Kyuwan Lee

Alternative messenger RNA (mRNA) splicing is a fundamental process of gene regulation, and errors in RNA splicing are known to be associated with a variety of different diseases. However, there is currently a lack of quantitative technologies for monitoring mRNA splice variants in cells. Here, we show that a combination of plasmonic dimer probes and hyperspectral imaging can be used to detect and quantify mRNA splice variants in living cells. The probes are made from gold nanoparticles functionalized with oligonucleotides and can hybridize to specific mRNA sequences, forming nanoparticle dimers that exhibit distinct spectral shifts due to plasmonic coupling. With this approach, we show that the spatial and temporal distribution of three selected splice variants of the breast cancer susceptibility gene, BRCA1, can be monitored at single-copy resolution by measuring the hybridization dynamics of the nanoplasmonic dimers. Our study provides insights into RNA and its transport in living cells, which could improve our understanding of cellular protein complexes, pharmacogenomics, genetic diagnosis and gene therapies.

Simultaneous measurement of surface‐enhanced Raman scattering (SERS) and localized surface plasmon resonance (LSPR) in nanoparticle dimers presents outstanding opportunities in molecular identification and in the elucidation of physical properties, such as the size, distance, and deformation of target species. SERS–LSPR instrumentation exists and has been used under limited conditions, but the extraction of SERS and LSPR readouts from a single measurement is still a challenge. Herein, the extraction of LSPR spectra from SERS signals is reported and a tool for measuring the interparticle distance from Raman enhancement data by the standardization of the SERS signal is proposed. The SERS nanoruler mechanism incorporates two important aspects (the LSPR scattering peak shift and the Raman shift for measuring interparticle distance), and signifies their exact one‐to‐one correspondence after spectral correction. The developed methodology is applied to calculate the interparticle distance between nanoparticle dimers from SERS signals, to detect and quantify DNA at the single‐molecule level in a base‐pair‐specific manner. It is also shown that the SERS nanoruler concept can be used in structural analysis for the specific detection of the interaction of immunoglobulin G (IgG) with its target from bianalyte Raman signals with identical shaping at single‐molecule resolution. The SERS profile shaping approach not only offers a new detection mechanism for single molecules, but also has excellent potential for studying protein interactions and the intracellular detection of mRNA.

Effective identification of breast cancer stem cells (CSC) benefits from a multiplexed approach to detect cell surface markers that can distinguish this subpopulation, which can invade and proliferate at sites of metastasis. We present a new approach for dual-mode sensing based on targeting using pointer and signal enhancement using enhancer particle networks for detection by surface plasmon resonance (SPR) and surface-enhanced Raman scattering (SERS). We demonstrate our concept to detect cell surface markers, CD44 and CD24, in three breast cancer cell lines to identify a CD44⁺/CD24⁻ subpopulation of CSCs. The designed network structure can be well-controlled and has improved sensitivity compared to conventional approaches with ability to detect a single target on the membrane of a living cell. We have also developed a fractal approach to model the dimension of the network structure and developed an empirical relationship to estimate the number of particles in the network and its size. The empirical equation was validated with experiments and finite-difference time-domain simulations, and the cell phenotyping results were found to be in good agreement with published data from conventional sorting by flow cytometry.

The designed gold nanoparticle dimer is employed to quantify label-free DNA. By using hyperspectral dark field spectroscopy, the measurement of characteristic spectra shows versatile quantification of label-free DNA up to atto molar concentration.

A multicomponent nanostructure comprising of gold nanorod–nanoparticle (AuNR–AuNP) composites was fabricated to detect thrombin at subnanomolar concentrations in diluted human blood serum. Simulation and experiments revealed that the strong electromagnetic coupling resonance at the nanorod–nanoparticle junction of these probes can be used to construct highly sensitive SERS aptasensors.