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Development of divergent-flow isoelectric focusing chip for separation of biological agents
Duša, Filip ; Šalplachta, Jiří ; Horká, Marie ; Lunerová, K. ; Rosenbergová, K. ; Kubíček, O.
Isoelectric focusing (IEF) is an efficient method for separation of different bacterium species. Divergent flow format of IEF (DF-IEF) enables continual separation of large volumes of samples and fractionation of the separated analytes for later analysis. The mentioned properties enabled development of DF-IEF chip for separation of H2O2 inactivated biological agents (B-agents). The chip was designed to perform separation in vertical alignment which solved issues connected with sedimentation of bacterial cells. The separation method was optimized using colored markers of isoelectric point and four different b-agents were separated using the DF-IEF chip. The obtained fractions were subsequently analyzed with matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The mass spectra proved that the developed DF-IEF chip is an effective tool for separation of inactivated B-agents.
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