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Updated: Sep 2, 2026
Aptamer-Based Target Detection Facilitated by a 3-Stage G-Quadruplex Isothermal Exponential Amplification Reaction
Published on: October 6, 2022
Dual-Signal Amplification: Synergistic Integration of Nanozyme-Catalyzed SERS Aptasensor with μHPLC Pre-enrichment
Jinxin Chi1, Qian Xie1,2, Shulun Xie1,2
1Institute of Analytical Technology and Smart Instruments, Xiamen Key Laboratory of Food and Drug Safety, College of Environment and Public Health, Xiamen Huaxia University, Xiamen361024, China.
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.
