Conjugatable water-soluble Pt(II) and Pd(II) porphyrin complexes: novel nano- and molecular probes for optical oxygen tension measurement in tissue engineering.

Measurement of oxygen tension in compressed collagen sheets was performed using matrix-embedded optical oxygen sensors based on platinum(II) and palladium(II) porphyrins supported on polyacrylamide nanoparticles. Bespoke, fully water-soluble, mono-functionalised Pt(II) and Pd(II) porphyrin complexes designed for conjugation under mild conditions were obtained using microwave-assisted metallation. The new sensors display a linear response (1/τ vs. O2) to varying oxygen tension over a biologically relevant range (7.0 × 10(-4) to 2.7 × 10(-1) mM) in aqueous solutions; a behaviour that is maintained following conjugation to polyacrylamide nanoparticles, and following embedding of the nanosensors in compressed collagen sheets, paving the way to innovative approaches for real-time resolution of oxygen gradients throughout 3D matrices useful for tissue regeneration.

Orthogonally bifunctionalised polyacrylamide nanoparticles: a support for the assembly of multifunctional nanodevices.

Polyacrylamide nanoparticles bearing two orthogonal reactive functionalities were prepared by reverse microemulsion polymerisation. Water-soluble photosensitisers and peptide or carbohydrate moieties were sequentially attached to the new nanospecies by orthogonal conjugations based on copper-catalysed azide-alkyne cycloaddition and isothiocyanate chemistry.

Quantum dots as enhancers of the efficacy of bacterial lethal photosensitization.

Because of the increasing resistance of bacteria to antibiotics there is considerable interest in light-activated antimicrobial agents (LAAAs) as alternatives to antibiotics for treating localized infections. The purpose of this study was to determine whether CdSe/ZnS quantum dots (QD) could enhance the antibacterial activity of the LAAA, toluidine blue O (TBO). Suspensions of Staphylococcus aureus and Streptococcus pyogenes were exposed to white light (3600 lux) and TBO (absorbance maximum = 630 nm) in the presence and absence of 25 nm diameter QD (emission maximum = 627 nm). When the TBO:QD ratio was 2667:1, killing of Staph. aureus was enhanced by 1.72log(10) units. In the case of Strep. pyogenes, an enhanced kill of 1.55log(10) units was achieved using TBO and QD in the ratio 267:1. Singlet oxygen and fluorescence measurements showed that QD suppress the formation of singlet oxygen from TBO and that QD fluorescence is significantly quenched in the presence of TBO (70-90%). Enhanced killing appears to be attributable to a non-Förster resonance energy transfer mechanism, whereby the QD converts part of the incident light to the absorption maximum for TBO; hence more light energy is harvested, resulting in increased concentrations of bactericidal radicals. QD may, therefore, be useful in improving the efficacy of antimicrobial photodynamic therapy.

Functionalization of solid surfaces with thermoresponsive protein-resistant films.

Pulsed plasma polymerization of N-isopropylacrylamide leads to the deposition of thermoresponsive films. The reversible (switching) behavior of these poly(N-isopropylacrylamide) surfaces has been exemplified by screening the adsorption of fibrinogen and fluorescein isothiocyanate labeled bovine serum albumin proteins by surface plasmon resonance (SPR) and fluorescence microscopy at low and elevated temperatures.

Photophysical properties of N-acetyl-menthyl anthranilate.

The results of a comprehensive investigation of the photophysical properties of the sunscreen analogue, N-acetyl menthyl anthranilate (NAMA), in various solvent systems are reported. Luminescence studies reveal that this compound is fluorescent (Phi(f)=0.16+/-0.01) in toluene and has a solvent dependent emission maximum in the range 363-370 nm. Phosphorescence has also been detected in low temperature glasses with an emission maximum at 420 nm in EPA, and a lifetime of 1.3 s; the triplet energy was found to be 311+/-3 kJ mol(-1). Kinetic UV-visible absorption measurements revealed a transient species with absorption maxima at 450 nm and solvent dependent lifetimes of 120-240 micros which are attributed to the triplet state. The triplet state is efficiently quenched by oxygen, leading to the formation of singlet oxygen in all of the solvent systems studied. The singlet oxygen quantum yields (Phi(Delta)), determined by time-resolved near-infrared luminescence measurements, were in the range 0.19-0.21.

