TW201209403A - LOC device for genetic analysis which performs nucleic acid amplification after sample preparation in a dialysis section - Google Patents
LOC device for genetic analysis which performs nucleic acid amplification after sample preparation in a dialysis section Download PDFInfo
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201209403 六、發明說明: 【發明所屬之技術領域】 本發明關於使用微系統技術(MST )之診斷裝置。特 別是本發明關於用於分子診斷之微流體和生化之處理及分 析。 【先前技術】 分子診斷已用於:可於病徵顯現之前,提供早期疾病 檢測預示之領域》分子診斷試驗係用於檢測: •遺傳病症 •後天病症 •傳染性疾病 •與健康有關情況之基因易致病因素 因高準確度及快速處理時間,分子診斷試驗得以減少 無效健康照護的發生、增進病患預後(patient outcome ) 、改進疾病管理及個體化患者照護。分子診斷的許多技術 係基於自生物樣本(諸如血液或唾液)萃取及擴增之特定 核酸(去氧核糖核酸(DNA )以及核糖核酸(RNA )兩者 )的檢測及辨識。核酸鹼基的互補特徵使得經合成DNA (寡核苷酸)短序列結合(雜交)至用於核酸試驗之特定 核酸序列。若發生雜交,則互補序列存在於樣本中。此使 得例如預測個人未來會得到的疾病、判定感染性病原體的 種類及致病性,或判定個人對藥物的反應成爲可能。 201209403 以核酸爲基礎之分子診斷試驗 以核酸爲基之試驗具有四個獨立步驟: 1. 樣本製備 2. 核酸萃取 3. 核酸擴增(選用的) 4. 檢測 許多樣本類型,諸如血液、尿液、痰和組織樣本,係 用於基因分析。診斷試驗判定所需的樣本類型,因並非所 有樣本代表疾病進程。這些樣本具有各種組分,但通常只 有其中之一受到關注。例如,在血液中,高濃度的紅血球 可抑制致病微生物的檢測。因此,於核酸試驗開始時經常 需要純化及/或濃縮步驟。 血液爲較常請求的樣本類型之一。其具有三種主要組 分:白血球、紅血球及血栓細胞(血小板)。血栓細胞促 進凝集且在體外維持活性。爲抑制凝聚作用,在純化及濃 縮之前令試樣與諸如乙二胺四乙酸(EDTA )之試劑混合 。通常自樣本移除紅血球以濃縮標靶細胞。在人體中,紅 血球佔細胞物質之約99%,但其不帶有DNA因彼不具細 胞核。此外,紅血球含有諸如血紅素之可能干擾下游核酸 擴增程序(描述於下)的成分。可藉由差示( differentially)溶胞於溶胞溶液中之紅血球來移除紅血球 ,而留下剩餘的完整細胞物質,其可接著使用離心而與樣 -6- 201209403 本分離。此提供自其萃取核酸之濃縮標靶細胞。 用於萃取核酸之確切規程(protocol)取決於樣本及 待實施之診斷分析。例如,用於萃取病毒RNA之規程與 用於萃取基因組DNA之規程相當不同。然而,自標靶細 胞萃取核酸通常包含細胞溶胞步驟及接續的核酸純化。細 胞溶胞步驟使細胞及細胞核膜破裂,而釋放出遺傳物質。 此經常使用溶胞清潔劑來完成,溶胞清潔劑係諸如十二烷 基硫酸鈉,其亦使存在於細胞中之大量蛋白質變性。 接著於清洗之前在高濃度的離液鹽(chaotropic salt )存在下,通常於分餾塔中的氧化矽基質、樹脂或順磁性 珠上,以酒精〔通常爲冰乙醇或異丙醇〕沉澱步驟,或是 經由固相純化步驟純化核酸,接著以低離子強度緩衝液進 行洗提。核酸沉澱之前之任意的步驟爲添加消化蛋白質之 蛋白酶,以進一步純化樣本。 其他的溶胞方法包括經由超音波振動之機械式溶胞以 及將樣本加熱至94°C以破壞細胞膜之熱溶胞。 標靶DNA或RNA可以極小量存在於經萃取之物質中 ,尤其是若標靶來自致病性來源。核酸擴增提供選擇性擴 增(即,複製)特定標靶(就可檢測程度而言爲低濃度者 )的能力。 最常使用之核酸擴增技術爲聚合酶連鎖反應(PCR) 。PCR係業界已知悉,以及於E. van Pelt-Verkuil等人之 Principles and Technical Aspects of PCR Amplification, Springer,2008中提供此類反應之綜合理解性描述。 201209403 PCR爲有用的技術,其相對複雜DNA背景而擴增標 靶DNA序列。若欲(藉由PCR )擴增RNA,則首先必須 使用名爲反轉錄酶之酵素將之轉錄爲cDNA (互補DNA) 。隨後,藉由PCR擴增得到的cDNA。 PCR爲指數型方法,只要維持反應的條件爲可接受的 則其可繼續進行。反應之成分爲:201209403 VI. Description of the Invention: TECHNICAL FIELD OF THE INVENTION The present invention relates to a diagnostic apparatus using microsystem technology (MST). In particular, the present invention relates to the processing and analysis of microfluidics and biochemicals for molecular diagnostics. [Prior Art] Molecular diagnostics have been used to provide areas for early detection of disease before the onset of symptoms. Molecular diagnostic tests are used to detect: • genetic disorders • acquired diseases • infectious diseases • genetically related diseases Pathogenic Factors Due to high accuracy and rapid processing time, molecular diagnostic tests can reduce the incidence of ineffective health care, improve patient outcomes, improve disease management, and individualized patient care. Many techniques for molecular diagnostics are based on the detection and identification of specific nucleic acids (both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)) extracted and amplified from biological samples such as blood or saliva. The complementary nature of the nucleobases allows for the binding (hybridization) of synthetic DNA (oligonucleotide) short sequences to specific nucleic acid sequences for nucleic acid assays. If hybridization occurs, the complementary sequence is present in the sample. This makes it possible, for example, to predict the disease that an individual will receive in the future, determine the type and pathogenicity of an infectious pathogen, or determine the individual's response to the drug. 201209403 Nucleic Acid-Based Molecular Diagnostic Tests Nucleic acid-based assays have four separate steps: 1. Sample preparation 2. Nucleic acid extraction 3. Nucleic acid amplification (optional) 4. Detection of many sample types, such as blood, urine , sputum and tissue samples are used for genetic analysis. Diagnostic tests determine the type of sample required, as not all samples represent disease progression. These samples have various components, but usually only one of them is of interest. For example, in the blood, high concentrations of red blood cells can inhibit the detection of pathogenic microorganisms. Therefore, purification and/or concentration steps are often required at the beginning of a nucleic acid assay. Blood is one of the more frequently requested sample types. It has three main components: white blood cells, red blood cells, and thrombocytes (platelets). Thrombotic cells promote agglutination and maintain activity in vitro. To inhibit coacervation, the sample is mixed with a reagent such as ethylenediaminetetraacetic acid (EDTA) prior to purification and concentration. Red blood cells are typically removed from the sample to concentrate the target cells. In the human body, red blood cells account for about 99% of cellular material, but they do not carry DNA because they do not have a nucleus. In addition, red blood cells contain components such as heme that may interfere with downstream nucleic acid amplification procedures (described below). The red blood cells can be removed by differentially lysing the red blood cells in the lysis solution, leaving the remaining intact cellular material, which can then be separated from the sample by centrifugation. This provides a concentrated target cell from which the nucleic acid is extracted. The exact protocol used to extract the nucleic acid depends on the sample and the diagnostic analysis to be performed. For example, the protocol used to extract viral RNA is quite different from the protocol used to extract genomic DNA. However, self-targeting cell extraction of nucleic acids typically involves a cell lysis step and subsequent nucleic acid purification. The cell lysis step ruptures the cell and nuclear membrane and releases the genetic material. This is often done using a lysing detergent such as sodium lauryl sulfate, which also denatures a large amount of protein present in the cells. The step of precipitating with alcohol (usually ice ethanol or isopropanol) in the presence of a high concentration of chaotropic salt, usually in the cerium oxide matrix, resin or paramagnetic beads in the fractionation column, prior to washing, Alternatively, the nucleic acid is purified via a solid phase purification step followed by elution with a low ionic strength buffer. Any step prior to precipitation of the nucleic acid is the addition of a protease that digests the protein to further purify the sample. Other lysis methods include mechanical lysis via ultrasonic vibration and heating of the sample to 94 °C to destroy the thermal lysis of the cell membrane. Target DNA or RNA can be present in the extracted material in very small amounts, especially if the target is from a pathogenic source. Nucleic acid amplification provides the ability to selectively amplify (i.e., replicate) a particular target (in the case of a low concentration in terms of detectability). The most commonly used nucleic acid amplification technique is the polymerase chain reaction (PCR). The PCR is known in the art and provides a comprehensive and comprehensible description of such reactions in E. van Pelt-Verkuil et al., Principles and Technical Aspects of PCR Amplification, Springer, 2008. 201209403 PCR is a useful technique for amplifying target DNA sequences against a relatively complex DNA background. If RNA is to be amplified (by PCR), it must first be transcribed into cDNA (complementary DNA) using an enzyme called reverse transcriptase. Subsequently, the obtained cDNA was amplified by PCR. PCR is an exponential method which can be continued as long as the conditions for maintaining the reaction are acceptable. The composition of the reaction is:
1. 引子對_具有約10-30個與E比鄰(flanking)標祀 序列區互補之核苷酸的短單股DN A 2. DNA聚合酶-合成DNA之熱穩定性酶 3. 去氧核糖核苷三磷酸(dNTP )-提供整合至新合 成之DNA股之核苷酸 4. 緩衝液-提供DNA合成之最佳化學環境 PCR —般包含將這些反應物置於含有經萃取之核酸的 小管(~1〇-5〇微升)》將管放置於聚合酶連鎖反應器( thermal cycler)中;一種令反應經受一連串不等量時間 之不同溫度的儀器。各熱循環的標準規程包括變性相、黏 著相及延伸相。延伸相有時代表引子延伸相。除了此三-步驟規程外,可採用二-步驟熱規程,於其中黏著及延伸 相合倂。變性相一般包含將反應溫度升溫至90-95 °C以使 DNA股變性:於黏著相中,將溫度降低至〜5 0-60°C以供 引子黏著:接著於延伸相中,將溫度升溫至最佳DN A聚 合酶活性溫度60-72°C,以供引子延伸。此方法重複循環 約2 0-40次,最終結果爲產生數百萬拷貝之引子間的標靶 -8 - 201209403 序列。 已發展出用於分子診斷之許多標準PCR規程之變體 ,其中包括諸如多引子組 PCR、聯結子引發(linker-primed) PCR、 直接 PCR、重複序列(tandem) PCR、即 時PCR以及反轉錄酶PCR。 多引子組PCR使用單一 PCR混合物中之多重引子組 以產生對不同DNA序列具特異性之不同大小之擴增子。 藉由一次標靶多個基因,由單一試驗可得到額外的資訊( 以其他方式則需要數次試驗)。最佳化多引子組PCR更 爲困難,因其需要選取具近似黏著溫度之引子及具近似長 度與鹼基組成之擴增子以確保各擴增子之擴增效率相等。 聯結子引發(linker-primed) PCR,又稱爲接合接合 子(ligation adaptor ) PCR,爲用於致能複雜DNA混合物 中實質上所有DNA序列之核酸擴增的方法,而不需要標 靶-特異性引子。此方法首先以合適的限制性內核酸酶( 酵素)來剪切(digest)標靶DNA群體。使用接合酶酵素 ,具有合適的懸伸(overhanging )端之雙股寡核苷酸聯 結子(亦稱爲接合子)接著與標靶DN A片段之端子接合 。接下來使用對聯結子序列具有特異性之寡核苷酸引子實 施核酸擴增。藉此,可擴增毗鄰聯結子寡核苷酸之DNA 來源的所有片段。 直接PCR描述一種直接於樣本上實施PCR而不需要 任何核酸萃取(或最少核酸萃取)之系統。長久以來認爲 ,PCR反應受到存在於未純化的生物樣本中之許多成分的 201209403 抑制,諸如血液中的原血紅素成分。傳統上,於製備反應 混合物之前,PCR需要加強純化標靶核酸。然而,利用化 學性質的適當變化及樣本濃縮,可以最少化DNA純化而 進行PCR或進行直接PCR。用於直接PCR之PCR化學性 質的調整包括加強緩衝液強度、使用高活性及進行性( processivity)之聚合酶及與潛在聚合酶抑制劑蜜合之添 加物。 重複序列PCR利用兩次獨立的核酸擴增以增進擴增 正確擴增子的機率。重複序列 PCR中的一類型爲巢式 PCR,其中使用兩對PCR引子,以於分別的核酸擴增進行 單一基因座擴增。第一對引子與標靶核酸序列外部區域的 核酸序列雜交。第二次擴增中所使用的第二對引子(巢式 引子)結合於第一 PCR產物中並且產生含有標靶核酸的 第二PCR產物(較第一PCR產物爲短)。此策略所運用 的論理爲:若於第一次核酸擴增期間因失誤而擴增錯誤的 基因座,由第二對引子再次擴增錯誤的基因座的機率非常 低,因此確保了特異性。 使用即時PCR或定量PCR以即時量測PCR產物之量 °藉使用含有螢光團之探針或螢光染料以及反應中的一套 參考標準,可測定樣本中之核酸的最初含量。此特別有用 於分子診斷,其中治療選擇可能取決於樣本中所載病原體 而有所不同。 反轉錄酶PCR ( RT-PCR )係用於自RNA來擴增DN A 。反轉錄酶爲將RNA反轉錄成互補DNA ( c DNA )之酵素 -10- 201209403 ,接著藉由PCR擴增cDNA。RT-PCR廣泛地用於表現型 態(expression profiling)以判定基因的表現或辨識RNA 轉錄本(包括轉錄起始及終止位址)之序列。其亦用於擴 增RNA病毒,諸如人類免疫缺乏病毒或C型肝炎病毒。 恆溫擴增爲另一種類型的核酸擴增,其不依靠擴增反 應期間之標靶DNA的熱變性,因此不需要複雜的機械。 恆溫核酸擴增方法可因此原始位置進行或於實驗室環境外 易於被操作。包括股取代擴增(Strand Displacement Amplification )、轉錄介導擴增(Transcription Mediated Amplification )、依賴核酸序列擴增(Nucleic Acid Sequence Based Amplification)、重組酵素聚合酶擴增( Recombinase Polymerase Amplification)、滾動循環擴增 (Rolling Circle Amplification )、分枝型擴增( Ramification Amplification )、解旋恆溫 DNA 擴增( Helicase-Dependent Isothermal DNA Amplification)及環 形恒溫擴增(Loop-Mediated Isothermal Amplification) 之一些恆溫核酸擴增方法已被敘述。 恆溫核酸擴增法不依賴模板DNA之持續加熱變性來 產生作爲進一步擴增之模板的單股分子,而是依賴諸如於 常溫下藉由特異性限制內核酸酶之DNA分子的酵素性切 割’或是利用酵素分開D N A股之其他方法。 股取代擴增(SDA )依賴特定限制性酵素的能力以切 割半修飾(hemi-modified) DNA之未經修飾股,及5,-3’ 外核酸酶-缺乏之聚合酶的能力以延伸並取代下游股。然 -11 - 201209403 後藉由偶合義(sense)與反義(antisense)反應而達成 指數性核酸擴增,其中來自義反應之股取代作爲反義反應 之模板。使用不以慣例方式切割DNA而是於DNA之一股 上產生切口之切口酶(諸如N. Alwl,N. BstNBl及Mlyl )係有用於此反應。藉使用熱穩定限制性酵素(/ να 1 )及 熱穩定性外-聚合酶(聚合酶)之組合已改進SDA »此 組合顯現出使反應的擴增效率由1〇8倍擴增增加至101()倍 擴增,以致可使用此技術來擴增獨特的單拷貝分子。 轉錄介導擴增(TMA)及依賴核酸序列擴增(NASBA )使用RNA聚合酶以複製RNA序列而非對應之基因組 DNA。此技術使用兩種引子及兩或三種酵素、RNA聚合酶 、反轉錄酶及任意的RNase Η (若反轉錄酶不具有RNase 活性)。一種引子含有供RNA聚合酶之啓動子序列。在 核酸擴增的第一步驟中,此引子於限定的位置與標靶核糖 體RNA ( rRNA )雜交。藉由自啓動子引子的3'端開始延 伸,反轉錄酶產生標靶rRN A之DNA拷貝。若存在另外 的RNase Η,則所得的RNA : DNA雙股中的RNA經由反 轉錄酶之RNase活性而被分解。接著,第二引子結合至 DNA拷貝。藉反轉錄酶自此引子的末端合成新的DNA股 而產生雙股DNA分子。RNA聚合酶辨識DNA模板中的啓 動子序列’並開始轉錄。各個新合成的RNA擴增子再進 入過程中並作爲新的複製之模板。 於重組酵素聚合酶擴增(RPA)中,藉結合相對的寡 核苷酸引子至模板DN A並且由DN A聚合酶將之延伸而達 -12- 201209403 成特定DNA片段之恆溫擴增。變性雙股 模板不需要熱。反之,RPA利用重組酵: 掃描dsDNA及促進同源(cognate)位置 由單股DNA結合蛋白與經取代模板股的 所得到的結構,因此防止引子因分支遷移 素分解離開可爲股取代DNA聚合酶 sub til is Pol I ( m )的大片段)所接近 端,且引子接著開始延伸。藉循環重複此 性核酸擴增。 解旋酶擴增(HDA)模擬活體內系統 中使用DNA解旋酶來產生用於引子雜交 著以DNA聚合酶延伸引子。於HDA反應 解旋酶穿過標靶DNA,破壞聯結兩股的 後由單股結合蛋白所結合。由解旋酶所暴 域使引子得以黏著。DNA聚合酶使用自 苷三磷酸(dNTP )以接著延伸各引子的 個DNA複製(replicate )。兩個複製的 進入下一個HD A循環’造成標靶序列之 〇 其他的基於DNA之恆溫技術包括 RCA ) ’於其中DNA聚合酶繞環狀〇ΝΑ 引子而產生由許多環狀重複拷貝所組成之 。藉由終止反應,聚合酶產生數千拷貝之 有栓繫至原始標粑DNA的拷貝鏈。此致 DNA ( dsDNA ) 素-引子錯合體來 :處的股交換。藉 交互作用來穩定 而放出。重組酵 (諸如 Bacillus 之寡核苷酸的31 步驟而達到指數 ,於活體內系統 之單股模板並接 的第一步驟中, 氫鍵,此二股隨 露之單股標靶區 由的去氧核糖核 3 ’端,以產生兩 dsDNA股獨立地 指數性核酸擴增 滾動循環擴增( 模板持續地延伸 .長的DNA產物 .環狀模板,其具 :使標靶之空間解 -13- 201209403 析度及信號之快速核酸擴增。於1小時內至多可產生ΙΟ12 拷貝之模板。分枝型擴增爲RCA之變體,並利用封閉的 環狀探針(C-探針)或扣鎖探針及具高進行性之DNA聚 合酶,以於常溫情況下指數地擴增C-探針。 環形恆溫擴增(LAMP )提供高選擇性且利用DNA聚 合酶及含有四個特別設計的引子之引子組,引子組辨識標 靶DNA上總共六個不同的序列。含有標靶DNA之義股及 反義股序列的內引子起始LAMP。由外引子引發之後續股 取代DNA合成釋出單股DNA。此作爲由第二內及外引子 所引發之DNA合成的模板,第二內及外引子與標靶之另 一端雜交,產生莖-環(stem-loop ) DNA結構。於接續的 LAMP循環中,內引子與產物上的環形雜交並起始取代 DNA合成,產生原始莖-環DN A及具有兩倍莖長度之新 莖-環DNA。於少於一小時內持續循環反應而聚積1〇9拷 貝之標靶。最終產物爲具有數個反相重複標靶之莖-環 DNA,以及具有多個環形(交替黏著相同股中之反相重複 標靶所形成)之花椰菜狀結構。 於完成核酸擴增之後,必須分析擴增的產物以判定是 否產生預期的擴增子(標靶核酸之擴增量)。分析產物的 方法有透過膠體電泳簡單測定擴增子的大小、使用DNA 雜交以識別擴增子之核苷酸組成。 膠體電泳爲檢查核酸擴增步驟使否產生預期之擴增子 之最簡單方式之一》膠體電泳利用施加至膠體基質之電場 來分離DNA片段。帶負電的DNA片段將以不同速率(主 -14- 201209403 要取決於其大小)移動通過基質。於電泳完成之後 色膠體中的片段使其成爲可見。於UV光下發螢光 乙菲錠爲最常用的染劑。 藉由與DNA大小標記(DNA標準片段(DNA ))相比較來判定片段的大小,DNA大小標記含 大小的DNA片段,其與擴增子一同跑膠。因寡核 子結合至毗鄰標靶DNA之特定位置,經擴增之產 小可被預測且以膠體上已知大小的帶被檢測。爲確 子爲何或若產生數種擴增子時,常利用DNA探針 子雜交。 DNA雜交意指藉由互補鹼基配對而形成雙股 用於特定擴增產物之正面識別的DNA雜交需使用 約20個核苷酸的DNA探針。若探針具有與擴增子 )DNA序列互補的序列,則雜交將於有利的溫度、 離子濃度條件下發生。若發生雜交,則表示關注的 DNA序列出現於原始樣本中。 光學檢測爲最常見之檢測雜交的方法。標記擴 是探針以經由發螢光或電致化學發光而發光。這些 引發產光部分之激發態的方式不同,但兩者同樣致 酸股之共價標記。於電致化學發光(ECL ),當以 激時,由發光團分子或錯.合體產生光。於發螢光時 成發射之激發光來發光。 使用發光源以檢測螢光,發光源提供波長爲螢 吸收之激發光以及檢測單兀。檢測單元包含光感測 ,可染 之溴化 ladder 有已知 苷酸引 物的大 認擴增 與擴增 DNA。 長度爲 (標靶 pH及 基因或 增子或 方法之 能核苷 電流刺 ,以造 光分子 器(諸 -15- 201209403 如光電倍增管或電荷耦合裝置(CCD )陣列)以檢測發射 的信號,以及防止激發光被包含於光感測器輸出之機構( 諸如波長-選擇濾波器)。回應激發光,螢光分子發射斯 托克斯位移(Stokes-shifted )光,以及此發射的光由檢 測單元收集。托克斯位移爲發射的光與吸收的激發光之間 之頻率差或波長差。 使用光感測器來檢測ECL發射,光感測器對於所採 用之ECL種類之發射波長爲敏感。例如,過渡金屬配位 錯合體發射可見波長的光,因而採用傳統光二極體及 CCD作爲光感測器。ECL之優勢爲,若排除周圍光線, ECL發射可爲檢測系統中唯一存在的光,因而增進靈敏度 〇 微陣列使數十萬的DNA雜交試驗得以同時進行。微 陣列爲有用的分子診斷工具,其可篩檢數千種遺傳疾病或 於單一試驗中檢測是否存在數種感染性病原體。微陣列由 許多不同的固定於基板上且成點狀之DNA探針所組成。 首先以螢光或發光分子標記標靶DNA (擴增子) (於核 酸擴增期間或之後),然後將其施加至探針陣列。於經控 制的溫度下、潮濕的環境中培養微陣列數小時或數天,此 時探針及擴增子之間發生雜交。於培養後,必須以一連串 緩衝液清洗微陣列以移除未經結合股。一旦清洗後,以氣 流(通常爲氮)乾燥微陣列表面。雜交及清洗的嚴格度很 重要。不夠嚴格可能導致高度非特異性結合。過度嚴格可 能導致無法適當進行結合而造成減低的靈敏度。藉由檢測 -16- 201209403 來自經標記之與互補探針形成雜交的擴增子之光發射而辨 識雜交。 使用微陣列掃描器檢測來自微陣列的螢光,微陣列掃 描器通常爲經電腦控制的反相掃描式螢光共軛焦顯微鏡, 其一般使用激發螢光染料的雷射及光感測器(諸如光電倍 增管或CCD )以檢測發射的信號。螢光分子發射經下轉 換的光(如上述),而光被檢測單元收集。 發射的螢光必須被收集、與未經吸收的激發波長分離 ,並被傳送至檢測器。於微陣列掃描器中常使用共軛焦配 置以藉由位於影像平面的共轭焦針孔來刪除失焦資訊。此 使得僅檢測光的聚焦部分。防止於物之焦點平面之上方或 下方的光進入檢測器,藉此增加信號對雜訊比。檢測器將 經檢測的螢光光子轉換成電能,電能並接著被轉換成數位 信號。此數位信號轉變成代表來自給定像素之螢光強度的 數字。陣列的各特徵係由一或多個此像素所構成。掃描的 最終結果爲陣列表面影像。由於已知微陣列上每一個探針 的確切序列及位置,因此可同時識別及分析雜交的標靶序 列。 可於下列找到更多有關螢光探針之資訊: http : "www.premierbiosoft.com/tech_notes/FRET_probe.html 以及 http : "www.invitrogen.com/site/us/en/home/References/Molecular -probes-The-Handbook/Technical-Notes-and-Product-1. A pair of short strands of DN A having about 10-30 nucleotides complementary to the E-flanking target sequence. 2. DNA polymerase-synthesis of DNA thermostable enzymes 3. Deoxyribose Nucleoside triphosphate (dNTP) - provides nucleotides integrated into newly synthesized DNA strands. 4. Buffer - The best chemical environment for DNA synthesis - PCR involves placing these reagents in small tubes containing extracted nucleic acids ( ~1〇-5〇μL) Place the tube in a polymer thermal cycler; an instrument that subjects the reaction to a series of different temperatures for varying amounts of time. Standard procedures for each thermal cycle include the denatured phase, the adhesive phase, and the extended phase. The extension phase sometimes represents the primer extension phase. In addition to this three-step procedure, a two-step thermal procedure can be employed in which the adhesion and extension are combined. The denatured phase generally involves ramping the reaction temperature to 90-95 ° C to denature the DNA strand: in the adhesive phase, the temperature is lowered to ~50-60 °C for adhesion of the primer: then in the extended phase, the temperature is raised The optimal DN A polymerase activity temperature is 60-72 ° C for extension of the primer. This method repeats the cycle for about 20-40 times, and the end result is a sequence of targets -8 - 201209403 that produce millions of copies of the primer. Variants of many standard PCR protocols for molecular diagnostics have been developed, including, for example, multiple primer set PCR, linker-primed PCR, direct PCR, tandem PCR, real-time PCR, and reverse transcriptase PCR. Multiple primer set PCR uses multiple primer sets in a single PCR mix to generate different sizes of amplicons specific for different DNA sequences. Additional information can be obtained from a single experiment by targeting multiple genes at once (in other ways, several trials are required). Optimizing multiple primer set PCR is more difficult because it requires the selection of primers with approximate adhesion temperatures and amplicon with approximate length and base composition to ensure equal amplification efficiency of each amplicon. Linker-primed PCR, also known as ligation adaptor PCR, is a method for enabling nucleic acid amplification of virtually all DNA sequences in complex DNA mixtures without the need for target-specific Sexual introduction. This method first digests the target DNA population with a suitable restriction endonuclease (enzyme). Using a zymase enzyme, a double-stranded oligonucleotide linker (also known as a zygote) with a suitable overhanging end is then ligated to the terminal of the target DN A fragment. Nucleic acid amplification is then carried out using oligonucleotide primers specific for the linker sequence. Thereby, all fragments of the DNA source adjacent to the linker oligonucleotide can be amplified. Direct PCR describes a system that performs PCR directly on a sample without any nucleic acid extraction (or minimal nucleic acid extraction). It has long been believed that the PCR reaction is inhibited by 201209403, a component of many components present in unpurified biological samples, such as the raw heme component in the blood. Traditionally, PCR requires enhanced purification of target nucleic acids prior to preparation of the reaction mixture. However, with appropriate changes in chemical properties and sample concentration, PCR can be performed or direct PCR can be performed with minimal DNA purification. The adjustment of PCR chemistry for direct PCR involves enhanced buffer strength, the use of high activity and processivity polymerases, and the addition of honey to potential polymerase inhibitors. Repeated sequence PCR utilizes two independent nucleic acid amplifications to increase the probability of amplifying the correct amplicon. Repetitive Sequence One type of PCR is nested PCR in which two pairs of PCR primers are used to perform single locus amplification for separate nucleic acid amplification. The first pair of primers hybridize to the nucleic acid sequence of the outer region of the target nucleic acid sequence. The second pair of primers (nested primers) used in the second amplification binds to the first PCR product and produces a second PCR product (short in the first PCR product) containing the target nucleic acid. The rationale used in this strategy is that if the wrong locus is amplified by mistakes during the first nucleic acid amplification, the probability of re-amplifying the wrong locus by the second pair of primers is very low, thus ensuring specificity. Instantaneous PCR or quantitative PCR is used to measure the amount of PCR product in real time. The initial amount of nucleic acid in a sample can be determined by using a probe containing a fluorophore or a fluorescent dye and a set of reference standards in the reaction. This is especially useful for molecular diagnostics where treatment options may vary depending on the pathogen contained in the sample. Reverse transcriptase PCR (RT-PCR) is used to amplify DN A from RNA. The reverse transcriptase is an enzyme that reverse transcribes RNA into complementary DNA (c DNA) -10- 201209403, and then the cDNA is amplified by PCR. RT-PCR is widely used in expression profiling to determine the expression of a gene or to identify sequences of RNA transcripts, including transcription initiation and termination sites. It is also used to augment RNA viruses such as human immunodeficiency virus or hepatitis C virus. Constant temperature amplification is another type of nucleic acid amplification that does not rely on thermal denaturation of the target DNA during the amplification reaction, and thus does not require complicated machinery. The thermostatic nucleic acid amplification method can be performed at the original location or easily outside the laboratory environment. Including Strand Displacement Amplification, Transcription Mediated Amplification, Nucleic Acid Sequence Based Amplification, Recombinase Polymerase Amplification, Rolling Cycle Expansion Some of the constant temperature nucleic acid amplification methods of Rolling Circle Amplification, Ramification Amplification, Helicase-Dependent Isothermal DNA Amplification, and Loop-Mediated Isothermal Amplification Has been described. The thermostatic nucleic acid amplification method does not rely on the continuous heat denaturation of the template DNA to produce a single-stranded molecule as a template for further amplification, but relies on an enzymatic cleavage such as a DNA molecule that specifically limits the endonuclease at normal temperature' or It is another way to separate DNA strands using enzymes. The ability of strand-substituted amplification (SDA) to rely on specific restriction enzymes to cleave unmodified strands of hemi-modified DNA, and 5,-3' exonuclease-deficient polymerases to extend and replace Downstream stocks. However, after -11 - 201209403, exponential nucleic acid amplification is achieved by a combination of sense and antisense, wherein the strand of the sense reaction is substituted as a template for the antisense reaction. A nicking enzyme (such as N. Alwl, N. BstNBl and Mlyl) which produces a nick in one of the DNA strands without cutting the DNA in a conventional manner is useful for this reaction. SDA has been improved by using a combination of thermostable restriction enzyme (/να 1 ) and thermostable exo-polymerase (polymerase). This combination appears to increase the amplification efficiency of the reaction from 1 to 8 fold amplification to 101. () amplification so that this technique can be used to amplify unique single copy molecules. Transcription-mediated amplification (TMA) and nucleic acid-dependent sequence amplification (NASBA) use RNA polymerase to replicate RNA sequences rather than corresponding genomic DNA. This technique uses two primers and two or three enzymes, RNA polymerase, reverse transcriptase, and any RNase Η (if the reverse transcriptase does not have RNase activity). One primer contains a promoter sequence for RNA polymerase. In the first step of nucleic acid amplification, the primer hybridizes to a target ribosomal RNA (rRNA) at a defined position. The reverse transcriptase produces a DNA copy of the target rRN A by extending from the 3' end of the promoter primer. If another RNase is present, the RNA in the obtained RNA:DNA double strand is decomposed by the RNase activity of the reverse transcriptase. Next, the second primer binds to the DNA copy. A double-stranded DNA molecule is produced by synthesizing a new DNA strand from the end of the primer by reverse transcriptase. RNA polymerase recognizes the promoter sequence in the DNA template and initiates transcription. Each newly synthesized RNA amplicon is reintroduced into the process and serves as a template for new replication. In recombinant enzyme polymerase amplification (RPA), a constant amplification of a specific DNA fragment is achieved by binding a relative oligonucleotide primer to the template DN A and extending it by DN A polymerase to -12-201209403. Denatured double-strand templates do not require heat. Conversely, RPA utilizes recombinant fermentation: scanning dsDNA and promoting the homologous (cognate) position of the structure obtained by the single-stranded DNA-binding protein and the substituted template strand, thus preventing the primer from being displaced by the branched mobilin to replace the DNA polymerase Sub til is a large segment of Pol I (m )) close to the end, and the primer then begins to extend. This nucleic acid amplification is repeated by circulation. The helicase amplification (HDA) mimics the in vivo system using DNA helicase to generate primers for hybridization with DNA polymerase extension primers. In the HDA reaction, the helicase passes through the target DNA, destroying the two strands and binding by a single-stranded binding protein. The primer is allowed to adhere by the heliostat. The DNA polymerase uses autologous triphosphate (dNTP) to subsequently replicate the DNA of each primer. Two replicates into the next HD A cycle 'cause the target sequence to be followed by other DNA-based thermostats including RCA' in which the DNA polymerase is formed by a number of circular repeats around the circular 〇ΝΑ primer. . By terminating the reaction, the polymerase produces thousands of copies of the copy strand tethered to the original target DNA. The resulting DNA (dsDNA)-introduction mismatch comes from: stock exchange. By interaction, it is stable and released. Recombinant fermentation (such as the 31 step of Bacillus oligonucleotides to reach the index, in the first step of the single-strand template merging in the in vivo system, the hydrogen bond, the deoxygenation of the two strands with the single-strand target area The 3' end of the ribonucleotide to generate two dsDNA strands independently exponentially nucleic acid amplification by rolling-cycle amplification (templates are continuously extended. Long DNA products. Circular template, which has: a spatial solution to the target-13 - 201209403 Rapid nucleic acid amplification of resolution and signal. Up to 12 copies of the template can be generated in 1 hour. Branched amplification is a variant of RCA and utilizes a closed circular probe (C-probe) or a lock Probes and highly progressive DNA polymerases to exponentially amplify C-probes at ambient temperatures. Circular thermostat amplification (LAMP) provides high selectivity and utilizes DNA polymerase and contains four specially designed primers In the introduction group, the primer group recognizes a total of six different sequences on the target DNA. The inner primer of the sense strand containing the target DNA and the antisense strand sequence starts the LAMP. The subsequent strand-initiated DNA synthesis release sheet is triggered by the external primer. Strand DNA. This is used as the second inner and outer primer The template for DNA synthesis initiated, the second inner and outer primers hybridize to the other end of the target to produce a stem-loop DNA structure. In the subsequent LAMP cycle, the inner primer hybridizes to the loop on the product. The initial substitution DNA synthesis produces the original stem-loop DN A and the new stem-loop DNA with twice the stem length. The cyclic reaction is continued for less than one hour to accumulate 1 〇 9 copies of the target. The final product has several Stem-loop DNA of an inverted repeat target, and a cauliflower-like structure with a plurality of loops (alternatingly adhering to inverted repeat targets in the same strand). After completion of nucleic acid amplification, the amplified product must be analyzed. To determine whether the expected amplicon (amplification amount of the target nucleic acid) is produced. The method of analyzing the product is to simply measure the size of the amplicon by colloidal electrophoresis and use DNA hybridization to identify the nucleotide composition of the amplicon. Electrophoresis is one of the simplest ways to check whether a nucleic acid amplification step produces the desired amplicon. Colloidal electrophoresis uses an electric field applied to a colloidal matrix to separate DNA fragments. Negatively charged DNA fragments will be at different rates ( The main-14-201209403 depends on its size) moving through the matrix. After the electrophoresis is completed, the fragments in the color colloid make it visible. Fluorescent phenanthrene in the UV light is the most commonly used dye. The size marker (DNA standard fragment (DNA)) is compared to determine the size of the fragment, and the DNA size label contains a DNA fragment of a size that is run along with the amplicon. Because the oligonucleon binds to a specific position adjacent to the target DNA, Amplification yields can be predicted and detected in bands of known size on the colloid. When so, or if several amplicons are produced, DNA probe hybridization is often used. DNA hybridization means complementary bases. DNA pairing to form a double strand for positive recognition of a particular amplification product requires the use of a DNA probe of about 20 nucleotides. If the probe has a sequence complementary to the amplicon DNA sequence, hybridization will occur under favorable temperature, ion concentration conditions. If hybridization occurs, it indicates that the DNA sequence of interest appears in the original sample. Optical detection is the most common method of detecting hybridization. The label extension is a probe that emits light by fluorescing or electrochemiluminescence. These trigger the excited states of the luminescent portion in different ways, but both are also covalently labeled with acid stocks. In electrochemiluminescence (ECL), when excited, light is generated by a luminophore molecule or a complex. When the fluorescent light is emitted, the emitted excitation light emits light. A luminescent source is used to detect fluorescence, and the illuminating source provides excitation light having a wavelength of fluorescence absorption and a detection unit. The detection unit contains photosensing, and the dyeable bromination ladder has a large amplification and amplification of DNA with known nucleotide primers. The length is (target pH and gene or enhancer or method of nucleoside current thorns to generate light molecules (the -15-201209403 such as photomultiplier tubes or charge coupled device (CCD) arrays) to detect the emitted signal, And a mechanism that prevents excitation light from being included in the output of the photosensor (such as a wavelength-selective filter). In response to the excitation light, the fluorescent molecules emit Stokes-shifted light, and the emitted light is detected. Unit collection. The Tox displacement is the frequency difference or wavelength difference between the emitted light and the absorbed excitation light. The light sensor is used to detect the ECL emission, and the light sensor is sensitive to the emission wavelength of the ECL type used. For example, a transition metal coordination complex emits light of a visible wavelength, thus using a conventional photodiode and a CCD as a photosensor. The advantage of ECL is that if the ambient light is excluded, the ECL emission can be the only light present in the detection system. Thus, the sensitivity of the microarray enables hundreds of thousands of DNA hybridization experiments to be performed simultaneously. Microarrays are useful molecular diagnostic tools that can screen thousands of genetic diseases or The presence or absence of several infectious pathogens is detected in a single assay. The microarray consists of a number of different DNA probes immobilized on a substrate and spotted. First, the target DNA (amplifier) is labeled with fluorescent or luminescent molecules. (during or after nucleic acid amplification), then applying it to the probe array. The microarray is cultured for several hours or days at a controlled temperature in a humid environment, where the probe and the amplicon occur. Hybridization. After incubation, the microarray must be washed with a series of buffers to remove unbound strands. Once cleaned, the surface of the microarray is dried with a stream of air (usually nitrogen). The stringency of hybridization and cleaning is important. This results in a highly non-specific binding. Excessive stringency may result in inability to properly bind to a reduced sensitivity. Hybridization is identified by detecting the light emission from the labeled amplicon that hybridizes with the complementary probe to detect-16-201209403. The microarray scanner detects fluorescence from the microarray, which is typically a computer controlled inverted scanning fluorescent conjugated focus microscope. A laser and photosensor (such as a photomultiplier tube or CCD) that excites the fluorescent dye is typically used to detect the emitted signal. The fluorescent molecules emit down-converted light (as described above) and the light is collected by the detection unit. The fluorescent light must be collected, separated from the unabsorbed excitation wavelength, and transmitted to the detector. A conjugate focal configuration is often used in microarray scanners to remove out-of-focus by conjugated focal pin holes located in the image plane. Information. This allows only the focused portion of the light to be detected. Light above or below the focal plane of the object is prevented from entering the detector, thereby increasing the signal-to-noise ratio. The detector converts the detected fluorescent photons into electrical energy, electrical energy. It is then converted to a digital signal. This digital signal is converted to a number representing the intensity of the fluorescence from a given pixel. Each feature of the array consists of one or more of these pixels. The final result of the scan is an image of the surface of the array. Since the exact sequence and position of each probe on the microarray is known, the hybrid target sequence can be identified and analyzed simultaneously. Find out more about fluorescent probes here: http : "www.premierbiosoft.com/tech_notes/FRET_probe.html and http : "www.invitrogen.com/site/us/en/home/References/ Molecular -probes-The-Handbook/Technical-Notes-and-Product-
