TWI470220B - Plastic microfluidic separation and detection platforms - Google Patents

Plastic microfluidic separation and detection platforms Download PDF

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TWI470220B
TWI470220B TW97121585A TW97121585A TWI470220B TW I470220 B TWI470220 B TW I470220B TW 97121585 A TW97121585 A TW 97121585A TW 97121585 A TW97121585 A TW 97121585A TW I470220 B TWI470220 B TW I470220B
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wafer
nucleic acid
cathode
micro flow
plastic
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TW200918888A (en
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Eugene Tan
Cheuk Wai Kan
Heung Chuan Lam
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Netbio Inc
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Description

塑料微型流動分離及檢測平台Plastic micro flow separation and detection platform

本發明係屬於在藉由雷射誘發之螢光檢測下藉由電泳進行核酸排序及片段尺寸測定的領域。分析係在塑料電泳晶片上執行。The present invention is in the field of nucleic acid sequencing and fragment size determination by electrophoresis by laser-induced fluorescence detection. The analysis was performed on a plastic electrophoresis wafer.

本申請案根據35 U.S.C.§119(e)主張2007年4月4日申請之美國臨時申請案第60/921,802號及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號的申請日期之權利，各申請案以全文引用的方式併入本文中。兩個在與本申請案同一日期申請之美國申請案：名為"快速多路擴增靶核酸之方法(METHODS FOR RAPID MULTIPLEXED AMPLIFICATION OF TARGET NUCLEIC ACIDS)"之代理人案號08－318－US及名為"綜合核酸分析(INTEGRATED NUCLEIC ACID ANALYSIS)"之代理人案號07－801－US，以全文引用的方式併入本申請案中。This application is based on US Provisional Application No. 60/921,802, filed on Apr. 4, 2007, and U.S. Provisional Application No. 60/964,502, filed on August 13, 2007, in The right to file the filing date of U.S. Provisional Application No. 61/028,073, filed on Feb. 12, the entire disclosure of which is hereby incorporated by reference. US Patent Application No. 08-318-US, entitled "METHODS FOR RAPID MULTIPLEXED AMPLIFICATION OF TARGET NUCLEIC ACIDS", filed on the same date as the present application. The agent's case number 07-801-US, entitled "INTEGRATED NUCLEIC ACID ANALYSIS", is incorporated herein by reference in its entirety.

自20世紀70年代DNA排序技術(Maxam及Gilbert,1977,Proc Natl Acad Sci USA 74：560－564；Sanger等人，1977,Proc Natl Acad Sci USA 74：5463－5467)出現以來，利用此等技術之廣泛應用已發展。同時，已引入愈加複雜之測試設備來實施DNA排序。舉例而言，在1986年，Applied Biosystems上市銷售一種自動DNA排序儀，其係基於由Sanger排序法所產生之DNA片段之分離；將DNA片段用一 組四種螢光染料標記且藉由毛細管電泳分離(Smith等人，1986,Nature 321：674－679)。因此，Sanger排序法已成為過去三十年最廣泛利用之排序技術。Since the advent of DNA sequencing technology in the 1970s (Maxam and Gilbert, 1977, Proc Natl Acad Sci USA 74: 560-564; Sanger et al., 1977, Proc Natl Acad Sci USA 74: 5463-5467), the use of such techniques Widespread application has been developed. At the same time, increasingly sophisticated test equipment has been introduced to implement DNA sequencing. For example, in 1986, Applied Biosystems marketed an automated DNA sequencer based on the separation of DNA fragments generated by the Sanger sequencing method; labeling DNA fragments with a set of four fluorescent dyes and by capillary electrophoresis Isolation (Smith et al., 1986, Nature 321 :674-679). Therefore, the Sanger ranking method has become the most widely used sorting technique in the past 30 years.

近年來，多種新的排序技術及相關測試設備已發展且繼續發展。稱為"下一代"方法(Metzker,2005,Genome Research 15：1767－1776中綜述)之此等化學方法包括焦磷酸排序法、連接反應排序法及單分子排序法。驅動研究下一代排序技術之主要目標一般為執行高產量基因組排序，且尤其為降低獲得完整基因組序列之成本。雖然下一代技術之每一鹼基對之成本在某些狀況下可比Sanger排序法之成本低，但所有此等方法(包括Sanger)均為高成本的且需要大量時間、人工及實驗室裝備。In recent years, a variety of new sequencing technologies and related test equipment have been developed and continue to evolve. These chemical methods, referred to as "next generation" methods (reviewed in Metzker, 2005, Genome Research 15: 1767-1776) include pyrosequic acid sequencing, ligation reaction sequencing, and single molecule sequencing. The primary goal of driving research into next-generation sequencing technologies is generally to perform high-volume genomic sequencing, and in particular to reduce the cost of obtaining complete genomic sequences. While the cost per base pair of next generation technology can be lower than the cost of the Sanger ranking in some cases, all of these methods, including Sanger, are costly and require significant time, labor, and laboratory equipment.

目前對自給定基因組獲得極大量之序列資料的強調並不否定快速獲得相對少量之基因組序列的價值。舉例而言，許多常見人類疾病可基於少於1000個鹼基對之DNA序列(比產生完整人類基因組所需之鹼基對少若干個數量級)來診斷。類似地，對藉由短串聯重複分析產生的成組少於20個特定DNA片段之尺寸的精確測定足以鑑別給定之個體。The current emphasis on obtaining a very large amount of sequence data from a given genome does not negate the value of rapidly obtaining a relatively small number of genomic sequences. For example, many common human diseases can be diagnosed based on DNA sequences of less than 1000 base pairs (a few orders of magnitude less than the base pairs required to produce the entire human genome). Similarly, an accurate determination of the size of fewer than 20 specific DNA fragments generated by short tandem repeat analysis is sufficient to identify a given individual.

對將允許聚焦核酸分析(focused nucleic acid analysis)(定義為快速鑑別給定人類、動物或病原體基因組之子集(藉由核酸排序或片段尺寸測定))之儀器及技術的發展存在未滿足之需要。聚焦核酸分析將使最終使用者能夠作出幾乎即時之臨床、法醫學或其他判定。視應用而定，聚焦核酸分析可在多種情形下執行，包括醫院實驗室、醫師診 所、床邊或在法醫學或環境應用之情況下在野外執行。There is an unmet need for the development of instruments and techniques that will allow for focused nucleic acid analysis, defined as the rapid identification of a subset of a given human, animal or pathogen genome (by nucleic acid sequencing or fragment sizing). Focused nucleic acid analysis will enable end users to make almost immediate clinical, forensic or other decisions. Depending on the application, focused nucleic acid analysis can be performed in a variety of situations, including hospital laboratories and physicians. Execution in the field, bedside or in the case of forensic or environmental applications.

關於核酸(DNA及RNA)排序，臨床應用包括診斷細菌性、真菌性及病毒性疾病(包括測定生物體之抗藥性概況)、癌症(包括測定對化學療法之反應)及遺傳性及其他常見疾病(包括測定對藥物之反應)。聚焦核酸序列亦非常適於藥物基因組分析及某些法醫學應用(包括(例如)線粒體DNA排序)。For nucleic acid (DNA and RNA) sequencing, clinical applications include the diagnosis of bacterial, fungal, and viral diseases (including measuring the drug resistance profile of organisms), cancer (including measuring the response to chemotherapy), and hereditary and other common diseases. (including the determination of the response to the drug). Focused nucleic acid sequences are also well suited for pharmacogenomic analysis and certain forensic applications including, for example, mitochondrial DNA sequencing.

關於核酸片段尺寸測定，聚焦核酸分析可用於法醫學及臨床應用中。舉例而言，一類人類鑑別法係基於短串聯重複(STR)分析(Edwards等人，1991,Am J Hum Genet 49(4)746：756)。在STR分析中，利用一系列引子來擴增含有變化數目之某些短串聯重複的某些基因組區。所得帶之尺寸藉由核酸片段尺寸測定(通常使用毛細管電泳)來測定，且該組STR等位基因之各成員之尺寸獨特地鑑別個體。STR分型已成為用於人類法醫學遺傳鑑別之全球標準，且為允許鑑別個體以及該個體之遺傳親屬之唯一生物測定技術。在臨床應用中，核酸片段尺寸測定可用於診斷給定病症(例如，(如)弗里德賴希共濟失調(Friedreich ataxia)中藉由搜索特徵缺失或***或測定核苷酸重複區之尺寸)(Pandolfo,M.,2006,Methods Mol.Med 126：197－216)。片段尺寸測定亦可用於鑑別傳染劑；DNA指紋法可用於病原體診斷中。For nucleic acid fragment size determination, focused nucleic acid analysis can be used in forensic and clinical applications. For example, one type of human identification method is based on short tandem repeat (STR) analysis (Edwards et al, 1991, Am J Hum Genet 49 (4) 746: 756). In STR analysis, a series of primers are used to amplify certain genomic regions containing varying numbers of short tandem repeats. The size of the resulting band is determined by nucleic acid fragment size determination (usually using capillary electrophoresis) and the size of each member of the set of STR alleles uniquely identifies the individual. STR typing has become the global standard for human forensic genetic identification and is the only biometric technique that allows for the identification of individuals and their genetic relatives. In clinical applications, nucleic acid fragment size determination can be used to diagnose a given condition (eg, in Friedrichich ataxia by searching for feature deletions or inserting or determining the size of a nucleotide repeat region). (Pandolfo, M., 2006, Methods Mol . Med 126: 197-216). Fragment size determination can also be used to identify infectious agents; DNA fingerprinting can be used in pathogen diagnosis.

聚焦核酸分析之應用不侷限於以上所討論之彼等應用。聚焦核酸分析可用於藉由排序及片段尺寸測定兩者來鑑別 臨床及環境樣品中之生物武器劑(biological weapons agent)。獸醫學及食物測試應用亦反映上述之彼等應用。獸醫學鑑別應用(諸如，賽馬繁育及追蹤、家畜繁育及寵物鑑別)亦在本發明之用途之範疇內。聚焦核酸分析之研究應用為數眾多。簡言之，聚焦核酸分析具有顯著轉變若干行業之潛能。The application of focused nucleic acid analysis is not limited to the applications discussed above. Focused nucleic acid analysis can be used to identify both by sorting and fragment size determination Biological weapons agent in clinical and environmental samples. The veterinary and food testing applications also reflect their respective applications. Veterinary identification applications, such as horse breeding and tracking, livestock breeding and pet identification, are also within the scope of the uses of the present invention. There are numerous research applications for focusing nucleic acid analysis. In short, focused nucleic acid analysis has the potential to significantly transform several industries.

現有高產量基於毛細管之排序儀及下一代排序儀不能夠以及時及成本有效之方式執行聚焦核酸分析。彼等技術所尋求之規模經濟受降低獲得及分析極大量序列資料之成本所驅動。為使能夠進行聚焦核酸分析之儀器及系統進入常規使用，其應經設計以具有某些"理想"性質及特徵。詳言之，該等儀器及系統應快速地產生結果(理想地，在幾分鐘內)以使可起訴之資料儘快產生。其應易於操作且試劑及消費品應為廉價的。此外，對於一些應用而言，在一次性物品中執行核酸分離為有用的；此顯著減少樣品污染之可能性。為實現此等性質，基於聚合物之生物晶片比其他材料(諸如，玻璃及矽)適合作為分離基板。Existing high throughput capillary based sorters and next generation sorters are not capable of performing focused nucleic acid analysis in a timely and cost effective manner. The economies of scale sought by their technology are driven by the reduced cost of obtaining and analyzing extremely large amounts of sequence data. Instruments and systems capable of performing focused nucleic acid analysis are designed to have certain "ideal" properties and characteristics. In particular, such instruments and systems should produce results quickly (ideally, within a few minutes) to enable prosecable information to be generated as quickly as possible. It should be easy to handle and reagents and consumer products should be inexpensive. Moreover, for some applications, it is useful to perform nucleic acid isolation in disposable articles; this significantly reduces the likelihood of sample contamination. To achieve these properties, polymer based biochips are suitable as separate substrates than other materials such as glass and germanium.

對在塑料晶片上實現DNA片段尺寸測定之嘗試藉由McCormick(Anal Chem 69(14)：2626 1997)報導，顯示ΦX174 RF DNA之Hae III限制片段之分離。雖然該等分離係在單個樣品下在單道晶片中執行，但其展示不良解析度分離及不良靈敏度。此外，該系統僅能夠檢測來自單螢光團之發射。雖然Sassi(J Chromatogr A ,894(1－2)：203 2000)報導由16個流動分離之分離道組成的丙烯酸晶片用於STR 尺寸測定之用途，但此方法亦顯示不良解析能力及低靈敏度。當執行同時16道分離及檢測時，此低系統靈敏度阻止等位基因階梯(法醫學分析中嚴格需要之內尺寸測定標準)之檢測。使用2 Hz掃描速率(代表嘗試增加系統信號雜訊比)引起解析能力及精確度兩者之降級。最終，該系統僅能夠檢測來自單螢光團之發射。Shi(Electrophoresis 24(19－20)：3371 2003及Shi,2006,Electrophoresis 27(10)：3703)報導在單個樣品單道塑料分離設備中2及4色分離及檢測。雖然報導4.5 cm通道提供單鹼基解析度，但實際上如藉由隔開一個鹼基對之等位基因之外觀所證明，解析度為不良的(THO1 9.3與10個等位基因之峰谷比接近1)。具有較長分離通道(6、10及18 cm)之設備用於此研究中以實現與4.5 cm設備相比更高之解析度以進行分析。當使用對解析度而言最佳之篩選基質組合物時，10及18 cm長之設備因設備分層而受到限制。An attempt to achieve DNA fragment size determination on plastic wafers was reported by McCormick ( Anal Chem 69 (14): 2626 1997), showing separation of Hae III restriction fragments of ΦX174 RF DNA. While these separations were performed in a single wafer under a single sample, they exhibited poor resolution separation and poor sensitivity. In addition, the system is only capable of detecting emissions from a single fluorophore. Although Sassi ( J Chromatogr A , 894(1-2): 203 2000) reported the use of an acrylic wafer consisting of 16 flow separation separators for STR size determination, this method also showed poor resolution and low sensitivity. This low system sensitivity prevents detection of the allelic ladder (a sizing standard that is strictly required in forensic analysis) when performing simultaneous 16 separations and detections. Using a 2 Hz scan rate (representing an attempt to increase the system signal to noise ratio) causes degradation in both resolution and accuracy. Ultimately, the system is only capable of detecting emissions from a single fluorophore. Shi ( electrophoresis 24 (19-20): 3371 2003 and Shi, 2006, Electrophoresis 27(10): 3703) reported 2 and 4 color separation and detection in a single sample single pass plastic separation apparatus. Although the 4.5 cm channel is reported to provide single base resolution, it is actually poorly resolved by the appearance of the alleles separated by one base pair (THO1 9.3 and 10 allele peaks and valleys) The ratio is close to 1). Equipment with longer separation channels (6, 10 and 18 cm) was used in this study to achieve higher resolution compared to 4.5 cm equipment for analysis. When using the best screening composition for resolution, 10 and 18 cm long equipment is limited by equipment stratification.

