Ultrasensitive SERS Sensors Of Virus Genes Via Nucleic Acid Amplification Strategy:Design,Preparation And Application

Virus is a kind of non-cellular organism that contains either deoxyribonucleic acid(DNA)or ribonucleic acid(RNA),which must be parasitized in living cells and proliferated by replication.Many diseases,such as Ebola virus disease,AIDS,dengue haemorrhagic fever,severe acute respiratory syndrome(SARS)and avian influenza,are caused by the virus.The rapid diagnosis of virus types and infections can effectively reduce the spread and infection of viral infectious diseases and provide important scientific basis for its prevention and control in time.However,the traditional virus detection methods generally have some problems such as harsh detection conditions,expensive,time-consuming or poor sensitivity.Therefore,it is urgent to develop a rapid,highly sensitive and specific detection technique for virus detection.In recent years,surface-enhanced Raman scattering(SERS)has been widely used in biological detection and analysis because of its ultra-high sensitivity and “fingerprint” characteristics.At the same time,more and more nucleic acid amplification technology are applied in detections to further improve the sensitivity.In this paper,ultra sensitive SERS sensors of virus genes via nucleic acid amplification strategies has been designed and prepared to dtecte influenza A virus and dengue virus which were slected as two virous models.The main research contents and innovations are as follows:(1)Ultrasensitive SERS determination of avian influenza A H7N9 virus via exonuclease III-assisted cycling amplificationInfluenza A virus H7N9 is one of the newly discovered highly pathogenic avian influenza viruses and poses a serious threat to public health and poultry industry.According to the requirement of H7N9 detection,an ultrasensitive SERS determination of H7N9 virus via Exo III-assisted signal amplification was proposed to meet the needs of rapid and accurate detection of H7N9-related genes.The two types of ss-DNAs fragments derived respectively from hemagglutinin(HA)and neuraminidase(NA)sequences from avian influenza A(H7N9)virus were chosen as the targets.The proposed SERS strategy to simultaneously determine the two targets was conducted by specially designing Capture,Replace,and Prober single-strand DNAs to realize Exo III-assisted cycling amplifications of the two targets,and then integrating the cyclic amplification with SERS-active substrate to achieve a SERS-based Exo III-assisted signal amplifications strategy specialized for detecting gene fragments of H7 and N9.After characterizations of the sensing mechanism and optimization of surface blocking and dosage of Exo III,a simultaneous and specific detection of H7 and N9 gene fragments was obtained with detection linear interval from 1 fM-100 pM and limit of detection low to several tens of atto moles.(2)Enzyme-free double nucleic acid signal amplifications integrated SERS detection strategy for DENV detectionDengue virus causes mild fever when it infects humans for the first time,and may develop into dengue hemorrhagic fever(DHF)and dengue shock syndrome(DSS),which can lead to death.According to the requirement of DENV detection,an enzyme-free ultrasensitive SERS determination combining LCHA and HCR has benn designed and proposed.Herein,a group of specific DNA sequences were designed based on characteristic fragment of DENV gene,i.e.two long strands L1 and L2,and four hairpins C1,C2,H1,H2,which can realize LCHA-assisted target DNA cycle amplification and HCR signal amplification of DENV,and then integrating the nucleic acid amplification products with SERS-active substrate to achieve a SERS-based enzyme-free double nucleic acid signal amplifications strategy for DENV detection.After characterizations of the sensing mechanism and optimum concentration of MCH,a rapid and specific SERS determination for DENV was obtained with detection linear interval from 1 fM-10 nM and limit of detection is 490 aM.