Release of the self-quenching of fluorescence near silver metallic surfaces

doi:10.1016/S0003-2697(03)00351-8

Analytical Biochemistry

Volume 320, Issue 1, 1 September 2003, Pages 13-20

https://doi.org/10.1016/S0003-2697(03)00351-8 Get rights and content

Abstract

Fluorescein is one of the most widely used fluorescent probes in microscopy, biotechnology, and clinical assays. One difficulty with fluorescein is its self-quenching, which results in decreased intensities with increasing labeling density. In this study we examined human serum albumin (HSA), which contained one to nine covalently linked fluorescein molecules per molecule of HSA. The occurrence of homo resonance energy transfer for labeling ratios greater than 1 were confirmed by decreases in the relative quantum yields, anisotropies, and lifetimes. We found that most of the self-quenching can be partially eliminated by proximity of the labeled protein to metallic silver particles. These results suggest the use of heavily labeled proteins and metallic colloids to obtain ultrabright reagents for use in immunoassays, imaging, and other applications.

Section snippets

Labeling of HSA

Human serum albumin (2 mg; Sigma) was dissolved in 1 ml of 0.1 M bicarbonate buffer (pH 9.2) and mixed with 5–90 μl of fluorescein-5-isothiocyanate (Molecular Probes) solution in DMSO (2 mg FITC/200 μl DMSO). The reaction mixture was incubated for 2 h at room temperature and the labeled protein was separated from the unreacted probe by passing over a Sephadex G-25 column equilibrated with 0.1× PBS.

Determining the degree of labeling

The ratio FITC/HSA in stock solution of labeled protein was determined by independent measurements of dye

Spectra properties of FITC–HSA in solution

While it is widely known that fluorescein bound to proteins is self-quenched, there is surprisingly little published data on this effect [8]. Hence we first examined the spectral properties of FITC–HSA with molar labeling ratios (L) ranging from 1-to-1 (L=1) to 1-to-9 (L=9). Emission spectra are shown in Fig. 1 for samples which had the same optical density at 490 nm. The relative intensity decreased progressively with increased labeling. The insert in Fig. 1 shows the intensities normalized to

Discussion

It is informative to consider how silver-enhanced fluorescence, particularly of a heavily labeled sample, can be used for improved assays. Fig. 12 shows emission spectra of a quartz plate coated with FITC–HSA to which we adjusted the concentration of rhodamine B (0.25 μM) to result in an approximate 1.5-fold larger RhB intensity. One can consider the RhB to be sample autofluorescence or any other interference signal. When the same conditions are used for FITC–HSA on silver with L=1 the

Acknowledgements

This work was supported by the NIH National Center for Research Resource, RR-08119. The authors thank Dr. Zygmunt Gryczynski for the photograph of the coated slides.

References (21)

R.S Knox
Theory of polarization quenching by excitation transfer
Physica
(1968)
L.W Runnels et al.
Theory and application of fluorescence homotransfer to melittin oligomerization
Biophys. J.
(1995)
J Karolin et al.
Donor-donor energy migration for determining intramolecular distances in proteins: I. Application of a model to the latent plasminogen activator inhibitor-1 (PAI-1)
Biophys. J.
(1998)
G.C Visor et al.
Fluorescence immunoassay
J. Pharm. Sci.
(1981)
T Vo-Dinh et al.
Immunosensors: principles and applications
Immunomethods
(1993)
J.R Lakowicz
Radiative decay engineering: biophysical and biomedical applications
Anal. Biochem.
(2001)
J.R Lakowicz et al.
Radiative decay engineering 2. Effects of silver island films on fluorescence intensity, lifetimes, and resonance energy transfer
Anal. Biochem.
(2002)
J Jablonski
Self-depolarization and decay of photoluminescence of solutions
Acta Phys. Pol.
(1955)
C Bojarski et al.
Investigations of the excitation energy transport mechanism in donor-acceptor systems
J. Fluoresc.
(1991)
P Bojarski et al.
Nonradiative excitation energy transport in one-component disordered systems
J. Fluoresc.
(1995)

There are more references available in the full text version of this article.

