Partition-Less Digital Immunoassay Using Configurable Topographic Nanoarrays for Extracellular Vesicle Diagnosis of...

Area of Science:

• Biotechnology
• Analytical Chemistry
• Nanotechnology

Background:

• Digital bioassays enhance bioanalysis and diagnostics through improved analytical performance.
• Current methods often rely on partitioning single molecules, requiring complex fabrication and workflows.
• There is a need for simpler, more adaptable digital bioassay platforms.

Purpose of the Study:

• To develop a partition-free digital enzyme-linked immunosorbent assay (dELISA) using microfluidics.
• To enhance single-molecule signal amplification for improved bioassay sensitivity.
• To demonstrate the utility of the developed assay in cancer diagnostics.

Main Methods:

• Developed microfluidic Topographically Intensified, Partition-less dELISA (μTIP-dELISA) utilizing a micropost device for nanogap array formation.
• Employed single-molecule signal amplification to enhance surface-bound enzymatic reactions.
• Adapted μTIP-dELISA for extracellular vesicle (EV)-based liquid biopsy to detect Ewing sarcoma (EWS) biomarkers.

Main Results:

• Achieved >300-fold improvement over conventional ELISA for detecting four EWS protein biomarkers.
• Demonstrated highly sensitive and specific detection of EWS cases using a machine-learning-defined four-marker EV signature.
• Classified control, pediatric, and adult subjects with 97% accuracy, assessing age as a prognostic factor.

Conclusions:

• μTIP-dELISA offers simplicity, adaptability, and integrates microfluidic advantages for improved assay performance, throughput, scalability, and automation.
• The μTIP single-molecule signal amplification strategy can advance the development of digital bioassays.
• μTIP-dELISA serves as a valuable tool for clinical diagnostics in various malignancies.