201017157 六、發明說明： 【發明所屬之技術領域】 本發明係關於一種流體檢測試片，特別是一種有關於用於定量檢測的流 體檢測試片。 【先前技術】 在生物檢體的定量的習知技術中，在進行生化檢測時，常利用特定酵素 與待測物質進行反應，再針對反應所產生的產物其化學或電學特性進行偵 Φ 測，例如：在溶氧電極上固定葡萄糖氧化騰（glucose oxidase)，利用葡萄 糖氧化晦與血糖（glucose)反應產生的過氧化氫所造成的電位或是電流改 變，來測定所生成的過氧化氫濃度，並推算出檢體中血糖濃度。由於此種電 化學檢驗方法只要微量的過氧化氫存在，即可造成電位或電流的改變，故所 需之檢體體積極少，且偵測速度快。但此種測量方式由於需要適當的酵素配 合以產生便於偵測的過氧化物，故目前僅適用於小分子的定量量測上，例如 血糖、總膽固醇、尿素、肌酸酐等。 另-種方式則是利用免疫分子具有專一性辨識能力的特定來進行測 量，例如傳統的酵素連結免疫吸附測試（enzyme linkedimmunQlsOTbent ELISA) ’多在96孔盤（96-well plate)中操作，並藉由_最終酵素反應 所發出之減’回推待_質之濃度。但是由於此_試，為了要避免免疫 分子與制射包含的其他雜質進行非專一性結合，所以必須在各個反應階 段加入清洗的轉’細絲與制物或是其錢疫奸結合之反應試劑自 96孔盤中清除，以免造成過多的酵素留置於各孔位（weU)中所造成的偽陽 性訊號，導致測試失敗。此種免疫測量方式具有專一性（spedfi(%)高及靈 敏度（sensitivity)佳等優點。 由於機電技術的發展，已有利用具有微流道結構之測試試片進行免疫该 測’簡化上述傳統測試方法中於各步驟後進行清洗等繁瑣流程。然目前已知 3 201017157 的測試試片，在進行_時仍需將反應試劑或反應所需之受質（substrate) 乂額外添加的;5γ^加人峨試#巾。此種反應試臟受質與檢職片分離存 放’於細時再行添加的方式乃是起因於其所需的反應試劑或受質 ’在室溫 中無法長期存放’需保存於特定環境中（如冷藏或避光保存），以避免因反 應物變質所導致酬試誤差。故此種免疫麵制祕作及赫上仍多有所 不便。 此外習知具錢道或微流道結翻流體檢職#，目流道周圍並非吸水 材質’且待啦體多為含有如蛋自魏是賴等絲度高之喊物，所以當 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， 不僅造成待啦體的浪費，更可能造成最㈣試結果的誤差。 此外，習知具有微流道結構的流體檢測試片在流體傳送方面，則是利用 微流道結構纽的毛細縣，紐魏過流道被動傳送至反應侧區域；另 一種方式則是在注入待測流體時即利用加壓等方式，給予流體一驅動力，使 得流體可主動通過流道，到達反應彳貞;躯域。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。 【發明内容】 為克服上述缺點，本發明提供一種流體檢測試片，包含有具有流道的基 板。流道上設置有依序連接之第一流體區、第二流體區與第三流體區，其中 第一流體區係供流體之注入。此外’流道中具有第一抗體、醣類材料、過氧 化物騰、第二抗體與受質反應試劑。第一抗體位於第一流體區中，用於辨識 待測物質。醣類材料及過氧化物腌則位於第一流體區或第二流體區中。第二 抗體則是固定(immobilized)於第二流體區中，且亦辨識同一待測物質，又第 二抗體及第一抗體係辨識相異之抗原決定位置(epitope)。受質反應試劑係共 同位於第三流體區中’受質反應試劑包含有一膽氧化晦。藉此，當包含有待 測物質之流體注入流道後，第一抗體、醣類材料及過氧化物晦係隨著流體流 4 201017157 Λ 動，部份之過氧化物腾會與第一抗體、待測物質及第二抗體結合並留置於第 二流體區中，未結合之過氧化物腺則隨流體流至該第三流體區，且醣氧化梅 會催化流至第三流體區之醣類材料進行氧化反應，並產生出過氧化氫 (Ηζ〇2)，且過鼠化氫會受到流至第三流體區的過氧化物晦催化進行反應，並 產生一電學訊號。 因此，本發明之主要目的，係提供一種流體檢測試片，由於具有所有反 應所需的試劑及材料，無須經由繁瑣之操作步驟即可直接測量最終反應訊號 進行定量偵測。 本發明之另一目的’係提供一種流體檢測試片，其中反應所須的試劑與 ® 材料，在反應前係以乾燥方式留存於試片中，故可長期保存，不致因試劑變 質而導致測試誤差。 本發明之另一目的，係提供一種流體檢測試片，可配合現有之電化學偵 測技術，兼具有偵測時間短、專一性高及靈敏度佳等優點。 本發明亦提供一種流體檢測檢測方法，主要包含下列步驟。（1)提供具有 待測物的趙》(2)提供—基板，紐包含至少流道，而流道包含依序連接之 第-流體區、第二流體區與第三流體區，第一流體區係供流體之注入。基板 上進一步包含有第一抗體，位於第一流體區中，用於辨識該流體内之該待測 φ 物質:醣類材料’位於第一流體區或第二流體區中；過氧化物騰，位於第一 流體區或第二流體區中；第二抗體，固定於第二流體區中，且亦辨識同一待 麟質而第_抗體及第一抗體係辨識相異之抗原決定位置；以及受質反應 試劑，位於第三流體區中’受質反應試劑包含有醣氧化酶。_流體加至流 道之第仙·體區’使第一抗體、醣類材料及該過氧化物酶隨著流體流動。（4) 使=測物質與第—抗體、第二抗體及部份之聽化物麟合㈣置於第二流 體，中’使流體帶著酿類材料、未結合之第—抗體及過氧化物晦流至第三流 體區聽類材料受到糖氧化梅之催錢行氧化反應，並產生出過氧化氮， 過氧化氫會觉到流至第三流體區的過氧化物梅催化進行反應 ，並產生一電學 5 201017157 訊號。（5)偵測產生之電學訊號。 因此’本發明之另-目的，係提供一種流體檢測方法，由於具有所有反 應所需的·及材料’無迦由繁狀操作姉卩可直制量最終反應訊號 進行定量摘測。 本發#目#係k供_種流體檢測^^法其巾反應所須的試酿 材料，在反應前係峨財式时於試片中，故可長祕存，不致因試劑變 質而導致測試誤差。 本發明之再-目的’倾供—種__，可配合财之電化學摘 測技術’兼具有偵測時間短、專—性高及靈敏度佳等優點。 【實施方式】 由於本發明係揭露-種流體檢測試片及其測試方法，其中所利用化學原 理及生物檢測技術，已為相關技術領域具有通常知識者所能明瞭，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 明特徵有關之不意，並未亦不需要依據實際情形完整♦製，合先敛明。 月參考第圖為本發明之第一較佳實施例流體檢測試片之示意圏。 參 流體檢測試片1包含有基板10及-電極層I9。基板10包含有流道u。流 道11上設置有依序連接之第一流體區lu、第二流體區112與第三流體區 113，其中第一流體區lu係供流體之注入。 請繼續參考第1B圖，為本發明第—較佳實施例流趙檢測試片流道中反 應材料分佈示意圖。第-流體區ηι中具有第一抗體仙膽類材料ιιΐ2 及過氧化物梅跡第二抗體區m十具有第二抗體mi，而第二抗體應 是固定在第二抗體區m之中。又，第一抗體而與第二抗趙ιΐ2ι都是辨 識流體中的同-種待測物質1101，但是第一抗體刖與第二抗體】⑵是各 自辨識待測物質蘭上*同的抗原決定位p所以在第—抗體丨⑴和第二 抗體1121的選用上，兩者均可為單株抗體（mAb)或多株抗體（㈣只 6 201017157 需要第一抗體1111和第一抗體1121是一組抗體配對（antibody pair)，在進 行抗原辨識時，不致發生互相干擾的狀況即可。第三流體區113之中則具有 受質反應試劑1132 ’受質反應試劑1132包含有醣氧化晦1133。其中，過氧 化物梅1113可以是辣根過氧化物騰(HRP, Horseradish Peroxidase)、抗壞血·酸 過氧化物晦（AP, Ascorbate Peroxidase)或是過氧化氣晦（hydrogen peroxidase)。在較佳的實施狀態中’醣類材料1112可為葡萄糖，於此同時， 醣氧化晦1133則是選用葡萄糖氧化酶。 _ 請繼續參考第lc至第1E圖’為本發明第一較佳實施例流體檢測試片 流道中反應材料在不同反應階段的分佈示意圖。首先請參考第1C圖，當含 有待測物質1101的流體注入流道U之後，第一抗體lm會辨識到待測物 質1101，並與其做結合。此外，由於第一抗體lni、醣類材料1112及過氧 化物晦1113並未固定於第一流體區中，所以此時第一抗趙丨m、與第一抗 體1111結合之待測物質110卜過氧化物臃1113，及醣類材料1112會隨著流 體一起繼續往第二流體區112流動。 請繼續參考第1D圖，當流體流至第二反應區112之後，第二抗體1121 會與待測物質1101結合。此時’由於第二抗體112ι是固定在第二流體區n2 • 之中，所以會將已經與待測物質1101結合的第一抗體1111以及在第一抗體 1111之上的過氧化物腺1113 —併留置於第二流體區112之中；而此時亦流 至第二流體區112的醣類材料m2則會和未與待測物質11〇1結合的第一抗 體1111和過氧化物梅1113則會隨著流體繼續向第三流體區ι13流動。 請繼續參考第ιέ圖，醣類材料m2和未與第一抗體lm、第二抗體 1112以及待測物質1101結合留置在第二流體區112之中的過氧化物腌1113 會流至第二流體區113。此時，醣類材料1112會與已經存在於第三流體區 113之中的醣氧化旖1133進行氧化反應，產生出過氧化氫（扣〇2)，而產生 出的過氧化風會受到流至第三反應區113的過氧化物晦m3進行催化，產 7 201017157 生電學訊號’利用電極層19上電極及電路之將電學信號傳出以供儀器偵測。 此時，可藉由偵測電學訊號的強度，推算出第三流體區113中產生出之 過氧化氫經過過氧化物腺1113催化消耗之後，反應完畢所剩餘之過氧化氫 的量，又由於在製作流體檢測試片1之時，要添加的醣類材料mi與醣氧 化梅1133兩者之含量均為已知的固定值，故當醣類材料mi隨流體流至第 二流體區113與醣氧化胯1133反應後所產生的過氧化氫其總量亦會是一可 推算之固定值。所以可藉由所偵測到的電學訊號強度推算之最終剩餘之過氧 化氫含量與預定產生之過氧化氫總量，其兩者之差即為在第三流體區113令 H 被流至此區的過氧化物腌1113所消耗之過氧化氫含量，再將此被消耗之過 氧化氫含量推算出參與反應之過氧化物晦1113知濃度，即可得知流至第三 流體區113中過氧化物梅m3之量。再加上，在製作流體檢測試片丨之時， 要添加的過氧化物晦1113含量也是一已知的固定值，所以留置於第二流體 區112之中的過氧化物晦m3其濃度亦可藉由「過氧化物酶1113總量」與 「流至第二流體區113之過氧化物酶含量」兩者簡單相減後推算得知。最 後，利用推算出的留置於第二流體區U2之中的過氧化物腌1113的濃度’ 可再推算出流體中所含有的待測物質U〇1的濃度，以達到定量檢測的目的。 在未反應前，第一抗體1111與過氧化物梅1113兩者的結合型態可以是 〇 I接形成共扼結合(eGnjugatiGn);或是第-抗體mi上共結合有生物素 (Biotin) ’而過氧化物晦1113則與親和素（Avidin)共軛結合利用生物素 與親和素會強力結合成-錯合物（AB eGmplex)的特性，使得在進行反應時， 當有第-抗體ini留置於第二流體區112時，也會將部份的過氧化物晦1113 固定留置於第二流體區112。上述之親合素可以是㈣素（Avidin)、鍵徽親 合素（Streptavidin)，或是中性鏈親和素（NeutrAvidin)。 凊繼續參考第IF ®至第1H圖，為本發明第—較佳實施織體檢測試 片流道中反應材料其他方式分佈示意圖。