*4.2. Infrared Spectroscopy*

Infrared (IR) spectroscopy is a well-established experimental technique for analyzing the secondary structures of proteins [83]. IR vibrational spectra contain a wealth of information about the structure and environment of amino acid side chains, as well as protein conformation and the polypeptide backbone. For in-situ investigation of the redox enzymes at the electrode surfaces, reflection-based methods, particularly attenuated total-reflectance infrared (ATR-IR) spectroscopy, are the most commonly used. The strongest features in the ATR-IR spectra of enzymes are typically amide bands (e.g., amide I at ~1650 cm−<sup>1</sup> and amide II at ~1550 cm−<sup>1</sup> ) that arise from the amide bonds in the polypeptide backbone and sensitively encode the secondary structure of the enzyme. Mao et al. developed an ATR-IR spectroscopy-based method for investigating the effects of organic solvents on the immobilization of *Tv*Lac on CNTs [84]. Six typical secondary structures in *Tv*Lac with and without ethanol treatment adsorbed on CNTs were resolved and compared. It has been found that the content of high-frequency (HF) β-sheets, which corresponds to the flexible β-extend, dropped

to just above zero after immobilization in the CNTs, while it increased by ~200% in ethanol-treated *Tv*Lac. In addition, a stronger amide II peak was obtained with ethanol-treated *Tv*Lac-CNT electrodes, showing an increased amount of detectable N–H in-plane bending events. The ratio of the amide I/II peak intensity was reduced at the ethanol-treated *Tv*Lac-CNT electrode, compared to that at the non-treated *Tv*Lac-CNT electrodes, indicating a significant orientation change of the adsorbed *Tv*Lac. With improved O2-reduction currents, ethanol-treated *Tv*Lac facilitating a favorable "end-on" orientation at the CNT surfaces for fast direct electron transfer was proposed. (Figure 6B) Based on a similar ATR-IR technique, the orientations of the small Lac in carbon nanomaterials were investigated [85]. Consequently, the effects of the surface curvature of carbon nanomaterials on the orientation of adsorbed small Lac in the view of DET-type bioelectrocatalysis was proposed.

**Figure 6.** (**A**) QCM-D response of naphthalene-functionalized CNTs modified gold surface before and after *Tr*Lac injection. The inset shows the QCM-D response of pristine CNTs modified gold surface before and after *Tr*Lac injection. Reprinted from [82] Copyright (2020) with permission from Elsevier. (**B**) Two orientations of β-strand at the surface of CNTs predicted from the Amide I/II peak intensity ratios. Inset: Amide I and II band regions on difference IR spectra (corrected by a linear baseline between 1700 and 1500 cm−<sup>1</sup> ) of the untreated *Tv*Lac-CNT film (black line) and Ethanol-treated *Tv*Lac-CNT film (red line). Yellow arrows represent incident light. Amide bonds (ball and sticks) are in the plane of β-strand. Reprinted from [85] Copyright (2017) with permission from American Chemical Society.
