TW201209158A - LOC device for genetic analysis with dialysis, chemical lysis and tandem nucleic acid amplification - Google Patents

LOC device for genetic analysis with dialysis, chemical lysis and tandem nucleic acid amplification Download PDF

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TW201209158A
TW201209158A TW100119228A TW100119228A TW201209158A TW 201209158 A TW201209158 A TW 201209158A TW 100119228 A TW100119228 A TW 100119228A TW 100119228 A TW100119228 A TW 100119228A TW 201209158 A TW201209158 A TW 201209158A
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nucleic acid
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pcr
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loc device
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TW100119228A
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Chinese (zh)
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Geoffrey Richard Facer
Alireza Moini
Kia Silverbrook
Mehdi Azimi
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Geneasys Pty Ltd
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Abstract

A lab-on-a-chip (LOC) device for genetic analysis of a biological sample, the LOC device having an inlet for receiving the sample, a supporting substrate, a dialysis section for separating cells larger than a predetermined threshold in the sample from smaller constituents, whereby the cells larger than a predetermined threshold include target cells containing genetic material for analysis, a plurality of reagent reservoirs, a lysis section downstream of the dialysis section for lysing the target cells to release the genetic material therein, the lysis section being in fluid communication with one of the reagent reservoirs containing a lysis reagent for lysing the target cells in the lysis section, a first nucleic acid amplification section downstream of the lysis section for amplifying first nucleic acid sequences in the genetic material, and, a second nucleic acid amplification section downstream of the first nucleic acid amplification section for amplifying second nucleic acid sequences in the amplicon from the first nucleic acid amplification section, wherein, the dialysis section, the lysis section, the first nucleic acid amplification section and the second nucleic acid amplification section are all supported on the supporting substrate.

Description

201209158 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 ) ' 別是,本發明有關用於分子診斷的微流體 析。 【先前技術】 分子診斷嚴然興起成爲一種熱門領域 能於症狀顯露之前)疾病偵測帶來希望。 用於偵測: •遺傳性疾病 •獲得性障礙 •傳染性疾病 •與健康狀況相關的基因易感性 分子診斷具有高精確度及快速的處理 少發生無效之健康照護服務、增進病患結 理及實現病患照護個人化的可能性。分子 係基於偵測與鑑定自生物樣本(例如血液 且擴增而得的特定核酸,包含去氧核糖核 糖核酸(RNA )兩者。核酸鹼基的互補天 (寡聚核苷酸)的短序列與特定核酸序列 可用於進行核酸檢驗。若發生雜合反應, 在互補序列。這使得例如預測個人未來將 定傳染性病源的本質與致病性或測定個人 之診斷裝置。特 及生化處理及分 ,其爲早期(可 分子診斷檢驗係 時間,且具有減 果、增進疾病管 診斷的許多技術 或唾液)中萃取 酸(DNA )與核 性允許合成DNA 結合(雜合)而 則表示樣本中存 罹患的疾病、測 對藥物的反應這 -5- 201209158 類事情將有可能達成。 以核酸爲基礎的分子診斷檢驗 以核酸爲基礎的檢驗具有四個不同步驟: 1. 樣本製備 2. 核酸萃取 3. 核酸擴增(隨意選用) 4. 偵測 許多樣本種類可用於基因分析,例如血液、尿液、唾 液與組織樣本。診斷檢驗決定所需的樣本種類,並非所有 樣本都具有病程代表性。此等樣本具有各種成分,但通常 這些成分中僅有一種成分才是關注對象。例如在血液中, 高濃度的紅血球可能抑制對病源微生物的偵測。因此,在 核酸檢驗的初期經常需要純化及/或濃縮步驟。 血液是最常尋求的樣本種類之一。血液具有三種主要 成分:白血球、紅血球及血小板。血小板幫助凝血且在體 外可保持活性。爲抑制凝結作用,於進行純化與濃縮之前 ,先使樣本與試劑(例如,乙二胺四乙酸(EDTA ))混 合。通常自樣本中去除紅血球以濃縮標靶細胞。在人類中 ,紅血球數量約佔細胞材料的99%,但由於紅血球不具細 胞核,因此紅血球未攜帶DN A。再者,紅血球含有多種成 分,例如血紅素,而血紅素可能干擾下游核酸擴增處理( 將說明於下)。藉著於溶胞溶液中有差別性地溶解紅血球 可達成紅血球之去除,且完整地保留其餘細胞材料,隨後 -6- 201209158 可利用離心從樣本中分離出其餘細胞材料。如此可提供標 靶細胞之濃縮,以從標靶細胞萃取出核酸。 用於萃取核酸的萃取法取決於樣本及欲執行之診斷性 分析檢驗法。例如,萃取病毒RN A之方法將與萃取基因體 DN A之方法有相當大的差異。然而,自標靶細胞中萃取核 酸通常涉及溶胞步驟,且於溶胞步驟之後接著進行核酸純 化。溶胞步驟使細胞與核膜破裂而釋出遺傳物質。通常使 用溶胞性清潔劑(例如,十二烷基硫酸鈉)達成此步驟, 溶胞性清潔劑亦使存在於細胞中的大量蛋白質變性( denature)。201209158 VI. Description of the Invention: TECHNICAL FIELD OF THE INVENTION The present invention relates to the use of microsystem technology (MST). In addition, the present invention relates to microfluidic analysis for molecular diagnostics. [Prior Art] Molecular diagnosis has emerged as a hot field. Before the symptoms are revealed, disease detection brings hope. For detection: • Hereditary diseases • Acquired disorders • Infectious diseases • Genetic susceptibility molecular diagnostics related to health status Highly accurate and rapid treatment of less effective health care services, improved patient outcomes and realization The possibility of patient care personalization. The molecular system is based on the detection and identification of specific nucleic acids derived from biological samples (eg, blood and amplified, including both deoxyribonucleic acid (RNA). Short sequences of complementary days (oligonucleotides) of nucleic acid bases) The specific nucleic acid sequence can be used for nucleic acid testing. If a heterozygous reaction occurs, it is in a complementary sequence. This makes it possible, for example, to predict the nature and pathogenicity of an individual's infectious agent in the future or to measure the individual's diagnostic device. In the early stage (many molecular diagnostic test system, and many techniques or saliva with reduced fruit, improved disease tube diagnosis), extracting acid (DNA) and nuclear nature allow synthetic DNA to bind (heterozygous), indicating that it is stored in the sample. The disease and the response to the drug will be possible. The nucleic acid-based molecular diagnostic test has four different steps: 1. Sample preparation 2. Nucleic acid extraction 3 Nucleic acid amplification (optional) 4. Detection of many sample types for genetic analysis, such as blood, urine, saliva and tissue samples. The type of sample required for the test decision, not all samples are representative of the disease course. These samples have various components, but usually only one of these components is the target of interest. For example, in the blood, high concentrations of red blood cells may inhibit the pair. Detection of pathogenic microorganisms. Therefore, purification and/or concentration steps are often required in the early stages of nucleic acid testing. Blood is one of the most commonly sought sample types. Blood has three main components: white blood cells, red blood cells, and platelets. The activity can be maintained in vitro. To inhibit coagulation, the sample is mixed with a reagent (eg, ethylenediaminetetraacetic acid (EDTA)) prior to purification and concentration. The red blood cells are typically removed from the sample to concentrate the target cells. Among them, the number of red blood cells accounts for about 99% of the cell material, but since the red blood cells do not have a nucleus, the red blood cells do not carry DN A. Furthermore, the red blood cells contain various components such as hemoglobin, and hemoglobin may interfere with downstream nucleic acid amplification processing (will explain In the next section. By dissolving red blood differentially in the lysis solution The ball can achieve red blood cell removal and retain the remaining cellular material intact, and then the remaining cell material can be separated from the sample by centrifugation from June to 201209158. This provides concentration of the target cells to extract nucleic acids from the target cells. The extraction method used to extract nucleic acids depends on the sample and the diagnostic assay to be performed. For example, the method of extracting the virus RN A will be quite different from the method of extracting the gene DN A. However, in the self-targeting cells Extraction of the nucleic acid typically involves a lysis step and subsequent purification of the nucleic acid following the lysis step. The lysis step ruptures the cell with the nuclear membrane to release the genetic material. A lytic detergent (eg, sodium lauryl sulfate) is typically used. To achieve this step, the lytic detergent also denatures a large amount of protein present in the cell.

接著以酒精沈澱步驟(通常使用冰冷的乙醇或異丙醇 )純化核酸,或藉由固相純化步驟純化核酸,通常於高濃 度離液鹽(chaotropic salt)存在下於管柱內的矽基質( silica matrix)、樹脂或在順磁珠上進行固相純化步驟, 之後進行清洗步驟,且隨後以低離子強度緩衝液進行沖提 (elution )。於沈澱核酸之前進行的選用步驟係添加可分 解蛋白質之蛋白酶以進一步純化該樣本。 其他溶胞方法包含藉由超音波振動的機械性溶胞法及 把樣本加熱至94°C以打破細胞膜的熱溶胞法。 存在於經萃取之材料中的標靶DN A或RN A可能含量極 小,特別是若標靶物是病源微生物的DNA或RNA時更是如 此。核酸擴增法提供選擇性地使低濃度之特定標靶核酸擴 增(即,複製)至可偵測之濃度的能力。 最常用的核酸擴增技術係聚合酶鏈鎖反應(PCR )。 201209158 PCR法在此領域中係廣爲人知,且於E. Van Pelt-Verkuil等 人所著且由Springer出版社於2008出版的《PCR擴增之原 理與技術體系(Principles and Technical Aspects of PCR amplification)》一書中提供此類反應之全面描述。 PCR是一項從複雜DNA背景中擴增標靶DNA序列的強 大技術。若欲(藉由PCR)擴增RNA,必需先使用被稱爲 反轉錄酶的酶使RNA轉錄成cDNA (互補DNA )。隨後,藉 由PCR擴增所產生的cDNA。 PCR是一種指數性方法,且只要用於維持反應的條件 可接受即可繼續進行。反應之成分如下: 1 ·引子對〜具有約10〜30個核苷酸的短單鏈DNA,該 短單鏈DNA與位於標靶序列兩側的區域互補。 2. DN A聚合酶〜一種合成DNA的熱穩定性酶。 3. 去氧核糖核苷三磷酸(dNTPs)〜提供倂入新合成 之DNA鏈中的核苷酸。 4. 緩衝液〜提供進行DNA合成之最佳化學環境。 PCR—般包含把此等反應物放入含有經萃取之核酸的 小試管中(約1 〇〜1 5微升)。把該小試管置於溫度循環器 中;溫度循環器係一種使該反應於一系列不同溫度下持續 不同時間的儀器。每個溫度循環的標準程序涉及變性階段 (denaturation phase)、黏合階段(annealing phase)及 延長階段(extension phase)。延長階段有時稱爲引子延 長階段。除了此種三步驟式程序之外,亦可採用兩步驟式 溫度程序,於兩步驟式溫度程序中係使黏合階段與延長階 201209158 段合倂。變性階段一般涉及使反應溫度升高至90〜95 °C以 使DNA鏈變性;於黏合階段中,使溫度降至約50~60°C以 使引子黏合;及隨後於延長階段中,使溫度升高至 6 0〜7 2 °C之DNA聚合酶最佳活性溫度以進行引子延長反應 。此過程重複循環約20〜40次,最終結果是創造出數百萬 個介於該等引子間之標靶序列的複製物。 標準PCR程序具有諸多變化型,例如爲進行分子診斷 已硏發出多重PCR、連接子-引子PCR( linker-primer PCR )、直接PCR、串接PCR、即時PCR及反轉錄酶PCR,等等The nucleic acid is then purified in an alcohol precipitation step (usually using ice-cold ethanol or isopropanol) or purified by a solid phase purification step, usually in the presence of a high concentration of chaotropic salt in the column ( The silica matrix), the resin or the solid phase purification step on the paramagnetic beads, followed by a washing step, and subsequent elution with a low ionic strength buffer. The optional step performed prior to precipitation of the nucleic acid is the addition of a protein-degrading protease to further purify the sample. Other lysis methods include mechanical lysis by ultrasonic vibration and thermal lysis of the cell membrane by heating the sample to 94 °C. The target DN A or RN A present in the extracted material may be extremely small, especially if the target is the DNA or RNA of the pathogenic microorganism. Nucleic acid amplification provides the ability to selectively amplify (i.e., replicate) a low concentration of a particular target nucleic acid to a detectable concentration. The most commonly used nucleic acid amplification technique is the polymerase chain reaction (PCR). The 201209158 PCR method is well known in the art and is based on "Principles and Technical Aspects of PCR amplification" by E. Van Pelt-Verkuil et al., published by Springer Press in 2008. A comprehensive description of such reactions is provided in the book. PCR is a powerful technique for amplifying target DNA sequences from complex DNA backgrounds. If RNA is to be amplified (by PCR), it is necessary to first transcribe RNA into cDNA (complementary DNA) using an enzyme called reverse transcriptase. Subsequently, the generated cDNA was amplified by PCR. PCR is an exponential method and can be continued as long as the conditions for maintaining the reaction are acceptable. The components of the reaction are as follows: 1 - Primer pair ~ Short single-stranded DNA of about 10 to 30 nucleotides complementary to the region flanking the target sequence. 2. DN A polymerase ~ a thermostable enzyme that synthesizes DNA. 3. Deoxyribonucleoside triphosphates (dNTPs) ~ provide nucleotides that are incorporated into the newly synthesized DNA strand. 4. Buffer ~ provides the best chemical environment for DNA synthesis. PCR generally involves placing such reactants in a small tube containing the extracted nucleic acid (about 1 〇 to 15 μl). The tube is placed in a temperature circulator; the temperature circulator is an instrument that allows the reaction to be carried out at a range of different temperatures for different periods of time. The standard procedure for each temperature cycle involves the denaturation phase, the annealing phase, and the extension phase. The extension phase is sometimes referred to as the primer extension phase. In addition to this three-step procedure, a two-step temperature program can be used to combine the bonding phase with the extended step 201209158 in a two-step temperature program. The denaturation phase generally involves raising the reaction temperature to 90 to 95 ° C to denature the DNA strand; in the bonding phase, the temperature is lowered to about 50 to 60 ° C to bond the primer; and then in the elongation phase, the temperature is made. The DNA polymerase optimal activity temperature was raised to 60 ° 7 to 2 ° C to carry out the primer extension reaction. This process is repeated for about 20 to 40 cycles, with the end result being the creation of millions of copies of the target sequence between the primers. Standard PCR programs are available in a variety of variations, such as for molecular diagnostics, multiplex PCR, linker-primer PCR, direct PCR, tandem PCR, real-time PCR, and reverse transcriptase PCR, etc.

多重PCR係於單個PCR反應混合物中使用多組引子以 製造出對不同DNA序列具有專一性之不同大小的擴增子( amplicon )。藉著一次鎖定多個基因,可從單次檢驗回合 得到額外的資訊,否則可能需要進行數次實驗方能得到額 外資訊。然而多重PCR之最佳化較爲困難且要求選擇具有 相似黏合溫度的引子’並且擴增子具有相似的長度及鹼基 組成以確保每個擴增子的擴增效率相等。 連接子-引子PCR亦稱爲接合轉接子PCR ( ngation adaptor PCR),其係一種能實質上對複雜DNA混合物中之 所有DNA序列進行核酸擴增而無需使用標靶物專一性引子 的方法。該方法首先涉及使用適當的限制核酸內切酶(酵 素)剪切該標耙DNA群。隨後利用接合酶(iigase enZyme )使具有適當懸臂末端之雙鏈寡聚核苷酸連接子(亦稱轉 接子)與標靶DNA斷片的末端接合。接著利用對該等連接 201209158 子序列具有專一性的寡聚核苷酸引子執行核酸擴增。如此 ,可擴增該DN A來源中被該等連接子寡聚核苷酸夾擊的所 有斷片。 直接PCR描述一種於無需任何核酸萃取或做最小限度 之核酸萃取下直接於樣本上執行PCR。長久以來一直認爲 存在於未經純化之生物樣本中的許多成分(例如,血液中 的血紅成分)會抑制PCR反應。傳統上,PCR要求於製備 反應混合物之前需先進行標靶核酸之大規模純化。然而, 藉由適當地改變化學試劑與樣本濃度,可能以最小限度的 DNA純化執行PCR或直接執行PCR。調整用於直接PCR的 PCR化學試劑包含提高緩衝強度、使用具有高活性及持續 性的聚合酶及可與潛在之聚合酶抑制劑螯合的添加劑。 串接PCR係利用兩種不同回合之核酸擴增反應以提高 擴增正確擴增子的或然率。其中一種形式的串接PCR係巢 式PCR,在巢式PCR中,使用兩對PCR引子以於兩個不同 回合之核酸擴增反應中擴增單一個基因座。第一對引子與 位於該標靶核酸序列之外側區域處的核酸序列雜合。用於 第二回合之核酸擴增反應中的第二對引子(巢式引子)係 結合於該第一 PCR產物之內且產生含有該標靶核酸序列的 第二PCR產物,該第二PCR產物將比第一 PCR產物要短。 此方法背後的原理邏輯在於若於第一回合之核酸擴增期間 出錯而擴增出錯誤的基因座,在藉由第二對引子進行第二 次擴增亦擴增出錯誤基因座的可能性極低,從而確保專一 性。 -10- 201209158 即時PCR (或定量PCR )係用於即時測量一種PCR產 物的量。藉由於反應中使用螢光染劑或含螢光發光基團之 探針且配合一套反應標準,可定量樣本中之核酸的起始量 '。此方法對於處理選項會隨樣本中之病源含量而有所不同 的分子診斷特別有用。 反轉錄酶PCR(RT-PCR)係用於從RNA擴增出DNA。 反轉錄酶係一種可把RNA反轉錄成互補DNA ( cDNA )的酶 ^ ’且隨後藉由PCR擴增該互補DNA。RT-PCR廣泛用於基因 表現硏究’藉以測定基因的表現或鑑定包含轉錄起始位置 及終止位置在內的RNA轉錄產物之序列。反轉錄酶PCR亦 用於擴增RNA病毒’例如人類免疫不全病毒或c型肝炎病 毒。 恆溫核酸擴增法無需仰賴持續加熱使模板DNA變性以 產生單鏈分子以作爲模板用於進行進一步擴增,因此不需 要精密複雜的機器。因此恆溫核酸擴增法可於原本所在位 置上進行或輕易地於實驗室環境以外的地方操作。目前已 . 揭示多種恆溫核酸擴增法,包含鏈置換擴增法、轉錄介導 擴增法、核酸序列依賴性擴增法、重組酶聚合酶擴增法' 滾環式擴增法、分枝型擴增法、解旋酶依賴性恆溫Dn A擴 增法及環形核酸介導擴增法。 恆溫核酸擴增法不依賴持續加熱使模板D N A變性以產 生單鏈分子作爲模板而用於進一步擴增,取代而之的是改 用替代方法,例如利用特定的限制核酸內切酶進行酶催化 剪切而於DN A分子上形成缺口或於恆溫下使用酶解開該 -11 - 201209158 DNA之雙鏈。 鏈置換擴增法(SDA )係仰賴某些限制酶具有於半修 飾DNA之未經修飾鏈上形成缺口的能力及5'_3f核酸內切酶 缺乏性聚合酶具有能延長和置換下游DNA鏈的能力。隨後 藉著結合同義反應與反義反應(在此等反應中源自同義反 應的鏈置換作用係做爲反義反應之模板)而達成指數性核 酸擴增。並非以傳統方式切斷DN A而是使用在DN A雙鏈之 其中一鏈上產生缺口的切口酶(例如N. Alwl、N. BstNBl 及Mlyl )對於此種反應而言係有用的。已藉由使用熱穩定 性限制酶(Χναΐ )與熱穩定性外聚合酶(5^聚合酶)之 組合改善鏈置換擴增法。此種組合經證明可使反應之擴增 效率自1〇8倍擴增提高至l〇IQ倍擴增,因此可能得以利用此 技術擴增特殊的單複製分子。 轉錄介導擴增法(TMA )和核酸序列依賴性擴增法( NASBA)使用RNA聚合酶以複製RNA序歹IJ,而非複製對應 的基因體DNA。該技術使用兩種引子及兩種或三種酶,分 別爲RN A聚合酶、反轉錄酶及(若反轉錄酶不具有rn A水 解酶活性時)可選用的RNA水解酶H(RNase H)。其中一 種引子含有供RN A聚合酶使用的啓動子序列。於核酸擴增 之第一步驟中,此引子於經界定之位置處與標靶核糖體 RNA ( rRNA)雜合。反轉錄酶藉著從該啓動子引子之3'端 開始延長而創造出該標靶rRNA的DNA複製物。藉由反轉錄 酶的RNA水解酶活性(若該反轉錄酶具有RNA水解酶活性 )或添加之RNase Η降解所產生之RNA:DNA雙鏈複合物中 -12- 201209158 的RN A。接著’第二種引子與該〇Ν A複製物結合。藉由反 轉錄酶從此引子之末端合成出新的DNA鏈而創造出雙鏈 DNA分子。RNA聚合酶辨識DNA模板中的啓動子序列且開 '始進行轉錄。每個新合成的RNA擴增子再進入上述程序中 且作爲模板以用於進行新一回合的複製反應。 於重組酶聚合酶擴增法(RPA)中,係藉著使相反序 列之寡聚核苷酸引子與模板DNA結合且利用DNA聚合酶延 φ 長該引子而達成特定DNA斷片之恆溫擴增。無需使用熱使 雙鏈DNA ( dsDNA )模板變性。取代的是,RPA採用重組 酶-引子複合物藉以掃描雙鏈DN A且幫助進行同源位置處 的鏈交換作用。藉由單鏈DNA結合蛋白與該經置換之模板 鏈交互作用而穩定所產生的結構,從而避免因支鏈遷移作 用而逐出引子。重組酶之拆解作用(disassembly)使該寡 聚核苷酸的3'端可與鏈置換DNA聚合酶接觸(該鏈置換 DNA聚合酶係例如枯草桿菌聚合酶1(5^)之大斷片)且 -φ 隨後進行引子延長反應。藉由重複循環此程序而達成指數 性核酸擴增。 解旋酶依賴性恆溫DN Α擴增法(HD A )係摹仿體內系 統使用DNA解旋酶藉以生成用於引子雜合反應的單鏈模板 且隨後藉由DN A聚合酶進行引子延長反應。於HD A反應的 第一步驟中,解旋酶沿著標靶DN.A行進且打斷連接兩條 DNA鏈的氫鍵,且隨後該兩條DNA鏈與該單鏈結合蛋白結 合。利用解旋酶暴露出該單鏈標靶區域露出允許引子與該 單鏈標靶區域黏合。隨後DNA聚合酶使用自由的去氧核糖 -13- 201209158 核苷三磷酸(dNTPs )延長每個引子之3'端以生成兩個 DNA複製物。該兩條經複製之雙鏈DNA各自進入下一個 HDA循環,而導致該目標序歹|J之指數性核酸擴增。 其他依賴DNA的恆溫技術包含滾環擴增法(RCA ) ’ 在滾環擴增法中,DNA聚合酶持續地繞著圓形DNA模板延 長引子,而生成由多個重複的該圓形模板複製物所組成之 長DN A產物。直至反應結束,該聚合酶產生數千個圓形模 板之複製物,且該等複製物之鏈長係與該原始標靶DN A有 關。此方法允許用於標靶物之空間解析及訊息之快速核酸 擴增。此方法可於1小時內生成高達1012個模板複製物。分 枝型擴增法係RCA法的一種變化型,且分枝型擴增法利用 —種封閉式環形探針(C-探針)或扣鎖探針(padlock probe )及一種具有高持續性之DNA聚合酶以於恆溫條件 下指數地擴增該C_探針。Multiplex PCR uses multiple sets of primers in a single PCR reaction mixture to create different sizes of amplicon that are specific for different DNA sequences. By locking multiple genes at once, additional information can be obtained from a single test round, otherwise additional experiments may be required to obtain additional information. However, optimization of multiplex PCR is difficult and requires selection of primers with similar binding temperatures' and the amplicon has similar length and base composition to ensure equal amplification efficiency for each amplicon. Linker-initiator PCR, also known as ngation adaptor PCR, is a method that allows nucleic acid amplification of virtually all DNA sequences in a complex DNA mixture without the use of a target-specific primer. The method first involves the cleavage of the marker DNA population using an appropriate restriction endonuclease (enzyme). The double-stranded oligonucleotide linker (also known as a adaptor) with the appropriate cantilever ends is then ligated to the ends of the target DNA fragment using a ligase (iigase enZyme). Nucleic acid amplification is then performed using oligonucleotide primers that are specific for the 201209158 subsequence. Thus, all fragments of the DN A source that are pinched by the linker oligonucleotides can be amplified. Direct PCR describes a method of performing PCR directly on a sample without any nucleic acid extraction or minimal nucleic acid extraction. It has long been believed that many components present in unpurified biological samples (e.g., blood red components in blood) inhibit the PCR reaction. Traditionally, PCR requires extensive purification of the target nucleic acid prior to preparation of the reaction mixture. However, by appropriately changing the chemical reagent and the sample concentration, it is possible to perform PCR with minimal DNA purification or directly perform PCR. Modification of PCR chemistries for direct PCR involves increasing buffer strength, using a polymerase with high activity and persistence, and additives that can be chelated with potential polymerase inhibitors. Tandem PCR utilizes two different rounds of nucleic acid amplification reactions to increase the probability of amplifying the correct amplicon. One form of tandem PCR is nested PCR in which two pairs of PCR primers are used to amplify a single locus in two different rounds of nucleic acid amplification reactions. The first pair of primers is hybridized to a nucleic acid sequence located at an exosome region of the target nucleic acid sequence. A second pair of primers (nested primers) for use in the second round of nucleic acid amplification reaction binds within the first PCR product and produces a second PCR product comprising the target nucleic acid sequence, the second PCR product Will be shorter than the first PCR product. The rationale behind this method is that if the wrong locus is amplified during the first round of nucleic acid amplification, the second amplification of the second pair of primers will also amplify the possibility of the wrong locus. Extremely low to ensure specificity. -10- 201209158 Real-time PCR (or quantitative PCR) is used to measure the amount of a PCR product in real time. The initial amount of nucleic acid in the sample can be quantified by using a fluorescent dye or a probe containing a fluorescent luminescent group in the reaction in conjunction with a set of reaction standards. This method is especially useful for molecular diagnostics where processing options vary with the source of the sample. Reverse transcriptase PCR (RT-PCR) is used to amplify DNA from RNA. A reverse transcriptase is an enzyme that reversely transcribes RNA into a complementary DNA (cDNA) and then amplifies the complementary DNA by PCR. RT-PCR is widely used in gene expression studies to determine the expression of a gene or to identify sequences of RNA transcripts including the start and end positions of transcription. Reverse transcriptase PCR is also used to amplify RNA viruses such as human immunodeficiency virus or hepatitis C virus. The thermostatic nucleic acid amplification method does not require denaturation of the template DNA by continuous heating to produce a single-stranded molecule for use as a template for further amplification, and thus does not require a sophisticated machine. Therefore, the thermostatic nucleic acid amplification method can be performed at the original location or easily operated outside the laboratory environment. At present, various thermostatic nucleic acid amplification methods have been disclosed, including strand displacement amplification, transcription-mediated amplification, nucleic acid sequence-dependent amplification, recombinase polymerase amplification, rolling circle amplification, and branching. Amplification method, helicase-dependent constant temperature Dn A amplification method and circular nucleic acid-mediated amplification method. The constant temperature nucleic acid amplification method does not rely on continuous heating to denature the template DNA to produce a single-stranded molecule as a template for further amplification, instead of using an alternative method, such as enzymatic cleavage using a specific restriction endonuclease The gap is formed on the DN A molecule or the enzyme is used to untwist the -11 - 201209158 DNA double strand. Chain displacement amplification (SDA) relies on the ability of certain restriction enzymes to form gaps in the unmodified strand of semi-modified DNA and the ability of 5'_3f endonuclease-deficient polymerase to extend and replace downstream DNA strands. ability. Exponential nucleic acid amplification is then achieved by combining a synonymous reaction with an antisense reaction (a strand displacement system derived from synonymous reactions in these reactions as a template for an antisense reaction). Instead of cutting DN A in a conventional manner, it is useful for such reactions to use nicking enzymes (e.g., N. Alwl, N. BstNBl, and Mlyl) that create a gap in one of the DN A duplexes. The strand displacement amplification method has been improved by using a combination of a thermostable restriction enzyme (Χναΐ) and a thermostable outer polymerase (5^ polymerase). This combination has been shown to increase the amplification efficiency of the reaction from 1-8 fold amplification to 1 〇IQ fold amplification, and thus it may be possible to amplify a particular single replica molecule using this technique. Transcription-mediated amplification (TMA) and nucleic acid sequence-dependent amplification (NASBA) use RNA polymerase to replicate the RNA sequence 歹IJ instead of replicating the corresponding genomic DNA. This technique uses two primers and two or three enzymes, RN A polymerase, reverse transcriptase, and (if the reverse transcriptase does not have rn A hydrolase activity) RNA hydrolase H (RNase H). One of the primers contains a promoter sequence for use by RN A polymerase. In the first step of nucleic acid amplification, the primer is hybridized to the target ribosomal RNA (rRNA) at a defined position. The reverse transcriptase creates a DNA replica of the target rRNA by prolonging from the 3' end of the promoter primer. RN A of -12-201209158 in RNA:DNA double-stranded complex produced by reverse transcriptase RNA hydrolase activity (if the reverse transcriptase has RNA hydrolase activity) or added RNase Η degradation. The second primer is then combined with the 〇ΝA replica. A double-stranded DNA molecule is created 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 re-entered into the above procedure and used as a template for a new round of replication reactions. In recombinant enzyme polymerase amplification (RPA), constant temperature amplification of a particular DNA fragment is achieved by binding the opposite sequence of the oligonucleotide primer to the template DNA and using the DNA polymerase to extend the primer. The double-stranded DNA (dsDNA) template is denatured without the use of heat. Instead, RPA uses a recombinase-introduction complex to scan double-stranded DN A and aid in strand exchange at homologous positions. The resulting structure is stabilized by interaction of the single-stranded DNA binding protein with the displaced template strand, thereby avoiding the priming of the primer due to branch migration. Disassembly of the recombinase allows the 3' end of the oligonucleotide to be contacted with a strand displacement DNA polymerase (a large fragment of the strand displacement DNA polymerase such as Bacillus subtilis polymerase 1 (5^)) And -φ is followed by an extension of the primer. Exponential nucleic acid amplification is achieved by repeating this procedure. The helicase-dependent thermoregulating DN Α amplification method (HD A) is a single-strand template in which the DNA helicase is used to generate a heterozygous reaction for primer hybridization and then the primer-extension reaction is carried out by DN A polymerase. In the first step of the HD A reaction, the helicase travels along the target DN.A and breaks the hydrogen bond connecting the two DNA strands, and then the two DNA strands bind to the single-stranded binding protein. Exposure of the single-stranded target region by helicase allows the primer to bind to the single-strand target region. The DNA polymerase then extended the 3' end of each primer using free deoxyribose-13-201209158 nucleoside triphosphates (dNTPs) to generate two DNA copies. The two replicated double-stranded DNAs each enter the next HDA cycle, resulting in exponential nucleic acid amplification of the target sequence. Other DNA-dependent thermostat techniques include rolling circle amplification (RCA). In rolling circle amplification, DNA polymerase continuously extends the primer around a circular DNA template, and the replication is replicated by multiple replicates of the circular template. The long DN A product consisting of the material. Until the end of the reaction, the polymerase produces replicas of thousands of circular templates, and the chain length of the replicas is related to the original target DN A . This method allows for spatial resolution of the target and rapid nucleic acid amplification of the message. This method can generate up to 1012 template replicas in one hour. The branching amplification method is a variant of the RCA method, and the branching amplification method utilizes a closed circular probe (C-probe) or a padlock probe and a high persistence The DNA polymerase exponentially amplifies the C_probe under constant temperature conditions.

環形核酸介導擴增法(LAMP )提供高度選擇性且採 用一種DNA聚合酶及一組四種經特殊設計之引子,該組引 子辨識該標靶DN A上總共六種不同的序列。以含有該標靶 DNA之同義鏈和反義鏈之序列的內側引子開始進行LAMP 。藉由外側引子引導進行的後續鏈置換DNA合成反應釋放 出單鏈DNA。此單鏈DNA係作爲模板以藉由第二內側引子 和第二外側引子引導進行DNA合成反應,該第二內側引子 及第二外側引子係與該標靶物之另一末端雜合而產生幹-環狀 DNA結構(stem-loop DNA structure)。於後續 LAMP 循環中,使內側引子與該產物上的環部雜合且開始進行置 -14- 201209158 換性DNA合成反應,而獲得該原始幹-環狀〇ΝΑ及一條具 有雙倍主幹長度的新幹-環狀DNA。該循環反應以一小時 內累積1〇9個標靶物複製物的速度持續進行。最終產物係 '具有數個反向重複之標靶序列,且該最終產物藉著同一條 鏈內的反向重複之標靶序列間交互黏合而形成具有多重環 部之花椰菜狀結構的幹-環狀DN A。 完成核酸擴增反應之後,必需分析該等擴增產物以判 ^ 斷是否生成預期的擴增子(標靶核酸之擴增量)。分析產 物之方法的範圍可從藉由凝膠電泳法簡單判斷擴增子之大 小到使用DN A雜合法鑑定該擴增子之核苷酸組成。 凝膠電泳法係用於確認核酸擴增處理是否生成預期擴 增子的最簡單方法之一。凝膠電泳法係藉由在凝膠基質( gel matrix )上施加電場以分離DNA斷片。帶負電之DNA斷 片以不同速率(該速率主要取決於DNA斷片的大小)移動 通過該基質。待電泳完成之後,可對凝膠中的該等斷片進 -φ 行染色以看見該等斷片。通常使用溴化乙啶進行染色,溴 化乙啶於紫外光下可釋放螢光。 可使DN Α尺寸標記與擴增子並排於凝膠上進行電泳, 且藉由與含有已知大小之DNA斷片的DNA尺寸標記(階梯 狀DNA條帶)做比較可判斷該等斷片之大小。由於該等寡 聚核苷酸引子結合至位於標靶DN A兩翼的特定位置處,因 此可預測該擴增產物之大小,且在凝膠上偵測到該擴增產 物呈現已知尺寸之條帶。爲確認該擴增子之一致性或若有 數種擴增子生成時,通常對該擴增子進行DN A探針雜合反 -15- 201209158 應。 DNA雜合反應係指藉由互補鹼基配對作用而形成雙鏈 DNA。用於特定擴增產物之陽性鑑定(positive identification )的DN A雜合反應需使用長度約20個核苷酸 的DN A探針。若該探針具有與該擴增子(標靶)DN A序列 互補的序列,在溫度、pH値與離子濃度適宜的條件下將發 生雜合反應。若發生雜合反應,則表示該原始樣本中存在 所關注的基因或DN A序列。 光學偵測法係用於偵測雜合反應的最普遍方法。擴增 子或探針之任一者係經標示(labeled)以透過螢光發光法 或電致化學發光法(electrochemiluminescence,ECL)而 發光。此等方法的差異在於產生激發態之發光基團的手段 不同,但兩種方法皆能對核苷鏈進行共價標示。於電致化 學發光法(ECL )中,係藉由電流刺激以使發光分子或發 光錯合物發光。於螢光發光法中,係利用可引起發光的激 發光進行照射。 係使用偵測單元和用於提供激發光(該激發光之波長 可被螢光分子吸收)之照明光源來偵測螢光。該偵測單元 包含用於偵測發射信號的光感測器(例如,光電倍增管或 電荷耦合元件(CCD )陣列)及用於避免激發光被納入光 感測器輸出信號中的機構(例如,波長選擇性濾波器)。 該等螢光分子回應該激發光而釋放出斯托克斯位移( stokes shifted)光線,且藉由該偵測單元收集此釋放光。 斯托克斯位移係釋放光與所吸收的激發光之間的頻率差異 -16- 201209158 或波長差異。 係使用對所採用之EC L物種的發光波長靈敏的光感測 器偵測EC L發光作用。例如,過渡金屬-配體錯合物( transition metal-ligand complexes)釋放出可見波長之光 線,因此可採用習知的光二極體和電荷耦合元件(CCD ) 作爲光感測器。ECL的優點在於,若排除周遭環境之光線 時,ECL光線可能是出現於偵測系統內的唯一光線,從而The circular nucleic acid-mediated amplification (LAMP) provides high selectivity and employs a DNA polymerase and a set of four specially designed primers that recognize a total of six different sequences on the target DN A. LAMP is initiated with an internal primer containing the sequence of the syntactic and antisense strands of the target DNA. The subsequent strand displacement DNA synthesis reaction guided by the outer primer releases a single-stranded DNA. The single-stranded DNA system serves as a template for DNA synthesis reaction guided by the second inner primer and the second outer primer, and the second inner primer and the second outer primer are hybridized with the other end of the target to generate a dry - a stem-loop DNA structure. In the subsequent LAMP cycle, the inner primer is hybridized to the loop on the product and the pro-DNA synthesis reaction is initiated from -14 to 201209158 to obtain the original dry-loop enthalpy and one having a double stem length. New stem-cyclic DNA. This cyclic reaction continues at a rate that accumulates 1 to 9 target replicas in one hour. The final product is a 'target sequence with several inverted repeats, and the final product is cross-linked by the reverse repeating target sequences in the same chain to form a dry-loop of broccoli-like structure with multiple loops. Form DN A. After completion of the nucleic acid amplification reaction, it is necessary to analyze the amplification products to determine whether or not the expected amplicon (amplification amount of the target nucleic acid) is generated. The method of analyzing the product can range from simply judging the size of the amplicon by gel electrophoresis to identifying the nucleotide composition of the amplicon using the DN A hybrid method. Gel electrophoresis is one of the simplest methods for confirming whether a nucleic acid amplification process produces an expected enhancer. Gel electrophoresis separates DNA fragments by applying an electric field on a gel matrix. Negatively charged DNA fragments move through the matrix at different rates, which are primarily dependent on the size of the DNA fragments. After the electrophoresis is completed, the fragments in the gel can be dyed with -φ lines to see the fragments. It is usually dyed with ethidium bromide, which emits fluorescence under ultraviolet light. The DN Α size marker and the amplicon can be electrophoresed side by side on the gel, and the size of the fragments can be judged by comparison with DNA size markers (stepped DNA bands) containing DNA fragments of known size. Since the oligonucleotide primers bind to a specific position on both wings of the target DN A, the size of the amplification product can be predicted, and the amplification product is detected on the gel to exhibit a strip of a known size. band. In order to confirm the identity of the amplicon or if several amplicons are generated, the amplicon is usually subjected to DN A probe hybridization -15-201209158. DNA hybridization refers to the formation of double-stranded DNA by complementary base pairing. The DN A hybridization reaction for positive identification of specific amplification products requires the use of a DN A probe of about 20 nucleotides in length. If the probe has a sequence complementary to the amplicon (target) DN A sequence, a heterozygous reaction will occur under conditions of suitable temperature, pH 値 and ion concentration. If a heterozygous reaction occurs, it indicates that the gene of interest or the DN A sequence is present in the original sample. Optical detection is the most common method for detecting heterozygous reactions. Any of the amplicons or probes are labeled to emit light by means of fluorescence luminescence or electrochemiluminescence (ECL). The difference in these methods is that the means for generating the luminescent group in the excited state are different, but both methods can covalently label the nucleoside chain. In electrochemiluminescence (ECL), current is stimulated to cause luminescent molecules or luminescent complexes to illuminate. In the luminescence method, irradiation is performed by laser light which causes luminescence. The detection unit and the illumination source for providing excitation light (the wavelength of which is absorbed by the fluorescent molecules) are used to detect fluorescence. The detecting unit includes a photo sensor for detecting a transmitted signal (for example, a photomultiplier tube or a charge coupled device (CCD) array) and a mechanism for preventing the excitation light from being incorporated into the photosensor output signal (for example) , wavelength selective filter). The fluorescent molecules should be excited to emit light to release stokes shifted light, and the emitted light is collected by the detecting unit. The Stokes shift is the difference in frequency between the light emitted and the absorbed excitation light -16- 201209158 or wavelength difference. The EC L luminescence is detected using a light sensor that is sensitive to the luminescence wavelength of the EC L species employed. For example, transition metal-ligand complexes emit light of visible wavelengths, so conventional photodiodes and charge coupled devices (CCDs) can be used as photosensors. The advantage of ECL is that if the ambient light is excluded, the ECL light may be the only light that appears in the detection system, thus

增進靈敏度。 微陣列允許同時執行數十萬個DNA雜合實驗。微陣列 係具有於單次檢驗中篩檢數千種遺傳疾病或偵測諸多感染 性病源之潛力的強力工具。一個微陣列係由諸多呈點狀固 定於基板上的不同探針所組成。首先於核酸擴增反應期間 或之後使用螢光分子或發光分子標示該標靶DNA (擴增子 ),且隨後於探針陣列上施用該等經標示之標靶DNA (擴 增子)。於溫度受控制且潮濕的環境下培育該微陣列持續 數小時或數天,此期間探針與擴增子之間發生雜合反應。 經培育(incubation )之後,必需以一系列的緩衝液清洗 該微陣列以去除未結合的DNA鏈。清洗後,使用空氣氣流 (通常是氮氣)乾燥該微陣列。雜合反應與清洗的嚴苛度 非常重要。不夠嚴苛可能導致高度的非專一性結合。過份 嚴苛可能導致無法適當結合,從而降低靈敏度。可藉由偵 測源自已與互補探針形成雜合體之經標示擴增子所發出的 光線來識別雜合反應。 使用微陣列掃瞄器偵測源自微陣列的螢光,該微陣列 -17- 201209158 掃瞄器通常係一種受電腦控制之倒立掃描式共軛焦螢光顯 微鏡(inverted scanning fluorescence confocal microscpe ,該種顯微鏡一般使用雷射以激發螢光染料),且使用光 感測器(例如,光電倍增管或CCD )偵測發光信號。該等 螢光分子釋放出如上述之斯托克斯位移光,且藉由該偵測 單元收集該斯托克斯位移光。 所釋放之螢光必需加以收集、與未被吸收的激發波長 分離且傳送給該偵測器。於微陣列掃瞄器中,通常使用共 軛焦配置以藉由於成像平面處設置共軛焦針孔的方式消除 失焦資訊。此方式允許僅會測得光線中的聚焦部分。源自 目標物之聚焦平面上方與下方的光線無法進入該偵測器, 從而提高信號雜訊比(signal to noise ratio )。利用該偵 測器把測得的螢光光子轉換成電能,且隨後把該電能轉換 成數位信號。此數位信號轉譯成代表源自指定像素之螢光 強度的數字。該陣列的每個特徵係由一個或一個以上的此 類像素所組成。掃描的最終結果係該陣列表面的影像。該 微陣列上每種探針的確切序列與位置係已知,因而可同時 鑑定與分析該等經雜合的標靶序列。 於下列網址可獲得有關螢光探針之更多資訊: http://www.premierbiosoft.com/tech_notes/FRET_probe.ht ml ;及 http://www.invitrogen.com/site/us/en/home/References/Improve sensitivity. Microarrays allow hundreds of thousands of DNA hybrid experiments to be performed simultaneously. Microarrays have powerful tools for screening thousands of genetic diseases or detecting the potential of many infectious agents in a single test. A microarray consists of a number of different probes that are spotted on a substrate. The target DNA (amplicon) is first labeled with a fluorescent molecule or a luminescent molecule during or after the nucleic acid amplification reaction, and then the labeled target DNA (amplifier) is subsequently applied to the probe array. The microarray is incubated for a period of hours or days in a temperature controlled and humid environment during which a heterozygous reaction occurs between the probe and the amplicon. After incubation, the microarray must be washed with a series of buffers to remove unbound DNA strands. After cleaning, the microarray is dried using an air stream (usually nitrogen). The severity of the heterozygous reaction and cleaning is very important. Not being harsh enough can lead to a high degree of non-specificity. Excessive rigor may result in inability to properly combine to reduce sensitivity. The heterozygous reaction can be identified by detecting the light emanating from the labeled amplicon that has formed a hybrid with the complementary probe. Fluorescence from a microarray is detected using a microarray scanner, which is typically a computer-controlled inverted scanning fluorescence confocal microscpe (inverted scanning fluorescence confocal microscpe) A laser is typically used to excite the fluorescent dye) and a light sensor (eg, a photomultiplier tube or CCD) is used to detect the luminescent signal. The fluorescent molecules emit Stokes shifted light as described above, and the Stokes shifted light is collected by the detecting unit. The released fluorescence must be collected, separated from the unabsorbed excitation wavelength and transmitted to the detector. In microarray scanners, a conjugate focal configuration is typically used to eliminate out-of-focus information by placing conjugated focal pinholes at the imaging plane. This mode allows only the focused portion of the light to be measured. Light from above and below the focus plane of the target cannot enter the detector, increasing the signal to noise ratio. The detector is used to convert the measured fluorescent photons into electrical energy and then convert the electrical energy into a digital signal. This digital signal is translated into a number representing the intensity of the fluorescence from the specified pixel. Each feature of the array consists of one or more such pixels. The final result of the scan is an image of the surface of the array. The exact sequence and position of each probe on the microarray is known so that the heterozygous target sequences can be identified and analyzed simultaneously. More information on fluorescent probes is available at: http://www.premierbiosoft.com/tech_notes/FRET_probe.ht ml ; and http://www.invitrogen.com/site/us/en/home /References/

Molecular-Probes-The-Handbook/Technical-Notes-and-Molecular-Probes-The-Handbook/Technical-Notes-and-

Product-Highlights/Fluorescence-Resonance-Energy- -18- 201209158Product-Highlights/Fluorescence-Resonance-Energy- -18- 201209158

Transfer-FRET.html。 重點照護之分子診斷 盡管分子診斷檢驗提供諸多優點,然而此種檢驗在臨 床檢驗上的發展低於預期且餘留在檢驗醫學上的少量實務 應用。導致此種情況的原因主要在於相較於依賴未涉及核 酸之方法的檢驗而言,核酸檢驗較複雜且相,費用較高。 分子診斷檢驗在臨床處置上的普遍適應性與儀器設備的發 展(顯著降低成本、從開始(樣本處理)到結束(生成結 果)提供快速且自動化之分析化驗及可於無人爲主要干預 下運作)息息相關。 重點照護技術用於診所、醫院臨床或甚至消費者在家 使用可帶來諸多優點,包括: •快速獲得結果而能利於做即時處理且增進照護品質Transfer-FRET.html. Molecular Diagnostics for Key Care Although molecular diagnostic tests offer many advantages, the development of such tests in clinical testing is less than expected and remains a small practical application of laboratory medicine. The main reason for this is that nucleic acid testing is more complicated and costly than testing that relies on methods that do not involve nucleic acids. The universal adaptability of molecular diagnostic tests in clinical disposition and the development of instruments and equipment (significantly reducing costs, providing rapid and automated analytical assays from the beginning (sample processing) to the end (generating results) and can operate without human intervention) It is closely related. Key care technologies for use in clinics, hospital clinics, or even consumers at home can bring many benefits, including: • Quick results to facilitate immediate treatment and improved care quality

•能藉由檢驗極少的樣本而獲得檢驗値。 •減少臨床工作量》 •藉由減少行政工作以減少檢驗工作量且增進辦公效 率。 •透過縮短住院時間長度、總結初診之門診病患的諮 詢及減少樣本之處理、儲存和運送過程而改善每位病患的 費用。 •幫助做出諸如感染控制及抗生素使用之臨床管理決 策0 -19- 201209158 以晶片上實驗室(LOC )爲基礎之分子診斷 以微流體技術爲基礎之分子診斷系統提供一種可用於 自動化且加速分子診斷分析化驗的工具。此系統具有較快 偵測時間之主要原因在於可於微流體裝置內進行極小體積 、自動化且經常性費用低廉之內建式串接裝置的診斷方法 步驟。毫微升和微升規模的體積亦減少試劑消耗與費用。 晶片上實驗室(LOC )裝置係微流體裝置之常見形式。 LOC裝置具有整合於單一載體基板(通常是矽)上以用於 流體處理之MS T層內的多個MS T結構。使用半導體工業之 超大型積體電路(VSLI)微影技術製造該L0C裝置可使每 個LOC裝置保持極低的單位成本。然而,爲了控制流體流 經該LOC裝置、添加試劑、控制反應條件及進行諸如此類 之作業需要龐大的外部管線系統和電子設備。把LOC裝置 有效地連接至此等外部裝置會限制L 0 C裝置在實驗室環境 中於分子診斷方面的用途。外部設備的費用與該等設備運 作之複雜度阻礙了 L0C式分子診斷學作爲用於重點照護環 境的實用選項。 鑒於上述理由’需要一種可用於重點照護之以晶片上 實驗室(L0C )裝置爲基礎的分子診療系統。 【發明內容】 現將於下述諸多段落中描述本發明之各種體系。 GCF011.1本發明之此體系提供—種用於生物樣本之 -20- 201209158 基因分析的晶片上實驗室(L〇c)裝置,該L〇c裝置包含 用於接收該樣本之入口; 載體基板;• Ability to obtain inspections by examining very few samples. • Reduce clinical workload • Reduce administrative workload and increase office efficiency by reducing administrative work. • Improve the cost per patient by reducing the length of hospital stay, summarizing the consultations of newly diagnosed outpatients, and reducing the handling, storage, and delivery of samples. • Helps make clinical management decisions such as infection control and antibiotic use. 0 -19- 201209158 On-wafer laboratory (LOC)-based molecular diagnostics Microfluidic-based molecular diagnostic systems provide an automated and accelerated molecule A tool for diagnostic analysis testing. The primary reason for the faster detection time of this system is the diagnostic method steps of a built-in tandem device that can be implemented in a microfluidic device with minimal volume, automation, and low cost. The volume of nanoliters and microliters also reduces reagent consumption and cost. On-wafer laboratory (LOC) devices are a common form of microfluidic devices. The LOC device has a plurality of MS T structures integrated into a single carrier substrate (typically helium) for use in fluid processing of the MS T layer. Fabrication of the LOC device using the ultra-large integrated circuit (VSLI) lithography technology of the semiconductor industry allows each LOC device to maintain an extremely low unit cost. However, large external piping systems and electronics are required to control the flow of fluid through the LOC unit, to add reagents, to control reaction conditions, and to perform such operations. Efficiently connecting the LOC device to such external devices limits the use of the L0C device for molecular diagnostics in a laboratory environment. The cost of external equipment and the complexity of the operation of such equipment hamper L0C molecular diagnostics as a practical option for a focused care environment. For the above reasons, there is a need for a molecular diagnostic system based on a wafer-on-a-lab (L0C) device that can be used for focused care. SUMMARY OF THE INVENTION Various systems of the present invention will now be described in the following paragraphs. GCF011.1 The system of the present invention provides a wafer-on-lab (L〇c) device for biological analysis of -20-201209158 gene analysis, the L〇c device comprising an inlet for receiving the sample; a carrier substrate ;

透析區段,該透析區段係使該樣本中尺寸大於預定臨 界値之細胞與較小成分分離,其中該等尺寸大於預定臨界 値之細胞包括含有用於分析之遺傳物質的標靶細胞; 複數個試劑貯存槽; 溶胞區段,該溶胞區段位於該透析區段下游且用於溶 解該等標靶細胞以釋出細胞內的遺傳物質,該溶胞區段係 與該等含有用於溶解該溶胞區段內之標靶細胞之溶胞試劑 的試劑貯存槽之一者流體連通; 培育區段,該培育區段位於該溶胞區段下游,且該培 育區段係與該等含有用於與遺傳物質進行酶催化反應之多 種酶的試劑貯存槽之一者流體連通:及 核酸擴增區段,該核酸擴增區段位於該培育區段下游 以用於從遺傳物質中擴增核酸序列;其中 其中透析區段、溶胞區段、培育區段及核酸擴增區段 係全部承載於載體基板上。 GCF01 1 .2較佳地,該第一核酸擴增區段係聚合酶鏈 鎖反應(PCR)區段。 GCF0 11.3較佳地,該LOC裝置亦包含雜合區段,該 雜合區段位於該PCR區段下游,且該雜合區段具有探針陣 列及光感測器,該探針陣列係用於與樣本中之標靶核酸序 -21 - 201209158 列雜合,且該光感測器係用於偵測該陣列中之任何探針的 雜合反應。 GCF0 1 1.4較佳地,該透析區段具有第一通道、第二 通道和複數個孔,該第一通道係與位於上游末端的入口流 體連通,該第二通道係與位於下游端的廢液通道流體連通 ,且該等孔小於該等標靶細胞且大於該等較小成分,第二 通道係藉由該等孔與第一通道流體連通,使得該等標靶細 胞留在第一通道內且同時該等較小成分流入第二通道 GCF01 1 .5較佳地,該第一通道和該第二通道係經建 構以藉由毛細作用而注滿樣本。 GCF0 11.6較佳地,該溶胞區段具有主動閥,該主動 閥係於溶胞期間使標靶細胞留在溶胞區段內,使得當打開 該主動閥時,流向培育區段的毛細驅動流體得以繼續。 GCF0 1 1 _7較佳地,該核酸擴增區段係恆溫核酸擴增 區段。 GCF011.8較佳地,該等試劑貯存槽各自具有使試劑 停留於該等試劑貯存槽內的表面張力閥,該表面張力閥具 有彎液面鋪,該彎液面錨係用於定住(p i η n i n g )試劑之彎 液面直到該試劑之彎液面與該樣本流接觸而去除該彎液面 以允許試劑從試劑貯存槽流出。 GCF011.9較佳地,該LOC裝置亦具有從入口到雜合 區段的流動路徑,其中該流動路徑係經建構以藉由毛細作 用把樣本從該入口引至該雜合區段。 GCF011.10較佳地,該LOC裝置亦具有互補金屬氧化 -22- 201209158 物半導體(CMOS )電路、溫度感測器及微系統技術( MST)層,該MST層包含該PCR區段,其中該CMOS電路係 設置於該載體基板和該MST層之間,該CMOS電路係經建 構以使用該溫度感測器之輸出達成該PC R區段的反饋控制 〇 GCF011.il較佳地,該PCR區段具有PCR微通道,該 PCR微通道係用於使該樣本進行熱循環以擴增核酸序列, ^ 該PCR微通道界定部份的樣本流動路徑,該PCR微通道具 有低於1 000 00平方微米的流動橫斷截面積。 GCF01 1.12較佳地,該LOC裝置亦具有至少一個長形 加熱器元件以用於加熱該長形PCR微通道中的核酸序列, 該長形加熱器元件係與該PCR微通道成平行地延伸。 GCF011.13較佳地,該PCR微通道之至少一個區段形 成長形PCR腔室。 GCF011.14較佳地,該PCR區段具有複數個長形PCR 腔室’且每個長形PCR腔室係由該PCR微通道之個別區段 . 所形成,該PCR微通道具有由一系列寬曲流道所形成之蜿 蜒構形,且每個寬曲流道係一個用於形成該等長形PCR腔 室之一者的通道區段。 GCF0 11.15較佳地,該培育區段具有加熱器以加熱 該遺傳物質和多種酶達預定之酶催化反應溫度。 GCF011.16較佳地,該LOC裝置亦包含用於容納探針 的雜合腔室陣列,使得每個雜合腔室內的該等探針係經配 置以與該等標靶核酸序列之一者雜合。 -23- 201209158 GCFO 1 1 .1 7較佳地,該光感測器係配 室設置而成的光二極體陣列。 GCFO 1 1.18 較佳地,該CMOS電路具有 數據界面,該數位記憶體係用於儲存源自該 出的雜合數據,且該數據界面係把該雜合數 裝置。 GCF011.19較佳地,該PCR區段具有主 閥係於熱循環期間使液體保留在該PCR區段 該CMOS電路之啓動信號而允許液體流向該夸 GCF01 1 .20較佳地,該主動閥係沸騰 沸騰啓動式閥具有彎液面錨及加熱器,該彎 成用於定住該阻止液體之毛細驅動流動的彎 熱器係用於使該液體沸騰以使該彎液面脫離 使得毛細驅動流動得以繼續。 該使用簡易、可量產且費用不高的基因 置係藉由該LOC裝置之樣本放置槽接收生物 儲存於該LOC裝置之試劑貯存槽內的試劑使 之透析區段分離樣本中所含的白血球、於該 學溶胞腔室中溶解白血球以釋出白血球的遺 LOC裝置之培育區段中對樣本之遺傳物質進 增標靶基因序列,及藉著與寡聚核苷酸探針 由該LOC裝置之整合式成像陣列(integral )感測該等探針之雜合反應以分析樣本之核 該透析區段之功能係從樣本中萃取附力口 準該等雜合腔 數位記憶體及 光感測器之輸 據傳輸給外部 動閥,該主動 內且回應源自 声雜合腔室。 啓動式閥,該 液面錨經建構 液面,且該加 該彎液面錨, I體分析LOC裝 樣本,且利用 用該LOC裝置 :LOC裝置之化 傳物質、於該 行預處理、擴 進行雜合且藉 imaging array 酸序列。 訊息且提高分 -24- 201209158 析檢驗系統之靈敏度、信號雜訊比和動態範圍。整合於裝 置中的透析區段可提供低的系統元件數量和簡約的製造程 序,從而獲得不昂貴的分析檢驗系統。 該溶胞方法係自樣本中的細胞萃取出分析和診斷之標 靶物且提供該等標靶物之連續性處理與分析。整合於該裝 置中的溶胞子單元可提供簡約的分析檢驗程序、低的系統 元件數量及簡約的系統製造程序,從而獲得不昂貴的分析a dialysis section, wherein the cells in the sample having a size greater than a predetermined threshold are separated from the smaller component, wherein the cells having a size greater than a predetermined threshold include target cells containing genetic material for analysis; a reagent storage tank; a lysis section located downstream of the dialysis section and configured to dissolve the target cells to release genetic material in the cell, the lysing section and the containing One of the reagent storage tanks for dissolving the lysis reagent of the target cells in the lysing section is in fluid communication; the incubation section is located downstream of the lysis section, and the cultivating section is And one of the reagent storage tanks containing a plurality of enzymes for enzymatically reacting with the genetic material: and a nucleic acid amplification section located downstream of the incubation section for use in genetic material The nucleic acid sequence is amplified; wherein the dialysis section, the lysis section, the incubation section, and the nucleic acid amplification section are all carried on the carrier substrate. GCF01 1.2 Preferably, the first nucleic acid amplification segment is a polymerase chain reaction (PCR) segment. GCF0 11.3 Preferably, the LOC device further comprises a hybrid segment located downstream of the PCR segment, and the hybrid segment has a probe array and a photo sensor, the probe array is used Hybridization with the target nucleic acid sequence-21 - 201209158 in the sample, and the photosensor is used to detect the heterozygous reaction of any of the probes in the array. GCF0 1 1.4 Preferably, the dialysis section has a first passage, a second passage and a plurality of holes, the first passage being in fluid communication with an inlet at an upstream end, the second passage being connected to a waste liquid passage at a downstream end Fluidly connected, and the pores are smaller than the target cells and larger than the smaller components, and the second channel is in fluid communication with the first channel by the pores such that the target cells remain in the first channel and At the same time, the smaller components flow into the second channel GCF01 1.5. Preferably, the first channel and the second channel are constructed to fill the sample by capillary action. GCF0 11.6 Preferably, the lysis zone has an active valve that retains the target cells within the lysis section during lysis so that when the active valve is opened, capillary drive to the incubation section The fluid can continue. GCF0 1 1 _7 Preferably, the nucleic acid amplification segment is a thermostatic nucleic acid amplification segment. GCF011.8 Preferably, the reagent storage tanks each have a surface tension valve for allowing the reagent to stay in the reagent storage tank, the surface tension valve having a meniscus, the meniscus anchor system for holding (pi The meniscus of the reagent is removed until the meniscus of the reagent contacts the sample stream to remove the meniscus to allow reagent to flow out of the reagent reservoir. GCF 011.9 Preferably, the LOC device also has a flow path from the inlet to the hybrid section, wherein the flow path is configured to direct the sample from the inlet to the hybrid section by capillary action. Preferably, the LOC device also has a complementary metal oxide-22-201209158 semiconductor (CMOS) circuit, a temperature sensor and a microsystem technology (MST) layer, the MST layer comprising the PCR segment, wherein the a CMOS circuit is disposed between the carrier substrate and the MST layer, the CMOS circuit being configured to achieve feedback control of the PC R segment using the output of the temperature sensor 〇GCF011.il preferably, the PCR region The segment has a PCR microchannel for thermal cycling of the sample to amplify the nucleic acid sequence, ^ the PCR microchannel defines a portion of the sample flow path, the PCR microchannel having less than 1 000 square microns The flow cross-sectional area. GCF01 1.12 Preferably, the LOC device also has at least one elongate heater element for heating the nucleic acid sequence in the elongate PCR microchannel, the elongate heater element extending parallel to the PCR microchannel. Preferably, at least one segment of the PCR microchannel is shaped into a PCR chamber. Preferably, the PCR segment has a plurality of elongate PCR chambers and each elongate PCR chamber is formed by an individual segment of the PCR microchannel having a series of A wide curved channel formed by a meandering configuration, and each wide curved channel is a channel section for forming one of the elongate PCR chambers. GCF0 11.15 Preferably, the incubation section has a heater to heat the genetic material and the plurality of enzymes to a predetermined enzyme catalytic reaction temperature. GCF011.16 preferably, the LOC device also includes an array of hybrid chambers for housing probes such that the probes within each hybrid chamber are configured to interact with one of the target nucleic acid sequences mixed. -23- 201209158 GCFO 1 1 .1 7 Preferably, the photosensor is an array of photodiodes arranged in a compartment. GCFO 1 1.18 Preferably, the CMOS circuit has a data interface for storing the hybrid data originating from the output, and the data interface is the heterogeneous device. Preferably, the PCR section has a main valve that maintains a liquid during the thermal cycle to maintain a start signal of the CMOS circuit in the PCR section to allow liquid to flow to the boast GCF01 1.20 preferably, the active valve The boiling boiling start valve has a meniscus anchor and a heater, and the curved heat exchanger for holding the capillary driving flow for blocking the liquid is used to boil the liquid to disengage the meniscus so that the capillary drive flows Can continue. The easy-to-use, mass-produced and low-cost gene set receives the white blood cells contained in the dialysis section separation sample by receiving the reagent stored in the reagent storage tank of the LOC device by the sample placement tank of the LOC device. The genetic material of the sample in the culture section of the LOC device that dissolves the white blood cells in the lysis chamber to release the white blood cells, and the target gene sequence is amplified by the LOC. The integrated imaging array of the device senses the hybrid reaction of the probes to analyze the core of the sample. The function of the dialysis section extracts the attached force from the sample. The hybrid cavity digital memory and light perception The data of the detector is transmitted to the external moving valve, and the response is derived from the acoustic hybrid chamber. The starter valve, the liquid level anchor is constructed through the liquid level, and the meniscus anchor is added, and the LOC is loaded with the sample, and the material is transferred by the LOC device: the LOC device, and the line is pretreated and expanded. Hybrid and borrow the acid sequence of the imaging array. Message and improve the score -24- 201209158 Analysis of the sensitivity, signal noise ratio and dynamic range of the inspection system. The dialysis section integrated into the unit provides a low number of system components and a simple manufacturing process, resulting in an inexpensive analytical inspection system. The lysis method extracts analytical and diagnostic targets from cells in the sample and provides continuity processing and analysis of the targets. The lysing subunit integrated in the unit provides a simple analytical test procedure, low system component count and simple system manufacturing procedures for inexpensive analysis

