本研究首先以熱裂解法製備疏水性之氧化鐵磁性奈米粒子 (MNP)，經AEAPTES進行配體交換後將粒子改質為親水性之粒子團簇 (MNP@AE)，再與3-MA進行醯胺化反應賦予粒子表面馬來醯亞胺官能基 (MNP@3-MA)，使粒子具有感測人類血清白蛋白之功能性。實驗產物經由XRD分析顯示MNP晶相組成為Fe3O4；由TEM影像可知MNP平均粒徑為12-13 nm之球狀粒子，MNP@AE與MNP@3-MA則為不規則粒子團簇；由DLS水合粒徑分析可知MNP在改質後聚集形成較大粒徑之團簇；由界面電位與可見光吸收度之沉降曲線可知，MNP之界面電位在改質後由負轉正，分散穩定性佳，在3-MA接枝後則轉為接近電中性，粒子沉降率增加；由FT-IR與XPS分析可觀察到對應官能基與元素之特徵峰，為改質與接枝階段之成功提供了有利證據。由SQUID可知MNP在改質前後皆接近超順磁性。
將MNP@3-MA修飾於金電極上，以電化學阻抗譜法 (EIS) 感測並計算吸附白蛋白後之阻抗變化率，修飾電極感測白蛋白濃度之線性範圍為1-8 g/dL，涵蓋人類血清中正常的白蛋白濃度，偵測極限為0.2 g/dL，整體感測時間為20 min；本研究之修飾電極之選擇性佳，然而再現性與穩定性仍有改善空間；以標準人類血清製備檢量線，並以修飾電極感測四個人類血清檢體，其回收率多介於88-105%，顯示本研究製備之修飾電極可用於感測人類血清檢體。

Over the past few decades, iron oxide nanoparticles (IONPs) have been widely applied to biosensors because of their superparamagnetic property. In this research, to detect human serum albumin (HSA), which is an indicator of liver diseases, surface-modified IONPs were deposited on gold electrodes as biosensors. First, magnetic iron oxide nanoparticles (MNPs) were synthesized by thermal decomposition, and further modified with AEAPTES ([3-(2-aminoethylamino)propyl]triethoxysilane) to convert hydrophobic MNPs into hydrophilic MNP@AE. Afterward, MNP@AE was grafted with 3-MA (3-maleimidopropionic acid) to provide maleimide end groups to adsorb HSA. By TEM images and XRD analysis, the morphologies, particle size distribution, and the crystal phases of MNPs series can be aquird. DLS spectrum provides the hydrodynamic diameters of MNP series. Zeta potential and UV-Vis were further applied to evaluate the dispersion stability of the MNP series. Characteristic functional groups and the change of surface elements of the MNP series can be identified by FT-IR and XPS spectrum. From M-H hysteresis curve, it is demonstrated that the MNP series are nearly superparamagnetic. In the end, MNP@3-MA modified electrodes were used to detect HSA by EIS (electrochemical impedance spectroscopy). After injection of HSA, the impedance increased with time. A calibration curve has been constructed between HSA concentration and impedance change ratio with a linear range covering the normal HSA level in human serum. Measurements of four real human serum samples from NCKU hospital were conducted, showing that the biosensor has the potential for clinic applications.

1. B Liu, J Liu. Sensors and biosensors based on metal oxide nanomaterials. Trends in Analytical Chemistry 121, 115690, 2019.
2. D Jiles. Introduction to magnetism and magnetic materials. 1st Ed., Springer Science Business Media, USA, 1991.
3. C Kittel. Introduction of solid state physics. 7th Ed., John Wiley & Sons Inc., USA, 1997.
4. AG Kolhatkar, AC Jamison , D Litvinov, RC Willson, TR Lee. Tuning the magnetic properties of nanoparticles. International Journal of Molecular Sciences 14, 15977-16009, 2013.
5. DK Cheng. Field and wave electromagnetics. 3rd Ed., Addison-Wesley, USA, 1989.
6. A Akbarzadeh, M Samiei, S Davaran. Magnetic nanoparticles: preparation, physical properties, and applications in biomedicine. Nanoscale Research Letters 7, 144, 2012.
7. JS Lee, JM Cha, HY Yoon, JK Lee, YK Kim. Magnetic multi-granule nanoclusters: A model system that exhibits universal size effect of magnetic coercivity. Scientific Report 5, 12135, 2015.
8. J Baumgartner, L Bertinetti, M Widdrat, AM Hirt, D Faivre. Formation of magnetite nanoparticles at low temperature: from superparamagnetic to stable single domain particles. Plos One 8, e57070, 2013.
9. S Laurent, AA Saei, S Behzadi, A Panahifar, M Mahmoudi. Superparamagnetic iron oxide nanoparticles for delivery of therapeutic agents: opportunities and challenges. Expert Opinion on Drug Delivery 11, 1449-1470, 2014.
