An Encapsulated Phosphate Selective MOF as Indicator Ionophore in Nucleic Acid Amplification Testing

During polymerase catalysed nucleic acid amplification testing (NAAT), inorganic pyrophosphate (PPi) is generated and deoxyribonucleotide triphosphate (dNTP) consumed. These present new opportunities for monitoring the outcome of NAAT, but require selective measurement between PPi and dNTPs. We have previously reported on preliminary results showing TbEu(BTC) is able to distinguish PPi and dNTPs under the conditions used in Loop-mediated amplification (LAMP), an isothermal nucleic acid amplification technique.

We now build on the initial study of the TbEu(BTC) sensor by encapsulation within in a hydrogel matrix to create a more effective LAMP monitoring tool capable of distinguishing between dNTPs and PPi. By overcoming the limitations of the original system, we aim to develop a more robust and scalable device for real-time monitoring of nucleic acid amplification, with potential applications in clinical diagnostics and other applications of nucleic acid amplification.

The europium(III)-based metal–organic framework (MOFs) (TbEu-BTC) was synthesised in situ in agarose. The lanthanide quenching by PPi and dNTPs is characterised due to their influence on the “antenna effect” by the organic ligands as a result of phosphate group coordination with the metal and direct interactions between the organic phosphate and the organic ligand. Agarose plays a pivotal role in the fluorescence energy transfer pathway, enhancing the fluorescence ratio's sensitivity to dNTP concentration.

Results are presented in nucleic acid amplification testing and the system demonstrated for Plasmodium malariae.