Bacterial surface layer proteins as a novel capillary coating material for capillary electrophoretic separations.

Area of Science:

• Biomaterials Science
• Analytical Chemistry
• Separation Science

Background:

• Capillary electrophoresis (CE) requires stable coatings to ensure reproducible separations.
• Existing CE coatings often lack long-term stability or are not suitable for challenging analytes.
• Surface layer proteins offer potential for novel biomimetic coatings.

Purpose of the Study:

• To develop and characterize a novel, stable coating for capillary electrophoresis using Surface layer protein A (SlpA).
• To evaluate the stability and performance of SlpA-coated capillaries in electrophoretic separations.
• To demonstrate the application of SlpA coatings for the separation of lipoproteins in human serum.

Main Methods:

• Extraction and purification of Surface layer protein A (SlpA) from Lactobacillus acidophilus.
• Coating of hydrophobized fused silica capillaries with SlpA.
• Contact angle measurements, atomic force microscopy (AFM), and fluorescence microscopy for surface characterization.
• Stability testing with NaOH treatment and evaluation of electroosmotic flow (EOF).
• Application in isotachophoresis (ITP) for lipoprotein separation in human serum.

Main Results:

• SlpA coating transformed hydrophobic silica surfaces to hydrophilic, forming a stable protein monolayer.
• AFM confirmed a uniform SlpA layer with low surface roughness (0.44 ± 0.02 nm).
• SlpA coatings exhibited exceptional stability, even after exposure to high pH (20 mM NaOH, pH 12.3).
• EOF in coated capillaries was partially suppressed (3.8 ± 0.5 x 10⁻⁹ m² V⁻¹ s⁻¹) compared to uncoated capillaries (5.9 ± 0.1 x 10⁻⁸ m² V⁻¹ s⁻¹).
• SlpA-coated capillaries demonstrated high repeatability (1.1–1.8% CV) and inter-capillary reproducibility (2–3% CV) for lipoprotein separation.
• The coating remained stable for over 100 runs at pH 9.40.

Conclusions:

• Surface layer protein A (SlpA) provides a novel, highly stable, and reproducible coating for capillary electrophoresis.
• SlpA-coated capillaries are suitable for challenging electrophoretic separations, including lipoprotein analysis.
• This biomimetic coating represents a significant advancement for long-term use in CE applications.