Fluorophore Distance Research Articles

Metal-induced energy transfer

Abstract Since about a decade, metal-induced energy transfer (MIET) has become a tool to measure the distance of fluorophores to a metal-coated surface with nanometer accuracy. The energy transfer from a fluorescent molecule to surface plasmons within a metal film results in the acceleration of its radiative decay rate. This can be observed as a reduction of the molecule’s fluorescence lifetime which can be easily measured with standard microscopy equipment. The achievable distance resolution is in the nanometer range, over a total range of about 200 nm. The method is perfectly compatible with biological and even live cell samples. In this review, we will summarize the theoretical and technical details of the method and present the most important results that have been obtained using MIET. We will also show how the latest technical developments can contribute to improving MIET, and we sketch some interesting directions for its future applications in the life sciences.

Simultaneous Surface Plasmon Resonance/Fluorescence Spectroelectrochemical in Situ Monitoring of Dynamic Changes on Functional Interfaces: A Study of the Electrochemical Proximity Assay Model System

Understanding the chemical composition and morphology of interfaces plays a vital role in the development of sensors, drug delivery systems, coatings for biomedical implants, and so forth. In many cases, the interface characterization can be performed by a combination of electrochemical and one of the optical techniques. In this study, we further enhanced capabilities in probing interfaces by combining electrochemical characterization with multiple optical techniques, that is, surface plasmon resonance (SPR) and fluorescence spectroscopy. This new combination was utilized to study the electrochemical proximity assay (ECPA)-a recently developed protein recognition strategy for the point-of-care test. The SPR/fluorescence spectroelectrochemical technique has achieved not only recognition of binding components involved in the ECPA model system, estimation of their thicknesses and surface coverages, but more importantly, highly reliable in situ monitoring of dynamic changes of components involved in interfacial binding via cross-validation and confirmation from three simultaneously generated signals-SPR, fluorescence, and electrochemistry. In addition, the obtained corresponding proportions among magnitudes of three signals provide crucial information for future studies on simultaneous characterization of multiple components in one step and differentiation of nonspecific binding events. Another advantage using this technique is that the excitation of fluorescence is not only confined by surface plasmons, but by photons, so the fluorescence information can be also gained as the distance of fluorophores from the surface exceeds the decay length of surface plasmons.

Synthesis and optical properties of water-soluble CdTe1−xSex quantum dots with ultra-long fluorescence lifetime

Water-soluble ternary-alloyed CdTe1−xSex quantum dots (QDs) have been synthesized via a simple one-pot approach in aqueous phase. The as-prepared CdTe1−xSex QDs show good crystallinity, excellent monodispersity, and a narrow size distribution with average size of 3.0 nm. Lattice parameters and band gaps of the ternary-alloyed CdTe1−xSex (0 ≤ x < 1) QDs are regulated respectively in linear and quadratic relationships with the Se content (x). By tuning composition, the photoluminescence (PL) emission wavelength of CdTe1−xSex can cover from 528.9 to 689.3 nm, and gradually moved to longer wavelengths with a decrease content of Se (x). The lifetime of CdTe1−xSex QDs is decreased gradually with the Se content (x) augment, attributing to the decreasing lattice spacing resulting from substitution of the larger Te atoms with the smaller Se atoms, bringing about a incremental plasmonic coupling effect at smaller inter-lattice fluorophore distance. However, the as-prepared CdTe1−xSex QDs (0 ≤ x < 1) have significantly longer fluorescence lifetimes of >80 ns with the maximum lifetime up to 174.64 ns. The longer life is good for the application of photoelectric conversion, fluorescent biomedical imaging and detecting.

Protein Detection: An Optical Nanoruler Based on a Conjugated Polymer−Silver Nanoprism Pair for Label‐Free Protein Detection (Adv. Mater. 39/2015)

An optical nanoruler system based on a conjugated polyelectrolyte (CP)-silver nanoprism (Ag NPR) pair is developed for label-free biological detection by taking advantage of the metal-enhanced fluorescence (MEF) effect of Ag nanostructures. The system described by L. Li, S. Wang, and co-workers on page 6040 effectively transduces and amplifies the change of the metal–fluorophore distance into the response of the CP fluorescence, which is used to monitor the presence of specific antigens and even mammalian cells with high sensitivity and good selectivity.

