Sensitive amperometric immunosensor for pathogen antigen based on MoS@AuNPs assembling dual-peptide as bioprobes with significant dual signal amplification.

It is crucial to timely and accurately identify the causative virus for early treatment and urgent prevention. Viral antigen detection can identify those people who are most likely at risk of spreading the disease, but most based on antibodies with limited stability and sensitivity. Peptides offer several advantages over antibodies, such as low cost, smaller size and good stability. The development of electrochemical immunoassay using specific peptide probes have the merits of good sensitivity and selectivity as well as good stability. Herein we report an amperometric immunosensor using peptides as capture probe and recognition probe. The molecular docking suggests that the two peptides of Pi (sequence: NFWISPKLAFALGGGKKKSC) and FK11 (sequence: WFLNDSELISML), bioscreened from phage display, bind to N-terminal domain of SARS-CoV-2 spike protein (SP). The peptide Pi is assembled on MoS@AuNPs modified electrode to capture SARS-CoV-2 SP, which is recognized by peptide FK11-displayed phage to form Pi/SARS-CoV-2 SP/FK11-phage sandwich. Then anti-M13 phage conjugated horseradish peroxidase (HRP) (anti M13-HRP) was introduced to recognize the phage capsid protein pVIII to form M13 phage/anti M13-HRP to enrich thousands of HRP, which can further electrochemically catalyze HO reduction at highly conductive MoS@AuNPs at - 0.35 V. Then amperometric immunosensor was constructed with linear range of 0.1-5000 pg/mL SARS-CoV-2 SP and detection limit of 0.074 pg/mL. The sensor also has good selectivity, batch reproducibility and stability, capable of detecting down to 10 transducing units/mL SARS-CoV-2 pseudoviruses. This work represents the first example of dual-peptide probes based sandwich-type electrochemical immunosensor integrated with dual signal amplification, which may provide a cost-effective assay platform in detecting real SARS-CoV-2 viruses for early diagnosis. The flexible and modular strategy can be extended to develop other type biosensors for a wide range of applications.