200804810 九、發明說明： 【發明所屬之技術領域】 本發明係關於一種生物感測器，其特別關於一種耦合波導 表面電漿共振生物感測器，藉由動態解析生物分子層的膜厚及 折射係數而去提升檢測效率。 【先前技術】 一省知的SPR生物感測器雖然具有高靈敏度、及不需進行 標示，便可即時、及定量生物分子的交互作舰息，但其 所，之實驗訊息僅能提供生物分子交互作用的動力學資料，無 法說明感測為表面生物分子層折射率及膜厚變化量之資訊，因 此缺乏對生物分子構形及結構改變的偵測能力，降低了對生物 分子辨識系統的精確度。 為了獲得生物分子層折射率及膜後變化量之資訊，分別有 =雙奸（tWG_e—)、雙緩衝介f (^七秦）、耦合電漿 11 (C〇UpledplaSm〇n_WaVeguideresonance，CPWR)、橢圓偏 術干涉術荨方法應運而生，但其中利用雙波長進行偵測 爲了解因光波色散產生之折射率變化效應；以雙介質進行 ίίΐΪ達到即時偵測的效果;而進行輕合電漿波導及橢圓 偏先術日讀測靈敏度會降低1〇_觸倍不等；採用干涉術時， =吏用的$ _統需時常進行校正，且祕細細亦受到本 貝相位量測所限制。 Μ2·號中揭露之_合電漿波導共振技術 二產或夕層金屬層（半導體層），在全反射下以稜鏡或 生表面共振波，雖可使用不同波長之光能進行激發，但 據判讀生贿綠絲差及無法經由數 5 200804810 综上所述，目前採用之 明的發日从有鐘於上述習知:二式都仍有待改善之處，本發 創作出-種能提升檢測效率缺失’ μ思改良而 測器。 之耦曰波導表面電巢共振生物感 【發明内容】 物感測n，可合料㈣《共振生 物分子辨識分子層的膜厚及折射係數，對生 識生物分子之動力學變化及構 他賴技術以建立生物分子作用之台有助於整合其 穿诱甘Jr皮表電漿共振之原理，使用光束 芦S if i偵測器讀取反射後之訊號，為具有至少五 二ίΐί iiii測器’其結構包括：稜鏡、上層金屬 曰波¥層、下層金屬層、生物分子層及緩衝液。 再者上述光束可為tm模態之雷射光束。 伊处上述之架構係同時結合麵合表面電漿共振，及波導 权怨並產生兩個共振吸收譜線。 另外，上述之架構，可使一雷射光束穿透棱鏡，產生一 入射角0，並將反射後的訊息經由偵測器顯示。 ^ ，，熟悉該項技藝人士瞭解本創作之目的、特徵及功 ^，茲藉由下述具體實施例，並配合所附之圖式，對本創作 砰加說明，說明如后： 6 200804810 【實施方式】 明芩考第一圖，本發明耦合波導表面電漿共振生物感測器 ^主要架構，本發明所述之生物感測器，係由六層膜層所構成， 分別包括稜鏡u( ε u)、上層金屬層12( ε 12，d12)、波導層13( ε ^’^^、、下層金屬層^㈠^^‘”生物分子層15^^，^^ 及緩衝液16( ε 10)，其中ε及d分別代表各膜層的介電常數及厚 度，，用已知波長之TM模態入射光進行激發時，會產生兩個 吸收瑨線，可藉此直接得到吸附於感測器表面微 的折射率及厚度變化。 卞層 第二圖說明傳統表面電漿共振(SPR)及耦合波導表面電漿 共振譜線（CWSPR)入射角及反射光強譜線關係圖，其中實線 所不係為耦合波導表面電漿共振之光強譜線，而虛線表示傳統 表面電漿共振（SPR)光強譜線，藉由此方法可達到即時動態的 偵測生物分子動力學及構形變化之目的。 “ 、研究生物分子吸附於表面之前如蛋白分子於CWSPR生物 感：則裔之表面，羧基端的 16_Mercaptohexadecanoic acid( HMA ) 必，先行固定於感測器的金層表面形成一層相同硫醇化的自200804810 IX. OBJECTS OF THE INVENTION: TECHNICAL FIELD The present invention relates to a biosensor, and more particularly to a coupled waveguide surface plasma resonance biosensor for dynamically analyzing the film thickness and refraction of a biomolecular layer Coefficient to improve detection efficiency. [Prior Art] Although the SPR biosensor of a province has high sensitivity and does not need to be labeled, it can instantly and quantitatively interact with biomolecules. However, the experimental information can only provide biomolecules. The kinetic data of the interaction cannot explain the information of the surface biomolecule layer refractive index and film thickness variation, so it lacks the ability to detect the biomolecule structure and structure changes, and reduces the accuracy of the biomolecular identification system. degree. In order to obtain information on the refractive index of the biomolecule layer and the amount of change in the film, there are = double rape (tWG_e-), double buffering f (^七秦), coupled plasma 11 (C〇UpledplaSm〇n_WaVeguideresonance, CPWR), ellipse The method of partial interferometry has emerged, but the use of dual-wavelength detection is used to understand the effect of refractive index change caused by light wave dispersion; the effect of instant detection is achieved by dual-medium media; and the light-coupled plasma waveguide is performed. The sensitivity of the ellipse first reading will be reduced by 1〇_touch times; when using interferometry, the $__ used for correction will be corrected frequently, and the secret is also limited by the phase measurement. Μ2· _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ According to the judgment of the bribery and the inability to pass the number 5 200804810 In summary, the current adoption of the Ming Dynasty