201017160 六、發明說明： 【發明所屬之技術領域】 本發明係關於-種紐檢測試>1’特別是—種·於驗定量 體檢測諸.κ。 【先前技術】 在生物檢體的定量的習知技術中，常利用免疫分子具有專一性辨識能力 的特定來騎測量’例如傳_料連結肢韻職（enzyme触^ ❹ imm皿〇1—袖as; ELISA) ’多在96孔盤（96_wdl 中操作，並藉 由侧最終酵素反應所發出之訊號，回推待測物質之濃度。但是由於此種測 試’為了要避免免疫分子與待測物令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人清洗的步驟，藉崎未與制物或是其他免疫分 子結合之反應試劑自96孔盤中清除，以免造成過多的酵素留置於各孔位 (well)中所造成的偽陽性訊號，導致測試失敗。 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镇 測’簡化上述傳統測試方法中於各步驟後進行清洗等繁項流程。然目前已知 _試試片，在進行_時仍需將反應試劑或反應所需之受質（subs謙） 以額外添加的方式加入測試試片中。此種反應試劑或受質與檢測試片分離存 放’於細時再獅加的方式75是起祕其所f的反應賴或受f，在室溫 中無法長期存放，需保存於特定環境中（如冷藏_光保存），以避免因反 應物變質所導致的測試誤差。故此種檢測試片在操作及保存上仍多有所不 便。 此外1知具有流道或微流道結構的流體檢測試#，因流道周圍並非吸水 材質’且制流體多為含有如蛋白質或是酶類等黏滞度高之組成物，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， 不僅造成待測流體的浪費，更可能造成最終測試結果的誤差。 3 201017160 此外’習知具有微流道結構的流體檢測试片在流體傳送方面，則是利用 微流道結構產生的毛細現象，將流體經過流道被動傳送至反應偵測區域；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，到達反應偵測區域◊但是無論是上述任一種方式， 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 參 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、畴類材料、過氧 化物晦、第二抗體、發光劑與受質反應試劑。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腾則位於第一流體區或第二流體區 中。第二抗體則是固定(immobHize句於第二流體區中，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(epit〇p0。發光劑與 受質反應試劑係制位於第三流舰中，受質反應試劑包含有—醣氧化晦。 藉此，當包含有待測物質之流體注入流道後，第一抗體、聽類材料及過氣化 〇 物腾係隨著流體流動，部份之過氧化物腌會與第-抗體、待測物質及第二抗 體結合並冑置於帛二讀1巾，未結合之過祕娜纖雜流至該第三流 體區，且醣氧化梅會催化流至第三流體區之聽類材料進行氧化反應，並產生 出過氧化氫既〇2)，且發光劑會受到流至第三流體區的過氧化物梅催化與過 氧化風進行反應，並產生一光學訊號。 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，無驗由繁权操作步聊可直·量最終反應訊號 進行定量偵測。 本發明之另-目的’係提供—種趟侧試片，其巾反應賴的試劑與 材料’在反猶細乾齡讀存於試# t，故可長期保存 ，不致因試劑變 4 201017160 質而導致測試誤差。201017160 VI. Description of the Invention: [Technical Field to Which the Invention Is Applicable] The present invention relates to a detection test of a kind of New Zealand>1', particularly a test for quantitative detection of κ. [Prior Art] In the conventional technique of quantification of biological specimens, it is often used to take a specific measurement of the specificity of the immune molecule. For example, the transmission of the limbs is carried out (enzyme touch ^ ❹ imm dish 〇 1 - sleeve As; ELISA) 'More in 96-well plates (96_wdl, and by the signal from the side of the final enzyme reaction, push back the concentration of the substance to be tested. But because of this test 'to avoid immune molecules and analytes In order to non-specifically combine other impurities involved, it is necessary to add a cleaning step at each reaction stage, and the reaction reagent that is not combined with the product or other immune molecules is removed from the 96-well plate to avoid excessive enzyme retention. The false positive signal caused by each well (well) leads to the test failure. Due to the development of electromechanical technology, the test strips with micro flow channel structure have been used for immunosampering 'simplified the above traditional test methods After the step, the complicated process such as cleaning is carried out. However, it is known that the test piece needs to be added to the reagent or the substrate required for the reaction. Adding to the test piece. The reaction reagent or the substrate is separated from the test piece. 'The method of adding the lion to the lion is 75. It is the reaction of the sputum or the f, and it cannot be stored for a long time at room temperature. It needs to be stored in a specific environment (such as refrigerated_light preservation) to avoid test errors caused by deterioration of the reactants. Therefore, such test strips are still inconvenient in operation and storage. The fluid detection test of the flow channel structure is because the fluid around the flow channel is not a water-absorbent material, and the fluid is mostly composed of a highly viscous composition such as a protein or an enzyme, so that when the fluid to be tested flows, it will be on the flow path. Residual, the fluid to be tested cannot be completely reacted, which not only causes waste of the fluid to be tested, but is more likely to cause errors in the final test results. 