Cascade Signal Amplification Strategy For Electrochemical Detection Of HPV16 DNA

Cervical cancer is a high-morbidity cancer with a high mortality rate.Persistent infection with high-risk human papillomavirus（HPV）especially the HPV16 subtype is the main reason for cervical cancer.A large number of studies have proved that the virulence and proliferation of HPV16 are mainly determined by the E6/E7 gene in the HPV genome,which plays the key role in physiological activity of HPV16.HPV16 E6/E7 DNA has been widely studied not only as a biomarker of cervical cancer but also the therapeutic target and development of vaccine.Traditional methods for HPV DNA detection exposed the disadvantages such as high cost,complicated operation,easy contamination and the need for radioactive labeling.Benefiting from the high sensitivity,good selectivity,low cost,and easy miniaturization,electrochemical biosensing technology has opened a new channel for the detection of HPV DNA.Meanwhile,the cascade signal amplification based nucleic acid hybridization strategy was also widely applied in DNA sensitive detection.Based on this,the following two aspects of research were carried out around the electrochemical detection of HPV16 E6/E7 DNA:1.Electrochemical detection of HPV16 E6/E7 DNA based on the cascade amplification of toehold-mediated strand displacement and hybridization chain reactionIn this work,a cascade amplification technology based on toehold-mediated strand displacement reaction（TSDR）and hybridization chain reaction（HCR）was designed for electrochemical detection of HPV16 E6/E7 DNA.In this sensing system,the DNA strands of L,A₁ and A₂were first annealed to form a double-stranded complex structure with a toehold₁at the 3’end of L.In the presence of target（T）,the T could bind with complex structure from toehold₁ to form a double strand structure with exposed toehold₂ and release A₁ competitively.Then the F strand could combine with toehold₂to release A₂and T.The released T could participate in the next cycle reaction that only a few T could cause the large amount of release of A₁ after multiple cycles.A₁ could open the capture probe（CP）modified on the electrode surface,and then HCR process between the hairpin probes H₁ and H₂ and formed a large amount of ds DNA.MB could insert into the ds DNA and generate a measurable electrical signal response.This sensing strategy could achieve the sensitive detection of HPV16 E6/E7 DNA down to 3.54 f M with good selectivity and reproducibility.The high recovery rate of this method is proved by the serum standard-up recovery experiment,which shows that the method provides a new idea for the detection of HPV DNA.2.Electrochemical detection of HPV16 E6/E7 DNA based on cascade nucleic acid signal amplification conjugated with enzymatic reactionIn this work,a sensitive HPV16 E6/E7 DNA sensing strategy was proposed by utilizing the toehold mediated strand displacement reaction（TSDR）,hybridization chain reaction（HCR）and enzymatic reaction.Compared with the first work,this work mainly achieved the following improvements:first,TSDR was designed to reduce the probes in the reaction system and improve the hybridization efficiency.Second,the hairpin-type capture probes（CP）anchored on the electrode surface was instead by DTN-assisted capture probes,which hold the good rigid structure to effectively avoid the non-specific adsorption.Finally,the enzymatic reaction with high efficiency is used to produce electrochemical signal to further improve the sensitivity.The main principle of this work was as follows.In the beginning,the strand L was hybridized with strand A₁ to form the double strand.In the presence of target（T）,the strand T could hybridize with L strand and competitively release A₁.When F probe was subsequently added,the T strand was competed by F probe and attend the next cycle.At the same time,A₁ could open the hairpin CP at the top of DTN and then trigger the HCR with biotin-labeled H₁ and H₂.Then,streptavidin-HRP was introduced to specifically bind with the biotin labelled HCR assembly,which then catalyze the TMB/H₂O₂ reaction and generate remarkable electrochemical signal.This sensing strategy could quantitatively detect HPV16 E6/E7 DNA down to 0.57 f M with a linear detection range from 1 f M to 10 p M.The standard-up recovery experiment in serum proved that this method has a higher recovery rate than work 1,indicating that the method is expected to be applied to the screening of cervical cancer.