Cited by (181)

Tracking metal enhanced fluorescence from Eschericcia coli nano-bio-assemblies within colloidal dispersions by static and 3D spectra emissions
2023, Microchemical Journal
It was studied the interactions of single Ultraluminescent 40 nm gold Core shell Nanoparticles (d40 Au@SiO₂-Fl) with Eschericcia Coli bacteria (Esc. Col). By this manner, it was tracked from initial Nano-Bio-assembly formation (Au@SiO₂-Fl-Esc. Col) towards inter-Nano-Biostructure interactions. In order to do that it was determined association constants between d40 Au@SiO₂-Fl and Biostructures by 3D Fluorescence, fluorescence spectroscopy measurements. The association constant was related with non-covalent Nano-Biostructure interactions of 45 ± 2 biostructure^-1; accompanied with a Quantum Yield ratio between the Bio-assembly and Nano-labeller of 3.6 ± 0.1 times, with the maximal concentration of bacteria addition. This phenomenon of interaction was tracked from the bulk of the colloidal dispersion. And, from these individual interactions, it was possible to generate Bioimaging by Laser Fluorescence Microscopy in real time of the dynamic of Bio-assembly formation as well as the by inter-Nano-Biostructures. These data leaded to the correlation of 3D Fluorescence spectroscopy within different bacteria concentrations. The recorded spectra showed increased values of intensities that fitted to a linear equation as model of the experimental data records. About analytical performances, it could be noted that optimized sensitivity by 3D Fluorescence was unless 65% higher than by static Fluorescence. Moreover, it should be mentioned that two distributions of emissions were recorded that correlated with Plasmonics coupling with natural emitters within the UV and MEF Nanolabeller emissions. Thus, optimizing conditions, LODs of 0.3x10 ³ bacterium*mL⁻¹ were determined. Moreover, by In Flow Cytometry was recorded different sensitivity values depending of the Laser excitation applied. These phenomena were explained by the development of different Nano-Bio-emitters species. In addition, in order to validate the methodology, it was quantified Biostructures from fortified aqueous samples. By 3D Fluorescence it was determined recoveries of (97 ± 8)x10³, and (93 ± 5)x10³ from upper and lower distributions of emissions recorded in 3D Fluorescence topographies. The reproducibility in all cases was by %CV of 98–99 %; therefore the proposed Bioassays showed interesting insights in Nano-Biotechnology with impact in varied Research fields.
In this way, it was showed and discussed about Bio-analytical applications for Eschericcia Coli detection and quantification within colloidal dispersion with relative simple, accessible and portable fluorescence spectroscopy set ups. Therefore, it was given insights for accessible Nano-Biodetection based on Ultra-sensitive MEF Nanoplatforms for Bio-labeling using standard spectroscopic and compared by In Flow Citometry analysis as well.
Evaluating the Effect of Dye-Dye Interactions of Xanthene-Based Fluorophores in the Fluorosequencing of Peptides
2022, Bioconjugate Chemistry
A peptide sequencing scheme utilizing fluorescence microscopy and Edman degradation to determine the amino acid position in fluorophore-labeled peptides was recently reported, referred to as fluorosequencing. It was observed that multiple fluorophores covalently linked to a peptide scaffold resulted in a decrease in the anticipated fluorescence output and worsened the single-molecule fluorescence analysis. In this study, we report an improvement in the photophysical properties of fluorophore-labeled peptides by incorporating long and flexible (PEG)₁₀ linkers at the peptide attachment points. Long linkers to the fluorophores were installed using copper-catalyzed azide–alkyne cycloaddition conditions. The photophysical properties of these peptides were analyzed in solution and immobilized on a microscope slide at the single-molecule level under peptide fluorosequencing conditions. Solution-phase fluorescence analysis showed improvements in both quantum yield and fluorescence lifetime with the long linkers. While on the solid support, photometry measurements showed significant increases in fluorescence brightness and 20 to 60% improvements in the ability to determine the amino acid position with fluorosequencing. This spatial distancing strategy demonstrates improvements in the peptide sequencing platform and provides a general approach for improving the photophysical properties in fluorophore-labeled macromolecules.
Inflow nano-optics from the near-to the far-field detection based on Metal-Enhanced Fluorescence signaling
2021, Microchemical Journal
