Chemiluminescence-based detection: principles and analytical applications in flowing streams and in immunoassays

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 microm 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 microm 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 microg/mL) with increasing mouse IgG (0-60 microg/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.

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

Luminol-type chemiluminescence derivatization reagents for liquid chromatography and capillary electrophoresis.

On-line electrogenerated Ru(bpy)33+ chemiluminescent detection of β-blockers separated with capillary electrophoresis

Chemiluminescence detection in capillary electrophoresis

On-line chemiluminescence detection of proteins separated by capillary zone electrophoresis

From a high performance capillary electrophoretic experiment in a phosphate buffer (pH 3.5), Eosine Y was found for the first time to migrate together with protein in a capillary tube as a supramolecular complex. This finding gave us not only the possibility to overcome the serious problems in protein estimation, but also the possibility to measure the Eosine Y in place of protein much more sensitively by several methods. However the results obtained so far were not so good

Junichi Yokogi

Papers

On-line chemiluminescence detection of proteins separated by capillary zone electrophoresis

Separation and detection of protein as a supramolecular complex using capillary electrophoresis