201015065 六、發明說明： 【發明所屬之技術領域】 本發明係關於一種流體的定量檢測方法，特別是一種有關於生化檢測與 免疫檢測所使用之流體的定量檢測方法。 【先前技術】 以流體檢測試片進行生化檢測與免疫檢測的習知技術中，流體檢測試片 在其基板或底材上設計有流道或微流道結構，而因流道周圍並非吸水材質， 且待測流體彡為含有如蛋自質歧_等黏滯度高之城物，卿當待測流 體流過後，會在流道上殘留，使得待測流體無法完全反應，如此一來，不僅 造成待測流體的浪費，更可能造成最終測試結果的誤差。 此外，習知技術的流體檢測試片在流體傳送方面，可設計有微流道結 構’並係利用微流道結構產生的毛細現象，將流馥經過流道被動傳送至反應 偵測區域；另-種方式則是在注入待測流體時即利用加壓等方式，給予流體 -驅動力，使得流體可絲通過流道，到達反應姻區域。但是無論是上述 任-種方式，待測流體注入流道後常常產生大小不一的氣泡使得流道阻塞， 造成實際測量上之誤差’甚至致使測試失敗。 最後’習知技術的檢測試片’在製作上多使用模鑄或射出成型的方式在 基板上做ih流道或微流道結構，所以必須仙聚乙烯(PE)、聚氣乙稀(Pvc) 或聚丙烯(PP)等價格較高之塑膠聚合物作為材質，進而造成試片之總體成本 的提高。 【發明内容】 為克服上述缺點’本發明提供一種流體檢測方法，主要包含下列步驟： ⑴提供-基板’自其上表面向下凹設至少—流道。流道包含依序連接之 第-流體區、第二紐區與第三流體區，第二流體區與第三流體區之底部形 成有靖化纖維層，且硝化纖維層包含有中空網狀構型，其中第二流體區係供 201015065 流體之傳送’第三流體區係供流體之反應，因此，第二流體區的確化纖維層 平均厚度不大於第三流體區硝化纖維層厚度，且第三流體區之硝化纖維廣可 吸收定量之流體。另外’有一反應材料形成於上述的硝化纖維層之中空網狀 構型中； 工 (2) 自基板的第一流體區注入一流體，使流體經由第二流體區進入第三流 體區； 一 (3) 使第三流體區的硝化纖維層吸收定量之流體；以及 (4) 藉由流體中的特定成份與第三流體區的反應材料交互作用而形成光 學反應而檢出。 本發明之主要目的，係提供-種麵檢測方法，其中，因所提供的基板 之流道具有可吸水_化_層’由於單位财_化_财量係為定 值，故可經由設定基板上硝化纖維層的體積，而提供流體的定量檢測。 本發明之另-目的，係提供-種紐檢測方法，其巾，因所提供的基板 之流道具有中空網狀_的硝化纖維層，由於流體流經中空網狀構型時，流 體中的氣齡被破壞，故可狄氣餘塞贿，而無敎且可靠的檢測結 果。 φ 【實施方式】 由於本發義郷-概體蚁量檢财法，其帽彻物理、化學原 理及溶液塗布技術，已為_技_域具㈣常知識者所_瞭，故以下文 中之說明’不再作完整描述。同時，以下文中所對照之圖式，係表達與本發 明特徵有關之示意，並未亦不需要依據實際情形完整綠製，合先敎明。 請^第i圖’係本發明之較佳實施例，為一種流體的定量檢測方法的 流程，係用於檢測流體中的特定成份，主要包含下列步驟. 步驟1:首先，提供-基板1G，請參考_，基板1G自其上表面1〇〇 凹叹至少-流道U ’流道u包含依序連接之第—流體區⑴、第二流 201015065 體區112與第三流體區113。在較佳的實施狀態中，基板10為生物相容 (biocompatible)材料。請繼續參考第3圖，為第2圖沿AA連線之剖面圖。 在第二流體區112與第三流體區113之底部均形成有令空網狀構型的硝化纖 維層1121與1131 ’其中第二流體區112係供流體之傳送，第三流體區U3 係供流體之反應。第二流體區的硝化纖維層1121平均厚度Da不大於第三 流體區之确化纖維層1131厚度Db，且第三流體區之硝化纖維層1131的吸 收液體量是固定的。又’硝化纖維層1121與1131的中空網狀構型中，包含 有反應材料，反應材料的組成係與流體中所含有的待測成份的種類有關。 步驟2 :自基板10的第一流體區ill中注入流體l(未圖示），使流體l 在注入第一流體區111後’經由第二流艘區112的傳送，到達第三流體區 113。 步驟3 :使第三流體區113的硝化纖維層1131吸收定量之流體l。 步驟4:藉由流體L中的特定成分與第三流體區113的反應材料交互作 用而形成一反應訊號而檢出，其中，前述之反應訊號可為冷光反應訊號、 螢光反應訊號、光吸收反應訊號，或是電化學反應訊號。 此外，為了降低流道與流體之間的毛細作用所造成的影響，本發明所 提出之流道並非習知技術所謂的微流道，且第二流趙區112與第三流體區 113的寬度Wa與Wb較佳至少為〇.3mm。 在製作上’琐化纖維層1121與1131的形成方式與反應材料形成於其中 的方式如下所述。先將硝化纖維粉末(nitr〇eellul〇se p〇W(jer)與含有酿類(ester) 和麵(ketone)的有機溶劑混合後形成一確化纖維溶液；再將硝化纖維溶液 澆注(casting)於第二流體區112與第三流體區113的底部，經乾燥後，於第 二流體區112底部則會形成硝化纖維層1121，而於第三流體區113的底部 則形成硝化麟層im。為達較佳喊注效果，流道u之表面粗輪度(如 值)以介於3微米至50微米之間為佳。 硝化纖維溶液乾燥後形成具有中空網狀構型的確化纖維層，為了調整 201015065 較佳的中空網狀構型’本發明的硝化纖維溶液中，硝化纖維粉末與含有醋 類和酮類的有機溶劑混合的較佳體積比例為1 : 9«由於單位體積的确化纖 維吸水量係為定值，故可由欲吸收之待測流體的體積推算出對應的頌化纖 維溶液的體積’之後再行澆注。如此可以固定檢測所需液體之體積量，並 適用於微量檢測。 待硝化纖維層1121與1131分別乾燥成形於第二流逋區112與第三流趙 區113的底部後，將含有反應材料的反應溶液注入硝化纖維層1121與 1131，經過風乾或是冷凍乾燥(lyophilization)後，以粉末狀的形式留存在硝 化纖維層1121與1131之中。 ® 上述硝化纖維層1121、1131與反應材料形成於其中的方式係以先形成 硝化纖維層之後再注入反應材料後的順序形成方式，另外，亦可將含有反 應材料的反應溶液’加入由硝化纖維粉末(nitrocellulose powder)與含有醋類 (ester)和酮類(ketone)的有機溶劑組成的硝化纖維溶液中；混合完畢之後，再 將混合好的溶液澆注(casting)於第二流體區112與第三流體區in的底部， 經過風乾或冷凍乾燥程序，同時將硝化纖維溶液形成硝化纖維層1121與 1131，以及將反應材料形成粉末狀留存在硝化纖維層1121與1131之中。 由於待測成份不同’檢測所需進行之反應亦有所差異；進而依反應種類 φ 的不同，產生出各種不同的訊號。例如進行生化檢測時，係用酵素催化流體 中的待測物質與化學試劑’進而產生出特定訊號以供偵測。所以要進行生化 檢測’反應材料則會包含酵素及相對應的化學試劑。另一方面，若要檢測檢 體中的某些蛋白質’例如：胎兒蛋白（-fetoprotein)是否存在，則是利用 具有專一性之抗體，與待測蛋白質進行專一性結合，再利用其他化學試劑與 已結合上待測蛋白質的抗體進行反應’發出可供偵測的訊號。所以要進行免 疫檢測’反應材料中則會包含有化學及抗體等免疫試劑。故，本發明所提供 之基板10，可用於各種生物檢體(如展液、血液等流體)中之各項待測成份的 檢測。 201015065 上述之較佳實施例係使用具有三個流體區域之基板，而根據本發明之流 體的定量檢财法’所制之基錢-何纽道㈣三舰1之後再加設 一第四流體區(未圖示），以供儲存流道中多餘之流體。