Biomedical subjects

J R Lakowicz

Publications and source records attributed to J R Lakowicz.

At least 55 records · Page 3Linked to original sources

PubMed1999

Polarization sensing of fluorophores in tissues for drug compliance monitoring.

We used a new method, polarization sensing, to monitor the concentration of the fluorophore rhodamine 800 in an intralipid suspension and in chicken tissue. Rhodamine 800 (Rh800) could be excited at 648 nm using a laser pointer. We developed a simple device for measuring the combined emission from a highly polarized reference film and the unpolarized or orthogonally polarized emission of Rh800 from the scattering intralipid or tissue. The concentration of Rh800 in this medium was revealed by large changes in the polarization (P) with values of P ranging from 0.8 to -0.9. It is possible to vary the sensitive Rh800 concentration range by variation of the detected emission wavelengths, orientation of the excitation polarizer, or fluorophore concentration in the reference film. Polarization sensing of fluorophores in tissue requires only steady-state detection, and can be accomplished with simple and/or portable electronics. Such devices may find use in electronic detection of ingested medicines based on transdermal detection of nontoxic long-wavelength fluorophores.

Animals↗

PubMed1999

Synthesis and spectral characterization of a long-lifetime osmium (II) metal-ligand complex: a conjugatable red dye for applications in biophysics.

There is a need for luminescent probes, which display both long excitation and emission wavelengths and long decay times. We synthesized and characterized an osmium metal-ligand complex which displays a mean decay time of over 100 ns when bound to proteins. [Os(1,10-phenanthroline)2(5-amino-1,10-phenanthroline)[(PF6)2 can be excited at wavelengths up to 650 nm, and displays an emission maximum near 700 nm. The probe displays a modest but useful maximum fundamental anisotropy near 0.1 for 488-nm excitation, and thus convenient when using an argon ion laser. [Os(phen)2(aphen)](PF6)2 is readily activated to the isothiocyanate for coupling to proteins. When covalently linked to bovine serum albumin the intensity decay is moderately heterogeneous with a mean decay time of 145 ns. The anisotropy decay of the labeled protein displays a correlation time near 40 ns. This relatively long lifetime luminophores can be useful as a biophysical probe or in clinical applications such as fluorescence polarization immunoassays.

Biophysics↗

PubMed1999

Sensing of carbon dioxide by a decrease in photoinduced electron transfer quenching.

We described a new approach to sensing of carbon dioxide based on photoinduced electron transfer (PET) quenching. Fluorophores like naphthalene and anthracene are known to be quenched by unprotonated amines by the PET mechanism. We examined the fluorescence spectral properties of two amine-containing fluorophores, 1-naphthylmetylamine (NMA) and 9-ethanolaminomethylanthracene (EAA). When dissolved in an organic solvent, both fluorophores displayed increased intensity when equilibrated with gaseous carbon dioxide. In the case of NMA, we found that the mean lifetime increased with increasing partial pressures of CO(2). The intensity and lifetime changes of NMA are completely reversible when CO(2) is removed by purging with argon. Our results are consistent with decreased quenching by the covalently linked amino groups when CO(2) is dissolved in the solution. At present, we are not certain whether the increased intensity is due to protonation of the amino groups or to carbamate formation. In either event, these results suggest that CO(2) can be detected directly using amine-containing fluorophores without the use of bicarbonate and a pH-sensitive fluorophore.

1-Naphthylamine↗

PubMed1999

Three-photon excitation of N-acetyl-L-tyrosinamide.

We observed emission from the tyrosine derivative N-acetyl-L-tyrosinamide (NATyrA) when excited with the fundamental output of a femtosecond Ti:Sapphire laser from 780 to 855 nm. The dependence on incident laser power indicates a three-photon process. The emission spectra and intensity decay in glycerol-water (30:70) at 5 degrees C were found to be identical for one- and three-photon excitation. Also the excitation spectrum of three-photon-induced fluorescence of NATyrA corresponds to the one-photon excitation spectrum. The time-zero or fundamental anisotropy spectrum was reconstructed from the frequency-domain anisotropy decays. The three-photon anisotropies are similar or larger than the one-photon anisotropies. These three-photon anisotropies are surprising given the near zero values known for tyrosine with two-photon excitation. The observations indicate that one- and three-photon excitation directly populates the same singlet excited states(s). However, the origin of the anisotropies with multi-photon excitation of tyrosine remain unclear and unpredictable.