3 201017160 In addition, the fluid detection test piece with micro flow path structure is known to be in fluid transfer. In one aspect, the capillary phenomenon generated by the microchannel structure is used to passively transfer the fluid to the reaction detection region through the flow channel; the other method is to use the pressurized method when injecting the fluid to be tested. The fluid is given a driving force so that the fluid can actively pass through the flow passage to reach the reaction detecting region. However, in any of the above manners, the fluid to be tested is often injected into the flow passage to generate bubbles of different sizes, causing the flow passage to block, resulting in actual The error in measurement 'even causes the test to fail. SUMMARY OF THE INVENTION To overcome the above disadvantages, the present invention provides a fluid detecting test piece comprising a ginseng plate having a flow path. The flow path is provided with a first fluid region sequentially connected a second fluid zone and a third fluid zone, wherein the first fluid zone is for fluid injection. Further, the flow channel has a first antibody, a domain material, a peroxide ruthenium, a second antibody, a luminescent agent, and a host reaction. The first antibody is located in the first fluid zone for identifying the substance to be tested, and the saccharide material and the peroxide are located in the first fluid zone or the second fluid zone. The second antibody is immobilized (immobHize in the second fluid region, and also identifies the same substance to be tested, and the second antibody and the first antibody system recognize different antigen-determining positions (epit〇p0. Luminescent agent and receptor) The reaction reagent system is located in the third flow ship, and the host reaction reagent contains saccharide ruthenium oxide. Thereby, after the fluid containing the substance to be tested is injected into the flow channel, the first antibody, the listening material and the gasified sputum As the fluid flows, part of the peroxide is combined with the first antibody, the test substance and the second antibody, and is placed on the second reading, and the unmixed misty flow to the first a three-fluid zone, and the sugar-oxidized plum catalyzes the oxidation of the auditory material flowing to the third fluid zone, and produces hydrogen peroxide (2), and the luminescent agent is subjected to peroxide flowing to the third fluid zone. Mei catalysis reacts with peroxidic wind and produces an optical signal. Therefore, the main object of the present invention is to provide a fluid test strip, which has no reagents and materials required for all reactions. Direct and quantitative final response signal Quantitative detection. Another object of the present invention is to provide a kind of side test piece, and the reagent and material of the towel reaction are stored in the test #t, so it can be stored for a long time without causing reagents. Change 4 201017160 quality and lead to test error.
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 待測物的麵》(2)触—純，基她含Μ流道，崎道包含依序連接之 第-流體區、第二流體區與第三趙區，第—流體區係供_之注入。基板 上進-步包含有第-抗體，位於第一流體區中，用於辨識該流體内之該待測 物質；聽類材料’位於第一流體區或第二流體區中；過氧化物晦，位於第一 流想區或第-流體區中，第—抗體，固定於第二流體區中，且亦辨識同一待 測物質’而第二抗體及第—紐係辨軸冑之抗原蚊位置；以及發光劑與 受質反應試劑，位於第三流贿中，受質反應試劑包含㈣氧化梅。⑶將流 體加至流道之第-流體區，使第-抗體、聽猶料及該過氧化物舰著流體 流動。(4)使制物質與第一抗體、第二抗體及部份之過氧化物梅結合並留置 於第二流躯中，使顧帶著_磐、未結合之第―抗體及魏化物職 至第三流躯，使_磐受_氧化狀絲進行氧化反應，並產生出過 氧化氫’發細衫顺至第三流舰的魏錄雜域魏化氫進行反 應’並產光學賴；。⑶細產生之光學峨。 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=需的及觀，無馳由繁狀操作步驟即可直制量最終反應訊號 進行定量1貞測。 本發明之再-目的’係提供_概體檢測綠，其_反應賴的試劑與材 料，在反應前係峨財式t存於試片巾，故可長祕存，不朗試刺變 質而導致測試誤差。 【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 理及生物檢顺術，已為梅技術領域具有通常知識者所能明瞭，故以下文 :之說明’ *再作完整描述。_ ’以下文中所職之圖式，縣達與本發 Η特徵有關之示意，並未亦不需要依據實際情形完整緣製，合先教明1 .201017160 ' 請參考帛1A ’為本發明之第一較佳實施例流體檢測試片之示意圖。 流體檢測試片1包含有基板1〇，基板1〇包含有流道1卜流道U上設置有 依序連接之第-流體區、第二流體區112與第三流趙區113，其中第一 流體區111係供流體之注入。 請繼續參考第1B圖，為本發明第—較佳實施例流體檢測試片流道中反 應材料分佈示意圖。第-流體區m中具有第一抗體冊、聽類材料1112 及過氧化物騰1113。第二流體區112中具有第二抗體U21，而第二抗體1121 疋固定在第二流體區112之中。又，第一抗體im與第二抗體1121都是辨 識流體中的同一麟測物質臟，但是第-抗艎1111與第二抗體1121是各 ® 自辨識待測物質1101上不同的抗原決定位置。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（mAb》或多株抗體（pAb)，只 需要第一抗體1111和第二抗體1121是一組抗體配對（antib〇dy 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 發光劑1131與受質反應試劑1132,受質反應試劑1132包含有醣氧化臃 1133 °其中’過氧化物酶m3可以是辣根過氧化物酶(HRp, H〇rseradish Peroxidase)、抗壞血酸過氧化物腾(AP，Ascorbate Peroxidase)或是過氧化氫腌 (hydrogen peroxidase)。在較佳的實施狀態中，發光劑1131是用3_氨基鄰苯 ❷ 二甲醯肼(5-Amino-2,3-dihydro-l，4-phthalazinec!ione)，而醣類材料 1112 則可 為葡萄糖’於此同時，醣氧化臃1133則是選用葡萄糖氧化臃。 