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Molecular Fluorescence, Phosphorescence, and Chemiluminescence Spectrometry Susmita Das,† Aleeta M. Powe,‡ Gary A. Baker,§ Bertha Valle,^ Bilal El-Zahab,z Herman O. Sintim,|| Mark Lowry,# Sayo O. Fakayode,r Matthew E. McCarroll,O Gabor Patonay,[ Min Li,0 Robert M. Strongin,# Maxwell L. Geng," and Isiah M. Warner*,† †
Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803, United States Department of Chemistry, University of Louisville, Louisville, Kentucky 40208, United States § Department of Chemistry, University of MissouriColumbia, Columbia, Missouri 65211-7600, United States ^ Department of Chemistry, Texas Southern University, Houston, Texas 77004, United States z Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, United States # Department of Chemistry, Portland State University, Portland, Oregon 97207, United States r Department of Chemistry, Winston-Salem State University, Winston-Salem, North Carolina 27110, United States O Department of Chemistry and Biochemistry, Southern Illinois University, Carbondale, Illinois 62901-4409, United States [ Department of Chemistry, Georgia State University, Atlanta, Georgia 30302-4098, United States 0 Process Development Center, Albemarle Corporation, Baton Rouge, Louisiana 70805, United States " Department of Chemistry, University of Iowa, Iowa City, Iowa 52242, United States
)
‡
’ CONTENTS Books, Reviews, and Chapters of General Interest Instrumentation and Laser Based Fluorescence Techniques Instrument Qualification and Standardization of Measurements Fluorescence Correlation Spectroscopy Single-Molecule Fluorescence Super-Resolution Imaging Improvements in Conventional Fluorescence Imaging Fluorescence Lifetime Imaging Resonance Energy Transfer Nonlinear Processes Sensors Sample Preparation, Quenching, and Related Phenomena Data Analyses Organized Media Low Temperature Luminescence Solid Surface Luminescence Luminescence in Chromatography, Electrophoresis, and Flow Systems Dynamic Luminescence Measurements Fluorescence Polarization, Molecular Dynamics, and Related Phenomena Chemiluminescence r 2011 American Chemical Society
Near-Infrared Fluorescence Luminescence Techniques in Biological and Clinical Analysis Reagents and Probes Green Chemistry Fluorescent Nanoparticles Total Luminescence and Synchronous Excitation Spectroscopies and Related Techniques Author Information Biographies References
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s a result of change in due date, this Review covers the one and two-third-year period since our last review1 from January 2010 through August 2011. Manuscripts outside this review period are only included if they have not been previously cited in the review. A computer search of Chemical Abstracts provided most of the references for this Review. A search for documents written in English containing the terms “fluorescence or phosphorescence or chemiluminescence” published in 2010 to August 2011 resulted in more than 75 000 hits. An initial screening reduced this number to approximately 18 000 publications that were considered for inclusion in this Review. Key word searches of this subset provided subtopics of manageable sizes. Other
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the fluorescence of nanosystems, polymers, and supermolecules, as well as the development and application of fluorescent probes.6 Practical Fluorescence Microscopy in Mammalian Cells: Protein Localization and Function offers a multidisciplinary approach to the application of fluorescence microscopy in cell imaging.7 In Single Molecule Biophysics: Experiments and Theories, T. Komatsuzaki et al. discuss single-molecule fluorescence based mainly on fluorescence resonance energy transfer (FRET), atomic force microscopy (AFM), and diffracted X-ray tracking (DXT), and their applications to important biological phenomena.8 Fluorescent Proteins II: Application of Fluorescent Protein Technology edited by G. Hung covers the fundamental properties and specific applications of fluorescent proteins in various areas.9 In addition to the above-mentioned books and book chapters, some useful review articles published since 2010 summarize and critically review the developments and applications of fluorescence techniques. Coverage here is limited to a small number of reviews of broader interest. Many other reviews that focus on narrower and more specific topics are included in the various sections of this manuscript. The Journal of Chemical Review, an ACS publication, dedicated an entire issue (Volume 110, Issue 5) to cover the latest development in molecular imaging including fluorescence and luminescence techniques. For example, Tor et al. reviewed the design, properties, and applications of fluorescent analogs for biomolecular building blocks.10 In another article, Kobayashi et al. summarized new strategies for fluorescent probe design in medical diagnostic imaging.11 Introduction and application of lanthanide luminescence for biomedical analyses, as well as cell and tissue imaging, are presented by B€unzli.12 Also, topics covered fluorescence lifetime measurements and fluorescence polarization/anisotropy along with their applications in diagnostics and biological imaging.13,14 Applications of fluorescence in areas other than biology and medicinal chemistry have also been reviewed in some useful articles. A tutorial review covers recent developments in the use of single-molecule fluorescence microscopy to study nanoscale catalysis.15 Another review article discusses the different conceptual approaches that have been developed to study molecular concentration and dynamics like diffusion and catalytic conversion at the micrometer and submicrometer levels.16 The research on label-free native fluorescence detection in analytical chemistry was reviewed, with particular focus on the instrumental requirements and applications.17 A tutorial review summarizes the progress of polymer-based fluorescent and colorimetric chemosensors.18 A critical review focuses on the design principles and the recent development of phosphorescent chemosensors for metal cations, anions, pH, oxygen, volatile organic compounds, and biomolecules detection.19
citations were found through individual searches by the various authors who wrote a particular section of this Review. In an effort to more effectively accomplish this goal, we have included authors who are knowledgeable in the various subtopics of this Review. Coverage is limited to articles that describe new developments in the theory and practice of molecular luminescence for chemical analysis in the ultraviolet, visible, and near-infrared region. We have included two new sections in this Review on fluorescence nanoparticles and green chemistry. Discussions of cited work are intended to be more critical and focused than in previous reviews. In general, citations are limited to journal articles and do not include patents, proceedings, reports, and dissertations. In an effort to reduce the length of this Review we have attempted to limit duplicate citations between sections by retaining the same reference number for a given citation which is cited under more than one heading. As in previous years, we are not able to provide extensive coverage of all developments of relevance to the extremely broad field of molecular fluorescence, phosphorescence, and chemiluminescence. Instead, we have focused on important advances of general interest and relevance to the field of analytical chemistry, rather than extensions of previous advances. In addition, we have attempted to balance inclusion of a sufficient number of highly relevant, high-impact references to adequately survey the field with ample descriptions of individual citations for better clarification. If you feel that we have omitted an important article published during the above referenced time period, please forward the reference to one of us and we will be certain to consider it for the next review.
