Clinical staining of the ocular surface: mechanisms and interpretations.

In this article we review the mechanism of ocular surface staining. Water-soluble dyes are excluded from the normal epithelium by tight junctions, the plasma membranes and the surface glycocalyx. Shed cells can take up dye. A proportion of normal corneas show sparse, scattered time-dependent, punctate fluorescein uptake, which, we hypothesise, is due to a graded loss of the glycocalyx barrier, permitting transcellular entry into pre-shed cells. In pathological staining, there is little evidence of 'micropooling' at sites of shedding and the term 'punctate erosion' may be a misnomer. It is more likely that the initial event involves transcellular dye entry and, in addition, diffusion across defective tight junctions. Different dye-staining characteristics probably reflect differences in molecular size and other physical properties of each dye, coupled with differences in visibility under the conditions of illumination used. This is most relevant to the rapid epithelial spread of fluorescein from sites of punctate staining, compared to the apparent confinement of dyes to staining cells with dyes such as lissamine green and rose bengal. We assume that fluorescein, with its lower molecular weight, spreads initially by a paracellular route and then by transcellular diffusion. Solution-Induced Corneal Staining (SICS), related to the use of certain contact lens care solutions, may have a different basis, involving the non-pathological uptake of cationic preservatives, such as biguanides, into epithelial membranes and secondary binding of the fluorescein anion. It is transient and may not imply corneal toxicity. Understanding the mechanism of staining is relevant to the standardisation of grading, to monitoring disease and to the conduct of clinical trials.

Sensitivity-Enhanced CMOS Phase Luminometry System Using Xerogel-Based Sensors.

We present the design and implementation of a phase luminometry sensor system with improved and tunable detection sensitivity achieved using a complementary metal-oxide semiconductor (CMOS) integrated circuit. We use sol-gel derived xerogel thin films as an immobilization media to house oxygen (O2) responsive luminescent molecules. The sensor operates on the principal of phase luminometry wherein a sinusoidal modulation signal is used to excite the luminophores encapsulated in the porous xerogel films and the corresponding phase shift of the emission signals is monitored. The phase shift is directly related to excited state lifetimes of the luminophores which in turn are related to the concentration of the target analyte species present in the vicinity of the luminophores. The CMOS IC, which consists of a 16 times 16 high-gain phototransistor array, current-to-voltage converter, amplifier and tunable phase shift detector, consumes an average power of 14 mW with 5-V power supply operating at a 38-kHz modulation frequency. The output of the IC is a dc voltage that corresponds to the detected luminescence phase shift with respect to the excitation signal. As a prototype, we demonstrate an oxygen sensor system by encapsulating the luminophore tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) within the xerogel matrices. The sensor system showed a fast response on the order of few seconds and we obtained a detection sensitivity of 118 mV per 1% change in O2 concentration. The system demonstrates a novel concept to tune and improve the detection sensitivity for specific concentrations of the target analyte in many biomedical monitoring applications.

Interaction of influenza virus fusion peptide with lipid membranes: effect of lysolipid.

The effect of lysophosphatidylcholine (LPC) on lipid vesicle fusion and leakage induced by influenza virus fusion peptides and the peptide interaction with lipid membranes were studied by using fluorescence spectroscopy and monolayer surface tension measurements. It was confirmed that the wild-type fusion peptide-induced vesicle fusion rate increased several-fold between pH 7 and 5, unlike a mutated peptide, in which valine residues were substituted for glutamic acid residues at positions 11 and 15. This mutated peptide exhibited a much greater ability to induce lipid vesicle fusion and leakage but in a less pH-dependent manner compared to the wild-type fusion peptide. The peptide-induced vesicle fusion and leakage were well correlated with the degree of interaction of these peptides with lipid membranes, as deduced from the rotational correlation time obtained for the peptide tryptophan fluorescence. Both vesicle fusion and leakage induced by the peptides were suppressed by LPC incorporated into lipid vesicle membranes in a concentration-dependent manner. The rotational correlation time associated with the peptide's tryptophan residue, which interacts with lipid membranes containing up to 25 mole % LPC, was virtually the same compared to lipid membranes without LPC, indicating that LPC-incorporated membrane did not affect the peptide interaction with the membrane. The adsorption of peptide onto a lipid monolayer also showed that the presence of LPC did not affect peptide adsorption.

