Effects of Metallic Silver Particles on Resonance Energy Transfer Between Fluorophores Bound to DNA

We examined the effects of metallic silver island films on resonance energy transfer (RET) between a donor and acceptor bound to double helical DNA. The donor was 4',6-diamidino-2-phenylindole (DAPI) and the acceptor was propidium iodide (PI). Proximity of the labeled DNA to the silver particles resulted in a dramatic increase in RET as seen from the emission spectra and the donor decay times. Proximity to silver particles results in an increase of the Förster distance from 35 Å an to an apparent value of 166 Å. These results suggest a new type of DNA hybridization assays based on RET over distances much longer than the free-space Forster distance.     

Optical properties of Rhodamine B and Rhodamine 6G on silver surfaces

The transmission spectra of Rhodamine B and Rhodamine 6G dyes coated on silver island films for various thickness were recorded. A strong coupling between the silver particle plasma resonance and the dye molecules is observed. The absorption of dye molecules increases when the absorption band of individual dyes and silver particles overlaps. In higher silver film thicknesses, transmission increases. The theoretical calculation using Maxwell-Garnett theory and Wang and Kerker’s results qualitatively supports the observed phenomenon. A red shift in the absorption peak of dyes on silver surfaces in comparison to solution phase absorption peak indicates photobleaching.     

Enhanced Fluorescence from Periodic Arrays of Silver Nanoparticles

Electron beam lithography was used to fabricate silver nanoparticle arrays and study the effects of geometrical properties of particles on metal-enhanced fluorescence. Nanoparticle size, shape, interparticle spacing, and nominal thickness were varied in a combinatorial pattern for investigation of the particle plasmon resonance effect on enhancement of fluorescence from three different fluorophores; Fluorescein, Cy3, and Cy5. A specific geometric property for optimal enhancement from each fluorophore was determined. For interparticle spacings greater or equal to 270 nm, the enhancement matched what is expected for a single-particle model. For those particles smaller than 210 nm, the enhancement was lower than for the larger spacing in the range studied. Triangular-shaped particles gave similar enhancement to those of square-shaped particles. This combinatorial pattern by e-beam lithography was found to be useful for studying how individual parameters enhance the fluorescence that are important for rational design of enhanced fluorescence sensors.     

Nucleobase-Specific Enhancement of Cy3 Fluorescence

We report on the role of dye–nucleobase interactions on the photophysical properties of the indocarbocyanine Cy3. The fluorescence efficiency and lifetime of Cy3 increase in the presence of all four nucleoside monophosphates. This behavior correlates with an increase in the activation energy for photoisomerization and a ∼4 nm red shift in the fluorescence spectrum. Changes are more dramatic for the purines (dAMP, dGMP) than the pyrimidines (dCMP, dTMP), and for the nonsulfonated cyanine (DiIC₂(3)) than the sulfonated dye (Cy3–SE). These results are consistent with a model in which Cy3–nucleoside π–π interactions decrease the efficiency of photoisomerization, increasing the efficiency of fluorescence. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fluorescence Enhancements on Silver Colloid Coated Surfaces

We observed a strong, more than 16-fold, enhancement of Texas Red-labeled BSA fluorescence emission when deposited on silver colloid coated surfaces (SCCS). The same labeled protein deposited on silver island films (SIFs) showed an approximate 8-fold fluorescence enhancement. The lifetimes of Texas Red-BSA fluorescence are significantly shorter on silvered surfaces than on uncoated quartz substrate indicating a strong change in radiative decay rate of the dyes. We also observed a 36-fold increased brightness of overlabeled fluorescein-HSA deposited on silver colloid coated surface. Stronger enhancement observed for overlabeled Fl-HSA protein indicates that presence of silver particles partially decreased self-quenching. Our results indicate that surfaces coated with silver colloids are valuable substrates for metal-enhanced fluorescence.     

