TWI684644B

TWI684644B - Integrated microfluidic systems, biochips and methods for the detection of nucleic acids and biological molecules by electrophoresis

Info

Publication number: TWI684644B
Application number: TW107118311A
Authority: TW
Inventors: 坦尤金; 藍興權; 李奧尼杜烕克包達諾夫維里瑞; 約翰凱洛格葛列格里; A 萊特約翰; 索曼歐里奇漢斯; F 塞登理查
Original assignee: 美商網路生物有限公司
Priority date: 2007-08-13
Filing date: 2008-06-10
Publication date: 2020-02-11
Also published as: TW200909794A; TW200911987A; TWI539001B; TW201530118A; TW201829784A; TWI393777B; TWI470220B; TW201350844A; TW201341783A; TWI409454B; TWI486572B; TW201311902A; TWI647439B; TWI504750B; TW200918888A; TW201527538A

Abstract

The present disclosure provides fully integrated microfluidic systems to perform nucleic acid analysis. These processes include sample collection, nucleic acid extraction and purification, amplification, sequencing, and separation and detection. The present disclosure also provides optical detection systems and methods for separation and detection of biological molecules. In particular, the various aspects of the invention enable the simultaneous separation and detection of a plurality of biological molecules, typically fluorescent dye-labeled nucleic acids, within one or a plurality of microfluidic chambers or channels. The nucleic acids can be labeled with at least 6 dyes, each having a unique peak emission wavelength. The present systems and methods are particularly useful for DNA fragment sizing applications such as human identification by genetic fingerprinting and DNA sequencing applications such as clinical diagnostics.

Description

利用電泳偵測核酸及生物分子之整合微流體系統、生物晶片及方法Integrated microfluidic system, biochip and method for detecting nucleic acids and biomolecules using electrophoresis

本發明係關於用於核酸分析之微流體學領域。本申請案根據35 U.S.C. § 119(e)規定主張以下申請案之申請日期之權利：2007年4月4日申請之美國臨時申請案第60/921,802號；2007年8月13日申請之美國臨時申請案第60/964,502號；及2008年2月12日申請之美國臨時申請案第61/028,073號，該等申請案之各者之全文以引用的方式併入本文中。亦將以下兩個美國專利申請案之全文以引用之方式併入本申請案中，該等申請案在同一天申請：第一個名為"Methods for Rapid Multiplexed Amplification of Target Nucleic Acids"，代理人檔案號碼08-318-US；及第二個名為"Plastic Microfluidic separation and detection platforms"，代理人檔案號碼07-865-US。The present invention relates to the field of microfluidics for nucleic acid analysis. This application claims the rights of the following application date according to 35 USC § 119(e): US Provisional Application No. 60/921,802 filed on April 4, 2007; US Provisional Application filed on August 13, 2007 Application No. 60/964,502; and US Provisional Application No. 61/028,073 filed on February 12, 2008, the entire contents of each of these applications are incorporated herein by reference. The full texts of the following two US patent applications are also incorporated into this application by reference, and these applications are applied on the same day: the first is named "Methods for Rapid Multiplexed Amplification of Target Nucleic Acids", attorney File number 08-318-US; and the second one is called "Plastic Microfluidic separation and detection platforms", agent file number 07-865-US.

對能允許完全整合(亦即樣品進入至結果輸出)之集中核酸分析(其定義為對給定人類、動物、植物或病原體基因組之子集之快速識別(藉由核酸測序或片段大小測定))的儀器及技術之研發存在未滿足之需要。集中核酸測序將使最終使用者能作出即時臨床判定、法醫判定或其他判定。例如，許多常見人類疾病可基於DNA序列之少於1000個的鹼基對(小於產生完整人類基因組所需之數量級)加以診斷。類似地，藉由短串聯重複序列分析產生的對少於20個之特異性DNA片段之集合之大小的精確測定足以識別給定個體。視應用而定，可在各種配置下，包括醫院實驗室、醫師辦公室、臨床(bedside)，或在法醫學應用或環境應用之情況下，在實地進行集中核酸分析。對改良之DNA測序及片段大小測定系統存在若干未滿足之需要。首先對易於使用且不需要高度受訓操作員之DNA測序及片段大小測定用儀器存在未滿足之需要。其次，對可消除所有手動處理之系統存在未滿足之需要。因此，僅需要最低限度之操作員訓練且系統應易於由受限於諸如(例如)穿戴防護服(haz-mat suit)之第一反應者所將面臨之挑戰性環境的個體操作。第三，對於不會犧牲對完整、精確及可靠資料之需要的超快分析存在未滿足之需要。對於人類識別應用而言，產生結果之適當時間為45分鐘或小於45分鐘，比使用習知技術所需之數天至數週少很多。對於臨床應用而言，諸如對感染物測序以確定適當之治療療法，90分鐘或小於90分鐘為合理之應答時間，從而使得用抗細菌及抗病毒藥物治療可在患者抵達急診室後即刻開始。不管應用如何，對產生即時有用之資料存在未滿足之需要。較短之應答時間亦允許樣品處理量之伴隨增加。第四，對小型化存在未滿足之需要。許多DNA分析系統需要整個實驗室及相關支持。例如，高處理量Genome Sequencer FLX(Roche Diagnostics Corp, Indianapolis, IN)DNA測序系統就安裝而言僅需要工作台，但需要大型實驗室進行所需文庫構建。小型化對於實驗室與現場即時照護(point-of-care)之用以及實地操作均為重要的。其對於每個樣品之成本降低亦為重要的。第五，對堅固耐用存在未滿足之需要。對於許多應用，尤其彼等在法醫學、軍事及國防中之應用而言，DNA分析儀器必須可在實地操作。相應地，不論由士兵揹運、由警車運送還是由直升機空投到戰場上，該儀器都必須能夠被輸送。類似地，該儀器必須能夠耐受包括溫度、濕度及空浮粒子(例如砂粒)之極限環境且能夠在該等極限環境下運作。第六，對能接受多種樣品類型且能並行進行高度多重分析之系統存在未滿足之需要。對大多數應用而言，分析來自單重反應中單一樣品類型之DNA之能力對於進行有意義之DNA分析為不可接受的。尋求將複雜之實驗室操作系列濃縮於生物晶片上的微流體學(亦稱作微全分析系統(μTAS)或晶片實驗室(lab-on-a-chip)技術，參見Manz等人， Sens. Actuators B 1990, 1, 244-248)研發者已明確認識到此等未滿足之需要，但至今尚未能設計出能進行所有可使微流體核酸分析滿足此等需要所必需或理想之生物化學與物理過程的整合生物晶片及儀器。因此，集中核酸分析在當今社會尚未進入廣泛使用。微流體系統之研發包括將微型裝配組件(諸如微量分離、反應、微型閥及泵)及各種偵測機制整合於全功能裝置內(參見，例如Pal等人，Lab Chip 2005, 5, 1024-1032)。自Manz等人(上述)在上個世紀90年代早期展示晶片上之毛細管電泳現象以來，其他人已尋求將其改良。若干群組已展示DNA處理功能與生物晶片分離及偵測之整合。已報導呈玻璃-PDMS(聚二甲基矽氧烷)混合結構之整合裝置(Blazej等人，Proc Natl Acad Sci U S A 2006, 103, 7240-5；Easley等人，Proc. Natl. Acad. Sci. USA 2006, 103, 19272-7；及Liu等人，Anal. Chem. 2007, 79, 1881-9)。Liu藉由短串聯重複(STR)大小測定將多重聚合酶鏈反應(PCR)、分離及四染料偵測法結合起來用於人類識別。Blazej將桑格測序反應(Sanger sequencing reaction)、桑格反應淨化、電泳分離及四染料偵測法結合用於pUC18擴增子(amplicon)之DNA測序。Easley將DNA之固相萃取、PCR、電泳分離及單色偵測結合以識別血中細菌感染之存在。由Burns(Pal, 2005,Id .)展示結合PCR、電泳分離及單色偵測之整合矽玻璃裝置。Huang報導將PCR之玻璃-PDMS部分與電泳分離之聚(甲基丙烯酸甲酯)(PMMA)部分及單色偵測結合用於識別細菌DNA之存在的混合裝置(Huang等人，Electrophoresis 2006, 27, 3297-305)。 Koh等人報導使PCR與生物晶片電泳分離及單色偵測結合用於識別細菌DNA之存在的塑料裝置(Koh等人，Anal. Chem. 2003, 75, 4591-8)。Asogawa報導結合DNA萃取、PCR擴增、生物晶片電泳分離及單色偵測之矽基裝置(Asogowa M,Development of portable and rapid human DNA Analysis System Aiming on-site Screening, 18th International Symposium on Human Identification, Poster, 2007年10月1-4日，Hollywood, CA, USA)。美國專利第7,332,126號(Tooke等人)描述使用離心力來實現核酸分離及循環測序所需之微流體操作。然而，此方法係基於小樣品體積(約一至幾μL之彼等者)。因此，其裝置並不適用於尤其以高度並行之方式處理供分離及分析核酸用的大量樣品，此係因為必須在靜止時將流體樣品施加於該裝置，即盤必須能夠在離心前含有操作所需之所有流體(對於高度並行裝置而言可能達數百mL)。其次，該裝置受限於細菌純系之樣品製備及循環測序(例如質體DNA)。在嘗試將DNA處理與生物晶片電泳分離整合之彼等裝置中存在若干缺陷。首先，偵測受到每一檢定資訊含量(大多數使用單色偵測器，不過有些具有達四色之偵測系統)或處理量(單樣品或兩樣品處理能力)之限制。第二，此等裝置不體現完整之樣品-應答整合，例如Blazej之裝置在循環測序前需要模板DNA之板外(off-board)擴增，而其他者使用需要某種前處理之樣品(例如Easley及Tooke需要將樣品在添加之前溶解)。第三，針對此等裝置作出之某些處理選擇對時間-應答產生負面影響：例如Blazej之基於雜交之方法需要超過20分鐘來淨化循環測序產物。第四，許多此等裝置部分或完全由玻璃或矽製造。使用此等基板及相關製造技術使得其固有地較費成本(Gardeniers等人，Lab-on-a-Chip (Oosterbroeck RE, van den Berg A，編)，Elsevier: London，第37-64頁(2003))且使其受限於必須進行該等裝置之再使用之應用；對於許多應用(諸如人類ID)而言此導致樣品污染之風險。最後，所展示之技術對於兩種應用而言為不適合的，即經由STR分析及測序進行人類識別。例如，Easley及Pal裝置均遇到不良解析之問題，其比單一鹼基解析差得多。片段大小測定應用(例如，藉由短串聯重複概況分析進行之人類識別)及測序均需要單一鹼基解析。除就微流體整合而言的先前技術之侷限性以外，關於螢光偵測之問題亦限制核酸分析在習知實驗室研究以外之廣泛應用。最廣泛使用之市售測序套組(BigDye^TM v3.1 [Applied Biosystems]及DYEnamic^TM ET [GE Healthcare Biosciences Corp, Piscataway, NJ])係基於存在二十年之四色偵測方法(參見，例如美國專利第4,855,225號；第5,332,666號；第5,800,996號；第5,847,162號；第5,847,162號)。此方法係基於將染料標記之核苷酸之發射信號解析為四種不同顏色，每一種顏色表示四種鹼基中之各者。此等四色染料系統具有若干缺點，包括螢光染料之低效激發、顯著之光譜重疊及發射信號之低效收集。該等四色染料系統尤其有問題，此係因為其限制可自測序產物之給定電泳(或其他)分離獲得之資訊量。對於能夠以電泳系統基於DNA片段之分離及偵測藉由片段大小及藉由顏色(染料波長)獲得高資訊含量檢定的系統存在未滿足之需要。可藉由電泳辨別之DNA片段之最大數目係由裝置之分離及解析之讀取長度決定。可偵測之顏色之最大數目係部分決定於螢光染料之可用性及偵測系統之波長辨別力。儘管已報導達四色之偵測，但通常現有生物晶片偵測系統限於單色。用於人類識別之STR分析為基於顏色多重性測DNA片段大小之實例且允許同時分析達16個位點(AmpFlSTR Identifiler套組，Applied Biosystems, Foster City, CA；及PowerPlex16套組，Promega Corporation, Madison, WI)。使用四種或五種螢光染料，單一分離通道可辨別各位點之許多對偶基因變異體之大小。若干測片段大小應用將需要在單一道(lane)上分離及偵測超過16個片段。例如，藉由指紋識別病原體(亦即分離及偵測大量特有DNA片段)及藉由測量整個人類基因組診斷非整倍體可藉由分別著眼於數打或數百個位點來達成。增加可在單一分離通道中偵測之位點之數目的一種方法為部分藉由增加額外位點之片段大小使所產生之片段大小範圍變寬。然而，對於額外位點使用較長片段為非理想的，此係因為較大片段之擴增對抑制劑及DNA降解更敏感，從而導致較長片段相對於較短片段之產量低。此外，較長片段之產生亦需要擴展時間之增加且因此增加總檢定時間。對於藉由增加可同時偵測之染料顏色之數目來增加給定分離通道中可偵測之位點之數目存在未滿足之需要。對於藉由增加在單一分離通道中可分析之DNA序列之數目來增加桑格測序分離能力(且因此縮減該方法之成本、勞動力及空間)存在未滿足之需要。此外，在某些應用中，對多個DNA片段測序產生難以讀取之"混合序列"資料；需要開發一種可正確解釋混合序列之方法。增加桑格分離通道之能力且開發解釋混合序列之能力之一種方法為增加測序反應中使用之染料顏色之數目。在DNA測序及片段大小測定中，可同時偵測用不同染料標記之多個片段。一般而言，相鄰染料之峰值發射波長之間的分離相對於染料之峰寬而言必須足夠大。因此，各分離通道之處理量可(例如)藉由在兩個獨立測序反應中使用兩套4種染料且合併產物並在單一通道上將其分離而加倍。此方法需要使用總共8種染料顏色，其中第一測序反應使用一套適用於標記二脫氧核苷酸終止子之4種染料顏色，且第二反應使用另一套適用於標記該等終止子之4種染料顏色；各套染料顏色為獨立的以便在兩個序列之解釋中可能無重疊。使用此相同方法，可使用一套12種染料以允許在單一通道中同時分析三個DNA片段之序列，一套16種染料允許分析四個序列，等等，此顯著增加桑格分離之資訊量。本申請案之新穎儀器及生物晶片滿足許多未滿足之需要，包括以上所列之彼等者。For centralized nucleic acid analysis (which is defined as the rapid identification of a subset of the genome of a given human, animal, plant, or pathogen (by nucleic acid sequencing or fragment size determination)) that allows full integration (i.e. sample entry to result output) There is an unmet need for the development of instruments and technologies. Centralized nucleic acid sequencing will enable end users to make immediate clinical judgments, forensic judgments, or other judgments. For example, many common human diseases can be diagnosed based on DNA sequences of less than 1000 base pairs (less than an order of magnitude needed to produce a complete human genome). Similarly, the precise determination of the size of the collection of less than 20 specific DNA fragments generated by short tandem repeat analysis is sufficient to identify a given individual. Depending on the application, centralized nucleic acid analysis can be performed in the field under various configurations, including hospital laboratories, physician offices, clinical (bedside), or in the case of forensic or environmental applications. There are several unmet needs for the improved DNA sequencing and fragment size determination system. First, there is an unmet need for DNA sequencing and fragment size instruments that are easy to use and do not require highly trained operators. Second, there is an unmet need for systems that can eliminate all manual processing. Therefore, only minimal operator training is required and the system should be easy to operate by individuals who are limited to the challenging environment that will be faced by first responders such as, for example, wearing haz-mat suits. Third, there is an unmet need for ultrafast analysis that does not sacrifice the need for complete, accurate, and reliable data. For human recognition applications, the appropriate time to produce results is 45 minutes or less, which is much less than the days to weeks required to use conventional techniques. For clinical applications, such as sequencing infectious agents to determine the appropriate treatment, 90 minutes or less is a reasonable response time, so that treatment with antibacterial and antiviral drugs can begin immediately after the patient arrives in the emergency room. Regardless of the application, there is an unmet need to produce information that is immediately useful. The shorter response time also allows a concomitant increase in sample throughput. Fourth, there is an unmet need for miniaturization. Many DNA analysis systems require the entire laboratory and related support. For example, the high-throughput Genome Sequencer FLX (Roche Diagnostics Corp, Indianapolis, IN) DNA sequencing system requires only a bench for installation, but requires a large laboratory for the required library construction. Miniaturization is important for point-of-care and laboratory operations. It is also important for the cost reduction of each sample. Fifth, there is an unmet need for ruggedness. For many applications, especially their applications in forensics, military and defense, DNA analysis instruments must be operable in the field. Correspondingly, the instrument must be able to be transported whether carried by soldiers, transported by police cars, or dropped by helicopter onto the battlefield. Similarly, the instrument must be able to withstand and operate in extreme environments including temperature, humidity, and airborne particles (such as sand particles). Sixth, there is an unmet need for systems that can accept multiple sample types and can perform highly multiplexed analyses in parallel. For most applications, the ability to analyze DNA from a single sample type in a single reaction is unacceptable for meaningful DNA analysis. Seeking microfluidics (also called micro-total analysis system (μTAS) or wafer laboratory (lab-on-a-chip) technology that concentrates a series of complex laboratory operations on biological wafers, see Manz et al., Sens. Actuators B 1990, 1 , 244-248) The developers have clearly recognized these unmet needs, but have not yet been able to design all the necessary or ideal biochemistry that can make microfluidic nucleic acid analysis meet these needs. Integrate biochips and instruments with physical processes. Therefore, concentrated nucleic acid analysis has not yet been widely used in today's society. The development of microfluidic systems includes the integration of micro-assembled components (such as micro-separation, reactions, micro-valves and pumps) and various detection mechanisms into a fully functional device (see, for example, Pal et al., Lab Chip 2005, 5, 1024-1032 ). Since Manz et al. (above) demonstrated capillary electrophoresis on wafers in the early 1990s, others have sought to improve it. Several groups have demonstrated the integration of DNA processing functions with biochip separation and detection. An integrated device with a glass-PDMS (polydimethylsiloxane) hybrid structure has been reported (Blazej et al., Proc Natl Acad Sci USA 2006, 103, 7240-5; Easley et al., Proc. Natl. Acad. Sci. USA 2006, 103, 19272-7; and Liu et al., Anal. Chem. 2007, 79, 1881-9). Liu combines multiple polymerase chain reaction (PCR), separation, and four-dye detection methods for human recognition by short tandem repeat (STR) size determination. Blazej combines Sanger sequencing reaction, Sanger purification, electrophoretic separation and four-dye detection methods for DNA sequencing of pUC18 amplicons. Easley combined DNA solid phase extraction, PCR, electrophoretic separation, and monochrome detection to identify the presence of bacterial infections in the blood. An integrated silica glass device combining PCR, electrophoretic separation, and monochrome detection was demonstrated by Burns (Pal, 2005, Id .). Huang reported a hybrid device combining the glass-PDMS part of PCR with the poly(methyl methacrylate) (PMMA) part separated by electrophoresis and monochrome detection to identify the presence of bacterial DNA (Huang et al., Electrophoresis 2006, 27 , 3297-305). Koh et al. reported a plastic device that combines PCR with biochip electrophoretic separation and monochrome detection for identifying the presence of bacterial DNA (Koh et al., Anal. Chem. 2003, 75, 4591-8). Asogawa reports a silicon-based device that combines DNA extraction, PCR amplification, biochip electrophoretic separation, and monochrome detection (Asogowa M, Development of portable and rapid human DNA Analysis System Aiming on-site Screening, 18th International Symposium on Human Identification, Poster , October 1-4, 2007, Hollywood, CA, USA). US Patent No. 7,332,126 (Tooke et al.) describes the use of centrifugal force to achieve the microfluidic operations required for nucleic acid separation and cycle sequencing. However, this method is based on small sample volumes (approximately one to several μL of others). Therefore, its device is not suitable for processing a large number of samples for the separation and analysis of nucleic acids, especially in a highly parallel manner, because the fluid sample must be applied to the device at rest, that is, the disk must be able to contain the operation site before centrifugation All fluids required (for highly parallel devices may reach hundreds of mL). Second, the device is limited to sample preparation and cycle sequencing (eg plastid DNA) of pure bacterial lines. There are several drawbacks in their devices that attempt to integrate DNA processing with biochip electrophoretic separation. First, the detection is limited by the information content of each verification (mostly monochrome detectors are used, but some have a four-color detection system) or throughput (single sample or two sample processing capabilities). Second, these devices do not reflect complete sample-response integration. For example, Blazej's device requires off-board amplification of template DNA before cycle sequencing, while others use samples that require some pretreatment (eg. Easley and Tooke need to dissolve the sample before addition). Third, certain processing choices made for these devices have a negative impact on time-response: For example, Blazej's hybridization-based method requires more than 20 minutes to purify cycle sequencing products. Fourth, many of these devices are partially or completely made of glass or silicon. The use of these substrates and related manufacturing techniques makes them inherently costly (Gardeniers et al., Lab-on-a-Chip (Oosterbroeck RE, van den Berg A, ed.), Elsevier: London, pages 37-64 (2003 )) and limit it to applications that must be reused for these devices; for many applications (such as human ID) this leads to the risk of sample contamination. Finally, the technology shown is not suitable for two applications, namely human identification via STR analysis and sequencing. For example, both the Easley and Pal devices encountered problems with poor resolution, which was much worse than single base resolution. Both fragment size determination applications (eg, human identification by short tandem repeat profiling) and sequencing require single base resolution. In addition to the limitations of the prior art in terms of microfluidic integration, the problem with fluorescent detection also limits the wide application of nucleic acid analysis outside of research in the conventional laboratory. The most widely used commercially available sequencing kits (BigDye ^TM v3.1 [Applied Biosystems] and DYEnamic ^TM ET [GE Healthcare Biosciences Corp, Piscataway, NJ]) are based on a four-color detection method that has existed for twenty years (see, for example US Patent Nos. 4,855,225; 5,332,666; 5,800,996; 5,847,162; 5,847,162). This method is based on parsing the emission signal of dye-labeled nucleotides into four different colors, each color representing each of the four bases. These four-color dye systems have several disadvantages, including inefficient excitation of fluorescent dyes, significant spectral overlap, and inefficient collection of emitted signals. These four-color dye systems are particularly problematic because they limit the amount of information that can be obtained from a given electrophoresis (or other) separation of sequencing products. There is an unmet need for a system that can obtain a high information content assay by fragment size and color (dye wavelength) based on the separation and detection of DNA fragments by an electrophoresis system. The maximum number of DNA fragments that can be identified by electrophoresis is determined by the read length of the device for separation and analysis. The maximum number of colors that can be detected is determined in part by the availability of fluorescent dyes and the wavelength discrimination of the detection system. Although four-color detection has been reported, the existing biochip detection system is usually limited to single color. STR analysis for human identification is an example of measuring DNA fragment size based on color multiplicity and allows simultaneous analysis of up to 16 sites (AmpFlSTR Identifiler kit, Applied Biosystems, Foster City, CA; and PowerPlex16 kit, Promega Corporation, Madison , WI). Using four or five fluorescent dyes, a single separation channel can identify the size of many dual gene variants at each site. Several segment size measurement applications will require separation and detection of more than 16 segments on a single lane. For example, identifying pathogens by fingerprints (ie, isolating and detecting a large number of unique DNA fragments) and diagnosing aneuploidy by measuring the entire human genome can be achieved by focusing on dozens or hundreds of sites, respectively. One way to increase the number of sites that can be detected in a single separation channel is to partially widen the range of generated segment sizes by increasing the segment size of additional sites. However, the use of longer fragments for additional sites is not ideal because the amplification of larger fragments is more sensitive to inhibitor and DNA degradation, resulting in lower yields of longer fragments relative to shorter fragments. In addition, the generation of longer segments also requires an increase in expansion time and therefore increases the total verification time. There is an unmet need to increase the number of detectable sites in a given separation channel by increasing the number of dye colors that can be detected simultaneously. There is an unmet need to increase the Sanger sequencing separation capacity (and thus reduce the cost, labor, and space of the method) by increasing the number of DNA sequences that can be analyzed in a single separation channel. In addition, in some applications, sequencing multiple DNA fragments produces "mixed sequence" data that is difficult to read; it is necessary to develop a method that can correctly interpret the mixed sequence. One way to increase the capacity of Sanger separation channels and develop the ability to interpret mixed sequences is to increase the number of dye colors used in sequencing reactions. In DNA sequencing and fragment size determination, multiple fragments labeled with different dyes can be detected simultaneously. In general, the separation between the peak emission wavelengths of adjacent dyes must be sufficiently large relative to the peak width of the dye. Therefore, the throughput of each separation channel can be doubled, for example, by using two sets of 4 dyes in two independent sequencing reactions and combining the products and separating them on a single channel. This method requires the use of a total of 8 dye colors, where the first sequencing reaction uses a set of 4 dye colors suitable for labeling dideoxynucleotide terminators, and the second reaction uses another set of dye colors suitable for labeling these terminators 4 dye colors; each set of dye colors is independent so that there may be no overlap in the interpretation of the two sequences. Using this same method, a set of 12 dyes can be used to allow simultaneous analysis of the sequence of three DNA fragments in a single channel, a set of 16 dyes allows the analysis of four sequences, etc., which significantly increases the amount of information for Sanger separation . The novel instruments and biochips of this application meet many unmet needs, including those listed above.