實際上，已發現塑料對在經設計以用於核酸排序及片段尺寸測定之生物晶片中之用途呈現若干主要障礙。塑料材料之自身螢光干擾450至800 nm之可見範圍中波長之檢測(Puriska,2005,Lab Chip 5(12)：1348；Wabuyele,2001Electrophoresis 22(18)：3939－48；Hawkins及Yager 2003Lab Chip, 3(4)：248－52)。In fact, plastics have been found to present several major obstacles to their use in biochips designed for nucleic acid sequencing and fragment size determination. The self-fluorescence of plastic materials interferes with the detection of wavelengths in the visible range of 450 to 800 nm (Puriska, 2005, Lab Chip 5(12): 1348; Wabuyele, 2001 Electrophoresis 22(18): 3939-48; Hawkins and Yager 2003 Lab Chip, 3(4): 248-52).

在Sanger排序及STR尺寸測定之商業套組中使用此等波長。此外，現有塑料設備具有對通常所用基板之低黏結強度及在通常所用之篩選基質下不良的效能結果。最終，通 道內表面與篩選基質及DNA樣品相互作用，由於電滲流及DNA與壁之相互作用而導致不良解析度(Kan,2004,Electrophoresis 25(21－22)：3564)。These wavelengths are used in commercial sets of Sanger sorting and STR size determination. In addition, existing plastic devices have the result of low bond strength to commonly used substrates and poor performance under commonly used screening matrices. Eventually, the inner surface of the channel interacts with the screening matrix and the DNA sample, resulting in poor resolution due to electroosmotic flow and interaction of DNA with the wall (Kan, 2004, Electrophoresis 25 (21-22): 3564).

因此，對能夠以高解析度及高信號雜訊比執行聚焦核酸分析之廉價、多道塑料生物晶片存在未滿足之實質性需要。Therefore, there is an unmet need for an inexpensive, multi-channel plastic biochip capable of performing focused nucleic acid analysis with high resolution and high signal to noise ratio.

本發明提供能夠以高解析度及高信號雜訊比執行聚焦核酸分析之廉價、多道塑料生物晶片及使用該等晶片之方法。The present invention provides an inexpensive, multi-channel plastic biochip capable of performing focused nucleic acid analysis with high resolution and high signal to noise ratio and methods of using the same.

在一第一態樣中，本發明提供塑料分離晶片及尤其電泳晶片，其包含一陽極部分、一陰極部分及該陽極部分與該陰極部分之間的一中心部分，其中陰極部分包含至少一個第一通孔；陽極部分包含至少一個第二通孔；且該中心部分包含複數個微型流動通道及一檢測窗口，各微型流動通道具有一分離區及一檢測區；其中各微型流動通道與至少一個第一通孔及至少一個第二通孔處於流體連通中；其中該複數個微型流動通道處於實質上同一平面中；複數個微型流動通道在該中心部分內彼此不相交；該檢測窗口包含薄塑料；且檢測窗口包含各微型流動通道之檢測區域。在檢測區外之晶片部分可具有相同厚度或具有比檢測區厚度大的厚度。In a first aspect, the present invention provides a plastic separation wafer and, in particular, an electrophoretic wafer comprising an anode portion, a cathode portion and a central portion between the anode portion and the cathode portion, wherein the cathode portion comprises at least one a through hole; the anode portion includes at least one second through hole; and the central portion includes a plurality of micro flow channels and a detection window, each of the micro flow channels has a separation area and a detection area; wherein each of the micro flow channels and at least one The first through hole and the at least one second through hole are in fluid communication; wherein the plurality of micro flow channels are in substantially the same plane; the plurality of micro flow channels do not intersect each other in the central portion; the detection window comprises a thin plastic And the detection window includes a detection area of each micro flow channel. The portion of the wafer outside the detection zone may have the same thickness or have a thickness greater than the thickness of the detection zone.

在一第二態樣中，本發明提供包含具有一頂面及一底面之一支撐體之設備，其包含一陽極部分、一陰極部分及在 該陽極部分與該陰極部分之間的一中心部分，其中該中心部分在檢測窗口上包含一孔隙，該陽極部分包含至少一個陽極孔，且該陰極部分包含至少一個陰極孔；該裝置進一步包含具有一頂面及一底面之根據第一態樣之一晶片，其中該晶片之頂面與支撐體之底面接觸，微型流動通道經由通孔與陰極孔及陽極孔處於流體連通中；且晶片固定附著於支撐體。In a second aspect, the present invention provides an apparatus comprising a support having a top surface and a bottom surface, comprising an anode portion, a cathode portion, and a central portion between the anode portion and the cathode portion, wherein the central portion includes an aperture on the detection window, the anode portion includes at least one anode aperture, and the cathode portion includes at least one cathode aperture; the apparatus further includes having a top surface and a bottom surface of the wafer according to the first aspect, wherein a top surface of the wafer is in contact with a bottom surface of the support body, and the micro flow channel is in fluid communication with the cathode hole and the anode hole via the through hole; and the wafer is fixedly attached On the support.

在一第三態樣中，本發明提供同時電泳分離及檢測複數個樣品之方法，其包含將複數個樣品提供至根據第一態樣之微晶片上之複數個微型流動通道中的每一者中；跨越複數個微型流動通道施加電位以將樣品注射至分離通道中且將包含複數個分析樣品之每一者的可檢測物質分離；且在檢測窗口上檢測包含複數個分離樣品之各可檢測物質。In a third aspect, the present invention provides a method of simultaneous electrophoretic separation and detection of a plurality of samples comprising providing a plurality of samples to each of a plurality of microfluidic channels on a microchip according to a first aspect Applying a potential across a plurality of microfluidic channels to inject a sample into the separation channel and separating the detectable substance comprising each of the plurality of analytical samples; and detecting each of the plurality of isolated samples that are detectable on the detection window substance.

本發明之特定較佳實施例將自以下某些較佳實施例之更詳細描述及申請專利範圍變得顯而易見。The detailed description of the preferred embodiments of the invention will be apparent from the

本發明提供能夠檢測尺寸相差約1個鹼基對且在至少1.0 ng之DNA模板濃度水平下之核酸物質分離的塑料分離晶片。The present invention provides a plastic separation wafer capable of detecting nucleic acid species separated by about 1 base pair in size and at a DNA template concentration level of at least 1.0 ng.

在多路PCR反應之前待分析用於STR分析之最低含量樣品包含具有少於800副本、少於400副本、少於200副本、少於100副本、少於50副本、少於30副本、少於10副本或1副本核酸模板之核酸模板。待分析以進行排序之最低濃度樣品包含具有少於0.5 pmole、少於0.1 pmole、少於0.01 pmole之核酸模板作為Sanger排序反應之輸入。The minimum amount of sample to be analyzed for STR analysis prior to the multiplex PCR reaction contains less than 800 copies, less than 400 copies, less than 200 copies, less than 100 copies, less than 50 copies, less than 30 copies, less than 10 copies or 1 copy of the nucleic acid template of the nucleic acid template. The lowest concentration sample to be analyzed for sorting contains less than 0.5 pmole, less than 0.1 pmole, less than 0.01 The pmole nucleic acid template serves as an input to the Sanger sequencing reaction.

如本文所用之短語"注射通道"意謂一相交通道，其允許樣品被引入與其相交之微型流動通道。交叉通道可處於單一交叉通道、單一T形連接或偏置雙重T形連接組態。The phrase "injection channel" as used herein means an intersecting channel that allows a sample to be introduced into a microfluidic channel that intersects it. The crossover channel can be in a single crossover channel, a single T-connection or an offset dual T-connection configuration.

如本文所用之短語"流體連通"係指含有流體之兩個腔室或其他組件或區域連接在一起以便流體可在該兩個腔室、組件或區域之間流動。因此，處於"流體連通"中之兩個腔室可(例如)藉由兩個腔室之間的微型流動通道連接在一起，以便流體可在兩個腔室之間自由流動。該等微型流動通道視情況可包括一或多個閥，該一或多個閥可閉合或關閉，以阻斷及/或另外控制腔室之間的流體連通。The phrase "fluid communication" as used herein refers to two chambers or other components or regions containing fluid that are connected together such that fluid can flow between the two chambers, components or regions. Thus, the two chambers in "fluid communication" can be connected together, for example, by a micro flow channel between the two chambers so that fluid can flow freely between the two chambers. The microfluidic channels may optionally include one or more valves that may be closed or closed to block and/or otherwise control fluid communication between the chambers.

如本文所用之短語"螢光染料"意謂在用光源激發後染料發出具有380－850 nm之波長的光。較佳地，染料發出具有在約450－800 nm之間的波長之光；更佳地，染料發出具有在約495－775 nm之間的波長之光。The phrase "fluorescent dye" as used herein means that the dye emits light having a wavelength of from 380 to 850 nm after excitation with a light source. Preferably, the dye emits light having a wavelength between about 450 and 800 nm; more preferably, the dye emits light having a wavelength between about 495 and 775 nm.

如本文所用之術語"自身螢光"意謂由除相關螢光團外之物質在光照射下產生的螢光。The term "self-fluorescence" as used herein means fluorescing produced by light other than a related fluorophore.

如本文所用之短語"基本上不發螢光"意謂當經受光照射(例如，在約350－500 nm、400－500 nm或450－500 nm之間的一或多個波長下；尤其在488 nm下；雷射照射)時來自所述目標(例如，固體或溶液)之背景螢光信號(例如，在約380－850 nm、400－800 nm、450－800 nm、500－800 nm或495－775 nm之間)具有低於來自由0.7 mm厚之borofloat玻璃組成的習知玻璃微型流動設備之螢光信號的背景水準。The phrase "substantially non-fluorescent" as used herein means when subjected to light illumination (eg, at one or more wavelengths between about 350-500 nm, 400-500 nm, or 450-500 nm; especially Background fluorescent signal from the target (eg, solid or solution) at 488 nm; for example, at about 380-850 nm, 400-800 nm, 450-800 nm, 500-800 nm Or between 495-775 nm) has a background level lower than that of a fluorescent signal from a conventional glass microfluidic device consisting of 0.7 mm thick borofloat glass.

如本文所用之術語"基於降冰片烯之聚合物"意謂自至少一種包含降冰片烯部分之單體製備之聚合物，其中含降冰片烯之單體係根據熟習此項技術者已知之方法根據開環複分解聚合來聚合(參見例如美國專利第4,945,135號、第5,198,511號、第5,312,940號及第5,342,909號)。The term "norbornene-based polymer" as used herein means a polymer prepared from at least one monomer comprising a norbornene moiety, wherein the single system containing norbornene is according to methods known to those skilled in the art. The polymerization is carried out according to the ring-opening metathesis polymerization (see, for example, U.S. Patent Nos. 4,945,135, 5,198,511, 5,312,940 and 5,342,909).

如本文所用之術語"聚(甲基丙烯酸甲酯)"或"PMMA"意謂甲基丙烯酸甲酯之合成聚合物，包括(但不限於)以商品名稱PlexiglasTM 、LimacrylTM 、R－CastTM 、PerspexTM 、PlazcrylTM 、AcrylexTM 、AcryliteTM 、AcrylplastTM 、AltuglasTM 、PolycastTM 及LuciteTM 銷售之彼等聚合物以及美國專利第5,561,208號、第5,462,995號及第5,334,424號(各以引用的方式併入本文中)中所述之彼等聚合物。As used herein the term "poly (methyl methacrylate)" or "PMMA" is meant a synthetic polymer of methyl methacrylate, including (but not limited to) the trade name Plexiglas TM, Limacryl TM, R- Cast TM , Perspex TM, Plazcryl TM, Acrylex TM, Acrylite TM, Acrylplast TM, Altuglas TM, Polycast TM Lucite TM and sale of their polymers, and U.S. Patent No. 5,561,208, No. 5,462,995 and No. 5,334,424 (each by reference These polymers are incorporated herein by reference.

如本文所用之術語"聚碳酸酯"意謂碳酸與二醇或二價酚之聚酯。該等二醇或二價酚之實例為對二甲苯二醇、2,2－雙(4－氧苯基)丙烷、雙(4－氧苯基)甲烷、1,1－雙(4－氧苯基)乙烷、1,1－雙(氧苯基)丁烷、1,1－雙(氧苯基)環己烷、2,2－雙(氧苯基)丁烷及其混合物，包括(但不限於)以商品名稱CalibreTM 、MakrolonTM 、PanliteTM 、MakroclearTM 、CyrolonTM 、LexanTM 及TuffakTM 銷售之彼等二醇或二價酚。The term "polycarbonate" as used herein means a polyester of carbonic acid and a diol or a divalent phenol. Examples of such diols or divalent phenols are p-xylene glycol, 2,2-bis(4-oxophenyl)propane, bis(4-oxophenyl)methane, 1,1-bis(4-oxo). Phenyl)ethane, 1,1-bis(oxyphenyl)butane, 1,1-bis(oxyphenyl)cyclohexane, 2,2-bis(oxyphenyl)butane, and mixtures thereof, including (but not limited to) the trade name Calibre TM, Makrolon TM, Panlite TM , Makroclear TM, Cyrolon TM, Lexan TM and sold by them Tuffak TM glycol or a divalent phenol.

