200533916 4 五·發明說明 [技術領域] 本發明係有關於分子探針晶片(molecular probe chips)，特 別是可快速準確進行分子樣本檢測定性(molecular sample testing, examination and characterization)，具有以共價鍵結 (covalent bonding)進行聯結(linking)之定錨化合物(anchoring compound)之分子探針晶片。 ί先前技藝】 利用定錨化合物(anchoring compound)將選定的分子探針 (molecular probe)固定化(immobilize)在具有伸展表面的基板 (substrate)上，可以製成分子探針晶片(molecular probe chips>針對特定標的物(target)的偵檢探測用途所設計製作的 分子探針晶片具有廣泛用途。例如，在基礎醫學及臨床醫學 的領域之中，一般習知為生物晶片(biochip)或生物感測晶片 (biological sensor chips)的分子探針晶片，可以快速而準確地 進行生醫樣本（biological sample)的檢測與診斷 (characterization and diagnosis)。在醫療用途中，進行檢測診 斷的樣本可能是分子，病毒，細菌或細胞樣本。在其他非醫 學領域的產業用途之中，所牽涉到的則可能是諸如食品或環 境取樣等的工業檢測定性樣本。 在生物晶片(biochip)的用途之中，基板表面固定化的探 200533916 針，可與檢測溶液中的目標分子進行相互作用(interaction reaction)。根據反應的結果可以偵測特定的生物分子。基板 表面固定化的探針可以是寡核甘酸(oligonucleic acid)、胜肽 (peptide)、蛋白質(protein)、抗體(antibody)、抗原(antigen) 或其他可以和偵測目標分子發生反應的細胞或組織表面生 物分子。此種利用基板表面聯結固定化分子作為擔體 (ligand>以便與生醫樣本中的生物分子(即待分析物(analyte)) 發生作用，藉由檢測兩者之間相互作用的結合量(binding concentration)與結合常數(binding constant)，便可以提供一種 本的數據化診斷與實驗參數。兩者之間的高親合性、高結 合容量與生物穩定度，在生物晶片的偵檢應用上是非常重要 的特性。 目前有多種生物鑑定的作法需要將生物分子如DNA或蛋 白質直接固定化在基板上。例如，西方點漬(western blotting) 係將蛋白質吸附在聚活化亞乙烯(poly vinylidene fluoride, PVDF)或硝化纖維素(nitrocellulose)薄膜基板上當作探針，可 與抗體或抗原蛋白質上的官能基互相作用，以測量樣品中是 否含有此種蛋白質。此外，南方及北方點漬(southern and northern blotting)亦分別利用類似的方式將DNA和RNA各自 吸附在硝化纖維素基板上，用以探測和探針具有互補性序列 的DNA和RNA。另一個例子是酵素連結免疫吸附分析法 (enzyme_linked immunosorbent assay, ELISA)，其係將特定抗 髋(或抗原)先吸附在聚苯乙烯(polystyrene)基板上，再將基板 200533916 ^ 暴露在檢體中。若此檢體含有對該些抗體(或抗原)具高親和 性的特定抗原(或抗體)，便會與之結合而被偵測出來。 例如，頒予 Fodor 等人的 U. S. Pat. No· 5,445,934, “Array of oligonucleotides on a solid substrate”中揭示了在基板上直接 合成聚核甘酸(polynucleotide)的方法。其係在玻璃載片上先 進行衍生化(derivatization)，以使其表面具有光保護基 (photoprotective group)化學物質，再利用光罩在適當位置進 行云保護，然後才與含有光保護基的核甘酸單體進行反應。 整個的製備過程必須重覆進行光罩、去保護(deprotection)和 反應(coupling)步驟，直至得到所需的聚核甘酸序列為止。另 外亦可以事先合成聚核甘酸，之後再利用諸如微點列(robotic printing)或噴墨(ink_jet printing)等技術，經由物理吸附作用， 光反應連結，或其他鍵結將其佈置於基板上。上述將生物分 子如DNA或蛋白質直接固定化在基板上的方法，其步騾複 雜費時，而且常需使用昂貴的設備。此外，生物分子大部份 係以微弱的物理吸附方式和基板連結，其穩定性低。此種作 法造成晶片的清洗與處理繁瑣費時，並且其再現性也低。200533916 4 V. Description of the invention [Technical field] The present invention relates to molecular probe chips, in particular, it can quickly and accurately perform molecular sample testing, examination and characterization, and has a covalent bond Covalent bonding is a molecular probe wafer of an anchoring compound that is linked. [Previous technology] An anchoring compound is used to immobilize a selected molecular probe on a substrate having an extended surface, and a molecular probe chip can be made. Molecular probe wafers designed for specific target detection applications have a wide range of uses. For example, in the fields of basic medicine and clinical medicine, biochips or biosensors are commonly known. Molecular probe chips of biological sensor chips can quickly and accurately characterize and diagnose biological samples. In medical applications, the samples used for detection and diagnosis may be molecules or viruses. , Bacteria or cell samples. Among other non-medical industrial applications, the qualitative samples involved in industrial testing such as food or environmental sampling may be involved. In biochip applications, the substrate surface is fixed Chemical probe 200533916, which can interact with the target molecule in the detection solution (inte raction reaction). Based on the results of the reaction, specific biomolecules can be detected. Probes immobilized on the substrate surface can be oligonucleic acid, peptide, protein, antibody, antigen (antigen) or other biomolecules on the surface of cells or tissues that can react with the detection target molecule. This kind of substrate uses immobilized molecules as a support (ligand) to interact with biomolecules in the biomedical sample (that is, the analyte (analyte)), by detecting the binding concentration and binding constant of the interaction between the two, it can provide a data-based diagnosis and experimental parameters. The high between the two Affinity, high binding capacity and biological stability are very important characteristics in the detection and application of biochips. At present, there are many biological identification methods that require biomolecules such as DNA or protein to be immobilized directly on the substrate. For example, Western blotting refers to the adsorption of proteins on poly vinylidene fluoride (PVDF) or nitrocellulose ( As a probe on a nitrocellulose film substrate, it can interact with functional groups on antibodies or antigenic proteins to measure whether the sample contains such proteins. In addition, southern and northern blotting also used similar methods to separately adsorb DNA and RNA on nitrocellulose substrates to detect and probe DNA and RNA with complementary sequences. Another example is the enzyme_linked immunosorbent assay (ELISA), which specifically adsorbs a specific anti-hip (or antigen) on a polystyrene substrate, and then exposes the substrate 200533916 ^ to the specimen . If the specimen contains a specific antigen (or antibody) with high affinity for these antibodies (or antigens), it will be detected by binding to it. For example, U.S. Pat. No. 5,445,934, "Array of oligonucleotides on a solid substrate" awarded to Fodor et al. Discloses a method for directly synthesizing polynucleotide on a substrate. It is first derivatized on a glass slide so that its surface has a photoprotective group chemical, and then uses a photomask to protect the cloud in place, and then reacts with riboic acid containing the photoprotective group. The monomers react. The entire preparation process must be repeated with the photomask, deprotection and coupling steps until the desired polynucleotide sequence is obtained. In addition, it is also possible to synthesize polynucleotide in advance, and then use technologies such as robotic printing or inkjet printing to arrange it on the substrate through physical adsorption, photoreactive bonding, or other bonding. The above method of directly immobilizing a biological molecule such as DNA or protein on a substrate is complicated and time-consuming, and often requires expensive equipment. In addition, most of the biomolecules are connected to the substrate by weak physical adsorption, and their stability is low. This method makes the cleaning and processing of the wafer cumbersome and time-consuming, and its reproducibility is also low.
