Sensitive detection of methylated DNA by elemental labeling inductively coupled plasma mass spectrometry using...

A highly sensitive method for the quantification of methylated DNA was developed by integrating gold nanoparticles (AuNPs) labeling with inductively coupled plasma mass spectrometry (ICP-MS), coupled with the cascade amplification of exponential isothermal amplification (EXPAR) and deoxyribozyme (DNAzyme). In this design, the target methylated DNA binds to a specific single-stranded DNA (ssDNA), forming a complex that is recognized and cleaved by the modification-dependent endonuclease FspEI. This cleavage releases a distinct X sequence, which then serves as a primer to initiate the EXPAR process. The amplified X sequences function as Deoxyribozyme (DNAzyme), cleaving substrate strands on magnetic beads (MB-DNA-AuNPs probe) and releasing AuNPs for subsequent ICP-MS detection.

The concentration of the target methylated DNA is quantitatively determined by measuring the released AuNPs via ICP-MS, while unmethylated DNA fails to trigger the cleavage and amplification cascade, ensuring high specificity. The proposed method achieved an exceptionally low limit of detection of 5.2 fmol L^-1 for methylated DNA, which is approximately four orders of magnitude lower than that obtained with conventional elemental labeling ICP-MS methods. Furthermore, this method is capable of detecting target methylated DNA with different lengths and distinguishing a methylation level as low as 0.1% in mixed samples.

The practical applicability of this method was validated through the analysis of methylated DNA in real serum samples, with recoveries ranging from 89.6% to 96.5% in spiking experiments. These results indicate its strong potential for the sensitive and reliable determination of methylated DNA in complex biological samples, offering a powerful tool for epigenetic research and clinical diagnostics.