Design and rapid prototyping of printed graphene electrochemical biosensors for sensitive monitoring of pesticide levels for agricultural use

While the use of pesticides (herbicides and insecticides) are critically important to meet the current and future food demands (increases crop yield by up to 40%), their overuse has shown long-term detrimental impacts on the environment from polluting watersheds used for drinking water to eutrophic “dead zones”. Current pesticide soil measurement methods (chromatography) are costly, require trained technicians, and take days to analyze; thus, farmers are taking an “over-application approach” which is pollution the environment and waterways. A disposable pesticide soil sensor would provide farmers the opportunity of precisely regulating the application of pesticides in an independent and economical fashion. Electrochemical biosensors provide the unique ability to quickly detect analytes with low-cost sensors; however, the detection limit and sensitivity of these biosensors are inadequate for current applications. This dissertation addresses this issue with the following focus in mind: 1) Increasing the enzymatic efficiency of organophosphate hydrolase by strategically functionalizing to nanomaterials [e.g., 17-fold increase in Vmax when functionalized to gold nanoparticles vs free enzyme]. 2) Develop a low-cost, rapid, and high-resolution manufacturing method to pattern solution-phase graphene [i.e., inkjet maskless lithography (IML), line resolution ~20 µm, sheet resistance ~ 0.7 kΩ/sq]. 3) Enhance the electroactive surface area by nano/microstructuring the graphene surface [3D petal-like graphene morphology] using laser annealing. 4) Increase the electrochemical surface area by incorporating macro and micro pores [2.2x with the inclusion of macropores] in the graphene surface. This work demonstrates the manufacturing of simple, low-cost electrochemical biosensors which suitable for rapid in-field detection of organophosphates. The fabricated graphene biosensors demonstrate high sensitivity, high linear sensing range, and ultra-low detection limits. Additionally, while this work is tailored towards a disposable pesticide sensor, the manufacturing techniques, sensor designs, and biosensor principle are a platform technology that could be amenable to other applications such as healthcare screening, drinking water monitoring, and even bioterror agent detection

Printed Graphene Electrochemical Biosensors Fabricated by Inkjet Maskless Lithography for Rapid and Sensitive Detection of Organophosphates

Solution phase printing of graphene-based electrodes has recently become an attractive low-cost, scalable manufacturing technique to create in-field electrochemical biosensors. Here, we report a graphene-based electrode developed via inkjet maskless lithography (IML) for the direct and rapid monitoring of triple-O linked phosphonate organophosphates (OPs); these constitute the active compounds found in chemical warfare agents and pesticides that exhibit acute toxicity as well as long-term pollution to soils and waterways. The IML-printed graphene electrode is nano/microstructured with a 1000 mW benchtop laser engraver and electrochemically deposited platinum nanoparticles (dia. ∼25 nm) to improve its electrical conductivity (sheet resistance decreased from ∼10 000 to 100 Ω/sq), surface area, and electroactive nature for subsequent enzyme functionalization and biosensing. The enzyme phosphotriesterase (PTE) was conjugated to the electrode surface via glutaraldehyde cross-linking. The resulting biosensor was able to rapidly measure (5 s response time) the insecticide paraoxon (a model OP) with a low detection limit (3 nM), and high sensitivity (370 nA/μM) with negligible interference from similar nerve agents. Moreover, the biosensor exhibited high reusability (average of 0.3% decrease in sensitivity per sensing event), stability (90% anodic current signal retention over 1000 s), longevity (70% retained sensitivity after 8 weeks), and the ability to selectively sense OP in actual soil and water samples. Hence, this work presents a scalable printed graphene manufacturing technique that can be used to create OP biosensors that are suitable for in-field applications as well as, more generally, for low-cost biosensor test strips that could be incorporated into wearable or disposable sensing paradigms

Enhanced enzymatic activity from phosphotriesterase trimer gold nanoparticle bioconjugates for pesticide detection