from the clock to the above-mentioned custom: the two styles still need to be improved, the creation of this type can improve The lack of detection efficiency is improved. Coupling 曰 waveguide surface electronoptical resonance biological sensation [Invention content] Sense sensing n, can be compounded (4) "Resonance biomolecule identification molecular layer film thickness and refractive index, the dynamics of biomolecules The technique to establish a biomolecule platform helps to integrate the principle of the plasma resonance of the Jr skin, and uses the beam reed S if i detector to read the reflected signal, which has at least five ίΐί iiii detector 'The structure includes: 稜鏡, upper metal chopping layer, lower metal layer, biomolecule layer and buffer. Furthermore, the beam may be a laser beam of the tm mode. The above-mentioned architecture is combined with surface-surface plasma resonance, and waveguide weights and two resonance absorption lines. In addition, the above structure enables a laser beam to penetrate the prism, generate an incident angle of 0, and display the reflected message via a detector. ^ , , familiar with the skilled person to understand the purpose, characteristics and merits of this creation, the following specific examples, together with the attached drawings, add a description to the creation, as explained below: 6 200804810 The first embodiment of the present invention is a coupled resonator surface acoustic resonance biosensor. The biosensor of the present invention is composed of six layers, including 稜鏡u ( ε u), upper metal layer 12 ( ε 12, d12), waveguide layer 13 ( ε ^ '^^, lower metal layer ^ (1) ^ ^ '" biomolecular layer 15 ^ ^, ^ ^ and buffer 16 ( ε 10), wherein ε and d respectively represent the dielectric constant and thickness of each film layer, and when excited by the TM mode incident light of a known wavelength, two absorption enthalpy lines are generated, thereby directly obtaining the sensation of adsorption The refractive index and thickness of the surface of the detector are slightly changed. The second layer of the 卞 layer illustrates the relationship between the incident surface angle and the reflected light intensity line of the conventional surface plasma resonance (SPR) and coupled waveguide surface plasma resonance line (CWSPR). The line is not tied to the intensity spectrum of the plasma resonance of the coupled waveguide surface, and the dotted line indicates the transmission. Surface plasmon resonance (SPR) intensity line, which can achieve real-time dynamic detection of biomolecular dynamics and configuration changes. ", research on biomolecules before adsorption on the surface such as protein molecules in CWSPR biological sense : On the surface of the genus, the 16-Mercaptohexadecanoic acid (HMA) at the carboxy end must be fixed on the surface of the gold layer of the sensor to form a layer of the same thiolated self.
