19 Bio-applications of NIR Spectroscopy
429
NIR spectroscopy is a potent tool in exploring the complex properties of proteins.
Protein research by NIR spectroscopy includes several significant contributions, e.g.,
analysis of the secondary structure [24]. Furthermore, this technique finds unique
usefulness in investigating hydration process of proteins. Monitoring changes that
occur in hydration as well as that in the protein secondary structure at the same time is
possible by NIR spectroscopy; in contrast, IR and Raman spectroscopies can hardly
investigate these properties simultaneously. The potential of NIR spectroscopy to
investigate proteins in aqueous environment and the appropriate methodology can
be presented on the example from literature [25, 26]. Murayama et al. [25] performed
a comprehensive comparison of the methods for analyzing NIR spectra that are suitable for investigating proteins in aqueous solution. Conventional spectral analysis
methods, chemometrics (PCA) and 2D-COS spectroscopy were evaluated in that
case. The study was based on the NIR spectra of human serum albumin (HSA) in
aqueous solutions within the concentration range of 0.5–5.0 wt%. It was concluded
that basic conventional methods of spectra pretreatment and analysis, such as secondderivative and difference spectra, remain critically important for analysis of protein
in relatively low concentration in water. For example, the difference spectra unveiled
that various species of water are responsible for the observed gradual concentrationdependent changes in the broad feature in the 7100–6500 cm
−1 . PCA is more resistant against spectral noise; however, 2D-COS is more informative on the correlations
between individual bands and also elucidates sequences of spectral changes. Therefore, the best approach is to combine various methods, as this yields highest potential
for interpretation of spectral variability.
With a similar aim, this study has been continued by Yuan et al. who compared
different methods for treating NIR spectra of bovine serum albumin (BSA) [26].
However, this time the source of spectral variability was the temperature perturbation
(45–85 °C), while concentration of protein was constant at 5.0 wt% and the pH
of the sample was 6.8. The evaluated methods were extended by the addition of
chemometric algorithm of evolving factor analysis (EFA). That study confirmed
the previous conclusions about the usefulness of conventional methods of spectral
analysis and the significance of combined use of various approaches. Namely, the
difference spectra were essential in finding the change in protein hydration that
occurs in the temperature range of 61–65 °C. However, this finding was supported by
analyzing the temperature profile through three-factor EFA in the 7400–6400 cm
−1
region. The investigation has also revealed that the structural variation of BSA in the
aqueous solution just precedes the change in the protein hydration, indicating that the
change in the hydration is initiated by the structural modifications in the protein itself.
For yielding these deeper insights from NIR spectra, application of EFA combined
together with the other methods was essential. Note, the spectral variability observed
in NIR region upon concentration change of HSA protein in water differs from the
one observed upon temperature change in aqueous solution of BSA.
NIR spectral bands of proteins can be used to follow complex biological processes
in vivo, such as embryonic development, [27] for example. The current state of the
art of protein research by NIR spectroscopy is exhaustively covered in a book chapter
by Ishigaki and Ozaki [28]. As mentioned in introduction and in the chapter referred
429
NIR spectroscopy is a potent tool in exploring the complex properties of proteins.
Protein research by NIR spectroscopy includes several significant contributions, e.g.,
analysis of the secondary structure [24]. Furthermore, this technique finds unique
usefulness in investigating hydration process of proteins. Monitoring changes that
occur in hydration as well as that in the protein secondary structure at the same time is
possible by NIR spectroscopy; in contrast, IR and Raman spectroscopies can hardly
investigate these properties simultaneously. The potential of NIR spectroscopy to
investigate proteins in aqueous environment and the appropriate methodology can
be presented on the example from literature [25, 26]. Murayama et al. [25] performed
a comprehensive comparison of the methods for analyzing NIR spectra that are suitable for investigating proteins in aqueous solution. Conventional spectral analysis
methods, chemometrics (PCA) and 2D-COS spectroscopy were evaluated in that
case. The study was based on the NIR spectra of human serum albumin (HSA) in
aqueous solutions within the concentration range of 0.5–5.0 wt%. It was concluded
that basic conventional methods of spectra pretreatment and analysis, such as secondderivative and difference spectra, remain critically important for analysis of protein
in relatively low concentration in water. For example, the difference spectra unveiled
that various species of water are responsible for the observed gradual concentrationdependent changes in the broad feature in the 7100–6500 cm
−1 . PCA is more resistant against spectral noise; however, 2D-COS is more informative on the correlations
between individual bands and also elucidates sequences of spectral changes. Therefore, the best approach is to combine various methods, as this yields highest potential
for interpretation of spectral variability.
With a similar aim, this study has been continued by Yuan et al. who compared
different methods for treating NIR spectra of bovine serum albumin (BSA) [26].
However, this time the source of spectral variability was the temperature perturbation
(45–85 °C), while concentration of protein was constant at 5.0 wt% and the pH
of the sample was 6.8. The evaluated methods were extended by the addition of
chemometric algorithm of evolving factor analysis (EFA). That study confirmed
the previous conclusions about the usefulness of conventional methods of spectral
analysis and the significance of combined use of various approaches. Namely, the
difference spectra were essential in finding the change in protein hydration that
occurs in the temperature range of 61–65 °C. However, this finding was supported by
analyzing the temperature profile through three-factor EFA in the 7400–6400 cm
−1
region. The investigation has also revealed that the structural variation of BSA in the
aqueous solution just precedes the change in the protein hydration, indicating that the
change in the hydration is initiated by the structural modifications in the protein itself.
For yielding these deeper insights from NIR spectra, application of EFA combined
together with the other methods was essential. Note, the spectral variability observed
in NIR region upon concentration change of HSA protein in water differs from the
one observed upon temperature change in aqueous solution of BSA.
NIR spectral bands of proteins can be used to follow complex biological processes
in vivo, such as embryonic development, [27] for example. The current state of the
art of protein research by NIR spectroscopy is exhaustively covered in a book chapter
by Ishigaki and Ozaki [28]. As mentioned in introduction and in the chapter referred