Cell-Free Bioassay for Measurement of Dioxins Based on Fluorescence Enhancement of Fluorescein Isothiocyanate-Labeled DNA Probe

Abstract

This study aims to develop a rapid and sensitive cell-free bioassay of dioxins. It is known that dioxin ligand can bind heterodimeric aryl hydrocarbon receptor (AhR) and triggers the formation of the complex of dioxin-AhR, AhR nuclear translocator (ARNT), and dioxin-responsive element (DRE) region of the DNA. The hypothesis of the proposed method is that if FITC were labeled at the DRE sequence, its fluorescence intensity would be enhanced when the complex forms because the interaction interface of the binding components (AhR, ARNT, and DRE) creates a rather hydrophobic condition that is in favor of FITC emission. Effects of modification site of FITC on the DNA probes on binding efficiency between the complex components and fluorescence emission enhancement were evaluated by surface plasmon resonance and fluorescence analysis, respectively. Results showed that the labeling site at the second base at the 5′ end apart from the core region (5′-TNGCGTG-3′) of DRE did not obviously interfere with the binding between the DNA probe and dioxinAhR/ARNT hybrid but presented significant fluorescence emission enhancement. 2,3,7,8-Tetrachlorodibenzo-pdioxin (TCDD) was used as the typical toxin in this study. The method had a linear range of 1-100 pM, with detection limit of 0.1 pM (0.64 fg/assay) and coefficient of variation of 5.6% (n ) 10, 50 pM TCDD in transformed cytosol). The whole detection cycle was ∼4 h. The method was also used to estimate the toxic equivalents (TEQ) of 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD) and 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (HxCDD). Measurement of TEQs of the mixture of TCDD, PeCDD, and HxCDD were highly consistent with the predicted data. The average recovery using fly ash extract was ∼93%.

Fluorescence Enhancement of Fluorescein Isothiocyanate-Labeled Protein A Caused by Affinity Binding with Immunoglobulin G in Bovine Plasma

“…It has been reported that the degree of fluorescence enhancement of FITC caused by affinity binding was 1.6-fold with protein A — IgG interaction in PBS [8] and 1.5-fold with DNA interaction [7]. On the other hand, glycerin treatment enhances fluorescence of immobilized FITC-labeled antibody by approximately 1.5-fold [9].…”

“…Fluorescence enhancement caused by affinity binding may result from the hydrophobic microenvironment [7,8]. The fluorescence enhancement of FITC-protein A after binding with IgG in bovine plasma gave almost the same relative fluorescence intensity as that in PBS, demonstrating relatively low difference in solvent effect on fluorescence enhancement between buffer solution and plasma.…”

“…Sample IgG from rabbit is known to bind with protein A strongly. Bovine plasma was selected as bulk solvent for the ease of use and weak binding ability of bovine IgG to protein A. Fluorescein isothiocyanate (FITC) was used for the fluorescence label because the fluorescence enhancement of FITC caused by micro-environmental change has been well established by many authors [7-9]. Other regents were used without further purification.…”

“…Some authors have reported the measurement of biomolecules and biologically active compounds by fluorescent enhancement [5-7]. This method is based on the enhanced fluorescence intensity of a fluorescent-labeled reagent which interacts with a target substance.…”

Functionalized polymer colloids bearing primary amino groups

“…This reaction leading to stable thiourea derivates can be carried out under mild, physiological conditions. In this way, e.g., fluorescence dyes are attached to biological macromolecules for labelling in biology or biomedicine [13,14]. In colloidal physics, fluorescently labelled particles are a prerequisite for optical tracer experiments like fluorescence correlation spectroscopy [15,16].…”