本發明提供完全整合微流體系統以進行核酸分析。此等過程包括樣品收集、DNA萃取與純化、擴增(其可為高度多重化)、測序及DNA產物之分離與偵測。本發明之分離及偵測模組係加固型且能夠比單一鹼基解析更好。其能夠偵測六種或六種以上顏色，且就此而論適用於自測序及測片段大小應用產生高資訊含量。生物晶片上之高度多重化快速PCR為在同一天申請、具有代理人檔案號碼MBHB 08-318-US且題為"METHODS FOR RAPID MULTIPLEXED AMPLIFICATION OF TARGET NUCLEIC ACIDS"之美國專利申請案的主題，該專利申請案之全文以引用之方式明確地併入本申請案中。此外，可在如題為"PLASTIC MICROFLUIDIC SEPARATION AND DETECTION PLATFORMS"、代理人檔案號碼07-865-US之美國專利申請案中所述之生物晶片內分離且偵測PCR產物，該專利申請案之全文以引用之方式明確地併入本申請案中。因此，在第一態樣中，本發明提供光學偵測器，其包含一或多個經定位用以照明一基板上之一或複數個偵測位置的光源；一或複數個經定位用以收集且引導自該基板上之該等偵測位置發出之光的第一光學元件；及一經定位以接收來自該等第一光學元件之光的光偵測器，其中該光偵測器包含一波長色散元件，其用於根據光波長分離來自該等第一光學元件之光且經定位以將一部分經分離之光提供至偵測元件，其中該等偵測元件之各者與一用於同時自該等偵測元件之各者收集偵測資訊之第一控制元件連通，且其中該光偵測器偵測來自標記一或多個生物分子之至少6種染料之螢光，各染料具有獨特之峰值發射波長。在第二態樣中，本發明提供用於分離及偵測生物分子之系統，其包含：一組件，其用於在一基板上之一或複數個通道中同時分離複數個生物分子，其中各通道包含一偵測位置；一或多個經定位用以照明該基板上之該等偵測位置的光源；一或複數個經定位用以收集且引導自該等偵測位置發出之光的第一光學元件；及一經定位以接收自該等第一光學元件引出之光的光偵測器，其中該光偵測器包含一波長色散元件，其用於根據光波長分離來自該等第一光學元件之光且經定位以將一部分經分離之光提供至偵測元件，其中該等偵測元件之各者與一用於同時自該等偵測元件之各者收集偵測資訊之第一控制元件連通，且其中該光偵測器偵測來自標記一或多個生物分子之至少6種染料之螢光，各染料具有獨特之峰值波長。在第三態樣中，本發明提供用於分離及偵測複數個生物分子之方法，其包含：將一或複數個分析樣品提供於一基板上之一或複數個微流體通道中，其中各微流體通道包含一偵測位置，且各分析樣品獨立地包含複數個生物分子，各生物分子獨立地經至少6種染料中之一種標記，各染料具有獨特之峰值波長；同時在各微流體通道中分離該等複數個經標記之生物分子；及藉由以下程序在各微流體通道中偵測該等複數個經分離之目標分析物：用一光源照明各偵測位置；收集自各偵測位置發出之光；將所收集之光引向一光偵測器；及(i)根據光波長分離所收集之光；及(ii)同時偵測來自標記一或多個生物分子之至少6種染料之螢光，各染料具有獨特之峰值波長。在第四態樣中，本發明提供整合生物晶片系統，其包含(a)一生物晶片，其包含一或複數個微流體系統，其中各微流體系統包含一與一分離室形成微流體連通之第一反應室，其中該第一反應室經調適用於核酸萃取、核酸純化、核酸擴增前淨化、核酸擴增、核酸擴增後淨化、核酸測序前淨化、核酸測序、核酸測序後淨化、反轉錄、反轉錄前淨化、反轉錄後淨化、核酸接合、核酸雜交或定量，且該分離室包含一偵測位置；及(b)一分離與偵測系統，其包含(i)一用於在該等分離室中同時分離複數個目標分析物之分離元件；(ii)一或多個經定位用以照明該生物晶片上之該等偵測位置的光源；(iii)一或複數個經定位用以收集且引導自該等偵測位置發出之光的第一光學元件；及(iv)一經定位以接收自該等第一光學元件引出之光的光偵測器，其中該光偵測器包含一波長色散元件，其用於根據光波長分離來自該等第一光學元件之光且經定位以將一部分經分離之光提供至至少六個偵測元件，其中該等偵測元件之各者與一用於同時自該等偵測元件之各者收集偵測資訊之第一控制元件連通，且其中該光偵測器偵測來自標記一或多個生物分子之至少6種染料之螢光，各染料具有獨特之峰值波長。在第五態樣中，本發明提供整合生物晶片系統，其包含(a)一生物晶片，其包含一或複數個微流體系統，其中各微流體系統包含一與一分離室形成微流體連通之第一反應室，其中該第一反應室經調適用於核酸萃取、核酸純化、核酸擴增前淨化、核酸擴增、核酸擴增後淨化、核酸測序前淨化、核酸測序、核酸測序後淨化、反轉錄、反轉錄前淨化、反轉錄後淨化、核酸接合、核酸雜交或定量，且該分離室包含一偵測位置；及(b)一分離與偵測系統，其包含(i)一用於在該等分離室中同時分離複數個包含DNA序列之生物分子之分離元件；(ii)一或多個經定位用以照明該生物晶片上之該等偵測位置的光源；(iii)一或複數個經定位用以收集且引導自該等偵測位置發出之光的第一光學元件；及(iv)一經定位以接收自該等第一光學元件引出之光的光偵測器，其中該光偵測器包含一波長色散元件，其用於根據光波長分離來自該等第一光學元件之光且經定位以將一部分經分離之光提供至至少六個偵測元件，其中該等偵測元件之各者與一用於同時自該等偵測元件之各者收集偵測資訊之第一控制元件連通，且其中該光偵測器偵測來自標記一或多個DNA序列之至少8種染料之螢光，各染料具有獨特之峰值波長，該等染料為至少兩個含有4種染料之子集之成員，以使該等染料集合能夠在一單一通道中偵測至少兩種DNA序列，其中染料數目為四的倍數，且欲偵測之DNA序列之數目等於該倍數，以使該等不同染料之各者存在於僅一個子集中。The present invention provides a fully integrated microfluidic system for nucleic acid analysis. These processes include sample collection, DNA extraction and purification, amplification (which can be highly multiplexed), sequencing, and separation and detection of DNA products. The separation and detection module of the present invention is a ruggedized type and can be better than a single base analysis. It can detect six or more colors, and is suitable for self-sequencing and size determination applications to generate high information content. The highly multiplexed rapid PCR on the biochip is the subject of a US patent application filed on the same day with the agent file number MBHB 08-318-US and entitled "METHODS FOR RAPID MULTIPLEXED AMPLIFICATION OF TARGET NUCLEIC ACIDS". The full text of the application is incorporated into this application by reference. In addition, PCR products can be isolated and detected in biochips as described in the US patent application titled "PLASTIC MICROFLUIDIC SEPARATION AND DETECTION PLATFORMS", attorney file number 07-865-US. The full text of the patent application The way of citing is expressly incorporated into this application. Therefore, in the first aspect, the present invention provides an optical detector including one or more light sources positioned to illuminate one or a plurality of detection positions on a substrate; one or a plurality of positioned light sources A first optical element that collects and guides light emitted from the detection positions on the substrate; and a photodetector positioned to receive light from the first optical elements, wherein the photodetector includes a A wavelength dispersive element, which is used to separate the light from the first optical elements according to the wavelength of the light and is positioned to provide a portion of the separated light to the detection element, wherein each of the detection elements is used simultaneously with a The first control element that collects detection information from each of these detection elements is in communication, and wherein the light detector detects the fluorescence of at least 6 dyes from one or more biomolecules, each dye has a unique The peak emission wavelength. In a second aspect, the present invention provides a system for separating and detecting biomolecules, which includes: a component for simultaneously separating a plurality of biomolecules in one or a plurality of channels on a substrate, each of which The channel includes a detection position; one or more light sources positioned to illuminate the detection positions on the substrate; one or a plurality of light sources positioned to collect and guide the light emitted from the detection positions An optical element; and a photodetector positioned to receive the light extracted from the first optical elements, wherein the photodetector includes a wavelength dispersive element for separating the first optical elements according to the wavelength of light The light of the element is positioned to provide a portion of the separated light to the detection element, wherein each of the detection elements and a first control for collecting detection information from each of the detection elements simultaneously The components are connected, and the light detector detects fluorescence from at least 6 dyes labeled with one or more biomolecules, each dye having a unique peak wavelength. In a third aspect, the present invention provides a method for separating and detecting a plurality of biomolecules, which includes: providing one or a plurality of analysis samples in one or a plurality of microfluidic channels on a substrate, each of which The microfluidic channel contains a detection position, and each analysis sample independently contains a plurality of biomolecules, each biomolecule is independently labeled with one of at least 6 dyes, each dye has a unique peak wavelength; Separate the plurality of labeled biomolecules; and detect the plurality of separated target analytes in each microfluidic channel by the following procedure: illuminate each detection location with a light source; collect from each detection location The emitted light; directs the collected light to a light detector; and (i) separates the collected light according to the light wavelength; and (ii) simultaneously detects at least 6 dyes from one or more biomolecules labeled Fluorescence, each dye has a unique peak wavelength. In a fourth aspect, the present invention provides an integrated biochip system, which includes (a) a biochip including one or more microfluidic systems, wherein each microfluidic system includes a microfluidic communication with a separation chamber The first reaction chamber, wherein the first reaction chamber is adapted for nucleic acid extraction, nucleic acid purification, purification before nucleic acid amplification, nucleic acid amplification, purification after nucleic acid amplification, purification before nucleic acid sequencing, nucleic acid sequencing, purification after nucleic acid sequencing, Reverse transcription, pre-reverse transcription purification, post-transcription purification, nucleic acid ligation, nucleic acid hybridization or quantification, and the separation chamber includes a detection location; and (b) a separation and detection system, which includes (i) a Separation elements that simultaneously separate a plurality of target analytes in the separation chambers; (ii) one or more light sources positioned to illuminate the detection locations on the biochip; (iii) one or a plurality of Positioning a first optical element for collecting and guiding the light emitted from the detection positions; and (iv) a light detector positioned to receive the light extracted from the first optical elements, wherein the light detection The device includes a wavelength dispersive element for separating light from the first optical elements according to the light wavelength and positioned to provide a portion of the separated light to at least six detection elements, wherein each of the detection elements Connected to a first control element for collecting detection information from each of these detection elements at the same time, and wherein the photodetector detects fluorescence from at least 6 dyes labeled with one or more biomolecules Light, each dye has a unique peak wavelength. In a fifth aspect, the present invention provides an integrated biochip system, which includes (a) a biochip including one or more microfluidic systems, wherein each microfluidic system includes a microfluidic communication with a separation chamber The first reaction chamber, wherein the first reaction chamber is adapted for nucleic acid extraction, nucleic acid purification, purification before nucleic acid amplification, nucleic acid amplification, purification after nucleic acid amplification, purification before nucleic acid sequencing, nucleic acid sequencing, purification after nucleic acid sequencing, Reverse transcription, pre-reverse transcription purification, post-transcription purification, nucleic acid ligation, nucleic acid hybridization or quantification, and the separation chamber includes a detection location; and (b) a separation and detection system, which includes (i) a In the separation chamber, a plurality of separation elements containing DNA sequences of biomolecules are simultaneously separated; (ii) one or more light sources positioned to illuminate the detection positions on the biochip; (iii) one or A plurality of first optical elements positioned to collect and guide the light emitted from the detection positions; and (iv) an optical detector positioned to receive light emitted from the first optical elements, wherein the The light detector includes a wavelength dispersive element for separating light from the first optical elements according to the light wavelength and positioned to provide a portion of the separated light to at least six detection elements, wherein the detection Each of the elements is in communication with a first control element for simultaneously collecting detection information from each of these detection elements, and wherein the light detector detects at least 8 species from one or more DNA sequences marked Fluorescence of dyes, each dye has a unique peak wavelength, these dyes are members of at least two subsets containing 4 dyes, so that these dye sets can detect at least two DNA sequences in a single channel, where The number of dyes is a multiple of four, and the number of DNA sequences to be detected is equal to this multiple, so that each of these different dyes exists in only one subset.

