Agarose-Droplet-Based Digital LAMP Assay for Counting Virus DNA in Single-Particle ICP-MS.

Inductively coupled plasma mass spectrometry (ICP-MS) has emerged as a promising analytical platform for the quantification of biomolecules using elemental tags; however, absolute quantification at extremely low concentrations by ICP-MS without a calibration curve remains challenging. Here, we developed a digital loop-mediated isothermal amplification (LAMP) assay for counting hepatitis B virus (HBV) DNA using single-particle (sp) ICP-MS. The sample and LAMP reagents were mixed and encapsulated in agarose droplets, which were generated by homemade centrifugal droplet generators. The agarose droplets were incubated at 65 °C for amplifying the virus DNA with LAMP primers and then cooled to 4 °C for generating "gel" particles during the temperature-dependent "sol-gel" transition. The LAMP amplicons were intercalated into the agarose particles using polyacrylamide-modified LAMP primers, enabling the labeling of dsDNA with [Ru(bpy) 2 dppz] 2+ and the removal of excess reagents. Only those agarose particles, containing virus DNA, could be labeled with 101 Ru and detected in spICP-MS. We also embedded the 153 Eu-containing polystyrene microspheres into agarose droplets as the internal standard for counting the total number of agarose droplets. The copy number of virus DNA could be counted from the 101 Ru/ 153 Eu pulse numbers in spICP-MS. We achieved the lowest quantification of 25 copy μL -1 virus DNA in one analysis without the need for a calibration curve. The developed assay can be easily tuned for counting multiple types of nucleic acid targets and extended for new possibilities of the spICP-MS-based digital assay.