3D superresolution microscopy by supercritical angle detection

We present a fundamentally new approach to 3D superresolution microscopy based on the principle of surface-generated fluorescence. This near-field fluorescence is strongly dependent on the distance of fluorophores from the coverslip and can therefore be used to estimate their axial positions. We established a robust and simple implementation of supercritical angle fluorescence detection for single-molecule localization microscopy, calibrated it using fluorescent bead samples, validated the method with DNA origami tetrahedra, and present proof-of-principle data on biological samples.

Role of Fluorophore–Metal Interaction in Photoinduced Electron Transfer (PET) Sensors: Time-Dependent Density Functional Theory (TDDFT) Study

The origins of fluorescence quenching by Hg(II) ion chelation and fluorescence enhancement by Zn(II) ion chelation to a PET sensor are investigated. Specifically, the fluorescence quenching and enhancing mechanisms associated with the ligand ADPA (N-(9-anthracenylmethyl)-N-(2-pyridinylmethyl)-2-pyridinemethanamine), protonated ADPA and metal bound (Zn(II) and Hg(II)) ADPA are studied via density functional theory (DFT) and time-dependent DFT (TDDFT) methods. The study found that a structural change in the excited state of ADPA induces reordering of the frontier molecular orbitals, and the S1 → S0 transition becomes a charge transfer transition from the fluorophore to the tertiary nitrogen of the dipicolylamine (DPA) unit, which is forbidden. Protonation on the tertiary amine or chelation of Zn(II) prevents such changes, and the HOMO-LUMO transition is contained within the fluorophore. Therefore, fluorescence is restored. The chelation of Hg(II), on the other hand, promotes extensive interaction between the Hg(II) ion and the fluorophore, which is reflected in the short Hg(II)-fluorophore distance (3.11 Å). A noticeable structural change upon the S0 → S1 transition is observed in the Hg(II)-ADPA system as well, where the resulting S1 → S0 transition becomes a charge transfer transition from mercury to the fluorophore and the fluorescence is thus quenched. Therefore, the present DFT/TDDFT calculations reproduce the fluorescence on-off behavior associated with the entire ADPA family of complexes, which illustrates that the combination of DFT and TDDFT calculations, including excited state geometry optimization, can be a valuable tool to uncover the detailed fluorescence sensing mechanisms.

Mechanical Nanosensor Based on FRET within a Thermosome: Damage‐Reporting Polymeric Materials

Under stress: Changes in stress of the polymer matrix in a protein–polymer hybrid material result in changes of conformation of the protein complex, thus resulting in a damage-reporting material (see picture). The reporter is an engineered chaperonin that covalently entraps a pair of fluorescent proteins. Deformation of the chaperonin leads to a change in fluorophore distance and a change in the fluorescene resonance energy transfer (FRET) signal.

Sulforhodamine Adsorbed Langmuir-Blodgett Layers on Silver Island Films: Effect of Probe Distance on the Metal-Enhanced Fluorescence.

Metal-Enhanced Fluorescence (MEF) has become an important method in biomedical sensing. In this paper, we present the distance-dependent MEF of sulforhodamine B (SRB) monolayer on silver island films (SIFs). SRB is electrostatically incorporated into the Langmuir-Blodgett (LB) layers of octadecylamine (ODA) deposited on glass and SIFs substrates. The distances between SRB and SIFs or glass surfaces are controlled by depositing a varied number of inert stearic acid (SA) spacer layers. SRB is incorporated into positively charged LB layers of ODA by immersing the ODA deposited substrates into aqueous solution of SRB. Dye incorporated ODA layers with 10 nm separation distance from the SIFs surface show maximum metal-enhanced fluorescence intensity; ~7-fold increase in intensity as compared to that from the glass surface. The corresponding enhancement factor is reduced with increasing or decreasing the probe distance from the SIFs surface. Additionally, SRB on SIF surfaces show reduced lifetimes. We observed the shortest lifetime from the SRB with 5 nm distance from the SIF surfaces and the lifetime increased consistently with increasing the distances between the fluorophore and the SIFs surface. These observed spectral changes, increase in fluorescence intensity and decreased fluorescence lifetimes, are in accordance with the expected effects due to near-field interactions between the silver nanoparticles and fluorophores. We have also analyzed the complex fluorescence heterogeneous decays on metallic nanostructured surfaces using continuous distributions of decay times. The decay-time distributions appear to be sensitive to the distance between the metal and fluorophore and represent the underlying heterogeneity of the samples. The present systematic study provides significant information on the effect of fluorophore distance on the metal-enhanced fluorescence phenomenon.