如本文所用之術語"核酸"意欲包含單鏈及雙鏈DNA及RNA，以及含有修飾鹼基、糖及主鏈之替代性核酸之任何及所有形式。因此，將理解術語"核酸"包括(但不限於)單鏈或雙鏈DNA或RNA(及其可為部分單鏈或部分雙鏈之形 式)、cDNA、適體、肽核酸("PNA")、2'－5' DNA(與DNA之A構形匹配的具有縮短主鏈之合成物質，該縮短主鏈含有一個鹼基間隔；2'－5' DNA通常將不與呈B形式之DNA雜交，但其將易於與RNA雜交)及鎖核酸("LNA")。核酸類似物包括具有類似或改良之結合、雜交之鹼基配對性質的天然核苷酸之已知類似物。嘌呤及嘧啶之"類似"形式在此項技術中熟知，且包括(但不限於)吖丙啶基胞嘧啶、4－乙醯基胞嘧啶、5－氟尿嘧啶、5－溴尿嘧啶、5－羧甲基胺基甲基－2－硫尿嘧啶、5－羧甲基胺基甲基尿嘧啶、肌苷、N6 －異戊烯基腺嘌呤、1－甲基腺嘌呤、1－甲基假尿嘧啶、1－甲基鳥嘌呤、1－甲基肌苷、2,2－二甲基鳥嘌呤、2－甲基腺嘌呤、2－甲基鳥嘌呤、3－甲基胞嘧啶、5－甲基胞嘧啶、N6 －甲基腺嘌呤、7－甲基鳥嘌呤、5－甲基胺基甲基尿嘧啶、5－甲氧基胺基甲基－2－硫尿嘧啶、β－D－甘露糖基Q核苷(beta－D－mannosylqueosine)、5－甲氧基尿嘧啶、2－甲硫基－N－6－異戊烯基腺嘌呤、尿嘧啶－5－氧基乙酸甲酯、假尿嘧啶、Q核苷、2－硫胞嘧啶、5－甲基－2－硫尿嘧啶、2－硫尿嘧啶、4－硫尿嘧啶、5－甲基尿嘧啶、尿嘧啶－5－氧基乙酸及2,6－二胺基嘌呤。本發明所提供之DNA主鏈類似物包括磷酸二酯、硫代磷酸酯、二硫代磷酸酯、甲基膦酸酯、胺基磷酸酯、烷基磷酸三酯、胺基磺酸鹽、3'－硫縮醛、亞甲基(甲基亞胺基)、3'－N－胺基甲酸酯、N－嗎啉基胺基甲酸酯及肽核酸(PNA)、甲基膦酸酯鍵或交替甲基膦酸酯及磷酸二酯鍵(Strauss－Soukup,1997,Biochemistry 36：8692－8698)及苯甲 基膦酸酯鍵，如US 6,664,057中所討論；亦參見OLIGONUCLEOTIDES AND ANALOGUES,A PRACITICAL APPROACH,F.Eckstein編輯，IRL Press,Oxford University Press(1991)；Antisense Strategies ,Annals of the New York Academy of Sciences，第600卷，Baserga及Denhardt編(NYAS 1992)；Milligan,1993,J.Med.Chem. 36：1923－1937；Antisense Research and Applications(1993,CRC Press)。本文之核酸可自細胞提取或根據熟習此項技術者已知之任何方式合成製備；舉例而言，在其他來源中，核酸可自cDNA或mRNA化學上合成或轉錄或逆轉錄。The term "nucleic acid" as used herein is intended to encompass both single-stranded and double-stranded DNA and RNA, as well as any and all forms of alternative nucleic acids containing modified bases, sugars, and backbones. Thus, it will be understood that the term "nucleic acid" includes, but is not limited to, single or double stranded DNA or RNA (and may be in the form of a partially single or partially double stranded), cDNA, aptamer, peptide nucleic acid ("PNA"). , 2'-5' DNA (a synthetic material with a shortened backbone that matches the A conformation of DNA, the shortened backbone contains one base spacing; 2'-5' DNA will usually not hybridize to DNA in B form But it will easily hybridize to RNA) and lock nucleic acids ("LNA"). Nucleic acid analogs include known analogs of natural nucleotides having similar or improved binding, hybridization base pairing properties. "Similar" forms of purines and pyrimidines are well known in the art and include, but are not limited to, aziridinyl cytosine, 4-ethenylcytosine, 5-fluorouracil, 5-bromouracil, 5-carboxyl Methylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, inosine, N 6 -isopentenyl adenine, 1-methyladenine, 1-methyl dummy Uracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5- Methylcytosine, N 6 -methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D -mannoside Q nucleoside (beta-D-mannosylqueosine), 5-methoxyuracil, 2-methylthio-N-6-isopentenyl adenine, uracil-5-oxyacetate , pseudouracil, Q nucleoside, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- Oxyacetic acid and 2,6-diaminoguanidine. The DNA backbone analogs provided by the present invention include phosphodiesters, phosphorothioates, dithiophosphates, methylphosphonates, aminophosphates, alkyl phosphates, amine sulfonates, 3 '-thioacetal, methylene (methylimido), 3'-N-carbamate, N-morpholinyl urethane and peptide nucleic acid (PNA), methylphosphonate Bond or alternating methylphosphonate and phosphodiester linkages (Strauss-Soukup, 1997, Biochemistry 36: 8692-8698) and phenylmethyl phosphonate linkages as discussed in US 6,664,057; see also OLIGONUCLEOTIDES AND ANALOGUES, A PRACITICAL APPROACH, edited by F. Eckstein, IRL Press, Oxford University Press (1991); Antisense Strategies , Annals of the New York Academy of Sciences, vol. 600, eds. Baserga and Denhardt (NYAS 1992); Milligan, 1993, J. Med Chem. 36: 1923-1937; Antisense Research and Applications (1993, CRC Press). Nucleic acids herein can be synthesized from cells or synthetically prepared according to any means known to those skilled in the art; for example, in other sources, nucleic acids can be chemically synthesized or transcribed or reverse transcribed from cDNA or mRNA.

如本文所用之術語"通孔"意謂在固體材料中所形成之透孔以允許材料頂面與底面之間的流體連接。The term "through hole" as used herein means a through hole formed in a solid material to allow a fluid connection between the top surface and the bottom surface of the material.

根據本發明之各種實施例之一例示性電泳晶片顯示於圖1 中。晶片(100 )包含一陽極部分(101 )、一陰極部分(102 )及在陽極部分與陰極部分之間的一中心部分(103 )。陰極部分包含至少一個第一通孔(104 )，且陽極部分包含至少一個第二通孔(105 )。中心部分包含複數個微型流動通道(106 )及一檢測窗口(107 )，各微型流動通道具有一分離區及一檢測區；其中各微型流動通道與至少一個第一通孔及至少一個第二通孔處於流體連通中。複數個微型流動通道實質上處於同一平面中且在中心部分內彼此不相交。各微型流動通道具有一樣品之激發及/或檢測可進行於其中之區域。其中包含複數個微型流動通道之激發及檢測區之該區域稱為檢測窗口，且此窗口包含薄塑料。An exemplary electrophoretic wafer in accordance with various embodiments of the present invention is shown in FIG . The wafer ( 100 ) includes an anode portion ( 101 ), a cathode portion ( 102 ), and a central portion ( 103 ) between the anode portion and the cathode portion. The cathode portion includes at least one first through hole ( 104 ), and the anode portion includes at least one second through hole ( 105 ). The central portion includes a plurality of micro flow channels ( 106 ) and a detection window ( 107 ), each micro flow channel having a separation area and a detection area; wherein each of the micro flow channels and the at least one first through hole and the at least one second pass The holes are in fluid communication. The plurality of micro flow channels are substantially in the same plane and do not intersect each other in the central portion. Each microfluidic channel has an area in which excitation and/or detection of a sample can occur. This region, which contains the excitation and detection zones of a plurality of microfluidic channels, is referred to as the detection window and this window contains thin plastic.

如本文所用之短語"薄塑料"意謂所述材料包含厚度(其最小尺寸)小於1 mm、小於750 μm、小於650 μm、小於500 μm、小於400 μm、小於300 μm、小於200 μm或小於100 μm之塑料；或所述材料包含厚度在25－2000 μm、25－1000 μm、25－750 μm、25－500 μm、25－400 μm、25－300 μm或25－200 μm之範圍內的塑料。雖然晶片經設計以在檢測窗口中為薄的，但在檢測區外之晶片部分可具有相同厚度或具有比檢測區厚度大的厚度。The phrase "thin plastic" as used herein means that the material comprises a thickness (its minimum dimension) of less than 1 mm, less than 750 μm, less than 650 μm, less than 500 μm, less than 400 μm, less than 300 μm, less than 200 μm or Plastics less than 100 μm; or the material contains thicknesses in the range of 25-2000 μm, 25-1000 μm, 25-750 μm, 25-500 μm, 25-400 μm, 25-300 μm or 25-200 μm Plastic. Although the wafer is designed to be thin in the detection window, the portion of the wafer outside the detection zone may have the same thickness or have a thickness greater than the thickness of the detection zone.

為說明起見，顯示圖1 之晶片具有四個微型流動通道，然而該揭示並不意欲限制之，實際上，熟習此項技術者將易於認識到晶片可含有交錯數目之微型流動通道(下文)，包括具有一個通道之晶片及具有兩個或兩個以上通道之晶片。如本文所用之術語"複數個"意謂兩個或兩個以上、四個或四個以上、八個或八個以上、16或16個以上、32個或32個以上、48個或48個以上、64個或64個以上、96個或96個以上、128個或128個以上、256個或256個以上、384個或384個以上、512個或512個以上或1024個或1024個以上；或2－4、2－8、2－16、2－32、2－48、2－64、2－96、2－128、2－384、2－512、2－1024個微型流動通道。For purposes of illustration, the wafer of Figure 1 is shown with four microfluidic channels, although the disclosure is not intended to be limiting. In fact, those skilled in the art will readily recognize that wafers may contain interlaced numbers of microfluidic channels (below). A wafer having one channel and a wafer having two or more channels are included. The term "plural" as used herein means two or more, four or more, eight or more, 16 or more, 32 or more than 32, 48 or 48. Above, 64 or more, 96 or 96, 128 or 128, 256 or 256, 384 or 384, 512 or 512 or 1024 or 1024 or more Or 2-4, 2-8, 2-16, 2-32, 2-48, 2-64, 2-96, 2-128, 2-384, 2-512, 2-1024 micro flow channels.

如圖3 中所示，晶片(250 )包含一基板層(360 )及一覆蓋層(370 )。將複數個溝槽(361 )圖案化至基板層中。一系列通孔(亦即，透孔)(371 、372 )形成於覆蓋層中以使流體進入微型流動通道，且可位於晶片之陽極及陰極部分中微型流動通道之末端。或者，通孔可在基板層中而非覆蓋層中形 成以實現相同功能。基板層之頂面與覆蓋層之底面黏結以形成微型流動通道。用於製造基於聚合物之微型流動系統之技術係藉由Becker及Gartner廣泛綜述(Becker,2000,Electrophoresis 21：12－26及Becker,2008,Electrophoresis 390(1)：89)，將其以全文引用方式併入本文中。任何數目之此等方法皆可用於製造本文所述之塑料分離晶片。As shown in Figure 3, the wafer (250) comprising a substrate layer (360) and a cover layer (370). A plurality of trenches ( 361 ) are patterned into the substrate layer. A series of through holes (i.e., through holes) ( 371 , 372 ) are formed in the cover layer to allow fluid to enter the micro flow channels and may be located at the ends of the micro flow channels in the anode and cathode portions of the wafer. Alternatively, vias may be formed in the substrate layer rather than in the cover layer to achieve the same function. The top surface of the substrate layer is bonded to the bottom surface of the cover layer to form a micro flow channel. The technology used to fabricate polymer-based microfluidic systems is extensively reviewed by Becker and Gartner (Becker, 2000, Electrophoresis 21: 12-26 and Becker, 2008, Electrophoresis 390(1): 89), which is cited in full. The manner is incorporated herein. Any number of such methods can be used to fabricate the plastic release wafers described herein.

詳言之，本發明之塑料分離晶片可藉以具有待製造結構之負型標準模熱壓印薄熱塑薄膜的方式製備。標準模可藉由使用電鑄來複製固體基板中所製備之設備來製備。固體基板可為玻璃板，其係藉由熟習此項技術者已知之標準光刻及化學蝕刻方法來圖案化。藉由施加熱及壓力，使基板與覆蓋層擴散黏結。In particular, the plastic release wafer of the present invention can be prepared by means of a negative standard embossed thin thermoplastic film having a structure to be fabricated. Standard molds can be prepared by using electroforming to replicate equipment prepared in a solid substrate. The solid substrate can be a glass sheet that is patterned by standard photolithography and chemical etching methods known to those skilled in the art. The substrate and the cover layer are diffusion bonded by applying heat and pressure.

晶片之基板及覆蓋層可建構自多種塑料基板，包括(但不限於)聚乙烯、聚(丙烯酸酯)(例如，聚(甲基丙烯酸甲酯))、聚(碳酸酯)及不飽和、部分不飽和或飽和環狀烯烴聚合物(COP)或不飽和、部分不飽和或飽和環狀烯烴共聚物(COC)(例如，ZEONORTM 、ZEONEXTM 或TOPASTM )。詳言之，對於本發明之晶片應用而言，較佳係COP及COC，因為其在光學上展示在可見光波長範圍中與其他聚合物相比固有之較低自身螢光。The substrate and cover layer of the wafer can be constructed from a variety of plastic substrates including, but not limited to, polyethylene, poly(acrylate) (eg, poly(methyl methacrylate)), poly(carbonate), and unsaturated, partially unsaturated or saturated cyclic olefin polymer (COP) or an unsaturated, partially unsaturated or saturated cyclic olefin copolymer (COC) (e.g., ZEONOR TM, ZEONEX TM or TOPAS TM). In particular, for wafer applications of the present invention, COP and COC are preferred because they optically exhibit lower self-fluorescence inherent in the visible wavelength range compared to other polymers.

保持本發明之方法中所利用之塑料基板及覆蓋層的厚度為薄的以將來自晶片之自身螢光降至最少。塑料基板及覆蓋層可各自獨立地具有小於2 mm、小於1 mm、小於750 μm、小於650 μm、小於500 μm、小於400 μm、小於300 μm、小於200 μm或小於100 μm之厚度；或塑料基板及覆蓋層可各自獨立地包含厚度在25－2000 μm、25－1000 μm、25－750 μm、25－650 μm、25－500 μm、25－400 μm、25－300 μm、25－200 μm或25－100 μm之範圍內的塑料。The thickness of the plastic substrate and cover layer utilized in the method of the present invention is kept thin to minimize self-fluorescence from the wafer. The plastic substrate and the cover layer may each independently have less than 2 mm, less than 1 mm, less than 750 μm, less than 650 μm, less than 500 μm, less than 400 μm, less than 300 a thickness of μm, less than 200 μm or less than 100 μm; or the plastic substrate and the cover layer may each independently comprise a thickness of 25-2000 μm, 25-1000 μm, 25-750 μm, 25-650 μm, 25-500 μm, Plastics in the range of 25-400 μm, 25-300 μm, 25-200 μm or 25-100 μm.

在一實施例中，如圖2 中所例示，晶片(250 )附著於具有頂面及底面之支撐體(201 )，其包含一陽極部分(202 )、一陰極部分(203 )及在陽極部分與陰極部分之間的中心部分(204 )，其中中心部分包含一檢測窗口(205 )，陽極部分包含至少一個陽極孔(206 )，且陰極部分包含至少一個陰極孔(207 )。具有向上通孔之晶片之頂面與支撐體之底面接觸，且晶片係固定附著於支撐體。晶片可根據熟習此項技術者已知之方法(例如，擴散黏結、溶劑黏結或黏著黏結)附著於支撐體。In one embodiment, as shown in Figure 2 illustrates a wafer (250) is attached to a support having a top surface and a bottom surface of (201), which comprises an anode portion (202), (203) and the anode portion a cathode portion A central portion ( 204 ) between the cathode portion and the cathode portion, wherein the central portion includes a detection window ( 205 ), the anode portion includes at least one anode aperture ( 206 ), and the cathode portion includes at least one cathode aperture ( 207 ). The top surface of the wafer having the upward via is in contact with the bottom surface of the support, and the wafer is fixedly attached to the support. The wafer can be attached to the support according to methods known to those skilled in the art (e.g., diffusion bonding, solvent bonding, or adhesive bonding).

支撐體層可建構自多種塑料基板，包括(但不限於)聚乙烯、聚(丙烯酸酯)(例如，聚(甲基丙烯酸甲酯))、聚(碳酸酯)及不飽和、部分不飽和或飽和環狀烯烴聚合物(COP)或不飽和、部分不飽和或飽和環狀烯烴共聚物(COC)(例如，ZEONORTM 、ZEONEXTM 或TOPASTM )。本發明之方法中所利用之塑料支撐體層的厚度足夠厚以提供結構剛性且提供足夠量之樣品及緩衝液在儲集層中。塑料支撐體之厚度將在100－15,000 μm之範圍內。The support layer can be constructed from a variety of plastic substrates including, but not limited to, polyethylene, poly(acrylate) (eg, poly(methyl methacrylate)), poly(carbonate), and unsaturated, partially unsaturated or saturated cyclic olefin polymer (COP) or an unsaturated, partially unsaturated or saturated cyclic olefin copolymer (COC) (e.g., ZEONOR TM, ZEONEX TM or TOPAS TM). The thickness of the plastic support layer utilized in the method of the present invention is sufficiently thick to provide structural rigidity and to provide a sufficient amount of sample and buffer in the reservoir. The thickness of the plastic support will be in the range of 100-15,000 μm.

