QCM analyses
Designer proteins (Bc3) 2, (Bc3) 4, (Bc3) 2T, and (Bc3) 4T (Figure 1 a) were synthesized and purified as described in MATERIALS AND METHODS. Using QCM, the mode of interactions between the crated artificial proteins and titanium surface was assessed. In the QCM assay, binding of molecules on the surface of the sensor decreases the resonance frequency of the sensor and detachment of molecules from the surface is observed as the resonance frequency increases. Steps of QCM measurements are schematically illustrated in Figure 2 a. First, the Ti sensor was equilibrated with water for 30–60 min and the measurement was started (time = 0). After 5 min, TBST (a solution buffer of the proteins) was injected 3 times (time = 5, 10, and 15 min). Then, protein solution was injected into the measurement chamber (time = 20 min). After 30 min, the sensor was washed with TBST 3 times (time = 50, 55, and 60 min), followed by washing with water 3 times (time = 65, 70, and 75 min). Results obtained from (Bc3)2 and (Bc3)2T are shown in Figure 2 b. Injections of the protein solutions rapidly decreased the resonance frequency of the sensor, indicating that both (Bc3)2 and (Bc3)2T have bound to the surfaces of Ti. Because TBST washing only slightly increased the frequency, the binding of these proteins to Ti is stable under the conditions. When the measurement sensor was washed with water (time = 65 min), an increase of frequencies was observed, which was due to buffer change (not detachment of proteins from the sensor). When the rates of frequency increase in water were compared between (Bc3)2and (Bc3)2T (time = 65–80 min), it can be recognized that (Bc3)2T was detaching faster than B(Bc3)2. Similar results were obtained from (Bc3)4 and (Bc3)4T (Figure 2 c).