In Vivo Real-Time Multiplex Detection of Plant Signalling Molecules Using Surface-Enhanced Raman Scattering Nanosensor

International food crisis caused by population growth, environment pollution and climate change has been emerged as one of humanity's the most important problems demanding new agricultural approaches such as smart farm. To understand the biological and chemical dynamics of plants is essential part in next-generation agriculture, and various sensor technologies are studied and applied. The chemicals as known as '<i>signalling molecules</i>' which are released against adverse stimuli to activate defense system of plants are drawing attention as a key to understand plant's biological status under stressors. As one of the analytical approaches to detect signalling molecules, surface-enhanced Raman scattering (SERS)-based optical nanosensor has shown strong potential for its non-invasiveness, water-transparency and capability of real-time detection of chemical dynamics from fingerprint spectra which depend on molecules' own vibration modes.<br/><br/>In our research, we fabricated PDDA-capped Ag bumpy nanoshell (AgNS@PDDA) which has high enhancement factor of <i>ca</i>. 10⁷ and introduced it into plants by infiltrating through stoma. Infiltrated AgNS@PDDA were found to be localized in intercellular space. Thanks to the plasmonic properties of AgNS@PDDA in near-IR window, it was possible to evade chlorophyll’s strong autofluorescence and obtain SERS signals with signal-to-noise ratio of up to 64 with an acquisition time of ~100 ms. PDDA polymer provided aqueous stability for maintaining colloidal stability and effectively attracted signalling molecules most of which are negative conjugate base form by coulombic interaction. As an example, adenosine triphosphate (ATP), known to play a critical role in plant autoimmune systems, is difficult to detect in SERS because of its poor affinity toward metal surface. However, with the PDDA chain forming hydrogen bonding, it was possible to detect with LOD of 10 nM in aqueous solution. Since the interaction between the polymer chain and analytes was reversible, AgNS@PDDA was found to be effective to monitor the change of the concentration of signalling molecules in real time. Also, for quantitative analysis in complicate mixture, we made mathematical models to calculate the concentration of each component from the intensities of SERS signals.<br/><br/>Then, we studied the plant's reaction against biotic and abiotic stress with SERS signal monitoring. When plants leaf was physically wounded, nasturlexin B, one of the phytoalexin, and eATP were detected and those molecules were successfully to monitor the change of concentration in real time for 1 hour after the wound occurred. In addition, glutathione could be detected in watercress exposed to non-freezing cold stress (4 °C, 24 h). The signals from both stresses showed statistically significant difference compared to the signals from the control group. Meanwhile, biotic stress was studied for the plants infected with fungal disease. <i>F. Graminearum</i> was cultured and injected into the plant, and the emerging of signaling molecules in the plant was monitored with nanosensors according to the elapsed days after injection. Even only 2 hours after the fungi injection, when no lesion was detectable, SERS signals of eATP and salicylic acid related to systemic acquired resistance (SAR) were successfully detected. Fungal signals were also detectable after day 2 when the lesion began to be visible. It indicated that the fungal infection was successfully diagnosed at a very early stage with SERS nanosensor, while real-time PCR analysis, an existing diagnostic method, could not confirmed fungal infection until day 1.<br/><br/>Based on our research, we expect that the SERS nanosensors provide novel approach for plant stress diagnosis and early diagnosis of diseases.