Highlights/Fluorescence-Resonance-Energy-Transfer- -17- 201209403 FRET.html 就地醫護分子診斷 儘管分子診斷試驗提供了優勢,臨床檢驗中此類型試 驗的成長不如預期且仍僅占檢驗醫學之實施的小部分。此 主要歸因於,與基於非關核酸方法之試驗相比,核酸試驗 相關之複雜度與成本。分子診斷試驗之於臨床處理的廣泛 適用性係與可顯著降低成本、提供自始(樣本處理)至終 (產生結果)之快速及自動化分析,以及不需大量人爲操 作之儀器發展息息相關。 用於醫師診所、鄰近的或基於使用者的醫院、家中之 就地醫護技術提供以下優點: •快速得到結果而致能快速促進治療及改進照護品質 〇 •經由試驗極少量樣本而得到檢驗値的能力。 •減少臨床工作量。 •減少實驗室工作量並因減少管理工作而增進工作效 率。 •因減少住院時間、門診病人於首次就診得知結果, 及簡化樣本的處理、儲存及運送而改善每個病人所需成本 〇 •促進臨床管理決策,諸如感染控制及抗生素使用》 以晶片上實驗室(LOC)爲基礎之分子診斷 -18- 201209403 以微流體技術爲基礎之分子診斷系統提供可自動化及 加速分子診斷分析之裝置。較短之檢測時間主要是因爲所 需之樣本體積極少、自動化及在微流體裝置內之低開銷內 置級聯式之診斷方法步驟。以奈升及微升爲規模之體積亦 減少試劑消耗及成本。晶片上實驗室(LOC )裝置係常見 之微流體裝置形式。LOC裝置具有在MST層內之MST結 構以用於將流體處理整合至單一支撐基材(通常爲矽)上 。利用半導體產業之VLSI (超大型積體電路)平版印刷 技術製造使各LOC裝置之單位成本非常低廉。然而,控 制流體流經LOC裝置、添加試劑、控制反應條件等等需 要大型之外部管路及電子裝置。連接LOC裝置至這些外 部裝置大幅地限制LOC裝置之分子診斷用途於實驗室環 境中。外部儀器之費用及其操作複雜性排除以LOC爲基 之分子診斷作爲就地醫護環境中之實用選擇。 鑒於上述,需要一種用於就地醫護之基於LOC裝置 之分子診斷系統。 【發明內容】 本發明各種樣態現在描述於下列數個段落。 GCF032.1本發明之樣態提供用於生物樣本之基因分 析的晶片上實驗室(LOC )裝置’該LOC裝置包含: 入口,用以接受該樣本; 支撐基材; 透析部,用以將該樣本中的小組份與較大組份分開; -19- 201209403 複數個試劑貯槽; 該透析部下游之核酸擴增部,用以擴增該樣本中核酸 序列;其中, 該透析部和該核酸擴增部兩者均被支撐在該支撐基材 上。 GCF03 2.2較佳地,該核酸擴增部係聚合酶連鎖反應 (PCR)部。 GCF03 2.3較佳地,該LOC裝置亦具有光感測器和 PCR部下游之雜交部,該雜交部具有用於與在該樣本中之 標靶核酸序列雜交之探針的陣列以形成探針-標靶雜交體 ,其中該光感測器係經配置以檢測該探針-標靶雜交體。 GCF03 2.4較佳地,該透析部具有與該入口流體連通 之第一通道,與該PCR部流體連通之第二通道,以及複 數個大於該等小組份且小於該等大組份之孔,該第二通道 與該第一通道經由該等孔流體連通,使得當該等較大組份 保留在該第一通道同時,該等小組份流入該第二通道。 GCF03 2.5較佳地,該第一通道、該第二通道和該等 複數個孔係經配置以藉由毛細作用以該樣本塡充。 GCF03 2.6較佳地,該第二通道係經配置以藉由毛細 作用吸引該樣本至該核酸擴增部。 GCF03 2.7較佳地,該核酸擴增部係恆溫核酸擴增部 〇 GCF0 3 2.8較佳地,該等試劑貯槽各具有用以保留試 劑在其中之表面張力閥,該表面張力閥具有彎液面固定器 -20- 201209403 ,其用以固定該試劑之彎液面直到與該樣本流之接觸 該彎液面以允許該等試劑自該試劑貯槽流出。 GCF032.9較佳地,該LOC裝置亦具有自該入 該雜交部之流動路徑,其中該流動路徑係經配置以藉 細作用自該入口吸取出該樣本至該雜交部。 GCF03 2.1 0 較佳地,該LOC裝置亦具有CMOS 、溫度感測器和微系統技術(MST)層,該MST層 該PCR部,其中該CMOS電路設置在該支撐基材 MST層之間,該CMOS電路係經配置以利用溫度感 出以回饋控制該PCR部。 GCF032.il較佳地,該PCR部具有PCR微通道 中在使用期間,該樣本係熱循環以擴增核酸序列,該 微通道界定部分該樣本流動路徑且具有橫跨該流之截 小於100,000平方微米。 GCF032.1 2較佳地,該LOC裝置亦具有至少一 長之加熱器元件以加熱該延長PCR微通道中之核酸 ,該延長之加熱器元件平行延伸到該PCR微通道。 GCF032.1 3較佳地,該PCR微通道之至少一個 成延長之PCR腔室。 GCF032.1 4 較佳地,該 PCR部具有複數個延 PCR腔室,這些腔室各藉由該PCR微通道之各自部 ,該微通道具有以一系列寬曲道形成之蜿蜒結構,該 曲道各係形成該等延長之PCR腔室之一者的通道部。 GCF032.1 5較佳地,該LOC裝置亦具有用以容 移除 口至 由毛 電路 包含 和該 測輸 ,其 PCR 面積 個延 序列 部形 長之 形成 等寬 持用 •21 · 201209403 於PCR之試劑的試劑貯槽;且 具有一孔之表面張力閥,其係經配置以固定該試劑之 彎液面,使得該彎液面將該試劑保留於該試劑貯槽中直到 與該液體樣本之接觸移除該彎液面。 GCF032.1 6 較佳地,該雜交部具有用於容納該等探 針之雜交腔室陣列。 GCF032.17較佳地,該光感測器係與該雜交腔室對 準配置之光二極體陣列。 GCF03 2.1 8 較佳地,該CMOS電路具有用於儲存來 自該光感測器輸出之雜交資料的數位記憶體和用於傳輸該 雜交資料至該外部裝置之資料介面。 GCF032.19較佳地,該PCR部具有用於在熱循環期 間保留液體於PCR部中且允許流體回應自該CMOS電路 之活化訊號而進入該雜交腔室之主動閥。 GCF032.20較佳地,該主動閥係具有經配置以固定 阻止該液體之毛細驅動流的彎液面之彎液面固定器的沸騰 引發閥,以及用以沸騰該液體以自該彎液面固定器解除該 彎液面使其收回毛細驅動流的加熱器。 此種LOC裝置設計使用比簡單過濾更好之方法,其 能降低阻塞且具有能以大小爲基礎將想要的樣本組份與不 想要的樣本組份分開之優點。此種LOC裝置具有對特定 序列擴增所提供之優點,包括:擴增所提供之靈敏度;寬 動態範圍;以及對標靶DNA序列之高特異性。 -22- 201209403 【實施方式】 較佳具體實施例之詳細說明 總論 此總論指明包含本發明具體實施例之分子診斷系 主要組件。該系統構造及操作之綜合細節於以下說明 說明。 參照圖1, 2,3,96和97,該系統具有下列最重 組件: 試驗模組10和1 1爲典型USB隨身碟的尺寸且 便宜可以製得。試驗模組1 0和1 1各包含微流體裝置 型地呈晶片上實驗室(LOC )裝置30的形式,且預 試劑以及典型地1 〇〇〇個以上之用於該分子診斷分析 針(見圖1和 96 )»當在圖96中之試驗模組1 1使 電致化學發光爲基礎的檢測技術同時,槪示於圖1之 模組1 〇使用以螢光爲基礎的檢測技術以辨識標靶分 該LOC裝置30具有用於螢光或電致化學發光檢測( 於下)之整合光感測器44。試驗模組1 0和1 1兩者 用用於電源、數據和控制之標準微型USB插頭14, 有印刷電路板(PCB ) 57,且均具有外部供電之電容彳 和電感器15。該試驗模組10和11兩者均爲僅供大 造之單一用途且以可供使用之無菌包裝分銷。 外殼13具有用於接收生物樣本之大容器24及可 之無菌密封帶22,其較佳具低黏性黏著劑,以於使 覆蓋大容器。具有膜防護件4 1 0之膜密封件408形成 統的 書中 要的 非常 , 典 載有 的探 用以 試驗 子。 詳述 皆使 均具 蓉32 量製 移除 用前 部份 -23- 201209403 外殻1 3以減少試驗模組中之抗濕性,而由小氣壓 供釋壓作用。膜防護件410保護膜密封件40 8免於 經由微型-USB埠1 6,試驗模組讀取器1 2供 驗模組1 〇或1 1。試驗模組讀取器1 2可爲許多不 ,及其選擇係描述於後。圖1、3及96中所示之 12版本爲智慧型電話之具體實施例。讀取器12之 係示於圖3中。處理器42執行來自程式儲存器43 軟體》處理器42亦與顯示螢幕18及使用者界面( 控螢幕17及按鈕19、蜂巢式無線電21、無線網 23,以及衛星導航系統25界接。蜂巢式無線電21 網路連接23係用於通訊。衛星導航系統25係用於 資料更新流行病學資料庫。替代性地,能夠以觸 P或按鈕19手動輸入位置資料。資料儲存器27 傳及診斷資訊、試驗結果、患者資訊、用於識別各 分析及探針數據及其陣列位置。資料儲存器27及 存器43可共享於共同記憶體設備。試驗模組讀取f 安裝的應用軟體提供結果分析與另外的試驗及診斷 爲執行診斷試驗,將試驗模組1 0 (或試驗模条 插入至試驗模組讀取器12上的微型-USB埠16。 密封帶22翻起並將生物樣本(呈液體形式)載入 大容器24中。按下開始按鈕20以藉由應用軟體來 驗。樣本流進LOC裝置30且以機載分析(0] assay )萃取、培養、擴增及以預合成的雜交-反應 音酸探針與樣本核酸(標靶)雜交。於試驗模組1 變動提 損傷。 電給試 同形式 讀取器 方塊圖 的應用 UI)觸 路連接 及無線 以位置 控螢幕 保有遺 探針之 程式儲 I 12中 資訊。 an) 將無菌 至樣本 起始試 n-board 性寡核 〇的情 -24 - 201209403 況中(其使用基於螢光的檢測),探針係經螢光標記且置 於殼13中的LED 26提供必要激發光以誘發自經雜交探 針的螢光發射(見圖1及2)。於試驗模組1 1中(其使 用基於電致化學發光(ECL)的檢測),LOC裝置30載 有ECL探針(如上述)且LED 26對於產生發光並非必要 。反之,電極860及8 70提供激發電流(見圖97)。使 用與各LOC裝置上之CMOS電路整合的光感測器44來檢 測發射(螢光或光致發光)。擴增所檢測的信號並將其轉 換成藉由試驗模組讀取器1 2分析之數位輸出。讀取器接 著顯示結果。 可本地儲存數據及/或將數據上傳至含有患者記錄之 網路伺服器。自試驗模組讀取器1 2移除試驗模組1 0或 1 1並將彼等適當處理。 圖1、3及96顯示組態成行動電話/智慧型電話28之 試驗模組讀取器1 2。於其他形式中,試驗模組讀取器爲 醫院、私人診所或實驗室中使用之膝上型電腦/筆記型電 腦101、專用讀取器1〇3、電子書讀取器107、平板電腦 109或桌上型電腦1〇5 (見圖98)。讀取器可與一些額外 的應用程式界接,諸如病患記錄、帳務、線上資料庫及多 使用者環境。其亦可與一些本地或遠端周邊設備界接,諸 如印表機及病患智慧卡。 參照圖99,透過讀取器12及網路125,由試驗模組 1〇產生之資料可用來更新用於流行病學資料111之主機 系統所載有之流行病學資料庫、用於遺傳資料1 1 3之主機 -25- 201209403 系統所載有之遺傳資料庫、用於電子化健康記錄(EHR) 115之主機系統所載有之電子化健康記錄、用於電子化醫 療記錄(EMR) 121之主機系統所載有之電子化醫療記錄 ,以及用於個人健康記錄(PHR) 123之主機系統所載有 之個人健康記錄。相反地,經由網路1 2 5及讀取器1 2 ’ 用於流行病學資料111之主機系統所載有之流行病學資料 、用於遺傳資料113之主機系統所載有之遺傳資料、用於 電子化健康記錄(EHR ) 115之主機系統所載有之電子化 健康記錄、用於電子化醫療記錄(EMR) 121之主機系統 所載有之電子化醫療記錄,以及用於個人健康記錄(PHR )123之主機系統所載有之個人健康記錄可用以更新試驗 模組10之LOC 30中之數位記憶體》 再次參照圖1、2、96及97,於行動電話組態中,讀 取器12使用電池電力。行動電話讀取器含有所有預載的 試驗及診斷資訊。經由一些無線或接觸界面亦可載入或更 新資料以致能與週邊裝置、電腦或線上伺服器連通。設置 微型-USB埠16以連接電腦或主要電力供應以再充電電池 〇 圖70顯示試驗模組1 0之具體實施例,其係用於僅需 要得知特定標靶存在與否之試驗,諸如試驗個人是否受到 例如A型流行性感冒病毒Η 1 N 1感染。僅作爲內建之僅供 USB電力/指示器之模組47爲適當的。不需要其他讀取器 或應用軟體。僅供ϋ S B電力/指示器之模組4 7上之指示 器45不出正或負結果。此組態非常適於大量飾檢β -26- 201209403 供應給系統的額外物件可包括含有供預處理特定樣本 之試劑的試驗管,及包含供樣本收集之壓舌板及刺血針。 爲便利之故,圖70顯示之具體實施例的試驗模組包括有 簧壓式可伸縮刺血針3 9 0及刺血針釋出按鈕3 9 2。可於遠 端地區使用衛星電話。 試驗模組電子裝置 圖2和9 7各自爲試驗模組1 〇和1 1中之電子組件的 方塊圖。整合於該LOC裝置30之該CMOS電路具有USB 裝置驅動器36、控制器34、USB相容LED驅動器29、計 時器33、電源調節器31、RAM3 8和程式及資料快閃記憶 體40。這些提供用於整個包括該光感測器44、該溫度感 測器1 7 0、該液體感測器1 7 4和各種加熱器1 5 2、1 5 4、 182、234之試驗模組10或11以及關聯的驅動器37和39 以及暫存器3 5和4 1的控制和記憶體。僅該LED26 (在試 驗模組1 0的例子中)、外部電源電容器32和該微型USB 插頭14在LOC裝置30的外部。該晶片上實驗室裝置30 包括用於連結至這些外部組份的黏合墊。該RAM38及該 程式和資料快閃記憶體40具有用於1 〇〇〇個探針之應用軟 體和診斷與檢測資訊(快閃/保全儲存,例如經由加密) 。在配置以ECL探測之試驗模組1 1的例子中,無LEd26 (見圖96和97 )。資料由該LOC裝置30加密以保全儲 存及與外部裝置通訊。該L0C裝置30以電致化學發光探 針和該雜交腔室負載’各具有ECL激發電極對860和870 -27- 201209403 試驗模組1 0的許多類型以一些檢測形式製造’準備 好可現成使用。該等檢測形式之不同在於試劑和探針之機 載分析。 快速以此系統鑑別的感染性疾病的一些例子包括: •流行性感冒-流行性感冒病毒A、B、C、傳染性鮭 魚貧血病毒、托高土病毒 •肺炎-呼吸道融合病毒(RSV )、腺病毒、間質肺 炎病毒、肺炎雙球菌、金黃色葡萄球菌 •結核病-結核分枝桿菌' 牛型分枝桿菌 '非洲分枝 桿菌、卡氏分枝桿菌和田鼠分枝桿菌 •惡性瘧原蟲、弓漿蟲和其他寄生性原生蟲病 •傷寒-傷寒桿菌 •伊波拉病毒 •人類免疫不全病毒(HIV ) •登革熱·黃病毒 •肝炎(A到E) •醫源性感染-例如難養芽孢梭菌、抗萬古黴素腸球 菌以及抗藥性金黃色葡萄球菌 •單純泡疹病毒(HSV ) •巨大細胞病毒(CMV ) •愛彼斯坦-巴爾病毒(EBV ) •腦炎-日本腦炎病毒、章地埔拉病毒 •百日咳-百日咳菌 -28- 201209403 •麻瘍-副黏液病毒 •腦膜炎-肺炎鏈球菌和腦膜炎雙球菌 •炭疽病-炭疽桿菌 以此系統鑑別的遺傳性疾病的一些例子包括: •囊性纖維變性 •血友病 •鐮狀細胞貧血病 •黑朦性白癡病 •血色素沉著症 •腦動脈病 •克隆氏病 •多囊性腎臓病 •先天性心臓病 •蕾特氏症 由該診斷系統鑑別之癌症的少數選擇包括: •卵巢癌 •結腸癌 •多發性內分泌腫瘤 •視網膜母細胞瘤 •透克氏症(Turcot syndrome) 上述清單並非詳盡無疑的,且該診斷系統可被配置以 使用核酸和蛋白質分析偵測許多不同疾病以及症狀。 系統組份的詳細結構 -29- 201209403 LOC裝置 L 0 C裝置3 0爲診斷系統之中心。其使用微流體 快速實施以核酸爲基礎之分子診斷分析的四個重要步 即樣本製備、核酸萃取、核酸擴增和檢測。LOC裝置 有替代的用途,並將詳述於下。如上述討論,試驗 1 〇及1 1可採取許多不同組態以檢測不同的標靶。同 ,LOC裝置30具有很多針對關注的標靶打造之不同 實施例。LOC裝置30之一種形式爲用於全血樣本之 體中的標靶核酸序列之螢光檢測之LOC裝置301。爲 述的目的,LOC裝置301的結構和操作係參考圖4 Ϊ 及27至57而詳細描述。 圖4爲LOC裝置301結構之圖式槪要。爲了便 ,顯示於圖4的處理階段係以相應於實施處理階段之 裝置301的功能部之元件符號表示。與各個以核酸爲 的分子診斷分析的主要步驟有關的處理階段亦表示: 輸入及製備288、萃取290、培養291、擴增292以 測2 94。LOC裝置30 1之各種貯槽、腔室、閥以及其 件將於以下更仔細的描述。 圖5爲 LOC裝置 301之透視圖。其使用高 CMOS和M ST (微系統技術)製造技術而製造。LOC 3 〇 1之層狀構造以圖1 2之槪要部分剖面圖(非按比 閫述。LOC裝置301具有支持COMS + MST晶片48之 板84,包含CMOS電路86和MST層87,以蓋46 MST層87。爲了本專利說明書目的,術語“ MS T層 平台 驟, 亦具 模組 樣地 具體 病原 了闡 g 26 利性 LOC 基礎 樣本 及檢 他組 容積 裝置 例) 矽基 覆蓋 ”關 -30- 201209403 於以不同試劑處理樣本之結構和層之集合。因此,這些結 構和組件經組態以定義具有特性尺寸的流動路徑,其支持 具處理期間之物理性質與樣本之物理性質相似之毛細作用 驅動之液體流。據此,MST層和組件通常使用面型微加 工技術和/或體型微加工技術製造。然而,其他製造方法 亦可製造針對毛細作用驅動之液體流及加工非常小容積而 尺寸化的結構和組件。描述於本說明書之特定具體實施例 顯示MST層爲支持在CMOS電路86上之結構和主動組件 ,但排除蓋46之特徵。然而,熟此技藝者將理解MST層 不需要下方的CMOS或甚至不需要上覆的蓋來使其處理該 樣本。 顯示於下列圖式的LOC裝置之整體尺寸爲1 760微米 X5824微米。當然,爲了不同應用而製造的LOC裝置可具 有不同的尺寸。 圖6顯示與蓋特徵疊置之MST層87的特徵。顯示於 圖6中之插圖AA至AD、AG和AH個別放大於圖13、14 、35、56、55和63中,且對LOC裝置301內之各個結構 的充分了解詳細描述於下。當圖11獨立顯示CMOS+ MST 裝置48結構時,圖7至10獨立顯示蓋46的特徵。 層狀構造 圖12和22爲槪略顯示該CMOS + MST裝置48之層狀 構造、該蓋46以及該兩者之間的流體交互作用之略圖。 該些圖表爲了圖示說明目的所以沒有依照比例繪製。圖 -31 - 201209403 12爲通過該樣本入口 68之槪要剖面圖且圖22爲通過該 貯槽 54之槪要剖面圖。如最佳顯示於圖 12’該 CMOS + MST裝置48具有矽基板84,其支撐著操作上述該 MST層87內之有效元件之該CMOS電路86»鈍化層88 密封且保護該CMOS層86免於流體流經該MST層87。 流體流經於該蓋層46及MST通道層100中之各自地 該蓋通道94及該MST通道90兩者(見例如圖7和16 ) 。當在該較小的MST通道90實施生化處理同時,細胞運 送發生在於該蓋46中製造之該較大的通道94中。細胞運 送通道按尺寸製作以便能運送該樣本中之細胞至該MST 通道90中之預定點。運送尺寸大於20微米的細胞(例如 ,某些白血球)需要通道尺寸大於20微米,且因此橫跨 該流的截面積大於4 00平方微米。特別在不需要運送細胞 的LOC中的位置之MST通道可以顯著地小。 將理解的是該蓋通道94和MST通道90爲同屬參考 且特別的MST通道90亦可根據其特定的功能參照爲(例 如)經加熱的微通道或透析MST通道。MST通道90藉由 蝕刻通過在該鈍化層8 8上沉積且以光阻劑圖案化之MST 通道層1〇〇形成。該MST通道90由頂部層66環繞,該 頂部層形成該CMOS + MST裝置48之頂部(相對於顯示於 圖中之方位)。 儘管有時作爲獨立的層顯示,該蓋通道層和該貯 存層78係自單一材料片形成。當然,該材料片亦可爲非 單一性。材料片係自兩邊蝕刻以形成蓋通道層與貯存 -32- 201209403 層78,在蓋通道層80中蝕刻該蓋通道94,在貯存層78 中鈾刻貯槽54、56、58、60和62。另外,該貯槽和該蓋 通道由微成形加工方法形成。蝕刻和微成形加工技術兩者 皆用以製造具有橫跨該流體的截面積與20,000平方微米 一樣大及與8平方微米一樣小的通道。 於該LOC裝置中不同位置有針對橫跨該流體之通道 的截面積之一系列適當的選擇。其中大量的樣本或具有大 組份的樣本係容納於該通道,高於20,00 0平方微米之截 面積(例如,在100微米厚之層中的200微米寬的通道) 是適合的。其中少量的液體或無大細胞存在的混合物係容 納於該通道,較佳係橫跨該流體之非常小的截面積。 下部密封64環繞該蓋通道94且該上密封層82環繞 該貯槽 54、56、58、60 和 62。 該五個貯槽54、56、58、60和62係預裝載分析特定 之試劑。此描述的具體實施例中,該貯槽預裝載下列試劑 ,但可簡易的以其他試劑取代: •貯槽54:抗凝血劑,其具有包括紅血球溶胞緩衝 液的選擇 •貯槽5 6 :溶胞試劑 •貯槽5 8 :限制酵素、接合酶和聯結子(用於聯結 子-引發 PCR (見圖 69,自 τ· Stachan et al.,Human Molecular Genetics 2,Garland Science, NY and London, 1 999節錄) •貯槽60:擴增混合物(去氧核苷酸三磷酸(ciNTPs -33- 201209403 )、引子、緩衝液)以及 •貯槽62 : DNA聚合酶 該蓋46和該CMOS + MST層48經由在該下部密封64 和該頂部層6 6中之相應的開口流體連通。該等開口係依 據流體是否自該MST通道90流至該蓋通道94或相反而 代表上管道96及下管道92。 LOC裝置操作 該LOC裝置301的操作係參考在血液樣本中之分析 病原體DNA逐步描述於下。當然,其他生物或非生物液 體的種類亦使用適當的試劑、檢測規程、L Ο C變體和偵測 系統之套組或組合分析。參考至圖4,分析生物樣本涉及 五個主要步驟,包含•樣本輸入和準備288、核酸萃取 290、核酸培養291、核酸擴增292和偵測及分析294。 該樣本輸入和準備步驟288涉及混合該血液與抗凝血 劑1 1 6且接著以該病原體透析部70將病原體與白血球和 紅血球分開。如最佳顯示於圖7和1 2中,該血液樣本經 由該樣本入口 68進入該裝置。毛細作用吸引該血液樣本 沿著該蓋通道94至該貯槽54。當該樣本血流開啓其表面 張力閥1 1 8時,抗凝血劑自該貯槽54釋出(見圖15和 22 )。該抗凝血劑可防止形成會阻塞流動的血凝塊。 如最佳顯示於圖22中,該抗凝血劑116藉由毛細作 用自該貯槽54抽出且經由該下管道92進入該MST通道 90。該下管道92具有毛細起始構造特徵(CIF) 102以形 -34- 201209403 成彎液面的幾何形狀,使其不固定在該下管道92的邊緣 。當該抗凝血劑116自該貯槽54抽出時,在該上部密封 82中之通氣孔122允許空氣取代該抗凝血劑〗16。 顯示於圖22之該MST通道90爲表面張力閥118的 —部分。該抗凝血劑116塡充該表面張力閥118且固定至 該上管道96之彎液面120於彎液面固定器98。在使用前 ’該彎液面120維持固定於該上管道96,使得該抗凝血 劑不會流入該蓋通道94。當該血液流經該蓋通道94至該 上管道96時,移除該彎液面1 20且將該抗凝血劑吸入該 流體。 圖15至21顯示插圖AE,其爲顯示於圖13之插圖 AA之一部分。如顯示於圖15、16和17,該表面張力閥 1 18具有三個獨立的MST通道90延伸於個別的下管道92 及上管道96之間。在表面張力閥中之這些MST通道90 可變化以改變進入該樣本混合物之試劑得流速。如由擴散 所混合在一起之該樣本混合物以及該些試劑,離開該貯槽 之流速決定在該樣本流中之試劑的濃度。因此,每各該貯 槽的該表面張力閥配置以符合該所需之試劑濃度。 該血液通過進入病原體透析部70(見圖4和15), 其中標靶細胞使用根據預定閥値制定大小之孔1 64的陣列 自該樣本濃縮。小於該閥値的細胞通過該些孔,而大細胞 不能通過該些孔。在該標靶細胞持續作爲分析的一部分同 時,不欲之細胞重新被導入廢料單元76。該不欲之細胞 爲經由該等孔1 64陣列阻擋之大細胞,或通過該等孔之小 -35- 201209403 細胞。 在描述於此之病原體透析部70中,自該全血 病原體濃縮以供微生物DNA分析。該些孔之陣列 體連通該蓋通道94中之輸入流至標靶通道74的許 米直徑的孔1 64而形成。該3微米直徑的孔1 64和 標靶通道74之該透析吸入孔1 68係由一系列的透 通道2 04連接(最佳顯示於圖15和21)。病原體 以經由該透析MST通道204通過該3微米直徑孔 塡充該標靶通道74。大於3微米的細胞諸如紅血 血球留在在該蓋46之該廢料通道72中,該廢料通 廢料貯槽76 (見圖7)。 其他孔形狀、大小和長寬比可用以分離特定病 其他標靶細胞諸如用於人類DNA分析的白血球。 供透析部和透析變體更詳細的詳情。 再參照圖6和7,該流體被吸入通過該標靶3 至該溶胞試劑貯槽56中之該表面張力閥128。該 力閥128具有七個MST通道90延伸於該溶胞試 5 6和該標靶通道74之間。當該彎液面由該樣本流 ,自所有七個該MST通道90之該流速將大於自該 劑貯槽54之流速,其中該表面張力閥118具有三1 通道90 (假設該流體的物理特性爲大致相等的) 在該樣本混合物中之溶胞試劑的比例係大於該抗凝 比例。 該溶胞試劑和標靶細胞在該化學溶胞部1 3 0內 樣本之 藉由流 多3微 用於該 析MST 小到足 164且 球和白 道通向 原體或 稍後提 I道74 表面張 劑貯槽 脫除時 抗凝血 圆MST 。因此 血劑之 之標靶 -36- 201209403 通道74中藉由擴散混合。沸騰引發閥126停止該流動直 到擴散和溶胞發生了足夠的時間,自該標靶細胞釋放該遺 傳物質(見圖6和7)。該沸騰引發閥之結構和操作參考 圖31和32詳細描述於下。其他主動閥種類(與被動閥相 反之諸如該表面張力閥118)亦已由申請人開發,其可用 於此以替代該沸騰引發閥。這些替代閥設計亦描述於下。 當該沸騰引發閥126開啓時,該經溶胞之細胞流入混 合部131以預擴增限制酶切(restriction digestion)以及 聯結子接合(linker ligation)。 參考圖13,當該流體移除在混合部131起始之表面 張力閥1 3 2上的彎液面時,限制酵素、聯結子和接合酶自 該貯槽5 8釋放。該混合物爲了擴散混合流經該混合部 131的長度。在該混合部131的末端爲通到該培養部114 之該培養器入口通道133的下管道134(見圖13)。該培 養器入口通道133將該混合物饋入經加熱之微通道210的 蜿蜒構造,其提供在限制酶切以及聯結子接合期間保留該 樣本之培養腔室(見圖13和14)。 圖23、24、25、26、27、28和29顯示在圖6之插圖 AB內的LOC裝置301之該等層。各個圖顯示形成該 CM0S + MST層48和該蓋46結構之該等層的連續附力口。 插圖AB顯示該培養部114的結束和該擴增部112的開始 。如最佳顯示於圖14和23,該流體塡入該培養部114之 該等微通道210直到抵達該沸騰引發閥106,其中該流體 在擴散發生同時停止。如上所討論,該沸騰引發閥1 06上 -37- 201209403 游之該微通道210成爲含有該樣本、限制酵素、接合 聯結子的培養腔室。該加熱器154之後啓動且維持穩 度以針對一段特定時間用於發生限制酶切和聯結子接 熟此技藝者將理解此培養步驟29 1(見圖4)爲 的且只需要於一些核酸擴增分析類型。再者,在一些 中,可能需要在該培養期間的末端具有一個加熱步驟 溫度增高到超過培養溫度。在進入該擴增部1 1 2前該 增高使該限制酵素和接合酶不活化。當使用恆溫核酸 時,限制酵素和接合酶的不活化具有特定關聯。 培養之後,該沸騰引發閥106啓動(開啓)且該 再流回該擴增部112。參考圖31和32,該混合物塡 經加熱微通道1 5 8之蜿蜒結構直到到達該沸騰引發閥 ,該等微通道形成一或更多擴增腔室。如最佳顯示 30之剖面示意圖,擴增混合物(dNTPs、引子、緩衝 自貯槽60釋放且聚合酶接著自貯槽62釋放進連接該 部和該擴增部(各爲1 1 4和1 1 2 )之該中介M S T通道 〇 圖35至51顯示在圖6之插圖AC中的LOC裝置 之層。各圖顯示連續疊加形成CMOS + MST裝置48 46結構之層。插圖AC係擴增部112的末端和雜交及 部5 2的起始。經培養的樣本、擴增混合物和聚合酶 微通道158而至沸騰引發閥1〇8。在擴散混合經足夠 後’啓動在微通道158中之加熱器154以供熱循環或 擴增。擴增混合物經歷預定數目的熱循環或預設之擴 酶和 定溫 合。 選擇 例子 以將 溫度 擴增 流體 充該 108 於圖 液) 培養 2 12 30 1 和蓋 檢測 流經 時間 恆溫 增時 -38- 201209403 間以擴增充分的標靶DNA。在核酸擴增程序 引發閥1 08開啓且流體再進入雜交及檢測部 發閥之操作更詳細描述於下。 如顯示於圖52,雜交及檢測部5 2具有雜 列110。圖52、53、54及56詳細顯示雜交 和個別雜交腔室180。雜交腔室180的入口 175,其在雜交期間防止標靶核酸、探針股和 雜交腔室180之間擴散,以防止錯誤的雜交檢 散屏障1 75之流動路徑長度足夠長以在探針和 及檢測訊號的時間內,防止標靶序列和探針從 散出且污染另一腔室,因此避免錯誤的結果。 另一防止錯誤讀取的機制是在一些該雜交 相同的探針。該CMOS電路86自相對於包含 之雜交腔室180之光二極體184導出單筆結果 筆結果中異常的結果可被忽略或給以不同比重 供給雜交所需的熱能係由CMOS控制加熱 供(更詳細描述於下)。在該加熱器啓動後, 互補標靶探針序列之間。在該CMOS電路86 驅動器29傳送訊息使位於該試驗模組1〇之 。彼等探針僅於當雜交發生時發螢光從而避免 以移除未繫結的股之清洗和乾燥步驟。雜交強 探針186之該莖-及-環結構打開,其允許該螢 應該LED激發光的螢光能量,詳述於下。螢 雜交腔室180下之該CMOS電路86中之光二 之後,沸騰 52。沸騰引 交腔室之陣 !室陣列1 1 0 爲擴散屏障 雜交探針於 測結果。擴 核酸雜交以 一個腔室擴 腔室中具有 相同的探針 。導出該單 〇 器182所提 雜交發生於 中之該LED LED26發光 通常需要用 丨制該 FRET 光團發射回 光由位於各 極體1 8 4所 -39- 201209403 偵測(見下面之雜交腔室敘述)》用於所有雜交腔室之該 光二極體184以及相關的電子裝置共同形成該光感測器 44 (見圖64 )。在其他實施例,該光感測器可爲電荷耦 合裝置陣列(CCD陣列)。自該光二極體1 84偵測之訊 號被放大且轉換成可以由該試驗模組讀取器1 2分析的數 位輸出。該偵測方法進一步的細節描述於下。 LOC裝置之其他詳細說明 模組化設計 LOC裝置301具有許多功能部,包括試劑貯槽54、 56、58、60及62、透析部70、溶胞部130、培養部114 及擴增部1 12、閥類型、增濕器及濕度感測器。於LOC裝 置之其他具體實施例,可省略此等功能部,可附加另外的 功能部或用於上述裝置之替代用途的功能部。 例如,可使用培養部1 1 4作爲重複序列擴增分析系統 之第一擴增部1 1 2,且使用化學溶胞試劑貯槽5 6來加入 引子、dNTP及緩衝液的第一擴增混合,並且使用試劑貯 槽58來添加反轉錄酶及/或聚合酶。若樣本需進行化學溶 胞,亦可添加化學溶胞試劑(連同擴增混合)至貯槽56 ,或替代性地,可藉由加熱樣本一段預定的時間以在培養 部中發生熱溶胞。在一些具體實施例中,若需要化學溶胞 並使化學溶胞試劑與此混合分離,可在用於引子、dNTP 及緩衝液的混合之貯槽5 8之毗連上游合倂另外的貯槽° 於一些情況中,欲省略諸如培養步驟2 9 1之步驟。於 -40- 201209403 此情況中,可特別地製造LOC裝置以免去試劑貯槽5 8及 培養部114或是貯槽可不止載有試劑,或若存在主動閥, 其不被啓動來分配試劑至樣本流中,及培養部單純成爲將 樣本自溶胞部130傳送至擴增部112之通道。加熱器係獨 立地操作,因此當反應仰賴熱時,諸如熱溶胞,令加熱器 不於此步驟期間啓動,確保熱溶胞不會發生在不需熱溶胞 之LOC裝置中。透析部70可位於微流體裝置內之流體系 統的開端,如圖4中所示者,或可位於微流體裝置內之任 何其他位置。於一些情況中,例如,於擴增階段292之後 ,雜交及檢測步驟2 94之前,進行透析以移除細胞碎片係 有利者。替代性地,可於LOC裝置上任何位置合倂二或 多個透析部。同樣地,可合倂另外的擴增部1 1 2以致能在 雜交腔室陣列1 1 〇中利用特定核酸探針進行檢測之前之多 標靶的同時或連續擴增。爲分析例如其中不需要進行透析 之全血液的樣本,簡單地於LOC設計之樣本輸入及製備 部2 88省略透析部70。於一些情況中,即便分析不需要 進行透析,不必要於LOC裝置省略透析部70。若透析部 的存在不會造成幾何性阻礙,仍可使用於樣本輸入及製備 部具有透析部70之LOC而不會損失所需之功能。 此外,檢測部294可包括蛋白質體室陣列,其係與雜 交腔室陣列相同但載有設計成與存在於非擴增之樣本中之 樣本標靶蛋白質共軛或雜交之探針,而不是設計用來與標 靶核酸序列雜交之核酸探針。 將了解的是’爲用於此診斷系統而製造之LOC裝置 201209403 係不同之根據特別LOC應用而選擇的功能部之組合。絕 大部分之功能部常見於許多LOC裝置,而針對新應用之 額外的LOC裝置之設計,有關於自現存LOC裝置中所使 用之大幅功能部選項中組構適當功能部之組合。 本說明中僅顯示少數LOC裝置,並顯示一些其他者 以闡述爲此系統所製造之LOC裝置的設計彈性。熟此技 藝者將可輕易地明白本文所示之LOC裝置並非窮舉,且 許多另外的LOC設計係關於組構適當功能部之組合。 樣本類型 LOC變體可接受及分析各種呈液體形式之樣本類型之 核酸或蛋白質內容,液體形式包括,但不限於,血液及血 液產物、唾液、腦脊髓液、尿液、精液、羊膜液、臍帶血 、母乳、汗液、肋膜積液、淚液、心囊液、腹腔液、環境 水樣本及飲料樣本。亦可使用LOC裝置分析得自巨觀核 酸擴增之擴增子;於此情況中,所有試劑貯槽將爲空的或 是係組態成不釋出其內容物,並僅使用透析、溶胞、培養 及擴增部來將樣本從樣本入口 6 8傳送至供核酸檢測之雜 交腔室1 8 0,如上所述。 針對一些樣本類型,需要預處理步驟,例如於輸入至 LOC裝置中之前,可能需要使精液液化及可能需以酵素預 處理黏液以減低黏性。 樣本輸入 參照圖1及1 2,添加樣本至試驗模組1 0之大容器24 -42 - 201209403 。大容器24爲截錐,其係藉毛細作用而饋入LOC裝置 301之入口 68。於此,其流至64μηι寬χ60μιη深之蓋通道 94中並亦藉由毛細作用而被吸引至抗凝劑貯槽54。 試劑貯槽 使用微流體裝置,諸如LOC裝置301 ’之分析系統所 需之小量試劑使得試劑貯槽含有生化處理之所有必須試劑 ,且各試劑貯槽爲小體積。此體積確實小於 1,0 0 0,0 0 0,0 0 0立方微米,於絕大多數的情況中係小於 300,000,000立方微米,普通小於70,000,000立方微米, 及於圖式中顯示的 LOC裝置 301的情況中係小於 20,000,000立方微米。 透析部 參照圖15至21、33及34,病原體透析部70係經設 計以濃縮來自樣本之病原體標靶細胞。如前述者,頂部層 66中呈直徑爲3微米之孔口 164之複數個孔口,過濾來 自大量樣本之標靶細胞。當樣本流經直徑爲3微米之孔口 164,微生物病原體通過孔而進入一系列透析MST通道 204並經由16μιη透析汲取孔168回流至標靶通道74中( 見圖33及34)。剩餘的樣本(紅血球等)滞留於蓋通道 94中。於病原體透析部70之下游,蓋通道94成爲通往 廢料儲器76之廢料通道72。針對產生相當廢物量之生物 樣本類型,試驗模組1 0之外殼1 3內之泡沫體(f0am )插 -43- 201209403 圖或其他多孔元件4 9係組態成與廢料儲器7 6呈流體連通 (見圖1 )。 病原體透析部70係皆以流體樣本之毛細作用運作。 位於病原體透析部70上游端之直徑爲3微米之孔口 164 具有毛細作用起始特徵(CIF ) 166 (見圖33 ),以致流 體被向下拉至下方的透析MST通道2〇4之中。用於標祀 通道74之第一吸入孔198亦具有CIF 202 (見圖15)以 防止流體輕易地固定彎液面於透析吸入孔168之上。 於圖7 4中槪要顯示之小組分透析部6 8 2可具有類似 於病原體透析部70之結構。藉由尺寸化(且成形,若必 要)適於允許小標靶細胞或分子通向標靶通道並繼續進一 步分析之孔口,小組分透析部分離樣本與任何小標靶細胞 或分子。大尺寸的細胞或分子被移除至廢料儲槽766。因 此,LOC裝置30 (見圖1及96 )並不受限於分離尺寸小 於3 μιη之病原體,而可用於分離任何所欲尺寸之細胞或 分子。 溶胞部 再次參照圖7、11及13,藉化學溶胞處理,樣本中 之遺傳物質自細胞釋出。如上述者,來自溶胞貯槽56之 溶胞試劑與用於溶胞貯槽56之表面張力閥128下游之標 靶通道74中的樣本流混合。然而,一些診斷分析較佳適 合熱溶胞處理,或甚至是標靶細胞之化學及熱溶胞的組合 ◊LOC裝置301容納此及培養部114之加熱的微通道210 -44- 201209403 。樣本流塡充培養部114並停止於沸騰引發閥106。培養 微通道210將樣本加熱至細胞膜破裂之溫度。 於一些熱溶胞應用中,化學溶胞部130中不需要酵素 反應,且熱溶胞全然取代化學溶胞部130中之酵素反應。 沸騰引發閥 如以上討論者,LOC裝置301具有三個沸騰引發閥 126、106及108。於圖6中顯示這些閥的位置。圖31爲 擴增部Π2之加熱的微通道158端部之獨立的沸騰引發閥 108之放大的平面圖。 藉由毛細作用,樣本流1 1 9沿加熱的微通道1 5 8被吸 引直至到達沸騰引發閥1 08爲止。樣本流之前沿的彎液面 120固定於閥入口 146之彎液面固定器98。彎液面固定器 9 8幾何使彎液面停止前進而阻止毛細作用流。如圖31及 32中所示者’彎液面固定器98係藉由自MST通道90至 蓋通道94之上管道開口而設置之孔口。彎液面丨2〇之表 面張力使閥保持閉合。環形加熱器1 52位於閥入口 1 46的 周圍。環形加熱器1 5 2經由沸騰引發閥加熱器接點1 5 3而 受C Μ Ο S控制。 爲打開閥,CMOS電路86發送電脈衝至閥加熱器接 點1 5 3。環形加熱器1 52電阻式地進行加熱直到液體樣本 1 19沸騰爲止。沸騰使彎液面120自閥入口 146脫除並開 始濕潤蓋通道94。一但開始濕潤蓋通道94,毛細作用恢 復。流體樣本119塡充蓋通道94且流經閥下管道15〇而 -45- 201209403 至閥出口 1 48,其中毛細作用驅動之液體流沿擴增部出口 通道160前進至雜交及檢測部52之中。液體感測器174 置於用於診斷的閥之前及之後。 將能了解的是,一但沸騰引發閥被打開,則不可能再 關上。然而’因LOC裝置301及試驗模組10爲單一用途 裝置’不需要再關閉閥。 培養部及核酸擴增部 圖 6、 7、 13、 14、 23、 24、 25、 35 至 45、 50 及 51 顯示培養部114及擴增部112。培養部114具有單一的、 加熱的培養微通道2 1 0,其係經蝕刻而成爲自下管道開口 134至沸騰引發閥1〇6之MST通道層100中的蜿蜒圖案 (見圖13及14)。控制培養部114的溫度致能更有效的 酵素性反應。同樣地,擴增部112具有從沸騰引發閥106 通向沸騰引發閥108之呈蜿蜒結構之加熱的擴增微通道 158(見圖6及14)。於混合、培養及核酸擴增發生時, 此等閥中止流動以將標靶細胞保留於加熱的培養或擴增微 通道210或158中。微通道之蜿蜒圖案亦促進(在某種程 度上)標靶細胞與試劑混合。 於培養部1 1 4及擴增部1 1 2中,樣本細胞及試劑經由 使用脈衝寬度調變(PWM)之CMOS電路86所控制的加 熱器154而被加熱。加熱的培養微通道210及擴增微通道 158之蜿蜒結構之每一個曲折具有三個獨立地可操作加熱 器1 54 (延伸於彼之個別加熱器接點156之間(見圖14 ) -46- 201209403 ),其提供輸入熱通量密度之二維控制。如最佳顯示於圖 51中者,加熱器154係支撐於頂部層66上並埋入下密封 64中。加熱器材料爲TiAl,但許多其他的傳導性金屬也 適用。伸長的加熱器154平行於形成蜿蜒狀的寬曲折之各 通道部的縱向長度。於擴增部112中,經由個別加熱器控 制,可操作各寬曲折以作爲獨立的PCR腔室。 使用微流體裝置,諸如LOC裝置30 1,之分析系統所 需之小體積的擴增子允許於擴增部112中擴增使用小體積 的擴增混合物。此體積大槪小於400奈升,於絕大多數情 況中小於170奈升,普通小於70奈升,及於LOC裝置 3 0 1的情況中,此體積係介於2奈升與3 0奈升之間。 加熱速率增加及較佳擴散混合 各通道部的小截面積增加擴增流體混合物的加熱速率 。所有流體與加熱器154保持相當短的距離。減少通道截 面積(即擴增微通道158截面)至小於100,0 00平方微米 ,而較“大規模”設備具有顯著較高之加熱速率。微影製造 技術使得擴增微通道158具有橫跨小於16,000平方微米 之實質上提供較高的加熱速率之流動路徑之截面。以微影 製造技術輕易地獲致1微米級尺寸特徵。若僅需要非常小 量的擴增子(如LOC裝置301中的情況)’可使截面縮 小至小於2,5 00平方微米。針對以LOC裝置上之1,000至 2,000個探針進行且於1分鐘內之“樣本入’答案出”所需 之診斷分析,橫跨流體之適當的截面積爲400平方微米及 -47- 201209403 1平方微米之間。 擴增微通道1 58中之加熱器元件以每秒大於80絕對 溫度(K)之速率加熱核酸序列,於大多數的情況中爲每 秒大於1 〇〇 K之速率。普通地,加熱器元件以每秒大於 1 000 K之速率加熱核酸序列,以及於許多情況中,加熱 器元件以每秒大於10, 〇〇〇 K之速率加熱核酸序列。通常 ,基於分析系統的需求,加熱器元件以每秒大於1 00,000 K、每秒大於1,000,000 K、每秒大於10,000,000 K'每秒 大於 20,000,000 K、每秒大於 40,000,000 K、每秒大於 80,000,000 K及每秒大於1 60,000,000 K之速率加熱核酸 序列。 小截面積通道亦有益於任何試劑與樣本流體之擴散性 混合。於擴散性混合完成之前,靠近兩液體間之界面處, 一種液體擴散至另一液體之擴散現象最顯著。現象發生密 度隨遠離界面距離而減少。使用具相當小截面積之橫跨流 體方向之微通道,而保持兩流體靠界面流動以快速擴散混 合。縮小通道截面至小於1〇〇,〇〇〇平方微米,獲致較“大 規模”設備具有顯著較高之擴散速率。微影製造技術使得 微通道具有橫跨小於1 6000平方微米之實質上提供較高的 混合速率之流動路徑的截面。若僅需要非常小量的擴增子 (如LOC裝置301中的情況),可使截面縮小至小於 2,500平方微米。針對以LOC裝置上之1,000至2,000個 探針進行且於1分鐘內之“樣本入,答案出”所需之診斷分 析,橫跨流體之適當的截面積爲40 0平方微米及1平方微 -48- 201209403 •米之間。 短的熱循環時間 使樣本混合物保持接近加熱器且使用極小流體量’致 使核酸擴增法期間之快速熱循環。針對至高1 5 〇鹼基對( bp)長之標祀序列,於30秒內完成各個熱循環(即,變 性、黏著及引子延伸)。在絕大多數之診斷分析中’個別 熱循環時間小於1 1秒,且大部分小於4秒。針對至高 150鹼基對(bp)長之標靶序列,用於一些最常見診斷分 析之LOC裝置30的熱循環時間爲0.45秒至K5秒之間。 此速度之熱循環使得試驗模組能在遠少於1 〇分鐘之內完 成核酸擴增程序;經常爲220秒之內。針對大多數分析, 擴增部於80秒之內由進入樣本入口的樣本流體產生充足 的擴增子。針對大部分的分析,於30秒內產生充足的擴 增子。 於完成預定數目擴增循環時,經由沸騰引發閥108將 擴增子饋入雜交及檢測部52。 雜交腔室 圖52、53、54、56及57顯示雜交腔室陣列11〇中的 雜交腔室180。雜交及檢測部52具有雜交腔室180之24 X 45陣列1 10,其各具有雜交-反應性FRET’探針186、加 熱器元件182及整合的光二極體184。併入光二極體184 以檢測得自標靶核酸序列或蛋白質與FRET探針1 8 6雜交 -49- 201209403 之螢光。藉由CMOS電路86獨立地控制各光二極體184 。對發射的光而言,FRET探針186及光二極體184之間 的任何物質必須爲透明。因此,探針1 8 6及光二極體1 8 4 之間的壁部97亦必須對發射的光呈光學透明。於LOC裝 置301中,壁部97爲二氧化矽之薄層(約〇.5微米)。 於各雜交腔室180之下直接地倂入光二極體184允許 使用極小體積之探針-標靶雜交體,卻仍產生可檢測的螢 光信號(見圖54 )。因爲小量而能使用小體積的雜交腔 室。於雜交之前,可檢測的探針-標靶雜交體量所需之探 針量大槪小於2 70微微克(pic〇gram ) (對應至900,000 立方微米),於大多數的情況中小於60微微克(對應至 200,000立方微米),普通小於 12微微克(對應至 40,000立方微米),並且於附圖中所示之LOC裝置301 的情況中爲小於2.7微微克(對應至腔室體積爲9,000立 方微米)。當然,縮小雜交腔室的尺寸容許較高的室密度 及因此更多的LOC裝置上的探針。於LOC裝置301中, 於1,500微米乘1,500微米的面積內,雜交部具有超過 1,〇〇〇個腔室(即,每個腔室小於2,250平方微米)。較 小的體積亦減少反應時間,使得雜交及檢測更快速。各個 腔室需求之小量探針的另一優點爲’於L0C裝置製造期 間,僅需要配置極小量的探針溶液至各個腔室中。根據本 發明之LOC裝置之具體實施例可使用有1奈毫升或更少 之探針溶液配置。 於核酸擴增之後,沸騰引發閥108被啓動且擴增子沿 -50- 201209403 流動路徑176流動並流進各雜交腔室180(見圖52及56 )。端點液體感測器178指示雜交腔室180塡充有擴增子 及可啓動加熱器182之時點。 於充分雜交時間後,啓動LED 26 (見圖2 )»各雜 交腔室180中之開口設有光學窗136以將FRET探針186 暴露於激發輻射(見圖52、54及56 ) 。LED 26發光持 續充分長的時間以誘發自探針之高強度的螢光信號。於激 發期間,光二極體184短路(shorted)。經預編程延遲 3〇〇 (見圖2)之後,於無激發光下,致能光二極體184 及檢測螢光發射。將光二極體1 84之有效區1 85上之入射 光(見圖54)轉換成可使用CMOS電路86測量之光電流 〇 各雜交腔室1 8 0載有用於檢測單一標靶核酸序列之探 針。若希望,則各雜交腔室180可載有檢測超過1,000種 不同標靶的探針。替代性地,許多或全部雜交腔室可載有 重複地檢測相同標靶核酸之相同探針。於雜交腔室陣列 1 1 〇中以此方式複製探針使得所得結果之可信度增加,以 及若希望,可藉由相鄰雜交腔室之光二極體來合倂所有結 果以得到單一結果。熟此技藝者將了解,依據分析明細, 於雜交腔室陣列110上可具有1至超過1,000種不同的探 針。 增濕器及濕度感測器 圖6的插圖AG指示增濕器196的位置。增濕器免於 -51 - 201209403 LOC裝置3 0 1操作期間之試劑及探針的蒸發。如最佳 於圖55之放大圖中者,水貯槽188係流體地連接至 蒸發器190。水貯槽188塡充有分子生物等級用水且 造期間爲密封的。如最佳顯示於圖55及67中者,藉 細作用,水被抽拉至三個下管道1 94且沿著個別水供 道192而到達蒸發器190之三個上管道193組。彎液 定於各個上管道193以保持水。蒸發器具有環形加 191,其環繞上管道193。藉由導熱柱3 76,環形加 191係連接至CMOS電路86而至頂金屬層195 (見| )。於啓動時,環形加熱器1 9 1加熱水而致使水蒸發 潤周圍的裝置。 於圖6中亦顯示濕度感測器232的位置。然而, 如顯示於圖63中之插圖AH的放大圖者,濕度感測 有電容式梳狀結構。經微影地蝕刻之第一電極296及 微影地蝕刻之第二電極298彼此相對,使得彼等之齒 。相對的電極形成電容器,其具有可藉由CMOS電g 來監測之電容。隨濕度增加,電極間之空氣隙的介電 增加,致使電容亦增加。濕度感測器23 2鄰接雜交腔 列1 1 〇 (最主要之濕度測量位置),以減緩含有暴露 針之溶液蒸發。 反饋感測器 溫度及液體感測器係倂入LOC裝置301整體以 置操作期間提供反饋及診斷。參照圖3 5,將九個溫 顯示 三個 於製 由毛 應通 面固 熱器 熱器 I 37 並濕 最佳 器具 與經 交插 ^ 86 常數 室陣 的探 於裝 度感 -52- 201209403 測器170分配至擴增部112整體。同樣地,培養部114亦 具有九個溫度感測器1 70。這些感測器各使用2x2陣列之 雙極接面電晶體(BJT )以監測流體溫度及提供反饋至 CMOS電路86。CMOS電路86利用此以準確地控制核酸 擴增處理期間的熱循環以及熱溶胞及培養期間之任何加熱 〇 於雜交腔室180中,CMOS電路86使用雜交加熱器 182作爲溫度感測器(見圖56)。雜交加熱器182之電阻 係溫度相依,且CMOS電路86利用此以得到各雜交腔室 1 8 0之溫度讀取。 LOC裝置301亦具有一些MST通道液體感測器174 及蓋通道液體感測器208。圖35顯示於經加熱的微通道 158中之每間隔曲折之一端的MST通道液體感測器174 之線。最佳如顯示於圖3 7中者,MST通道液體感測器 174爲藉由CMOS結構86中之頂金屬層195之暴露的區 域所形成之一對電極。液體封閉電極間的電路以指示其存 在於感測器的位置。 圖25顯示蓋通道液體感測器208之放大透視圖。相 對的TiAl電極對218及220係沉積於頂部層66上。電極 218及220之間爲間隙222,以於缺少液體的情況中保持 電路爲開路。液體存在時使電路閉合及CMOS電路86利 用此反饋以監測流動。 重力自主(GRAVITATIONAL INDEPENDENCE) 試驗模組10爲方向自主。其不需被緊固至平穩表面 -53- 201209403 而操作。因毛細作用驅動之流體流以及缺少至 外部管路,使得模組確實爲可攜式並可簡易地 的可攜式手持讀取器,諸如行動電話。重力自 試驗模組亦加速度性地獨立於所有實用範圍。 耐振動並能於移動的載具上或是於攜帶的行動Highlights/Fluorescence-Resonance-Energy-Transfer- -17- 201209403 FRET. Html In-situ Nursing Molecular Diagnostics Despite the advantages offered by molecular diagnostic tests, this type of test in clinical tests has grown less than expected and still accounts for only a small portion of the implementation of laboratory medicine. This is mainly due to the complexity and cost associated with nucleic acid testing compared to experiments based on non-amino acid methods. The broad applicability of molecular diagnostic tests to clinical treatment is closely related to the rapid and automated analysis that can significantly reduce costs, provide initial (sample processing) to final (results), and the development of instruments that do not require extensive human manipulation. For physicians' clinics, proximity or user-based hospitals, home-based in-home healthcare technologies offer the following advantages: • Quick results are obtained to quickly promote treatment and improve care quality • Tested by testing a very small number of samples ability. • Reduce clinical workload. • Reduce laboratory workload and increase work efficiency by reducing management efforts. • Improve the cost per patient by reducing hospital stays, getting results from first visits, and simplifying the handling, storage, and delivery of samples. • Promoting clinical management decisions, such as infection control and antibiotic use. Laboratory (LOC)-based molecular diagnostics-18- 201209403 Microfluidic-based molecular diagnostic systems provide devices that automate and accelerate molecular diagnostic analysis. The shorter detection time is primarily due to the fact that the required sample volume is less active, automated, and low-cost built-in cascaded diagnostic method steps within the microfluidic device. The volume of nanoliters and microliters also reduces reagent consumption and cost. On-wafer laboratory (LOC) devices are in the form of common microfluidic devices. The LOC device has an MST structure within the MST layer for integrating fluid processing onto a single support substrate (typically helium). The unit cost of each LOC device is very low by using the VLSI (Ultra Large Integrated Circuit) lithography technology of the semiconductor industry. However, controlling the flow of fluid through the LOC unit, adding reagents, controlling reaction conditions, and the like requires large external piping and electronics. Connecting the LOC devices to these external devices greatly limits the molecular diagnostic use of the LOC devices in the laboratory environment. The cost of external instruments and their operational complexity excludes LOC-based molecular diagnostics as a practical option in a local healthcare setting. In view of the above, there is a need for a molecular diagnostic system based on LOC devices for in situ care. SUMMARY OF THE INVENTION Various aspects of the invention are now described in the following paragraphs. GCF032. 