Rational use of stacking principles for signal enhancement in capillary electrophoretic separations of poliovirus...

The use of an earlier developed capillary electrophoresis (CE) method, either to investigate poliovirus (PV) samples with a low viral-purity level or to study the less abundant sub-viral particles, revealed the necessity for an intra-column signal enhancement strategy. Although intra-column signal enhancement is a very popular approach to assay small molecules, it is less straightforward for the analysis of biological macromolecules or particles. A reason could be that, for a proper signal enhancement approach, these samples have to be thoroughly studied to understand the factors affecting the separation process. For the investigated PV samples, a screening design revealed that injecting larger sample plugs significantly enhanced the analytical signal, but also significantly decreased the separation efficiency. A subsequently executed central composite design determined the largest sample plug that can be injected without compromising the separation. Finally, the sample dilution and the length of the injected plug were used for tuning the intensity of the analytical response. Two combinations of sample dilution and injected plug size, at extreme values, were investigated in detail to define the best procedure for PV analysis using CE. In both situations, PV was effectively separated and quantified in rather complex samples, showing a good repeatability, an acceptable linearity for the PV particles and a decreased limit of detection in comparison with the existing method. In conclusion, intra-column signal enhancement can be successfully applied for viral suspensions, extending the applicability of CE methods to samples with lower virus concentrations, and/or allowing a significant reduction in the minimum required volume of sample. For PV samples, 5μl of sample is necessary instead of the previous 20μl, while the analytical signal was enhanced up to 14 times. The results of this study can provide a basis for the development of routine CE methods for viral particle analysis, especially when rational and reproducible signal enhancement is required.