1295730 九、發明說明： 【發明所屬之技術領域】 =明係提供-種可用於檢體中待測物含量分析之微流體 的=及、方法，主要係利用晶片上細長微流道作為待測物含量八 析使檢體内之待測物與前述微流道内複數侧定物質進行二 :’並糟由反應調節部調節檢體中待測物與固定物質之 待測物含量分析區之反應起始點起依序累積反應，達到 二二圍集中之效果。晶片使用者可藉由前述微流道内產生反應 ::圍大小，參照微流道的精徵或外部刻度而得知待測物： 【先前技術】 許多臨床生化檢測應用均著眼於特定生化物質或病原體的 摘，，藉由偵測的結果可以反映出患病程度、患者的健康狀況或 醫藥處理的療效4外，生物或化學物質的侧也可用於藥物檢 測、工業製程監測、環境監測、植物或動物的檢測等應用。生化 檢測的檢體選擇視需求而定，可以來自人或動物的血液、尿液、 唾液或血清等體液、製程或環境中的樣本等。而待測物（analytes) 則疋包含於鈿述檢體内的化學物質、蛋白質、胜肤、配體、核酸 或病毒、細菌之類的病原體。 待測物含量分析可分為兩種，一種為定性測試（qualitative test)，另一種為半定量/定量分析（quantitative assay)。定性測試的 原理在於檢測目標待測物是否存在或達到某一門檻值，然後給予 一陽性（positive)或陰性（negative)的結果。一個簡單的例子 就是市面上可購得之驗孕試片，測試受試者尿液檢體中的hCG (human chorionic gonadotropin，人類絨毛膜促性腺激素）含量是否 超過一門檻值，例如·· 25 mIU/m卜如果超過此含量，則驗孕試 1295730 片的結果就為陽性結果。此種利用傳統試片變色的方式，讓受試 者可以自行採樣進行居家檢測，此法雖然方便迅速，然而畢竟只 能用於定性檢測。本發明之重點在於開發一個能做定性、半定量 或定量分析的方法及工具，而不限於定性分析的層次。 以反應原理來區分生化待測物含量分析，可運用的原理包括： 光譜法（spectrometry)、電分析化學法（Electroanalytical chemistry)、層析分離法（chromatographic separation)、電泳法 (electromigration methods)以及免疫分析法（immunoassay)等。有些 生化分析的方法需將待測物加以標示才能定量，若以標示方法作 區分又可分為以螢光（fluorescence)、冷光、酵素（eilZyme)、放射 性(radioactive)元素、奈米微粒（nanoparticles)、磁性粒子（magnetic bead)等方式做定量者。無論是使用上述何種原理所開發的生化檢 測工具，大部份檢測方法都有一個共同的需求，就是必需具備有 一個量測待測物訊號的儀器，例如使用光度計（ph〇t〇meters)量測 特定波長的吸收光，或使用光電倍增管量測放出的螢光强度，或 使用電極及電壓或電流量測儀器量測反應後所得的微小的電訊 號，或以掃描器或CCD攝影器量測顏色的改變量等。 可攜式即時生化檢測器材可以在小診所、醫院之醫護點、居 家或缺乏醫療資源的偏遠地區進行檢測，並能卽時得到檢測結 果。可攜式即時生化檢測器材具備以下的特色：（a)不需要用到 昂貴、笨重的機臺，（b)簡易而自動化的操作方式，不需經過專 業訓練也能得到準確而可靠的結果，（c)使用低成本零件，卽用 卽丟，以避免汚染。 目前生化檢測所使用的器材有大型醫院或檢驗中心所使用的 大型機臺、個人使用的掌上型機臺或簡單的檢測試片等。掌上型 的儀器雖然價位較低且易於攜帶，以血糖、尿酸或總膽固醇等幾 1295730 種待測物的量測較常見。若要擴大應用至包含LDL (低密度膽固 醇Low density lipoprotein)等各種不同種類的待測物，則由於檢體 中其他成份的存在對訊号的干擾，使得不論是光學式或電化學式 的檢測原理都仍面臨瓶頸需要克服。因此，目前市面上使用於血 糖、尿酸或總膽固醇以外的半定量/定量分析，通常使用大型檢測 儀器。這類大型機臺多受限於醫院或檢驗中心進行，不適於可攜 式即時生化檢測。簡單的檢測試片雖然價格低，但檢測待測物含 量的準確性較差，適用於定性而非半定量/定量檢測，能檢測待測 物的種類就受限制。 近年來，以晶片做為分析工具的研究漸多，例如繞射感測 (diffraction-based sensing)、石英晶體微平衡（Quartz Crystal Microbalance)、表面電漿共振（surface piasrn〇n resonance )等， 這些方法共同的特色是除了晶片上必需設計製作與分析原理相 關的機制外，還必需搭配可用於定出待測物含量的貴重儀器，使 用者也必需具有專業操作技巧，有些方法甚至必需加上複雜的檢 體前處理流程，因此不適於可攜式即時生化檢測。 微流體晶片由於具有可以處理微量流體的功能，其發展漸受 重視’例如利用毛細電泳晶片以檢測檢體中的成份。但毛細電泳 晶片必需在晶片以外另以機臺輔助其訊号量測，才能達到定量之 功能。其他不以毛細電泳為原理的微流體晶片，雖然也有可能用 於生化分析’但也無法直接由晶片完成待測物定量的工作。例如 美國專利公開第2002/0127740號，其方法為導入特定體積的檢體 於反應區’至於待測物的含量則無晶片上的量測機制，必須外加 光學檢測儀器讀取結果。 生化檢測試片雖然價格低，但較適用於定性而非定量檢測。 1295730 例如前述檢測hCG的驗孕試片原理為橫向流動（lateral flow) ’以 具有吸拉液體(wicking)作用的試紙為主體，讓生化反應在試紙上 進行。由於結構簡單，能控制反應的設計參數很少’較適合做定 性量測，若用於半定量/定量分析則準確度不夠。美國專利第 5,559,041號免疫檢測試片，或是其他利用奈米金微粒辅助顯色效 果的生化檢測試片等，即是生化檢測試片應用於定性分析的例 子01295730 IX. Description of the invention: [Technical field to which the invention belongs] = The system provides a microfluid that can be used for the analysis of the content of the analyte in the sample, and the method mainly uses the elongated microchannel on the wafer as the test The content of the material is such that the analyte in the sample and the plurality of side substances in the microchannel are subjected to two: 'and the reaction regulation unit adjusts the reaction between the analyte and the analyte in the analysis region of the analyte. The starting point accumulates the reaction in order to achieve the effect of the concentration of the second and the second. The wafer user can know the object to be tested by the reaction in the micro flow channel: the size of the microchannel, or the external scale of the microchannel: [Prior Art] Many clinical biochemical detection applications focus on specific biochemical substances or The pathogen extract can be used to detect the degree of disease, the health of the patient or the efficacy of medical treatment. The side of the biological or chemical substance can also be used for drug testing, industrial process monitoring, environmental monitoring, plants. Or applications such as animal testing. The biochemical test sample selection depends on the needs, and can be derived from human or animal blood, urine, saliva or serum body fluids, processes or samples in the environment. The analytes are the pathogens contained in the chemical substances, proteins, skins, ligands, nucleic acids or viruses, bacteria, etc. contained in the test. The analyte content analysis can be divided into two types, one is a qualitative test and the other is a semi-quantitative/quantitative assay. The principle of qualitative testing is to detect whether a target analyte is present or reaches a certain threshold and then give a positive or negative result. A simple example is a commercially available test strip that tests whether the content of hCG (human chorionic gonadotropin, human chorionic gonadotropin) in a urine sample exceeds a threshold, for example, 25 If the mIU/mbu exceeds this content, the result of the test of 1295730 tablets is a positive result. This method of using the color change of the traditional test piece allows the subject to self-sample for home detection. Although this method is convenient and rapid, it can only be used for qualitative detection. The focus of the present invention is to develop a method and tool that can be used for qualitative, semi-quantitative or quantitative analysis, and is not limited to the level of qualitative analysis. The principle of reaction is used to distinguish the content of biochemical analytes. The principles that can be applied include: spectrometry, electroanalytical chemistry, chromatographic separation, electromigration methods, and immunization. Analytical method (immunoassay) and the like. Some biochemical analysis methods need to label the analyte to be quantified. If it is differentiated by labeling method, it can be divided into fluorescence, luminescence, eilZyme, radioactive elements, and nanoparticles. ), magnetic beads (magnetic bead) and other methods to do quantitative. Regardless of the biochemical detection tools developed using the above principles, most of the detection methods have a common need, that is, it is necessary to have an instrument for measuring the signal to be tested, for example, using a photometer (ph〇t〇meters) Measure the absorbed light at a specific wavelength, or measure the fluorescence intensity emitted by using a photomultiplier tube, or measure the tiny electrical signal obtained after the reaction using an electrode and a voltage or current measuring instrument, or take a scanner or CCD The device measures the amount of change in color, and the like. Portable real-time biochemical testing equipment can be tested in small clinics, hospital care points, homes or remote areas where medical resources are lacking, and can be tested at times. Portable instant biochemical testing equipment has the following features: (a) no need for expensive, cumbersome machines, and (b) simple and automated operation, accurate and reliable results without professional training. (c) Use low-cost parts and use them to avoid contamination. At present, the equipment used for biochemical testing is a large machine used by a large hospital or inspection center, a palm-sized machine for personal use, or a simple test strip. Although the handheld instrument is low in price and easy to carry, it is more common to measure 12,957,30 kinds of analytes such as blood sugar, uric acid or total cholesterol. In order to expand the application to various types of analytes including LDL (Low density lipoprotein), the interference of signals due to the presence of other components in the sample makes the optical or electrochemical detection principle Still facing bottlenecks need to be overcome. Therefore, semi-quantitative/quantitative analysis other than blood sugar, uric acid or total cholesterol is currently available on the market, and large-scale detection instruments are usually used. Such large-scale machines