利用兩相性多醣體包覆之量子點為偵測細菌含量之探針

Abstract

量子點 (QD) 擁有傳統有機染料與螢光蛋白所沒有之敏感性與穩定性，在科學和技術上有其特殊的優勢。在生物醫學領域，量子點已逐漸被應用於生物分子螢光標示、DNA鑑定與疾病診斷上。在本研究中，先用過碘酸鈉 (NaIO4) 氧化幾丁聚醣而得到醛基化幾丁聚醣。接著在醛基化幾丁聚醣上鍵結直鏈烷基胺，進而合成具有疏水和親水端的兩相性幾丁聚醣。另外用氫氧化鈉使海藻酸丙二酯 (PGA) 在與直鏈烷基胺混合時水解，直鏈烷基胺會與PGA分子鏈上的丙二醇基產生置換反應，進而形成具有疏水和親水端的兩相性海藻酸丙二酯（兩相性PGA）。將上述兩相性多醣體藉由疏水交互作用 (hydrophobic interaction) 包覆LumidotTM 量子點並使其從有機相轉至水相中均勻分散，發現幾丁聚醣接枝正十六胺 (Chitosan-C16) 有較佳包覆量子點成功率與更高的螢光發光效率，故選用Chitosan-C16做為後續研究之材料。進一步研究兩相性幾丁聚醣，發現醛基化幾丁聚醣上的醛基化GlcN只有14%能與正十六胺鍵結，但已經足以包覆量子點。本研究中也嘗試自行製備硒化鎘�硫化鋅量子點。在製備核層（硒化鎘）的反應時間控制下，適於合成發黃色螢光或波長更長的量子點。以兩相性幾丁聚醣包覆自製硒化鎘�硫化鋅量子點並分散於0.1 M MES-NaOH buffer中，小於pH值5.3的酸鹼度是較適用的範圍。由於兩相性幾丁聚醣包覆之自製量子點粒子表面帶有胺基，擁有帶正電的介面電位；細菌細胞膜表面帶有負電的介面電位，且因具有帶羧基的蛋白質，量子點可與細菌表面以靜電吸引力結合或以EDC做偶合聯結。本研究嘗試結合量子點與大腸桿菌細胞膜，並以此測定菌液內之菌量。雖然實驗並未成功，但如果能在材料的製備和材料與細菌結合方式的部分進行改進的話，也許還有再嘗試的可能。

Quantum dot (QD) has better sensitivity and stability compared with traditional organic dye and fluorescent protein. It has special advantages in science and technology. In the biomedical field, QD has been increasingly used in fluorescent labeling of biological molecules, DNA identification and disease diagnosis. In this study, we first used sodium periodate (NaIO4) to oxidize chitosan for the formation of aldehyde chitosan. Then, alkyl chains were bonded to aldehyde chitosan to form amphiphilic chitosan which has both hydrophobic and hydrophilic features. In addition, alkyl amines were grafted with propylene glycol alginate (PGA) by reacting with propylene glycol groups in the presence of sodium hydroxide. The alkylated PGA also has amphiphilic characteristics. These two amphiphilic polysaccharides could make LumidotTM QDs converted from the organic phase into aqueous phase dispersion by hydrophobic interaction. Our results showed that LumidotTM QDs capped with chitosan conjugated with 1-hexadecylamine revealed much better quantum yield than the ones made by amphiphilic PGA. This is probably due to the merit of positively charged chitosan resulting in robust electrostatic interaction on the surface of LumidotTM QDs. Although aldehyde chitosan has only 14% of aldehyde GlcN grafted with 1-hexadecylamine, yet it is sufficient to encapsulate LumidotTM QDs. This study also attempted to prepare CdSe/ZnS QDs. It was suitable for yellow and longer wavelength fluorescence QDs by reaction time control of the nuclear layer (CdSe). CdSe/ZnS QDs were encapsulated by amphiphilic chitosan dispersed in 0.1 M MES-NaOH buffer. If pH is higher than 6, the dispersion of particles clearly becomes unstable. However, the stability can be maintained if pH is less than 5.3. Since QDs encapsulated with amphiphilic chitosan have positive charges, they can attach to negatively charged bacterial surface by electrostatic attraction, or by EDC coupling to cell membrane’s proteins with carboxyl groups. In this study, QDs were tried to label on E. coli for detecting the amount of bacteria in suspension. Although the experiment did not succeed, if QD synthesis and the method of QD-bacteria association can be improved, it remains worthwhile for further exploring.