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. 2023 Dec 4;39(50):18410–18423. doi: 10.1021/acs.langmuir.3c02567

Figure 4.

Figure 4

Quantitative tuning of the surface density of two biotinylated proteins of different size (under flow). (A) QCM-D dissipation shifts ΔD (top) and frequency shifts ΔF (bottom; for overtone i = 5) obtained for b-ZZ mixed with free biotin at distinct molar ratios (as indicated). Conditions: cb-zz + cbiotin = 0.625 μM was maintained constant, with cb-zz and cbiotin determined according to eq 6 (εflow < 0.007); b-ZZ/biotin incubation—3 min, starting from 0 min; during remaining times, plain working buffer was flown over the sensor surface, flow rate—200 μL/min. b-ZZ only was incubated at a different flow rate (20 μL/min) and therefore is not displayed in the graph; see Figure S6A for details of the SAv-on-SLB sensor functionalization and b-ZZ grafting. (B) Frequency shifts at saturation (ΔFb-ZZ,sat; i = 5) vs the predicted fractional b-ZZ surface coverage (Γb-ZZ,sat/Γas). (C) Standard curve relating frequency shifts to b-ZZ molar surface density, obtained through a combined QCM-D/SE experiment (see Figure S7 for details). The dashed line is an empirical fit to the data, with Γb-ZZ = (α – 1) / [Mb-ZZ(β + C–1ΔF–1)] and α = 0.8389 ± 0.0012, β = 7.83 ± 0.14 × 10–4 cm2/ng, the mass sensitivity constant C = 18.0 ng/(cm2 Hz), and the molecular mass Mb-zz = 16.2 kDa. (D) Plot of the measured b-ZZ surface density as a function of Γb-ZZ,sat/Γas for two distinct flow rate regimes (200 μL/min—purple spheres; 10 or 20 μL/min—gray spheres, see Figure S6B for details). The black dashed line is a linear fit through the purple data (200 μL/min), with a slope of 6.1 ± 0.2 pmol/cm2. b-ZZ surface densities were determined from panel (B) and Figure S6C, respectively, using the empirical fit from panel (C).