本研究使用共沉澱法合成鐵奈米粒子，利用Thioglycolic acid改質鐵奈米粒子的表面，並得到大小約為27 nm的鐵奈米粒子，再加入EDC/NHS與Protein G反應形成醯胺鍵(Amide bond)，接著透過Protein G與IgG的生物親和性結合之後，使抗體與抗原反應，達到快速抓取並純化HDL的功能。
本研究成功合成每毫克鐵奈米粒子接枝0.7908 μg抗體，並且達到 49.46 μg/mg HDL之穩定抓取，與先前實驗室所合成之鐵奈米粒子HDL抓取率比較，以APTES 、L-Cystein作為保護基改質之鐵奈米粒子分別只有 3.5 μg/mL、12.5 μg/mL HDL之抓取率，因此本篇研究以Thioglycolic acid作為保護基改質之鐵奈米粒子的HDL抓取率最佳。
最後再將臨床取得之10名病人血漿檢體分別以磁珠純化以及傳統離心純化做DHR 123螢光抗氧化能力檢測作統計分析得出Pearson's 相關係數為0.92801，呈現強相關；電化學循環伏安法抗氧化能力檢測，也與傳統離心純化之HDL螢光抗氧化數值，統計分析得出Pearson's 相關係數為0.91082，呈現強相關。
最後將各分析而得的抗氧化數值與心血管疾病相關因子內皮前驅幹細胞菌落形成單位數量(Colony-Forming Units of Endothelial progenitor cell, EPC-CFUs)作分析，其呈現趨勢與文獻趨勢一致。
對此，本研究尋求一個能取代舊有耗時的純化HDL方式，藉由磁珠分離的方式與電化學分析的方式，來達到快速且能檢驗大量樣本之特性，期望能在未來做出可快速篩檢的檢測HDL功能的檢測工具。

In the study, we applied co-precipitation method to synthesize iron oxide magnrtite (Fe3O4) nanoparticles, followed by modifying Thioglycolic acid on FeNPs surface. Then, we used EDC/NHS crosslinking of carboxylates and protein G to form an amide bond. Finally, we synthesized Ig-G@Protein G@Thioglycolic acid@FeNPs structure with protein G bio-affinity, and antibodies will react with antigen then. Therefore, this study applied modified magnetic beads to rapidly purify HDL.
This study successfully synthesized the modified magnetic beads with 0.7908 μg of antibody on surface. Compared to APTES or L-Cystein modified magnetic beads, the thioglycolic acid modified magnetic beads could reach to higher capturing capacity of 49.46 μg/ml per mg of magnetic beads. However, the magnetic beads modified by APTES or L-Cystein only achieved 3.5 or 12.5 μg/mL per mg of magnetic beads. Therefore, the magnetic beads modified by thioglycolic acid in this study would have the best HDL capturing capacity.
Next, it was applied for 10 clinical specimens obtained by the Taipei Medical University. The magnetic beads purification and traditional centrifugal purification were used to make DHR 123 fluorescent antioxidant capacity. For statistical analysis, Pearson's r correlation coefficient is 0.92801, which showed high-strength correlation. In addition, the analysis between the capacity detection of electrochemical cyclic voltammetry antioxidant and traditional centrifugal purification of HDL fluorescence antioxidant values resulted in the Pearson's r correlation which is 0.91082, and it showed the high-strength correlation.
Finally, we conducted a further study between the oxidation value and the cardiovascular disease-related factors endothelial progenitor stem cells concentration, and the trend was consistent with literature trend.
To sum up, in this study, we aim to provide a new novel method to take place of ultracentrifugation method which takes time-consuming operation with separate pure HDL from serum and to replace the fluorescence detection method with electrochemical cyclic voltammetry. In these results, applying magnetic beads represents an effective method for HDL quality examination.

1. Rader, D.J., Molecular regulation of HDL metabolism and function: implications for novel therapies. The Journal of clinical investigation, 2006. 116(12): p. 3090-3100.
2. Navab, M., et al., A cell-free assay for detecting HDL that is dysfunctional in preventing the formation of or inactivating oxidized phospholipids. Journal of lipid research, 2001. 42(8): p. 1308-1317.
3. Rosenson, R.S., et al., Dysfunctional HDL and atherosclerotic cardiovascular disease. Nature Reviews Cardiology, 2016. 13(1): p. 48-60.
4. Hafiane, A. and J. Genest, High density lipoproteins: Measurement techniques and potential biomarkers of cardiovascular risk. BBA Clinical, 2015. 3: p. 175-188.
5. Kelesidis, T., et al., A biochemical fluorometric method for assessing the oxidative properties of HDL. 2011. 52(12): p. 2341-2351.
