臺灣博碩士論文加值系統

　　萊克多巴胺是一種β受體激動劑，常用於豬肉來增進豬肉品質，但同時它也會對人體帶來危害。本篇研究透過將Nafion-銀奈米粒子修飾在玻璃碳電極表面（Nafion-Ag GCE），應用於偵測豬肝樣品中的萊克多巴胺。透過循環伏安法實驗結果可以得知，Nafion-Ag GCE對萊克多巴胺氧化反應明顯具有電催化效果，增加萊克多巴胺的氧化電流值。此外，帶負電的Nafion-Ag可以透過庫倫吸引力將在酸性環境中帶正電的萊克多巴胺吸附在電極表面，增加Nafion-Ag GCE對萊克多巴胺偵測靈敏度及選擇性。透過Nernst equation及Laviron’s equation計算，可以得知Nafion-Ag GCE與萊克多巴胺之氧化反應為兩個電子與兩個質子參與下發生。最佳化條件下（6 uL of 0.10 % Nafion-0.5 X AgNPs溶液，pH＝1.8 B-R緩衝溶液，預濃縮電位0 V，預濃縮時間300 s），線性範圍為0.0075 ppm到1.000 ppm與偵測極限為1.6 ppb。以相對標準偏差（relative standard deviation, RSD %)來評估Nafion-Ag GCE的再現性與穩定性，分別為1.51 % （n = 5）及1.56 % （n = 10）。本研究方法的可行性則是成功檢測出豬肝樣品中的萊克多巴胺含量，添加回收率為95.2-101.8 %，相對標準偏差為0.55-4.83 %。

　　β agonist ractopamine is frequently added to pork to enhance the quality of the meat, but it can potentially be detrimental to humans. This work used Nafion-silver nanoparticle- modified glassy carbon electrode (Nafion-Ag GCE) to detect ractopamine in samples of pork liver. The findings of the cyclic voltammetry experiments demonstrated that the Nafion-Ag GCE had an electrocatalytic impact on the oxidation of ractopamine and boosted the oxidation current of ractopamine. Additionally, the positively charged ractopamine can be adsorbed by the negatively charged Nafion-Ag via Coulomb attraction, increasing the sensitivity and selectivity of Nafion-Ag GCE for ractopamine detection. The Nernst equation and Laviron equation can be used to calculate the two electrons and two protons involved in the oxidation reaction between ractopamine and Nafion-Ag GCE. Under optimum conditions (6 uL of 0.10 % Nafion-0.5X AgNPs modifier, pH 1.8 Britton-Robinson buffer solution, accumulation potential 0 V, accumulation time 300 s), the linearity ranges from 0.0075 ppm to 1.000 ppm and the detection limit is 1.6 ppb. The reproducibility and stability of Nafion-Ag GCE were assessed using relative standard deviation (RSD), which were 1.51 % (n = 5) and 1.56 % (n = 10), respectively. The content of ractopamine in pork liver samples was successfully detected, and the spiked recovery and RSD were 95.2-101.8 % and 0.55-4.83 %, respectively, indicating the feasibility of this proposed method.