而第四流體區的硝化 纖維層之構型、形成方式、使狀雜纖維溶液之成份與較佳關、反應材 料之組成，均與前述之較佳實施例相同，在此不再重複贅述。 以上所述僅為本發明較佳實施例而已，並非用以限定本發明申請專利權 矛J，同時以上的描述對於熟之本技術領域之專門人士應可明瞭與實施，因此 其他未脫離本發明所揭示之精神下所完成的等效改變或修飾，均應包含於下 述之申請專利範圍。 【圖式簡單說明】 第1圖，為本發明較佳實施例流體檢測方法之流程圖。 第2圖，為本發明較佳實施例流體檢測方法所提供之基板之示意圖。 第3圖’為本發明較佳實施例流體檢測方法所提供之基板之剖面示意 圖0 【主要元件符號說明】 步驟 1 ' 2'3'4 基板 10 上表面 100 流道 11 第一流艘區 111 第二流體區 112 第三流體區 113 硝化纖維層 1121 ' 1131 硝化纖維層1121厚度 Da 201015065 硝化纖維層1131厚度 第二流體區的寬度 第二流體區的寬度201015065 VI. Description of the Invention: [Technical Field] The present invention relates to a method for quantitatively detecting a fluid, and more particularly to a method for quantitatively detecting a fluid used in biochemical detection and immunoassay. [Prior Art] In the conventional technique of performing biochemical detection and immunodetection using a fluid detecting test piece, the fluid detecting test piece is designed with a flow path or a micro flow path structure on the substrate or the substrate, and the surrounding material is not a water absorbing material. , and the fluid to be tested is a city with a high viscosity such as egg self-quality, and when the fluid to be tested flows, it will remain on the flow path, so that the fluid to be tested cannot be completely reacted, so that not only The waste of the fluid to be tested is more likely to cause errors in the final test results. In addition, the fluid detecting test piece of the prior art can be designed with a micro flow channel structure in terms of fluid transfer and utilizes the capillary phenomenon generated by the micro flow path structure to passively transport the flow through the flow path to the reaction detection area; In the same way, when the fluid to be tested is injected, the fluid-driving force is given by means of pressurization or the like, so that the fluid can pass through the flow path to reach the reaction area. However, in any of the above modes, the fluid to be tested is often injected into the flow path to cause bubbles of different sizes to block the flow path, causing an error in the actual measurement, which even causes the test to fail. Finally, the 'testing test piece of the prior art' uses iM flow path or micro flow path structure on the substrate by means of die casting or injection molding, so it is necessary to make polyethylene (PE) and polyethylene (PE). ) A higher-priced plastic polymer such as polypropylene (PP) is used as the material, which in turn causes an increase in the overall cost of the test piece. SUMMARY OF THE INVENTION To overcome the above disadvantages, the present invention provides a fluid detecting method mainly comprising the following steps: (1) Providing a substrate - at least a flow path recessed downward from an upper surface thereof. The flow channel comprises a first fluid zone, a second contact zone and a third fluid zone connected in sequence, a bottom layer of the second fluid zone and the third fluid zone is formed with a layer of Jingjing fiber, and the nitrocellulose layer comprises a hollow network structure Type, wherein the second fluid zone is for the transfer of the fluid of 201015065 'the third fluid zone is for the reaction of the fluid, therefore, the average thickness of the confirmed fiber layer of the second fluid zone is not greater