Intracellular Delivery of Therapeutic Protein via Ultrathin Layered Double Hydroxide Nanosheets.

The therapeutic application of biofunctional proteins relies on their intracellular delivery, which is hindered by poor cellular uptake and transport from endosomes to cytoplasm. Herein, we constructed a two-dimensional (2D) ultrathin layered double hydroxide (LDH) nanosheet for the intracellular delivery of a cell-impermeable protein, gelonin, towards efficient and specific cancer treatment. The LDH nanosheet was synthesized via a facile method without using exfoliation agents and showed a high loading capacity of proteins (up to 182%). Using 2D and 3D 4T1 breast cancer cell models, LDH-gelonin demonstrated significantly higher cellular uptake efficiency, favorable endosome escape ability, and deep tumor penetration performance, leading to a higher anticancer efficiency, in comparison to free gelonin. This work provides a promising strategy and a generalized nanoplatform to efficiently deliver biofunctional proteins to unlock their therapeutic potential for cancer treatment.

Optical-based microbubble for on-demand droplet release from static droplet array (SDA) for dispensing one droplet into one tube.

Static droplet array (SDA) is a pivotal tool for high-capacity screening assays, yet extraction and collection the target droplets that contain unique analytes or cells from the SDA remains one major technical bottleneck that limits its broader application. Here we present an optical-based on-demand droplet release (OODR) system by incorporating a 1064 nm laser-responsive indium tin oxide (ITO) layer into a chamber array-based droplet microfluidic chip. By focusing the 1064 nm laser onto the ITO layer, microbubbles can be created via local heating to selectively push-out the droplets from the chamber. Then the released droplet is readily exported in a one-droplet-one-tube (ODOT) manner by the inherent capillary force into pipette tip. Releasing of the droplets containing fluorescein sodium demonstrated ∼100% successful rate (9 out of 6400 droplets were successfully released) and low residual (only ∼5% of the droplet volume remains in the chamber). White or fluorescence image-based releasing of single-cell-droplets directly after cell loading or multi-cells-droplets derived from on-chip single-cell cultivation for both E. coli and yeast cells further demonstrated the wide applicability of OODR. The present system is user-friendly and has the potential to be applied in various high-throughput screening assays, including single molecule/cell analysis, drug screening, and phenotype-based cell sorting.

Effect of Multiple Structural Parameters on the Performance of a Micromixer with Baffles, Obstacles, and Gaps.

As an essential component of chip laboratories and microfluidic systems, micromixers are widely used in fields such as chemical and biological analysis. In this work, a square cavity micromixer with multiple structural parameters (baffles, obstacles, and gaps) has been proposed to further improve the mixing performance of micromixers. This study examines the comprehensive effects of various structural parameters on mixing performance. The impact of baffle length, obstacle length-to-width ratio, gap width, and obstacle shape on the mixing index and pressure drop were numerically studied at different Reynolds numbers (Re). The results show that the mixing index increases with baffle length and obstacle length-to-width ratio and decreases with gap width at Re = 0.1, 1, 10, 20, 40, and 60. The mixing index can reach more than 0.98 in the range of Re ≥ 20 when the baffle length is 150 µm, the obstacle length-to-width ratio is 600/100, and the gap width is 200 µm. The pressure drop of the microchannel is proportional to baffle length and obstacle length-to-width ratio. Combining baffles and obstacles can further improve the mixing performance of square cavity micromixers. A longer baffle length, larger obstacle length-to-width ratio, narrower gap width, and a more symmetrical structure are conducive to improving the mixing index. However, the impact of pressure drop must also be considered comprehensively. The research results provide references and new ideas for passive micromixer structural design.

Auto Flow-Focusing Droplet Reinjection Chip-Based Integrated Portable Droplet System (iPODs).

Droplet microfluidics provides powerful tools for biochemical applications. However, precise fluid control is usually required in the process of droplet generation and detection, which hinders droplet-based applications in point-of-care testing (POCT). Here, we present a droplet reinjection method capable of droplet distribution without precise fluid control and external pumps by which the droplets can be passively aligned and detected one by one at intervals. By further integrating the surface-wetting-based droplet generation chip, an integrated POrtable Droplet system (iPODs) is developed. The iPODs integrates multiple functions such as droplet generation, online reaction, and serial reading. Using the iPODs, monodisperse droplets can be generated at a flow rate of 800 Hz with a narrow size distribution (CV <2.2%). Droplets are kept stable, and the fluorescence signal can be significantly identified after the reaction. The spaced droplet efficiency in the reinjection chip is nearly 100%. In addition, we validate digital loop-mediated isothermal amplification (dLAMP) within 80 min with a simple operation workflow. The results show that iPODs has good linearity (R2 = 0.999) at concentrations ranging from 101 to 104 copies/µL. Thus, the developed iPODs highlights its potential to be a portable, low-cost, and easy-to-deploy toolbox for droplet-based applications.