Ishii 等人之 US· Pat. No. 5,474,895，“Non-isotopic detection of nucleic acids using a polystyrene support-based sandwich hybridization assay and compositions useful therefor” 中提出利用三明治雜交分析(sandwich hybridization analysis) 來取代螢光偵測。基板經適當的官能基修飾或連結以使其帶 有活性基，即諸如醇基、羰基、胺基、醛基、環氧基或硫醇 200533916 基等的官能基。其表面可以和聚核甘酸形成共價或非共價連 結。又如，Van Ness 等人之 U.S. Pat. No. 5,514,785 “Solid suppqrt for nucleic acid hybridization assays”案中提出利用共 價鍵結將聚核甘酸連結在尼隆(nylon)支撐物上。再如Rampal 於 U.S· Pat. No. 6,013,789，“Covalent attachment of biomolecules to derivatized polypropylene supports” 案中揭 示，聚丙烯薄膜先經過氨化，之後再與其未端含有磷咪唑烷 (phosphorimidazolide)的聚核甘酸反應，如此，聚核甘酸便可 經由磷咪唑烷(phosphoramidate)鍵結而與聚丙烯產生共價連 結。 另外，利用在氧化銦錫(indium tin oxide, ITO)電極表面上 進行分子的自組裝而形成單層薄膜(self_assembled monolayers)，亦可以提供一種新的界面，可用以探討某些生 物分子特殊的氧化還原反應。有機矽可以和表面具有氫氧基 的基板反應(例如二氧化矽)，形成單層或雙層的有機矽薄 膜。例如，Chrisey 等人於 U.S. Pat No.5,688,642 號”Selective attachment of nucleic acid molecules to patterned self-assembled surfaces”案中提出具有兩個活性基的有機矽化合 物，利用一端的活性基和基板表面的氫氧基產生連結，再由 另一端的活性基連結生物分子，以使生物分子得以被固定化 在基板上。 這些方法雖然已有廣泛應用，但卻有分子探針製備繁瑣 費時及不穩定等，商業大量製備(commercial mass production) 200533916 上的限制。這是由於要在諸如PVDF，硝化纖維素或顯微玻 璃(microscopic glass)等常見較適用的基板上固定化生物分 子，通常所依賴的物理吸附(physical adsorption)作用乃是一種 不穩定而且耗時的過程。整個固定化處理程序通常需要至少 六至八小時的安置(incubation)時間。其典型的處理方法係先 將生物分子探針加至晶片基板上並靜置晶片。待分子探針與 基材之間進行一定長時間(通長約三至四小時)的物理吸附反 應之後，連續操作數次的清洗程序。清洗之後再加入填補物 (blocking reagent)，並再靜置一段長時間(通常約三至四小時） @ 後，此晶片才得以作為生物晶片使用。 [概要内容] 因此有需要提供一種分子探針晶片，具有共價鍵結定錨 化合物，其製備簡單且快速，適於低成本大量生產。 另亦有需要提供一種分子探針晶片，具有共價鍵結定錨 化合物;具有儲存及使用上之高穩定性。. · 為達成前述及其他目的，本發明提供一種分子探針晶 片，具有共價鍵結定錨化合物，其包括有：一基板，該基板 一表面上佈覆有一金屬薄膜層；複數個的定錨化合物分子， 大致具有延伸之長形分子構形，每一個該些定錨化合物分子 各係以其長形構形之一第一端共價鍵結於該金屬薄膜層 上；與複數個的探針分子，每一個該些探針分子各係共價鍵 結於該些定錨分子化合物中對應一定錨分子化合物反對於 /3 200533916 長形構形之該第一端之一第二端。 本發明並提供分子探針晶片之一^種無探針半成品晶片基 板，可預先儲存以在需要時聯結該分子探針晶片進行偵檢感 测所需之探針分子，該無探針半成品晶片基板包括有：一基 板，該基板一表面上佈覆有一金屬薄膜層；與複數個的定錨 化合物分子，大致具有延伸之長形分子構形，每一個該些定 錨化合物分子各係以其長形構形之一第一端共價鍵結於該 金屬薄膜層上，且其反對於該長形構形之該第一端之一第二 端，可於該需要情況下與該探針分子共價鍵結，以完整形成 該分子探針晶片。 本發明並提供一種分子探針晶片之製作方法，其步驟包 括有先於一基板之一表面上佈覆一金屬薄膜層；再將複數個 大致具有延伸且實質為長形之分子構形的定錨化合物分 子，其各自長形構形之一第一端，共價鏈結於該基板之佈覆 金屬薄膜層上。本發明之方法亦可再於每一個該些定錨分子 化合物對應於該第一端之第二端分別共價鍵結一探針分 子。 [寳施方式] 本發明具有共價鍵結定錨化合物之分子探針晶片，其晶 片之基板係可採用一般的砂(silicon)，氮化砍(silicon nitride)，石英(quartz)或玻璃(glass)質的片形板材。基板表面 上佈覆一層金屬，其最佳係利用，例如，蒸鍍的方式進行金 200533916 屬薄膜層的佈覆。適用的金屬包含金，銀，鉻及鎳等。以生 物晶片之用途為例，較佳之金屬應為金或銀。 本發明之探針晶片若作為感測晶片之用途，可適用於諸 如電化學生物感測分析方式的偵檢測驗。在另一種用途之 中，本發明之探針晶片係被當作振盪晶片使用，可應用於諸 如壓電(piezoelectric)生物感測分析方式的偵測。在又另一種 應用之中，本發明之探針晶片係被當作表面電漿共振晶片使 用，適用於諸如光學生物感測1分析方式的偵測。 依據本發明，晶片基板上所佈覆之金屬薄膜層若依特別 設計並經特定製程處理之後，可以成為具透光性的基板，例 如，依特定形態陣列排列，整體透光之金屬薄膜層。此種具 透光性之金屬佈覆感測元件可在生物探針標示螢光物或呈 色劑後，使用於應用了光學偵測原理的掃描器或顯微鏡系統 上。 依據本發明，晶片基板上所佈覆之金屬薄膜層，其化學 修飾係採用共價鍵結的形式，因此增加了分子探針晶片基板 表面上被固定化的分子探針的穩定性。在生物晶片的應用用 途之中，本發明共價鍵結所達成的探針固定化，可在晶片以 緩衝液(buffer)進行處理，於晶片表面上探針與標的物間進行 行相互作用過程中的解離(dissociation)，結合(association)與 再生(regeneration)步驟時，晶片上以共價鍵結方式所固定化 的生物分子探針即不致因緩衝液的沖洗而脫落流失。 依據本發明，晶片基板上所佈覆之金屬薄膜層，其化學 200533916 修飾方式提供一種新的形式，可將分子探針迅速地固定化於 晶片基板表面上，縮短探針的固定化所需時間。本發明具有 共價鍵結定錨化合物之分子探針晶片，在某些生物晶片的應 用用途之中，生物分子探針的固定化所需時間，可從習知技 術依賴傳統物理吸附(physical adsorption)作用進行處理所需 的六至八小時，大幅縮減到三至五分鐘的程度。 本發明之分子探針晶片，典型係可適用於生物晶片之用 途。依據本發明之方法所製備之生物晶片能迅速且高效率地 與受檢生物分子，例如DNA、RNA、胺基酸或蛋白質中的 某些特定官能基進行共價鍵結，形成具有專一性的生物探 針。利用此等具專一性生物探針，可針對特定的生醫檢測樣 品進行快速偵測。 依據本發明一較佳實施例，生物晶片基板可以是一般光 學儀器所使用的載玻片，其上塗佈諸如金或銀等的一層金屬 薄膜。此種塗佈有金屬的生物晶片基板，本身可以當作電極 片使用，適用於某些電化學分析形式的偵測。由於晶片基板 材質屬於光穿透性元件，因此亦可應用於一般以光學檢測原 理為基礎的偵測儀器上。此外，若對所加入檢體的生物分子 上進行螢光物質的標示，此種塗佈有金屬的透光生物晶片基 板，亦可應用在目前的螢光檢測系統上。 依據本發明之方法，生物晶片基板上的化學修飾係採用 共價鍵結的形式，因此增加了生物晶片基板表面固定化探針 的穩定性。停留晶片表面但未有反應的物質分子，利用溶劑 200533916 予以沖洗便可去除。如此便可以降低雜質的干擾，因而提高 探計和生醫檢測樣品中之標的物，兩者間之親合性與結合容 量，進一步提昇偵測的靈敏度。 對於各種不同的檢測源生物分子，依據本發明，可分別 提供多樣的，經化學修飾之不同功能性的生物晶片，分別進 行針對性的高準確度檢測。針對檢測源生物分子其結構上的 差異，依據本發明可以選用不同功能性的生物晶片，以達到 探針和標的物分子最佳的專一性結合。依據本發明之生物晶 片，因其對於檢測源生物分子的較佳針對性及專一性，便可 以提供高可信度的檢測資料。 ，依據本發明，要在生物晶片上聯結固定化的生物分子探 針，首先須令選定的有機分子和晶片基板上的金屬，兩者之 間生成共價鍵結，連接在晶片基板表層上。有機分子另外必 須預留一官能基，其可以和欲連結的生物分子探針進行化學 反應。例如，依據本發明之一較佳實施例，於塗佈有金或銀 的晶片基板上，可以採用硫醇基作為主要定錨分子。這是由 於硫原子極易與金或銀發生硫醇基化反應而形成共價鍵 結。 在本發明之一較佳實施例之中，首先可在基板上放置氫 硫基焼胺（mercaptoalkylamine)，其分子結構為 [HS(CH2)nNH2]，其中n=2〜16。氫硫基烷胺之硫原子會與金 或銀發生硫醇基化反應而形成共價鍵結，而有機分子末端的 胺基則可保留，作為親核劑以利於進行化合物的衍生處理。 200533916 利用前述以金或銀金屬塗佈為基礎的生物晶片或生物感 测晶片，進行檢測時，由於晶片是固態而待檢測物則為液態 (諸如血液，尿液，體液或唾液等)，倒此偵測的靈敏度和極 限係依晶片上固定化探針分子的立體障礙性(steric hindrance) 與自由度而定。定錨分子聯結化合物原子間除需保持良好的 自由度之外，定錨分子和探針兩者之間亦必須維持一特定距 離長鹿以模擬探針以待測物在液-液相中的真實相互作用。 因此，若採用較短亞甲基的硫基烷胺（例如n=2的硫基乙胺) 時，則基板上的固定化定錨分子必須先進行鏈長的延伸，之 後才可與探針分子進行化學反應，產生含特定功能基的生物 晶片。另一方面，若使用較長的硫基烷胺（n=8〜16)，則可 直接和探針分子進行化學反應，產生含特定功能基的生物晶 片。 在採用較短亞甲基的硫基烷胺來進行化學修飾的基板之 實例之中，在其基板上的固定化定錨分子，由於必須進行鏈 長的延伸，因此首先可令此基板和烷二醛(alkyl dialdehyde) 進行化合物反應。此化合物所具有的醛基可以和基板中的胺 基進行偶合反應，以生成共價鍵結。其所產生的衍生物具有 末端醛基，可以進一步再和AH3-胺基丙基)-1，3-丙二胺化合 物中的一級胺基進行偶合反應。此時所生成的長鏈衍生物， 其末端具有親核性的一級胺基，可進一步和探針分子進行化 學反應，產生含特定功能基的生物晶片。 AH3-胺基丙基)-1，3-丙二胺化合物係為一種高極性(highly 200533916 polarized)分子。若利用此化合物來進行基板上有機分子鏈長 的衍生化，可以使整個化學修飾後的基板表面，具有較高的 親水性質。由於此等化合物具有一個相當的長度，當此類型 的生物晶片探針和待測檢體溶液中之標的物分子相互作用 時，探針分子與基板表面之間便得以維持一個較長的距離。 由於探針分子同時亦擁有較高的自由度，當與標的物互相辨 識時，晶片的立體障礙性便較低。利用此類型晶片所求算得 出之標的物濃度與結合常魏因此即相當趨近於液-液相中的 真實值。 可連結生物探針之基本生物晶片基板之製備 圖1，2及3之化學反應分別顯示依據本發明一較佳實施 例，於塗佈有金或銀的基板表面上，逐步利用化學修飾處理 而可固定化選定長度之基本有機分子。圖3之化學反應所得 結果為可連結各式生物探針之基本生物晶片基板，可供連結 至各種特定生物分子檢測所需用之生物探針分子，以進行相 關生醫，食品或環保樣本之檢測。 首先，在一較佳實施例之中，如圖1之化學反應所顯示 的，先在塗佈金或銀金屬薄膜層120之晶片基板11〇上放置 氫硫基乙胺。此可以利用將塗佈金或銀之晶片基板底材浸入 濃度約為20 mM的氫硫基乙胺水溶液，或者浸入含有約1〇 mM氫硫基乙胺的磷酸鹽中性(pH值約為7.2)緩衝溶_PBS) 之中，反應大約二小時。之後，利用諸如乙醇和蒸餾水清洗 200533916 基板，即可得到己經氫硫基乙胺進行表面修飾的基板。 圖2之化學反應顯示，圖1修飾反應所得基板102,在與 濃度約2.5%，酸鹼度保持在pH約為7.的戊二醛水溶液反應 大約一小時，進行戊二醛的偶合反應之後，便可獲得如圖2 中所顯示反應結果之基板1〇3。 接著，圖3之化學反應顯示，圖2反應所得基板，利用 和醛基[-CHO]進行反應，即可轉換成末端為胺基[-NH2]的衍 生化合物： a. 首先，圖2反應所得含有醛基的基板103,利用pH 約為7，濃度約為0.1M的磷酸鈉（sodium phosphate)水溶液 進行清洗。由於毒性考量，反應最好必須在抽風櫥内進行。US Pat. No. 5,474,895 by Ishii et al., “Non-isotopic detection of nucleic acids using a polystyrene support-based sandwich hybridization assay and compositions useful therefor” proposes to use sandwich hybridization analysis instead of fluorescent detection Measurement. The substrate is modified or linked with an appropriate functional group so that it carries a reactive group, that is, a functional group such as an alcohol group, a carbonyl group, an amine group, an aldehyde group, an epoxy group, or a thiol 200533916 group. Its surface can form covalent or non-covalent bonds with polynucleotide. In another example, U.S. Pat. No. 5,514,785 to Van Ness et al. Proposed the use of covalent bonding to link polynucleotide to a nylon support using a solid suppqrt for nucleic acid hybridization assays. For another example, Rampal in US Pat. No. 6,013,789, "Covalent attachment of biomolecules to derivatized polypropylene supports" revealed that the polypropylene film is first ammoniated, and then polyglycolic acid containing phosphorimidazolide at its end. In this way, polynucleotide can be covalently linked to polypropylene via phosphoramidate bonding. In addition, the use of molecular self-assembly on the surface of indium tin oxide (ITO) electrodes to form single-layer films (self_assembled monolayers) can also provide a new interface for exploring the specific oxidation of certain biomolecules Reduction reaction. Organic silicon can react with substrates with hydroxyl groups on the surface (such as silicon dioxide) to form single or double-layered organic silicon