TW201017163A - Apparatus for identifying a single biomolecule - Google Patents

Abstract

A bioassay system is disclosed. The bioassay system may include a plurality of optical detection apparatuses, each of which includes a substrate having a light detector, and a linker site formed over the light detector, the linker site being treated to affix the biomolecule to the linker site. The linker site is proximate to the light detector and is spaced apart from the light detector by a distance of less than or equal to 100 micrometers. The light detector collects light emitted from the biomoiecule within a solid angle of greater than or equal to 0.8 SI steridian. The optical detection apparatus may further include an excitation light source formed over the substrate so as to provide light source for exciting a fluorophore attached to the biomolecule.

Description

201017163 六、發明說明： 【發明所屬之技術領域】 本發明係關於一包括複數個光學偵測裝置之生物分析 系統及用於偵測與分析生物分子之生物分析系統的使用， 而生物分子，例如核酸。在一實施例中，本發明係關於一 生物分析系統，其包括至少一萬個之光學偵測裝置用以監 測，在一些實施例中’大量的螢光團（fluorophore)分子用以 平行地偵測與分析生物分子。 【先前技術】 人類基因體計畫（The Human Genome Project，HGP)刺 激定序處理量的大量增加且導致定序成本相對降低。與每 個基因體13年及近三十億美元之成本相比，定序成本已顯 著降低一確切地，兩個獨立之基因體最近已被完成 (McGuire et al.，317:1687 (2007))。個人基因體代表 在對於病患與健康照護提供者兩者之醫學治療中的典範轉 移(Paradigm shift)。藉由管理疾病之基因風險因子，健康照 護提供者可更立即實施預防性藥物與提供定做治療。由於 具有經完成之基因體大銀行，藥物設計與投藥可為更加有 效’將藥物基因學(pharmacogenetics)的初期領域往前推進。 為了使對於個人之定做藥物普及化，美國國家衛生研 究院（NIH)國家人類基因研究所(NHGRI)確定了每個基因 體定序成本自千萬降低到近一千美元之基準。然而，一般 V, 一、· 南通量（high-throughput)毛細電泳與自動化基因體定序 技術不能滿足對於個人基因體定序之增加的需求。此外， 201017163 現灯之&序方法需要複雜與錯誤傾向（e__p·)之影像 擷取與分析㈣。例如’為了獲得多重影像，許多現行技 術需要不是陣列就是偵測系統來移動。之後可將所產生之 影像依序排列、對齊與分析。影像棘、處理與分析步驟 皆為傾向於錯誤、花費額外時間且需要昂貴的設備。相反 地，不包括移動絲之現行系_常被限制 ，由於不大多 數目之價測單。最後’現行元件不將要被制之分子置 於接近鄰近於-對應之偵測單元，其實質上限制了债測訊 號的強度。 因此’對於減少核酸定序成本之裝置的需要存在。為 了達到“$麵基因體，，範例，元件需能平行定序多個分 子、、具有_化之設計與製造製程及避免需要現行元件與 方法之複雜與錯誤傾向的掃_與影像分析處理。另外，元 件需要具有定序單一分子的能力以避免於群集定序 (clustered sequencing)方法之擴大（例如介於理想複製模板 之序列間的飄移）與定序（在定序模板間之逐步定序反應 (stepwise sequencing reaction)的去相（dephasing))兩者步驟 中之已知不同時期的困難。 【發明内容】 本發明實施例提供一包括複數個光學偵測裝置之生物 分析系統及使用用於核酸偵測之生物分析系統的方法，例 如定序。實施例所提供之生物分析系統具有大規模平行定 序反應（large-scale parallel sequencing reaction)之能力， 201017163 即，同時將大量不同的核酸模板進行定序。各定序反應使 用一單一分子為模板（即’單一分子定序）。所提供之元 件也具有經簡化之設計-排除了需要目前元件之複雜、昂貴 與錯誤傾向的掃猫與彳貞測步驟。本發明所提供之經簡化之 設計與系統功能，係部分基於要被偵測之核酸所附著（不 是直接就是例如’藉由一聚合酶分子）之連結位（linker Site) 的直接聯繫(direct correspondence)與一或多個摘測單元（例 如’光偵測器），及部分基於介於連結位與債測單元間之 短距離。在一些實施例中，藉由一偵測之大的立體角（s〇lid angle)顯示了介於核酸與偵測單元間的此短距離。 在一方面，提供了在一偵測單元鑑定一單一生物分子 的生物分析系統。生物分析系統可包括複數個光學偵測裝 置，各裝置可包括一具一光偵測器之基板，基板被設置來 偵測散發自生物分子的光。在一些實施例中，光偵測器可 包括電荷耦合元件（charged-coupled device)、互補金屬氧化 半導體（complementary metal-oxide semiconductor, CMOS) 感測器或光二極體(photodiode)其中之一。光學j貞測裝置可 更包括—遮蔽薄板(blind sheet)於基板上，遮蔽薄板包括具 有一直杈的一針孔，及一濾層被提供於遮蔽薄板與基板之 間且被提供於針孔之下，濾層為非平面且被設置來過濾散 發自生物分子的光。在—些實施例中，非平面㈣之部分 可為弧線形、球形、橢圓形或弓形。光學偵測裝置可更包 括一連結位，其被提供接近於針孔，連結位被處理以將該 生物分子置於接近針孔。 在另一方面，提供了在一偵測單元鑑定一單一生物分 201017163 子的生物分析系統。生物分析***可包括複數個光學偵測 裝置，各裝置可包括一具一光偵測器之基板，基板被設置 來偵測散發自生物分子的光。光學偵測裝置可更包括一光 散發層形成於基板上，其中光散發層包括一凹處，及包括 一濾層，其被提供於凹處與基板之間且於一針孔之下，濾 層為非平面且被設置來過濾散發自生物分子的光。在一些 實施例中’非平面濾層之部分可為弧線形、球形、橢圓形 或弓形。光學偵測裝置也可包括—連結位(linker site)，其 被提供接近於凹處與針孔，連結位被處理以將生物分子置 於接近針孔。光學偵測裝置可更包括與基板結合之一激發 光源以提供一用以激發一附著於生物分子之螢光團 (fluorophore)的光源。 在另一方面’另一實施例提供一藉由連結至少一核酸 至一實施例所提之光學偵測裝置的一連結位（不是直接 地’就是藉由與結合至連結位之核酸聚合酶結合）並在— 對應之光彳貞測器上偵測核酸之债測核酸的方法。在一些實 施例中’偵測核酸藉由與，例如一經標誌(labeled)的探針雜 合。在一些實施例中，藉由於光學偵測裝置上執行核酸定 序來偵測核酸。在一些實施例中，核酸定序方法係擇自驗 基延伸定序(base-extension sequencing)、末端標諸碟酸鹽定 序（terminally-labeled phosphate sequencing)與擺動定序 (wobble sequencing)。在其他實施例中，定序反應為一驗基 延伸定序反應。在其他實施例中，鹼基延伸定序反應更包 括將經阻礙(blocked)與經標諸、之核苷酸加至光學偵測巢置 的步驟。在其他實施例中，核苷酸為經螢光標誌的。 201017163 在另一方面，實施例也提供偵測一樣本分子的方法。 在一些實施例中，這些方法包括將經標誌之樣本附著至本 發明所提供之光學偵測裝置上的連結位及在一對應之光偵 測器上偵測樣本分子的步驟。在一些實施例中，樣本分子 藉由一連結分子附著至一連結位。在一些實施例中，連結 分子包括適合與樣本分子結合之捕獲分子（capture molecule)與2) 一核酸標籤。在其他實施例中，將樣本分 子提供於實施例所提供之連結分子已附著於連結位上的光 ^偵測裝置。在其他實施射，允許—樣本分子與一連結 刀子。且之後將結合複合物提供於光學 許其附著至-連結位。在其他實施财，樣本分子^並^ 物分子，例如—多胜肽、核酸、脂質、 ： (metabolite)。 / 代謝物 目的、特禮丈、和優點能更 ，並配合所附圖示，作詳 為了讓本發明之上述和其他 明顯易懂，下文特舉較佳實施例 細說明如下： 【實施方式】 1·生物分析系統 生物分析系統可用來平行監測大量（例如， 施例中，大於_〇〇)之單一分子。生物分析=實 複數個光學偵測裝置。各奸偵測裝置可檢測出於=括 子上之螢光__Ph⑽)的存在，藉由偵測散發自=1 的光子。藉由平行操作光學偵測裝置，生物分析系鸷光團 可 201017163 高通量確認，例如一基因體之序列或於一組織樣本中被表 現之基因的輪廓(pr〇fne)。 參見第1圖，顯示一生物分析系統1。生物分析系統i 可包括生物分析基板1〇與複數個光學偵測裝置20形成於 基板10上。可獨立操作各光學偵測裝置2〇以偵測並鑑定 附著於其之一單一生物分子。例如，藉由連續執行一鹼基 延伸與使用光學偵測震置20偵測散發自與經延伸之驗基 結合之一螢光團的光可確認一單股DNA之序列。藉由將大 • 量之光學偵測裝置20整合於基板10上，可平行偵測並確 認大量之單一生物分子。根據設計選擇，生物分析系統1 可包括至少’例如一萬（1〇,〇〇〇)、二十五萬（250,000)、二百 萬（2,000,000)、或甚至一千萬（ι〇,〇〇〇,〇〇〇)或更多個光學偵 測裝置20形成於基板1〇上。 生物分析系統1可更包括一與基板10耦合之偵測與記 錄系統2，用以控制光學偵測裝置20的操作與用以記錄擷201017163 VI. Description of the Invention: [Technical Field] The present invention relates to a bioanalytical system including a plurality of optical detecting devices and a bioanalytical system for detecting and analyzing biomolecules, for example, biomolecules, for example Nucleic acid. In one embodiment, the present invention is directed to a bioanalysis system that includes at least 10,000 optical detection devices for monitoring, in some embodiments, 'a large number of fluorophore molecules are used to detect in parallel Measuring and analyzing biomolecules. [Prior Art] The Human Genome Project (HGP) stimulates a large increase in the amount of sequencing processing and leads to a relatively low ordering cost. Compared to the cost of 13 years and nearly $3 billion per genomic body, the cost of sequencing has been significantly reduced. To be exact, two separate genomes have recently been completed (McGuire et al., 317:1687 (2007) ). The personal genome represents a paradigm shift in medical treatment for both patients and health care providers. By managing the genetic risk factors for disease, health care providers can implement preventive drugs and provide customized treatments more immediately. Thanks to the completed genomic banks, drug design and drug delivery can be more effective in advancing the initial field of pharmacogenetics. In order to popularize custom-made drugs for individuals, the National Institute of Human Genetics (NIH) National Human Genome Research Institute (NHGRI) has determined that the cost per sequencing of each gene has been reduced from a few million to nearly a thousand dollars. However, in general, V, I, · high-throughput capillary electrophoresis and automated genomic sequencing techniques do not meet the increased demand for individual genome sequencing. In addition, the 201017163 current light & method requires complex and error-prone (e__p·) image capture and analysis (4). For example, in order to obtain multiple images, many current technologies require either an array or a detection system to move. The resulting images can then be arranged, aligned and analyzed sequentially. Image spines, processing, and analysis steps are all prone to errors, extra time, and expensive equipment. Conversely, the current system that does not include moving silk is often limited because it does not have a large number of price orders. Finally, the current component does not place the molecule to be fabricated close to the adjacent-corresponding detection unit, which substantially limits the strength of the debt measurement signal. Therefore, there is a need for a device that reduces the cost of nucleic acid sequencing. In order to achieve the "$ genomic body, the paradigm, the components need to be able to sequence multiple molecules in parallel, have a design and manufacturing process, and avoid sweeping and image analysis processing that requires the complexity and error of the current components and methods. In addition, components need to have the ability to sequence a single molecule to avoid amplification of clustered sequencing methods (eg, drift between sequences of ideal replica templates) and sequencing (step-by-step sequencing between sequencing templates) The dephasing of the stepwise sequencing reaction is difficult in the known different periods. The invention provides a biological analysis system including a plurality of optical detecting devices and is used for Methods of bioassay systems for nucleic acid detection, such as sequencing. The bioanalytical systems provided in the examples have the capability of large-scale parallel sequencing reactions, 201017163, ie, a large number of different nucleic acid templates simultaneously Sequencing. Each sequencing reaction uses a single molecule as a template (ie, 'single molecule sequencing'). The components are also provided with a simplified design - eliminating the need for sweeping cats and speculations that require the complexity, cost, and error of the current components. The simplified design and system functionality provided by the present invention is based in part on being detected The nucleic acid to which the nucleic acid is attached (not directly, for example, a direct link to a linker site by a polymerase molecule) and one or more excision units (eg, a 'photodetector), and Partly based on the short distance between the link and the debt detection unit. In some embodiments, this short between the nucleic acid and the detection unit is indicated by a detected large s〇lid angle In one aspect, a biometric system for identifying a single biomolecule in a detection unit is provided. The bioanalysis system can include a plurality of optical detection devices, each device can include a substrate with a photodetector, the substrate Configuring to detect light emitted from biomolecules. In some embodiments, the photodetector can include a charge-coupled device, a complementary metal oxide semiconductor (com A plementary metal-oxide semiconductor (CMOS) sensor or one of a photodiode. The optical j-measuring device may further include a blind sheet on the substrate, the masking sheet including a needle having a constant flaw A hole, and a filter layer are provided between the masking sheet and the substrate and are provided below the pinhole, the filter layer being non-planar and arranged to filter light emitted from the biomolecule. In some embodiments, non-planar Part (4) may be curved, spherical, elliptical or arcuate. The optical detection device can further include a link that is provided proximate to the pinhole and the link is processed to place the biomolecule close to the pinhole. In another aspect, a bioanalytical system for identifying a single biological sub-item 201017163 is provided in a detection unit. The bioanalysis system can include a plurality of optical detection devices, each of which can include a substrate having a photodetector disposed to detect light emanating from the biomolecule. The optical detecting device may further include a light emitting layer formed on the substrate, wherein the light emitting layer includes a recess, and includes a filter layer provided between the recess and the substrate and under a pinhole, filtering The layers are non-planar and are arranged to filter light that is emitted from biomolecules. In some embodiments the portion of the 'non-planar filter layer can be curved, spherical, elliptical or arcuate. The optical detection device can also include a linker site that is provided proximate to the recess and the pinhole that is processed to place the biomolecule close to the pinhole. The optical detection device can further include an excitation light source coupled to the substrate to provide a light source for exciting a fluorophore attached to the biomolecule. In another aspect, another embodiment provides a link (not directly) by binding at least one nucleic acid to an optical detection device of an embodiment by combining with a nucleic acid polymerase that binds to a linker And a method of detecting the nucleic acid of the nucleic acid on the corresponding photodetector. In some embodiments, the detection nucleic acid is hybridized with, for example, a labeled probe. In some embodiments, the nucleic acid is detected by performing nucleic acid sequencing on the optical detection device. In some embodiments, the nucleic acid sequencing method is selected from the group consisting of base-extension sequencing, terminally-labeled phosphate sequencing, and wobble sequencing. In other embodiments, the sequencing reaction is an experimental extension of the sequencing reaction. In other embodiments, the base extension sequencing reaction further includes the step of adding blocked and labeled nucleotides to the optical detection nest. In other embodiments, the nucleotides are fluorescently labeled. 201017163 In another aspect, the embodiments also provide methods for detecting the same molecule. In some embodiments, the methods include the step of attaching the marked sample to a link on the optical detection device provided by the present invention and detecting sample molecules on a corresponding photodetector. In some embodiments, the sample molecules are attached to a linking site by a linker molecule. In some embodiments, the linker molecule comprises a capture molecule suitable for binding to the sample molecule and 2) a nucleic acid tag. In other embodiments, the sample molecules are provided to the photodetecting device to which the linking molecules provided in the examples have been attached to the linking sites. In other implementations, the allowable - sample molecules are linked to a knife. The binding complex is then provided to the optical attachment to the -linking position. In other implementations, the sample molecule is a molecule of, for example, a multi-peptide, a nucleic acid, a lipid, or a metabolite. The metabolites, the features, and the advantages of the metabolites, and the accompanying drawings, in order to make the above and other aspects of the present invention obvious and obvious, the following detailed description of the preferred embodiments are as follows: 1. Bioanalytical System The Bioanalytical System can be used to parallel monitor a large number (eg, in the example, greater than _〇〇) of a single molecule. Bioanalysis = Real Multiple optical detection devices. The detection device can detect the presence of the fluorescent __Ph(10) on the brackets by detecting photons emitted from =1. By operating the optical detection device in parallel, the bioanalytical system can be confirmed by high throughput, such as the sequence of a gene or the outline of a gene (pr〇fne) expressed in a tissue sample. Referring to Figure 1, a bioanalysis system 1 is shown. The bioanalysis system i may include a bioanalytical substrate 1A and a plurality of optical detecting devices 20 formed on the substrate 10. Each optical detection device can be operated independently to detect and identify a single biomolecule attached to it. For example, the sequence of a single strand of DNA can be confirmed by performing one base extension continuously and detecting the light emitted from one of the fluorophores bound to the extended assay using the optical detection transducer 20. By integrating a large number of optical detecting devices 20 on the substrate 10, a large number of single biomolecules can be detected and confirmed in parallel. Depending on the design choice, the bioanalysis system 1 may include at least 'for example, 10,000 (1, 〇〇〇), 250,000 (250,000), 2 million (2,000,000), or even 10 million (ι〇, 〇〇 〇, 〇〇〇) or more optical detecting devices 20 are formed on the substrate 1A. The biometric system 1 can further include a detection and recording system 2 coupled to the substrate 10 for controlling the operation of the optical detection device 20 and for recording defects.

