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 107 – 108. 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.

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 cm2/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.

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.

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.

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.