為達縣發明的定量制之目的， 流體檢測試片1中醣類材料1112及過氧化物腌1113在流道u中的分佈方 8 201017157 式除如第1B圖所示之均存在於第一流道111的分佈方式之外，還可以分佈 在第二流道112之中。 首先，請參考第1F圖，第一抗體1111和過氧化腾m3位於第一流體 區111中，而醣類材料1112則位於第二流體區112中，此時，第一抗禮im 與過氧化物腌1113兩者的結合型態方式則與上述之直接共輛結合，或是利 用生物素與親和素結合成一錯合物的特性，第一抗體1111與過氧化物腌13 各自以結合有生物素與親和素的方式存在。流道11中其餘之反應材料第二 抗趙1121、受質反應试劑1132 ’及聽氧化晦II33的分佈方式則與第1B圖201017157 VI. Description of the Invention: [Technical Field] The present invention relates to a fluid detecting test piece, and more particularly to a fluid detecting test piece for quantitative detection. [Prior Art] In the conventional technique of quantification of a biological sample, in the biochemical detection, a specific enzyme is often used to react with a substance to be tested, and then the chemical or electrical properties of the product produced by the reaction are detected. For example, a glucose oxidase is immobilized on a dissolved oxygen electrode, and a potential or a current change caused by hydrogen peroxide generated by the reaction of glucose gluconate with glucose is used to measure the concentration of hydrogen peroxide generated. And calculate the blood glucose concentration in the sample. Since such an electrochemical test method can cause a change in potential or current as long as a trace amount of hydrogen peroxide is present, the required sample body is less active and the detection speed is fast. However, this type of measurement is currently only suitable for quantitative measurement of small molecules, such as blood sugar, total cholesterol, urea, creatinine, etc., because of the need for proper enzymes to produce peroxides that are easy to detect. Another way is to use the specificity of the immune molecule to identify the specificity of the measurement, such as the traditional enzyme linked immunosorbent assay (enzyme linkedimmunQlsOTbent ELISA) 'more in the 96-well plate (96-well plate) operation, and borrow The concentration emitted by the _ final enzyme reaction is reduced. However, due to this test, in order to avoid non-specific binding of immune molecules and other impurities contained in the injection, it is necessary to add a cleaning reaction to the filaments and the preparations or the combination of the money and the traits in each reaction stage. Cleared from the 96-well plate to avoid false positive signals caused by excessive enzymes remaining in each well (weU), resulting in a test failure. This type of immunological measurement has the advantages of specificity (spedfi (%) high and sensitivity (sensitivity). Due to the development of electromechanical technology, the test piece with micro flow channel structure has been used to immunize the test' to simplify the above traditional test. In the method, the cumbersome process such as cleaning after each step is carried out. However, it is known that the test piece of 3 201017157 is required to additionally add the reaction reagent or the substrate required for the reaction when performing _;峨 峨 # 巾 巾 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 It needs to be stored in a specific environment (such as refrigerated or protected from light) to avoid the error of the test due to the deterioration of the reactants. Therefore, this kind of immunization mask is still inconvenient. Micro-flow channel junction fluid inspection job #, the flow around the road is not a water-absorbing material' and the body is mostly a high-spirited thing such as the egg from Wei Wei Lai, so when the fluid to be tested flows, it will flow. Remaining on the road, making it to be The fluid cannot be completely reacted, which not only causes waste of the body, but is more likely to cause errors in the most (four) test results. In addition, the fluid test piece with the micro-channel structure is used in fluid transfer. In the capillary channel of the micro-channel structure, the Newwei over-flow channel is passively transmitted to the reaction side region; the other method is to apply a driving force to the fluid when the fluid to be tested is injected, so that the fluid can actively pass. The flow path reaches the reaction enthalpy; the body. However, no matter the above-mentioned __ mode, the fluid to be tested often injects into the flow channel and often causes bubbles of different sizes to block the flow path, causing errors in actual measurement and even causing the test to fail. SUMMARY OF THE INVENTION In order to overcome the above disadvantages, the present invention provides a fluid detecting test piece comprising a substrate having a flow path, wherein the flow path is provided with a first fluid zone, a second fluid zone and a third fluid zone sequentially connected, The first fluid zone is for the injection of fluid. In addition, the flow channel has the first antibody, the saccharide material, the peroxide, the second antibody and the receptor reaction test. The first