I. 整合及整合系統 A. 整合之一般描述 利用微流體學允許在單一生物晶片上製造發揮一種以上之功能的特徵。該等功能中之兩種或兩種以上功能可呈微流體聯繫以使樣品之連續處理能夠實現；此結合稱為整合。儘管對於任何給定之應用而言並非必須實施所有過程，但存在一系列必須加以整合以達成任何給定之應用的可能功能或組成過程。因此，所選擇之整合方法必須適用於以不同順序有效地聯繫若干不同組成過程。可整合之過程包括(但不限於)以下項： 1.樣品***； 2.移除外來物質(例如諸如粉塵、纖維之大顆粒)； 3.細胞分離(亦即，移除除含有欲分析之核酸之彼等細胞以外的細胞，諸如自含有欲分析之微生物核酸的臨床樣品中移除人類細胞(且相應地移除人類基因組DNA))； 4.濃縮含有所關注之核酸之細胞； 5.溶解細胞且萃取核酸； 6.純化來自溶胞物之核酸；同時有可能將核酸濃縮至較小體積； 7.擴增前核酸淨化； 8.擴增後淨化； 9.測序前淨化； 10.測序； 11.測序後淨化(例如用以移除會干擾電泳的未合併之經染料標記之終止子及離子)； 12.核酸分離； 13.核酸偵測； 14. RNA之反轉錄； 15.反轉錄前淨化； 16.反轉錄後淨化； 17.核酸接合； 18.核酸定量； 19.核酸雜交；及 20.核酸擴增(例如PCR、滾環擴增、鏈置換擴增及多重置換擴增)。可將某些此等過程組合之許多方法中之一種方法為藉由STR分析進行人類識別的整合系統。此類系統可需要結合DNA萃取、人類特異性DNA定量、添加定量DNA至PCR反應中、多重PCR擴增及分離與偵測(視情況亦可併入用以移除反應成分或引子之淨化步驟)。可藉由諸如擦拭之技術收集全血、乾血、面頰內表面、指紋、性攻擊、接觸或其他法醫學上相關之樣品的一或多種樣品(參見Sweet等人，J.Forensic Sci . 1997, 42, 320-2)。暴露於溶胞物(視情況在攪動存在下)自拭子釋放DNA至試管中。B. 整合組件及其用途之一般描述 1. 樣品收集及初始處理 對於許多應用而言，將以下離散組件有利地整合至生物晶片中：樣品***、移除外來物質、移除干擾性核酸及濃縮所關注之細胞。一般而言，生物晶片之預處理組件接受樣品，進行顆粒及含有外來核酸之細胞的初始移除，且濃縮所關注之細胞至較小體積。一種方法為使用可容納拭子(例如類似"Q-尖端")且充滿溶解溶液之樣品管來進行溶解及萃取步驟。可將拭子與若干含細胞之位點(包括血跡、指紋、水、空氣過濾器)或臨床位點(例如頰部拭子、傷口拭子、鼻拭子)接觸放置。此等管與生物晶片之其他組件之界面可包括用於移除外來物質之過濾器。另一方法為使用大體積之血或環境樣品採集濾筒，其處理1-100 mL樣品。在血液之情況下，當通過含有所關注之核酸之微生物時白血球減少介質可移除人類白血球及干擾DNA。對於環境樣品而言，可使用大網眼過濾器來移除粉塵及汙跡，而小網眼過濾器(例如＜20 μm、＜10 μm、＜5 μm、＜2.5 μm、＜1 μm、＜0.5 μm、＜0.2 μm、＜0.1 μm之過濾器)可用來來捕集微生物，將其濃縮成小體積。此等預處理組件可為獨立消耗品或在製造時附著於整合晶片上。或者可將生物晶片加以設計以進行差異溶解，從而根據類型分離細胞(例如來自***上皮細胞之***或來自細菌之紅血球)。2. 溶解及萃取 可使用各種溶解及萃取方法。例如，一種典型程序包括在將樣品與小量降解酶(諸如蛋白酶-K，其分解細胞壁且釋放核酸)混合後施加熱。其他可用之方法為音波處理及超音波處理，其中之任一者或兩者有時均在珠粒存在下進行。例如，可對含有10⁶ 個細胞或10⁶ 個以下之細胞之樣品進行溶解及萃取。視應用而定，可在本發明之生物晶片及方法中使用較小數目之起始細胞，少於10⁵ 個、少於10⁴ 個、少於10³ 個、少於10² 個、少於10個，且在當欲分析多複本序列之情況下，少於1個。 3. 核酸之純化 核酸純化之一種形式可藉由將純化介質***輸入通道與輸出通道之間而獲得。此純化介質可基於二氧化矽纖維且使用離液-鹽試劑(chaotropic-salt reagent)來溶解生物樣品，暴露DNA(及RNA)且使DNA(及RNA)與該純化介質結合。接著將溶胞物經由輸入通道傳遞通過純化介質以結合核酸。將經結合之核酸藉由基於乙醇之緩衝液洗滌以移除污染物。此舉可藉由使洗滌試劑經由輸入通道流經純化膜而實現。接著將經結合之核酸藉由合適之低鹽緩衝液之流動自該膜溶離(例如Boom美國5,234,809)。此方法之一種變體包括使用不同組態之固相。例如，可使用矽膠來結合核酸。可使用順磁性二氧化矽珠粒，且在結合、洗滌及溶離步驟期間利用其磁性將其固定於通道或室壁上。亦可使用非磁化二氧化矽珠粒，其被裝填於緻密'管柱'(在該處其係以玻璃料所固持)內(通常被製造於裝置之塑料中，但此等者亦可在裝配過程中***)，或在其操作之特定階段期間"游離"。可將游離珠粒與核酸混合且接著使之在裝置中相對於玻璃料或堰流動以將其捕集，以使其不干擾下游過程。其他形式包括分布於凝膠介質中的具有二氧化矽顆粒之溶膠-凝膠及具有包括二氧化矽顆粒之聚合物單體，其中為了較大機械穩定性使載體交聯。基本上，在習知配置下起作用之任何核酸純化方法均可適用於本發明之整合生物晶片。 4. 核酸擴增 可使用各種核酸擴增方法，諸如PCR及反轉錄PCR，其在至少兩個溫度且更通常為三個溫度之間需要熱循環。可使用諸如鏈置換擴增之等溫方法，且對於全基因組擴增可使用多重置換擴增。在同一天申請之題為"METHODS FOR RAPID MULTIPLEXED AMPLIFICATION OF TARGET NUCLEIC ACIDS"之美國專利申請案(代理人檔案號碼08-318-US)的教示之全文以引用之方式併入本文中(如上所述)。 5. 核酸定量 以微流體格式定量之一種方法係基於即時PCR。在此定量方法中，在輸入通道與輸出通道之間製造一反應室。將該反應室與熱循環器耦接，且將光學激發與偵測系統耦接至該反應室以允許來自反應溶液之螢光得以量測。樣品中DNA之量與來自每一循環之反應室之螢光強度有關。參見，例如Heid等人，Genome Research 1996, 6, 986-994。其他定量方法包括在擴增前或後使用諸如picoGreen、SYBR或溴化乙錠之***染料，接著可使用螢光或吸光度偵測該等染料。 6. 二次純化 對於STR分析，可直接將經多重擴增及經標記之PCR產物用於分析。然而，可藉由純化產物以移除PCR所必需但會干擾分離或其他後續步驟之離子而極大地改良電泳分離效能。類似地，繼循環測序或其他核酸處理後的純化可為適用的。總體而言，繼核酸之初始萃取或純化後之任何純化步驟可視為二次純化。可使用各種方法，包括超音處理，其中驅使小離子/引子/未併入之染料標記通過過濾器，使所要之產物留於過濾器上，該產物隨後可被溶離且直接應用於分離或後續模組中。超濾介質包括聚醚碸及再生纖維素"編織"過濾器，以及軌跡侵蝕膜(其中在極薄(1-10 µm)之膜中形成高度均一大小之孔)。後者具有收集大小大於過濾器表面上之孔大小之產物而非捕集表面下一些深度之產物的優點。亦可使用與上述相同之方法(亦即，典型之二氧化矽固相純化)來純化經擴增之核酸。其他方法包括使用水凝膠、具有孔大小可變性之性質的交聯聚合物，亦即孔之大小響應於諸如熱及pH值之環境變量而變化。在一種情況下，該等孔為緻密的且PCR產物不能通過。當孔膨大時，產物之水動力或電泳流可能穿過該等孔。另一方法為使用雜交，產物非特異性雜交至固定於一表面(諸如珠粒之表面)上之隨機DNA或特異性雜交(其中產物上之序列標籤之補體係位於固體表面上)。在此方法中，使所關注之產物經由雜交固定且藉由洗滌移除不想要之物質；隨後加熱熔化雙鏈體且釋放經純化之產物。7. 循環測序反應 典型之循環測序需要熱循環，與PCR幾乎一樣。較佳方法為彼等使用經染料標記之終止子的方法，以使各延伸產物帶有對應於延伸反應之最終鹼基之單一螢光標記。 8. 注入、分離及偵測 可以各種方式進行電泳通道中之經標記之核酸片段之注入、分離及偵測，此已在同一天申請之題為"PLASTIC MICROFLUIDIC SEPARATION AND DETECTION PLATFORMS"(給定之代理人檔案號碼為07-865-US)之美國專利申請案中描述，該案之全文以引用之方式併入本文中。首先，如其中所討論之交叉注入器可用於注入樣品之一部分。在一替代實施例中，可使用電動注入("EKI")。在任一種情況下，在載入通道(在交叉注入情況下)或分離通道(在EKI情況下)之開口端附近之測序產物的進一步濃縮可藉由電極近旁靜電濃縮產物來進行。在圖14中展示在晶片之電泳部分上之兩電極樣品孔。兩個電極均塗覆有一滲透層，其阻止DNA與電極金屬接觸，但允許離子及水進入樣品孔與電極之間。該等滲透層可由交聯聚丙烯醯胺形成(參見美國專利申請公開案US 2003-146145-A1)。距通道開口最遠之電極為分離電極，而距通道開口最近之電極為反電極。藉由對反電極相對於分離電極通正電，會將DNA吸至反電極且在接近分離通道之開口處濃縮。藉由使反電極浮動且在分離通道之遠端使用分離電極及陽極注入，電動注入濃縮之產物。C. 整合方法 生物晶片亦含有用於整合功能性模組之若干不同構件。此等構件涉及自生物晶片上之一點至另一點輸送液體、對於具流速依賴性之過程(例如，某些洗滌步驟、顆粒分離及溶離)控制流動速率、閘控生物晶片上之流體運動之時間及間隔(例如經由使用某些形式之閥)，及流體之混合。可使用各種方法用於流體運輸及受控流體流動。一種方法為正置換泵送，其中在運動過程中，與流體或介入氣體或流體接觸之柱塞驅動流體移動一精確距離，該距離係基於由柱塞置換之體積。此類方法之一實例為注射泵。另一方法為使用氣動、磁力致動或其他方式致動之整合彈性膜。單獨而言，此等膜可用作閥以使流體容納於界定之空間中及/或阻止流體之過早混合或傳遞。然而，當串聯使用時，此等膜可形成類似於蠕動泵之泵。藉由膜之同步、連續致動，流體可自其後側"推出"，此係因為在前側之膜被打開以接受移動流體(且排空裝置之通道中的任何置換空氣)。用於致動此等膜之較佳方法為氣動致動。在該等裝置中，生物晶片由流體層構成，該等流體層中之至少一者具有膜，該等膜之一側暴露於裝置之流體通道及室中。膜之另一側暴露於垂直於壓力源之氣動歧管層。藉由應用壓力或真空使該等膜打開或閉合。可使用通常為打開或通常為閉合之閥，在壓力或真空之應用下改變狀態。注意到任何氣體均可用於致動，此係因為氣體不與分析下之流體接觸。驅動流體及控制流動速率之另一方法為藉由改變流體之前彎月面、後彎月面或兩彎月面處之壓力而在流體自身上直接施加真空或壓力。施加適當之壓力(通常在0.05-3 psig範圍內)。流動速率亦可藉由使流體通道之大小合適來控制，此係因為流動速率與流體兩端之壓差及水力直徑之四次方成正比，且與通道或液體塞之長度及其黏度成反比。可使用各種主動閥達成流體閘控。前述者可包括壓電閥或電磁閥，其可直接併入晶片中，或施加於生物晶片以使主晶片體上之埠與該等閥連通，引導流體進入該等閥，且隨後返回晶片中。此等類型之閥之一缺點為對於許多應用而言，其可能難於製造且對於併入拋棄式整合裝置而言過於昂貴。如上所述，較佳方法為使用膜作為閥。例如可使用由10 psig致動之膜來成功地容納經受PCR之流體。在某些應用中，毛細管微型閥(其為被動閥)可為較佳的。基本上，微型閥係流動路徑中的緊縮。在微型閥中，當施加於流體之壓力低於稱為破裂壓力之臨界值時，表面能及/或諸如尖銳邊緣之幾何特徵可用於阻止流動，其中該破裂壓力常由以下關係式給出： P_閥 α (γ/d_H )*sin(θ_c )，其中γ為液體之表面張力，d_H 為閥之水力直徑(定義為4×(截面積)/截面周長)，且θ_c 為液體與閥表面之接觸角。使得被動閥對於某些應用為較佳之性質包括：極低之死體積(通常在皮升(picoliter)範圍內)及小的物理範圍(physical extent)(各者僅略大於通向閥及離開閥之通道)。小的物理範圍允許在生物晶片之給定表面上有高密度之閥。此外，某些毛細管閥非常易於製造，其基本上由經表面處理或未經表面處理之塑料片中之小孔組成。毛細管閥之恰當使用可減少所需膜閥之總數，簡化總體製造及形成穩固系統。在本發明裝置中建構之毛細管閥有兩種類型：平面內閥，其中閥之小通道及尖銳角落係藉由在一層中形成"槽"且將此層接合至無特徵蓋子(通常為裝置之另一層)而形成；及通孔閥，其中在裝置之兩個流體載運層之間的中間層中製造小(通常250 µm或250 µm以下)孔。在兩種情況下，可使用氟聚合物處理來增大流體與閥接觸之接觸角。圖7展示在氟聚合物處理之情況下此等閥對於所關注之液體(即去離子水及循環測序試劑)之閥調效能與閥大小的函數關係。在兩種情況下，觀測到閥調壓力對閥尺寸之預期依賴性(壓力約1/直徑)。通孔閥具有顯著優於平面內閥之優點。首先，其易於製造，此係因為小通孔可易於在製造閥層後藉由繞柱成形(molding around post)、打孔、模切、鑽孔或雷射鑽孔在塑料片中形成。平面內閥需要相當精確的製造，且極精細之閥(具有高閥調壓力)必需使用微影技術來製造所需成型或壓印工具。其次，通孔閥可在 "所有側"上較為完全地以氟聚合物塗覆。將低表面張力之氟聚合物溶液應用於孔導致藉由毛細管作用完全塗覆該孔之內壁。塗覆平面內閥之所有側需要將氟聚合物應用於閥以及密封於閥上之匹配層之區域。因此，在閥之"頂部"無塗覆之情況下形成典型平面內閥。在經機械加工之原型中，通孔閥既易於實施又展現較大閥調壓力，如圖7中所示。可以各種方式達成混合。首先，可藉由將兩種流體共注入單一通道中利用擴散來混合流體，其中該通道通常具有小的橫向尺寸及足夠的長度以使得在給定流動速率下滿足擴散時間： t_D =(寬度)² /(2×擴散常數) 不幸的是，此類混合對於快速混合大的體積通常不夠，此係因為擴散或混合時間與通道寬度之平方成比例。混合可以多種方式增強，諸如層壓，其中流體液流被分開且重組(Campbell及GrzybowskiPhil. Trans. R. Soc. Lond. A 2004, 362, 1069-1086)；或經由使用精細微結構在流動通道內形成紊亂平流(Stroock等人，Anal. Chem. 2002, 74, 5306-5312)。在使用主動泵及閥之系統中，混合可藉由多次循環裝置上之兩點之間之流體而達成。最後，後者亦可在使用毛細管閥之系統中達成。安置於兩個通道或室之間的毛細管閥充當流體流之樞軸；當流體經由毛細管自一通道流入另一通道時，若使用足夠低之壓力抽吸流體，則後彎月面被截獲。壓力之逆轉驅使流體返回至第一通道中，且再次使其阻止於毛細管處。可使用多次循環來有效地混合成分。以微流體格式分離及偵測之方法描述於同一天申請之題為"PLASTIC MICROFLUIDIC SEPARATION AND DETECTION PLATFORMS"(代理人檔案號碼MBHB 07-865-US)之美國專利申請案中，該案之全文以引用之方式併入本文中(參見例如其中之段落68-79，94-98)。圖13之上半部分展示自兩個組件構建整合生物晶片(1301 )，該等兩個組件在製造中或製造期間接合。第一組件為將圖1之生物晶片之溶解、擴增及測序特徵與圖11之生物晶片之測序產物純化特徵結合的16-樣品生物晶片(1302 )，且第二組件為16-道塑料分離生物晶片(1303 )。亦可在分離前電動注入純化測序產物。D. 製造方法 本發明之裝置可主要由塑料構成。可用之塑料類型包括(但不限於)：環烯烴聚合物(COP)；環烯烴共聚物(COC)；(當具有足夠分子量時，兩者均具有極佳光學性質、低吸濕性及高操作溫度)；聚(甲基丙烯酸甲酯)(PMMA)(易於加工且可獲得具極佳光學性質者)；及聚碳酸酯(PC)(高度可成形且具有良好抗衝擊性及高操作溫度)。關於材料及製造方法之更多資訊包含於題為"METHODS FOR RAPID MULTIPLEXED AMPLIFICATION OF TARGET NUCLEIC ACIDS" (代理人檔案號碼08-318-US)之美國專利申請案中，該案之全文以引用之方式併入本文中(如上所述)。可使用各種方法來製造生物晶片之個別零件且將其裝配於最終裝置中。因為生物晶片可由一或多種類型之塑料構成，有可能包括***組件，所關注之方法係關於個別零件之製造、繼之以零件之後處理及裝配。可以若干方式製造塑料組件，包括射出成形、熱壓印及機械加工。射出成形零件可由總體特徵(諸如流體儲集器)以及精細特徵(諸如毛細管閥)構成。在某些情況下，較佳在一套零件上製造精細特徵且在另一套上製造較大特徵，此係因為此等不同尺寸的特徵之射出成形之方法可變化。對於大型儲集器(在一側上量測為若干(約1-50 mm)毫米且深度為若干毫米(約1-10 mm)且能夠容納數百微升)而言，可使用加工型射出成形工具或藉由使用石墨電極燒進鋼或其他金屬中而製造之工具來使用習知成形，其中該石墨電極已經加工成該工具之負極。對於精細特徵，工具製造及成形製程均可變化。通常在所關注之基板上使用微影製程(例如，玻璃之各向同性蝕刻，或矽上之深度反應性離子蝕刻或其他製程)製造工具。接著可用鎳電鍍基板(通常在鉻層之沈積後以促進黏著)且例如藉由在酸中蝕刻來移除基板。此鎳"子體"鍍層為射出成形工具。該成形製程亦可稍不同於上述者。對於精細、狹窄特徵而言，已發現壓縮射出成形(其中該模具在塑料注入空腔後略經物理壓縮)比標準射出成形在逼真度、精確度及重現性方面更佳。對於熱壓印而言，關於如上所述之總體及精細特徵之類似問題需控制，且可如上所述製造工具。在熱壓印中，可將塑料樹脂以球粒形式，或作為經由成形或壓印製造之材料之預成形坯施加於工具表面或一平坦基板上。接著可在精確控制之溫度及壓力下接觸第二工具以將塑料溫度提高超過其玻璃轉移溫度且導致材料流動以填充該(等)工具之空腔。在真空下之壓印可避免空氣捕集於工具及塑料之間的問題。亦可使用機械加工來製造零件。可使用高速電腦數控(CNC)機每日自成形、擠壓或溶劑澆鑄塑料製造許多個別零件。銑床、操作參數及切割工具之合理選擇可獲得高表面品質(在COC之高速銑削下，可獲得50 nm之表面粗糙度(Bundgaard等人，Proceedings of IMechE Part C: J. Mech. Eng. Sci. 2006, 220,1625-1632))。亦可使用銑削來製造可能難於以成形或壓印獲得之幾何形狀且容易地混合單一零件上之特徵尺寸(例如可將大的儲集器及精細毛細管閥加工於同一基板中)。銑削優於成形或壓印之另一優點為無需使用脫模劑自成形工具中脫出所製成之零件。個別零件之後處理包括光學檢驗(其可為自動的)、用以移除諸如毛邊或懸掛塑料之缺陷的清潔操作，及表面處理。若在加工塑料中需要光學品質表面，可利用適於塑料之溶劑蒸氣拋光。例如，對於PMMA，可使用二氯甲烷，而對於COC及COP，可使用環己烷或甲苯。在裝配前，可應用表面處理。可進行表面處理以促進或減少潤濕(亦即改變零件之親水性/疏水性)；抑制微流體結構內氣泡之形成；增加毛細管閥之閥調壓力；及/或抑制蛋白吸附至表面。減少可濕性之塗層包括氟聚合物及/或具有氟部分之分子，其中當分子被吸附或結合於裝置之表面時氟部分可暴露於液體中。塗層可被吸附或者沈積，或其可共價鍵接至表面。可用於製造該等塗層之方法包括浸漬塗佈、經由裝配之裝置之通道傳遞塗佈試劑、上墨、化學氣相沈積及噴墨沈積。在塗佈分子與表面之間的共價鍵可藉由用氧或其他電漿或UV-臭氧處理而形成以形成活性表面，且隨後使表面處理分子沈積或共沈積於表面上(參見Lee等人，Electrophoresis 2005, 26, 1800-1806；及Cheng等人，Sensors and Actuators B 2004, 99, 186-196)。可以各種方式進行組件零件於最終裝置中之裝配。諸如過濾器之***裝置可被模切且接著用取置機放置。熱擴散接合可用於(例如)相同材料之兩層或兩層以上之接合，其中各層為均一厚度。一般而言，可將零件堆疊，且將該堆疊置於熱壓機中，其中溫度可提高至包含該等零件之材料之玻璃轉移溫度附近，以導致零件之間的界面融合。此方法之一優點在於該接合為"一般的"，亦即，不論層之內部結構怎樣，皆可使具大致相同尺寸之層的任何兩個堆疊接合，此係因為可將熱及壓力均一地施加於該等層上。熱擴散接合亦可藉由使用特定製造之接合托架用於接合更複雜之零件，諸如在接合或相反表面上不為平面之彼等者。該等托架與欲接合之層之外表面一致。其他接合變體包括溶劑輔助熱接合，其中諸如甲醇之溶劑部分溶解塑料表面，從而在較低之接合溫度下增強接合強度。另一變體為使用低分子量材料之旋轉塗佈層。例如，可將與基板成分化學結構相同但較低分子量之聚合物旋轉塗佈於欲接合之至少一個層上，裝配該等組件，且藉由擴散接合將接合所得之堆疊。在熱擴散接合過程中，低分子量成分可在比該等成分低之溫度下經歷玻璃轉移溫度，且擴散至基板塑料中。可使用黏著劑及環氧樹脂來接合不同材料且黏著劑及環氧樹脂很可能在以不同方式製造接合組件時使用。黏著劑膜可被模切且放置於組件上。亦可經由旋轉塗佈塗覆液體黏著劑。可成功地利用黏著劑於結構化零件上之上墨(諸如在奈米接觸印刷中)將黏著劑塗覆於結構化表面而不需將黏著劑"引導"至特定區域上。在一實例中，本發明之生物晶片可如圖6中所示裝配。層1及2可藉由所包括之特徵(例如銷及插座)對準；分別地，層3及4可類似地藉由所包括之特徵對準。層1加層2之堆疊可被倒置並施加於層3加層4之堆疊，且接著可結合成整個堆疊。E. 實例實例 1 用於核酸萃取及擴增之整合生物晶片 圖1中展示用於DNA萃取及PCR擴增之整合生物晶片。此4-樣品裝置整合以下功能：試劑分配及計量；試劑與樣品之混合；樣品至晶片之熱循環部分之傳遞；及熱循環。在下文之實例2中使用相同之生物晶片且具有對於循環測序效能而言額外之結構。生物晶片如圖2-5中所示由4層熱塑性塑料構建。該等4層為經加工之PMMA且分別具有0.76 mm、1.9 mm、0.38 mm及0.76 mm之層厚度，且生物晶片之側尺寸為124 mm×60 mm。一般而言，三層或三層以上之生物晶片允許使用在多個檢定之間分配的不定數目之常見試劑：兩個流體層及一至少含有通孔之層，使得外層中之流體通道能夠彼此'交叉'。(應認識到存在特殊情況-諸如在多個樣品中使用僅一種常見試劑-使三層構造並不為必需)。選擇4層使其與用於其他功能(諸如超音處理，實例3)之晶片構建相容且完全整合(實例4)。生物晶片之通道之截面尺寸在127 μm×127 μm至400 μm×400 μm範圍內，而儲集器之截面在400 µm×400 µm至1.9×1.6 mm範圍內；通道及儲集器延伸之距離為0.5 mm至數十毫米短。在生物晶片中使用之毛細管閥為："平面內"閥之尺寸為127 μm×127 μm且通孔毛細管閥之直徑為100 µm。將四個加工層之某些通道、儲集器及毛細管閥用疏水/疏油材料PFC 502A(Cytonix, Beltsville, MD)處理。藉由用濕潤之Q-尖端塗佈，隨後藉由在室溫下空氣乾燥來進行表面處理。乾燥之氟聚合物層藉由光學顯微鏡術測出厚度小於10 μm。表面處理用於兩種目的：阻止液體內氣泡之形成，尤其在諸如循環測序試劑之低表面張力液體內(當液體快速潤濕通道或室之壁時此可能會出現(且在空氣可被置換前"封閉"氣泡))；及在毛細管閥抵抗液體流動處增強毛細管破裂壓力。未處理之區域為PCR及循環測序之熱循環室。在表面處理後，如圖6中所示連接該等層。使用熱擴散連接進行連接，其中在壓力下加熱組件之堆疊至接近塑料之玻璃轉移溫度(T_g )之溫度。在由7.5分鐘內自環境溫度上升至130℃、在130℃下保持7.5分鐘以及快速冷卻至室溫組成之熱連接概況過程中在整個11.5平方吋生物晶片上施加45磅(lbs)之力達15分鐘。開發氣動儀器用於驅動本發明之生物晶片內之流體。兩個小蠕動泵提供壓力及真空。正壓力輸出在具有約0.05-3 psig範圍內之三個調節器之間分配。將真空轉移至具有約(-0.1)-(-3) psig之輸出真空的調節器。將第四、較高之壓力自N₂ 汽缸或者自高容量泵傳至另一調節器。將正或負壓力施加於一系列8個壓力選擇器模組。各模組裝備有電磁閥，該等閥可自5個輸入中選擇欲傳輸至生物晶片之輸出壓力。輸出壓力管線終止於至少一個氣動界面。此界面用位於晶片之輸入側上之晶片埠上的O型環夾持於晶片上(該埠沿該等特徵之頂部)。接受來自壓力選擇器模組之輸出壓力管線的額外電磁閥(亦即閘閥；每界面8個)恰好在生物晶片埠上。非常接近於晶片的此等閥在壓力管線與晶片之間提供低死體積之界面(約13 μL)。當施加壓力以移動其他液體時，低死體積之界面可阻止生物晶片上之某些流體之無意運動(例如，當施加壓力時由於氣體之壓縮，在液體塞與關閉閥之間之小氣體體積決定塞可移動之最大限度)。所有壓力選擇器閥及閘閥係使用基於指令之LabView^TM 程式在電腦控制下操作。此系統之一重要特徵為短壓力循環時間為可能的。可執行一些流體控制事件，其需要短至30毫秒之壓力脈衝，且/或可使用複雜壓力概況，其中壓力可快速自一值至另一值變換(亦即一調節器至另一調節器)(亦即時間滯後不超過10-20毫秒)。樣品由約10⁶ 個細胞/mL之藉由pGEM測序質體***(pUC18測序目標)轉型之大腸桿菌(E. coli )DH5的細菌懸浮液組成。PCR試劑由dNTP KOD Taq聚合酶(Novagen, Madison, WI)(濃度0.1 μM)組成。將1.23 μL細菌懸浮液之樣品添加至四個埠104 之各者，各自在層1及2中分別構成通孔202 及336 。接著樣品存在於層2中之樣品通道303 中。接著，將10 μL PCR試劑添加至埠105 ，其構成在層1及2中之通孔217 及306 。接著PCR試劑存在於層2中之室307 中(參見圖8a)。用於排空針對PCR試劑而言之置換空氣之埠為埠107，其構成109 及通孔203 +305 。在操作中，由樣品及下游過程(諸如試劑之計量、流體之混合)置換之空氣經由晶片之輸出端上之埠108 排出，該埠由通孔227 構成。PCR反應之終體積可如所希望增加或減少。將生物晶片置於上述氣動歧管中。執行以下之自動化壓力概況，其中在步驟之間無延遲。除非另有說明，否則氣動界面閥(對應於沿晶片之輸入側之埠)在所有步驟過程中為關閉的。將0.12 psig之壓力施加於埠104 上達15秒以將樣品沿著通道303 驅至通孔304 。樣品通過通孔304 且出現於層1之樣品室204 中之層2的另一側上且被驅至第一混合接合點205 。在第一混合接合點，將樣品藉由毛細管閥210 持留(參見圖8b-c)。將0.12 psig之壓力施加至埠105 達10秒以驅使PCR試劑通過通孔320 。PCR試劑出現於分配通道208 中之層2的另一側上，且移動至計量室209 中，其限定試劑體積等於樣品體積，其中其藉由毛細管閥211 持留於混合接合點205 處。(參見圖8d)。將0.12 psig之壓力施加至埠107 (由通孔203 及305 構成 )(其中埠105 對大氣開放)達3秒以使通道208 排空(參見圖8e)。將0.8 psig之壓力施加至埠107 及105 達0.03秒且同時將0.7 psig之壓力施加至埠104 達0.03秒而藉由使液體頂破毛細管閥210 及211 並通過該等閥來 將樣品與PCR試劑初始混合(參見圖8f)。將0.12 psig之壓力施加至埠104 及107 達10秒以將樣品及PCR試劑泵送至混合通道214 中，且滯留於毛細管閥210 及211 處。通過混合球形物212 進入收縮部分213 構成對液流之額外水力阻力，從而減低由上述高壓脈衝賦予之高速度。將0.7 psig之壓力施加至埠104 及107 達0.03秒以將液體自毛細管閥210 及211 中分離(參見圖8g)。將0.12 psig之壓力施加至埠104 及107 達3秒以經由混合通道214 將液體泵送至毛細管閥219 ，液體在其中持留(參見圖8h)。將0.7 psig之壓力施加至埠104 及107 達0.1秒以將樣品與PCR試劑之混合物經由通孔315 及402 且經由層2及3之主體驅至PCR室502 中(參見圖8i)。將0.12 psig之壓力施加至埠104 及107 達3秒以達成將樣品與PCR試劑之混合物泵送至室502 中。接著使樣品與PCR試劑之混合物之前緣通過通孔403 及316 ，出現於層1中，且在毛細管閥220 處阻止(參見圖8j)。接著將生物晶片加壓至30 psig N₂ 且經由帕耳帖效應(Peltier)使用氣囊(gas bladder)壓縮機制使其熱循環以進行PCR擴增，此如與本案同時申請之題為"METHODS FOR RAPID MULTIPLEXED AMPLIFICATION OF TARGET NUCLEIC ACIDS"(代理人檔案號碼08-318-US)之美國專利申請案及2008年2月6日申請之題為"DEVICES AND METHODS FOR THE PERFORMANCE OF MINIATURIZED IN VITRO ASSAYS"之國際專利申請案第PCT/US08/53234號(代理人檔案號碼07-084-WO)中所述，該等申請案之各者之全文以引用的方式併入本文中。選擇之樣品、試劑體積及PCR室大小使得液體填充閥219 與閥220 之間的區域。因此，小截面積(通常127 μm×127 μm)之液體/蒸氣界面位於距層4之熱循環底面約3 mm處。在熱循環期間施加壓力抑制樣品中溶解之氧之脫氣。液體/蒸氣界面之小截面積及距Peltier表面之距離均抑制蒸發。在循環過程中所觀測到之生物晶片頂部之溫度從不超過60℃，且因此液體/蒸氣界面處之蒸氣壓比若該等界面在PCR室中所將具有之蒸氣壓顯著較低。對於2 μL樣品，其中1.4 μL在室502 中且其餘者在通孔及毛細管閥中，在PCR 40次循環中所觀測到之蒸發量小於0.2 μL。未循環之流體體積(在此情況下為0.6 μL)可藉由選擇較小直徑之通孔來減小。使用以下溫度概況執行PCR： • 在98℃下將細菌熱溶解3分鐘 • 以下項之40次循環： o 在98℃下變性5秒 o 在65℃下退火15秒 o 在72℃下延伸4秒 o 在72℃下最終延伸(2分鐘) PCR產物係藉由用約5 µL去離子水沖洗室502 回收且藉由平板凝膠電泳分析。PCR產量高達40 ng/反應，比隨後之測序反應所需之量多得多。在本申請案中，僅藉由溶解細菌產生細菌核酸。可使核酸經受所需之純化，亦即可改良擴增、測序及其他反應之效率之過程。實例 2 用於循環測序試劑分配、與 PCR 產物之混合及循環測序之整合生物晶片 使用如實例1中所述之生物晶片。將使用實例1中所述之方案於試管中產生之PCR產物添加至如上所述生物晶片之樣品及PCR試劑埠中。將50 μL循環測序試劑(BigDye^TM 3.1/BDX64, MCLab, San Francisco)添加至埠106 (由通孔215 及308 構成)及室309 。在安裝兩個氣動界面(一者用於晶片輸入端且一者用於晶片輸出端)後，將PCR產物如實例1中所述處理直至PCR室，但無PCR熱循環步驟。流體在晶片中之處置如圖9a中所示。使用氣動系統軟體執行以下壓力概況；除非另有說明，否則所有對應於晶片埠之電磁閥均被關閉： 1.將0.1 psig之壓力施加至埠106 (其中埠109 對大氣開放)達10秒以將循環測序試劑泵入通道310 中(參見圖9b)。 2.將0.7 psig之壓力施加至埠106 及108 上達0.2秒(由通孔216 及314 構成)，以驅使循環測序試劑自通道304 經由通孔311 、經由層2之主體且進入層1上之循環測序試劑計量室218 中(參見圖9c)。 3.將0.1 psig之壓力施加至埠106 (其中埠109 對大氣開放)，將循環測序試劑驅至毛細管閥221 ，循環測序試劑在其中持留(參見圖9d)。 4.將0.1 psig之壓力施加至埠108 (其中埠106 對大氣開放)達1秒，以驅使過量循環測序試劑返回室101 中，使通道310 為空(參見圖9e)。 5.將0.7 psig之壓力施加至埠104 及107 達0.1秒(其中埠109 對大氣開放)，以驅使PCR產物通過毛細管閥220 且進入通孔317 ，通過層2之主體及層3中之通孔404 ，且進入層4之循環測序室503 中(參見圖9f)。 6.將0.1 psig之壓力施加至埠109 (其中埠104 及107 對大氣開放)達5秒，以驅使PCR樣品返回通孔。毛細管作用將液體持留於通孔之入口處，防止PCR產物與室503 之間出現捕集之空氣泡(參見圖9g)。 7.將0.7 psig之壓力施加至埠108 (其中埠109 對大氣開放)達0.2秒，以將循環測序試劑驅至室503 中，而同時將0.1 psig施加至埠104 及107 ，以使PCR產物與測序試劑接觸(參見圖9h)。 8.將0.1 psig之壓力施加至埠104 、107 及108 達10秒(其中埠109 對大氣開放)，以將PCR產物及桑格試劑驅入室中。PCR產物之後彎月面及測序試劑之後彎月面被阻止於毛細管閥220 及221 (參見圖9i)。 9.將0.25 psig真空及持續期間0.1秒之5個真空脈衝施加至埠108 (其中埠109 對大氣開放)，以將兩種液體部分吸回試劑計量室218 中(參見圖9j)。 10.將0.1 psig之壓力施加至埠104 、107 及108 (其中埠109 對大氣開放)達10秒以將混合物泵回至室503 ，其中後彎月面被阻止於如步驟8中之毛細管閥(參見圖9k)。再額外重複步驟9-10兩次以實現測序試劑與PCR產物之混合。接著將生物晶片加壓至30 psig N₂ 且使用以下溫度概況執行熱循環： • 95℃/1分鐘初始變性 o 以下項之30次循環 o 在95℃下變性5秒 o 在50℃下退火10秒 o 在60℃下延伸1分鐘樣品(參見圖9l)係藉由乙醇沈澱回收且純化並藉由如下文(部分II，實例5)中所述以Genebench^TM 儀器電泳分離及雷射誘發螢光偵測加以分析。Phred品質分析產生408 +/- 57 QV20鹼基/樣品。實例 3 在 4- 樣品生物晶片中之超濾 如實例1中所述，構建四層適於測序產物純化之效能之4-樣品生物晶片，且展示於圖11中。在構造中之一額外元件為超濾(UF)過濾器1116 ，其在熱連接前被切割成合適大小且置於層3與4之間。圍繞UF過濾器之良好連接的形成必需使用層3。層3及4圍繞過濾器形成不間斷周邊，此係因為所有通向過濾器及離開過濾器之通道均在層2之底部((例如)在通道越過過濾器之狀況下，直接在層2與4之間之連接導致對過濾器之不良連接)。在此實例中，使用分子量截留(MWCO)為30 kD之再生纖維素(RC)過濾器(Sartorius, Goettingen, Germany)。當具有交替材料聚醚碸(Pall Corporation, East Hills, NY)時，已檢驗多種其他MWCO(10 kD、50 kD及100 kD)。 1.將使用pUC18模板及KOD酶在試管反應中產生之四種10 μL循環測序產物樣品添加至第一層中之埠1104 且經由第二層中之通道1105 將其驅至第二層中之室1106 。將200 μL去離子水添加至埠1120 (第一層中之通孔)至第二層中之儲集器1121 。接著將生物晶片安裝於兩個氣動界面之間。使用氣動系統軟體執行以下之壓力概況。除非另有說明，否則所有對應於生物晶片埠之電磁閥均被關閉。 2.將0.09 psig之壓力施加至埠1104 (其中埠1119 對大氣開放)達5秒將測序產物驅至層1中之毛細管閥1108 ，測序產物在其中持留。 3.將0.6 psig之壓力施加至埠1104 (其中埠1119 對大氣開放)達0.1秒以使樣品膨脹通過層1中之毛細管閥1108 且將其經層2中之通孔1111 傳遞入層2中之UF輸入室1112 。 4.將0.09 psig之壓力施加至埠1104 (其中埠1119 對大氣開放)達10-30秒(在不同實驗中使用不同時間)以完成測序產物至室1112 之傳遞。測序產物藉由層2中之毛細管閥1113 持留(參見圖12a及12b)。 5.將0.8 psig之壓力施加至埠1124 (其中埠1119 及1104 對大氣開放)達0.5秒，以驅動測序產物經由閥1113 進入過濾室1115 中。此亦清除保持之液體之輸入毛細管閥1108 。 6.將0.09 psig之壓力施加至埠1124 (其中埠1119 對大氣開放)達10-30秒以完成測序產物至室1115 之傳遞。測序產物保持於閥1113 處(參見圖12c)。 7.將7.5 psig之壓力緩慢施加於用於超濾之晶片之所有埠。在超濾期間，當經由過濾器1116 驅動液體時，測序產物彎月面保持在1113 受阻止而液體之前緣"回縮"。10 µL測序產物需要約120秒用於過濾。在過濾後壓力得以釋放(參見圖12c及12d)。 8.將0.09 psig之壓力施加至埠1120 (其中埠1124 對大氣開放)達3秒以驅使水進入通道1122 (層4中)且部分填充溢流室1123 (參見圖12e)。 9.將0.8 psig之壓力施加至埠1120 及1124 (其中埠1119 對大氣開放)以驅使水經由通道1122 中之通孔毛細管閥1110 進入室1112 。 10.將0.09 psig之壓力施加至埠1120 (其中埠1119 對大氣開放)達10-30秒以完成液體至室1112 之傳遞，該液體藉由閥1113 保持於其中(參見圖12f)。 11.將0.09 psig之壓力施加至埠1124 (其中埠1120 開口)以驅使室1123 及通道1122 中之水返回室1121 (參見圖12g)。 12.將0.8 psig之壓力施加至埠1124 (其中埠1119 及1104 對大氣開放)達0.5秒以將水經由閥1113 驅至過濾室1115 中。此亦清除保持之液體之輸入毛細管閥1108 。 13.將0.09 psig之壓力施加至埠1124 (其中埠1119 對大氣開放)達10-30秒以完成水至室1115 之傳遞。測序產物保持於閥1113 (參見圖12h)。如以上之步驟6，驅使水通過UF過濾器，完成第一洗滌。將步驟8-13額外重複一次。重複步驟8-12以部分填充室1115 ，其中水之最終體積用於溶離(參見圖12k)。將1.6 psig之真空施加至埠1104 ，其中所有其他埠被關閉1秒，自室1115 吸取一些水至室1112 中(最大運動由與液體之彎月面與對應於埠1119 之電磁閥之間的死空間之相同數量級之真空形成所決定)(參見圖12l)。使埠1104 對大氣開放達1秒，由於在液體與對應於埠1119 之閥之間產生的部分真空使液體移回至室1115 (參見圖12m)。將16-17重複50次以產生50次溶離循環。將0.09 psig之壓力施加至埠1124 達10秒(其中埠1119 對大氣開放)以驅動液體使其後彎月面在1113 處受阻。將0.7 psig/0.05秒之壓力施加至埠1124 (其中埠1119 對大氣開放)以分離溶離物(參見圖12n)。回收樣品且直接用如所述之Genebench^TM 運行，產生多達479 QV20個鹼基。實例 4 用於核酸萃取、模板擴增、循環測序、測序產物純化及純化產物之電泳分離與偵測的完全整合生物晶片 圖13說明16-樣品生物晶片1301 之實施例，其結合圖1之生物晶片之溶解及萃取、模板擴增及循環測序功能；圖11之晶片之超濾；及電泳分離及偵測。藉由子組件1302 進行超濾過程且可如實例1、2及3中所述進行；使1302 之底面上之輸送點1304 對準分離子組件1303 上之輸入孔1305 。使用反電極以預濃縮步驟電動執行注入。圖14中所說明之輸入孔1305 由液體接收孔1401 、主要分離電極1402 及反電極1403 組成。分離通道1306 通向孔儲集器1401 之底部。分離電極通常經鉑或金塗覆，且較佳為平面鍍金電極，其大體上覆蓋1401 之內表面之1、3或4。反電極為薄金、鋼或鉑導線(通常直徑為0.25 mm)，其已經一薄層(約10 µm)交聯聚丙烯醯胺塗覆。此在電極上形成水凝膠保護層。在面板d上，可將純化之測序產物(1401 中之黑點)轉移至孔中。在1402 與1403 之間施加正電位，帶負電之測序產物被引至1403 ，如面板c-d中一樣。1403 上之水凝膠層防止測序產物與金屬電極接觸且因此防止測序產物之電化學及損害。接著使反電極1403 相對於1402 浮動。接著在主要分離電極1402 與分離通道1306 之遠端的陽極(未圖示)之間施加正電位。此允許產物注入(面板e)且沿著1306 電泳以供分離及偵測(面板f)。如圖14中所示，此流程允許測序產物在通道1306 之端附近之濃度相對於自超濾傳遞之測序產物顯著增加。雖然此濃度對於一些應用而言為理想的，但在所有情況下並非為必需。在該等情況下，可使用不具有反電極1403 之圖14之孔來直接進行EKI。或者，在載入孔中之單一電極可為交叉-T或雙倍-T注入器之二分之一(參見，例如與本案同時申請之題為"PLASTIC MICROFLUIDIC SEPARATION AND DETECTION PLATFORMS"、代理人檔案號碼07-865-US之美國專利申請案)。分離在分離通道1306 中發生，且偵測經由雷射誘發之螢光在偵測區1307 中發生。在此生物晶片中，提供凹處1308 以使(例如)帕耳帖塊(未圖示)匹配1301 之低面以提供PCR及循環測序之熱循環。在儀器內之氣動界面(未圖示)夾持於晶片端部以提供微流體控制。II. 分離 (DEPARATION) 及偵測系統 A. 分離與偵測組件及其用途之詳細描述 1. 分離儀器 用如美國專利申請公開案第US 2006-0260941-A1號中所述之生物晶片及儀器進行DNA分離。分離晶片可為玻璃(參見美國專利申請公開案第US 2006-0260941-A1號)或塑料(與本案同時申請之題為"Plastic Microfluidic separation and detection platforms"、代理人檔案號碼07-865-US之美國專利申請案)，該等案之全文以引用之方式併入本文中。 2. 激發及偵測儀器 該儀器包含激發及偵測子系統用於與樣品相互作用且對樣品作訊問。樣品通常包括一或多個經染料(例如螢光染料)標記之生物分子(包括(但不限於)DNA、RNA及蛋白質)。激發子系統包含激發源及激發光束路徑，其中光學元件包括透鏡、針孔、鏡及物鏡，以調節並聚焦激發/偵測窗口中之激發源。樣品之光學激發可藉由一系列雷射器類型來完成，其中發射波長在400至650 nm之間的可見區內。固態雷射器可提供約460 nm、488 nm及532 nm之發射波長。此等雷射器包括(例如)來自Coherent (Santa Clara, CA)之Compass、Sapphire及Verdi產品。氣體雷射器包括發射約488 nm、514 nm、543 nm、595 nm及632 nm之可見波長的氬離子及氦氖雷射器。具有可見區發射波長之其他雷射器可自CrystaLaser(Reno, NV)購得。在一實施例中，可使用488 nm固態雷射器Sapphire 488-200(Coherent, Santa Clara, CA)。在另一實施例中，可使用波長超過可見光範圍之光源來激發具有超過可見光範圍之吸收及/或發射光譜的染料(例如，紅外或紫外發射染料)。或者可藉由使用具有適於染料激發之發射波長之非雷射器光源(包括發光二極體及燈)獲得光學激發。偵測子系統包含一或多個光學偵測器、波長色散裝置(其進行波長分離)及一或一系列光學元件，該或該等光學元件包括(但不限於)透鏡、針孔、鏡及物鏡以自在激發/偵測窗口處存在之經螢光團標記之DNA片段收集發射之螢光。所發射之螢光可來自單一染料或染料組合。為辨別信號以確定其來自發射染料之貢獻，可使用螢光之波長分離。此可藉由使用二向色鏡及帶通過濾器元件(可自包括Chroma, Rockingham, VT及Omega Optical, Brattleboro, VT之眾多供應商購得)達成。在此組態中，發射之螢光通過一系列二向色鏡，其中波長之一部分將被鏡反射以繼續沿著路徑行進，而其他部分將通過。一系列離散光偵測器(各者定位於二向色鏡之末端)將偵測特定範圍波長之光。可將一帶通過濾器定位於二向色鏡與光偵測器之間以在偵測前進一步使波長範圍變窄。可用以偵測波長-分離信號之光學偵測器包括光電二極體、崩潰光電二極體、光電倍增管模組及CCD攝影機。此等光學偵測器可購自諸如Hamamatsu(Bridgewater, NJ)之供應商。在一實施例中，藉由使用二向色鏡及帶通過濾器分離波長成分，且用光電倍增管(PMT)偵測器(H7732-10, Hamamatsu)偵測此等波長成分。可選擇二向色鏡及帶通組件以使PMT之各者上之入射光由對應於螢光染料之發射波長之窄波長帶組成。該帶通通常經選擇以具有波長範圍在1與50 nm之間之帶通的螢光發射峰為中心。該系統能夠進行八種顏色偵測且可經設計具有八個PMT及相應之一套二向色鏡與帶通過濾器以將發射之螢光分成八種不同顏色。藉由應用額外之二向色鏡、帶通過濾器及PMT可偵測八種以上染料。圖15展示離散帶通過濾器及二向色過濾器實施例之光束路徑。此波長辨別及偵測組態之一種整合形式為H9797R, Hamamatsu, Bridgewater, NJ。辨別構成螢光信號之染料之另一方法包括使用諸如稜鏡、繞射光柵、透射光柵(可自包括ThorLabs, Newton, NJ；及Newport, Irvine, CA之眾多供應商購得)；及攝譜儀(可自包括Horiba Jobin-Yvon, Edison, NJ之眾多供應商購得)之波長色散元件及系統。在此操作模式中，螢光之波長成分分散於物理空間。沿此物理空間置放之偵測器元件偵測光且使偵測器元件之物理位置與波長具相關性。適用於此功能之偵測器為基於陣列的且包括多元件光電二極體、CCD攝影機、後端薄型CCD攝影機、多陽極PMT。熟習該項技術者將能夠應用波長色散元件及光學偵測器元件之組合以產生能夠辨別來自系統中所使用之染料之波長的系統。在另一實施例中，使用攝譜儀替代二向色及帶通過濾器自激發螢光分離波長成分。攝譜儀設計之細節可在John James,Spectrograph Design Fundamental , Cambridge, UK: Cambridge University Press, 2007中獲得。在本申請案中使用具有凹面全像光柵，光譜範圍為505-670 nm之攝譜儀P/N MF-34(P/N 532.00.570)(HORIBA Jobin Yvon Inc, Edison, NJ)。可用線性32-元件PMT偵測器陣列(H7260-20, Hamamatsu, Bridgewater, NJ)完成偵測。收集之螢光在針孔上成像、反射、色散、及藉由凹面全像光柵成像於線性PMT偵測器上，該偵測器安裝於攝譜儀之輸出端口處。基於PMT之偵測器之使用利用PMT偵測器之低的暗雜訊、高敏感性、高動態範圍及快速響應特徵。用於偵測激發螢光之攝譜儀及多元件PMT偵測器之使用允許可應用於該等系統中及道中之染料數目及染料之發射波長之彈性，無需物理重組態儀器之偵測系統(二向色、帶通及偵測器)。自此組態收集之資料為橫越對於各道之各掃描而言可見波長範圍之波長依賴性光譜。每掃描產生之完整光譜提供根據染料發射波長及可存在於樣品中之染料數目之染料彈性。此外，當陣列中所有PMT元件平行讀出時，光譜儀及線性多元件PMT偵測器之使用亦允許極快讀出速率。圖16展示多元件PMT及攝譜儀實施例之光束路徑。儀器可使用操作之起始模式，以同時偵測多道且同時偵測多波長。在一組態中，激發光束同時衝擊於所有道上。來自此之螢光藉由諸如CCD攝影機或陣列之二維偵測器收集。在此收集之起始模式中，使用波長色散元件。偵測器之一維表示物理波長分離，而另一維表示空間或道-道分離。對於多樣品之同時激發及偵測而言，使用掃描鏡系統(62)(P/N 6240HA, 67124-H-0及6M2420X40S100S1, Cambridge technology, Cambridge MA)以控制激發及偵測光束路徑以使生物晶片之道之各者成像。在此操作模式中，掃描鏡控制光束路徑，自道至道自第一道至最後道連續掃描，且再次自第一道至最後道再重複該過程。使用諸如美國專利申請公開案第US 2006-0260941-A1號之尋找道之演算法來識別道之位置。用於同時多道及多種染料偵測之光學偵測系統之實施例展示於圖16中。螢光激發及偵測系統40 藉由經由一部分微通道之各者掃描能源(例如雷射器光束)來激發藉由DNA樣品(例如含有在一套STR位置擴增後之DNA片段)之電泳分離之成分，同時收集並傳遞來自染料之誘發螢光至一或多個光偵測器用於記錄，且最終分析。在一實施例中，螢光激發及偵測總成40 包括一雷射器60 、一掃描器62 、一或多個光偵測器64 及各個鏡68 、攝譜儀及用於經由開口42 將自雷射器60 發出之雷射光束傳遞至測試模組55 且返回至光偵測器64 之透鏡72 。掃描器62 將入射雷射光束移動至相對於測試模組55 之各種掃描位置。特定言之，掃描器62 將雷射光束移動至測試模組55 中之各微通道之有關部分以偵測各別分離成分。多元件PMT64 收集來自測試模組55 之資料(例如來自變化長度之DNA片段之螢光信號)且將該資料經由連接至埠75 之電纜電子提供至位於保護層50 外之資料獲得及儲存系統。在一實施例中，資料獲得及儲存系統可包括可購自Option Industrial Computers(l3 audreuil-Dorion, Quebec, Canada)之加固電腦。在另一實施例中("起始模式")，激發源同時入射至所有偵測點上，且來自所有偵測點之螢光經同時收集。同時可經兩維偵測器陣列進行光譜色散(偵測螢光之波長光譜)及空間色散(偵測點)。在此組態中，該2維偵測器陣列定位於該系統中以使光譜成分橫越陣列偵測器之一維(列)成像並偵測，而空間成分橫越陣列偵測器之另一維成像並偵測。較佳儀器使用操作之掃描模式，而非"起始"模式。在掃描模式中，當掃描器與欲訊問之道符合且在其入射在另一通道上之前，各通道之信號需要被收集、整合、及讀出。具有快速讀出之偵測器允許最佳光收集及整合，轉譯成較高之信號-雜訊效能。理想地，偵測器之讀出時間應顯著小於掃描器與通道符合之總時間。多元件PMT可在小於0.7 ms之時間讀出且此讀出時間遠小於各個別通道之偵測的整合時間。針孔上之螢光入射光可經光柵根據其波長組成色散且聚焦於線性多陽極PMT偵測器陣列上。該偵測器提供32個電流輸出，陣列中每元件中之一者對應於入射於元件上之光子之數目。在多樣品(或道)偵測過程中，當雷射器在適當位置激發所選擇之道，積分電路將整合PMT輸出電流至產生與整合PMT電流成比例之輸出電壓。同時，使用Analog Devices(Norwood, MA)微分驅動器IC(P/N SSM2142)將單一端輸出電壓轉換成微分模式。在整合時間(藉由掃描速率及道之數目確定)結束時，資料獲得系統將讀出微分信號且在其緩衝器中保存資料。當保存資料後，系統將移動掃描器以將雷射光束轉移至另一選擇之道，同時重設積分電路。各單一元件PMT模組具有其自身之積分電路。對於8顏色偵測系統而言，存在8個PMT模組及8個積分電路。使用相應數目之PMT模組及積分電路可添加額外顏色。由於PMT元件(H77260-20, Hamamatsu, Japan)之各者具有與單一PMT管(H7732-10, Hamamatsu, Japan)類似或更快之信號反映，且平行讀出，因此能夠非常快速地操作此偵測器。當與光譜儀耦合時，此光譜儀及多陽極偵測器系統能提供橫越可見光譜(450 nm至650 nm)之完全光譜掃描，讀出時間小於0.1 ms。提供快速更新率之能力允許將此光譜儀/偵測器系統應用於在單一運行中連續偵測多道之掃描模式實施例。基於PMT之偵測器之使用提供低雜訊、高敏感性及高動態範圍及快速響應。具有凹面全像光柵(Horiba Jobin-Yvon)及多陽極PMT偵測器之140 mm光譜儀為H7260-20偵測器(Hamamatsu, Japan)。其他光譜儀組態及多陽極PMT偵測器亦可用於本申請案。使用信號處理演算法修正、過濾及分析資料來獲得測定電泳圖之核苷酸鹼基。此過程由定位一可呼叫信號、修正信號基線、過濾雜訊、移除顏色串擾(cross-talk)、識別信號峰，及測定相關之鹼基。進行定位可呼叫信號以自信號之開始與結束移除無關資料且藉由使用臨限值來完成。接著，自信號移除背景，因此該信號對於所有偵測之顏色具有通用基線。最終，應用低通濾波器以自信號移除高頻雜訊。為消除偵測之顏色歧義，計算一加權矩陣且應用於信號以擴增核苷酸-染料光譜之顏色-空間。此顏色分離矩陣之計算使用Li等人，Electrophoresis 1999, 20, 1433-1442之方法完成。在此適應中，自相關之檢定中所使用之染料數目"m"及偵測器元件數目之"n"計算"m×n"顏色分離矩陣。來自偵測器空間(PMT元件)之信號轉化成染料空間係藉由如下所述之矩陣運算來進行：D=CSM×PMT，其中D為m染料之各者之染料空間中之信號，CSM為顏色分離矩陣，且PMT為具有來自偵測器之n元件之各者之信號之矩陣。其次，使用零交叉濾波器及頻率分析之組合識別顏色分離信號中之峰。最終，對於測片段大小之應用，修正之迹線為等位基因-對應的(allele-called)以識別各片段且基於測大小標準分配片段大小。對於DNA測序應用而言，修正之迹線為鹼基-對應的(base-called)以將四種核苷酸之一者與迹線中各峰相關聯。鹼基對應之詳細描述可在Ewing等人，Genome Research , 1998, 8, 175-185，及Ewing等人，Genome Research , 1998, 8, 186-194，該等揭示案之全文以引用之方式併入本文中。 3. 染料標記 連接於寡核苷酸及經修飾之寡核苷酸上之染料標記可為合成的或購買的(例如Operon Biotechnologies、Huntsville、Alabama)。大量染料(大於50種)可應用於螢光激發應用中。此等染料包括彼等來自螢光素、若丹明AlexaFluor、Biodipy、Coumarin及Cyanine染料家庭。此外，亦可使用抑止劑用於標記寡序列以最小化背景螢光。可購得且可使用具有410 nm(Cascade Blue)至775 nm(Alexa Fluor 750)之發射最大值的染料。在500 nm至700 nm範圍之間之染料具有在可見光譜中且可使用習知光電倍增管偵測之優點。可購得之染料之廣泛範圍允許選擇具有發射波長遍及偵測範圍之染料組。已報導能夠辨別許多染料之偵測系統用於流式細胞儀應用(參見Perfetto等人， Nat. Rev. Immunol. 2004, 4, 648-55；及Robinson等人， Proc of SPIE 2005, 5692, 359-365)。螢光染料具有自其峰發射波長藍移通常20至50 nm之峰激發波長。因此，使用大範圍發射波長之染料可需要使用多重激發源，其中在發射波長範圍內激發波長達成染料之有效激發。或者，可使用能量轉移染料以使具有單一發射波長之單一雷射器適用於激發所關注之所有染料。此藉由將能量轉移部分連接至染料標記而達成。此部分通常為另一螢光染料，其具有與光源(例如雷射器)之激發波長相容之吸收波長。非常接近於發射器之此吸收器之放置使得吸收能量自吸收器轉移至發射器，允許長波長染料之更有效激發(Ju等人， Proc Natl Acad Sci U S A 1995, 92, 4347-51)。經染料標記之二脫氧核苷酸可購自Perkin Elmer，(Waltham, MA)。 B. 實例實例 5. 核酸之六色分離及偵測 以下實例說明經6種螢光染料標記之核酸片段之分離及偵測，且展示光譜儀/多元件激發/偵測系統之顏色解析能力。藉由在多重PCR擴增反應中應用經螢光標記之引子以6-FAM、VIC、NED、PET染料標記DNA片段。在此反應中，在根據製造商所推薦之條件下使1 ng人類基因DNA (9947A)在25 μL反應中擴增(AmpFlSTR Identifiler, Applied Biosystems)。移除2.7 μL PCR產物且與0.3 μL GS500-LIZ測大小標準物(Applied Biosystems)及0.3 μL HD400-ROX測大小標準物相混合。添加HiDi(Applied Biosystems)至總共13 μL且將樣品***分離生物晶片之樣品孔中且經受電泳。使用由一系列四操作組成之Genebench進行DNA之電泳分離：預電泳、載入、注入及分離。此等操作在微流體生物晶片上進行，將該生物晶片加熱至50℃之均一溫度。生物晶片含有16通道系統用於多次分離及偵測，各由注入器通道及分離通道組成。用於分析之DNA藉由DNA之電泳輸送經由篩分基質沿分離通道分離。生物晶片之分離長度在160至180 mm範圍內。第一步驟為預電泳，其藉由沿通道長度施加160 V/cm場達六(6)分鐘來實現。將分離緩衝液(TTE1X)注入陽極、陰極及廢物孔中。將用於分析之樣品注入樣品孔中且自樣品孔向廢物孔施加175 V達18秒，隨後橫越樣品及廢物孔施加175 V，且在陰極施加390 V達72秒，將樣品載入分離通道中。樣品之注入藉由沿分離通道長度施加160 V/cm場完成，同時分別橫越樣品及廢物孔施加50 V/cm及40 V/cm之場。用注入電壓參數30分鐘來繼續分離，其中在該30分鐘期間，光學系統偵測DNA之分離帶。此資料收集速率為5 Hz且PMT增益設置為-800 V。載入含有擴增DNA之16個樣品用於同時分離及偵測。收集來自32-元件PMT之各者之信號作為時間之函數以產生電泳圖。所得電泳圖(圖17)展示對應於16道之一者的激發/偵測窗口處之DNA片段之存在的峰。此外，對於各峰而言，32-元件PMT之各元件的相對信號強度對應於與DNA片段相關之染料之光譜含量(或染料，若一種以上染料存在於偵測窗口)。圖18展示所偵測之染料的發射光譜，及基板之背景光譜。將基板背景光譜自該光譜中減去得到峰之各者。進行此實踐導致識別6種不同染料光譜。6種染料之光譜在相同曲線上疊置。將此資料與實際公開之染料光譜的相比較顯展示該等染料之相對值類似於公開之資料。此實例展示說明該系統能偵測且分辨反應溶液中之6種染料。此之光譜輸出用於產生顏色修正矩陣且將信號自偵測器空間轉換至染料空間表示(圖19及20)。實例 6. 核酸之八色分離及偵測 在此實例中，展示經螢光染料標記之酸的8種染料分離及偵測。對於8位點之前向引子及反向引子對序列係自公開之序列中選擇(Butler等人， J Forensic Sci 2003, 48, 1054-64)。雖然文件中所述之任何位點及因此之引子對亦可用於本實例，但所選擇之位點為CSF1P0、FGA、THO1、TPOX、vWA、D3S1358、D5S818及D7S820。對於引子對而言，前向引子之各者係經獨立之螢光染料標記(Operon Biotechnologies, Huntsville, Alabama)。所選擇之用於連接至引子之染料包括Alexa Fluor Dyes 488、430、555、568、594、633、647及Tamra。眾多其他染料為可購得的且亦可用作標記。各位點係根據(Butler, 2003,Id. )之PCR反應方案獨立地擴增以產生具有經相應染料標記之片段之反應溶液。PCR反應之模板為1 ng人類基因DNA(來自Promega, Madison WI之類型9947A)。各PCR反應物係藉由經PCR淨化管柱淨化而純化，其中將引子(經標記及經染料標記之引子)及酶移除且藉由DI溶離劑交換PCR緩衝液。淨化之所得產物為DI水中經標記DNA片段之溶液。使用MinElute^TM 管柱(Qiagen, Valencia, CA)根據Smith方案進行經染料標記之產物之淨化。共進行八次反應。將八次淨化之PCR反應物以產生相等信號強度之峰之比率混合在一起，從而產生含有經8種不同染料標記之片段的混合物。或者，可將8個位點之引子混合在一起以形成主要引子混合物以供多重擴增。用如實例1中所述之儀器及方案分離及偵測此溶液。調節攝譜儀之光柵以使8種染料之發射落於偵測器元件之32像素範圍內。調節所裝載以供分析之樣品數量以使所偵測之信號屬於偵測系統之動態範圍。實例 7. 光譜儀 / 多元件 PMT 系統以下實例說明用圖16之光譜儀/多元件PMT系統對經標記DNA片段之分離/偵測，特定言之用於識別DNA模板之序列。在此反應中，根據所推薦之反應條件用GE Amersham BigDye^TM 測序套組使0.1 pmol DNA模板M13及M13測序引子擴增。藉由乙醇沈澱且再懸浮於13 μL DI水中來淨化反應混合物。在如實例5中所述之電泳分離條件下分離樣品。改變樣品裝載條件且藉由將175 V越過樣品孔施加至廢物孔達105秒來進行。圖21展示DNA序列之電泳圖，其中有色迹線表示對應於所使用之4種染料各者之光譜最大值的偵測器元件。所獲得之序列為519個鹼基的對應於Phred品質分數＞20之鹼基及435個鹼基之QV30(圖22)。實例 8. 兩種測序反應產物之同時分離及偵測 在此實例中，同時在單一分離通道中進行來自兩個DNA模板循環測序之片段之分離及偵測。可藉由如下所述之經染料標記之終止子反應或經染料標記之引子反應製備循環測序反應：對於經染料標記之終止子反應： 準備各模板片段之循環測序反應，其中模板片段由以下各物組成：適用於所關注之模板序列之測序引子；及用於進行DNA測序之試劑，包括循環測序緩衝劑、聚合酶、寡核苷酸、二脫氧核苷酸及經標記之二脫氧核苷酸。使用八種不同染料用於標記。在第一循環測序反應中，使用一組經4種染料標記之二脫氧核苷酸。在第二循環測序反應中，使用另一組經4種染料標記之二脫氧核苷酸(其中發射波長不同於彼等在第一循環測序反應中所使用之四種染料)。根據使各反應多次熱循環之方案獨立進行各循環測序反應。各熱循環包括變性、退火及延伸步驟，其中溫度及次數遵循桑格方案(參見，桑格等人， Proc Natl Acad Sci U S A 1977, 74, 5463-7)。合併來自兩反應之循環測序產物以形成來自兩個DNA模板之各者的由經總共八種獨特之染料標記之DNA片段組成的樣品。對於經染料標記之引子反應： 或者，用於分離及偵測之樣品可藉由使用經引子標記之循環測序製造。對於各DNA模板進行四個循環測序反應。各反應為由經標記之測序引子及進行DNA測序之試劑(包括循環測序緩衝劑、聚合酶、寡核苷酸)所組成之循環測序反應。此外，各反應將包括二脫氧核苷酸(ddATP、ddTTP、ddCTP或ddGTP)及一經標記引子中之一者。各與引子締合之染料具有獨特之發射波長且與循環測序溶液中之二脫氧核苷酸之類型(ddATP、ddTTP、ddCTP或ddGTP)相關。根據使各反應多次熱循環之方案獨立進行各循環測序反應。各熱循環包括變性、退火及延伸步驟，其中溫度及次數遵循桑格方案(參見，桑格，1977,Id. )。對於循環測序第二DNA模板，使用另一組4種染料(其中發射波長不同於在第一循環測序反應中所使用之四種染料)。將所有八個反應(各具有不同染料)之產物混合在一起以形成由來自兩種DNA模板之各者之DNA片段組成的樣品。用於分離及偵測之樣品： 藉由乙醇沈澱淨化測序反應之各者。樣品之分離及偵測遵循實例8之方案。分離及偵測之結果為生成兩種不同DNA序列，對應於兩種模板DNA片段之各者。本實例之方法可經改良以允許使用四之倍數之染料來偵測單一分離通道中成該倍數之DNA序列(例如用於同時偵測3種序列之12種染料、用於同時偵測4種序列之16種染料、用於同時偵測5種序列之20種染料，等等)。最終，經標記片段之分離不需受限於電泳。實例 9 在單一通道中之 500 個或 500 個以上的位點之分離及偵測 存在若干種可應用於臨床診斷之核酸分析之應用，包括DNA及RNA測序及片段大小測定。在此實例中，同時偵測10種顏色之使用使得訊問多達500個位點。例如，可使用大量片段之大小分析來識別病原體或表徵個體基因組內之許多位點。在產前及胚胎植入前遺傳診斷之背景下，目前藉由核型及藉由螢光原位雜交(FISH)診斷非整倍體。在FISH研究中，每細胞兩個信號之存在表明在該細胞中存在給定位點之兩複本，一個信號表明單體性或部分單體性，且三個信號表明三體性或部分三體性。FISH通常使用約10個探針以分析細胞是否含有正常染色體補體。然而此方法不允許對整個基因組之詳細檢視，且由FISH得知表現為正常之細胞很可能具有不能為該技術所偵測之主要異常。本發明之教示使用多色分離及偵測以允許約500個染色體位點廣泛分散遍及所有欲分析之染色體，從而允許對染色體結構更詳細之分析。在此實例中，自公開之序列中選擇約500個位點之引子對序列，其中各位點作為每單倍體基因組之單一複本存在。此外，選擇10組50個引子對以使各組界定相應之DNA片段組，以使該等片段中無相同大小者。對於各組而言，將引子對之前向引子以一種螢光染料標記，且無兩組共有相同染料。所選擇之用於連接至引子之染料為Alexa Fluor Dyes 488、430、555、568、594、633、647、680、700及Tamra。眾多其他染料為可購的且亦可用作標記。位點可在一或若干平行PCR反應中擴增，此如上述"METHODS FOR RAPID MULTIPLEXED AMPLIFICATION OF TARGET NUCLEIC ACIDS"中所述。使用本文中所述之方法分離及偵測經擴增之引子。在單一分離通道中，可根據大小精確識別所有500個片段，每十種染料識別50個片段。位點、染料及分離通道之數目可基於所想要之應用變化。若需要可藉由使用較小數目之染料標記或每標記產生較少DNA片段來偵測較小數目之片段；因此，小於500、小於400、小於300、小於200、小於100、小於75、小於50、小於40、小於30或小於20個片段可如所希望被偵測。每道可識別之位點之最大數目係基於分離系統之讀出長度及解析(例如20至1500鹼基對範圍內之DNA片段之單一鹼基對解析導致數百片段)乘以可偵測之不同染料之數目(如上所述，可獲得數打)。因此，在單一分離通道中可識別數以千計之位點，且當開發額外之染料時，該數目將增加。 I. Integration and integration system A. General description of integration The use of microfluidics allows the fabrication of features that perform more than one function on a single biochip. Two or more of these functions can be in microfluidic connection to enable continuous processing of the sample; this combination is called integration. Although it is not necessary to implement all processes for any given application, there is a series of possible functions or constituent processes that must be integrated to achieve any given application. Therefore, the selected integration method must be suitable for effectively connecting several different constituent processes in different orders. The processes that can be integrated include (but are not limited to) the following items: 1. Sample insertion; 2. Remove foreign substances (such as large particles such as dust and fibers); 3. Cell separation (ie, remove those that contain substances to be analyzed Cells other than those of the nucleic acid, such as removing human cells from clinical samples containing the microbial nucleic acid to be analyzed (and correspondingly removing human genomic DNA)); 4. Concentrating cells containing the nucleic acid of interest; 5. Lyse cells and extract nucleic acids; 6. Purify nucleic acids from lysates; At the same time, it is possible to concentrate nucleic acids to a smaller volume; 7. Purify nucleic acids before amplification; 8. Purify after amplification; 9. Purify before sequencing; 10. Sequencing; 11. Purification after sequencing (for example to remove unmerged dye-labeled terminators and ions that interfere with electrophoresis); 12. Nucleic acid isolation; 13. Nucleic acid detection; 14. Reverse transcription of RNA; 15. Purification before reverse transcription; 16. Purification after reverse transcription; 17. Nucleic acid ligation; 18. Nucleic acid quantification; 19. Nucleic acid hybridization; and 20. Nucleic acid amplification (such as PCR, rolling circle amplification, strand displacement amplification and multiple displacement amplification) increase). One of many methods that can combine some of these processes is an integrated system for human recognition by STR analysis. Such systems may require combining DNA extraction, human-specific DNA quantification, adding quantitative DNA to PCR reactions, multiplex PCR amplification and separation and detection (optionally incorporating purification steps to remove reaction components or primers ). One or more samples of whole blood, dried blood, inner cheek surfaces, fingerprints, sexual assault, contact, or other forensic related samples can be collected by techniques such as wiping (see Sweet et al., J. Forensic Sci . 1997, 42, 320-2). Exposure to lysate (optionally in the presence of agitation) releases DNA from the swab into the test tube. B. General description of integrated components and their uses 1. Sample collection and initial processing For many applications, the following discrete components are advantageously integrated into biochips: sample insertion, removal of foreign substances, removal of interfering nucleic acids, and concentration The cell of interest. In general, the pre-processing component of the bio-wafer receives a sample, performs the initial removal of particles and cells containing foreign nucleic acids, and concentrates the cells of interest to a smaller volume. One method is to use a sample tube that can hold a swab (such as a "Q-tip") and is filled with a dissolution solution to perform the dissolution and extraction steps. The swab can be placed in contact with several cell-containing sites (including blood stains, fingerprints, water, air filters) or clinical sites (eg, buccal swabs, wound swabs, nasal swabs). The interface between these tubes and other components of the biochip may include filters for removing foreign materials. Another method is to use a large volume of blood or environmental sample collection cartridges, which process 1-100 mL samples. In the case of blood, leukocyte reduction mediators can remove human leukocytes and interfere with DNA when passed through microorganisms containing the nucleic acid of interest. For environmental samples, large mesh filters can be used to remove dust and dirt, while small mesh filters (eg, <20 μm, <10 μm, <5 μm, <2.5 μm, <1 μm, < 0.5 μm, <0.2 μm, <0.1 μm filters) can be used to trap microorganisms and concentrate them into a small volume. These pretreatment components can be independent consumables or attached to the integrated wafer during manufacturing. Alternatively, the biochip can be designed for differential lysis to separate cells according to type (eg sperm from vaginal epithelial cells or red blood cells from bacteria). 2. Dissolution and extraction Various dissolution and extraction methods can be used. For example, a typical procedure involves applying heat after mixing the sample with a small amount of degrading enzymes (such as proteinase-K, which breaks down the cell wall and releases nucleic acids). Other available methods are sonic treatment and ultrasonic treatment, either or both of which are sometimes performed in the presence of beads. For example, for ¹⁰⁶ samples containing ¹⁰⁶ cells or the cell of the dissolution and extraction. Depending on the application, can be used in the biochip of the present invention and method of a small number of starting cells, less than ^105, less than ^104, less than ^103, less than ^102, less than 10, and less than 1 in the case of multi-copy sequence. 3. Purification of nucleic acid A form of nucleic acid purification can be obtained by inserting a purification medium between the input channel and the output channel. This purification medium may be based on silica fibers and use chaotropic-salt reagents to dissolve the biological sample, expose DNA (and RNA) and bind the DNA (and RNA) to the purification medium. The lysate is then passed through the purification medium via the input channel to bind the nucleic acid. The bound nucleic acids are washed with an ethanol-based buffer to remove contaminants. This can be achieved by passing the washing reagent through the input channel through the purification membrane. The bound nucleic acid is then dissociated from the membrane by flow of a suitable low-salt buffer (for example, Boom US 5,234,809). A variation of this method involves the use of different configurations of solid phases. For example, silica gel can be used to bind nucleic acids. Paramagnetic silicon dioxide beads can be used, and their magnetic properties are used to secure them to the channel or chamber wall during the bonding, washing, and dissolution steps. Non-magnetized silica beads can also be used, which are packed in a dense'pillar' (where it is held by glass frit) (usually manufactured in the plastic of the device, but these can also be Inserted during assembly), or "free" during a specific stage of its operation. Free beads can be mixed with nucleic acids and then flowed in the device relative to the glass frit or weir to trap them so that they do not interfere with downstream processes. Other forms include sol-gels with silica particles distributed in the gel medium and polymer monomers with silica particles, where the carrier is cross-linked for greater mechanical stability. Basically, any nucleic acid purification method functioning in the conventional configuration can be applied to the integrated biochip of the present invention. 4. Nucleic acid amplification Various nucleic acid amplification methods can be used, such as PCR and reverse transcription PCR, which require thermal cycling between at least two temperatures and more usually three temperatures. Isothermal methods such as strand displacement amplification can be used, and multiple displacement amplification can be used for whole genome amplification. The full text of the teaching of the US patent application (Agent File Number 08-318-US) entitled "METHODS FOR RAPID MULTIPLEXED AMPLIFICATION OF TARGET NUCLEIC ACIDS" that was filed on the same day is incorporated herein by reference (as described above) ). 5. Quantification of nucleic acids One method for quantification in a microfluidic format is based on real-time PCR. In this quantitative method, a reaction chamber is made between the input channel and the output channel. The reaction chamber is coupled to a thermal cycler, and an optical excitation and detection system is coupled to the reaction chamber to allow the fluorescence from the reaction solution to be measured. The amount of DNA in the sample is related to the fluorescence intensity from the reaction chamber of each cycle. See, for example, Heid et al., Genome Research 1996, 6, 986-994. Other quantitative methods include the use of intercalating dyes such as picoGreen, SYBR, or ethidium bromide before or after amplification, which can then be detected using fluorescence or absorbance. 6. Secondary purification For STR analysis, multiple amplified and labeled PCR products can be used directly for analysis. However, electrophoretic separation performance can be greatly improved by purifying the product to remove ions that are necessary for PCR but interfere with separation or other subsequent steps. Similarly, purification following cycle sequencing or other nucleic acid processing may be applicable. In general, any purification steps following the initial extraction or purification of nucleic acids can be considered as secondary purification. Various methods can be used, including ultrasound treatment, where small ions/primers/unincorporated dye labels are driven through the filter, leaving the desired product on the filter, which can then be dissolved and used directly for separation or subsequent In the module. Ultrafiltration media include polyether ballast and regenerated cellulose "woven" filters, as well as track erosion membranes (where pores of uniform height are formed in extremely thin (1-10 µm) membranes). The latter has the advantage of collecting products that are larger than the pore size on the surface of the filter rather than trapping products at some depth below the surface. The same method as described above (ie, typical silica solid phase purification) can also be used to purify the amplified nucleic acid. Other methods include the use of hydrogels, cross-linked polymers with pore size variability, that is, the pore size changes in response to environmental variables such as heat and pH. In one case, the pores are dense and the PCR product cannot pass through. When the pores are enlarged, the hydrodynamic or electrophoretic flow of the product may pass through the pores. Another method is to use hybridization, where the product hybridizes non-specifically to random DNA immobilized on a surface (such as the surface of a bead) or specific hybridization (where the complementary system of sequence tags on the product is located on a solid surface). In this method, the product of interest is immobilized by hybridization and unwanted substances are removed by washing; the duplex is then heated to melt and release the purified product. 