取自光學偵測裝置20的資料。此外，生物分析系統1可更 A 胃包括一激發光源（未顯示）。激發光源可產生激發光，以 便誘導螢光團散發螢光。在一實施例中，激發光源可離開 光學偵測裝置20或生物分析基板10單獨設置。在替代實 施例中，可將激發光源與光學偵測裝置20或生物分析基板 10進行整合。 於此實施例中，如第1圖中所示，當自上方觀看時， 光學偵測裝置20可具有一圓形的形狀。可以瞭解的是，光 學偵測裝置20可具有其他幾何形狀，例如一正方形的形 狀、一多邊形的形狀、一卵形的形狀與其類似。另外，第 201017163 1圖顯示複數個光學偵測裝置20被配置於一方格圖案中。 可以瞭解的是，光學偵測裝置20可被配置於其他圖案中’ 例如三角格圖案、一蜂巢格圖案與其類似。 由於生物分析系統1之複數個光學偵測裝置20為玎獨 立地操作，將於以下依照各種實施例描述要被使用之，光 學偵測裝置20。雖然將描述一光學偵測裝置20,但可理解 的是’於生物分析系統1中不同之光學偵測裝置20沒有必 要相同。根據設計選擇，根據不同實施例可建構不同形式 之光學偵測裝置20。 參見第2圖，其顯示沿著第1圖之線A-A，依照〆實 施例之光學偵測裝置20的一剖面圖。如第2圖中所示，光 學偵測裝置20包括一光偵測器210形成於基板10上與/ 連結位220形成於光偵測器210上。此外，光學偵測装ί 20可更包括一控制電路215形成於基板1〇上，用以控制 光感測器210之操作。控制電路215可與偵測與記錄系統 2耦合以便接受來自偵測與記錄系統2之控制指示並傳送 經偵測之訊號至偵測與記錄系統2。在一些實施例中，蒸 板10可為一玻璃基板、一半導體基板（例如，石夕）或〆塑 膠基板。在一些實施例中，一或更多之控制電路215町相 當於各光偵測器210。 在一些實施例中’光偵測器210可包括一單一光電導 (photoconductive)光子偵測器或一組之光電導光子偵測 器，例如一或更多之電荷麵合元件（charged-coupled device) 或互補金屬氧化半導體（complementary metal-oxide semiconductor, CMOS)感測器。在替代實施例中，光偵測器 201017163 210可包括一單一光電壓（photovoltaic)光子偵測器或一組 之光電壓光子偵測器。在替代實施例中，光偵測器210可 包括一皁一累崩光二極體（avalanche photodiode)或一組之 累崩光二極體。在替代實施例中，光偵測器210可包括一 單一光電晶體(phototransistor)或一組之光電晶體。 在一實施例中’光學偵測裝置20可更包括一遮蔽薄板 (blind sheet)230於光偵測器210上。遮蔽薄板230可包括 一針孔235。在一實施例中，針孔235可具有一圓形之形 ❹ 狀且可具有小於或等於10,000、1,000、500、300、200、 150或100 nm之一直徑D1。可以理解的是，針孔235可 具有其他形狀，例如一卵形之形狀、一正方形之形狀與其 類似。在一實施例中’遮蔽薄板230可包括一不透光材料， 以便阻擋不期望之光到達光偵測器210。因此，所期望之 光線可經由針孔235到達光偵測器210。 可將連結位(linker site)220形成接近於針孔235。於顯 示在此圖中之實施例中，連結位220形成於針孔235之内。 在一實施例中，藉由一小於或等於1 〇〇 μιη之距離H1可將 形成接近於針孔235之連結位220與光偵測器210隔開。 在替代實施例中，距離Η1可小於或等於75、50、25、15、 10、5 或 3 μιη。 光學偵測裝置20可更包括一濾層240 (光學）與一微 透鏡(microlens)250 (光學）介於光偵測器210與遮蔽薄板 230之間。雖然第2圖顯示濾層240係形成在微透鏡250 上，可以理解的是’濾層240可形成在微透鏡250之下。 在一些實施例中，濾層240可包括一單一透明層或複數個 11 201017163 具有不同折射率(refractive index)之透明次層（sublayer)。 如於第3a圖中所示，於一些實施例中，複數個次層可 為非平面。非平面可包括，但不限於，半球形、橢圓形、 弓形或除此之外弧線形之形狀及/或表面。當 濾、層240包括 複數個弧線形次層時，藉由連續沈積次層於基板1〇上可形 成^慮層240 °於操作中，複數個弧線形次層可具有光學傳 送特性’其為一特定波長或波長範圍之高度地反射及有關 於一不同波長或波長範圍之逆向地傳達。例如，於一實施 例中之複數個弧線形濾膜可反射波長 633 nm之光，而發射 ❿ 具有680 nm之波長的光。在一些實施例中，可將一具有較 南折射率之次層失在具有較低之折射率之兩次層之間。或 者’可將一具有較低折射率之次層夾在具有較高之折射率 之兩次層之間。在一些實施例中，濾層240可包括具有單 一區域的一層或具有複數個次區域的一層，而複數個次區 域具有對不同波長範圍不同之透明度。 回到第2圖，可對連結位22〇進行處理以使生物分子 30附著於其。在一實施例中，生物分子30可包括一單股 · DNA分子32及與單股DNA分子32結合之一末端引子(end primer)34。藉由末端連結引子34，生物分子30可附著於 連結位220。再者，可以一螢光團36來標誌DNA分子32。 當被一第一波長λΐ之激發光激發時，螢光團36可散發一 第二波長λ2之螢光。在一些實施例中，第一波長λΐ比第 二波長λ2短。在一些實施例中’第一波長λΐ比第二波長 λ2長’例如於多光子激發(multi_photon excitation)中。之後 光偵測器210偵測散發自螢光團36之螢光以便鑑定螢光團 12 201017163 36所附著之鹼基形式，藉此連續地測定DNA分子32的序 列。 參見第3b圖，其顯示根據一實施例之光學偵測裝置 20的一剖面圖。如於第3b圖中所示’遮蔽薄板230形成 於光彳貞測器210之上，且藉由距離H1與光感測器210垂 直地間隔。具有厚度T之遮蔽薄板230包括具有半徑R1 (即’直徑D1之一半）之針孔235。於此實施例中，連結 位220可形成於針孔235中以與一生物分子（未顯示）結 • 合。 當螢光團36設置在位於連結位220上之一第一位置 36A且藉由一距離H2與連結位220分開時，具有半徑R2 之光偵測盗210可在一第一立體角（s〇Hd angie)Q 1内收集散 發自螢光團36的螢光。當螢光團36設置在一第二位置36B 且幾乎與連結位220接觸（即，距離H2接近零或小於1 pm) 時，光偵測器210之後可在一第二立體角θ2内收集散發自 螢光團36的螢光。第二立體角Θ2大於第一立體角Θ1且提 供一實質上較強的訊號。 為了使光偵測器210經由針孔235暴露於散發自螢光 團36之螢光’光偵測器21〇之半徑r2必須大於或等於相 當於投射在光偵測器210上表面上的第二立體角02的半 徑。藉由使遮蔽薄板230 (或連結位220 )更接近光偵測器 210 (即，藉由減少距離Η1)，光偵測器之後可自一立體 角内收集更集中之光（即，一較強之光訊號）。在一實施 例中’藉由一小距離Η1來將遮蔽薄板230(或連結位220) 與光偵測器210分開，以使第二立體角θ2為至少〇 8 SI球 13 201017163 面度（steradian)。 參見第4圖，其顯示沿著第1圖之線A-A，依照另一 實施例之光學偵測裝置20的一剖面圖。於此實施例中，將 一激發光源40與光學偵測裝置20進行整合。如第4圖中 所示，激發光源40形成於光學偵測裝置20之遮蔽薄板230 之上。在一實施例中，激發光源40可包括一 p型與一 η型 半導體層（410與430)，及一光散發層420介於層410與 層430間的接面區之間。層410與層430可連接至一電源。 根據用於層410、420與430之材料及/或材料之物理與原 ❹ 子結構，激發光源40可為一發光二極體(light emitting diode, LED)、一發光雷射二極體(light emitting laser diode，LD)、 一有機發光二極體(organic light emitting diode, OLED)或一 高分子發光二極體(polymer light emitting diode, PLED)。無 機材料’例如、神化鎵、填化銦、錄化鎵與氮化鎵，或有 機材料，例如具有聚對苯乙烯 poly(para-pheneylene-vinylene)骨架之共輛高分子 (conjugated polymer) ’皆為可用來製造散發光之接面二極 φ 體(junction diode)之半導體材料的例子。 在其他實施例中，激發光源40可形成遮蔽薄板230或 可被形成於遮敝薄板230之内。在一些實施例中，與光學 偵測裝置20整合之激發光源4〇可散發一波長帶或複數個 波長帶的光。激發光源40可間歇或持續地散發光。激發光 源40可一次散發一波長帶的光或者同時散發數個波長帶 的光。 再次參見第4圖’激發光源40可包括一凹處450於其 14 201017163 一中央部分以便露出針孔235。在此實施例中，連結位22〇 可不形成於針孔235中。當然，連結位22〇可形成於凹處 450中且接近於針孔235。在實施例中，其中激發光源4〇 形成遮蔽薄板230或被形成於遮蔽薄板23〇之内，凹處45〇 形成針孔235或被形成於針孔235之内。在一些實施例中， ^孔放可形成在層41〇與遮蔽薄板23〇兩者之一中央部 藉由例如，使用適合的製程來餘刻層41 〇盥遮蔽薄 板230 。 八The data taken from the optical detecting device 20. In addition, the bioanalysis system 1 can include an excitation source (not shown). The excitation source produces excitation light to induce the fluorophore to emit fluorescence. In one embodiment, the excitation source can be separately disposed from the optical detection device 20 or the bioanalytical substrate 10. In an alternate embodiment, the excitation source can be integrated with optical detection device 20 or bioanalytical substrate 10. In this embodiment, as shown in FIG. 1, the optical detecting device 20 may have a circular shape when viewed from above. It will be appreciated that the optical detection device 20 can have other geometric shapes, such as a square shape, a polygonal shape, an oval shape, and the like. Further, the figure 201017163 1 shows that a plurality of optical detecting devices 20 are arranged in a one-segment pattern. It will be appreciated that the optical detection device 20 can be configured in other patterns, such as a triangular lattice pattern, a honeycomb pattern, and the like. Since the plurality of optical detecting devices 20 of the bioanalytical system 1 operate independently of each other, the optical detecting device 20 will be described below in accordance with various embodiments. Although an optical detecting device 20 will be described, it will be understood that the different optical detecting devices 20 in the biological analysis system 1 are not necessarily the same. Depending on the design choice, different forms of optical detection device 20 can be constructed in accordance with various embodiments. Referring to Fig. 2, there is shown a cross-sectional view of the optical detecting device 20 in accordance with the embodiment of the line A-A of Fig. 1. As shown in FIG. 2, the optical detecting device 20 includes a photodetector 210 formed on the substrate 10 and/or a connecting portion 220 formed on the photodetector 210. In addition, the optical detecting device 20 further includes a control circuit 215 formed on the substrate 1 to control the operation of the photo sensor 210. Control circuitry 215 can be coupled to detection and recording system 2 for accepting control indications from detection and recording system 2 and transmitting the detected signals to detection and recording system 2. In some embodiments, the evaporation plate 10 can be a glass substrate, a semiconductor substrate (e.g., Shi Xi) or a plastic substrate. In some embodiments, one or more control circuits 215 are associated with each photodetector 210. In some embodiments, the photodetector 210 can include a single photoconductive photon detector or a set of photoconductive photodetectors, such as one or more charge-coupled devices. Or a complementary metal-oxide semiconductor (CMOS) sensor. In an alternate embodiment, photodetector 201017163 210 may include a single photon photon detector or a set of photovoltage photon detectors. In an alternate embodiment, photodetector 210 can include a avalanche photodiode or a set of discrete photodiodes. In an alternate embodiment, photodetector 210 can comprise a single phototransistor or a group of optoelectronic crystals. In an embodiment, the optical detecting device 20 may further include a blind sheet 230 on the photodetector 210. The masking sheet 230 can include a pinhole 235. In an embodiment, the pinhole 235 can have a circular shape and can have a diameter D1 that is less than or equal to one of 10,000, 1,000, 500, 300, 200, 150, or 100 nm. It will be appreciated that the pinhole 235 can have other shapes, such as an oval shape, a square shape, and the like. In one embodiment, the masking sheet 230 can include an opaque material to block unwanted light from reaching the photodetector 210. Therefore, the desired light can reach the photodetector 210 via the pinhole 235. A linker site 220 can be formed proximate to the pinhole 235. In the embodiment shown in this figure, the joint location 220 is formed within the pinhole 235. In one embodiment, the bond 220 forming the pinhole 235 is separated from the photodetector 210 by a distance H1 less than or equal to 1 〇〇 μη. In an alternate embodiment, the distance Η1 can be less than or equal to 75, 50, 25, 15, 10, 5, or 3 μηη. The optical detecting device 20 may further include a filter layer 240 (optical) and a microlens 250 (optical) interposed between the photodetector 210 and the shielding sheet 230. Although FIG. 2 shows that the filter layer 240 is formed on the microlens 250, it is understood that the 'filter layer 240 may be formed under the microlens 250. In some embodiments, filter layer 240 can comprise a single transparent layer or a plurality of transparent layers of 2010-11163 having a different refractive index. As shown in Figure 3a, in some embodiments, the plurality of sub-layers can be non-planar. Non-planar shapes may include, but are not limited to, hemispherical, elliptical, arcuate or otherwise curved shapes and/or surfaces. When the filter layer 240 includes a plurality of arc-shaped sub-layers, a plurality of arc-shaped sub-layers may have optical transmission characteristics by continuously depositing a sub-layer on the substrate 1 于. The high degree of reflection of a particular wavelength or range of wavelengths and the inverse of a different wavelength or range of wavelengths. For example, a plurality of arc filters in an embodiment can reflect light having a wavelength of 633 nm, and emit light having a wavelength of 680 nm. In some embodiments, a sub-layer having a lower index of refraction can be lost between two layers having a lower index of refraction. Alternatively, a sublayer having a lower refractive index may be sandwiched between two layers having a higher refractive index. In some embodiments, filter layer 240 can include a layer having a single region or a layer having a plurality of sub-regions, while the plurality of sub-regions have different transparency for different wavelength ranges. Returning to Figure 2, the link 22 can be treated to attach the biomolecule 30 thereto. In one embodiment, biomolecule 30 can include a single strand of DNA molecule 32 and an end primer 34 that binds to single stranded DNA molecule 32. The biomolecule 30 can be attached to the bonding site 220 by the terminal linking primer 34. Further, a fluorescent cluster 36 can be used to mark the DNA molecule 32. When excited by the excitation light of a first wavelength λ , , the fluorophore 36 can emit fluorescence of a second wavelength λ 2 . In some embodiments, the first wavelength λ 短 is shorter than the second wavelength λ 2 . In some embodiments the 'first wavelength λ 长 is longer than the second wavelength λ 2 ' for example in multi_photon excitation. The photodetector 210 then detects the fluorescence emitted from the phosphor group 36 to identify the base form to which the fluorophore 12 201017163 36 is attached, thereby continuously determining the sequence of the DNA molecule 32. Referring to Figure 3b, a cross-sectional view of optical detection device 20 in accordance with an embodiment is shown. The masking sheet 230 is formed over the photodetector 210 as shown in Fig. 3b and is vertically spaced from the photo sensor 210 by a distance H1. The masking sheet 230 having a thickness T includes a pinhole 235 having a radius R1 (i.e., one half of the diameter D1). In this embodiment, the bonding site 220 can be formed in the pinhole 235 to interface with a biomolecule (not shown). When the luminescent group 36 is disposed at a first position 36A located at the joint position 220 and separated from the joint position 220 by a distance H2, the light detecting thief 210 having the radius R2 may be at a first solid angle (s〇 Hd angie) Q 1 collects fluorescence emitted from the fluorescent group 36. When the luminescent group 36 is disposed at a second position 36B and is almost in contact with the bonding position 220 (ie, the distance H2 is near zero or less than 1 pm), the photodetector 210 can then collect the emission within a second solid angle θ2. Fluorescence from the fluorescent cluster 36. The second solid angle Θ 2 is greater than the first solid angle Θ 1 and provides a substantially stronger signal. In order to expose the photodetector 210 to the fluorescent light detector 21 散 emitted from the fluorescent ray 36 via the pinhole 235, the radius r2 must be greater than or equal to the equivalent of the projection on the upper surface of the photodetector 210. The radius of the two solid angles 02. By bringing the masking sheet 230 (or the joint location 220) closer to the photodetector 210 (ie, by reducing the distance Η1), the photodetector can then collect more concentrated light from a solid angle (ie, a comparison) Strong light signal). In one embodiment, the masking sheet 230 (or the joint location 220) is separated from the photodetector 210 by a small distance Η1 such that the second solid angle θ2 is at least SI8 SI sphere 13 201017163 面度 (steradian ). Referring to Fig. 4, there is shown a cross-sectional view of optical detecting device 20 in accordance with another embodiment along line A-A of Fig. 1. In this embodiment, an excitation source 40 is integrated with the optical detection device 20. As shown in Fig. 4, the excitation light source 40 is formed on the masking sheet 230 of the optical detecting device 20. In one embodiment, the excitation source 40 can include a p-type and an n-type semiconductor layer (410 and 430), and a light-emitting layer 420 is interposed between the junction regions between the layer 410 and the layer 430. Layer 410 and layer 430 can be connected to a power source. The excitation light source 40 can be a light emitting diode (LED), a light emitting diode (light), depending on the physical and primary structure of the materials and/or materials used for layers 410, 420, and 430. An emitter laser diode (LD), an organic light emitting diode (OLED) or a polymer light emitting diode (PLED). Inorganic materials 'for example, deuterated gallium, filled indium, recorded gallium and gallium nitride, or organic materials, such as conjugated polymers with poly-para-pheneylene-vinylene backbones' It is an example of a semiconductor material that can be used to fabricate junction junction bipolar φ body (junction diode). In other embodiments, the excitation source 40 can form a masking sheet 230 or can be formed within the concealing sheet 230. In some embodiments, the excitation source 4A integrated with the optical detection device 20 can emit light in a wavelength band or a plurality of wavelength bands. The excitation light source 40 can emit light intermittently or continuously. The excitation light source 40 can emit light of one wavelength band at a time or simultaneously emit light of several wavelength bands. Referring again to Fig. 4, the excitation source 40 can include a recess 450 at a central portion of its 14 201017163 to expose the pinhole 235. In this embodiment, the joint 22 〇 may not be formed in the pinhole 235. Of course, the joint 22 〇 can be formed in the recess 450 and close to the pinhole 235. In an embodiment, wherein the excitation light source 4 is formed into a masking sheet 230 or formed within the masking sheet 23, the recess 45 is formed into a pinhole 235 or formed within the pinhole 235. In some embodiments, the holes can be formed in the central portion of either the layer 41 and the masking sheet 23 by masking the sheet 230 by, for example, using a suitable process to repose the layer 41. Eight

，此外，經由—形成於下層410上之金屬接觸415與一 形成於上層430上之金屬接觸435，—激發光源仙可愈一 =44=合。電源_可單獨設置且藉由偵測與記錄系 統來控制，或可與债測與記錄系統2整合。 激發光源4〇之光散發層働可散發激發先 420*上之=戶：’而水平方如於第4圖中繪於光散發層Further, through the metal contact 415 formed on the lower layer 410 and the metal contact 435 formed on the upper layer 430, the excitation source can be more than one = 44 = combined. The power source _ can be set separately and controlled by the detection and recording system, or can be integrated with the debt measurement and recording system 2. The excitation light source 4 〇 light emission layer 働 can be ignited first 420* on = household: ' and the horizontal side as shown in Figure 4 on the light emission layer

平行於遮蔽薄板2:之!光沿著實質上 先可以不干涉到達 :激發 此可比-般元件更準確_定生物:子先。仏_裝置因 2·核酸偵測 :使用生物分析系統（包括’例如不是單 ，置就是複數個此種裝置）成先子偵測 ，摘測之方法或製程中，子偵測 序。此生物分析系統及利用其之方法盥程：如核酸定 肩應用為有用的。這些應用可為個人的、公^的: 15 201017163 商業的或工業的。 在一些實施例中，生物分析系統適合核酸之大量平行 定序。部分由於連結位與生物分析系統之光偵測器的直接 聯繫及/或連結位與光偵測器的緊密接近（在一些實施例 中，顯示為一大的立體角），可使用實施例中所提供之生 物分析系統來對核酸進行定序，而不需昂貴、複雜與錯誤 傾向之掃猫與分析系統，例如一移動掃瞎透鏡（moving scanning lens)或一移動元件台（moving device stage)與隨後 的影像分析’因此減少錯誤與成本。生物分析系統可偵測 ❹ 具有實質上經改善之訊號強度的訊號，其使單一分子分析 變成可能。 可以廣泛變化之定序形式來使用生物分析系統，且生 物分析系統適合將單一分子進行定序。另外，與現行生物 晶片元件相較，光學偵測元件具有經簡化之設計、裝配及 製造。例如’可將要被定序之核酸附著於陣列上之隨機的 連結位’避免對於在預定位置沈積或合成核酸之時間消耗 與昂責機器的使用。 參 可使用生物分析系統為在生物分子偵測之方法與程序 中之一系統的部分，包括核酸雜合或對於，例如整個基因 體疋序之疋序、轉錄表現(transcriptional profiling)、競爭轉 錄表現或基因鑑定。生物分子偵測也可包括結合相互作 用，例如蛋白質/蛋白質、抗體/抗原、受器/配體及核酸/蛋 白質之偵測及/或測量。這些應用對於分析與診斷程序與方 法為有用的。 適s於本發明所提供之系統上偵測之核酸，於一些實 16 201017163 施例中，為連結分子之一部分，其將一適合於分析結合相 互作用之分子，例如蛋白質、其他核酸、碳水化合物部分 或小分子附著至一實施例所提供之元件上的連結位。在— 些實施例中，連結分子可更包括一捕獲分子，其由於結合 之相互作用與要被分析之分子結合。在一連結分子中之核 酸’藉由’例如直接定序或雜合，作為連結分子之捕獲分 子的一鑑定標籤。 實施例所提供之方法包括將要被偵測之分子附著於實 響 施例所知供之元件的一位址陣列（address array)的一步驟。 在一些實施例中’位址陣列可包括具有複數個針孔235之 遮蔽薄板230 ’與可形成在針孔235中或環繞針孔235之 連接位220。參見’例如第1與2圖。因此生物分析系統 可同時讀取數百萬之核酸片段。若各片段為，例如1000鹼 基長’一單一元件可獲得數十億位元(bit)之序列資料，而 使整個基因體之定序與再定序(resequencing)為可能。 2.1要被偵測之分子 適合以實施例所提供之方法來偵測之核酸可包括任何 核酸，包括，例如 DNA、RNA 或 PNA (peptide nucleic acid (胜肽核酸）），且可含有任何序列-已知與未知，包括自 然發生或人工序列。核酸可被自然取得、重組產生或化學 合成。核酸可包括自然發生核苷酸、不存在於自然之核苷 酸類似物或經修飾之核苷酸。基於實際應用，被偵測之核 酸長度可多樣化。在一些實施例中，核酸包括至少10、20、 50、100、200、500、1000、2000、5000、10000、20000 17 201017163 個驗基或更多。在一些實施例中，核酸河為自至20、 自10至50、自10至100、自50至100、自50至500、自 50 至 1000、自 5〇 至 5000、自 500 至 2000、自 500 至 5000, 或自1000至5000個鹼基。 用以偵測之核酸可為單 股核酸模板，藉由本技術 社⑽板板可來自一雙 或ζ·« Wiro合成，也可產生 或其他化學處理。藉由例如化/知的方法，例如加熱或鹼 單股核酸模板。 予 在一些實施例中，要被價 —連結位。在—些實施例中，^之核酸於其5,或3,端附著 酸之5,端、3,端或5，端與3,广可更包括—或多個與核 在其他實施财，一末端連^者結合之末端連結引子。 使用末端I㈣子=至嫌之3,端。可 連結位，並提供一對於一或多 有社兀忏上之 例如—定序引子。 烟偵測之引子之互補序列， 2·1·1末端連結引子 末知》連結弓丨子為短的核峻分 ^ 苷酸所組成。在-政實施例中刀子，通吊由少於100個核 少5、10、15、20、25、30、％末端連結引子之長度為至 在督故"士 ^ 75、9〇個核苷酸或更多。 仕貫施例中，末端連結引子 又夕 加、自H)至30,或自1〇至二 8至25、自1〇至 中，士 〇個核皆酸。在一此實施你丨 其可為經分支的。 的’然而’在其他實施例中， 可使用末端連結引子來將 ^要破偵測之核酸附著至於位 18 201017163 置陣列上之連結位。在一些實施例中，末端連結引子可將 核酸直接地連結至陣列表面，例如藉由共價連結（例如酯 (ester)或硫醇(thiol)連結）或非共價連結，例如抗原/抗體或 生物素(biotin)/抗生物素蛋白（avidin)結合。參見，例如第6 圖、第7圖與第8圖。在一些實施例中，末端連結引子可 將核酸非直接地連結至陣列表面，例如藉由與一居中分子 (intermediate molecule)結合，而居中分子，例如一聚合酶。Parallel to the masking sheet 2: the light along the ground can be reached without interference first: excitation This is more accurate than the general component _ set the creature: the child first.仏 _ device due to 2 · nucleic acid detection: the use of biological analysis systems (including 'for example, not single, set a number of such devices) into the first detection, extraction method or process, sub-detection sequence. This bioanalytical system and methods of using the same are useful for nucleic acid shoulder applications. These applications can be personal, public: 15 201017163 Commercial or industrial. In some embodiments, the bioanalytical system is adapted to a large number of parallel sequencing of nucleic acids. In part, due to the direct connection of the link to the photodetector of the bioanalytical system and/or the close proximity of the link to the photodetector (in some embodiments, shown as a large solid angle), embodiments may be used. A bioanalytical system is provided to sequence nucleic acids without the need for expensive, complicated, and error-prone sweeping cats and analysis systems, such as a moving scanning lens or a moving device stage. With subsequent image analysis 'then reduce errors and costs. The bioanalytical system detects signals with substantially improved signal intensities that make single molecule analysis possible. Bioanalytical systems can be used in a widely varying sequence, and bioanalytical systems are suitable for sequencing a single molecule. In addition, optical sensing elements have been simplified in design, assembly, and fabrication as compared to current biochip components. For example, 'a random link to the nucleic acid to be sequenced attached to the array' avoids the time spent on depositing or synthesizing nucleic acids at predetermined locations and the use of the machine. The bioanalytical system can be used as part of a system in methods and procedures for biomolecule detection, including nucleic acid heterozygous or for, for example, whole genome sequencing, transcriptional profiling, competitive transcriptional expression Or genetic identification. Biomolecular detection can also include binding interactions such as protein/protein, antibody/antigen, receptor/ligand, and nucleic acid/protein detection and/or measurement. These applications are useful for analysis and diagnostic procedures and methods. Nucleic acids detectable on the system provided by the present invention, in some embodiments of 201016163, are a part of a linker molecule, which will be a molecule suitable for analyzing binding interactions, such as proteins, other nucleic acids, carbohydrates Some or small molecules are attached to the bonding sites on the elements provided by an embodiment. In some embodiments, the linker molecule can further comprise a capture molecule that binds to the molecule to be analyzed due to the interaction of the bond. The nucleic acid in a linked molecule 'by direct' or direct hybridization serves as an identification tag for the capture molecules of the linked molecule. The method provided by the embodiment includes the step of attaching the molecule to be detected to an address array of the component known to the actual embodiment. In some embodiments, the address array can include a masking sheet 230' having a plurality of pinholes 235 and a connection location 220 that can be formed in or around the pinholes 235. See, for example, Figures 1 and 2. So bioanalytical systems can read millions of nucleic acid fragments simultaneously. If each fragment is, for example, 1000 bases long, a single element can obtain billions of bits of sequence data, and it is possible to sequence and resequence the entire genome. 2.1 The molecule to be detected The nucleic acid to be detected by the method provided in the examples may include any nucleic acid, including, for example, DNA, RNA or PNA (peptide nucleic acid), and may contain any sequence - Known and unknown, including naturally occurring or artificial sequences. Nucleic acids can be obtained naturally, recombinantly, or chemically synthesized. Nucleic acids can include naturally occurring nucleotides, nucleoside analogs that are not found in nature, or modified nucleotides. The length of the nucleic acid being detected can be varied based on the actual application. In some embodiments, the nucleic acid comprises at least 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10000, 20000 17, 201017163 bases or more. In some embodiments, the nucleic acid river is from 20 to 50, from 10 to 50, from 10 to 100, from 50 to 100, from 50 to 500, from 50 to 1000, from 5 to 5000, from 500 to 2000, from 500 to 5000, or from 1000 to 5000 bases. The nucleic acid to be detected may be a single-stranded nucleic acid template, and the plates of the present technology (10) may be derived from a pair of ζ·« Wiro synthesis, or may be produced or otherwise chemically treated. For example by heating/knowing, for example, heating or alkali single-stranded nucleic acid template. In some embodiments, the price is to be linked. In some embodiments, the nucleic acid of the nucleic acid at its 5, or 3, end is attached to the 5, the end, the 3, the end, or the 5, the end and the 3, and the end may further include - or a plurality of cores in other implementations, One end is connected to the end of the linker. Use the end I (four) sub = to the 3, the end. It can be linked and provided for one or more of the social, for example, sequencing primers. The complementary sequence of the smoke detection primer, the 2·1·1 end-linking primer, the end-of-sense, the scorpion scorpion is composed of a short nuclear nucleus. In the governmental example, the knives are hanged by less than 100 nucleus with 5, 10, 15, 20, 25, 30, and % end-linked primers to the length of the ruling to the ruling. Glycosyl or more. In the Shishi example, the end-linking primer is added, from H) to 30, or from 1〇 to 2-8 to 25, from 1〇 to the middle, and the 〇 〇 核 核 。 。. In this case, you can implement it as a branch. In other embodiments, an end-linking primer can be used to attach the nucleic acid to be detected to the linkage on the array of bits 18 201017163. In some embodiments, the end-linking primer can bind the nucleic acid directly to the surface of the array, such as by covalent attachment (eg, ester or thiol linkage) or non-covalent linkage, eg, antigen/antibody or Biotin/avidin binding. See, for example, Figure 6, Figure 7, and Figure 8. In some embodiments, the end-linking primer can bind the nucleic acid non-directly to the surface of the array, for example by binding to an intermediate molecule, such as a polymerase.

