TW201102654A - Assay device - Google Patents

Abstract

The present invention relates to the use of surfaces that exhibit different surface energies wherein the difference in surface energies is configured to disrupt capillary laminar flow of a fluid travelling between the two surfaces. The invention further relates to the use of such surfaces in assay methods including a device utilising same.

Description

201102654 六、發明說明： 【發明所屬之技術領域】 概言之，本發明係關於尤其用於照護點 驗之用即棄型檢驗測試裝置領域。本發明進一步係關於該 等裝置（包括包含該等裝置之套組）促進血液中各種脂蛋白 之含s之準確量測的用途。測試套組可包含一或多種檢驗 測試裝置且視情況包括說明書、刺血針裴置、消毒酒精拭 子或膠黏繃帶。 【先前技術】 在本申請案中提及數個出版物及專利文件以更全面地描 述本發明所屬技術領域之現狀。該等出版物中之每一者的 揭示内容均以引用方式併入本文中。 側向流動檢驗裝置及方法已為熟習此項技術者所習知。 以前，已研發該等裝置來測試容易大量獲得之試樣。通 常，該等裝置包含諸如硝化纖維素基質等側向流動基質， 其上施加流體試樣。試樣内之流體沿基質行進且試樣内之 一或多種分析物與側向流動基質内所含有之試劑反應。通 常二該等試劑中之至少一種在基質内固定不動，此使得能 夠藉由例如目測來檢測基質特定區域上與分析物之任一反 應。 當測試試樣係血液或血液組份時，不—定能夠或不方便 採集到大置試樣，例如在照護點（例如藥房或醫生診所）。 =外，側向流動裝置通常不適於少量流體試樣，例如用於 刀析手指刺取（finger prick)」體積之血液。在此情形 148680.doc 201102654 下’在檢驗完成之前膜可能已經乾燥，《者在實施量測之 前流體不足以行進測試裝置之長度。而且，隨著裝置（在 其中處理流體)規模減小，出現一個顯著缺點即該等流 體有保持穩疋層流之趨勢。此使得液體及(例如)試劑難以 有效奶σ ’限制了在不増加裝置複雜性的情況下檢驗裝置 之規杈可成功降低之程度。圖）提供業内現有複雜檢驗裝 置之實例。 因此，备刚茜要簡單且有效的能夠快速測試同時能夠分 析較低或較小體積試樣之裝置。該等裝置可能有用之一個 領域係膽固醇及血液脂質測試領域。 【發明内容】 在第心樣中，本發明提供包含兩個展示不同表面能之 至少部分對立表面之檢驗裝置，纟中表面能差異經設置以 破壞行進於兩個表面之間之流體的毛細管層流。 當將液體置於表面上時，其在表面上之鋪展程度由表面 張力決定。在親水性表面上，液體被吸引至表面上且鋪展 成薄膜。在疏水性表面上，液體鋪展較差且可能「成珠 (bead)」。表面能通常與表面疏水性人親水性密切相關。铁 而表面能描述與多種材料之相互作用，而表面疏水性通常 僅描述與水之相互作用Hjc具有巨大㈣㈣U 1 具有高表面能（即高鍵結潛力）之材料可與水達成更高相互 作用且由此更具親水性。因此，疏水性通常隨表面：增加 而降低《因此，親水性表面（例如玻璃）具有高表面能，曰而 疏水性表面（例如P 丁 F E或聚苯乙烯）具有低表面能。 148680.doc 201102654 用於評價物質之親水性/疏水性性質且由此其表面能之 適且里度係各表面上液體之接觸角（0)。201102654 VI. Description of the Invention: [Technical Field to Which the Invention Is Applicable] In summary, the present invention relates to the field of disposable inspection test devices, particularly for use in care inspection. The invention further relates to the use of such devices, including kits comprising such devices, to promote accurate measurement of the s-containing of various lipoproteins in the blood. The test kit may contain one or more test test devices and, as appropriate, instructions, lancet devices, sterile alcohol swabs or adhesive bandages. [Prior Art] Several publications and patent documents are mentioned in this application to more fully describe the state of the art to which the invention pertains. The disclosures of each of these publications are incorporated herein by reference. Lateral flow testing devices and methods are well known to those skilled in the art. Previously, such devices have been developed to test samples that are readily available in large quantities. Typically, such devices contain a lateral flow matrix such as a nitrocellulose matrix to which a fluid sample is applied. The fluid within the sample travels along the substrate and one or more analytes in the sample react with the reagents contained within the lateral flow matrix. Typically, at least one of the agents is immobilized within the matrix, which enables detection of any reaction with the analyte on a particular region of the substrate by, for example, visual inspection. When the test sample is a blood or blood component, it is not possible or convenient to collect a large sample, such as at a point of care (eg, a pharmacy or a doctor's office). In addition, lateral flow devices are generally not suitable for small fluid samples, such as for the analysis of finger prick volume of blood. In this case 148680.doc 201102654, the film may have dried before the test is completed, and the fluid is not sufficient to travel the length of the test device before the measurement is performed. Moreover, as the scale of the apparatus in which the fluid is treated is reduced, a significant disadvantage is that the fluids tend to maintain a steady laminar flow. This makes it difficult for the liquid and, for example, the reagent to be effective, to limit the extent to which the gauge of the test device can be successfully reduced without adding complexity to the device. Figure) Provides examples of existing complex inspection equipment in the industry. Therefore, it is simple and effective to quickly test devices that can simultaneously analyze lower or smaller volume samples. One area in which such devices may be useful is in the field of cholesterol and blood lipid testing. SUMMARY OF THE INVENTION In a first embodiment, the present invention provides an inspection apparatus comprising two at least partially opposing surfaces exhibiting different surface energies, wherein the difference in surface energy is configured to disrupt a capillary layer of fluid traveling between the two surfaces. flow. When a liquid is placed on a surface, its spread on the surface is determined by the surface tension. On the hydrophilic surface, the liquid is attracted to the surface and spreads into a film. On hydrophobic surfaces, the liquid spreads poorly and may "bead". Surface energy is often closely related to surface hydrophobic human hydrophilicity. Iron and surface energy describe the interaction with a variety of materials, while surface hydrophobicity usually only describes the interaction with water. Hjc has a large (IV) (IV) U 1 material with high surface energy (ie high bonding potential) can achieve higher interaction with water. And thus more hydrophilic. Therefore, the hydrophobicity generally decreases as the surface: increases. Therefore, a hydrophilic surface (e.g., glass) has a high surface energy, and a hydrophobic surface (e.g., P F or polystyrene) has a low surface energy. 148680.doc 201102654 is used to evaluate the hydrophilic/hydrophobic nature of a substance and thus its surface energy is suitable and the degree of contact of the liquid on each surface (0).

接觸角係與液滴表面相切之線與上方形成小滴之表面之 平面之間形成的角。疏水性表面與液滴產生較小介面張 力’且產生較大接觸角。接觸角之量測已為熟習此項技術 者所熟知，例如，接觸角可如Robert j Good及R〇bert LThe contact angle is the angle formed between the line tangent to the surface of the droplet and the plane above the surface on which the droplet is formed. The hydrophobic surface creates a smaller interface tension with the droplets and produces a larger contact angle. Measurement of contact angles is well known to those skilled in the art, for example, contact angles such as Robert j Good and R〇bert L

Str〇mberg 編輯，「Surface and Colloid Science _ Experimental Methods」，第 Π卷，（Plenum Press，1979)， 第63-70頁中所述來測定。圖2a提供展示接觸角之量測之 通用圖。 當比較兩個表面日寺，術語r較差疏水性」意指第一表面 上液滴之接觸角小於（smaller或less than)第二表面上類似 液滴之接觸角°反之’該術語亦可指（例如）展示類似差異 之兩個親水性表面，即一個表面比另一個表面更具親水 性。術語「疏水性」係指在空氣中表面具有至少9〇。之液 體接觸角。反之’術語「親水性」係指在空氣中材料具有 小於90。之液體接觸角。圖2b提供展示疏水性表面與親水 性表面之間之接觸角差異的通用圖。 個由於表面能最^地使用接觸角來定量，故該兩 ^刀#立表面應呈現至少1。至5。、約10。、至少2〇。 或等於及大於30。之流體接觸角差異。通常，具有較低表 “之表面應具有至少9〇。、較佳地至少· 少110。、最佳地至少⑶。之流體接觸i 更么地至 通常，微或毛細管通道中之藉由毛細管流動之液體移動 148680.doc 201102654 係層流而非奈流，且因此不利於試劑，試樣混合。令人驚 ，的是，本發明者已發現，兩個至少部分對立表表面 能差異促進試樣流體中之試劑混合且不減小流體沿流動路 =行進時由層流提供之填充速度。此加上微通道(其中擴 散混合具有顯著益處）大小的優點克服了檢驗裝置之大小/ 規模減小時達成奈流混合甚為齦難或困難的大問題。 通常，「毛細管流動」或「側向流動」係指由可經由例 如表面張力弓丨導液體流動之毛細管勢能梯度或表面勢能梯 度引起之液體的被動流動。毛細管流動速率係指當將液體 施加於端時試樣沿流動路徑（例如膜條）向前移動之速 度。然而，隨著液體沿長度行進，速率以指數方式衰減。、 因此，通常量測液體沿界定長度之膜條或毛細管移動且填 充：條或毛細管所需要之時間並稱為毛細管流動時間。毛 々B 動時間通常以sec/cm表示且與流動速率逆相關。該 等術語通常係、指流體在表面上之移動，其中流體以特定方 向或沿特定路徑流動/行進。應注意，術語「側向流動」 意=為描述性的以具有限制意義，此乃因可以其他方式 構k裝置且具有相同效果。例如’使用所述普遍原理可容 易地，想出徑向或垂直流動。在特定實施例中，隨流體流 動或灯進’其肖各種試劑料反應。 ' 可包3熟習此項技術者所習知之材料或由該等 材料組成，例如卡紙板、玻璃、矽、塑膠及諸如此類：較 佳地„亥材料包含疏水性材料或由疏水性材料組成，或藉 由例如塗敷適宜材料或與適宜材料層壓在一起而具有疏水 I48680.doc 201102654 性區域，兮势 等適且材料包括（但不限 聚合物（例如，⑯〃 鼠化材料，例如說 』如，聚四氟乙埽（PTFE))及氯 ^ 適於改蠻矣& 軋鼠祆合物。可證實 …支表面能之其他材料包括煙 蠟及諸如此類。 J如壙舳、礼I、石 該兩個至少部分對立表面實f上起切 驟的作^，例如-㈣化學以 序之各步 t、呼笪主 應邊專反應可在該等表面 _表面中或於料表面之間發生1此，包含該等As described in Str〇mberg, "Surface and Colloid Science _ Experimental Methods", Vol., (Plenum Press, 1979), pp. 63-70. Figure 2a provides a generalized diagram showing the measurement of the contact angle. When comparing two surface Japanese temples, the term r is poorly hydrophobic means that the contact angle of the droplets on the first surface is smaller than (smaller or less than) the contact angle of similar droplets on the second surface. Conversely, the term may also refer to (for example) showing two hydrophilic surfaces with similar differences, ie one surface is more hydrophilic than the other. The term "hydrophobic" means having at least 9 Å in the surface of the air. The liquid contact angle. Conversely the term "hydrophilic" means that the material has less than 90 in air. Liquid contact angle. Figure 2b provides a generalized diagram showing the difference in contact angle between a hydrophobic surface and a hydrophilic surface. Since the surface energy is most quantitatively measured using the contact angle, the two surfaces of the two knives should exhibit at least one. To 5. About 10 At least 2 baht. Or equal to and greater than 30. The difference in fluid contact angle. Generally, the surface having the lower table "should have at least 9 〇., preferably at least 110., optimally at least (3). The fluid contact i is more commonly to the capillary tube in the micro or capillary channel. Flowing liquid movement 148680.doc 201102654 laminar flow rather than flow, and thus detrimental to reagents, sample mixing. Surprisingly, the inventors have discovered that two at least partially opposite surface energy differences promote the test The reagents in the sample fluid mix and do not reduce the fill velocity provided by the laminar flow along the flow path = travel. This plus the advantage of the size of the microchannels (where diffusion mixing has significant benefits) overcomes the size/scale reduction of the test device An hour is a big problem that is difficult or difficult to mix. In general, "capillary flow" or "lateral flow" refers to a capillary potential energy gradient or surface potential energy gradient that can be caused by, for example, a surface tension bow. Passive flow of liquid. Capillary flow rate refers to the rate at which a sample moves forward along a flow path (e.g., a strip of film) when liquid is applied to the end. However, as the liquid travels along its length, the rate decays exponentially. Thus, the time required for the liquid to move along a defined length of film strip or capillary and fill: strip or capillary is typically referred to as capillary flow time. The 々B movement time is usually expressed in sec/cm and is inversely related to the flow rate. These terms are generally used to refer to the movement of a fluid on a surface in which the fluid flows/traves in a particular direction or along a particular path. It should be noted that the term "lateral flow" means = descriptive to have a limiting meaning, since the device can be constructed in other ways and has the same effect. For example, it is easy to use the general principle to think of radial or vertical flow. In a particular embodiment, the various reagent materials react with the fluid flow or lamp. ' can be used by or familiar to those skilled in the art, such as cardboard, glass, enamel, plastic, and the like: preferably the material contains or consists of a hydrophobic material, or By, for example, coating a suitable material or laminating with a suitable material to have a hydrophobic I48680.doc 201102654 region, the potential is suitable and the material includes (but is not limited to a polymer (eg, 16 鼠 ratified material, for example, 』 For example, polytetrafluoroethylene (PTFE) and chlorine are suitable for changing the sputum & squirrel chelating compound. It can be confirmed that other materials supporting surface energy include smoky wax and the like. The two at least partially opposite surfaces of the stone are cleaved, for example, - (4) chemically in each step t, the main response of the snoring can be in the surface _ surface or between the surface of the material 1 occurs, including these

