新型金屬奈米粒子與螢光探針分子之開發並應用於抗菌材料和螢光感測器

Abstract

近年來，奈米材料被應用於許多領域中，本論文旨在探討所開發出的多肽鍵高分子水膠自組裝行為。藉由討論銀粒子對於此多肽鍵水膠成膠過程的影響，並分別探討1. 此多肽鍵水膠在螢光化學感測器之性能表現。2. 此多肽鍵水膠與官能機化之金奈米粒子抗菌力探討。3. 此多肽鍵水膠金屬離子檢測能力。4. 此多肽鍵水膠對於藥物克倫特羅之檢測能力。 本研究第一章節中，我們優先探討銀奈米粒子對於此多肽鍵水膠成膠過程有何影響。從實驗結果我們發現到當銀奈米粒子被此多肽鍵水膠包覆時，此多肽鍵水膠具有抗菌的效果。我們也接著探討此多肽鍵水膠對於官能機化的金奈米粒子應用於螢光化學感測器，深入研究其發光機制。發現此多肽鍵水膠對於不同金屬離子具有度辨識性與辨識能力，對於重金屬污染的檢測具有一定的應用潛力。我們也進一步的發現，此多肽鍵水膠對於藥物克倫特羅也具有檢測能力。 本研究第二章節中，我們透過液相方法合成出具有生物相容性且含有 N-terminally 2-(Naphthalen-6-yl) acetic acid 保護基的 Nap-FFC 多肽，藉由討論此 Nap-FFC 多肽的自組裝過程來了解其成膠能力。在此研究中，我們發現含銀奈米粒子磺化的 Nap-FFC 多肽所形成的奈米複合材料 AgNPs @ Nap-FFC 經過硼氫化鈉還原反應後能夠獲得分散性極佳的銀奈米粒子，主要是因為 Nap-FFC 在反應過程中扮演安定劑的角色。更有趣的是我們也發現到 AgNPs @ Nap-FFC 此複合材料對於耐甲氧西林金黃色葡萄球菌還有動桿菌皆具表現出抗菌作用，尤其是對 HeLa 細胞顯示有著極佳的生物相容性。因此，在本研究中所製備出的 AgNPs @ Nap-FFC 奈米複合材料對於未來的傷口抗菌敷料與生物醫學相關的應用是具有一定的潛力。 本研究第三章節中，我們透過一鍋式反應合成出結構簡單的希夫鹼衍生物 PE。我們也透過紫外光可見光光譜、螢光光譜儀、穿透式顯微鏡、動態光散射儀成功地發現PE具有特殊的聚集誘導發光現象。其中有趣的是我們發現透過加入少量的 CH3CN，PE即可和Fe3+，Cr3+ 和Al3+離子產生 PE-PE* 準分子，藉此準分子聚集誘導發光現象放出螢光時檢測Fe3+, Cr3+ 和Al3+離子，同時我們也由傅立葉轉換紅外線光譜、質譜儀還有核磁共振光譜計算 PE:金屬離子化學劑量為2:1，在通過計算標準偏差與線型回歸找出偵測極限與解離常數同時由密度泛函理論計算出各金屬離子複合物之量子產率。最重要的是我們發現到此聚集誘導發光現象可用來檢測 Raw264.7 活體細胞中是否含有Fe3+, Cr3+ 和Al3+金屬離子。 本研究第四章節中，我們透過簡單的有機合成反應合成出結構簡單的希夫鹼衍生物 AP。在此也透過 PET 基底成功地發現 AP 也具有特殊的聚集誘導發光現象。我們也由螢光滴定實驗與質譜儀 AP:金屬離子化學劑量為1:1。有趣的是我們發現 AP 可和 Cu2+ 離子產生 AP-Cu2+ 錯合物。同時，在通過計算標準偏差與線型回歸找出偵測極限與解離常數並藉由密度泛函理論計算出量子產率。更重要的是我們發現到此聚集誘導發光現象可有效得用來檢測 Raw264.7 活體細胞中是否含有 Cu2+ 金屬離子。 本研究第五章節中，我們直接藉由以 NaBH4 還原劑來還原接上 Glu 與 PE 的奈米金粒子進而得到超穩定的奈米金粒子；直接與克倫特羅（CLB）的比色裸眼法進行聚集誘導發光現象測定。實驗中令人印象深刻的是， PE-Glu-AuNP 在室溫下其穩定性可達6個月，通過 PE-Glu-AuNP 的 CLB 的比色測定已經在 pH 為 5的情況下在 300 nM 開始檢測克倫特羅，直接透過肉眼觀察反應與我們了解到通過計算標準偏差與線型回歸找出的滴定結果相近。通過穿透式顯微鏡與動態光散射儀我們了解到 PE-Glu-AuNPs 原先大小為 84.8 ± 52.3 nm，與克倫特羅滴定後觀察到聚集誘導發光現象而此時 PE-Glu-AuNPs:CLB 大小為84.8 ± 52.3 nm。實驗過程中 PE-Glu-AuNPs 的 CLB 檢測不受任何其它干擾物的影響， PE-Glu-AuNPs 對克倫特羅的偵測選擇性也在尿液樣品研究中得到了很好的證明。

