20 research outputs found
Surface Plasmon Resonance Enhanced Ellipsometry for Biodetection
Biosensors enable scientists to learn more about biomolecular interactions, and are often used as detectors to indicate the presence of specific analytes. Currently, many detection methods require what are called labels, molecules that bind to an analyte of interest and can be easily detected. Radioactive isotopes are often used for this purpose, but labels such as these have the potential to interfere with the processes and molecules being studied. This poses a problem for medical screening, as labels may lead to an incorrect diagnosis. Therefore, label-free bio sensing techniques are in demand. Surface Plasmon Resonance Imaging (SPR), is one such method. A phenomenon derived from Maxwell’s Equations, SPR occurs at the interface between a dielectric and a metal thin film (Fig. 1). In the metal surface, electrons are not tied to particular atoms and are free to move throughout the material. This “sea” of free electrons can be modeled as a simple harmonic oscillator. In the presence of a drive force—in this case, the electric field in a light wave—electrons will oscillate. Unless the drive force is very close to the resonance frequency, little energy will be transferred to the oscillator. If the driving force matches the resonant frequency however, total energy transfer and SPR will occur. In other words, when a light beam has the correct wavelength to excite the plasmon, it will be absorbed by the metal. One important property of SPR is that the resonant frequencies can be found in the visible light spectrum. The particular frequency required depends largely on the dielectric constant of the metal used. Most importantly, this constant is sensitive to the refractive index of the metal’s surroundings. Consider a glass-gold interface submerged in water (Fig. 1). This system will oscillate at some frequency. If foreign particles are introduced, they raise the refractive index of the water and redshift the plasmon frequency. Thus, the plasmon surface can “detect” refractive index changes and foreign particles with high levels of sensitivity
Carbon-deuterium bonds as an infrared probe of protein dynamics, local electrostatics and folding
The new technique being developed in the Romesberg laboratory of incorporating carbon-deuterium bonds into proteins and using them as infrared probes is further explored. Carbon-deuterium bonds are incorporated into horse heart cytochrome c through its semi-synthesis in which only the C-terminal 39 residues are accessible. Chapter 3 describes a project investigating redox-liked differences in cytochrome c by the incorporation of C-D bonds at six residues throughout the protein. It is found that when the protein is oxidized, there are both electrostatic changes as well as a greater amount of unfolded protein present only on the proximal side of the heme. The lack of consistent linewidth changes, indicating greater flexibility of the protein in the oxidized state, along with distinct changes in the amount of unfolded protein present suggests an alternative explanation for the difference in the two redox states of cytochrome c. The data indicates that there is in fact no difference in flexibility between the reduced and oxidized states of the protein, but rather a change in the unfolding equilibrium, giving rise to more unfolded protein in the oxidized state. Subsequent chapters describe the development of using C-D bonds as infrared probes for protein folding. Six residues throughout the protein were characterized as cytochrome c was unfolded in both GnHCl and Urea. In GnHCl, the unfolding of the protein is cooperative with the exception of the Met80 loop, which undergoes an intermediate most likely due to misligation. In urea, the unfolding mechanism is quite different, and a sequential unfolding pathway similar to that observed in the amide- exchange NMR studies is presented. Along with a sequential unfolding pathway, some new observations on the folding of cytochrome c in urea have resulted. Although a similar misligated intermediate involving the Met80 loop is observed in urea, some notable and interesting differences from the GnHCl data are discussed. The possibility of a new cooperative folding unit containing the Met80 loop and the 60's helix also presents itself when the protein is unfolded in urea. Lastly, the high resolution data revealing the sequential unwinding of the C-terminal helix from the C-terminus is presente
Collagen-Gold Nanoparticle Conjugates for Versatile Biosensing
Integration of noble metal nanoparticles with proteins offers promising potential to create a wide variety of biosensors that possess both improved selectivity and versatility. The multitude of functionalities that proteins offer coupled with the unique optical properties of noble metal nanoparticles can allow for the realization of simple, colorimetric sensors for a significantly larger range of targets. Herein, we integrate the structural protein collagen with 10 nm gold nanoparticles to develop a protein-nanoparticle conjugate which possess the functionality of the protein with the desired colorimetric properties of the nanoparticles. Applying the many interactions that collagen undergoes in the extracellular matrix, we are able to selectively detect both glucose and heparin with the same collagen-nanoparticle conjugate. Glucose is directly detected through the cross-linking of the collagen fibrils, which brings the attached nanoparticles into closer proximity, leading to a red-shift in the LSPR frequency. Conversely, heparin is detected through a competition assay in which heparin-gold nanoparticles are added to solution and compete with heparin in the solution for the binding sites on the collagen fibrils. The collagen-nanoparticle conjugates are shown to detect both glucose and heparin in the physiological range. Lastly, glucose is selectively detected in 50% mouse serum with the collagen-nanoparticle devices possessing a linear range of 3–25 mM, which is also within the physiologically relevant range
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
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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
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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
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<sup>–17</sup> 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