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12th International Symposium on Bioluminescence & Chemiluminescence |
Symposium abstracts:
Roda, B.1, Guardigli, M.2, Zattoni, A.1, Melucci, D.1, Reschiglian, P.1, Roda, A.2
1. Department of Chemistry "G. Ciamician", University of Bologna, Via Selmi 2, 40126 Bologna, Italy
Email: barbara@ciam.unibo.it
2. Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy
Separation of micronsize particles of biological and diagnostic interest can
be performed by gravitational field-flow fractionation (GFFF). A drawback of
GFFF for bio-applications lies in the detection of separated particles, which
is usually performed through UV-Vis detectors, with relative low sensitivity.
Chemiluminescence (CL) offers ultrasensitive detection tool.
In this work, horseradish peroxidase (HRP) was chemically immobilized on polystyrene
beads (PS) and used as a model for real-time GFFF-CL imaging system. The GFFF
channel was placed in front of a back-illuminated double Peltier cooled CCD
(Night Owl, Berthold Systems, Aliquippa, PA). Separation was visualized while
particles were swept down the separation channel. Image was acquired through
the light emitted from the H2O2/luminol/enhancer CL system triggered by HRP-PS.
The possibility to visualize the overall separation pattern at very low detection
limits, down to few molecules/pixel, allows not only to simultaneously detect
separated fractions (i.e. free HRP vs. HRP bound on PS of different size) but
also to optimize the separation mechanism. The kinetic profile and shape of
the eluting bands are markers of separation linearity. Band shape analysis allows
to get more insights into GFFF basics and, therefore, to improve GFFF-CL by
mobile phase/CL cocktail /channel wall composition optimization.
This
is a preprint of an article accepted for publication in Luminescence: Copyright
2001 John
Wiley & Sons, Ltd (Wiley website)