Plant Bioelectronics and Biohybrids: The Growing Contribution of Organic Electronic and Carbon-Based Materials.

Life in our planet is highly dependent on plants as they are the primary source of food, regulators of the atmosphere, and providers of a variety of materials. In this work, we review the progress on bioelectronic devices for plants and biohybrid systems based on plants, therefore discussing advancements that view plants either from a biological or a technological perspective, respectively. We give an overview on wearable and implantable bioelectronic devices for monitoring and modulating plant physiology that can be used as tools in basic plant science or find application in agriculture. Furthermore, we discuss plant-wearable devices for monitoring a plant's microenvironment that will enable optimization of growth conditions. The review then covers plant biohybrid systems where plants are an integral part of devices or are converted to devices upon functionalization with smart materials, including self-organized electronics, plant nanobionics, and energy applications. The review focuses on advancements based on organic electronic and carbon-based materials and discusses opportunities, challenges, as well as future steps.

Implantable Organic Electronic Ion Pump Enables ABA Hormone Delivery for Control of Stomata in an Intact Tobacco Plant.

Electronic control of biological processes with bioelectronic devices holds promise for sophisticated regulation of physiology, for gaining fundamental understanding of biological systems, providing new therapeutic solutions, and digitally mediating adaptations of organisms to external factors. The organic electronic ion pump (OEIP) provides a unique means for electronically-controlled, flow-free delivery of ions, and biomolecules at cellular scale. Here, a miniaturized OEIP device based on glass capillary fibers (c-OEIP) is implanted in a biological organism. The capillary form factor at the sub-100 µm scale of the device enables it to be implanted in soft tissue, while its hyperbranched polyelectrolyte channel and addressing protocol allows efficient delivery of a large aromatic molecule. In the first example of an implantable bioelectronic device in plants, the c-OEIP readily penetrates the leaf of an intact tobacco plant with no significant wound response (evaluated up to 24 h) and effectively delivers the hormone abscisic acid (ABA) into the leaf apoplast. OEIP-mediated delivery of ABA, the phytohormone that regulates plant's tolerance to stress, induces closure of stomata, the microscopic pores in leaf's epidermis that play a vital role in photosynthesis and transpiration. Efficient and localized ABA delivery reveals previously unreported kinetics of ABA-induced signal propagation.

Single nucleotide polymorphism detection using gold nanoprobes and bio-microfluidic platform with embedded microlenses.

The use of microfluidics platforms combined with the optimal optical properties of gold nanoparticles has found plenty of application in molecular biosensing. This paper describes a bio-microfluidic platform coupled to a non-cross-linking colorimetric gold nanoprobe assay to detect a single nucleotide polymorphism associated with increased risk of obesity fat-mass and obesity-associated (FTO) rs9939609 (Carlos et al., 2014). The system enabled significant discrimination between positive and negative assays using a target DNA concentration of 5 ng/µL below the limit of detection of the conventionally used microplate reader (i.e., 15 ng/µL) with 10 times lower solution volume (i.e., 3 µL). A set of optimization of our previously reported bio-microfluidic platform (Bernacka-Wojcik et al., 2013) resulted in a 160% improvement of colorimetric analysis results. Incorporation of planar microlenses increased 6 times signal-to-loss ratio reaching the output optical fiber improving by 34% the colorimetric analysis of gold nanoparticles, while the implementation of an optoelectronic acquisition system yielded increased accuracy and reduced noise. The microfluidic chip was also integrated with a miniature fiber spectrometer to analyze the assays' colorimetric changes and also the LEDs transmission spectra when illuminating through various solutions. Furthermore, by coupling an optical microscope to a digital camera with a long exposure time (30 s), we could visualise the different scatter intensities of gold nanoparticles within channels following salt addition. These intensities correlate well to the expected difference in aggregation between FTO positive (none to small aggregates) and negative samples (large aggregates).

Continuous iontronic chemotherapy reduces brain tumor growth in embryonic avian in vivo models.