antibody is located in the first fluid zone for identifying the substance to be tested. The saccharide material and the peroxide salt are located in the first fluid zone or the second fluid zone. The second antibody is immobilized on the first In the two-fluid region, the same substance to be tested is also identified, and the second antibody and the first anti-system identify different epitopes. The host-reactive reagents are co-located in the third fluid region. A cholesteric oxide is included. Thereby, after the fluid containing the substance to be tested is injected into the flow channel, the first antibody, the saccharide material and the peroxide lanthanum are moved with the fluid flow 4 201017157, and some of the peroxide is oxidized. Will bind to the first antibody, the test substance and the second antibody and remain in the second fluid zone, the unbound peroxide gland flows with the fluid to the third fluid zone, and the sugar oxidized plum will catalyze the flow to the first The saccharide material in the three-fluid zone undergoes an oxidation reaction and produces hydrogen peroxide (Ηζ〇2), and the hydrogen peroxide is catalyzed by the peroxide ruthenium flowing to the third fluid zone to generate an electrical signal. . Accordingly, it is a primary object of the present invention to provide a fluid test strip which, by having all the reagents and materials required for the reaction, can directly measure the final reaction signal for quantitative detection without cumbersome steps. Another object of the present invention is to provide a fluid detecting test piece in which a reagent and a material required for the reaction are stored in a test piece in a dry manner before the reaction, so that the test can be stored for a long period of time without causing deterioration due to deterioration of the reagent. error. Another object of the present invention is to provide a fluid detecting test piece which can be combined with the existing electrochemical detecting technology, and has the advantages of short detection time, high specificity and good sensitivity. The invention also provides a fluid detection and detection method, which mainly comprises the following steps. (1) Providing Zhao with the object to be tested (2) provides a substrate, the button includes at least a flow channel, and the flow channel includes a first fluid region, a second fluid region and a third fluid region, which are sequentially connected, the first fluid The zone is supplied with fluid. The substrate further comprises a first antibody located in the first fluid region for identifying the substance to be tested in the fluid: the saccharide material is located in the first fluid region or the second fluid region; Located in the first fluid zone or the second fluid zone; the second antibody is fixed in the second fluid zone, and also identifies the same antigenic-determining position; and the antigen-determining position of the first antibody and the first antibody system are different; The chromogenic reagent, located in the third fluid zone, contains a sugar oxidase. The fluid is applied to the first body region of the channel to cause the first antibody, the carbohydrate material, and the peroxidase to flow with the fluid. (4) placing the test substance with the first antibody, the second antibody, and a portion of the transcript (4) in the second fluid, in which the fluid carries the brewed material, the unconjugated antibody, and the peroxide The turbulent flow to the third fluid zone is oxidized by the sugar-oxidized plum and produces nitrogen peroxide, which is catalyzed by the peroxide-mei catalyzed by the hydrogen peroxide. Generate an electrical 5 201017157 signal. (5) Detecting the electrical signal generated. Therefore, another object of the present invention is to provide a fluid detecting method which is capable of performing quantitative measurement by directly processing the final reaction signal due to the fact that it has all the reactions and materials required for the reaction. This hair #目#系k _ kind of fluid detection ^^ method of the towel reaction required for the material, in the test before the 峨 峨 于 in the test piece, it can be long secret, not caused by reagent deterioration Test error. The re-purpose of the present invention is the same as that of the electrochemical electrochemical extraction technology, which has the advantages of short detection time, high specificity and high sensitivity. [Embodiment] Since the present invention discloses a fluid detecting test piece and a test method thereof, the chemical principle and the biological detecting technology used therein have been known to those having ordinary knowledge in the related art, and therefore, the description below is not A full description is given. At the same time, the drawings in the following texts express the unintentional