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

ABSTRACT 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 > 10 6 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 64 5 ≈ 1.1 × 10 9 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.