Automated dual olfactory device for studying head/tail chemosensation in Caenorhabditis elegans.

The correct interpretation of threat and reward is important for animal survival. Often, the decisions underlying these behavioral programs are mediated by volatile compounds in the animal's environment, which they detect and discriminate with specialized olfactory neurons along their body. Caenorhabditis (C.) elegans senses chemical stimuli with neurons located in the head and the tail of the animal, which mediate either attractive or aversive behaviors. How conflicting stimuli are processed in animals navigating different chemical gradients is poorly understood. Here, we conceived, created, and capitalized on a novel microfluidic device to enable automated and precise stimulation of head and tail neurons, either simultaneously or sequentially, while reading out neuronal activity in sensory and interneurons using genetically encoded calcium indicators. We achieve robust and programmable chemical pulses through the modulation of inlet pressures. To evaluate the device performance, we synchronized the flow control with microscopy data acquisition and characterized the flow properties in the fabricated devices. Together, our design has the potential to provide insight into the neural circuits and behavior of C. elegans simulating the experience of natural environments.

Integration of spatially opposing cues by a single interneuron guides decision-making in C. elegans.

The capacity of animals to respond to hazardous stimuli in their surroundings is crucial for their survival. In mammals, complex evaluations of the environment require large numbers and different subtypes of neurons. The nematode C. elegans avoids hazardous chemicals they encounter by reversing their direction of movement. How does the worms' compact nervous system process the spatial information and direct motion change? We show here that a single interneuron, AVA, receives glutamatergic excitatory and inhibitory signals from head and tail sensory neurons, respectively. AVA integrates the spatially distinct and opposing cues, whose output instructs the animal's behavioral decision. We further find that the differential activation of AVA stems from distinct localization of inhibitory and excitatory glutamate-gated receptors along AVA's process and from different threshold sensitivities of the sensory neurons. Our results thus uncover a cellular mechanism that mediates spatial computation of nociceptive cues for efficient decision-making in C. elegans.

Neural engineering with photons as synaptic transmitters.

Neuronal computation is achieved through connections of individual neurons into a larger network. To expand the repertoire of endogenous cellular communication, we developed a synthetic, photon-assisted synaptic transmission (PhAST) system. PhAST is based on luciferases and channelrhodopsins that enable the transmission of a neuronal state across space, using photons as neurotransmitters. PhAST overcomes synaptic barriers and rescues the behavioral deficit of a glutamate mutant with conditional, calcium-triggered photon emission between two neurons of the Caenorhabditis elegans nociceptive avoidance circuit. To demonstrate versatility and flexibility, we generated de novo synaptic transmission between two unconnected cells in a sexually dimorphic neuronal circuit, suppressed endogenous nocifensive response through activation of an anion channelrhodopsin and switched attractive to aversive behavior in an olfactory circuit. Finally, we applied PhAST to dissect the calcium dynamics of the temporal pattern generator in a motor circuit for ovipositioning. In summary, we established photon-based synaptic transmission that facilitates the modification of animal behavior.

Sexually dimorphic architecture and function of a mechanosensory circuit in C. elegans.

How sensory perception is processed by the two sexes of an organism is still only partially understood. Despite some evidence for sexual dimorphism in auditory and olfactory perception, whether touch is sensed in a dimorphic manner has not been addressed. Here we find that the neuronal circuit for tail mechanosensation in C. elegans is wired differently in the two sexes and employs a different combination of sex-shared sensory neurons and interneurons in each sex. Reverse genetic screens uncovered cell- and sex-specific functions of the alpha-tubulin mec-12 and the sodium channel tmc-1 in sensory neurons, and of the glutamate receptors nmr-1 and glr-1 in interneurons, revealing the underlying molecular mechanisms that mediate tail mechanosensation. Moreover, we show that only in males, the sex-shared interneuron AVG is strongly activated by tail mechanical stimulation, and accordingly is crucial for their behavioral response. Importantly, sex reversal experiments demonstrate that the sexual identity of AVG determines both the behavioral output of the mechanosensory response and the molecular pathways controlling it. Our results present extensive sexual dimorphism in a mechanosensory circuit at both the cellular and molecular levels.

