Triplexed CEA-NSE-PSA Immunoassay Using Time-Gated Terbium-to-Quantum Dot FRET

Area of Science:

• Biomedical Engineering
• Analytical Chemistry
• Nanotechnology

Background:

• Time-gated Förster resonance energy transfer (TG-FRET) using terbium (Tb) complexes and semiconductor quantum dots (QDs) offers unique photophysical properties for multiplexed biosensing.
• Current multiplexed Tb-to-QD FRET immunoassays show promise for in vitro diagnostics but often lack the performance required for clinical applications.

Purpose of the Study:

• To develop a homogeneous TG-FRET immunoassay capable of simultaneously quantifying three clinically relevant biomarkers: carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), and prostate-specific antigen (PSA) from a single serum sample.
• To evaluate the performance and clinical relevance of this multiplexed assay.

Main Methods:

• Conjugation of Tb-IgG antibody donor with compact QD-F(ab')2 antibody acceptor conjugates emitting at 605, 650, and 705 nm.
• Development of a sandwich immunoassay format where antigen binding triggers TG-FRET sensitization of QD acceptors by Tb donors.
• Quantification of antigen concentrations based on the specific increase in FRET ratios of QD and Tb TG luminescence intensities.

Main Results:

• Successful simultaneous quantification of CEA, NSE, and PSA from a single 50 µL serum sample.
• Achieved limits of detection (LoDs) of 3.6 ng/mL for CEA, 3.5 ng/mL for NSE, and 0.3 ng/mL for PSA, which are within the clinically relevant range.
• Demonstrated the feasibility of multiplexed Tb-to-QD FRET immunoassays for clinical diagnostics.

Conclusions:

• The developed homogeneous TG-FRET immunoassay enables simultaneous detection of multiple cancer biomarkers from a single sample.
• The assay exhibits clinically relevant sensitivity and demonstrates the potential for translation into routine clinical diagnostic platforms.
• This multiplexed biosensing approach offers a promising advancement for in vitro diagnostics.