表面之檢驗裝置可採取多 D 種形式’例如，卡片、晶片、卡 1 ，且可為單—整體元件或構成多組件殼體之-部 分0 該兩個至少部分對立表面可為基本上平行的表面。因 此’在-個實施例中，該兩個至少部分對立表面之—以例 如凹陷浮雕（SUnken re㈣或凸起浮雕（raised灿⑷方式界 定一通道，該兩個至少部分對立表面之另一者形成蓋或密 封件以封閉通道頂端。或者，該等表面以相對於彼此成小 於180之角度定位。因此’在一些實施例中，第一表面與 第二表面可彼此相鄰且相對於彼此成一定角度，以形成呈 「V」形之有角度通道。在再一些其他實施例中，該兩個 至v郤刀對立表面之一通常係平面的，且該等至少部分對 立表面之另-者可在絲面上以例#約9〇。或更小之角度 形成柱狀物或脊狀物，以形成「L」形。熟習此項技術者 應明瞭该專變體並不互斥且可以進行組人。 該兩個至少部分對立表面界定至少一條流體流動路徑。 流體流動路徑係用於將流體試樣自例如裝置之第一區域轉 148680.doc 201102654 移至裝置之第二區域。 在/實轭例中，该兩個至少部分對立表面界定開放的 s、、該毛細官通道具有足以產生開放通道之截面 幾何結構’該開放通道經構造以與該兩個至少部分對立表 面之第二表面形成封閉的毛細管通道。 匕該至夕一條流體流動路徑可為包含實體側壁且至 少部分由該兩個部分科W本 欣 丨刀對立表面界定之通道、凹槽、毛細 管、執道或路徑及諸如此類。 在另B轭例中，間隔元件經構造以使該兩個至少部分 對立表面相隔—定距離以在該等表面之間產生毛細管通 道。 該流動路徑具有p高開的頂部與底部表面但$具有實體側 壁，且取決於用於液體容納之疏水性區域可為開放的（例 如對於大氣）。因此，在該實施例中，表面通常係由間 隔體隔開之平面的、基本上平行之表面，且具有介於其間 或於其上界定之親水性路徑。在該情形下，試樣流體將沿 "玄親水f生路彳望藉由毛細管力於兩個表面之間（且與兩個表 面接觸）行進’但仍党疏水性區域/區限制。因 此”亥兩個至少部分對立表面及流體流動路徑可以多種方 式進行構造。 與構造無關，當試樣流體藉由毛細管力沿兩個表面（且 與兩個表面接觸）行進時，令人驚奇地，兩個表面之間的 表面能差異及進而疏水性/親水性差異使得試樣流體行進 時紊流之趨勢增加且不明顯減小流動速率。在某些實施例 148680.doc 201102654 中，且相對而言，在該兩個至少部分對立表面之一係上表 :且：兩個至少部分對立表面之另一者係下表面時，可設 、曰上表©係疏水性較差或親水性表面而下表面係親水 性較差或疏水性表面時，流體之紊流或「環流」可能進一 步增強。 在檢驗裝置之實施例中，該裝置進—步包含與裝置第一 區域流體連通以引入流體試樣之施加區域。… 施加p域係、設備上引人水性試樣之區域，且與流體流動 =流體連通。通常’存在單—施加區域，可自此獲取至 — 兩伤一伤、四份或更多份水性流體試樣分液用 於貫施檢驗。或者，諸多不同流體試樣或相同試樣之不同 分液可具有複數個單獨施加區域。 本文所用之術語「水性試樣」係指任何液體試樣，較佳 =可檢測其t之分析物之液體試樣。該等試樣之非限制性 實例包括全血、血清或血漿試樣、尿、腦脊趙液（⑶）、 淋巴、漿液狀滲出物或其他生物流體'動物組織勻漿、脫 去蛋白質之_”、乳汁、生蛋、發酵液、動物飼料及 海洋飼料。應瞭解，試樣僅需要在任—檢驗之至少實質部 分呈液體或水性形式。因此’熟f此項技術者可設想，檢 驗亦可在高於例如在室溫下通常呈固態之試樣(例如蠟)之 炫點的溫度下實施。 檢驗裝置可在該兩個至少部分對立表面之一中包含孔洞 以將流體試樣引入至施加區域。 3 / 該兩個至少部分對立表面之一或另一者可包含—或多個 148680.doc 201102654 孔洞以助於或能夠將試樣施加至施加區域同係與施加 區域流體連通之開口（opening)，且可為模製或鑽製開口或 簡單的由該兩個至少部分對立表面界定之開口。 檢驗裝置可進一步包含至少一個排氣口，其較佳位於裝 置之第二區域中。 排氣口通常係小孔洞，其較佳位於流體流動路徑之另一 端，即遠離第一區域，且使毛細管通道與裝置周圍之大氣 連接。排氣口容許空氣或流體自裝置内部移動至裝置外部 (或反之）以使裝置與大氣之間壓力平衡，否則該壓力會抑 制毛細管流動。 檢驗裝置可在裝置之第一區域與第二區 終止結，其經構造以中斷或中止行進於兩個表面 體的毛細管流體流動。 流動終止結係毛細管通道中在不施加來自外部的外力下 水性試樣通常不流動至其中的區域。該結不具有移動部 件，其通常依賴於來自液體試樣之表面張力的背壓來終止 流體流動。應瞭解，該背壓可以多種方式產生。例如，在 某些實施例中’背壓係藉由增加液體流動路徑(即毛細管 通道）之截面積來產生。截面積之增加可能較突然（例如當 小截面積U動路徑進入較大的非毛細管室時）或者係逐 漸的(例如’當流體流動路徑之壁在一段距離内呈錐形、 v刀岔或變寬時)。或者’結可藉由使用壁或屏障或藉由改 變裝置内部之表面特性（例如藉由物理及/或化學處理）以降 低水性試樣與流動路徑之壁之間的黏著力而例如形成較該 148680.doc 201102654 兩個至少部分對立表面中之任一者實質上更具疏水性之區 域來產生。流動終止結可在剩餘檢驗期限内完全終止流體 流動，或者其可僅使液體流動暫時延遲需要的時間段。 在某些實施例中，檢驗裝置進一步包含佈置於施加區域 與至少一條流體流動路徑之間的濾膜。 全血之細胞組份（例如紅細胞）可能干擾分析物檢驗（例如 膽固醇檢驗）。因此，當水性試樣係血液時，較佳地，在 分析之前將該等細胞組份自血清或血漿分離出來。理想 地’錄驗之第-步之前實施分離且無需手動處理步驟被 動地貫施。因& ’濾膜或濾墊起隨液體藉由毛細管作用行 進通過過濾器而移除水性試樣中之微粒（例如血細胞）之作 用。較佳地，餘包含至少_種吸收性材料或由至少一種 吸收性材料組成。 過：器通常容許在將水性試樣與用於實施檢驗之試劑組 合之前過渡試樣。過渡器可佈置於位於兩個表面之一中的 孔洞内且與施加區域流體 ••瓶埂逋/接觸。或者，其可位於施 加區域與流體流動路徑之間。 過渡器可由纖維性其睹加』、 基貝$成以阻止（例如）紅細胞移動。 適宜材料包括發泡體、玻 圾埽、、戴維（例如硼矽酸鹽）、溶膠過 慮益、層析介質，例如请 …，氏或濾膜，例如硝化纖維素、聚 砜或聚知。在一些實施例 過濾益可包含結合水性試樣 體二: 其移除的試劑，例如結合劑，例如抗 過渡器亦可包含—或多種預 表面活性劑/洗滌劑、檢 w 檢驗喊劑、染料、阻斷劑或配體結 148680.doc [ -11· 201102654 合抑制劑。阻斷劑及配體結合抑制劑可限制檢驗時水性試 樣之特定組份（例如人類血清白蛋白（HSA))之干擾。因 此込濾器可包含HSA提取構件（extraction means)(例如， 抗-HSA抗體）或利用例如沉;殿、固定及諸如此類之任何其 他構件。 ' 通系檢驗裝置之流體流動路徑包含至少一種選自由下 列組成之群之試劑：兩親（amphipathic)聚合物、染料、探 針、轉或配體結合抑制劑。 流動路徑可包含檢驗所需要之試劑。例如，染料、藥 物、發光團、抗體、核苦酸、核酸探針、引物、酶及諸如 此類。不同流動路徑可包含不同試劑或試劑組合。流動路 «W長度包含不同試劑’例如，容許在整個水性試 樣毛細管或側向流動期間依序添加試劑。 流體流動路徑可包含一或多種選自由Ampiex Red'K37 及尼羅紅（Nile Red)組成之群之染料或探針。The inspection device for the surface may take the form of multiple D's, for example, a card, a wafer, a card 1, and may be a single-unitary component or a component that constitutes a multi-component housing. The two at least partially opposing surfaces may be substantially parallel. surface. Thus, in an embodiment, the two at least partially opposing surfaces define a channel in a manner such as a recessed relief (Sunken re (4) or a raised relief (4), the other of the two at least partially opposing surfaces being formed a cover or seal to close the channel tip. Alternatively, the surfaces are positioned at an angle of less than 180 relative to each other. Thus, in some embodiments, the first surface and the second surface may be adjacent to each other and be opposite each other An angle to form an angled passage in the shape of a "V." In still other embodiments, one of the two opposing surfaces of the knife is generally planar, and the at least partially opposing surfaces are otherwise Columns or ridges are formed on the surface of the filament by an angle of about 9 〇 or smaller to form an "L" shape. Those skilled in the art should understand that the variants are not mutually exclusive and can be performed. The at least partially opposing surfaces define at least one fluid flow path. The fluid flow path is for moving the fluid sample from, for example, the first region of the device to 148680.doc 201102654 to the second region of the device. In the case of a solid yoke, the at least partially opposing surfaces define an open s, the capillary channel having a cross-sectional geometry sufficient to create an open channel, the open channel being configured to be opposite the at least partially opposite surface The two surfaces form a closed capillary channel. The fluid flow path may be a channel, a groove, a capillary, an obstruction or a path that includes a solid sidewall and is at least partially defined by the opposing surfaces of the two portions. And in another B yoke example, the spacer element is configured such that the at least partially opposing surfaces are spaced apart to create a capillary channel between the surfaces. The flow path has a top and bottom surface that is p open. However, $ has a solid sidewall and may be open depending on the hydrophobic region for liquid containment (e.g., for the atmosphere). Thus, in this embodiment, the surface is typically planar, substantially parallel, separated by a spacer. a surface having a hydrophilic path defined therebetween or on it. In this case, the sample fluid will be along the " It is expected that by capillary force to travel between two surfaces (and in contact with both surfaces), but still the party hydrophobic area/zone limitation. Therefore, at least some of the opposing surfaces and fluid flow paths can be constructed in various ways. Regardless of the construction, when the sample fluid travels along both surfaces (and in contact with both surfaces) by capillary forces, surprisingly, the difference in surface energy between the two surfaces and thus the difference in hydrophobicity/hydrophilicity Increasing the tendency of turbulent flow as the sample fluid travels and not significantly reducing the flow rate. In certain embodiments 148680.doc 201102654, and relatively speaking, one of the two at least partially opposing surfaces is tabulated: : When the other of the two at least partially opposite surfaces is the lower surface, the turbulent flow of the fluid may be set when the surface is less hydrophobic or hydrophilic and the lower surface is less hydrophilic or hydrophobic. The circulation may be further enhanced. In an embodiment of the testing device, the device further includes fluidly contacting the first region of the device to introduce an application region of the fluid sample. ... applying a p-domain system, an area on the device that introduces an aqueous sample, and is in fluid communication with fluid flow =. Usually there is a single-application zone from which it can be taken to - two wounds, one injury, four or more parts of the aqueous fluid sample for liquid testing. Alternatively, a plurality of different fluid samples or different liquids of the same sample may have a plurality of separate application zones. As used herein, the term "aqueous sample" means any liquid sample, preferably a liquid sample from which an analyte of t can be detected. Non-limiting examples of such samples include whole blood, serum or plasma samples, urine, cerebrospinal fluid ((3)), lymph, serous exudate or other biological fluid 'animal tissue homogenate, deproteinized_ ", milk, raw eggs, fermentation broth, animal feed and marine feed. It should be understood that the sample only needs to be at least a substantial part of the test in liquid or aqueous form. Therefore, the skilled person can imagine that the test can also Executing at a temperature above the glare of a sample, such as a wax, which is typically solid at room temperature. The inspection device may include a hole in one of the two at least partially opposing surfaces to introduce a fluid sample to the application Zone 3 / one of the at least partially opposing surfaces or the other may include - or a plurality of 148680.doc 201102654 holes to assist or enable application of the sample to the opening of the application zone in fluid communication with the application zone ( Opening, and may be a molded or drilled opening or simply an opening defined by the two at least partially opposing surfaces. The inspection device may further comprise at least one vent, preferably located at the device In the second region, the vent is typically a small hole that is preferably located at the other end of the fluid flow path, ie away from the first region, and that connects the capillary channel to the atmosphere surrounding the device. The vent allows for air or fluid self-assembly Internally moved to the outside of the device (or vice versa) to balance the pressure between the device and the atmosphere, otherwise the pressure will inhibit capillary flow. The inspection device may terminate the junction at the first and second regions of the device, which is configured to interrupt or abort Capillary fluid flow that travels between the two surface bodies. The flow terminates in the capillary channel in an area where the aqueous sample does not normally flow without external force applied from the outside. The junction does not have moving parts, which typically depend on the liquid The back pressure of the surface tension of the sample terminates the fluid flow. It will be appreciated that the back pressure can be produced in a variety of ways. For example, in certain embodiments the 'back pressure system' increases the cross-sectional area of the liquid flow path (ie, the capillary channel). The increase in cross-sectional area may be abrupt (for example, when the small cross-sectional area U-path enters a larger non-capillary chamber) Or gradual (eg 'when the wall of the fluid flow path is tapered, v-cut or widened over a distance). Or 'the knot can be changed by using a wall or barrier or by changing the surface properties of the interior of the device ( For example, by physical and/or chemical treatment to reduce the adhesion between the aqueous sample and the wall of the flow path, for example, forming substantially more hydrophobic than either of the at least partially opposing surfaces of the 148680.doc 201102654 An area of sexuality is created. The flow termination knot may completely terminate fluid flow for the remainder of the inspection period, or it may only temporarily delay the liquid flow for a desired period of time. In certain embodiments, the inspection device further includes an arrangement region and A filter between at least one fluid flow path. Cellular components of whole blood (eg, red blood cells) may interfere with analyte testing (eg, cholesterol testing). Therefore, when the aqueous sample is blood, preferably, the cell components are separated from the serum or plasma prior to analysis. Ideally, the separation was performed prior to the first step of the recording and no manual processing steps were required. The &' filter or filter pad acts to remove particles (e. g., blood cells) from the aqueous sample as it passes through the filter by capillary action. Preferably, the remainder comprises or consists of at least one absorbent material. The pass: typically allows for the transition of the sample prior to combining the aqueous sample with the reagent used to perform the test. The transitioner can be disposed in a bore in one of the two surfaces and fluid/contact with the application area. Alternatively, it may be located between the application zone and the fluid flow path. The transitioner can be made up of fiber, which is made to prevent, for example, red blood cell movement. Suitable materials include foams, rubies, davids (e.g., borosilicates), sol-overs, chromatographic media, such as, for example, filters or membranes such as nitrocellulose, polysulfone or poly. In some embodiments, the filtration benefits may include binding to the aqueous sample body 2: the agent it removes, such as a binding agent, such as an anti-transition device may also include - or a plurality of pre-surfactants / detergents, test agents, dyes , blocker or ligand knot 148680.doc [ -11· 201102654 combined inhibitor. Blockers and ligand binding inhibitors can limit the interference of specific components of the aqueous sample, such as human serum albumin (HSA), at the time of testing. Thus, the sputum filter can contain HSA extraction means (e. g., anti-HSA antibodies) or utilize any other components such as sinks, temples, and the like. The fluid flow path of the line-testing device comprises at least one agent selected from the group consisting of: an amphipathic polymer, a dye, a probe, a transfer or a ligand binding inhibitor. The flow path can include reagents required for testing. For example, dyes, drugs, luminophores, antibodies, nucleotides, nucleic acid probes, primers, enzymes, and the like. Different flow paths can include different reagents or combinations of reagents. The flow path «W length contains different reagents', for example, allows for the sequential addition of reagents throughout the capillary or lateral flow of the aqueous sample. The fluid flow path may comprise one or more dyes or probes selected from the group consisting of Ampiex Red'K37 and Nile Red.

Amplex Red (10_乙醯基_3,7_二經基吩。惡嗪）尤其可自 mvitrogen購得（目錄號為A12222及A22m)，立盥過氛化 氫(h2〇2)以Η化學計算比反應產生高螢光性試齒靈 Ο—in)。Κ37 (4-二甲基胺基米二氟曱基_石黃醢基-亞节 本發明者揭示於其先前國際專利申請案 PCT/GB2005/004757 中。尼羅 尼羅紅係親脂性染色劑，亦稱為 尼羅藍（Nile blu&惡嘻嗣’可自Invhr〇gen購得（目錄號為 NU42)或可藉由煮沸含有硫酸之尼羅藍溶液來產生。尼罹 紅將細胞㈣質小滴染成紅色’且亦具有強烈營光性，當 148680.doc 201102654 處於富含脂質環境中時發射強烈的黃-金色光。 流體流動路徑可包含一或多種選自由膽固醇酯酶、麟固 醇氧化酶及辣根過氧化物酶組成之群的酶。 膽固醇酯酶（固醇酯醯基水解酶，登記號：Ec 丨13) 係催化膽固醇酯及一些其他固醇酯水解以釋放膽固醇及脂 肪酸陰離子之酶。膽固醇氧化酶（膽固醇：氧氣氧化還原 酶，登兄號· EC 1.1.3.6)係催化膽固醇在分子氧存在下氧 化成4-膽留稀-3 -酮及過氧化氫的酶。辣根過氧化物酶 (Sigma Aldrich，登記號：EC ^丨丨丄乃係過氧化氫氧化還 原酶。熟習此項技術者亦習知其他等效過氧化氫氧化還原 酶，且可自例如大豆獲得。 在其他實施例中，流體流動路徑包含一或多種選自由鹼 金屬辛酸鹽及辛酸組成之群的配體結合抑制劑。適宜鹼金 屬辛酸鹽包括辛酸鈉及辛酸鉀。 本發明者已確定，染料/發光團可結合人類血清白蛋白 (HSA)之疏水性結合位點/結構域，且可在結合hsa時發螢 光。HSA係重要的血清組份，其濃度為約3〇_5〇 ^^/以丨。本 發明者認為，此額外螢光可造成顯著背景信號，其可能會 使量測失真並導致脂蛋白濃度測定具有誤差。 已知HSA具有至少兩類能夠結合各種配體之結合位點。 第一類在本文中稱為「疏水性結構域」，其結合脂肪酸， 而第二類結構域在本文中稱為「藥物結合結構域」（其由 兩種或更多種結構域組成）。此等結構域已為熟習此項技 術者所習知，並於 Nature Structural Bi〇1〇gy (V5 ] 148680.doc -13- 201102654 (1998))之一篇論文中彼此區別《此論文確定疏水性結構域 為可結合脂肪酸之結構域而藥物結合結構域能夠結合許多 可與HSA締合之藥物。 因此，在較佳實施例中，在試樣分析之前藉由添加配體 結合抑制劑來阻斷發光團可結合之HSA之疏水性結合位 點。 配體結合抑制劑可為疏水性的。抑制劑可為兩親性的。 配體結合抑制劑可包含脂肪酸或其功能衍生物以及其他疏 水性为子。可阻斷H S A之疏水性結合位點之適宜脂肪酸衍 生物的實例可包含脂肪酸、其酯、醯鹵、羧酸酐或醯胺 等。較佳脂肪酸衍生物係脂肪酸酯。 脂肪酸或其衍生物可包含C1-C20脂肪酸或其衍生物。較 佳地’脂肪酸或其衍生物可包含C3_C18脂肪酸或其衍生 物，更佳為C5-C14脂肪酸或其衍生物，且甚至更佳為匸7_ C9脂肪酸或其衍生物。尤佳地，配體結合抑制劑包含辛酸 (C8)或其衍生物，例如，辛酸鹽：較佳地，配體結合抑制 劑以鹼金屬辛酸鹽、較佳第〖族鹼金屬辛酸鹽（例如，辛酸 鈉或辛酸鉀）形式添加。 較佳地，在分析之前向試樣中添加約1〇_4〇〇 的配體 釔合抑制劑，更佳地添加約2〇_2〇〇 mM的配體結合抑制 劑，且甚至更佳地添加約30_80 mM的配體結合抑制劑。尤 佳地，添加約5〇 mM最終濃度之抑制劑。因此，在該方法 之-較佳實施例中，可在分析之前向試樣中添加約Μ感 辛酸納。 148680.doc •14- 201102654 在特定實施例中，亦可使用配體來阻斷HSA之藥物結合 結構域。HS A之藥物結合結構域之配體包括諸如下述藥物 刀子.曱狀腺素、布洛芬（ibuprofen)、***（diazepam)、 類固醇激素及其衍生物（藥物）、血紅素、膽紅素、親脂性 前藥、華法林（warfarin)、基於香豆素之藥物、麻醉劑、地 西泮、布洛芬及抗抑鬱藥（例如，噻噸）、苯曱酸或其衍生 物（例如三氣苯甲酸或三碘笨甲酸）、梭鏈孢酸（fusidic acid)或其衍生物。或者，可藉由使用HSA提取構件（例 如，抗-HSA抗體）來阻斷或移除hsa結合結構域。 檢驗裝置通常包含至少一個檢測區，其經構造以量測至 少一部分流體試樣與至少一種試劑間之反應的結果及/或 進程。 檢測區可為裝置之離散區域或區，或可僅為例如流體流 動路徑之一個區、點或區域，在其中測定或量測一部分水 性試樣（例如分析物）與至少一種試劑間之反應的結果及/或 進程。該至少一條流體流動路徑可將試樣流體自施加區域 轉移至檢測區。該至少—條流體流動路徑經構造以與至少 一個施加區域及檢測區流體連通。 在某些實施例中，設備包含複數條流體流動路徑及複數 個檢測區。通常，該至少—條流動路徑藉由例如流體連通 使施加區域或區與至少—個檢測區連接。通常，設備包含 單-施加區域及-條至人條、三條至五條或至多四條流動 路徑，該等流動路徑通向至少一個至八個、三個至五個或 至少四個檢測區。應清楚’該裝置可包含之流動路徑及檢 148680.doc 15 201102654 測區的數量可以變化，且該等流動路徑及檢測區可以具有 不同長度及/或體積。施加區域、流動路徑及/或檢測區通 常界定為毛細管通道之一部分或全部。 可藉助光學量測來測定發生於檢驗裝置上/檢驗裝置中 之反應或檢驗之結果及/或進程。或者，可藉由目測（例如 比色法）來測定反應之結果及/或進程。在其他實施例中， 藉助螢光來測定反應之結果及/或進程。 當藉由螢光來測定反應之結果及/或進程時，檢測區可 佈置成與檢驗讀出器之激發構件光學接觸。檢測區應進一 步佈置成可藉由檢驗讀出器之檢測構件來檢測自檢驗產生 之螢光。相同檢驗之不同態樣或不同檢驗可具有單獨檢測 區。通¥，檢測區在裝置之第二區域内或朝向裝置之第二 區域。 在一個實施例中，檢驗裝置包含至少三條流體流動路徑 及至少二個檢測區，其中，第一流動路徑與施加區域及第 一檢測區流體連通，第二流體流動路徑與施加區域及第二 檢測區流體連通且第二流體流動路徑與施加區域及第三檢 測區流體連通。 當檢驗裝置係用於量測膽固醇及血液脂質時，通常，第 一流體流動路徑將包含第一染料（例如Amplex Red)，第二 流體流動路徑將包含第二染料（例如K37)且第三流體流動 路徑將包含第三染料（尼羅紅）。在此情形下，第一流體流 動路徑可進一步包含膽固醇酯酶、膽固醇氧化酶及辣根過 氧化物酶。 148680.doc •16- 201102654 在膽固醇及血液脂質檢驗中使用酶較為有利，此乃因膽 固醇通常以酯化狀態發現，因此較佳使用膽固醇酯酶來水 解膽固醇酯以釋放膽固醇。隨後可藉由膽固醇氧化酶之作 用將游離膽固醇轉化成膽错_4_烯_3_酮，在該過程中會產 生過氧化氫。有利地，藉由辣根過氧化物酶將Amplex尺以 及過氧化氫轉化成試鹵靈及水。試_靈隨後可作為螢光化 合物進行檢測，其最大吸收在約563 nm處且峰值發射波長 為587 nm。總膽固醇含量可藉由在約485 nm下激發試樣並 在約600 nm下量測所得螢光來量測。 在一些實施例中，流體流動路徑可包含兩親聚合物。 本發明者已發現，可使用兩親聚合物來塗敷表面（例 如，塑膠或玻璃或疏水性材料）以進一步增強流體流動。 兩親聚合物係具有親水性及疏水性二者之聚合物。該化 合物亦可稱為兩親（amphiphiHc)化合物或非離子型親水性 聚S物在特疋Λ施例中，兩親聚合物係可溶於水及寬範 圍有機溶劑之物質。 可使用兩親聚合物來促進流體流動及/或水溶液與例如Amplex Red (10_Ethyl _3,7-di- thiophene. Oxazine) is commercially available from mvitrogen (catalog numbers A12222 and A22m) and is hydrogenated over hydrogen chloride (h2〇2). Calculate the specific reaction to produce a high-fluorescence test tooth Ο—in). Κ37 (4-Dimethylaminomethane difluoroindolyl) is disclosed in its prior international patent application PCT/GB2005/004757. Nile Nile red lipophilic coloring agent, also Nile blu & 嘻嗣 嘻嗣 can be purchased from Invhr〇gen (catalog number NU42) or can be produced by boiling a solution of Nile Blue containing sulfuric acid. Dyed in red' and also strongly luminescent, emitting intense yellow-gold light when 148680.doc 201102654 is in a lipid-rich environment. The fluid flow path may comprise one or more selected from the group consisting of cholesterol esterase, linalol oxidation An enzyme consisting of a group of enzymes and horseradish peroxidase. Cholesterol esterase (sterol ester thiol hydrolase, accession number: Ec 丨 13) catalyzes the hydrolysis of cholesterol esters and some other sterol esters to release cholesterol and fatty acid anions. The enzyme, cholesterol oxidase (cholesterol: oxygen oxidoreductase, Deng Xi, EC 1.1.3.6) is an enzyme that catalyzes the oxidation of cholesterol into 4-cholesteric 3-ketone and hydrogen peroxide in the presence of molecular oxygen. Root peroxidase (Sigma Aldrich, The symbol: EC ^ is a hydrogen peroxide reductase. Other equivalent hydrogen peroxide reductases are also known to those skilled in the art and are available, for example, from soybeans. In other embodiments, the fluid flow path Containing one or more ligand binding inhibitors selected from the group consisting of alkali metal octoate and octanoic acid. Suitable alkali metal octoates include sodium octanoate and potassium octoate. The inventors have determined that the dye/luminescent group can bind to human serum albumin. Hydrophobic binding site/domain of (HSA), and can be fluorescent when bound to hsa. The important serum component of HSA is about 3〇_5〇^^/丨. The inventors believe that This additional fluorescence can cause significant background signals that can distort the measurement and cause errors in lipoprotein concentration determination. It is known that HSA has at least two types of binding sites capable of binding to various ligands. Known as the "hydrophobic domain", which binds to fatty acids, and the second type of domain is referred to herein as the "drug binding domain" (which consists of two or more domains). These domains have been Familiar It is known to the skilled artisan and differs from each other in a paper by Nature Structural Bi〇1〇gy (V5) 148680.doc -13- 201102654 (1998). "This paper identifies hydrophobic domains as bindable fatty acids. The domain and the drug binding domain are capable of binding to a number of drugs that can be associated with HSA. Thus, in a preferred embodiment, the HLA of the luminescent group can be blocked by the addition of a ligand binding inhibitor prior to sample analysis. Hydrophobic binding sites. Ligand binding inhibitors can be hydrophobic. Inhibitors can be amphiphilic. Ligand binding inhibitors can comprise fatty acids or functional derivatives thereof, as well as other hydrophobic agents. Examples of suitable fatty acid derivatives which block the hydrophobic binding site of H S A may include fatty acids, esters thereof, hydrazine halides, carboxylic anhydrides or decylamines and the like. Preferred fatty acid derivatives are fatty acid esters. The fatty acid or derivative thereof may comprise a C1-C20 fatty acid or a derivative thereof. Preferably, the fatty acid or derivative thereof may comprise a C3_C18 fatty acid or a derivative thereof, more preferably a C5-C14 fatty acid or a derivative thereof, and even more preferably a 匸7_C9 fatty acid or a derivative thereof. More preferably, the ligand binding inhibitor comprises caprylic acid (C8) or a derivative thereof, for example, an octanoate: preferably, the ligand binding inhibitor is an alkali metal octoate, preferably a basal alkali metal octoate (eg , sodium octanoate or potassium octoate) is added. Preferably, about 1 〇 4 〇〇 of the ligand chelating inhibitor is added to the sample prior to the analysis, more preferably about 2 〇 2 〇〇 mM of the ligand binding inhibitor, and even better. A ligand binding inhibitor of about 30-80 mM was added. More preferably, an inhibitor of a final concentration of about 5 mM is added. Thus, in a preferred embodiment of the method, about sensitizing sensitized sodium can be added to the sample prior to analysis. 148680.doc • 14- 201102654 In certain embodiments, ligands can also be used to block the drug binding domain of HSA. Ligands of the drug binding domain of HS A include, for example, the following drug knives: scorpion spleen, ibuprofen, diazepam, steroid hormones and their derivatives (drugs), heme, biliary Erythramine, lipophilic prodrug, warfarin, coumarin-based drugs, anesthetics, diazepam, ibuprofen and antidepressants (eg, thioxanthene), benzoic acid or its derivatives ( For example, tris-benzoic acid or triiodobenzoic acid), fusidic acid or a derivative thereof. Alternatively, the hsa binding domain can be blocked or removed by using an HSA extraction construct (e. g., an anti-HSA antibody). The testing device typically includes at least one detection zone configured to measure the result and/or progression of a reaction between at least a portion of the fluid sample and at least one reagent. The detection zone can be a discrete area or zone of the device, or can be, for example, only a zone, point or zone of a fluid flow path in which a reaction between a portion of an aqueous sample (eg, an analyte) and at least one reagent is measured or measured. Results and / or processes. The at least one fluid flow path transfers sample fluid from the application zone to the detection zone. The at least one fluid flow path is configured to be in fluid communication with the at least one application zone and the detection zone. In some embodiments, the apparatus includes a plurality of fluid flow paths and a plurality of detection zones. Typically, the at least one flow path connects the application zone or zone to at least one detection zone by, for example, fluid communication. Typically, the apparatus includes a single-application zone and a strip to strip, three to five or up to four flow paths leading to at least one to eight, three to five or at least four detection zones. It should be clear that the flow path and inspection that can be included in the device can vary, and that the flow paths and detection zones can have different lengths and/or volumes. The application zone, flow path and/or detection zone are generally defined as part or all of the capillary channel. Optical measurements can be used to determine the results and/or progress of reactions or tests occurring on/in the test device. Alternatively, the results and/or progress of the reaction can be determined by visual inspection (e.g., colorimetry). In other embodiments, the results and/or progress of the reaction are determined by means of fluorescence. When the result and/or progress of the reaction is determined by fluorescence, the detection zone can be placed in optical contact with the excitation member of the test reader. The detection zone should be further arranged to detect the fluorescence produced by the test by examining the detection means of the reader. Different aspects of the same test or different tests may have separate detection zones. The detection zone is in the second zone of the device or towards the second zone of the device. In one embodiment, the inspection device includes at least three fluid flow paths and at least two detection regions, wherein the first flow path is in fluid communication with the application region and the first detection region, the second fluid flow path and the application region, and the second detection The zone is in fluid communication and the second fluid flow path is in fluid communication with the application zone and the third detection zone. When the assay device is used to measure cholesterol and blood lipids, typically, the first fluid flow path will comprise a first dye (eg, Amplex Red) and the second fluid flow path will comprise a second dye (eg, K37) and a third fluid The flow path will contain a third dye (Nile Red). In this case, the first fluid flow path may further comprise cholesterol esterase, cholesterol oxidase, and horseradish peroxidase. 148680.doc •16- 201102654 It is advantageous to use enzymes in cholesterol and blood lipid tests because cholesterol is usually found in an esterified state, so cholesterol esterase is preferred to hydrolyze cholesterol esters to release cholesterol. The free cholesterol can then be converted to the cholestyl-4_ene-3-one by the action of cholesterol oxidase, during which hydrogen peroxide is produced. Advantageously, the Amplex ruler and hydrogen peroxide are converted to resorufin and water by horseradish peroxidase. The test can then be detected as a fluorescent compound with a maximum absorption at about 563 nm and a peak emission wavelength of 587 nm. The total cholesterol content can be measured by exciting the sample at about 485 nm and measuring the resulting fluorescence at about 600 nm. In some embodiments, the fluid flow path can comprise an amphiphilic polymer. The inventors have discovered that amphiphilic polymers can be used to coat surfaces (e.g., plastic or glass or hydrophobic materials) to further enhance fluid flow. The amphiphilic polymer is a polymer having both hydrophilicity and hydrophobicity. The compound may also be referred to as an amphiphie compound or a nonionic hydrophilic poly S. In an exemplary embodiment, the amphiphilic polymer is soluble in water and a wide range of organic solvents. Amphiphilic polymers can be used to promote fluid flow and/or aqueous solutions with, for example

