Development Of Aptamer-based Fluorescence Resonance Energy Transfer Biosensor For The Detection Of Hemagglutinin Of Influenza A Virus

Influenza A viruses are respiratory pathogens responsible for seasonal flu,which pose considerable clinical and public health challenges.The development of rapid and accurate new diagnostic tools is essential for proper diagnosis and prompt effective treatment of these infections.The technology currently used has significant shortcomings,which cause deficiencies in the efficiency and sensitivity of clinical diagnosis.Due to HA identity among different virus subtypes is not high,the detection of HA is considered,that is both one of the diagnostic criteria in subtype and ideal targets for IAVs detection.Fluorescence resonance energy transfer(FRET)spectroscopy is the distance-dependent transfer of energy from the excited state of a donor to an acceptor fluorophore.FRET is a physical phenomenon that occurs only over very short distances,and hence FRET technology is widely used to investigate molecular interactions.Graphene oxide(GO)has an excellent performance in the adsorption of the nucleic acid of their large surface area,surface adsorption characteristics and chemical stability.In the current study,we employed the IAVs HA aptamer to engineer a fluorescence-based aptasensor.Aptamers are functional nucleic acid ligands that are single-stranded oligonucleotides with specific binding toward the target antigen.The reversible folding of the aptamer can monitor the change between donor and acceptor.In this study,two forms of aptamer-based biosensor were constructed:1.We applied these concepts by constructing an aptamer biosensor to detect simultaneous changes of H5N1 HA and H1N1 HA.The two sets of aptamer can be labeled with two different fluorescent dyes.DNaseⅠ was used to achieve the signal amplification of the fluorescence signal.At the same time,Fluorescence signal intensity measurements were linearly correlated with H5N1 HA and H1N1 HA.The limits of detection(LOD)for HA were 0.73 ng/m L and 0.43 ng/m L,respectively.2.Upconversion Nanoparticles(UCNPs)and GO act as a donor and acceptor,respectively,which satisfies a prerequisite for designing the FRET-based biosensor for H5N1 HA.Here,we synthesized sandwich-structured UCNPs(SWUCNPs for short)with high energy transfer efficiency,which improves the efficiency of energy transfer.The detection limit was 60.9 pg/ml.This study proposed a SWUCNPs based biosensor system and realized the quick,sensitive,and selective detection of H5N1 HA.In this work,we employed the fluorescent dyes or UCNPs as fluorescence donor,using the chemical and optical properties of GO,constructed the homogeneous detection methods based on FRET.