Quantum Dot Digital Immunoassays on a Recyclable High-Refractive-Index Bead Array Imaged with a Low-Numerical-Aperture Objective

In digital immunoassays (DIAs), imaging detection has relied on high-magnification (≥60×), high-numerical-aperture (NA, ≥0.6) oil objectives, which impede high-throughput automated imaging and instrument miniaturization. Here, we present a simple and cost-effective method for fabricating a high-refractive-index bead array (h-RIBA) substrate through the self-assembly of barium titanate glass microspheres (BTGMs) and their embedding in a PDMS membrane. Functioning like a sticky note, the h-RIBA substrate can be easily attached to material surfaces and enables clear imaging of individual quantum dots (QDs) at the single-molecule level using a 20×/0.4 NA air objective. As a proof of concept, we successfully performed magnetic bead-based DIAs for carcinoembryonic antigen (CEA) using this substrate and objective, achieving a linear range of 0–1.5 pM and a limit of detection (LOD) of 88 fM. Measured CEA concentrations in plasma are consistent with the values provided by the hospital, validating that the h-RIBA substrate is suitable for clinical testing. The h-RIBA substrate is characterized by low cost, user-friendly operation, and broad compatibility with various fluorophores and nanoparticles. Its integration with low-NA objectives provides a practical path toward automating single-molecule fluorescence imaging, thereby accelerating the translation and commercialization of the DIA technology.