物除去了對於支揮載體（例如膜）的 膜等支撐載體，其 L液體。使用兩親聚合 需要，且具有如下效 148680.doc •17- 201102654 果.液體沿（例如）微管、表 行進較藉由單獨毛細管作用 因此，與藉由單獨毛細管作 親聚合物毛細管流動速率增 離。 面、疏水性表面及諸如此類之 之行進距離更遠或速度更大。 用所預期的相比，通過使用兩 加及/或流體流動/行進更遠距 人可使用兩親聚合物來塗敷流體流動路徑。或者，兩親聚 口物可呈流動路徑表面上之塗層或薄膜形式，或可呈流動 路徑空間（V〇id)或空腔（⑽ity)内之粉末、小球、微顆粒、 奈米顆粒、微微顆粒或填料形式。當兩親聚合物係空腔内 之真料時’其可完全填充空腔或可為具有例如縫隙之部分 填充。兩親聚合物可形成呈例如疏水性表面上之軌道或路 控形式（沿其發生流體流動）之流動路徑。例如，兩親聚合 物可印刷（例如藉由喷墨印刷或氣泡式噴墨印刷）、塗抹、 喷塗或施加至表面（例如平坦表面）上，以形成例如「軌 運」及/或層。可藉由例如混合將試劑與兩親聚合物組 合，或可將試劑佈置成位於兩親聚合物上方、下方或附近 的層。 t乙一醇（PEG)係兩親聚合物。peg之有用分子量包括 約600 Da至10,000 Da及約1〇〇〇 Da至3 000 Da。聚乙二醇亦 稱為聚環氧乙烷（PEO)或聚氧乙烯（p0E)，其係環氧乙院之 募聚物或聚合物。PEG之分子量可介於3〇〇 g/m〇1至 10,000,000 g/mol的寬範圍内。PEG具有以下一般結構： H0-(CH2-CH2-0-)n-H 〇 通常包括於PEG名稱中之數字表示其平均分子量。例 148680.doc -18- 201102654 如，n=80之PEG的平均分子量為約3500道爾頓，且稱為 PEG 3500 。 通常’ PEG包括具有一系列分子量之分子。儘管具有不 同分子量之PEG由於物理特性（例如黏性）不同而用於各種 不同應用’但其化學特性幾乎相同。端視聚合過程中所用 之起始劑，亦可得到不同形式之PEG，例如單官能團曱基 醚PEG(甲氧基聚（乙二醇）），縮寫為mPEG。亦可得到具有 不同幾何結構之PEG。具支鏈PEG具有3個至1〇個自中央核 心基團發散出之PEG鏈。星形PEG具有10個至1〇〇個自中央 核心基團發散出之PEG鏈。梳形PEG具有多個通常接枝至 聚合物主鏈之PEG鏈。亦可在稱為聚乙二醇化之過程中使 PEG共價偶合至其他分子，例如，此在利用peg之流體流 動特性用於試劑混合時可能有利。較佳地，當兩親聚合物 係聚乙二醇（PEG)時，其具有約1000 Da至20,000 Da、更尤 佳約1000 Da至6000 Da且再尤佳約1000 Da至3000 Da之分 子量。尤其有用之PEG 包括 PEG2000、PEG6000、PEG12000 及PEG20000 ° 兩親聚合物之另一實例係兩親多肽，即多肽具有二級結 構，由此多肽具有親水性面及疏水性面二者。兩親肽結構 (例如α-螺旋多肽）之設計已為熟習此項技術者所習知。例 如，Grell 等人，（2001) J Pept Sci 7(3):146-51 ; Chen 等 人，（2002) J Pept Res 59(1):18-33 ; Iwata等人，（1994) J boil Chem 269(7):4928-33 ; Cornut等人，（1994) FEBS Lett 349(1):29-33 ; Negrete等人，（1998) Protein Sci 7(6):1368- 148680.doc •19- 201102654 79。其他兩親或非離子型聚合物包括聚乙烯醇（pvA) (Sigma Aldrich: 360627-25G)、羧甲基纖維素（Sigma:匕 5678) ; 雙（2-胺基乙基）pEG 2〇〇〇(聚氧乙烯雙 (胺））（Aldrich Chemistry: 14501)&pEG 曱基醚 5〇〇〇 ⑷如化The support carrier for a support carrier (e.g., a membrane) is removed, and the liquid is L liquid. The use of amphiphilic polymerization is required and has the following effect: 148680.doc • 17- 201102654. The liquid travels along (for example) microtubules, the surface is moved by capillary action alone, and the capillary flow rate is increased by the capillary tube alone. from. Faces, hydrophobic surfaces, and the like travel a greater distance or speed. The amphiphilic polymer can be used to coat the fluid flow path by using two additions and/or fluid flow/travel further than would be expected. Alternatively, the amphiphilic agglomerate may be in the form of a coating or film on the surface of the flow path, or may be in the form of a flow path space (V〇id) or a cavity ((10)ity) of powder, pellets, microparticles, nanoparticles. , in the form of microparticles or fillers. When the amphiphilic polymer is in the cavity of the cavity, it can completely fill the cavity or can be filled with a portion having, for example, a slit. The amphiphilic polymer can form a flow path in the form of, for example, a track or a controlled flow on the hydrophobic surface along which fluid flow occurs. For example, the amphiphilic polymer can be printed (e.g., by ink jet printing or bubble jet printing), smeared, sprayed, or applied to a surface (e.g., a flat surface) to form, for example, "rail" and/or layers. The reagent may be combined with the amphiphilic polymer by, for example, mixing, or the reagent may be disposed as a layer above, below or adjacent to the amphiphilic polymer. t-Ethyl alcohol (PEG) is an amphiphilic polymer. Useful molecular weights for peg include from about 600 Da to 10,000 Da and from about 1 Da to 3 000 Da. Polyethylene glycol, also known as polyethylene oxide (PEO) or polyoxyethylene (p0E), is a polymer or polymer of epoxy. The molecular weight of PEG can range from 3 〇〇 g/m 〇 1 to 10,000,000 g/mol. PEG has the following general structure: H0-(CH2-CH2-0-)n-H 〇 The number generally included in the PEG name indicates its average molecular weight. Example 148680.doc -18- 201102654 For example, a PEG having n = 80 has an average molecular weight of about 3500 Daltons and is referred to as PEG 3500. Typically 'PEG comprises a molecule having a range of molecular weights. Although PEGs having different molecular weights are used in a variety of different applications due to differences in physical properties (e.g., viscosity), their chemical properties are almost the same. The PEG can be obtained in a different form depending on the initiator used in the polymerization, for example, a monofunctional mercaptoether PEG (methoxypoly(ethylene glycol)), abbreviated as mPEG. PEGs with different geometries are also available. Branched PEG has from 3 to 1 PEG chains emanating from a central core group. Star PEG has 10 to 1 PEG chains emanating from a central core group. Comb shaped PEG has a plurality of PEG chains which are typically grafted to the polymer backbone. It is also possible to covalently couple PEG to other molecules during a process known as pegylation, for example, which may be advantageous when utilizing the fluid flow characteristics of peg for reagent mixing. Preferably, when the amphiphilic polymer is polyethylene glycol (PEG), it has a molecular weight of from about 1000 Da to 20,000 Da, more preferably from about 1000 Da to 6000 Da, and even more preferably from about 1000 Da to 3000 Da. Another useful example of PEG comprising PEG 2000, PEG 6000, PEG 12000 and PEG 20000 ° amphiphilic polymers is an amphiphilic polypeptide, i.e., the polypeptide has a secondary structure whereby the polypeptide has both a hydrophilic side and a hydrophobic side. The design of amphiphilic peptide structures (e.g., alpha-helical polypeptides) is well known to those skilled in the art. For example, Grell et al., (2001) J Pept Sci 7(3): 146-51; Chen et al., (2002) J Pept Res 59(1): 18-33; Iwata et al., (1994) J boil Chem 269(7): 4928-33; Cornut et al., (1994) FEBS Lett 349(1): 29-33; Negrete et al., (1998) Protein Sci 7(6): 1368-148680.doc • 19- 201102654 79. Other amphiphilic or nonionic polymers include polyvinyl alcohol (pvA) (Sigma Aldrich: 360627-25G), carboxymethylcellulose (Sigma: 匕 5678); bis(2-aminoethyl)pEG 2〇〇 〇 (polyoxyethylene bis(amine)) (Aldrich Chemistry: 14501) & pEG decyl ether 5 〇〇〇 (4)

Chemistry: 81323-250G)及其他離子型聚合物。 使用兩親聚合物（尤其與所述實施例組合）可進—步幫助 將檢驗所花費之時間自數小時或甚至數天縮短至短至一分 鐘左右。 在特定實施例中，冑用該檢驗$置來製作脂f >佈1 或實施膽固醇及脂蛋白檢驗。 可使用該檢驗裝置藉由自患者取樣且隨後在取樣地點杏 施檢驗來獲得患者的脂質分佈。因此，錄驗裝置應適^ 可為任何生物流體夕钟接，Α.Ι »_ .Chemistry: 81323-250G) and other ionic polymers. The use of amphiphilic polymers (especially in combination with the described examples) can further help reduce the time taken for testing from hours or even days to as short as about one minute. In a particular embodiment, the test is used to make a fat f > cloth 1 or to perform a cholesterol and lipoprotein test. The test device can be used to obtain a lipid profile of a patient by sampling from a patient and then performing an apricot test at the sampling site. Therefore, the recording device should be suitable for any biological fluid, Α.Ι »_ .

