Continuous-flow protease assay based on fluorescence resonance energy transfer

A homogeneous continuous-flow assay using fluorescence resonance energy transfer (FRET) for detection was developed to measure the hydrolysis of HIV Protease Substrate 1 (to which two choromophores, EDANS and DABCYL are covalently attached) by a protease (e.g. Subtilisin Carlsberg) and the influence of inhibitors. In the continuous-flow assay, an inhibitor solution and an enzyme solution were first eluted into the system and allowed to react with each other in a reaction coil. Subsequently, the substrate solution was added to an enzyme-inhibitor mixture in a second reaction coil and incubated for 1 min. Finally, the fluorescence intensity was monitored.The system was also utilized to measure the inhibition of the protease by two weak acidity inhibitors which are 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) and ethylenediaminetetraacetic acid (EDTA). Using the obtained optimum conditions for AEBSF, a detection limit of 0.3 mmol/l was achieved and the relative standard deviation was below 3.7% in the 2.5-7.5 mmol/l range. For EDTA, which required a 20 times higher substrate concentration than AEBSF, a detection limit of 0.2 mmol/l was obtained and the relative standard deviation was below 9.6% in the 0.5-7.5 mmol/l range.The optimization of pH, substrate concentration, enzyme concentration, reaction time and temperature are described. Organic modifier effects were also investigated. Methanol, acetonitrile and DMSO could be tolerated up to 30%.     

Two-photon fluorescence excitation using an integrated optical microcavity: a promising tool for biosensing of natural chromophores

Application of an integrated optics (IO) microcavity (MC) for evanescent excitation of two-photon excited fluorescence (TPF) is demonstrated. The MC provides a high local intensity, which is required for the TPF, because of resonant enhancement of the intracavity power and a strong two-dimensional confinement of the guided mode. Numerical estimations show a large increase, by more than a factor of 10⁴ of the TPF intensity at the MC compared to a conventional straight waveguide. This will lead to a significant improvement of the detection limits of UV-absorbing chromophores (down to 10⁻⁸ M) when using the MC as a biosensor. Feasibility of TPF excitation using an IO MC is confirmed experimentally for the first time.     

Electro‐optical switches based on polymer and dendrimer filled nematics

Two‐phase systems which can be switched electrically between a light scattering and a transparent state can be prepared based on either a dispersion of well‐defined sub‐micron sized crosslinked polymeric particles in a liquid crystal (LC) matrix (Polymer Filled Nematics, PFN's) or on a dispersion of palmitoyl‐functionalised poly(propylene imine) dendrimers in an LC (Dendrimer Filled Nematics). The present paper describes the preparation of both systems and their properties. The PFN's can be electrically switched between a scattering and a transparent state by an appropriate choice of materials and refractive indices. The preparation of the disperse polymeric phase is separated from the preparation of the polymer/LC blend, which enables control over the morphology of the system. Rather surprisingly, it is found that, due to the rheological properties of the PFN blends, stable films of the blends can be simply produced by conventional coating processes. The dendrimer‐stabilised nematics require extremely low switching voltages to switch from a scattering to a highly transparent state. Moreover, the switching process is totally reversible and hysteresis effects appear to be absent.     

Enantioselective room temperature phosphorescence detection of non-phosphorescent analytes based on interaction with beta-cyclodextrin/1-bromonaphthalene complexes

Menthol (MT) induces strong room temperature phosphorescence (RTP) of 1-bromonaphthalene (1BrN) in aqueous β-cyclodextrin (β-CD) suspensions, even under non-deoxygenated conditions. Interestingly, (−)-MT and (+)-MT enantiomers give rise to different phosphorescence intensities, the difference being 19 ± 3%. It is argued that the signal can be mainly ascribed to the formation of ternary complexes β-CD/1BrN/MT which show different RTP lifetimes, i.e. 4.28 ± 0.06 and 3.71 ± 0.06 ms for (−)-MT and (+)-MT, respectively. Most probably, the stereochemical structure of (−)-MT provides a better protection of 1BrN against quenching by oxygen than (+)-MT. This interpretation is in line with the observation that under deoxygenated conditions the phosphorescence intensity difference for the two complexes becomes very small, i.e. only about 4%.The lifetime difference under aerated conditions enables the direct determination of the MT stereochemistry. For mixtures, in view of the 0.06 ms uncertainty in the lifetime, enantiomeric purity can be determined down to 10%. Furthermore, in the case of MT the concentration of the least abundant enantiomer should be at least 3 × 10⁻⁴ M, since otherwise complex dissociation would obscure the lifetime difference.     

Fast excited-state intramolecular proton transfer and subnanosecond dynamic stokes shift of time-resolved fluorescence spectra of the 5-methoxysalicylic acid/diethyl ether complex

Excited-state intramolecular proton transfer (ESIPT) occurring in the salicylic acid (SA) derivative 5-methoxysalicylic acid (5-MeOSA) in an apolar solvent (cyclohexane) and in the presence of the hydrogen bond accepting agent diethyl ether (DEE) is investigated. Analysis of the directly measured subnanosecond time-resolved emission spectra (TRES) together with conventional steady-state fluorescence and time-correlated single-photon-counting (TCSPC) decays indicates that ESIPT in this system occurs much faster than fluorescence, and that the equilibrium between normal and tautomeric excited states is established before the emission from both states takes place. However, changes in time- and frequency-resolved fluorescence of the 5-MeOSA/DEE complex are observed due to structural relaxation within the complex, which is reflected in the dynamic Stokes shift of the tautomeric fluorescence band. The normal fluorescence band of 5-MeOSA/DEE does not exhibit marked changes within the investigated time range. A single-exponential relaxation time of 460 ps was determined for the dynamic Stokes shift of the tautomeric band, and it is attributed to a geometric change within the 5-MeOSA/DEE complex upon excitation. Since both tautomeric and normal emission bands are well resolved and exhibit different time-dependent behaviors, a double-well potential appears to be adequate to describe the excited state of the system studied.     

Pre-organization induced synthesis of a crossed alkene-bridged nisin Z DE-ring mimic by ring-closing metathesis

An alkene-bridged mimic of the complex DE-bisthioether-ring system of the antibiotic nisin was prepared in one step from the linear precursor.     

Thermochemical behavior of mixtures ofN,N-dimethylformamide with dimethylsulfoxide,acetonitrile, andN-methylformamide: Volumes and heat capacities

Densities and molar heat capacities have been measured for mixtures ofN,N-dimethylformamide with dimethylsulfoxide, acetonitrile, andN-methylformamide at 25°C over the complete mole fraction range. From these data the apparent molar volumes and heat capacities have been calculated for both components. These quantities, as a function of the mole fraction, deviate very little from their molar values, indicating that the mixtures can be regarded as almost ideal.