In vivo dynamics of indole- and phenol-derived plant hormones: Long-term, continuous, and minimally invasive phytohormone sensor

Abstract

Specific phytohormone combinations regulate plant growth and responses to environmental stimuli. Monitoring their distribution is key for understanding signaling cross-talk and detecting plant stress early. However, typical means of monitoring these chemicals are often laborious, destructive, or limited to model plants. In this study, we present an amperometric and minimally invasive sensing platform that can be attached to plant leaves for the simultaneous detection of two key phytohormones, auxin [indole-3-acetic acid (IAA)] and salicylic acid (SA). The platform incorporates magnetized microneedles coated with superparamagnetic Fe₃O₄ intercalated into a scaffold of multiwalled carbon nanotubes (MWCNTs). It achieves detection limits of 1.41 μM (IAA) and 1.15 μM (SA) with a strong correlation (R² ≥ 0.7) to ultrahigh-performance liquid chromatography-tandem mass spectrometry measurements. Furthermore, implementing cyclical amperometric cleaning extends the sensor lifespan by preventing electrode passivation. Last, the sensor's capability to monitor the real-time plant responses to several stressors is validated, showcasing its potential for phytodiagnostics and precision farming.

Fig. 1.. Overview of the working principle of the proposed device.

(A) A schematic image depicting the arrangement of the three electrodes around the leaf. (B) The postulated reaction mechanism for the electro-oxidation of IAA and SA by Fe₃O₄@MWCNTs. (C) Attachment of the sensor to the leaf using 3M micropore tape for breathability. (D) Outline of the fabrication process of the sensors. Briefly, (1) a template of the electrodes is fabricated using dip-in laser lithography. (2) The template is then embossed onto PDMS to obtain a negative mold. Subsequently, (3) a polyimide varnish is poured onto the cavities of the negative mold and cured at 250°C. A varnish mixed with ferromagnetic microparticles of NdFeB is used for the working electrode. (4) The substrates are then sputtered with a Ti adhesion layer followed by either Pt (for the working and counter electrodes) or Ag (for the reference electrode). (5) The polyimide replica of the working electrode is then magnetized in a 2 T magnetic field. (6) The working electrode is coated with the Fe₃O₄@MWCNTs. (7) The silver paste interconnects are added and insulated with PDMS. (8) The reference electrode is chlorinated and the electrochemical cell is assembled.

Fig. 2.. Characterization of the Fe₃O₄@MWCNT layer.

(A) Transmission electron microscopy (TEM) image of the Fe₃O₄ intercalated MWCNTs. (B) X-ray powder diffraction (XRD) spectra of the MWCNTs (black), Fe₃O₄ (blue), and the Fe₃O₄-decorated MWCNTs (red). The prominent crystal planes’ peaks are annotated on the plot. The presence of the characteristic peaks of the MWCNTs and the Fe₃O₄ in the combined sample confirms the successful intercalation of the MWCNTs. (C and D) Magnetic hysteresis loops of the Fe₃O₄-decorated MWCNTs and Fe₃O₄, respectively. (E) The Fe₃O₄-decorated MWCNTs being pulled out of a solution of ethanol (70% v/v) toward a magnet. (F) Scanning electron microscopy images of the working electrode (bottom) and the counter electrode (top). The Fe₃O₄@MWCNTs decorating the MNs of the working electrode coat the tip and side lengths of the pyramidal needles. Furthermore, the top view images show partial coverage of the base of the electrode.

Fig. 3.. Analytical testing of the performance of the device.

(A and D) Normalized square wave voltammetry tests for the detection of IAA and SA spiked 0.1 M PBS stocks, respectively, using a reference of Ag/AgCl electrode. (B and E) MPA detection of IAA and SA spiked 0.1 M PBS stocks, respectively. An intermediary cleaning potential of −0.5 V is applied to regenerate the electrodes between the detection potentials. (C and F) Current densities as a function of the concentration of IAA and SA in a background of 0.1 M PBS, respectively. (G) Normalized square wave voltammetry tests for the combined detection of IAA and SA in 0.1 M PBS using a reference of Ag/AgCl electrode. (H) Combined detection of IAA and SA in a background of 0.1 M PBS using MPA detection. Equal concentrations of IAA and SA were used in these tests.

