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Controlled enzyme-immobilisation on capillaries for microreactors for peptide mapping

Bossi, A., Guizzardi, L., D’Acunto, M. R., Righetti, P. G.
Analytical and bioanalytical chemistry 2004 v.378 no.7 pp. 1722-1728
electrophoresis, immobilized enzymes, peptide mapping, proteins, screening, silica, trypsin
In the present paper, the covalent immobilisation of the digesting enzyme trypsin has been achieved through photo-immobilisation on a portion of a silica capillary, thus leading to the construction of a capillary electrophoretic (CE)-microreactor for peptide mapping. The CE-microreactor is characterised by being a single piece, thus ensuring no fluidic or electrical leakage. The enzyme was immobilised with a surface density of 15.8 μg/cm², the stability was high (80% after 38 days) and the rate of conversion was 0.2 ng/s. On-line protein mapping was tested with proteins of different dimensions, showing competitiveness in terms of time (completed map within 15 min) and exhaustive maps of small proteins. The results of the CE-microreactor and the potential to immobilise biocomponents easily on a desired portion of the capillary indicate further developments towards the construction of a variety of miniaturised enzymatic screening devices for high-throughput screening analysis.