7. Cycle sequencing reaction Typical cycle sequencing requires thermal cycling, which is almost the same as PCR. The preferred method is that they use dye-labeled terminators so that each extension product bears a single fluorescent label corresponding to the final base of the extension reaction. 8. Injection, separation and detection The injection, separation and detection of labeled nucleic acid fragments in the electrophoresis channel can be performed in various ways. This has been applied on the same day and is entitled "PLASTIC MICROFLUIDIC SEPARATION AND DETECTION PLATFORMS" (given agent US file application number 07-865-US) is described in the US patent application, the entire text of which is incorporated herein by reference. First, the cross injector as discussed therein can be used to inject a portion of the sample. In an alternative embodiment, electric injection ("EKI") may be used. In either case, further concentration of the sequencing product near the open end of the loading channel (in the case of cross-injection) or separation channel (in the case of EKI) can be performed by electrostatically concentrating the product near the electrode. The two electrode sample holes on the electrophoresis portion of the wafer are shown in FIG. Both electrodes are coated with a permeable layer, which prevents DNA from contacting the electrode metal, but allows ions and water to enter between the sample well and the electrode. Such permeable layers may be formed from cross-linked polypropylene amide (see US Patent Application Publication US 2003-146145-A1). The electrode furthest from the channel opening is the separation electrode, and the electrode closest to the channel opening is the counter electrode. By applying positive electricity to the counter electrode relative to the separation electrode, DNA will be sucked to the counter electrode and concentrated near the opening of the separation channel. By floating the counter electrode and using the separation electrode and anode injection at the far end of the separation channel, the concentrated product is injected electrically. C. Integration method The biochip also contains several different components for integrating functional modules. These components involve transporting liquid from one point on the biowafer to another, controlling the flow rate for processes that depend on flow rate (e.g., certain washing steps, particle separation and dissolution), and gate the time of fluid movement on the biowafer And spacing (eg by using some form of valve), and fluid mixing. Various methods can be used for fluid transportation and controlled fluid flow. One method is positive displacement pumping, where during movement, the plunger in contact with the fluid or intervening gas or fluid drives the fluid to move a precise distance based on the volume displaced by the plunger. An example of such a method is a syringe pump. Another method is to use pneumatic, magnetic or other means to actuate the integrated elastic membrane. Individually, these membranes can be used as valves to contain fluid in a defined space and/or to prevent premature mixing or transfer of fluid. However, when used in series, these membranes can form a pump similar to a peristaltic pump. By synchronized, continuous actuation of the membrane, the fluid can be "pushed" from its rear side, because the membrane on the front side is opened to accept the moving fluid (and any replacement air in the channel of the evacuation device). The preferred method for actuating these membranes is pneumatic actuation. In these devices, the bio-wafer is composed of a fluid layer, at least one of the fluid layers has a membrane, and one side of the membranes is exposed to the fluid channel and chamber of the device. The other side of the membrane is exposed to a layer of pneumatic manifold perpendicular to the pressure source. The membranes are opened or closed by applying pressure or vacuum. Valves that are normally open or normally closed can be used to change state under pressure or vacuum applications. Note that any gas can be used for actuation because the gas is not in contact with the fluid under analysis. Another method of driving the fluid and controlling the flow rate is to directly apply a vacuum or pressure on the fluid itself by changing the pressure at the front meniscus, back meniscus, or both meniscus of the fluid. Apply appropriate pressure (usually in the range of 0.05-3 psig). The flow rate can also be controlled by making the size of the fluid channel suitable. This is because the flow rate is proportional to the differential pressure of the two ends of the fluid and the fourth power of the hydraulic diameter, and is inversely proportional to the length of the channel or liquid plug and its viscosity . Various active valves can be used to achieve fluid gating. The foregoing can include piezoelectric valves or solenoid valves, which can be directly incorporated into the wafer, or applied to a bio-wafer to connect ports on the main wafer body to the valves, direct fluid into the valves, and then return to the wafer . One of the disadvantages of these types of valves is that for many applications, it may be difficult to manufacture and too expensive to incorporate a disposable integrated device. As mentioned above, the preferred method is to use a membrane as the valve. For example, a membrane actuated by 10 psig can be used to successfully contain the fluid subjected to PCR. In some applications, capillary microvalves (which are passive valves) may be preferred. Basically, microvalves are deflated in the flow path. In microvalves, when the pressure applied to the fluid is below a critical value called burst pressure, surface energy and/or geometric features such as sharp edges can be used to prevent flow, where the burst pressure is often given by the following relationship: P _valve α (γ/d _H )*sin(θ _c ), where γ is the surface tension of the liquid, d _H is the hydraulic diameter of the valve (defined as 4×(cross-sectional area)/section perimeter), and θ _c is The contact angle between the liquid and the valve surface. The properties that make passive valves better for certain applications include: extremely low dead volume (usually in the picoliter range) and small physical extent (each of which is only slightly larger than the access valve and the exit valve Channel). The small physical range allows high-density valves on a given surface of the biological wafer. In addition, some capillary valves are very easy to manufacture, and they basically consist of small holes in plastic sheets that are surface-treated or not. Proper use of capillary valves can reduce the total number of membrane valves required, simplify overall manufacturing and form a stable system. There are two types of capillary valves constructed in the device of the present invention: in-plane valves, where the small channels and sharp corners of the valve are formed by forming a "groove" in one layer and joining this layer to a featureless cover (usually the device Another layer); and a through-hole valve, in which small (usually 250 µm or less) holes are made in the intermediate layer between the two fluid-carrying layers of the device. In both cases, fluoropolymer treatment can be used to increase the contact angle of fluid to valve contact. Figure 7 shows the valve regulation performance of these valves for the liquids of interest (ie, deionized water and cycle sequencing reagents) as a function of valve size under fluoropolymer treatment. In both cases, the expected dependence of the valve regulation pressure on the valve size (pressure about 1/diameter) was observed. Through-hole valves have significant advantages over in-plane valves. First, it is easy to manufacture because small through holes can be easily formed in the plastic sheet by molding around post, punching, die cutting, drilling, or laser drilling after manufacturing the valve layer. In-plane valves require fairly precise manufacturing, and very fine valves (with high valve adjustment pressure) must use lithography technology to manufacture the required forming or stamping tools. Second, the through-hole valve can be more completely coated with fluoropolymer on "all sides". Applying a low surface tension fluoropolymer solution to the hole results in complete coating of the inner wall of the hole by capillary action. Coating all sides of the valve in the plane requires the application of fluoropolymer to the valve and the area of the matching layer sealed on the valve. Therefore, a typical in-plane valve is formed without coating the "top" of the valve. In the machined prototype, the through-hole valve is easy to implement and exhibits a larger valve pressure regulation, as shown in Figure 7. Mixing can be achieved in various ways. First, the fluids can be mixed by diffusion by co-injecting two fluids into a single channel, where the channel usually has a small lateral dimension and sufficient length to satisfy the diffusion time at a given flow rate: t _D = (width ) ² / (2 × diffusion constant) Unfortunately, this type of mixing is usually not sufficient for rapid mixing of large volumes because the diffusion or mixing time is proportional to the square of the channel width. Mixing can be enhanced in various ways, such as lamination, where the fluid streams are separated and reorganized (Campbell and Grzybowski Phil. Trans. R. Soc. Lond. A 2004, 362, 1069-1086); or by using fine microstructures in the flow Turbulent advection is formed in the channel (Stroock et al., Anal. Chem. 2002, 74, 5306-5312). In systems using active pumps and valves, mixing can be achieved by circulating the fluid between two points on the device multiple times. Finally, the latter can also be achieved in systems using capillary valves. The capillary valve placed between the two channels or chambers acts as a pivot for the fluid flow; when the fluid flows from one channel to another through the capillary tube, if a sufficiently low pressure is used to draw the fluid, the posterior meniscus is intercepted. The reversal of pressure drives the fluid back into the first channel and again prevents it from capillary. Multiple cycles can be used to effectively mix the ingredients. The method of separation and detection in microfluidic format is described in the US patent application entitled "PLASTIC MICROFLUIDIC SEPARATION AND DETECTION PLATFORMS" (agent file number MBHB 07-865-US) filed on the same day. The way of citation is incorporated herein (see, for example, paragraphs 68-79, 94-98 therein). The upper part of FIG. 13 shows the construction of an integrated biochip ( 1301 ) from two components that are joined during or during manufacturing. The first component is a 16-sample biochip ( 1302 ) combining the dissolution, amplification, and sequencing features of the biochip of FIG. 1 with the sequencing product purification feature of the biochip of FIG. 11, and the second component is a 16-channel plastic separation Biochip ( 1303 ). The sequencing product can also be injected electrically before separation. D. Manufacturing method The device of the present invention may be mainly composed of plastic. Available plastic types include (but are not limited to): cycloolefin polymer (COP); cycloolefin copolymer (COC); (when they have sufficient molecular weight, both have excellent optical properties, low hygroscopicity and high handling Temperature); poly(methyl methacrylate) (PMMA) (easy to process and obtain excellent optical properties); and polycarbonate (PC) (highly formable with good impact resistance and high operating temperature) . More information about materials and manufacturing methods is included in the US patent application entitled "METHODS FOR RAPID MULTIPLEXED AMPLIFICATION OF TARGET NUCLEIC ACIDS" (Agent file number 08-318-US), the full text of which is cited by reference Incorporated herein (as described above). Various methods can be used to manufacture individual parts of the bio-wafer and assemble it in the final device. Because biochips can be composed of one or more types of plastics, and may include interposer components, the method of interest is related to the manufacture of individual parts, subsequent processing and assembly of the parts. Plastic components can be manufactured in several ways, including injection molding, hot stamping, and machining. Injection molded parts can be composed of general features (such as fluid reservoirs) and fine features (such as capillary valves). In some cases, it is better to manufacture fine features on one set of parts and larger features on another, because the method of injection molding of these features of different sizes can vary. For large reservoirs (measured at several (approximately 1-50 mm) millimeters on one side and a depth of several millimeters (approximately 1-10 mm) and capable of holding hundreds of microliters), process-type injection can be used Forming tools or tools manufactured by burning graphite into steel or other metals using graphite electrodes are formed using conventional forming, where the graphite electrodes have been processed into the negative electrode of the tool. For fine features, tool manufacturing and forming processes can vary. Typically, a lithography process (eg, isotropic etching of glass, or deep reactive ion etching on silicon or other processes) is used to fabricate tools on the substrate of interest. The substrate can then be plated with nickel (usually after deposition of the chromium layer to promote adhesion) and the substrate removed, for example, by etching in acid. This nickel "sub-body" coating is an injection molding tool. The forming process can also be slightly different from the above. For fine, narrow features, compression injection molding (where the mold is slightly compressed physically after the plastic is injected into the cavity) has been found to be better than standard injection molding in terms of fidelity, precision, and reproducibility. For hot embossing, similar issues regarding the general and fine features as described above need to be controlled, and tools can be manufactured as described above. In hot stamping, the plastic resin can be applied to the tool surface or a flat substrate in the form of pellets, or as a preform of material manufactured by forming or stamping. The second tool can then be contacted under precisely controlled temperature and pressure to raise the temperature of the plastic beyond its glass transition temperature and cause the material to flow to fill the cavity of the tool(s). Embossing under vacuum can avoid the problem of trapping air between tools and plastics. Mechanical machining can also be used to manufacture parts. A high-speed computer numerical control (CNC) machine can be used to manufacture many individual parts by self-forming, extruding, or solvent casting plastic daily. Reasonable selection of milling machines, operating parameters and cutting tools can achieve high surface quality (under COC high-speed milling, surface roughness of 50 nm can be obtained (Bundgaard et al. Proceedings of IMechE Part C: J. Mech. Eng. Sci. 2006, 220,1625-1632)). Milling can also be used to manufacture geometries that may be difficult to obtain by forming or embossing and easily mix feature sizes on a single part (for example, large reservoirs and fine capillary valves can be processed in the same substrate). Another advantage of milling over forming or embossing is that there is no need to use a mold release agent to remove the parts made from the forming tool. Post-processing of individual parts includes optical inspection (which can be automated), cleaning operations to remove defects such as burrs or hanging plastic, and surface treatment. If optical quality surfaces are required in processing plastics, solvent vapor polishing suitable for plastics can be used. For example, for PMMA, dichloromethane can be used, while for COC and COP, cyclohexane or toluene can be used. Before assembly, surface treatment may be applied. Surface treatment can be carried out to promote or reduce wetting (ie to change the hydrophilicity/hydrophobicity of the part); to inhibit the formation of bubbles in the microfluidic structure; to increase the pressure regulation of the capillary valve; and/or to inhibit protein adsorption to the surface. The wettability-reducing coating includes fluoropolymers and/or molecules with fluorine moieties, where the fluorine moieties can be exposed to the liquid when the molecules are adsorbed or bound to the surface of the device. The coating can be adsorbed or deposited, or it can be covalently bonded to the surface. Methods that can be used to manufacture such coatings include dip coating, delivery of coating reagents through channels of assembled devices, ink application, chemical vapor deposition, and inkjet deposition. The covalent bond between the coated molecule and the surface can be formed by treatment with oxygen or other plasma or UV-ozone to form an active surface, and then surface treatment molecules are deposited or co-deposited on the surface (see Lee et al. People, Electrophoresis 2005, 26, 1800-1806; and Cheng et al., Sensors and Actuators B 2004, 99, 186-196). The assembly of the component parts in the final device can be carried out in various ways. An insertion device such as a filter can be die cut and then placed with a pick and place machine. Thermal diffusion bonding can be used, for example, to bond two or more layers of the same material, where each layer has a uniform thickness. In general, the parts can be stacked and placed in a hot press, where the temperature can be increased to around the glass transition temperature of the material containing the parts to cause the interface between the parts to fuse. One of the advantages of this method is that the bonding is "general", that is, regardless of the internal structure of the layer, any two stacks of layers with approximately the same size can be bonded, because heat and pressure can be uniformly Applied on these layers. Thermal diffusion bonding can also be used to join more complex parts by using specially manufactured bonding brackets, such as those that are not planar on the bonding or opposite surfaces. These brackets are consistent with the outer surface of the layer to be joined. Other bonding variants include solvent-assisted thermal bonding, where a solvent such as methanol partially dissolves the plastic surface, thereby enhancing the bonding strength at lower bonding temperatures. Another variant is to use a spin-coating layer of low molecular weight material. For example, a polymer with the same chemical structure as the substrate composition but a lower molecular weight can be spin-coated on at least one layer to be joined, the components are assembled, and the resulting stack is joined by diffusion bonding. During the thermal diffusion bonding process, low molecular weight components can undergo glass transition temperature at a temperature lower than these components and diffuse into the substrate plastic. Adhesives and epoxy resins can be used to join different materials and the adhesives and epoxy resins are likely to be used when manufacturing joint components in different ways. The adhesive film can be die cut and placed on the assembly. Liquid adhesive can also be applied via spin coating. Adhesives can be successfully used to ink structured parts (such as in nano-contact printing) to apply the adhesive to the structured surface without "guiding" the adhesive onto specific areas. In one example, the biochip of the present invention can be assembled as shown in FIG. 6. Layers 1 and 2 can be aligned by included features (such as pins and sockets); respectively, layers 3 and 4 can be similarly aligned by included features. The stack of layer 1 plus layer 2 can be inverted and applied to the stack of layer 3 plus layer 4, and then can be combined into the entire stack. E. Examples Example 1 Integrated biochip for nucleic acid extraction and amplification Figure 1 shows an integrated biochip for DNA extraction and PCR amplification. This 4-sample device integrates the following functions: reagent distribution and metering; mixing of reagent and sample; transfer of sample to the thermal cycle part of the wafer; and thermal cycle. The same biochip is used in Example 2 below and has an additional structure for cycle sequencing performance. The biochip is constructed from 4 layers of thermoplastic as shown in Figure 2-5. These 4 layers are processed PMMA and have layer thicknesses of 0.76 mm, 1.9 mm, 0.38 mm, and 0.76 mm, respectively, and the side dimensions of the bio-wafer are 124 mm×60 mm. In general, three or more layers of biochips allow the use of an indefinite number of common reagents distributed between multiple tests: two fluid layers and a layer containing at least through holes, so that the fluid channels in the outer layer can communicate with each other 'cross'. (It should be recognized that there are special circumstances-such as the use of only one common reagent in multiple samples-making the three-layer configuration unnecessary.) The 4 layers were chosen to be compatible and fully integrated with the chip construction used for other functions (such as ultrasonic processing, Example 3) (Example 4). The cross-sectional dimensions of the channel of the biochip are in the range of 127 μm×127 μm to 400 μm×400 μm, and the cross-section of the reservoir is in the range of 400 μm×400 μm to 1.9×1.6 mm; the distance between the channel and the reservoir extends It is 0.5 mm to tens of millimeters short. The capillary valve used in the biochip is: the size of the "in-plane" valve is 127 μm × 127 μm and the diameter of the through-hole capillary valve is 100 μm. Some channels, reservoirs and capillary valves of the four processing layers were treated with hydrophobic/oleophobic material PFC 502A (Cytonix, Beltsville, MD). Surface treatment was performed by coating with a wet Q-tip, followed by air drying at room temperature. The thickness of the dried fluoropolymer layer measured by optical microscopy was less than 10 μm. Surface treatment is used for two purposes: to prevent the formation of air bubbles in the liquid, especially in low surface tension liquids such as circulating sequencing reagents (this may occur when the liquid quickly wets the walls of the channel or chamber (and the air can be replaced Before "closing" the bubble)); and enhance the capillary rupture pressure where the capillary valve resists liquid flow. The untreated area is the thermal cycling chamber for PCR and cycle sequencing. After surface treatment, the layers are connected as shown in FIG. 6. The connection is made using a thermal diffusion connection, where the stack of components is heated under pressure to a temperature close to the glass transition temperature (T _g ) of the plastic. Apply 45 pounds (lbs) of force to the entire 11.5 square inch biochip during a thermal connection profile consisting of a rise from ambient temperature to 130°C in 7.5 minutes, holding at 130°C for 7.5 minutes, and rapid cooling to room temperature 15 minutes. Pneumatic instruments were developed to drive the fluid in the biochip of the present invention. Two small peristaltic pumps provide pressure and vacuum. The positive pressure output is divided among three regulators with a range of about 0.05-3 psig. Transfer the vacuum to a regulator with an output vacuum of about (-0.1)-(-3) psig. Pass the fourth and higher pressure from the N ₂ cylinder or from the high capacity pump to another regulator. Apply positive or negative pressure to a series of 8 pressure selector modules. Each module is equipped with solenoid valves, which can select the output pressure to be transmitted to the biochip from 5 inputs. The output pressure line terminates in at least one pneumatic interface. This interface is clamped to the wafer with an O-ring on the wafer port on the input side of the wafer (the port is along the top of the features). An additional solenoid valve (ie, gate valve; 8 per interface) that accepts the output pressure line from the pressure selector module happens to be on the biochip port. These valves, very close to the wafer, provide a low dead volume interface (about 13 μL) between the pressure line and the wafer. When applying pressure to move other liquids, the low dead volume interface can prevent unintentional movement of certain fluids on the biochip (for example, due to the compression of the gas when applying pressure, the small gas volume between the liquid plug and the closing valve Decide to the maximum extent that the plug can move). All pressure selector valves and gate valves are operated under computer control using the command-based LabView ^TM program. An important feature of this system is that short pressure cycle time is possible. Some fluid control events can be performed, which require pressure pulses as short as 30 milliseconds, and/or complex pressure profiles can be used, where the pressure can be quickly transformed from one value to another (ie, one regulator to another regulator) (That is, the time lag does not exceed 10-20 milliseconds). Samples of about ¹⁰⁶ cells / mL pGEM sequencing by mass of insertion (target pUC18 sequencing) Transformation of Escherichia coli (E. coli) DH5 bacteria suspension composition. The PCR reagent consisted of dNTP KOD Taq polymerase (Novagen, Madison, WI) (concentration 0.1 μM). A sample of 1.23 μL of bacterial suspension was added to each of the four ports 104 , each forming through holes 202 and 336 in layers 1 and 2, respectively. The sample then exists in the sample channel 303 in layer 2. Next, 10 μL of PCR reagent is added to port 105 , which constitutes through holes 217 and 306 in layers 1 and 2. Then the PCR reagent is present in the chamber 307 in layer 2 (see Fig. 8a). The port for evacuating the replacement air for the PCR reagent is port 107, which constitutes 109 and through holes 203 + 305 . In operation, displacement of the sample and the downstream processes (such as the reagent of the measurement, the mixed fluid) through the air outlet port on the output terminal wafer 108, the port 227 is constituted by a through hole. The final volume of the PCR reaction can be increased or decreased as desired. Place the biochip in the pneumatic manifold described above. Perform the following automated stress profile with no delay between steps. Unless otherwise stated, the pneumatic interface valve (corresponding to the port along the input side of the wafer) is closed during all steps. A pressure of 0.12 psig was applied to the port 104 for 15 seconds to drive the sample along the channel 303 to the through hole 304 . The sample passes through the via 304 and appears on the other side of layer 2 in the sample chamber 204 of layer 1 and is driven to the first mixing junction 205 . At the first mixing junction, the sample is retained by the capillary valve 210 (see Figures 8b-c). A pressure of 0.12 psig was applied to port 105 for 10 seconds to drive the PCR reagent through the through hole 320 . The PCR reagent appears on the other side of layer 2 in the distribution channel 208 and moves into the metering chamber 209 , which defines the reagent volume equal to the sample volume, where it is held at the mixing junction 205 by the capillary valve 211 . (See Figure 8d). 0.12 psig pressure is applied to the port 107 (composed of a through hole 305 and 203) (where the atmosphere opening port 105 pairs) for 3 seconds to allow the evacuation passage 208 (see FIG. 8e). Apply a pressure of 0.8 psig to ports 107 and 105 for 0.03 seconds and at the same time apply a pressure of 0.7 psig to port 104 for 0.03 seconds . The sample and PCR were applied by bursting the liquid through capillary valves 210 and 211 and through these valves The reagents are initially mixed (see Figure 8f). 0.12 psig of pressure applied to the ports 104 and 107 for 10 seconds, and the PCR reagents to the sample is pumped to the mixing channel 214, and is retained in the capillary tube 211 and valve 210. The mixing of the spherical object 212 into the constricted portion 213 constitutes an additional hydraulic resistance to the liquid flow, thereby reducing the high velocity imparted by the above-mentioned high pressure pulse. A pressure of 0.7 psig was applied to ports 104 and 107 for 0.03 seconds to separate the liquid from capillary valves 210 and 211 (see FIG. 8g). A pressure of 0.12 psig is applied to ports 104 and 107 for 3 seconds to pump liquid through the mixing channel 214 to the capillary valve 219 , where the liquid is retained (see Figure 8h). A pressure of 0.7 psig was applied to ports 104 and 107 for 0.1 seconds to drive the mixture of sample and PCR reagent through through holes 315 and 402 and through the body of layers 2 and 3 into PCR chamber 502 (see FIG. 8i). A pressure of 0.12 psig was applied to ports 104 and 107 for 3 seconds to achieve pumping of the mixture of sample and PCR reagent into chamber 502 . The leading edge of the mixture of sample and PCR reagent is then passed through through holes 403 and 316 , appears in layer 1, and is blocked at capillary valve 220 (see FIG. 8j). Then pressurize the biochip to 30 psig N ₂ and use the gas bladder compression mechanism of the Peltier effect to thermally circulate it for PCR amplification. This is called "METHODS FOR" RAPID MULTIPLEXED AMPLIFICATION OF TARGET NUCLEIC ACIDS (Agent File Number 08-318-US) and the international patent application entitled "DEVICES AND METHODS FOR THE PERFORMANCE OF MINIATURIZED IN VITRO ASSAYS" on February 6, 2008 As described in Patent Application No. PCT/US08/53234 (Agent file number 07-084-WO), the full text of each of these applications is incorporated herein by reference. The selected sample, reagent volume, and PCR chamber size are such that liquid fills the area between valve 219 and valve 220 . Therefore, the liquid/vapor interface with a small cross-sectional area (usually 127 μm×127 μm) is located approximately 3 mm from the bottom surface of the layer 4 thermal cycle. Applying pressure during the thermal cycle suppresses degassing of dissolved oxygen in the sample. The small cross-sectional area of the liquid/vapor interface and the distance from the Peltier surface both inhibit evaporation. The temperature of the top of the bio-wafer observed during cycling never exceeds 60°C, and therefore the vapor pressure at the liquid/vapor interface is significantly lower than the vapor pressure that such an interface would have in the PCR chamber. For a 2 μL sample, of which 1.4 μL is in the chamber 502 and the rest are in the through-hole and capillary valve, the evaporation observed in 40 cycles of PCR is less than 0.2 μL. The volume of uncirculated fluid (0.6 μL in this case) can be reduced by choosing a smaller diameter through hole. Perform PCR using the following temperature profile: • Thermally dissolve bacteria at 98°C for 3 minutes • 40 cycles of: o Denaturation at 98°C for 5 seconds o Annealing at 65°C for 15 seconds o Extension at 72°C for 4 seconds o The final extension (2 minutes) of the PCR product at 72°C was recovered by rinsing the chamber 502 with approximately 5 µL of deionized water and analyzed by slab gel electrophoresis. The PCR yield is as high as 40 ng/reaction, which is much more than the amount required for subsequent sequencing reactions. In this application, bacterial nucleic acid is produced only by dissolving bacteria. The nucleic acid can be subjected to the required purification, that is, the process of improving the efficiency of amplification, sequencing and other reactions. Example 2 Integrated biochip for cycle sequencing reagent distribution, mixing with PCR products and cycle sequencing The biochip as described in Example 1 was used. The PCR product generated in the test tube using the protocol described in Example 1 was added to the sample and PCR reagent port of the biochip as described above. 50 μL of cycle sequencing reagent (BigDye ^™ 3.1/BDX64, MCLab, San Francisco) was added to port 106 (consisting of through holes 215 and 308 ) and chamber 309 . After installing two pneumatic interfaces (one for the wafer input and one for the wafer output), the PCR product was processed as described in Example 1 up to the PCR chamber, but there was no PCR thermal cycling step. The treatment of the fluid in the wafer is shown in Figure 9a. Use the pneumatic system software to perform the following pressure profiles; unless otherwise stated, all solenoid valves corresponding to the chip ports are closed: 1. Apply 0.1 psig pressure to port 106 (where port 109 is open to the atmosphere) for 10 seconds or more The cycle sequencing reagent is pumped into channel 310 (see Figure 9b). 2. Apply a pressure of 0.7 psig to ports 106 and 108 for 0.2 seconds (consisting of through holes 216 and 314 ) to drive the circulating sequencing reagent from channel 304 through through hole 311 , through the body of layer 2, and into layer 1 Cycle sequencing reagent metering chamber 218 (see Figure 9c). 3. Apply a pressure of 0.1 psig to port 106 (where port 109 is open to the atmosphere), drive the cycle sequencing reagent to capillary valve 221 , where the cycle sequencing reagent is retained (see Figure 9d). 4. Apply a pressure of 0.1 psig to port 108 (where port 106 is open to the atmosphere) for 1 second to drive excess circulating sequencing reagent back into chamber 101 , leaving channel 310 empty (see Figure 9e). 5. Apply a pressure of 0.7 psig to ports 104 and 107 for 0.1 seconds (where port 109 is open to the atmosphere) to drive the PCR product through capillary valve 220 and into through hole 317 , through the body of layer 2 and the passage in layer 3 Well 404 , and enter the layer 4 cycle sequencing chamber 503 (see Figure 9f). 6. Apply a pressure of 0.1 psig to port 109 (where ports 104 and 107 are open to the atmosphere) for 5 seconds to drive the PCR sample back to the through hole. Capillary action retains the liquid at the entrance of the through-hole, preventing trapped air bubbles from appearing between the PCR product and the chamber 503 (see Figure 9g). 7. Apply a pressure of 0.7 psig to port 108 (where port 109 is open to the atmosphere) for 0.2 seconds to drive the cycle sequencing reagent into chamber 503 , while simultaneously applying 0.1 psig to ports 104 and 107 to cause the PCR product Contact with sequencing reagents (see Figure 9h). 8. Apply pressure of 0.1 psig to ports 104 , 107, and 108 for 10 seconds (where port 109 is open to the atmosphere) to drive the PCR product and Sanger reagent into the chamber. The meniscus after the PCR product and the sequencing reagent are blocked by the capillary valves 220 and 221 (see FIG. 9i). 9. Apply 0.25 psig vacuum and 5 vacuum pulses with a duration of 0.1 second to port 108 (where port 109 is open to the atmosphere) to partially draw the two liquids back into the reagent metering chamber 218 (see Figure 9j). 10. Apply a pressure of 0.1 psig to ports 104 , 107, and 108 (where port 109 is open to the atmosphere) for 10 seconds to pump the mixture back to chamber 503 , where the back meniscus is blocked by the capillary valve as in step 8. (See Figure 9k). Repeat steps 9-10 twice more to achieve mixing of sequencing reagents and PCR products. Next pressurize the bio-wafer to 30 psig N ₂ and perform thermal cycling using the following temperature profile: • 95°C/1 minute initial denaturation o 30 cycles of the following o Denaturation at 95°C for 5 seconds o Annealing at 50°C 10 Second o Sample extended at 60°C for 1 minute (see Figure 9l) was recovered and purified by ethanol precipitation and separated by Genebench ^™ instrument electrophoresis and laser induced fluorescence as described below (Part II, Example 5) Detect and analyze. Phred quality analysis produces 408 +/- 57 QV20 bases/sample. Example 3 Ultrafiltration in 4- Sample Biowafers As described in Example 1, four - sample biowafers were constructed with four layers suitable for the efficiency of sequencing product purification, and are shown in FIG. 11. One additional element in the configuration is an ultrafiltration (UF) filter 1116 , which is cut to size and placed between layers 3 and 4 before thermal connection. Layer 3 must be used to form a good connection around the UF filter. Layers 3 and 4 form an uninterrupted perimeter around the filter. This is because all channels leading to and leaving the filter are at the bottom of layer 2 (for example, when the channel crosses the filter, directly on layer 2 and The connection between 4 leads to poor connection to the filter). In this example, a regenerated cellulose (RC) filter with a molecular weight cut-off (MWCO) of 30 kD (Sartorius, Goettingen, Germany) was used. When having the alternate material polyether ballast (Pall Corporation, East Hills, NY), a variety of other MWCOs (10 kD, 50 kD, and 100 kD) have been tested. 1. Add the four 10 μL cycle sequencing product samples generated in the test tube reaction using the pUC18 template and the KOD enzyme to the port 1104 in the first layer and drive it to the second layer via the channel 1105 in the second layer Room 1106 . Add 200 μL of deionized water to port 1120 (through hole in the first layer) to the reservoir 1121 in the second layer. The biochip is then installed between two pneumatic interfaces. Use the pneumatic system software to perform the following pressure profile. Unless otherwise stated, all solenoid valves corresponding to the biochip ports are closed. 2. Apply a pressure of 0.09 psig to port 1104 (where port 1119 is open to the atmosphere) for 5 seconds to drive the sequencing product to capillary valve 1108 in layer 1, where the sequencing product is retained. 3. Apply a pressure of 0.6 psig to port 1104 (where port 1119 is open to the atmosphere) for 0.1 seconds to expand the sample through capillary valve 1108 in layer 1 and pass it into layer 2 through through hole 1111 in layer 2 The UF input room 1112 . 4. Apply a pressure of 0.09 psig to port 1104 (where port 1119 is open to the atmosphere) for 10-30 seconds (using different times in different experiments) to complete the delivery of the sequencing product to chamber 1112 . The sequencing product is retained by the capillary valve 1113 in layer 2 (see Figures 12a and 12b). 5. Apply a pressure of 0.8 psig to port 1124 (where ports 1119 and 1104 are open to the atmosphere) for 0.5 seconds to drive the sequencing product into filter chamber 1115 through valve 1113 . This also clears the retained capillary valve 1108 of liquid. 6. Apply a pressure of 0.09 psig to port 1124 (where port 1119 is open to the atmosphere) for 10-30 seconds to complete the delivery of the sequencing product to chamber 1115 . The sequencing product is held at valve 1113 (see Figure 12c). 