are mostly restricted to hospitals or inspection centers and are not suitable for portable real-time biochemical testing. Although the simple test piece has a low price, the accuracy of detecting the content of the test object is poor, and it is suitable for qualitative rather than semi-quantitative/quantitative detection, and the type of the test object can be detected. In recent years, there have been more researches on wafers as analytical tools, such as diffraction-based sensing, Quartz Crystal Microbalance, surface piasrn〇n resonance, etc. The common feature of the method is that in addition to the mechanism that must be designed and fabricated on the wafer, it must be matched with a valuable instrument that can be used to determine the content of the analyte. The user must also have professional operation skills, and some methods must even add complexity. The pre-sample processing procedure is therefore not suitable for portable real-time biochemical testing. Microfluidic wafers have received increasing emphasis due to their ability to handle trace amounts of fluids, e.g., using capillary electrophoretic wafers to detect components in the sample. However, the capillary electrophoresis wafer must be machine-assisted in addition to the wafer to achieve its quantitative function. Other microfluidic wafers that are not based on capillary electrophoresis, although they may be used for biochemical analysis, are not able to perform the quantification of the analyte directly from the wafer. For example, U.S. Patent Publication No. 2002/0127740, the method of which is to introduce a specific volume of the sample in the reaction zone' to the content of the analyte to be measured without a wafer, must be read by an optical detector. Although biochemical test strips are low in price, they are more suitable for qualitative rather than quantitative testing. 1295730 For example, the principle of the test pregnancy test for detecting hCG is that the lateral flow is mainly performed on a test paper having a wicking action, and the biochemical reaction is carried out on the test paper. Due to its simple structure, the design parameters that can control the reaction are few, which is more suitable for qualitative measurement. If it is used for semi-quantitative/quantitative analysis, the accuracy is not enough. U.S. Patent No. 5,559,041, an immunoassay test piece, or other biochemical test strips that utilize nano-nanoparticles to assist in color development, is an example of biochemical test strips for qualitative analysis.
Se-Hwan Paek的研究，以及美國專利第5,340,539號，都是 試圖把橫向流動（lateral flow)生化檢測試片推廣到半定量/定量分 析的例子。其原理以具有吸拉液體(wicking)作用的試紙為基材， 藉著讀取試紙上反應區域的大小（例如變色長度的長短）來判新 待測物的量。由於吸取檢體體積可能有誤差、樣品在試片内流速 不均、反應區域太短、反應範圍分散造成訊號強弱不一等因素， 使付待測物含置分析結果誤差較大。以Se_Hwan Paek在試紙上所 做的待測物含量分析實驗為例，第一圖0)顯示液態檢體由生化檢 測試片的下方吸入至反應區反應後，再往上流出反應區的測試結 果。由左到右排列者為相同試片用於不同待測物濃度之結果。第 一圖（b)顯示檢體反應後試片上反應區域影像放大圖。此習知技術 雖可用於檢體中待测物的定性測試，但不適於直接做半定 二 分析，理由如下·· 里疋里 1.由於試紙式的生化檢測試片有材料及製程上的限制，使得 反應區域寬度較寬，無法做到非常細長。當待測物的詈俞 /在寬的試片上變色長度愈短，以目視法依據變色 的長短來推估待測物在檢體中的量時，讀值誤差愈大。 2·由於試紙式的生化檢測試片的反應區域寬度較I檢體往 上吸入時的路徑及速度無法控制一致，使得被載取的待測 1295730 物有時左邊量較多，有時偏中或偏右，造成變色區域的前 緣常會呈現有凹有凸的不規則形狀，如第一圖（b)所示。此 外，當液態檢體由下方吸入至反應區反應再往上流出的過 程中，由於沒有任何控制機制，使得有些待測物跑得快有 些跑得慢，造成了有反應作用者可能分散在反應區内各處 的現象。這可從第一圖（b)之局部放大圖中反應範圍的前緣 顯色有各種層次的灰階看出。在此狀況之下，一般使用者 若以肉眼直接判斷變色範圍的長短，到底應該讀到前緣的 凹處或凸處，顏色要多深或多淺才算是有反應的範圍，這 些都將造成讀值上極大的困擾及誤差。 3. 試紙式的生化檢測試片必需使用能吸入液體檢體之材質 製作成試片，材質及架構受限，因此難以直接在此材質上 附加’控制流入檢體體積’的功能。以濃度量測為例，若想 依據變色範圍的長短來推估檢體中待測物的濃度，前提之 一是每次進入檢測試片中的檢體必需控制為事先設定的 體積，否則，進入反應區的檢體多寡不一，就算得知變色 範圍的長短也無法準確換算出待測物的濃度。試紙式的生 化檢測試片難以在現有材質及架構上整合這項功能，將有 礙於分析時的準確度提升。 4. 試紙式的生化檢測試片難以直接在此材質上附加各種有 助於待測物含量分析的功能，例如：檢體成份的分離、檢 體流經反應區的速度控制、試劑（例如抗凝劑、反應呈色 用之標記等）的添加及均勻混合、單一檢體進行多項測 試、或反應後的清洗等步驟，都有助於增加分析的方便性 與準確性，但由於材質及架構受限，這些功能難以整合在 這種單純的架構上。 1295730 因此，如何開發一種可有效處理檢體、簡化使用者操作流 程、準確量得待測物含量、並方便讀取結果、甚至不必外加複雜 的量測儀器之檢測分析晶片，為目前本領域亟待突破之處。 【發明内容】 有鑑於習知檢測分析技術之缺失，本發明之目的在於提供 一可用於待測物含量分析之微流體晶片，其係包括：一檢體通 入口，用以通入檢體；一待測物含量分析區，具一微流道，其 中該微流道表面佈放複數個固定物質，且該微流道一端接檢體 通入口；以及一反應調節部，用以調節檢體内待測物與複數個 固定物質之接觸機會，使待測物與固定物質由反應起始點開始 依序累積反應，達到反應範圍集中之效果。藉由前述微流道内 產生集中反應的範圍大小，可得知該檢體中之待測物含量。 上列敘述中： • 「固定物質」指的是固定在待測物含量分析區微流道表 面，不隨檢體流走，且能與檢體内的待測物反應的物 質，例如：核酸、配體、受體、抗原、抗體、酵素、胜 肽、蛋白質或其他可與待測物反應之生物或化學物質。 • 「反應起始點」係指微流道接檢體通入口之一端佈放固 定物質之起點。 • 「依序累積反應」指的是每一個待測物粒子進入反應起 始點後，在行進的過程中有足夠的時間及碰撞機會與在 微流道内最先遇到的，且尚未作用的固定物質反應。也 就是說，待測物質不會因為流速過快或沒有碰撞機會而 錯過尚未作用的固定物質，卻已隨檢體行進到微流道内 的下一個位置。所有進入微流道内的待測物質由反應起 始點開始依序與固定物質相遇產生反應，因此反應的範 1295730 圍將「集中」在從反應起始點開始的待測物含量分析 區。一旦檢體内的待測物被前段的固定物質反應完，則 後段的固定物質雖遇到剩餘檢體流過，但檢體内已無待 測物可與之反應。由於有待測物/固定物質產生反應的 範圍集中在微流道前段，而待測物耗盡後未發生反應的 固定物質位於後段，因此檢體内待測物的量將正比於前 段有反應範圍的面積大小。若流道的寬度設計具有規則 性，則待測物的量將正比於流道内反應區域的長度。 φ 「反應調節部」指的是能使檢體内的待測物在微流道内 行進的過程中有足夠的時間及碰撞機會與固定物質反 應的機制。實現反應調節部的方法包括控制檢體流動的 速度及/或在流道内增設各種有助於擾動檢體、增加反 應面積及接觸機率的設計。 本發明之另一目的係關於一種待測物含量分析方法，係包 含：提供檢體；將前述檢體由檢體通入口導入待測物含量分析區 之微流道’其中該微流道表面修飾複數個固定物質；調節該檢體 中待測物與該固定物質之反應，使自反應起始點依序累積反應， 達到反應範圍集中之效果；及藉由前述微流道内產生反應的範圍 大小，可得知該檢體中之待測物含量。 前述待測物含量分析方法係可利用本發明之分析晶片達成。 【貫施方式】 本發明之待測物含量分析微流體晶片之一實施態樣係如第 二圖所示’該待測物含量分析微流體晶片1主要係包括：一檢體 通入口2 ’用以通入檢體；一待測物含量分析區3，具一微流道4， 其中該微流道4表面佈放複數個固定物質（以微流道上的網狀花紋 表示），且該微流道4一端接檢體通入口；以及一反應調節部，用 以調節檢體内待測物與微流道4内複數個固定物質之接觸機會， 11 1295730 使待測物與固定物質由反應起始點41開始依序累積反應，達到反 應範圍集中之效果。藉由前述微流道4累積於前段的待測物/固定 物質反應範圍（微流道4之黑色實線區域），並參照微流道4的幾何 形狀特徵或外部標記刻度6，可得知該檢體中之待測物含量。於 本實施態樣中，前述反應調節部為一流速控制裝置51，例如：可 控制流速的幫浦，係連結微流道4與檢體通入口相反之另一端。 於本發明之另一實施態樣中，前述反應調節部也可以是待測 物含量分析區的微流道材質，藉由微流道材質之親/疏水性來控制 檢體前進的速度，達成累積依序反應的需求。在一基板上製作各 種剖面形狀的凹槽，再與另一平面或具有凹槽的基板接合，兩面 基板間的凹槽即形成微流道，可供微量流體在流道内進行各項任 務。微流體在流道内流動的方式受凹槽表面狀況（即兩片基板的材 貝）所衫響。基板材質則可以選自親水性或疏水性材質，上/下美 板的材質組合可以是：親水性/親水性、親水性/疏水性、疏水 親水性或疏水性/疏水性。前述基板材質較佳係包含：聚二甲基矽 氧烧（polydimethylsiloxane，PDMS)、聚碳酸酯（ρ〇^_〇η^， PC)、環烯烴聚合物（cyclic olefin copolymers，c〇C)、聚苯乙稀 (polystyrene， PS)、玻璃或聚甲基丙烯酸曱西旨 (polymethylmethacrylate，PMMA) 〇 第三圖係顯示本發明之又-實施態樣，其μ述本發明待測 物含量分析微流體晶片1之反應調節部也可以是在前述微流道4 表面局部或全面修飾的物質52 (斜線部分），例如，但不限於： 特定官能基、親水性物質及/或疏水性物質，以進一步局部調整檢 體在流道内不同區段的前進速度，達成累積依序反應的需求。 第四係顯示本發明之再—實施態樣’前述本發明待測物 含量分析難體晶片1之反應㈣部也可以是待測物含量分析區 之微流道4截面形狀改變及寬窄不-的内徑53，以控制檢體的前 12 i29573〇 ^速度或流動模式。第四B圖係顯示第四A圖之微流道4狹窄部分 的剖面放大圖。 於本發明之另一實施態樣中，其中前述待測物含量分析微流 口曰曰1之反應調節部係包含在微流道之内表面設置凸出物，以增 口固疋物質之佈放面積，並有助於微流體以擾流方式前進，有效 促使待測物與固定物質接觸機率提高。 第五A圖係顯示於本發明之一實施態樣中，前述待測物含量 刀析# 1的微流道4的反應範 圍係可參照微流道4的幾何 特徵而得知檢體中之待測物含量，前述微流道4之幾何特徵係為 數個連、績的幫道構造，藉由觀察反應範圍到達第幾個彎道構造可 以對應得知該檢體中之待測物含量 ，例如：低量、中量或高量等。 第五B圖係顯示於前述待測物含量分析微流體晶片1微流道4的幾 何特徵為複數個更密集的連續彎道構造，可以更精確地定量。唯 本發明之微流道4的幾何特徵並不限於本實施態樣之彎道形構 造，其形狀和長度係可視實際檢測需要而調整，例如··矩形彎道、 螺旋形彎道等並不受限制，也可以利用其他種幾何特徵達到方便 辨識及得知待測物含量之功效。 第六A圖與第六B圖顯示本發明的待測物含量分析微流體晶 片1，其微流道4之管道係可對應設置標記刻度6，該標記刻度6可 以設置於微流道旁，亦可設置於微流道4上（圖未顯示），藉由觀察 反應範圍所對應的刻度而得知檢體中之待測物含量。 剷述微流道4之邊何特徵與標§己刻度6係可一併役置或單獨 設置以幫助得知檢體中之待測物含量。本發明之另一實施態樣係 為，經由前述微流道4之幾何特徵與標記刻度6讀取之數值，可進 一歩藉由一校正曲線或實驗對照表換算出實際檢體中待測物之 含量。 13 1295730 於本發明之又一實施態樣中，待測物含量分析微流體晶片係 可藉由串聯或並聯的方式連結多組檢體通入口、多組待測物含量 分析區或多組反應調節部，其可連結的檢體通入口、待測物含量 分析區或反應調節部的數目係不受限制，每一組待測物含量分析 區可佈放相同或不同固定物質，可做為比對用或量測不同待測 物。第七A圖係顯示本發明之待測物含量分析微流體晶片1串聯結 合一檢體通入口 2、複數個待測物含量分析區3與一反應調節部， 其中該反應調節部係以流速控制裝置51為例，當然該反應調節部 亦可以是微流道材質、微流道表面局部或全面修飾的物質、微流 道内表面所設置的凸出物及/或微流道寬窄不一的内徑或微流道 截面形狀改變。另外第七B圖係顯示本發明之待測物含量分析微 流體晶片1並聯結合一檢體通入口2、複數個待測物含量分析區3 與一反應調節部，其中該反應調節部係以流速控制裝置51為例， 當然該反應調節部亦可以是微流道材質、微流道表面局部或全面 修飾的物質、微流道内表面所設置的凸出物及/或微流道寬窄不一 的内徑或微流道截面形狀改變。 本發明待測物含量分析微流體晶片之微流道上所佈放的固 定物質係包括：核酸、配體、受體、抗原、抗體、酵素、胜肽、 蛋白質或其他可與待測物反應之生物或化學物質。 本發明之待測物含量分析微流體晶片，係可進一步包含一體 積調節區，該體積調節區可以是一液體截流元件，用以截取事先 設定的檢體體積送入反應區，例如，但不限於：姆指幫浦。 本發明之待測物含量分析微流體晶片，係可進一步包含一排 氣元件，用以排放檢體中所含的氣體，避免由於氣泡存在造成量 取檢體體積的誤差。 本發明之待測物含量分析微流體晶片，係可進一步包含一前 處理區，用於進行檢體成份分離、分解、催化、改變狀態、修飾、 14 1295730 標記、試m抗凝@、抗凝▲、抗降解、抗 燥、濃縮、升降溫、調整pH值或清洗等步驟 ^二稀釋、乾 包括’例如，但不限於：„制物標記反應區，=前處理區可 物加以標記。其巾前述制物標記係可湘包括：2内之待測 冷光、奈米微粒或其他具有呈色效果之物質，如·啟、螢光 接之抗體或其他具有呈色效果之試劑。 、呈色顆粒相 本發明之待測物含量分析微流體晶片，係可進勺人, 處理區，用於進行標記、添加試劑、清洗、乾燥或升降後 其中榣汜係指利用酵素、螢光、冷光、奈米微粒或其他具有^呈色 效果之物質，如：與呈色顆粒相接之抗體或其他具有呈多、丄 試劑。 /、壬邑效果之 本發明之另一目的亦提供一種待測物含量分析方法，係包 含·提供檢體；將前述檢體由一檢體通入口導入待測物含量分析 區之微流道，其中該微流道表面修飾複數個固定物質；調節=檢 體中待測物與該固定物質由反應起始點依序累積反應，達二 範圍集中之效果；及藉由前述微流道内產生反應的範圍大小，$ 得知該檢體中待測物的含量。故本發明之方法可以不需要使用 CCD或其他影像分析軟體以辨識變色區域。 刚述之待測物含量分析方法，其中前述調節該檢體中待測物 與該固定物質之反應係藉由一流速控制裝置以控制檢體前進的 速度，達成累積依序反應的要求，亦可藉由前述微流道所包含之 上、下兩基板材質特性來控制檢體前進的速度，達成累積依序反 應的要求，其中該基板材質係分別選自親水性或疏水性材質。 前述調節該檢體中待測物與該固定物質之反應亦可利用微 流道之内徑具有寬窄不一或截面形狀改變之設計，以調整檢體前 進的速度及流動的模式。 前述調節該檢體中待測物與該固定物質之反應係藉由在微 15 1295730 流道之内表面設置凸出物，以增加固定物貝之佈放面積，並有助 於微流雜以擾流方式前進，有效促使符’則物與固定物質接觸機率 提高。 一 ^ 另外，前述調節該檢體中待測物與^固定物質之反應係藉由 在微流道表面的局部或金面修飾特定自肥基親水性物質及/或疏 水性物質來㈣檢體前進的速度，達成累積依序反應的需求。前 述之本發明待測物含量分析方法，係玎進一步包含一體積調節步 驟、一排氣步驟、一前處理步驟及/或/後處理步驟。 其中前述前處理步騍係包括檢體成份分離、分解、催化、改 變狀態、修飾、標記、試劑混合、抗凝固、抗凝血、抗降解、抗 退化、豨釋、乾燥、濃縮、升降溫、調整PH值或清洗等步驟。 其中前述之後處理步驟係包括：標記、添加試劑、清洗、乾燥或 升降溫等步驟。 前述之標記步驟係可藉由加入包括·酵素、榮光、冷光、奈 米微粒、與呈色顆粒相接之抗體、或其他具有呈色效果之物質或 試劑以達到標記之目的。 本發明所提供之待測物含量分析方法，係可利用本發明之待 測物含量分析晶片來達成。 第八圖係相較於顯示習知技術之試紙層析生化檢測試片，本 發明之待測物含量分析晶片為何讀值解析度可以提高的原理，一 般生化檢測試片寬度較寬，所以變色區域前緣常會呈現不規則形 狀及不同層次之灰階，影響讀值的準確性，本發明之微流體晶片 上之待測物含量分析區為細長的微流道，也就是將生化檢測試片 之訊號區域中之變色不規則形狀面積（正方形框線所示）展開成微 流體官道，如此，不規則形狀的變色區域面積大小便可轉換為微 流道内產生反應的範圍大小。例如，假設將習知技術之變色面積 定為長度10單位寬度10單位，本發明若將寬度減少為十分之一 16 1295730 (即1單位），而將長度增長為十倍（即100單位），則可以根據待測 物含量分析區微管道内有待測物反應的長短，例如87單位，就 能更精確定出待測樣品的量，增加解析度，這是習知檢測試片所 無法達到的。 本發明之另一優點為可以整合’控制流入檢體體積’的功 能於同一晶片上。以濃度量測為例，若想依據變色範圍的長短來 推估檢體中待測物的濃度，前提之一是每次進入檢測試片中的檢 體必需控制為事先設定的體積，才能準確換算出待測物的濃度。 本發明可用各種高分子、矽、玻璃或其他材質製作晶片，並在晶 片上整合控制流入檢體體積的功能，因此有助於待測物含量分析 時的準確度提升。除此之外，其他功能例如試劑的添加及均勻混 合、單一檢體進行多項測試、或反應後的清洗等步驟，都可以整 合在同一晶片上，有助於增加待測物含量分析的方便性與準確 性。相較於前述試紙層析生化檢測試片難以整合各式複雜附加功 能的情形，本發明具有進步性。 本發明藉由檢體流動方式的特殊設計，使得有反應之變色範 圍由反應區域的起點開始累積，達到反應範圍集中的效果。如前 所述，此種技術可用於不需外加儀器而直接讀取待測物的含量的 應用。然而，對於必需外加儀器以進行待測物定量的方式而言， 此種技術也很有用。例如，想以儀器量測待測物上所標定的螢光 亮度作為待測物含量的依據，若任由待測物分散在反應區内的任 意各處起反應，單位面積内的待測物含量低，則所使用的儀器靈 敏度必需夠高才足以偵測到有標記的待測物。由於本發明可促使 反應範圍集中，因此所需觀察及測量的反應範圍縮小，單位面積 内的訊號强度提高，使得儀器靈敏度不必太高仍能得到準確的量 17 1295730 、J放果。因此本發明也可適用於待測物的量少，或量測準確度要 /車交兩專’需以儀器輔助待測物含量分析之狀況。 土 除了半定量/定量分析，本發明也適用於定性分析。藉由檢體 机=方式的控制，使得反應範圍集中，有助於檢體中待測物含量 低B守的定性分析判新是否超過門檻值。 ^總而言之，本發明之晶片與試紙層析生化檢測試片相較，本 ^月可用於定性、半定量及定量檢測，且其顯色及讀值解析度 呵，1測較準確，並能附加檢體前處理/反應後處理功能等優點。 二’、他刀析晶片相較，則有不必使用複雜儀器即能檢測待測物含 量的優點。 有關本發明之前述及其他技術内容、特點與功效，在以下配合參 考圖式之較佳實施例的詳細說明中，將可清楚的明白。以下實施 例係用於進一步闡述本發明之優點，並非用於限制本發明之申請 專利範圍。 實施例 本發明係關於一種於微流體晶片上進行檢體内待測物含量 刀析之裝置及其方法，其特徵係為使待測物與修飾於晶片上微管 道表面之固定物質反應，藉由微管道内產生集中反應，再根據反 應之範圍大小以判斷檢體内待測物的含量。 實施例1.本發明之待測物分析微流髏晶片以流速控制的方式調節 反應，用於待測物含董分析 本實施例係利用微機電製程(MEMS process)以厚光阻梦程 以及可撓性高分子材料製作本發明之微流體晶片，並分別測試檢 1295730 體中不同待測物含量。 該晶片上之基板具有形成微流道之凹槽，材質為生物相容性 之可撓性高分子材質，用以規範檢體行經路徑；下板基材材質為 玻璃，上下基板組合形成具有微流道之晶片。於晶片上之檢體通 入口中加入摻有抗凝血劑EDTA之全血，利用外加幫浦作為流速 控制裝置，可成功使全血在寬與深皆為1〇0微米之細長微流道中 流動，並且其流動速度可根據施加於幫浦的壓力大小來控制。 利用如第二圖所示之待測物含量分析微流體晶片1進行實 驗，晶片上之待測物含量分析區3上之微管道4之流道寬100// m、深100//m，且U型微流道4每段直線長度L為8mm，其上 佈有固定物質山羊抗老鼠免疫球蛋白G (Goat-anti-mouse-IgG，濃 度5.6mg/ml)。將檢體（含有經過奈米金粒子標示之待測物 Mouse-IgG C)由檢體通入口 2導入待測物含量分析區3。另外藉 由流速控制裝置51控制樣品於微流道4中推進，速度為 0.8mm/min，以使檢體内的待測物與佈放於微管道4表面之固定 物質充分反應。 一般待測物含量分析的應用都是以相同體積的檢體内含有不 同濃度的待測物做測試。本實施例中，為了易於驗證實驗結果， 改用相同待測物濃度，但不同體積的檢體作測試，加入檢體體積 多者所含待測物的量也成正比例增多。本實施例以兩個相同的晶 片做實驗。晶片一及晶片二係分別擷取1.5 /z 1及3 //1之檢體（内 含相同》農度的待測物Mouse-IgG CGC，OD540=50) ’導入待測物含 量分析區3。檢體流經微流道4後，管壁上的固定物質 (Goat-anti-mouse_IgG)將與待測物進行抗原抗體反應，捉住待測 物。由於待測物已結合有紫色的奈米金粒子作為標示，因此與固 定物質反應結合而停留於流道之管壁後仍呈紫色。從反應起始點 19 1295730 開始， ，有此種反應之區域會呈現唤aSe-Hwan Paek's research, and U.S. Patent No. 5,340,539, are examples of attempts to extend lateral flow biochemical test strips to semi-quantitative/quantitative analysis. The principle is based on a test paper having a wicking action as a substrate, and the amount of the test object is judged by reading the size of the reaction area on the test paper (for example, the length of the color change length). Due to the possible errors in the volume of the sample taken, the uneven flow rate of the sample in the test piece, the reaction area being too short, and the dispersion of the reaction range, the signal strength is different, and the error in the analysis result of the sample to be tested is large. Taking Se_Hwan Paek's test content analysis on the test paper as an example, the first figure 0) shows the test result of the liquid sample being inhaled from the bottom of the biochemical test strip to the reaction zone and then flowing upwards into the reaction zone. . Arranged from left to right are the results of the same test piece for different concentrations of the analyte. The first figure (b) shows an enlarged image of the reaction area on the test piece after the sample reaction. Although this conventional technique can be used for the qualitative test of the test object in the sample, it is not suitable for direct semi-fixed analysis. The reasons are as follows: · Li Lili 1. Because the test strip biochemical test strip has materials and processes The restriction makes the reaction area wider and cannot be very slender. When the length of the discoloration of the object to be tested/the width of the test piece is shorter, the reading error is larger when the amount of the object to be tested is estimated by the visual method according to the length of the discoloration. 2. Because the reaction zone width of the test strip biochemical test strip is inconsistent with the path and speed of the I specimen when it is sucked up, the 1295730 to be tested is sometimes loaded with a large amount on the left side, sometimes in the middle. Or to the right, the leading edge of the discolored area often presents a concave and convex irregular shape, as shown in the first figure (b). In addition, when the liquid sample is sucked from the bottom to the reaction zone and then flows upward, there is no control mechanism, so that some of the analytes run faster and run slower, causing the reaction to be dispersed in the reaction. The phenomenon of the whole area. This can be seen from the front edge of the reaction range in the partial enlargement of the first figure (b). Under this circumstance, if the general user directly judges the length of the discoloration range by the naked eye, it should read the concave or convex part of the leading edge, and how deep or shallow the color is to be a reactive range, all of which will result in Great troubles and errors in reading. 3. The test strip biochemical test strip must be made of a material that can be inhaled into the liquid sample. The material and structure are limited, so it is difficult to directly add the function of 'controlling the volume of the inflow sample' to the material. Taking concentration measurement as an example, if one wants to estimate the concentration of the analyte in the sample according to the length of the color change range, one of the premise is that each time the sample entering the test piece must be controlled to a previously set volume, otherwise, The number of specimens entering the reaction zone varies, and even if the length of the discoloration range is known, the concentration of the analyte can not be accurately converted. The test strip type bioassay test piece is difficult to integrate this function on existing materials and structures, which will hinder the accuracy of analysis. 4. Test strip biochemical test strips are difficult to directly attach a variety of functions to the material to be analyzed, such as separation of sample components, speed control of the sample flowing through the reaction zone, reagents (eg anti- The addition and uniform mixing of the coagulant, the reaction coloring mark, etc., the multiple test of a single sample, or the cleaning after the reaction all contribute to the convenience and accuracy of the analysis, but due to the material and structure. Limited, these features are difficult to integrate on this simple architecture. 