Qtools

A QCM-D instrument (Q-sense E1, Q-sense, Gothenburg, Sweden) was used to monitor the interaction between cyt c and CL. An SiO₂-coated quartz crystal (QSX 303, Q-sense) sensor was first cleaned by immersion in 2% SDS for 30 min, followed by UV ozone treatment for 10 min, before being installed into the QCM-D chamber. To form lipid bilayers on the surface of the SiO₂ sensors, a CL-containing liposomal solution (1 mg/mL, equilibrated in 50 mM Tris–HCl buffer, pH 7.4) was pumped (200 µL/min) over the SiO₂ sensor surface for 2 h. Subsequently, the flow liquid was changed to 50 mM Tris–HCl buffer to remove excess liposomes. Then, either a ferric or ferrous cyt c solution (50 µM) was drawn (200 µL/min) over the CL-containing lipid bilayer for 4 h. Nonspecifically adsorbed cyt c was subsequently removed using the Tris–HCl buffer (50 mM, pH 7.4). QCM-D measurement was performed at overtones 1, 3, 5, 7, 9, 11, and 13 of a fundamental frequency in 5 MHz at 25 °C. The dynamic shifts on both frequency and dissipation were acquired. The hydrated mass and shear modulus were estimated using Q-tools (Biolin Scientific, Gothenburg, Sweden) software based on the Kelvin–Voigt model, with densities of the lipid membrane and protein of 1.1 and 1.35 g/cm³, respectively^{61 (link)}.

QCM-D experiments were conducted to track bicelle adsorption kinetics on silicon dioxide-coated sensor chips by using a Q-Sense E4 instrument (Biolin Scientific AB, Stockholm, Sweden), as previously described^{43 (link),44 (link)}. Immediately before experiment, the QCM-D sensor chips were repeatedly rinsed with water and ethanol, and then air dried with nitrogen gas before oxygen plasma treatment for 1 min in a vacuum chamber (PDC-002, Harrick Plasma, Ithaca, NY). Liquid samples were introduced into the measurement chambers under continuous flow conditions at a volumetric rate of 50 µL min⁻¹, as regulated by a Reglo Digital MS-4/6 peristaltic pump (Ismatec, Glattsburg, Switzerland). Measurement data were collected with a time resolution of 1 Hz at the 3rd–11th odd overtones by the Q-Soft software package (BiolinScientific AB), and the overtone-normalized data from the 5th odd overtone are reported. Data processing was completed using the Q-Tools (Biolin Scientific AB) and OriginPro (OriginLab, Northampton, MA) software programs.

QCM-D measurements were performed using a Q-Sense E4 instrument (Biolin Scientific AB, Gothenburg, Sweden), as previously described [25 (link)]. Prior to each round of experiments, TiO₂-coated sensor chips (model no. QSX 310, Biolin Scientific AB) were cleaned with deionized water and ethanol and then dried with nitrogen, followed by 1 min oxygen plasma treatment in a CUTE-1MPR machine (Femto Science Inc., Hwaseong, Republic of Korea). During the experiments, the sample solutions were added into the chambers using a peristaltic pump (Reglo Digital, Ismatec, Glattbrugg, Switzereland) at a defined flow rate of 100 μL/min. The QSoft (version no. 2.5.28.732) and QTools (version no. 3.1.33) software programs (Biolin Scientific AB, Gothenburg, Sweden) were used to complete data collection at multiple overtones and data processing, respectively. All presented QCM-D data were collected from the 5th overtone, and at least three independent replicates were performed per condition. For statistical analysis, two-tailed Student’s t-tests were performed using the GraphPad Prism software package (version no. 10.1.2; Boston, MA, USA), and p < 0.05, p < 0.01, and p < 0.001 indicate the levels of statistical significance (*, **, ***).