Preparation and methodology for chemical mapping of sol–gel thin films containing lysozyme

Enzyme distribution and secondary structure of sol–gel immobilized glucose oxidase by micro-attenuated total reflection FT-IR spectroscopy

“…To overcome these drawbacks some authors have recently proposed to use Fourier Transform Infrared (FT-IR) microscopy for the quantitative determination of an immobilized protein's spatial distribution throughout supporting media without the need for protein modification or staining. FT-IR microscopy indeed is able to identify chemical groups in heterogeneous compounds [19][20][21][22][23]. In addition, the content of GOD in a matrix could be related to the overall intensity of the FT-IR peaks.…”

ATR FT-IR spectroscopy on Vmh2 hydrophobin self-assembled layers for Teflon membrane bio-functionalization

“…According to different authors [18,19,21] the amount of protein detectable in different samples was estimated by evaluating the area of amide I and amide II bands in the 1700-1500 cm −1 region since this area results to be proportional to the amount of protein [18]. Given the adopted approach (macro-ATR) for spectra collection the amide content value results to be averaged on the entire examined surface of the layer investigated by attenuated total reflection method that interrogate the first layers of the sample (typically 5-6 m).…”

“…In particular second-derivative spectra were obtained with Savitsky-Golay derivative function algorithm for a seven data point window. The area of each absorption band was assumed to be proportional to the relative amount of the structure type [18,19,21,[30][31][32][33]. All the data treatment was performed using GRAMS software.…”

“…To give quantitative details about the dependence of the characteristics of self-assembled layers on the adopted purification procedure the amount of protein detectable in different samples was estimated by evaluating the area of amide I and amide II bands in the 1700-1500 cm −1 region [18][19][20][21]. Relative lipid content in different samples was determined by calculating the ratio of the Table 2 Results of the detected amide content, lipid/amide ratio and carbohydrate/amide ratio analysis for layers prepared using hydrophobin from raw extract and from the two different procedures of protein purification.…”

“…For this study we adopted Attenuated-Total-Reflected Fourier-Transformed Infrared (ATR FT-IR) spectroscopy, a powerful technique in chemical and structural characterization of very thin interfaces [16,17]. In order to identify the conditions to obtain uniform protein layers with low amount of contaminants we estimated the amide content [18][19][20][21], the lipid/amide and carbohydrate/amide ratios [22,23] of layers obtained by means of different preparation processes, namely two different protein purification procedures and various coating conditions. In fact both these steps can obviously influence the characteristics of hydrophobin layers and the macro-ATR FT-IR geometry adopted for spectra acquisition can be extremely useful to obtain averaged information on the entire examined surface of the layer.…”