Exosome And MiRNA-21 Electrochemical Sensor Based On Gold Nanoparticle Signal Amplification

Exosomes are a 30-150 nm extracellular nanovesicle secreted by all cell types,it can be found in most body fluids,such as blood,urine,ascites and saliva.They carry various molecular components from their original cells,including membrane and cytoplasmic proteins,DNA,micro RNA,and more.Exosomes have been shown to play important roles in a variety of cellular physiological and pathological processes,and they act as mediators to transfer and transfer membrane and cytoplasmic molecules between cells.The concentration and phenotype of exosomes is dynamically linked to many diseases,such as cancer,making it an ideal biomarker for early detection of cancer.It is worth noting that exosomal proteins can predict the differentiation of maternal cancer cells of origin and slight changes in protein patterns between different exosomes.Analyzing and detecting exosomal proteins can provide more information about disease progression.Therefore,it is an urgent to develop highly sensitive methods for analyzing exosomal proteins,in the non-invasive early diagnosis and monitoring of cancer.We establish an electrochemical aptasensor for exosomal proteins profiling was fabricated based on the DNA NTH,the CD63 aptamer-modified DNA NTHs were immobilized on the gold electrode through three vertices of the DNA NTH by Au-S bonds.The fourth vertex appended with an CD63 aptamer design to capture the exosomal CD63 protein.After the capture of the target exosomal protein by the aptamer,the Au NPs-DNA could also bind to the exosomal protein,the Au NPs-DNA including poly adenine aptamer（poly A-aptamer）and poly adenine biotin-DNA（poly A biotin-DNA）,poly A-aptamer can identified the exosomal protein,poly A biotin-DNA containg a random sequence with biotin,the avidin-horseradish peroxidase conjugation（avidin-HRP）could be immobilized on the poly A biotin-DNA by the biotin-tagged report probe,which can be used to catalyze the reduction of hydrogen peroxide（H₂O₂）in the3,3’,5,5’-tetramethylbenzidine（TMB）substrate,and thus amplified the amperometric signal.Since the poly A-aptamer could be easily changed according to different exosomal proteins,this strategy could be used to profile the exosomal proteins derived from different cancer cells with high sensitivity and specificity.MicroRNAs（miRNAs）are a class of short noncoding RNAs（18-25 nucleotides）acting as sequence-specific post-transcriptional regulators.They play critical roles in the cellular network of animals、plants and protozoa.miRNAs participate in various biological processes such as cell differentiation,proliferation,apoptosis,stress-specific cellular outcome,and tissue development.As a promising biomarker for early diagnosis and prognosis of non-invasive diseases（especially cancer）,miRNA provides important information for understanding the occurrence,metastasis and further biomedical applications of cancer.Recent years,miRNA have attracted more attention,it have shown that the expression levels of miRNA are closely related to a variety of cancer diseases.We design a novel electrochemical aptasensor for miRNA assay based on bridge DNA-gold nanoparticles（Au NPs）.Target miRNA-21 triggers the hairpin H1modified on the gold electrode.Then hairpin H2 can hybridize with the hairpin H1 to replace miRNA-21,meanwhile,miRNA-21 is released,which triggers more hairpin H1,finally the electrode captured bridge DNA-gold nanoparticles,because the Au NPs with large surface area are attached with numerous DNA probes,which absorb Ru Hex via electrostatic interaction,significant electrochemical response can be obtained to indicate the initial level of miRNA-21.The electrochemical sensor is easy to operate and has high detection sensitivity,which has a good application prospect in miRNA detection.