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1.
Increased resonance energy transfer between fluorophores bound to DNA in proximity to metallic silver particles 总被引:1,自引:0,他引:1
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. 相似文献
2.
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. 相似文献
3.
Lakowicz JR Shen Y D'Auria S Malicka J Fang J Gryczynski Z Gryczynski I 《Analytical biochemistry》2002,301(2):261-277
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. 相似文献
4.
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. 相似文献
5.
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. 相似文献
6.
Lakowicz JR Shen B Gryczynski Z D'Auria S Gryczynski I 《Biochemical and biophysical research communications》2001,286(5):875-879
High sensitivity detection of DNA is essential for genomics. The intrinsic fluorescence from DNA is very weak and almost all methods for detecting DNA rely on the use of extrinsic fluorescent probes. We show that the intrinsic emission from DNA can be enhanced many-fold by spatial proximity to silver island films. Silver islands are subwavelength size patches of metallic silver on an inert substrate. Time-resolved measurements show a decreased lifetime for the intrinsic DNA emission near the silver islands. These results of increased intensity and decreased lifetime indicate a metal-induced increase in the radiative rate decay of the DNA bases. The possibility of increased radiative decay rates for DNA bases and other fluorophores suggest a wide variety of DNA measurements and other biomedical assays based on metal-induced increases in the fluorescence quantum yield of weakly fluorescent substances. 相似文献
7.
Metal-enhanced fluorescence immunoassays using total internal reflection and silver island-coated surfaces 总被引:2,自引:0,他引:2
Matveeva E Gryczynski Z Malicka J Gryczynski I Lakowicz JR 《Analytical biochemistry》2004,334(2):303-311
We present a generic immunoassay platform that uses enhanced total internal reflection fluorescence in the proximity of silver island films (SIFs), a surface coating consisting of metal (silver) particles. This platform is used with a model immunoassay where a protein antigen, rabbit immunoglobulin G, was immobilized on the SIF-coated glass surface. The signal from a fluorescent dye-labeled anti-rabbit antibody binding to the surface antigen was detected; different color dyes have been tested. Close placement of the fluorophore to surface-bound silver nanostructures results in dramatic signal enhancement (up to 40-fold) on the SIFs as compared with the glass slides. Use of the total internal reflection mode of excitation has significant advantages (over classic front-face excitation) for practical assay development. The limited evanescent wave excitation volume makes it possible to minimize the background signal and use the immunoassay with no need for any washing steps. 相似文献
8.
PicoGreen (PG) is a fluorescent probe for both double-stranded DNA (dsDNA) detection and quantification based on its ability to form a luminescent complex with dsDNA as compared with the free dye in solution. To expand the sensitivity of PG detection, we have studied the spectral properties of PG, both free and in complex with DNA in solution, when the fluorophore is in proximity to silver nanoparticles. We show that for a broad range of PG concentrations (20 pM-3.5 μM), it does not form dimers/oligomers and it exists in a monomeric state. On binding to DNA in the absence of silver, PG fluorescence increases approximately 1100-fold. Deposition of PG/DNA complex onto silver island films (SiFs) increases fluorescence approximately 7-fold due to the metal-enhanced fluorescence (MEF) effect, yielding fluorescence enhancement of 7700-fold as compared with the free dye on glass. In contrast to PG in complex with DNA, the free dye on SiFs demonstrates a decrease in brightness approximately 5-fold. Therefore, the total enhancement of PG on binding to DNA on silver reaches a value of approximately 38,000 as compared with free PG on SiFs. Consequently, the metal-enhanced detection of PG fluorescence is likely to find important utility for amplified dsDNA quantification. 相似文献
9.
Kindzelskii AL Yang Z Nabel GJ Todd RF Petty HR 《Journal of immunology (Baltimore, Md. : 1950)》2000,164(2):953-958
Previous studies have shown that Ebola virus' secretory glycoprotein (sGP) binds to Fc gamma RIIIB (CD16b) and inhibits L-selectin shedding. In this study, we test the hypothesis that sGP interferes with the physical linkage between CR3 and Fc gamma RIIIB. Neutrophils were stained with rhodamine-conjugated anti-CD16b mAb (which does not inhibit sGP binding) and fluorescein-conjugated anti-CR3 mAb reagents and then incubated in media with or without sGP. Physical proximity between fluorochrome-labeled CR3 and Fc gamma RIIIB on individual cells was measured by resonance energy transfer (RET) imaging, quantitative RET microfluorometry, and single-cell imaging spectrophotometry. Cells incubated with control supernatants displayed a significant RET signal, indicative of physical proximity (<7 nm) between CR3 and Fc gamma RIIIB. In contrast, cells exposed to sGP showed a significant reduction in the CR3-Fc gamma RIIIB RET signal using these methods. Interestingly, colocalization and cocapping of CR3 and Fc gamma RIIIB were not affected, suggesting that the proximity of these two receptors is reduced without triggering dissociation. Thus, sGP alters the physical linkage between Fc gamma RIIIB and CR3. 相似文献
10.
