1270673 九、發明說明： [發明所屬之技術領域] 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)之定錯化合物(anchoring compound)之分 子探針晶片。 [先前技術] 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針，可 與檢測溶液中的目標分子進行相互作用(interaction reaction)。根據 7 1270673 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)，便可以提供一種基本 • 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稀(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， I 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 immuno-sorbent assay，ELISA)，其係 將特定抗體(或抗原)先吸附在聚苯乙烯(polystyrene)基板上，再將 基板暴露在檢體中。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。 8 1270673 例如，頒予 Fodor 等人的 U· S· Pat· No· 5,445,934，’’Array of oligonucleotides on a solid substrate’’中揭不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去保護，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(robQtic 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，其步驟複 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。1270673 IX. Description of the Invention: [Technical Field of the Invention] The present invention relates to molecular probe chips, and in particular, to rapid and accurate molecular sample testing, examination and characterization. Codon bonding A molecular probe wafer of an anchoring compound that is linked. [Prior Art] Molecular probe chips can be fabricated by immobilizing a selected molecular probe on a substrate having an extended surface using an anchoring compound. . Molecular probe wafers designed for the detection and detection of specific targets have a wide range of uses. For example, in the field of basic medicine and clinical medicine, a molecular probe wafer, which is generally known as a biochip or a biological sensor chip, can quickly and accurately perform a biological sample. ) characterization and diagnosis. In medical use, the sample to be tested for diagnosis may be a molecular, viral, bacterial or cellular sample. Among other industrial uses in the non-medical field, it may involve qualitative samples of industrial tests such as food or environmental sampling. In the use of biochips, probes immobilized on the surface of the substrate can interact with target molecules in the detection solution. Specific biomolecules can be detected based on the results of the 7 1270673 reaction. The probe immobilized on the surface of the substrate may be an oligonucleic acid, a peptide, a protein, an antibody, an antigen or other cells which can react with a target molecule or Tissue surface biomolecules. Such a substrate is used to bond an immobilized molecule as a ligand to interact with biomolecules (ie, analytes) in a biomedical sample, by detecting the amount of interaction between the two ( The binding concentration) and the binding constant provide a basic data diagnosis and experimental parameters. The high affinity, high binding capacity and biostability between the two are very important features in biochip detection applications. A variety of bioassays currently require the immobilization of biomolecules such as DNA or proteins directly onto substrates. For example, western blotting adsorbs proteins on polyvinylidene fluoride (PVDF) or nitrocellulose membrane substrates as probes, and functional groups on antibodies or antigenic proteins. Interaction to measure whether the sample contains such protein. In addition, southern and northern blotting also use DNA and RNA to adsorb and DNA and RNA with complementary sequences in a similar manner. Another example is an enzyme-linked immuno-sorbent assay (ELISA), which adsorbs a specific antibody (or antigen) onto a polystyrene substrate and exposes the substrate to the sample. in. If the specimen contains a specific antigen (or antibody) with high affinity for the antibodies (or antigens), it will be detected in combination with it. 8 1270673 For example, U.S. Pat. No. 5,445,934, '’Array of oligonucleotides on a solid substrate', to Fodor et al., discloses a method of directly synthesizing polynucleotide on a substrate. It is derivatized on a glass slide to have a photoprotective group of chemical substances on its surface, which is then deprotected by a photomask and then with a photoprotective group. The monomer is reacted. The entire preparation process must be repeated for the reticle, deprotection and coupling steps until the desired polynucleotide sequence is obtained. Alternatively, the polynucleic acid may be synthesized in advance, and then placed on the substrate by physical adsorption, photoreaction bonding, or other bonding using techniques such as robQtic printing or inkjet printing. The above method of directly immobilizing a biomolecule 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 a weak physical adsorption method, and the stability is low. This practice causes the cleaning and handling of the wafer to be cumbersome and time consuming, and its reproducibility is also low.
Ishii 等人之 U.S. Pat. No. 5,474,895，"Non_isotopic detection of nucleic acids using a polystyrene support-based sandwich hybridization assay and compositions uscfUl ώ6Γβ〇Γπψ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、環氧基或硫醇棊等的官能基。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. 1270673Ishii et al., U.S. Pat. No. 5,474,895, "Non_isotopic detection of nucleic acids using a polystyrene support-based sandwich hybridization assay and compositions uscfUl ώ6Γβ〇Γπψ proposed the use of sandwich hybridization analysis instead of fluorescence detection. The substrate is modified or linked by a suitable functional group to carry a reactive group, i.e., a functional group such as an alcohol group, a carbonyl group, an amine group, an aldehyde group, an epoxy group or a thiol group. The surface may form a covalent or non-covalent linkage with the polynucleotide. Another example is Van Ness et al. U.S. Pat. 1270673
