Ratiometric Two-Photon Near-Infrared Probe to Detect DPP IV in Human Plasma, Living Cells, Human Tissues, and Whole Organisms Using Zebrafish.

DPP IV, otherwise known as CD26 lymphocyte T surface antigen, is a transmembrane glycoprotein also found in circulation in the blood. It plays an important role in several processes like glucose metabolism and T-cell stimulation. Moreover, it is overexpressed in renal, colon, prostate, and thyroid human carcinoma tissues. It can also serve as a diagnostic in patients with lysosomal storage diseases. The biological and clinical importance of having readouts for the activity of this enzyme, in physiological and disease conditions, has led us to design a near-infrared (NIR) fluorimetric probe that also has the characteristics of being ratiometric and excitable by two simultaneous NIR photons. The probe consists of assembling an enzyme recognition group (Gly-Pro) (Mentlein, 1999; Klemann et al., 2016) on the two-photon (TP) fluorophore (derivative of dicyanomethylene-4H-pyran, DCM-NH2) disturbing its NIR characteristic internal charge transfer (ICT) emission spectrum. When the dipeptide group is released by the DPP IV-specific enzymatic action, the donor-acceptor DCM-NH2 is restored, forming a system that shows high ratiometric fluorescence output. With this new probe, we have been able to detect, quickly and efficiently, the enzymatic activity of DPP IV in living cells, human tissues, and whole organisms, using zebrafish. In addition, due to the possibility of being excited by two photons, we can avoid the autofluorescence and subsequent photobleaching that the raw plasma has when it is excited by visible light, achieving detection of the activity of DPP IV in that medium without interference.

Coupled Excited-State Dynamics in N-Substituted 2-Methoxy-9-Acridones.

Fluorophores of the acridone family have been widely employed in many applications, such as DNA sequencing, the detection of biomolecules, and the monitoring of enzymatic systems, as well as being the bases of intracellular sensors and even antitumoral agents. They have been widely used in fluorescence imaging due to their excellent photophysical properties, in terms of quantum yield and stability. However, frequently, the fluorescence emission data from acridones are not easily interpretable due to complex excited-state dynamics. The formation of π-stacking aggregates and excimers and excited-state proton transfer (ESPT) reactions usually result in emission features that are dependent on the experimental conditions. Therefore, an in-depth understanding of the dynamics involved in the excited-state transients of these dyes is mandatory for their appropriate application. Herein, we synthesized and fully characterized different 2-methoxy-9-acridone dyes. Their transient fluorescence emission spectra exhibited a complex dynamic behavior that can be linked to several excited-state reactions. We performed a thorough study of the excited-state dynamics of these dyes by means of time-resolved fluorimetry supported by computational calculations. All this allowed us to establish a multistate kinetic scheme, involving an ESPT reaction coupled to an excimer formation process. We have unraveled the rich dynamics behind this complex behavior, which provides a better understanding of the excited states of these dyes.

Klotho Lacks an FGF23-Independent Role in Mineral Homeostasis.

