Decoupling Fabrication From Encoding: DNA-Addressable Template Microparticles for Large, User-Defined Optical Barcode...

Optical barcodes for pooled high-throughput screening must support large libraries while remaining decodable in a single imaging step. Existing approaches often trade design control for manufacturability: deterministic barcodes often require per-code redesign of particle fabrication, whereas stochastic combinatorial barcodes are difficult to generate as predefined batches. Here we introduce a chemically programmable barcoding architecture that decouples particle fabrication from barcode assignment. Using a contact-free multilaminar flow lithography platform with all-around three-dimensional sheathing, we continuously fabricate a universal hydrogel scaffold containing five spatially segregated DNA-addressable domains at rates > 10⁶ particles/h. Chosen barcode identities are subsequently written on demand onto the same template batch by domain-selective DNA hybridization. Single-domain measurements resolved 64 candidate optical states, indicating an experimentally informed theoretical upper bound of 64⁵ ≈ 1.1 × 10⁹ barcodes. We further implemented a predefined 59,049-code library by split-pool labeling, achieving an 83.6% recovery of decoded beads at a stringent posterior threshold (≥ 0.95). After 11 days, >7,800 beads were correctly re-identified at ≥ 0.95 accuracy in matched fields of view. This strategy provides a highly scalable, chemically programmable route to build large, user-defined optical barcode libraries with single-image optical readout and longitudinal traceability.