分佈至條的#品收集區域。一例示 性條材料為獲自Whatman之F]65_25a玻璃纖維過遽器，其 具有約0.2 gm/cm3之充填密度及約〇 12 mm之厚度。 八 如圖所示’ |置5G亦包括反應桿7G，其包含伸長支樓件 72a，及位於該支#τ表面之—個或多個複合可濕性吸收反 應測試條74、76、78及8〇。元件取86形成如本文所述之 膜層合物，其中74為HDL分析膜且86為試劑膜。 支撑件72呈透明或其具有允許可透過支樓件觀察到概塾 々固或開σ用於特殊分析的每_測試墊包含分析物相 關之試劑，其能有效地在襯塾令產生可以熟知方式摘測到 的分析物相關之變化。 藉由安裝構件將反應桿安裝於支樓件52，該安裝構件能 ，效地：⑷維持該裝置處於樣品分佈位置，其中測試臈/ 试劑臈層合物與樣品分佈矩陣間隔開；及⑻將該裝置轉移 至一測試位置，其中測續腹μ 、八膜/4劑膜層合物及樣品分佈矩陣 95372.doc -22- 200526960 成流體流通。當所要量的樣品已進入測試墊以後，及/或經 過了一確定的接觸時間以後，安裝構件亦可用於破壞該流 體連通，此藉由將裝置自測試位置轉移至測試條不與樣品 分佈矩陣成流體流通的位置（其可與"樣品分佈"位置相同） 而達成。如共同擁有之美國專利第5，114,35〇號所述，可藉 由監視能反映出濕潤程度的測試墊頂部表面處的反射來控 制此轉移。或者，當已知襯墊材料之樣品吸收的吸收能力 及比率時，可藉由使用預定接觸時間來充分精確地控制樣 品數量。 例如，安裝構件可包括諸如彈性材料塊82、84之一對彈 性T，其用於偏壓測試膜/試劑膜層合物使其朝向不轉移或 袠刀佈位置，在此位置層合物與樣品分佈矩陣間隔開。 藉由壓縮或釋放彈性膜，可選擇性地建立並分離樣品分佈 矩陣62與層合物之間的流體流通。該流體流通可經由直接 接觸或藉由一中間元件。可藉由彈簣或活塞類物件&縮該 等支挣塊。或者’外部機械裝置可喷合主體52及/或支推件 62並:多動一個朝向另-個。-例示性系統為Ch〇lestech LDX〇分析器’其為可有利地用於諸如本文所述之分析裝置 的自給式自動化分析器。 實例 斤揭示藉由凹版印刷塗佈來製備例示性分 析層口物’且使用該等層合物來進行關於已知之標準的 =DL刀析:貧料展示了極好的精確性，其說明了所揭示之 以塗覆受控且均—量的分析試劑的程序的有效性。 95372.doc •23· 200526960 所運用之膜層合物為兩個B T S - 8 3之聚磯不對稱膜的層合 物，其藉由使一個膜之小的有孔（明亮）側接觸第二膜之大的 有孑L (黯淡）側層合而成，且其由San Diego CA之Pall公司製 備而成。用於塗佈之寬度高達約5英吋。 使用標準沈澱及HDL分析試劑混合物。沈澱溶液包括3.9 mg/ml 硫酸葡聚糖（500,000 MW)及 7.9 mM 之 Mg(OAc)2 水溶 液。亦添加了界面活性劑（由BASF生產之普盧蘭尼克 (Pluronic)L64, 0.05%含量）來便利膜上之吸收。HDL分析試 劑溶液包含80.3 U/ml膽固醇氧化酶、473 U/ml膽固醇酯 酶、440 U/ml辣根過氧物酶、4.14 mg/ml 4-胺基安替比林及 26.5 mg/ml TOOS(3-[乙基（3-甲基苯基）胺基]-2-羥基丙烷磺 酸）水溶液，其中添加了 3 mg/ml CHAPS作為界面活性劑。 使用一具有凹版印刷式圓柱體之膠印機以將一恒定用量 的試劑溶液塗覆至一與膜層合物（平凹版印刷程序）接觸的 橡皮捲筒。用於此試驗之圓柱體為65線/英吋，30 BCM之捲 筒，其中BCM係指每平方英吋表面之十億立方微米數或微 升數。 首先將沈澱試劑塗覆至第一膜的黯淡面。繼塗覆之後， 使用IR乾燥吹風器來乾燥膜。接著以相似的方式將HDL比 色分析試劑塗覆至層合物之第二膜的明亮面，並使用IR乾 燥吹風器對其進行乾燥。 以下HDL分析資料來自如上所述製得的層合式膜，其被 切成0.22英吋截面且被組裝成分析盒，諸如上述美國申請 公開案第20030166291號及美國申請案序列號第 95372.doc -24- 200526960 分析。在LDX⑧ 10/410，67 1號中所描述。根據標準程序來執^一 分析器中分析樣品（8祖標準）且為每―標準求得8個盒之 平均數。獲得極好的精確性及與標準值的相關性 HDL標準（mg/dL) LDX®平均結果 32.6 33.9 42.5 40.8 76.2 75.5 62.3 58.9 53.2 52.4 45.7 47.7 49.5 53.0 在用於上述試驗的平凹版印刷程序中，將高達4遍的試劑 溶液塗覆至膜的每一面。由塗佈溶液之重量損耗來判定每 遍之塗佈重I，且自一遍至另一遍可觀察到良好的一致 性〇 發現一直接凹版印刷程序比平版程序能更有效地塗覆溶 液’該平版程序通常重複此系統不需要的遍數。在一較佳 程序中，凹版印刷式圓柱體在與網膜饋入方向相對的方向 上自轉（意即，如圖2中所示，反向凹版印刷），且該網膜無 需壓印圓柱體便可接觸該圓柱體（如圖2之42處所示，”親吻，， 凹版印刷）。選擇具有若干井之一圖案的圓柱體，該圓柱體 能有效地藉由該給定塗佈系統來塗覆一均一塗層。 使用反向/親吻凹版印刷程序來塗佈膜，其中網膜速度為 5-1〇英尺/分鐘且圓柱體旋轉速度對沈澱試劑塗佈而言約為 95372.doc -25- 200526960 網膜速度的兩倍且對HDL分析試劑塗佈而言約與網膜速戶 相同。在此等運行中所塗覆至膜的溶液量對於沈澱試劑而 言通常為飽和用量的約65%，且對於HDL分析試劑而士 J β為飽 和用量的約45%。 【圖式簡單說明】 圖1為根據本發明之一實施例可塗覆有試劑之兩個層人 式不對稱膜的橫截面視圖； θ合 圖2為根據本發明之一實施例用以展示將試劑涂"Membranes". It can be purchased in a variety of pore sizes and pore size ratios. Manufacturing materials include polysulfone, polyethersulfone, polyamine, polyetheramide, polyurethane, cellulose acetate, polyvinylpyrrolidone, polybenzene Ethylene and modified polystyrene as well as blends, copolymers, and laminated composites. For further description of the preferred film types, reference may be made to, for example, the aforementioned US Application Publication No. 20030166291 and the US Application Serial No. 10 / 41〇, 671. Exemplary asymmetric membranes are polysulfone or polyethersulfonium membranes, such as the BTS 83 membrane provided by Pall Corporation (San Diego, CA). In the example, the HDL analysis layer In the composition, each film usually has a thickness of about 100-15 ^^ m and an absorption capacity of about 80 μΐ / sq in (about 12.4 y / cm2). The minimum pore size I ranges from 4 to 0.001. Change to 丨 〇, the maximum / minimum pore size ratio is as high as 100 or more. In one embodiment, the laminate