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

<jats:p> The formation of magnesium pyrophosphate (Mg <jats:sub>2</jats:sub> P <jats:sub>2</jats:sub> O <jats:sub>7</jats:sub> ) in nucleic acid amplification and cell-free transcription systems has attracted considerable attention, since Mg <jats:sub>2</jats:sub> P <jats:sub>2</jats:sub> O <jats:sub>7</jats:sub> serves as a reliable indicator of reaction efficiency. However, real-time monitoring of Mg <jats:sub>2</jats:sub> P <jats:sub>2</jats:sub> O <jats:sub>7</jats:sub> remains challenging, relying largely on time-consuming analytical techniques or end-point detection methods. Here, we report a Mg <jats:sub>2</jats:sub> P <jats:sub>2</jats:sub> O <jats:sub>7</jats:sub> -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 <jats:sub>2</jats:sub> O <jats:sub>7</jats:sub> <jats:sup>4-</jats:sup> ) and magnesium (Mg <jats:sup>2+</jats:sup> ) ions. Real-time fluorescence profiles revealed that the GSH-AuNCs/Mg <jats:sub>2</jats:sub> P <jats:sub>2</jats:sub> O <jats:sub>7</jats:sub> 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 <jats:sub>4</jats:sub> ) <jats:sub>2</jats:sub> SO <jats:sub>4</jats:sub> ), deoxynucleotides (dNTPs) and Bst polymerase, on Mg <jats:sub>2</jats:sub> P <jats:sub>2</jats:sub> O <jats:sub>7</jats:sub> 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 <jats:sup>2</jats:sup> -10 <jats:sup>8</jats:sup> copies/reaction. The resulting one-tube, contamination-free assay was shown to have a response time of &lt;25 min even in a crude saliva sample. Beyond diagnostics, this crystallization-activated fluorescence strategy may also support real-time investigation of Mg <jats:sub>2</jats:sub> P <jats:sub>2</jats:sub> O <jats:sub>7</jats:sub> formation in other biotechnological processes, including in vitro transcription and Mg <jats:sub>2</jats:sub> P <jats:sub>2</jats:sub> O <jats:sub>7</jats:sub> -bioorganic composites synthesis. </jats:p>