201213803 六、發明說明： 【發明所屬之技術領域】 本發明是有關於一種質譜分析方法，特別是一種整合 式質譜分析方法。 【先前技術】 在現在生物醫學的領域中，生物樣本中特定分子（如， 代謝物、蛋白質或多胜肽、具藥理學活性的分子等）的定 ® 性與定量分析扮演了非常重要的角色。然而，由於生物樣 本中含有的分子種類眾多，每一種分子的含量與特性又各 不相同，傳統的分析技術通常涉及了許多階段，需要耗費 大量人力與時間。 以血液樣本中蛋白質分子的分析為例，目前常用的方 法之一是二維聚丙烯醯胺膠體電泳-質譜分析法 (two-dementional polyacrylamide gel electrophoresis/mass spectrometry，簡稱2D-PAGE/MS )，茲將其分析方法概述 φ 如下。 血液樣本與其他的生物樣本中，往往含有許多待測物 分子（analyte molecules)以外的成分或細胞，因此，在實 際進行分析之前，必須先進行血液樣本的前處理，以將待 測物分子和其他分子分離開來。一般來說，可利用過濾、 離心、蒸餾、沈澱、萃取與稀釋等手段，將待測物分子和 其他成分分離開來；或者是可利用前分群（prefracti〇nation) 法’先依照待測物分子量的大小、待測物在細胞中的位置、 組織中細胞的種類或細胞中胞器的種類等差異，以得到具 3 201213803 有相似特定性質的待測物分子。卜 此外’由於某些待測物分 1 ” 叫彳貞測方法可制之敏感值 的下限，因此在前處理階段亦料對此類制物分子進行 擴增（amplification)或濃縮，以使得其含量至少能符合债 測方法之敏感度。 接著’利用2D-PAGE原理來分離樣本中的多種待測物 (蛋白質）分子。由於蛋白質分子會隨著其胺基酸序列與 轉譯後修飾或構形的不同而具有不同的等電點（isoelectric • POMP1)，在2D-pAGE中’將蛋白質分子置於長條狀的等 電點梯度膠體（immobilized pH gradient gel，IPG)中，以 使蛋白質分子在電場的作用下在第一個方向（第一維）中 移動到膠體上相等於各蛋白質等電點的位置上。接著，將 蛋白質膠分子轉移到聚丙烯醯胺膠體上，並使蛋白質分子 在電場的作用下，依照其體積大小而在第二方向（第二維） 移動至特定位置。如此一來，即可將樣本中的多種蛋白質 分子予以分離。接著，利用染色劑來染色位於膠體上的蛋 φ 白質，藉以在膠體上呈現出蛋白質分子的分布情形；此外， 還可以利用各蛋白質分子染色的深度來進行相對定量。 其後，在質譜轉移階段，需利用切膠手法由膠體中取 出含有目標蛋白質分子的膠體片段，並將蛋白質分子由膠 體中溶析或萃取出來（如：利用電析法（electro-elution ) 或溶劑萃取（solvent extraction)等）；而後再將所得到之 ' 蛋白質分子轉移到質譜分析儀中，以運用於下一階段的質 譜分析^ 利用質譜分析技術搭配電腦資料庫搜尋演算法（如： 201213803 MS-Fit資料庫、Mascot資料庫、ProFound資料庫等），能 夠進行蛋白質的定性分析。目前已發展出多種質譜分析記 術，如：基質輔助雷射脫附游離法（matrix-assisted laser desorption/ionization, MALDI )、無介質雷射脫附離子化 (matrix-free laser desorption/ ionization，MFLDI)質譜分 析儀、電喷灌離子化（electrospray ionization，ESI)質譜儀 等等。201213803 VI. Description of the Invention: [Technical Field of the Invention] The present invention relates to a mass spectrometry method, and more particularly to an integrated mass spectrometry method. [Prior Art] In the field of biomedicine today, the qualitative and quantitative analysis of specific molecules (such as metabolites, proteins or peptides, pharmacologically active molecules, etc.) in biological samples plays a very important role. . However, due to the large variety of molecules contained in biological samples, the content and characteristics of each molecule are different. Traditional analytical techniques usually involve many stages and require a lot of manpower and time. Taking the analysis of protein molecules in blood samples as an example, one of the commonly used methods is two-dementional polyacrylamide gel electrophoresis/mass spectrometry (2D-PAGE/MS). An overview of its analytical method φ is as follows. Blood samples and other biological samples often contain many components or cells other than the analyte molecules. Therefore, before the actual analysis, the blood sample must be pretreated to the molecules of the analyte. Other molecules are separated. In general, the molecules of the analyte and other components can be separated by means of filtration, centrifugation, distillation, precipitation, extraction and dilution; or the prefracti〇nation method can be used first. The molecular weight, the position of the analyte in the cell, the type of cells in the tissue, or the type of organelle in the cell, etc., to obtain a molecule of the test object having similar specific properties to 3 201213803. In addition, because some of the analytes are divided into 1 by the method of speculation, the lower limit of the sensitive value can be made, so in the pretreatment stage, the molecules of such materials are also expected to be amplified or concentrated so that The content can at least meet the sensitivity of the debt measurement method. Then use the 2D-PAGE principle to separate the various analytes (proteins) in the sample. Since the protein molecules will be modified or conformated with their amino acid sequence and after translation. Different from different isoelectric points (isoelectric • POMP1), in 2D-pAGE, 'protein molecules are placed in long strips of immobilized pH gradient gel (IPG) to make protein molecules Under the action of the electric field, it moves to the position on the colloid which is equal to the isoelectric point of each protein in the first direction (first dimension). Then, the protein gel molecules are transferred to the colloidal gel of polyacrylamide, and the protein molecules are Under the action of the electric field, it moves to a specific position in the second direction (second dimension) according to its volume. Thus, a plurality of protein molecules in the sample can be separated. Next, the dye is used to dye the egg φ white matter on the colloid, thereby exhibiting the distribution of protein molecules on the colloid; in addition, the relative depth of dyeing of each protein molecule can be used for relative quantification. The colloidal fragment containing the target protein molecule is taken out from the colloid by a gel-cutting method, and the protein molecule is eluted or extracted from the colloid (for example, by electro-elution or solvent extraction). ); then transfer the resulting 'protein molecules to the mass spectrometer for mass spectrometry in the next stage. ^ Use mass spectrometry to match the computer database search algorithm (eg 201213803 MS-Fit database, Mascot) Database, ProFound database, etc., capable of qualitative analysis of proteins. A variety of mass spectrometry techniques have been developed, such as matrix-assisted laser desorption/ionization (MALDI), no media Laser-free laser desorption/ionization (MFLDI) mass spectrometry Analyzers, electrospray ionization (ESI) mass spectrometers, and the like.
然而，在利用上述方法進行生物樣本的定性與定量分 析時’往往需要專業的技術人員投入大量的時間，以 2D-PAGE為例，完成一個週期的分析通常費時工至2天。 此外，樣本在各階段之間轉移時常出現損耗逸失；而某些 偵測技術的靈敏度又較低，因此可能無法識別出這些微量 或逸失的待測物分子。 有鑑於上述問題’相關領於亟思提出整合式的質譜分 析方法。然而，目前提出的整合式質譜分析方法往往侷限 於分離原理（如2D-PAGE)的整合或是前處理和分離方法 的整合’前者如將2D-PAGE結合於單一晶片上（2D_pAGE on a chip );後者如將固態萃取（s〇Hd phase extracti〇n )方 法和高效能液相層析（high_perf〇rmance liquid chromatography，HPLC)整合於單一晶片上。 因此，就整體定量定性分析的流程來看，仍有進一步 整合的空間。 【發明内容】 發明内谷曰在提供本揭示内容的簡化摘要，以使閱讀 201213803 者對本揭示内容具備基本的理解。此發明内容並非本揭示 内容的完整概述，且其用意並非在指出本發明實施例的重 要/關鍵元件或界定本發明的範圍。 本發明之一實施態樣係有關於一種整合式質譜分析方 法，可用以分離與分析一待測樣本中的多種待測物分子。 根據本發明此一態樣，可利用單一晶片來萃取與分離待測 樣本中的多種待測物分子，且此一晶片相容於多種質譜分 析方法；因此可以進一步簡化既有的整合式生物樣本檢測 方法。 依據本發明一實施例，上述方法包含以下步驟。首先， 提供整合式晶片，此晶片包含一基材，且在基材的上表面 上有一奈米微結構。接著，將待測樣本引入至晶片上表面 的一基線上；並使晶片接觸一分離溶液系統，且分離溶液 系統的液面低於該基線，藉使不同種類的待測物分子彼此 分離。其後，移除分離溶液系統，藉使得待測物分子吸附 於奈米微結構之外表面上。將其上吸附了待測物分子之晶 片置於質譜分析儀中，以進行待測物分子的質譜分析。 本發明之另一實施態樣係有關於一種整合式質譜分析 套組，可用以分離與分析一待測樣本中的多種待測物分 子。根據本發明此一實施態樣，提供了具有萃取、分離功 能的單一晶片，且此一晶片相容於多種質譜分析方法；因 此可以進一步簡化既有的整合式生物樣本檢測套組中所用 的套件。 依據本發明實施例，上述套組包含一晶片與一分離溶 液系統。上述晶片包含基材，且在基材的上表面上有一奈 201213803 米微結構；舉例來說，此一奈米微結構可以是奈米孔洞、 奈米層片或奈米線狀結構。