Chemiluminescence detection in integrated post‐separation reactors for microchip‐based capillary electrophoresis and affinity electrophoresis

Abstract

Chemiluminescence (CL) detection based on the horseradish peroxidase (HRP) catalyzed reaction of luminol with peroxide was investigated as a post-separation detection scheme for microchip-based capillary electrophoresis. An integrated injector, separator and post-separation reactor was fabricated on planar glass wafers. The fluorescein conjugate of HRP (HRP-F1) was used as a sample for optimization of the CL detector response. In devices etched 10 μm deep, with an aluminum mirror integrated onto the backside of the detection zone to enhance collection efficiency, the detection limit, estimated at 3 standard deviations (SD) above background noise, for 1 nL injected sample plugs was 35 nM in HRP-F1. In devices etched 40 μm deep, 8 nL plugs gave a detection limit of 7 nM. Separation and CL detection of the products of an immunological reaction of a F(ab')₂ fragment of the HRP conjugate of goat anti-mouse immunoglobulin G (IgG) with mouse IgG was performed on-chip. A linear calibration curve was obtained for the decrease in peak height of the HRP conjugate (53 μg/mL) with increasing mouse IgG (0–60 μg/mL). When microperoxidase was used as an internal standard, the R² value of a linear least-squares fit was 0.9867, and the relative errors in the slope and intercept were ± 5.8 and ± 1.3%, respectively.