An enzymatic ratiometric fluorescence assay for 6-mercaptopurine by using MoS₂ quantum dots

A ratiometric fluorescence method is described for the determination of the anticancer drug 6-mercaptopurine (6-MP). The method is based on the use of fluorescent MoS₂ quantum dots (MQDs) and of the enzyme horseradish peroxidase (HRP). In the absence of 6-MP, HRP catalyzes the oxidation of o-phenylenediamine (OPD) by H₂O₂ to form 2,3-diaminophenazine (DAP). This leads to quenching of the violet fluorescence of MQDs (measured at excitation/emission wavelengths of 360/415 nm), while the strong yellow fluorescence of DAP (peaking at 560 nm) becomes increasingly strong. In the presence of 6-MP, however, it will be preferentially oxidized by the HRP/H₂O₂ system to form a disulfide dimer. Hence, less H₂O₂ is available for the oxidation of OPD and less DAP will be formed. This results in the recovery of the violet fluorescence and a decrease of the yellow fluorescence. The ratio of the two signals can be used to quantify either H₂O₂ or 6-MP. Linear responses are observed for H₂O₂ in 0.5-140 μM concentration range, and for 6-MP in the 0.5-70 μM concentration range, with detection limits of 0.1 μM and 0.29 μM, respectively. The method was applied to the determination of 6-MP in spiked human urine and gave satisfactory results. Graphical Abstract Schematic of an enzymatic fluorometric method for determination of 6-mercaptopurine (6-MP). It is based on the presence of 6-MP that can inhibit the HRP-catalyzed oxidation of o-phenylenediamine (OPD) to form 2,3-diaminophenazine (DAP). Hence, the fluorescence resonance energy transfer (FRET) between DAP and MoS₂ quantum dots (MQDs) is suppressed.