Polarization-Based Sensing with a Self-Referenced Sample

Abstract

We describe a new method of fluorescence sensing based on fluorescence polarization. The sensor consists of two compartments, both of which contain the sensing fluorophore. One side of the sensor contains a constant concentration of analyte, and the other contains the unknown concentration. Emission from both sides is observed through polarizers, with the polarization from the sample being rotated 90° from that of the reference. Changes in the fluorescence intensity of the sample result in changes in the measured polarization for the combined emission. We show that this approach can be used to measure glucose and calcium using fluorophores which show analyte-dependent intensity changes, and no change in the spectral shape. Only a single fluorophore is required, this being the sensing fluorophore in both sides of the sensor. We also show that polarization sensing of glucose and calcium can be performed with visual detection of the polarization. In this case the only electronic component is the light source. These simple schemes can be used with a variety of analytes. The only requirement is a change in fluorescence intensity in response to the analyte.

Optical Determination of Glutamine Using a Genetically Engineered Protein

“…An additional characterization of the GlnBP S179C-acrylodan system was made using a new method recently been developed in our lab that allows for quantitative polarization measurements on any fluorophore displaying changes in emission intensity to be made (33)(34)(35). These measurements do not give the actual polarization of the samples, but rather code a sample and reference emission by orthogonally polarized light.…”

Molecular-Size Fluorescence Emitters

“…2.1). This was done with double labeling in glucose sensor (Shav-Tal et al 2004) and in glutamine sensor (Lakowicz et al 1999) based on bacterial periplasmic glucose and glutamine binding proteins correspondingly, In these sensors the organic environment-sensitive dye Acrylodan was the responsive dye reacting to the target binding by quenching its emission. Ruthenium bis-(2,2′-bipyridyl)-1,10-phenanthroline-9-isothiocyanate served as a nonresponsive long-lived reference.…”

“…Commonly Φ is measured by a reference method comparing in the same experimental conditions the fluorescence spectra the solutions of the studied sample and the reference (Brouwer 2011). The tabulated Φ values for the references covering the whole spectral range are available (Lakowicz 2006).Primarily, Φ depends on the dye structure, and it is not easy to predict it based on calculations. We can only estimate the efficiency of non-emissive channels of excited-state decay, such as internal vibrations.…”

A Novel Fluorescence Competitive Assay for Glucose Determinations by Using a Thermostable Glucokinase from the Thermophilic Microorganism Bacillus stearothermophilus

“…This can be accomplished using a polarized reference film (61,62) or as in the current paper using a protein sample in the absence of analyte (63). The principle of the measurement is shown in Scheme 1.…”