Fluorescence Polarization↗

PubMed1999

Lifetime-based pH sensors: indicators for acidic environments.

We characterized the pH-dependent intensity decays of three fluorophores, Oregon green 514 carboxylic acid, Cl-NERF, and DM-NERF, using frequency-domain fluorometry, with the objective of identifying lifetime-based sensors for low pH values. These three probes were originally designed as dual excitation wavelength-ratiometric probes, with high photostability and high quantum yields in aqueous solutions. We found that their fluorescence intensity decays were strongly dependent on pH. Moreover, global intensity decays analysis reveals that these probes have double exponential intensity decays at intermediate pH values and that the decay time amplitudes are greatly dependent on pH. The longer lifetime components originated from the unprotonated forms and the shorter components from the protonated forms. Both forms can emit fluorescence at intermediate pH values. The apparent pKa values were also determined from the titration curves of phase angles and modulations versus pH for the purpose of pH sensing. The apparent pKa values range from pH 3 to 5, a range where lifetime-based sensors are not presently reported. Since these probes show low pKa values and display substantial phase and modulation changes with pH, they are suitable as lifetime-based pH sensors to monitor the pH changes in acidic environments. One potential application of these probes is to trace the pH in different cellular compartments.

Fluoresceins↗

PubMed1999

Polarization sensing with visual detection.

We describe a new approach to fluorescence sensing which relies on visual determination the polarization. The sensing device consists of a fluorescent probe, which changes intensity in responses to the analyte, and an oriented fluorescent film, which is not affected by the analyte. An emission filter is selected to observe the emission from both the film and the sensing fluorophore. Changes in the probe intensity result in changes in the polarization of the combined emission from the sensor and reference. The degree of polarization can be detected visually using a dual polarizer with adjacent sections oriented orthogonally to each other. The emission passing through the dual polarizer is viewed with a second analyzing polarizer. This analyzer is rotated manually to yield equal intensities from both sides of the dual polarizer. This approach was used to measure the concentration of RhB in intralipid and to measure pH using 6-carboxyfluorescein. The analyzer angle is typically accurate to 1 degree, providing pH values accurate to +/- 0.1 pH unit at the midpoint of the titration curve. We also describe a method of visual polarization sensing that does not require an oriented film and that can use the same fluorophore for the sample and reference. These approaches to visual sensing are generic and can be applied to a wide variety of analytes for which fluorescent probes are available. Importantly, the devices are simple, with the only electronic component being the light source.

Algorithms↗

PubMed1999

Anisotropy-based sensing with reference fluorophores.

We describe a new approach to fluorescence sensing based on measurements of steady-state anisotropies in the presence of reference fluorophores with known anisotropies. The basic concept is that the anisotropy of a mixture reflects a weighted average of the anisotropies of the emitting species. By use of reference fluorophores the starting anisotropy can be near zero, or near 0.9 for oriented films which contain the reference fluorophore. Changing intensities of the analyte result in changes in anisotropy. A wide dynamic range of anisotropies is available because of the freedom to select high or low starting values. Anisotropy-based sensing was demonstrated for pH using 6-carboxyfluorescein and for protein affinity or immunoassay using an oriented film with high anisotropy and a protein labeled with a metal-ligand complex. The latter measurements were performed with a simple light-emitting diode excitation source without an excitation polarizer. The sensitive range of the assay can be adjusted by changing the intensity of the reference fluorophore. Anisotropy-based sensing can have numerous applications in clinical and analytical chemistry.

Anisotropy↗

PubMed1999

Glucose sensor for low-cost lifetime-based sensing using a genetically engineered protein.

