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Cells in their native microenvironment are subjected to varying combinations of biochemical cues and mechanical cues in a wide range. Although many signaling pathways have been found to be responsive for extracellular cues, little is known about how biochemical cues crosstalk with mechanical cues in a complex microenvironment. Here, we introduced heterogeneous droplets on a microchip, which were rapidly assembled by combining wettability-patterned microchip and programmed droplet manipulations, for a high-throughput cell screening of the varying combinations of biochemical cues and mechanical cues. This platform constructed a heterogeneous droplet/microgel array with orthogonal gradual chemicals and materials, which was further applied to analyze the cellular Wnt/β-catenin signaling in response to varying combinations of Wnt ligands and substrate stiffness. Thus, this device provides a powerful multiplexed bioassay platform for drug development, tissue engineering, and stem cell screening.

Optogenetic method is widely used for dissecting the neuronal function and connectivity in a specific neural circuit, which can help understanding how the animal process information and generate behavior. The nematode C. elegans has a simple but complete nervous system, making it an attractive model to study the dynamics signals of neural circuits. However, in vivo analysis on neural circuits usually rely on the complex and expensive optical equipment to allow optogenetic stimulating the neuron while recording its activities in such a freely moving animal. Hence, in this paper we reported a portable optofluidic platform that works based on optical fiber illumination and functional imaging for worm optogenetic manipulation. A light beam from LED laser pen crossing the 3D-printed optical fiber channel is used to activate the neurons specific-expressed with light sensitive proteins ChR-2. The imaging light path is perpendicular to the stimulation light, which allows activating neuron precisely and measuring cellular signals simultaneously. By using such an easy-to-assemble device, optical stimulation of the specific neurons and detection of dynamic calcium responses of other neurons could be proceeded simultaneously. Thus, the developed microfluidic platform puts forward a simple, rapid and low-cost strategy for further neural circuits studies.

Immune response to environmental pathogen invasion is a complex process to prevent host from further damage. For quantitatively understanding immune responses and revealing the pathogenic environmental information, real-time monitoring of such a whole dynamic process with single-animal resolution in well-defined environments is highly desired. In this work, an integrated microfluidic device coupled with worm-based biosensor was proposed for in vivo studies of dynamic immune responses and antibiotics interference in infected C. elegans. Individual worms housed in chambers were exposed to the various pathogens and discontinuously manipulated for imaging with limited influence on physiological activities. The expression of immune responses gene (irg-1) was time-lapse measured in intact worms during pathogen infection. Results demonstrated that irg-1 gene could be induced in the presence of P. aeruginosa strain PA14 in a dose-dependent manner, and the survival of infected worm could be rescued under gentamicin or erythromycin treatments. We expect it to be a versatile platform to facilitate future studies on pathogenesis researches and rapid drug screen using C. elegans disease model.

C. elegans is a leading model organism for addressing many fundamental biology issues. Sufficient quantities of the worms with specific developmental stage are needed in C. elegans studies due to the size-dependent features shown in C. elegans. In this work, we presented a simple and feasible microfluidic chip to realize simultaneous sorting of C. elegans with different sizes by graduated microfilters. The micro-device was fabricated with PDMS-agarose hybrid layers consisting of 5 arrays of geometrically optimized square columns as microfilters for separating worms of different sizes. Fluid flow and electric field were simultaneously applied to drive the worms to rapidly and directionally pass through the microfilters, resulting in increased sorting efficiency and accuracy. Further, the sorted worms could be easily collected from 4 grooves of the agarose layer by slipping the PDMS layer to the top of grooves. With this sorting strategy, we demonstrated the feasibility of simultaneously sorting different sizes of C. elegans with high efficiency and accuracy (>95%) and throughput of hundreds of worms per minute. Therefore, the micro-device could be a promising tool to C. elegans assays that need a large number of stage-synchronized worms. It could also be useful for body size-based mutant screening and taxis-response studies in C. elegans.

C. elegans is a commonly used model organism for studying the basic mechanisms of lifespan extension by dietary restriction (DR). Progress has been limited, however, by the lack of an automated system for quantitative analysis of this effect of bacterial diet. In this work, multifunctional logarithmic gradient-customizing microfluidic device, which takes advantage of hydrodynamics, was developed to long-term maintain worm culture on the chip with parallel live imaging and generate logarithmic concentration gradient of bacteria. We successfully employed this platform to investigate the longevity and stress response of the worms in response to various concentrations of bacterial food. The results indicated that the lifespan extension of the worms was induced by DR when bacteria density was 10 8 cells mL −1 and further dilutions would reduce the lifespan, which proposed a new dietary restriction regime in C. elegans . In addition, imaging analysis showed that DR regulated the subcellular localization DAF-16 in a way different from starvation that reduced lifespan of the worms. The platform allows not only horizontal studies of C. elegans in response to various environments over a wide range of concentrations, but also multiparameter evaluation of longevity in the worms with high temporal resolution, providing clues to better understand the aging process and help age-related drug screen.

