近年來有許多研究指出胭脂紅色素會引起過敏及哮喘的症狀，而其被大量廣泛應
用於日常食品中，因此食用色素的毒性研究逐漸引起大眾的關注；斑馬魚 (Danio rerio)
因其培養周期短以及與人類基因相似等諸多優點，在藥物以及毒物測試領域中已逐漸
成為主流動物模型；早期對於斑馬幼魚的研究大多使用瓊脂糖作為固定的機制，其方
法不僅實驗效率低且會對斑馬魚造成永久性的損傷。本研究提出利用 SMA 微致動器
結合微流體裝置控制流道壁面來有效的達到固定斑馬幼魚的目的，並探討胭脂紅色素
溶液對其行為的影響。實驗中選用 4、5、6 d.p.f. (Days Post Fertilization) 的斑馬幼魚
作為實驗動物模型，並且分為 Control 組以及 Red 0.2‰組，量化斑馬幼魚尾鰭擺動時
造成的環流量變化。實驗數據顯示，5 d.p.f.- Red 0.2 ‰組的環流量平均小於 Control
組 40%，6 d.p.f.- Red 0.2‰組的環流量平均大於 Control 組 53%，且透過統計學方法
比較發現在 5、6 d.p.f.暴露在胭脂紅色素中的斑馬幼魚與控制組間存在顯著性差異
(p<0.05) 。本研究的成果在於證實了胭脂紅色素對斑馬幼魚的行為影響，提供了未來
在評估胭脂紅食用色素時的參考依據，並且未來將此微流體裝置結合其他觀測的設計
來達到更全面的評估各項仍未測定的藥物、毒物測試等等。

Recent studies have highlighted that food additives induce mutation and highly carcinogenic effects on humans. Cochineal red, which has been widely used on a daily basis in the form of food colors, cosmetics, and other products, has been shown to be harmful to human health and requires further examination. The zebrafish (Danio rerio), which has several benefits such as rapid growth, a short rearing period, and a high genetic resemblance to humans, has emerged as a prominent animal model for drug testing. The conventional approach of partially immobilizing zebrafish larvae with agarose, on the other hand, causes persistent defects, reduces experiment effectiveness, and causes zebrafish larvae morbidity. To advent the zebrafish larvae experiment, this research proposes microfluidics with shape memory alloy (SMA) actuation mechanism towards rapid immobilization effective control of zebrafish. The effectiveness of the proposed device for drug testing was further investigated by evaluating the effects of cochineal red (2%) on behavioral parameters of zebrafish larvae. The experiment data showed the circulation of 5 d.p.f.- Red 0.02‰ group decreased by 40%, but the circulation of 6 d.p.f.- Red 0.02‰ group significantly increased by 53% compared to the control group. Thus, we can find Cochineal Red’s effects on zebrafish is more significant in the early stages. The result is supported by a statistically significant difference (*p<0.05) in the 5, 6 d.p.f. groups. Further, the proposed microfluidic device can be used towards various biomedical research focusing drug discovery and toxicity evaluation.

[1] R. L. Valluzzi, V. Fierro, S. Arasi, M. Mennini, V. Pecora, and A. Fiocchi, "Allergy to food additives," Current Opinion in Allergy and Clinical Immunology, vol. 19, no. 3, pp. 256-262, 2019.

[2] B. Mepham, "Food additives: an ethical evaluation," British Medical Bulletin, vol. 99, no. 1, pp. 7-23, 2011.

[3] A. J. Zillich, R. J. Kuhn, and T. J. Petersen, "Skin discoloration with blue food coloring," (in eng), Ann Pharmacother, vol. 34, no. 7-8, pp. 868-870, 2000 Jul-Aug 2000.

[4] P. Amchova, H. Kotolova, and J. Ruda-Kucerova, "Health safety issues of synthetic food colorants," Regulatory Toxicology and Pharmacology, vol. 73, no. 3, pp. 914-922, 2015/12/01/ 2015.

[5] N. Mehedi et al., "Reproductive toxicology of tartrazine (FD and C Yellow No. 5) in Swiss albino mice," (in English), American Journal of Pharmacology and Toxicology, vol. 4, no. 4, pp. 130-135, 2009.

[6] D. K. Doguc, B. M. Ceyhan, M. Ozturk, and F. Gultekin, "Effects of maternally exposed colouring food additives on cognitive performance in rats," Toxicology and Industrial Health, vol. 29, no. 7, pp. 616-623, 2013/08/01 2012.

[7] J. F. Borzelleca, K. Depukat, and J. B. Hallagan, "Lifetime toxicity/carcinogenicity studies of FD & C blue No. 1 (Brilliant blue FCF) in rats and mice," Food and Chemical Toxicology, vol. 28, no. 4, pp. 221-234, 1990/01/01/ 1990.

[8] Y. Gibert, M. Trengove, and A. Ward, "Zebrafish as a genetic model in pre-clinical drug testing and screening," Current medicinal chemistry, vol. 20, no. 19, pp. 2458-2466, 2013.

