Digital analysis of single plasmonic nanoparticles for ultrasensitive biosensing

Abstract

Abstract: A major hurdle in the development of next-generation biosensors is the ability detect single molecules and the use of this detection to form an appropriate assessment of the larger biological system. Once, this has been achieved, it will be possible to build new and more efficient biosensors that can detect subtle concentration changes in early disease stages and, biosensors that no longer require calibration. The unique optical properties of plasmonic nanoparticles offer the possibility to develop single nanoparticle assays that are capable of these ultra-low detection limits. Current spectroscopic techniques can sensitively analyse spectral shifts of these nanoparticles; however, these nanoparticles need to be analysed in a high-throughput manner to obtain a useful assessment of a biological system. This main objective of this thesis was to develop a technique for the sensitive and high-throughput analysis of plasmonic nanoparticles, and the construction of a biosensing interface capable of ultra-sensitive detection. The first stage in this goal was achieved by developing a technique for high-throughput single nanoparticle analysis. The technique utilises a consumer-grade camera and a dark-field microscope to analyse the λmax of several thousands of nanoparticles within a second, without the need for a spectrometer. The development of this digital analysis technique enabled single nanoparticle assays to be performed rapidly. However, to detect at very low concentrations it is necessary to employ a signal enhancement step. Within this work, two signal amplification and detection strategies were explored: i) enzyme-catalysed nanoparticle growth and ii) core-satellite formation for multiplexed detection of DNA. The enzyme-catalysed system was able to grow the nanoparticles and cause a change in LSPR. However, for integration into single nanoparticle assays the enzyme-based amplification strategy needs to be optimised to minimise the background signal. A second amplification technique based on plasmon coupling was fabricated and used to construct a single nanoparticle assay. This single-satellite nanosensor was used to demonstrate the ability to count single DNA. Finally, DNA-directed core-satellite formation was used with the digital analysis technique to show the ability to perform multiplexed single nanoparticle assays in-parallel.