meanings associated with the features of the present invention, and do not need to be completely based on the actual situation. The monthly reference figure is a schematic diagram of a fluid detecting test piece according to a first preferred embodiment of the present invention. The fluid detecting test piece 1 includes a substrate 10 and an -electrode layer I9. The substrate 10 includes a flow path u. The flow path 11 is provided with a first fluid zone lu, a second fluid zone 112 and a third fluid zone 113 which are sequentially connected, wherein the first fluid zone lu is for fluid injection. Please refer to FIG. 1B for a schematic diagram of the distribution of the reaction material in the flow path of the test strip according to the first preferred embodiment of the present invention. The first fluid region ηι has a first antibody scented material ιιΐ2 and a peroxide smear second antibody region m s having a second antibody mi, and the second antibody is immobilized in the second antibody region m. Further, the first antibody and the second anti-Zhao ΐ2 2 are both the same test substance 1101 in the identification fluid, but the first antibody 刖 and the second antibody 】 (2) are antigenic determinations each identifying the substance to be tested. Therefore, in the selection of the first antibody (1) and the second antibody 1121, both of them can be monoclonal antibodies (mAb) or multiple antibodies ((4) only 6 201017157 requires the first antibody 1111 and the first antibody 1121 is a The antibody pair is not affected by mutual recognition when the antigen is recognized. The third fluid region 113 has a substrate reaction reagent 1132'. The reagent reagent 1132 contains glycocalyx 1133. Among them, the peroxide plum 1113 may be Horseradish Peroxidase, Ascorbate Peroxidase or Hydrogen Peroxidase. In a preferred embodiment, the saccharide material 1112 may be glucose, and at the same time, the glucose oxidized cesium 1133 is selected from glucose oxidase. _Continue to refer to the first lc to the first embodiment of the present invention. Body test Schematic diagram of the distribution of the reaction materials in the flow path at different reaction stages. First, referring to FIG. 1C, after the fluid containing the substance to be tested 1101 is injected into the flow channel U, the first antibody lm recognizes and combines with the substance 1101 to be tested. In addition, since the first antibody lni, the saccharide material 1112, and the peroxide 晦1113 are not fixed in the first fluid region, the first anti-Zhao M, the test substance 110 combined with the first antibody 1111 is at this time. The peroxide 臃 1113, and the saccharide material 1112 will continue to flow along the fluid to the second fluid zone 112. Referring further to Figure 1D, after the fluid flows to the second reaction zone 112, the second antibody 1121 will The test substance 1101 is combined. At this time, since the second antibody 112 is fixed in the second fluid region n2, the first antibody 1111 which has been bound to the test substance 1101 and the first antibody 1111 will be over. The oxide gland 1113 - and remains in the second fluid region 112; and the saccharide material m2 which also flows to the second fluid region 112 at this time and the first antibody 1111 which does not bind to the test substance 11 〇 1 Peroxide Mei 1113 will follow The body continues to flow to the third fluid region ι13. Please continue to refer to the figure ι, the saccharide material m2 and the first antibody lm, the second antibody 1112, and the test substance 1101 are not retained in the second fluid region 112. The oxide salt 1113 will flow to the second fluid zone 113. At this time, the saccharide material 1112 will be oxidized with the glucoside ruthenium 1133 already present in the third fluid zone 113 to produce hydrogen peroxide (Buckling 2 The peroxidized wind generated is catalyzed by the peroxide 晦m3 flowing to the third reaction zone 113, and the electrical signal is transmitted from the upper electrode and the circuit of the electrode layer 19 for supply. Instrument detection. At this time, by detecting the intensity of the electrical signal, the amount of hydrogen peroxide remaining after the reaction of the hydrogen peroxide generated in the third fluid region 113 through the peroxide gland 1113 can be estimated, and At the time of preparation of the fluid test strip 1, the content of both the saccharide material mi and the oxidized plum 1133 to be added is a known fixed value, so when the saccharide material mi flows with the fluid to the second fluid region 113 and The total amount of hydrogen peroxide produced after the reaction of glucosinolate 1133 will also be an inferred fixed value. Therefore, the difference between the final remaining hydrogen peroxide content and the predetermined total amount of hydrogen peroxide generated