Multiplexed Homogeneous Immunoassay Based on Counting Single Immunocomplexes together with Dark-Field and Fluorescence Microscopy

The development of multiplexed immunoassays is impeded by the difficulty in distinguishing labeled immunocomplexes from free probes and nonspecifically bound probes. Here, we attempted to overcome this issue by counting core–satellite-structured immunocomplexes simultaneously using dark-field and fluorescence microscopy. The tumor biomarkers of carcinoembryonic antigen (CEA), α-fetoprotein (AFP), and prostate-specific antigen (PSA) were chosen as model targets. Gold nanoparticles (AuNPs) with diameters of 70 nm were coated with the detection antibodies of the three targets. Quantum dot (QD) 525, QD 585, and QD 655 were modified with the capture antibodies of CEA, AFP, and PSA, respectively. Then, an immunocomplex containing one AuNP and one or several QDs was formed, whereas free and nonspecifically bound probes had either one AuNP or one QD. When observed with a transmission grating-based spectral microscope, the immunocomplexes had overlapping scattering and fluorescent spectral images and were therefore identified and quantified precisely. The biomarkers inside the immunocomplexes were recognized on the basis of the fluorescent first-order streaks of the QDs. Model biomarkers in buffer and in 12.6% blank plasma were quantified for validation. The limits of detection for CEA, PSA, and AFP in buffer were in dozens of femtomolar and were close to those in blank plasma. The results demonstrated that our approach worked well in distinguishing immunocomplexes from free and nonspecifically bound probes. The successful quantification of the three targets in five human plasma samples verified the reliability of our method in clinical applications.