A near-infrared, surface-enhanced, fluorophore-linked immunosorbent assay

Area of Science:

• Biochemistry
• Immunotechnology
• Analytical Chemistry

Background:

• Enzyme-linked immunosorbent assay (ELISA) is widely used but has limited linear range and multiplexing capabilities.
• Fluorophore-linked immunosorbent assay (FLISA) offers better linearity and multiplexing but lacks sensitivity.
• A need exists for immunoassays combining high sensitivity, broad linear range, and multiplexing potential.

Purpose of the Study:

• To develop a near-infrared, surface-enhanced fluorophore-linked immunosorbent assay (SE-FLISA) with sensitivity comparable to ELISA.
• To improve the limit of detection (LOD) and linear range of FLISA.
• To validate the SE-FLISA technique on both a model system and a clinically relevant biomarker.

Main Methods:

• Implementation of a near-infrared, surface-enhanced approach to a traditional FLISA protocol.
• Utilized rabbit immunoglobulin-G as a model system for direct assay validation.
• Applied the SE-FLISA technique to detect alpha-fetoprotein (AFP) in a clinically relevant assay.

Main Results:

• Achieved a 59-fold enhancement in sensitivity and an 8-fold improvement in LOD for rabbit IgG.
• Demonstrated a 42-fold enhancement in sensitivity and a 16-fold improvement in LOD for alpha-fetoprotein detection.
• The SE-FLISA method requires only two additional steps to traditional FLISA protocols.

Conclusions:

• The developed SE-FLISA provides a significant improvement in sensitivity and LOD over traditional FLISA.
• This technique maintains the advantages of FLISA (linearity, multiplexing) while achieving ELISA-level sensitivity.
• SE-FLISA shows great promise for biomarker research and clinical diagnostics requiring sensitive quantitation over an extended range.