by the detected electrical signal strength is that the difference between the two is that the third fluid region 113 causes H to flow to the region. The peroxide is salted and the hydrogen peroxide content consumed by the 1113 is used, and then the consumed hydrogen peroxide content is deduced to calculate the concentration of the peroxide 晦1113 participating in the reaction, and the flow to the third fluid region 113 is known. The amount of oxide plum m3. In addition, when the fluid detecting test piece is prepared, the content of the peroxide 晦 1113 to be added is also a known fixed value, so the concentration of the peroxide 晦 m3 remaining in the second fluid region 112 is also It can be estimated by simply subtracting both the "peroxidase 1113 total amount" and the "peroxidase content flowing to the second fluid region 113". Finally, the concentration of the substance to be tested U〇1 contained in the fluid can be further calculated by using the calculated concentration of the peroxide salt 1113 remaining in the second fluid region U2 to achieve the purpose of quantitative detection. Before the unreacted, the binding form of the first antibody 1111 and the peroxide plum 1113 may be 〇I to form a conjugated (eGnjugatiGn); or the first antibody may be bound to biotin (Biotin). Conjugate 晦1113 is conjugated with avidin (Avidin), and biotin and avidin bind strongly to the characteristics of AB-Gmplex, so that when the reaction is carried out, when the first antibody ini is indwelled In the second fluid zone 112, a portion of the peroxide crucible 1113 is also retained in the second fluid zone 112. The avidin described above may be Avidin, Streptavidin, or NeutrAvidin.凊Continuing to refer to the IF ® to 1H drawings, which are schematic diagrams showing other modes of distribution of the reaction materials in the flow path of the texture detecting test piece of the first preferred embodiment of the present invention. For the purpose of the quantitative system of Daxian invention, the distribution of the sugar material 1112 and the peroxide salt 1113 in the fluid detection test piece 1 in the flow path u is in the first flow except that the first flow is shown in FIG. 1B. In addition to the manner in which the tracks 111 are distributed, they may also be distributed in the second flow path 112. First, referring to FIG. 1F, the first antibody 1111 and the peroxidation m3 are located in the first fluid region 111, and the saccharide material 1112 is located in the second fluid region 112. At this time, the first resistance im and peroxidation The combination of the two types of pickled 1113 is combined with the above-mentioned direct combination, or the combination of biotin and avidin into a complex, the first antibody 1111 and the peroxide are marinated 13 respectively to bind the organism The way of avidin and avidin exists. The remaining reaction materials in the flow channel 11 are distributed in the second anti-Zhao 1121, the photoreactive reagent 1132 ′ and the yttrium oxide II33.
中相同。採用此種分佈之設置狀態，在流體流過第二流體區112後，醣類材 料im-樣會隨著流體流至第三流體區113,故其後之各反應階段中反應材 料的流動分佈情形及所進行之反應則與第lc圖至第1E圓所示，在此不再 重複贅述。 請再參考第1G圖，此種分佈設置下，第—抗體mi是位於 m中，而過氧化㈣13與醣類材料1112則位於第二流體區ιΐ2中二而在 第1H圖所代表的分佈方式，則是第一抗體而與糖類材料⑴2位 流體區m、過氧化梅1113麻於帛二流體區m卜流道丨丨 1132 * 1133 行之反應難第1Γ之各反紐段巾反雜料的_分料形及所進 行之反應顺第lc圖至第1Ε圖_，在此砰重複贊述。 在第=311圖，為第1Α圖沿ΑΑ連線的剖面示意圖。如圖所示， 在第趟£ m之底部包含有纖維層111〇 …此使得在流體注入第== 體=體^)3:紐-械至第二流體區⑴。而在第二流 化纖維層113〇。第一;二3自具有、第二確化纖維層_與第三胡 之中，而受質反二=? b圖)係固定於第，維_。 應趣1(1132，第1Μ)係形成於第三硝化纖維層1130 9 201017157 中ο 上述的第二硝化纖維層1120與第三硝化纖維層1130可以是以一層硝化 纖維膜(NC membrane)的形式鋪設在第二流體區112以及第三流體區113的 底部。 另一種較佳的製作形式，則可以將硝化纖維溶液以澆注（casting)的方 式，洗注在第二流體區112以及第三流體區113的底部，再經過風乾或是冷 凍乾燥的步驟’藉此各自形成具有中空網狀構型的第二硝化纖維層U2〇與 第三硝*化纖維層1130。此外，為了降低流道與流體之間的毛細作用所造成 的影響’此種以澆注方式所製成之流道並非習知技術所謂的微流道，且第二 流體區112與第三流體區113的最小寬度較佳為〇 3 _，而基板1〇則可採 用生物相容材料。為達較佳的洗注效果，流道11的表面粗糙度較佳範圍（Ra 值）在3微米至50微米之間，而第二硝化纖維層112〇的平均厚度等於第三 硝化纖維層1130厚度。此外，流道u可進一步包括第四流體區（未圖示）， 第四流趙區之底部亦形成有硝化纖維層，供多餘流體之貯存，且此硝化纖維 層具有中空網狀構型。 上述之硕化纖維溶液係將硝化纖維粉末與包含有酯類及酮類溶劑混合 後製成’而硝化纖維粉末與酯類及酮類溶劑所混合的較佳體積比例為丨：9。 又’在此種以澆注方式形成硝化纖維層的狀態下，可在第二硝化纖維層112〇 與第二视纖維層113〇乾燥後，將第二抗體1121以溶液的方式注入第二石肖 化纖維層im ’再經频^或冷絲制過程，使帛二抗體1121以粉末狀 的方式留存於第二硝化纖維層112〇之中。 除上述先形成硝化纖維層於流體區底部後再將第二抗體1121及受質反 應試劑形成於其巾的方式之外，還可鄕二抗㈣21麟液的方式注入預 先製備好的硝化纖維溶液，混合均勻後，再躲於第二流體區ιΐ2的底部， 再經風乾或冷綠魏程，同時將硝化_絲形成第二舰娜層測 及第二抗體1121以粉末狀的方式留存其中。 201017157 此外，受質反應試劑1132形成於第三硝化纖維層113〇中的方式亦與上 述第二抗體1121形成於第二靖化纖維層1120的方式大致相同，可採用先形 成第三硝化纖維層1130後再注入受質反應試劑並乾燥，或是與硝化纖維溶 液混合後一同注入第三流體區113底部乾燥成型的兩種方式，故在此不再重 複贅述。 除上述的第一較佳實施例之外，本發明亦提供一種流體檢測方法，如下 之第二較佳實施例中所述’其中檢測方法中所採用之檢測試片，其構造特徵 參 與第一較佳實施例中大致相同’故不再重複贅述，且其中提及之檢測試片元 件編號，請參考第1B圖至第1H圖。 睛參考第2圖，為本發明第二較佳實施例流趙檢測方法之流程示意圖。 流體檢測方法2包含以下步驟： 步驟21 :提供一流體，流體包含有待測物質u〇1，請參考第m圖。 步驟22 :提供基板10，基板10包含至少有流道11。流道11包含依序 連接之第一流體區11卜第二流體區112與第三流體區113，第一流艎區m 係供流體之注入。第一流體區ill中具有第一抗體llu。第二抗體區112中 具有第二抗體U21，且第二抗體1121是固定在第二抗體區112之中。又， β 第一抗體1111與第二抗體1121都是辨識流體中的同一種待測物質11〇1，但 是第抗體ini與第二抗體1121是各自辨識待測物質丨丨。！上不同的抗原 決疋位置。