’ BOOKS, REVIEWS, AND CHAPTERS OF GENERAL INTEREST A number of books and book chapters published in the last two years have summarized and discussed the mechanisms, developments, and applications of fluorescence techniques. These books either are comprehensive or tutorial, covering a broad spectrum of areas including principles and applications, or focus on specific areas in which fluorescence techniques are employed. For example, Reviews in Fluorescence in 2009 and 2010, the sixth and seventh volumes of the book serial edited by Geddes,2,3 provide a comprehensive collection of current trends and emerging topics in the field of fluorescence and closely related disciplines. Another book, Lanthanide Luminescence: Photophysical, Analytical and Biological Aspects compiled by P. H€anninen et al. contains comprehensive information about the photophysical basics and relevant lanthanide probes or materials and also describes instrumentation-related aspects including chemical and physical sensors.4 The uses of lanthanides in bioanalysis and medicine such as assays for in vitro diagnostics and research are also outlined. Luminescence Applied in Sensor Science edited by L. Prodi et al. summarizes luminescence signaling systems and luminescence-based devices since fluorescence measurements are usually very sensitive, low cost, versatile, easily performed, and offer submicrometer visualization and submillisecond temporal resolution.5 Recently, fluorescence technique has increasingly found interesting and important applications in nanotechnology, biotechnology, and medicinal chemistry. Numerous books or book chapters provide outlines on significant progress about the use of luminescence technology in these areas. For instance, Fluorescence of Supermolecules, Polymers, and Nanosystems edited by M. N. Berberan-Santos focuses on
’ INSTRUMENTATION AND LASER BASED FLUORESCENCE TECHNIQUES In previous editions of this Review, the “General Instrumentation” and “Laser-based Techniques” sections were separate. Recently, they were combined in an effort to eliminate overlap and improve continuity. We, again, combine these sections as it has become increasingly difficult to differentiate these topics. In an attempt to improve flow, this combined section is divided into eight subtopics. A small number of examples5 are included for each subtopic. Emphasis was placed on references that focus on the instrumentation or fundamentals of a particular technique. 598
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the application and potential of FCS and fluorescence crosscorrelation spectroscopy (FCCS) in vivo.25 Practical issues and sources of artifacts when performing measurements in living cells were discussed. Cross-talk that results from spectral overlap can reduce accuracy and limit potential applications of FCS. In an effort to overcome this drawback, Lee et al. developed an instrument for spectral cross-talk-free dual-color FCCS.26 Two spectrally distinct fluorophores, (Cy3 and IRD800) were excited simultaneously using two different excitation sources (532 and 780 nm) with the fluorescence information processed on two different color channels monitored with single-photon avalanche diodes (SPADs). No color cross-talk (cross-excitation and/or cross-emission) and/or fluorescence resonance energy transfer (FRET) was observed, which significantly improved data quality. FCS can be performed on commercial devices, which makes the technique more accessible. Two examples follow. Total internal reflection-fluorescence correlation spectroscopy (TIR-FCS) has numerous applications in biology, physics, and material science; however, use of TIR-FCS is still rather limited largely because of the need for a complex, in-house constructed optical setup whose assembly and adjustment is difficult. No commercial instrument is available, but Yordanov et al. demonstrate that the proper combination of commercial devices for confocal fluorescence correlation spectroscopy and for total internal reflection microscopy may enable TIR-FCS without the need for any special optical alignments.27 Capabilities of the setup were demonstrated by measuring the diffusion coefficient of single dye molecule and quantum dots in proximity of a waterglass interface. Despite the fact that FCS was developed using analog detectors, there is now a widespread belief that photon counting detectors and avalanche photodiodes are essential to perform FCS experiments. However, FCS using analog detection on a commercial microscope is possible. Moens et al. report FCS, raster image correlation spectroscopy, and number and brightness on a commercial confocal laser scanning microscope with analog detectors (Nikon C1).28 The authors noted that each analog instrument has its own idiosyncrasies that need to be understood before using it for FCS. Care should be taken in selecting the acquisition parameters to avoid possible artifacts due to the detector noise. Single-Molecule Fluorescence. The number of reports per year on single-molecule imaging continues to grow roughly exponentially. In a recent review, Moerner and co-workers discussed single-molecule spectroscopy and imaging of biomolecules in living cells.29 It is impossible to survey comprehensively this growing field. Only some recent highlights are included below. Single-molecule fluorescence spectroscopy was first demonstrated at near-absolute zero temperatures but now include roomtemperature as well as native in vivo observations. Measurements at temperatures above 37 C have been difficult. The indexmatching fluids used with high-numerical-aperture objective lenses can conduct heat from the sample to the lens which can cause the lens to fail. Schwartz et al. overcame this issue using TiO2 colloids with diameters of 2 μm and a high refractive index which can act as lenses allowing real-time single-molecule measurements of mesophilic and thermophilic enzymes at 70 C.30 Xie et al. demonstrated the use of vertically aligned silicon dioxide nanopillars to achieve below-the-diffraction-limit observation volume in vitro and inside live cells.31 The highly confined illumination volume allowed in vitro single-molecule detection at high fluorophore concentrations. Also, the vertical nanopillars
Instrument Qualification and Standardization of Measurements. There is an ongoing effort to standardize fluores-
