An Ultrasensitive Electrochemical Plant Sensor Based on Redox Cycling Amplification Using Paranitrophenol

Abstract

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 reduction. From zero potential and in the positive range, the generator’s current rises to a plateu, due to 4-hydroxyl-amino-phenol oxidation. The collector electrode shows a reduction behavior which is a mirror image of the generator’s oxidation. From this behavior we conclude that at small overpotentials, the generator reaction is limited by its kinetics. At larger overpotentials, the process is mass transport limited, though, unlike generic three electrodes systems, does not characterize in a peak. The mass transport is almost constant since the majority of the oxidized molecules are reduced at the collector and return to the generator at a constant rate, dependent on the diffusion characteristics of the IDA electrodes. The redox amplification factor is 5, calculated as the current at the generator divided by the current at standard three electrodes measurements.Dependency on switching potentialWe have shown that by enabling the initial reduction of p-nitrophenol and scanning the potentials down to a switching potential more negative than its reduction potential, the reversible redox cycling reaction was carried out. When performing a similar measurement with a switching potential less negative than -0.75 V, the redox cycling signal was not observed.pH dependencyThe solution’s pH was adjusted from 4 to 6. It was shown that with increasing the pH, the potential in which the plateau was reached has become less positive, and the plateau current has decreased. These two results indicate that the process is proton dependent in this pH range.Summary and conclusionsFollowing the successful feasibility study and encouraging initial results, the redox cycling phenomena in direct contact to the plant’s leaves and stems will be tested in order to develop an early detection, fully integrated redox cycling chip-on-plant soft and flexible sensor based on our supersonic cluster beam deposition and femtosecond laser micro-processing previously reported novel process. Figure 1