Parallel frequency-domain detection of molecular affinity kinetics by single nanoparticle plasmon sensors

Based on frequency-domain optical measurement using single nanoparticle plasmon sensors (NanoSPR), a versatile multiplex molecular affinity kinetics detection method is proposed. To improve the detection precision and throughput, a single-color imaging NanoSPR method (SI-NanoSPR) is used to obtain the light scattering signals of thousands of gold nanorod sensors over time under the configuration of a total internal reflection dark-field microscope. The frequency-domain power spectral density analysis of the fluctuation signal extracts the characteristic frequency f_c, by which the molecular affinity kinetics manifest the identifiable measurand. By measuring the kinetics of two different aptameric affinity systems in the same microscope field of view, the obtained equilibrium dissociation constants (K_D values) are demonstrated to be in agreement with previous studies, which were measured by traditional techniques. We expect that our NanoSPR method may pave the way for a deeper understanding of the physiological essence of biological affinity systems by accurately quantifying multiple affinity constants. The high-throughput biosensing potential is of great significance in further biomedical and pharmaceutical applications.