Dual-Signal Amplification: Synergistic Integration of Nanozyme-Catalyzed SERS Aptasensor with μHPLC Pre-enrichment for Ultrasensitive ATP Monitoring

Abstract

The precise quantification of adenosine triphosphate (ATP) at physiologically critical femtomolar levels in complex biological fluids remains a formidable challenge, constrained by irreconcilable demands for extreme sensitivity, specificity, and operational robustness. Conventional integrated platforms, such as LC-SERS, often suffer from functional fragmentation, in which separation and detection operate sequentially without synergy, thereby limiting overall performance. Stand-alone amplification strategies, meanwhile, are intrinsically hampered by matrix interference. Herein, we introduce a versatile biosensing paradigm that overcomes these limitations through the synergistic integration of two orthogonal amplification mechanisms within a unified microfluidic platform. Our dual-signal amplification microfluidic platform (2Amp-MFP) integrates online micro high-performance liquid chromatography (μHPLC) for target pre-enrichment and interference removal with a nanozyme-catalyzed SERS aptasensor (NC-SERS aptasensor) for ATP recognition and catalytic signal generation. The upstream amino-silica monolith (ASM) provides phosphate-dependent retention, allowing adenosine diphosphate (ADP), adenosine monophosphate (AMP), and matrix components to be removed during loading and washing, while triphosphate species are retained and subsequently eluted with a Mg2+-containing mobile phase. In the downstream aptasensor, ATP is selectively recognized, triggering the displacement of a fraction of the aptamer-conjugated Au@Pt nanozymes from the sensor interface. After washing, the Au@Pt@Aptamer NPs remaining hybridized on the monolith catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to generate Raman-active reporter oxTMB, yielding a SERS intensity that decreases with increasing ATP concentration. Under optimized conditions, the 2Amp-MFP achieved a limit of detection (LOD) of 63 fM for ATP and enabled specific quantification of spiked ATP in human serum and urine samples. This synergistic dual-signal amplification strategy provides a promising platform for trace ATP analysis in complex biological matrices.