films. For example, Chrisey et al., In US Pat No. 5,688,642 "Selective attachment of nucleic acid molecules to patterned self-assembled surfaces" proposed an organosilicon compound with two active groups, using the active group at one end and the oxygen on the substrate surface. The radicals are linked, and the biomolecules are linked by the active group at the other end, so that the biomolecules can be immobilized on the substrate. Although these methods have been widely used, the preparation of molecular probes is cumbersome, time-consuming, unstable, and the like, and commercial mass production (200533916) is limited. This is due to the immobilization of biomolecules on common and more suitable substrates such as PVDF, nitrocellulose or microscopic glass, and the physical adsorption effect usually relied on is a kind of unstable and time-consuming the process of. The entire immobilization process typically requires at least six to eight hours of incubation time. The typical processing method is to first add a biomolecule probe to a wafer substrate and let the wafer stand. After performing a physical adsorption reaction between the molecular probe and the substrate for a certain period of time (approximately three to four hours in length), the cleaning procedure is continuously performed several times. After cleaning, adding blocking reagent, and let it stand for a long period of time (usually about three to four hours), then the wafer can be used as a biochip. [Summary] Therefore, there is a need to provide a molecular probe wafer with a covalently bonded anchor compound, which is simple and fast to prepare and suitable for mass production at low cost. There is also a need to provide a molecular probe wafer having a covalently bonded anchor compound; having high stability in storage and use. In order to achieve the foregoing and other objectives, the present invention provides a molecular probe wafer having a covalently bonded anchoring compound, which includes: a substrate, a metal thin film layer is coated on one surface of the substrate; The anchor compound molecules generally have an elongated elongated molecular configuration, and each of the anchor compound molecules is covalently bonded to the metal thin film layer with a first end of one of its elongated configurations; and a plurality of Each of the probe molecules is covalently bonded to each of the anchor molecule compounds corresponding to a certain anchor molecule compound against / 3 200533916 one of the first ends of the long configuration. The invention also provides one kind of semi-finished wafer substrate without probes, which can be stored in advance to connect the molecular probe wafers with probe molecules required for detection and sensing when needed. The semi-finished wafer without probes The substrate includes: a substrate, a metal thin film layer is coated on one surface of the substrate; and a plurality of anchor compound molecules, which generally have an extended elongated molecular configuration, and each of the anchor compound molecules is based on its A first end of the elongated configuration is covalently bonded to the metal thin film layer, and it opposes a second end of the first end of the elongated configuration, and can be connected with the probe if necessary. The molecules are covalently bonded to complete the molecular probe wafer. The invention also provides a method for manufacturing a molecular probe wafer. The method comprises the steps of: firstly coating a metal thin film layer on a surface of a substrate; and then fixing a plurality of molecular structures with substantially extended and substantially elongated molecular configurations. The anchor compound molecules, each of which has a first end of one of the elongated configurations, are covalently linked to the cloth-coated metal film layer of the substrate. The method of the present invention can also covalently bond a probe molecule to each of the anchor molecules corresponding to the second end of the first end. [Bao Shi mode] In the molecular probe wafer of the present invention having a covalently bonded anchor compound, the substrate of the wafer can be ordinary silicon, silicon nitride, quartz or glass ( glass) quality sheet-shaped plate. A layer of metal is coated on the surface of the substrate, and the best method is, for example, gold 200533916 which is a thin film layer. Suitable metals include gold, silver, chromium and nickel. Taking biochip applications as an example, the preferred metal should be gold or silver. If the probe chip of the present invention is used as a sensing chip, it can be applied to detection such as electrochemical biosensor analysis. In another application, the probe chip of the present invention is used as an oscillating chip, and can be applied to detection such as piezoelectric biosensor analysis. In yet another application, the probe wafer of the present invention is used as a surface plasma resonance wafer, and is suitable for detection such as optical biosensor 1 analysis method. According to the present invention, the metal thin film layer coated on the wafer substrate can be a light transmissive substrate if it is specially designed and processed by a specific process, for example, the metal thin film layer is arranged in an array in a specific form and has overall light transmission. This translucent metal cloth-covered sensing element can be used on a scanner or microscope system that uses the principle of optical detection after the biological probe is labeled with a fluorescent substance or a coloring agent. According to the present invention, the chemical modification of the metal thin film layer coated on the wafer substrate is in the form of covalent bonding, thereby increasing the stability of the molecular probes immobilized on the surface of the molecular probe wafer substrate. In the application of biological wafers, the probes achieved by the covalent bonding of the present invention can be immobilized, and the wafer can be processed with a buffer, and the interaction between the probes and the target can be performed on the wafer surface During the dissociation, association, and regeneration steps, the biomolecule probes immobilized by the covalent bonding method on the wafer will not fall off due to the washing of the buffer solution. According to the present invention, the chemical film 200533916 modification method of the metal thin film layer provided on the wafer substrate provides a new form, which can quickly immobilize the molecular probe on the surface of the wafer substrate and shorten the time required for the immobilization of the probe. . In the molecular probe wafer having a covalently bonded anchor