Immunoassay employing surface-enhanced Raman spectroscopy 总被引:4,自引:0,他引:4
Surface-enhanced Raman scattering (SERS) was used to measure binding between biomolecules with mutual affinity, including antigen-antibody interactions. The conjugation of nitro groups onto bovine serum albumin enhanced their specific SERS activity 10(4)-fold. A dye, 2-[4'-hydroxyphenylazo]benzoic acid (HABA), with a major absorption at the Raman excitation frequency, demonstrated surface-enhanced resonance Raman scattering (SERRS) when captured from solution by avidin-coated silver films. Individual peak intensities showed a logarithmic relationship to the HABA concentration in solution over the range 10(-8) to 10(-5) M. Another resonance dye, p-dimethylaminoazobenzene (DAB) was covalently attached to an antibody directed against human thyroid stimulating hormone (TSH), without loss of antibody activity. The resultant conjugate was used in a sandwich immunoassay for TSH antigen: silver surfaces coated with anti-TSH antibody captured TSH antigen which in turn captured the DAB-anti-TSH antibody conjugate. A linear relationship was observed between the intensity of the resultant SERRS signals and the TSH antigen concentration over a range of from 4 to 60 microIU/ml. These results demonstrate the potential utility of the SERRS effect as a readout in a one-step, no wash immunoassay system. 相似文献
11.
Resonance energy transfer from a cylindrical distribution of donors to a plane of acceptors. Location of apo-B100 protein on the human low-density lipoprotein particle. 总被引:1,自引:0,他引:1
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P Bastiaens A de Beus M Lacker P Somerharju M Vauhkonen J Eisinger 《Biophysical journal》1990,58(3):665-675
The resonance energy transfer (RET) from a cylindrical assembly of donors to acceptors in a plane was investigated, and the dependence the average RET rate (kT) on the cylinder's size, shape, and proximity to the acceptor plane was determined. This geometry provides a model for the RET from a donor-containing protein to acceptors embedded in an associated phospholipid mono- or bilayer. The determination of kT for a series of acceptors at different levels in the phospholipid layer is shown to provide information on the protein's relationship to the phospholipid layer. Two models for the donor (D) and acceptor (A) distributions are employed: (a) The D's and A's are uniformly distributed in the cylinder and the plane, respectively, and analytical expressions for kT in terms of experimental parameters are derived. (b) The RET rates between all D, A pairs within the cylinder and in the plane are calculated and averaged for a large number of random D and A distributions. The average transfer rates obtained by the two approaches are in agreement and the width of the frequency distribution of kT for the latter provides an estimate of the error to be expected when, as is usually the case, the true D and A locations are unknown. This methodology is illustrated by analyzing RET from the 37 tryptophan residues of the apo-B100 protein to a series of pyrenylphosphatidylcholine acceptors inserted in the phospholipid monolayer of the human low-density lipoprotein particle, and it is concluded that significant portions of the protein penetrate the phospholipid layer. 相似文献
12.
The detection of submonolayers of proteins based on native fluorescence is a potentially valuable approach for label-free detection. We have examined the possibility of using silver nanostructures to increase the emission of tryptophan residues in proteins. Fluorescence spectra, intensities, and lifetimes of multilayers and submonolayers of proteins deposited on the surfaces of silver island films were measured. Increased fluorescence intensities from two- to three-fold and similar decreases in lifetimes were observed in the presence of the silver nanoparticles compared with the proteins on the surface of the bare quartz. The observed spectral effects of silver nanoparticles on tryptophan fluorescence indicates the possibility for the design of analytical tools for the detection of proteins without traditional labeling by extrinsic fluorophores. 相似文献
13.
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. 相似文献
14.
