Bead-free digital enzyme-linked immunoassay on PDMS microwell array.

Area of Science:

• Biomarker Discovery
• Assay Development
• Microfluidics

Background:

• Low-abundance protein biomarkers are crucial for disease diagnosis but challenging to measure.
• Conventional assays lack sensitivity, while advanced methods are costly and complex.
• A need exists for cost-effective, ultrasensitive protein detection methods.

Purpose of the Study:

• To develop a cost-effective, bead-free digital ELISA platform for ultrasensitive protein detection.
• To overcome limitations of existing ultrasensitive assays, such as high costs and complex fabrication.

Main Methods:

• Fabrication of a polydimethylsiloxane (PDMS) microwell array platform using soft lithography.
• Development of a bead-free digital ELISA within PDMS microwells for immunocomplex formation and separation.
• Modification of the PDMS chip for large-scale femtoliter droplet arrays with minimized evaporation and cross-contamination.

Main Results:

• Achieved ultrasensitive protein detection using the PDMS microwell array digital ELISA.
• Demonstrated significantly reduced costs compared to bead-based digital ELISA methods like SiMoA.
• Successfully applied the platform for single-molecule enzyme and cytokine analysis, with detection limits of 1.57 fM (SβG) and 12.3 fg/ml (IL-6).

Conclusions:

• A straightforward, low-cost PDMS microwell array for digital ELISA was successfully developed.
• The platform enables mass production with minimal batch-to-batch variation.
• This cost-effective and highly sensitive platform shows promise for early disease screening.