This paper reports the in situ detection of C-reactive protein (CRP) and antibody-coated nanobead interaction by measuring the variation of the Brownian diffusion of the nanobeads. The measurements are carried out by using total internal reflective fluorescence-enhanced (TIRF-enhanced) micro-particle-tracking velocimetry (micro-PTV), which is highly sensitive for analyzing nanobead movements in the near-wall region. The characteristics of evanescent wave penetration of various liquid samples, which have different refractive indices, are investigated for nanobead illumination. The Brownian velocities of the nanobeads in various concentrations of CRPs within glycerol solution and serum are measured in real time. The detectable CRP concentration range of 1–6 mg/ml in human serum is found, and the detecting process can be finished in 10 min. From the results, the dissociation constant of CRP and anti-CRP in various viscosity solutions can be determined through the developed kinetic analysis.
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