1 The aspect of the invention provides a wafer-on-lab (LOC) device for genetic analysis of a biological sample. The LOC device comprises: an inlet for receiving the sample; a support substrate; a dialysis portion for the sample The group is separated from the larger component; -19- 201209403 a plurality of reagent storage tanks; a nucleic acid amplification section downstream of the dialysis section for amplifying the nucleic acid sequence in the sample; wherein the dialysis section and the nucleic acid amplification section Both are supported on the support substrate. GCF03 2. Preferably, the nucleic acid amplification unit is a polymerase chain reaction (PCR) unit. GCF03 2. Preferably, the LOC device also has a photodetector and a hybridization portion downstream of the PCR portion, the hybrid portion having an array of probes for hybridizing to the target nucleic acid sequence in the sample to form a probe-label A target hybrid, wherein the photosensor is configured to detect the probe-target hybrid. GCF03 2. Preferably, the dialysis portion has a first passage in fluid communication with the inlet, a second passage in fluid communication with the PCR portion, and a plurality of holes larger than the plurality of components and smaller than the large components. The two channels are in fluid communication with the first channel via the apertures such that when the larger components remain in the first channel, the plurality of components flow into the second channel. GCF03 2. Preferably, the first channel, the second channel, and the plurality of apertures are configured to buffer the sample by capillary action. GCF03 2. Preferably, the second channel is configured to attract the sample to the nucleic acid amplification portion by capillary action. GCF03 2. Preferably, the nucleic acid amplification unit is a thermostatic nucleic acid amplification unit 〇 GCF0 3 2. Preferably, the reagent storage tanks each have a surface tension valve for retaining the reagent therein, the surface tension valve having a meniscus holder -20-201209403 for fixing the meniscus of the reagent until The sample stream contacts the meniscus to allow the reagents to flow out of the reagent reservoir. GCF032. Preferably, the LOC device also has a flow path from the hybridization portion, wherein the flow path is configured to aspirate the sample from the inlet to the hybridization portion. GCF03 2. Preferably, the LOC device also has a CMOS, temperature sensor and micro system technology (MST) layer, the MST layer of the PCR portion, wherein the CMOS circuit is disposed between the support substrate MST layers, the CMOS The circuitry is configured to utilize temperature sensing to feedback control of the PCR section. GCF032. Il preferably, the PCR portion has a PCR microchannel that, during use, thermally cycles to amplify a nucleic acid sequence that defines a portion of the sample flow path and has a cross-section that is less than 100,000 square microns across the flow. GCF032. Preferably, the LOC device also has at least one elongated heater element for heating the nucleic acid in the extended PCR microchannel, the extended heater element extending in parallel to the PCR microchannel. GCF032. Preferably, at least one of the PCR microchannels is an extended PCR chamber. GCF032. Preferably, the PCR portion has a plurality of extended PCR chambers, each of which has a respective portion of the PCR microchannel, the microchannel having a meandering structure formed by a series of wide curved tracks. Each line forms a channel portion of one of the extended PCR chambers. GCF032. Preferably, the LOC device also has a function for receiving the removal port to be included in the hair circuit and the measurement and transmission, and the PCR area is formed in a length of the sequence length. • 21 · 201209403 reagent for PCR a reagent reservoir; and a surface tension valve having a bore configured to secure a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the liquid sample removes the Meniscus. GCF032. Preferably, the hybridization portion has an array of hybridization chambers for receiving the probes. GCF032. Preferably, the photosensor is an array of photodiodes arranged in alignment with the hybridization chamber. GCF03 2. Preferably, the CMOS circuit has a digital memory for storing the hybrid data output from the photo sensor and a data interface for transmitting the hybrid data to the external device. GCF032. Preferably, the PCR portion has an active valve for retaining liquid in the PCR portion during thermal cycling and allowing fluid to respond to activation signals from the CMOS circuit into the hybridization chamber. GCF032. Preferably, the active valve has a boiling initiation valve configured to secure a meniscus retainer that blocks the meniscus of the liquid drive flow, and to boil the liquid from the meniscus holder The heater that lifts the meniscus to retract the capillary drive flow. This LOC device design uses a better method than simple filtering, which reduces clogging and has the advantage of being able to separate the desired sample components from the unwanted sample components on a size basis. Such LOC devices have the advantages provided by amplification of a particular sequence, including: sensitivity provided by amplification; wide dynamic range; and high specificity for the target DNA sequence. -22-201209403 [Embodiment] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Overview This general specification indicates the main components of the molecular diagnostic system including the specific embodiments of the present invention. The details of the construction and operation of this system are explained below. Referring to Figures 1, 2, 3, 96 and 97, the system has the following heaviest components: Test modules 10 and 11 are typical USB flash drives and are inexpensive to manufacture. The test modules 10 and 1 each comprise a microfluidic device in the form of a lab-on-lab (LOC) device 30, and pre-reagents and typically more than one or more of these are used for the molecular diagnostic assay needle (see Figures 1 and 96)»When the test module 1 in Fig. 96 makes electrochemiluminescence-based detection technology, the module 1 shown in Fig. 1 uses a fluorescence-based detection technique to identify Targeting The LOC device 30 has an integrated light sensor 44 for fluorescence or electrochemiluminescence detection (below). Both test modules 10 and 1 1 are provided with a standard micro USB plug 14 for power, data and control, a printed circuit board (PCB) 57, and each having an externally supplied capacitor 彳 and inductor 15. Both test modules 10 and 11 are for a single use for large-scale use and are distributed in sterile packaging for use. The outer casing 13 has a large container 24 for receiving a biological sample and a sterile sealing tape 22 which preferably has a low viscosity adhesive to cover the large container. The membrane seal 408 having the membrane guard 4 10 is formed in a very general book, and the test sample is used. The details are all reduced to 32. Remove the front part -23- 201209403 Shell 1 3 to reduce the moisture resistance of the test module, and the pressure is released by the small pressure. The membrane guard 410 protects the membrane seal 40 8 from the test module 1 or the test module 1 2 via the micro-USB 埠1. The test module reader 12 can be many, and its selection is described later. The 12 version shown in Figures 1, 3 and 96 is a specific embodiment of a smart phone. The reader 12 is shown in Figure 3. The processor 42 executes from the program storage 43 software processor 42 and also interfaces with the display screen 18 and the user interface (control screen 17 and button 19, cellular radio 21, wireless network 23, and satellite navigation system 25. Honeycomb The radio 21 network connection 23 is used for communication. The satellite navigation system 25 is used for the data update epidemiological database. Alternatively, the location data can be manually input by touching the P or the button 19. The data storage 27 transmits and diagnoses the information. , test results, patient information, used to identify each analysis and probe data and their array location. Data storage 27 and memory 43 can be shared in a common memory device. Test module reads f installed application software provides results analysis In addition to the test and diagnosis to perform a diagnostic test, the test module 10 (or the test strip is inserted into the micro-USB port 16 on the test module reader 12). The sealing band 22 is turned up and the biological sample is presented. The liquid form is loaded into the large container 24. The start button 20 is pressed to be tested by the application software. The sample flows into the LOC device 30 and is extracted, cultured, amplified and analyzed by onboard analysis (0) assay. The synthesized hybrid-reactive acid probe hybridizes with the sample nucleic acid (target). The damage is detected in the test module 1. The application UI of the same type reader block diagram is connected to the touch screen and wireless to position the screen. The information stored in the program is stored in the I. 12. An) sterilized to the sample to start the test of n-board oligonuclear -24-24 - 201209403 (using fluorescence-based detection), the probe is The LEDs 26 that are fluorescently labeled and placed in the shell 13 provide the necessary excitation light to induce fluorescence emission from the hybridized probe (see Figures 1 and 2). In test module 1 1 (which uses electrochemiluminescence (ECL) based detection), LOC device 30 carries an ECL probe (as described above) and LED 26 is not necessary to produce illumination. Conversely, electrodes 860 and 870 provide an excitation current (see Figure 97). A light sensor 44 integrated with a CMOS circuit on each LOC device is used to detect the emission (fluorescence or photoluminescence). The detected signal is amplified and converted to a digital output analyzed by the test module reader 12. The reader then displays the result. Data can be stored locally and/or uploaded to a web server containing patient records. The test module 10 or 1 1 is removed from the test module reader 1 2 and processed as appropriate. 1, 3 and 96 show a test module reader 12 configured as a mobile/smartphone 28. In other forms, the test module reader is a laptop/notebook 101, a dedicated reader 1〇3, an e-book reader 107, a tablet 109 used in a hospital, a private clinic, or a laboratory. Or a desktop computer 1〇5 (see Figure 98). The reader can be interfaced with additional applications such as patient records, accounting, online databases and multi-user environments. It can also interface with some local or remote peripheral devices, such as printers and patient smart cards. Referring to FIG. 99, the data generated by the test module 1 through the reader 12 and the network 125 can be used to update the epidemiological database contained in the host system for the epidemiological data 111 for genetic data. 1 1 3 Host-25- 201209403 The genetic database contained in the system, the electronic health record contained in the host system for Electronic Health Record (EHR) 115, for electronic medical records (EMR) 121 The electronic medical record contained in the host system and the personal health record contained in the host system for Personal Health Record (PHR) 123. Conversely, the epidemiological data contained in the host system for epidemiological data 111 via the network 1 2 5 and the reader 1 2 ', the genetic data contained in the host system for the genetic material 113, Electronic health records contained in the host system for Electronic Health Record (EHR) 115, electronic medical records contained in the host system for Electronic Medical Record (EMR) 121, and for personal health records (PHR) 123 The personal health record contained in the host system can be used to update the digital memory in the LOC 30 of the test module 10. Referring again to Figures 1, 2, 96 and 97, in the mobile phone configuration, read The device 12 uses battery power. The mobile phone reader contains all preloaded test and diagnostic information. Data can also be loaded or updated via some wireless or contact interface to enable communication with peripheral devices, computers or online servers. A micro-USB port 16 is provided to connect a computer or a primary power supply to recharge the battery. Figure 70 shows a specific embodiment of the test module 10 for testing that only needs to know the presence or absence of a particular target, such as an experiment. Whether the individual is infected with, for example, influenza A virus Η 1 N 1 . It is only suitable as a built-in module 47 for USB power/indicator only. No other readers or application software is required. For example, the indicator 45 on the module 4 of the S B power/indicator does not produce positive or negative results. This configuration is ideal for a large number of inspections. Additional items supplied to the system by beta -26-201209403 may include test tubes containing reagents for pre-treatment of specific samples, and tongue depressors and lancets containing sample collection. For convenience, the test module of the embodiment shown in Fig. 70 includes a spring-loaded retractable lancet 309 and a lancet release button 392. Satellite phones can be used in remote areas. Test Module Electronics Figures 2 and 9 are each a block diagram of the electronic components of test modules 1 and 11. The CMOS circuit integrated in the LOC device 30 has a USB device driver 36, a controller 34, a USB compatible LED driver 29, a timer 33, a power conditioner 31, a RAM 38, and a program and data flash memory 40. These are provided for the test module 10 including the photo sensor 44, the temperature sensor 170, the liquid sensor 174 and the various heaters 1 5 2, 1 5 4, 182, 234. Or 11 and associated drivers 37 and 39 and the control and memory of registers 35 and 41. Only the LED 26 (in the example of the test module 10), the external power supply capacitor 32, and the micro USB plug 14 are external to the LOC device 30. The on-wafer laboratory device 30 includes an adhesive pad for joining to these external components. The RAM 38 and the program and data flash memory 40 have application software and diagnostic and detection information for one probe (flash/security storage, such as via encryption). In the example of the test module 1 1 configured with ECL detection, there is no LEd 26 (see Figures 96 and 97). The data is encrypted by the LOC device 30 to preserve storage and communicate with external devices. The L0C device 30 is fabricated in a number of test forms with electrochemiluminescence probes and the hybridization chamber load 'each having many types of ECL excitation electrode pairs 860 and 870 -27-201209403 test module 10' ready for ready use . These test formats differ in the onboard analysis of reagents and probes. Some examples of infectious diseases that are quickly identified by this system include: • Influenza-influenza virus A, B, C, infectious salmon anemia virus, tocovirus • pneumonia-respiratory fusion virus (RSV), gland Virus, interstitial pneumonia virus, pneumococci, Staphylococcus aureus • tuberculosis - Mycobacterium tuberculosis ' Mycobacterium bovis 'Mycobacterium tuberculosis, Mycobacterium vaccae and Mycobacterium vaccae • Plasmodium falciparum, Toxoplasma and other parasitic protozoa • Typhoid- typhoid • Ebola • Human immunodeficiency virus (HIV) • Dengue fever • Flavivirus • Hepatitis (A to E) • Iatrogenic infections – such as refractory spores Bacteria, vancomycin-resistant enterococci and drug-resistant Staphylococcus aureus • Herpes simplex virus (HSV) • Giant cell virus (CMV) • Epstein-Barr virus (EBV) • Encephalitis - Japanese encephalitis virus, chapter Deppa virus • Pertussis-Bertus bacillus -28- 201209403 • Ma Yang-paramyxovirus • Meningitis - Streptococcus pneumoniae and meningococcus • Anthracnose - Bacillus anthracis Some examples of sexually transmitted diseases include: • Cystic fibrosis • Hemophilia • Sickle cell anemia • Black scrophular idiots • Hemochromatosis • Cerebral arterial disease • Crohn's disease • Polycystic renal rickets • Congenital Cardiac Disease • The few options for the diagnosis of cancer by Leier's disease include: • Ovarian cancer • Colon cancer • Multiple endocrine neoplasms • Retinoblastoma • Turcot syndrome The above list is not exhaustive. And the diagnostic system can be configured to detect many different diseases and symptoms using nucleic acid and protein analysis. Detailed structure of the system components -29- 201209403 LOC device L 0 C device 30 is the center of the diagnostic system. It uses microfluidics to rapidly perform four important steps in nucleic acid-based molecular diagnostic analysis: sample preparation, nucleic acid extraction, nucleic acid amplification, and detection. The LOC device has an alternative use and will be detailed below. As discussed above, trials 1 and 1 can take many different configurations to detect different targets. Again, the LOC device 30 has a number of different embodiments for targeting the target of interest. One form of LOC device 30 is a LOC device 301 for fluorescence detection of a target nucleic acid sequence in a body of a whole blood sample. For the purposes of this description, the structure and operation of the LOC device 301 are described in detail with reference to Figures 4 and 27 through 57. 4 is a schematic diagram showing the structure of the LOC device 301. For convenience, the processing stages shown in Fig. 4 are represented by the component symbols corresponding to the functional portions of the apparatus 301 that implements the processing stage. The processing stages associated with each of the major steps in the diagnostic analysis of nucleic acids are also indicated: Input and Preparation 288, Extraction 290, Culture 291, and Expansion 292 to determine 2 94. The various reservoirs, chambers, valves, and components of the LOC unit 30 1 will be described more closely below. Figure 5 is a perspective view of the LOC device 301. It is manufactured using high CMOS and M ST (microsystem technology) manufacturing techniques. The layered structure of LOC 3 〇1 is a partial cross-sectional view of Fig. 12 (not to be described in detail. LOC device 301 has a board 84 supporting COMS + MST wafer 48, including CMOS circuit 86 and MST layer 87, to cover 46 MST layer 87. For the purposes of this patent specification, the term "MS T layer platform, also has a specific sample of the pathogens. The basic sample of the LOC and the sample of the volume of the inspection group" 30- 201209403 A collection of structures and layers of samples treated with different reagents. Thus, these structures and components are configured to define a flow path having a characteristic size that supports a capillary action driven liquid flow having physical properties similar to the physical properties of the sample during processing. Accordingly, MST layers and components are typically fabricated using surface micromachining techniques and/or bulk micromachining techniques. However, other manufacturing methods can also produce structures and assemblies that are sized for liquid flow driven by capillary action and that are processed to a very small volume. The particular embodiment described in this specification shows that the MST layer is a structural and active component supported on CMOS circuitry 86, but excluding the features of cover 46. However, those skilled in the art will appreciate that the MST layer does not require the underlying CMOS or even the overlying cover to handle the sample. The overall dimensions of the LOC device shown in the following figures are 1 760 microns X 5824 microns. Of course, LOC devices made for different applications can have different sizes. Figure 6 shows the features of the MST layer 87 overlaid with the cover features. The illustrations AA to AD, AG and AH shown in Fig. 6 are individually enlarged in Figs. 13, 14, 35, 56, 55 and 63, and a sufficient understanding of the respective structures in the LOC device 301 will be described in detail below. When FIG. 11 independently shows the structure of the CMOS+ MST device 48, FIGS. 7 through 10 independently show the features of the cover 46. Layered Structure Figures 12 and 22 are schematic views showing the layered configuration of the CMOS + MST device 48, the cover 46, and the fluid interaction therebetween. The figures are not drawn to scale for illustrative purposes. Figures -31 - 201209403 12 are schematic cross-sectional views through the sample inlet 68 and Figure 22 is a cross-sectional view through the sump 54. As best shown in FIG. 12', the CMOS + MST device 48 has a germanium substrate 84 that supports the CMOS circuit 86»passivation layer 88 that operates the active components in the MST layer 87 described above to seal and protect the CMOS layer 86 from Fluid flows through the MST layer 87. Fluid flows through both the cover channel 94 and the MST channel 90 in the cap layer 46 and the MST channel layer 100 (see, for example, Figures 7 and 16). While the biochemical treatment is being performed on the smaller MST channel 90, cell transport occurs in the larger channel 94 made in the cover 46. The cell transport channel is sized to carry the cells in the sample to a predetermined point in the MST channel 90. Transporting cells larger than 20 microns in size (e.g., certain white blood cells) requires channel sizes greater than 20 microns, and thus cross-sectional areas across the flow are greater than 400 square microns. The MST channel, particularly at locations in the LOC where transport of cells is not required, can be significantly small. It will be understood that the cover channel 94 and the MST channel 90 are of the same reference and that the particular MST channel 90 can also be referred to as, for example, a heated microchannel or a dialysis MST channel depending on its particular function. The MST channel 90 is formed by etching through an MST channel layer 1 deposited on the passivation layer 88 and patterned with a photoresist. The MST channel 90 is surrounded by a top layer 66 that forms the top of the CMOS + MST device 48 (relative to the orientation shown in the figure). Although sometimes shown as a separate layer, the cover channel layer and the reservoir layer 78 are formed from a single piece of material. Of course, the sheet of material may also be non-uniform. The sheet of material is etched from both sides to form a lid channel layer and a reservoir -32 - 201209403 layer 78, which is etched in the lid channel layer 80, and uranium engraved tanks 54, 56, 58, 60 and 62 are stored in the reservoir layer 78. Further, the sump and the cover passage are formed by a micro-forming process. Both etching and microforming techniques are used to fabricate channels having a cross-sectional area across the fluid that is as large as 20,000 square microns and as small as 8 square microns. There are a series of suitable choices for one of the cross-sectional areas of the channel across the fluid at different locations in the LOC device. A large number of samples or samples having a large component are accommodated in the channel, and a cross-sectional area of more than 20,00 square micrometers (e.g., a 200 micrometer wide channel in a layer of 100 micrometers thick) is suitable. A small amount of liquid or a mixture free of large cells is contained in the channel, preferably across a very small cross-sectional area of the fluid. A lower seal 64 surrounds the cover passage 94 and the upper seal layer 82 surrounds the sump 54, 56, 58, 60 and 62. The five reservoirs 54, 56, 58, 60 and 62 are preloaded to analyze specific reagents. In the particular embodiment described, the reservoir is preloaded with the following reagents, but can be easily replaced with other reagents: • Storage tank 54: anticoagulant with a choice of red blood cell lysis buffer • Storage tank 5 6 : Dissolution Reagents • Storage tank 5 8 : Restriction of enzymes, ligases and junctions (for junction-priming PCR (see Figure 69, from τ· Stachan et al.) , Human Molecular Genetics 2, Garland Science, NY and London, 1 999 excerpt) • Storage tank 60: amplification mixture (deoxynucleotide triphosphate (ciNTPs -33 - 201209403), primer, buffer) and • storage tank 62: The DNA polymerase cover 46 and the CMOS + MST layer 48 are in fluid communication via respective openings in the lower seal 64 and the top layer 66. The openings represent the upper conduit 96 and the lower conduit 92 depending on whether fluid flows from the MST passage 90 to the cover passage 94 or vice versa. LOC Device Operation The operation of the LOC device 301 is described step by step with reference to the analysis of pathogen DNA in a blood sample. Of course, other types of biological or non-biological fluids are also analyzed using appropriate reagents, assay protocols, L Ο C variants, and detection systems. Referring to Figure 4, the analysis of biological samples involves five major steps, including • sample input and preparation 288, nucleic acid extraction 290, nucleic acid culture 291, nucleic acid amplification 292, and detection and analysis 294. The sample input and preparation step 288 involves mixing the blood with the anticoagulant 1 16 and then separating the pathogen from the white blood cells and red blood cells by the pathogen dialysis unit 70. As best shown in Figures 7 and 12, the blood sample enters the device via the sample inlet 68. Capillary action draws the blood sample along the lid channel 94 to the sump 54. When the sample blood flow opens its surface tension valve 1 18, the anticoagulant is released from the sump 54 (see Figures 15 and 22). The anticoagulant prevents the formation of blood clots that can block flow. As best shown in Figure 22, the anticoagulant 116 is withdrawn from the sump 54 by capillary action and enters the MST channel 90 via the lower conduit 92. The lower duct 92 has a capillary initiation configuration feature (CIF) 102 to form a meniscus geometry of -34 - 201209403 such that it is not fixed to the edge of the lower duct 92. When the anticoagulant 116 is withdrawn from the sump 54, the vent 122 in the upper seal 82 allows air to replace the anticoagulant. The MST channel 90 shown in Figure 22 is part of the surface tension valve 118. The anticoagulant 116 fills the surface tension valve 118 and is secured to the meniscus 120 of the upper conduit 96 to the meniscus holder 98. The meniscus 120 remains fixed to the upper conduit 96 prior to use such that the anticoagulant does not flow into the lid passage 94. As the blood flows through the lid channel 94 to the upper conduit 96, the meniscus 110 is removed and the anticoagulant is drawn into the fluid. Figures 15 through 21 show an inset AE which is part of the inset AA shown in Figure 13. As shown in Figures 15, 16 and 17, the surface tension valve 18 has three separate MST passages 90 extending between the individual lower conduits 92 and the upper conduits 96. These MST channels 90 in the surface tension valve can be varied to vary the flow rate of the reagent entering the sample mixture. The flow rate of the sample mixture in the sample stream is determined by the flow rate of the sample mixture as it is mixed by diffusion and the reagents. Thus, the surface tension valve for each of the sump is configured to meet the desired reagent concentration. The blood passes through the pathogen dialysis section 70 (see Figures 4 and 15), wherein the target cells are concentrated from the sample using an array of wells 1 64 sized according to a predetermined valve. Cells smaller than the valve pass through the holes, and large cells cannot pass through the holes. While the target cells continue to be part of the analysis, the unwanted cells are reintroduced into the waste unit 76. The undesired cells are large cells that are blocked by the array of such holes 1 64, or small cells that pass through the holes -35-201209403. In the pathogen dialysis section 70 described herein, the whole blood pathogen is concentrated for microbial DNA analysis. The array of apertures is formed by communicating the input flow in the cover channel 94 to the aperture 1 64 of the target channel 74. The 3 micron diameter hole 1 64 and the dialysis suction port 168 of the target channel 74 are connected by a series of through channels 206 (best shown in Figures 15 and 21). The pathogen is filled with the target channel 74 through the dialysis MST channel 204 through the 3 micron diameter aperture. Cells larger than 3 microns, such as red blood cells, remain in the waste channel 72 of the lid 46, which passes through the waste reservoir 76 (see Figure 7). Other pore shapes, sizes, and aspect ratios can be used to isolate specific target cells such as white blood cells for human DNA analysis. More detailed details for the dialysis department and dialysis variants. Referring again to Figures 6 and 7, the fluid is drawn through the target 3 to the surface tension valve 128 in the lysis reagent reservoir 56. The force valve 128 has seven MST channels 90 extending between the lysis test and the target channel 74. When the meniscus flows from the sample, the flow rate from all seven of the MST channels 90 will be greater than the flow rate from the agent sump 54, wherein the surface tension valve 118 has three 1 channel 90 (assuming the physical properties of the fluid are The ratio of the lysing reagent in the sample mixture is substantially equal to the anticoagulation ratio. The