C. elegans was widely used as a prominent model organism in the study of physiological response to gaseous environment, thanks to its suitability for in vivo optical imaging of neuronal sensing. Immobilization of the active worms is a key procedure to obtain high-resolution imaging and gas stimulations. In this work, we presented a novel microfluidic approach for immobilizing C. elegans due to the fact that its movement could be eliminated under the condition of gentle surface-dehydration. Nitrogen gas was directly injected into the microfluidic chip to dehydrate the surface of worm appropriately, resulting in rapid and repetitive immobilization of C. elegans. The imaging analysis of subcellular distribution of DAF-16, a well-known transcription factor regulating different stress responses, indicated that this dehydration-based immobilization method would not induce obvious stress response of the worm within 20 min. Furthermore, this microfluidic device could generate gas stimulation to the immobilized worm without the interference resulted from the switch of liquid-gas. Finally, the developed micro-device clearly demonstrated that URX neuronal responses were induced by oxygen gas with increasing levels of 0-20%, indicating that this method could be used for imaging analysis of gas-evoked neuronal sensing, especially to the pressure-change sensitive neurons in C elegans. (C) 2017 Elsevier B.V. All rights reserved.

Hand-powered centrifugal microfluidics combined with isothermal nucleic acid amplification testing (NAAT) have been one of the most promising rapid detection platforms in resource-limited settings. However, current hand-powered centrifuges still suffer from customized instrument-based operation and low rotation rate; and most isothermal NAAT were conducted with complicated reaction systems for DNA detection and required an additional step for RNA detection. Herein, we built a fully hand-powered centrifugal miniaturized NAAT platform inspired by buzzer toys, which embedded sample preparation, strand exchange amplification (SEA) and visual fluorescence detection together. The centrifugal disc was easily fabricated, and operated the mixing in 1 min by simply dragging the looped rope through it with a mean input force of 16.5 N, enabling its rotation rate reach 5000 rpm. In addition, SEA was an ultra-simple one-step DNA or RNA detection method initiated by Bst DNA polymerase and a pair of primers, and thus we took all its merits and integrate it into microfluidic systems firstly. Furthermore, taking Vibrio parahemolyticus as an example, the microfluidic platform achieved DNA or RNA detection within 1 h; and the detection limit of the microchip for artificially spiked oysters was 103 CFU/g without cumbersome sample preparation, and reached to 100 CFU/g after enrichment. Therefore, we provided an ultra-simple and non-instrumental microfluidic platform powered merely by hands, performing general potential in sample-to-answer NAAT for versatile pathogens in remote regions.

Liquid metal nanodroplets not only share similar metallic properties and nanoscale effect with solid metal nanoparticles, but also possess the additional uniqueness in nonvolatile fluidity and ambient sintering ability into continuous conductors. In most cases, liquid metal nanodroplets are encapsulated into ultrathin and fragile shells of oxides and amphiphile monolayers, and may be hindered from incorporating homogeneously into various composites through conventional processing methods. In this study, ring-opening polymerization is found to be initiated by sonicating the liquid metal EGaIn in fluidic lactones. By this in situ polymerization, EGaIn nanodroplets are encapsulated into polylactone shells with tunable thickness, which can further be dried into a solid powder. Besides high chemical stability and dispersibility in organic solvents, the powder of the EGaIn capsules combines the exceptional properties of the EGaIn droplets (e.g., photothermal effect) and the polylactone shells (e.g., biocompatibility, biodegradability, and compatibility with different polymer matrixes), being capable of being introduced into thermoplastic composites through liquid casting and thermal- or photomolding for the notch-insensitive tearing property, sintering-induced electric conductivity, and photothermal effect. Thus, the EGaIn initiator of ring-opening polymerization may start a pathway to produce stable andthermal/photomoldable powders of EGaIn capsules and their multifunctionalcomposites, applicable in biomedicines, soft electronics, and smart robots.

During the past decades, few micro-devices for analysis of associative learning behavior have been reported. In this work, an agarose-PDMS hybridized micro-chip was developed to establish a new associative learning model between mechanosensation and food reward in C. elegans. The micro-chip consisted of column arrays which mimicked mechanical stimulation to C. elegans. After trained by pairing bacterial food and mechanical stimuli in the chip, the worms exhibited associative learning behavior and gathered in the regions where there was food during training. The key research findings include: (1) Associative learning behavior of C. elegans could be generated and quantitatively analyzed by this developed micro-chip. (2) Associative learning behavior could be enhanced by extending the training time and developmental stage. (3) Mechanosensation-related genes and neurotransmitters signals had effects on the learning behavior. (4) The associative learning ability could be strengthened by exogenous dopamine in both wild type and mutants. We validated that the design of the micro-chip was useful and convenient for the study of learning behavior based on mechanosensation.