組裝單分子層(self-assembled monolayer，SAM)。在形成 SAM 時，先將折射率為1.361的乙醇溶液打入CWSPR感測裝置的 反應槽内，之後，再使含有i mM的洛Mercapt〇hexadec_ic acid的乙醇溶液流入反應槽内，待至spR角度飄移量增加至 乾和後(約為六小時後）’再將原先的乙醇溶液注入以清除表面 上未發生鍵結的分子。 弟二圖表示以波長632.8 nm的TM-mode入射光波下，自 ，層16_Mercaptohexadecanoic add固定化過程前後反射光強 譜線變化的原始實驗數據，虛線為固定化前所偵測得之光強譜 線，實線為固定化後所偵測得之光譜線。 7 200804810 第四圖表示自組層16-Mercaptohexadecanoic add之折射 率及膜厚關係圖。利用Fresnel方程式為基礎的擬合方法，可 將反射光強譜線的吸收譜線個別求解，分別得到二組單分 ^斤射率與厚度的解集合，因絲-組解集合會在折射率-厚 度平面上構成一條曲線，所以將無法得到唯一解，因此，經由 射率-厚度平面此二條曲線的交點可獲得sam的折=率 ^厚度，此單一分子層的折射率與厚度分別為1464及2 % 理的。i1此，藉由CWSPR的二個在反射光強譜線的 rmw碏線可计异出SAM的折射率與厚度並作為隨後以此 CWSPR的二個吸收譜線直接測定生物分子層的折射率與厚度 的校準依據。 /、 ^人類血清白蛋白（human serum albumin，HSA)為例， f應鈾為使蛋白質吸附於感測器表面。在蛋白吸附過程，折射 J J I·334的磷酸鹽溶液(PBS，pH 74)以小於5〇 μ1/ιηίη的流 入其反應槽内，並設定反應溫度固定維持於30(rc，然 ,使含有1 μΜ的人類血清白蛋白的磷酸鹽溶液流入反應槽 巧亚即時量測CWSPR生物感測器的反射光強譜線變化情形， =灸再將原先賴魏狀以清絲面未發生專— 生物分子。 一第五圖顯示HSA分子吸附於感測器表面所造成CWSpR ，一個吸收睹線的共振角度增加情形，虛線為第一個CWSpR ，態，實線為第二個CWSPR模態。於第二個cwspR吸收譜 共振角度增加量較第一個CWspR吸收譜線的共振角度增 ^量多是因為第二個CWSPR模態耦合SPs的百分比例較第一 =CWSPR模態來的高所致，藉由前述以方程式為基 礎，擬合方法在不同量測時間下估算CWSpR的二個在反射光 ，瑨線的吸收譜線可動態的測定出HSA吸附過程中所造成的 感測層的折射率與厚度變化情形。 8 200804810 主，弟六圖表示感測層的折射率（實線）與厚度（虛線）變化 十月形，以此二個CWSPR吸收譜線解得的關於生物分子層 率及厚度的即時資料可用來作為動態酬 ^ 時產生的生物分子構形變化。 对又互作用 ^藉由約離子誘導組織性轉麩氨梅（tissue tranSglutaminase，tTG)分子結構改變來驗證此發展的cwspR 生物感測裔可直接動態監測生物分子構形變化的能力，首先將 50 mM 的 Tris-HCl(150 mM NaCl)緩衝液以小於 5〇 μ1/ιηίη 的流 速流入已經上述SAM改質後的感測器表面，並設定 固定維持於2赋，再使具有344·65爾的仰分子的 流入，最後以原先Tris-HCl緩衝液沖洗掉未發生鍵結的⑽ 分子，使感測器表面固定上一層tTG單分子作為引起分子構形 變化的標地分子，然後再將誘發tTG分子構形變化的活化因子 鈣，子(5 mM)通入，同時藉由感測器即時記錄其變化情形，待 訊號至穩定後以原先的TriS-HCl緩衝液沖洗掉誘發構形變化 的鈣離子，使tTG分子結構再行回復。 第七圖是經由CWSPR生物感測器監測並以前述Fresnd 方程式為基礎的擬合方法動態的量測tTG分子因分子構形改 變導致其感測層的折射率及厚度變化情形，很明顯可發現^離 子的效應促使其感測層的厚度增加且折射率減小，主要是因為 鈣離子會促使的tTG分子活化使得其分子半徑增加所致。… 弟八圖為監測此tTG單分子層的折射率及厚度改變的詳 細數據。 " 利用此CWSPR架構同時麵合波導層中波導模態及在下層 金屬層與緩衝液界面的SPR模態所建構的生物感測器具有直 接偵測發生生物分子交互作用時伴隨其生物分子構形變化的 能力。藉由增加波導層厚度以增加增加光波於波導層的光程， 200804810 度位置。在單一波 譜線的^物/¾ >丨4、，衝介質下’此具有二個CWSPR吸收 證實具杨(||撕⑽ ^早分子層的折神及厚度，_子誘發tTG分子構形 ’這發展的cwspr生物感測器可提供 马直接研九生物分子構形變化的重要的工具。 太名if由^上較佳具體實施例之詳述，歸望能更加清楚描述 徵巧神’而並非以上述所揭露的較佳具體實施例 錄_ ϋτ加。相反地’其目的是希望能涵蓋各 *交，、相等性的安排於本創作所欲申請之專利範圍的範 臀内。 【圖式簡單說明】 第一圖為本發明耦合波導表面電漿共振生物感測器之主 要架構； 第二圖為傳統表面電漿共振(SPR)及耦合波導表面電漿共 振譜線（CWSPR)入射角及反射光強譜線關係圖； 第三圖為16-Mercaptohexadecanoic acid固定化前後反射 光強譜線關係圖； 第四圖為 16-Mercaptohexadecanoic acid 自組層（SAM) 折射率及膜厚變化圖； 第五圖為1 μΜ的HSA分子吸附於感測器表面時第一及 第二CWSPR模態變化情形； 第六圖為HAS單分子層的折射率及厚度變化情形； 第七圖為動態監測tTG分子吸附及鈣離子誘發分子構形 變化的tTG單分子層折射率及厚度變化情形；以及 第八圖為tTG單分子層及鈣離子誘發過程其折射率及厚 度變化情形。 200804810 【主要元件符號說明】 1 ： 雷射光束 2 ： 偵測器 Θ 雷射入射角 11 棱鏡 12 上層金屬層 13 波導層 14 下層金屬層 15 生物分子層 16 緩衝液 11A self-assembled monolayer (SAM) is assembled. In the formation of SAM, the ethanol solution with a refractive index of 1.361 is first introduced into the reaction tank of the CWSPR sensing device, and then the ethanol solution containing i mM Locapcap 〇hexadec_ic acid is flowed into the reaction tank until the spR angle The amount of drift increased to dry and after (about six hours later)' and the original ethanol solution was injected to remove molecules that did not bond on the surface. The second figure shows the original experimental data of the intensity spectrum of the reflected light before and after the immobilization process of the layer 16_Mercaptohexadecanoic add under the TM-mode incident light wave with a wavelength of 632.8 nm. The dotted line is the intensity spectrum detected before the immobilization. The solid line is the spectral line detected after the immobilization. 