Multiplexed sensing and imaging for nucleic acids using lanthanide probes and time-resolved photoluminescence

<p dir="ltr">Accurate and rapid detection of specific nucleic acids plays a critical role in disease diagnostics, especially in recent years as demands in public health soar during the COVID-19 infection. To improve efficiency and reduce false negatives, simultaneous detection of multiple nucleic acids targets in a single test is becoming increasingly important. Since most nucleic acid tests rely on fluorescent dyes for readout, multiplexing is largely restricted to colour/intensity combinations, which are prone to spectral crosstalk. </p><p dir="ltr">By contrast, lifetime multiplexing presents a novel strategy to overcome this limitation. However, existing probes for lifetime multiplexing are largely based on nanoparticles, imposing hurdles on bio-applications due to the influence of large size, the complexity in surface modification, the issues of sedimentation as well as aggregation, and potential toxicity. </p><p dir="ltr">This thesis advances time-domain multiplexed sensing and imaging for nucleic acids using molecular probes based on lanthanide complexes in conjunction with a new lifetime imaging system, aiming to achieve ultrasensitive detection, accurate quantification and precise localization at high throughput. </p><p dir="ltr">The first part of this thesis implemented the time-gating technique to realize luminescence lifetime imaging microscopy that enabled the detection of lifetimes in the microsecond region. It complements existing fluorescence lifetime imaging microscopy (FLIM) based on timecorrelated single photon counting (TCSPC), which offers precise measurement for lifetimes in the nanosecond region but has limited practicality for multiplexing because of the inability to accommodate lifetime populations in the small timescale. The extended lifetime range (μs– ms) is necessary to apply high-level multiplexing, which was successfully achieved with simultaneously visualization and identification of three cancer cell lines stained with different europium complexes. </p><p dir="ltr">The second part of this thesis demonstrated the application of luminescence lifetime imaging microscopy to a practical biosensing and imaging technique: luminescence in situ hybridisation (LISH). Instead of trying to tune the lifetime of a particular probe, we followed an alternative strategy by surveying existing lanthanide complexes that potentially provide different lifetimes for multiplexing, and summarising some key factors that lead to their different lifetimes. Specifically, we selected three europium complexes with different ligand molecules, and conjugated with oligonucleotides to develop luminescence in situ hybridisation probes. The lifetime imaging system was then used for the imaging and identification of infectious bacteria species in biofilms with these conjugated oligonucleotides probes. </p><p dir="ltr">The last part of this thesis further extended lifetime multiplexing to digital PCR assays. We adopted the principle of luminescence resonance energy transfer (LRET) to continuously tune the luminescence lifetimes of an acceptor organic dye based on its distance to a donor europium complex. Accordingly, a series of lifetime-encoded probes were designed for the detection of multiple PCR amplicons. Using the lifetime imaging microscopy, we acquired pseudo-colour images of microfluidic-generated droplets contain three PCR amplicons to demonstrate the lifetime-encoded multiplexed digital PCR detection. We finally demonstrated this technique for simultaneous detection of multiple nucleic acid sequences upon a single emission colour band, which provided a new strategy for the coding of digital PCR with the possibility of ultrahigh coding capacity and detection sensitivity. </p><p dir="ltr">This thesis demonstrates the practicality of lifetime multiplexing in detection of nucleic acids using lanthanide complexes in a range of analytical platforms and assay formats, which are expected to facilitate further research and development towards accurate and high-throughput analysis and diagnosis.</p>