A bipolar-electrochemiluminescence split-type immunoassay based on a cathodic amplification strategy

A bipolar electrochemiluminescence (ECL) detection of myoglobin (Myo) in a split-type mode was designed using a cathodic signal amplification strategy. Specifically, a sandwich immunoreaction was firstly constructed in a 96-well plate with the target Myo as the middle unit to immobilize the phosphatase labeled probes, which could catalyze 2-phosphate to generate ascorbic acid (AA). In ECL detection procedure, the AA was introduced into the cathodic pole of the bipolar ECL system and the in-situ reduction of HAuCl 4 to Au nanoparticles on the cathodic pole occurred. The improvement conductivity of cathodic pole generated an enhanced ECL response of the bipolar system. Because the amount of AA generated depends on the concentration of Myo, the conductivity of cathodic pole is positive correlation with the Myo concentration, resulting in different output of the anode reporting ECL signal. The introduction of split-type mode in the signal amplification strategy and anodic pole without any modification as the signal output end could effectively avoid the weakness caused destruction of the protein activity and the reduced light transmittance in the traditional mode, generating an improved sensitivity and accuracy. The detection limits of Myo by using photomultiplier tube and charge-coupled device imaging are as low as 3.0 × 10 −13 and 5.0 × 10 −13 g mL −1 , respectively. Furthermore, it showed excellent performance in the determination of Myo in human serum samples. The developed cathodic signal amplification strategy based bipolar ECL biosensor could be a potential analytical tool for future trace analysis of biomarkers.