參見，例如第8圖。因此，末端連結引子可包含經修飾之 核酸或以其他方式修飾以協助附著至一連結位，藉由本技 術領域已知的方法，例如，雙硫、硫酯、醯胺、磷酸二醋 或醋連結；或藉由，例如抗體/抗原，或生物素/抗生物素蛋 白結合，例如末端連結引子包含一包括一抗原區域之核苷 酸或一經生物素化(biotinylated)之核苷酸。在其他實施例 中，一經修飾之核苷酸位於一末端連結引子之3,端上。在 一些實施例中’一末端連結引子之5,端包含一經修飾之核 苷酸。 末端連結引子也可做為一用以债測核酸之一或多個引 子的互補物(complement)，例如，一定序序列。在一些實施 例中’引子係藉由雜合用來偵測核酸，例如，引子包含: 可偵測之標誌，例如一螢光或放射同位素標誌。在/痤貫 施例中’末端連結引子的5’端包括與一定序引子立補之/ 列。在一些實施例中，與定序引子互補之末端連結弓丨子係 被定位以使定序引子之3,端直接與要被定序之核酸中的第 一個核苷酸鄰接。 例如，第6圖為要被定序之序列附著裏/光學價川 19 201017163 置20之一實施例的圖解顯示。一單股核酸32、末端連結 引子 34 與黏合定序引子（anneaied sequcncing prjmer)346 附 著於一經處理具有反應性官能基之連結位220,連結位220 與於末端連結引子34上之一經修飾之核苷酸344結合。在 一些實施例中，核酸32經由其5,端可附著至連結位220， 而末端連結引子34可附著至核酸32之3,端以作為定序引 子346之一互補物。 在一些實施例中’藉由一連接酶(ligase)，例如一 DNA 連接酶將末端連結引子加至要被偵測之核酸的末端。在一❹ 些實施例中’在連接(ligati〇n)前，末端連結引子與要被偵 測之核酸兩者皆為單股。在其他實施例中，兩者皆為雙股。 在另外其他實施例中，一者為單股，而另一者為雙股。連 接為本技術領域所熟知。例如，在聚合群落定序方法(p〇1〇ny sequencing method)中，Shendure et al. (defence， 309:1728-1732(2005))以新英格蘭生物實驗室（New England Biolab’，NEB)快速連接套組(Quick Ligation kit)將一 T30 末 端連結引子(32 bp)連結至一樣本DNA片段。其中，連結反 ❹ 應溶液包括0.26 pMole之DNA、0.8 pMole之T30末端連 結引子、4.0 μΐ T4 DNA連接酶於lx快速連接緩衝溶液 (Quick Ligation buffer)中。於混合後，於室溫培養反應溶液 約10分鐘，且之後置於冰上。藉由將樣本加熱至65°c，1 〇 分鐘來停止連接反應。 在其他實施例中’可將末端連結引子合成於要被偵測 之核酸上。例如’末端連結引子可為一藉由例如末端轉移 酶(terminal transferase)所加入之均聚物（h〇in〇p〇iymer)。例 20 201017163 如 ’ Harris et al.〇Sc/⑼ce, 320:106-109(2008))將一多腺嗓呤 尾巴(poly A tail)加至DNA模板，其做為於一病毒基因體 之早一分子定序中之一多胸腺嘴唆(P〇ly T)定序引子的互 補物。 2·1·2定序引子 一定序引子為一與要被偵測之核酸的一片段或其之連 結的末端連結引子互補之單股募核苷酸。在一些實施例 ® 中，定序引子的長度為至少8、10、15、20、25、30、35、 40、45、50個核苷酸或更多。在特定實施例中，定序引子 之長度可為自8至25、自10至20、自1〇至30，或自10 至50個核苷酸。定序引子可由任何形式之核苷酸所形成， 包括自然發生核苷酸、不存在於自然之核苷酸類似物或經 修飾之核苷酸。在實施例中，在一定序引子與包括一或多 個末端連結分子之要被定序的核酸雜合後，可修飾定序引 _ 子之5’端，以促進其與位置陣列上之一連結位結合。 在一些實施例中，一定序引子包含經修飾之核苷酸， 例如鎖核酸(locked nucleic acid, LNA)(經修飾之核糖核甘 酸’其在一聚核酸(polynudeic acid)中提供增強之驗基堆疊 相互作用（base stacking interaction))。如鎖核酸之效用解 說，Levin et al. (Nucleic Acid Research 34(20):142(2006)) 顯示一含鎖核酸之引子具有經改善之專一性及顯示較強之 結合相對於對應之未鎖引子(unlocked primer)。製造MCP1 引子（5’-cttaaattttcttgaat-3’）之三種變化，包含3個鎖核酸核 苷酸（於蓋（cap)中）於引子中之不同位置： 21 201017163 MCP1 -L AN-3 ’（5 ’ -cttaaattttCtTgaAt-3，） ； MCP1 -L AN-5 ’ (5’-CtTaAattttcttgaat-3’） ； 與 MCPl-LAN-even (5’-ctTaaatTttctTgaat-3’）。所有經鎖核酸取代之引子具有提 高之溶解溫度（melting temperature, Tm)，而 MCP1-LNA-5’ . 引子顯示特別提高之定序準確度（Phred Q30 counts)。因 此，在其他實施例中，定序引子可包含至少一鎖核苷酸於 其5’端區域，即，定序引子之5’端一半、三分之一或四分 之一。 在被提供至一光學偵測元件之前，可將定序引子與單 ❹ 股樣本核酸（即，一包括至少一末端連結引子之要被偵測 的核酸）雜合。可將定序引子與樣本核酸雜合，藉由將樣 本核酸與一莫耳過剩（molar excess)之定序引子混合於一含 鹽溶液中，例如 5xSSC(或 5xSSPE)、0.1% Tween20(或 0.1% SDS)與0.1% BSA緩衝溶液。可將混合物加熱65°C 至少5分鐘且缓慢冷卻至室溫以允許引子/模板黏合。藉由 適合之方法可將殘餘之引子去除，例如一分子篩（molecular sieve) ° · 藉由適合的方法，包括序列之視覺檢閱或電腦協助之 引子設計，可設計引子，包括末端連結與定序引子兩者。 許多軟體套組為可用來協助引子設計，包括DNAStar™ (DNAstar, Inc.，Madison, WI)、OLIGO 4.0 (National Biosciences, Inc.)、Vectoe NTI®(Invitrogen)、Primer Premier 5 (Premierbiosoft)與 Primer3 (Whitehead Institute for Biomedical Research, Cambridge, MA)。引子設計，考慮到 例如，要被定序之分子、專一性、長度、所需的熔解溫度、 22 201017163 二級結構、引子二聚體、GC含量、緩衝溶液的pH與離子 強度及所使用之酵素（即，聚合酶或連接酶）。參見，例 k〇 Joseph Sambrook and David Russell, Molecular Cloning: ' A Laboratory Manual Cold Spring Harbor Laboratory Press, 3rd edition (2001)。 2.1.3結合至陣列表面 在將定序引子與包括一或多個末端連結引子之要被定 鲁序的核酸黏合後，製備此複合物於一合適的緩衝溶液中、 將其提供至一位置陣列的表面並允許其結合。在一些實施 例中，將樣本核酸（要被偵測之核酸與一或多個末端連結 引子）附著至連結位，且之後提供定序或偵測引子。在其 他實施例中，在被提供至一元件之前，先將複合物進行雜 合。只有一個核酸樣本結合之連結位被瞭解為有效位置。 在實施例中，提供複合物至光學偵測元件且樣本核酸附著 ^ 至在位置陣列上之隨機的連結位。在其他實施例中，可提 供樣本核酸至在位置陣列上之預定的連結位，藉由適合的 方法’例如藉由機器或液體處理系統。 將核酸附著至一固體支持物之適合的方法為本技術領 域所熟知。在一些實施例中，可將樣本核酸直接附著至一 連結位，藉由共價連結，例如雙硫、硫酯、醯胺、磷酸二 酯或酯連結；或藉由非共價連結，例如抗體/抗原，或生物 素/抗生物素蛋白結合。在一些實施例中，藉由一介於中間 的分子可將樣本核酸附著至一連結位。在一些實施例中， 川於中間的分子可為—聚合酶，例如一 DNA聚合酶。 23 201017163 如一直接、共價之核酸附著的說明例子，Adeesietal. (Nucleic Add Research，28:87 (2000))修飾一引子的 5,端以 包括一 SH官能基。根據Adeesi et al·之方法，可製備一核 酸於 50μΜ磷酸缓衝鹽(PBS)(NaPi: 〇] MNaH2p〇4pH6 5， · 0.1 MNaCl)中。之後可將u5 μ1之引子溶液提供至一經鹽 化之玻璃載片，且將其培養於一濕度控制盒於室溫中約5 小時以使引子結合至晶片表面。在結合反應完成後，以pBS 溶液於室溫震動清洗兩次，每次5分鐘，以移除未結合之 DNA。在清潔後’將 1〇 mM β-硫氫乙醇（p_mercapt〇ethan〇1) φ 加至一 PBS溶液且於室溫下用來沖洗位置陣列，以將未結 合之DNA的硫醇基去活化。再來，清洗陣列表面，例如一 次以5xSSC、0.1% Tween與一次以5xSSC緩衝溶液。因 此，在一些實施例中，Adeesi etal.所使用之方法可使用於 本發明所提供之方法中以將樣本核酸複合物，例如經由一 定序引子或樣本核酸之5’端附著至一連結位。 在一替代實施例中，樣本核酸可包括，例如一經生物 素化之核苷酸，且可與在連結位表面上之抗生物素蛋白結 _ 合。在另一實施例中，樣本核酸可包括一抗原部分，例如 BrdU或洋地黃毒(digoxigenin)，其藉由一抗體（或其片段） 被結合於連結位上。藉由“抗體，，，可以瞭解的是，此措辭 包括免疫球蛋白分子之片段，包括，例如一或多個CDR 區；或可變之重或可變之輕片段。抗體可為自然發生、重 組或合成。抗體也可包括，例如多株（p〇lyCl〇ne)與軍株 (monoclone)變形。在一些實施例中，抗體以至少1〇6、1〇7、 10、10 Μ或更高之結合常數(associati〇n constant)結合至 24 201017163 其抗原。抗體之結構、功能與產生為本技術領域所熟知。 參見’例如 Gary Howard and Matthew Kasser,See, for example, Figure 8. Thus, the end-linking primer may comprise a modified nucleic acid or otherwise modified to assist in attachment to a linking site, by methods known in the art, for example, disulfide, thioester, guanamine, phosphodiacetate or vinegar linkage. Or by, for example, antibody/antigen, or biotin/avidin binding, for example, a terminal linker comprises a nucleotide comprising an antigenic region or a biotinylated nucleotide. In other embodiments, a modified nucleotide is located on the 3' end of the terminating primer. In some embodiments, the terminus lends to the 5th of the primer and the terminus comprises a modified nucleotide. The end-linking primer can also be used as a complement for one or more of the primers, for example, a sequence. In some embodiments, the primer is used to detect nucleic acid by hybridization. For example, the primer comprises: a detectable marker, such as a fluorescent or radioisotope marker. In the /those embodiment, the 5' end of the 'end-linking primer' includes a column that is complementary to a certain order primer. In some embodiments, the terminally linked scorpion line that is complementary to the sequencing primer is positioned such that the 3' end of the sequencing primer is directly contiguous with the first nucleotide in the nucleic acid to be sequenced. For example, Figure 6 is a graphical representation of one embodiment of a sequence of attachments to be sequenced/optical price 19 201017163. A single-stranded nucleic acid 32, an end-linking primer 34 and an anneaied sequcncing prjmer 346 are attached to a link 220 having a reactive functional group, and a linker 220 is attached to a modified core of the terminal linker 34. Glycoside 344 binds. In some embodiments, nucleic acid 32 can be attached via its 5' end to linker position 220, while end linker 34 can be attached to the 3' end of nucleic acid 32 as one of the complements of sequencing primer 346. In some embodiments, an end-linking primer is added to the end of the nucleic acid to be detected by a ligase, such as a DNA ligase. In some embodiments, the end-linking primer and the nucleic acid to be detected are both single-stranded prior to ligation. In other embodiments, both are double stranded. In still other embodiments, one is a single share and the other is a double share. Connections are well known in the art. For example, in the 群落1〇ny sequencing method, Shendure et al. (defence, 309:1728-1732 (2005)) is fast in New England Biolab' (NEB). A Quick Ligation kit links a T30 end linker (32 bp) to the same DNA fragment. Among them, the conjugated reaction solution included 0.26 pMole of DNA, 0.8 pMole of T30 end ligated primer, and 4.0 μM of T4 DNA ligase in lx Quick Ligation buffer. After mixing, the reaction solution was incubated at room temperature for about 10 minutes and then placed on ice. The ligation reaction was stopped by heating the sample to 65 ° C for 1 〇 minutes. In other embodiments, an end-linking primer can be synthesized on the nucleic acid to be detected. For example, the 'end-linking primer' can be a homopolymer (h〇in〇p〇iymer) added by, for example, a terminal transferase. Example 20 201017163 As in 'Harris et al. 〇 Sc/(9) ce, 320: 106-109 (2008)), a poly A tail is added to a DNA template as an early viral genome. One of the one-molecular sequence is the complement of a multi-thymidine (P〇ly T) sequencing primer. 2·1·2 Sequencing Primer A pre-sequence primer is a single-stranded nucleotide complementary to a fragment of a nucleic acid to be detected or a ligated end-linking primer thereof. In some embodiments, the sequencing primers are at least 8, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides or more in length. In particular embodiments, the sequencing primers can be from 8 to 25, from 10 to 20, from 1 to 30, or from 10 to 50 nucleotides in length. The sequencing primer can be formed from any form of nucleotide, including naturally occurring nucleotides, nucleotide analogs that are not found in nature, or modified nucleotides. In an embodiment, after the sequenced primer is hybridized to the nucleic acid to be sequenced comprising one or more terminal linker molecules, the 5' end of the sequence can be modified to facilitate its alignment with one of the positional arrays. The link is combined. In some embodiments, the sequence primer comprises a modified nucleotide, such as a locked nucleic acid (LNA) (modified ribonucleotide, which provides an enhanced assay in a polynudeic acid) Base stacking interaction). As explained by the effect of locked nucleic acids, Levin et al. (Nucleic Acid Research 34(20): 142 (2006)) showed that a primer containing a locked nucleic acid has improved specificity and a stronger binding relative to the corresponding unlocked Unlocked primer. Three variations of the MCP1 primer (5'-cttaaattttcttgaat-3') were made, including three locked nucleic acid nucleotides (in the cap) at different positions in the primer: 21 201017163 MCP1 -L AN-3 '(5 '-cttaaattttCtTgaAt-3,) ; MCP1 -L AN-5 ' (5'-CtTaAattttcttgaat-3') ; with MCPl-LAN-even (5'-ctTaaatTttctTgaat-3'). All primers substituted with a locked nucleic acid have an elevated melting temperature (Tm), while MCP1-LNA-5'. The primers show a particularly improved sequence accuracy (Phred Q30 counts). Thus, in other embodiments, the sequencing primer may comprise at least one locked nucleotide in its 5' end region, i.e., one-half, one-third or one-quarter of the 5' end of the sequencing primer. The sequencing primer can be hybridized to a single-stranded sample nucleic acid (i.e., a nucleic acid to be detected comprising at least one end-linking primer) prior to being provided to an optical detection element. The sequencing primer can be hybridized to the sample nucleic acid by mixing the sample nucleic acid with a molar excess of the sequencing primer in a saline solution, such as 5xSSC (or 5xSSPE), 0.1% Tween20 (or 0.1). % SDS) with 0.1% BSA buffer solution. The mixture can be heated to 65 ° C for at least 5 minutes and slowly cooled to room temperature to allow the primer/template to bond. Residual primers can be removed by suitable methods, such as a molecular sieve. • Primers can be designed, including end-linking and sequencing primers, by suitable methods, including sequence visual inspection or computer-assisted primer design. Both. Many software kits are available to assist with primer design, including DNAStarTM (DNAstar, Inc., Madison, WI), OLIGO 4.0 (National Biosciences, Inc.), Vectoe NTI® (Invitrogen), Primer Premier 5 (Premierbiosoft), and Primer3 (Whitehead Institute for Biomedical Research, Cambridge, MA). Primer design, taking into account, for example, the molecularity to be sequenced, specificity, length, desired melting temperature, 22 201017163 secondary structure, primer dimer, GC content, pH and ionic strength of the buffer solution, and used Enzyme (ie, polymerase or ligase). See, for example, k〇 Joseph Sambrook and David Russell, Molecular Cloning: 'A Laboratory Manual Cold Spring Harbor Laboratory Press, 3rd edition (2001). 2.1.3 Binding to the surface of the array After binding the sequencing primer to the nucleic acid to be sequenced, including one or more end-linking primers, the complex is prepared in a suitable buffer solution and supplied to a location. The surface of the array and allow it to bond. In some embodiments, the sample nucleic acid (the nucleic acid to be detected and one or more end-linking primers) is attached to the conjugation site, and then sequencing or detection primers are provided. In other embodiments, the composite is first hybridized prior to being provided to a component. Only one binding site of a nucleic acid sample is known to be a valid position. In an embodiment, the complex is provided to the optical detection element and the sample nucleic acid is attached to a random linkage at the position array. In other embodiments, the sample nucleic acid can be provided to a predetermined junction on the array of locations by a suitable method', e.g., by a machine or liquid handling system. Suitable methods for attaching nucleic acids to a solid support are well known in the art. In some embodiments, the sample nucleic acid can be directly attached to a linker by covalent attachment, such as disulfide, thioester, guanamine, phosphodiester or ester linkage; or by non-covalent linkage, eg, antibody /antigen, or biotin/avidin binding. In some embodiments, the sample nucleic acid can be attached to a conjugation site by an intermediate molecule. In some embodiments, the molecule in the middle can be a polymerase, such as a DNA polymerase. 23 201017163 As an illustrative example of direct, covalent nucleic acid attachment, Adeesietal. (Nucleic Add Research, 28:87 (2000)) modifies the 5' end of a primer to include an SH functional group. According to the method of Adeesi et al., a nucleic acid can be prepared in 50 μM phosphate buffered saline (PBS) (NaPi: 〇) MNaH2p〇4 pH6 5 · · 0.1 MNaCl). The u5 μ1 primer solution can then be supplied to a salted glass slide and cultured in a humidity control box at room temperature for about 5 hours to allow the primer to bind to the wafer surface. After the binding reaction was completed, the pBS solution was shaken twice at room temperature for 5 minutes each time to remove unbound DNA. After cleaning, 1 mM β-thioethanol (p_mercapt〇ethan〇1) φ was added to a PBS solution and used to rinse the position array at room temperature to deactivate the thiol group of the unbound DNA. Again, the array surface is cleaned, for example once with 5xSSC, 0.1% Tween and once with 5xSSC buffer solution. Thus, in some embodiments, the method used by Adeesi et al. can be used in the methods provided herein to attach a sample nucleic acid complex, e.g., via a 5' end of a sequencing primer or sample nucleic acid, to a linker. In an alternate embodiment, the sample nucleic acid can comprise, for example, a biotinylated nucleotide and can be associated with avidin on the surface of the linker. In another embodiment, the sample nucleic acid can comprise an antigenic moiety, such as BrdU or digoxigenin, which is bound to the binding site by an antibody (or a fragment thereof). By "antibody," it will be appreciated that this phrase includes fragments of immunoglobulin molecules, including, for example, one or more CDR regions; or variable heavy or variable light fragments. Antibodies may occur naturally, Recombinant or synthetic. Antibodies may also include, for example, multiple strains (p〇lyCl〇ne) and military strains (monoclone). In some embodiments, the antibodies are at least 1 , 6, 1, 7, 10, 10 or more. The high binding constant (associati〇n constant) binds to its antigen in 24 201017163. The structure, function and production of antibodies are well known in the art. See, for example, Gary Howard and Matthew Kasser,