The rapid detection of organophosphates (OPs), a class of strong neurotoxins, is critically important for monitoring acute insecticide exposure and potential chemical warfare agent use. Herein, we improve the enzymatic activity of a phosphotriesterase trimer (PTE3), an enzyme that selectively recognizes OPs directly, by conjugation with distinctly sized (i.e., 5, 10, and 20 nm diameter) gold nanoparticles (AuNPs). The number of enzymes immobilized on the AuNP was controlled by conjugating increasing molar ratios of PTE3 onto the AuNP surface via metal affinity coordination. This occurs between the PTE3-His6 termini and the AuNP-displayed Ni2+-nitrilotriacetic acid end groups and was confirmed with gel electrophoresis. The enzymatic efficiency of the resultant PTE3–AuNP bioconjugates was analyzed via enzyme progress curves acquired from two distinct assay formats that compared free unbound PTE3 with the following PTE3–AuNP bioconjugates: (1) fixed concentration of AuNPs while increasing the bioconjugate molar ratio of PTE3 displayed around the AuNP and (2) fixed concentration of PTE3 while increasing the bioconjugate molar ratio of PTE3–AuNP by decreasing the AuNP concentration. Both assay formats monitored the absorbance of p-nitrophenol that was produced as PTE3 hydrolyzed the substrate paraoxon, a commercial insecticide and OP nerve agent simulant. Results demonstrate a general equivalent trend between the two formats. For all experiments, a maximum enzymatic velocity (Vmax) increased by 17-fold over free enzyme for the lowest PTE3–AuNP ratio and the largest AuNP (i.e., ratio of 1[thin space (1/6-em)]:[thin space (1/6-em)]1, 20 nm dia. AuNP). This work provides a route to improve enzymatic OP detection strategies with enzyme–NP bioconjugates

Fabrication of High-resolution Graphene-based Flexible Electronics via Polymer Casting

In this study, a novel method based on the transfer of graphene patterns from a rigid or flexible substrate onto a polymeric film surface via solvent casting was developed. The method involves the creation of predetermined graphene patterns on the substrate, casting a polymer solution, and directly transferring the graphene patterns from the substrate to the surface of the target polymer film via a peeling-off method. The feature sizes of the graphene patterns on the final film can vary from a few micrometers (as low as 5 µm) to few millimeters range. This process, applied at room temperature, eliminates the need for harsh post-processing techniques and enables creation of conductive graphene circuits (sheet resistance: ~0.2 kΩ/sq) with high stability (stable after 100 bending and 24 h washing cycles) on various polymeric flexible substrates. Moreover, this approach allows precise control of the substrate properties such as composition, biodegradability, 3D microstructure, pore size, porosity and mechanical properties using different film formation techniques. This approach can also be used to fabricate flexible biointerfaces to control stem cell behavior, such as differentiation and alignment. Overall, this promising approach provides a facile and low-cost method for the fabrication of flexible and stretchable electronic circuits

Enabling Inkjet Printed Graphene for Ion Selective Electrodes with Postprint Thermal Annealing

Inkjet printed graphene (IPG) has recently shown tremendous promise in reducing the cost and complexity of graphene circuit fabrication. Herein we demonstrate, for the first time, the fabrication of an ion selective electrode (ISE) with IPG. A thermal annealing process in a nitrogen ambient environment converts the IPG into a highly conductive electrode (sheet resistance changes from 52.8 ± 7.4 MΩ/□ for unannealed graphene to 172.7 ± 33.3 Ω/□ for graphene annealed at 950 °C). Raman spectroscopy and field emission scanning electron microscopy (FESEM) analysis reveals that the printed graphene flakes begin to smooth at an annealing temperature of 500 °C and then become more porous and more electrically conductive when annealed at temperatures of 650 °C and above. The resultant thermally annealed, IPG electrodes are converted into potassium ISEs via functionalization with a poly(vinyl chloride) (PVC) membrane and valinomycin ionophore. The developed potassium ISE displays a wide linear sensing range (0.01–100 mM), a low detection limit (7 μM), minimal drift (8.6 × 10–6 V/s), and a negligible interference during electrochemical potassium sensing against the backdrop of interfering ions [i.e., sodium (Na), magnesium (Mg), and calcium (Ca)] and artificial eccrine perspiration. Thus, the IPG ISE shows potential for potassium detection in a wide variety of human fluids including plasma, serum, and sweat

Electrical Differentiation of Mesenchymal Stem Cells into Schwann‐Cell‐Like Phenotypes Using Inkjet‐Printed Graphene Circuits

Graphene-based materials (GBMs) have displayed tremendous promise for use as neuro-interfacial substrates as they enable favorable adhesion, growth, proliferation, spreading and migration of immobilized cells. Herein we report the first case of the differentiation of Mesenchymal Stem Cells (MSCs) into Schwann Cell (SC) like phenotypes through the application of electrical stimuli from a graphene-based electrode. Electrical differentiation of MSCs into SC like phenotypes is carried out on a flexible, inkjet-printed graphene interdigitated electrode (IDE) circuit that is made highly conductive (sheet resistance \u3c 1 kΩ/☐) via a post-print pulse-laser annealing process. MSCs immobilized on the graphene printed IDEs and electrically stimulated/treated (etMSCs) displayed significant enhanced cellular differentiation and paracrine activity above conventional chemical treatment strategies [~85% of the etMSCs differentiated into SCs like phenotypes with ~80 ng/mL of nerve growth factor (NGF) secretion vs. 75% and ~55 ng/mL for chemically treated MSCs (ctMSCs)]. These results help pave the way for in vivo peripheral nerve regeneration where the flexible This article is protected by copyright. All rights reserved. 3 graphene electrodes could conform to the injury site and provide intimate electrical simulation for nerve cell regrowth