Flow Control in Porous Media: From Numerical Analysis to Quantitative µPAD for Ionic Strength Measurements.

Microfluidic paper-based analytical devices (µPADs) are a promising technology to enable accurate and quantitative in situ assays. Paper's inherent hydrophilicity drives the fluids without the need for external pressure sources. However, controlling the flow in the porous medium has remained a challenge. This study addresses this problem from the nature of the paper substrate and its design. A computational fluid dynamic model has been developed, which couples the characteristics of the porous media (fiber length, fiber diameter and porosity) to the fluidic performance of the diffusion-based µPAD sensor. The numerical results showed that for a given porous membrane, the diffusion, and therefore the sensor performance is affected not only by the substrate nature but also by the inlets' orientation. Given a porous substrate, the optimum performance is achieved by the lowest inlets' angle. A diffusion-based self-referencing colorimetric sensor was built and validated according to the design. The device is able to quantify the hydronium concentration in wines by comparison to 0.1-1.0 M tartaric acid solutions with a 41.3 mM limit of detection. This research showed that by proper adjustments even the simplest µPADs can be used in quantitative assays for agri-food applications.

Numerical and experimental analysis of a high-throughput blood plasma separator for point-of-care applications.

Blood plasma separation from undiluted blood is an essential step in many diagnostic procedures. This study focuses on the numerical optimization of the microfluidic blood plasma separator (BPS) and experimental validation of the results to achieve portable blood plasma separation with high purity and reasonable yield. The proposed design has two parts: a microchannel for blood processing and a tank below the aforementioned main channel for plasma collection. The study uses 3D computational fluid dynamic analysis to investigate the optimal ratio of heights between the top microchannel and the tank and their geometry at various flow rates. Thereafter, the results are compared with the experimental findings of the fabricated devices. These results are contrasted with some recent reported works to verify the proposed device's contribution to the improvement in the quality and quantity of the extracted plasma. The optimized design is capable of achieving a 19% yield with purity of 77.1%, depending on the requirement of the point-of-care (POC) application. These amounts could be tuned, for instance to 100% pure plasma, but the yield would decrease to 9%. In this study, the candidate application is hemostasis; therefore, the BPS is integrated to a biomimetic surface for hemostasis evaluation near the patients.

Portable 3D-printed sensor to measure ionic strength and pH in buffered and non-buffered solutions.

A miniaturized 3D-printed device has been designed, manufactured and validated to perform as a low-cost sensor for compositional analysis of buffered and non-buffered solutions in industrial or remote areas. The proposed sensor takes advantage of the transport phenomenon and colorimetric measurements. The novel design can simultaneously detect the ionic strength of the solution by measuring the diffusion width of the ions and the pH by image analysis of the pH indicator color change. The results showed that it can detect pH variations of 0.25 and ionic measure difference of 0.1 M in non-buffer solutions. In addition, the design showed its adaptability to be used as a self-referencing sensor. The 3D-printed sensor presented here is not only successful in the evaluation of some important chemical characteristics but also brings flexibility, cost-effectiveness, swiftness and user-friendliness.

Cost-effective microfabrication of sub-micron-depth channels by femto-laser anti-stiction texturing.

Micro Electro Mechanical Systems (MEMS) and microfluidic devices have found numerous applications in the industrial sector. However, they require a fast, cost-effective and reliable manufacturing process in order to compete with conventional methods. Particularly, at the sub-micron scale, the manufacturing of devices are limited by the dimensional complexity. A proper bonding and stiction prevention of these sub-micron channels are two of the main challenges faced during the fabrication process of low aspect ratio channels. Especially, in the case of using flexible materials such as polydimethylsiloxane (PDMS). This study presents a direct laser microfabrication method of sub-micron channels using an infrared (IR) ultrashort pulse (femtosecond), capable of manufacturing extremely low aspect ratio channels. These microchannels are manufactured and tested varying their depth from 0.5 µm to 2 µm and width of 15, 20, 25, and 30 µm. The roughness of each pattern was measured by an interferometric microscope. Additionally, the static contact angle of each depth was studied to evaluate the influence of femtosecond laser fabrication method on the wettability of the glass substrate. PDMS, which is a biocompatible polymer, was used to provide a watertight property to the sub-micron channels and also to assist the assembly of external microfluidic hose connections. A 750 nm depth watertight channel was built using this methodology and successfully used as a blood plasma separator (BPS). The device was able to achieve 100% pure plasma without stiction of the PDMS layer to the sub-micron channel within an adequate time. This method provides a novel manufacturing approach useful for various applications such as point-of-care devices.

