Recombinase Polymerase Amplification Coupled with Graphene Oxide Nanomaterial-Assisted Purification and Target Signal Selection for Highly Sensitive and Rapid Harmful Algae Detection

Abstract

Harmful algal blooms (HABs) pose serious ecological and health risks, creating an urgent need for rapid, sensitive, and accurate detection of harmful algae. Here, we developed a graphene oxide (GO)- and Mg2+-regulated recombinase polymerase amplification assay (G-M-RPA) for rapid and ultrasensitive detection of Heterosigma akashiwo, Prorocentrum minimum, and Akashiwo sanguinea. Using conserved internal transcribed spacer sequences, we designed species-specific primers labeled with 6-carboxyfluorescein for fluorescence readout. Innovative one-step purification of RPA products was achieved using self-synthesized GO-wrapped magnetic nanocomposites instead of traditional purification kits, reducing purification time from 15 to 2 min (∼86.7%). The fluorescence resonance energy transfer property of the GO nanosheets was applied to quench the fluorescence of excess single-stranded primers, significantly lowering background levels. Mg2+ further enhanced primer–GO interactions, likely through electrostatic screening and Mg2+-mediated interactions. The entire detection process was completed within ∼30 min, achieving a genomic DNA detection limit of 3.3 fg/μL, corresponding to ∼7 cells/mL for H. akashiwo. The assay also showed reliable performance in mixed-DNA and seawater matrices, with favorable reproducibility and short-term stability. Overall, the proposed G-M-RPA strategy provides a rapid, sensitive, and practical platform for multispecies harmful-algae detection, nearshore environmental monitoring, and early warning of HABs.