Fibroblast growth factor-23 (FGF23) is a bone-derived hormone regulating vitamin D hormone production and renal handling of minerals by signaling through an FGF receptor/αKlotho (Klotho) receptor complex. Whether Klotho has FGF23-independent effects on mineral homeostasis is a controversial issue. Here, we aimed to shed more light on this controversy by comparing male and female triple knockout mice with simultaneous deficiency in Fgf23 and Klotho and a nonfunctioning vitamin D receptor (VDR) (Fgf23/Klotho/VDR) with double (Fgf23/VDR, Klotho/VDR, and Fgf23/Klotho) and single Fgf23, Klotho, and VDR mutants. As expected, 4-week-old Fgf23, Klotho, and Fgf23/Klotho knockout mice were hypercalcemic and hyperphosphatemic, whereas VDR, Fgf23/VDR, and Klotho/VDR mice on rescue diet were normocalcemic and normophosphatemic. Serum levels of calcium, phosphate, and sodium did not differ between 4-week-old triple Fgf23/Klotho/VDR and double Fgf23/VDR or Klotho/VDR knockout mice. Notably, 3-month-old Fgf23/Klotho/VDR triple knockout mice were indistinguishable from double Fgf23/VDR and Klotho/VDR compound mutants in terms of serum calcium, serum phosphate, serum sodium, and serum PTH, as well as urinary calcium and sodium excretion. Protein expression analysis revealed increased membrane abundance of sodium-phosphate co-transporter 2a (NaPi-2a), and decreased expression of sodium-chloride co-transporter (NCC) and transient receptor potential cation channel subfamily V member 5 (TRPV5) in Fgf23/Klotho/VDR, Fgf23/VDR, and Klotho/VDR mice, relative to wild-type and VDR mice, but no differences between triple and double knockouts. Further, ex vivo treatment of live kidney slices isolated from wild-type and Klotho/VDR mice with soluble Klotho did not induce changes in intracellular phosphate, calcium or sodium accumulation assessed by two-photon microscopy. In conclusion, our data suggest that the main physiological function of Klotho for mineral homeostasis in vivo is its role as co-receptor mediating Fgf23 action. © 2017 American Society for Bone and Mineral Research.

Photophysics of a Live-Cell-Marker, Red Silicon-Substituted Xanthene Dye.

Dyes with near-red emission are of great interest because of their undoubted advantages for use as probes in living cells. In-depth knowledge of their photophysics is essential for employment of such dyes. In this article, the photophysical behavior of a new silicon-substituted xanthene, 7-hydroxy-5,5-dimethyl-10-(o-tolyl)dibenzo[b,e]silin-3(5H)-one (2-Me TM), was explored by means absorption, steady-state, and time-resolved fluorescence. First, the near-neutral pH, ground-state acidity constant of the dye, pKN-A, was determined by absorbance and steady-state fluorescence at very low buffer concentrations. Next, we determined whether the addition of phosphate buffer promoted the excited-state proton-transfer (ESPT) reaction among the neutral and anion form of 2-Me TM in aqueous solutions at near-neutral pH. For this analysis, both the steady-state fluorescence method and time-resolved emission spectroscopy (TRES) were employed. The TRES experiments demonstrated a remarkably favored conversion of the neutral form to the anion form. Then, the values of the excited-state rate constants were determined by global analysis of the fluorescence decay traces recorded as a function of pH, and buffer concentration. The revealed kinetic parameters were consistent with the TRES results, exhibiting a higher rate constant for deprotonation than for protonation, which implies an unusual low value of the excited-state acidity constant pK*N-A and therefore an enhanced photoacid behavior of the neutral form. Finally, we determined whether 2-Me TM could be used as a sensor inside live cells by measuring the intensity profile of the probe in different cellular compartments of HeLa 229 cells.

New Dual Fluorescent Probe for Simultaneous Biothiol and Phosphate Bioimaging.

The simultaneous detection of relevant metabolites in living organisms by using one molecule introduces an approach to understanding the relationships between these metabolites in healthy and deregulated cells. Fluorescent probes of low toxicity are remarkable tools for this type of analysis of biological systems in vivo. As a proof of concept, different naturally occurring compounds, such as biothiols and phosphate anions, were the focus for this work. The 2,4-dinitrobenzenesulfinate (DNBS) derivative of 9-[1-(4-tert-butyl-2-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (Granada Green; GG) were designed and synthesized. This new sulfinyl xanthene derivative can act as a dual sensor for the aforementioned analytes simultaneously. The mechanism of action of this derivative implies thiolysis of the sulfinyl group of the weakly fluorescent DNBS-GG by biological thiols at near-neutral pH values, thus releasing the fluorescent GG moiety, which simultaneously responds to phosphate anions through its fluorescence-decay time. The new dual probe was tested in solution by using steady-state and time-resolved fluorescence and intracellularly by using fluorescence-lifetime imaging microscopy (FLIM) in human epithelioid cervix carcinoma (HeLa) cells.

