Single-Particle ICP−MS Enabled Multiplex Digital Detection of Nucleic Acids via Feedback Recombinase Polymerase Amplification

Abstract Digital droplet assays, with their single-molecule sensitivity, hold great promise for early disease diagnosis by enabling precise biomarker quantification and the generation of patient-specific molecular profiles. Inductively coupled plasma mass spectrometry (ICP−MS) has shown its great potential in the quantification of biomolecules with elemental labeling strategies. However, the development of ICP−MS-based digital assays has been limited, primarily due to the challenge of efficiently anchoring elemental labels within droplets. To address this, we developed a feedback recombinase polymerase amplification (F-RPA)-based digital analyzer via a facile droplet generator and combined it with single particle (sp)-ICP−MS for the quantitative analysis of human papillomavirus (HPV) genetic markers. In this system, HPV DNA molecules are randomly partitioned across tens of thousands of agarose droplets, following a Poisson distribution. Individual encapsulated target DNA molecules initiate on-demand F-RPA amplification and anchoring of elemental tags for subsequent signal transduction. Leveraging the discrete event counting characteristics and multi-element analytical capabilities of sp-ICP−MS, the digital F-RPA assay achieved highly sensitive and multiplexed analysis, with detection limits as low as 20 copies/μL for HPV16 and 9 copies/μL for HPV18 DNA. The method was validated by quantifying the HPV DNA load in cervical cancer cell lines and further applied to clinical cervical swab samples, where it identified 26 HPV16-positive and 20 HPV18-positive cases with 100% precision. This digital F-RPA sp-ICP−MS system emerges as a robust diagnostic platform for trace biomarker analysis, significantly advancing the role of sp-ICP−MS in digital droplet detection and clinical diagnosis.