Through applications of fluorescence correlation spectroscopy, our research has provided a unique view of HIV virion-antibody interactions. We developed a novel FRET-FCS based assay to identify how neutralizing and non-neutralizing epitopes are expressed on single virions. Most of our methods can be expanded for application to studies of in vitro primary infection systems. Recently, we developed a quantitative, intrinsic, label-free, and minimally invasive method based on two-photon fluorescence lifetime (FLT) imaging microscopy (2p-FLIM) for imaging NADH metabolism of virally infected cells and tissue sections.
Herein we describe promising results from the combination of fluorescent lifetime imaging microscopy (FLIM) and diffusion reflection (DR) medical imaging techniques. Three different geometries of gold nanoparticles (GNPs) were prepared: spheres of 20nm diameter, rods (GNRs) of aspect ratio (AR) 2.5, and GNRs of AR 3.3. Each GNP geometry was then conjugated using PEG linkers estimated to be 10nm in length to each of 3 different fluorescent dyes: Fluorescein, Rhodamine B, and Sulforhodamine B. DR provided deep-volume measurements (up to 1cm) from within solid, tissue-imitating phantoms, indicating GNR presence corresponding to the light used by recording light scattered from the GNPs with increasing distance to a photodetector. FLIM imaged solutions as well as phantom surfaces, recording both the fluorescence lifetimes as well as the fluorescence intensities. Fluorescence quenching was observed for Fluorescein, while metal-enhanced fluorescence (MEF) was observed in Rhodamine B and Sulforhodamine B – the dyes with an absorption peak at a slightly longer wavelength than the GNP plasmon resonance peak. Our system is highly sensitive due to the increased intensity provided by MEF, and also because of the inherent sensitivity of both FLIM and DR. Together, these two modalities and MEF can provide a lot of meaningful information for molecular and functional imaging of biological samples.
In the past several years we have demonstrated the metal-enhanced fluorescence (MEF) and the significant
changes in the photophysical properties of fluorophores in the presence of metallic nanostructures and nanoparticles
using ensemble spectroscopic studies. In the represented study, we explored the distance effect on intrinsic fluorescence
of proteins adsorbed on our layer-by-layer assembled metallic nanostructures. The study is expected to provide more
information on the importance of positioning the proteins at a particular distance for enhanced fluorescence from
metallic structures. For the present study, we considered using easy and inexpensive LbL technique to provide welldefined
distance from metallic surface. The explored proteins were adsorbed on different numbers of alternate layers of
poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH). SA and BSA were electrostatically attached to
the positively charged PAH layer. We obtained a maximum of ~11-fold and 9-fold increase in fluorescence intensity
from SA and BSA, respectively. And also we observed ~3-fold decrease in BSA lifetime on metallic nanostructures than
those on bare control quartz slides. This study reveals the distance dependence of protein fluorescence.
We consider the possibility of using aluminum nanostructures for enhancing the intrinsic emission of biomolecules. We
used the finite-difference time-domain (FDTD) method to calculate the effects of aluminum nanoparticles on nearby
fluorophores that emit in the ultra-violet (UV). We find that the radiated power of UV fluorophores is significantly
increased when they are in close proximity to aluminum nanostructures. We show that there will be increased localized
excitation near aluminum particles at wavelengths used to excite intrinsic biomolecule emission. We also examine the
effect of excited-state fluorophores on the near-field around the nanoparticles. Finally we present experimental evidence
showing that a thin film of amino acids and nucleotides display enhanced emission when in close proximity to aluminum
nanostructured surfaces. Our results suggest that biomolecules can be detected and identified using aluminum
nanostructures that enhance their intrinsic emission. We hope this study will ignite interest in the broader scientific
community to take advantage of the plasmonic properties of aluminum and the potential benefits of its interaction with
biomolecules to generate momentum towards implementing fluorescence-based bioassays using their intrinsic emission.
