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.