On-line protein capture on magnetic beads for ultrasensitive microfluidic immunoassays of cancer biomarkers

• A modular microfluidic system for multiple protein detection is described that incorporates on-line magnetic bead capture of proteins.
• Massively labeled, antibody laden magnetic beads capture the proteins and deliver them to a sensor array.
• Detection of two proteins in serum was achieved with detection limits of 5 fg mL−1.
• Good correlations were found with standard ELISA for two proteins in conditioned media of oral cancer cell lines.
• Microfluidic chambers are fabricated with non-lithographic procedures and coupled to inexpensive commercial components.

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.