UC Irvine

Cancer is complex and heterogeneous. Molecular profiling can richly characterize this tumor heterogeneity, uncovering diverse cell types and functional states for improved cancer diagnostics. In particular, extensive protein profiling can provide functional and spatial information, such as biomarker distribution, dynamic cell signaling pathways, and tissue morphology. Nanomaterial-based probes (NPs) are uniquely suited for molecular profiling applications due to their signal strength, narrow emission windows, and tunable fluorescence lifetime properties. The goal of this work is to develop a nanomaterial-based platform for molecular profiling that offers sensitive, multiplexed detection of functional molecules, while maintaining cellular resolution.

The ability to directly detect low-level expression biomarkers requires exquisitely sensitive molecular analysis. In order to achieve a high degree of sensitivity, we employ the rapid bioorthogonal reaction between trans-cyclooctene (TCO) and tetrazine (Tz). In this two-step procedure, TCO-modified antibodies bind to biomarker targets, followed by covalent attachment of numerous Tz-NPs, resulting in chemical amplification. Despite the improved sensitivity, we demonstrated that the majority of TCOs conjugated to antibodies via standard amine-coupling are non-reactive, masked by hydrophobic interactions with the antibody. We engineered linkers that restore the reactivity of antibody-bound TCO by increasing solubility and physically preventing burying within the antibody. Consequently, these TCO-modified antibodies support a higher degree of chemical amplification, resulting in greater than 5-fold signal enhancement, which can facilitate direct detection of even low-level expression markers. We further optimized linkers to maximize reactivity of a wide range of bioorthogonal reactants after antibody bioconjugation.

In addition to improving detection sensitivity, we also explored the fluorescence lifetime properties of NPs. The phasor approach to fluorescent lifetime imaging (FLIM) utilizes a simple graphical representation to resolve distinct fluorescent lifetimes. Utilizing phasor analysis, we uncovered unique lifetime features of NPs that were leveraged for enhanced multiplexed detection. We performed FLIM and phasor analysis using four different fluorescent probes, which emit light within the same emission window but have unique fluorescent lifetimes, for simultaneous detection of functional molecules. The resulting phasor map displayed lifetimes corresponding to all four fluorescent probes, and can be readily resolved. Future work will focus on expanding this approach to other spectral windows.