Versatile, reusable and highly sensitive SERS-based point-of-care testing microplatform for reliable ATP detection.

The advancement in miniaturized Raman spectrometers, coupled with the single-molecule-level sensitivity and unique fingerprint identification capability of surface-enhanced Raman scattering (SERS), offers great potential for point-of-care testing (POCT). Despite this, accurately quantifying analyte molecules, particularly in complex samples with limited sample volumes, remains difficult. Herein, we present a versatile and reusable SERS microplatform for highly sensitive and reliable quantitative detection of adenosine triphosphate (ATP) in biological fluids. The platform utilizes gold-Prussian blue core-shell nanoparticles modified with polyethyleneimine (Au@PB@PEI NPs), embedded within gold nanoparticle-immobilized capillary-based silica monolithic materials. PB acts as an internal standard, while PEI enhances molecular capture. The periodic, bimodal porous structure of the silica monolithic materials provides uniform and abundant sites for nanoparticle attachment, facilitating rapid liquid permeation, intense SERS enhancement, and efficient enrichment. The platform regulates ATP capture and release through magnesium ions in the liquid phase, eliminating matrix interferences and enabling platform reuse. Integrating efficient molecular enrichment, separation, an interference-free internal standard, a liquid flow channel, and a detection chamber, our platform offers simplicity in operation, exceptional sensitivity and accuracy, and rapid analysis (∼10 min). Employing PB as an internal calibration standard, ratiometric Raman signals (I/I) facilitate precise ATP quantification, achieving a remarkable limit of detection down to 0.62 pM. Furthermore, this platform has been proven to be highly reproducible and validated for ATP quantification in both mouse cerebrospinal fluid and human serum, underscoring its immense potential for POCT applications.