Using Antibodies: A Practical Handbook CRC Press; 1st edition (2006)。 在又另一實施例中，藉由一聚合酶，例如DNA聚合 酶’可將樣本核酸附著至連結位。熟悉此技藝人士可理解， 為了保留酵素功能，可得之資訊，例如酵素之一級、二級 與三級結構應被考慮。例如，Taq與Phi29聚合酶之結構為 本技術領域所知，分別參見：Kim et al·， 376:612-616(1995)與 Kamtekar et al.，Mo/. Cell, 16:609-618(2004)。將一聚合酶固定至一表面，而保持活性 之方法為本技術領域所知，且敘述於，例如美國專利公開 號 2008/0199932’ 公開於 2008 年 8 月 21 日與 Korlach et al. iWAS 105:1176-1181(2008)。第8圖為一實施例之圖解表 示，其中樣本核酸（即，要被定序之核酸32、末端連結引 子34與定序引子346)藉由一聚合酶38結合至一連結位 220 ’且聚合酶38係已結合至連結位220，藉由方法384， 例如直接非共價吸附、一抗體、生物素或化學連結，例如 酿胺鍵。 在一些實施例中，一連結位之經乙醛(aldehyde)修飾之 表面係以含乙醛之矽烷(silane)試劑處理。乙醛很快地與在 蛋白質上之一級胺反應以形成一 Schiffs鹼基連結。由於 除了 一般於NH2端之更加反應性的α-胺外，很多蛋白質顯 露離胺酸於其表面上，所以其可以各種之方向附著至載 片，以允許蛋白質之不同側與於溶液中之其他蛋白質或小 25 201017163 分子反應。在另一實施例中，藉由uv光活化 (photoactivation)，一光 NHS(photoNHS) (— N_羥基琥珀醢 亞胺叛酸鹽分子（N-hydroxy succimido carboxylate)以一石炭 鏈連結器（linker)，連結至一疊氮硝基苯 . (azidonitrobenzene))附著至一於元件上之經胺修飾之表 面。在這些實施例中，藉由消除氮，UV光激起一疊氮硝基 苯部分以產生高反應性氮稀(nitrene)。氮稀立即與元件表面 上之NH2反應以形成一聯氨(hydrazine)鍵。連結器之另一 端為NHS羧酸鹽’其與聚合酶表面上之離胺酸反應以產生 ❹ 一酿胺共價鍵。在另一實施例中’於緩衝環境下’ NHS敌 酸鹽部分與元件表面上之一級胺反應。UV光用來活化一疊 氮石肖基苯部分且形成一高反應性氮燁為一電子不足基團 (electron deficient group)且立即與聚合酶表面上之離胺酸 殘基的一級胺反應。這些方法更詳細敘述於以下之實施例 4中。 2.2定序形式 ❷ 實施例所提供之生物分析系統可用來偵測與定序核 酸’藉由本技術領域已知的方法，如回顧於，例如美國專 利號 6，946,249 與 Shendure et al.，Ato 及ev 5:335-44(2004)。在一些實施例中，定序方法依靠不是DNA 聚合S#就是DN A連接酶的專一性，且包括，例如驗基延伸 定序（base extension sequencing)(單一鹼基逐步延伸（single base stepwise extension))、藉由合成之多鹼基定序（包括， 例如以經末端標誌之核苷酸來定序）與擺動定序（w〇bWe 26 201017163 sequencing) ’其為根據連結。所有方法通常需要一單股樣 本核酸，包括至少一要被附著至一連結位（不是直接就是 - 間接）之末端連結引子。之後定序開始實施於—定序引子 , (連接酶定序—般指錨定引子（anchorprimer)，其斜定皮2丨 子提供類似物用途）。 對於所有定序形式而言，實施例提供可再定序之單一 分子。例如，於定序讀取完成後，可將定序引子與經延伸 之核苷酸自樣本核酸刪除、清洗元件並重複定序。在各種 • 實施例中，藉由相同或不同方法可重新執行再定序。藉由 將相同分子再定序，預期定序錯誤會下降成定序讀取=數 之次方。例如，若單次讀取每個鹼基之錯誤為1〇_3，之後 在兩次讀取後，其下降成（1〇-3)2，即，1〇-6。此為單一分子 定序之特別的優點，由於用以定序之經修飾的核普酸可失 去其標誌或阻礙基團產生，例如假的刪除。 2.2.1鹼基延伸定序：逐步延伸 •在一些實施例中，實施例所提供之光偵測裝置可用來 執行鹼基延伸定序’如’例如美國專利號5,302,509中所揭 露。在一些實施例中，藉由將包括一要被定序之單股核酸 32、一與要被定序之單股核酸32之3,端結合之末端連結引 子34與黏合至其之一定序引子346的一部份雙重複樣本核 酸附著至一連結位220來開始鹼基延伸定序，如第6圖中 所示。在一些實施例中，聚合酶38與經修飾之核苷酸之後 被提供至光偵測元件於適合之緩衝溶液中。在一些實施例 中，藉由在連結位之聚合酶，將樣本核酸複合物附著至連 27 201017163 結位。在-些實施例中’核苷酸包括1價連結之可偵測 標誌，例如一螢光標誌，與一阻礙基團以避免任何二次延 伸。因此，在一單一核普酸加入至定序引子346 一 彳I㈣㈣。 之3’端之 P圖為-驗基延伸反應之1施例的第—步 解顯示。將具有螢光阻礙基團364之核脊酸362 聚合酶38加至定序引子346之3,蠕。在—些實施财 光標誌扮演阻礙基團。在其他實施例巾a 00 4 r 匕們為分開之部 參 分。-早-核苷酸合併於定序引子346之3,端 由立 之標灿對應之光㈣器2Π)來•。之後移除 ς 與阻礙基團，例如，藉由化學或酵素分解，以允許驗基2 伸之額外循環。在實施例巾，可_或連續地財任ς順 序中移除標諸與阻礙基團。藉由編輯鹼基加入順序，於3'， 至5,方向’-次-個驗基推論出樣本核酸之序列。第9圖 顯示平行地將一些樣本核酸延伸、偵測與去阻礙/去標誌的 一循環。 一般而言，在逐步延伸時有兩個方式來辨認所加入之 核苷酸。於一第一例子中，四種核苷酸具有相同之可偵測 標誌，但以一預定之順序一次加入一個。經延伸之核苷酸 的鑑定藉由於延伸反應中所加入之核苷酸的順序來確認。 在於延伸時辨認經整合之鹼基的第二模式中，四種不同之 核苷酸同時加入，且各與一有區別可偵測之標誌結合。在 不同實施例中，標誌之激發或散發光譜及/或強度可不同。 藉由可偵測標誌之強度及/或波長（即，激發或散發光譜） 來確認於延伸中加入之核苷酸強度。這兩個方法論之實施 28 201017163 例呈現於實施例5中。 2.2.2藉由合成之定序：多步驟延伸（multi-step extension) 在一些實施例中，藉由合成之定序可藉由多重不中斷 延伸（multiple uninterrupted extension)，例如沒有使用阻礙 基團來執行。在這些實施例中，藉由偵測在核三磷酸 (nucleoside triphosphates)水解後之焦填酸（pyrophosphate) • 釋放，即，β與γ磷酸鹽複合物之釋放，來監測聚合反應。 可直接偵測複合物，例如藉由在複合物上之螢光部分，或 間接偵測複合物，藉由將焦磷酸與一化學或生物冷光偵測 系統結合。 在一些實施例中，藉由使用經末端-磷酸鹽標誌之核苷 酸（terminal-phosphate-labeled nucleotide)將核酸樣本定 序。經末端-磷酸鹽標誌之核苷酸與其使用方法之實施例序 述於，例如美國專利號7,361,466與美國專利公開號 2007/0141598，公開於2007年6月21日。簡單地說，將 核苷酸提供至實施例所提供之裝置，當於聚合中水解時， 藉由對應之光偵測器偵測經標誌之磷酸鹽。在一些實施例 中，所有之四種核苷酸包括有區別之標誌且可被同時加 入。在一些實施例中，核苷酸包括無法區別，即，相同之 標誌’且被以預定之順序連續地加入。連續、猶環的加入 具有無法區別之標誌之核苷酸，依然允許多重、不中斷聚 合步驟，例如於均聚體序列中。 29 201017163 2.2.3 連接酶定序（Ligase-Based Sequencing) 在其他實施例中，藉由連接酶定序，將一樣本核酸於 實施例所提供之光學偵測裝置上進行定序。連接酶定序方 法敘述於，例如美國專利號5,750,341、PCT公開w〇Using Antibodies: A Practical Handbook CRC Press; 1st edition (2006). In yet another embodiment, the sample nucleic acid can be attached to the conjugation site by a polymerase, such as DNA polymerase. Those skilled in the art will appreciate that in order to preserve the function of the enzyme, information such as one, two and three levels of enzymes should be considered. For example, the structure of Taq and Phi29 polymerase is known in the art, see: Kim et al., 376:612-616 (1995) and Kamtekar et al., Mo/. Cell, 16:609-618 (2004). ). A method of immobilizing a polymerase to a surface while maintaining activity is known in the art and is described, for example, in U.S. Patent Publication No. 2008/0199932' issued on August 21, 2008 and Korlach et al. iWAS 105: 1176-1181 (2008). Figure 8 is a graphical representation of an embodiment in which sample nucleic acids (i.e., nucleic acid 32 to be sequenced, end-linking primer 34, and sequencing primer 346) are bound by a polymerase 38 to a linker 220' and aggregated. The enzyme 38 has been bound to the linker 220 by method 384, such as direct non-covalent adsorption, an antibody, biotin or chemical linkage, such as a chiral linkage. In some embodiments, an acetaldehyde-modified surface of a linker is treated with an acetaldehyde-containing silane reagent. Acetaldehyde quickly reacts with a primary amine on the protein to form a Schiffs base linkage. Since many proteins are exposed to the surface of the amine in addition to the more reactive alpha-amines generally at the NH2 end, they can be attached to the slide in a variety of orientations to allow the different sides of the protein to be in the solution. Protein or small 25 201017163 molecular response. In another embodiment, by photoactivation of a UV light, a light NHS (photoNHS) (N-hydroxy succimido carboxylate is a carbon charcoal chain linker (linker) ), attached to azidonitrobenzene, attached to an amine-modified surface on the element. In these examples, by eliminating nitrogen, the UV light excites the monoazide nitrobenzene moiety to produce a highly reactive nitrene. Nitrogen is immediately reacted with NH2 on the surface of the element to form a hydrazine bond. The other end of the linker is an NHS carboxylate which reacts with the amine acid on the surface of the polymerase to produce a covalent bond of the oxime amine. In another embodiment, the <RTI ID=0.0>> The UV light is used to activate a stack of nitrosyl Schottylbenzene moieties and form a highly reactive nitrogen oxime which is an electron deficient group and reacts immediately with the primary amine of the amino acid residue on the surface of the polymerase. These methods are described in more detail in Example 4 below. 2.2 Sequencing Forms 生物 The bioanalytical system provided in the Examples can be used to detect and sequence nucleic acids 'by methods known in the art, as reviewed, for example, in U.S. Patent No. 6,946,249 and Shendure et al., Ato and Ev 5: 335-44 (2004). In some embodiments, the sequencing method relies on the specificity of not being DNA polymerized S# or DN A ligase, and includes, for example, base extension sequencing (single base stepwise extension) ), by synthetic multi-base sequencing (including, for example, sequencing with terminally labeled nucleotides) and swinging sequence (w〇bWe 26 201017163 sequencing) 'which is a link. All methods typically require a single strand of sample nucleic acid, including at least one end-linking primer to be attached to a linker (not directly or indirectly). Sequencing is then carried out in the -sequence primer, (ligase sequencing - generally referred to as an anchor primer, which is used for analog purposes). For all sequencing forms, the examples provide a single molecule that can be reordered. For example, after the sequencing reads are completed, the sequencing primers and the extended nucleotides can be deleted from the sample nucleic acid, the components washed, and the sequence repeated. In various embodiments, re-sequencing can be re-executed by the same or different methods. By reordering the same molecules, it is expected that the sequencing error will fall to the power of the sequential reading = number. For example, if the error of reading each base in a single time is 1〇_3, then after two readings, it falls to (1〇-3)2, that is, 1〇-6. This is a particular advantage of single molecule sequencing, since the modified nucleotides used for sequencing can lose their signature or hinder the generation of groups, such as false deletions. 2.2.1 Base Extension Sequence: Progressive Extensions • In some embodiments, the light detection device provided by the embodiments can be used to perform base extension sequencing as disclosed in, for example, U.S. Patent No. 5,302,509. In some embodiments, the end-linking primer 34 comprising a single-stranded nucleic acid 32 to be sequenced, a 3, and a single-stranded nucleic acid 32 to be sequenced, and a certain sequence of primers bonded thereto A portion of the double repeat sample nucleic acid of 346 is attached to a linker 220 to initiate base extension sequencing, as shown in FIG. In some embodiments, the polymerase 38 and the modified nucleotide are then provided to a photodetecting element in a suitable buffer solution. In some embodiments, the sample nucleic acid complex is attached to the junction by a polymerase at the conjugation site. In some embodiments, the nucleotides include a detectable marker of a monovalent linkage, such as a fluorescent marker, and a barrier group to avoid any secondary extension. Therefore, a single nucleotide is added to the sequencing primer 346 彳I(4)(d). The P diagram at the 3' end is shown in the first step of the example of the assay extension reaction. A nuclear ridge acid 362 polymerase 38 having a fluorescent hindrance group 364 is added to the 3 of the sequencing primer 346, which is creeped. In some cases, the implementation of the financial symbol plays a blocking group. In other embodiments, the towel a 00 4 r is divided into separate parts. - The early-nucleotide is incorporated into the 3rd of the ordering primer 346, and the end is made up of the corresponding light (4) 2). The ruthenium and hindrance groups are then removed, for example, by chemical or enzymatic decomposition, to allow for an additional cycle of the base 2 extension. In the example wipes, the target and hindrance groups can be removed in a sequential manner. By editing the base addition sequence, the sequence of the sample nucleic acid is deduced from 3' to 5, direction '-times-test bases. Figure 9 shows a cycle in which some sample nucleic acids are extended, detected, and deblocked/de-marked in parallel. In general, there are two ways to recognize the nucleotides added during the stepwise extension. In a first example, the four nucleotides have the same detectable signature, but one at a time in a predetermined order. The identification of the extended nucleotide is confirmed by the order of the nucleotides added in the extension reaction. In the second mode of identifying the integrated base upon extension, four different nucleotides are added simultaneously and each binds to a distinguishable detectable marker. In various embodiments, the excitation or emission spectrum and/or intensity of the markers can vary. The strength of the nucleotide added to the extension is confirmed by the intensity and/or wavelength of the detectable marker (i.e., the excitation or emission spectrum). Implementation of these two methodologies 28 201017163 The example is presented in Example 5. 2.2.2 Sequence by synthesis: multi-step extension In some embodiments, the sequence by synthesis can be by multiple uninterrupted extensions, such as without the use of barrier groups. To execute. In these examples, the polymerization was monitored by detecting the release of pyrophosphates after hydrolysis of the nucleoside triphosphates, i.e., the release of the beta and gamma phosphate complexes. The complex can be detected directly, for example by combining the fluorescent moiety on the complex, or indirectly detecting the complex, by combining pyrophosphate with a chemical or biological luminescence detection system. In some embodiments, the nucleic acid sample is sequenced by using a terminal-phosphate-labeled nucleotide. Examples of end-phosphate-labeled nucleotides and methods of use thereof are described in, for example, U.S. Patent No. 7,361,466 and U.S. Patent Publication No. 2007/0141598, issued Jun. 21, 2007. Briefly, nucleotides are provided to the apparatus provided in the examples, and when hydrolyzed in the polymerization, the labeled phosphate is detected by a corresponding photodetector. In some embodiments, all four nucleotides include a distinctive signature and can be added simultaneously. In some embodiments, the nucleotides include indistinguishable, i.e., the same flag' and are continuously added in a predetermined order. The addition of a continuous, quaternary ring of nucleotides with indistinguishable markers still allows for multiple, uninterrupted polymerization steps, such as in homopolymer sequences. 29 201017163 2.2.3 Ligase-Based Sequencing In other embodiments, the same nucleic acid is sequenced on an optical detection device provided by the examples by ligase sequencing. The ligase sequencing method is described, for example, in U.S. Patent No. 5,750,341, PCT Publication.

06/073504 與 Shendure et al. 309:1728-1732 (2005) 中。在Shendure et al.的方法中，例如，一未知單股DNA 樣本可位於兩個末端連結引子的侧面，且固定於一固體支 持物上。於未知序列之一特定位置（即，接近於一特定末 參 端連結引子之nth鹼基），可藉由將一所謂錨定引子（其為 一定序引子之類似物）與末端連結引子黏合，且之後將4 個退化之九聚體的聯合提供至混合物來檢視。四個九聚體 皆具有有區別之螢光標誌，且除了質問位置（query position) 其在所有位置皆為退化的，在質問位置各九聚體以一可區 別之鹼基—A、C、G或T來質問。將樣本清洗、螢光掃瞄 且鑑疋質問之鹼基。之後自樣本核酸去除錨定引子與經連 結之九聚體、清洗元件且重複步驟，以質問一不同位置。06/073504 and Shendure et al. 309:1728-1732 (2005). In the method of Shendure et al., for example, an unknown single-strand DNA sample can be located on the side of the two end-linking primers and immobilized on a solid support. At a specific position in an unknown sequence (ie, an nth base close to a specific terminal end-linking primer), a so-called anchoring primer (which is an analog of a certain order primer) can be bonded to the end-linking primer. The combination of 4 degenerate 9-mers is then provided to the mixture for review. Each of the four 9-mers has a distinctive fluorescent signature and is degraded at all positions except for the query position, where the 9-mers are distinguished by a base - A, C, G or T to question. The sample is cleaned, scanned, and the base of the question is examined. The anchored primer and the ligated 9-mer are then removed from the sample nucleic acid, the components are washed and the steps are repeated to challenge a different location.