compound of the present invention, in the application of some biochips, the time required for the immobilization of the biomolecular probe can depend on conventional physical adsorption from conventional techniques. ) The time required for processing is six to eight hours, which is greatly reduced to three to five minutes. The molecular probe wafer of the present invention is typically applicable to the use of biological wafers. The biochip prepared according to the method of the present invention can quickly and efficiently perform covalent bonding with certain specific functional groups in a test biomolecule, such as DNA, RNA, amino acid, or protein to form a specific Biological probe. With these specific bioprobes, you can quickly detect specific biomedical test samples. According to a preferred embodiment of the present invention, the bio-wafer substrate may be a glass slide used in general optical instruments, and a metal thin film such as gold or silver is coated thereon. This kind of metal-coated bio-wafer substrate can be used as an electrode itself, which is suitable for detection in some forms of electrochemical analysis. Since the material of the wafer substrate is a light transmissive element, it can also be applied to detection instruments generally based on optical detection principles. In addition, if fluorescent substances are labeled on the biomolecules added to the specimen, such metal-coated light-transmitting biochip substrates can also be applied to current fluorescent detection systems. According to the method of the present invention, the chemical modification on the substrate of the biological wafer is in the form of covalent bonding, thereby increasing the stability of the immobilized probe on the surface of the substrate of the biological wafer. Molecules remaining on the wafer surface but not reacted can be removed by rinsing with solvent 200533916. In this way, the interference of impurities can be reduced, and the target substance in the probe and biomedical test samples can be improved. The affinity and binding capacity between the two can further improve the detection sensitivity. For a variety of different detection source biomolecules, according to the present invention, a variety of chemically modified biochips with different functionalities can be separately provided for targeted high-accuracy detection. Aiming at the difference in the structure of the detection source biomolecule, according to the present invention, biochips with different functions can be selected to achieve the best specific combination of the probe and the target molecule. The biological wafer according to the present invention can provide highly reliable detection data because of its better pertinence and specificity for detecting the source biomolecules. According to the present invention, in order to connect the immobilized biomolecule probes on a biochip, first, a selected organic molecule and a metal on the wafer substrate must be covalently bonded to each other and connected to the surface layer of the wafer substrate. Organic molecules must additionally have a functional group that can chemically react with the biomolecule probe to be linked. For example, according to a preferred embodiment of the present invention, on a wafer substrate coated with gold or silver, a thiol group can be used as the main anchor molecule. This is because the sulfur atom easily reacts with gold or silver to form a thiol reaction to form a covalent bond. In a preferred embodiment of the present invention, first, mercaptoalkylamine can be placed on the substrate, and its molecular structure is [HS (CH2) nNH2], where n = 2 ~ 16. The sulfur atom of the hydrogenthioalkylamine will undergo a thiolation reaction with gold or silver to form a covalent bond, while the amine group at the end of the organic molecule can be retained as a nucleophile to facilitate the derivatization of the compound. 200533916 Using the above-mentioned gold or silver metal-coated bio-wafer or bio-sensing wafer for testing, because the wafer is solid and the test object is liquid (such as blood, urine, body fluid or saliva, etc.), The sensitivity and limit of this detection are determined by the steric hindrance and degree of freedom of the immobilized probe molecules on the wafer. In addition to maintaining a good degree of freedom between the anchor molecule and the compound atom, a certain distance must be maintained between the anchor molecule and the probe to simulate the probe to test the object in the liquid-liquid phase. Real interaction. Therefore, if a shorter methylene thioalkylamine (such as thioethylamine with n = 2) is used, the immobilized anchor molecule on the substrate must be extended by the chain length before it can be used with the probe. The molecules react chemically to produce a biochip containing specific functional groups. On the other hand, if a longer thioalkylamine (n = 8 to 16) is used, a chemical reaction with the probe molecule can be directly performed to generate a biological wafer containing a specific functional group. In the case of using a shorter methylene thioalkylamine to chemically modify a substrate, the anchor molecule on the substrate must be extended by a chain length, so this substrate and the alkyl Dialdehydes carry out compound reactions. The aldehyde group of this compound can be coupled with the amine group in the substrate to form a covalent bond. The resulting derivative has a terminal aldehyde group, which can be further coupled with the primary amine group in AH3-aminopropyl) -1,3-propanediamine compound. The long-chain derivative produced at this time has a nucleophilic primary amine group at the end, which can further chemically react with the probe molecule to generate a biochip containing a specific functional group. AH3-aminopropyl) -1,3-propanediamine compound is a highly polarized molecule. If this compound is used to derivatize the organic molecular chain length on the substrate, the entire surface of the substrate after chemical modification can have higher hydrophilic properties. Because these compounds have a considerable length, when this type of biochip probe interacts with the target molecule in the test solution, a longer distance is maintained between the probe molecule and the substrate surface. Since the probe molecule also has a high degree of freedom, when the target is recognized by the target, the stereoscopic obstacle of the wafer is low. The concentration of the target substance calculated with this type of wafer and the combination of Chang Wei are therefore quite close to the true value in the liquid-liquid phase. Preparation of a basic biochip substrate that can be connected with a biological