Recent advances in nanomaterials-assisted assays by ourselves (Rusling et al., 2014) and others (Zhang et al., 2013; Meissner et al., 2015; Das and Kelley, 2011; Kelley et al., 2014; Lam et al., 2013) have improved multiplexed protein sensitivity up to 1000-fold compared to earlier established commercial assays. Graphene, Biomarker proteins, Immunoarray, Microfluidics 1. Introduction Accurate, sensitive, cost-effective measurements of multiple proteins in patient samples are critical for progress in clinical detection and monitoring of cancer (Kulasingam and Diamandis, 2008; de Gramont et al., 2015). Recent advances in nanomaterials-assisted assays by ourselves (Rusling et al., 2014) and others (Zhang et al., 2013; Meissner et al., 2015; Das and Kelley, 2011; Kelley et al., 2014; Lam et al., 2013) have improved multiplexed protein sensitivity up to 1000-fold compared to earlier established commercial assays. However, cost and assay complexity still raise barriers to translation of effective protein-based cancer diagnostics into widespread clinical and point-of-care (POC) use (Rusling, 2013). Enzyme-linked immunosorbent assays PSI-7409 [ELISA] have long been the gold standard for clinical protein determinations, and typically achieve detection limits of 1C10 pg/mL for serum proteins (Lequin, 2005). ELISA employs enzyme labels attached to PSI-7409 detection antibodies that have been pre-captured on an antibody-decorated well plate to measure proteins using optical detection of a colored enzyme reaction product. Many variations on this sandwich assay format, often utilizing magnetic beads, have been used in more modern, multiplexed commercial protein detection kits (Zhang et al., Rabbit Polyclonal to MRPS18C 2013; Rusling et al., 2014; Dixit et al., 2016). In our recent work, magnetic beads loaded with massive numbers of horseradish peroxidase (HRP) labels and detection antibodies were used to achieve ultrasensitive multiplexed protein detection at levels as low as 5 fg/mL (Otieno et al., 2014; Krause et al., 2013). Iron oxide (Fe3O4) nanoparticles have peroxidase-like activity for catalysis of hydrogen peroxide (H2O2) reduction, which can be optically monitored by following the H2O2Cassisted oxidation of 3,3,5,5-tetramethylbenzidine (TMB) or o-phenylendiamine (OPD) (Zhang et al., 2008; Chang et al., 2009; Liu et al., 2014; Wei and Wang, 2008). Peroxidase-like activity of Fe3O4 nanoparticles for electrochemical detection of hydrogen peroxide has been enhanced by incorporation with other materials like platinum (Ma et al., 2013), graphene derivatives (Fang et al., 2014; Liu et al., 2011; Yang et al., 2014), platinum/palladium (Sun et al., 2012), and gold (Sun et al., 2013). Chitosan coated Fe3O4 nanoparticles were used in colorimetric ELISA to detect of carcinoembryonic antigen with 1 ng/mL LOD (Gao et PSI-7409 al., 2008) and thrombin with LOD of 1 1 nM (Zhang et al., 2010). Dumbbell-like gold-Fe3O4 was used for electrochemical detection of prostate specific antigen (PSA) with 5 pg/mL LOD and dynamic range of 0.01C10 ng/mL (Wei et al., 2010). Loading Fe3O4 nanoparticles onto graphene oxide (GO) nanosheets improved wettability and dispersion of the composite material (Dong et al., 2012; Wu et al., 2013). Fe3O4 loaded on GO was previously synthesized and utilized for removal of cobalt (Liu et al., 2011), hydrocarbons (Han et al., 2012) and organic dyes (Jiao et al., 2015) from environmental samples. Electrostatic interactions between negatively charged graphene oxide sheets and Fe3O4 nanoparticles coated with positively charged poly(diallydimethylammonium chloride) (PDDA) were used to assemble core-shell Fe3O4@GO particles (Wei et al., 2012). In this paper, we describe the first preparation and use of multiple-Fe3O4 nanoparticles assembled onto graphene oxide nanosheets and decorated with antibodies (Ab2) to first isolate biomarker proteins from the sample under magnetic control, and then electrochemically detect them at ultra-high sensitivity using the intrinsic peroxidase activity. Electrostatic interactions between intact GO sheets and PDDA-coated Fe3O4 nanoparticles (NP) provide precise control over the number of Fe3O4 NPs per GO sheet, and can be used to optimize the dynamic range of the assay. Here, Ab2-Fe3O4@GO particles were evaluated as substitutes for HRP-Ab2-magnetic beads (MB) (1 m diam.) in an 8-sensor microfluidic system featuring off-line capture of PSA and prostate specific membrane antigen (PSMA) on magnetic particles,.