有利地，此方法為非漸次的，即，驗基不需依次被質問。 因此，錯誤不會累計。此外，此方法可自不是5,就是3,方 向來質問核苷酸，即，不需標準之5，—3，DNa合成。藉由 此方法，可通常將總共約13個驗基之一樣本核酸進行定 序。 2·3應用 生物分析系統可同時偵測數百萬之核酸片段。若各片 段為，例如1000個鹼基長，一單一元件一次可獲得超過數 30 201017163 十億個鹼基序列。此處所提供之元件的額外應用與方法將 於以下討論。 2.3.1整個基因體定序 生物分析系統可用來執行，例如病毒、細菌、真菌、 真核生物或脊椎動物，例如哺乳動物’例如人類，之整個 或部分基因體定序。 為了定序’可將基因體DNA切割成至少2〇、50、1〇〇、 • 200、300、500、800、1200、1500個核苷酸或更長之片段。 在一些實施例中，經切割之基因體DNA可為自20至50、 自 20 至 100、自 20 至 500、自 20 至 1000、自 5〇〇 至 1200 或自500至1500個核苦酸長。在一些實施例中，為了如上 述之定序’具有經結合之末端連結引子之要被定序的核 酸’被製成單股、與定序引子黏合且被提供至實施例所提 供之元件。 〇 2.3.2 基因表現研究(Gene Expression Profiling) 在其他實施例中，為了基因表現研究，生物分析系統 可用來將cDNA定序。例如，藉由測量於元件上被偵測之 特定序列的相關頻率，可將mRNA程度進行定量。可將數 百萬cDNA分子平行定序於實施例所提供之一元件上。若 一細胞平均包含350,〇〇〇個mRNA分子，於一百萬個定序 反應中，預期將存在於正好每個細胞一複製的一轉錄物 (transcript)定序三次。因此，實施例所提供之元件適合具有 單一數目靈敏度之單一分子定序。 31 201017163 cDNA合成為本技術領域所熟知，且一般包括總rna 萃取與視需要而^之mRNA豐富。藉由步驟，包括，例如： 為了第股合成之反轉錄；RNAse處理以移除殘餘rna ;Advantageously, the method is non-progressive, i.e., the test base need not be challenged in turn. Therefore, the error will not accumulate. In addition, this method can be used to question nucleotides from 5, or 3, ie, without the need for standard 5,-3, DNa synthesis. By this method, a sample nucleic acid of a total of about 13 test groups can usually be sequenced. 2. 3 Applications The Bioanalytical System detects millions of nucleic acid fragments simultaneously. If each fragment is, for example, 1000 bases long, a single element can obtain more than 30 201017163 billion base sequences at a time. Additional applications and methods for the components provided herein will be discussed below. 2.3.1 Whole genomic sequencing The bioanalytical system can be used to perform whole or partial genomic sequencing of, for example, viruses, bacteria, fungi, eukaryotes or vertebrates, such as mammals such as humans. For sequencing, the genomic DNA can be cleaved into fragments of at least 2, 50, 1 〇〇, 200, 300, 500, 800, 1200, 1500 nucleotides or longer. In some embodiments, the cleaved genetic DNA can be from 20 to 50, from 20 to 100, from 20 to 500, from 20 to 1000, from 5 to 1200, or from 500 to 1500 nucleotides long. . In some embodiments, the nucleic acid' to be sequenced with the bound end-linking primers as described above is made into a single strand, bonded to a sequencing primer, and provided to the elements provided by the examples. 〇 2.3.2 Gene Expression Profiling In other embodiments, bioanalytical systems can be used to sequence cDNA for gene expression studies. For example, the degree of mRNA can be quantified by measuring the relative frequency of a particular sequence detected on the element. Millions of cDNA molecules can be sequenced in parallel to one of the elements provided in the examples. If a cell contains an average of 350, mRNA mRNA molecules, in one million sequencing reactions, it is expected that a transcript that replicates exactly one cell will be sequenced three times. Thus, the elements provided by the examples are suitable for single molecule sequencing with a single number of sensitivities. 31 201017163 cDNA synthesis is well known in the art and generally includes total RNA extraction and mRNA enrichment as needed. By steps including, for example: reverse transcription for the synthesis of the strand; RNAse treatment to remove residual rna;

第一股之隨機六聚體起始(priming)與藉由DNA聚合酶之 第二股合成，來由mRNA產生cDNA。合成之cDNA適合 在實施例所提供之元件上進行定序。分離與製備DNA與 RNA兩者之方法為本技術領域所熟知。參見’ j〇seph Sambrook and David Russell, Molecular Cloning: AThe first strand of random hexamer priming and the second strand of DNA polymerase are used to generate cDNA from mRNA. The synthesized cDNA is suitable for sequencing on the elements provided in the examples. Methods for isolating and preparing both DNA and RNA are well known in the art. See also ' j〇seph Sambrook and David Russell, Molecular Cloning: A

Laboratory Manual Cold Spring Harbor Laboratory Press, 3rd ❹ edition (2001)。 在一些實施例中’藉由於美國專利號6,812,005與 7,361，488中所述方法可將cDNA進行定序。簡短來說， cDNA與接合物（adapter)聚核酸連結，以專一限制酵素來處 理接合物，且最後經處理之核酸與附著於實施例所提供元 件之連結位的互補寡核皆酸結合。在實施例中，接合物分 子為末端連結引子。 在一些實施例中，HiRNA之多腺嘌呤尾巴（poly A tail) ❹ 可做為一適合之末端連結引子，其與一多胸腺嘴淀(poly T tail)定序引子互補。 2.3.3偵測及/或測量結合之相互作用 在其他實施例中，生物分析系統可用來偵測各種結合 相互作用，包括，例如DNA/DNA、RNA/RNA或DNA/RNA 鹼基配對、核酸/蛋白質相互作用、抗原/抗體、受器/配體 結合與酵素/受質結合。一般而言，一樣本分子附著於包括 32 201017163 一鏗定核酸標籤（identifying nucleic acid tag，id)之連乡士八 子。在一些實施例中，連結分子更包括一與樣本分子結合 之捕獲分子。連結分子也包括用以與連結位結合之工具， 例如一促進共價化學連結之部份，例如，雙硫、硫酯二醯 胺、填酸二酯或酯連結；或藉由非共價連結，例如抗體/抗 原或生物素/抗生物素蛋白結合。在一些實施例申，藉由^ 標鐵將連結分子附著至陣列。Laboratory Manual Cold Spring Harbor Laboratory Press, 3rd ❹ edition (2001). In some embodiments, the cDNA can be sequenced by the methods described in U.S. Patent Nos. 6,812,005 and 7,361,488. Briefly, the cDNA is ligated to the adapter polynucleic acid to specifically conjugate the enzyme to the conjugate, and the final treated nucleic acid is combined with the complementary oligonuclear acid attached to the junction of the elements provided in the examples. In an embodiment, the conjugate molecule is an end-linking primer. In some embodiments, the poly A tail of the HiRNA can be used as a suitable end-linking primer that is complementary to a poly T tail sequencing primer. 2.3.3 Detection and/or Measurement of Binding Interactions In other embodiments, bioanalytical systems can be used to detect various binding interactions, including, for example, DNA/DNA, RNA/RNA or DNA/RNA base pairing, nucleic acids. /protein interactions, antigen/antibody, receptor/ligand binding combined with enzyme/substrate. In general, the same molecule is attached to the Lianxiangshi Bazi including 32 201017163 identifying nucleic acid tag (id). In some embodiments, the linker molecule further comprises a capture molecule that binds to the sample molecule. Linking molecules also include means for binding to the linking sites, such as a moiety that promotes covalent chemical bonding, for example, disulfide, thioester diamine, acid diester or ester linkage; or by non-covalent linkage For example, antibody/antigen or biotin/avidin binding. In some embodiments, the linker molecules are attached to the array by the iron.

，一樣本分子提供至一元件，且藉由其之連結分子將 ’、附著於卩通機連結位，例如藉由與一位於連結分子上之 ，獲分子結合。在—些實施射，將樣本分子與連結分子 奶口允許其結合、且之後提供至實施例所提供之一元件。 ^1些2_巾’連結分子先被提供至元件，允許其附著 人之而之後提供樣本分子。再來，藉由鑑定經結 =個揭II的方法來㈣ID(例如’藉由雜合或定序）。 = 子種類可附著至相同之陣列，且藉由其之標 ’ ^使用與其結合之捕獲分子獨特 的ID，可以其 妙#二用為特徵。因此，在一些實施例中，偵測一 :::::本分子的方法包括，藉由包括-核酸標籤(m) ττ_ ^刀 來連結一樣本分子至一元件之連結位、執行 ID之核酸定序| 、 中 ， 、，、彳貞測經標遠、之樣本分子的步驟。在實施例 >核it定序為驗基延伸定序 。在一些實施例中，核酸定 糸擇自連接酶定序或經末端 -磷酸鹽標誌之核苷酸定序。 9由使用核脊酸“小段(bits)，，，上至4n個具區別之捕獲 缺：：被附著於與鑑定於生物分析系統上，其中η為-自 然數字顯示被定^ &序之ID的長度。例如，5個核苷酸可提供 33 201017163 超過一千個獨特ID，而12個核苷酸提供超過一千六百萬 個組合。例如，連結分子附著至一元件而其位置藉由偵測 其對應之ID標籤來確認。之後連結分子作為探針以，例如 調查與一或多個經標誌之樣本分子的結合相互作用。即， 具有一或多個連結分子附著至其的一元件可作為一探針陣 列。 在實施例中，一經標誌、之樣本分子為經螢光標諸。當 與連結分子之捕獲分子結合時，藉由對應於被連結分子附 著之連結位的光偵測器，來偵測經標諸之樣本分子。因此， ⑩ 在一些實施例中，方法可更包括提供一經標誌之樣本分子 至一元件及偵測經標誌之樣本分子的步驟。在實施例中， 元件具有附著至連結位之連結分子，而連結分子包括一附 著至連結位之核酸標籤（ID)。可將多重標誌樣本分子同時 提供至一探針陣列且可藉由其標誌來區分，例如藉由其螢 光標諸之強度及/或波長。在一經標誌質問分子之所給予的 濃度下，基於動力學（例如，接合（docking)/與未接合 (undocking))與統計學（例如，在任何給予之時間，樣本 ® 分子之部分為結合或為結合之狀態）兩者可干擾介於樣本 分子與經標誌質問分子之間的結合相互作用的分離常數。 在一些實施例中，一連結分子之ID為至少5、10、15、 20、25、30、40、50、75、90、100、150、200 或更多個 核苷酸長。在一些實施例中，ID為自5至10、20、40、80 或160 ;或自10至20或50 ;或自20至35個核苷酸長。 ID包含一獨特之核酸序列，即，一要被偵測之核酸。在實 施例中，獨特之核酸序列可為至少1、2、4、6、8、10、 34 201017163 12、14、ι6、2 例中，獨特> 4 4、3〇或更多個核苷酸長。在一些實施 10至20個桉^序列為自4至1〇、12、15或20;或自 ID包含—序=長。1D包括至少-末端連結引子，即， 中，為經修飾的、與—定序引子互補，此序列在-些實施例 生物素化2核昝龄以附著至一連結位，例如藉由包含一經 部分為3,蠕。在—些實施例中，1；0之末端連結引子 至獨特核i轉列、核酸相。在—些實施例中，其為5,端 於獨特之核_的=另〜實Γ中，末端連結引子出現 在實施例中，播:端兩 化合物、r質 樣本分子與捕獲分子包括係擇自一碳水 有機分子Γ例如2質、胜肽、抗原、核酸、荷爾蒙、小 這些部分可為自t予上的）或維他命部分或其組合之部分。 如經化學合&咬=發生（例如經生物化學純化）或合成（例 包含一些或全部雜魬產生）。此外，這些基質可沒有包含、 保護基團等）。在天然成分（例如非天然胺基酸、阻礙或 白質，例如生長因實施例中，一樣本分子或捕獲分子為蛋 許多結合核酸子、、胜肽抗原、抗體或受器。 域所知，如回顧於 '至連結分子或連結位的方法為本技術領 ‘33丨公開揭露形成例如美國專利公開號2004/0038331中。 上。美國專利號4蛋白質养核苷酸結合物於一固態支持物 端的一實施例。其中’111長1供結合蛋白質至一核酸之3 ’ 至一分子之3,部分’末端轉移酶首先用來將核糖殘基加 應之後產生一3， 過碟酸氧化(periodate oxidation)反 蛋白質之醯胺基基團於一核酸上’ 3,乙醛基團之後與 $成共價鍵結。當蛋白質結合至ID之3, 35 201017163 端時，係經由ID之5’端附著至連結位。 在一些實施例中，一捕獲分子，例如一蛋白質，連結 至一 ID之5,端。在這些實施例中，IDi 3,端或一定序引 子之5,端係用來將捕獲分子附著至—連結位。美國專利號 6,013,434，例如揭露寡核苷酸聚醯胺結合物，其中連接係 藉由募核苷酸之5端。美國專利號6,丨97,513揭露pNA與 DNA兩者經由核酸之5端結合至一具有竣酸部分之分子， 例如蛋白質。PNA與DNA分子包含芳香胺(arylamine)或氮 氧乙醯(ammooxyacetyl)部分。美國專利號6，153,737揭露⑩ 包含至少一 2’經官能化之核苷的寡核苷酸，適合與多種分 子結合。 2.3.4額外之偵測方法The same molecule is provided to a component, and the linker molecule is attached to the linker, for example by binding to a molecule on the linker molecule. In some implementations, the sample molecules are allowed to bind to the linked molecular milk mouth and are then provided to one of the elements provided in the examples. ^1 2 towel's linker molecules are first supplied to the component, allowing it to attach to the person and then providing the sample molecule. Again, (4) ID (e.g., by heterozygous or sequencing) by identifying the method of merging. The sub-category can be attached to the same array, and its unique identifier, which is unique to the capture molecule to which it is associated, can be characterized by its use. Therefore, in some embodiments, the method of detecting a ::::: molecule comprises: linking the nucleic acid of the same molecule to a binding position of the element by including a nucleic acid tag (m) ττ_^ The steps of sequencing |, , , , , , and measuring the sample molecules of the standard. In the example > nuclear it is sequenced as the basis extension sequence. In some embodiments, the nucleic acid is selected from ligase sequencing or nucleotide sequencing via a terminal-phosphate label. 9 by using nuclear spine "bits,", up to 4n differential capture:: is attached to and identified on the biological analysis system, where η is - natural digital display is determined ^ & The length of the ID. For example, 5 nucleotides can provide 33 201017163 with more than a thousand unique IDs, while 12 nucleotides provide more than 16 million combinations. For example, a linker molecule attaches to a component and its position is borrowed. Confirmed by detecting its corresponding ID tag. The linker molecule is then used as a probe to, for example, investigate the binding interaction with one or more labeled sample molecules. That is, one having one or more linker molecules attached thereto The component can serve as a probe array. In an embodiment, the labeled sample molecule is a fluoroscopic cursor. When combined with the capture molecule of the linker molecule, the light is detected by a link corresponding to the attached molecule. For detecting the labeled sample molecules. Thus, in some embodiments, the method may further comprise the steps of providing a labeled sample molecule to a component and detecting the labeled sample molecule. In an embodiment The element has a linker molecule attached to the linker, and the linker molecule comprises a nucleic acid tag (ID) attached to the linker. The multiplexer sample molecule can be simultaneously provided to a probe array and can be distinguished by its flag, for example By virtue of the intensity and/or wavelength of its cursor, based on the concentration given by the marker molecule, based on kinetics (eg, docking/undocking) and statistics (eg, at At any given time, the portion of the sample® molecule is bound or in a bound state. Both can interfere with the separation constant between the sample molecule and the labeled interaction between the labeled interrogating molecules. In some embodiments, a linker molecule The ID is at least 5, 10, 15, 20, 25, 30, 40, 50, 75, 90, 100, 150, 200 or more nucleotides in length. In some embodiments, the ID is from 5 to 10 , 20, 40, 80 or 160; or from 10 to 20 or 50; or from 20 to 35 nucleotides in length. The ID comprises a unique nucleic acid sequence, ie a nucleic acid to be detected. In an embodiment The unique nucleic acid sequence can be at least 1, 2, 4, 6 8, 10, 34 201017163 12, 14, ι6, 2 cases, unique > 4 4, 3 〇 or more nucleotides long. In some implementations 10 to 20 桉 ^ sequence is from 4 to 1 〇, 12, 15 or 20; or self-ID inclusion - order = length. 1D includes at least - end-linking primers, ie, in the modified, complementary to - sequencing primers, this sequence is biotinylated in some embodiments 2 The core age is attached to a link, for example by including a portion of 3, in some embodiments, the end of 1; 0 is linked to the unique core i-transform, nucleic acid phase. In some embodiments In the case of 5, which is in the unique nucleus _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ For example, a moiety such as a substance, a peptide, an antigen, a nucleic acid, a hormone, or a small portion may be a part of a vitamin moiety or a combination thereof. Such as chemical synthesis & bite = occurrence (such as biochemical purification) or synthesis (including some or all of the production of chowder). Furthermore, these matrices may contain no, protecting groups, etc.). In the case of natural ingredients (eg, unnatural amino acids, barriers or white matter, such as growth examples, the present molecule or capture molecule is an egg, a plurality of binding nucleic acids, a peptide antigen, an antibody, or a receptor. A review of the method of "to a linker molecule or a linker" is disclosed in US Patent Publication No. 2004/0038331. U.S. Patent No. 4 protein-nuclear nucleotide conjugate at a solid support end An embodiment wherein '111 is 1 for binding a protein to 3' of a nucleic acid to 3, and a portion of the 'terminal transferase is first used to add a ribose residue to produce a 3, peracid oxidation. The anti-protein guanamine group is on a nucleic acid '3, and the acetaldehyde group is covalently bonded to $. When the protein binds to ID 3, 35 201017163, it is attached via the 5' end of the ID. In some embodiments, a capture molecule, such as a protein, is linked to the 5' end of an ID. In these embodiments, IDi 3, 5 or a sequence of primers, is used to capture Molecular attachment to U.S. Patent No. 6,013,434, for example, discloses an oligonucleotide polyamine conjugate in which the linkage is by the 5th end of the nucleotide. U.S. Patent No. 6, 丨97,513 discloses both pNA and DNA via the 5th end of the nucleic acid. Binding to a molecule having a capric acid moiety, such as a protein. PNA and DNA molecules comprise an arylamine or ammooxyacetyl moiety. U.S. Patent No. 6,153,737 discloses that 10 comprises at least one 2' functionalized A nucleoside oligonucleotide suitable for binding to a variety of molecules. 2.3.4 Additional detection methods

2.3.4.1FRET 在一些實施例中，於一本發明所提供之光偵測裝置上 偵測一分子’藉由Forster共振能量轉移(F〇rster res〇nance φ energy transfer，FRET)，有時也知為螢光共振能量轉移 (Fluorescence resonance energy transfer, FRET)。如本技術 領域所知，當一經激發之提供者分子非放射性轉移能量至 一接文者分子時，其散發能量，一般為光時，FRET發生。 FRET可幫助減低背景訊號，藉由，例如提供對於要偵測分 子之介於有效激發與散發波長之間的較大光譜間隔。fret 常用來偵測接近之分子相互作用，由於其功效衰敗隨著介 於提供者與接受者分子間之距離的第六能量。例如，Zhang 36 201017163 et al_ (漏㈣施初油，4:826_31 (2〇〇5))藉由fret偵測核 酸雜合。^中，—經生物素化之核酸標的與—覆蓋抗生物 素蛋白之《子點(quantum dot)提供者結合，其之後激 經⑺、结合之驗探針。在本發明一些實施例中，二經 標諸之分子與經標認之樣本分子可形成—用以藉由 FRET來_之提供者/接受者（歧之減）配對。2.3.4.1 FRET In some embodiments, a molecule is detected on a photodetection device provided by the present invention by F 〇 rster res〇nance φ energy transfer (FRET), sometimes Known as Fluorescence resonance energy transfer (FRET). As is known in the art, FRET occurs when an excited donor molecule non-radioactively transfers energy to an electroner molecule, which emits energy, typically light. FRET can help reduce the background signal by, for example, providing a larger spectral separation between the effective excitation and emission wavelengths for the molecules to be detected. Fret is often used to detect near-molecular interactions due to its efficacy decaying with the sixth energy between the donor and the acceptor molecule. For example, Zhang 36 201017163 et al_ (leak (four) initial oil, 4:826_31 (2〇〇5)) detects nuclear heterozygosity by fret. ^, - the biotinylated nucleic acid target is combined with the "quantum dot" provider covering the avidin, which is then stimulated (7), combined with the probe. In some embodiments of the invention, the labeled molecules are formed with the labeled sample molecules - for pairing with the provider/recipient of FRET.