Most of the applications of fluorescence require the use of labeled drugs and labeled biomolecules. Due to the
need of labeling biomolecules with extrinsic fluorophores, there is a rapidly growing interest in methods which provide
label-free detection (LFD). Proteins are highly fluorescent, which is due primarily to tryptophan residues. However,
since most proteins contain tryptophan, this emission is not specific for proteins of interest in a biological sample. This is
one of the reasons of not utilizing intrinsic tryptophan emission from proteins to detect specific proteins. Here, we
present the intrinsic fluorescence for several proteins bound to the silver or aluminum metal nanostructured surfaces. We
demonstrate the metal enhanced fluorescence (MEF) of proteins with different numbers of tryptophan residues. Large
increases in fluorescence intensity and decreases in lifetime provide the means of direct detection of bound protein
without separation from the unbound. We present specific detection of individual types of proteins and measure the
binding kinetics of proteins such as IgG and streptavidin. Additionally, specific detection of IgG and streptavidin has
been accomplished in the presence of large concentrations of other proteins in sample solutions. These results will allow
design of surface-based assays with biorecognitive layer that specifically bind the protein of interest and thus enhance its
intrinsic fluorescence. The present study demonstrates the occurrence of MEF in the UV region and thus opens new
possibilities to study tryptophan-containing proteins without labeling with longer wavelength fluorophores and provides
an approach to label-free detection of biomolecules.
Fluorescence detection is a central component in biological research. In recent years there has been a growing
interest in the interactions of fluorophores with metallic surfaces or particles. A single-stranded oligonucleotide was
chemically bound to a single 50 nm diameter silver particle and a Cy5-labeled complementary single-stranded
oligonucleotide was hybridized with the particle-bound oligonucleotide. The bound Cy5 molecules on the silver particles
were spatially separated from the silver surface by the hybridized DNA duplex chains, which were about 8 nm in length,
to reduce the competitive quenching. We use fluorescence lifetime correlation spectroscopy (FLCS) with picosecond
time-resolved detection to separate the fluorescence correlation spectroscopy (FCS) contributions from fluorophores and
metal-conjugated fluorophores. The single Cy5-labeled 50 nm silver particles displayed a factor of 15-fold increase in
emission signal and 5-fold decrease in emission lifetimes in solution relative to the Cy5-DNA in the absence of metal.
Lifetime measurements support the near-field interaction mechanism between the fluorophore and silver nanoparticle. In
this study, FLCS is being applied to a system where the brightness and the fluorescent lifetime of the emitting species
are significantly different. Our measurements suggest that FLCS is a powerful method for investigating the metal-fluorophore
interaction at the single molecule level and to separate two different species from a mixture solution
emitting at the same wavelength. Additionally, the highly bright Cy5-DNA-Ag molecules offer to be excellent probes in
high background biological samples.
In the past several years we have demonstrated the metal-enhanced fluorescence (MEF) and the significant changes
in the photophysical properties of fluorophores in the presence of metallic nanostructures and nanoparticles using
ensemble spectroscopic studies. Here, in the present study, we explored the new insights of these interactions using
single-molecule fluorescence spectroscopy. The single molecule study is expected to provide more information,
especially on the heterogeneity in the fluorescence enhancement and decrease in lifetimes associated with fluorophore-metal
interactions, which is otherwise not possible to observe using ensemble measurements. For the present study, we
considered using CdTe nanocrystals (QDots) prepared using modified Weller method as the fluorophores under
investigation. QDots having few nanometer sizes, tunable absorption and fluorescence spectral properties, and high
photo-stabilities are of important class of fluorescent probes. Because of these unique features Qdots are widely used as
probes in various fields, including biological labeling and imaging. These CdTe nanocrystals show characteristic spectral
features in solution and on the solid substrate. The CdTe nanocrystals dispersed in PVA and spin-casted on SiFs surface
show ~5-fold increase in fluorescence intensity and ~3-fold decrease in lifetimes compared to on glass substrate. The
data obtained using ensemble and single molecule spectroscopy are complimentary to each other. Additionally as
anticipated we have seen increased heterogeneity in the plasmon induced fluorescence modulations. Moreover single
molecule spectroscopic study revealed significant reduction in blinking of CdTe nanocrystals on plasmonic
nanostructures. Subsequently, we present these important findings on metal-fluorophore interactions of CdTe
nanocrystals (QDots) on plasmonic nanostructures.
