Simplified Gluten Detection Approach in the Presence of a Black Hole Quencher (BHQ)

Optical biosensors based in the fluorescence resonance energy transfer (FRET) phenomenon require of fluorophores. The use of fluorophores requires of a complex optical system that often leads to signal loss. Thus, a new approach based in a Black Hole Quencher (BHQ) that further simplifies the biosensor is sought. This approach potentially increases the sensitivity and accuracy. For the development of this approach, the gliadin conjugate with BHQ-10 must be established first. In the present work, we used the physical phenomenon of FRET to study the gliadin conjugation with BHQ-10 molecule. We performed an experiment with a fluorophore (6-Carboxyfluorescein or 6-FAM) labeled aptamer and non-covalently attached to graphene oxide (GO). The gliadin from gluten was conjugated with BHQ using two different cross-linking reagents present in BHQ: BHQ-10 succinimidyl ester and BHQ-10 carboxylic acid. BHQ-10 with carboxylic acid as the cross-linking reagent demonstrated to be an efficient reaction for gliadin conjugation with BHQ-10. The average quenching efficiency obtained was 62% in comparison to the gliadin as a control experiment. This sets the basis for the breakthrough development of a simplified gluten detection biosensor based on absorbance measurements instead of different wavelength ranges as fluorophores do. The aim is to develop a point-of-care microfluidic system that measures gliadin from gluten in a sensitive, accurate and cost-efficient manner.