Mechanistic Insights into Magnesium Pyrophosphate Formation in the Presence of Gold Nanoclusters Enable Genetic Analysis via Co-Aggregation-Induced Fluorescence Enhancement

The formation of magnesium pyrophosphate (Mg ₂ P ₂ O ₇ ) in nucleic acid amplification and cell-free transcription systems has attracted considerable attention, since Mg ₂ P ₂ O ₇ serves as a reliable indicator of reaction efficiency. However, real-time monitoring of Mg ₂ P ₂ O ₇ remains challenging, relying largely on time-consuming analytical techniques or end-point detection methods. Here, we report a Mg ₂ P ₂ O ₇ -driven co-aggregation mechanism involving glutathione-capped gold nanoclusters (GSH-AuNCs) that induces fluorescence enhancement, enabling real-time crystal formation monitoring. The mechanism was first investigated in simplified mixtures containing pyrophosphate (P ₂ O ₇ ^4- ) and magnesium (Mg ²⁺ ) ions. Real-time fluorescence profiles revealed that the GSH-AuNCs/Mg ₂ P ₂ O ₇ co-aggregation can be correlated with crystal formation/growth/solubilization and solution turbidity, while distinct kinetic patterns can be indicative of the crystal size at the end of the reaction. As a next level of complexity, we examined the effects of common components in an enzymatic amplification reaction, i.e. , dithiothreitol (DTT), ammonium sulfate ((NH ₄ ) ₂ SO ₄ ), deoxynucleotides (dNTPs) and Bst polymerase, on Mg ₂ P ₂ O ₇ formation through real-time GSH-AuNCs fluorescence variations. Guided by the above results, we studied and selected the experimental parameters for the design of an optimized qualitative (end-point) or quantitative (real-time) genetic test. Finally, the loop-mediated isothermal amplification (LAMP) was used as a platform to demonstrate the quantification of Influenza A RNA within the range of 10 ² -10 ⁸ copies/reaction. The resulting one-tube, contamination-free assay was shown to have a response time of <25 min even in a crude saliva sample. Beyond diagnostics, this crystallization-activated fluorescence strategy may also support real-time investigation of Mg ₂ P ₂ O ₇ formation in other biotechnological processes, including in vitro transcription and Mg ₂ P ₂ O ₇ -bioorganic composites synthesis.