或者，可藉由在固體支撐體上圖案化該等溝槽以將晶片基板與支撐體結構形成在一起來製造晶片。可將覆蓋層與支撐體黏結以完成結構。在此組態中，保持與微型流動通 道之檢測部分一致的支撐體及晶片之檢測窗口之厚度為薄的以將自身螢光降至最少。此晶片部分之厚度係少於1000 μm、少於750 μm、少於500 μm或少於250 μm；或在25－1000 μm、25－750 μm或25－500 μm之範圍內。Alternatively, the wafer can be fabricated by patterning the trenches on a solid support to form the wafer substrate with the support structure. The cover layer can be bonded to the support to complete the structure. In this configuration, keep the micro flow The thickness of the support and the detection window of the wafer, which are consistent with each other, is thin to minimize self-fluorescence. The thickness of the wafer portion is less than 1000 μm, less than 750 μm, less than 500 μm or less than 250 μm; or in the range of 25-1000 μm, 25-750 μm or 25-500 μm.

複數個微型流動通道中之每一者可具有至少10 μm、50 μm、100 μm、200 μm、500 μm或1 mm之深度；或具有在1－1000 μm、10－100 μm、10－50 μm或25－50 μm之範圍內的深度。複數個微型流動通道可具有至少25 μm、50 μm、100 μm、200 μm、500 μm或1 mm之寬度；或具有在25－1000 μm、25－200 μm或50－200 μm之範圍內的寬度。各通道之微通道橫截面可具有實質上正方形、矩形、圓形、半圓形、橢圓形、三角形或梯形橫截面。熟習此項技術者將認識到微型流動通道之深度、寬度及橫截面可能一致或可能不一致。Each of the plurality of micro flow channels may have a depth of at least 10 μm, 50 μm, 100 μm, 200 μm, 500 μm, or 1 mm; or have a density of 1-1000 μm, 10-100 μm, 10-50 μm Or depth in the range of 25-50 μm. A plurality of micro flow channels may have a width of at least 25 μm, 50 μm, 100 μm, 200 μm, 500 μm or 1 mm; or have a width in the range of 25-1000 μm, 25-200 μm or 50-200 μm . The microchannel cross section of each channel can have a substantially square, rectangular, circular, semi-circular, elliptical, triangular or trapezoidal cross section. Those skilled in the art will recognize that the depth, width and cross-section of the micro flow channels may or may not be uniform.

複數個微型流動通道(106 )中之每一者包含一分離區(108 )及一檢測區(109 )。分離區通常具有分離長度為約2－50 cm、10－50 cm、2－25 cm、10－25 cm之通道。分離長度定義為樣品注射點與樣品檢測點之間的通道部分。分離長度通常小於跨越陰極與陽極儲集層之間的分離通道之總長度。Each of the plurality of microfluidic channels ( 106 ) includes a separation zone ( 108 ) and a detection zone ( 109 ). The separation zone typically has channels separated by a length of about 2-50 cm, 10-50 cm, 2-25 cm, and 10-25 cm. The separation length is defined as the portion of the channel between the sample injection point and the sample detection point. The separation length is typically less than the total length of the separation channel spanning between the cathode and anode reservoirs.

同時分析複數個樣品可藉由將獨立分離通道中之各樣品注射及堆疊至本文所述之任何分離晶片中來執行。如熟習此項技術者所熟悉，視(例如)通道表面上所存在之電荷而定(下文)，沿分離通道施加電場引起樣品沿通道自分離通 道之陰極部分朝陽極部分或自陽極部分朝陰極部分移動。樣品穿過篩選基質之移動基於尺寸來分離物質。Simultaneous analysis of a plurality of samples can be performed by injecting and stacking each sample in the separate separation channels into any of the separate wafers described herein. As is familiar to those skilled in the art, depending on, for example, the charge present on the surface of the channel (below), applying an electric field along the separation channel causes the sample to separate from the channel. The cathode portion of the track moves toward the anode portion or from the anode portion toward the cathode portion. The movement of the sample through the screening matrix separates the material based on size.

當經分離之樣品通過檢測窗口時，可激發附著於樣品內之各物質之染料標記且可檢測所得螢光。在各通道之分離區之末端，檢測窗口通常與複數個微通道中之每一者的檢測區重疊。通常，複數個微型流動通道中之每一者的檢測區處於沿該等通道實質上相同之位置，使得檢測窗口可處於支撐體之中心部分中的單一位置處。When the separated sample passes through the detection window, the dye mark attached to each substance attached to the sample can be excited and the resulting fluorescence can be detected. At the end of the separation zone of each channel, the detection window typically overlaps the detection zone of each of the plurality of microchannels. Typically, the detection zones of each of the plurality of microfluidic channels are at substantially the same location along the channels such that the detection window can be at a single location in the central portion of the support.

較佳地，為晶片提供用於將複數個樣品同時注射至複數個樣品或緩衝液孔中之注射器以能夠同時進行多重樣品分離及檢測。該等注射器將(例如)複數個樣品中之一樣品提供至複數個微型流動通道之一微型流動通道中。注射器可根據熟習此項技術者已知之任何方法(例如)藉由電泳輸送、氣體傳動或液體傳動經由將樣品連接至分離通道之針或管或通道將樣品引入通道中。Preferably, the wafer is provided with a syringe for simultaneously injecting a plurality of samples into a plurality of samples or buffer wells to enable simultaneous multiple sample separation and detection. The injectors provide, for example, one of a plurality of samples to one of a plurality of microfluidic channels. The syringe can be introduced into the channel via a needle or tube or channel that connects the sample to the separation channel by any method known to those skilled in the art, for example, by electrophoretic delivery, gas delivery, or liquid transfer.

在某些實施例中，樣品可經由晶片之陰極儲集層載入晶片中。可根據熟習此項技術者已知之方法經由陰極孔之一者引入注射量之各樣品。舉例而言，可經由對分離通道及/或分離通道之交叉通道與樣品及廢液孔加偏壓以便將樣品孔中之一部分樣品(亦即，注射量)提供至分離通道中來注射樣品。在樣品注射之後，將額外緩衝溶液引入各陰極孔中；可提供足夠量以稀釋孔中任何剩餘樣品。舉例而言，將約至少5、10、25、50或100倍於樣品注射量之量的緩衝液引入陰極孔中。或者，將在約5－100倍、5－50倍或10－50 倍於樣品注射量之範圍內之量的緩衝液引入陰極孔中。In some embodiments, the sample can be loaded into the wafer via a cathode reservoir of the wafer. Each sample of the injection amount can be introduced through one of the cathode holes according to a method known to those skilled in the art. For example, the sample can be injected via biasing the separation channel and/or the separation channel with the sample and waste well to provide a portion of the sample well (ie, the amount of injection) into the separation channel. After the sample is injected, an additional buffer solution is introduced into each of the cathode wells; a sufficient amount can be provided to dilute any remaining sample in the well. For example, a buffer of about at least 5, 10, 25, 50, or 100 times the amount of sample injected is introduced into the cathode well. Or, it will be about 5-100 times, 5-50 times or 10-50 An amount of buffer in the range of the injection amount of the sample is introduced into the cathode hole.

在其他實施例中，複數個微型流動通道中之每一者進一步包含一用於引入樣品之注射通道。舉例而言，參考圖4 ；其中顯示晶片(400 )之透視圖，顯示陰極部分(401 )及中心部分(403 )之鄰接部分。陰極部分包含至少一個第二通道(405 )，且中心部分包含複數個微型流動通道(406 )。在晶片之陰極部分內，對於各微型流動通道而言，各微型流動通道進一步包含一包含樣品孔(409 )及廢液孔(410 )之注射通道(408 )。In other embodiments, each of the plurality of microfluidic channels further comprises an injection channel for introducing a sample. For example, reference is made to Figure 4 ; there is shown a perspective view of the wafer ( 400 ) showing the abutting portions of the cathode portion ( 401 ) and the central portion ( 403 ). The cathode portion includes at least one second channel ( 405 ) and the central portion includes a plurality of microfluidic channels ( 406 ). Within the cathode portion of the wafer, for each microfluidic channel, each microfluidic channel further includes an injection channel ( 408 ) including a sample well ( 409 ) and a waste liquid ( 410 ).

注射通道可處於單一交叉通道(如圖4 中所說明)、單一T形連接或偏移雙重T形連接組態。在一些實施例中，注射通道為偏移雙重T形連接組態，其將樣品注射量減至最小，藉此改良分離解析度。樣品自注射通道注射至微型流動通道可根據熟習此項技術者已知之方法來達成，該等方法包括經由在樣品、廢液、陽極及陰極孔中施加適當電位之電泳注射。In a single injection channel may be cross-channels (as illustrated in FIG. 4), or a single T-shaped connector connected to an offset double T-shaped configuration. In some embodiments, the injection channel is an offset dual T-junction configuration that minimizes sample injection, thereby improving separation resolution. Injection of the sample from the injection channel to the microfluidic channel can be accomplished according to methods known to those skilled in the art, including electrophoretic injection via application of an appropriate potential in the sample, waste, anode, and cathode wells.

微型流動分離及檢測晶片之一替代性實施例在圖5 中說明。晶片(500 )包含一陽極部分(501 )、一陰極部分(502 )及一中心部分(503 )。針對各微型流動通道(506 )，陰極部分包含一第一通孔(504 )，且針對各微型流動通道(506 )，陽極部分包含至少一個第二通孔(505 )。中心部分包含複數個微型流動通道(506 )及一檢測窗口(507 )，各微型流動通道具有一分離區及一檢測區；其中各微型流動通道與一第一通孔及一第二通孔處於流體連通中。複數個微型流動通道 基本上處於同一平面，且在中心部分內彼此不相交。檢測窗口包含薄塑料且與各微型流動通道之檢測區重疊。An alternative embodiment of a microfluidic separation and detection wafer is illustrated in FIG . The wafer ( 500 ) includes an anode portion ( 501 ), a cathode portion ( 502 ), and a central portion ( 503 ). For each micro flow channel ( 506 ), the cathode portion includes a first via ( 504 ), and for each microfluid channel ( 506 ), the anode portion includes at least one second via ( 505 ). The central portion includes a plurality of micro flow channels ( 506 ) and a detection window ( 507 ), each of the micro flow channels has a separation area and a detection area; wherein each of the micro flow channels is in a first through hole and a second through hole In fluid communication. The plurality of micro flow channels are substantially in the same plane and do not intersect each other in the central portion. The detection window contains a thin plastic and overlaps the detection zone of each micro flow channel.

在此情況下，省略注射通道以利於各微型流動通道之陽極(第二)及陰極(第一)通孔。根據熟習此項技術者已知之方法(上文)經由陰極通孔之一者引入注射量之各樣品。在注射樣品之後，將額外緩衝溶液引入各陰極緩衝液孔中；較佳地，提供足夠量以稀釋孔中任何剩餘樣品，藉此調節自長期樣品注射引入之任何背景信號且改良檢測窗口上所觀測到之信號雜訊比。舉例而言，將約至少5、10、25、50或100倍於樣品注射量之量的緩衝液引入陰極孔中。或者，將在約5－100倍、5－50倍或10－50倍於樣品注射量之範圍內之量的緩衝液引入陽極緩衝液孔中。In this case, the injection channels are omitted to facilitate the anode (second) and cathode (first) vias of each micro flow channel. Each sample of the injection amount is introduced via one of the cathode through holes according to a method known to those skilled in the art (above). After the sample is injected, an additional buffer solution is introduced into each of the cathode buffer wells; preferably, a sufficient amount is provided to dilute any remaining sample in the well thereby adjusting any background signal introduced from the long-term sample injection and improving the detection window Observed signal noise ratio. For example, a buffer of about at least 5, 10, 25, 50, or 100 times the amount of sample injected is introduced into the cathode well. Alternatively, an amount of buffer in the range of about 5-100 times, 5-50 times, or 10-50 times the amount of sample injected is introduced into the anode buffer well.

藉由跨越微晶片上之微通道施加電位差來提供微型流動通道內樣品之電泳分離。通常藉由將陰極及陽極分別置放於陰極孔及陽極孔中，沿微型流動通道之分離部分建立電場，且將樣品(例如，核酸)自陰極末端經由分離部分移動至檢測部分且最終至陽極，可跨越微通道之末端施加高壓。有效分離所需之電場通常在50 V/cm至600 V/cm之範圍內。來自電源之電壓經由電極施加，且陽極及陰極儲集層中使用緩衝液以提供電極與篩選聚合物之間的電接觸。Electrophoretic separation of the sample within the microfluidic channel is provided by applying a potential difference across the microchannels on the microchip. Usually, by placing the cathode and the anode in the cathode hole and the anode hole, respectively, an electric field is established along the separated portion of the micro flow channel, and the sample (for example, nucleic acid) is moved from the cathode end to the detecting portion via the separating portion and finally to the anode. A high voltage can be applied across the end of the microchannel. The electric field required for efficient separation is typically in the range of 50 V/cm to 600 V/cm. The voltage from the power source is applied via the electrodes, and a buffer is used in the anode and cathode reservoirs to provide electrical contact between the electrodes and the screening polymer.

使用與陰極及陽極孔中之緩衝液接觸之電極將樣品分離所需之高壓施加至分離通道。由於與緩衝液接觸之電極上存在高壓，所以緩衝液水分子進行水解，導致OH－ 、H＋ 及氫氣之形成。此形成導致緩衝液之pH值隨時間而改變，且 緩衝液內形成氣泡。緩衝液之pH值變化可經由在陽極及陰極儲集層中使用足夠緩衝溶液(例如，1X TTE；Amresco)而減弱以提供電極與篩選基質之間的接觸。緩衝液內所形成之氣泡具有移動至篩選基質中之傾向，阻斷通道，導致不良的核酸分離。The high pressure required to separate the sample is applied to the separation channel using an electrode in contact with the buffer in the cathode and anode wells. Due to the high pressure on the electrode in contact with the buffer, the buffer water molecules undergo hydrolysis, resulting in the formation of OH - , H + and hydrogen. This formation causes the pH of the buffer to change over time and bubbles form in the buffer. The pH change of the buffer can be attenuated by using sufficient buffer solution (eg, 1X TTE; Amresco) in the anode and cathode reservoirs to provide contact between the electrode and the screening substrate. The bubbles formed in the buffer have a tendency to move into the screening matrix, blocking the channels, resulting in poor nucleic acid separation.

可藉由使用以下方法之一者或組合來防止電極上形成之氣泡移動至通道中。首先，可升高儲集層內之電極以移動氣泡產生來源(電極)遠離通道中之入孔。其次，玻璃料、聚合物玻璃料或聚合物膜或聚合物過濾器可***陰極入孔與電極末端之間。詳言之，聚合物玻璃料(例如，聚醚醚酮(PEEK))可***陰極入孔與電極末端之間。The movement of the bubbles formed on the electrodes into the channels can be prevented by using one or a combination of the following methods. First, the electrodes in the reservoir can be raised to move the bubble generation source (electrode) away from the entry aperture in the channel. Second, a frit, polymer frit or polymer film or polymer filter can be inserted between the cathode inlet and the electrode tip. In particular, a polymeric frit (eg, polyetheretherketone (PEEK)) can be inserted between the cathode inlet and the electrode tip.

