Detection Of Tumor Markers And Tumor Cells By Biosensor Based On DNA Probe

MicroRNAs are small non-coding RNA molecules that have crucial roles in the regulation of gene expression and post-transcriptional regulators of the immune response.Among the large number of microRNAs,the excessive expression of microRNA-21 has been found in the serum of cancer patients,including medulloblastoma,glioblastoma,colon cancer,hepatocellular cancer,and breast cancer,when compared to the corresponding normal tissues.Recently,the increasing of microRNA-21 expression had been verified in the early head and neck squamous cell carinoma tissue samples.N6-methyladenosine?m6A?has been identified as the most frequent messenger RNA?mRNA?modification which is the reversible process of RNA methylation modification and related to biological rhythm regulation.MicroRNA is closely related to tumor,and there may be some interaction between microRNA and epigenetics in epigenetics.The current research of microRNA in tumor epigenetics is mainly about abnormal methylation of microRNA,including hypermethylation inactivation of tumor suppressor gene Cp G island and activation of hypomethylation of oncogene,which leads to abnormal expression of downstream target gene.Some microRNA methylation has tumor specificity.A microRNA can regulate multiple target genes.If these microRNA are used as therapeutic targets to develop new drugs for treatment,the efficiency may be much higher than that for a single target gene.Therefore,the study of microRNA in tumor epigenetics will open up a new field for tumor diagnosis,treatment and prevention.Thus,developing a sensitive,specific,and convenient method for microRNA,microRNA methylation and tumor cell analysis is urgently needed and significantly important.?1?It is extremely important for quantifying trace microRNAs in the biomedical applications.In this study,an ultrasensitive,rapid and efficient label-free fluorescence method was proposed and applied for detecting microRNA-21 in serum of gastric cancer patients based on DNA hybridization chain reaction?HCR?.DNA H1 and DNA H2 were designed and used as hairpin probes,the HCR was proceeded in the presence of target microRNAs.Amounts of SYBR Green?dyes were used as signal molecules to intercalate long DNA concatemers from HCR,which guaranteed the model of label-free fluorescence and strong fluorescence density.The detection method showed a wide linear region from 1 fM to 10⁵ f M,and the limit of detection was 0.2554 f M?at S/N=3?for microRNAs.The results showed that this method had an excellent specificity and reproducibility.Furthermore,the label-free fluorescence strategy exhibited a sensitive response to microRNA-21 in real serum samples of gastric cancer patients and the results obtained were in accordance with reference method?R=0.994?.Overall,the proposed strategy could be satisfactory for rapid,ultrasensitive and efficient detection of microRNA-21,and held great potentials in clinic diagnosis of gastric cancer.?2?N6-methyladenosine?m6A?has been identified as the most frequent messenger RNA modification which is the reversible process of RNA methylation modification and related to biological rhythm regulation.In this work,an electrochemical immunosensor was developed for RNA methylation detection via using gold nanoparticles modified Au electrode?AuNPs/Au?as substrate electrode.Firstly,the hairpin DNA probe was assembled on the surface of AuNPs/Au via Aue S bond.In the presence of the hairpin DNA,which is the template of assistant DNA and target RNA,the ligase can selectively ligate them as the template of the hairpin DNA to form a long DNA:RNA heteroduplex strand.Successively,anti-N6-methyladenosine antibody could be specifically conjugated onto the RNA methylation site,and the horseradish peroxidase labeled goat anti mouse IgG?HRP-IgG?was further conjugated onto the anti-m6A antibody.In the detection buffer solution with H₂O₂ and hydroquinone,HRP-IgG can catalyze hydroquinone oxidation to generate benzoquinone,resulting in a highly electrochemical reduction signal.This developed immunosensor shows a wide linear range for methylated RNA from 10 f M to 10 nM,low detection limit of 3.35 f M?S/N=3?,high specificity and satisfactory reproducibility.And this immunosensing method owns great potentials for providing the assay of the expression level of methylated RNA in micro-biological system.?3?Sensitive detection of cancer cells at extremely low concentrations would greatly facilitate the screening and early diagnosis of cancer.Herein,we present a novel nanopore-based strategy for ultrasensitive detection of Ramos cells?human Burkitt's lymphoma cells?,by combining the enzymatic signal amplification with an aerolysin nanopore sensor.In this assay,an aptamer for Ramos cells was prehybridized with a short complementary DNA.The presence of target cells causes the target-aptamer complex to unwind to free the complementary DNA,which would subsequently trigger the enzymatic cycling amplification.This process eventually generated a large number of output DNA,which could quantitatively produce characteristic current events when translocated through aerolysin.The proposed method exhibits excellent sensitivity,and as few as 5 Ramos cells could be detected.With good selectivity,the approach can allow for the determination of cancer cells in human serum,offering a powerful tool for biomedical research and clinical diagnosis.