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• A 3D micromixer is created to address flow instability issues in a 2D mixer. • The highest-resolution image for the focused sample stream was obtained in the 3D mixer. • The early folding kinetics of G-quadruplex was investigated, and a lag phase was found. Hydrodynamic flow focusing-based micromixers (laminar micromixers) have been widely applied to the investigation of the folding kinetics of biomacromolecules, due to their capability to resolve the folding events in a microsecond time scale. Although most existing laminar micromixers attempted to reduce mixing time, our work focused on developing a system that can generate high-resolution images under a more stable flow, which results in more accurate kinetic analysis. Specifically, we developed a simple three-dimensional (3D) laminar micromixer, which distributes the sample stream to a 3D profile to address flow-instability issues caused by Dean vortices in a two-dimensional mixer. In the 3D mixer, the sample stream was successfully focused to a one-pixel width at the resolution of ˜200 nm/pixel, which approached the resolving power of an optical microscope and represented the highest imaging resolution ever achieved using a 3D mixer. Furthermore, we used the device to investigate the early folding kinetics of G-quadruplex under various sodium cation concentrations. Interestingly, a lag phase was found before the exponential phase during the folding process. Moreover, we obtained the G-quadruplex's observed kinetics of (1.51 ± 0.02) ×104 s−1, (1.68 ± 0.04) ×104 s−1 and (2.01 ± 0.02) ×104 s−1 at 50-, 100- and 200 mM NaCl, respectively. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • A novel microfluidic device was proposed to achieve cell pairing at single cell level. • Highly efficient cell trapping (93%) and pairing (84%) were realized by using hydrodynamics, without external equipment. • The result showed that HUVEC could enhance the proliferation of HeLa. Abstract Understanding the mechanism of cell-cell communication is the key to unravel many physiological and pathological processes of multicellular organisms, but limited by lacking of highly efficient analytical approach, particularly at single cells resolution. In this work, we proposed a novel microfluidic-based method to achieve cell pairing for cell-cell communication investigation. Based on the only application of hydrodynamics, two types of cells were trapped by face-to-face heart-shaped micro-protrusions with high efficiency and single-cell accuracy. Oil-isolated micro-chambers were used to reduce cross-talk and spatial confinement on cells for long-term cell culture with high viability. The efficiency of 93% and 84% for single cells trapping and cells pairing were realized, respectively. To demonstrate the capability of the proposed platform for probing intercellular communication, the proliferation of cancer cell co-cultured with stromal cell was further discussed at single cells resolution. The result showed that the HUVEC could enhance the proliferation of HeLa cell. We believed that this microfluidic device as a flexible and reliable tool would have wide applications in the study of intercellular communication. [ABSTRACT FROM AUTHOR]

Highlights • A simple, inexpensive and fully integrated device was demonstrated for detecting gene mutations. • Integration of lateral flow test allowed the detection of gene mutation by naked eyes. • The L858R mutation in lung adenocarcinoma cells was detected with high specificity and sensitivity. Abstract Paper-based diagnostics have been successfully explored for nucleic acid detection. However, multi-step integration (such as nucleic acid extraction, washing, amplification and detection) into a single paper chip remains a huge challenge. Here, a simple, inexpensive, and fully integrated paper chip was demonstrated for detecting gene mutations, which could potentially play a guiding role in individualized treatments to improve the survival rate of lung cancer patients. To simplify the pipetting and manipulation of samples, we incorporated DNA extraction, purification, amplification and detection into a single chip. Further integration of lateral flow test allowed the detection of gene mutation by naked eye. The gene mutation in the epidermal growth factor receptor (EGFR L858R) in lung adenocarcinoma cells was successfully detected with high specificity and sensitivity within 90 min by the proposed fully integrated chip. This low cost, disposable and user-friendly chip provided a new and rapid molecular diagnostic platform, especially suitable for molecular diagnostics in resource-limited areas. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • A novel SU-8 mesh chip was fabricated for high-throughput 3D wound healing assay. • "Wound on a chip" model was constructed for wound healing assay by real-time monitoring the cells growth processes. • This 3D cell culture method provided more complex cellular processes and some tissue-specific properties. Abstract The dynamic process of cells is the basis of many biological problems in living organisms and has been a major research subject over several decades. In this paper, we present a novel SU-8 mesh chip (SMC) with thousands of chamber arrays for high-throughput 3D wound healing assay, which could directly and efficiently evaluate 3D cell growth process and cellular drug responses in vitro. Four kinds of adherent cells (HeLa, HepG2, HUVEC, NIH-3T3) were successfully seeded into the SMC to form three-dimensional hollow cell aggregates models with precisely pre-defined sizes and shapes, followed by monitored their spontaneous growth process in real time. Compared with traditional 2D wound healing assay, this 3D model provided more complex cellular processes and some tissue-specific properties, which conferred a high degree of clinical and biological relevance to in vitro models. NIH-3T3 and HUVEC cell aggregates in this chip that termed as "wound on a chip" model had been developed and further investigated the drug response in these 3D wound healing processes by applying different concentrations of candidate drugs. [ABSTRACT FROM AUTHOR]