cence measurements. Recently, two excellent resources became available to aid this effort. An IUPAC Technical Report on fluorescence standards was released.20 Chromophore-based fluorescence standards for the characterization of photoluminescence systems and the determination of relevant fluorometric quantities were classified according to their scope and area of application. Recommendations were given on the selection, use, and development of fluorescence standards. In another effort, ASTM International (ASTM) released a Standard Guide for Fluorescence, a clear and concise reference geared for users of fluorescence instrumentation at all levels of experience. A review of this document was published by P.C. DeRose and U. Resch-Genger.21 The guide focused on steady-state fluorometry including available standards and instrument characterization procedures. It covered the most relevant instrument properties that need qualification (i.e., linearity and spectral responsivity of the detection system, spectral irradiance reaching the sample, wavelength accuracy, sensitivity or limit of detection for an analyte, and day-to-day performance verification). Procedures for the determination of other relevant fluorometric quantities including fluorescence quantum yields and fluorescence lifetimes were also introduced briefly. Quantum yield measurements received a great deal of additional attention. Resch-Genger and co-workers compared two relative and one absolute fluorometric method for determination of the fluorescence quantum yields of quinine sulfate dihydrate, coumarin 153, fluorescein, rhodamine 6G, and rhodamine 101.22 Complete uncertainty budgets for the resulting quantum yield values were derived for each method, and standard operation procedures were provided. The work simultaneously resulted in a set of assessed quantum yield standards. In related work, Rurack and Spieles recently determined the fluorescence quantum yields of a series of red and near-infrared emitting dyes with absorption/ excitation and emission ranges of 520900 and 6001000 nm.23 The quantum yields were measured relative to the National Institute of Standards and Technology’s standard reference material (SRM) 936a (quinine sulfate, QS) on a traceably characterized fluorometer, that employs a chain of transfer standard dyes. Recently, the fluorescence and phosphorescence quantum yields of standard solutions were re-evaluated based on an absolute method using an integrating sphere equipped with a multichannel spectrometer.24 The technical aspects in the determination of absolute emission quantum yields for lanthanide complexes and those of organic crystals of anthracene were examined. It was found that special care must be taken for the accurate determination for lanthanide complexes, because of their narrow absorption and emission bands. Fluorescence Correlation Spectroscopy. Fluctuation-based techniques including fluorescence correlation spectroscopy (FCS) and its tandem methods including dual-color cross-correlation, total internal reflection fluorescence correlation, and fluorescence lifetime correlation spectroscopy continue to receive a great deal of attention. The scope of this topic is far greater than can be covered comprehensively in this space. Two informative review articles that place an emphasis on applications of the technique are provided, first followed by a limited number of representative reports that focus on instrumentation. FCS is a powerful technique that can accurately probe the dynamics, local concentration, and photophysics of single molecules both in vitro and in vivo. Schwille and co-workers reviewed 599
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interfaced tightly with live cells and functioned as highly localized light sources inside the cell. Single molecule localization and tracking received increased attention. Moerner and co-workers reported a widefield fluorescence microscope exhibiting a double-helix point spread function (DH-PSF) for three-dimensional nanoscale localization (10 nm localization capability along x, y, and z) and tracking of dim single emitters.32 Single quantum dots in aqueous solution and a quantum dot-labeled structure inside a living cell in three dimensions were tracked using the DH-PSF. Smith et al. reported an iterative algorithm for determining the maximum likelihood estimate of the position and intensity of a single fluorophore that achieves theoretically minimum uncertainty.33 The technique enables real-time data analysis for super-resolution imaging. Super-Resolution Imaging. The field of super-resolution imaging continues to expand by providing valuable information about never-before-seen phenomena that occur outside the reach of conventional imaging techniques. Only a decade ago, reports were relatively rare, but today hundreds of reports that take advantage of and advance the technique are reported each year. Only a small sampling can be included. Huang et al. reviewed the principles of various super-resolution approaches and described recent applications of super-resolution microscopy in cells.34 It was demonstrated that super-resolution microscopy provided new insights into cell biology, microbiology, and neurobiology. Sauer and co-workers reported a stepwise protocol for dSTORM imaging in fixed and living cells on a widefield fluorescence microscope with standard fluorescent probes.35 The protocol focuses on the photoinduced adjustment of the ratio of fluorophores in the “on” and “off” states. Data acquisition and data processing can be performed in seconds to minutes. Fast super-resolution fluorescence imaging of live cells with high spatiotemporal resolution was reported using stochastic optical reconstruction microscopy (STORM) via labeling proteins with photoswitchable dyes.36 Two-dimensional (2D) and 3D super-resolution images of living cells were obtained with clathrin-coated pits and transferrin cargo as model systems. Bright, fast-switching probes enabled spatial resolutions of 25 nm and temporal resolutions as fast as 0.5 s. Photoswitchable dyes with distinct emission wavelengths also allowed two-color 3D superresolution imaging in live cells. In another interesting report, Hell and co-workers used fast stimulated emission depletion (STED) microscopy for the dynamic imaging of colloidal-crystal nanostructures at 200 frames per second.37 The technique was used to visualize the annealing of potential point defects during the formation of the colloidal crystal. Improvements in Conventional Fluorescence Imaging. Many advances were reported in conventional fluorescence imaging. Betzig and co-workers addressed a key challenge when imaging living cells, i.e., how to noninvasively extract the most spatiotemporal information possible, using a form of planeillumination microscopy.38 Plane-illumination limits excitation to the information-rich vicinity of the focal plane, which provides effective optical sectioning and high speed while reducing photobleaching and minimizing out-of-focus background. Scanned Bessel beams were used in conjunction with structured illumination and/or two-photon excitation to create thinner light sheets better suited to three-dimensional (3D) subcellular imaging (resolution down to similar to 0.3 μm, speeds up to nearly 200 image planes per second).
