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Biomedical subjects

Ignacy Gryczynski

Publications and source records attributed to Ignacy Gryczynski.

54 records · Page 3Linked to original sources
PubMed2003

DNA hybridization assays using metal-enhanced fluorescence.

We describe a new approach to DNA hybridization assays using metal-enhanced fluorescence. Thiolated oligonucleotides were bound to silver particles on a glass substrate. Addition of a complementary fluorescein-labeled oligonucleotide resulted in a dramatic time-dependent 12-fold increase in fluorescence intensity during hybridization. Proximity to silver particles resulted in a decreased fluorescence lifetime. This effect is thought to be the result of enhanced fluorescence from fluorescein near metallic silver particles. Hybridization could thus be measured from the decay kinetics of the emission, which can be measured independently from the emission intensity. These results suggest the use of silver particles as a general approach to measure DNA hybridization as a method to increase the sensitivity of DNA detection.

Base Sequence↗
PubMed2003

Fluorescence spectral properties of cyanine dye-labeled DNA oligomers on surfaces coated with silver particles.

We examined the fluorescence spectral properties of DNA oligomers, labeled with Cy3 or Cy5, when bound to quartz surfaces coated with metallic silver particles. Prior to binding of labeled DNA the surfaces were treated with polylysine or 3-aminopropyl triethoxysilane or were coated with avidin for binding of biotinylated oligomers. The fluorescence intensities were increased an average of 8-fold on these surfaces. Despite the increased emission intensity, the photostability of the labeled DNA was the same or higher on the silver-coated surfaces than on the uncoated slides. The time-integrated intensities, that is the area under the intensity plots with continuous illumination, increased an average of 6-fold. In all cases the lifetimes were dramatically shortened on the silver particles, indicating an over 100-fold increase in the radiative decay rates. These results suggest the use of substrates containing silver particles for increased sensitivity of DNA detection on DNA arrays.

Avidin↗
PubMed2003

Increased resonance energy transfer between fluorophores bound to DNA in proximity to metallic silver particles.

We examined the effects of metallic silver particles on resonance energy transfer (RET) between fluorophores covalently bound to DNA. A coumarin donor and a Cy3 acceptor were positioned at opposite ends of a 23-bp double helical DNA oligomer. In the absence of silver particles the extent of RET is near 9%, consistent with a Forster distance R(0) near 50 A and a donor to acceptor distance near 75 A. The transfer efficiency increased when the solution of AMCA-DNA-Cy3 was placed between two quartz plates coated with silver island films to near 64%, as determined by both steady-state and time-resolved measurements. The apparent R(0) in the presence of silver island films increases to about 110 A. These values of the transfer efficiency and R(0) represent weighted averages for donor-acceptor pairs near and distant from the metallic surfaces, so that the values at an optimal distance are likely to be larger. The increased energy transfer is observed only between two sandwiched silvered slides. When we replaced one silvered slide with a quartz plate the effect vanished. Also, the increased energy transfer was not observed for silvered slides separated more than a few micrometers. These results suggest the use of metal-enhanced RET in PCR, hybridization, and other DNA assays, and the possibility of controlling energy transfer by the distance between silver surfaces.

Base Sequence↗
PubMed2003

Effects of fluorophore-to-silver distance on the emission of cyanine-dye-labeled oligonucleotides.

We examined the fluorescence spectral properties of Cy3- and Cy5-labeled oligonucleotides at various distances from the surface of silver island films. The distance to the surface was controlled by alternating layers of biotinylated bovine serum albumin (BSA) and avidin, followed by binding of a biotinylated oligonucleotide. The maximum enhancement of fluorescence near a factor of 12 was observed for the first BSA-avidin layer, with the enhancement decreasing to 2-fold for six layers. The minimum lifetimes were observed for the first BSA-avidin layer, and were about 25-fold shorter than on quartz slides without silver, with the lifetimes being about 2-fold shorter for six BSA-avidin layers. These results suggest that maximum fluorescence enhancements occur about 90A from the silver surface, a distance readily obtained by one or two layers of proteins.

Animals↗
PubMed2003

Fluorescence spectral properties of cyanine dye labeled DNA near metallic silver particles.

