This work presents an in vivo stem-mounted sensor for Nicotiana tabacum plants and an in situ cell suspension sensor for Solanum lycopersicum cells. Stem-mounted sensors are mechanically stable and less sensitive to plant and air movements than the previously demonstrated leaf-mounted sensors. Interdigitated-electrode-arrays with a dual working electrode configuration were used with an auxiliary electrode and an Ag/AgCl quasi-reference electrode. Signal amplification by redox cycling is demonstrated for a plant-based sensor responding to enzyme expression induced by different cues in the plants. Functional biosensing is demonstrated, first for constitutive enzyme expression and later, for heat-shock-induced enzyme expression in plants. In the cell suspension with redox cycling, positive detection of the enzyme β-glucuronidase (GUS) was observed within a few minutes after applying the substrate (pNPG, 4-Nitrophenyl β-D-glucopyranoside), following redox reactions of the product (p-nitrophenol (pNP)). It is assumed that the initial reaction is the irreversible reduction of pNP to p-hydroxylaminophenol. Next, it can be either oxidized to p-nitrosophenol or dehydrated and oxidized to aminophenol. Both last reactions are reversible and can be used for redox cycling. The dual-electrode redox-cycling electrochemical signal was an order of magnitude larger than that of conventional single-working electrode transducers. A simple model for the gain is presented, predicting that an even larger gain is possible for sub-micron electrodes. In summary, this work demonstrates, for the first time, a redox cycling-based in vivo plant sensor, where diffusion-based amplification occurs inside a tobacco plant’s tissue. The technique can be applied to other plants as well as to medical and environmental monitoring systems.
The ability to detect small signals in plants is highly important since it can provide early stage detection of plant stress by micro metabolites sensing. This work presents a first demonstration of redox cycling amplification in plant sensors improving signal level. The concept and the setup is discussed demonstrating a five-fold redox cycling amplification by using on-chip interdigitated microelectrodes arrays (IDA).The concept of plant sensors refers to any sensor which is used to monitor the plant’s status or correlated parameters in the plant’s environment. In this work, the plant sensor monitors signals which are expressed by the plant itself, i.e. whether it is well hydrated, is it experiencing a heat shock, etc. In this work, modified plants were tailored to generate β-glucuronidase (GUS), a bio-signaling molecule, due to an induced stress effect. The expressed enzyme then reacts with PNPG (4-Nitrophenyl β-D-glucopyranoside) to produce p-nitrophenol which acts as the electroactive species.P-nitrophenol electrochemical characterization show that the molecule is reduced at -0.75 V, associated with a four-electron reaction of the nitro group reduction into hydroxylamine species. Subsequent reduction and oxidation peaks at +0.15 V and +0.18 V occurs due to 4-hydroxyl-amino-phenol to 4-nitrosophenol oxidation and the subsequent reversible reduction.When using redox cycling amplification, two closely packed working electrodes are individually biased at different potentials. In the first working electrode, referred to as the generator, the initial oxidation (or reduction) occurs. The second working electrode, the collector, subsequently reduces (or oxidizes) the oxidized (or reduced) form back to its initial state. The target molecule transfers charges during every successive reaction, effectively amplifying the detected current per molecule. The collection efficiency of the redox cycling process depends on the reversibility of the reaction, therefore only the reversible oxidation of the hydroxylamine species and the reduction of the NO group.In our work, we have demonstrated five-fold redox amplification in cyclic voltammetry related to the reduction of p-nitrophenol, the biologic reaction’s product.Suspension-cultured Msk8 tomato cells with constitutive expression of GUS enzyme were derived from plant tissue and suspended in 0.1 M phosphate buffer (PB) of pH 5.8. The cells were stirred in a glass with 0.13M p-nitrophenyl beta-D glucuronide (PNPG) for 30 minutes. The measurement was conducted in a batch cell (Micrux, Spain) using a gold IDA chip with 5mm electrodes and 5mm spacings, and an Ag/AgCl thin film electrodeposited electrode. The collector electrode (WE2) was biased at -0.8 V and the generator electrode (WE1) was scanned from -1 V to +1 V in a cyclic voltammetry measurement.Cycling voltammetry of redox cyclingAs shown in the attached figure, in the negative values of the voltammetric measurement, the generator electrode exhibits a reduction slope, which relates to the electroactive product’s reductio...
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