Mengfang Chang

Instrument Response Standard in Time-Resolved Fluorescence Spectroscopy at Visible Wavelength: Quenched Fluorescein Sodium

Time-resolved fluorescence properties of quenched fluorescein sodium, including self-quenching and collisional quenching by iodide, have been studied by using a picosecond time-correlated single-photon counting (TCSPC) apparatus, together with an upconversion spectrophotofluorometer with a time resolution better than 300 fs. The steady-state fluorescence intensity of fluorescein sodium reached the maximum when its concentration was 510 μM with pH > 9. Both the fluorescence intensity and lifetime decreased with increasing concentrations of NaI quencher. When the NaI concentration was 12.2 M, a monoexponential decay with a lifetime as short as 17 ps was exactly determined for the first time using the femtosecond-resolved upconversion system. Picosecond time-resolved fluorescence measurements of circular permuted green and yellow fluorescent proteins (cpGFP and cpYFP) were reported, demonstrating that the fluorescence decay of quenched fluorescein sodium is a better approximation of the instrument response function (IRF) needed for the accurate deconvolution of fluorescence lifetime data, particularly for detectors used in the visible spectral region. We believe that this picosecond lifetime standard will find wide applications in fluorescence lifetime imaging microscopy (FLIM).

Sensitive Detection of Polycyclic Aromatic Molecules: Surface Enhanced Raman Scattering via π-π Stacking.

We report silver nanoparticles (Ag NPs) with high stability, sensitivity, and no surface enhanced Raman scattering (SERS) background. The Ag NPs were synthesized via a one-step process with polysodium styrenesulfonate (PSSS) templates, and they could efficiently adsorb polycyclic aromatic molecules via π-π stacking. The adsorption mechanisms and applicability were systematically studied by experimental measurements and theoretical simulations. When the polycyclic aromatic analytes were adsorbed on the PSSS-templated Ag NPs, the vibrations of π-π stacking-bound moieties were attenuated, yet those of the other unbound aromatic moieties increased. Most importantly, when the analytes had more than two π-π stacking binding sites, the PSSS-templated Ag NPs could trap the analytes by focusing through the optical force induced or via the simultaneously formed analyte-Ag NPs aggregates. This afforded high SERS intensity and very low detection limits.

Dual emission fluorescent silver nanoclusters for sensitive detection of the biological coenzyme NAD+/NADH.

Fluorescent silver nanoclusters (Ag NCs) displaying dual-excitation and dual-emission properties have been developed for the specific detection of NAD(+) (nicotinamide adenine dinucleotide, oxidized form). With the increase of NAD(+) concentrations, the longer wavelength emission (with the peak at 550 nm) was gradually quenched due to the strong interactions between the NAD(+) and Ag NCs, whereas the shorter wavelength emission (peaking at 395 nm) was linearly enhanced. More important, the dual-emission intensity ratio (I395/I550), fitting by a single-exponential decay function, can efficiently detect various NAD(+) levels from 100 to 4000 μM, as well as label NAD(+)/NADH (reduced form of NAD) ratios in the range of 1-50.

The phosphorescence and excitation-wavelength dependent fluorescence kinetics of large-scale graphene oxide nanosheets

In this study, phosphorescence emission and a strong excitation-wavelength dependent fluorescence has been found in large-scale graphene oxide (GO) nanosheets. GO was covalently functionalized with triphenylamine (GO-CONH-TPA) in order to enhance the GO fluorescence quantum yield to 18%. The intersystem crossing (ISC) dynamics were studied using a femtosecond transient absorption technique, which appeared in the same timescale as the fluorescence dynamics of GO-CONH-TPA in both a polar solvent and solid film. Therefore, both, the solvation (several hundreds of picoseconds) and the intersystem crossing (ISC) gave rise to the strong excitation-wavelength dependent fluorescence. Moreover, the phosphorescence emission of the GO-CONH-TPA film at room temperature has been described for the first time in this report, and the lifetime of phosphorescence was found to be 6.95 μs. The fluorescence kinetics of GO-CONH-TPA were attributed to the aromatic hydrocarbon-carboxylic domain structure of GO.