Fluorescence enhancement of a fluorescein derivative upon adsorption on cellulose.

9-[1-(2-Methyl-4-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (2-Me-4-OMe TG) is a fluorescein derivative dye whose photophysical properties show a remarkable pH dependence. In aqueous solution the fluorescence quantum yield (Φf) of its anionic species is nearly a hundred times higher than that of its neutral species. Such a large difference in Φf makes 2-Me-4-OMe TG useful as an "on-off" pH indicator. Here we report that adsorption on the surface of microcrystalline cellulose exerts a profound effect upon the photophysical properties of 2-Me-4-OMe TG. On the solid only the dye neutral species is observed and its Φf is 0.31 ± 0.10, which is approximately thirty times higher than the value found for the neutral species in aqueous solution (Φf = 0.01). 2-Me-4-OMe TG and Dabcyl (DB) were co-adsorbed on the surface of microcrystalline cellulose to study the transfer of excitation energy from the former to the latter. In the absence of the dye, the formation of DB aggregates is observed at concentrations greater than 0.34 µmol per gram of cellulose, while in the presence of 2-Me-4-OMe TG the formation of DB aggregates is thoroughly inhibited. The quenching of fluorescence of 2-Me-4-OMe TG by DB reaches efficiencies as high as 90% for the most concentrated samples.

Interaction of YOYO-3 with different DNA templates to form H-aggregates.

Homodimeric cyanine dyes are DNA intercalators that display a large enhancement of fluorescence emission when bound to double-stranded DNA. However, other different interaction modes are possible, such as H-type molecular aggregates of the dye, templated by the nucleic acid. In this paper, we study in depth the formation of nonfluorescent H-aggregates of the cyanine homodimer YOYO-3 with two different DNA templates using absorption and both steady-state and time-resolved fluorescence spectroscopy. First, a nonfluorescent YOYO-3 H-aggregate complex was found to form in single-stranded polycytidine chains, resulting in the appearance of a new absorption band at approximately 500 nm. The specific interaction of cytosine bases suggests the involvement of the C-rich i-motif in facilitating the formation of the H-aggregate complex. Second, the interaction of YOYO-3 with double-stranded poly(A·T) tracts also led to the appearance of a new absorption band at approximately 500 nm, and hence of a different type of H-aggregate. We found that the aggregate is formed mainly in double-stranded regions with consecutive adenine bases in the same strand (and thymine bases in the complementary strand). These poly(A·T) tracts provide narrow minor grooves and enhanced electrostatic negative potential to promote the aggregation of the negatively charged cyanine. As the YOYO-3 H-aggregates are nonfluorescent, our results provide an important basis to quantitatively understand the fluorescence emission of this cyanine dye in the presence of DNA strands.

8-HaloBODIPYs and their 8-(C, N, O, S) substituted analogues: solvent dependent UV-vis spectroscopy, variable temperature NMR, crystal structure determination, and quantum chemical calculations.

The UV-vis electronic absorption and fluorescence emission properties of 8-halogenated (Cl, Br, I) difluoroboron dipyrrin (or 8-haloBODIPY) dyes and their 8-(C, N, O, S) substituted analogues are reported. The nature of the meso-substituent has a significant influence on the spectral band positions, the fluorescence quantum yields, and lifetimes. As a function of the solvent, the spectral maxima of all the investigated dyes are located within a limited wavelength range. The spectra of 8-haloBODIPYs display the narrow absorption and fluorescence emission bands and the generally quite small Stokes shifts characteristic of classic difluoroboron dipyrrins. Conversely, fluorophores with 8-phenylamino (7), 8-benzylamino (8), 8-methoxy (9), and 8-phenoxy (10) groups emit in the blue range of the visible spectrum and generally have larger Stokes shifts than common BODIPYs, whereas 8-(2-phenylethynyl)BODIPY (6) has red-shifted spectra compared to ordinary BODIPY dyes. Fluorescence lifetimes for 6, 8, and 10 have been measured for a large set of solvents and the solvent effect on their absorption and emission maxima has been analyzed using the generalized Catalán solvent scales. Restricted rotation about the C8-N bond in 7 and 8 has been observed via temperature dependent (1)H NMR spectroscopy, whereas for 10 the rotation about the C8-O bond is not hindered. The crystal structure of 8 demonstrates that the short C8-N bond has a significant double character and that this N atom exhibits a trigonal planar geometry. The crystal structure of 10 shows a short C8-O bond and an intramolecular C-H···π interaction. Quantum-chemical calculations have been performed to assess the effect of the meso-substituent on the spectroscopic properties.

