Amperometric DNA biosensor for Mycobacterium tuberculosis detection using flower-like carbon nanotubes-polyaniline nanohybrid and enzyme-assisted signal amplification strategy

The authors described a universal amperometric DNA biosensor for rapid and ultrasensitive detection of the specific IS6110 DNA sequence of Mycobacterium tuberculosis (MTB). Surprisingly, when tufted carbon nanotubes (CNTs) doped with polyaniline (PAN), a unique flower-like structure with large surface area, abundant active groups and efficient redox activity was obtained. Accordingly, CNTs-PAN nanohybrid was designed both as novel redox nanoprobe and nanocarrier to decorate signal probe, forming tracer label for the generation and amplification of electrochemical signal. In addition, functionalized fullerene (C-60) nanoparticles (FC60) were employed as sensor platform to accelerate electron transfer and increase the loading of capture probe (CP). Moreover, enzyme-assisted target recycling amplification including three-way DNA junction could resolve the restriction of target sequences and make this method universal for other analytes. In the presence of target DNA, the assistant probe (AP), together with target DNA, could hybridize with CP and open its hairpin structure to form the Y-shaped junction and recognition sequence for nicking endonuclease. Once the CP was cleaved, the released AP and target DNA could hybridize with another CP to trigger the next cycle of cleavage, resulting in a large number of cleaved CP. After hybridization between cleaved CP and tracer label, a significantly enhanced electrochemical signal of CNTs-PAN could be easily read out. Based on the multiple signal amplification strategy, a wide detection linear range from 1 fM to 10 nM was obtained for target DNA of MTB. More importantly, the universal DNA biosensor also showed high specificity and sensitivity for MTB detection in clinical samples, which may provide a pragmatic tool for MTB testing and hold a great potential for other analytes.