lysis reagent and the target cell are used in the chemical lysing portion of the sample by using 3 micrometers for the MST to be small to the foot 164 and the ball and the white channel are led to the original body or the surface of the channel 74 is later introduced. Anticoagulant circle MST when the agent storage tank is removed. Therefore, the target of the blood agent -36- 201209403 is mixed by diffusion in channel 74. The boiling initiation valve 126 stops the flow until diffusion and lysis occur for a sufficient time to release the genetic material from the target cells (see Figures 6 and 7). The structure and operation of the boiling initiation valve are described in detail below with reference to Figs. 31 and 32. Other active valve types (as opposed to passive valves such as the surface tension valve 118) have also been developed by the applicant, which can be used in place of the boiling initiation valve. These alternative valve designs are also described below. When the boiling initiation valve 126 is opened, the lysed cells flow into the mixing portion 131 for pre-amplification restriction digestion and linker ligation. Referring to Fig. 13, when the fluid is removed from the meniscus on the surface tension valve 132 starting from the mixing portion 131, the restriction enzyme, the linker and the ligase are released from the reservoir 58. The mixture flows through the length of the mixing portion 131 for diffusion mixing. At the end of the mixing portion 131 is a lower duct 134 (see Fig. 13) leading to the incubator inlet passage 133 of the cultivating portion 114. The feeder inlet channel 133 feeds the mixture into the crucible configuration of the heated microchannel 210, which provides a culture chamber that retains the sample during restriction enzyme digestion and junction assembly (see Figures 13 and 14). Figures 23, 24, 25, 26, 27, 28 and 29 show the layers of the LOC device 301 in the inset AB of Figure 6. The various figures show the continuous attachment of the layers forming the CM0S + MST layer 48 and the structure of the cover 46. The inset AB shows the end of the culture portion 114 and the start of the amplification portion 112. As best shown in Figures 14 and 23, the fluid breaks into the microchannels 210 of the culture portion 114 until the boiling initiation valve 106 is reached, wherein the fluid stops while diffusion occurs. As discussed above, the boiling initiation valve 106 on the -37-201209403 swims the microchannel 210 into a culture chamber containing the sample, restriction enzymes, and junction junctions. The heater 154 is then activated and maintains stability for the occurrence of restriction enzyme digestion and coupling for a specific period of time. Those skilled in the art will understand that this incubation step 29 1 (see Figure 4) is only required for some nucleic acid amplification. Increase the type of analysis. Furthermore, in some cases, it may be desirable to have a heating step at the end of the incubation period to increase the temperature above the culture temperature. This increase before entering the amplification section 1 1 2 causes the restriction enzyme and ligase to be inactivated. When thermostated nucleic acids are used, there is a specific association between restriction enzymes and ligase inactivation. After the cultivation, the boiling initiation valve 106 is activated (turned on) and flows back to the amplifying portion 112. Referring to Figures 31 and 32, the mixture is heated to the structure of the microchannel 1 58 until the boiling initiation valve is reached, the microchannels forming one or more amplification chambers. As best shown in cross-sectional view of 30, the amplification mixture (dNTPs, primers, buffers are released from storage tank 60 and polymerase is then released from storage tank 62 into the junction and the amplification portion (each 1 1 4 and 1 1 2 ) The intermediate MST channels are shown in Figures 35 through 51 as layers of the LOC device in the inset AC of Figure 6. Each of the figures shows a layer that is continuously stacked to form a CMOS + MST device 48 46. The end of the AC line amplification portion 112 is illustrated. Hybridization and initiation of the moiety 52. The cultured sample, amplification mixture and polymerase microchannel 158 are passed to the boiling initiation valve 1〇8. After the diffusion mixing is sufficient, the heater 154 in the microchannel 158 is activated. Heating cycle or amplification. The amplification mixture undergoes a predetermined number of thermal cycles or preset enzyme expansion and temperature stabilization. Select an example to charge the temperature amplification fluid to the liquid) culture 2 12 30 1 and cover detection The flow time is constantly increased from -38 to 201209403 to amplify sufficient target DNA. The operation of the nucleic acid amplification procedure to initiate valve 108 opening and fluid re-entry into the hybridization and detection section is described in more detail below. As shown in Fig. 52, the hybridization and detection section 52 has a matrix 110. Figures 52, 53, 54, and 56 show hybridization and individual hybridization chambers 180 in detail. The inlet 175 of the hybridization chamber 180 prevents diffusion between the target nucleic acid, the probe strand, and the hybridization chamber 180 during hybridization to prevent the erroneous hybridization detection barrier 175 from having a flow path length long enough for the probe and During the time of detecting the signal, the target sequence and the probe are prevented from escaping and contaminating another chamber, thus avoiding erroneous results. Another mechanism to prevent erroneous reading is in some of the same probes that hybridize. The CMOS circuit 86 derives a single result from the photodiode 184 containing the hybridization chamber 180. The result of the anomaly in the result can be ignored or the thermal energy required to supply the hybridization with different specific gravity is controlled by CMOS heating (more) Detailed description is given below). After the heater is activated, it is complementary between the target probe sequences. The CMOS circuit 86 driver 29 transmits a message to the test module 1 . These probes only fluoresce when hybridization occurs to avoid washing and drying steps to remove unbound strands. The stem-and-loop structure of the hybrid strong probe 186 is opened, which allows the fluorescent energy of the LED to excite the light, as detailed below. After the light in the CMOS circuit 86 under the hybridization chamber 180, the light is boiled 52. The boiling chamber is introduced into the chamber. The chamber array 1 1 0 is the diffusion barrier. The hybrid probe is used to test the results. The nucleic acid hybridization has the same probe in one chamber expansion chamber. Deriving the hybridization of the single-shot device 182, the LED LED 26 emits light, and usually needs to be used to detect the FRET light group to emit light. The detection is performed by the respective polar bodies 1 - 8 - 39 - 201209403 (see the hybrid cavity below). The photodiode 184 and associated electronics for all of the hybridization chambers together form the photosensor 44 (see Figure 64). In other embodiments, the photosensor can be a charge coupled device array (CCD array). The signal detected by the photodiode 1 84 is amplified and converted to a digital output that can be analyzed by the test module reader 12. Further details of this detection method are described below. Other Detailed Description of the LOC Device The modular design LOC device 301 has a number of functional components including reagent reservoirs 54, 56, 58, 60 and 62, a dialysis section 70, a lysis section 130, a culture section 114, and an amplification section 1 12, Valve type, humidifier and humidity sensor. In other embodiments of the LOC device, such functional portions may be omitted and additional functional portions or functional portions for alternative uses of the above-described devices may be added. For example, the culture portion 1 14 can be used as the first amplification portion 1 1 2 of the repeated sequence amplification analysis system, and the chemical lysis reagent storage tank 56 can be used to add the first amplification mixture of the primer, the dNTP, and the buffer. A reagent reservoir 58 is used to add reverse transcriptase and/or polymerase. If the sample is to be chemically lysed, a chemical lysis reagent (along with amplification mix) may be added to the sump 56, or alternatively, the sample may be heated for a predetermined period of time to cause thermal lysis in the culture. In some embodiments, if chemical lysis is required and the chemical lysis reagent is mixed with the mixture, additional storage tanks may be combined upstream of the sump for mixing the primers, dNTPs, and buffers. In the case, the steps such as the cultivation step 291 are omitted. In the case of -40-201209403, the LOC device can be specially manufactured to prevent the reagent storage tank 58 and the culture portion 114 or the storage tank from carrying more than the reagent, or if there is an active valve, it is not activated to dispense the reagent to the sample flow. The medium and the culture unit are simply passages for transferring the sample from the lysis unit 130 to the amplification unit 112. The heater operates independently, so when the reaction relies on heat, such as hot lysis, the heater is not activated during this step, ensuring that hot lysis does not occur in LOC devices that do not require hot lysis. The dialysis section 70 can be located at the beginning of the flow system within the microfluidic device, as shown in Figure 4, or can be located at any other location within the microfluidic device. In some cases, for example, after the amplification phase 292, prior to the hybridization and detection step 2 94, dialysis is performed to remove the cell debris system. Alternatively, two or more dialysis sections can be combined at any location on the LOC device. Similarly, additional amplifications 1 1 2 can be combined to enable simultaneous or sequential amplification of multiple targets prior to detection using specific nucleic acid probes in the hybridization chamber array 1 1 . To analyze, for example, a sample of whole blood in which dialysis is not required, the dialysis portion 70 is simply omitted from the sample input and preparation portion 2 88 of the LOC design. In some cases, even if the analysis does not require dialysis, it is not necessary to omit the dialysis section 70 from the LOC device. If the presence of the dialysis section does not cause geometrical obstruction, the sample input and preparation portion can still have the LOC of the dialysis section 70 without losing the desired function. In addition, the detection portion 294 can include a protein body array array that is identical to the hybridization chamber array but carries a probe that is designed to conjugate or hybridize to a sample target protein present in the non-amplified sample, rather than a design A nucleic acid probe for hybridization to a target nucleic acid sequence. It will be appreciated that the LOC device 201209403, which is manufactured for use in this diagnostic system, differs in the combination of functional components selected for the particular LOC application. Most of the functional components are common to many LOC devices, and the design of additional LOC devices for new applications has a combination of appropriate functional components in the bulk of the functional options used in existing LOC devices. Only a few LOC devices are shown in this description, and some others are shown to illustrate the design flexibility of the LOC devices manufactured for this system. Those skilled in the art will readily appreciate that the LOC devices shown herein are not exhaustive, and that many additional LOC designs are related to the combination of appropriate functional components. Sample Type LOC Variants can accept and analyze a variety of nucleic acid or protein contents in liquid form, including, but not limited to, blood and blood products, saliva, cerebrospinal fluid, urine, semen, amniotic fluid, umbilical cord Blood, breast milk, sweat, pleural effusion, tears, pericardial fluid, peritoneal fluid, environmental water samples and beverage samples. Amplicon derived from meganucleic acid amplification can also be analyzed using a LOC device; in this case, all reagent reservoirs will be empty or configured to not release their contents, and only use dialysis, lysis The culture and amplification section delivers the sample from the sample inlet 6 8 to the hybridization chamber for nucleic acid detection 180, as described above. For some sample types, a pre-treatment step is required, for example, prior to input into the LOC device, it may be necessary to liquefy the semen and possibly pre-treat the mucus with enzyme to reduce stickiness. Sample Input Refer to Figures 1 and 12 to add the sample to the large container 24-42 - 201209403 of the test module. The large container 24 is a truncated cone that is fed into the inlet 68 of the LOC unit 301 by capillary action. Here, it flows into the 64 μηι wide 60 μπη deep cover channel 94 and is also attracted to the anticoagulant storage tank 54 by capillary action. Reagent Tanks The use of microfluidic devices, such as the small amount of reagents required by the analysis system of the LOC device 301', allows the reagent reservoir to contain all of the necessary reagents for biochemical treatment, and each reagent reservoir is in a small volume. This volume is indeed less than 1,0 0 0,0 0 0,0 0 0 micron, in most cases less than 300,000,000 cubic microns, generally less than 70,000,000 cubic microns, and shown in the figure In the case of LOC device 301, it is less than 20,000,000 cubic microns. Dialysis section Referring to Figures 15 to 21, 33 and 34, the pathogen dialysis section 70 is designed to concentrate the pathogen target cells from the sample. As previously described, a plurality of apertures in the top layer 66 are apertures 164 having a diameter of 3 microns, filtering the target cells from a large number of samples. As the sample flows through a 3 micron diameter orifice 164, the microbial pathogen passes through the well into a series of dialysis MST channels 204 and is returned to the target channel 74 via a 16 [mu] dialysis extraction well 168 (see Figures 33 and 34). The remaining sample (red blood cells, etc.) is retained in the cover channel 94. Downstream of the pathogen dialysis section 70, the cover channel 94 becomes a waste channel 72 to the waste reservoir 76. The foam (f0am) insert-43-201209403 or other porous elements in the outer casing 13 of the test module 10 are configured to be fluid with the waste reservoir 76 for a type of biological sample that produces a relatively large amount of waste. Connected (see Figure 1). The pathogen dialysis unit 70 operates with the capillary action of the fluid sample. An orifice 164 having a diameter of 3 microns at the upstream end of the pathogen dialysis section 70 has a capillary action initiation feature (CIF) 166 (see Fig. 33) such that the fluid is pulled downward into the dialysis MST channel 2〇4 below. The first suction port 198 for the standard channel 74 also has a CIF 202 (see Figure 15) to prevent fluid from easily securing the meniscus above the dialysis suction port 168. The small component dialysis section 682 shown in Fig. 7 4 may have a structure similar to that of the pathogen dialysis section 70. The small component dialysis section separates the sample from any small target cells or molecules by sizing (and shaping, if necessary) to allow small target cells or molecules to pass to the target channel and continue to further analyze the well. Large size cells or molecules are removed to waste reservoir 766. Thus, LOC device 30 (see Figures 1 and 96) is not limited to isolation of pathogens smaller than 3 μηη, but can be used to isolate cells or molecules of any desired size. Lysis Department Referring again to Figures 7, 11, and 13, the chemical species in the sample are released from the cells by chemical lysis. As described above, the lysis reagent from the lysis tank 56 is mixed with the sample stream in the target channel 74 downstream of the surface tension valve 128 of the lysis tank 56. However, some diagnostic assays are preferably suitable for hot lysis treatment, or even a combination of chemical and thermal lysis of target cells. The LOC device 301 houses the heated microchannels 210-2012-201209403 of the culture portion 114. The sample stream is flooded with the culture portion 114 and stopped at the boiling initiation valve 106. The culture microchannel 210 heats the sample to the temperature at which the cell membrane ruptures. In some hot lysis applications, the enzyme reaction is not required in the chemical lysis unit 130, and the hot lysis completely replaces the enzyme reaction in the chemical lysis unit 130. Boiling Initiating Valve As discussed above, LOC unit 301 has three boiling inducing valves 126, 106 and 108. The position of these valves is shown in Figure 6. Figure 31 is an enlarged plan view of the independent boiling initiation valve 108 at the end of the heated microchannel 158 of the amplification section Π2. By capillary action, the sample stream 1 1 9 is drawn along the heated microchannel 158 until it reaches the boiling initiation valve 108. The meniscus 120 at the leading edge of the sample stream is secured to the meniscus holder 98 of the valve inlet 146. The meniscus holder 9 8 geometry stops the meniscus and prevents capillary flow. As shown in Figures 31 and 32, the meniscus holder 98 is an orifice provided by the opening of the pipe from the MST passage 90 to the cover passage 94. The surface tension of the meniscus 使 2〇 keeps the valve closed. Ring heater 1 52 is located around valve inlet 1 46. The ring heater 1 5 2 is controlled by C Μ Ο S via the boiling induced valve heater contact 1 5 3 . To open the valve, CMOS circuit 86 sends an electrical pulse to valve heater contact 1 53. The ring heater 1 52 is resistively heated until the liquid sample 1 19 is boiled. Boiling removes meniscus 120 from valve inlet 146 and begins to wet cover passage 94. Once the lid passage 94 is wetted, the capillary action is restored. The fluid sample 119 is filled with the passage 94 and flows through the lower valve conduit 15 and -45-201209403 to the valve outlet 1 48, wherein the capillary-driven liquid flow advances along the expansion outlet passage 160 to the hybridization and detection portion 52. . The liquid sensor 174 is placed before and after the valve for diagnosis. It will be understood that once the boiling trigger valve is opened, it is impossible to close it again. However, since the LOC device 301 and the test module 10 are single-purpose devices, it is not necessary to close the valve. Culture section and nucleic acid amplification section The culture section 114 and the amplification section 112 are shown in Figs. 6, 7, 13, 14, 23, 24, 25, 35 to 45, 50, and 51. The culture portion 114 has a single, heated culture microchannel 210 that is etched to form a ruthenium pattern in the MST channel layer 100 from the lower conduit opening 134 to the boiling initiation valve 〇6 (see Figures 13 and 14). ). Controlling the temperature of the culture portion 114 enables a more efficient enzyme reaction. Similarly, the amplifying portion 112 has an amplifying microchannel 158 (see Figs. 6 and 14) which is heated from the boiling inducing valve 106 to the boiling initiation valve 108. Upon mixing, culture, and nucleic acid amplification, the valves stop flow to retain the target cells in the heated culture or amplification microchannels 210 or 158. The microchannel 蜿蜒 pattern also promotes (to some extent) the target cells to be mixed with the reagents. In the culture unit 1 14 and the amplification unit 1 1 2, the sample cells and reagents are heated via a heater 154 controlled by a pulse width modulation (PWM) CMOS circuit 86. Each of the turns of the heated culture microchannel 210 and the amplification microchannel 158 has three independently operable heaters 1 54 extending between the individual heater contacts 156 (see Figure 14). 46- 201209403), which provides two-dimensional control of the input heat flux density. As best shown in Figure 51, heater 154 is supported on top layer 66 and buried in lower seal 64. The heater material is TiAl, but many other conductive metals are also suitable. The elongated heater 154 is parallel to the longitudinal length of each of the channel portions forming the meandering meandering. In the amplification section 112, each of the wide meanders can be operated as an independent PCR chamber via individual heater control. The use of a microfluidic device, such as the LOC device 30, requires a small volume of amplicons required for amplification in the amplification portion 112 to use a small volume of amplification mixture. This volume is less than 400 nanoliters, in most cases less than 170 nanoliters, less than 70 nanoliters, and in the case of LOC device 310, this volume is between 2 nanoliters and 30 nanoliters. between. Increased heating rate and better diffusion mixing The small cross-sectional area of each channel portion increases the heating rate of the amplification fluid mixture. All fluids are kept at a relatively short distance from the heater 154. The channel cross-sectional area (i.e., the cross section of the augmented microchannel 158) is reduced to less than 100,00 square microns, while the "high scale" device has a significantly higher heating rate. The lithography manufacturing technique allows the amplifying microchannel 158 to have a cross-section that spans a flow path that provides a relatively high heating rate of less than 16,000 square microns. The 1 micron size feature is easily achieved with lithography manufacturing techniques. If only a very small number of amplicons (as is the case in LOC device 301) are required, the cross section can be reduced to less than 2,500 square microns. For a diagnostic analysis of 1,000 to 2,000 probes on a LOC device and a "sample into" answer within 1 minute, the appropriate cross-sectional area across the fluid is 400 square microns and -47- 201209403 1 square micron. The heater element in the amplification microchannel 1 58 heats the nucleic acid sequence at a rate of greater than 80 absolute temperatures (K) per second, which in most cases is greater than 1 〇〇 K per second. Typically, the heater element heats the nucleic acid sequence at a rate greater than 1 000 K per second, and in many cases, the heater element heats the nucleic acid sequence at a rate greater than 10, 〇〇〇 K per second. Typically, based on the requirements of the analytical system, the heater elements are greater than 100,000 K per second, greater than 1,000,000 K per second, greater than 10,000,000 K per second, greater than 20,000,000 K per second, and greater than 40,000,000 K per second. The nucleic acid sequence is heated at a rate greater than 80,000,000 K per second and greater than 1 60,000,000 K per second. A small cross-sectional area channel is also beneficial for the diffusive mixing of any reagent with the sample fluid. The diffusion of one liquid to another is most pronounced near the interface between the two liquids before the diffusion mixing is completed. The density of the phenomenon decreases with distance from the interface. A microchannel with a relatively small cross-sectional area across the flow direction is used while keeping the two fluids flowing through the interface for rapid diffusion mixing. Reducing the cross-section of the channel to less than 1 〇〇, 〇〇〇 square micron, results in a significantly higher diffusion rate than the "large scale" devices. The lithography manufacturing technique allows the microchannels to have a cross-section that spans a flow path that provides a higher mixing rate of less than 16,000 square microns. If only a very small amount of amplicons are required (as is the case in LOC unit 301), the cross section can be reduced to less than 2,500 square microns. For a diagnostic analysis of 1,000 to 2,000 probes on a LOC device and "sample entry, answer out" within 1 minute, the appropriate cross-sectional area across the fluid is 40 0 square microns and 1 square. Micro-48- 201209403 • Between meters. A short thermal cycle time keeps the sample mixture close to the heater and uses a very small amount of fluid' to cause rapid thermal cycling during the nucleic acid amplification process. Each thermal cycle (i.e., variability, adhesion, and primer extension) is completed in 30 seconds for a sequence of up to 15 〇 base pairs (bp) long. In most diagnostic analyses, the individual thermal cycle times are less than 11 seconds and most are less than 4 seconds. For a target sequence of up to 150 base pairs (bp) long, the thermal cycle time of the LOC device 30 for some of the most common diagnostic analyses is zero. 45 seconds to K5 seconds. This rate of thermal cycling allows the test module to perform nucleic acid amplification procedures in less than one minute; often within 220 seconds. For most analyses, the amplification section produces sufficient amplicons from the sample fluid entering the sample inlet within 80 seconds. For most of the analysis, sufficient amplicon is generated in 30 seconds. Upon completion of the predetermined number of amplification cycles, the amplicon is fed to the hybridization and detection section 52 via the boiling initiation valve 108. Hybridization Chambers Figures 52, 53, 54, 56 and 57 show hybridization chambers 180 in the hybridization chamber array 11A. Hybridization and detection portion 52 has a 24 X 45 array 110 of hybridization chambers 180 each having a hybrid-reactive FRET' probe 186, a heater element 182, and an integrated photodiode 184. Photodiode 184 is incorporated to detect fluorescence from a target nucleic acid sequence or protein that hybridizes to FRET probe 186 -49-201209403. Each of the photodiodes 184 is independently controlled by the CMOS circuit 86. For the emitted light, any material between the FRET probe 186 and the photodiode 184 must be transparent. Therefore, the wall portion 97 between the probe 186 and the photodiode 1 8 4 must also be optically transparent to the emitted light. In the LOC device 301, the wall portion 97 is a thin layer of cerium oxide (about 〇. 5 microns). Direct intrusion of photodiode 184 under each hybridization chamber 180 allows the use of a very small volume of probe-target hybrid, yet still produces a detectable fluorescent signal (see Figure 54). A small volume of hybridization chamber can be used because of the small amount. Prior to hybridization, the amount of probe required to detect the probe-target hybrid is greater than 2 70 picograms (corresponding to 900,000 cubic micrometers), in most cases less than 60 pico G (corresponding to 200,000 cubic microns), typically less than 12 picograms (corresponding to 40,000 cubic micrometers), and less than 2. in the case of the LOC device 301 shown in the figures. 