Picogram level electrochemical impedimetric immunosensor for monitoring Mycobacterium tuberculosis based on specific and sensitive ESAT-6 monoclonal antibody

Tuberculosis (TB) as a global epidemic disease is caused by Mycobacterium tuberculosis (M. tb). A small early secreted antigenic target 6-kDa protein (ESAT-6) secreted by M. tb has become a major virulence factor for detection of M. tb infection. Here, the recombinant protein His-CFP10-ESAT-6 (designated as rHis-CE) was expressed and purified as the immunogen to develop stable and specific ESAT-6 monoclonal antibodies (MAbs), which can recognize both recombinant and natural ESAT-6 protein. Three resultant ESAT-6 MAbs (named 4E1, 4D7 and 4G8) were characterized and screened in detail. An ultrasensitive electrochemical impedance ESAT-6 immunosensor for label-free detection of M. tb infection was proposed by the immobilization of the screening 4D7 ESAT-6 MAbs on the N-doped graphene oxide (N-GO) functionalized electrode. The structure and surface state of N-GO and ESAT-6 immunosensor were characterized by various characterization instruments such as transmission electron microscopy, X-ray photoelectron spectroscopy, contact angle measurement, and cyclic voltammetry. The quantitative detection of ESAT-6 was mainly achieved by recording the relative change of impedance before and after specificity reaction. Under the optimized conditions, ESAT-6 could be assayed in a range of 0.01-100 ng/mL with a very low detection limit of 7.0 pg/mL. Compared with the traditional assay methods, this immunosensor showed much higher sensitivity, wider linear range and simpler operation. More-over, the ESAT-6 immunosensor showed good specificity, high reproducibility and excellent stability.