Voltammetric immunoassay for Mycobacterium tuberculosis secretory protein MPT64 based on a synergistic amplification strategy using rolling circle amplification and a gold electrode modified with graphene oxide, FeO and Pt nanoparticles.

The authors describe an electrochemical immunoassay for ultrasensitive determination of the Mycobacterium tuberculosis (MTb) secretory protein MPT64 which is an antigen for early diagnosis of infection with MTb. Protein G was used to immobilize antibodies against MPT64 on a gold electrode. Graphene oxide with its large surface area was used as a carrier to anchor magnetite (FeO) and platinum (Pt) nanoparticles. The nanocomposite of type GO@FeO@Pt was used as a signal reporter with excellent catalytic activity and recyclability. The nanocomposite exhibits peroxidase-like activity for hydrogen peroxide, best at -0.25 V (vs. Ag/AgCl). It works over a wide range of pH values (2-10) and temperatures (25-65 °C). Further signal amplification strategy was accomplished by rolling circle amplification. This voltammetric assay has a linear response to the logarithm of MPT64 concentration in the range from 5.0 fg·mL to 1.0 ng·mL and a detection limit of 0.34 fg·mL (at a signal to noise ratio of 3, for n = 10). The assay can be completed within 4 h. It was successfully applied to the determination of MPT64 in spiked serum samples. Conceivably, the assay has a large potential in providing laboratory evidence for rapid diagnosis of MTb infection. Graphical abstract An electrochemical biosensor was developed for rapid detection of Mycobacterium tuberculosis secretory protein MPT64. The detection signal was synergistically amplified by GO@FeO@Pt and rolling circle amplification. Phi29: phi29 DNA polymerase; BSA: bovine serum albumin; GO: graphene oxide.