Establishment Of Exosomal MicroRNAs Electrochemical Sensor Based On Strand Displacement-triggered G-quadruplex/Rolling Circle Amplification Strategy

The liquid biopsy is a kind of non-invasive method for tumor diagnosis which takes the patient's body fluid as the biopsy sample.As a crucial part of liquid biopsy,microRNAs play an important role in the process of tumorigenesis,including cell proliferation,invasion,metastasis and drug resistance.Compared to cell-free microRNAs,exosomal microRNAs possess distinctive advantages in terms of their quantity and stability in serum.In addition,exosomal microRNAs are integrated into exosomes through a specific sorting mechanism,which enables exosomal microRNAs to reflect certain genetic properties of tumor cells more specifically.Emerging evidences suggest that the exosomal microRNAs are related to the tumorigenesis and development of breast cancer,lung cancer,ovarian cancer,colorectal cancer and many other cancers.The level of exosomal microRNAs in carcinoma tissue increases abnormally.Therefore,the exosomal microRNAs are potential biomarkers for early diagnosis,drug efficacy prediction,and prognostic assessment of several cancers.However,there are still some technical and biological limitations that hinder the clinical detection of exosomal microRNAs.Reverse transcription-PCR(RT-PCR)is the most widely used method in clinical practice.But it has high requirements for experimental environment and personnel,and it's easy to cause cross contamination between pores.When there are trace microRNAs reverse transcription products in the sample,or it contains inhibitory substances,it will also lead to false negative results.Biosensors,an interdisciplinary high technology which are cost-effective,simple,and require lower sample volumes,show greater utility for clinical nucleic acid and protein detection,even applying to develop military field diagnostic equipment.Electrochemical biosensor is a class of novel sensing technology which can transform the concentration of the target to measurable electrical signal through the biomolecular recognition element fixed on the electrode surface,which has the characteristics of economy,rapidity,simplicity and so on.Recently,it has become a hot spot for researchers to explore.Since electrochemical biosensor is more suitable for molecular detection of low concentration and complicated biological system,it has been widely employed in clinical nucleic acid and protein detection,food safety detection,environmental monitoring and other fields.The purpose of this study is to construct an electrochemical biosensor with high specificity,high sensitivity and convenient operation for the detection of exosomal microRNAs.In this method,the locked nucleic acid(LNA)-labeled toehold mediated strand displacement reaction(TMSDR)is initiated by the target microRNA-21,and through the G-quadruplex/rolling circle amplification(RCA)strategy,we realize the ultra-sensitive electrochemical sensing of exosomal microRNAs and preliminarily explore the accuracy of the biosensor in clinical application.1.Introduction.This section briefly introduces the important clinical significance and methodological status of exosomal microRNAs detection.Meanwhile,the advantages and application progress of biosensors are presented,especially focusing on electrochemical biosensors.Moreover,the research goal of this paper was expounded.2.Establishment of electrochemical biosensor based on Strand displacement-triggered G-quadruplex/rolling circle amplification strategy and its preliminary application in clinical detection of exosomal microRNAs.To successfully detect trace microRNAs,various biosensors have been fabricated based on different signal amplification strategies.Among these amplification strategies,RCA causes trace targets to produce high-molecular weight linear products to improve the magnifying power of the signal.Importantly,RCA requires no labeling and has no specific temperature requirements,making it highly suitable for clinical practice.Moreover,G-quadruplex,as one of the most widely used DNAzymes,is formed by a G-rich DNA sequence with the participation of K~+.It possesses efficient binding capacity with signal indicators such as methylene blue(MB)compared to double-stranded DNA due to end-stacking of the guanine quartet with a?-system to obtain the final desired signal strength.Thus,the RCA-assisted G-quadruplex strategy is likely to dramatically improve signal amplification.In addition,TMSDR,which is initiated by a short-overhang single-stranded region,can efficiently accomplish the specific identification of a target sequence at a constant temperature and displacement.TMSDR has been used as a specific recognition strategy in the fabrication of various biosensors.Moreover,the addition of LNA can markedly enhance the melting temperature(Tm)differences between the base-mismatched sequence and the complementary duplex,further improving the specificity and accuracy of the system,even to the resolution of a single base.Thus,the magnetic bead separation technique with LNA-assisted TMSDR will be helpful for the specific detection of exosomal microRNAs.Here,we design a strand displacement-initiated G-quadruplex/RCA strategy for highly specific and sensitive electrochemical sensing of exosomal microRNAs.In the presence of exosomal microRNA-21,the LNA-labeled TMSDR is initiated,releasing output DNA probe to trigger the subsequent RCA reaction by hybridizing with the C-rich circular template.Then the obtained G-rich RCA products can bind to the probe anchored on the surface of gold electrode and generate G-quadruplex conformations.The final electrochemical signal is obtained through efficiently embedding the signal indicator MB into the G-quadruplex structures for the successful ultra-sensitive biosensing of exosomal microRNAs.Based on the TMSDR-triggered G-quadruplex/RCA strategy,a good linear relationship was observed between the peak current of each response and the logarithm of the microRNA-21concentration within a range of 10 fM to 10 nM with a correlation coefficient(R~2)of 0.9972.And the detection limit of this electrochemical biosensor is down to 2.75 fM.Moreover,our biosensor exhibits excellent repeatability,stability,and high consistency compared to RT-PCR for clinical detection.In conclusion,this assay is expected to provide a hopeful strategy for the early non-invasive diagnosis and prognostic estimation of cancer.