Redox Dye Mediated Fluorescence Energy Transfer of Carbon Nanotube Based Nanosensors

Single-walled carbon nanotubes (SWCNTs) are attractive materials for biochemical sensing and imaging, due to their stable photoluminescence in the tissue-transparent, near-infrared (NIR) region. Certain single-stranded DNA (ssDNA) biopolymers have been known to effectively quench the fluorescence of SWCNTs, a process made reversible by the introduction of biomolecules such as catecholamines. These nanosensors have enabled visualization of dopaminergic neuromodulation, yet with limitations in 1- selectivity of dopamine over other catecholamines, and 2- the necessity to screen for molecular recognition. These limitations motivated us to devise nanosensors with the capability to detect a broader spectrum of biomolecules based on an engineered quenching phenomenon.<br/><br/>Apart from ssDNA, certain redox dyes can quench SWCNT fluorescence via surface binding. Excitingly, we find that catecholamines do not reverse redox dye-based quenching, instead, fluorescence de-quenching can be selectively reversed upon removal of the dye molecules from the SWCNT surface. Motivated by this finding, we sought to design and develop rationally-designed, reversible dye-quenching mediated nanosensors capable of selectively detecting target biomolecules[1].<br/><br/>As a proof-of-concept, we designed the SWCNT corona where DNA hybridization is linked to the dynamic movement of methylene blue (MB), a representative dye molecule used in this study, on the SWCNT surface; thus, selectively modulating SWCNT fluorescence emission. In particular, (GT)₁₅T₂₀-wrapped SWCNTs were designed as a model to hybridize with the target sequence, A₂₀. To show that the movement of MB on the surface modulates SWCNT fluorescence, we designed multiple variations, where MB was conjugated either to the 3’-end or between (GT)₁₅ and T₂₀ of (GT)₁₅T₂₀, or to the 5’-end or 3’-end of A₂₀. All ssDNA-wrapped SWCNTs were prepared by general sonication-based dispersion. Importantly, we prepared these nanosensors under conditions where ssDNA does not quench the fluorescence; thus, eliminating any contribution from non-specific turn-on responses.<br/><br/>When MB was conjugated to (GT)₁₅T₂₀, fluorescence intensity specifically increased upon incubation with A₂₀. As expected, the increment was significantly stronger when MB was conjugated to the 3’-end of the sequence than between (GT)₁₅ and T₂₀, because the hybridization does not affect the position of MB in the latter case. For the other design where MB was conjugated to A₂₀, A₂₀-MB (MB conjugated to 3’-end) more effectively quenched the fluorescence of (GT)₁₅T₂₀-SWCNT than MB-A₂₀, which was also expected from the unidirectional nature of DNA hybridization. Overall, these results strongly suggest that the movement of MB away from the SWCNT surface modulates SWCNT fluorescence intensity. Notably, the normalized change in fluorescence intensity reached 100% upon optimization for (GT)₁₅T₂₀-MB-SWCNT. These nanosensors’ high turn-on ratio can be beneficial in developing NIR fluorescence nanosensors for DNA and RNA detection, and possibly in the future for other biomolecules. Correspondingly, we used our design to develop nanosensors capable of detecting viral RNA fragments in plants, using tobacco mosaic virus as a model pathogen. Thus, our results demonstrate the potential of using redox-dye movement relative to the SWCNT surface as a fluorescence mediator to develop rationally-designed, selective nanosensors for the detection and imaging of a broad range of biomolecules.<br/><br/>1. Nishitani S, Ao K, Jalil A, Arias-Soto OI, Moudi A, Chen F, et al. Redox dye-mediated fluorescence energy transfer of carbon nanotube based nanosensors. ChemRxiv. 2024