No· 5,514,785 "Solid support 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，之後再與其秦末端含有磷咪唑烷 (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遺原反 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. Νο·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 m ~ 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)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 10 1270673 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ti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。 [發明内容] 因此有需要提供一種分子探針晶片，具有共價鏈結定錨化合 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；複數個的定鋪化合物分子，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fi1偵檢感測所需之探 11 1270673 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，共價鍵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。 [實施方式] . 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)，氮化砂(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 12 1270673 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 13 1270673 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^•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，因而提高探針和生醫檢測樣品 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 14 1270673 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 醇基作為主要定銷分子。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烷 胺(mercaptoalkylamine)，其分子結構為[HS(CH2)nNH2]，其中 n=2〜16。氫硫基焼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。 利用前述以金或銀金屬塗佈為基礎的生物晶片或生物感測晶 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， 15 1270673 尿液，體液或唾液等),.因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。定錯分 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度，以模擬探針以待測物在 液-液相中的真實栢互作用。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，貝ij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fi1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酸(alkyl dialdehyde)進行化合物反應。 此化合物所具有的酸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 ΛΚ3-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。 ΛΚ3-胺基丙基丙二胺化合物係為一種高極性(highly 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。 16 1270673 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 面之問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數，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110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)緩衝溶液(PBS)之中，反應大約二小時。 之後，利用諸如乙醇和蒸餾水清洗基板，即可得到已經氫硫基乙 胺進行表面修飾的基板。 17 1270673 圖2之化學反應顯示，圖1修飾反應所得基板1〇2,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103。 接著，圖3之化學反應顯示，圖2反應所得基板，利甩和醛基 [-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， _ 濃度約為〇·1Μ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。 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)的尽(3_胺基丙基)_1，3_丙二胺（iV«(3_Aminopropyl) -l，3-propanediamine)，簡稱 APPDA。APPDA 之結構為[H2N(CH2)3 nh(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100毫升。最後加入磷酸鈉鹽，以配製濃度約為0.1M的緩衝 18 1270673 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 I·2 克的氰硼焼鈉(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。 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中之定錯化合物而_，其係為末端為胺基[-NHJ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100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甩途所需的分子探針。此種半成品基 板100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鏈結處理可 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， 1270673No. 5,514,785 "Solid support for nucleic acid hybridization assays" proposes the use of covalent bonding to link the polynucleotide to the nylon support. Another example is Rampal on US Pat. No. 6,013,789," In the case of Covalent attachment of biomolecules to derivatized polypropylene supports, the polypropylene film is first subjected to ammoniation, and then reacted with a polynucleotide containing a phosphorimidazolide at the end of the Qin, so that the polynucleotide can be passed through the phosphoimidazolium. (phosphoramidate) bonding and covalent attachment to polypropylene. _ In addition, the use of self-assembly of molecules on the surface of indium tin oxide (ITO) electrodes to form self-assembled monolayers can also provide a new interface for exploring certain biomolecules. Special oxidation of the original reaction. The organic ruthenium may be reacted with a substrate having a hydroxyl group on the surface (e.g., ruthenium dioxide) to form a single or double layer organic ruthenium film. For example, Chrisey et al., in the "Selective attachment of nucleic acid molecules to patterned self-assembled surfaces", US Pat. 5,688,642, proposes an organic m~ ruthenium compound having two reactive groups, utilizing one end of the active group and the substrate surface. The hydroxyl group is linked, and the biomolecule is linked by the active group at the other end so that the biomolecule can be immobilized on the substrate. Although these methods have been widely used, there are limitations in commercial mass production due to the cumbersome and time-consuming preparation of molecular probes. This is due to the fact that biomolecules are immobilized on commonly used substrates such as PVDF, nitrocellulose or microscopic glass, and the physical 10 1270673 physical adsorption effect is usually unstable. Time consuming process. The entire immobilization process typically requires at least six to eight hours of incubation time. A typical treatment method is to first add a raw ti molecular probe to a wafer substrate and to hold the wafer. After a physical adsorption reaction between the molecular probe and the substrate for a certain period of time (about three to four hours), the cleaning procedure is continuously performed several times. After the cleaning, the blocking reagent is added and allowed to stand for a long time (usually about three to four hours), the wafer can be used as a biochip. [Experiment] It is therefore necessary to provide a molecular probe wafer. It has a covalent chain-binding anchor compound, which is simple and rapid to prepare, and is suitable for low-cost mass production. There is also a need to provide a molecular probe wafer with a covalent bond-forming anchor compound, which has high stability in storage and use. To achieve the foregoing and other