所以在第一抗體1111和第二抗體1121的選用上，兩者均可為單 株抗體或多株抗體’只需要第一抗體1111和第二抗體1121是一組抗體配 對，在進行抗原辨識時，不致發生互相干擾的狀況即可。第三流體區113之 中則具有受質反應試劑1132，受質反應試劑1132包含有醣氧化梅1133 ^其 中，過氧化物晦1113可以是辣根過氧化物晦、抗壞血酸過氧化物臃或是過 氧化氫晦。在較佳的實施狀態中，醣類材料1112則可為葡萄糖，於此同時， 醣氧化晦1133則是選用葡萄糖氧化晦。此外，醣類材料1112及過氧化物晦 11 201017157 1113在流道11的各種分佈情形，則如第一較佳實施例中所述（請參考第IB 圖、第IF圖、第1G圖及第1H圖）。 步驟23 :將流艎加至流道11之第一流體區ill。流體會沿著流道11依 序由第一流體區111流經第二流體區112，流至第三流體區113，並藉使存 在於流道11中的第一抗體1111、醣類材料1112及過氧化物腌1113隨著流 體流動。 步驟24:待測物質1101會隨流體流至第二流體區112,使待測物質1101 與第一抗體1111、第二抗體1121及部份之過氧化物晦m3結合並一同留置 於第二流體區112中。流體繼續帶著醣類材料1112、未結合之第一抗體mi 及過氧化物酶1113流至第三流體區113。在第三流體區中醣類材料m2受 到醣氧化晦1133之催化進行氧化反應’並產生出過氧化氫(h2〇2)。而過氧 化氫會受到流至第三流體區113的過氧化物晦m3催化與進行反應，並產 生一電學訊號，利用電極層19上電極及電路之將電學信號傳出以供儀器偵 測》 步驟25 :债測由步驟24產生之電學訊號。此時，可藉由偵測電學訊號 的強度，推算出第三流體區113中產生出之過氧化氳經過過氧化物腺1113 催化消耗之後，反應完畢所剩餘之過氧化氫的量，又由於在製作流體檢測試 片1之時’要添加的醣類材料1111與醣氧化酶1133兩者之含量均為已知的 固定值，故當醣類材料1111隨流體流至第三流體區113與醣氧化腾1133反 應後所產生的魏錢其總量亦會是—可推算之JU定值。所財藉由所摘測 到的電學訊號強度推算之最終剩餘之過氧化氫含量與預定產生之過氧化氫 總量，其兩者之差即為在第三流體區113中被流至此區的過氧化物腌ιιΐ3 所消耗之過氧域含量’再將此被雜之魏化氫含錄算岭與反應之過 氧化物酶1113知濃度，即可得知流至第三流體區113中過氧化物酶ιιΐ3之 量:再加上，在製作越檢職片丨之時，要添加的過氧化鱗ιιΐ3含量 也是-已知的固定值’所以留置於第二流體區112之中的過氧化物晦ιιΐ3 12 201017157 其濃度亦可藉由「過氧化物腌1113總量」與「流至第三流體區113之過氧 化物腌含量」兩者簡單相減後推算得知。最後，利用推算出的留置於第二流 體區m之中的過氧化物腌1113的濃度，可再推算出流體中所含有的^ 物質1101的濃度，以達到定量檢測的目的。 4 此外，根據本發明之流體檢測方法，其中第一抗體llu與過氧化物梅 1113的結合方式與較佳選用種類、各流體區的構造組成、硝化纖維層之構 型、形成方式、使用之硝化纖維溶液之成份與較佳比例、各項反應材料之組 成及形成方式，均與前述之第一較佳實施例相同，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 權利；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1A圖，為本發明之第一較佳實施例流體檢測試片之示意圖。 第1B圖，為本發明第一較佳實施例流體檢測試片流道中反應材料分佈 示意圖。 第ic圖至第1E圖，為本發明第一較佳實施例流體檢測試片流道中反 應材料在不同反應階段的分佈示意圖。 第1F圖至第1H圖，為本發明第一較佳實施例流體檢測試片流道中反 應材料其他方式分佈示意圖。 第II圖’為本發明第一較佳實施例流體檢測試片剖面示意圖。 第2圖，為本發明第二較佳實施例流體檢測方法之流程示意圖。 【主要元件符號說明】 流體檢測試片 13 201017157 基板 10 流道 11 待測物質 1101 第一流體區 111 纖維層 1110 第一抗體 1111 醣類材料 1112 過氧化物梅 1113 第二流體區 112 第二硝化纖維層 1120 第二抗體 1121 第三流體區 113 第三硝化纖維層 1130 受質反應試劑 1132 醣氧化酶 1133Same in the middle. With such a distributed state, after the fluid flows through the second fluid region 112, the sugar material im-like flows with the fluid to the third fluid region 113, so the flow distribution of the reactive material in each subsequent reaction phase The situation and the reaction performed are shown in the figures lc to 1E, and the details are not repeated here. Please refer to the 1G map. Under this distribution setting, the first antibody mi is located in m, and the peroxidation (tetra) 13 and the sugar material 1112 are located in the second fluid region ι 2 and the distribution pattern represented by the 1H map. , is the first antibody and the saccharide material (1) 2 fluid zone m, peroxidized plum 1113 麻 帛 帛 流体 流体 流体 m 132 132 132 132 132 132 132 132 132 132 132 132 132 132 132 132 132 132 132 132 132 132 132 132 132 132 132 132 132 The _substrate shape and the reaction carried out are in accordance with the lc diagram to the first diagram _, and are repeated here. In the figure = 311, it is a schematic cross-sectional view of the first line along the ΑΑ line. As shown, at the bottom of the 趟£ m, there is a fibrous layer 111 〇 ... which causes the fluid to be injected into the == body = body ^) 3: the neo-mechanical to the second fluid zone (1). In the second fluidized fiber layer 113, 〇. The first; the second three are self-contained, the second confirmed fiber layer _ and the third hum, and the tempering anti-two = ? b map) is fixed at the first, dimension _. The first nitrocellulose layer 1120 and the third nitrocellulose layer 1130 may be in the form of a layer of nitrocellulose membrane (NC membrane). The second nitrocellulose layer 1120 and the third nitrocellulose layer 1130 may be formed in the form of a layer of nitrocellulose membrane (1,1,132). Laying at the bottom of the second fluid zone 112 and the third fluid zone 113. In another preferred form, the nitrocellulose solution can be smeared in the second fluid zone 112 and the bottom of the third fluid zone 113 by casting, and then air-dried or freeze-dried. This each forms a second nitrocellulose layer U2 and a third nitrocellulose layer 1130 having a hollow network configuration. In addition, in order to reduce the influence of the capillary action between the flow path and the fluid, the flow path formed by the casting method is not a so-called micro flow channel of the prior art, and the second fluid region 112 and the third fluid region The minimum width of 113 is preferably 〇3 _, and the substrate 1 可采用 can be made of a biocompatible material. For better washing effect, the surface roughness of the flow passage 11 is preferably in the range of 3 to 50 micrometers, and the average thickness of the second nitrocellulose layer 112 is equal to the third nitrocellulose layer 1130. thickness. Further, the flow path u may further include a fourth fluid zone (not shown), and a bottom of the fourth flow zone is also formed with a nitrocellulose layer for storage of excess