請繼續參考第1C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11之後，第一抗體1111會辨識到待測物 質1101，並與其做結合。此外，由於第一抗趙1111、酷類材料1112及過氧 化物晦1113並未固定於第一流體區中，所以此時第一抗體lm、與第一抗 體1111結合之待測物質1101、過氧化物臃1113，及醣類材料1112會隨著流 6 201017160 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1121是固定在第二流體區112 之中，所以會將已經與待測物質1101結合的第一抗體mi以及在第一抗體 1111之上的過氧化物晦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113流動。 請繼續參考第1E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之中的過氧化物臃1113 會流至第二流體區113。此時，醣類材料m2會與已經存在於第三流趙區 113之中的醣氧化梅1133進行氧化反應，產生出過氧化氫，而產生出的過氧 化氫以及發光劑1131會受到流至第三反應區113的過氧化物腌1113進行催 化，產生光學訊號。此時，可藉由偵測光學訊號的強度，推算出參與反應的 酵素濃度，亦即可藉由偵測光學訊號的強度推算出有多少過氧化物梅1113 流至第二流體區113 ’且由於在製作流體檢測試片1之時，要添加的過氧化 物腌1113含量係為一已知的固定值，所以留置於第二流體區112之中的過 氧化物晦1113其濃度亦可藉由兩者簡單相減後推算得知。最後，利用已知 Ο 的留置於第二流體區I12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1101的濃度，以達到定量檢測的目的。 在未反應則，第一抗體1111與過氧化物腾1113兩者的結合型態可以是 直接形成共軛結合(conjugation);或是第一抗體1111上共軛結合有生物素 (Biotin)，而過氧化物晦1Π3則與親和素（Avidin)共輛結合，利用生物素 與親和素會強力結合成一錯合物（AB complex)的特性，使得在進行反應時， 當有第一抗體1111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卵白素（Avidin)、鏈黴親 合素（Streptavidin) ’或是中性鏈親和素（NeutrAvidin)。 7 201017160 請繼續參考第IF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。為達成本發明的定量檢測之目的， 流體檢測試片1中醣類材料1112及過氧化物腌1113在流道11中的分佈方 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和過氧化梅1113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，第一抗體mi 與過氧化物酶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（AB complex)的特性，第一抗體1111 ® 與過氧化物臃1113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體1121、發光劑1131、受質反應試劑1132，及聽氧化 梅1133的分佈方式則與第1B圖中相同。採用此種分佈之設置狀態，在流艘 流過第二流體區112後，醣類材料1112 一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1C圖至第1E圖所示，在此不再重複贅述。 請再參考第1G圖，此種分佈設置了，第一抗體llu是位於第一流體區 111中’而過氧化酶1113與聽類材料1112則位於第二流體區112中。而在 ® 帛1㈣所代表的分佈方式’則是第一抗體1111與聽類材料1112位於第- 流體區m、過氧化梅1113則位於第二流體區112中。流道时其餘之反 應材料第二抗體im、發細lm、受f反應試劑1132，及聽氧化梅ιΐ33 的分佈方式則與第1B圖中相同，且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m圖所示，在此不再重複費述。 咕繼續參考第_，為第1A圖沿从連線的剖面示意圖。如圖所示， 在第-流體區ill之底部包含有纖維層麗，而第—抗體⑴第出圖） j形成於麟層1110之巾，如此餅在趙注人第_流體區⑴之後第 抗體（111卜第1B圖）會隨流體-起流至第二流趙區112。而在第二流 8 201017160 體區112與第三流魅113的底部各自具有第二槪纖維層⑽與第 化纖維層113G。第二抗體（mi，第1B圖）係固定於第二硝化纖維層⑽ 之中，而受質反應試劑（1132，第1B圖）係、形成於第三确化纖維層⑽ 中。 '上述的第二硝化纖維層1120與第三硝乂匕織維層113〇可以是以一層硝化 纖維膜(NC membrane)的形式鋪設在第二流舰112以及第三流體區 底部。 的 另-種較佳的製作形式，則可以將硝化纖維溶液以洗注（casting)的方 式’逄注在第二流體區112以及第三流體1 113的底部，再經過風乾或是冷 來乾燥的步驟，藉此各自形成具有中空峨構型的第二舰纖維層㈣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，此種以組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職，而基板⑺則可採 用生物相容材料。未達較佳喊注效果，流道u的表面織度較絲圍㈤ 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mo厚度。此外，流道u可進一步包括第四流體區（未圖示「 第四流體區之底部亦形成有確化纖維層，供多餘流體之貯存，且此石肖化纖維 Φ 層具有中空網狀構型。 /上述之概纖維溶祕將辄_粉末與包含有賴及_溶劑混合 後製成，㈣化_粉末無臟_鋪賴合驗麵積_為i: 9。 在此種以洗/主方式形成硝化纖維層的狀態下，可在第二確化纖維層1⑽ 與第三硝化纖維層113〇乾燥後，將第二抗體1121以驗的方式注入第二靖 化纖維層1120，再經過風乾或冷雜_雕，鄕二抗體ιΐ2ι以粉末狀 的方式留存於第二硝化纖維層 1120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 應試劑形成於其中的方式之外，還可將第二抗體mi以溶液的方式注入預 201017160 先製備好的硝化纖維溶液，混合均勻後，再潦注於第二流體區ιΐ2的底部， 再經風乾或冷絲燥過程’同時將硝化_溶液形成第二舰纖維層 及第二抗體1121以粉末狀的方式留存其中。 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形成於第二硝化纖維層112〇的方式大致綱，可採用先形 成第三雜纖維層後再注人受f反應試舰乾燥，或是與硝化纖維溶 液混合後-同注人第三流體區113底部錢成型的兩種方式，故在此不再 複贅述。 除上述的第-較佳實施例之外，本發明亦提供__種越檢測方法，如下 之第二較佳實蘭巾騎，其巾檢财法帽制之檢職#，其構造特徵 與第-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1Β圖至第1Η圖。 凊參考第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。 流體檢測方法2包含以下步驟： 步驟21 ’長1供一流體’流體包含有待測物質1101，請參考第1Β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^第—流體區1U、第二流體區⑴與第三流體區113，第-流體區in 係供机體之左入。第一流體區m巾具有第一抗體.。第二抗體區112中 具^第二抗體1121，且第二抗體1121是固定在第二抗體區112之中。又， 第抗體1111與第二抗體都是辨識流體中的同一種待測物質^⑼但 是f抗體1111與第二抗體1121是各自辨識待測物質11〇1上不同的抗原 '、疋位置所以在第—抗體和第二抗體的選用上，兩者均可為單 對抗=或f株抗體，只需要第一抗體1111和第二抗體1121是一組抗體配 中，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 則具有發糊11S1與受質反應賴⑴2,受質反應細1132包含有酿氧 201017160 化梅1133。其中，過氧化物腺m3可以是辣根過氧化物臃、抗壞血酸過氧 化物腌或是過氧化氫梅。在較佳的實施狀態中，發光劑1131是用3-氨基鄰 苯二甲醯肼，而醣類材料1112則可為葡萄糖，於此同時，醣氧化腾1133則 是選用葡萄糖氧化梅。此外，醣類材料1112及過氧化物晦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1B圖、第1F圖、 第1G圖及第1H圖）。 步驟23 :將流體加至流道u之第一流體區m。流體會沿著流道u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Π11、醣類材料1112及過氧化物酶1113隨著流 體流動。 步驟24:待測物質iioi會隨流體流至第二流艎區112，使待測物質u〇1 與第一抗體1111、第二抗鱧1121及部份之過氧化物酶1113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lm 及過氧化物腺1113流至第三流體區Π3。在第三流體區中醣類材料1112受 到醣氧化酶1133之催化進行氧化反應，並產生丨過氧化氫^而發細1131 會受到流至第三流體區113的過氧化物晦1113催化與過氧化氫進行反應， 並產生一光學訊號。 