奈米酵素之合成與應用於蛋白質、重金屬和
陰離子檢測

奈米酵素除了具有催化特定反應，它還擁有獨特特性如具有大表面積可以進一步的修飾辨識分子等。在本研究中，我們合成奈米酵素並應用於生物分子、重金屬離子和陰離子檢測。論文分為四章，第一章為回顧奈米酵素的種類、發展和應用。第二章是利用鉍離子 (Bi3+)和金之間的親金作用力 (aurophilic interaction)，使得Bi3+可以沉積於金奈米粒子 (gold nanoparticles, Au NPs)表面來合成鉍–金奈米粒子 (Bi–Au NPs)。此種奈米酵素具過氧化酶 (peroxidase)活性，可以催化過氧化氫 (H2O2)和Amplex Red (AR)反應而生成具有螢光的試鹵靈 (resorufin)，藉此來偵測H2O2含量。進一步我們將纖維蛋白原 (fibrinogen, Fib)修飾在Bi–Au NPs後 (Fib-Bi–Au NPs)，在凝血酶 (thrombin)作用下，其表面的Fib會形成網狀結構的纖維蛋白 (fibrin)，而使得其peroxidase活性降低，藉此可用於偵測thrombin，並可進一步用作抗凝血藥的篩選。於第三章中，我們將不同的金屬離子 (銀離子，Ag+；鉍離子，Bi3+；鉛離子，Pb2+；鉑離子，Pt4+；汞離子，Hg2+) 沉積於Au NPs，使其具有不同的酵素活性 [如過氧化酶 (peroxidase)、氧化酶 (oxidase) 和過氧化氫酶 (catalase)]，並進一步以金屬離子 (Hg2+/Bi3+、Pt4+/Hg2+、Pb2+/Hg2+和Ag+/Bi3+)當作輸入，Au NPs的酵素活性當做輸出，構建出OR、AND、INHIBIT和XOR邏輯閘 (logic gate)。第四章介紹用我們建構的兩個logic gate [Pt4+/Pb2+(AND)Au NPPOX和Bi3+/Hg2+(INHIBIT)Au NPPOX]來選擇性的偵測Pb2+和Hg2+。當Au NPs在Pt4+和Pb2+共存下，其會藉由Pt和Pb原子/離子之間的金屬作用 (metallophilic interaction))及Au和Pb/Pt原子/離子之間的親金作用(aurophilic interaction)，使得整個奈米酵素的peroxidase活性增加 (AND logic gate)，藉此來偵測Pb2+。在Bi3+的存在下，Au NPs的表面價態會改變(Au+/Au0)，而使得整個奈米酵素的peroxidase活性增加。但是當Bi3+和Hg2+共存下，較強的金汞齊作用力會使得整個奈米酵素的peroxidase活性下降 (INHIBIT logic gate)，藉此來偵測Hg2+。另外我們用產生紅色的resorufin和氧氣氣泡來構建出邏輯迴路(logic circuit)，並可用視覺來判別有無Pb2+和Hg2+的存在。第五章中，我們利用一步合成的方式合出良好分散性且具有peroxidase催化活性的非晶相氫氧化/氧化鈷修飾之氧化石墨烯 (cobalt hydroxide/oxide-modified graphene oxide, CoOxH-GO)。發現氰離子 (CN−)會顯著抑制CoOxH-GO奈米複合物的催化活性，以此構建出CN−探測器來檢測水樣和實驗室廢液中的CN−濃度。另外我們通過在帶正電荷的多孔尼龍膜 (positively charged and porous nylon membrane, N+M)上製備CoOxH-GO薄膜進而製造出了一種紙基的CoOxH-GO/N+M探針，此探針運作原理是CN−會抑制膜上CoOxH-GO催化H2O2氧化AR形成紅色resorufin的活性，故可由視覺來判斷CN−的濃度。隨著CN−濃度的增加，膜的紅色強度降低，故可以容易地以肉眼檢測濃度達奈莫耳 (nanomolar)等級的CN−。