10. SH Sun, H Zeng. Size-controlled synthesis of magnetite nanoparticles. Journal of the American Chemical Society 124, 8204-8205, 2002.
11. W Wu, ZH Wu, TK Yu, CZ Jiang, WS Kim. Recent progress on magnetic iron oxide nanoparticles: synthesis, surface functional strategies and biomedical applications. Science and Technology of Advanced Materials 16, 023501, 2015.
12. R Hufschmid, H Arami, RM Ferguson, M Gonzales, E Teeman, LN Brush, et al. Synthesis of phase-pure and monodisperse iron oxide nanoparticles by thermal decomposition. Nanoscale 7, 11142-11154, 2015.
13. AM Jubb, HC Allen. Vibrational spectroscopic characterization of hematite, maghemite, and magnetite thin films produced by vapor deposition. ACS Applied Materials & Interfaces 2, 2804-2812, 2010.
14. AS Teja, PY Koh. Synthesis, properties, and applications of magnetic iron oxide nanoparticles. Progress in Crystal Growth and Characterization of Materials 55, 22-45, 2009.
15. L Gloag, M Mehdipour, DF Chen, RD Tilley, JJ Gooding. Advances in the application of magnetic nanoparticles for sensing. Advanced Materials 31, 1904385-1904410, 2019.
16. R Massart. Preparation of aqueous magnetic liquids in alkaline and acidic media. IEEE Transactions on Magnetics 17, 1247-1248, 1981.
17. DS Wang, YD Li. Effective octadecylamine system for nanocrystal synthesis. Inorganic Chemistry, 50, 5196-5202, 2011.
18. TG Hyeon. Chemical synthesis of magnetic nanoparticles. Chemical Communications 927-934, 2003.
19. JN Park, KJ An, YS Hwang, JG Park, HJ Noh, JY Kim, et al. Ultra-large-scale syntheses of monodisperse nanocrystals. Nature Materials 3, 891-895, 2004.
20. TG Hyeon, SS Lee, JN Park, YH Chung, HB Na. Synthesis of highly crystalline and monodisperse maghemite nanocrystallites without a size-selection process. Journal of the American Chemical Society 123, 12798-12801, 2001.
21. W Wu, QG He, CZ Jiang. Magnetic iron oxide nanoparticles: synthesis and surface functionalization strategies. Nanoscale Research Letters 3, 397-415, 2008.
22. W Wu, CZ Jiang, VAL Roy. Designed synthesis and surface engineering strategies of magnetic iron oxide nanoparticles for biomedical applications. Nanoscale 8, 19421, 2016.
23. MT Nguyen, KYu, T Tokunaga, K Boonyaperm, S Kheawhom, M Arita, et al. Green synthesis of size-tunable iron oxides and iron nanoparticles in a salt matrix. Applied Catalysis B: Environmental 7, 17697-17705, 2019.
24. TJ Daou, G Pourroy, S Bégin-Colin, JM Grenèche, C Ulhaq-Bouillet, P Legaré, et al. Hydrothermal synthesis of monodisperse magnetite nanoparticles. Chemistry of Materials 18, 4399-4404, 2006.
25. W Wu, QG He, CZ Jiang. Magnetic iron oxide nanoparticles: synthesis and surface functionalization strategies. Nanoscale Research Letters 3, 397-415, 2008.
26. S Santra, R Tapec, N Theodoropoulou, J Dobson, A Hebard, W Tan. Synthesis and characterization of silica-coated iron oxide nanoparticles in microemulsion: the effect of nonionic surfactants. Langmuir 17, 2900-2906, 2001.
27. W Wu, ZH Wu, TK Yu, CZ Jiang, WS Kim. Recent progress on magnetic iron oxide nanoparticles: synthesis, surface functional strategies and biomedical applications. Science and Technology of Advanced Materials 16, 023501, 2015.
28. A Heuer-Jungemann, N Feliu, I Bakaimi, M Hamaly, A Alkilany, I Chakraborty, et al. The role of ligands in the chemical synthesis and applications of inorganic nanoparticles. Chemical Reviews 119, 4819-4880, 2019.
29. BI Kharisov, HVR Dias, OV Kharissova, A Vázquez, Y Peña, IGómez. Solubilization, dispersion and stabilization of magnetic nanoparticles in water and non-aqueous solvents: recent trends. RSC Advances 4, 45354-45381, 2014.
30. YP Bao, TL Wen, ACS Samia, A Khandhar, KM Krishnan. Magnetic nanoparticles: material engineering and emerging applications in lithography and biomedicine. Journal of Materials Science 51, 513-553, 2016.
