Quantum Dot-Based FRET Immunoassay for HER2 Using Ultrasmall Affinity Proteins.

Area of Science:

• Biotechnology
• Nanotechnology
• Biochemistry

Background:

• Engineered scaffold affinity proteins offer alternatives to antibodies in biological applications.
• Their small size is advantageous for nanoparticle conjugation and Förster resonance energy transfer (FRET) applications.
• However, engineered affinity proteins have been underutilized in nanobiosensing.

Purpose of the Study:

• To investigate the self-assembly of engineered affinity proteins with quantum dots (QDs) for nanobiosensing.
• To evaluate the performance of these novel conjugates in detecting clinically relevant biomarkers.
• To demonstrate the potential of ADAPT-QD conjugates in a benchtop immunoassay analyzer.

Main Methods:

• Utilized histidine-tagged albumin-binding domain-derived affinity proteins (ADAPTs) for conjugation to quantum dots (QDs).
• Characterized the size of ADAPT-QD conjugates compared to traditional antibody-based conjugates.
• Employed time-gated Tb-to-QD FRET detection for quantifying human epidermal growth factor receptor 2 (HER2) in serum samples using the KRYPTOR analyzer.

Main Results:

• ADAPTs efficiently self-assembled onto zwitterionic ligand-coated QDs, forming significantly smaller conjugates than those using IgG, Fab', or nanobodies.
• Achieved a limit of detection for HER2 down to 40 × 10^-12 m (≈8 ng mL^-1) in serum.
• Demonstrated immediate applicability in a clinical setting using a benchtop immunoassay analyzer.

Conclusions:

• ADAPT-QD conjugates represent a promising, miniaturized platform for nanobiosensing.
• These conjugates offer superior detection limits compared to existing antibody-based assays for HER2.
• The study highlights the potential of engineered affinity proteins in advancing clinical diagnostics.