[9] R. Geisler, A. Köhler, T. Dickmeis, and U. Strähle, "Archiving of zebrafish lines can reduce animal experiments in biomedical research," EMBO reports, vol. 18, no. 1, pp. 1-2, 2017.

[10] S. Guo, "Using zebrafish to assess the impact of drugs on neural development and function," Expert opinion on drug discovery, vol. 4, no. 7, pp. 715-726, 2009.

[11] A. M. Stewart, O. Braubach, J. Spitsbergen, R. Gerlai, and A. V. Kalueff, "Zebrafish models for translational neuroscience research: from tank to bedside," Trends in neurosciences, vol. 37, no. 5, pp. 264-278, 2014.

[12] P. McGrath and C.-Q. Li, "Zebrafish: a predictive model for assessing drug-induced toxicity," Drug discovery today, vol. 13, no. 9-10, pp. 394-401, 2008.

[13] P. Asharani, Y. L. Wu, Z. Gong, and S. Valiyaveettil, "Toxicity of silver nanoparticles in zebrafish models," Nanotechnology, vol. 19, no. 25, p. 255102, 2008.

[14] P. K. Chan, C. C. Lin, and S. H. Cheng, "Noninvasive technique for measurement of heartbeat regularity in zebrafish (Danio rerio) embryos," BMC biotechnology, vol. 9, no. 1, pp. 1-10, 2009.

[15] A. Nady, A. R. Peimani, G. Zoidl, and P. Rezai, "A microfluidic device for partial immobilization, chemical exposure and behavioural screening of zebrafish larvae," (in English), Lab on a Chip, Article vol. 17, no. 23, pp. 4048-4058, Dec 2017.

[16] C. EUR, "New risks from ancient food dyes: cochineal red allergy," European annals of allergy and clinical immunology, p. 232, 2014.

[17] J. F. D'Mello, Food safety: contaminants and toxins. CABI, 2003.

[18] E. Current, "approved additives and their E Numbers," Food Standards Agency, 2014.

[19] K. Howe et al., "The zebrafish reference genome sequence and its relationship to the human genome," Nature, vol. 496, no. 7446, pp. 498-503, 2013.

[20] R. Jones, "Let sleeping zebrafish lie: a new model for sleep studies," PLoS Biol, vol. 5, no. 10, p. e281, 2007.

[21] G. J. Lieschke and P. D. Currie, "Animal models of human disease: zebrafish swim into view," Nature Reviews Genetics, vol. 8, no. 5, pp. 353-367, 2007.

[22] K. Dooley and L. I. Zon, "Zebrafish: a model system for the study of human disease," Current opinion in genetics & development, vol. 10, no. 3, pp. 252-256, 2000.

[23] R. Willemsen, S. v. t. Padje, J. C. van Swieten, and B. A. Oostra, "Zebrafish (Danio rerio) as a Model Organism for Dementia," in Animal Models of Dementia, P. P. De Deyn and D. Van Dam, Eds. Totowa, NJ: Humana Press, 2011, pp. 255-269.

[24] P. K. Tan et al., "Evaluation of gene expression measurements from commercial microarray platforms," (in eng), Nucleic Acids Res, vol. 31, no. 19, pp. 5676-5684, 2003.

[25] Z. Yang et al., "A highly conserved regulatory element controls hematopoietic expression of GATA2 in zebrafish," BMC Developmental Biology - BMC DEV BIOL, vol. 7, pp. 97-13, 08/20 2007.

[26] C.-H. Chen et al., "Transient laminin beta 1a induction defines the wound epidermis during zebrafish fin regeneration," PLoS Genet, vol. 11, no. 8, p. e1005437, 2015.

[27] Y. Roussel, M. Paradis, S. F. Gaudreau, B. W. Lindsey, and T. V. Bui, "Spatiotemporal transition in the role of synaptic inhibition to the tail beat rhythm of developing larval zebrafish," Eneuro, vol. 7, no. 1, 2020.

[28] L. Godinho, "Imaging zebrafish development," Cold Spring Harbor Protocols, vol. 2011, no. 7, p. pdb. prot5647, 2011.

[29] P. J. Rombough, "Ontogenetic changes in the toxicity and efficacy of the anaesthetic MS222 (tricaine methanesulfonate) in zebrafish (Danio rerio) larvae," Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, vol. 148, no. 2, pp. 463-469, 2007/10/01/ 2007.

[30] J. D. Jontes and M. R. Emond, "Fluorescence imaging of transgenic zebrafish embryos," Cold Spring Harbor Protocols, vol. 2012, no. 5, p. pdb. prot069245, 2012.

[31] Y.-c. Shen et al., "A student team in a University of Michigan biomedical engineering design course constructs a microfluidic bioreactor for studies of zebrafish development," Zebrafish, vol. 6, no. 2, pp. 201-213, 2009.

[32] J. Akagi et al., "Miniaturized embryo array for automated trapping, immobilization and microperfusion of zebrafish embryos," PloS one, vol. 7, no. 5, p. e36630, 2012.

