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).