Recent studies have demonstrated that silver metallic particles can increase the quantum yield and decrease the lifetimes of nearby fluorophores. These studies are extended to double stranded DNA oligomers labeled with N,N'-(dipropyl)-tetramethylindocarbocyanine (Cy3) or N,N-(dipropyl)-tetramethylindodicarbocyanine (Cy5). The proximity to silver particles increases the apparent quantum yields and decreases the lifetimes of the double helical DNA 23-mer labeled individually with Cy3 or Cy5. The decreased lifetimes are accompanied by apparently increased photostability of the labeled oligomers near silver particles. Because of spatial averaging across the sample these results are likely to significantly underestimate the effects of silver particles on labeled DNA localized at an optimal distance from the metallic surface. These results suggest that DNA arrays fabricated on substrates with silver particles can display increased sensitivity and photostability in the analysis of gene expression.

Base Sequence↗
PubMed2003

Fluorescence properties of labeled proteins near silver colloid surfaces.

The fluorescence properties of a monolayer of labeled avidin molecules were studied near silver island films. We first adsorbed a monolayer of biotinylated-BSA as a base that was used to capture labeled avidin molecules. For labeled avidin on silver island films, we observed an increase of the fluorescence intensity of between 18 and 80 with one-photon excitation and up to several hundredfold or larger with two-photon excitation. The probes were moderately more photostable in the presence of silver islands. There was also a dramatic decrease in the lifetimes with the amplitude-weighted values decreasing from 7- to 35-fold. The data suggest that these spectral changes are due to both increased rates of excitation near the metallic particles and increases in the rates of radiative decay. Because these silver island surfaces are very heterogeneous, we are hopeful that larger increases in intensity and photostability can be obtained for probes situated at an optimal distance from the ideal island surfaces.

Avidin↗
PubMed2003

Effects of metallic silver island films on resonance energy transfer between N,N'-(dipropyl)-tetramethyl- indocarbocyanine (Cy3)- and N,N'-(dipropyl)-tetramethyl- indodicarbocyanine (Cy5)-labeled DNA.

Resonance energy transfer (RET) is typically limited to distances below 60 A, which can be too short for some biomedical assays. We examined a new method for increasing the RET distances by placing donor- and acceptor-labeled DNA oligomers between two slides coated with metallic silver particles. A N,N'-(dipropyl)-tetramethylindocarbocyanine donor and a N,N'-(dipropyl)-tetramethylindodicarbocyanine acceptor were covalently bound to opposite 5' ends of complementary 23 base pair DNA oligomers. The transfer efficiency was 25% in the absence of silver particles or if only one slide was silvered, and it increased to an average value near 64% between two silvered slides. The average value of the Forster distance increased from 58 to 77 A. The energy transfer data were analyzed with a model assuming two populations of donor-acceptor pairs: unaffected and affected by silver island films. In an affected fraction of about 28%, the apparent energy transfer efficiency is near 87% and the Forster distance increases to 119 A. These results suggest the use of metallic silver particles to increase the distances over which RET occurs in biomedical and biotechnology assays.

Carbocyanines↗
PubMed2003

Fluorescence-sensing methods.

Novel approaches to sensor design, based on the use of an internal standard with appropriate spectral properties, provide new possibilities for designing simple devices for fluorescence sensing. Detection of combined emission from the reference and an analyte-sensitive fluorophore has been achieved in numerous measurements in cuvettes, tissues, and high-throughput formats. These methods have been used with a long-lifetime reference to measure pH, O2, pCO2, glucose, and calcium by means of modulation-sensing methods as well as by the use of oriented films as the reference for polarization sensing of glucose, pH, oxygen, and lactate. Polarization sensing has also been developed with visual detection to measure the concentration of rhodamine B and pH. Modulation and polarization sensing was found to be effective in highly scattering media such as Intralipid or tissue. The applicability of these technologies to transdermal diagnostics depends on the availability of red fluorophores that can be used in vivo. One dye that could possibly be used is indocyanine green (IcG), which absorbs and emits at wavelengths above 700 nm. Furthermore, IcG has already been approved for use in humans for monitoring burn severity and it has been detected through the skin. It appears likely that modern optics and electronic technology will allow the development of practical devices for biomedical use as shown in Scheme 1.