Effects of fluorescent probe structure on the dynamics at cysteine-34 within bovine serum albumin: evidence for probe-dependent modulation of the cybotactic region.

We have prepared a series of bovine serum albumins (BSA) that have been site-selectively labeled at cysteine-34 with one of four different sulfhydryl-selective boron dipyrromethene difluoride (BODIPY) fluorescent probes (BODIPY FL IA, BODIPY FL C(1) IA, BODIPY 530/550 IA, and BODIPY 493/503 MB). We determine how the choice of extrinsic probe structure dictates the recovered BSA-BODIPY dynamics under thermal (10-80 degrees C) and chemical (0-5M guanidine hydrochloride) denaturation conditions. The results of these experiments show that the global protein dynamics are sensed equally by each fluorescent probe; however, the probe itself influences the local probe dynamics within the cybotactic region that surrounds cysteine-34. Thus, it seems inappropriate to think of these extrinsic fluorescent probes as passive, nonparticipatory viewers of local protein dynamics.

Optical sensor array and integrated light source.

We report a new, solid-state, integrated optical array sensor platform. The optical sensor array and integrated light source (OSAILS) is demonstrated for quenchometric O2 detection. The OSAILS requires low voltages (3-5 V dc), it is stable (< or = 5% RSD over the course of several hours of continuous operation), it is reproducible and reversible (< or =3% RSD), it exhibits a rapid response time (<30 s), and it provides good detection limits (0.2% O2). The OSAILS opens the door to a number of new optical sensor array strategies.

Dendrimeric organochalcogen catalysts for the activation of hydrogen peroxide: improved catalytic activity through statistical effects and cooperativity in successive generations.

Dendrimeric polyphenylsulfides, -selenides, and -tellurides are prepared in high yield using propyloxy spacers to connect the phenylchalcogeno groups to the dendrimeric core. The selenides and tellurides catalyze the oxidation of bromide with hydrogen peroxide to give positive bromine species that can be captured by cyclohexene in two-phase systems. The corresponding sulfides show no catalytic activity. The increase in the rate of catalysis followed statistical effects for 1, 6, and 12 phenyltelluro groups. However, the increase in the rate of catalysis exceeds statistical contributions for the first few generations with 1, 3, 6, and 12 phenylseleno groups and suggested cooperativity among phenylseleno groups. The increase in catalytic rate was lost upon replacing all but one phenylseleno group with phenoxy groups. On the basis of H2O2 consumed, the dendrimer with 12 phenylseleno groups has a turnover number of >60 000 mol of H2O2 consumed per mole of catalyst.

Extending the reach of immunoassays to optically dense specimens by using two-photon excited fluorescence polarization.

Fluorescence anisotropy/polarization measurements represent a powerful tool for quantifying biomolecule/ligand complexation. These types of measurements are also at the heart of a wide variety of commercial homogeneous fluoroimmunoassays. In this note, we demonstrate the power of two-photon excited fluorescence anisotropy (2-PEFA) measurements as a tool for quantifying hapten/antibody association in the presence of a strongly absorbing, nonfluorescent dye. The results of these experiments show that 2-PEFA measurements are intrinsically more sensitive when compared to traditional one-photon excited fluorescence anisotropy (1-PEFA) strategies and 2-PEFA-based measurements allow one to perform accurate hapten/antibody binding measurements in strongly absorbing samples directly under conditions where 1-PEFA measurements fail completely. Overall, the 2-PEFA approach offers significant advantages when compared to traditional 1-PEFA methods especially in strongly absorbing samples. 2-PEFA also opens the door to perform more rapid and reliable polarization/anisotropy-based measurements with minimal sample preparation.

Effects of ethanol volume percent on fluorescein-labeled spinach apo- and holocalmodulin.