The directed motility of organisms in response to fluid velocity, which is called rheotaxis, is important in the life cycle of C. elegans, enabling them to navigate their environment and maintain their positions in the presence of adverse flow. Thus, to study the mechanism underlying rheotaxis behavior and reveal information on parasitic diseases, the profile analysis of the rheotaxis response in worm populations with high resolution in well-defined fluid environments is highly desirable. In this work, we presented a rapid and robust microfluidic approach to quantitatively analyze the rheotaxis behavior of worms in response to velocity. The flow-based microfluidic chip contained six helical spline microchannels for generating six flow streams with different flow velocities. Since the worms loaded in the chip would swim upstream into channels, the distribution of the worms in response to the different flow velocities was successfully monitored for the quantitative analysis of their rheotaxis behavior using this microfluidic chip. The results indicated that the rate range of around 50 μm s−1 was the most favorable flow velocity for the wild-type worms. Further, we analyzed ASH neuron-blocked worms and found that the functionally defective ASH neurons inhibited their sensitivity to flow rate. In addition, the rheotaxis analysis of the mutant worms indicated that TRP mechanosensory channels and serotonin signals also play a regulatory role in the rheotaxis response of these worms. Thus, our microfluidic method provides a useful platform to study the rheotaxis behaviors in C. elegans and can be further applied for anti-parasitic drug tests.

Gas embolism is the abnormal emergence of bubble in the vascular system, which can induce local ischemic symptoms. For studying the mechanism underlying gas embolism and revealing local ischemic diseases information, novel technique for analyzing cells response to bubble contact with high controllability is highly desired. In this paper, we present an integrated microfluidic device for the precise generation and control of microbubble based on the gas permeability of polydimethysiloxane (PDMS) to study the effect of bubble's mechanical contact on cells. Cell viability analysis demonstrated that short-term (15 min) bubble contact was generally non-lethal to cultured endothelial cells. The significant increase in intracellular calcium of the microbubble-contacted cells and cell-to-cell propagation of calcium signal in the adjacent cells were observed during the process of bubble expansion. In addition, the analysis of intercellular calcium signal in the cells treated with suramin and octanol revealed that cell-released small nucleotides and gap junction played an important role in regulating the propagation of calcium wave triggered by bubble contact. Thus, our microfluidic method provides an effective platform for studying the effect of gas embolism on cultured adherent cells and can be further needed for anti-embolism drugs test.

Studies of chemo-sensing in C. elegans to fluctuating gaseous cues are limited due to the lack of a method of precise gas control. In this paper, we describe a microfluidic-based on-demand gas segmented flow generator for performing fluctuating gaseous stimulations to worms. This highly versatile and programmable micro-device integrated with pneumatic valves for flexible and stable gas flow control and worm immobilization enabled us to examine the temporal features of neuronal response to multiple gas pulses with sub-second precision. As a result, we demonstrated the capability of the micro-device to generate repetitive gaseous chemical pulses with varying durations. By characterizing intracellular calcium signals, we showed that URX sensory neurons were sensitive to O2 pulses with duration of more than 0.5 s. Furthermore, URX neuronal adaptation and recovery in response to gaseous chemical pulses were investigated by varying the durations and intervals. The developed microfluidic system is shown to be a useful tool for studying the dynamics of in vivo gas-evoked neuronal responses and revealing the temporal properties of environmental stimulations.

Caenorhabditis elegans, one of the widely studied model organisms, sense external chemical cues and perform relative chemotaxis behaviors through its simple chemosensory neuronal system. To study the mechanism underlying chemosensory behavior, a rapid and reliable method for quantitatively analyzing the worms' behaviors is essential. In this work, we demonstrated a microfluidic approach for investigating chemotaxis responses of worms to chemical gradients. The flow-based microfluidic chip was consisted of circular tree-like microchannels, which was able to generate eight flow streams containing stepwise chemical concentrations without the difference in flow velocity. Worms' upstream swimming into microchannels with various concentrations was monitored for quantitative analysis of the chemotaxis behavior. By using this microfluidic chip, the attractive and repellent responses of C. elegans to NaCl were successfully quantified within several minutes. The results demonstrated the wild type-like repellent responses and severely impaired attractive responses in grk-2 mutant animals with defects in calcium influx. In addition, the chemotaxis analysis of the third stage larvae revealed that its gustatory response was different from that in the adult stage. Thus, our microfluidic method provided a useful platform for studying the chemosensory behaviors of C. elegans and screening of chemosensation-related chemical drugs.