關鍵字

奈米酵素；金屬離子；凝血酶；氰離子；邏輯閘

並列摘要

Nanozymes can catalyze specific reactions. In addition, nanozymes possess unique features such as large surface area for further conjugation of multiple molecules for biorecognition. In this study, we synthesized nanozymes and applied them in the detection of biomolecules and heavy metal ions. This dissertation is structured in four chapters. In Chapter 1, the background of nanozymes including their classification, developments, and applications is reviewed. In Chapter 2, Bismuth–gold nanoparticles (Bi–Au NPs) were prepared through self-deposition of bismuth ions (Bi3+) on Au NPs as a result of their aurophilic interactions. Bi–Au NPs possess intrinsic peroxidase-like activity, which in the prescence of hydrogen peroxide (H2O2) could catalytically oxidize Amplex Red (AR) to produce highly fluorescent resorufin, applicable for the detection of H2O2. Then, the as-prepared Bi–Au NPs were further modified with fibrinogen (Fib) to form fibrinogen-adsorbed Bi–Au NPs (Fib-Bi–Au NPs). In the presence of thrombin, soluble fibrinogen converts into insoluble strands of fibrin on Bi–Au NPs' surfaces, causing a decrease in peroxidase-like activity of Bi–Au NPs, which can be employed in the detection of thrombin and further screening of anticoagulant drugs for thrombin. In Chapter 3, we have successfully demonstrated that Au NPs can be programmed to regulate their peroxidase (POX)-, oxidase (OX)- and catalase (CAT)-like activities through deposition of various metal ions (Ag+, Bi3+, Pb2+, Pt4+, Hg2+). Furthermore, we used metal ions (i.e., Hg2+/Bi3+, Pt4+/Hg2+, Pb2+/Hg2+, and Ag+/Bi3+, respectively) as inputs and the enzyme-like activity of the Au NPs as the output for the construction of OR, AND, INHIBIT, and XOR logic gates. In Chapter 4, we used as constructed “Pt4+/Pb2+(AND)Au NPPOX” and “Bi3+/Hg2+(INHIBIT)Au NPPOX” logic gates for the selective detection of Pb2+ and Hg2+. When Pt4+ and Pb2+ co-exist, strong metallophilic interactions (between Pt and Pb atoms/ions) and aurophilic interactions (between Au and Pb/Pt atoms/ions) result in significant enhanced peroxidase-like activity (AND logic gate) which were then employed in the detection of Pb2+. High peroxidase-like activity of Au NPs in the presence of Bi3+ is a result of the various valence (oxidation) states of Bi3+ and Au (Au+/Au0) atoms on the nanoparticle’s surface. When Bi3+ and Hg2+ co-exist, strong Hg–Au amalgamation results in a large decrease in the peroxidase-like activity (INHIBIT logic gate) of the Au NPs which were then employed in the detection of Hg2+. In addition, an integrated logic circuit based on the color change (formation of reddish resorufin product) and generation of O2 bubbles from these two probes has been constructed, allowing visual detection of Pb2+ and Hg2+ in aqueous solution. In Chapter 5, we used a simple one-step synthesis of well-dispersed amorphous cobalt hydroxide/oxide-modified graphene oxide (CoOxH-GO) possessing peroxidase-like catalytic activity. Interestingly, cyanide ions (CN–) significantly inhibited the catalytic activity of CoOxH-GO nanocomposite, which allows for the construction of a probe for the detection of CN– in water samples and laboratory wastes. We fabricated a paper-based CoOxH-GO probe for the visual detection of CN– by preparing a thin film of CoOxH-GO on a positively charged and porous nylon membrane (N+M), which operates on the principle that CN– inhibited CoOxH-GO catalyzed H2O2 mediated oxidation of AR to reddish resorufin on membrane. The intensity of the red color of membrane decreases with the increase in CN– concentration, which can be easily observed with naked eye in nanomolar concentrations.

並列關鍵字

nanozyme ； metal ion ； thrombin ； cyanide ion ； logic gate

參考文獻

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[3] Nagel, Z. D.; Klinman, J. P. Tunneling and Dynamics in Enzymatic Hydride Transfer. Chem. Rev. 2006, 106, 3095–3118.

[4] Bruice, T. C.; Benkovic, S. J. Chemical Basis for Enzyme Catalysis. Biochemistry 2000, 39, 6267–6274.

[5] Raynal, M.; Ballester, P.; Vidal-Ferran, A.; van Leeuwen, P. W. N. M. Supramolecular Catalysis. Part 2: Artificial Enzyme Mimics. Chem. Soc. Rev. 2014, 43, 1734–1787.

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奈米酵素之合成與應用於蛋白質、重金屬和陰離子檢測

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