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This study presents a simple route to fabricate shape imprinted microspheres and their application toward multiplex immunoassay. Specifically, a photocurable fluid (polyethylene glycol diacrylate, [PEGDA]) confined in a micromold results in drop formation due to mold swelling and capillarity upon addition of immiscible wetting fluid; during this formation, part of the photocurable fluid is entrapped at the swollen open end, thereby producing polymeric microspheres with controlled imprinted shapes on their surface upon photopolymerization. This approach is able to produce highly uniform microspheres with different imprinted shapes depending on mold geometry in a consistent manner; their size can also be tuned by varying mold dimensions. Furthermore, we impart amine‐reactive functional groups to the microsphere, which allows them to be functionalized efficiently. Lastly, we conjugate different capture antibodies on microspheres with different imprinted shapes, and these microspheres were demonstrated to be shape‐encoded suspension arrays for multiplex immunoassays. This technique provides high selectivity and a simple decoding method for biosensing applications.

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