Development Of Lable-Free Electrochemical Biosensor For Biomarkers Detection

Biomarkers are becoming more and more important in our daily lives such routine blood tests and cholesterol markers.With the increasing demand for biomarkers detection technology,the sensitivity of biomarkers detection is in urgent need of improvement.Label-free electrochemical biosensor has gradually become a powerful biomarker detection technology due to its low cost and easy operation.We combined the amplification approach with the electrochemical methods to develop a simple and rapid electrochemical biosensor with low background,high sensitivity and good specificity,and it can detect 5-hydroxymethylcytosine and microRNA in lung cancer cells.Lung cancer is the world's leading malignancy in terms of mortality,with 80%of cases of non-small cell lung cancer?NSCLC?.It is reported that microRNA-486-5p is expressed lowly in non-small cell lung cancer and the expression level in tumor tissues decreases with the increase of NSCLC stage.Therefore,microRNA-486-5p is a potential biomarker that can be used for early diagnosis,tumor staging and prognosis of NSCLC,and it also provides a potential therapeutic target for NSCLC research.We constructed a label-free and substrate-free electrocatalysis-assisted biosensor for accurate quantification of microRNA in lung cancer cells.The thiolated capture probes are immobilized on the FeCN-and AuNP-modified GCE electrode by layer-by-layer self-assembly via the Au–S bond.Then the capture probes hybridize with the target microRNAs,and the resultant surface-bound microRNAs are subsequently polyadenylated at the 3'-OH termini by poly?A?polymerase to form the poly?A?tails which can further hybridize with the T-rich assistance probes to form the double-stranded DNAs.Iron-embedded nitrogen-rich carbon nanotubes?FeCN?enables the electrocatalysis reduction of thionine intercalated into the dsDNA grooves in the absence of H₂O₂,and the introduction of abundant thionines greatly enhances the electrochemical signal.This label-free electrochemical biosensor exhibits excellent selectivity and high sensitivity,and it has a large dynamic range of 6 orders of magnitude and a detection limit of 0.853fM.It can be further applied for accurate quantification of microRNA in lung cancer cells.Known as the"sixth base",5-hydroxymethylcytosine is an intermediate in the process of DNA demethylation,which is closely related to leukemia,melanoma or other diseases.Therefore,5-hydroxymethylcytosine is an important and potential biomarker.Sensitive detection of 5-hydroxymethylcytosine is still a great challenge because of its similar structure and low abundance.We constructed a label-free and immobilization-free electrochemical magnetobiosensor using screen printed carbon electrode?SPCE?for sensitive detection of 5?hydroxymethylcytosine based on a dual signal amplification strategy combined with terminal deoxynucleotidyl transferase?TDT?and Ru?III?redox cycling.DNA is fixed onto magnetic beads by specific modification of 5-hydroxymethylcytosine sites and specific biotin-streptavidin interaction.Extending the DNA squence by TDT and Ru?III?redox cycling can achieve a dual signal amplification.This electrochemical magnetobiosensor can achieve high sensitivity?LOD=9.06 fM?and wide dynamic range,without fixing the DNA to the electrode by adding a magnet.This biosensor may find wide applications in the study of the 5-hydroxymethylcytosine methylation/demethylation mechanism and the clinical diagnosis of epigenetic-associated diseases.