Multiplexed electrochemical immunoassay of biomarkers using chitosan nanocomposites

• Electrochemical simultaneous detection of AFP and CEA was successfully performed.
• CHIT-PB-AuNPs-Ab2,1 and CHIT-Fc-AuNPs-Ab2,2 were used as signal tags.
• The detection limit was 0.03 ng mL−1 for AFP and 0.02 ng mL−1 for CEA.
• This method performed very well on the detection of clinical serum specimens.

In this work, a novel and sensitive multiplexed immunoassay protocol for simultaneous electrochemical determination of alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) was designed using functionalized chitosan composites. The immunosensing platform was prepared via immobilizing capture anti-AFP and anti-CEA on chitosan-Au nanoparticles (AuNPs) through EDC/NHS linking. The signal tags were fabricated by immobilizing electroactive redox probes – Prussian blue (PB) and ferrocenecarboxylic acid (Fc) on chitosan (CHIT), following by absorbing AuNPs to immobilize labeled anti-AFP and anti-CEA, respectively. A sandwich-type immunoassay format was employed for the simutaneous detection of AFP and CEA. The assay was based on the electrochemical oxidation/reduction of the redox species in signal tags, which has a relationship with the concentration of analytes. Experimental results revealed that the multiplexed electrochemical immunoassay enabled the simutaneous monitoring of AFP and CEA with a wide range of 0.05–100 ng mL−1 for both AFP and CEA. The detection limits (LOD) was 0.03 ng mL−1 for AFP and 0.02 ng mL−1 for CEA (S/N=3). The assay results of serum samples with the proposed method were in a good agreement with the reference values from standard ELISA method. And the negligible cross-reactivity between the two analytes makes it possesses potential promise in clinical diagnosis.