Light-controlled molecular switches modulate nanocrystal fluorescence

Spiropyran dyes were attached to fluorescent core-shell CdSe/ZnS nanocrystals via thiol-containing linkers. Photoisomerization of the dye to its merocyanine form by UV irradiation caused a dramatic loss in the intrinsic nanoparticle fluorescence, which was regained upon reversing the isomerization with visible light. The fluorescence quenching efficiency increased with increasing spectral overlap of fluorescence emission and merocyanine adsorption bands, consistent with FRET as the quenching mechanism. Typically, complete quenching required at least 80 bound dye molecules per particle.     

Alkaline phosphatase assay using a near-infrared fluorescent substrate merocyanine 700 phosphate

Alkaline phosphatase (ALP) is a phosphomonoester hydrolase that is commonly used as a conjugating enzyme in biological research. A wide variety of substrates have been developed to assay its activity. In this study, we developed an ALP assay method utilizing merocyanine 700 (MC700) based substrate MC700 phosphate (MC700p). MC700 is a near-infrared fluorescent merocyanine dye, and has excitation/emission maxima at 686 nm/722 nm in ALP assay buffer. Upon hydrolysis by ALP, MC700p is converted to MC700. The fluorescence of MC700 is dependent on the pH and detergent concentration in the buffer. The fluorescence signal produced by MC700p hydrolysis is linearly related to the ALP amount and substrate concentration. A stop solution containing EDTA could be used to stop the ALP/MC700p reaction. It was also demonstrated that MC700p could substitute pNpp as the ALP substrate in a commercial 17β-Estradiol enzyme immunoassay kit.     

Fluorescent Dimers of Merocyanine 540 (MC540) in the Gel Phase of Phosphatidylcholine Liposomes

Abstract— Fluorescence emission from merocyanine 540 (MC540) dimers was observed in dipalmitoylphosphatidylcholine (DPPC) vesicles. This unusual behavior was observed only for vesicles in the gel-phase state. No dimer fluorescence was observed either in monopalmitoylphosphati-dylcholine (C₁₆PC) micelles or in liquid-crystalline DPPC vesicles, indicating that dimer fluorescence efficiency increases in highly packed interfaces. The excitonic theory of Kasha was used to interpret the spectral features. The overall fluorescence quantum yield (φ_r) decreases with decreasing lipid: probe ratio, not only because of the presence of a weakly fluorescent dimer that absorbs a high fraction of the total absorbed light but also due to quenching of monomer emission. This suggests the existence of probe domains. The dimer fluorescence quantum yields (φ_m) were estimated in DPPC large unilamellar vesicles (LUV) and DPPC multilamellar vesicles. The dependence of φ_r with probe concentration is compatible with values of φ_m lower than 0.05. The dimerization equilibrium of MC540 in C₁₆PC micelles and DPPC-LUV was also studied. Apparent dimerization equilibrium constants, K_dapp and dimer absorption spectrum were calculated in C₁₆PC micelles for the first time. The dimerization equilibrium constant in DPPC-LUV was calculated and discussed in terms of the fraction of volume occupied by the lipid phase.     

Photophysics of anthracene-indole systems in unilamellar vesicles of DMPC and POPC: Exciplex formation and temperature effects

The quenching of anthracene fluorescence by indole (IN), 1,2-dimethylindole (DMI), tryptophan (Trp) and indole 3-acetic acid (IAA) in dimiristoylphophatidylcholine (DMPC) and palmitoyloleoylphosphatidylcholine (POPC) lipid bilayers was investigated. The studies were carried out at 25 degrees C in POPC vesicles and below (15 degrees C) and above (35 degrees C) the phase transition temperature (24 degrees C) of DMPC. A very efficient quenching of the anthracene fluorescence by IN and DMI in the lipid membrane is observed in all cases. It is less efficient in the case of Trp and IAA. Stern-Volmer plots are linear for DMI but present a downward curvature for the other quenchers. This was interpreted as an indication of the presence of an inaccessible fraction of anthracene molecules. By a modified Stern-Volmer analysis the fraction accessible to the quenchers and the quenching constant were determined. Partition constants of the quenchers were obtained from the changes in the fluorescence emission of the indole moiety caused by the presence of the phospholipid. Using the partition constants bimolecular quenching rate constants were determined in terms of the local concentration of quencher in the lipid bilayer. These corrected rate constants are lower than those in homogeneous solvents. In the case of DMPC values the gel phase are higher than in the liquid-crystalline phase. In the quenching by IN and DMI a new, red shifted, emission band appears which could be assigned to an exciplex emission. The exciplex band is absent in the quenching by IAA and Trp.     