檢驗系統。 於培育區段中,遺傳物質接受各種預處理,例如DNA 限制剪切及轉接子引子之接合反應,藉以爲後續分析階段 提供最適宜或必要之條件、提高分析結果的訊息含量且提 高該分析檢驗系統之靈敏度、信號雜訊比和可靠度。 標靶基因序列之擴增反應可提高該分析檢驗系統之靈 敏度與信號雜訊比。 探針雜合區段係藉由提供雜合反應達成該等標靶物之 分析。整合式探針雜合區段提供一種使用簡易、可量產、 不昂貴且具有低系統元件數量的整合式解決方案。 該整合式成像感測器免除了對昂貴之外部成像系統的 需求且提供具有低系統元件數量、可量產且不昂貴的整合 式解決方案,此整合式解決方案係一種小巧、質輕且極方 便攜帶之系統。該整合式成像感測器因受益於大的光線收 集角度而提高讀取靈敏度且免除在光學收集系統中使用諸 多光學元件之需求。 整合於該LOC裝置中且把持該分析檢驗之所有試劑需 -25- 201209158 求的該等試劑貯存槽可提供低的系統元件數量和簡約的製 造程序,從而獲得不昂貴的分析檢驗系統。 GCF 0 1 2.1本發明之此體系提供一種用於生物樣本之 基因分析的晶片上實驗室(LOC )裝置,該LOC裝置包含 用於接收該樣本之入口; 載體基板; 透析區段,該透析區段係使該樣本中尺寸大於預定臨 界値之細胞與較小成分分離,其中該等尺寸大於預定臨界 値之細胞包含含有用於分析之遺傳物質的標靶細胞; 複數個試劑貯存槽; 溶胞區段,該溶胞區段位於該透析區段下游且用於溶 解該等標靶細胞以釋出細胞內的遺傳物質,該溶胞區段係 與該等含有用於溶解溶胞區段內之標靶細胞之溶胞試劑的 試劑貯存槽之一者流體連通: 培育區段,該培育區段位於該溶胞區段下游,且該培 育區段係與該等含有用於與該遺傳物質進行酶催化反應之 多種酶的試劑貯存槽之—者流體連通; 第一核酸擴增區段,該第一核酸擴增區段位於該培育 區段下游以用於擴增該遺傳物質中的核酸序列;及 第二核酸擴增區段,該第二核酸擴增區段係位於該培 育區段下游以用於與該第一核酸擴增區段並行地擴增該遺 傳物質中的核酸序列;其中, 該透析區段、該溶胞區段、該培育區段 '該第一核酸 -26- 201209158 擴增區段和該第二核酸擴增區段皆承載於該載體基板上。 GCF0 12.2較佳地,該第一核酸擴增區段係第一聚合 酶鏈鎖反應(PCR)區段,且該二核酸擴增區段係第二聚 合酶鏈鎖反應(PCR)區段。 GCF012.3較佳地,該第一PCR區段具有第一組引子 對且該第一組引子對係用於黏合第一組互補核酸序列,及 該第二PCR區段具有第二組引子對且該第二組引子對係用 於黏合第二組互補核酸序列,該第一組互補核酸序列與該 第二組互補核酸序列不同。 GCF012.4 較佳地,該第一PCR區段和該第二PCR區 段係經建構而可用不同擴增參數進行操作,該等擴增參數 係下列參數之至少一者: 反轉錄酶種類; 聚合酶種類; 去氧核糖核苷三磷酸濃度;Inspection system. In the culturing section, the genetic material undergoes various pretreatments, such as DNA restriction shearing and conjugation of the primers, to provide the most appropriate or necessary conditions for subsequent analysis stages, to increase the message content of the analysis results, and to improve the analysis. Verify system sensitivity, signal-to-noise ratio, and reliability. The amplification reaction of the target gene sequence can increase the sensitivity and signal to noise ratio of the analytical test system. The probe hybrid segment achieves analysis of the targets by providing a hybrid reaction. The integrated probe hybrid section provides an integrated solution that is easy to use, mass-produced, inexpensive, and has a low number of system components. The integrated imaging sensor eliminates the need for expensive external imaging systems and offers an integrated solution with low system component count, mass production and low cost. This integrated solution is compact, lightweight and extremely compact. Easy to carry system. The integrated imaging sensor increases read sensitivity and eliminates the need to use multiple optical components in an optical collection system by benefiting from large light collection angles. The reagent storage tanks that are integrated into the LOC device and that hold all of the reagents for the analysis require -25-201209158 provide a low system component count and a simple manufacturing procedure, resulting in an inexpensive analytical inspection system. GCF 0 1 2.1 This system of the invention provides a wafer-on-lab (LOC) device for genetic analysis of a biological sample, the LOC device comprising an inlet for receiving the sample; a carrier substrate; a dialysis section, the dialysis zone The segment separates cells of the sample having a size greater than a predetermined threshold 与 from the smaller component, wherein the cells having a size greater than the predetermined threshold 包含 comprise target cells containing genetic material for analysis; a plurality of reagent storage tanks; a segment, the lysis segment being located downstream of the dialysis section and for dissolving the target cells to release genetic material within the cell, the lysing segment being associated with the lysing segment One of the reagent storage tanks of the lysis reagent of the target cell is in fluid communication: a cultivation section, the cultivation section is located downstream of the lysis section, and the cultivation section is associated with the genetic material a reagent storage tank for performing a plurality of enzymes for enzymatically catalyzing fluid communication; a first nucleic acid amplification section, the first nucleic acid amplification section being located downstream of the incubation section for amplifying the genetic material And a second nucleic acid amplification segment downstream of the incubation segment for amplifying the genetic material in parallel with the first nucleic acid amplification segment a nucleic acid sequence; wherein the dialysis section, the lysis section, the incubation section 'the first nucleic acid-26-201209158 amplification section and the second nucleic acid amplification section are all carried on the carrier substrate. GCF0 12.2 Preferably, the first nucleic acid amplification segment is a first polymerase chain reaction (PCR) segment and the second nucleic acid amplification segment is a second polymerase chain reaction (PCR) segment. Preferably, the first PCR segment has a first set of primer pairs and the first set of primer pairs is used to bind the first set of complementary nucleic acid sequences, and the second PCR segment has a second set of primer pairs. And the second set of primer pairs is used to bind a second set of complementary nucleic acid sequences that differ from the second set of complementary nucleic acid sequences. GCF012.4 Preferably, the first PCR segment and the second PCR segment are constructed and operable with different amplification parameters, the amplification parameters being at least one of the following parameters: a reverse transcriptase species; Polymerase species; concentration of deoxyribonucleoside triphosphate;

熱循環時間; 熱循環重複次數;及 於PCR之一特定階段期間內的溫度。 GCF012.5 較佳地,該LOC裝置亦包含第一雜合區段 和第二雜合區段及光感測器,該第一雜合區段位於該第一 PCR區段下游且該第一雜合區段具有用於與第一標靶核酸 序列雜合的第一探針陣列,及該第二雜合區段位於第二 PCR區段下游且該第二雜合區段具有用於與第二標靶核酸 -27- 201209158 序列雜合的第二探針陣列,及該光感測器係用於偵測該第 一陣列和第二陣列中之任何探針的雜合反應。 GCF0 12.6較佳地,該透析區段具有第一通道、第二 通道和複數個孔,該第一通道係與位於上游末端的入口流 體連通,該第二通道係與位於下游端的廢液通道流體連通 ’且該等孔小於該等標祀細胞且大於該等較小成分’該第 二通道係藉由該等孔與該第一通道流體連通,使得該等標 靶細胞留在第一通道內且同時該等較小成分流入第二通道 GCF012.7較佳地,該第一通道和該第二通道係經建 構以藉由毛細作用而注滿樣本。 GCF 0 1 2.8較佳地,該溶胞區段具有主動閥’該主動 閥係於溶胞期間使標靶細胞留在溶胞區段內,使得當打開 該主動閥時,流向培育區段的毛細驅動流體得以繼續。 GCF012.9較佳地,該第一核酸擴增區段係第一恆溫 核酸擴增區段,且該第二核酸擴增區段係第二恆溫核酸擴 增區段。 GCF012.10較佳地,該等試劑貯存槽各自具有用於 使試劑留在該等試劑貯存槽內的表面張力閥,該表面張力 閥具有彎液面錨,該彎液面錨係用於定住試劑之彎液面直 到該試劑之彎液面與該樣本流體接觸而去除該彎液面以允 許試劑從試劑貯存槽流出。 GCF012.il較佳地,該LOC裝置亦具有互補金屬氧化 物半導體(CMOS )電路、溫度感測器及微系統技術( -28- 201209158 MST)層,該MST層包含該第一 PCR區段和該第二PCR區 段,其中該CMOS電路係設置於該載體基板和該MST層之 間,該CMOS電路係經建構以使用該溫度感測器之輸出達 成該第一 PCR區段和該第二PCR區段之反饋控制。 GCF012.12較佳地,該PCR區段具有用於使該樣本進 行熱循環的PCR微通道,該PCR微通道界定具有低於 1 00000平方微米之流動橫斷截面積的流動路徑。 GCF012.13 較佳地,該PCR微通道具有至少一個長形 加熱器元件,該長形加熱器元件係與該PCR微通道成平行 地延伸。 GCF012.14較佳地,該PCR區段具有複數個長形PCR 腔室,且每個長形PCR腔室係由該PCR微通道之個別區段 形成,該PCR微通道具有由一系列寬曲流道所形成之蜿蜒 構形,且每個寬曲流道係一個用於形成該等長形PCR腔室 之一者的通道區段。Thermal cycle time; number of thermal cycle repetitions; and temperature during a particular phase of PCR. Preferably, the LOC device also includes a first hybrid segment and a second hybrid segment and a photo sensor, the first hybrid segment being located downstream of the first PCR segment and the first hybrid The segment has a first probe array for hybridization with the first target nucleic acid sequence, and the second hybrid segment is located downstream of the second PCR segment and the second hybrid segment has a second and second hybrid segment Target Nucleic Acid-27-201209158 A heterozygous second probe array, and the photosensor is used to detect the heterozygous reaction of any of the probes in the first array and the second array. GCF0 12.6 Preferably, the dialysis section has a first passage, a second passage and a plurality of orifices, the first passage being in fluid communication with an inlet at an upstream end, the second passage being associated with a waste liquid passage at a downstream end Connecting 'and the holes are smaller than the target cells and larger than the smaller components'. The second channel is in fluid communication with the first channel by the holes such that the target cells remain in the first channel At the same time, the smaller components flow into the second channel GCF 012.7. Preferably, the first channel and the second channel are configured to fill the sample by capillary action. GCF 0 1 2.8 Preferably, the lysis section has an active valve that retains the target cells within the lysis section during lysis, such that when the active valve is opened, flow to the incubation section The capillary drive fluid continues. Preferably, the first nucleic acid amplification segment is a first thermostatic nucleic acid amplification segment and the second nucleic acid amplification segment is a second thermostatic nucleic acid amplification segment. Preferably, the reagent storage tanks each have a surface tension valve for retaining reagents in the reagent storage tanks, the surface tension valve having a meniscus anchor, the meniscus anchor system for holding The meniscus of the reagent is removed until the meniscus of the reagent contacts the sample fluid to remove the meniscus to allow reagent to flow out of the reagent reservoir. GCF012.il preferably, the LOC device also has a complementary metal oxide semiconductor (CMOS) circuit, a temperature sensor and a microsystem technology (-28-201209158 MST) layer, the MST layer comprising the first PCR segment and The second PCR segment, wherein the CMOS circuit is disposed between the carrier substrate and the MST layer, the CMOS circuit being configured to use the output of the temperature sensor to achieve the first PCR segment and the second Feedback control of the PCR segment. Preferably, the PCR segment has a PCR microchannel for thermal cycling of the sample, the PCR microchannel defining a flow path having a cross-sectional cross-sectional area of less than 100,000 square microns. GCF012.13 Preferably, the PCR microchannel has at least one elongate heater element extending parallel to the PCR microchannel. Preferably, the PCR segment has a plurality of elongate PCR chambers, and each elongate PCR chamber is formed by individual segments of the PCR microchannel having a series of wide curves The channel formed by the flow path, and each wide curved channel is a channel section for forming one of the elongate PCR chambers.

GCF0 12.15較佳地,該培育區段具有加熱器以加熱 該遺傳物質和酶達預定之酶催化反應溫度。 GCF012.16較佳地,該L0C裝置亦包含用於容納該等 第一探針的第一雜合腔室陣列,使得每個雜合腔室內的該 等第一探針係經配置以與該等第一標靶核酸序列之一者雜 合。 GCF012.17較佳地,該光感測器係配準該等雜合腔 室設置而成的光二極體陣列。 GCF0 12.18較佳地,該CMOS電路具有數位記億體及 -29- 201209158 數據界面,該數位記憶體係用於儲存源自該光感測器之輸 出的雜合數據,且該數據界面係傳輸該雜合數據至外部裝 置。 GCF012.19較佳地,該第一PCR區段具有主動閥,該 主動閥係於熱循環期間使液體保留在該第一 PC R區段內且 回應源自該CMOS電路之啓動信號而允許液體流至該第一 雜合腔室陣列。 GCF012.20 較佳地,該主動閥係沸騰啓動式閥,該 沸騰啓動式閥具有彎液面錨及加熱器,該彎液面錨係經建 構以定住該阻止液體之毛細驅動流動的彎液面,且該加熱 器係用於使該液體沸騰以使彎液面脫離該彎液面錨,使得 毛細驅動流動得以繼續。 該使用簡易、可量產且費用不高的基因體分析LOC裝 置係藉由該LOC裝置之樣本放置槽接收生物樣本,且利用 儲存於該LOC裝置之試劑貯存槽內的試劑使用該LOC裝置 之透析區段分離樣本中所含的白血球、於該LOC裝置之化 學溶胞腔室中溶解白血球以釋出白血球之遺傳物質、於該 LOC裝置之培育區段中對樣本之遺傳物質進行預處理、擴 增標靶基因序列,及藉著與寡聚核苷酸探針進行雜合且藉 由該L Ο C裝置之整合式成像陣列感測該等探針之雜合反應 以分析樣本之核酸序列。 該透析區段之功能係從樣本中萃取附加訊息且提高分 析檢驗系統之靈敏度、信號雜訊比和動態範圍。整合於裝 置中的透析區段可提供低的系統元件數量和簡約的製造程 -30- 201209158 序’從而獲得不昂貴的分析檢驗系統。 該溶胞方法係自樣本中的細胞萃取出分析和診斷之標 靶物且提供該等標靶物之連續性處理與分析。整合於該裝 置中的溶胞子單元可提供簡約的分析檢驗程序、低的系統 元件數量及簡約的系統製造程序,從而獲得不昂貴的分析 檢驗系統。GCF0 12.15 Preferably, the incubation section has a heater to heat the genetic material and enzyme to a predetermined enzyme catalyzed reaction temperature. Preferably, the LOC device also includes a first hybrid chamber array for accommodating the first probes such that the first probes within each hybrid chamber are configured to One of the first target nucleic acid sequences is heterozygous. Preferably, the photosensor is associated with an array of photodiodes arranged in the hybrid chambers. GCF0 12.18 Preferably, the CMOS circuit has a digital interface and a -29-201209158 data interface, the digital memory system is configured to store the hybrid data originating from the output of the photo sensor, and the data interface transmits the Hybrid data to an external device. GCF012.19 Preferably, the first PCR section has an active valve that retains liquid within the first PC R section during thermal cycling and allows liquid in response to an activation signal originating from the CMOS circuit Flow to the first hybrid chamber array. GCF012.20 Preferably, the active valve is a boiling start valve having a meniscus anchor and a heater, the meniscus anchor being constructed to hold the meniscus that prevents the capillary drive flow of the liquid And the heater is used to boil the liquid to disengage the meniscus from the meniscus anchor so that the capillary drive flow continues. The genomic analysis LOC device that is simple to use, mass-produced, and inexpensive is to receive a biological sample by using a sample placement tank of the LOC device, and using the reagent stored in the reagent storage tank of the LOC device to use the LOC device. The dialysis section separates the white blood cells contained in the sample, dissolves the white blood cells in the chemical lysis chamber of the LOC device to release the genetic material of the white blood cells, and pretreats the genetic material of the sample in the incubation section of the LOC device, Amplifying the target gene sequence and analyzing the nucleic acid sequence of the sample by heterozygous with the oligonucleotide probe and sensing the heterozygous reaction of the probes by the integrated imaging array of the L Ο C device . The function of the dialysis section is to extract additional information from the sample and to increase the sensitivity, signal to noise ratio, and dynamic range of the analytical inspection system. The dialysis section integrated into the unit provides a low number of system components and a simple manufacturing process -30-201209158 order to obtain an inexpensive analytical inspection system. The lysis method extracts analytical and diagnostic targets from cells in the sample and provides continuity processing and analysis of the targets. The lysate unit integrated in the unit provides a simple analytical inspection program, low system component count, and minimal system manufacturing procedures for an inexpensive analytical inspection system.

於培育區段中,遺傳物質接受各種預處理,例如DNA 限制剪切及轉接子引子之接合反應,藉以爲後續分析階段 提供最適宜或必要之條件、提高分析結果的訊息含量且提 高該分析檢驗系統之靈敏度、信號雜訊比和可靠度。 標靶基因序列之擴增反應可提高該分析檢驗系統之靈 敏度與信號雜訊比。再者,該等並聯之擴增腔室允許不同 的標靶物或標靶物群1且各自適當地使用不同的引子對或引 子群組且亦允許使用各自不同的最佳擴增參數,從而提高 分析檢驗之靈敏度、信號雜訊比和可靠度。 該探針雜合區段係藉由提供雜合反應達成該等標靶物 之分析。該整合式探針雜合區段提供一種使用簡易、可量 產、不昂貴且具有低系統元件數量的整合式解決方案。 該整合式成像感測器免除對昂貴之外部成像系統的需 求且提供具有低系統元件數量、可量產且不昂貴的整合式 解決方案’此整合式解決方案係一種小巧、質輕且極方便 攜帶之系統。該整合式成像感測器因受益於大的光線收集 角度而提高讀取靈敏度且免除在光學收集系統中使用諸多 光學元件之需求。 -31 - 201209158 整合於該LOC裝置中且把持該分析 求的該等試劑貯存槽可提供低的系統元 造程序,從而獲得不昂貴的分析檢驗系 GCF013.1本發明之此體系提供一 基因分析的晶片上實驗室(LOC )裝置 用於接收該樣本之入口; 載體基板: 透析區段,該透析區段係使該樣本 界値之細胞與較小成分分離,其中該等 値之細胞包括含有用於分析之遺傳物質 複數個試劑貯存槽: 溶胞區段,該溶胞區段位於該透析 解該等標靶細胞以釋出細胞內的遺傳物 與該等含有用於溶解溶胞區段內之標靶 試劑貯存槽之一者流體連通; 培育區段,該培育區段位於該溶胞 育區段係與該等含有用於與遺傳物質進 種酶的試劑貯存槽之一者流體連通; 第一核酸擴增區段,該第一核酸擴 區段下游以用於擴增該遺傳物質中的第 第二核酸擴增區段,該第二核酸擴 一核酸擴增區段下游以用於擴增源自該 之擴增子內的第二核酸序列;其中’ 檢驗之所有試劑需 件數量和簡約的製 統。 種用於生物樣本之 ,該L0C裝置包含 中尺寸大於預定臨 尺寸大於預定臨界 的標靶細胞; 區段下游且用於溶 質,該溶胞區段係 細胞之溶胞試劑的 區段下游,且該培 行酶催化反應之多 增區段位於該培育 一核酸序列;及 增區段係位於該第 第一核酸擴增區段 -32- 201209158 該透析區段、該溶胞區段、該培育區段、該第一核酸 擴增區段和該第二核酸擴增區段皆承載於該載體基板上。 GCF013.2較佳地,該第一核酸擴增區段係第一聚合 酶鏈鎖反應(PCR )區段,且該二核酸擴增區段係第二聚 合酶鏈鎖反應(PCR)區段。 GCF013.3較佳地,該第一 PCR區段具有第一組引子 對且該第一組引子對係用於黏合第一組互補核酸序列,及 該第二PCR區段具有第二組引子對且該第二組引子對係用 於黏合第二組互補核酸序列,該第一組互補核酸序列與該 第二組互補核酸序列不同。 GCF013.4 較佳地,該第一PCR區段和該第二PCR區 段係經建構而可用不同擴增參數進行操作,該等擴增參數 係下列參數之至少一者: 反轉錄酶種類; 聚合酶種類;In the culturing section, the genetic material undergoes various pretreatments, such as DNA restriction shearing and conjugation of the primers, to provide the most appropriate or necessary conditions for subsequent analysis stages, to increase the message content of the analysis results, and to improve the analysis. Verify system sensitivity, signal-to-noise ratio, and reliability. The amplification reaction of the target gene sequence can increase the sensitivity and signal to noise ratio of the analytical test system. Furthermore, the parallel amplification chambers allow for different targets or target groups 1 and each suitably uses a different primer pair or group of primers and also allows the use of different optimal amplification parameters, thereby allowing Improve sensitivity, signal noise ratio and reliability of analytical tests. The probe hybrid segment achieves analysis of the targets by providing a hybrid reaction. The integrated probe hybrid section provides an integrated solution that is easy to use, productive, inexpensive, and has a low number of system components. The integrated imaging sensor eliminates the need for expensive external imaging systems and offers an integrated solution with low system component count, mass production and low cost. This integrated solution is compact, lightweight and extremely convenient. Carrying system. The integrated imaging sensor increases read sensitivity and eliminates the need to use many optical components in an optical collection system by benefiting from large light collection angles. -31 - 201209158 The reagent storage tanks integrated in the LOC device and holding the analysis can provide a low system of metaprogramming, thereby obtaining an inexpensive analytical test system GCF013.1. The system of the present invention provides a genetic analysis. a wafer on-lab (LOC) device for receiving an inlet of the sample; a carrier substrate: a dialysis section that separates cells of the sample boundary from smaller components, wherein the cells of the cells include a plurality of reagent storage tanks for analysis: a lysis section, the lysis section is located in the dialysis solution to the target cells to release the genetic material in the cells and the contents are used to dissolve the lysed section One of the target reagent storage tanks is in fluid communication; the incubation section is located in the lysate section and is in fluid communication with one of the reagent storage tanks containing the enzyme for seeding the genetic material a first nucleic acid amplification section downstream of the first nucleic acid expansion section for amplifying a second nucleic acid amplification section in the genetic material, the second nucleic acid being expanded downstream of the nucleic acid amplification section for use Expansion Derived from the second subset of the nucleic acid sequence of the amplification; wherein all reagents required parts' number of inspection systems and simple system. For use in a biological sample, the LOC device comprises a target cell having a medium size greater than a predetermined proximity size greater than a predetermined threshold; downstream of the segment and for a solute, the lysis segment being downstream of a segment of the cell lysing reagent, and The multi-increasing segment of the enzyme-catalyzed reaction is located in the incubation nucleic acid sequence; and the extension segment is located in the first nucleic acid amplification segment -32 - 201209158, the dialysis section, the lysis segment, the incubation The segment, the first nucleic acid amplification segment, and the second nucleic acid amplification segment are all carried on the carrier substrate. Preferably, the first nucleic acid amplification segment is a first polymerase chain reaction (PCR) segment, and the second nucleic acid amplification segment is a second polymerase chain reaction (PCR) segment. . GCF013.3 preferably, the first PCR segment has a first set of primer pairs and the first set of primer pairs is used to bind the first set of complementary nucleic acid sequences, and the second PCR segment has a second set of primer pairs And the second set of primer pairs is used to bind a second set of complementary nucleic acid sequences that differ from the second set of complementary nucleic acid sequences. GCF013.4 Preferably, the first PCR segment and the second PCR segment are constructed and operable with different amplification parameters, the amplification parameters being at least one of the following parameters: a reverse transcriptase species; Type of polymerase;

去氧核糖核苷三磷酸濃度: 緩衝溶液; 熱循環時間; 熱循環重複次數;及 於PCR之一特定階段期間內的溫度。 GCF013.5較佳地,該LOC裝置亦具有雜合區段,該 雜合區段位於該第二PCR區段下游,且該雜合區段具有探 針陣列及光感測器’該探針陣列係用於與標靶核酸序列雜 合且該光感測器係用於偵測該陣列中之任何探針的雜合反 -33- 201209158 應。 GCF013.6較佳地,該透析區段具有第一通道、第二 通道及複數個孔,該第一通道係與位於上游末端之該入口 流體連通,該第二通道係與位於下游端之廢液通道流體連 通,該等孔小於該等標靶細胞且大於該等較小成分,該第 二通道藉由該等孔與該第一通道流體連通而使該等標靶細 胞留在該第一通道內且同時使該等較小成分流入該第二通 道。 GCF0 13.7較佳地,該第一通道和該第二通道係經建 構以藉由毛細作用注滿該樣本。 GCF0 13.8較佳地,該溶胞區段具有主動閥,該主動 閥係於溶胞期間使該等標靶細胞保留於該溶胞區段內,使 得當打開該主動閥時,流向該培育區段的毛細驅動流得以 繼續。 GCF013.9較佳地,該第一核酸擴增區段係第一恆溫 核酸擴增區段,且該第二核酸擴增區段係第二恆溫核酸擴 增區段。 _ GCF0 1 3 . 1 0 較佳地,該等試劑貯存槽各自具有使試 劑留在該等試劑貯存槽內的表面張力閥,該表面張力閥具 有彎液面錨,該彎液面錨係用於定住該試劑之彎液面直到 該試劑之彎液面與該樣本流接觸而去除該彎液面以允許該 試劑從該試劑貯存槽流出。 GCF013.il 較佳地,該LOC裝置亦具有CMOS電路、 溫度感測器及微系統技術(MST )層,該MST層包含該第 -34- 201209158 一 PC R區段和該第二PC R區段,其中該CMOS電路係設置於 該載體基板和該MST層之間,該CMOS電路係經建構以使 用該溫度感測器之輸出達成該第一 PCR區段和該第二PCR '區段的反饋控制。 GCF013.12較佳地,該第一PCR區段具有PCR微通道 ,該PCR微通道係使該樣本進行熱循環,該PCR微通道界 定具有低於1 00000平方微米之流動橫斷截面積的流動路徑 〇 GCF013.13較佳地,該PCR微通道具有至少一個長形 加熱器元件,該長形加熱器元件與該PCR微通道成平行地 延伸。Deoxyribonucleoside triphosphate concentration: buffer solution; thermal cycle time; number of cycles of thermal cycling; and temperature during a particular phase of PCR. GCF013.5 Preferably, the LOC device also has a hybrid segment located downstream of the second PCR segment, and the hybrid segment has a probe array and a photo sensor 'the probe The array is used to hybridize to the target nucleic acid sequence and the photosensor is used to detect heterozygous anti-33-201209158 of any probe in the array. Preferably, the dialysis section has a first passage, a second passage, and a plurality of holes, the first passage being in fluid communication with the inlet at the upstream end, the second passage being associated with the waste at the downstream end The liquid channel is in fluid communication, the holes being smaller than the target cells and larger than the smaller components, the second channel being in fluid communication with the first channel by the holes to leave the target cells in the first The smaller components are introduced into the second channel within the channel and simultaneously. GCF0 13.7 Preferably, the first channel and the second channel are constructed to fill the sample by capillary action. GCF0 13.8 Preferably, the lysis section has an active valve that retains the target cells in the lysis section during lysis, such that when the active valve is opened, the flow is directed to the culturing zone The capillary drive flow of the segment continues. Preferably, the first nucleic acid amplification segment is a first thermostatic nucleic acid amplification segment and the second nucleic acid amplification segment is a second thermostatic nucleic acid amplification segment. _ GCF0 1 3 . 1 0 Preferably, the reagent storage tanks each have a surface tension valve for leaving reagents in the reagent storage tanks, the surface tension valve having a meniscus anchor, the meniscus anchor system The meniscus of the reagent is retained until the meniscus of the reagent contacts the sample stream to remove the meniscus to allow the reagent to flow out of the reagent reservoir. GCF013.il Preferably, the LOC device also has a CMOS circuit, a temperature sensor and a micro system technology (MST) layer, the MST layer comprising the -34-201209158-PC R segment and the second PC R region a segment, wherein the CMOS circuit is disposed between the carrier substrate and the MST layer, the CMOS circuit being configured to use the output of the temperature sensor to achieve the first PCR segment and the second PCR segment Feedback control. GCF013.12 Preferably, the first PCR segment has a PCR microchannel that thermally cycles the sample, the PCR microchannel defining a flow having a cross-sectional cross-sectional area of less than 100,000 square microns Path 〇GCF013.13 Preferably, the PCR microchannel has at least one elongated heater element that extends parallel to the PCR microchannel.

GCF013.14較佳地,該PCR區段具有複數個長形PCR 腔室,且每個長形PCR腔室係由該PCR微通道之個別區段 形成,該PCR微通道具有由一系列寬曲流道所形成之蜿蜒 構形,且每個寬曲流道係一個用於形成該等長形PCR腔室 -φ 之一者的通道區段。 . GCF0 13.15較佳地,該培育區段具有加熱器以用於 加熱該遺傳物質和該等酶達預定之酶催化反應溫度。 GCF013.16較佳地,該LOC裝置亦包含用於容納該等 探針的雜合腔室陣列,使得每個雜合腔室內的該等探針係 經配置以與該等標靶核酸序列之一者雜合。 G C F 0 1 3 · 1 7較佳地,該光感測器係配準該等雜合腔 室設置而成的光二極體陣列。 GCF013.18較佳地,該CMOS電路具有數位記憶體及 -35- 201209158 數據界面,該數位記億體係用於儲存源自該光感測器之輸 出的雜合數據,且該數據界面係傳輸該雜合數據至外部裝 置。 GCF013_19較佳地,該第一 PCR區段具有主動閥,該 主動閥係於熱循環期間使液體保留在該第一 PCR區段內且 回應源自該CMOS電路之啓動信號而允許液體流至該第一 雜合腔室陣列。 GCF013.20較佳地,該主動閥係沸騰啓動式閥,該 沸騰啓動式閥具有彎液面錨及加熱器,該彎液面錨係經建 構以定住該阻止液體之毛細驅動流動的彎液面,且該加熱 器係用於使液體沸騰以使彎液面脫離彎液面錨,使得毛細 驅動流動得以繼續。 該使用簡易、可量產且費用不高的基因體分析L0C裝 置係藉由該LOC裝置之樣本放置槽接收生物樣本,且利用 儲存於該LOC裝置之試劑貯存槽內的試劑使用該LOC裝置 之透析區段分離樣本中所含的白血球、於該LOC裝置之化 學溶胞腔室中溶解白血球以釋出白血球之遺傳物質、於該 LOC裝置之培育區段中對樣本之遺傳物質進行預處理、擴 增標靶基因序列,及藉著與寡聚核苷酸探針進行雜合且藉 由該LOC裝置之整合式成像陣列感測該等探針之雜合反應 以分析樣本之核酸序列。 該透析區段之功能係從樣本中萃取附加訊息且提高分 析檢驗系統之靈敏度、信號雜訊比和動態範圍。整合於裝 置中的透析區段可提供低的系統元件數量和簡約的製造程 -36- 201209158 序,從而獲得不昂貴的分析檢驗系統。 該溶胞方法係自樣本中的細胞萃取出分析和診斷之標 靶物且提供該等標靶物之連續性處理與分析。整合於該裝 置中的溶胞子單元可提供簡約的分析檢驗程序、低的系統 元件數量及簡約的系統製造程序,從而獲得不昂貴的分析 檢驗系統。 於培育區段中,遺傳物質接受各種預處理,例如DNA φ 限制剪切及轉接子引子之接合反應,藉以爲後續分析階段 提供最適宜或必要之條件、提高分析結果的訊息含量且提 高該分析檢驗系統之靈敏度、信號雜訊比和可靠度。 標靶基因序列之擴增反應可提高該分析檢驗系統之靈 敏度與信號雜訊比。再者,該等串接之擴增腔室允許對該 擴增處理之早期循環和晚期循環做分段式的局部最佳化, 從而提高分析檢驗之靈敏度、信號雜訊比和可靠度。 該探針雜合區段係藉由提供雜合反應達成該等標靶物 -φ 之分析。整合式探針雜合區段提供一種使用簡易、可量產 ^ 、不昂貴且具有低系統元件數量的整合式解決方案。 該整合式成像感測器免除對昂貴之外部成像系統的需 求且提供具有低系統元件數量、可量產且不昂貴的整合式 解決方案,此整合式解決方案係一種小巧、質輕且極方便 攜帶之系統。該整合式成像感測器因受益於大的光線收集 角度而提高讀取靈敏度且免除在光學收集系統中使用諸多 光學元件之需求。 整合於該LOC裝置中且把持該分析檢驗之所有試劑需 -37- 201209158 求的該等試劑貯存槽可提供低的系統元件數量和簡約的製 造程序,從而獲得不昂貴的分析檢驗系統。 GCF014.1本發明之此體系提供一種用於生物樣本之 基因分析的晶片上實驗室(LOC )裝置,該LOC裝置包含 用於接收該樣本之入口; 載體基板; 透析區段,該透析區段係使該樣本中大於預定臨界値 之細胞與較小成分分離,其中該等大於預定臨界値之細胞 包括含有用於分析之遺傳物質的標靶細胞 複數個試劑貯存槽; 溶胞區段,該溶胞區段係位於該透析區段下游以用於 溶解該等標靶細胞而釋出該等標靶細胞內的遺傳物質,該 溶胞區段係與該等含有用於溶解該溶胞區段中之該等標靶 細胞之溶胞試劑的試劑貯存槽之一者流體連通;及 核酸擴增區段,該核酸擴增區段係位於該溶胞區段下 游以用於自該遺傳物質擴增核酸序列;其中 該透析區段、該溶胞區段及該核酸擴增區段皆承載於 該載體基板上。 GCF0 14.2較佳地,該核酸擴增區段係聚合酶鏈鎖反 應(P C R )區段。 GCF014.3較佳地,該LOC裝置亦具有雜合區段,該 雜合區段位於該PCR區段下游,且該雜合區段具有探針陣 列及光感測器,該探針陣列係用於與標靶核酸序列雜合, -38- 201209158 且該光感測器係用於偵測該陣列中之任何探針的雜合反應 GCF014.4較佳地,該透析區段具有第一通道、第二 通道及複數個孔,該第一通道係與位於上游末端之該入口 流體連通,該第二通道係與位於下游端之廢液通道流體連 通,該等孔小於該等標靶細胞且大於該等較小成分,該第 二通道係藉由該等孔與該第一通道流體連通而使該等標靶 細胞留在該第一通道內同時使該等較小成分流入該第二通 道。 GCF014.5較佳地,該第一通道和該第二通道係經建 構以藉由毛細作用注滿該樣本。 GCF014.6較佳地,該溶胞區段具有主動閥,該主動 閥係於溶胞期間使該等標靶細胞留在該溶胞區段內,使得 當打開該主動閥時,流向該培育區段的毛細驅動流動得以 繼續。Preferably, the PCR segment has a plurality of elongate PCR chambers, and each elongate PCR chamber is formed by individual segments of the PCR microchannel having a series of wide curves The crucible is formed by the flow path, and each wide curved channel is a channel section for forming one of the elongate PCR chambers -φ. GCF0 13.15 Preferably, the incubation section has a heater for heating the genetic material and the enzymes to a predetermined enzyme catalyzed reaction temperature. Preferably, the LOC device also includes an array of hybrid chambers for holding the probes such that the probes within each hybrid chamber are configured to interact with the target nucleic acid sequences One is heterozygous. G C F 0 1 3 · 1 7 Preferably, the photosensor is associated with an array of photodiodes arranged in the hybrid chambers. GCF013.18 Preferably, the CMOS circuit has a digital memory and a data interface of -35-201209158, the digital system is used for storing the hybrid data originating from the output of the photo sensor, and the data interface is transmitted. The hybrid data is sent to an external device. GCF013_19 preferably, the first PCR section has an active valve that retains liquid within the first PCR section during thermal cycling and allows liquid to flow to the activation signal in response to the activation signal from the CMOS circuit The first hybrid chamber array. GCF013.20 Preferably, the active valve is a boiling start valve having a meniscus anchor and a heater, the meniscus anchor being constructed to hold the meniscus that prevents the capillary drive flow of the liquid The heater is used to boil the liquid to disengage the meniscus from the meniscus anchor so that the capillary drive flow continues. The genomic analysis L0C device that is simple to use, mass-produced, and inexpensive is to receive a biological sample by using a sample placement tank of the LOC device, and using the reagent stored in the reagent storage tank of the LOC device to use the LOC device. The dialysis section separates the white blood cells contained in the sample, dissolves the white blood cells in the chemical lysis chamber of the LOC device to release the genetic material of the white blood cells, and pretreats the genetic material of the sample in the incubation section of the LOC device, The target gene sequence is amplified and the nucleic acid sequence of the sample is analyzed by heterozygous with the oligonucleotide probe and sensing the heterozygous reaction of the probes by an integrated imaging array of the LOC device. The function of the dialysis section is to extract additional information from the sample and to increase the sensitivity, signal to noise ratio, and dynamic range of the analytical inspection system. The dialysis section integrated into the unit provides a low number of system components and a simple manufacturing process, resulting in an inexpensive analytical inspection system. The lysis method extracts analytical and diagnostic targets from cells in the sample and provides continuity processing and analysis of the targets. The lysate unit integrated in the unit provides a simple analytical inspection program, low system component count, and minimal system manufacturing procedures for an inexpensive analytical inspection system. In the culturing section, the genetic material undergoes various pretreatments, such as DNA φ restriction shearing and conjugation of the primer primers, to provide the most appropriate or necessary conditions for subsequent analysis stages, to increase the message content of the analysis results, and to increase the Analyze the sensitivity, signal-to-noise ratio, and reliability of the inspection system. The amplification reaction of the target gene sequence can increase the sensitivity and signal to noise ratio of the analytical test system. Furthermore, the cascaded amplification chambers allow for segmental local optimization of the early and late cycles of the amplification process, thereby increasing sensitivity, signal to noise ratio and reliability of the assay. The probe hybrid segment achieves the analysis of the target -φ by providing a hybrid reaction. The integrated probe hybrid section provides an integrated solution that is easy to use, mass-produced, inexpensive, and has a low number of system components. The integrated imaging sensor eliminates the need for expensive external imaging systems and offers an integrated solution with low system component count, mass production and low cost. This integrated solution is compact, lightweight and extremely convenient. Carrying system. The integrated imaging sensor increases read sensitivity and eliminates the need to use many optical components in an optical collection system by benefiting from large light collection angles. The reagent storage tanks that are integrated into the LOC device and that hold all of the reagents for the analysis require -37-201209158 provide a low system component count and a simple manufacturing procedure to obtain an inexpensive analytical inspection system. GCF014.1 This system of the invention provides a wafer on-lab (LOC) device for genetic analysis of a biological sample, the LOC device comprising an inlet for receiving the sample; a carrier substrate; a dialysis section, the dialysis section The cells in the sample greater than the predetermined threshold are separated from the smaller components, wherein the cells greater than the predetermined threshold include a plurality of reagent storage tanks containing the target cells for analysis of the genetic material; a lysis segment, a lytic segment located downstream of the dialysis section for dissolving the target cells to release genetic material within the target cells, the lysing segments being associated with the lysate for dissolving the lysing zone One of the reagent storage tanks of the lysis reagent of the target cells in the segment is in fluid communication; and the nucleic acid amplification segment is located downstream of the lysis segment for use from the genetic material Amplifying the nucleic acid sequence; wherein the dialysis section, the lysis section, and the nucleic acid amplification section are all carried on the carrier substrate. GCF0 14.2 Preferably, the nucleic acid amplification segment is a polymerase chain reaction (P C R ) segment. GCF014.3 Preferably, the LOC device also has a hybrid segment located downstream of the PCR segment, and the hybrid segment has a probe array and a photo sensor, the probe array For hybridization with a target nucleic acid sequence, -38-201209158 and the photosensor is used to detect a heterozygous reaction of any probe in the array. GCF014.4 preferably, the dialysis section has a first a channel, a second channel, and a plurality of holes, the first channel being in fluid communication with the inlet at an upstream end, the second channel being in fluid communication with a waste channel at a downstream end, the holes being smaller than the target cells And greater than the smaller components, the second channel is in fluid communication with the first channel by the holes to leave the target cells in the first channel while allowing the smaller components to flow into the second aisle. GCF 014.5 Preferably, the first channel and the second channel are constructed to fill the sample by capillary action. Preferably, the lysis zone has an active valve that retains the target cells within the lysis zone during lysis so that when the active valve is opened, the flow is directed to the incubation The capillary drive flow of the section continues.

GCF014.7較佳地,該核酸擴增區段係恆溫核酸擴增 區段。 GCF014.8較佳地,該等試劑貯存槽各自具有使試劑 留在該等試劑貯存槽內的表面張力閥’該表面張力閥具有 彎液面錨,該彎液面錨係用於定住該試劑之彎液面直到該 試劑之彎液面與該樣本流接觸而去除該彎液面以允許該試 劑從該試劑貯存槽流出。 GCF014.9較佳地,該L〇C裝置亦具有從入口到雜合 區段的流動路徑,其中該流動路徑係經建構以藉由毛細作 -39- 201209158 用把樣本從該入口引至該雜合區段。 GCF014.10較佳地,該l〇C裝置亦具有CMOS電路、 溫度感測器及微系統技術(MST )層,該MST層包含該 PCR區段’其中該CMOS電路係設置於該載體基板和該MST 層之間’該CMOS電路係經建構以使用該溫度感測器之輸 出達成該PCR區段的反饋控制。 GCF014.il較佳地,該PCR區段具有PCR微通道,該 PCR微通道係使該樣本進行熱循環以擴增核酸序列,該 PCR微通道界定部份的樣本流動路徑,且該pCR微通道具 有低於1 00000平方微米的流動橫斷截面積。 GCF014.12較佳地,該PCR微通道具有至少一個長形 加熱器元件以用於加熱該長形PCR微通道內的核酸序列, 該長形加熱器元件係與該PCR微通道呈平行延伸。 GCF014.13 較佳地,該PCR微通道之至少一個區段形 成長形PCR腔室。 GCF014.14較佳地,該PCR區段具有複數個長形PCR 腔室,且每個長形PCR腔室係由該PCR微通道之個別區段 形成,該PCR微通道具有由一系列寬曲流道所形成之蜿蜒 構形,每個寬曲流道係一個用於形成該等長形PCR腔室之 —者的通道區段。GCF014.7 Preferably, the nucleic acid amplification segment is a thermostatic nucleic acid amplification segment. GCF014.8 Preferably, the reagent storage tanks each have a surface tension valve for leaving reagents in the reagent storage tanks. The surface tension valve has a meniscus anchor, and the meniscus anchor is used to hold the reagent. The meniscus is removed until the meniscus of the reagent contacts the sample stream to remove the meniscus to allow the reagent to flow out of the reagent reservoir. GCF014.9 preferably, the L〇C device also has a flow path from the inlet to the hybrid section, wherein the flow path is constructed to direct the sample from the inlet to the capillary by capillary-39-201209158 Hybrid section. GCF014.10 Preferably, the device also has a CMOS circuit, a temperature sensor and a micro system technology (MST) layer, the MST layer includes the PCR segment, wherein the CMOS circuit is disposed on the carrier substrate and The CMOS circuit is constructed between the MST layers to achieve feedback control of the PCR segment using the output of the temperature sensor. GCF014.il preferably, the PCR segment has a PCR microchannel that thermally cycles the sample to amplify a nucleic acid sequence, the PCR microchannel defining a portion of the sample flow path, and the pCR microchannel Has a cross-sectional cross-sectional area of less than 100,000 square microns. Preferably, the PCR microchannel has at least one elongate heater element for heating the nucleic acid sequence within the elongate PCR microchannel, the elongate heater element extending parallel to the PCR microchannel. GCF014.13 Preferably, at least one segment of the PCR microchannel is shaped into a growth PCR chamber. Preferably, the PCR segment has a plurality of elongate PCR chambers, and each elongate PCR chamber is formed by individual segments of the PCR microchannel having a series of wide curves The crucible configuration formed by the flow channels, each wide curved channel being a channel section for forming the elongate PCR chamber.

GCF014.15較佳地,該L0C裝置亦具有容納用於PCR 之試劑的試劑貯存槽:及 具有孔之表面張力閥,該具有孔之表面張力閥係經建 構以定住該試劑之彎液面’如此該彎液面使該試劑保留在 -40- 201209158 該試劑貯存槽內直到該彎液面與該流體樣本接觸而去除該 彎液面且該試劑流出該試劑貯存槽。 GCF014.16較佳地,該LOC裝置亦具有用於容納該等 探針的雜合腔室陣列,使得每個雜合腔室內的該等探針係 經配置以與該等標靶核酸序列之一者雜合。 GCF014.17較佳地,該光感測器係配準該等雜合腔 室設置而成的光二極體陣列。 GCF014.18較佳地,該CMOS電路具有數位記憶體及 數據界面,該數位記憶體係用於儲存源自該光感測器之輸 出的雜合數據,且該數據界面係傳輸該雜合數據至外部裝 置。GCF014.15 Preferably, the LOC device also has a reagent storage tank containing reagents for PCR: and a surface tension valve having a hole, the surface tension valve having a hole configured to hold the meniscus of the reagent' Thus, the meniscus retains the reagent in the reagent storage tank from -40 to 201209158 until the meniscus contacts the fluid sample to remove the meniscus and the reagent flows out of the reagent reservoir. Preferably, the LOC device also has an array of hybrid chambers for holding the probes such that the probes within each hybrid chamber are configured to interact with the target nucleic acid sequences One is heterozygous. GCF014.17 Preferably, the photosensor is associated with an array of photodiodes arranged by the hybrid chambers. GCF014.18 Preferably, the CMOS circuit has a digital memory and a data interface, the digital memory system is configured to store the hybrid data originating from the output of the photo sensor, and the data interface transmits the hybrid data to External device.

GCF014.19較佳地,該PCR區段具有主動閥,該主動 閥係於熱循環期間使液體保留在該PCR區段內且回應源自 該CMOS電路之啓動信號而允許液體流至該等雜合腔室》 GCF014.20較佳地,該主動閥係沸騰啓動式閥,該 沸騰啓動式閥具有彎液面錨及加熱器,該彎液面錨係經建 構以定住該阻止液體之毛細驅動流動的彎液面,且該加熱 器係用於使該液體沸騰以使該彎液面脫離該彎液面錨,使 得毛細驅動流動得以繼續。 該使用簡易、可量產且費用不高的基因體分析LOC裝 置係藉由該LOC裝置之樣本放置槽接收生物樣本,且利用 儲存於該LOC裝置之試劑貯存槽內的試劑使用該LOC裝置 之透析區段分離樣本中所含的白血球、於該LOC裝置之化 學溶胞腔室中溶解白血球以釋出白血球之遺傳物質、擴增 -41 - 201209158 標靶基因序列,及藉著與寡聚核苷酸探針進行雜合且藉由 該L 0 C裝置之整合式成像陣列感測該等探針之雜合反應以 分析樣本之核酸序列。 該透析區段之功能係從樣本中萃取附加訊息且提高分 析檢驗系統之靈敏度、信號雜訊比和動態範圍。整合於裝 置中的透析區段可提供低的系統元件數量和簡約的製造程 序,從而獲得不昂貴的分析檢驗系統。 該溶胞方法係自樣本中的細胞萃取出分析和診斷之標 靶物且提供該等標靶物之連續性處理與分析。整合於該裝 置中的溶胞子單元可提供簡約的分析檢驗程序、低的系統 元件數量及簡約的系統製造程序,從而獲得不昂貴的分析 檢驗系統。 標靶基因序列之擴增反應可提高該分析檢驗系統之靈 敏度與信號雜訊比。 該探針雜合區段係藉由提供雜合反應達成該等標靶物 之分析。整合式探針雜合區段提供一種使用簡易、可量產 、不昂貴且具有低系統元件數量的整合式解決方案。 該整合式成像感測器免除對昂貴之外部成像系統的需 求且提供具有低系統元件數量、可量產且不昂貴的整合式 解決方案,此整合式解決方案係一種小巧、質輕且極方便 攜帶之系統。該整合式成像感測器因受益於大的光線收集 角度而提高讀取靈敏度且免除在光學收集系統中使用諸多 光學元件之需求。 整合於該LOC裝置中且把持該分析檢驗之所有試劑需 -42- 201209158 求的該等試劑貯存槽可提供低的系統元件數量和簡約的製 造程序,從而獲得不昂貴的分析檢驗系統。 GCF01 5.1本發明之此體系提供一種用於生物樣本之 ' 基因分析的晶片上實驗室(LOC )裝置,該LOC裝置包含 用於接收該樣本之入口; 載體基板; 透析區段,該透析區段係使該樣本中大於預定臨界値 之細胞與較小成分分離,其中該等大於預定臨界値之細胞 包括含有用於分析之遺傳物質的標靶細胞; 複數個試劑貯存槽; 溶胞區段,該溶胞區段係位於該透析區段下游以用於 溶解細胞而釋出細胞內的遺傳物質,該溶胞區段係與該等 含有用於溶解該溶胞區段中之該等標靶細胞之溶胞試劑的 試劑貯存槽之一者流體連通;GCF014.19 preferably, the PCR section has an active valve that retains liquid within the PCR section during thermal cycling and allows liquid to flow to the impurities in response to an activation signal originating from the CMOS circuit Preferably, the active valve is a boiling start valve having a meniscus anchor and a heater, the meniscus anchor being constructed to hold the capillary drive to prevent liquid The flowing meniscus is used and the heater is used to boil the liquid to disengage the meniscus from the meniscus anchor so that the capillary drive flow continues. The genomic analysis LOC device that is simple to use, mass-produced, and inexpensive is to receive a biological sample by using a sample placement tank of the LOC device, and using the reagent stored in the reagent storage tank of the LOC device to use the LOC device. The dialysis section separates the white blood cells contained in the sample, dissolves the white blood cells in the chemical lysis chamber of the LOC device to release the genetic material of the white blood cells, amplifies the sequence of the -41 - 201209158 target gene, and uses the oligomeric nucleus The glycoside probe is hybridized and the hybridization reaction of the probes is sensed by the integrated imaging array of the L0C device to analyze the nucleic acid sequence of the sample. The function of the dialysis section is to extract additional information from the sample and to increase the sensitivity, signal to noise ratio, and dynamic range of the analytical inspection system. The dialysis section integrated into the unit provides a low number of system components and a simple manufacturing process, resulting in an inexpensive analytical inspection system. The lysis method extracts analytical and diagnostic targets from cells in the sample and provides continuity processing and analysis of the targets. The lysate unit integrated in the unit provides a simple analytical inspection program, low system component count, and minimal system manufacturing procedures for an inexpensive analytical inspection system. The amplification reaction of the target gene sequence can increase the sensitivity and signal to noise ratio of the analytical test system. The probe hybrid segment achieves analysis of the targets by providing a hybrid reaction. The integrated probe hybrid section provides an integrated solution that is easy to use, mass-produced, inexpensive, and has a low number of system components. The integrated imaging sensor eliminates the need for expensive external imaging systems and offers an integrated solution with low system component count, mass production and low cost. This integrated solution is compact, lightweight and extremely convenient. Carrying system. The integrated imaging sensor increases read sensitivity and eliminates the need to use many optical components in an optical collection system by benefiting from large light collection angles. The reagent storage tanks that are integrated into the LOC device and that hold all of the reagents for the analysis require -42-201209158 provide a low system component count and a simple manufacturing procedure, resulting in an inexpensive analytical inspection system. GCF01 5.1 This system of the invention provides a wafer on-lab (LOC) device for 'gene analysis of biological samples, the LOC device comprising an inlet for receiving the sample; a carrier substrate; a dialysis section, the dialysis section The cells in the sample greater than the predetermined threshold 分离 are separated from the smaller components, wherein the cells greater than the predetermined threshold 包括 include target cells containing genetic material for analysis; a plurality of reagent storage tanks; a lysis section, The lytic segment is located downstream of the dialysis section for lysing cells to release genetic material within the cell, the lysing segment being associated with the target for dissolving the lysed segment One of the reagent storage tanks of the lysis reagent of the cells is in fluid communication;

第一核酸擴增區段,該第一核酸擴增區段係位於該溶 胞區段下游以用於擴增該遺傳物質內的核酸序列;及 第二核酸擴增區段,該第二核酸擴增區段係位於該溶 胞區段下游以用於與該第一核酸擴增區段並行地擴增該遺 傳物質中的核酸序列;其中, 該透析區段、該溶胞區段、該第一核酸擴增區段及該 第二核酸擴增區段皆承載於該載體基板上。 GCF015.2較佳地,該第一核酸擴增區段係第一聚合 酶鏈鎖反應(PCR)區段,且該二核酸擴增區段係第二聚 -43- 201209158 合酶鏈鎖反應(PCR)區段。 GCF015.3 較佳地,該第一PCR區段具有第一組引子 對且該第一組引子對係用於黏合第一組互補核酸序列,及 該第二PCR區段具有第二組引子對且該第二組引子對係用 於黏合第二組互補核酸序列,該第一組互補核酸序列與該 第二組互補核酸序列不同。 GCF015.4 較佳地,該第一 PCR區段和該第二PCR區 段係經建構以使用不同擴增參數進行操作,該等擴增參數 係下列參數之至少一者: 反轉錄酶種類; 聚合酶種類; 去氧核糖核苷三磷酸濃度; 緩衝溶液; 熱循環時間; 熱循環重複次數;及 於PCR之一特定階段期間內的溫度。 GCF015.5較佳地,該LOC裝置亦包含第一雜合區段 和第二雜合區段及光感測器,該第一雜合區段位於該第一 PCR區段下游且該第一雜合區段具有用於與第一標靶核酸 序列雜合的第一探針陣列,及該第二雜合區段位於第二 PCR區段下游且該第二雜合區段具有用於與第二標靶核酸 序列雜合的第二探針陣列,及該光感測器係用於偵測該第 一陣列和第二陣列中之任何探針的雜合反應。 GCF0 15.6較佳地,該透析區段具有第一通道、第二 -44- 201209158 通道及複數個孔,該第一通道係與位於上游末端之該入口 流體連通’該第二通道係與位於下游端之廢液通道流體連 通,該等孔小於該等標靶細胞且大於該等較小成分,該第 二通道藉由該等孔與該第一通道流體連通而使該等標靶細 胞留在該第一通道內同時使該等較小成分流入該第二通道 GCF015.7較佳地,該第一通道和該第二通道係經建 構以藉由毛細作用注滿該樣本。 GCF0 15.8較佳地,該溶胞區段具有主動閥,該主動 閥係於溶胞期間使該等標靶細胞留在該溶胞區段內,使得 當打開該主動閥時,流向該培育區段的毛細驅動流動得以 繼續。 GCF0 15.9較佳地,該第一核酸擴增區段係第一恆溫 核酸擴增區段,且該第二核酸擴增區段係第二恆溫核酸擴 增區段。a first nucleic acid amplification segment, the first nucleic acid amplification segment being located downstream of the lysis segment for amplifying a nucleic acid sequence within the genetic material; and a second nucleic acid amplification segment, the second nucleic acid An amplification segment is located downstream of the lysis segment for amplifying a nucleic acid sequence in the genetic material in parallel with the first nucleic acid amplification segment; wherein the dialysis section, the lysis segment, the The first nucleic acid amplification segment and the second nucleic acid amplification segment are both carried on the carrier substrate. GCF015.2 Preferably, the first nucleic acid amplification segment is a first polymerase chain reaction (PCR) segment, and the second nucleic acid amplification segment is a second poly-43-201209158 synthase chain reaction (PCR) section. GCF015.3 Preferably, the first PCR segment has a first set of primer pairs and the first set of primer pairs is used to bind the first set of complementary nucleic acid sequences, and the second PCR segment has a second set of primer pairs And the second set of primer pairs is used to bind a second set of complementary nucleic acid sequences that differ from the second set of complementary nucleic acid sequences. GCF015.4 Preferably, the first PCR segment and the second PCR segment are constructed to operate using different amplification parameters, the amplification parameters being at least one of the following parameters: a reverse transcriptase species; Polymerase species; deoxyribonucleoside triphosphate concentration; buffer solution; thermal cycle time; number of cycles of thermal cycling; and temperature during a particular phase of PCR. Preferably, the LOC device further comprises a first hybrid segment and a second hybrid segment and a photo sensor, the first hybrid segment being located downstream of the first PCR segment and the first hybrid The segment has a first probe array for hybridization with the first target nucleic acid sequence, and the second hybrid segment is located downstream of the second PCR segment and the second hybrid segment has a second and second hybrid segment A second probe array that is hybridized to the target nucleic acid sequence, and the photosensor is used to detect a heterozygous reaction of any of the probes in the first array and the second array. Preferably, the dialysis section has a first passage, a second -44 - 201209158 passage, and a plurality of holes, the first passage being in fluid communication with the inlet at the upstream end - the second passage is downstream The waste channel of the end is in fluid communication, the holes are smaller than the target cells and larger than the smaller components, and the second channel is in fluid communication with the first channel by the holes to leave the target cells in The first channel and the second channel are simultaneously configured to fill the sample by capillary action. Preferably, the lysis zone has an active valve that retains the target cells within the lysis zone during lysis so that when the active valve is opened, the flow is directed to the culturing zone The capillary drive flow of the segment continues. Preferably, the first nucleic acid amplification segment is a first thermostatic nucleic acid amplification segment and the second nucleic acid amplification segment is a second thermostatic nucleic acid amplification segment.