Jose?Luis?Elechiguerra Justin?L?Burt Jose?R?Morones Alejandra?Camacho-Bragado Xiaoxia?Gao Humberto?H?Lara Miguel?Jose?Yacaman
The interaction of nanoparticles with biomolecules and microorganisms is an expanding field of research. Within this field,
an area that has been largely unexplored is the interaction of metal nanoparticles with viruses. In this work, we demonstrate
that silver nanoparticles undergo a size-dependent interaction with HIV-1, with nanoparticles exclusively in the range of
1–10 nm attached to the virus. The regular spatial arrangement of the attached nanoparticles, the center-to-center distance
between nanoparticles, and the fact that the exposed sulfur-bearing residues of the glycoprotein knobs would be attractive
sites for nanoparticle interaction suggest that silver nanoparticles interact with the HIV-1 virus via preferential binding
to the gp120 glycoprotein knobs. Due to this interaction, silver nanoparticles inhibit the virus from binding to host cells,
as demonstrated in vitro. 相似文献
15.
Forster Resonance Energy Transfer has served as a molecular ruler that reports conformational changes and intramolecular distances of single biomolecules. However, such rulers suffer from low and fluctuating signal intensities, limited observation time due to photobleaching, and an upper distance limit of approximately 10 nm. Noble metal nanoparticles have plasmon resonances in the visible range and do not blink or bleach. They have been employed as alternative probes to overcome the limitations of organic fluorophores, and the coupling of plasmons in nearby particles has been exploited to detect particle aggregation by a distinct color change in bulk experiments. Here we demonstrate that plasmon coupling can be used to monitor distances between single pairs of gold and silver nanoparticles. We followed the directed assembly of gold and silver nanoparticle dimers in real time and studied the kinetics of single DNA hybridization events. These "plasmon rulers" allowed us to continuously monitor separations of up to 70 nm for >3,000 s. 相似文献
16.
Robert E. Ricklefs Julia Dodge Gray Steven C. Latta Maria Svensson‐Coelho 《Journal of avian biology》2011,42(6):570-584
We compared the haemosporidian parasite faunas (Plasmodium and Haemoproteus) of small land birds on the islands of St Lucia, St Vincent and Grenada in the southern Lesser Antilles. The islands differ in distance from the South American source of colonists, proximity to each other, and similarity of their avifaunas. On each island, we obtained 419–572 blood samples from 22–25 of the 34–41 resident species. We detected parasite infection by PCR and identified parasite lineages by sequencing a portion of the mitochondrial cytochrome b gene. Parasite prevalence varied from 31% on St Lucia to 22% on St Vincent and 18% on Grenada. Abundant parasite lineages differed between the three islands in spite of the similarity in host species. As in other studies, the geographic distributions of the individual parasite lineages varied widely between local endemism and broad distribution within the West Indies, including cases of long‐distance disjunction. St Vincent was unusual in the near absence of Plasmodium parasites, which accorded with low numbers of suitable mosquito vectors reported from the island. Parasites on St Vincent also tended to be host specialists compared to those on St Lucia and Grenada. Similarity in parasite assemblages among the three islands varied in parallel with host assemblage similarity (but not similarity of infected hosts) and with geographic proximity. Parasite prevalence increased with host abundance on both St Lucia and St Vincent, but not on Grenada; prevalence did not vary between endemic and more widespread host species. In addition, the endemic host species harbored parasites that were recovered from a variety of non‐endemic species as well. These results support the individualistic nature of haemosporidian parasite assemblages in evolutionarily independent host populations. 相似文献
17.
Molecular proximity of Kv1.3 voltage-gated potassium channels and beta(1)-integrins on the plasma membrane of melanoma cells: effects of cell adherence and channel blockers
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Tumor cell membranes have multiple components that participate in the process of metastasis. The present study investigates the physical association of beta1-integrins and Kv1.3 voltage-gated potassium channels in melanoma cell membranes using resonance energy transfer (RET) techniques. RET between donor-labeled anti-beta1-integrin and acceptor-labeled anti-Kv1.3 channels was detected on LOX cells adherent to glass and fibronectin-coated coverslips. However, RET was not observed on LOX cells in suspension, indicating that molecular proximity of these membrane molecules is adherence-related. Several K(+) channel blockers, including tetraethylammonium, 4-aminopyridine, and verapamil, inhibited RET between beta1-integrins and Kv1.3 channels. However, the irrelevant K(+) channel blocker apamin had no effect on RET between beta1-integrins and Kv1.3 channels. Based on these findings, we speculate that the lateral association of Kv1.3 channels with beta1-integrins contributes to the regulation of integrin function and that channel blockers might affect tumor cell behavior by influencing the assembly of supramolecular structures containing integrins. 相似文献
18.
