Electrochemiluminescent Imaging for Multi-immunoassay Sensitized by Dual DNA Amplification of Polymer Dot Signal.

A true-color electrochemiluminescent (ECL) imaging strategy was designed for a multi-immunoassay of proteins by coupling highly efficient polymer dots (Pdots) with dual DNA amplification. The Pdots were prepared by nanoprecipitation of poly[(9,9-dioctylfuorenyl-2,7-diyl)- alt-co-(1,4-benzo-{2,1',3}-thiadiazole)] in the presence of poly(styrene- co-maleic anhydride) and functionalized with DNA1 that hybridized with black hole quencher-labeled DNA2 to self-quench the ECL emission. The Pdots modified Au/ITO electrode showed 100-fold stronger ECL emission than the Pdots modified ITO electrode. After the capture antibody immobilized on Au/ITO slide recognized the target protein and then reacted with biotin-labeled antibody, streptavidin and biotin-labeled oligonucleotide, respectively, a large number of DNA1 functionalized Pdots could be introduced onto the slide surface by rolling circle amplification of the oligonucleotide to trigger the enzymatically cyclic release of the Pdots from the self-quenched probes to solution in the presence of Exo III. The dual DNA amplification produced a greatly amplified ECL signal for true-color ECL imaging. Using carcinoembryonic antigen, cytokeratin-19-fragment, and neuron-specific enolase as a lung cancer-specific biomarker panel, the ECL imaging-based multi-immunoassay exhibited excellent performance with a linear range of 1 pg mL-1 to 500 ng mL-1 and limits of detection of 0.17, 0.12, and 0.22 pg mL-1, respectively. The proposed method could accurately detect these biomarkers in clinical human serum samples for lung cancer screening. The Pdots-based true-color ECL imaging approach possessed the advantages of visual analysis along with wide detection range and high sensitivity and thus has great potential in clinical application.