Key Role of Ionic Hydrogen Bonding in Nonspecific Protein Adsorption on a Hydrophobic Surface

Nonspecific protein adsorption on the channel walls is a frequently encountered problem in microfluidic chips, resulting in substantial sample loss and low device performance. However, the molecular mechanisms underlying strong interaction of proteins with solid surfaces are largely unknown. Here, we studied the spontaneous adsorption of an ionic complementary peptide EAK16-II [(Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys)2] and its quaternized derivative QEAK16-II [(Ala-Glu-Ala-Glu-Ala-LysMe3-Ala-LysMe3)2] from solution to a poly(methyl methacrylate) (PMMA) surface. We found that EAK16-II with free ε-amino groups can readily self-organize into a complete coating layer predominantly composed of α-helixes and β-sheets, which efficiently suppress nonspecific adsorption of standard proteins or proteins from human whole blood. Once the free ε-amino groups in EAK16-II are quaternized, QEAK16-II sparsely adsorbs on the PMMA surface with less than 10% coverage, which can be easily substituted by proteins and lead to serious nonspecific protein adsorption similarly to that observed on the pristine PMMA surface. These results clearly indicated the critical role of the ionic hydrogen bonding and eliminate other forces such as hydrophobic, electrostatic, and hydrogen-bonding interactions in the strong adsorption of peptides and proteins on the PMMA surface. Expectedly, the high-performance separations of amino acids, peptides, and proteins were achieved in the PMMA microchannels dynamically coated with EAK16-II and its analogues with different basic amino acid residues in sequence including EAR16-II [(Ala-Glu-Ala-Glu-Ala-Arg-Ala-Arg)2] and EAKR16-II [(Ala-Glu-Ala-Glu-Ala-Arg-Ala-Lys)2]. The present study provides significant insights into the mechanism underlying strong interaction of proteins and peptides with a solid surface.