GCF015.10較佳地,該等試劑貯存槽各自具有使試 劑留在該等試劑貯存槽內的表面張力閥,該表面張力閥具 有彎液面錨,該彎液面錨係用於定住該試劑之彎液面直到 該試劑之彎液面與該樣本流接觸而去除該彎液面以允許該 試劑從該試劑貯存槽流出。 GCF015.11較佳地,該LOC裝置亦具有CMOS電路、 溫度感測器及微系統技術(MST )層,該MS T層包含該第 —PCR區段和該第二PCR區段,其中該CMOS電路係設置於 該載體基板和該MST層之間,該CMOS電路係經建構以使 -45 - 201209158 用該溫度感測器之輸出達成該第一 PCR區段和該第二PCR 區段的反饋控制。 GCF015.12較佳地,該第一PCR區段具有PCR微通道 ,該PCR微通道係使該樣本進行熱循環,該PCR微通道界 定具有低於1 00 000平方微米之流動橫斷截面積的流動路徑 〇 GCF015.13 較佳地,該PCR微通道具有至少一個長形 加熱器元件,該長形加熱器元件係與該PCR微通道呈平行 延伸。 GCF015.14 較佳地,該PCR區段具有複數個長形PCR 腔室,且每個長形PCR腔室係由該PC R微通道之個別區段 形成,該PCR微通道具有由一系列寬曲流道所形成之蜿蜒 構形,且每個寬曲流道係一個用於形成該等長形PCR腔室 之一者的通道區段。 GCF015.15較佳地,該LOC裝置亦具有容納用於PCR 之試劑的試劑貯存槽;及 具有孔之表面張力閥,該具有孔之表面張力閥係經建 構以定住該試劑之彎液面,如此該彎液面使該試劑保留在 該試劑貯存槽內直到該彎液面與該流體樣本接觸而去除該 彎液面且該試劑流出該試劑貯存槽。 GCF015.16較佳地,該LOC裝置亦具有用於容納該等 第一探針的第一雜合腔室陣列,使得每個雜合腔室內的該 等第一探針係經配置以與該等第一標靶核酸序列之一者雜 合。 -46- 201209158 G CFO 15. 17較佳地,該光感測器係配準該等雜合腔 室設置而成的光二極體陣列。 GCF0 15. 18較佳地,該CMOS電路具有數位記憶體及 數據界面,該數位記憶體係用於儲存源自該光感測器之輸 出的雜合數據,且該數據界面係傳輸該雜合數據至外部裝 置。 GCF015.19較佳地,該第一PCR區段具有主動閥,該 主動閥係於熱循環期間使液體保留在該第一PCR區段內且 回應源自該CMOS電路之啓動信號而允許液體流至該第一 雜合腔室陣列。 GCF015.20較佳地,該主動閥係沸騰啓動式閥,該 沸騰啓動式閥具有彎液面錨及加熱器,該彎液面錨係經建 構以定住該阻止液體之毛細驅動流動的彎液面,且該加熱 器係用於使該液體沸騰以使該彎液面脫離該彎液面錨,使 得毛細驅動流動得以繼續。Preferably, the reagent storage tanks each have a surface tension valve for retaining the reagents in the reagent storage tanks, the surface tension valve having a meniscus anchor, the meniscus anchor system for holding the reagent The meniscus is removed until the meniscus of the reagent contacts the sample stream to remove the meniscus to allow the reagent to flow out of the reagent reservoir. Preferably, the LOC device also has a CMOS circuit, a temperature sensor and a microsystem technology (MST) layer, the MS T layer comprising the first PCR segment and the second PCR segment, wherein the CMOS a circuit is disposed between the carrier substrate and the MST layer, the CMOS circuit is constructed such that -45 - 201209158 uses the output of the temperature sensor to achieve feedback of the first PCR segment and the second PCR segment control. GCF015.12 Preferably, the first PCR segment has a PCR microchannel that thermally cycles the sample, the PCR microchannel defining a cross-sectional area of flow having a flow cross-section of less than 1 000 000 micrometers Flow Path 〇 GCF015.13 Preferably, the PCR microchannel has at least one elongated heater element that extends parallel to the PCR microchannel. GCF015.14 Preferably, the PCR segment has a plurality of elongate PCR chambers, and each elongate PCR chamber is formed by individual segments of the PC R microchannel having a series of widths The meandering path formed by the meandering channel, and each wide curved channel is a channel section for forming one of the elongate PCR chambers. Preferably, the LOC device also has a reagent storage tank containing reagents for PCR; and a surface tension valve having a hole configured to hold the meniscus of the reagent, The meniscus thus retains the reagent in the reagent reservoir until the meniscus contacts the fluid sample to remove the meniscus and the reagent exits the reagent reservoir. Preferably, the LOC device also has a first hybrid chamber array for accommodating the first probes such that the first probes within each hybrid chamber are configured to One of the first target nucleic acid sequences is heterozygous. -46- 201209158 G CFO 15. 17 Preferably, the photo sensor is associated with an array of photodiodes arranged in the hybrid chambers. GCF0 15.18 Preferably, the CMOS circuit has a digital memory and a data interface, the digital memory system is configured to store the hybrid data originating from the output of the photo sensor, and the data interface transmits the hybrid data To an external device. GCF015.19 Preferably, the first PCR section has an active valve that retains liquid within the first PCR section during thermal cycling and allows liquid flow in response to an activation signal originating from the CMOS circuit To the first hybrid chamber array. GCF015.20 Preferably, the active valve is a boiling start valve having a meniscus anchor and a heater, the meniscus anchor being constructed to hold the meniscus that prevents capillary movement of the liquid And the heater is used to boil the liquid to disengage the meniscus from the meniscus anchor, such that the capillary drive flow continues.

該使用簡易、可量產且費用不高的基因體分析LOC裝 置係藉由該LOC裝置之樣本放置槽接收生物樣本,且利用 儲存於該LOC裝置之試劑貯存槽內的試劑使用該LOC裝置 之透析區段分離樣本中所含的白血球、於該LOC裝置之化 學溶胞腔室中溶解白血球以釋出白血球之遺傳物質、擴增 標靶基因序列’及藉著與寡聚核苷酸探針進行雜合且藉由 該'LOC裝置之整合式成像陣列感測該等探針之雜合反應以 分析樣本之核酸序列。 該透析區段之功能係從樣本中萃取附加訊息且提高分 -47- 201209158 析檢驗系統之靈敏度、信號雜訊比和動態範圍。整合於裝 置中的透析區段可提供低的系統元件數量和簡約的製造程 序,從而獲得不昂貴的分析檢驗系統。 該溶胞方法係自樣本中的細胞萃取出分析和診斷之標 靶物且提供該等標靶物之連續性處理與分析。整合於該裝 置中的溶胞子單元元件可提供簡約的分析檢驗程序、低的 系統元件數量及簡約的系統製造程序,從而獲得不昂貴的 分析檢驗系統。 標靶基因序列之擴增反應可提高該分析檢驗系統之靈 敏度與信號雜訊比。再者,該等並行之擴增腔室允許不同 的標靶物或標靶物群組可適當地使用各自不同的引子對或 引子群組且亦允許使用各自不同的最佳擴增參數,從而提 高分析檢驗之靈敏度、信號雜訊比和可靠度。 該探針雜合區段係藉由提供雜合反應達成該等標靶之 分析。整合式探針雜合區段提供一種使用簡易、可量產、 不昂貴且具有低系統元件數量的整合式解決方案。 該整合式成像感測器免除對昂貴之外部成像系統的需 求且提供具有低系統元件數量、可量產且不昂貴的整合式 解決方案,此整合式解決方案係一種小巧、質輕且極方便 攜帶之系統。該整合式成像感測器因受益於大的光線收集 角度而提高讀取靈敏度且免除在光學收集系統中使用諸多 光學元件之需求。 整合於該LOC裝置中且把持該分析檢驗之所有試劑需 求的該等試劑貯存槽可提供低的系統元件數量和簡約的製 -48-The genomic analysis LOC device that is simple to use, mass-produced, and inexpensive is to receive a biological sample by using a sample placement tank of the LOC device, and using the reagent stored in the reagent storage tank of the LOC device to use the LOC device. The dialysis section separates the white blood cells contained in the sample, dissolves the white blood cells in the chemical lysis chamber of the LOC device to release the genetic material of the white blood cells, and amplifies the target gene sequence' and by using the oligonucleotide probe Hybridization is performed and the hybridization reaction of the probes is sensed by the integrated imaging array of the 'LOC device to analyze the nucleic acid sequence of the sample. The function of the dialysis section is to extract additional information from the sample and to improve the sensitivity, signal-to-noise ratio and dynamic range of the test system. The dialysis section integrated into the unit provides a low number of system components and a simple manufacturing process, resulting in an inexpensive analytical inspection system. The lysis method extracts analytical and diagnostic targets from cells in the sample and provides continuity processing and analysis of the targets. The lysed subunit elements integrated into the unit provide a simple analytical inspection program, low system component count, and simple system manufacturing procedures for an inexpensive analytical inspection system. The amplification reaction of the target gene sequence can increase the sensitivity and signal to noise ratio of the analytical test system. Furthermore, the parallel amplification chambers allow different target or target groups to suitably use different primer pairs or primer groups and also allow for the use of different optimal amplification parameters, thereby Improve sensitivity, signal noise ratio and reliability of analytical tests. The probe hybrid segment achieves analysis of the targets by providing a hybrid reaction. The integrated probe hybrid section provides an integrated solution that is easy to use, mass-produced, inexpensive, and has a low number of system components. The integrated imaging sensor eliminates the need for expensive external imaging systems and offers an integrated solution with low system component count, mass production and low cost. This integrated solution is compact, lightweight and extremely convenient. Carrying system. The integrated imaging sensor increases read sensitivity and eliminates the need to use many optical components in an optical collection system by benefiting from large light collection angles. The reagent storage tanks integrated into the LOC device and holding all of the reagents required for the analysis can provide a low number of system components and a simple system.

201209158 造程序,從而獲得不昂貴的分析檢驗系統。 GCF0 16_1本·發明之此體系提供一種用於生物樣 基因分析的晶片上實驗室(LOC )裝置,該LOC裝置 用於接收該樣本之入口; 載體基板; 透析區段,該透析區段係使該樣本中大於預定臨 之細胞與較小成分分離,其中該等大於預定臨界値之 包括含有用於分析之遺傳物質的標靶細胞; 複數個試劑貯存槽; 溶胞區段,該溶胞區段係位於該透析區段下游以 溶解該等標靶細胞以釋出該等標靶細胞內的遺傳物質 溶胞區段係與該等含有用於溶解該溶胞區段中之該等 細胞之溶胞試劑的試劑貯存槽之一者流體連通; 第一核酸擴增區段,該第一核酸擴增區段係位於 胞區段下游以用於擴增該遺傳物質內的第一核酸序列 第二核酸擴增區段,該第二核酸擴增區段係位於 一核酸擴增區段下游以用於擴增源自該第一核酸擴增 之擴增子內的第二核酸序列;其中, 該透析區段、該溶胞區段、該第一核酸擴增區段 第二核酸擴增區段皆承載於該載體基板上。 GCF016.2較佳地,該第一核酸擴增區段係第一 酶鏈鎖反應(PCR )區段,且該二核酸擴增區段係第 合酶鏈鎖反應(PCR)區段。 本之 包含 界値 細胞 用於 ,該 標靶 該溶 :及 該第 區段 及該 聚合 二聚 -49- 201209158 GCF016.3較佳地,該第一 PCR區段具有第一組引子 對且該第一組引子對係用於黏合第一組亙補核酸序列,及 該第二PCR區段具有第二組引子對且該第二組引子對係用 於黏合第二組互補核酸序列,該第一組互補核酸序列與該 第二組互補核酸序列不同。 GCF016.4 較佳地,該第一PCR區段和該第二PCR區 段係經建構以用不同擴增參數進行操作,該等擴增參數係 下列參數之至少一者: 反轉錄酶種類; 聚合酶種類; 去氧核糖核苷三磷酸濃度; 緩衝溶液; 熱循環時間; 熱循環重複次數;及 於P C R之一特定階段期間內的溫度。 GCF016.5較佳地,該LOC裝置亦具有雜合區段,該 雜合區段位於該第二PCR區段下游,且該雜合區段具有探 針陣列及光感測器,該探針陣列係用於與標靶核酸序列雜 合且該光感測器係用於偵測該陣列中之任何探針的雜合反 應。 GCF016.6較佳地,該透析區段具有第一通道、第二 通道及複數個孔,該第一通道係與位於上游末端之該入口 流體連通,該第二通道係與位於下游端之廢液通道流體連 通,該等孔小於該等標靶細胞且大於該等較小成分,該第 -50- 201209158 二通道藉由該等孔與該第一通道流體連通而使該等標靶細 胞留在該第一通道內同時使該等較小成分流入該第二通道 〇 GCF016.7較佳地,該第一通道和該第二通道係經建 構以藉由毛細作用注滿該樣本。 GCF0 16.8較佳地,該溶胞區段具有主動閥,該主動 閥係於溶胞期間使該等標靶細胞留在該溶胞區段內,使得 當打開該主動閥時,流向該培育區段的毛細驅動流動得以 繼續。 GCF0 16.9較佳地,該第一核酸擴增區段係第一恆溫 核酸擴增區段,且該第二核酸擴增區段係第二恆溫核酸擴 增區段。 GCF0 16.10較佳地,該等試劑貯存槽各自具有使試 劑留在該等試劑貯存槽內的表面張力閥,該表面張力閥具 有彎液面錨’,該彎液面錨係用於定住該試劑之彎液面直到 該試劑之彎液面與該樣本流接觸而去除該彎液面以允許該 試劑從該試劑貯存槽流出。 GCF016.il較佳地,該LOC裝置亦具有CMOS電路、 溫度感測器及微系統技術(MST )層,該MST層包含該第 —PCR區段和該第二PCR區段,其中該CMOS電路係設置於 該載體基板和該MST層之間,該CMOS電路係經建構以使 用該溫度感測器之輸出達成該第一PCR區段和該第二PCR 區段的反饋控制。 GCF01 6.1 2較佳地,該第一PCR區段具有PCR微通道 -51 - 201209158 ,該PCR微通道係使該樣本進行熱循環,該PCR微通道界 定具有低於1 00000平方微米之流動橫斷截面積的流動路徑 〇 GCF016.13 較佳地,該PCR微通道具有至少一個長形 加熱器元件,該長形加熱器元件係與該PCR微通道呈平行 延伸。 GCF016.14 較佳地,該PCR區段具有複數個長形PCR 腔室,且每個長形PCR腔室係由該PCR微通道之個別區段 形成,該PCR微通道具有由一系列寬曲流道所形成之蜿蜒 構形,且每個寬曲流道係一個用於形成該等長形PCR腔室 之一者的通道區段。 GCF016.15較佳地,該LOC裝置亦具有容納用於PCR 之試劑的試劑貯存槽;及 具有孔之表面張力閥,該具有孔之表面張力閥係經建 構以定住該試劑之彎液面,如此該彎液面使該試劑保留在 該試劑貯存槽內直到該彎液面與流體樣本接觸而去除該彎 液面且該試劑流出該試劑貯存槽。 GCF016.16較佳地,該LOC裝置亦具有用於容納該等 探針的雜合腔室陣列,使得每個雜合腔室內的該等探針係 經配置以與該等標靶核酸序列之一者雜合。 GCF0 16.17較佳地,該光感測器係配準該等雜合腔 室設置而成的光二極體陣列。 GCF016.18較佳地,該CMOS電路具有數位記憶體及 數據界面,該數位記億體係用於儲存源自該光感測器之輸 -52- 201209158 出的雜合數據,且該數據界面係傳輸該雜合數據至外部裝 置。 GCF016.19較佳地,該第一 PCR區段具有主動閥,該 主動閥係於熱循環期間使液體保留在該第一 PCR區段內且 回應源自該CMOS電路之啓動信號而允許液體流至該第一 雜合腔室陣列。 GCF016.20較佳地,該主動閥係沸騰啓動式閥,該 沸騰啓動式閥具有彎液面錨及加熱器,該彎液面錨係經建 構以定住該阻止液體之毛細驅動流動的彎液面,且該加熱 器係用於使該液體沸騰以使該彎液面脫離該彎液面錨,使 得毛細驅動流動得以繼續。 該使用簡易、可量產且費用不高的基因體分析LOC裝 置係藉由該L0C裝置之樣本放置槽接收生物樣本,且利用 儲存於該L0C裝置之試劑貯存槽內的試劑使用該LOC裝置 之透析區段分離樣本中所含的白血球、於該L0C裝置之化 學溶胞腔室中溶解白血球以釋出白血球之遺傳物質、於該 LOC裝置之培育區段中對樣本之遺傳物質進行預處理、擴 增標靶基因序列,及藉著與寡聚核苷酸探針進行雜合且藉 由該L Ο C裝置之整合式成像陣列感測該等探針之雜合反應 以分析樣本之核酸序列。 該透析區段之功能係從樣本中萃取附加訊息且提高分 析檢驗系統之靈敏度、信號雜訊比和動態範圍。整合於裝 置中的透析區段可提供低的系統元件數量和簡約的製造程 序,從而獲得不昂貴的分析檢驗系統。 -53- 201209158 該溶胞方法係自樣本中的細胞萃取出欲進行分析和診 斷之標靶物且提供該等標靶物之連續性處理與分析。整合 於該裝置中的溶胞子單元元件可提供簡約的分析檢驗程序 、低的系統元件數量及簡約的系統製造程序,從而獲得不 昂貴的分析檢驗系統。 標靶基因序列之擴增反應可提高該分析檢驗系統之靈 敏度與信號雜訊比。再者,該等串接之擴增腔室允許對該 擴增處理之早期循環和晚期循環做分段式的局部最佳化, 從而提高分析檢驗之靈敏度、信號雜訊比和可靠度。 該探針雜合區段係藉由提供雜合反應達成該等標靶物 之分析。整合式探針雜合區段提供一種使用簡易、可量產 、不昂貴且具有低系統元件數量的整合式解決方案。 該整合式成像感測器免除對昂貴之外部成像系統的需 求且提供具有低系統元件數量、可量產且不昂貴的整合式 解決方案,此整合式解決方案係一種小巧、質輕且極方便 攜帶之系統。該整合式成像感測器因受益於大的光線收集 角度而提高讀取靈敏度且免除在光學收集系統中使用諸多 光學元件之需求。 整合於該LOC裝置中且把持該分析檢驗之所有試劑需 求的該等試劑貯存槽可提供低的系統元件數量和簡約的製 造程序’從而獲得不昂貴的分析檢驗系統。 【實施方式】 綜述 •54- 201209158 此綜述指出納入本發明具體實施例之分子診斷系統的 主要構件。該系統結構與操作之全面性細節將稍後陳述於 本案說明書中。 參閱第1、2、3、139和140圖,該系統具有下列首要 構件:201209158 Created a program to obtain an inexpensive analytical inspection system. GCF0 16_1 This invention provides a wafer-on-lab (LOC) device for biological sample gene analysis, the LOC device for receiving an inlet of the sample; a carrier substrate; a dialysis section, the dialysis section The cells in the sample larger than the predetermined cells are separated from the smaller components, wherein the cells are larger than the predetermined threshold, including the target cells containing the genetic material for analysis; a plurality of reagent storage tanks; a lysis section, the lysis zone a segment is located downstream of the dialysis section to dissolve the target cells to release a genetic material lysing segment within the target cells and the cells for dissolving the cells in the lysing segment One of the reagent storage tanks of the lysis reagent is in fluid communication; a first nucleic acid amplification section, the first nucleic acid amplification section being located downstream of the cell section for amplifying the first nucleic acid sequence in the genetic material a second nucleic acid amplification segment, the second nucleic acid amplification segment being located downstream of a nucleic acid amplification segment for amplifying a second nucleic acid sequence derived from the amplicon of the first nucleic acid amplification; The dialysis section, the Cell segments, the first nucleic acid amplification of a second nucleic acid segment amplification segments are supported on the carrier substrate. Preferably, the first nucleic acid amplification segment is a first enzyme chain reaction (PCR) segment and the second nucleic acid amplification segment is a synthase chain reaction (PCR) segment. The inclusion of the boundary cell for the target is: and the first segment and the polymeric dimerization -49 - 201209158 GCF016.3 preferably, the first PCR segment has a first set of primer pairs and the The first set of primer pairs is used to bind the first set of complementary nucleic acid sequences, and the second set of primers has a second set of primer pairs and the second set of primer pairs is used to bind the second set of complementary nucleic acid sequences, the first A set of complementary nucleic acid sequences differs from the second set of complementary nucleic acid sequences. GCF016.4 Preferably, the first PCR segment and the second PCR segment are constructed to operate with different amplification parameters, the amplification parameters being at least one of the following parameters: a reverse transcriptase species; Polymerase species; deoxyribonucleoside triphosphate concentration; buffer solution; thermal cycle time; number of cycles of thermal cycling; and temperature during a particular phase of PCR. GCF016.5 Preferably, the LOC device also has a hybrid segment located downstream of the second PCR segment, and the hybrid segment has a probe array and a photo sensor, the probe The array is used to hybridize to a target nucleic acid sequence and the photosensor is used to detect a heterozygous reaction of any of the probes in the array. GCF016.6 preferably, the dialysis section has a first passage, a second passage, and a plurality of holes, the first passage being in fluid communication with the inlet at the upstream end, the second passage being associated with the waste at the downstream end The liquid channel is in fluid communication, and the holes are smaller than the target cells and larger than the smaller components, and the second channel of the -50-201209158 is in fluid communication with the first channel to allow the target cells to remain Simultaneously flowing the smaller components into the second channel 〇GCF016.7 in the first channel. Preferably, the first channel and the second channel are configured to fill the sample by capillary action. Preferably, the lysis zone has an active valve that retains the target cells in the lysis zone during lysis so that when the active valve is opened, the flow is directed to the culturing zone The capillary drive flow of the segment continues. GCF0 16.9 Preferably, the first nucleic acid amplification segment is a first thermostatic nucleic acid amplification segment and the second nucleic acid amplification segment is a second thermostatic nucleic acid amplification segment. GCF0 16.10 Preferably, the reagent storage tanks each have a surface tension valve for retaining reagents in the reagent storage tanks, the surface tension valve having a meniscus anchor, the meniscus anchor system for holding the reagent The meniscus is removed until the meniscus of the reagent contacts the sample stream to remove the meniscus to allow the reagent to flow out of the reagent reservoir. GCF016.il preferably, the LOC device also has a CMOS circuit, a temperature sensor and a microsystem technology (MST) layer, the MST layer comprising the first PCR segment and the second PCR segment, wherein the CMOS circuit And being disposed between the carrier substrate and the MST layer, the CMOS circuit being configured to use the output of the temperature sensor to achieve feedback control of the first PCR segment and the second PCR segment. GCF01 6.1 2 Preferably, the first PCR segment has PCR microchannels -51 - 201209158, the PCR microchannels thermally cycling the sample, the PCR microchannel defining a flow cross-section having less than 100,000 square microns Flow path of the cross-sectional area 〇GCF016.13 Preferably, the PCR microchannel has at least one elongated heater element that extends parallel to the PCR microchannel. GCF016.14 Preferably, the PCR segment has a plurality of elongate PCR chambers, and each elongate PCR chamber is formed by individual segments of the PCR microchannel having a series of wide curves The channel formed by the flow path, and each wide curved channel is a channel section for forming one of the elongate PCR chambers. Preferably, the LOC device also has a reagent storage tank containing reagents for PCR; and a surface tension valve having a hole configured to hold the meniscus of the reagent, The meniscus thus retains the reagent in the reagent reservoir until the meniscus contacts the fluid sample to remove the meniscus and the reagent exits the reagent reservoir. Preferably, the LOC device also has an array of hybrid chambers for accommodating the probes such that the probes within each hybrid chamber are configured to interact with the target nucleic acid sequences One is heterozygous. GCF0 16.17 Preferably, the photosensor is associated with an array of photodiodes arranged in the hybrid chambers. GCF016.18 Preferably, the CMOS circuit has a digital memory and a data interface, and the digital system is used to store the hybrid data derived from the optical sensor - 52 - 201209158, and the data interface is The hybrid data is transmitted to an external device. GCF016.19 preferably, the first PCR section has an active valve that retains liquid within the first PCR section during thermal cycling and allows liquid flow in response to an activation signal originating from the CMOS circuit To the first hybrid chamber array. GCF016.20 Preferably, the active valve is a boiling start valve having a meniscus anchor and a heater, the meniscus anchor being constructed to hold the meniscus that prevents the capillary drive flow of the liquid And the heater is used to boil the liquid to disengage the meniscus from the meniscus anchor, such that the capillary drive flow continues. The genomic analysis LOC device that is simple to use, mass-produced, and inexpensive is to receive a biological sample by using a sample placement tank of the LOC device, and using the reagent stored in the reagent storage tank of the LOC device to use the LOC device. The dialysis section separates the white blood cells contained in the sample, dissolves the white blood cells in the chemical lysis chamber of the LOC device to release the genetic material of the white blood cells, and pretreats the genetic material of the sample in the incubation section of the LOC device, Amplifying the target gene sequence and analyzing the nucleic acid sequence of the sample by heterozygous with the oligonucleotide probe and sensing the heterozygous reaction of the probes by the integrated imaging array of the L Ο C device . The function of the dialysis section is to extract additional information from the sample and to increase the sensitivity, signal to noise ratio, and dynamic range of the analytical inspection system. The dialysis section integrated into the unit provides a low number of system components and a simple manufacturing process, resulting in an inexpensive analytical inspection system. -53- 201209158 This lysis method extracts the target to be analyzed and diagnosed from the cells in the sample and provides continuity processing and analysis of the targets. The lysed subunit elements integrated in the device provide a simple analytical test procedure, a low number of system components, and a simple system manufacturing process to obtain an inexpensive analytical inspection system. The amplification reaction of the target gene sequence can increase the sensitivity and signal to noise ratio of the analytical test system. Furthermore, the cascaded amplification chambers allow for segmental local optimization of the early and late cycles of the amplification process, thereby increasing sensitivity, signal to noise ratio and reliability of the assay. The probe hybrid segment achieves analysis of the targets by providing a hybrid reaction. The integrated probe hybrid section provides an integrated solution that is easy to use, mass-produced, inexpensive, and has a low number of system components. The integrated imaging sensor eliminates the need for expensive external imaging systems and offers an integrated solution with low system component count, mass production and low cost. This integrated solution is compact, lightweight and extremely convenient. Carrying system. The integrated imaging sensor increases read sensitivity and eliminates the need to use many optical components in an optical collection system by benefiting from large light collection angles. The reagent reservoirs integrated into the LOC device and holding all of the reagents required for the assay can provide a low number of system components and a simple manufacturing procedure' to obtain an inexpensive analytical inspection system. [Embodiment] Summary • 54-201209158 This review indicates the main components of the molecular diagnostic system incorporating the specific embodiment of the present invention. The full details of the structure and operation of the system will be described later in this specification. Referring to Figures 1, 2, 3, 139 and 140, the system has the following primary components:

檢驗模組1 0和檢驗模組1 1係典型USB記憶鍵的尺寸且 該等模組之造價極低。檢驗模組1 0和檢驗模組1 1各自包含 一個微流體裝置,該微流體裝置通常爲晶片上實驗室( LOC)裝置30之形態,該LOC裝置30已預先裝入用於分子 診斷之分析檢驗的試劑和通常1 000種以上之探針(見第1 和139圖)。第1圖中槪要顯示之檢驗模組10使用以螢光爲 基礎之偵測技術以鑑定標靶分子,第1 39圖中之檢驗模組 1 1使用以電致化學發光爲基礎之偵測技術。該LOC裝置30 具有整合式光感測器44以用於螢光偵測或電致化學發光偵 測(此等偵測方法將詳述於下檢驗模組1 〇和檢驗模組 1 1兩者皆使用標準微型USB插頭14以用於供電、資料傳輸 及控制,該兩模組皆具有印刷電路板(PCB ) 5 7 ’且該兩 模組皆具有外部供電電容器32和電感器15。檢驗模組1〇和 檢驗模組11兩者皆爲單次使用性以達成採即用型滅菌包裝 進行批量生產及銷售。 外殼13具有用於接收生物樣本的大放置槽24及於使用 前遮蓋該大放置槽24的可撕式滅菌密封膠帶22 (膠帶22較 佳具有低黏性黏著劑)。具有薄膜護片410之密封膜408形 成部份的外殻1 3以降低該檢驗模組內的除濕作用且同時釋 -55- 201209158 放因氣壓小幅波動所造成之壓力。該薄膜護片410保護該 密封膜408免於受損。 檢驗模組讀取器12經由微型USB插槽16供電給該等檢 驗模組1 〇或檢驗模組1 1。檢驗模組讀取器1 2可採用許多不 同形式,且於稍後說明此等形式之選擇。第1、3和1 3 9圖 中所顯示的讀取器12之版本係一種智慧型手機具體實施例 。第3圖中顯示此讀取器12之方塊圖。處理器42.運行源自 程式儲存器43的應用軟體。該處理器42亦連結顯示螢幕18 及使用者介面(UI)觸控螢幕17和按鍵19、蜂巢式無線電 21、無線網路連接器23及衛星導航系統25。蜂巢式無線電 21和無線網路連接器23係用於通訊。衛星導航系統25係用 於更新附帶位置資料之流行病學資料庫。或可選擇經由觸 控螢幕17或按鍵19手動輸入該位置資料。資料儲存器27容 納遺傳和診斷訊息、檢驗結果、病患資料、用於識別每每 種探針和該探針之陣列位置的分析檢驗與探針資料。資料 儲存器27與程式儲存器43可被分享給一個共用的記憶設備 。安裝於該檢驗模組讀取器1 2上的應用軟體提供結果分析 且隨附額外的檢驗與診斷訊息。 爲進行診斷性檢驗,係使檢驗模組1 0 (或檢驗模組1 1 )插入該檢驗模組讀取器12上的微型USB插槽16。掀起該 滅菌密封膠帶2 2且把(液體形式之)生物樣本注入該樣本 大放置槽24。按下啓動鈕20開始藉由該應用軟體進行檢驗 。該樣本流入該LOC裝置30,且該載板上之分析檢驗係對 該樣本核酸(該標靶物)進行萃取、培育、擴增且利用預 -56- 201209158 先合成之雜合反應性寡聚核苷酸探針與該樣本核酸進行雜 合。於檢驗模組1 0之情況下(檢驗模組1 1使用以電致化學 發光爲基礎之偵測法),該等探針係經螢光標示,且容納 於該外殼13內的發光二極體(LED ) 26提供必要的激發光 以誘發該等經雜合之探針發出螢光(見第1和2圖)。於檢 驗模組1 1之情況下(檢驗模組1 1使用以電致化學發光爲基 礎之偵測法),該LOC裝置30中裝有上述ECL探針,且不 φ 必使用發光二極體(LED ) 26產生冷光之發光作用。改用 電極860和電極870提供激發電流(見第140圖)。使用經 整合於各個LOC裝置之CMOS電路中的光感測器44偵測該 發光作用(螢光或冷光)。所測得之信號係經放大且轉換 成數位輸出値,且藉由該檢驗模組讀取器1 2分析該數位輸 出値。隨後該讀取器顯示結果。 該資料可就地儲存且/或上傳至含有病患記錄的網路 伺服器。從該檢驗模組讀取器1 2上移除該檢驗模組1 0或檢 φ 驗模組1 1且妥善處置該檢驗模組。 . 第1、3及139圖顯示建構成行動電話/智慧型手機28般 的檢驗模組讀取器1 2。在其他形態方面,該檢驗模組讀取 器係可用於醫院、私人開業診所或實驗室中的手提式個人 電腦/筆記型電腦1 0 1、專用讀取器1 03、電子書讀取器1 〇7 、平板電腦109或桌上型電腦105 (見第141圖)。該讀取 器可連結諸多的附加應用用途,例如病患記錄、帳單、線 上資料庫及多人使用環境。該讀取器亦可連接諸多的當地 週邊設備或遠端週邊設備,例如印表機及病患之智慧卡。 •57- 201209158 參閱第142圖,可經由讀取器12與網路125使用由該檢 驗模組1 〇所生成的資料更新流行病學資料之主機系統1 1 1 內建的流行病學資料庫、遺傳資料之主機系統1 1 3內建的 遺傳資料庫、電子健康記錄(EHR)之主機系統115內建 的電子健康記錄、電子醫療記錄(EMR)之主機系統121 內建的電子醫療記錄及個人健康記錄(PHR)之主機系統 123內建的個人健康記錄。反之,可經由網路125和讀取器 1 2使用流行病學資料之主機系統1 1 1內建的流行病學資料 庫、遺傳資料之主機系統113內建的遺傳資料庫、電子健 康記錄(EHR )之主機系統115內建的電子健康記錄、電 子醫療記錄(EMR)之主機系統121內建的電子醫療記錄 及個人健康記錄(PHR)之主機系統123內建的個人健康 記錄更新該檢驗模組10之LOC裝置30中的數位記億體。 回到第1、2、1 3 9和1 4 0圖’該讀取器1 2使用行動電話 配置內'的電池供電。該行動電話讀取器包含已預先上傳的 所有檢驗和診斷資訊。亦可經由一些無線界面或接觸界面 裝載或更新資料而能與周邊裝置、電腦或線上服務器進行 通訊。微型USB插槽16係供連接電腦或連接主電源供應器 以供電池充電之用。 第7 1圖顯示該檢驗模組1 〇的一個具體實施例,該檢驗 模組1 〇係用於僅需取得特定標靶物之陽性或陰性結果的檢 驗,諸如用於檢驗個人是否感染例如H1N1 A型流感病毒。 爲特定目而建造之僅靠USB供電和作爲指示器的模組47可 勝任此任務。無需其他的讀取器或應用軟體。僅靠USB供 -58- 201209158 電和作爲指示器之模組47上的指示器45發出信號以顯示陽 性或陰性結果。此種結構配置非常適用於大量篩檢。 隨該系統提供的附加工具可包括含有試劑之檢驗試管 (該等試劑係用於某些樣本之預處理)且附帶用於採集樣 本之刮勺和刺血針。第7 1圖顯示一種爲方便使用而包含彈 壓伸縮式刺血針3 90及採血針釋放按鈕3 92的檢驗模組之具 體實施例。衛星電話可用於偏遠地區。 檢驗模組之電子構件The inspection module 10 and the inspection module 1 1 are typical USB memory keys and the cost of such modules is extremely low. The inspection module 10 and the inspection module 1 1 each comprise a microfluidic device, typically in the form of a wafer-on-lab (LOC) device 30, which has been pre-loaded for analysis of molecular diagnostics. The reagents tested and usually more than 1 000 probes (see Figures 1 and 139). The test module 10 to be displayed in Fig. 1 uses a fluorescence-based detection technique to identify target molecules, and the test module 1 in Fig. 39 uses detection based on electrochemiluminescence. technology. The LOC device 30 has an integrated photo sensor 44 for fluorescence detection or electrochemiluminescence detection (the detection methods will be described in detail in both the lower inspection module 1 and the inspection module 1 1). Both standard micro USB plugs 14 are used for power supply, data transfer and control. Both modules have a printed circuit board (PCB) 5 7 ' and both modules have an external supply capacitor 32 and an inductor 15. Both the Group 1 and the inspection module 11 are single-use for mass production and sale in a ready-to-use sterilization package. The housing 13 has a large placement slot 24 for receiving biological samples and covers the large area prior to use. The tearable sterilizing sealing tape 22 of the groove 24 is placed (the tape 22 preferably has a low-adhesive adhesive). The sealing film 408 having the film protective sheet 410 forms part of the outer casing 13 to reduce dehumidification in the inspection module. Acting and simultaneously releasing -55-201209158 due to the pressure caused by small fluctuations in air pressure. The film protector 410 protects the sealing film 408 from damage. The test module reader 12 is powered by the micro USB slot 16 to the same. Inspection module 1 检验 or inspection module 1 1. Inspection The module reader 12 can take many different forms, and the selection of such forms will be described later. The versions of the reader 12 shown in Figures 1, 3 and 139 are a smart phone implementation. The block diagram of the reader 12 is shown in Fig. 3. The processor 42 runs the application software from the program storage 43. The processor 42 also connects the display screen 18 and the user interface (UI) touch screen. 17 and a button 19, a cellular radio 21, a wireless network connector 23, and a satellite navigation system 25. The cellular radio 21 and the wireless network connector 23 are used for communication. The satellite navigation system 25 is used to update the attached location data. Epidemiological database. Alternatively, the location data can be manually entered via touch screen 17 or button 19. Data storage 27 holds genetic and diagnostic information, test results, patient data, and is used to identify each probe and probe. The analysis of the array position of the needle and the probe data. The data storage 27 and the program storage 43 can be shared to a shared memory device. The application software installed on the inspection module reader 12 provides result analysis. Additional diagnostic and diagnostic messages are included. For diagnostic testing, the test module 10 (or test module 1 1) is inserted into the micro USB slot 16 of the test module reader 12. The sealing tape 2 2 is sterilized and a biological sample (in liquid form) is injected into the sample large placement slot 24. Pressing the start button 20 begins the inspection by the application software. The sample flows into the LOC device 30, and the carrier plate The assay is performed by extracting, culturing, and amplifying the sample nucleic acid (the target) and hybridizing the sample nucleic acid with a hybrid reactive oligonucleotide probe synthesized in advance-56-201209158. In the case of the inspection module 10 (the inspection module 1 1 uses an electrochemiluminescence-based detection method), the probes are fluorescently labeled and housed in the housing 13 Body (LED) 26 provides the necessary excitation light to induce the heterozygous probe to emit fluorescence (see Figures 1 and 2). In the case of the inspection module 1 1 (the inspection module 1 1 uses a detection method based on electrochemiluminescence), the LOC device 30 is equipped with the above ECL probe, and the light emitting diode must be used for the φ φ (LED) 26 produces a luminescent effect of luminescence. Switching electrode 860 and electrode 870 are used to provide an excitation current (see Figure 140). The illuminating effect (fluorescent or luminescent) is detected using a photo sensor 44 integrated in a CMOS circuit of each LOC device. The measured signal is amplified and converted to a digital output 値, and the digital output 値 is analyzed by the test module reader 12. The reader then displays the result. This information can be stored locally and/or uploaded to a web server containing patient records. The inspection module 10 or the inspection module 1 1 is removed from the inspection module reader 1 2 and the inspection module is properly disposed. Figures 1, 3, and 139 show the construction of a test module reader 12 that constitutes a mobile phone/smartphone 28. In other aspects, the test module reader can be used in a portable personal computer/notebook computer in a hospital, a private practice clinic or a laboratory, a dedicated reader 101, an electronic book reader 1 〇7, tablet 109 or desktop computer 105 (see Figure 141). The reader can be used to link a variety of additional application uses, such as patient records, bills, online databases, and multi-person environments. The reader can also be connected to a variety of local peripherals or remote peripherals such as printers and patient smart cards. • 57- 201209158 Referring to Figure 142, the epidemiological database built into the host system 1 1 1 that can update epidemiological data via the reader 12 and the network 125 using the data generated by the test module 1 〇 The host system of the genetic data system 1 1 3 built-in genetic database, electronic health record (EHR) host system 115 built-in electronic health record, electronic medical record (EMR) host system 121 built-in electronic medical records and A personal health record built into the personal health record (PHR) host system 123. On the other hand, the epidemiological database built into the host system 1 1 1 of the epidemiological data, the genetic database built in the host system 113 of the genetic data, and the electronic health record can be used via the network 125 and the reader 12. The electronic health record built in the host system 115 of the EHR), the electronic medical record built into the host system 121 of the electronic medical record (EMR), and the personal health record built into the host system 123 of the personal health record (PHR) update the test mode. The digits in the LOC device 30 of group 10 are recorded in billions. Returning to the 1, 2, 1 3 9 and 1 4 0 diagrams, the reader 1 2 is powered by the battery in the mobile phone configuration. The mobile phone reader contains all the inspection and diagnostic information that has been pre-uploaded. It can also be loaded or updated via some wireless interface or contact interface to communicate with peripheral devices, computers or online servers. The micro USB slot 16 is for connecting to a computer or to a mains power supply for charging the battery. Figure 7 1 shows a specific embodiment of the test module 1 , for testing that only needs to obtain positive or negative results for a particular target, such as for testing whether an individual is infected, for example, H1N1 Influenza A virus. Modules 47 powered solely by USB and as indicators for specific purposes are sufficient for this task. No other readers or application software is required. USB-only -58-201209158 and the indicator 45 on the module 47 as an indicator signal a positive or negative result. This configuration is ideal for large-scale screening. Additional tools provided with the system may include test tubes containing reagents (for the pretreatment of certain samples) with a spatula and lancet for collecting samples. Fig. 7 shows a specific embodiment of an inspection module including a compression telescopic lancet 3 90 and a lancet release button 3 92 for convenience of use. Satellite phones can be used in remote areas. Inspection module electronic component

第2和140圖分別是檢驗模組10及檢驗模組1 1內部之電 子構件的方塊圖。整合於LOC裝置30內的CMOS電路具有 USB裝置驅動器36、控制器34、USB相容式LED驅動器29 、時鐘3 3、功率調節器3 1、隨機存取記憶體(RAM ) 3 8和 程式及資料快閃記憶體40。此等元件爲整個檢驗模組1 0或 檢驗模組1 1 (包含光感測器44、溫度感測器1 70、液體感 測器174、各種加熱器152、154、182、234連同相關驅動 器37和39及記錄器35和41 )提供控制和記憶功能。僅發光 二極體(LED ) 26 (如檢驗模組10之例子)、外部供電電 容器32和微型USB插頭14位於該LOC裝置30之外部。該 LOC裝置30包含多個用於與此等外部構件連接的焊墊( bond pad)。該隨機存取記憶體(RAM) 38和該程式及資 料快閃記億體40具有該應用軟體和用於1〇〇〇個以上之探針 的診斷及檢驗資訊(例如經加密之快閃/安全性儲存)。 於經建構以進行ECL偵測之檢驗模組1 1的情況中不需發光 -59- 201209158 二極體(LED) 26(見第139和140圖)。藉由該LOC裝置 3 0加密資料以達到安全儲存且與外部裝置進行安全通訊。 LOC裝置30裝有電致化學發光探針,且該等雜合腔室各自 具有一對ECL激發電極860和870。 多種檢驗模組1 〇係經製造成諸多檢驗類型以供現成使 用。該等檢驗方式之間的差異取決於該等試劑和探針所進 行之載板上分析檢驗。 利用此系統快速鑑定的一些傳染性疾病具體實例包含 •流感一 A型流感病毒、B型流感病毒、C型流感病毒 、傳染性鮭魚貧血病毒(Isavirus)、托高土病毒( Thogotovirus ) •肺炎—呼吸道細胞融合性病毒(R s V )、腺病毒、 間質肺炎病毒、肺炎鏈球菌、金黃色葡萄球菌 •結核病-結核分枝桿菌、牛分枝桿菌、非洲分枝桿 菌、卡氏分枝桿菌及田鼠分枝桿菌 •惡性瘧疾原蟲、弓漿蟲和其他原生動物寄生蟲 •傷寒-腸道性傷寒沙門氏菌血清變異型( salmonella enterica serovar typhi) •伊波拉病毒 •人類免疫不全病毒(HIV ) •登革熱-黃病毒 • A型、B型、C型、D型、E型之肝炎 •院內感染-例如困難梭狀桿菌、萬古黴素抗藥性腸 -60- 201209158 球菌及二甲苯青黴素抗藥性金黃色葡萄球菌 •單純皰疹病毒(HSV ) •巨細胞病毒(CMV ) •艾普斯坦-巴爾二氏病毒(EBV) •腦炎—日本腦炎病毒、錢德普病毒(Chandipura virus ) •百日咳一百日咳博氏菌 •麻疼-副黏液病毒 •腦膜炎一肺炎鏈球菌及腦膜炎雙球菌 •炭疽熱_炭疽桿菌 可利用此系統鑑定的一些遺傳性疾病具體實例包含: •囊狀纖維化 •血友病 •鐮刀型貧血症 •泰薩二氏症(Tay-Sachs disease,又稱家族性黑朦 癡呆症) •血色素沉著症 •白腦病(cerebral arteriopathy) •克隆氏症(crohn's disease) •多囊性腎臟病 •先天性心臟病 •蕾特式症(Rett syndrome ) -61 - 201209158 可藉由該診斷系統鑑定之少數癌症選項包含: •卵巢腫瘤 •大腸癌 •多發性內分泌贅瘤 •視網膜胚細胞瘤 •透克氏症(turcot syndrome) 上述選項尙未詳盡列出’且該診斷系統可經建構以利 用核酸及蛋白質體分析技術偵測遠勝於上述種類之各種疾 病與身體狀況。 系統構件之細節構造 L0C裝置 LOC裝置30係該診斷系統的核心。該LOC裝置使用微 流體平臺快速地執行依賴核酸之分子診斷性分析檢驗的四 個主要步驟,即製備樣本、萃取核酸、擴增核酸與偵測。 該LOC裝置亦具有不同用途,且將於稍候詳細說明此等用 途。如上述,檢驗模組1 0和檢驗模組1 1可採用不同之結構 配置以偵測不同標靶物。同樣地,該LOC裝置30具有許多 針對所關注之標靶物量身定做的不同具體實施例。該L0C 裝置30之其中一種形式係用於對全血樣本內之病源中的標 靶核酸序列進行螢光偵測的L0C裝置3 0 1。爲達成說明目 的,現將參照第4~26及27〜57圖詳細描述L0C裝置301之結 構和操作。 第4圖係該L0C裝置301之構造的槪要表示圖。爲求方 -62- 201209158 便’係使用與該LOC裝置3 0 1用於執行該方法階段之功能 區段相應的元件符號標示出第4圖中所示之該等方法階段 。與核酸分子診斷性分析檢驗之各個主要步驟相關的該等 方法階段亦標示爲:樣本之置入與製備階段288、萃取階 段290 '培育階段291、擴增階段292及偵測階段294。稍候 將更詳細地描述該LOC裝置301之各種貯存槽、腔室、閥 和其他構件。 | 第5圖係LOC裝置301之透視圖。該LOC裝置301係使用 CMOS及MST (微系統技術)大量製造技術所製造而成。 第12圖之槪要(未按比例)局部剖面圖中繪示該LOC裝置 301之層狀結構。該LOC裝置301具有用於承載CMOS + MST 晶片48之矽基板84,該CMOS+ MST晶片4 8包含互補金屬氧 化物半導體(CMOS)電路86和微系統技術(MST)層87 ,且具有覆蓋於該MST層87上的蓋層46。針對本專利說明 書之該等目的,「微系統技術(MST )層」一詞係指一種 使用各種試劑處理樣本之結構與膜層的集合體。因此,此 . 等結構與構件係經建構以界定多條具有特性尺寸的流動路 徑,該等特性尺寸可於處理期間支持液體(該液體與該樣 本具有類似之物理性質)進行毛細驅動流動。有鑒於此, —般使用面型(surface)微機械加工技術及/或體型(bulk )微加工技術製造該等MST層和構件。然而,其他製造方 法亦可製造出尺寸經設計以達成毛細驅動流動且可處理極 小量體積的結構和構件。本案說明書中所描述之特疋具體 實施例顯示該MST層係承載於該CMOS電路86上但排除該 -63- 201209158 蓋層46之特徵以外的多個結構與主動構件。然而’所屬領 域中熟悉該項技藝者將明白爲使該MST層能夠處理樣本, 該MST層不必具有下層的CMOS或確實不具有上方的蓋層 〇 下列圖式中所顯示之LOC裝置的整體尺寸係1760微米 x 5 8 24微米。當然,針對不同應用製成的LOC裝置可能具 有不同尺寸。 第6圖顯示與該蓋層之特徵重疊的該MST層87之特徵 。第6圖中所示之插圖AA~AD和插圖AG〜AH分別放大顯示 於第13、14、35、56、55和63圖中,且對該等插圖AA〜AD 和插圖AG~AH做詳細說明如下以供全面性地瞭解該LOC裝 置301中之各個結構。第7〜10圖單獨顯示該蓋層46之特徵 ,同時第11圖單獨顯示該CMOS + MST裝置48之結構。 層狀結構 第12和22圖係圖示該CMOS + MST裝置48之層狀結構、 蓋層46和兩者間之流體互動關係的槪略圖。該等圖式未按 比例繪示,目的是作爲圖解說明之用。第12圖係穿過樣本 入口 68的槪要剖面圖,且第22圖係穿過貯存槽54的槪要剖 面圖。最佳如第12圖所示般,該CMOS + MST裝置48具有矽 基板84,該矽基板84支撐著CMOS電路86,且該CMOS電路 86操作位於上方該MST層87內的主動元件。鈍化層88密封 且保護該CMOS層8 6不受流經該MST層8 7之流體影響。 流體流經分別位於該蓋層46及MS T通道層100中的該 -64- 201209158 等蓋層通道94和該等微系統技術(MST )通道90 (見第7 和16圖)。細胞運送作用發生在製造於該蓋層46中的該等 較大通道94內,同時生化處理係於該等較小的MST通道90 內進行。細胞運送通道之尺寸係建構成能把樣本中的細胞 運送至該等MST通道90內之預定位置處。運送尺寸大於20 微米的細胞(例如某些白血球)需要大於2 0微米之通道尺 寸,因此流動橫斷截面積係大於400平方微米。MST通道 (特別是位於該LOC裝置內無需運送細胞之位置處的MST 通道)可明顯較小。 將可理解蓋層通道94與MST通道90係統稱,且特別是 M ST通道9 0亦可依據通道的特殊功能而例如稱爲經加熱之 微通道或透析MST通道。係利用光阻進行圖案化且蝕刻穿 透沉積於該鈍化層88上的MS Τ通道層100而形成MS Τ通道90 。藉由頂層66蓋住該等MST通道90,且該頂層66形成該 CMOS + MST裝置48之頂部(參考圖式中顯示之方位)。Figures 2 and 140 are block diagrams of the electronic components inside the inspection module 10 and the inspection module 1 1 , respectively. 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 clock 3 3, a power conditioner 31, a random access memory (RAM) 38, and a program. Data flash memory 40. These components are the entire inspection module 10 or inspection module 1 1 (including photosensor 44, temperature sensor 170, liquid sensor 174, various heaters 152, 154, 182, 234 along with associated drivers) 37 and 39 and recorders 35 and 41) provide control and memory functions. Only the light emitting diode (LED) 26 (as in the example of the test module 10), the external power supply capacitor 32 and the micro USB plug 14 are located outside of the LOC device 30. The LOC device 30 includes a plurality of bond pads for connection to such external components. The random access memory (RAM) 38 and the program and data flashing body 40 have the application software and diagnostic and verification information for more than one probe (eg, encrypted flash/safe Sexual storage). No illumination is required in the case of an inspection module 1 1 constructed for ECL detection -59- 201209158 Diode (LED) 26 (see Figures 139 and 140). The data is encrypted by the LOC device 30 to achieve secure storage and secure communication with external devices. The LOC device 30 is equipped with electrochemiluminescent probes, and each of the hybrid chambers has a pair of ECL excitation electrodes 860 and 870. A variety of inspection modules 1 are manufactured in a variety of inspection types for off-the-shelf use. The difference between these assays depends on the assays on the plates performed by the reagents and probes. Some examples of infectious diseases that are rapidly identified using this system include • Influenza A, Influenza B, Influenza C, Isavirus, Thogotovirus • Pneumonia – Respiratory Cell Fusion Virus (R s V ), Adenovirus, Interstitial Pneumonia Virus, Streptococcus pneumoniae, Staphylococcus aureus • Tuberculosis - Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium tuberculosis, Mycobacterium tuberculosis Mycobacterium vaccae • Plasmodium falciparum, Toxoplasma gondii and other protozoan parasites • Salmonella enteric serovar typhi • Ebola virus • Human immunodeficiency virus (HIV) • Dengue-flavor virus • Type A, B, C, D, and E hepatitis • In-hospital infections – such as Clostridium difficile, vancomycin resistant bowel -60- 201209158 Cocci and xylene penicillin resistant golden yellow Staphylococcus • Herpes Simplex Virus (HSV) • Cytomegalovirus (CMV) • Epstein-Barr's Virus (EBV) • Encephalitis – Japanese Brain Inflammatory virus, Chandipura virus • Pertussis I. pneumoniae • Hemp pain - paramyxovirus • Meningitis - Streptococcus pneumoniae and meningococcus • Anthrax _ Anthrax can be identified by this system Specific examples of hereditary diseases include: • Cystic fibrosis • Hemophilia • Sickle anemia • Tay-Sachs disease (also known as familial sputum dementia) • Hemochromatosis • White encephalopathy ( Cerebral arteriopathy) • Crohn's disease • Polycystic kidney disease • Congenital heart disease • Rett syndrome -61 - 201209158 A few cancer options that can be identified by this diagnostic system include: • Ovary Tumors • Colorectal cancer • Multiple endocrine neoplasia • Retinal blastoma • Turcot syndrome The above options are not exhaustively listed and the diagnostic system can be constructed to detect distant using nucleic acid and proteomic analysis techniques. It is better than the various diseases and physical conditions of the above categories. Detail Construction of System Components L0C Device The LOC device 30 is the core of the diagnostic system. The LOC device uses a microfluidic platform to rapidly perform four major steps in nucleic acid-dependent molecular diagnostic assays: preparing samples, extracting nucleic acids, amplifying nucleic acids, and detecting. The LOC device also has different uses and will be described in detail later. As described above, the inspection module 10 and the inspection module 1 1 can be configured in different configurations to detect different targets. As such, the LOC device 30 has a number of different specific embodiments tailored to the target of interest. One of the L0C devices 30 is a LOC device 301 for fluorescence detection of a target nucleic acid sequence in a pathogen within a whole blood sample. For the purpose of explanation, the structure and operation of the LOC device 301 will now be described in detail with reference to Figures 4 to 26 and 27 to 57. Fig. 4 is a schematic diagram showing the construction of the LOC device 301. For the sake of the method -62-201209158, the method symbols corresponding to the functional sections of the LOC device 310 for performing the method stages are labeled with the method stages shown in Fig. 4. The method stages associated with each of the major steps in the diagnostic assay of nucleic acid molecules are also indicated as: sample insertion and preparation stage 288, extraction stage 290 'cultivation stage 291, amplification stage 292, and detection stage 294. The various reservoirs, chambers, valves and other components of the LOC device 301 will be described in more detail later. 5 is a perspective view of the LOC device 301. The LOC device 301 is manufactured using mass production techniques of CMOS and MST (Microsystem Technology). The layered structure of the LOC device 301 is illustrated in a partial cross-sectional view of Fig. 12 (not to scale). The LOC device 301 has a germanium substrate 84 for carrying a CMOS + MST wafer 48, the CMOS + MST wafer 48 comprising a complementary metal oxide semiconductor (CMOS) circuit 86 and a microsystem technology (MST) layer 87, and having A cap layer 46 on the MST layer 87. For the purposes of this patent specification, the term "microsystem technology (MST) layer" refers to an assembly of structures and layers that process samples using various reagents. Thus, the structures and components are constructed to define a plurality of flow paths having characteristic dimensions that support the liquid (which has similar physical properties to the sample) for capillary drive flow during processing. In view of this, the MST layers and members are typically fabricated using surface micromachining techniques and/or bulk micromachining techniques. However, other manufacturing methods can also produce structures and components that are sized to achieve capillary drive flow and that can handle very small volumes. The specific embodiment described in the description of the present invention shows that the MST layer is carried on the CMOS circuit 86 but excludes a plurality of structures and active components other than the features of the cover layer 46 of the -63-201209158. However, it will be understood by those skilled in the art that in order for the MST layer to be able to process samples, the MST layer does not have to have an underlying CMOS or indeed does not have an overlying cap layer. The overall dimensions of the LOC device shown in the following figures It is 1760 microns x 5 8 24 microns. Of course, LOC devices made for different applications may have different sizes. Figure 6 shows the features of the MST layer 87 that overlap the features of the cap layer. The illustrations AA to AD and the illustrations AG to AH shown in Fig. 6 are enlarged and displayed in the figures 13, 13, 35, 56, 55 and 63, respectively, and the illustrations AA to AD and the illustrations AG to AH are detailed. The following is provided for a comprehensive understanding of the various structures in the LOC device 301. The features of the cap layer 46 are shown separately in Figures 7 through 10, while the structure of the CMOS + MST device 48 is shown separately in Figure 11. Layered Structures Figures 12 and 22 are schematic diagrams showing the layered structure of the CMOS + MST device 48, the cap layer 46, and the fluid interaction between the two. These drawings are not drawn to scale and are intended to be illustrative. Fig. 12 is a cross-sectional view through the sample inlet 68, and Fig. 22 is a cross-sectional view through the storage tank 54. Preferably, as shown in Fig. 12, the CMOS + MST device 48 has a germanium substrate 84 that supports the CMOS circuit 86 and that operates the active components located above the MST layer 87. The passivation layer 88 seals and protects the CMOS layer 86 from the fluid flowing through the MST layer 87. The fluid flows through the capping channel 94 of the -64-201209158 and the microsystem technology (MST) channel 90 located in the cap layer 46 and the MS T channel layer 100, respectively (see Figures 7 and 16). Cell transport occurs in the larger channels 94 fabricated in the cap layer 46 while biochemical processing is performed within the smaller MST channels 90. The cell transport channel is sized to transport cells in the sample to predetermined locations within the MST channels 90. Cells that are larger than 20 microns in size (e.g., certain white blood cells) require channel sizes greater than 20 microns, so the flow cross-sectional area is greater than 400 square microns. The MST channel (especially the MST channel located at the location within the LOC device where no cells need to be transported) can be significantly smaller. It will be understood that the capping channel 94 and the MST channel 90 system, and in particular the M ST channel 90, may also be referred to as a heated microchannel or a dialysis MST channel depending on the particular function of the channel. The MS Τ channel 90 is formed by patterning with a photoresist and etching through the MS Τ channel layer 100 deposited on the passivation layer 88. The MST channels 90 are covered by a top layer 66 and the top layer 66 forms the top of the CMOS + MST device 48 (refer to the orientation shown in the figures).

