Synthesis Of Ascorbic Acid-releasing Nucleotide Probes And Applications In Bioanalysis

After the outbreak of the novel coronavirus(COVID-19),establish a new rapid and sensitive pathogen detection method has attracted more and more attention.RT-PCR is considered to be the golden method for pathogen detection,however,this method requires complicated sample preparation,professional instruments and operators,and take a lot of time.There is an urgent need for a method that relies on simple instruments to achieve rapid detection of COVID-19 virus.In this paper,a method based on synthetic nucleotide probes combined with chemiluminescence system to achieve rapid detection of pathogen nucleic acid is established.The main contents are list at below:Firstly,a new chimeric nucleotide probe dAP4AA was designed and synthesized.The probe couples deoxyadenosine triphosphate(dATP)with 2-phospho-L-ascorbic acid(AAP)through a pyrophosphate bond.The dAP4AA nucleotide probe was synthesized by N,N-dimethylformamide(CDI)to activate the phosphate group,and the yield was 45%.It is expected that dAP4AA can produce easily detectable ascorbic acid(AA)under the action of DNA polymerase(Kf exo^-)and alkaline phosphatase(r SAP).Combined with changes in the luminescence signal in the Luminol-H₂O₂-CoOOH chemiluminescence system to realize the detection of pathogen nucleic acids in high sensitive.Experiments have shown that dAP4AA probe is a substrate of DNA polymerase and can be used in the amplification of DNA strands.At the same time,alkaline phosphatase cannot catalyze the hydrolysis of dAP4AA,but can hydrolyze the product of DNA polymerase reaction AAP3 to generate AA.By the detection of AA,the detection of target nucleic acid molecules is achieved.The detection of target fragments of DNA can be achieved using magnetic nanoparticles.This method can effectively detect the 60 fM hepatitis B virus(HBV)nucleic acid fragment.In combination with loop-mediated isothermal amplification(LAMP),dAP4AA can be used to detect 10 copies of COVID-19 DNA.Compared with the use of traditional fluorescent dyes,the sensitivity can be increased by 1-2 orders of magnitude.3minutes reaction can get an obvious detection signal,which can be used in rapid detection of pathogens.Based on this experiment,we continue to optimize the reaction conditions and develop a small hand-held test instrument to achieve more convenient pathogen detection.