Hybrid phospholipid bilayer coatings for separations of cationic proteins in capillary zone electrophoresis

Abstract

Protein separations in CZE suffer from nonspecific adsorption of analytes to the capillary surface. Semipermanent phospholipid bilayers have been used to minimize adsorption, but must be regenerated regularly to ensure reproducibility. We investigated the formation, characterization, and use of hybrid phospholipid bilayers (HPBs) as more stable biosurfactant capillary coatings for CZE protein separations. HPBs are formed by covalently modifying a support with a hydrophobic monolayer onto which a self-assembled lipid monolayer is deposited. Monolayers prepared in capillaries using 3-cyanopropyldimethylchlorosilane (CPDCS) or n-octyldimethylchlorosilane (ODCS) yielded hydrophobic surfaces with lowered surface free energies of 6.0 ± 0.3 or 0.2 ± 0.1 mJ m(-2) , respectively, compared to 17 ± 1 mJ m(-2) for bare silica capillaries. HPBs were formed by subsequently fusing vesicles comprised of 1,2-dilauroyl-sn-glycero-3-phosphocholine or 1,2-dioleoyl-sn-glycero-3-phosphocholine to CPDCS- or ODCS-modified capillaries. The resultant HPB coatings shielded the capillary surface and yielded reduced electroosmotic mobility (1.3-1.9 × 10(-4) cm(2) V(-1) s(-1) ) compared to CPDCS- and ODCS-modified or bare capillaries (3.6 ± 0.2 × 10(-4) cm(2) V(-1) s(-1) , 4.8 ± 0.4 × 10(-4) cm(2) V(-1) s(-1) , and 6.0 ± 0.2 × 10(-4) cm(2) V(-1) s(-1) , respectively), with increased stability compared to phospholipid bilayer coatings. HPB-coated capillaries yielded reproducible protein migration times (RSD ≤ 3.6%, n ≥ 6) with separation efficiencies as high as 200 000 plates/m.

Keywords: CZE; Capillary coatings; Hybrid bilayers; Phospholipids; Proteins.

Figure 1

Scheme illustrating formation of HPB coatings. Bare silica (A) is reacted with CPDCS or ODCS to generate a covalently-bound, hydrophobic monolayer (B) onto which DLPC or DOPC can self-assemble, resulting in a HPB (C). Schematics not to scale.

Figure 2

Stability and reproducibility of HPB coatings. Coating reproducibility for CPDCS-(A) and ODCS-modified (B) capillaries in the presence and absence of the HPB. Each plot shows the mean μ_eof of 10 consecutive runs in bare (diamonds), CPDCS- or ODCS-modified (squares), DLPC HPB (triangles), and DOPC HPB (circles) capillaries (n ≥ 3 capillaries). Coatings were not regenerated between separations.

Figure 3

Protein separations in HPB-coated capillaries. Electropherograms showing multiple separations of Lyso, α-Chymo, and Myo in capillaries with HPBs formed from CPDCS/DOPC (A), CPDCS/DLPC (B), ODCS/DOPC (C), and ODCS/DLPC (D). (E) Protein separations in bare silica (i), CPDCS-modified (ii), and ODCS-modified (iii) capillaries show broad peaks that are not resolved. (F) Separations of Lyso and α-Chymo in capillaries coated with a DLPC PLB are presented for comparison of protein peak symmetry and efficiency. Coatings were not regenerated between any protein separations. Electropherograms offset for clarity.