[33] J. Akagi et al., "Fish on chips: Microfluidic living embryo array for accelerated in vivo angiogenesis assays," Sensors and Actuators B: Chemical, vol. 189, pp. 11-20, 2013.

[34] W. Wang, X. Liu, D. Gelinas, B. Ciruna, and Y. Sun, "A fully automated robotic system for microinjection of zebrafish embryos," PloS one, vol. 2, no. 9, p. e862, 2007.

[35] L. L. Bischel, B. R. Mader, J. M. Green, A. Huttenlocher, and D. J. Beebe, "Zebrafish Entrapment By Restriction Array (ZEBRA) device: a low-cost, agarose-free zebrafish mounting technique for automated imaging," Lab on a Chip, vol. 13, no. 9, pp. 1732-1736, 2013.

[36] X. Lin et al., "High-throughput mapping of brain-wide activity in awake and drug-responsive vertebrates," Lab on a chip, vol. 15, no. 3, pp. 680-689, 2015.

[37] M. Newman, E. Ebrahimie, and M. Lardelli, "Using the zebrafish model for Alzheimer’s disease research," Frontiers in genetics, vol. 5, p. 189, 2014.

[38] F. Yang, C. Gao, P. Wang, G.-J. Zhang, and Z. Chen, "Fish-on-a-chip: Microfluidics for zebrafish research," Lab on a Chip, vol. 16, no. 7, pp. 1106-1125, 2016.

[39] H.-S. A. P. M. Alloys, "Properties and selection: nonferrous alloys and special-purpose materials," 1990.

[40] M. Kohl, B. Krevet, and E. Just, "SMA microgripper system," Sensors and Actuators A: Physical, vol. 97-98, pp. 646-652, 2002/04/01/ 2002.

[41] M. H. Elahinia and H. Ashrafiuon, "Nonlinear Control of a Shape Memory Alloy Actuated Manipulator," Journal of Vibration and Acoustics, vol. 124, no. 4, pp. 566-575, 2002.

[42] S. Maeda, K. Abe, K. Yamamoto, O. Tohyama, and H. Ito, "Active endoscope with SMA (Shape Memory Alloy) coil springs," in Proceedings of Ninth International Workshop on Micro Electromechanical Systems, 1996, pp. 290-295.

[43] C. Liang and C. Rogers, "Design of shape memory alloy actuators," 1992.

[44] J. M. Jani, M. Leary, A. Subic, and M. A. Gibson, "A review of shape memory alloy research, applications and opportunities," Materials & Design (1980-2015), vol. 56, pp. 1078-1113, 2014.

[45] A. J. Hill, H. Teraoka, W. Heideman, and R. E. Peterson, "Zebrafish as a model vertebrate for investigating chemical toxicity," (in English), Toxicol. Sci., Review vol. 86, no. 1, pp. 6-19, Jul 2005.

[46] R. L. Vaz, T. F. Outeiro, and J. J. Ferreira, "Zebrafish as an animal model for drug discovery in Parkinson’s disease and other movement disorders: a systematic review," Frontiers in neurology, vol. 9, p. 347, 2018.

[47] U. Strähle et al., "Zebrafish embryos as an alternative to animal experiments—a commentary on the definition of the onset of protected life stages in animal welfare regulations," Reproductive Toxicology, vol. 33, no. 2, pp. 128-132, 2012.

[48] Y.-F. Wang et al., "Edible additive effects on zebrafish cardiovascular functionality with hydrodynamic assessment," Scientific reports, vol. 10, no. 1, pp. 1-8, 2020.

[49] S. Subendran, C.-W. Kang, and C.-Y. Chen, "Comprehensive Hydrodynamic Investigation of Zebrafish Tail Beats in a Microfluidic Device with a Shape Memory Alloy," Micromachines, vol. 12, no. 1, p. 68, 2021.

[50] M. A. Eddings, M. A. Johnson, and B. K. Gale, "Determining the optimal PDMS–PDMS bonding technique for microfluidic devices," Journal of Micromechanics and Microengineering, vol. 18, no. 6, p. 067001, 2008.

[51] R. J. Adrian, "Twenty years of particle image velocimetry," Experiments in fluids, vol. 39, no. 2, pp. 159-169, 2005.

[52] E. G. Drucker and G. V. Lauder, "Locomotor forces on a swimming fish: three-dimensional vortex wake dynamics quantified using digital particle image velocimetry," Journal of Experimental Biology, vol. 202, no. 18, pp. 2393-2412, 1999.

[53] C. D. Meinhart, S. T. Wereley, and J. G. Santiago, "PIV measurements of a microchannel flow," Experiments in fluids, vol. 27, no. 5, pp. 414-419, 1999.

[54] R. Lindken, M. Rossi, S. Große, and J. Westerweel, "Micro-particle image velocimetry (µPIV): recent developments, applications, and guidelines," Lab on a Chip, vol. 9, no. 17, pp. 2551-2567, 2009.

[55] L. Adrian, R. J. Adrian, and J. Westerweel, Particle image velocimetry (no. 30). Cambridge university press, 2011.