Mertz and co-workers presented HiLo microscopy, a simple wide-field imaging technique that is capable of optically sectioned images in real time with comparable quality to confocal laser scanning microscopy.39 Raw images acquired with speckle illumination were fused with others from standard uniform illumination to give optically sectioned images of varying thicknesses from the same raw data. The technique is capable of near video rate imaging over larger fields of view than attainable with a standard confocal microscope. In another interesting advance, Muller and Enderlein combined the resolving power of confocallaser scanning microscopy with that of a wide-field imaging microscopy that uses conventional confocal-laser scanning microscopy with fast wide-field CCD detection.40 The technique doubled the lateral optical resolution. Advances can come also in the form of reduced cost or complexity. Richards-Kortum and co-workers reported a fiber-optic fluorescence microscope that uses a consumer-grade digital camera for in vivo cellular imaging.41 The portable, inexpensive unit, including an LED light, an objective lens, a fiber-optic bundle, and a consumer-grade digital camera, has potential for use at point-of-care in low-resource settings. In other work, Zhu et al. demonstrate wide-field fluorescent and darkfield imaging on a cell phone.42 The cost-effective and compact imaging platform attached to a cell phone could be quite useful especially for resource-limited settings. Fluorescence Lifetime Imaging. There has been an increase in applications of lifetime imaging in recent years. This is largely the result of technique maturation followed by the commercial availablility of instrumentation. Berezin and Achilefu recently reviewed fluorescence lifetime measurements and biological imaging including fluorescence lifetime imaging applications.13 Some interesing reports that focus on both instrumentation and applications are included below. Rapid acquisition of time-resolved fluorescence data is essential for microscopy applications, such as fluorescence lifetime imaging. With this in mind, McLoskey et al. investigated a high (100 MHz) repetition rate semiconductor laser excitation source optimally matched to low dead-time counting electronics for fast time-correlated single-photon counting fluorescence acquisition.43 The fastest time to acquire data, with possible FLIM applications in mind, was estimated and demonstrated using a representative dye. Leray et al. compared the Polar (or phasor) approach to the least square fitting method in time domain FLIM image analysis.44 It was found that, in the presence of as little as 5% fluorescence background, the least square fitting does not provide the best estimator of the lifetime parameter for fluorophores that exhibit monoexponential intensity decays. It was demonstrated that in living cells the polar approach distinguished two fluorophores undetectable with usual time-domain least-squares fitting software. Wide-field multi-parameter FLIM (WFMP-FLIM) was reported for long-term minimally invasive observation of proteins in living cells under minimum light intensity.45 Vitali et al. demonstrated efficiency WFMP-FLIM both in FRET analysis of simultaneously recorded donor and acceptor fluorescence in living HeLa cells and in the tracking of mitochondrial transport combined with fluorescence lifetime analysis in neuronal processes. Use of lanthanide coordination complexes or other probes with long emission lifetimes eliminates short-lifetime autofluorescence background from biological specimens; however, lanthanide complexes emit far fewer photons per unit time than conventional fluorescent probes. It is difficult to acquire rapidly 600
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Analytical Chemistry high quality images at probe concentrations that are relevant to live cell experiments. To address this, Gahlaut et al. described the development and characterization of a time-resolved luminescence (TRL) microscope with pulsed light-emitting diode (LED) epi-illumination and an intensified charge-coupled device (ICCD) camera for gated, widefield detection.46 TRL microscopy was sufficiently sensitive and precise to allow high-resolution and quantitative imaging of lanthanide luminescence in living cells under physiologically relevant experimental conditions. Resonance Energy Transfer. Forster resonance energy transfer (FRET) is a powerful technique used to investigate the detailed mechanisms of biological systems at the molecular level. Sahoo recently reviewed the basic principles and applications of FRET in chemistry, biology, and physics.47 The following are a sampling of the exciting advances reported during this review period. Powerful as it may be, conventional two-color FRET cannot capture the intrinsic complexity of many biological systems. In recent years, three-color FRET was developed. Additionally, four-color FRET now is being used in even more complex systems. Lee et al. determined in real time six interfluorophore FRET efficiencies single-molecule four-color FRET.48 The technique was used to probe the correlated motion of the four arms of the Holliday junction and to assess correlatation of RecAmediated strand exchange events. Recently, Stein et al. used single-molecule four-color FRET with alternating laser excitation to sort subpopulations and to visualize the control of energytransfer paths on DNA Origami.49 Uphoff et al. described a new method called “switchable FRET” which combines single-molecule fluorescence resonance energy transfer (FRET) with the reversible photoswitching of fluorophores.50 Switchable FRET sequentially analyzes FRET between a single donor and spectrally identical photoswitchable acceptors. This reduces complexity by enabling direct monitoring of multiple distances. DNA molecules, proteinDNA complexes, and dynamic Holliday junctions are systems that were used for showcasing switchable FRET for studying dynamic, multicomponent biomolecules. Rajapakse et al. used time-resolved luminescence resonance energy transfer (LRET) to enable live-cell imaging of protein protein interactions.51 A luminescent terbium complex, TMPLumi4, was introduced into cultured cells and bound specifically to transgenically expressed Escherichia coli dihydrofolate reductase (eDHFR) fusion proteins. LRET between the eDHFR-bound terbium complex and green fluorescent protein (GFP) was detected as long-lifetime, sensitized GFP emission. Background signals were eliminated using a time delay between excitation and detection. Nonlinear Processes. Several advances in multiphoton probes and proteins, instrumentation, and applications were reported. Only a small fraction can be included. Bunzli investigated multiphoton-excited luminescent lanthanide bioprobes52 The feasibility of [Eu2(LC5)3] as a multiphoton luminescence bioprobe was demonstrated by two-photon scanning microscopy imaging experiments on HeLa cells. In another probe-related report, Drobizhev et al. reviewed the two-photon absorption properties of a wide variety of fluorescent proteins and provided a comprehensive guide to choosing the appropriate protein and excitation wavelength in two-photon applications.53 A wide range of excitation wavelengths has been used in multiphoton microscopy (MPM): however, the emission has been limited to visible wavelengths. An interesting instrument allowed MPM with near-infrared contrast agents. Yazdanfar et al. reported an all-NIR system with nonlinear excitation at 1550 nm
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which expands the range of available MPM fluorophores and virtually eliminates background autofluorescence.54 The system allows for use of fiber-based, turnkey ultrafast lasers developed for telecommunications. Kirkby et al. reported a high speed 3D acousto-optic lens microscope (AOLM) for femtosecond 2-photon imaging.55 Numerous features, including 2-photon imaging at 10-fold lower power than previously reported, high speed 2D raster-scan, and 3D random access, are likely to make the AOLM a useful tool for studying fast physiological processes distributed in 3D space. In other work, Truong et al. combined nonlinear excitation with the orthogonal illumination of light-sheet microscopy to image two times deeper and ten times faster than point-scanning two-photon microscopy.56 The performance was demonstrated through live imaging of fruit fly and zebrafish embryos.
