Abstract
"因在台灣與水產生物科技相關的奈米計劃付之闕如，因此本計畫的執行將有起先導之效應， 以本校水產生物研究成果為根基，加上光電、奈米、材料等領域的整合與協助，將研究進一 步拓展至奈米科技的水產生物研究領域，其成果勢必是可期的。本計畫的架構(如下圖所示) 是以表面電漿共振光學感測技術(子計畫3)為主要檢測技術針對水產毒素(子計畫1)、病源菌 (子計畫2)進行奈米檢測技術研發，並以幾丁聚醣/氧化矽奈米複合材質(子計畫4)與分子拓印 聚合物(子計畫5)的方式提升檢測靈敏度與識別性，各子計畫之研究內容概述如下： 水產毒素(河魨毒和麻痺性貝毒)之奈米檢測技術研發：水產毒素河魨毒和麻痺性貝毒為神經 性劇毒，在國內常引起食物中毒而導致死亡事件，因此甚被重視。已知廣存在河魨類、蝦虎 類、織紋螺類、玉螺類、榧螺類、扁蟹類、二枚貝類和海星類之水產動物中。其毒素之檢測 目前依賴生物毒性檢測法和高效能液相層析儀法，前法粗糙，後法又須繁雜精製純化技術， 耗時不經濟。而且中毒患者之血液、尿液毒素量微量，目前尚無快速可信賴之定性、定量檢 測法，因此急需開發精製純化水產毒素之奈米技術及可定性定量之奈米檢測方法，以快速精 確省時的檢測微量水產毒素。此技術之研發為國際首創，將創新簡化水產毒素之檢測科技， 因應醫療需求，並對水產毒素之科技研究助益宏大。本第一子計畫第一年目標以標準品河魨 毒和麻痹性貝毒數種主要成分，分別添加入血液或生物檢體中，探討簡易純化後，即以液相 層析儀配合質譜儀之微量定性定量分析法；其次製備大量標準品供研究群使用。並進行鈉腔 體之製備。第二年目標為利用廖若川教授和張克亮教授研發之奈米顆粒 (一為chitosan 和 silica 之結合體，另一為分子拓印聚合物) 作為生物體或血液中河魨毒和麻痹性貝毒之純化 和分析管柱樹脂用之探討，以開發奈米顆粒作為海洋生物毒素純化、精製和檢測之素材，並 與傳統HPLC 和LC-MS 法做比較。同時進行鈉腔體層析管柱樹脂之研發。第三年應用廖若 川教授或張克亮教授研發之奈米顆粒，結合不同金屬離子，探討表面電漿共振光學感測技術 之開發，合併第二年奈米顆粒純化精製之技術及表面電漿共振光學感測技術，開發海洋生物 之奈米技術檢測法，並與傳統之HPLC 及LC-MS 法做比較。另外探討鈉腔體溶合表面電漿 共振光學感測技術之研發。 奈米科技於病原菌檢測技術上之應用：血清型O157:H7 大腸桿菌是人類重要病原菌之一， 其會造成人們嚴重腹痛與血便。約7% 病人會併發血尿症狀，其中約5%病人可因急性腎衰 竭而死亡。由於此病菌感染劑量很低，以致於其可因交叉污染或交叉感染而造成人們生病， 因而許多食品，甚至水均可能成為其傳染來源。也因為此病菌感染劑量很低，需要快速且靈 敏的方法來偵測此菌。雖然已有許多免疫方法被開發出來偵測此菌，然而其靈敏度均不足， 以致於無法直接偵測使用；而且部分會產生偽陽性反應。近年來表面電漿共振光譜分析技術 由於靈敏快速，顯示可用於快速檢測病原菌。當菌體表面抗原與固定在金表面之抗體結合 時，會造成共振角的偏移，其可由光譜分析顯示出來。將幾丁聚醣與矽酸鹽之複合材料做成 奈米粒，將其表面接上抗體，可與金表面抗體結合之菌體結合，以增大共振角的偏移，因而 增加靈敏度。由於專一性與靈敏度是目前對此菌之偵測方法所需克服的二大要點，此計畫預 計第一年生產對此病原菌專一性很高的單株抗體，並評估其專一性。此外，將提供此抗體給 子計畫3 與4，提供菌體給子計畫5。第二年將此抗體分別固定在金表面與奈米粒表面，以 建立分析此病原菌之免疫表面電漿共振分析法。第三年將此方法應用於分析食品中此菌污染 情形。 表面電漿共振光學感測技術在生物晶片的應用：自從B. Liedberg 在1983 年將SPR 原理應用 於氣體成分檢測後，以SPR 為基礎之分析方法，已成功且廣泛地應用在各類研究領域。利 用表面電漿共振於生物感測為量測生物分子在固體與液體或固體與氣體界面發生交互作用 時，所造成界面之介電常數的微小改變，可在無須對生物分子做任何的標記下，即時地分析 生物分子間的交互作用。由於SPR 技術具有快速地、靈敏度高以及大量平行篩檢等優點， 可預期將被廣泛地應用在很多生物分子診斷的領域上，諸如抗原與抗體之交互作用、蛋白質 分子的非特定吸附、薄膜與蛋白質間交互反應或DNA 雜交等。因此，利用SPR 原理設計的 生物晶片，可達到對生物分子進行大量平行篩檢的目的。金屬奈米粒子(Metallic Nanoparticles) 在生物醫學上的應用最近幾年備受重視，在光學偵測的應用上有許多優異之處。金的奈米粒 子現在已廣泛地應用在生化材料上，最近，有人利用金奈米粒子產生的表面增強拉曼散射效 應，可以將最低濃度偵測極限推至20fM。因此，若能將SPR 生物感測技術結合金屬奈米粒 子的表面增強效應，將可能有效增加生物晶片的靈敏度。 在過去幾年中，我們建立了一個理論模型，利用電子－聲子散射以及電子－電子散射機制， 來解釋自由電子金屬的光學性質隨溫度的變化。我們也利用此模型來了解表面增益拉曼散射 的現象中，基底溫度對於此現象的影響。我們也利用此模型來探討表面電漿共振光學感應器 在增溫環境操作下的靈敏度限制，並將研究結果發表於國際期刊中，有關實驗的部份，我們 也建立了角度探測、波長探測及外差干涉相位偵測系統。基於我們過去幾年來持續在溫變表 面電漿共振效應的理論及實驗的研究成果，我們預計在此三年計畫中研究下列三項課題：(A) 應用相位調變表面電漿共振技術於水產生物檢測，(B)利用奈米金屬顆粒的表面電漿共振來 建造並應用表面增強拉曼散射光譜，(C)利用近場光學掃描顯微術偵測奈米金屬顆粒的表面 電漿共振。同時，亦進行奈米金屬顆粒的改質與各項奈米材質的材料分析：如TEM, STM 及 AFM，以了解並建立其微觀組織與性質的關係。 以仿生模板法合成幾丁聚醣/氧化矽奈米複合材質及其在水產毒素與E. coli 之分離和檢測之 應用：本子計畫4 之目的在開發製造具有生物分離與生物檢測功能之中孔洞奈米複合材質的 技術。第一年擬採用幾丁聚醣等生物分子做為模板，開發出具有不同孔洞與表面性質之中孔 洞幾丁聚醣/氧化矽奈米粒。第二年擬配合子計畫1，調整奈米粒之孔洞大小、形態及表面性 質，以改進其在水產毒素之吸附及層析檢驗上之應用。並配合子計畫2，探討奈米粒特性及 抗體固定方法對E. coli O157 檢測之靈敏度和專一性的影響。第三年擬探討製備幾丁聚醣/ 氧化矽自組單層之方法，以配合子計畫1 至3 開發SPR 生物感測上之方法。另外擬開發可 發出螢光之幾丁聚醣/氧化矽奈米粒，並評估其在生物檢測上之應用效益。 分子拓印分析法檢測：本研究將進行以分子拓印(molecularly imprinting) 技術合成可識別生 化分子之聚合物。