Fluorescence emission control and switching of oxymethylcrowned spirobenzopyrans by metal ion

Oxymethylcrowned spirobenzopyran 1 and pyrenylspirobenzopyran 2 were synthesized, and fluorescence emission of their corresponding merocyanine form was examined in the presence of metal ions. For 2, fluorescence emission derived from the pyrene moiety was completely quenched by photoinduced electron transfer (PET) of the nitrogen atom when the merocyanine form was not produced, namely, without metal ions. However, when 2 was converted to the merocyanine form by the complexation of its crown ether with a metal ion, fluorescence resonance energy transfer (FRET) from the pyrene to the merocyanine moieties took place to produce fluorescence emission. This result demonstrates that the spirobenzopyran isomerization can function as a fluorescence emission switch. Fluorescence quantum yield measurement for 1 and 2 showed that fluorescence emission depends on the binding metal ion in which the fluorescence quantum yield generally increased with the increase of metal ion radius.     

Manipulating FRET with polymeric vesicles: development of a "mix-and-detect" type fluorescence sensor for bacterial toxin

A simple "mix-and-detect" type of fluorescence sensor for cholera toxin (CT) is reported. The sensor consists of a BODIPY lipid dye and polydiacetylene (PDA) vesicles and utilizes the lipid insertion and FRET mechanism to offer a direct and fluorescence "turn-on" detection of the analyte. BODIPY conjugated GM1, dissolved in a Tris buffer through aggregate formation, demonstrated substantial fluorescence quenching with addition of PDA vesicle solution. The close proximity of the dye molecules to the conjugated chains as a result of lipid insertion enables energy transfer from dye to the polymer backbone, yielding the observed phenomenon. When CT is present, the binding of BO-GM1 to CT results in formation of a complex that prohibits it from membrane insertion, leading to the blocking of the quenching process. The fluorescence signal was found to be proportional to the CT concentration. The method is very simple and allows specific and sensitive detection of the protein toxin with just a few mixing steps. It can be further developed into a general sensing strategy for detection of other proteins with amplified FRET mechanism.     

Laser-induced fluorescence and hole-burning spectra of functional dyes in supersonic jets: spectra of a merocyanine dye

In LIF (laser-induced fluorescence) excitation and hole-burning spectra of a merocyanine dye, EtMD, in a supersonic jet, bands due to one of the four possible isomers were observed. Only lower-frequency vibronic bands were observed in the LIF-excitation spectra, while broad and deep dips were observed in the higher energy region of the hole-burning spectra where no LIF bands were observed. The LIF bands were found to consist of two transition systems, which are presumably due to the molecules in the ground and low-lying excited vibrational states. Possible relaxation processes of EtMD in the excited electronic state were discussed.     

Temperature-dependent vesicle formation of aqueous solutions of mixed cationic and anionic surfactants

The phase behavior of aqueous solutions of mixed cetyltrimethylammonium bromide (CTAB) and sodium octyl sulfate (SOS) was examined at different temperatures (20, 30, 40, and 50 degrees C). While stable vesicles were formed in a narrow composition range on the SOS-rich side at 20 degrees C, the range widened remarkably when the temperature was raised to 30 degrees C. Thus, the vesicle region extended to cover almost the entire composition range, CTAB:SOS = 0.5:9.5-5.0:5.0, at the total surfactant concentrations of 50-70 mM on the SOS-rich side. To analyze the temperature dependence of this phase behavior of the mixed surfactant system, DSC and fluorescence polarization measurements were performed on the system. The experimental findings obtained revealed that pseudo-double-tailed CTAB/SOS complex, the major component of the bimolecular membrane formed by the surfactant mixture, undergoes a gel (Lbeta)-liquid crystal (Lalpha) phase transition at about 26 degrees C. This phenomenon was interpreted as showing that the bimolecular membrane has no curvature and is rigid and easy to precipitate at temperatures below the phase transition point, whereas it has a curvature and is loose enough to disperse in the solution as vesicles at temperatures above the phase transition point. Vesicles formed by the anionic/cationic surfactant complex were then stable at temperatures above the phase transition temperature of the complex.     