’ SENSORS The development and application of fluorescent systems for sensing or reporting on the concentration of a particular analyte continues to be an extremely active area of research. A somewhat restrictive search still reported nearly 600 publications during the time period covered by this Review. Here, we report a smaller number of papers that we feel represent important and interesting developments. It should be noted the term sensor often is broadly used to describe systems that may not fit a rigorous definition. For the purposes of this Review, we used the broader sense of the term and focused on developments that rely on a molecule (or nanoparticle) as a reporter moiety that communicates presence of an analyte via modulation of a fluorescence signal. As was the case in our last review, the detection of explosives continues to be the focus of much work. Several papers focused on the perturbation or quenching of fluorescence following interaction of a fluorescent species with an explosive molecule, such as trinitrotoluene (TNT). For example, an interesting paper from Chen’s group looked at the selective response of nanoparticles to nitroaromatic derivatives.57 The particles were composed of pyrene functionalized Ru nanoparticles, bound through olefin metathesis reaction by Rudcarbene bonds. Fluorescence quenching as a function of analyte concentration was examined for five different nitroaromatic compounds. The detection was based on fluorescence quenching of pyrene in the presence of nitroaromatic compounds. Two different olefins were examined, and while both exhibited a response toward various nitroaromatic compounds, the vinylpyrene system was significantly more selective toward TNT and dinitrotoluene (DNT) compounds. Sensitivity was demonstrated with detection limits reaching the nM concentration range. Also along the lines of the use of fluorescence quenching as a response mechanism was a paper published by Peng et al. that describes a molecular-based system that was developed for the detection of 2,4,5-trinitrophenol (TNP).58 The detection of TNP is important as an explosive, yet it has not received as much attention as TNT. The fluorescent sensor is a derivatized form of anthracene that has an emission maximum around 483 nm, which decreases significantly in intensity with the addition of TNP. Further, the response was found to be extremely selective toward TNP when screened against solutions that contain various other explosives, such as TNT, DNT, and RDX. The authors went a step further and ascribed the response to the formation of a hostguest complex between the fluorophore and the analyte. The solid-state precipitates were investigated and 601
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by Hudson et al.64 This work details further computational studies of a PET based probe for Zn2+. The new studies used B3LYP and time-dependent B3LYP calculations to determine the HOMO and LUMO energy levels using four different basis sets that showed good concurrence. A useful result was the finding that computational predictions were the same either when the whole molecule was considered or when the electron receptor and fluorophore moieties were calculated separately. Another divalent metal ion of significant interest is Hg2+, both for its importance as a toxic metal in the environment and because it is a challenging ion for which to design an effective fluorescence sensor. An interesting approach where the signal transduction results from pyrene excimer emission is represented in a paper published by Zhou et al.65 The fluorophore is devised such that in the free form the pyrene moieties are separate and only express a weak fluorescence signal. However, the probe is designed so that when it coordinates with the mercury ion the pyrene moieties collapse into a face-to-face orientation which results in excimer fluorescence. The selectivity toward Hg2+ was quite good when tested against a number of other metal ions. A detection limit of 0.2 μM was reported for this system. Page et al. also reported developments in the area of mercury detection.66 They developed a novel strategy for the use of quantum dots coupled to a Zn responsive chromophore that acts as a FRET pair with the quantum dot. The dye chromophore moiety, a thiosemicarbazide-functionalized rhodamine B, is adsorbed to the quantum dot. Following exposure to mercuric ions and the subsequent desulfurization reaction, the dye returns to its normal fluorescent state, where it acts as an energy acceptor from the quantum dot. The combination of FRET-based fluorescence of the chromophore and the quantum dot emission results in a ratiometric response with a reported detection limit of 79 ppb. Also, there were a significant number of manuscripts detailing sensors for various reactive oxygen species. Ganea et al. developed a nanoprobe system for the detection of hydroxyl radical.67 The biocompatible nanoparticles were developed from molecular micelles composed of lysine-coumarin 3-carboxylic acid, which is responsive to hydroxyl radical. The reference dye, neutral red, was encapsulated within the core of the particle. The analytical signal was derived from the ratio of 7-hydroxy coumarin 3-carboxylic acid and neutral red dye. The system was shown to be selective toward hydroxyl radical, and the system was used to detect hydroxyl radicals in vitro in viable breast cancer cells exposed to oxidative stress. Srikun et al. reported a new approach for imaging hydrogen peroxide in living cells.68 Their approach takes advantage of SNAP-tag technology for site-specific protein labeling of the surface or interior of the cells. The sensing response is based on the chemoselective hydrogen peroxide deprotection of functionalized fluorophores with boronate esters. This combination allowed selective labeling in the cellular system and selective spectral response to hydrogen peroxide. Organelletargeted detection and imaging of H2O2 was demonstrated for several types of living cells with subcellular resolution. An additional common topic was the development of pH sensitive fluorescent probes. Best et al. detailed an investigation of pyronin B derivatives that operate as photoinduced electrontransfer (PET) sensors for pH.69 Three of the four derivatives were quenched as a function of pH through a spyrocyclic quenching mechanism, and the fourth was found to operate through a PET based mechanism. Another system based on a phosphorescent metallophrphyrin dye was reported.70 In this system, a dual response was reported for dissolved oxygen and pH.
the analyteligand complex was found to have a bright red color, compared to the yellow color observed for the ligand (fluorescence sensor) alone. Analysis of the solid complex confirmed that the sensing response was a result of organized self-assembly during the sensing process. Also relying on the formation of hostguest complexes for a sensing response was a paper published by Anslyn and Ponnu.59 The system is based on the formation of a cyclodextrin inclusion complex formed by 9,10-bis(phenylethynyl)anthracene (BPEA), the cyclodextrin, and the nitroaromatic compounds. While some response was observed in the absence of cyclodextrin, the response was enhance and more selective with cyclodextrin. SternVolmer analysis of the response was shown to capably classify aromatic and nonaromatic explosives. Another interesting application that was published during the review period is based on the dual-emission of hybrid quantum dots. Zhang et al. reported the development of sensors based on dual emission quantum dots that respond to the presence of TNT.60 The system is based on nanoparticles that are composed of two different sized CdTe quantum dots in a silica nanoparticle matrix. The larger, red emitting quantum dots are imbedded through the core of the silica particle, and the smaller green emitting quantum dots are tethered to the surface. The net color that is observed changes when the green surface-bound quantum dots are quenched through interaction with TNT. Then, the system changes from green to red, which allows naked eye detection of TNT when illuminated with UV light. The authors went a step further by demonstrating a practical application using a contact-lift method for sampling. The TNT particulates were detected on the surface of various materials at concentrations ranging from 5 to 1000 ng/mm2. There were a large number of papers detailing the detection of ionic species that use fluorescence probes or sensors. A common theme continued from the last review period is the search for effective zinc sensors, especially for applications in imaging cellular systems. Xu et al. reported the development of a system capable of selective zinc sensing with good cell permeability.61 The molecule was determined to undergo an amide-imidic tautomerization process when bound to M2+ ion, with excellent selectivity toward Zn2+. The fluorescence was enhanced 22-fold and red-shifted from 283 to 514 nm. The system was shown to successfully image intracellular Zn2+ ions during the development of living zebrafish embryos. Another interesting system for the detection of zinc was reported by Nagano’s group.62 The response mechanism of this system is based on internal charge transfer (ICT), which results in a remarkable fluorescence enhancement and a blueshift in the absorption maxima. This is a significant aspect of the reported research, as it addresses the common problem of the fluorophore still fluorescing in the “off” state. Because the fluorescence “on” state is accompanied by the shift in absorption maxima, the amount of fluorescence signal in the “off” state is greatly minimized due to the decreased absorptivity of the “off” state molecule. Use of the probe in imaging Zn2+ ions in HeLa cells was demonstrated. Du and Lippard reported the development of another system for the detection of zinc.63 The base fluorophore of this system is rhodamine B, which is known to have good quantum yields and stability. Following interaction with Zn2+, the nonfluorescent form undergoes a 220fold increase in fluorescence intensity due to the opening of the spirolactam ring. The response was reported to be highly specific with no significant response from other competitive cations. Live cell imaging of HeLa cells was demonstrated also for this system. Another computationally based work involving zinc was published 602
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The phosphorescence of the dye is quenched by oxygen, and protonation of the dye results in a significant shift in the spectrum. The dye was immobilized in a polymer membrane, where the system operated satisfactorily between pH range from 6 to 8 and 0200 μM O2. In a very different approach to imaging intracellular pH, Tantama et al. demonstrated a genetically encoded red fluorescent protein (RFP) sensor.71 Due to excited-state proton transfer, the probe has an extremely long stokes shift that is pH dependent. In addition, it was shown that the fluorescence lifetime was pH dependent, such that fluorescence lifetime imaging microscopy could be used to image intracellular pH. The probe’s abilities to image pH were evaluated by imaging energydependent changes in cytosolic and mitochondrial pH.
