| 摘要: | 生物感測器( biosensor )的主要組成可分為一具選擇性的生物分子以及ㄧ訊號轉換器( transducer )所組成，其中又以將生物分子固定於訊號轉換器表面最為重要，近年來，自組裝單分子層膜技術( self assembly monolayer, SAM )大量的被應用在生物感測器表面的辨識分子的固定化程序上，自組裝單分子層主要是有機分子在溶液中自發性的排列聚集而吸附在基材上行成單分子層膜，其優點有簡單製備、薄膜瑕疵少、具有多樣的官能基種類能在不同的基材上可供生物分子固定使用，本研究藉由自組裝單分子層技術，設計並建構生物感測器之感測介面來檢測生物分子，並探討生物分子與其辨識分子間之交互作用機制。研究之系統包含表面電漿共振儀( surface plasmon resonance, SPR )、共振波導檢測儀( guided mode resonance, GMR )及電梳式阻抗感測器( interdigitated electrode, IDE )等三種不同的感測器。 在表面電漿共振儀的研究系統中利用 Langmuir 等溫吸附模式及相關動力學參數來分析硼酸分子衍生物與糖化血紅素間的交互作用關係， 實驗結果顯示糖化血紅素( HbA1c )與硼酸分子間的交互作用， pH 值的改變是影響硼酸及雙醇類物質形成配位酯鍵的最主要因素之一。當 pH 值大於硼酸分子的pKa 值時，硼酸分子以陰離子的形式存在，且較易與雙醇類分子產生鍵結，添加路易士鹼可使硼酸分子的立體結構發生改變，並可降低硼酸分子對 pH 值的敏感性。此外在高鹽的環境下非專一性吸附行為會增加，使硼酸分子與 HbA1c 間的專一性作用則會降低。由此所得之結果將有助於糖尿病的診斷準確率，進而提供檢測糖化血紅素創新、簡易及靈敏的另ㄧ選擇。 SPR 的研究亦延伸至以 DNA-protein 共聚物建構生物感測器，研究方面利用單股DNA與蛋白質(抗體)形成 DNA-protein conjugate ，其蛋白質活性並不會因與DNA形成複合物而失去活性，除此之外，利用 PEG-thiol 與 carboxylic acid terminal thiol 以 mixed SAMs 來建構的 DNA 晶片，具有良好的抗非特異性吸附能力並保留 DNA 的雜交能力，並成功的將 DNA 晶片轉換成蛋白質晶片，並用來檢測蛋白質，而此種方可法則應用在藥物篩選、生物感測及疾病檢測上有相當的發展潛力。 GMR 是一種光學濾波器，其週期性的波導層能形成一個非常窄小零穿透濾波效果，被濾去的波長即為共振波長，利用此特性將生物分子固定於次波長光柵結構上，量測共振波長的改變，便可作為生物分子的檢測。本研究主要利用 GMR 提供一新穎的生物檢測器，並且透過即時的觀察來研究分子間的交互作用機制，在DNA的檢測上，對於不同長度的 DNA 20 mer、40mer、60mer的去氧核醣核酸之雜交行為皆可分辨並可及時偵測，除此之外，本研究亦將凝血酶的適合體( aptamer )固定於波導共振元件之表面，並於實驗中即時檢測不同濃度之凝血酶，檢測範圍從 0.25 μM 到 1.5 μM 之濃度與其訊號飄移量呈線性關係。此外，在即時檢測的資料中亦提供動力學資訊，結果顯示 aptamer對凝血酶的平衡吸附常數（ equilibrium association constant ） Ka 達 106M-1。 電梳式感測器是基於生物分子於溶液中的生化反應中能使溶液中的阻抗產生變化，其中亦包含生物分子吸附至電極表面所造成的阻抗變化，在電梳式感測器的研究方面，利用( PEG )及仿生性染料( Cibacron Blue F3GA, CB )開發出對於人類血清白蛋白具有專一性的生物感測層，對於人類白蛋白捕捉檢測上，成功的檢測出人類白蛋白，並得到檢測區間為0.1 mg/ml到0.4 mg/ml，亦證明利用阻抗量測生物分子的可行性。 藉由相關的應用研究，可以了解到不同的環境因子對於生物間的辨識行為之影響，研究結果亦可經由動力學的分析得到生物辨識作用機制的基礎資訊，作為生物感測條件最佳化的依據。除此之外，我們亦證明了電阻抗量測於生物分子的應用上具有相當大的發展潛力。 Biosensor is a device with the ability to detect an analyte of its biological component together with the physicochemical detector component. It consists of 2 parts: the sensitive biological recognizing element, and the transducer and/or detector element. In recent years, self-assembled monolayers (SAMs) have been widespread employed as bases of the biological recognizing elements to be immobilized onto sensor surface. SAMs are ordered molecular assemblies formed by the adsorption of active surfactant molecules on solid surface. The advantages are the ease for preparation and provided wide ranges of functional groups for immobilizing the biomolecular assemblies on different substrates. This makes SAMs inherently attractive for biosensor fabrication. This study concentrates on the functionalized SAMs on gold and glass (SiO2) surfaces for developing optical biosensors, surface plasmon resonance (SPR), guided mode resonance (GMR) and electrode sensor, and interdigitated electrode (IDE) biosensors. The study of SPR investigated the effect of Lewis bases and salt concentrations with different pH values on the mechanism of glycated hemoglobin (HbA1c) and phenylboronic acid (PBA) interaction. The results of the interaction between HbA1c and phenylboronic acid exhibited the pH value is an important factor affecting HbA1c and PBA, forming the complex and Lewis