選擇不導電且具有阻止電極上形成之氣泡通過孔之孔徑的玻璃料、膜或過濾器。由於在電極與篩選基質之間***玻璃料、聚合物膜或過濾器，因此可防止自電解過程形成之氣泡進入通道中。此實施可減少及/或消除因氣泡堵塞通道所造成的故障。A frit, film or filter that is non-conductive and has an aperture that blocks the passage of bubbles formed in the electrode through the aperture is selected. Since a frit, a polymer film or a filter is interposed between the electrode and the screening substrate, bubbles formed from the electrolysis process can be prevented from entering the channel. This implementation can reduce and/or eliminate failures caused by air clogging of the passage.

電連接用於由複數個樣品組成之同時分析之分離設備以便共同電源可用於同時加偏壓於複數個通道。此外，晶片及儀器之實體限制通常將不允許所有通道在長度、深度及寬度方面具有相同實體布局。Electrically connected separate devices for simultaneous analysis consisting of a plurality of samples so that a common power source can be used to simultaneously bias a plurality of channels. In addition, physical limitations of wafers and instruments will generally not allow all channels to have the same physical layout in terms of length, depth and width.

為實現複數個微型流動通道中之每一者實質上相同之電泳注射及分離條件，個別設備之各通道區段應具有基本上相同之電阻且因此具有基本上相同之電場。實質上相同之電場(亦即，其中跨越複數個微型流動通道中之每一者的 電場相差不超過約＋/－5%)可藉由同時調整複數個微型流動通道中之每一者的長度、寬度及深度以調整通道之各區段之電阻來建立。各區段之電阻R 可藉由以下關係式來描述： 其中ρ 為電阻率，l 為長度且A 為通道之橫截面積。To achieve substantially the same electrophoretic injection and separation conditions for each of the plurality of microfluidic channels, each channel segment of the individual device should have substantially the same electrical resistance and thus have substantially the same electric field. Substantially the same electric field (i.e., wherein the electric field across each of the plurality of microfluidic channels differs by no more than about +/- 5%) can be adjusted by simultaneously adjusting the length of each of the plurality of microfluidic channels The width and depth are established by adjusting the resistance of each section of the channel. The resistance R of each segment can be described by the following relationship: Where ρ is the resistivity, l is the length and A is the cross-sectional area of the channel.

存在於分離晶片之通道壁上的表面電荷可導致電滲透及樣品與壁之相互作用。可藉由將表面塗層塗覆至微型流動通道之內壁將此等影響降至最低。該等表面塗層及改質可經由熟習此項技術者已知之方法實現(例如Ludwig及Belder,2003Electrophoresis 24(15)：2481－6)。Surface charges present on the walls of the separation wafer can cause electro-osmosis and sample-wall interaction. This effect can be minimized by applying a surface coating to the inner wall of the micro flow channel. Such surface coatings and modifications can be accomplished by methods known to those skilled in the art (e.g., Ludwig and Belder, 2003 Electrophoresis 24(15): 2481-6).

可利用大量用於表面改質之候選者，包括羥丙基甲基纖維素(HPMC)、聚(氧化乙烯)(PEO)、聚(乙烯醇)(PVA)、聚二甲基丙烯醯胺(PDMA)、聚(乙烯基吡咯啶酮)、二甲基丙烯醯胺(DMA)、二乙基丙烯醯胺(DEA)、聚(二乙基丙烯醯胺)(PDEA)及其混合物，諸如PDMA：PDEA。A large number of candidates for surface modification can be utilized, including hydroxypropyl methylcellulose (HPMC), poly(ethylene oxide) (PEO), poly(vinyl alcohol) (PVA), polydimethyl methacrylate ( PDMA), poly(vinylpyrrolidone), dimethyl methacrylate (DMA), diethyl acrylamide (DEA), poly(diethyl acrylamide) (PDEA), and mixtures thereof, such as PDMA :PDEA.

此外，為用於電泳應用，複數個微型流動通道中之每一者較佳係充滿篩選基質。在非限制性實例中，該等篩選基質可包含線性聚丙烯醯胺(PAA)、聚二甲基丙烯醯胺(PDMA)、聚二乙基丙烯醯胺(PDEA)、聚乙烯基吡咯啶酮(PVP)及其組合，包括(例如)PVP：PAA、PDMA：PAA、PDEA：PAA、PDEA：PDMA：PAA。在某些實施例中，篩選基質包含0.1－50 wt%之聚丙烯醯胺。大量此等篩選基質亦 具有動態自塗能力。如使用本發明之電泳分離晶片之此等實施例所實施般，核酸自陽極經由篩選基質電泳移動至陰極末端，且在其中按尺寸分離。如上文所述，通道之內壁可經塗佈以將電滲透及核酸與壁之相互作用的影響降至最低。Moreover, for use in electrophoretic applications, each of the plurality of microfluidic channels is preferably filled with a screening matrix. In a non-limiting example, the screening matrices may comprise linear polyacrylamide (PAA), polydimethyl acrylamide (PDMA), polydiethyl acrylamide (PDEA), polyvinylpyrrolidone (PVP) and combinations thereof, including, for example, PVP: PAA, PDMA: PAA, PDEA: PAA, PDEA: PDMA: PAA. In certain embodiments, the screening matrix comprises 0.1-50 wt% polyamidoguanamine. a large number of these screening substrates are also With dynamic self-coating ability. As embodied in such embodiments using the electrophoretic separation wafer of the present invention, the nucleic acid is electrophoretically moved from the anode to the cathode end via a screening matrix and is separated therein by size. As described above, the inner wall of the channel can be coated to minimize the effects of electroosmosis and interaction of the nucleic acid with the wall.

解析度(本文特定為電泳解析度)為明確區分兩個及時分離之峰的能力(或藉由鹼基尺寸)。解析度(R)藉由以下方程式定義 其中t 為第n個峰之移動時間，hw 為第n個峰之全寬及半最大值，且△b 為兩個峰之間的鹼基數目之差。單鹼基對解析度定義在R大於0.4之點。視覺上，兩個峰在峰谷比大於0.7時可彼此區分。R及峰：谷要求兩者必須均滿足以具有高解析度，且亦可認為解析度為一系列片段尺寸之特徵。在STR分析中等位基因之片段尺寸的範圍在90至400 bp之範圍內，且跨越此範圍之片段尺寸的單鹼基對解析度為STR分析所需。排序分析之片段尺寸範圍高達1200 bp。實現每一道長閱讀長度及資料產量之能力部分地藉由晶片能夠產生單鹼基對解析度之範圍來測定。The resolution (herein specified as electrophoretic resolution) is the ability to clearly distinguish between two timely separated peaks (or by base size). Resolution (R) is defined by the following equation Wherein t is an n-th peak of the moving time, the n-th HW is the full width at half maximum of peaks of and, and △ b the difference between the number of bases of the two peaks. The single base pair resolution is defined at a point where R is greater than 0.4. Visually, the two peaks can be distinguished from each other when the peak-to-valley ratio is greater than 0.7. R and peak: The valley requires that both must be satisfied to have high resolution, and the resolution can be considered as a feature of a series of segment sizes. The fragment size of the alleles in the STR analysis ranged from 90 to 400 bp, and the single base pair resolution across the fragment size of this range was required for STR analysis. The segment size of the sort analysis is up to 1200 bp. The ability to achieve each length of read length and data yield is determined in part by the extent to which the wafer is capable of producing a single base pair resolution.

光學檢測系統之檢測極限由信號雜訊比(SNR)限定。此比率定義為信號功率與破壞信號之雜訊功率(雜訊功率之標準偏差)之比率。高SNR表明信號存在之更高確定性。3之信號雜訊比一般定義為可為確信鑑別信號存在所接受之 信號雜訊比(Gilder,2007,J Forensic Sci .52(1)：97)。The detection limit of an optical inspection system is limited by the signal to noise ratio (SNR). This ratio is defined as the ratio of the signal power to the noise power of the corrupted signal (the standard deviation of the noise power). A high SNR indicates a higher certainty of the presence of the signal. The signal to noise ratio of 3 is generally defined as the acceptable signal to noise ratio for the presence of a confident identification signal (Gilder, 2007, J Forensic Sci. 52(1): 97).

當分析及檢測核酸樣品中之複數個核酸物質時，來自晶片檢測窗口中之塑料的自身螢光對螢光背景有強烈影響。本發明之電泳分離晶片之有利特徵在於使用薄檢測窗口以將來自塑料之背景螢光降至最小。將此背景水準與borofloat(其為用於製造微型流動分離晶片之常用基板)相比。藉由使用薄塑料窗口，在PCR方法中可檢測到產生用於分析之螢光標記之片段的最少1000個副本、300個副本、100個副本、30個副本、10個副本、1個副本之模板核酸。亦可檢測到用於排序反應之最少0.5 pmole、0.1 pmole、0.01 pmole或0.001 pmole之核酸模板。When analyzing and detecting a plurality of nucleic acid species in a nucleic acid sample, the self-fluorescence from the plastic in the wafer detection window has a strong influence on the fluorescent background. An advantageous feature of the electrophoretic separation wafer of the present invention is the use of a thin detection window to minimize background fluorescence from the plastic. Use this background level with borofloat (Compared to a common substrate for manufacturing micro flow separation wafers). By using a thin plastic window, a minimum of 1000 copies, 300 copies, 100 copies, 30 copies, 10 copies, 1 copy of the fragments that produce the fluorescent markers for analysis can be detected in the PCR method. Template nucleic acid. Nucleic acid templates for a minimum of 0.5 pmole, 0.1 pmole, 0.01 pmole, or 0.001 pmole for sequencing reactions can also be detected.

分離及檢測晶片應用Separation and inspection of wafer applications

本發明之各種態樣之應用廣泛擴展用於核酸鑑別及排序兩者。用於人類鑑別中之實例包括刑事法醫學及國家安全，例如在軍用檢查點、邊界及港口、機場及大規模災難地點鑑別。獸醫學鑑別應用(包括，賽馬繁育及追蹤、家畜繁育及寵物鑑別)亦在本發明之電泳晶片之用途範疇內。The various aspects of the invention are widely used for both nucleic acid identification and sequencing. Examples for human identification include criminal forensics and national security, such as identification at military checkpoints, borders and ports, airports, and large-scale disaster locations. Veterinary identification applications (including horse breeding and tracing, livestock breeding and pet identification) are also within the scope of use of the electrophoretic wafers of the present invention.

此外，本發明之儀器可加固且藉此運用於結果可即時使用之領域中。因而，該等儀器可用於軍用檢查點、邊界及港口、機場及大規模傷亡地點。Furthermore, the apparatus of the present invention can be reinforced and utilized in the field where the results can be used immediately. As a result, these instruments can be used for military checkpoints, borders and ports, airports and mass casualties.

核酸排序技術之應用可分成四個領域：人類臨床診斷學，包括(例如)細菌性感染及抗體敏感性、病毒性感染(鑑別及抗藥性概況分析)、遺傳性疾病、複雜病症(哮喘、心 臟病、糖尿病)及藥物基因組學；獸醫學臨床診斷學；研究排序，包括再排序及完成；生物武器劑鑑別，包括(例如)炭疽芽孢桿菌(B.anthracis )及艾博拉病毒(Ebola virus )檢測；及食品安全。一些實例如下。The application of nucleic acid sequencing technology can be divided into four areas: human clinical diagnostics, including, for example, bacterial infections and antibody susceptibility, viral infections (identification and drug resistance profiling), hereditary diseases, complex conditions (asthma, heart) disease, diabetes), and pharmacogenomics; Veterinary Clinics of diagnostics; sorting studies, including complete and reordering; identify biological weapons agents, including (e.g.) B. anthracis (B.anthracis) and Yi Bola viruses (Ebola virus) Testing; and food safety. Some examples are as follows.

一患有HIV之患者需要抗藥性測試。目前，可花費數週來確立抗性。抗藥菌株可在此時間期間控制。對當患者在醫師診所中等待時可在1－2小時內提供回答之儀器及系統存在未滿足之需要。使用根據本發明之電泳分離晶片允許頻繁抗藥性監測、臨床上及成本上更有效之使用抗病毒劑及更好之患者結果。A patient with HIV needs a drug resistance test. Currently, it can take weeks to establish resistance. Drug resistant strains can be controlled during this time. There is an unmet need for instruments and systems that provide answers within 1-2 hours while the patient is waiting in the physician's office. The use of electrophoretic separation of wafers in accordance with the present invention allows for frequent drug resistance monitoring, clinical and cost effective use of antiviral agents and better patient outcomes.

一患有菌血症之患者處於休克中。目前，可花費數天來確定病原體對抗生素是否有抗性及其身份。在此期間，必須用廣譜抗菌素(broad－spectrum antibiotics)來治療患者，廣譜抗菌素可引起對患者之嚴重副作用且促進目前盛行之抗菌藥抗性增加。此外，該等治療可為次佳的。使用根據本發明之電泳分離晶片允許在1－2小時中鑑別病原體之抗生素抗性概況，產生更有效之靶向治療、減少之抗生素毒性及更佳之患者結果。對患者及公眾健康之益處受人稱讚。A patient with bacteremia is in shock. Currently, it can take several days to determine if a pathogen is resistant to antibiotics and its identity. In the meantime, patients must be treated with broad-spectrum antibiotics, which can cause serious side effects in patients and promote the current prevalence of antimicrobial resistance. In addition, such treatments may be sub-optimal. The use of electrophoretic separation of wafers in accordance with the present invention allows identification of pathogen antibiotic resistance profiles in 1-2 hours, resulting in more effective targeted therapies, reduced antibiotic toxicity, and better patient outcomes. The benefits to patients and public health are praised.

一患有癌症之患者正進行手術。目前，當患者在手術台上時取出腫瘤樣品至病理學檢查。基於簡單組織病理學菌株之結果，作出關於外科醫生應如何進取之決定。使用根據本發明之電泳分離晶片可用在不足1小時內癌症之明確核酸診斷代替組織病理學，允許作出基於更好資訊之外科 手術決定。A patient with cancer is undergoing surgery. Currently, tumor samples are taken to the pathology examination when the patient is on the operating table. Based on the results of a simple histopathological strain, a decision is made as to how the surgeon should make progress. The use of an electrophoretic separation wafer according to the present invention can replace the histopathology with a clear nucleic acid diagnosis of cancer in less than one hour, allowing for a basis for better information. The operation is decided.

以下實例說明本發明之特定實施例及其各種用途。其闡述僅為達成說明之目的，且不應理解為限制本發明。The following examples illustrate specific embodiments of the invention and its various uses. The description is for the purpose of illustration only and is not to be construed as limiting.