Synthesis and photophysics of a new family of fluorescent 9-alkyl-substituted xanthenones.

9-Alkyl xanthenones with different aliphatic pendant groups have been easily prepared by means of nucleophilic addition of the corresponding Grignard derivative to a tert-butyldimethylsilyl ether (TBDMS)-protected 3,6-dihydroxy-xanthenone. The photophysical behavior of the new dyes has been explored by using absorption, steady-state-, and time-resolved fluorescence measurements. We determined the equilibrium constants, visible spectral characteristics, fluorescence quantum yield, and decay times. Remarkably, they retain similar fluorescent properties of fluorescein including the characteristic phosphate-mediated excited-state proton-transfer (ESPT) reaction. 6-Hydroxy-9-isopropyl-3H-xanthen-3-one (5) was investigated in living cells; it presented a good permeability and efficient accumulation inside the cytosol. For the first time, we reported that the requirement of an aryl group at C-9 is no longer needed and new fluorescent sensors can be therefore easily developed.

Fluorescence lifetime imaging microscopy for the detection of intracellular pH with quantum dot nanosensors.

While the use of quantum dot (QD) nanoparticles for bioimaging and sensing has been improved and exploited during the last several years, most studies have used emission intensity-based techniques. Fluorescence lifetime imaging microscopy (FLIM) can also be employed for sensing purposes, overcoming many of the limitations of the aforementioned systems. Herein, we show that the photoluminescence (PL) lifetime of mercaptopropionic acid-capped QDs (MPA-QDs) collected from FLIM images can be used to determine intracellular pH. The PL average lifetime of MPA-QDs varied from 8.7 ns (pH < 5) to 15.4 ns (pH > 8) in media mimicking the intracellular environment. These long decay times of QD nanoparticles make them easily distinguishable from intrinsic cell autofluorescence, improving selectivity in sensing applications. We demonstrate, for the first time, the successful detection of changes in the intracellular pH of different cell types by examining the PL decay time of QDs. In particular, the combination of FLIM methodologies with QD nanoparticles exhibits greatly improved sensitivity compared with other fluorescent dyes for pH imaging. A detailed description of the advantages of the FLIM technique is presented.

Real-time phosphate sensing in living cells using fluorescence lifetime imaging microscopy (FLIM).

Phosphate ions play important roles in signal transduction and energy storage in biological systems. However, robust chemical sensors capable of real-time quantification of phosphate anions in live cells have not been developed. The fluorescein derivative dye 9-[1-(2-methyl-4-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (2-Me-4-OMe TG) exhibits the characteristic excited-state proton-transfer (ESPT) reaction of xanthenic derivatives at approximately physiological pH resulting in the dependence of the dye's nanosecond fluorescence decay time on the phosphate buffer concentration. This allows the 2-Me-4-OMe TG dye to be used with fluorescence lifetime imaging microscopy (FLIM) as a real-time phosphate intracellular sensor in cultured cells. This methodology has allowed the time course of cellular differentiation of MC3T3-E1 murine preosteoblast cells to be measured on the basis of the decrease in the decay time of 2-Me-4-OMe TG. These changes were consistent with increased alkaline phosphatase activity in the extracellular medium as a marker of the differentiation process.

