Fig. 4
pH controls the peptide concentration and orientation. a Sulfur content, as measured by X-ray photoelectron spectroscopy (XPS), indicates increasing surface peptide with solution concentration but is undetectable for concentrations below 5 µg mL−1. b Peptide titration shows that enzyme-linked immunosorbent assay (ELISA) is discriminatory for peptide concentrations down to at least 0.5 µg mL−1 but saturates at approximately 20 µg mL−1 due to the large footprint of the primary and secondary antibodies compared to the peptide. Hence, ELISA is not capable of discerning immobilized peptide surface density for high densities while XPS is not capable of detecting low peptide surface concentrations. c XPS sulfur atomic concentration of peptide-coated surfaces compared to uncoated controls for immobilization pH values of 5.4 and 9.8. The sulfur content shows surface peptide at pH 5.4, but no detectable peptide at pH 9.8. Presence of peptide after Tween 20 or sodium dodecyl sulfate (SDS) washing indicates covalent attachment. d The absorbance measured with ELISA for peptide-coated samples for immobilization pH values of 5.4 and 9.8 compared with uncoated controls. ELISA shows that peptide is present at both pH values. e Time of flight secondary ion mass spectrometry (ToF-SIMS) normalized mass fragments of the hydrophobic residues indicate higher number of FLAGDOWN peptide for pH 9.8, suggesting that a greater proportion of peptide for pH = 5.4 is in the FLAGUP orientation. Error bars are s.d. and P-values are from the Student’s two-tailed t-test: **P < 0.01, ***P < 0.001 (b, d and e). XPS error bars are calculated from the background noise (a, c)
