Interest is increasing in the multiplexed bead-based method to detect nucleic acid quantities, using oligonucleotides (oligos) coupled to micrometre-sized polystyrene beads. This paper describes an evaluation of this coupling step by examining different buffers at various pHs and the effect of changing the oligo concentration. The use of 2-(N-morpholino)ethanesulfonic acid (MES)-borate buffer (pH 4.5) during the effective coupling step yielded the best coupling efficiency. The addition of 15 pmol of oligo to 105 beads resulted in the highest number of coupled oligos per bead (similar to 336 +/- 28 oligos mu m(-2)). In addition, an alternative method to calibrate the fluorescence intensity by using FloSense Rainbow Calibration Particles was evaluated. In this way, it was possible to compare data independently of the type of flow cytometer used or the photomultiplier tube settings. (c) 2005 Society of Chemical Industry