6. Van Roosbroeck, R., et al., Synthetic antiferromagnetic nanoparticles as potential contrast agents in MRI. 2014. 8(3): p. 2269-2278.
7. Hafiane, A. and J.J.B.c. Genest, High density lipoproteins: measurement techniques and potential biomarkers of cardiovascular risk. 2015. 3: p. 175-188.
8. Benjamin, E.J., P. Muntner, and M.S. Bittencourt, Heart disease and stroke statistics-2019 update: a report from the American Heart Association. Circulation, 2019. 139(10): p. e56-e528.
9. World Health Organization 2013-2020年預防和控制非傳染性疾病全球計畫. https://apps.who.int/gb/ebwha/pdf_files/WHA66/A66_R10-ch.pdf?ua=1].
10. Ministry of Health and Welfare 107年國人死因統計結果. 2019; https://www.mohw.gov.tw/cp-16-48057-1.html].
11. Goldstein, L. and S. Brown, The low-density lipoprotein pathway and its relation to atherosclerosis. Annual review of biochemistry, 1977. 46(1): p. 897-930.
12. Assmann, G. and A.M. Gotto Jr, HDL cholesterol and protective factors in atherosclerosis. Circulation, 2004. 109(23_suppl_1): p. III-8-III-14.
13. Taiwan Society of Lipids & Atheroscierosis 2009年台灣血脂異常防治共識-血指異常預防及診療臨床指引.
14. Padró, T., et al., Detrimental effect of hypercholesterolemia on high-density lipoprotein particle remodeling in pigs. Journal of the American College of Cardiology, 2017. 70(2): p. 165-178.
15. Bonroy, K., et al., Comparison of random and oriented immobilisation of antibody fragments on mixed self-assembled monolayers. Journal of Immunological Methods, 2006. 312(1): p. 167-181.
16. Navab, M., et al., Normal high density lipoprotein inhibits three steps in the formation of mildly oxidized low density lipoprotein: step 1. 2000. 41(9): p. 1481-1494.
17. Navab, M., et al., A cell-free assay for detecting HDL that is dysfunctional in preventing the formation of or inactivating oxidized phospholipids. 2001. 42(8): p. 1308-1317.
18. Navab, M., et al., Mechanisms of disease: proatherogenic HDL—an evolving field. Nature clinical practice Endocrinology & metabolism, 2006. 2(9): p. 504-511.
19. Ansell, B.J., et al., Inflammatory/antiinflammatory properties of high-density lipoprotein distinguish patients from control subjects better than high-density lipoprotein cholesterol levels and are favorably affected by simvastatin treatment. Circulation, 2003. 108(22): p. 2751-2756.
20. Undurti, A., et al., Modification of high density lipoprotein by myeloperoxidase generates a pro-inflammatory particle. Journal of Biological Chemistry, 2009. 284(45): p. 30825-30835.
21. Patel, S., et al., Reconstituted high-density lipoprotein increases plasma high-density lipoprotein anti-inflammatory properties and cholesterol efflux capacity in patients with type 2 diabetes. Journal of the American College of Cardiology, 2009. 53(11): p. 962-971.
22. Thévenot, D.R., et al., Electrochemical biosensors: recommended definitions and classification. Analytical Letters, 2001. 34(5): p. 635-659.
23. Kelesidis, T., et al., A high throughput biochemical fluorometric method for measuring lipid peroxidation in HDL. 2014. 9(11): p. e111716.
24. Villamena, F.A., Chapter 4 - Fluorescence Technique, in Reactive Species Detection in Biology, F.A. Villamena, Editor. 2017, Elsevier: Boston. p. 87-162.
25. Castelli, W.P., et al., Distribution of triglyceride and total, LDL and HDL cholesterol in several populations: a cooperative lipoprotein phenotyping study. 1977. 30(3): p. 147-169.
26. Sarafian, M.H., et al., Objective set of criteria for optimization of sample preparation procedures for ultra-high throughput untargeted blood plasma lipid profiling by ultra performance liquid Chromatography–Mass spectrometry. 2014. 86(12): p. 5766-5774.
27. Wieland, H. and D.J.J.o.L.R. Seidel, A simple specific method for precipitation of low density lipoproteins. 1983. 24(7): p. 904-909.
28. Seidel, D., P. Alaupovic, and R.J.T.J.o.c.i. Furman, A lipoprotein characterizing obstructive jaundice. I. Method for quantitative separation and identification of lipoproteins in jaundiced subjects. 1969. 48(7): p. 1211-1223.
29. Chen, J.K., S. Labrake‐Farmer, and D.B.J.J.o.c.p. McClure, Purified HDL‐apolipoproteins, A‐I and C‐III, substitute for HDL in promoting the growth of SV40‐transformed REF52 cells in serum‐free medium. 1986. 128(3): p. 413-420.
