1245895 A7 B7 五、發明説明（1 ) 發明領域 本發明可廣泛的應用於製藥、民生及國防工業、環境 監控及科學研究。本發明涉及藉由與其他合成或天然分子 之專一性結合及其偵測的分子之偵測及分析方法。 發明背景 核酸雜交反應、抗原一抗體反應及受體一配位體結合 是分子相互作用的實例，因為相互作用的專一性，該等相 互作用的實例對鑑別或偵測此等物質有很大的價值。一實 例為利用專一性抗體進行食物及水中的生物製劑和毒素之 偵測，以及利用雜交反應技術，進行對特定微生物具專一 性之核酸序列的偵測。在有或無放大技術（可應用於核酸） 之下，專一性地偵測此類物質的能力，允許進行推定劑或 物質的鑑別。 發明概述 在本文中揭露一種偵測及鑑別微量的分子目標物之方 法，其係利用目標物與二個分子探針之間的專一***互作 用來進行，該方法包含： 將該分子探針中之一者附著於導電性珠粒上， 將該探針中另一者固定於二電極之間的間隙中， 對該電極施予一電位差，以及 監測由電極中之一者到電極中之另一者之電流增加 量，其可發生於若該導電性珠粒藉由該專一性之交互作用 拉入該間隙時。 一般來說，探針中之一者係物理性地結合至電極間的” 本紙張尺度適用中國國家標準（CNS) Α4規格（210X297公釐） -4- (請先閲讀背面之注意事項再填寫本頁) 、τ 可線- 1245895 A7 B7 五、發明説明（2 ) (請先閲讀背面之注意事項再填寫本頁) 井”中。如果目標物存在，該目標物會在合適的條件下結合 至此探針。攜帶導電性珠粒的另一探針接著結合至該目標 物的另一部分。 較佳地，導電性珠粒為鐵珠粒。 較佳地，導電性珠粒在附著於該分子探針中之一者之 前先被消磁。 較佳地，該消磁作用是藉由在一經屏障以與地球及其 他磁場相隔的環境下加熱來進行。 在電極之間導電性珠粒的物理定位作用造成電路封 閉。 探針與目標物之間反應的專一性是偵測的基礎。 此外，此過程可被設計用於偵測經微處理器控制的微 陣列中之多作用劑/分子或分析特定物質之濃度。 在本說明書中更進一步的揭露一種偵測微量之分子目 標物的方法，該方法利用目標物及二分子探針之間的專一 ***互作用來進行，該方法包含下述步驟： (a) 藉由加入氣體或固體至溶液中製備一樣本或製備 一待鑑別的作用劑， (b) 將樣本引入具有二個緊接地置放的電極之偵測裝 置中’在該電極之間的間隔中結合有探針’並容許結合/ 雜交反應發生， (c) 在步驟(b)進行前、進行期間或進行後，加入第二 個探針（超過目標物的量），其結合至導電性珠粒並容許專 一性之結合/雜交反應發生，以及 本紙張尺度適用中國國家標準（CNS) A4規格（210X297公釐） 1245895 A7 B7 五、發明説明（3 ) (e)藉由偵測該電極間任何電流的改變，測定導電性 粒子結合至該間隙是否已發生。 較佳地，步驟（C)中應用的環境無法對生鏽的鐵珠粒導 電，例如載體流體之先前去氧化作用。 較佳地，步驟（e)係比下述步驟優先進行： (d)調整溶液的化學性質及/或溫度以使反應條件 最適化。較佳地，步驟（e)中使用微處理器。 較佳地，步驟（a)包括使細胞物理及/或化學地縮小（破 碎）成其成分並釋出其内容物/組成分以供偵測。 此方法可被設計為二或三度空間的微陣列且用來偵測 具有不同化學本質的多種分子，包括但不限定於核酸、蛋 白質、碳水化合物、脂質及無機分子。 此方法可包括内裝的二重覆或三重覆以供品質控制。 此方法也可包括與負控制一起進行之電子式自我檢查 及/或預分析試驗。 此方法也可包括與正控制一起進行的後分析試驗，此 試驗的結果應該是負的。 在本說明書中更進一步的揭露了 一種用於分析溶液中 特定物質之濃度的方法，包含： 提供一個別晶片的陣列，每個晶片包含一封閉的電 路，該電路包括界於一對電極之間的結合探針之分析物及 導電性珠粒，其中晶片在電極之間的間隙尺寸及經結合之 分析物的量上不同，及因此該珠粒之量不同， 導入含有一未知濃度之分析物的樣本到該微陣列中， -6- (請先閲讀背面之注意事項再填寫本頁) 本紙張尺度適用中國國家標準（CNS) A4規格（210X297公釐） 1245895 A7 B7 五、發明説明（4 ) (請先閲讀背面之注意事項再填寫本頁) 藉此該分析物競爭地置取代晶片中經珠粒結合之探 針，該晶片為含有特定量或少量之經結合的分析物，而非 含有大量經結合之分析物的晶片，以及具有充分經取代之 珠粒之晶片將轉換為一開放的電路。 較佳地，利用已知濃度之標準品先進行校正，容許進 行樣品中分析物之濃度分析。 本發明更揭露一種分析物質之濃度的方法，其藉由提 供比分析物少之結合“井”的探針，該分析物比結合珠粒 之探針多，且在無先前結合珠粒（關閉）之下，容許反應 在具有數千個晶片之微陣列中進行。加入分析物後開始的 晶片之積分比率，可以用來計算分析物的濃度。當分析物 無法或太貴而不能純化或製造以供用於電生物晶片時，此 表現方法具有優於習知方法的優點。 在本發明中更進一步揭露一種分析一特定物質之濃度 的方法，包含： 提供一相同晶片之陣列，在二電極之間有一小間隙， 該電極僅接受一導電性珠粒及結合井之探針， 將含有未知量分析物的樣品引入位在卡匣内之該微陣 列中，該卡匣含有比樣品中的分析物量還要少的已知量之 經添加的結合珠粒之探針，藉此 游離的分析物與結合分析物之結合珠粒之探針（形成 於引入之分析物與卡匣内側之結合珠粒的探針反應後），競 爭結合該電生物晶片之有限數量的該結合井的探針。 較佳地，此方法包含利用先前已知之結合珠粒之探針 本紙張尺度適用中國國家標準（CNS) A4規格（210X297公釐） 1245895 五、發明説明（ 的里，计异樣品中分析物的濃度，藉由微處理機記錄”開啟，， 至”六關閉”訊號的比例及利用已知濃度之分析物的標準口預 在本說明書中更進一步的揭露一種用於偵測微量分子 目標物的裝置，其係藉由利用目標物與二分子探針之S的 專一***互作用來進行，該裝置包含： 曰、 井其具有彼此相隔以形成一間隙的二個電極，以 及該探針中之一者附著於該井， 用於對該電極施加一電位差的構件，以及 用於監測由該電極中之-者至另—者之電流增加的構 件，其可發生於若該導電性珠粒藉由該專一性之交互作用 拉入該間隙時。 較佳地，該裝置係與多數其他相同之裝置一起收容在 一微陣列中。 較佳地，該微陣列係收容入一卡匣中。 較it地，上述係位在一建構有可容納該卡之溝槽之 可以攜帶的裝置中。 9 較佳地，該組合進一步包括一微處理器，其讀取來自 。又置於ό亥卡匡上的鑑別之該卡匣的内容。 定義 如本說明書中所使用者，下述術語係意圖具有下述一 般意義： 核酸意指DNA、RNA，其單股或雙股以及任何化學改 質形式。改質包括但不限制於提供其他化學基團者。 本紙張尺度適用中國國家標準（CNS) Α4規格（210X297公釐） (請先閲讀背面之注意事項再填寫本頁) •、盯| 1245895 A7 B7 五、發明説明（6 ) “配位體”意指專一性地結合至受體並藉此在細胞中 誘導一訊息的分子，例如：荷爾蒙係一配位體，當其與受 體結合時，會引發階梯式的細胞反應，造成細胞的生長或 其他反應。 在本說明書中使用之“雜交反應”一詞意指二條單一 互補DNA股之接合（DNA/DNA之雜交反應）），或互補 之DNA及RNA股之接合（DNA/RNA之雜交反應）。 “分析物”意指存在病患血液或體液中的一種物質。 分析物的濃度一般而言會隨著代謝或病理狀態而改變，且 為臨床醫生管理病患健康的資訊。 “抗原”意指具有分子表面結構之物質，該結構引發 免疫反應物質，亦即抗體之產生，及/或與（其）專一性 的抗體反應（抗原/抗體反應）。 “抗體”為一種蛋白質（免疫球蛋白）），其識別及結 合至抗原，為免疫反應的一部分。 “分子探針”意指任何核酸、蛋白質分子或其他分 子，具有與其他相同或不同類型分子專一性地結合的能 力。一般來說，核酸專一性地結合至呈現序列互補性的核 酸。因此，具有下述序列一A-G-G-C-G-T-A(由5’端到3’ 端）一之探針（在此例子中為一核酸分子）將專一性地與另 一股DNA結合，該另一股DNA含有具有下述序列 T-A-C-G-C-C-T(由5’端到3’端）一的區域，其中A，T，G 及C分別代表腺嘌呤，胸腺嘧啶，鳥嘌呤，胞嘧啶。一抗 原的抗體可作為相對應此抗原的分子探針。 -9- (請先閲讀背面之注意事項再填寫本頁) r線| 本紙張尺度適用中國國家標準（CNS) A4規格（210X297公釐） 1245895 A7 B7 五、發明説明（7 ) “抗原決定部位”意指抗原分子中結合至一抗體的部 分。一抗原可具有很多不同的抗原決定部位，其可與不同 的抗體結合。 圖式之簡要說明 本發明之較佳形式將藉由實施例，參考後附圖式來說 明，其中： 第1圖為應用於測試裝置之電路的概要圖， 第2圖為顯示二個分別含有附著的探針及附著的抗體 之晶片的概要圖’ 第3圖描述添加經結合之鐵珠粒之第二探針的概要圖， 第4圖描述一可能之微陣列設計的概要圖， 第5圖描述一定量分析物方法背後的原理， 第6圖描述使用一微陣列定量分析物之概要圖，該微陣 列含有事先經結合之珠粒，該珠粒具有夾在探針之間的目 標物， 第7圖為描述另一個分析物的定量方法之原理的概要 圖，此方法利用競爭性的結合，而非事先結合之珠粒， 第8圖為描述第二種分析物的定量方法之概要圖，此方 法係在過量分析物存在下利用微陣列， 第9圖為描述用於亨丁頓氏基因（Huntingtin gene )之 重覆區域的二探針的設計之概要圖， 第10圖為描述另一組探針之概要圖，此探針之重覆組 合數目，比存在於既定病患中之重覆的正確數目還少。該 探針之二端無接合作用發生，以及 -10- (請先閱讀背面之注意事項再填寫本頁) 本紙張尺度適用中國國家標準（CNS) A4規格（210X297公釐） 1245895 A7 B7 五、發明説明（8 ) 第11圖為描述另一組探針之概要圖，該探針之重覆組 合數目，比存在既定病患中重覆的正確數目還多，造成懸 突之探針及接合作用的失敗。 較佳的具體實施例之描述 在第1圖中，基本單元包含導線1、開關2、電池3、安 培計、燈泡或微處理器4，及反應”井”5，該井中含有二個 電極6及該等電極間的小間隙7。電路原是開啟的，但當鐵 珠粒位於電極間時，接著打開開關2使電路完整並造成電 流，此可藉由安培計4偵測。 在第2圖中，晶片8是反應”井”加上第1圖中所提及的電 極。在此晶片的中央是’’井”5，其可有一點凹陷，其壁上有 共價結合的分子探針9，該探針對待尋找/分析之分子（目標 物）具專一性。此探針可為核酸（左面板）或抗體（右面板）。 相對於”井”之任一壁的是二個電極6。當在一微陣中製造多 個晶片時，該”井”不必一定要凹陷（參見第4圖）。具有平面 的”井”之優點為於反應終點時，較易藉由應用磁場，去除 未結合的過量之結合鐵珠粒之探針(描述於第3圖中）。 在第3圖中，例如鐵珠粒10之某些導電珠粒係位於兩電 極6間，被經夾住之目標物分子11結合，該目標物分子係附 著於結合”井”之第一探針9以及結合鐵珠粒的第二探針 12。在左面板中，此探針係核酸分子，可辨識目標核酸的 不同部位。在右面板中，該探針係為抗體，其對分析物上 不同的區域（抗原決定部位）有專一性，通常為蛋白質分子。 在第4圖中，該晶片係小型化的8，且各自設計為偵測 -11- (請先閲讀背面之注意事項再填寫本頁) 本紙張尺度適用中國國家標準（CNS) A4規格（210X297公釐） 1245895 A7 """"""'" -----— B7 五、發明説明（9 ) ~ --— 同：/、他的特異分子。出口及入口可以引入作用劑及樣 至晶片。在此方法中’微處理器具有計算能力，多個製 劑/分子可在—個便於攜帶的裝置中，同時（多重）债測出 “承載u陣列的筒g13是拋棄式的，且可更換成另一者 =同者以測定不同組分子。可内嵌二重複或三重複以供 貝保af °而且’與負控制—起進行的預試驗及與正控制 一起進打的後試驗（若試驗結果為負），確保試驗的 性。 在第5圖中，分析物丨丨係於試驗，，井”5中，夾在二個探 針之間，此探針中之一9是結合至，，井，，以及另一個12是結合 至鐵珠粒10，該鐵珠粒係與電極6相接觸。加入含足夠濃度 的分析物之試驗樣本，造成附著鐵珠粒的第二探針之競爭 性結合及取代，因此阻斷了此電路（關閉）。 曰在第6圖中，在電極間的被夾住之分析物及鐵珠粒的 里，在一系列製定成陣列之晶片中逐漸改變，允許如下述 解說般偵測未知濃度的分析物。 在第7圖中，未利用事先結合的珠粒。結合珠粒之探針 的存在量比分析物還少。此結果係結合分析物及結合粒子 之探針15，與游離的分析物11競爭結合有限的結合，，井，，之 採針9。在電極間的間隙係縮小至僅容納一個珠粒。左面板 顯示完全被分析物佔據的結合，，井，，之探針，而無附著的結 合珠粒之探針附著（關閉）。右面板顯示結合珠粒之探針， 其結合附著於結合，，井，，之探針的分析物分子（開啟）。 在第8圖中，具有數千個此種晶片的微陣列，記錄，，開 本紙張尺度適用中國國家標準（CNS) A4規格（210X297公爱） (請先閲讀背面之注意事項再填寫本頁) 、\吞· -12- 1245895 A7 B7 五、發明説明（1G ) 啟”訊號的總數，為結合分析物及結合粒子之探針與結合” 井”之探針結合的結果。結合”井”之探針被未附著結合珠粒 之探針的游離分析物佔據之晶片，會記錄”關閉”的訊號。” 開啟”相對於”關閉”訊號的比率取決於結合分析物及結合 粒子之探針及游離分析物之相對濃度。筒匣13之放大圖顯 示一些晶片是開啟的，同時有些晶片是關閉的。事先已知 結合珠粒之探針的莫耳濃度允許計算分析物的濃度。 在第9圖中，說明測量亨丁頓氏疾病中CAG重覆之數 目的原理。在實施例9中可以找到此方法之描述。結合珠粒 之探針17係設計成在探針探測區域具有以下的序列： CTGGAAGGA，以及結合”井”之探針18的探針探測區域具 有以下的序歹|J : GGTGGCGGCTGTTGCTGCTGCTG。圖 中，上股19是存在於此種特定病患中之帶有四組”CAG”重 複的目標基因。只有此與探針互補之區域被描述為具有實 際核酸序列。其他的端點（5, 20及3’ 21)藉由箭頭表示。雜 交的結合珠粒17與結合”井”之探針18係利用相同的前述方 式描述。探針之劃底線的部分（圖上方）標示出鄰接重複區 域的未重複部分。圖中，此二個探針具有與目標物（正確 為4 )相符的重複之組合數目。因此，此二個探針的5’端 及3’端便集合在一起，且可藉由酶(DNA接合酶）接合。 第10圖中，僅解釋說明一不同的結合”井”之探針22的 序列，因為結合珠粒之探針是相同的。此結合”井”之探針 22係放置於同一微陣列的另一個電生物晶片中。在此所描 述的結合”井”之探針22只有二個重複，在與目標物雜交時 本紙張尺度適用中國國家標準（CNS) A4規格（210X297公釐） -13 - (請先閱讀背面之注意事項再填寫本頁) -口 _ r線丨 1245895 A7 B7 五、發明説明（11 ) 會在二個探針間造成一個間隙。此二個探針無法被接合。 在第11圖中，另一個結合”井”之探針23具有過多重複 (5個）。因此，過量的部分在雜交後會有”懸突”24。同樣也 無接合作用。 樣本白勺取得 可使含有推定目標物之空氣起泡通過適當的溶質。固 體或液體可溶解於溶液中。要求完整的細胞及組織被破裂 打開或經製備以釋出待測分子。 裝置（電生物晶片） 在其最簡單的設計中，此電生物晶片係由一個小的測 試”井”所組成，該井内部含有二電線，在該二電線之間有 一小的間隙。該間隙上結合了分子探針，該探針對推定的 目標物有專一性。另一元件為第二專一性探針，其係與電 導體的小珠粒結合。 反應 將樣本加入晶片。加入超過目標物量的第二探針（在加 入樣本之前、其間或之後）。容許反應發生。反應後，未結 合的探針及鐵珠粒藉由產生適當的磁場來吸走。最後，記 錄結果。 定量 二個電極之間的間隙寬度及結合”井”之探針的量，以 及因此經夾住之結合”井”之探針/目標物/結合珠粒之探 針，在晶片之相反設計（記錄”開啟”訊號而非記錄”關閉” 訊號）陣列中可改變，以供測量待測樣本中目標物存在量 本紙張尺度適用中國國家標準（CNS) Α4規格（210X297公釐） -14- (請先閱讀背面之注意事項再填寫本頁) 、τ. 一線- 1245895 A7 B71245895 A7 B7 V. Description of the invention (1) Field of the invention The present invention can be widely used in pharmaceutical, people's livelihood and national defense industries, environmental monitoring and scientific research. The present invention relates to a method for detecting and analyzing molecules by specifically binding and detecting them with other synthetic or natural molecules. BACKGROUND OF THE INVENTION Nucleic acid hybridization reactions, antigen-antibody reactions, and receptor-ligand binding are examples of molecular interactions. Because of the specificity of the interactions, the examples of such interactions are very important for identifying or detecting such substances. value. One example is the use of specific antibodies to detect biological agents and toxins in food and water, and the use of hybridization technology to detect specific nucleic acid sequences specific to specific microorganisms. The ability to specifically detect such substances with or without amplification techniques (which can be applied to nucleic acids) allows the identification of putative agents or substances. SUMMARY OF THE INVENTION Disclosed herein is a method for detecting and identifying a small amount of molecular target, which is performed using a specific interaction between the target and two molecular probes. The method includes: One of the electrodes is attached to the conductive beads, the other of the probes is fixed in the gap between the two electrodes, a potential difference is applied to the electrode, and the monitoring is performed from one