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Aberrations in the miRNA levels have been the cause behind various diseases, including periodontitis. Therefore, sensitive, specific, and accurate detection of disease\u2010associated miRNAs is vital to early diagnosis and can facilitate inhibitor screening and drug design. In this study, we developed a label\u2010free, real\u2010time sensing method for the detection of miR31, which has been frequently linked to periodontitis, using an engineered protein nanopore and in the presence of a complementary ssDNA as a molecular probe. Our method is rapid and highly sensitive with nanomolar concentration of miR31 that could be determined in minutes. Furthermore, our sensor showed high selectivity toward the target miR31 sequence even in the presence of interfering nucleic acids. In addition, artificial saliva and human saliva samples were successfully analyzed. 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