Federico Ossi Stefano Focardi Bryony A. Tolhurst Gian Pietro Picco Amy L. Murphy Davide Molteni Noemi Giannini Jean-Michel Gaillard Francesca Cagnacci 《The Journal of wildlife management》2022,86(1):e22151
Automated contact detection by means of proximity loggers permits the measurement of encounters between individuals (animal-animal contacts) and the time spent by individuals in the proximity of a focal resource of interest (animal-fixed logger contacts). The ecological inference derived from contact detection is intrinsically associated with the distance at which the contact occurred. But no proximity loggers currently exist that record this distance and therefore all distance estimations are associated with error. Here we applied a probabilistic approach to model the relationship between contact detection and inter-logger distance, and quantify the associated error, on free-ranging animals in semi-controlled settings. The probability of recording a contact declined with the distance between loggers, and this decline was steeper for weaker radio transmission powers. Even when proximity loggers were adjacent, contact detection was not guaranteed, irrespective of the radio transmission power. Accordingly, the precision and sensitivity of the system varied as a function of inter-logger distance, radio transmission power, and experimental setting (e.g., depending on animal body mass and fine-scale movements). By accounting for these relationships, we were able to estimate the probability that a detected contact occurred at a certain distance, and the probability that contacts were missed (i.e., false negatives). These calibration exercises have the potential to improve the predictability of the study and enhance the applicability of proximity loggers to key wildlife management issues such as disease transmission rates or wildlife use of landscape features and resources. 相似文献
19.
The intrinsic fluorescence of the catalytic portion of the chloroplast ATP synthase (CF1) is quenched when cysteine 322, the penultimate amino acid of the gamma subunit, is specifically labeled with pyrene maleimide (PM). The epsilon subunit of CF1 contains the only two residues of tryptophan, which dominate the intrinsic fluorescence of unlabeled CF1. CF1 deficient in the epsilon subunit (CF1-epsilon) was reconstituted with mutant epsilon subunits in which phenylalanine replaced tryptophan at position 15 (epsilonW15F) and position 57 (epsilonW15/57F). CF1(epsilonW15F) containing a single tryptophan, epsilonW57, was labeled with PM at gammaC322. Resonance energy transfer (RET) from epsilonW57 to PM on gammaC322 occurred with an efficiency of energy transfer of 20%. RET was also observed from epsilonW57 to PM attached to the disulfide thiols of the gamma subunit (gammaC199,205) with an efficiency of approximately 45%. The R(o) (the distance at which the efficiency of energy transfer is 50%) for the epsilonW57 and PM donor/acceptor pair is 30 A, indicating that both gammaC322 and gammaC199,205 must be within 40 A of epsilonW57. These RET measurements show that both gammaC322 and gammaC199,205 are located near the base of the alpha/beta hexamer. This places the C-terminus of CF1 gamma much closer to epsilon than hypothesized based on homology to crystal structures of mitochondrial F1. These new RET measurements also allow the alignment of the predicted epsilon subunit structure. The orientation is similar to that predicted from cross-linking and mutational studies for the epsilon subunit of Escherichia coli F1. 相似文献
20.
Víctor H Pérez-Luna Saipeng Yang Emmanuil M Rabinovich Tione Buranda Larry A Sklar Philip D Hampton Gabriel P López 《Biosensors & bioelectronics》2002,17(1-2):71-78
A new fluorescence-based biosensor is presented. The biosensing scheme is based on the fact that a fluorophore in close proximity to a metal film (<100 A) experiences strong quenching of fluorescence and a dramatic reduction in the lifetime of the excited state. By immobilizing the analyte of interest (or a structural analog of the analyte) to a metal surface and exposing it to a labeled receptor (e.g. antibody), the fluorescence of the labeled receptor becomes quenched upon binding because of the close proximity to the metal. Upon exposure to free analyte, the labeled receptor dissociates from the surface and diffuses into the bulk of the solution. This increases its separation from the metal and an increase of fluorescence intensity and/or lifetime of the excited state is observed that indicates the presence of the soluble analyte. By enclosing this system within a small volume with a semipermeable membrane, a reversible device is obtained. We demonstrate this scheme using a biotinylated self-assembled monolayer (SAM) on gold as our surface immobilized analyte analog, fluorescently labeled anti-biotin as a receptor, and a solution of biotin in PBS as a model analyte. This scheme could easily be extended to transduce a wide variety of protein-ligand interactions and other biorecognition phenomena (e.g. DNA hybridization) that result in changes in the architecture of surface immobilized biomolecules such that a change in the separation distance between fluorophores and the metal film is obtained. 相似文献