Visible absorption and fluorescence spectroscopy of conformationally constrained, annulated BODIPY dyes.

Six conformationally restricted BODIPY dyes with fused carbocycles were synthesized to study the effect of conformational mobility on their visible electronic absorption and fluorescence properties. The symmetrically disubstituted compounds (2, 6) have bathochromically shifted absorption and fluorescence spectral maxima compared to those of the respective asymmetrically monosubstituted dyes (1, 5). Fusion of conjugation extending rings to the α,ß-positions of the BODIPY core is an especially effective method for the construction of boron dipyrromethene dyes absorbing and emitting at longer wavelengths. The fluorescence quantum yields Φ of dyes 1-6 are high (0.7 ≤ Φ ≤ 1.0). The experimental results are backed up by quantum chemical calculations of the lowest electronic excitations in 1, 2, 5, 6, and corresponding dyes of related chemical structure but without conformational restriction. The effect of the molecular structure on the visible absorption and fluorescence emission properties of 1-6 has been examined as a function of solvent by means of the recent, generalized treatment of the solvent effect, proposed by Catalán (J. Phys. Chem. B 2009, 113, 5951-5960). Solvent polarizability is the primary factor responsible for the small solvent-dependent shifts of the visible absorption and fluorescence emission bands of these dyes.

Bulk and single-molecule fluorescence studies of the saturation of the DNA double helix using YOYO-3 intercalator dye.

We report a thorough photophysical characterization of the interactions between double-stranded DNA (dsDNA) and the trimethine cyanine homodimer dye YOYO-3. The fluorescence emission of this dye is enhanced by intercalation within the DNA double helix. We have explored the saturation of the dsDNA by bound YOYO-3 at the single-molecule level by studying the single-pair Förster resonance energy transfer (FRET) from an energy donor, Alexa Fluor 488, tagged at the 5' end of the double helix and the energy acceptor, YOYO-3, bound to the same DNA molecule. The spontaneous binding of YOYO-3 gives rise to an effective distribution of different FRET efficiencies and, therefore, donor-acceptor (D-A) distances. These distributions reveal the existence of multiple states of YOYO-3. Steady-state and time-resolved fluorescence and circular dichroism confirmed the presence of a DNA-bound aggregate of YOYO-3, conspicuous at high dye/base pair ratios. The spectral features of the aggregate suggest that it may have the structure of a parallel H-aggregate.

Effects of the anion salt nature on the rate constants of the aqueous proton exchange reactions.

The proton-transfer ground-state rate constants of the xanthenic dye 9-[1-(2-methyl-4-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (TG-II), recovered by Fluorescence Lifetime Correlation Spectroscopy (FLCS), have proven to be useful to quantitatively reflect specific cation effects in aqueous solutions (J. M. Paredes, L. Crovetto, A. Orte, J. M. Alvarez-Pez and E. M. Talavera, Phys. Chem. Chem. Phys., 2011, 13, 1685-1694). Since these phenomena are more sensitive to anions than to cations, in this paper we have accounted for the influence of salts with the sodium cation in common, and the anion classified according to the empirical Hofmeister series, on the proton transfer rate constants of TG-II. We demonstrate that the presence of ions accelerates the rate of the ground-state proton-exchange reaction in the same order than ions that affect ion solvation in water. The combination of FLCS with a fluorophore undergoing proton transfer reactions in the ground state, along with the desirable feature of a pseudo-dark state when the dye is protonated, allows one unique direct determination of kinetic rate constants of the proton exchange chemical reaction.

A chloride ion nanosensor for time-resolved fluorimetry and fluorescence lifetime imaging.