Using Pyridinium Styryl Dyes as the Standards of Time-Resolved Instrument Response

In this paper, two pyridinium styryl dyes, [2-(4-dimethylamino-phenyl)-vinyl]-1-methylpyridinium iodide (DASPMI), were synthesized and characterized by steady state fluorescence spectroscopy as well as picosecond and femtosecond time-resolved fluorescence spectroscopies. Both dyes exhibit large Stokes shifts and fluorescence decays equivalent to the instrument response function (IRF) standards employed in time-correlated single-photon counting. Due to their styryl and pyridinium moieties, DASPMIs have higher peak fluorescence intensity and shorter excited-state lifetimes than iodide ion-quenched fluorophores. The fluorescence lifetimes of o-DASPMI and p-DASPMI were measured to be 6.6 ps and 12.4 ps, respectively. The fluorescence transients of these DASPMIs were used as the IRFs for iterative reconvolution fitting of the time-resolved fluorescence decay profiles of Rhodamine B (RhB), sulforhodamine B (SRB), and the SRB-SRB2m RNA aptamer complex. The quality of the fits employing the DASPMI-derived IRFs are consistently equivalent to those employing IRFs obtained from light scattering. These results indicate that DASPMI-derived IRFs may be suited for a broad range of applications in time-resolved spectroscopy and fluorescence lifetime imaging microscopy (FLIM), especially in the visible emission range.

Using Fractional Intensities of Time-resolved Fluorescence to Sensitively Quantify NADH/NAD + with Genetically Encoded Fluorescent Biosensors

In this paper, we propose a novel and sensitive ratiometric analysis method that uses the fractional intensities of time-resolved fluorescence of genetically encoded fluorescent NADH/NAD+ biosensors, Peredox, SoNar, and Frex. When the conformations of the biosensors change upon NADH/NAD+ binding, the fractional intensities (αiτi) have opposite changing trends. Their ratios could be exploited to quantify NADH/NAD+ levels with a larger dynamic range and higher resolution versus commonly used fluorescence intensity and lifetime methods. Moreover, only one excitation and one emission wavelength are required for this ratiometric measurement. This eliminates problems of traditional excitation-ratiometric and emission-ratiometric methods. This method could be used to simplify the design and achieve highly sensitive analyte quantification of genetically encoded fluorescent biosensors. Wide potential applications could be developed for imaging live cell metabolism based on this new method.

Time-Resolved Fluorescence of Water-Soluble Pyridinium Salt: Sensitive Detection of the Conformational Changes of Bovine Serum Albumin.

In this paper, we report a pyridinium salt “turn-on” fluorescent probe, 4-[2-(4-Dimethylamino-phenyl)-vinyl]-1-methylpyridinium iodide (p-DASPMI), and applied its time-resolved fluorescence (TRF) to monitor the protein conformational changes. Both the fluorescence lifetime and quantum yield (QY) of p-DASPMI were increased about two orders of magnitude after binding to the protein bovine serum albumin (BSA). The free p-DASPMI in solution presents an ultrashort fluorescence lifetime (12.4 ps), thus it does not interfere the detection of bound p-DASPMI which has nanosecond fluorescence lifetime. Decay-associated spectra (DAS) show that p-DASPMI molecules bind to subdomains IIA and IIIA of BSA. The TRF decay profiles of p-DASPMI can be described by the multi-exponential decay function ( ∑ α n exp ( - t / τ n ) ), and the obtained parameters, such as lifetimes ( τ n ), fractional amplitudes ( α n ), and fractional intensities ( α n τ n ), may be used to deduce the conformational changes of BSA. The pH and Cu2+ induced conformational changes of BSA were investigated through the TRF of p-DASPMI. The results show that the p-DASPMI is a candidate fluorescent probe in studying the conformational changes of proteins through TRF spectroscopy and microscopy in the visible range.

Fluorescence Dynamics of LicT Protein by Time-Resolved Spectroscopy

In this paper, the fluorescence dynamics of tryptophan residues in LicT protein is investigated by time-resolved fluorescence method combined with UV absorption and steady-state fluorescence spectroscopy. The local microenvironment and structural changes of LicT protein before and after activation are studied. The activated LicT protein AC 141 prevents the antitermination of gene transcription involved in carbohydrate utilization to accelerate the bodys metabolism. The structural properties and microenvironment of activated protein AC 141 and wild-type protein Q 22 were determined by different fluorescence emissions and lifetimes of tryptophan residues. The interaction between tryptophan residues and solvent is elucidated by decay associated spectroscopy (DAS) and time-resolved emission spectra (TRES), indicating that upon activation, the structure of AC 141 is more compact than that of wild-type Q 22. In addition, TRES also showed that tryptophan residues in the protein had a continuous spectral relaxation process. Anisotropy results illustrated the conformational motions of residues and whole proteins, suggesting that tryptophan residues had independent local motions in the protein system, and that the motions were more intense in the activated protein.