溶液系統包含下列溶液之一或 其混合物：水、曱醇、乙腈、醋酸、三氟醋酸、醋酸銨、 丙酮、碳酸鈉、碳酸氫鈉、磷酸鈉、磷酸二氫鈉、磷酸鉀、 磷酸二氫鉀以及三羥曱基胺基甲烧。 - 具體來說，運用上述本發明各實施態樣或下文實施方 式中提出的具體實施例，至少可以實現以下效果和/或優 點。 _ 首先，可將帶有待測物分子的晶片直接運用於質譜分 析儀中，因此可以略去先前所使用的質譜轉移步驟。另外， 由於不需進行質譜轉移步驟，可以降低待測物分子在轉移 過程間耗損或逸失的機率，還可以省略以往常用的待測物 分子擴增/濃縮步驟。 其次，相較於先前技術，此處提出的整合式方法對人 力操作的需求較低’適用於更加自動化的操作流程，因此 也具有高通量的優點。又，將多種步驟整合於單一晶片上， # 可以減少套組中套件的數目亦可降低操作時間，而能夠降 低成本。 此外’由於本發明之矽奈米結構能有效吸收質譜儀之 雷射能量並取代傳統有機基質，故此一整合式質譜分析方 法可不需要使用額外的有機基質，因此在進行質譜分析的 時候不會產生基質雜訊的問題，而能夠提升偵測敏感度且 有利於小分子或低濃度分子的分析。 在參閱下文實施方式後，本發明所屬技術領域中具有 通常知識者當可輕易暸解本發明之基本精神及其他發明目 201213803 的，以及本發明所採用之技術手段與實施態樣。 【實施方式】 為了使本揭示内容的敘述更加詳盡與完備，下文中參 照圖式以針對本發明的實施態樣與具體實施例提出了說明 ' 性的描述；但這些描述並非實施或運用本發明具體實施例 的唯一形式。當可理解，雖然實施方式中涵蓋了多個具體 實施例的特徵以及用以建構與操作這些具體實施例的方法 • 步驟與其順序；但亦可利用其他具體實施例來達成相同或 均等的功能與步驟順序。因此，除非上下文另有相反的明 示，下文所提出的各步驟可以同時進行，或可調整其進行 的先後順序。 為了進一步整合既有的生物樣本定量與定性分析方 法，本發明的一目的在於將分析的前端作業（即，樣本前 處理）與分析作業（即，樣本中多種待測物分子的分離與 質譜分析）結合於單一晶片上，並據以發展出整合式質譜 φ 分析方法與整合式質譜分析套組。 本發明之一實施態樣係有關於一種整合式質譜分析方 法，可用以分離與分析一待測樣本中的多種待測物分子。 第1圖所示的概要圖式中，闡明了根據本發明一具體 實施例的操作流程；而第2圖為可運用於本發明具體實施 例中之一種例示性整合式晶片的電子顯微照片。 第1A至1D圖繪示了根據本發明一具體實施例的整合 式質譜分析方法的部分步驟。 首先，提供整合式晶片100,第1A圖為整合式晶片 201213803 100的概要前視圖。根據本發明的原理與精神，整合式晶 片100包含一基材（第1A圖中未繪示），且在基材的上表 面上有一奈米微結構（第1A圖中未繪示）。 適用於本方法之基材的材料包括（但不限於）：矽、玻 璃、石英、二氧化矽、高分子聚合物以及上述材料的組合 物。 此處所述的奈米微結構係指結構特徵的尺寸屬於微米 (micron)、次微米（submicron)或奈米（nano)等級。在 φ 可用以實現本方法的通常情況中，適用的奈米微結構之實 施例包括（但不限於）：奈米孔洞、奈米層片或奈米線狀結 構。 目前已發展出多種方法與技術以在基材的表面上形成 奈米微結構；例如：依循氣-液-固（vapor-liquid-solid，VLS) 成長機制的化學氣相沉積法（chemical vapor deposition， CVD )、分子束蠢晶（molecular beam epitaxy )與雷射消炫 法（laser ablation );依循固-液-固（solid-liquid-solid, SLS ) 成長機制的直接加熱冷卻法；以及電化學钱刻法。 以金屬辅助蝕刻法為例，可利用光罩在基材表面上形 成一圖樣化的金屬層以作為姓刻層，而後利用氫氣酸 (Hydrofluoric acid，HF)混合溶液（如 HF/H202/Et0H 混 合溶液）來進行自發性電化學蝕刻；如此一來，即可在基 材的表面上形成奈米線狀微結構。相似地，亦可利用金屬 輔助蝕刻法來製備層片狀或孔洞狀的奈米微結構。 第2圖的電子顯微照片呈現了一種適用於本方法的 例示性晶片200的剖面，在照片中可以清楚地看出，晶片 201213803 200具有基材搬，且在基材2(^一表面上形成了奈米線 204的結構。此-晶片2()()係利用上文所述的金屬輔助餘 刻法所製成。 和不具有奈米微結構的基材相較之下，具有奈米微結 •構的基材之一特徵在於其具有較高的高寬比（aspect ratio)，以奈米線狀結構為例，若其奈米線的寬度為約5〇〇 nm，且其深度（咼度）為約2 μιη，則此奈米線的高寬比為 4 (2000/500=4)。因此，若在基材上密集地形成奈米線狀 鲁結構，則可以大幅地増加晶片之表面積與單位基材截面積 的比例（以下稱為「表面積/面積比」）。此處所述的具有較 高之高寬比與較高表面積/面積比的特徵，有利於後續的分 離/萃取、無基質質譜分析步驟。 舉例來說’奈米線的寬度可為約1 nm至約500 nm ; 更具體而言’其寬度可為約1、2、3、4、5、6、7、8、9、 10、11、12、13、14、15、16、17、18、19、20、30、40、 50、60、70、80、90、100、110、120、130、140、150、 I 160 、 170 、 180 、 190 、 200 、 204 、 220 、 230 、 240 、 250 、 260 、 270 、 280 、 290 、 300 、 310 、 320 、 330 、 340 、 350 、 360、370、380、390、400、410、420、430、440、450、 460、470、480、490或500 nm。而奈米線的深度為約〇.1 μπι 至約10 μπι ;更具體而言，其深度可為約0.1、0.2、0.3、 0.4、0.5、0.6、0.7、0.8、0.9、卜 2、3、4、5、6、7、8、 9或10 μιη。此外，在本發明任選的實施例中，奈米線的高 寬比可為約 2-500，如約 2、3、4、5、6、7、8、9、10、 20、30、40、50、60、70、80、90、100、150、200、250、 201213803 300、350、400、450、500 ° 具有不同奈米微結構之基材的表面積/面積比之計算 方式各不相同。一般來說，具有奈米線狀微結構的基材之 表面積/面積比會受到奈米線的高寬比以及奈米微結構在 • 基材上的密度等因素的影響。至於奈米孔洞與奈米層片則 分別取決於孔洞的深度以及層片數目。根據本發明任選的 實施例，具有奈米微結構的基材之表面積/面積比約為4至 2000 ;具體來說 4、5、6、7、8、9、10、20、30、40、50、 φ 60、70、80、90、100、150、200、250、300、350、400、 450、500、550、600、650、700、750、800、850、900、 950、1〇〇〇、11〇〇、1200、1300、1400、1500、1600、1700、 1800 、 1900 或 2000 。 此外，在任選的實施例中，可對晶片100表面進行特 殊處理，例如以含氟高分子來修飾晶片100表面，以使得 晶片100具有更佳的疏水性。 接著’請參照第1B圖；在此步驟中，將待測樣本引入 φ 至晶片1〇〇之上表面的基線105位置，以使待測樣本no 與晶片100的奈米微結構接觸。舉例來說，可以利用微量 滴管吸取待測樣本，並將其滴至晶片的上表面。此一步驟 亦可利用機械手臂或其他自動化設備來完成。 之後’如第1C圖所示，將此晶片100與分離溶液系統 115接觸’以分離待測樣本110中的多種待測物分子（如： 120a、120b、120c 與 i2〇d)。 根據本發明的原理與精神，此處所述的待測樣本11 〇 可以是任何生物樣本，例如取自生物體血液、唾液、尿液 201213803 或各種細胞和組織的樣本。此類生物樣本中，可能含有各 式各樣的蛋白質（或多胜肽）、RNA、DNA、具藥理學活性 的分子、胞器等等。 根據本發明一實施例，此處所述的待測物分子 (120a-d )係指測生物樣本中的蛋白質（或多胜肽）成分。 在本步驟中，所謂「分離」多種待測物分子係指依據待測 物分子的至少一種物理特性，將具有不同性質的待測物分 子分離開來。 根據本發明的原理與精神，分離溶液系統115中可以 選用一或多種適當的有機溶劑或緩衝溶液；甚至在某些實 施例中也可以使用水（如，HPLC等級的水或去離子水） 作為分離溶液系統115。 作為例示而非限制，分離溶液系統115中可包含一或 多種下列有機溶劑：曱醇、乙醇、丙醇、異丙醇、乙腈、 丙酮、正己烧、環己炫、乙驗與醋酸乙酯。 另外’可用於分離溶液系統115的例示性緩衝溶液可 包含以下一或多種溶液：、醋酸、三氟醋酸、醋酸銨、丙 酮、碳酸納、碳酸氫納、磷酸鈉、填酸二氫納、璘酸_、 峨酸二氫钟以及二經曱基胺基曱烧（tris-hydroxymethyl amino methane, TRIS )。 在本發明中，由於所形成的奈米微結構具有液體通透 性（permeability)’因此分離溶液系統115中的溶液可在壓 力或電場的作用下由下方往上方移動；在溶液移動的過程 中，待測樣本11〇中的某些待測物分子（如12〇ad)會藉 由溶液展開的帶動而在奈米微結構上分離並向上移動，^ 12 201213803 而達到了將不同種類之待測物分子彼此分離之目的。 根據本發明的原理與精神，可利用各種適當的方式使 得晶片100與分離溶液系統115接觸。在一實施例中，可 使晶片100的一端（如，靠近基線105的那一端）與分離 溶液系統115接觸。例如，請參見第1C圖，可以將晶片 100的一端浸泡到分離溶液系統115中。或者是，在另一 實施例中，可將分離溶液系統115由晶片1〇〇的一端（如 罪近基線105的那一端）注入。又或者是，在另一實施例 中’可將晶片100整個浸泡於分離溶液系統115中。 在一任選的實施例中’分離溶液系統115的液面高度 低於基線105的高度（如第1 c圖所示）；亦即，待測樣本 11〇不可直接浸泡於分離溶液系統115中。在此情形下，分 離溶液系統115中的溶液會因為基材100本身的通透性， 而在毛細現象的作用下接觸到待測樣本11〇,並進而帶動 其中的待測物分子12〇a-d在晶片1〇〇中移動。 在實施此一分離步驟時，必須使得晶片1〇()與分離溶 液系統115接觸達一定的時間，以允許待測樣本則中的 : = …120a_d)能夠有效地彼此分離。此一接觸 測樣本與待測物分子身的性質、晶片的分 離月b力和7或分離溶液系統的種類。 …=i在進行本步驟時，也可任選地控制環境的溫度、 Π 乂利待測物分子12〇“的彼此分離。舉例來 s ，可在恆溫恆濕機+進行分 液系蛴4考疋可在分離溶 液以加速分離溶液系統115中的溶 (連门樣本m中的待測物分子120a_d)在晶片1〇〇中 t S1 13 201213803 的移動。 相較於先前技術，本發明所提出的整合式晶片可藉由 奈米微結構來進行樣本中待測物分子的彼此分離，且分離 後的待測物樣本可直接留置於奈米微結構的表面上。理論 上’在本發明的任選實施例中，可運用多種既有的分離原 理與技術（如，層析法（包含管柱式層析與平板式層析） 與電泳分離法），來進行多種待測物分子之間的分離。 作為例示而非限制，常用的分離方法包含：液相層析 (liquid phase chromatography, LC )、反相液相層析 (reversed-phase liquid chromatography，RPLC )、薄層層析 (thin-layer chromatography，TLC )、尺寸篩除層析術（size exclusion chromatography, SEC )、微胞電動力層析（micellar electrokinetic chromatography, MEKC )、等電聚焦 (isoelectric focusing，IEF )、毛細管帶狀電泳分離（capillary zone electrophoresis，CZE )以及毛細管電泳分離（capillary electrophoresis, CE)模式等。下文將針對這些不同分離技 術於本發明中之應用，提出進一步的說明。 當利用平板式層析與電泳分離法來進行此分離步驟 時，經分離的待測物分子120a-d會停留在晶片100上的特 定位置。值得注意的是，傳統上在進行管柱式層析法時， 是讓混合物由管柱的一端進入，不同的成分在不同的時間 自管柱的另一端流出；然而，當利用本發明提出的整合式 晶片來進行管柱式層析時，可利用奈米微結構來取代傳統 的管柱；此外，經分離的待測物分子會因為隨後進行吸附 步驟而附著於奈米微結構的表面上，而不會被析出。 201213803 在此本步驟中，將待測物分子保留於奈米微結構的表 面而不將其析出的優點之一在於待測物分子不需經過額外 的分離與轉置步驟。如此一來，可將晶片直接送入各種質 譜分析儀器中進行質譜分析（詳如後述），不但大幅簡化分 析流程，也可避免待測物分子在轉置過程中逸失或被被污 染的可能性，同時也可提升偵測靈敏度。 其後，在第1D圖所示的步驟中，移除分離溶液系統 115，藉使待測物分子（如120a-d)吸附於奈米微結構（第 1D圖中未繪示）之外表面上。 在本步驟中，所謂的移除分離溶液系統115包含兩個 部分。第一部分是指將晶片100自溶液系統115中取出， 而使得晶片100不再與溶液系統115相接觸。第二個部分 是乾燥晶片100，以移除存在於晶片/奈米微結構中的溶 液，而使得待測物分子（如120a-d)被固定在奈米微結構 的外表面上。此一固定步驟在某種程度上相當於先前技術 所使用的萃取技術，只不過先前技術中，必須先進行樣本 的前處理，將樣本中的待測物分子「萃取」出來，而後再 進行待測物分子之間的分離；而本發明所提出的方法，僅 藉由施用與移除分離溶液系統，即可達成由待測樣本中萃 取出待測物分子與分離多種待測物分子兩種目的。 一般來說，在此一步驟中常用的手段包含洗滌、乾燥、 真空乾燥、加熱；或者是可混用上述步驟中的多種步驟。 舉例來說，當待測樣本110與溶液系統115中不含雜 質或有機成分等對於後續偵測會造成負面影響的成分時， 可以利用風乾、真空乾燥或加熱乾燥等手法直接乾燥晶 15 201213803 片，以移除位於晶片/奈米微結構中的溶液。需注意，生物 樣本中許多待測物分子對於溫度非常敏感；因此，若是選 用加熱乾燥法，必須選擇適當的加熱溫度，以免影響或破 壞待測物分子的性質，而影響分析的準確性。 另一方面，若是待測樣本11()與溶液系統115中可能 ' 含有雜質或有機成分（例如細胞碎片、鹽類、緩衝液、其 他物染物等），則必須先進行洗滌步驟（例如，以去離子水 沖洗），而後再乾燥晶片。 • 當晶片10〇完全乾燥後，將其上吸附了待測物分子（如 120a-d)之晶片1〇〇置於質譜分析儀中，以對待測物分子 (如120a-d)進行質譜分析。在此步驟中，可搭配各種電 腦資料庫搜尋演算法（如：MS-Fit資料庫、Mascot資料庫、 ProFound資料庫等）’將質譜分析的結果與資料庫進行比 對，以對待測物分子進行定性分析。 根據本發明的原理與精神，本發明所提出的方法可運 用多種既有的質譜分析技術。此外，此處所提出的晶片1〇〇 _ 可直接運用於各種質譜分析儀中，而不需進行先前技術所 必須的質譜轉移步驟。 