.，、淋巴等。 例如膽固醇、 一或多者的濃 、沿pi 楚., lymph, etc. Such as cholesterol, one or more of the thick, along the pi Chu

( ，赝穿透構件， ，例如習用刺血針裝 148680.doc -20- 201102654 置，其較佳包含安全套筒。或者，針可為包括筒體及活塞 之習用注射器組件之一部分；（iii)本發明第一態樣之檢驗 裝置；（iv)用以覆蓋皮膚刺傷傷口之紗布或絆創膏 (adhesive pUster) ; (v)提供有關裝置使用之細節的說明 書，（v〇用以避免血液接觸之用即棄型手套；（vii)檢驗讀 出器。套組組件可於包裝構件（例如間隔化的卡紙板或塑 膠外殼，其較佳具有可氣密性密封的蓋子）中提供。 【實施方式】 ' 圖3a展示可用於例如測定血液中之脂蛋白含量之本發明 檢驗裝置的一個實施例。 在此實施例中，檢驗裝置包含第一支撐表面（1)。支撐 表=常包含不透明材料或由不透明材料組成例如納入 疋里顏料（例如炭黑）之塑膠材料。在此情形下，支撐表 面係由與螢光、發光或光度計量測相容之醫藥級聚合物形 成。：狀烯烴聚合物通常具有出色之機械特性、低自發螢 ^门V透射。適且聚合物包括環狀烯烴共聚物，例如 丁啊⑧ C〇C(CAS 號為 2_7-43-2)、Ze_r® COP、 eonex® COP 或 Udel® 聚碾（CAS 號為 25 135_517)。在此 例中支係由包含1%炭黑之T〇pAs 形成。使用 黑具有額外優點，例如’在雷射焊接期間，對功率之要 :低、增加了裝置之操作簡易性並保護反應室中之熱不 m ^使用厌黑亦可具有與熱耗散/絕緣有關之益處。 向』膠中納八顏料（例如炭黑）會改變該等塑膠之表面 及進而其疏水性/親水性。此具有以下優點：$需要表 148680.doc 201102654 修飾（例如化學修飾）來在第一支撐表面（1)與第二表面 (2)(在此貫例中為遮盍部件）之間產生表面能差異。 該實施例之支樓表面係模製得到此項技 術者所習知之任一標準模製或機械加工技術來形成。儘管 其大致平坦，但其包含異型區域（profiled area)，在此凹陷 浮雕（sunken-relief)實例中，其形成沿施加區域（4)之圓周 佈置之（在此情形下四個）毛細管通道（3)。在此情形下，四 個通道中之每一者以約90。角度等距離彼此間隔。 所模製支撐表面形成施加區域（4)、流體流動路徑（5)及 檢測區（6)。 距離施加區域（4)最遠之流體流動路徑（5 )的各末端之特 徵在於一或多個毛細管通道壁逐漸呈錐形。此錐形之寬度 及/或深度增加且進而毛細管流動通道之截面積增加，形 成流動終止結（7)而阻止進一步毛細管流體流動。此處，通 道形成鱗莖狀末端’但亦可採取多種其他形式。界定施加 區域、流體流動路徑、檢測區及流動終止結之毛細管通道 的結構更精密地顯示於圖扑中。熟習此項技術者應瞭解， 通道可以任何形式佈置或呈不同形狀以適應具體檢驗。例 如且作為非限制性實例，通道可呈卵形或「淚滴」形、梯 形、三角形、圓柱形或管形。更多實例顯示於圖4中。對 於醫院、實驗室或大體積使用，可構想在光碟大小及形狀 之檢驗裝置上佈置1〇_2〇條、2〇_4〇條、4〇 6〇條或5〇1〇〇條 毛細管通道。 圖5展示流動終止結之更多實施例。在圖中，在朝向 148680.doc -22- 201102654 达私變乍或收纟但以形成截面積減小之流體流動路經部八 (13)之前’流體流動路徑（5)沿實質上其全部長度保持同等 截面積。此流體流動路徑部分又與室（14)相鄰且流體連 通。該室由低壁或屏障（15)分割成區—在此情形下分割成兩 部分（15a)及（15b)。使用時，流體進入室〇4)填充部分 (15a) 〇壁（15)或更準確而言作用於壁區域中之試樣流體之 毛細管力阻止流體流至室之第二部分（15b)中。在圖外中， 壁由凸起部分(16)代替。再次用於凸起部分(16)區域 中之試樣流體之毛細管力阻止流體填充整個室。熟習此項 技術者應認識到，室（14)及壁（15)或凸起部分（16)之構造 (例如大小、形狀、高度）可採取多種形式，包括不 =(赝, a penetrating member, such as a conventional lancet device 148680.doc -20- 201102654, preferably comprising a safety sleeve. Alternatively, the needle may be part of a conventional syringe assembly including a barrel and a piston; a test device according to a first aspect of the invention; (iv) a gauze or an adhesive pUster for covering a wound of a skin; (v) providing instructions on the details of the use of the device, (v) for avoiding blood contact A disposable glove; (vii) an inspection reader. The kit assembly can be provided in a package member, such as a compartmentalized cardboard or plastic casing, preferably having a hermetically sealable lid. Figure 3a shows an embodiment of the testing device of the invention that can be used, for example, to determine the amount of lipoprotein in the blood. In this embodiment, the testing device comprises a first support surface (1). The support table = often contains opaque material or Composition of an opaque material such as a plastic material incorporating a pigment (such as carbon black). In this case, the support surface is a medical grade polymerization compatible with fluorescence, luminescence or photometric measurement. Formation: The olefin polymer usually has excellent mechanical properties and low spontaneous V-transmission. Suitable polymers include cyclic olefin copolymers such as Ding 8 C〇C (CAS No. 2_7-43-2) ), Ze_r® COP, eonex® COP or Udel® poly-grinding (CAS number 25 135_517). In this case the branch is formed from T〇pAs containing 1% carbon black. The use of black has additional advantages, such as 'in thunder During the welding process, the power requirement is low, the operation ease of the device is increased, and the heat in the reaction chamber is protected. The use of blackening may also have the benefits related to heat dissipation/insulation. Pigments (such as carbon black) change the surface of the plastic and thus its hydrophobicity/hydrophilicity. This has the following advantages: $ 148 680.doc 201102654 modification (eg chemical modification) to the first support surface (1) A difference in surface energy occurs between the second surface (2) (in this case, the concealing member). The surface of the floor of this embodiment is molded to obtain any standard molding or machining known to those skilled in the art. Technology to form. Although it is roughly flat, it contains different A profiled area, in this example of a sunken-relief, which forms (in this case four) capillary channels (3) arranged along the circumference of the application zone (4). In this case, four Each of the channels is spaced apart from each other by an angle of about 90. The molded support surface forms an application zone (4), a fluid flow path (5), and a detection zone (6). The farthest distance from the application zone (4) Each end of the fluid flow path (5) is characterized in that one or more capillary channel walls are gradually tapered. The width and/or depth of the cone increases and thus the cross-sectional area of the capillary flow channel increases, forming a flow termination junction ( 7) Prevent further capillary fluid flow. Here, the channels form bulbous ends' but a variety of other forms are also possible. The structure of the capillary channel defining the application zone, the fluid flow path, the detection zone, and the flow termination junction is more closely shown in the map. Those skilled in the art will appreciate that the channels can be arranged in any form or in different shapes to accommodate a particular inspection. For example and by way of non-limiting example, the channels may be oval or "teardrop" shaped, trapezoidal, triangular, cylindrical or tubular. Further examples are shown in Figure 4. For hospitals, laboratories or large-volume use, it is conceivable to arrange 1〇_2〇, 2〇_4〇, 4〇6〇 or 5〇1〇〇 capillary channels on the inspection device of the size and shape of the disc. . Figure 5 shows a more embodiment of a flow termination junction. In the figure, before the 148680.doc -22- 201102654 reaches the enthalpy or shrinks but forms the cross-sectional area of the reduced fluid flow path through the eight (13) 'the fluid flow path (5) along substantially all of it The length maintains the same cross-sectional area. This fluid flow path portion is in turn adjacent to the chamber (14) and is in fluid communication. The chamber is divided into zones by a low wall or barrier (15) - in this case divided into two parts (15a) and (15b). In use, fluid enters chamber 〇4) fill portion (15a) The capillary wall (15) or more precisely the capillary force of the sample fluid acting in the wall region prevents fluid from flowing into the second portion (15b) of the chamber. In the outside of the figure, the wall is replaced by a raised portion (16). The capillary force applied again to the sample fluid in the region of the raised portion (16) prevents the fluid from filling the entire chamber. Those skilled in the art will recognize that the configuration (e.g., size, shape, height) of chamber (14) and wall (15) or raised portion (16) can take a variety of forms, including not =

以使裝置流體流動路徑内之壓力平衡。 支樓表面亦包香多個對準柱⑻則吏支撐表面與第二表 面與第二表To balance the pressure within the fluid flow path of the device. The surface of the branch is also covered with a plurality of alignment posts (8), the support surface and the second surface and the second table