In this communication we evaluated the detection and tracking of Ultraluminescent gold core–shell nanoplatforms based on Metal-Enhanced Fluorescence (MEF) within colloidal dispersion by In-Flow Cytometry and Imaging Flow Cytometry (IFC). The enhanced properties were developed by the incorporation of Rhodamine B (RhB) as a laser dye reporter at variable controlled silica spacer lengths (Au@SiO₂-RhB) from a gold core template. Thus, SiO₂ was varied from 6 to 7 nm to 25 nm in order to place the laser dye sensitizer at different lengths of the higher electromagnetic fields surrounding the 45-nm gold core diameter. By Laser Fluorescence Microscopy, high intense emissions were shown at shorter SiO₂ lengths, while lower emissions were found towards longer ones. These characteristics related to MEF were evaluated by in-flow cytometry with dual laser excitation and fluorescence detection, and by an IFC set-up. By In-flow cytometry, different scattered-light distributions were shown, depending on the nanoparticle concentrations in the colloidal dispersion and size of the Au@SiO₂-RhB. Moreover, varied fluorescent event detections and counting were recorded according to the SiO₂ lengths applied. A ratio of fluorescent event detection counting of core–shell/core-less of 40–42 was established for optimal MEF Enhancement Factors (MEF_EF) of 41–43 determined by Laser Fluorescence Microscopy. Core-less nanoparticles showed a marked decrease of fluorescent event detection counting caused by higher photobleaching and absence of the MEF effect. Hence, an improved detection effect was shown within in-flow based on MEF from single nanoplatforms. Therefore, the Ultraluminescence emission of core–shell nanoarchitectures within in-flow techniques was shown and discussed. A non-classical light in real time within colloidal gold core–shell nanoparticles was generated by accurate control of their architectures.
Fluorophore Multimerization as an Efficient Approach towards Bright Protein Labels
2021, European Journal of Organic Chemistry
Cell analysis techniques like flow cytometry and fluorescence microscopy are widely used to explore cells in real-time and provide important insights into a variety of cellular processes. These techniques require bright and specific staining reagents to generate strong fluorescence signal and enable reliable read-out, making the choice of fluorescent labels a key aspect of experimental design. Much research has been done in the field of fluorophore development to generate bright small-molecule dyes, fluorescent proteins, and emitting nanoparticles. However, it remains challenging to modify biomolecules with multiple emitters and avoid self-quenching of such fluorophores at the same time. Herein, we present advances in multimerization-based methods for the generation of fluorescent labels with high brightness and discuss their advantages and limitations in the context of flow cytometry and fluorescence microscopy applications.
Controllably prepared molecularly imprinted core-shell plasmonic nanostructure for plasmon-enhanced fluorescence assay
2019, Biosensors and Bioelectronics
Plasmon-enhanced fluorescence (PEF) is an emerging technology for sensitive detection. It relies on the plasmonic effect of a noble metal nanostructure to dramatically enhance the fluorescence of target fluorophores around the metal surface. Because there is a compromise between plasmonic enhancement and fluorescence quenching, it is critical to control the distance between the fluorophore and the metal surface to an appropriate range. This makes the fabrication of plasmonic nanostructures for PEF assays a challenging task. Herein, we report a controllably prepared core-shell plasmonic nanostructure coated with molecularly imprinted polymer (MIP) for sensitive and specific PEF assay. Riboflavin (RF) was used as a test compound in this study. RF-imprinted Ag@SiO₂ nanoparticles were prepared in a controllable manner, providing an optimal distance between the metal surface and RF molecules. The obtained hybrid nanostructure allowed for sensitive detection and specific recognition towards the target. Based on the plasmonic hybrid nanostructure, a sensitive and specific PEF assay of RF was developed and successfully applied to the determination of RF in human urine. Thus, the study paved the way for controllable preparation of molecularly imprinted plasmonic nanostructures for sensitive and specific PEF assays.
Aggregation-induced emission of azines: An up-to-date review
2019, Journal of Molecular Liquids
Luminogenic materials which feature the unique photophysical phenomenon of aggregation-induced emission (AIE) has attracted growing attention. Research on AIEgens have immensely expanded since 2001. Salicylaldehyde azine derivatives connected through NN single bond have strong AIE fluorescence properties in aggregate/solid state and has stimulated many novel developments in various technological fields, extending from optoelectronics, chemo sensing, bio imaging to stimuli-responsive devices. This review article presents an integrated report that focuses on the mechanism and applications of AIE. It also provides an evidence-based discussion on representative azine systems featuring AIE, enabling them for potential technological applications. This comprehensive review would benefit researchers who are actively engaged in the area of luminogens or planning to enter the fascinating field of AIE research.

View all citing articles on Scopus

View full text

Release of the self-quenching of fluorescence near silver metallic surfaces

Abstract

Section snippets

Labeling of HSA

Determining the degree of labeling

Spectra properties of FITC–HSA in solution

Discussion

Acknowledgements

Physica

Biophys. J.

Biophys. J.

J. Pharm. Sci.

Immunomethods

Anal. Biochem.

Anal. Biochem.

Self-depolarization and decay of photoluminescence of solutions

Acta Phys. Pol.

Investigations of the excitation energy transport mechanism in donor-acceptor systems

J. Fluoresc.

Nonradiative excitation energy transport in one-component disordered systems

J. Fluoresc.