實例Instance 實例1Example 1

晶片設計及電泳 實例1A：晶片設計 本發明之設備之特定實施例的示意圖在圖6 中說明。此微型流動設備由16個微通道組成，各微通道具有雙重T交叉注射器。通道之橫截面尺寸(90 μm寬及40 μm深)及陽極與交叉注射器之間的通道長度(25 cm)對所有通道而言均為相等的。各通道之分離長度(交叉點與激發/檢測窗口之間的距離)在16至20 cm長之範圍內。陰極孔與注射器之間的通道橫截面積經調整以便陰極與交叉點之間的所有電阻及因此電場在偏壓下基本上相等。此確保與載入樣品之分離通道無關，樣品所經受之電場相同。所有通道之交叉點電壓基本上相同。用於注射樣品之樣品入口及樣品廢液支管均為2.5 mm長。兩個通道之間的偏距為500 μm。Wafer Design and Electrophoresis Example 1A: Wafer Design A schematic of a particular embodiment of the apparatus of the present invention is illustrated in FIG . This microfluidic device consists of 16 microchannels, each with a dual T-cross syringe. The cross-sectional dimensions of the channel (90 μm wide and 40 μm deep) and the channel length (25 cm) between the anode and the cross-injector are equal for all channels. The separation length of each channel (the distance between the intersection and the excitation/detection window) is in the range of 16 to 20 cm long. The channel cross-sectional area between the cathode aperture and the injector is adjusted such that all of the electrical resistance between the cathode and the intersection and thus the electric field are substantially equal under bias. This ensures that the sample is subjected to the same electric field regardless of the separation channel from which the sample is loaded. The intersection voltages of all channels are substantially the same. The sample inlet and sample waste branch for the injection of the sample are both 2.5 mm long. The offset between the two channels is 500 μm.

實例1B：晶片及支撐體製造 藉由熱壓印、鑽孔以形成入孔且擴散黏結以密封通道，將晶片圖案化。在玻璃中藉由光刻使用化學濕式蝕刻方法製造母片(master)。接著此玻璃母片用於藉由電鑄來製造鎳鈷壓印工具以產生玻璃母片之負型複製物。ZenorTM －1420R薄膜板(尺寸為5"×2"及厚度為188 μm)用作基板材 料。在此等板上，陰極、陽極、樣品及廢液入孔藉由鑽孔來形成。繼此之後，在壓印工具上將晶片設計特徵熱壓印至基板中。如圖7 中所說明，藉由將堆疊置放於135℃下及1250 psi壓縮壓力下經加熱之水壓機中15分鐘來實現壓印。將堆疊保持在1250 psi之壓縮壓力下且在釋放之前使其冷卻至38℃。用含有降冰片烯單體之薄熱塑性聚合物製造此晶片導致在激發及檢測窗口產生低背景螢光。實現高黏結強度擴散黏結允許使用高黏度篩選基質。Example 1B: Wafer and Support Fabrication Wafers were patterned by hot stamping, drilling to form in-holes and diffusion bonding to seal the vias. A master is produced by photolithography using a chemical wet etching method in glass. This glass master is then used to make a nickel cobalt embossing tool by electroforming to produce a negative replica of the glass master. A Zenor TM -1420R film sheet (size 5" x 2" and thickness 188 μm) was used as the substrate material. On these plates, the cathode, anode, sample, and waste liquid inlet holes are formed by drilling. Following this, the wafer design features are thermally embossed into the substrate on the embossing tool. As illustrated in FIG. 7, a hydraulic press by heating for 15 minutes to achieve the platen by the stacked and placed in a 135 deg.] C under a compression pressure of 1250 psi. The stack was held at a compression pressure of 1250 psi and allowed to cool to 38 °C prior to release. Fabrication of this wafer with a thin thermoplastic polymer containing norbornene monomer results in low background fluorescence at the excitation and detection windows. Achieving high bond strength diffusion bonding allows the use of high viscosity screening substrates.

藉由將一ZenorTM －1420R薄膜板(尺寸為5"×2"及厚度為188 μm)在基板上對準且使此堆疊經受熱及壓力來實現基板之擴散黏結。在薄膜板之間不塗覆黏著劑；黏結完全藉由熱及壓力來實現。晶片之最終厚度為約376 μm。藉由此方法製造之分離晶片經測試且證明在損壞之前能夠經受至少830 psi之壓力。The Zenor TM -1420R by a thin plate (size 5 "× 2" and a thickness of 188 μm) on the substrate and aligned so that this stack is subjected to heat and pressure to achieve diffusion bonding of the substrate. No adhesive is applied between the film sheets; the bonding is achieved entirely by heat and pressure. The final thickness of the wafer is about 376 μm. The discrete wafers fabricated by this method were tested and demonstrated to withstand a pressure of at least 830 psi prior to damage.

圖8 說明藉由CNC銑削自3/8"厚之丙烯酸板(GE Plastic)製造的晶片支撐體。晶片支撐體由三個主要部分組成：陰極板、中心部分及陽極板。陰極板含有陰極孔、樣品及廢液孔及對準孔。陽極板含有陽極孔及對準孔。陰極板及陽極板均為3/8"厚度以提供足夠樣品量進行樣品注射及提供足夠緩衝液量進行電泳。中心部分為0.04"厚度且具有作為用於微通道中雷射誘發螢光檢測之"檢測窗口"的開口。在此組態下，來自分離晶片之自身螢光藉由約376 μm厚之基板來控制。使用雙面壓敏性黏著劑將分離晶片附著於晶片支撐體。選擇對分離緩衝液及篩選基質而言為惰性之黏 著劑。使用壓敏性環氧樹脂將支撐體與分離晶片連接。激發及檢測區中塑料之厚度藉由在此區域中載體上製造切口來降至最小。 Figure 8 illustrates a wafer support manufactured by CNC milling from a 3/8" thick acrylic sheet (GE Plastic). The wafer support consists of three main parts: a cathode plate, a central portion, and an anode plate. The cathode plate contains a cathode hole. The sample and the waste hole and the alignment hole. The anode plate contains the anode hole and the alignment hole. The cathode plate and the anode plate are both 3/8" thick to provide a sufficient sample amount for sample injection and provide sufficient buffer amount for electrophoresis. The central portion is 0.04" thick and has an opening as a "detection window" for laser-induced fluorescence detection in microchannels. In this configuration, the self-fluorescence from the separate wafer is by a substrate of approximately 376 μm thick. Control. The separation wafer is attached to the wafer support using a double-sided pressure sensitive adhesive. An adhesive that is inert to the separation buffer and the screening substrate is selected. The support is attached to the separation wafer using a pressure sensitive epoxy. The thickness of the plastic in the excitation and detection zone is minimized by making slits in the carrier in this zone.

ZenorTM －1420R之光學發射光譜具有在570 nm下之拉曼(Raman)發射峰，該發射峰限制螢光染料之螢光檢測。圖9 證明與典型玻璃分離晶片(玻璃1.4 mm及玻璃0.7 mm)相比，塑料晶片(Pchip1及PChp2)之低自身螢光。藉由選擇COP聚合物且將檢測區中設備之厚度降至最小且藉由用薄膜製造該設備來實現塑料晶片之低自身螢光。Zenor TM -1420R having the optical emission spectrometer (RAMAN) of the Raman emission peak at 570 nm, the fluorescence emission peak detection limit of the fluorescent dye. Figure 9 demonstrates the low self-fluorescence of plastic wafers (Pchip1 and PChp2) compared to typical glass-separated wafers (glass 1.4 mm and glass 0.7 mm). The low self-fluorescence of the plastic wafer is achieved by selecting the COP polymer and minimizing the thickness of the device in the detection zone and by fabricating the device with a film.

實例1C：表面改質及篩選基質 藉由最初用去離子水預處理微通道表面，接著用1 MNaOH處理微通道表面來實現表面改質。應用吹氣處理(nitrogen flush)自通道移除流體。繼表面處理之後，使0.1%(w/v)羥丙基甲基纖維素(HPMC)溶液流動穿過通道，接著在室溫下培育隔夜。使用高純度氮湧過通道以移除通道內部之流體。Example 1C: Surface Modification and Screening of Substrates Surface modification was achieved by initially pretreating the microchannel surface with deionized water followed by treatment of the microchannel surface with 1 M NaOH. The fluid is removed from the channel using a nitrogen flush. Following surface treatment, a 0.1% (w/v) hydroxypropyl methylcellulose (HPMC) solution was flowed through the channel and then incubated overnight at room temperature. High purity nitrogen is used to surge through the channel to remove fluid from inside the channel.

用於此等實驗之篩選基質為7 M脲及1X TTE(Amresco)緩衝液中4%線性聚丙烯醯胺(LPA)。The screening substrates used in these experiments were 7 M urea and 4% linear polyacrylamide (LPA) in 1X TTE (Amresco) buffer.

實例1D：電泳STR尺寸測定 核酸分析之電泳分離及分析在Genebench－FXTM 系列100(Network Biosystems,Inc.,Woburn,MA)上執行。此儀器經組態以接受塑料分離晶片及晶片支撐體以考慮到晶片與儀器之間的優良光學、電學及熱耦合。在整個操作中，腔室溫度維持在50℃下。Example 1D: Size Determination of STR electrophoresis and electrophoretic separation of nucleic acid analysis performed on a series Genebench-FX TM 100 (Network Biosystems, Inc., Woburn , MA). The instrument is configured to accept plastic separation wafers and wafer supports to allow for superior optical, electrical, and thermal coupling between the wafer and the instrument. The chamber temperature was maintained at 50 ° C throughout the run.

對於DNA尺寸測定實驗而言，將人類基因組DNA用ABI AmpFISTR套組(Applied Biosystems Inc.,Foster City,CA)擴增。將PCR產物(2.7 μL)與0.3 μL尺寸測定標準及10 μL甲醯胺混合，且載入樣品孔中進行分析。檢定由以下組成：在156 V/cm下執行預電泳6分鐘，接著藉由在陽極孔施加3900 V之電位差且將陰極孔接地來引入樣品。藉由歷時18秒施加350 V/cm之電場，接著藉由跨越樣品及廢液孔施加350 V/cm之電場且同時跨越陰極及陽極孔施加15.6 V/cm之電場進行1.2分鐘之雙負載來引入DNA樣品。注射樣品後，藉由跨越陰極及陽極孔施加156 V/cm之電場同時將800 V之阻擾電壓(pullback voltage)維持40分鐘來執行電泳DNA分離。For DNA size determination experiments, human genomic DNA was amplified using an ABI AmpFISTR kit (Applied Biosystems Inc., Foster City, CA). The PCR product (2.7 μL) was mixed with 0.3 μL size measurement standard and 10 μL of methotrexate and loaded into the sample well for analysis. The assay consisted of performing pre-electrophoresis at 156 V/cm for 6 minutes, then introducing the sample by applying a potential difference of 3900 V to the anode aperture and grounding the cathode aperture. An electric field of 350 V/cm was applied over 18 seconds, followed by an electric field of 350 V/cm across the sample and waste holes while applying an electric field of 15.6 V/cm across the cathode and anode holes for a 1.2 minute double load. A DNA sample was introduced. After the sample was injected, electrophoretic DNA separation was performed by applying an electric field of 156 V/cm across the cathode and the anode well while maintaining a pullback voltage of 800 V for 40 minutes.

對於DNA排序實驗而言，將M13質體用GE Amersham DYEnamicTM ET染料終止子循環排序套組(GE Healthcare)來循環排序，乙醇沈澱且再懸浮於10 μL去離子水中。分離檢定由以下組成：在156 V/cm下執行預電泳6分鐘，接著藉由在陽極孔施加3900 V之電位差且將陰極孔接地來引入樣品。藉由歷時60秒施加350 V/cm之電場來引入DNA樣品。注射樣品後，藉由跨越陰極及陽極孔施加156 V/cm之電場同時維持400 V之阻擾電壓60分鐘來執行電泳DNA分離。藉由自Peakfit提取峰資訊(峰間隔及峰寬)來計算DNA分離解析度。For DNA sequencing experiments, M13 plastid sorting Terminator Cycle kit (GE Healthcare) is circulated for sorting GE Amersham DYEnamic TM ET dyes, ethanol precipitated and resuspended in 10 μL of deionized water. The separation assay consisted of performing pre-electrophoresis at 156 V/cm for 6 minutes, then introducing the sample by applying a potential difference of 3900 V to the anode aperture and grounding the cathode aperture. A DNA sample was introduced by applying an electric field of 350 V/cm for 60 seconds. After the sample was injected, electrophoretic DNA separation was performed by applying an electric field of 156 V/cm across the cathode and the anode well while maintaining a blocking voltage of 400 V for 60 minutes. By from Peakfit The peak information (peak interval and peak width) is extracted to calculate the DNA separation resolution.

在塑料晶片中之16道中同時實現成功之分離。圖10 顯示來自5色標記套組(ABI AmpFlSTR Identifiler套組)之等位 基因階梯的等位基因呼叫概況。此等結果證明本發明之設備能夠在塑料晶片中用5種顏色分離且清楚解析等位基因，包括間隔等於僅單鹼基對之距離的等位基因(THO 1，等位基因9.3及10)。圖11 顯示9947A人類基因組DNA之等位基因呼叫STR概況，顯示在1.0 ng DNA模板下實現全部概況。圖12 顯示對於多達480個bp而言R>0.4之解析度，證明單鹼基解析度至多為480個bp。圖13 藉由顯示間隔1個核苷酸之2個等位基因可毫不含糊地清楚解析來說明此解析度。圖14及15 顯示證明單鹼基對解析度之DNA排序概況。Successful separation is achieved simultaneously in 16 lanes in the plastic wafer. Figure 10 shows an allelic call profile from the allelic ladder of the 5-color marker set (ABI AmpFlSTR Identifiler kit). These results demonstrate that the apparatus of the present invention is capable of separating and clearly interpreting alleles in five colors in a plastic wafer, including alleles spaced apart by a distance of only a single base pair (THO 1, alleles 9.3 and 10). . Figure 11 shows the STR profile of the alleles of the 9947A human genomic DNA, showing that the overall profile was achieved under a 1.0 ng DNA template. Figure 12 shows a resolution of R > 0.4 for up to 480 bp, demonstrating a single base resolution of at most 480 bp. Figure 13 illustrates this resolution by showing that the two alleles separated by one nucleotide are clearly and unambiguously resolved. Figures 14 and 15 show DNA sequencing profiles demonstrating single base pair resolution.

實例2Example 2

電動注射塑料晶片 實例2A：晶片設計 本發明之電泳分離晶片之另一組態使用單通道進行分離。藉由電動樣品注射將各樣品引入分離通道中。此替代性方法允許使用小樣品量且顯著簡化分離過程。用於電動樣品注射之晶片設計的示意圖顯示於圖16 中之分離圖，顯示支撐體及分離晶片部分。該設備由16個分離長度有效為20 cm之微通道組成。各通道在各末端具有一入孔。該等通道90 μm寬及40 μm深。Electric Injection Plastic Wafer Example 2A: Wafer Design Another configuration of the electrophoretic separation wafer of the present invention uses a single channel for separation. Each sample was introduced into the separation channel by injection of a motorized sample. This alternative method allows the use of small sample volumes and significantly simplifies the separation process. A schematic of a wafer design for electric sample injection is shown in the separation diagram of Figure 16 , showing the support and the separated wafer portion. The device consists of 16 microchannels separated by a length of 20 cm. Each channel has an access hole at each end. These channels are 90 μm wide and 40 μm deep.

