A novel dynamic coating medium for single stranded DNA separation in capillary electrophoresis: Thermosensitive hydrogel nanoparticles

Nowadays, research on single-stranded DNA (ssDNA) requires a quick, effective and easy operation method to separate small ssDNA fragments from the organism and ssDNA production process to realize their application in gene therapy and drug diagnosis. Herein, a novel separation matrix of thermosensitive hydrogel nanoparticles (NPs) was proposed, which has been successfully applied to the isolation of biomacromolecules such as enzymes, peptides and proteins, based on the desirable properties of various monomers and advantages, such as high sieving capacity, tunability of hydrophilic and hydrophobic performance and non-toxicity. The flexible particles were prepared by precipitation polymerization method in aqueous solution while N-isopropylacrylamide (NIPAm), acrylic acid (AAc) and N-t-butyl acrylamide (TBAm) was performed as co-monomer, which were first used as the dynamic coating to separate small fragments of ssDNA in capillary electrophoresis (CE). The synthesized NPs were bound to the inner wall of the capillary through non-covalent interactions forming stable dynamic coating to affect the electroosmotic flow and improve the separation reproducibility of ssDNA fragments. Based on the formulation design of NPs and condition optimization of electrophoresis, the running buffer TG (25 mM Tris, 192 mM glycine, pH 8.3) was modified by 4 mg·mL⁻¹ optimized NPs aqueous suspension addition while its molar feed ratio of NIPAm /AAc /TBAm /BIS was 83/5/10/2 and particle size was about 214 nm. Both the formulation design and temperature sensitivity of hydrogel nanoparticles contributed to the DNA separation efficiency. The designed NPs with low viscosity provide an approach to increase their capability as the separation medium for small fragment DNA with important physiological functions great potential and promise.