SCIENCE/TECHNOLOGY
Dynamic Fluorescence Spectroscopy Gains Attention of More Chemists Innovations in phase- and modulation-resolved fluorescence may allow use of technique to extract new analytical information Chemists have begun using ad vanced techniques to extract new analytical chemical information from fluorescence spectroscopy. Dy namic, multidimensional methods such as phase- and modulationresolved fluorescence have long been used by biologists to gain in formation from complex systems. Now these methods may lead to renewed interest in fluorometry by analytical and clinical chemists as well. The progress being made in dy namic fluorescence spectroscopy was the focus of talks by several speak ers early this month in Atlanta at the Pittsburgh Conference & Expo sition on Analytical Chemistry & Applied Spectroscopy (C&EN, March 20, page 26). The problem in fluorescence spec troscopy is that the same fluoresc ing species may be located in dif ferent environments of the same sample, causing them to have dif ferent lifetimes. Or lifetimes of flu orescing species may be identical, t h o u g h they move at different speeds in individual environments. Enhanced methods of modulating laser excitation light together with improved computing power have allowed chemists to devise ways of resolving very similar fluorescences. Perhaps the pioneer in applying phase- and modulation-resolved flu orescence spectroscopy to purely chemical problems is analytical chemistry professor Linda B. Mc
Gown of Duke University. Her for mer graduate student, now analyti cal chemistry professor Frank V. Bright of the State University of New York, Buffalo, has gone on to achieve his own innovations in the field. In this form of spectroscopy, the investigator applies a sinusoidal field to the excitation laser light. This causes the light to alternate at a certain frequency between "in tense" and "not-so-intense." The sample then emits fluorescent light at the same frequency. But depending on the lifetime of the fluorescence, the phase of the oscillating emitted light will be shifted by a certain number of de grees of angle. Also, at any one fre quency, the longer the lifetime, the more t h e emitted light will be demodulated. This is because longlived species cannot "keep up with" high-frequency excitation. Demod ulation appears as shallower waves, and a lower ratio of the "intense" to the "not-so-intense" fluorescent light.
(Lifetime is the time needed for the concentration of excited-state molecules to decrease to a fraction 1/e of their original concentration. The dependence o n e — t h e base of natural logarithms—arises from the first-order kinetics involved.) In Atlanta, graduate student David W. Millican of Duke described a band-pass filter that he developed with McGown, based on lifetimes rather than wavelength. In work supported by the Department of En ergy, he demonstrated qualitative analysis of mixtures of very similar compounds, benzo[fc]fluorene (life time 8 nanoseconds) and benzo[b]fluorene (lifetime 29 nanoseconds), with light modulated at 6, 18, and 30 MHz. His three-dimensional maps of ex citation wavelength versus emission wavelength versus phase-resolved fluorescence intensity showed the 8-nanosecond species predominating at 30 MHz. As he adjusted frequen cies, the 29-nanosecond compound became evident at 18 MHz and predominated at 6 MHz.
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March 27, 1989 C&EN 27
Science/Technology
Electrochemical Surface Science Molecular Phenomena at Electrode Surfaces major work bringing together the three interrelated disciplines of electrochemistry, surface science, and metal-cluster chemistry. Emphasizes the most recent and critical advancements in electrochemical research. Offers detailed descriptions on the use of ultrahigh vacuum surface spectroscopic methods in the study of the electrode-solution interface. Reports recent advances in the adaptation of new experimental techniques, from scanning tunneling microscopy to electrochemical problems. Investigates the critical aspects of in situ vibrational spectroscopy and reviews various electrode processes such as the electrochemical reactivity of polymer deposits, C02 methanation, and surface organometallic chemistry. Provides 36 chapters covering a broad but balanced range of important topics written by recognized experts in the field. An invaluable resource for students and active researchers in electrochemistry, surface science, chemical engineering, and catalysis. Manuel P. Soriaga, Editor, Texas A&M University
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March 27, 1989 C&EN
Graduate student W. Tyler Cobb of Duke described detection of peaks in high-performance liquid chromatography according to lifetimes of the components. This approach allows identification of compounds in unresolved peaks. The phase shift and the demodulation each give an independent value of the lifetime. If these are different, then the user knows that the cause is an unresolved chromatographic peak. Bright described applications of phase- and modulation-resolved fluorescence spectroscopy both to improvement of clinical fluoroimmunoassays and to studies of solvation by supercritical fluids. He worked with graduate students Thomas A. Betts, Gino C. Catena, Huang Jingfran, and Kevin S. Litwiler, and undergraduate student David P. Paterniti. The work was supported by the National Institutes of Health, the Petroleum Research Fund, and 3M Co. Immunoassays depend on competitive binding to an antibody by an unlabeled antigen—such as drug molecules in a patient's serum—and a labeled antigen added to the reaction mixture. Bright used the drugs phenytoin or theophylline covalently bound to fluorescein. With little or no drug in the sample, the technician detects relatively large amounts of labeled drug in the antigen-antibody complex. For samples containing greater amounts of unlabeled drug, a lesser proportion of labeled drug appears in the complex. Immunoassays may be heterogeneous or homogeneous. In heterogeneous immunoassays, the technician separates the antigen-antibody complex from excess added labeled drug before measuring label intensities. Faster, cheaper homogeneous immunoassays would eliminate the separation step. The Buffalo chemists' approach to distinguishing labeled complexes from unbound labeled antigen in homogeneous immunoassays was to measure the rotation times of each. Phenytoin-fluorescein molecules have molecular weights of only several hundred daltons and fast rotation times. Antibody complexes have molecular weights of 150,000 daltons and rotate much more slowly.
