Laura Sagle

Effects of Hofmeister Anions on the Phase Transition Temperature of Elastin-like Polypeptides

The modulation of the lower critical solution temperature (LCST) of two elastin-like polypeptides (ELPs) was investigated in the presence of 11 sodium salts that span the Hofmeister series for anions. It was found that the hydrophobic collapse/aggregation of these ELPs generally followed the series. Specifically, kosmotropic anions decreased the LCST by polarizing interfacial water molecules involved in hydrating amide groups on the ELPs. On the other hand, chaotropic anions lowered the LCST through a surface tension effect. Additionally, chaotropic anions showed salting-in properties at low salt concentrations that were related to the saturation binding of anions with the biopolymers. These overall mechanistic effects were similar to those previously found for the hydrophobic collapse and aggregation of poly(N-isopropylacrylamide), PNIPAM. There is, however, a crucial difference between PNIPAM and ELPs. Namely, PNIPAM undergoes a two-step collapse process as a function of temperature in the presence of sufficient concentrations of kosmotropic salts. By contrast, ELPs undergo collapse in a single step in all cases studied herein. This suggests that the removal of water molecules from around the amide moieties triggers the removal of hydrophobic hydration waters in a highly coupled process. There are also some key differences between the LCST behavior of the two ELPs. Specifically, the more hydrophilic ELP V5A2G(3)-120 construct displays collapse/aggregation behavior that is consistent with a higher concentration of anions partitioning to polymer/aqueous interface as compared to the more hydrophobic ELP V(5)-120. It was also found that larger anions could bind with ELP V5A2G(3)-120 more readily in comparison with ELP V(5)-120. These latter results were interpreted in terms of relative binding site accessibility of the anion for the ELP.

Advances in localized surface plasmon resonance spectroscopy biosensing

In recent years, localized surface plasmon resonance (LSPR) spectroscopy advancements have made it a sensitive, flexible tool for probing biological interactions. Here, we describe the basic principles of this nanoparticle-based sensing technique, the ways nanoparticles can be tailored to optimize sensing, and examples of novel LSPR spectroscopy applications. These include detecting small molecules via protein conformational changes and resonance LSPR spectroscopy, as well as coupling LSPR with mass spectrometry to identify bound analytes. The last few sections highlight the advantages of single nanoparticle LSPR, in that it lowers limits of detection, allows multiplexing on the nanometer scale, and enables free diffusion of sensors in solution. The cases discussed herein illustrate creative ways that LSPR spectroscopy has been improved to achieve new sensing capabilities.

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.

Hydrogen Bonding of β-Turn Structure Is Stabilized in D2O

The lower critical solution temperature (LCST) of elastin-like polypeptides (ELPs) was investigated as a function of ELP chain length and guest residue chemistry. These measurements were made in both D(2)O and H(2)O. Differences in the LCST values with heavy and light water were correlated with secondary structure formation of the polypeptide chains. Such structural information was obtained by circular dichroism and infrared measurements. Additional thermodynamic data were obtained by differential scanning calorimetry. It was found that there is a greater change in the LCST value between H(2)O and D(2)O for those polypeptides which form the greatest amount of beta-turn/beta-aggregate structure. Moreover, these same molecules were the least hydrophobic ELPs. Therefore, hydrogen bonding rather than hydrophobicity was the key factor in the stabilization of the collapsed state of ELPs in D(2)O compared with H(2)O.

Methyl Groups of Trimethylamine N-Oxide Orient Away from Hydrophobic Interfaces

The molecular orientation of trimethylamine N-oxide (TMAO), a powerful protein stabilizer, was explored at aqueous/hydrophobic interfaces using vibrational sum frequency spectroscopy (VSFS). The systems studied included the octadecyltrichlorosilane (OTS)/water interface, which represents an aqueous solution in direct contact with a hydrophobic medium. Surprisingly, the measurements revealed that the methyl groups of TMAO pointed into the aqueous phase and away from the OTS. This orientation may arise from the more hydrophilic nature of methyl groups attached to a strongly electron-withdrawing atom such as a quaternary nitrogen. Additional studies were performed at the air/water interface. This interface showed a high degree of TMAO alignment, but the dangling OH from water was present even at 5 M TAMO. Moreover, the addition of this osmolyte modestly increased the surface tension of the interface. This meant that this species was somewhat depleted at the interface compared to the bulk solution. These findings may have implications for the stabilizing effect of TMAO on proteins. Specifically, the strong hydration required for the methyl groups as well as the oxide moiety should be responsible for the osmolytes depletion from hydrophobic/aqueous interfaces. Such depletion effects should help stabilize proteins in their folded and native conformations on entropic grounds, although orientational effects may play an additional role.

Characterization of alkaline transitions in ferricytochrome c using carbon-deuterium infrared probes.

The alkaline-induced structural transitions of ferricytochrome c have been studied intensively as a model for how changes in metal ligation contribute to protein function and folding. Previous studies have demonstrated that multiple non-native species accumulate with increasing pH. Here, we used a combination of experiments and simulations to provide a high-resolution view of the changes associated with increasing alkaline conditions. Alkaline-induced transitions were characterized under equilibrium conditions by following changes in the IR absorptions of carbon-deuterium chromophores incorporated at Leu68, Lys72, Lys73, Lys79, and Met80. The data suggest that at least four intermediates are formed as the pH is increased prior to complete unfolding of the protein. The first alkaline transition observed appears to be driven by a single deprotonation and occurs with a midpoint of pH 8.8, but surprisingly, the intermediate formed does not appear to be one of the well-characterized lysine misligates. At higher pH, second and third deprotonations, with a combined apparent midpoint pH of 10.2, induce transitions to Lys73- or Lys79-misligated species. Interestingly, the lysine misligates appear to undergo iron reduction by the coordinated amine. A transition from the lysine misligates to another intermediate, likely a hydroxide-misligated species, is associated with a fourth deprotonation and a midpoint of pH 10.7. Finally, the protein loses tertiary structure with a fifth deprotonation that occurs with a midpoint of pH 12.7. Native topology-based models with enforced misligation are employed to help understand the structures of the observed intermediates.

Capping Agent-Free Gold Nanostars Show Greatly Increased Versatility and Sensitivity for Biosensing

We report the first assessment of the plasmonic biosensing capabilities of capping agent-free gold nanostars. Capping agent removal was carried out using aqueous solutions of sodium borohydride, which yielded a refractive index sensitivity of 474 nm/RIU for the polyvinylpyrrolidone (PVP)-free nanostars compared with 98 nm/RIU for PVP-coated gold nanostars. Following PVP removal, biotinylated thiol and streptavidin protein were added to the nanostars, which resulted in red shifts as large as 51 nm and a limit of detection as low as 0.1 pM. Refractive index-based sensing of prostate specific antigen (PSA) both in buffer and serum was then carried out and was shown to yield shifts as large as 127 nm and have a limit of detection of 100 pM in serum. Last, a sandwich assay involving PSA was developed to aggregate the nanostars together for greater sensitivity. The sandwich assay did, indeed, give shifts close to 200 nm and was capable of detecting 10(-17) M PSA in serum. The greatly increased sensitivity and amenability to functionalization of PVP-free gold nanostars should prove useful in applications ranging from catalysis to drug delivery.

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.

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.

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.