In this work, the first CdSe/ZnS quantum dot (QD) photoluminescence lifetime based chloride ion nanosensor is reported. The acridinium dication lucigenin was self-assembled on the surface of negatively charged mercaptopropionic acid capped QDs to achieve QD-lucigenin conjugates. Upon attachment, a drastic decrease of the photoluminescence lifetime of both QD nanoparticles and lucigenin is observed by virtue of a charge transfer mechanism. Since lucigenin is a chloride-sensitive indicator dye, the photoluminescence decay of QD-lucigenin conjugates changes by adding chloride ion. The photoluminescence lifetime of the QDs in the conjugate increases after reacting with Cl(-), but also shows a concomitant decrease in the lucigenin lifetime immobilized on the surface. The photoluminescence lifetime of QD-lucigenin nanosensors shows a linear response in the Cl(-) concentration range between 0.5 and 50 mM. Moreover, the ratio τ(ave)(QD)/τ(ave)(luc) can be used as an analytical signal since the lifetime ratio presents a linear response in the same Cl(-) concentration range. The system also shows good selectivity towards most of the main anions and molecules that can be found in biological fluids. These nanosensors have been satisfactorily applied for Cl(-) determination in simulated intracellular media with high sensitivity and high selectivity. Finally, we demonstrate the potential application of the proposed nanosensor in confocal fluorescence lifetime imaging (FLIM). These results show the promising application of the QD-lucigenin nanosensors in FLIM, particularly for intracellular sensing, with the invaluable advantages of the time-resolved fluorescence techniques.

Photophysics of the interaction between a fluorescein derivative and Ficoll.

Ficoll has been widely used as a crowding agent to mimic intracellular media because it is believed to be noninteracting and is composed of mixed sizes such that smaller and larger diffusing solutes can be studied. Due to the interest that the fluorescent dye 9-[1-(2-methyl-4-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (TG-II) as a fluorometric probe of phosphate ions in intracellular media could generate, we describe the spectral characteristics of the system TG-II-Ficoll in aqueous solution by means of absorption spectroscopy, steady-state fluorescence, time-resolved fluorescence, time-resolved emission spectroscopy, and fluorescence lifetime correlation spectroscopy. The spectral characteristics found are consistent with the formation of an adsorption complex on the surface of Ficoll, probably due to hydrogen bonding between TG-II and Ficoll. In addition, the diffusion coefficient calculated for the association was similar to the diffusion coefficient previously recovered for Ficoll in the same experimental conditions. Therefore, our overall data clearly demonstrate that Ficoll is not an inert crowding agent when in the presence of fluorescein derivative dyes.

Rational design, synthesis, and spectroscopic and photophysical properties of a visible-light-excitable, ratiometric, fluorescent near-neutral pH indicator based on BODIPY.

A visible-light-excitable, ratiometric, brightly fluorescent pH indicator for measurements in the pH range 5-7 has been designed and synthesized by conjugatively linking the BODIPY fluorophore at the 3-position to the pH-sensitive ligand imidazole through an ethenyl bridge. The probe is available as cell membrane permeable methyl ester 8-(4-carbomethoxyphenyl)-4,4-difluoro-3-[2-(1H-imidazol-4-yl)ethenyl]-1,5,7-trimethyl-3a,4a-diaza-4-bora-s-indacene (I) and corresponding water-soluble sodium carboxylate, sodium 8-(4-carboxylatophenyl)-4,4-difluoro-3-[2-(1H-imidazol-4-yl)ethenyl]-1,5,7-trimethyl-3a,4a-diaza-4-bora-s-indacene (II). The fluorescence quantum yield Φ(f) of ester I is very high (0.8-1.0) in the organic solvents tested. The fluorescence lifetime (ca. 4 ns) of I in organic solvents with varying polarity/polarizability (from cyclohexane to acetonitrile) is independent of the solvent with a fluorescence rate constant k(f) of 2.4×10(8) s(-1). Probe I is readily loaded in the cytosol of live cells, where its high fluorescence intensity remains nearly constant over an extended time period. Water-soluble indicator II exhibits two acid-base equilibria in aqueous solution, characterized by pK(a) values of 6.0 and 12.6. The Φ(f) value of II in aqueous solution is high: 0.6 for the cationic and anionic forms of the imidazole ligand, and 0.8 for neutral imidazole. On protonation-deprotonation in the near-neutral pH range, UV/Vis absorption and fluorescence spectral shifts along with isosbestic and pseudo-isoemissive points are observed. This dual-excitation and dual-emission pH indicator emits intense green-yellow fluorescence at lower pH and intense orange fluorescence at higher pH. The influence of ionic strength and buffer concentration on the absorbance and steady-state fluorescence of II has also been investigated. The apparent pK(a) of the near-neutral acid-base equilibrium determined by spectrophotometric and fluorometric titration is nearly independent of the added buffer and salt concentration. In aqueous solution in the absence of buffer and in the pH range 5.20-7.45, dual exponential fluorescence decays are obtained with decay time τ(1)=4.3 ns for the cationic and τ(2)=3.3 ns for the neutral form of II. The excited-state proton exchange of II at near-neutral pH becomes reversible on addition of phosphate (H(2)PO(4)(-)/HPO(4)(2-)) buffer, and a pH-dependent change of the fluorescence decay times is induced. Global compartmental analysis of fluorescence decay traces collected as a function of pH and phosphate buffer concentration was used to recover values of the deactivation rate constants of the excited cationic (k(01)=2.4×10(8) s(-1)) and neutral (k(02)=3.0×10(8) s(-1)) forms of II.