7 picograms (corresponding to a chamber volume of 9,000 cubic microns). Of course, reducing the size of the hybridization chamber allows for higher chamber densities and therefore more probes on the LOC device. In the LOC device 301, the hybrid has more than one chamber (i.e., less than 2,250 square microns per chamber) in an area of 1,500 microns by 1,500 microns. The smaller volume also reduces reaction time, making hybridization and detection faster. Another advantage of the small number of probes required for each chamber is that during the manufacture of the L0C device, only a very small amount of probe solution needs to be dispensed into each chamber. A specific embodiment of the LOC device according to the present invention may be configured using a probe solution of 1 nanoliter or less. After nucleic acid amplification, the boiling initiation valve 108 is activated and the amplicon flows along the -50-201209403 flow path 176 and into each of the hybridization chambers 180 (see Figures 52 and 56). The endpoint liquid sensor 178 indicates the point at which the hybridization chamber 180 is filled with the amplicon and the heater 182 can be activated. After sufficient hybridization time, the LED 26 (see Figure 2) is activated. » The opening in each of the hybrid chambers 180 is provided with an optical window 136 to expose the FRET probe 186 to the excitation radiation (see Figures 52, 54 and 56). The LED 26 illumination continues for a sufficiently long period of time to induce a high intensity fluorescent signal from the probe. The photodiode 184 is shorted during the excitation. After a preprogrammed delay of 3 〇〇 (see Figure 2), the photodiode 184 is enabled and the fluorescent emission is detected under no excitation light. The incident light on the active region 1 85 of the photodiode 1 84 (see FIG. 54) is converted into a photocurrent that can be measured using the CMOS circuit 86. Each hybridization chamber 180 carries a probe for detecting a single target nucleic acid sequence. needle. If desired, each hybridization chamber 180 can carry probes that detect more than 1,000 different targets. Alternatively, many or all of the hybridization chambers may carry the same probe that repeatedly detects the same target nucleic acid. Copying the probes in this manner in the hybrid chamber array 1 1 使得 increases the confidence of the results obtained, and if desired, all results can be combined by photodiodes of adjacent hybridization chambers to obtain a single result. Those skilled in the art will appreciate that there may be from 1 to over 1,000 different probes on the hybridization chamber array 110, depending on the analysis details. Humidifier and Humidity Detector The inset AG of Figure 6 indicates the position of the humidifier 196. The humidifier is free of -51 - 201209403 LOC device 3 0 1 evaporation of reagents and probes during operation. As best seen in the enlarged view of Fig. 55, the water sump 188 is fluidly coupled to the evaporator 190. The water storage tank 188 is filled with molecular biological grade water and sealed during manufacture. As best shown in Figures 55 and 67, by the effect, water is drawn to the three lower conduits 1 94 and along the individual water supply passages 192 to the three upper conduits 193 of the evaporator 190. The meniscus is placed on each of the upper tubes 193 to hold the water. The evaporator has a ring-shaped addition 191 which surrounds the upper pipe 193. With a thermally conductive post 3 76, a ring-shaped 191 is connected to the CMOS circuit 86 to the top metal layer 195 (see |). At startup, the ring heater 191 heats the water causing the water to evaporate around the device. The position of the humidity sensor 232 is also shown in FIG. However, as shown in the enlarged view of the inset AH shown in Fig. 63, the humidity sensing has a capacitive comb structure. The lithographically etched first electrode 296 and the lithographically etched second electrode 298 are opposite each other to make their teeth. The opposing electrodes form a capacitor with a capacitance that can be monitored by CMOS electricity g. As the humidity increases, the dielectric of the air gap between the electrodes increases, causing the capacitance to increase. Humidity sensor 23 2 is adjacent to the hybridization chamber 1 1 〇 (the most important humidity measurement location) to slow the evaporation of the solution containing the exposed needle. The feedback sensor temperature and liquid sensor system is integrated into the LOC device 301 to provide feedback and diagnostics during operation. Referring to Figure 3 5, the nine temperatures are displayed in three by the hair-in-the-surface heat exchanger I 37 and the wet optimal instrument and the interpolated ^ 86 constant chamber array of the sense of installation -52-201209403 170 is distributed to the entire amplification unit 112. Similarly, the culture portion 114 also has nine temperature sensors 170. These sensors each use a 2x2 array of bipolar junction transistors (BJT) to monitor fluid temperature and provide feedback to CMOS circuitry 86. The CMOS circuit 86 utilizes this to accurately control the thermal cycling during the nucleic acid amplification process and any heating during thermal lysis and incubation in the hybridization chamber 180, which uses the hybridization heater 182 as a temperature sensor (see Figure 56). The resistance of hybrid heater 182 is temperature dependent and CMOS circuit 86 utilizes this to obtain a temperature reading of each hybridization chamber 180. The LOC device 301 also has a number of MST channel liquid sensors 174 and a cover channel liquid sensor 208. Figure 35 shows the line of the MST channel liquid sensor 174 at one of the ends of each of the heated microchannels 158. Preferably, as shown in Figure 37, the MST channel liquid sensor 174 is a pair of electrodes formed by the exposed regions of the top metal layer 195 in the CMOS structure 86. The liquid closes the circuit between the electrodes to indicate where they are located in the sensor. Figure 25 shows an enlarged perspective view of the lid channel liquid sensor 208. Pairs of TiAl electrode pairs 218 and 220 are deposited on top layer 66. A gap 222 is provided between the electrodes 218 and 220 to keep the circuit open in the absence of liquid. The circuit is closed when the liquid is present and the CMOS circuit 86 uses this feedback to monitor the flow. The GRAVITATIONAL INDEPENDENCE test module 10 is self-directed. It does not need to be fastened to a smooth surface -53- 201209403 to operate. The fluid flow driven by capillary action and the lack of access to the external tubing make the module a portable and easily portable handheld reader such as a mobile phone. The gravity self test module is also acceleration independent of all practical ranges. Vibration-resistant and mobile-carrying vehicles or carrying actions
核酸擴增變體 直接PCR 傳統上,於製備反應混合物之前,PC R需 標靶DNA。然而,適當地改變化學及樣本濃 最少量的DNA純化實施核酸擴增,或進行直 以PCR進行核酸擴增時,此方法便稱做直3 LOC裝置中於經控制的常溫下實施核酸擴增時 直接恆溫擴增。當用於LOC裝置時,尤其是 體設計的簡化時,直接核酸擴增技術具相當多 接PCR或是直接恆溫擴增之擴增化學調整包 液強度、使用高活性及高進行性之聚合酶及與 抑制劑螯合之添加物。稀釋樣本中存在之抑制 的。 爲利用直接核酸擴增技術,LOC裝置設計 外的特徵。第一特徵爲試劑貯槽(例如,圖 5 8 )’其經適當地尺寸化以供應充分量之擴增 稀釋劑,使得可能干擾擴增化學之樣本成分的 輔助設備之 插入至類似 主操作代表 其耐衝擊及 電話上操作 要大量純化 度,可利用 接擴增。當 妾PCR 〇於 ,此方法爲 關於所需流 的優勢。直 括增加緩衝 潛在聚合酶 劑亦爲重要 倂入兩個額 8中的貯槽 反應混合或 最終濃度足 -54- 201209403 夠低以成功地進行核酸擴增。非細胞樣本成分的所欲稀釋 度爲5倍至20倍。當適度確認標靶核酸序列的濃度被維 持於足夠高以用於擴增及檢測時,使用不同的LOC結構 ,例如圖4中的病原體透析部70。於此具體實施例中( 進一步於圖6中說明),於樣本萃取部290之上游使用有 效地濃縮足夠小而得以進入擴增部292之病原體的濃度並 將較大細胞排出至廢料貯槽76之透析部。於另外的具體 實施例中,使用透析部以選擇性地去除血漿中之蛋白質及 鹽而保留關注的細胞。 支持直接核酸擴增之第二LOC結構性特徵爲設計通 道的深寬比以調整樣本及擴增混合成分之間的混合比。例 如,爲確保經由單一混合步驟之相關於樣本之抑制劑的稀 釋爲較佳的5倍-20倍範圍中,設計樣本及試劑通道之長 度與截面,以使混合起始位置之上游的樣本通道構成之流 組抗較試劑混合物流動之通道的流組抗高出4倍-1 9倍。 經由控制設計幾合而容易地控制微通道中之流組抗。針對 恆定截面積,微通道之流組抗隨通道長度而線性地增加。 對於混合設計而言爲重要的是,微通道中之流組抗較多取 決於最小截面積尺寸。例如,當深寬比極爲不均一時,方 形截面之微通道的流組抗與最小垂直尺寸之立方成反比。 反轉錄酶PCR ( RT-PCR) 當分析或萃取之樣本核酸種類爲RNA時,諸如來自 RNA病毒或信使RNA,於PCR擴增之前必須先將RNA反 -55- 201209403 轉錄爲互補DNA(cDNA)。可於與PCR相同之腔室中實 施反轉錄反應(一步驟RT-PCR ),或是其可爲分別的起 始反應(二步驟RT-PCR )。於此所述之LOC變體中,可 藉由添加反轉錄酶及聚合酶至試劑貯槽62以及程式化加 熱器154以先循環反轉錄步驟並接續進行核酸擴增步驟, 而簡單地實施一步驟RT-PCR。藉由利用試劑貯槽58來儲 存及分配緩衝液、引子、dNTP及反轉錄酶,以及利用培 養部1 1 4以用於反轉錄步驟,接著於擴增部丨丨2中以普通 方式進行擴增’亦可簡單地完成二步驟RT-PCR。 恆溫核酸擴增 針對一些應用’較佳之核酸擴增方法爲恆溫核酸擴增 ’因此不需於各種溫度循環重複地循環反應成分,而是將 擴增部維持於常溫下,普通爲約37°C至41〇C。已描述__ 些恆溫核酸擴增方法,包括股取代擴增(SDA )、轉錄介 導擴增(TMA )、依賴核酸序列擴增(NASBA )、重組酵 素聚合酶擴增(RPA )、解旋恆溫DNA擴增(HDA )、 滾動循環擴增(RCA)、分枝型擴增(RAM)及環形,丨§溫 擴增(LAMP ),以及此等之任何或其他恆溫擴增方法可 用於本文之LOC裝置之特定具體實施例中。 爲實施恆溫核酸擴增,鄰接擴增部之試劑貯槽6 〇及 62將載有用於特定恆溫方法之適當的試劑而不是載有 PCR擴增混合及聚合酶。例如,針對SDA,試劑貯槽6〇 含有擴增緩衝液、引子及dNTP,以及試劑貯槽62含有適 -56- 201209403 當的核酸內切酶及外切_DNA聚合酶。針對RPA,試劑貯 槽60含有擴增緩衝液、引子、dNTP及重組酶蛋白,及試 劑貯槽62含有股取代DNA聚合酶,諸如。同樣地, 針對HDA ’試劑貯槽60含有擴增緩衝液、引子及dNTP ’以及試劑貯槽62含有適當的DN A聚合酶及解旋酶(而 非使用熱)以解開雙股DNA。熟此技藝者將了解以任何 適用於核酸擴增法之方式,可將必要試劑分配於兩個試劑 貯槽。 針對自RNA病毒,諸如HIV或C型肝炎病毒之病毒 核酸的擴增,NASBA或TMA係適當的因其不需先將RNA 轉錄成cDNA。於此實例中,試劑貯槽60塡充有擴增緩 衝液、引子及dNTP,以及試劑貯槽62塡充有RNA聚合 酶、反轉錄酶及任意的RNase Η。 針對一些恆溫核酸擴增類型,於維持恆溫核酸擴增之 溫度以利反應續行之前,必須採用初始變性循環以分開雙 股DNA模板。因可藉擴增微通道158中之加熱器154嚴 密地控制擴增部1 1 2中之混合的溫度,於本文中描述之 LOC裝置之所有具體實施例中均可輕易完成此變性循環( 見圖1 4 )。 恆溫核酸擴增對於樣本中潛在的抑制劑之耐受性較高 ,因而通常適用於自所欲樣本之直接核酸擴增。因此,恆 溫核酸擴增尤其有用於分別顯示於圖75、76及77中之 LOC 變體 XLIII 673、LOC 變體 XLIV 674 及 LOC 變體 XLVII 677。直接恆溫擴增‘亦可與如圖75及77中所示之 -57- 201209403 一或多個預擴增透析步驟70、686或682及/或如圖76中 所示之預-雜交透析步驟682組合,以分別於核酸擴增之 前有助於樣本中之標靶細胞的部份濃縮或是於樣本進入雜 交腔室陣列1 1 〇前移除不想要的細胞碎片。熟此技藝者將 了解可使用預-擴增透析及預-雜交透析之任何組合。 亦可以平行的擴增部,諸如,圖71、72及73中所槪 述者,實施恆溫核酸擴增。多工及一些恆溫核酸擴增方法 ,諸如LAMP,係與初始反轉錄步驟相容以擴增RNA » 螢光檢測系統之另外的細節 圖58及59顯示雜交·反應性FRET探針23 6。此等經 常被稱爲分子信標及係爲由單股核酸產生之莖-及-環探針 ,並於與互補核酸雜交時發螢光。圖58顯示於與標靶核 酸序列238雜交之前之單一 FRET探針236。探針具有環 240、莖242、於5'端之螢光團246及於31端之淬熄劑248 。環240包含與標靶核酸序列23 8互補之序列。探針序列 兩側的互補序列黏著在一起以形成莖242。 於缺少互補標靶序列時,如圖5 8中所示者,探針維 持閉合。莖242保持螢光團-淬熄劑對彼此相當接近,使 得大量的共振能量可於彼此間傳輸,而當以激發光244照 射時實質地消除螢光團發螢光團的能力。 圖59顯示呈開放或經雜交組態的FRET探針236。於 與互補標靶核酸序列2 3 8雜交時,莖-及-環結構被破壞, 螢光團及淬熄劑於空間上分離,因此恢復螢光團246發螢 -58- 201209403 光的能力。光學檢測地螢光發射2 5 〇以作爲探針已雜 指標。 探針以極高專一性與互補標靶雜交,因探針之莖 係設計成較具單一不互補核苷酸之探針-標靶螺旋穩 因雙股DNA相對堅固’立體上探針-標靶螺旋與莖螺 可能共存。 引子-聯結的探針 引子-聯結的莖-及-環探針及引子-聯結的線性探 亦稱作蠍子型探針,爲分子信標之替代物且可用於 裝置之即時及定量核酸擴增。及時擴增可直接實施於 裝置之雜交腔室中。使用引子-聯結的探針之優點爲 元件實體地聯結至引子,因此於核酸擴增期間僅需單 交事件而不需要分別的引子雜交及探針雜交。此確保 有效地反應且當使用分別的引子及探針時產生更強的 、更短的反應時間,具有更佳的識別度。於製造期間 針(與聚合酶及擴增混合)將沉積於雜交腔室180中 需LOC裝置上之獨立的擴增部。替代性地,擴增部 使用或用於其他反應。 引子-聯結的線性探針 圖78及79分別顯示首輪核酸擴增期間之引子-的線性探針692及於後續核酸擴增期間之雜交的組態 照圖78,引子-聯結的線性探針692具有雙股莖區段 交的 螺旋 定。 旋不 針, LOC LOC 探針 次雜 即時 信號 ,探 且不 未被 聯結 。參 -59- 242 201209403 。其中一股結合引子聯結的探針序列696 ’其係與標靶核 酸696上的區域同源且以螢光團246標記其5'端,以及經 由擴增阻斷物694聯結其3’端至寡核苷酸引子700。以淬 熄劑部分248標記莖242之另外一股的3·端。於完成首輪 核酸擴增之後,利用目前爲互補的序列698,探針可環繞 且雜交至延伸的股。於首輪核酸擴增期間,寡核苷酸引子 700黏著至標靶DN A 238 (圖78)並接著延伸而形成含有 探針序列及擴增產物兩者之DNA股。擴增阻斷物694防 止聚合酶之讀取通過及拷貝探針區域696。於接續的變性 時,雜交之延伸的寡核苷酸引子700/模板及引子-聯結的 線性探針之雙股莖242分離,因此釋出淬熄劑248。一但 用於黏著及延伸步驟的溫度降低,引子聯結的線性探針之 引子聯結的探針序列696捲曲並與延伸的股上之擴增的互 補序列698雜交,以及檢測出的螢光指出標靶DNA存在 。未延伸的引子-聯結的線性探針保留其雙股莖且螢光保 持淬熄。此檢測方法特別適於快速檢測系統,因其依賴單 —分子製程。 引子-聯結的莖-及-環探針 圖80A至80F顯示引子-聯結的莖-及-環探針7〇4之 操作。參照圖80A,引子-聯結的莖-及-環探針704具有互 補雙股DNA之莖242及合倂探針序列的環24〇。以螢光 團246標記其中一個莖股708之5’端。以3'-端淬熄劑 248標記另一股710’且另一股710帶有擴增阻斷物694 -60- 201209403 及寡核苷酸引子700兩者。於初始變性相(見圈 標靶核酸23 8之股及引子聯結的莖242分開莖-704。當溫度冷卻以用於黏著相時(見圖80C) 結的莖-及-環探針704上之寡核苷酸引子70 0與 序列238雜交。於延伸期間(見圖80D ),合成 序列23 8之互補706以形成含有探針序列704及 物兩者之DNA股。擴增阻斷物694防止聚合酶 過及拷貝探針區域704。變性之後,當接著黏著 引子-聯結的莖-及-環探針之環區段240之探針 圖80F)黏著至延伸的股上之互補序列706。此 螢光團246與淬熄劑248相距甚遠,造成螢光發 增強。 控制探針 雜交腔室陣列1 1 〇包括具有用於分析品質 負控制探針之一些雜交腔室180。圖92及93槪 螢光團之負控制探針796,以及圖94及95描述 之正控制探針798。正及負控制探針具有如前述 針之莖-及-環結構。然而,不論探針雜交成爲開 保持封閉,將永遠自正控制探針7 9 8發射螢光信 負控制探針796從不發射螢光信號250。 參照圖92及93,負控制探針796不具螢光 具有或不具有淬熄劑248 )。因此,不論標靶 23 8與探針雜交(見圖93 )或是探針保持其莖. 3 8 0B ), 及-環探針 ,引子·聯 L標靶核酸 :標靶核酸 .擴增的產 :之讀取通 探針時, 序列(見 組態使得 射的顯著 制之正及 要說明無 無淬熄劑 FRET 探 放組態或 號25 0且 :團(及可 核酸序列 及-環組態 61 - 201209403 (見圖92 ),可忽略對激發光244之回應。替代性地, 可設計負控制探針796使得其永遠保持淬熄。例如,藉由 合成環240而得到將不會與所硏究的樣本中之任何核酸序 列雜交之探針序列,探針分子之莖242將與其自身重新雜 交,及螢光團及淬熄劑將保持緊密相鄰且將不會發射可見 的螢光》此負控制信號對應於來自雜交腔室180的低階發 射,於雜交腔室1 80中探針未經雜交但是淬熄劑未淬熄來 自指示劑的所有發射。 相反地,建構無淬熄劑之正控制探針798,如圖94 及95中所示者。回應激發光244,不論正控制探針798 是否與標靶核酸序列2 3 8雜交,無物質使來自螢光團246 之螢光發射2 5 0淬熄。 圖52顯示雜交腔室陣列1 1 0中的正及負控制探針( 分別爲3 78及3 80 )之可行分佈。控制探針3 78及3 80係 置於雜交腔室180中並定位成橫越雜交腔室陣列110之線 。然而,陣列內之控制探針的配置係任意的(如同雜交腔 室陣列1 1 0之組態)。 蛋白質檢測變體 LOC裝置之一些具體實施例使用均質蛋白質檢測分析 以偵測粗細胞溶胞產物中之特定蛋白質。許多均質蛋白質 檢測分析已發展用於LOC裝置之這些具體實施例。通常 這些分析利用抗體或適體來擷取標靶蛋白質。 在一種分析類型中,結合至特定蛋白質142的適體 -62- 201209403 141以兩種不同的螢光團或發光團143 作爲螢光共振能量轉移(FRET )或電 轉移(ERET)反應中的予體和受體 )。予體143和受體144皆連接至相同 合至標靶蛋白質142時,構形的改變造 如,在缺乏標靶之適體141形成予體和 形(見圖1 〇 〇 A );當結合至標靶時, 和受體間較大的間隔(見圖1 〇 〇 B )。 予體爲發光團,結合至標靶的效應爲 862 (見圖 1 00B )。Nucleic Acid Amplification Variants Direct PCR Traditionally, PC R requires target DNA prior to preparation of the reaction mixture. However, when appropriately changing the chemical and sample concentration, the minimum amount of DNA purification is performed for nucleic acid amplification, or when PCR is performed for nucleic acid amplification, the method is referred to as performing a nucleic acid amplification at a controlled normal temperature in a straight 3 LOC device. When directly heated at a constant temperature. When used in LOC devices, especially in the simplification of bulk design, direct nucleic acid amplification technology has a large number of PCR or direct constant amplification amplification chemical adjustment package strength, using high activity and high progressive polymerase And an additive that chelate with the inhibitor. Dilute the presence of inhibition in the sample. In order to utilize direct nucleic acid amplification techniques, features of the LOC device are designed. A first feature is a reagent reservoir (eg, Figure 58) that is appropriately sized to supply a sufficient amount of amplification diluent such that the insertion of an auxiliary device that may interfere with the sample component of the amplification chemistry is representative of a similar primary operation A large amount of purification is required for impact resistance and telephone operation, and amplification can be utilized. When 妾PCR is used, this method is an advantage over the required flow. Adding buffers directly to potential polymerases is also important. Into two tanks in the 8 tanks, the reaction mixture or the final concentration is -54-201209403 low enough to successfully perform nucleic acid amplification. The desired dilution of the non-cellular sample component is 5 to 20 times. When the concentration of the target nucleic acid sequence is moderately confirmed to be sufficiently high for amplification and detection, a different LOC structure, such as the pathogen dialysis section 70 of Figure 4, is used. In this particular embodiment (further illustrated in Figure 6), the concentration of the pathogen sufficient to enter the amplification portion 292 is effectively concentrated upstream of the sample extraction portion 290 and the larger cells are discharged to the waste storage tank 76. Dialysis department. In another specific embodiment, a dialysis section is used to selectively remove proteins and salts in plasma while retaining cells of interest. A second LOC structural feature that supports direct nucleic acid amplification is to design the aspect ratio of the channel to adjust the mixing ratio between the sample and the amplified mixture. For example, to ensure that the dilution of the inhibitor associated with the sample via a single mixing step is in the range of preferably 5 to 20 times, the length and cross section of the sample and reagent channels are designed such that the sample channel upstream of the mixing start position The composition of the flow group is 4 to 1 times higher than that of the channel through which the reagent mixture flows. The flow group resistance in the microchannel is easily controlled by controlling the design. For a constant cross-sectional area, the flow resistance of the microchannel increases linearly with the length of the channel. It is important for the hybrid design that the flow group resistance in the microchannel is more dependent on the minimum cross-sectional area size. For example, when the aspect ratio is extremely non-uniform, the flow resistance of the microchannels of the square cross section is inversely proportional to the cube of the smallest vertical dimension. Reverse Transcriptase PCR (RT-PCR) When the sample nucleic acid species analyzed or extracted is RNA, such as from RNA virus or messenger RNA, RNA anti-55-201209403 must be transcribed into complementary DNA (cDNA) prior to PCR amplification. . The reverse transcription reaction (one-step RT-PCR) can be carried out in the same chamber as the PCR, or it can be a separate initiation reaction (two-step RT-PCR). In the LOC variant described herein, a reverse transcription step and a subsequent step of performing a nucleic acid amplification step can be performed by adding a reverse transcriptase and a polymerase to the reagent storage tank 62 and the stylized heater 154, and simply performing a step. RT-PCR. The reagent storage tank 58 is used to store and dispense buffers, primers, dNTPs, and reverse transcriptase, and the culture portion 1 1 4 is used for the reverse transcription step, followed by amplification in the amplification unit 丨丨2 in a conventional manner. 'Two-step RT-PCR can also be done simply. Thermostatic Nucleic Acid Amplification For some applications, the preferred nucleic acid amplification method is constant temperature nucleic acid amplification. Therefore, it is not necessary to repeatedly circulate the reaction components at various temperature cycles, but the amplification portion is maintained at normal temperature, usually about 37 ° C. To 41〇C. __ Some thermostatic nucleic acid amplification methods, including strand-substituted amplification (SDA), transcription-mediated amplification (TMA), nucleic acid sequence-dependent amplification (NASBA), recombinant enzyme polymerase amplification (RPA), unwinding Constant temperature DNA amplification (HDA), rolling cycle amplification (RCA), branched amplification (RAM) and circular, 丨 ̄ ̄ temperature amplification (LAMP), and any other or other isothermal amplification methods available for this article In a specific embodiment of the LOC device. To perform a constant temperature nucleic acid amplification, reagent reservoirs 6 and 62 adjacent to the amplification section will carry appropriate reagents for a particular constant temperature method rather than carrying PCR amplification mixes and polymerases. For example, for SDA, the reagent reservoir 6A contains amplification buffer, primers, and dNTPs, and the reagent reservoir 62 contains an endonuclease and an exo-DNA polymerase suitable for -56-201209403. For RPA, reagent reservoir 60 contains amplification buffer, primers, dNTPs, and recombinase proteins, and reagent reservoir 62 contains a stock-substituted DNA polymerase, such as. Similarly, the HDA' reagent reservoir 60 contains amplification buffer, primer and dNTP' and reagent reservoir 62 contains appropriate DN A polymerase and helicase (without heat) to unwind the double stranded DNA. Those skilled in the art will appreciate that the necessary reagents can be dispensed into two reagent reservoirs in any manner suitable for nucleic acid amplification. For amplification of viral viruses, such as HIV or hepatitis C virus nucleic acids, NASBA or TMA is appropriate because it does not require the transcription of RNA into cDNA. In this example, the reagent reservoir 60 is filled with amplification buffer, primer, and dNTP, and the reagent reservoir 62 is filled with RNA polymerase, reverse transcriptase, and any RNase. For some types of thermostatic nucleic acid amplification, an initial denaturation cycle must be employed to separate the double-stranded DNA template before maintaining the temperature of the thermostated nucleic acid amplification for continued reaction. Since the temperature of the mixing in the amplification section 112 can be tightly controlled by the heater 154 in the amplification microchannel 158, this denaturation cycle can be easily accomplished in all of the specific embodiments of the LOC apparatus described herein (see Figure 1 4). Thermostatic nucleic acid amplification is more tolerant to potential inhibitors in the sample and is therefore generally suitable for direct nucleic acid amplification from a desired sample. Thus, constant temperature nucleic acid amplification is particularly useful for LOC variant XLIII 673, LOC variant XLIV 674 and LOC variant XLVII 677, respectively, shown in Figures 75, 76 and 77. Direct thermostatic amplification can also be combined with one or more preamplification dialysis steps 70, 686 or 682 as shown in Figures 75 and 77 and/or a pre-hybridization dialysis step as shown in Figure 76. The combination of 682 facilitates partial concentration of the target cells in the sample prior to nucleic acid amplification, respectively, or removal of unwanted cellular debris prior to entry of the sample into the hybridization chamber array 1 1 . Those skilled in the art will appreciate that any combination of pre-amplification dialysis and pre-hybrid dialysis can be used. Thermostatic nucleic acid amplification can also be performed in parallel amplification sections, such as those described in Figures 71, 72, and 73. Multiplex and some constant temperature nucleic acid amplification methods, such as LAMP, are compatible with the initial reverse transcription step to amplify RNA. Additional details of the fluorescence detection system. Figures 58 and 59 show hybridization-reactive FRET probes 23 6 . These are often referred to as molecular beacons and are stem-and-loop probes produced from single-stranded nucleic acids and fluoresce when hybridized to complementary nucleic acids. Figure 58 shows a single FRET probe 236 prior to hybridization to the target nucleic acid sequence 238. The probe has a ring 240, a stem 242, a fluorophore 246 at the 5' end, and a quencher 248 at the 31 end. Loop 240 comprises a sequence that is complementary to the target nucleic acid sequence 238. The complementary sequences flanking the probe sequence are joined together to form stem 242. In the absence of a complementary target sequence, as shown in Figure 58, the probe remains closed. The stems 242 maintain the fluorophore-quenching agents in close proximity to each other such that a large amount of resonant energy can be transmitted between each other and substantially eliminate the ability of the fluorophore to emit fluorophores when illuminated by the excitation light 244. Figure 59 shows a FRET probe 236 in an open or hybridized configuration. Upon hybridization with the complementary target nucleic acid sequence 2 3 8 , the stem-and-loop structure is destroyed, and the fluorophore and quencher are spatially separated, thereby restoring the ability of the fluorophore 246 to emit -58-201209403 light. The optical detection of the ground fluorescence emission is 2 5 〇 as a probe impurity indicator. The probe hybridizes to the complementary target with extremely high specificity, because the stem of the probe is designed to be a probe with a single non-complementary nucleotide - the target is stable due to the relatively strong 'stereo-probe-label The target helix and the snail may coexist. Primer-Linked Probes-Linked Stem-and-Ring Probes and Probe-Linked Linear Probes, also known as scorpion-type probes, are alternatives to molecular beacons and can be used for both immediate and quantitative nucleic acid amplification of devices. . Timely amplification can be performed directly into the hybridization chamber of the device. The advantage of using a primer-coupled probe is that the element is physically linked to the primer, so that only one event is required during nucleic acid amplification without the need for separate primer hybridization and probe hybridization. This ensures an effective response and produces a stronger, shorter reaction time with better identification when using separate primers and probes. The needle (mixed with the polymerase and amplification) will be deposited in the hybridization chamber 180 during manufacture to require a separate amplification portion on the LOC device. Alternatively, the amplification section is used or used for other reactions. Primer-Linked Linear Probes Figures 78 and 79 show the introduction of the linear probe 692 during the first round of nucleic acid amplification and the hybridization during subsequent nucleic acid amplification, as shown in Figure 78, the primer-linked linear probe 692. A spiral with a double stem section. The needle is not rotated, and the LOC LOC probe is mixed with the instant signal, and the probe is not uncoupled. Ref. -59- 242 201209403. One of the primer-binding probe sequences 696' is homologous to the region on the target nucleic acid 696 and is labeled 5' to its fluorophore 246, and its 3' end is coupled via amplification blocker 694 to Oligonucleotide primer 700. The other end of the stem 242 is labeled with a quencher portion 248. After completion of the first round of nucleic acid amplification, using the currently complementary sequence 698, the probe can wrap around and hybridize to the extended strand. During the first round of nucleic acid amplification, oligonucleotide primer 700 is attached to target DN A 238 (Fig. 78) and then extended to form a DNA strand containing both the probe sequence and the amplification product. Amplification blocker 694 prevents the polymerase from reading through and copying probe region 696. Upon subsequent denaturation, the hybridized extended oligonucleotide primer 700/template and the primer-coupled linear probe of the double stem 242 are separated, thereby releasing the quencher 248. Once the temperature for the adhesion and extension steps is reduced, the primer-linked probe sequence 696 of the primer-joined linear probe is crimped and hybridized to the amplified complementary sequence 698 on the extended strand, and the detected fluorescent light indicates the target. DNA exists. The unextended primer-linked linear probe retains its double stem and the fluorescence remains quenched. This test method is particularly suitable for rapid detection systems because it relies on a single-molecule process. Primer-Linked Stem-and-Ring Probes Figures 80A through 80F show the operation of the primer-coupled stem-and-loop probes 7〇4. Referring to Fig. 80A, the primer-ligated stem-and-loop probe 704 has a stem 242 which complements the double stranded DNA and a loop 24〇 of the combined probe sequence. The 5' end of one of the stem strands 708 is marked with a fluorophore 246. Another strand 710' is labeled with 3'-end quencher 248 and the other strand 710 carries both amplification blockers 694-60-201209403 and oligonucleotide primer 700. In the initial denaturing phase (see the strand of the target nucleic acid 23 8 and the stem 242 linked by the primer separate stem -704. When the temperature is cooled for the adhesive phase (see Figure 80C), the stem-and-loop probe 704 is attached. The oligonucleotide primer 70 is hybridized to sequence 238. During extension (see Figure 80D), the complement 706 of sequence 23 is synthesized to form a DNA strand containing both probe sequence 704 and both. Amplification blocker 694 The polymerase is prevented from passing and copying the probe region 704. After denaturation, the probe sequence 706 on the extended strand is adhered to the probe (Fig. 80F) of the loop segment 240 of the stem-and-loop probe followed by adhesion. This fluorophore 246 is far removed from the quencher 248, resulting in enhanced fluorescence. Control Probes The hybridization chamber array 1 1 includes some hybridization chambers 180 having analytical negative control probes. Figures 92 and 93 负 the negative control probe 796 of the fluorophore, and the positive control probe 798 depicted in Figures 94 and 95. The positive and negative control probes have a stem-and-loop structure as described above. However, regardless of whether the probe hybridization is on and off, the fluorescent signal negative control probe 796 will never be emitted from the positive control probe 798. Referring to Figures 92 and 93, negative control probe 796 is not fluorescent with or without quencher 248). Thus, regardless of whether the target 23 8 hybridizes to the probe (see Figure 93) or the probe retains its stem. 3 8 0B ), and the - loop probe, the primer L-target nucleic acid: the target nucleic acid. Production: When reading the probe, the sequence (see configuration makes the shot significant and correct without the quenching agent FRET probe configuration or No. 25 0: group (and nucleic acid sequence and - ring) Configuration 61 - 201209403 (see Figure 92), the response to excitation light 244 can be ignored. Alternatively, negative control probe 796 can be designed such that it remains quenched forever. For example, by synthesizing ring 240 it will not The probe sequence that hybridizes to any of the nucleic acid sequences in the sample of interest, the stem 242 of the probe molecule will rehybridize with itself, and the fluorophore and quencher will remain in close proximity and will not emit visible fluorescein This negative control signal corresponds to the low order emission from the hybridization chamber 180 where the probe is not hybridized but the quencher does not quench all of the emission from the indicator. Conversely, the construction is not quenched. The positive control probe 798 of the extinguishing agent, as shown in Figures 94 and 95, responds to the excitation light 244, Whether or not the control probe 798 is hybridized to the target nucleic acid sequence 2 3 8 , the no substance quenches the fluorescent emission from the fluorophore 246. Figure 52 shows the positive and negative in the hybrid chamber array 1 1 0 A feasible distribution of control probes (3 78 and 3 80 respectively). Control probes 3 78 and 380 are placed in hybridization chamber 180 and positioned across the line of hybridization chamber array 110. However, within the array The configuration of the control probes is arbitrary (as is the configuration of the hybrid chamber array 1 1 0.) Some examples of protein detection variant LOC devices use homogeneous protein detection assays to detect specific proteins in crude cell lysates Many homogeneous protein detection assays have been developed for these specific embodiments of LOC devices. Typically these assays utilize antibodies or aptamers to capture target proteins. In one type of assay, aptamers that bind to a particular protein 142-62- 201209403 141 uses two different fluorophores or luminophores 143 as the precursor and acceptor in the fluorescence resonance energy transfer (FRET) or electrotransfer (ERET) reaction. Both the host 143 and the acceptor 144 are connected to the same Target to target At protein 142, the conformational changes result in the formation of a conformation and shape in the absence of the target aptamer 141 (see Figure 1 〇〇A); when bound to the target, a larger separation from the receptor (see Figure 1 〇〇B) The precursor is a luminophore and the effect of binding to the target is 862 (see Figure 1 00B).
