A Monolith-Based μHPLC-SERS Integrated Microplatform Featuring Mg-Regulated Dual-Capture Mechanism for Real-Time Online ATP Monitoring.

Adenosine triphosphate (ATP), a universal energy currency and biomarker of cellular viability, undergoes dynamic fluctuations critical for neurological health, food safety, and clinical diagnostics. Real-time monitoring of ATP in cerebrospinal fluid (CSF) remains challenging due to limited sample volumes, ultralow concentrations and matrix complexity. Current analytical platforms often struggle to balance separation efficiency, detection sensitivity, and real-time capability. Surface-enhanced Raman scattering (SERS) offers single-molecule sensitivity, but specificity remains a concern, especially when distinguishing ATP from its metabolites such as adenosine diphosphate (ADP) and adenosine monophosphate (AMP). Traditional liquid chromatography-SERS (LC-SERS) platforms are limited by memory effects from nanoparticle aggregation, insufficient hotspot density of conventional substrates, nonspecific adsorption of interfering molecules, and peak broadening caused by packed-bed columns, hindering their application in complex biological matrices. Herein, we introduce a novel micro high-performance liquid chromatography-SERS (μHPLC-SERS) platform featuring a Mg-regulated dual-capture mechanism for online ATP monitoring. This platform utilizes an amino-silica monolith (ASM) with a bimodal pore structure for rapid permeation and efficient ATP entrapment. It integrates an affinity-based SERS detection segment and an electrostatic pre-enrichment segment. The detection segment features a 1 cm SERS aptasensor, functionalized with ATP aptamer-modified gold nanoparticles, which captures ATP through Mg-triggered conformational changes, enabling dynamic detection under flow conditions. The 10 cm pre-enrichment column selectively retains ATP via phosphate-amine interactions in Mg-free conditions, effectively excluding metabolites like ADP and AMP due to their fewer phosphate groups. This results in high specificity and enhanced sensitivity, with a limit of detection of 1.03 × 10 mol/L. Validated in mouse models, this study introduces a microplatform that integrates online enrichment, separation, and SERS detection, offering a novel solution for real-time monitoring of ATP levels in CSF. This advancement ensures precise online ATP quantification in complex matrices under continuous flow, paving the way for applications in food safety, clinical diagnostics, and personalized medicine.