Local and long-lasting administration of potent chemotherapeutics is a promising therapeutic intervention to increase the efficiency of chemotherapy of hard-to-treat tumors such as the most lethal brain tumors, glioblastomas (GBM). However, despite high toxicity for GBM cells, potent chemotherapeutics such as gemcitabine (Gem) cannot be widely implemented as they do not efficiently cross the blood brain barrier (BBB). As an alternative method for continuous administration of Gem, we here operate freestanding iontronic pumps - "GemIPs" - equipped with a custom-synthesized ion exchange membrane (IEM) to treat a GBM tumor in an avian embryonic in vivo system. We compare GemIP treatment effects with a topical metronomic treatment and observe that a remarkable growth inhibition was only achieved with steady dosing via GemIPs. Daily topical drug administration (at the maximum dosage that was not lethal for the embryonic host organism) did not decrease tumor sizes, while both treatment regimes caused S-phase cell cycle arrest and apoptosis. We hypothesize that the pharmacodynamic effects generate different intratumoral drug concentration profiles for each technique, which causes this difference in outcome. We created a digital model of the experiment, which proposes a fast decay in the local drug concentration for the topical daily treatment, but a long-lasting high local concentration of Gem close to the tumor area with GemIPs. Continuous chemotherapy with iontronic devices opens new possibilities in cancer treatment: the long-lasting and highly local dosing of clinically available, potent chemotherapeutics to greatly enhance treatment efficiency without systemic side-effects. SIGNIFICANCE STATEMENT: Iontronic pumps (GemIPs) provide continuous and localized administration of the chemotherapeutic gemcitabine (Gem) for treating glioblastoma in vivo. By generating high and constant drug concentrations near the vascularized growing tumor, GemIPs offer an efficient and less harmful alternative to systemic administration. Continuous GemIP dosing resulted in remarkable growth inhibition, superior to daily topical Gem application at higher doses. Our digital modelling shows the advantages of iontronic chemotherapy in overcoming limitations of burst release and transient concentration profiles, and providing precise control over dosing profiles and local distribution. This technology holds promise for future implants, could revolutionize treatment strategies, and offers a new platform for studying the influence of timing and dosing dependencies of already-established drugs in the fight against hard-to-treat tumors.

Biohybrid Energy Storage Circuits Based on Electronically Functionalized Plant Roots.

Biohybrid systems based on plants integrate plant structures and processes into technological components targeting more sustainable solutions. Plants' biocatalytic machinery, for example, has been leveraged for the organization of electronic materials directly in the vasculature and roots of living plants, resulting in biohybrid electrochemical devices. Among other applications, energy storage devices were demonstrated where the charge storage electrodes were seamlessly integrated into the plant tissue. However, the capacitance and the voltage output of a single biohybrid supercapacitor are limited. Here, we developed biohybrid circuits based on functionalized conducting roots, extending the performance of plant based biohybrid energy storage systems. We show that root-supercapacitors can be combined in series and in parallel configuration, achieving up to 1.5 V voltage output or up to 11 mF capacitance, respectively. We further demonstrate that the supercapacitors circuit can be charged with an organic photovoltaic cell, and that the stored charge can be used to power an electrochromic display or a bioelectronic device. Furthermore, the functionalized roots degrade in composting similarly to native roots. The proof-of-concept demonstrations illustrate the potential of this technology to achieve more sustainable solutions for powering low consumption devices such as bioelectronics for agriculture or IoT applications.

Long-distance turgor pressure changes induce local activation of plant glutamate receptor-like channels.

In Arabidopsis thaliana, local wounding and herbivore feeding provoke leaf-to-leaf propagating Ca2+ waves that are dependent on the activity of members of the glutamate receptor-like channels (GLRs). In systemic tissues, GLRs are needed to sustain the synthesis of jasmonic acid (JA) with the subsequent activation of JA-dependent signaling response required for the plant acclimation to the perceived stress. Even though the role of GLRs is well established, the mechanism through which they are activated remains unclear. Here, we report that in vivo, the amino-acid-dependent activation of the AtGLR3.3 channel and systemic responses require a functional ligand-binding domain. By combining imaging and genetics, we show that leaf mechanical injury, such as wounds and burns, as well as hypo-osmotic stress in root cells, induces the systemic apoplastic increase of L-glutamate (L-Glu), which is largely independent of AtGLR3.3 that is instead required for systemic cytosolic Ca2+ elevation. Moreover, by using a bioelectronic approach, we show that the local release of minute concentrations of L-Glu in the leaf lamina fails to induce any long-distance Ca2+ waves.

Flexible Organic Electronic Ion Pump for Flow-Free Phytohormone Delivery into Vasculature of Intact Plants.

Plant vasculature transports molecules that play a crucial role in plant signaling including systemic responses and acclimation to diverse environmental conditions. Targeted controlled delivery of molecules to the vascular tissue can be a biomimetic way to induce long distance responses, providing a new tool for the fundamental studies and engineering of stress-tolerant plants. Here, a flexible organic electronic ion pump, an electrophoretic delivery device, for controlled delivery of phytohormones directly in plant vascular tissue is developed. The c-OEIP is based on polyimide-coated glass capillaries that significantly enhance the mechanical robustness of these microscale devices while being minimally disruptive for the plant. The polyelectrolyte channel is based on low-cost and commercially available precursors that can be photocured with blue light, establishing much cheaper and safer system than the state-of-the-art. To trigger OEIP-induced plant response, the phytohormone abscisic acid (ABA) in the petiole of intact Arabidopsis plants is delivered. ABA is one of the main phytohormones involved in plant stress responses and induces stomata closure under drought conditions to reduce water loss and prevent wilting. The OEIP-mediated ABA delivery triggered fast and long-lasting stomata closure far away from the delivery point demonstrating systemic vascular transport of the delivered ABA, verified delivering deuterium-labeled ABA.

