Triple signal amplification using gold nanoparticles, bienzyme and platinum nanoparticles functionalized graphene as enhancers for simultaneous multiple electrochemical immunoassay

• Triple signal amplification strategy was constructed for simultaneously detecting CEA and AFP.
• This method performed very well on the detection of clinical serum specimens.
• The one working-electrode platform without deoxygenation simplified the operation.
• The method could be modified and extended to the detection of other multiple targets.

Here we demonstrated an ultrasensitive electrochemical immunoassay employing graphene, platinum nanoparticles (PtNPs), glucose oxidase (GOD) and horseradish peroxidase (HRP) as enhancers to simultaneously detect carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP). This immunosensor is based on the observation that multiple-labeled antibodies (thionine-labeled anti-CEA and ferrocene-labeled anti-AFP) recognition event yielded a distinct voltammetric peak through “sandwich” immunoreaction, whose position and size reflected the identity and level of the corresponding antigen. Greatly enhanced sensitivity for cancer markers is based on a triple signal amplification strategy. Experimental results revealed that the immunoassay enabled simultaneous determination of CEA and AFP in a single run with wide working ranges of 0.01–100 ng mL−1. The detection limits reached 1.64 pg mL−1 for CEA and 1.33 pg mL−1 for AFP. No obvious cross-talk was observed during the experiment. In addition, through the analysis of clinical serum samples, the proposed method received a good correlation with ELISA as a reference. The signal amplification strategy could be easily modified and extended to detect other multiple targets.