Raman photostability of off-resonant gap-enhanced Raman tags

Surface-enhanced Raman scattering (SERS) nanoprobes show promising potential for biosensing and bioimaging applications due to advantageous features of ultrahigh sensitivity and specificity. However, very limited research has been reported on the SERS photostability of nanoprobes upon continuous laser irradiation, which is critical for high-speed and time-lapse microscopy. The core–shell off-resonant gap-enhanced Raman tags (GERTs) with built-in Raman reporters, excited at near-infrared (NIR) region but with a plasmon resonance at visible region, allow decoupling the plasmon resonance behaviors with the SERS performance and therefore show ultrahigh Raman photostability during continuous laser irradiation. In this work, we have synthesized five types of off-resonant GERTs with different embedded Raman reporters, numbers of shell layer, or nanoparticle shapes. Via thorough examination of time-resolved SERS trajectories and quantitative analysis of photobleaching behaviors, we have demonstrated that double metallic-shell GERTs embedded with 1,4-benzenedithiol molecules show the best photostability performance, to the best of our knowledge, among all SERS nanoprobes reported before, with a photobleaching time constant up to 4.8 × 10⁵ under a laser power density of 4.7 × 10⁵ W cm⁻². Numerical calculations additionally support that the local plasmonic heating effect in fact can be greatly minimized using the off-resonance strategy. Moreover, double-shell BDT-GERTs are highly potential for high-speed and high-resolution Raman-based cell bioimaging.

Off-resonant gap-enhanced Raman tags (GERTs) show ultrahigh Raman enhancement and photostabilities and therefore can be used as ideal highly photostable nanoprobes for high-speed and high-resolution Raman bioimaging.

The surface-enhanced Raman scattering (SERS) effect strongly boosts the Raman signal of reporter molecules adsorbed on the surface of metallic plasmonic nanoparticles with the intense electromagnetic field enhancement.^1–7 With the unique fingerprint spectral feature, SERS nanoprobes, namely, metallic nanoparticles together with molecules as Raman reporters, have been extensively investigated for the biomedical applications including biosensing and bioimaging similar to the fluorescent nanoprobes.^8–14 In contrast to fluorophores, SERS nanoprobes exhibit a much larger multiplexing capability due to the narrow spectral linewidth. In addition, SERS nanoprobes show better stability than fluorophores since fluorophores easily suffer the photobleaching issue caused by modification of covalent bonds or non-specific reactions between the fluorophores and surrounding molecules upon singlet state-triplet state transition,^15,16 which is especially problematic in time-lapse microscopy.¹⁷ Typically the photobleaching in SERS nanoprobes does not follow this process and is much less problematic than that in fluorophores. It can be further minimized by decreasing the laser power and prolonging the laser exposure time. However, the photobleaching is still not favorable for high-contrast SERS-based bioimaging, which recently shows great potential for intraoperative precise identification of tumor margins and microscopic tumor invasion^18–21 and inevitably requires high-speed and a number of imaging cycles.Recently a new type of SERS nanoprobes, namely, gap-enhanced Raman tags (GERTs), have been reported to show excellent SERS enhancement,^7,22,23 which is favorable for high-speed SERS imaging.^13,22,24 GERTs are composed of plasmonic Au core–shell nanomatryoshka structures^25–27 with a uniform and nanometer-sized interior gap between the metallic core and the shell in addition to an external mesoporous silica layer if needed.²² Such nanoprobes show strong near-infrared (NIR) Raman enhancement due to the combined near-field electromagnetic and chemical enhancement in the subnanometer core–shell junction geometry while they only present one localized surface plasmon resonance (LSPR) in the visible range in the far-field spectrum.²⁷ Therefore GERTs with the built-in nanogap geometry allow decoupling the LSPR spectrum with the SERS performance. This off-resonance NIR excitation strategy is able to minimize the excitation laser induced photo-thermal effect to GERTs, leading to their ultrahigh SERS photostability during 30 min continuous cell and tumor SERS imaging without being photobleached.²² The off-resonant NIR GERTs as imaging probes are also favorable for generating minimal photothermal damage to the biological samples during the imaging process, as demonstrated by monitoring the changes in mitochondrial membrane potential of cancer cells during imaging.²⁸ The core–shell structure of GERTs additionally offers a variety of embedded Raman reporters and the numbers of shell layer,²⁹ but it remains a question and a challenge to understand how these factors of nanoprobe composition and morphology affect their SERS photostability.In this work, we synthesized five types of off-resonant GERTs either with different embedded Raman reporters (including 1,4-benzenedithiol (BDT), 4,4′-biphenyldithiol (BPDT), 4,4′-terphenyldithiol (TPDT), and 4-nitrobenzenethiol (NBT)), numbers of shell layer, or nanoparticle (NP) shapes. We have compared their particle morphologies, optical properties, and SERS photostability under continuous laser irradiation. Careful examination of time-resolved SERS trajectories and quantitative analysis of photobleaching behaviors indicate that double metallic-shell GERTs embedded with BDT molecules show the best photostability performance to the best of our knowledge. Numerical calculations are additionally performed to estimate the local laser-induced lattice temperature change of GERTs at on-resonance and off-resonance conditions. Further investigations of Raman-based cell imaging have demonstrated that those double-shell GERTs are great nanoprobes for high-speed and high-resolution Raman bioimaging.