1295730 Therefore, how to develop a test and analysis wafer that can effectively process the sample, simplify the user's operation process, accurately measure the content of the sample to be tested, and conveniently read the result without even adding a complicated measuring instrument is urgently needed in the field. Breakthroughs. SUMMARY OF THE INVENTION In view of the lack of conventional detection and analysis techniques, the object of the present invention is to provide a microfluidic wafer that can be used for analyte content analysis, which includes: a sample inlet opening for accessing a sample; a sample content analysis area having a micro flow channel, wherein a plurality of fixed substances are disposed on the surface of the micro flow channel, and one end of the micro flow channel is connected to the inlet of the sample; and a reaction regulating portion is used for adjusting the sample The contact opportunity between the test object and a plurality of fixed substances causes the test object and the fixed substance to sequentially accumulate reactions from the reaction starting point to achieve the effect of concentration of the reaction range. The content of the analyte in the sample can be known by the range of the concentration of the concentrated reaction in the microchannel. In the above description: • “Fixed substance” refers to a substance that is fixed on the surface of the microchannel of the analyte content analysis area, does not flow with the sample, and can react with the analyte in the sample, for example: nucleic acid. , a ligand, a receptor, an antigen, an antibody, an enzyme, a peptide, a protein, or other biological or chemical substance that can react with a test substance. • “Reaction starting point” refers to the starting point at which the fixed substance is placed at one end of the inlet of the microfluidic channel. • “Sequential Cumulative Response” means that after each particle of the analyte enters the reaction initiation point, there is sufficient time and collision opportunity in the process of traveling and the first encountered in the microchannel, and it has not yet been applied. Fixed substance reaction. That is to say, the substance to be tested does not miss the immobilized substance because the flow rate is too fast or there is no chance of collision, but has proceeded to the next position in the microchannel with the sample. All the substances to be tested entering the microchannel are reacted with the fixed substance in order from the starting point of the reaction, so the reaction range 1295730 will be "concentrated" in the analyte content analysis area from the reaction starting point. Once the test object in the test body is reacted by the fixed substance in the front stage, the fixed substance in the back stage flows through the remaining sample, but no analyte in the test body can react with it. Since the range of reaction of the analyte/fixed substance is concentrated in the front of the microchannel, and the immobilized substance that has not reacted after the sample is exhausted is located in the back stage, the amount of the analyte in the sample will be proportional to the reaction in the front stage. The size of the area. If the width of the flow channel is designed to be regular, the amount of analyte will be proportional to the length of the reaction zone within the flow channel. φ "Reaction regulation section" refers to a mechanism that allows sufficient time and collision opportunity to react with a fixed substance during the process of the object to be tested in the microchannel. The method of realizing the reaction adjusting unit includes controlling the speed of the sample flow and/or adding various designs in the flow path to help disturb the sample, increase the reaction area, and contact probability. Another object of the present invention relates to a method for analyzing the content of a sample to be tested, comprising: providing a sample; and introducing the sample from the inlet of the sample into the microchannel of the analyte content analysis area, wherein the surface of the microchannel Modifying a plurality of fixed substances; adjusting the reaction between the analyte and the immobilized substance in the sample, so that the reaction is sequentially accumulated from the starting point of the reaction to achieve the concentration of the reaction range; and the range of the reaction generated by the aforementioned microchannel The size can be used to know the content of the analyte in the sample. The foregoing method for analyzing the content of the analyte can be achieved by using the analysis wafer of the present invention. [Appropriate Method] The content of the sample to be tested of the present invention is as shown in the second figure. The content of the analyte-containing microfluidic wafer 1 mainly includes: a sample inlet 2' For measuring the sample; the analyte content analysis area 3 has a micro flow channel 4, wherein the surface of the micro flow channel 4 is provided with a plurality of fixed substances (represented by a mesh pattern on the micro flow channel), and One end of the microchannel 4 is connected to the inlet of the sample; and a reaction regulating portion is used to adjust the contact opportunity between the sample to be tested and the plurality of fixed substances in the microchannel 4, 11 1295730, the object to be tested and the fixed substance are The reaction starting point 41 starts to accumulate the reaction in order to achieve the effect of concentration of the reaction range. The microfluidic channel 4 is accumulated in the front object of the analyte/fixed substance reaction range (the black solid line region of the microchannel 4), and can be known by referring to the geometrical feature of the microchannel 4 or the external marker scale 6. The content of the analyte in the sample. In the present embodiment, the reaction adjusting unit is a flow rate controlling device 51, for example, a pump that can control the flow rate, and connects the other end of the microchannel 4 opposite to the sample inlet. In another embodiment of the present invention, the reaction adjusting portion may also be a microchannel material of the analyte content analysis region, and the velocity of the sample is controlled by the affinity/hydrophobicity of the microchannel material to achieve Accumulate the need for sequential reactions. Grooves of various cross-sectional shapes are formed on one substrate, and then joined to another flat surface or a substrate having grooves. The grooves between the two substrates form a micro flow path for the micro fluid to perform various tasks in the flow path. The manner in which the microfluid flows in the flow channel is affected by the surface condition of the groove (i.e., the material of the two substrates). The material of the substrate may be selected from hydrophilic or hydrophobic materials, and the material combination of the upper/lower plates may be: hydrophilic/hydrophilic, hydrophilic/hydrophobic, hydrophobic hydrophilic or hydrophobic/hydrophobic. Preferably, the substrate material comprises: polydimethylsiloxane (PDMS), polycarbonate (ρ〇^_〇η^, PC), cyclic olefin copolymers (c〇C), Polystyrene (PS), glass or polymethylmethacrylate (PMMA) 〇 third figure shows a further embodiment of the present invention, which describes the content of the sample to be tested in the present invention. The reaction adjusting portion of the fluid wafer 1 may also be a substance 52 (hatched portion) partially or completely modified on the surface of the micro flow channel 4, such as, but not limited to, a specific functional group, a hydrophilic substance, and/or a hydrophobic substance, Further, the advancement speed of the