Materials Goat anti-rabbit IgG and normal rabbit IgG-biotin were purchased from Sigma-Aldrich (St. Louis, MO) and Santa Cruz Biotechnology, Inc (Santa Cruz, CA), respectively. Proteins for the isomagnetophoretic immunoassays using three types of breast cancer biomarkers such as estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) were all purchased from R&D Systems (Minneapolis, MN). MCF-7 and SK-BR-3 breast cancer cell lines were provided from

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... TCC (Manassas, VA) and Korean cell line bank (Seoul, Korea), respectively. Superparamagnetic nanoparticle solution was obtained from Miltenyi Biotec (Bergisch Gladbach, Germany). Streptavidin/anti-biotin were conjugated with magnetic nanoparticles (MNPs) which consist of iron oxide with a diameter of about 50 nm, including polymer coating and proteins on their surface. 10 μm carboxylated microbead for isomagnetophoretic immunoassay of rabbit IgG-biotin and 6 μm fluorescent microbeads (yellowgreen: excitation-441 nm, emission-486 nm; yellow-orange: excitation-529 nm, emission-546 nm; bright blue: excitation-360 nm, emission-407 nm) for that of breast cancer biomarkers were bought from Polysciences, Inc (Warrington, PA). Gadolinium paramagnetic diethylenetriamine pentaacetic acid (Gd-DTPA) solution and Tween 20 were obtained from Sigma-Aldrich. Design and fabrication of an isomagnetophoretic immunoassay microchannel A chemical gradient in solution plays an important role in a wide range of biological and chemical applications. 1−7 During isomagnetophoresis, the gradient of Gd-DTPA solution is a key factor and should be maintained through an entire microchannel for a long time. Considered for this aspect, the microchannel for isomagnetophoresis was designed almost symmetrically for generating a linear concentration gradient. 6 The microchannels were fabricated by using a conventional poly(dimethylsiloxane) (PDMS) (Sylgard 184; Dow Corning, Midland, MI) molding process, and Ni microstructures were electroplated on a Pyrex glass wafer. Detailed process was followed according to the previously reported method. 8 Briefly, the negative photoresist SU-8 2025 (MicroChem Corp., Newton, MA) was used for a PDMS mold. The isomagnetophoretic immunoassay microchannel was designed to have one main microchannel Electronic Supplementary Material (ESI) for Lab on a Chip This journal is