Intraspecific competition in populations of Helix aspersa with different histories of exposure to lead.

A range of invertebrates have become adapted to certain toxic metals, such that, in the presence of the contaminant, some measure of their performance, typically growth or reproduction, is superior to that of an unexposed population. Under such a toxic stress, a population with a history of exposure might outperform a naïve population in competition for limited resources. This study compared the shell growth of laboratory-bred juveniles from six populations of Helix aspersa with different histories of exposure to Pb. In 10 trials using various combinations of two populations, the snails competed for a limited supply of food that contained either no Pb or 500 micrograms/g-1 Pb, for 98 days. Each trial consisted of 10 juveniles, five from each population and was replicated four times. Nearly all of the food provided was consumed quickly after presentation. The total amount of shell growth within each replicate (the sum of the mean growth of the two populations) was highly consistent between trials so that the total amount of shell built was limited by food availability. The presence of Pb in the diet caused no measurable depression of shell growth and exposure history did not appear to confer any competitive advantage or disadvantage in any of the trials. One population consistently grew faster than its competitors in every trial of which it was a part. Shell growth tended to be greater in smaller juveniles. Snail activity is known to be inhibited at high densities and this may have contributed to the lower incremental growth in individuals kept at the higher densities. The competitive advantage enjoyed by one population may be primarily determined by their activity or perhaps their Ca metabolism.

Protonation of tetrasulfonated zinc phthalocyanine in aqueous acetonitrile solution.

The phenomenon of protonation of phthalocyanines (Pc) and its effect upon their photophysical properties has seen considerable neglect in the literature. The work reported here clearly shows that tetrasulfonated zinc Pc, a known photodynamic therapy (PDT) agent, is strongly susceptible to protonation at the azomethine bridges. Absorption and fluorescence spectra demonstrate the absolute dependence of the redshifted peak on the pH of the solution. The fluorescence spectra and lifetimes of the protonated Pc are reported, and the potential application of this phenomenon to the development of a PDT agent with increased selectivity is discussed.

Photochemistry of the pi-extended 9,10-bis(1,3-dithiol-2-ylidene)-9,10-dihydroanthracene system: generation and characterisation of the radical cation, dication, and derived products.

Flash photolysis of bis[4.5-di(methylsulfanyl) 1,3-dithiol-2-ylidene]-9,10(-dihydroanthracene (1) in chloroform leads to formation of the transient radical cation species 1.+ which has a diagnostic broad absorption band at lambdamax approximately 650 nm. This band decays to half its original intensity over a period of about 80 micros. Species 1.+ has also been characterised by resonance Raman spectroscopy. In degassed solution 1.+ disproportionates to give the dication 1(2+), whereas in aerated solutions the photodegradation product is the 10-[4,5-di(methylsulfanyl) 1,3-dithiol-2-ylidene]anthracene-9(10 H)one (2). The dication 1(2+) has been characterised by a spectroelectrochemical study [lambdamax (CH2Cl2) = 377, 392, 419, 479 nm] and by an X-ray crystal structure of the salt 1(2-) (ClO4)2, which was obtained by electrocrystallisation. The planar anthracene and 1,3-dithiolium rings in the dication form a dihedral angle of 77.2 degrees; this conformation is strikingly different from the saddle-shaped structure of neutral 1 reported previously.

What do sentinels stand for?