probe The chemical reactions in Figures 1, 2 and 3 respectively show that according to a preferred embodiment of the present invention, the surface of a substrate coated with gold or silver is gradually modified by chemical modification. Basic organic molecules of selected lengths can be immobilized. The result of the chemical reaction in Figure 3 is a basic biochip substrate that can be connected to various types of bioprobes, and can be connected to the bioprobe molecules required for the detection of various specific biomolecules for related biomedical, food or environmental protection samples. Detection. First, in a preferred embodiment, as shown in the chemical reaction of FIG. 1, hydrogenthioethylamine is first placed on a wafer substrate 11 coated with a gold or silver metal thin film layer 120. This can be done by immersing a gold or silver coated wafer substrate in an aqueous solution of hydrothioethylamine at a concentration of about 20 mM, or by immersing it in a phosphate-neutral phosphate containing about 10 mM hydrothioethylamine (pH is about 7.2) Buffered PBS) and react for about two hours. After that, the substrate of 200533916 is washed with such as ethanol and distilled water to obtain a substrate which has been surface-modified with hydrogenthioethylamine. The chemical reaction of FIG. 2 shows that the substrate 102 obtained by the modification reaction of FIG. 1 reacts with an aqueous solution of glutaraldehyde having a concentration of about 2.5% and a pH of about 7. for about one hour. After the coupling reaction of glutaraldehyde is performed, A substrate 103 having a reaction result as shown in FIG. 2 can be obtained. Next, the chemical reaction in FIG. 3 shows that the substrate obtained in the reaction in FIG. 2 can be converted into a derivative compound with an amine group [-NH2] at the end by reacting with the aldehyde group [-CHO]: a. First, the reaction obtained in FIG. 2 The substrate 103 containing the aldehyde group is cleaned with a sodium phosphate aqueous solution having a pH of about 7 and a concentration of about 0.1M. Due to toxicity considerations, the reaction must preferably be carried out in a fume hood.
b. 以100毫升經由去離子水所製得的碎冰為準，小心加 入約20克（21.3毫升)的#-(3_胺基丙基)丙二胺（ΛΗ3-Aminopropyl)-l，3-propanediamine)，簡稱 APPDA。APPDA 之結構為[H2N(CH2)3NH(CH2)3NH2]，其一末端的一級胺基可 與烷二醛進行偶合反應，另一末端的一級胺基則預留作為親 和劑，保留供反應後進行化合物的衍生。 同樣基於安全考量，進行此反應時須配載安全眼鏡及手 套。之後，緩慢滴入約8-10毫升的濃鹽酸。此時，碎冰可 以預防大量的煙霧產生，並減緩溫度的上升。其後加入攪拌 子進行攪拌，測量酸鹼值，並持續逐滴加入濃鹽酸，直至溶 液的酸鹼值大約為7時止。此時冰應該已完全溶化。其後加 入去離子水，直至溶液體積約為1〇〇毫升。最後加入磷酸鈉 200533916 鹽，以配製濃度約為0·1Μ的緩衝溶液，並再次調整溶液的 酸鹼值到7.0,亦即中性酸鹼值。 c·將清洗後含有醛基的基板放入APPDA水溶液中。此 時應須攪拌或搖晃反應槽。 d. 加入約1.2克的氛棚烧納（cyanoborohydride， [NaB(CN)H3])，並持續攪拌至少約四小時以上。 e. 反應完成後，所得含有APPDA的基板，利用大量的 蒸餾水清洗，之後使用濃度約為1M的氯化鈉水溶液加以清 洗，最後再以蒸餾水清洗，即可得如圖3反應結果之基板 100〇 注意到圖3中之基板100係屬分子探針晶片之一種半成 品晶片。以圖3中之定錨化合物而言，其係為末端為胺基[-NH2]的一種衍生化合物，亦即，其長形分子構形遠離基板110 之金屬佈覆層120之末端是為一胺基依據本發明，基板1〇〇 係為分子探針晶片的一種半成品。這是由於此基板100上的 每一定錨化合物分子的胺基尚未聯結任何偵檢感測用途所 需的分子探針。此種半成品基板1⑻可以在適當的條件之下 儲存一段長時間。典型可達至少三個月之久。當有需要時， 半成品基板100便可以進一步快速地進行處理，聯結適合與 胺基共價鍵結的多種分子探針中的任何一種，以應用於特定 的偵檢感測用途。 依據本發明，半成品基板聯結分子探針所需的共價鍵結 處理可以極為快速。典型的處理可以快至約三分鐘的共價鍵 200533916 結反應時間的範圍。在醫學用途之中，當類如嚴重急性呼吸 道症候群(SARS，Severe Acute Respiratory Syndrome)類型的 疫病爆發時，依據本發明之分子探針設計觀念，諸如圖3中 所顯示之半成品基板100即可由庫存之中取出，快速進行探 針聯結處理，以便快速而大量地產出檢測生物晶片，應用於 疫病病毒的即時追蹤散佈調察。 涵3A顯不圖3中分子探針晶片半成品100之一^重簡化表 例1: 如同前述，圖3之化學反應所得結果，即如圖3A之簡化 表示，係為可適於連結各式生物探針之基本生物晶片基板， 可供連結至各種特定生物分子檢測所需用之生物探針分 子，以進行相關生醫檢測。圖4之化學反應即顯示，在本發 明一實施例之中，圖3A之基本生物晶片基板100表面，利 用丁二酸酐(succinic anhydride)對定錨有機分子之胺基[-NH2] 進行尽烷基化反應，以轉換成末端為酸基[-COOH]的衍生化 合物。丁二酸酐之分子結構係如圖4之反應式中所顯示。此 化合物所具有的活性羰基可以和基板中的胺基反應，其開環 後會形成末端酸基，可與具有胺基的生物分子結合： a. 利用100毫升的水清洗含有APPDA的基板，然後再 浸泡於100毫升的水中。 b. 攪拌之下同時緩慢地加入約10克的丁二酸酐。 200533916 C.在室溫下反應約一小時。習知相關技術建議反應中應 利用氫氧化鈉將反應溶液的酸鹼值保持在6左右，然而，依 據本發明，反應期間並不必須控制酸鹼值。在沒有氫氧化鈉 參與反應之下，整個反應過程快速且產率高。 d.反應完成後利用大量的水清洗基板，之後使用濃度約 為1M的氯化鈉水溶液清洗，最後再以水清洗，以便移除未 反應的丁二酸。此時即可獲得圖4中所顯示末端為酸基[-COOH]衍生化合物的基板400。 此類型的生物晶片，其定錨有機分子末端所具有的酸 基，可與具有胺基的生物分子，諸如蛋白質、胜肽、核酸、 醣類、脂類等結合，利於相關生醫檢測。 例2: 圖5之化學反應顯示，在圖3A之基本生物晶片基板100 表面，利用硫化丁二酸亞醯氨-馬林亞醯氨苯基-丁酸 (sulfosuccinimidyl-4-(p-maleimidophenyl)-butyrate),簡稱 siTifo-SMPB，對定錨有機分子之胺基[-NH2]進行#•烷基化反 應，以轉換成末端為4-(p-馬林亞醯氨苯基)-丁酸基(4-(p-maleimidophenyl)-butyrate group) 〇CO(CH2)3(CH) 6N(C0)2(CH)2]的衍生化合物。sulfo_SMPB之結構係如圖5之 反應式中所顯7F。此化合物的酯基和基板中的胺基可進行7V-烷基化反應，其所生成的化合物可以和具有硫醇基的生物分 子進行反應： 200533916 將含有APPDA的基板放入含有約2mM sulfo-SMPB的磷 酸鹽緩衝水溶液(PBS)中反應約一小時。在一較佳實施例之 中，PBS係利用約137mM的氯化鈉[NaCl]，2.7mM氯化鉀 [KC1]，10mM磷酸鈉[Na2P04]，以及1.8mM磷酸二氫鉀 [KH2P04]配製而成，其酸鹼值約為7.4。之後，使用PBS沖 洗基板，即可獲得圖5中所顯示末端為sulfo-SMPB衍生化 合物的基板500。此基板最好須保存於4°C之下。 此類型的生物晶片，其定錨有機分子末端所具有的反應 基，可與具有硫醇基的生物分子結合，利於進行相關生醫檢 測。 例3: 圖6之化學反應顯示，在圖3A之基本生物晶片基板100 表面，利用 2_亞胺五ί哀硫(2_iminothiolane hydrochloride，其 結構係如化學反應式中所顯示)對定錨有機分子之胺基[-NH2] 進行硫醇基化反應時會開環，以轉換形成末端為硫醇基[-SH] 的衍生化合物： 於反應槽中加入約0.05M三乙基胺、0.15M氯化鈉水溶 液及ImM乙二胺四醋酸(EDTA)，並調控溶液酸鹼值為約 8Ό。首先將溶液中的氣體移除，其後再將含有APPDA的基 板浸入反應槽中。之後加入約5當量的2-亞胺五環硫。在一 較佳實施例之中，整個反應係在氮氣環境中於室溫下進行約 45分鐘，再使用酸鹼值約為7.2的磷酸鹽緩衝水溶液(pbs) 200533916 進行清洗。最後，基板被保存於PBS中，並置放於室溫下。 此時即可獲得圖6中所顯示末端為硫醇基[-SH]的衍生化合 物的基板600。 此類型的生物晶片，其定錨有機分子末端所具有的硫醇 基，可與具有雙硫鍵的生物分子結合，適於進行相關生醫檢 測。 例4: 圖7之化學反應顯示在圖3A之基本生物晶片基板100表 面，利用氰尿醯氯(cyanuric chloride，其結構係如化學反應式 中所顯示)對定錨有機分子之胺基[-NH2]進行親電子性加成 取代反應，以轉換成末端為氰尿醯氯-活化物質的衍生化合 物： 首先配製約0.01M氰尿醯氯的乙腈溶液，加入約2M鹼 性的二異丙基乙基胺(A^V_diisopropylethylamine)。在約 0-5°C 之下，將含有APPDA的基板浸入其中反應約三小時之後， 取出基板利用乙腈溶劑及丙酮沖洗，以移除基板上未反應的 氰尿醢氯及二異丙基乙基胺。此時即可獲得圖7中所顯示末 端為氰尿醯氯-活化物質的衍生化合物的基板700。 此類型的生物晶片，其定錨有機分子末端所具有的氰尿 醯氯活化基，可與具有醇基、胺基的生物分子結合，適於進 行相關生醫檢測。 200533916 例5: 圖8之化學反應顯示在圖3A之基本生物晶片基板100表 面，利用二咪唾基碳醯(carbonyldiimidazol，其結構係如化學 反應式中所顯示）具有活性的咪唑基碳醯基對定錨有機分子 之胺基|>NH2]進行偶合反應，以轉換成末端為咪唑基碳醯-活 化物質[-CON2C3H3]的衍生化合物： a. 依序利用約30%丙酮水溶液及70%丙酮水溶液清洗含 有APPDA的基板。同樣基於安全考量，此等清洗應於通風 櫥内進行。之後使用丙酮溶劑進行沖洗，以便移除吸附在玻 璃上的水。注意到在清洗的過程之中應保持不讓基板乾掉。 b. 將清洗過的基板放入反應槽中，加入約1克/20毫升 的二咪唑基碳醯，並在室溫之下攪拌約一小時。 c. 反應後使用丙酮進行沖洗，以將反應中所產生的咪唑 化合物予以移除。此時即可獲得圖8中所顯示末端為咪唑基 碳醯-活化物質[-CON2C3H3]的衍生化合物的基板800>此等含 咪唑基碳醯-活化物質的基阪在氮氣下無水丙酮之中可以保 存一年，亦可直接進行下一步的偶合反應。 此類型的生物晶片，其定錨有機分子末端所具有的咪唑 基碳醯活化基，可與具有醇基的生物分子結合，適於進行相 關生醫檢測。 圖9之化學反應顯示依據本發明另一較佳實施例，於塗 佈有金或銀金屬薄膜層920的基板910表面上利用化學修飾 200533916 處理，固定化選定長度之基本有機分子，以供連結需用之生 物探針分子之基本生物晶片基板。圖9中之基板900，與圖3 中之基板100 —樣，亦係屬分子探針晶片之一種半成品晶 片^以圖9中之定錨化合物而言，其亦係為末端為胺基[_NH2] 的--種衍生化合物，亦即，其長形分子構形遠離基板910之 金屬佈覆層920之末端是為一胺基。基板900係為本發明分 子探針晶片的一種半成品，其每一定錨化合物分子的胺基尚 未聯結任何偵檢感測用途所需的分子探針。半成品基板900 可以在適當的條件之下長時間儲存。當有需要時，半成品基 板900便可以進一步快速地進行處理，聯結上適合與胺基共 價鍵結的多種分子探針中的任何一種，以應用於特定的偵檢 感測用途。 圖9中所顯不之基本晶片基板900與圖3中所顯示者(100) 相較之下，具有較長的定錨有機分子(n=8〜16)。除此之外， 圖9中所顯示之基本晶片基板900同樣亦可應用於前述例1 至5之生物晶片用途之中。如同前述，定錨分子和探針兩者 之間必須維持一特定距離長度及自由度，以適於檢測反應時 的相互作用特性。圖10至14中分別顯示前述例1至5中以 較長之氫硫基焼胺，其中n=8〜16作為定鋪有機分子，分別 製作生物晶片之實施例，及分子探針晶片100ft 110ft 1200， 1300 及 1400。 雖然本發明已配合圖式以較佳實施例揭示如上，然其並 非用以限定本發明。例如，本發明之說明雖然係以生物晶片 200533916 為例進行詳細說明，但本發明具有共價鍵結定錨化合物之分 子探針晶片，如同可以理解的，同樣亦可適用於其他使用了 金或銀當作生物晶片上感測薄膜之不同用途的處理。其應用 係例如光學感測晶片，電化學感測晶片，及壓電感測晶片 等。因此，任何熟習此技藝者，在不脫離本發明之精神和範 圍之情況下，當可進行此類更動與變化，因此本發明之保護 範圍當以後附之申請專利範圍所界定者為準。 