Attomolar protein detection in complex sample matrices with semi-homogeneous fluidic force discrimination assays

Area of Science:

• Biotechnology
• Assay Development
• Biosensing

Background:

• Fluidic force discrimination (FFD) assays previously combined microbead labels with microarray formats for multiplexed detection.
• Existing methods faced limitations in sensitivity and speed for certain applications.

Purpose of the Study:

• To develop a simplified, semi-homogenous (SH) implementation of the FFD assay.
• To enhance assay sensitivity and speed for detecting analytes in complex matrices.

Main Methods:

• Implemented a semi-homogenous approach mixing microbeads directly with samples for efficient target capture.
• Utilized controlled fluidic forces to remove unbound beads from a microarray surface.
• Counted remaining beads on capture spots for target identification and quantification.

Main Results:

• Achieved a 1000-fold improvement in sensitivity, reaching attomolar concentrations (e.g., 35 aM staphylococcal enterotoxin B).
• Demonstrated assay adaptability for analyte extraction, preconcentration, and identification in serum and whole blood.
• Developed a kinetic model showing solution-phase capture provides a significant advantage at low concentrations.

Conclusions:

• The SH FFD assay offers a rapid (10 min), highly sensitive, and adaptable method for analyte detection.
• This approach significantly improves detection limits, particularly in complex biological samples.
• The findings highlight the kinetic benefits of solution-phase capture in microfluidic assays.