Rapid and Label-free Detection of Avian Influenza Virus H5N1 using a Target-responsive Hydrogel based Fluorescence Aptasensor

Abstract

Abstract. The purpose of this study was to develop a target-responsive hydrogel based fluorescent aptasensor for rapid and label-free detection of avian influenza virus (AIV) H5N1. A specific aptamer against H5N1 with high affinity was selected. Two different single-stranded DNAs (ssDNA 1 and ssDNA 2 ) were designed to be partially complementary to two ends of the aptamer. Both aptamer and ssDNA 1 were functionalized with acrydite at the 5’-terminal for hydrogel synthesis by polymerization. Quantum dot (QD) used as a fluorescence reporter was labeled at the 5’-terminal of ssDNA 2 and a quencher for the QD was conjugated at the 3’-terminal of the aptamer. Hybridization between the aptamer and ssDNAs formed the crosslinker to obtain 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 ssDNA 2 -QD. The response of the aptamer in hydrogel upon target binding was demonstrated by the quartz crystal microbalance (QCM) method. The aptamer concentration was optimized to be 1 µM and the operating procedure was simplified. The results indicated fluorescent changes at the emission peak were correlated to the titers of H5N1 (y=21.0x+562.4, R 2 =0.94). The total detection time from sampling to results was 45 min. The lowest detection limit was 0.2 HAU. This developed hydrogel based aptasensor was capable of rapid detection of target AIV H5N1 in the range of 2 -2.3 to 2 6 HAU/20 uL.