Metal-enhanced fluorescence (MEF) is useful in single molecule detection (SMD) by increasing the photostability,
brightness and increase in radiative decay rates of fluorophores. We have investigated MEF from an individual
fluorophore tethered to a single silver nanoparticle and also a single fluorophore between a silver dimer. The
fluorescence lifetime results revealed a near-field interaction mechanism of fluorophore with the metal particle. Finite-difference
time-domain (FDTD) calculations were employed to study the distribution of electric field near the metal
monomer and dimer. The coupling effect of metal particles on the fluorescence enhancement was studied. We have also
investigated the photophysics of FRET near metal nanoparticles and our preliminary results suggest an enhanced FRET
efficiency in the presence of a metal nanoparticle. In total, our results demonstrate improved detectability at the single
molecule level for a variety of fluorophores and quantum dots in proximity to the silver nanoparticles due to the near-field
metal-fluorophore interactions.
Thermal evaporation was used to deposit particulate aluminum films of varied thicknesses on quartz substrates. These
substrates were characterized by scanning electron microscopy (SEM), which reveal that with an increase in aluminum
thickness, the films progress from particulate towards smooth surfaces. Until now, metal-enhanced fluorescence (MEF)
has primarily been observed in the visible-NIR wavelength region using silver or gold island films and roughened
surfaces. We now report that fluorescence can also be enhanced in the ultraviolet-blue region of the spectrum using
nano-structured aluminum films. We used two probes, one in the ultraviolet (a DNA base analogue 2-aminopurine: 2-
AP) and another one in blue spectral region (a coumarin derivative: 7-HC) for the present study. We observed increased
emission, decrease in fluorescence lifetime and increase in photostability of the dyes in a 10 nm spin-casted polyvinyl
alcohol film on the Al nanostructured surfaces. We observe that the fluorescence enhancement factor depends on the
thickness of the Al films because the size of the nanostructures formed varies with Al thickness. These studies indicate
that Al nano-structured substrates can potentially find widespread use in MEF applications particularly in the UV - blue
spectral regime. Finite-Difference Time-Domain (FDTD) calculations were performed that revealed enhanced near-fields
induced around aluminum nanoparticles by a radiating fluorophore emitting at the emission wavelength of 2-AP.
The effect of such enhanced fields on the fluorescence enhancement observed is also discussed.
Fluorescence is widely used in biological research. Future advances in biology and medicine often depend on the advances in the capabilities of fluorescence measurements. In this overview paper we describe how a combination of fluorescence, and plasmonics, and nanofabrication can fundamentally change and increase the capabilities of fluorescence technology. This change will be based on the use of surface plasmons which are collective oscillations of
free electrons in metallic surfaces and particles. Surface plasmon resonance is now used to measure bioaffinity reactions. However, the uses of surface plasmons in biology are not limited to their optical absorption or extinction. We have shown that fluorophores in the excited state can create plasmons which radiate into the far field; additionally fluorophores in the ground state can interact with and be excited by surface plasmons. These interactions suggest that the
novel optical absorption and scattering properties of metallic nanostructures can be used to control the decay rates, location and direction of fluorophore emission. We refer to this technology as plasmon-controlled fluorescence. We predict that plasmon-controlled fluorescence (PCF) will result in a new generation of probes and devices. PCF is likely to allow design of structures which enhance emission at specific wavelengths and the creation of new devices which control and transport the energy from excited fluorophores in the form of plasmons, and then convert the plasmons back to light.
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