/實施綱提供之核酸定相-㈣_中，對於一 附著至-聚合酶或連接酶之提供者帶色團⑽麵叩h㈣， 螢光標純苷酸扮演1受者帶色目。因此，於這些實施 或連接酶上之提供者帶色團激發-核酸 ^的^者▼色團’而核酸要被聚合於或連接至樣本核 在FRET錢巾之快速下降，*接近聚合酶之 核皆酸不被激發。在—些實施例中，提供者分子為，例如 另一榮光®，例如—量子點。量子點，例如，半導體量子 點為本技術領域所知且敘述於，例如國際公開號wo 03/003015。結合量子點至，例如生物分子的方法為本技術 領所知’如回顧於’例如Mednhz et al (偏騰施⑽油， 4:235-46 (2005))與美國專利公開號2〇〇6/〇_皿盥 2〇〇8/_7843分別公開於屬年3月20日與細8年/月 Π日。在-些實施例中，量子點與—舰聚合酶分子結 合，其更進-步敘述於以下之實施例3。如先前所討論， 為了將酵素與連結位結合，熟悉此技藝人士可毫無疑問地 理解田將螢光團結合至，例如一 DNA聚合酶或連接酶時 必需小心，以藉由減輕結合螢光團至酵素之一級、二級與 二級結構上的任何影響來保持酵素功能。 37 201017163 2.3.4.2多光子激發 在一些實施例中’由兩個或更多之光子激發一帶色 團。例如，在一些實施例中’於FRET中，不是提供者就 是接受者帶色團之激發係經由兩個或更多之光子。兩個光 子與多光子激發更進一步敘述於’例如美國專利號 6,344,653 與 5,034,613 中。 2.3.4.3 時間解析债測(Time Resolved Detection) 在一些實施例中，可調整本發明所提供之光源與光偵 測器以具有一特徵相移（characteristic phase shift)。使用本 技術領域已知的方法’例如’如於美國專利公開號 2008/0037〇08公開於2008年2月14日，揭露了，散發自 於實施例所提供之元件上被偵測之分子的光，可藉由一對 應光偵測器來測量，而無來自激發光源的干擾。 魯 3.使用生物分析系統之生物分子分析服務 β實施例也提供一藉由使用根據本發明生物分析系統來 ^供生物分子分析服務的方法。在—些實施例中，方法包 ^服務，求者提供—包括—要被分析之生物分子的樣 分折供者，與服務請求者接受來自服務提供者之 結果了驟其中藉由使用本發明所提供之元件產生 :服務費：ϊ實施例中，為了報酬之考慮來執行方法，例 服務nL㈣服務協定。此外，縣可被直接運送於 務响未者與服務提供者之間，或藉由一賣主(ven㈣居中 38 201017163 運送。在一些實施例中，服務提供者或賣主可地理性位於 美國之外的領土，例如於另一國家。 除非以其他方式指出，使用於說明書，包括申請專利 範圍中之成分、反應條件等的所有數字表現量，可被理解 為在所有情況下藉由措辭“約，，來修飾。 除非以其他方式指出，發出一系列要素之措辭“至少，， 可被瞭解為意指於系列中之每一要素。 ❹ 【實施例】 實施例1:一高通量生物分析系統之建構 藉由參閱第1-4圖，製造一生物分析系統丨之方法將 會於以下進行詳述。首先，藉由商業上可得之用於一般邏 輯與光學元件的0.25 μιη半導體製造製程一起提供一石夕基 板10與形成於基板1〇上表面上之複數個光偵測器21〇。 光4貞測器210為光二極體光子债測器，各具有一 24 之 直徑與一 452 μιη2之露出面積。將各光偵測器排列成彼此 鄰接以便光偵測器210之512行與512列的陣列形成於基 板10上。 複數個控制電路215形成於基板1〇之上表面之沒有形 成光偵測器210的位置上。於此實施例中，一控制電路215 對應於一光偵測器210，以便控制其對應之光偵測器210 之操作及控制介於光偵測器210與偵測記錄系統2間之通 訊。 39 201017163 在此實施例中，一濾層240形成於光偵測器210之上 表面與控制電路215上。在形成濾層240前，對光偵測器 210之上表面與控制電路215實施全面平坦化（global planarization)製程。遽層240包括複數個次層。在此實施 例中，濾、層240可包括形成於一基板材料(base substrate material)上之大量次層，其可藉由形成（forming)、放置 (placing)或沈積一基板材料，例如Si02於基板1〇上來提 供。在一實施例中，可實施半導體製造或沈積方法。例如， 沈積方法可包括，但不限於’物理沈積、化學沈積、分子 束蠢晶（molecular beam epitaxy，MBE)與原子層沈積（atomic layer deposition, ALD) ° 在一些實施例中，基板材料可包括一或多個凹陷形成 於一表面上。在一些情況中，凹陷可為非平面，例如具有 一經定義之半徑的半球形或弧線形。例如，參見第3a圖， 於一實施例中，最接近基板1 〇之濾層240之次層的半徑可 為約6.1 μιη，且可自1 μιη變化至100 μιη。於一系統中之 凹陷的尺寸可根據系統之設計與應用及所對應之針孔的尺 寸來變化。在一實施例中，根據許多考量可變化半徑例如， 系統之應用、設計、特徵、光債測器及/或光源與要被偵測 之生物分子的特徵。在一些實施例中’凹陷可為基板形成 製程之部分或藉由使用一般半導體製程移除技術來移除基 板材料的一部份來形成。例如，移除方法可包括渥姓刻、 乾蝕刻或其方法。在其他實施例中，可在基板材料形成時， 使用壓力或擠壓來形成凹陷。 ' —旦如上述形成渡層240之基板材料’系列之次層可 201017163 2形料之頂部上以產生如第3a圖所示之濾層 替代層可开Γ成列Γ居中，轉由沈積具有不同介電特性之材料的 且右^古;久層。例如，藉由首先於基板材料上沈積一 ^Γ丨#、率之次層於經平坦化之光偵測器210之上表 面與控制電路1·-^ 上可形成濾層240。之後’一具有較低 =率二次層沈積於已形成之具有較高折射率之次層上： ^ = f例中，具有較高折射率之次層可包含Nb205，/ Nucleic acid phase-providing provided by the protocol - (4), for a provider attached to the -polymerase or ligase with a chromophore (10) face 叩h (d), the fluorescein pure glycine acts as a recipient of the color. Thus, the provider on these implementations or ligases carries a chromophore-producing chromophore of the nucleic acid' and the nucleic acid is polymerized or attached to the sample nucleus at a rapid drop in the FRET,* close to the polymerase Nucleic acid is not activated. In some embodiments, the donor molecule is, for example, another glory®, such as a quantum dot. Quantum dots, for example, semiconductor quantum dots are known in the art and are described, for example, in International Publication No. WO 03/003015. Methods of binding quantum dots to, for example, biomolecules are known in the art as reviewed in 'For example, Mednhz et al (Pentium (10) Oil, 4: 235-46 (2005)) and U.S. Patent Publication No. 2, 6 /〇_皿盥2〇〇8/_7843 are published on March 20th and 8th/Month of the year respectively. In some embodiments, the quantum dots are combined with a -ship polymerase molecule, which is further described in Example 3 below. As discussed previously, in order to bind an enzyme to a binding site, those skilled in the art will undoubtedly understand that binding of a fluorophore to a DNA polymerase or ligase, such as a DNA polymerase or ligase, must be taken to mitigate binding fluorescence. Any effect on the primary, secondary and secondary structure of the enzyme to the enzyme maintains the function of the enzyme. 37 201017163 2.3.4.2 Multiphoton Excitation In some embodiments, a band of chromophores is excited by two or more photons. For example, in some embodiments, in FRET, it is not the provider that the recipient's chromophore excitation system passes two or more photons. Two photon and multiphoton excitations are further described in, for example, U.S. Patent Nos. 6,344,653 and 5,034,613. 2.3.4.3 Time Resolved Detection In some embodiments, the light source and light detector provided by the present invention can be adjusted to have a characteristic phase shift. A method known in the art is used, for example, as disclosed in U.S. Patent Publication No. 2008/0037, filed on Feb. 14, 2008, the disclosure of which is incorporated herein by reference. Light can be measured by a corresponding photodetector without interference from the excitation source. Lu 3. Biomolecular Analysis Services Using Bioanalytical Systems The beta embodiment also provides a method for providing biomolecular analysis services by using a bioanalytical system according to the present invention. In some embodiments, the method includes a service, the requestor provides - including - a sample share of the biomolecule to be analyzed, and the service requester accepts the result from the service provider by using the present invention The components provided are: service fee: In the embodiment, the method is implemented for the consideration of compensation, for example, the service nL (four) service agreement. In addition, the county may be shipped directly between the whistleblower and the service provider, or by a vendor (ven) at 38 201017163. In some embodiments, the service provider or vendor may be geographically located outside of the United States. Territory, for example in another country. Unless otherwise stated, all numerical expressions used in the specification, including the ingredients, reaction conditions, etc. in the scope of the patent application, can be understood to be understood in all cases by the wording "about," Modifications. Unless otherwise stated, the wording of a series of elements is "at least, can be understood to mean each element of the series." [Examples] Example 1: A high-throughput bioanalytical system Construction The method of fabricating a bioanalytical system by referring to Figures 1-4 will be described in detail below. First, it is provided by a commercially available 0.25 μm semiconductor fabrication process for general logic and optics. a plurality of photodetectors 21 形成 formed on the upper surface of the substrate 1 . The photo Detector 210 is a photodiode photon debt detector, each having a The diameter of 24 is an exposed area of 452 μm 2. The photodetectors are arranged adjacent to each other so that an array of 512 rows and 512 columns of photodetectors 210 is formed on the substrate 10. A plurality of control circuits 215 are formed on the substrate 1. In the embodiment, a control circuit 215 corresponds to a photodetector 210 for controlling the operation and control of the corresponding photodetector 210. The communication between the photodetector 210 and the detection recording system 2. 39 201017163 In this embodiment, a filter layer 240 is formed on the upper surface of the photodetector 210 and the control circuit 215. Before forming the filter layer 240 A global planarization process is performed on the upper surface of the photodetector 210 and the control circuit 215. The germanium layer 240 includes a plurality of sublayers. In this embodiment, the filter layer 240 may include a substrate material. A plurality of sublayers on the base substrate material, which may be provided by forming, placing, or depositing a substrate material, such as SiO 2 on the substrate 1. In one embodiment, semiconductor fabrication or Deposition Methods. For example, deposition methods can include, but are not limited to, 'physical deposition, chemical deposition, molecular beam epitaxy (MBE), and atomic layer deposition (ALD). In some embodiments, substrate materials One or more depressions may be included on a surface. In some cases, the depressions may be non-planar, such as hemispherical or arcuate with a defined radius. For example, see Figure 3a, in one embodiment, The sublayer of the filter layer 240 closest to the substrate 1 may have a radius of about 6.1 μηη and may vary from 1 μηη to 100 μηη. The size of the recess in a system can vary depending on the design and application of the system and the size of the corresponding pinhole. In one embodiment, the radius of the system can be varied, for example, based on the application, design, characteristics, optical fingerprint detector, and/or source of the biomolecule to be detected. In some embodiments, the recess can be formed as part of the substrate formation process or by removing portions of the substrate material using conventional semiconductor process removal techniques. For example, the removal method can include engraving, dry etching, or a method thereof. In other embodiments, pressure or extrusion may be used to form the depressions as the substrate material is formed. The sub-layer of the series of substrate materials forming the ferrosilicon layer 240 can be topped on the top of the 201017163 2-shaped material to produce a filter layer as shown in Fig. 3a, which can be reclaimed into a column, and is deposited by deposition. The material of different dielectric properties is right-handed; For example, the filter layer 240 can be formed by first depositing a sub-layer on the substrate material on the surface of the planarized photodetector 210 and the control circuit 1·-^. Thereafter, a secondary layer having a lower = rate is deposited on the formed sublayer having a higher refractive index: in the case of ^ = f, the sublayer having a higher refractive index may include Nb205,

高折射= 層可具有⑽2。藉由接連地沈積較 、衩低折射率之次層，形成濾層240直到一大量 =已，積於基板材料上。在此實施射，滤層240包 ^ *人層。基於各種考量，例如系統之應用、設計、特 徵光债測器及/或光源，可改變次層之數目、其折射率、 其材料及/或其他參數。High refraction = the layer can have (10)2. The filter layer 240 is formed by successively depositing a lower, lower refractive index sub-layer until a large amount of it has accumulated on the substrate material. Here, the filter layer 240 is coated with a human layer. The number of sublayers, their refractive indices, their materials, and/or other parameters can be varied based on various considerations, such as system applications, designs, characteristic optical debt detectors, and/or light sources.