200533916 ' 圖式簡單說明 圖1，2及3之化學反應分別顯示依據本發明一較佳實施 例，於塗佈有金或銀的基板表面上利用化學修飾(chemical modification)處理之後將特定長度之基本有機分子固定化， 以供連結需用之生物探針分子之基本分子探針晶片基板。 圖3A顯示圖3中分子探針晶片之一種簡化表示。 圖4之化學反應顯示在圖3A之分子探針晶片基板表面， 利用丁二酸酐(succinic anhydride)對定錨有機分子之胺基卜 NH2]進行烷基化(A^alkylation)反應，以轉換成末端為酸基 [-COOH]的衍生化合物。 圖5之化學反應顯示在圖3A之分子探針晶片基板表面， 利用硫化丁二酸亞醯氨-馬林亞醯氨苯基-丁酸 (sulfosuccinimidyl-4-(p-maleimidophenyl)-butyrate， sulfo-SMPB)對定錨有機分子之胺基[-NH2]進行N-焼基化反應(N-alkylation)，以轉換成末端為4-(p-馬林亞醯氨苯基)-丁酸基 (4-(p-maleimidophenyl)-butyrate group) [-CO(CH2)3(CH) 6N(CO)2(CH)2]的衍生化合物。 圖6之化學反應顯示在圖3A之分子探針晶片基板表面， 利用2-亞胺五環硫（2-iminothiolane hydrochloride)對定鋪有 機分子之胺基[-NH2]進行硫醇基化(thiolation)反應，以轉換成 末端為硫醇基[-SH]的衍生化合物。 圖7之化學反應顯示在圖3A之分子探針晶片基板表面， 200533916 利用氰尿醯氯(cyanuric chloride)對定錨有機分子之胺基[-NH2]進行氰尿醯氯化(cyanuric chlorination)反應，以轉換成末 端為氰尿酿氯活化(cyanuric chloride-activated)物質的衍生化 合物。 圖8之化學反應顯示在圖3A之分子探針晶片基板表面， 利用二咪挫基碳酿(carbonyldiimidazol)對定錨有機分子之胺 基[-NH2]進行二咪挫基碳醯化(carbonyldiimidazolization)反 應，以轉換成末端為咪唑基碳醯-活化(carbonyldiimidazol· activated)物質[-CON2C3H3]的衍生化合物。 圖9之化學反應顯示依據本發明另一較佳實施例，於塗 佈有金或銀的基板表面上利用化學修飾處理，固定化選定長 度之基本有機分子，以供連結需用之生物探針分子之基本分 子探針晶片基板。 圖10之化學反應顯示在圖9之分子探針晶片基板表面， 利用了二酸酐(succinic anhydride)對定鋪有機分子之胺基[-NH2]進行烷基化反應，以轉換成末端為酸基[-COOH]的衍生 化合物。 圖11之化學反應顯示在圖9之分子探針晶片基板表面， 利用sulfo-SMPB對定錨有機分子之胺基[-NH2]進行烷基化 反應，以轉換成末端為4-(p-馬林亞醯氨苯基:l·丁酸基[-C0(CH2)3(CH)6N(C0)2(CH)2]的衍生化合物。 圖12之化學反應顯示在圖9之分子探針晶片基板表面， 利用2-亞胺五環硫對定錨有機分子之胺基[-NH2]進行硫醇基 200533916 化反應，以轉換成末端為硫醇基[-SH]的衍生化合物。 圖13之化學反應顯示在圖9之分子探針晶片基板表面， 利用氰尿醯氯對定錨有機分子之胺基[-NH2]進行氰尿醯氯化 反應，以轉換成末端為氰尿醯氯-活化物質的衍生化合物。 圖14之化學反應顯示在圖9之分子探針晶片基板表面， 利用二咪唑基碳醯對定錨有機分子之胺基[-NHJ進行二咪唑 基碳醯化反應，以轉換成末端為咪唑基碳醯-活化物質l· C〇N2C3H3]的衍生化合物。b. Based on 100 ml of crushed ice made with deionized water, carefully add about 20 g (21.3 ml) of #-(3-aminopropyl) propanediamine (ΛΗ3-Aminopropyl) -1,3 -propanediamine), referred to as APPDA. The structure of APPDA is [H2N (CH2) 3NH (CH2) 3NH2]. The primary amine group at one end can be coupled with alkanedialdehyde, and the primary amine group at the other end is reserved as an affinity agent. Derivatization of the compounds is performed. Also based on safety considerations, safety glasses and gloves are required to carry out this reaction. After that, about 8-10 ml of concentrated hydrochloric acid was slowly added dropwise. At this time, crushing ice can prevent the generation of a large amount of smoke and slow down the temperature rise. Then add a stir bar to stir, measure the pH, and continue to add concentrated hydrochloric acid dropwise until the pH of the solution is about 7. The ice should now be completely dissolved. Thereafter, deionized water was added until the solution volume was about 100 ml. Finally, sodium phosphate 200533916 salt was added to prepare a buffer solution with a concentration of about 0.1M, and the pH value of the solution was adjusted to 7.0 again, which is the neutral pH value. c. Place the substrate containing the aldehyde group after cleaning in an APPDA aqueous solution. At this time, the reaction tank should be stirred or shaken. d. Add about 1.2 grams of cyanoborohydride ([NaB (CN) H3]) and continue stirring for at least about four hours. e. After the reaction is completed, the obtained substrate containing APPDA is washed with a large amount of distilled water, and then washed with an aqueous solution of sodium chloride having a concentration of about 1M, and finally washed with distilled water to obtain a substrate 100 as shown in the reaction result in FIG. 3. Note that the substrate 100 in FIG. 3 is a semi-finished wafer of a molecular probe wafer. Taking the anchor compound in FIG. 3 as an example, it is a derivative compound with an amine group [-NH2] at the end, that is, the end of the long molecular configuration away from the metal cloth coating 120 of the substrate 110 is a Amine group According to the present invention, the substrate 100 is a semi-finished product of a molecular probe wafer. This is because the amine group of each anchor compound molecule on the substrate 100 has not been connected to any molecular probe required for detection and sensing applications. Such a semi-finished substrate 1⑻ can be stored for a long time under appropriate conditions. It typically lasts at least three months. When necessary, the semi-finished substrate 100 can be further processed quickly, and any one of a variety of molecular probes suitable for covalent bonding with an amine group can be connected to be applied to a specific detection and sensing application. According to the present invention, the covalent bonding process required for the semi-finished substrate to connect the molecular probes can be extremely fast. Typical processing can be as fast as about three minutes of covalent bond 200533916 junction reaction time range. In medical applications, when an epidemic such as Severe Acute Respiratory Syndrome (SARS) occurs, according to the molecular probe design concept of the present invention, such as the semi-finished substrate 100 shown in FIG. It can be taken out quickly, and the probe connection processing can be performed quickly, in order to quickly and mass produce detection biochips, which can be used for the real-time tracking and dissemination of disease viruses. Han 3A shows one of the semi-finished products of the molecular probe wafer 100 in Figure 3. ^ Simplified Table Example 1: As mentioned above, the result obtained by the chemical reaction in Figure 3, which is a simplified representation as shown in Figure 3A, is suitable for connecting various types of organisms. The basic biochip substrate of the probe can be connected to the bioprobe molecules required for the detection of various specific biomolecules for related biomedical detection. The chemical reaction of FIG. 4 shows that, in an embodiment of the present invention, the surface of the basic biochip substrate 100 of FIG. 3A is succinic anhydride used to exhaust the amine group [-NH2] of the anchor organic molecule. Glycation reaction to convert to a derivative compound with an acid group [-COOH] at the end. The molecular structure of succinic anhydride is shown in the reaction formula of FIG. 4. The active carbonyl group of this compound can react with the amine group in the substrate, and the terminal acid group will be formed after ring opening, which can be combined with biomolecules with amine group: a. Wash the substrate containing APPDA with 100 ml of water, and then Soak in 100 ml of water. b. While stirring, slowly add about 10 grams of succinic anhydride. 