Use of Spectral Scanning Fluoromer and Spectral Analysis to Optimize Assays with Multiple Fluorometric Labels

Jorma Lampinen, Hanna Granö, Vuokko Kytoniemi and Arja Lamberg
Thermo Fisher Scientific, Vantaa, Finland

Introduction

When fluorometric multianalyte assays are developed it is extremely important to optimize excitation and emission wavelengths for each label to achieve the best possible assay resolution. This paper shows the principle how wavelength selection optimization can be easily performed with the spectral scanning fluorometer and analysis of the fluorescence spectra. During the optimization procedure the fluorescence excitation and emission spectra of each label is measured separately and in combination with all labels within the same well. Spectra are analyzed by calculating the ratios between the emission signal of each label and the summarized signal of interfering labels. These signal/interference signal ratios were calculated separately for each label and for both excitation and emission spectra. The maxima of the ratio curves are used for measurement of the label in the label mixture.

The optimization of multilabel assay parameters was performed with four common fluorescent labels: Methylumbelliferone (MeU), Alexa Fluor 488, 6-Carboxy-X-Rhodamine (6-ROX) and Alexa Fluor 680. Optimization is performed with the Varioskan® spectral scanning fluorometer connected to Skanlt® Software and with 96- well black microtiter strip plates (Thermo Microtiter, prod. no. 95029450, Thermo Fisher Scientific, Vantaa, Finland). The results show that using this optimization principle it is possible to design new multianalyte assays quite easily with optimal label resolution. The results will also show that the optimal wavelengths for multianalyte assays differ from commonly used wavelengths as well as from the spectral maxima of these labels.