includes at least one heat-sensing reagent such that a film containing the reagent is laminated to another membrane in the reagent Harmful changes may occur. The harmful changes may include reagent form 95372.doc 200526960 in the distribution or actual chemical structure of any or all changes. These laminates can be advantageously prepared by the reagent coating method described herein. III. Analytical device is convenient for use, generally Membrane laminates such as the ancestral analysis laminates described herein are used in diagnostic analysis for diagnostic analysis. Therefore, the present invention provides an analysis device comprising a membrane laminate as described herein. For example, An analysis device for analyzing HDL in a blood or serum sample includes: a rhenium substrate having a sample receiving well for receiving an aliquot of a blood fluid sample, and a rhenium-membrane laminate including a bonding layer The first laminated film and the second laminated film at the interface of the composite, and the plutonium-cavity converter located between the sample receiving well and the first film of the laminate, which is used when the sample migrates from the sample receiving well to The red blood cells in the sample are removed during the lamination. The membrane laminate of the tritium is the laminate described above, in which the first membrane is soaked with dye to effectively precipitate the non-ship (arc and several calls) in the sample. Reagents for lipoprotein particles, Preventing the coffee and v particles from flowing through the laminate interface by passing through it; soaking the second film full can effectively produce an optically detectable signal proportional to the amount of HDL-related cholesterol in the second film And the reagents in at least one of the membranes do not exist in the lamination area of the adjacent membrane adjacent to the laminate interface, so that the reagents in the two membranes can be physically separated by at least the lamination area It is particularly preferred that the Hdl analysis reagent does not exist in the laminated region of the second membrane adjacent to the laminate interface. 95372.doc -20- 200526960 As above, the concentration of each reagent is in the membrane containing the reagent. ， 见见 上 上 I 均一 #. The membrane is preferably an asymmetric membrane oriented as described above, wherein the outer surface of the reagent membrane is a large perforated (dim) surface, and the outer surface of the membrane is a small perforated (bright) surface. The plutonium-plutonium sample wells, filters, and laminates are placed in such a way as to allow fluid communication with each other. For example, these elements can be on a common substrate, or they can be on one or more separate substrates and begin fluid flow during the analysis. Different embodiments of this analysis device include those described in the above-mentioned ws et al., Wu Guozhong Eye Publication No. 66291 and Lamex Application Serial No. 10 / 410,671. An embodiment is shown in FIG. 3. The device 50 includes a body or support 52 that may define a sample well 54. The well is in fluid contact with a screen 60 which can be brought into a notch area 58 formed in the upper edge of the support. The fluid contact may be a direct contact or, as in the device shown in Fig. 3, the fluid contact is provided by a capillary tube 56 formed in a plate at the base of the well. The