Sentinel species are biological monitors that accumulate a pollutant in their tissues without significant adverse effects. Primarily used to measure the amount of a pollutant that is biologically available, they may also increase the sensitivity of an analytical procedure or summarise a complex pollution signal. This paper examines the validation of sentinels, referring particularly to the use of invertebrates in monitoring toxic metal pollution in terrestrial ecosystems. Few studies measure the capacity of a sentinel to quantify changes in ambient levels. Definitions of 'bioavailability' also differ between workers and few note that this may be a highly species-specific measure. Validation requires that the temporal and spatial scales over which a sentinel integrates a pollution signal are known. The sentinel has to be calibrated against source concentrations and this relationship shown to be consistent over the normal range of exposure. This requires some consideration of the environmental and biological determinants of pollutant assimilation. Differences between populations can confound simple comparisons between sites based on native populations. Transplanted individuals, matched for age, sex and physiological state, might be used when the aim is not to assess bioavailability to the resident population. A simple measure of their capacity to detect differences in ambient pollutant levels is proposed to evaluate candidate species, to assess their consistency and capacity to equilibrate with their source. A small survey of earthworms from a well-defined gradient of Pb pollution is used to illustrate problems of interpreting tissue concentrations in sentinels.

The photophysical properties of menthyl anthranilate: a UV-A sunscreen.

The results of a comprehensive investigation of the photophysical properties of the sunscreen agent menthyl anthranilate in various solvent systems are reported. Luminescence studies reveal that this ester is highly fluorescent (phi f = 0.64 +/- 0.06 in ethanol) and has a solvent-dependent emission maximum in the range of 390-405 nm. Phosphorescence has also been detected in low-temperature glasses with an emission maximum at 445 nm and a lifetime of 2.5 s. Kinetic UV-visible absorption measurements revealed a transient species with absorption maxima at 480 nm and solvent-dependent lifetimes of 26-200 microseconds that are attributed to the triplet state. The triplet state is efficiently quenched by oxygen, leading to the formation of singlet oxygen in all of the solvent systems studied. The singlet-oxygen quantum yields (phi delta) determined by time-resolved near-infrared luminescence measurements were determined to be in the range 0.09-0.12 for all systems.

Luminescence imaging microscopy and lifetime mapping using kinetically stable lanthanide(III) complexes.

The sensitised luminescence from stable lanthanide complexes (1 and 2) bearing a phenanthridine antenna has been used to generate time-resolved images of silica particles. The millisecond order luminescent lifetime of these complexes is utilised to demonstrate time-gated imaging of the sample from a fluorescent background and to facilitate lifetime mapping over the area of the sample.

Variation in the mineral composition of eggs of the snail, Helix aspersa between populations exposed to different levels of metal contamination.

The eggs of terrestrial gastropods represent a major investment of resources by the parent, not least in the provision of essential metals. In this experiment 11 populations of Helix aspersa were collected from habitats with different histories of lead exposure and allowed to reproduce in the laboratory. After 4 months on a low metal diet, five four-egg samples were taken from a single clutch of each population and analysed for their metal content. Within-clutch and between population variation in the eggs were measured. No Pb or Cd was detected in any of the eggs. Both trace and essential metals show considerable variation between eggs within the same clutch. Cu levels were consistent across populations while egg Zn was closely correlated with soil Zn. Median Ca and Mg levels show little relation to soil concentrations. However, Ca provision declines with soil Pb across the populations. While the metal content of the eggs appears to be closely regulated, an interaction between Pb and Ca in the parental tissues may mediate the supply of Ca to the eggs.

Excited triplet state photophysics of the sulphonated aluminium phthalocyanines bound to human serum albumin.

