合成奈米材料於類酵素生化感測器與水裂解光催化劑之應用

Synthesis of Nanomaterials as Enzyme Mimicking Biosensors and Water Splitting Photocatalysts

王成一(Chen-I Wang)

指導教授 ： 張煥宗

國立臺灣大學/理學院/化學研究所/博士(2013年)

https://doi.org/10.6342/NTU.2013.00730

摘要

本論文主要是利用合成的奈米材料來偵測樣品中重金屬離子(汞、鉛)、生化分子乙醯膽鹼(Acetylcholine, ACh)與DNA濃度，且應用奈米材料之光觸媒活性進行水裂解產氫(water splitting)。論文分成七個章節，第一章內容包括介紹以奈米材料作為感測器的背景、以類酵素活性的概念設計之奈米材料生物感測器，及半導體型光觸媒應用於綠色能源的介紹。第二章為利用金奈米粒子(Gold nanoparticles, Au NPs)之過氧化酶-類酵素催化活性(peroxidase-mimicking catalytic activity)，來建立具高靈敏度和選擇性之重金屬離子－汞(Mercury, Hg)與鉛(Lead, Pb)螢光感測器。當有過氧化氫(H2O2)存在時，Hg(或Pb)離子與Au NPs所形成合金(alloys)對過氧化酶之受質－Amplex UltraRed(AUR)具有極高的催化活性，此催化機制可應用於Hg與Pb離子濃度的偵測。對兩者的偵測極限(雜訊比=3)和線性範圍分別是(4 nM；0.05–1 μM)和(4 nM；0.05–5 μM)，其偵測效果優於其他由Au NPs所製備的奈米材料。此系統亦成功應用於湖水與血液樣品中目標重金屬之偵測。第三章為利用具有過氧化酶-類酵素催化特性之金/銀奈米粒子(Au/Ag NPs)，以建立具高靈敏度和選擇性之乙醯膽鹼(Acetylcholine, ACh)螢光感測器。利用Au/Ag NPs於H2O2存在時對AUR具有極高的催化活性，結合乙醯膽鹼酶(Acetylcholinesterase, AChE)與膽鹼氧化酶(Choline oxidase, ChOx)將ACh氧化為H2O2之反應機制，可應用於樣品中ACh濃度的偵測。對於水溶液中ACh之偵測極限(雜訊比=3)和線性範圍為0.21 nM和1–100 nM，其偵測效果優於其他光學感測器與電化學分析方法，也顯示對ACh偵測具有高度選擇性。此系統亦成功應用於血漿與血液樣品中ACh之偵測。第四章為利用活性氧化物(Reactive oxygen species, ROS)可以熄滅碳奈米/石墨烯(Carbon-dots@Reduced graphene oxide, C-dots@RGO)螢光的特性，來建立具高靈敏度和選擇性ACh之螢光感測器。ROS來自於ACh氧化(經AChE與ChOx反應)所生成之H2O2，而C-dots@RGO對於ROS的產生亦扮演重要角色。對水溶液中ACh之偵測極限(雜訊比=3)和線性範圍為30 pM和0.05–10 nM。此系統不僅成功應用於血漿與全血液樣品中ACh之偵測，更提供ACh偵測的另一個選擇。第五章為利用亞甲基藍(Methylene blue, MB)作為C-dots@RGO螢光的熄滅劑(Quencher)，來驗證對DNA偵測的實用性。相較於單股DNA(Single-strand DNA, ssDNA)，MB與雙股DNA(Double-strand DNA, dsDNA)展現出更強的作用力與選擇性，使C-dots@RGO螢光能更有效地回復(turn-on)，此機制可應用於目標DNA的偵測。對於抑癌基因BRCA1之偵測極限(雜訊比=3)和線性範圍為14.6 nM和25–250 nM。而固相偵測平台的成功使用，更證明其在高通量分析的應用價值。第六章為利用量子點(Quantum dots, QDs)來製作光敏化電極(TiO2/CdZnS/CdZnSe)並應用於水裂解產氫。當使用太陽照度為100 mW cm-2的光照射時，TiO2/CdZnS/CdZnSe電極提供的光電流密度與產氫效率分別為9.7 mA cm-2(電位為-0.9 V，飽和甘汞電極為參考電極)與7.3 ± 0.1%(水裂解產氫速率為172.8 mmol•h-1•g-1)。由入射光子電流轉換效率(Incident photon to current conversion efficiency, IPCE)的頻譜顯示，在500 nm處的光電轉換量子效率值為80%。該電極展現極佳的產氫效率，相信未來有極大的潛力應用於綠色能源的開發上。最後一章則是總結前面章節的結果，並說明其未來的發展方向。

