Lara González-Cabaleiro

Accurate multiplexed immunophenotyping is essential for understanding cellular heterogeneity. Dual-mode detection strategies, such as surface-enhanced Raman scattering (SERS)-fluorescence, offer synergistic advantages and cross-validation to reduce false positives. We present a method for fabricating dual-mode nanoprobes using metal-phenolic networks (MPNs) as functional coatings on hollow plasmonic nanocapsules. Unlike conventional silica shells, MPNs enable the one-step conjugation of biorecognition elements via metal-phenolic coordination, integrating distinct Raman reporters and fluorescently labeled antibodies within a single probe. These plasmonic MPN nanoprobes achieved multiplex detection of the epidermal growth factor receptor and CD44 in cultured cells using SERS and flow cytometry. Application to mixed HER14 and HEK-293 cultures revealed population distributions of ∼63% HER14 and ∼37% HEK-293 as determined by SERS, corroborated by flow cytometry. This work highlights the potential of dual-mode plasmonic MPNs for accurate, scalable immunophenotyping, offering a robust platform for targeted cell imaging and diagnostics.

Lateral flow immunoassays (LFIAs) are easy‐to‐use antigen tests that provide different signal readouts, with colorimetric readouts being the most commonly used. However, these analytical devices have relatively low sensitivity and produce semiquantitative results, limiting their diagnostic applications. Herein, we address these challenges by implementing a digital surface‐enhanced Raman spectroscopy (SERS)‐based LFIA for the accurate and ultrasensitive quantitative detection of SARS‐CoV‐2 nucleocapsid (N) protein. Compared with average SERS intensity measurements, the digital approach allowed to overcome fluctuations in Raman scattering signals, thereby increasing the analytical sensitivity of the assay. Our method exhibited a quantification range of the viral protein in nasal swabs from 0.001 to 10 pg mL⁻¹, and a limit of detection down to 1.9 aM (0.9 fg mL⁻¹), improving colorimetric LFIAs and conventional‐SERS‐based LFIAs by several orders of magnitude. Importantly, this approach shows an analytical sensitivity of 0.03 TCID₅₀ mL⁻¹, which is greater than that reported by other immunoassays. In conclusion, we successfully demonstrate the robust detection and quantification of SARS‐CoV‐2N protein in nasal swabs at ultralow concentrations. The improvement in the sensitivity of LFIA by digital SERS may pave the way to translate this technology into the diagnostic arena for the ultrasensitive detection of microbes and disease biomarkers.