具標靶含磷酸根代謝物及凝集素的蛋白質官能化金奈米探針之合成與應用

蛋白質官能化金奈米粒子和金奈米團簇等金奈米材料是目前應用在分析化學方法開發上被廣為使用的探針。然而，因為一般具功能化的蛋白質價格昂貴且取得不易，因此也影響到蛋白質官能化探針使用的普及性。而價格親民的雞蛋蛋白富含蛋白質，因此已被選為製造蛋白質官能化金奈米探針的首選。蛋白質官能化金奈米探針可以一鍋反應進行合成，生成具有雞蛋白蛋白質官能基修飾的金奈米粒子以及金奈米團簇。亦即可用雞蛋白蛋白質來做為合成金奈米探針的還原劑以及包覆劑，而合成好的金奈米探針表面大部分會以修飾上佔雞蛋白蛋白質含量為百分之五十四的雞蛋卵白蛋白為主。目前大部分的研究都著重於利用雞蛋白蛋白質官能化的金奈米探針的光學性質為應用方向。然而，蛋卵白蛋白上的醣基配體可以用做探針用以標靶如蛋白質毒素等凝集素之類的目標物。例如，蓖麻中的蓖麻毒蛋白和大腸桿菌O78：H11產生的熱不穩定腸毒素等蛋白質毒素的B亞基可以和半乳糖(14)葡萄糖基的配體具有辨識能力。由於發展快速偵測此類毒素的方法有相當重要性，因此本論文利用這些金奈米探針發展出可快速檢測這兩種毒素的方法。此外，含磷酸根的代謝物可當作用於指示健康狀況的生物指標，所以本論文的第一部分使用會放射紅色螢光以及粒徑大小在2.1 ± 0.3 nm左右的雞蛋白蛋白質官能化金奈米團簇為感測具磷酸根的腺苷-5'-三磷酸和焦磷酸等代謝物的探針。感測的機制為利用銅離子會抑制雞蛋白蛋白質官能化金奈米團簇的螢光放光的特性，但由於銅離子會和磷酸根有高度親和作用，因此加入具磷酸根代謝物後，雞蛋白蛋白質官能化金奈米團簇的螢光會被重新打亮，此方法對腺苷-5'-三磷酸的偵測極限在19 μM左右，而對焦磷酸偵測極限則可低至5 μM。在論文的第二部分，我應用了雞蛋白蛋白質官能化金奈米團簇上的醣基配體(半乳糖(14)葡萄醣基)為辨識蓖麻毒蛋白B亞基的探針，並使用螢光光譜儀和基質輔助雷射脫附游離質譜儀為偵測工具，結果顯示此法對蓖麻毒蛋白B亞基的偵測極限可低至8 nM左右。在論文的第三部分則進一步使用粒徑為9.1±1.3 nm的雞蛋白蛋白質官能化金奈米粒子為標靶如伴刀豆球蛋白A、香蕉凝集素，及蓖麻毒蛋白B亞基等多種凝集素的親和探針，並使用基質輔助雷射脫附游離質譜法為偵測方法，結果顯示對於這些凝集素的偵測極限可低至nM。除此之外，由於雞蛋白蛋白質上的醣基結構上僅具有相當有限的半乳糖配體，因此為了增加雞蛋白蛋白質表面上半乳糖的數量以提高所製備的奈米探針對標靶具半乳糖配體受體的凝集素之捕獲能力，雞蛋白蛋白質先使用美拉德反應進行乳糖(半乳糖(14)葡萄醣基)修飾反應以增加雞卵白蛋白上半乳糖的數量，由反應結果得知雞卵白蛋白上半乳糖數目平均增加了約17個左右。進一步使用所得的雞卵白蛋白產物合成了粒徑為~8.72.0 nm的金奈米粒子。實驗結果顯示利用此半乳糖修飾的雞卵白蛋白所合成的金奈米粒子可對熱不穩定腸毒素B亞基具良好的辨識力，也據此發展了靈敏的比色偵測法，利用肉眼可辨識的偵測限可低至31 nM左右，而使用基質輔助雷射脫附游離質譜法做為檢測方法時，則偵測限可再低至4 nM左右，這些結果均較現有方法所得到的偵測限還低。因此，我們相信在本論文所提出的方法應有可用於實際應用的潛力。

