Spectrally Resolved Surface-Enhanced Raman Scattering Imaging Reveals Plasmon-Mediated Chemical Transformations.

Challenges investigating molecules on plasmonic nanostructures have limited understanding of these interactions. However, the chemically specific information in the surface-enhanced Raman scattering (SERS) spectrum can identify perturbations in the adsorbed molecules to provide insight relevant to applications in sensing, catalysis, and energy conversion. Here, we demonstrate spectrally resolved SERS imaging, to simultaneously image and collect the SERS spectra from molecules adsorbed on individual nanoparticles. We observe intensity and frequency fluctuations in the SERS signal on the time scale of tens of milliseconds from n-mercaptobenzoic acid (MBA) adsorbed to gold nanoparticles. The SERS signal fluctuations correlate with density functional theory calculations of radicals generated by the interaction between MBA and plasmon-generated hot electrons. Applying localization microscopy to the data provides a super-resolution spectrally resolved map that indicates the plasmonic-induced molecular charging occurs on the extremities of the nanoparticles, where the localized electromagnetic field is reported to be most intense.

Super-resolution Surface-Enhanced Raman Scattering Imaging of Single Particles in Cells.

The ability to locate and identify molecular interactions in cells has significant importance for understanding protein function and molecular biology. Functionalized metallic nanoparticles have been used as probes for protein tracking and drug delivery because of their ability to carry therapeutic agents and readily functionalized surfaces. In this work, we present a super-resolution surface-enhanced Raman scattering (SERS) approach for imaging and tracking membrane receptors interacting with peptide-functionalized gold nanostars (AuNS). The αvß3 integrin receptors in colon cancer cells are successfully targeted and imaged using AuNS with the high-affinity amino acid sequence arginine-glycine-aspartic acid-phenylalanine-cysteine (RGDFC) attached. The RGDFC peptide interaction with the integrin receptor provides a bright and fluctuating SERS signal that can be analyzed with localization microscopy algorithms. Additionally, the observed SERS spectrum is used to confirm protein-peptide interaction. Experiments with functionalized and bare AuNS illustrate specific and nonspecific binding events. Specific binding is monitored with a localization precision of ∼6 nm. The observed spatial resolution is associated with tight binding, which was confirmed by the slower diffusion coefficient measured from 4.4 × 10-11 cm2/s for the AuNS-RGDFC compared to 7.8 × 10-10 cm2/s for the bare AuNS. Super-resolution SERS images at different focal planes show evidence of internalized particles and suggest insights into protein orientation on the surface of cells. Our work demonstrates super-resolution SERS imaging to probe membrane receptor interactions in cells, providing chemical information and spatial resolution with potential for diverse applications in life science and biomedicine.

High-speed imaging of surface-enhanced Raman scattering fluctuations from individual nanoparticles.

The concept of plasmonic hotspots is central to the interpretation of the surface-enhanced Raman scattering (SERS) effect. Although plasmonic hotspots are generally portrayed as static features, single-molecule SERS (SM-SERS) is marked by characteristic time-dependent fluctuations in signal intensity. The origin of those fluctuations can be assigned to a variety of dynamic and complex processes, including molecular adsorption or desorption, surface diffusion, molecular reorientation and metal surface reconstruction. Since each of these mechanisms simultaneously contributes to a fluctuating SERS signal, probing their relative impact in SM-SERS remains an experimental challenge. Here, we introduce a super-resolution imaging technique with an acquisition rate of 800,000 frames per second to probe the spatial and temporal features of the SM-SERS fluctuations from single silver nanoshells. The technique has a spatial resolution of ~7 nm. The images reveal short ~10 µs scattering events localized in various regions on a single nanoparticle. Remarkably, even a fully functionalized nanoparticle was 'dark' more than 98% of the time. The sporadic SERS emission suggests a transient hotspot formation mechanism driven by a random reconstruction of the metallic surface, an effect that dominates over any plasmonic resonance of the particle itself. Our results provide the SERS community with a high-speed experimental approach to study the fast dynamic properties of SM-SERS hotspots in typical room-temperature experimental conditions, with possible implications in catalysis and sensing.

Digital Protocol for Chemical Analysis at Ultralow Concentrations by Surface-Enhanced Raman Scattering.

Single molecule surface-enhanced Raman spectroscopy (SM-SERS) has the potential to revolutionize quantitative analysis at ultralow concentrations (less than 1 nM). However, there are no established protocols to generalize the application of this technique in analytical chemistry. Here, a protocol for quantification at ultralow concentrations using SM-SERS is proposed. The approach aims to take advantage of the stochastic nature of the single-molecule regime to achieved lower limits of quantification (LOQ). Two emerging contaminants commonly found in aquatic environments, enrofloxacin (ENRO) and ciprofloxacin (CIPRO), were chosen as nonresonant molecular probes. The methodology involves a multivariate resolution curve fitting known as non-negative matrix factorization with alternating least-squares algorithm (NMF-ALS) to solve spectral overlaps. The key element of the quantification is to realize that, under SM-SERS conditions, the Raman intensity generated by a molecule adsorbed on a "hotspot" can be digitalized. Therefore, the number of SERS event counts (rather than SERS intensities) was shown to be proportional to the solution concentration. This allowed the determination of both ENRO and CIPRO with high accuracy and precision even at ultralow concentrations regime. The LOQ for both ENRO and CIPRO were achieved at 2.8 pM. The digital SERS protocol, suggested here, is a roadmap for the implementation of SM-SERS as a routine tool for quantification at ultralow concentrations.