囊泡型荧光纳米界面的构建及其对CD44蛋白的比例型传感

本文设计合成了两亲性Eu(Ⅲ)配合物(Eu L^3+)、两亲性香豆素衍生物(CA)以及荧光素修饰的透明质酸(HA-FA).Eu L^3+和CA可在水中共组装形成带正电荷的囊泡型荧光纳米界面(Eu L^3+/CA).HA-FA可通过静电引力络合在Eu L^3+/CA表面,促使CA与FA之间发生有效的荧光共振能量转移,体系的荧光发射以荧光素的绿色荧光为主.当肿瘤细胞标识物CD44蛋白与络合在囊泡表面上的透明质酸发生特异相互作用后,降低了CA与FA之间的能量转移效率,体系的荧光发射从绿色转变为蓝色.据此,实现了对CD44的高灵敏检测(DL=1.79×10^-7g/m L),而所测试的氨基酸、蛋白质等生物分子几乎不对荧光纳米界面的荧光性质产生影响.基于此,我们成功地将Eu L^3+/CA/HA-FA用于人乳腺癌细胞MCF-7和MDA-MB-231悬浮液中CD44蛋白的高效检测,该工作为构建新型CD44蛋白荧光探针提供了思路,为癌症早期诊断和治疗奠定了基础.     

Phospholipid bilayer free volume analysis employing the thermal ring-closing reaction of merocyanine molecular switches

The free volume properties of phospholipid bilayers have been determined using a new assay that applies the photochromic and solvatochromic properties of merocyanines. The orientation and embedding depth of the merocyanines in the bilayer are controlled using substitution on the merocyanine indole moiety. The free volume changes at the aqueous interface (region 1), the phospholipid headgroup (region 2), and the aliphatic interior (region 3) of the bilayer are compared by analyzing the rate constants for the merocyanine ring-closing reaction. Free volume variations during the P(beta)(')(gel) <--> L(alpha)(liquid) phase transition are observed in region 1, in accordance with large structural rearrangements between the gel and the liquid phases in this region. The largest free volume is found in region 3, and the smallest is found in region 2. This distribution of free volume in the bilayer agrees with computational studies of these systems. Comparison of the free volume in region 2 of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipids shows that this method is sensitive to small structural differences between lipids. In region 2, the free volume is found to be approximately 2 times larger in DPPC bilayers, which could be related to different merocyanine interactions with the two phosphatidylcholines. Free volume properties determined on picosecond and second time scales are compared based on an analysis of merocyanine formation and decoloration reactions.     

FLUIDITY AND OXYGEN PENETRATION OF LIPID VESICLES STUDIED BY FLUORESCENCE PROBES

Abstract. –An analysis of the temperature dependence of trans -stilbene fluorescence yield in dipalmitoyl lecithin vesicles is used to obtain activation energies. The results are interpreted in terms of bilayer fluidity through and above the phase transition. Oxygen quenching of the fluorescence of pyrenebutyric acid (incorporated in dipalmitoyl lecithin and egg lecithin vesicles) is reported as a function of temperature and bulk oxygen concentration. Above the bilayer phase transition, quenching rates (determined by oxygen quenching) decrease with decreasing temperature. A reduction in oxygen quenching is observed through the dipalmitoyl lecithin phase transition.     

Preparation and properties of vesicles from condensable amphiphilic amino acids

Three double‐chain amphiphiles with amino acid groups as hydrophilic moiety were synthesized. These amphiphiles can be easily dispersed in buffer solution to form transparent dispersion. Examination of the dispersion by transmission electron microscopy (TEM) showed the formation of stable vesicular aggregates, which was also confirmed by the ability to encapsulate water‐soluble dyes. Since amino acid groups are located on the surface of the vesicles, water‐soluble carbodiimide can induce the condensation of these groups to form peptide. The phase transition temperatures of these vesicles were estimated by differential scanning calorimetry (DSC), and a decrease of phase transition temperature was observed after polycondensation due to the disturbance of the ordered arrangement of the hydrophobic chains. The leakage rate of the vesicles before and after condensation was studied by monitoring the increase of fluorescence intensity of water‐soluble dye. These vesicles belong to the least permeable ones and the leakage rate can be controlled by varying the degree of condensation or the temperature.     