200533916 C. Reaction at room temperature for about one hour. It is suggested in the related art that sodium hydroxide should be used to maintain the pH value of the reaction solution at about 6, but according to the present invention, it is not necessary to control the pH value during the reaction. Without the participation of sodium hydroxide, the whole reaction process is fast and the yield is high. d. After the reaction is completed, the substrate is washed with a large amount of water, and then washed with an aqueous solution of sodium chloride having a concentration of about 1M, and finally washed with water to remove unreacted succinic acid. At this time, a substrate 400 having an acid group [-COOH] -derived compound as shown in FIG. 4 can be obtained. This type of biochip, which anchors the acid group at the end of the organic molecule, can be combined with biomolecules with amine groups, such as proteins, peptides, nucleic acids, sugars, lipids, etc., which is conducive to relevant biomedical testing. Example 2: The chemical reaction of FIG. 5 shows that on the surface of the basic biological wafer substrate 100 of FIG. 3A, sulfosuccinimidyl-4- (p-maleimidophenyl) is used. -butyrate), abbreviated as siTifo-SMPB, perform # • alkylation reaction on the amine group [-NH2] of the anchored organic molecule to convert it to 4- (p-marininamidophenyl) -butyric acid (4- (p-maleimidophenyl) -butyrate group) oCO (CH2) 3 (CH) 6N (C0) 2 (CH) 2]. The structure of sulfo_SMPB is shown as 7F in the reaction formula of Fig. 5. The ester group of this compound and the amine group in the substrate can be subjected to 7V-alkylation reaction, and the resulting compound can react with the biomolecules having a thiol group: 200533916 Place the substrate containing APPDA into a substrate containing about 2mM sulfo- SMPB was reacted in phosphate buffered saline (PBS) for about one hour. In a preferred embodiment, PBS is formulated using approximately 137 mM sodium chloride [NaCl], 2.7 mM potassium chloride [KC1], 10 mM sodium phosphate [Na2P04], and 1.8 mM potassium dihydrogen phosphate [KH2P04]. Cheng, its pH value is about 7.4. Thereafter, the substrate was washed with PBS to obtain a substrate 500 having a sulfo-SMPB derivative at the end shown in FIG. 5. This substrate must be stored below 4 ° C. This type of biochip, which anchors the reactive group at the end of the organic molecule, can be combined with the biomolecule with a thiol group, which is conducive to relevant biomedical testing. Example 3: The chemical reaction of FIG. 6 shows that on the surface of the basic biochip substrate 100 of FIG. 3A, 2_iminothiolane hydrochloride (the structure of which is shown in the chemical reaction formula) is used to anchor the organic molecules. When the amine group [-NH2] undergoes thiolation reaction, it will be ring-opened to transform to form a derivative compound with a thiol group [-SH] at the end: Add about 0.05M triethylamine and 0.15M chlorine to the reaction tank Sodium chloride aqueous solution and ImM ethylenediaminetetraacetic acid (EDTA), and the pH value of the solution was adjusted to about 8%. The gas in the solution was removed first, and then the substrate containing APPDA was immersed in the reaction tank. After that, about 5 equivalents of 2-imine pentacyclic sulfur were added. In a preferred embodiment, the entire reaction is performed in a nitrogen environment at room temperature for about 45 minutes, and then washed with a phosphate buffered saline (pbs) 200533916 having a pH of about 7.2. Finally, the substrate was stored in PBS and placed at room temperature. At this time, a substrate 600 having a derivatized compound having a thiol group [-SH] at the end as shown in FIG. 6 can be obtained. This type of biochip, which has a thiol group at the end of the anchoring organic molecule, can be combined with a biomolecule with a double sulfur bond, which is suitable for relevant biomedical testing. Example 4: The chemical reaction of FIG. 7 is shown on the surface of the basic bio-wafer substrate 100 of FIG. 3A. The amine group of the anchored organic molecule is made of cyanuric chloride (the structure is shown in the chemical reaction formula) [- NH2] to perform an electrophilic addition substitution reaction to convert to a derivative compound with a terminal cyanuric chloride-activated substance: firstly prepare an acetonitrile solution of about 0.01M cyanuric chloride and add about 2M basic diisopropyl Ethylamine (A ^ V_diisopropylethylamine). After the substrate containing APPDA was immersed in the reaction at about 0-5 ° C for about three hours, the substrate was taken out and washed with acetonitrile solvent and acetone to remove unreacted cyanuric chloride and diisopropylethyl on the substrate. Based amine. At this time, a substrate 700 having a derivative of a cyanuric chloride-activated substance as shown in FIG. 7 can be obtained. This type of biochip, which anchors the cyanuric chloride and chloride activating group possessed at the end of the organic molecule, can be combined with biomolecules with alcohol and amine groups, which is suitable for relevant biomedical testing. 200533916 Example 5: The chemical reaction of Fig. 8 is shown on the surface of the basic biochip substrate 100 of Fig. 3A. The use of carbonyldiimidazol (the structure of which is shown in the chemical reaction formula) has an active imidazolylcarbamyl group. Coupling reaction of the amine group of anchored organic molecule | > NH2] to convert to a derivative compound whose terminal is imidazolylcarbamidine-activated substance [-CON2C3H3]: a. Sequentially use about 30% acetone aqueous solution and 70% The substrate containing APPDA was washed with an acetone aqueous solution. Also for safety reasons, such cleaning should be performed in a fume hood. Rinse with acetone solvent to remove water adsorbed on the glass. Note that the substrate should be kept dry during cleaning. b. Put the cleaned substrate into the reaction tank, add about 1 g / 20 ml of diimidazolylcarbonamidine, and stir at room temperature for about one hour. c. After the reaction, rinse with acetone to remove the imidazole compounds produced in the reaction. At this time, a substrate 800 having an imidazolylcarbamidine-activated substance [-CON2C3H3] -derived compound shown in FIG. 8 at the end can be obtained. These bases containing imidazolylcarbamidine-activated substance in anhydrous acetone under nitrogen It can be stored for one year, or the next coupling reaction can be directly carried out. This type of biochip has an imidazolyl carbamate activating group at the end of the anchoring organic molecule, which can be combined with biomolecules with alcohol groups, which is suitable for relevant biomedical testing. The chemical reaction in FIG. 9 shows that according to another preferred embodiment of the present invention, the surface of the substrate 910 coated with the gold or silver metal thin film layer 920 is treated with chemical