步驟25 :細由步驟24產生之光學域μ時，可藉由侧光學訊號 的強度’轉出參與反應的酵素濃度’亦即可藉由偵測光學訊號的強度推算 出有多少過氧化物梅11B流至第三流體區⑴’且由於在製作流體檢測試片 1之時’要添加的過氧化物晦1113含量係為一已知的固定值，所以留置於第 =流體區m之中的過氧化_ 1113其濃度亦可藉由兩者簡單相減後推算 2。最後，利用已知的留置於第二流體區112之中的過氧化物晦1113的 =度’可再推算出流體中所含有的待測物質醒的濃度，以達到定量 的目的。 此外，滅本發明之流體檢測方法，其中第一抗體lm與過氧化物晦 201017160 1113的結合方式與較佳_義、各雜區的構造組成舰纖維層之構 型、形成方式、使用之硝 1 纖維溶液之成份與較伽例、各項反應材料之植 成及形成方式，均與前述之第一較佳實施例相同，在此不再重複贊述。 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—較佳實施織體檢測試片之示意圖。 -第1B圖’為本發明第—触實補趙制試腫道巾反應材料分佈 示意圖。 第1C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麵測試片流道中反 應材料其他方式分佈示意圖^ ❹ 第11 ®，為本發明第·'較佳實施嫌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 1 基板 10 流道 11 待測物質 1101 第一流體區 111 纖維層 1110 12 201017160 第一抗體 1111 醣類材料 1112 過氧化物腌 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 發光劑 1131 受質反應試劑 1132 醣氧化酶 1133 步驟 21 ' 22 > 23'24'25The invention also provides a fluid detection and detection method, which mainly comprises the following steps. (1) Providing the face with the object to be tested" (2) Touch-pure, the base contains the turbulent flow channel, and the shovel channel includes the first-fluid zone, the second fluid zone and the third Zhao zone, the first fluid The district is for the injection of _. The substrate further comprises a first antibody in the first fluid region for identifying the substance to be tested in the fluid; the hearing material is located in the first fluid region or the second fluid region; In the first flow zone or the first fluid zone, the first antibody is immobilized in the second fluid zone, and the same test substance is also identified, and the second antibody and the first antibody are located at the position of the antigen mosquito; And the luminescent agent and the accepting reagent, located in the third briber, and the receptor reagent comprises (4) oxidized plum. (3) The fluid is applied to the first fluid zone of the flow channel to cause the first antibody, the listener, and the peroxide ship to flow. (4) combining the substance with the first antibody, the second antibody and a part of the peroxide plum and leaving it in the second body, so that the _磐, unbound ―antibody and Wei s The third flow body, so that _ _ _ oxidized filaments undergo oxidation reaction, and produce hydrogen peroxide 'hair trousers to the third stream of the Wei Lu miscellaneous domain Wei hydrogen to react 'and produce optical lass; (3) Finely produced optical defects. Therefore, another object of the present invention is to provide a method for detecting a fluid. Since all the reactions are required and required, the final reaction signal can be directly quantified by a complicated operation step. The re-purpose of the present invention provides a general detection green, and the reagents and materials of the reaction are stored in the test piece before the reaction, so that the long-term secret can be preserved. Lead to test errors. [Embodiment] Since the present invention discloses a fluid detecting test piece and a test method thereof, which utilizes the chemistry and biopsy, which has been known to those skilled in the art of the art, the following description: * Make a full description. _ 'The following diagrams of the position, the county and the characteristics of the hairpin, do not need to complete the system according to the actual situation, the first teaching 1 .201017160 'Please refer to 帛 1A 'for the invention A schematic view of a fluid detecting test piece of the first preferred embodiment. The fluid detecting test piece 1 includes a substrate 1 〇, and the substrate 1 〇 includes a flow path 1 on which a first fluid region, a second fluid region 112, and a third fluid region 113 are sequentially disposed. A fluid zone 111 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 fluid detecting test piece according to the first preferred embodiment of the present invention. The first fluid region m has a first antibody book, a listening material 1112, and a peroxide 1113. The second fluid region 112 has a second antibody U21 therein, and the second antibody 1121 is fixed in the second fluid region 112. Further, both the first antibody im and the second antibody 1121 are identifiable to the same cytoplasm in the fluid, but the first anti-sputum 1111 and the second antibody 1121 are different antigen-determining positions on the respective self-identifying test substance 1101. Therefore, in the selection of the first antibody 1111 and the second antibody 1121, both of them can be monoclonal antibodies (mAbs) or multiple antibodies (pAbs), and only the first antibody 1111 and the second antibody 1121 are a pair of antibody pairs. (antib〇dy pair), in the case of antigen recognition, there is no possibility of mutual interference. The third fluid region 113 has a luminescent agent 1131 and a substrate reaction reagent 1132, and the receptor reaction reagent 1132 contains glucose oxidation.