14 research outputs found
Biocompatible noble metal nanoparticle substrates for bioanalytical and biophysical analysis of protein and lipids
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
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 ×
10<sup>6</sup> 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 CX<sub>3</sub>CX<sub>2</sub>C motif coordinating
the AdoMet binding [4Fe-4S] cluster and a C-terminal CX<sub>2</sub>CX<sub>22</sub>C 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