雖然圖中有時顯示蓋層通道層80和貯存槽層78是不同 的膜層,但該蓋層通道層80和該貯存槽層78係由整片的材 料片所形成。當然,該材料片也可能不是一整片。此材料 片之兩面皆經蝕刻以形成蓋層通道層80 (與此層中蝕刻出 該等蓋層通道94 )及貯存槽層7 8 (於此層中蝕刻出該等貯 存槽54、56、58、60和62)。或可藉由微鑄模法形成該等 貯存槽與該等蓋層通道。蝕刻技術及微鑄模技術兩者係用 於製造具有高達約2 0 0 0 0平方微米且小至約8平方微米之流 動橫斷截面積的通道。 -65- 201209158 於該LOC裝置中之不同位置處,該等通道之流動橫斷 截面積可能具有適當的選擇範圍。當該通道中含有大 本或該通道中所含樣本之成分眾多時,適合使用高胃 20000平方微米之截面積(例如於100微米之厚層中具有· 2〇〇微米寬的通道)。當該通道中含有小量液體或不含大 型細胞之混合物時,較佳使用極小的流動橫斷截面積。 下密封層64封住該等蓋層通道94,且上密封層82蓋住 該等貯存槽54、56、58、60和62。 該等五個貯存槽54、56、58、60和62係預先裝入分析 檢驗專用之試劑。於本案所述之具體實施例中,該等貯存 槽係預先裝入下列試劑,但亦可容易地置換成其他試劑, 該等試劑如下: •貯存槽5 4 :抗凝血劑且可隨意地包含紅血球溶胞緩 衝液 •貯存槽5 6 :溶胞試劑 •貯存槽5 8 :限制酶、接合酶與連接子(用於進行連 接子引子PCR,見第70圖及參閱T. Stanchan等人著作且於 1999年於紐約和倫敦由Gariancj Science出版社出版之《人 類分子遺傳學2》)。 •貯存槽60 :擴增混合物(去氧核糖核苷三磷酸( dNTPs)、引子、緩衝液),及 •貯存槽62 : DNA聚合酶。 該蓋層46與該CMOS + MST層48藉由位於下密封膜64與 頂層66中的對應開口而流體連通。此等開口係根據流體是 -66 - 201209158 從該等MST通道90流向該等蓋層通道94或反向流動而稱爲 上吸孔96與下吸孔92 ° LOC裝置之操作Although the cover channel layer 80 and the reservoir layer 78 are sometimes shown as different film layers, the cover channel layer 80 and the reservoir layer 78 are formed from a single piece of material. Of course, the piece of material may not be a whole piece. Both sides of the sheet of material are etched to form a capping channel layer 80 (and the capping channels 94 are etched in the layer) and a reservoir layer 7 8 (the reservoirs 54, 56 are etched in this layer, 58, 60 and 62). Alternatively, the storage tanks and the cover channels may be formed by micro-molding. Both the etching technique and the micro-molding technique are used to fabricate channels having a flow cross-sectional area of up to about 2,000 square microns and as small as about 8 square microns. -65- 201209158 At different locations in the LOC device, the flow cross-sectional area of the channels may have an appropriate range of choice. When the channel contains a large number of samples or a large number of components contained in the channel, it is suitable to use a cross-sectional area of 20,000 square micrometers of high stomach (for example, a channel having a width of 2 μm in a thick layer of 100 μm). When the channel contains a small amount of liquid or a mixture containing no large cells, it is preferred to use a very small flow cross-sectional area. The lower sealing layer 64 seals the capping channels 94 and the upper sealing layer 82 covers the reservoirs 54, 56, 58, 60 and 62. The five storage tanks 54, 56, 58, 60, and 62 are pre-loaded with reagents for analysis and inspection. In the specific embodiments described in the present invention, the storage tanks are pre-filled with the following reagents, but can be easily replaced with other reagents, such as: • Storage tank 5 4: anticoagulant and optionally Contains red blood cell lysis buffer • Storage tank 5 6 : Lysis reagent • Storage tank 5 8 : Restriction enzyme, ligase and linker (for PCR of linker primer, see Figure 70 and see T. Stanchan et al. And in 1999, in New York and London, published by Gariancj Science, "Human Molecular Genetics 2"). • Storage tank 60: amplification mixture (deoxyribonucleoside triphosphates (dNTPs), primers, buffer), and • storage tank 62: DNA polymerase. The cap layer 46 and the CMOS + MST layer 48 are in fluid communication by corresponding openings in the lower sealing film 64 and the top layer 66. These openings are referred to as upper suction holes 96 and lower suction holes 92 ° LOC devices depending on whether the fluid flows from the MST passages 90 to the cover channels 94 or in the opposite direction depending on whether the fluid is -66 - 201209158.

以下係以分析血液樣本中之病源DNA爲例採逐步方式 說明該LOC裝置301之操作。當然’亦可使用一組適當的 試劑或試劑組合、檢驗程序、LOC裝置變化型與偵測系統 對其他種類之生物性或非生物性流體進行分析。回到第4 圖,分析生物樣本涉及五個主要步驟,該五個主要步驟包 含樣本之置入與製備步驟288、核酸萃取步驟290、核酸培 育步驟291、核酸擴增步驟292及偵測與分析步驟294。 該樣本之置入與製備步驟28 8涉及使血液與抗凝血劑 1 1 6混合且隨後利用該病源透析區段70使病源與白血球和 紅血球分離。最佳如第7和1 2圖所示般,該血液樣本經由 樣本入口 68進入該裝置內。毛細作用吸引該血液樣本沿著 該蓋層通道94前往貯存槽54。當血液樣本流體使表面張力 閥118開啓時,從貯存槽54中釋出抗凝血劑(見第15和22 圖)。抗凝血劑避免形成可能阻斷流體流動的凝結塊。 最佳如第22圖所示,藉由毛細作用從貯存槽54吸出抗 凝血劑1 1 6,且抗凝血劑1 1 6經由下吸孔9 2流入該M S T通道 90。下吸孔92具有毛細作用引動特徵(C IF) 102用於塑造 該彎液面之幾何形狀,使得該彎液面不會停泊在該下吸孔 92之邊緣。當從貯存槽54中吸出抗凝血劑116時,位於該 上密封層82中之通氣孔丨22允許空氣進入而取代抗凝血劑 -67- 201209158 116° 第22圖所示之MST通道90係表面張力閥118的一部分 。抗凝血劑116塡滿該表面張力閥U8且使彎液面120定住 於該上吸孔96之彎液面錨98處。於使用前,彎液面120保 持定住於該上吸孔96處,使得抗凝血劑不會流入該蓋層通 道94。當血液流經該蓋層通道94抵達該上吸孔96時會去除 該彎液面120且把該抗凝血劑吸入該血液流體中。 第I5〜21圖顯示插圖AE,該插圖AE係第13圖所示之插 圖A A的一部分。如第1 5、1 6和1 7圖所示,表面張力閥1 1 8 具有三個獨立之MST通道90,該三個MST通道90延伸於各 自的下吸孔92與上吸孔96之間。表面張力閥中之MST通道 90的數量可變化以改變流入樣本混合物中的試劑流率。當 該樣本混合物與該等試劑藉由擴散作用混合在一起時,流 出貯存槽的流率決定該樣本體流中的試劑濃度。因此,每 個貯存槽的表面張力閥係經建構以符合期望之試劑濃度。 血液流入病源透析區段70 (見第4和15圖),於該病 源透析區段70中使用由多個按預定臨界値製造尺寸之孔 1 64所組成的陣列濃縮該等標靶細胞。小於該臨界値的細 胞可通過該等孔’同時較大的細胞無法通過該等孔。不想 要的細胞可能是被該孔陣列攔下的較大細胞或通過該等孔 的較小細胞之任一者,且引導該等不想要之細胞轉向而前 往廢料單元7 6 ’同時該等標靶細胞繼續進行分析檢驗。 於本案所述之病源透析區段7 0中,源自全血樣本中的 病源係經濃縮以進行微生物D N A分析。該孔陣列係由眾多 -68- 201209158 直徑3微米的孔164所形成,且該等孔164使該蓋層通道94 中的入料流體可流體連通地連接至標靶物通道74。該等直 徑3微米的孔164係透過一系列透析MST通道204而與該標 靶物通道74的該等透析上吸孔168連接(最佳顯示於第15 和2 1圖)。病源夠小而可通過該等直徑3微米之孔1 64,且 病源係經由該等透析MST通道204注入該標靶物通道74。 大於3微米之細胞(例如,紅血球與白血球)留在該蓋層 46中的廢液通道72內,該廢液通道72係通往廢料貯存槽76 (見第7圖)。 可使用其他的孔造形、尺寸及深寬比以分離特定之病 源或其他標靶細胞’例如用於進行人類DNA分析之白血球 。稍候提供該透析區段及透析法之變化型的更詳細描述。 再次參閱第6和7圖,該流體受牽引而通過該標靶物通 道74抵達該溶胞試劑貯存槽56的表面張力閥128。表面張 力閥128具有七個MST通道90,該七個MS T通道90延伸於該The following is an example of analyzing the operation of the LOC device 301 by analyzing the pathogenic DNA in the blood sample as an example. Of course, other types of biological or abiotic fluids can also be analyzed using a suitable set of reagents or reagent combinations, assay procedures, LOC device variants and detection systems. Returning to Figure 4, the analysis of the biological sample involves five major steps, including sample insertion and preparation steps 288, nucleic acid extraction step 290, nucleic acid incubation step 291, nucleic acid amplification step 292, and detection and analysis. Step 294. The sample placement and preparation step 28 8 involves mixing the blood with the anticoagulant 1 16 and then using the pathogenic dialysis section 70 to separate the pathogen from the white blood cells and red blood cells. Preferably, as shown in Figures 7 and 12, the blood sample enters the device via sample inlet 68. Capillary action draws the blood sample along the cover channel 94 to the reservoir 54. When the blood sample fluid causes the surface tension valve 118 to open, the anticoagulant is released from the reservoir 54 (see Figures 15 and 22). Anticoagulants avoid the formation of agglomerates that may block fluid flow. Preferably, as shown in Fig. 22, the anticoagulant 116 is aspirated from the reservoir 54 by capillary action and the anticoagulant 116 is introduced into the M S T channel 90 via the lower suction port 92. The lower suction aperture 92 has a capillary action priming feature (CIF) 102 for shaping the geometry of the meniscus such that the meniscus does not park at the edge of the lower suction aperture 92. When the anticoagulant 116 is aspirated from the reservoir 54, the vent 22 in the upper sealing layer 82 allows air to enter instead of the anticoagulant -67-201209158 116° MST channel 90 shown in Fig. 22. A portion of the surface tension valve 118. The anticoagulant 116 fills the surface tension valve U8 and positions the meniscus 120 at the meniscus anchor 98 of the upper suction hole 96. Prior to use, the meniscus 120 remains settled at the upper suction aperture 96 such that the anticoagulant does not flow into the cover layer passage 94. When the blood flows through the cap channel 94 to the upper suction port 96, the meniscus 120 is removed and the anticoagulant is drawn into the blood fluid. Figures I5 to 21 show an illustration AE which is part of the insert A A shown in Fig. 13. As shown in Figures 15, 5 and 17 , the surface tension valve 1 18 has three separate MST channels 90 extending between the respective lower suction holes 92 and the upper suction holes 96. . The number of MST channels 90 in the surface tension valve can be varied to vary the flow rate of the reagent flowing into the sample mixture. When the sample mixture is mixed with the reagents by diffusion, the flow rate out of the reservoir determines the concentration of the reagent in the sample body stream. Therefore, the surface tension valve of each storage tank is constructed to meet the desired reagent concentration. Blood flows into the source dialysis section 70 (see Figures 4 and 15) in which the target cells are concentrated using an array of a plurality of wells 1 64 of a predetermined threshold size. Cells smaller than the critical enthalpy can pass through the holes' while larger cells cannot pass through the holes. Unwanted cells may be any of the larger cells that are blocked by the array of holes or smaller cells that pass through the holes, and direct the unwanted cells to turn to the waste unit 7 6 'at the same time The target cells continue to be analyzed for analysis. In the pathogenic dialysis section 70 described herein, the source derived from the whole blood sample is concentrated for microbial D N A analysis. The array of holes is formed by a plurality of -68-201209158 3 micron diameter holes 164 that connect the feed fluid in the cap layer channel 94 to the target channel 74 in fluid communication. The 3 micron diameter holes 164 are coupled to the dialysis uptake holes 168 of the target channel 74 through a series of dialysis MST channels 204 (best shown in Figures 15 and 21). The source of the disease is small enough to pass through the 3 micron diameter 1 64 and the source is injected into the target channel 74 via the dialysis MST channel 204. Cells larger than 3 microns (e.g., red blood cells and white blood cells) remain in the waste channel 72 in the cover layer 46, which leads to the waste storage tank 76 (see Figure 7). Other pore shapes, sizes, and aspect ratios can be used to isolate a particular source or other target cell', such as leukocytes for human DNA analysis. A more detailed description of the dialysis section and variations of the dialysis method is provided later. Referring again to Figures 6 and 7, the fluid is drawn through the target passage 74 to the surface tension valve 128 of the lysis reagent reservoir 56. The surface tension valve 128 has seven MST channels 90 extending from the

溶胞試劑貯存槽56與該標靶物通道74之間。當藉由該樣本 流體去除該等彎液面時,假設該等流體之物理性質大致相 等,源自全部七個MST通道90的流率將大於源自抗凝血劑 貯存槽54 (其表面張力閥118具有三個MST通道90)之流 率。因此,該樣本混合物中之溶胞試劑所占比例大於抗凝 血劑所占比例。 該溶胞試劑與該等標靶細胞於化學溶胞區段1 3 0內的 標靶物通道7 4中藉由擴散作用而混合。沸騰啓動式閥1 2 6 使該流體停止一段時間’該段時間足以進行擴散和溶胞作 -69- 201209158 用以釋出該等標靶細胞內的遺傳物質(見第6和7圖)。以 下參照第3 1與3 2圖更詳細地描述該沸騰啓動式閥之結構與 操作。本案申請人亦已硏發出可用本發明中以替代該沸騰 式啓動閥的其他主動閥類型(主動閥係相對於諸如表面張 力閥1 1 8之被動閥而言)。此等替代性之閥設計亦稍候做 說明。 當沸騰啓動式閥1 26開啓時,已溶解之細胞流入混合 區段1 3 1以進行擴增之前的限制剪切反應和連接子接合反 應。 參閱第1 3圖,當該流體解除位於該混合區段1 3 1起始 點處之該表面張力閥132處的彎液面時,從貯存槽58釋出 限制酶、連接子和接合酶。該混合物流經該混合區段1 3 1 之全長以進行擴散混合。位於該混合區段1 3 1之終點處的 下吸孔1 3 4係通往培育區段1 1 4之培育室入口通道1 3 3 (見 第13圖)。培育室入口通道】33把該混合物餽入由多個經 加熱之微型通道210組成的蜿蜒構形中,該等微型通道210 之蜿蜒構形可提供培育腔室以於進行限制剪切反應及連接 子之接合反應期間用於容納該樣本(見第1 3及1 4圖)。 第23、24、25、26、27、28和29圖顯示第6圖之插圖 AB中該LOC裝置301的該等膜層。第23〜2 9圖各自顯示形成 CMOS + MST層48和蓋層46之該等膜層依序遞增的情形。插 圖AB顯示培育區段1 14之終點和擴增區段1 12之起點。最佳 如第1 4和2 3圖所示,該液流注入培育區段1 1 4之該等微通 道210,直到該流體抵達該沸騰啓動式閥1〇6’於該沸騰啓 -70- 201209158 動式閥106處該流體停止流動且同時進行擴散。如上述, 位於沸騰啓動式閥106上游的微通道210成爲含有樣本、限 制酶、接合酶和連接子的培育腔室。隨後該等加熱器1 54 係經啓動且維持恆定溫度持續一段特定時間以供進行限制 剪切反應和連接子接合反應。 熟悉該項技藝者將理解此培育步驟291 (見第4圖)係 選用性步驟,且僅有某些類型的核酸擴增分析檢驗需要此 φ 培育步驟29 1。此外,在某些情況下,於培育階段的終點 時可能需要加熱步驟以提高溫度至高於該培育溫度。於流 體進入擴增區段112之前提高溫度係使該等限制酶和接合 ' 酶失去活性。當將採用恆溫核酸擴增法時,使限制酶和接 合酶的失活作用具有密切相關性。 於培育後,沸騰啓動式閥1 06係經啓動(開啓)且該 流體繼續流動而進入擴增區段112。參閱第31和32圖,多 個微通道158形成一個或一個以上之擴增腔室,該混合物 'φ 注入該等經加熱之微通道158的蜿蜒構形中直到該混合物 . 抵達沸騰啓動式閥108。最佳如第30圖之槪要剖面圖所示 ’自貯存槽60釋出擴增混合物(dNTP、引子、緩衝液)且 隨後自貯存槽62釋出聚合酶而流入連接該培育區段1 14和 擴增區段1 12的中間MST通道2 1 2。 第35至51圖顯示第6圖之插圖AC中的LOC裝置301之該 等膜層。第35至51圖各自顯示形成CMOS + MST裝置48和蓋 層46之該等膜層依序遞增的情形。插圖AC係位於該擴增區 段112之終點和該雜合與偵測區段5 2之起點處。經培育之 -71 - 201209158 樣本、擴增混合物及聚合酶流經該等微通道158而到達沸 騰啓動式閥1 08。經過足夠時間以進行擴散混合之後,該 等微通道158中的該等加熱器1 5 4係經啓動以用於進行熱循 環或恆溫擴增。該擴增混合物經歷預定之熱循環次數或經 過預設之擴增時間以擴增充足的標靶DNA。於該核酸擴增 處理之後,沸騰啓動式閥1 08開啓,且流體繼續流入該雜 合與偵測區段5 2。該等沸騰啓動式閥之操作係於稍候做進 一步詳細說明。 如第52圖所示,該雜合與偵測區段52具有雜合腔室陣 列110。第52、53、54和56圖顯示該雜合腔室陣列110及各 別雜合腔室1 80之細節。擴散阻障器1 75係位於雜合腔室 180之入口處,該擴散阻障器175防止於雜合期間在該等雜 合腔室180之間發生該等標靶核酸、探針鏈與已雜合之探 針的擴散作用,從而避免發生錯誤的雜合偵測結果。該等 擴散阻障器1 7 5提供一段流動路徑長度,該流動路徑長度 係夠長而足以於供探針與核酸分子雜合且偵測信號的時間 內防止該等標靶序列和探針擴散出一個腔室且污染另一個 腔室,從而避免得到錯誤的結果。 另一種用於防止錯誤讀値的機制係使多個該等雜合腔 室中具有相同探針。多個光二極體1 84對應於該等含有相 同探針之雜合腔室180’且CMOS電路86自該等光二極體 1 84得到單一結果。於單一結果之推導過程中異常的結果 可忽略不列入計算或採差別加權計算。 藉由受CMOS控制之加熱器1 82提供雜合反應所需之熱 -72- 201209158The lysis reagent reservoir 56 is between the target channel 74. When the meniscus is removed by the sample fluid, assuming that the physical properties of the fluids are substantially equal, the flow rate from all seven MST channels 90 will be greater than the source of anticoagulant storage 54 (the surface tension) Valve 118 has a flow rate of three MST passages 90). Therefore, the proportion of the lysing reagent in the sample mixture is greater than the proportion of the anticoagulant. The lysis reagent is mixed with the target cells by diffusion in the target channel 74 of the chemical lysis section 1130. The boiling start valve 1 2 6 stops the fluid for a period of time sufficient for diffusion and lysis - 69-201209158 to release the genetic material within the target cells (see Figures 6 and 7). The structure and operation of the boiling start valve will be described in more detail below with reference to Figures 31 and 32. The Applicant has also issued other active valve types that can be used in the present invention to replace the boiling start valve (active valve trains are relative to passive valves such as surface tension valve 118). These alternative valve designs are also described later. When the boiling start valve 126 is opened, the dissolved cells flow into the mixing section 133 to perform the limiting shear reaction and the linker engagement reaction before amplification. Referring to Fig. 13 3, when the fluid releases the meniscus at the surface tension valve 132 at the starting point of the mixing section 133, the restriction enzyme, linker and ligase are released from the storage tank 58. The mixture flows through the entire length of the mixing section 133 for diffusion mixing. The lower suction hole 134 at the end of the mixing section 133 is the cultivating chamber inlet passage 1 3 3 leading to the cultivating section 141 (see Fig. 13). The chamber inlet channel 33 feeds the mixture into a crucible configuration consisting of a plurality of heated microchannels 210, the crucible configuration of which provides an incubation chamber for limiting shearing reactions And the linker is used to accommodate the sample during the junction reaction (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 23th to 29th views each show the case where the film layers forming the CMOS + MST layer 48 and the cap layer 46 are sequentially increased. Panel AB shows the endpoint of the incubation section 1 14 and the beginning of the amplification section 112. Preferably, as shown in Figures 14 and 23, the liquid stream is injected into the microchannels 210 of the incubation section 1 14 until the fluid reaches the boiling start valve 1〇6' at the boiling start-70- At 201209158, the fluid stops flowing at the valve 106 and simultaneously diffuses. As described above, the microchannel 210 upstream of the boiling start valve 106 becomes an incubation chamber containing a sample, a restriction enzyme, a ligase, and a linker. The heaters 154 are then activated and maintained at a constant temperature for a specified period of time for limiting the shear reaction and the linker ligation reaction. Those skilled in the art will appreciate that this incubation step 291 (see Figure 4) is an optional step and that only certain types of nucleic acid amplification assays require this φ incubation step 291. In addition, in some cases, a heating step may be required at the end of the incubation phase to raise the temperature above the incubation temperature. Increasing the temperature before the fluid enters the amplification section 112 deactivates the restriction enzymes and the junction 'enzyme. When a thermostatic nucleic acid amplification method is to be employed, there is a close correlation between the restriction enzyme and the inactivation of the binding enzyme. After incubation, the boiling start valve 106 is activated (turned on) and the fluid continues to flow into the amplification section 112. Referring to Figures 31 and 32, a plurality of microchannels 158 form one or more amplification chambers, and the mixture 'φ is injected into the crucible configuration of the heated microchannels 158 until the mixture. Arrives at the boiling start Valve 108. Preferably, as shown in the cross-sectional view of Fig. 30, the amplification mixture (dNTP, primer, buffer) is released from the storage tank 60 and then the polymerase is released from the storage tank 62 to flow into the incubation section 1 14 And the intermediate MST channel 2 1 2 of the amplification section 1 12 . Figures 35 through 51 show the layers of the LOC device 301 in the illustration AC of Figure 6. Figures 35 through 51 each show the case where the layers of the CMOS + MST device 48 and the cap layer 46 are sequentially incremented. The illustration AC is located at the end of the amplification section 112 and at the beginning of the hybrid and detection section 52. The incubated -71 - 201209158 sample, amplification mixture and polymerase flow through the microchannels 158 to the boiling start valve 108. After sufficient time has elapsed for diffusion mixing, the heaters 154 in the microchannels 158 are activated for thermal cycling or isothermal amplification. The amplification mixture undergoes a predetermined number of thermal cycles or a predetermined amplification time to amplify sufficient target DNA. After the nucleic acid amplification process, the boiling start valve 108 is opened and fluid continues to flow into the hybrid and detection section 52. The operation of these boiling start valves will be further detailed later. As shown in Fig. 52, the hybrid and detection section 52 has a hybrid chamber array 110. Figures 52, 53, 54 and 56 show details of the hybrid chamber array 110 and the respective hybrid chambers 180. A diffusion barrier 1 75 is located at the entrance of the hybrid chamber 180, and the diffusion barrier 175 prevents the occurrence of the target nucleic acid, the probe strand and the already between the hybrid chambers 180 during hybridization. The diffusion of heterozygous probes to avoid erroneous hybrid detection results. The diffusion barriers 175 provide a length of flow path that is long enough to prevent hybridization of the probes to the nucleic acid molecules and to prevent diffusion of the target sequences and probes during the detection of signals. One chamber is left and the other chamber is contaminated to avoid erroneous results. Another mechanism for preventing erroneous readings is to have the same probe in a plurality of such hybrid chambers. A plurality of photodiodes 1 84 correspond to the hybrid chambers 180' containing the same probes and the CMOS circuit 86 obtains a single result from the photodiodes 1 84. Outlier results in the derivation of a single result can be ignored or not included in the calculation or differential weighting calculation. Providing the heat required for the hybrid reaction by the CMOS controlled heater 182 -72-201209158

能(以下進一步詳細描述該等加熱器182)。該等加熱器 經啓動之後,互補的標靶序列與探針序列之間發生雜合反 應。該CMOS電路86中的LED驅動器29發送信號給位於該 檢驗模組1 〇中的發光二極體(LED ) 26以使LED發光。此 等探針僅於已發生雜合反應時才會發光,因而可免除一般 用於去除未結合之探針鏈的清洗和乾燥步驟。雜合反應迫 使該等螢光共振能量轉移(FRET)探針186的幹-環狀結構 打開,以允許螢光發光基團回應LED的激發光而發出螢光 能量,此作用將於稍候做更詳細描述。藉由位於各個雜合 腔室180下方之CMOS電路86中的光二極體184偵測螢光( 有關雜合腔室之說明請見下文)。用於所有雜合腔室之該 等光二極體184和相關電子構件全體共同形成該光感測器 44 (見第65圖)。於其他具體實施例中,該光感測器可能 是電荷耦合元件陣列(CCD陣列)。自光二極體184所測 得的信號係經放大且轉換成數位輸出値,且藉由該檢驗模 組讀取器1 2分析該數位輸出値。該偵測方法之進一步細節 於稍候說明。 LOC裝置之附加細節 設計之模組化 該LOC裝置301具有許多功能區段,包括該等試劑貯 存槽54、56、58、60和62、透析區段70、溶胞區段130、 培育區段1 1 4及擴增區段1 1 2、多種類型之閥、增濕器和濕 度感測器。於LOC裝置之其他具體實施例中,此等功能區 -73- 201209158 段可能省略,且可添加額外的功能區段’或該等功能可用 於達成除上述用途以外之不同用途。 例如,培育區段1 1 4可作爲串接擴增分析檢驗系統的 第一擴增區段1 1 2,且該化學溶胞試劑貯存槽5 6係用於添 加由引子、dNTP和緩衝液組成之第一擴增混合物,及試劑 貯存槽58係用於添加反轉錄酶及/或聚合酶。若期望對該 樣本進行化學溶胞,化學溶胞試劑亦可隨著該擴增混合物 —同加入該貯存槽56中,或者,藉著加熱該樣本持續一段 預定時間於該培育區段中以進行熱溶胞。於某些具體實施 例中,若需要進行化學溶胞且期望該引子、dNTP和緩衝液 之混合物與化學溶胞試劑分開時,可於緊鄰貯存槽5 8之上 游處倂入一個附加貯存槽以用於容納該引子、dNTP和緩衝 液之混合物。 於某些情況中,可能期望省略某一步驟,例如培育步 驟291。於此情況下,LOC裝置可經特殊製造以省略該試 劑貯存槽5 8和培育區段1 1 4,或可簡單地不把試劑裝入該 貯存槽,或者若具有主動閥時可使該等主動閥不啓動而不 把該等試劑分配至該樣本流體中,且此時簡單地使該培育 區段轉爲通道以把該樣本從溶胞區段130輸送至擴增區段 1 1 2。該等加熱器可獨立操作,且因此反應的進行取決於 熱能(例如熱溶胞反應係取決於熱能)時,可編程該等加 熱器使該等加熱器於此步驟期間不啓動以確保在不需要熱 溶胞反應的LOC裝置內不會發生熱溶胞反應。透析區段70 可位於如第4圖所示之微流體裝置內之流體系統的起始點 -74- 201209158 處,或透析區段70可位於該微流體裝置內的其他任何地方 。例如,於某些情況下,於擴增階段292之後進行透析以 於該雜合與偵測步驟294之前去除細胞殘渣可能是有益的 。或者,可於整個LOC裝置的任何位置處倂入兩個或兩個 以上的透析區段。類似地,該LOC裝置可倂入多個附加的 擴增區段112以確保以並聯或串聯方式擴增多個標靶物, 隨後於雜合腔室陣列1 1 0中利用特定核酸探針偵測該等標 靶物。對不需透析的樣本(例如全血樣本)進行分析時, 可簡單地從該LOC裝置設計的該樣本之置入與製備區段 2 8 8中省略該透析區段70。於某些情況下,即使該項分析 不需透析,也無需從該LOC裝置中省略該透析區段70。若 透析區段的存在不會對該分析檢驗造成幾何性障礙,仍可 使用在該樣本之置入與製備區段中具有透析區段70的LOC 裝置而不會損及必要功能。 再者,該偵測區段294可包含多個蛋白質體腔室陣列 ,該等蛋白質體腔室陣列係與雜合腔室陣列相同,但於該 等蛋白質體腔室陣列中裝入經設計以與存在於未經擴增之 樣本中的樣本標靶蛋白結合或雜合之探針,而非裝入經設 計以於與標靶核酸序列雜合的核酸探針。 將可理解,爲用於此診斷系統中而製造的該LOC裝置 係根據特定LOC用途選出多個功能區段之不同組合。對於 該等LOC裝置中之許多LOC裝置而言皆具有大多數的功能 區段,且藉著從現有LOC裝置內所使用之功能區段的廣泛 選項中彙整出由多個功能區段組成之適當組合可設計出用 -75- 201209158 於新用途的額外LOC裝置。 本案說明書中僅出示少數的LOC裝置,更有一 裝置僅爲槪要繪示以圖解說明針對此系統所製造之 置的設計靈活性。所屬技術領域中熟悉該項技藝者 易地理解本案說明書中所出示的該等L0C裝置尙未 出,且藉著從現有L0C裝置內所使用之功能區段的 項中彙整出由多個功能區段組成之適當組合可做出 外L0C設計。 樣本種類 多種LOC裝置變化型可接受且分析液態之各種 類中的核酸成分或蛋白質成分,該等液態樣本種類 不限於血液和血液製品、唾液、腦脊髓液、尿液、 羊水、臍帶血、母乳、汗液、胸膜滲出液、淚液、 、腹水、環境水樣及飲料樣本。亦可使用該LOC裝 自大量核酸擴增所取得之擴增子;於此種情況下, 試劑貯存槽將被清空或配置成不會釋放出貯存槽中 分’且該等透析區段、溶胞區段、培育區段及擴增 僅用於把樣本從該樣本入口 6 8輸送到該等雜合腔1 進行如上述之核酸偵測。 對於某些樣本種類而言需要預處理步驟,例如 置入該L 0 C裝置中之則’精液可能需要經過液化, 需要利用多種酶對黏液進行預處理以降低黏度。 些L0C LOC裝 將可輕 詳盡列 廣泛選 許多額 樣本種 包含但 精液、 心囊液 置分析 所有的 所含成 區段將 ί 1 8 0 以 把樣本 及可能 -76- 201209158 樣本之置入 參與第1和1 2圖’把該樣本加到該檢驗模組〗〇之大放 置槽24中。大放置槽24係截頭狀圓錐體,以藉由毛細作用 把該樣本饋入該LOC裝置301之入口 68。該樣本於此處流 入6 4微米寬X 6 0微米深的蓋層通道9 4,且於該蓋層通道9 4 中藉由毛細作用吸引該樣本朝向抗凝血劑貯存槽54流動。 試劑貯存槽 使用微流體裝置(例如LOC裝置3〇1 )之分析檢驗系 統需要小體積的試劑,故允許該等試劑貯存槽容納用於進 行生化處理所需要的所有試劑,每個試劑貯存槽皆具有小 體積。此體積約小於1,000,000,000立方微米,且在大多數 的情況下此體積小於300,000,000立方微米,通常小於 70,000,000立方微米,且於該等圖式所示之LOC裝置301的 例子中,此體積小於20,000,000立方微米。 透析區段 參閱第15〜21、33和34圖,病源透析區段70係經設計 以自該樣本中濃縮病原標靶細胞。如先前所述,於頂層66 中具有複數個孔,該等孔之形狀係呈直徑3微米之孔164, 該複數個孔從大量樣本中濾出標靶細胞。當該樣本流過該 等直徑3微米之孔164,微生物病源通過該等孔而進入—系 列透析MST通道204,且該等微生物病源經由16微米之透 析上吸孔168向上流流回該標靶物通道74中(見第33與34 -77- 201209158 圖)。該樣本之剩餘部分(如紅血球等等)留在該蓋層通 道94中。於病源透析區段70之下游處,該蓋層通道94轉爲 通往廢料貯存槽76的廢液通道72。對於產生相當數量之廢 料的生物樣本種類,係於檢驗模組1 0之外殼1 3內配置泡棉 狀插入物或其他多孔性元件4 9以與該廢料貯存槽7 6流體連 通(見第1圖)。 病源透析區段70的運作完全仰賴流體樣本之毛細作用 。位於病源透析區段7 0之上游末端處的該等直徑3微米之 孔164具有毛細作用引動特徵(CIF) 166 (見第33圖), 以便吸引該流體向下流入下方的該透析MST通道204。用 於該標靶物通道74之第一上吸孔198亦具有毛細作用引動 特徵(CIF ) 202 (見第15圖),以避免該流體輕易地使彎 液面定住在該等透析上吸孔1 68上。 第Π5圖槪要示出之小成分透析區段682可具有類似於 該病源透析區段70的結構。該小成分透析區段藉著塑造孔 之尺寸(且如有必要,可塑造孔之形狀)使該等孔適合讓 小標靶細胞或分子通過,而從樣本中分離該等小標靶細胞 或分子,以使該等小標靶細胞或分子進入標靶物通道中且 繼續進行進一步分析。較大尺寸之細胞或分子被送往廢料 貯存槽766。因此,該LOC裝置30(見第1和39圖)不僅限 於用以分離尺寸小於3微米之病源,也可用於分離任何期 望尺寸之細胞或分子。 溶胞區段 -78- 201209158 回到第7、1 1和1 3圖,藉由化學溶胞法自細胞內釋出 該樣本中之遺傳物質。如上述般,源自溶胞試劑貯存槽56 的溶胞試劑於該溶胞試劑貯存槽56之表面張力閥128下游 處的標靶物通道74內與該樣本流體混合。然而,某些診斷 性分析檢驗更適合使用熱溶胞法,或甚至對標靶細胞使用 化學溶胞法兼熱溶胞法。該LOC裝置301提供該培育區段 1 14之經加熱的微通道210以供熱溶胞之用。該樣本流體注 入培育區段1 14且停止於該沸騰啓動式閥106之處。培育微 通道210加熱該樣本以達到使細胞膜破裂之溫度。 於某些熱溶胞應用中,化學溶胞區段130內的酶催化 反應並非必要,且熱溶胞反應完全取代該化學溶胞區段 1 3 0內的酶催化反應。 沸騰啓動式閥Yes (the heaters 182 are described in further detail below). After activation of the heaters, a hybrid reaction occurs between the complementary target sequence and the probe sequence. The LED driver 29 in the CMOS circuit 86 sends a signal to a light emitting diode (LED) 26 located in the test module 1 to cause the LED to emit light. These probes only illuminate when a heterozygous reaction has taken place, thus eliminating the cleaning and drying steps typically used to remove unbound probe strands. The hybrid reaction forces the dry-loop structure of the fluorescent resonance energy transfer (FRET) probe 186 to open, allowing the fluorescent luminescent group to emit fluorescent energy in response to the excitation light of the LED, which will be performed later. More detailed description. Fluorescence is detected by photodiodes 184 located in CMOS circuitry 86 below each of the hybrid chambers 180 (see below for a description of hybrid chambers). The photodiode 184 and associated electronic components for all of the hybrid chambers collectively form the photosensor 44 (see Figure 65). In other embodiments, the photosensor may be a charge coupled device array (CCD array). The signal measured from the photodiode 184 is amplified and converted to a digital output 値, and the digital output 値 is analyzed by the test module reader 12. Further details of this detection method are given later. Modularization of additional details of the LOC device The LOC device 301 has a number of functional sections including the reagent reservoirs 54, 56, 58, 60 and 62, the dialysis section 70, the lysis section 130, the incubation section 1 1 4 and the amplification section 1 1 2. Various types of valves, humidifiers and humidity sensors. In other embodiments of the LOC device, such functional areas -73-201209158 may be omitted and additional functional sections may be added' or such functionality may be used to achieve different uses than those described above. For example, the incubation section 1 14 can serve as the first amplification section 1 1 2 of the tandem amplification assay assay system, and the chemical lysis reagent storage tank 56 is used for the addition of primers, dNTPs, and buffers. The first amplification mixture, and reagent storage tank 58, is used to add reverse transcriptase and/or polymerase. If chemical lysis of the sample is desired, the chemical lysis reagent may also be added to the storage tank 56 along with the amplification mixture, or by heating the sample for a predetermined period of time in the incubation section. Hot lysis. In some embodiments, if chemical lysis is desired and a mixture of the primer, dNTP, and buffer is desired to be separated from the chemical lysis reagent, an additional storage tank can be inserted immediately upstream of the storage tank 58 Used to hold a mixture of the primer, dNTP and buffer. In some cases, it may be desirable to omit a certain step, such as incubation step 291. In this case, the LOC device may be specially manufactured to omit the reagent storage tank 58 and the incubation section 112, or may simply not load the reagent into the storage tank, or may have such an active valve if it has an active valve The active valve is not activated without dispensing the reagents into the sample fluid, and at this point the incubation section is simply turned into a channel to deliver the sample from the lysis section 130 to the amplification section 112. The heaters can operate independently, and thus the reaction proceeds depending on thermal energy (eg, the thermal lysis reaction depends on thermal energy), the heaters are programmable such that the heaters do not start during this step to ensure that they are not A hot lysis reaction does not occur in a LOC device that requires a hot lysis reaction. The dialysis section 70 can be located at a starting point -74 - 201209158 of the fluid system within the microfluidic device as shown in Figure 4, or the dialysis section 70 can be located anywhere else within the microfluidic device. For example, in some cases it may be beneficial to perform dialysis after the amplification phase 292 to remove cell debris prior to the hybridization and detection step 294. Alternatively, two or more dialysis sections can be inserted at any location throughout the LOC device. Similarly, the LOC device can incorporate a plurality of additional amplification segments 112 to ensure amplification of multiple targets in parallel or in series, followed by utilization of specific nucleic acid probes in the hybrid chamber array 1 1 0 The targets were measured. When analyzing a sample that does not require dialysis (e.g., a whole blood sample), the dialysis section 70 can simply be omitted from the placement and preparation section of the sample designed for the LOC device. In some cases, the dialysis section 70 need not be omitted from the LOC device even if the analysis does not require dialysis. If the presence of the dialysis section does not create a geometrical impediment to the analytical test, the LOC device with the dialysis section 70 in the insertion and preparation sections of the sample can still be used without compromising the necessary function. Furthermore, the detection section 294 can comprise a plurality of protein body chamber arrays that are identical to the hybrid chamber array, but are loaded in the protein body chamber array to be designed to exist The probe in the unamplified sample binds or hybridizes to the probe, rather than the nucleic acid probe designed to hybridize to the target nucleic acid sequence. It will be appreciated that the LOC device manufactured for use in this diagnostic system selects different combinations of multiple functional segments depending on the particular LOC application. Most of the LOC devices in these LOC devices have most of the functional segments, and are suitably composed of multiple functional segments by extensive options from the functional segments used within existing LOC devices. The combination can be designed with additional LOC units for new applications from -75 to 201209158. Only a few LOC devices are shown in this specification, and a device is only shown to illustrate the design flexibility created for this system. Those skilled in the art will readily appreciate that the L0C devices shown in the description of the present specification are not eliminated, and that a plurality of functional regions are merged from items of functional segments used in existing L0C devices. An appropriate combination of segment components can be used to make an external L0C design. Sample types A variety of LOC device variants are acceptable and analyze nucleic acid components or protein components in various classes of liquids. Such liquid sample types are not limited to blood and blood products, saliva, cerebrospinal fluid, urine, amniotic fluid, cord blood, breast milk. , sweat, pleural exudate, tears, ascites, environmental water samples and beverage samples. The LOC can also be used to amplify amplicon obtained from a large amount of nucleic acid amplification; in this case, the reagent storage tank will be emptied or configured so as not to release the fraction in the storage tank and the dialysis section is dissolved Cell segments, incubation segments, and amplification are only used to deliver samples from the sample inlets 6 to the hybrid chambers 1 for nucleic acid detection as described above. For some sample types, a pretreatment step is required, such as placement in the L0C device. Semen may need to be liquefied, and multiple enzymes are required to pretreat the mucus to reduce viscosity. Some L0C LOC packs will be available in a wide range of sample sizes, but semen and pericardial fluids will be analyzed. All included segments will be included in the sample and possibly -76-201209158 samples. Figures 1 and 12 show the sample being added to the large placement slot 24 of the inspection module. The large placement slot 24 is a frustoconical cone for feeding the sample into the inlet 68 of the LOC device 301 by capillary action. The sample is here introduced into a 64 μm wide X 60 μm deep capping channel 94, and the sample is attracted to the anticoagulant storage tank 54 by capillary action in the capping channel 94. Reagent storage tanks Analytical inspection systems using microfluidic devices (eg, LOC devices 3〇1) require small volumes of reagents, allowing the reagent storage tanks to contain all reagents required for biochemical processing, each reagent storage tank Has a small volume. This volume is less than about 1,000,000,000 cubic microns, and in most cases the volume is less than 300,000,000 cubic microns, typically less than 70,000,000 cubic microns, and in the example of LOC device 301 shown in the figures, this volume is less than 20,000,000. Cubic micrometers. Dialysis section Referring to Figures 15 to 21, 33 and 34, the pathogenic dialysis section 70 is designed to concentrate pathogenic target cells from the sample. As previously described, there are a plurality of apertures in the top layer 66 that are shaped as apertures 164 having a diameter of 3 microns that filter the target cells from a large number of samples. As the sample flows through the 3 micron diameter holes 164, microbial pathogens pass through the holes into the series of dialysis MST channels 204, and the microbial sources flow upwardly back to the target via the 16 micron dialysis uptake 168. In channel 74 (see Figures 33 and 34 -77 - 201209158). The remainder of the sample (e.g., red blood cells, etc.) remains in the cap layer channel 94. Downstream of the pathogenic dialysis section 70, the cap channel 94 is turned into a waste channel 72 to the waste reservoir 76. For the type of biological sample that produces a significant amount of waste, a foam-like insert or other porous element 49 is disposed in the outer casing 13 of the test module 10 to be in fluid communication with the waste storage tank 76 (see paragraph 1). Figure). The operation of the source dialysis section 70 relies entirely on the capillary action of the fluid sample. The 3 micron diameter holes 164 at the upstream end of the source dialysis section 70 have a capillary action priming feature (CIF) 166 (see Figure 33) to attract the fluid down into the dialysis MST channel 204 below. . The first upper suction aperture 198 for the target passageway 74 also has a capillary action priming feature (CIF) 202 (see Figure 15) to prevent the fluid from easily resting the meniscus on the dialysis suction orifice. 1 68 on. The small component dialysis section 682 to be shown in Fig. 5 may have a structure similar to the pathogenic dialysis section 70. The small component dialysis section separates the small target cells from the sample by shaping the size of the pores (and, if necessary, shaping the shape of the pores) such that the pores are adapted to allow small target cells or molecules to pass through or Molecules, such that the small target cells or molecules enter the target channel and continue for further analysis. Larger sized cells or molecules are sent to waste storage tank 766. Thus, the LOC device 30 (see Figures 1 and 39) is not limited to isolation of pathogens having a size of less than 3 microns, but can also be used to isolate cells or molecules of any desired size. Lysis segment -78- 201209158 Returning to Figures 7, 1 and 1 3, the genetic material in the sample is released from the cell by chemical lysis. As described above, the lysis reagent from the lysis reagent reservoir 56 is mixed with the sample fluid in the target channel 74 downstream of the surface tension valve 128 of the lysis reagent reservoir 56. However, some diagnostic assays are more suitable for use with the hot lysis method, or even for the target cells using the chemical lysis method and the hot lysis method. The LOC device 301 provides heated microchannels 210 of the incubation section 14 for thermal lysis. The sample fluid is injected into the incubation section 14 and stops at the boiling start valve 106. The microchannel 210 is incubated to heat the sample to reach a temperature at which the cell membrane is broken. In some hot lysis applications, the enzymatic reaction in the chemical lysis section 130 is not necessary, and the hot lysis reaction completely replaces the enzyme catalyzed reaction within the chemical lysis section 130. Boiling start valve

