Single‐Microdroplet Imprinting Enables Specific Nanocavities for Scarce Bioanalyte Recognition and Label‐Free SERS Detection

ABSTRACT Molecularly imprinted polymers (MIPs) offer a more affordable, durable yet equally practical alternative to natural antibodies. However, current syntheses of MIPs face challenges the need for large amounts of pure target, low yields, a lengthy process, and the resulting MIPs exhibiting nonspecific recognition in complex samples. Here, we propose a single microdroplet imprinting (SMI) strategy to create high‐affinity nanocavities over surface‐enhanced Raman scattering (SERS) substrates for the selective recognition of extracellular vesicles (EVs), used here as a model class of cancer‐associated bioanalytes. Experimentally, the SMI is reproducible (coefficient of variation <4%), and high‐yielding (285‐times less reagent volume and 6‐times faster imprinting process), enabling the imprinting of diverse analytes and dummy molecules with precise control over imprinted layer characteristics. Furthermore, a label‐free SERS‐based detection mechanism is developed that scrutinizes the permeability of nanocavities post‐EVs recognition, utilizing a Raman reporter to quantify EVs, thereby achieving high specificity. This platform enables the reproducible quantitation of EVs in 35 µL untreated urine samples, with a sensitivity as low as 317 EVs particles µL −1 . The entire workflow is streamlined into three simple steps, dropping, dipping, and sensing. The SMI approach and detection mechanism are readily adaptable to a broad range of other bioanalytes and MIP‐based platforms.