摘要 i
Abstract ii
致謝 iii
目錄 iv
表目錄 vii
圖目錄 viii
第一章 緒論 1
1-1 萊克多巴胺（Ractopamine） 1
1-2 電化學方式偵測萊克多巴胺 2
1-3 金屬奈米電化學感測器應用於檢測瘦肉精文獻探討 3
1-4 全氟酸樹酯（Nafion） 4
1-5 銀奈米粒子 4
1-6 循環伏安法（Cyclic Voltammetry） 5
1-7 差分脈衝伏安法（Differential pulse voltammetry） 7
1-8 電化學阻抗（Electrochemical impedance spectroscopy） 8
1-9 研究目的與動機 9
第二章 實驗藥品與儀器 10
2-1 實驗藥品 10
2-2 實驗儀器 12
第三章 實驗方法 13
3-1 銀奈米粒子的製備 13
3-1-1 配製藥品 13
3-1-2 合成銀奈米粒子（AgNPs）的步驟 13
3-2 製備Nafion-Ag複合材料 14
3-3 製備Nafion-Ag修飾玻璃碳電極 15
3-4 電化學分析步驟 16
3-5 真實樣品處理 18
第四章 結果與討論 19
4-1 銀奈米粒子與Nafion-Ag溶液材料鑑定 19
4-1-1 穿透式電子顯微鏡 19
4-1-2 吸收光譜 20
4-1-3 粒徑大小與Zeta電位 21
4-2 電化學偵測萊克多巴胺 22
4-3 工作電極之電化學阻抗分析與表現 24
4-4 最佳化條件 28
4-4-1 不同Nafion濃度與有/無添加AgNPs對電訊號的影響 28
4-4-2 最佳Nafion濃度與不同AgNPs濃度對電訊號的影響 32
4-4-3 材料Nafion-Ag溶液體積對電訊號的影響 34
4-4-4 緩衝溶液的pH值對電訊號的影響 36
4-4-5 預濃縮處理電位與時間對電訊號的影響 40
4-4-6 稀釋Nafion溶液的溶劑選擇 44
4-5 電化學反應機制探討 46
4-5-1 掃描速率對電訊號的影響 46
4-5-2 電化學反應機制 50
4-6 萊克多巴胺檢量線製備與偵測極限 51
4-7 選擇性與化學干擾容忍度 54
4-8 再現性與穩定性 56
4-9 檢測真實樣品中的萊克多巴胺 59
第五章 結論與未來展望 61
第六章 參考文獻 62

表目錄
表 2 1、實驗藥品與來源 10
表 2 2、實驗儀器與型號 12
表 3 1、電化學分析步驟 17
表 4 1、粒徑大小與Zeta電位 21
表 4 2、不同電極之EIS模擬電路參數 25
表 4 3、不同Nafion濃度有/無添加AgNPs的最終濃度 28
表 4 4、0.1 % Nafion濃度與不同濃度AgNPs配置方法 32
表 4 5、我國與各國萊克多巴胺容許量比較 51
表 4 6、Nafion-Ag GCE與不同的萊克多巴胺電化學感測器比較 53
表 4 7、偵測豬肝中的萊克多巴胺 60

圖目錄
圖 1 1、萊克多巴胺的結構 1
圖 1 2、Nafion的結構 4
圖 1 3、(a) 循環伏安法三角波訊號、(b) 循環伏安法的簡化電路 5
圖 1 4、循環伏安法示意圖 6
圖 1 5、(a) 差分脈衝伏安法的電位波形、(b) 差分脈衝伏安法示意圖 7
圖 3 1、Nafion-Ag複合材料製備流程與方法 14
圖 3 2、Nafion-Ag GCE修飾電極的製備流程 15
圖 3 3、三電極系統裝置圖 16
圖 3 4、真實樣品處理流程 18
圖 4 1、(A) Nafion (B) AgNPs (C,D) Nafion-Ag之TEM圖 19
圖 4 2、AgNPs、Nafion-Ag之吸收光譜圖 20
圖 4 3、不同電極在有/無含有萊克多巴胺溶液中的CV圖 23
圖 4 4、Nafion-Ag GCE在萊克多巴胺溶液中不同次循環的CV圖 23
圖 4 5、Nyquist圖分析不同工作電極 24
圖 4 6、不同工作電極使用CV於[Fe(CN)63－/4－]溶液中掃描 27
圖 4 7、不同工作電極使用CV於[Ru(NH3)62+/3+]溶液中掃描 27
圖 4 8、改變Nafion濃度修飾之電極的CV圖 29
圖 4 9、不同Nafion濃度+0.5 X AgNPs的修飾電極之CV圖 30
圖 4 10、14種修飾電極Rac Ipa統計圖表（n＝3） 31
圖 4 11、0.1 % Nafion +不同濃度AgNPs修飾電極之CV圖 33
圖 4 12、0.1 % Nafion +不同濃度AgNPs修飾電極CV圖Rac Ipa統計圖（n＝3） 33
圖 4 13、滴加不同Nafion-Ag體積修飾電極之CV圖 35
圖 4 14、滴加不同Nafion-Ag體積修飾電極之CV圖Rac Ipa統計圖表（n＝3） 35
圖 4 15、萊克多巴胺酸性與鹼性環境中質子化與去質子化情形 36
圖 4 16、Nafion-Ag GCE在不同pH值環境之CV圖 37
圖 4 17、Nafion-Ag GCE在不同pH值環境CV圖Rac Ipa統計圖表（n＝3） 37
圖 4 18、Nafion-Ag GCE在不同pH值＋1.000 ppm Rac之pH值與Rac Epa線性關係圖 39
圖 4 19、Nafion-Ag GCE改變預濃縮處理電位之CV圖 41
圖 4 20、Nafion-Ag GCE在不同預濃縮處理電位CV圖Rac Ipa統計圖表（n＝3） 41