如上述討論般,LOC裝置301具有三個沸騰啓動式閥 126、106和108。此等閥之位置係顯示於第6圖中。第31圖 係單獨位於擴增區段112之該等經加熱之微通道158末端處 的沸騰啓動式閥1〇8之放大平面圖。 藉由毛細作用吸引該樣本流體1 1 9沿著該等經加熱之 微通道1 58流動,直到該樣本流體抵達該沸騰啓動式閥1 08 。該樣本流體之領先彎液面120定住在該閥入口 146處之彎 液面錨98處。彎液面錨98之幾何形狀使該領先彎液面停止 以中止該毛細流動。如第31和32圖所示,彎液面錨98係藉 由從MST通道90通往該蓋層通道94的上吸孔所提供之開孔 -79- 201209158 。彎液面120之表面張力使該閥保持關閉。環形加熱器152 係位於該閥入口 146之周長邊緣處。該環形加熱器152係經 由該沸騰啓動式閥之加熱器接觸點1 53而受CMOS控制。 爲打開該閥,CMOS電路86傳送電脈衝給該閥之加熱 器接觸點153。環形加熱器152以電阻方式加熱該液體樣本 1 1 9直到該液體樣本沸騰。沸騰作用使該彎液面1 20脫離該 閥入口 1 46且開始潤濕該蓋層通道94。一旦開始潤濕該蓋 層通道94,再度進行毛細流動。流體樣本119注入蓋層通 道94且流經該閥之下吸孔150而前往該閥出口 148,於該閥 出口 148處,該毛細驅動流體沿著該擴增區段離開通道160 繼續流動而進入該雜合與偵測區段5 2。多個液體感測器 1 74設置於該閥前後處以供判斷之用。 將可理解,一旦該等沸騰啓動式閥係經開啓,該等沸 騰啓動式閥無法再關閉。然而,當LOC裝置301與檢驗模 組1 〇係單次使用裝置,便無需再次關閉該等閥。 培育區段及核酸擴增區段 第 6、7、13、14、23、24、25、35 〜45、50 與 51 圖顯 示培育區段114與擴增區段112。該培育區段114具有單條 經加熱之培育微通道2 1 0,該經加熱之培育微通道2 1 0係位 於該MST通道層100中且經蝕刻成從該下吸孔134通到該沸 騰啓動式閥1〇6的蜿蜒圖案(見第13與Μ圖)。控制該培 育區段1 1 4之溫度能夠以更高效率進行酶催化反應。同樣 地,擴增區段Π2具有從該沸騰啓動式閥1〇6通往該沸騰啓 -80- 201209158 動式閥108且呈蜿蜒構形的經加熱之擴增微通道158 (見第 6與1 4圖)。此等閥中止該流體之流動以使該等標靶細胞 留在該經加熱之培育微通道210或擴增微通道158中且同時 進行混合、培育與核酸擴增。該等微通道之蜿蜒圖案於某 種程度上亦有利於混合該等標靶細胞與試劑。 於培育區段1 1 4和擴增區段1 1 2中,使用脈衝寬度調變 (PWM)藉由CMOS電路86控制該等加熱器154加熱該等樣 本細胞與試劑。該經加熱之培育微通道2 1 0和擴增微通道 158之蜿蜒構形的每個寬曲流道皆具有三個可獨立操作的 加熱器1 54,該等加熱器1 54延伸於該等加熱器各自的加熱 器接觸點156之間(見第14圖),該等加熱器接觸點156提 供輸入熱通量密度之二維控制。最佳如第5 1圖所示,該等 加熱器154係承載於該頂層66上且包埋於該下密封層64內 。該加熱器材料係鈦鋁合金(TiAl ),但多種其他導電性 材料亦適用。該等長形加熱器154係與每個通道區段(每 個通道區段形成該蜿蜒造形之該等寬曲流道)之長度成平 行。於擴增區段1 1 2中,藉由個別加熱器控制使每一個寬 曲流道皆可作爲獨立的PCR腔室而運作。 使用微流體裝置(例如LOC裝置301 )的該分析檢驗 系統需要小體積的擴增子,因此允許於擴增區段1 1 2內以 低的擴增混合物體積進行擴增反應。此體積大約小於400 微毫升(nanoliter ),大多數的情況下小於170微毫升, 一般小於70微毫升且於LOC裝置301的情況下此體積介於2 微毫升至30微毫升之間。 -81 - 201209158 加熱速率提高與更高的擴散混合作用 每個通道區段的小截面提高該擴增流體混合物之加熱 速率。所有流體與該加熱器1 5 4相距一段相對短的距離》 可看出使該通道截面(即該擴增微通道158之截面)縮小 至小於100,000平方微米所達成之加熱速率比利用較大規 格之設備所提供的加熱速率更高。微影製造技術允許製造 出具有低於16,000平方微米之流動路徑橫斷截面積的擴增 微通道158,該低於16,000平方微米之流動路徑橫斷截面 積可提供實質較高的加熱速率。利用微影技術可輕易達成 1微米程度的特徵尺寸。若需要極少的擴增子(如LOC裝 置301的情況),該截面積可縮小至低於2,5 00平方微米。 爲了達到於該LOC裝置上使用1000種至2000種探針進行診 斷分析檢驗且於一分鐘內完成「置入樣本、輸出結果」的 要求,介於400平方微米至1平方微米之間的流動橫斷截面 積可滿足此要求。 該擴增微通道158內的加熱器元件以每秒80凱氏溫度 (K)以上之速率加熱該等核酸序列,且於大多數的情況 下係以高於每秒1 00 K之速率加熱該等核酸序列。一般而 言,該加熱器元件以每秒1,〇〇〇 K以上之速率加熱該等核 酸序列,且於許多情況下,該加熱器元件以每秒1 0,000 K 以上之速率加熱該等核酸序列。通常依據該分析檢驗系統 之需求,該加熱器元件係以每秒1 00,000 K以上之速率、 每秒1,000,000 K以上之速率、每秒1 0,000,000 K以上之速 -82- 201209158 率、每秒20,000,000 1<:以上之速率、每秒40,000,000〖以上 之速率、每秒80,000,000 1C以上之速率及每秒1 60,000,000 K以上之速率加熱該等核酸序列。 小截面積之通道亦有益於任何試劑與該樣本流體的擴 散混合作用。於完成擴散混合之前,一種液體擴散進入另 一種液體的擴散作用係以靠近兩液體間之界面處的擴散作 用最大。濃度會隨著遠離該界面之距離而遞減。使用具有 相對較小之流動方向橫斷截面積的微通道可確保兩種流體 緊鄰著界面流動以達到更快地擴散混合。可看出使該通道 截面縮小至低於100,〇〇〇平方微米所達成之混合速率高於 利用較大規格之設備所提供的混合速率。微影製造技術允 許製造具有低於1 60 00平方微米之流動路徑橫斷截面積的 微通道,該低於1 60 00平方微米之截面積可提供明顯較高 的混合速率。若需要極小的體積(如LOC裝置301的情況 )’該截面積可縮小至低於25 00平方微米。爲了達到於該 LOC裝置上使用1000種至2000種探針進行診斷性分析檢驗 且於一分鐘內完成「置入樣本、輸出結果」的要求,介於 400平方微米至1平方微米之間的流動橫斷截面積係可滿足 此要求。 短的熱循環時間 使該樣本混合物保持靠近該等加熱器且使用極小流體 體積允許於核酸擴增過程期間進行快速熱循環。對於長度 高達150個鹼基對(bp)的標靶序列而言,每個熱循環( 83 - 201209158 即,變性、黏合與引子延長)係於30秒內完成。於大多數 的診斷性分析檢驗中,個別的熱循環時間係在1 1秒以內’ 且大部分熱循環的時間係在4秒以內。用於進行一部分最 常用之診斷性分析檢驗的LOC裝置30對於長度高達150個 鹼基對的標靶序列而言具有介於約0.45秒至1.5秒之間的熱 循環時間。以此速率進行熱循環允許該檢驗模組完成核酸 擴增程序的時間遠少於10分鐘,且通常少於220秒。對於 多數分析檢驗而言,該擴增區段可從該樣本流體進入該樣 本入口起算80秒以內產生足夠的擴增子。對於絕大多數的 分析檢驗而言,係於3 0秒內產生足夠的擴增子。 當完成預定次數之擴增循環後,使該等擴增子經由沸 騰啓動式閥1 0 8饋入該雜合與偵測區段5 2。 雜合腔室 第52、53、54、56與57圖顯示該雜合腔室陣列110中 的該等雜合腔室180。該雜合與偵測區段52具有由雜合腔 室180組成24x25的陣列110,且每個雜合腔室具有雜合敏 感性FRET探針186、加熱器元件182和整合式光二極體ι84 。該光一極體1 8 4係經倂入以用於偵測標祀核酸序列或蛋 白質與該等FRET探針186之雜合反應所產生的螢光。藉由 CMOS電路86獨立地控制每個光二極體184。介於該等 F R E T探針1 8 6與光二極體1 8 4之間的任何材料皆必需可讓 釋放光通過。因此,位於該等探針186與光二極體I"之間 的隔牆區段97對於該釋放光而言亦爲可透光性。於該L〇c -84- 201209158 裝置301中,該隔牆區段97係二氧化矽薄層(約0.5微米) 〇 於每一個雜合腔室180正下方倂入一個光二極體184允 許該等探針一標靶物雜合體的體積達到極小同時仍能發出 可偵測的螢光信號(見第54圖)。小量的探針-標靶物雜 合體允許使用小體積之雜合腔室。欲達到可偵測量之探針 -標靶物雜合體於雜合前所需要的探針量係低於約270皮 φ 克(相當於900,000立方微米),於多數情況下低於約60 皮克(相當於200,000立方微米),通常約低於12皮克( 相當於40,000立方微米)且於該等附圖中所示之L〇c裝置 301的情況下低於約2.7皮克(相當於9,000立方微米之腔室 體積)。當然’縮小該等雜合腔室之尺寸允許於該LOC裝 置上具有更高的腔室密度且從而可具有更多探針。於LOC 裝置301中’該雜合區段於1500微米乘1500微米的面積中 具有1 000個以上的腔室(即,每個腔室之面積係低於225 0 φ 平方微米)。較小的體積亦可縮短反應時間,使得雜合與 偵測更快速。每個腔室中需要小量探針的附加優點係於 LOC裝置之製造過程期間僅需要在每個腔室中點入極小量 的探針溶液。本發明之LOC裝置具體實施例係使用1皮升 ' 或低於1皮升的探針溶液體積點製而成。 , 於核酸擴增之後,沸騰啓動式閥1 08係經啓動,且該 擴增子沿著流動路徑1 7 6流動且流入每一個雜合腔室1 8 〇 ( 見第5 2和5 6圖)。終點液體感測器1 7 8指示何時把該擴增 子注入該等雜合腔室180及何時可啓動該等加熱器182。 -85- 201209158 經過足夠的雜合時間之後,該發光二極體(LED ) 26 (見第2圖)係經啓動。位於每個雜合腔室1 8 0中的開孔提 供一個光學窗口 136以用於使該等FRET探針186暴露於激 發光下(見第52、54和56圖)。該發光二極體(LED) 26 持續發光足夠長的時間以誘使該等探針發出高強度之螢光 信號。於激發期間,係使該等光二極體184短路。經過一 段預先編程的(p re-pro gemmed)延遲時間300之後(見第 2圖),使該光二極體184運作且於無激發光下偵測螢光發 光作用。於該光二極體184之主動區185上的入射光(見第 5 4圖)係經轉換成光電流,隨後可使用c Μ 0 S電路8 6測量 該光電流。 該等雜合腔室180係各自裝有用於偵測單一種標靶核 酸序列之探針。如有需要,可於每個雜合腔室180中裝入 探針以偵測超過1 0 0 0個以上之不同標靶物。或者,可於多 個雜合腔室或所有雜合腔室中裝入相同探針以重覆偵測相 同之標靶核酸。以此種方式於整個雜合腔室陣列110中重 複裝入該等探針導致提高所獲得之結果的可信度,且如有 需要1可使藉由與該等雜合腔室相鄰之該等光二極體所測 得的多個結果合倂以提供單一個結果。所屬技術領域中熟 悉該項技藝者將理解根據該分析檢驗之規格要求,該雜合 腔室陣列110上可能具有一種至1〇〇〇種以上的不同探針。 增濕器及濕度感測器 第6圖之插圖AG標示該增濕器196之位置。該增濕器 -86- 201209158 防止於該L O C裝置3 0 1之操作期間的該等試劑與探針之蒸 發作用。最佳係如第5 5圖之放大圖所示’水貯存槽1 8 8係 與三個蒸發器190流體連通。於製造期間’係以分子生物 級的水注入該水貯存槽1 8 8且密封該水貯存槽1 8 8。最佳係 如第5 5與6 8圖所示,藉由毛細作用使水被吸入三個下吸孔 1 94且沿著各自的水供應通道1 92流至位於該等蒸發器1 90 處三個爲一組的上吸孔1 93 »彎液面定住於各個上吸孔1 93 之處以留住水。該等蒸發器具有環形加熱器191,該等環 形加熱器環繞該等上吸孔193。該等環形加熱器191藉由通 往頂部金屬層195 (見第37圖)的導電柱3 76而連接該 CMOS電路86。當啓動時,環形力[]熱器191力Π熱該水,而造 成水份蒸發且使該裝置環境增濕。 濕度感測器232之位置亦顯示於第6圖中。然而,最佳 係如第63圖中的插圖ΑΗ之放大圖所示,該濕度感測器具 有電容梳狀結構。經微影蝕刻之第一電極2 96和經微影蝕 刻之第二電極298面向彼此,使得第一電極296與第二電極 298的齒交錯穿插。該等相對的電極形成具有電容量之電 容器’且可藉由CMOS電路86控制該電容器。當濕度提高 時’該等電極間之氣隙的電容率隨之提高,使得電容量亦 隨之增加。濕度感測器2 3 2鄰接該雜合腔室陣列1 1 〇,於雜 合腔室陣列1 1 〇處進行濕度測量對於延緩溶液(含有經曝 光之探針)之蒸發作用極爲重要。 反饋感測器 -87- 201209158 溫度感測器與液體感測器係納入該L O C裝置3 0 1各處 ,藉以於裝置運作期間提供反饋與診斷。參閱第35圖,九 個溫度感測器1 70分佈於該擴增區段1 12各處。同樣地,該 培育區段Π 4亦具有九個溫度感測器1 70。此等感測器各自 使用2x2的雙極接面電晶體(BJT)以監測該流體溫度且提 供反饋給該CMOS電路86。CMOS電路86使用此反饋以精確 地控制該核酸擴增程序期間之熱循環及精確地控制熱溶胞 和培育期間的任何加熱作用。 於該雜合腔室180中,CMOS電路86使用該等雜合加熱 器182作爲溫度感測器(見第56圖)。該等雜合加熱器182 之電阻係溫度依賴性,且CMOS電路86利用該電阻是溫度 依賴性這點推導出該等雜合腔室180之每個腔室的加熱器 讀値。 LOC裝置301亦具有複數個MST通道液體感測器174和 蓋層通道液體感測器20 8。第35圖出示一列MST通道液體 感測器1 74,該列MST通道液體感測器1 74係位於該經加熱 之微通道158的每隔一個曲流道之一末端處。最佳係如第 37圖所示,該等MST通道液體感測器174係一對電極,該 對電極係由CMOS結構86中的頂部金屬層195之暴露區域所 形成。液體使該等電極間之電路斷路以指示液體出現於該 感測器之位置處。 第25圖顯示蓋層通道液體感測器208之放大透視圖。 多對相對的鈦鋁(TiAl )電極21 8和220係設置於該頂層66 上。間隙222係介於該等電極21 8與220之間,以於無液體 -88- 201209158 時使電路保持開路。液體的存在係使該電路斷路,且 CMOS電路8 6使用此反饋信號以監測該流體。 重力非依賴性 檢驗模組1 〇係位向非依賴性。無需爲了進行操作而使 該檢驗模組1 0固定於平坦穩定之表面上。毛細驅動流體流 動且無通往輔助設備之外部配管,因而允許該等模組方便 攜帶且可輕易地插入同爲手提可攜式讀取器(例如行動電 話)中。具有重力非依賴性之操作方式表示該檢驗模組於 所有實際情況下亦爲加速非依賴性。該等檢驗模組耐衝擊 且耐震,且該等檢驗模組將可於正在移動中的交通工具上 或正手持行動電話時進行操作。 透析之變化型 白血球標靶細胞As discussed above, LOC device 301 has three boiling start valves 126, 106 and 108. The position of these valves is shown in Figure 6. Figure 31 is an enlarged plan view of a boiling start valve 1 〇 8 located at the end of the heated microchannels 158 of the amplification section 112, respectively. The sample fluid 1 1 9 is attracted by capillary action along the heated microchannels 1 58 until the sample fluid reaches the boiling start valve 108. The leading meniscus 120 of the sample fluid settles at the meniscus anchor 98 at the valve inlet 146. The geometry of the meniscus anchor 98 stops the leading meniscus to stop the capillary flow. As shown in Figures 31 and 32, the meniscus anchor 98 is provided by an opening provided from the MST passage 90 to the upper suction opening of the cover passage 94 -79-201209158. The surface tension of the meniscus 120 keeps the valve closed. A ring heater 152 is located at the peripheral edge of the valve inlet 146. The ring heater 152 is CMOS controlled by the heater contact point 153 of the boiling start valve. To open the valve, CMOS circuit 86 delivers an electrical pulse to heater contact 153 of the valve. The annular heater 152 heats the liquid sample 1 1 9 until the liquid sample boils. The boiling action causes the meniscus 1 20 to disengage from the valve inlet 1 46 and begin to wet the cap channel 94. Once the cover channel 94 begins to wet, capillary flow is again performed. A fluid sample 119 is injected into the capping channel 94 and through the lower suction port 150 to the valve outlet 148 where the capillary drive fluid continues to flow along the adiabatic section away from the channel 160. The hybrid and detection section 52. A plurality of liquid sensors 1 74 are provided at the front and rear of the valve for judgment. It will be appreciated that once the boiling start valves are opened, the boiling start valves can no longer be closed. However, when the LOC device 301 and the test module 1 are single-use devices, it is not necessary to close the valves again. The culture section and the nucleic acid amplification section 6, 6, 7, 14, 23, 24, 25, 35 to 45, 50 and 51 show the incubation section 114 and the amplification section 112. The incubation section 114 has a single heated incubation microchannel 210 that is located in the MST channel layer 100 and etched from the lower suction port 134 to the boiling start The 蜿蜒 pattern of the valve 1〇6 (see 13 and )). Controlling the temperature of the culture section 112 can enable an enzymatic reaction with higher efficiency. Similarly, the amplification section Π2 has heated amplifying microchannels 158 from the boiling start valve 1〇6 to the boiling start-80-201209158 valve 108 and in a 蜿蜒 configuration (see section 6). With 1 4 figure). These valves terminate the flow of the fluid to allow the target cells to remain in the heated incubation microchannel 210 or amplification microchannel 158 while mixing, culturing, and nucleic acid amplification. The enthalpy pattern of the microchannels also facilitates mixing of the target cells and reagents to some extent. In the incubation section 112 and the amplification section 112, the heaters 154 are controlled by CMOS circuitry 86 to heat the sample cells and reagents using pulse width modulation (PWM). Each wide curved flow path of the heated incubation microchannel 210 and the amplification microchannel 158 has three independently operable heaters 1 54 extending from the heater 1 54 Between the respective heater contact points 156 of the heaters (see Figure 14), the heater contacts 156 provide two-dimensional control of the input heat flux density. Preferably, as shown in Fig. 51, the heaters 154 are carried on the top layer 66 and embedded in the lower sealing layer 64. The heater material is titanium aluminum alloy (TiAl), but a variety of other conductive materials are also suitable. The elongate heaters 154 are parallel to the length of each of the channel sections (each of which forms the wide curved path of the profile). In the amplification section 112, each of the wide flow channels can be operated as a separate PCR chamber by individual heater control. This analytical assay system using a microfluidic device (e.g., LOC device 301) requires a small volume of amplicons, thus allowing amplification reactions to be performed in the amplification section 1 1 2 with a low volume of amplification mixture. This volume is less than about 400 microliters (Nanoliter), in most cases less than 170 microliters, typically less than 70 microliters and in the case of LOC device 301 this volume is between 2 microliters and 30 microliters. -81 - 201209158 Increased heating rate and higher diffusion mixing The small cross section of each channel section increases the heating rate of the augmented fluid mixture. All fluids are at a relatively short distance from the heater 154. It can be seen that the heating rate achieved by reducing the cross-section of the channel (i.e., the cross-section of the amplifying microchannel 158) to less than 100,000 square microns is greater than that of utilization. Large format equipment provides a higher heating rate. The lithography manufacturing technique allows the fabrication of an augmented microchannel 158 having a cross-sectional cross-sectional area of a flow path of less than 16,000 square microns that provides a substantially higher heating rate across a cross-sectional profile of a flow path of 16,000 square microns. Feature sizes of up to 1 micron can be easily achieved using lithography. If very few amplicons are required (as is the case with LOC unit 301), the cross-sectional area can be reduced to less than 2,500 square microns. In order to achieve the diagnostic analysis test using 1000 to 2000 probes on the LOC device and complete the "implant sample, output result" requirement within one minute, the flow cross between 400 square micrometers and 1 square micrometer The cross-sectional area meets this requirement. The heater elements within the amplification microchannel 158 heat the nucleic acid sequences at a rate above 80 Kelvin (K) per second, and in most cases heat the rate at a rate greater than 100 K per second. And other nucleic acid sequences. In general, the heater element heats the nucleic acid sequences at a rate of greater than 1, 〇〇〇K per second, and in many cases, the heater element heats the nucleic acid sequences at a rate of more than 10,000 K per second. . Usually according to the requirements of the analysis and inspection system, the heater element is at a rate of more than 100,000 K per second, a rate of 1,000,000 K per second or more, a rate of more than 10,000,000 K per second -82-201209158 rate, per second. 20,000,000 1 <: above rate, 40,000,000 per second 〖the above rate, a rate of 80,000,000 1 C per second or more and a rate of 1 60,000,000 K per second or more to heat the nucleic acid sequences. The passage of the small cross-sectional area is also beneficial for the diffusion mixing of any reagent with the sample fluid. The diffusion of one liquid into the other prior to the diffusion mixing is maximized by the diffusion near the interface between the two liquids. The concentration will decrease with distance from the interface. The use of microchannels with a relatively small cross-sectional area of flow direction ensures that the two fluids flow next to the interface for faster diffusion mixing. It can be seen that the cross-section of the channel is reduced to less than 100, and the mixing rate achieved by 〇〇〇 square micron is higher than the mixing rate provided by a larger specification device. The lithography manufacturing technique allows the fabrication of microchannels having a cross-sectional area of the flow path of less than 1 60 square microns, which provides a significantly higher mixing rate than the cross-sectional area of 1 60 square microns. If a very small volume is required (as is the case with LOC device 301), the cross-sectional area can be reduced to less than 20,000 square microns. In order to achieve a diagnostic analysis test using 1000 to 2000 probes on the LOC device and complete the "put sample, output result" requirement within one minute, the flow between 400 square microns and 1 square micron The cross sectional area can meet this requirement. A short thermal cycle time keeps the sample mixture close to the heaters and uses a very small fluid volume to allow for rapid thermal cycling during the nucleic acid amplification process. For target sequences up to 150 base pairs (bp) in length, each thermal cycle (83 - 201209158, denaturation, adhesion, and primer extension) is completed in 30 seconds. In most diagnostic assays, individual thermal cycling times are within 11 seconds' and most of the thermal cycling time is within 4 seconds. The LOC device 30 for performing a portion of the most commonly used diagnostic assays has a thermal cycle time of between about 0.45 seconds and 1.5 seconds for a target sequence of up to 150 base pairs in length. Thermal cycling at this rate allows the assay module to complete the nucleic acid amplification procedure for much less than 10 minutes, and typically less than 220 seconds. For most analytical assays, the amplified segment can produce sufficient amplicons within 80 seconds from the entry of the sample fluid into the sample inlet. For most analytical tests, sufficient amplicons are generated within 30 seconds. After completing the predetermined number of amplification cycles, the amplicon is fed into the hybrid and detection section 52 via a boiling start valve 108. Hybrid Chambers Figures 52, 53, 54, 56 and 57 show the hybrid chambers 180 in the array of hybrid chambers 110. The hybrid and detection section 52 has an array 110 of 24x25 composed of a hybrid chamber 180, and each hybrid chamber has a hybrid sensitive FRET probe 186, a heater element 182, and an integrated photodiode ι 84 . The photoreceptor 1 8 4 is introgressed for detecting fluorescence generated by a heterozygous reaction of the target nucleic acid sequence or protein with the FRET probes 186. Each of the photodiodes 184 is independently controlled by a CMOS circuit 86. Any material between the F R E T probes 1 8 6 and the photodiodes 1 8 4 must allow light to pass through. Therefore, the partition wall section 97 between the probes 186 and the photodiode I" is also permeable to the emitted light. In the apparatus 301 of the L〇c-84-201209158, the partition section 97 is a thin layer of cerium oxide (about 0.5 micrometer), and a photodiode 184 is inserted directly under each of the hybrid chambers 180 to allow The volume of the probe-target hybrid is minimal while still producing a detectable fluorescent signal (see Figure 54). A small amount of probe-target hybrid allows the use of a small volume of hybrid chamber. The amount of probe required to achieve a detectable amount of probe-target hybrid prior to hybridization is less than about 270 picograms (equivalent to 900,000 cubic micrometers), and in most cases less than about 60 skins. Grams (equivalent to 200,000 cubic microns), typically less than about 12 picograms (equivalent to 40,000 cubic micrometers) and less than about 2.7 picograms in the case of the L〇c device 301 shown in the figures (equivalent to 9,000 cubic micron chamber volume). Of course, reducing the size of the hybrid chambers allows for a higher chamber density on the LOC device and thus more probes. In the LOC device 301, the hybrid section has more than 1 000 chambers in an area of 1500 microns by 1500 microns (i.e., the area of each chamber is less than 225 0 φ square microns). Smaller volumes also reduce reaction time, making hybridization and detection faster. An additional advantage of requiring a small amount of probe in each chamber is that only a minimal amount of probe solution needs to be dispensed into each chamber during the manufacturing process of the LOC device. A specific embodiment of the LOC device of the present invention is made using a volume of probe solution of 1 picoliter or less than 1 picoliter. After the nucleic acid amplification, the boiling start valve 108 is activated, and the amplicon flows along the flow path 176 and flows into each of the hybrid chambers 1 8 〇 (see Figures 5 and 5) ). The endpoint liquid sensor 178 indicates when the amplicon is injected into the hybrid chambers 180 and when the heaters 182 can be activated. -85- 201209158 After enough mixing time, the light-emitting diode (LED) 26 (see Figure 2) is activated. The openings in each of the hybrid chambers 180 provide an optical window 136 for exposing the FRET probes 186 to lasing (see Figures 52, 54 and 56). The light emitting diode (LED) 26 continues to illuminate for a sufficient period of time to induce the probes to emit high intensity fluorescent signals. The photodiodes 184 are shorted during the excitation. After a pre-programmed (p re-pro gemmed) delay time of 300 (see Figure 2), the photodiode 184 operates and detects fluorescent light emission without excitation light. Incident light (see Figure 5 4) on the active region 185 of the photodiode 184 is converted to photocurrent, which can then be measured using a c Μ 0 S circuit 8.6. The hybrid chambers 180 are each equipped with a probe for detecting a single target nucleic acid sequence. If desired, probes can be placed in each of the hybrid chambers 180 to detect more than 100 different targets. Alternatively, the same probe can be loaded into multiple hybrid chambers or all hybrid chambers to repeatedly detect the same target nucleic acid. Repeated loading of the probes throughout the hybrid chamber array 110 in this manner results in increased confidence in the results obtained, and if desired, can be adjacent to the hybrid chambers. The multiple results measured by the photodiodes are combined to provide a single result. Those skilled in the art will appreciate that depending on the specifications of the analytical test, the hybrid chamber array 110 may have from one to more than one different probes. Humidifier and Humidity Detector The illustration AG of Figure 6 indicates the location of the humidifier 196. The humidifier -86-201209158 prevents evaporation of the reagents and probes during operation of the L O C device 310. Preferably, the water storage tank 18 8 is in fluid communication with the three evaporators 190 as shown in the enlarged view of FIG. During the manufacturing period, molecular water-grade water is injected into the water storage tank 1 8 8 and the water storage tank 1 8 8 is sealed. Preferably, as shown in Figures 5 and 6 8, water is drawn into the three lower suction holes 1 94 by capillary action and flows along the respective water supply channels 1 92 to the evaporators 1 90 at three A set of upper suction holes 1 93 » The meniscus is fixed at each upper suction hole 1 93 to retain water. The evaporators have annular heaters 191 that surround the upper suction holes 193. The ring heaters 191 are connected to the CMOS circuit 86 by conductive posts 3 76 to the top metal layer 195 (see Figure 37). When activated, the ring-shaped force [] heater 191 heats the water, causing moisture to evaporate and humidifying the device environment. The position of the humidity sensor 232 is also shown in Figure 6. However, as shown in the enlarged view of the illustration in Fig. 63, the humidity sensing device has a capacitive comb structure. The lithographically etched first electrode 2 96 and the microetched second electrode 298 face each other such that the teeth of the first electrode 296 and the second electrode 298 are interleaved. The opposing electrodes form a capacitor having a capacitance' and the capacitor can be controlled by CMOS circuitry 86. As the humidity increases, the permittivity of the air gap between the electrodes increases, resulting in an increase in capacitance. Humidity sensor 2 3 2 is adjacent to the hybrid chamber array 1 1 〇, and humidity measurement at the hybrid chamber array 1 1 极为 is extremely important for retarding the evaporation of the solution (containing the exposed probe). Feedback Sensor -87- 201209158 Temperature sensor and liquid sensor are included in the LOC unit 301 to provide feedback and diagnosis during device operation. Referring to Fig. 35, nine temperature sensors 1 70 are distributed throughout the amplification section 112. Similarly, the incubation section Π 4 also has nine temperature sensors 170. Each of these sensors uses a 2x2 bipolar junction transistor (BJT) to monitor the temperature of the fluid and provide feedback to the CMOS circuit 86. The CMOS circuit 86 uses this feedback to precisely control the thermal cycling during the nucleic acid amplification procedure and to precisely control any heating during hot lysis and incubation. In the hybrid chamber 180, the CMOS circuit 86 uses the hybrid heaters 182 as temperature sensors (see Figure 56). The resistance of the hybrid heaters 182 is temperature dependent, and the CMOS circuit 86 derives the heater readings for each of the hybrid chambers 180 using the resistance to be temperature dependent. The LOC device 301 also has a plurality of MST channel liquid sensors 174 and a capping channel liquid sensor 208. Figure 35 shows a list of MST channel liquid sensors 1 74 located at one of the ends of every other meandering channel of the heated microchannel 158. Preferably, as shown in Fig. 37, the MST channel liquid sensors 174 are a pair of electrodes formed by exposed regions of the top metal layer 195 in the CMOS structure 86. The liquid breaks the circuit between the electrodes to indicate that liquid is present at the location of the sensor. Figure 25 shows an enlarged perspective view of the capping channel liquid sensor 208. A plurality of pairs of opposing titanium aluminum (TiAl) electrodes 21 8 and 220 are disposed on the top layer 66. A gap 222 is interposed between the electrodes 21 8 and 220 to keep the circuit open without liquid -88 - 201209158. The presence of liquid breaks the circuit and the CMOS circuit 86 uses this feedback signal to monitor the fluid. Gravity-independent test module 1 〇 system position independent. The inspection module 10 is not required to be fixed to a flat, stable surface for operation. The capillary drive fluid flows without external piping to the accessory, thereby allowing the modules to be easily carried and easily inserted into a portable portable reader (e.g., a mobile phone). A gravity-independent operation means that the test module is also acceleration-independent in all practical situations. The inspection modules are shock and shock resistant, and the inspection modules will operate on the moving vehicle or while holding the mobile phone. Dialysis-modified white blood cell target cell

上述LOC裝置301內之透析設計係針對病源所設計。 第64圖係透析區段3 2 8之蓋要剖面圖,該透析區段32 8係設 計用於濃縮血液樣本中之白血球以進行人類DN A分析。將 可理解該透析區段328之結構係與上述之病源標靶細胞之 透析區段70實質相同’只除了該透析區段328之該等孔165 是直徑7.5微米之孔’藉以限制白血球自該蓋層通道94進 入該等透析MST通道204。當欲分析之樣本爲血液樣本且 源自紅血球中的血紅素會干擾後續反應步驟時,隨同該貯 存槽54 (見第22圖)中之抗凝血劑一同加入紅血球溶胞緩 -89- 201209158 衝液將確保可於此透析步驟期間去除該樣本中大部份的已 溶胞之紅血球(從而去除血紅素)。常用的紅血球溶胞緩 衝液係含0.15M之氯化銨(NH4C1) 、10mM之碳酸氫鉀( KHC03 ) 、O.lmM 之乙二胺四乙酸(EDTA)且 pH 7_2~7_4 之溶液,但所屬技術領域中熟悉該項技藝者將理解任何可 有效溶解紅血球之緩衝液皆可使用。 於白血球透析區段328下游處的蓋層通道94轉成標靶 物通道74,使得白血球繼續進行該分析檢驗。再者’於此 情況中,該等透析上吸孔丨68通往廢液通道72 ’以去除該 樣本中所有較小的細胞和成分。應注意此透析變化型僅降 低該標靶物通道7 4中之非所欲之樣本的濃度。 第116圖槪要地說明大成分透析區段686,該大成分透 析區段686亦可從樣本中分離大的標靶成分。此透析區段 中之該等孔係經製造而具有經量製之尺寸與形狀’藉以阻 檔該標靶物通道內所關注的大標靶成分以用於進一步分析 。若使用如上述之白血球透析區段,大部份(但非全部) 尺寸較小之細胞、有機體或分子流入廢料貯存槽768中。 因此,該LOC裝置之其他具體實施例不僅限於用以分離尺 寸大於7.5微米的白血球,也可用於分離任何期望尺寸的 細胞、有機物或分子。 ο 8 段L 7 區的4 析IIII7 透V第 的型於 道化示 通變顯 1 置18 3M3 流裝5 之0C 落 L 陷爲0 i稱j 氣係 止述’ 防下 〇〇 有51 具 例 該 且 置 裝 施中 實圖 澧)3 具1 置 裝 和 -90- 201209158 。此LOC裝置具有可注入流體樣本又不會留下陷落於通道 內之空氣氣泡的透析區段。LOC裝置變化型VIII 518亦具 有一層附加材料層,該材料層稱爲界面層5 94。該界面層 594係位於該蓋層通道層80與該CMOS+ MST裝置48之MST 通道層100之間。該界面層594允許於該等試劑貯存槽與該 MST層87之間具有更複雜的流體互連結構且不會增加矽基 板84之尺寸。 參閱第78圖,旁通渠道600係經設計以於該流體樣本 從該界面廢液通道6 04流到該界面標靶物通道602時導入一 段時間延遲。此時間延遲允許該流體樣本流經該透析MST 通道204而到達使彎液面定住的透析上吸孔168。利用位於 從該旁通渠道600通往該界面標靶物通道602之上吸孔處的 毛細作用引動特徵(CIF ) 202,使該樣本流體從源自透析 MST通道204的所有透析上吸孔168之上游端注入該界面標 靶物通道602。 無旁通渠道600時,樣本流體仍會從該上游端注入界 面標靶物通道602,但最後向前推進的彎液面抵達且通過 屬於尙未塡滿之MS T通道的上吸孔,而導致空氣陷落於該 點位置。該陷落的空氣會減少通過該白血球透析區段328 的樣本流率。 核酸擴增變化型 並聯式聚合酶鏈鎖反應 LOC裝置之數種變化型具有多個以並聯方式運作的擴 -91 - 201209158 增區段。例如,第72圖顯示之LOC裝置變化型VII 492具有 多個並聯的擴增區段1 1 2 . 1〜1 1 2.4,該等並聯的擴增區段 112.1〜112.4允許同時執行多個核酸擴增分析檢驗。 第130圖顯示之LOC裝置變化型XI 746亦具有多個並聯 的擴增區段112.1〜112.4,但附加地具有多個並聯之培育區 段114.1〜114.4,使得該樣本可於進行擴增之前先經不同處 理。其他LOC裝置變化型(例如第1〇5圖槪要顯示之LOC裝 置變化型XIV 641 )說明該複數個並聯的擴增區段可能爲 「X」個,此數目僅受限於LOC裝置之尺寸大小。LOC裝 置越大可容納越多個並聯的擴增反應區段。 該等分開的擴增區段可經配置以針對特定標靶大小或 特定之擴增混合物組成實施不同的循環次數及/或溫度。 藉由數個平行運行的擴增區段,該L0C裝置可於各個區段 中執行多重式核酸擴增法或單重式核酸擴增法。於多重式 核酸擴增中,係使用一對以上之引子擴增一個以上的標靶 序列。具有「m」個腔室的並聯式核酸擴增系統可執行相 當於η重的擴增反應,其中n = n(l) + n(2) + _..n(i) + ...+ n(m),且n(i)係表示在用於該多重式擴增反應 之不同引子對中欲用於腔室「i」之引子號碼,且需謹記 該並聯式擴增系統中之信號雜訊比(SNR )係高於在單腔 室系統中執行η重式擴增反應之信號雜訊比。當於n ( i ) =1的特殊情況下,腔室[i]中的擴增反應正好成爲單重式擴 增反應。 -92 - 201209158 串接式聚合酶鏈鎖反應 該等圖式中之第106、107、108、111和114圖槪要說 明裝置內之擴增區段112.1和11 2.2採串聯運作的L0C裝置 。該第一擴增區段1 12.1包含擴增混合物之試劑貯存槽60.1 及聚合酶之試劑貯存槽62.1 °加於該初始區段之後的每個 擴增區段亦包含兩個試劑貯存槽,擴增混合物之貯存槽 60.2和聚合酶之貯存槽62.2。 φ 串聯的多個擴增區段允許進行串接式PCR分析檢驗, 使得該第一擴增區段11 2 · 1係用於進行預擴增反應以提高 於區段1 1 2.2中執行後續核酸擴增反應之靈敏度。串聯之 • 多個擴增區段亦可用於進行巢式聚合酶鏈鎖反應(PCR) 〇 在用於預擴增之串接式PCR中,第一擴增區段112.1係 擴增該含有標靶序列之樣本內的核酸序列。此擴增反應無 需對該標靶序列具有專一性(例如,全基因體之擴增反應 ) ’但此擴增反應確實提高該標靶序列之濃度。進行預擴 增反應之後,使該樣本與源自貯存槽6 0.2和貯存槽6 2.2的 試劑混合且隨後使該樣本混合物流入第二擴增區段1 1 2.2 。儲存於貯存槽6 0 ·2中的該等試劑包含僅會擴增該經預擴 增之樣本混合物中之標靶序列的專一性探針。應注意亦可 • 採用如第一擴增階段或第二擴增階段中以恆溫技術取代 PCR的類似方法以達到預擴增之諸多優點》 巢式PCR係一種特殊形式的串接式PCr法,此方法具 有高標靶專一性之附加優點。於巢式P C R法中,第一擴增 -93- 201209158 區段1 1 2.1內的核酸擴增步驟係藉著使用引子擴增長度比 該最終標靶序列要大的序列,該等引子係形成該貯存槽 6 0.1內所儲存之擴增混合試劑的一部分,且該等引子係與 標靶序列外側處的區域互補。第一擴增區段1 1 2. 1中之反 應生成由標靶序列加上夾擊區序列(flanking section)所 組成的擴增子。此經擴增之混合物係與源自貯存槽60.2之 試劑和源自貯存槽62.2之聚合酶混合。儲存於貯存槽60.2 中的該等試劑包含與該標靶序列兩末端處之位置(即,源 自第一擴增階段之擴增子的子區序列)互補的引子。當於 第二擴增區段11 2.2中執行核酸擴增反應時,由於源自第 —擴增階段的擴增子濃度遠大於原始樣本分子之濃度,因 此在與該標靶物無關之序列位置處發生擴增反應的機率大 幅減小。當利用序列專一性恆溫擴增技術取代該等PCR擴 增階段之其中一個階段或兩個階段時,亦能達成巢式PCR 法的靈敏度與專一性之優點。 分開儲存聚合酶且單獨地添加聚合酶於該樣本混合物 中的優點在於可爲預擴增步驟和最終核酸擴增步驟選用不 同的聚合酶。例如,此做法允許爲預擴增步驟選用一種低 錯誤率(例如,可校讀)的聚合酶以防止創造出含錯誤之 標靶序列或不正確之標靶序列,同時允許於最終擴增步驟 中使用較高速或更耐溫的聚合酶。 直接式聚合酶鏈鎖反應 傳統上,於製備反應混合物之前,PCR需要進行標祀 -94- 201209158 DN A的大規模純化。然而,藉著適當改變化學試劑與樣本 濃度,可能藉由最小程度的DNA純化便能執行核酸擴增反 應或可直接進行擴增。當核酸擴增方法是PCR法時,此種 方法稱爲直接式PCR。於受控制之恆定溫度下執行核酸的 LOC裝置中’該方法係直接恆溫擴增法。於L〇c裝置中使 用直接核酸擴增技術(特別是有關簡化所需流體設計方面 )具有相當多的優點。針對直接PCR或直接恆溫擴增法之 擴增化學試劑的調整包括提高緩衝強度、使用具有高活性 和高持續性之聚合酶及可與聚合酶抑制劑螯合之添加劑。 稀釋存在於樣本中的抑制劑亦相當重要。 爲利用直接核酸擴增技術之優勢,該L O C裝置設計納 入兩個附加特徵。第一種特徵係試劑貯存槽(例如第8圖 之貯存槽58),該貯存槽具有適當尺寸以供應足量的擴增 反應混合物或稀釋劑,使得可能干擾擴增化學試劑之樣本 成分的最終濃度夠低以允許成功地進行核酸擴增。非細胞 性樣本成分的期望稀釋程度係介於5倍至20倍。可於適當 時機使用不同的LOC結構(例如第4圖中之病源透析區段 7 0 )以確保標靶核酸序列的濃度維持在可進行擴增和偵測 之夠高濃度。於此具體實施例中(進一步繪示於第6圖) ,係於該樣本萃取區段290之上游處採用透析區段,該透 析區段係有效濃縮該些小到足以進入擴增區段2 9 2的病源 且剔除較大細胞而使該較大細胞進入廢料貯存槽76。於另 一具體實施例中,透析區段係用於選擇性地剔除血漿中的 蛋白質和鹽類,同時留下所關注之細胞。 -95- 201209158 該第二種LOC結構特徵(用於支援直接核酸擴增之特 徵)係通道深寬比之設計,以調整該樣本與該等擴增混合 物成分之間的混合比例。例如,爲確保可通過單次混合步 驟使該樣本帶來之抑制劑較佳稀釋5倍〜20倍,該等樣本通 道與試劑通道的長度和截面係經設計,使得位於該混合作 用啓始位置上游的該樣本通道之流動阻抗比該試劑混合物 所流經之通道的流動阻抗要大4倍〜1 9倍。可透過控制該設 計之幾何結構而輕易地達到控制微通道中之流動阻抗。對 於恆定截面而言,微通道之流動阻抗係隨著通道長度呈線 性遞增。混合設計的重點是,微通道中的流動阻抗更主要 取決於最小之截面尺寸。例如,當微通道之深寬比不一致 ,具有矩形截面之微通道的流動阻抗係與最小垂直尺寸之 立方成反比。 反轉錄酶-聚合酶鏈鎖反應(RT-PCR) 當欲分析或萃取之樣本核酸物種係RN A時,例如源自 RNA病毒之RNA或訊息RNA,進行PCR擴增之前,首先需 使該RNA反轉錄成互補DNA ( cDNA )。可在與進行PCR相 同的腔室中執行反轉錄反應(單步驟式RT-PCR ),或該 反轉錄反應可作爲獨立初始反應(雙步驟式RT-PCR )而 執行。於本案所述之該等LOC裝置變化型中,可藉著使反 轉錄酶連同聚合酶一同加入試劑貯存槽6 2中,且程式化該 等加熱器1 54藉以執行先進行反轉錄步驟且隨後進行核酸 擴增步驟之循環,便可簡單地執行RT_PCR。藉著利用該 -96- 201209158 培育區段1 1 4及利用該試劑貯存槽5 8儲存且分配該等緩衝 液' 引子、dNTP和反轉錄酶進行反轉錄步驟,且隨後利用 一般方法於擴增區段1 1 2中進行擴增,亦可輕易地達成雙 步驟式RT-PCR。 恆溫核酸擴增法 對於某些應用而言,恆溫核酸擴增法係較佳的核酸擴 增方法,因此免除使該等反應成分重複循環經歷各種溫度 循環的需要,取而代之的是使該擴增區段保持一個恆定溫 度(通常約3 7°C ~4 1 °C )。前述已揭示多種恆溫核酸擴增方 法,包括鏈置換擴增法(SDA)、轉錄介導擴增法(TMA )、核酸序列依賴性擴增法(NASBA)、重組酶聚合酶擴 增法(R P A )、解旋酶依賴性®溫D N A擴增法(H D A )、 滾環擴增法(RCA )、分枝擴增法(ramification amplification,RAM )和環形核酸介導擴增法(LAMP ), 且於本案所述之LOC裝置的特定具體實施例中可採用此等 方法之任意一者或其他恆溫擴增方法。 爲執行恆溫核酸擴增,與擴增區段鄰接的該等試劑貯 存槽60與試劑貯存槽62將裝入適用於進行所述恆溫法的試 劑’以替代PCR擴增混合物和聚合酶。例如,爲進行鏈置 換擴增法(SDA ),試劑貯存槽60含有擴增緩衝液、引子 和dNTP’且試劑貯存槽62含有適當的切口酶(nickase) 和外聚-DNA聚合酶(exo-DNA polymerase)。爲進行重組 酶聚合酶擴增法(RPA ),試劑貯存槽60含有擴增緩衝液 -97- 201209158 、引子、dNTP和重組酶,且試劑貯存槽62含有鏈置換dna 聚合酶(例如,万5 μ )。问樣地’爲進行解旋酶依賴性'區 溫DNA擴增法(HDA ) ’試劑貯存槽60含有擴增緩衝液、 引子和dNTP ’且試劑貯存槽62含有適合的DNA聚合酶和取 代加熱以解開雙股DNA鏈的解旋酶。熟悉該項技藝者將理 解可於該兩個試劑貯存槽之間採任何適合進行核酸擴增法 的方式分配該等必要試劑。 由於核酸序列依賴性擴增法(NASBA)或轉錄介導擴 增法(TMA)無需先把RNA轉錄成cDNA,因此NASBA法 或TMA法係合適用於擴增源自RNA病毒(例如HIV病毒或C 型肝炎病毒)的病毒核酸。於此範例中,試劑貯存槽60係 裝入擴增緩衝液、引子和dNTP,且試劑貯存槽62裝.入RNA 聚合酶 '反轉錄酶和選用性的RNA水解酶H (RNase Η)。 對於某些形式的恆溫核酸擴增法而言,於維持用於進 行恆溫核酸擴增法的溫度之前,可能需要具有初始變性循 環以使該雙鏈DN Α模板分開。由於可藉由該等擴增微通道 158內的加熱器154謹慎地控制該擴增區段112內的混合物 之溫度(見第14圖),因此於本案所述LOC裝置之所有具 體實施例中皆可輕易達成此步驟。 恆溫核酸擴增法更能忍受該樣本中潛在的抑制劑,且 如上述般,恆溫核酸擴增法通常適用於當希望以該樣本進 行直接核酸擴增反應之時。因此,恆溫核酸擴增法有時特 別適用於LOC裝置變化型XLIII 673、LOC裝置變化型XLIV 674和LOC裝置變化型XLVII 677,該等變化型673、674和 -98- 201209158 6 7 7係分別顯示於第1 1 7、1 1 8和1 1 9圖。直接式恆溫擴增法 亦可與如第117和119圖所示之一個或一個以上的擴增前透 析步驟70、686或682及/或如第118圖所示之雜合前透析步 驟682合倂使用,以分別於進行核酸擴增反應之前幫助部 分濃縮該樣本中的標靶細胞或於該樣本進入雜合腔室陣列 110之前先去除不想要的細胞殘渣。所屬技術領域中熟悉 該項技藝者將理解可使用上述擴增前透析步驟和雜合前透 析步驟之任意組合。 亦可於並聯的多個擴增區段(如第72、104和105圖槪 要繪示之擴增區段)內執行恆溫核酸擴增法,許多或某些 恆溫核酸擴增方法(例如,環形核酸介導擴增法(LAMP ))係與初始的反轉錄步驟兼容以用於擴增RNA。 螢光偵測系統之附加細節The dialysis design within the LOC device 301 described above is designed for the source of the disease. Figure 64 is a cross-sectional view of the dialysis section 3 2 8 designed to concentrate white blood cells in a blood sample for human DN A analysis. It will be understood that the structure of the dialysis section 328 is substantially identical to the dialysis section 70 of the above-described pathogenic target cells 'only the holes 165 of the dialysis section 328 are holes of 7.5 micrometers in diameter' to limit white blood cells from The capping channel 94 enters the dialysis MST channel 204. When the sample to be analyzed is a blood sample and the hemoglobin derived from the red blood cells interferes with the subsequent reaction step, the anti-coagulant in the storage tank 54 (see Fig. 22) is added together with the red blood cell lysis-89-201209158 The flushing will ensure that most of the lysed red blood cells in the sample are removed during the dialysis step (thus removing hemoglobin). The commonly used red blood cell lysis buffer is a solution containing 0.15M ammonium chloride (NH4C1), 10 mM potassium hydrogencarbonate (KHC03), O.lmM ethylenediaminetetraacetic acid (EDTA) and pH 7_2~7_4, but belongs to Those skilled in the art will understand that any buffer that effectively dissolves red blood cells can be used. The capping channel 94 downstream of the leukocyte dialysis section 328 is converted to the target channel 74 such that the leukocyte continues the assay. Further, in this case, the dialysis upper suction port 68 leads to the waste channel 72' to remove all of the smaller cells and components in the sample. It should be noted that this dialysis variant only reduces the concentration of undesired samples in the target channel 74. Figure 116 schematically illustrates a large component dialysis section 686 which also separates large target components from the sample. The pores in the dialysis section are manufactured to have a sized and shaped shape to block the large target component of interest within the target channel for further analysis. If a leukocyte dialysis section as described above is used, most, but not all, of the smaller cells, organisms or molecules flow into the waste reservoir 768. Thus, other embodiments of the LOC device are not limited to separating white blood cells having a size greater than 7.5 microns, but can also be used to separate cells, organisms or molecules of any desired size. ο 8 segment L 7 zone 4 analysis IIII7 V V type in the Daohua show change 1 set 18 3M3 flow pack 5 0C fall L trap 0 0 i said j gas system stop description There are examples of this and the installation of the real map 3) 3 with 1 set and -90- 201209158. This LOC device has a dialysis section that can inject a fluid sample without leaving air bubbles trapped within the channel. The LOC device variant VIII 518 also has a layer of additional material referred to as the interface layer 5 94. The interfacial layer 594 is between the capping channel layer 80 and the MST channel layer 100 of the CMOS+ MST device 48. The interfacial layer 594 allows for a more complex fluid interconnect structure between the reagent reservoirs and the MST layer 87 without increasing the size of the crucible substrate 84. Referring to Fig. 78, the bypass channel 600 is designed to introduce a time delay as the fluid sample flows from the interface waste channel 206 to the interface target channel 602. This time delay allows the fluid sample to flow through the dialysis MST channel 204 to the dialysis uptake 168 that holds the meniscus. The sample fluid is passed from all of the dialysis uptake holes 168 originating from the dialysis MST channel 204 using a capillary action priming feature (CIF) 202 located at the suction port from the bypass channel 600 to the interface target channel 602. The upstream end is injected into the interface target channel 602. Without the bypass channel 600, the sample fluid will still be injected into the interface target channel 602 from the upstream end, but the final advancing meniscus arrives and passes through the upper suction hole belonging to the unfilled MS T channel. Causes air to fall at this point. This trapped air reduces the sample flow rate through the leukocyte dialysis section 328. Nucleic Acid Amplification Variants Parallel Polymerase Chain Reactions Several variants of the LOC device have multiple extensions operating in parallel - 91 - 201209158. For example, the LOC device variant VII 492 shown in Fig. 72 has a plurality of parallel amplification segments 1 1 2 2 . 1 to 1 1 2.4, and the parallel amplification segments 112.1 to 112.4 allow simultaneous execution of multiple nucleic acid amplifications. Increase analytical testing. The LOC device variant XI 746 shown in Fig. 130 also has a plurality of parallel amplification sections 112.1 to 112.4, but additionally has a plurality of parallel incubation sections 114.1 to 114.4, so that the sample can be expanded prior to amplification. Treated differently. Other LOC device variants (eg, LOC device variant XIV 641 to be displayed in Figure 1-5) indicate that the plurality of parallel amplified segments may be "X", which is limited only by the size of the LOC device. size. The larger the LOC device, the more multiple amplification reaction segments can be accommodated in parallel. The separate amplification segments can be configured to perform different number of cycles and/or temperatures for a particular target size or a particular amplification mixture composition. The L0C device can perform a multiplex nucleic acid amplification method or a single nucleic acid amplification method in each segment by a plurality of amplification sections operating in parallel. In multiplex nucleic acid amplification, more than one target sequence is amplified using one or more primers. A parallel nucleic acid amplification system having "m" chambers can perform an amplification reaction equivalent to η weight, where n = n(l) + n(2) + _..n(i) + ...+ n(m), and n(i) represents the primer number to be used for the chamber "i" in the different primer pairs used for the multiplex amplification reaction, and it is necessary to keep in mind that the parallel amplification system The signal-to-noise ratio (SNR) is higher than the signal-to-noise ratio of the η-type amplification reaction performed in a single-chamber system. In the special case of n ( i ) =1, the amplification reaction in the chamber [i] just becomes a single-fold amplification reaction. -92 - 201209158 Cascading Polymerase Chain Reactions Figures 106, 107, 108, 111 and 114 in these figures illustrate the amplification sections 112.1 and 11 2.2 in the device and the L0C devices operating in series. The first amplification section 1 12.1 includes a reagent storage tank 60.1 of the amplification mixture and a reagent storage tank of the polymerase 62.1 °. Each amplification section after the initial section also includes two reagent storage tanks. The storage tank 60.2 of the mixture and the storage tank 62.2 of the polymerase are added. Multiple amplification segments of φ in series allow for tandem PCR analysis to be performed such that the first amplification segment 11 2 1 is used to perform a pre-amplification reaction to enhance subsequent nucleic acid execution in segment 1 1 2.2 Sensitivity of the amplification reaction. Multiplexed segments can also be used for nested polymerase chain reaction (PCR). In cascade PCR for preamplification, the first amplified segment 112.1 is amplified. A nucleic acid sequence within a sample of the target sequence. This amplification reaction does not require specificity for the target sequence (e.g., amplification of the whole genome)' but this amplification reaction does increase the concentration of the target sequence. After the pre-amplification reaction, the sample is mixed with the reagents from the storage tank 6 0.2 and the storage tank 6 2.2 and then the sample mixture is flowed into the second amplification section 1 1 2.2. The reagents stored in reservoir 60<2> contain specific probes that only amplify the target sequences in the pre-amplified sample mixture. It should be noted that • a similar method of replacing PCR with a constant temperature technique in the first amplification stage or the second amplification stage can be used to achieve the advantages of pre-amplification. Nested PCR is a special form of tandem PCr method. This method has the added advantage of high target specificity. In the nested PCR method, the first amplification-93-201209158 segment 1 1 2.1 nucleic acid amplification step is performed by using a primer to amplify a sequence having a length greater than the final target sequence, and the primers are formed. A portion of the amplification mixing reagent stored in the reservoir 6 0.1, and the primers are complementary to the region at the outside of the target sequence. The reaction in the first amplification segment 1 1 2. 1 generates an amplicon consisting of the target sequence plus a flanking section. This amplified mixture is mixed with a reagent derived from storage tank 60.2 and a polymerase derived from storage tank 62.2. The reagents stored in storage tank 60.2 comprise primers that are complementary to the position at both ends of the target sequence (i.e., the sequence of the sub-regions derived from the amplicon of the first amplification stage). When the nucleic acid amplification reaction is performed in the second amplification section 11 2.2, since the concentration of the amplicon derived from the first amplification stage is much larger than the concentration of the original sample molecule, the sequence position unrelated to the target The probability of an amplification reaction occurring is greatly reduced. The sensitivity and specificity of the nested PCR method can also be achieved when the sequence-specific thermostatic amplification technique is used to replace one or both of these PCR amplification stages. The advantage of separately storing the polymerase and separately adding the polymerase to the sample mixture is that different polymerases can be selected for the pre-amplification step and the final nucleic acid amplification step. For example, this approach allows for a low error rate (eg, readable) polymerase for the preamplification step to prevent the creation of a faulty target sequence or an incorrect target sequence while allowing for the final amplification step. Use a higher speed or temperature resistant polymerase. Direct Polymerase Chain Reactions Traditionally, PCR requires extensive purification of the standard -94-201209158 DN A prior to preparation of the reaction mixture. However, by appropriately changing the chemical reagent and sample concentration, it is possible to perform a nucleic acid amplification reaction or directly perform amplification by minimal DNA purification. When the nucleic acid amplification method is a PCR method, this method is called direct PCR. In a LOC device that performs nucleic acid at a controlled constant temperature, the method is a direct isothermal amplification method. The use of direct nucleic acid amplification techniques in L(R) devices, particularly with respect to simplifying the required fluid design, has considerable advantages. Adjustments to amplification chemistries for direct PCR or direct isothermal amplification include increasing buffer strength, using polymerases with high activity and high persistence, and additives that can be chelated with polymerase inhibitors. It is also important to dilute the inhibitor present in the sample. To take advantage of direct nucleic acid amplification techniques, the L O C device design incorporates two additional features. The first feature is a reagent storage tank (e.g., storage tank 58 of Figure 8) having an appropriate size to supply a sufficient amount of amplification reaction mixture or diluent to cause interference with the sample components of the amplification chemical agent. The concentration is low enough to allow for successful nucleic acid amplification. The desired dilution of the non-cellular sample components is between 5 and 20 times. Different LOC structures (e. g., the source dialysis section 70 in Figure 4) can be used at the appropriate time to ensure that the concentration of the target nucleic acid sequence is maintained at a sufficiently high concentration for amplification and detection. In this particular embodiment (further depicted in FIG. 6), a dialysis section is employed upstream of the sample extraction section 290, the dialysis section being effective to concentrate the small enough to enter the amplification section. The source of 2 and the larger cells are removed to allow the larger cells to enter the waste storage tank 76. In another embodiment, the dialysis section is used to selectively reject proteins and salts in the plasma while leaving the cells of interest. -95- 201209158 This second LOC structural feature (characteristic for supporting direct nucleic acid amplification) is a channel aspect ratio design to adjust the mixing ratio between the sample and the components of the amplification mixture. For example, to ensure that the inhibitor of the sample is preferably diluted 5 to 20 times by a single mixing step, the length and cross section of the sample channel and the reagent channel are designed such that the mixing action is initiated. The flow impedance of the upstream sample channel is 4 to 19 times greater than the flow impedance of the channel through which the reagent mixture flows. The flow impedance in the control microchannel can be easily achieved by controlling the geometry of the design. For a constant cross section, the flow impedance of the microchannel increases linearly with the length of the channel. The focus of the hybrid design is that the flow impedance in the microchannel is more dependent on the smallest cross-sectional dimension. For example, when the aspect ratios of the microchannels are inconsistent, the flow impedance of the microchannel having a rectangular cross section is inversely proportional to the cube of the smallest vertical dimension. Reverse transcriptase-polymerase chain reaction (RT-PCR) When the sample nucleic acid species RN A to be analyzed or extracted, such as RNA or RNA derived from RNA virus, prior to PCR amplification, the RNA is first Reverse transcription into complementary DNA (cDNA). The reverse transcription reaction (single-step RT-PCR) can be performed in the same chamber as the PCR, or the reverse transcription reaction can be performed as an independent initial reaction (two-step RT-PCR). In the variants of the LOC device described in the present application, the reverse transcriptase can be added to the reagent storage tank 6 2 together with the polymerase, and the heaters 1 54 can be programmed to perform the reverse transcription step first and then RT_PCR can be simply performed by performing a cycle of the nucleic acid amplification step. The reverse transcription step is carried out by using the -96-201209158 incubation section 1 14 and using the reagent storage tank 58 to store and dispense the buffers' primers, dNTPs and reverse transcriptase, and then using the general method for amplification Amplification in the segment 1 1 2 can also easily achieve two-step RT-PCR. Thermostatic Nucleic Acid Amplification Method For certain applications, the constant temperature nucleic acid amplification method is a preferred nucleic acid amplification method, thereby eliminating the need to repeatedly cycle the reaction components through various temperature cycles, and instead, the amplification region is replaced. The section is maintained at a constant temperature (typically about 3 7 ° C ~ 4 1 ° C). A variety of thermostatic nucleic acid amplification methods have been disclosed, including strand displacement amplification (SDA), transcription-mediated amplification (TMA), nucleic acid sequence-dependent amplification (NASBA), and recombinant enzyme polymerase amplification (RPA). ), helicase-dependent® warm DNA amplification (HDA), rolling circle amplification (RCA), ramification amplification (RAM), and circular nucleic acid-mediated amplification (LAMP), and Any of these methods or other isothermal amplification methods can be employed in particular embodiments of the LOC device described herein. To perform thermostatic nucleic acid amplification, the reagent storage tank 60 and reagent storage tank 62 adjacent to the amplification section will be loaded with a reagent 's suitable for performing the constant temperature method' in place of the PCR amplification mixture and the polymerase. For example, to perform strand displacement amplification (SDA), reagent storage tank 60 contains amplification buffer, primers, and dNTP' and reagent storage tank 62 contains appropriate nickase and exo-DNA polymerase (exo- DNA polymerase). For recombinase polymerase amplification (RPA), reagent storage tank 60 contains amplification buffer -97 - 201209158, primer, dNTP, and recombinase, and reagent storage tank 62 contains strand displacement dna polymerase (eg, 10,000 μ). The sample 'for the helicase-dependent' region temperature DNA amplification method (HDA) 'reagent storage tank 60 contains amplification buffer, primer and dNTP' and reagent storage tank 62 contains suitable DNA polymerase and substituted heating To unwind the helicase of the double stranded DNA strand. Those skilled in the art will appreciate that such necessary reagents can be dispensed between any of the two reagent storage tanks in any manner suitable for nucleic acid amplification. Since nucleic acid sequence-dependent amplification (NASBA) or transcription-mediated amplification (TMA) does not require transcription of RNA into cDNA first, the NASBA method or the TMA method is suitable for amplifying RNA viruses (eg HIV viruses or Viral nucleic acid of hepatitis C virus). In this example, reagent reservoir 60 is loaded with amplification buffer, primers, and dNTPs, and reagent reservoir 62 is loaded with RNA polymerase 'reverse transcriptase and optional RNA hydrolase H (RNase®). For some forms of thermostatic nucleic acid amplification, it may be desirable to have an initial denaturation cycle to separate the double-stranded DN Α template prior to maintaining the temperature for the thermostatic nucleic acid amplification process. Since the temperature of the mixture within the amplification section 112 can be carefully controlled by the heaters 154 in the amplification microchannels 158 (see Figure 14), in all of the specific embodiments of the LOC apparatus described herein This step can be easily reached. The thermostatic nucleic acid amplification method is more tolerant of potential inhibitors in the sample, and as described above, the thermostatic nucleic acid amplification method is generally suitable when it is desired to perform a direct nucleic acid amplification reaction with the sample. Therefore, the thermostatic nucleic acid amplification method is sometimes particularly suitable for the LOC device variant XLIII 673, the LOC device variant XLIV 674, and the LOC device variant XLVII 677, which are separately 673, 674, and -98-201209158 6 7 7 respectively. Shown on pages 1 1 7 , 1 1 8 and 1 1 9 . The direct isothermal amplification method can also be combined with one or more pre-amplification dialysis steps 70, 686 or 682 as shown in Figures 117 and 119 and/or a pre-hybridization dialysis step 682 as shown in Figure 118. The hydrazine is used to help partially concentrate the target cells in the sample prior to performing the nucleic acid amplification reaction or to remove unwanted cell debris prior to entering the hybrid chamber array 110. Those skilled in the art will appreciate that any combination of the pre-amplification dialysis step described above and the pre-hybridization dialysis step can be used. Thermostatic nucleic acid amplification, multi- or some thermostatic nucleic acid amplification methods can also be performed in multiple amplification sections in parallel (such as the amplification sections to be depicted in Figures 72, 104, and 105) (eg, Circular Nucleic Acid Mediated Amplification (LAMP) is compatible with the initial reverse transcription step for amplification of RNA. Additional details of the fluorescence detection system

