銀離子修飾二硫化鉬奈米點雙模式感測器應用於檢測鹼性磷酸?及酵素免疫分析法

Modification of MoS2 Nanodots with Silver Ion as a Dual-mode Sensor for Sensitive Detection of Alkaline Phosphatase: Application to ELISA

趙建閔(CHIEN-MIN CHAO)

指導教授 ： 謝明穆

國立高雄師範大學/理學院/化學系/碩士(2018年)

摘要

本篇透過基於特定的鹼性磷酸?（Alkaline phosphatase）放大二硫化鉬奈米點 （Monolayer-Molybdenum disulfide Nano-dots）及銀奈米粒子 （Silver nanoparticles） 複合體的獨特光學性質，開發具有比色和螢光雙模式的新型傳感器。在此策略中，鹼性磷酸?能夠從磷酸化抗壞血酸 (Ascorbic acid-phosphate)中去除磷酸基團產生抗壞血酸 (Ascorbic acid) ，而銀離子 (Silver ion) 可以通過靜電作用力 (Electrostatic interaction) 附著在二硫化鉬奈米點表面，透過抗壞血酸誘導銀離子於二硫化鉬奈米點表面上進行原位性生成 (In-situ growth) 銀奈米粒子，同時伴隨著銀奈米粒子的表面電將共振(Surface Plasmon Resonance，SPR)顯著增加使二硫化鉬奈米點有明顯的螢光消光現象。其中銀奈米粒子扮演一種奈米消光基團 (Nano quencher) 的重要角色，通過二硫化鉬奈米點作為供體 (Donor) 到銀奈米粒子作為受體 (Acceptor) 產生螢光共振能量轉移效應 (Fluorescence resonance energy transfer, FRET)導致二硫化鉬奈米點的螢光淬滅。
最佳化條件後，該奈米感測器在比色法及螢光測定法下對鹼性磷酸?的偵測極限 ( LOD ) 分別為0.03 U/L及0.003 U/L，而透過鹼性磷酸?修飾的免疫球蛋白抗體可將前述酵素系統應用於免疫分析方法之開發並成功應用於偵測目標抗原免疫球蛋白，除了高選擇性外，藉由銀奈米粒子生成所導致的顏色變化能透過肉眼有效的快速辨識免疫球蛋白抗原75 pg/mL以上存在於系統中，且由於感測器具有雙模式的偵測，在螢光系統下偵測極限更能達到8 pg/mL，並能利用人類血漿模擬血液基質的情況下不受基質干擾的進行兔子免疫球蛋白抗原的偵測。在具有高選擇性、高辨識能力及高靈敏度的多重優點下，可依不同需求透過ALP修飾不同抗體及抗原的組合進行多樣的酵素免疫分析檢測。

關鍵字

二硫化鉬 ； 奈米點 ； 銀奈米粒子 ； 原位生成 ； 雙模式感測器 ； 酵素免疫分析法 ； 免疫球蛋白抗原

並列摘要

We report a new dual-mode sensor for determination of alkaline phosphatase based on monolayer-molybdenum disulfide Nano-dots and silver Nano-particles hybrid. Through the ascorbic acid-phosphate is hydrolyzed and produced ascorbic acid by alkaline phosphatase. On the other hand, the silver ions interact with the surfaces of monolayer-molybdenum disulfide Nano-dots via electrostatic interaction and reduced by ascorbic acid to reduce silver Nano-particles, which in-situ growth on the surface of monolayer-molybdenum disulfide Nano-dots. As a result, obviously increase in the surface plasmon resonance band of silver nanoparticles and the fluorescence of monolayer-molybdenum disulfide Nano-dots is effectively quenched.
In the novel dual-mode sensor, silver Nano-particles act as a“Nano-quencher” to decrease monolayer-molybdenum disulfide Nano-dots fluorescence by fluorescence resonance energy transfer from monolayer-molybdenum disulfide Nano-dots (donor) to silver Nano-particles (acceptor).
The limit of detection of Alkaline phosphatase activity are respectively as low as 0.03 U/L and 0.003 U/L by colorimetric and fluorometric method under optimal conditions. Combine with Alkaline phosphatase sensing strategy, enzyme-modified anti-IgG antibody could be applied to detect target antigen in an enzyme-linked immunosorbent assay (ELISA) platform. The limit of detection of rabbit Immunoglobulin G is respectively as low as 75 pg/mL and 8 pg/mL by naked-eye and fluorometric method under optimal conditions. More importantly, the developing dual-sensor allowed the rabbit Immunoglobulin G been detected in 25-1000 pg/mL of human serum samples.

並列關鍵字

Alkaline phosphatase ； MoS2 Nano-dost ； Dual-mode sensor ； ELISA

參考文獻

1. Rissin, D. M.; Kan, C. W.; Campbell, T. G.; Howes, S. C.; Fournier, D. R.; Song, L.; Piech, T.; Patel, P. P.; Chang, L.; Rivnak, A. J., Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations. Nat. Biotechnol. 2010, 28, 595.

2. Liang, J.; Yao, C.; Li, X.; Wu, Z.; Huang, C.; Fu, Q.; Lan, C.; Cao, D.; Tang, Y., Silver nanoprism etching-based plasmonic ELISA for the high sensitive detection of prostate-specific antigen. Biosens. Bioelectron. 2015, 69, 128-134.

3. De La Rica, R.; Stevens, M. M., Plasmonic ELISA for the ultrasensitive detection of disease biomarkers with the naked eye. NAT NANOTECHNOL 2012, 7, 821.

4. Tian-Tian, L.; ZHANG, L.-Y.; Zhen-Long, Y.; Xiao-Chi, M.; Sa, D., Synthesis of Boronic Acid-functionalized Soluble Dendrimers and its Application in Detection of Human Liver Microsomal Glycoprotein Based on Enzyme-linked Immunosorbent Assay. CHINESE J ANAL CHEM 2017, 45, 1259-1263.

5. Sheng, W.; Sun, C.; Fang, G.; Wu, X.; Hu, G.; Zhang, Y.; Wang, S., Development of an Enzyme-Linked Immunosorbent Assay for the Detection of Tyramine as an Index of Freshness in Meat and Seafood. J. Agric. Food. Chem. 2016, 64, 8944-8949.