第二種分析類型使用必須獨立連接 抗原表位或標靶蛋白質142區域的兩個 體 141 (見圖 101A, 101B,102A 和 145或適體141以不同螢光團或發光團 其可作爲螢光共振能量轉移(FRET ) 能量轉移(ERET )反應中的予體和受 團143和144形成一部分之經由長彈性 抗體或適體的短互補寡核苷酸147對( )。一旦抗體145或適體141連結至標 補寡核苷酸147互相碰上並彼此雜交( )。如此使予體和受體1 43和1 44彼此 可用作標靶蛋白質偵測之訊號的FRET 爲了確定沒有,或非常少的背景訊 個抗體145或適體141的寡核苷酸147 和144標示,其可 化學發光共振能量 L圖 100A 和 100B ]適體141,且當結 成間隔的改變。例 受體位於近端之構 新的構形造成予體 當受體爲淬熄劑且 I加光發射250或 至不同且無重疊之 抗體145或兩個適 102B)。這些抗體 143和144標示, 或電化學發光共振 體。螢光團或發光 聯結子1 4 9接合至 見圖101A和102A !靶蛋白質142,互 見圖101B和102B 靠近,導致有效之 250 或 ERET 862。 號起因於接合至兩 在沒有接合至蛋白 -63- 201209403 質1 42的情況下彼此雜交’需要小心地選擇互補寡核苷酸 147的序列和長度,使得雙股的解離常數(kd)相當高( 〜5 μΜ)。因此’當以這些寡核苷酸標示之游離抗體或適 體以毫微莫耳濃度混合時,恰巧低於其kd之莫耳濃度, 雙股形成的機率和產生之FRET 25 0或ERET 8 62訊號係 可忽略。然而’當抗體145兩者或適體141兩者皆接合至 標IE蛋白質142,寡核苷酸147的區域濃度將遠高於其kd ’造成幾乎完全雜交並且產生可偵測之FRET 250或 ERET 862 訊號。 當設計一個均質蛋白質檢測分析時,螢光團或發光團 的選擇爲重要的考量。粗細胞溶胞產物通常爲混濁的,且 可能包含自動發螢光的物質。在這種情況下,需要使用具 有長效螢光或電化學發光的分子以及最適化以發出最大 FRET 2 5 0或ERET 862之予體-受體對143和144。一個 此予體-受體對爲銪螯合物和Cy5,當與其他的予體-受體 對相比時’此種予體-受體對先前已顯示在此種系統能顯 著增進訊號-背景比,其藉由在干擾背景螢光、電化學發 光或散射光衰減後使訊號被讀取。銪螯合物和AlexaFluor 647或Μ蜜合物和Fluorescein FRET或 ERET對亦可良 好作用。這個方法的靈敏度和特異性與酵素連結免疫吸附 法(ELIS As )相似,但不須對樣本進行操控。 在LOC裝置的一些具體實施例,抗體145之一者或 適體141之一者係接合至蛋白質體分析腔室124(例如見 圖103和104),且蛋白質溶胞產物係在溶胞期間於化學 -64- 201209403 溶胞部130內與另一抗體145或適體141結合以促進 入蛋白質體分析腔室124之前接合至第一抗體145或 141。當在蛋白質體分析腔室中僅需要一個接合或雜 件,此可增加接續之速度和產生的可偵測訊號。 螢光團設計 需要具長螢光壽命之螢光團以允許激發光具足夠 來衰變至較致能光感測器44時之螢光發射的強度爲 強度,藉此提高充分的信號對雜訊比。而且,較長的 壽命代表較大之整合的螢光子計數。 螢光團246(見圖59)之螢光壽命大於100奈秒 常大於200奈秒、更常見爲大於3 00奈秒,以及於大 的情況中爲大於4 0 0奈秒。 以過渡金屬或鑭系金屬爲底的金屬-配位子錯合 長壽命(自數百奈秒至毫秒)、適當的量子產率,以 熱、化學及光化學穩定性,此等特性均爲相關於螢光 系統需求之有利特性。 以過渡金屬離子釕(Ru ( II ))爲底之經特別 底硏究之金屬-配位子錯合物爲參(2,2'-聯吡啶)釕 )([Ru(bpy)3]2+),彼之壽命爲約1μ3。此錯合物可 Biosearch Technologies,其商品名爲 Pulsar 650。 在進 適體 交事 時間 低之 螢光 、經 多數 物具 及高 檢測 地徹 (II 購自 -65- 201209403The second type of analysis uses two bodies 141 that must be joined independently of the epitope or target protein 142 region (see Figures 101A, 101B, 102A and 145 or aptamer 141 with different fluorophores or luminescent groups that can be used as fluorescence Resonance Energy Transfer (FRET) Energy Transfer (ERET) reactions in which the donor and acceptor groups 143 and 144 form part of a pair of short complementary oligonucleotides 147 via a long elastic antibody or aptamer ( ). Once antibody 145 or aptamer 141 is linked to the complementary oligonucleotide 147 to each other and hybridize to each other ( ) such that the precursor and the receptors 1 43 and 1 44 can be used as FRET for the target protein detection signal in order to determine no, or very A few background antibodies 145 or aptamer 141 oligonucleotides 147 and 144 are indicated, which are chemiluminescent resonance energy L maps 100A and 100B] aptamer 141, and when the formation is changed in intervals. The receptor is located at the proximal end. The new configuration results in a donor when the acceptor is a quencher and I add a light emission of 250 or to a different and non-overlapping antibody 145 or two suitable 102B). These antibodies 143 and 144 are labeled, or electrochemiluminescent resonators. The fluorophore or luminescent linker 14 9 is bonded to see Figure 101A and 102A! Target protein 142, which is adjacent to Figures 101B and 102B, resulting in a valid 250 or ERET 862. No. Because the ligation to the two hybridizes to each other without binding to the protein-63-201209403 11 42 'The sequence and length of the complementary oligonucleotide 147 need to be carefully selected so that the dissociation constant (kd) of the double strand is quite high (~5 μΜ). Therefore, 'when the free antibodies or aptamers labeled with these oligonucleotides are mixed at a nanomolar concentration, they happen to be lower than the molar concentration of their kd, the probability of double-strand formation and the FRET 25 0 or ERET 8 62 produced. The signal is negligible. However, when both antibody 145 or aptamer 141 are conjugated to the standard IE protein 142, the regional concentration of oligonucleotide 147 will be much higher than its kd' causing almost complete hybridization and producing detectable FRET 250 or ERET 862 signal. The choice of fluorophores or luminophores is an important consideration when designing a homogeneous protein assay. Crude cell lysates are typically turbid and may contain substances that autofluorescent. In this case, it is desirable to use molecules with long-acting fluorescence or electrochemiluminescence as well as donor-acceptor pairs 143 and 144 that are optimized to emit maximum FRET 250 or ERET 862. One such donor-receptor pair is a ruthenium chelate and Cy5, when compared to other host-receptor pairs, 'this type of receptor-receptor has previously been shown to significantly enhance the signal in such systems- The background ratio is such that the signal is read by attenuating background fluorescence, electrochemiluminescence, or scattered light attenuation. The ruthenium chelate and the AlexaFluor 647 or ruthenium complex and the Fluorescein FRET or ERET pair also work well. The sensitivity and specificity of this method is similar to that of enzyme-linked immunosorbent assay (ELIS As), but does not require manipulation of the sample. In some embodiments of the LOC device, one of the antibodies 145 or one of the aptamers 141 is ligated to the proteomic analysis chamber 124 (see, for example, Figures 103 and 104), and the protein lysate is lysed during lysis Chemistry -64 - 201209403 The lysate 130 binds to another antibody 145 or aptamer 141 to facilitate binding to the first antibody 145 or 141 prior to entry into the proteomic analysis chamber 124. When only one bond or miscellaneous is required in the proteomic analysis chamber, this increases the speed of the connection and the detectable signal produced. The fluorophore design requires a fluorophore with a long fluorescence lifetime to allow the excitation light to be sufficiently attenuated to the intensity of the fluorescent emission when the photosensor 44 is enabled, thereby increasing the sufficient signal to the noise. ratio. Moreover, a longer lifetime represents a larger integrated fluorescence count. Fluorescence lifetime of fluorophore 246 (see Figure 59) is greater than 100 nanoseconds, often greater than 200 nanoseconds, more typically greater than 300 nanoseconds, and greater than 4000 nanoseconds in large cases. Metal-coordination-based long-life (from hundreds of nanoseconds to milliseconds) based on transition metals or lanthanide metals, appropriate quantum yields, thermal, chemical, and photochemical stability, all of which are characterized by thermal, chemical, and photochemical stability. A beneficial feature related to the needs of the fluorescent system. The metal-coordination complex based on the transition metal ion ruthenium (Ru(II)) is a ruthenium (2,2'-bipyridyl) ruthenium ([Ru(bpy)3]2 +), the lifetime of his is about 1μ3. This complex is available from Biosearch Technologies under the trade name Pulsar 650. Fluorescent, versatile and high-detection in the accommodating time of the aptamer (II purchased from -65- 201209403
表1 : Pulsar 65 0 (釕螯合物)之光物理性質 參數 符號 値 單元 吸收波長 λ a b s 460 n m 發射波長 λ e m 650 nm 吸光係數 E 1 4800 M'1 cm'1 營光壽命 Tf 1 .0 量子產率 H ^ 1 (去氧的) N/A 鑭系金屬-配位子錯合物,铽螯合物,已成功地顯示 作爲FRET探針系統中的螢光指示劑,且具有1 600μ5之 長壽命。 表2:铽螯合物之光物理性質Table 1: Photophysical properties of Pulsar 65 0 (钌 chelate) Parameter symbol 値 Unit absorption wavelength λ abs 460 nm Emission wavelength λ em 650 nm Absorption coefficient E 1 4800 M'1 cm'1 Camp light lifetime Tf 1.0 Quantum yield H ^ 1 (deoxygenated) N/A lanthanide metal-coordination complex, ruthenium chelate, has been successfully shown as a fluorescent indicator in the FRET probe system with 1 600 μ5 Long life. Table 2: Photophysical properties of ruthenium chelate
參數 符號 値 單元 吸收波長 λ a b s 330-350 n m 發射波長 λ e m 548 nm 吸光係數 E 1 3 800 Qabs,及配位子相依,可高至 30000 @ X c = 340 nm) 螢光壽命 tf 1600 (雜交的探針) μδ 量子產率 H 1 (配位子相依) N/A LOC裝置3 0 1所使用的螢光檢測系統不利用濾鏡來移 除不想要的背景螢光。若淬熄劑248無天然發射以增加信 號·對-雜訊比,則因此具有優勢。無天然發射,則淬熄劑 24 8不貢獻至背景螢光。高淬熄效率亦爲重要者,此使得 -66 - 201209403 雜父發生前沒有蛋光。購自加州Novato市之Biosearch Technologies, Inc.的黑洞淬熄劑(BHq)不具有天然發射 及具有高淬熄效率,以及係用於系統之合適的淬熄劑。 BHQ-1之最大吸收値發生於534 nm及淬熄範圍爲480-580 nm,使得其爲用於Tb-螯合螢光團之合適的淬熄劑。 BHQ-2之最大吸收値發生於579 nm及淬熄範圍爲5 60-670 nm使得其爲用於Pulsar 650之合適的淬熄劑。 購自愛荷華州 Coralville市之 Integrated DNA Technologies的愛荷華黑淬熄劑(Iowa Black FQ及RQ) 爲適合的具有少許或無背景發射之替代性淬熄劑。Iowa Black FQ之淬熄範圍爲420-620 nm,於53 1 nm具有最大 吸收値,並因此爲用於Tb-螯合螢光團之合適的淬熄劑。 Iowa Black RQ於656 nm具有最大吸收値及淬熄範圍爲 500-700 nm,使得其爲用於Pulsar 65 0之理想淬熄劑。 於本文所述之具體實施例中,淬熄劑248爲初始時即 附著於探針之功能部分,但於其他具體實施例中,淬熄劑 可爲游離於溶液中之分離的分子。 激發源 在本文描述之螢光偵測爲基礎的具體實施例中,因爲 低功率消耗、低成本和小尺寸,LED係選做替代雷射二極 體、高功率電燈或雷射的激發源。參照圖81,LED26係 直接安置於LOC裝置301之外部表面上之來自各腔室之 雜交腔室陣列1 1 〇上。在雜交腔室陣列1 1 〇之對側爲光感 -67- 201209403 測器44,其由用於偵測螢光訊號之光二極體1 84的陣列 所組成(見圖53、54和64 )。 圖82、83和84槪述用於將探針暴露於激發光之其他 具體實施例。在顯示於圖82之LOC裝置30中,由激發 LED26所產生之激發光244係由透鏡254導向雜交腔室陣 列110之上。脈衝激發激發LED26且由光感測器44偵測 螢光發射。 在圖83所顯示之LOC裝置30中,由激發LED26所 產生之激發光244係由透鏡254、第一光稜鏡712和第二 光稜鏡714導向雜交腔室陣列110之上。脈衝激發激發 LED26且由光感測器44偵測螢光發射。 同樣地,顯示於圖84之LOC裝置30,由激發 LED26所產生之激發光244係由透鏡254、第一鏡面716 和第二鏡面718導向雜交腔室陣列110之上。再次脈衝激 發激發LED26且由光感測器44偵測螢光發射。 LED26的激發波長係倚賴螢光染料的選擇。Philips LXK2-PR14-R00爲針對Pulsar 650染料之合適的激發源 。SET UVT0P3 3 5T039BL LED係針對铽螯合物標記之合 適的激發源。 -68- 201209403Parameter symbol 値 unit absorption wavelength λ abs 330-350 nm emission wavelength λ em 548 nm absorption coefficient E 1 3 800 Qabs, and ligand-dependent, up to 30000 @ X c = 340 nm) fluorescence lifetime tf 1600 (hybridization Probes) μδ Quantum Yield H 1 (Coordination Dependent) N/A LOC Device 3 0 1 The fluorescence detection system used does not use filters to remove unwanted background fluorescence. It is therefore advantageous if the quencher 248 has no natural emission to increase the signal-to-noise ratio. Without natural emission, the quencher 24 8 does not contribute to background fluorescence. High quenching efficiency is also important, which makes -66 - 201209403 no egg light before the occurrence of the father. The black hole quencher (BHq), available from Biosearch Technologies, Inc. of Novato, Calif., does not have natural emissions and has high quenching efficiency, as well as suitable quenchers for use in systems. The maximum absorption enthalpy of BHQ-1 occurs at 534 nm and the quenching range is 480-580 nm, making it a suitable quencher for Tb-chelating fluorophores. The maximum absorption enthalpy of BHQ-2 occurs at 579 nm and the quenching range is 5 60-670 nm making it a suitable quencher for Pulsar 650. Iowa Black Quench (Iowa Black FQ and RQ) from Integrated DNA Technologies of Coralville, Iowa, is an alternative quenching agent with little or no background emission. Iowa Black FQ has a quenching range of 420-620 nm with a maximum absorption enthalpy at 53 1 nm and is therefore a suitable quencher for Tb-chelating fluorophores. Iowa Black RQ has a maximum absorption enthalpy at 656 nm and a quenching range of 500-700 nm, making it an ideal quencher for Pulsar 65 0 . In the specific embodiments described herein, quenching agent 248 is a functional portion that is initially attached to the probe, but in other embodiments, the quenching agent can be a separate molecule that is free of solution. Excitation Sources In the specific embodiment based on the fluorescence detection described herein, LEDs are selected as excitation sources instead of laser diodes, high power lamps, or lasers because of low power consumption, low cost, and small size. Referring to Fig. 81, LEDs 26 are disposed directly on the outer surface of the LOC device 301 on the array of hybridization chambers 1 1 from each chamber. On the opposite side of the hybridization chamber array 1 1 is a light-sensing -67-201209403 detector 44 consisting of an array of photodiodes 1 84 for detecting fluorescent signals (see Figures 53, 54 and 64). . Figures 82, 83 and 84 illustrate other specific embodiments for exposing the probe to excitation light. In the LOC device 30 shown in FIG. 82, the excitation light 244 generated by the excitation LED 26 is directed by the lens 254 onto the hybridization chamber array 110. The pulse excitation excites the LED 26 and the photodetector 44 detects the fluorescent emission. In the LOC device 30 shown in Fig. 83, the excitation light 244 generated by the excitation LED 26 is directed by the lens 254, the first aperture 712 and the second aperture 714 onto the hybridization chamber array 110. The pulse excites the excitation LED 26 and the photodetector 44 detects the fluorescent emission. Similarly, the LOC device 30 shown in FIG. 84, the excitation light 244 generated by the excitation LED 26 is directed by the lens 254, the first mirror 716, and the second mirror 718 onto the hybridization chamber array 110. The LED 26 is pulse activated again and the fluorescent emission is detected by the photo sensor 44. The excitation wavelength of LED 26 relies on the choice of fluorescent dye. Philips LXK2-PR14-R00 is a suitable excitation source for Pulsar 650 dyes. SET UVT0P3 3 5T039BL LED is the appropriate excitation source for the ruthenium chelate label. -68- 201209403
表格 3: Philips LXK2-PR14-R00 LED 規格 參數 符號 値 單位 波長 λ e X 460 nm 發射頻率 ^ e m 6.52 ( 1 0) 14 Hz 輸出功率 Pl 0.5 1 5 (分鐘)@ 1 A W 輻射圖案 Lambertian 數據圖 N/ATable 3: Philips LXK2-PR14-R00 LED Specifications Parameter 値 Unit wavelength λ e X 460 nm Transmit frequency ^ em 6.52 ( 1 0) 14 Hz Output power Pl 0.5 1 5 (minutes) @ 1 AW Radiation pattern Lambertian Data sheet N /A
表格 4: SET UVT0P3 3 4T039BL LED 規格 參數 符號 値 單位 波長 λ e 340 nm 發射頻率 Ve 8.82(10)14 Hz 功率 Pi 0.000240(分鐘)@ 20mA W 脈衝正向電流 I 200 m A 輻射圖案 Lambertian N/A 紫外光激發光 矽在UV光譜中吸收少量光。因此,使用UV激發光 是有利的。可使用UV LED激發源,但LED26之寬光譜 降低此方法之效果。爲了說明此,使用經過濾的UV LED 。隨意地,UV雷射可爲激發源,除非相當高的雷射花費 對於特定的試驗模組市場不實用。 LED驅動器 LED驅動器29針對所需的持續時間在固定電流下驅 動LED26。低功率USB2.0認證裝置可在至多1單位負載 (1 〇〇毫安培)以最小操作電壓4·4伏特得到。標準電力 調節電路係用於此目的。 -69- 201209403 光二極體 圖54顯示光二極體184,其合倂於L〇c裝置3〇1之 CMOS電路86。光二極體184係在沒有額外遮罩或步驟下 製成CMOS電路86之一部分。這是CMOS光二極體優於 CCD之一項顯著的優點,Ccd爲另一種感測技術,其可 使用非標準式加工步驟整合到同一晶片上或者製於相鄰晶 片上。晶片上偵測係花費低廉且降低分析系統的尺寸。較 短光學路徑長度降低來自週遭環境的雜訊以有效收集該螢 光訊號’以及抑制對於透鏡及濾鏡之傳統光學總成之需求 〇 光二極體184之量子效率爲光子衝撞其有效區185之 分率’光子係有效轉換成光電子。對於標準矽處理,對於 可見光該量子效率根據處理參數(諸如覆蓋層之數量及吸 收性質)係在0.3至0.5的範圍中。 光二極體1 8 4之偵測閥値決定可被偵測之螢光訊號的 最小強度。偵測閥値亦決定光二極體1 84的尺寸大小以及 在雜交及檢測部52中之雜交腔室180數目(見圖52)。 腔室的尺寸大小和數量爲技術參數,其由LOC裝置的尺 寸(LOC裝置301的實例中,其尺寸爲1:760微米 X 5 8 24微米)所限制,且合倂其他功能性模組(諸如病原 體透析部70及擴增部1 1 2 )之後可用之不動物件的尺寸 所限制。 對於標準矽處理,光二極體1 84最少偵測5個光子。 然而,爲了確認可信賴的偵測,最小値可設爲十個光子。 -70- 201209403 因此以量子效率範圍在0.3至0.5(如上所討論),來自 等探針之螢光發射爲最少17個光子,而針對可靠偵測30 個光子包含的的合適誤差界線。 校準腔室 光二極體184的電學特性之不均勻性、自動螢光和尙 未完全衰減之剩餘激發光子通量將背景雜訊引入並偏移至 輸出訊號。使用一或多種校準訊號將背景自各輸出訊號移 除。校準訊號藉由將在陣列中之一或多種校準光二極體 1 84暴露於各自的校準源而產生。低校準源用來判斷標靶 尙未與探針反應之負結果。高校準源代表自探針-標靶複 合物造成的正結果。在本文所描述的具體實施例,低校準 光源由在雜交腔室陣列110中之校準腔室382所提供,其 不含任何探針; 包含不具有螢光指示劑的探針;或 包含具有指示劑的探針和配置使得總是預期發生淬熄 的淬熄劑。 來自此種校準腔室3 82之輸出訊號非常接近來自在 LOC裝置中之所有雜交腔室的輸出訊號中的雜訊和偏差。 自其他雜交腔室所產生的輸出訊號減去校準訊號大體上移 除了背景和留下由螢光發射產生的訊號(若有產生任何訊 號的話)。自腔室陣列之區域中的環境光線產生的訊號亦 被去除。 -71 - 201209403 可理解的是參考圖92至95之上述負控制組探針可用 於校準腔室。然而’如圖86和87所示,其爲顯示於圖 85之LOC變體X728的插圖DG和DH之放大圖,另一選 項爲將校準腔室382與擴增子流體隔離。當雜交由流體隔 離阻止時,背景雜訊和偏差可由將流體隔離之腔室淨空或 藉由包含缺少指示劑的探針或確切具有指示劑與淬熄劑兩 者的任何“標準”探針來判斷。 校準腔室3 82可提供高校準源以於對應的光二極體產 生高訊號。高訊號對應在已雜交腔室中的所有探針。以指 示劑且無淬熄劑或僅以指示劑點樣探針將一致地提供近似 雜交腔室訊號之訊號,主要數量之探針已於雜交腔室內雜 交。將可理解校準腔室3 82可用以代替控制探針或加至控 制探針上。 遍布雜交腔室陣列的校準腔室382的數量和安排是隨 意的。然而,若光二極體184由相對最近的校準腔室382 校準,則校準較準確。參考圖56,雜交腔室陣列110對 於每八個雜交腔室180具有一個校準腔室3 82。也就是說 ,校準腔室3 82係安置於每個三乘三之正方形雜交腔室 180的中間。在這個配置中,雜交腔室180係由緊接鄰近 的雜交腔室3 82所校準。 由於從周圍雜交腔室180之自螢光訊號的激發光,圖 91顯示用以自對應校準腔室3 82之光二極體184減除訊 號的差分成像器電路788。差分成像器電路78 8自像素 790和“虛擬”像素792取樣訊號。在一個具體實施例中 -72- 201209403 ,“虛擬”像素792係被遮住以防光照射,所以其輸出訊 號提供暗參考像素。或者,“虛擬”像素792可和陣列的 其餘部分暴露於激發光。在一個具體實施例中,“虛擬” 像素792是可以接受光的,自腔室陣列之區域中的環境光 線產生的訊號亦可被減除。來自像素790的訊號是微弱的 (例如,接近暗訊號),且沒有參考暗訊號位準會很難分 辨背景値與非常微弱的訊號。 在使用期間,啓動 “read_row” 794 和 “read_row_d” 795且開啓M4 797和 MD4 80 1電晶體。關閉開關807 Γ 和8 09使得來自像素790及“虛擬”像素792的輸出各自 儲存在像素電容器803及虛擬像素電容器805上。在像素 訊號被儲存後,停用開關 8 07和 809。然後關閉 “read_col”開關81 1和虛擬“read_c〇l”開關813,且在輸出 的該轉換的電容器放大器815放大差分訊號817。 光二極體之抑制及致能 於LED 26激發期間必須抑制光二極體1 84及於螢光 期間必須致能光二極體184。圖65爲單一光二極體184 之電路圖及圖66爲光二極體控制信號之時序圖。電路具 有光二極體184及六個MOS電晶體,MshurU 394、M,x 396 、Mreset 3 98、Msf 400、Mread 402 及 Mbias 404。於激發循 環開始時,藉由拖曳(pulling) Mshunt閘極384及重設 閘極388爲高而開啓tl、電晶體Mshunt 3 94及Mreset 398 。於此期間,激發光子於光二極體184中產生載子。當產 -73- 201209403 生的載子量可充分使光二極體184飽和時,此等載子必須 被移除。於此循環期間,因電晶體的洩漏或因基板中之激 發-產生的載子擴散,Mshunt 3 94直接地移除光二極體184 中所產生的載子,而Mreset 3 98重設累積於節點‘NS’ 406 之任何載子。於激發之後,於t4開始俘獲循環。於此循 環中,來自螢光團之發射的回應被俘獲並整合入節點‘NS’ 4 06上的電路。此藉由拖曳tx閘極3 86爲高而達成,此開 啓電晶體Mtx 3 96及轉移光二極體184上任何累積的載體 至節點‘NS’ 406。俘獲循環期間可如螢光發射般長。來自 雜交腔室陣列110中之所有光二極體184的輸出同時被俘 獲。 於結束俘獲循環t5與開始讀取循環t6之間具有延遲 。此延遲肇因於,在俘獲循環之後,分別讀取雜交腔室陣 列110中之各光二極體184的需求(見圖52)。待讀取 的第一光二極體184於讀取循環之前將具有最短的延遲, 而最後光二極體184於讀取循環之前將具有最長的延遲。 於讀取循環期間,藉由拖曳閘極393爲高而開啓電晶體 Mread 402。使用源極-隨耦器電晶體Msf 400來緩衝及讀 出‘NS’節點406之電壓。 以下討論另外之任意的致能或抑制光二極體之方法: 1 抑制方法 圖88、89及90顯示用於Mshunt電晶體394之三種可 行的組態778、7 80 ' 7 82。於激發期間被致能之最大値 -74- 201209403 |h|= 5 V時,Mshunt電晶體394具有非常高的關閉比。如 圖88中所示者,Mshunt閘極3 84係組態成位於光二極體 184之緣上。任意地,如圖89中所示者,Mshunt閘極384 係可組態成環繞光二極體184。第三個選擇爲將Mshunt閘 極384組構於光二極體184之內,如圖90中所示者。依 此第三選擇,光二極體有效區185較少。 這三種組態7 7 8、7 8 0及7 8 2降低自光二極體1 8 4中 所有位置至Mshunt閘極3 84之平均路徑長度。於圖88中 ,Mshunt閘極3 84係於光二極體184之一側上。此爲用以 製造之最簡單且對於光二極體有效區1 85衝擊最小的組態 。然而,滯留於光二極體184遠端之任何載子需要較長時 間以擴散通過Mshunt閘極3 84。 於圖89中,Mshunt閘極384環繞光二極體184。此進 一步降低光二極體184中之載子至Mshunt閘極3 84之平均 路徑長度。然而,繞光二極體184周圍而延伸Mshunt閘極 384造成光二極體有效區185大幅縮減。於圖90中之組 態782將Mshunt閘極384定位於有效區185中。此提供了 至Mshunt閘極3 84的最短平均路徑長度及因此得到最短過 渡時間。然而,對於有效區185之衝擊最大。其亦造成較 寬的洩漏路徑。 2.致能方法 a. 觸發器光二極體以固定的延遲來驅動並聯電晶體。 b. 觸發器光二極體以可程控的延遲來驅動並聯電晶體。 -75- 201209403 c. 由LED驅動脈衝以固定的延遲來驅動並聯電晶體 d. 如2c般但以可程控的延遲來驅動並聯電晶體。 圖68爲透過雜交腔室180顯示埋入於CMOS電丨 中之光二極體184及觸發器光二極體187之槪略剖視 以觸發器光二極體187取代光二極體184之角落中的 積。因相較於螢光發射時激發光的強度爲高,具小面 觸發器光二極體187係充分的。觸發器光二極體187 激發光244爲敏感。觸發器光二極體187顯示激發光 已熄滅並於短暫延遲At 300之後啓動光二極體184 ( 2 )。此延遲使得螢光光二極體1 84得以於沒有激 244時檢測來自FRET探針186之螢光發射。此致能 及增進信號對雜訊比。 於各雜交腔室180下,光二極體184及觸發器光 體1 87兩者均位於CMOS電路86中。光二極體陣列 當電子組件合倂以形成光感測器44 (見圖64 )。光 體184爲CMOS結構製造期間所製成的pn接面而不 外的遮罩或步驟。於MST製造期間,光二極體184 的介電層(未顯示)係利用標準MST光蝕刻技術而 地薄化以使更多螢光照射光二極體184的有效區185 二極體184具有視場,使得來自雜交腔室180內之右 標靶雜交體的螢光信號入射至感測器表面上。轉換螢 爲接著可使用CMOS電路86而被測量的光電流。 替代性地’一或多個雜交腔室180可僅專用於觸 珞86 圖。 小面 積之 係對 244 見圖 發光 檢測 二極 與適 二極 需另 之上 任意 。光 請-光成 發器 -76- 201209403 光二極體187。可使用這些選擇於此等與上述之2a及2b 的組合中。 螢光的延遲偵測 下述推導說明針對上述之LED/螢光團組合之使用長 生命週期螢光團的螢光延遲偵測。在由圖60顯示之時間 ίι和Ο之間的固定強度Ie之理想脈衝激發之後,螢光強 度推導爲時間的函數。 令[S1 ] ( 〇於時間t等於激發態的強度,然後在激發 期間及之後,每單位體積每單位時間的激發態數量由下面 微分方程式描述: dt tf hve 其中C爲螢光團的莫耳濃度,ε爲莫耳消光係數,1;6爲 激發頻率,且h = 6.62606896 ( 10) -34 JS爲普朗克常數 〇 此微分方程式具有一般式: ~- + p(x)y = q(x)Table 4: SET UVT0P3 3 4T039BL LED Specifications Parameter 値 Unit wavelength λ e 340 nm Transmit frequency Ve 8.82(10)14 Hz Power Pi 0.000240 (minutes) @ 20mA W Pulse forward current I 200 m A Radiation pattern Lambertian N/A The ultraviolet excitation pupil absorbs a small amount of light in the UV spectrum. Therefore, it is advantageous to use UV excitation light. A UV LED excitation source can be used, but the broad spectrum of LED 26 reduces the effectiveness of this method. To illustrate this, a filtered UV LED is used. Optionally, a UV laser can be an excitation source unless a relatively high laser cost is not practical for a particular test module market. LED Driver The LED driver 29 drives the LED 26 at a fixed current for the desired duration. The low-power USB 2.0 certified device is available with a minimum operating voltage of 4·4 volts for up to 1 unit load (1 〇〇 mA). Standard power conditioning circuits are used for this purpose. -69- 201209403 Photodiode Figure 54 shows an optical diode 184 that is integrated into a CMOS circuit 86 of the L〇c device 3〇1. Light diode 184 is formed as part of CMOS circuit 86 without additional masking or steps. This is a significant advantage of CMOS photodiodes over CCDs, which are another sensing technique that can be integrated onto the same wafer or fabricated on adjacent wafers using non-standard processing steps. The on-wafer detection system is inexpensive and reduces the size of the analysis system. The shorter optical path length reduces noise from the surrounding environment to efficiently collect the fluorescent signal' and suppresses the need for conventional optical assemblies for lenses and filters. The quantum efficiency of the photodiode 184 is photon collision with its active region 185. The fractional 'photon system is effectively converted into photoelectrons. For standard enthalpy treatment, the quantum efficiency is in the range of 0.3 to 0.5 depending on processing parameters such as the number of capping layers and the absorptive properties for visible light. The detection valve of the photodiode 1 8 4 determines the minimum intensity of the fluorescent signal that can be detected. The detection valve 値 also determines the size of the photodiode 184 and the number of hybridization chambers 180 in the hybridization and detection section 52 (see Figure 52). The size and number of chambers are technical parameters that are limited by the size of the LOC device (in the example of LOC device 301, which is 1:760 microns X 5 8 24 microns) and incorporates other functional modules ( The size of the non-animal pieces that can be used after the pathogen dialysis section 70 and the amplification section 1 1 2) are limited. For standard 矽 processing, photodiode 1 84 detects at least 5 photons. However, to confirm reliable detection, the minimum 値 can be set to ten photons. -70- 201209403 Thus with a quantum efficiency range of 0.3 to 0.5 (as discussed above), the fluorescence emission from the probes is a minimum of 17 photons, and the appropriate error bounds for reliable detection of 30 photons are included. Calibration Chamber The non-uniformity of the electrical characteristics of the photodiode 184, autofluorescence, and residual excitation photon flux that are not fully attenuated introduces background noise and shifts to the output signal. Use one or more calibration signals to remove the background from each output signal. The calibration signal is generated by exposing one or more of the calibration photodiodes 1 84 in the array to respective calibration sources. A low calibration source is used to determine the negative result of the target 尙 not reacting with the probe. A high calibration source represents a positive result from the probe-target complex. In the specific embodiments described herein, the low calibration source is provided by a calibration chamber 382 in the hybridization chamber array 110, which does not contain any probes; includes a probe that does not have a fluorescent indicator; or contains an indication The probe and configuration of the agent is such that the quenching quenching agent is always expected to occur. The output signal from such a calibration chamber 382 is very close to the noise and bias in the output signal from all of the hybridization chambers in the LOC device. The output signal from the other hybridization chamber minus the calibration signal substantially removes the background and leaves the signal generated by the fluorescent emission (if any signal is generated). Signals generated by ambient light in the area of the array of chambers are also removed. -71 - 201209403 It will be appreciated that the above negative control group probes with reference to Figures 92 through 95 can be used in the calibration chamber. However, as shown in Figures 86 and 87, which is an enlarged view of the inset DG and DH of the LOC variant X728 shown in Figure 85, another option is to isolate the calibration chamber 382 from the amplicon fluid. When hybridization is prevented by fluid isolation, background noise and bias can be cleared by chambers that isolate the fluid or by any probe that contains a missing indicator or any "standard" probe that has both an indicator and a quencher. Judge. The calibration chamber 3 82 provides a high calibration source to generate a high signal for the corresponding photodiode. The high signal corresponds to all probes in the hybridized chamber. The indicator, with no quencher or only the indicator spotting probe, will consistently provide a signal that approximates the hybridization chamber signal, and the majority of the probes have been hybridized within the hybridization chamber. It will be appreciated that the calibration chamber 382 can be used in place of or in addition to the control probe. The number and arrangement of calibration chambers 382 throughout the array of hybrid chambers is arbitrary. However, if the photodiode 184 is calibrated by the relatively closest calibration chamber 382, the calibration is more accurate. Referring to Figure 56, hybridization chamber array 110 has a calibration chamber 382 for every eight hybridization chambers 180. That is, the calibration chamber 382 is disposed in the middle of each of the three by three square hybridization chambers 180. In this configuration, the hybridization chamber 180 is calibrated by the adjacent hybridization chambers 382. Due to the excitation light from the fluorescent signal from the surrounding hybridization chamber 180, Figure 91 shows a differential imager circuit 788 for subtracting the signal from the photodiode 184 of the corresponding calibration chamber 382. Differential imager circuit 78 8 samples the signal from pixel 790 and "virtual" pixel 792. In one embodiment -72-201209403, the "virtual" pixel 792 is shielded from light illumination, so its output signal provides a dark reference pixel. Alternatively, the "virtual" pixel 792 can be exposed to the excitation light and the remainder of the array. In one embodiment, the "virtual" pixel 792 is light absorbing, and the signal generated by the ambient light in the region of the array of cells can also be subtracted. The signal from pixel 790 is weak (e.g., close to the dark signal), and it is difficult to distinguish between background and very weak signals without reference to the dark signal level. During use, start "read_row" 794 and "read_row_d" 795 and turn on the M4 797 and MD4 80 1 transistors. The switches 807 and 809 are turned off such that the outputs from pixel 790 and "virtual" pixel 792 are each stored on pixel capacitor 803 and virtual pixel capacitor 805. After the pixel signal is stored, switches 8 07 and 809 are disabled. The "read_col" switch 81 1 and the dummy "read_c〇l" switch 813 are then turned off, and the converted capacitor amplifier 815 is amplified at the output of the differential signal 817. The suppression and enabling of the photodiode must be such that the photodiode 184 must be suppressed during the excitation of the LED 26 and the photodiode 184 must be enabled during the luminescence. 