Measurement of dimensions of pentagonal doughnut-shaped C-reactive protein using an atomic force microscope and a dual polarisation interferometric biosensor

In order to develop the C-reactive protein (CRP) sensor chips for clinical detection of atherosclerosis and coronary heart disease, we used an atomic force microscope (AFM) and a dual polarization interferometric (DPI) biosensor to probe the surface ultrastructure and to measure the dimensions of CRP. A single pentagonal structure was directly visualized by AFM, and quantitative measurements of the dimensions of the protein were provided. The average height calculated for each pentagonal CRP particle was approximately 3.03 ± 0.37 nm, which basically corresponds to that (36 Å in protomer diameter) previously obtained from the structure of CRP determined by X-ray crystallography. Moreover, a experiment using dual polarization interferometric (DPI) as a biosensor was then performed, and the average monolayer thickness value (3.18 ± 0.43 nm) that was calculated basically corresponds to that obtained from the experimental value (3.03 ± 0.37 nm) of the height measured by an AFM method for CRP. Further investigations will be performed to study the surface ultrastructure of a single pentagonal CRP molecule, and for this purpose a CRP sample (at low concentration) was scanned in vacuum by AFM. The higher-resolution images clearly revealed the presence of doughnut-shaped CRP molecules. In addition, phase images of CRP molecules were captured simultaneously with their height images, and the lateral dimensions of the doughnut-shaped CRP molecules were then measured. It was found that the average values calculated for the outer diameter (11.13 ± 1.47 nm) and pore diameter (3.52 ± 0.42 nm) are respectively close to those (102 Å in outer diameter and 30 Å in pore diameter) previously obtained from the structure of CRP determined by X-ray crystallography. This study represents the first direct characterization of the surface ultrastructure and dimensional measurement of the CRP molecule on the sensor chip.