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

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

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

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

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

Surface Enhanced Raman Spectroscopy of a Au@Au Core–Shell Structure Containing a Spiky Shell

We have synthesized a novel Au@Au core–shell structure containing a spiky shell and characterized the surface enhanced Raman spectroscopy (SERS) of such a structure. The experimental and calculated SERS intensities for probe molecules residing between the core and shell are considerably higher in this structure containing a spiky shell, when compared to the smooth shell counterpart. Moreover, the SERS intensities for probe molecules residing on the outside of the spiky core–shell structure are comparable to those measured for the dye residing between the core and shell and similar to that of a gold nanostar. Finite-difference time-domain calculations in combination with hybridization theory are able to predict the extinction spectral features and show that the field enhancements are considerably larger than the smooth core–shell structure throughout the visible and infrared regions of the spectrum. This spiky Au@Au core–shell system shows excellent promise as a next generation SERS nanotag, in which encapsulated probe molecules yield increased SERS signal and the increased surface area of the shell allows for a greater degree of functionalization

Biochemical and Kinetic Characterization of Radical <i>S</i>‑Adenosyl‑l‑methionine Enzyme HydG

The radical <i>S</i>-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 <i>p</i>-cresol. <i>Clostridium acetobutylicum</i> 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 <i>p</i>-cresol but are necessary for conversion of a tyrosine-derived intermediate to cyanide and CO