參見第5圖，其顯示一概括濾層24〇之一實施例建構 的表格。在第5 ®中’―較低減之:欠層提供-較接近慮 層240之底部表面的次層，而一較高編號之次層提供一較 接近濾層240之上表面的次層。第5圖中所示，於此實施 例中，濾層240之奇數次層由，例如五氧化二銳(Nb2〇5)所 製成，其具有一較高之折射率。偶數次層由例如二氧化矽 (SiOJ所製成，其具有一較低之折射率。次層可藉由使用 一錢鍍系統來形成，濺鍍系統之例子包括Model No. RAS 110 of Radical Assisted Sputtering Series，由 Shincron Co.， 1^(1.(81^1^§&〜&-1〇1，1'〇1^〇，认？八]^)所製造。於此實施例中 之各層之厚度也提供於第5圖之表格中。合成之濾、層240 對於螢光團Cy5之螢光為高度透明，而對於散發自氦氖 41 201017163 (Helium-Neon)雷射之光在約633 nm波長為低穿透度，氣 氖雷射做為一外部光源以激發螢光團Cy5。 再次參見第2與4圖，具有針孔235之遮蔽薄板230 形成於濾層240上。在—實施例中，針孔μ5之尺寸f為 約0.2μιη，且可自0>1 μιη變化至丨μιη。基於系統之設計 與應用及對應之濾層的尺寸可改變於系統中之針孔尺寸。 在一實施例中，基於許多考量’例如系統之應用、設計、 特徵、光偵測器及/或光源與要被偵測之生物分子的特徵可 改變半徑。製造具有針孔235之遮蔽薄板MO於爐層MO籲 或基板10上的一製程將於以下詳述。 首先，藉由’例如旋塗一光阻材料於濾層24〇上，形 成一光阻層於濾層240上（若濾層24〇為視需要形成於光 偵測器210之上表面與控制電路215上）或於經平坦化之 光债測器210之上表面與控制電路215上（若不形成慮層 240)。之後，將光阻顯影以在針孔區形成光阻圖案。藉由 使^光罩覆蓋針孔區，且將光阻曝光以使只有由光罩覆蓋 之區域維持於濾層240或經平坦化之光偵測器21〇之上表❹ 面與控制電路215上來形成光阻圖案。 之後，金屬層沈積於已形成光阻圖案之濾層24〇上。 在此實施例中’金屬層可包括鉻(Cr)，藉由執行—磁控濺 鍍製程（magnetron sputtering process)，其沈積於滅層 或經平坦化之光偵測器210之上表面與控制電路上。 接著，移除於針孔區上之金屬層部分與於針孔區。 光阻圖案，藉此形成具有針孔235之遮蔽薄板23〇。之 或者，藉由首先沈積一金屬層（例如，cr)於 ^於濾層240 42 201017163 上，之後形成一罩幕於金屬層上，藉此將金屬層之上表面 之部分曝光，來形成遮蔽薄板230。之後將金屬層曝光之 部分進打钱刻，直到露出濾層240，藉此形成針孔於金屬 層上。之後，將罩幕自金屬層移除且形成具有針孔2%之 遮蔽薄板230於濾層240上。在一些實施例中，以以〇2填 滿由介於遮蔽薄板230與最接近遮蔽薄板23〇之次層之間 的空間所形成之半球’且在形成遮蔽薄板23()冑，將半球 磨光以平坦表面。而上述金屬層具有形成於據層24〇之半 _ 球中央上的針孔235。 再次參見第2與4圖，於此實施例中，藉由在針孔说 或凹處45〇填入-支持材料來形成連結位22〇。支持材料 $為對於散發自螢麵36之螢光為透明之聚合物或無機 材料。 再次參見第1圖’雖然只顯示12個光學偵測裝置20。 可以瞭解的是’至少:萬個光學偵測裝置2〇可形成於基板 • 10上。例如，於此實施例中，各光學偵測裝置2〇呈有一 半經為約—或更小之圓形形狀，其可佔據約HKW之 面積。對於具有1平方英对（即，2.54Cm乘以2.54cm) 之面積的基板1〇而言，建構大於六百萬個光學制裝置 2〇於基板H)上為可_。藉由㈣操作那些六 學偵測裝置20，可以高通量來债測生物分子。 實施例2 :以 與偵測 向通量生物分析系統之生物分子的附著 43 201017163 一經螢光染劑Cy5標誌之核酸用來測試偵測系統。Cy5 與生物素分別附著至一 60員（mer)之寡核苷酸的3,與5, 端。將經標誌'與生物素化之DNA溶解於TrisMg (10 mM Tris，10 mMNaCl，100 mMMgCl2, pH8)緩衝溶液中、將其 沈積於位置陣列上’並將其培養於一保濕箱（humid chamber) 中。約30分鐘後，以Tris緩衝溶液將未結合之DNA清洗 掉。 經由一 635 nm光散發二極體來提供激發光，635 nm 光散發二極體可形成於遮蔽薄板上。為了讀取來自各像素 之訊號，將激發光開啟約1-5秒，記錄來自各像素訊號， 且將此循環重複一百輪。之後相應地計算各像素之代表平 均訊號與對應之標準差。將DNA樣本沈積前與後之訊號進 行比較，且具有大於3倍標準差總和之平均訊號差異的像 素視為正(positive)像素，即， (Avg 後-Avg 前)> 3X(STD 後-STD 前)。 實施例3 :連結量子點至聚合酶 以下為將經官能化之量子點結合至一聚合酶分子上之 一級胺的兩種方法’弟一種使用胺活化點（amine-activated dot)，第二種使用叛基活化點（carboxyl-activated dot)。 3.1胺EVITAGtm與DNA聚合酶之結合 經胺 EVITAGtm (例如 Evident Technologies, cat# E2-C11-AM2-0620 ; EVITAG™ 套組之 QD 產品也在 201017163 eBioscience，Inc” San Diego，CA 之 eFluor™ 標誌下販售） 官能化之量子點（QD)藉由BS3 (雙琥珀醯亞胺辛二酸酯納' 鹽（Bis(sulfosuccinimidyl) suberate))、一 同質雙功能 (homobifunctional)水可溶交聯劑來活化，其包含一胺反應 性N-羥基琥珀醯亞胺羧酸鹽（NHS)酯於一 8碳延長臂 (spacer arm)之各末端。在pH 7-9，NHS酯與QD表面上之 一級胺反應以形成穩定之醯胺鍵，且釋放NHS離去基團 (leaving group)。Taq DNA 聚合酶或 Phi29 DNA 聚合酶具 • 有許多一級胺（例如，離胺酸(K)殘基與各多胜肽之N端）， 其為可得之NHS酯交聯之標的。 3.1.1量子點之表面活化 以 25 μΐ 10 mM BS3(Bis(sulfosuccinimidyl) suberate)， Pierce, part# 21580)與 25 μΐ lOxPBS (磷酸緩衝鹽，pH 7.4) 將2.0 nmol之EVITAGtm活化於一具有dH2〇之終體積250 μΐ中。在培養30分鐘後，使用一 P10管柱(Amersham Biosciences,產品編號17-0851-01)將溶液去鹽且以lxPBS 洗提。有顏色之蛋白質包含經活化之QD。 3.1.2DNA聚合酶結合 藉由將於0.1 Μ碳酸鈉緩衝溶液中（pH 9.2)之100 pg 之DNA聚合酶加入至混合物中來結合聚合酶。在混合均勻 後，培養於4°C並傾斜旋轉2小時。 3.1.3QD結合之聚合酶的純化 45 201017163 藉由以 30K Microspin filter (Pall Corporation，part# OD100C33)在6000 i*pm離心5-10分鐘，將結合物濃縮至總 體積〜200 μΐ。藉由30K Microspin filter以dH20清洗結合 物兩次。 再來，以 Suprdex 30/75 Resin (GE Healthcare, part# 17-0902-10或17-1044-10用於小蛋白質與胜肽）藉由尺寸 排除來純化結合物。在將經濃縮之結合混合物載至管柱且 允許其進入柱床(column bed)前，以dH20預先平衡管柱。 在黑光激發下以dH20洗提這些樣本，並收集螢光片段。 ❹ 將螢光片段加在一起且藉由以100K Microspin filter在 6000 rpm離心5-10分鐘濃縮至總體積〜100 μΐ。經純化與 濃縮之結合物可儲存於4°C。 3.2羧基EVITAGtm舆DNA聚合酶之結合 經叛基 EVITAG™(例如 Evident Technologies，cat# E2-C11-CB2-0620)官能化之QD經由ECD居中之硫-NHS 酯偶合反應來活化。胺反應性硫-NHS酯與例如在TaqDNA ❹ 聚合酶或Phi29 DNA聚合酶上之離胺酸(K)之侧鏈中之一 級胺反應。 3.2.1量子點之表面活化 將 2.0 nmol 之 EVITAG™ 稀釋於 25 mM MES pH 5.0 緩衝溶液中。在立即使用前，EDC ( 1-乙基-(3-二甲基氨基 丙基）碳醯 二亞胺 鹽酸鹽 (l-(3-Dimethylaminopropyl)-3-ethylcarbodiimide 46 201017163 hydrochloride))溶解於冷MES pH 5.0中以至濃度50 mg/m卜平行地，相似地製備於25 mMMESpH5.0中之50 mg/ml 的硫-NHS(Pierce, part# 24525)溶液。 之後’將50 μΐ之EDC溶液與50 μΐ之硫-NHS溶液加 至EVITAG™溶液。將混合物混合均勻並以慢傾斜旋轉於 室溫 30 分鐘。使用一 pi〇 管柱(Amersham Biosciences,產 品編號17-0851-01)將溶液去鹽且以ο」μ碳酸鈉緩衝溶液 (pH 9.2)來洗提。收集含經活化之qd的有顏色部分。 3.2.2DNA聚合.酶結合 將於0.1 Μ碳酸鈉緩衝溶液中（pH 9.2)之100 pg的 DNA聚合酶加入至混合物中。在混合均勻後，將樣本培養 於4°C並傾斜旋轉2小時。 3·2·3舆QD結合之聚合酶的純化 藉由以 30Κ Microspin filter (Pall Coi*poration, part# OD100C33)在6000 rpm離心5-10分鐘，將結合物濃縮至總 體積〜200 μ卜藉由30K Microspin filter以dH20清洗結合 物兩次。再來，以Suprdex 3 0/75 Resin藉由尺寸排除來純 化結合物。 簡單地說’以dH20預先平衡管柱。之後將經濃縮之 結合混合物載至管柱且允許其進入柱床(c〇lurnn bed)。在黑 光激發下以dH2〇洗提管柱，並收集螢光片段。將螢光片 段加在一起且藉由以100K Microspin filter在6000 rpm離 心5-10分鐘將其濃縮至總體積〜100 μ1。經純化與濃縮之 47 201017163 結合物可儲存於4°C。 實施例4 :連結聚合酶至元件 使用光一 NHS(photoNHS) (N-羥基琥珀醯亞胺羧酸鹽 分子（N-hydroxy succimido carboxylate)以一碳鏈連結器連 結至一疊氮确基苯(azidonitrobenzene))以將一酵素，例如 聚合酶附著至一元件的兩種方法被描述。Referring to Figure 5, there is shown a table summarizing the construction of one of the filter layers 24'. In the 5th ® 'lower minus: the underlying layer provides a sublayer closer to the bottom surface of the layer 240, and the higher numbered sublayer provides a sublayer closer to the upper surface of the filter layer 240. As shown in Fig. 5, in this embodiment, the odd-numbered layers of the filter layer 240 are made of, for example, Nb2〇5, which has a relatively high refractive index. The even-numbered layers are made of, for example, cerium oxide (SiOJ, which has a lower refractive index. The sub-layer can be formed by using a money plating system, and examples of the sputtering system include Model No. RAS 110 of Radical Assisted Sputtering Series, manufactured by Shincron Co., 1^(1.(81^1^§&~&-1〇1,1'〇1^〇, 八?)). In this embodiment The thickness of each layer is also provided in the table of Figure 5. The synthetic filter, layer 240 is highly transparent to the fluorescence of the fluorophore Cy5, and for the light emitted from the 氦氖41 201017163 (Helium-Neon) laser The wavelength of about 633 nm is low penetration, and the gas-jet laser acts as an external source to excite the fluorophore Cy5. Referring again to Figures 2 and 4, a masking sheet 230 having pinholes 235 is formed on the filter layer 240. - In the embodiment, the size f of the pinhole μ5 is about 0.2 μm, and can vary from 0 1 μm to 丨 μιη. The size of the filter layer based on the design and application of the system and the corresponding filter layer can be changed in the system. In one embodiment, based on a number of considerations, such as system applications, designs, features, photodetectors, and/or light sources The radius can be changed with the characteristics of the biomolecule to be detected. A process for manufacturing the masking sheet MO having the pinholes 235 on the furnace layer MO or the substrate 10 will be described in detail below. First, by, for example, spin coating The photoresist material is formed on the filter layer 24 to form a photoresist layer on the filter layer 240 (if the filter layer 24 is formed on the upper surface of the photodetector 210 and the control circuit 215 as needed) or planarized The upper surface of the optical debt detector 210 is connected to the control circuit 215 (if the layer 240 is not formed). Thereafter, the photoresist is developed to form a photoresist pattern in the pinhole region. By covering the pinhole region with the mask, And exposing the photoresist so that only the area covered by the reticle is maintained on the filter layer 240 or the surface of the planarized photodetector 21 and the control circuit 215 to form a photoresist pattern. Thereafter, the metal layer is deposited. In the filter layer 24 on which the photoresist pattern has been formed. In this embodiment, the 'metal layer may include chromium (Cr), by performing a magnetron sputtering process, which is deposited on the layer or the The upper surface of the planarized photodetector 210 is on the control circuit. Next, remove a portion of the metal layer on the pinhole region and the pinhole region. A photoresist pattern, thereby forming a masking sheet 23 having a pinhole 235. Alternatively, by first depositing a metal layer (eg, cr) On the filter layer 240 42 201017163, a mask is formed on the metal layer, thereby exposing a portion of the upper surface of the metal layer to form the mask sheet 230. After that, the exposed portion of the metal layer is punched until the filter is exposed. Layer 240, thereby forming a pinhole on the metal layer. Thereafter, the mask is removed from the metal layer and a masking sheet 230 having 2% pinholes is formed on the filter layer 240. In some embodiments, the hemisphere is formed by filling the hemisphere formed by the space between the masking sheet 230 and the sublayer closest to the masking sheet 23〇 with 〇2 and forming the masking sheet 23(). With a flat surface. The metal layer has pinholes 235 formed in the center of the half-ball of the layer 24. Referring again to Figures 2 and 4, in this embodiment, the joint 22 22 is formed by filling the support material in a pinhole or recess 45. The support material $ is a polymer or inorganic material that is transparent to the fluorescent light emitted from the face 36. Referring again to Fig. 1 'only 12 optical detecting devices 20 are shown. It can be understood that at least: 10,000 optical detecting devices 2 can be formed on the substrate 10 . For example, in this embodiment, each of the optical detecting devices 2 has a circular shape of about half or less, which can occupy an area of about HKW. For a substrate 1 having an area of 1 square inch (i.e., 2.54 cm by 2.54 cm), it is possible to construct more than six million optical devices 2 on the substrate H). By operating the six-sense detection device 20 (4), it is possible to measure biomolecules with high throughput. Example 2: Attachment to Biomolecules for Detection to Flux Bioassay Systems 43 201017163 A fluorescent dye Cy5-labeled nucleic acid was used to test the detection system. Cy5 and biotin are attached to the 3, and 5, respectively, of a 60-mer (mer) oligonucleotide. The labeled 'biotinylated DNA was dissolved in TrisMg (10 mM Tris, 10 mM NaCl, 100 mMMgCl2, pH 8) buffer solution, deposited on a position array' and cultured in a humid chamber in. After about 30 minutes, the unbound DNA was washed away with a Tris buffer solution. Excitation light is provided via a 635 nm light emitting diode, and a 635 nm light emitting diode can be formed on the masking sheet. In order to read the signal from each pixel, the excitation light is turned on for about 1-5 seconds, the signal from each pixel is recorded, and this cycle is repeated for one hundred rounds. Then, the representative average signal of each pixel and the corresponding standard deviation are calculated accordingly. The signal before and after deposition of the DNA sample is compared, and the pixel having an average signal difference greater than 3 times the standard deviation is regarded as a positive pixel, that is, (Avg post-Avg front) > 3X (after STD - Before STD). Example 3: Bonding Quantum Dots to a Polymerase The following are two methods for binding a functionalized quantum dot to a primary amine on a polymerase molecule. One uses an amine-activated dot, the second A carboxyl-activated dot is used. 3.1 Amine EVITAGtm Binding to DNA Polymerase The amine EVITAGtm (eg Evident Technologies, cat# E2-C11-AM2-0620; EVITAGTM kit QD product is also under the eFluorTM logo of 201017163 eBioscience, Inc” San Diego, CA Soldering) Functionalized quantum dots (QD) by BS3 (Bis (sulfosuccinimidyl suberate)), a homobifunctional water-soluble crosslinker Activated, which comprises an amine-reactive N-hydroxysuccinimide carboxylate (NHS) ester at each end of an 8-carbon spacer arm. At pH 7-9, the NHS ester is on the surface of the QD. The amine reacts to form a stable indoleamine bond and releases the NHS leaving group. Taq DNA polymerase or Phi29 DNA polymerase has a number of primary amines (eg, lysine (K) residues and The N-terminal of the multi-peptide, which is the target of the cross-linking of available NHS esters. 3.1.1 Surface activation of quantum dots with 25 μΐ 10 mM BS3 (Bis (sulfosuccinimidyl) suberate), Pierce, part # 21580) and 25 Μΐ lOxPBS (phosphate buffer, pH 7.4) Activate 2.0 nmol of EVITAGtm The final volume of dH2 was 250 μM. After 30 minutes of incubation, the solution was desalted and eluted with lxPBS using a P10 column (Amersham Biosciences, product number 17-0851-01). The colored protein contained activated QD. 3.1.2 DNA polymerase binding polymerase was added to the mixture by adding 100 pg of DNA polymerase in 0.1 Μ sodium carbonate buffer solution (pH 9.2). After mixing, culture at 4 ° C And tilting for 2 hours. 3.1.3 Purification of QD-bound polymerase 45 201017163 Concentrate the conjugate to total volume by centrifugation at 6000 i*pm for 5-10 minutes with a 30K Microspin filter (Pall Corporation, part # OD100C33) 200 μΐ. The conjugate was washed twice with dH20 by a 30K Microspin filter. Then, Suprdex 30/75 Resin (GE Healthcare, part # 17-0902-10 or 17-1044-10 for small proteins and peptides) The conjugate is purified by size exclusion. The column was pre-equilibrated with dH20 before loading the concentrated binding mixture onto the column and allowing it to enter the column bed. These samples were eluted with dH20 under black light excitation and the fluorescent fragments were collected.萤 Add the fluorescent fragments together and concentrate to a total volume of ~100 μΐ by centrifugation at 6000 rpm for 5-10 minutes with a 100K Microspin filter. The purified and concentrated combination can be stored at 4 °C. 3.2 Binding of Carboxy EVITAGtm(R) DNA Polymerase The QD functionalized by Rebel EVITAGTM (e.g., Evident Technologies, cat# E2-C11-CB2-0620) is activated via the ECD-centered sulfur-NHS ester coupling reaction. The amine-reactive sulfur-NHS ester is reacted with, for example, a primary amine in the side chain of the lysine (K) on Taq DNA 聚合酶 polymerase or Phi29 DNA polymerase. 3.2.1 Surface Activation of Quantum Dots Diluted 2.0 nmol of EVITAGTM in 25 mM MES pH 5.0 buffer solution. EDC (1-ethyl-(3-dimethylaminopropyl)-3-ethylcarbodiimide 46 201017163 hydrochloride) was dissolved in cold before use immediately. A 50 mg/ml solution of sulfur-NHS (Pierce, part # 24525) in 25 mM MES pH 5.0 was prepared similarly in MES pH 5.0 up to a concentration of 50 mg/m. Thereafter, 50 μL of EDC solution and 50 μL of sulfur-NHS solution were added to the EVITAGTM solution. The mixture was mixed well and spun at room temperature for 30 minutes at a slow tilt. The solution was desalted using a pi(R) column (Amersham Biosciences, product number 17-0851-01) and eluted with a ο" sodium carbonate buffer solution (pH 9.2). The colored portion containing the activated qd is collected. 3.2.2 DNA Polymerization. Enzyme Binding 100 pg of DNA polymerase in 0.1 Μ sodium carbonate buffer solution (pH 9.2) was added to the mixture. After mixing well, the samples were incubated at 4 ° C and tilted for 2 hours. The purification of the 3·2·3舆QD-binding polymerase was carried out by centrifugation at 6000 rpm for 5-10 minutes with a 30 μM Microspin filter (Pall Coi*poration, part# OD100C33), and the conjugate was concentrated to a total volume of ~200 μb. The conjugate was washed twice with dH20 by a 30K Microspin filter. Again, the conjugate was purified by size exclusion with Suprdex 3 0/75 Resin. Simply put 'pre-equilibrate the column with dH20. The concentrated binding mixture is then loaded onto the column and allowed to enter the bed. The column was eluted with dH2 黑 under black light excitation, and the fluorescent fragments were collected. The fluorophores were added together and concentrated to a total volume of ~100 μl by centrifugation at 6000 rpm for 5-10 minutes with a 100K Microspin filter. Purified and concentrated 47 201017163 The conjugate can be stored at 4 °C. Example 4: Linking a polymerase to a component using N-hydroxysuccinamium carboxylate (N-hydroxy succimido carboxylate) linked to azidonitrobenzene by a carbon chain linker )) Two methods of attaching an enzyme, such as a polymerase, to a component are described.

