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Ultrasensitive mediator-free electrochemical detection for biomarker proteins was achieved at low cost using a novel composite of Fe 3 O 4 nanoparticles loaded onto graphene oxide (GO) nano-sheets (Fe 3 O 4 @GO). This paramagnetic Fe 3 O 4 @GO composite (1µm size range) was decorated with antibodies against prostate specific antigen (PSA) and prostate specific membrane antigen (PSMA), and then used to first capture these biomarkers and then deliver them to an 8-sensor detection chamber of a microfluidic immunoarray. Screen-printed carbon sensors coated with electrochemically reduced graphene oxide (ERGO) and a second set of antibodies selectively capture the biomarker-laden Fe 3 O 4 @GO particles, which subsequently catalyze hydrogen peroxide reduction to detect PSA and PSMA. Accuracy was confirmed by good correlation between patient serum assays and enzyme-linked immuno-sorbent assays (ELISA). Excellent detection limits (LOD) of 15 fg/mL for PSA and 4.8 fg/mL for PSMA were achieved in serum. The LOD for PSA was 1000-fold better than the only previous report of PSA detection using Fe 3 O 4 . Dynamic ranges were easily tunable for concentration ranges encountered in serum samples by adjusting the Fe 3 O 4 @GO Concentration. Reagent cost was only $0.85 for a single 2-protein assay., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Accurate, sensitive, multiplexed detection of biomarker proteins holds significant promise for personalized cancer diagnostics. Here we describe the incorporation of a novel on-line chamber to capture cancer biomarker proteins on magnetic beads derivatized with 300,000 enzyme labels and 40,000 antibodies into a modular microfluidic immunoarray. Capture and detection chambers are produced from PDMS on machined molds and do not require lithography. Protein analytes are captured from serum or other biological samples in the stirred capture chamber on the beads held in place magnetically. The beads are subsequently washed free of sample components, and wash solutions sent to waste. Removal of the magnet and valve switching sends the magnetic bead-protein bioconjugates into a detection chamber where they are captured on 8 antibody-decorated gold nanoparticle-film sensors and detected amperometrically. Most steps in the immunoassay including protein capture, washing and measurement are incorporated into the device. In simultaneous assays, the microfluidic system gave ultralow detection limits of 5 fg mL(-1) for interleukin-6 (IL-6) and 7 fg mL(-1) for IL-8 in serum. Accuracy was demonstrated by measuring IL-6 and IL-8 in conditioned media from oral cancer cell lines and showing good correlations with standard ELISAs. The on-line capture chamber facilitates rapid, sensitive, repetitive protein separation and measurement in 30 min in a semi-automated system adaptable to multiplexed protein detection., (© 2013 Elsevier B.V. All rights reserved.)