Introducing a portable electrochemical biosensor for Mycobacterium avium subsp. paratuberculosis detection using graphene oxide and chitosan.

In this contribution, a novel, low-cost, high throughput, and ultra-selective electrochemical DNA nanobiosensor was developed for accurate on-site detection of Mycobacterium avium subspecies paratuberculosis (MAP) in real media for practical diagnosis of Johne's disease (JD). The method was designed based on the immobilization of graphene oxide and chitosan biopolymer on the surface of a glassy carbon electrode, modified by electrochemical immobilization of graphene oxide and chitosan biopolymer, followed by activation of biopolymer via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxy succinimide (EDC/NHS) coupling system. Afterward, the commercial probe DNA (ssDNA) was stabilized on the activated electrode surface to prepare an ultra-selective ssDNA-stabilized nanobiosensor for MAP sensing called "ssDNA-stabilized GO-CH-EDC/NHS-modified electrode". Several characterization methods distinguished the bioelectrode. The DNA hybridization between the nanobiosensor and target DNA was confirmed by cyclic voltammetry and differential pulse voltammetry. "At optimal experimental conditions, the nanobiosensor showed a linear range of 1.0 × 10-15 -1.0 × 10-12  mol L-1 , a detection limit as low as 1.53 × 10-13  mol L-1 , and a repeatability with a relative standard deviation (%RSD) of 4.7%. The reproducibility was also appropriate, with a %RSD of about 10%. It was used to diagnose MAP in real samples with highly accurate results. Therefore, the developed nanobiosensor can be used for clinical diagnosis of MAP.