We describe a glucose sensor based on a mutant glucose/galactose binding protein (GGBP) and phase-modulation fluorometry. The GGBP from Escherichia coli was mutated to contain a single cysteine residue at position 26. When labeled with a sulfhydryl-reactive probe 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid, the labeled protein displayed a twofold decrease in intensity in response to glucose, with a dissociation constant near 1 microM glucose. The ANS-labeled protein displayed only a modest change in lifetime, precluding lifetime-based sensing of glucose. A modulation sensor was created by combining ANS26-GGBP with a long-lifetime ruthenium (Ru) metal-ligand complex on the surface of the cuvette. Binding of glucose changed the relative intensity of ANS26-GGBP and the Ru complex, resulting in a dramatic change in modulation at a low frequency of 2.1 MHz. Modulation measurements at 2.1 MHz were shown to accurately determine the glucose concentration. These results suggest an approach to glucose sensing with simple devices.

Bacterial Proteins↗

PubMed1999

Long-lifetime lipid rhenium metal-ligand complex for probing membrane dynamics on the microsecond timescale.

We report the luminescence and spectral properties of a phospholipid analogue containing a long-lifetime luminescent rhenium metal-ligand complex (MLC) covalently linked to the amino group of phosphatidyl ethanolamine. When incorporated into synthetic membranes, this lipid probe displays intensity decay times near 3 microseconds. Importantly, the probe displays highly polarized emission with a maximal fundamental anisotropy of 0.33. This probe is expected to have numerous applications for studies of microsecond diffusion and dynamics of membranes.

Diffusion↗

PubMed1999

Resonance energy transfer study using a rhenium metal-ligand lipid conjugate as the donor in a model membrane.

We measured steady state and time-resolved resonance energy transfer between donors and acceptors in model membranes. The donor was a long lifetime rhenium-lipid complex, which displayed a mean lifetime of 1 microsecond and lifetime components as long as 3 microseconds in the labeled DOPC membranes. The transfer efficiencies were found to be substantially larger than those predicted without consideration of lateral diffusion. The larger transfer efficiencies are consistent with a mutual lateral diffusion coefficient in the membrane near 2 x 10(-8) cm2/s. These results demonstrate that lateral diffusion in membranes can be detected with microsecond lipid probes.

Energy Transfer↗

PubMed1999

Polarization-based oxygen sensor.

A new approach to oxygen sensing based on the luminescence polarization observed from a novel type of sensor is described. The oxygen sensor consists of an oxygen-sensitive silicone film containing tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) chloride [Ru(dpp)3Cl2] and an oxygen-insensitive film of Styryl 7 in poly(vinyl alcohol). Polarizers are used to select orthogonally polarized emission components from Ru(dpp)3Cl2 and Styryl 7. The polarization of the combined emission was found to be highly sensitive to the partial pressure of oxygen. This method of polarization sensing is generic and can be used with any fluorophore which displays an analyte-dependent change in intensity.

Fluorescence Polarization↗

PubMed1999

beta-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus: structure and activity in the presence of alcohols.

beta-Glycosidase from the extreme thermophilic archaeon Sulfolobus solfataricus is a tetrameric protein with a molecular mass of 240 kDa, stable in the presence of detergents, and with a maximal activity at temperatures above 95 degrees C. Understanding the structure-activity relationships of the enzyme under different conditions is of fundamental importance for both theoretical and applicative purposes. In this paper we report the effect of methanol, ethanol, 1-propanol, and 1-butanol on the activity of S. solfataricus beta-glycosidase expressed in Escherichia coli. The alcohols stimulated the enzyme activity, with 1-butanol producing its maximum effect at a lower concentration than the other alcohols. The structure of the enzyme was studied in the presence of 1-butanol by circular dichroism, and Fourier-transform infrared and fluorescence spectroscopies. Circular dichroism and steady-state fluorescence measurements revealed that at low temperatures the presence of the alcohol produced no significant changes in the tertiary structure of the enzyme. However, time-resolved fluorescence data showed that the alcohol modifies the protein microenvironment, leading to a more flexible enzyme structure, which is probably responsible for the enhanced enzymatic activity.