圖 4 21、Nafion-Ag GCE改變預濃縮處理時間之CV圖 43
圖 4 22、Nafion-Ag GCE在不同預濃縮處理時間CV圖Rac Ipa統計圖表（n＝3） 43
圖 4 23、最佳化條件下改變稀釋Nafion的溶劑其CV圖與Rac Ipa統計圖表（n＝3）45
圖 4 24、Nafion-Ag GCE改變不同掃描速率之CV圖 48
圖 4 25、掃描速率與Rac Ipa線性關係圖 48
圖 4 26、掃描速率的對數值與Rac Epa線性關係圖 49
圖 4 27、反應機制圖 50
圖 4 28、萊克多巴胺於電極表面推測化學反應方程式 50
圖 4 29、Nafion-Ag GCE偵測不同濃度萊克多巴胺之DPV圖 52
圖 4 30、Rac Ipa與萊克多巴胺濃度的關係圖（n＝3） 52
圖 4 31、Nafion-Ag GCE對於萊克多巴胺的選擇性測試 54
圖 4 32、Nafion-Ag GCE對於萊克多巴胺的化學干擾容忍度測試 55
圖 4 33、Nafion-Ag GCE再現性測試 56
圖 4 34、Nafion-Ag GCE重複性測試DPV圖 57
圖 4 35、Nafion-Ag GCE穩定性測試 58
圖 4 36、偵測豬肝中萊克多巴胺之DPV圖（n = 3） 60

1.Feddern, V., et al., Ractopamine analysis in pig kidney, liver and lungs: A validation of the method scope extension using QuEChERS as a sample preparation step. Journal of Chromatography B, 2018. 1091: p. 79-86.
2.Naves Aroeira, C., et al., Growth promoters in cattle and pigs: A review of legislation and implications for human health. Food Reviews International, 2021: p. 1-23.
3.Armstrong, T., et al., The effect of dietary ractopamine concentration and duration of feeding on growth performance, carcass characteristics, and meat quality of finishing pigs. Journal of Animal Science, 2004. 82(11): p. 3245-3253.
4.Rajkumar, M., Y.-S. Li, and S.-M. Chen, Electrochemical detection of toxic ractopamine and salbutamol in pig meat and human urine samples by using poly taurine/zirconia nanoparticles modified electrodes. Colloids and Surfaces B: Biointerfaces, 2013. 110: p. 242-247.
5.Simon, T., et al., Development of extremely stable dual functionalized gold nanoparticles for effective colorimetric detection of clenbuterol and ractopamine in human urine samples. Analytica Chimica Acta, 2018. 1023: p. 96-104.
6.Han, Q., et al., Quantitative determination of ractopamine in swine urine using Fourier transform infrared (FT-IR) spectroscopy analysis. Infrared Physics & Technology, 2021. 113: p. 103653.
7.Shelver, W.L. and D.J. Smith, Determination of ractopamine in cattle and sheep urine samples using an optical biosensor analysis: comparative study with HPLC and ELISA. Journal of Agricultural and Food Chemistry, 2003. 51(13): p. 3715-3721.
8.Han, S., et al., Gold nanoparticle-based colorimetric ELISA for quantification of ractopamine. Mikrochim Acta, 2018. 185(4): p. 210.