Single molecule FRET detection in CdSe-QD donor and Cy5-labeled molecular chaperone acceptor complex by imaging microscopy

We report single molecule spectroscopic evidence of FRET in CdSe quantum dot (QD) conjugated with Cy5-labeled molecular chaperone systems in buffer solutions. Donor QDs are core-shell type nanocrystals covered with organic surfactants on their outermost surfaces, i.e. CdSe/ZnS/TOPO’s. As prototype molecular chaperones, we adopt prefoldins (PFDs), on which Cy5’s are labeled as acceptors. Donor QDs possess two-fold degenerate emission dipoles perpendicular to the c-axis, due to their Wurtzite crystal structures, while acceptor Cy5’s possess linear absorption and emission dipoles. Thus, their combination provides novel features to those in conventional FRET systems. PFDs are jellyfish-shaped hexameric co-chaperones of group II chaperonins, which recognize hydrophobic portions of denatured proteins and encapsulate them within their central cavities. Hence, PFDs will also capture the CdSe/ZnS/TOPO QDs due to its surface similarity to the denatured proteins. By introducing simple microscope setup for single QD-PFD-Cy5 spectroscopy, we have successfully captured the emission spectra in FRET regime. We also have observed peculiar features in time evolution profiles of single QD emissions conjugated with Cy5-labeled PFDs under polarization modulation measurements. Notable point of our hybrid conjugates is that they are biochemically in living action. We describe our present results in relation to possible protein reactions.     

Effects of Silver Island Films on the Luminescent Intensity and Decay Times of Lanthanide Chelates

We examined the emission intensity and decay times of chelates Tb³⁺ and Eu³⁺ in micron thick samples between films of sub-wavelength size silver particles. We observed modest increases in emission intensities for the complexes between the silver particles as compared to between unsilvered quartz plates. The intensity decay times were dramatically decreased by the silver particles, which was in part mediated by diffusion toward the silver particles. These results indicated that luminescent lanthanides in close proximity to silver particles display increased rates of radiative decay. The use of luminophore-metallic surface interactions provides new opportunities for creation of luminescent probes with novel spectral properties.     

Annealed Silver-Island Films for Applications in Metal-Enhanced Fluorescence: Interpretation in Terms of Radiating Plasmons

The effects of thermally annealed silver island films have been studied with regard to their potential applicability in applications of metal-enhanced fluorescence, an emerging tool in nano-biotechnology. Silver island films were thermally annealed between 75 and 250^∘C for several hours. As a function of both time and annealing temperature, the surface plasmon band at ≈420 nm both diminished and was blue shifted. These changes in plasmon resonance have been characterized using both absorption measurements, as well as topographically using Atomic Force Microscopy. Subsequently, the net changes in plasmon absorption are interpreted as the silver island films becoming spherical and growing in height, as well as an increased spacing between the particles. Interestingly, when the annealed surfaces are coated with a fluorescein-labeled protein, significant enhancements in fluorescence are osbserved, scaling with annealing temperature and time. These observations strongly support our recent hypothesis that the extent of metal-enhanced fluorescence is due to the ability of surface plasmons to radiate coupled fluorophore fluorescence. Given that the extinction spectrum of the silvered films is comprised of both an absorption and scattering component, and that these components are proportional to the diameter cubed and to the sixth power, respectively, then larger structures are expected to have a greater scattering contribution to their extinction spectrum and, therefore, more efficiently radiate coupled fluorophore emission. Subsequently, we have been able to correlate our increases in fluorescence emission with an increased particle size, providing strong experiment evidence for our recently reported metal-enhanced fluorescence, facilitated by radiating plasmons hypothesis.     

Optical absorption of submonolayer silver films: Size effect of ϵfree and ϵbound in small island particles

Optical absorption spectra of submonolayer silver films deposited on a fused quartz ATR prism were measured. Optical plasma resonance absorption peaks both for p- and s-polarized light were observed at near 3.5 eV. The observed spectra were interpreted well by the model of two-dimensionally distributed ellipsoids of rotation applying the Drude formula and the classical size effect to the free electron part of the dielectric constants of silver island particles ?_free and the bulk value shifted to the higher energy region to the bound electron part ?_bound. It was found that the amount of the shift increases with decrease of mean thickness of silver.     