7. Slowly apply 7.5 psig pressure to all ports of the chip used for ultrafiltration. During ultrafiltration, when the liquid is driven through the filter 1116 , the sequencing product meniscus remains at 1113 while the liquid front "retracts". The 10 µL sequencing product requires about 120 seconds for filtration. The pressure is released after filtration (see Figures 12c and 12d). 8. Apply a pressure of 0.09 psig to port 1120 (where port 1124 is open to the atmosphere) for 3 seconds to drive water into channel 1122 (in layer 4) and partially fill overflow chamber 1123 (see Figure 12e). 9. Apply a pressure of 0.8 psig to ports 1120 and 1124 (where port 1119 is open to the atmosphere) to drive water into chamber 1112 through through-hole capillary valve 1110 in channel 1122 . 10. Apply a pressure of 0.09 psig to port 1120 (where port 1119 is open to the atmosphere) for 10-30 seconds to complete the transfer of liquid to chamber 1112 , which is held by valve 1113 (see Figure 12f). 11. Apply a pressure of 0.09 psig to port 1124 (where port 1120 is open) to drive water in chamber 1123 and channel 1122 back to chamber 1121 (see FIG. 12g). 12. Apply a pressure of 0.8 psig to port 1124 (where ports 1119 and 1104 are open to the atmosphere) for 0.5 seconds to drive water through the valve 1113 into the filter chamber 1115 . This also clears the retained capillary valve 1108 of liquid. 13. Apply a pressure of 0.09 psig to port 1124 (where port 1119 is open to the atmosphere) for 10-30 seconds to complete the transfer of water to chamber 1115 . The sequencing product remains at the valve 1113 (see Figure 12h). As in step 6 above, the water is driven through the UF filter to complete the first wash. Repeat steps 8-13 one more time. Repeat steps 8-12 to partially fill chamber 1115 , with the final volume of water used for dissociation (see Figure 12k). Apply a vacuum of 1.6 psig to port 1104 , where all other ports are closed for 1 second, draw some water from chamber 1115 into chamber 1112 (the maximum motion is caused by the dead between the meniscus with the liquid and the solenoid valve corresponding to port 1119 It is determined by the vacuum formation of the same order of space) (see Figure 12l). The port 1104 was opened to the atmosphere for 1 second, and the liquid was moved back to the chamber 1115 due to the partial vacuum generated between the liquid and the valve corresponding to the port 1119 (see FIG. 12m). Repeat 16-17 50 times to produce 50 dissolution cycles. A pressure of 0.09 psig is applied to port 1124 for 10 seconds (where port 1119 is open to the atmosphere) to drive the liquid so that its posterior meniscus is blocked at 1113 . A pressure of 0.7 psig/0.05 seconds is applied to port 1124 (where port 1119 is open to the atmosphere) to separate the dissolved matter (see FIG. 12n). Samples were recovered and run directly with Genebench ^™ as described, producing up to 479 QV20 bases. Example 4 Fully integrated biochip for nucleic acid extraction, template amplification, cycle sequencing, sequencing product purification, and electrophoretic separation and detection of purified products FIG. 13 illustrates an embodiment of 16-sample biochip 1301 , which incorporates the organism of FIG. 1 The functions of chip dissolution and extraction, template amplification and cycle sequencing; ultrafiltration of the chip of Figure 11; and electrophoretic separation and detection. The ultrafiltration process is performed by the subassembly 1302 and can be performed as described in Examples 1, 2, and 3; align the delivery point 1304 on the bottom surface of 1302 with the input hole 1305 in the separation subassembly 1303 . The counter electrode is used to perform the injection electrically in a pre-concentration step. The input hole 1305 illustrated in FIG. 14 is composed of a liquid receiving hole 1401 , a main separation electrode 1402, and a counter electrode 1403 . The separation channel 1306 leads to the bottom of the hole reservoir 1401 . The separation electrode is usually coated with platinum or gold, and is preferably a planar gold-plated electrode, which substantially covers 1, 3, or 4 of the inner surface of 1401 . The counter electrode is a thin gold, steel or platinum wire (typically 0.25 mm in diameter), which has been coated with a thin layer (approximately 10 µm) of cross-linked polypropylene amide. This forms a hydrogel protective layer on the electrode. On panel d, the purified sequencing product (black dots in 1401 ) can be transferred to the wells. A positive potential is applied between 1402 and 1403 , and the negatively charged sequencing product is directed to 1403 , as in the panel cd. The hydrogel layer on 1403 prevents the sequencing product from coming into contact with the metal electrode and thus prevents the electrochemistry and damage of the sequencing product. Next, the counter electrode 1403 is floated relative to 1402 . Next, a positive potential is applied between the main separation electrode 1402 and the anode (not shown) at the distal end of the separation channel 1306 . This allows product injection (panel e) and electrophoresis along 1306 for separation and detection (panel f). As shown in FIG. 14, this procedure allows the concentration of sequencing products near the end of channel 1306 to be significantly increased relative to the sequencing products delivered from ultrafiltration. Although this concentration is ideal for some applications, it is not necessary in all cases. In such cases, the hole of FIG. 14 without the counter electrode 1403 can be used for direct EKI. Alternatively, the single electrode in the loading hole may be one-half of the cross-T or double-T injector (see, for example, the title of "PLASTIC MICROFLUIDIC SEPARATION AND DETECTION PLATFORMS" applied at the same time as the case, the agent file US Patent Application No. 07-865-US). Separation occurs in the separation channel 1306 , and detection of laser-induced fluorescence occurs in the detection zone 1307 . In this bio-wafer, a recess 1308 is provided to match, for example, a Peltier block (not shown) to the lower surface of 1301 to provide thermal cycling for PCR and cycle sequencing. A pneumatic interface (not shown) inside the instrument is clamped to the end of the wafer to provide microfluidic control. II. Separation (DEPARATION) and detection system A. Detailed description of separation and detection components and their use 1. Separation instruments use biological chips and instruments as described in US Patent Application Publication No. US 2006-0260941-A1 Perform DNA isolation. The separation chip can be glass (see US Patent Application Publication No. US 2006-0260941-A1) or plastic (the title of "Plastic Microfluidic separation and detection platforms" applied at the same time as this case, the file number of the agent file 07-865-US US patent applications), the full text of which is incorporated by reference. 2. Excitation and detection instrument The instrument contains an excitation and detection subsystem for interacting with and interrogating the sample. The sample usually includes one or more biomolecules (including, but not limited to, DNA, RNA, and protein) labeled with dyes (such as fluorescent dyes). The excitation subsystem includes an excitation source and an excitation beam path, wherein the optical elements include a lens, a pinhole, a mirror, and an objective lens to adjust and focus the excitation source in the excitation/detection window. The optical excitation of the sample can be accomplished by a series of laser types, where the emission wavelength is in the visible region between 400 and 650 nm. Solid-state lasers can provide emission wavelengths of approximately 460 nm, 488 nm and 532 nm. These lasers include, for example, Compass, Sapphire and Verdi products from Coherent (Santa Clara, CA). Gas lasers include argon ion and helium-neon lasers that emit visible wavelengths of about 488 nm, 514 nm, 543 nm, 595 nm, and 632 nm. Other lasers with emission wavelengths in the visible region can be purchased from CrystaLaser (Reno, NV). In one embodiment, a 488 nm solid-state laser Sapphire 488-200 (Coherent, Santa Clara, CA) can be used. In another embodiment, a light source with a wavelength that exceeds the visible range can be used to excite dyes with absorption and/or emission spectra that exceed the visible range (eg, infrared or ultraviolet emitting dyes). Or optical excitation can be obtained by using non-laser light sources (including light emitting diodes and lamps) with emission wavelengths suitable for dye excitation. The detection subsystem includes one or more optical detectors, a wavelength dispersion device (which performs wavelength separation), and one or a series of optical elements, including or not limited to lenses, pinholes, mirrors and The objective lens collects the emitted fluorescence from the fluorophore-labeled DNA fragments present at the excitation/detection window. The emitted fluorescence can come from a single dye or a combination of dyes. To distinguish the signal to determine its contribution from the emission dye, wavelength separation of fluorescence can be used. This can be achieved by using dichroic mirrors and bandpass filter elements (available from many suppliers including Chroma, Rockingham, VT and Omega Optical, Brattleboro, VT). In this configuration, the emitted fluorescent light passes through a series of dichroic mirrors, where part of the wavelength will be reflected by the mirror to continue to travel along the path, while the other part will pass. A series of discrete light detectors (each positioned at the end of the dichroic mirror) will detect light in a specific range of wavelengths. A band pass filter can be positioned between the dichroic mirror and the light detector to further narrow the wavelength range before detection. Optical detectors that can be used to detect wavelength-separated signals include photodiodes, collapsed photodiodes, photomultiplier tube modules, and CCD cameras. These optical detectors can be purchased from suppliers such as Hamamatsu (Bridgewater, NJ). In one embodiment, the wavelength components are separated by using a dichroic mirror and a band-pass filter, and these wavelength components are detected by a photomultiplier tube (PMT) detector (H7732-10, Hamamatsu). The dichroic mirror and bandpass components can be selected so that the incident light on each of the PMTs consists of a narrow wavelength band corresponding to the emission wavelength of the fluorescent dye. The bandpass is usually selected to be centered on a fluorescent emission peak with a bandpass in the wavelength range between 1 and 50 nm. The system can detect eight colors and can be designed with eight PMTs and a corresponding set of dichroic mirrors and band-pass filters to separate the emitted fluorescence into eight different colors. More than eight dyes can be detected by applying additional dichroic mirrors, band pass filters and PMT. Figure 15 shows the beam path of an embodiment of a discrete band pass filter and a dichroic filter. An integrated form of this wavelength discrimination and detection configuration is H9797R, Hamamatsu, Bridgewater, NJ. Another method to identify the dyes that make up the fluorescent signal includes the use of materials such as prisms, diffraction gratings, and transmission gratings (available from many suppliers including ThorLabs, Newton, NJ; and Newport, Irvine, CA); and photogrammetry Apparatus (available from many suppliers including Horiba Jobin-Yvon, Edison, NJ) wavelength dispersion components and systems. In this mode of operation, the wavelength components of the fluorescent light are dispersed in the physical space. A detector element placed along this physical space detects light and makes the physical position of the detector element correlate with the wavelength. Detectors suitable for this function are array-based and include multi-element photodiodes, CCD cameras, back-end thin CCD cameras, and multi-anode PMT. Those skilled in the art will be able to apply a combination of wavelength dispersive elements and optical detector elements to produce a system that can discern the wavelength of the dye used in the system. In another embodiment, a spectrograph is used instead of dichroic and bandpass filters to separate wavelength components from self-excited fluorescence. Spectrograph design details can be obtained in John James, Spectrograph Design Fundamental , Cambridge, UK: Cambridge University Press, 2007. In this application, a spectrograph P/N MF-34 (P/N 532.00.570) (HORIBA Jobin Yvon Inc, Edison, NJ) with a concave holographic grating and a spectral range of 505-670 nm is used. Linear 32-element PMT detector arrays (H7260-20, Hamamatsu, Bridgewater, NJ) can be used for detection. The collected fluorescence is imaged on the pinhole, reflected, dispersive, and imaged by a concave holographic grating on a linear PMT detector, which is installed at the output port of the spectrograph. The use of PMT-based detectors utilizes the low dark noise, high sensitivity, high dynamic range, and fast response characteristics of PMT detectors. The use of spectrographs and multi-element PMT detectors for the detection of excited fluorescence allows the flexibility of the number of dyes and the emission wavelength of the dyes that can be applied in these systems and channels, without the need for physical reconfiguration of the instrument detection System (dichroic, bandpass and detector). The data collected from this configuration is the wavelength-dependent spectrum across the visible wavelength range for each scan of each track. The complete spectrum produced by each scan provides dye flexibility according to the dye emission wavelength and the number of dyes that can be present in the sample. In addition, when all PMT elements in the array are read out in parallel, the use of spectrometers and linear multi-element PMT detectors also allows extremely fast readout rates. 16 shows the beam path of an embodiment of a multi-element PMT and spectrograph. The instrument can use the initial mode of operation to simultaneously detect multiple channels and multiple wavelengths simultaneously. In one configuration, the excitation beam hits all tracks simultaneously. The fluorescence from this is collected by a two-dimensional detector such as a CCD camera or array. In the initial mode of this collection, wavelength dispersive elements are used. One dimension of the detector represents physical wavelength separation, while the other dimension represents space or track-to-track separation. For simultaneous excitation and detection of multiple samples, a scanning mirror system (62) (P/N 6240HA, 67124-H-0 and 6M2420X40S100S1, Cambridge technology, Cambridge MA) is used to control the excitation and detection beam path to enable biological Each of the Way of Wafer imaging. In this mode of operation, the scanning mirror controls the beam path, scanning continuously from track to track from the first track to the last track, and repeating the process again from the first track to the last track. An algorithm such as a track finding algorithm such as US Patent Application Publication No. US 2006-0260941-A1 is used to identify the position of the track. An embodiment of an optical detection system for simultaneous multi-channel and multi-dye detection is shown in FIG. 16. The fluorescent excitation and detection system 40 excites the electrophoretic separation by DNA samples (e.g. containing DNA fragments amplified at a set of STR positions) by scanning energy through each of the microchannels (e.g. laser beam) At the same time, the components of the dye are collected and transferred to one or more light detectors for recording and final analysis. In one embodiment, the fluorescent excitation and detection assembly 40 includes a laser 60 , a scanner 62 , one or more optical detectors 64 and various mirrors 68 , a spectrograph, and a passage 42 The laser beam emitted from the laser 60 is transmitted to the test module 55 and returned to the lens 72 of the light detector 64 . The scanner 62 moves the incident laser beam to various scanning positions relative to the test module 55 . In particular, the scanner 62 moves the laser beam to the relevant part of each microchannel in the test module 55 to detect the separate components. The multi-element PMT 64 collects data from the test module 55 (such as fluorescent signals from DNA fragments of varying lengths) and provides the data electronically to the data acquisition and storage system located outside the protective layer 50 via a cable connected to port 75 . In one embodiment, the data acquisition and storage system may include a ruggedized computer available from Option Industrial Computers (13 Audreuil-Dorion, Quebec, Canada). In another embodiment ("initial mode"), the excitation source is incident on all detection points simultaneously, and the fluorescence from all detection points is collected simultaneously. At the same time, the spectral dispersion (detecting the wavelength spectrum of fluorescent light) and the spatial dispersion (detecting points) can be performed through the two-dimensional detector array. In this configuration, the 2D detector array is positioned in the system so that the spectral components are imaged and detected across one dimension (row) of the array detector, while the spatial components cross the other of the array detector One-dimensional imaging and detection. The preferred instrument uses the scan mode of operation rather than the "start" mode. In the scanning mode, when the scanner matches the path to be interrogated and before it is incident on another channel, the signals of each channel need to be collected, integrated, and read out. A detector with fast readout allows optimal light collection and integration, which translates into higher signal-to-noise performance. Ideally, the readout time of the detector should be significantly less than the total time the scanner meets the channel. Multi-component PMT can be read in less than 0.7 ms and the read time is much shorter than the integration time of the detection of each channel. The fluorescent incident light on the pinhole can be dispersed by the grating according to its wavelength and focused on the linear multi-anode PMT detector array. The detector provides 32 current outputs, one of each element in the array corresponds to the number of photons incident on the element. In the multi-sample (or channel) detection process, when the laser excites the selected channel at an appropriate position, the integrating circuit will integrate the PMT output current to generate an output voltage proportional to the integrated PMT current. At the same time, use Analog Devices (Norwood, MA) differential driver IC (P/N SSM2142) to convert the single-ended output voltage into differential mode. At the end of the integration time (determined by the scan rate and the number of tracks), the data acquisition system will read the differential signal and save the data in its buffer. After saving the data, the system will move the scanner to transfer the laser beam to another option, and reset the integration circuit. Each single-component PMT module has its own integration circuit. For the 8-color detection system, there are 8 PMT modules and 8 integration circuits. Additional colors can be added using the corresponding number of PMT modules and integrating circuits. Since each of the PMT components (H77260-20, Hamamatsu, Japan) has a signal reflection similar to or faster than a single PMT tube (H7732-10, Hamamatsu, Japan), and is read out in parallel, it is possible to operate this reconnaissance very quickly Tester. When coupled with a spectrometer, this spectrometer and multi-anode detector system can provide a complete spectral scan across the visible spectrum (450 nm to 650 nm) with a readout time of less than 0.1 ms. The ability to provide a fast update rate allows this spectrometer/detector system to be applied to a scan mode embodiment that continuously detects multiple channels in a single run. The use of PMT-based detectors provides low noise, high sensitivity, high dynamic range and fast response. The 140 mm spectrometer with a concave holographic grating (Horiba Jobin-Yvon) and a multi-anode PMT detector is the H7260-20 detector (Hamamatsu, Japan). Other spectrometer configurations and multi-anode PMT detectors can also be used in this application. Use signal processing algorithms to modify, filter, and analyze data to obtain nucleotide bases for electropherogram determination. This process consists of locating a callable signal, correcting the signal baseline, filtering noise, removing color cross-talk, identifying signal peaks, and determining related bases. The positioning callable signal is to remove irrelevant data from the beginning and end of the signal and is done by using thresholds. Next, the background is removed from the signal, so the signal has a common baseline for all detected colors. Finally, a low-pass filter is applied to remove high-frequency noise from the signal. To eliminate the detected color ambiguity, a weighting matrix is calculated and applied to the signal to amplify the color-space of the nucleotide-dye spectrum. The calculation of this color separation matrix is done using the method of Li et al., Electrophoresis 1999, 20, 1433-1442. In this adaptation, the “m×n” color separation matrix is calculated from the number of dyes “m” used in the relevant verification and the number “n” of detector elements. The conversion of the signal from the detector space (PMT element) into the dye space is performed by the matrix operation described below: D=CSM×PMT, where D is the signal in the dye space of each of the m dyes, CSM is The color separation matrix, and the PMT is a matrix with signals from each of the n elements of the detector. Second, a combination of zero-crossing filters and frequency analysis is used to identify the peaks in the color-separated signal. Finally, for the application of size measurement, the modified trace is allele-called to identify each fragment and allocate the size of the fragment based on the size measurement criteria. For DNA sequencing applications, the modified trace is base-called to associate one of the four nucleotides with each peak in the trace. A detailed description of the base correspondence can be found in Ewing et al., Genome Research , 1998, 8, 175-185, and Ewing et al., Genome Research , 1998, 8, 186-194. The full text of these disclosures is incorporated by reference Into this article. 3. Dye label The dye label attached to the oligonucleotide and the modified oligonucleotide can be synthetic or purchased (eg, Operan Biotechnologies, Huntsville, Alabama). A large number of dyes (more than 50) can be used in fluorescent excitation applications. These dyes include those from the luciferin, rhodamine Alexa Fluor, Biodipy, Coumarin and Cyanine dye families. In addition, inhibitors can also be used to label oligo sequences to minimize background fluorescence. Dyes with emission maximums from 410 nm (Cascade Blue) to 775 nm (Alexa Fluor 750) are commercially available and can be used. Dyes in the range of 500 nm to 700 nm have the advantage that they are in the visible spectrum and can be detected using conventional photomultiplier tubes. The wide range of commercially available dyes allows the selection of dye sets with emission wavelengths throughout the detection range. A detection system capable of discriminating many dyes has been reported for flow cytometry applications (see Perfetto et al., Nat. Rev. Immunol. 2004, 4, 648-55; and Robinson et al., Proc of SPIE 2005, 5692, 359 -365). Fluorescent dyes have a peak excitation wavelength that is blue shifted from its peak emission wavelength, usually 20 to 50 nm. Therefore, the use of dyes with a wide range of emission wavelengths may require the use of multiple excitation sources, where the excitation wavelengths within the emission wavelength range achieve effective excitation of the dye. Alternatively, energy transfer dyes can be used to make a single laser with a single emission wavelength suitable for exciting all dyes of interest. This is achieved by connecting the energy transfer part to the dye label. This part is usually another fluorescent dye, which has an absorption wavelength compatible with the excitation wavelength of the light source (such as a laser). The placement of this absorber very close to the emitter allows absorption energy to be transferred from the absorber to the emitter, allowing more efficient excitation of long-wavelength dyes (Ju et al., Proc Natl Acad Sci USA 1995, 92, 4347-51). The dye-labeled dideoxynucleotide can be purchased from Perkin Elmer, (Waltham, MA). B. Examples Example 5. Six-color separation and detection of nucleic acids The following examples illustrate the separation and detection of nucleic acid fragments labeled with 6 fluorescent dyes, and demonstrate the color resolution capabilities of spectrometer/multi-element excitation/detection systems. DNA fragments were labeled with 6-FAM, VIC, NED, PET dyes by using fluorescently labeled primers in multiplex PCR amplification reactions. In this reaction, 1 ng of human gene DNA (9947A) was amplified in a 25 μL reaction under conditions recommended by the manufacturer (AmpFlSTR Identifiler, Applied Biosystems). The 2.7 μL PCR product was removed and mixed with 0.3 μL GS500-LIZ sizing standard (Applied Biosystems) and 0.3 μL HD400-ROX sizing standard. HiDi (Applied Biosystems) was added to a total of 13 μL and the sample was inserted into the sample well of the separation biochip and subjected to electrophoresis. Genebench consists of a series of four operations for DNA electrophoretic separation: pre-electrophoresis, loading, injection and separation. These operations are performed on a microfluidic biochip, which is heated to a uniform temperature of 50°C. The biochip contains a 16-channel system for multiple separations and detections, each consisting of an injector channel and a separation channel. The DNA used for analysis is separated along the separation channel by the electrophoretic transport of DNA through the screening matrix. The separation length of the biochip is in the range of 160 to 180 mm. The first step is pre-electrophoresis, which is achieved by applying a 160 V/cm field along the length of the channel for six (6) minutes. Inject separation buffer (TTE1X) into the anode, cathode and waste wells. Inject the sample for analysis into the sample well and apply 175 V from the sample well to the waste well for 18 seconds, then apply 175 V across the sample and waste well, and apply 390 V at the cathode for 72 seconds to load the sample into the separation In the channel. The injection of the sample is accomplished by applying a 160 V/cm field along the length of the separation channel, while applying 50 V/cm and 40 V/cm fields across the sample and the waste hole, respectively. The separation is continued with the injection voltage parameter for 30 minutes, during which the optical system detects the separation band of DNA. The data collection rate is 5 Hz and the PMT gain is set to -800 V. Load 16 samples containing amplified DNA for simultaneous separation and detection. The signals from each of the 32-element PMT are collected as a function of time to generate an electropherogram. The resulting electropherogram (FIG. 17) shows the peak corresponding to the presence of the DNA fragment at the excitation/detection window of one of the 16 channels. In addition, for each peak, the relative signal intensity of each element of the 32-element PMT corresponds to the spectral content of the dye associated with the DNA fragment (or dye, if more than one dye is present in the detection window). Fig. 18 shows the emission spectrum of the detected dye and the background spectrum of the substrate. Subtract the peak of the substrate background spectrum from the spectrum. Performing this practice resulted in the identification of 6 different dye spectra. The spectra of the 6 dyes are superimposed on the same curve. Comparing this information with the actual published spectrum of dyes shows that the relative values of these dyes are similar to published information. This example shows that the system can detect and distinguish 6 dyes in the reaction solution. This spectral output is used to generate a color correction matrix and convert the signal from the detector space to the dye space representation (Figures 19 and 20). Example 6. Eight-color separation and detection of nucleic acids In this example, eight dye separation and detection of fluorescent dye-labeled acids are shown. For the 8-position forward primer and reverse primer pair sequences were selected from published sequences (Butler et al., J Forensic Sci 2003, 48, 1054-64). Although any of the sites described in the document and therefore the primer pairs can also be used in this example, the selected sites are CSF1P0, FGA, THO1, TPOX, vWA, D3S1358, D5S818, and D7S820. For primer pairs, each of the forward primers is labeled with an independent fluorescent dye (Operon Biotechnologies, Huntsville, Alabama). The dyes selected for attachment to the primer include Alexa Fluor Dyes 488, 430, 555, 568, 594, 633, 647, and Tamra. Many other dyes are commercially available and can also be used as markers. Each site was independently amplified according to the PCR reaction protocol of (Butler, 2003, Id. ) to produce a reaction solution having fragments labeled with the corresponding dyes. The template for the PCR reaction was 1 ng of human genetic DNA (type 9947A from Promega, Madison WI). Each PCR reaction was purified by purification through a PCR purification column, in which primers (labeled and dye-labeled primers) and enzyme were removed and the PCR buffer was exchanged by DI dissolving agent. The purified product is a solution of labeled DNA fragments in DI water. A MinElute ^™ column (Qiagen, Valencia, CA) was used to purify the dye-labeled product according to the Smith protocol. A total of eight reactions were carried out. The eight cleaned PCR reactions were mixed together at a ratio that produced peaks with equal signal intensities, resulting in a mixture containing fragments labeled with 8 different dyes. Alternatively, 8-site primers can be mixed together to form a main primer mixture for multiple amplification. This solution was separated and detected using the equipment and protocol as described in Example 1. Adjust the grating of the spectrograph so that the emission of 8 dyes falls within 32 pixels of the detector element. Adjust the number of samples loaded for analysis so that the detected signal belongs to the dynamic range of the detection system. Example 7. Spectrometer / multi-element PMT system The following example illustrates the separation/detection of labeled DNA fragments using the spectrometer/multi-element PMT system of FIG. 16, specifically to identify sequences of DNA templates. In this reaction, 0.1 pmol DNA template M13 and M13 sequencing primers were amplified with GE Amersham BigDye ^™ sequencing kit according to the recommended reaction conditions. The reaction mixture was purified by ethanol precipitation and resuspended in 13 μL DI water. The samples were separated under electrophoretic separation conditions as described in Example 5. Change the sample loading conditions and proceed by applying 175 V across the sample hole to the waste hole for 105 seconds. Figure 21 shows an electropherogram of a DNA sequence, in which colored traces represent detector elements corresponding to the maximum spectral value of each of the four dyes used. The sequence obtained was 519 bases corresponding to Phred quality score> 20 bases and 435 base QV30 (Figure 22). Example 8. Simultaneous Separation and Detection of Two Sequencing Reaction Products In this example, the separation and detection of fragments from two DNA template cycle sequencing are performed simultaneously in a single separation channel. The cycle sequencing reaction can be prepared by the dye-labeled terminator reaction or the dye-labeled primer reaction as follows: For the dye-labeled terminator reaction: prepare a cycle sequencing reaction of each template fragment, in which the template fragment consists of the following Composition: sequencing primers suitable for the template sequence of interest; and reagents for DNA sequencing, including cycle sequencing buffers, polymerases, oligonucleotides, dideoxynucleotides and labeled dideoxynucleosides acid. Eight different dyes are used for marking. In the first cycle sequencing reaction, a group of dideoxynucleotides labeled with 4 dyes was used. In the second cycle sequencing reaction, another group of dideoxynucleotides labeled with 4 dyes (where the emission wavelength is different from the four dyes they used in the first cycle sequencing reaction) was used. Each cycle sequencing reaction was performed independently according to the scheme of multiple thermal cycles for each reaction. Each thermal cycle includes denaturation, annealing, and extension steps, where the temperature and number follow the Sanger scheme (see, Sanger et al., Proc Natl Acad Sci USA 1977, 74, 5463-7). The cycle sequencing products from the two reactions were combined to form a sample consisting of DNA fragments labeled with a total of eight unique dyes from each of the two DNA templates. For dye-labeled primer reactions: Alternatively, samples for separation and detection can be manufactured by using primer-labeled cycle sequencing. Four cycle sequencing reactions were performed for each DNA template. Each reaction is a cycle sequencing reaction composed of labeled sequencing primers and reagents (including cycle sequencing buffer, polymerase, oligonucleotide) for DNA sequencing. In addition, each reaction will include one of dideoxynucleotide (ddATP, ddTTP, ddCTP, or ddGTP) and a labeled primer. Each dye associated with the primer has a unique emission wavelength and is related to the type of dideoxynucleotide (ddATP, ddTTP, ddCTP, or ddGTP) in the circulating sequencing solution. Each cycle sequencing reaction was performed independently according to the scheme of multiple thermal cycles for each reaction. Each thermal cycle includes denaturation, annealing, and extension steps, where the temperature and number of times follow the Sanger scheme (see, Sanger, 1977, Id. ). For the second DNA template for cycle sequencing, another set of 4 dyes is used (where the emission wavelength is different from the four dyes used in the first cycle sequencing reaction). The products of all eight reactions (each with a different dye) are mixed together to form a sample consisting of DNA fragments from each of the two DNA templates. Samples for separation and detection: Purify each of the sequencing reactions by ethanol precipitation. The separation and detection of samples follow the scheme of Example 8. The result of the separation and detection is to generate two different DNA sequences, corresponding to each of the two template DNA fragments. The method of this example can be modified to allow the use of a multiple of four dyes to detect the DNA sequence of that multiple in a single separation channel (eg 12 dyes for simultaneous detection of 3 sequences and 4 for simultaneous detection) 16 dyes in sequence, 20 dyes for simultaneous detection of 5 sequences, etc.). Finally, the separation of labeled fragments need not be limited to electrophoresis. Example 9 Isolation and detection of 500 or more sites in a single channel There are several applications of nucleic acid analysis that can be used in clinical diagnosis, including DNA and RNA sequencing and fragment size determination. In this example, the simultaneous detection of the use of 10 colors allows interrogation of up to 500 sites. For example, size analysis of a large number of fragments can be used to identify pathogens or to characterize many loci within an individual's genome. In the context of prenatal and pre-implantation genetic diagnosis, aneuploidy is currently diagnosed by karyotype and by fluorescence in situ hybridization (FISH). In the FISH study, the presence of two signals per cell indicates that there are two copies of a given site in the cell, one signal indicates monosomy or partial monosomy, and three signals indicate trisomy or partial trisomy . FISH usually uses about 10 probes to analyze whether the cell contains normal chromosomal complement. However, this method does not allow a detailed examination of the entire genome, and cells known to be normal by FISH are likely to have major abnormalities that cannot be detected by this technique. The teachings of the present invention use multicolor separation and detection to allow approximately 500 chromosomal sites to be widely dispersed throughout all chromosomes to be analyzed, thereby allowing more detailed analysis of the chromosome structure. In this example, a primer pair sequence of about 500 sites is selected from the published sequences, where each site exists as a single copy per haploid genome. In addition, 10 sets of 50 primer pairs are selected so that each set defines a corresponding set of DNA fragments, so that none of the fragments have the same size. For each group, the primer pair is labeled with a fluorescent dye, and no two groups share the same dye. The dyes selected for attachment to the primers are Alexa Fluor Dyes 488, 430, 555, 568, 594, 633, 647, 680, 700 and Tamra. Many other dyes are commercially available and can also be used as markers. Sites can be amplified in one or several parallel PCR reactions, as described in "METHODS FOR RAPID MULTIPLEXED AMPLIFICATION OF TARGET NUCLEIC ACIDS" above. The methods described herein are used to isolate and detect amplified primers. In a single separation channel, all 500 fragments can be accurately identified according to size, and 50 fragments can be identified for every ten dyes. The number of sites, dyes and separation channels can vary based on the desired application. If necessary, a smaller number of fragments can be detected by using a smaller number of dye labels or generating fewer DNA fragments per label; therefore, less than 500, less than 400, less than 300, less than 200, less than 100, less than 75, less than 50, less than 40, less than 30 or less than 20 fragments can be detected as desired. The maximum number of recognizable sites per lane is based on the read length and resolution of the separation system (e.g. a single base pair analysis of a DNA fragment in the range of 20 to 1500 base pairs results in hundreds of fragments) multiplied by the detectable The number of different dyes (as mentioned above, dozens are available). Therefore, thousands of sites can be identified in a single separation channel, and this number will increase when additional dyes are developed.