以分子拓印聚合物模擬生物抗體，連結表面液裝共振 (surface plasmon resonance)方法分析水產生物毒素如 tetrodotoxin 及引致食品中毒子微生物如 E. coli H157:O57 檢驗上之應用。" "The recent advent of the nanotechnology has attracted great attention to the fundamental and applied research in Taiwan and abroad due to their potential use in engineering applications. However, a lack of research efforts in Taiwan on marine biotechnology-related nanotechnology has regretfully lost great opportunities for taking their advantages into applications while research and development in other fields are booming for exploring new nanotechnologies and their potential applications. Therefore, this study is directed toward a fundamental research and development of nanotechnology for being used in the marine biotechnology. The project proposed herein is based on the surface plasmon resonance analytical technique (sub-project 3) to detect marine toxin (sub-project 1) and pathogen (sub-project 2). The detection sensitivity will be enhanced through materials improvement using chitosan and silica nanocomposites (sub-project 4) and molecularly imprinted polymer assay (sub-project 5), as shown below. A schematic showing project themes and inter-relationship among the sub-projects In the first sub-project, development of nano detection technique for marine toxin (TTX and PSP) is to be performed. The marine toxins including tetrodotoxin (TTX) and paralytic shellfish poisons (PSP) are known as notorious neurotoxins to induce serious food poisoning incidents in Taiwan. These marine toxins are widely distributed in the marine animals, such as puffers, gastropods, xanthid crabs, gobies, bivalve and starfishes. The popular availably detecting methods are bioassay and high performance liquid chromatagraphy. The former method is not accuate and not valid, and the latter one needs complicated purification processes. Therefore, the simple, rapid and reliable nano-purification techniques and determining techniques are demanded to develop. These new techniques will promote the advancing progression for marine toxin research and revolve the requirement of rapid clinical examination for food poisoning victims. The aims of the first year in the first sub-project are focused on development of LC-MS method to assay TTX and PSP in the toxic marine samples and blood serum. Furthermore, large amounts of TTX and PSP will be prepared for supporting other researches. The preparation of sodium channel will also be undertaken. The aims of second year are focused to utilize nano-materials developed by Professors K. L. Chang and Y. C. Liu (one is nanocomposite of chitosan and silica and another is nano-product of molecularly imprinted polymer). The simple purifying and identifying processes for TTX and PSP to use above nano-materials will be studied. The results will be compared to those from HPLC and LC-MS. Meanwhile, utilization of sodium channel for purifying TTX and PSP will also be developed. The aims of third year are to develop detecting method of the surface plasmon resonance (SPR) combining metal nanoparticles (SPR-MN) for TTX and PSP by using above nano-materials. The results will be also compared to those HPLC and LC-MS. The overall goal of the first sub-project is to develop new nano-technique method for detecting trace TTX and PSP in toxic marine animals and blood serum. The second sub-project will focus on the detection of pathogen using the SPR