31. R Narayan, UY Nayak, AM Raichur, S Garg. Mesoporous silica nanoparticles: a comprehensive review on synthesis and recent advances. Pharmaceutics 10, 118, 2018.
32. B Arkles. Hydrophobicity, hydrophilicity and silane surface modification. Gelest, Inc., 2011.
33. M Rostami, M Mohseni, Z Ranjbar. Investigating the effect of pH on the surface chemistry of an amino silane treated nano silica. Pigment & Resin Technology 40, 363-373, 2011.
34. FD Osterholtz, ER Pohl. Kinetics of the hydrolysis and condensation of organofunctional alkoxysilanes: a review. Journal of Adhesion Science and Technology 6, 127-149, 1992.
35. D Liu, AM Pourrahimi, LKH Pallon, RL Andersson, MS Hedenqvist, UW Gedde, et al. Morphology and properties of silica-based coatings with different functionalities for Fe3O4, ZnO and Al2O3 nanoparticles. RSC Advances 5, 48094-48103, 2015.
36. MJ Zhu, MZ Lerum, W Chen. How to prepare reproducible, homogeneous, and hydrolytically stable aminosilane-derived layers on silica. Langmuir 28, 416-423, 2012.
37. J Bart, R Tiggelaar, M Yang, S Schlautmann, H Zuilhof, H Gardeniers. Room-temperature intermediate layer bonding for microfluidic devices. Lab on a Chip, 9, 3481-3488, 2009.
38. ThermoScientific. Instructions NHS and Sulfo-NHS. Technical Report 24500, Pierce Biotechnology, Rockford, IL, 2011. https://tools.thermofisher.com/content/sfs/manuals/MAN0011309_NHS_SulfoNHS_UG.pdf
39. MP Wickramathilaka, BY Tao. Characterization of covalent crosslinking strategies for synthesizing DNA-based bioconjugates. Journal of Biological Engineering 13, 63, 2019.
40. MA Rothschild, M Oratz, S Schreiber. Serum Albumin. Hepatology 8, 385-401, 1988.
41. G Fanali, A di Masi, V Trezza, M Marino, M Fasano, P Ascenzi. Human serum albumin: from bench to bedside. Molecular Aspects of Medicine 33, 209-290, 2012.
42. JF Xu, YS Yang, AQ Jiang, HL Zhu. Detection methods and research progress of human serum albumin. Critical Reviews in Analytical Chemistry 52, 72-92, 2020.
43. MT Shaha, E Alveroglua. Facile synthesis of nanogels modified Fe3O4@Ag NPs for the efficient adsorption of bovine & human serum albumin. Materials Science & Engineering C 118, 111390, 2021.
44. F Nakashima, T Shibata, K Kamiya, J Yoshitake, R Kikuchi, T Matsushita, et al. Structural and functional insights into S-thiolation of human serum albumins. Scientific Reports 8, 932, 2018.
45. A Bocedi, G Cattani, L Stella, R Massoud, G Ricci. Thiol disulfide exchange reactions in human serum albumin: the apparent paradox of the redox transitions of Cys34. The FEBS Journal 285, 3225-3237, 2018.
46. WM Huang, X Wu, X Gao, YF Yu, H Lei, ZS Zhu, et al. Maleimide-thiol adducts stabilized through stretching. Nature Chemistry 11, 310-319, 2019.
47. JMJM Ravasco, H Faustino, A Trindade, PMP Gois. Bioconjugation with Maleimides: A Useful Tool for Chemical Biology. Chemistry — A European Journal 25, 43-59, 2019.
48. EM Materón, CM Miyazaki, O Carr, N Joshi, PHS Picciani, CJ Dalmaschio, et al. Magnetic nanoparticles in biomedical applications: A review. Applied Surface Science Advances 6, 100163, 2021.
49. Wahajuddin, S Arora. Superparamagnetic iron oxide nanoparticles: magnetic nanoplatforms as drug carriers. International Journal of Nanomedicine 7, 3445-3471, 2012.
50. JH Lee, JT Jang, JS Choi, SH Moon, SH Noh, JW Kim, et al. Exchange-coupled magnetic nanoparticles for efficient heat induction. Nature Nanotechnology 6, 418-422, 2011.
51. S Chandra, K Arora, D Bahadur. Impedimetric biosensor based on magnetic nanoparticles for electrochemical detection of dopamine. Materials Science and Engineering 177, 1531-1537, 2012.
52. N Chauhan, S Chawla, CS Pundir, U Jain. An electrochemical sensor for detection of neurotransmitter-acetylcholine using metal nanoparticles, 2D material and conducting polymer modified electrode. Biosensors and Bioelectronics 89, 377-383, 2017.