We report the effects of EtOH volume percent (0-70%) on spinach apo- and holocalmodulin that have been site-selectively labeled with fluorescein (F). In these experiments, calmodulin (CaM) has one F reporter group attached to Cys-26, and this site is located immediately adjacent to one of the four primary Ca(2+)-binding sites (EF hands). The optimum analytical CaM-F sensitivity to Ca2+ occurs between approximately 10 and 30% EtOH. Our results also show that added EtOH causes changes in CaM and these changes are surprisingly different for apo- and holo-CaM. Apo-CaM-F appears to lose one of its two waters of hydration at approximately 20% EtOH and retains one water of hydration between approximately 20 and 70% EtOH. In apo-CaM-F, the semiangle that describes the range over which the fluorescein reporter group can precess remains essentially constant (42 +/- 2 degrees) between 0 and 70% EtOH. This shows that the fluorescein reporter group precessional freedom in apo-CaM-F is not affected significantly by EtOH. Holo-CaM-F also appears to lose one water of hydration at approximately 20-30% EtOH but then appears to denature as the EtOH volume percent increases. The fluorescein reporter group semiangle within holo-CaM-F decreases from 43 +/- 1 degrees in neat aqueous buffer to 36 +/- 1 degrees at 70% EtOH. This shows that holo-CaM-F is less nativelike and the EF hand "closes down" about the fluorescein reporter group in holo-CaM-F as the EtOH volume percent increases.

Linkage and redox isomerism in ruthenium complexes of catecholate, semiquinone, and o-acylphenolate ligands derived from 1,2-dihydroxy-9,10-anthracenedione (alizarin) and related species: syntheses, characterizations, and photophysics.

The complexes Ru(CO)2L2(AL-2H) (AL = alizarin; L = PPh3, PCyc3, PBu3, P(m-NaSO3C6H4)3), Ru(CO)(dppe)(PBu3)(AL-2H), and RuH(CO)L2(AL-H) (L = PPh3, PCyc3), and Ru(CO)2L2(AR-2H) (AR = anthrarobin; L = PBu3) were prepared by reactions of Ru3(CO)12, L, and AL, and the complexes RuH(CO)(PPh3)2(AL-H), RuH(CO)(PPh3)2(QN-H) (QN = quinizarin), and RuH(CO)(PPh3)2(LQN-H) (LQN = leucoquinizarin) are prepared by reactions of RuH2(CO)(PPh3)3 with AL or QN. The AL-2H and AR-2H ligands act as 1,2-catecholates, whereas the AL-H, QN-H, LQN-H ligands are 1,9-o-acylphenolate ligands. RuH(CO)(PPh3)2(AL-H) is characterized by X-ray crystallography. The electrochemistry of these complexes is examined, and the semiquinone complexes [Ru(CO)2L2(AL-2H)]+ (L = PPh3, PCyc3, PBu3) and [Ru(CO)(dppe)(PBu3)(AL-2H)]+ are generated by chemical oxidation and were characterized by EPR and IR spectroscopy. The photophysical properties are also reported.

DNA-induced conformational changes in bacteriophage 434 repressor.

Although bacteriophage 434 repressor binds to its specific DNA sites only as a dimer, formation of the dimers in solution occurs at concentrations three orders of magnitude higher than those needed to bind the 434 operator DNA. Our results suggest that both specific and non-specific DNA induce conformational changes in repressor that lead to formation of repressor dimers. The repressor conformational changes induced by DNA occur at concentrations much lower than those needed for binding of repressor, suggesting that the alternative conformations of repressor persist even if the protein is not in direct contact with DNA. Hence, DNA acts in a "catalytic" fashion to induce a steady-state amount of an alternative repressor conformation that has an enhanced affinity for its specific binding site. These findings suggest that the repressor conformer induced by non-specific DNA is the form of the repressor that is optimized for searching for DNA binding sites along non-specific DNA. Upon finding a binding site, the repressor protein undergoes an additional conformational change that allows it to "lock-on" to its specific site.

Kinetics and thermodynamics of free flavins and the flavin-based redox active site within glucose oxidase dissolved in solution or sequestered within a sol-gel-derived glass.