fluid, and the nitrocellulose layer has a hollow network configuration. The above-mentioned master fiber solution is obtained by mixing nitrocellulose powder with a solvent containing a ketone and a ketone, and the preferred volume ratio of the nitrocellulose powder to the ester and the ketone solvent is 丨:9. Further, in the state in which the nitrocellulose layer is formed by casting, the second antibody 1121 can be injected into the second diatom as a solution after the second nitrocellulose layer 112 〇 and the second visor layer 113 are dried. The chemical fiber layer im 're-frequency or cold wire process, so that the second antibody 1121 remains in the powder form in the second nitrocellulose layer 112 。. In addition to the above method of forming the nitrocellulose layer at the bottom of the fluid zone and then forming the second antibody 1121 and the substrate reaction reagent in the towel, the pre-prepared nitrocellulose solution may be injected in the manner of the second antibody (4) 21 lin liquid. After mixing evenly, it is hidden in the bottom of the second fluid zone ιΐ2, and then air-dried or cold green, while the nitrification-filament is formed into the second naval layer and the second antibody 1121 is retained in a powder form. 201017157 In addition, the manner in which the reagent reagent 1132 is formed in the third nitrocellulose layer 113〇 is also substantially the same as the manner in which the second antibody 1121 is formed on the second lining fiber layer 1120, and the third nitrocellulose layer can be formed first. After the 1130, the reagent is further injected and dried, or mixed with the nitrocellulose solution and injected into the bottom of the third fluid region 113 to form a dry form. Therefore, the description thereof will not be repeated here. In addition to the first preferred embodiment described above, the present invention also provides a fluid detecting method, as described in the second preferred embodiment below, wherein the detecting test piece used in the detecting method has its structural features participating in the first In the preferred embodiment, the description is omitted, and the description of the test piece element number mentioned therein is referred to the drawings 1B to 1H. FIG. 2 is a schematic flow chart of a method for detecting a flow detection according to a second preferred embodiment of the present invention. The fluid detecting method 2 comprises the following steps: Step 21: Providing a fluid containing the substance to be tested u〇1, please refer to the mth figure. Step 22: Providing a substrate 10 comprising at least a flow channel 11. The flow path 11 includes a first fluid zone 11 connected in sequence, a second fluid zone 112 and a third fluid zone 113, and the first flow zone m is for fluid injection. The first fluid region ill has a first antibody 11u. The second antibody region 112 has a second antibody U21, and the second antibody 1121 is immobilized in the second antibody region 112. Further, both the β first antibody 1111 and the second antibody 1121 are the same test substance 11〇1 in the identification fluid, but the first antibody ini and the second antibody 1121 are each identifying the substance to be tested. ! Different antigens on the decision site. Therefore, in the selection of the first antibody 1111 and the second antibody 1121, both of them can be monoclonal antibodies or multiple antibodies. Only the first antibody 1111 and the second antibody 1121 are required to be a pair of antibodies, and when antigen identification is performed. It is not necessary to interfere with each other. The third fluid zone 113 has a matrix reaction reagent 1132, and the matrix reagent reagent 1132 comprises a sugar oxidized plum 1133^ wherein the peroxide oxime 1113 can be horseradish peroxide strontium, ascorbyl peroxide or Bismuth hydrogen peroxide. In a preferred embodiment, the saccharide material 1112 can be glucose. At the same time, the glucoside ruthenium oxide 1133 is selected from the group consisting of glucose ruthenium oxide. In addition, the various distributions of the saccharide material 1112 and the peroxide 晦11 201017157 1113 in the flow channel 11 are as described in the first preferred embodiment (please refer to the IB diagram, the IF diagram, the 1G diagram, and the 1H picture). Step 23: The flow is added to the first fluid zone ill of the flow path 11. The fluid will sequentially flow from the first fluid zone 111 to the third fluid zone 112 along the flow path 11 to the third fluid zone 113, and the first antibody 1111 and the saccharide material 1112 present in the flow channel 11 And peroxide marinated 1113 as the fluid flows. Step 24: The test substance 1101 will flow with the fluid to the second fluid region 112, and the test substance 1101 is combined with the first antibody 1111, the second antibody 1121 and a