objects, the present invention provides a molecular probe wafer having a covalently bonded anchoring compound comprising: a substrate having a metal film layer on one surface thereof; Placing a compound molecule having an elongated elongated molecular configuration, each of the anchoring compound molecules being covalently bonded to the metal thin film layer at one end of the elongated configuration; and a plurality of a probe molecule, each of which is covalently bonded to the anchor molecule compound corresponding to a certain anchor molecule compound against the elongated configuration One of the second ends. The present invention also provides a probeless semi-finished wafer substrate of a molecular probe wafer, which can be pre-stored to couple the molecular probe wafer to the probe 1 1270673 needle required for fi1 detection sensing when needed. The probeless semi-finished wafer substrate comprises: a substrate having a metal film layer on a surface thereof; and a plurality of anchoring compound molecules having substantially elongated elongated molecular configurations, each of which Each of the anchoring compound molecules is covalently bonded to the metal thin film layer at one end of one of its elongated configurations, and is opposed to the second end of the first end of the elongated configuration, If necessary, covalently bonding with the probe molecule to completely form the molecular probe wafer. The present invention also provides a method for fabricating a molecular probe wafer, the steps of which include first > on one surface of a substrate Laying a metal film layer; and then arranging a plurality of anchoring compound molecules having an extended and substantially elongated molecular configuration, one of the first ends of each of the elongated configurations, covalently bonded to the substrate Metal coated film In the method of the present invention, each of the anchor molecules may be covalently bonded to a probe molecule corresponding to the second end of the first end. [Embodiment] The present invention has a covalent bond. For the molecular probe wafer of the anchor compound, the substrate of the wafer may be a general silicon, silicon nitride, quartz or glass plate. The metal is coated with a layer of metal, for example, by vapor deposition to cover the metal film layer. Suitable metals include gold, silver, chromium and nickel. For the use of biochips, for example, the preferred metal should be It is gold or silver. If the probe wafer of the present invention is used as a sensing wafer, it can be applied to detection such as electrochemical biosensing analysis. In another application, the 12 1270673 probe wafer of the present invention is used. It is used as an oscillating wafer and can be applied to detection such as piezoelectric biosensing analysis. In yet another application, the probe wafer of the present invention is used as a surface plasma resonant wafer for detection such as optical biosensing 1 analysis. According to the present invention, the metal thin film layer coated on the wafer substrate can be a light-transmissive substrate, for example, a specific light-transmissive metal thin film layer, if it is specially designed and processed by a specific process. Such a light-transmissive metal-clad sensing element can be used on a scanner or microscope system to which optical detection principles are applied after the bio-probe is labeled with a phosphor or color former. According to the present invention, the metal thin film layer coated on the wafer substrate is chemically modified in a covalently bonded form, thereby increasing the stability of the molecular probe immobilized on the surface of the molecular probe wafer substrate. In the application of the biochip, the probe immobilization by the covalent bonding of the present invention can be processed in the buffer of the wafer, and the interaction process between the probe and the target on the surface of the wafer In the dissociation, association and regeneration steps, the biomolecule probe immobilized on the wafer by covalent bonding is not lost due to buffer washing. According to the present invention, the metal film layer coated on the wafer substrate is chemically modified to provide a new form for rapidly immobilizing the molecular probe on the surface of the wafer substrate, thereby shortening the time required for the immobilization of the probe. The present invention has a molecular probe wafer with a covalently bonded anchor compound. In some applications of biochips, the time required for immobilization of the bio-13 1270673 sub-probe can be dependent on conventional physical adsorption from conventional techniques. (physical adsorption) The six to eight hours required for treatment are greatly reduced to three to five minutes. The molecular probe wafer of the present invention is typically applicable to the use of biochips. The biochip prepared according to the method of the present invention can rapidly and efficiently covalently bond with certain specific functional groups in the biological molecule to be tested, such as DNA, RNA, amino acid or protein, to form a specific one. Sexual biological probe. These specific bioprobe probes allow for rapid detection of specific biomedical test samples. According to a preferred embodiment of the present invention, the bio-wafer substrate may be a glass slide used in a general optical instrument to which a metal thin film such as gold or silver is applied. The metal coated bio-wafer substrate itself can be used as an electrode sheet and is suitable for the detection of certain electrochemical analysis forms. Since the material of the wafer substrate is a light-transmitting element, it can also be applied to a detecting instrument based on the principle of optical detection. Further, if a fluorescent substance is labeled on a biomolecule to which a sample is added, such a light-transmitting bio-wafer