Paper QC+AS+BI+MN-ThM13
Characterization of the Conformation of Linker-Suspended Proteins at Surfaces through Acoustic Ratio Measurements

Thursday, November 13, 2014, 12:00 pm, Room 317

Characterization of protein shape and orientation following surface binding is an area of great interest in biophysics with many applications in chemistry and nano/biotechnology. Techniques such as ellipsometry and AFM have been extensively used for providing such information. A lot of effort has also been put with acoustic sensors; results in this case though depend greatly on the data interpretation model employed. An important question is always the preservation of protein integrity/form.

In this work we employ acoustic devices based on a QCM geometry at 35 MHz. The acoustic ratio ΔD/ΔF, i.e., the dissipation over frequency change of the shear wave has been employed in our analysis. We have previously shown¹ that as a tool, this ratio provides valuable information regarding the conformation of surface attached DNA molecules; we have also employed this approach in the design of DNA assays for diagnostic purposes, including detection of sequence targets in real samples².

Here we expand this methodology in proteins; streptavidin is used as a case study for characterizing spherical protein immobilization on an acoustic device. Good control of the binding mode was achieved by changing the distance of the protein from the surface, ranging from zero (direct physisorption) to several nm, using anchor molecules. In this way we can manipulate the degree of surface interference to the protein structure. Our results clearly show that direct protein adsorption is a multistep process resulting in very low acoustic ratio, in agreement with the literature. However, we show for the first time that suspending the protein away from the surface from a single point through a variable-length linker, gives an entirely different picture; the process is a single-step event, as judged from D-F plots, and the resulting acoustic ratio is much higher (order of magnitude) than that obtained in physisorption. The effect of the linker length on the apparent acoustic ratio is analyzed. This approach gives more reliable and different information regarding the protein shape than do simple physisorption protocols and interpretation models involving notions such as ‘film’ formation etc.

References:

1. A. Tsortos, et al., Biosens. Bioelectron. 2008, 24:836; A. Tsortos et al., Biophys. J. 2008, 94:2706