Triple-amplification-assisted visual miRNA sensing for portable identification of medicinal plants and productions

The development of colorimetric biosensors offers a promising tool for on-site visual analysis of medicinal plants and related products in market regulation. However, compared to fluorescent or electrochemical sensing strategies, colorimetric sensors often suffer from weak signals and limited sensitivity, making it challenging to detect low-abundance RNA targets in commercial samples. In response to this issue, here we design a triple signal amplification strategy to achieve ultrasensitive colorimetric signal output for visible microRNA (miRNA) detection in medicinal plants and products, and the smartphone-based imaging enabled portable quantitative analysis for on-site market regulation. The specifically expressed miRNA in ginseng is chosen as the target strand to initiate the first amplification (hybridization chain reaction) for DNAzyme activation, catalyzing the successive cleavage of substrate strands for the liberation of trigger strands that drive rolling circle amplification. This process produces a high abundance of repetitive G-quadruplex motifs, which function as catalytic scaffolds to accelerate a colorimetric reaction, ultimately enabling sensitive and reliable detection by the naked eye. Combining three amplification approaches into a unified cascade, this strategy generates distinguishable colorimetric signals with naked eyes, and the quantitative analysis could be achieved using a smartphone software, offering a wide dynamic range (1-10 5 pM) and a low detection limit of 651 fM. Successful application in medicinal plant ginseng and related products demonstrates the practicality for real sample analysis. The modular amplification design holds broad potential for detecting other molecular targets, underscoring its promise for portable biosensing and on-site regulatory applications.