9.Peng, C., et al., In-tube solid phase microextraction and determination of ractopamine in pork muscle samples using amide group modified polysaccharide-silica hybrid monolith as sorbent prior to HPLC analysis. Food Chemistry, 2021. 355: p. 129662.
10.Tang, Y., et al., Determination of ractopamine in pork using a magnetic molecularly imprinted polymer as adsorbent followed by HPLC. Food chemistry, 2016. 201: p. 72-79.
11.He, L., et al., Determination of ractopamine and clenbuterol in feeds by gas chromatography–mass spectrometry. Animal Feed Science and Technology, 2007. 132(3-4): p. 316-323.
12.Wang, J., et al., Development of immunoaffinity sample-purification for GC–MS analysis of ractopamine in swine tissue. Chromatographia, 2006. 64(9): p. 613-617.
13.Gressler, V., et al., Development of a readily applied method to quantify ractopamine residue in meat and bone meal by QuEChERS-LC-MS/MS. Journal of Chromatography B, 2016. 1015-1016: p. 192-200.
14.Zhang, L., et al., Simultaneous Determination of Salbutamol, Ractopamine, and Clenbuterol in Animal Feeds by SPE and LC—MS. Journal of chromatographic science, 2009. 47(4): p. 324-328.
15.林俞廷, et al., 禽畜產品中乙型受體素多重殘留檢驗方法之精進. 食品藥物研究年報, 2018(9): p. 52-65.
16.Shen, Y., et al., Electrochemical detection of Sudan red series azo dyes: Bibliometrics based analysis. Food and Chemical Toxicology, 2022. 163: p. 112960.
17.Orooji, Y., et al., An electrochemical strategy for toxic ractopamine sensing in pork samples; twofold amplified nano-based structure analytical tool. Journal of Food Measurement and Characterization, 2021. 15(5): p. 4098-4104.
18.Roushani, M., M. Ghanbarzadeh, and F. Shahdost-Fard, Fabrication of an electrochemical biodevice for ractopamine detection under a strategy of a double recognition of the aptamer/molecular imprinting polymer. Bioelectrochemistry, 2021. 138: p. 107722.
19.Sun, D., et al., One-Step Electrodeposition of Silver Nanostructures on 2D/3D Metal-Organic Framework ZIF-67: Comparison and Application in Electrochemical Detection of Hydrogen Peroxide. ACS Applied Materials & Interfaces, 2020. 12(37): p. 41960-41968.
20.Bai, Y., X. Chen, and H. Qi, Characterization and bioactivity of phlorotannin loaded protein-polysaccharide nanocomplexes. LWT, 2022. 155: p. 112998.
21.Vatandost, E., et al., Green tea extract assisted green synthesis of reduced graphene oxide: Application for highly sensitive electrochemical detection of sunset yellow in food products. Food chemistry: X, 2020. 6: p. 100085.
22.Mehmandoust, M., et al., Voltammetric carbon nanotubes based sensor for determination of tryptophan in the milk sample. Journal of Food Measurement and Characterization, 2021. 15(6): p. 5288-5295.
23.He, Q., et al., Facile Electrochemical Sensor for Nanomolar Rutin Detection Based on Magnetite Nanoparticles and Reduced Graphene Oxide Decorated Electrode. Nanomaterials, 2019. 9(1): p. 1-16.
24.Duan, J., et al., Glassy carbon electrode modified with gold nanoparticles for ractopamine and metaproterenol sensing. Chemical Physics Letters, 2013. 574: p. 83-88.
25.Wei, Q., et al., Formation of flowerlike gold nanostructure on ordered mesoporous carbon electrode and its application in electrochemical determination of ractopamine. Materials Letters, 2015. 147: p. 58-60.
26.Liu, L., et al., Glassy carbon electrode modified with Nafion–Au colloids for clenbuterol electroanalysis. Electrochimica Acta, 2010. 55(24): p. 7240-7245.
27.Cao, L., et al., Biochar-Supported Cu(2+)/Cu(+) Composite as an Electrochemical Ultrasensitive Interface for Ractopamine Detection. ACS Applied Bio Materials, 2021. 4(2): p. 1424-1431.
