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Emerging evidence indicates that the conformation of C-reactive protein (CRP) plays important roles in human inflammation and cardiovascular disease (CVD). The different conformations in the structure of CRP under different pH conditions remain an important issue to be investigated for explaining various functions of CRP under certain physiologic and pathologic conditions. We directly measured the pH-induced conformational changes in the structure of CRP by dual polarization interferometry (DPI). The CRP was attached to an aldehyde-functionalized DPI sensor chip at a concentration of 50 μg/ml, and attained 2.019 ng/mm2 to form a surface coverage with a 1.71×10(-14) mol/mm2 CRP monolayer. A pentagonal structure with an average monolayer thickness value of 5.70±0.12nm and a layer density of 0.374±0.058 g/cm2 was obtained at pH 7.0. Moreover, the DPI biosensor signals directly reflected the considerable structural parameters and phenomena of conformational changes of CRP in a pH range of 2.0-10.0. The results obtained showed that the pentameric structure of CRP might dissociated into monomers or monomer aggregates as the pH shifts toward both acidic and alkaline conditions, but only partial rearrangements of CRP subunits might occur at extremely acidic physiological conditions. Considering the proinflammatory effect and subclinical chronic inflammation, pH-induced conformational changes in the structure of CRP between monomeric and pentameric formations may strongly relate to vascular atherosclerosis and subsequent CVD., (Copyright © 2010 Elsevier B.V. All rights reserved.)

A new technique is developed to measure the nanoparticles' brownian motions by employing microparticle-tracking velocimetry (micro-PTV) in evanescent wave field, which can provide high signal-to-noise ratio images for analyzing nanoparticles' movements. This method enables real-time detection of C-reactive proteins (CRPs) during the rapid interaction between CRPs and anti-CRP-coated nanobeads as CRP concentrations are related to the nanobeads' brownian velocity in the equilibrium state. The smallest observable nanobeads with 185 nm were utilized in this experiment to detect CRP concentrations as low as 0.1 microg/mL even in a high-viscosity solution. Further, the dissociation constant, K(D), can be evaluated based on the experimental results.

A novel bio-sensing technique based on the measurement of nanobeads' Brownian motion using a micro-particle tracking velocimetry (micro-PTV) has been successfully developed to detect antigen-antibody interactions. The rapid interaction between antigens (C-reactive proteins, CRPs) and nanobeads with conjugated antibodies (anti-CRPs) has enabled real-time detection of CRPs to be easily carried out. During the binding process of CRPs to nanobeads, the mean value of the beads' diameters increases so that the Brownian velocity decreases with the increase of time. Moreover, higher CRP concentration leads to a lower Brownian velocity of the nanobeads in the equilibrium state. From the results, the limit of detection 0.1microg/ml was observed and could be further improved by using smaller nanobeads. Moreover, based on kinetic analysis, the average dissociation constant K(D)=(6.48+/-1.43)x10(-7) found by this technique for anti-CRP was shown to be in close agreement with the literature values obtained by dual-polarization interferometry (DPI) and indirect competition enzyme-linked immunosorbent assay (ELISA). This simple sensing method can further be used to detect other bio-molecules and viruses.

We used dual-polarization interferometry (DPI) to study the interaction kinetics between a 'homopolyvalent' antigen (Ag) and a monoclonal antibody (Ab). A model system, which uses a monoclonal Ab against a homopentameric Ag, C-reactive protein (CRP), is presented with principle and experiments for the study of the interactions between an Ab and an Ag that has multiple identical epitopes. This allows evaluation of the dissociation constant (K(D)) and of the binding stoichiometry by DPI based on measurements of phase changes of Ab-Ag complexes in the transverse magnetic (TM) and transverse electric (TE) polarization modes. The average experimental value of K(D) found by the DPI technique for anti-CRP Ab was shown to be in close agreement with the value obtained by an indirect competition-enzyme-linked immunosorbent assay (ELISA). Moreover, the total number of Ab combining sites on the DPI sensor chip was calculated, and the binding stoichiometry of the surface Ag-Ab complex was obtained. This study illustrates the advantages of the DPI method in biosensing in its capacity for simultaneous evaluation of the thickness and refractive index (density, mass) of adsorbed layers. This allowed a comprehensive analysis of affinity reactions between an Ab having two binding sites and a multi-sited Ag.

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 A 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 A in outer diameter and 30 A 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.