We report on the steady-state and time-resolved fluorescence from the redox active site flavine adenine dinucleotides (FADs) that are bound to glucose oxidase (GOx) when this enzyme is dissolved in aqueous solution or sequestered within a sol-gel-derived glass. To the best of our knowledge, this represents the first report on the actual dynamics of an enzyme active site when the enzyme is part of a sol-gel-derived glass. The results from these experiments show that the "free" FAD intramolecular folding/unfolding kinetics are slowed 3-10-fold within the glass vs solution. The intramolecular exciplex formation event (i.e., excited-state FAD residue folding/unfolding) is completely arrested for the GOx-bound FAD if the enzyme is sequestered within a glass in the absence of glucose. This is significantly different from the behavior of GOx dissolved in solution. However, despite this difference in behavior, the GOx molecules that are sequestered within the glasses continue to function somewhat like GOx dissolved in aqueous solution if they are challenged with glucose. We also found that the GOx molecules do not leach from the glass and they exhibit rotational mobility that is only 2-fold less than GOx dissolved in aqueous solution at 20 degrees C. In aqueous solution or within these glasses, the enzyme pocket that hosts the FAD redox sites opens up by 25-30% when GOx is challenged with glucose. Finally, we present preliminary analytical results for film-based sol-gel-derived biosensors that contain GOx, L-amino acid oxidase or cholesterol oxidase wherein the intrinsic FAD fluorescence produces the analytical signal.

A parallel multiharmonic frequency-domain fluorometer for measuring excited-state decay kinetics following one-, two-, or three-photon excitation.

We report on the performance of a new, multiharmonic frequency-domain instrument that uses the high harmonic content of a passively mode-locked, pulse-picked femto-second Ti-sapphire laser as the excitation source for the determination of one-, two-, or three-photon excited time-resolved fluorescence anisotropy and intensity decay kinetics. In operation, the new instrument can provide a complete frequency-domain data set at 100 modulation frequencies in less than 1 min. The new instrument exhibits 5-10-ps measurement precision and it can rapidly and accurately recover complex excited-state fluorescence anisotropy and intensity decay kinetics under one-, two-, or three-photon excitation for dilute or optically dense samples that exhibit single or multiexponential decay kinetics. This latter aspect of the instrument is demonstrated by successfully determining the excited-state intensity decay kinetics for a dilute aqueous solution of rhodamine 6G dissolved in a high concentration of bromocresol green. This approach is extended by determining the excited-state fluorescence intensity decay kinetics of dilute fluorescein directly in undiluted, whole blood as a function of pH under two-photon excitation conditions. The high-speed capabilities of the new instrument are exploited by performing two-photon excited fluorescence anisotropy decay experiments on the fly for site-selectively labeled bovine serum albumin as it undergoes enzymatic digestion by trypsin.

Unfolding of acrylodan-labeled human serum albumin probed by steady-state and time-resolved fluorescence methods.

Steady-state and time-resolved fluorescence spectroscopy was used to follow the local and global changes in structure and dynamics during chemical and thermal denaturation of unlabeled human serum albumin (HSA) and HSA with an acrylodan moiety bound to Cys34. Acrylodan fluorescence was monitored to obtain information about unfolding processes in domain I, and the emission of the Trp residue at position 214 was used to examine domain II. In addition, Trp-to-acrylodan resonance energy transfer was examined to probe interdomain spatial relationships during unfolding. Increasing the temperature to less than 50 degrees C or adding less than 1.0 M GdHCl resulted in an initial, reversible separation of domains I and II. Denaturation by heating to 70 degrees C or by adding 2.0 M GdHCl resulted in irreversible unfolding of domain II. Further denaturation of HSA by either method resulted in irreversible unfolding of domain I. These results clearly demonstrate that HSA unfolds by a pathway involving at least three distinct steps. The low detection limits and high information content of dual probe fluorescence should allow this technique to be used to study the unfolding behavior of entrapped or immobilized HSA.

Accessibility of the fluorescent reporter group in native, silica-adsorbed, and covalently attached acrylodan-labeled serum albumins.

