Glyphosate inhibits melanization and increases susceptibility to infection in insects.

Melanin, a black-brown pigment found throughout all kingdoms of life, has diverse biological functions including UV protection, thermoregulation, oxidant scavenging, arthropod immunity, and microbial virulence. Given melanin's broad roles in the biosphere, particularly in insect immune defenses, it is important to understand how exposure to ubiquitous environmental contaminants affects melanization. Glyphosate-the most widely used herbicide globally-inhibits melanin production, which could have wide-ranging implications in the health of many organisms, including insects. Here, we demonstrate that glyphosate has deleterious effects on insect health in 2 evolutionary distant species, Galleria mellonella (Lepidoptera: Pyralidae) and Anopheles gambiae (Diptera: Culicidae), suggesting a broad effect in insects. Glyphosate reduced survival of G. mellonella caterpillars following infection with the fungus Cryptococcus neoformans and decreased the size of melanized nodules formed in hemolymph, which normally help eliminate infection. Glyphosate also increased the burden of the malaria-causing parasite Plasmodium falciparum in A. gambiae mosquitoes, altered uninfected mosquito survival, and perturbed the microbial composition of adult mosquito midguts. Our results show that glyphosate's mechanism of melanin inhibition involves antioxidant synergy and disruption of the reaction oxidation-reduction balance. Overall, these findings suggest that glyphosate's environmental accumulation could render insects more susceptible to microbial pathogens due to melanin inhibition, immune impairment, and perturbations in microbiota composition, potentially contributing to declines in insect populations.

On-chip dilution in nanoliter droplets.

Droplet microfluidics is enabling reactions at nano- and picoliter scale, resulting in faster and cheaper biological and chemical analyses. However, varying concentrations of samples on a drop-to-drop basis is still a challenging task in droplet microfluidics, primarily limited due to lack of control over individual droplets. In this paper, we report an on-chip microfluidic droplet dilution strategy using three-valve peristaltic pumps.

An open-source, battery-powered, low-cost, and dual-channel pneumatic pulse generator for microfluidic cell-stretch assays.

Cells in the body are regularly subjected to mechanical forces that influence their biological fate in terms of morphology, gene expression, and differentiation. The current gold standard method to replicate these effects in vitro is to culture cells on devices with elastic substrates and to impart mechanical stretch using mechanical or pneumatic pull-push methods. Microfluidic device designs offer several advantages in this context for general uniform and controlled stretching. However, the experimental setups are bulky, not user-friendly, and often involve several components that reside outside of the tissue culture incubator. Given the wide utility of mechanical stimulation in in-vitro research, our aim was to create a turn-key research tool that bioengineers can deploy in their cell-stretch assays, without having to deal with the complexity and nuances of ad hoc experimental setups. Here, we present an open-source, battery-powered, dual-channel cyclic pneumatic pulse generator box that can reside within an incubator and is compatible with custom microfluidic cell stretch devices. Our method depends on generating pressure-vacuum pulses simply using a linear miniature pneumatic air cylinder actuated using a continuous servo motor. To the best our knowledge, this is a first example of a completely battery-powered, standalone system that doesn't have any peripherals residing out of the incubator. We provide a detailed list of different components as well as the step-by-step assembly process. We validate its performance in a cell stretch assay using a commercially available microfluidic chip. Our results show an acute stimulation of cyclic stretching over 8 h on human umbilical vein endothelial cells (HUVECs) resulted in preferential alignment of cells perpendicular to the axis of stretch.

Low Cost, Ease-of-Access Fabrication of Microfluidic Devices Using Wet Paper Molds.

Rapid prototyping methods enable the widespread adoption of microfluidic technologies by empowering end-users from non-engineering disciplines to make devices using processes that are rapid, simple and inexpensive. In this work, we developed a liquid molding technique to create silicone/PDMS microfluidic devices by replica molding. To construct a liquid mold, we use inexpensive adhesive-backed paper, an acetate backing sheet, and an off-the-shelf digital cutter to create paper molds, which we then wet with predetermined amounts of water. Due to the immiscibility of water and PDMS, mold patterns can be effectively transferred onto PDMS similarly to solid molds. We demonstrate the feasibility of these wet paper molds for the fabrication of PDMS microfluidic devices and assess the influence of various process parameters on device yield and quality. This method possesses some distinct benefits compared to conventional techniques such as photolithography and 3D printing. First, we demonstrate that the shape of a channel's cross-section may be altered from rectangular to semicircular by merely modifying the wetting parameters. Second, we illustrate how electrical impedance can be utilized as a marker for inspecting mold quality and identifying defects in a non-invasive manner without using visual tools such as microscopes or cameras. As a proof-of-concept device, we created a microfluidic T-junction droplet generator to produce water droplets in mineral oil ranging in size from 1.2 µL to 75 µL. We feel that this technology is an excellent addition to the microfluidic rapid prototyping toolbox and will find several applications in biological research.

A Hydrogel-Based Microfluidic Nerve Cuff for Neuromodulation of Peripheral Nerves.