激光刻蚀银纳米粒子岛膜的SERS特性

利用激光刻蚀的方法,通过改变激光脉冲的能量,制备出一系列的纳米金属银胶体。将银胶体沉积在铝基底上形成一层银岛膜。用紫外—可见吸收光谱、SEM及拉曼光谱对银胶体和银岛膜进行了表征。研究结果表明,刻蚀所用激光脉冲的能量影响胶体中银颗粒的分布;不同能量下制备的胶体所形成的银岛膜具有不同的表面增强效果;该岛膜具有增强效果优异、制备简便等特点。     

Absorption and photoluminescence properties of silver clusters in SiO<Subscript>2</Subscript> matrix

Absorption and luminescence properties of silver nanoclusters embedded in SiO₂ matrixes were studied experimentally. Thin SiO₂ films with different amount of silver were produced by co-deposition of Ag and SiO₂ onto the silica substrates in vacuum. The thus obtained films possess three peaks in absorption spectra at 297, 329 and 401 nm and two peaks in luminescence spectra at about 500 and 650 nm. We ascribed these spectral features to silver nanoclusters of different sizes that present in the film. Thermal annealing transforms both absorption and emission spectra of the films. Lager clusters that are formed after annealing possess one absorption band at 350–450 nm and one luminescence band at 510 nm. The luminescence was observed only in samples with the silver content of less than 2.2%. Quenching of the luminescence in samples with higher concentration of silver is due to the presence of larger particles with plasmonic properties.     

Time-resolved and integrated angular distributions of plume ions from silver at low and medium laser fluence

Laser impact on metals in the UV regime results in a significant number of ablated plume ions even at moderate fluence (0.7–2.4 J/cm²). The ablated particles are largely neutrals at the lowest fluence, but the fraction of ions increases strongly with fluence. The ion flow in different directions from a silver target irradiated by a laser beam at a wavelength of 355 nm in vacuum was measured with a hemispherical array of Langmuir probes. The time-of-flight spectra in all directions, as well as the total angular yield were determined. The angular distribution peaks strongly in forward direction with increasing fluence and can be well approximated by Anisimov’s model. Typically, the spectra of silver ions peak from 70 eV up to 145 eV in a direction close to the normal of the target surface with increasing fluence. With increasing observation angle, the time-of-flight spectra exhibit a peak at longer flight times, i.e., at a lower kinetic energy. At the highest fluence, the ionized fraction of the ablated particles in the plume increases up to 0.5.     

表面增强拉曼光谱研究胞嘧啶在银胶上的吸附行为

为探讨胞嘧啶(Cytosine,Cy)在基底银表面的吸附特性和规律,采用表面增强拉曼散射(SERS)光谱对其吸附行为进行分析,并结合量子化学密度泛函理论(DFT)/B3LYP计算方法对Cy分子的常规拉曼光谱(NRS)及Cy与Ag团簇吸附的SERS光谱进行计算,与测定结果进行比对且对其拉曼峰进行系统指认及归属,理论计算结合实测值探讨了Cy在基底Ag上的增强效应和吸附行为。考察了Cy分子在Ag纳米粒子上的不同吸附时间、浓度、pH等条件对SERS光谱的影响及优化,发现pH影响最大,在中性和强碱性条件下的增强效应明显优于酸性。Cy分子存在2种不同的异构体和3种不同的存在形态,并随酸度变化相互转化而达动态平衡。基于Cy在不同pH时的形态分布和相应的SERS变化规律,结合DFT算得的Cy分子中的电荷分布及在银基底表面的吸附机制,详细探讨了酸碱对Cy分子的SRES光谱影响的内因和吸附机理,指出在中性和弱碱性时,是Cy中的N3和O与Ag形成配位吸附;在pH大于11时,N与Ag形成配位吸附,而O与Ag形成共价吸附。     

Improvement of frequency sensitivity of series photodetector frequency circuit system and its application for Cy5 fluorescence detection

