Amplification-Free Dye-Labeled Digital Immunoassay: From Subpicomolar to Attomolar

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biotechnology

Background:

• Digital immunoassays (DIA) are crucial for single-molecule counting.
• Dye molecules offer advantages over traditional labels like enzymes or nanoparticles in DIA.
• Existing dye-labeled DIA methods lack the sensitivity for detecting low-abundance neurological biomarkers in blood.

Purpose of the Study:

• To develop an ultrasensitive, amplification-free DIA using dye labeling and synergistic sedimentation (SynSed).
• To evaluate the assay's performance for detecting glial fibrillary acidic protein (GFAP), a neurological biomarker.
• To assess the potential of this assay for diagnosing neurodegenerative diseases.

Main Methods:

• Development of a novel dye-labeled DIA system incorporating synergistic sedimentation (SynSed).
• Optimization of the assay for GFAP detection in plasma samples.
• Validation of assay sensitivity, dynamic range, limit of detection (LOD), and precision.

Main Results:

• The SynSed-assisted DIA achieved an ultrasensitive, amplification-free detection of GFAP.
• Demonstrated a wide dynamic range (0.015–10 fM) with excellent linearity (R² > 0.99) and a low LOD of 5.7 aM.
• Significantly elevated GFAP levels were observed in plasma from Alzheimer's and Parkinson's disease patients compared to healthy controls, with distinct differences between patient groups.

Conclusions:

• The developed SynSed-assisted dye-labeled DIA offers high sensitivity, stability, and specificity.
• This assay shows significant promise for early screening of neurodegenerative diseases like Alzheimer's and Parkinson's.
• The technology supports therapeutic monitoring and risk stratification for neurological disorders.