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Aisha Javed,1 Shah Rukh Abbas,1 Muhammad Uzair Hashmi,1 Noor Ul Ain Babar,2 Irshad Hussain2 1Department of Industrial Biotechnology, Atta-ur-Rahman School of Applied Biosciences, National University of Science and Technology, Islamabad, 44000, Pakistan; 2Department of Chemistry, Syed Babar Ali School of Science and Engineering, Lahore University of Management Sciences, Lahore, 54792, PakistanCorrespondence: Shah Rukh AbbasDepartment of Industrial Biotechnology, Atta-ur-Rahman School of Applied Biosciences, National University of Sciences and Technology, H-12, Islamabad, PakistanTel +92 3355449622; +92 51-9085-6125Email sabbas@asab.nust.edu.pkBackground: Mycobacterium tuberculosis’ rapid detection is still a formidable challenge to have control over the lethal disease. New diagnostic methods such as LED fluorescence microscopy, Genexpert, Interferon Gamma Release Assay (IGRA) are limited on efficacy spectrum owing to their high cost, time-intensive and laborious nature, in addition their low sensitivity hinders their robustness and portability. Electroanalytical methods are now being considered as an excellent alternative, being currently employed for efficient detection of the analytes with the potential of being portable. This report suggests label-free electrochemical detection of Mycobacterium tuberculosis (Mtb) via its marker, insertion sequence (IS6110).Methods: In this pursuit, graphene oxide-chitosan nanocomposite (GO-CHI), a biocompatible matrix, having a large electroactive area with an overall positively charged surface, is fabricated and characterized. The obtained GO-CHI nanocomposite is then immobilized on the ITO surface to form a positively functionalized electrochemical sensor for the detection of Mtb. DNA probe, specific for the IS6110, was electrostatically anchored on a positively charged electrode surface and the resistance of charge transfer was investigated for the sensitive and specific (complementary vs non-complementary) detection of Mtb by cyclic voltammetry and differential pulse voltammetry techniques.Results: The cyclic voltammetry was found to be diffusion controlled facilitating the absorption of analyte on the electrode surface. The label-free “genosensor” was found to detect a hybridization efficiency with a limit of detection of 3.4 pM, and correlation coefficient R2=0.99 when analysed over a range of concentrations of DNA from 7.86 pM to 94.3pM. The genosensor was also able to detect target DNA from raw sputum samples of clinical isolates without DNA purification.Conclusion: This electrochemical genosensor provides high sensitivity and specificity; thus offering a promising platform for clinical diagnosis of TB and other infectious diseases in general.Keywords: label-free detection, DNA genosensor, Mycobacterium detection, tuberculosis, graphene oxide nanocomposites, GO nanocomposites electrochemical sensing