Chen et al. used photoluminescence (PL) quenching and electrogenerated chemiluminescence (ECL) to study the electrochemical oxidation waves of conjugated polymers.77 They demonstrated that wave speeds of PL quenching can be augmented by increasing the anion concentration, decreasing the anion size, or increasing the electrochemical potentials. Their results lend more information for understanding the electrochemical oxidation wave phenomenon. Xie et al. tailored the efficiencies and spectra of white organic light-emitting diodes (WOLEDs) based on complementary blue and yellow phosphors.78 The electrical and optical characteristics of the WOLEDs could be manipulated easily by the insertion of an ultrathin interlayer between the two emitters and are dependent significantly on the selection of the interlayers. In related work, Zhang et al. developed highly efficient nondoped all-polymer white-light-emitting diodes. (PWLEDs).79 The polymer diode contained three layers: (1) a fluorescent three-color, white single polymer as an emissive layer; (2) an ethanol-soluble phosphonate-functionalized polyfluorene (PF-EP) as an electron-injection/electron-transport layer; and (3) a LiF/Al as a cathode layer. The authors note that this kind of PWLED shows excellent color stability, achieves high brightness at low voltages, is well suitable for solution-processing technology, and provides great potential of low-cost, large-area manufacturing for PWLEDs. Fluorescence quenching has many bioanalytical applications. The quenching of fluorophores by the same proteins that they label covalently is a phenomenon that is not well-known or well-characterized. The Webb group reported that Alexa dyes are quenched by interactions with Trp, Tyr, His, and Met residues through a combination of static and dynamic quenching mechanisms.80 Because of these findings, the authors suggest that the potential effects of intramolecular quenching should be considered in the interpretation of data that involves quantitative measurements of fluorescence intensity in proteins. The Geddes group studied the interaction of dsDNA with the fluorescent probe PicoGreen to determine the origin and mode of PicoGreen/DNA interaction.81 They reported that the quenching of PicoGreen in the free state results from its intramolecular, dynamic fluctuations. However, on binding to DNA, intercalation and electrostatic interactions immobilize the dye molecule. This immobilization results in a >1000-fold enhancement in its fluorescence. On the basis of the results, the authors proposed a model of PicoGreen/DNA complex formation. Lin et al. investigated two-photon excited fluorescence.82 Theoretical and experimental data show that the fluorescence is enhanced and quenched via surface plasmons (SPs) that are excited by total internal reflection with a silver film. Using various theoretical models, SPs were analyzed at different dielectric spacer thicknesses between the fluorescence dye and the metal film. The maximum fluorescence enhancement in the surface plasmon-total internal reflection fluorescence (SP-TIRF) configuration can be increased up to 30-fold with a suitable dielectric thickness SiO2 spacer. The experimental results demonstrate that the fluorescence lifetimes and the trend of the enhancements are consistent with the theoretical analysis. Tan et al. investigated the quenching properties of a conjugated polyelectrolyte (poly(phenylene ethynylene) containing anionic alkoxyl sulfonate side groups, PPE-SO3) by dye-doped silica nanoparticles and found that the polyelectrolyte is better quenched in the nonaggregated state.83 The analyses involved aqueous solutions of tris(2,2-bipyridyl)dichlororuthenium(II) (Rubpy)doped silica nanoparticles (SiNPs) in water, methanol, and the surfactant Triton X-100. The SiNPs showed hyper-efficient
’ SAMPLE PREPARATION, QUENCHING, AND RELATED PHENOMENA Various fluorescence methods can necessitate the use of myriad sample preparation techniques. The sample preparation tasks are variable and depend on the particular application. Numerous articles on sample preparation and quenching were published during the survey period. The review article offered by Zhu et al. summarized recent progress in the development of fluorescence biosensors that integrate the quenching property of single-walled carbon nanotubes (SWNTs) and the recognition property of functional nucleic acids (aptamers).72 The authors advocate exploiting the strengths and properties of both SWNTs and aptamers to create a series of fluorescence biosensors for high selectivity and sensitivity detection of a broad range of analytes. In another review, Hwang et al. overviewed the main photophysical features of conjugated polymers.73 In particular, they detail the way electronic, excited states evolve on various time scales in terms of exciton relaxation, localization, and electronic energy transfer. The Bright Group demonstrated a new method that uses the quenching and filtering effect of a gold film for the enhancement of luminescence from solgel based sensors.74 The authors noted that the gold film improved all sensor characteristics via the suppression of stray-light interference and the quenching of luminophores that are within 10 nm of the film. Apellaniz et al. reported an approach to compare two mechanisms (a graded single-vesicle infiltration versus an all-ornone vesicle infiltration) of lipid bilayer permeabilization by peptides.75 The two HIV-derived peptides (CpreTM and NpreTM) permeabilize large unilamellar vesicles by the all-or-none and graded mechanism, respectively. This important quantitative analysis of vesicle population distribution allowed the assessment of cholesterol effects and the identification of mixed mechanisms of membrane permeabilization. The study of quantum dots (QDs) continues to reveal novel and exciting information. Because of their unique chemical, physical, and optical properties, QDs have been applied widely in biological and biomedical research. Zhang et al. presented a new approach to study the effect of physiological cations (Ca2+, Mg 2+, Na+, K+) upon QDs using single-particle detection.76 Their results showed that divalent cations selectively can induce the self-assembly of QDs in a concentration- and time-dependent manner. In comparison with conventional TEM, SEM, and AFM measurements, singleparticle detection has significant advantages of simple or no sample preparation, rapid analysis, low cost, and easy measurement in the native environment. The single particle detection technique could be useful to characterize early protein assembly states and to study enzyme-responsive bimolecular assembly. 603
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quenching which was approximately 10010 000 times more efficient compared to the Rubpy dye alone. Quite surprisingly, quenching by SiNPs was found to be more efficient when the polymer existed in a nonaggregated state. Jayaraj et al. presented a strategy that allowed recording the phosphorescence from a number of thioketones in aqueous solution at room temperature.84 The method involves encapsulation of thioketone molecules in close proximity within a closed nanocontainer. The close proximity prevents self-quenching and allows the observance of phosphorescence.
