Corrections for matrix effects on fluorescence measurement of a multi-platform optical sensor

Abstract

The LEDIF (LED-induced fluorescence) is an in situ optical instrument that utilizes fluorescence, absorbance, and scattering to identify and quantify substances in water bodies. In this study, matrix effects on fluorescence signals caused by inner filtering, temperature, intramolecular deactivation, turbidity, and pH were investigated, and compensation equations developed to correct measured values and improve accuracy. Multiple simultaneous matrix effect corrections were demonstrated with a laboratory sample subjected to known interferences and physical conditions. In general, compensation was found to be important to improve the accuracy of fluorescence measurements.

A Multi-Platform Optical Sensor for In Vivo and In Vitro Algae Classification

“…The absorptivity for 523 nm excitation and the 580 nm emission wavelengths was computed as 2.0719 × 10 −7 and 1.9743 × 10 −7 (cells/mL) −1 ·cm −1 , with excitation and emission optical path lengths of 8.7 and 9 mm, respectively {Beer-Lambert [17] equation, $A=\epsilon lC$ was used, where $A$ is absorbance (absorbance unit), $\epsilon$ represents absorptivity, $l$ denotes optical path length, and $C$ is the concentration}. The inner-filtering effect was then corrected {[15] equation, $I_{corr}=I_{obs}antilo{g}_{10}false[{\left(O{D}_{ex}\right)}_{l,corr}+{\left(O{D}_{em}\right)}_{l,corr}false]$ was used, where $I_{corr}$ and $I_{obs}$ are the corrected and observed fluorescence peak intensities, respectively, $antilo{g}_{10}$ denotes antilog base 10, and ${\left(O{D}_{ex}\right)}_{l,corr}$ and ${\left(O{D}_{em}\right)}_{l,corr}$ represent the path-length-corrected excitation and emission optical densities, respectively}. By taking the ratio of the fluorescence peak intensity of phycoerythrin over phycocyanin at each cell concentration, a scaling factor ($S_{\beta }$) can be derived, allowing the prediction of unknown phycocyanin peak intensity from known phycoerythrin peak intensity of Porp.…”

“…The qualification (i.e., characterization) of the LEDIF’s spectrometer has been described by [ 15 ]. Figure 2 a shows the measured center wavelength and full width at half maximum (FWHM) bandwidth of the six excitation sources instrumented in the LEDIF for this work.…”

“…The fluorescence peak intensity is a linear function of concentration up to 3 mg/L and the fluorescence peaks were 444, 499, and 522 nm. At concentration higher than 3 mg/L, the inner filtering compensation procedure of [ 15 ] can be adapted to correct for non-linearity. No shift of fluorescence peak with excitation wavelengths is observed for all dissolved pigments.…”

“…The LEDIF is also capable of measuring other biological pigments such as chlorophyll b in Chlorophytes and Euglenophytes that were not presented in this paper. Matrix effects in these measurements were not investigated, which, could be important and made possible by adapting the compensation techniques of [ 15 ]. Finally, a spectrum separation algorithm can be applied to improve the quantification of algae mixtures.…”

“…Ng et al developed and field tested a multi-platform optical sensing technology utilizing multi-excitation fluorescence, broadband absorbance, and scattering to observe both spatial pattern and temporal trend of contaminants and natural substances in the water [ 11 , 12 , 13 , 14 , 15 ]. This work extends and characterizes the technology for in vivo algae classification to provide a comprehensive spectrum of water quality information.…”