support is preferably a plastic plate and the wells, notched areas, and / or capillary tubes are formed by standard molding or machining methods. The sieving mat 60, which is brought into the region 58 by the bottom of the Shitosoda sample and migrates through the pad matrix from the bottom to the top, partially removes large particulate matter (bags, fields, etc.). The pad 60 is preferably formed of a matrix of glass fibers of material, which is designed to draw water flow by surface wetting and to delay the movement of blood cells when a blood sample is drawn through the matrix. An exemplary pad is a break-out, cut-off filter, such as supplied by Whatmai ^ or a filter. The pad is sized to absorb one of the sample fluids, for example, 95372.doc 200526960, such as about 15-25 μΐ. The sieve mat 60 may additionally contain a red blood cell capture reagent such as exogenous lectin, an antibody specific for a red blood cell surface membrane protein, fibrin, or an ion exchanger. The sieve mat 60 contacts the extended mating matrix 62 extending along the upper edge of the plate 52. The strip can also be supported by a foam cushion or other support. The matrix 62 is used to distribute the sample fluid from the central sample coating area 64 in contact with the fluid generated by the pad 60 to a sample collection area such as µ, 68 within the matrix. The matrix is preferably formed of glass fibers. The packing density and thickness of the matrix can allow the amount of sample fluid (eg, 10-25 μΐ) supplied to the sample coating area of the strip to be absorbed and distributed to the ## collection area of the strip. An exemplary strip material is a F] 65_25a glass fiber converter from Whatman, which has a filling density of about 0.2 gm / cm3 and a thickness of about 012 mm. As shown in the figure, | 5G also includes a response rod 7G, which includes an elongated branch member 72a, and one or more composite wettable absorption test strips 74, 76, 78, and 74 located on the surface of the branch # τ. 80. The element takes 86 to form a film laminate as described herein, where 74 is an HDL analysis membrane and 86 is a reagent membrane. The support member 72 is transparent or has a structure that allows observing the solids or openings through the branch pieces. Each test pad used for special analysis contains analyte-related reagents, which can effectively generate Analyte-related changes detected by the method. The reaction rod is mounted on the supporting member 52 by a mounting member, which can effectively and effectively: ⑷ maintain the device in a sample distribution position, in which the test 臈 / reagent 臈 laminate is spaced from the sample distribution matrix; and ⑻ The device was transferred to a test position, in which the continuous μ, eight membranes / 4 doses of film laminate, and the sample distribution matrix 95372.doc -22- 200526960 were flowed into fluid. After the required amount of sample has entered the test pad, and / or after a certain contact time has elapsed, the mounting member can also be used to disrupt the fluid communication by transferring the device from the test position to the test strip without the sample distribution matrix Into the fluid flow location (which can be the same as the "sample distribution" location). As described in commonly owned U.S. Patent No. 5,114,350, this transfer can be controlled by monitoring reflections at the top surface of the test pad