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.

Ex Vivo Detection of Circulating Tumor Cells from Whole Blood by Direct Nanoparticle Visualization.

The detection of circulating tumor cells (CTCs) from blood samples can predict prognosis, response to systemic chemotherapy, and metastatic spread of carcinoma. Therefore, approaches for CTC identification is an important aspect of current cancer research. Here, a method for the direct visualization of nanoparticle-coated CTCs under dark field illumination is presented. A metastatic breast cancer cell line (4T1) was transduced with a non-native target protein (Thy1.1). Positive 4T1-Thy1.1 cells incubated with antibody-coated metallic nanoshells appeared overly bright at low magnification, allowing a quick screening of samples and easy visual detection of even single isolated CTCs. The use of a nontransduced cell line as control creates the ideal scenario to evaluate nonspecific binding. A murine metastatic tumor model with the 4T1-Thy1.1 cell line was also implemented. Blood was drawn from mice over the course of one month, and CTCs were successfully detected in all positive subjects. This work validates the use of metallic nanoshells as labels for direct visualization of CTCs while providing guidelines to a systematic development of nanotechnology-based detection systems for CTCs.

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.

Improved synthesis of gold and silver nanoshells.

Metallic nanoshells have been in evidence as multifunctional particles for optical and biomedical applications. Their surface plasmon resonance can be tuned over the electromagnetic spectrum by simply adjusting the shell thickness. Obtaining these particles, however, is a complex and time-consuming process, which involves the preparation and functionalization of silica nanoparticles, synthesis of very small metallic nanoparticles seeds, attachment of these seeds to the silica core, and, finally, growing of the shells in a solution commonly referred as K-gold. Here we present synthetic modifications that allow metallic nanoshells to be obtained in a faster and highly reproducible manner. The main improved steps include a procedure for quick preparation of 2.3 ± 0.5 nm gold particles and a faster approach to synthesize the silica cores. An investigation on the effect of the stirring speed on the shell growth showed that the optimal stirring speeds for gold and silver shells were 190 and 1500 rpm, respectively. In order to demonstrate the performance of the nanoshells fabricated by our method in a typical plasmonic application, a method to immobilize these particles on a glass slide was implemented. The immobilized nanoshells were used as substrates for the surface-enhanced Raman scattering from Nile Blue A.