substrate coated with a metal can also be applied to a current fluorescent detection system. According to the method of the present invention, the chemical modification on the bio-wafer substrate is in the form of covalent bonding, thereby increasing the stability of the immobilized probe on the surface of the bio-wafer substrate. The molecules of the material that remain on the surface of the wafer but are not reactive can be removed by washing with a solvent. In this way, the interference of impurities can be reduced, thereby increasing the affinity of the probe and the target in the biomedical test sample, and the affinity and binding capacity between the two, further improving the sensitivity of the detected spirit 14 1270673. For various detection source biomolecules, according to the present invention, a plurality of chemically modified different functional bio-discs can be separately provided for targeted high-accuracy detection. In view of the structural differences of the detection source biomolecules, different functional biochips can be selected according to the present invention to achieve the best specific combination of the probe and the target molecule. The biochip according to the present invention can provide high-confidence detection data because of its better pertinence and specificity for detecting source biomolecules. According to the present invention, in order to bond an immobilized biomolecule probe on a biochip, the selected organic molecule and the metal on the wafer substrate must first be covalently bonded to each other and attached to the surface of the wafer substrate. The organic molecule must additionally have a functional group that can be chemically reacted with the biomolecule probe to be linked. For example, in accordance with a preferred embodiment of the present invention, a thiol group can be employed as the primary anchoring molecule on a wafer substrate coated with gold or silver. This is because the sulfur atom is highly susceptible to thiolation with gold or silver to form a covalent bond. In a preferred embodiment of the invention, a mercaptoalkylamine having a molecular structure of [HS(CH2)nNH2] wherein n = 2 to 16 can be first placed on the substrate. The sulfur atom of the thiocarbaguanamine undergoes a thiolation reaction with gold or silver to form a covalent bond, and the amine group at the terminal of the organic molecule can be retained as a nucleophile to facilitate the derivatization of the compound. Using the aforementioned bio-wafer or bio-sensing wafer based on gold or silver metal coating, when the wafer is solid and the object to be detected is liquid (such as blood, 15 1270673 urine, body fluid or saliva, etc.), Therefore, the sensitivity and limit of detection depend on 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 atoms of the wrong molecular coupling compound, the anchor molecule and the probe must also be maintained for a certain distance length to simulate the probe in the liquid-liquid phase of the analyte. Real cypress interaction. Therefore, if a shorter methylene thioalkylamine (for example, n=2 thioethylamine) is used, the immobilized anchor molecules on the Bayij substrate must first be extended by the chain length before being explored. The needle molecules undergo a chemical reaction to produce a biochip containing specific functional groups. On the other hand, if a longer thioalkylamine (n = 8 to 16) is used, it can directly react with the probe molecule into fi1 to produce a biochip containing a specific functional group. In the case of a substrate which is chemically modified with a shorter methylene thioalkylamine, the immobilized anchor molecule on its substrate, since the chain length must be extended, the substrate and the alkane can be first made. The dicarboxylic acid (alkyl dialdehyde) is subjected to a compound reaction. The acid group possessed by this compound can be coupled with an amine group in the substrate to form a covalent bond. The derivative produced has a terminal aldehyde group which can be further subjected to a coupling reaction with a primary amine group in the ΛΚ3-aminopropyl)-1,3-propanediamine compound. The long-chain derivative formed at this time has a nucleophilic primary amine group at the end, and can further chemically react with the probe molecule to produce a biochip containing a specific functional group. The ΛΚ3-aminopropyl propylenediamine compound is a highly polarized molecule. If the compound is used to derivatize the organic molecular chain length on the substrate, the surface of the entire chemically modified substrate can have a high hydrophilic property. 16 1270673 Since these compounds have a considerable length, when the biochip probe of this type interacts with the target molecules in the sample solution to be tested, the probe molecules and the surface of the substrate are maintained for a longer period of time. distance. Since the probe molecules also have a high degree of freedom, the steric hindrance of the wafer is low when it is recognized from the target. The concentration of the target and the binding constant calculated by this type of wafer are thus relatively close to the true value in the liquid-liquid phase. Preparation of the basic bio-wafer substrate to which the bioprobe can be attached. The chemical reactions of Figures 1, 2 and 3 respectively show the stepwise chemical modification treatment on the surface of the substrate coated with gold or silver according to a preferred embodiment of the present invention. A basic organic molecule of a selected length can be immobilized. The result of the chemical reaction of Figure 3 is a basic bio-wafer substrate that can be linked to various bioprobes, and can be linked to various biomolecule molecules for specific biomolecule detection for related biomedical, food or environmental samples. Detection. First, in a preferred embodiment, as shown by the chemical reaction of Figure 1, thioethylamine is first placed on a wafer substrate 110 coated with a gold or silver metal film layer 120. This can be achieved by immersing the coated gold or silver wafer substrate with an aqueous solution of thioethylamine at a concentration of about 20 mM, or by immersing it in a phosphate containing about 1 mM of thioethylamine (pH is about 7.2) In a buffer solution (PBS), the reaction is about two hours. Thereafter, the substrate is washed with, for example, ethanol and distilled water to obtain a substrate which has been surface-modified with thioethylamine. 