that reflects the degree of wetting. Alternatively, when the absorption capacity and ratio of the sample absorption of the cushioning material are known, the number of samples can be sufficiently accurately controlled by using a predetermined contact time. For example, the mounting member may include a pair of elastic T, such as one of the elastic material blocks 82, 84, which is used to bias the test film / reagent film laminate toward the non-transfer or trowel cloth position where the laminate and The sample distribution matrix is spaced. By compressing or releasing the elastic membrane, fluid flow between the sample distribution matrix 62 and the laminate can be selectively established and separated. The fluid flow can be through direct contact or through an intermediate element. You can earn them by impeaching or plunging objects. Alternatively, the 'external mechanical device can spray the main body 52 and / or the pusher 62 and: move one more toward the other. -An exemplary system is a Cholestech LDXo Analyzer 'which is a self-contained automated analyzer that can be advantageously used in an analysis device such as described herein. The example reveals that an exemplary analytical layer was prepared by gravure coating and that these laminates were used to perform a known standard = DL knife analysis: Lean material demonstrated excellent accuracy, which illustrates that The effectiveness of the disclosed procedure for applying a controlled and uniform amount of analytical reagents. 95372.doc • 23 · 200526960 The membrane laminate used is a laminate of two BTS-8 3 polyisocyanate asymmetric membranes, which is made by contacting the small perforated (bright) side of one membrane with the second The large L (dim) side of the film is laminated and it is made by Pall Corporation of San Diego CA. For coating widths up to about 5 inches. A standard precipitation and HDL analysis reagent mixture was used. The precipitation solution included 3.9 mg / ml dextran sulfate (500,000 MW) and a 7.9 mM aqueous solution of Mg (OAc) 2. A surfactant (Pluronic L64, 0.05% by BASF) was also added to facilitate absorption on the membrane. The HDL analysis reagent solution contains 80.3 U / ml cholesterol oxidase, 473 U / ml cholesterol esterase, 440 U / ml horseradish peroxidase, 4.14 mg / ml 4-aminoantipyrine, and 26.5 mg / ml TOOS ( 3- [Ethyl (3-methylphenyl) amino] -2-hydroxypropanesulfonic acid) aqueous solution to which 3 mg / ml CHAPS was added as a surfactant. An offset printing machine with a gravure printing cylinder was used to apply a constant amount of the reagent solution to a rubber roll in contact with the film laminate (flat gravure printing process). The cylinder used for this test was a 65-line / inch, 30 BCM roll, where BCM refers to one billion cubic micrometers or microliters per square inch of surface. The precipitating agent is first applied to the dull side of the first film. Following coating, the film was dried using an IR drying blower. HDL colorimetric analysis reagent was then applied to the bright side of the second film of the laminate in a similar manner and dried using an IR drying blower. The following HDL analysis data comes from the laminated film prepared as described above, which was cut into a 0.22 inch cross section and assembled into an analysis box, such as the above-mentioned U.S. Application Publication No. 20030166291 and U.S. Application Serial No. 95372.doc- 24- 