臃1133 ° where 'peroxidase m3 can be horseradish peroxidase (HRp, H〇rseradish Peroxidase), ascorbate peroxidase (AP), or hydrogen peroxidase. In a preferred embodiment, the illuminant 1131 is 3-amino-2-pyrazine (5-Amino-2,3-dihydro-l, 4-phthalazinec!ione), and the saccharide material 1112 is At the same time, glucose oxidized cesium 1133 is selected from glucose cerium oxide. Please continue to refer to Figures 1C to 1E for the distribution of the reaction materials in the flow path of the fluid detecting test strip in the first preferred embodiment of the present invention. Schematic. Please refer to the 1C first. After the fluid containing the substance to be tested 1101 is injected into the flow channel 11, the first antibody 1111 recognizes and combines with the substance to be tested 1101. In addition, since the first anti-Zhao 1111, the cool material 1112 and the peroxide 晦1113 is not fixed in the first fluid region, so at this time, the first antibody lm, the test substance 1101 combined with the first antibody 1111, the peroxide 臃1113, and the saccharide material 1112 will follow the flow 6 201017160 Continue to flow to the second fluid zone 112. Please continue to refer to Figure 1D, after the fluid flows to the second reaction zone 112, the second antibody 1121 will bind to the test substance 1101. At this time, since the second antibody 1121 is fixed at In the second fluid region 112, the first antibody mi that has been bound to the substance to be tested 1101 and the peroxide 晦 1113 above the first antibody 1111 are left in the second fluid region 112; The saccharide material m2 which also flows to the second fluid region 112 at this time and the first antibody 1111 and the peroxide plum 1113 which are not combined with the substance to be tested 11〇1 continue to the third fluid region 113 as the fluid continues. Flow. Please continue to refer to Figure 1E, sugar materials M2 and the peroxide 臃1113 which is not retained in the second fluid region 112 in combination with the first antibody lm, the second antibody ©1112, and the test substance 1101, will flow to the second fluid region 113. At this time, the saccharide material M2 will be oxidized with the sugar oxidized plum 1133 already present in the third stream Zhao 113 to produce hydrogen peroxide, and the generated hydrogen peroxide and the illuminant 1131 will be flowed to the third reaction zone 113. The peroxide salted 1113 is catalyzed to produce an optical signal. At this time, by measuring the intensity of the optical signal, the concentration of the enzyme involved in the reaction can be estimated, and by detecting the intensity of the optical signal, it is estimated how much peroxide plum 1113 flows to the second fluid region 113' and Since the peroxide 1113 content to be added is a known fixed value at the time of preparation of the fluid test strip 1, the concentration of the peroxide 晦 1113 remaining in the second fluid region 112 can also be borrowed. It is estimated by simple subtraction between the two. Finally, the concentration of the test substance 1101 contained in the fluid can be further calculated by using the concentration of the peroxide salt 1113 remaining in the second fluid region I12, which is known to be Ο, for the purpose of quantitative detection. In the case of unreacted, the binding form of the first antibody 1111 and the peroxide 1113 may be direct formation of conjugation; or the first antibody 1111 is conjugated to biotin (Biotin). The peroxide 晦1Π3 is combined with avidin (Avidin), and biotin and avidin are strongly combined to form a complex of AB complex, so that when the reaction is carried out, when the first antibody 1111 is left In the second fluid zone 112, a portion of the peroxide crucible 1113 is also retained in the second fluid zone 112. The above avidin may be Avidin, Streptavidin or NeutrAvidin. 