Flow-LAMP: Label-Free Digital LAMP Using Scatter-Based Flow Cytometry on Vortex-Generated Polydisperse Gel Beads

Accurate nucleic acid quantification is vital for clinical diagnostics, yet the widespread adoption of digital PCR remains limited due to its reliance on fluorescence detection and specialized microfluidics. We present Flow-LAMP, a label-free digital assay integrating loop-mediated isothermal amplification with scatter-based flow cytometric analysis of agarose gel beads. Polydisperse gel beads are formed by vortex emulsification and retain magnesium pyrophosphate precipitate in positive reactions. Flow cytometry enables volume and amplification readouts via forward (FSC) and side scattering (SSC) signals, respectively. We confirmed that SSC was strongly correlated with amplification products, while FSC-Height accurately reflected the bead volume. Using Epstein-Barr virus plasmid, Flow-LAMP achieved accurate quantification with a limit of detection of 38.15 copies/μL. Results from testing clinical plasma samples correlated well with qPCR and digital PCR. By eliminating fluorescent labeling and microfluidics, Flow-LAMP offers a cost-effective and accessible platform for digital nucleic acid detection using standard lab equipment.