1-Butanol↗

PubMed1999

Fluorescence spectral properties of the anticancer drug topotecan by steady-state and frequency domain fluorometry with one-photon and multi-photon excitation.

Topotecan is an antitumor agent with activity against a variety of cancers. We examined the steady-state and time-resolved fluorescence spectral properties of topotecan with one- and two-photon excitation. Topotecan was found to display a high two-photon cross section near 20 GM for wavelengths within the fundamental output of a Ti:sapphire laser, 800-880 nm. In frozen solution the anisotropies of topotecan are near the theoretical maxima for one-photon and two-photon excitation with colinear electronic transitions. The intensity and anisotropy decays of topotecan fluorescence were found to be homogeneous (single exponentials) in phosphate-buffered saline and propylene glycol. The steady-state and time-resolved data indicate that topotecan binds to a double-helical DNA oligomer d(AT)10 resulting in increased anisotropies and multiexponential intensity and anisotropy decays. Subnanosecond components in the anisotropy decay of the DNA-topotecan complex suggest loose binding of the drug to DNA. Loose binding of topotecan to DNA is also revealed by accessibility of topotecan to collisional quenching by iodide.

Antineoplastic Agents↗

PubMed1998

Low-frequency modulation sensors using nanosecond fluorophores.

We describe a new approach to fluorescence sensing based on a mixture of fluorophores, one of which is sensitive to the desired analyte. If a long-lifetime analyte-insensitive fluorophore is mixed with a short-lifetime analyte-sensitive fluorophore, the modulation of the emission at conveniently low frequencies becomes equal to the fractional fluorescence intensity of the sensing fluorophore. Under these conditions, the modulation can be used to determine the analyte concentration. This can be used with any fluorophore that changes intensity in response to analyte and does not require the sensing fluorophore to display a change in lifetime. The feasibility of modulation-based sensing was demonstrated using mixtures of 6-carboxyfluorescein and [Ru 2,2'-(bipyridyl)3]2+ as a pH sensor and of the calcium probe Fluo-3 and [Ru 2,2'-(bipyridyl)3]2+ as a calcium sensor.

2,2'-Dipyridyl↗

PubMed1998

Time-resolved and steady-state fluorescence quenching of N-acetyl-L-tryptophanamide by acrylamide and iodide.

We examined the time-resolved and steady-state fluorescence quenching of N-acetyl-L-tryptophanamide (NATA) by acrylamide and iodide, over a range of viscosities in propylene glycol. The quenching of NATA by acrylamide and iodide results in heterogeneity of the intensity decay which increases with the quencher concentration. We attribute the complex decays of NATA to transient effects in diffusion and the nature of the fluorophore-quencher interaction. These data were compared using the phenomenological radiation boundary condition (RBC) and distance-dependent quenching (DDQ) models for collisional quenching. We used global analysis of the time-resolved frequency-domain and steady-state data to select between the models. Consideration of both the frequency-domain and steady state data demonstrate that the quenching rate depends exponentially on the fluorophore-quencher distance, indicating the validity of the DDQ model. The rate constants for acrylamide and iodide quenching, at the constant distance of 5 A, were found to be near 10(13) s-1 and 10(9) s-1, respectively. These rates reflect electron transfer and exchange interactions as the probable quenching mechanisms, respectively.

Acrylamide↗

PubMed1998

A long-lifetime Ru(II) metal-ligand complex as a membrane probe.