7 201017160 Please continue to refer to the IF diagram to the 1H diagram, which is a schematic diagram showing other modes of distribution of the reaction materials in the flow path of the fluid detecting test piece according to the first preferred embodiment of the present invention. For the purpose of quantitative detection of the present invention, the distribution manner of the saccharide material 1112 and the peroxide salt 1113 in the fluid detecting test piece 1 in the flow path 11 is present in the first flow path 111 except as shown in FIG. 1B. In addition to the distribution mode, it may also be distributed in the second flow path 112. First, please refer to FIG. 1F. The first antibody 1111 and the peroxidized plum 1113 are located in the first fluid zone 111, and the saccharide material 1112 is located in the second fluid zone 112. At this time, the first antibody mi and the peroxide The binding mode of the enzyme 1113 is directly combined with the above-mentioned, or the combination of biotin and avidin into a complex of AB complex, the first antibody 1111 ® and the peroxide 臃 1113 are respectively It exists in a way that combines biotin and avidin. The distribution pattern of the remaining reaction material second antibody 1121, illuminant 1131, substrate reaction reagent 1132, and listener oxide 1133 in the flow channel 11 is the same as in Fig. 1B. With this distributed state, after the flow through the second fluid zone 112, the sugar material 1112 will flow with the fluid to the third fluid zone 113, so the flow distribution of the reactive material in each subsequent reaction stage. The situation and the reaction performed are shown in FIGS. 1C to 1E, and the description thereof will not be repeated here. Referring again to Figure 1G, such a distribution is such that the first antibody 11u is located in the first fluid zone 111 and the peroxidase 1113 and the hearing material 1112 are located in the second fluid zone 112. The distribution pattern represented by ® 帛1 (4) is that the first antibody 1111 and the hearing material 1112 are located in the first fluid zone m and the peroxidized plum 1113 is located in the second fluid zone 112. The remaining reaction materials of the second antibody im, the fine lm, the f-reactive reagent 1132, and the oxidized plum ιΐ33 are distributed in the same manner as in the first embodiment, and the flow of the reaction material in each subsequent reaction stage. The distribution situation and the reaction performed are shown in the figures lc to m, and will not be repeated here.咕 Continue to refer to the _, which is a schematic diagram of the section along the line of the 1A diagram. As shown in the figure, the fiber layer is contained at the bottom of the first fluid region ill, and the first antibody (1) is formed in the towel of the lining layer 1110, so that the cake is after the _ fluid zone (1) of the Zhao injection The antibody (111b, Figure 1B) will flow with the fluid to the second flow zone 112. In the second stream 8 201017160, the body region 112 and the bottom portion of the third flow charm 113 each have a second fiber layer (10) and a second fiber layer 113G. The second antibody (mi, Fig. 1B) is immobilized in the second nitrocellulose layer (10), and the receptor reagent (1132, Fig. 1B) is formed in the third confirmed fiber layer (10). The second nitrocellulose layer 1120 and the third nitrocellulose layer 113 上述 may be laid in the form of a layer of nitrocellulose membrane (NC membrane) at the bottom of the second flow ship 112 and the third fluid zone. In another preferred form, the nitrocellulose solution can be injected in the second fluid zone 112 and the bottom of the third fluid 113, and then dried by air drying or cold. The steps of forming a second shipboard layer (4) and a third nitrocellulose layer 1130 having a hollow 峨 configuration. Further, in order to reduce the influence of capillary action between the flow path and the fluid, such a flow path formed in a group manner is not a so-called micro flow path of the prior art, and the second fluid region 112 and the third fluid region The minimum width of 113 is