Distance-Dependent Metal-Enhanced Fluorescence from Langmuir−Blodgett Monolayers of Alkyl-NBD Derivatives on Silver Island Films

We described the effect of fluorophore distance from the silver island films (SIFs) on the metal-enhanced fluorescence (MEF) from two newly developed long-chain nitrobenzoxadiazole derivatives (NBD-C16 and NBD-C18). The well-established Langmuir-Blodgett technique is used to deposit the fluorophores at defined distances from the SIFs surface, and an inert amphiphilic stearic acid is used to control the distance. NBD probes deposited directly on the SIFs surface show the highest metal-enhanced fluorescence of approximately 32-fold, and both of the probes that were studied show a consistent decrease in metal-enhanced fluorescence when increasing the distance from the fluorophore to the SIFs surface. The lowest fluorescence enhancement of approximately 4-fold is observed for the probes located 90 nm from the SIFs surface. Additionally, we also have noticed the shortest fluorescence lifetimes for the NBD probes deposited directly onto the SIFs surface, and the lifetimes are consistently increased when increasing the distances between the fluorophore and SIFs surfaces. These contrasting spectral changes, enhanced fluorescence, and decreased fluorescence lifetimes are in accordance with an increase in the rate of radiative decay for fluorophores near the silver particles. The present study provides significant information on the effect of fluorophore distance on the metal-enhanced fluorescence phenomenon.

A biosensor for theophylline based on fluorescence detection of ligand-induced hammerhead ribozyme cleavage.

Recently, Breaker and coworkers engineered hammerhead ribozymes that rearrange from a catalytically inactive to an active conformation upon allosteric binding of a specific ligand. To monitor cleavage activity in real time, we have coupled a donor-acceptor fluorophore pair to the termini of the substrate RNA of such a hammerhead ribozyme, modified to cleave in trans in the presence of the bronchodilator theophylline. In the intact substrate, the fluorophores interact by fluorescence resonance energy transfer (FRET). The specific FRET signal breaks down as the effector ligand binds, the substrate is cleaved, and the products dissociate, with a rate constant dependent on the concentration of the ligand. Our biosensor cleaves substrate at 0.46 min(-1) in 1 mM theophylline and 0.04 min(-1) without effector, and discriminates against caffeine, a structural relative of theophylline. We have measured the theophylline-dependence profile of this biosensor, showing that concentrations as low as 1 microM can be distinguished from background. To probe the mechanism of allosteric regulation, a single nucleotide in the communication domain between the catalytic and ligand-binding domains was mutated to destabilize the inactive conformation of the ribozyme. As predicted, this mutant shows the same activity (0.3 min(-1)) in the presence and absence of theophylline. Additionally, time-resolved FRET measurements on the biosensor ribozyme in complex with a noncleavable substrate analog reveal no significant changes in fluorophore distance distribution upon binding of effector.

Imaging transmitter release. II. A practical guide to evanescent-wave imaging.

Total internal reflection of a laser beam at an interface between two media of different refractive index sets up an evanescent wave field in the medium with lower refractive index. This near field decays over a distance of approximately lambda/5, lambda denoting the wavelength of light, and thus provides a convenient means for the confinement of fluorescence excitation to the near-interface region. Evanescent-wave excitation thereby permits, for example, the observation of individual fluorophores at the surface despite the presence of high concentrations in bulk solution. Although evanescent-wave excitation of fluorescence and the related technique of surface-plasmon resonance have a long record in the study of chemical reactions at surfaces, adsorption kinetics or spectroscopy, their potential for biomedical studies is only gradually emerging. Evanescent-wave microscopy provides high-contrast images of the near-membrane region of cells grown on a glass substrate at unprecedented resolution. At present, no commercial equipment is available for evanescent-wave microscopy. This review aims at readers who want to modify their fluorescence microscope to include an evanescent-wave illumination mode. Starting from the point that every objective exceeding a certain numerical aperture is generating evanescent waves, we demonstrate how the optical path can be modified to suppress the far-field excitation, and how one can switch easily between these types of illumination. The phenomena resulting from interactions of evanescent waves with cells are reviewed. The ways in which systematic variations of the angle of incident light can be used to obtain quantitative information on fluorophore distance, distribution and concentration are also discussed.