適用於本方法的質譜分析方法包括：脫附電噴灑離子 化（desorption electrospray ionization，DESI)質譜分析法、 各種雷射脫附離子化（laserdesorption/ionization, LDI)質 譜分析法與石夕材表面直接遊離（desorption/ionization on silicon，DIOS)質譜分析法等等。 值得注意的是’由於本發明所採用的整合式晶片具有 高表面積/面積比之奈米微結構，此種奈米微結構能夠有效 201213803 地吸收質f#儀所發射出的能量（如雷射能量），並可將此能 量轉移至待測物分子使其離子化，以供進行質譜分析。因 此，在分析過程中不需要添加額外的有機基質來輔助生物 樣本中待測物分子的離子化。換句話說，本發明所提出的 •方法能夠實現無基質質譜分析（matrix_free mass .sPectrometir)。由於不需添加額外的有機基質，根據本發 明之方法的另-項優點在於可以降低或排除有機基質所引 起的雜訊問題，進而提升分析的靈敏度。 # 另一方面’此處所提出的整合式晶片幾乎可用於各種 質譜分析儀中，包括：脫附電喷灑離子化（DESI)質譜儀、 無介質雷射脫附離子化（matrix-free LDI，MFLDI)質譜分 析儀、基質輔助雷射脫附離子化（matrix-assisted LDI, MALDI )質譜分析儀、表面增強雷射脫附離子化 (surface-enhanced LDI，SELDI)質譜分析儀、表面輔助雷 射脫附離子化（surface-assisted LDI，SALDI)質譜分析儀 以及矽材表面直接遊離（DIOS)質譜分析儀。舉例來說， • MALDI質譜分析儀是目前使用最廣泛的一種質譜分析 儀’當將根據本方法將整合式晶片運用於MALDI質譜分析 儀之中時’可不需額外添加有機基質，而直接進行質譜分 析。 . 第3A-3D圖繪示了根據本發明一實施例，利用液相層 析法來分離多種待測物分子的原理。在本實施例中，所採 用的方法與材料大致上與參照第1A-第1D圖所述的方法與 材料相同；此處為求簡潔，不再贅述相似的部分，而僅針 對明顯的差異點進行說明。 17 201213803 第3A圖繪示了根據本發明一實施例的整合式晶片3〇〇 的概要剖面圖，晶片300有一基材302與位於基材上表面 上之奈米微結構304(在本實施例中是奈米線）。舉例來說’ 此處所示的晶片300可以是第2圖所示的晶片200。此外， 晶片300上有一預定義的基線3〇5位置。 • 在第3B圖中’將待測樣本310引入至晶片300的基線 305位置上。 隨後在第3C圖中，將含有待測樣本31〇的晶片3〇〇 • 整個浸泡於分離溶液系統315中，以分離待測樣本310中 的多種待測物分子（如：320a、320b與320c)。在本實施 例中，刀離溶液系統315可包令—或多種上文所述的有機 溶劑或水；或者是’分離溶液系統315也可以僅包含水。 在本實施例中’分離溶液系統315中的有機溶劑係作 為液態流動相（mobile phase)，而整合式晶片300與其上 的奈米微結構304則可作為固態相（stati〇nary phase)。當 分離溶液系統315中的液體（液態相）與具有高表面積/面 • 積比與通透性的奈米微結構304 (固態相）接觸時，樣本 310中的待測物分子32〇a-c會經過洗滌作用而和奈米微結 構壁面相互作用，並產生流體阻滯現象（retenti〇n)。樣本 310中與奈米微結構壁面作用力較強的待測物分子（如 320c)流動的速度較為緩慢；相對地，樣本31〇中與奈米 微結構壁面作用力較弱的待測物分子（如320a)流動的迷 ’ 度則較快；如此一來’可在奈米微結構上造成分離現象， 而使得不同的待測物分子320a-c彼此分離。 之後，如第3D圖所示，移除分離溶液系統315，藉使 18 201213803 待測物分子（如320a-c)吸附於奈米微結構3〇4之外表面 最後，將第3D圖中所示的晶片3〇〇連同其上的待測 物分子320a-c —併送入適當的質譜分析儀中，並根據上文 所述的方法進行待測物分子32〇a_c的質譜分析。However, in the qualitative and quantitative analysis of biological samples using the above methods, it is often necessary for a professional technician to invest a large amount of time. Taking 2D-PAGE as an example, it is usually time-consuming to complete a cycle analysis to 2 days. In addition, loss of loss often occurs when samples are transferred between stages; and some detection techniques are less sensitive, so these trace or loss of analyte molecules may not be recognized. In view of the above problems, the related experts have proposed an integrated mass spectrometry method. However, the currently proposed integrated mass spectrometry methods are often limited to the integration of separation principles (such as 2D-PAGE) or the integration of pretreatment and separation methods. The former combines 2D-PAGE on a single wafer (2D_pAGE on a chip). The latter is integrated into a single wafer by a solid-state extraction (s〇Hd phase extracti〇n) method and high-performance liquid chromatography (HPLC). Therefore, there is still room for further integration in terms of the overall quantitative qualitative analysis process. SUMMARY OF THE INVENTION The present invention provides a simplified summary of the disclosure in order to provide a basic understanding of the present disclosure. This Summary is not an extensive overview of the disclosure, and is intended to be illustrative of the scope of the invention. One embodiment of the present invention relates to an integrated mass spectrometry method for separating and analyzing a plurality of analyte molecules in a sample to be tested. According to this aspect of the invention, a single wafer can be used to extract and separate a plurality of analyte molecules in a sample to be tested, and the wafer is compatible with a plurality of mass spectrometry methods; thus, the existing integrated biological sample can be further simplified. Detection method. According to an embodiment of the invention, the method comprises the following steps. First, an integrated wafer is provided which comprises a substrate and has a nanostructure on the upper surface of the substrate. Next, the sample to be tested is introduced onto a baseline of the upper surface of the wafer; and the wafer is brought into contact with a separation solution system, and the liquid level of the separation solution system is lower than the baseline, so that different kinds of analyte molecules are separated from each other. Thereafter, the separation solution system is removed by allowing the analyte molecules to adsorb on the outer surface of the nanostructure. A wafer on which molecules of the analyte are adsorbed is placed in a mass spectrometer for mass spectrometric analysis of the molecules of the analyte. Another embodiment of the present invention is directed to an integrated mass spectrometry kit for separating and analyzing a plurality of analyte molecules in a sample to be tested. According to this embodiment of the present invention, a single wafer having an extraction and separation function is provided, and the wafer is compatible with a plurality of mass spectrometry methods; thus, the kit used in the existing integrated biological sample detection kit can be further simplified. . In accordance with an embodiment of the invention, the kit includes a wafer and a separate solution system. The wafer comprises a substrate and has a 201213803 square microstructure on the upper surface of the substrate; for example, the nanostructure may be a nanopore, a nanopore or a nanowire structure. The solution system comprises one of the following solutions or a mixture thereof: water, decyl alcohol, acetonitrile, acetic acid, trifluoroacetic acid, ammonium acetate, acetone, sodium carbonate, sodium hydrogencarbonate, sodium phosphate, sodium dihydrogen phosphate, potassium phosphate, dihydrogen phosphate Potassium and trishydroxymethylaminocarbyl. In particular, at least the following effects and/or advantages can be achieved by using the above-described embodiments of the present invention or the specific embodiments set forth in the following embodiments. _ First, the wafer with the analyte molecules can be directly used in the mass spectrometer, so the mass spectrometry step previously used can be omitted. In addition, since the mass spectrometry transfer step is not required, the probability of loss or loss of the molecules of the analyte during the transfer process can be reduced, and the conventional molecular amplification/concentration step of the analyte can be omitted. Secondly, compared to the prior art, the integrated approach proposed here has a lower demand for human operation', which is suitable for more automated operational processes and therefore has the advantage of high throughput. In addition, by integrating multiple steps on a single wafer, # can reduce the number of kits in