之激發/發射波長。 遮蓋部件（2)亦係模製得到，且 洞。遮蓋部件可以支撐部件為底座 於其間之間隔元件。 且通常平坦且包含多個孔 1座’視情況包含塾圈或位 孔洞（9)與施加區域對準且容許使用者 148680.doc -23- 201102654 向施加區域中施加流體試樣。更多-系列小孔洞用作排氣 口 （10)。 在所示實施例中，對準孔（11)藉由容納對準柱（8)而相對 於支撐部件來定位遮蓋部件。在替代實施例中，孔洞及對 準孔提供於支撐部件中，冑蓋部件僅僅發揮遮蓋作用。 。對準7C件可簡單地為凸起部件與對應對準孔或凹槽。支 撐部件與遮蓋部件可藉由例如機械方式（例如藉由使曰用螺 絲釘、鉚釘、螺栓或接頭片）固定或聯結在一起。或者， 支㈣件與遮蓋部件可藉由摩擦配合而結合在一起。在其 貫包例中it蓋部件與支樓部件係、藉由例如使用膠水、 溶劑、膠帶及諸如此類而聯結在一起。在此情形下，遮蓋 部件與支撐料係藉φ使用熱焊接及雷射焊接而聯結在一 起。 支撑部件及遮蓋部件之一或二者可包含識別構件，以提 供唯-標識及/或提供一種手段以供檢驗讀出器用以例如 監測檢驗裝置之已使用次數或試樣之施加時間以度過合適 檢驗時間。該等識別構件亦可含有說明書或諸如校準或品 質控制數據等數據。識別構件可採取多種形式，包括（但 不限於）文字、盲人點字、數字數據、一維條碼、二維條 碼' RFID標滅及諸如此類。 過渡器（12)穩固地保持在支樓部件與遮蓋部件之間，與 施加區域（4)及孔洞（9)及流體流動路徑相鄰並流體連通。 時將未經稀釋或經稀釋試樣經由孔洞（9)直接施 加至過濾器中。 1486S0.doc •24- 201102654 該檢驗裝置可用以測試全血、靜脈血，其例如藉由手指 刺取獲得或在新生兒情形下藉由足跟刺取來獲得。此係打 開小傷口之方式，例如在指尖，此產生不多於數滴血液 (約小於50 μΐ ’例如約1〇 μΐ至2〇 μΐ)。在形成血滴後，可 直接將其施加至檢驗裝置之施加區域或藉由吸管抽吸並相 應地隨後施加。使用藉由例如手指刺取所獲得之血液的能 力直接代表優於熟習此項技術者所習知之檢驗的顯著優 點。 或者，可使用檢驗裝置來測試源自血漿之試樣，在此情 形下可對試樣實施1:80稀釋。通常，利稀釋劑係構酸鹽 緩衝鹽水(PBS)’且其可包含至少一種配體結合抑制劑。 稀釋劑亦可包含兩種配體結合抑制劑，一種用於疏水性結 合位點且一種用於藥物結合位點。 試樣中之流體移動通過過濾器，且隨此進行，紅細 (在全企情形下）及較大微粒藉由被動過渡而被移除。由 降低了由該等細胞或較大微粒而弓丨起之背景干擾。在該 例中，過慮器進-步包含繼提取構件，其包含如前文 述移除試樣令之HSA的抗.脱抗體，再次降低背景。 六流體藉助毛細管流動自施加區域（4)行進至與施加區 ^體連通之流體流動路郎）中。隨其行進，液體使乾燥 ^试雜如螢光染料及酶）水合並與其混合1此， 如側向流動而移動，並不需要施加外朴 幫為）來移動水性試樣。 所用術語「乾燥形式」係指組份保持通常實質上不含」 S ] 148680.doc -25- 201102654 弄空液體或水分之形式；即其在藉由實施檢驗本身而重構 時、而非在檢驗程序之前及獨立於檢驗程序而重構時呈溶 液形式。因此，水性試樣本身重構乾燥試劑，由此消除了 對於單獨重構緩衝液及步驟之需要。 當使用酶或其他試料，尤其#以乾燥形式使用時，較 佳穩定化該等酶或其他試劑。在本發明之上下文中，「經 穩定化試劑」係就例如儲存穩定性、熱穩定性等而言具有 改良之穩疋f生之„式劑。因此，在特定實施例中，所用試劑 I 3穩疋劑具體穩疋化方法揭示於國際專利申請案第 WO 9_05182號及第w〇 91/〇14773號中其内容以弓|用 方式併入本文中。其他適宜穩定劑包括乙婦基料。定綱盘 甲基丙烯酸二甲基胺基乙基醋之共聚物，其例如由 International Speciality Products^ ^ Qafquat (RTM) ώ 售。GafquaKCAS 登記號：53633_54_8 ; 7732魯5)係一系 列水浴性共聚物（例如聚四級銨鹽_丨〗）之名稱。 流體試樣所接觸之支#部件與遮蓋部件之間表面能之差 異使流體與乾燥試劑之混合增強。 隨著前面流體向前移動，通過排氣口排出等體積空氣， 裝置内塵力平衡。在流體到達流動終止結後，表面張力阻 止進一步毛細管流動。 在此Ρό /又，可將裝置放置於適宜檢驗讀出器内，並量測 分析物（在此情形下為膽固醇及血液脂質）之含量。 &檢驗裝置之殼體通常經調整能夠放置得與檢驗讀出器功 能連通。例如，可將檢驗裝置***讀出器中、放置於讀出 I48680.doc •26- 201102654 器上或附接至讀出器，且讀出器可包含塢站構件（d〇cking means)(例如插槽）或對準構件以使檢驗裝置能夠適當地插 入、放置或附接。在此實施例中，檢驗裝置之遮蓋部件中 具有「V」形切口，此有助於檢驗裝置與讀出器對準。通 苇，本發明檢驗裝置係用即棄型，但是讀出器通常可重複 使用。 該檢驗裝置可用於多種檢驗方法或反應，例如免疫檢驗 及螢光檢驗，包括贍固醇、脂蛋白或三酸甘油酯檢驗。 免疫檢驗係量測水性試樣（例如血清或尿）中之物質之濃 度的生物化學測試。該檢驗利用抗體與其抗原之反應利用 抗體與其抗原之特異性結合。較佳使用單株抗體，此乃因 其結合特定分子之一個位點，可提供特定及準確測試。可 量測抗原或抗體二者之存在，例如，當檢測感染時，可量 測對抗病原體之抗體的存在。或者，當量測諸如激素及諸 如此類等生物分子時，激素生物分子可作為抗原。可將所 量測水性流體之響應與習知濃度之標準物進行比較，例 如在圖上!會製標準曲、線。抗體或抗原之數量的檢測可藉 由多種方法來達成，例如對抗原或抗體進行標記。作為非 限制性實例，標記可由酶（EIA或EUSA)、放射性同位素 (例如1-125)、磁性標記或發光或螢光標記組成。 有利地’本發明裝置依賴於毛細管流動來實施低體積試 樣之流體傳送，且因此不需要使用移動部件。因此，該裝 置克服了先前技術中所遇到之規模、經濟、製造及機械故 障專問題。 148680.doc •27- 201102654 與本發明檢驗裝置—起^吏用之檢驗讀出器可適於接受例 如來自多名患者或用於來自個別患者之水性試樣之多項測 試的兩個或三個檢驗裝置。該讀出器可包含兩個(或更多 個）能夠與檢驗裝置之檢測區對準之激發構件。「激發構 件」可操作以激發檢測中之試樣，例如，使其發螢光。該 。又備亦包含至少一種可操作以檢測例如在檢測區由試樣發 射之螢光的檢測構件。 通常’激發構件包含可操作以在約4〇〇 nm_6〇〇 nm下照 射武樣之照射源。因此，光源較佳能夠在約400 nm 600 nm下照射試樣。照射源可包含一或多個燈泡、或一或多個 LED、或其他來源（例如一或多個雷射器）。激發波長可使 用至夕種干擾過遽斋來改變。激發構件亦可包含可操作 以偏振由照射源所產生之光的偏振構件。激發構件亦可進 步包合適於將光聚焦於試樣上之聚焦構件。聚焦構件可 包含透鏡或光導裝置，例如光纖絲或光學薄膜（3M)。 檢測構件可包含光電二極體' CCD、或光電倍增管或光 學感測器（其較佳對黃色-紅色敏感）。試樣發射之營光端視 所用染料可在包括約440 nm-650 nm在内之範圍内檢測。 檢測構件應能夠檢測在約490 nm、約495 nm、約57〇 nm、 約600 nm及約610 nm下發射之螢光。螢光可藉由第二透鏡 收集，且可通過偏振器。散射之激發光可藉由截止過濾器 或帶通過濾器來移除。量測螢光強度時，來自光電二極體 之電流或來自光電倍增管之計數率可自電流計、電壓計或 汁數率計模塊讀出。熟習此項技術者易知其他方式。 M8680.doc •28- 201102654 讀出器亦可包含激發修正系統，以將光源之波動考慮在 /内」亥《又備可包含至少一種用於在測定分析物濃度之前進 行杈準之螢光標準物。標準物可為内標準物。 在特定實施例中’檢驗讀出器經構造可在檢驗裝置進入 讀出器時或在此後-定時間同時或依次檢測及量測單個或 複數個檢驗之螢光強度。 胃出ϋ亦可包含適於基於所檢測之螢光來敎試樣中之 分析物之濃度的處理構件。 讀出器可進-步包含用於顯示自試樣所測定之量測值的 顯示構件，較佳以讀出形式。例如，顯示構件可包含lcd 螢幕，或可依賴於電腦來供電及/或計算及/或顯示。以其 最基本形式’顯不構件可簡單地為顯示指示器讀數或量測 值之窗口。 通笔，檢驗項出器係便攜式的，且有利地，檢驗裝置及 璜出器可用以簡單、快速且同時地實施檢驗，以測定諸如 生物流體等水性試樣之分析物的存在/不存在或濃度。例 如，具有膽固醇、脂蛋白及HDL濃度知識之臨床醫師可使 用該裝置來決定有效治療過程。另外，檢驗裝置及讀出器 係便攜式的，且可由GP或出診護士使用，或甚至可以測試 套組形式供家庭使用。 在此情形下，處理構件適於基於螢光分析直接測定試樣 中例如膽固醇、三酸甘油酯、HDL、LDL、VLDL& mL中 之一或多者的濃度。或者，處理構件可適於基於總脂蛋 白、膽固醇及HDL之濃度計算試樣中、VLDL& ΙΓ)[的 [S] 148680.doc 201102654 濃度。 裝置在膽固醇及脂質分析中之用途 以下實例闡述使用本發明檢驗裝置來量測水性生物試樣 中之脂蛋白的方法。 在該實例中，給流體流動路徑塗敷以兩親聚合物PEQ以 加速流體傳送。對於脂質分佈，亦將兩親聚合物與諸如 Amplex Red、K3 7或尼羅紅等螢光染料及/或諸如酶等其他 試劑組合，塗佈或印刷在流體流動路徑内或檢測區内。 螢光染料及/或其他試劑可以一或多個微微升小滴陣列 形式印刷。適宜地，該陣列可包含沿第一軸線之約15〇個 至4500個小滴乘以沿第二軸線之25個至1〇〇個小滴。具體 陣列大小包括每爪!!!2約3400個x65個小滴、約”㈧個乂“個 小滴、約1500個χ65個小滴、約19〇〇個χ65個小滴、6〇〇個 Χ65個小滴、450個χ65個小滴或約4〇〇個χ65個小滴。熟習 此項技術者應瞭解，該等小滴密度之變化對檢驗性能具有 極小影響，但亦應瞭解，反過來說，亦可對該等密度進行 最優化以改良檢驗性能。例如，該等小滴亦可以奈升或毫 微微升小滴形式施加，以重疊ρ車列形式施加以一個（或 多個）接一個（或多個）摞在一起之形式施加，以間隔分離之 離散個別陣列形式施加或以形成較大大小陣列之數個陣列 之區塊形式（即累積）施加。亦可針對例如特定試劑或染料 濃度對陣列大小進行最優化。 * 17刷」時，可將螢光染料小滴以約〇.丨mM至3 〇 福、更適宜地約0.3囊至2.5福、且甚至更適宜地約〇·5 148680.doc -30- 201102654 祕至2.0 mM之濃度施加至裂置。或者，當I置係用於經 稀釋血液試樣時，螢光染料之濃度可為約〇8 2 -M。螢光染料之可用濃度係1〇邊’且對於未經稀釋之 血液試樣，可用濃度係約2.G mM。隨後乾燥㈣，然後使 用0 使用時，試樣流體使兩親聚合物水合並將螢光染料稀 釋，螢光染料隨後可與試樣中之脂蛋白結合。如此結合 時，染料在適當激發下發螢光。試樣中之總脂蛋白濃度可 利用螢光分析來測定。 該方法通常包含： ⑴使水性生物試樣與至少一種染料或發光團及視情況 至少一種兩親聚合物接觸’其中，該至少一種染料或發光 團結合水性生物試樣中之至少一種脂蛋白，且當與其結合 時在適當激發下發螢光； (II)在約400 nm-620 nm間之激發波長下激發來自步驟⑴ 之產物； (iii)在約440 nm-650 nm間之波長下量測步驟（H)之後的螢 光發射。 可使用該方法來製作水性生物試樣之脂質分佈。 所用術語「總脂蛋白」意指至少VLDL、HDL、LDL、 IDL及乳糜微粒之總體濃度，換言之，試樣中三酸甘油酯 及總膽固醇之濃度的和。所用術語「總膽固醇」意指試樣 中膽固醇之總濃度。所用術語「脂質分佈」意指試樣中脂 質組份（即總脂蛋白及總膽固醇及三酸甘油酯）之濃度或相 [ 148680.doc •31 · 201102654 對濃度》 2在於血液或血清試樣中之大多數脂質結合脂蛋白。臨 床只驗至中貫知之習用測試不量測總脂蛋白。因此，按照 慣例’需I首先測定膽固醇及膽固醇醋之濃度及三酸甘油 s旨之濃度，並隨後相加來測定總脂蛋白濃度。臨床實驗室 中2酸甘油醋之習用量測具有相當大誤差，此乃因其依賴 於里測血液中天然循環之甘油。有利地，由於直接量測脂 蛋白顆粒之數量（體積）來測定總脂蛋白濃度（等於總脂質濃 度）’故本發明之膽固醇檢驗不具有該誤差。因此，可避 免由试樣中之循環甘油引起之例如三酸甘油自旨濃度的誤 差。 在先則國際專利申請案PCT/GB2005/004757(以WO 006/061646公佈）中’本發明者提出基於使用例如7來 里測生物大分子中之脂蛋白的簡化檢驗，其在臨床醫師希 望快速且有效地獲得脂質分佈的情況下尤其有用。對於使 用Κ37螢光里測來測定血液試樣中總脂蛋白（即、 L IDL& VLDL)之濃度而言，本發明者認識到，對於 …疋總知蛋白濃度，結合各脂蛋白種類之κ37的螢光響應 (即總月曰蛋白濃度)可能基本上相同，而與其組成(即，試 樣中HDL:LDL:IDL:VLDL之比率）無關。因此，較佳之情 /係以如下方式使用K3 7 :螢光強度響應在臨床測試中所 遇到之試樣的整個所預期脂蛋白分子濃度範圍内實質上呈 線性。 不々人又限於任何假設，吾人認為來自螢光染料之螢光強 148680.doc -32· 201102654 度取決於其對試樣中 伤干特疋月曰蛋白分子（HDL、LDL、IDL或 )之親和力。螢光之量子產額取決於脂蛋白分子複合 物内之^兄以及由緊密堆積在-起的探針分子間之能量轉 移引起之螢光猝減程度。因&，在先前申請案中，本發明 者推：、’可選擇可用於藉由簡單螢光量測來進行準確總脂 蛋白里測之適宜探針物質濃度及激發及發射波長。本發明 者進一步認朗，由於探針對HDL具有較高親和力，故與 及LDL相比s亥探針濃度可較佳地平衡中之Kg 7較 咼量子產額，且由此平衡HDL内之較高猝滅程度以使所 有脂蛋白顆粒產生恆定螢光信號響應。 本發明者實施了-系列實驗以研究能否獲得探針物質 K37之螢光與每一脂蛋白顆粒種類（HDL、ldl&vldl)之 脂蛋白濃度（在f⑨A清臨床試樣中可&遇到之脂蛋白漢 度範圍内）間的線性及對等關係。令人驚奇的是，他們發 現，在限疋K37濃度及特定激發及發射波長下K37之螢光 與脂蛋白濃度間存在線性關係。因此，使用先前專利申請 案（PCT/GB2005/004757)之方法，熟習此項技術者可識別 出亦展示該與脂蛋白濃度之關係的其他適宜染料。 在膽固醇檢驗中使用酶較為有利，此乃因膽固醇通常以 酯化狀態發現，因此較佳使用膽固醇酯酶來水解膽固醇酯 以釋放膽固醇。隨後可藉由膽固醇氧化酶之作用將游離膽 固醇轉化成膽错-4-烯-3-酮，在該過程中會產生過氧化 氫。有利地，藉由辣根過氧化物酶將Amplex Red及過氧化 氫轉化成試_靈及水。試齒靈隨後可作為螢光化合物進行 148680.doc 33- 201102654 檢測，其最大吸收在約563 nm處且峰值發射波長為587 nm。總膽固醇含量可藉由在約485 nm下激發試樣並在約 600 nm下量測所得螢光來量測。 例如，當使用酶時，其可以超過用於量測例如酶Km之 比率許多倍之量提供。與業内使用之約1:1 〇〇〇比率相比， 該比率可能非常高。令人驚奇的是，裝置表面能差異外加 使用乾燥之經穩定化酶及視情況兩親聚合物之組合使得能 夠使用極少量之試樣及試劑。不欲受限於理論，吾人認為 兩個至少部分對立表面之表面能差異會在水性試樣之層流 内產生圓周運動，從而達成更有效混合。由於該效率提 高，故可在較小反應體積中使用較大量的酶，從而達成較 先前技術方法中以前可達成之反應更有效及快速之反應。 該方法之第二步驟可包含： (ii) 在約400 nm-5 20 nm間之激發波長下激發來自步驟 ⑴之產物。 激發波長可為約420 nm-480 nm或約440 nm-470 nm。所 用激發波長取決於檢驗中所用之具體螢光染料。對於 Amplex red，激發波長係約480 nm ;對於K37，激發波長 係約440 nm ;且對於尼羅紅，激發波長係約580 nm。 該方法之第三步驟包含： (iii) 在約490 nm-650 nm間之波長下量測榮光發射。 或者，螢光發射可在約520 nm-620 nm間之波長下量 測。在約540 nm或更高發射波長下，可觀測到用於測定總 脂蛋白濃度（即HDL、IDL、LDL及VLDL之濃度、以及（若 148680.doc 34· 201102654 存在）乳糜微粒之濃度）之更準確讀數。然而，所量測之較 佳螢光發射波長取決於檢驗中所用之具體螢光染料。對於 Amplex Red’螢光係在約600 nm下量測；對於&37,榮光 係在約495 nm下量測；且對於尼羅紅，螢光係在約61〇 nm 下量測。 應瞭解’激發及發射波長無需在各具體染料之最佳波長 下量測。可選擇當組合或平行使用染料時（例如當在單一 檢驗裝置中同時實施檢驗時）給出最佳分離或性能之波 長。應瞭解，步驟（ii)及激發及檢測亦可基本上同時 實施。 另外，三酸甘油酯之濃度可藉由自總脂蛋白濃度減去總 膽固醇濃度來計算。因此，由此產生對臨床醫師有用之由 總脂蛋白濃度、總膽固醇濃度以及三酸甘油酯濃度組成之 試樣的更詳細脂質分佈。 本發明者先前已發現，許多染料會結合脂蛋白並呈現取 決於=結合特定脂蛋白之不同螢光㈣。對該等染料實施 螢光量測能夠區別存在於試樣中之各類脂蛋白。此係藉由 將由月日蛋白晃合物中之—種脂蛋白引起之螢光增強或減弱 與預期來自其他脂蛋白（於不存在特定有產—㈣㈣脂 蛋下）之螢光進行比較來實施，如自總脂蛋白含量之校 準曲線及已知值所敎。例如，螢光染料尼羅紅在狐中 較在諸如LDUVLDL等其他脂蛋白中呈現顯著較高之營 〜 b可使用其他螢光染料（例如尼羅紅、K3 7或對特 定脂蛋白顯示特異性、戎 1螢光増強或減弱之任何其他親脂 [ 14S6S0.doc •35· 201102654 性探針）來區別試樣中之脂蛋白種類或亞類。 因此，本發明方法能夠使用螢光分析來測定試樣中特定 月曰蛋白種類或亞類之濃度。通常，此涉及使用第二及，或 第三螢光染料藉由對脂蛋白具有特異性之染料之榮光響應 的偏移來測定特定脂蛋白種類或亞類之濃度。 作為實例’為使用尼羅紅測定試樣中之hdl濃度，對尼 羅紅因HDL存在而發出的過量榮光實施計算。f先，藉由 K3 7螢光與月曰蛋白濃度之線性關係（如藉由步驟⑴所測定） 量測總脂蛋白濃度(量測值為「A」”其次，隨後使用 LDL(及/或VLDL ’因為螢光對濃度響應必須基本上相同） 於不同濃度下校準尼羅紅勞光以獲得具有斜率「χ」及截 距γ」之杈準曲線。熟習此項技術者應知道如何製備一 定濃度範圍之LDL(及/或VLDL)並測定每一濃度之對應螢 光。 可構建一系列濃度之H D L及值定濃度之L D L的校準曲線 以獲得斜率「2」。已知總脂蛋白濃度量測值「A」（自 K37可知)及未知試樣之過量尼羅紅螢光％，可藉由下 列公式測定該未知試樣中hdl之濃度「c」c=(b (ax Y))/Z。 _ 應瞭解’在實❹可使用預先製作之校準曲線或標準校 準曲線。此外，本發明檢驗裝置或用於與所研發之該等裝 置-起使用以獲得脂質分佈之檢驗讀出器可包含内標準物 及/或具有容許在無使用者干預下自動計算脂蛋白濃度之 處理構件。 又 148680.doc -36- 201102654 因此’應瞭解’可利用螢光量測來測定hdl、凡队⑽ 由:算）、LDL(藉由計算）、總脂蛋白、三酸甘油_(藉: 計算）以及總膽輯之濃度。所有該等參數均可藉由在類 似波長範圍内激發及量測螢光來同時平行測定。如上文所 論述，此健於習用檢驗之顯著改良，檢驗必須單獨 且通常在專用實驗室中實施，導致結果產生延遲。另外， 多個脂質參數可同時量測之事實大大簡化了實施量測所需 要之儀器使用。 所產生之脂質分佈包括測定結合試樣中之LDL之膽固醇 的浪度。由於LDL膽固醇高度致動脈粥樣硬化，因此獲知 LDL膽固醇之濃度尤其有利。因&，該方法提供試樣中脂 質組成之至少三個 '較佳四個或五個、或更多參數之多個 讀出。此外，可自三酸甘油自旨濃度計算/評估膽固醇_ VLDL-膽固醇之濃度’此乃因通常假定大多數三酸甘油酯 攜贡於VLDL t且VLDL之膽固醇組份佔2〇%。此對於幫助 臨床醫師決定適宜治療過程尤其有利。 實驗概覽 以下實驗細節描述如上所述本發明之一個實施例之完全 說明。 實例1 -總膽固醇檢驗 該檢驗使用能夠將一個膽固醇或膽固醇酯分子轉化成過 氧化氫分子（H2〇2)之三重酶系統（triple enzyme system) °Excitation/emission wavelength. The cover member (2) is also molded and has a hole. The cover member can support the member as a spacer element between the bases. And generally flat and comprising a plurality of apertures 1 ' optionally including a loop or a bit (9) aligned with the application area and allowing the user 148680.doc -23- 201102654 to apply a fluid sample to the application area. More - Series holes are used as vents (10). In the illustrated embodiment, the alignment aperture (11) positions the cover member relative to the support member by receiving the alignment post (8). In an alternative embodiment, the apertures and alignment apertures are provided in the support member and the flip cover member only serves as a covering. . The alignment 7C member can simply be a raised component with a corresponding alignment hole or groove. The support member and the cover member can be fixed or coupled together by, for example, mechanical means (e.g., by using a screw, rivet, bolt, or tab). Alternatively, the support member and the cover member may be joined together by a friction fit. In its case, the cover member and the branch member are joined together by, for example, using glue, solvent, tape, and the like. In this case, the covering member and the supporting material are joined together by heat welding and laser welding. One or both of the support member and the cover member may include an identification member to provide a unique identification and/or a means for the test reader to, for example, monitor the number of times the test device has been used or the application time of the sample to spend Suitable inspection time. The identification members may also contain instructions or data such as calibration or quality control data. The identification component can take a variety of forms including, but not limited to, text, blind braille, digital data, one-dimensional bar codes, two-dimensional bar codes 'RFID tagging, and the like. The transition device (12) is held securely between the branch member and the cover member adjacent to and in fluid communication with the application region (4) and the aperture (9) and the fluid flow path. The undiluted or diluted sample is applied directly to the filter via the hole (9). 1486S0.doc • 24-20112654 This test device can be used to test whole blood, venous blood, which is obtained, for example, by finger sticking or by heel puncture in the case of a newborn. This is the way to open a small wound, such as at the fingertip, which produces no more than a few drops of blood (about less than 50 μΐ ', for example about 1 μ μ μ to 2 μ μ μ). After the blood droplets are formed, they can be applied directly to the application area of the test device or by suction through a pipette and subsequently applied accordingly. The ability to use blood obtained by, for example, finger pricking directly represents a significant advantage over tests well known to those skilled in the art. Alternatively, a test device can be used to test a sample derived from plasma, in which case the sample can be diluted 1:80. Typically, the diluent is a tetherate buffered saline (PBS)' and it may comprise at least one ligand binding inhibitor. The diluent may also comprise two ligand binding inhibitors, one for the hydrophobic binding site and one for the drug binding site. The fluid in the sample moves through the filter and, as a result, the red (in the case of the entire situation) and the larger particles are removed by passive transition. Background interference caused by the cells or larger particles is reduced. In this example, the filter further includes a subsequent extraction member comprising the anti-de-antibody of the HSA from which the sample is removed as described above, again reducing the background. The six fluids travel from the application zone (4) to the fluid flow path in communication with the application zone by means of capillary flow. As it progresses, the liquid combines the dry and the mixed dyes, such as fluorescent dyes and enzymes, with water, such as moving laterally, without the need to apply external aid to move the aqueous sample. The term "dry form" as used herein means that the component remains generally substantially free of "S] 148680.doc -25- 201102654 in the form of a liquid or moisture; that is, when it is reconstituted by performing the test itself, rather than It is in the form of a solution before the test procedure and when reconstituted independently of the test procedure. Thus, the aqueous sample itself reconstitutes the dry reagent, thereby eliminating the need for separate reconstitution buffers and steps. When enzymes or other samples are used, especially when used in dry form, it is preferred to stabilize the enzymes or other agents. In the context of the present invention, "stabilized reagent" has an improved stability in terms of, for example, storage stability, thermal stability, etc. Thus, in a particular embodiment, reagent I 3 is used. The specific stabilization method of the stabilizer is disclosed in the International Patent Application No. WO 9_05182 and the disclosure of the disclosure of the entire disclosure of the entire disclosure of A copolymer of dimethylaminoethyl methacrylate methacrylate, which is sold, for example, by International Speciality Products ^ Qafquat (RTM). Gafqua KCAS Accession No.: 53633_54_8; 7732 Lu 5) is a series of water-bath copolymers ( For example, the name of polytetra-ammonium salt 丨 。. The difference in surface energy between the part and the cover part that the fluid sample contacts makes the mixing of the fluid and the dry reagent enhanced. The air outlet discharges an equal volume of air, and the dust balance in the device. After the fluid reaches the flow termination knot, the surface tension prevents further capillary flow. Here, the device can be placed in a suitable test reading. And measuring the amount of analyte (in this case cholesterol and blood lipids). & The housing of the test device is typically adjusted to be placed in functional communication with the test reader. For example, the test device can be inserted In the reader, placed on the readout I48680.doc • 26-201102654 or attached to the reader, and the reader may include docking components (eg slots) or alignment components In order to enable the inspection device to be properly inserted, placed or attached. In this embodiment, the cover member of the inspection device has a "V" shaped cutout which facilitates alignment of the inspection device with the reader. By the way, the inspection apparatus of the present invention is disposable, but the reader is usually reusable. The test device can be used in a variety of test methods or reactions, such as immunoassays and fluorescent tests, including steroid, lipoprotein or triglyceride assays. An immunoassay is a biochemical test that measures the concentration of a substance in an aqueous sample, such as serum or urine. This assay utilizes the specific binding of an antibody to its antigen by the reaction of the antibody with its antigen. Monoclonal antibodies are preferred because they bind to a specific molecule and provide specific and accurate testing. The presence of both antigens or antibodies can be measured, for example, when an infection is detected, the presence of antibodies against the pathogen can be measured. Alternatively, hormone biomolecules can be used as antigens when measuring biomolecules such as hormones and the like. The response of the measured aqueous fluid can be compared to a standard of known concentration, such as on the graph! Standard chords, lines are produced. Detection of the amount of antibody or antigen can be accomplished by a variety of methods, such as labeling an antigen or antibody. As a non-limiting example, the label may consist of an enzyme (EIA or EUSA), a radioisotope (e.g., 1-125), a magnetic label, or a luminescent or fluorescent label. Advantageously, the device of the present invention relies on capillary flow to effect fluid transfer of a low volume sample, and thus does not require the use of moving parts. Therefore, the device overcomes the problems of scale, economy, manufacturing, and mechanical failure encountered in the prior art. 148680.doc • 27- 201102654 The test reader for use with the test device of the present invention may be adapted to receive two or three tests, for example, from multiple patients or for multiple tests of aqueous samples from individual patients. Inspection device. The reader can include two (or more) excitation members that can be aligned with the detection zone of the inspection device. The "exciting member" is operable to excite the sample under test, for example, to fluoresce it. The. Also included is at least one detection member operable to detect, for example, fluorescence emitted by the sample in the detection zone. Typically the 'excitation member' contains an illumination source operable to illuminate at approximately 4 〇〇 nm _ 6 〇〇 nm. Therefore, the light source is preferably capable of illuminating the sample at about 400 nm to 600 nm. The illumination source can include one or more light bulbs, or one or more LEDs, or other sources (eg, one or more lasers). The excitation wavelength can be changed to the time of the disturbance. The excitation member can also include a polarizing member operable to polarize light generated by the illumination source. The excitation member can also be further adapted to focus the focusing member that focuses the light onto the sample. The focusing member can comprise a lens or light guide, such as a fiber optic or optical film (3M). The sensing member may comprise a photodiode 'CCD, or a photomultiplier tube or an optical sensor (which is preferably sensitive to yellow-red). The dye emitted by the sample can be detected in a range including about 440 nm to 650 nm. The detection member should be capable of detecting fluorescence emitted at about 490 nm, about 495 nm, about 57 〇 nm, about 600 nm, and about 610 nm. Fluorescence can be collected by a second lens and passed through a polarizer. The scattered excitation light can be removed by a cut filter or a belt pass filter. When measuring the intensity of the fluorescence, the current from the photodiode or the count rate from the photomultiplier tube can be read from the ammeter, voltmeter or juice meter module. Those skilled in the art will be familiar with other methods. M8680.doc •28- 201102654 The reader may also include an excitation correction system to account for fluctuations in the source of light. It may also include at least one fluorescent standard for accurate measurement prior to analyte concentration. Things. The standard can be an internal standard. In a particular embodiment, the verification reader is configured to detect and measure the fluorescence intensity of a single or a plurality of assays simultaneously or sequentially as the inspection device enters the reader or thereafter. The gastric effusion may also comprise a processing member adapted to lick the concentration of the analyte in the sample based on the detected fluorescence. The reader may further comprise a display member for displaying the measured value from the sample, preferably in readout form. For example, the display member can include an lcd screen or can be powered and/or calculated and/or displayed depending on the computer. In its most basic form, the component is simply a window that displays indicator readings or measurements. In general, the test item is portable and, advantageously, the test device and the extractor can be used to perform tests simply, quickly and simultaneously to determine the presence/absence of an analyte of an aqueous sample, such as a biological fluid, or concentration. For example, clinicians with knowledge of cholesterol, lipoprotein, and HDL concentrations can use the device to determine an effective treatment process. In addition, the testing device and the reader are portable and can be used by a GP or a visiting nurse, or even a test kit for home use. In this case, the processing means is adapted to directly determine the concentration of one or more of the samples such as cholesterol, triglyceride, HDL, LDL, VLDL & mL based on fluorescence analysis. Alternatively, the treatment member can be adapted to calculate the concentration of [S] 148680.doc 201102654 in the sample, based on the concentration of total lipoprotein, cholesterol and HDL, VLDL & Use of the device in cholesterol and lipid analysis The following examples illustrate the use of the assay device of the present invention to measure lipoproteins in aqueous biological samples. In this example, the fluid flow path is coated with an amphiphilic polymer PEQ to accelerate fluid transfer. For lipid distribution, the amphiphilic polymer is also combined with a fluorescent dye such as Amplex Red, K3 7 or Nile Red and/or other reagents such as enzymes, coated or printed in or within the fluid flow path. Fluorescent dyes and/or other reagents can be printed in the form of one or more picolite liter droplet arrays. Suitably, the array can comprise from about 15 to 4500 droplets along the first axis multiplied by 25 to 1 droplets along the second axis. The specific array size includes about 3400 x 65 droplets per jaw!!!2, about "(eight) 乂" droplets, about 1500 χ65 droplets, about 19 〇〇 χ 65 droplets, 6 〇〇 Χ 65 droplets, 450 χ 65 droplets or about 4 χ χ 65 droplets. Those skilled in the art will appreciate that variations in these droplet densities have minimal impact on inspection performance, but it should also be understood that, conversely, the densities can be optimized to improve inspection performance. For example, the droplets may also be applied in the form of nanoliters or femtoliter droplets, applied in the form of overlapping ρ trains, applied in the form of one (or more) entanglements together, separated by spacing. The discrete individual array forms are applied or applied in the form of blocks (ie, cumulative) that form a plurality of arrays of larger sized arrays. The array size can also be optimized for, for example, a particular reagent or dye concentration. * 17 brushes", the fluorescent dye droplets may be from about 丨.丨mM to 3 〇, more preferably from about 0.3 capsules to 2.5 福, and even more suitably about 〇·5 148680.doc -30- 201102654 Secret to a concentration of 2.0 mM applied to the split. Alternatively, the concentration of the fluorescent dye may be about 〇 8 2 -M when I is used to dilute the blood sample. The available concentration of the fluorescent dye is 1 ’' and for the undiluted blood sample, the available concentration is about 2. G mM. Subsequent drying (4), and then using 0, the sample fluid combines the amphiphilic polymer water to dilute the fluorescent dye, which in turn can bind to the lipoprotein in the sample. When so combined, the dye fluoresces under appropriate excitation. The total lipoprotein concentration in the sample can be determined by fluorescence analysis. The method generally comprises: (1) contacting an aqueous biological sample with at least one dye or luminophore and, optionally, at least one amphiphilic polymer, wherein the at least one dye or luminophore binds to at least one lipoprotein in the aqueous biological sample, And when combined with it, emit fluorescence under appropriate excitation; (II) excite the product from step (1) at an excitation wavelength between about 400 nm and 620 nm; (iii) at a wavelength between about 440 nm and 650 nm. Fluorescence emission after step (H) is measured. This method can be used to make lipid profiles for aqueous biological samples. The term "total lipoprotein" as used herein means the total concentration of at least VLDL, HDL, LDL, IDL and chylomicrons, in other words, the sum of the concentrations of triglycerides and total cholesterol in the sample. The term "total cholesterol" is used to mean the total concentration of cholesterol in a sample. The term "lipid distribution" as used herein means the concentration or phase of the lipid component (ie, total lipoprotein and total cholesterol and triglyceride) in the sample [ 148 680.doc • 31 · 201102654 versus concentration 2 ) in blood or serum samples. Most of the lipids bind to lipoproteins. The clinical test only does not measure total lipoprotein. Therefore, according to the convention, it is necessary to first measure the concentration of cholesterol and cholesterol vinegar and the concentration of triglyceride, and then add them to determine the total lipoprotein concentration. There is considerable error in the measurement of 2-acid glycerin in clinical laboratories because it relies on the natural circulation of glycerol in the blood. Advantageously, the total lipoprotein concentration (equal to total lipid concentration) is determined by measuring the amount (volume) of lipoprotein particles directly. Thus, the cholesterol test of the present invention does not have this error. Therefore, an error in the concentration of, for example, triglyceride caused by circulating glycerin in the sample can be avoided. In the first international patent application PCT/GB2005/004757 (published in WO 006/061646), the present inventors propose a simplified test based on the use of, for example, 7 to measure lipoproteins in biological macromolecules, which It is especially useful in the case where the lipid distribution is effectively obtained. For the determination of the concentration of total lipoprotein (i.e., L IDL & VLDL) in a blood sample using Κ37 fluorescence spectroscopy, the inventors have recognized that for a protein concentration of 疋37, binding to κ37 of each lipoprotein species The fluorescence response (i.e., the total mencidin concentration) may be substantially the same regardless of its composition (i.e., the ratio of HDL:LDL:IDL:VLDL in the sample). Therefore, it is preferred to use K3 7 in such a manner that the fluorescence intensity response is substantially linear over the entire range of expected lipoprotein molecules of the sample encountered in the clinical test. Not limited to any hypothesis, we believe that the fluorescence intensity from fluorochrome 148680.doc -32· 201102654 depends on its affinity for the testicular scorpion protein molecule (HDL, LDL, IDL or). . The quantum yield of fluorescence depends on the degree of fluorescence reduction in the lipoprotein molecular complex and the energy transfer between the closely packed probe molecules. In the prior application, the present inventors have selected: 'optional probe concentration and excitation and emission wavelengths for accurate total lipoprotein measurement by simple fluorescence measurement. The inventors further recognize that since the probe has a higher affinity for HDL, the concentration of the probe can be better balanced than the LDL compared to the LDL, and thus the balance in the HDL is balanced. The degree of high quenching is such that all lipoprotein particles produce a constant fluorescent signal response. The present inventors carried out a series of experiments to investigate whether the fluorescence of the probe substance K37 and the lipoprotein concentration of each lipoprotein particle type (HDL, ldl & vldl) can be obtained (in the f9A clear clinical sample & Linearity and equivalence between the lipoprotein Hanta range. Surprisingly, they found a linear relationship between K37 fluorescence and lipoprotein concentrations at concentrations of K37 and specific excitation and emission wavelengths. Thus, using the method of the prior patent application (PCT/GB2005/004757), those skilled in the art will recognize other suitable dyes that also exhibit this relationship to lipoprotein concentrations. It is advantageous to use an enzyme in a cholesterol test because cholesterol is usually found in an esterified state, so cholesterol esterase is preferably used to hydrolyze cholesterol ester to release cholesterol. The free cholesterol can then be converted to cholest-4-en-3-one by the action of cholesterol oxidase, during which hydrogen peroxide is produced. Advantageously, Amplex Red and hydrogen peroxide are converted to test water and water by horseradish peroxidase. The test tooth can then be tested as a fluorescent compound 148680.doc 33- 201102654 with a maximum absorption at about 563 nm and a peak emission wavelength of 587 nm. The total cholesterol content can be measured by exciting the sample at about 485 nm and measuring the resulting fluorescence at about 600 nm. For example, when an enzyme is used, it can be provided in excess of the ratio used to measure, for example, the enzyme Km many times. This ratio can be very high compared to the approximately 1:1 ratio used in the industry. Surprisingly, the difference in surface energy of the device plus the use of a combination of dry stabilized enzymes and optionally amphiphilic polymers allows the use of very small amounts of samples and reagents. Without wishing to be bound by theory, it is believed that the difference in surface energy between at least partially opposing surfaces creates a circular motion within the laminar flow of the aqueous sample, resulting in more efficient mixing. Because of this increased efficiency, larger amounts of enzyme can be used in smaller reaction volumes to achieve a more efficient and rapid response than previously achievable in prior art methods. The second step of the method can comprise: (ii) exciting the product from step (1) at an excitation wavelength between about 400 nm and 5 20 nm. The excitation wavelength can be from about 420 nm to 480 nm or from about 440 nm to 470 nm. The excitation wavelength used depends on the specific fluorescent dye used in the assay. For Amplex red, the excitation wavelength is about 480 nm; for K37, the excitation wavelength is about 440 nm; and for Nile Red, the excitation wavelength is about 580 nm. The third step of the method comprises: (iii) measuring the glory emission at a wavelength between about 490 nm and 650 nm. Alternatively, the fluorescence emission can be measured at a wavelength between about 520 nm and 620 nm. At the emission wavelength of about 540 nm or higher, the total lipoprotein concentration (ie, the concentration of HDL, IDL, LDL, and VLDL, and the concentration of chylomicrons (if 148680.doc 34·201102654)) can be observed. More accurate readings. However, the preferred fluorescence emission wavelength measured depends on the particular fluorescent dye used in the assay. The Amplex Red' fluorescence system was measured at approximately 600 nm; for &37, the glory system was measured at approximately 495 nm; and for Nile Red, the fluorescence system was measured at approximately 61 〇 nm. It should be understood that the excitation and emission wavelengths need not be measured at the optimum wavelength of each particular dye. It is optional to give the optimum separation or performance wavelength when the dyes are used in combination or in parallel (e. g., when testing is performed simultaneously in a single test apparatus). It should be understood that step (ii) and excitation and detection can also be carried out substantially simultaneously. Alternatively, the concentration of triglyceride can be calculated by subtracting the total cholesterol concentration from the total lipoprotein concentration. Thus, a more detailed lipid profile of the sample consisting of total lipoprotein concentration, total cholesterol concentration, and triglyceride concentration useful to the clinician is thereby produced. The inventors have previously discovered that many dyes bind to lipoproteins and exhibit different fluorescence depending on = binding to a particular lipoprotein (4). Fluorescence measurements of these dyes distinguish the various types of lipoproteins present in the sample. This is achieved by comparing the fluorescence enhancement or attenuation caused by the lipoprotein in the protein of the moon protein to the fluorescence expected from other lipoproteins (in the absence of a specific production - (4) (4) fat egg). , such as the calibration curve from the total lipoprotein content and known values. For example, the fluorescent dye Nile Red is significantly higher in foxes than in other lipoproteins such as LDMVLDL. b can use other fluorescent dyes (such as Nile Red, K3 7 or specific for specific lipoproteins).戎1 Fluorescent or weakened any other lipophilic [14S6S0.doc •35· 201102654 probe) to distinguish the lipoprotein species or subclasses in the sample. Thus, the method of the invention enables the use of fluorescence analysis to determine the concentration of a particular scorpion protein species or subclass in a sample. Typically, this involves the use of a second and/or third fluorescent dye to determine the concentration of a particular lipoprotein species or subclass by offsetting the glory response of a dye specific for lipoproteins. As an example, the calculation of the hdl concentration in the sample using Nile Red was carried out for the excess glory emitted by Nile Red due to the presence of HDL. f First, the total lipoprotein concentration (measured as "A") is measured by the linear relationship between K3 7 fluorescence and lunar protein concentration (as determined by step (1)), followed by LDL (and/or VLDL 'Because the fluorescence response to the concentration must be essentially the same) Calibrate Nile Red Light at different concentrations to obtain a calibration curve with slope "χ" and intercept γ". Those skilled in the art should know how to prepare LDL (and / or VLDL) in the concentration range and determine the corresponding fluorescence for each concentration. A series of concentrations of HDL and a constant concentration of LDL can be constructed to obtain a slope of "2". Total lipoprotein concentration is known. For the measured value "A" (known from K37) and the excess Nile red fluorescence % of the unknown sample, the concentration "h"c = (b (ax Y)) / Z of the hdl in the unknown sample can be determined by the following formula. _ It should be understood that 'pre-made calibration curves or standard calibration curves can be used. In addition, the inspection device of the present invention or the test reader for use with the devices developed to obtain lipid distribution may be included Standards and / or have allowed no user intervention The processing component that automatically calculates the lipoprotein concentration. 148680.doc -36- 201102654 Therefore, 'should understand' can use fluorescence measurement to determine hdl, Fan (10) by: calculation, LDL (by calculation), total lipoprotein , triglyceride _ (borrow: calculation) and the concentration of total gallbladder. All of these parameters can be measured simultaneously in parallel by exciting and measuring fluorescence over a similar wavelength range. As discussed above, this is a significant improvement over the practice test, which must be performed separately and usually in a dedicated laboratory, resulting in delays in the results. In addition, the fact that multiple lipid parameters can be measured simultaneously greatly simplifies the instrumentation required to perform the measurements. The resulting lipid profile includes the determination of the degree of calcium binding to the LDL in the sample. Since LDL cholesterol is highly atherogenic, it is particularly advantageous to know the concentration of LDL cholesterol. For &, the method provides for multiple reads of at least three 'better four or five, or more parameters of the lipid composition of the sample. Further, the concentration of cholesterol_VLDL-cholesterol can be calculated/evaluated from the triglyceride concentration. This is because it is generally assumed that most of the triglycerides carry tribute to VLDL t and the cholesterol component of VLDL accounts for 2%. This is especially beneficial for helping clinicians decide the appropriate treatment process. EXPERIMENTAL OVERVIEW The following experimental details describe a complete description of one embodiment of the invention as described above. Example 1 - Total Cholesterol Test This test uses a triple enzyme system capable of converting a cholesterol or cholesterol ester molecule into a hydrogen peroxide molecule (H2〇2).