preferably 〇3, while the substrate (7) is made of biocompatible material. Without a better screaming effect, the surface texture of the flow path u is between 3 micrometers and 50 micrometers by the wire circumference (five value) and the average thickness of the second nitrocellulose fiber layer 112 is equal to the thickness of the third nitrocellulose fiber layer mo. In addition, the flow path u may further include a fourth fluid region (not shown) "the bottom of the fourth fluid region is also formed with a layer of a definitive fiber for storage of excess fluid, and the stone Φ layer has a hollow mesh configuration. / The above-mentioned fiber dissolves 辄 _ powder is mixed with the Dependent and _ solvent, (4) _ powder without dirt _ shop area _ is i: 9. In this way of washing / main mode In the state in which the nitrocellulose layer is formed, the second antibody 1121 can be injected into the second layer of the jinglon fiber 1120 after being dried by the second varnish layer 1 (10) and the third nitrocellulose layer 113, and then air-dried or cold. The second antibody 1102 and the substrate-reactive reagent are formed in the powdery manner in the second nitrocellulose layer 1120. In addition, the second antibody mi can be injected into the pre-201017160 prepared nitrocellulose solution in a solution manner, and then uniformly mixed, then injected into the bottom of the second fluid zone ιΐ2, and then air-dried or cold-dried. Process 'simultaneous nitrification _ dissolution The second ship fiber layer and the second antibody 1121 are formed to remain in a powder form. Further, the manner in which the substrate reaction reagent 1132 is formed in the third nitrocellulose layer 113 is also formed in the second nitrification with the second antibody 1121. The manner of the fiber layer 112〇 is roughly as follows, and the third fiber layer can be formed first and then injected by the f-reaction test ship, or mixed with the nitrocellulose solution, and then injected into the bottom of the third fluid zone 113. There are two ways, so I won't repeat them here. In addition to the above-described first-preferred embodiment, the present invention also provides a method for detecting the second method, the following second preferred real towel ride, and the towel is checked. The structure of the cap system is substantially the same as that of the first preferred embodiment, so the description of the test piece component mentioned in the above is not repeated, and reference is made to the first to the first. Referring to Fig. 2, there is shown a flow chart of a fluid detecting method according to a second preferred embodiment of the present invention. The fluid detecting method 2 comprises the following steps: Step 21 'Long 1 for a fluid' fluid containing the substance to be tested 1101, please refer to paragraph 1 Fig. Step 22. Provide base The 10' substrate 10 includes at least a flow path 11. The flow path 11 includes a sequential-connecting first-fluid zone 1U, a second fluid zone (1) and a third fluid zone 113, and the first fluid zone is for the left-hand side of the body The first fluid region m towel has a first antibody. The second antibody region 112 has a second antibody 1121, and the second antibody 1121 is immobilized in the second antibody region 112. Further, the first antibody 1111 and the second antibody The antibodies are all the same test substance in the identification fluid ^ (9) but the f antibody 1111 and the second antibody 1121 are each distinct from the test substance 11 〇 1 different antigen ', 疋 position so in the first antibody and the second antibody Alternatively, both of them can be single-antibody=or f-antibody antibodies, and only the first antibody 1111 and the second antibody 1121 are required to be a group of antibodies, and it is not necessary to interfere with each other when performing antigen recognition. The third fluid zone 113 has a hair spray 11S1 and a substrate reaction reaction (1) 2, and the host reaction reaction 1132 contains a brewing oxygen 201017160. Among them, the peroxide gland m3 may be horseradish peroxide strontium, ascorbic acid peroxide salt or hydrogen peroxide plum. In a preferred embodiment, the luminescent agent 1131 is 3-aminophthalic acid, and the saccharide material 1112 is