Flow Field Flow Fractionation Method Development for Applied Bioanalysis
Flow field flow fractionation (F4) and asymmetric F4 (AF4) are open channel separation instruments relying on an axial-flow and a perpendicular cross-flow to separate analytes based on hydrodynamic radius. The cross-flow provides enough force to simultaneously separate non-binding analytes based on size and remove non-specific binding giving the F4 potential as a method for bioanalysis. With the desire to apply F4 in bioanalysis, a need to understand how a complex sample, containing a variety of biomolecules and nanomaterials, would behave in the system was created. Changes in carrier solution (CS) and binding buffer (BB) composition such as ionic strength, cation type, and pH affect on particle and protein recoveries, and on the binding between protein and a ssDNA were investigated.
Proteins required higher ionic strength to prevent adsorption in the channel, while nanoparticles had the opposite trend with increased ionic strength causing lower recoveries. No effect was seen from varying the BB, but the CS had played a significant role. During AF4's unique focusing step, the BB is replaced with the CS rapidly, indicating that on-line incubation is a possibility for F4 bioanalysis reducing the amount of sample handling time. The presence of MgCl2 in the CS, which plays an important role in DNA folding, was necessary for binding to occur. An experimental detection limit of 33 nM was achieved for immunoglobulin E, limited by the labeling of one fluorophore per protein.
The study on protein-ssDNA analysis was expanded to calculate the dissociation constant (Kd) of the model system which was in very good agreement with Kd values obtained by other methods. A variety of ssDNA was investigated to determine the affect different lengths and secondary structures had on the recovery and retention times in F4. With the ability to possibly bind both intramoleculary and intermoleculary, ssDNA showed unique elution profiles from more globular analytes. Folding equilibriums were calculated for ssDNA with known secondary structure by comparing the elution times of linear DNA that was unlikely to form intramolecular bonds. This work has shown the versatility F4/AF4 could have for future applications of ssDNA as labeling agents.