Fluorescence quenching techniques are used to investigate the accessibility of a model biorecognition element-reporter group system when in buffer, surface-adsorbed, and covalently attached to a silica surface. The site-selective fluorescent reporter group, 6-acryloyl(dimethylamino)naphthalene (acrylodan, Ac), is attached covalently (at cysteine-34) to bovine and human serum albumin (BSA and HSA, respectively) and serves as a surrogate recognition element-reporter group system. Molecular oxygen is used to quench the Ac fluorescence and the accessibility, in the form of bimolecular rate constants (kq), in each model system is quantified. Although one might expect these systems to exhibit similar behavior, differences in quenching characteristics are observed, such as wavelength dependency of the Stern-Volmer quenching constant (KSV) for the native proteins in buffer. BSA-Ac exhibits wavelength dependent KSV values as well as a blue-shifted emission spectrum on O2 addition. Physisorption of BSA-Ac onto a fused-silica optical fiber lowers the accessibility of Ac to O2, whereas covalent attachment of BSA-Ac to APTES/glutaraldehyde-modified silica enhances the accessibility of the Ac reporter group to O2.

Selective removal of ribonucleases from solution with covalently anchored macromolecular inhibitor.

Poly[2'-O-(2,4-dinitrophenyl)]poly(A)[DNP-poly(A)] has been found to be a potent inhibitor in solution for RNases A, B, S, T1, T2 and H as well as phosphodiesterases I and II. Kinetic measurements with RNase B and RNase T1 showed DNP-poly(A) to be a reversible competitive inhibitor with K1 equal to 1.03 and 1.05 microM, respectively. Data on the quenching of fluorescence of RNase T1 by DNP-poly(A) indicate the existence of more than one RNase-binding site in each DNP-poly(A) molecule. By attaching each DNP-poly(A) molecule at one end covalently to oxirane acrylic beads, an affinity column was prepared for selective removal of RNases from aqueous solutions by simple filtration. It was found that a 1000-fold reduction in RNase concentration can be obtained by passing either 7.0 microM or 7.0 nM RNase A solution through a 5-cm-long column. The column can be saturated by passing through a concentrated RNase solution and subsequently regenerated by washing with salt solution. The regenerated column can be used repeatedly with no significant decrease in RNase-binding affinity and capacity. By titration of the derivatized beads with RNase, the first dissociation constant (Kd) and binding capacity for the bound enzyme can be determined. The (Kd) was found to be 0.66 microM for RNase B and 0.48 microM for RNase T1; the corresponding binding capacities were found to be 21.0 x (10)-8 and 9.6 x (10)-8 mol/g, respectively.

Biosensor for the nonspecific determination of ionic surfactants.

We report the analytical figures of merit for the first biosensor for ionic surfactant quantification. The biosensor consists of a silanized silica optical fiber, onto which acrylodan-labeled bovine serum albumin (BSA-Ac) is immobilized. Recent work from our laboratory on native BSA-Ac (Lundgren, J. S.; Bright, F. V. J. Phys. Chem. 1996, 100, 8580-8586) has shown that ionic surfactants dehydrate the local environment surrounding the lone acrylodan residue, open up the pocket hosting the acrylodan reporter group, and dramatically increase the segmental mobility of domain I in BSA. In the current work, we use BSA-Ac as an actual biorecognition element for surfactant detection and quantification. We also compare several BSA-Ac immobilization strategies and determine the analytical figures of merit for the BSA-Ac-based biosensor to a prototypical analyte, cetyltrimethylammonium bromide (CTAB). The biosensor linear dynamic range extends from 5 to 60 µM, and the t(90) response time (90% of the total response) is less than 30 s. Biosensor response precision (relative standard deviation) during 34 sensing cycles is 2.5%. On the down side, biosensor performance decreases 38% after 25 days of storage; however, this drift can be compensated. This work also demonstrates the utility of BSA-Ac as a model biorecognition element-reporter group system for grading the suitability of different surface immobilization strategies.