nanomaterial-assisted chemiluminescence emission for protein sensing and cell discrimination was investigated.91 Hammami et al. employed PCA and factorial discriminant analysis of front face fluorescence spectroscopy (FFFS) and synchronous fluorescence spectroscopy (SFS) for pattern recognition and discimination of sheep milk samples based primarily on lactation periods and diet compositions.92 In general, compared to SFS, better discriminations of milk samples were observed with FFFS. The utility of hierarchical cluster analysis (HCA) in conjuction with LDA for accurate discrimination of 20 volatile organic compounds of unknown sample compositions at sample concentrations of 0.2%, 0.6%, and 1.0% with an accuracy of 96.7% or better was reported.93 The use of PARAFAC for mapping steady-state fluorescence excitation emission matrixes (EEMs) for various studies were demonstrated and continued to be an active area of research during the review period. For example, Chen and Kenny reported the first application of PARAFAC of EEMs and time-resolved fluorescence for the analysis of biphenyl and 2,5-diphenyloxazole distribution constants between the bulk aqueous and sodium dodecyl sulfate micellar phases.94 To validate the accuracy of the method, the developed technique was used to determine the distribution constants of anthracene, phenanthrene, naphthalene, and pyrene. The values of distribution constants obtained for anthracene, phenanthrene, naphthalene, and pyrene in the validation study were in close agreement with previously reported known value distribution constants in the literature, demonstrating the accuracy of the technique. In a related study, the combine use of PARAFAC unfolded partial least-squares coupled to residual bilinearization (U-PLS/RBL) for spectrofluorimetry determination of galantamine in organized media in both artificial and natural water samples was reported.95 The use of PARAFAC and U-PLS/RBL strategy in this study eliminated typical uncalibrated spectra interferences, which allowed the determination of galantamine at the ng/mL concentration without sample separation. Also, the utility of PARAFAC of fluorescence EEMs in other environmnetal studies were reported. Fellman et al. reported the use of PARAFAC of fluorescence EEMs for the examination of the impact of glacier runoff on the biodegradability and biochemical composition of terrigenous dissolved organic matter (DOM) in near-shore marine ecosystems.96 The results of the analysis suggest that terrigenous DOM from glacial runoff, is a considrable source of carbon and nutrients to nearshore coastal zones of southeast Alaska. Parallel factor analysis of fluorescence EEMs was explored for simultaneous determination of two widely used fungicides (thiabendazole and fuberidazole) in a very interfering environment.97 The use of PARAFAC allowed the determination of both fungicides at parts-per-billion levels in the presence of high concentrations of carbaryl, carbendazim, and 1-naphthylacetic acid spectral interferences. Obviously, data reduction techniques in fluorescence spectroscopy will continue to be a considerable area of active research in clinical, biomedical, environmental, and agricultural studies. It is envisioned that future direction includes new innovation of data reduction strategy that will improve considerably analyte detection limits, reduction of the analysis times, and minimization of analyte separation and/or extraction in a complex sample matrix. In addition, many papers that involve the use of data reduction to rapidly obtain the maximum useful information from fluorescence data for effective pattern recognition and accurate analyte classification in diverse sample matrixes will be published.
’ DATA ANALYSES The practical application of various data reduction strategies, including partial-least- squares (PLS) regression, principal component analysis (PCA), linear discriminant analysis (LDA), and parallel factor analysis (PARAFAC) in fluorescence spectroscopy for spectra calibration, sample analysis, pattern recognition, and classification continues to generate considerable interests in medical and biomedical, pharmaceutical, food and agriculture, and environmental studies during the review period. For example, the use of a nonlinear variable-angle synchronous fluorescence in conjuction with PLS regression for simultaneous determination of protoporphyrin IX, uroporphyrin III, and coproporphyrin III in human whole blood was reported.85 In addition to fast analysis time and good analyte recovery, low detection limits of 0.18, 0.29, and 0.24 nmol/L, respectively, were reported for protoporphyrin IX (PP), uroporphyrin III (UP) and coproporphyrin III (CP). According to the authors, this techniqe has potential practical utility for rapid and routine determination of porphyrins in whole blood and differential diagnosis of porphyria in a large number of samples. In another study, PLS regression was utilized to correlate changes in peroxyoxalate chemiluminescence emission with metal ion concentrations for simultaneous determination of Cu2+, Ni2+, and Zn2+ ions in a stopped-flow system of water samples.86 This method was found not only to be simple but also to be accurate, with good precision. In a related study, the capability of PLS regression analysis of chemiluminescence emission for accurate and simultaneous determination of pharmaceutical formulations and plasma samples was demonstrated by Ensafi et al.87 Numerous research papers that demonstrate the utility of PCA and LDA of fluorescence data for effective pattern recognition and classification of diverse sample complexity in various studies were published also during the review period. Al-Salhi et al. reported the use of PCA of native fluorescence spectra of body fluids in the detection and descrimination of lung cancer from normal control patients with 90% discrimination accuracy.88 In a related study, Welsher et al. reported the utility of PCA of deep-tissue fluorescence anatomical imaging of mice using carbon nanotube fluorophores for effective discrimination of organs, including the pancreas.89 Other new innovations involving the use of PCA in conjunction with n-way partial least-squaresdiscriminant analysis (NPLS-DA) and linear discriminant analysis (LDA) of fluorescence data were reported also. For example, the combined use of multiway robust principal component analysis (MROBPCA) and NPLS-DA of fluorescence data for accurate determination and discrimination of various cell culture media compositions was investigated by Ryan et al.90 According to the authors, the technique is robust and reliable, with potential applications in biopharmaceutical quality control and analysis. In another study, the use of LDA of sensor arrays based on 604