200526960 Analysis. Described in LDX⑧ 10/410, 67 No. 1. Analyze samples (8 ancestor standards) in the analyzer according to standard procedures and obtain the average of 8 boxes for each standard. Obtain excellent accuracy and correlation with standard values HDL standard (mg / dL) LDX® average results 32.6 33.9 42.5 40.8 76.2 75.5 62.3 58.9 53.2 52.4 45.7 47.7 49.5 53.0 In the planographic gravure printing process used for the above tests, The reagent solution was applied up to 4 times to each side of the membrane. The coating weight I of each pass is determined by the weight loss of the coating solution, and good consistency can be observed from one pass to another. It was found that a direct gravure printing process can coat the solution more efficiently than a lithographic process. The program usually repeats a number of passes that are not needed by this system. In a preferred procedure, the gravure-printed cylinder rotates in a direction opposite to the feeding direction of the omentum (meaning, as shown in FIG. 2, reverse gravure printing), and the screen does not need to emboss the cylinder. Touch the cylinder (shown at 42 in Figure 2, "Kiss ,, gravure printing"). Select a cylinder with a pattern of several wells, which can effectively coat a cylinder with a given coating system. Uniform coating. Film was applied using a reverse / kiss gravure printing process with a web speed of 5-10 feet per minute and a cylinder rotation speed of approximately 95372.doc -25- 200526960 for Shendian reagent coating. It is twice as fast and is about the same as the omentum for the application of HDL analysis reagents. The amount of solution applied to the membrane in these runs is usually about 65% of the saturated amount for the precipitation reagents, and for HDL Analyze the reagent and let J β be about 45% of the saturated amount. [Brief description of the figure] FIG. 1 is a cross-sectional view of a two-layer human-type asymmetric membrane that can be coated with a reagent according to an embodiment of the present invention; Fig. 2 is an implementation according to the present invention. The reagents are applied to show
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圖3為根據本發明之實施例用以合倂膜層合物 置之側視圖。 【主要元件符號說明】 10 層合物 12 第一膜 14 第二膜 16 外部第一膜表面 18 層合式第一膜表面 20 層合式第二膜表面 22 外部第二膜表面 24 較大的孔 26 較小的孔 30 凹版印刷式印刷系統 32 層合膜或網膜 34 支撐件 95372.doc -26- 200526960 36 凹版印刷式圓 38 試劑腔室 40 管 41 刮漿刀 42 張緊圓柱體 44 乾燥器 46 張緊捲筒 50 診斷分析 52 主體 54 樣品接收井 5 6 毛細管管道 58 缺口區域 60 襯墊 62 分佈矩陣 64 樣品塗覆區域 66, 68 樣品收集區域 70 反應桿 72 伸長支撐件 74, 86 膜層 76, 78, 80 測試條 82, 84 彈性材料塊 95372.doc -27-Fig. 3 is a side view of a composite film laminate arrangement according to an embodiment of the present invention. [Description of Symbols of Main Components] 10 Laminate 12 First film 14 Second film 16 External first film surface 18 Laminated first film surface 20 Laminated second film surface 22 External second film surface 24 Larger holes 26 Smaller holes 30 Gravure printing system 32 Laminated film or omentum 34 Support 95372.doc -26- 200526960 36 Gravure circle 38 Reagent chamber 40 Tube 41 Squeegee 42 Tightening cylinder 44 Dryer 46 Tensioning reel 50 Diagnostic analysis 52 Body 54 Sample receiving well 5 6 Capillary tube 58 Notch area 60 Gasket 62 Distribution matrix 64 Sample coating area 66, 68 Sample collection area 70 Response rod 72 Elongated support 74, 86 Membrane layer 76 , 78, 80 Test strips 82, 84 Blocks of elastic material 95372.doc -27-