Sensing of gene expression in live cells using electrical impedance spectroscopy and DNA-functionalized gold nanoparticles.

A novel electrical impedance spectroscopy-based method for non-destructive sensing of gene expression in living cells is presented. The approach used takes advantage of the robustness and responsiveness of electrical impedance spectroscopy and the highly specific and selective nature of DNA hybridization. The technique uses electrical impedance spectroscopy and gold nanoparticles functionalized with single-stranded DNA complementary to an mRNA of interest to provide reliable, real-time, and quantifiable data on gene expression in live cells. The system was validated by demonstrating specific detection of the uidA mRNA, which codes for the ß-glucuronidase (GUS) enzyme, in Solanum lycopersicum MsK8 cells. Gold nanoparticles were functionalized with single-stranded DNA oligonucleotides consisting of either a sequence complementary to uidA mRNA or an arbitrary sequence. The DNA-functionalized gold nanoparticles were mixed with cell suspensions, allowing the gold nanoparticles to penetrate into the cells. The impedance spectra of suspensions of cells with gold nanoparticles inserted within them were then studied. In suspensions of uidA-expressing cells and gold nanoparticles functionalized with the complementary single-stranded DNA oligonucleotide, the impedance magnitude in the frequency range of interest was significantly higher (146 %) in comparison to all other controls. Due to its highly selective nature, the methodology has the potential to be used as a precision agricultural sensing system for accurate and real-time detection of markers of stress, viral infection, disease, and normal physiological activities.

In Vivo Plant Bio-Electrochemical Sensor Using Redox Cycling.

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 ß-glucuronidase (GUS) was observed within a few minutes after applying the substrate (pNPG, 4-Nitrophenyl ß-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.