Acoustic sensors for biophysical studies
Funding: EU Horizon2020 (2018), HFSP (2008)
Combined SPR and Love Wave -SAW system for biophysical studies
A wide variety of biosensing techniques are currently available for studying phenomena at biointerfaces. Despite the great advances in the field of biosensors, there is still an ongoing demand for more accurate analytical information. In this respect, we have developed a platform that combines two different real-time, label-free and quantitative biosensing technologies. Specifically, the platform facilitates the integration of an optical technique the Surface Plasmon Resonance (SPR), with an acoustic one, a Love wave - surface acoustic wave (LW-SAW) (Fig. 1). Upon binding of biomolecules on the sensor surface, SPR measures resonance angle changes while in parallel LW-SAW measures acoustic wave phase and amplitude changes. The data obtained from the two techniques can be used to provide complementary information on biomolecular interactions not accessible with a single technique e.g., it is possible to extract the optical “dry” mass, acoustic “wet” mass, film thickness, water content, conformational status and viscoelastic properties (Sensors 2020).
(A) Hybrid SPR/LW-SAW sensor device (not drawn to scale); The Love wave geometry consists of a quartz substrate where a set of interdigitated electrodes is deposited on the surface, covered by a polymer layer for acoustic waveguiding. For the SPR response an Au sensing layer is deposited on top of the polymer layer. (B) Assembly of the developed system; first, the SPR/LW-SAW sensor device (2) is placed on the mount of the SPR instrument (1). Then, the PDMS chip (3) is fixed to the 3D-printed holder (4) and applied on the device surface.
Combined QCM-D/ellipsometry for biophysical studies
We use a system that combines the acoustic biosensor Quartz crystal microbalance with dissipation monitoring (QCM-D) with the optical technique Spectroscopic Ellipsometry (SE). Combined QCM-D/SE measurements are performed in order to simultaneously track the film formation on the sensor surface (Fig. 1). As molecules adsorb on the sensor, QCM-D records changes in the wave’s frequency (ΔF) and energy (ΔD); in parallel SE records changes in the polarization state of the reflected light as Psi (Ψ) and Delta (Δ) shifts (Fig. 2). The obtained signals can be modeled in order to get an insight into the film properties such as thickness, refractive index, “wet” mass, “dry” mass and water content.
Schematic representation of a combined QCM-D/Ellipsometry experiment.
Both techniques are: label-free, fast, sensitive (ng/cm2) and non-invasive. At the end of a combined experiment, the surface-adsorbed molecules can be further studied by other biophysical techniques such as infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and atomic force microscopy (AFM). We have used this combined system to track the adsorption/binding of various molecules such as proteins, polymers and DNA in biotechnology applications (Chem Comm 2017).
Left: Signals recorded by the SE during protein adsorption on gold sensor; Psi (red line) and Delta (purple line) angle shifts are recorded at different wavelengths (370 – 1000 nm).
Right: Frequency (black line) and energy dissipation (blue line) signals are recorded by the QCM-D during protein adsorption on gold sensor.
Study of the conformation of surface-attached DNA molecules
A major focus of our research is the elucidation of the way by which acoustic waves interact with biological molecules attached to the device surface and specifically, the molecular mechanisms involved in the interaction processes leading to acoustic energy dissipation. A novel theory developed in the group has shown that acoustic measurements can be directly related to DNA intrinsic viscosity [η] (i.e., ΔD/ΔF ~ [η]) which, in turn, can be used to provide quantitative data on the shape and size (Biophys. J 2008, Biosens. Bioelectron. 2008, Anal. Chem. 2016) of the molecules. The theory was recently analysed more thoroughly by elaboration of the initial ‘discrete molecule’ binding theory. Essentially, by combining the mechanics of damped harmonic oscillators with hydrodynamic considerations we derived explicit expressions for QCM-D energy dissipation and frequency change as functions of the analyte properties (Phys. Rev. Applied 2019).
The theory has been tested in resolving conformational changes of pre-designed model double stranded dsDNA molecules of same shape (rod) but different size and also molecules of the same size (90 bp) but various conformations (‘straight’, ‘curved’ at various positions along the chain, ‘triangle’-shaped). Experimentally more challenging DNA molecules with a pre-determined triple helical straight and bent conformation have also been used to test the validity of the model. The theory is currently being extended to the study of conformational changes of other surface-attached biological molecules.
DNA hybridization (Anal. Chem. 2012)
DNA/protein interaction (FEBS Lett. 2010, Anal. Chem. 2017)
DNA Holliday-junction transition (mediated by magnesium) between an ‘open’ and ‘closed’; the size of the structure is ~ 10 nm and the recorded change is ~ 1.5 nm (Nano Lett. 2010)