Researchers at the Institute of Materials Science and Applied Research at Vilnius University in Lithuania have used deep-ultraviolet LEDs to perform frequency domain fluorescence measurements for the real-time identification of hazardous biological compounds. The 340- and 280-nm LEDs were modulated at frequencies as high as 200 MHz to measure fluorescence lifetimes in four basic biological autofluorophores: the coenzymes nicotinamide adenine dinucleotide (NADH) and riboflavin, and the amino acids tyrosine and tryptophan.Sensor Electronic Technology Inc. of Columbia, S.C., fabricated the AlGaN multiple-quantum-well LEDs employed in the study. The university group chose these devices because the wavelengths are short enough to excite fluorescence in coenzymes and proteins. LEDs consume low power and are small, lightweight and economical, so they are suited for use in fluorescence sensors.The method used was developed years ago for measurements of fluorescence lifetime, but this is the first time it has been applied to sense all basic biofluorophores using LEDs, said Arturasù Zukauskas, director of the institute. The investigators performed spectral and frequency domain measurements using the 340-nm LEDs to characterize NADH and riboflavin and the 280-nm emitters for tyrosine and tryptophan. They measured phase and modulation depth of both the excitation and fluorescence signals with a radio-frequency lock-in amplifier, a method earlier reported by a team at the University of Maryland, College Park.Isolated signalsTo determine the difference between phase shifts and the modulation-depth ratio, they optically isolated the fluorescence and excitation signals with an additional long-pass filter placed before a Hamamatsu photomultiplier. They extracted fluorescence lifetime by fitting the measured frequency dependencies of the modulation ratio and phase shift to a single-exponent model. The experimental results were in good agreement with models, thus validating the method.The researchers found that, by using a set of UV LEDs with different wavelengths, it should be possible to perform real-time characterization of fluorescence decay in most biological agents. This work could result in the development of a variety of UV-fluorescence-based tools designed to sense airborne biological pathogens and to use in medical applications or in any biological investigation where live organisms are present.Applied Physics Letters, Aug. 22, 2005, 084106.