This study elucidated the frequency characteristics of series photodetector frequency circuit system for detection of DNA probe ET996 marked with fluorescence dye Cy5. We developed 48 MHz series photodetector frequency circuit system with good sensitivity for fluorescence detection. In accordance with the theory of series photodetector frequency circuit system, the frequency sensitivity can be improved by adjusting circuit parameters such as A (tan θ), C_q, C₀, and C_p. In this research of A adjustment, the capacitance parameter C_m of 48 MHz series photodetector frequency circuit system was adjusted to improve the frequency sensitivity for detection of fluorescence dye concentration; moreover, the bias of photodetector was also adjusted to improve the frequency sensitivity. In the optimal conditions of capacitance match and photodetector bias, the detection limit of ET996-Cy5 fluorescence dye concentration 2 pmol/L can be measured by 48 MHz sensor system. The results of fluorescence experiment also demonstrated that the frequency shift of 48 MHz sensor system was linearly related to the logarithm of fluorescence dye concentration from 200 nmol/L to 2 pmol/L. The frequency method can be applied simply and the detection limit of ET996-Cy5 fluorescence dye concentration was lower than the conventional fluorescence technique by 2 orders.     

Ratiometric FRET-based detection of DNA and micro-RNA on the surface using TIRF detection

A new FRET-based method for the ratiometric detection of DNA oligomers on a surface using TIRF detection mode is reported. The dual-labeled system consisting of two hybridized oligomers, Cy3oligoY:Cy5oligoX was immobilized on the surface, and the total internal reflection fluorescence (TIRF) was used to detect emission signals from the surface. Two signals, green and red, which originated from the green donor Cy3 and the red acceptor Cy5, have been simultaneously detected. When the target single-stranded complimentary oligomer was present in the solution, this oligomer replaced the Cy3oligoY in the donor:acceptor complex on the surface and the ratio of red-to-green signal was dramatically changed. This detection scheme is generally applicable to the detection of DNA or RNA on a surface.     

Tunable surface-plasmon-resonance wavelength of silver island films

This paper experimentally and theoretically investigates the effect of the underlayer medium on tuning of the surface plasmon resonance (SPR) wavelength of silver island films,and the effect of substrate temperature on the morphologies and optical properties of the films.From the absorption spectra of single Ag with various thickness and overcoated (Ag/TiO 2) films deposited on glass substrates at various substrate temperatures by RF magnetron sputtering,we demonstrate that the surface plasmon resonance wavelength can be made tunable by changing the underlayer medium,the thickness of metal layer and the substrate temperature.By varying substrate temperatures,the interparticle coupling effects on plasmon resonances of nanosilver particles enhance as the spacing between the particles reduces.When the substrate temperature is up to 500 C,the absorption peak decreases sharply and shifts to shorter wavelength side due to the severe coalescence between silver islands in the film.     

Plasmonic Enhancement of Single-Molecule Fluorescence Near a Silver Nanoparticle

In this short paper, we reported the enhanced fluorescence from a single fluorophore bound to a 50nm silver nanoparticle. We found that on average the Cy5 molecules bound to metal nanoparticles are approximately 15-fold brighter than that of free dyes, and that single molecule lifetimes are shorter as compared to free fluorophores. The increased emission rate is primarily the result of local plasmon enhancement. These results demonstrate that the use of fluorophore-metal interactions can increase the brightness and photostability of fluorophores for single molecule detection.     

Study of Langmuir-Blodgett polydiacetylene polymer films by surface enhanced Raman scattering

Large shifts (～50 cm^-1) to lower energies in the C=C and C≡C stretching modes of the Raman spectra of Langmuir-Blodgett deposted thin films of 15-8 polydiacetylene polymer are observed as the film thickness changes from a monolayer or one bilayer to two or more bilayers. The spectra of two or more bilayers are found to be identical to bulk material. These large shifts, observed by surface enhanced Raman scattering from the silver island substrate on which the films are deposited are attributed to delocalization of the electronic states in the backbone of the polydiacetylene polymer.     

Cy3B™: Improving the Performance of Cyanine Dyes

The spectral properties of a rigidified trimethine cyanine dye, Cy3B have been characterised. This probe has excellent fluorescent properties, good water solubility and can be bioconjugated. The emission properties of this fluorophore have also been investigated upon conjugation to an antibody. This study compared the conjugated emission properties of Cy3B with other commercially available fluorophores emitting at similar wavelengths.