Digital Immunoassays for Sensitive Quantification of Blood Biomarkers Using Solid-State Nanopores

Area of Science:

• Biomolecular detection
• Nanopore sensing
• Digital immunoassays

Background:

• Digital immunoassays offer absolute quantification of biomolecules.
• Nanopore sensors excel at nucleic acid counting but face challenges with protein detection due to translocation variability and limited recognition.
• Precise protein quantification using nanopore platforms remains a significant challenge.

Purpose of the Study:

• To develop a nanopore-based digital immunoassay for sensitive and precise protein quantification.
• To overcome limitations in nanopore protein sensing through molecular amplification and DNA-based signal conversion.
• To validate the assay's performance using a clinically relevant biomarker.

Main Methods:

• Adaptation of a digital immunoassay scheme for nanopore sensing.
• Utilizing gold nanoparticle (AuNP)-mediated molecular amplification with a single-molecule readout.
• Employing a DNA NanoLock probe and paramagnetic bead-based immunocapture for protein-to-DNA signal conversion.
• Incorporating AuNPs with DNA proxy reporters for signal amplification.

Main Results:

• Demonstrated translation of protein recognition into a quantifiable DNA signal.
• Achieved signal amplification of 2 orders of magnitude using AuNPs, enhancing sensitivity.
• Successfully detected glial fibrillary acidic protein (GFAP) in plasma samples with femtomolar sensitivity (∼40 pg/mL).
• Reduced assay times to hours and extended dynamic range to 3-log.

Conclusions:

• The developed nanopore digital immunoassay provides a robust and reproducible method for precise protein quantification.
• The system effectively overcomes previous challenges in nanopore protein sensing, offering improved sensitivity and reduced assay times.
• Self-calibration eliminates inter-device variability, making the assay suitable for various nanopore platforms.