Surface-enhanced Raman spectroscopy (SERS) has become an essential ultrasensitive analytical tool for biomolecular analysis of small molecules, macromolecular proteins, and even cells. SERS enables label-free, direct detection of molecules through their intrinsic Raman fingerprint. In particular, protein and lipid bilayers are dynamic three-dimensional structures that necessitate label-free methods of characterization. Beyond direct detection and quantitation, the structural information contained in SERS spectra also enables deeper biophysical characterization of biomolecules near metallic surfaces. Therefore, SERS offers enormous potential for such systems, although making measurements in a nonperturbative manner that captures the full range of interactions and activity remains a challenge. Many of these challenges have been overcome through advances in SERS substrate development, which have expanded the applications and targets of SERS for direct biomolecular quantitation and biophysical characterization. In this review, we will first discuss different categories of SERS substrates including solution-phase, solid-supported, tip-enhanced Raman spectroscopy (TERS), and single-molecule substrates for biomolecular analysis. We then discuss detection of protein and biological lipid membranes. Lastly, biophysical insights into proteins, lipids and live cells gained through SERS measurements of these systems are reviewed.

Rohit Chikkaraddy opened the discussion of the Introductory Lecture: Regarding quantifying the chemical enhancement, you showed a systematic change in the SERS enhancement for halide substituted molecules due to charge transfer from the metal. Is the extra enhancement due to an inherent increase in the Raman cross-section of the molecule? How do you go about referencing, as the charge transfer changes the vibrational frequency? Richard Van Duyne answered: The extra enhancement is not due to an increase in the Raman cross section, as that is ratioed out in the calculation of the enhancement factor. The charge transfer (CT) process does not transfer a complete electron, it is a fractional degree of CT. Thus the change in vibrational frequency is small. DFT calculations that provide eigenvectors allow one to reference the vibrational modes of the free molecule with those of the adsorbed molecule. Sylwester Gawinkowski asked: You have shown that the enhancement factor curve is redshifted relative to the plasmon resonance band and has a maximum at about 800 nm. This means that the SERS signal should be strongest for excitations in the near infrared spectral region. Why do most SERS reports, particularly related to single molecule SERS, have the excitation in the green or red spectral range and not in the near infrared? Richard Van Duyne replied: The SERS excitation spectrum for isolated nanoparticles (e.g. the NSL nanotriangles that I showed in Fig. 1 of the introductory lecture 1 ) is redshifted with respect to the localized surface plasmon resonance (LSPR) by half the Stokes frequency of the vibrational mode. As the nanoparticle size is decreased the LSPR shifts to the blue so it is only for a specific size that one gets an LSPR maximum at 800 nm. Essentially all single molecule SERS experiments are done with dye molecules and the laser excitation wavelength is chosen to get maximum resonance Raman (RR) as well as SERS enhancement. For Rhodamine 6G (R6G) the laser excitation wavelength of 532 nm is close to the absorption maximum of R6G. SMSERS should be possible in the NIR for a wide range of dye molecules with absorption maxima in that spectra region. 1 A.-I. Henry, T. W. Ueltschi, M. O. McAnally and R. P. Van Duyne, Faraday Discuss., 2017, DOI: 10.1039/c7fd00181a. Marc Porter asked: Why is the oxidized form of nitrobenzene (I may not have the name of the reactant correct; my notes are a bit fuzzy, which I blame on jet lag) more sensitive to the local environment than its reduced from. Does the supporting electrolyte play a role here? Richard Van Duyne replied: The redox system you are referring to is the dye Nile Blue. The oxidized form is positively charged and the adsorption has electrostatic character. Hence it is more sensitive to the electrostatics of the local environment than the neutral reduced form. Sumeet Mahajan commented: In your work on surface-enhanced FSRS with a high rep rate laser why does the signal to noise not increase when there are 10× more pulses with the 1 MHz setup compared to the 100 kHz...

Although great strides have been made in recent years toward making highly enhancing surface-enhanced Raman spectroscopy (SERS) substrates, the biological compatibility of such substrates remains a crucial problem. To address this issue, liposome-based SERS substrates have been constructed in which the biological probe molecule is encapsulated inside the aqueous liposome compartment, and metallic elements are assembled using the liposome as a scaffold. Therefore, the probe molecule is not in contact with the metallic surfaces. Herein we report our initial characterization of these novel nanoparticle-on-mirror substrates, both experimentally and theoretically, using finite-difference time-domain calculations. The substrates are shown to be structurally stable to laser irradiation, the liposome compartment does not rise above 45 °C, and they exhibit an analytical enhancement factor of 8 × 10 for crystal violet encapsulated in 38 liposomes sandwiched between a 40 nm planar gold mirror and 80 nm gold colloid.

For applications ranging from medical diagnostics and drug screening to chemical and biological warfare detection, inexpensive, rapid-readout, portable devices are required. Localized surface plasmon resonance (LSPR) technologies show substantial promise toward meeting these goals, but the generation of portable, multiplexed and/or microfluidic devices incorporating sensitive nanoparticle arrays is only in its infancy. Herein, we have combined photolithography with Hole Mask Colloidal lithography to pattern uniform nanoparticle arrays for both microfluidic and multiplexed devices. The first proof-of-concept study is carried out with 5- and 7-channel microfluidic devices to acquire one-shot binding curves and protein binding kinetic data. The second proof-of-concept study involved the fabrication of a 96-spot plate that can be inserted into a standard plate reader for the multiplexed detection of protein binding. This versatile fabrication technique should prove useful in next generation chips for bioassays and genetic screening.

Selectivity is often a major obstacle for localized surface plasmon resonance-based biosensing in complex biological solutions. An additional degree of selectivity can be achieved through the incorporation of shape complementarity on the nanoparticle surface. Here, we report the versatile fabrication of substrate-bound Au-Ag nanobowl arrays through the galvanic ion replacement of silver nanodisk arrays. Both localized surface plasmon resonance (LSPR) and surface enhanced Raman spectroscopy (SERS) were carried out to detect the binding of analytes of varying size to the nanobowl arrays. Large increases in the LSPR and SERS response were measured for analytes that were small enough to enter the nanobowls, compared to those too large to come into contact with the interior of the nanobowls. This size-selective sensing should prove useful in both size determination and differentiation of large analytes in biological solutions, such as viruses, fungi, and bacterial cells.