Dynamics of acrylodan-labeled bovine and human serum albumin sequestered within aerosol-OT reverse micelles.

We investigate the effects of hydration on acrylodan-labeled bovine and human serum albumin (BSA-Ac and HSA-Ac) in aerosol-OT (AOT) reverse micelles solubilized in n-heptane. Time-resolved fluorescence intensity decay experiments reveal a dipolar relaxation process surrounding the acrylodan cybotactic region. This process is best described by a two-term rate law wherein the average relaxation increases with increased hydration. However, the actual rate constants describing the relaxation process either remain unchanged or actually decrease with increased hydration. The results illustrate that the fractional contribution associated with the individual relaxation pathways causes the observed changes in relaxation dynamics. The recovered rotational reorientation dynamics of the acrylodan residue are also affected by the extent of protein hydration. As hydration is increased, the semiangle through which the acrylodan residue precesses increases by 10 degrees for both protein systems. Interestingly, the recovered semiangles for the native proteins equal those recovered at lower hydration when the proteins are sequestered within the AOT reverse micelle. These results demonstrate the importance of hydration on protein behavior in environments where water is limited (e.g., biosensor interfaces and sol--gel-derived biocomposites).

Dynamics of acrylodan-labeled bovine and human serum albumin entrapped in a sol-gel-derived biogel.

We investigate acrylodan-labeled bovine and human serum albumin (BSA-Ac and HSA-Ac) entrapped within a tetramethylorthosilane-derived biogel composite. The effects of biogel aging and drying were studied by following the acrylodan steady-state and time-resolved emission, the decay of anisotropy, and the dipolar relaxation kinetics as a function of ambient storage time. The results indicate that there is a substantial amount of nanosecond and subnanosecond dipolar relaxation within the local environment surrounding cysteine-34 in both proteins, even when they are fully encapsulated in a dry biogel. Time-resolved anisotropy experiments show that the acrylodan residue and the protein are able to undergo nanosecond motion within the biogel. The semiangle through which the acrylodan can process is the same for a freshly formed biogel and the native protein in buffer. However, once the biogel begins to dry, the semiangle increases (approximately 20 degrees and 10 degrees for BSA-Ac and HSA-Ac, respectively). This suggests that the "pocket" hosting the acrylodan reporter group opens as the biogel dries.

Synthesis and characterization of fluoropolymeric substrata with immobilized minimal peptide sequences for cell adhesion studies. I.

In this work, poly(tetrafluoroethylene-co-hexafluoropropylene) (also known as fluorinated ethylene propylene; FEP) was functionalized at the surface using a radio frequency glow discharge plasma. This particular surface modification produced controlled densities of hydroxyl functionality on the FEP surface. These surface hydroxyl groups provided sites for the covalent attachment of minimal peptide sequences, that are specific for neuronal attachment. FSCA, ATR-FTIR, ToF-SIMS, and fluorescence spectroscopy were used to evaluate peptide reaction efficiencies and to verify that intact peptide sequences were covalently attached to the FEP surfaces. These modified substrata were then used to study the cell attachment and response to covalently bound minimal peptide sequences. Cell attachment and differentiation results using NG108-15 and PC12 neuronal cell lines are presented in the adjoining paper by Ranieri et al.

Dynamics surrounding Cys-34 in native, chemically denatured, and silica-adsorbed bovine serum albumin.

We report the steady-state and time-resolved fluorescence of 6-acryloyl(dimethylamino)naphthalene (acrylodan) covalently attached to Cys-34 in bovine serum albumin (BSA). For this conceptually simple system, complicated fluorescence intensity and anisotropy decay kinetics are observed. The steady-state and time-resolved results demonstrate the presence of an excited-state reaction for the BSA-acrylodan system. Additional analysis shows that dipolar relaxation of the environment surrounding acrylodan within BSA is responsible for most of the observed time-dependent evolution of the emission spectrum. The effects of temperature, chemical denaturation, and protein adsorption to a bare silica substrate are also investigated. These results demonstrate the complexity of the changes within a protein/biorecognition element that affect the signal from a single fluorescent reporter group.