than the thickness of the nitrocellulose layer of the third fluid zone, and the third The nitrocellulose in the fluid zone absorbs a large amount of fluid. In addition, a reaction material is formed in the hollow network configuration of the nitrocellulose layer; (2) injecting a fluid from the first fluid region of the substrate to allow fluid to enter the third fluid region via the second fluid region; 3) causing the nitrocellulose layer of the third fluid zone to absorb a quantity of fluid; and (4) detecting an optical reaction by interacting with a specific component of the fluid and a reactive material of the third fluid zone. The main object of the present invention is to provide a method for detecting a type of surface, wherein the flow path of the substrate provided has a water-absorptive layer, which is a fixed value, and can be set via a setting substrate. The volume of the upper nitrocellulose layer provides a quantitative detection of the fluid. Another object of the present invention is to provide a method for detecting a seed, which has a hollow mesh-like nitrocellulose layer in the flow path of the substrate provided, and the fluid flows through the hollow network configuration. The age of the gas is destroyed, so it can be used to bury the gas, and the results are innocent and reliable. φ [Embodiment] Because of the 郷----------------------------------------------------------------------------- The description 'is no longer fully described. At the same time, the drawings in the following texts are indicative of the features relating to the features of the present invention, and do not require a complete green system according to the actual situation. The preferred embodiment of the present invention is a flow of a quantitative method for detecting a fluid, which is used for detecting a specific component in a fluid, and mainly comprises the following steps. Step 1: First, provide a substrate 1G, Please refer to _, the substrate 1G is slanted from its upper surface by at least - the flow path U' flow path u includes the first fluid region (1), the second flow 201015065 body region 112 and the third fluid region 113. In a preferred embodiment, substrate 10 is a biocompatible material. Please continue to refer to Figure 3, which is a cross-sectional view along line AA of Figure 2. At the bottom of the second fluid zone 112 and the third fluid zone 113, there are formed nitrocellulose layers 1121 and 1131 ' of an air network configuration, wherein the second fluid zone 112 is for fluid transfer, and the third fluid zone U3 is for The reaction of the fluid. The average thickness Da of the nitrocellulose layer 1121 of the second fluid zone is not greater than the thickness Db of the confirmed fiber layer 1131 of the third fluid zone, and the amount of absorbed liquid of the nitrocellulose layer 1131 of the third fluid zone is fixed. Further, in the hollow network configuration of the nitrocellulose layers 1121 and 1131, a reaction material is included, and the composition of the reaction material is related to the type of the component to be tested contained in the fluid. Step 2: Injecting a fluid 1 (not shown) from the first fluid zone ill of the substrate 10 