High-efficiency bioaffinity separation of cells and proteins using novel thermoresponsive biotinylated magnetic nanoparticles |
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Authors: | Noriyuki Ohnishi Hirotaka Furukawa Hata Hideyuki Jing-Ming Wang Chung-Il An Eiichiro Fukusaki Kazunori Kataoka Katsuhiko Ueno Akihiko Kondo |
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Institution: | (1) Magnabeat Inc., 5-1Goi-kaigan, Ichihara, 290-8551 Chiba, Japan;(2) Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, 565-0871 Osaka, Japan;(3) Department of Materials Sciences Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, 113-8656 Tokyo, Japan;(4) Japan Industrial Technology Association AIST Central 4-10, Higashi, Tsukuba, 305-0046 Ibaraki, Japan;(5) Department of Chemical Sciences, Engineering Kobe University, 1-1 Rokkodai, Nada, 657-8501 Kobe, Japan;(6) Department of Chemical Sciences, Engineering Kobe University, 1-1 Rokkodai, Nada, 657-8501 Kobe, Japan;(7) Department of Biotechnology Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, 565-0871 Osaka, Japan;(8) Present address: Department of Biomedical Engineering, University of California, 431 E. Health Sciences Dr., 95616 Davis, CA |
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Abstract: | Thermoresponsive magnetic nanoparticles with an upper critical solution temperature (UCST) in aqueous solution were synthesized
for the first time. Named Therma-Max, the material was synthesized by redox copolymerization of N-acryloyl glycinamide with a monomer form of biotin using methacrylated dextran-magnetite. While the resulting Therma-Max
was completely dispersed at temperatures above the UCST (18°C) and could not be separated by a permanent magnet, it was rapidly
flocculated when the temperature fell below the UCST and was easily separated by a permanent magnet. The flocculated particles
dispersed completely when the temperature was raised to above the UCST. Because biotin was immobilized on the Therma-Max,
avidin and antibodies were subsequently immobilized with good efficiency. Furthermore, transiently transfected Arabidopsis protoplasts, which have surface display of CD4 antigen, were efficiently captured and enriched by using a biotinylated anti-CD4
antibody in combination with avidin-conjugated Therma-Max. Also, the silkworm storage protein (SP2) was efficiently separated
from the silkworm hemolymph by using biotinylated anti-IgG antibody and anti-SP2 antibody in combination with avidin-conjugated
Therma-Max. In both cases, it was confirmed that specificity and adsorption capacity were markedly improved by converting
the conventional micro-size fine magnetic particles to nano-size particles. These results show the potential of Therma-Max
with a UCST in bioaffinity separation of cells and biomolecules. |
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Keywords: | Magnetic separation bioaffinity separation magnetic nanoparticles thermoresponsive polymer avidin biotin |
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