of the electrodes to the other in the electrode. The amount of current increase can occur when the conductive beads are pulled into the gap by the specific interaction. Generally speaking, one of the probes is physically bonded to the electrodes. ”This paper size applies to the Chinese National Standard (CNS) A4 specification (210X297 mm) -4- (Please read the precautions on the back before filling This page), τ OK line-1245895 A7 B7 V. Description of the invention (2) (Please read the precautions on the back before filling this page). If the target is present, the target will bind to the probe under appropriate conditions. Another probe carrying conductive beads is then bound to another part of the target. Preferably, the conductive beads are iron beads. Preferably, the conductive beads are demagnetized before attaching to one of the molecular probes. Preferably, the degaussing effect is performed by heating in an environment separated from the earth and other magnetic fields through a barrier. The physical positioning of the conductive beads between the electrodes causes the circuit to close. The specificity of the reaction between the probe and the target is the basis of detection. In addition, this process can be designed to detect multiple agents / molecules in a microprocessor-controlled microarray or analyze the concentration of specific substances. In this specification, a method for detecting trace molecular targets is further disclosed. The method is performed by using a specific interaction between a target and a two-molecule probe. The method includes the following steps: (a) borrowing Prepare a sample by adding a gas or solid to the solution or prepare an agent to be identified, (b) introduce the sample into a detection device with two electrodes placed close to each other, 'combined in the space between the electrodes There are probes' and allow the binding / hybridization reaction to occur, (c) Add a second probe (exceeding the amount of the target) before, during or after step (b), which binds to the conductive beads And allows specific binding / hybridization reaction to occur, and this paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) 1245895 A7 B7 V. Description of the invention (3) (e) By detecting any The change in current determines whether or not the bonding of conductive particles to the gap has occurred. Preferably, the environment applied in step (C) is unable to conduct electricity to the rusted iron beads, such as the previous deoxidation of the carrier fluid. Preferably, step (e) is performed in preference to the following steps: (d) The chemical properties and / or temperature of the solution are adjusted to optimize the reaction conditions. Preferably, a microprocessor is used in step (e). Preferably, step (a) includes physically and / or chemically shrinking (shattering) the cells into their components and releasing their contents / components for detection. This method can be designed as a two- or three-dimensional microarray and is used to detect a variety of molecules with different chemical properties, including but not limited to nucleic acids, proteins, carbohydrates, lipids, and inorganic molecules. This method may include built-in double or triple repeats for quality control. This method may also include electronic self-examination and / or pre-analysis tests performed with negative control. This method may also include a post-analysis test performed with positive control, and the results of this test should be negative. This specification further discloses a method for analyzing the concentration of a specific substance in a solution, including: providing an array of other wafers, each wafer containing a closed circuit, the circuit comprising a pair of electrodes Probe-bound analytes and conductive beads, in which the wafers differ in the size of the gap between the electrodes and the amount of bound analytes, and therefore the amount of the beads is different, introducing an analyte containing an unknown concentration Sample into this microarray, -6- (Please read the precautions on the back before filling this page) This paper size is applicable to China National Standard (CNS) A4 specification (210X297 mm) 1245895 A7 B7 V. Description of the invention (4 ) (Please read the precautions on the back before filling out this page.) This analyte competitively replaces the bead-bound probe in the wafer, which contains a specific amount or a small amount of the bound analyte instead of A wafer containing a large number of bound analytes, and a wafer with sufficiently replaced beads will be converted into an open circuit. Preferably, calibration is performed using a standard of known concentration to allow analysis of the concentration of the analyte in the sample. The present invention further discloses a method for analyzing the concentration of a substance by providing fewer probes that bind to a "well" than an analyte, the analyte being more than a bead-bound probe, and without a previously bound bead (close ), The reaction is allowed to proceed in a microarray with thousands of wafers. The integration ratio of the wafer starting after the analyte is added can be used to calculate the concentration of the analyte. This performance method has advantages over conventional methods when the analyte cannot or is too expensive to be purified or manufactured for use in electrobiochips. A method for analyzing the concentration of a specific substance is further disclosed in the present invention, including: providing an array of the same wafer with a small gap between two electrodes, the electrode receiving only a conductive bead and a probe of a binding well A sample containing an unknown amount of analyte is introduced into the microarray in a cassette containing a known amount of added bead-bound probe that is less than the amount of analyte in the sample. The free analyte and the bead-bound probe that binds the analyte (formed after the introduced analyte reacts with the bead-bound probe on the inside of the cassette), competes to bind a limited number of the bindings of the electrobiochip Well probe. Preferably, the method includes the use of a previously known bead-bound probe. The paper dimensions are in accordance with Chinese National Standard (CNS) A4 (210X297 mm) 1245895. 5. Description of the invention Concentration, the ratio of the signal "on" to "six off" is recorded by the microprocessor and the standard port of the analyte with a known concentration is used in this specification to further disclose a method for detecting trace molecular targets. A device is performed by utilizing a specific interaction of a target with S of a two-molecule probe. The device includes: a well having two electrodes spaced apart from each other to form a gap, and One is attached to the well, a means for applying a potential difference to the electrode, and a means for monitoring an increase in current from one of the electrodes to the other, which may occur if the conductive beads borrow When pulled into the gap by the specific interaction. Preferably, the device is housed in a microarray with most other identical devices. Preferably, the microarray is housed in More specifically, the above is located in a portable device constructed with a slot that can accommodate the card. 9 Preferably, the combination further includes a microprocessor that reads from. The contents of the cassette identified on the Haika Kuang. Definitions as used in this specification, the following terms are intended to have the following general meanings: Nucleic acid means DNA, RNA, its single or double strands, and any Chemical modification form. Modification includes but is not limited to those who provide other chemical groups. This paper size applies to China National Standard (CNS) A4 specification (210X297 mm) (Please read the precautions on the back before filling this page) • 1245895 A7 B7 V. Description of the invention (6) "Ligand" means a molecule that specifically binds to a receptor and thereby induces a message in the cell. For example, a hormone is a ligand. When bound to a receptor, it triggers a stepwise cellular response, causing cell growth or other reactions. As used in this specification, the term "hybridization reaction" means the joining of two single complementary DNA strands (hybridization reaction of DNA / DNA) ) , Or the joining of complementary DNA and RNA strands (hybrid reaction of DNA / RNA). "Analyte" means a substance that is present in the patient's blood or body fluids. The concentration of the analyte generally varies with metabolic or pathological conditions. It is changed and it is information for clinicians to manage patients' health. "Antigen" means a substance with a molecular surface structure that triggers an immune response substance, that is, the production of antibodies, and / or specific antibodies Reaction (antigen / antibody reaction). An "antibody" is a protein (immunoglobulin)) that recognizes and binds to an antigen as part of an immune response. "Molecular probe" means any nucleic acid, protein molecule, or other molecule, Has the ability to specifically bind to other molecules of the same or different type. In general, nucleic acids specifically bind to nucleic acids that exhibit sequence complementarity. Therefore, a probe (in this example, a nucleic acid molecule) having the following sequence-AGGCGTA (from the 5 'end to the 3' end)-specifically binds to another strand of DNA, which contains The region of the following sequence TACGCCT (from the 5 'end to the 3' end), where A, T, G, and C represent adenine, thymine, guanine, and cytosine, respectively. Antibodies to primary antibodies can be used as molecular probes for this antigen. -9- (Please read the notes on the back before filling in this page) r line | This paper size is applicable to Chinese National Standard (CNS) A4 (210X297 mm) 1245895 A7 B7 V. Description of the invention (7) "Antigen determining site "" Means the portion of an antigen molecule that binds to an antibody. An antigen can have many different epitopes, which can bind to different antibodies. Brief description of the drawings The preferred form of the present invention will be described by way of examples with reference to the following drawings, where: Figure 1 is a schematic diagram of a circuit applied to a test device, and Figure 2 is a diagram showing two Overview of attached probe and antibody-attached wafers' Figure 3 depicts a schematic of a second probe with bound iron beads added, Figure 4 depicts a schematic of a possible microarray design, and Figure 5 Figure depicts the principle behind a certain amount of analyte method. Figure 6 depicts a schematic diagram for quantifying an analyte using a microarray containing pre-bound beads with the target sandwiched between probes. Figure 7 is a schematic diagram describing the principle of another method for quantifying an analyte. This method uses a competitive combination rather than a pre-bound bead. Figure 8 is a summary of the method for quantifying a second analyte. This method uses a microarray in the presence of excess analytes. Figure 9 is a schematic diagram describing the design of a two-probe for the overlapping region of Huntingtin gene. Figure 10 is a description Another group An overview of the probe. The number of repeat combinations for this probe is less than the correct number of repeats present in a given patient. There is no bonding effect on the two ends of the probe, and -10- (Please read the precautions on the back before filling this page) This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) 1245895 A7 B7 V. Description of the Invention (8) Figure 11 is a schematic diagram describing another set of probes. The number of repeated combinations of the probes is more than the correct number of repeats in a given patient, causing overhanging probes and joints. Failure of function. Description of the preferred embodiment In the first figure, the basic unit comprises a wire 1, a switch 2, a battery 3, an ammeter, a light bulb or a microprocessor 4, and a reaction "well" 5, which contains two electrodes 6 And a small gap 7 between the electrodes. The circuit was originally open, but when the iron beads were located between the electrodes, then the switch 2 was turned on to complete the circuit and cause current, which can be detected by the ammeter 4. In Figure 2, the wafer 8 is a reaction "well" plus the electrode mentioned in Figure 1. In the center of this wafer is a "well" 5, which may be a little recessed, with a covalently bound molecular probe 9 on the wall, which is specific to the molecule (target) to be found / analyzed. The needle can be a nucleic acid (left panel) or an antibody (right panel). Two electrodes 6 are opposite to any wall of the "well". When manufacturing multiple wafers in a microarray, the "well" does not have to be recessed (See Figure 4.) The advantage of having a flat "well" is that at the end of the reaction, it is easier to remove unbound excess iron-bound probes (described in Figure 3) by applying a magnetic field. In Fig. 3, for example, some conductive beads of iron beads 10 are located between two electrodes 6 and are bound by sandwiched target molecules 11 which are attached to the first probe of the binding "well". Needle 9 and a second probe 12 bound to iron beads. In the left panel, this probe is a nucleic acid molecule, which can identify different parts of the target nucleic acid. In the right panel, the probe is an antibody, which is used for the analyte Specific regions (antigenic regions) are specific, usually protein In Figure 4, the chip is a miniaturized 8 and each is designed to detect -11- (Please read the precautions on the back before filling this page) This paper size applies to China National Standard (CNS) A4 specifications (210X297 mm) 1245895 A7 " " " " " " '" ------ B7 V. Description of Invention (9) ~ --- Same as: /, his specific molecule. Export And the inlet can introduce agents and samples to the wafer. In this method, the 'microprocessor has computing power, and multiple agents / molecules can be measured in a portable device at the same time (multiple) debt. The tube g13 is disposable and can be replaced with another = the same to determine different groups of molecules. Two or three repetitions can be embedded for pre-failure af ° and ‘pre-test with negative control and post-test with positive control (if the test result is negative) to ensure the test performance. In Figure 5, the analyte is tied to the test, well "5", sandwiched between two probes. One of the probes 9 is bound to, the well, and the other 12 is bound. To the iron beads 10, the iron beads are in contact with the electrode 6. Adding a test sample containing a sufficient concentration of the analyte causes the competitive binding and substitution of the second probe attached to the iron beads, thus blocking this Circuit (closed). In Figure 6, the analytes and iron beads sandwiched between the electrodes are gradually changed in a series of arrayed wafers, allowing the detection of unknown concentrations as explained below. In Figure 7, the pre-bound beads were not used. The amount of beads-bound probes was less than that of the analytes. This result is a combination of the analyte-bound particle probe 15 and the free Analyte 11 competes with limited binding, wells, and needles 9. The gap between the electrodes is reduced to accommodate only one bead. The left panel shows the bindings that are completely occupied by the analytes, wells, and probes. , Without the attachment of the bead-bound probe (closed). Right The panel shows bead-bound probes, which bind to the analyte molecules attached to the bound, well, and probe (on). In Figure 8, a microarray with thousands of such wafers is recorded, The format of the paper is applicable to the Chinese National Standard (CNS) A4 specification (210X297 public love) (Please read the precautions on the back before filling this page), \ Tan · -12- 1245895 A7 B7 V. Description of the invention (1G) Kai " The total number of signals is the result of binding of the analyte-binding particle-bound probe to the "well" -bound probe. A "closed" signal is recorded on a wafer that is bound to a "well" probe by a free analyte that is not attached to a bead-bound probe. The ratio of the "on" to "off" signals depends on the relative concentration of the probe and free analyte bound to the analyte and the bound particles. The enlarged view of the cartridge 13 shows that some wafers are open, while some wafers are closed. The known molar concentration of the bead-bound probe allows calculation of the analyte concentration. In Figure 9, the principle of measuring the number of CAG repeats in Huntington's disease is illustrated. A description of this method can be found