Surface functionalized magnetic nanoclusters for bioseparations.

摘要

A new immunomagnetic separation process, that uses protein-A coated magnetic nanoclusters (PACMAN) as the separation vehicles, has been developed. These nanoclusters are produced by sonicating egg yolk phosphatidylcholine and the transmembrane Fc receptor protein-A in a buffered aqueous ferrofluid suspension. The separation is demonstrated on a model system consisting of a mixture of chicken (CRBC) and antibody bound sheep red blood cells (SRBC). Using a negative selection procedure, an initial mixture containing an equal number of chicken and sheep red blood cells is concentrated to 80% CRBCs in a single stage, while in three stages the number concentration of CRBCs was increased to over 99%.; Another magnetic separation process, that uses streptavidin coated magnetic nanoclusters (ACMAN) as the separation vehicles, has been developed. The nanoclusters are produced by sonicating a mixture of egg yolk phosphatidylcholine and N-(biotinoyl)dipalmitoyl-L-{dollar}alpha{dollar}-phosphatidylethanolamine, triethylammonium salt (biotinylated DPPE) in a buffered aqueous ferrofluid suspension. The separation is demonstrated on a model system consisting of a mixture of {dollar}lambda{dollar}DNA and biotinylated pTC45. Exposure of this mixture to a magnetic field gradient causes selective migration of the 'magnetized' pTC45 to the high magnetic field region, thus affecting a separation. Preliminary results show that the purity of pTC45 obtained is nearly 100% and the recovery is 15-20%.; We have developed a rapid method for cassette isolation that provides DNA preparations free of contaminating plasmid sequences by constructing DNA expression vectors containing triple helix forming sequences which can be digested from the plasmid, incubated with biotinylated triple helix forming oligonucleotide and captured by magnetic streptavidin separation. Two plant expression cassettes, p538 (4.61 kb) and p539 (4.61 kb), are constructed using the triple helix forming sequence from pTC45 and a CaMV 35S/BAR/Tr7 insert from p165. The purity of the insert DNA isolation at the optimized conditions is found to be almost 100%. The recovery of the insert is found to vary between 60 and 95% depending on the freshness of the components. Scale up of the above procedure gives insert recovery between 55 and 90%, while, the purity is almost 100%.