to cause the fluid 1 to pass through the second flow zone 112 after being injected into the first fluid zone 111 to reach the third fluid zone 113. . Step 3: The nitrocellulose layer 1131 of the third fluid zone 113 is caused to absorb the quantitative fluid l. Step 4: detecting a reaction signal by interacting with a specific component of the fluid L and the reaction material of the third fluid region 113, wherein the reaction signal may be a cold light reaction signal, a fluorescent reaction signal, or a light absorption. Reaction signal, or electrochemical reaction signal. Further, in order to reduce the influence of the capillary action between the flow path and the fluid, the flow path proposed by the present invention is not a so-called micro flow path of the prior art, and the width of the second flow region 112 and the third fluid region 113 Wa and Wb are preferably at least 〇3 mm. The manner in which the 'trimmed fiber layers 1121 and 1131 are formed and the reaction material are formed therein are as follows. First, a nitration fiber powder (nitr〇eellul〇se p〇W (jer) is mixed with an organic solvent containing an ester and a ketone to form a gel solution; the nitrocellulose solution is cast on the After the second fluid zone 112 and the bottom of the third fluid zone 113 are dried, a nitrocellulose layer 1121 is formed at the bottom of the second fluid zone 112, and a nitrification layer im is formed at the bottom of the third fluid zone 113. For better screaming effect, the surface roughness of the flow path u (such as the value) is preferably between 3 micrometers and 50 micrometers. The nitrocellulose solution is dried to form a layer of corroded fibers having a hollow network configuration, in order to Adjusting 201015065 Preferred hollow mesh configuration 'In the nitrocellulose solution of the present invention, the preferred volume ratio of the nitrocellulose powder to the organic solvent containing vinegar and ketone is 1:9« due to the volumetric water absorption of the unit fiber The quantity is a fixed value, so the volume of the corresponding deuterated fiber solution can be deduced from the volume of the fluid to be tested to be absorbed, and then the casting can be carried out. This can fix the volume of the liquid required for detection and is suitable for micro-detection. Nitrogen After the fiber layers 1121 and 1131 are respectively dried and formed at the bottoms of the second flowing region 112 and the third flowing region 113, the reaction solution containing the reactive material is injected into the nitrocellulose layers 1121 and 1131, and air-dried or lyophilized. Thereafter, it remains in the form of powder in the nitrocellulose layers 1121 and 1131. The nitrocellulose layer 1121, 1131 and the reaction material are formed in the order of forming the nitrocellulose layer and then injecting the reaction material. Alternatively, the reaction solution containing the reaction material may be added to a nitrocellulose solution consisting of a nitrocellulose powder and an organic solvent containing an ester and a ketone; after mixing, The mixed solution is cast in the bottom of the second fluid zone 112 and the third fluid zone in, and is