Figure 6.

Analysis of concentration and anchor contamination of GAG samples. (A) Schematic showing the surface functionalization steps for postchromatographic QCM-D analysis of the GAG samples: ① SLB formation, ② SAv monolayer formation, and ③ binding of biotinylated GAGs and/or free alkoxyamine-EG₄-biotin. (B) QCM-D responses (ΔF—bottom; ΔD—top; i = 5) for the binding of biotinylated CS-D GAG (GAG-b) from eluate fraction 3. Data for all other eluate fractions (EF) are shown in Figure S8C. Conditions: GAG-b incubation, started at 0 min and proceeded for 16 min (during other times, plain working buffer was flown over the sensor); flow rate—20 μL/min; GAG-b concentration—the eluate fractions as retrieved from the size-exclusion column were diluted 15-fold for QCM-D analysis. The steepest slope in the ΔF_GAG-b vs time graph (max rate, −ΔF_GAG-b/Δt; indicated by the tilted red dashed line) reflects the rate of steady-state mass-transport-limited binding. The frequency shift at saturation, ΔF_GAG-b,sat (max response; indicated by the horizontal red dashed line), is a measure of the fraction of biotin binding sites occupied by biotinylated GAGs. (C) Max rate, −ΔF_GAG-b/Δt, as a function of the eluate fraction. (D) GAG-b concentration in the eluate fractions, calculated from −ΔF_GAG-b/Δt and reference data for another GAG-b of known concentration and similar size (see Figure S8B for details). (E) Max response, −ΔF_GAG-b,sat, as a function of the eluate fraction. The gray background highlights the fractions for which binding did not saturate during the set incubation time. (F) Concentration of residual free alkoxyamine-EG₄-biotin in the eluate fractions, estimated through eq 8 based on the data in (D,E), and R_GAG-b / R_{b-alkoxyamine} ≈ 12.