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. Moreover, the ESAT-6 immunosensor showed good specificity, high reproducibility and excellent stability.
A label-free electrochemical aptasensor is reported for sensitive detection of the 6-kDa early secreted antigenic target (ESAT-6). For the first time, the bimetallic organic framework (b-MOF) of Zr-MOF-on-Ce-MOF was decorated with nitrogen-doped graphene (NG) and applied as the matrix for electroactive toluidine blue (Tb) to form the NG@Zr-MOF-on-Ce-MOF@Tb nanohybrid. The prepared nanohybrid with excellent hydrophilicity, dispersibility, and large specific surface exhibited significant electrochemical response. This nanohybrid could be directly used for anchoring ESAT-6 binding aptamers (EBA) through the interaction between the 5′-phosphate group (PO 4 3− ) of EBA and Zr 4+ of Zr-MOF. The signal response before and after incubating the ESAT-6 antigen has been evaluated by cyclic voltammetry at a scan rate of 100 mV s −1 from − 0.7 to 0.3 V (vs. SCE). Under optimal conditions, the proposed aptasensor displayed a wide linear range from 100 fg mL −1 to 10 ng mL −1 with a limit of detection (LOD) of 12 fg mL −1 . The developed method showed good reproducibility with a relative standard deviation (RSD) of 2.27%. The aptasensor showed favorable results in the analysis of the real samples. With these merits, the aptasensor has exceptional potential as a diagnostic tool for tuberculosis in clinical practice. Graphical abstract
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