The SUNY workers determined amounts of species of different rotation times by use of modulated polarized light. Only molecules whose electric dipoles were oriented parallel to the plane of polarization would absorb the light. Fluorescent light from molecules that rotated little before fluorescence would also be polarized mostly parallel to the plane. Light from fasterrotating species would contain a certain amount oriented perpendicular to the plane of polarization. Bright's group measured phase shifts and extents of demodulation of fluorescent light emitted parallel and perpendicular to the plane of polarization. The researchers used these data to calculate concentrations of species of different rotation times. The investigators ascribed an observed rotation time of 1 nanosecond to fluorescein rotating rapidly about the bond connecting it to the unbound drug. A 1-nanosecond lifetime also appeared for fluorescein rotating rapidly within the antibody complex. They assigned a 28-nanosecond rotation time to the antibody complex itself. Interestingly, this was not a rotation of the entire complex, which was expected at 100 nanoseconds, and which was too slow to observe. One flexible arm of the antibody apparently rotates within the complex, producing the 28-nanosecond time. Bright has also demonstrated optical fibers for transmitting modulated light to the sample and demodulated, phase-shifted light from sample to detector. This feature may be useful for remote-sensing clinical instruments. It may also lead to studies in which antibodies immobilized on fibers change fluorescent parameters on complexing antigens. The Buffalo team also studied solvation of fluorescent 4-amino-Nmethylphthalimide (4-AMP) in supercritical carbon dioxide with a small amount of 2-propanol added as a modifier. Supercritical carbon dioxide is a very dense gas (about 0.5 g per mL), made by heating and compressing the compound above its critical temperature of 31 °C. Thus it is a bridge between gases and liquids for studies of solvation. Such studies are significant, be-
cause supercritical fluids have become increasingly important in recent years in research and industry. Supercritical carbon dioxide is used both as a mobile phase in chromatography and for such extractions as caffeine from coffee. The SUNY researchers expected that solvation would alter the fluorescence lifetime of 4-AMP as the solvent relaxed the excited molecules on a picosecond time scale. By measuring phase shifts and demodulations of exciting light modulated at many different frequencies, they calculated fluorescence lifetimes present. The best fit to the data was a spectrum of lifetimes, which they ascribed to 4-AMP molecules complexed with anywhere from one to 14 molecules each of 2-propanol. Also calculated from the data were equilibrium formation constants and enthalpies, entropies, and free energies of 4-AMP/2-propanol complexes. Stephen Stinson
Research on ligninases takes big step forward Efforts to harness for practical purposes the lignin-degrading enzymes of the white rot fungus Phanerochaete chrysosporium have been brought somewhat closer to reality as a result of recently reported research from scientists at Oregon Graduate Center. Michael H. Gold, professor and chairman of the Beaverton-based graduate center's department of chemical and biological sciences, and coworkers have made major contributions toward understanding the biodégradation of lignin, which is the second most abundant natural polymer. Lignin is a random phenylpropanoid matrix that makes up 20 to 30% of woody plants and retards microbial depolymerization of cellulose. Two extracellular enzymes produced by P. chrysosporium, lignin peroxidase and manganese peroxidase, are responsible for the breakdown of lignin. Lignin peroxidase (LiP) was discovered independently in 1983 in Gold's lab and in the labo-
ratory of T. Kent Kirk at the U.S. Forest Products Laboratory in Madison, Wis. Gold's group discovered manganese peroxidase (MnP) the following year. With coworkers Margaret Alic, Janet R. Kornegay, and David G. Pribnow, Gold has now developed the first DNA transformation system for P. chrysosporium [AppL & Environ. Microbiol. 55, 406 (1989)]. The system represents "an important step toward the production of large amounts of ligninases," Gold says. It will also facilitate studies on the regulation and expression of the genes that encode LiP and MnP as well as genetic approaches to structure-function studies of the enzymes. Gold's research is supported by the Department of Energy, Department of Agriculture, and National Science Foundation. Additionally, Gold, Pribnow, and coworkers Mary B. Mayfield, Valerie J. Nipper, and Julie A. Brown have characterized for the first time a
cDNA (that is, a deoxyribonucleic acid copy of a messenger ribonucleic acid) that encodes MnP [/. Biol. Chem., 264, 5036 (1989)]. Combined with the P. chrysosporium transformation system, as well as research by other groups, Gold says, the results of the research on the MnP gene should allow the Oregon Graduate Center scientists to produce large amounts of recombinant LiP and MnP in the near future. LiP and MnP, or modified organisms that produce these enzymes themselves, could find use in a number of bioprocessing applications, Gold says. There are a number of points in the production of paper, for example, where the enzymes might be used in place of traditional chemistry. Also, numerous possibilities exist for the use of these enzymes as nonspecific oxidative reagents for degrading aromatic environmental pollutants such as chlorophenols and dyes. Rudy Baum
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March 27, 1989 C&EN
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