in Example 9. The bead-bound probe 17 is designed to have the following sequence in the probe detection area: CTGGAAGGA, and the probe detection area of the "well" probe 18 has the following sequence: J: GGTGGCGGCTGTTGCTGCTGCTG. In the figure, the upper strand 19 is the target gene with four sets of “CAG” repetitions present in this particular patient. Only this region that is complementary to the probe is described as having the actual nucleic acid sequence. The other endpoints (5, 20 and 3 '21) are indicated by arrows. The hybrid binding beads 17 and the "well" probe 18 are described in the same manner as previously described. The underlined portion of the probe (above the figure) indicates the unrepeated portion of the adjacent repeating region. In the figure, these two probes have a combined number of repetitions corresponding to the target (4 correct). Therefore, the 5 'and 3' ends of the two probes are grouped together and can be ligated by an enzyme (DNA ligase). In Figure 10, only the sequence of a different "well" -bound probe 22 is explained because the beads-bound probes are the same. The "well" probe 22 is placed in another electrobiochip in the same microarray. The “well” probe 22 described here has only two repetitions. When hybridizing with the target, the paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) -13-(Please read the Note for this page, please fill in this page again)-口 _ r 线 丨 1245895 A7 B7 V. Description of the invention (11) will create a gap between the two probes. These two probes cannot be joined. In Fig. 11, another probe "23" that incorporates "wells" has too many repetitions (5). Therefore, the excess portion will have "overhangs" 24 after hybridization. It also has no bonding effect. Obtaining the sample allows the air containing the putative target to foam through the appropriate solute. Solid or liquid can be dissolved in solution. Requires intact cells and tissues to be ruptured, opened or prepared to release the molecules to be tested. Device (Electro-Biochip) In its simplest design, this electro-biochip consists of a small test "well" containing two wires inside it with a small gap between the two wires. A molecular probe is bound to the gap, and the probe is specific to the putative target. The other element is a second specific probe, which is bound to the beads of the electrical conductor. Reaction Add the sample to the wafer. Add a second probe (before, during, or after adding the sample) in excess of the target amount. Allow the reaction to occur. After the reaction, the unbound probe and iron beads are sucked away by generating an appropriate magnetic field. Finally, record the results. Quantify the width of the gap between the two electrodes and the amount of the "well" probe, and therefore the sandwiched "well" probe / target / bead-bound probe is designed on the opposite side of the wafer ( Record “on” signal instead of recording “off” signal) The array can be changed to measure the amount of target in the sample to be measured. Please read the precautions on the back before filling out this page), τ. First Line-1245895 A7 B7
發明説明（12 (競爭)）。含有目標物的待測樣本的添加只會 之棟針的競泰糾έ士人A & 、、5珠粒 ^1±、纟。合及取代Ί含有超過特定主的、、Ό〇井”之探針/目標物/結合珠粒探針維 啟，，乂他的晶片是，，關閉”的，因為藉由力…析物與結二其 之才木針的競爭性結合及取代。湘已知的標準品= 正’允許待測樣本的正確定量。 又 、旦另一種定量的方法涉及在具有數千個晶片❸微陣列中 測里，先韵關閉”的晶片（無具有經夾住之目標物（在二探 針之間）的預先結合之珠粒），其在超過結合珠粒之探針2 未知》辰度分析物的存在下，會被，，開啟，，。佥子探針的事诰 首先製造一對具有專一性的分子探針。核酸探針可利 用已知之目標物的序列來建構。這些序列的資訊通常可以 在例如 Entre-Genome (National Center for Biotechnology Information，National Library of Medicine，National Institutes of Health，US A)之資料庫中找到。具有與目標物 之二端序列互補的探針可商業化地合成。此外，探針可庆 下述方式設計：當與目標物雜交作用時，使探針的二端具 有物理近似性，以致於DNA接合酶（共價結合經集合之 DNA股的酶）可以接合該二端，以供加強鐵珠粒及”井”之間 的結合。抗體可以由暴露在抗原下之實驗室動物來生產。因 如此，抗原的來源是需要的。單一電生物晶片的佈局 為 (請先閲讀背面之注意事項再填寫本頁) 、^τ— ▼線- 本紙張尺度適用中國國家標準（™s) Α4規格（210X297公爱） -15 - 1245895 A7 B7 五、發明説明（13 ) 核酸探針或抗體可以共價結合到不同的物質上。第一 抗體沿著界於一個開啟電路之二端間的小間隙，結合在容 器的壁上（第1及2圖）。該二個電極藉由晶片上的導線連接 至微處理器，該微處理器形成及/或監測其餘的電路。第二 專一性探針藉由相似的技術與小的游離鐵珠粒結合。當目 標物存在及在恰當的條件下，該目標物結合及夾在二探針 間（第3圖）。藉由第一探針的位置，使結合至第二探針的該 鐵珠粒與電極接觸並關閉電路，允許當電池提供電位差 時，通過電流。目標物的濃度範圍很廣，從單一分子到如 同結合珠粒之探針一般多，提供設計之健全性。然而，靈 敏度無法被妥協。此裝配在理論上可偵測單一分子之存 在。此外，當用於偵測特異核酸序列時，無需先前的放大 作用。此方法係多用途的且可用於偵測DNA、RNA、蛋白 質及其他大分子。 微陣列的製造 多個單一晶片可以製造成微陣列（第4圖）。這些個別的 晶片可製造成偵測不同的分子。相同晶片的二重複或三重 複可製造在同一微陣列上，以達品質保證的目的。 具有經結合之鐵珠粒的第二抗體收容在含有微陣列的 卡匣中。卡匣中係設計成稍真空（其具有可膨脹一些的部分 以供容納更多的樣本），為了吸入預定體積的樣本。該卡匣 的進/出口容許將樣本引入卡匣中。 讀取裝置 一個可攜帶的裝置建構成有一可容納卡匣的溝槽。插 本紙張尺度適用中國國家標準（CNS) A4規格（210X297公釐） -16- (請先閲讀背面之注意事項再填寫本頁)Invention description (12 (competition)). The addition of the sample to be tested containing the target object will only increase the number of jewelers A &, 5 beads ^ 1 ±, 纟. Combining and replacing the probes / targets / binding beads probes that contain more than a specific master, and the wells are “open,” “his chip is, close”, because by force ... Competitive combination and replacement of talents. The standard known in Hunan = positive ’allows the correct quantification of the sample to be measured. Moreover, another quantitative method involves measuring in a microarray with thousands of wafers, and the first rhyme is turned off. (There is no pre-bound bead with a clamped target (between two probes). Particles), in the presence of more than 2 probes that are bound to the beads, the unknown analytes, will be ,, open ,,, etc. The matter of the 佥 子 探针 produce a pair of molecular probes with specificity first. Nucleic acid probes can be constructed using sequences of known targets. Information about these sequences can often be found in databases such as Entre-Genome (National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, US A) .Probes with complementary sequences to the two ends of the target can be synthesized commercially. In addition, the probe can be designed in such a way that when hybridized with the target, the two ends of the probe have physical similarity so that DNA ligase (an enzyme that covalently binds the aggregated DNA strands) can join the two ends to enhance the binding between the iron beads and the "well". The antibody can be exposed to the antigen Produced by laboratory animals. Because of this, the source of the antigen is needed. The layout of a single electrobiochip is (please read the precautions on the back before filling this page), ^ τ— ▼ line-This paper size applies to Chinese national standards (™ s) Α4 size (210X297 public love) -15-1245895 A7 B7 V. Description of the invention (13) Nucleic acid probes or antibodies can be covalently bound to different substances. The first antibody is bounded by an open circuit. The small gap between the two ends is bonded to the wall of the container (Figures 1 and 2). The two electrodes are connected to the microprocessor by wires on the chip, which form and / or monitor the remaining circuits. The second specificity probe is bound to small free iron beads by similar techniques. When the target is present and under proper conditions, the target binds and is sandwiched between the two probes (Figure 3). The position of the first probe brings the iron beads bound to the second probe into contact with the electrode and closes the circuit, allowing current to be passed when the battery provides a potential difference. The concentration of the target ranges from a single molecule to binding Of beads There are usually many needles, providing robustness of the design. However, sensitivity cannot be compromised. This assembly can theoretically detect the presence of a single molecule. In addition, when used to detect specific nucleic acid sequences, no previous amplification is required. This method It is versatile and can be used to detect DNA, RNA, proteins and other large molecules. Manufacturing of microarrays Multiple single wafers can be manufactured into microarrays (Figure 4). These individual wafers can be manufactured to detect different molecules . Two or three duplicates of the same wafer can be manufactured on the same microarray for quality assurance purposes. A secondary antibody with bound iron beads is housed in a cassette containing the microarray. The cartridge is designed to be slightly vacuumed (it has a section that can be expanded to accommodate more samples) in order to suck in a predetermined volume of sample. The cassette entry / exit allows samples to be introduced into the cassette. Reading device A portable device is constructed with a slot for receiving a cassette. The dimensions of this paper are applicable to Chinese National Standard (CNS) A4 (210X297 mm) -16- (Please read the precautions on the back before filling this page)