Ik後使用所產生之過氧化氫來氧化染料Amplex Red(不具 有螢光性）以產生高度螢光性產物試鹵靈。 [ 148680.doc -37· 201102654 將酶穩定化至塑膠上 總膽固醇檢驗使用以下酶及染料： 膽固醇酯酶（3.1.1.13) 膽固醇氧化酶（1.1.3.6) 辣根過氧化物酶（1.11 · 1 · 7)The resulting hydrogen peroxide is used to oxidize the dye Amplex Red (without fluorescence) after Ik to produce a highly fluorescent product, resorufin. [ 148680.doc -37· 201102654 Stabilize the enzyme to the total cholesterol test on the plastic using the following enzymes and dyes: cholesterol esterase (3.1.1.13) cholesterol oxidase (1.1.3.6) horseradish peroxidase (1.11 · 1 · 7)

AmplexRed: 10-乙醯基_3,7_二羥基吩噁嗪 使用Gafquat作為穩定劑將酶穩定化至塑膠上。將三種 酶添加至0.01M磷酸鉀緩衝液溶液（pH 7 〇)中。各酶之最終 活性經畺測為200 U/ml緩衝液。隨後將溶液用Gafquat(帶 大量正電荷之聚合物）調配物以1:1進行稀釋，並將5 μ1所 得溶液沈積至塑膠表面上’並在30亡下於矽勝存在下乾燥 2小時。s玄過程獲得乾燥酶生物表面’各種酶沈積〇 5 口。 檢驗程序-使用1/80試樣稀釋物 採取兩種途徑：a)使膽固醇試樣（血漿）與乾燥酶及 Amplex Red在溶液中反應；b)使膽固醇試樣與乾燥酶及乾 燥的經穩定化之Amplex Red染料反應： (a)膽固醇試樣（血漿）與乾燥酶及Ampiex Recj在溶液中 之反應。 稀釋緩衝液A : 4.16 mM Amplex Red、1 〇 mM膽酸、 0.2% Triton X-100存於 Dulbecco填酸鹽緩衝鹽水（pH 7.2) 中ο 首先將待檢驗試樣以1份比80份稀釋於稀釋緩衝液Α中， 隨後使用50 μΐ稀釋試樣在試樣檢驗室中重構及‘激活乾燥三 重酶混合物（預先如上文所述實施穩定化）。 148680.doc -38- 201102654 膽固醇含量係藉由在480 nm下激發試樣混合物並在600 nm下量測所得螢光來量測。膽固醇濃度係通過量測4〇秒後 之穩態螢光或Vmax(最大受質產生速率）直接進行測定。藉 由參照檢驗標準數據來實施各項評價。 (b)膽固醇試樣（血漿）與乾燥酶及AmplexRed之反應。 稀釋緩衝液B : 1〇 mM膽酸、〇_2°/〇 Triton X-100存於AmplexRed: 10-Ethyl _3,7-dihydroxyphenoxazine Stabilizes the enzyme onto the plastic using Gafquat as a stabilizer. Three enzymes were added to a 0.01 M potassium phosphate buffer solution (pH 7 〇). The final activity of each enzyme was determined to be 200 U/ml buffer. The solution was then diluted in a 1:1 ratio with a solution of Gafquat (a polymer with a large amount of positive charge) and a solution of 5 μl of the solution was deposited onto the surface of the plastic' and dried for 30 hours in the presence of 30 s. The sinuous process obtained the surface of the dried enzyme bio-deposited enzymes. Test Procedure - Two routes were taken using 1/80 sample dilutions: a) reacting cholesterol sample (plasma) with dry enzyme and Amplex Red in solution; b) stabilizing cholesterol sample with dried enzyme and drying Amplex Red dye reaction: (a) Reaction of cholesterol sample (plasma) with dried enzyme and Ampiex Recj in solution. Dilution buffer A: 4.16 mM Amplex Red, 1 〇 mM cholic acid, 0.2% Triton X-100 in Dulbecco's sulphate buffered saline (pH 7.2) ο Firstly dilute the sample to be tested in 1 part to 80 parts Diluted in buffer Α, then reconstituted and 'activated dry triple enzyme mixture (pre-stabilized as described above) using a 50 μΐ dilution sample in the sample assay chamber. 148680.doc -38- 201102654 Cholesterol content was measured by exciting the sample mixture at 480 nm and measuring the fluorescence at 600 nm. The cholesterol concentration was measured directly by measuring steady-state fluorescence or Vmax (maximum yield generation rate) after 4 sec. The evaluations were carried out by reference to the inspection standard data. (b) Reaction of a cholesterol sample (plasma) with a dry enzyme and Amplex Red. Dilution Buffer B : 1 〇 mM cholic acid, 〇 2 ° / 〇 Triton X-100