the kit and reduce operating time, while reducing costs. In addition, since the nanostructure of the present invention can effectively absorb the laser energy of the mass spectrometer and replace the traditional organic matrix, the integrated mass spectrometry method does not require the use of an additional organic matrix, and thus does not occur during mass spectrometry. The problem of matrix noise can improve the detection sensitivity and facilitate the analysis of small molecules or low concentration molecules. After referring to the embodiments below, those skilled in the art to which the present invention pertains can readily understand the basic spirit of the invention and other inventions 201213803, as well as the technical means and implementations of the invention. BRIEF DESCRIPTION OF THE DRAWINGS In order to make the description of the present disclosure more detailed and complete, the following description is made with reference to the embodiments of the present invention, and the description The only form of the specific embodiment. It will be understood that the features of the various embodiments, and the methods, steps, and sequences thereof, which are used to construct and operate the embodiments, can be utilized in the embodiments, but other specific embodiments may be utilized to achieve the same or equivalent functions. Step sequence. Therefore, unless the context clearly dictates otherwise, the steps set forth below may be performed simultaneously, or the order in which they are performed may be adjusted. In order to further integrate existing quantitative and qualitative analysis methods of biological samples, one object of the present invention is to analyze the front-end work of the analysis (ie, sample preparation) and the analysis operation (ie, separation and mass spectrometry analysis of multiple analyte molecules in the sample). ) Combines on a single wafer and develops an integrated mass spectrometry φ analysis method and an integrated mass spectrometry suite. One embodiment of the present invention relates to an integrated mass spectrometry method for separating and analyzing a plurality of analyte molecules in a sample to be tested. In the schematic diagram shown in FIG. 1, an operational flow according to an embodiment of the present invention is illustrated; and FIG. 2 is an electron micrograph of an exemplary integrated wafer that can be used in a specific embodiment of the present invention. . Sections 1A through 1D illustrate some of the steps of an integrated mass spectrometry method in accordance with an embodiment of the present invention. First, an integrated wafer 100 is provided, and Fig. 1A is a schematic front view of the integrated wafer 201213803 100. In accordance with the principles and spirit of the present invention, integrated wafer 100 includes a substrate (not shown in Figure 1A) and has a nanostructure (not shown in Figure 1A) on the upper surface of the substrate. Materials suitable for the substrate of the method include, but are not limited to, ruthenium, glass, quartz, ruthenium dioxide, high molecular weight polymers, and combinations of the foregoing. The nanostructures referred to herein are those in which the dimensions of the structural features are of the micron, submicron or nano scale. In the general case where φ can be used to carry out the method, embodiments of suitable nanostructures include, but are not limited to, nanopores, nanosheets or nanowire structures. A variety of methods and techniques have been developed to form nanostructures on the surface of substrates; for example, chemical vapor deposition in accordance with a vapor-liquid-solid (VLS) growth mechanism. , CVD ), molecular beam epitaxy and laser ablation; direct heating and cooling according to the solid-liquid-solid (SLS) growth mechanism; and electrochemistry Money engraving. Taking the metal-assisted etching method as an example, a patterned metal layer can be formed on the surface of the substrate by using a photomask as a surname layer, and then a mixed solution of hydrofluoric acid (HF) (such as HF/H202/Et0H mixing) can be used. The solution) is subjected to spontaneous electrochemical etching; thus, a nanowire-like microstructure can be formed on the surface of the substrate. Similarly, metal-assisted etching can also be used to prepare layered or porous nanostructures. The electron micrograph of Fig. 2 presents a cross section of an exemplary wafer 200 suitable for use in the method. It is clear from the photograph that the wafer 201213803 200 has a substrate transfer and is on the surface of the substrate 2 The structure of the nanowire 204 is formed. This wafer 2 () () is made by the metal assisted residual method described above. Compared with the substrate without the nano microstructure, One of the bases of the rice micro-structure is characterized by a high aspect ratio, and a nanowire-like structure is exemplified, if the width of the nanowire is about 5 〇〇 nm, and The depth (twist) is about 2 μηη, and the aspect ratio of this nanowire is 4 (2000/500=4). Therefore, if the nanowire-like Lu structure is densely formed on the substrate, it can be greatly increased. The ratio of the surface area of the wafer to the cross-sectional area of the unit substrate (hereinafter referred to as "surface area/area ratio"). The characteristics of the higher aspect ratio and higher surface area/area ratio described herein are advantageous for subsequent separation. /Extraction, no matrix mass spectrometry step. For example, the width of the nanowire can be from about 1 nm to about 5 00 nm; more specifically 'the width can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, I 160 , 170 , 180 , 190 , 200 , 204 , 220 , 230 , 240 , 250 , 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 or 500 nm The depth of the nanowire is about 11. 