65 is a circuit diagram of a single photodiode 184 and FIG. 66 is a timing diagram of an optical diode control signal. The circuit has a photodiode 184 and six MOS transistors, MshurU 394, M, x 396, Mreset 3 98, Msf 400, Mread 402 and Mbias 404. At the beginning of the excitation cycle, tl, transistor Mshunt 3 94, and Mreset 398 are turned on by pulling Mshunt gate 384 and reset gate 388 high. During this period, the photons are excited to generate carriers in the photodiode 184. When the amount of carrier produced at -73-201209403 is sufficient to saturate the photodiode 184, these carriers must be removed. During this cycle, Mshunt 3 94 directly removes the carriers generated in photodiode 184 due to leakage of the transistor or due to excitation-generated carrier diffusion in the substrate, while Mreset 3 98 resets the accumulation at the node. Any carrier of 'NS' 406. After excitation, the capture cycle begins at t4. In this loop, the response from the emission of the fluorophore is captured and integrated into the circuit on node 'NS' 608. This is achieved by dragging the tx gate 386 high, which turns on any accumulated carrier on the transistor Mtx 3 96 and the transfer photodiode 184 to node 'NS' 406. The capture cycle can be as long as a fluorescent emission. The outputs from all of the photodiodes 184 in the hybrid chamber array 110 are simultaneously captured. There is a delay between the end of the capture cycle t5 and the start of the read cycle t6. This delay is due to the need to read the respective photodiodes 184 in the hybridization chamber array 110 after the capture cycle (see Figure 52). The first photodiode 184 to be read will have the shortest delay before the read cycle, and the last photodiode 184 will have the longest delay before the read cycle. During the read cycle, the transistor Mread 402 is turned on by dragging the gate 393 high. The source-slaffer transistor Msf 400 is used to buffer and read the voltage of the 'NS' node 406. Any other method of enabling or suppressing the photodiode is discussed below: 1 Suppression Method Figures 88, 89 and 90 show three possible configurations 778, 7 80 ' 7 82 for Mshunt transistor 394. The maximum 値-74- 201209403 |h|= 5 V is enabled during the excitation period, and the Mshunt transistor 394 has a very high shutdown ratio. As shown in FIG. 88, the Mshunt Gate 3 84 is configured to be located on the edge of the photodiode 184. Optionally, as shown in FIG. 89, the Mshunt gate 384 can be configured to surround the photodiode 184. A third option is to group the Mshunt gate 384 within the photodiode 184, as shown in FIG. According to the third option, the photodiode effective area 185 is less. These three configurations 7 7 8 , 7 8 0 and 7 8 2 reduce the average path length from all positions in the photodiode 1 8 4 to the Mshunt gate 3 84. In Fig. 88, the Mshunt gate 3 84 is attached to one side of the photodiode 184. This is the simplest configuration to make and the smallest impact on the active area of the photodiode 1 85. However, any carrier remaining at the distal end of the photodiode 184 requires a longer time to diffuse through the Mshunt gate 3 84. In FIG. 89, the Mshunt gate 384 surrounds the photodiode 184. This further reduces the average path length of the carriers in the photodiode 184 to the Mshunt gate 3 84. However, extending the Mshunt gate 384 around the photodiode 184 causes the photodiode active area 185 to be substantially reduced. The configuration 782 in Figure 90 positions the Mshunt gate 384 in the active area 185. This provides the shortest average path length to the Mshunt gate 3 84 and thus the shortest transition time. However, the impact on the active area 185 is greatest. It also creates a wide leak path. 2. Enabling method a. The flip-flop photodiode drives the parallel transistor with a fixed delay. b. The flip-flop photodiode drives the shunt transistor with a programmable delay. -75- 201209403 c. Driving the parallel transistor with a fixed delay by the LED drive pulse d. Drive the parallel transistor as a 2c but with a programmable delay. 68 shows a schematic cross-sectional view of the photodiode 184 and the flip-flop photodiode 187 embedded in the CMOS cell through the hybridization chamber 180. The flip-flop photodiode 187 replaces the product in the corner of the photodiode 184. . Since the intensity of the excitation light is higher than that of the fluorescent emission, the facet trigger photodiode 187 is sufficient. The trigger photodiode 187 excitation light 244 is sensitive. The flip-flop photodiode 187 shows that the excitation light has extinguished and activates the photodiode 184 (2) after a brief delay of At 300. This delay allows the fluorescent photodiode 1 84 to detect the fluorescent emissions from the FRET probe 186 when there is no excitation 244. This enables and enhances the signal-to-noise ratio. Under each of the hybridization chambers 180, both the photodiode 184 and the flip-flop light 187 are located in the CMOS circuit 86. Photodiode Array The electronic components are merged to form a photosensor 44 (see Figure 64). The light body 184 is a mask or step other than the pn junction made during the fabrication of the CMOS structure. During the MST fabrication, the dielectric layer (not shown) of the photodiode 184 is thinned by standard MST photolithography techniques to allow more of the active area 185 of the fluorescent illuminating diode 184 to have a field of view. The fluorescent signal from the right target hybrid within the hybridization chamber 180 is incident on the surface of the sensor. The conversion flicker is the photocurrent that can then be measured using the CMOS circuit 86. Alternatively, one or more of the hybridization chambers 180 may be dedicated only to the contact 86 map. The small area of the pair of 244 is shown in the illuminating detection of the two poles and the appropriate two poles need to be any other. Light Please - Light into the hair -76- 201209403 Light diode 187. These choices can be used in combination with 2a and 2b above. Fluorescent Delay Detection The following derivation shows the use of long-life fluorophore fluorescence delay detection for the above LED/fluorescent combination. After an ideal pulse excitation of the fixed intensity Ie between times ίι and Ο shown in Figure 60, the fluorescence intensity is derived as a function of time. Let [S1] (when the time t is equal to the intensity of the excited state, then during and after the excitation, the number of excited states per unit volume per unit time is described by the following differential equation: dt tf hve where C is the fluorophore Concentration, ε is the molar extinction coefficient, 1; 6 is the excitation frequency, and h = 6.62606896 (10) -34 JS is the Planck constant 〇 This differential equation has the general formula: ~- + p(x)y = q (x)
OX 其有解法:OX has a solution:
\e^P{x)dx q{x)dxJrk ΛΧ) = ——.. 現在使用此來解答式 [sim=!^iL+ke-^ hve -77- 201209403 然後於時間 [S\] ( n ) = 0,且式(3 )之 A:: k ηεοτ L〇''lzi hve 將(4 ) 代入(3 ) [剛 \εοτ{ IeecTf ^_(,_,i)/r/ hve hve 於時間t2, 剛2)\e^P{x)dx q{x)dxJrk ΛΧ) = ——.. Now use this to solve the equation [sim=!^iL+ke-^ hve -77- 201209403 and then at time [S\] ( n ) = 0, and A:: k ηεοτ L〇''lzi hve of equation (3) substituting (4) into (3) [just\εοτ{ IeecTf ^_(,_,i)/r/ hve hve at time T2, just 2)
eSCTf Ie€CT f a-^h)/rf hv„ h\ 於/ 2 G,激發態以指數衰減且以式(6 )描述 [51](〇 = [51](/2)e-(,-,j)/r^ ... ( 6 ) 將(5 )代入(6 ): ...(7 ) [51](〇 = ^^[1- g-(hWl )/r/ ]e_(iW2)/r/ hv„ 螢光強度由下列等式得到: j {〇 = _d^mhv ^ ... ( 8 ) αχ 其中ν/爲螢光頻率,η爲量子產率,且1爲光學路徑 長度 於是由(7 )可知: ^1](0 丨1召-"-'2)/1·, ( 9 ) dt " hve … 將(9 )代入(8 ): IAt) = /efc/7—[1 -e'(,2',,)/r/>'(,',ί)/Γ/ ... ( 10) -78- 201209403 因爲 If(t) ieSclv^J-e'{,~,l)lTfeSCTf Ie€CT f a-^h)/rf hv„ h\ at / 2 G, the excited state is exponentially decayed and described by equation (6) [51] (〇= [51](/2)e-(, -,j)/r^ (6) Substituting (5) into (6): ...(7) [51](〇= ^^[1- g-(hWl )/r/ ]e_( iW2)/r/ hv„ The fluorescence intensity is obtained by the following equation: j {〇= _d^mhv ^ ... ( 8 ) αχ where ν/ is the fluorescence frequency, η is the quantum yield, and 1 is the optical path The length is then known from (7): ^1](0 丨1call-"-'2)/1·, (9) dt " hve ... Substituting (9) into (8): IAt) = /efc/ 7—[1 -e'(,2',,)/r/>'(,',ί)/Γ/ ... ( 10) -78- 201209403 because If(t) ieSclv^J-e' {,~,l)lTf
τί K 因此’我們可以寫出下列的近似式,該式描述在足夠 長的激發脈衝(>> Tf)後之螢光強度衰減:對於 l^t2 If{t)^Iesc^^-eH,~'l)lTf ...(11) 在上一節,我們針對>> Tf作的情況做總結, 而對於 t > tl Ι^ΐπΐη'ε-㈣〜。 從上述的等式,我們可以導出下列式子: «/(0 = ηΒεοΙηβ~{'~,ι)ΙΤ/ ... ( 12) 其中 = 爲每單位面積每單位時間之螢光光子數且 Κ =4~爲每單位面積每單位時間之激發光子數。 因此, 〇〇 nfit)=\fif{t)dt ... ( 13 ) ,3 其中七爲每單位面積之螢光光子數且〖3爲光二極體開 啓的時間點。將 (12) 代入 (13): «/ = ^ηεεοΙηβΛ,~,ι)ΙΤ/ dt ...(14) 目前,每單位面積每單位時間到達光二極體之螢光光 子數,&(〇,係由下式獲得: ^(〇 = «>(〇^0 ... ( 15 ) -79- 201209403 其中彳。爲光學系統之光收集效率。 將(1 2 )代入(1 5 )我們發現 = Φοη,εαΙηβ'0''1'11^ …(16) 螢光光子 同樣地,每單位螢光面積t到達光二極體之 數將如下述: 〇〇 圮⑺汾且代入(16)並積分: ^3 ns = φΰηΒεοΙητ fe<h~h)'Tf 因此, ns = φ^ιΒεοΙητfe~^'11 ... ( 17) 體184內 之電子率 快更多。 子率爲: ί3的理想値係於當因螢光光子產生於光二極 之電子率等於由激發光子產生於光二極體184內 時,因爲激發光子通量衰減比螢光光子通量衰減 由於螢光之每單位螢光面積的感測器輸出電 β/{ί) = φίη!{ί) 其中0 /爲在螢光波長之感測器的量子效率。 代入(1 7 )我們得到: έ}(〇 = φ/φ0ηΒεαΙηβΗ,-,ι)Ι^ ... ( 18) 感測器輸 同樣地,由於激發光子之每單位螢光面積的 出電子率爲: ee(0 = ΦΑε ...(19) -80- 201209403 其中久爲在激發波長之感測器的量子效率,且τβ爲相 對於激發LED之F切斷』特性的時間常數。在時間t2之 後’ LED之衰減光子通量增加螢光訊號的強度且延長其衰 減時間,但我們假設對I f ( t )有可忽略的影響,因此我 們採取保守的方法。 目前,如先前所提及,/3的理想値爲當: ·έ>⑹=研3) 因此,由(1 8 )和(1 9 ) 我們得到: 並且重整之後我們得到: ln(£c/7—) h-t2=—i~~- ( 20) T/ 由上面兩段,我們得到下列兩個工作等式: ns =Φ〇ΚΡτ/β~6"τί …(2 1 ) = ... ( 22) 其中尸= fc/?7且Δ,= ί3-,我們亦了解,實際上,。 用於螢光偵測的理想時間及使用Philips LXK2-PR1 4-R00 LED和Pulsar 650染料偵測的螢光光子數決定如下 201209403 理想偵測時間係使用式(22 )決定: 回想擴增子的濃度,且假設所有擴增子雜交,然後發 螢光的螢光團濃度爲:c = 2.89(10)_6mol/L。 腔室的高度爲光學路徑長度1 = 8(10)'6m。 已將螢光區域視爲等同於光二極體區域,然而實際的 螢光區域大體上大於光二極體區域:因此可大槪假設 卢。=0.5爲光學系統之光採集效率。光二極體的特性,^ = 10 fc 爲在螢光波長之光二極體量子效率對在激發波長之光二極 體的量子效率之比的極保守値》 以典型的LED衰減生命週期之&= 0.5奈秒和使用Τί K Therefore 'we can write the following approximation, which describes the attenuation of the fluorescence intensity after a sufficiently long excitation pulse (>> Tf): for l^t2 If{t)^Iesc^^-eH ,~'l)lTf ...(11) In the previous section, we summarize the situation for >> Tf, and for t > tl Ι^ΐπΐη'ε-(four)~. From the above equation, we can derive the following formula: «/(0 = ηΒεοΙηβ~{'~, ι)ΙΤ/ ... ( 12) where = is the number of fluorescent photons per unit area per unit time and Κ =4~ is the number of excitation photons per unit area per unit time. Therefore, 〇〇 nfit)=\fif{t)dt ... ( 13 ) , 3 where seven is the number of fluorescent photons per unit area and 3 is the time point at which the photodiode is turned on. Substituting (12) into (13): «/ = ^ηεεοΙηβΛ,~,ι)ΙΤ/ dt (14) At present, the number of fluorescent photons per unit area per unit time reaches the photodiode, & , obtained by: ^(〇= «>(〇^0 ... ( 15 ) -79- 201209403 where 彳 is the light collection efficiency of the optical system. Substituting (1 2 ) into (1 5 ) Found = Φοη, εαΙηβ'0''1'11^ (16) Fluorescent photons Similarly, the number of photodiodes per unit of fluorescent area t will be as follows: 〇〇圮(7)汾 and substituted into (16) Integral: ^3 ns = φΰηΒεοΙητ fe<h~h)'Tf Therefore, ns = φ^ιΒεοΙητfe~^'11 ... ( 17) The electron rate in body 184 is much faster. Sub-rate: ideal for ί3 When the electron rate generated by the photon generated by the photon is equal to that generated by the excitation photon in the photodiode 184, since the excitation photon flux decays more than the fluorescence photon flux is attenuated due to the fluorescence per unit area of the fluorescence. The sensor output is electrically β/{ί) = φίη!{ί) where 0 / is the quantum efficiency of the sensor at the fluorescent wavelength. Substituting (1 7 ) we get: έ}(〇= φ/φ0ηΒεαΙηβΗ,-,ι)Ι^ (18) Sensor output is the same, because the electron emission rate per unit of fluorescent area of the excited photon : ee(0 = ΦΑε ...(19) -80- 201209403 where is the quantum efficiency of the sensor at the excitation wavelength for a long time, and τβ is the time constant with respect to the F-cut characteristic of the excited LED. At time t2 Then the 'attenuated photon flux of the LED increases the intensity of the fluorescent signal and extends its decay time, but we assume a negligible effect on I f ( t ), so we take a conservative approach. Currently, as mentioned earlier, The ideal of /3 is as follows: ·έ>(6)=研3) Therefore, from (1 8 ) and (1 9 ) we get: and after reforming we get: ln(£c/7—) h-t2= —i~~- ( 20) T/ From the above two paragraphs, we get the following two working equations: ns =Φ〇ΚΡτ/β~6"τί ...(2 1 ) = ... ( 22) where corpse = Fc/?7 and Δ,= ί3-, we also understand, in fact,. The ideal time for fluorescence detection and the number of fluorescent photons detected using the Philips LXK2-PR1 4-R00 LED and Pulsar 650 dye is determined as follows: 201209403 The ideal detection time is determined using equation (22): Recall the amplicon Concentration, and assuming that all amplicons hybridize, then the fluorescence concentration of the fluorescing is: c = 2.89 (10) _ 6 mol / L. The height of the chamber is the optical path length 1 = 8 (10) '6 m. The fluorescent region has been considered to be equivalent to the photodiode region, but the actual fluorescent region is substantially larger than the photodiode region: therefore, it is assumed that Lu is a large factor. =0.5 is the light collection efficiency of the optical system. The characteristics of the photodiode, ^ = 10 fc is the extremely conservative ratio of the quantum efficiency of the photodiode at the fluorescence wavelength to the quantum efficiency of the photodiode at the excitation wavelength. With a typical LED decay life cycle & 0.5 nanoseconds and use
Pulsar650規格,可決定△/: F = [1.48(10)6][2.89(10)-6][8(10)'6](1) =3.42(10)'5 A. ln([3.42(10)-s](10)(0.5))Pulsar650 specification, can be determined △ /: F = [1.48 (10) 6] [2.89 (10) -6] [8 (10) '6] (1) = 3.42 (10) '5 A. ln ([3.42 ( 10)-s](10)(0.5))
Ar =-— 1 1(10)-6 _ 0.5(10)-9 =4.34(10)-9 s 偵測到的光子數目係使用等式(21)決定。首先’每 單位時間發射的激發光子數目乂係由檢測照明幾何而定。Ar =-— 1 1(10)-6 _ 0.5(10)-9 =4.34(10)-9 s The number of photons detected is determined using equation (21). First, the number of excitation photons emitted per unit time is determined by the detection illumination geometry.
Philips LXK2-PR14-R00 LED 具有 Lambertian 輻射模 式,因此: h) = nl0 cos(0) …(23 ) 其中A;爲與該LED的向前軸線方向之角度爲0之每單位 -82- 201209403 立體角每單位時間發射的光子數目,且〜爲β在該向前軸 線方向之値。 • 由該LED每單位時間所發射的光子之總數爲:Philips LXK2-PR14-R00 LED has Lambertian radiation mode, therefore: h) = nl0 cos(0) ... (23) where A; is the unit with the angle of the forward axis of the LED is 0 -82 - 201209403 The number of photons emitted per unit time, and ~ is the β in the direction of the forward axis. • The total number of photons emitted by the LED per unit time is:
Ω ,0 cos ⑼ ί/Ω 現在, ΑΩ = 2π[1 — cos(0 + Α0)] - 2π[1 — cos(0)] ΔΩ = 2n[cos{0) - cos(9 + Δ ^)] 4;rsin(0)cos[·^ |sin Αθ_ Cl j 47rcos(0)sin2Ω ,0 cos (9) ί/Ω Now, ΑΩ = 2π[1 — cos(0 + Α0)] - 2π[1 — cos(0)] ΔΩ = 2n[cos{0) - cos(9 + Δ ^)] 4;rsin(0)cos[·^ |sin Αθ_ Cl j 47rcos(0)sin2
LG = 27rsin(^)ii?0 代入(24) 重新排列,我們得到:LG = 27rsin(^)ii?0 Substitute (24) Rearrange, we get:
...(26 ) LED的輸出功率爲0.515瓦且ve = 6.52(10)14赫茲 ,因此: hve -83- ...(27 ) 201209403 =_0515_ ~ [6.63(10)-34][6.52(10)14] =1 .19(10)18 光子 /秒 將此値帶入(26 )我們得到: ... 1.19(10)18 η'0=~ΊΓ~ =3.79(10)17光子/秒/球面度 參照圖61,光學中心252和LED26之透鏡254係如 示意圖所示。光二極體爲16微米χ16微米,且對於在陣 列中間的光二極體,自LED26所發射至光二極體184的 光錐的立體角(Ω)係大約: Ω =感測器面積/r2 [16 (10)'6] [16(10)-6] 2.825(10)-3]2 =3.2 1 ( 1 〇Γ5 球面度 將理解該光二極體陣列44之中央光二極體184爲用 於這些計算之用途。位於該陣列邊緣的光感測器在雜交事 件時僅接收2%之少量光子用於Lambertian激發源強度分 佈。 每單位時間發射的激發光子數: hc = η,ςΐ ... ( 28 ) =[3.79(10),7][3.21(10)·5] =1 .22 ( 1 0 ) 13 光子 /秒 -84- 201209403 現在參考等式(29): ns =</>^eFTfe'A,lt/ ns = (0.5)11.22(10)^^3.42(10)-^^(10)-6^-43400)^^0^ =208每感測器之光子 因此,使用 Philips LXK2-PR14-R00 LED 和 Pulsar 65 0螢光團,我們可以輕易地偵測任何造成被激發之光子 數目的雜交事件。 該SET LED照明幾何顯示於圖62中。在Id = 20毫 安培,LED具有最小光學功率輸出Pl = 240微瓦,波長中 心於λε = 3 40奈米(铽螯合物之吸收波長)。驅動LED 於ID = 2 0 0毫安培線性增加該輸出功率至ρι = 2.4毫瓦。 藉由將LED的光學中心25 2置於離雜交腔室陣列110距 離17.5毫米處,我們大約集中輸出通量於具有最大直徑 爲2毫米的圓點大小。 在雜交陣列平面之2毫米直徑點中的光子通量由等式 27得到。 η,'·...(26) The output power of the LED is 0.515 watts and ve = 6.52 (10) 14 Hz, therefore: hve -83- ...(27 ) 201209403 =_0515_ ~ [6.63(10)-34][6.52( 10)14] =1.19(10)18 photons/sec. Bring this 値 into (26) we get: ... 1.19(10)18 η'0=~ΊΓ~ =3.79(10)17 photons/second /Sphericality Referring to Figure 61, the optical center 252 and the lens 254 of the LED 26 are as shown in the schematic. The photodiode is 16 microns χ 16 microns, and for the photodiode in the middle of the array, the solid angle (Ω) of the light cone emitted from the LED 26 to the photodiode 184 is approximately: Ω = sensor area / r2 [16 (10) '6] [16(10)-6] 2.825(10)-3]2 =3.2 1 (1 〇Γ5 Sphericality will understand that the central photodiode 184 of the photodiode array 44 is used for these calculations The photo sensor located at the edge of the array receives only 2% of the photons for the Lambertian excitation source intensity distribution during the hybridization event. The number of excitation photons emitted per unit time: hc = η, ςΐ ... (28 ) =[3.79(10),7][3.21(10)·5] =1 .22 ( 1 0 ) 13 Photons/sec -84- 201209403 Now refer to equation (29): ns =</>^ eFTfe'A,lt/ ns = (0.5)11.22(10)^^3.42(10)-^^(10)-6^-43400)^^0^ =208 photons per sensor So, use Philips LXK2 -PR14-R00 LED and Pulsar 65 0 fluorophore, we can easily detect any hybridization event that causes the number of excited photons. The SET LED illumination geometry is shown in Figure 62. At Id = 20 mA, the LED has a minimum optical power output of Pl = 240 microwatts and a wavelength center of λε = 3 40 nm (the absorption wavelength of the ruthenium chelate). The drive LED linearly increases the output power to ρι = 2.4 mW at ID = 205 mA. By placing the optical center 25 2 of the LED at a distance of 17.5 mm from the hybridization chamber array 110, we concentrated the output flux to a dot size having a maximum diameter of 2 mm. The photon flux in the 2 mm diameter dot of the hybrid array plane is obtained from Equation 27. η,'·
Pi hve 2.4(10)- [6.63(10)-34] [8.82(10)14] 4· 10 ( 10 ) 15 光子 /秒 使用等式2 8,我們得到: ne = -85- 201209403 4.10(10)15 [16(10)6]2 对 1(10).3]2 光子/秒 3.34(10) 現在,再呼叫等式22及使用先前列舉的Tb螯合物特 性, ln[(6.94(10)-5)(10)(0.5)] 1(10)-3 — 0.5(10)-9 =3.98 ( 10 ) ·9 秒 現在自等式2 1 : ns = (0.5)[3.34(10)"][6.94(10)-5][1(10)-3>'398(,0),/1(,0)'3 =1 1,600每感測器之光子 由雜交事件使用SET LED和铽螯合物系統所發射之 光子理論數値係可簡單的偵測得到且遠超過3 0個光子數 之低限値,此低限値爲用於上述所指示之光感測器之可信 賴的偵測所需。 探針與光二極體間之最大間隔 雜交之晶片上偵測避免以共焦顯微鏡(見本發明的背 景)偵測之需要。背離傳統偵測技術爲節省與系統有關的 時間和成本之重要的因素。傳統偵測需要必須使用透鏡和 彎曲鏡面之成像光學。藉由採用非成像光學,診斷系統避 免複雜及笨重的光學元件串之需求。將光二極體放置於非 常靠近探針具有極高收集效率的優點。當在探針和光二極 -86- 201209403 體間的材料厚度爲1微米的等級時,發射光之收集角 達173°。此角度藉由考慮自最靠近光二極體之雜交 表面中心的探針發射的光來計算,光二極體具有平行 室表面的平面活性表面區。光可以於其內由光二極體 之發射角錐係定義爲在其頂點和在其平面之周圍上的 器角落具有發射探針。對於16微米xl6微米的感測 此錐體的頂角爲1 70° ;在光二極體經擴充使得其面 合29微米xl 9.75微米之雜交腔室面積的限制例中, 爲173°。在腔室表面和光二極體活性表面之間的分隔 微米或更小是容易達成的。 應用非成像光學方法需要光二極體184非常靠近 腔室以收集螢光輻射之足夠光子。在光二極體和探針 的最大間隔係參照如下圖54所決定。 利用铽螯合物螢光團和 SET UVT0P3 3 5 T039BL ,我們計算自個別雜交腔室180到達16微米X 16微 光二極體184的11600個光子。在實施此計算時,我 設雜交腔室180之光收集區域具有與光二極體有效區 相同的底面積,且雜交光子之總數的一半到達光二 1 84。即光學系統之光收集效率爲A =0.5。 更精確我們可以寫出么=[(雜交腔室之光收集區 底面積)/ (光二極體區域)ΠΩ/4π] ’其中由在雜交 之基底於代表點之光二極體所對向的Ω =立體角。對 確的正方錐幾何: Ω = 4 ar c s in ( a2/ ( 4d。2 + a2 )),其中 d〇 =在 係商 腔室 於腔 吸收 感測 器, 積符 頂角 爲1 雜交 之間 LED 米之 們假 185 極體 域的 腔室 於正 腔室 -87- 201209403 與光二極體之間的距離,且α爲光二極體尺寸。 各雜交腔室釋放23200個光子,經選擇的光二極體具 有偵測低限値爲1 7個光子,因此,所需的最小光學效率 爲 · φ0= 1 7/23200 = 7.3 3 X 1 Ο'4 雜交腔室180之光收集區域的底面積爲29微米 X 19.75 微米。 解出dQ,將得到在雜交腔室及光二極體1 84之間的 最大限制距離爲dQ = 249微米。在此限制中,如上所定義 之收集錐角僅爲〇 . 8 °。應注意的是此分析忽略了折射之可 忽略的影響。Pi hve 2.4(10)- [6.63(10)-34] [8.82(10)14] 4· 10 ( 10 ) 15 Photons/sec Using Equation 2 8, we get: ne = -85- 201209403 4.10(10 ) 15 [16(10)6]2 vs. 1(10).3]2 photons/sec 3.34(10) Now, call equation 22 again and use the previously listed Tb chelate properties, ln[(6.94(10) )-5)(10)(0.5)] 1(10)-3 — 0.5(10)-9 =3.98 ( 10 ) ·9 seconds Now theorem 2 1 : ns = (0.5)[3.34(10)" ;][6.94(10)-5][1(10)-3>'398(,0),/1(,0)'3 =1 1,600 photons per sensor are used by hybrid events using SET LEDs and 铽The photon theory number emitted by the chelate system can be easily detected and is well below the lower limit of 30 photons, which is the reliable for the above-mentioned photosensors. Detection required. The maximum spacing between the probe and the photodiode is detected on the wafer to avoid detection by a confocal microscope (see the background of the present invention). Deviating from traditional detection techniques is an important factor in saving time and cost associated with the system. Conventional detection requires imaging optics that must use lenses and curved mirrors. By using non-imaging optics, the diagnostic system avoids the need for complex and cumbersome strings of optical components. Placing the photodiode in a very close proximity to the probe has the advantage of extremely high collection efficiency. When the material thickness between the probe and the photodiode -86-201209403 is 1 micron, the emission angle of the emitted light is 173°. This angle is calculated by considering the light emitted from the probe closest to the center of the hybridization surface of the photodiode, which has a planar active surface region of the parallel chamber surface. The light may be defined therein by an emission pyramid of the photodiode having a radiation probe at its apex and at a corner of the periphery of its plane. The apex angle of this cone was 1 70° for a sensing of 16 μm x 16 μm; it was 173 ° in the case where the photodiode was expanded to face a hybrid chamber area of 29 μm x 9.75 μm. A separation of micrometers or less between the surface of the chamber and the active surface of the photodiode is easily achieved. Applying a non-imaging optical method requires the photodiode 184 to be very close to the chamber to collect enough photons of the fluorescent radiation. The maximum spacing between the photodiode and the probe is determined as shown in Figure 54 below. Using the ruthenium chelate fluorophore and SET UVT0P3 3 5 T039BL, we calculated 11600 photons from individual hybridization chambers 180 to 16 micron X 16 low-light diodes 184. In carrying out this calculation, I set the light collection region of the hybridization chamber 180 to have the same bottom area as the active region of the photodiode, and half of the total number of hybrid photons reaches the light II 84. That is, the light collection efficiency of the optical system is A = 0.5. More precise, we can write out = [(the bottom area of the light collection area of the hybrid chamber) / (photodiode area) Π Ω / 4π] 'the Ω which is opposed by the photodiode at the representative point of the hybrid = solid angle. For the true square pyramid geometry: Ω = 4 ar cs in ( a2 / ( 4d. 2 + a2 )), where d 〇 = in the chamber chamber in the cavity absorption sensor, the apex angle is 1 hybrid The distance between the chamber of the LED meter and the 185 pole body is in the positive chamber -87- 201209403 and the photodiode, and α is the size of the photodiode. Each hybridization chamber releases 23,200 photons, and the selected photodiode has a detection low limit of 17 photons. Therefore, the minimum optical efficiency required is φ0 = 1 7/23200 = 7.3 3 X 1 Ο ' 4 The bottom area of the light collection region of the hybridization chamber 180 is 29 microns x 19.75 microns. Solving dQ will result in a maximum limiting distance between the hybridization chamber and the photodiode 1 84 of dQ = 249 microns. In this limitation, the collecting cone angle as defined above is only 〇 8 °. It should be noted that this analysis ignores the negligible effects of refraction.