Dulbecco磷酸鹽緩衝鹽水（1^7.2)中。 該程序與上文過程類似。然而，在該過程中，Amplex Red染料係在流動路徑中與三重酶混合物一起乾燥。首 先’如上所述將檢驗室之一個界定區域塗敷以〇5 u膽固 醇酯酶（3.1.1.13)、〇.5 U膽固醇氧化酶（1.1.3.6)及0.5 U辣 根過氧化物酶（1.11 · 1 7)。隨後將檢驗室之第二及單獨區域 塗敷以10 μΐ Amplex Red / PEG2000溶液，並在30eC下於 石夕膠存在下乾燥2 hr。染料塗敷溶液係由存於二曱基亞石風 (DMSO)中之 5.35 mg/ml Amplex Red、5% w/v PEG2000構 成。 將待檢驗試樣以1份比80份稀釋於稀釋緩衝液B中，並使 用50 μΐ稀釋試樣在試樣檢驗室中重構及激活乾燥Ampiex Red染料及三重酶混合物。 膽固醇含量係藉由在480 nm下激發試樣混合物並在6〇〇 nm下量測所得螢光來量測。膽固醇濃度再次係通過量測 Vmax(最大受質產生速率）或4〇秒後之穩態螢光直接進行測 定。藉由參照檢驗標準數據來實施各項評價。 經測定’兩種檢驗均能夠測定2_i i 之臨床相關膽固 148680.doc -39· 201102654 醇含量。 檢驗程序-使用未經稀釋試樣 純態生物試樣係藉由將試樣施加至位於可消耗裝置内之 灌注有抗-HS A抗體之硼矽酸鹽過濾器中來檢驗，該過濾 益用以過濾及引導試樣至合適的2〇〇 μιη深之讀數或檢測區 域。將讀數區域首先每mm2預塗敷4〇〇個χ65個酶試劑微微 升小滴、繼之每mm2 600個χ65個染料試劑微微升小滴及每 mm2 450個χ65個抑制劑試劑微微升小滴，此有助於使試樣 快速流動至讀數區域中。隨後在48〇 nm下（10 nm帶通）激 發試樣，產生螢光’其可通過6〇〇 nm(i〇 nm帶通）過濾器 來檢測，以容許藉由參照適宜標準量測來測定試樣之總膽 固醇含量。 酶試劑：將膽固醇酯酶、膽固醇氧化酶及辣根過氧化物 酶以200個單位/ml各自溶解於Gafquai穩定劑混合物中。 洗條劑溶液··將1.63 g膽酸、1 〇 g聚乙二醇2〇〇〇、800 μΐ Triton Χ-100及253.3 μΐ馬來酸二乙酯溶解於二甲基甲醯胺 (DMF)中，並將最終體積調節至4〇 。 染料試劑：將5 mg Ampliflu Red固體添加至480 μΐ洗滌 劑溶液中。 抑制劑試劑：將260 mg疊氮化鈉及912.92 mg磷酸氫二 鉀三水合物溶解於水中’並將最終體積調節至4〇爪卜 實例2 -總脂質檢驗 將K37染料溶解於DMF中，使最終濃度為〗〇 mM。然 後，將5% w/v PEG 2000溶解於染料溶液中，並將6〇奈升 148680.doc -40- 201102654 所知洛液沈積至塑膠表面上，並藉由移除溶劑在真空中於 室溫下在黑暗中乾燥1小時。 檢驗程序-使用經稀釋試樣 . 首先，將待檢驗試樣（血漿）以1份比80份稀釋於含有50 mM辛酸鈉之磷酸鹽緩衝鹽水（ρΗ 7·4)中。 將5 μ 1經稀釋血聚試樣施加至乾燥染料中，其自發水 合。總脂質含量係藉由在440 ηΓητ(1() nm帶通）激發試樣 並量測通過495 nm過濾器（1〇 nm帶通）之所得螢光來量 測。根據經驗藉由參照已知標準物來測定總脂質含量。 檢驗程序-使用未經稀釋試樣 砘態生物试樣係藉由將試樣施加至位於可消耗裝置内之 灌注有抗_HSA抗體之硼矽酸鹽過濾器中來檢驗，該過濾 态用以過濾及引導試樣至合適的2〇〇 μιη深之讀數區域。將 讀數區域預塗敷3〇〇〇個χ65個存MDMF中之2 K3 7/5/。（w/v) PEG2〇〇〇的微微升小滴，此有助於使試樣快 迷流動至讀數區域中，並將染料自發分配至試樣中所含有 之月曰蛋白中。隨後在440 nmT(1〇 nm帶通）激發試樣，產 生螢光，其可通過495 nm(l〇 nm帶通）過濾器來檢測，以 容許藉由參照適宜標準量測來測定試樣之總脂質含量。 實例3 - HDL膽固醇檢驗 將尼羅紅溶解於DMF中，使最終濃度為〇5mM。然後， =5% w/v PEG 2〇〇〇溶解於染料溶液中，並將6〇奈升所得 心液沈積至塑膠表面上，並藉由移除溶劑在真空中於室溫 下在黑暗中乾燥1小時。 148680.doc 201102654 檢驗程序-使用經稀釋試樣 首先，將待檢驗試樣（血漿）以丄份比和份稀釋於含有^ πιΜ辛酸鈉之磷酸鹽緩衝鹽水（pH7.4)*。 將5 μΐ經稀釋血漿試樣施加至乾燥染料中，其自發水 ^。HDL膽固醇含量係藉由在· nmir(i〇nm帶通）激發試 樣並量測通過610 nm過濾器（10 nm帶通）之所得螢光來量 測。HDL膽固醇含量係藉由使用闡述於主要說明中之算法 來計算。亦可使用未經稀釋之全血實施等效檢驗。 檢驗程序-使用未經稀釋試樣 、’屯I、生物试樣係藉由將試樣施加至位於可消耗裝置内之 灌注有抗_HSA抗體之硼矽酸鹽過濾器中來檢驗，該過濾 器用以過濾及引導試樣至合適的200 μιη深之讀數區域。將 §賣數區域每mm2預塗敷34〇0個χ65個存於DMF中之〇.5 mM 尼羅紅/5% (w/v) PeG2000的微微升小滴，此有助於使試 樣快速流動至讀數區域中，並將染料自發分配至試樣中所 含有之脂蛋白中。隨後在580 nm下（10 nm帶通）激發試 樣’產生螢光，其可通過610 nm( 10 nm帶通）過濾器來檢 測’以容許藉由參照適宜標準量測來測定試樣之HDL-c含 對來自闡述於實例1、2及3中之三個測試的數據進行處 理’提供以下結果： 總膽固醇之量測值-即測試（1) 總脂質濃度之量測值-即測試（2) HDL膽固醇之量測值-即測試（3) 148680.doc -42- 201102654 三酸甘油酯之計算值-即測試（2)減去（1) VLDL·之計算值—即三酸甘油酯的值/2.2 LDL之計算值-即藉由Friedwald公式測定 實例4 -使用PEG來增強近水平毛細管中之側向流體流動 長100 mm且具有2 mm、1 mm及0·5 mm内部直徑之玻璃 毛細管保持未經處理、經洗滌劑處理或塗敷以PEG。經洗 蘇劑處理之毛細管係藉由用virkon及Triton XI 00 5%溶液 洗滌、隨後乾燥來準備。經PEG處理之毛細管係藉由使存 於氯仿中之5% (w/v) PEG流過毛細管、使過剩物排空隨後 乾燥來準備。 將經處理及未經處理之毛細管固定於近水平位置（約10° 向上流動角），並將毛細管之尖端浸沒於水中。量測移動 至各毛細管中之水的行進距離及流速： 未經處理之毛細管： 2 mm -在約30秒内到達20 mm 1 mm -在15秒内到達90 mm 0·5 mm -在1 8秒内到達管末端（100 mm) 經洗蘇劑處理之毛細管 2 mm -在約20秒内到達20 mm 1 mm -在12秒内到達90 mm 0.5 mm -在15秒内到達管末端（100 mm) 塗敷有PEG之毛細管： 2 mm -在約20秒内到達80 mm 1 mm -在1 -2秒内到達末端 14S680.doc -43- 201102654 0.5 mm -在1 -2秒内到達管末端（1 〇〇 mm) 該數據顯示，塗敷有PEG之毛細管中之毛細管流體流動 速度係經洗滌劑處理之毛細管的約四倍且係未經處理毛細 管的約六倍。 藉由用矽化劑（二曱基二氣矽烷）處理並在120°C下烘烤 使毛細管具有疏水性。水未進入該等毛細管之内腔。給疏 水性毛細管塗敷以PEG(如上文）使流體流動狀況恢復到與 塗敷有PEG而未經石夕化（silation)之毛細管相當。在PEG表 面塗層不連續之一些實驗中，流體流動在PEG塗層中斷處 停止。 實驗5 -使用PEG來增強垂直毛細管中之流體流動 如上所述對長度為100 mm且直徑為2 mm、1 mm或0.5 mm之毛細管進行處理。將毛細管固定於垂直位置，並將 毛細管之尖端浸沒於水中。量測垂直毛細管中水到達之高 度： 未經處理之管： 2 mm 直徑-9 mm 1 mm直徑-22 mm 0.5 mm直徑-51 mm 經洗滌劑處理之管 2 mm 直徑-1 0 mm 1 mm直徑-22 mm 0.5 mm直徑-53 mm 塗敷有PEG之管 148680.doc • 44- 201102654 2 mm直徑-11 mm 1 mm直徑-25 mm 0.5 mm直徑-54 mm 水完全未進入疏水性含石夕毛細管。藉由給含石夕毛細管塗 敷以PEG使毛細管流動高度得以恢復且與彼等不含矽之塗 敷有PEG的毛細管接近。 使用公式h = 2yCos0/pgr(其中h係高度（m) ; γ係表面張 力；Θ係接觸角；ρ係密度；g係由重力引起之加速度；且r 係管的半徑（m))計算之海平面處之理論最大高度係： 1 4 mm -直徑為2 mm 2 8 mm -直徑為1 mm 56 mm -直徑為 0.5 mm 實例6 -轉移再現性及長期穩定性 如下量測疏水性分子（即染料）/兩親聚合物混合物之轉移 再現性及長期穩定性： 將分子量為2000 Da之PEG溶解於疏水性染料（尼羅紅或 K37)以5% w/v濃度存於二曱基曱醯胺（DMF)中之溶液中。 PEG/染料薄膜係藉由將25 μΐ存於DMF中之PEG/染料溶液 ' 沈積於5 ml玻璃小瓶中來製造。使溶液在小瓶底部鋪展並 • 隨後於真空室中放置1小時以蒸發溶劑。 轉移再現性係藉由比較添加至脂蛋白溶液中之存於DMF 中之染料的螢光強度與再溶解染料/PEG薄膜之相同脂蛋白 溶液之螢光強度來量測。再現性係藉由獲得存於脂蛋白溶 液中之10種染料/PEG薄膜之螢光強度的變異係數（CVs)來 148680.doc -45- 201102654 計算。 穩定性係藉由以下進行評價：塗佈薄膜以用於長期儲 存，並量測不同儲存時間後再溶解於脂蛋白溶液中之該等 薄膜之螢光強度。將薄膜在黑暗中於一系列條件下儲存： 在空氣中無乾燥劑、在空氣中存在矽膠及在真空中存在矽 膠。將薄膜在該等條件下於20°C及37°C溫度下儲存。 測定溶解於DMF中或存於PEG薄膜中之脂蛋白溶液中之 染料的螢光強度： K37 尼羅紅 來自DMF 268000 297000 來自PEG薄膜 269000 296000 PEG薄膜所獲得之螢光強度讀數與添加溶解於DMF中之 染料時所獲得之螢光強度讀數相同（在〇_5%内）。計算含有 脂蛋白之染料/PEG薄膜之螢光讀數的再現性： K37 尼羅紅 薄膜1 6.23E+05 4.27E+05 薄膜2 6.25E+05 4.27E+05 薄膜3 6.24E+05 4.29E+05 薄膜4 6.25E+05 4.26E+05 薄膜5 6.24E+05 4.27E+05 薄膜6 6.26E+05 4.30E+05 薄膜7 6.23E+05 4.30E+05 薄膜8 6.25E+05 4.28E+05 薄膜9 6.22E+05 4.28E+05 薄膜10 6.22E+05 4.26E+05 平均值 6.24E+05 4.28E+05 SD 1370.32 1475.73 CV (%) 0.22 0.34 148680.doc -46· 201102654 所獲得兩種染料之CV小於0.5%。 穩定性量測 時間 K37，於 DMF 中， 20°C K37，於 PEG 中， 20°C，在 空氣中 K37，於 PEG 中， 20°C，在 空氣中， 在矽膠上 K37，於 PEG 中， 20°C，在 真空中， 在矽膠上 K37，於 PEG 中， 37°C，在 空氣中 K37，於 PEG 中， 37〇C，在 空氣中， 在矽膠上 K3.7，於 PEG 中， 37〇C，在 真空中， 在矽膠上 0 138630 139320 137241 138624 137440 138317 136942 1週 137500 137113 137840 138801 137640 136922 136817 4週 134890 134641 133980 134202 135016 135412 134097 8週 135254 136021 135972 136671 135926 136201 135552 %CV 1.31 1.44 1.25 1.56 0.92 0.90 0.98 時間 NR，於 DMF 中， 20°C NR，於 PEG 中， 20°C，在 空氣中 NR，於 PEG 中， 20°C，在 空氣中， 在矽膠上 NR，於 PEG 中， 20°C，在 真空中， 在矽膠上 NR，於 PEG 令， 37°C，在 空氣中 NR，於 PEG 中， 37°C，在 空氣中， 在矽膠上 NR，於 PEG 中， 37°C，在 真空中， 在矽膠上 0 495197 496130 494590 495760 494118 494241 494829 1週 495002 495600 494802 494400 494636 493907 494621 4週 493128 492686 493440 493662 494017 492506 492906 8週 493492 494620 494525 493986 493920 492500 493650 %CV 0.21 0.31 . 0.12 0.19 0.06 0.19 0.18 結論： (1) 當使用水溶液重構時，納入至PEG薄膜中之染料完全 再溶解，且獲得與於有機溶劑中添加之染料相同之螢光強 度。 (2) 染料/PEG薄膜具有再現性，且以相同方式構成之薄膜 達成相同螢光強度。 (3) 當於所測試最苛刻儲存條件（37°C，無乾燥劑）下儲存 -47- 148680.doc 201102654 時，染料/PEG薄膜可在至少52週内保持穩定。 實例7 -兩親/非離子型聚合物之比較 藉由及取7 mm存於各種溶劑中之5%聚合物溶液來塗敷 删石夕酸鹽毛細管（長100 mm ’直徑為1 mm ’來自CompositeDulbecco phosphate buffered saline (1^7.2). This procedure is similar to the above process. However, in this process, the Amplex Red dye is dried in the flow path with the triple enzyme mixture. First, a defined area of the test chamber is coated with 〇5 u cholesterol esterase (3.1.1.13), U.5 U cholesterol oxidase (1.1.3.6) and 0.5 U horseradish peroxidase (1.11). · 1 7). The second and individual areas of the test chamber were then coated with 10 μΐ Amplex Red / PEG2000 solution and dried at 30 °C for 2 hr in the presence of Shiqi gum. The dye coating solution consisted of 5.35 mg/ml Amplex Red, 5% w/v PEG2000 in diterpene sulphur (DMSO). The test sample was diluted in dilution buffer B in 1 part to 80 parts, and the dried Ampiex Red dye and triple enzyme mixture were reconstituted and activated in a sample test chamber using a 50 μL diluted sample. The cholesterol content was measured by exciting the sample mixture at 480 nm and measuring the resulting fluorescence at 6 〇〇 nm. The cholesterol concentration was measured directly by measuring Vmax (maximum yield generation rate) or steady-state fluorescence after 4 sec. The evaluations were carried out by referring to the inspection standard data. It has been determined that both tests are capable of determining the clinically relevant cholesterol of 2_i i 148680.doc -39· 201102654 alcohol content. Inspection Procedure - Use of undiluted sample Pure biological sample is tested by applying the sample to a borosilicate filter infused with an anti-HS A antibody in a consumable device. To filter and direct the sample to a suitable 2 〇〇μηη deep reading or detection area. The reading area is first pre-coated with 4 χ65 enzyme reagent microliter liters per mm2, followed by 600 χ65 dye reagent microliters per mm2 and 450 χ65 inhibitor reagents per mm2 This helps to quickly flow the sample into the reading area. The sample is then excited at 48 〇 nm (10 nm bandpass) to produce fluorescence, which can be detected by a 6 〇〇 nm (i〇nm bandpass) filter to allow for determination by reference to appropriate standard measurements. The total cholesterol content of the sample. Enzyme reagent: cholesterol esterase, cholesterol oxidase and horseradish peroxidase were each dissolved in a Gafquai stabilizer mixture at 200 units/ml. Stripping solution··················································· Medium and adjust the final volume to 4〇. Dye Reagent: 5 mg Ampliflu Red solid was added to the 480 μL detergent solution. Inhibitor reagent: dissolve 260 mg sodium azide and 912.92 mg dipotassium hydrogen phosphate trihydrate in water 'and adjust the final volume to 4 〇 claws. Example 2 - Total lipid test dissolves K37 dye in DMF The final concentration is 〇 mM. Then, 5% w/v PEG 2000 was dissolved in the dye solution, and 6 liters of 148680.doc -40 - 201102654 known solution was deposited onto the plastic surface, and the chamber was removed in a vacuum by removing the solvent. Dry in the dark for 1 hour under warm conditions. Test Procedure - Use of diluted sample. First, the sample to be tested (plasma) was diluted in 1 part to 80 parts in phosphate buffered saline (ρΗ 7.4) containing 50 mM sodium octanoate. A 5 μl diluted blood aggregate sample was applied to the dried dye, which spontaneously hydrated. The total lipid content was measured by exciting the sample at 440 η Γητ (1 () nm bandpass) and measuring the fluorescence obtained through a 495 nm filter (1 〇 band pass). The total lipid content was determined empirically by reference to known standards. Inspection Procedure - Use of an undiluted sample The sputum biological sample is tested by applying the sample to a borosilicate filter infused with an anti-HSA antibody in a consumable device. Filter and direct the sample to the appropriate 2 〇〇μηη deep reading area. The reading area is pre-coated with 2 K3 7/5/ of 3 χ 65 storage MDMFs. (w/v) A slight liter of droplets of PEG2〇〇〇, which helps to quickly flow the sample into the reading zone and spontaneously dispense the dye into the sputum protein contained in the sample. The sample is then excited at 440 nmT (1 〇 band pass) to produce fluorescence, which can be detected by a 495 nm (10 带 band pass) filter to allow for the determination of the sample by reference to an appropriate standard measurement. Total lipid content. Example 3 - HDL Cholesterol Test Nile Red was dissolved in DMF to a final concentration of 〇5 mM. Then, =5% w/v PEG 2〇〇〇 is dissolved in the dye solution, and 6 liters of the resulting cardiosol is deposited onto the plastic surface and removed in a vacuum at room temperature in the dark by removing the solvent. Dry for 1 hour. 148680.doc 201102654 Inspection procedure - use of diluted sample First, the sample to be tested (plasma) is diluted in a ratio of parts and parts to phosphate buffered saline (pH 7.4)* containing sodium π Μ Μ octanoate. A 5 μΐ diluted plasma sample was applied to the dry dye with spontaneous water ^. The HDL cholesterol content was measured by exciting the sample in nmir (i〇nm bandpass) and measuring the fluorescence obtained through a 610 nm filter (10 nm band pass). The HDL cholesterol content is calculated by using the algorithm set forth in the main description. Equivalent tests can also be performed using undiluted whole blood. Inspection Procedure - Use of an undiluted sample, '屯I, biological sample is tested by applying the sample to a boron phthalate filter infused with an anti-HSA antibody in a consumable device, the filtration The device is used to filter and guide the sample to the appropriate 200 μm deep reading area. The § sell area is pre-coated with 34 〇 0 χ 65 微 5 5 mM Nile Red / 5% (w / v) PeG2000 micro-liter droplets in the DMF, which helps to make the sample Flow quickly into the reading zone and spontaneously dispense the dye into the lipoprotein contained in the sample. The sample is then excited at 580 nm (10 nm bandpass) to generate fluorescence, which can be detected by a 610 nm (10 nm bandpass) filter to allow HDL determination of the sample by reference to appropriate standard measurements. -c contains data from the three tests set forth in Examples 1, 2 and 3 'Providing the following results: Measured total cholesterol - ie test (1) Measured total lipid concentration - ie test ( 2) HDL cholesterol measurement - that is, test (3) 148680.doc -42- 201102654 Triglyceride calculated value - that is, test (2) minus (1) VLDL · calculated value - that is, triglyceride Value of /2.2 LDL calculated - ie by Friedwald formula example 4 - PEG is used to enhance the lateral fluid flow in the near horizontal capillary 100 mm long and has an internal diameter of 2 mm, 1 mm and 0·5 mm The capillaries remain untreated, detergent treated or coated with PEG. The detergent-treated capillary was prepared by washing with virkon and Triton XI 00 5% solution followed by drying. The PEG-treated capillary was prepared by flowing 5% (w/v) PEG in chloroform through a capillary, evacuating the excess and then drying. The treated and untreated capillaries are held in a near horizontal position (about 10° upward flow angle) and the tip of the capillary is immersed in water. Measure the travel distance and flow rate of water moving into each capillary: Untreated capillary: 2 mm - reach 20 mm in about 30 seconds 1 mm - reach 90 mm in 15 seconds 0. 5 mm - at 1 8 The end of the tube reaches the end of the tube (100 mm). The capillary treated with the desiccant 2 mm - reaches 20 mm in about 20 seconds 1 mm - reaches 90 mm in 12 seconds 0.5 mm - reaches the end of the tube within 15 seconds (100 mm Capillary coated with PEG: 2 mm - reaches 80 mm in about 20 seconds 1 mm - reaches the end in 1-2 seconds 14S680.doc -43- 201102654 0.5 mm - reaches the end of the tube in 1-2 seconds ( 1 〇〇mm) This data shows that the capillary fluid flow rate in a capillary coated with PEG is about four times that of a detergent-treated capillary and about six times that of an untreated capillary. The capillary was rendered hydrophobic by treatment with a deuterating agent (dimercaptodioxane) and baking at 120 °C. Water does not enter the lumen of the capillaries. Coating the hydrophobic capillary with PEG (as above) restores fluid flow to the equivalent of a capillary coated with PEG without silation. In some experiments where the PEG topcoat was discontinuous, fluid flow stopped at the break of the PEG coating. Experiment 5 - Use of PEG to enhance fluid flow in vertical capillaries Capillaries of length 100 mm and having a diameter of 2 mm, 1 mm or 0.5 mm were treated as described above. Secure the capillary in a vertical position and immerse the tip of the capillary in water. Measure the height of the water in the vertical capillary: Untreated tube: 2 mm diameter - 9 mm 1 mm diameter - 22 mm 0.5 mm diameter - 51 mm Detergent treated tube 2 mm diameter - 1 0 mm 1 mm diameter -22 mm 0.5 mm diameter -53 mm PEG coated tube 148680.doc • 44- 201102654 2 mm diameter -11 mm 1 mm diameter -25 mm 0.5 mm diameter -54 mm water does not enter the hydrophobic rock-bearing capillary . The capillary flow height was restored by coating the shi-containing capillary with PEG and was in close proximity to their PEG-free capillaries. Use the formula h = 2yCos0/pgr (where h is the height (m); the γ-series surface tension; the lanthanide contact angle; the ρ-system density; the g-based acceleration due to gravity; and the radius of the r-tube (m)) Theoretical maximum height at sea level: 1 4 mm - diameter 2 mm 2 8 mm - diameter 1 mm 56 mm - diameter 0.5 mm Example 6 - Transfer reproducibility and long-term stability The following measurements of hydrophobic molecules (ie Transfer reproducibility and long-term stability of dyes/amphiphilic polymer mixtures: PEG with a molecular weight of 2000 Da was dissolved in a hydrophobic dye (Nile Red or K37) at a concentration of 5% w/v in dimercaptopurine In a solution in an amine (DMF). The PEG/dye film was made by depositing 25 μl of the PEG/dye solution stored in DMF in a 5 ml glass vial. The solution was spread on the bottom of the vial and then placed in a vacuum chamber for 1 hour to evaporate the solvent. The transfer reproducibility was measured by comparing the fluorescence intensity of the dye added to the DMF added to the lipoprotein solution to the fluorescence intensity of the same lipoprotein solution of the redissolved dye/PEG film. Reproducibility was calculated by obtaining the coefficient of variation (CVs) of the fluorescence intensity of the 10 dye/PEG films present in the lipoprotein solution 148680.doc -45- 201102654. The stability was evaluated by coating the film for long-term storage and measuring the fluorescence intensity of the films dissolved in the lipoprotein solution after different storage times. The film was stored in the dark under a range of conditions: no desiccant in air, silicone in air and silicone in vacuum. The film was stored under the conditions of 20 ° C and 37 ° C. Determination of the fluorescence intensity of the dye dissolved in DMF or in the lipoprotein solution in the PEG film: K37 Nile Red from DMF 268000 297000 Fluorescence intensity reading from PEG film 269000 296000 PEG film added and dissolved in DMF The fluorescence intensity readings obtained for the dyes in the middle are the same (within 〇 5%). Calculate the reproducibility of fluorescence readings of lipoprotein-containing dyes/PEG films: K37 Nile Red Film 1 6.23E+05 4.27E+05 Film 2 6.25E+05 4.27E+05 Film 3 6.24E+05 4.29E+ 05 Film 4 6.25E+05 4.26E+05 Film 5 6.24E+05 4.27E+05 Film 6 6.26E+05 4.30E+05 Film 7 6.23E+05 4.30E+05 Film 8 6.25E+05 4.28E+ 05 Film 9 6.22E+05 4.28E+05 Film 10 6.22E+05 4.26E+05 Average 6.24E+05 4.28E+05 SD 1370.32 1475.73 CV (%) 0.22 0.34 148680.doc -46· 201102654 The dye has a CV of less than 0.5%. Stability measurement time K37, in DMF, 20 ° C K37, in PEG, 20 ° C, in air K37, in PEG, 20 ° C, in air, on silica gel K37, in PEG, 20 ° C, in vacuum, K37 on silica gel, in PEG, 37 ° C, in air K37, in PEG, 37 ° C, in air, on silica gel K3.7, in PEG, 37 〇C, in vacuum, on silicone 0 0 138630 139320 137241 138624 137440 138317 136942 1 week 137500 137113 137840 138801 137640 136922 136817 4 weeks 134890 134641 133980 134202 135016 135412 134097 8 weeks 135254 136021 135972 136671 135926 136201 135552 %CV 1.31 1.44 1.25 1.56 0.92 0.90 0.98 time NR, in DMF, 20 ° C NR, in PEG, 20 ° C, NR in air, in PEG, 20 ° C, in air, on ruthenium NR, in PEG, 20 ° C, in vacuum, on ruthenium NR, on PEG, 37 ° C, in air NR, in PEG, 37 ° C, in air, on silica gel NR, in PEG, 37 ° C In vacuum, on silicone 0 0 495197 496130 49 4590 495760 494118 494241 494829 1 week 495002 495600 494802 494400 494636 493907 494621 4 weeks 493128 492686 493440 493662 494017 492506 492906 8 weeks 493492 494620 494525 493986 493920 492500 493650 % CV 0.21 0.31 . 0.12 0.19 0.06 0.19 0.18 Conclusion: (1) When using an aqueous solution At the time of reconstitution, the dye incorporated into the PEG film was completely redissolved, and the same fluorescence intensity as that of the dye added in the organic solvent was obtained. (2) The dye/PEG film is reproducible, and the film constructed in the same manner achieves the same fluorescence intensity. (3) The dye/PEG film may remain stable for at least 52 weeks when stored at -47-148680.doc 201102654 under the most stringent storage conditions tested (37 ° C, no desiccant). Example 7 - Comparison of Amphiphilic/Nonionic Polymers A dephosphazate capillary (length 100 mm 'diameter 1 mm') was obtained by taking a 7 mm solution of 5% polymer solution in various solvents. Composite