17 1270673 The chemical reaction of Figure 2 shows that the substrate 1〇2 obtained by the modification reaction of Figure 1 is reacted with an aqueous solution of glutaraldehyde having a concentration of about 2.5% and a pH of about 7 for about one hour to carry out the coupling reaction of glutaraldehyde. Thereafter, the substrate 103 as shown in the reaction shown in Fig. 2 can be obtained. Next, the chemical reaction of FIG. 3 shows that the substrate obtained by the reaction of FIG. 2 can be converted into a derivative compound having an amine group [-NH2] at the end by reaction with aldehyde group [-CHO]. The aldehyde group-containing substrate 103 obtained by the reaction was washed with an aqueous solution of sodium phosphate having a pH of about 7, and a concentration of about 0.1 Torr. Due to toxicity considerations, the reaction must preferably be carried out in a hood. b/According to 100 ml of crushed ice made via deionized water, carefully add about 20 g (21.3 ml) of (3_aminopropyl)_1,3-propylenediamine (iV«(3_Aminopropyl) -l,3-propanediamine), referred to as APPDA. The structure of APPDA is [H2N(CH2)3 nh(ch2)3nh2], and the primary amine group at one end can be coupled with an alkanedialdehyde, and the primary amine group at the other end is reserved as a affinity agent. Derivatization of the compound is then carried out. Also for safety reasons, safety glasses and gloves must be loaded for this reaction. Thereafter, about 8-10 ml of concentrated hydrochloric acid was slowly added dropwise. At this time, crushed ice can prevent a large amount of smoke generation and slow down the temperature rise. Thereafter, a stir bar was added for stirring, and the pH was measured, and concentrated hydrochloric acid was continuously added dropwise until the pH of the solution was about 7. At this point the ice should have completely dissolved. Thereafter, deionized water was added until the solution volume was about 100 ml. Finally, sodium phosphate salt was added to prepare a buffered 18 1270673 solution having a concentration of about 0.1 M, and the pH value of the solution was again adjusted to 7.0, that is, a neutral pH value. c. The substrate containing the aldehyde group after washing is placed in an aqueous solution of APPDA. At this point, the reaction tank should be stirred or shaken. d. Add about 1.2 grams of sodium cyanoborohydride (NaB(CN)H3)) and continue to stir for at least about four hours. After completion of the reaction, the obtained APPDA-containing substrate was washed with a large amount of distilled water, then washed with an aqueous solution of sodium chloride having a concentration of about 1 M, and finally washed with distilled water to obtain a substrate 100 as a result of the reaction shown in Fig. 3. It is noted that the substrate 100 of Figure 3 is a semi-finished wafer of molecular probe wafers. Taking the wrong compound in FIG. 3 as a derivative compound having an amine group [-NHJ at the end, that is, the end of the metal coating layer 120 whose elongated molecular configuration is away from the substrate 110 is An 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 certain anchor compound molecule on the substrate 100 has not been coupled to any molecular probe required for detection of the stimuli. Such a semi-finished substrate 100 can be stored under suitable conditions for a long period of time. Typical for at least three months. The semi-finished substrate 100 can be further processed, if desired, to bond any of a variety of molecular probes suitable for covalent bonding to an amine group for use in a particular detection sensing application. According to the present invention, the covalent linkage treatment required for the semi-finished substrate to bond molecular probes can be extremely fast. Typical treatments can range up to about three minutes of covalent bonding reaction time. Among medical uses, such as severe acute respiratory syndrome (SARS, 1270673)
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。 圖3A顯不圖3中分子探針晶片半成品100之一種簡化表示。 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[·ΝΗ2]進行N-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。 c. 在室溫下反應約一小時。習知相關技術建議反應中應利 1270673 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_(/7-maleimidophenyl)-butyrate)， 簡稱 sulfo-SMPB，對定錨有機分子之胺基[-NH2]進行N-烷基化反 應，以轉換成末端為4-(p-馬林亞醯氡苯基)-丁酸基 (4-(p-maleimidophenyl)-butyrate group) [-CO(CH2)3(CH)6N(CO)2 (CH)2]的衍生化合物。sulfo-SMPB之結構係如圖5之反應式中 所顯示。此化合物的酯基和基板中的胺基可進行N-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 21 1270673 應： 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[KH2P〇4]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-亞胺五環硫(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[-νη2]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0.05Μ三乙基胺、0.15Μ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8.0。 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 22 1270673 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)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[-NHd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 的二異两基乙基胺(AyV»diisopropylethylamine)。在約 0-5°C之 下，將含有APPDA的基板浸入其中反鹰約三小時之後，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醯 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:f結合，適於進行相 關生醫檢測。 23 1270673 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，利用二咪唑基碳醯(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[-!