RESEARCH ARTICLE A comprehensive study of protein–mesoporous– macroporous silica interactions by extended canonical variate analysis of Raman spectra Diogo Videira‐Quintela 1 | Sofia F. Prazeres 1 | F. E. Ortega‐Ojeda 1,2 | G. Montalvo‐García 1,2 1 Department of Analytical Chemistry, Physical Chemistry and Chemical Engineering, University of Alcalá, Alcalá de Henares, Spain 2 University Institute of Research in Police Sciences, Alcalá de Henares, Spain Correspondence Montalvo‐García, Department of Analytical Chemistry, Physical Chemistry and Chemical Engineering, University of Alcalá, Ctra. Madrid‐Barcelona Km. 33.6, 28,805 Alcalá de Henares, Madrid, Spain. Email: gemma.montalvo@uah.es Funding information People Programme (Marie Curie Actions) of the European Union's SeventhFramework Programme FP7/ 2007‐2013/, Grant/Award Number: REA grant agreement n°606713 ‐ BIBAFOODS; University of Alcal Project, Grant/Award Number: CCG2018/EXP‐039 Abstract Understanding the protein–support interactions is of major importance when manufacturing bionanomaterials to a certain application. These interactions can be the cause for enhanced properties or denaturation phenomena in the target protein. Raman spectroscopy was applied to a bionanomaterial comprehending the protein β‐galactosidase immobilized by physical adsorption into a mesoporous–macroporous silica material, with a nanoporous network consisting of 9‐nm mesopores and 200‐nm macropores. Raman spectra of the bionanomaterial evidenced a complex amount of differences related to the Raman shifts, intensities, band enlargement, appearance of new bands, and overlapping, in comparison with the silica support and the protein spectra. To help in the analysis of the Raman spectra and in the inspection of possible protein–support interactions, ECVA (extended canonical variate analysis) was used as a chemometric complementary tool, dividing the spectra into four seg- ments: 1 (3,100 to 2,800 cm -1 ), 2 (1,800 to 1,500 cm -1 ), 3 (1,500 to 1,200 cm -1 ), and 4 (1,200 to 900 cm -1 ). Major alterations in the Amide I band (1,800 to 1,500 cm -1 ) and the amino acid band regions demonstrated possible structure alter- ations to a non‐native form of the protein β‐galactosidase. Also, other minor alterations were observed in other spectral regions (3,100 to 2,800 cm -1 ,1,500 to 1,200 cm -1 , and 1,200 to 900 cm -1 ) also representative of protein structure alteration due to protein–support interactions. KEYWORDS β‐galactosidase, extended canonical variate analysis, meso‐macroporous silica, protein, support interactions, Raman spectroscopy 1 | INTRODUCTION The creation of bionanomaterials in which proteins are immobilized or encapsulated into adequate supports is an enormous field with vast technological applications. It is an effective method to ensure protein stability in the presence of a wide range of temperature, pH, and ionic strength conditions, reusability and reduction of costs in industrial processes. [1,2] The hierarchical porous silica, characterized by having mesopores and macropores of adjustable pore size/volume, are beginning to appear as good available support alternatives for Received: 22 July 2019 Revised: 22 September 2019 Accepted: 30 September 2019 DOI: 10.1002/jrs.5773 J Raman Spectrosc. 2019;1–11. © 2019 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jrs 1