Metal Services有限公司_ cvl〇12)，並搖動直至乾燥。所 用溶劑端視溶解性而定，其中氯仿由於具有高蒸發速率而 較有利。 經塗敷毛細管之實驗以一式三份實施，且未經塗敷毛細 s母次均作為參照實施。藉由用於各毛細管之切入卡片中 之2個插槽將4個毛細管垂直保持在具線紋參照卡片上。毛 細管之末端恰好延伸超過卡片底部7 mm。 使用連接至Winnov 500050G V1000 + 〇v PC卡片之Metal Services Ltd. _ cvl〇 12) and shake until dry. The solvent used depends on the solubility, and chloroform is advantageous because of its high evaporation rate. The experiments in which the capillaries were applied were carried out in triplicate, and the uncoated capillaries were used as a reference. The four capillaries are held vertically on the textured reference card by the two slots in the cut-in card for each capillary. The end of the capillary extends just over 7 mm from the bottom of the card. Use to connect to the Winnov 500050G V1000 + 〇v PC Card

Sunkwang高解析度低照度彩色相機為該實驗錄像且使用 程式Videum Capture來捕獲及分析數據。除非另有說明， 否則訊框速率設置為每秒5個訊框（以避免丟失訊框p平底 觀察玻璃中含有流動液體(存於水中之1〇_4 M孟加拉玟魂 紅（Rose Bengal))，並簡單地降低卡片末端，直至卡片底 部停靠在觀察玻璃壁上。將毛細管浸沒於流動液體中至2 議深度。此產生距卡片底部5嶋之間距、自卡片底部至 第一條線9麵之間距及自第—條線沿卡片向上至第二條線 之又-6匪間距。藉由計數在卡片底部與第一條線間之9 mm行程上所經過之訊框數來計算流逮。 148680.doc -48- 201102654 表ι·聚合物塗層/溶劑系統之水柱高度及毛細管流速方面 的性能。* =來自單一毛細管之結果，（）=來自3個毛細管 中之2個毛細管之結果，當毛細管具有低於卡片第一條線 之柱高度時對結果進行估計。 塗層 溶劑 平均水 柱1¾度 mm 柱尚度之 STDEV 平均流速 mm/s 平均流速之 STDEV mm/s 最快速 度 mm/s PEG2000 氯仿 17.8 0.76 21.9 3.3 25.7 PEG2000 DMF 19.8 1.89 27.6 28.1 60 PEG2000 水 13.5 5.77 29.7 37.2 72 PEG6000 氯仿 19.3 0.76 21.5 4.8 25.7 PEG12,000 氯仿 16.5 0.87 7.6 3.3 9.5 PEG20,000 氯仿 15.3 1.04 3.2 1.6 4.9 Triton XI00 水 14.2 1.04 7.9 2.2 10 羧曱基纖維素 水 18.0 * 180 丰 180 雙(2-胺基乙 基)聚乙二醇2000 氯仿 17.0 3.5 38.8(52.5) 25.0(10.6) 60 PEG曱基醚5000 氯仿 17.8 1.26 2.4 0.2 2.6 圖7展示PEG分子量對垂直流速之影響。 雖然本發明之某些較佳實施例已在上文闡述及具體例 示，但本發明並不意欲限於該等實施例。可對其進行各種 修改，此並不背離以下申請專利範圍中所述之本發明之範 缚及精神。 【圖式簡單說明】 圖la及lb係顯示接觸角（0)之量測之通用圖。 圖2提供先前技術檢驗裝置之實例。 圖3展示使用「淚滴」形流動終止結之檢驗裝置的一個 148680.doc -49- 201102654 實施例。 圖4展示替代檢驗設計/形式。 圖5及6展示流體流動路徑、流動終止結及另一裝置實施 例之替代構造。 圖7展示PEG分子量對垂直流速之影響。 【主要元件符號說明】 1 第一支撐表面 2 第二表面-遮蓋部件 3 毛細管通道 4 施加區域 5 流體流動路徑 6 檢測區 7 流動終止結 8 對準柱 9 孔洞 10 排氣口 11 對準孑L 12 過濾器 13 截面積減小之流體流動路徑部分 14 室 15 低壁或屏障 15a 室之部分 15b 室之第二部分或遠端區域 16 凸起部分 148680.doc -50-The Sunkwang high-resolution, low-light color camera captures and analyzes the data for the experimental video and using the program Videum Capture. Unless otherwise stated, the frame rate is set to 5 frames per second (to avoid losing frames p flat bottom observation glass containing flowing liquid (1〇_4 M in the water) (Rose Bengal) And simply lower the end of the card until the bottom of the card rests on the viewing glass wall. Immerse the capillary in the flowing liquid to a depth of 2 inches. This creates a distance of 5 inches from the bottom of the card, from the bottom of the card to the first line. The distance between the distance and the distance from the first line to the second line of the second line is calculated by counting the number of frames passing through the 9 mm stroke between the bottom of the card and the first line. 148680.doc -48- 201102654 Table 1 · Polymer coating / solvent system performance in terms of water column height and capillary flow rate. * = results from a single capillary, () = results from 2 of the 3 capillaries Estimate the result when the capillary has a column height lower than the first line of the card. Coating solvent average water column 13⁄4 degrees mm STDEV average flow rate mm/s Average flow rate STDEV mm/s fastest speed mm/ s PEG2000 Chloroform 17.8 0.76 21.9 3.3 25.7 PEG2000 DMF 19.8 1.89 27.6 28.1 60 PEG2000 Water 13.5 5.77 29.7 37.2 72 PEG6000 Chloroform 19.3 0.76 21.5 4.8 25.7 PEG12,000 Chloroform 16.5 0.87 7.6 3.3 9.5 PEG 20,000 Chloroform 15.3 1.04 3.2 1.6 4.9 Triton XI00 Water 14.2 1.04 7.9 2.2 10 Carboxymethyl cellulose water 18.0 * 180 Feng 180 bis(2-aminoethyl) polyethylene glycol 2000 Chloroform 17.0 3.5 38.8 (52.5) 25.0 (10.6) 60 PEG thiol ether 5000 Chloroform 17.8 1.26 2.4 0.2 2.6 Figure 7 shows the effect of PEG molecular weight on vertical flow rate. While certain preferred embodiments of the invention have been described and illustrated in the foregoing, the invention is not intended to be limited to the embodiments. This does not depart from the spirit and spirit of the invention as set forth in the following claims. Brief Description of the Drawings Figures la and lb show a general view of the measurement of the contact angle (0). Figure 2 provides a prior art test. An example of a device. Figure 3 shows an embodiment of an inspection device using a "teardrop" shaped flow termination knot 148680.doc - 49 - 201102654. Figure 4 shows an alternative inspection design/form. Figures 5 and 6 show alternative configurations of fluid flow paths, flow termination knots, and another device embodiment. Figure 7 shows the effect of PEG molecular weight on vertical flow rate. [Main component symbol description] 1 First support surface 2 Second surface - Covering member 3 Capillary channel 4 Application area 5 Fluid flow path 6 Detection area 7 Flow termination junction 8 Alignment column 9 Hole 10 Exhaust port 11 Alignment 孑L 12 Filter 13 Reduced cross-sectional area of fluid flow path 14 chamber 15 low wall or barrier 15a chamber portion 15b chamber second or distal region 16 raised portion 148680.doc -50-

Claims (1)

201102654 七、申請專利範圍： 1. 一種檢驗裝置，其包含兩個展示不同表面能之至少部分 對立表面，其中表面能差異經設置以破壞行進於該兩個 表面之間之流體的毛細管層流。 二求項1之k驗裝置，其中該兩個至少部分對立表面 界疋至少-條流體流動路經以將流體試樣自該裝置之第 一區域轉移至該裝置之第二區域。 3·如請求項2之檢驗裝置’其中該兩個至少部分對立表面 之-界定開放的毛細管通道’該毛細管通道具有足以產 生開放通道之截面幾何結構，該開放通道經構造以與該 兩個至少部分對立表面之笛_ I , 刀耵JL衣囟之第一表面形成封閉的毛細管 道。 4. 如吻求項2之檢驗裝置，其中間隔元件經構造以使該兩 個至少冑分對立表面相隔一定距離以在該等表面之間產 生毛細管通道。 5. 如請求項2、3或4中任一項之檢驗裝置，其進一步包含 與該裝置之第一區域流體連通以引入流體試樣之施加區 域0 6.如請求項5之檢驗裝置，其在該兩個至少部分對立表面 之中包含孔洞以將流體試樣引入至該施加區域。 7'如請求項5或6之檢驗裝置，其進—步包含至少一個位於 該裝置之第二區域中之排氣口。 8.如請求項5、6或7之檢驗裝置，其在該裝置之第一區域 與第二區域之間進一步包含流動終止結，該流動終止結 Γ si 148680.doc 201102654 經構造以中斷或中止行進於該兩個表面之間之流體的毛 細管流體流動。 9·如請求項8之檢驗裝置，其中該裝置進一步包含佈置於 該施加區域與該至少一條流體流動路徑之間的濾膜。 10.如請求項2至9中任一項之檢驗装置，其中該流體流動路 ‘ 徑包含至少一種選自由下列組成之群之試劑：兩親聚合 物、染料、探針、酶或配體結合抑制劑。 π.如請求項之檢驗裝置，其中該流體流動路徑包含一或 多種選自由Amplex Red、Κ：37及尼羅紅（Nile Red)組成之 群之染料或探針。 12. 如請求項1〇或11之檢驗裝置，其中該流體流動路徑包含 一或多種選自由膽固醇酯酶、膽固醇氧化酶及辣根過氧 化物酶組成之群的酶。 13. 如請求項1〇、11或12之檢驗裝置，其中該流體流動路徑 包含一或多種選自由鹼金屬辛酸鹽及辛酸組成之群的配 體結合抑制劑。 14. 如請求項10至13中任一項之檢驗裝置’其進一步包含至 少一個檢測區’該檢測區經構造以量測至少一部分流體 試樣與該至少一種試劑間之反應的結果及/或進程。 - 15. 如請求項14之檢驗裝置，其包含至少三條流體流動路徑 及至少三個檢測區，其中，第一流動路徑與該施加區域 及第一檢測區流體連通，第二流體流動路徑與該施加區 域及第二檢測區流體連通且第三流體流動路徑與該施加 區域及第三檢測區流體連通。 148680.doc 201102654 16.如請求項15之檢驗裝置，其中該第—流體流動路徑包含 Amplex Red，該第二流體流動路徑包含K37且該第三流 體流動路徑包含尼羅紅。 17·如請求項16之檢驗裝置’其中該第一流體流動路徑進一 步包含膽固醇酯酶、膽固醇氧化酶及辣根過氧化物酶。 18. 如請求項2至17中任一項之檢驗裝置，其中該流體流動 路徑包含兩親聚合物。 19. 一種組件套組’其包含至少一個如請求項1至丨8之檢驗 裝置且進一步包含以下中的一或多者：（丨）用於對患者皮 膚進行殺菌之構件；（Π)皮膚穿透構件；（iii)紗布；（iv) 綷創膏（adhesive plaster) ; (v)提供有關該裝置使用之細 節的說明書；（vi)用即棄型手套及（vii)檢驗讀出器。 20_種如請求項1至18中任一項之檢驗裝置的用途’其用 於里測流體試樣中膽固醇或至少一種脂質之含量。 148680.doc201102654 VII. Patent Application Range: 1. An inspection apparatus comprising two at least partially opposing surfaces exhibiting different surface energies, wherein the surface energy difference is set to disrupt capillary laminar flow of fluid traveling between the two surfaces. The apparatus of claim 1, wherein the at least two opposing surfaces define at least one fluid flow path to transfer the fluid sample from the first region of the device to the second region of the device. 3. The test device of claim 2, wherein the at least two opposing surfaces define an open capillary channel, the capillary channel having a cross-sectional geometry sufficient to create an open channel, the open channel being configured to interact with the at least two Part of the opposite surface of the flute _ I, the first surface of the JL 形成 形成 forms a closed capillary channel. 4. The test device of claim 2, wherein the spacer element is configured such that the two at least two opposing surfaces are spaced apart to create a capillary channel between the surfaces. 5. The testing device of any of claims 2, 3 or 4, further comprising an application region in fluid communication with the first region of the device to introduce a fluid sample. 6. The inspection device of claim 5, A hole is included in the at least partially opposing surfaces to introduce a fluid sample to the application area. 7' The test device of claim 5 or 6, wherein the step further comprises at least one vent located in the second region of the device. 8. The testing device of claim 5, 6 or 7, further comprising a flow termination knot between the first region and the second region of the device, the flow termination knot si 148680.doc 201102654 configured to interrupt or abort Capillary fluid flow of fluid traveling between the two surfaces. 9. The testing device of claim 8, wherein the device further comprises a filter disposed between the application region and the at least one fluid flow path. 10. The testing device of any one of claims 2 to 9, wherein the fluid flow path comprises at least one reagent selected from the group consisting of amphiphilic polymers, dyes, probes, enzymes or ligands. Inhibitor. π. The test apparatus of claim 1, wherein the fluid flow path comprises one or more dyes or probes selected from the group consisting of Amplex Red, Κ:37, and Nile Red. 12. The test device of claim 1 or 11, wherein the fluid flow path comprises one or more enzymes selected from the group consisting of cholesterol esterase, cholesterol oxidase, and horseradish peroxidase. 13. The test device of claim 1, 11 or 12, wherein the fluid flow path comprises one or more ligand binding inhibitors selected from the group consisting of alkali metal octoates and octanoic acid. 14. The testing device of any one of claims 10 to 13 further comprising at least one detection zone configured to measure a result of a reaction between at least a portion of the fluid sample and the at least one reagent and/or process. 15. The inspection device of claim 14, comprising at least three fluid flow paths and at least three detection zones, wherein the first flow path is in fluid communication with the application zone and the first detection zone, and the second fluid flow path is The application zone and the second detection zone are in fluid communication and the third fluid flow path is in fluid communication with the application zone and the third detection zone. 16. The inspection device of claim 15, wherein the first fluid flow path comprises Amplex Red, the second fluid flow path comprises K37 and the third fluid flow path comprises Nile Red. 17. The test device of claim 16, wherein the first fluid flow path further comprises cholesterol esterase, cholesterol oxidase, and horseradish peroxidase. 18. The testing device of any one of claims 2 to 17, wherein the fluid flow path comprises an amphiphilic polymer. 19. A kit of parts comprising at least one test device as claimed in claims 1 to 8 and further comprising one or more of the following: (丨) means for sterilizing the skin of the patient; (Π) skin wear (iii) gauze; (iv) adhesive plaster; (v) instructions for the details of use of the device; (vi) disposable gloves and (vii) inspection reader. The use of the test device of any one of claims 1 to 18, which is used for measuring the content of cholesterol or at least one lipid in a fluid sample. 148680.doc