modification 200533916 to fix the basic organic molecules of a selected length for connection. Basic bio-wafer substrate for the bio-probe molecules to be used. The substrate 900 in FIG. 9 is the same as the substrate 100 in FIG. 3, and is also a semi-finished wafer of a molecular probe wafer ^ For the anchor compound in FIG. 9, it is also an amine group at the end [_NH2 ] —A derivative compound, that is, the end of the elongated molecular configuration away from the metal cloth coating 920 of the substrate 910 is a monoamine group. The substrate 900 is a semi-finished product of the molecular probe wafer of the present invention, and the amine group of each anchor compound molecule has not yet been connected to any molecular probe required for detection and sensing applications. The semi-finished substrate 900 can be stored for a long time under appropriate conditions. When necessary, the semi-finished substrate 900 can be further processed quickly, and any of a variety of molecular probes suitable for covalent bonding with an amine group can be linked to be applied to specific detection and sensing applications. The basic wafer substrate 900 shown in FIG. 9 has a longer anchoring organic molecule (n = 8 to 16) compared with the one shown in FIG. 3 (100). In addition, the basic wafer substrate 900 shown in FIG. 9 can also be applied to the bio-wafer applications of the foregoing Examples 1 to 5. As mentioned above, a certain distance and degree of freedom must be maintained between the anchor molecule and the probe to be suitable for detecting the interaction characteristics during the reaction. Figs. 10 to 14 respectively show the examples of the previous examples 1 to 5 in which a long hydrogen sulfanilamide is used, wherein n = 8 to 16 are used as fixed organic molecules to prepare biochips, and molecular probe chips 100ft 110ft 1200, 1300 and 1400. Although the present invention has been described above with reference to the preferred embodiments, it is not intended to limit the present invention. For example, although the description of the present invention is described in detail using biochip 200533916 as an example, the molecular probe wafer having a covalently bonded anchor compound of the present invention, as can be understood, can also be applied to other gold or Silver is treated as a different application for sensing films on biochips. Its applications are, for example, optical sensing wafers, electrochemical sensing wafers, and piezoelectric sensing wafers. Therefore, anyone skilled in this art can make such changes and changes without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be defined by the scope of the attached patent application. 200533916 '' Brief description of the diagram The chemical reactions of Figures 1, 2 and 3 respectively show that according to a preferred embodiment of the present invention, a specific length of the substrate is chemically modified on the surface of the substrate coated with gold or silver. The basic organic molecule is immobilized for connecting the basic molecular probe wafer substrate of the biological probe molecule to be used. FIG. 3A shows a simplified representation of the molecular probe wafer of FIG. 3. The chemical reaction of FIG. 4 is shown on the surface of the molecular probe wafer substrate of FIG. 3A. The succinic anhydride is used to perform an A ^ alkylation reaction on the anchored organic molecule NH2] to convert it into Derived compound with an acid group [-COOH] at the end. The chemical reaction of FIG. 5 is shown on the surface of the molecular probe wafer substrate of FIG. 3A. The sulfosuccinimidyl-4- (p-maleimidophenyl) -butyrate, sulfo -SMPB) N-alkylation of the amine group [-NH2] of the anchored organic molecule to convert it to 4- (p-marininamidoaminophenyl) -butanoate Derivative of (4- (p-maleimidophenyl) -butyrate group) [-CO (CH2) 3 (CH) 6N (CO) 2 (CH) 2]. The chemical reaction of FIG. 6 is shown on the surface of the molecular probe wafer substrate of FIG. 3A. The 2-iminothiolane hydrochloride is used to thiolation the amine group [-NH2] of the organic molecule. ) Reaction to convert to a derivatized compound terminated with a thiol group [-SH]. The chemical reaction of FIG. 7 is shown on the surface of the molecular probe wafer substrate of FIG. 3A. 200533916 A cyanuric chlorination reaction is performed on the amine group [-NH2] of the anchored organic molecule using cyanuric chloride. To convert to a derivative compound with a cyanuric chloride-activated substance at the end. The chemical reaction of FIG. 8 is shown on the surface of the substrate of the molecular probe wafer of FIG. 3A. The amide [-NH2] of the anchored organic molecule is subjected to carbonyldiimidazolization using carbonyldiimidazol. Reaction to convert to a derivative compound with a terminal carbonyldiimidazol-activated substance [-CON2C3H3]. The chemical reaction shown in FIG. 9 shows that according to another preferred embodiment of the present invention, a basic organic molecule of a selected length is immobilized by chemical modification treatment on the surface of a substrate coated with gold or silver for connection with a biological probe required. Molecules of the basic molecular probe wafer substrate. The chemical reaction of FIG. 10 is shown on the surface of the molecular probe wafer substrate of FIG. 9. A succinic anhydride is used to alkylate the amine group [-NH2] of the organic molecule to be converted into an acid group at the end. [-COOH] Derived compounds. The chemical reaction in FIG. 11 is shown on the surface of the molecular probe wafer substrate in FIG. 9. The amine [-NH2] of the anchored organic molecule is alkylated with sulfo-SMPB to convert the terminal to 4- (p-Ma Lin Yamin aminophenyl: l · butanoic acid derivative of [-C0 (CH2) 3 (CH) 6N (C0) 2 (CH) 2]. The chemical reaction shown in Figure 12 is shown in the molecular probe chip of Figure 9 On the surface of the substrate, the amine group [-NH2] of the anchored organic molecule was subjected to a thiol group 200533916 using 2-imine pentacyclic sulfur to convert it into a derivative compound with a thiol group [-SH] at the end. The chemical reaction is shown on the surface of the molecular probe wafer substrate in FIG. 9. The cyanuric chloride reaction of the amine group [-NH2] of the anchored organic molecule is performed using cyanuric chloride to convert the terminal to cyanuric chloride. Derivative compounds of the substance. The chemical reaction of FIG. 14 is shown on the surface of the molecular probe wafer substrate of FIG. 9. The resulting terminal compound is derived from imidazolylcarbamidine-activated substance l · CON2C3H3].
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