Lateral flow assay with near-infrared dye for multiplex detection

Area of Science:

• Biomedical Diagnostics
• Assay Development
• Point-of-Care Testing

Background:

• Lateral flow assays (LFAs) are widely used point-of-care diagnostic tools due to their speed and ease of use.
• Traditional LFAs often suffer from limited analytical sensitivity, lack quantitative output, and face challenges in multiplexing.
• These limitations hinder their application in precise biomarker measurement.

Purpose of the Study:

• To develop a quantitative, multiplex lateral flow assay (LFA) utilizing near-infrared (NIR) detection.
• To enhance analytical sensitivity and enable simultaneous measurement of multiple biomarkers.
• To demonstrate the feasibility of NIR detection for improved LFA performance.

Main Methods:

• Conjugated NIR dye to antibodies and integrated them into LFAs for singleplex and duplex assays.
• Optimized singleplex NIR-LFAs for interleukin-6 (IL-6) detection (0–200 pg/mL).
• Developed duplex NIR-LFAs for simultaneous IL-6 (0–100 pg/mL) and C-reactive protein (CRP) (50–2500 ng/mL) measurement, validated with spiked samples against ELISA.

Main Results:

• NIR-LFAs achieved a limit of detection of 4 pg/mL for IL-6 in a plasma matrix with <7% CV.
• Duplex NIR-LFAs accurately quantified both IL-6 and CRP simultaneously.
• Assay results showed strong correlation with ELISA, with R² values of 0.9825 for IL-6 and 0.9711 for CRP.

Conclusions:

• NIR-LFAs provide quantitative measurements at picogram-per-milliliter concentrations with high signal-to-background ratios.
• The developed assays can detect biomarkers at vastly different concentrations in a multiplex format.
• NIR-LFAs represent a promising advancement for biomarker evaluation in point-of-care settings, offering comparable performance to ELISAs.