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’ ORGANIZED MEDIA Numerous articles have described fluorescence research using organized media. Only a very small, selective sample of the work performed can be included in this article. During the survey period, a number of reviews were published that contained exciting information. Cheng et al. offered a critical review that focused on the design of biocompatible dendrimers for cancer diagnosis and therapy.98 The authors discussed (1) nanotoxicity, long-term circulation, degradation, and other safety aspects of dendrimers; (2) the construction of novel dendrimers with biocompatible components; and (3) the challenges of developing dendrimerbased nanoplatforms for targeted cancer diagnosis and therapy. Also, during the review period, much work was published on the possibility of the use of organized macromolecules for drug delivery. Barick et al. demonstrated a new paradigm for precise control of targeted, on-demand drug delivery that uses ultrasound irradiation.99 Using a soft-chemical approach, the authors fabricated highly mesoporus, spherical 3D ZnO nanoassemblies composed of stable, well-defined nanocrystals. They discussed a method for entrapping drugs and suggested that drug release is dependent on the pH of the medium, the construction of the nanocrystals, and the externally applied ultrasound. Much research was performed on manipulating the growth and properties of molecular architectures. The Kang group reported the use of two active enzymes to control the assembly of 3D bioarchitecture.100 By coupling a bilayer of avidin/biotin-lactate oxidase (biotin-LOD) with a bilayer of avidin/biotin-horseradish peroxidase (biotin-HRP), the authors can control the position and height of nanostructures on a prepatterned surface. This layerby-layer construction yields bienzyme structures that are highly functional and viable for biosensing applications. Li et al. demonstrated controllable growth of monolayer-to-multilayer microstripes of an organic semiconductor by adjusting the pulling speed in a dip-coating process.101 Results confirmed that lower pulling speeds yielded mixed multilayers, while higher pulling speeds gave pure monolayer and bilayer microstripes. Their work provides powerful information for molecular design in controlling molecular structures. Exciting research with DNA continued to be reported. The Yeung group investigated porous alumina membranes as entrapment vesicles for nanoparticles and individual DNA molecules.102 For optimal entrapment, the pore diameter must be larger than the analyte particle diameter and the depthdistrubution of particles does not comply to one-dimensional diffusion. Their results provide novel insights into membrane separations with conventional liquid chromotography and sizeexclusion chromatography. Much work was done on the nanometer scale. Haper et al. found that yeast cells, when deposited on a weakly condensed lipid/silica thin film, actively direct their integration into a solid-state three-dimensional architecture.103 The integration process: (1) catalyzes silica deposition; (2) catalyzes the formation of a coherent interface between the cell and surrounding silica matrix; and (3) selects for live cells over apoptotic cells. The authors assess that this is the first demonstration of active cell-directed integration into a nominally solidstate three-dimensional architecture. This process promises a new way to integrate “bio” with “nano” into platforms to manipulate cellular behavior at the individual cell level and to interface living organisms with electronics, photonics, and fluidics. In application-related work, Calzaferri et al. designed dye-nanochannel antenna materials for light harvesting, transport, and trapping.104 In a three-step process, the authors incorporate chromophores
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into one-dimensional nanochannels and organize the latter in precise ways. The first step involves the arrangement of dyes inside zeolite channels (ZC). The second stage consists of coupling an extermal acceptor or donor fluorophore at the ends of the ZC, while the last step interfaces the material to an external device. Investigations in the field of lipids and membranes continues to give exciting information and augment our knowledge. Mager et al. presented a single-step process to reconstitute cell membranes on solid supports.105 They created the technique to support lipid bilayers from whole cell lipids without solvent extraction or the use of detergents. Fluorescence microscopy determined that both leaflets of the cell membrane, but not the cytoskeleton, were transferred to the support to create a bilayer fluid over an area much larger than a single cell. This method offers the capacity to create fluid, biologically relevant bilayers to study membrane-related processes. Sarles et al. developed a technique (regulated attachment method, RAM) that enables precise control over the size of the bilayer.106 Their method uses a flexible, deformable substrate to open and close an aperture that subdivides aqueous volumes that are submersed in an organic solvent. The RAM allows lipid bilayers to be unzipped completely and permits the introduction of certain species into the preformed bilayers. Thus, controlling the size of the interface through indirect interactions with the supporting substrate presents a new platform for assembling durable lipid bilayers. The authors envision this technology can be scaled to larger dimensions to create lipid networks and smaller dimensions for lipids to host single proteins. Harris’s group used fluorescence microscopy to characterize individual vesicles that are produced by extrusion.107 Generally, extrusion of hydrated lipid suspensions forms vesicles of uniform size. Though bulk analysis methods give information on the average size of vescicles, their technique obtains information on the size, lamellarity, and structure of individual vesicles. The results show that extruded vesicles exhibit a wide distribution of size, lamellarity, and structure. Bile salts are natural surfactants that fulfill vital biological roles in solubilizing cholesterol, lipids, and fat-soluble vitamins. Tolbert’s group reported the binding and turn-on of numerous analogs by bile salt aggregates.108 Their observations could lead to novel tools to study trafficking in biological systems. Work on ionic liquids (IL) and micelles yielded surprising information. Rai et al. found that the addition of the ionic liquid, 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim][PF(6)]), drastically increased the micellar aggregate size within aqueous sodium dodecybenzene sulfonate (SDBS).109 The authors propose that the aromatic nature of the ionic IL cation and the presence of largely aliphatic (butyl or longer) alkyl chains on the IL appear to be crucial for this dramatic critical expansion in self-assembly dimensions within aqueous SDBS.
’ LOW TEMPERATURE LUMINESCENCE Many research articles involving the use of low temperature luminescence and related techniques were published during the review period. In particular, many articles reported the use of low temperature luminescence and related techniques for the investigation of photophysical properties, dynamics, and conformational changes of proteins, DNA, polymers, and supramolecules. For instance, van Stokkum et al. reported the photophysical properties of reaction between phototropin flavin mononucleotide (FMN) cofactor and cysteine in light, oxygen, or voltage domains in aqueous solutions at low temparatures.110 According 605
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to the authors, fluorescence lifetimes of FMN are highly pH dependent, with 2.7 ns obtained at pH 2 and 3.94.1 ns recorded at pH 38. In a related study, the thermal activation of phosphorescence quenching, photophysics, dynamics, and confomational changes of protein in the millisecond range was examined at low temperature (8 to 273 K).111 In this study, the phosphorescence quenching property of a Zn-protoporphyrin substituting for the heme in the beta-subunits of human hemoglobin (ZnHbA) or tryptophan residues of Zn-HbA and human myoglobin proteins was used as probes to monitor the reaction mechanism. The excited-state double proton transfer in 7-azaindole (7AI) dimers using picosecond time-resolved fluorescence spectroscopy was reported also.112 In general, the reaction mechanism was found to be highly dependent on the conformations of 7AI dimers during the excitation. The planar conformers were reported to rapidly tautomerize (