bases. Lewis bases could change the stereo-structure of phenylboronic acid to form B(OH)3 and therefore would easily bind with saccharine. In addition, a linear response ranging from 0.43 to 3.49 μg/ml appeared in HbA1c, and the detection limit was 0.01 μg/ml. The SPR study in this thesis was extended to the protein biosensor fabrication via protein–DNA conjugates. The ssDNA probe surface was built up by following the mixed self-assembled monolayers (mixed SAMs) method. The results represented that the protein-DNA conjugates were immobilized on the chip surface with a sequence complementary to the ssDNA on the surface via sequence specific hybridization. In addition, the chip had high specificities to human serum albumin (HSA). The results demonstrated that the DNA chip was successfully converted into protein chip. This strategy would be useful and could be extensively employed for more versatile applications, such as drug screening, biosensors, and medical diagnostics. In the GMR research, GMR served as an optical filter which reflects specific wavelength in transmission light. GMR periodic waveguides produced sharp spectra when the illuminating wave matched to a leaky waveguide mode. Since the coupling range was typically small, these resonating elements exhibited high parametric sensitivity. They could thus be extremely responsive to small amounts of the change of biological molecules on chip interface. Recent efforts for improvement of resonant sensor had employed amplitude mode where the wavelength shift of the reflectance was measured directly. The purpose of this case study was to use GMR to provide a new method for investigating the interactions between the biomolecular. In this case, we presented a simple design of GMR for DNA and protein detection. The results demonstrated that it was successful to obtain the DNA hybridization by analysis of the surface wavelength peak shift using different DNA mass of 20 mer, 40 mer, and 60 mer. The study also stretched to immobilize the thrombin aptamer on GMR device for detecting the thrombin. The results also showed that the linear relationship of the concentrations of thrombin from 0.25μM to 1.5μM and the equilibrium association constant (Ka) is about 106 M-1. The investigation of interdigitated electrode sensor for biosensing is based on the fact that many biochemical reactions in a solution produce changes in the electrical resistance. Impedance measurements would reflect the changes of the resistance with the biomolecular assemblies attached to the sensor surface. This study showed an interdigitated electrode sensor for detection of Human Serum Albumin (HSA). The blue dye Cibacron Blue F3GA (CB), with a high affinity for HSA, was used as a captor. Experimental results showed a linear trend in the concentrations of HSA from 0.1 mg/ml to 0.4 mg/ml. In this research, we studied the SPR, GMR and IDE biosensors. The SPR and GMR could provide useful knowledge to understand the mechanism of biomolecular recognition. The impedance sensor, IDE, demonstrated a great potential for detection of specific sites on various biomolecular species. |
