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 degrees C for amplifying the virus DNA with LAMP primers and then cooled to 4 degrees 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 Ru-101 and detected in spICP-MS. We also embedded the Eu-153-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 Ru-101/Eu-153 pulse numbers in spICP-MS. We achieved the lowest quantification of 25 copy mu L-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.