Response surface methodology optimized electrochemical DNA biosensor based on HAPNPTs/PPY/MWCNTs nanocomposite for detecting Mycobacterium tuberculosis.

An important issue in the prognosis of tuberculosis (TB) is a short period between correct diagnosis and start the suitable antibiotic therapy. So, a rapid and valid method for detection of Mycobacterium tuberculosis (M. tb) complex is considered as a necessity. Herein, a rapid, low-cost, and PCR-free DNA biosensor was developed based on multi-walled carbon nanotubes (MWCNTs), polypyrrole (PPy), and hydroxyapatite nanoparticles (HAPNPs) for highly sensitive and specific recognition of M.tb. The biosensor consisted of M.tb ssDNA probe covalently attached to the HANPs/PPy/MWCNTs/GCE surface that hybridized to a complementary target sequence to form a duplex DNA. The M.tb target recognition was based on the oxidation signal of the electroactive Methylene Blue (MB) on the surface of the modified GCE using differential pulse voltammetry (DPV) method. It is worth to mention that for the first time Plackett-Burman (PB) screening design and response surface method (RSM) based on central composite design (CCD) was applied as a powerful and an efficient approach to find optimal conditions for maximum M.tb biosensor performance leading to simplicity and rapidity of operation. The proposed DNA biosensor exhibits a wide detection range from 0.25 to 200.0 nM with a low detection limit of 0.141 nM. The performance of designed biosensor for clinical diagnosis and practical applications was revealed through hybridization between DNA probe-modified GCE and extracted DNA from sputum clinical samples.