The prime objective of this thesis is to design and synthesis of short self-assembled peptide-based hydrogels mineralized with silver nanoparticles, fluorescent chemosensor probes and dual functionalized gold nanoparticles towards antibacterial studies, metal ions and clenbuterol detections, respectively. The Chapter 1 describes the significance of silver nanoparticle entrapped short peptide in antibacterial applications. Then, highlighted significance of designed fluorescent chemosensors and functionalized gold nanoparticles and mechanism of signal transduction of several probes in metal ions and clenbuterol detections. In chapter 2, we have focused on a biocompatible N-terminally 2-(Naphthalen-6-yl) acetic acid (Nap) protected Phe-Phe-Cys peptide (Nap-FFC) synthesized by liquid phase method. This Nap-FFC peptide was used to design supramolecular hydrogel via self-assembling process. In addition, the mineralized silver based Nap-FFC nanocomposites (AgNPs@Nap-FFC) has been prepared by utilizing sodium borohydride, where Nap-FFC acts as scaffolds for the mineralization of silver nanoparticles (AgNPs) as well as plays the key role of stabilizing agents for the synthesized AgNPs. As prepared, AgNPs@Nap-FFC nanocomposites have a high monodispersity, long term stability, and functional flexibility in comparison to other AgNPs based nanocomposites. More interestingly, AgNPs@Nap-FFC has exhibited antibacterial effect against both Gram-positive (Methicillin-resistant Staphylococcus aureus), Gram-negative (Acinetobacter baumannii) bacteria and showed favorable biocompatibility to HeLa cells. Hence, this study implies that AgNPs@Nap-FFC based nanocomposites prepared by simple and cost-effective manner, will be more effective to be used in future antibacterial wound dressing and biomedical application without side effects. Chapter 3 discusses about a simple pyrene based schiff base derivative 2-((pyren-1-ylmethylene) amino) ethanol (PE) has been synthesized via one-pot reaction and its nano-aggregation induced emission (AIEE) characteristics were reported through UV/PL and supported by TEM and DLS studies. Interestingly, PE in CH3CN revealed the naked eye and fluorescence turn-on sensing responses towards Fe3+, Cr3+ and Al3+ ions, via PET and PE-PE* excimer formation. The 2:1 stoichiometry of sensor complexes PE---M3+ (M = Fe/ Cr/ Al) were calculated from job’s plots based on UV-Vis absorption titrations and supported by FTIR and ESI (+Ve) mass analysis. In addition, the binding sites of sensor complex PE---M3+ were well established from the 1H NMR titrations. Furthermore, the fluorescence reversibility of PE---M3+ complexes, via consequent additions of M3+ ions and PMDTA, respectively. The detection limits (LODs) and the association constants (Kas) values of PE---M3+ complexes were calculated by standard deviation and linear fittings. Furthermore, the quantum yield (Φf), TEM analysis, pH effect and density functional theory (DFT) studies were investigated for the PE---M3+ sensor complexes. More importantly, confocal fluorescence microscopy imaging in Raw264.7 cells showed that PE could be used as an effective fluorescent probe for detecting Fe3+, Cr3+ and Al3+ ions in living cells. In Chapter 4, we have demonstrated a novel anthracene and pyridine comprising schiff base derivative 2-(2-(anthracen-9-ylmethylene)hydrazinyl)pyridine (AP) has been synthesized via one-pot reaction and its fluorescent “OFF-ON” detection of Cu2+ ions, via PET based mechanism was reported firstly. The 1:1 stoichiometry of AP---Cu2+ sensor complex was calculated from job’s plot based on PL titrations and supported by ESI (+Ve) mass analysis. In addition, the binding sites of sensor conjugate AP---Cu2+ was buttressed by the 1H NMR titration. The detection limit (LOD) and association constant (Ka) of AP---Cu2+ complex was scrutinized by standard deviation and linear fittings. Furthermore, quantum yield (Φf), SEM analysis, pH effect, FTIR interpretation and density functional theory (DFT) studies were investigated for the AP---Cu2+ sensing conjugate. More decisively, confocal fluorescence microscopy imaging from Raw264.7 cells indicated that AP could be used as an effective fluorescent probe for the analysis of Cu2+ ions in living cells. Chapter 5 reports, a new ultra stable dual functionalized gold nanoparticles (AuNPs) were developed and utilized in colorimetric naked eye assay of clenbuterol (CLB). The simple NaBH4 reduction method has been employed to dual functionalize the AuNPs by glutamic acid (Glu) and polyethylenimine (PE). Impressively, the above gold nanoparticles (PE-Glu-AuNPs) were found to be extremely stable at room temperature up to six months. Remarkably, the colorimetric assay of CLB by PE-Glu-AuNPs has been initiated at 300 nM at pH 5 and visualized through naked eyes visualized. Moreover, the UV-vis titration based sub nanomolar CLB detection has been estimated from standard deviation and linear fittings. Further to note, the aggregation induced mechanism on CLB detection was well verified through Transmission electron microscopy (TEM) and dynamic light scattering (DLS) studies. The DLS studies calculated the initial size of PE-Glu-AuNPs as 12.8 ± 8.6 nm, which was later aggregated as 84.8 ± 52.3 nm with CLB. Additionally, the TEM studies supports the above aggregation induced CLB detection. Demonstrably, the PE-Glu-AuNPs based CLB detection was not affected any other interferences. More excitingly, the sensor selectivity of PE-Glu-AuNPs to clenbuterol was also well demonstrated in human urine sample studies.