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Constructing a Fluorescence Lifetime Nanoprobe Library to Advance Lifetime-Based Multiplexing


Despite progress of the modern biomedical revolution, efficient personalized cancer cures remain largely elusive due to the vast amount of potential biomarkers to identify. This diverse pool of receptors is a product of a complex ecosystem of distinct key cell types within a tumor which may help promote proliferation. This intra-tumor heterogeneity has necessitated development of methods to identify key cell type receptors, such as molecular probes that target biomarkers. Probes harboring fluorescent species are among the most successful and couple distinct-spectra fluorescent species to a targeting protein, such as an antibody. However, existing probe libraries are limited to only 10 simultaneous detection channels, severely reducing the amount of ascertainable molecular information. Fluorescence lifetime imaging microscopy (FLIM) has been proposed as an expansion to conventional fluorescence imaging by adding an additional dimension of channels to analyze, but has been limited by the complexities of curve fitting decay half-lives. Recently, the phasor approach to FLIM provided an elegant means to analyze species mixtures on a phasor plot. Here, we demonstrate the construction of a lifetime probe library encapsulating a controlled loading ratio of fluorescent species and analyzed with the phasor approach to FLIM. Using a reverse microemulsion synthesis method, organic dyes, dark quenchers, and quantum dots have been incorporated into probe cores to unlock a variety of distinct phasor positions within a single spectral window. Probe surfaces may be modified with polyethylene glycol chains to help prevent in-vitro protein adsorption and a functional loss of targeting specificity. Bioorthogonal click chemistry linkers may be conjugated onto chain termini to allow highly specific coupling to analogous premodified antibodies via rapid in-vitro two-step targeting of tumor biomarkers, or through the formation of immunoconjugates for direct targeting. Subsequently, the presence of key receptors may be inferred using the phasor approach. With the success of our preliminary probes, multiplexed targeting assays will follow. Ultimately, we intend to use our lifetime probe library in clinical settings to efficiently characterize tumors via simultaneous detection of up to 80 pre-selected personalized biomarkers.

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