A passive portable microfluidic blood-plasma separator for simultaneous determination of direct and indirect ABO/Rh blood typing.

The blood typing test is mandatory in any transfusion, organ transplant, and pregnancy situation. There is a lack of point-of-care (POC) blood typing that could perform both direct and indirect methods using a single droplet of whole blood. This study presents a new methodology combining a passive microfluidic blood-plasma separator (BPS) and a blood typing detector for the very first time, leading to a stand-alone microchip which is capable of determining the blood group from both direct and indirect methods simultaneously. The proposed design separates blood cells from plasma by applying hydrodynamic forces imposed on them, which overcomes the clogging issue and consequently maximizes the volume of the extracted plasma. An axial migration effect across the main channel is responsible for collecting the plasma in plasma collector channels. The BPS novel design approached 12% yield of plasma with 100% purity in approximately 10 minutes. The portable BPS was designed and fabricated to perform ABO/Rh blood tests based on the detection of agglutination in both antigens of RBCs (direct) and antibodies of plasma (indirect). The differences between agglutinated and non-agglutinated samples were distinguishable by the naked eye and also validated by particle analysis of microscopic pictures. The results of this passive BPS in ABO/Rh blood grouping verified the quality and quantity of the extracted plasma in practical applications.

Hemostasis-On-a-Chip: Impedance Spectroscopy Meets Microfluidics for Hemostasis Evaluation.

In the case of vascular injury, a complex process (of clotting) starts, involving mainly platelets and coagulation factors. This process in healthy humans is known as hemostasis, but when it is deregulated (thrombosis), it can be the cause of important cardiovascular diseases. Nowadays, the aging of the population and unhealthy lifestyles increase the impact of thrombosis, and therefore there is a need for tools to provide a better understanding of the hemostasis mechanisms, as well as more cost-effective diagnosis and control devices. This study proposes a novel microflow chamber, with interchangeable biomimetic surfaces to evaluate global hemostasis, using reduced amounts of blood sample and reagents, and also a minimized time required to do the test. To validate the performance of this novel device, a study on the new oral anticoagulant Apixaban (APIX) has been performed and compared to previous conventional techniques. The test shows an excellent agreement, while the amount of the required sample has been reduced (only 100 µL is used), and the amount of reagent as well. An imprinted electrode embedded in the chamber in order to measure the impedance during the coagulation process. This approach distinguishes the impedance behavior of plasma poor in platelets (PPP) and plasma rich in platelets (PRP) for the first time.

Novel Variable Radius Spiral⁻Shaped Micromixer: From Numerical Analysis to Experimental Validation.

A novel type of spiral micromixer with expansion and contraction parts is presented in order to enhance the mixing quality in the low Reynolds number regimes for point-of-care tests (POCT). Three classes of micromixers with different numbers of loops and modified geometries were studied. Numerical simulation was performed to study the flow behavior and mixing performance solving the steady-state Navier⁻Stokes and the convection-diffusion equations in the Reynolds range of 0.1⁻10.0. Comparisons between the mixers with and without expansion parts were made to illustrate the effect of disturbing the streamlines on the mixing performance. Image analysis of the mixing results from fabricated micromixers was used to verify the results of the simulations. Since the proposed mixer provides up to 92% of homogeneity at Re 1.0, generating 442 Pa of pressure drop, this mixer makes a suitable candidate for research in the POCT field.