^12]進行偶合反應，以轉換成末端為咪唑基碳醯-活化物質 [-C0N2C3H3]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 〆 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-活化物質的基板，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。 24 1270673 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]的一種衍生化合物，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fi1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中所顯示之基本晶片基板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 作為定錨有機分子，分別製作生物晶片之實施例，及分子探針晶 25 l27〇673 片 1000， 1100, 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。 [囷式簡單說明] 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) φ 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3Α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3Α之分子探針晶片基板表面，利用 丁二酸酐(succinic anhydride)對定鋪有機分子之胺基[-ΝΗ2]進行尽 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 26 1270673 硫化丁二酸亞醯氨-馬林亞醯氨苯基-丁酸(sulfosuccinimidyl-4-(p-maleimidophenyl)-butyrate，sulfo-SMPB)對定鋪有機分子之月女 基[-NH2]進行烷基化反應(Alkylation)，以轉換成末端為4-(p-馬林亞醯氨苯基)_ 丁酸基(4-(/?-maleimidophenyl)-biityrate 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)對定錨有機分子之胺 基[-NHJ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，利用 氰尿醯氯(cyanuric chloride)對定鋪有機分子之胺基[-NH2]進行氰 尿醯氯化(cyanuric chlorination)反應，以轉換成末端為氰尿醯氯-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 二咪哩基碳醯(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 為咪唾基碳醯活化(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。 27 1270673 . 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 丁二酸酐(succinic anhydride)對定錨有機分子之胺基[-NH2]進行焼 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)-丁酸基[-CO(CH2)3(CH)6 N(CO)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。 k 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[-NH2]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[-CON2C3H3]的衍生化 合物。 [主要元件符號說明] 基板(分子探針晶片) 基板(分子探針晶片) 基板(製程階段） 基板 100, 400, 500, 600, 800, 900, 1000, 1100, 1200, 1300, 1400 102,103 110,910 28 1270673 金屬佈覆層 120, 920 29In the case of an outbreak of the Severe Acute Respiratory Syndrome type, according to the molecular probe design concept of the present invention, the semi-finished substrate 100 such as that shown in FIG. 3 can be taken out of the stock, and the probe coupling process can be quickly performed for rapid and large-scale real estate. The detection of bio-chips is applied to the real-time tracking and dissemination of the disease virus. Figure 3A shows a simplified representation of the molecular probe wafer blank 100 of Figure 3. Example 1: As mentioned above, the result of the chemical reaction of Figure 3, as shown in simplified form in Figure 3A, is a basic bio-wafer substrate that can be adapted to link various bioprobes for linkage to various specific biomolecules. Bioprobe molecules are used for related biomedical testing. The chemical reaction of Fig. 4 shows that, in an embodiment of the invention, the surface of the basic biochip substrate 100 of Fig. 3A is subjected to N-alkane by using succinic anhydride for the amine group [·ΝΗ2] of the anchoring organic molecule. The base reaction is converted to a derivative compound having an acid group [-COOH] at the end. The molecular structure of succinic anhydride is shown in the reaction scheme of Figure 4. The active carbonyl group of this compound can react with the amine group in the substrate, and after opening, it forms a terminal acid group, which can be combined with a biomolecule having an amine group: a. The substrate containing APPDA is washed with 100 ml of water, and then Soak in 100 ml of water. b. Slowly add about 10 grams of succinic anhydride while stirring. c. React at room temperature for about one hour. The related art suggests that the reaction should be maintained at 1270673. The pH of the reaction solution is maintained at about 6 with sodium hydroxide. However, according to the present invention, it is not necessary to control the pH value during the reaction. In the absence of sodium hydroxide to participate in the reaction, the entire 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, then washed with an aqueous solution of sodium chloride having a concentration of about 1 M, and finally washed with water to remove unreacted succinic acid. At this time, the substrate 400 having the acid group [-COOH] derivative compound shown in Fig. 4 was obtained. This type of biochip, which has an acid group at the end of the anchoring organic molecule, can be combined with biomolecules having an amine group, such as proteins, peptides, nucleic acids, sugars, lipids, etc., to facilitate related biomedical testing. Example 2: The chemical reaction of Figure 5 shows that on the surface of the basic biochip substrate 100 of Figure 3A, sulfosuccinimidyl_4_(/7-maleimidophenyl)-butyrate is used. ), referred to as sulfo-SMPB, for the N-alkylation reaction of the amine group [-NH2] of the anchoring organic molecule to convert to a 4-(p-marinal phenyl)-butyric acid group at the end ( 4-(p-maleimidophenyl)-butyrate group) Derivative compound of [-CO(CH2)3(CH)6N(CO)2(CH)2]. The structure of sulfo-SMPB is shown in the reaction scheme of Figure 5. The ester group of the compound and the amine group in the substrate can be subjected to an N-alkylation reaction, and the resulting compound can be reacted with a biomolecule having a thiol group. 