Spectral filtering of light-emitting diodes for fluorescence detection.

The use of light-emitting diodes (LEDs) for fluorescence detection has recently gained much interest. The broad wavelength emission of LEDs requires spectral filtering that is not necessary when using a laser. For instance, filtering the LED light using a bandpass filter improves the signal-to-background ratio for riboflavin by a factor of 70. The bandwidth of the necessary bandpass filters affects both the signal and the background in these measurements. Fluorescence signal can be maximized with wider-bandpass high-transmittance filters. Background is governed by scattering of the LED emission light transmitted by two bandpass filters. When there is large crosstalk between the filters, the LED intensity is linearly related to the background. By estimating and optimizing the crosstalk between excitation and emission filters with a method presented here, the signal-to-background can be optimized. Bandpass filters should be selected with sharp on-off transition, strong blocking outside their transmitting region and the widest bandwidth with minimal crosstalk. Using optimized spectral filtering and capillary electrophoresis analysis, LODs of 50, 3 and 20nM are obtained for riboflavin, fluorescein and eosin Y, respectively. These results are superior to those reported in the literature for 5mW LEDs.