Multifunctional microfluidic chip for optical nanoprobe based RNA detection - application to Chronic Myeloid Leukemia.

Many diseases have their treatment options narrowed and end up being fatal if detected during later stages. As a consequence, point-of-care devices have an increasing importance for routine screening applications in the health sector due to their portability, fast analyses and decreased cost. For that purpose, a multifunctional chip was developed and tested using gold nanoprobes to perform RNA optical detection inside a microfluidic chip without the need of molecular amplification steps. As a proof-of-concept, this device was used for the rapid detection of chronic myeloid leukemia, a hemato-oncological disease that would benefit from early stage diagnostics and screening tests. The chip passively mixed target RNA from samples, gold nanoprobes and saline solution to infer a result from their final colorimetric properties. An optical fiber network was used to evaluate its transmitted spectra inside the chip. Trials provided accurate output results within 3 min, yielding signal-to-noise ratios up to 9 dB. When compared to actual state-of-art screening techniques of chronic myeloid leukemia, these results were, at microscale, at least 10 times faster than the reported detection methods for chronic myeloid leukemia. Concerning point-of-care applications, this work paves the way for other new and more complex versions of optical based genosensors.

Hybrid Microfluidic Platform for Multifactorial Analysis Based on Electrical Impedance, Refractometry, Optical Absorption and Fluorescence.

This paper describes the development of a novel microfluidic platform for multifactorial analysis integrating four label-free detection methods: electrical impedance, refractometry, optical absorption and fluorescence. We present the rationale for the design and the details of the microfabrication of this multifactorial hybrid microfluidic chip. The structure of the platform consists of a three-dimensionally patterned polydimethylsiloxane top part attached to a bottom SU-8 epoxy-based negative photoresist part, where microelectrodes and optical fibers are incorporated to enable impedance and optical analysis. As a proof of concept, the chip functions have been tested and explored, enabling a diversity of applications: (i) impedance-based identification of the size of micro beads, as well as counting and distinguishing of erythrocytes by their volume or membrane properties; (ii) simultaneous determination of the refractive index and optical absorption properties of solutions; and (iii) fluorescence-based bead counting.

Bio-microfluidic platform for gold nanoprobe based DNA detection--application to Mycobacterium tuberculosis.

We have projected and fabricated a microfluidic platform for DNA sensing that makes use of an optical colorimetric detection method based on gold nanoparticles. The platform was fabricated using replica moulding technology in PDMS patterned by high-aspect-ratio SU-8 moulds. Biochips of various geometries were tested and evaluated in order to find out the most efficient architecture, and the rational for design, microfabrication and detection performance is presented. The best biochip configuration has been successfully applied to the DNA detection of Mycobacterium tuberculosis using only 3 µl on DNA solution (i.e. 90 ng of target DNA), therefore a 20-fold reduction of reagents volume is obtained when compared with the actual state of the art.

Inkjet printed and "doctor blade" TiO2 photodetectors for DNA biosensors.

A dye sensitized TiO(2) photodetector has been integrated with a DNA detection method based on non-cross-linking hybridization of DNA-functionalized gold nanoparticles, resulting in a disposable colorimetric biosensor. We present a new approach for the fabrication of dye sensitized TiO(2) photodetectors by an inkjet printing technique-a non-contact digital, additive, no mask and no vacuum patterning method, ideal for cost efficient mass production. The developed biosensor was compared against a dye sensitized photodetector fabricated by the traditional "doctor blade" method. Detection of gold nanoparticle aggregation was possible for concentrations as low as 1.0 nM for the "doctor blade" system, and 1.5 nM for the inkjet printed photodetector. The demonstrated sensitivity limits of developed biosensors are comparable to those of spectrophotometric techniques (1.0 nM). Our results show that a difference higher than 17% by traditional photodetector and 6% by inkjet printed in the photoresponses for the complementary and non-complementary gold nanoprobe assays could be attained for a specific DNA sequence from Mycobacterium tuberculosis, the etiologic agent of human tuberculosis. The decrease of costs associated with molecular diagnostic provided by a platform such as the one presented here may prove of paramount importance in developing countries.