1 μπι to about 10 μπι; more specifically, the depth can be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, Bu 2, 3 4, 5, 6, 7, 8, 9 or 10 μηη. Further, in an optional embodiment of the present invention, the aspect ratio of the nanowires may be about 2 to 500, such as about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 201213803 300, 350, 400, 450, 500 ° with different The surface area/area ratio of the substrate of the m-microstructure is calculated differently. Generally speaking Factors having a surface area of the substrate microstructure nano wire / nanowire area than would be the aspect ratio and density of the nano-microstructure • like on a substrate. As for the nanopore and the nanolayer, it depends on the depth of the hole and the number of layers. According to an optional embodiment of the invention, the surface area to area ratio of the substrate having the nanostructures is from about 4 to about 2000; specifically, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 50, φ 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1〇 〇〇, 11〇〇, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000. Moreover, in an optional embodiment, the surface of the wafer 100 can be specially treated, for example, with a fluoropolymer to modify the surface of the wafer 100 to provide better hydrophobicity of the wafer 100. Next, please refer to FIG. 1B; in this step, the sample to be tested is introduced into φ to the baseline 105 position on the upper surface of the wafer 1 , so that the sample to be tested is in contact with the nano microstructure of the wafer 100. For example, a sample to be tested can be aspirated using a micropipette and dropped onto the upper surface of the wafer. This step can also be done with a robotic arm or other automated equipment. Thereafter, as shown in Fig. 1C, the wafer 100 is brought into contact with the separation solution system 115 to separate a plurality of analyte molecules (e.g., 120a, 120b, 120c, and i2〇d) in the sample 110 to be tested. In accordance with the principles and spirit of the present invention, the sample 11 to be tested described herein can be any biological sample, such as a sample taken from biological blood, saliva, urine 201213803, or various cells and tissues. Such biological samples may contain a wide variety of proteins (or polypeptides), RNA, DNA, pharmacologically active molecules, organelles, and the like. According to an embodiment of the invention, the analyte molecule (120a-d) described herein refers to a protein (or multi-peptide) component in a biological sample. In this step, the so-called "separation" of a plurality of analyte molecules means that the analyte molecules having different properties are separated according to at least one physical property of the molecules to be tested. In accordance with the principles and spirit of the present invention, one or more suitable organic solvents or buffer solutions may be employed in the separation solution system 115; even in certain embodiments, water (e.g., HPLC grade water or deionized water) may be used as The solution system 115 is separated. By way of illustration and not limitation, the separation solution system 115 may comprise one or more of the following organic solvents: decyl alcohol, ethanol, propanol, isopropanol, acetonitrile, acetone, n-hexanol, cyclohexyl, ethyl acetate, and ethyl acetate. Further, an exemplary buffer solution that can be used in the separation solution system 115 can comprise one or more of the following solutions: acetic acid, trifluoroacetic acid, ammonium acetate, acetone, sodium carbonate, sodium hydrogencarbonate, sodium phosphate, dihydrogen sodium, hydrazine Acid _, dihydrogen citrate and tris-hydroxymethyl amino methane (TRIS). In the present invention, since the formed nano microstructure has liquid permeability, the solution in the separation solution system 115 can be moved from below to below by pressure or electric field; during the movement of the solution Some of the analyte molecules (such as 12〇ad) in the sample to be tested will be separated on the nanostructure by the expansion of the solution and move up, ^ 12 201213803 to achieve different kinds of treats The purpose of separating the analyte molecules from each other. In accordance with the principles and spirit of the present invention, wafer 100 can be brought into contact with separation solution system 115 in a variety of suitable manners. In one embodiment, one end of the wafer 100 (e.g., the end near the baseline 105) can be contacted with the separation solution system 115. For example, referring to Figure 1C, one end of the wafer 100 can be immersed in the separation solution system 115. Alternatively, in another embodiment, the separation solution system 115 can be injected from one end of the wafer 1 (as the end of the baseline near the baseline 105). Still alternatively, in another embodiment, the wafer 100 can be entirely immersed in the separation solution system 115. In an optional embodiment, the liquid level of the separation solution system 115 is lower than the height of the baseline 105 (as shown in FIG. 1c); that is, the sample 11 to be tested cannot be directly immersed in the separation solution system 115. . In this case, the solution in the separation solution system 115 contacts the sample to be tested 11 〇 under the action of the capillary phenomenon due to the permeability of the substrate 100 itself, and further drives the analyte molecule 12 〇ad therein. Move in the wafer 1〇〇. In carrying out this separation step, the wafer 1 must be brought into contact with the separation solution system 115 for a certain period of time to allow the : = 120a_d in the sample to be tested to be effectively separated from each other. This contact measures the nature of the sample and the molecular body of the analyte, the separation of the wafer, and the type of the separation solution system. ...=i When performing this step, it is also possible to optionally control the temperature of the environment, and to separate the molecules of the analyte 12" from each other. For example, s can be performed in a constant temperature and humidity machine + a liquid separation system 蛴 4 The solution may be separated in order to accelerate the dissolution of the solution in the separation solution system 115 (the analyte molecules 120a-d in the gate sample m) in the wafer 1 t t S1 13 201213803. Compared to the prior art, the present invention The proposed integrated wafer can separate the molecules of the analyte in the sample by the nano microstructure, and the separated sample of the sample can be directly deposited on the surface of the nano microstructure. Theoretically, the present invention In an alternative embodiment, a variety of existing separation principles and techniques (eg, chromatography (including column chromatography and plate chromatography) and electrophoretic separation) can be employed to perform interaction between various analyte molecules As an example and not limitation, commonly used separation methods include: liquid phase chromatography (LC), reversed-phase liquid chromatography (RPLC), thin layer chromatography (thin- Layer chromatography,TL C), size