Construction of a target-stimulated nanoparticle-assembly homogeneous sensing platform for multiplexed DNA methylation assay

Detection of multiple tumor-associated DNA methylation variations can shed light on the pathogenesis and physiological characteristics underlying disease development. Herein, we constructed a target-stimulated gold nanoparticles (AuNPs) -assembly homogeneous sensing platform for multiple DNA methylation assays via single-particle inductively coupled plasma mass spectrometry (SP-ICP-MS). The introduction of target methylated DNA could initiate the DNA glycosylase-assisted specific digestion of the Linker probe, thus leading to a reduction in the agglomeration of AuNPs. The AuNP aggregates with varying size distributions can be well discriminated by the intensity and frequency of the Au pulse signal utilizing SP-ICP-MS, thereby enabling dual-mode detection of methylated DNA with good accuracy. Through the modular design of Linker probes, this homogeneous sensing system is feasible for multiplexed DNA methylation analysis, showing good universality. Taking methylated MLH1 and methylated MGMT as representatives, the limit of quantification under the two quantification modes are 25.0 pmol L^-1 and 10.0 pmol L^-1 for methylated MLH1 and methylated MGMT, respectively. The proposed method was successfully applied to direct analysis of methylated MLH1 and methylated MGMT in 34% human serum samples, demonstrating the robust resistance of the proposed method to biological matrices and its suitability for real biological sample analysis. The programmability and expandability of the homogeneous SP-ICP-MS strategy render it a promising candidate for disease-relevant biomolecule diagnostics and pathogenic mechanism research.