specimens in different sections of the flow channel is further adjusted to achieve the demand for cumulative sequential reaction. The quaternary system shows the re-implementation of the present invention. The reaction of the analyte content of the present invention is analyzed. The fourth portion of the reaction can also be the change in the cross-sectional shape of the microchannel 4 of the analyte content analysis region. The inner diameter of 53 is to control the front 12 i29573 速度 speed or flow pattern of the specimen. The fourth B diagram shows an enlarged cross-sectional view of the narrow portion of the microchannel 4 of the fourth A diagram. In another embodiment of the present invention, the reaction regulating portion of the microfluid port 1 of the sample to be tested comprises a protrusion disposed on the inner surface of the microchannel to increase the content of the solid material. The area is increased, and the microfluid is promoted in a spoiler manner, which effectively promotes the contact probability of the object to be tested and the fixed substance. The fifth A diagram is shown in an embodiment of the present invention, and the reaction range of the microchannel 4 of the sample to be measured can refer to the geometric characteristics of the microchannel 4 to know the sample. The geometrical characteristics of the microfluidic channel 4 are a plurality of gangway structures of the test object. By observing the reaction range to reach the first curve structure, the content of the test object in the sample can be correspondingly known. For example: low, medium or high. The fifth B-picture is shown in the foregoing analysis of the content of the analyte. The geometric characteristics of the microfluidic wafer 1 microchannel 4 are a plurality of more dense continuous curve configurations, which can be more accurately quantified. The geometrical features of the microchannel 4 of the present invention are not limited to the curve-shaped structure of the present embodiment, and the shape and length thereof can be adjusted according to the actual detection needs, for example, a rectangular curve, a spiral curve, etc. Restricted, other geometric features can also be used to facilitate the identification and knowledge of the content of the analyte. 6A and 6B show the microfluidic wafer 1 of the analyte content of the present invention, and the pipeline of the microchannel 4 can be correspondingly provided with a marking scale 6, which can be disposed beside the microchannel. It can also be disposed on the microchannel 4 (not shown), and the content of the analyte in the specimen can be known by observing the scale corresponding to the reaction range. Shovel the side of the micro-channel 4 and the characteristics of the standard 6.1 scale can be used together or separately to help know the content of the sample in the sample. According to another embodiment of the present invention, the value of the microfluidic channel 4 and the value read by the mark scale 6 can be used to convert the object to be tested in the actual sample by using a calibration curve or an experimental comparison table. The content. 13 1295730 In another embodiment of the present invention, the analyte content analysis microfluidic wafer system can connect multiple sets of sample inlets, multiple sets of analyte content analysis zones or multiple sets of reactions by series or parallel connection. The adjustment portion, the number of connectable sample inlets, the analyte content analysis area or the reaction adjustment unit is not limited, and each group of the analyte content analysis area can be laid with the same or different fixed substances, and can be used as Compare or measure different analytes. 7A is a graph showing the content of the analyte to be tested according to the present invention. The microfluidic wafer 1 is coupled in series with a sample inlet 2, a plurality of analyte content analysis regions 3 and a reaction regulating portion, wherein the reaction regulating portion is at a flow rate. The control device 51 is taken as an example. Of course, the reaction adjusting portion may also be a micro-channel material, a partially or completely modified substance on the surface of the micro-channel, a protrusion provided on the inner surface of the micro-channel, and/or a narrow flow path of the micro-channel. The inner diameter or microchannel cross-sectional shape changes. In addition, the seventh B diagram shows the analyte content analysis of the present invention. The microfluidic wafer 1 is coupled in parallel with a sample inlet 2, a plurality of analyte content analysis regions 3 and a reaction adjustment portion, wherein the reaction adjustment portion is The flow rate control device 51 is taken as an example. Of course, the reaction adjusting portion may also be a micro flow channel material, a partially or completely modified substance on the surface of the micro flow channel, a protrusion provided on the inner surface of the micro flow channel, and/or a narrow flow path of the micro flow channel. The inner diameter or microchannel cross-sectional shape changes. The analyte content of the microfluidic wafer of the present invention is characterized by: a nucleic acid, a ligand, a receptor, an antigen, an antibody, an enzyme, a peptide, a protein or the like which can react with the analyte. Biological or chemical substance. The microfluidic wafer of the present invention may further comprise a volume adjustment zone, which may be a liquid intercepting component for intercepting a predetermined volume of the sample and feeding it into the reaction zone, for example, but not Limited to: thumb finger pump. The microfluidic wafer of the present invention can further comprise a gas venting element for discharging the gas contained in the sample to avoid the error of the volume of the sample due to the presence of the bubble. The analyte content analysis microfluidic wafer of the present invention may further comprise a pre-treatment zone for separating, decomposing, catalyzing, changing state, modifying the sample, 14 1295730 labeling, testing m anti-coagulation@, anticoagulation ▲, anti-degradation, anti-drying, concentration, temperature rise, pH adjustment or cleaning steps, etc. 2 dilution, dry including 'for example, but not limited to: „product labeling reaction zone, = pre-treatment zone can be marked. The above-mentioned product marking system can include: cold light to be tested in 2, nano particles or other substances having a coloring effect, such as an antibody, or a fluorescent agent, or other reagent having a coloring effect. Particle phase The content of the analyte in the present invention is analyzed by a microfluidic wafer, which can be used as a scooping person, a treatment zone, for labeling, adding reagents, washing, drying or lifting, wherein the sputum refers to the use of enzymes, fluorescent light, luminescent light, and nai. Rice particles or other substances having a coloring effect, such as an antibody that is in contact with a coloring particle or other agent having a poly- and hydrazine agent. Another effect of the present invention is to provide a substance to be tested. The analysis method comprises: providing a sample; and the sample is introduced into the microchannel of the analyte content analysis area from a sample inlet, wherein the surface of the microchannel is modified with a plurality of fixed substances; The test substance and the immobilized substance are cumulatively reacted by the reaction starting point in order to achieve the effect of the concentration of the two ranges; and by the range