portion of the peroxide 晦m3 and left together in the second fluid. In area 112. The fluid continues to flow to the third fluid zone 113 with the carbohydrate material 1112, the unbound primary antibody mi, and the peroxidase 1113. In the third fluid zone, the saccharide material m2 is catalyzed by the oxidation of ruthenium sulphate 1133 to produce hydrogen peroxide (h2 〇 2). The hydrogen peroxide is catalyzed and reacted by the peroxide 晦m3 flowing to the third fluid region 113, and an electrical signal is generated, and the electrical signals are transmitted from the electrodes and circuits on the electrode layer 19 for detection by the instrument. Step 25: Debt test the electrical signal generated by step 24. At this time, by detecting the intensity of the electrical signal, it is possible to estimate the amount of hydrogen peroxide remaining after the reaction of the peroxide generated in the third fluid region 113 through the peroxide gland 1113, and When the fluid detecting test piece 1 is produced, the content of both the saccharide material 1111 and the sugar oxidase 1133 to be added is a known fixed value, so when the saccharide material 1111 flows with the fluid to the third fluid region 113 and The total amount of Wei Qianqi produced after the reaction of sugar oxidizing 1133 will also be the estimated value of JU. The difference between the final remaining hydrogen peroxide content and the predetermined total amount of hydrogen peroxide generated by the measured electrical signal strength is the difference between the two in the third fluid zone 113. The peroxygen content consumed by the peroxide-washed ιιΐ3 is further known as the peroxidase content of the mixed-digested hydrogen-containing calculus and the reaction, and the peroxide is introduced into the third fluid region 113. The amount of the enzyme ιιΐ3: In addition, the content of the peroxidized scale ιι 3 to be added is also a known fixed value at the time of making the vacant film, so the peroxide 留 remaining in the second fluid region 112 Ιιΐ3 12 201017157 The concentration can also be estimated by simply subtracting both the "peroxide salt 1113" and the "peroxide salt content of the third fluid zone 113". Finally, by using the calculated concentration of the peroxide salt 1113 remaining in the second fluid region m, the concentration of the substance 1101 contained in the fluid can be further calculated to achieve the purpose of quantitative detection. In addition, according to the fluid detecting method of the present invention, the combination of the first antibody 11u and the peroxide plum 1113 and the preferred type, the structural composition of each fluid region, the configuration of the nitrocellulose layer, the formation manner, and the use thereof The composition and the preferred ratio of the nitrocellulose solution, the composition of the various reactive materials, and the manner of formation thereof are the same as those of the first preferred embodiment described above, and the detailed description thereof will not be repeated here. The above is only the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. The above description should be understood and implemented by those skilled in the art, and thus the other embodiments are not disclosed. Equivalent changes or modifications made in the spirit of the invention are to be included in the scope of the claims below. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1A is a schematic view showing a fluid detecting test piece according to a first preferred embodiment of the present invention. Fig. 1B is a schematic view showing the distribution of a reaction material in a flow path of a fluid detecting test piece according to a first preferred embodiment of the present invention. 1 to 1E are schematic views showing the distribution of the reaction materials in the flow paths of the fluid detecting test piece in different reaction stages according to the first preferred embodiment of the present invention. 1F to 1H are schematic views showing other modes of distribution of the reaction material in the flow path of the fluid detecting test piece according to the first preferred embodiment of the present invention. Fig. II is a schematic cross-sectional view showing a fluid detecting test piece according to a first preferred embodiment of the present invention. Fig. 2 is a flow chart showing a fluid detecting method according to a second preferred embodiment of the present invention. [Main component symbol description] Fluid detecting test piece 13 201017157 Substrate 10 Flow path 11 Test substance 1101 First fluid area 111 Fiber layer 1110 First antibody 1111 Sugar material 1112 Peroxide Mei 1113 Second fluid region 112 Second nitrification Fiber layer 1120 Second antibody 1121 Third fluid zone 113 Third nitrocellulose layer 1130 Receptor reagent 1132 Sugar oxidase 1133