30. Goux, A., et al., Capillary gel electrophoresis analysis of apolipoproteins AI and A-II in human high-density lipoproteins. 1994. 218(2): p. 320-324.
31. Asahara, T., et al., Isolation of putative progenitor endothelial cells for angiogenesis. Science, 1997. 275(5302): p. 964-7.
32. Urbich, C. and S. Dimmeler, Endothelial progenitor cells: characterization and role in vascular biology. Circ Res, 2004. 95(4): p. 343-53.
33. Dzau, V.J., et al., Therapeutic potential of endothelial progenitor cells in cardiovascular diseases. Hypertension, 2005. 46(1): p. 7-18.
34. Pletcher, D. and F.C. Walsh, Industrial electrochemistry. 2012: Springer Science & Business Media.
35. Peljo, P. and H.H. Girault, Electrochemical potential window of battery electrolytes: the HOMO–LUMO misconception. Energy & Environmental Science, 2018. 11(9): p. 2306-2309.
36. Bockris, J.O.M. and A.K. Reddy, Modern electrochemistry 2B: electrodics in chemistry, engineering, biology and environmental science. Vol. 2. 2000: Springer Science & Business Media.
37. Potentiometric and Voltammetric Study of the
Ferricyanide/Ferrocyanide Redox Couple in Aqueous
Solution of Potassium Chloride. Physical Chemistry Laboratory II Chemistry Degree, 2019-2020.
38. Thévenot, D.R., et al., Electrochemical Biosensors: Recommended Definitions and Classification*. Analytical Letters, 2007. 34(5): p. 635-659.
39. Luo, X., et al., Application of Nanoparticles in Electrochemical Sensors and Biosensors. Electroanalysis, 2006. 18(4): p. 319-326.
40. Bakker, E. and M. Telting-Diaz, Electrochemical Sensors. Analytical Chemistry, 2002. 74(12): p. 2781-2800.
41. Shao, Y., et al., Graphene Based Electrochemical Sensors and Biosensors: A Review. Electroanalysis, 2010. 22(10): p. 1027-1036.
42. Chevion, S., M.A. Roberts, and M. Chevion, The use of cyclic voltammetry for the evaluation of antioxidant capacity. Free Radical Biology and Medicine, 2000. 28(6): p. 860-870.
43. Heineman, P.T.K.W.R., Cyclic Voltammetry. Journal of Chemical Education: p. 772.
44. Lasia, A., Electrochemical impedance spectroscopy and its applications, in Modern aspects of electrochemistry. 2002, Springer. p. 143-248.
45. Orazem, M.E. and B. Tribollet, Electrochemical impedance spectroscopy. 2017: John Wiley & Sons.
46. Zhu, C., et al., Electrochemical Sensors and Biosensors Based on Nanomaterials and Nanostructures. Analytical Chemistry, 2015. 87(1): p. 230-249.
47. Campuzano, S., et al., Motion-driven sensing and biosensing using electrochemically propelled nanomotors. Analyst, 2011. 136(22): p. 4621-30.
48. Wang, Y., et al., Electrochemical sensors for clinic analysis. Sensors, 2008. 8(4): p. 2043-2081.
49. Yang, Y., et al., Aptamer-functionalized carbon nanomaterials electrochemical sensors for detecting cancer relevant biomolecules. Carbon, 2018. 129: p. 380-395.
50. Wang, J., et al., Performance of screen-printed carbon electrodes fabricated from different carbon inks. 1998. 43(23): p. 3459-3465.
51. Sun, S. and H.J.J.o.t.A.C.S. Zeng, Size-controlled synthesis of magnetite nanoparticles. 2002. 124(28): p. 8204-8205.
52. Friák, M., A. Schindlmayr, and M. Scheffler, Ab initio study of the half-metal to metal transition in strained magnetite. New journal of physics, 2007. 9(1): p. 5.
53. Cullity, B.D. and C.D. Graham, Introduction to magnetic materials. 2011: John Wiley & Sons.
54. Bychkov, Y.A. and E.I.J.J.o.p.C.S.s.p. Rashba, Oscillatory effects and the magnetic susceptibility of carriers in inversion layers. 1984. 17(33): p. 6039.
55. Meissner, W. and R. Ochsenfeld, Ein neuer effekt bei eintritt der supraleitfähigkeit. Naturwissenschaften, 1933. 21(44): p. 787-788.
56. Kittel, C., Introduction to Solid State Physics, 6th edn., 1986. Wiley.
57. Ngo, A., et al., Nanoparticles of: Synthesis and superparamagnetic properties. 1999. 9(4): p. 583-592.
58. Xuan, S., et al., Tuning the grain size and particle size of superparamagnetic Fe3O4 microparticles. 2009. 21(21): p. 5079-5087.
