A heparin sodium-based one-tube RPA/SCas12a amplification strategy and smartphone-read phosphorothioate-modified G-quadruplex-powered colorimetric detector for on-site detection of monkeypox virus DNA in wastewater

Detection of MPXV DNA in urban wastewater is crucial for early warning, rapid response, and effective outbreak control, offering a non-invasive approach to strengthen epidemic preparedness and public health defense. Based on heparin sodium-assisted one-tube recombinase polymerase amplification (RPA)/split gRNA with Cas12a (SCas12a) and phosphorothioate-modified hairpin G-quadruplex (psHG4), we have developed a colorimetric biosensing platform to achieve sensitive and on-site detection of MPXV DNA in urban wastewater (called hoRPA/SCas12a). The MPXV DNA initiates a one-tube RPA/SCas12a reaction facilitated by heparin sodium, subsequently inducing the DNase activity of SCas12a. The loop DNA of psHG4 is cleaved by active SCas12a to release the pre-blocked psG4 sequence, enabling the formation of a psG4/hemin complex with K + and hemin. The complex is capable of initiating the oxidation of 3,3′, 5,5′-tetramethylbenzidine (TMB) to generate the colorimetric substrate TMBox. The portable smartphone was employed to record the RGB value changes of the sample. The hoRPA/SCas12a platform displays a linear range of 10 aM to 100 fM, with a detection limit of 3 aM. Moreover, this platform can selectively and accurately detect MPXV DNA in urban wastewater. The hoRPA/SCas12a platform is vital for early warning, effective public health response, and strengthening wastewater-based epidemiology in emerging infectious disease control.