Time-resolved total internal reflection fluorescence study on hybridization of complementary single-stranded DNAs at a water/oil interface

Hybridization of complementary single-stranded DNAs (ssDNA) at a water/CCl4 interface was studied on the basis of picosecond total internal reflection fluorescence spectroscopy. Complementary ssDNAs dissolved in water were shown to produce the relevant double-stranded DNA (dsDNA) at a water/CCl4 interface in the presence of octadecylamine (ODA) in the oil phase, while hybridization between ssDNAs did not proceed in the water phase, as demonstrated by the fluorescence dynamics of ethidium bromide as a probe for the DNA structure. The structures of dsDNA and the roles of ODA in hybridization of ssDNA at the interface were discussed.     

Time-resolved total internal reflection fluorometry study on polarity at a liquid/liquid interface

The polarity of a water/oil (oil: cyclohexane, carbon tetrachloride, toluene, chlorobenzene, o-dichlorobenzene, or 1,2-dichloroethane) interface was investigated by means of time-resolved total-internal-reflection (TIR) fluorescence spectroscopy of a polarity-sensitive probe: sulforhodamine B (SRB). In bulk solutions, the nonradiative decay rate constant of SRB increased with an increase in a solvent polarity parameter [ET(30)], and this relationship was used to estimate the polarities of water/oil interfaces. For the oil having a relatively low solvent polarity [ET(30) < 35 kcal/mol], the polarity of the water/ oil interface agreed with that of the arithmetic average of the polarities of the two phases [ET(30)calc]. For water/odichlorobenzene and water/1,2-dichloroethane interfaces [ET(30) of the oil > 35 kcal/mol], on the other hand, the interfacial polarity determined by TIR spectroscopy was lower than the ET(30)calc. The results are discussed in terms of thickness/roughness of the water/oil interface.     

A Comparative Study of Rotational Relaxations of an Eosin-protein Complex Using Delayed Fluorescence and Phosphorescence

Abstract

The slow rotational diffusion of a macromolecule bovine serum albumin in solution has been investigated by measuring the decay of dichroism in laser-pulse induced transient emissions from triplet probes covalently bound to the protein. A comparison of the anisotropy parameters of phosphorescence and delayed fluorescence from eosin-bound bovine serum albumin is described. The results indicate that the measurement of delayed fluorescence is often the preferable option for investigating the rotational diffusion of the eosin-bound macromolecules.     

Electrically controlled fluorescence in a nematic liquid crystal doped by a chiral fluorophore

We have discovered a strong electric field sensitivity of the fluorescence intensity while studying the diffusion processes of a chiral fluorescent molecule (CFM) in a nematic liquid crystal (NLC) host. The experimental and theoretical study of this phenomenon indicates that the alignment of the CFM along two privileged orientation directions (with asymmetric distribution with respect to the NLC’s optical axis) is in the origin of this phenomenon. As a result, the obtained guest–host system demonstrates noticeable dichroism. Thus, the application of an electric field allows the reorientation of the anisotropy axis of the host, the change of CFM’s absorption (at the wavelength of excitation) and the dynamic electric control of its fluorescence intensity. The study of these phenomena allows also the identification of the angular distribution of guest CFMs suggesting that the elastic energy of orientation of the host molecules might be in the origin of the asymmetric angular distribution of CFM.     

Excitation energy migration processes in various multi-porphyrin assemblies

The electronic interactions and excitation energy transfer (EET) processes of a variety of multi-porphyrin arrays with linear, cyclic and box architectures have been explored. Directly meso-meso linked linear arrays (Z(N)) exhibit strong excitonic coupling with an exciton coherence length of approximately 6 porphyrin units, while fused linear arrays (T(N)) exhibit extensive π-conjugation over the whole array. The excitonic coherence length in directly linked cyclic porphyrin rings (CZ(N)) was determined to be approximately 2.7 porphyrin units by simultaneous analysis of fluorescence intensities and lifetimes at the single-molecule level. By performing transient absorption (TA) and TA anisotropy decay measurements, the EET rates in m-phenylene linked cyclic porphyrin wheels C12ZA and C24ZB were determined to be 4 and 36 ps(-1), respectively. With increasing the size of C(N)ZA, the EET efficiencies decrease owing to the structural distortions that produce considerable non-radiative decay pathways. Finally, the EET rates of self-assembled porphyrin boxes consisting of directly linked diporphyrins, B1A, B2A and B3A, are 48, 98 and 361 ps(-1), respectively. The EET rates of porphyrin boxes consisting of alkynylene-bridged diporphyrins, B2B and B4B, depend on the conformation of building blocks (planar or orthogonal) rather than the length of alkynylene linkers.     

