Real-time and Simultaneous Detection of Salicylic Acid and ROS Production in Living Plants Upon Biotic and Abiotic Stresses Using Single-Walled Carbon Nanotube Based Sensors

Salicylic acid (SA) is an important plant hormone, which controls various aspects of development and mediates plant response to mainly biotic stress. In this work, we developed a SA plant nanobionic sensor, which enables non-destructive and real-time monitoring of SA in living plants. The SA nanosensor is discovered by <i>in vitro</i> screening of single-walled carbon nanotubes (SWNTs) wrapped with different cationic co-polymers, against a list of common plant hormones analytes. This sensing strategy is known as corona phase molecular recognition (CoPhMoRe) which relies on amphiphilic co-polymers acting as synthetic antibodies that bind to specific target analytes, as well as disperse SWNTs in aqueous medium<i>. </i>Upon binding to SA, the SA nanosensor achieved a selective 35% near-infrared (nIR) fluorescence quenching response whereas nIR fluorescence remains relatively invariant upon addition of other key plant hormones. <i>In planta</i> validation of the sensor was achieved in <i>Arabidopsis thaliana </i>(A. thaliana) mutants with altered SA levels, and in non-model <i>Brassica rapa subsp. chinensis</i> (Pak Choy) vegetable infected with black rot causing bacteria. To unravel the interplay between SA and redox signals during biotic and abiotic stress responses, the SA nanosensor is further multiplexed with a H₂O₂ nanosensor in the same leaf to monitor the unique ROS and SA signaling waveforms simultaneously, in response to bacterial infection and wounding. This resolves a longstanding goal of the plant biology field to uncover the sequential links between different plant signaling pathways with the nanosensors providing spatiotemporal information as the plant mounts an integrated stress response.