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

Abstract

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.

Homogeneous immunoassay utilizing fluorescence resonance energy transfer from quantum dots to tyramide dyes deposited on full immunocomplexes

“…The underlying reasons for this behavior have been extensively discussed in the literature. 31,32 In summary, when the target concentration is low, the amount of the sandwich-structured immunocomplex increases, leading to an increase in ΔFI. However, when the target concentration exceeds a certain threshold, half of the immunocomplex (either DA-target or target-CA) begins to form.…”

Digital detection of proteins

“…They extended this approach to enable homogeneous assays where coincidence detection of AuNPs modified with detection antibodies and quantum dots modified with capture antibodies were detected using dark-field and fluorescence microscopy, respectively. 113 Gite et al 114 developed a digital immunoassay where one antibody was modified with 500 nm fluorescent particles and the other antibody was modified with a magnetic particle. After the formation of sandwich immunocomplexes in solution, the complexes were pulled down to a surface using a magnet and the individual fluorescent particles counted using a wide FOV imager.…”

Merging microfluidics with luminescence immunoassays for urgent point-of-care diagnostics of COVID-19

“…Based on whether labels are required to be isolated, immunoassays are divided into two major types: heterogeneous and homogeneous immunoassays [ 108 ]. The signal can be outputted directly without washing in homogeneous immunoassays [ 109 ].…”