Chemical selectivity of self-assembled monolayers of calix[4]resorcinarene

Selective molecular interactions at an interface formed by self-assembly of a macrocyclic synthetic host, calix[4]resorcinarene with four thiol groups (R4SH), are investigated. The recognition of guest adsorbates from aqueous solutions is monitored using surface plasmon resonance (SPR) and the orientation of the guest-molecule is probed using polarization modulation infrared absorption spectroscopy (PM-IRRAS). The experiments reported here demonstrate that the chemical selectivity of self-assembled monolayers (SAMs) of host molecules such as calix[4]resorcinarenes extends to isomers of several different guest molecules. By using structural isomers of guest molecules such as bipyridine and nitrophenol that are multidentate hydrogen bond acceptors, it is shown that geometric match between guest and host molecules is an integral aspect of the recognition phenomena. Results from SPR and PM-IRRAS experiments reported here highlight the interplay between steric size and forces such as hydrogen bonding and hydrophobic interactions. Competitive and sequential adsorption of guest molecules such as -hydroxy-γ-butyrolactone and 4,4′-bipyridine shows that these guests compete for the same binding sites on the surface and that the interplay between steric size and molecular forces underlies the preferential selectivity of one guest molecule over another.     

High-pressure total internal reflection fluorescence apparatus

Important interfacial processes in disciplines ranging from medicine to the separations sciences occur over a wide range of pressures, temperatures, and time scales. In this paper we report a new high-pressure total internal reflection fluorescence (HP-TIRF) apparatus that allows rapid fluorescence measurements of sub-monolayers in contact with liquids and supercritical fluids between 293 K and 353 K and up to 250 bar with picosecond time resolution. We use the HP-TIRF system to study the in-plane rotational reorientation dynamics of the fluorescent probe BODIPY 494/503 (C(2v) symmetry) covalently attached to silica surfaces that have been silanized with n-propyltrimethoxysilane (C(3)-TMOS) or 3,3,3-trifluoropropyltrimethoxysilane (CF(3)-TMOS) when the interface is subjected to pure supercritical carbon dioxide (scCO(2)). The in-plane BODIPY 494/503 rotational reorientation dynamics are assessed by using the Debye-Stokes-Einstein expression. As the scCO(2) density increases the local microviscosity surrounding the tethered BODIPY 494/503 molecule decreases. The terminal group (CH(3) versus CF(3)) within the silane monolayer governs the onset and absolute magnitude of the observed viscosity changes. The results are explained in terms of the wellknown solubility of fluorine-containing species in scCO(2).     

Development of a total internal reflection ultrafast transient lens method for studying molecular dynamics on an interface

We have developed the total internal reflection ultrafast transient lens (TIR-UTL) method to detect nonradiative chemical processes at interfaces and surfaces with subpicosecond time resolution. In the TIR-UTL measurements, the evanescent field of a pump beam irradiated under the TIR condition generates a refractive index change. The refractive index change is attributed to changes of the molecular electronic state, of density by molecular orientation/structure change, and of temperature by vibrational relaxation processes. The refractive index change is detected as a change of the power intensity of the probe beam adjusted coaxially with the pump beam. At first, we discuss a theoretical principle of a coaxial configuration in the TIR-UTL measurement. This configuration has an advantage of versatility over the established TIR configuration. Then, we evaluate time resolution of TIR-UTL and obtain a value of less than 400 fs. We measure the ultrafast molecular dynamics of the cationic chromophore Auramine O (AuO) at a silica/water interface. Two slow time constants originating from AuO adsorbed on the silica surface are detected by TIR-UTL. These are attributed to AuO, whose twisting motion is strongly hindered by adsorption on a silica surface.     