21 1270673: The substrate containing the APPDA is placed in an amount of about 2 mM. The sulfo-SMPB was reacted in phosphate buffered water (PBS) for about one hour. In a preferred embodiment, the PBS utilizes about 137 mM sodium chloride [NaCl], 2.7 mM potassium chloride [KC1], 10 mM sodium phosphate [Na2P04], and 1.8 mM potassium dihydrogen phosphate [KH2P〇4]. Formulated to have a pH of approximately 7.4. Thereafter, the substrate was washed with PBS to obtain a substrate 500 having a sulfo-SMPB-derived compound at the end shown in Fig. 5. > This substrate should preferably be stored below 4 °C. This type of biochip has a reactive group at the end of the anchoring organic molecule that can be combined with a biomolecule having a thiol group to facilitate related biomedical testing. Example 3: The chemical reaction of Figure 6 shows the anchoring of 2-iminothiolane hydrochloride (the structure is shown in the chemical reaction formula) on the surface of the basic biochip substrate 100 of Figure 3A. When the amide group of the organic molecule [-νη2] undergoes thiolation, it is opened to convert to form a derivative compound having a terminal thiol group [-SH]: about 0.05 Μ triethylamine, 0.15 加入 is added to the reaction tank. An aqueous solution of sodium chloride and 1 mM ethylenediaminetetraacetic acid (EDTA) were used to adjust the pH value of the solution to about 8.0. The gas in the solution is first removed, and then the substrate containing the APPDA is immersed in the reaction tank. About 5 equivalents of 2-imine pentacyclic sulfur are then added. In a preferred embodiment 22 1270673, the entire reaction is carried out in a nitrogen atmosphere at room temperature for about 45 minutes and then washed with a phosphate buffered aqueous solution (PBS) having a pH of about 7.2. Finally, the substrate was stored in PBS and placed at room temperature. At this time, the substrate 600 of the derivative compound shown in Fig. 6 which is a thiol group [-SH] can be obtained. This type of biochip has a thiol group at the end of the anchoring organic molecule which can be combined with a biomolecule having a disulfide bond and is suitable for related biomedical testing. Example 4·· The chemical reaction of Fig. 7 is shown on the surface of the basic biochip substrate 100 of Fig. 3A, using cyanuric chloride (the structure is as shown in the chemical reaction formula) to anchor the amine group of the organic molecule [ -NHd undergoes an electrophilic addition substitution reaction to convert to a derivative compound having a terminal cyanuric chloride-activating substance: First, an acetonitrile solution of about 0.01 M cyanuric chloride is prepared, and about 2 M basic diisomeric base is added. Ethylamine (AyV»diisopropylethylamine). After immersing the substrate containing APPDA in the anti-eagle for about three hours at about 0-5 ° C, the substrate was taken out and rinsed with acetonitrile solvent and acetone to remove unreacted cyanuric chloride and diisopropyl on the substrate. Ethylamine. At this time, the substrate 700 of the derivative compound having a cyanuric chloride-activating material at the end shown in Fig. 7 can be obtained. This type of biochip, which has a cyanuric chloride-activated group at the end of the anchoring organic molecule, can be combined with an alcohol group and an amine group: f, which is suitable for related biomedical tests. 23 1270673 Example 5: The chemical reaction of Figure 8 is shown on the surface of the basic biochip substrate 100 of Figure 3A, using an imidazolyl carbon fluorenyl group having a diimidazolium carbon carbonyl (carbonyldiimidazol whose structure is as shown in the chemical reaction formula) The coupling reaction of the amine group [-!^12] of the anchoring organic molecule is carried out to convert to a derivative compound having an imidazolium-based carbonium-activating substance [-C0N2C3H3]: a. sequentially using about 30% aqueous acetone solution and 70 The substrate containing APPDA was washed with a % acetone solution. Also for safety reasons, such cleaning should be carried out in a fume hood. It is then rinsed with an acetone solvent to remove water adsorbed on the glass. Note that the substrate should not be allowed to dry during the cleaning process. b. Place the cleaned substrate in a reaction tank, add about 1 g / 20 ml of diimidazolium-based carbonium, and stir at room temperature for about one hour. c. After the reaction, rinse with acetone to remove the imidazole compound produced in the reaction. At this time, the substrate 800 of the derivative compound shown in Fig. 8 which is an imidazole-based carbonium-activating substance [-CON2C3H3] can be obtained. These substrates containing an imidazole-based carbonium-activated substance can be stored for one year in anhydrous acetone under nitrogen, or directly subjected to the next coupling reaction. This type of biochip, which has an imidazole-based carbonium activating group at the end of the anchoring organic molecule, can be combined with a biomolecule having an alcohol group, and is suitable for related biomedical testing. 24 1270673 The chemical reaction of FIG. 9 shows that, according to another preferred embodiment of the present invention, a basic organic molecule of a selected length is immobilized on a surface of a substrate 910 coated with a gold or silver metal thin film layer 920 by chemical modification. A basic bio-wafer substrate that links the desired bioprobe molecules. The substrate 900 of FIG. 9, like the substrate 100 of FIG. 3, is also a semi-finished wafer of molecular probe wafers. In the anchor compound of FIG. 9, it is also a derivative compound having an amine group [-NH2] at the end, that is, the end of the metal coating layer 920 whose elongated molecular configuration is away from the substrate 910 is An amine group. 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 been coupled to any molecular probe required for detection of sensing applications. The semi-finished substrate 900 can be stored for a long time under appropriate conditions. When desired, the semi-finished substrate 900 can be further rapidly processed into fi1 to bond any of a variety of molecular probes suitable for covalent bonding with an amine for application to a particular detection sensing application. The basic wafer substrate 900 shown in Fig. 9 has longer anchoring organic molecules (n = 8 to 16) as compared with the one shown in Fig. 3. In addition, the basic wafer substrate 900 shown in Fig. 9 can also be applied to the biochip applications of the foregoing examples 1 to 5. As described above, a certain distance length and degree of freedom must be maintained between the anchoring molecule and the probe to be suitable for detecting the interaction characteristics at the time of the reaction. 