Biomedical subjects

Zygmunt Gryczynski

Publications and source records attributed to Zygmunt Gryczynski.

8 recordsLinked to original sources

PubMed2003

Directional surface plasmon-coupled emission: A new method for high sensitivity detection.

Fluorescence emission is nearly isotropic in space. With typical optical components the collection efficiency is 1% or less. In this preliminary report, we describe a novel approach to transforming the normally isotropic emission into directional emission with a collection efficiency near 50%. This can be accomplished for fluorophores located near a semi-transparent silver film on a glass substrate. The emission couples with the surface plasmon resonance on the silver surface and enters the transparent substrate at a sharply defined angle, the surface plasmon angle for the emission wavelength. We estimate that 40-70% of the total emission enters the substrate at the plasmon angle and can thus be directed towards a detector. Background emission from fluorophores distant from the silver does not couple with the plasmon and is not detected. Different emission wavelengths couple at different angles allowing spectral discrimination without additional optics. Surface plasmon-coupled emission represents a new technology which can be used for high detection efficiency with microfluidic and/or surface-bound assay formats.

Fluorescent Dyes↗

PubMed2003

Effects of fluorophore-to-silver distance on the emission of cyanine-dye-labeled oligonucleotides.

We examined the fluorescence spectral properties of Cy3- and Cy5-labeled oligonucleotides at various distances from the surface of silver island films. The distance to the surface was controlled by alternating layers of biotinylated bovine serum albumin (BSA) and avidin, followed by binding of a biotinylated oligonucleotide. The maximum enhancement of fluorescence near a factor of 12 was observed for the first BSA-avidin layer, with the enhancement decreasing to 2-fold for six layers. The minimum lifetimes were observed for the first BSA-avidin layer, and were about 25-fold shorter than on quartz slides without silver, with the lifetimes being about 2-fold shorter for six BSA-avidin layers. These results suggest that maximum fluorescence enhancements occur about 90A from the silver surface, a distance readily obtained by one or two layers of proteins.

Animals↗

PubMed2003

Fluorescence-sensing methods.

Novel approaches to sensor design, based on the use of an internal standard with appropriate spectral properties, provide new possibilities for designing simple devices for fluorescence sensing. Detection of combined emission from the reference and an analyte-sensitive fluorophore has been achieved in numerous measurements in cuvettes, tissues, and high-throughput formats. These methods have been used with a long-lifetime reference to measure pH, O2, pCO2, glucose, and calcium by means of modulation-sensing methods as well as by the use of oriented films as the reference for polarization sensing of glucose, pH, oxygen, and lactate. Polarization sensing has also been developed with visual detection to measure the concentration of rhodamine B and pH. Modulation and polarization sensing was found to be effective in highly scattering media such as Intralipid or tissue. The applicability of these technologies to transdermal diagnostics depends on the availability of red fluorophores that can be used in vivo. One dye that could possibly be used is indocyanine green (IcG), which absorbs and emits at wavelengths above 700 nm. Furthermore, IcG has already been approved for use in humans for monitoring burn severity and it has been detected through the skin. It appears likely that modern optics and electronic technology will allow the development of practical devices for biomedical use as shown in Scheme 1.

Fluorescence↗

PubMed2003

Metal-enhanced fluorescence: potential applications in HTS.

Metallic surfaces and particles can have dramatic effects on fluorescence, including localized excitation, increased quantum yields, increased photostability and increased distances for resonance energy transfer (RET), and directional emission. While all these effects have not yet been realized in a single system, metal-enhanced fluorescence promises to provide the next generation of high sensitivity fluorescence assays for low copy number detection of biochemical species.

Algorithms↗

PubMed2002

Polarization oxygen sensor: a template for a class of fluorescence-based sensors.

We describe a novel oxygen polarization sensor based on a single molecule that changes the observed anisotropy of its emission with variation of the oxygen concentration. The approach is demonstrated both in solution and in solid films. A simple mathematical description of the sensor is included and experimentally verified. The technique demonstrated here creates opportunity for development of a new class of polarization-based dyes and sensors.

Biosensing Techniques↗

PubMed2002

A novel fluorescence competitive assay for glucose determinations by using a thermostable glucokinase from the thermophilic microorganism Bacillus stearothermophilus.