Fluorescence↗
PubMed2003

Metal-enhanced emission from indocyanine green: a new approach to in vivo imaging.

Indocyanine green (ICG) is widely used in medical imaging and testing. Its complex spectral behavior and low quantum yield limits some applications. We show that proximity of ICG to a metallic silver particle increases its intensity approximately 20-fold and decreases the decay time. Since the rate of photobleaching is not increased, our results suggest that ICG-silver particle complexes can yield at least 20-fold more photons per ICG molecule for improved medical imaging.

Computer Simulation↗
PubMed2003

Increased intensities of YOYO-1-labeled DNA oligomers near silver particles.

DNA detection is usually performed using fluorescence probes. Using a DNA oligomer stained with the widely used dye 1,1'-[1,3-propanediylbis[(dimethylimino)-3,1-propanediyl]]bis[4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]]-quinolinum tetraiodide (YOYO-1), we show that a substrate containing silver particles can lead to a greater than 10-fold increase in the fluorescence intensity. Proximity to silver particles also increases the photostability of YOYO-1-DNA. These results suggest that substrates or gels containing silver particles may be used for increased sensitivity in DNA detection.

Base Sequence↗
PubMed2003

Silver particles enhance emission of fluorescent DNA oligomers.

Here we describe a new opportunity in methodology for increasing the detectability of fluorescently labeled DNA on solid substrates. We show that the use of glass substrates coated with metallic silver particles results in an approximate 5-fold increase in the intensity of Cy3- or Cy5-labeled DNA oligomers. Proximity to these silver particles also increases the photostability of Cy3- and Cy5-labeled oligomers. These results suggest the use of DNA array substrates with silver particles for increased sensitivity in genetic analysis.

Carbocyanines↗
PubMed2003

Metal-enhanced fluorescence: potential applications in HTS.

Metallic surfaces and particles can have dramatic effects on fluorescence, including localized excitation, increased quantum yields, increased photostability and increased distances for resonance energy transfer (RET), and directional emission. While all these effects have not yet been realized in a single system, metal-enhanced fluorescence promises to provide the next generation of high sensitivity fluorescence assays for low copy number detection of biochemical species.

Algorithms↗
PubMed2002

Effects of metallic silver particles on resonance energy transfer in labeled bovine serum albumin.

Resonance energy transfer (RET) is widely used to detect proximity between biomolecules. In transparent solution the maximum donor-to-acceptor distance for RET is about 70 A. We measured the effects of metallic silver island films on RET from the intrinsic tryptophan of a protein to a bound probe as the acceptor. These preliminary experiments revealed a dramatic increase in the apparent Förster distance increasing from 28.6 to 63 A. These results suggest the use of silver island films for detecting long range proximity between biomolecules and for biotechnology applications based on RET.

Anilino Naphthalenesulfonates↗
PubMed2002

Radiative decay engineering. 2. Effects of Silver Island films on fluorescence intensity, lifetimes, and resonance energy transfer.

Metallic surfaces can have unusual effects on fluorophores such as increasing or decreasing the rates of radiative decay and the rates of resonance energy transfer (RET). In the present article we describe the effects of metallic silver island films on the emission spectra, lifetimes, and energy transfer for several fluorophores. The fluorophores are not covalently coupled to the silver islands so that there are a range of fluorophore-to-metal distances. We show that proximity of fluorophores to the silver islands results in increased fluorescence intensity, with the largest enhancement for the lowest-quantum-yield fluorophores. Importantly, the metal-induced increases in intensity are accompanied by decreased lifetimes and increased photostability. These effects demonstrate that the silver islands have increased the radiative decay rates of the fluorophore. For solvent-sensitive fluorophores the emission spectra shifted to shorted wavelengths in the presence of the silver islands, which is consistent with a decrease of the apparent lifetime for fluorophores near the metal islands. We also observed an increased intensity and blue spectral shift for the protein human glyoxalase, which displays a low quantum yield for its intrinsic tryptophan emission. In this case the blue shift is thought to be due to increased emission from a buried low-quantum-yield tryptophan residue. Increased intensities were also observed for the intrinsic emission of the nucleic acid bases adenine and thymine and for single-stranded 15-mers poly(T) and poly(C). And finally, we observed increased RET for donors and acceptors in solution and when bound to double-helical DNA. These results demonstrate that metallic particles can be used to modify the emission from intrinsic and extrinsic fluorophores in biochemical systems.

