Application of the concept of an electrophoretic ratchet.

Fractionation via a gel electrophoretic ratchet has previously succeeded for comparatively large (radius R > or = 95 nm) spheres (Serwer, P, Griess, G.A., Anal. Chim. Acta 1998, 372, 299-306). The electrical oscillations are the following electrical field pulses: high field --> low field --> high field, etc. The field is inverted after each pulse; the time-integral of the field can be zero. Response to the ratchet is caused by steric trapping in the high field-direction, but not in the low field-direction. Trapping and, therefore, response to the ratchet decrease as R decreases. The smaller spheres do not respond to the ratchet. In the present study, spheres with R values smaller than 95 nm are made, for the first time, to respond to a similar gel electrophoretic ratchet. To achieve this objective, the heterogeneity of pore size is increased for the gel used. The heterogeneity of pore size is increased by (i) forming the gel with degraded hydroxyethyl agarose, and (ii) gelling at comparatively high temperature. If a particle still does not respond to the ratchet (because the particle is too small), this particle has a net migration in the high field-direction, when the above-described pulsed field is biased in the high field-direction. If a particle does respond to the improved ratchet, the particle has a net migration in the low field-direction. Here, the R of ratchet-responding spheres is reduced to 30-50 nm. These ratchet-responding spheres include both intact bacteriophage particles (R = 30 nm) and latex spheres. The smaller ratchet-responding spheres have an electrophoretic mobility that decreases in magnitude as the electrical field increases in magnitude. A ratchet-based procedure is developed here to achieve continuous preparative gel electrophoresis.