We describe the use of a thermostable glucokinase in a novel competitive fluorescence assay for glucose. Glucokinase from Bacillus stearothermophilus (BSGK) was found to retain enzymatic activity in solution for over 20 days. The single cysteine residue in BSGK, which is near the active site, was labeled with a fluorescent probe, 2-(4-iodoacetamidoanilino)naphthalene-6-sulfonic acid. The ANS-labeled BSGK displayed a modest 25% decrease in the emission intensity upon binding glucose but no change in lifetime. To obtain a larger spectral change we developed a competitive assay for glucose using the intrinsic tryptophan fluorescence from BSGK and a resonance energy transfer (RET) acceptor-labeled sugar. The sugar-labeled acceptor quenched the BSGK tryptophan emission, and the quenching was reversed upon addition of glucose. The use of RET as the sensing mechanism can be easily extended to longer wavelengths for a more practical glucose sensor.

Enzyme Stability↗

PubMed2002

Radiative decay engineering. 2. Effects of Silver Island films on fluorescence intensity, lifetimes, and resonance energy transfer.

Metallic surfaces can have unusual effects on fluorophores such as increasing or decreasing the rates of radiative decay and the rates of resonance energy transfer (RET). In the present article we describe the effects of metallic silver island films on the emission spectra, lifetimes, and energy transfer for several fluorophores. The fluorophores are not covalently coupled to the silver islands so that there are a range of fluorophore-to-metal distances. We show that proximity of fluorophores to the silver islands results in increased fluorescence intensity, with the largest enhancement for the lowest-quantum-yield fluorophores. Importantly, the metal-induced increases in intensity are accompanied by decreased lifetimes and increased photostability. These effects demonstrate that the silver islands have increased the radiative decay rates of the fluorophore. For solvent-sensitive fluorophores the emission spectra shifted to shorted wavelengths in the presence of the silver islands, which is consistent with a decrease of the apparent lifetime for fluorophores near the metal islands. We also observed an increased intensity and blue spectral shift for the protein human glyoxalase, which displays a low quantum yield for its intrinsic tryptophan emission. In this case the blue shift is thought to be due to increased emission from a buried low-quantum-yield tryptophan residue. Increased intensities were also observed for the intrinsic emission of the nucleic acid bases adenine and thymine and for single-stranded 15-mers poly(T) and poly(C). And finally, we observed increased RET for donors and acceptors in solution and when bound to double-helical DNA. These results demonstrate that metallic particles can be used to modify the emission from intrinsic and extrinsic fluorophores in biochemical systems.

Adenine↗

PubMed2002

High probability of disrupting a disulphide bridge mediated by an endogenous excited tryptophan residue.

It is well known that ultraviolet (UV) radiation may reduce or even abolish the biological activity of proteins and enzymes. UV light, as a component of sunlight, is illuminating all light-exposed parts of living organisms, partly composed of proteins and enzymes. Although a considerable amount of empirical evidence for UV damage has been compiled, no deeper understanding of this important phenomenon has yet emerged. The present paper presents a detailed analysis of a classical example of UV-induced changes in three-dimensional structure and activity of a model enzyme, cutinase from Fusarium solani pisi. The effect of illumination duration and power has been investigated. A photon-induced mechanism responsible for structural and functional changes is proposed. Tryptophan excitation energy disrupts a neighboring disulphide bridge, which in turn leads to altered biological activity and stability. The loss of the disulphide bridge has a pronounced effect on the fluorescence quantum yield, which has been monitored as a function of illumination power. A general theoretical model for slow two-state chemical exchange is formulated, which allows for calculation of both the mean number of photons involved in the process and the ratio between the quantum yields of the two states. It is clear from the present data that the likelihood for UV damage of proteins is directly proportional to the intensity of the UV radiation. Consistent with the loss of the disulphide bridge, a complex pH-dependent change in the fluorescence lifetimes is observed. Earlier studies in this laboratory indicate that proteins are prone to such UV-induced radiation damage because tryptophan residues typically are located as next spatial neighbors to disulphide bridges. We believe that these observations may have far-reaching implications for protein stability and for assessing the true risks involved in increasing UV radiation loads on living organisms.

Carboxylic Ester Hydrolases↗