subjected to an air drying or freeze-drying process, and the nitrocellulose solution is formed into the nitrocellulose layers 1121 and 1131, and the reaction material is formed. The powder remains in the nitrocellulose layers 1121 and 1131. Due to the different components to be tested, the reaction required for the detection is also Differently, depending on the type of reaction φ, various signals are generated. For example, in biochemical detection, the enzyme and the chemical reagent in the fluid are catalyzed by the enzyme to generate a specific signal for detection. Biochemical detection 'reactive materials will contain enzymes and corresponding chemical reagents. On the other hand, to detect the presence of certain proteins in the sample, such as the presence of fetal protein (-fetoprotein), the use of specific antibodies , specifically bind to the protein to be tested, and then use other chemical reagents to react with the antibody that has been bound to the protein to be tested' to emit a signal for detection. Therefore, it is necessary to carry out an immunoassay. The reaction material contains immunological reagents such as chemicals and antibodies. Therefore, the substrate 10 provided by the present invention can be used for detecting various components to be tested in various biological samples (such as fluids such as liquids and blood). 201015065 The preferred embodiment described above uses a substrate having three fluid regions, and the base money of the fluid according to the present invention, the Heiway (four) three ships 1 and then a fourth fluid Zone (not shown) for storing excess fluid in the runner. The configuration of the nitrocellulose layer in the fourth fluid zone, the formation mode, the composition of the fiber-like fiber solution, and the composition of the preferred material and the reaction material are the same as those of the preferred embodiment described above, and the detailed description thereof will not be repeated here. The above description is only for the preferred embodiment of the present invention, and is not intended to limit the scope of the present application, and the above description should be understood and implemented by those skilled in the art, so that the other embodiments are not deviated from the present invention. Equivalent changes or modifications made in the spirit of the disclosure are intended to be included in the scope of the claims below. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a flow chart showing a fluid detecting method according to a preferred embodiment of the present invention. 2 is a schematic view of a substrate provided by a fluid detecting method according to a preferred embodiment of the present invention. 3 is a schematic cross-sectional view of a substrate provided by a fluid detecting method according to a preferred embodiment of the present invention. [Main element symbol description] Step 1 '2'3'4 Substrate 10 Upper surface 100 Flow path 11 First flow area 111 Two fluid zone 112 third fluid zone 113 nitrocellulose layer 1121 ' 1131 nitrocellulose layer 1121 thickness Da 201015065 nitrocellulose layer 1131 thickness second fluid zone width second fluid zone width
Db Wa WbDb Wa Wb
99