LOC 變體 XLI 顯示於圖74之LOC變體XLI 671係用於分析樣本, 其中感興趣的是該較小、大致上可溶的組份。樣本被加入 樣本入口 68。固體或粉狀樣本與用於毛細流體至表面張 力閥118的樣本入口 68中適合的液體結合。在貯槽54中 之試劑經由表面張力閥118與樣本混合且流體接著流入至 小組份透析部682。低於特定尺寸低限値之樣本組份,諸 如鹽類、代謝物、DNA和蛋白質類,留存於樣本中。大 組份諸如細胞、病原體和碎片係轉至廢料貯槽766。經純 化的樣本接著進入其他功能單元(如於前述LOC裝置所 描述)以用於進一步處理諸如培養、核酸擴增及雜交684 -88- 201209403 結論 描述於本文之裝置、系統及方法促進分子診斷試驗成 爲低花費、快速且成爲重點照護試驗。描述於上文之系統 及其組件已完全說明,且於此領域之技藝工作者將可容易 地識別不偏離主要發明槪念之精神與範疇的許多變化和修 改。 【圖式簡單說明】 現將藉由僅參照附圖之實例描述本發明之較佳具體實 施例,其中: 圖1顯示經配置而用於螢光檢測之試驗模組和試驗模 組讀取器。 圖2爲經配置而用於螢光檢測之試驗模組中之電子組 件的圖式槪要。 圖3爲試驗模組讀取器中之電子組件的圖式槪要。 圖4爲LOC裝置之結構的圖式槪要。 圖5爲LOC裝置之透視圖。 圖6爲具有來自彼此疊加之所有層之特徵和結構之 LOC裝置的平面圖。 圖7爲具有獨立顯示之蓋結構之LOC裝置的平面圖 〇 圖8爲具有以虛線顯示之內通道和貯槽之蓋的頂部透 -89- 201209403 視圖。 圖9爲具有以虛線顯示之內通道和貯槽之蓋的爆炸頂 部透視圖。 圖10爲顯示頂部通道配置之蓋的底部透視圖。 圖11爲獨立顯示CMOS + MST裝置結構之LOC裝置 的平面圖。 圖12爲在樣本入口之LOC裝置的剖面示意圖。 圖13爲圖6所示之插圖AA的放大視圖。 圖14爲圖6所示之插圖AB的放大視圖。 圖15爲圖13所示之插圖AE的放大視圖。 圖16爲圖解在插圖AE內部之LOC裝置的層狀構造 之部分透視圖。 圖17爲圖解在插圖AE內部之LOC裝置的層狀構造 之部分透視圖。 圖18爲圖解在插圖AE內部之LOC裝眞的層狀構造 之部分透視圖。 圖19爲圖解在插圖AE內部之LOC裝置的層狀構造 之部分透視圖》 圖20爲圖解在插圖AE內部之LOC裝置的層狀構造 之部分透視圖。 圖21爲圖解在插圖AE內部之LOC裝置的層狀構造 之部分透視圖。 圖22爲顯示在圖2 1之該溶胞試劑貯槽之剖面示意圖 -90- 201209403 圖23爲圖解在插圖AB內部之LOC裝置的層狀構造 之部分透視圖。 圖24爲圖解在插圖AB內部之LOC裝置的層狀構造 之部分透視圖。 圖25爲圖解在插圖AI內部之LOC裝置的層狀構造 之部分透視圖。 圖26爲圖解在插圖AB內部之LOC裝置的層狀構造 之部分透視圖。 圖27爲圖解在插圖AB內部之L0C裝置的層狀構造 之部分透視圖。 圖28爲圖解在插圖AB內部之LOC裝置的層狀構造 之部分透視圖。 圖29爲圖解在插圖AB內部之LOC裝置的層狀構造 之部分透視圖。 Η 3 0爲擴增混合貯槽及聚合酶貯槽之剖面示意圖。 圖31顯示獨立之沸騰引發閥的特徵。 圖32爲顯示於圖31行經線32_32之沸騰引發閥之剖 面示意圖。 圖33爲顯示於圖15之插圖AF的放大圖。 圖34爲顯示於圖33行經線34_34之沸騰引發閥之剖 面示意圖。 圖35爲顯示於圖6之插圖AC的放大圖。 圖36爲顯示擴增部之插圖AC內部之進一步放大圖 -91 - 201209403 圖37爲顯示擴增部之插圖AC內部之進一步放大圖 〇 圖38爲顯示擴增部之插圖AC內部之進一步放大圖 〇 圖39爲顯示於圖38之插圖AK內部之進一步放大圖 〇 圖40爲顯示擴增腔室之插圖AC內部之進一步放大 圖。 圖41爲顯示擴增部之插圖AC內部之進一步放大圖 〇 圖42爲顯示擴增腔室之插圖AC內部之進一步放大 圖。 圖43爲顯示於圖42之插圖AL內部之進一步放大圖 〇 圖44爲顯不擴增部之插圖AC內部之進一步放大圖 0 圖45爲顯示於圖44之插圖AM內部之進一步放大圖 〇 圖46爲顯示擴增腔室之插圖AC內部之進一步放大 圖。 圖47爲顯示於圖46之插圖AN內部之進一步放大圖 〇 圖48爲顯示擴增腔室之插圖AC內部之進一步放大 圖。 -92- 201209403 圖49爲顯示擴增腔室之插圖AC內部之進一步放大 圖。 圖50爲顯不擴增部之插圖AC內部之進一'步放大圖 〇 圖5 1爲擴增部之剖面示意圖。 圖52爲雜交部之放大平面圖。 圖53爲兩個獨立之雜交腔室之進一步放大平面圖。 圖54爲單個雜交腔室之剖面示意圖。 圖55爲顯示於圖6之插圖AG中闡述之增濕器之放 大圖。 圖56爲顯示於圖52之插圖AD之放大圖。 圖57爲在插圖AD中之LOC裝置的爆炸透視圖。 圖58爲在封閉組態中FRET探針的圖。 圖59爲呈開放且雜交組態中FRET探針的圖。 圖60爲激發光密度隨著時間改變的曲線圖。 圖61爲雜交腔室陣列之激發照明幾何(excitation illumination geometry)的圖。 圖62爲感測器電子技術LED照明幾何的圖示。 圖63爲顯示於圖6之插圖AH之濕度感測器的放大 平面圖。 .圖64爲顯示部分光感測器之光二極體陣列之槪要圖 〇 圖65爲單一光二極體之電路圖。 圖66爲光二極體控制訊號之時序圖。 -93- 201209403 圖67爲顯示於圖55之插圖AP之蒸發器的放大圖。 圖68爲以偵測光二極體和觸發光二極體通過雜交腔 室之剖面示意圖。 圖69爲連接子-引發PCR之圖。 圖7 0爲表示具有刺血針之試驗模組的槪要圖。 圖71爲LOC變體VII之結構的圖形表示。 圖72爲LOC變體VIII之結構的圖形表示。 圖73爲LOC變體XIV之結構的圖形表示。 圖74爲LOC變體XLI之結構的圖形表示。 圖75爲LOC變體XLIII之結構的圖形表示。 圖76爲LOC變體XLIV之結構的圖形表示。 圖77爲LOC變體XLVII之結構的圖形表示。 圖7 8爲在初次擴增期間之引子聯結之線性螢光探針 之圖。 圖79爲在接續擴增循環期間之引子聯結之線性螢光 探針之圖。 圖80A至80F圖示說明引子-連結之螢光莖-及-環探 針的熱循環。 圖81爲關於雜交腔室陣列及光二極體之激發LED的 槪要說明。 圖82爲引導光進入LOC裝置之雜交腔室陣列的激發 LED和光學透鏡之槪要說明。 圖83爲用於引導光進入LOC裝置之雜交腔室陣列的 激發LED、光學透鏡和光學稜鏡之槪要說明。 -94- 201209403 圖84爲用於引導光進入LOC裝置之雜交腔室陣列的 激發LED、光學透鏡和鏡子排列之槪要說明。 圖85爲顯示彼此重疊之所有特徵並顯示插圖da至 DK的位置之平面圖。 圖86爲顯示於圖85之插圖DG的放大圖。 圖87爲顯示於圖85之插圖DH的放大圖。 圖88顯示用於光二極體之並聯電晶體之一個具體實 施例。 圖89顯示用於光二極體之並聯電晶體之一個具體實 施例。 圖90顯示用於光二極體之並聯電晶體之一個具體實 施例。 圖91爲示差成像器之電路圖。 圖92槪略說明呈莖-及-環結構中之負控制螢光探針 〇 圖93槪略說明呈開放構造中之圖92的負控制螢光探 針。 圖94槪略說明呈莖-及-環結構中之正控制螢光探針 〇 圖95槪略說明呈開放構造中之圖94的正控制螢光探 針。 圖96顯示經配置和ECL偵測一起使用之試驗模組和 試驗模組讀取器。 圖97爲經配置和ECL偵測一起使用之試驗模組中之 -95- 201209403 電子組件的圖示槪要。 圖98顯示試驗模組與替代的試驗模組讀取器。 圖99顯示試驗模組和試驗模組讀取器以及容納各種 資料庫之主機系統。 圖100A和100B爲說明將適體連結到蛋白質以製造 可偵測訊號的圖。 圖101A和101B爲說明將兩個適體連結到蛋白質以 製造可偵測訊號的圖。 圖102A和102B爲說明將兩個適體連結到蛋白質以 製造可偵測訊號的圖。 圖103爲顯示彼此重疊之所有特徵與顯示插圖GA至 GL之位置的LOC變形L的平面圖。 圖104爲顯示於圖103之插圖GD的放大圖。 【主要元件符號說明】 1 0 :試驗模組 1 1 :試驗模組 1 2 :試驗模組讀取器 1 3 :外殼 14 :微型-USB插頭 1 5 :感應器 16 :微型-USB埠 1 7 :觸控螢幕 1 8 :顯示螢幕 -96- 201209403 1 9 :按鈕 2 0 :開始按鈕 21 :蜂巢式無線電 22 :無菌密封帶 23 :無線網路連接 24 :大容器 25 :衛星導航系統 26 :發光二極體 2 7 :資料儲存器 2 8 :電話 29 : LED驅動器 30 : LOC裝置 3 1 :電源調節器 32 :電容器 3 3 :計時器 34 :控制器 35 :暫存器 36 :微型USB裝置1 . 1或2.0 3 7 :驅動器 3 8 :隨機存取記憶體 39 : ECL激發驅動器 40 :程式和資料快取 41 : ECL激發暫存器 42 :處理器 -97- 201209403 43 :程式儲存器 44 :光感測器 45 :指示器 46 :蓋 4 7 :模組 48 : CMOS + MST 裝置 49 :多孔元件 5 2 :雜交及檢測部 54 :抗凝血劑貯槽 56、 56.1、 56.2、 56.3:貯槽 5 7 :印刷電路板 58、58.1、58.2:貯槽 60、60.1-60.12、60.X:貯槽 62, 62.1、 62.2、 62.3、 62.4、 62.X:貯槽 64 :下部密封 66 :頂部層 6 8 :樣本入口 70 :透析部 72 :廢料通道 74 :標靶通道 76 :廢料貯槽 78 :貯槽層 80 :蓋通道層 8 2 :上密封層 -98- 201209403 84 :矽基板 86 : CMOS 電路 87 : MST 層 8 8 :鈍化層 90 : MST通道 92 :下管道 94 :蓋通道 96 :上管道 97 :壁部 98:彎液面固定器 I 00 : MST通道層 101 :膝上型電腦/筆記本 102 :毛細作用起始特徵 103 :專用讀取器 105 :桌上型電腦 106 :沸騰引發閥 107 :電子書讀取器 108 :沸騰引發閥 109 :平板電腦 110、110.1-110.12、110.X:雜交室陣列 II 1 :流行病學資料 112、112.1-112.12、112.X:擴增部 I 1 3 :遺傳資料 II 4 :培養部 -99- 201209403 1 1 5 :電子化健康記錄 1 1 6 :抗凝血劑 1 1 8 :表面張力閥 1 1 9 :液體樣本 1 2 0 :彎液面 1 2 1 :電子化醫療記錄 1 2 2 :通氣孔 123 :個人健康記錄 1 2 5 :網路 126 :沸騰引發閥 128、128.2、128.3:表面張力閥 1 3 0、1 3 0.1 - 1 3 0.3 :溶胞部 1 3 1 :混合部 132、132.1、1 32.3 :表面張力閥 1 3 3 :培養器入口通道 134 :下管道 1 36 :光學窗 141 :適體 1 4 2 :標靶蛋白質 1 43 :予體 144 :受體 145 :抗體 1 4 6 :閥入口 1 4 7 :互補寡核苷酸 -100- 201209403 148 : 149 : 150 : 152 : 153 : 154 : 156 : 158: 160 : 164 : 166 : 168 : 170 : 174 : 175 : 176 : 178 : 180 : 182: 184 : 185: 186 : 187 : 188 : 閥出口 聯結子 閥下管道 環形加熱器 閥加熱器接點 加熱器 加熱器接點 微通道 出口通道 孔口 毛細作用起始特徵 透析吸入孔 溫度感測器 液體感測器 擴散屏障 流動路徑 液體感測器 雜交腔室 加熱器 光二極體 有效區 探針 光二極體 水貯槽 -101 - 201209403 190 :蒸發器 1 9 1 :環形加熱器 192 :水供應通道 193 :上管道 194 :下管道 1 9 5 :頂金屬層 196 :增濕器 1 9 8 :吸入孔 202 :毛細作用起始特徵 204 : MST 通道 208 :液體感測器 210 :微通道 2 1 2 : MST 通道 2 18 :電極 220 :電極 222 :間隙 2 3 2 :濕度感測器 2 3 4 :加熱器 236 : FRET 探針 23 8 :標靶核酸序列 240 :環 242 :莖 244 :激發光 246 :螢光團 201209403 2 4 8 :淬熄劑 25 0 :螢光信號 2 5 2 :光學中心 2 5 4 :透鏡 28 8 :樣本輸入及製備 290 :萃取階段 291 :培養階段 292 :擴增階段 294 :檢測階段 2 9 6 :第一電極 298 :第二電極 3 00 :可程式化延遲 30 1 : L 0 C 裝置 3 7 6 :導熱柱 3 7 8 :正控制探針 3 8 0 :負控制探針 3 82 :校準室 3 8 4 :閘極 3 8 6 :聞極 3 8 8 :閘極 3 90 :可伸縮刺血針 3 92 :刺血針釋出按鈕 3 9 3 :鬧極 3 94 : MOS電晶體 201209403 3 9 6 : Μ O S電晶體 398: MOS電晶體 400: MOS電晶體 402: MOS電晶體 404: MOS電晶體 406 :節點 40 8 :膜密封件 4 1 0 :膜防護件 647 : AlexaFluor 67 1: LOC 變體 673 : LOC 變體 674 : LOC 變體 677 : LOC 變體 6 8 2 :透析部 684:核酸擴增及雜交 6 8 6 :透析步驟 692 :引子-聯結的線性探針 694 :擴增阻斷物 6 9 6 .探針區域 698 :互補序列 7〇〇 :寡核苷酸引子 704 :莖-及-環探針 706 :互補序列 708 :莖股 -104 - 201209403 710 : 712 : 714 : 716 : 718 : 766 : 778 : 780 : 7 82 : 788 : 790 : 792 : 794 : 795 : 796 : 797 : 79 8 ·· 801 : 803 : 805 : 8 07 ·· 809 : 8 11: 813 : 股 第一光稜鏡 第二光稜鏡 第一鏡 第二鏡 廢料貯槽 組態 組態 組態 差分成像器電路 像素 虛擬像素 讀取_列 讀取_列 負控制探針 (電晶體) 正控制探針 (電晶體) 像素電容器 虛擬像素電容器 開關 開關 開關 開關 -105 201209403 8 1 5 :電容器放大器 8 1 7 :示差訊號 860 : ECL激發電極 87 0 : ECL激發電極LOC Variant XLI The LOC variant XLI 671 shown in Figure 74 is used to analyze the sample, of which the lesser, substantially soluble component is of interest. The sample is added to the sample entry 68. The solid or powdered sample is combined with a suitable liquid for the capillary fluid to the sample inlet 68 of the surface tension valve 118. The reagent in the sump 54 is mixed with the sample via the surface tension valve 118 and the fluid then flows into the sub- dialysis section 682. Sample components below the lower limit of a particular size, such as salts, metabolites, DNA, and proteins, are retained in the sample. Large components such as cells, pathogens, and debris are transferred to waste storage tank 766. The purified sample is then passed to other functional units (as described in the aforementioned LOC apparatus) for further processing such as culture, nucleic acid amplification and hybridization. 684-88-201209403 Conclusions The devices, systems, and methods described herein facilitate molecular diagnostic assays. Become a low-cost, fast and a focused care test. The system and its components described above are fully described, and those skilled in the art will be able to readily recognize many variations and modifications without departing from the spirit and scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS Preferred embodiments of the present invention will now be described by way of example only with reference to the accompanying drawings in which: FIG. 1 shows a test module and a test module reader configured for fluorescence detection. . Figure 2 is a schematic representation of the electronic components in a test module configured for fluorescence detection. Figure 3 is a schematic diagram of the electronic components in the test module reader. Figure 4 is a schematic view of the structure of the LOC device. Figure 5 is a perspective view of the LOC device. Figure 6 is a plan view of an LOC device having features and structures from all of the layers superimposed on each other. Figure 7 is a plan view of a LOC device having a lid structure that is independently shown. Figure 8 is a top view through the -89-201209403 with a lid of the inner channel and the sump shown in phantom. Figure 9 is an exploded top perspective view of the cover with the inner passage and the sump shown in phantom. Figure 10 is a bottom perspective view showing the cover of the top channel configuration. Figure 11 is a plan view of the LOC device showing the structure of the CMOS + MST device independently. Figure 12 is a schematic cross-sectional view of the LOC device at the sample inlet. Figure 13 is an enlarged view of the inset AA shown in Figure 6. Figure 14 is an enlarged view of the inset AB shown in Figure 6. Figure 15 is an enlarged view of the inset AE shown in Figure 13. Figure 16 is a partial perspective view illustrating the layered configuration of the LOC device inside the inset AE. Figure 17 is a partial perspective view illustrating the layered configuration of the LOC device inside the inset AE. Figure 18 is a partial perspective view illustrating the layered configuration of the LOC mount inside the inset AE. Figure 19 is a partial perspective view illustrating the layered configuration of the LOC device inside the inset AE. Figure 20 is a partial perspective view illustrating the layered configuration of the LOC device inside the inset AE. Figure 21 is a partial perspective view illustrating the layered configuration of the LOC device inside the inset AE. Figure 22 is a schematic cross-sectional view showing the lysing reagent reservoir of Figure 21 -90 - 201209403. Figure 23 is a partial perspective view showing the layered configuration of the LOC device inside the inset AB. Figure 24 is a partial perspective view illustrating the layered configuration of the LOC device inside the inset AB. Figure 25 is a partial perspective view illustrating the layered configuration of the LOC device inside the illustration AI. Figure 26 is a partial perspective view illustrating the layered configuration of the LOC device inside the inset AB. Figure 27 is a partial perspective view illustrating the layered configuration of the LOC device inside the inset AB. Figure 28 is a partial perspective view illustrating the layered configuration of the LOC device inside the inset AB. Figure 29 is a partial perspective view illustrating the layered configuration of the LOC device inside the inset AB. Η 30 is a schematic cross-sectional view of the amplified mixed storage tank and the polymerase storage tank. Figure 31 shows the characteristics of a separate boiling initiation valve. Figure 32 is a schematic cross-sectional view showing the boiling initiation valve of the line 32_32 of Figure 31. Figure 33 is an enlarged view of the inset AF shown in Figure 15. Figure 34 is a cross-sectional view showing the boiling initiation valve of the line 34_34 of Figure 33. Figure 35 is an enlarged view of the inset AC shown in Figure 6. Fig. 36 is a further enlarged view showing the inside of the illustration AC of the amplification section - 91 - 201209403. Fig. 37 is a further enlarged view showing the inside of the illustration AC of the amplification section. Fig. 38 is a further enlarged view showing the inside of the illustration AC of the amplification section. 39 is a further enlarged view of the inside of the illustration AK shown in FIG. 38. FIG. 40 is a further enlarged view showing the inside of the illustration AC of the amplification chamber. Fig. 41 is a further enlarged view showing the inside of the illustration AC of the amplification section. Fig. 42 is a further enlarged view showing the inside of the illustration AC of the amplification chamber. Figure 43 is a further enlarged view of the inside of the illustration AL shown in Figure 42. Figure 44 is a further enlarged view of the inside of the AC of the display portion. Figure 45 is a further enlarged view of the inside of the illustration AM shown in Figure 44. 46 is a further enlarged view showing the inside of the illustration AC of the amplification chamber. Figure 47 is a further enlarged view of the inside of the illustration AN shown in Figure 46. Figure 48 is a further enlarged view showing the inside of the illustration AC of the amplification chamber. -92- 201209403 Figure 49 is a further enlarged view showing the inside of the illustration AC of the amplification chamber. Fig. 50 is a diagram showing the enlargement of the inside of the AC of the display portion of the display portion. Fig. 51 is a schematic cross-sectional view of the amplification portion. Figure 52 is an enlarged plan view of the hybridization section. Figure 53 is a further enlarged plan view of two separate hybridization chambers. Figure 54 is a schematic cross-sectional view of a single hybridization chamber. Figure 55 is an enlarged view of the humidifier illustrated in the inset AG of Figure 6. Figure 56 is an enlarged view of the inset AD shown in Figure 52. Figure 57 is an exploded perspective view of the LOC device in the inset AD. Figure 58 is a diagram of a FRET probe in a closed configuration. Figure 59 is a diagram of a FRET probe in an open and hybrid configuration. Figure 60 is a graph of excitation light density as a function of time. Figure 61 is a graph of excitation illumination geometry of a hybrid chamber array. Figure 62 is a graphical representation of the sensor electronics electronics LED illumination geometry. Figure 63 is an enlarged plan view showing the humidity sensor of the inset AH of Figure 6. Figure 64 is a schematic diagram showing an array of photodiodes of a portion of the photosensors. Figure 65 is a circuit diagram of a single photodiode. Figure 66 is a timing diagram of the photodiode control signal. -93- 201209403 Figure 67 is an enlarged view of the evaporator of the illustration AP shown in Figure 55. Figure 68 is a schematic cross-sectional view showing the detection of the photodiode and the triggering photodiode through the hybridization chamber. Figure 69 is a diagram of linker-priming PCR. Figure 70 is a schematic view showing a test module having a lancet. Figure 71 is a graphical representation of the structure of LOC Variant VII. Figure 72 is a graphical representation of the structure of LOC variant VIII. Figure 73 is a graphical representation of the structure of the LOC variant XIV. Figure 74 is a graphical representation of the structure of the LOC variant XLI. Figure 75 is a graphical representation of the structure of the LOC variant XLIII. Figure 76 is a graphical representation of the structure of the LOC variant XLIV. Figure 77 is a graphical representation of the structure of the LOC variant XLVII. Figure 7 is a diagram of the primer-linked linear fluorescent probe during the initial amplification. Figure 79 is a diagram of a linear fluorescent probe coupled to a primer during a subsequent amplification cycle. Figures 80A through 80F illustrate the thermal cycling of the primer-linked fluorescent stem-and-loop probe. Figure 81 is a schematic illustration of the excitation LED for the hybrid chamber array and photodiode. Figure 82 is a schematic illustration of the excitation LED and optical lens that direct light into the hybridization chamber array of the LOC device. Figure 83 is a schematic illustration of an excitation LED, an optical lens, and an optical raft for directing light into the hybrid chamber array of the LOC device. -94- 201209403 Figure 84 is a schematic illustration of the excitation LEDs, optical lenses, and mirror arrays used to direct light into the hybrid chamber array of the LOC device. Figure 85 is a plan view showing all the features overlapping each other and showing the positions of the insets da to DK. Figure 86 is an enlarged view of the inset DG shown in Figure 85. Figure 87 is an enlarged view of the inset DH shown in Figure 85. Figure 88 shows a specific embodiment of a parallel transistor for an optical diode. Figure 89 shows a specific embodiment of a parallel transistor for an optical diode. Fig. 90 shows a specific embodiment of a parallel transistor for an optical diode. Figure 91 is a circuit diagram of the differential imager. Figure 92 schematically illustrates the negative control fluorescent probe in the stem-and-loop configuration. Figure 93 illustrates the negative control fluorescent probe of Figure 92 in an open configuration. Figure 94 is a schematic illustration of a positive control fluorescent probe in a stem-and-loop configuration. Figure 95 illustrates a positive control fluorescent probe of Figure 94 in an open configuration. Figure 96 shows the test module and test module reader configured for use with ECL detection. Figure 97 is a graphical representation of the -95-201209403 electronic component in a test module that is configured for use with ECL detection. Figure 98 shows the test module and an alternative test module reader. Figure 99 shows the test module and test module reader and the host system that houses the various databases. Figures 100A and 100B are diagrams illustrating the attachment of an aptamer to a protein to make a detectable signal. Figures 101A and 101B are diagrams illustrating the attachment of two aptamers to a protein to make a detectable signal. Figures 102A and 102B are diagrams illustrating the attachment of two aptamers to a protein to make a detectable signal. Figure 103 is a plan view showing LOC deformation L of all the features overlapping each other and the positions at which the insets GA to GL are displayed. Figure 104 is an enlarged view of the inset GD shown in Figure 103. [Main component symbol description] 1 0 : Test module 1 1 : Test module 1 2 : Test module reader 1 3 : Case 14 : Micro-USB plug 1 5 : Sensor 16: Micro-USB埠1 7 : Touch Screen 1 8 : Display Screen - 96 - 201209403 1 9 : Button 2 0 : Start Button 21 : Honeycomb Radio 22 : Aseptic Sealing Band 23 : Wireless Network Connection 24 : Large Container 25 : Satellite Navigation System 26 : Illuminated Diode 2 7 : Data storage 2 8 : Telephone 29 : LED driver 30 : LOC device 3 1 : Power conditioner 32 : Capacitor 3 3 : Timer 34 : Controller 35 : Register 36 : Micro USB device 1 1 or 2.0 3 7 : Driver 3 8 : Random Access Memory 39 : ECL Excitation Driver 40 : Program and Data Cache 41 : ECL Excitation Register 42 : Processor - 97 - 201209403 43 : Program Memory 44 : Light sensor 45: indicator 46: cover 4 7 : module 48 : CMOS + MST device 49 : porous element 5 2 : hybridization and detection portion 54 : anticoagulant storage tank 56, 56.1, 56.2, 56.3: storage tank 5 7: printed circuit board 58, 58.1, 58.2: storage tank 60, 60.1-60.12, 60.X: storage tank 62, 62.1, 62.2, 62.3, 62.4, 62.X: storage 64: lower seal 66: top layer 6 8 : sample inlet 70: dialysis section 72: waste channel 74: target channel 76: waste storage tank 78: sump layer 80: cover channel layer 8 2: upper sealing layer - 98 - 201209403 84 : 矽 substrate 86 : CMOS circuit 87 : MST layer 8 8 : passivation layer 90 : MST channel 92 : lower pipe 94 : cover channel 96 : upper pipe 97 : wall portion 98 : meniscus holder I 00 : MST channel layer 101 : Laptop/Notebook 102: Capillary Action Starting Feature 103: Dedicated Reader 105: Desktop Computer 106: Boiling Initiating Valve 107: E-Book Reader 108: Boiling Initiating Valve 109: Tablet PC 110, 110.1 -110.12, 110.X: Hybridization chamber array II 1 : Epidemiological data 112, 112.1-112.12, 112.X: Amplification section I 1 3 : Genetic data II 4: Culture department -99- 201209403 1 1 5 : Electronics Health record 1 1 6 : Anticoagulant 1 1 8 : Surface tension valve 1 1 9 : Liquid sample 1 2 0 : Meniscus 1 2 1 : Electronic medical record 1 2 2 : Vent 123 : Personal health record 1 2 5 : Network 126 : boiling initiation valve 128, 128.2, 128.3: surface tension valve 1 3 0, 1 3 0.1 - 1 3 0.3 : lysis unit 1 3 1 : mixing portion 132 , 132.1, 1 32.3 : Surface tension valve 1 3 3 : Incubator inlet channel 134 : Lower tube 1 36 : Optical window 141 : Aptamer 1 4 2 : Target protein 1 43 : Host 144 : Receptor 145 : Antibody 1 4 6 : valve inlet 1 4 7 : complementary oligonucleotide -100- 201209403 148 : 149 : 150 : 152 : 153 : 154 : 156 : 158 : 160 : 164 : 166 : 168 : 170 : 174 : 175 : 176 : 178 : 180 : 182 : 184 : 185 : 186 : 187 : 188 : Valve outlet coupling sub valve down pipe ring heater valve heater contact heater heater contact microchannel outlet channel orifice capillary action starting characteristic dialysis inhalation Hole temperature sensor liquid sensor diffusion barrier flow path liquid sensor hybrid chamber heater light diode active area probe light diode water storage tank -101 - 201209403 190 : evaporator 1 9 1 : ring heater 192 : Water supply channel 193: Upper pipe 194: Lower pipe 1 9 5: Top metal layer 196: Humidifier 1 9 8 : Suction hole 202: Capillary action Starting feature 204: MST Channel 208: Liquid sensor 210: Micro Channel 2 1 2 : MST Channel 2 18 : Electrode 220: electrode 222: gap 2 3 2 : humidity sensor 2 3 4 : heater 236 : FRET probe 23 8 : target nucleic acid sequence 240 : ring 242 : stem 244 : excitation light 246 : fluorophore 201209403 2 4 8: quencher 25 0 : fluorescent signal 2 5 2 : optical center 2 5 4 : lens 28 8 : sample input and preparation 290 : extraction stage 291 : culture stage 292 : amplification stage 294 : detection stage 2 9 6 : First electrode 298: second electrode 3 00: programmable delay 30 1 : L 0 C device 3 7 6 : heat conducting column 3 7 8 : positive control probe 3 8 0 : negative control probe 3 82 : calibration chamber 3 8 4 : Gate 3 8 6 : Smell 3 8 8 : Gate 3 90 : Retractable lancet 3 92 : Lancet release button 3 9 3 : No. 3 94 : MOS transistor 201209403 3 9 6 : Μ OS transistor 398: MOS transistor 400: MOS transistor 402: MOS transistor 404: MOS transistor 406: node 40 8 : film seal 4 1 0 : membrane guard 647 : AlexaFluor 67 1: LOC variant 673 : LOC Variant 674 : LOC Variant 677 : LOC Variant 6 8 2 : Dialysate 684 : Nucleic Acid Amplification and Hybridization 6 8 6 : Dialysis Step 692 : Primer-Linked Linear Probe 694 : Amplification Blocker 6 9 6 . Probe region 698: Complementary sequence 7〇〇: Oligonucleotide primer 704: Stem-and-loop probe 706: Complementary sequence 708: Stem-104 - 201209403 710: 712: 714: 716: 718 : 766 : 778 : 780 : 7 82 : 788 : 790 : 792 : 794 : 795 : 796 : 797 : 79 8 ·· 801 : 803 : 805 : 8 07 ·· 809 : 8 11: 813 : The first light edge Mirror second aperture first mirror second mirror scrap storage tank configuration configuration configuration differential imager circuit pixel virtual pixel reading_column read_column negative control probe (transistor) positive control probe (transistor Pixel capacitor virtual pixel capacitor switch switch switch -105 201209403 8 1 5 : Capacitor amplifier 8 1 7 : differential signal 860 : ECL excitation electrode 87 0 : ECL excitation electrode
Claims (1)
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US35601810P | 2010-06-17 | 2010-06-17 | |
US43768611P | 2011-01-30 | 2011-01-30 |
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TW201209403A true TW201209403A (en) | 2012-03-01 |
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TW100119241A TW201211533A (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for simultaneous detection of multiple conditions in a patient |
TW100119254A TW201209407A (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with reagent mixing proportions determined by number of active outlet valves |
TW100119224A TW201209402A (en) | 2010-06-17 | 2011-06-01 | Apparatus for loading oligonucleotide spotting devices and spotting oligonucleotide probes |
TW100119249A TW201211534A (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with PCR section and diffusion mixer |
TW100119251A TW201209159A (en) | 2010-06-17 | 2011-06-01 | Genetic analysis LOC with non-specific nucleic acid amplification section and subsequent specific amplification of particular sequences in a separate section |
TW100119245A TW201209405A (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with flow-channel structure having active valve for capillary-driven fluidic propulsion without trapped air bubbles |
TW100119246A TW201209406A (en) | 2010-06-17 | 2011-06-01 | Test module with microfluidic device having LOC and dialysis device for separating pathogens from other constituents in a biological sample |
TW100119231A TW201211539A (en) | 2010-06-17 | 2011-06-01 | LOC device for pathogen detection and genetic analysis with chemical lysis, incubation and tandem nucleic acid amplification |
TW100119237A TW201209404A (en) | 2010-06-17 | 2011-06-01 | LOC device for genetic analysis which performs nucleic acid amplification before removing non-nucleic acid constituents in a dialysis section |
TW100119223A TW201219770A (en) | 2010-06-17 | 2011-06-01 | Test module incorporating spectrometer |
TW100119228A TW201209158A (en) | 2010-06-17 | 2011-06-01 | LOC device for genetic analysis with dialysis, chemical lysis and tandem nucleic acid amplification |
TW100119234A TW201211540A (en) | 2010-06-17 | 2011-06-01 | LOC device for pathogen detection and genetic analysis with dialysis and nucleic acid amplification |
TW100119253A TW201219776A (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with conductivity sensor |
TW100119248A TW201211243A (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with dialysis section having stomata tapering counter to flow direction |
TW100119226A TW201211240A (en) | 2010-06-17 | 2011-06-01 | LOC device for pathogen detection with dialysis, thermal lysis, nucleic acid amplification and prehybridization filtering |
TW100119235A TW201209403A (en) | 2010-06-17 | 2011-06-01 | LOC device for genetic analysis which performs nucleic acid amplification after sample preparation in a dialysis section |
TW100119238A TW201211532A (en) | 2010-06-17 | 2011-06-01 | LOC device with parallel incubation and parallel DNA and RNA amplification functionality |
TW100119243A TW201211242A (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for genetic and mitochondrial analysis of a biological sample |
TW100119252A TW201219115A (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module with flexible membrane for internal microenvironment pressure-relief |
TW100119232A TW201211241A (en) | 2010-06-17 | 2011-06-01 | LOC device for pathogen detection, genetic analysis and proteomic analysis with dialysis, chemical lysis, incubation and tandem nucleic acid amplification |
TW100119227A TW201211538A (en) | 2010-06-17 | 2011-06-01 | LOC device for pathogen detection with dialysis, chemical lysis and tandem nucleic acid amplification |
TW100119250A TW201211244A (en) | 2010-06-17 | 2011-06-01 | Test module with diffusive mixing in small cross sectional area microchannel |
Family Applications Before (15)
Application Number | Title | Priority Date | Filing Date |
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TW100119241A TW201211533A (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for simultaneous detection of multiple conditions in a patient |
TW100119254A TW201209407A (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with reagent mixing proportions determined by number of active outlet valves |
TW100119224A TW201209402A (en) | 2010-06-17 | 2011-06-01 | Apparatus for loading oligonucleotide spotting devices and spotting oligonucleotide probes |
TW100119249A TW201211534A (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with PCR section and diffusion mixer |
TW100119251A TW201209159A (en) | 2010-06-17 | 2011-06-01 | Genetic analysis LOC with non-specific nucleic acid amplification section and subsequent specific amplification of particular sequences in a separate section |
TW100119245A TW201209405A (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with flow-channel structure having active valve for capillary-driven fluidic propulsion without trapped air bubbles |
TW100119246A TW201209406A (en) | 2010-06-17 | 2011-06-01 | Test module with microfluidic device having LOC and dialysis device for separating pathogens from other constituents in a biological sample |
TW100119231A TW201211539A (en) | 2010-06-17 | 2011-06-01 | LOC device for pathogen detection and genetic analysis with chemical lysis, incubation and tandem nucleic acid amplification |
TW100119237A TW201209404A (en) | 2010-06-17 | 2011-06-01 | LOC device for genetic analysis which performs nucleic acid amplification before removing non-nucleic acid constituents in a dialysis section |
TW100119223A TW201219770A (en) | 2010-06-17 | 2011-06-01 | Test module incorporating spectrometer |
TW100119228A TW201209158A (en) | 2010-06-17 | 2011-06-01 | LOC device for genetic analysis with dialysis, chemical lysis and tandem nucleic acid amplification |
TW100119234A TW201211540A (en) | 2010-06-17 | 2011-06-01 | LOC device for pathogen detection and genetic analysis with dialysis and nucleic acid amplification |
TW100119253A TW201219776A (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with conductivity sensor |
TW100119248A TW201211243A (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with dialysis section having stomata tapering counter to flow direction |
TW100119226A TW201211240A (en) | 2010-06-17 | 2011-06-01 | LOC device for pathogen detection with dialysis, thermal lysis, nucleic acid amplification and prehybridization filtering |
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Application Number | Title | Priority Date | Filing Date |
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TW100119238A TW201211532A (en) | 2010-06-17 | 2011-06-01 | LOC device with parallel incubation and parallel DNA and RNA amplification functionality |
TW100119243A TW201211242A (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for genetic and mitochondrial analysis of a biological sample |
TW100119252A TW201219115A (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module with flexible membrane for internal microenvironment pressure-relief |
TW100119232A TW201211241A (en) | 2010-06-17 | 2011-06-01 | LOC device for pathogen detection, genetic analysis and proteomic analysis with dialysis, chemical lysis, incubation and tandem nucleic acid amplification |
TW100119227A TW201211538A (en) | 2010-06-17 | 2011-06-01 | LOC device for pathogen detection with dialysis, chemical lysis and tandem nucleic acid amplification |
TW100119250A TW201211244A (en) | 2010-06-17 | 2011-06-01 | Test module with diffusive mixing in small cross sectional area microchannel |
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CA2887206A1 (en) * | 2012-10-05 | 2014-04-10 | Rustem F. Ismagilov | Methods and systems for microfluidics imaging and analysis |
TWI512286B (en) * | 2013-01-08 | 2015-12-11 | Univ Nat Yunlin Sci & Tech | Microfluidic bio-sensing system |
US11318479B2 (en) | 2013-12-18 | 2022-05-03 | Berkeley Lights, Inc. | Capturing specific nucleic acid materials from individual biological cells in a micro-fluidic device |
EP3167045A4 (en) * | 2014-07-11 | 2018-01-17 | Advanced Theranostics Inc. | Point of care polymerase chain reaction device for disease detection |
JP2016148574A (en) * | 2015-02-12 | 2016-08-18 | セイコーエプソン株式会社 | Electronic component carrier device and electronic component inspection apparatus |
AU2016250580B2 (en) * | 2015-04-22 | 2021-02-25 | Berkeley Lights, Inc. | Culturing station for microfluidic device |
EP3275992A1 (en) * | 2016-07-29 | 2018-01-31 | Bayer Aktiengesellschaft | Adapter for cell-culture vessel |
JP2019050798A (en) * | 2017-07-05 | 2019-04-04 | 株式会社Screenホールディングス | Sample container |
TWI671397B (en) * | 2017-07-14 | 2019-09-11 | 國立中興大學 | Granular body extraction device |
WO2019013825A1 (en) * | 2017-07-14 | 2019-01-17 | Hewlett-Packard Development Company, L.P. | Microfluidic valve |
MX2020005123A (en) * | 2017-11-17 | 2020-11-06 | Siemens Healthcare Diagnostics Inc | Sensor assembly and method of using same. |
CN111601660B (en) * | 2018-01-11 | 2022-08-16 | 纳诺卡夫公司 | Microfluidic cellular devices and methods of use thereof |
CN111656162B (en) | 2018-02-01 | 2023-09-01 | 东丽株式会社 | Device for evaluating particles in liquid and method for operating same |
EP3560593B1 (en) * | 2018-04-25 | 2024-06-05 | OPTOLANE Technologies Inc. | Cartridge for digital real-time pcr |
US11366109B2 (en) * | 2018-12-06 | 2022-06-21 | Winmems Technologies Co., Ltd. | Encoded microflakes |
TWI711822B (en) * | 2019-10-18 | 2020-12-01 | 國立成功大學 | Miniature and intelligent urine sensing system |
TWI739318B (en) * | 2020-02-24 | 2021-09-11 | 國立陽明交通大學 | An impedance-type chip for measuring instantaneous sweat pressure, a micro-control system for sweat pressure measurement, and a measuring method of instantaneous sweat pressure |
WO2021216627A1 (en) * | 2020-04-21 | 2021-10-28 | Roche Sequencing Solutions, Inc. | High-throughput nucleic acid sequencing with single-molecule sensor arrays |
TWI795735B (en) * | 2021-02-26 | 2023-03-11 | 行政院農業委員會水產試驗所 | Automatic portable detection system for pathogenic bacteria |
CN115494833A (en) * | 2021-06-18 | 2022-12-20 | 广州视源电子科技股份有限公司 | Robot control method and device |
TWI793671B (en) * | 2021-07-09 | 2023-02-21 | 中國醫藥大學 | Tumor microenvironment on chip and production method thereof |
TWI814499B (en) * | 2021-07-22 | 2023-09-01 | 中央研究院 | Device for continuously producing and analyzing rna, and methods of producing rna by using the device |
TWI800964B (en) * | 2021-10-29 | 2023-05-01 | 宏碁股份有限公司 | Power supply device and electronic system |
TWI805205B (en) * | 2022-01-26 | 2023-06-11 | 國立中正大學 | High stability waveguide analog resonance biosensing system with self-compensation |
TWI818596B (en) * | 2022-06-22 | 2023-10-11 | 嘉碩生醫電子股份有限公司 | Shear-mode liquid-phase sensor having groove structure and methods of manufacturing and using the same |
TWI840226B (en) * | 2023-05-17 | 2024-04-21 | 博錸生技股份有限公司 | Automatic sample preparation system |
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2011
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- 2011-06-01 TW TW100119254A patent/TW201209407A/en unknown
- 2011-06-01 TW TW100119224A patent/TW201209402A/en unknown
- 2011-06-01 TW TW100119249A patent/TW201211534A/en unknown
- 2011-06-01 TW TW100119251A patent/TW201209159A/en unknown
- 2011-06-01 TW TW100119245A patent/TW201209405A/en unknown
- 2011-06-01 TW TW100119246A patent/TW201209406A/en unknown
- 2011-06-01 TW TW100119231A patent/TW201211539A/en unknown
- 2011-06-01 TW TW100119237A patent/TW201209404A/en unknown
- 2011-06-01 TW TW100119223A patent/TW201219770A/en unknown
- 2011-06-01 TW TW100119228A patent/TW201209158A/en unknown
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- 2011-06-01 TW TW100119226A patent/TW201211240A/en unknown
- 2011-06-01 TW TW100119235A patent/TW201209403A/en unknown
- 2011-06-01 TW TW100119238A patent/TW201211532A/en unknown
- 2011-06-01 TW TW100119243A patent/TW201211242A/en unknown
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TW201209407A (en) | 2012-03-01 |
TW201209406A (en) | 2012-03-01 |
TW201211539A (en) | 2012-03-16 |
TW201211242A (en) | 2012-03-16 |
TW201209159A (en) | 2012-03-01 |
TW201209402A (en) | 2012-03-01 |
TW201211240A (en) | 2012-03-16 |
TW201211533A (en) | 2012-03-16 |
TW201209404A (en) | 2012-03-01 |
TW201209158A (en) | 2012-03-01 |
TW201211241A (en) | 2012-03-16 |
TW201211538A (en) | 2012-03-16 |
TW201211540A (en) | 2012-03-16 |
TW201219770A (en) | 2012-05-16 |
TW201219115A (en) | 2012-05-16 |
TW201209405A (en) | 2012-03-01 |
TW201211534A (en) | 2012-03-16 |
TW201219776A (en) | 2012-05-16 |
TW201211243A (en) | 2012-03-16 |
TW201211532A (en) | 2012-03-16 |
TW201211244A (en) | 2012-03-16 |
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