This work analyzes some key aspects of the behavior of sensors based on piezoelectric Thickness Shear Mode (TSM) resonators to study and monitor microbial biofilms. The operation of these sensors is based on the analysis of their resonance properties (both resonance frequency and dissipation factor) that vary in contact with the analyzed sample. This work shows that different variations during the microorganism growth can be detected by the sensors and highlights which of these changes are indicative of biofilm formation. TSM sensors have been used to monitor in real time the development of Staphylococcus epidermidis and Escherichia coli biofilms, formed on the gold electrode of the quartz crystal resonators, without any coating. Strains with different ability to produce biofilm have been tested. It was shown that, once a first homogeneous adhesion of bacteria was produced on the substrate, the biofilm can be considered as a semi-infinite layer and the quartz sensor reflects only the viscoelastic properties of the region immediately adjacent to the resonator, not being sensitive to upper layers of the biofilm. The experiments allow the microrheological evaluation of the complex shear modulus (G* = G′ + jG″) of the biofilm at 5 MHz and at 15 MHz, showing that the characteristic parameter that indicates the adhesion of a biofilm for the case of S. epidermidis and E. coli, is an increase in the resonance frequency shift of the quartz crystal sensor, which is connected with an increase of the real shear modulus, related to the elasticity or stiffness of the layer. In addition both the real and the imaginary shear modulus are frequency dependent at these high frequencies in biofilms.
Acknowledgments
We acknowledge to Zaira Martin, Eduardo Díaz and Jose Luis García, from the Centro de Investigaciones Biológicas (CIB, CSIC) for their valuable help. This work was supported by the projects DPI2013-46915-C2-1-R and DPI2016-80254-R, and the scholarship JAEPREDOC2011-058 (JAE-CSIC Program/European Social Fund).