Fluorescence quenching of anthracene by indole derivatives in phospholipid bilayers

The quenching of anthracene fluorescence by indole, 1,2-dimethylindole (DMI), tryptophan (Trp) and indole 3-acetic acid (IAA) in palmitoyloleoylphosphatidylcholine (POPC) lipid bilayers was investigated. A very efficient quenching of the anthracene fluorescence in the lipid membrane is observed. Stern-Volmer plots are linear for DMI but present a downward curvature for the other quenchers. This was interpreted as an indication of the presence of an inaccessible fraction of anthracene molecules. By a modified Stern-Volmer analysis the fraction accessible to the quenchers and the quenching constant were determined. The changes in the fluorescence emission spectrum of indole and DMI have been used to calculate the partition constants of these probes into the membranes, and bimolecular quenching rate constants were determined in terms of the local concentration of quencher in the lipid bilayer. The rate constants are lower than those in homogeneous solvents, which may be ascribed to a higher viscosity of the bilayer. No changes in the emission spectra of Trp and IAA are observed in the presence of vesicles, indicating that these probes locate preferentially in the aqueous phase, or in close proximity to the vesicular external interface in a medium resembling pure water. In these cases quenching rate constants were determined in terms of the analytical concentration. In the quenching by DMI a new, red shifted, emission band appears; it is similar to that observed in non-polar solvents and it is ascribable to an exciplex emission. The exciplex band is absent in the quenching by IAA and Trp and only very weakly present when the quencher is indole. From the position of the maximum of the exciplex emission, a relatively high local polarity could be estimated for the region of the bilayer where the quenching reaction takes place.     

ROLE OF MEROCYANINE DYE AS A PROTON PUMP IN PHOTOVOLTAGE GENERATION

A novel photoelectrochemical cell using a proton pump mechanism in the aggregated planar structure of oxidised cholesterol incorporating merocyanine dyes is reported. Lipid dye binding, as verified from spectral studies and photoisomerisation of the dye, is responsible for this photovoltage generation whose magnitude and storage duration are related to the equilibrium constant of dye-lipid binding through an empirical formula.     

Effect of pressure on the Prodan fluorescence in bilayer membranes of phospholipids with varying acyl chain lengths

The fluorescence spectra of 6-propionyl-2-(dimethylamino)naphthalene (Prodan) were observed as a function of pressure for the bilayer membrane systems of dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC). The wavelength of the emission maximum, lambdamax, was found to be 480, 430, and 500 nm for the liquid crystalline (Lalpha), ripple gel (P'beta), and pressure-induced interdigitated gel (LbetaI) phase, respectively. Since the lambdamax reflects the solvent property around the probe molecules, we could speculate on the location of the Prodan molecules in the bilayer membranes; in the Lalpha phase of the lipid bilayer, the Prodan molecules distribute around the phosphate of the lipids (i.e. the polar region). The Lalpha/P'beta phase transition caused the Prodan molecules to move into the less polar region near the glycerol backbone. The fluorescence intensity of the Prodan in the P'beta phase was dependent on the chain length of the lipids and on pressure; the shorter the chain length of the lipid, the stronger the fluorescence intensity of the Prodan. Moreover, for the DLPC bilayer membrane system, the fluorescence intensity at 430 nm increased with increasing pressure, indicating that the partition of Prodan into the DLPC bilayer membrane is promoted by applying pressure. In the case of the DPPC and DSPC bilayers, as the pressure increased further, the pressure-induced interdigitation caused the Prodan molecules to squeeze out of the glycerol backbone region and to move the hydrophilic region near the bilayer surface. The ratio of fluorescence intensity at 480 nm to that at 430 nm, F480/F430, showed a sharp change at the phase-transition pressure. In the case of the DPPC and DSPC bilayers, the values of F480/F430 showed an abrupt increase above a certain pressure higher than the Lalpha/P'beta transition pressure, which corresponds to the interdigitation from the P'beta to the LbetaI phase. The plot of F480/F430 versus pressure is available for recognition of the bilayer phase transitions, especially the bilayer interdigitation.     

Partition and location of nimesulide in EPC liposomes: a spectrophotometric and fluorescence study

