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Updated: Sep 28, 2025
High-Throughput Automated Multiplex Immunofluorescence Assays for Translational Research
Published on: June 10, 2025
Multiplexed Homogeneous Immunoassay Based on Counting Single Immunocomplexes together with Dark-Field and
Xiaojun Liu1, Xinyi Lin1, Xiaoyan Pan2
1School of Chemistry and Materials Science, Jiangsu Normal University, Xuzhou 221116, Jiangsu, China.
Insights
This study introduces a novel method for accurately counting multiplexed immunocomplexes using dark-field and fluorescence microscopy. The technique precisely distinguishes target biomarkers like CEA, AFP, and PSA from interfering probes for reliable quantification.
Area of Science:
- Biotechnology and Biomedical Engineering
- Nanotechnology for Diagnostics
- Advanced Microscopy Techniques
Background:
- Multiplexed immunoassays face challenges in differentiating specific immunocomplexes from free or non-specifically bound probes.
- Accurate detection and quantification of tumor biomarkers are crucial for early diagnosis and monitoring.
Purpose of the Study:
- To develop a method for simultaneously counting core-satellite-structured immunocomplexes.
- To overcome limitations in distinguishing specific immunocomplexes from background noise in multiplexed assays.
- To validate the quantification of tumor biomarkers carcinoembryonic antigen (CEA), α-fetoprotein (AFP), and prostate-specific antigen (PSA).
Main Methods:
- Utilized dark-field and fluorescence microscopy combined with a transmission grating-based spectral microscope.
- Constructed core-satellite immunocomplexes using 70 nm gold nanoparticles (AuNPs) and target-specific quantum dots (QDs).
- Differentiated immunocomplexes based on overlapping scattering and fluorescent spectral images.
Main Results:
- Achieved precise identification and quantification of immunocomplexes by analyzing spectral images.
- Demonstrated femtomolar limits of detection for CEA, AFP, and PSA in buffer and blank plasma.
- Successfully quantified three target biomarkers in human plasma samples, confirming method reliability.
Conclusions:
- The developed method effectively distinguishes specific immunocomplexes from free and non-specifically bound probes.
- The core-satellite immunocomplex counting strategy offers a reliable approach for multiplexed biomarker quantification.
- This technique shows significant potential for accurate clinical diagnostics and biomarker analysis.
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.
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