關鍵字

類酵素 ； 重金屬離子 ； 乙醯膽鹼 ； 碳奈米點 ； 石墨烯 ； DNA突變 ； 水裂解

並列摘要

This thesis focuses on the synthesis of nanomaterials for the detection of heavy metal ions (mercury and lead), biomolecules (acetylcholine (ACh) and DNA), and photoelectrochemical water splitting. My thesis is divided into seven parts. Chapter one introduces the background of nanomaterials as sensors with the concept of enzyme mimics for fluorescence biosensing. An introduction to semiconductor photocatalysts with unique properties offered by photocatalytic nanomaterials for the production of green energy has also been provided. A fluorescence assay for the highly sensitive and selective detection of Hg2+ and Pb2+ ions using a Au NP-based probe, which exhibits peroxidase-mimicking catalytic activity in the H2O2-mediated oxidation of Amplex UltraRed (AUR) has been described in chapter two. The corresponding limit of detection (LOD) values (S/N = 3) of Hg2+ and Pb2+ ions detection when using Au NP-based probe are 4 nM (linear range = 0.05–1 μM) and 4 nM (linear range = 0.05–5 μM) respectively, which is better than other ligand–Au NP conjugates that are being used in the detection of Hg2+ and Pb2+. The practicality of the probe has been validated through determination of the concentrations of Hg2+ and Pb2+ ions in a lake water and blood sample. The third chapter describes the fluorescent assay for the detection of ACh based on enzyme mimics of Au/Ag NPs. The sensing strategy involves reacting ACh with acetylcholinesterase (AChE) and choline oxidase (ChOx) to produce betaine and H2O2, which reacts with AUR in the presence of bimetallic NPs catalyst to form a fluorescent product. The sensing system exhibits excellent sensitivity and selectivity for ACh than other optical biosensors and electrochemical detection methods. This probe provides the detection of ACh over a linear range of 1–100 nM, with an LOD (S/N = 3) of 0.21 nM. The practicality of this assay has been validated by the determination of concentrations of ACh in plasma and blood samples. The fourth chapter describes a PL-quenching assay for the detection of ACh using carbon-dots@reduced graphene oxide (C-dots@RGO). AChE and ChOx converts ACh to H2O2 that generates the ROS. The as-produced ROS induces PL quenching of the C-dots@RGO through an etching process. The results revealed that RGO plays a critical role in ROS production. The PL intensity of the C-dots@RGO is inversely proportional to the concentration of ACh over a range of 0.05–10 nM, with a LOD (S/N = 3) of 30 pM. The practicality of this assay has been assessed by the determination of the concentrations of ACh in plasma and blood samples. This study opens an avenue for the detection of various analytes using C-dots@RGO in conjunction with different enzymes, substrates, and/or inhibitors. The fifth chapter deals with the practicality of this stable C-dots@RGO for the detection of DNA, based on analyte-induced “turn-on” PL when using methylene blue (MB) as a quencher. Relative to single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) intercalates more strongly with MB and interacts more weakly with RGO; as a result, the combination of the C-dots@RGO, MB, and a DNA probe was selective for perfectly matched DNA over mismatched DNA. Using this probe, we detected the tumor suppressor gene BRCA1 over concentrations ranging from 25 to 250 nM, with a LOD (S/N = 3) of 14.6 nM. The successful use of the C-dots@RGO probe for the detection of DNA in a solid phase suggests its potential for application in high-throughput analysis. In the sixth chapter, TiO2/CdZnS/CdZnSe electrodes have been prepared and employed for photochemical water splitting. The TiO2/CdZnS/CdZnSe electrodes have light absorption over the wavelength 400-700 nm and a band gap of 1.87 eV. Upon one sun illumination of 100 mW cm-2, the TiO2/CdZnS/CdZnSe electrodes provides a significant photocurrent density of 9.7 mA cm-2 at -0.9 V vs. a saturated calomel electrode (SCE). Incident photon to current conversion efficiency (IPCE) spectrum of the electrodes displays a maximum IPCE value of 80% at 500 nm. Moreover, the TiO2/CdZnS/CdZnSe electrodes prepared from three different batches provide a remarkable photon-to-hydrogen efficiency of 7.3 ± 0.1% (the rate of the photocatalytically produced H2 by water-splitting is about 172.8 mmol•h-1•g-1), which is the most efficient quantum dots based photocatalyst used in solar water splitting. The last chapter summaries the whole results from chapter two to six with future prospects.

並列關鍵字

Enzyme-mimics ； Heavy metal ion ； Acetylcholine ； Carbon-dots ； Reduced graphene oxide ； DNA mutation ； Water splitting

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