Fig. 4.. Selectivity and fouling analysis of the device.

(A) Normalized square wave voltammetry tests for the cross-detection of interfering analytes in 0.1 M PBS. (B) Stacked bar plot for the oxidation currents recorded at +0.5 V (top) and +1.1 V (bottom). (C and D) Continuous flow-cell oxidation test output to evaluate the sensor’s lifetime for SA and IAA, respectively, with a cleaning pulse (blue) and without a cleaning pulse (red).

Fig. 5.. Mechanical testing of the MNs and correlation of the readings to standard methods.

(A) The testing setup for the crash test with an inset showing the flat metal head compressing the Pt-coated MNs. The load versus compression plot for the three compression rates tested. The inset shows the state of the MNs after mechanical failure. (B and C) The correlation between the sensor’s readings of foliar SA and IAA levels, respectively, in tobacco leaves. A linear regression dashed line is plotted alongside the 95% confidence bands and the R² value. To ensure the acquisition of varied readings, half of the recordings were conducted at night and the other under light exposure. The sensor’s readings of foliar levels of SA (D) and IAA (E) with the corresponding measurements of the hormones in Arabidopsis thaliana using UHLPC-MS/MS are plotted. To ensure the acquisition of varied readings, half of the recordings were conducted at night and the other under light exposure. The levels of foliar SA are markedly high as these specimens were infected with Pst. DC3000 bacteria.

Fig. 6.. Influence of light exposure on the foliar concentration of IAA and SA.

The foliar levels of IAA (blue) and SA (red) measured for young (A) and old (B) tobacco leaves under short day conditions. The age of the leaf influences the cycling behavior of both IAA and SA under short day conditions. The cycling behavior is maintained under long day conditions (C) but the rise and fall in the foliar levels of IAA and SA is rendered more gradual. After constant light exposure (D), the levels of IAA decrease while the levels of SA increase, which might mark a phyto-protective effect against buildup of reactive oxygen species. The yellow shading of the plots indicates the length of the photoperiod whilst the gray shading indicates the corresponding length of the dark period.

Fig. 7.. Influence of mechanical stimulation on the foliar concentration of IAA and SA.

(A) The testing setup for the automated stimulation of the plant leaf. A flat metal surface applies a gentle force of 0.04 N on the leaf at the desired rate. (B) Changes in the levels of foliar IAA (top) and SA (bottom) over time during mechanical stimulation. The insets show the initial minutes after the start of the test (the x axis of the inset is in minutes). A bar plot (n = 5) of the final oxidation currents of IAA (blue) and SA (red) in the three mechanical stimulation conditions is shown alongside images of the leaf after each test.

Fig. 8.. Influence of pathogens on the levels of foliar IAA and SA.

The tobacco leaves used for infection via Botrytis and DC 3000 bacteria inoculation. An aliquot of 1 ml of the infiltration buffer containing the desired fungi or bacteria concentration was pipetted on the leaf. An adjacent leaf was used as a control to eliminate the effect (if any) of the Vogel buffer from the results. (A) The foliar levels of IAA (top) and SA (bottom) for the control (blue) and infected leaves (red) with Botrytis. A magnified image of tissue necrosis due to Botrytis infection on the infected tobacco leaf. (B) The foliar levels of IAA (top) and SA (bottom) for the control (blue) and infected leaves (red) with DC3000. Tissue necrosis at high pathogen concentrations limits the overproduction of foliar SA and IAA leading to a convergence in the readings between the infected leaf and adjacent control leaf. A magnified image of tissue necrosis due to Botrytis and DC3000 infection on the infected tobacco leaves is shown.