nanotechnology. E. coli O157:H7 is now recognized as one of the most important Detection sensitivity improvement: Chitosan and silica nanocomposites/fluorescent nanoparticles (sub-project 4) Molecularly imprinted polymer SPR-MN technique (sub-project 3) assay (sub-project 5) Marine toxin (sub-project 1) Pathogen (sub-project 2) enteropathogens. Bloody diarrhea accompanied by severe cramping abdominal pain is the major symptom for infection with E. coli O157:H7. About 7% of cases may have serious complication of hemolytic uremic syndrome (HUS), among which about 5% of HUS patients die in the acute phase of the disease. The low infectious dose for this pathogen ensures that cross-contamination and cross-infection occurs readily and can involve a diverse range of vehicles, including those such as water sources. Therefore, a very sensitive detection method is needed. Various immunoassays have been developed for rapid detection of E. coli O157:H7. However, their sensitivities are not high enough to directly detect this pathogen. Recently, SPR spectrometry shows potential as a sensitive method for rapid detecting pathogens. The binding of antigens on cell with antibody immobilized on gold surface will result in a shift of the resonance angle (Δθres) that can be measured directly. The antibody-coated chitosan-silicate composite nanoparticle can bind with cell and thus, may improve the sensitivity of immuno-SPR assay. The commercial available antibody against E. coli O157:H7 is still lack of specificity. Therefore, in the first year the monoclonal antibody (MAb) against E. coli O157:H7 will be produced and its specificity will be evaluated. In addition, we’ll provide the MAb and E. coli O157:H7 cell to sub-projects 3 & 4, and sub-project 5, respectively. In the second year, MAb will be immobilized on gold surface, and on the surface of chitosan-silica nanoparticles to establish the immuno-SPR assay. The sensitivity and specificity of the established assay will be evaluated and compared with several commercial available kits for this pathogen. In the third year, this assay will be applied to detect E. coli O157:H7 in various foods. The third sub-project will deal with the application development of SPR in the marine biotechnology. Since the first pioneering work of B. Liedberg et al in 1983 that demonstrated the use of SPR as an optical biosensensor, the first commercial device was made available. This resulted in a wide spread of SPR to a wide spectrum of biological applications, including immunological analysis, studies of protein-protein interactions, epitope mapping, molecular-biological studies on the mechanisms of gene expression, signal transduction and cell-cell interactions, and the examination of kinetics of binding. The success of using SPR techniques for biological applications is due to the two basic factors: (1) SPR allows the measurement of kinetics of biomolecular interactions in real time with high sensitivity; and (2) no labeling of the biomolecules is necessary for their detection. In the last several years, we have established a theoretical model which can account