## 3 Results and Discussion  
### Effect of TFE on the Thermal Denaturation of Collagen  
Figure 2 shows the phase difference \(\varphi\) as a function of temperature. The \(T_{\rm d}\) of the native collagen (as a control) is 40.2 \({}^{\circ}\)C from the onset of the significant drop in the phase difference. Figure 3 shows the effect of TFE concentration on the phase difference during the heating process. It can be seen that the onset of the significant drop in the phase difference is shifted to a lower temperature, indicating that the \(T_{\rm d}\) of the TFE-mixed collagen is decreased from 37.3 \({}^{\circ}\)C to 28.4 \({}^{\circ}\)C. In this stage, the addition of TFE increases the heterogeneity of collagen in the aqueous buffer solutions due to the formation of unordered peptide carbonyl groups (-C=O) [4,17]. As the TFE concentration is higher than 40% (_v/v_), the significant drop in the phase difference diminishes and reveals that the collagen has been completely destructed. It has been suggested that the variation in the optical properties can be used to explore the thermal degradation of TFE-mixed collagen and the resulting structural destabilization [4]. Thus, we recorded the initial optical rotation as a function of TFE concentration from the displacement-enhanced heterodyne measurement, as shown in Figure 4a. It can be seen that initial optical rotation is reduced by 0.1\({}^{\circ}\) as TFE concentration increased to 15% (_v/v_), while this value is significantly reduced to less than 0.4\({}^{\circ}\). It has been suggested that TFE introduces the \(\alpha\)-helical structure in the aqueous TFE solution, and then causes the disruption of the inter-strand hydrogen boding (_i.e._, the bifurcated hydrogen bonding for C=O chains between the N-H and O-H group of TFE). As the TFE concentration further increases (40%-80%, _v/v_), the initial optical rotation of the TFE-mixed collagen remains almost unchanged, indicating that the inter-strand hydrogen bonding between the N-H Gly and neighboring C=O Xaa groups has been destructed and the fibrils in the collagen have been completely inhibited [4,18-20]. In this way, the triple helices have become destabilized by transforming the helical structure into an unordered structure, as proposed by [PERSON] [4] and [PERSON] [18]. The dependence of the denaturation temperature on the TFE concentration is illustrated in Figure 4b, revealing that \(T_{\rm d}\) of the TFE-mixed collagen decreased proportionally to the TFE concentration in the solution.  
Figure 2: The phase difference of collagen as a function of temperature. (Collagen concentration: 0.341 mg/mL).  
Figure 4: **(a)** Initial optical rotation and **(b)** denaturation temperature (\(T_{\rm d}\)) as a function of TFE concentration (%, _v/v_). (Collagen concentration: 0.341 mg/mL).  
Figure 3: Variation of phase difference with heating temperature under different Trifluoroethanol (TFE) concentration (_v/v_): **(a)** 5% (=0.157 mL); **(b)** 10% (=0.333 mL); **(c)** 15% (=0.529 mL); **(d)** 20% (=0.75 mL); **(e)** 40% (=2 mL) and **(f)** 80% (=12 mL). (Collagen concentration: 0.341 mg/mL) The inset \(T_{\rm d}\) represents the denaturing temperature.