Quantitative estimation of Pyrophosphate ions released during loop-mediated isothermal amplification

Abstract

The increasing demand for rapid, sensitive, and instrument-free nucleic acid detection in clinical and field diagnostics necessitates innovative approaches for monitoring DNA amplification. Pyrophosphate ions (PPi) represent crucial targets generated during DNA and RNA polymerization, serving a pivotal role in monitoring real-time amplification processes and indirectly detecting various microorganisms. The Loop-Mediated Isothermal Amplification (LAMP) technique, known for its high sensitivity and user-friendly attributes, operates under isothermal conditions, eliminating the risk of PPi hydrolysis encountered in PCR. In this study, carbon dots were employed for the detection of PPi ions through an oxidation reaction involving o-Phenylenediamine (OPD) and hydrogen peroxide (H₂O₂). Blue-fluorescent carbon dots were synthesized using a hydrothermal method. Synthesized CDs acquired partial graphitic crystallinity with d-spacing values of 0.33 and 0.50 nm. Carbon and nitrogen were found in abundance in the CD structure. OPD (o-Phenylenediamine) and H₂O₂ react to form
2,3-Diaminophenazine (2,3-DAP) which is dark yellow while PPi blocks the electron transfer between these reactants and color change does not occur. Under UV light, it was found that fluorescence was quenched when PPi was not present between this reaction. While, in the presence of PPi, fluorescence was not quenched depicting that PPi blocks the formation of 2,3-DAP. The limit of Detection (LOD) and Limit of Quantitation (LOQ) for this technique was found to be around 50 nM and 190 nM. A standard plot, constructed using diverse concentrations of inorganic PPi, facilitated the quantification of PPi generated during LAMP at various time intervals and DNA concentrations.