glucose. At the same time, the sugar oxidizing 1133 is glucose oxidized plum. In addition, various distributions of the saccharide material 1112 and the peroxide 晦1113 in the flow channel 11 are as described in the first preferred embodiment (please refer to FIG. 1B, FIG. 1F, FIG. 1G, and FIG. 1H). ). Step 23: Add fluid to the first fluid zone m of the flow path u. The fluid flows along the flow path u sequentially from the first fluid region 111 through the second fluid region 112 to the third fluid region 113, and the first antibody Π11, the saccharide material 1112 present in the flow channel 11 And peroxidase 1113 follows the fluid flow. Step 24: The test substance iioi will flow with the fluid to the second flow area 112, and the test substance u〇1 is combined with the first antibody 1111, the second anti-slurry 1121 and a part of the peroxidase 1113 and left together. In the second fluid zone 112. The fluid continues to flow with the saccharide material 1112, the unbound first antibody lm, and the peroxide gland 1113 to the third fluid zone Π3. In the third fluid zone, the saccharide material 1112 is oxidized by the oxidation of the sugar oxidase 1133, and the hydrazine hydrogen peroxide is generated, and the fine electrode 1131 is catalyzed by the peroxide 晦 1113 flowing to the third fluid region 113. Hydrogen peroxide reacts and produces an optical signal. Step 25: When the optical domain μ generated by the step 24 is fine, the intensity of the side optical signal can be 'transferred out of the enzyme concentration participating in the reaction', and the amount of the peroxide plum can be estimated by detecting the intensity of the optical signal. 11B flows to the third fluid zone (1)' and since the content of the peroxide 晦 1113 to be added at the time of the preparation of the fluid test strip 1 is a known fixed value, it is left in the first fluid region m. The concentration of peroxidation _ 1113 can also be estimated by simply subtracting the two. Finally, the concentration of the test substance contained in the fluid can be recalculated using the known degree = of the peroxide 晦 1113 remaining in the second fluid zone 112 for quantitative purposes. In addition, the method for detecting a fluid according to the present invention, wherein the first antibody lm and the peroxide 晦201017160 1113 are combined with each other, and the configuration of each of the miscellaneous regions constitutes a configuration of the ship fiber layer, a formation mode, and a used nitrate. 1 The composition of the fiber solution and the galvanic case, the formation and formation of each reaction material are the same as the first preferred embodiment described above, and the description is not repeated here. The above description is only for the preferred embodiment of the present invention, and is not intended to limit the present invention. The above description is to be understood 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 of a first embodiment of the present invention. - Figure 1B is a schematic view showing the distribution of the reaction material of the scalp towel of the first method of the invention. 1C to 1E are schematic views showing the distribution of the reaction materials in the flow paths of the fluid detecting test piece at different reaction stages in the first preferred embodiment of the present invention. 1F to 1H are schematic views showing other modes of distribution of reactive materials in the flow path of the flow test piece according to the first preferred embodiment of the present invention. ❹ 11th, which is a schematic view of the cross section of the preferred embodiment of the present invention. . Fig. 2 is a schematic flow chart showing a fluid detecting method according to a second preferred embodiment of the present invention. [Main component symbol description] Fluid test strip 1 Substrate 10 Flow path 11 Test substance 1101 First fluid area 111 Fiber layer 1110 12 201017160 First antibody 1111 Sugar material 1112 Peroxide salt 1113 Second fluid area 112 Second Nitrocellulose layer 1120 Second antibody 1121 Third fluid zone 113 Third nitrocellulose layer 1130 Luminescent agent 1131 Receptor reagent 1132 Sugar oxidase 1133 Step 21 ' 22 > 23'24'25
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