Fluorescent Probe Encapsulated in Avidin Protein to Eliminate Nonspecific Fluorescence and Increase Detection Sensitivity in Blood Serum

Abstract

Quantitative detection of trace amounts of a biomarker in protein rich human blood plasma using fluorescent probes is a great challenge as the real signal is usually obscured by nonspecific fluorescence. This problem occurs because most of the fluorescent dyes bind very tightly with blood proteins to produce a large fluorescence increase, resulting in overestimation of the biomarker concentrations and false positive diagnosis. In this paper, we report that biotinylated fluorescent probes encapsulated in avidin protein can generate very specific fluorescence in blood serum by blocking out nonspecific dye-protein interactions. We applied our novel probe design to detect two different types of biomolecules, hydrogen sulfide and nitroreductase. Our Avidin conjugated probes achieved quantitative analyte detection in blood serum; whereas concentrations were overestimated up to 320-fold when bare fluorescent probes were employed. As compared to conventional approaches where fluorescent probes are encapsulated into polymers and nanoparticles, our simple approach successfully overcomes many key issues such as dye leakage, long preparation steps, inconsistent dye-host ratios, difficulty in constructing in situ in a complex medium, and limited application to detect only small metabolites.

Target-activated streptavidin–biotin controlled binding probe

“…Previously, these three moieties have been used for the detection of nitroreductase and fluoride and Cu( i ) ions. 31 – 33 …”

Amplified Expansion Stimulated Emission Depletion Microscopy

“…Therefore, a single biotin molecule on the target can be made to bind to a highly amplified number of avidin molecules through repetition of the reaction cycles of avidin and the biotin‐conjugated proteins (Scheme A). Biotin–avidin signal amplification that employs fluorophore‐conjugated avidin has thus been widely used as a way to amplify immunofluorescence signals …”

Cellular metabolic activity marker via selective turn-ON detection of serum albumin using an NBD-based fluoroprobe

“…Fluorescence spectroscopy is one of the widely used and efficient tools to study the interaction between drug molecules and SA because of its rapidness, good selectivity, as well as high sensitivity and especially real-time visualization by naked-eye. [13][14] Several fluorescent dyes are already known for SA binding, but the binding site and stoichiometry is not explored in most of the cases. [14][15][16][17] Quite a few numbers of fluorescent probes have been developed based on their sensitivity towards local polarity and viscosity, which causes a significant change in emissive states upon binding to the multiple hydrophobic pockets present in SA.…”

“…[13][14] Several fluorescent dyes are already known for SA binding, but the binding site and stoichiometry is not explored in most of the cases. [14][15][16][17] Quite a few numbers of fluorescent probes have been developed based on their sensitivity towards local polarity and viscosity, which causes a significant change in emissive states upon binding to the multiple hydrophobic pockets present in SA. 18 Nevertheless, limitations arise due to the absorbance of the probes in the near-UV region resulting to the interference by the auto-fluorescence of protein molecules, change in the secondary structure of protein upon ligand binding and lower selectivity of the sensor while other relevant analytes are present.…”