The binding of the sulphonated aluminum phthalocyanines to human serum albumin (HSA) in aqueous phosphate buffer solution at 25 degrees C has been studied by measuring the properties of the triplet excited states of these dyes. The triplet lifetimes were measured by triplet-triplet absorption flash photolysis. The triplet lifetime of the disulphonated AlS2Pc (2.5 microM) varies from 500 +/- 30 microseconds in the absence of protein to 1.100 microseconds and longer with HSA concentrations above 100 microM. Under identical conditions, the maximum triplet lifetimes of the mono-, tri- and tetrasulphonated compounds bound to HSA are shorter than those for the disulphonated species. The increase in the triplet state lifetimes is attributed to the ability of the bulk aqueous phase to interact with the sensitizer at the site of binding; the site of binding being dependent on the degree of sulphonation. For AlS2Pc and AlS3Pc at all HSA concentrations, and regardless of the degree of sulphonation, all the triplet state decay profiles follow simple pseudo-first-order kinetics. The exponential decay of the triplet phthalocyanine at all HSA concentrations is ascribed to the rapid association and dissociation of the phthalocyanine-HSA complex on the time-scales of the triplet state lifetimes. A simplified one-step binding model is utilized to describe the results. The association of AlS1Pc with HSA results in substantial quenching of the triplet state quantum yield, and a more complex model is required to analyze the results. The tetrasulphonated compound (AlS4Pc) binds to the protein at a site where it experiences some protection from the aqueous phase.

Characterisation of the photochemotherapeutic agent disulphonated aluminium phthalocyanine and its high-performance liquid chromatographic separated components.

Disulphonated aluminium phthalocyanine (AlPcS2), a potential clinical photosensitiser, has been synthesised in a reproducible form and shown by reversed-phase HPLC to consist of at least eight components which are believed to be individual AlPcS2 regioisomers. These components have been isolated either as single bands or mixtures of two using preparative reversed-phase HPLC methods. The number and position of sulphonate groups per phthalocyanine macromolecule for each component has been determined using a chemical degradation and HPLC assay. Results suggest that the bulk AlPcS2 material consists mostly (> 60%) of an amphiphilic alpha-, alpha-disubstituted regioisomer, with both sulphonate groups substituted to the same side of the molecule (adjacent form). Possible structures for some of the other separated components of AlPcS2 are also presented.

A retrospective analysis of 94 patients with CIN and false negative cervical smears taken at colposcopy.

This report investigates the reasons for false negative cervical cytology in 94 out of 630 patients (15%) in whom cervical intraepithelial neoplasia (CIN) was diagnosed on colposcopically directed biopsy. Cervical smears were taken immediately before biopsy and the cases with false negative cytology were compared with those whose cytology was abnormal. Patients with false negative cytology were more likely to have been younger (P < 0.01), to have had fewer pregnancies (P < 0.001), to have had a less severe grade of dyskaryosis on their referral smear (P < 0.001), to have had no endocervical cells on the smear (P < 0.05), to have had a less severe grade of CIN on biopsy (P < 0.001), to have had no punctation visible at colposcopy (P < 0.01), and to have had no mosaic pattern seen at colposcopy (P < 0.05). We found no effect attributable to the patient's menstrual history, the interval between referral smear and colposcopy clinic visit, the smear taker or the type of spatula used to take the smear.

The time resolved fluorescence and anisotropy of subtilisins BPN' and Carlsberg.

Time-resolved emission and anisotropy have been measured for the tryptophan (Trp) residues of two closely related subtilisin proteins. The single Trp of subtilisin Carlsberg shows complex lifetime properties, and anisotropy consistent with a fast (ca. 200 ps) segmental motion, on the "wobbling in a cone model" the semi angle is in the range 38 to 47 degrees. The lifetime and anisotropy properties for this single Trp residue suggest that the predominant state is that of an effectively non-emitting statically quenched fluorophore. This fast component is also resolved in the anisotropy of subtilisin BPN' but with relatively low amplitude, due to the dominant emission of the other Trp residues. The diversity of the photophysical properties is not readily correlated with the structure of the proteins, though the observed complexity is consistent with the likely heterogeneity of environment due to the surface location of all the Trp residues.

Reproducibility of ultrasonic measurement of fetal cardiac haemodynamics.

Two observers made independent estimates of volume flow across all four heart valves in 27 fetuses using a combination of cross-sectional and Doppler echocardiography. There were considerable discrepancies between observers in volume flow estimation arising from inconsistency both in cross-sectional and in Doppler measurements. One observer also produced a second estimate by making new measurements from the original recordings. There was considerable within-observer variability which was mainly due to errors in cross-sectional measurements. The problems of applying this technique to fetal cardiac haemodynamics are discussed.