40‧‧‧螢光激發及偵測總成42‧‧‧開口50‧‧‧保護層55‧‧‧測試模組60‧‧‧雷射器62‧‧‧掃描器/掃描鏡系統64‧‧‧光偵測器/多元件PMT68‧‧‧鏡72‧‧‧透鏡75‧‧‧埠101‧‧‧室104‧‧‧埠105‧‧‧埠106‧‧‧埠107‧‧‧埠108‧‧‧埠109‧‧‧埠202‧‧‧通孔203‧‧‧通孔204‧‧‧樣品室205‧‧‧第一混合接合點208‧‧‧分配通道209‧‧‧計量室210‧‧‧毛細管閥211‧‧‧毛細管閥212‧‧‧混合球形物213‧‧‧收縮部分214‧‧‧混合通道215‧‧‧通孔216‧‧‧通孔217‧‧‧通孔218‧‧‧循環測序試劑計量室219‧‧‧毛細管閥220‧‧‧毛細管閥221‧‧‧毛細管閥227‧‧‧通孔303‧‧‧樣品通道304‧‧‧通孔/通道305‧‧‧通孔306‧‧‧通孔307‧‧‧室308‧‧‧通孔309‧‧‧室310‧‧‧通道311‧‧‧通孔314‧‧‧通孔315‧‧‧通孔316‧‧‧通孔317‧‧‧通孔320‧‧‧通孔336‧‧‧通孔402‧‧‧通孔403‧‧‧通孔404‧‧‧通孔502‧‧‧PCR室503‧‧‧循環測序室1104‧‧‧埠1105‧‧‧通道1106‧‧‧室1108‧‧‧毛細管閥1110‧‧‧通孔毛細管閥1111‧‧‧通孔1112‧‧‧UF輸入室1113‧‧‧毛細管閥1115‧‧‧過濾室1116‧‧‧超濾(UF)過濾器1119‧‧‧埠1120‧‧‧埠1121‧‧‧儲集器/室1122‧‧‧通道1123‧‧‧溢流室1124‧‧‧埠1301‧‧‧整合生物晶片1302‧‧‧16-樣品生物晶片/子組件1303‧‧‧16-道塑料分離生物晶片/分離子組件1304‧‧‧輸送點1305‧‧‧輸入孔1306‧‧‧分離通道1307‧‧‧偵測區1308‧‧‧凹處1401‧‧‧液體接收孔/孔儲集器1402‧‧‧主要分離電極1403‧‧‧反電極40‧‧‧ Fluorescence excitation and detection assembly 42‧‧‧ opening 50‧‧‧ protective layer 55‧‧‧ test module 60‧‧‧ laser 62‧‧‧ scanner/scan mirror system 64‧‧ ‧Photodetector/Multi-element PMT68‧‧‧Mirror 72‧‧‧Lens 75‧‧‧Port 101‧‧‧Room 104‧‧‧ Port 105‧‧‧Port 106‧‧‧Port 107‧‧‧‧108 108 ‧‧Port 109‧‧‧port 202‧‧‧through hole 203‧‧‧through hole 204‧‧‧sample chamber 205‧‧‧ first mixing junction 208‧‧‧distribution channel 209‧‧‧measuring chamber 210‧‧ ‧Capillary valve 211‧‧‧Capillary valve 212‧‧‧ Mixed spherical object 213‧‧‧Constriction part 214‧‧‧Mixing channel 215‧‧‧Through hole 216‧‧‧Through hole 217‧‧‧Through hole 218‧‧‧ Cycle sequencing reagent metering chamber 219‧‧‧Capillary valve 220‧‧‧Capillary valve 221‧‧‧Capillary valve 227‧‧‧Through hole 303‧‧‧Sample channel 304‧‧‧Through hole/channel 305‧‧‧Through hole 306 ‧‧‧Through hole 307‧‧‧Room 308‧‧‧Through hole 309‧‧‧Room 310‧‧‧Channel 311‧‧‧Through hole 314‧‧‧Through hole 315‧‧‧Through hole 316‧‧‧Through hole 317‧‧‧Through hole 320‧‧‧Through hole 336‧‧‧Through hole 402‧‧‧Through hole 403‧‧‧Through hole 404‧‧‧Through hole 502‧‧‧‧PCR chamber 503‧‧‧Cycling sequencing room 1104 ‧‧‧Port 1105‧‧‧Channel 1106‧‧‧Room 1108‧‧‧Capillary valve 1110‧‧‧Through hole capillary valve 1111‧‧‧Through hole 1112‧‧‧UF input chamber 1113‧‧‧Capillary valve 1115‧‧ ‧Filter chamber 1116‧‧‧Ultrafiltration (UF) filter 1119‧‧‧ port 1120‧‧‧ port 1121‧‧‧reservoir/chamber 1122‧‧‧channel 1123‧‧‧ overflow chamber 1124‧‧‧ port 1301‧‧‧ Integrated biochip 1302‧‧‧16-Sample biochip/subassembly 1303‧‧‧‧16-channel plastic separation biochip/separation subassembly 1304‧‧‧Transport point 1305‧‧‧Input hole 1306‧‧‧ Separation channel 1307‧‧‧detection area 1308‧‧‧recess 1401‧‧‧liquid receiving hole/hole reservoir 1402‧‧‧main separation electrode 1403‧‧‧counter electrode

