Gel green fluorescence ssDNA aptasensor based on carbon nanotubes for detection of anthrax protective antigen

Bacillus anthracis, the causative agent of anthrax, is a harmful pathogen with potential ability as a biological weapon which persuades scientists to develop novel methods to detect anthrax from infected resources. In this study, a multi-walled carbon nanotube (MWCNTs)-based fluorescence aptasensor was fabricated to detect the recombinant protective antigen domain 4 (rPAD4) of Bacillus anthracis as the most important key factor in development of anthrax. First, PAD4 was recombinant expressed in E. coli and purified by Ni-NTA column. Second, the affinity of aptamer to rPAD4 was confirmed by ELAA assay. In aptasensor design, the aptamer was labeled with Gel Green and immobilized on MWCNTs. Upon the adsorption of labeled aptamer on MWCNTs, fluorescence emission was quenched. In contrast, by adding rPAD4 to hybridization reaction and incubation for 10 min, the fluorescence emission was significantly recovered to 85% compared to the control. Detection limit for the sensitivity and specificity of the aptasensor was determined 20 ng/ml and 62.5 ng/ml purified and unpurified rPAD4 protein, respectively. Also, applicability of aptasensor was showed in mouse serum sample. Finally, results indicated that nanosensor has the potential to be developed as a high-sensitive, cost-effective and fast-acting system for measuring of PA in anthrax diagnostic tests.