第58和59圖顯示雜合敏感性FRET探針236。此等探針 通常稱爲分子信標且爲幹·環狀探針,此等探針係由單鏈 核酸生成,並且當該等探針與互補之核酸雜合時會發出螢 光。第58圖顯示與標靶核酸序列23 8雜合前的單個FRET探 -針236。該探針具有環部240、主幹242、位於5'端的螢光 發光基團246及位於V端的消光基團248。該環部240係由 與該標靶核酸序列2 3 8互補的序列所組成。位於該探針序 列兩側上的互補序列黏合在一起而形成該主幹242。 當缺乏互補之標靶序列時,該探針保持如第5 8圖所示 之閉合狀。該主幹242使該螢光發光基團與消光基團之配 -99- 201209158 對保持彼此靠近,使得該螢光發光基團與該消光基團之間 可發生顯著的共振能量轉移,而實質消除該螢光發光基團 受激發光244照射時的發光能力。 第59圖顯示FRET探針2 3 6處於打開或已雜合之結構。 當與標靶核酸序列23 8雜合時,該幹-環狀結構會瓦解,該 螢光發光基團246和消光基團2 48係呈空間上分離,因而恢 復該營光發光基團246可發出螢光之能力。光學測得該螢 光釋放250係代表該探針已經雜合。 由於該探針之主幹螺旋結構係經設計以使該主幹螺旋 結構比該具有單個核酸不互補的探針-標靶物螺旋結構更 穩定,因此該等探針以非常高的專一性與互補標靶序列雜 合。由於雙鏈DNA係相對較剛硬,因此雙鏈DNA於立體結 構上不可能發生該探針-標靶物螺旋結構與該主幹螺旋結 構共同存在的情況。 接有引子之探針 接有引子之幹-環狀探針及接有引子之線性探針(或 稱蠍型探針)係分子信標之替代物,且該等探針可用於該 L Ο C裝置中以進行即時且定量性核酸擴增。即時擴增法可 於該LOC裝置的該等雜合腔室內直接執行。使用接有引子 之探針的益處在於該探針分子係與該引子物理性連接,因 此於核酸擴增期間期間僅需發生單次雜合事件,而無需使 引子之雜合反應與探針之雜合反應分開進行。此可確保反 應有效地即時進行,且相較於分開使用引子與探針而言, -100- 201209158 該接有引子之探針可得到更強信號、更短的反應時間和更 佳的識別力。可於製造期間把該等探針(連同聚合酶和擴 增混合物)置入該等雜合腔室180內,且無需於該LOC裝 置上另闢獨立的擴增區段。或者,該擴增區段可保留但不 使用或用於進行其他反應。 接有引子之線性探針 第120和121圖分別顯示於初始回合的核酸擴增反應期 間內一種接有引子之線性探針692及於後續回合之核酸擴 增反應期間該線性探針經雜合的結構。參閱第1 2 0圖,該 接有引子之線性探針692具有一個雙鏈主幹部位242。該雙 鏈中之一鏈包含該接有引子之探針序列696,該探針序列 696係與該標靶核酸序列696上的一段區域相同,該探針序 列696之V端上標記螢光發光基團246且該探針序列696之Y 端係透過擴增阻斷子694連接寡聚核苷酸引子700。於該主 幹242之另一條鏈的Y端處標記消光基團248。於初始回合 之核酸擴增反應完成後,該探針可圈成環狀且利用現已互 補之序列69 8與該經延長之核酸鏈雜合。於初始回合之核 酸擴增反應期間,該寡聚核苷酸引子700黏合於該標靶 DNA23 8上(見第120圖)且隨後該引子700係經延長而形 成包含該探針序列與該擴增產物兩者的DNA鏈。該擴增阻 斷子694防止聚合酶讀取和複製該探針區域696。當進行接 續之變性反應時,該經延長之寡聚核苷酸引子700與模板 之雜合體係解離,且該接有引子之線性探針的雙鏈主幹 -101 - 201209158 242亦會解離而釋出該消光基團248。一旦溫度降低以進行 黏合步驟和延長步驟時,該接有引子之線性探針的接有引 子之探針序列696會捲起且與該經延長之核酸鏈上的該經 擴增之互補序列6 9 8雜合,且測得螢光係表示存在該標祀 DN A。未延長的接有引子之線性探針保有本身的雙鏈主幹 且仍保持消光狀態。由於此種偵測方法係憑藉單分子處理 (single molecular process),因此此偵測方法特別適用 於快速偵測系統。 接有引子之幹-環狀探針 第122A至122F圖顯示接有引子之幹-環狀探針704的操 作。參閱第122A圖,接有引子之幹-環狀探針7 〇4具有由互 補雙鏈DNA組成之主幹242和含有該探針序列之環部240。 該等主幹鏈708中之一主幹鏈的5'端上標記螢光發光基團 246。該另一條主幹鏈710係於V端標記消光基團248,且 該主幹鏈710同時攜帶擴增阻斷子694和寡聚核苷酸引子 700兩者。於初始變性階段期間(見第122B圖),該標靶 核酸238之該等核酸鏈係如同該接有引子之幹-環狀探針 7 〇4之主幹242般分離。當冷卻溫度以進行黏合階段時(見 第122C圖),該接有引子之幹-環狀探針704上的寡聚核苷 酸引子700與該標靶核酸序列2 3 8雜合。於延長階段期間( 見第122D圖),合成出與該標靶核酸序列2 3 8互補的互補 序列7 0 6而形成同時含有該探針序列7 〇 4和擴增產物兩者的 DN A鏈。擴增阻斷子6 94防止聚合酶讀取和複製該探針區 -102- 201209158 域7 〇4。當該探針於變性步驟(見第122E圖)之後接著進 行黏合,該接有引子之幹-環狀探針之環部240的探針序列 (見第122F圖)與該延長鏈上的互補序列706黏合。此種 結構使該螢光發光基團246相對遠離該消光基團248,得以 明顯提高發光作用。 對照探針 · φ 雜合腔室陣列110包含一些含有陽性對照探針及陰性 對照探針的雜合腔室1 80,該等探針係用於分析檢驗之品 質控制。第135及136圖槪要說明不含螢光發光基團之陰性 對照探針796,且第137及138圖係不含消光基團之陽性對 照探針7 9 8的槪要圖。陽性對照探針和陰性對照探針具有 類似上述FRET探針的幹-環狀結構。然而,不論該等探針 是否雜合而成爲打開結構或保持閉合狀態,陽性對照探針 79 8將一直發出螢光信號25 0,且陰性對照探針796則永遠 φ 不會發出螢光信號250。 參閱第135及136圖,陰性對照探針796不具螢光發光 基團(且可具有或可能不具有消光基團248)。因此,不 論是該標靶核酸序列23 8是否與該探針雜合(見第1 3 6圖) ‘’或是該探針維持本身的幹-環狀結構(見第135圖),該 陰性對照探針對激發光244的反應皆微不足道。或者,陰 性對照探針796可經設計’而使該陰性對照探針796永遠保 持消光。例如’藉著人工合成該環部240使該環部具有一 段將不會與硏究中之樣本內任何核酸序列雜合的探針序列 -103- 201209158 ,該探針分子之主幹2 42將重新與該探針分子本身雜合, 且螢光發光基團與消光基團將保持靠近且將不會偵測到可 見之螢光信號。此陰性對照信號可相當於源自雜合腔室 1 80的低度釋放光(該些腔室中之探針未經雜合但消光基 團未能消除源自報導基團(reporter )之所有釋放光)。 反之,如第1 3 7和1 3 8圖所示,該陽性對照探針798係 經建構成不含消光基團。不論該陽性對照探針7 9 8是否與 該標靶核酸序列238雜合,皆無法消除該螢光發光基團246 回應激發光244所釋放的螢光250。 第5 2圖顯示陽性對照探針及陰性對照探針於該雜合腔 室陣列1 1 0各處的可能分佈情形(分別爲3 7 8和3 8 0 )。於 穿越該雜合腔室陣列110配置成一排的多個雜合腔室180中 放置該等對照探針3 7 8和3 8 0。然而,該等對照探針於該陣 列中的配置方式係隨意配置(依據雜合腔室陣列1 1 〇當時 的配置形態而定)。 螢光發光基團之設計 具有長時間發光壽命週期的螢光發光基團係必要的, 以允許有足夠的時間以供激發光衰減至低於螢光發光的強 度(此時使該光感測器44運作)’從而提供足夠的信號雜 訊比。此外,蛋光發光壽命週期越長’轉換成積分營光光 子計數越大。 螢光發光基團246 (見第59圖)具有大於1 00奈秒的螢 光壽命週期,通常大於200奈秒、更常大於300奈秒且最常 -104- 201209158 爲大於400奈秒。 以過渡金屬或鑭系元素爲基礎之金屬-配體錯合物具 有長壽命週期(從數百奈秒至數百毫秒)、充分的量子產 率及高的溫度穩定性、化學穩定性和光化學穩定性’這些 性質係與螢光偵測系統要求有關的有利性質。 經充分硏究之以過渡金屬離子釕(Ru ( II ))爲基礎 的金屬-配體錯合物係三(2,2f-聯吡啶)釕(Π ) ( [Ru ( bpy) 3]2+),此錯合物具有約1微秒之壽命週期。此錯合 物係Biosearch Technology公司旗下品名爲Pulsar 650之商 品0 表一、Pulsar 650(釕螯合物)之光物理性質 參數 符號 數値 單位 吸收波長 λ a b s 460 奈米 發光波長 λ e m 650 奈米 消光係數 E 1 4800 螢光壽命週期 Tf 1.0 微秒 量子產率 H 1(脫氧) 無(n/a) 铽螯合物(terbium chelate)係一種鑭系金屬-配體錯 合物,已成功證明鉞螯合物可作爲FRET探針系統中的螢 光報導基團,且鉱蠻合物亦具有1600微秒之長帚命週期。 -105- 201209158 表二、銶螯合物之光物理性質 參數 符號 數値 單位 吸收波長 Labs 330-350 奈米 發光波長 ^em 548 奈米 消光係數 E 13800 (依據xabs和配體,於Xe=340奈 米處,消光係數可高達30000) NfW1 螢光壽命週期 Tf 1600 (已雜合之探針) 微秒 量子產率 Η 1 (依據配體而定) 無(N/A) LOC裝置301使用的螢光偵測系統未使用濾波器去除 不想要的背景螢光。因此若消光基團24 8不具有原生光( native emission)是有益的,藉以提高信號雜訊比。不具 原生光,便沒有源自消光基團248之背景螢光。高消光率 亦很重要,如此可防止在發生雜合反應之前發出螢光。黑 洞式消光基團(BHQ)不具有原生光且具有高消光率(黑 洞式消光基團可購自美國加州諾瓦多市(Novato )之 Biosearch Technologies公司),是適用於該系統的消光基 團。黑洞式消光基團BHQ-1具有534奈米之最大吸收波長 ,且消光範圍介於480奈米〜5 80奈米,使得BHQ-1可作爲 適用於Tb-螯合物螢光發光基團的消光基團。黑洞式消光 基團BHQ-2具有5 79奈米之最大吸收波長,且消光範圍介 於560奈米〜670奈米,使得BHQ-2可作爲適用於螢光發光 基團Pulsar 650的消光基團。 愛荷華黑消光基團(Iowa Black FQ與RQ,可購自美 -106- 201209158 國愛荷華州科勒爾維爾市(Coralville)之Integrated DNA Technologies公司)係具有少許背景光或無背景光之適用 的替代消光基團。愛荷華黑FQ具有介於420奈米〜620奈米 之消光範圍且具有531奈米之最大吸收波長,因此愛荷華 黑FQ係適用於Tb-螯合物螢光發光基團的消光基團。愛荷 華黑RQ具有65 6奈米之最大吸收波長,且消光範圍介於500 奈米〜700奈米,使得愛荷華黑RQ係用於螢光發光基團 Pulsar 650的理想消光基團。 於本案所述實施例中,消光基團248係一種於最初便 接附於該探針上的官能性基團,但於其他可行之實施例中 該消光基團係自由存在於溶液中的獨立分子。 激發光來源Figures 58 and 59 show the hybrid sensitive FRET probe 236. Such probes are commonly referred to as molecular beacons and are dry-loop probes that are produced from single-stranded nucleic acids and that emit fluorescence when the probes are hybridized to complementary nucleic acids. Figure 58 shows a single FRET probe 236 prior to hybridization with the target nucleic acid sequence 23 8 . The probe has a ring portion 240, a stem 242, a fluorescent luminescent group 246 at the 5' end, and a matting group 248 at the V end. The loop portion 240 is composed of a sequence complementary to the target nucleic acid sequence 238. Complementary sequences located on either side of the probe sequence are bonded together to form the stem 242. In the absence of a complementary target sequence, the probe remains closed as shown in Figure 58. The stem 242 maintains the phosphorescent group and the matting group-99-201209158 pair close to each other, so that significant resonance energy transfer can occur between the fluorescent group and the extinction group, and substantial elimination occurs. The luminescent group of the luminescent group is illuminated by the excitation light 244. Figure 59 shows the structure in which the FRET probe 2 3 6 is open or hybrid. When hybridized with the target nucleic acid sequence 23 8 , the dry-loop structure is collapsed, and the fluorescent luminescent group 246 and the extinction group 2 48 are spatially separated, thereby restoring the camping luminescent group 246 The ability to emit fluorescent light. Optically measuring the fluorescence release 250 indicates that the probe has been hybridized. Since the backbone helical structure of the probe is designed such that the backbone helical structure is more stable than the probe-target helical structure that is not complementary to a single nucleic acid, the probes have very high specificity and complementarity The target sequence is heterozygous. Since the double-stranded DNA system is relatively rigid, it is unlikely that the double-stranded DNA is stereostructured to coexist with the probe-target helix structure and the backbone helix structure. The probe with the primer is followed by a dry-loop probe with a primer and a linear probe (or 蝎 probe) with a primer, which is a substitute for the molecular beacon, and the probe can be used for the L Ο C device for immediate and quantitative nucleic acid amplification. The instant amplification method can be performed directly in the hybrid chambers of the LOC device. The benefit of using a probe with a primer is that the probe molecule is physically linked to the primer, so that only a single heterozygous event occurs during the nucleic acid amplification period, without the need for a hybrid reaction of the primer and the probe. The heterozygous reaction proceeds separately. This ensures that the reaction is carried out efficiently and in real time, and that the probe with the primer gives a stronger signal, shorter reaction time and better discrimination than the separate use of the primer and the probe -100-201209158 . The probes (along with the polymerase and the amplification mixture) can be placed into the hybrid chambers 180 during manufacture without the need for separate amplification sections on the LOC device. Alternatively, the amplified segment can be retained but not used or used for other reactions. The linear probes with primers are shown in Figures 120 and 121, respectively, showing a linear probe 692 with primers during the initial round of the nucleic acid amplification reaction and the linear probe during the subsequent round of nucleic acid amplification reactions. Structure. Referring to Figure 1 20, the linear probe 692 with the primer has a double-stranded stem portion 242. One of the double strands comprises the probe sequence 696 with the primer, the probe sequence 696 is identical to a region on the target nucleic acid sequence 696, and the fluorescent sequence is labeled on the V-terminus of the probe sequence 696. The group 246 and the Y-terminus of the probe sequence 696 are ligated to the oligonucleotide primer 700 via amplification blocker 694. A matting group 248 is marked at the Y end of the other chain of the backbone 242. Upon completion of the nucleic acid amplification reaction of the initial round, the probe can be looped and hybridized to the extended nucleic acid strand using the now complementary sequence 698. During the nucleic acid amplification reaction of the initial round, the oligonucleotide primer 700 is adhered to the target DNA 23 8 (see FIG. 120) and then the primer 700 is extended to form the probe sequence and the extension. The DNA strand of both products is increased. The amplification blocker 694 prevents the polymerase from reading and replicating the probe region 696. When the subsequent denaturation reaction is carried out, the extended oligonucleotide primer 700 is dissociated from the hybrid system of the template, and the double-stranded backbone-101 - 201209158 242 with the linear probe of the primer is also dissociated. The matting group 248 is exited. Once the temperature is lowered for the binding step and the extension step, the primer-like probe sequence 696 of the primer-attached linear probe is rolled up and the amplified complementary sequence 6 on the extended nucleic acid strand 9 8 is heterozygous, and the measured fluorescence indicates the presence of the standard DN A. The unexpanded linear probe with primers retains its own double-stranded backbone and remains in a matte state. Since this detection method relies on a single molecular process, this detection method is particularly suitable for fast detection systems. Dry-ring probe with primers The 122A to 122F diagrams show the operation of the stem-loop probe 704 with the primer attached. Referring to Fig. 122A, the stem-loop probe 7 〇4 with primer has a stem 242 composed of complementary double-stranded DNA and a loop 240 containing the probe sequence. Fluorescent luminescent groups 246 are labeled on the 5' end of one of the backbone chains 708. The other backbone strand 710 is linked to a V-terminally labeled extinction group 248, and the backbone strand 710 carries both the amplification blocker 694 and the oligonucleotide primer 700. During the initial denaturation phase (see Figure 122B), the nucleic acid strands of the target nucleic acid 238 are separated as the stem 242 of the stem-loop probe 7 〇4 with the primer introduced. When the temperature is cooled to carry out the bonding stage (see Figure 122C), the oligonucleotide primer 700 on the stem-loop probe 704 with the primer is hybridized to the target nucleic acid sequence 2 3 8 . During the elongation phase (see Figure 122D), the complementary sequence 760, which is complementary to the target nucleic acid sequence 298, is synthesized to form a DN A chain containing both the probe sequence 7 〇4 and the amplification product. . Amplification blocker 6 94 prevents the polymerase from reading and replicating the probe region -102- 201209158 domain 7 〇4. When the probe is followed by a denaturation step (see Figure 122E) followed by adhesion, the probe sequence of the loop portion 240 of the stem-loop probe with the primer (see Figure 122F) is complementary to the extension chain. Sequence 706 is bonded. This configuration allows the fluorescent luminescent group 246 to be relatively far from the extinction group 248 to significantly enhance luminescence. Control Probes φ Hybrid Chamber Array 110 contains a number of hybrid chambers 1 80 containing positive control probes and negative control probes for quality control of analytical assays. Sections 135 and 136 illustrate a negative control probe 796 that does not contain a fluorescent luminescent group, and lines 137 and 138 are schematic representations of a positive control probe 798 without an extinction group. The positive control probe and the negative control probe have a dry-loop structure similar to the above FRET probe. However, regardless of whether the probes are hybridized to become an open structure or remain closed, the positive control probe 79 8 will always emit a fluorescent signal 25 0, and the negative control probe 796 will always emit a fluorescent signal 250. . Referring to Figures 135 and 136, the negative control probe 796 does not have a fluorescent luminescent group (and may or may not have an extinction group 248). Thus, whether or not the target nucleic acid sequence 23 8 is hybridized to the probe (see Figure 136), or the probe maintains its own dry-loop structure (see Figure 135), the negative The response of the control probe to excitation light 244 was negligible. Alternatively, the negative control probe 796 can be designed to keep the negative control probe 796 permanently matted. For example, by artificially synthesizing the loop 240, the loop has a probe sequence that will not be hybridized to any nucleic acid sequence in the sample under investigation -103-201209158, and the backbone of the probe molecule 2 42 will be re It is hybridized to the probe molecule itself, and the fluorescent luminescent group and the extinction group will remain close and no visible fluorescent signal will be detected. This negative control signal can correspond to a low release of light originating from the hybrid chamber 180 (the probes in the chambers are not heterozygous but the extinction group fails to eliminate all of the reporters from the reporter) Release light). Conversely, as shown in Figures 137 and 138, the positive control probe 798 is constructed to contain no matting groups. Whether or not the positive control probe 798 is heterozygous to the target nucleic acid sequence 238 does not eliminate the fluorescent light 250 released by the fluorescent luminescent group 246 back to the stress luminescence 244. Figure 5 2 shows the possible distribution of the positive control probe and the negative control probe throughout the hybrid chamber array 110 (3 7 8 and 380, respectively). The control probes 3 7 8 and 380 are placed in a plurality of hybrid chambers 180 arranged in a row across the hybrid chamber array 110. However, the arrangement of the control probes in the array is arbitrarily configured (depending on the configuration of the hybrid chamber array 1 1 〇 at the time). The design of the fluorescent luminescent group is necessary to have a long-term luminescent lifetime of the fluorescent luminescent group to allow sufficient time for the excitation light to decay below the intensity of the fluorescent luminescence (the light sensing is now performed) The device 44 operates) to provide sufficient signal to noise ratio. In addition, the longer the egg light luminescence life cycle, the greater the conversion to the integral camp photon count. Fluorescent luminescent group 246 (see Figure 59) has a fluorescence lifetime of greater than 100 nanoseconds, typically greater than 200 nanoseconds, more often greater than 300 nanoseconds, and most often -104 to 201209158 is greater than 400 nanoseconds. Metal-ligand complexes based on transition metals or lanthanides have long lifetimes (from hundreds of nanoseconds to hundreds of milliseconds), sufficient quantum yield and high temperature stability, chemical stability and photochemistry Stability 'These properties are of an advantageous nature in relation to the requirements of the fluorescence detection system. Metal-ligand complex based on transition metal ion ruthenium (Ru(II)) is fully studied. Tris(2,2f-bipyridyl) ruthenium (Π) ( [Ru ( bpy) 3] 2+ The complex has a life cycle of about 1 microsecond. This complex is a product of Biosearch Technology under the name Pulsar 650. Table 1 , Pulsar 650 (钌 chelate) photophysical property parameter number 値 unit absorption wavelength λ abs 460 nm emission wavelength λ em 650 nm Extinction coefficient E 1 4800 Fluorescence lifetime Tf 1.0 Microsecond quantum yield H 1 (deoxidation) None (n/a) Terbium chelate is a lanthanide metal-ligand complex that has been successfully demonstrated The ruthenium chelate can serve as a fluorescent reporter group in the FRET probe system, and the ruthenium complex also has a long life cycle of 1600 microseconds. -105- 201209158 Table 2. Photophysical properties of ruthenium chelate parameters Symbol number 値 unit absorption wavelength Labs 330-350 nm luminescence wavelength ^em 548 nm extinction coefficient E 13800 (according to xabs and ligand, at Xe=340 At the nanometer, the extinction coefficient can be as high as 30,000) NfW1 Fluorescence lifetime Tf 1600 (probe of hybrid probe) Microsecond quantum yield Η 1 (depending on the ligand) None (N/A) used by LOC device 301 The fluorescence detection system does not use a filter to remove unwanted background fluorescence. Therefore, it is beneficial if the extinction group 24 8 does not have a native emission, thereby increasing the signal to noise ratio. Without native light, there is no background fluorescence from the extinction group 248. High extinction is also important to prevent fluorescence from occurring before a heterozygous reaction occurs. The black hole type matting group (BHQ) does not have primary light and has a high extinction ratio (black hole type extinction group available from Biosearch Technologies of Novato, California, USA), which is an extinction group suitable for the system. . The black hole type matting group BHQ-1 has a maximum absorption wavelength of 534 nm, and the extinction range is from 480 nm to 580 nm, making BHQ-1 suitable as a fluorescent luminescent group for Tb-chelate. Extinction group. The black hole type matting group BHQ-2 has a maximum absorption wavelength of 5 79 nm, and the extinction range is from 560 nm to 670 nm, making BHQ-2 an extinction group suitable for the fluorescent luminescent group Pulsar 650. . Iowa Black Matting Group (Iowa Black FQ and RQ, available from US-106-201209158, Integrated DNA Technologies, Coralville, Iowa) with little or no background light Suitable alternative extinction groups. Iowa Black FQ has an extinction range of 420 nm to 620 nm and has a maximum absorption wavelength of 531 nm, so Iowa Black FQ is suitable for the extinction base of Tb-chelate fluorescent group. group. Iowa Black RQ has a maximum absorption wavelength of 65 6 nm, and the extinction range is from 500 nm to 700 nm, making Iowa Black RQ an ideal extinction group for the fluorescent luminescent group Pulsar 650. In the embodiments described herein, the matting group 248 is a functional group that is initially attached to the probe, but in other feasible embodiments the matting group is freely present in solution. molecule. Source of excitation light

於本案所述之基於螢光偵測的實施例中,由於發光二 極體(LED )具有低功率消耗、低成本且小尺寸,故選擇 LED取代雷射二極體、高功率燈或雷射作爲激發光來源。 參閱第123圖,LED 26係設置於該LOC裝置301之外表面上 且位於該雜合腔室陣列1 1 0之正上方。該光感測器44則位 於雜合腔室陣列110之反面上,該光感測器44係由多個光 二極體184所組成之陣列(見第53、54和65圖)以用於偵 測源自每個腔室的螢光信號。 第124、125和126圖槪要圖解說明使探針暴露於激發 光下的其他實施例。於第124圖所示之LOC裝置30中,由 激發LED 26產生之激發光244係藉由透鏡254引導至雜合腔 -107- 201209158 室陣列1 10上。激發LED 26係經脈衝’且利用光感測器44 偵測螢光釋放光。 於第125圖所示之LOC裝置30中,由激發LED 26產生 之激發光2 44係藉由透鏡254、第一光學稜鏡712和第二光 學稜鏡71 4引導至雜合腔室陣列110上。激發LED 26係經脈 衝,且利用光感測器44偵測螢光釋放光。 同樣地,於第126圖所示之LOC裝置30中,由激發LED 26產生之激發光244係藉由透鏡2 54、第一反射鏡716和第 二反射鏡7 1 8引導至雜合腔室陣列1 1 0上。同樣地,激發 LED 26係經脈衝,且利用光感測器44偵測螢光釋放光。 LED 26之激發光波長係取決於螢光染劑之選擇而有所 不同。飛利浦LXK2-PR14-R00型LED係適用於Pulsar 650染 齊!J之激發光源。SET UVT0P 3 3 5 T039BL型LED係適用於Tb-螯合物標示物之激發光源。 表三、飛〗 FIJ 浦 LXK2-PR14-R00型 LED之規格 參數 符號 數値 單位 波長 λ e X 460 奈米 發射頻率 Vem 6.52(10)14 赫茲(Hz) 輸出功率 Pl 每1安培0.5 1 5分鐘 瓦特(w) 輻射分佈圖 朗伯分佈輪廓 無(N/A) -108- 201209158 表四、SET UVT0P334T039BL LED之規格 參數 符號 數値 單位 波長 λε 340 奈米 發射頻率 Ve 8.82(10)14 赫茲(Hz) 功率 Pi 每20毫安培0·000240分鐘 瓦特(w) 脈衝順向電流 I 200 毫安培(mA) 輻射分佈圖 朗伯分佈 無(N/A) 紫外線激發光 矽吸收紫外線(UV )光譜中的極少光線。因此,使 用紫外線激發光是有利的。雖可使用紫外線LED激發光源 ’但LED 26的廣範圍光譜會降低此方法之功效。爲解決此 問題,可使用經濾波之紫外線LED。除非雷射之相對高成 本無法於特定檢驗模組市場上推行,否則可隨意地使用紫 外線雷射作爲激發光源。 LED驅動器 LED驅動器29以固定電流驅動該LED 26持續所需之時 間。低功率USB 2.0認證驅動器可提供最高1單位負載( 100毫安培)且具有4.4伏特之最小操作電壓。標準功率調 節電路係用於達成此目的。 光二極體 第54圖顯示整合於L〇C裝置301之CMOS電路86中的光 二極體184。光二極體I84係製成CMOS電路86的一部分, 且無需額外的遮罩或步驟。此爲CMOS光二極體明顯勝於 -109- 201209158 CCD的優勢(CCD係一種替代性的感測技術,使用非標準 處理步驟可把CCD整合於同一個晶片上或製造於鄰近的晶 片上)。晶片上偵測法係低成本且可縮小該分析檢驗系統 之尺寸。較短之光學路徑長度可減小源自周遭環境之雜訊 ,以達到有效收集螢光信號且免除對於由透鏡和濾波器所 組成之傳統光學組件的需求。 光二極體184之量子效率係光子撞擊於於該光二極體 184之主動區185上而有效轉換成光電子之比例。對於標準 矽製程而言,視製程參數(例如,覆蓋層之數量和吸收性 質)而定,針對可見光之量子效率係介於0.3至0.5。 光二極體1 84之偵測臨界値決定所能偵測到之螢光信 號的最小強度。該偵測臨界値亦決定光二極體1 84之尺寸 且從而決定該雜合與偵測區段52中的雜合腔室180之數目 (見第5 2圖)。腔室的尺寸和數目係技術性參數,該等技 術性參數受限於LOC裝置之尺寸(於LOC裝置301之例子中 ,該尺寸係1 760微米X 5 8 24微米)和納入其他功能模組( 例如病源透析區段70及擴增區段11 2 )之後可獲得的實際 佔地面積。 對於標準矽製程而言,光二極體1 84偵測最少5個光子 。然而,爲確保可靠地偵測,該最少値可設定爲1 〇個光子 。因此,如上述具有介於0.3〜0.5的量子效率而言,源自該 等探針的螢光釋放光必需最少爲17個光子,但爲了達成可 靠偵測可納入適當誤差範圍而爲30個光子。 -110- 201209158 校準腔室 光二極體184之電特性、自發螢光及尙未完全衰退之 殘餘激發光子通量的不一致性會於該輸出信號中導入把背 景雜訊和偏移。係使用一個或一個以上之校準信號去除每 個輸出信號中的背景値。係藉著使該陣列中的一個或一個 以上之校準光二極體184暴露於各自的校準光源以生成校 準信號。使用低校準光源用於決定陰性結果(陰性結果係 標靶物未與探針反應)。高校準光源係表示源自於探針-標靶複合物之陽性結果。於本案所述實施例中,係由雜合 腔室陣列1 10中的多個校準腔室3 82提供低校準光源,該等 校準腔室3 8 2係: 不含任何探針; 含有不具螢光報導基團之探針;或 含有報導基團和消光基團但該消光基團係經配置而預 期消光作用會一直發生的探針。In the fluorescence detection based embodiment described in the present application, since the light emitting diode (LED) has low power consumption, low cost, and small size, the LED is selected to replace the laser diode, the high power lamp or the laser. As a source of excitation light. Referring to Fig. 123, LEDs 26 are disposed on the outer surface of the LOC device 301 and directly above the hybrid chamber array 110. The photo sensor 44 is located on the opposite side of the hybrid chamber array 110. The photo sensor 44 is an array of a plurality of photodiodes 184 (see Figures 53, 54 and 65) for detection. The fluorescent signal originating from each chamber is measured. Embodiments 124, 125, and 126 illustrate other embodiments for exposing the probe to excitation light. In the LOC device 30 shown in Fig. 124, the excitation light 244 generated by the excitation LED 26 is directed by the lens 254 to the hybrid cavity - 107 - 201209158 chamber array 1 10 . The excitation LED 26 is pulsed' and the photodetector 44 is used to detect the fluorescence release light. In the LOC device 30 shown in FIG. 125, the excitation light 2 44 generated by the excitation LED 26 is directed to the hybrid chamber array 110 by the lens 254, the first optical 稜鏡 712, and the second optical 稜鏡 71 4 . on. The excitation LED 26 is pulsed and the photodetector 44 is used to detect the fluorescent light. Similarly, in the LOC device 30 shown in FIG. 126, the excitation light 244 generated by the excitation LED 26 is guided to the hybrid chamber by the lens 2 54 , the first mirror 716 and the second mirror 7 18 . Array 1 1 0. Similarly, the excitation LED 26 is pulsed and the photodetector 44 is used to detect the fluorescent light release. The wavelength of the excitation light of LED 26 varies depending on the choice of fluorescent dye. Philips LXK2-PR14-R00 LED system is suitable for Pulsar 650 dyeing! J excitation source. SET UVT0P 3 3 5 The T039BL LED is suitable for the excitation source of the Tb-chelate label. Table 3, Fei〗 FIJ Pu LXK2-PR14-R00 type LED specification parameter number 値 unit wavelength λ e X 460 nm emission frequency Vem 6.52 (10) 14 Hz (Hz) output power Pl per 1 amp 0.5 1 5 minutes Watt (w) Radiation Profile Lambert Distribution Profile None (N/A) -108- 201209158 Table IV, SET UVT0P334T039BL LED Specifications Parameter Number 値 Unit Wavelength λε 340 Nano Transmit Frequency Ve 8.82(10)14 Hertz (Hz Power Pi per 20 milliamperes 0·000240 minutes watts (w) pulse forward current I 200 milliamperes (mA) radiation profile Lambertian distribution no (N/A) UV excitation pupils absorb ultraviolet (UV) spectra Very little light. Therefore, it is advantageous to use ultraviolet light to excite light. Although UV LEDs can be used to excite the light source', the broad spectrum of LED 26 reduces the effectiveness of this method. To solve this problem, a filtered UV LED can be used. Unless the relatively high cost of the laser cannot be implemented in the specific inspection module market, the ultraviolet laser can be used as the excitation source at will. LED Driver The time required for the LED driver 29 to drive the LED 26 at a fixed current for a sustained period of time. The low-power USB 2.0 certified driver provides up to 1 unit load (100 mA) with a minimum operating voltage of 4.4 volts. Standard power conditioning circuitry is used for this purpose. Photodiode Figure 54 shows the photodiode 184 integrated in the CMOS circuit 86 of the L〇C device 301. Light diode I84 is made part of CMOS circuit 86 without the need for additional masking or steps. This is the advantage of CMOS photodiodes over the -109-201209158 CCD (CCD is an alternative sensing technique that uses non-standard processing steps to integrate CCDs on the same wafer or on adjacent wafers). On-wafer detection is low cost and reduces the size of the analytical inspection system. The shorter optical path length reduces noise from ambient environments for efficient collection of fluorescent signals and eliminates the need for conventional optical components consisting of lenses and filters. The quantum efficiency of the photodiode 184 is caused by photons impinging on the active region 185 of the photodiode 184 to be efficiently converted into photoelectrons. For standard tantalum processes, the quantum efficiency for visible light ranges from 0.3 to 0.5 depending on process parameters (for example, the number of overlays and the absorptive properties). The detection threshold of the photodiode 1 84 determines the minimum intensity of the fluorescent signal that can be detected. The detection threshold also determines the size of the photodiode 184 and thereby determines the number of hybrid chambers 180 in the hybrid 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 case of LOC device 301, which is 1 760 microns X 5 8 24 microns) and incorporated into other functional modules. The actual footprint available after (eg, pathogenic dialysis section 70 and amplification section 11 2 ). For standard 矽 process, photodiode 1 84 detects a minimum of 5 photons. However, to ensure reliable detection, the minimum 値 can be set to 1 光 photons. Therefore, as described above with a quantum efficiency of 0.3 to 0.5, the fluorescence-derived light from the probes must have a minimum of 17 photons, but 30 photons can be included in the appropriate error range for reliable detection. . -110- 201209158 Calibration Chamber The inconsistencies in the residual excitation photon flux of the electrical characteristics, spontaneous fluorescence, and incomplete decay of the photodiode 184 introduce background noise and offset into the output signal. The background 値 in each output signal is removed using one or more calibration signals. A calibration signal is generated by exposing one or more of the calibration photodiodes 184 in the array to respective calibration sources. A low calibration source is used to determine the negative result (the negative result is that the target is not reacting with the probe). A highly calibrated light source indicates a positive result derived from the probe-target complex. In the embodiment of the present invention, the low calibration light source is provided by a plurality of calibration chambers 382 in the hybrid chamber array 110, the calibration chambers 3 8 2: without any probes; A probe of a light reporter group; or a probe comprising a reporter group and an extinction group but the matte group is configured to anticipate that matting will occur all the time.

源自此等校準腔室3 82之輸出信號幾乎接近源自該 LOC裝置中所有雜合腔室之輸出信號的雜訊和偏移。由其 他雜合腔室所產生的輸出信號減去該校準信號可實質地去 除背景値且留下由螢光釋放光(若有螢光)所產生之信號 。亦可扣除由該腔室陣列區域中之周遭光線所引起的信號 將理解以上參照第135〜138圖所示之陰性對照探針可 用於校準腔室中。然而,如第128及129圖所示’第128及 129圖係第127圖中之LOC裝置變化型X 728之插圖DG和插 -111 - 201209158 圖DH的放大圖,另一種選擇係使該等校準腔室3 82與擴增 子流體隔離。藉著清空該等經流體隔離之腔室,或使該等 經流體隔離之腔室包含不具報導基團之探針,或因藉由流 體隔離使得雜合反應無法發生而使該等腔室確實包含任意 一種同時具有報導基團和消光基團兩者之「正常」探針, 可決定該背景雜訊和偏移。 該等校準腔室382可提供高校準光源以產生於對應的 光二極體中產生高信號。該高信號對應於腔室中所有已進 行雜合之探針。點製具有報導基團但無消光基團的探針或 僅點製消光基團將可恆定地提供接近雜合腔室內之大多數 探針已經雜合時的信號。亦將瞭解該等校準腔室382可用 於取代對照探針,或可除對照探針以外附加使用該等校準 腔室3 82。 該等校準腔室382於整個雜合腔室陣列中之數目和配 置係可隨意決定。然而,若藉由相對最接近的校準腔室 382校準該等光二極體184,該校準係更加精確。參閱第56 圖,雜合腔室陣列1 10具有一個校準腔室3 82以供全部八個 雜合腔室1 80使用。即是,校準腔室3 82係位於由該等雜合 腔室1 80所組成之3 X 3方形陣列的中央。於此種配置中,係 藉由緊鄰的校準腔室382校準該等雜合腔室180。 第134圖顯示一種微分成像電路788,該微分成像電路 78 8係使源自周圍該等雜合腔室丨80的螢光信號減去激發光 造成與校準腔室3 82所對應之光二極體184發出的信號。該 微分成像電路7 8 8抽樣源自像素7 9 0和「假」像素7 9 2之信 -112- 201209158 號。於具體實施例中,「假」像素7 9 2係經遮蔽而免受光 線照射,因此假像素792之輸出信號提供暗參考値(dark reference )。或者,「假」像素792會與該陣列之其餘部 分一同暴露於激發光下。於「假」像素792接受光線照射 的具體實施例中,由該腔室陣列區域中之周遭光線引起的 信號亦經扣除。源自像素790之信號很小(即,接近暗信 號),且若不參考暗信號則難以區分背景値和極小之信號 〇 使用期間,「讀行線(read_r〇w )」794及「假像素 讀行線(read_row_d)」795係經啓動,且M4電晶體797及 MD4電晶體801係經開啓。切換器807和切換器809關閉, 使得源自像素790及「假」畫素792的輸出値分別儲存於像 素電容器8 03與假像素電容器805上。該等像素信號經儲存 之後,使切換器807和切換器809停止運作。隨後,「讀列 線(read_C〇l)」切換器811及假像素「讀列線」切換器 813係經關閉,且位於該輸出端處之該等經切換之電容器 放大器8 1 5放大該微分信號8 1 7。 光二極體之抑制與啓動 激發期間需藉由LED26抑制該光二極體184且於螢光 發光期間啓動光二極體184。第66圖係單一光二極體184之 電路圖,且第67圖係用於二極體控制信號之時序圖。該電 路具有光二極體184和六個金屬氧化物半導體(M0S )電 晶體 Mshunt 394、Mtx 396、Mreset 398、Msf 400、Mread 402 -113- 201209158 和Mbias 4 04。於激發循環之初始期間tl,藉由拉高該分流 電晶體(Mshunt)閘極3 84和該重設閘極3 8 8使該分流電晶 體Mshunt 3 94及重設電晶體Mreset 3 98啓動。於此期間內, 該等激發光子於光二極體184中生成載子。當所產生之載 子數量可能足以使光二極體1 84飽和,則需去除此等載子 。由於電晶體內漏電或由於基板內經激發所生成之載子的 擴散作用造成於此循環期間電晶體Mshunt 394直接去除光 二極體184中所生成之載子,同時電晶體Mreset 3 98重設累 積在節點「NS」4 06上的任何載子。於激發後,於時間t4 處開始進行捕捉循環(capture cycle )。於此捕捉循環期 間,由螢光發光基團所發出之應答係經捕捉且於節點「NS 」406上的電路中進行積分。可藉著拉高電晶體Mtx閘極 3 86而啓動該電晶體M,x3 96且把累積於光二極體184上的任 何載子傳送給節點「NS」406而達成此動作。於該捕捉循 環之期間可與螢光發光基團的發光時間一樣長。源自雜合 腔室陣列1 10中之所有光二極體184的輸出値係經同時捕捉 〇 於該捕獲循環t5尾聲及該讀取循環t6初始之間具有延 遲。此延遲係由於需要接續該捕獲循環之後個別讀取該雜 合腔室陣列11〇(見第52圖)中之每個光二極體184所造成 。欲讀取之第一個光二極體184將具有在該讀取循環之前 最短的延遲,同時最後一個光二極體184將具有在該讀取 循環之前最長的延遲。於該讀取循環期間,藉由拉高該讀 取閘極3 9 3而啓動該電晶體Μ “ a d 4 0 2。使用源極追隨電晶 -114- 201209158 體Msf 400緩衝且讀出該「NS」節點406之電壓。 尙具有用於啓動或抑制光二極體之附加選用方法,且 該等方法係討論如下: 1 ·抑制方法 第13 1、132及133圖顯示三種用於分流電晶體(Mshun )3 94之可行的結構配置。(778、780、782)分流電晶體 (Mshunt) 394於激發期間所使用之最大|Κω| = 5伏特處具有 極高的關閉電流比(off ratio)。如第13 1圖所示,分流電 晶體(Mshunt)閘極3 84係經配置以位於該光二極體184之 邊緣上。可隨意地如第132所示般,分流電晶體(Mshunt ) 閘極3 84可經配置成環繞著光二極體184 »第三種選擇係如 第1 3 3圖所示般把該分流電晶體(Mshunt )閘極3 84建構在 光二極體1 84之內側處。選用第三種選擇時,將具有較少 的光二極體主動區185。 此等三種結構配置7 7 8 ' 7 8 0和7 8 2縮短從該光二極體 184中之所有位置到該分流電晶體(Mshunt)閘極3 84的平 均路徑長度。於第131圖中’分流電晶體(Mshunt )閘極 3 84係位於該光二極體184之一側上。此種結構配置於製造 上最爲簡單且侵占最少的該光二極體主動區丨85。然而, 殘留於光二極體1 84之遠側上的任何載子可能耗費更長的 時間傳播至分流電晶體(Mshunt)鬧極384。 於第132圖中,分流電晶體(Mshunt)閘極3 84環繞著 光二極體184。此種配置進一步縮短光二極體184中之載子 -115- 201209158 前往分流電晶體(Mshunl )閘極3 84之平均路徑長度。然而 ,使分流電晶體(Msh unt )閘極3 8 4延長到光二極體1 8 4之 周長附近更迫使該光二體主動區185更大幅地縮減。第133 圖中之配置782係使該分流電晶體(Mshunt )閘極3 84位於 該主動區1 85內。此種配置提供通往分流電晶體(Mshunt ) 閘極384之最短路徑長度,且因而提供最短的變遷時間。 然而,對主動區185之衝擊最大。該配置782亦具有較寬的 漏電路徑。 2.啓動方法 a.觸發光二極體係使用一固定之延遲來驅動該分流 電晶體。 _b.觸發光二極體係使用一可編程之延遲來驅動該分 流電晶體。 c. 由該LED區段脈衝以一固定延遲來驅動該分流電晶 體。 d. 如2 c中所示但改用可編程之延遲來驅動該分流電 晶體。 第69圖係橫斷雜合腔室1 80之槪要剖面圖,該剖面圖 顯示包埋於CMOS電路86中之光二極體184和觸發光二極體 187。把該光二極體184之角落內的小面積置換成觸發光二 極體187。具有小面積之觸發光二極體18 7係足夠用於當激 發光之強度高到將堪比螢光發光之強度之時。觸發光二極 體187對激發光244敏感。該觸發光二極體187記錄該激發 -116- 201209158 光244已消退且經過一段短時間延遲Δί 3 00 (見第2圖)之 後啓動該光二極體184。此延遲允許該螢光之光二極體184 在無激發光244之情況下偵測源自該等FRET探針186之螢 光釋放光。此種配置允許進行偵測且增進信號雜訊比。 光二極體184和觸發二極體187兩者皆位於每個雜合腔 室180下方的CMOS電路86內。由多個光二極體組成之陣列 聯合適當的電子元件共同形成該光感測器44 (見第65圖) 。該等光二極體184係於CMOS結構製造期間無需使用額外 之遮罩或步驟下所製成的pn-接合面。於MST製造期間,係 隨意地使用標準MST光微影技術使位於該等光二極體184 上方之介電層(圖中未示出)薄化,以允許更多螢光照射 該光二極體184之主動區185。該光二極體184具有一個視 野,使得源自雜合腔室180內之該等探針-標靶物雜合體 的螢光信號入射在該感測器之感測面上。該螢光經轉換成 光電流,且隨後可使用CMOS電路86測量該光電流。 或者,一個或一個以上之雜合腔室180可僅專用觸發 光二極體187。此等選擇可用於此等2c體系中且與上述2a 和2b體系併用。 螢光之延遲偵測 下列導函數說明於上述LED/螢光發光基團組合中使用 長壽命週期之螢光發光基團的延遲螢光偵測。如第60圖所 示,藉著時間^與時間>2間之理想的恆定強度脈衝/e推導出 激發後的螢光強度之時間函數。 -117- 201209158 令[幻](〇等於處於時間t處的激發態之密度,則於激發 期間和激發之後,藉由下列微分方程式描述每單位體積之 單位時間內的激發態數目。 幽丛…⑴ dt tf hve 其中c係螢光發光基團之莫耳濃度,ε係莫耳消光係數 ve係激發頻率,且普朗克常數A = 6.62606896(1 〇y34焦耳秒( )° 該微分方程式具有下列通式: + p(x)y = Φ) αχ 該方程式之解係: -.(2) [e^P()dx q{x)dx +k ΛΧ) = —— 現使用此式求解方程式(1 ) ’ [sm)=l^iL+ke-^ ...ο) hVe 於時間6,且由式(3)可得: k = -i^lLe'、…...(4) hve 把式(4 )代入式(3 ): 於時間時: …(5) hve hve -118- 201209158 對於&匕,該等經激發之狀態呈指數衰退且以下述方 程式描述= [51](〇 = [51](i2)e-(,-,2)/^ …⑹ 把式(5 )代入式(6 ):The output signals from such calibration chambers 3 82 are nearly as close to the noise and offset from the output signals of all of the hybrid chambers in the LOC device. Subtracting the calibration signal from the output signal produced by the other hybrid chamber substantially removes the background and leaves the signal produced by the fluorescent light (if there is fluorescence). It is also possible to subtract the signal caused by the ambient light in the array of cells. It will be understood that the negative control probes shown in Figures 135 to 138 above can be used in the calibration chamber. However, as shown in Figures 128 and 129, '128 and 129 are diagrams 127 of the LOC device variant X 728 in Fig. 127 and an enlarged view of Fig. DH. Another option is to make these The calibration chamber 382 is fluidly isolated from the amplicon. The chambers are indeed emptied by emptied the fluid-isolated chambers, or the fluid-isolated chambers contain probes that do not have a reporter group, or because the fluidization does not allow the hybridization reaction to occur. The inclusion of any "normal" probe with both a reporter group and a matting group can determine the background noise and offset. The calibration chambers 382 can provide a high calibration source to produce a high signal in the corresponding photodiode. This high signal corresponds to all probes in the chamber that have been hybridized. Pointing a probe with a reporter group but no extinction group or a dot-only matting group will constantly provide a signal near the time when most of the probes in the hybrid chamber have been hybridized. It will also be appreciated that the calibration chambers 382 can be used in place of the control probes or that the calibration chambers 382 can be additionally used in addition to the control probes. The number and configuration of the calibration chambers 382 in the entire array of hybrid chambers can be determined at will. However, if the photodiodes 184 are calibrated by the relatively closest calibration chamber 382, the calibration is more accurate. Referring to Fig. 56, the hybrid chamber array 110 has a calibration chamber 382 for use by all eight hybrid chambers 180. That is, the calibration chamber 382 is located in the center of the 3 x 3 square array comprised of the hybrid chambers 180. In this configuration, the hybrid chambers 180 are calibrated by the adjacent calibration chamber 382. Figure 134 shows a differential imaging circuit 788 that subtracts the excitation light from the fluorescent signals surrounding the hybrid chambers 80 to cause the photodiodes corresponding to the calibration chambers 382. Signal sent by 184. The differential imaging circuit 768 samples the letter from the pixel 709 and the "false" pixel 7 9 2 -112-201209158. In a specific embodiment, the "false" pixel 7 9 2 is shielded from light, so the output signal of the dummy pixel 792 provides a dark reference. Alternatively, the "false" pixel 792 will be exposed to the excitation light along with the rest of the array. In a particular embodiment where the "false" pixel 792 is exposed to light, the signal caused by ambient light in the array of cells is also subtracted. The signal from pixel 790 is small (ie, close to the dark signal), and it is difficult to distinguish between background 値 and very small signal without reference to dark signal 「 during use, "read line (read_r〇w)" 794 and "false pixel" The read row line (read_row_d) 795 is activated, and the M4 transistor 797 and the MD4 transistor 801 are turned on. Switch 807 and switch 809 are turned off so that the output 源自 from pixel 790 and "false" pixel 792 are stored on pixel capacitor 803 and dummy pixel capacitor 805, respectively. After the pixel signals are stored, the switch 807 and the switch 809 are stopped. Subsequently, the "read column line (read_C)" switch 811 and the dummy pixel "read column line" switch 813 are turned off, and the switched capacitor amplifiers 8 1 5 at the output end amplify the differential Signal 8 1 7. Inhibition and Activation of the Photodiode The photodiode 184 is inhibited by the LED 26 during excitation and the photodiode 184 is activated during the illumination. Figure 66 is a circuit diagram of a single photodiode 184, and Figure 67 is a timing diagram for a diode control signal. The circuit has a photodiode 184 and six metal oxide semiconductor (MOS) transistors Mshunt 394, Mtx 396, Mreset 398, Msf 400, Mread 402-113-201209158 and Mbias 4 04. During the initial period t1 of the excitation cycle, the shunt transistor Mshunt 3 94 and the reset transistor Mreset 3 98 are activated by pulling up the shunt transistor (Mshunt) gate 3 84 and the reset gate 38 8 . During this period, the excitation photons generate carriers in the photodiode 184. When the number of carriers produced may be sufficient to saturate the photodiode 1 84, these carriers need to be removed. The transistor Mshunt 394 directly removes the carrier generated in the photodiode 184 during the cycle due to leakage inside the transistor or diffusion due to the excitation of the carrier generated in the substrate, while the reset of the transistor Mreset 3 98 is accumulated. Any carrier on node "NS" 4 06. After the excitation, a capture cycle is started at time t4. During this capture cycle, the response from the luminescent group is captured and integrated in the circuit on node "NS" 406. This operation can be achieved by pulling the transistor Mtx gate 3 86 to activate the transistor M, x3 96 and transferring any carriers accumulated on the photodiode 184 to the node "NS" 406. The period of the capture cycle may be as long as the luminescence time of the fluorescent luminescent group. The output chirp from all of the photodiodes 184 in the hybrid chamber array 1 10 is delayed by simultaneously capturing the end of the capture cycle t5 and the beginning of the read cycle t6. This delay is caused by the individual reading of each photodiode 184 in the hybrid chamber array 11 (see Figure 52) after the capture cycle needs to be continued. The first photodiode 184 to be read will have the shortest delay before the read cycle, while the last photodiode 184 will have the longest delay before the read cycle. During the read cycle, the transistor Μ "ad 4 0 2 is activated by pulling up the read gate 3 3 3 . The source is followed by the transistor - 114 - 201209158 body Msf 400 buffer and read out" NS" node 406 voltage.尙 has an additional method for starting or suppressing the photodiode, and the methods are discussed as follows: 1 · Suppression method Figures 13 1 , 132 and 133 show three feasible methods for shunting transistor (Mshun ) 3 94 Structure configuration. (778, 780, 782) shunt transistors (Mshunt) 394 have a very high off-rate ratio at the maximum |Κω| = 5 volts used during excitation. As shown in Figure 13, a shunt transistor (Mshunt) gate 3 84 is configured to be located on the edge of the photodiode 184. Optionally, as shown in FIG. 132, the shunt transistor (Mshunt) gate 3 84 can be configured to surround the photodiode 184. The third option is to divide the shunt transistor as shown in FIG. The (Mshunt) gate 3 84 is constructed at the inner side of the photodiode 1 84. When the third option is selected, there will be fewer photodiode active regions 185. These three structural configurations 7 7 8 ' 7 8 0 and 7 8 2 shorten the average path length from all positions in the photodiode 184 to the shunt transistor (Mshunt) gate 3 84. In Fig. 131, a shunt transistor (Mshunt) gate 3 84 is located on one side of the photodiode 184. This configuration is configured to be the simplest and least encroaching of the photodiode active region 丨85. However, any carriers remaining on the far side of the photodiode 1 84 may take longer to propagate to the shunt transistor (Mshunt). In Fig. 132, a shunt transistor (Mshunt) gate 3 84 surrounds the photodiode 184. This configuration further shortens the average path length of the carrier in the photodiode 184 from -115 to 201209158 to the shunt transistor (Mshunl) gate 3 84. However, extending the shunt transistor (Msh unt) gate 3 8 4 to the vicinity of the perimeter of the photodiode 1 8 4 forces the photoreceptor active region 185 to be more substantially reduced. The configuration 782 in Figure 133 is such that the shunt transistor (Mshunt) gate 3 84 is located within the active region 185. This configuration provides the shortest path length to the shunt transistor (Mshunt) gate 384 and thus provides the shortest transition time. However, the impact on the active zone 185 is greatest. This configuration 782 also has a wider leakage path. 2. Startup Method a. The trigger photodiode system uses a fixed delay to drive the shunt transistor. The _b. trigger photodiode system uses a programmable delay to drive the shunt transistor. c. The shunting electron crystal is driven by the LED segment pulse with a fixed delay. d. Drive the shunt transistor as shown in 2 c but with a programmable delay. Figure 69 is a cross-sectional view of the cross-hybrid cavity 1 80 showing the photodiode 184 and the triggering photodiode 187 embedded in the CMOS circuit 86. A small area in the corner of the photodiode 184 is replaced with a trigger photodiode 187. A small-area triggering photodiode 18 7 is sufficient for when the intensity of the laser is high enough to be comparable to the intensity of the fluorescent light. The trigger photodiode 187 is sensitive to the excitation light 244. The trigger photodiode 187 records that the excitation -116-201209158 light 244 has subsided and the photodiode 184 is activated after a short delay Δί 3 00 (see Figure 2). This delay allows the fluorescent photodiode 184 to detect fluorescent light emission from the FRET probes 186 without excitation light 244. This configuration allows for detection and improves signal to noise ratio. Both photodiode 184 and trigger diode 187 are located within CMOS circuitry 86 below each hybrid cavity 180. An array of a plurality of photodiodes is combined with appropriate electronic components to form the photosensor 44 (see Figure 65). The photodiodes 184 do not require the use of additional masks or pn-junctions formed during the fabrication of the CMOS structure. During the MST fabrication, a standard MST photolithography technique is used to thin the dielectric layer (not shown) above the photodiode 184 to allow more phosphor to illuminate the photodiode 184. Active area 185. The photodiode 184 has a field of view such that fluorescent signals originating from the probe-target hybrids within the hybrid chamber 180 are incident on the sensing surface of the sensor. The fluorescent light is converted to a photocurrent, and the photocurrent can then be measured using a CMOS circuit 86. Alternatively, one or more of the hybrid chambers 180 may only exclusively trigger the photodiode 187. These choices can be used in these 2c systems and in combination with the above 2a and 2b systems. Fluorescence Delay Detection The following derivative functions illustrate the use of delayed fluorescence detection of long lifetime fluorescent light-emitting groups in the above LED/fluorescent group combination. As shown in Fig. 60, the time function of the intensity of the fluorescence after excitation is derived by the ideal constant intensity pulse /e between time & time > -117- 201209158 Let [phantom] (〇 is equal to the density of the excited state at time t, then the number of excited states per unit volume per unit volume is described by the following differential equation during and after excitation. (1) dt tf hve where m is the molar concentration of the fluorescent luminescent group, ε is the molar extinction coefficient ve is the excitation frequency, and the Planck constant A = 6.62606896 (1 〇 yyyy joules sec ( ) ° The differential equation has the following General formula: + p(x)y = Φ) αχ The solution of this equation: -.(2) [e^P()dx q{x)dx +k ΛΧ) = —— Now use this formula to solve the equation ( 1) '[sm)=l^iL+ke-^ ... ο) hVe is at time 6, and is obtained by equation (3): k = -i^lLe', ... (4) hve Substituting (4) into equation (3): at time: ...(5) hve hve -118- 201209158 For &匕, the excited states are exponentially decayed and described by the following equation = [51] (〇= [51](i2)e-(,-,2)/^ (6) Substituting equation (5) into equation (6):

J FCTJ FCT

[51](〇 = -~^[l-e-(,2',l)/r/]g-(^)/r/ …⑺ hve 該螢光強度係寫成下列方程式:[51](〇 = -~^[l-e-(,2',l)/r/]g-(^)/r/ (7) hve The fluorescence intensity is written as the following equation:

其中v/系螢光頻率,;7係量子產率,且/係光學路徑長 度。 現由式(7 )得到: 4^1](〇_ Ieec 己-(’2-’1)"/ 1己-(卜,2)"/ dt hve 把式(9 )代入式(8 ),得到: IAt) = Iesc^^[\-e~{,1-h)lTf ]eHt~,l)lTf ...(10) 對於 / (〇_>/〆咖',叫)"/Where v/ is the fluorescence frequency, 7 is the quantum yield, and / is the optical path length. Now obtained by equation (7): 4^1](〇_ Ieec 己-('2-'1)"/ 1己-(卜,2)"/ dt hve Substituting equation (9) into equation (8) ), get: IAt) = Iesc^^[\-e~{,1-h)lTf ]eHt~,l)lTf ...(10) For / (〇_>/〆咖', call) &quot ;/