Implantable neuromodulation devices typically have metal in contact with soft, ion-conducting nerves. These neural interfaces excite neurons using short-duration electrical pulses. While this approach has been extremely successful for multiple clinical applications, it is limited in delivering long-duration pulses or direct current (DC), even for acute term studies. When the charge injection capacity of electrodes is exceeded, irreversible electrochemical processes occur, and toxic byproducts are discharged directly onto the nerve, causing biological damage. Hydrogel coatings on electrodes improve the overall charge injection limit and provide a mechanically pliable interface. To further extend this idea, we developed a silicone-based nerve cuff lead with a hydrogel microfluidic conduit. It serves as a thin, soft and flexible interconnection and provides a greater spatial separation between metal electrodes and the target nerve. In an in vivo rat model, we used this cuff to stimulate and record from sciatic nerves, with performance comparable to that of metal electrodes. Further, we delivered DC through the lead in an acute manner to induce nerve block that is reversible. In contrast to most metallic cuff electrodes, which need microfabrication equipment, we built this cuff using a consumer-grade digital cutter and a simplified molding process. Overall, the device will be beneficial to neuromodulation researchers as a general-purpose nerve cuff electrode for peripheral neuromodulation experiments.

Nerve cuff electrode pressure estimation via electrical impedance measurement.

OBJECTIVE: Implantable neuromodulation devices that have cuff electrodes are known to exert mechanical pressure on the target nerves. The amount of pressure exerted by cuff enclosures is one of the key determinants of physiological safety of these devices since excess pressures can cause neural damage. Because direct measurements of pressure on a nerve are challenging, the current cuff design approaches rely heavily on theoretical models or numerical computations for pressure predictions. An experimental approach to test these devices for pressure can complement existing theoretical models and can also serve as a quality control step to screen cuff electrode designs before implantation. APPROACH: We hypothesize that the pressure exerted on a nerve by a cuff can be estimated by measuring the resulting changes to the nerve's electrical impedance. MAIN RESULTS: We investigated ten 1 cm-long explanted rat sciatic nerves: five that were used within an hour after surgery, and five after 50 h of storage in physiological saline. For each experiment we applied variable pressure on the nerve ex vivo and measured the resulting changes in its impedance. We found a strong correlation between the external pressure on the nerve and its impedance and generated a pressure-impedance calibration curve. At the upper limit of physiologically safe pressure, the nerve impedance increased by ~2 kΩ, whereas, a rise of ~3 kΩ corresponded to pressure value that onsets irreversible nerve damage. SIGNIFICANCE: As a proof-of-concept, we used this protocol to generate a pressure-impedance calibration curve for a monkey tibial nerve and estimated pressure exerted by a commercial silicone cuff electrode on the explanted nerve. This single-point measurement was in an agreement with an independent estimate of the pressure measured using a mechanical pull test within 3 mmHg.

Normally closed plunger-membrane microvalve self-actuated electrically using a shape memory alloy wire.

Various microfluidic architectures designed for in vivo and point-of-care diagnostic applications require larger channels, autonomous actuation, and portability. In this paper, we present a normally closed microvalve design capable of fully autonomous actuation for wide diameter microchannels (tens to hundreds of µm). We fabricated the multilayer plunger-membrane valve architecture using the silicone elastomer, poly-dimethylsiloxane (PDMS) and optimized it to reduce the force required to open the valve. A 50-µm Nitinol (NiTi) shape memory alloy wire is incorporated into the device and can operate the valve when actuated with 100-mA current delivered from a 3-V supply. We characterized the valve for its actuation kinetics using an electrochemical assay and tested its reliability at 1.5-s cycle duration for 1 million cycles during which we observed no operational degradation.

Miniature Elastomeric Valve Design for Safe Direct Current Stimulator.

For safety reasons, commercial neural implants use charge-balanced biphasic pulses to interact with target neurons using metal electrodes. Short biphasic pulses are used to avoid irreversible electrochemical reactions at the electrode-tissue interfaces. Biphasic pulses are effective at exciting neurons, but quite limited in inhibiting their activity. In contrast, direct current can both excite and inhibit neurons, however delivered to metal electrodes, it causes toxic electrochemical reactions. We recently introduced Safe Direct Current Stimulator (SDCS) technology, which can excite or inhibit neurons without violating the safety criteria. Instead of direct current, SDCS generates an ionic direct current (iDC) from a biphasic input signal using a network of fluidic channels and mechanical valves. A key enabler towards transforming SDCS concept from a benchtop design to an implantable neural prosthesis is the design of a miniature valve. In this work, we present poly-dimethylsiloxane (PDMS) based elastomeric valves, squeeze valve (SV) and plunger valve (PV) capable of being actuated using a shape memory alloy wire.

Optoelectrical microfluidics as a promising tool in biology.

Noncontact robotic particle grippers with trapping, manipulation, and release functions are highly desired in cell biology and microfluidics. Optoelectric techniques combine optical and electrokinetic effects to create thousands of such individually addressable traps. By projecting reconfigurable light patterns, these techniques can concentrate molecules, as well as manipulate, sort, and electroporate cells in a programmable manner. We describe the underlying physical mechanisms and discuss applications in biology and future prospects of these devices.

Software-programmable continuous-flow multi-purpose lab-on-a-chip.

Current lab-on-a-chip (LoC) devices are assay-specific and are custom-built for each single experiment. Performing an experiment requires scientists or engineers to go through the time-consuming process of designing, fabricating, and testing a chip before conducting the actual experiment. This prolonged cycle can take months to complete, increasing effort and cost and reducing productivity. Similarly, minor modifications to an assay protocol re-incur the overheads of the design cycle. In this paper, we develop a multi-purpose, software-programmableLab-on-a-Chip (SPLoC), where the user simply writes or downloads a program for each experiment. We describe the components necessary to realize the SPLoC, which include a high-level programming language, an abstract instruction set, a runtime and control system, and a microfluidic device. We describe two key features of our high-level language compiler, and describe a novel variable-volume variable-ratio mixer. Finally, we demonstrate our SPLoC on four diverse, real-world assays.