of the range of the reaction generated in the micro flow channel, the amount of the test object in the sample is known. The method of the invention may not require the use of a CCD or other image analysis software to identify a color change region. The method for analyzing the content of the analyte to be tested, wherein the aforementioned adjustment of the reaction between the analyte and the immobilized substance in the sample is controlled by a flow rate The device controls the advancement speed of the sample to achieve the requirement of cumulative sequential reaction, and the speed of the sample advancement can be controlled by the material properties of the upper and lower substrates included in the micro flow channel to achieve the cumulative sequential response requirement. The material of the substrate is respectively selected from a hydrophilic or hydrophobic material. The adjustment of the reaction between the analyte and the immobilized substance in the sample may also be performed by using the inner diameter of the microchannel. The design of the narrowness or the change of the cross-sectional shape to adjust the speed of the sample and the mode of flow. The above-mentioned adjustment of the reaction between the analyte and the immobilized substance in the sample is set by the inner surface of the flow path of the micro 15 1295730 Protrusions, to increase the area of the fixed object shell, and help the microfluids to advance in a turbulent manner, effectively promoting the probability of contact with the fixed substance. In addition, the aforementioned adjustment of the sample The reaction between the analyte and the immobilized substance is achieved by modifying the specific self-fermenting hydrophilic substance and/or hydrophobic substance on the surface or the gold surface of the surface of the microchannel to (4) the speed at which the sample advances, and the demand for cumulative sequential reaction. The foregoing method for analyzing the content of the analyte according to the present invention further comprises a volume adjustment step, a venting step, a pre-processing step and/or/a post-processing step. wherein the pre-treatment step comprises separating the sample components. , decomposition, catalysis, state change, modification, labeling, reagent mixing, anti-coagulation, anti-coagulation, anti-degradation, anti-degradation, release, drying, concentration, temperature rise, pH adjustment or cleaning Step. The foregoing post-processing steps include steps of labeling, adding reagents, washing, drying or temperature rise and fall. The labeling step described above can be carried out by adding a substance or reagent including an enzyme, glory, luminescence, nanoparticle, an antibody which is in contact with the colored particles, or other coloring effect. The method for analyzing the content of the analyte to be provided by the present invention can be achieved by using the analyte content analysis wafer of the present invention. The eighth figure is based on the test paper chromatographic biochemical test piece showing the conventional technique, and the content of the test object of the present invention is analyzed on the principle that the read value of the wafer can be improved, and the general biochemical test piece has a wide width, so the color is changed. The front edge of the region often presents an irregular shape and different levels of gray scale, which affects the accuracy of the reading. The analyte analysis area on the microfluidic wafer of the present invention is an elongated microchannel, that is, a biochemical test strip. The color-changing irregular shape area (shown by the square frame line) in the signal area is expanded into a microfluidic official track, so that the size of the irregularly shaped color-changing area can be converted into the range of the reaction generated in the micro-channel. For example, assuming that the color-changing area of the prior art is set to 10 units in length and 10 units in width, the present invention reduces the width to one tenth of 16 1295730 (ie, 1 unit), and the length is increased by ten times (ie, 100 units). According to the content of the analyte, the length of the reaction of the analyte in the microchannel, for example, 87 units, can more accurately determine the amount of the sample to be tested and increase the resolution, which is impossible for the conventional test strip. Achieved. Another advantage of the present invention is that the ability to integrate & control the flow of sample volume onto the same wafer can be integrated. Taking concentration measurement as an example, if one wants to estimate the concentration of the analyte in the sample according to the length of the color change range, one of the premise is that each time the sample entering the test piece must be controlled to a previously set volume, it can be accurate. Convert the concentration of the analyte. The invention can be used to fabricate wafers from various polymers, enamels, glass or other materials, and integrates the function of controlling the volume of the inflow sample on the wafer, thereby contributing to the improvement of the accuracy of the analyte content analysis. In addition, other functions such as the addition and uniform mixing of reagents, multiple tests of a single sample, or cleaning after the reaction can be integrated on the same wafer, which helps to increase the convenience of analysis of the analyte content. With accuracy. The present invention is progressive in comparison with the case where the aforementioned test paper chromatography biochemical test piece is difficult to integrate various complex additional functions. According to the special design of the sample flow mode, the color change range of the reaction is accumulated from the starting point of the reaction zone, and the effect of the concentration of the reaction range is achieved. As mentioned earlier, this technique can be used for applications that directly read the content of the analyte without the need for an external instrument. However, this technique is also useful in ways that require additional instrumentation to quantify the analyte. For example, if you want to measure the brightness of the fluorescent light on the test object as the basis of the content of the test object, if the test object is dispersed in any part of the reaction zone, the test object per unit area If the content is low, the sensitivity of the instrument used must be high enough to detect the labeled analyte. Since the present invention can promote the concentration of the reaction range, the reaction range required for observation and measurement is reduced, and the signal intensity per unit area is increased, so that the sensitivity of the instrument does not have to be too high, and an accurate amount can be obtained. 