7 200804810 The fourth graph shows the relationship between the refractive index and film thickness of the self-assembled layer 16-Mercaptohexadecanoic add. By using the Fresnel equation-based fitting method, the absorption lines of the reflected light intensity lines can be solved individually, and the solution sets of the two groups of single-shots and thicknesses are obtained respectively. - A curve is formed on the thickness plane, so a unique solution cannot be obtained. Therefore, the intersection of the two curves can be obtained by the intersection of the two curves of the luminosity-thickness plane. The refractive index and thickness of the single molecular layer are respectively 1464. And 2% rational. I1, by using the two rmw碏 lines of the CWSPR in the reflected light intensity line, the refractive index and thickness of the SAM can be calculated and the refractive index of the biomolecule layer can be directly determined by the two absorption lines of the CWSPR. The basis for the calibration of the thickness. /, ^ Human serum albumin (HSA) as an example, f should be uranium for protein adsorption on the surface of the sensor. In the protein adsorption process, the phosphate solution (PBS, pH 74) of JJI·334 is refracted into the reaction tank at less than 5〇μ1/ιηίη, and the reaction temperature is set to be fixed at 30 (rc, then, 1 μΜ is contained. The phosphate solution of human serum albumin flows into the reaction tank to measure the reflected light intensity line of the CWSPR biosensor. The moxibustion will not have the special-biomolecule in the original shape. A fifth graph shows the CWSpR caused by the adsorption of HSA molecules on the surface of the sensor. The resonance angle of an absorption enthalpy increases. The dotted line is the first CWSpR, and the solid line is the second CWSPR mode. The increase in the resonance angle of the cwspR absorption spectrum is greater than the resonance angle of the first CWspR absorption line because the percentage of the second CWSPR mode coupled SPs is higher than that of the first = CWSPR mode. Based on the equation above, the fitting method estimates the two reflected light in CWSpR under different measurement times. The absorption line of the 瑨 line can dynamically determine the refractive index and thickness of the sensing layer caused by HSA adsorption. Change situation. 8 200804810 The main and younger six figures show that the refractive index (solid line) and thickness (dashed line) of the sensing layer change in the shape of the moon, and the instantaneous data on the layer rate and thickness of the biomolecule obtained by the two CWSPR absorption lines can be used. As a dynamic regenerative biomolecule configuration change. Interaction and interaction ^ The tissue-transformed tissue transglutaminase (tTG) molecular structure changes to verify the development of cwspR biosensing The ability to dynamically monitor changes in biomolecular configuration by first flowing 50 mM Tris-HCl (150 mM NaCl) buffer into the surface of the sensor after modification of the above SAM at a flow rate of less than 5 μμl/ιηίη, and setting the immobilization Maintaining at 2, and then letting the influx of 347·65 Å of the upstream molecule, and finally rinsing the unbonded (10) molecule with the original Tris-HCl buffer, and fixing the surface of the sensor with a layer of tTG single molecule as the causing molecule The target molecule of the configuration change, and then the activation factor calcium (5 mM), which induces the change of the tTG molecule configuration, is introduced, and the change is immediately recorded by the sensor, and the signal