28.Heitner-Wirguin, C., Recent advances in perfluorinated ionomer membranes: structure, properties and applications. Journal of Membrane Science, 1996. 120(1): p. 1-33.
29.Ren, X., E. Gobrogge, and F. Beyer, States of water in recast Nafion® films. Journal of Membrane Science, 2021. 637: p. 119645.
30.Lim, S.H., et al., A glucose biosensor based on electrodeposition of palladium nanoparticles and glucose oxidase onto Nafion-solubilized carbon nanotube electrode. Biosensors and Bioelectronics, 2005. 20(11): p. 2341-2346.
31.Sasikumar, G., J. Ihm, and H. Ryu, Optimum Nafion content in PEM fuel cell electrodes. Electrochimica Acta, 2004. 50(2-3): p. 601-605.
32.Zhou, Z.-L., et al., Electrochemical sensor for formaldehyde based on Pt–Pd nanoparticles and a Nafion-modified glassy carbon electrode. Microchimica Acta, 2009. 164(1): p. 133-138.
33.Rao, C.R., et al., Metal nanoparticles and their assemblies. Chemical Society Reviews, 2000. 29(1): p. 27-35.
34.Daraee, H., et al., Application of gold nanoparticles in biomedical and drug delivery. Artif Cells Nanomed Biotechnol, 2016. 44(1): p. 410-422.
35.Garcia-Segura, S., et al., Opportunities for nanotechnology to enhance electrochemical treatment of pollutants in potable water and industrial wastewater–a perspective. Environmental Science: Nano, 2020. 7(8): p. 2178-2194.
36.Amin, H.M., et al., Tailoring the Electrocatalytic Activity of Pentlandite FexNi9‐XS8 Nanoparticles via Variation of the Fe: Ni Ratio for Enhanced Water Oxidation. ChemElectroChem, 2021. 8(20): p. 3863-3874.
37.Campbell, F.W. and R.G. Compton, The use of nanoparticles in electroanalysis: an updated review. Analytical and Bioanalytical Chemistry, 2010. 396(1): p. 241-259.
38.Kumar, K.K., et al., Green synthesis of curcumin-silver nanoparticle and its modified electrode assisted amperometric sensor for the determination of paracetamol. Chemosphere, 2022: p. 134994.
39.Verma, D., et al., Development of MWCNT decorated with green synthesized AgNps-based electrochemical sensor for highly sensitive detection of BPA. Journal of Applied Electrochemistry, 2021. 51(3): p. 447-462.
40.Hwa, K.-Y., T.S.K. Sharma, and A. Ganguly, Design strategy of rGO–HNT–AgNPs based hybrid nanocomposite with enhanced performance for electrochemical detection of 4-nitrophenol. Inorganic Chemistry Frontiers, 2020. 7(10): p. 1981-1994.
41.Suherman, A.L., et al., Electrochemical detection of ultratrace (picomolar) levels of Hg2+ using a silver nanoparticle-modified glassy carbon electrode. Analytical chemistry, 2017. 89(13): p. 7166-7173.
42.de Lima, C.A., et al., Silver nanoparticle-modified electrode for the determination of nitro compound-containing pesticides. Analytical and Bioanalytical Chemistry 2016. 408(10): p. 2595-2606.
43.Shivakumar, M., et al., Electrochemical detection of nitrite using glassy carbon electrode modified with silver nanospheres (AgNS) obtained by green synthesis using pre‐hydrolysed liquor. Electroanalysis, 2017. 29(5): p. 1434-1442.
44.P.S, J. and D.S. Sutrave, A Brief Study of Cyclic Voltammetry and Electrochemical Analysis. International Journal of ChemTech Research, 2018. 11(9): p. 77-88.
45.Venton, B.J. and Q. Cao, Fundamentals of fast-scan cyclic voltammetry for dopamine detection. Analyst, 2020. 145(4): p. 1158-1168.
46.Laviron, E., General expression of the linear potential sweep voltammogram in the case of diffusionless electrochemical systems. Journal of Electroanalytical Chemistry and Interfacial Electrochemistry, 1979. 101(1): p. 19-28.