圖1為針對4個個別樣品之溶解及模板擴增而言的整合生物晶片之實施例的圖示。圖2為圖1之生物晶片的第一層之實施例的圖示。圖3為圖1之生物晶片的第二層之實施例的圖示。圖4為圖1之生物晶片的第三層之實施例的圖示。圖5為圖1之生物晶片的第四層之實施例的圖示。圖6為圖1之生物晶片的裝配及連接之實施例的圖示。圖7為說明針對兩種閥(平面內閥及通孔閥)而言的去離子水及循環測序試劑之閥的毛細管閥調壓力與反向水力直徑之函數關係的圖。圖8為展示針對PCR模板擴增而言的圖1之生物晶片的流體步驟之實施例的圖示。圖8a為展示已將樣品及PCR試劑裝載於本發明之生物晶片中的圖示。圖8b為展示經由通道至樣品室傳遞樣品之圖示(其係沿樣品通道以不同位置展示以說明流動路徑)。圖8c為展示樣品室中之樣品的圖示。圖8d為展示將PCR試劑傳遞至試劑室之圖示。圖8e為展示將過量PCR試劑抽出之圖示。圖8f及8g為展示藉由第一組毛細管閥進行液體之初始混合步驟及滯留之圖示。圖8h至8j為展示將混合液體傳遞至PCR室之圖示，其中在該點處開始熱循環。圖9為展示整合生物晶片之流體步驟之實施例的圖示。圖9a至9e為展示將循環測序試劑傳遞至層1中之計量室且自該等室附近移除過量試劑的圖示。圖9f及9g為展示將PCR產物引入桑格反應室之圖示。圖9h-9k為展示藉由往復運動將桑格試劑與PCR產物混合之圖示。圖9l為展示循環產物可加以移除以供分析之圖示。圖10為對圖1之生物晶片中產生之產物測序的測序迹線(電泳圖)。圖11為展示針對循環測序產物之超濾效能而言的整合生物晶片之實施例的圖示。除在層3與4之間添加超濾(UF)過濾器1116 外，該晶片裝配類似於生物晶片1之裝配。圖12為展示在測序產物之純化期間圖11之生物晶片之流體步驟的圖示。圖12a及12b為展示將桑格產物傳遞至UF輸入室之圖示。圖12c為展示已將測序產物傳遞至過濾室之圖示。圖12d為展示測序產物幾乎完全過濾之圖示。圖12e至12g為展示將洗滌劑傳遞至UF輸入室且接著自傳遞通道移除過量洗滌劑之圖示。圖12h為展示第一洗滌循環開始之圖示；其後為如圖12d中之過濾及隨後之洗滌循環。圖12i及12j為展示傳遞至UF輸入室之溶離液體(與洗滌劑相同之液體)的圖示。圖12k到12m為展示對UF輸入室加壓且關閉輸出埠並接著釋放壓力導致往復運動之單一循環的圖示。圖12n為展示為進一步處理或移除而準備之純化產物的圖示。圖13為展示針對模板擴增、循環測序、測序產物淨化、藉由電泳分離及藉由雷射誘發螢光偵測之效能而言的整合生物晶片之實施例的圖示。圖14為展示藉由反電極使經標記之核酸片段濃縮且注入分離通道的圖示。圖15為展示激發及偵測系統之實施例的圖示。圖16為展示激發及偵測系統之實施例的圖示。圖17為分離及偵測6-染料樣品所產生之電泳圖。圖中各迹線表示來自32-陽極光電倍增管(PMT)之32個元件中之各者的信號。各迹線相對於彼此偏移以允許資料易於被檢視。圖18為展示自電泳圖提取的6種染料之各者之染料光譜的圖；亦展示背景螢光光譜。圖19為展示6-FAM、VIC、NED、PET及LIZ染料之染料發射光譜的圖。圖20為展示5-FAM、JOE、NED及ROX染料之染料發射光譜的圖。圖21為分離及偵測4種染料樣品所產生之電泳圖。圖中各迹線表示來自32-陽極PMT之32個元件中之各者的信號。各迹線相對於彼此偏移以允許資料易於被檢視。圖22為測序迹線。Figure 1 is an illustration of an embodiment of an integrated biochip for dissolution and template amplification of 4 individual samples. FIG. 2 is an illustration of an embodiment of the first layer of the biochip of FIG. 1. FIG. FIG. 3 is an illustration of an embodiment of the second layer of the biochip of FIG. 1. FIG. 4 is an illustration of an embodiment of the third layer of the bio-wafer of FIG. FIG. 5 is an illustration of an embodiment of the fourth layer of the biochip of FIG. 1. FIG. 6 is an illustration of an embodiment of the assembly and connection of the biochip of FIG. 7 is a graph illustrating the function of capillary valve pressure regulation and reverse hydraulic diameter for two types of valves (in-plane valve and through-hole valve) for deionized water and circulating sequencing reagent valves. 8 is a diagram showing an embodiment of the fluid steps of the bio-wafer of FIG. 1 for PCR template amplification. FIG. 8a is a diagram showing that a sample and PCR reagent have been loaded into the bio-wafer of the present invention. Figure 8b is a diagram showing the transfer of samples through the channel to the sample chamber (which are shown in different positions along the sample channel to illustrate the flow path). Figure 8c is a diagram showing the sample in the sample chamber. Figure 8d is a diagram showing the delivery of PCR reagents to the reagent chamber. Figure 8e is a diagram showing the extraction of excess PCR reagent. 8f and 8g are diagrams showing the initial mixing step and retention of liquid by the first set of capillary valves. 8h to 8j are diagrams showing the transfer of the mixed liquid to the PCR chamber, where the thermal cycle starts at this point. FIG. 9 is a diagram showing an embodiment of the fluid step of integrating a biochip. 9a to 9e are diagrams showing the delivery of cycle sequencing reagents to the metering chambers in layer 1 and the removal of excess reagents from near the chambers. 9f and 9g are diagrams showing the introduction of PCR products into the Sanger reaction chamber. 9h-9k are diagrams showing the mixing of Sanger reagent and PCR products by reciprocating motion. Figure 91 is a diagram showing that the circulating product can be removed for analysis. FIG. 10 is a sequencing trace (electrophoresis diagram) for sequencing products produced in the biochip of FIG. 1. FIG. 11 is a diagram showing an embodiment of an integrated biochip in terms of ultrafiltration performance of cycle sequencing products. The wafer assembly is similar to that of biological wafer 1 except that an ultrafiltration (UF) filter 1116 is added between layers 3 and 4. FIG. 12 is a diagram showing the fluid steps of the biochip of FIG. 11 during purification of sequencing products. 12a and 12b are diagrams showing the delivery of Sanger products to the UF input chamber. Figure 12c is a diagram showing that the sequencing product has been delivered to the filtration chamber. Figure 12d is a diagram showing that the sequencing products are almost completely filtered. 12e to 12g are diagrams showing delivery of detergent to the UF input chamber and then removal of excess detergent from the delivery channel. Fig. 12h is a diagram showing the start of the first washing cycle; followed by filtration and subsequent washing cycles as in Fig. 12d. 12i and 12j are diagrams showing the dissolved liquid (the same liquid as the detergent) delivered to the UF input chamber. 12k to 12m are diagrams showing a single cycle of pressurizing the UF input chamber and closing the output port and then releasing the pressure to cause reciprocating motion. Figure 12n is a diagram showing the purified product prepared for further processing or removal. 13 is a diagram showing an embodiment of an integrated biochip for template amplification, cycle sequencing, purification of sequencing products, separation by electrophoresis, and fluorescence-induced fluorescence detection by laser. 14 is a diagram showing that the labeled nucleic acid fragments are concentrated and injected into the separation channel by the counter electrode. 15 is a diagram showing an embodiment of an excitation and detection system. Figure 16 is a diagram showing an embodiment of an excitation and detection system. Figure 17 is an electropherogram generated by separating and detecting 6-dye samples. Each trace in the figure represents the signal from each of the 32 elements of the 32-anode photomultiplier tube (PMT). The traces are offset relative to each other to allow the data to be easily viewed. Fig. 18 is a graph showing the dye spectrum of each of the six dyes extracted from the electrophoresis chart; the background fluorescence spectrum is also shown. FIG. 19 is a graph showing the dye emission spectra of 6-FAM, VIC, NED, PET, and LIZ dyes. FIG. 20 is a graph showing the dye emission spectra of 5-FAM, JOE, NED, and ROX dyes. Figure 21 is an electropherogram generated by separating and detecting four kinds of dye samples. Each trace in the figure represents the signal from each of the 32 elements of the 32-anode PMT. The traces are offset relative to each other to allow the data to be easily viewed. Figure 22 is a sequencing trace.