is stabilized. After that, the original TriS-HCl buffer is used to wash away the calcium ions that induce the conformational change, and the tTG molecular structure is restored. The seventh figure is the dynamics of the fitting method based on the above-mentioned Fresnd equation monitored by the CWSPR biosensor. The measurement of the refractive index and thickness of the sensing layer caused by the change of the molecular structure of the tTG molecule clearly shows that the effect of the ion promotes the thickness of the sensing layer and decreases the refractive index, mainly because of the calcium ion. The activation of the tTG molecule is caused by an increase in its molecular radius. ... The Eight Diagrams are detailed data for monitoring the change in refractive index and thickness of this tTG monolayer. " Using this CWSPR architecture to simultaneously cover the waveguide mode in the waveguide layer and the SPR mode constructed at the interface of the underlying metal layer and the buffer interface, the biosensor constructed directly detects the biomolecule interaction with its biomolecular structure The ability to change shape. By increasing the thickness of the waveguide layer to increase the optical path of the optical wave to the waveguide layer, the position of 200804810 degrees. In the single-spectrum line of ^^/3⁄4 >丨4, under the rushing medium, this has two CWSPR absorptions to confirm the structure and thickness of the yang (||Tear (10) ^ early molecular layer, _ sub-induced tTG molecular structure This development of the cwspr biosensor can provide an important tool for the horse to directly study the changes in the configuration of the nine biomolecules. Taiming if by the detailed description of the preferred embodiment, the hope can more clearly describe the genius 'It is not the preferred embodiment disclosed above that _ 加 τ 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。. BRIEF DESCRIPTION OF THE DRAWINGS The first figure is the main structure of the coupled resonator surface resonator biosensor of the present invention; the second figure is the conventional surface plasma resonance (SPR) and coupled waveguide surface plasma resonance line (CWSPR) The relationship between the incident angle and the reflected light intensity line; the third picture shows the relationship between the intensity spectrum of the 16-Mercaptohexadecanoic acid before and after immobilization; the fourth picture shows the refractive index and film thickness of the 16-Mercaptohexadecanoic acid self-organized layer (SAM). Figure 5 shows the 1 μΜ HS The first and second CWSPR modes change when A molecules are adsorbed on the surface of the sensor; the sixth picture shows the change of refractive index and thickness of the HAS monolayer; The seventh picture shows the dynamic monitoring of tTG molecule adsorption and calcium ion induced molecules The change in the refractive index and thickness of the tTG monolayer in the configuration change; and the eighth figure shows the change in the refractive index and thickness of the tTG monolayer and the calcium ion induced process. 200804810 [Description of main components] 1 : Laser beam 2 : Detector Θ Laser incident angle 11 Prism 12 Upper metal layer 13 Waveguide layer 14 Lower metal layer 15 Biomolecular layer 16 Buffer 11