Fabrication of Two-Dimensional and Three-Dimensional High-Resolution Binder-Free Graphene Circuits Using a Microfluidic Approach for Sensor Applications

In this study, a simple microfluidic method, which can be universally applied to different rigid or flexible substrates, was developed to fabricate high-resolution, conductive, 2D and 3D microstructured graphene-based electronic circuits. The method involves controlled and selective filling of microchannels on substrate surfaces with a conductive binder-free graphene nanoplatelets (GNP) solution. The ethanol-thermal reaction of GNP solution at low temperatures (~75 °C) prior to microchannel filling (pre-heating) further reduces GNP, enhances conductivity, reduces sheet resistance (~0.05 kΩ sq-1), enables room temperature fabrication and eliminates harsh post-processing, which makes this fabrication technique compatible with degradable substrates. This method can also be used in combination with 3D printing to fabricate 3D circuits. The feature sizes of the graphene patterns can range from a few micrometers (down to ~15 µm in width and ~5 µm in depth) to a few millimeters and use very small amounts of GNP solution (~2.5 mg of graphene to obtain ~0.1 kΩ sq-1 of sheet resistance for 1 cm2). This microfluidic approach can also be implemented using other conductive liquids, such as conductive graphene-silver solutions. This technology has the potential to pave the way for low-cost, disposable and biodegradable circuits for a range of electronic applications including near field communication antennas, pressure or strain sensors

All-graphene-based open fluidics for pumpless, small-scale fluid transport via laser-controlled wettability patterning

Open microfluidics have emerged as a low-cost, pumpless alternative strategy to conventional microfluidics for delivery of fluid for a wide variety of applications including rapid biochemical analysis and medical diagnosis. However, creating open microfluidics by tuning the wettability of surfaces typically requires sophisticated cleanroom processes that are unamenable to scalable manufacturing. Herein, we present a simple approach to develop open microfluidic platforms by manipulating the surface wettability of spin-coated graphene ink films on flexible polyethylene terephthalate via laser-controlled patterning. Wedge-shaped hydrophilic tracks surrounded by superhydrophobic walls are created within the graphene films by scribing micron-sized grooves into the graphene with a CO2 laser. This scribing process is used to make superhydrophobic walls (water contact angle ∼160°) that delineate hydrophilic tracks (created through an oxygen plasma pretreatment) on the graphene for fluid transport. These all-graphene open microfluidic tracks are capable of transporting liquid droplets with a velocity of 20 mm s−1 on a level surface and uphill at elevation angles of 7° as well as transporting fluid in bifurcating cross and tree branches. The all-graphene open microfluidic manufacturing technique is rapid and amenable to scalable manufacturing, and consequently offers an alternative pumpless strategy to conventional microfluidics and creates possibilities for diverse applications in fluid transport

Stamped multilayer graphene laminates for disposable in-field electrodes: application to electrochemical sensing of hydrogen peroxide and glucose

A multi-step approach is described for the fabrication of multi-layer graphene-based electrodes without the need for ink binders or post-print annealing. Graphite and nanoplatelet graphene were chemically exfoliated using a modified Hummers’ method and the dried material was thermally expanded. Expanded materials were used in a 3D printed mold and stamp to create laminate electrodes on various substrates. The laminates were examined for potential sensing applications using model systems of peroxide (H2O2) and enzymatic glucose detection. Within the context of these two assay systems, platinum nanoparticle electrodeposition and oxygen plasma treatment were examined as methods for improving sensitivity. Electrodes made from both materials displayed excellent H2O2sensing capability compared to screen-printed carbon electrodes. Laminates made from expanded graphite and treated with platinum, detected H2O2 at a working potential of 0.3 V (vs. Ag/AgCl [0.1 M KCl]) with a 1.91 μM detection limit and sensitivity of 64 nA·μM−1·cm−2. Electrodes made from platinum treated nanoplatelet graphene had a H2O2 detection limit of 1.98 μM (at 0.3 V), and a sensitivity of 16.5 nA·μM−1·cm−2. Both types of laminate electrodes were also tested as glucose sensors via immobilization of the enzyme glucose oxidase. The expanded nanographene material exhibited a wide analytical range for glucose (3.7 μM to 9.9 mM) and a detection limit of 1.2 μM. The sensing range of laminates made from expanded graphite was slightly reduced (9.8 μM to 9.9 mM) and the detection limit for glucose was higher (18.5 μM). When tested on flexible substrates, the expanded graphite laminates demonstrated excellent adhesion and durability during testing. These properties make the electrodes adaptable to a variety of tests for field-based or wearable sensing applications