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

Abstract

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.

Keywords: Cancer biomarkers; Immunoarray; Magentic beads; Microfluidics; Protein detection.

Fig. 1

Photographs of microfluidic system for on-line protein capture and detection using magnetic beads. (A) Capture chamber in which target proteins are captured on-line from the sample by heavily labeled HRP-antibody-magnetic beads to form protein-bead bioconjugates. These are washed, and then flowed into the detection chamber (B) in the modular microfluidic system (C). The magnet (D) traps bioconjugate beads in the channel during injection of sample and washing, and is removed for transfer of beads to the detection chamber.

Fig. 2

Conceptual strategy for ultrasensitive amperometric detection by microfluidic immunoarray. Protein analytes are captured on-line on Ab₂-MP-HRP bioconjugates in capture chamber. The protein-Ab₂-MP-HRP is then magnetically separated and washed in the chamber before being transported into the detection chamber.

Fig. 3

Amperometric responses for individual standard solutions of IL-6 and IL-8 in undiluted calf serum at −0.2 V vs. Ag/AgCl developed by injecting a mixture of 1 mM HQ and 0.1 mM H₂O₂ for (A) IL-6 and (C) IL-8, also showing calibration plots for (B) IL-6 and (D) IL-8 standards. Error bars represent standard deviations (n=8) for the eight electrodes of a single immunoarray.

Fig. 4

Amperometric responses for standard protein mixtures in undiluted calf serum at −0.2 V vs Ag/AgCl developed by injecting a mixture of 1 mM HQ and 0.1 mM H₂O₂ for (A) IL-6 and (C) IL-8, also showing calibration plots for (B) IL-6 and (D) IL-8. Error bars represent standard deviations (n=4) for the four IL-6 or IL-8 antibody-modified electrodes on a single immunoarray.

Fig. 5

Correlation plots of immunoarray assay results for conditioned media of HaCat, HN12, HN13 and Cal27 cell lines vs. standard ELISA assays for (A) IL-6 and (B) IL-8 (also see Fig. S6, SI). Error bars represent standard deviations for the immunoarray for n=4, and where not apparent are smaller than the point size.