Advances in surface-enhanced Raman spectroscopy (SERS) substrates for lipid and protein characterization: sensing and beyond.

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.

Ultrasensitive Plasmonic Platform for Label-Free Detection of Membrane-Associated Species.

Lipid membranes and membrane proteins are important biosensing targets, motivating the development of label-free methods with improved sensitivity. Silica-coated metal nanoparticles allow these systems to be combined with supported lipid bilayers for sensing membrane proteins through localized surface plasmon resonance (LSPR). However, the small sensing volume of LSPR makes the thickness of the silica layer critical for performance. Here, we develop a simple, inexpensive, and rapid sol-gel method for preparing thin conformal, continuous silica films and demonstrate its applicability using gold nanodisk arrays with LSPRs in the near-infrared range. Silica layers as thin as ∼5 nm are observed using cross-sectional scanning transmission electron microscopy. The loss in sensitivity due to the thin silica coating was found to be only 16%, and the biosensing capabilities of the substrates were assessed through the binding of cholera toxin B to GM1 lipids. This sensor platform should prove useful in the rapid, multiplexed detection and screening of membrane-associated biological targets.

Tunable Au-Ag nanobowl arrays for size-selective plasmonic biosensing.

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.

Patterned Plasmonic Nanoparticle Arrays for Microfluidic and Multiplexed Biological Assays.

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.

Localized Surface Plasmon Resonance Biosensing: Current Challenges and Approaches.

Localized surface plasmon resonance (LSPR) has emerged as a leader among label-free biosensing techniques in that it offers sensitive, robust, and facile detection. Traditional LSPR-based biosensing utilizes the sensitivity of the plasmon frequency to changes in local index of refraction at the nanoparticle surface. Although surface plasmon resonance technologies are now widely used to measure biomolecular interactions, several challenges remain. In this article, we have categorized these challenges into four categories: improving sensitivity and limit of detection, selectivity in complex biological solutions, sensitive detection of membrane-associated species, and the adaptation of sensing elements for point-of-care diagnostic devices. The first section of this article will involve a conceptual discussion of surface plasmon resonance and the factors affecting changes in optical signal detected. The following sections will discuss applications of LSPR biosensing with an emphasis on recent advances and approaches to overcome the four limitations mentioned above. First, improvements in limit of detection through various amplification strategies will be highlighted. The second section will involve advances to improve selectivity in complex media through self-assembled monolayers, "plasmon ruler" devices involving plasmonic coupling, and shape complementarity on the nanoparticle surface. The following section will describe various LSPR platforms designed for the sensitive detection of membrane-associated species. Finally, recent advances towards multiplexed and microfluidic LSPR-based devices for inexpensive, rapid, point-of-care diagnostics will be discussed.

Biochemical and kinetic characterization of radical S-adenosyl-L-methionine enzyme HydG.

The radical S-adenosyl-L-methionine (AdoMet) enzyme HydG is one of three maturase enzymes involved in [FeFe]-hydrogenase H-cluster assembly. It catalyzes L-tyrosine cleavage to yield the H-cluster cyanide and carbon monoxide ligands as well as p-cresol. Clostridium acetobutylicum HydG contains the conserved CX3CX2C motif coordinating the AdoMet binding [4Fe-4S] cluster and a C-terminal CX2CX22C motif proposed to coordinate a second [4Fe-4S] cluster. To improve our understanding of the roles of each of these iron-sulfur clusters in catalysis, we have generated HydG variants lacking either the N- or C-terminal cluster and examined these using spectroscopic and kinetic methods. We have used iron analyses, UV-visible spectroscopy, and electron paramagnetic resonance (EPR) spectroscopy of an N-terminal C96/100/103A triple HydG mutant that cannot coordinate the radical AdoMet cluster to unambiguously show that the C-terminal cysteine motif coordinates an auxiliary [4Fe-4S] cluster. Spectroscopic comparison with a C-terminally truncated HydG (ΔCTD) harboring only the N-terminal cluster demonstrates that both clusters have similar UV-visible and EPR spectral properties, but that AdoMet binding and cleavage occur only at the N-terminal radical AdoMet cluster. To elucidate which steps in the catalytic cycle of HydG require the auxiliary [4Fe-4S] cluster, we compared the Michaelis-Menten constants for AdoMet and L-tyrosine for reconstituted wild-type, C386S, and ΔCTD HydG and demonstrate that these C-terminal modifications do not affect the affinity for AdoMet but that the affinity for L-tyrosine is drastically reduced compared to that of wild-type HydG. Further detailed kinetic characterization of these HydG mutants demonstrates that the C-terminal cluster and residues are not essential for L-tyrosine cleavage to p-cresol but are necessary for conversion of a tyrosine-derived intermediate to cyanide and CO.

Surface-Enhanced Raman Spectroscopy of Fluid-Supported Lipid Bilayers.

Supported lipid bilayers are essential model systems for studying biological membranes and for membrane-based sensor development. Surface-enhanced Raman spectroscopy (SERS) stands to add considerably to our understanding of the dynamics and interactions of these systems through direct chemical information. Despite this potential, SERS of lipid bilayers is not routinely achieved. Here, we carried out the first measurements of a solid-supported lipid bilayer on a SERS-active substrate and characterized the bilayer using SERS, atomic force microscopy, surface plasmon resonance spectroscopy, ellipsometry, and fluorescence recovery after photobleaching (FRAP). The creation of a fluid, SERS-active supported lipid bilayer was accomplished through use of a novel silica-coated silver film-over-nanosphere substrate. These substrates offer a powerful new platform to couple common surface techniques that are challenging on the nanoscale, for example, ellipsometry and FRAP, with SERS for studying biological membranes and their dynamics.

The facile removal of CTAB from the surface of gold nanorods.

A common capping agent for gold nanorods, Cetyl trimethylammonium bromide (CTAB), is particularly problematic for biological studies because of its cytotoxicity. Several procedures have been developed to remove the CTAB from the surface of the gold nanorods, but most are lengthy, involving many steps, and use expensive reagents. Here, we present a simple, one-pot method for the complete removal of CTAB from the surface of gold nanorods, so that particles can be more effectively utilized in biological in vivo studies. The procedure involves first adding sodium borohydride to remove the CTAB, quickly followed by a replacement ligand, such as mercaptoundecanoic acid (MUA). Both the CTAB removal and MUA replacement were monitored by FTIR, surface enhanced Raman spectroscopy (SERS) and X-Ray Photoelectron Spectroscopy (XPS) and compared to commercially available citrate-capped gold nanorods. The procedure presented herein is shown to be as effective at removing CTAB and replacing it with MUA as commercially available gold nanorod samples.

Novel Liposome-Based Surface-Enhanced Raman Spectroscopy (SERS) Substrate.

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 × 106 for crystal violet encapsulated in 38 liposomes sandwiched between a 40 nm planar gold mirror and 80 nm gold colloid.