exclusion chromatography (SEC), micellar electrokinetic chromatography (MEKC), isoelectric focusing (IEF), capillary zone electrophoresis (capillary zone electrophoresis) , CZE) and capillary electrophoresis (CE) mode, etc. Further explanation will be provided below for the application of these different separation techniques in the present invention. This separation step is carried out by using plate chromatography and electrophoresis separation. The separated analyte molecules 120a-d will remain at a particular location on the wafer 100. It is worth noting that, in the case of column chromatography, the mixture is allowed to enter from one end of the column, The composition flows out from the other end of the column at different times; however, when the integrated wafer proposed by the present invention is used for column chromatography, the nano microstructure can be used to replace the conventional column; The separated analyte molecules will adhere to the surface of the nanostructure due to the subsequent adsorption step, and will not be 201213803 In this step, one of the advantages of retaining the molecule of the analyte on the surface of the nano-structure without precipitating it is that the molecule of the analyte does not need to undergo additional separation and transposition steps. The wafer can be directly sent to various mass spectrometry instruments for mass spectrometry (as described later), which not only greatly simplifies the analysis process, but also avoids the possibility that the analyte molecules will escape or be contaminated during the transposition process. Improve detection sensitivity. Thereafter, in the step shown in FIG. 1D, the separation solution system 115 is removed, so that the molecules of the analyte (eg, 120a-d) are adsorbed on the surface of the nanostructure (not shown in FIG. 1D). on. In this step, the so-called removal separation solution system 115 contains two parts. The first portion refers to the removal of the wafer 100 from the solution system 115 such that the wafer 100 is no longer in contact with the solution system 115. The second part is to dry the wafer 100 to remove the solution present in the wafer/nano microstructure so that the analyte molecules (e.g., 120a-d) are immobilized on the outer surface of the nanostructure. This fixed step is somewhat equivalent to the extraction technique used in the prior art, except that in the prior art, the sample must be pre-processed to "extract" the analyte molecules in the sample and then proceed. The separation between the analyte molecules; and the method proposed by the present invention can achieve the extraction of the analyte molecules from the sample to be tested and the separation of the plurality of analyte molecules by the application and removal of the separation solution system. of. In general, the means commonly used in this step include washing, drying, vacuum drying, heating; or mixing various steps in the above steps. For example, when the sample to be tested 110 and the solution system 115 do not contain impurities or organic components, which may adversely affect subsequent detection, the crystals may be directly dried by air drying, vacuum drying or heat drying. To remove the solution located in the wafer/nano microstructure. It should be noted that many analyte molecules in biological samples are very sensitive to temperature; therefore, if the heating drying method is selected, the appropriate heating temperature must be selected to avoid affecting or destroying the properties of the analyte molecules and affecting the accuracy of the analysis. On the other hand, if the sample to be tested 11() and the solution system 115 may contain impurities or organic components (such as cell debris, salts, buffers, other dyes, etc.), the washing step must be performed first (for example, Rinse with deionized water and then dry the wafer. • When the wafer 10 is completely dried, the wafer on which the analyte molecules (such as 120a-d) are adsorbed is placed in a mass spectrometer to perform mass spectrometry on the analyte molecules (such as 120a-d). . In this step, you can use a variety of computer database search algorithms (such as: MS-Fit database, Mascot database, ProFound database, etc.) to compare the results of mass spectrometry with the database to treat the analyte molecules. Conduct a qualitative analysis. In accordance with the principles and spirit of the present invention, the method of the present invention utilizes a variety of existing mass spectrometry techniques. In addition, the wafers 1 _ presented herein can be directly used in various mass spectrometers without the need for mass spectrometry steps necessary in the prior art. Mass spectrometry methods suitable for the method include: desorption electrospray ionization (DESI) mass spectrometry, various laser desorption ionization (LDI) mass spectrometry and direct Desorption/ionization on silicon (DIOS) mass spectrometry and the like. It is worth noting that 'the integrated wafer used in the present invention has a nano-structure with a high surface area/area ratio, and the nano-structure can effectively emit energy (such as laser energy) from the absorption device of 201213803. ), and this energy can be transferred to the analyte molecule for ionization for mass spectrometry analysis. Therefore, no additional organic matrix needs to be added during the analysis to aid in the ionization of the analyte molecules in the biological sample. In other words, the method proposed by the present invention enables matrix-free mass spectrometry (matrix_free mass.sPectrometir). Since there is no need to add an additional organic matrix, another advantage of the method according to the invention is that the noise problems caused by the organic matrix can be reduced or eliminated, thereby increasing the sensitivity of the analysis. #应用'The integrated wafer proposed here can be used in almost all kinds of mass spectrometers, including: desorption electrospray ionization (DESI) mass spectrometer, medium-free laser-desorption ionization (matrix-free LDI, MFLDI) mass spectrometer, matrix-assisted laser desorption ionization (MADI) mass spectrometer, surface-enhanced laser desorption ionization (surface-enhanced LDI, SELDI) mass spectrometer, surface-assisted laser A surface-assisted LDI (SALDI) mass spectrometer and a coffin surface direct free (DIOS) mass spectrometer. For example, • The MALDI mass spectrometer is currently the most widely used mass spectrometer. 