101‧‧‧室 101‧‧‧

104‧‧‧埠 104‧‧‧ port

105‧‧‧埠 105‧‧‧ port

106‧‧‧埠 106‧‧‧ port

107‧‧‧埠 107‧‧‧ port

108‧‧‧埠 108‧‧‧ port

109‧‧‧埠 109‧‧‧ port

Claims

一種用於多種樣品DNA分析之方法，其包含：將自生物晶片上之第一樣品室之第一樣品提取的第一模板DNA，經由第一通道注入該生物晶片之第一子組件中之第一反應儲集器；將自該生物晶片上之第二樣品室之第二樣品提取的第二模板DNA，經由第二入口注入該生物晶片之第一子組件中之第二反應儲集器，該第二樣品室係與該第一樣品室分開；在該生物晶片之第一子組件引發熱循環以進行DNA片段之PCR擴增，該第一子組件至少包括選定用於基於第一樣品進行PCR擴增之第一反應儲集器，以及選定用於基於第二樣品進行PCR擴增之第二反應儲集器；引發液體流動以分別將第一擴增DNA片段自第一反應儲集器移至該生物晶片之第二子組件中之第一分離單位，以及將第二擴增DNA片段自第二反應儲集器移至該生物晶片之第二子組件中之第二分離單位；在該第一分離單位中引發電場，以在該生物晶片上之第一分離通道中根據大小分離該第一擴增DNA片段；在該第二分離單位中引發電場，以在該生物晶片上之第二分離通道中根據大小分離該第二擴增DNA片段，該第二分離通道係與第一分離通道流體分離；及偵測經分離之DNA片段。 A method for DNA analysis of multiple samples, comprising: injecting the first template DNA extracted from the first sample of the first sample chamber on the biological wafer into the first sub-assembly of the biological wafer through the first channel The first reaction reservoir; the second template DNA extracted from the second sample in the second sample chamber on the bio-wafer is injected into the second reaction reservoir in the first sub-assembly of the bio-wafer through the second inlet The second sample chamber is separated from the first sample chamber; thermal cycling is initiated in the first sub-assembly of the bio-wafer for PCR amplification of DNA fragments. The first sub-assembly includes at least A first reaction reservoir for PCR amplification of a sample, and a second reaction reservoir selected for PCR amplification based on the second sample; initiating liquid flow to separate the first amplified DNA fragments from the first The reaction reservoir is moved to the first separation unit in the second subassembly of the biochip, and the second amplified DNA fragment is moved from the second reaction reservoir to the second in the second subassembly of the biochip Separation unit; an electric field is induced in the first separation unit to separate the first amplified DNA fragment according to size in the first separation channel on the biochip; an electric field is induced in the second separation unit to cause the The second amplified DNA fragment is separated according to size in a second separation channel on the wafer, the second separation channel is fluidly separated from the first separation channel; and the separated DNA fragment is detected.

如請求項1之方法，其進一步包含：自該第一樣品提取該第一模板DNA；及自該第二樣品提取該第二模板DNA。 The method of claim 1, further comprising: extracting the first template DNA from the first sample; and extracting the second template DNA from the second sample.

如請求項1之方法，其進一步包含：將自該生物晶片上之第三樣品室之第三樣品提取的第三模板DNA，經由第三通道注入該生物晶片之第一子組件中之第三反應儲集器，該第三樣品室係與該第一及第二樣品室分開；將自該生物晶片上之第四樣品室之第四樣品提取的第四模板DNA，經由第四入口注入該生物晶片之第一子組件中之第四反應儲集器，該第四樣品室係與該第一、第二及第三樣品室分開；在該生物晶片之第一子組件引發熱循環以進行DNA片段之PCR擴增，該第一子組件至少包括選定用於基於第三樣品進行PCR擴增之第三反應儲集器，以及選定用於基於第四樣品進行PCR擴增之第四反應儲集器；引發液體流動以分別將第三擴增DNA片段自第三反應儲集器移至該生物晶片之第二子組件中之第三分離單位，以及將第四擴增DNA片段自第四反應儲集器移至該生物晶片之第二子組件中之第四分離單位；在該第三分離單位中引發電場，以在該生物晶片上之第三分離通道中根據大小分離該第三擴增DNA片段；在該第四分離單位中引發電場，以在該生物晶片上之第四分離通道中根據大小分離該第四擴增DNA片段，該第四分離通道係與第一、第二及第三分離通道流體分離；及偵測經分離之DNA片段。 The method of claim 1, further comprising: injecting the third template DNA extracted from the third sample of the third sample chamber on the bio-wafer into the third of the first sub-assembly of the bio-wafer through the third channel The reaction reservoir, the third sample chamber is separated from the first and second sample chambers; the fourth template DNA extracted from the fourth sample of the fourth sample chamber on the bio-wafer is injected into the via the fourth inlet The fourth reaction reservoir in the first sub-assembly of the bio-wafer, the fourth sample chamber is separated from the first, second, and third sample chambers; thermal cycling is initiated in the first sub-assembly of the bio-wafer to proceed For PCR amplification of DNA fragments, the first subassembly includes at least a third reaction reservoir selected for PCR amplification based on a third sample, and a fourth reaction reservoir selected for PCR amplification based on a fourth sample Collector; inducing liquid flow to move the third amplified DNA fragment from the third reaction reservoir to the third separation unit in the second subassembly of the biochip, and the fourth amplified DNA fragment from the fourth The reaction reservoir moves to the fourth separation unit in the second subassembly of the biochip; an electric field is induced in the third separation unit to separate the third expansion according to size in the third separation channel on the biochip Increase the DNA fragment; trigger an electric field in the fourth separation unit to separate the fourth amplified DNA fragment according to the size in the fourth separation channel on the biochip, the fourth separation channel is connected to the first, second and Fluid separation in the third separation channel; and Detect the separated DNA fragments.

如請求項2之方法，其進一步包含：將第一試劑及該第一模板DNA注入該第一反應儲集器中；及將第二試劑及該第二模板DNA注入該第二反應儲集器中。 The method of claim 2, further comprising: injecting a first reagent and the first template DNA into the first reaction reservoir; and injecting a second reagent and the second template DNA into the second reaction reservoir in.

如請求項1之方法，其中用於偵測該經分離之DNA片段之系統進一步包含：一或多個經定位用以照明該第一分離通道上之第一偵測位置及該第二分離通道上之第二偵測位置的光源；用於在偵測位置之間連續掃描該一或多個光源的鏡；一或複數個經定位用以收集及引導自該等偵測位置發出之光的第一光學元件；及經定位以接收來自該一或複數個第一光學元件之光的光偵測器。 The method of claim 1, wherein the system for detecting the isolated DNA fragment further comprises: one or more positioned to illuminate the first detection position on the first separation channel and the second separation channel The light source at the second detection position on the top; the mirror used to continuously scan the one or more light sources between the detection positions; one or more positioned to collect and guide the light emitted from these detection positions A first optical element; and a photodetector positioned to receive light from the one or more first optical elements.

如請求項5之方法，其中該光偵測器包含波長色散元件，其根據光波長將來自該一或複數個第一光學元件之光色散為至少6個波長成分，且該波長色散元件經定位以將經色散為至少6個波長成分之至少一部分提供至至少6個偵測元件，其中該等偵測元件之各者與用於同時自該等偵測元件之各者收集偵測資訊之第一控制元件連通，且其中該光偵測器偵測來自標記一或多個生物分子之至少6種染料之螢光，各染料具有獨特之峰值發射波長。 The method of claim 5, wherein the photodetector includes a wavelength dispersion element that disperses light from the one or more first optical elements into at least 6 wavelength components according to the wavelength of the light, and the wavelength dispersion element is positioned To provide at least a portion of at least 6 wavelength components with dispersion to at least 6 detection elements, wherein each of these detection elements and the first for collecting detection information from each of these detection elements simultaneously A control element is connected, and wherein the light detector detects fluorescence from at least 6 dyes labeled with one or more biomolecules, each dye having a unique peak emission wavelength.

如請求項1之方法，其中引發液體流動以分別將第一擴增DNA片段自第一反應儲集器移至生物晶片之第二子組件中之第一分離單位，以及將第二擴增DNA片段自第二反應儲集器移至生物晶片之第二子組件中之第二分離單位之步驟進一步包含：引發液體流動以將具有該第一擴增DNA片段之第一PCR混合物自第一反應儲集器移至第一稀釋儲集器；以第一稀釋劑稀釋該第一PCR混合物；引發液體流動以將具有該第二擴增DNA片段之第二PCR混合物自第二反應儲集器移至第二稀釋儲集器；及以第二稀釋劑稀釋該第二PCR混合物。 The method of claim 1, wherein a liquid flow is initiated to move the first amplified DNA fragments from the first reaction reservoir to the first separation unit in the second subassembly of the biochip, respectively, and the second amplified DNA The step of moving the fragments from the second reaction reservoir to the second separation unit in the second subassembly of the biochip further includes: initiating a liquid flow to remove the first PCR mixture with the first amplified DNA fragments from the first reaction The reservoir is moved to the first dilution reservoir; the first PCR mixture is diluted with the first diluent; liquid flow is initiated to move the second PCR mixture with the second amplified DNA fragment from the second reaction reservoir To a second dilution reservoir; and diluting the second PCR mixture with a second diluent.

如請求項1之方法，其中在第二分離單位中引發電場，以在生物晶片上之第二分離通道中根據大小分離第二擴增DNA片段之步驟進一步包含：在該第一分離單位引發電場的同時在該第二分離單位引發電場，以同時分離該第二擴增DNA片段及該第一擴增DNA片段。 The method of claim 1, wherein the step of initiating the electric field in the second separation unit to separate the second amplified DNA fragments according to size in the second separation channel on the biochip further comprises: initiating the electric field in the first separation unit At the same time, an electric field is induced in the second separation unit to simultaneously separate the second amplified DNA fragment and the first amplified DNA fragment.

如請求項6之方法，其中該光源係單一雷射器，該波長色散元件係稜鏡、繞射光柵、透射光柵、攝譜儀或全像繞射光柵，以及該至少6個偵測元件各為線性多陽極光電倍增管。 The method according to claim 6, wherein the light source is a single laser, the wavelength dispersive element is a laser, a diffraction grating, a transmission grating, a spectrograph, or a holographic diffraction grating, and the at least six detection elements are each It is a linear multi-anode photomultiplier tube.