for the temperature variation of the optical properties of metal. We achieved this from the consideration of the phonon-electron and electron-electron interactions in the metal. We have applied this model to the study of the substrate temperature effects on surface-enhanced Raman scattering (SERS). We have also very recently applied the model to the study of the temperature-dependent sensitivity of optical sensors based on SPR and optical spectroscopy for single-molecules near a microstructure. We have also been working on the experimental confirmation of our modeling results in our laboratory. Based on the lasting works of theoretical modeling and experimental instrumentation for the temperature-dependent SPR effect, we propose to extend our research to the following three issues: (A) applying phase-modulation technique in SPR experiment to marine-biological applications, (B) construction and applications of Surface Enhanced Raman Scattering (SERS) by using SPR in metallic nanoparticles and (C) using Near-Fields Scanning Optical Microscopy (NSOM) to measure the SPR in single metallic nanoparticles. In addition, material characterizations will be carried out, including TEM, SEM, STM and AFM, in order to establish better correlations between the structure/microstructure and properties. In the forth sub-project, we propose a biomimetic approach to synthesize chitosan-silica nanocomposite for the separation and detection of marine toxins and E. coli O157. We will develop a novel synthesis method using chitosan and small biomolecules as the template. Mesoporous chitosan/silica composite nanoparticles with different porous and surface properties are to be made in the first year. The goals of the second year include modifying the pore size, morphology and surface properties of the nanoparticles to improve their functionalities in the sorption and separation of marine toxins (sub-project 1). We will also work with sub-project 2 to investigate the effects of nanoparticle characteristics and antibody immobilization methods on the sensitivity and selectivity of E. coli detection. For the third year, we will explore the conditions to form chitosan/silica self-assembled monolayer and apply in the SPR biosensors for marine toxins and E. coli O157 (sub-projects 1 to 3). In addition, we plan to make fluorescent chitosan/silica composite nanoparticles and evaluate its effectiveness in biosensing applications. The fifth sub-project will use marine toxin and food-borne microorganisms as template to synthesize imprinted polymer. The resulting polymers can act as artificial antibodies to link with surface plasmon resonance. The binding activity and assay efficiency against marine food toxin and micro-organism will be studied."
Keyword(s)
水產毒素
微生物
病原菌
血清型O157:H7 大腸桿菌
酵素結合免疫吸附分析
_x000d_ 單株抗體
免疫磁性分離
表面電漿共振
奈米金屬顆粒
表面增強拉曼散射
幾丁聚醣/_x000d_ 氧化矽奈米複合材質
螢光奈米粒
分子拓印
marine toxin (TTX and PSP)
pathogen
Escherichia coli O157:H7
enzyme-linked_x000d_ immunosorbent assay
monoclonal antibody
immunomagnetic separation
surface plasmon_x000d_ resonance
nanocomposites
fluorescent nanoparticles
molecularly imprinting