59. Valenzuela, R., et al., Influence of stirring velocity on the synthesis of magnetite nanoparticles (Fe3O4) by the co-precipitation method. Journal of Alloys and Compounds, 2009. 488(1): p. 227-231.
60. Rani, S. and G.D. Varma, Superparamagnetism and metamagnetic transition in Fe3O4 nanoparticles synthesized via co-precipitation method at different pH. Physica B: Condensed Matter, 2015. 472: p. 66-77.
61. Xu, H., et al., Development of High Magnetization Fe3O4/Polystyrene/Silica Nanospheres via Combined Miniemulsion/Emulsion Polymerization. Journal of the American Chemical Society, 2006. 128(49): p. 15582-15583.
62. Zhou, K., et al., Preparation and application of mediator‐free H2O2 biosensors of graphene‐Fe3O4 composites. 2011. 23(4): p. 862-869.
63. Rusmini, F., Z. Zhong, and J. Feijen, Protein Immobilization Strategies for Protein Biochips. Biomacromolecules, 2007. 8(6): p. 1775-1789.
64. Feeney, R.E., G. Blankenhorn, and H.B. Dixon, Carbonyl-amine reactions in protein chemistry, in Advances in protein chemistry. 1975, Elsevier. p. 135-203.
65. Rusmini, F., Z. Zhong, and J.J.B. Feijen, Protein immobilization strategies for protein biochips. 2007. 8(6): p. 1775-1789.
66. Yan, Q., et al., EDC/NHS activation mechanism of polymethacrylic acid: anhydride versus NHS-ester. RSC Advances, 2015. 5(86): p. 69939-69947.
67. Wang, C., et al., Different EDC/NHS activation mechanisms between PAA and PMAA brushes and the following amidation reactions. Langmuir, 2011. 27(19): p. 12058-68.
68. Scientific, T., EDC reaction chemistry Crosslinking Technical Handbook. p. 4.
69. MacCallum, R.M., A.C.R. Martin, and J.M. Thornton, Antibody-antigen Interactions: Contact Analysis and Binding Site Topography. Journal of Molecular Biology, 1996. 262(5): p. 732-745.
70. Edelman, G.M.J.S., Antibody structure and molecular immunology. 1973. 180(4088): p. 830-840.
71. Arora, M., Cell culture media: a review. Mater Methods, 2013. 3(175): p. 24.
72. Antigen-binding site. Available from: https://www.britannica.com/science/antigen-binding-site.
73. Compton, S.J. and C.G.J.A.b. Jones, Mechanism of dye response and interference in the Bradford protein assay. 1985. 151(2): p. 369-374.
74. Baicu, S.C. and M.J. Taylor, Acid-base buffering in organ preservation solutions as a function of temperature: new parameters for comparing buffer capacity and efficiency. (0011-2240 (Print)).
75. Bae, Y.M., et al., Study on orientation of immunoglobulin G on protein G layer. Biosensors and Bioelectronics, 2005. 21(1): p. 103-110.
76. Dias, A.M.G.C., et al., A biotechnological perspective on the application of iron oxide magnetic colloids modified with polysaccharides. Biotechnology Advances, 2011. 29(1): p. 142-155.
77. Jannah, N.R. and D. Onggo, Synthesis of Fe3O4 nanoparticles for colour removal of printing ink solution. Journal of Physics: Conference Series, 2019. 1245.
78. Wilson, D. and M.A. Langell, XPS analysis of oleylamine/oleic acid capped Fe3O4 nanoparticles as a function of temperature. Applied Surface Science, 2014. 303: p. 6-13.
79. Ouyang, L., et al., A surface-enhanced Raman scattering method for detection of trace glutathione on the basis of immobilized silver nanoparticles and crystal violet probe. Anal Chim Acta, 2014. 816: p. 41-9.
80. Gruian, C., et al., FTIR and XPS studies of protein adsorption onto functionalized bioactive glass. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2012. 1824(7): p. 873-881.
81. Rossi, F., et al., HDL cholesterol is a strong determinant of endothelial progenitor cells in hypercholesterolemic subjects. Microvascular research, 2010. 80(2): p. 274-279.
82. Gordts, S.C., et al., Lipid lowering and HDL raising gene transfer increase endothelial progenitor cells, enhance myocardial vascularity, and improve diastolic function. PLoS One, 2012. 7(10).
83. Shih, C.-M., et al., Dysfunctional high density lipoprotein failed to rescue the function of oxidized low density lipoprotein-treated endothelial progenitor cells: a novel index for the prediction of HDL functionality. Translational Research, 2019. 205: p. 17-32.