實例2B：設備製造 圖16 之設備係根據以上部分中所述之程序來製造。概括言之，在厚度為188 μm之COP薄膜(ZeonorTM －1420R)中形成入孔(直徑為1 mm)。接著藉由熱壓印形成通道圖案(90 μm寬及40 μm深)。將COP蓋(ZeonorTM －1420R)擴散黏結至 基板以密封通道。Example 2B: Equipment Manufacturing The equipment of Figure 16 was fabricated according to the procedure described in the previous section. In summary, the formation of the hole (diameter 1 mm) in the COP film (Zeonor TM -1420R) in a thickness of 188 μm. A channel pattern (90 μm wide and 40 μm deep) was then formed by hot stamping. The COP lid (Zeonor TM -1420R) diffusion bonding to the substrate to seal the passage.

實例2C：電泳 藉由如以上部分中所述將表面改質應用於通道來製備用於分離之設備。繼此之後，用篩選基質填充通道。將樣品載入樣品/陰極儲集層。經由電極將注射場施加至樣品以將帶負電之DNA注射至分離通道中。在將DNA注射至通道後，將緩衝液(1X TTE；Ameresco)以10倍於樣品量之量添加至樣品/陰極儲集層中。跨越陰極及陽極施加電場以將DNA自注射塞沿分離通道向下分離。添加用以稀釋樣品/陰極中之樣品且不必在載入緩衝液之前移除樣品。分離及檢測在Genebench－FXTM 系列100儀器上執行，且使用先前實例中所述之軟體執行資料分析。Example 2C: Electrophoresis An apparatus for separation was prepared by applying surface modification to a channel as described in the section above. Following this, the channel is filled with a screening matrix. The sample is loaded into the sample/cathode reservoir. An injection field is applied to the sample via an electrode to inject negatively charged DNA into the separation channel. After the DNA was injected into the channel, a buffer (1X TTE; Ameresco) was added to the sample/cathode reservoir in an amount 10 times the amount of the sample. An electric field is applied across the cathode and anode to separate the DNA from the injection plug down the separation channel. The sample is added to dilute the sample/cathode and it is not necessary to remove the sample prior to loading the buffer. Separation and detection is performed on a series Genebench-FX TM 100 instrument, and the use of the previously described data analysis software running instance.

實例3Example 3

DNA排序 對於DNA排序分析而言，在由以下各物組成之反應混合物中擴增DNA模板：PCR酶SpeedSTAR HS(Takara,Madison,WI)(U/μL)：0.025，Fast緩衝劑1：1x，dNTPs：0.25 mM，引子(正向)：250 nM及引子(反向)：250 nM。將所需含量之模板DNA添加至混合物中。將DI水或TE緩衝液(Tris 10 mM或EDTA 0.1 mM)添加至反應混合物中，至10 μL之總量。根據廠商推薦之方案，PCR反應混合物之熱循環由以下組成：95℃下60秒之熱起始活化、30次變性循環、退火及擴展(98℃下5秒、55℃下10－15秒及72℃下5－10秒/kbp)及最終在72℃下擴展60秒。DNA sorting For DNA sequencing analysis, DNA templates were amplified in reaction mixtures consisting of: PCR enzyme SpeedSTAR HS (Takara, Madison, WI) (U/μL): 0.025, Fast buffer 1:1 x, dNTPs: 0.25 mM, primer (forward): 250 nM and primer (reverse): 250 nM. The desired amount of template DNA is added to the mixture. DI water or TE buffer (Tris 10 mM or EDTA 0.1 mM) was added to the reaction mixture to a total amount of 10 μL. According to the manufacturer's recommended protocol, the thermal cycling of the PCR reaction mixture consists of 60 seconds of hot initiation activation at 60 ° C, 30 denaturation cycles, annealing and expansion (5 seconds at 98 ° C, 10-15 seconds at 55 ° C and 5-10 sec/kbp at 72 ° C and finally 60 sec at 72 ° C.

根據廠商之方案，整個PCR產物藉由使用30K MWCO UF過濾器(Pall,East Hills,NY)來淨化。由DI水中DNA組成之經淨化之產物經稀釋或整體用作排序反應之模板。The entire PCR product was purified by using a 30K MWCO UF filter (Pall, East Hills, NY) according to the manufacturer's protocol. The purified product consisting of DNA in DI water is diluted or used as a template for the sorting reaction.

使用DYEnamicTM ET終止子循環排序套組(GE Amersham Biosciences)在半強度反應下使用以下反應混合物來執行PCR模板之循環排序。排序預混合物：4 μL，稀釋緩衝液：4 μL，引子(10 μM)：5 pmol。將DNA模板添加至排序反應混合物中。將DI水添加至反應混合物中，至20 μL之總量。根據廠商推薦之循環方案，所用循環條件由(95℃下20秒、50℃下15秒、60℃下60秒)30次循環組成。Using DYEnamic TM ET terminator cycle sorting kit (GE Amersham Biosciences) to sort execution cycle of PCR template using the following reaction mixture at half intensity of the reaction. Sort the premix: 4 μL, dilution buffer: 4 μL, primer (10 μM): 5 pmol. The DNA template is added to the sequencing reaction mixture. DI water was added to the reaction mixture to a total amount of 20 μL. According to the manufacturer's recommended circulation scheme, the cycle conditions used consisted of 30 cycles of 20 seconds at 95 ° C, 15 seconds at 50 ° C, and 60 seconds at 60 ° C.

藉由乙醇沈澱來淨化排序反應混合物。將經沈澱之產物再懸浮於13 μL DI水中且用作分離及檢測之樣品。The sequencing reaction mixture was purified by ethanol precipitation. The precipitated product was resuspended in 13 μL of DI water and used as a sample for separation and detection.

對於STR分析，擴增係在具有由以下各物組成之以下反應混合物之10 μL反應中進行：PCR酶SpeedSTAR HS(Takara,Madison,WI)(U/μL)：0.0315，Fast緩衝劑1：1x，引子設置：2 μl，Fast緩衝劑1：1X，dNTPs：200 μM，引子(正向/反向)：2 μL，來自AmpFlSTR ProfilerTM 、COFilerTM 或IdentifilerTM (Applied Biosystems,Foster City,CA)。For STR analysis, the amplification was performed in a 10 μL reaction with the following reaction mixture consisting of: PCR enzyme SpeedSTAR HS (Takara, Madison, WI) (U/μL): 0.0315, Fast buffer 1:1x , primer setting: 2 μl, Fast buffer 1:1X, dNTPs: 200 μM, primer (forward/reverse): 2 μL from AmpFlSTR Profiler TM , COFiler TM or Identifiler TM (Applied Biosystems, Foster City, CA) .

循環方案根據酶廠商條件，其由以下組成：95℃下60秒之熱起始活化、接著28次變性循環、退火及擴展(98℃下4 s、59℃下15 s、72℃下5 s)及最終在72℃下擴展60秒。PCR產物用作分離及檢測之樣品。或者，PCR產物亦可經純化且用作分離及檢測之樣品。The cycle protocol consisted of enzyme manufacturer conditions consisting of 60 minutes of hot initiation activation at 95 °C followed by 28 denaturation cycles, annealing and expansion (4 s at 98 °C, 15 s at 59 °C, 5 s at 72 °C) And finally extended at 72 ° C for 60 seconds. The PCR product was used as a sample for separation and detection. Alternatively, the PCR product can also be purified and used as a sample for separation and detection.

應瞭解上文揭示內容強調本發明之某些特定實施例，且其所有修改或替代性等效物在如隨附申請專利範圍中所述之本發明之精神及範疇內。It is to be understood that the above disclosure is intended to be in the nature of the invention and the invention

100‧‧‧晶片100‧‧‧ wafer

101‧‧‧陽極部分101‧‧‧Anode section

102‧‧‧陰極部分102‧‧‧ cathode part

103‧‧‧在陽極部分與陰極部分之間的一中心部分103‧‧‧ a central part between the anode part and the cathode part

104‧‧‧至少一個第一通孔104‧‧‧At least one first through hole

105‧‧‧至少一個第二通孔105‧‧‧At least one second through hole

106‧‧‧複數個微型流動通道106‧‧‧Multiple micro flow channels

107‧‧‧檢測窗口107‧‧‧Detection window

108‧‧‧分離區108‧‧‧Separation zone

109‧‧‧檢測區109‧‧‧Detection area

201‧‧‧具有頂面及底面之支撐體201‧‧‧Supports with top and bottom surfaces

202‧‧‧陽極部分202‧‧‧Anode part

203‧‧‧陰極部分203‧‧‧ cathode part

204‧‧‧在陽極部分與陰極部分之間的一中心部分204‧‧‧ a central part between the anode part and the cathode part

205‧‧‧檢測窗口205‧‧‧Detection window

206‧‧‧至少一個陽極孔206‧‧‧At least one anode hole

207‧‧‧至少一個陰極孔207‧‧‧At least one cathode hole

250‧‧‧晶片250‧‧‧ wafer

360‧‧‧基板層360‧‧‧ substrate layer

361‧‧‧複數個溝槽361‧‧‧Multiple grooves

370‧‧‧覆蓋層370‧‧‧ Coverage

371‧‧‧通孔371‧‧‧through hole

372‧‧‧通孔372‧‧‧through hole

400‧‧‧晶片400‧‧‧ wafer

401‧‧‧陰極部分401‧‧‧ cathode part

403‧‧‧中心部分403‧‧‧ central part

405‧‧‧至少一個第二通道405‧‧‧ at least one second channel

406‧‧‧複數個微型流動通道406‧‧‧Multiple micro flow channels

408‧‧‧包含樣品孔及廢液孔之注射通道408‧‧‧Injection channel containing sample wells and waste liquid holes

409‧‧‧樣品孔409‧‧‧ sample hole

410‧‧‧廢液孔410‧‧‧ Waste hole

500‧‧‧晶片500‧‧‧ wafer

501‧‧‧陽極部分501‧‧‧Anode part

502‧‧‧陰極部分502‧‧‧ cathode part

503‧‧‧中心部分503‧‧‧ central part

504‧‧‧第一通孔504‧‧‧First through hole

505‧‧‧第二通孔505‧‧‧second through hole

506‧‧‧複數個微型流動通道506‧‧‧Multiple micro flow channels

507‧‧‧檢測窗口507‧‧‧Detection window

圖1說明根據本發明之各種實施例之一微型流動分離及檢測晶片。1 illustrates a microfluidic separation and detection wafer in accordance with various embodiments of the present invention.

圖2說明可用於建構根據本發明之各種實施例之一微型流動分離及檢測晶片的分離支撐體及晶片層。2 illustrates a separate support and wafer layer that can be used to construct a microfluidic separation and detection wafer in accordance with various embodiments of the present invention.

圖3說明可用於建構根據本發明之各種實施例之一微型流動分離及檢測晶片的分離設備層。3 illustrates a separation device layer that can be used to construct a microfluidic separation and detection wafer in accordance with various embodiments of the present invention.

圖4說明根據本發明之各種實施例之一微型流動分離及檢測晶片的陰極部分透視圖。4 illustrates a perspective view of a cathode portion of a microfluidic separation and detection wafer in accordance with various embodiments of the present invention.

圖5說明根據本發明之各種實施例之具有注射通道的一微型流動分離及檢測晶片。Figure 5 illustrates a microfluidic separation and detection wafer having an injection channel in accordance with various embodiments of the present invention.

圖6為根據本發明之各種實施例之一微型流動分離及檢測晶片的示意圖。6 is a schematic illustration of a microfluidic separation and detection wafer in accordance with various embodiments of the present invention.

圖7說明用於壓印之堆疊。Figure 7 illustrates a stack for imprinting.

圖8說明藉由CNC銑削自3/8"厚之丙烯酸板(GE Plastic)製造的晶片支撐體；(頂)俯視圖；底(側視圖)。Figure 8 illustrates a wafer support fabricated from a 3/8" thick acrylic sheet (GE Plastic) by CNC milling; (top) top view; bottom (side view).

圖9為證明與典型玻璃分離晶片相比，塑料晶片具有低自身螢光的螢光光譜；(a)經組裝之塑料晶片(Pchip2)；(b)經組裝之塑料晶片(Pchip1)；(c)僅塑料覆蓋層；(d)玻璃晶片，1.4mm厚；(e)玻璃晶片，0.7mm厚；(f)僅塑料基板。Figure 9 is a graph showing the fluorescence spectrum of a plastic wafer having a low self-fluorescence compared to a typical glass separation wafer; (a) assembled plastic wafer (Pchip2); (b) assembled plastic wafer (Pchip1); ) only plastic cover; (d) glass wafer, 1.4 mm thick; (e) glass wafer, 0.7 mm thick; (f) plastic substrate only.

圖10為來自5色標記套組(ABI AmpFlSTR Identifiler套組)之等位基因階梯的等位基因呼叫概況(allele－called profile)；上至下：藍色、綠色、黃色、紅色、橙色檢測器信號。Figure 10 is an allele-called profile of the allelic ladder from the 5-color marker set (ABI AmpFlSTR Identifiler kit); top to bottom: blue, green, yellow, red, orange detector signal.

圖11為9947A人類基因組DNA之等位基因呼叫STR概況，上至下：藍色、綠色、黃色、紅色、橙色檢測器信號；全部概況在1.0 ng DNA模板上實現。Figure 11 shows the STR profile of the alleles of the 9947A human genomic DNA, top to bottom: blue, green, yellow, red, and orange detector signals; all profiles were performed on a 1.0 ng DNA template.

圖12顯示對於達480個bp而言R>0.4之解析度，證明單鹼基解析度達480個bp；上至下：藍色、綠色、黃色、紅色、橙色檢測器信號。Figure 12 shows a resolution of R > 0.4 for up to 480 bp, demonstrating a single base resolution of 480 bp; top to bottom: blue, green, yellow, red, orange detector signals.

圖13顯示藉由1個核苷酸分離之2個等位基因(THO1 9.3及10)之解析度。Figure 13 shows the resolution of two alleles (THO1 9.3 and 10) separated by one nucleotide.

圖14為pGEM片段之DNA排序分析；上至下：藍色、綠色、黃色及紅色檢測器信號。Figure 14 is a DNA sequencing analysis of pGEM fragments; top to bottom: blue, green, yellow, and red detector signals.

圖15為顯示pGEM片段之DNA排序分析的複合四個鹼基對圖。Figure 15 is a composite four base pair map showing DNA sequencing analysis of pGEM fragments.

圖16為用於直接電動樣品注射之晶片設計的分離示意圖，其顯示支撐體(上)及晶片(底)層。Figure 16 is a schematic exploded view of a wafer design for direct motor sample injection showing the support (top) and wafer (bottom) layers.