Dynamics of water-in-oil nanoemulsions revealed by fluorescence lifetime correlation spectroscopy.

The size, diffusional properties, and dynamics of reverse water-in-oil nanoemulsions, or reverse micelles (RMs), have been widely investigated because of interest in this system as a model for biological compartmentalization. Here, we have employed fluorescence lifetime correlation spectroscopy (FLCS) to reveal the dynamics and sizes of aerosol-OT (AOT)/isooctane RMs using a fluorescent xanthene derivative called Tokyo Green II (TG-II). The dye undergoes a partition and a shift in its tautomeric equilibrium such that the TG-II anion remains in the inner micellar aqueous core, and the neutral quinoid form lies in the interfacial region. By applying FLCS, we specifically obtained the lifetime filtered autocorrelation curves of the anionic TG-II, which shows a characteristic lifetime of approximately 4 ns. Analysis of the FLCS curves provides the diffusion coefficient and hydrodynamic radius of the RMs as well as micelle dynamics in the same experiment. The FLCS curves show dynamics in the microsecond time range, which represents an interconversion rate that changes the distribution of the TG-II neutral and anionic forms in the hydrophobic interface and the water core.

Effect of surface modification on semiconductor nanocrystal fluorescence lifetime.

Semiconductor nanocrystals, namely, quantum dots (QDs), present a set of unique photoluminescence properties, which has led to increased interest in using them as advantageous alternatives to conventional organic dyes. Many applications of QDs involve surface modification to enhance the solubility or biocompatibility of the QDs. One of the least exploited properties of QDs is the very long photoluminescence lifetime that usually has complex kinetics owing to the effect of quantum confinement. Herein, we describe the effect of different surface modifications on the photoluminescence decay kinetics of QDs. The different surface modifications were carefully chosen to provide lipophilic or water-soluble QDs with either positive or negative surface net charges. We also survey the effect on the QD lifetime of several ligands that interact with the QD surface, such as organic chromophores or fluorescent proteins. The results obtained demonstrate that time-resolved fluorescence is a useful tool for QD-based sensing to set the basis for the development of time-resolved-based nanosensors.

Quantum dot photoluminescence lifetime-based pH nanosensor.

The first CdSe/ZnS quantum dot photoluminescence lifetime-based pH nanosensor has been developed. The average lifetime of mercaptopropionic acid-capped QD nanosensors showed a linear response in the pH range of 5.2-6.9. These nanosensors have been satisfactorily applied for pH estimation in simulated intracellular media, with high sensitivity and high selectivity toward most of the intracellular components.