Adenine↗
PubMed2002

Lateral diffusion coefficients in membranes measured by resonance energy transfer and a new algorithm for diffusion in two dimensions.

We describe measurements of lateral diffusion in membranes using resonance energy transfer. The donor was a rhenium (Re) metal-ligand complex lipid, which displays a donor decay time near 3 micros. The long donor lifetime resulted in an ability to measure lateral diffusion coefficient below 10(-8) cm(2)/s. The donor decay data were analyzed using a new numerical algorithm for calculation of resonance energy transfer for donors and acceptors randomly distributed in two dimensions. An analytical solution to the diffusion equation in two dimensions is not known, so the equation was solved by the relaxation method in Laplace space. This algorithm allows the donor decay in the absence of energy transfer to be multiexponential. The simulations show that mutual lateral diffusion coefficients of the donor and acceptor on the order of 10(-8) cm(2)/s are readily recovered from the frequency-domain data with donor decay times on the microsecond timescale. Importantly, the lateral diffusion coefficients and acceptor concentrations can be recovered independently despite correlation between these parameters. This algorithm was tested and verified using the donor decays of a long lifetime rhenium lipid donor and a Texas red-lipid acceptor. Lateral diffusion coefficients ranged from 4.4 x 10(-9) cm(2)/s in 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPG) at 10 degrees C to 1.7 x 10(-7) cm(2)/s in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) at 35 degrees C. These results demonstrated the possibility of direct measurements of lateral diffusion coefficients using microsecond decay time luminophores.

Algorithms↗
PubMedDate not supplied

Directional surface plasmon-coupled emission from a 3 nm green fluorescent protein monolayer.

High-sensitivity detection schemes are of great interest for a number of applications. Unfortunately, such schemes are usually high-cost. We demonstrate a low-cost approach to a high-sensitivity detection scheme based on surface plasmon-coupled emission (SPCE). The SPCE of a monomolecular layer of green fluorescent protein (GFP) is reported here. The protein was electrostatically attached to a thin, SiO(2)-protected silver film deposited on a quartz substrate. The visible, directional emission of GFP was observed at a sharp, well-defined angle of 47.5 degrees from the normal to the coupling prism, and the spectrum corresponded to that of GFP. The SPCE resulting from the reverse Kretschmann configuration showed a 12-fold enhancement over the free space fluorescence. The directional emission was 97% p-polarized. The directionality and high polarization can be coupled with the intrinsic spectral resolution of SPCE to be used in the design miniaturized spectrofluorometers. The observation of SPCE in the visible region of the spectrum from a monolayer of protein opens up new possibilities in protein-based sensing.

Biotechnology↗
PubMedDate not supplied

Surface plasmon-coupled polarized emission of N-acetyl-l-tryptophanamide.

We report an observation of ultraviolet (UV) surface plasmon-coupled emission (SPCE) of N-acetyl-l-tryptophanamide (NATA). The sample was spin coated from poly(vinyl alcohol) (PVA) solution on 20 nm aluminum film deposited on a quartz substrate. The directional UV SPCE occurs within a well-defined narrow angle at 52 degrees from the normal to the coupling hemicylinder quartz prism. The NATA directional emission is highly p polarized as expected for surface plasmon-coupled radiation. The 10 nm protective SiO2 layer deposited on top of the aluminum film significantly neutralized the fluorophore quenching by the metal surface. SPCE of NATA demonstrates a remarkable intrinsic dispersive property-the maximum of the emission spectrum depends on the observation angle. The efficient spectral resolution of SPCE can be used in the construction of miniaturized spectrofluorometers. The observation of SPCE of tryptophan opens a new possibility for the study of many unlabeled proteins with the technique complementary to surface plasmon resonance analysis.

Surface Plasmon Resonance↗