Simple visualized readout of suppressed coffee ring patterns for rapid and isothermal genetic testing of antibacterial resistance

We present a facile method based on the coffee ring effect that can rapidly detect antibiotic-resistant bacteria, as an affordable genetic testing platform. When a colloidal solution of particles is dropped onto a substrate surface, an outward capillary flow upon evaporation induces the migration of the particles to the periphery of the droplet, forming a characteristic ring pattern. Herein, we utilize capture DNA microbeads which in the presence of target nucleic acid, form suppressed ring patterns by hybridization-induced crosslinking of the microbeads. The coffee ring-based assay is integrated with isothermal amplification based on rolling circle amplification (RCA), to produce long, single-stranded target DNA and induce hybridization, via a one-step procedure (i-CoRi assay). The resultant ring patterns can be simply observed with the naked eye or recorded with a standard mobile device for readout. The i-CoRi assay was validated for the rapid and specific detection of the antibiotic resistance gene mecA for MRSA, showing that detection was possible at the sub-zeptomolar range (similar to 0.2 zM) with the specificity of distinguishing 2 mismatched bases. The spatial patterns of the microbeads were characterized, showing the dense packing of the microbeads at the center of the droplet and thinning of the ring pattern for the MRSA target, which were distinct from the negative controls MSSA, E. coil, and P. aeruginosa. The images of the microbead patterns were also processed by a simple readout algorithm to discriminate the presence or absence of the coffee ring, to enable diagnostic decision making. The current method provides a rapid and versatile platform for the specific identification of bacterial pathogens and multidrug resistance, especially for diagnosis in resourcelimited settings.