關鍵字

金奈米團簇；金奈米粒子；具標靶含磷酸根代謝物；凝集素；雞蛋白；蛋白質

並列摘要

Protein functionalized Au nanoprobes, i.e. Au nanoparticles (NPs) and Au nanoclusters (NCs), have been widely used in the method development in analytical chemistry. Nevertheless, high cost and availability of functional proteins have limited the popularity of such functional nanoprobes. Chicken egg whites (CEW) derived from low-cost chicken eggs are protein rich natural-products. Thus, CEW proteins have become a choice when functionalizing Au nanoprobes (Au@CEW). Au@CEW NPs and Au@CEW NCs can be easily generated from one-pot reactions. Namely, CEW proteins are used as reducing and capping agents in the generation of Au nanoprobes. The generated nanoprobes are mainly functionalized by chicken ovalbumin (COA), which dominates ~54% of CEW proteins. Most of the existing studies using Au@CEW nanoprobes are focused on the use of the optical properties of the nanoprobes. Nevertheless, the glycan ligands on COA can be used as the probes to target theircorresponding target species, i.e. lectins, including some protein toxins such as ricin and heat labile enterotoxin (LT). Ricin and LT generated fromRicinus communis and Escherichia coli O78:H11, respectively. The B subunits of these two lectins, ricin B and LT-B, possess binding moieties withGalβ(14)Glc ligands. Owing to the importance for developing rapid detection methods for such toxins, these two lectins are selected as the main targets in this study. In addition, phosphate-containing metabolites are important biomarkers for indication of health conditions. Thus, in the first part of this thesis, Au@CEWNCs with reddishphotoluminescenceand the particle size of ~2.1±0.3 nmwere used as sensing probes for phosphate-containing metabolites, i.e. adenosine-5′-triphosphate (ATP) and pyrophosphate (PPi). The sensing mechanism was based on the use of the high affinity arising between Cu2+ and phosphate contain-metabolites. Given that the as-prepared Au NCs were quenched in the presence of Cu2+, the photoluminescence of Au@CEW NCs was restored in the presence of phosphate–containing species. The limits of detection (LODs)of the sensing method toward ATP and PPi were ~19 and ~5 μM, respectively. In the second part of the dissertation,I employed the glycan ligands, i.e. Galβ(14)GlcNAc, on the surface of the as-generated Au@CEW NCsas probes to interact with ricin B, while fluorescence spectroscopy and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) were used as the detection tools. TheLOD toward ricin B was as low as ~8nM. In the third part of this thesis, Au@CEW NPs (particle size: 9.1 ± 1.3 nm) dominated by Au@COANPs were used as affinity probes for multiple-lectins including concanavalin A (Con A), banana lectin (BanLec), and ricin B based on the interaction between the glycan ligands on COA with the glycan moieties on these lectins. The LODs towardCon A, banana lectin, and ricin B were in low nM rangewhen using MALDI-MS as the detection tool. CEWproteins only contain a limited number of galactose ligands on their glycan structure. Thus, to increase the number of galactose on the surface of CEW proteins can improve the trapping capacity of the as-prepared nanoprobes toward their target lectins. Thus, CEW proteins were reacted with -lactose (Galβ(14)Glc) through the Maillard reaction to increase the number of galactose on COA. The resultant product with additional ~17 lactose was used to generate lac-COA capped Au NPs (Au@lac-CEW NPs) (particle size: ~8.7  2.0 nm) through one-pot reactions. The results showed that the as-prepared Au NPs are suitable colorimetric sensing nanoprobes for LT-B.The LOD toward LT-B by the naked eyes was ~31nM, while it was as low as ~4 nM when using MALDI-MS as the detection tool. The obtained LOD is lower than those obtained from the existing methods. Thus, the proposed methodsmay have potential to be used in real world applications.