香豆素寡聚合物、金及二氧化矽奈米粒子應用在醣受體的感測器開發

Applications of Coumarin Oligomer, Silica and Gold Nanoparticle to Sensors for Saccharide Receptors

廖元正(Yuan-Cheng Liao)

指導教授 ： 陳昭岑

國立臺灣大學/理學院/化學研究所/博士(2004年)

https://doi.org/10.6342/NTU.2004.10260

摘要

摘要
本論文主要是在開發醣受體 (蛋白質)感測分子，分別利用香豆素單體分子及寡聚合物、金及二氧化矽奈米粒子來當感測分子架構中的訊號讀出單元，再將醣殘基連結在這些架構上，利用醣殘基與蛋白質的專一結合性。一旦使感測器分子與分析蛋白質結合，可藉由所設計的信號讀出，達到辨識偵測效果。此感測分子系統因為本身具有信號讀出單元，所以欲分析的蛋白質不必先作標識即可達到偵測目的，對於微量樣品的蛋白質分析，提供了另一方便快速的方法，亦可將所發展的醣受體感測分子用到蛋白質體學醣受體種類研究。
我們設計合成具有norbornene-香豆素-醣殘基三種組成單元的感測分子單體，藉由開環歧化聚合反應 (ROMP) 有效率建構出具有多價性 (multivalence) 的醣-香豆素寡聚合物。在聚合過程中引入冠醚烯類衍生物進行開環共聚合反應，以增加寡聚合物的水溶性及降低醣殘基的立體擁擠性；寡聚合物具有多價醣殘基及烷醚親水的骨幹，所以有較佳的水溶性，同時可增強與蛋白質的結合力。此感測分子利用醣殘基與含有多結合部位的凝集素專一結合，造成寡聚合物間的交錯聚集 (aggregation) 及沈澱，同時紫外光-可見光吸收值大幅下降，達到簡易方便的偵測的目的。
關於金奈米粒子應用在醣受體感測器的開發部分，我們建構具有微陣列形式的感測器，這部份的研究主要是在玻璃基材上製備金奈米粒子的自我組裝單層，再將所設計合成的醣硫醇衍生物固定化在金奈米粒子單層上，即可得到醣受體感測器。當有偵測的生化分子存在且結合時，藉由量測玻璃上金奈米粒子自我組裝單層吸收光譜訊號的差異，便可即時瞭解此分子與醣分子作用與否。我們成功建構出對於甘露糖、半乳糖及葡萄糖胺有專一結合的蛋白質感測器，且進一步在玻璃上建構兩種不同醣配基 (patterning)，測試其與蛋白質的結合分析，結果顯示此感測器具有不錯的訊號偵測及專一辨識性。
關於二氧化矽奈米粒子應用在醣受體感測分子的開發部分，我們將二氧化矽奈米粒子表面修飾香豆素醣類衍生物，當有多結合部位的蛋白質或細菌與表面修飾醣配基的二氧化矽奈米粒子專一結合時，便會造成二氧化矽奈米粒子與偵測的生物分子產生交錯連結 (cross linking)，進而造成沈澱現像，藉由肉眼的判斷即可輕易檢測蛋白質或細菌的存在。我們利用具有甘露糖殘基的二氧化矽奈米粒子檢測鼠傷寒沙門桿菌 (Salmonella typhimurium) 及黏質沙雷氏桿菌(Serratia marcescens)，此感測粒子對於上述桿菌的偵測靈敏度約105隻/毫升。

關鍵字

凝集素 ； 金奈米粒子 ； 香豆素 ； 二氧化矽奈米粒子

並列摘要

Abstract
This dissertation goal is development of sugar receptor(protein) sensors. The skeleton of sensors include monomer and oligomer of coumarin, gold nanoparticle and silica nanoparticle. These skeleton further combine with some sugar residue, then sensor can enable to bind with analytic protein via sugar and protein specific binding ability, finally achieve the detective goal through readout unit. Because of the sensor system have signal readout unit themselves, so can ease to detect for analytic protein that label fluorescent molecule unnecessary. We develop these sensor system for improving the proteomic research efficiency.
We design and synthesis of sensor monomer consist of three components, including norbornene, coumarin and sugar moiety. We can construct of multivalent sugar-coumarin oligomer high efficiency via norbornene ring opening metathesis polymerization (ROMP). Alkene crown derivative lead into copolymer for increasing water solubility of oligomer and decreasing steric crowded of sugar residue. As a result of multivalent sugar residue and triethylene glycol construction for oligomer then enhance to bind with protein ability and raise detective limitation. The sensors utilize sugar residue to bind with multi-binding site of lectin specifically, then make interoligomeric cross-aggregation and precipitation. Consequently, we can reach detective goal by UV-visible absorbance value go down substantially.
In a part of gold nanoparticle application in sensor of sugar receptor, we expect to construct microarray characteristic sensor. This research focus on fabricate self-assemble gold nanoparticle monolayer on cover glass slide, then we design and synthesis of sugar disulfide derivatives immobilize on gold nanoparticle monolayer that can get sugar receptor sensor. When biomolecule attach and bind with sugar moiety that further measure absorbance signal variation of gold nanoparticle monolayer on glass. So we can use these sensor to determine protein binding with sugar moiety or not in real time. We successful construct sugar receptor sensors of binding with mannose, galactose and glucosamine specifically. We also fabricate two different sugar ligand area on identical glass, similarly, have good detective ability and specific recognition for two different lectins.
In a part of silica nanoparticle application in sensor of sugar receptor, we modified sugar-coumarin derivatives on silica nanoparticle surface. Equally, when multi-binding site of lectin and bacteria bind with sugar residue specifically of modification on silica nanoparticle surface, then produce silica nanoparticle cross-linking with biomolecule and make precipitable phenomenon in assay solution. We can ease diagnose protein and bacteria existence via naked eye. The silica nanoparticle surface modified mannose residue that can diagnose Salmonella typhimurium and Serratia marcescens. This silica nanoparticle detective limitation is about 105/mL for above bacillus.

並列關鍵字

silica nanoparticle ； coumarin ； gold nanoparticle ； lectin

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