A luminescent metal-ligand complex, [Ru(bpy)2(dppz)]2+, (where dppz is dipyrido[3,2-a:2',3'-c] phenazine), was used as a photoluminescence probe for investigating submicrosecond lipid dynamics in a dipalmitoyl-L-alpha-phosphotidylglycerol (DPPG) model bilayer system. The luminescence of [Ru(bpy)2(dppz)]2+ in buffer is completely quenched but becomes luminescent when intercalated into DPPG vesicles. The experimental results show that the emission intensity of [Ru(bpy)2(dppz)]2+ intercalated into DPPG vesicles increases dramatically as temperature is increased towards the lipid phase transition temperature. This effect is abolished in bilayers containing a high concentration (> 30 mol%) of cholesterol, suggesting this probe is sensitive to the membrane composition. Frequency-domain emission intensity decays, measured as a function of increasing temperature towards the lipid phase transition temperature (2 to 57 degrees C), display two major lifetime components. The short lifetime disappears at temperatures well above the phase transition temperature. A comparison of oxygen quenching with iodide quenching suggests the heterogeneity of probe location at temperatures well below the lipid phase transition temperature and the homogeneity of probe location at temperature well above the lipid phase transition temperature. [Ru(bpy)2(dppz)]2+ displays polarized emission, enabling the study of membrane dynamics. The long decay time displayed by this probe allows measurement of the overall rotational correlation time of lipid vesicles on the microsecond time-scale. Because of the long lifetime, polarized emission, and background free nature of the photoluminescence measurements, [Ru(bpy)2(dppz)]2+ has numerous applications in the biophysical studies of membranes.

Anisotropy↗

PubMed1998

Long-lifetime Ru(II) complexes for the measurement of high molecular weight protein hydrodynamics.

We describe the synthesis and characterization of two asymmetrical ruthenium(II) complexes, [Ru(dpp)2(dcbpy)]2+ and [Ru(dpp)2(mcbpy)]2+, as well as the water soluble sulfonated derivatives [Ru(dpp(SO3Na)2)2(dcbpy)]2+ and [Ru(dpp(SO3Na)2)2(mcbpy)]2+ (dpp is 4,7-diphenyl-1,10-phenanthroline, dcbpy is 4,4'-dicarboxylic acid-2,2'-bipyridine, mcbpy is 4-methyl,4'-carboxylic acid-2,2'-bipyridine, and dpp(SO3Na)2 is the disulfonated derivative of dpp) as probes for the measurement of the rotational motions of proteins. The spectral (absorption, emission, and anisotropy) and photophysical (time-resolved intensity and anisotropy decays) properties of these metal-ligand complexes were determined in solution, in both the presence and absence of human serum albumin (HSA). These complexes display lifetimes ranging from 345 ns to 3.8 microseconds in deoxygenated aqueous solutions under a variety of conditions. The carboxylic acid groups on these complexes were activated to form N-hydroxysuccinimide (NHS) esters which were used to covalently lable HSA, and were characterized spectroscopically in the same manner as above. Time-resolved anisotropy measurements were performed to demonstrate the utility of these complexes in measuring long rotational correlation times of bioconjugates between HSA and antibody to HSA. The potential usefulness of these probes in fluorescence polarization immunoassays was demonstrated by an association assay of the Ru(II)-labeled HSA with polyclonal antibody.

Anisotropy↗

PubMed1998

Use of a long-lifetime Re(I) complex in fluorescence polarization immunoassays of high-molecular-weight analytes.

We describe a new class of fluorescence polarization immunoassays based on the luminescence from a Re(I) metal-ligand complex. Re(I) complexes are extremely photostable and possess useful photophysical properties including long lifetimes, high quantum yields, and high emission polarization in the absence of rotational diffusion. In the present study, a conjugatable, highly luminescent Re(I) metal-ligand complex, [Re(bcp)(CO)3(4-COOHPy)](ClO4), where bcp is 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline and 4-COOHPy is isonicotinic acid, has been evaluated for use in fluorescence polarization immunoassays (FPIs) with high-molecular-weight antigens. This Re(I) complex (Re) displays highly polarized emission (with a maximum anisotropy near 0.3) in the absence of rotational diffusion and a long average lifetime (2.7 microseconds) when bound to human serum albumin (HSA) in oxygenated aqueous solution. The emission polarization of the Re-HSA conjugate is sensitive to the binding of anti-HSA, resulting in a significant increase in anisotropy. The labeled HSA was also used in a competition immunoassay where unlabeled HSA was also used as an antigen. These experimental results, combined with theoretical predictions, demonstrate the potential of this Re(I) metal-ligand complex as a luminescence probe in FPIs of high-molecular-weight analytes (10(5)-10(8) Da).

Antigens↗