53. TT Xu, Bo Chi, Fan Wu, SS Ma, SY Zhan, MH Yi, et al. A sensitive label-free immunosensor for detection α-fetoprotein in whole blood based on anticoagulating magnetic nanoparticles. Biosensors and Bioelectronics 95, 87-93, 2017.
54. YG Wang, Y Zhang, D Wu, HM Ma, XH Pang, DW Fan, et al. Ultrasensitive label-free electrochemical immunosensor based on multifunctionalized graphene nanocomposites for the detection of alpha fetoprotein. Scientific Reports 7, 42361, 2017.
55. BH Sun, XJ Ni, YH Cao, GQ Cao. Electrochemical sensor based on magnetic molecularly imprinted nanoparticles modified magnetic electrode for determination of Hb. Biosensors and Bioelectronics 91, 354-358, 2017.
56. S Kumar, M Umar, A Saifi, S Kumar, S Augustine, S Srivastava, et al. Electrochemical paper based cancer biosensor using iron oxide nanoparticles decorated PEDOT:PSS. Analytica Chimica Acta 1056, 135-145, 2019.
57. SL Lv, JL Sheng, SY Zhao, MC Liu, LH Chen. The detection of brucellosis antibody in whole serum based on the low-fouling electrochemical immunosensor fabricated with magnetic Fe3O4@Au@PEG@HA nanoparticles. Biosensors and Bioelectronics 117, 138-144, 2018.
58. LT Tufa, SJ Oh, VT Tran, JH Kim, KJ Jeong, TJ Park. Electrochemical immunosensor using nanotriplex of graphene quantum dots, Fe3O4, and Ag nanoparticles for tuberculosis. Electrochimica Acta 290, 369-377, 2018.
59. 郭蘋萱。多功能性超順磁氧化鐵奈米粒子的製備、鑑定與生醫應用。國立成功大學化工系碩士學位論文，2016。
60. 陳筑雯。製備超順磁氧化鐵奈米粒應用於人類血清白蛋白之免疫專一性結合與檢測。國立成功大學化工系碩士學位論文，2017。
61. 黃宜茵。以不同的表面修飾氧化鐵奈米粒子用於偵測人類血清白蛋白。國立成功大學化工系碩士學位論文，2018。
62. 陳瑩瑩。以表面接枝之磁性奈米團簇修飾金電極用於電化學阻抗式感測血清白蛋白。國立成功大學化工系碩士學位論文，2020。
63. LY Zhao, JJ Fei, HZ Lian, L Mao, XB Cui. Development of a novel amine- and carboxyl-bifunctionalized hybrid monolithic column for non-invasive speciation analysis of chromium. Talanta 212, 127099, 2020.
64. IOPD Berti, MV Cagnoli, G Pecchi, JL Alessandrini, SJ Stewart, JF Bengoa, et al. Alternative low-cost approach to the synthesis of magnetic iron oxide nanoparticles by thermal decomposition of organic precursors. Nanotechnology 24, 175601, 2013.
65. RJ Yang, CC Tseng, WJ Ju, HL Wang, LM Fu. A rapid paper-based detection system for determination of human serum albumin concentration. Chemical Engineering Journal 352, 241-246, 2018.
66. RJ Yang, CC Tseng, WJ Ju, LM Fu, MP Syu. Integrated microfluidic paper-based system for determination of whole blood albumin. Sensors and Actuators B: Chemical 273, 1091-1097, 2018.
67. Z Luo, T Lv, K Zhu, Y Li, L Wang, JJ Gooding, et al. Paper-based ratiometric fluorescence analytical devices towards point-of-care testing of human serum albumin. Angewandte Chemie International Edition 59, 3131-3136, 2020.
68. WE Owen, WL Roberts. Performance characteristics of an HPLC assay for urinary albumin. American Society for Clinical Pathology 124, 219-225, 2005.
69. P Yomthiangthae, O Chailapakul, W Siangproh. Rapid urinary albumin detection using a simple redox cycling process coupled with a paper-based device. Journal of Electroanalytical Chemistry 911, 116230, 2022.
70. BY Liao, CJ Chang, CF Wang, CH Lu, JK Chen. Controlled antibody orientation on Fe3O4 nanoparticles and CdTe quantum dots enhanced sensitivity of a sandwich-structured electrogenerated chemiluminescence immunosensor for the determination of human serum albumin. Sensors and Actuators B: Chemical 336, 129710, 2021.
71. AF Ogata, JM Edgar, S Majumdar, JS Briggs, SV Patterson, MX Tan. Virus-enabled biosensor for human serum albumin. Analytical chemistry 89, 1373-1381, 2017.
72. YH Chuang, YT Chang, KL Liu, HY Chang, TR Yew. Electrical impedimetric biosensors for liver function detection. Biosensors and Bioelectronics 28, 368-372, 2011.