Xf Ve 因此,吾等可寫出下列描述經過夠長激發脈衝 (,2 - G » Γ,)之後的近似方程式: 對於 ,/,(〇 =/e£c/77Ye_('-'2)/r/ ...(11) 於先前段落中,吾等可推論當»、, 對於 ,/,(i) = /esc/7-^e_(’_’2)/r/。 由上述方程式,吾等可獲得下式: -119- 201209158 h'f(t) = nesc^e'(l',l)lT, ...(12) 其中, I Μ) = 係每單位面積之單位時間的螢光光子數,及 \ 係每單位面積之單位時間的激發光子數。 hVe 結果係, cc nf{t) = \nf{t)dt ...(13) 其中心係每單位面積之螢光光子數,且ί3係光二極體 開啓時之瞬時時間。把式(1 2 )代入式(1 3 ):Xf Ve Therefore, we can write the following approximate equation after a sufficiently long excitation pulse (, 2 - G » Γ,): For, /,(〇=/e£c/77Ye_('-'2)/ r/ ...(11) In the previous paragraph, we can infer when »,, for, /, (i) = /esc/7-^e_('_'2)/r/. From the above equation, We can obtain the following formula: -119- 201209158 h'f(t) = nesc^e'(l',l)lT, ...(12) where, I Μ) = is the unit time per unit area The number of fluorescent photons, and \ is the number of excitation photons per unit area per unit time. The hVe result is cc nf{t) = \nf{t)dt (13) the number of fluorescent photons per unit area of the center system, and the instantaneous time when the ί3 light diode is turned on. Substituting equation (1 2 ) into equation (1 3 ):

CO 此時,抵達該光二極體的每單位面積之單位時間內的 螢光光子數)Ϊ,(0係寫成下式: «;(0 = «/(0^〇 ...(15) 其中么係該光學系統之集光效率。 把式(12)代入式(15),吾等可得: ns{t) = φ0ήεεεΙηβ'(,~'ι)ΙΤί ...(16) 同樣地,抵達該光二極體的每單位螢光面積之螢光光 子數乂將如下式: co 纪=ί&(〇並代入式(1 6 )中且積分得到: '3CO At this time, the number of fluorescent photons per unit area of the photodiode is Ϊ, (0 is written as: «; (0 = «/(0^〇...(15) where What is the light collection efficiency of the optical system. Substituting equation (12) into equation (15), we can get: ns{t) = φ0ήεεεΙηβ'(,~'ι)ΙΤί (16) Similarly, arrival The number of fluorescent photons per unit of fluorescence area of the photodiode will be as follows: co 纪 = ί & (〇 and substituted into equation (16) and the integral is obtained: '3

Hs = φ^η(εα1ητ fe~(h~,:t)lTf 因此可得: 〜=认εάητ〆1^” ...(17) -120- 201209158 若激發光子通量的衰退速率遠快於螢光光子通量的衰 退速率,該最佳値ί3係當螢光光子於光二極體184中生成電 子的速率等於激發光子在光二極體184中生成電子的速率 之時刻。 由螢光所造成每單位螢光面積之感測器輸出電子的速 率係: '^) = φ/ήχί) I 其中^係該感測器於該螢光波長下之量子產率。 把上式代入式(1 7 ),吾等得到: ^(0 = ii^〇«>/77^(W2,/r/ -(18) 同樣地,由激發光子所造成每單位螢光面積之感測器 輸出電子的速率係: …(π) 其中九係該感測器於該激發光波長下之量子產率’且 &係對應於該激發LED之「關閉」性質的時間常數。經時 # 間ί2之後,該LED之衰退中的光子通量可能提高螢光信號 之強度且延長該光子通量的衰退時間’但吾等假設此狀況 對於If (t)的影響微乎其微’因此吾等採取保守方法 〇 此時,如先前所述’該(3之最佳値係當: 因此’由式(18)及式(19) ’吾等得: 且重新整理後’吾等得到: -121 - ...(20)201209158Hs = φ^η(εα1ητ fe~(h~,:t)lTf Therefore, we can get: ~= recognize εάητ〆1^” (17) -120- 201209158 If the rate of decay of the excited photon flux is much faster than The rate of decay of the fluorescent photon flux, which is the time at which the rate at which the fluorescent photons generate electrons in the photodiode 184 is equal to the rate at which the excited photons generate electrons in the photodiode 184. Caused by fluorescence The rate at which the sensor outputs electrons per unit of fluorescence area is: '^) = φ/ήχί) I where ^ is the quantum yield of the sensor at the wavelength of the fluorescence. Substituting the above formula into (1 7 ), we get: ^(0 = ii^〇«>/77^(W2,/r/ -(18) Similarly, the rate of electron output from the sensor per unit of fluorescence area caused by the excitation photons The system: ... (π) where the quantum yield of the sensor at the wavelength of the excitation light 'and & corresponds to the time constant of the "off" property of the excitation LED. After the time interval #ί2, the The photon flux in the decay of the LED may increase the intensity of the fluorescent signal and extend the decay time of the photon flux', but we assume the effect of this condition on If (t) It is very small' therefore we have adopted a conservative approach. At this time, as mentioned earlier, 'the best of 3's: therefore' by equations (18) and (19) 'we have: and after rearranging Wait until: -121 - ...(20)201209158

\η{εεΙη\η{εεΙη

TeTe

Tf 由前述兩段說明,吾等得到下列兩個運算式: ns = φ^ι^τ!…(21)Tf is explained by the two paragraphs above. We get the following two expressions: ns = φ^ι^τ!...(21)

At =At =

In F Φ/Φο y --(22) 其中=江/77且= 6 _ G。吾等亦知實際上丨2 — (1 » Γ/。 螢光偵測之最佳時間及使用飛利浦[乂]<:2-?1114-11〇〇型 LED與Pulsar 650螢光染劑所測得之螢光光子數係由下列 方式決定。 係使用方程式(2 2 )決定該最佳偵測時間: 回憶該擴增子之濃度,且假設所有擴增子皆雜合,則 螢光發光基團之濃度係:c= 2.89(10)4莫耳/公升。 該腔室之高度係光學路徑長度/ = 8(10)_6公尺。 吾等係使該螢光面積等於光二極體面積,但實際螢光 面積係實質大於光二極體之面積;因此吾等可大致假設吾 等之光學系統的集光效率& =〇.5。由該等光二極體特性可 知, f = l〇係該光二極體於螢光波長下之量子產率比上激 Φα 發波長下之量子產率的比値之極保守値。 利用一般LED之衰退壽命週期re= 0.5奈秒且使用Pulsar 650之規格,可決定△/: •122- 201209158 F = [1.48(10)6 ][2.89(10) *6 ][8(10)'6 ] (1) = 3.42(10)-5 A. ln([3.42(10)-5](10)(0.5)) 1 1 1(10)-6 —0.5(10)-9 = 4.34(10)-9 秒 使用方程式(21)決定所測得之光子數。首先,藉由 檢查照明幾何而決定每單位時間所發出之激發光子數之。 飛利浦 LXK2-PR14-R00 型 LED 具有朗伯(Lambertian # )輻射圖形,因此: n, = nl0 cos(0) -.-(23) 其中%係偏離LED之順軸方向Θ角之每單位立體角之單 位時間所發出的光子數,且、係順軸方向上之&的閥値( valve) 。LED每單位時間所發出之光子總數係: ή, =j η,c/Ω Ω ...(24)In F Φ / Φο y -- (22) where = Jiang / 77 and = 6 _ G. We also know that 丨2 — (1 » Γ/. The best time for fluorescent detection and the use of Philips [乂]<:2-?1114-11〇〇 LEDs and Pulsar 650 Fluorescent Dyestuffs The measured number of fluorescent photons is determined by the following method: The optimal detection time is determined using equation (2 2 ): recalling the concentration of the amplicon, and assuming that all amplicons are heterozygous, the fluorescent emission The concentration of the group is: c = 2.89 (10) 4 m / liter. The height of the chamber is the optical path length / = 8 (10) _ 6 m. We make the area of the phosphor equal to the area of the photodiode However, the actual area of the phosphor is substantially larger than the area of the photodiode; therefore, we can roughly assume the light collection efficiency of our optical system & = 〇.5. From the characteristics of these photodiodes, f = l〇 The ratio of the quantum yield of the photodiode at the fluorescence wavelength is much more conservative than the quantum yield at the wavelength of the excitation Φα. The decay life cycle of the general LED is re=0.5 nanoseconds and the Pulsar 650 is used. Specifications can be determined △ /: •122- 201209158 F = [1.48(10)6 ][2.89(10) *6 ][8(10)'6 ] (1) = 3.42(10)-5 A. ln( [3.42(10) -5](10)(0.5)) 1 1 1(10)-6 —0.5(10)-9 = 4.34(10)-9 seconds Use equation (21) to determine the number of photons measured. First, by Check the illumination geometry to determine the number of excitation photons emitted per unit time. The Philips LXK2-PR14-R00 LED has a Lambertian # radiation pattern, so: n, = nl0 cos(0) -.-(23) % is the number of photons emitted per unit time of the solid angle of the off-axis angle of the LED, and is the valve of the & in the direction of the axis. The photon emitted by the LED per unit time The total number is: ή, =j η,c/Ω Ω ...(24)

=J )ϊ/0 cos(^)JQ Ω=J )ϊ/0 cos(^)JQ Ω

此時, ΑΩ = 2π[1 - cos(0 + △ 0)] — 2河1 - cos(0)] ΔΩ = 2^[cos(0) - cos(^ + Δ0)] :4;rsin(0)cos .(ΑΘ) L 2夕 l 2 J + 4;rcos(0)sin2 ΆΘ' dQ. = 2nsm{6)de 把此式代入式(24),得到: 123 201209158 ή, = j 2τίηΙΰ cos(0)sin(9)d0 ο =碑0 */0 重新整理後,吾等得到: ' …(26) 該LED之輸出功率係0.515瓦特(W)且K =6.52(10)14赫 因此:At this time, ΑΩ = 2π[1 - cos(0 + △ 0)] — 2 river 1 - cos(0)] ΔΩ = 2^[cos(0) - cos(^ + Δ0)] :4;rsin(0 )cos .(ΑΘ) L 2 夕 l 2 J + 4; rcos(0)sin2 ΆΘ' dQ. = 2nsm{6)de Substituting this formula into equation (24) yields: 123 201209158 ή, = j 2τίηΙΰ cos( 0) sin(9)d0 ο = monument 0 */0 After rearranging, we get: ' ...(26) The output power of this LED is 0.515 watts (W) and K = 6.52 (10) 14 Hz.

Ave 茲 ..•(27) __0.515_ ~ [6.63(10)-34 ][6.52(10)14] = 1.19(10)18 光子渺 把此値代入式(2 6 ),吾等得到: ... 1.19(10)18 ηιο = π =3.79(10)17 光子 /秒 參閱第61圖,圖中槪要顯示該LED26之光學中心252 和透鏡254。該等光二極體係16微米χ16微米,且對位於陣 列中央的光二極體而言,自LED 26發出且抵達光二極體 1 84之光線的錐形立體角(Ω )係約: Ω=感測器面積/r2 [16(10)~6][16(10)~63 [2.825(10)-3]2~ = 3·21(10)·5 秒 將可理解光二極體陣列44之中央光二極體184的目的 係用於此等計算。當發生朗伯激發光源強度分佈之雜合事 -124- 201209158 件時,位於該陣列之邊緣處的感測器可能僅收到低於2%的 光子。 每單位時間發出之激發光子數係: ne = ήμ ...(28) =[3.79(10),7][3.21(10)'5] = 1.22(10)13 光子/# 現回到式(2 9 ): ns =Φ〇ήβΓτ/β'Δ,/Τ/ π, = (0.5)[1.22(10)l3][3.42(10)-5][l(10)_6]e'434(l〇r,/1(1〇r6 = 208光子/每個感測器 因此,使用飛利浦LXK2-PR14-R00型LED和Pulsar 650 螢光發光基團,吾等可輕易地偵測任何產生此發射光子數 目的雜合事件。 第62圖中所示之SET型LED的照明幾何。於ID = 20毫安 培時,該LED具有中心波長= 340奈米(铽螯合物之吸收 波長)之最小光學功率輸出Pi = 240微瓦特(μΨ )。以 ID = 2 00毫安培驅動該LED可線性地提高輸出功率達Pl = 2.4 毫瓦特(mW)。藉著把該LED之光學中心252設置在與該 雜合腔室陣列110相距17.5毫米處,吾等可使此輸出通量 集中在尺寸係具有2毫米之最大直徑的圓形點內。 位於該雜合遠離平面(hybridization away plane)處 的該2毫米直徑之點內的光子通量係以方程式27表示。 ή _ Pi 1 hve _ 2.4(10)-3 [6.63(10)-34 ][8.82(10)14] -125- 201209158 = 4.10(10)15 光子/秒 使用方程式2 8,吾等得到: he = η,Ω. =4.10(10)15 [16(10)~6]2 4K10)'3]2 = 3.34(10)"光子 /秒 現回到方程式22且使用先前列出之铽(Tb )螯合物@ 性質,可得下式: 1η[(6·94(10)-5)(10)(0.5)]Ave ..•(27) __0.515_ ~ [6.63(10)-34 ][6.52(10)14] = 1.19(10)18 Photon 渺 Substituting this 入 into equation (2 6 ), we get: . .. 19.19(10)18 ηιο = π =3.79(10)17 Photons/sec Refer to Figure 61 for the optical center 252 and lens 254 of the LED 26. The photodiode system is 16 micrometers χ 16 micrometers, and for the photodiode in the center of the array, the cone solid angle (Ω) of the light emitted from the LED 26 and reaching the photodiode 1 84 is about: Ω = sensing Area /r2 [16(10)~6][16(10)~63 [2.825(10)-3]2~ = 3·21(10)·5 seconds will understand the central light of the photodiode array 44 The purpose of the polar body 184 is for such calculations. When a heterodyne of the Lambertian excitation source intensity distribution occurs -124-201209158, the sensor at the edge of the array may only receive less than 2% of the photons. The number of excitation photons emitted per unit time: ne = ήμ ... (28) = [3.79(10), 7] [3.21(10)'5] = 1.22(10)13 Photon /# 2 9 ): ns =Φ〇ήβΓτ/β'Δ, /Τ/ π, = (0.5)[1.22(10)l3][3.42(10)-5][l(10)_6]e'434(l 〇r, /1 (1〇r6 = 208 photons per sensor) Therefore, using Philips LXK2-PR14-R00 LED and Pulsar 650 fluorescent illuminating group, we can easily detect any generated photons Number of hybrid events. The illumination geometry of the SET-type LED shown in Figure 62. At ID = 20 mA, the LED has a minimum optical power of center wavelength = 340 nm (absorption wavelength of ruthenium chelate) Output Pi = 240 microwatts (μΨ). Driving the LED at ID = 200 mA can linearly increase the output power to Pl = 2.4 milliwatts (mW) by setting the optical center 252 of the LED to The chamber arrays 110 are spaced 17.5 mm apart, and we can concentrate this output flux in a circular point having a maximum diameter of 2 mm. The 2 mm located at the hybridization away plane The photon flux in the point of the diameter is The program 27 indicates. ή _ Pi 1 hve _ 2.4(10)-3 [6.63(10)-34 ][8.82(10)14] -125- 201209158 = 4.10(10)15 photons/second using equation 2 8, Etc. get: he = η, Ω. = 4.10(10)15 [16(10)~6]2 4K10)'3]2 = 3.34(10)"Photons/sec is now back to Equation 22 and is listed previously After the (Tb) chelate @ nature, the following formula can be obtained: 1η[(6·94(10)-5)(10)(0.5)]

Af =-Γ i 1(10)·3 ~ 0.5(10)-9 =3.98(10)·9 秒 現從方程式2 1得到: ns = (0.5)[3.34(10)M][6.94(10)-5][l(10)-3]e_3 98<1〇r,/I(I〇r3 = 11600光子/感測器 使用S E T型LE D和铽螯合物系統係可輕易偵測到雜合 事件所發出之光子理論數目,且該光子理論數目係超過藉 由上述光感應器進行可靠偵測所需要30個光子之最小値。 探針與光二極體間之最大間距 雜合反應之晶片上偵測法免除需藉由共軛焦顯微鏡( 見先前技術之段落)進行偵測的需要。此種脫離傳統偵測 技術之偵測方式係本發明系統節約時間和節省成本之重要 因素。傳統偵測法要求必需用到諸多透鏡或曲面反射鏡的 成像光學技術。藉由採用非成像光學技術,該診斷系統免 除對複雜且體積龐大之光學元件串的需要。將該光二極體 -126- 201209158 置於極靠近_該等探針之處具有極高集光效率的優點:當該 等探針與該光二極體之間的材料厚度係1微米時,釋放光 之收集角度係最高可達1 7 3。。此角度係依照位於該雜合腔 室最靠近該光二極體之表面的質心處之探針所發出的光線 計算而得,其中該光二極體具有與該腔室表面平行的平坦 主動表面區。於發光角之圓錐體內光線能被光二極體吸收 ,且該發光角之圓錐體係界定爲在該平坦腔室面之周長上 的感測器角落處及該圓錐體之頂點處具有發光探針。對於 16微米X 16微米之感測器而言,此圓錐體之頂角係170。; 在光二極體擴大到其面積與29微米χ19.75微米之雜合腔室 相當的極限情況下,該頂角係1 73 °。可輕易達到使該腔室 表面與光二極體之主動表面之間的分隔距離爲1微米或低 於1微米。 採用非成像光學技術之體系必需使光二極體184極靠 近該雜合腔室以收集充足的螢光光子。可參閱第54圖如下 述方法般決定光二極體與探針之間的最大間距。 利用铽螯合物螢光發光基團及SET UVT0P3 3 5T039BL 型LED,吾等計算出有11600個源自各別雜合腔室180的光 子抵達16微米X 16微米之光二極體184。執行此計算時,吾 等假設雜合腔室180之集光區具有與光二極體主動區185相 等之基底面積,且該等雜合光子總數中有半數光子抵達光 二極體1 84。即是,該光學系統的集光效率係九=0.5。 更精確地,吾等可寫成公式么=[(雜合腔室之集光區 )/ (光二極體面積)][Ω/4π],其中Ω=位於雜合腔室基底 -127- 201209158 上一個代表點處之光二極體所正對的立體角。 對於正四棱錐之幾何形狀而言: n = 4arcsin(fl2/(4i^2 + fl2)) ’其中 <=該腔室與該光二極體之 間的距離,且《係該光二極體之尺寸。 每個雜合腔室釋出23200個光子。選定之光二極體具 有1 7個光子之偵測臨界値;因此,所需的最小光學效率係 ^0 = 17/23200 = 7.33 x1ο-4 該雜合腔室180之集光區的基底面積係29微米χ19.75 微米。 解出4,吾等將得到該雜合腔室之底部與光二極體 1 8 4之間的最大限制距離係4 = 249微米。於此限制條件下 ,上述界定之集光圓錐角係僅0 · 8度。應注意此分析忽略 該微不足道的折射效應。 LOC裝置變化型 以上詳細描述及圖解說明之LOC裝置301僅爲眾多可 行的L Ο C裝置設計中之一者。現將描述及/或以槪要流程圖 (從樣本入口到偵測)圖示多種使用不同組合之上述各種 功能性區段的LOC裝置變化型,以說明該等可行組合中之 一部分組合。該等流程圖係經適當分割成樣本之置入與製 備階段2 8 8、萃取階段290、培育階段291、擴增區段2 92、 預雜合階段2 93及偵測階段294。對於以上簡要描述或僅以 槪略形式顯示之所有LOC裝置,基於清晰及簡潔之理由, -128- 201209158 故不顯示完整佈局之附圖。亦爲求清晰,圖中未出示較小 的功能性單元(例如,液體感測器及溫度感測器),但將 可理解此等較小之功能性單元係已倂入每個下列LOC裝置 設計中之適當位置處。Af = -Γ i 1(10)·3 ~ 0.5(10)-9 =3.98(10)·9 seconds is now obtained from Equation 2 1 : ns = (0.5)[3.34(10)M][6.94(10) -5][l(10)-3]e_3 98<1〇r,/I(I〇r3 = 11600 photon/sensor can be easily detected using SET type LE D and ruthenium chelate system The number of photon theories emitted by the event, and the number of photon theories exceeds the minimum of 30 photons required for reliable detection by the light sensor. The maximum spacing between the probe and the photodiode is heterozygous on the wafer. The detection method eliminates the need for detection by a conjugate focal microscope (see the paragraph of the prior art). This detection method from the conventional detection technology is an important factor saving time and cost in the system of the present invention. The measurement method requires the use of imaging optics of many lenses or curved mirrors. By using non-imaging optics, the diagnostic system eliminates the need for complex and bulky optical component strings. The photodiode -126 - 201209158 The advantage of having very high collection efficiency when placed very close to the probes: when the probes and the photodiode When the thickness of the material is 1 micron, the collection angle of the released light is up to 173. This angle is based on the probe located at the centroid of the surface of the hybrid chamber closest to the surface of the photodiode. The light is calculated, wherein the photodiode has a flat active surface region parallel to the surface of the chamber. The light in the cone of the illuminating angle can be absorbed by the photodiode, and the cone of the illuminating angle is defined as the flat a luminescent probe at the sensor corner on the perimeter of the chamber face and at the apex of the cone. For a 16 micron X 16 micron sensor, the apex angle of the cone is 170. The pole body is enlarged to a limit equivalent to a hybrid chamber of 29 micrometers and 19.75 micrometers, which is 173 degrees. It can be easily achieved between the surface of the chamber and the active surface of the photodiode. The separation distance is 1 micron or less. The system using non-imaging optical technology must have the photodiode 184 very close to the hybrid chamber to collect sufficient fluorescent photons. Refer to Figure 54 to determine the light II as follows. Polar body and probe The maximum spacing. Using the ruthenium chelate luminescent group and the SET UVT0P3 3 5T039BL type LED, we calculated that 11600 photons from the respective hybrid chamber 180 reached the 16 micron x 16 micron photodiode. 184. When performing this calculation, we assume that the collection region of the hybrid chamber 180 has a substrate area equal to the photodiode active region 185, and that half of the total number of such photons arrive at the photodiode 184. That is, the light collection efficiency of the optical system is nine = 0.5. More precisely, we can write a formula = [(light collecting area of the hybrid chamber) / (photodiode area)] [Ω / 4π] , where Ω = the solid angle directly opposite the photodiode at a representative point on the hybrid chamber substrate -127-201209158. For the geometry of a regular pyramid: n = 4arcsin(fl2/(4i^2 + fl2)) 'where <= the distance between the chamber and the photodiode, and the size of the photodiode . Each hybrid chamber releases 23,200 photons. The selected photodiode has a detection threshold of 17 photons; therefore, the minimum optical efficiency required is ^0 = 17/23200 = 7.33 x1ο-4 the substrate area of the collection region of the hybrid chamber 180 29 microns χ 19.75 microns. Solution 4, we will get the maximum limit distance between the bottom of the hybrid chamber and the photodiode 1 8 4 is 4 = 249 microns. Under this constraint, the above-defined light collecting cone angle is only 0 · 8 degrees. It should be noted that this analysis ignores this negligible refraction effect. LOC Device Variations The LOC device 301, described and illustrated above, is only one of many possible L Ο C device designs. A variety of LOC device variations of the various functional segments described above using different combinations will now be described and/or illustrated in a schematic flow diagram (from sample entry to detection) to illustrate some of the combinations of such possible combinations. The flow diagrams are suitably divided into sample placement and preparation stages 28 8 , extraction stage 290, incubation stage 291, amplification section 2 92, pre-hybridization stage 2 93, and detection stage 294. For all LOC devices that are briefly described above or only in the form of abbreviations, for the sake of clarity and conciseness, -128-201209158 does not show the drawings of the complete layout. Also for clarity, the smaller functional units (eg, liquid sensors and temperature sensors) are not shown, but it will be appreciated that such smaller functional units have been incorporated into each of the following LOC devices. At the right place in the design.

LOC裝置變化型VIII 第73~77及78〜104圖顯示LOC裝置變化型VIII 518。圖 中係以相同元件符號表示與LOC裝置301中所示之等效特 徵及結構對應的該等特徵和結構。未與先前所述特徵對應 之特徵係以新的元件符號表示。 如第104圖槪略顯示般,此LOC裝置之變化型518使用 12個不同的擴增區段112.1〜112.12萃取290、培育291、擴 增292及偵測294人類DNA。LOC裝置變化型VIII 518使用 多個擴增區段以提高分析檢驗之靈敏度且增進所偵測之螢 光的信號雜訊比。 參閱第73、74及75圖,血液樣本進入樣本入口 68,且 毛細作用沿著蓋層通道94把該血液樣本吸引至該抗凝血劑 表面張力閥Π 8。蓋層46係經製造而具有用於替代該下密 封層64之替代層。於此設計中,界面層594係位於該 CMOS + MST裝置48之蓋層通道層80與MST通道層100之間 。界面層594允許於該等試劑貯存槽與MST層87之間具有 更複雜的流體互連結構又不會增加矽基板84之尺寸。第75 圖重疊地顯示該等貯存槽、該等頂部通道及該等界面通道 ,藉以說明利用該界面層594所達成之更精密複雜的配管 -129- 201209158 工程。 最佳係如第103圖所示’界面層594要求抗凝血劑表面 張力閥118具有兩個界面通道5 96和5 98。貯存槽側之界面 通道596把該貯存槽出口與該等下吸孔92連接在一起’且 樣本側之界面通道598把該等上吸孔96與蓋層通道94連接 在一起。源自貯存槽5 4的抗凝血劑經由該貯存槽側之界面 通道596流過MST通道90以使彎液面定在該等上吸孔96處 。沿著蓋層通道9 4流動之樣本流體浸濕該樣本側之界面通 道5 9 8以去除彎液面,使得當樣本流體繼續流向白血球透 析區段3 2 8時,使抗凝血劑與該血液樣本結合。· 參閱第78及103圖,白血球透析區段3 28包含旁通渠道 600,該旁通渠道600係用於塡充該等流體通道結構又不會 擄獲空氣氣泡。血液樣本流經蓋層通道94而抵達界面標靶 物通道602之上游末端。該界面標靶物通道602係經由外觀 呈直徑7.5微米之孔狀的多個孔165而與多個透析MST通道 204流體連通。該等透析MST通道204之每一者係從直徑7.5 微米之孔165通往各自的透析上吸孔168。該等透析上吸孔 168係對該界面廢液通道604開放。然而,該等上吸孔經配 置以用於定住彎液面而不允許毛細驅動流動繼續進行。 反之,位於白血球透析區段3 28之極上游末端處的旁 通渠道600具有毛細引動特徵(ciF ) 202,以促進毛細驅 動流體從旁通渠道600流入界面廢液通道604 (見第78及 103圖)。該旁通渠道亦具有—個寬曲流道以延長自該界 面標靶物通道602至該界面廢液通道604間的流動路徑。較 -130- 201209158 長的流動路徑延遲該樣本流體,使得於最上游透析MST通 道204處形成該等彎液面後,該樣本流體注入界面廢液通 道604。該樣本流體始於該上游末端處,且當該樣本流體 沿著該界面廢液通道604朝下游移動時,該樣本流體解除 位於每個透析上吸孔1 68處的彎液面。當樣本流體注入該 透析區段時此做法可確保所有的透析MST通道充滿樣本流 體。 無該旁通渠道6 0 0或無配置用於定住彎液面之透析上 吸孔168時,某些透析MST通道204可能無法塡滿。同樣地 ,可能於界面廢液通道6 04中形成空氣氣泡。於上述任一 種情況下皆可能扼止流經該透析區段之流動。 回到第74及75圖,界面廢液通道604饋入該流向廢料 貯存槽76之廢液通道72。界面標靶物通道602饋入標靶通 到74。含有標靶細胞之樣本流體係沿著標靶物通道74被吸 至該溶胞液試劑表面.張力閥128。 若具有上述之抗凝血劑表面張力閥118,該溶胞試劑 表面張力閥128具有溶胞試劑貯存槽側之界面通道606和溶 胞樣本側之界面通道608 (見第75圖)。溶胞試劑係自貯 存槽56經由蓋層通道94流向該溶胞試劑貯存槽側之界面通 道606。該試劑流入該等下吸孔92、通過MST通道90而流 向該等上吸孔96,且該等試劑於該等上吸孔96處定住彎液 面(見第74圖)。源自標靶物通道74之樣本流體注入該溶 胞樣本側之界面通道608。該樣本流體去除該等上吸孔96 處的彎液面,且當該樣本流體流入化學溶胞區段1 30時, -131 - 201209158 該溶胞試劑與該樣本合倂。 於化學溶胞區段1 3 0中’溶胞試劑擴散混合於該樣本 流體各處以溶解該等標靶細胞且釋出標靶細胞內的遺傳物 質。該樣本流體停止於該混合區段出口閥206處。最佳係 如第78及103圖所示,該混合區段入口閥係沸騰啓動式閥 206。該經溶胞之樣本經由界面導管6 1 0流入混合區段出口 下吸孔6 1 2。該樣本持續沿著M S T通道9 0流向沸騰啓動式 閥2 06,且當彎液面定在頂層66中的閥上吸孔151處時,該 樣本停止於沸騰啓動式閥206處(特別是參見第80 Α及81 A 圖)。位於該閥上游的液體感測器1 74提供該樣本流體係 大致抵達該閥上吸孔151的反饋。若CMOS電路86係經程式 化而寫入一段延遲時間以確保該等標靶細胞被完全溶解, 則該液體感測器之反饋會開始執行該段延遲時間。 於該延遲時間之後,藉由該等加熱器接觸點1 5 6 (見 第81 A及81B圖)使環形加熱器152啓動。位於閥上吸孔151 處的樣本液體沸騰且該彎液面解除。該樣本流入沸騰啓動 式閥界面凹槽616(見第82圖)且流出閥下吸孔150(見第 8 1 B圖)。下游之液體感測器1 7 4指示該流體已沿著μ S T通 道90再度開始流動。 該經溶胞之樣本流體持續流向該限制酶、接合酶和連 接子之表面張力閥132的上吸孔96(見第78圖)。參閱第 80Α、81Α、82、83及84圖’該貯存槽58中的限制酶、接 合酶和連接子-引子流入該蓋層通道94且前往該限制酶、 接合酶和連接子之閥界面通道6 1 4。該限制酶、接合酶和 -132- 201209158 引子之閥界面通道614對三個上吸孔96開放(藉由彎液面 使酶和連接子-引子停留於該三個上吸孔96處)。MST通道 90中的該經溶胞之樣本流體通過該等上吸孔96且去除該等 彎液面’使得限制酶、接合酶和連接子-引子與該樣本流 體混合。 參閱第78圖’該樣本、限制酶、接合酶和連接子-引 子流經MST通道混合區段丨3丨以進行擴散混合且之後進入 培育區段1 1 4之經加熱的微通道中。培育區段n 4係由蜿蜒 之微通道210所組成(見第79圖),且藉由承載於上方頂 層66上之各自的加熱器154 (見第81 A圖)加熱該蜿蜒微通 道210。該等加熱器154延伸於與該CMOS電路86連接之各 對加熱器接觸點1 5 6之間。 參閱第85圖,該樣本流體係停止於培育室出口閥207 處。該培育室出口閥207係沸騰啓動式閥,該沸騰啓動式 閥類似於混合區段出口閥206。緊鄰該培育室出口閥207之 上游的液體感測器1 74指示該樣本流體大約何時會停止於 該閥上吸孔151處(見第85、87及88B圖)。CMOS電路86 回應該液體感測器而開始實施培育時間延遲(如需要)。 經充分培育後,該環形加熱器1 52使位於該閥上吸孔 1 5 1處的液體沸騰藉以脫離彎液面。流體再次開始流入該 沸騰啓動式閥界面凹槽616(見第89圖)且流出該閥下吸 孔150 (見第87圖)。源自閥下吸孔150之樣本沿著MST培 育出口通道630(見第85圖)流向聚合酶表面張力閥140( 見第74及75圖)。當樣本流體行經該擴增輸入通道632之 -133- 201209158 蜿蜒路徑時,源自貯存槽6 2之聚合酶與該樣本流體合倂。 回到第85圖,擴增注入通道63 2引導該樣本流體通過 該十二個擴增混合物表面張力閥138。每個擴增混合物貯 存槽60.1 -6 0. 1 2 (見第75圖)中的擴增混合物流經各自的 蓋層通道94 (見第90及91圖)和各自的擴增界面導管 6 18〜629 (見第89圖)以使該等彎液面定住於該該等擴增 混合物表面張力閥138處(見第95圖)。該樣本流體依次 打開該等表面張力閥之每一者,且源自個別擴增混合物貯 存槽60.1〜60.12 (見第75圖)之擴增混合物與該樣本流體 —同進入該12個擴增區段112.1〜112.12之每一者各自的經 加熱微通道1 5 8。 參閱第92圖,該等12個擴增區段112.1〜112.12之每一 者各自具有該等擴增出口閥108之一者。該樣本流體停止 於每個擴增出口閥108之閥上吸孔151處。經熱循環後,該 閥加熱器1 52使位於閥上吸孔1 5 1處的液體沸騰(最佳係顯 示於第96B圖),並且該樣本流向該閥界面凹槽616(見第 97圖)且自i閥下吸孔150流出。 位於該等擴增出口閥108之下游處係由多個雜合腔室 iso組成之多個獨立的雜合腔室陣列,該等雜 合腔室陣列110.1〜110.12係分別用於該12種不同擴增子之 —者(見第9 2及1 0 0圖)。該樣本係沿著該流動路徑1 7 6流 經每個獨立的雜合腔室陣列1 1 〇 . 1〜1 1 〇 . 1 2且經由各自的擴 散阻障器入口 175流入個別之雜合腔室180中。參閱第1〇〇 圖’當該樣本流體到達該終點液體感測器〗78,使該等雜 -134- 201209158 合加熱器182通電以進行最適宜之探針—標靶物雜合反應 〇 參閱第77及100〜102圖,沉積於頂層66上之氮化鈦( TiN )條狀物環繞著濕度感測器2 32及雜合與偵測區段52。 該氮化鈦條狀物爲激發LED 26 (見第3圖)提供LED晶片 載體表面634。激發LED係被密封於該LED晶片載體表面 634,且透過位於MST層87中之該等通氣孔122和通氣通道 636使該等雜合腔室180內的空氣壓力等於大氣壓(見第 1 00 及 1 02 圖)。 參閱第104圖,用於該12種擴增子之每一者的該等雜 合腔室陣列110.1〜110.12係於該下方CMOS電路86 (見第12 圖)內具有單個光感測器44。於該等雜合腔室180之任一 者中的探針一標靶物雜合體發出螢光信號,且藉由對應的 光二極體1 84偵測該螢光信號。該等雜合腔室陣列 110.卜110.12之每一者具有至少一個校準腔室382,該至少 一個校準腔室3 82與該樣本流體隔離,使得無擴增子進入 校準腔室382。如本案說明書之他處所描述般,該等校準 腔室3 82係用於校準該等光二極體之輸出値,以根據讀値 誤差而針對系統雜訊做調整。LOC Device Variant VIII Figures 73-77 and 78-104 show LOC device variant VIII 518. The features and structures corresponding to the equivalent features and structures shown in LOC device 301 are denoted by the same reference numerals in the drawings. Features that do not correspond to the previously described features are denoted by new component symbols. As shown schematically in Figure 104, the variant 518 of the LOC device uses 12 different amplification segments 112.1 to 112.12 to extract 290, incubate 291, expand 292, and detect 294 human DNA. The LOC device variant VIII 518 uses multiple amplification segments to increase the sensitivity of the assay and to increase the signal to noise ratio of the detected fluorescence. Referring to Figures 73, 74 and 75, the blood sample enters the sample inlet 68 and capillary action draws the blood sample along the cap channel 94 to the anticoagulant surface tension valve Π 8. The cover layer 46 is manufactured to have an alternative layer for replacing the lower seal layer 64. In this design, interface layer 594 is located between capping channel layer 80 and MST channel layer 100 of the CMOS + MST device 48. The interface layer 594 allows for a more complex fluid interconnect structure between the reagent reservoirs and the MST layer 87 without increasing the size of the germanium substrate 84. Figure 75 overlays the storage tanks, the top channels, and the interface channels to illustrate the more sophisticated piping -129 - 201209158 project achieved with the interface layer 594. The preferred system, as shown in Figure 103, of the interface layer 594 requires the anticoagulant surface tension valve 118 to have two interface channels 5 96 and 5 98. The storage tank side interface 596 connects the storage tank outlet to the lower suction holes 92, and the sample side interface passage 598 connects the upper suction holes 96 with the cover layer passage 94. The anticoagulant from the reservoir 5 4 flows through the MST channel 90 via the interface channel 596 on the reservoir side to position the meniscus at the upper suction holes 96. The sample fluid flowing along the capping channel 94 wets the interface channel 594 on the sample side to remove the meniscus such that when the sample fluid continues to flow to the white blood cell dialysis section 3 2 8 , the anticoagulant is Blood samples are combined. • Referring to Figures 78 and 103, the white blood cell dialysis section 3 28 includes a bypass channel 600 for accommodating the fluid channel structures without capturing air bubbles. The blood sample flows through the capping channel 94 to the upstream end of the interface target channel 602. The interface target channel 602 is in fluid communication with a plurality of dialysis MST channels 204 via a plurality of apertures 165 that are shaped like holes having a diameter of 7.5 microns. Each of the dialysis MST channels 204 leads from a 7.5 micron diameter orifice 165 to a respective dialysis uptake orifice 168. The dialysis upper suction holes 168 are open to the interface waste liquid passage 604. However, the upper suction holes are configured to hold the meniscus without allowing the capillary drive flow to continue. Conversely, the bypass channel 600 at the extreme upstream end of the leukocyte dialysis section 3 28 has a capillary priming feature (ciF) 202 to facilitate capillary flow of fluid from the bypass channel 600 into the interface waste channel 604 (see pages 78 and 103). Figure). The bypass channel also has a wide curved flow path to extend the flow path from the interface target channel 602 to the interface waste channel 604. The sample fluid is delayed by a longer flow path than -130-201209158 such that the sample fluid is injected into the interface waste channel 604 after the meniscus is formed at the most upstream dialysis MST channel 204. The sample fluid begins at the upstream end, and as the sample fluid moves downstream along the interface waste channel 604, the sample fluid releases the meniscus at each dialysis upper suction port 168. This ensures that all dialysis MST channels are filled with sample fluid when the sample fluid is injected into the dialysis section. Some dialysis MST channels 204 may not be full when there is no bypass channel 600 or no dialysis upper suction port 168 configured to hold the meniscus. Similarly, air bubbles may form in the interface waste channel 604. In either case, it is possible to stop the flow through the dialysis section. Returning to Figures 74 and 75, the interface waste channel 604 feeds into the waste channel 72 that flows to the waste reservoir 76. Interface target channel 602 is fed to the target to 74. A sample flow system containing target cells is drawn along the target channel 74 to the surface of the lysate reagent. Tension valve 128. If the anticoagulant surface tension valve 118 is provided, the lysis reagent surface tension valve 128 has an interface channel 606 on the lysis reagent storage tank side and an interface channel 608 on the lysis sample side (see Fig. 75). The lysis reagent flows from the reservoir 56 through the capping channel 94 to the interface channel 606 on the lysis reagent storage tank side. The reagent flows into the lower suction holes 92, through the MST passage 90, to the upper suction holes 96, and the reagents hold the meniscus at the upper suction holes 96 (see Fig. 74). A sample fluid originating from the target channel 74 is injected into the interface channel 608 on the lysis sample side. The sample fluid removes the meniscus at the upper suction holes 96, and when the sample fluid flows into the chemical lysis section 130, the lysis reagent is combined with the sample. In the chemical lysis section 130, a lysis reagent is diffused and mixed throughout the sample fluid to dissolve the target cells and release the genetic material within the target cells. The sample fluid stops at the mixing section outlet valve 206. Best Modes As shown in Figures 78 and 103, the mixing section inlet valve is a boil-start valve 206. The sample of the lysed cells flows into the mixing section outlet lower suction hole 6 1 2 via the interface conduit 61. The sample continues to flow along the MST channel 90 to the boiling start valve 206, and when the meniscus is positioned at the valve upper suction port 151 in the top layer 66, the sample stops at the boiling start valve 206 (see especially 80th and 81A)). A liquid sensor 1 74 located upstream of the valve provides feedback that the sample flow system generally reaches the suction port 151 of the valve. If the CMOS circuit 86 is programmed to write a delay time to ensure that the target cells are completely dissolved, the feedback from the liquid sensor will begin to perform the delay time. After the delay time, the ring heater 152 is activated by the heater contact points 156 (see Figures 81A and 81B). The sample liquid located at the suction hole 151 of the valve boils and the meniscus is released. The sample flows into the boiling start valve interface groove 616 (see Figure 82) and out of the valve lower suction hole 150 (see Figure 8 1 B). The downstream liquid sensor 174 indicates that the fluid has started to flow again along the μ S T channel 90. The lysed sample fluid continues to flow to the upper suction port 96 of the restriction enzyme, ligase, and linker surface tension valve 132 (see Figure 78). Referring to Figures 80, 81, 82, 83 and 84, the restriction enzymes, ligase and linker-introducers in the reservoir 58 flow into the capping channel 94 and proceed to the valve interface channel of the restriction enzyme, ligase and linker. 6 1 4. The restriction enzyme, ligase, and valve interface channel 614 of the -132-201209158 primer are open to the three upper suction holes 96 (the enzyme and linker-inducers are retained at the three upper suction holes 96 by the meniscus). The lysed sample fluid in the MST channel 90 passes through the upper suction holes 96 and removes the meniscus' such that the restriction enzyme, ligase, and linker-introduction are mixed with the sample fluid. Referring to Figure 78, the sample, restriction enzyme, ligase, and linker-primer flow through the MST channel mixing section 丨3丨 for diffusion mixing and then into the heated microchannel of the incubation section 112. The incubation section n 4 is comprised of a microchannel 210 of tantalum (see Figure 79) and is heated by a respective heater 154 (see Figure 81A) carried on the upper top layer 66 (see Figure 81A). 210. The heaters 154 extend between respective pairs of heater contacts 156 connected to the CMOS circuit 86. Referring to Figure 85, the sample flow system is stopped at the incubation chamber outlet valve 207. The chamber exit valve 207 is a boil-start valve that is similar to the mixing section outlet valve 206. A liquid sensor 1 74 immediately upstream of the chamber exit valve 207 indicates when the sample fluid will stop at the valve upper suction port 151 (see Figures 85, 87 and 88B). The CMOS circuit 86 begins to implement the incubation time delay (if needed) in response to the liquid sensor. After sufficient incubation, the ring heater 152 boils the liquid at the suction port 157 of the valve to escape the meniscus. The fluid again begins to flow into the boiling start valve interface groove 616 (see Figure 89) and out of the valve lower suction port 150 (see Figure 87). Samples from the lower valve suction port 150 flow along the MST culture outlet channel 630 (see Figure 85) to the polymerase surface tension valve 140 (see Figures 74 and 75). When the sample fluid travels through the -133-201209158 蜿蜒 path of the amplification input channel 632, the polymerase originating from reservoir 6 is fluidly conjugated to the sample. Returning to Fig. 85, the amplification injection channel 63 2 directs the sample fluid through the twelve amplification mixture surface tension valves 138. The amplification mixture in each of the amplification mixture storage tanks 60.1 -6 0. 1 2 (see Figure 75) flows through the respective capping channels 94 (see Figures 90 and 91) and the respective augmentation interface conduits 6 18 ~ 629 (see Figure 89) to position the meniscus at the surface tension valve 138 of the amplification mixture (see Figure 95). The sample fluid sequentially opens each of the surface tension valves, and the amplification mixture from the individual amplification mixture storage tanks 60.1 to 60.12 (see Figure 75) and the sample fluid enter the 12 amplification zones. Each of the segments 112.1 to 112.12 is heated by a microchannel 1 5 8 . Referring to Fig. 92, each of the twelve amplification sections 112.1 to 112.12 has one of the amplification outlet valves 108. The sample fluid is stopped at the valve upper suction hole 151 of each of the expansion outlet valves 108. After thermal cycling, the valve heater 152 boils the liquid at the suction port 151 of the valve (best shown in Figure 96B) and the sample flows toward the valve interface groove 616 (see Figure 97). And flowing out from the i-port lower suction hole 150. Located downstream of the amplification outlet valve 108 is a plurality of independent hybrid chamber arrays composed of a plurality of hybrid chambers iso, the hybrid chamber arrays 110.1 to 110.12 are used for the 12 different types, respectively. Of the amplicon (see Figures 9 2 and 1 0 0). The sample flows along the flow path 167 through each of the individual hybrid chamber arrays 1 1 〇 1 1 1 1 〇 1 2 and flows into the individual hybrid chambers via respective diffusion barrier inlets 175. In the room 180. Refer to Figure 1 'When the sample fluid reaches the endpoint liquid sensor〗 78, energizing the hybrid-134-201209158 heater 182 for the most appropriate probe-target hybrid reaction. In the 77th and 100th to 102th, the titanium nitride (TiN) strip deposited on the top layer 66 surrounds the humidity sensor 2 32 and the hybrid and detection section 52. The titanium nitride strip provides an LED wafer carrier surface 634 for the excitation LED 26 (see Figure 3). The excitation LEDs are sealed to the LED wafer carrier surface 634, and the air pressure in the hybrid chambers 180 is equal to atmospheric pressure through the vents 122 and venting channels 636 located in the MST layer 87 (see 00 and 00 1 02 picture). Referring to Fig. 104, the hybrid chamber arrays 110.1 to 110.12 for each of the 12 ampersons have a single photosensor 44 within the lower CMOS circuit 86 (see Fig. 12). The probe-target hybrid in any of the hybrid chambers 180 emits a fluorescent signal and the fluorescent signal is detected by the corresponding photodiode 184. Each of the hybrid chamber arrays 110.110.12 has at least one calibration chamber 382 that is fluidly isolated from the sample such that no amplicon enters the calibration chamber 382. As described elsewhere in this specification, the calibration chambers 3 82 are used to calibrate the output ports of the photodiodes to adjust for system noise based on read errors.

LOC裝置變化型XXIII 第109圖之LOC裝置變化型XXIII 650係用於進行基因 分析且使用白血球透析區段328以實質降低樣本中之紅血 球濃度。於化學溶胞區段1 3 0中,源自貯存槽5 6之溶胞試 -135- 201209158 劑釋出白血球中的遺傳物質。於化學溶胞區段1 3 0下游處 ,該樣本與源自貯存槽58之限制酶、接合酶和連接子-引 子混合且注入培育區段1 1 4。經培育後,緊鄰培育區段1 ! 4 之下游處的沸騰啓動式閥1 08開啓以使樣本流入擴增區段 1 1 2且最終流入雜合腔室陣列1 1 〇。LOC Device Variant XXIII The LOC Device Variant XXIII 650 of Figure 109 is used for genetic analysis and uses a white blood cell dialysis section 328 to substantially reduce the red blood cell concentration in the sample. In the chemical lysis section 130, the lysate-135-201209158 agent from the storage tank 56 releases the genetic material in the white blood cells. Downstream of the chemical lysis section 130, the sample is mixed with restriction enzymes, ligase and linker-derived from storage tank 58 and injected into the incubation section 1 14 . After incubation, the boiling start valve 108 immediately downstream of the incubation section 1 ! 4 opens to allow the sample to flow into the amplification section 1 1 2 and ultimately into the hybrid chamber array 1 1 〇.

LOC裝置變化型XXIV LOC裝置變化型XXIV 651係一種具有白血球透析區段 3 2 8、化學溶胞區段1 3 0和限制酶、接合酶和連接子之培育 區段114的基因分析LOC裝置(見第11〇圖)。該LOC裝置 變化型XXIV 651使用多個並聯之擴增區段112.ι、 1 12.2…1 12· X及個別雜合腔室陣列1 lo.i、1丨0.2... 1 10.X。LOC Device Variant XXIV LOC Device Variant XXIV 651 is a genetically analyzed LOC device having a white blood cell dialysis section 328, a chemical lysis section 1 30 and a restriction enzyme, ligase and linker incubation section 114 ( See picture 11). The LOC device variant XXIV 651 uses a plurality of parallel amplification sections 112. ι, 1 12.2...1 12·X and individual hybrid chamber arrays 1 lo.i, 1 丨 0.2... 1 10.X.

LOC裝置變化型XXV LOC裝置變化型XXV 652 (見第111圖)係基因分析 LOC裝置之具體實施例。該LOC裝置變化型XXV 652使用 白血球透析區段3 2 8、化學溶胞區段1 3 0和限制酶、接合酶 及連接子之培育區段114。接著使該樣本饋入串接之擴增 腔室112.1與擴增腔室112.2,且之後於單一個雜合腔室陣 列1 1 〇中進行偵測。LOC Device Variant XXV LOC Device Variant XXV 652 (see Figure 111) is a specific example of a genetic analysis LOC device. The LOC device variant XXV 652 uses a white blood cell dialysis section 3 28, a chemical lysis section 1 30 and a restriction enzyme, ligase and linker incubation section 114. The sample is then fed into the cascaded amplification chamber 112.1 and the amplification chamber 112.2, and then detected in a single hybrid chamber array 1 1 .

LOC裝置變化型XXVI 第112圖所示之LOC裝置變化型XXVI 653係用於進行 基因分析且使用白血球透析區段328以實質降低樣本中之 -136- 201209158 紅血球濃度。於化學溶胞區段1 3 0中,源自貯存槽5 6之溶 胞試劑釋出白血球內的遺傳物質。於化學溶胞之後,緊鄰 化學溶胞區段130之下游處的沸騰啓動式閥1〇8開啓以使樣 本流入擴增區段Π 2且最終流入雜合腔室陣列丨丨〇。LOC Device Variant XXVI The LOC Device Variant XXVI 653 shown in Figure 112 is used for genetic analysis and uses a white blood cell dialysis section 328 to substantially reduce the red blood cell concentration of -136 - 201209158 in the sample. In the chemical lysis section 130, the lysis reagent derived from the storage tank 56 releases the genetic material in the white blood cells. After chemical lysis, the boiling start valve 1 〇 8 immediately downstream of the chemical lysis section 130 is opened to cause the sample to flow into the amplification section Π 2 and ultimately into the hybrid chamber array 丨丨〇.

LOC裝置變化型XXVIILOC device variant XXVII

第113圖所示之LOC裝置變化型XXVII 654係一種具有 白血球透析區段328和化學溶胞區段130的基因分析LOC裝 置。該LOC裝置變化型XXVII 654接著使用多個並聯之擴 增區段112.1、112.2…112 .X及個別雜合腔室陣列nn ' 1 1 0.2 ... 1 1 0.X。The LOC device variant XXVII 654 shown in Fig. 113 is a genetic analysis LOC device having a white blood cell dialysis section 328 and a chemical lysis zone 130. The LOC device variant XXVII 654 then uses a plurality of parallel expansion sections 112.1, 112.2...112.X and individual hybrid chamber arrays nn ' 1 1 0.2 ... 1 1 0.X.