100‧‧‧晶片100‧‧‧ wafer

101‧‧‧陽極部分101‧‧‧Anode section

102‧‧‧陰極部分102‧‧‧ cathode part

103‧‧‧在陽極部分與陰極部分之間的一中心部分103‧‧‧ a central part between the anode part and the cathode part

104‧‧‧至少一個第一通孔104‧‧‧At least one first through hole

105‧‧‧至少一個第二通孔105‧‧‧At least one second through hole

106‧‧‧複數個微型流動通道106‧‧‧Multiple micro flow channels

107‧‧‧檢測窗口107‧‧‧Detection window

Claims (32)

一種用於分離及檢測以至少4種螢光染料標記之核酸片段的塑料電泳晶片，其包含：一具有頂面及底面之基板層，其進一步包含一陽極部分、一陰極部分及在該陽極部分與該陰極部分之間的中心部分，一具有頂面及底面之覆蓋層，藉此該基板層之頂面與該覆蓋層之底面黏結以形成至少一個微型流動通道，位於該陽極之第一通孔，其與至少一個微型流動通道處於流體連通中，位於該陰極之第二通孔，其與至少一個微型流動通道處於流體連通中，一檢測窗口，其包含塑料，該塑料係選自於由聚乙烯、聚(碳酸酯)、不飽和、部分不飽和或飽和環狀烯烴聚合物(COP)、不飽和、部分不飽和或飽和環狀烯烴共聚物(COC)及降冰片烯熱聚物組成之群組，且具有少於2mm之厚度，其中在電泳時，該核酸片段通過該檢測窗口並以大於3之信號雜訊比檢測。 A plastic electrophoretic wafer for separating and detecting a nucleic acid fragment labeled with at least four fluorescent dyes, comprising: a substrate layer having a top surface and a bottom surface, further comprising an anode portion, a cathode portion and the anode portion a central portion between the cathode portion and a cover layer having a top surface and a bottom surface, whereby a top surface of the substrate layer is bonded to a bottom surface of the cover layer to form at least one micro flow channel, the first pass of the anode a hole in fluid communication with the at least one micro flow channel, in a second through hole of the cathode, in fluid communication with the at least one micro flow channel, a detection window comprising plastic, the plastic being selected from Polyethylene, poly(carbonate), unsaturated, partially unsaturated or saturated cyclic olefin polymer (COP), unsaturated, partially unsaturated or saturated cyclic olefin copolymer (COC) and norbornene thermal polymer Groups, and having a thickness of less than 2 mm, wherein upon electrophoresis, the nucleic acid fragments pass through the detection window and are detected with a signal to noise ratio greater than three. 如請求項1之晶片，其中該檢測窗口之塑料具有少於約300μm之厚度。 The wafer of claim 1, wherein the plastic of the detection window has a thickness of less than about 300 μm. 如請求項1之晶片，其中該檢測窗口之塑料具有少於約500μm之厚度。 The wafer of claim 1, wherein the plastic of the detection window has a thickness of less than about 500 μm. 如請求項1之晶片，其中該檢測窗口之塑料具有1mm或少於1mm之厚度。 The wafer of claim 1, wherein the plastic of the detection window has a thickness of 1 mm or less. 如請求項1之晶片，其中各微型流動通道具有2cm至50cm之分離長度。 A wafer according to claim 1, wherein each of the micro flow channels has a separation length of from 2 cm to 50 cm. 如請求項1之晶片，其中各微型流動通道進一步包含一注射通道。 The wafer of claim 1, wherein each of the micro flow channels further comprises an injection channel. 如請求項1之晶片，其中當在約450與500nm之間的波長下激發時，該塑料不發波長在500與800nm之間的螢光。 The wafer of claim 1, wherein the plastic does not emit fluorescence having a wavelength between 500 and 800 nm when excited at a wavelength between about 450 and 500 nm. 如請求項7之晶片，其中在約488nm之波長下激發該塑料。 The wafer of claim 7, wherein the plastic is excited at a wavelength of about 488 nm. 如請求項1之晶片，其中以用於PCR擴增之核酸模板之單一副本開始，可以檢測片段尺寸測定應用所產生之核酸樣品中之複數種核酸物質。 The wafer of claim 1, wherein a plurality of nucleic acid species in the nucleic acid sample produced by the fragment size determination application can be detected starting with a single copy of the nucleic acid template for PCR amplification. 如請求項1之晶片，其中以用於PCR擴增之核酸模板之單一副本開始，可以檢測核酸排序應用所產生之核酸樣品中之複數個核酸物質。 The wafer of claim 1, wherein the plurality of nucleic acid species in the nucleic acid sample produced by the nucleic acid sequencing application can be detected starting with a single copy of the nucleic acid template for PCR amplification. 如請求項1之晶片，其中各微型流動通道進一步包含一表面塗層。 The wafer of claim 1, wherein each of the micro flow channels further comprises a surface coating. 如請求項11之晶片，其中該表面塗層為羥丙基甲基纖維素(HPMA)、聚(氧化乙烯)(PEO)、聚(乙烯醇)(PVA)、聚(二甲基丙烯醯胺)(PDMA)、聚(乙烯基吡咯啶酮)、二甲基丙烯醯胺(DMA)、二乙基丙烯醯胺(DEA)、聚(二乙基丙烯醯胺)或其混合物。 The wafer of claim 11, wherein the surface coating is hydroxypropylmethylcellulose (HPMA), poly(ethylene oxide) (PEO), poly(vinyl alcohol) (PVA), poly(dimethyl methacrylate) (PDMA), poly(vinylpyrrolidone), dimethyl methacrylate (DMA), diethyl acrylamide (DEA), poly(diethyl acrylamide) or a mixture thereof. 如請求項11之晶片，其中各該微型流動通道進一步包含篩選基質。 The wafer of claim 11, wherein each of the micro flow channels further comprises a screening matrix. 如請求項13之晶片，其中該篩選基質包含直鏈或交聯聚(N,N-二烷基丙烯醯胺)、直鏈聚丙烯醯胺、聚二甲基丙烯醯胺、聚乙烯基吡咯啶酮或其組合。 The wafer of claim 13, wherein the screening substrate comprises a linear or cross-linked poly(N,N-dialkyl acrylamide), a linear polydecylamine, a polydimethyl acrylamide, a polyvinylpyrrole Pyridone or a combination thereof. 如請求項14之晶片，其中該篩選基質包含1-50wt%之聚丙烯醯胺。 The wafer of claim 14, wherein the screening matrix comprises from 1 to 50% by weight of polyacrylamide. 如請求項1之晶片，其進一步包含至少一個陰極孔，且在各陰極孔與各對應之微型流動通道之間進一步包含一多孔層，其中該多孔層能夠實質上阻斷氣泡自該等陰極孔進入各微型流動通道中。 The wafer of claim 1, further comprising at least one cathode aperture, further comprising a porous layer between each cathode aperture and each of the corresponding micro flow channels, wherein the porous layer is capable of substantially blocking bubbles from the cathodes The holes enter each of the micro flow channels. 如請求項16之晶片，其中該多孔層包含玻璃料、聚合物玻璃料、聚合物膜或聚合物過濾器。 The wafer of claim 16, wherein the porous layer comprises a frit, a polymer frit, a polymer film, or a polymer filter. 如請求項1之晶片，其中可在核酸樣品之電泳分析後，在單鹼基解析度下檢測該核酸樣品中之複數種核酸物質。 The wafer of claim 1, wherein the plurality of nucleic acid species in the nucleic acid sample are detectable at a single base resolution after electrophoretic analysis of the nucleic acid sample. 如請求項1之晶片，其包含複數個微型流動通道，其中該複數個微型流動通道中之每一者進一步包含一用於同時注射複數個要分析之樣品之注射器，每一個樣品係注射至該複數個微型流動通道中之每一者。 The wafer of claim 1, comprising a plurality of micro flow channels, wherein each of the plurality of micro flow channels further comprises a syringe for simultaneously injecting a plurality of samples to be analyzed, each sample being injected into the sample Each of a plurality of micro flow channels. 如請求項1之晶片，其中該第一通孔及第二通孔係包含於該覆蓋層中。 The wafer of claim 1, wherein the first through hole and the second through hole are included in the cover layer. 如請求項1之晶片，其中該檢測窗口之塑料具有約25μm至2000μm之厚度。 The wafer of claim 1, wherein the plastic of the detection window has a thickness of about 25 μm to 2000 μm. 一種裝置，其包含一支撐體，其包含第二檢測窗口；，及 如請求項1之晶片，其中該晶片係附著於該支撐體，且其中該第二檢測窗口與該晶片之檢測窗口之位置重疊。 A device comprising a support comprising a second detection window; The wafer of claim 1, wherein the wafer is attached to the support, and wherein the second detection window overlaps with a position of the detection window of the wafer. 如請求項22之裝置，其中該第一通孔及第二通孔係包含於該覆蓋層中。 The device of claim 22, wherein the first through hole and the second through hole are included in the cover layer. 一種同時電泳分離及檢測複數個樣品之方法，其包含將複數個樣品提供至如請求項1之包含複數個微型流動通道之晶片上的複數個微型流動通道中之每一者；跨越該複數個微型流動通道施加電位以將包含該複數個分析樣品之每一者的可檢測物質分離；在該晶片之檢測窗口檢測包含該複數個分離樣品之該等可檢測物質中之每一者。 A method for simultaneous separation and detection of a plurality of samples by electrophoresis, comprising providing a plurality of samples to each of a plurality of microfluidic channels on a wafer comprising a plurality of microfluidic channels of claim 1; spanning the plurality of The micro flow channel applies a potential to separate a detectable substance comprising each of the plurality of analytical samples; each of the detectable substances comprising the plurality of separated samples is detected at a detection window of the wafer. 如請求項24之方法，其進一步包含將跨越該複數個微型流動通道中之每一者之電場維持實質上相同的步驟。 The method of claim 24, further comprising the step of maintaining substantially the same electric field across each of the plurality of microfluidic channels. 如請求項25之方法，其中藉由平衡該複數個微型流動通道中之每一者之各部分的電阻而使跨越該複數個微型流動通道中之每一者之電場維持實質上相同。 The method of claim 25, wherein the electric field across each of the plurality of microfluidic channels is maintained substantially the same by balancing the electrical resistance of each of the plurality of microfluidic channels. 如請求項24之方法，其中該等可檢測物質包含核酸。 The method of claim 24, wherein the detectable substances comprise nucleic acids. 如請求項27之方法，其中該等可檢測物質包含官能性連接於該等核酸之染料。 The method of claim 27, wherein the detectable substances comprise a dye functionally linked to the nucleic acids. 一種用於分離及檢測以至少4種螢光染料標記之核酸片段的系統，其包含(i)一塑料電泳晶片，其包含：一具有頂面及底面之基板層，其進一步包含一陽極部 分、一陰極部分及在該陽極部分與該陰極部分之間的中心部分，一具有頂面及底面之覆蓋層，藉此該基板層之頂面與該覆蓋層之底面黏結以形成至少一個微型流動通道，位於該陽極之第一通孔，其與至少一個微型流動通道處於流體連通中，位於該陰極之第二通孔，其與至少一個微型流動通道處於流體連通中，一檢測窗口，其包含塑料，該塑料係選自於由聚乙烯、聚(碳酸酯)、不飽和、部分不飽和或飽和環狀烯烴聚合物(COP)、不飽和、部分不飽和或飽和環狀烯烴共聚物(COC)及降冰片烯熱聚物組成之群組，且具有少於2mm之厚度，及(ii)一電泳儀器，其包含一電力系統及一光學系統，該電力系統具有一陽極及一陰極，該陽極與該基板層之陽極部分接觸，且該陰極與該基板層之陰極部分接觸，藉此當該電力系統由該陰極傳送電壓至該陽極時，可將該經標記之核酸片段由該陰極部分移動至該檢測窗口，其中該經標記之核酸片段係由該光學系統以大於3之信號雜訊比檢測，且該核酸樣品中之複數種核酸物質可在該核酸樣品之電泳分析後在單鹼基解析度下檢測。 A system for separating and detecting nucleic acid fragments labeled with at least four fluorescent dyes, comprising: (i) a plastic electrophoretic wafer comprising: a substrate layer having a top surface and a bottom surface, further comprising an anode portion a portion, a cathode portion, and a central portion between the anode portion and the cathode portion, a cover layer having a top surface and a bottom surface, whereby a top surface of the substrate layer is bonded to a bottom surface of the cover layer to form at least one micro a flow channel, located in the first through hole of the anode, in fluid communication with the at least one micro flow channel, in the second through hole of the cathode, in fluid communication with the at least one micro flow channel, a detection window, Containing a plastic selected from the group consisting of polyethylene, poly(carbonate), unsaturated, partially unsaturated or saturated cyclic olefin polymers (COP), unsaturated, partially unsaturated or saturated cyclic olefin copolymers ( a group of COC) and a norbornene thermal polymer, and having a thickness of less than 2 mm, and (ii) an electrophoresis apparatus comprising an electrical system and an optical system having an anode and a cathode, The anode is in contact with an anode portion of the substrate layer, and the cathode is in contact with a cathode portion of the substrate layer, whereby the labeled core can be used when the power system transmits a voltage from the cathode to the anode The acid fragment is moved from the cathode portion to the detection window, wherein the labeled nucleic acid fragment is detected by the optical system at a signal noise ratio greater than 3, and a plurality of nucleic acid species in the nucleic acid sample are available in the nucleic acid sample After electrophoresis analysis, it was detected at a single base resolution. 一種用於分離及檢測以至少4種螢光染料標記之核酸片段的塑料電泳晶片，其包含：一具有頂面及底面之基板層，其進一步包含一陽極部 分、一陰極部分及在該陽極部分與該陰極部分之間的中心部分，一具有頂面及底面之覆蓋層，藉此該基板層之頂面與該覆蓋層之底面黏結以形成至少一個微型流動通道，位於該陽極之第一通孔，其與至少一個微型流動通道處於流體連通中，位於該陰極之第二通孔，其與至少一個微型流動通道處於流體連通中，一檢測窗口，其包含具有厚度少於2mm之塑料，其中在經電泳分析後，可以大於3之信號雜訊比且單鹼基對檢測用於碎片尺寸測量應用之核酸樣品中之複數個核酸種類。 A plastic electrophoretic wafer for separating and detecting nucleic acid fragments labeled with at least four fluorescent dyes, comprising: a substrate layer having a top surface and a bottom surface, further comprising an anode portion a portion, a cathode portion, and a central portion between the anode portion and the cathode portion, a cover layer having a top surface and a bottom surface, whereby a top surface of the substrate layer is bonded to a bottom surface of the cover layer to form at least one micro a flow channel, located in the first through hole of the anode, in fluid communication with the at least one micro flow channel, in the second through hole of the cathode, in fluid communication with the at least one micro flow channel, a detection window, A plastic having a thickness of less than 2 mm is included, wherein after electrophoretic analysis, a plurality of nucleic acid species in a nucleic acid sample for fragment size measurement applications can be detected with a signal to noise ratio greater than three and a single base pair. 如請求項30之晶片，其中各微型流動通道進一步包含一表面塗層。 The wafer of claim 30, wherein each of the micro flow channels further comprises a surface coating. 如請求項31之晶片，其中該表面塗層為羥丙基甲基纖維素(HPMA)、聚(氧化乙烯)(PEO)、聚(乙烯醇)(PVA)、聚(二甲基丙烯醯胺)(PDMA)、聚(乙烯基吡咯啶酮)、二甲基丙烯醯胺(DMA)、二乙基丙烯醯胺(DEA)、聚(二乙基丙烯醯胺)或其混合物。 The wafer of claim 31, wherein the surface coating is hydroxypropyl methylcellulose (HPMA), poly(ethylene oxide) (PEO), poly(vinyl alcohol) (PVA), poly(dimethyl methacrylate) (PDMA), poly(vinylpyrrolidone), dimethyl methacrylate (DMA), diethyl acrylamide (DEA), poly(diethyl acrylamide) or a mixture thereof.
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