47.Vilas-Boas, Â., et al., Evaluation of total polyphenol content of wines by means of voltammetric techniques: Cyclic voltammetry vs differential pulse voltammetry. Food chemistry, 2019. 276: p. 719-725.
48.Deshmukh, M.A., et al., EDTA-modified PANI/SWNTs nanocomposite for differential pulse voltammetry based determination of Cu (II) ions. Sensors and Actuators B: Chemical, 2018. 260: p. 331-338.
49.Ciucci, F., Modeling electrochemical impedance spectroscopy. Current Opinion in Electrochemistry, 2019. 13: p. 132-139.
50.Orazem, M.E. and B. Tribollet, Electrochemical impedance spectroscopy. New Jersey, 2008: p. 383-389.
51.Guo, S., et al., Three-dimensional Pt-on-Au bimetallic dendritic nanoparticle: one-step, high-yield synthesis and its bifunctional plasmonic and catalytic properties. The Journal of Physical Chemistry C, 2010. 114(36): p. 15337-15342.
52.Zhou, S., et al., Enhanced CO tolerance for hydrogen activation in Au− Pt dendritic heteroaggregate nanostructures. Journal of the American Chemical Society, 2006. 128(6): p. 1780-1781.
53.Mohammadi, S. and G. Khayatian, Silver nanoparticles modified with thiomalic acid as a colorimetric probe for determination of cystamine. Microchimica Acta, 2016. 184(1): p. 253-259.
54.Yuan, Y., et al., Silver nanoparticle based label-free colorimetric immunosensor for rapid detection of neurogenin 1. Analyst, 2012. 137(2): p. 496-501.
55.Kamau, G., Surface preparation of glassy carbon electrodes. Analytica Chimica Acta, 1988. 207: p. 1-16.
56.Elgrishi, N., et al., A Practical Beginner’s Guide to Cyclic Voltammetry. Journal of Chemical Education, 2017. 95(2): p. 197-206.
57.Paramelle, D., et al., A rapid method to estimate the concentration of citrate capped silver nanoparticles from UV-visible light spectra. Analyst, 2014. 139(19): p. 4855-61.
58.Goldburg, W.I., Dynamic light scattering. American Journal of Physics, 1999. 67(12): p. 1152-1160.
59.Lu, G.W. and P. Gao, CHAPTER 3 - Emulsions and Microemulsions for Topical and Transdermal Drug Delivery, in Handbook of Non-Invasive Drug Delivery Systems, V.S. Kulkarni, Editor. 2010, William Andrew Publishing: Boston. p. 59-94.
60.Smith, D., The pharmacokinetics, metabolism, and tissue residues of β-adrenergic agonists in livestock. Journal of animal science, 1998. 76(1): p. 173-194.
61.Sairi, M. and D.W.M. Arrigan, Liquid / Liquid Interface-Based Electrochemical Sensing of Ractopamine and Salbutamol. Procedia Chemistry, 2016. 20: p. 76-80.
62.Walczak, M.M., et al., pH dependent redox couple: An illustration of the Nernst equation. Journal of chemical education, 1997. 74(10): p. 1195.
63.Guo, H., et al., Electrochemical determination of dopamine and uric acid with covalent organic frameworks and Ox-MWCNT co-modified glassy carbon electrode. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2022. 648: p. 1434-1442.
64.Ma, X., et al., A novel sensitive electrochemical method for the detection of ractopamine in meat food via polycitrulline-modified electrode. Food Additives & Contaminants: Part A 2020. 37(9): p. 1459-1466.
65.Poo-arporn, Y., et al., The development of disposable electrochemical sensor based on Fe3O4-doped reduced graphene oxide modified magnetic screen-printed electrode for ractopamine determination in pork sample. Sensors and Actuators B: Chemical, 2019. 284: p. 164-171.
66.Yang, X., et al., Electrochemical determination of toxic ractopamine at an ordered mesoporous carbon modified electrode. Food chemistry, 2014. 145: p. 619-624.
67.Wu, C., et al., Electrochemical sensor for toxic ractopamine and clenbuterol based on the enhancement effect of graphene oxide. Sensors and Actuators B: Chemical, 2012. 168: p. 178-184.