A target-responsive and size-dependent hydrogel aptasensor embedded with QD fluorescent reporters for rapid detection of avian influenza virus H5N1

Abstract

A target-responsive hydrogel based fluorescent aptasensor was developed for rapid detection of avian influenza virus (AIV) H5N1. A specific aptamer against H5N1 was selected and two single-stranded DNAs (ssDNA1 and ssDNA2) were designed to be partially complementary to two ends of the aptamer. Both aptamer and ssDNA1 were functionalized with acrydite at 5’-terminal for hydrogel synthesis by polymerization. Quantum dots (QDs), used as fluorescence reporters, were labeled at 5’-terminal of ssDNA2 and quenchers for QDs were conjugated at 3’-terminal of the aptamer. Hybridization between aptamer and ssDNAs formed the crosslinker to complete the QD-aptamer hydrogel. Initially, without targets, the prepared hydrogel remained in a shrunken state because of the crosslinking in the polymer network and the QD was quenched. With target AIVs, the crosslinking was dissociated due to the binding reaction between the aptamer and target, resulting in the abrupt swelling of the hydrogel and the release of aptamer-quencher and ssDNA2-QD. The response of aptamer upon target binding was demonstrated by quartz crystal microbalance (QCM) method and the microstructure of hydrogel was characterized by SEM. Spectrophotometric results indicated fluorescent changes at the emission peak were correlated to the titers of H5N1. The total detection time from sampling to results was 30min. The lowest detection limit was 0.4HAU with a detecting range of 2−1.2 to 26 HAU 20μL−1. The size-dependent property of this aptasensor was examined quantitatively and qualitatively. This simple, low-cost, specific, and label-free aptasensor has great potential for the in-field rapid detection of AIV H5N1.