Influence of Structural Parameters on the Performance of an Asymmetric Rhombus Micromixer with Baffles.

As an important part of lab-on-a-chip and micro-total analysis systems, micromixers have a wide range of applications in biochemical analysis, pharmaceutical preparation and material synthesis. In the work, a novel rhombic separation and recombination micromixer with baffles was presented to further improve the performance of the micromixer and study the effect of multiple structural parameters on mixing. The effects of the rhombic angle, the width ratio of sub-channel and the size and relative positions of baffles on the mixing index were studied numerically at different Reynolds numbers (Re), and the sensitivity of the mixing index to various structures was also investigated. The results showed that the mixing index increased with the subchannel's width ratio and slowly decreased after reaching the peak value in the range of Re from 0.1 to 60. The maximum mixing index appeared when the width ratio was 6.5. The pressure drops in the microchannel were proportional to the width ratio. The mixing effect can be further improved by adding baffle structure to asymmetric rhombus micromixer, and more baffle quantity and larger baffle height were beneficial to the improvement of the mixing index. The research results can provide reference and new ideas for the structure design of passive micromixers.

A Palm Germ-Radar (PaGeR) for rapid and simple COVID-19 detection by reverse transcription loop-mediated isothermal amplification (RT-LAMP).

The current COVID-19 pandemic caused by SARS-CoV-2 is raging, seriously threatening people's lives. The establishment of rapid and accurate pathogen detection technology is not only critical in this epidemic, but also a reminder that we must always be prepared for possible future outbreaks. Therefore, we developed a Palm Germ-Radar (PaGeR) device for rapid and simple detection of COVID-19 from extracted patient sample RNA by RT-LAMP. The whole procedure of rapid COVID-19 detection is based on 4 simple steps: inactivation, extraction, amplification, and detection. SARS-CoV-2 down to 1 copy/µL could be detected selectively with naked-eye. Three detection methods (colorimetric, fluorometric and lateral dipstick readout) could be performed in PaGeR instrument. By employing the PaGeR, we successfully detected SARS-CoV-2 in clinical RNA samples isolated from swab specimens. The results showed that 15 out of 17 COVID-19 patients were diagnosed as positive while all 55 normal samples were diagnosed as negative. Therefore, the developed PaGeR instrument can realize the detection of COVID-19 with easily visualized results, providing a promising instrument for rapid detection in the community as well as at home.

Rapid generation of hybrid biochemical/mechanical cues in heterogeneous droplets for high-throughput screening of cellular responses.

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/ß-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.

A low-cost microfluidic platform coupled with light emitting diode for optogenetic analysis of neuronal response in C. elegans.

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.

A fully integrated hand-powered centrifugal microfluidic platform for ultra-simple and non-instrumental nucleic acid detection.

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 Initiator of Ring-Opening Polymerization: Self-Capsulation into Thermal/Photomoldable Powder for Multifunctional Composites.

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.

A dual-stimulation strategy in a micro-chip for the investigation of mechanical associative learning behavior of C. elegans.

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.

An integrated microfluidic device for studying controllable gas embolism induced cellular responses.

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.

Profile analysis of C. elegans rheotaxis behavior using a microfluidic device.

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.

Real-time monitoring of immune responses under pathogen invasion and drug interference by integrated microfluidic device coupled with worm-based biosensor.

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.

An on-demand gas segmented flow generator with high spatiotemporal resolution for in vivo analysis of neuronal response in C. elegans.

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.

Quantitative analysis of Caenorhabditis elegans chemotaxis using a microfluidic device.

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.

Highly efficient microfluidic sorting device for synchronizing developmental stages of C. elegans based on deflecting electrotaxis.

C. elegans as a powerful model organism has been widely used in fundamental biological studies. Many of these studies frequently need a large number of different stage-synchronized worms due to the stage-specific features of C. elegans among 4 distinct larval stages and the adult stage. In this work, we present an interesting and cost-effective microfluidic approach to realize simultaneous sorting of C. elegans of different developmental stages by deflecting electrotaxis. The microfluidic device was fabricated using PDMS consisting of symmetric sorting channels with specific angles, which was further hybridized to an agarose plate. While applying an electric field, different stages of C. elegans would crawl to the negative pore with different angles due to their deflecting electrotaxis. Thus, the worms were separated and synchronized by stages. lon-2 mutant was further used to study this electrotactic response and the results indicated that the body size plays a key role in determining the deflecting angle in matured adult worms. In addition to discriminating wild-type hermaphrodites, it could also be employed to sort mutants with abnormal development sizes and males. Therefore, our device provided a versatile and highly efficient platform for sorting C. elegans to meet the requirement of large numbers of different stage-synchronized worms. It can also be further used to investigate the neuronal basis of deflecting electrotaxis in worms.