'When the integrated wafer is used in a MALDI mass spectrometer according to this method', the mass spectrometry can be performed without additional organic matrix. analysis. 3A-3D illustrate the principle of separating a plurality of analyte molecules by liquid phase stratification according to an embodiment of the present invention. In the present embodiment, the method and material used are substantially the same as the methods and materials described with reference to FIGS. 1A-1D; here, for the sake of brevity, similar parts will not be described, but only for obvious differences. Be explained. 17 201213803 FIG. 3A is a schematic cross-sectional view showing an integrated wafer 3 according to an embodiment of the present invention. The wafer 300 has a substrate 302 and a nanostructure 304 on the upper surface of the substrate (in this embodiment). In the middle is the nanowire). For example, the wafer 300 shown here may be the wafer 200 shown in FIG. In addition, wafer 300 has a predefined baseline 3〇5 position. • Introduce the sample 310 to be tested to the baseline 305 position of the wafer 300 in Figure 3B. Then, in FIG. 3C, the wafer containing the sample 31〇 to be tested is entirely immersed in the separation solution system 315 to separate a plurality of analyte molecules in the sample 310 to be tested (eg, 320a, 320b, and 320c). ). In this embodiment, the knife-off solution system 315 may comprise - or a plurality of the organic solvents or waters described above; or the 'separation solution system 315 may also comprise only water. In the present embodiment, the organic solvent in the separation solution system 315 is used as a liquid phase, and the integrated wafer 300 and the nanostructures 304 thereon can be used as a solid phase. When the liquid (liquid phase) in the separation solution system 315 is in contact with the nanostructure 304 (solid phase) having a high surface area/surface ratio and permeability, the analyte molecule 32 〇ac in the sample 310 will After washing, it interacts with the wall of the nano-structure and produces a fluid block phenomenon (retenti〇n). In sample 310, the molecules of the analyte (such as 320c) with strong interaction with the surface of the nano-microstructures flow slowly; relatively, the molecules of the sample with weak interaction with the nano-structures in the sample 31〇 (eg, 320a) The flow of the fan's degree is faster; thus, 'the separation phenomenon can be caused on the nano-structure, and the different analyte molecules 320a-c are separated from each other. Thereafter, as shown in FIG. 3D, the separation solution system 315 is removed, so that 18 201213803 analyte molecules (eg, 320a-c) are adsorbed on the outer surface of the nanostructures 3〇4, and finally, in the 3D map. The wafer 3 is shown together with the analyte molecules 320a-c thereon and fed into an appropriate mass spectrometer, and mass spectrometric analysis of the analyte molecules 32〇a_c is performed according to the method described above.
第4A-4D圖繪示了根據本發明另一實施例，利用電泳 2離法來分離多種待測物分子的原理。在本實施例中所 心用的方法與材料大致上與參照第1A_第1D圖與第3A-3D 圖=的方法與材料相同；此處為求簡潔，不再贅述相似 的°卩刀而僅針對明顯的差異點進行說明。 第4A圖繪示之整合式晶片4〇〇與上文所述之晶片3〇〇 大致相同，装 具具有基材402與位於基材上表面上之奈米線 404。此外，曰μ 曰日片400亦上有一預定義的基線405位置。 jr ^ λ 405位置上Β圖中，將待測樣本410引入至晶片400的基線 整二1在第4C圖中，將含有待測樣本410的晶片40C 的夕=姓於分離溶液系統415中，以分離待測樣本410中 例；，物分子（如：420a、420b與420〇。在本實施 _ 離’谷液系統415可包含一或多種上文所述的緩衝 0 iW- al ,、，士 s u卜’將一外接電壓源430連接至晶片4〇〇兩端， 的 中形成一電場。在此種情形下，樣本400中 動 a乃子420a-c會在緩衝溶液的帶動下而在電場中移 。田利用等電聚焦原理來進行分離時，待測物分子 42〇a-dc备a泰… ， 太半，站隹電场的作用下向正極或負極移動分別移動到 丁只微、。構404中的等電點（iso-electric point，PI)位置。 19 201213803 或者是，當利用毛細管電泳原理來進行分離時，待測物分 子420a-c會根據其電荷/大小比（q/r)而彼此分離。 接著，可利用上文參照第1D圖所述的方法，來移除 分離溶液系統415，藉使待測物分子（如420a-c)吸附於 奈米微結構404之外表面上，如第4D圖所示。 最後，將第4D圖中所示的晶片400連同其上的待測 物分子420a-c —併送入適當的質譜分析儀中，並根據上文 所述的方法進行待測物分子420a-c的質譜分析。 本發明之另一實施態樣係有關於一種整合式質譜分析 套組，可用以分離與分析一待測樣本中的多種待測物分子。 依據本發明實施例，上述套組包含一晶片（如上文所 述的晶片100或晶片200 )與一分離溶液系統（如上文所 述的分離溶液系統115)。上述晶片包含基材，且在基材的 上表面上有一奈米微結構；舉例來說，此一奈米微結構可 以是奈米孔洞、奈米層片或奈米線狀結構。溶液系統包含 下列溶液之一或其混合物：水、甲醇、乙醇、丙醇、異丙 醇、乙腈、丙酮、正己烷、環己烷、乙醚、醋酸乙酯、醋 酸、三氟醋酸、醋酸銨、丙酮、碳酸鈉、碳酸氫鈉、磷酸 鈉、磷酸二氫鈉、磷酸鉀、磷酸二氫鉀以及三羥曱基胺基 甲烷（TRIS )。 根據本發明各種實施例，可選用矽、玻璃、石英、二 氧化石夕或高分子聚合物或混合兩種以上的上述材料，來製 備本套組中的基材。 此外，在本發明不同的實施例中，可將此套組中的晶 片運用於不同的質譜分析儀中；例如：脫附電喷灑離子化 201213803 (DESI)質譜儀、無介質雷射脫附離子化（MFLDI)質譜 分析儀、表面增強雷射脫附離子化（SELDI)質譜分析儀、 表面輔助雷射脫附離子化（SALDI)質譜分析儀、或矽材 表面直接遊離（DIOS)質譜分析儀。 ' 由上述本發明實施方式可知，本發明提出了一種更為 • 簡單的整合式質譜分析方法/套組，且其整合程度更甚先前 技術；因此不論在進行質譜分析時，對於人力、時間、物 力的需求都比先前技術來得低，能夠提升分析效率並降低 ^ 成本。 此外，運用本發明之質譜分析方法/套組時，可以不需 進行質譜轉移與其他轉移步驟，亦不需使用有機基質。不 但可以降低待測物分子在轉移過程間耗損或逸失的機率， 還可以省略以往常用的待測物分子擴增/濃縮步驟。這些特 點可以提升本發明之質譜分析方法/套組的偵測敏感度；且 有利於小分子或低濃度分子的分析。 舉例來說，在癌症患者的體細胞内，往往存有多種低 φ 濃度的生物標記（biomarker )蛋白質，且這些生物標記的 含量可能會隨著患者對治療的反應性而改變。若能及早、 準確地偵測到這些生物標記存在或濃度的改變，對於癌症 的確診與治療將有非常大的助益。在其他牽涉到患者體内 特定蛋白質成分改變的疾病中，亦存在著類似的現象。因 * 此，本發明的質譜分析方法/套組可望對癌症與這些疾病的 ' 診斷與治療開啟新的契機。 雖然上文實施方式中揭露了本發明的具體實施例，然 其並非用以限定本發明，本發明所屬技術領域中具有通常 21 201213803 知識者，在不悖離本發明之原理與精神的情形下，當可對 其進行各種更動與修飾，因此本發明之保護範圍當以附 申請專利範圍所界定者為準。 【圖式簡單說明】 為讓本發明的上述與其他目的、特徵、優點與實施 能更明顯易懂，所附圖式之說明如下：4A-4D are diagrams illustrating the principle of separating a plurality of analyte molecules by electrophoresis 2 ionization according to another embodiment of the present invention. The methods and materials used in this embodiment are substantially the same as the methods and materials referred to in FIGS. 1A-1D and 3A-3D =; here, for the sake of brevity, the similar files are not described again. Only the obvious differences are explained. The integrated wafer 4A shown in Fig. 4A is substantially identical to the wafer 3' described above, and has a substrate 402 and a nanowire 404 on the upper surface of the substrate. In addition, the 曰μ 曰 片 400 also has a predefined baseline 405 position. In the jr ^ λ 405 positional top view, the sample to be tested 410 is introduced to the baseline of the wafer 400. In FIG. 4C, the wafer 40C containing the sample 410 to be tested is stored in the separation solution system 415. In order to separate the sample to be tested 410; the object molecules (eg, 420a, 420b, and 420〇. In the present embodiment, the liquid solution system 415 may include one or more of the buffers described above, iW-al, , An external voltage source 430 is connected to both ends of the wafer 4 to form an electric field. In this case, the moving a is 420a-c in the sample 400 is driven by the buffer solution and is in the electric field. When the field is separated by the isoelectric focusing principle, the analyte molecule 42〇a-dc is prepared as a..., too, and the movement of the electric field to the positive or negative electrode is moved to Ding Wei, respectively. The isoelectric point (PI) position in the structure 404. 