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.
Finite-difference time-domain calculations are used to study how fluorescence is modified when fluorophores are located
in proximity to various metal nanoparticle systems. The fluorophore is modeled as a radiating point dipole with
orientation defined by its polarization. The angle-resolved far-field distributions of the emission in a single plane are
computed. The emission patterns show interesting intensity variations and angular profiles depending on the dipole
orientation, size of the metal particles and the metal-dipole spacing. We also compute changes in the total radiated power
through a closed volume containing the fluorophore and metal nanoparticles relative to an isolated fluorophore. This
change in total radiated power is proportional to changes in the relative radiative decay rates of the fluorophore-metal
system. Our results suggest a high dependence of the radiated power on the fluorophore orientation, particle size, metalfluorophore
distance and particularly the presence of metal nanoparticle dimers. We examined the effect of a fluorophore
on the near-fields around silver nanoparticles. The fields can be enhanced compared to the isolated fluorophore and
exhibit interesting spatial variations around the nanoparticle that can be useful for applications involving molecular
spectroscopy.
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.
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.
Current research has revealed the importance of a class of cell surface proteins called integrins in various vital physiological functions such as blood clotting, regulation of blood pressure, tissue blood flow, and vascular remodeling. The key to integrin functionality is its ability to mediate force transmission by interacting with the extracellular matrix and cytoskeleton. In addition, they play a role in signal transduction via their connection with the proteins in focal adhesion (FA) points. To understand the complex mechanism of cell-cell and cell-extracellular matrix (ECM) adhesion that is responsible for these diverse biochemical interactions, it is necessary to identify the integrins on cells and monitor their interaction with various ligands. To this end, for the first time, we employ surface-enhanced Raman spectroscopy (SERS) to detect integrins. The results show the capability using SERS to detect the integrins to the nanomolar concentration regime and to distinguish between two different kinds of integrins, V3 and 51, that are present in vascular smooth muscle cells (VSMCs). It is anticipated that the SERS approach will potentially help elucidate the mechanism of integrin-ligand interactions in a variety of phenomena of physiological importance.
This study reports on current work involving the use of Surface Enhanced Raman Spectroscopy (SERS) for the intracellular detection of cell constituents in mouse fibroblast cells using gold nanoshells. Gold nanoshells were acquired from Nanospectra Biosciences that are based on a silica dielectric core and an outer gold shell layer. They
have the unique property of a tunable surface plasmon resonance wavelength from the visible through the near infrared which allows control of the electromagnetic field strength on its surface. Hence gold nanoshells can serve as SERS substrates with plasmonic properties that are not aggregation dependent and thus can be expected to overcome the reproducibility problem that is generally associated with aggregation based colloidal metal nanoparticles. These results represent the first steps in the development of a nanoshell-based SERS probe to detect cell organelles and/or intracellular biochemicals with the goal of ultimately improving the ability to monitor intracellular biological processes in real time.
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.
KEYWORDS: Gold, Control systems, Nanoparticles, Particles, Absorption, Absorption spectroscopy, Target recognition, Plasmons, Tellurium, Chemical elements
Transposable elements (TEs) or transposons are mobile segments of DNA that are capable of being excised and moved from one chromosomal location to another by a process known as transposition. This process requires an enzyme called the transposase that performs the excision reaction, recognizes specific target site sequences and then promotes insertion of the TE at the target site (transposition). This study provides new clues towards unraveling the causes behind the preferential affinity of the Hermes transposable element for certain insertion sites compared to other sequences which also contain recognizable target sites. The technique consists of a rapid, simple and reproducible assay that can be used to detect differences in the ability of various oligonucleotides to influence the aggregation of colloidal gold nanoparticles. The aggregation of the gold nanoparticles is monitored through UV-Visible absorption spectroscopy. Single isolated colloidal gold particles have a surface plasmon resonance manifested as a single absorbance peak at approximately 520 nm and aggregated gold complexes develop new red-shifted peaks/shoulders depending on the nature and extent of the aggregated complex. A simple ratiometric study of the area under the single and aggregated plasmon resonance peaks gives information about the extent of the aggregation. It is postulated that differences in dynamic flexibility of the oligonucleotides affect their influence on the aggregation state of the gold nanoparticles. Therefore such differences in dynamic flexibility between various insertion sites could directly or indirectly contribute to the observed target site preferences of the Hermes transposable element.
A rapid, simple, and reproducible assay is described that can be used to detect differences in the ability of oligonucleotides to influence the aggregation of colloidal gold nanoparticles. The aggregation reaction of the gold colloid was monitored through UV-visible absorption spectroscopy. Single isolated colloidal gold particles have a surface plasmon resonance manifested as a single absorbance peak at approximately 520 nm, and aggregated gold complexes develop new red-shifted peaks/shoulders depending on the nature and extent of the aggregated complex. A simple ratiometric study of the area under the single and aggregated plasmon resonance peaks thus gives information about the extent of the aggregation. It is postulated that differences in dynamic flexibility of the oligonucleotides affect their influence on the aggregation state of the gold nanoparticles. The results of this study provide new clues toward unraveling the causes behind the preferential affinity of the Hermes transposable element for certain insertion sites compared to other sequences that also contain recognizable target sites. The technique is robust and thus can potentially be used to study similar questions for numerous transposable elements and target sequences.
Integrins play an important role in the adhesion of cells to extracellular matrix and to other cells around them, more specifically fibronectin. The ultimate goal of this research is to detect these integrins on the surface of the cell with a combined atomic force microscopy (AFM) system coupled with a surface enhanced Raman spectroscopy (SERS) system. For this paper the focus was on identifying whether SERS is capable of being used to generate a unique spectrum for integrins. This was done using silver colloidal particles and the integrins a5B1 and aVB3. It was shown that a unique spectrum could be identified for each of these integrins at the nanomolar level.
Traditional Raman spectroscopy while extremely sensitive to structure and conformation, is an ineffective tool for the detection of bioanalytes at the sub milimolar level. Surface Enhanced Raman Spectroscopy (SERS) is a technique developed more recently that has been used with applaudable success to enhance the Raman cross-section of a molecule by factors of 106 to 1014. This technique can be exploited in a nanoscale biosensor for the detection of pathogenic proteins and DNA in foods by using a biorecognition molecule to bring a target analyte in close proximity to the mental surface. This is expected to produce a SERS signal of the target analyte, thus making it possible to easily discriminate between the target analyte and possible confounders. In order for the sensor to be effective, the Raman spectra of the target analyte would have to be distinct from that of the biorecognition molecule, as both would be in close proximity to the metal surface and thus be subjected to the SERS effect. In our preliminary studies we have successfully used citrate reduced silver colloidal particles to obtain unique SERS spectra of α-helical and β-sheet bovine serum albumin (BSA) that served as models of an α helical antiobiody (biorecognition element) and a β-sheet target protein (pathogenic prion). In addition, the unique SERS spectra of double stranded and single stranded DNA were also obtained where the single stranded DNA served as the model for the biorecognition element and the double stranded DNA served as themodel for the DNA probe/target hybrid. This provides a confirmation of the feasibility of the method which opens opportunities for potentially wide spread applications in the detection of food pathogens, biowarefare agents, andother bio-analytes.
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