Multiplex electrochemical origami immunodevice based on cuboid silver-paper electrode and metal ions tagged nanoporous silver–chitosan

• A 3D multiplex electrochemical origami immunodevice was developed based on dual-signal amplification strategy.
• The novel CS-PWE was fabricated via a seed-mediated growth approach as sensing platform.
• Different metal ions functionalized NSC hybrids were synthesized as signal amplification label.
• This proposed strategy on the µ-ECOI described here could be easily extended for the detection of other relative biomarkers in our future work.

A 3D microfluidic electrochemical origami immunodevice (denoted as µ-ECOI in this work) for sensitive detection of tumor markers was designed. High sensitivity was achieved by using novel cuboid silver modified paper working electrode (CS-PWE) as sensor platform and different metal ions-coated nanoporous silver–chitosan (NSC) as labels. The CS-PWE was fabricated through a seed-mediated growth approach and served as a promising platform for antibodies attachment. The metal ions could be detected directly through square wave voltammetry without metal preconcentration, and each biorecognition event produced a distinct voltammetric peak, whose position and size reflected the identity and amount of the corresponding antigen. The large number of metal ions loading on the NSC greatly amplified the detection signals, and the good biocompatibility of CS-PWE retained good stability for the sandwich-type immunoassay. Using cancer antigen 125 (CA125) and carcinoma antigen 199 (CA199) as model analytes, the simultaneous multiplex immunoassay showed linear ranges of over 4 orders of magnitude with the detection limits down to 0.02 and 0.04 mU mL−1, respectively. Moreover, this strategy accurately detected the concentrations of CA125 and CA199 in human serum samples. The detection limits of CA125 and CA199 were 0.08 and 0.10 mU mL−1, respectively. This facile biosensing µ-ECOI exhibited high sensitivity and specificity with excellent stability, reproducibility, and accuracy, indicating its wide range of potential applications in point-of-care testing.