19 201213803 Alternatively, when the separation is performed by the capillary electrophoresis principle, the analyte molecules 420a-c are based on their charge/size ratio (q/). r) separated from each other. Next, the method described above with reference to FIG. 1D can be used to remove The solution system 415 is separated by adsorbing molecules of the analyte (e.g., 420a-c) onto the outer surface of the nanostructure 404, as shown in Fig. 4D. Finally, the wafer 400 shown in Fig. 4D is associated therewith. The analyte molecules 420a-c on the substrate are sent to an appropriate mass spectrometer and mass spectrometric analysis of the analyte molecules 420a-c is performed according to the method described above. Another embodiment of the present invention is An integrated mass spectrometry kit can be used to separate and analyze a plurality of analyte molecules in a sample to be tested. According to an embodiment of the invention, the kit comprises a wafer (such as wafer 100 or wafer 200 as described above). And a separate solution system (such as the separation solution system 115 described above). The wafer comprises a substrate and has a nano microstructure on the upper surface of the substrate; for example, the nano microstructure can be Michole, nanosheet or nanowire structure. The solution system contains one of the following solutions or a mixture thereof: water, methanol, ethanol, propanol, isopropanol, acetonitrile, acetone, n-hexane, cyclohexane, ether , ethyl acetate, acetic acid, three Acetic acid, ammonium acetate, acetone, sodium carbonate, sodium hydrogencarbonate, sodium phosphate, sodium dihydrogen phosphate, potassium phosphate, potassium dihydrogen phosphate, and trishydroxyalkylaminomethane (TRIS). According to various embodiments of the present invention, optional The substrate in the kit is prepared by using ruthenium, glass, quartz, silica dioxide or a polymer or a mixture of two or more of the above materials. Further, in various embodiments of the present invention, the kit may be used. The wafers are used in different mass spectrometers; for example: desorption electrospray ionization 201213803 (DESI) mass spectrometer, medium-free laser desorption ionization (MFLDI) mass spectrometer, surface-enhanced laser desorption ion (SELDI) mass spectrometer, surface assisted laser desorption ionization (SALDI) mass spectrometer, or coffin surface direct free (DIOS) mass spectrometer. As can be seen from the above-described embodiments of the present invention, the present invention proposes a more simple integrated mass spectrometry method/set, and the degree of integration is more advanced than that of the prior art; therefore, for mass spectrometry, for manpower, time, The demand for material resources is lower than that of the prior art, which can improve the efficiency of analysis and reduce the cost. In addition, the mass spectrometry method/set of the present invention eliminates the need for mass spectrometry transfer and other transfer steps, and does not require the use of an organic matrix. Not only can the probability of loss or loss of the analyte molecules during the transfer process be reduced, but also the conventional molecular amplification/concentration step of the test object can be omitted. These features can enhance the detection sensitivity of the mass spectrometry method/set of the present invention; and facilitate the analysis of small molecules or low concentration molecules. For example, in the somatic cells of cancer patients, there are often a variety of low-φ concentrations of biomarker proteins, and the content of these biomarkers may change with the patient's response to treatment. If the presence or concentration of these biomarkers is detected early and accurately, it will be of great help for the diagnosis and treatment of cancer. A similar phenomenon exists in other diseases involving changes in specific protein components in patients. Because of this, the mass spectrometry method/set of the present invention is expected to open up new opportunities for the diagnosis and treatment of cancer and these diseases. The specific embodiments of the present invention are disclosed in the above embodiments, which are not intended to limit the present invention, and the present invention has the general knowledge of 21 201213803, without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the scope of the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS In order to make the above and other objects, features, advantages and embodiments of the present invention more obvious, the description of the drawings is as follows:
第1A圖至第1D圖為方法流程圖，概要繪示了根據本 發明一具體實施例之整合式質譜分析方法的部分實施流 程； 、第2圖為電子顯微照片，闡明了可用於本發明之整合 式質譜分析方法/套組中的例示性晶片的剖面照片； 口 第3Α圖至第3D圖為方法流程圖，概要繪示了根據本 發明一具體實施例利用液相層析法來進行整合式質譜分析 方法的部分實施流程；以及 第4Α圖至第4D圖為方法流程圖，概要繪示了根據本 發明另一具體實施例利用電泳分離法來進行整合式質譜分 析方法的部分實施流程。 曰刀 【主要元件符號說明】 100 晶片 105 基線 110 待測樣本 115 分離溶液系統 t S3 22 201213803 120a、120b、120c、120d 待測物分子 200 晶片 202 基材 204 奈米線 300 晶片 302 基材 304 奈米線 305 基線 310 待測樣本 315 分離溶液系統 320a、320b、320c 待測物分子 400 晶片 402 基材 404 奈米線 405 基線 410 待測樣本 415 分離溶液系統 420a、420b、420c 待測物分子 430 外接電壓源 231A to 1D are process flow diagrams, schematically showing a partial implementation flow of an integrated mass spectrometry method according to an embodiment of the present invention; and FIG. 2 is an electron micrograph illustrating the use of the present invention. Cross-sectional photograph of an exemplary wafer in an integrated mass spectrometry method/set; Ports 3D through 3D are method flow diagrams schematically depicting the use of liquid chromatography in accordance with an embodiment of the present invention Partial implementation flow of the integrated mass spectrometry method; and FIG. 4 to FIG. 4D are method flow diagrams, schematically showing a partial implementation flow of the integrated mass spectrometry method using electrophoresis separation method according to another embodiment of the present invention .曰 【 [Main component symbol description] 100 wafer 105 baseline 110 sample to be tested 115 separation solution system t S3 22 201213803 120a, 120b, 120c, 120d analyte molecule 200 wafer 202 substrate 204 nanowire 300 wafer 302 substrate 304 Nanowire 305 Baseline 310 Samples to be tested 315 Separation solution system 320a, 320b, 320c Datum of molecules 400 Wafer 402 Substrate 404 Nanowire 405 Baseline 410 Sample to be tested 415 Separation solution system 420a, 420b, 420c Molecular molecules to be tested 430 external voltage source 23