Study On Graphene Oxide-based Biosensor For Detecting MicroRNA With Helicase-assisted Signal Amplification

Nowadays molecular biology studies are revolutionizing the science related diagnosis of human diseases which can help physicians better understand each patient's clinical conditions such as disease state,prediction of future disease states,drug response and treatment prognosis.The essence of molecular diagnostics lies in the use of biomarkers,including proteins,carbohydrates,or nucleic acids that are associated with pathological cells.As a group of endogenous noncoding RNAs,microRNAs(miRs,18-25 nucleotides)play significant roles in cellular processes of various diseases.Notably,the frequent aberrant expression and functional implication of miRs in many human diseases have lifted these small cellular components to the ranks of cancer biomarkers.Thus,the way of sensitive and efficient detection of miRs is crucial to better understand these relatively lesser nucleic acids and to further validate their functions in clinical molecular diagnoses.However,due to their mlsmall size and highly homologous properties,the sensitive and economical detection of these small molecules is still a left challenge.Recently,various novel methods have been developed with a special focus on sensitivity and specificity,such as nanoparticle-derived probes,isothermal amplification,electrochemical methods,etc.Among these new findings,the cyclic amplifications of most isothermal-amplification-based methods rely on DNAzymes for miR detection,such as polymerases,endonuclease or nicking enzyme and duplex-specific nuclease.Helicases are an important class of DNAzymes that move directionally along a nucleic acid phosphodiester backbone,separating two annealed nucleic acid strands(i.e.,DNA,RNA,or RNA-DNA hybrid)using energy derived from ATP hydrolysis,which have great potential to be used as catalytic agents in corresponding molecular diagnostic systems,especially in hybridization systems.In this study,the mechanism and enzymatic activity of Escherichia coli RecQ helicase(RecQE)expressed by p ET24a-RecQE recombinant plasmid in graphene oxide(GO)–basedsensors via different hybridization reactions for microRNA(miR)detection were studied.The roles of RecQE in the sensing platform based on 1:1 hybridization reaction and hybridization chain reaction(HCR)were systematically explored.The RecQE assisted GO sensor and RecQE assisted HCR/GO sensor were developed and further researched,the main contents are as follows:(1)One-step RecQE enzyme assisted GO sensing platform based on equivalent hybridization modes was developed to study the molecular mechanism of helicase to the hybridization reaction.In this assay,the fluorescence signal quenching and recovery of labeled fluorescent groups on GO surface were achieved by simple DNA/RNA hybridization,directly reflecting the molecular mechanism of RecQE.Compared with the non-enzymatic system,the fluorescence signal of RecQE assisted system coupling with ATP increased by 22.56 %,indicating that RecQE had an obvious promoting effect on GO surface hybridization.The results of this assay strategy for detecting the specificity and sensitivity of the let-7a miR showed that the sensing platform could distinguish all kinds of base-mismatched miRs with high specificity and accurately detect the corresponding target of the system,with a good detection limit of 0.18 nM for mi R based on 3? rule.These results showed that the sensitivity and specificity of GO sensing platform had not been affected by the introduction of RecQE,which verified the excellent compatibility of the GO sensing platform with RecQE.Additionally,the annealing of ?blocking? DNA strand(BDNA)to FDNA could effectively reduce the non-specific binding of RecQE and other small molecular proteins with FDNA,reducing the background fluorescence signal caused by external interference.(2)A GO-based Biosensor for detecting miR with augmented sensitivity through RecQE-assisted signal amplification of hybridization chain reaction(HCR)was futher developed with the conclusions obtained from the RecQE assisted FDNA-BDNA-GO sensor.Compared to the fluorescence intensity displayed by HCR/GO sensor,it was found that the helicase-assisted HCR/GO sensor showed by an overall 244.3% rise of the fluorescence intensity,and the new detection platform shortened dramatically the miR detection time from 4 h to 50 min.After optimizing the RecQE assisted HCR/GO sensor,the application of this assay strategy for detecting the sensitivity and specificity of the let-7a miR was investigated,the results showed that a good detection linear relationship could be obtained in the concentration range from 10 fM to 2000 fM and the detection limit of the method was calculated to be 4.2 fM for miR based on 3? rule.The sensitivity of the helicase-assisted HCR/GO sensing platform was 2 orders of magnitude higher than common HCR/GO sensing platform without enzyme.Meanwhile,the sensor could distinguish all kinds of base-mismatched miRs with high specificity,and accurately detected the corresponding target of the system.For the first time,we successfully applied the helicase to the GO sensing platform to detect miR with high sensitivity,improving the low sensitivity and efficiency of the enzyme-free HCR/GO sensing platform,which provides a reliable and sensitive miR quantitative method for biomedical research and clinical early diagnosis.