Digital Duplex Homogeneous Immunoassay by Counting Immunocomplex Labeled with Quantum Dots.

Digital multiplexed homogeneous immunoassay is supposed to have the advantages of high sensitivity, high analytical throughput, small sampling errors, and low consumption. We present a spectral imaging-based multiplex, homogenous immunoassay by counting sandwich-structured immunocomplexes in the form of quantum dot (QD) aggregates. As a proof of concept, the method was utilized to detect two tumor biomarkers: carcino-embryonic antigen (CEA) and α-fetoprotein (AFP). The immunocomplex induced by CEA contained QD 655 and QD 585 and were recognized by the spectral pattern of dual-color QD aggregates under a transmission-grating-based spectral imaging microscope. Immunocomplexes induced by AFP were labeled with the QD 585 aggregate and were identified by the spectral blue-shift pattern of same-color QD aggregates. Limits of detection for AFP and CEA were calculated to be 0.02 and 0.10 pM at a signal-to-noise ratio of 3, respectively. Further successful quantification of the model proteins in human plasma demonstrated the accuracy and reliability of our approach.