1245895 A7 B7_____ 五、發明説明（14 ) 入该卡E ’經由該讀取裝置之電子零件，連接多個位於卡 E底部（或旁邊）的小電路（第*圖）與微處理器上。該微處理 恭言買取卡£中的内容，係由一個獨特的鑑別器如卡匣底部 的條碼及將其本身的程式，以供解釋開啟/關閉的訊號並 以生物製劑”方式顯示結果或呈報分析物的濃度。 因此’為”讀取”結果，該卡匣被***此裝置中。微陣 列的電路接著與收容在讀取裝置中的微處理器之電子零件 相接觸。該微處理器使反應溫度最適化、計時反應，及控 制微小電磁圈的陣列，該電磁圈產生一個變化的磁場以供 達成溫和攪拌反應物之效果來促進反應。在反應終點，該 微處理器產生一垂直（相對於電極的軸）的磁場，其可將未 反應的結合鐵珠粒之探針吸走以去除偽訊號。該電極應該 以銅或其他導電性的金屬製造，其不會被磁場所感應。該 心支處理器接著解來自微陣列的個別晶片上記錄之開啟/關 閉訊號，及產生結果，以顯示在液晶銀幕（LCD)上的文字， 點字法或合成的聲音之方式。控制面板上的按鈕，允許操 作員操縱該指令單及依所需實行不同功能。該結果也可以 藉由收音機傳遞到遠處或以預先建構的印表機印出。 分子分折 體液中已知之生物分子通常需要濃度分析。一例子為 曱狀腺中毒或曱狀腺機能衰退中的曱狀腺贺爾蒙分析。 本發明之理論容許照顧點（p〇int-〇f_care)分析，其係 藉由在床側或在臨床上，利用體液或血液的微:樣本^適 時方式進行，且毋需使用佔空間的複雜機械。晶片（基本 本紙張尺度適用中國國家標準（CNS) A4规格（210X297公釐） (請先閲讀背面之注意事項再填寫本頁) 、言 -線丨 -17- 1245895 A71245895 A7 B7_____ V. Description of the invention (14) Enter the card E 'through the electronic parts of the reading device, and connect a plurality of small circuits (pictured *) at the bottom (or next to) the card E with the microprocessor. This microprocessing congratulations to buy the contents of the card is made by a unique discriminator such as the bar code at the bottom of the cassette and its own program for interpreting the on / off signal and displaying the result or report in the form of biological agent " The concentration of the analyte. Therefore the cartridge is inserted into the device 'for a' read 'result. The circuit of the microarray is then in contact with the electronic parts of the microprocessor housed in the reading device. The microprocessor enables Optimize the reaction temperature, time the reaction, and control the array of tiny electromagnetic coils that generate a changing magnetic field for the effect of gently stirring the reactants to promote the reaction. At the end of the reaction, the microprocessor generates a vertical (relative to (The axis of the electrode), which can remove unreacted probes that bind iron beads to remove false signals. The electrode should be made of copper or other conductive metal, which will not be induced by the magnetic field. The The heart support processor then decodes the on / off signals recorded on individual chips from the microarray and generates the results to display text, braille on the liquid crystal screen (LCD) Or synthesizing the sound. The buttons on the control panel allow the operator to manipulate the instruction sheet and perform different functions as needed. The results can also be transmitted remotely via the radio or printed on a pre-built printer The molecular analysis of known biomolecules in body fluids usually requires concentration analysis. An example is the analysis of glandular hormones in glandular poisoning or glandular function decline. The theory of the present invention allows points of care (pint- 〇f_care) analysis, which is performed on the side of the bed or clinically by using micro: sample ^ of body fluids or blood, in a timely manner, without the need to use space-consuming complex machinery. Wafers (basically this paper size applies Chinese national standards (CNS) A4 specification (210X297 mm) (Please read the precautions on the back before filling this page), Word-line 丨 -17- 1245895 A7
士二）,係设汁成攜帶夹住之分析物，其I一性㈤附著至結 ^ 井之彳木針以及結合鐵珠粒之探針，其量足以封閉二 在此結構巾，除非結合鐵珠粒之探針被 取代，否則電路總是“開啟，，。 、S足夠濃度之未結全分析物添加至晶片中時，結合至 鐵珠粒之抵針可被取代。此可達到係因未結合之分析物與 經結合之分析物，競爭該結合鐵珠粒之探針或結合“井” 之探針，造成預先定位之電路之鐵珠粒被取代。現在結果 為一開放電路（關閉）。 可製造一微陣列，在該微陣列上為多數晶片，各自在 二電極之間的寬度，以及排列之經夾住的分析物/結合鐵 珠粒之探針的量上稍微改變。未知量之分析物的添加造成 一些結合鐵珠粒之探針被取代。雖然此不會影響具有較寬 間隙及更多結合鐵珠粒之探針（維持在“開啟”）的晶片、， 但具有較少間隙及較小數目之結合鐵珠粒之探針的^曰片將 關閉。界於一系列“開啟”晶片及一系列“關閉，，晶片之 間的位置，產生試驗材料中分析物之濃度的正確評估。經 取代的珠粒在讀取前，藉由磁場吸走。要求利用具有已: 濃度之標準品預先校正。因為核酸目標物在實驗室中 成容易度，當測量核酸時，此方法是令人滿咅的。、口 當為了製造預先之電生物晶片來純化或合成分 (例如蛋白質或其他大分子）是太困難或太昂貴時， 能利用本發明來分析體液或血液中一分析物的、農产 製造含有數千個電生物晶片的微陣列，各二人 結合井之探針。此等探針係同樣地為抗體，但亦可 核酸（特別當分析病毒裝填物時）。製造結合至導電H 粒之不同探針。在分析物存在下，使用較小量（莫耳對莫JII), the system is designed to carry the clamped analyte, and its I is attached to the knot of the wood needle and the probe bound to the iron beads, and the amount is sufficient to seal the two in this structure, unless bound The iron bead probe is replaced, otherwise the circuit is always “on.” When the full analyte of sufficient concentration is added to the wafer, the pin that is bound to the iron bead can be replaced. This can be achieved Because the unbound analyte and the bound analyte compete for the iron bead-bound probe or the "well" probe, the pre-positioned iron bead is replaced. Now the result is an open circuit ( Close). A microarray can be fabricated with a large number of wafers on it, each slightly varying in width between the two electrodes, and in the amount of arrayed clamped analytes / probes that bind iron beads. The addition of an unknown amount of analyte caused some of the probes to bind to the iron beads to be replaced. Although this will not affect wafers with wider gaps and more probes that are bound to the iron beads (maintained "on"), but With fewer gaps and smaller numbers of knots ^ Probe iron beads of said sheet closes. Boundary in a series of "on" and the wafer number ",, between a closed position of the wafer, the test material to produce the correct analysis of the assessment of the concentration of analyte. The substituted beads are sucked away by a magnetic field before reading. Requires pre-calibration using standards with an already: concentration. Because nucleic acid targets are easily accessible in the laboratory, this method is very satisfying when measuring nucleic acids. When it is too difficult or expensive to purify or synthesize components (such as proteins or other macromolecules) in order to manufacture a prior electrobiochip, the present invention can be used to analyze an analyte in body fluids or blood. Thousands of microarrays of electrobiochips, two of them combined with well probes. These probes are similarly antibodies, but they can also be nucleic acids (especially when analyzing viral loads). Fabricate different probes bound to conductive H particles. Use smaller amounts in the presence of the analyte (Moore vs Mo
本紙張尺度適用中國國家標準（CNS) Α4規格 1245895 A7 _______B7__ 五、發明説明（16 ) ^ 耳）之第二結合珠粒之探針。藉由競爭數千個但數量相 上仍遠不及（與游離及結合至結合珠粒之探針的分析物相 比較）的晶片，以及利用已知量之結合珠粒之探針，分^ 物之濃度可以藉由微處理器測量之方式，藉由開啟曰 曰曰 的比率來計算。在微陣列中必須有多數的電生物晶片以供 獲得準確的結果。亦製造晶片以致僅接受一導電性珠粒來 增進準確性。為了不擾亂動力學平衡，未附著至結合“井，， 之探針的結合珠粒之探針不藉由磁場吸走。進行一系列的 測量並平均以得到最後結果。 實施例 實施例1 結核桿菌（Mycobacterium tuberculosis )(造成結核 病）之核糖核蛋白體核糖核酸（rRNA) 在AMPLIFIED™ 結核桿菌直接試驗（Mycobacterium Tuberculosis Direct Test)中為偵測目標物（參考文獻ι-ίο)。 雖然在上述試驗中要求放大作用（轉錄中介之放大作 用（TMA))，本發明之方法僅需要破裂細菌細胞壁之簡單 方法來釋出rRNA。 利用本發明之電生物晶片，可偵測到少至如一份複製 物的量。不需要預先的放大作用。 實施例2 急性心肌梗塞（心臟病）之適時實驗室診斷具有潛在 救命特性，因為可設置治療干涉。此等干涉本身並非無危 險性且指令一精確試驗。 直至目前，試驗不是敏感度不夠，就是非專一性。仃 如，最早的心肌梗塞指示劑為血漿肌血球素之上升，其可 在梗塞後6小時可偵測（參考文獻11-14)。然而，肌血球 -19- f請先閱讀背面之注意事項再填寫本頁) 本紙張尺度適用中國國家標準（CNS) A4規格（210X297公釐） 1245895 A7 ____B7______ 五、發明説明（17 ) 素亦存在於骨骼肌中且其上升對於心肌傷害並非專一性 的，故要求血漿肌鈣蛋白之第二分析的確認 (http://demapoc.mah.roche.eom/content/products/c read/c _read.htm)，其為比肌血球素慢上升的標誌物。 在急性心肌梗塞之最早階段偵測微量心臟肌鈣蛋白T 之能力同時要求專一性及敏感性。現在使用我們的電生物 晶片使此要求變為可能。 在此應用中，係運用如上述“定量”部分中描述之競 爭性結合的原理。分析物為心臟肌鈣蛋白τ以及二探針為 升高以抗心臟肌鈣蛋白τ之抗體。抗體將偏妤地結合至心 臟肌鈣蛋白T上，二不相同的抗原決定部位，且彼此不互 相干擾（抗原決定部位不會太靠近以致於干擾二抗體之結 合）。除了可以偵測先前技術無法偵測之急性心肌梗塞發 作早期的循環心臟肌鈣蛋白τ量之外（其可能在急性心肌 梗塞發作後，在遠短於6小時之時間内即存在）’此應用 亦容許定量此蛋白質之血漿濃度（藉由已知濃度之心臟肌 鈣蛋白τ的連續稀釋液，可立即達到儀器之校炎）。 實施例3 在科學研究中，科學家通常需要研究基因表現。在多 細胞有機體中，帶有相同組基因的細胞係特定化以供承擔 許多身體的功能，例如覆蓋體表面（外皮或皮膚）、吸收 流體以及電解質（腸）以及與外部世界交互作用（神經系 統）。結果’細胞需要表現於此等特定化細胞内的工具’ 該特定化細胞未表現於其他分化的細胞。直i目前，基因 表現係藉由螢光探針或其他方法，個別地或應用最近常被 討論的微陣列來進行，該微陣列係經印刷或附著分子探 針，該探針可與信使RNA雜交並產生存在或不存在之定性 -20- (請先閱讀背面之注意事項再填寫本頁) 本紙張尺度適用中國國家標準（CNS) A4規格（210X297公釐） 18 !245895 五 、發明説明 …果。不用說，此等微陣列不具有定量mRNA的能力， mRNA可在低量下表現。低量表現亦為重要的，因為我們 $知道低量表現必須與產生極少量蛋白質同義，因為細胞 蛋白質之濃度是動態的且代表生產及破壞之間的平衡。 利用本發明，mRNA之定量及對應之蛋白質是簡 =，如上述“定量”部分所描述者。 貫施例4 大變偵測為發現特定疾病之方法，其係在生殖細胞中 代代相傳（遺傳性）。 藉由以基因區域之放大作用為基礎的習知方法，進行 基因中單點突變（有時候非常大）的偵測會有大片段無法 放大的缺點，並因此限制經濟地及系統地研究一特定人之 基因的點突變之能力。 利用本發明，成對的探針（結合“井”及結合珠粒） 可設計成具有已知序列的未突變基因（在公眾資料庫發現 的）。 成對之採針係設計為序列之連續伸長互補。已研究依 需‘要，設計許多對以覆蓋基因之整個長度，且不同之結合 之探針附著至個別的電生物晶片，可探查之區域 2在卡II之獨特鑑別器中。以此方式及利用裝置之特殊 :構’使得其可再使用’可進行基因突變之快速、經濟且 糸統的研究。 實施例 許多癌症係藉由-基因之一部分的移位至另一基因或 ^另-基因内（嵌合性基因）。例子係多到無法列出且包 3曱狀腺之濾泡癌、特定急性骨趙白血病’許多軟組織肉 瘤，例如滑液肉瘤以及外骨骼黏液軟骨肉瘤。 本紙張尺度適用中國國家標準（CNS) A4規格（210X297公爱） -21 - 1245895 A7 B7 五、發明説明（19 ) 雖然此等嵌合性基因之mRNA轉錄物（嵌合性轉錄物） 之鑑別，可容易地藉由習知用於放大之聚合酶鏈反應以及 以經放大之產物為基礎之用於鑑定的電泳來進行，此方法 是慢且費力的。 利用本發明，可製得雜交至嵌合性轉錄物之二成分的 一對探針。一探針為結合至“井”之探針以及另一探針為 結合至導電性珠粒之探針。