Role of the V/III precursor ratio on exciton dynamics in GaN MOCVD epilayers

Time-resolved photoluminescence spectroscopy is performed on strained epilayers of GaN, grown by MOVPE on Al₂O₃ substrates. As the V/III precursor ratio R is increased, the decay time of the donor–accepter pair (DAP) recombinations at 3.22 and 3.29 eV is changed from 1 ns to much more than 20 ns. Coherently with this change, the X_A and X_B free excitons and a donor-bound exciton D^oX lying 5.7 meV below X_A are subject to significant variations. For R8000, DAP transitions are fast and the D^oX recombination dominates the spectrum at T=8 K, with decay times of 35–45 ps, while free excitons decay on a 25 ps time scale. For R10000, the DAP transitions are very slow and all excitonic recombinations become faster: times of up to 280 ps are obtained for D^oX and 35 ps for the X_A line, which now dominates the time-integrated spectrum. Exciton dynamics in GaN epilayers can thus be controlled by the V/III precursor ratio, which affects both the density of vacancies and extrinsic impurity incorporation.     

Near IR scanning angle total internal reflection Raman spectroscopy at smooth gold films

Total internal reflection (TIR) Raman and reflectivity spectra were collected for nonresonant analytes as a function of incident angle at sapphire or sapphire/smooth 50 nm gold interfaces using 785 nm excitation. For both interfaces, the Raman signal as a function of incident angle is well-modeled by the calculated interfacial mean square electric field (MSEF) relative to the incident field times the thickness of the layer being probed in the Raman measurement (D(RS)). The Raman scatter was reproducibly enhanced at the interface containing a gold film relative to the sapphire interface by a factor of 4.3-4.6 for aqueous pyridine or 2.2-3.7 for neat nitrobenzene, depending on the analyzed vibrational mode. The mechanism for the increased Raman signal is the enhanced MSEF at incident angles where propagating surface plasmons are excited in the metal film. The background from the TIR prism was reduced by 89-95% with the addition of the gold film, and the percent relative uncertainty in peak area was reduced from 15 to 1.7% for the 1347 cm(-1) mode of nitrobenzene. Single monolayers of benzenethiol (S/N = 6.8) and 4-mercaptopyridine (S/N = 16.5) on gold films were measured by TIR Raman spectroscopy with 785 nm excitation (210 mW) without resonant enhancement in 1 min.     

Ultrafast spin dynamics in colloidal ZnO quantum dots

Time-resolved Faraday rotation measurements in the ultraviolet have been performed to reveal the ultrafast spin dynamics of electrons in colloidal ZnO quantum dots. Oscillating Faraday rotation signals are detected at frequencies corresponding to an effective g factor of g = 1.96. Biexponential oscillation decay is observed that is due to (i) rapid depopulation of the fundamental exciton (tau = 250 ps) and (ii) slow electron spin dephasing ( T 2 = 1.2 ns) within a metastable state formed by hole-trapping at the quantum dot surface.     

Specific recognition and formation of two- dimensional streptavidin domains in monolayers: applications to molecular devices

By virtue of the high-affinity specific interaction between the vitamin, biotin, and the protein, streptavidin, monolayers of synthetic lipids with biotin headgroups can tightly bind streptavidin at the lipid-water interface. Through this specific recognition fluorescently-labelled streptavidin spontaneously organizes in the plane of the interface to form large protein domains, directly visible in situ by fluorescence microscopy and exhibiting optical anisotropy. Further structural characterization has shown that these domains are two-dimensional protein crystals. Correlation with the known three-dimensional crystal structure of streptavidin indicates that two of streptavidin's four biotin binding sites are free for further derivatization to create multilayered organized protein molecular devices.     