4.1UV表面活化 藉由以UV光之光活化，一光NHS用來將一聚合酶附 著至於一裝置上之一經胺修飾的表面。UV光藉由排除氮激 起一疊氮硝基苯部分以產生高反應性氮烯(nitrene)基團。氮 缚與元件表面上之NH2反應以形成一聯氨(hydrazine)鍵。 連結器之另一端為NHS羧酸鹽，其與聚合酶上之離胺 酸殘基反應以產生一醯胺共價鍵。 4·1·1表面製備 將 1 mM 光 NHS (Sigma，Art No. Α3407，分子量 390.35)之溶液製備於95%乙醇中。清洗經胺修飾之表面 以碳酸鹽緩衝溶液且之後以95%乙醇。接著，將光NHS 溶液提供至經胺修飾之表面。254_365 nm UV光照射於表 面3分鐘，之後以95%乙醇沖洗三次。 4.1.2胺之末端蓋(end-cap) 48 201017163 將10mM乙酸善號轴酸亞胺醋（N_acet〇xysucdnimide) 之溶液製備於碳酸鹽緩衝溶液(pH 9.3)中且提供至表面以 末端蓋住任何未經反應之胺。於室溫以溫和搖晃培養此裝 置2小時。接著清洗表面三次，每次以碳酸鹽緩衝溶液及 蒸德去離子水。 4.1.3DNA聚合酶結合 再來，將於碳酸鹽缓衝溶液(PH9.3)中之imM聚合酶 ❿提供至表面且於持續溫和搖晃下在室溫培養2小時。接著 清洗表面二次，母次以碳酸鹽緩衝溶液及pH 7.4 PBS (鱗 酸缓衝鹽）。經聚合酶結合之表面可儲存在4。〇。 4.2經緩衝之表面活化 在另一實施例中，在緩衝環境下，可將*NHS活化與 結合至表面。之後在聚合酶存在下使用uv光以活化疊氮 硝基苯部分。再次，高反應性氮烯在uv光下形成為一電 • 子不足基團（electron deficient group)且立即與聚合酶表面 上之一級胺反應，於表面上形成共價鍵結。 4.2.1表面製備 將 1 mM 光 NHS (Sigma, Art No. A3407，分子量 390.35 )之溶液製備於碳酸鹽緩衝溶液中。經胺修飾之表 面以碳酸鹽緩衝溶液(pH 9.3)沖洗。將光NHS溶液提供至 經胺修飾之表面且於持續溫和搖晃下培養2小時。表面之 後以碳酸鹽緩衝溶液沖洗三次。 49 201017163 4·2·2胺之末端蓋 將製備於碳酸鹽緩衝溶液(ρΗ93)中之iQmM乙酸·& 綱醯亞㈣溶液提供至表面以末端蓋住任何未經反應之 胺基團。於室溫在持續溫和搖晃下培養此溶液2小時，且 之後以PBS緩衝溶液(1>117 4)沖洗表面三次。 4.2.3DNA聚合酶結合 將1 mM DNA聚合酶之溶液製備於而缓衝溶液(ρΗ 7.4) 且提供至經末端蓋住之卜11(1_(^1)1^(1)表面。將^乂光 (254-365 nm)照射於表面3分鐘。之後以pBS緩衝溶液(ρΗ 7.4) 沖洗表面三次。經聚合酶結合之表面可之後儲存在 4°C。 實施例5 :鹼基延伸定序形式 如上所討論，有兩種一般方法來辨認於逐步延伸時所 ❹ 加之核苷酸：連續加入4種經相同性標誌之核苷酸或同時 加入4種經差別性標諸之核苷酸。各個這些形式的例子於 以下提供。 5.1連續加入4種具有相同性標誌、之核普酸 5.1.1腺嘌呤(A)分子延伸：加入經阻礙與標諸之腺η票 呤與合適之聚合酶於一定序反應緩衝溶液（例如，40 mM Tris-HClpH9.1，lmMMgCl2)中。只有當胸腺嘧啶(T)為在 50 201017163 被定序核酸中與末端連結引子之5’端鄰接之核苷酸時，將 腺嘌呤加至定序引子之3’端。若核酸之核苷酸最接近引子 之3’端為鳥嘌呤(G)、胞嘧啶(C)或腺嘌呤(A)，之後沒有延 伸會發生。 5.1.2延伸反應清潔步驟：在延伸反應完成之後，清洗 陣列晶片一次使用5xSSC與0.1% SDS，而一次使用 5xSSC以移除腺嘌呤與未反應之溶液。 5.1.3螢光偵測與記錄：於連結位讀取螢光訊號以確認 是否腺嘌呤已被延伸，其指出一對應之胸腺嘧啶於被定序 之核酸中。 5.1.4移除保護與螢光基團：於偵測後，藉由化學或酵 素裂解來移除保護與螢光基團。 ® 5.1.5清潔步驟：清洗陣列晶片一次使用5xSSC與0.1 %SDS，而一次使用5xSSC以移除經裂解之保護與標誌基 團。 5.1.6校對與記錄：確認自先前延伸步驟成功移除保護 與螢光基團。若具有殘餘保護與螢光基團偵測，例如於偵 測與記錄系統2上之分析軟體會記錄位置。僅在下個清潔 步驟中確認保護與螢光基團移除時，定序反應之記錄才可 繼續。 51 201017163 5.1.7重複5.1.1-5.1.6，此次使用鳥嘌呤來用於延伸反 應。 5.1.8重複5.1.1-5.1.6，此次使用胞嘧咬來用於延伸反 應。 5.1.9重複5.1.1-5.1.6，此次使用胸腺嘴咬來用於延伸 反應。 5.1.10使用A、G、C、T之每四個延伸反應為一循環。 藉由重複循環，於一逐步方式中確認一核酸之序列。 5.2同時加入4種具有區別之標誌的核苷酸 5.2.1鹼基延伸反應：將4種經阻礙與有區別之標諸的 DNA核苷酸（A、G、C、T)與核酸聚合酶加入至一在陣 列上的定序緩衝溶液。延伸反應可只發生在定序引子之3’ 參 端。與最接近連結引子5’端之被定序核酸的核苷酸互補的 核苷酸，可被加至定序引子之3’端，參見，例如第6圖。 5·2·2延伸反應清潔步驟：在延伸反應完成之後，一次 以5xSSC與0.1% SDS，且一次以5xSSC來清洗晶片，以 移除在反應溶液中之殘餘材料。 5·2·3螢光偵測與記錄：於各連結位偵測各有區別之榮 52 201017163 光訊號以確認所加入之核苷酸。 5.2.4移除保護與螢光基團：於偵測後，藉由化學或酵 素裂解來移除保護與螢光基團。 5.2.5清潔步驟：清洗晶片一次使用5xssC與0.1% SDS，而一次使用5xSSC以移除經裂解之保護與螢光基團。 5.2.6校對與記錄：確認自先前延伸步驟成功移除保護 與螢光基團。若具有殘餘保護與螢光基團偵測，例如於偵 測與記錄系統2上之分析軟體會記錄位置。僅在下個清潔 步驟中確認保護與螢光基團移除時，定序反應之記錄才可 繼續。 5.2.7重複5.2.1-5.2.6。重複反應循環，以確認核酸之 序列。 實施例6:已知核酸之定序 一化學合成之60員募核苷酸，具有已知序列：生物素 -5’-tcag tcag tcag tcag tcag tcag tcag tcag tcag tcag tcag tc ACACGGAGGTTCTA-3’，作為一定序模板。此定序模板與 一 14員之寡核苷酸定序引子（5’-TAGAACCTCCGTGT-3，) 結合。模板之5’端經修飾以包括一生物素分子。將模板附 著至一含有卵白素（streptavidin)之反應器表面。定序反應使 53 201017163 用一 DNA聚合酶以執行一驗基延伸反應於具有15 mM DTT之1χ定序缓衝溶液中。各延伸反應步驟只有增加一形 式之核苷酸，其具有二級延伸保護（阻礙）基團與一螢光 標諸（例如，Cy5 )。若與定序引子之3’端鄰接之DNA模 板的核苷酸為與加入之核芽酸互補’則之後加入螢光標諸 驗基。於清洗掉未反應之驗基材料後’偵測瑩光訊號。若 所加入之驗基並非互補’則有螢光訊號會被彳貞測到。在4貞 測後，化學移除螢光基團’且使用一含鹽溶液（例如 5xSSC ; 0.1% SDS)更進一步清洗陣列，且再偵測一次以 鲁 確認螢光標誌之移除。在移除與清洗步驟之後，對於沒有 偵測到螢光訊號之位置而言，其被認為螢光標誌已移除。 於下個反應循環中獲得之螢光訊號，之後被視為下個延伸 定序之訊號。在移除與清洗之後，對於仍然維持螢光訊號 之位置而言，其使用軟體被記錄為具有不完全移除反應。 來自位置之訊號可持續以被記錄，只要確認於下個循環中 之移除步驟。基於此方法，可連續加入四種形式之反應驗 基材料’並循環地執行反應。因此，確認了模板之序列。❹ 雖然本發明已以較佳實施例揭露如上’然其並非用以 限定本發明，任何熟習此技藝者，在不脫離本發明之精 神和範圍内’當可作些許之更動與潤飾，因此本發明之 保護範圍當視後附之申請專利範圍所界定者為準。 54 201017163 【圖式簡單說明】 第1圖為一平面圖，其顯示一包括光學偵測裝置的一 陣列的生物分析系統。 第2圖為沿著第1圖之綠A-A之一剖面圖，其顯示一 實施例之光學偵測裝置。 第3a圖為一剖面圖’其顯示根據一實施例之光學偵測 裝置的細部。 第3b圖為一剖面圖，其顯示根據另一實施例之光學偵 測裝置的細部。 第4圖為沿著第1圖之線A_A的一剖面圖，其顯示另 一實施例之光學偵測裝置。 第5圖為一表格，其顯示一實施例之一濾層的構成。 第6圖顯示一連結於一元件之連結位的核酸。 第7圖顯示，在具有經阻礙與標誌之核苷酸的鹼基延 伸一循環後，一連結於一元件之連結位的核酸。 第8圖顯示於第7圖中所示之鹼基延伸定序反應的一 替代實施例。 第9圖顯示，藉由鹼基延伸定序將許多核酸平行定序 的一循環。 【主要元件符號說明】 1〜生物分析系統； 2〜偵測與記錄系統； 55 201017163 l 〇〜生物分析基板； 20〜光學偵測裝置； 210〜光偵測器； 220〜連結位； 215〜控制電路； 230〜遮蔽薄板； 235〜針孔； Η1〜距離； Η2〜距離； 240〜濾、層； 250〜微透鏡； 30〜生物分子； 32〜單股DNA分子； 344〜經修飾之核苷酸； 346〜黏合定序引子； 34〜末端引子； 36〜螢光團； 3 62〜核苦酸； 364〜螢光阻礙基團； 36Α〜第一位置； 36Β〜第二位置； 38〜聚合酶； 384〜方法； Θ1〜第一立體角； Θ2〜第二立體角； 201017163 40〜激發光源； 410〜p型半導體層； 420〜光散發層； 430〜η型半導體層； 450〜凹處； 415、435〜金屬接觸； 440〜電源。4.1 UV Surface Activation By activation with UV light, a photoNHS is used to attach a polymerase to an amine-modified surface on a device. The UV light excites the monoazide nitrobenzene moiety by excluding nitrogen to produce a highly reactive nitrene group. Nitrogen binds to NH2 on the surface of the element to form a hydrazine bond. The other end of the linker is an NHS carboxylate which reacts with an amino acid residue on the polymerase to produce a guanamine covalent bond. 4.1.1 Surface preparation A solution of 1 mM light NHS (Sigma, Art No. Α3407, molecular weight 390.35) was prepared in 95% ethanol. The amine modified surface was washed with a carbonate buffer solution followed by 95% ethanol. Next, a photo NHS solution is provided to the amine modified surface. 254_365 nm UV light was applied to the surface for 3 minutes and then washed three times with 95% ethanol. 4.1.2 End-cap of amine 48 201017163 A solution of 10 mM acetic acid acetanilide (N_acet〇xysucdnimide) was prepared in a carbonate buffer solution (pH 9.3) and provided to the surface to cover the end. Any unreacted amine. The apparatus was incubated for 2 hours at room temperature with gentle shaking. The surface was then washed three times, each time with a carbonate buffer solution and distilled deionized water. 4.1.3 DNA polymerase binding Further, imM polymerase in a carbonate buffer solution (pH 9.3) was supplied to the surface and incubated at room temperature for 2 hours under gentle shaking. The surface was then washed twice with a mother's carbonate buffer solution and pH 7.4 PBS (salt buffer). The surface bound by the polymerase can be stored at 4. Hey. 4.2 Buffered Surface Activation In another embodiment, *NHS can be activated and bound to the surface in a buffered environment. The uv light is then used in the presence of the polymerase to activate the azide nitrobenzene moiety. Again, the highly reactive nitrene forms an electron deficient group under uv light and immediately reacts with a primary amine on the surface of the polymerase to form a covalent bond on the surface. 4.2.1 Surface preparation A solution of 1 mM light NHS (Sigma, Art No. A3407, molecular weight 390.35) was prepared in a carbonate buffer solution. The amine-modified surface was rinsed with a carbonate buffer solution (pH 9.3). The light NHS solution was supplied to the amine-modified surface and incubated for 2 hours under gentle shaking. The surface was rinsed three times with a carbonate buffer solution. 49 201017163 4·2·2 amine end cap The iQmM acetic acid·& 醯 醯 (4) solution prepared in a carbonate buffer solution (ρΗ93) was supplied to the surface to cover any unreacted amine groups at the ends. The solution was incubated for 2 hours at room temperature under gentle shaking, and then the surface was washed three times with a PBS buffer solution (1 > 117 4). 4.2.3 DNA polymerase binding A solution of 1 mM DNA polymerase was prepared in a buffer solution (ρΗ 7.4) and supplied to the surface of the end of the 11 (1_(^1)1^(1). Light (254-365 nm) was applied to the surface for 3 minutes. The surface was then rinsed three times with pBS buffer solution (ρΗ 7.4). The polymer-bound surface was then stored at 4 ° C. Example 5: Base Extension Sequence As discussed above, there are two general methods for identifying nucleotides added during a stepwise extension: consecutive additions of four nucleotides of the same identity or simultaneous addition of four differentially labeled nucleotides. Examples of these forms are provided below. 5.1 Continuous addition of 4 nucleotides with the same identity, nucleotides 5.1.1 Adenine (A) Molecular extension: addition of hindered and labeled glandular sputum and appropriate polymerase In a sequence reaction buffer (eg, 40 mM Tris-HCl pH 9.1, lmMMgCl2), only when thymine (T) is a nucleotide adjacent to the 5' end of the terminal linker in the 50 201017163 sequenced nucleic acid Add adenine to the 3' end of the sequencing primer. If the nucleotide of the nucleic acid is closest to the introduction The 3' end is guanine (G), cytosine (C) or adenine (A), and no extension will occur afterwards. 5.1.2 Extension reaction cleaning step: After the extension reaction is completed, the array wafer is cleaned once using 5xSSC and 0.1% SDS, and 5xSSC is used at a time to remove adenine and unreacted solution. 5.1.3 Fluorescence detection and recording: Fluorescent signals are read at the link to confirm whether adenine has been extended, indicating a corresponding Thymidine in the sequenced nucleic acid. 5.1.4 Removal of protective and fluorescent groups: After detection, the protective and fluorescent groups are removed by chemical or enzymatic cleavage. : Cleaning the array wafers using 5xSSC and 0.1% SDS at a time, and using 5xSSC at a time to remove the cleaved protection and labeling groups. 5.1.6 Proofreading and Recording: Confirmation that the protective and fluorescent groups were successfully removed from the previous extension step. If there is residual protection and fluorophore detection, for example, the analysis software on the detection and recording system 2 will record the position. Recording of the sequencing reaction is only confirmed in the next cleaning step when the protection and the fluorophore removal are confirmed. Can continue. 51 201017163 5. 1.7 Repeat 5.1.1-5.1.6, this time use guanine for extension reaction. 5.1.8 Repeat 5.1.1-5.1.6, this time use cytosine bite for extension reaction. 5.1.9 Repeat 5.1 .1-5.1.6, this time using the thymus mouth bite for the extension reaction. 5.1.10 Using each of the four extension reactions of A, G, C, T is a cycle. By repeating the cycle, in a stepwise manner Confirm the sequence of a nucleic acid. 5.2 Add four nucleotides with different markers at the same time. 5.2.1 Base extension reaction: Four kinds of DNA nucleotides that are blocked and differentiated (A, G, C, T) is added to the sequencing buffer solution on the array with the nucleic acid polymerase. The extension reaction can occur only at the 3' terminus of the sequencing primer. A nucleotide complementary to the nucleotide of the sequenced nucleic acid closest to the 5' end of the linker can be added to the 3' end of the sequencing primer, see, for example, Figure 6. 5.2.2 Extension reaction cleaning step: After the completion of the extension reaction, the wafer was washed with 5xSSC and 0.1% SDS once, and once with 5xSSC to remove residual material in the reaction solution. 5·2·3 Fluorescence detection and recording: Detection of each difference in each link 52 201017163 Optical signal to confirm the added nucleotide. 5.2.4 Removal of Protected and Fluorescent Groups: After detection, the protective and fluorescent groups are removed by chemical or enzymatic cleavage. 5.2.5 Cleaning Step: Clean the wafer using 5xssC and 0.1% SDS at a time, and use 5xSSC at a time to remove the cleavage of the protective and fluorescent groups. 5.2.6 Proofreading and Recording: Confirm that the protection and fluorescent groups were successfully removed from the previous extension step. If there is residual protection and fluorophore detection, for example, the analysis software on the detection and recording system 2 will record the location. The recording of the sequencing reaction can only continue if the protection and fluorophore removal are confirmed in the next cleaning step. 5.2.7 Repeat 5.2.2 - 5.2.6. The reaction cycle is repeated to confirm the sequence of the nucleic acid. Example 6: Sequencing of known nucleic acids - 60 chemically synthesized nucleotides with known sequence: biotin-5'-tcag tcag tcag tcag tcag tcag tcag tcag tcag tcag tcag tc ACACGGAGGTTCTA-3', as A certain order template. This sequencing template was combined with a 14-member oligonucleotide sequencing primer (5'-TAGAACCTCCGTGT-3,). The 5' end of the template is modified to include a biotin molecule. The template is attached to a reactor surface containing streptavidin. The sequencing reaction allowed 53 201017163 to perform a primer extension reaction using a DNA polymerase in a 1 χ sequencing buffer solution with 15 mM DTT. Each extension reaction step is augmented with a single form of nucleotide having a secondary extension protecting (obstructing) group and a fluorescent label (e.g., Cy5). If the nucleotide of the DNA template adjacent to the 3' end of the sequencing primer is complementary to the added nucleate, then the fluorescing cursor is added. After the unreacted test substrate is washed away, the fluorescent signal is detected. If the test base is not complementary, then a fluorescent signal will be detected. After 4 measurements, the fluorescent group was chemically removed and the array was further cleaned using a saline solution (e.g., 5xSSC; 0.1% SDS) and the detection of the fluorescent marker was removed again. After the removal and cleaning steps, it is considered that the fluorescent marker has been removed for the location where the fluorescent signal is not detected. The fluorescent signal obtained in the next reaction cycle is then treated as the next extended sequence signal. After removal and cleaning, the software used is recorded as having an incomplete removal reaction for the position where the fluorescent signal is still maintained. The signal from the location can be recorded as long as it is confirmed in the removal step in the next cycle. Based on this method, four types of reaction test materials can be continuously added and the reaction can be carried out cyclically. Therefore, the sequence of the template is confirmed. Although the present invention has been disclosed in the above preferred embodiments, it is not intended to limit the invention, and it is intended that the invention may be modified and modified without departing from the spirit and scope of the invention. The scope of the invention is defined by the scope of the appended claims. 54 201017163 [Simple description of the drawings] Fig. 1 is a plan view showing a bioanalysis system including an array of optical detecting devices. Fig. 2 is a cross-sectional view along the green A-A of Fig. 1, showing an optical detecting device of an embodiment. Figure 3a is a cross-sectional view showing details of an optical detecting device in accordance with an embodiment. Figure 3b is a cross-sectional view showing details of an optical detecting device in accordance with another embodiment. Fig. 4 is a cross-sectional view along line A_A of Fig. 1 showing an optical detecting device of another embodiment. Figure 5 is a table showing the construction of a filter layer of one embodiment. Figure 6 shows a nucleic acid linked to the junction of an element. Fig. 7 shows a nucleic acid linked to a junction of a member after a cycle in which the base of the nucleotide which is hindered from the marker is stretched. Figure 8 shows an alternative embodiment of the base extension sequence reaction shown in Figure 7. Figure 9 shows a cycle in which many nucleic acids are sequenced in parallel by base extension sequencing. [Main component symbol description] 1~Bioanalysis system; 2~Detection and recording system; 55 201017163 l 〇~ biological analysis substrate; 20~ optical detection device; 210~photodetector; 220~link bit; 215~ Control circuit; 230~ masking sheet; 235~ pinhole; Η1~distance; Η2~distance; 240~filter, layer; 250~microlens; 30~ biomolecule; 32~ single stranded DNA molecule; 344~ modified core Glycosyl acid; 346~bonding sequence primer; 34~end primer; 36~fluorescent group; 3 62~nucleotidic acid; 364~fluorescent hindering group; 36Α~first position; 36Β~second position; 38~ Polymerase; 384~method; Θ1~first solid angle; Θ2~second solid angle; 201017163 40~excitation light source; 410~p type semiconductor layer; 420~ light emitting layer; 430~n type semiconductor layer; 450~ concave Where; 415, 435 ~ metal contact; 440 ~ power.

Claims (1)

201017163 七、申請專利範圍： 1. 一種鑑定單一生物分子之裝置，包括： 一基板’具有一光偵測器’該基板被設置來偵測散發 自該生物分子的光； 一遮蔽薄板(blind sheet)於該基板上，該遮蔽薄板包括 一針孔，該針孔具有一直徑； 一濾層，其被提供於該遮蔽薄板與該基板之間且被提 供於該針孔之下，該濾層為非平面且被設置來過濾散發自 該生物分子的光；以及 參 一連結位(linker site) ’其被提供接近於該針孔，該連 結位被處理以將該生物分子置於接近該針孔。 2. 如申請專利範圍第1項所述之鑑定單一生物分子之 裝置，其中該光偵測器係擇自電荷耦合元件 (charged-coupled device)、互補金屬氧化半導體 (complementary metal-oxide semiconductor, CMOS)感測器 或光二極體(photodiode)其中之一。 3. 如申請專利範圍第1項所述之鑑定單一生物分子之 _ 裝置，其中該非平面包括弧線形、球形、橢圓形或弓形其 中之一。 4. 如申請專利範圍第1項所述之鑑定單一生物分子之 裝置，其中該針孔具有一小於或等於約l，〇〇〇nm之直獲。 5. 如申請專利範圍第1項所述之鑑定單一生物分子之 裝置，其中該針孔具有一小於或等於約200 nm之直徑。 6. —種鑑定單一生物分子之裝置，包括： 58 201017163 一基板，具有一光偵測器，該基板被設置來偵測散發 自該生物分子的光； 一光散發層形成於該基板上，其中該光散發層包括一 凹處； 一濾層，其被提供於該凹處與該基板之間且於一針孔 之下，該濾層為非平面且被設置來過濾散發該生物分子的 光；以及 一連結位，其被提供接近於該凹處與該針孔，該連結 • 位被處理以將該生物分子置於接近該針孔。 7.如申請專利範圍第6項所述之鑑定單一生物分子之 裝置，其中該光散發層穿過該凹處沿著平行於該光偵測器 之一表面的水平方向散發來自一激發光源的光至該連結 位。 8. 如申請專利範圍第6項所述之鑑定單一生物分子之 *置’更包括一遮蔽薄板於該基板上’該遮蔽薄板包括該 針孔’該針孔具有一直徑。 9. 如申請專利範圍第6項所述之鑑定單一生物分子之 裝置’其中該非平面包括弧線形、球形、橢圓形或弓形其 中之一 〇 10. 如申請專利範圍第6項所述之鑑定單一生物分子之 裝置’其中該針孔具有一小於或等於約1，〇〇〇 nm之直徑。 11·如申請專利範圍第6項所述之鑑定單一生物分子之 裝置，其中該針孔具有一小於或等於約200 nm之直徑。 12.如申請專利範圍第6項所述之鑑定單一生物分子之 裝置’其中該激發光源係擇自/發光二極體(light emitting 59 201017163 diode, LED)、一有機發光二極體（〇rganic Ught emitting diode, OLED)、一 1¾ 分子發光二極體(p〇lymer emitting diode, PLED)與雷射二極體(iaser 出0如，LD)。 13. 一種光學偵測系統，包括至少10,000個如申請專利 範圍第1項所述之裝置。 14. 一種光學偵測系統，包括至少25〇,〇〇〇個如申請專 利範圍第1項所述之裝置。201017163 VII. Patent application scope: 1. A device for identifying a single biomolecule, comprising: a substrate 'having a photodetector' configured to detect light emitted from the biomolecule; a blind sheet On the substrate, the shielding sheet comprises a pinhole having a diameter; a filter layer is provided between the shielding sheet and the substrate and is provided under the pinhole, the filter layer Non-planar and arranged to filter light emitted from the biomolecule; and a linker site 'which is provided proximate to the pinhole, the junction being processed to place the biomolecule close to the needle hole. 2. The device for identifying a single biomolecule according to claim 1, wherein the photodetector is selected from a charge-coupled device, a complementary metal-oxide semiconductor (CMOS). One of a sensor or a photodiode. 3. The device for identifying a single biomolecule as described in claim 1, wherein the non-planar comprises one of an arc, a sphere, an ellipse or an arc. 4. The device for identifying a single biomolecule according to claim 1, wherein the pinhole has a direct value of less than or equal to about 1, 〇〇〇nm. 5. The device for identifying a single biomolecule of claim 1, wherein the pinhole has a diameter of less than or equal to about 200 nm. 6. A device for identifying a single biomolecule, comprising: 58 201017163 a substrate having a photodetector disposed to detect light emitted from the biomolecule; a light emitting layer formed on the substrate Wherein the light emitting layer comprises a recess; a filter layer is provided between the recess and the substrate and under a pinhole, the filter layer is non-planar and arranged to filter and emit the biomolecule Light; and a link that is provided proximate to the recess and the pinhole, the link being processed to place the biomolecule close to the pinhole. 7. The device for identifying a single biomolecule according to claim 6, wherein the light emitting layer passes through the recess and emits light from an excitation source in a horizontal direction parallel to a surface of the photodetector. Light to the link. 8. The identification of a single biomolecule as described in claim 6 further includes a masking sheet on the substrate. The masking sheet includes the pinhole. The pinhole has a diameter. 9. The device for identifying a single biomolecule according to item 6 of the patent application, wherein the non-planar comprises one of an arc, a sphere, an ellipse or an arch. 〇 10. The single identification as described in claim 6 The device of biomolecules wherein the pinhole has a diameter less than or equal to about 1, 〇〇〇nm. 11. The device for identifying a single biomolecule of claim 6, wherein the pinhole has a diameter of less than or equal to about 200 nm. 12. The device for identifying a single biomolecule as described in claim 6 wherein the excitation source is a light emitting diode (light emitting 59 201017163 diode, LED), an organic light emitting diode (〇rganic) Ught emitting diode (OLED), a 13⁄4 molecular light emitting diode (PLED) and a laser diode (iaser 0, LD). 13. An optical detection system comprising at least 10,000 devices as described in claim 1 of the scope of the patent application. 14. An optical detection system comprising at least 25 turns, such as the device of claim 1 of the patent application. 15. -種光學债測***，包括至少、2,〇〇〇，麵個如申請 專利犯圍第1項所述之裂置。 16. -種光學偵測系統，包括至少1()，麵，_個如 專利範圍第1項所述之裝置。 17. —種鑑定生物分子之裝置，包括： 一基板，具有一光偵測器； 一遮蔽薄板於該基板上，該喊薄板包括一針孔之陣 ;慮層，形成於該針孔之陣 供 個之至少—4b 』 〒被徒供於15. An optical debt measurement system, including at least 2, 〇〇〇, face, such as the application of the patent stipulations mentioned in item 1. 16. An optical detection system comprising at least 1 (), a face, a device as described in the first item of the patent scope. 17. A device for identifying biomolecules, comprising: a substrate having a photodetector; a masking sheet on the substrate, the shingling sheet comprising a matrix of pinholes; and a layer formed in the array of pinholes For at least - 4b 』 一、…針 濾層結構為非平面；以及 一連、，㈣賴攸各料 生物分子卩愤賴結位。 。㈣處理以使 18.如申請專利範圍第 置，其中該先偵測器係擇自電==定生物分子之 半導艘感測器或光二㈣其中讀、互補金屬氧 L其中麵㈣祕_、球形、_形或弓形其 60 201017163 20. 如申請專利範圍第17項所述之鑑定生物分子之裝 置，其中各針孔具有一小於或等於約1，〇〇〇 nm之直徑。 21. 如申請專利範圍第17項所述之鑑定生物分子之裝 置，其中各針孔具有一小於或等於約200 nm之直徑。 22. —種光學偵測系統，包括至少10,000個如申請專利 範圍第6項所述之裝置。 23. —種光學偵測系統，包括至少250,000個如申請專 利範圍第6項所述之裝置。 ❹ 24.—種光學偵測系統，包括至少2,000,000個如申請 專利範圍第6項所述之裝置。 25.—種光學偵測系統，包括至少10,000,000個如申請 專利範圍第6項所述之裝置。 61First, the needle filter structure is non-planar; and one, (4) Lai 攸 various materials biomolecules angered the knot. . (4) Processing to make 18. If the scope of the patent application is set, wherein the first detector is selected from the self-conducting == semi-conducting sensor of the biomolecule or the light two (four) of which the reading, complementary metal oxygen L (4) secret _ And a device for identifying a biomolecule according to claim 17, wherein each pinhole has a diameter of less than or equal to about 1, 〇〇〇nm. 21. The device for identifying biomolecules of claim 17, wherein each pinhole has a diameter of less than or equal to about 200 nm. 22. An optical detection system comprising at least 10,000 devices as described in claim 6 of the scope of the patent application. 23. An optical detection system comprising at least 250,000 devices as described in claim 6 of the patent application. ❹ 24. An optical detection system comprising at least 2,000,000 devices as described in claim 6 of the scope of the patent application. 25. An optical detection system comprising at least 10,000,000 devices as described in claim 6 of the scope of the patent application. 61