因此可達到正面的鑑別，即使 係利用藉由微細的針自腫瘤或血液（若腫瘤為白血病或在 疾病之早期或晚期可容易進入血流者）取出之微量樣品。 許多不同的癌症在此方式中可同時篩檢出來。 實施例6 許多疾病具有病毒病因。一例子為HIV (由人類免疫 不全病毒所感染）。雖然此疾病可藉由抗病毒劑來控制， 但該等藥劑非常昂貴。因為病毒容易突變，在不同時期下， 並非所有病患對相同藥劑皆有反應。監測病毒負荷為決定 藥物效力及疾病狀態的一方法。 利用此方法，使病毒負荷研究高度準確、簡單、快速 以及便宜。 先前已描述過分析的原理。倘若基因序列已知的話， 利用此方法可研究任何病毒。 實施例7 許多感染性疾病具有類似的表癥。例如炭疽病、流行 性感冒、登革熱、天花、一般感冒、玫瑰疹等，所具有之 最初表癥包含違和（一般感覺不舒服）、發燒、肌肉痛以 及非特定性發疹。為了使主治醫師可準確地鑑定此等具有 類似表癥但結果有極大不同之多數疾病，本發明可適用於 在臨床上、經濟地且快速地進行感染作用劑之整個面板微 -22- (請先閲讀背面之注意事項再填寫本頁) 本紙張尺度適用中國國家標準（CNS) A4規格（210X297公釐） 1245895 A7 B7 五、發明説明 量偵測（因此在早期）。許多病患甚至不需要住院觀察且 可因病因之正面性鑑別結果而被送回家，同時節省金錢及 避免對病人造成危險（若其等需要在可能隱藏有害微生物 之醫院中被看護）。 f施例8 監測飲用水及食品（或動物飼料）之有害物質，例如 狂牛病作用劑（牛海綿狀腦病變（BSE))係受到試驗之高成 本及低敏感性的阻礙（參考文獻15-20)。例如，具有bse 之牛在腦液（腦脊髓液）中具有微量之作用物。敏感性試 驗不僅可早期债測疾病，且可免於大屠殺動物。因為食用 生病的牛而感染到此疾病的人類亦可彳貞測。利用其他研究 者發現的專一性抗體，本發明可大幅度地削減成本及時間 來鑑別致病作用物。 實施例8 許多遺傳性疾病係由於隱匿重覆序列之特定區域過長 所造成。例如杭廷頓氏病（Huntington’s disease)，一種 不變的致命疾病，係因為位於染色體4中的亨丁頓氏基因 (Huntingtin gene )中，存在之“CAG”重覆超過36倍 (http://www.ncbi.nlm.nih.gQv/entrez/dispomim>cgi?id:::=141 100) 〇 為了測量人亨丁頓氏基因（Huntingtin gene )中重覆 的數目，習用方法係應用PCR。 本發明簡化重覆之數目的測量。因此，探針係設計成 鄰接鄰近重覆序列之基因的不變部分 (http://www.ncbi.nlm.iiih.gov/entrez/cuierv.fcgi?cmd=Retri eve&db=nucleotide&list uids=450395&dopt=GenBank) 〇 除了鄰近重覆之不變序列之外，結合“井”之探針攜 -23 - (、請先閱讀背面之注意事項再填寫本頁} 本紙張尺度適用中國國家標準（CNS) A4規格（210X297公釐） 1245895 A7 B7 五、發明説明（21 ) 帶不同之重覆的數目。結合珠粒之探針攜帶在重覆之相對 側上的不變區域且為一或二重覆。具有特定數目之重覆的 人，例如5 “CAG”重覆，若結合井之探針含有4重覆且結 合珠粒之探針含有1重覆，當雜交時，將具有二探針完美 地端對端排列。其他測量超過4 (例如5或以上）或小於 4 (例如3或以下）之含有結合“井”之探針的井，將雜 交但將不會產生完美的端部排列。利用DNA接合酶（共價 地結合二DNA股，該等股係排列在互補股上且端部緊密相 鄰），此二探針可共價地接合。具有超過5之組合數目之 重覆的探針將具有懸突股，其無法與另一股接合且具有低 於5之組合重覆的探針亦同，因為在二探針之間有大間 隙。於雜交及接合反應後，反應條件的調整造成探針及目 標物分離（變性作用），造成具有其他非4CAG重覆之結 合“井”之探針的電生物晶片的“關閉”。 結果是人亨丁頓氏基因（Huntingtin gene)中，CAG 重覆的數目之快速且準確的測量。本發明之方法適用於任 何種類之具有改變之重覆數目的基因性疾病。 -24- (請先閱讀背面之注意事項再填寫本頁) 本紙張尺度適用中國國家標準（CNS) A4規格（210X297公釐） 1245895 A7 B7 五、發明説明（22 ) 參考文獻： 1. Behr? M.A. S.A. Warren, H. Salamon, P.C. Hopewell, A. Ponce de Leon, C.L. Daley- and P.M. Small. 1999 Transmission of Mycobacterium tuberculosis from patients smear-negative for acid-fast bacilli. Lancet 353:444-449. 2. Bermann, J.S G. Yuoh5 G. Fish and G.L. Woods. 1999 Clinical evaluation of the enhanced Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test for rapid diagnosis of tuberculosis in prison inmates. J Clin. Microbiol. 37:1419-1425· 3. Bermann，J.S and G.L. Woods. 1999 Enhanced mycobacterium tuberculosis direct test for detection of M. tuberculosis complex in positive ESP II broth cultures of nonrespiratory specimens Diag. Microbiol. Infect. Dis. 35:245-248. 4. Chedore, P. and F.B. Jamieson. 1999. Routine use of the Gen-Probe MTD2 amplification test for detection of Mycobacterium tuberculosis in clinical specimens in a large public health mycobacteriology laboratory Diag. Microbiol. Infect. Dis. 35: 185-191. 5. Della-Latta， P and S. Whittier 1998. Comprehensive evaluation of performance, laboratory application， and clinical usefulness of two direct -25- (請先閲讀背面之注意事項再填寫本頁) 本紙張尺度適用中國國家標準（CNS) A4規格（210X297公釐） 1245895 A7 _B7_ 五、發明説明（23 ) amplification technologies for the detection of Mycobacterium tuberculosis complex. Am. J Clin. Pathol. 110:301-310. 6. Della-Latta，P and Vivian Jonas. 1999. Inhibitory effect of Alpha-Tec XPR-Plus Phosphate Buffer on the enhanced Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test. J Clin. Microbiol 37:1234-1235. 7. Gamboa，F C Fernandez，E. Padilla，J.M. Manterola，J lonca，P J Cardona，L. Matas，and V Ausina. 1998. Comparative Evaluation of initial and new versions of the Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test for direct detection of Mycobacterium tuberculosis in respiratory and nonrespiratory specimens. J Clin. Microbiol. 36:684-689. 8. Moore， D.F and Curry. J I 1999. Reduction in turnaround time for detection and identification of Mycobacterium tuberculosis from pulmonary specimens using nucleic acid amplification tests Presented at the 99th General Meeting of the American Society of Microbiology Chicago, Illinois. 9. Piersimoni，C. A. Callegaro，C. Scarparo，V Penati，D· Nista, S. Bornigia, C. Lacchini, M. Scagnelli, G. Santini, and G. De Sio. 1998 Comparative evaluation of the new -26 - (請先閲讀背面之注意事項再填寫本頁) 本紙張尺度適用中國國家標準（CNS) A4規格（210X297公釐） 1245895 A7 B7 五、發明説明（24 )The size of this paper applies to China National Standard (CNS) A4 specification 1245895 A7 _______B7__ V. Description of the invention (16) ^ ear) The second bead-bound probe. Fragments are separated by competing for thousands of wafers that are still far behind in number (compared to analytes that are free and bound to bead-bound probes), and using known amounts of bead-bound probes. The concentration can be calculated by the microprocessor and by the ratio of the opening date. There must be a large number of electrobiochips in a microarray for accurate results. Wafers are also manufactured so as to accept only one conductive bead to increase accuracy. In order not to disturb the kinetic equilibrium, the bead-bound probes that are not attached to the bound "wells" are not sucked away by the magnetic field. A series of measurements were performed and averaged to obtain the final results. Example Example 1 Nodules The ribonucleoprotein ribonucleic acid (rRNA) of Mycobacterium tuberculosis (causing tuberculosis) is the target of detection in the AMPLIFIED ™ Mycobacterium Tuberculosis Direct Test (Reference ι-ίο). Amplification is required (Transcriptional Amplification (TMA)). The method of the present invention only requires a simple method to rupture the bacterial cell wall to release rRNA. Using the electrobiochip of the present invention, as few as one copy can be detected No prior magnification is required. Example 2 A timely laboratory diagnosis of acute myocardial infarction (heart disease) has potential life-saving characteristics because therapeutic interventions can be set up. Such interventions are not inherently non-hazardous and require an accurate test. Until now, the tests have either been insufficiently sensitive or non-specific. For example, the earliest myocardium The plug indicator is an increase in plasma myoglobin, which can be detected 6 hours after infarction (References 11-14). However, myoblasts-19-f (please read the precautions on the back before filling this page) Paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm) 1245895 A7 ____B7______ V. Description of the invention (17) The element also exists in skeletal muscle and its rise is not specific for myocardial injury, so plasma troponin is required Confirmation of the second analysis (http: //demapoc.mah.roche.eom/content/products/c read / c_read.htm), which is a marker of slower rise than myosin. Earliest in acute myocardial infarction The ability to detect trace cardiac troponin T at the same time requires both specificity and sensitivity. This requirement is now made possible with our electrobiochip. In this application, competition as described in the "Quantitative" section above is used The principle of sexual binding. The analyte is cardiac troponin τ and the second probe is an antibody raised against cardiac troponin τ. The antibody will preferentially bind to cardiac troponin T. Two different antigens determine And do not interfere with each other (the epitopes are not too close to interfere with the binding of the secondary antibodies). In addition to detecting the circulating cardiac troponin τ in the early stages of acute myocardial infarction that cannot be detected by the prior art ( It may exist after the onset of acute myocardial infarction in a time period much shorter than 6 hours) 'This application also allows the quantification of the plasma concentration of this protein (through serial dilutions of cardiac troponin τ of known concentration, Reach the inflammation of the instrument immediately.) Example 3 In scientific research, scientists usually need to study gene performance. In multicellular organisms, cell lines with the same set of genes are specialized to perform many body functions, such as covering the surface of the body (the outer skin or skin), absorbing fluids and electrolytes (the intestine), and interacting with the outside world (the nervous system). ). As a result, "the cell needs a tool that appears in these specialized cells" The specialized cell does not appear in other differentiated cells. At present, gene expression is performed by fluorescent probes or other methods, individually or using a recently-discussed microarray, which is a printed or attached molecular probe that can interact with messenger RNA Hybridization and qualitative existence or non-existence -20- (Please read the precautions on the back before filling this page) This paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm) 18! 