10 to 14 show examples of the above-mentioned Examples 1 to 5, wherein the longer hydrogen thioguanamine, wherein n = 8 to 16 as anchoring organic molecules, respectively, for producing a biochip, and the molecular probe crystal 25 l27 〇 673 tablets 1000, 1100, 1200, 1300 and 1400. Although the present invention has been described above in connection with the preferred embodiments, it is not intended to limit the invention. For example, although the description of the present invention is described in detail by taking a biochip as an example, the present invention has a molecular probe wafer having a covalently bonded anchor compound, and as can be understood, it can also be applied to other uses of gold or silver. Treated as a different application for sensing the film on a biochip. Applications include, for example, optical sensing wafers, electrochemical sensing wafers, and piezoelectric sensing wafers. Therefore, any person skilled in the art can make such changes and changes without departing from the spirit and scope of the invention, and the scope of the invention is defined by the scope of the appended claims. [Simplified Explanation of the Formulas] The chemical reactions of Figures 1, 2 and 3 respectively show that after treatment with a chemical modification φ on the surface of a substrate coated with gold or silver, according to a preferred embodiment of the present invention, The basic organic molecules of the length are immobilized for the basic molecular probe wafer substrate to which the desired bioprobe molecules are attached. Figure 3A shows a simplified representation of the molecular probe wafer of Figure 3. The chemical reaction of Figure 4 is shown on the surface of the molecular probe wafer substrate of Figure 3, using succinic anhydride to carry out the alkylation reaction of the amine group [-ΝΗ2] of the organic molecule. To convert to a derivative compound whose end is an acid group [-COOH]. The chemical reaction of Figure 5 is shown on the surface of the molecular probe wafer substrate of Figure 3A, using sulfosuccinimidyl-4-(p-maleimidophenyl)-butyrate using 26 1270673 sulfosuccinate-butyric acid (sulfosuccinimidyl-4-(p-maleimidophenyl)-butyrate , sulfo-SMPB) Alkylation of the moon-based [-NH2] of the organic molecules to be converted to 4-(p-marinal hydrazine)-butyric acid group 4-(/?-maleimidophenyl)-biityrate group) Derivative compound of [-CO(CH2)3(CH)6N(CO)2(CH)2]. The chemical reaction of Figure 6 is shown on the surface of the molecular probe wafer substrate of Figure 3A, using 2-iminothiolane hydrochloride to thiolate the amine group of the anchoring organic molecule [-NHJ] The reaction is converted to a derivative compound having a terminal thiol group [-SH]. The chemical reaction of Figure 7 is shown on the surface of the molecular probe wafer substrate of Figure 3A, using cyanuric chloride to carry out cyanuric chlorination reaction on the amine group [-NH2] of the organic molecule. A derivative compound that is converted to a cyanuric chloride-activated material at the end. The chemical reaction of Figure 8 shows the carbonyldiimidazolization reaction of the amine group [_NH2] of anchoring organic molecules by using carbonyldiimidazol on the surface of the molecular probe wafer substrate of Figure 3A. a derivative compound that is converted to a carbonyldiimidazol_activated substance [_CON2C3H3]. The chemical reaction of Figure 9 shows that according to another preferred embodiment of the present invention, a chemical modification treatment is applied to the surface of a substrate coated with gold or silver to immobilize a basic organic molecule of a selected length for attachment to a biological probe for use. A molecular molecular probe wafer substrate of molecules. 27 1270673. The chemical reaction of Figure 10 is shown on the surface of the molecular probe wafer substrate of Figure 9, using succinic anhydride to thiolate the amine group [-NH2] of the anchoring organic molecule to convert to the end. A derivative compound of an acid group [-COOH]. The chemical reaction of Figure 11 is shown on the surface of the molecular probe wafer substrate of Figure 9, using sulfo-SMPB to alkylate the amine group [-NH2] of the anchoring organic molecule to convert to a terminal 4-(p-horse Derivative compound of linoleyl-butyric acid [-CO(CH2)3(CH)6 N(CO)2(CH)2]. ^ The chemical reaction of Figure 12 is shown on the surface of the molecular probe wafer substrate of Figure 9, using a 2-imine pentacyclic sulfur to thiolize the amine group [-NH2] of the anchoring organic molecule to convert to the end. Derivative compound of thiol group [-SH]. The chemical reaction of Figure 13 is shown on the surface of the molecular probe wafer substrate of Figure 9. The cyanide chlorination reaction of the amine group [-NH2] of the anchoring organic molecule is carried out by cyanuric chloride to convert the end to cyanuric chloride. - a derivative compound of an activating substance. k The chemical reaction of Figure 14 is shown on the surface of the molecular probe wafer substrate of Figure 9. The diimidazolyl carbon oxime is used to carry out the diimidazolium carbonization reaction of the amine group [-NH2] of the anchoring organic molecule to convert it to the terminal imidazole. Derivative compound of the base carbon-activated substance [-CON2C3H3]. [Major component symbol description] Substrate (Molecular Probe Wafer) Substrate (Molecular Probe Wafer) Substrate (Processing Stage) Substrate 100, 400, 500, 600, 800, 900, 1000, 1100, 1200, 1300, 1400 102, 103 110, 910 28 1270673 metal cloth covering 120, 920 29