Time-resolved surface-enhanced ellipsometric contrast imaging for label-free analysis of biomolecular recognition reactions on glycolipid domains

We have applied surface-enhanced ellipsometry contrast (SEEC) imaging for time-resolved label-free visualization of biomolecular recognition events on spatially heterogeneous supported lipid bilayers (SLB). Using a conventional inverted microscope equipped with total internal reflection (TIR) illumination, biomolecular binding events were monitored with a lateral resolution near the optical diffraction limit at an acquisition rate of ～1 Hz with a sensitivity in terms of surface coverage of ～1 ng/cm(2). Despite the significant improvement in spatial resolution compared to alternative label-free surface-based imaging technologies, the sensitivity remains competitive with surface plasmon resonance (SPR) imaging and imaging ellipsometry. The potential of the technique to discriminate local differences in protein binding kinetics was demonstrated by time-resolved imaging of anti-GalCer antibodies binding to phase-separated lipid bilayers consisting of phosphatidylcholine (POPC) and galactosylceramide (GalCer). A higher antibody binding capacity was observed on the GalCer-diluted fluid region in comparison to the GalCer-rich gel phase domains. This observation is tentatively attributed to differences in the presentation of the GalCer epitope in the two phases, resulting in differences in availability of the ligand for antibody binding. The complementary information obtained by swiftly switching between SEEC and fluorescence (including TIR fluorescence) imaging modes was used to support the data interpretation. The simplicity and generic applicability of the concept is discussed in terms of microfluidic applications.     

Radiative exciton recombination dynamics in QD-tagged polystyrene microspheres

Fluorescent polystyrene microspheres are prepared by the incorporation of fluorescent CdSe/CdS core/shell semiconductor nanocrystals (quantum dots, QDs) using the emulsification/solvent evaporation method. The radiative exciton recombination dynamics is investigated by nanosecond time-resolved fluorescence spectroscopy at ambient conditions. The time constants of fast and slow fluorescence decay in QDs, dispersed in toluene, were 3.5 and 17.8 ns, respectively. For the QD-tagged microspheres, the time constants of fast and slow processes were ~2–3 and ~11–12 ns, respectively, and did not depend significantly on the QD-content of the microspheres. The fast decay component could be attributed to the recombination of delocalized exciton in the internal core states, and the slow component was attributed to the localized exciton in the surface states. It was found that the ratio of amplitudes of the fast and slow processes also changed after incorporation of QDs in microspheres. The observed differences in fluorescence decay between non-entrapped QDs and QD-tagged microspheres were probably due to energy transfer between the nanocrystals, which were in close proximity inside the microspheres. The obtained fluorescent QD-tagged microspheres are characterized by the other methods as well, which makes them of value for various applications as optical materials.     

Interface and temperature dependent magnetic properties in permalloy thin films and tunnel junction structures

Magnetization dynamics and field dependent magnetization of different devices based on 25-30 nm thick Permalloy (Py) films: such as single Py layers (Py/MgO; Py/CoFeB/Al2O3) and Py inserted as a magnetic layer in magnetic tunnel junctions (Py/CoFe/Al2O3/CoFe; Py/CoFeB/Al2O3/CoFe; Py/MgO/Fe) have been extensively studied within a temperature range between 300 K down to 5 K. The dynamic response was investigated in the linear regime measuring the ferromagnetic resonance response of the Py layers using broadband vector network analyzer technique. Both the static and the dynamic properties suggest the possible presence of a thermally induced spin reorientation transition in the Py interface at temperatures around 60 K in all the samples investigated. It seems, however, that the details of the interface between Py and the hardening ferromagnet/insulator structure, the atomic structure of Py layers (amorphous vs. textured) as well as the presence of dipolar coupling through the insulating barrier in the magnetic tunnel junction structures could strongly influence this low temperature reorientation transition. Our conclusions are indirectly supported by structural characterization of the samples by means of X-Ray diffraction and high resolution transmission electron microscopy techniques. Micromagnetic simulations indicate the possibility of strongly enhanced surface anisotropy in thin Py films over CoFe or CoFeB underlayers. Comparison of the simulations with experimental results also shows that the thermally-induced spin reorientation transition could be influenced by the presence of strong disorder at the surface.     

Effect of host–guest interactions on the photophysical properties of a monocrown ether substituted phthalocyanine

The photophysical properties of supramolecular systems consisting of a monocrown ether substituted phthalocyanine CRPc (1) and potassium salts of p-nitrobenzoic acid (2), picric acid (3) and porphyrin monocarboxylic acid (4) are described. The fluorescence from CRPc is quenched by complexation with (2), (3) and (4) giving downward curving Stern–Volmer plots. The quenching constants and the fraction of CRPc accessible to quenching are calculated using the modified Stern–Volmer equation. Times-resolved fluorescence measurements for the systems show biexponential decay profiles. The lifetimes for long-lived and short-lived components determined for the each system remain constant. The results show that host–guest complexation not only are important in the supramolecular preorganization of donor–acceptor dyads based on noncovalent interaction but also may directly mediate electron transfer process.     

