Improving the Sensitivity of a Multiplexed Digital Immunoassay Based on Extremely High Bead Analysis Efficiency

Area of Science:

• Biotechnology
• Analytical Chemistry
• Clinical Diagnostics

Background:

• Ultrasensitive protein quantification is crucial for early disease detection and monitoring.
• Traditional digital enzyme-linked immunosorbent assays (dELISA) suffer from bead loss and limited sensitivity.
• Existing methods struggle to achieve high sensitivity and efficiency in protein detection.

Purpose of the Study:

• To develop an ultrasensitive, stable, and user-friendly detection methodology for subfemtogram protein quantification.
• To overcome the limitations of traditional dELISA, such as bead loss and multistep reaction inefficiencies.
• To introduce a novel digital immunoassay with extremely high bead analysis efficiency (HiBeA).

Main Methods:

• Integration of a bead transfer strategy in multistep immunoreaction processing.
• Utilizing a flow cytometry detection mode for enhanced bead analysis.
• Development of the highly efficient bead analysis (HiBeA) digital immunoassay.

Main Results:

• Achieved over 95% bead analysis ratio, ensuring high sensitivity, efficiency, and stability.
• Demonstrated multiplexed detection of IL-10 and IL-6 with detection limits of 5.9 and 8.8 fg/mL, respectively.
• Showcased a 3- to 4-fold sensitivity enhancement using only 1% of beads compared to the commercial single-molecule array (SiMoA) system.

Conclusions:

• HiBeA offers ultrasensitive and robust detection with high bead analysis efficiency.
• The method provides stable, multiplexed detection capabilities for clinical applications.
• HiBeA presents significant potential for advancing ultrasensitive clinical diagnostics.