Ultrasensitive electrochemical biosensor for the simultaneous detection of the two monkeypox virus antigens M1R and A29 using reduced graphene oxide-ZIF-8 nanocomposite

• A novel electrochemical immunosensor was engineered for the simultaneous and selective detection of MPXV proteins M1R and A29. • Three morphologically distinct GO/ZIF-8 composites were synthesized, characterized, and evaluated for their electrochemical performance for the first time. • rGO/ZIF-8 nanoparticles were established as the optimal biosensing platform, enabling efficient antibody immobilization and signal transduction. • The immunosensor achieved ultrasensitive detection limits of 3.4 pg/mL for M1R and 5.1 pg/mL for A29, demonstrating its potential for clinical and environmental monitoring applications. Monkeypox virus (MPXV) has become a public health emergency of international concern, and its transmission occurs through respiratory droplets, direct contact with skin lesions, fomites, and sexual contact. However, there are no widely accessible diagnostic tests for MPXV in decentralized settings. Here, a novel electrochemical biosensor is presented for the simultaneous detection of MPXV proteins, M1R, and A29. Nanocomposites of graphene oxide and Zeolitic Imidazolate Framework-8 (ZIF-8) with three different morphological structures were synthesized and characterized using scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, and X-ray photoelectron spectroscopy. The composites were deposited onto dual-carbon screen-printed electrodes (SPEs) and electrochemically reduced to form reduced graphene oxide-ZIF-8/SPE (rGO-ZIF-8/SPE). Monoclonal antibodies targeting the A29 and M1R antigens of the MPXV were immobilized on the rGO-ZIF-8/SPE to develop a multiplexed immunosensor. Detection was performed using differential pulse voltammetry, demonstrating excellent sensitivity for MPXV antigens within a dynamic range of 1 pg/mL to 10 µg/mL, with detection limits of 5.1 pg/mL for A29 and 3.4 pg/mL for M1R. The biosensor demonstrated high sensitivity and selectivity, with no cross-reactivity to other respiratory virus antigens, making it ideal for point-of-care MPXV testing. The electrochemical immunosensor was tested in spiked serum samples, and the results were compared to commercial ELISA kits, showing high recovery percentages ranging from 90 to 104 %. The dual electrochemical biosensing platform also offers excellent reproducibility and scalability, making it suitable for frequent, decentralized use.