17 1295730, J. Therefore, the present invention is also applicable to the situation that the amount of the object to be tested is small, or the measurement accuracy is required to be analyzed by the instrument. Soil In addition to semi-quantitative/quantitative analysis, the invention is also applicable to qualitative analysis. By the control of the sample machine = mode, the reaction range is concentrated, which helps the qualitative analysis of the sample in the sample to be low, and whether the new value exceeds the threshold value. ^ In summary, the wafer of the present invention can be used for qualitative, semi-quantitative and quantitative detection compared with the test paper chromatography biochemical test strip, and the color rendering and reading resolution are good, 1 is more accurate, and can be attached Advantages of pre-sample processing/post-reaction processing. Second, when he analyzes the wafer, there is an advantage that the content of the analyte can be detected without using a complicated instrument. The above and other technical contents, features, and advantages of the present invention will become apparent from the Detailed Description of the <RTIgt; The following examples are intended to further illustrate the advantages of the invention and are not intended to limit the scope of the invention. EMBODIMENT OF THE INVENTION The present invention relates to an apparatus for performing a method for analyzing a sample content in a sample on a microfluidic wafer, and a method thereof, which is characterized in that a substance to be tested is reacted with a fixed substance modified on a surface of a microchannel on a wafer, A concentrated reaction is generated in the micro-pipe, and the content of the test object in the test body is judged according to the range of the reaction. Example 1. Analysis of the analyte of the present invention The microfluidic wafer adjusts the reaction in a flow rate controlled manner for use in the analyte analysis. This embodiment utilizes a MEMS process to achieve a thick photoresist process and The flexible polymer material is used to fabricate the microfluidic wafer of the present invention, and the contents of different analytes in the 1295730 body are separately tested. The substrate on the wafer has a groove for forming a micro flow channel, and the material is a biocompatible flexible polymer material for standardizing the path of the sample; the substrate of the lower plate is made of glass, and the upper and lower substrates are combined to form a micro The wafer of the runner. The whole blood containing anticoagulant EDTA is added to the inlet of the sample on the wafer, and the external pump is used as the flow rate control device, and the whole blood can be successfully made in the elongated microchannel with the width and the depth of 1 〇 0 μm. Flow, and its flow rate can be controlled according to the amount of pressure applied to the pump. The experiment was carried out by analyzing the microfluidic wafer 1 as the content of the analyte to be tested as shown in the second figure, and the flow path of the microchannel 4 on the analyte content analysis area 3 on the wafer was 100//m and 100/m deep. The U-shaped microchannel 4 has a straight line length L of 8 mm, and is provided with a goat Go anti-mouse-IgG (concentration: 5.6 mg/ml). The sample (containing the test substance labeled Mouse-IgG C indicated by the nano gold particles) is introduced into the analyte content analysis zone 3 from the sample inlet 2 . Further, the flow rate controlling means 51 controls the sample to be advanced in the microchannel 4 at a speed of 0.8 mm/min so that the object to be tested in the sample sufficiently reacts with the fixing substance placed on the surface of the microchannel 4. Generally, the application of the analyte content analysis is carried out by testing the same volume of the sample with different concentrations of the analyte. In this embodiment, in order to easily verify the experimental results, the concentration of the same analyte is used instead, but the specimens of different volumes are tested, and the amount of the analyte to be tested is increased in proportion to the volume of the sample. This example was experimented with two identical wafers. The wafer 1 and the wafer second system respectively take 1.5/z 1 and 3 //1 samples (with the same "agronomic test substance" Mouse-IgG CGC, OD540=50) 'Import the analyte content analysis area 3 . After the sample flows through the microchannel 4, the immobilized substance (Goat-anti-mouse_IgG) on the tube wall will react with the analyte to perform antigen-antibody reaction and catch the analyte. Since the test object has been combined with purple nano gold particles as a label, it is purple after being reacted with the fixed substance and staying in the wall of the flow channel. Starting from the reaction starting point 19 1295730, the area with such a reaction will appear a
析區之微流道4產生反應的紫色範圍， 檢體中待測物的含量。 ，隨著待測物被反應耗盡， 1月。藉由觀察待測物含量分 ，透過實驗對照表即可估算 之微流道4長度約為88mm 實驗結果顯示··晶片一中有反麻 4咖’晶片二之有反應之微流道4長度約為16〇腿+/_ 8醜。晶片 二之待測物含量為晶片-之兩倍，測試結果其微管道反應之範圍 大小也大約兩倍’此即顯示微流道反應區域之長度可直接反映出 待測物之含I多募。由此Μ明藉由微米級之微流道並配合檢體流 速控制之反應㈣機制可達_敍應，㈣#巾的效果，用於 半定量/定量分析。 實施例2·本發明之待測物含董分析微流醴晶片以流速控制及流道 幾何形狀變化的方式調節反應，用於待測物含量分析The microchannel 4 of the analysis zone produces a purple range of reaction, and the content of the analyte in the sample. , as the analyte is exhausted, January. By observing the content of the analyte, the length of the microchannel 4 can be estimated by the experimental comparison table to be about 88 mm. The experimental results show that there is a microchannel 4 length of the reaction in the wafer 1 About 16 〇 leg + / _ 8 ugly. The content of the test object of the wafer 2 is twice that of the wafer, and the range of the reaction of the microchannel is also about twice as large as the test result. This indicates that the length of the reaction region of the microchannel directly reflects the inclusion of the analyte. . Therefore, it is possible to achieve the effect of semi-quantitative/quantitative analysis by using the micro-flow microchannel and the reaction of the sample flow rate control (4) mechanism. Example 2: The test object of the present invention contains the analysis of the microfluidic wafer, and the reaction is adjusted in the manner of flow rate control and flow path geometry for analysis of the analyte content.
本實施例目的在測試檢體中待測物含量不同時之量測結 果。微流體晶片的材質、製程與實施例一相同，U型微流道4每 段直線長度L為8mm，但流道的幾何形狀改採寬窄不一的變化， 寬度300 um的微流道每隔2mm距離卽單側非對稱變窄為150 um —次。微流道上佈放有固定物質山羊抗老鼠免疫球蛋白G (Goat-anti-mouse_IgG，濃度5.6mg/ml)。為縮短檢測時間，此例 流速由外加幫浦控制為12mm/min，較實施例一快。 本實施例以兩個相同的晶片做實驗。晶片一所測試的檢體為 含有經過奈米金粒子標示待測物（Mouse-IgG CGC，OD540=50) 4ul之檢體，而晶片二所使用的檢體為待測物（M〇use_IgG CGC， OD540=50) 2ul再加上2ul的水，因此濃度為晶片一内檢體濃度的 20 1295730 測試方法與實施例一相同，檢體流經微流道4後’待測物與微 流道内佈置之固定物質反應結合而停留於流道之管壁呈紫色。觀 察待測物含量分析區之微流道4產生反應的紫色範圍，配合實驗 對照表即可估算檢體中待測物的含量。晶片〆的紫色區域長度為 136 mm +/_ 20mm，晶片二的紫色區域長度為72 mm仏16mm， 與預期結果接近，濃度與長度接近比例關係，但由於流速比實施 例一快，誤差範圍中稍大。 實施例3.本發明之待測物含量分析微流髏晶片以流速控制及流道 增設凸出擾流結構的方式調節反應，用於待測物含量分 析測試 本實施例目的在測試檢體中待測物含量不同時之量測結 果。微流體晶片的材質、製程與實施例二相同，u型微流道4每 段直線長度L為8mm，流道亦採寬窄不一的變化（單側非對稱變 窄，寬度15〇um〜300um間規則性交替變化），但流道内增設凸出 物，使流道内的液體以擾流的方式前進。微流道上佈放有固定物 質山羊抗老鼠免疫球蛋白 G (Goat-anti-mouse-IgG，濃度 5.6mg/ml)。此例流速由外加幫浦控制為12mm/min，與實施例二 相同。 本實施例以兩個相同的晶片做實驗。晶片一所測試的檢體為 含有經過奈米金粒子標示待測物（Mouse-IgG CGC，OD540=50) 6ul之檢體，而晶片二所使用的檢體為待測物（Mouse-IgG CGC， OD540=50) 3ul再加上3ul的水’因此漢度為晶片一待測物濃度的 -—〇 測試方法與實施例二相同’檢體流經微流道4後，待測物與微 流道内佈置之固定物質反應結合而停留於流道之管壁呈紫色。觀 21 1295730 察待測物含量分析區之微流道4有產生反應的紫色範圍即可得知 檢體中待測物的含量。晶片一的紫色區域長度為112 mm +/- 8mm， 晶片二的紫色區域長度為64 mm +/- 8mm，與預期結果接近，濃 度與長度接近比例關係，且由於有效改變流動模式，流速雖與實 施例二相同，但誤差範圍較小。 由前述實施例可得知本發明之微流體晶片可作為待測物含 量分析之工具，並且藉由待測物含量分析區上的微管道之幾何特 徵及/或標記刻度，可直接得知檢體中待測物的量，卽時得到檢測 結果，不需借助其他影像分析軟體或儀器。 雖然本發明已以較佳實施例揭露如上，然其並非用以限定本 發明，任何熟悉此技藝者，在不脫離本發明之精神和範圍内，當 可作各種之更動與潤飾，因此，本發明之保護範圍，當視後附之 申請專利範圍所界定者為準。 22 1295730 【圖式簡單說明】 第一圖顯示習知技術試紙層析生化檢測試片示意圖及其部 分訊號影像放大圖。 第二圖為本發明待測物含量分析之微流體晶片其反應調節 部為一流速控制裝置之實施態樣。 第三圖為本發明待測物含量分析之微流體晶片其反應調節 部為微流道表面修飾之實施態樣。 第四A圖為本發明待測物含量分析之微流體晶片其反應調節 部為微流道寬窄不一的内徑之實施態樣。 第四B圖為第四A圖之微流道局部放大。 第五A圖為本發明待測物含量分析之微流體晶片以微流道的 幾何特徵作為讀值辨識參考之一實施例。 第五B圖為本發明待測物含量分析之微流體晶片以微流道的 幾何特徵作為讀值辨識參考之另一實施例。 第六A圖為本發明待測物含量分析之微流體晶片微流道的標 記刻度之一實施例。 第六B圖為本發明待測物含量分析之微流體晶片微流道的標 記刻度之另一實施例。 第七A圖係顯示本發明待測物含量分析之微流體晶片串聯複 數組檢體通入口、待測物含量分析區及反應調節部。 第七B圖係顯示本發明待測物含量分析之微流體晶片並聯複 數組檢體通入口、待測物含量分析區及反應調節部。 第八圖係說明本發明之待測物含量分析晶片較習知生化檢 測試片有較高解析度原理。 