Study of the mechanism of action of anti-inflammatory drugs (NSAIDs) and their side-effects may fall in the domain of membranology. In this work the extent of the interaction between an NSAID (nimesulide) and membrane phospholipids was quantified by the partition coefficient, K_p , using egg phosphatidylcholine (EPC) liposomes as cell membrane models. The liposome/aqueous phase partition coefficients were determined under physiological conditions, by derivative spectrophotometry and fluorescence quenching. Derivative spectrophotometry allows elimination of background signal effects (light scattering) due to the presence of liposomes. Theoretical models, accounting for simple partition of the NSAID between two different media, were used to fit the experimental data, allowing the determination of K_p in multilamellar vesicles (MLVs) and large unilamellar vesicles (LUVs). The location of nimesulide in MLVs and LUVs was evaluated by fluorescence quenching using spectroscopic probes located at different sites on the membrane. All n-AS probes were quenched and the relative quenching efficiencies were ordered as 2-AS<6-AS9-AS<12-AS; this suggests the drug is deeply buried in the membrane. Fluorescence quenching using the 12-AS probe was also used to determine the partition coefficient of the drug in MLVs and LUVs. The two techniques yield similar results. Finally, measurement of zeta-potential in the presence of different concentrations of nimesulide was performed to investigate possible changes in the zwitterionic phosphatidylcholine membranes. The membrane surface potential was not altered, which seems to be an indication that nimesulide binds to lipid bilayer mostly by hydrophobic interactions.     

A nanoscale vesicular polydiacetylene sensor for organic amines by fluorescence recovery

We report a nanoscale lipid membrane-based sensor of conjugated polydiacetylene (PDA) vesicles for fluorescence detection of organic amines. The vesicle sensor was constructed by incorporation of a BODIPY fluorescent dye into the PDA vesicles. The fluorescent properties of the resulting vesicles can be manipulated by adjusting lipid components, and are controlled by environmental and solution conditions. The fluorescence of the BODIPY dye was significantly quenched in the polymerization of diacetylene lipid vesicles by a UV irradiation process. However, it was sufficiently recovered by external stimuli such as a hike of solution pH. The fluorescence recovery process was reversible, and a decrease in solution pH resulted in repeated quenching. The reported system transforms an external stimulus into a large fluorescence intensity change, demonstrating great potential in developing new signal reporting method for biosensor design. The quench-recovery phenomenon of the BODIPY-PDA is believed to be related to the energy transfer between the dye and the PDA conjugate backbone. The vesicle sensor was applied for detecting an organic amine, triethylamine (TEA) and a large linear relationship was obtained between the increase in fluorescence intensity and the concentrations of TEA. The detection limit of TEA by vesicle sensors using fluorescence recovery was found to be 10 μM.     

Microstructure, morphology, and ultrafast dynamics of a novel edible microemulsion

An edible microemulsion (ME) composed of Tween 80/butyl lactate/isopropyl myristate (IPM)/water has been formulated. Pseudoternary phase diagram of the system contains a large single isotropic region. The phase behavior of the system is also studied at low pH (2.6) and in 0.9% NaCl solution. Conductivity, viscosity, ultrasonic velocity, and compressibility studies find consistent results in the structural transition (from water-in-oil (w/o) to bicontinuous, and from bicontinuous to oil-in-water (o/w)) behavior of the ME. Dynamic light scattering studies reveal the size of the MEs. The absorption and steady state emission spectra of 4-(dicyanomethylene)-2-methyl-6-(p-dimethylamino-styryl)-4H-pyran (DCM) successfully probe the polarity of the ME at its solvation shell and shows the efficacy of hosting model drug molecules. The rotational anisotropy of the dye has been studied to ascertain the geometrical restriction of the probe molecule. Picosecond-resolved fluorescence spectroscopy applies well to study the relaxation dynamics of water in the solvation shell of the MEs. The study finds strong correlation in the relaxation dynamics of water with the structure of host assembly and offers an edible ME system which could act as a potential drug delivery system and nontoxic nanotemplate for other applications.     

Efficacious Anticancer Drug Delivery Mediated by a pH‐Sensitive Self‐Assembly of a Conserved Tripeptide Derived from Tyrosine Kinase NGF Receptor

We present herein a short tripeptide sequence (Lys–Phe–Gly or KFG) that is situated in the juxtamembrane region of the tyrosine kinase nerve growth factor (Trk NGF) receptors. KFG self‐assembles in water and shows a reversible and concentration‐dependent switching of nanostructures from nanospheres (vesicles) to nanotubes, as evidenced by dynamic light scattering, transmission electron microscopy, and atomic force microscopy. The morphology change was associated with a transition in the secondary structure. The tripeptide vesicles have inner aqueous compartments and are stable at pH 7.4 but rupture rapidly at pH≈6. The pH‐sensitive response of the vesicles was exploited for the delivery of a chemotherapeutic anticancer drug, doxorubicin, which resulted in enhanced cytotoxicity for both drug‐sensitive and drug‐resistant cells. Efficient intracellular release of the drug was confirmed by fluorescence‐activated cell sorting analysis, fluorescence microscopy, and confocal microscopy.