In vivo multiplex molecular imaging of vascular inflammation using surface-enhanced Raman spectroscopy.

Vascular immune-inflammatory responses play a crucial role in the progression and outcome of atherosclerosis. The ability to assess localized inflammation through detection of specific vascular inflammatory biomarkers would significantly improve cardiovascular risk assessment and management; however, no multi-parameter molecular imaging technologies have been established to date. Here, we report the targeted in vivo imaging of multiple vascular biomarkers using antibody-functionalized nanoparticles and surface-enhanced Raman scattering (SERS). Methods: A series of antibody-functionalized gold nanoprobes (BFNP) were designed containing unique Raman signals in order to detect intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and P-selectin using SERS. Results: SERS and BFNP were utilized to detect, discriminate and quantify ICAM-1, VCAM-1 and P-selectin in vitro on human endothelial cells and ex vivo in human coronary arteries. Ultimately, non-invasive multiplex imaging of adhesion molecules in a humanized mouse model was demonstrated in vivo following intravenous injection of the nanoprobes. Conclusion: This study demonstrates that multiplexed SERS-based molecular imaging can indicate the status of vascular inflammation in vivo and gives promise for SERS as a clinical imaging technique for cardiovascular disease in the future.

Surface-Enhanced, Spatially Offset Raman Spectroscopy (SESORS) in Tissue Analogues.

Surface-enhanced, spatially offset Raman spectroscopy (SESORS) combines the remarkable enhancements in sensitivity afforded by surface-enhanced Raman spectroscopy (SERS) with the non-invasive, subsurface sampling capabilities of spatially offset Raman spectroscopy. Taken together, these techniques show great promise for in vivo Raman measurements. Herein, we present a step forward for this technique, demonstrating SESORS through tissue analogues of six known and varied thicknesses, with a large number of distinct spatial offsets, in a backscattering optical geometry. This is accomplished by spin-coating SERS-active nanoparticles (NPs) on glass slides and monitoring the relative spectral contribution from the NPs and tissue sections, respectively, as a function of both the tissue thickness and the spatial offset of the collection probe. The results show that SESORS outperforms SERS alone for this purpose, the NP signal can be attained at tissue thicknesses of >6.75 mm, and greater tissue thicknesses require greater spatial offsets to maximize the NP signal, all with an optical geometry optimized for utility. This demonstration represents a step forward toward the implementation of SESORS for non-invasive, in vivo analysis.

Bioanalytical Measurements Enabled by Surface-Enhanced Raman Scattering (SERS) Probes.

Since its discovery in 1974, surface-enhanced Raman scattering (SERS) has gained momentum as an important tool in analytical chemistry. SERS is used widely for analysis of biological samples, ranging from in vitro cell culture models, to ex vivo tissue and blood samples, and direct in vivo application. New insights have been gained into biochemistry, with an emphasis on biomolecule detection, from small molecules such as glucose and amino acids to larger biomolecules such as DNA, proteins, and lipids. These measurements have increased our understanding of biological systems, and significantly, they have improved diagnostic capabilities. SERS probes display unique advantages in their detection sensitivity and multiplexing capability. We highlight key considerations that are required when performing bioanalytical SERS measurements, including sample preparation, probe selection, instrumental configuration, and data analysis. Some of the key bioanalytical measurements enabled by SERS probes with application to in vitro, ex vivo, and in vivo biological environments are discussed.

Plasmonic color analysis of Ag-coated black-Si SERS substrate.

Red-Green-Blue (RGB) dark-field imaging can direct the choice of laser excitation for Raman enhancements on nanostructured plasmonic surfaces. Here we demonstrate that black silicon (b-Si) is a structured surface that has been shown to effectively absorb broad wavelengths of light, but also enables surface enhanced Raman scattering (SERS) when coated with silver (Ag). Coating b-Si with increasing amounts of Ag results in increased dark-field scattering at discrete frequencies associated with localized plasmon resonances. The dark-field scattering was monitored by collecting a far-field image with an inexpensive complementary metal oxide semiconductor (CMOS) camera, similar to what is available on most mobile phones. Color analysis of the RGB pixel intensities correlates with the observed SERS intensity obtained with either green (532 nm) or red (633 nm) laser excitation in SERS experiments. Of particular note, the SERS response at 633 nm showed low spectral variation and a lack of background scattering compared to SERS at 532 nm. The difference in background suggests sub-radiant (dark or Fano resonances) may be associated with the SERS response at 633 nm and a non-resonant character of SERS. These results indicate that b-Si serves a template where Ag nucleates during physical vapor deposition. Increased deposition causes the deposits to coalesce, and at larger Ag thicknesses, bulk scattering is observed. Comparison with a high enhancement Ag SERS substrate further illustrates that a high density of plasmonic junctions, or hotspots, is important for maximizing the SERS response. The randomness of the b-Si substrate and the corresponding Ag nano-features contributes to a broadband spectral response and enhancement in SERS. Metal-coated b-Si is a promising SERS substrate due to its performance and facile fabrication.

Surface enhanced Raman correlation spectroscopy of particles in solution.

Surface enhanced Raman correlation spectroscopy (SERCS) is shown as a label-free, chemically specific method for monitoring individual polymer beads and lipid vesicles interacting with a 2-D planar surface enhanced Raman (SERS) substrate in solution. The enhancement afforded by the SERS substrate allows for spectral data to be acquired in series at rates between 31 and 83 Hz. Auto- and cross-correlation of spectral data facilitates the measurement of diffusion constants for particles ranging in radius from 50 to 500 nm while discriminating signal associated with the target analyte from extraneous fluctuations. The measured diffusion coefficients are on the order of 10(-10)-10(-11) cm(2)/s, a factor of 40 times slower than predicted from the Stokes-Einstein equation, suggesting that particles are experiencing hindered diffusion at the surface. The enhanced signals appear to originate from particles less than 5 nm of the SERS substrate, consistent with adsorption to the surface. This work provides a means to measure and monitor surface interactions and demonstrates the utility and limits of SERS detection in solution over planar SERS substrates.

Label-free in situ detection of individual macromolecular assemblies by surface enhanced Raman scattering.

We demonstrate label-free detection of lipid vesicles and polystyrene beads freely diffusing in aqueous solution using surface enhanced Raman scattering (SERS). The signals observed enable real-time identification and monitoring of individual particles interacting with the SERS substrate. SERS is demonstrated as a label-free method capable of monitoring transient species in solution on the millisecond time scale.

Characterization of hotspots in a highly enhancing SERS substrate.

Vapor deposition of silver and gold onto a porous anodized aluminum oxide template is shown to produce a SERS substrate with an average surface enhancement factor of 10(7)-10(8). The high level of enhancement is explored using a combination of dark-field Rayleigh scattering and Raman spectroscopy and imaging. The scattering spectrum of the surface indicates a Plasmon resonance at 633 nm and dark-field imaging shows a relatively uniform scattering intensity at this wavelength. These measurements are consistent with the uniform enhanced Raman intensity observed in Raman maps of the substrate. Scanning electron microscopy shows the surface exhibits heterogeneous nanostructures with diameters of approximately 100 nm, the size of the pores in the template. Our measurements indicate that interactions between adjacent structures forming junctions and crevices likely give rise to a high density of hotspots, which provide the extraordinary SERS enhancement. The advantage of substrates prepared in this way is the reproducibly dense distribution of hotspots across the surface, increasing the likelihood that an analyte will experience the largest enhancement.