245895 V. Description of the invention ... fruit. Needless to say, these microarrays do not have the ability to quantify mRNA, and mRNA can be expressed at a low amount. Low-volume performance is also important because we know that low-volume performance must be synonymous with the production of very small amounts of protein because the concentration of cellular protein is dynamic and represents a balance between production and disruption. With the present invention, the quantification of mRNA and the corresponding protein are simplified, as described in the "Quantification" section above. Example 4 The detection of major changes is a method for detecting specific diseases, which is passed down from generation to generation (hereditary) in germ cells. With the conventional method based on the amplification of gene regions, the detection of single point mutations (sometimes very large) in genes has the disadvantage that large fragments cannot be amplified, and therefore restricts the economic and systematic study of a specific The ability to point mutations in human genes. With the present invention, paired probes (binding "wells" and binding beads) can be designed as unmutated genes (found in public databases) with known sequences. The paired needles are designed to complement the continuous elongation of the sequence. It has been researched on demand that many pairs are designed to cover the entire length of the gene, and different bound probes are attached to individual electrobiochips, and the detectable area 2 is in the unique discriminator of Card II. In this way, and using the device's special: configuration 'makes it reusable', it can carry out fast, economical and systematic research on gene mutation. EXAMPLES Many cancer lines are translocated to another gene or to another gene (chimeric gene). Examples are too many follicular carcinomas that cannot be listed and include 3 sacral glands, specific acute osteoblastic leukemia 'and many soft tissue sarcomas, such as synovial sarcoma and exoskeleton myelochondrosarcoma. This paper size applies Chinese National Standard (CNS) A4 (210X297 public love) -21-1245895 A7 B7 V. Description of the invention (19) Although the identification of mRNA transcripts (chimeric transcripts) of these chimeric genes It can be easily performed by the conventional polymerase chain reaction for amplification and electrophoresis for identification based on the amplified product. This method is slow and laborious. According to the present invention, a pair of probes that hybridize to two components of the chimeric transcript can be prepared. One probe is a probe bound to a "well" and the other probe is a probe bound to a conductive bead. Therefore, a positive identification can be achieved, even if trace samples are taken from the tumor or blood (if the tumor is leukemia or those who can easily enter the bloodstream at an early or late stage of the disease) by means of fine needles. Many different cancers can be screened simultaneously in this way. Example 6 Many diseases have viral causes. An example is HIV (infected with a human immunodeficiency virus). Although this disease can be controlled by antiviral agents, these agents are very expensive. Because the virus is susceptible to mutation, not all patients respond to the same agent at different times. Monitoring viral load is one way to determine drug efficacy and disease status. With this method, viral load research is made highly accurate, simple, fast, and cheap. The principle of analysis has been described previously. If the gene sequence is known, this method can be used to study any virus. Example 7 Many infectious diseases have similar manifestations. For example, anthracnose, influenza, dengue fever, smallpox, common cold, roseola, etc. The initial symptoms include contradiction (generally uncomfortable), fever, myalgia, and non-specific rash. In order to allow the attending physician to accurately identify the majority of these diseases with similar symptoms but with greatly different results, the present invention can be applied to the entire panel micro-22- (please Please read the notes on the back before filling this page) This paper size is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) 1245895 A7 B7 5. Detecting the amount of invention description (so early). Many patients do not even need to be hospitalized for observation and can be sent home because of the positive identification of the cause, while saving money and avoiding danger to the patient (if they need to be cared for in a hospital that may hide harmful microorganisms). fExample 8 Monitoring of harmful substances in drinking water and food (or animal feed), such as mad cow disease agents (bovine spongiform encephalopathy (BSE)) is hindered by the high cost and low sensitivity of the test (Reference 15 -20). For example, cattle with bse have trace amounts of substances in the brain fluid (cerebrospinal fluid). Sensitivity tests not only detect diseases early, but they are also exempt from animal slaughter. Humans infected with the disease by eating sick cattle can also be tested. By using specific antibodies discovered by other researchers, the present invention can significantly reduce costs and time to identify pathogenic agents. Example 8 Many hereditary diseases are caused by too long a specific region of a hidden repeat sequence. For example, Huntington's disease, a constant fatal disease, is caused by the presence of "CAG" in the Huntingtin gene located on chromosome 4 more than 36 times (http: / /www.ncbi.nlm.nih.gQv/entrez/dispomim>cgi?id:::=141 100) o In order to measure the number of repeats in the human Huntingtin gene, the conventional method is to use PCR. The invention simplifies the measurement of the number of iterations. Therefore, the probe is designed to be adjacent to the invariant part of a gene adjacent to the overlapping sequence (http://www.ncbi.nlm.iiih.gov/entrez/cuierv.fcgi?cmd=Retri eve & db = nucleotide & list uids = 450395 & dopt = GenBank) 〇In addition to the invariant sequence adjacent to the repeat, combined with the "well" probe to carry -23-(Please read the precautions on the back before filling this page} This paper size applies to China Standard (CNS) A4 specification (210X297 mm) 1245895 A7 B7 V. Description of the invention (21) Number of different repeats. Probes that bind beads carry the same area on the opposite side of the repeat and are one Or duplicates. A person with a specific number of repeats, such as 5 "CAG" repeats, if the probe that binds the well contains 4 repeats and the probe that binds the beads contains 1 repeat, when hybridized, it will have The two probes are perfectly arranged end-to-end. Other wells containing probes that bind "wells" that measure more than 4 (such as 5 or more) or less than 4 (such as 3 or less) will hybridize but will not produce perfect End arrangement. Using DNA ligase (covalently binding two DNA strands, the strands are aligned Complementary strands with closely adjacent ends), the two probes can covalently join. Repeated probes with a combined number of more than 5 will have overhanging strands that cannot be joined to another strand and have a lower than 5 The same combination of repeated probes is the same, because there is a large gap between the two probes. After the hybridization and ligation reaction, the adjustment of the reaction conditions caused the probe and target to separate (denaturation), resulting in other non-4CAG weights. The "shut-off" of the electrobiochip combined with the "well" probe. The result is a fast and accurate measurement of the number of CAG repeats in the human Huntingtin gene. The method of the present invention is applicable to Any kind of genetic disease with a repeated number of changes. -24- (Please read the notes on the back before filling out this page) This paper size applies to China National Standard (CNS) A4 (210X297 mm) 1245895 A7 B7 V. Invention Description (22) References: 1. Behr? MASA Warren, H. Salamon, PC Hopewell, A. Ponce de Leon, CL Daley- and PM Small. 1999 Transmission of Mycobacterium tuberculosis from patie nts smear-negative for acid-fast bacilli. Lancet 353: 444-449. 