Advancing time-gated luminescence techniques for ultrasensitive and high-throughput biodetection
Creators: name: Lu, Yiqing
Publisher: name: Macquarie University
Year: 2022
Subjects: Other education not elsewhere classified

description: "Detection, quantification, or localisation of particular cells or molecules quickly, sensitively and accurately, is fundamental to many areas of modern biomedical research and industry: from understanding sub-cellular processes and biochemical pathways to the early detection of diseases in clinical settings. However, the targets-of-interest are often in a tiny minority within a surrounding abundance of biological and biochemical substances, which presents significant problems for rapid detection of trace of biological analytes in large-volume samples. Current fluorescence detection based on fluorescent biolabels is seriously limited by autofluorescence from non-target detritus and organisms and spectral overlapping associated with multichannel detection in the spectral domain. These limits can be addressed using time-gated luminescence detection techniques based on molecular probes with luminescence lifetimes in a μs-to-ms region rather than the nanosecond lifetimes of standard fluorescent labels, so that autofluorescence background can be effectively suppressed. This thesis explores novel time-gated luminescence strategies to develop a new generation of analytical systems - time-domain scanning cytometry - for high-throughput, ultrasensitive detection, quantification and localization of trace biomolecules and rare-event cells in biological samples. The first part of this thesis presents two practical scanning strategies for fully automated time-gated luminescence detection of rare cell types in 2-dimensional samples. We first report a "step-by-step" scanning scheme incorporating the time-gated detection with wide-field scanning followed by imaging confirmation. This minimises the requirements for image acquisition, storage and processing to specific areas-of-interest identified as containing target analytes. We then present a more sophisticated "on-the-fly" scanning scheme employing continuous-motion scanning which involves X-axis scanning for rapid identification of target cells and orthogonal Y-axis scanning for accurate localisation and quantitative measurement of cells. This accelerates the scanning speed of a microscopy slide by a factor of 20, reducing whole-sample scanning time from 1 hour to ~ 3 minutes, and enables ultrasensitive quantification of low-abundance biomolecules on rare-event cells. The second part of this thesis further explores the principle, feasibility and techniques of using the different luminescence lifetimes as distinguishable tags for high-throughput detection of multiple bioanalytes simultaneously. We first investigate the fast-fitting algorithm for real-time measurement of the luminescence lifetimes in the μs region. This extends the capability of the scanning cytometry system to rapid identification of microspheres with different luminescence lifetimes. This powerful time-resolved measurement capability has enabled development of novel lifetime-tunable upconversion nanocrystals, which we have named "τ-dots". The lifetimes of these "τ-dots" can be engineered to predetermined values on the tens-hundreds of microsecond range for both microspheres containing many individual nanoparticles and for single nanoparticles. The applications of τ-dots in fast scanning cytometry demonstrates how the temporal dimension can be used in luminescence detection, instead of multi-channel spectral detection used in conventional fluorescence techniques. The advances brought to the time-domain luminescence detection in this thesis are expected to stimulate new discoveries in analytical biotechnology for public health and safety. Robust time-domain scanning cytometry systems can meet the increasing demands of many critical areas of clinical diagnosis, environmental monitoring, bioinformatic research and personalised medicine, where ultrasensitive and high-throughput analytical tools are needed to cope with the biological complexity. This thesis is by publication. Results are presented in the form of five major journal papers." -- Abstract.