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Methods based on Förster (or fluorescence) resonance energy transfer (FRET) are widely used in various areas of bioanalysis and molecular biology, such as fluorescence microscopy, quantitative real-time polymerase chain reaction (PCR), immunoassays, or enzyme activity assays, just to name a few. In the last years, these techniques were successfully implemented to multiplex biomolecular screening on microarrays. In this review, some fundamental considerations and practical approaches are outlined and it is demonstrated how this very sensitive (and distance-dependent) method can be utilized for microarray-based high-throughput screening (HTS) with a focus on protein microarrays. The advantages and also the demands of this dual-label technique in miniaturized multiplexed formats are discussed with respect to its potential readout modes, such as intensity, dual wavelength, and time-resolved FRET detection.

Chemical sensing, imaging and microscopy based on the use of fluorescent probes has so far been limited almost exclusively to the detection of a single parameter at a time. We present a scheme that can overcome this limitation by enabling optical sensing of two parameter simultaneously and even at identical excitation and emission wavelengths of two probes provided (a) their decay times are different enough to enable two time windows to be recorded, and (b) the emission of the shorter-lived probe decays to below the detectable limit while that of the other still can be measured. We refer to this new scheme as the dual lifetime determination (DLD) method and show that it can be widely varied by appropriate choice of probes and experimental settings. DLD is demonstrated to work by sensing oxygen and temperature independently from each other by making use of two probes, one for oxygen (a platinum porphyrin dissolved in polystyrene), and one for temperature [a europium complex dissolved in poly(vinyl methylketone)]. DLD was applied to monitor the consumption of oxygen in the glucose oxidase-catalyzed oxidation of glucose at varying temperatures. The scheme is expected to have further applications in cellular assays and biophysical imaging. © 2008 Springer-Verlag.

An optical dual sensor for oxygen and temperature is presented that is highly oxygen sensitive and covers a broad temperature range. Dual sensing is based on luminescence lifetime measurements. The novel sensor contains two luminescent compounds incorporated into polymer films. The temperature-sensitive dye (ruthenium tris-1,10-phenanthroline) has a highly temperature-dependent luminescence and is incorporated in poly(acrylonitrile) to avoid cross-sensitivity to oxygen. Fullerene C70 was used as the oxygen-sensitive probe owing to its strong thermally activated delayed fluorescence at elevated temperatures that is extremely oxygen sensitive. The cross-sensitivity of C70 to temperature is accounted for by means of the temperature sensor. C70 is incorporated into a highly oxygen-permeable polymer, either ethyl cellulose or organosilica. The two luminescent probes have different emission spectra and decay times, and their emissions can be discriminated using both parameters. Spatially resolved sensing is achieved by means of fluorescence lifetime imaging. The response times of the sensor to oxygen are short. The dual sensor exhibits a temperature operation range between at least 0 and 120°C, and detection limits for oxygen in the ppbv range, operating for oxygen concentrations up to at least 50 ppmv. These ranges outperform all dual oxygen and temperature sensors reported so far. The dual sensor presented in this study is especially appropriate for measurements under extreme conditions such as high temperatures and ultralow oxygen levels. This dual sensor is a key step forward in a number of scientifically or commercially important applications including food packaging, for monitoring of hyperthermophilic microorganisms, in space technology, and safety and security applications in terms of detection of oxygen leaks. © 2008 American Chemical Society.

A novel kind of composite material for simultaneous luminescent determination of air pressure and temperature is presented. The dual sensor consists of a fluorinated platinum porphyrin complex (PtTFPP) as an oxygen-sensitive probe, and of the highly temperature-sensitive europium complex Eu(tta)3(dpbt) as temperature probe. Both are incorporated into different polymer microparticles to control response characteristics and to avoid interferences. Encapsulation of PtTFPP in poly(styreneco-acrylonitrile) (PSAN) results in a broad dynamic range from 0.05 to 2.00 bar for pressure measurements. The europium complex was incorporated into polyvinyl chloride) to reduce the cross sensitivity towards oxygen. This system represents a new class of luminescent sensor system, where the signals are separated via the different luminescence lifetimes of the indicators. It is possible to monitor the emission of the temperature-sensitive probe by means of time-resolved fluorescence imaging without interferences, because the luminescence lifetime of the temperature indicator is tenfold longer than that of the oxygen indicator. The temperature image can then be used to compensate cross sensitivity of the pressure indicator towards temperature. In combination with an appropriate time-resolved measurement technique, this material enables simultaneous imaging of pressure (or oxygen partial pressure) and temperature distributions on surfaces. It is distinguished from other approaches of dual pressure and temperature sensitive paints because it avoids the need of signal separation by application of different cameras or by use of different optical filters or light sources. © 2008 WILEY-VCH Verlag GmbH & Co. KGaA.

(Figure Presented) Ultratrace quantities of oxygen can be determined at single spots as well as spatially resolved, and over a temperature range of more than 100 K, by exploiting the extremely efficient quenching of the delayed fluorescence of the fullerene C70 incorporated into matrices of organosilica or ethyl cellulose. © 2007 Wiley-VCH Verlag GmbH & Co. KGaA.

A sensitive and broad-ranged optical thermoraeter, based on the thermally activated delayed fluorescence of fullerene C-70, is presented. It consists of C-70 molecularly dispersed in a polymer film. Several polymer matrices were investigated. In the absence of oxygen the fluorescence intensity increases markedly with temperature. At 25 degrees C the fluorescence intensity of C-70 increases maximally by a factor of between 17 and 22, depending on the polymer, whereas at 100 degrees C the fluorescence intensity can be 79 times higher. In the absence of oxygen and for temperatures above 20 degrees C, the red fluorescence of C-70 in the films is so intense that it is easily perceived by the naked eye. For the systems studied, the fluorescence intensity is very sensitive to temperature. This results in a working range from -80 to at least 140 degrees C in the case of C-70 in poly(tert-butyl methacrylate) (PtBMA). Perylene was incorporated into the film as an internal reference in order to enable ratiometric measurements. The sensitivity of the lifetime of the delayed fluorescence to temperature is also high and results in an even wider working range. The performance of the C-70/PtBMA film was measured against a well-known optical temperature probe, [Ru(phen)(3)] (phen=phenanthroline). The results show that the C-70/PtBMA film is a very good system for optical temperature-sensing over a wide range of temperatures, outperforming known standards.

Microarray formats have become an important tool for parallel (or multiplexed) monitoring of biomolecular interactions. Surface-immobilized probes like oligonucleotides, cDNA, proteins, or antibodies can be used for the screening of their complementary targets, covering different applications like gene or protein expression profiling, analysis of point mutations, or immunodiagnostics. Numerous reviews have appeared on this topic in recent years, documenting the intriguing progress of these miniaturized assay formats. Most of them highlight all aspects of microarray preparation, surface chemistry, and patterning, and try to give a systematic survey of the different kinds of applications of this new technique. This review places the emphasis on optical technologies for microarray analysis. As the fluorescent read out of microarrays is dominating the field, this topic will be the focus of the review. Basic principles of labeling and signal amplification techniques will be introduced. Recent developments in total internal reflection fluorescence, resonance energy transfer assays, and time-resolved imaging are addressed, as well as non-fluorescent imaging methods. Finally, some label-free detection modes are discussed, such as surface plasmon microscopy or ellipsometry, since these are particularly interesting for microarray development and quality control purposes. © Springer-Verlag 2006.