2. Bermann, JS G. Yuoh5 G. Fish and GL Woods. 1999 Clinical evaluation of the enhanced Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test for rapid diagnosis of tuberculosis in prison inmates. J Clin. Microbiol. 37: 1419-1425 · 3. Bermann, JS and GL Woods. 1999 Enhanced mycobacterium tuberculosis direct test for detection of M. tuberculosis complex in positive ESP II broth cultures of nonrespiratory specimens Diag. Microbiol Infect. Dis. 35: 245-248. 4. Chedore, P. and FB Jamieson. 1999. Routine use of the Gen-Probe MTD2 amplification test for detection of Mycobacterium tuberculosis in clinical specimens in a large public health mycobacteriology laboratory Diag. Microbiol. Infect. Dis. 35: 185-191. 5. Della-Latta, P and S. Whittier 1998. Comprehensive evaluation of performance, laboratory application, and clinical usefulness of two direct -25- (Please read the notes on the back first (Fill in this page again) Degree applies to Chinese National Standard (CNS) A4 specification (210X297 mm) 1245895 A7 _B7_ V. Description of invention (23) amplification technologies for the detection of Mycobacterium tuberculosis complex. Am. J Clin. Pathol. 110: 301-310. 6. Della-Latta, P and Vivian Jonas. 1999. Inhibitory effect of Alpha-Tec XPR-Plus Phosphate Buffer on the enhanced Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test. J Clin. Microbiol 37: 1234-1235. 7. Gamboa, FC Fernandez , E. Padilla, JM Manterola, J lonca, PJ Cardona, L. Matas, and V Ausina. 1998. Comparative Evaluation of initial and new versions of the Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test for direct detection of Mycobacterium tuberculosis in respiratory and nonrespiratory specimens. J Clin. Microbiol. 36: 684-689. 8. Moore, DF and Curry. JI 1999. Reduction in turnaround time for detection and identification of Mycobacterium tuberculosis from pulmonary specimens using nucleic acid amplification tests Presented at the 99 th General Meeting of the American Society of Microbiology Chicago, Illinois. 9. Piersimoni, CA Callegaro, C. Scarparo, V Penati, D. Nista, S. Bornigia, C. Lacchini, M. Scagnelli, G. Santini, and G. De Sio. 1998 Comparative evaluation of the new -26-(Please read the notes on the back before filling out this page) This paper size is applicable to China National Standard (CNS) A4 (210X297 mm) 1245895 A7 B7 V. Description of the invention ( twenty four )
Gen-Probe Mycobacterium tuberculosis Direct Test and the semiautomated Abbott LCx Mycobacterium tuberculosis assay for direct detection of Mycobacterium tuberculosis complex in respiratory and extrapulmonary specimens J Clin. Microbiol 36:3601-3604· 10. Wang5 S.X. and L.Tay 1999. Evaluation of three nucleic acid amplification methods for direct detection of Mycobacterium tuberculosis complex in respiratory specimens J Clin. Microbiol. 37:1932-1934. 11· Bakker AJ5 Loelemey MJ Gorgels JP- von Vlies B5 Smits R，Tijssen JG，Haagen FD: Troponin T and myoglobin at admission: value of early diagnosis of acute myocardial infraction. Eur Heart J 1994. 12. De Winter RJ? Koster RW5 Sturk A5 Sanders GT: Value of myoglobin，troponin T，and CK_MB Mass in ruling out an acute myocardial infarction in the emergency room. Circulation. 1995; 92: 3401 - 3407. 13. Hamm CW? Katus HA: New biochemical markers for myocardial cell injury Current Opinion in Cardiology. 1995; 10: 355 - 360. 14. Tucker JF Collins PA，Anderson AJ Hess M, Farley IM， Hagemann DA，Harkins HJ，Zwicke D: Value of serial -27- (請先閲讀背面之注意事項再填寫本頁) 本紙張尺度適用中國國家標準（CNS) A4規格（210X297公釐） 1245895 A7 __B7_ 五、發明説明（25 ) myoglobin levels in the early diagnosis of patients admitted for acute myocardial infarction. Ann Emerg Med 1994; 24: 704 - 708· 15. P. Brown et al.，“Bovine spongiform encephalopathy and variant Creutzfeldt-JakOb disease: background， evolution, and current concerns，” Emerging Infectious Diseases, 7[1] :6-16, January-February 2001. 16. J Bieschke et al·， “Ultra-sensitive detection of pathological prion protein aggregates by dualcolor scanning for intensely fluorescent targets， Proceedings of the National Academy of Sciences (PNAS)，97:5468-73, 2000. 17. M.R. Scott et al. “Identification of a prion protein epitope modulating transmission of bovine spongiform encephalopathy to transgenic mice，PNAS，94:14279-84, 1997. 18. The Evaluation of Tests for the Diagnosis of Transmissible Spongiform Encephalopathy in Bovines, European Commission， Directorate General XXIV. Consumer Policy and Consumer Health Protection, July 8, 1999. 19. J. Safar et al·，“Eight prion strains have PrPSc molecules with different conformations, Nature Medicine, -28- (請先閲讀背面之注意事項再填寫本頁) 本紙張尺度適用中國國家標準（CNS) A4規格（210X297公釐） 1245895 A7 B7 五、發明説明（26 ) 4:1157-65, 1998. (請先閲讀背面之注意事項再填寫本頁) 20. BSE Surveillance, U.S. Department of Agriculture, Animal and Plant Health Inspection Service. 本紙張尺度適用中國國家標準（CNS) A4規格（210X297公釐） _ 29 - 1245895 A7 B7 五、發明説明（27 ) 元件符號對照表： 1 導線 22 探針 2 開關 23 探針 3 電池 24 懸突 4 安培計、燈泡或微處理 器 5 井 6 電極 7 間隙 8 晶片 9 探針 10 鐵珠粒 11 目標物分子 12 探針 13 筒匣 14 測試樣本 15 探針 17 探針 18 探針 19 上股 20 5,端 21 3,端 (請先閲讀背面之注意事項再填寫本頁) 本紙張尺度適用中國國家標準（CNS) A4規格（210X297公釐） -30-Gen-Probe Mycobacterium tuberculosis Direct Test and the semiautomated Abbott LCx Mycobacterium tuberculosis assay for direct detection of Mycobacterium tuberculosis complex in respiratory and extrapulmonary specimens J Clin. Microbiol 36: 3601-3604 · 10. Wang5 SX and L.Tay 1999. Evaluation of three nucleic acid amplification methods for direct detection of Mycobacterium tuberculosis complex in respiratory specimens J Clin. Microbiol. 37: 1932-1934. 11 · Bakker AJ5 Loelemey MJ Gorgels JP- von Vlies B5 Smits R, Tijssen JG, Haagen FD: Troponin T and myoglobin at admission: value of early diagnosis of acute myocardial infraction. Eur Heart J 1994. 12. De Winter RJ? Koster RW5 Sturk A5 Sanders GT: Value of myoglobin, troponin T, and CK_MB Mass in ruling out an acute myocardial infarction in the emergency room. Circulation. 1995; 92: 3401-3407. 13. Hamm CW? Katus HA: New biochemical markers for myocardial cell injury Current Opinion in Cardiology. 1995; 10: 355-360. 14. Tucker JF Co llins PA, Anderson AJ Hess M, Farley IM, Hagemann DA, Harkins HJ, Zwicke D: Value of serial -27- (Please read the precautions on the back before filling out this page) This paper size applies to Chinese National Standard (CNS) A4 Specifications (210X297 mm) 1245895 A7 __B7_ V. Description of the invention (25) myoglobin levels in the early diagnosis of patients admitted for acute myocardial infarction. Ann Emerg Med 1994; 24: 704-708 · 15. P. Brown et al., "Bovine spongiform encephalopathy and variant Creutzfeldt-JakOb disease: background, evolution, and current concerns," Emerging Infectious Diseases, 7 [1]: 6-16, January-February 2001. 16. J Bieschke et al ·, "Ultra-sensitive detection of pathological prion protein aggregates by dualcolor scanning for intensely fluorescent targets, Proceedings of the National Academy of Sciences (PNAS), 97: 5468-73, 2000. 17. MR Scott et al. "Identification of a prion protein epitope modulating transmission of bovine spongiform encephalopathy to transgenic mice, PNAS, 94: 14279-84, 1997. 18. The Evaluation of Tests for the Diagnosis of Transmissible Spongiform Encephalopathy in Bovines, European Commission, Directorate General XXIV. Consumer Policy and Consumer Health Protection, July 8, 1999. 19. J. Safar et al ·, "Eight prion strains have PrPSc molecules with different conformations, Nature Medicine, -28- (Please read the precautions on the back before filling out this page) This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) 1245895 A7 B7 V. Description of the Invention (26) 4: 1157-65, 1998. (Please read the notes on the back before filling this page) 20. BSE Surveillance, US Department of Agriculture, Animal and Plant Health Inspection Service. Paper size Applicable to China National Standard (CNS) A4 specification (210X297 mm) _ 29-1245895 A7 B7 V. Description of the invention (27) Component symbol comparison table: 1 lead 22 probe 2 switch 23 probe 3 battery 24 overhang 4 amp meter , Bulb or microprocessor 5 well 6 electrode 7 gap 8 chip 9 probe 10 iron bead 11 Target molecule 12 Probe 13 Cartridge 14 Test sample 15 Probe 17 Probe 18 Probe 19 Upper strand 20 5, End 21 3, End (Please read the precautions on the back before filling this page) This paper size applies China National Standard (CNS) A4 specification (210X297 mm) -30-