Total internal reflected resonance light scattering determination of chlortetracycline in body fluid with the complex cation of chlortetracycline-europium-trioctyl phosphine oxide at the water/tetrachloromethane interface

A highly selective method of chlortetracycline (CTC) is proposed on the basis of the measurements of total internal reflected resonance light scattering (TMR-RLS) at water/tetrachloromethane (H20/CCl4) interfaces. In the pH range of 7.54-8.14, the interaction of the binary complex of Eu(III)/CTC in the presence of trioctyl phosphine oxide (TOPO) occurs at the H20/CCl4 interface, resulting in greatly enhanced TIR-RLS signals with the maximum peak located at 340 nm. The enhanced TIR-RLS intensity is in proportion to the CTC concentration in the range 0.98 to approximately 20.0 x 10(-7) mol/L. The limit of detection is 9.8 x 10(-9) mol/L. Synthetic samples and body fluid samples including human urine, human serum, and fresh milk were determined with the recovery of 95.4-106.4% and RSD of 2.9-3.9%.     

Fluorescence lifetimes and correlated photon statistics from single CdSe/oligo(phenylene vinylene) composite nanostructures

We present measurements of fluorescence intensity trajectories and associated excited-state decay times from individual CdSe/oligo(phenylene vinylene) (CdSe-OPV) quantum dot nanostructures using time-tagged, time-resolved (TTTR) photon counting techniques. We find that fluorescence decay times for the quantum dot emitter in these composite systems are at least an order of magnitude shorter than ZnS-capped CdSe quantum dot systems. We show that both the blinking suppression and associated lifetime/count rate behavior can be described by a modified version of the diffusive reaction coordinate model which couples slow fluctuations in quantum dot electron (1Se, 1Pe) energies to Auger-assisted hole trapping processes, hence modifying both blinking statistics and excited-state decay rates.     

Fast-gated intensified charge-coupled device camera to record time-resolved fluorescence spectra of tryptophan

The possibilities of a 200 ps gated intensified charge-coupled device (CCD) camera to record time-resolved fluorescence were explored using the fluorescing amino acid tryptophan and its derivative Nacetyl-tryptophan amide (NATA) as model compounds. The results were compared to complementary data from time-correlated single-photon counting (TCSPC) experiments. If a spectral resolution of 1-2 nm is desired, the fast-gated intensified CCD (ICCD) camera is the method of choice. For a 10(-5) M tryptophan solution, time-resolved emission spectra and intensity decays (measured over 12 ns at 25 ps resolution) could be obtained in typically 10 minutes, giving the well-known lifetimes of 0.5 and 3 ns. In addition, a longer lifetime of 7 ns was found at the red edge of the spectrum. The very short gate time of the ICCD camera allowed us to observe a shift in the emission maximum of tryptophan even within the first nanosecond of decay of the fluorescence emission. As expected from the tryptophan rotamer model, such a shift is not observed in NATA. Using amplitudes obtained by global analysis, decay-associated spectra of these lifetimes were constructed.     

Characterization of chiral interactions using fluorescence anisotropy

This work details a study whereby the characterization of chiral selectors and identification of optimal separation conditions is evaluated by steady-state fluorescence anisotropy measurements. Earlier studies in our laboratory have shown fluorescence anisotropy to be an effective tool in evaluating chiral recognition, and in this study, the feasibility of characterizing chiral separation systems by the technique is evaluated. Four chiral selectors were examined under various conditions to explore correlation between chiral separation ability and differences in the steady-state fluorescence anisotropy of the enantiomers measured under similar conditions. A good correlation between the fluorescence anisotropy data and separation data was observed with R2 values ranging from 0.9279 to 0.9959. The fluorescence anisotropy measurements were examined under conditions that mimicked chiral separation conditions and the feasibility of a priori optimization of chiral separations is discussed.