摘要： Subwavelength nanohole arrays have been very attractive for label-free biosensing applications as they offer simplicity and flexibility in read-out scheme. Recently, platforms employing imaging-based devices integrated to custom-made light sources and plasmonic nanohole array substrates have been proposed as strong candidates to increase throughput by allowing simultaneous evaluation of binding interactions. Despite their high-throughput and multiplexed nature, these platforms dramatically suffer from sensitivity compared to classical spectrometer-based systems. In this article, we introduced a highly sensitive and plasmonic imaging-based platform that can work with very low analyte concentrations. The system employs a tunable optic filter integrated to a CMOS camera that records diffraction intensity patterns of the transmitted light from a plasmonic biochip composed of periodic nanohole arrays. Monitoring diffraction field intensity variations that correspond to transmission values at different wavelengths within the spectrum, we have successfully reconstructed the transmission spectrum of nanohole arrays. Using bulk solutions, we achieved spectral shifts within the reconstructed spectrum that yields refractive index sensitivities very close to the one calculated from the original spectrum obtained with a spectrometer. Similarly, we showed that our platform yields spectral shift amounts very close to the original one upon the attachment of protein mono- and bilayers. By monitoring plasmonic diffraction field intensity images, created through a very sharp illumination light source overlapping with the plasmonic mode of interest, we experimentally achieved sub-1 ng/mL limit-of-detection. Integrating the plasmonic biochip to a microfluidic chamber, we could monitor protein binding kinetics and determined the associated binding parameters very close to the ones obtained through the classical spectrometer-based analyses. Simultaneously monitoring multiple sensing spots in real-time within the same plasmonic biochip, we demonstrated the high-throughput capability of our plasmonic imaging-based technique. Our results showed the possibility of developing plasmonic read-out platforms that could provide high-throughput and multiplexed biosensing without losing sensitivity when integrating large-scale plasmonic chips with multiple sensing locations to imaging-based devices.