【元件符號對照說明】 1-—待測物含量分析微流體晶片 23 1295730 2— 檢體通入口 3— 待測物含罝分析區 4— —微流道 6…標記刻度 41…反應起始點 51 —流速控制裝置 52…微流道表面修飾的物質 53…微流道寬窄不一的内徑 參考資料： [1] Se-Hwan Paek， et. al·， Development of Rapid One-Step Immunochromatographic Assay，Methods 22, ρ·53-ρ.60, 2000 24The purpose of this embodiment is to measure the results when the contents of the test object are different in the test sample. The material and process of the microfluidic wafer are the same as those in the first embodiment. The length L of each segment of the U-shaped microchannel 4 is 8 mm, but the geometry of the flow channel is changed by a wide and narrow variation, and the micro flow channel having a width of 300 μm is used every other time. The 2mm distance 卽 unilateral asymmetric narrowing is 150 um-times. A fixed substance goat anti-mouse immunoglobulin G (Goat-anti-mouse_IgG, concentration 5.6 mg/ml) was placed on the microchannel. In order to shorten the detection time, the flow rate of this example was controlled by an external pump to be 12 mm/min, which was faster than that of the first embodiment. This example was experimented with two identical wafers. The sample to be tested on the wafer is a sample containing 4 ul of the object to be tested (Mouse-IgG CGC, OD540=50), and the sample used for the wafer 2 is the analyte (M〇use_IgG CGC). , OD540=50) 2ul plus 2ul of water, so the concentration is the internal concentration of the sample in the wafer. 12 1295730 The test method is the same as in the first example. After the sample flows through the microchannel 4, the sample and the microchannel are in the test. The fixed substance disposed in the reaction combines and the wall of the tube that stays in the flow channel is purple. Observe the purple range of the reaction generated by the microchannel 4 in the analyte analysis area, and use the experimental comparison table to estimate the content of the analyte in the sample. The purple region of the wafer has a length of 136 mm + / _ 20 mm, and the purple region of the wafer has a length of 72 mm 仏 16 mm, which is close to the expected result. The concentration is close to the length, but since the flow rate is faster than that of the first embodiment, the error range is Slightly larger. Example 3. Analysis of the content of the analyte according to the present invention The microfluidic wafer is adjusted in such a manner that the flow rate is controlled and the flow channel is provided with a convex spoiler structure, and is used for the analyte content analysis test. The purpose of this embodiment is to test the sample. The measurement result when the content of the analyte is different. The material and process of the microfluidic wafer are the same as those in the second embodiment. The linear length L of each segment of the u-shaped microchannel 4 is 8 mm, and the flow path is also varied in width and width (unilateral asymmetric narrowing, width 15 〇 um~300 um). Regularly alternating between the two, but the addition of protrusions in the flow channel, so that the liquid in the flow channel advances in a turbulent manner. A fixed substance goat anti-mouse immunoglobulin G (Goat-anti-mouse-IgG, concentration 5.6 mg/ml) was placed on the microchannel. The flow rate of this example was controlled by an external pump to be 12 mm/min, which was the same as in the second embodiment. This example was experimented with two identical wafers. The sample to be tested on the wafer is a sample containing 6 ul of the object to be tested (Mouse-IgG CGC, OD540=50), and the sample used for the wafer 2 is the analyte (Mouse-IgG CGC). , OD540=50) 3ul plus 3ul of water 'so the Handu is the wafer-testant concentration--〇 test method is the same as the second example. 'The sample flows through the micro-channel 4, the object to be tested and the micro-flow The fixing material arranged in the flow channel reacts and the wall of the tube which stays in the flow channel is purple. View 21 1295730 The microchannel 4 of the analyte content analysis area has a purple range in which the reaction is generated to know the content of the analyte in the sample. The purple area of wafer one is 112 mm +/- 8 mm long, and the purple area of wafer two is 64 mm +/- 8 mm, which is close to the expected result. The concentration is close to the length, and the flow rate is due to the effective change of flow pattern. The second embodiment is the same, but the error range is small. It can be known from the foregoing embodiments that the microfluidic wafer of the present invention can be used as a tool for analyzing the content of the analyte, and can be directly detected by the geometric features and/or mark scale of the micro-pipe on the analyte analysis region. The amount of the object to be tested in the body, when the test results are obtained, without the need for other image analysis software or instruments. While the present invention has been described above in terms of the preferred embodiments thereof, it is not intended to limit the invention, and the invention may be modified and modified without departing from the spirit and scope of the invention. The scope of protection of the invention is defined by the scope of the appended claims. 22 1295730 [Simple description of the diagram] The first figure shows the schematic diagram of the chromatographic biochemical test strip of the conventional technical test paper and its enlarged image of the partial signal. The second figure is an embodiment of the flow rate control device of the microfluidic wafer in which the content of the analyte is analyzed. The third figure is an embodiment of the microfluidic surface of the microfluidic wafer in which the content of the analyte is analyzed in the present invention. The fourth A is an embodiment in which the reaction regulating portion of the microfluidic wafer in which the content of the analyte is analyzed is a narrow inner diameter of the microchannel. The fourth B is a partial enlargement of the microchannel of the fourth A diagram. The fifth A is an embodiment of the microfluidic wafer in which the content of the analyte is analyzed according to the geometric characteristics of the microchannel as a reference for reading the value. The fifth B is another embodiment of the microfluidic wafer of the present invention for analyzing the content of the analyte to have the geometric characteristics of the microchannel as the reference for reading the value. Fig. 6A is an embodiment of the marking scale of the microfluidic wafer microchannel of the analysis of the content of the analyte of the present invention. Fig. 6B is another embodiment of the marking scale of the microfluidic wafer microchannel of the analysis of the content of the analyte of the present invention. Fig. 7A is a view showing the microfluidic wafer series complex array sample inlet, the analyte content analysis region, and the reaction adjustment portion of the present invention. Fig. 7B is a view showing the microfluidic wafer parallel array detector inlet, the analyte content analysis region and the reaction adjustment portion of the present invention. The eighth figure illustrates the fact that the analyte content analysis wafer of the present invention has a higher resolution principle than the conventional biochemical test wafer. [Component symbol comparison description] 1--Test object content analysis Microfluidic chip 23 1295730 2 - Sample inlet 3 - Test object containing 罝 analysis area 4 - Micro flow path 6... Mark scale 41... Reaction start point 51 - Flow rate control device 52... Microfluidic surface modified material 53... Microfluid channel width and narrow inner diameter Reference: [1] Se-Hwan Paek, et. al., Development of Rapid One-Step Immunochromatographic Assay, Methods 22, ρ·53-ρ.60, 2000 24