Fig 1: Development of multiplexed magneto-nanosensor cytokine assays. (A) Optical image of an 8 × 8 array of magneto-nanosensor chip. The chip consists of 64 individually-accessible magneto-nanosensors. Inset: optical image of an individual magneto-nanosensor. (B) Schematic of sandwich protein immunoassays using magneto-nanosensors. (1) Different capture antibodies that specifically capture their target proteins were immobilized on different magneto-nanosensors. Bovine serum albumin (BSA) and biotinylated BSA (Biotin, not shown here) were also immobilized on different sensors as negative and positive controls, respectively. (2) Serum containing target proteins was added to the magneto-nanosensor chip, and allowed to bind to the corresponding capture antibodies. (3) After washing unbound proteins, biotinylated detection antibodies were added to the chip, and allowed to bind to the bound proteins to form sandwich complexes. (4) After unbound detection antibodies were washed, the chip was loaded into a reader station, and streptavidin-coated MNPs were introduced. The binding signals of MNPs to the sandwich complexes were monitored and recorded by the reader station. (C) Real-time monitoring of binding signals of MNPs. The baseline signals were measured after the chip was loaded into the reader station. Then, MNPs were introduced to the chip as indicated by the gray arrow. The binding signals were monitored until they reached their plateaus. Six recombinant proteins (GCSF: 1 ng/mL, TNF-α: 1 ng/mL, Eotaxin: 1 ng/mL, FLT3LG: 1 ng/mL, IL-6: 0.1 ng/mL, and VEGF: 0.1 ng/mL) were used as a test sample. The error bars represent standard deviations of 4 identical magneto-nanosensors. (D) Titration curves of 6-plex protein assays measured by multiplexed magneto-nanosensors. Six recombinant proteins were mixed to be at indicated final concentrations (10 and 1 ng/mL, and 100, 10, and 1 pg/mL), and measured using multiplexed magneto-nanosensor protein assays. Each data point is the average signal of 4 identical magneto-nanosensors, and titration curves (solid lines) were calculated using four parameter logistic (4-PL) regression (IL-6: R2 = 0.9997, GCSF: R2 = 0.9892, Eotaxin: R2 = 0.9936, TNF-α: R2 = 0.9950, FLT3LG: R2 = 0.9524, and VEGF: R2 = 0.9969).