Mannitol solution

The IP was evaluated by providing an enteral bolus of 15 mL/kg of a 5% lactulose (Sigma-Aldrich, Copenhagen, Denmark) and 5% mannitol solution (Sigma-Aldrich, Copenhagen, Denmark) exactly 3 h before euthanasia. Urine was collected at the time of euthanasia and stored at −20 °C until assayed. Concentrations of lactulose and mannitol were measured in urine by an enzymatic spectrophotometric method (Pentra 400, Irvine, CA, USA). In the presence of mannitol dehydrogenase, mannitol was oxidized by NAD into the fructose and NADH. The amount of NADH was detected by spectrophotometry at 340 nm. lactulose was also hydrolyzed into galactose and fructose. Fructose was then catalyzed into fructose-6-phosphate and to glucose-6-phosphate by phosphoglucoisomerase. Glucose-6-phosphate was dehydrogenated by adding glucose-6-phosphate-dehydrogenase in the presence of NADP to form NADPH. The concentration of NADPH is proportional to the concentration of lactulose and can be measured spectrophotometrically at 340 nm. L/M ratios quantified by enzymatic assay are provided in the Supplementary Materials.

After synthesis of SA sensor, S3 free polymer that is unbound to SWNTs were removed by centrifugal filtration using the Amicon® Ultra-4 Centrifugal Filter tubes with 100 kDa MWCO. Complete removal of free polymer is confirmed by the absence of the polymer UV-Vis absorbance band (λ_max = 370 nm) in the filtrate. The SA sensor solution was then diluted to 1.25 mg/L and infiltrated into N. benthamiana/pak choi leaves. Infiltrated plants were kept in dark at 28 °C for 1 h. Plasmolysis of leaf was induced by treatment with 0.8 M Mannitol solution (Sigma-Aldrich, Singapore). All leaf samples were viewed with an inverted confocal microscope (SP8, Leica, Germany). For confocal imaging, the excitation laser and detector wavelength ranges used for each fluorophore is as follows, SA sensor: 405 nm (415—440 nm); Cy3-(GT)₁₅ SWNT: 552 nm (565—580 nm); Chlorophyll autofluorescence: 594 nm (640—700 nm). Confocal images were analyzed using the ImageJ software.