The sensitive and accurate detection of copper ions is crucial for public health, medical research, and environmental monitoring. In this study, we developed a sensor based on template-assembly activation of the primer exchange reaction (PER) for the on-site detection of copper ions in blood. Copper ions triggered the assembly of two template fragments into a hairpin structure via a click-chemistry reaction, activating the PER. Polymerase then repeatedly guided the polymerization of multiple primers along the assembled PER template, generating amplified products containing G-triplex sequences. These G-triplex structures specifically enhanced the fluorescence of thioflavin T and exhibited peroxidase-like activity, serving as dual-functional signal reporters for both fluorescence and colorimetric outputs. The sensor effectively amplified a single copper ion signal into multiple G-triplex signals, achieving high sensitivity with detection limits of 68.5 nM in fluorescence mode and 70.4 nM in colorimetric mode. The system also demonstrated strong selectivity, due to the high specificity of the click-chemistry reaction for copper ions. The dual-mode detection minimized false signals and improved accuracy through cross-verification. Additionally, both fluorescence and colorimetric signals were easily measurable without complex instrumentation, enabling flexible application. The method was successfully applied to blood samples, showcasing its robust performance. This sensor offers a promising alternative for copper ion detection with broad potential applications in public health, medical research, and environmental monitoring.
Keywords: Copper ion; Fluorescence and colorimetric signals; G-triplex; Primer exchange reaction; Signal amplification; Template-assembly activation.
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