LOC裝置變化型XXVIII 第114圖所示之LOC裝置變化型XXVIII 65 5係基因分析 LOC裝置之具體實施例。該LOC裝置變化型XXVIII 655使 用白血球透析區段328和化學溶胞區段130。接著使該樣本 饋入串接之擴增腔室112.1與擴增腔室112.2,且之後於單 —個雜合腔室陣列1 1 〇中進行偵測。 結論 本案所述之裝置、系統和方法有利於以低價、高速及 重點照護方式進行分子診斷檢驗。 上述之系統與該系統之構件純爲解說之用,且所屬技 術領域中熟悉該項技藝者將可輕易地領悟許多不偏離本案 -137- 201209158 廣義發明槪念之精神與範圍的變化體系與修飾態樣。 【圖式簡單說明】 現參照附圖所描述之本發明的多個較佳具體實施例係 僅供示範之用,該等附圖如下: 第1圖顯示經建構以用於螢光偵測之檢驗模組及檢驗 模組讀取器。 第2圖係該經建構以用於螢光偵測之檢驗模組內的電 子構件之槪要總覽圖。 第3圖係該檢驗模組讀取器內之電子構件的槪要總覽 圖。 第4圖係該LOC裝置之構造的槪要表示圖。 第5圖係該LOC裝置之透視圖。 第6圖係具有源自所有彼此重疊膜層之特徵與結構的 該LOC裝置之平面圖。 第7圖係單獨顯示該蓋層結構的該LOC裝置之平面圖 〇 第8圖係具有內部通道及貯存槽之該蓋層的俯視透視 圖,該等內部通道及貯存槽係以虛線繪示。 第9圖係具有內部通道及貯存槽之該蓋層的俯視分解 透視圖,該等內部通道及貯存槽係以虛線繪示。 第1 〇圖係該蓋層之仰視透視圖,該仰視透視圖顯示該 等頂部通道的結構配置。 第11圖係該LOC裝置之平面圖,該平面圖單獨顯示該 -138- 201209158 CMOS + MST裝置的結構。 第12圖係該LOC裝置於樣本入口處的槪要剖面圖° 第13圖係第6圖所τκ之插圖AA的放大圖。 第14圖係第6圖所不之插圖AB的放大圖。 第15圖係第13圖所示之插圖AE的放大圖。 第16圖係繪示插圖AE中之該LOC裝置的層狀結構之部 分透視圖。 第17圖係繪示插圖AE中之該LOC裝置的層狀結構之部 分透視圖。 第18圖係繪示插圖AE中之LOC裝置的層狀結構之部分 透視圖。 第19圖係繪示插圖AE中之LOC裝置的層狀結構之部分 透視圖。 第20圖係繪示插圖AE中之LOC裝置的層狀結構之部分 透視圖。 第21圖係繪示插圖AE內之該LOC裝置的層狀結構之部 分透視圖。 第22圖係第21圖所示之溶胞試劑貯存槽的槪要剖面圖 〇 第23圖係繪示插圖AB中之該LOC裝置的層狀結構之部 分透視圖。 第2 4圖係繪示插圖A B中之該L Ο C裝置的層狀結構之部 分透視圖。 第25圖係繪示插圖AI中之該LOC裝置的層狀結構之部 -139- 201209158 分透視圖。 第26圖係繪示插圖AB內之該LOC裝置的層狀結構之部 分透視圖。 第27圖係繪示插圖AB內之該LOC裝置的層狀結構之部 分透視圖。 第28圖係繪示插圖AB內之LOC裝置的層狀結構之部分 透視圖。 第29圖係繪示插圖AB內之LOC裝置的層狀結構之部分 透視圖。 第30圖係該擴增混合物貯存槽及該聚合酶貯存槽之槪 要剖面圖。 第31圖單獨繪示沸騰啓動式閥之特徵。 第3 2圖係沿第3 1圖所示之線段3 3 - 3 3取得該沸騰啓動 式閥的槪要剖面圖。 第33圖係第15圖所示之插圖AF的放大圖。 第34圖係沿第33圖中所示之線段3 5 -3 5取得該透析區 段之上游末端的槪要剖面圖。 第35圖係第6圖所示之插圖AC的放大圖。 第36圖係顯示該擴增區段之插圖AC內部的進一步放大 圖。 第37圖係顯不該擴增區段之插圖AC內部的進一步放大 圖。 第38圖係顯示該擴增區段之插圖ACR部的進—步放大 圖。 -140- 201209158 第39圖係第38圖所示之插圖AK的進一步放大圖。 第40圖係顯示該擴增腔室之插圖AC內部的進一步放大 圖。 第41圖係顯示該擴增區段之插圖AC內部的進一步放大 圖。 第42圖係顯示該擴增腔室之插圖AC內部的進一步放大 圖。 第43圖係第42圖所示之插圖AL內部的進一步放大圖^ 第44圖係顯示該擴增區段之插圖AC內部的進一步放大 圖。 第45圖係第44圖所示之插圖AM的進一步放大圖。 第46圖係顯示該擴增腔室之插圖AC內部的進一步放大 圖。 第47圖係第46圖所示之插圖AN的進一步放大圖。 第48圖係顯示該擴增腔室之插圖AC內部的進一步放大 圖。 第49圖係顯示該擴增腔室之插圖AC內部的進一步放大 圖。 第50圖係顯示該擴增區段之插圖AC內部的進一步放大 圖。 第5 1圖係該擴增區段之槪要剖面圖》 第52圖係該雜合區段之放大平面圖。 第53圖係單獨顯示兩個雜合腔室的進一步放大剖面圖 -141 - 201209158 第54圖係單個雜合腔室之槪要剖面圖。 第55圖係第6圖所示之插圖AG內繪示的增濕器之放大 圖。 第56圖係第52圖所示之插圖AD的放大圖。 第5 7圖係插圖AD內之LOC裝置的分解透視圖。 第5 8圖係處於閉合結構之FRET探針圖解。 第59圖係處於打開且經雜合之結構的FRET探針圖解 〇 第60圖係激發光強度隨時間變化之線圖。 第61圖係雜合腔室陣列的激發光幾何學之圖解。 第62圖係感測器電子技術之LED發光幾何學的圖解。 第6 3圖係第6圖之插圖A Η內所示之濕度感測器的放大 平面圖。 第64圖係白血球標靶透析區段之槪要剖面圖。 第65圖係光感測器之光二極體陣列的槪要局部示圖。 第66圖係單個光二極體之電路圖。 第67圖係光二極體控制信號之時間圖。 第68圖係第55圖之插圖ΑΡ內所示的蒸發器之放大圖。 第69圖係穿過具有偵測光二極體及觸發光二極體之雜 合腔室的槪要剖面圖。 第70圖係連接子-引子PCR法(linker-primed PCR)。 第7 1圖係配備有刺胳針之檢驗模組的槪要表示圖。 第72圖係LOC裝置變化型VII之構造的圖解表示圖。 第73圖係LOC裝置變化型VIII之透視圖。 -142- 201209158 第74圖係具有所有彼此重疊膜層之特徵與結構的L0C 裝置變化型VIII之平面圖。 第75圖係單獨示出該蓋層結構的LOC裝置變化型VHI 之平面圖。 第76圖係用於LOC裝置變化型VIII之該等蓋層通道的 仰視透視圖。 第77圖係該LOC裝置變化型VIII之平面圖,該平面圖 單獨顯示該CMOS + MST裝置之結構。 第78圖係第74圖所示之插圖CA的放大圖。 第79圖係部分透視圖,該部分透視圖繪示第74圖所示 之插圖CA內的LOC裝置變化型VIII之層狀結構。 第80圖係部分透視圖,該部分透視圖繪示第74圖所示 之插圖CA內的LOC裝置變化型VIII之層狀結構。 第81圖係部分透視圖,該部分透視圖繪示第74圖所示 之插圖CA內的LOC裝置變化型VIII之層狀結構。 第82圖係部分透視圖,該部分透視圖繪示第74圖所示 之插圖CA內的LOC裝置變化型VIII之層狀結構。 第83圖係部分透視圖,該部分透視圖繪示第74圖所示 之插圖CA內的LOC裝置變化型VIII之層狀結構。 第84圖係部分透視圖,該部分透視圖繪示第74圖所示 之插圖CA內的LOC裝置變化型VIII之層狀結構。 第85圖係第74圖所示之插圖CB的放大圖。 第8 6圖係部分透視圖,該部分透視圖繪示第74圖所示 之插圖CB內的LOC裝置變化型VIII之層狀結構。 -143- 201209158 第8 7圖係部分透視圖,該部分透視圖繪示第7 4圖所示 之插圖CB內的LOC裝置變化型VIII之層狀結構。 第88A及88B圖係部分透視圖,該等部分透視圖繪示第 74圖所示之插圖CB內的LOC裝置變化型VIII之層狀結構。 第8 9圖係部分透視圖,該部分透視圖繪示第74圖所示 之插圖CB內的LOC裝置變化型VIII之層狀結構。 第90圖係部分透視圖,該部分透視圖繪示第74圖所示 之插圖CB內的LOC裝置變化型VIII之層狀結構。 第9 1圖係部分透視圖,該部分透視圖繪示第74圖所示 之插圖CB內的LOC裝置變化型VIII之層狀結構。 第92圖係第74圖所示之插圖CC的放大圖。 第9 3圖係部分透視圖,該部分透視圖繪示第74圖所示 之插圖CC內的LOC裝置變化型VIII之層狀結構。 第94圖係部分透視圖,該部分透視圖繪示第74圖所示 之插圖CC內的LOC裝置變化型VIII之層狀結構。 第95圖係部分透視圖,該部分透視圖繪示第74圖所示 之插圖CC內的LOC裝置變化型VIII之層狀結構。 第96A及96B圖係部分透視圖,該等部分透視圖繪示第 74圖所示之插圖CC內的LOC裝置變化型VIII之層狀結構。 第97圖係部分透視圖,該部分透視圖繪示第74圖所示 之插圖CC內的LOC裝置變化型VIII之層狀結構。 第98圖係部分透視圖,該部分透視圖繪示第74圖所示 之插圖CC內的LOC裝置變化型VIII之層狀結構。 第99圖係部分透視圖,該部分透視圖繪示第74圖所示 -144- 201209158 之插圖CC內的LOC裝置變化型VIII之層狀結構。 第100圖係第74圖所示之插圖CD的放大圖。 第101圖係第74圖所示之插圖CD的放大透視圖。 第102圖係第74圖所示之插圖CD的分解圖。 第103圖係第78圖所示之插圖CE的放大圖。 第104圖係LOC裝置變化型VIII之構造的圖解表示圖。 第105圖係LOC裝置變化型XIV之構造的槪要圖。 第106圖係LOC裝置變化型XV之構造的槪要圖。 第107圖係LOC裝置變化型XVIII之構造的槪要圖。 第108圖係LOC裝置變化型XXII之構造的槪要圖。 第109圖係LOC裝置變化型XXIII之構造的槪要圖。 第1 10圖係LOC裝置變化型XXIV之構造的槪要圖。 第111圖係LOC裝置變化型XXV之構造的槪要圖。 第112圖係LOC裝置變化型XXVI之構造的槪要圖。 第113圖係LOC裝置變化型XXVII之構造的槪要圖。 第114圖係LOC裝置變化型XXVIII之構造的槪要圖。 第115圖係LOC裝置變化型XLI之構造的槪要圖。 第116圖係LOC裝置變化型XLII之構造的槪要圖。 第117圖係LOC裝置變化型XLIII之構造的槪要圖。 第118圖係LOC裝置變化型XLIV之構造的槪要圖。 第119圖係LOC裝置變化型XLVII之構造的槪要圖。 第1 20圖係接有引子之線性探針於擴增反應之初始回 合期間之圖解。 第1 2 1圖係接有引子之線性探針於後續擴增循環期間 -145- 201209158 之圖解。 第122A至122F圖圖解說明一種接有引子之螢光幹-環 狀探針的熱循環。 第123圖係與該雜合腔室陣列及該等光二極體有關的 激發LED之槪要圖。 第124圖係用於把光線引導至該LOC裝置之雜合腔室 上的激發LED及光學鏡片之槪要圖。 第125圖係用於把光線引導至該LOC裝置之雜合腔室 上的激發LED '光學鏡片及光學稜鏡之槪要圖。 第126圖係用於把光線引導至該LOC裝置之雜合腔室 上的激發LED、光學鏡片及鏡面配置之槪要圖。 第127圖係一平面圖’該平面圖顯示所有彼此重疊之 特徵且顯示插圖DA〜DK之位置。 第128圖係第127圖所示之插圖DG的放大圖。 第129圖係第127圖所示之插圖DH的放大圖。 第130圖係LOC裝置變化型XI之構造的圖解表示圖。 第131圖係用於該等光二極體之分流電晶體的具體實 施例。 第1 3 2圖係用於該等光二極體之分流電晶體的具體實 施例。 第1 3 3圖係用於該等光二極體之分流電晶體的具體實 施例。 第134圖係微分成像器之電路圖。 第1 3 5圖槪要圖解處於幹-環狀結構的陰性對照組螢光 -146- 201209158 探針。 第1 3 6圖槪要圖解處於打開結構的第1 3 5圖之陰性對照 組螢光探針。 第1 3 7圖槪要圖解處於幹-環狀結構的陽性對照組營光 探針。 第1 3 8圖槪要圖解處於打開結構的第1 3 7圖之陽性對照 組螢光探針。 第139圖顯示與ECL偵測倂用之檢驗模組及檢驗模組讀 取器。 第1 40圖係與螢光偵測倂用之檢驗模組內的電子構件 之槪要總覽圖。 第1 4 1圖顯示檢驗模組及多種可供選擇的檢驗模組讀 取器。 第I42圖顯示搭配儲存各種資料庫之主機系統的檢驗 模組及檢驗模組讀取器。 【主要元件符號說明】 1 〇 :檢驗模組 1 1 :檢驗模組 1 2 :檢驗模組讀取器 13 :外殻 1 4 :微型U S B插頭 15 :電感器 16 :微型USB插槽 -147- 201209158 1 7 :觸控螢幕 18 :顯示螢幕 1 9 :按鍵 20 :啓動鈕 2 1 :蜂巢式無線電 22 :可撕式滅菌密封膠帶 23 :無線網路連接器 24 :大放置槽 25 :衛星導航系統 26 :發光二極體(LED ) 2 7 :資料儲存器 28 :行動電話/智慧型手機 29 : LED驅動器 30 :晶片上實驗室(LOC )裝置 3 1 :功率調節器 32 :電容器 3 3 :時鐘 3 4 :控制器 3 5 :記錄器 36: USB裝置驅動器 3 7 :驅動器 38 :隨機存取記憶體(RAM ) 3 9 :驅動器 40 :程式及資料快閃記億體 -148 - 201209158 4 1 :記錄器 42 :處理器 43 :程式儲存器 44 _·光感測器/光感測器陣列 4 5 :指示器 46 :蓋層 47 :僅靠USB供電和作爲指示器之模組 48 : CMOS + MST 晶片LOC Device Variant XXVIII LOC Device Variant XXVIII 65 5 Line Genetic Analysis Example 114 shows a specific embodiment of the LOC device. The LOC device variant XXVIII 655 uses a white blood cell dialysis section 328 and a chemical lysis zone 130. The sample is then fed into the converging amplification chamber 112.1 and the amplification chamber 112.2, and then detected in a single hybrid chamber array 1 1 . Conclusion The devices, systems, and methods described in this case facilitate molecular diagnostic testing at low cost, high speed, and focused care. The above-mentioned system and the components of the system are purely illustrative, and those skilled in the art will be able to easily comprehend many changes and modifications of the spirit and scope of the general inventive concept without deviating from the present case-137-201209158 Aspect. BRIEF DESCRIPTION OF THE DRAWINGS A number of preferred embodiments of the present invention, which are described with reference to the drawings, are for illustrative purposes only, and the drawings are as follows: Figure 1 shows the construction for fluorescence detection. Inspection module and inspection module reader. Figure 2 is a summary view of the electronic components within the inspection module constructed for fluorescence detection. Figure 3 is a schematic overview of the electronic components within the test module reader. Figure 4 is a schematic representation of the construction of the LOC device. Figure 5 is a perspective view of the LOC device. Figure 6 is a plan view of the LOC device having features and structures derived from all of the overlapping film layers. Figure 7 is a plan view of the LOC device showing the cover structure separately. Figure 8 is a top perspective view of the cover layer having internal passages and storage tanks, the internal passages and storage tanks being shown in dashed lines. Figure 9 is a top exploded perspective view of the cover layer having internal passages and storage tanks, the internal passages and storage tanks being shown in dashed lines. The first drawing is a bottom perspective view of the cover, the bottom perspective showing the structural configuration of the top channels. Figure 11 is a plan view of the LOC device showing the structure of the -138-201209158 CMOS + MST device separately. Fig. 12 is a schematic cross-sectional view of the LOC device at the entrance of the sample. Fig. 13 is an enlarged view of the figure AA of τκ in Fig. 6. Figure 14 is an enlarged view of the illustration AB of Figure 6. Fig. 15 is an enlarged view of the illustration AE shown in Fig. 13. Figure 16 is a partial perspective view showing the layered structure of the LOC device in the inset AE. Figure 17 is a partial perspective view showing the layered structure of the LOC device in the inset AE. Figure 18 is a partial perspective view showing the layered structure of the LOC device in the illustration AE. Figure 19 is a partial perspective view showing the layered structure of the LOC device in the illustration AE. Figure 20 is a partial perspective view showing the layered structure of the LOC device in the illustration AE. Figure 21 is a partial perspective view showing the layered structure of the LOC device in the inset AE. Figure 22 is a schematic cross-sectional view of the lysis reagent storage tank shown in Figure 21 〇 Figure 23 is a partial perspective view showing the layered structure of the LOC device in the illustration AB. Fig. 24 is a partial perspective view showing the layered structure of the L Ο C device in the illustration A B. Figure 25 is a partial perspective view of the layered structure of the LOC device in the illustration AI - 139 - 201209158. Figure 26 is a partial perspective view showing the layered structure of the LOC device in the inset AB. Figure 27 is a partial perspective view showing the layered structure of the LOC device in the inset AB. Figure 28 is a partial perspective view showing the layered structure of the LOC device in the inset AB. Figure 29 is a partial perspective view showing the layered structure of the LOC device in the inset AB. Figure 30 is a cross-sectional view of the amplification mixture storage tank and the polymerase storage tank. Figure 31 is a separate illustration of the characteristics of a boiling start valve. Figure 3 is a schematic cross-sectional view of the boiling start valve taken along line 3 3 - 3 3 shown in Figure 31. Fig. 33 is an enlarged view of the illustration AF shown in Fig. 15. Figure 34 is a cross-sectional view of the upstream end of the dialysis section taken along line 35-35 of Figure 33. Fig. 35 is an enlarged view of the illustration AC shown in Fig. 6. Figure 36 is a further enlarged view showing the inside of the illustration AC of the amplified section. Figure 37 is a further enlarged view of the inside of the illustration AC of the amplification section. Figure 38 is a further enlarged view showing the ACR portion of the illustration of the amplified segment. -140- 201209158 Figure 39 is a further enlarged view of the illustration AK shown in Figure 38. Figure 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 enlarged section. Figure 42 is a further enlarged view showing the inside of the illustration AC of the amplification chamber. Fig. 43 is a further enlarged view of the inside of the illustration AL shown in Fig. 42. Fig. 44 is a further enlarged view showing the inside of the illustration AC of the enlarged section. Fig. 45 is a further enlarged view of the illustration AM shown in Fig. 44. Figure 46 is a further enlarged view showing the inside of the illustration AC of the amplification chamber. Fig. 47 is a further enlarged view of the illustration AN shown in Fig. 46. Figure 48 is a further enlarged view showing the inside of the illustration AC of the amplification chamber. Figure 49 is a further enlarged view showing the inside of the illustration AC of the amplification chamber. Fig. 50 is a further enlarged view showing the inside of the illustration AC of the enlarged section. Fig. 51 is a schematic cross-sectional view of the enlarged section. Fig. 52 is an enlarged plan view of the hybrid section. Figure 53 is a further enlarged cross-sectional view showing two hybrid chambers separately -141 - 201209158 Figure 54 is a cross-sectional view of a single hybrid chamber. Fig. 55 is an enlarged view of the humidifier shown in the illustration AG shown in Fig. 6. Fig. 56 is an enlarged view of the illustration AD shown in Fig. 52. Figure 5 is an exploded perspective view of the LOC device in the illustration AD. Figure 58 is a FRET probe diagram in a closed configuration. Figure 59 is a diagram of the FRET probe in an open and heterozygous structure. Figure 60 is a line graph of excitation light intensity over time. Figure 61 is a graphical representation of the excitation light geometry of a hybrid chamber array. Figure 62 is an illustration of the LED illumination geometry of the sensor electronics. Figure 6 is an enlarged plan view of the humidity sensor shown in Figure A of Figure 6. Figure 64 is a schematic cross-sectional view of the dialysis section of the white blood cell target. Figure 65 is a schematic partial view of an optical diode array of a photosensor. Figure 66 is a circuit diagram of a single photodiode. Figure 67 is a time diagram of the photodiode control signal. Figure 68 is an enlarged view of the evaporator shown in the illustration of Figure 55. Figure 69 is a cross-sectional view through a hybrid chamber having a detection photodiode and a trigger photodiode. Figure 70 is a linker-primed PCR. Figure 7 is a schematic representation of a test module equipped with a lancet. Figure 72 is a graphical representation of the construction of the LOC device variant VII. Figure 73 is a perspective view of a variation VIII of the LOC device. - 142 - 201209158 Figure 74 is a plan view of a LOC device variant VIII having all of the features and structures of the overlapping layers. Fig. 75 is a plan view showing the LOC device variation type VHI of the cap layer structure alone. Figure 76 is a bottom perspective view of the cover channels for the LOC device variant VIII. Figure 77 is a plan view of the LOC device variant VIII, which shows the structure of the CMOS + MST device separately. Fig. 78 is an enlarged view of the illustration CA shown in Fig. 74. Fig. 79 is a partial perspective view showing the layered structure of the LOC device variation VIII in the inset CA shown in Fig. 74. Fig. 80 is a partial perspective view showing the layered structure of the LOC device variation VIII in the inset CA shown in Fig. 74. Fig. 81 is a partial perspective view showing the layered structure of the LOC device variation VIII in the inset CA shown in Fig. 74. Fig. 82 is a partial perspective view showing the layered structure of the LOC device variation VIII in the inset CA shown in Fig. 74. Fig. 83 is a partial perspective view showing the layered structure of the LOC device variation VIII in the inset CA shown in Fig. 74. Fig. 84 is a partial perspective view showing the layered structure of the LOC device variation VIII in the inset CA shown in Fig. 74. Fig. 85 is an enlarged view of the illustration CB shown in Fig. 74. Fig. 8 is a partial perspective view showing the layered structure of the LOC device variation VIII in the inset CB shown in Fig. 74. - 143 - 201209158 Figure 8 7 is a partial perspective view showing the layered structure of the LOC device variant VIII in the inset CB shown in Figure 74. Parts 88A and 88B are partial perspective views showing the layered structure of the LOC device variation VIII in the inset CB shown in Fig. 74. Fig. 8 is a partial perspective view showing the layered structure of the LOC device variation VIII in the inset CB shown in Fig. 74. Fig. 90 is a partial perspective view showing the layered structure of the LOC device variation VIII in the inset CB shown in Fig. 74. Fig. 91 is a partial perspective view showing the layered structure of the LOC device variation VIII in the inset CB shown in Fig. 74. Fig. 92 is an enlarged view of the illustration CC shown in Fig. 74. Fig. 9 is a partial perspective view showing the layered structure of the LOC device variation VIII in the inset CC shown in Fig. 74. Fig. 94 is a partial perspective view showing the layered structure of the LOC device variation VIII in the inset CC shown in Fig. 74. Fig. 95 is a partial perspective view showing the layered structure of the LOC device variation VIII in the inset CC shown in Fig. 74. Sections 96A and 96B are partial perspective views showing the layered structure of the LOC device variant VIII in the inset CC shown in Fig. 74. Fig. 97 is a partial perspective view showing the layered structure of the LOC device variation VIII in the inset CC shown in Fig. 74. Fig. 98 is a partial perspective view showing the layered structure of the LOC device variation VIII in the inset CC shown in Fig. 74. Fig. 99 is a partial perspective view showing the layered structure of the LOC device variation VIII in the illustration CC of Fig. 74-144-201209158. Figure 100 is an enlarged view of the illustration CD shown in Figure 74. Figure 101 is an enlarged perspective view of the illustration CD shown in Figure 74. Figure 102 is an exploded view of the illustration CD shown in Figure 74. Fig. 103 is an enlarged view of the illustration CE shown in Fig. 78. Figure 104 is a graphical representation of the construction of the LOC device variant VIII. Figure 105 is a schematic diagram of the construction of the variable XIV of the LOC device. Fig. 106 is a schematic diagram showing the construction of the variable XV of the LOC device. Figure 107 is a schematic diagram of the construction of the LOC device variant XVIII. Figure 108 is a schematic diagram of the construction of the LOC device variant XXII. Figure 109 is a schematic diagram of the construction of the LOC device variant XXIII. Figure 10 is a schematic diagram of the construction of the LOC device variant XXIV. Figure 111 is a schematic diagram of the construction of the LOC device variant XXV. Figure 112 is a schematic diagram of the construction of the LOC device variant XXVI. Figure 113 is a schematic diagram of the construction of the LOC device variant XXVII. Figure 114 is a schematic diagram of the construction of the LOC device variant XXVIII. Figure 115 is a schematic diagram of the construction of the LOC device variant XLI. Figure 116 is a schematic diagram of the construction of the LOC device variant XLII. Figure 117 is a schematic diagram of the construction of the LOC device variant XLIII. Figure 118 is a schematic diagram of the construction of the LOC device variant XLIV. Figure 119 is a schematic diagram of the construction of the LOC device variant XLVII. Figure 20 is a graphical representation of the linear probe attached to the primer during the initial round of the amplification reaction. Figure 1 2 1 is an illustration of a linear probe with primers during the subsequent amplification cycle -145 - 201209158. Panels 122A through 122F illustrate the thermal cycling of a fluorescent dry-loop probe with primers attached thereto. Figure 123 is a schematic diagram of the excitation LED associated with the array of hybrid chambers and the photodiodes. Figure 124 is a schematic diagram of an excitation LED and an optical lens for directing light onto a hybrid chamber of the LOC device. Figure 125 is a schematic diagram of an excitation LED 'optical lens and optical aperture' for directing light onto a hybrid chamber of the LOC device. Figure 126 is a schematic diagram of the excitation LED, optical lens, and mirror configuration for directing light onto the hybrid chamber of the LOC device. Fig. 127 is a plan view 'The plan view shows all the features overlapping each other and the positions of the illustrations DA to DK are displayed. Figure 128 is an enlarged view of the illustration DG shown in Fig. 127. Fig. 129 is an enlarged view of the illustration DH shown in Fig. 127. Figure 130 is a graphical representation of the construction of the LOC device variant XI. Figure 131 is a specific embodiment of a shunt transistor for the photodiodes. Fig. 1 2 2 is a specific embodiment of a shunt transistor for the photodiodes. The first 133 diagram is a specific embodiment of the shunt transistor for the photodiodes. Figure 134 is a circuit diagram of a differential imager. Figure 135 shows a negative control fluorophore -146-201209158 probe in a dry-loop configuration. Figure 1 3 6 illustrates the negative control set of fluorescent probes in Figure 135 of the open configuration. Figure 1 3 7 illustrates the positive control camp light probe in a dry-loop configuration. Figure 1 3 8 illustrates the positive control set of fluorescent probes in Figure 137 of the open configuration. Figure 139 shows the test module and test module reader for ECL detection. Figure 1 40 is an overview of the electronic components in the inspection module for fluorescence detection. Figure 1 4 1 shows the inspection module and a variety of optional inspection module readers. Figure I42 shows the inspection module and inspection module reader of the host system with various databases. [Main component symbol description] 1 〇: inspection module 1 1 : inspection module 1 2 : inspection module reader 13 : housing 1 4 : micro USB plug 15 : inductor 16 : micro USB slot - 147- 201209158 1 7 : Touch screen 18 : Display screen 1 9 : Button 20 : Start button 2 1 : Honeycomb radio 22 : Tornable sealing tape 23 : Wireless network connector 24 : Large slot 25 : Satellite navigation system 26: Light-emitting diode (LED) 2 7 : Data storage 28: Mobile phone/Smartphone 29: LED driver 30: On-wafer laboratory (LOC) device 3 1 : Power conditioner 32: Capacitor 3 3: Clock 3 4 : Controller 3 5 : Recorder 36 : USB device driver 3 7 : Drive 38 : Random access memory (RAM ) 3 9 : Driver 40 : Program and data flashing billion body -148 - 201209158 4 1 : Record 42: Processor 43: Program Memory 44 _· Light Sensor/Photo Sensor Array 4 5: Indicator 46: Cover 47: Powered by USB and as a module 48: CMOS + MST Wafer

49 :泡綿狀插入物/多孔性元件 5 2 :雜合與偵測區段 5 4 :貯存槽 5 6 :貯存槽 5 7 :印刷電路板 5 8 :貯存槽 60 :貯存槽 60.1 :貯存槽 60.2 :貯存槽 60.3 :貯存槽 6 〇 . 4 :貯存槽 6 0.5 :貯存槽 6 0.6 :貯存槽 6 〇 . 7 :貯存槽 6 〇 . 8 :貯存槽 60.9 :貯存槽 -149- 201209158 60.10 :貯存槽 6 〇 . 1 1 :貯存槽 6 0 . 1 2 :貯存槽 6 0 . X :貯存槽 6 2 :貯存槽 6 2 . 1 :貯存槽 6 2.2 :貯存槽 6 2.3 :貯存槽 6 2.4 :貯存槽 6 2 . X :貯存槽 6 4 :下密封層 66 :頂層 6 8 :樣本入口 70 :透析區段/透析步驟 72 :廢液通道 74 :標靶通道 76 :廢料單元/廢料貯存槽 7 8 :貯存槽層 8 0 :蓋層通道層 8 2 :上密封層 8 4 :矽基板 (CMOS)電路 86 :互補金屬氧化物半導體 87 :微系統技術(MST)層 8 8 :鈍化層 -150- 201209158 90 :微系統技術(MST )通道 92 :下吸孔 94 :蓋層通道 96 :上吸孔 97 :隔牆區段 9 8 :彎液面錨 100 :微系統技術(MST )通道層 1 01 :手提式個人電腦/筆記型電腦 102 :毛細作用引動特徵(CIF ) 103 :專用讀取器 105 :桌上型電腦 106 :沸騰啓動式閥 107 :電子書讀取器 108 :沸騰啓動式閥/擴增出口閥 109 :平板電腦 1 1 〇 :雜合腔室陣列 1 1 〇. 1 :雜合腔室陣列 1 1 〇 . 2 :雜合腔室陣列 1 1 0.3 :雜合腔室陣列 I 10.4 :雜合腔室陣列 II 〇. 5 :雜合腔室陣列 1 10.6 :雜合腔室陣列 1 10.7 :雜合腔室陣列 1 1 〇 . 8 :雜合腔室陣列 -151 201209158 1 1 ο . 9 :雜合腔室陣列 1 1 0 . 1 0 :雜合腔室陣列 1 1 〇. 1 1 :雜合腔室陣列 1 1 0.1 2 :雜合腔室陣列 1 1 0 . X :雜合腔室陣列 1 1 1 :流行病學資料之主機系統 1 1 2 :擴增區段 1 1 2 . 1 :擴增區段 1 1 2.2 :擴增區段 1 1 2.3 :擴增區段 1 12.4 :擴增區段 1 1 2.5 :擴增區段 1 1 2.6 :擴增區段 1 1 2.7 :擴增區段 1 1 2.8 :擴增區段 1 1 2.9 :擴增區段 1 1 2.1 0 :擴增區段 1 1 2.1 1 :擴增區段 112.12:擴增區段 1 1 2 . X :擴增區段 1 1 3 :遺傳資料之主機系統 1 1 4 :培育區段 1 1 4 · 1 :培育區段 1 1 4.2 :培育區段 -152 - 201209158 1 1 4.3 :培育區段 1 1 4.4 :培育區段 1 1 5 :電子健康記錄(EHR )之主機系統 1 1 6 :抗凝血劑 1 1 8 :表面張力閥 1 1 9 :樣本流體 1 2 0 :彎液面 121 :電子醫療記錄(EMR)之主機系統 122 :通氣孔 123 :個人醫療記錄(PHR)之主機系統 125 :網路 126 :沸騰啓動式閥 1 2 8 :表面張力閥 1 3 0 :(化學)溶胞區段 1 3 1 :混合區段 1 3 2 :表面張力閥 1 3 3 :培育室入口通道 1 3 4 :下吸孔 136 :光學窗口 1 3 8 :擴增混合物表面張力閥 1 3 8 . 1 :擴增混合物表面張力閥 138.2 :擴增混合物表面張力閥 1 3 8.3 :擴增混合物表面張力閥 1 3 8.4 :擴增混合物表面張力閥 -153- 201209158 1 3 8 · X :擴增混合物表面張力閥 140:聚合酶表面張力閥 140.1 :聚合酶表面張力閥 M0.2:聚合酶表面張力閥 140.3 :聚合酶表面張力閥 140.4:聚合酶表面張力閥 140.X:聚合酶表面張力閥 1 4 6 :閥入口 1 4 8 :閥出口 1 5 0 :下吸孔 1 5 1 :閥上吸孔 1 5 2 :加熱器 1 5 3 :沸騰啓動式閥加熱器接觸點 1 5 4 :加熱器 1 5 6 :加熱器接觸點 158 :微通道 160 :擴增區段離開通道 164 :孔 165 :孔 166 :毛細作用引動特徵(CIF ) 1 6 8 :透析上吸孔 1 7 0 :溫度感測器 174 :液體感測器 175 :擴散阻障器 -154- 201209158 1 7 6 :流動路徑 178 :終點液體感測器 180 :雜合腔室 1 8 2 :加熱器 184 :光二極體 18 5 ·主動區 186 :螢光共振能量轉移(FRET)探針 187:觸發式光二極體 1 8 8 :水貯存槽 190 :蒸發器 1 9 1 :環形加熱器 192 :水供應通道 1 9 3 :上吸孔 194 :下吸孔 1 9 5 :頂部金屬層之暴露區域 1 9 6 :增濕器 197 :蓋層通道液體感測器 1 9 8 :第一上吸孔 202 :毛細作用引動特徵(CIF ) 204 :透析MST通道 2 06 :混合區段出口閥/沸騰啓動式閥 2 07 :培育室出口閥 2 0 8 :混合區段出口閥 2 1 0 :經加熱之微通道 -155- 201209158 2 1 2 :中間MST通道 2 1 8 :欽銘電極 2 2 0 :欽銘電極 222 :間隙 23 2 :濕度感測器 2 3 4 :加熱器 23 6 :螢光共振能量轉移探針(FRET)探針49: foam insert/porous element 5 2 : hybrid and detection section 5 4 : storage tank 5 6 : storage tank 5 7 : printed circuit board 5 8 : storage tank 60 : storage tank 60.1 : storage tank 60.2 : Storage tank 60.3 : Storage tank 6 〇. 4 : Storage tank 6 0.5 : Storage tank 6 0.6 : Storage tank 6 〇. 7 : Storage tank 6 〇. 8 : Storage tank 60.9 : Storage tank -149- 201209158 60.10 : Storage Tank 6 〇. 1 1 : Storage tank 6 0 . 1 2 : Storage tank 6 0 . X : Storage tank 6 2 : Storage tank 6 2 . 1 : Storage tank 6 2.2 : Storage tank 6 2.3 : Storage tank 6 2.4 : Storage Slot 6 2 . X : Storage tank 6 4 : Lower sealing layer 66 : Top layer 6 8 : Sample inlet 70 : Dialysis section / Dialysis step 72 : Waste liquid channel 74 : Target channel 76 : Waste unit / waste storage tank 7 8 : Storage tank layer 80: Cover channel layer 8 2: Upper sealing layer 8 4: 矽 substrate (CMOS) circuit 86: Complementary metal oxide semiconductor 87: Microsystem technology (MST) layer 8 8 : Passivation layer - 150- 201209158 90 : Microsystem Technology (MST) Channel 92: Lower Suction Hole 94: Cover Channel 96: Upper Suction Hole 97: Partition Wall Section 9 8: Meniscus Anchor 100: Microsystem Technology (MST) Channel Layer 1 01 : Portable Human Computer/Notebook 102: Capillary Actuation Feature (CIF) 103: Dedicated Reader 105: Desktop Computer 106: Boiling Start Valve 107: E-book Reader 108: Boiling Start Valve/Amplification Exit Valve 109: Tablet PC 1 1 〇: Hybrid Chamber Array 1 1 〇. 1 : Hybrid Chamber Array 1 1 〇. 2 : Hybrid Chamber Array 1 1 0.3 : Hybrid Chamber Array I 10.4 : Hybrid Chamber Array II 〇. 5: Hybrid Chamber Array 1 10.6: Hybrid Chamber Array 1 10.7: Hybrid Chamber Array 1 1 〇. 8: Hybrid Chamber Array - 151 201209158 1 1 ο . 9 : Miscellaneous Pool array 1 1 0 . 1 0 : Hybrid chamber array 1 1 〇. 1 1 : Hybrid chamber array 1 1 0.1 2 : Hybrid chamber array 1 1 0 . X : Hybrid chamber array 1 1 1 : host system of epidemiological data 1 1 2 : amplification section 1 1 2 2 : amplification section 1 1 2.2 : amplification section 1 1 2.3 : amplification section 1 12.4 : amplification zone Segment 1 1 2.5: Amplified segment 1 1 2.6 : Amplified segment 1 1 2.7 : Amplified segment 1 1 2.8 : Amplified segment 1 1 2.9 : Amplified segment 1 1 2.1 0 : Amplified segment 1 1 2.1 1 : Amplification section 112.12: Amplification section 1 1 2 . X : Amplification section 1 1 3 : host system of genetic data 1 1 4 : incubation section 1 1 4 · 1 : cultivation section 1 1 4.2 : cultivation section -152 - 201209158 1 1 4.3 : cultivation section 1 1 4.4 : cultivation section 1 1 5 : Host system of electronic health record (EHR) 1 1 6 : Anticoagulant 1 1 8 : Surface tension valve 1 1 9 : Sample fluid 1 2 0 : Meniscus 121 : Host of electronic medical record (EMR) System 122: Vent 123: Personal Medical Record (PHR) Host System 125: Network 126: Boiling Start Valve 1 2 8 : Surface Tension Valve 1 3 0 : (Chemical) Cell Segment 1 3 1 : Mixing Zone Section 1 3 2 : Surface tension valve 1 3 3 : Incubator inlet channel 1 3 4 : Lower suction hole 136: Optical window 1 3 8 : Amplification mixture surface tension valve 1 3 8 . 1 : Amplification mixture surface tension valve 138.2 : Amplification mixture surface tension valve 1 3 8.3 : Amplification mixture surface tension valve 1 3 8.4 : Amplification mixture surface tension valve -153- 201209158 1 3 8 · X : Amplification mixture surface tension valve 140: Polymerase surface tension valve 140.1: Polymerase Surface Tension Valve M0.2: Polymerase Surface Tension Valve 140.3: Polymerase Surface Tension Valve 140.4: Polymerase Surface Sheet Valve 140.X: Polymerase surface tension valve 1 4 6 : Valve inlet 1 4 8 : Valve outlet 1 5 0 : Lower suction hole 1 5 1 : Valve upper suction hole 1 5 2 : Heater 1 5 3 : Boiling start type Valve heater contact point 1 5 4 : Heater 1 5 6 : Heater contact point 158 : Microchannel 160 : Amplification section leaves channel 164 : Hole 165 : Hole 166 : Capillary action priming feature (CIF ) 1 6 8 : Dialysis upper suction hole 170: temperature sensor 174: liquid sensor 175: diffusion barrier -154 - 201209158 1 7 6 : flow path 178: end point liquid sensor 180: hybrid chamber 1 8 2 : heater 184 : photodiode 18 5 · active area 186 : fluorescent resonance energy transfer (FRET) probe 187 : triggered photodiode 1 8 8 : water storage tank 190 : evaporator 1 9 1 : ring heater 192: water supply channel 1 9 3 : upper suction hole 194 : lower suction hole 1 9 5 : exposed area of the top metal layer 1 9 6 : humidifier 197 : cover channel liquid sensor 1 9 8 : first on Suction hole 202: capillary action priming feature (CIF) 204: dialysis MST channel 2 06: mixing section outlet valve/boiling start valve 2 07: incubation chamber outlet valve 2 0 8 : mixing section outlet valve 2 1 0 : plus Microchannel-155- 201209158 2 1 2 : Intermediate MST channel 2 1 8 : Chin Ming electrode 2 2 0 : Chin Ming electrode 222 : Gap 23 2 : Humidity sensor 2 3 4 : Heater 23 6 : Fluorescence resonance Energy Transfer Probe (FRET) Probe

2 3 8 :標靶核酸序列/標靶DNA 240 :環部 242 :主幹 244 :激發光 246 :螢光發光基團 248 :消光基團 25 0 :釋放螢光/螢光信號 2 5 2 :光學中心 2 5 4 :透鏡 2 8 8 :樣本之置入與製備階段 290 :萃取階段(萃取區段) 291 :培育階段/培育步驟 292 :擴增階段/擴增步驟/擴增區段 293 :預雜合階段 294 :偵測階段/偵測區段 296 :第一電極 2 9 8 :第—電極 -156- 201209158 3 00 :預設延遲時間 301 :晶片上實驗室(LOC)裝置 3 2 8 :透析區段 3 7 6 :導電柱 3 78 :對照探針 3 80 :對照探針 3 82 :校準腔室 3 84 :分流電晶體(Mshunt)閘極 3 86 :電晶體Mtx閘極 3 8 8 :重設閘極 3 90 :彈壓伸縮式刺血針 3 92 :刺血針釋放按鈕 3 93 :讀取閘極 394:金屬氧化物電晶體,Mshunt 396:金屬氧化物電晶體,Mtx 3 98 :金屬氧化物電晶體,Mreset 400 :源極追隨電晶體,Msf 402:金屬氧化物電晶體,Mread 404 :金屬氧化物電晶體,Mbias 4 0 6 :節點 408 :密封膜 410 :薄膜護片2 3 8 : Target nucleic acid sequence / target DNA 240 : Ring portion 242 : stem 244 : excitation light 246 : fluorescent luminescent group 248 : extinction group 25 0 : release fluorescent / fluorescent signal 2 5 2 : optical Center 2 5 4 : Lens 2 8 8 : Sample placement and preparation stage 290 : Extraction stage (extraction stage) 291 : Cultivation stage / incubation step 292 : Amplification stage / amplification step / amplification section 293 : Pre Hybridization phase 294: detection phase/detection section 296: first electrode 2 9 8 : first electrode - 156 - 201209158 3 00: preset delay time 301: on-wafer laboratory (LOC) device 3 2 8 : Dialysis section 3 7 6 : Conductive column 3 78 : Control probe 3 80 : Control probe 3 82 : Calibration chamber 3 84 : Shunt transistor (Mshunt) gate 3 86 : Transistor Mtx gate 3 8 8 : Reset gate 3 90: spring-loaded telescopic needle 3 92 : lancet release button 3 93 : read gate 394: metal oxide transistor, Mshunt 396: metal oxide transistor, Mtx 3 98: metal Oxide transistor, Mreset 400: source follower transistor, Msf 402: metal oxide transistor, Mread 404: metal oxide transistor, Mbias 4 0 6 : node 408: dense Film 410: protective film sheet

492: LOC裝置變化型VII 5 1 8 : L Ο C裝置變化型V 111 -157- 201209158 594 :界面層 5 96 :界面通道 5 9 8 :界面通道 600 :旁通渠道 602 :界面標靶物通道 604 :界面廢液通道 606 :溶胞試劑貯存槽側之界面通道 608 :溶胞試劑貯存槽側之界面通道 610 :界面導管 6 1 2 :混合區段出口下吸孔 614 :閥界面通道 6 1 6 :閥界面凹槽 6 1 8 :擴增界面導管 619 :擴增界面導管 620 :擴增界面導管 621 :擴增界面導管 622 :擴增界面導管 623 :擴增界面導管 624 :擴增界面導管 62 5 :擴增界面導管 626 :擴增界面導管 627 :擴增界面導管 62 8 :擴增界面導管 629 :擴增界面導管 -158- 201209158492: LOC device variant VII 5 1 8 : L Ο C device variant V 111 -157- 201209158 594 : interface layer 5 96 : interface channel 5 9 8 : interface channel 600 : bypass channel 602 : interface target channel 604: interface waste liquid channel 606: interface channel 608 on the side of the lysis reagent storage tank: interface channel 610 on the side of the lysis reagent storage tank: interface conduit 6 1 2: mixing section outlet lower suction hole 614: valve interface channel 6 1 6: valve interface groove 6 1 8 : amplification interface conduit 619 : amplification interface conduit 620 : amplification interface conduit 621 : amplification interface conduit 622 : amplification interface conduit 623 : amplification interface conduit 624 : amplification interface conduit 62 5 : Amplification interface catheter 626 : amplification interface catheter 627 : amplification interface catheter 62 8 : amplification interface catheter 629 : amplification interface catheter -158 - 201209158

630: MST 培育出口通道 63 2 :擴增注入通道 634 : LED晶片載體表面 63 6 :通氣通道 6 3 8 :熱溶胞腔室 641 : LOC裝置變化型XIV 650: LOC裝置變化型XXIII 651 : LOC裝置變化型XXIV 652: LOC裝置變化型XXV 65 3 : LOC裝置變化型XXVI 654: LOC裝置變化型XXVII 655: LOC裝置變化型XX VIII630: MST incubation outlet channel 63 2 : Amplification injection channel 634 : LED wafer carrier surface 63 6 : Ventilation channel 6 3 8 : Thermal lysis chamber 641 : LOC device variant XIV 650 : LOC device variant XXIII 651 : LOC Device variant XXIV 652: LOC device variant XXV 65 3 : LOC device variant XXVI 654: LOC device variant XXVII 655: LOC device variant XX VIII

673 : LOC裝置變化型XLIII673 : LOC device variant XLIII

674 : LOC裝置變化型XLIV 677: LOC裝置變化型XLVII 6 82 :透析區段/透析步驟/雜合前透析步驟 686 :擴增前透析步驟 692 :接有引子之線性探針 694 :擴增阻斷子 696 :接有引子之探針序列 69 8 :經擴增之互補序列 700 :寡聚核苷酸引子 7 04 :接有引子之幹-環狀探針/探針區域 706 :互補序列 -159- 201209158 708 :主幹鏈 710 :主幹鏈 712 :第一光學稜鏡 714:第二光學稜鏡 7 1 6 :第一反射鏡 7 1 8 :第一反射鏡674: LOC device variant XLIV 677: LOC device variant XLVII 6 82: dialysis section / dialysis step / hybrid pre-dialysis step 686: pre-amplification dialysis step 692: linear probe 694 with primer: amplification resistance Breaker 696: Probe sequence with primers 69 8 : Amplified complementary sequence 700: Oligonucleotide primer 7 04: Dry-loop probe/probe region 706 with complementary primers: Complementary sequence - 159- 201209158 708: trunk chain 710: trunk chain 712: first optical 稜鏡 714: second optical 稜鏡 7 1 6 : first mirror 7 1 8 : first mirror

728: LOC裝置變化型X728: LOC device variant X

746: LOC裝置變化型XI 766 :廢料貯存槽 76 8 :廢料貯存槽 7 8 2 :配置 788:微分成像電路 7 9 0 :像素 792 :假像素 794 :讀行線 7 9 5 :假像素讀行線 796 :陰性對照探針 797 : M4電晶體 798 :陽性對照探針 8 0 1 : M D 4電晶體 8 〇 3 :像素電容器 8 0 5 :假像素電容器 8 0 7 :切換器 80 9 :切換器 -160- 201209158 8 1 1 :讀列線切換器 8 1 3 :假像素讀列線切換器 815 :電容器放大器 8 1 7 :微分信號 860 :電極 870 :電極746: LOC device variant XI 766: waste storage tank 76 8 : waste storage tank 7 8 2 : configuration 788: differential imaging circuit 7 9 0 : pixel 792 : dummy pixel 794 : read line 7 9 5 : dummy pixel read line Line 796: Negative control probe 797: M4 transistor 798: Positive control probe 8 0 1 : MD 4 transistor 8 〇 3 : Pixel capacitor 8 0 5 : Fake pixel capacitor 8 0 7 : Switcher 80 9 : Switcher -160- 201209158 8 1 1 : Read line switch 8 1 3 : Fake pixel read line switch 815 : Capacitor amplifier 8 1 7 : Differential signal 860 : Electrode 870 : Electrode

-161 --161 -

Claims (1)

201209158 七、申請專利範園: 1.—種用於生物樣本之基因分析之晶片上實驗室( LOC)裝置,該LOC裝置包含: 用於接收該樣本之入口; 載體基板; 透析區段,該透析區段係使該樣本中大於預定臨界値 之細胞與較小成分分離,其中該等大於預定臨界値之細胞 包括含有用於分析之遺傳物質的標靶細胞; 複數個試劑貯存槽; 溶胞區段,該溶胞區段係位於該透析區段下游以用於 溶解該等標靶細胞以釋出該等標靶細胞內的遺傳物質,該 溶胞區段係與該等含有用於溶解該溶胞區段中之該等標靶 細胞之溶胞試劑的試劑貯存槽之一者流體連通; 第一核酸擴增區段,該第一核酸擴增區段係位於該溶 胞區段下游以用於擴增該遺傳物質內的第一核酸序列;及 第二核酸擴增區段,該第二核酸擴增區段係位於該第 一核酸擴增區段下游以用於擴增源自該第一核酸擴增區段 之擴增子內的第二核酸序列:其中, 該透析區段、該溶胞區段、該第一核酸擴增區段及該 第二核酸擴增區段皆承載於該載體基板上。 2. 如申請專利範圍第1項之LOC裝置,其中該第一核酸 擴增區段係第一聚合酶鏈鎖反應(PCR)區段,且該二核 酸擴增區段係第二PCR區段。 3. 如申請專利範圍第2項之LOC裝置,其中該第一 PCR -162- 201209158 區段具有第一組引子對且該第一組引子對係用於黏合第一 組互補核酸序列,且該第二PCR區段具有第二組引子對且 該第二組引子對係用於黏合第二組互補核酸序列,該第一 組互補核酸序列與該第二組互補核酸序列不同。 4. 如申請專利範圍第3項之LOC裝置,其中該第一 PCR 區段和該第二PCR區段係經建構而以不同之擴增參數操作 ,該等擴增參數係下列之至少一者: 反轉錄酶種類; 聚合酶種類; 去氧核糖核苷三磷酸濃度; 緩衝溶液; 熱循環時間; 熱循環重複次數;及 於PC R之一特定階段期間內的溫度。 5. 如申請專利範圍第4項之LOC裝置,該LOC裝置進一 步包含雜合區段,該雜合區段係位於該第二PCR區段下游 ,且該雜合區段具有探針陣列及光感測器,該探針陣列係 用於與標靶核酸序列雜合且該光感測器係用於偵測該陣列 中之任何探針的雜合反應。 6. 如申請專利範圍第3項之LOC裝置,其中該透析區段 具有第~通道、第二通道及複數個孔,該第一通道係與位 於上游末端之該入口流體連通,該第二通道係與位於下游 端之廢液通道流體連通,該等孔小於該等標靶細胞且大於 該等較小成分,該第二通道係藉由該等孔與該第一通道流 -163- 201209158 體連通,以使該等標靶細胞留在該第一通道內且同時該等 較小成分流入該第二通道。 7.如申請專利範圍第6項之LOC裝置,其中該第一通道 和該第二通道係經建構以藉由毛細作用注滿該樣本。 8 ·如申請專利範圍第1項之L Ο C裝置,其中該溶胞區段 具有主動閥’該主動閥係於溶胞期間使該等標靶細胞停留 於該溶胞區段內,使得當打開該主動閥時,流向培育區段 的毛細驅動流得以繼續。 9. 如申請專利範圍第1項之LOC裝置,其中該第一核酸 擴增區段係第一恆溫核酸擴增區段,且該第二核酸擴增區 段係第二恆溫核酸擴增區段。 10. 如申請專利範圍第1項之LOC裝置,其中該等試劑 貯存槽各自具有用於使試劑留在該等試劑貯存槽內的表面 張力閥,該表面張力閥具有彎液面錨,該彎液面錨係用於 定住該試劑之彎液面直到該試劑之彎液面與該樣本流接觸 而去除該彎液面以允許該試劑從該試劑貯存槽流出》 1 1.如申請專利範圍第4項之LOC裝置,該LOC裝置進 一步包含CMOS電路、溫度感測器及微系統技術(MST ) 層,該MST層包含該第一PCR區段和該第二PCR區段,其 中該CMOS電路係設置於該載體基板與該MST層之間,該 CMOS電路係經建構而以使用該溫度感測器之輸出以達成 該第一PCR區段和該第二PCR區段的反饋控制。 12.如申請專利範圍第1 1項之LOC裝置,其中該第一 PCR區段具有PCR微通道,該PCR微通道係用於使該樣本 -164- 201209158 進行熱循環’該PCR微通道係界定具有低於100000平方微 米之流動橫斷截面積的流動路徑。 13. 如申請專利範圍第12項之LOC裝置,其中該PCR微 通道具有至少一個長形加熱器元件’該長形加熱器元件係 與該PCR微通道呈平行延伸。 14. 如申請專利範圍第13項之LOC裝置,其中該PCR區 段具有複數個長形PCR腔室,且每個該長形PCR腔室係由 該PCR微通道之個別區段形成,該PCR微通道具有由一系 列寬曲流道所形成之蜿蜒構形,且每個該寬曲流道係形成 該等長形PCR腔室之一者的通道區段。 15. 如申請專利範圍第14項之LOC裝置,該LOC裝置進 一步包含: 容納用於PCR之試劑的試劑貯存槽;及 具有孔之表面張力閥,該具有孔之表面張力閥係經建 構以定住該試劑之彎液面,如此該彎液面使該試劑保留在 該試劑貯存槽內直到該彎液面與該流體樣本接觸而去除該 彎液面且該試劑流出該試劑貯存槽。 16. 如申請專利範圍第14項之LOC裝置,該LOC裝置進 一步包含用於容納該等探針的雜合腔室陣列,使得每個雜 合腔室內的該等探針係經配置以與該等標靶核酸序列之一 者雜合。 1 7 ·如申請專利範圍第1 6項之L Ο C裝置,其中該光感測 器係配準該等雜合腔室設置而成的光二極體陣列。 18.如申請專利範圍第16項之LOC裝置,其中該CMOS -165- 201209158 電路具有數位記憶體及數據界面,該數位記憶體係用於儲 存源自該光感測器輸出的雜合數據,且該數據界面係傳輸 該雜合數據至外部裝置。 19. 如申請專利範圍第16項之LOC裝置,其中該第一 PCR區段具有主動閥’該主動閥係於熱循環期間使液體保 留在該第一PCR區段內且回應源自該CMOS電路之啓動信 號而允許液體流至該第一雜合腔室陣列。 20. 如申請專利範圍第19項之LOC裝置,其中該主動閥 係沸騰啓動式閥,該沸騰啓動式閥具有彎液面錨及加熱器 ,該彎液面錨係經建構以定住該阻止該液體之毛細驅動流 的彎液面,且該加熱器係用於使該液體沸騰以使該彎液面 脫離該彎液面錨,使得毛細驅動流動得以繼續。 -166 -201209158 VII. Application for Patent Park: 1. A wafer-on-lab (LOC) device for genetic analysis of biological samples, the LOC device comprising: an inlet for receiving the sample; a carrier substrate; a dialysis section, The dialysis section separates cells in the sample larger than the predetermined threshold 与 from the smaller components, wherein the cells larger than the predetermined threshold 包括 include target cells containing the genetic material for analysis; a plurality of reagent storage tanks; lysis a segment, the lysis segment being located downstream of the dialysis section for dissolving the target cells to release genetic material within the target cells, the lysing segments being associated with the cells for dissolution One of the reagent storage tanks of the lysis reagent of the target cells in the lysing section is in fluid communication; the first nucleic acid amplification section, the first nucleic acid amplification section is located downstream of the lysis section For amplifying a first nucleic acid sequence within the genetic material; and a second nucleic acid amplification segment downstream of the first nucleic acid amplification segment for amplification derived The first nucleic acid amplification a second nucleic acid sequence in the amplicon of the segment: wherein the dialysis section, the lysis section, the first nucleic acid amplification section, and the second nucleic acid amplification section are all carried on the carrier substrate . 2. The LOC device of claim 1, wherein the first nucleic acid amplification segment is a first polymerase chain reaction (PCR) segment and the second nucleic acid amplification segment is a second PCR segment . 3. The LOC device of claim 2, wherein the first PCR-162-201209158 segment has a first set of primer pairs and the first set of primer pairs is used to bind the first set of complementary nucleic acid sequences, and the The second PCR segment has a second set of primer pairs and the second set of primer pairs is used to bind a second set of complementary nucleic acid sequences that differ from the second set of complementary nucleic acid sequences. 4. The LOC device of claim 3, wherein the first PCR segment and the second PCR segment are constructed to operate with different amplification parameters, the amplification parameters being at least one of : reverse transcriptase species; polymerase species; deoxyribonucleoside triphosphate concentration; buffer solution; thermal cycle time; number of thermal cycle repeats; and temperature during a particular phase of PC R. 5. The LOC device of claim 4, further comprising a hybrid segment located downstream of the second PCR segment, the hybrid segment having a probe array and light A sensor, the probe array is for hybridization with a target nucleic acid sequence and the photosensor is for detecting a heterozygous reaction of any of the probes in the array. 6. The LOC device of claim 3, wherein the dialysis section has a first channel, a second channel, and a plurality of holes, the first channel being in fluid communication with the inlet at the upstream end, the second channel And being in fluid communication with the waste liquid channel at the downstream end, wherein the holes are smaller than the target cells and larger than the smaller components, and the second channel is connected to the first channel by the holes -163-201209158 Connected to allow the target cells to remain in the first channel while the smaller components flow into the second channel. 7. The LOC device of claim 6, wherein the first channel and the second channel are configured to fill the sample by capillary action. 8. The apparatus of claim 1, wherein the lysis section has an active valve that causes the target cells to remain in the lysis section during lysis, such that When the active valve is opened, the capillary drive flow to the incubation section continues. 9. The LOC device of claim 1, wherein the first nucleic acid amplification segment is a first thermostatic nucleic acid amplification segment and the second nucleic acid amplification segment is a second thermostatic nucleic acid amplification segment . 10. The LOC device of claim 1, wherein the reagent storage tanks each have a surface tension valve for retaining reagents in the reagent storage tanks, the surface tension valve having a meniscus anchor, the bend The liquid level anchor is used to hold the meniscus of the reagent until the meniscus of the reagent contacts the sample stream to remove the meniscus to allow the reagent to flow out of the reagent reservoir. 1 1. As claimed in the patent application a LOC device of 4, the LOC device further comprising a CMOS circuit, a temperature sensor and a microsystem technology (MST) layer, the MST layer comprising the first PCR segment and the second PCR segment, wherein the CMOS circuit system And disposed between the carrier substrate and the MST layer, the CMOS circuit is configured to use the output of the temperature sensor to achieve feedback control of the first PCR segment and the second PCR segment. 12. The LOC device of claim 11, wherein the first PCR segment has a PCR microchannel for thermal cycling of the sample -164 - 201209158 'the PCR microchannel system A flow path having a cross-sectional cross-sectional area of less than 100,000 square microns. 13. The LOC device of claim 12, wherein the PCR microchannel has at least one elongated heater element' that extends parallel to the PCR microchannel. 14. The LOC device of claim 13, wherein the PCR segment has a plurality of elongate PCR chambers, and each of the elongate PCR chambers is formed by an individual segment of the PCR microchannel, the PCR The microchannel has a crucible configuration formed by a series of wide curved channels, and each of the wide curved channels forms a channel section of one of the elongate PCR chambers. 15. The LOC device of claim 14, wherein the LOC device further comprises: a reagent storage tank containing a reagent for PCR; and a surface tension valve having a hole, the surface tension valve having the hole being constructed to be fixed The meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until the meniscus contacts the fluid sample to remove the meniscus and the reagent exits the reagent reservoir. 16. The LOC device of claim 14, wherein the LOC device further comprises an array of hybrid chambers for receiving the probes such that the probes within each hybrid chamber are configured to One of the target nucleic acid sequences is heterozygous. 1 7 · The L Ο C device of claim 16 of the patent application, wherein the photo sensor is associated with an array of photodiodes arranged by the hybrid chambers. 18. The LOC device of claim 16, wherein the CMOS-165-201209158 circuit has a digital memory and a data interface for storing hybrid data originating from the photosensor output, and The data interface transmits the hybrid data to an external device. 19. The LOC device of claim 16, wherein the first PCR segment has an active valve that retains liquid within the first PCR segment during thermal cycling and is responsive to the CMOS circuit The activation signal allows liquid to flow to the first hybrid chamber array. 20. The LOC device of claim 19, wherein the active valve is a boiling start valve having a meniscus anchor and a heater, the meniscus anchor being constructed to hold the block The capillary of the liquid drives the meniscus of the flow, and the heater is used to boil the liquid to disengage the meniscus from the meniscus anchor such that capillary drive flow continues. -166 -
TW100119228A 2010-06-17 2011-06-01 LOC device for genetic analysis with dialysis, chemical lysis and tandem nucleic acid amplification TW201209158A (en)

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TW100119249A TW201211534A (en) 2010-06-17 2011-06-01 Microfluidic device with PCR section and diffusion mixer
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
TW100119250A TW201211244A (en) 2010-06-17 2011-06-01 Test module with diffusive mixing in small cross sectional area microchannel
TW100119253A TW201219776A (en) 2010-06-17 2011-06-01 Microfluidic device with conductivity sensor
TW100119227A TW201211538A (en) 2010-06-17 2011-06-01 LOC device for pathogen detection with dialysis, chemical lysis and tandem nucleic acid amplification
TW100119243A TW201211242A (en) 2010-06-17 2011-06-01 Microfluidic device for genetic and mitochondrial analysis of a biological sample
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
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
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
TW100119252A TW201219115A (en) 2010-06-17 2011-06-01 Microfluidic test module with flexible membrane for internal microenvironment pressure-relief
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
TW100119234A TW201211540A (en) 2010-06-17 2011-06-01 LOC device for pathogen detection and genetic analysis with dialysis and nucleic acid amplification
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
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
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
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
TW100119241A TW201211533A (en) 2010-06-17 2011-06-01 Microfluidic device for simultaneous detection of multiple conditions in a patient
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
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TW100119249A TW201211534A (en) 2010-06-17 2011-06-01 Microfluidic device with PCR section and diffusion mixer
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
TW100119250A TW201211244A (en) 2010-06-17 2011-06-01 Test module with diffusive mixing in small cross sectional area microchannel
TW100119253A TW201219776A (en) 2010-06-17 2011-06-01 Microfluidic device with conductivity sensor
TW100119227A TW201211538A (en) 2010-06-17 2011-06-01 LOC device for pathogen detection with dialysis, chemical lysis and tandem nucleic acid amplification
TW100119243A TW201211242A (en) 2010-06-17 2011-06-01 Microfluidic device for genetic and mitochondrial analysis of a biological sample
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
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
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
TW100119252A TW201219115A (en) 2010-06-17 2011-06-01 Microfluidic test module with flexible membrane for internal microenvironment pressure-relief
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
TW100119234A TW201211540A (en) 2010-06-17 2011-06-01 LOC device for pathogen detection and genetic analysis with dialysis and nucleic acid amplification
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
TW100119223A TW201219770A (en) 2010-06-17 2011-06-01 Test module incorporating spectrometer

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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
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
TW100119241A TW201211533A (en) 2010-06-17 2011-06-01 Microfluidic device for simultaneous detection of multiple conditions in a patient
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
TW100119238A TW201211532A (en) 2010-06-17 2011-06-01 LOC device with parallel incubation and parallel DNA and RNA amplification functionality

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