Cell signaling has been recognized as a highly dynamic process. How to capture such dynamics with changes of chemical microenvironment at single cell resolution among cells population without interfering with adjacent cells has been still an immense challenge. Here, we proposed a precision guided microfluidic chemical perfusion strategy for investigating chemical-activated cell signaling via dynamic imaging with high temporal (∼12 ms), spatial resolutions and precision. Theory for the microfluidic operation was first established and validated by both numerical simulations and experiments. A computer-aided multi-channel pressure control system was constructed to automatically implement chemical perturbations with various microperfusion times, relaxation times and number of cycles. The width and the position of the chemical microflow could also be precisely controlled with high reliability and accuracy. Investigations of intracellular and intercellular calcium signaling were successfully demonstrated using the proposed method. These results revealed that the precision guided microfluidic chemical perfusion strategy provided a versatile means for cell signaling analysis, which is potentially meaningful in chemical biology, cell biology and pharmacology. [ABSTRACT FROM AUTHOR]

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 . [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Paper-based microfluidic chips have shown great potential for use in rapid clinical diagnostics. In this work, we present a paper-based device that integrates an electromagnetically actuated valve and an adjustable time controller for the simultaneous detection of multiple tumor biomarkers. The time controller consists of a timing channel and two movable conductive iron bands that function as an electric switch. Once the conductive iron bands are connected by the fluid flow, the circuit would be closed to actuate the magnetic valve, initiating chemiluminiscent immunoassay (CLIA) reactions. Using a HRP- O -phenylenediamine-H 2 O 2 detection system, simultaneous detection of different concentrations of carcinoembryonic antigens (CEA) was demonstrated. The resulting CLIA peak intensity exhibited a good linear relationship with the logarithm of the CEA concentration from 0.1–80 ng mL −1 . Further experiments showed that the developed device was capable of detecting multiple tumor biomarkers simultaneously. We can program different incubation times for the detection of different biomarkers by moving the conductive bands on this paper-based device. In this study, only 16 min was required to complete CEA detection. This strategy greatly shortens detection time compared to the conventional chemiluminiscence technique for CEA detection (nearly 2 h). This paper-based device provides a simple and inexpensive platform for immunoassay-based detection of disease-related biomarkers. [ABSTRACT FROM AUTHOR]

Cell-based biosensor (CBB) has shown potential to provide a rapid and sensitive measurement means for monitoring specific biological substances. However, the detecting frequency and reusability remain a challenge for further application of CBB. In this paper, a microfluidic cell-based biosensor (μCBB) was developed, which consisted of a localized millisecond chemical pulse as sample introduction for pulsing individual cells without interfering with adjacent cells and single HeLa cells expressing endogenous P2Y receptors as the sensing elements. Gated injection of pinched flow was used to generate millisecond ATP pulses with predefined width. By integrating localized ATP pulse with predefined width, multiple sensing elements could be used to detect ATP pulses without impact on their functionality. Thus, the detecting frequency of the developed μCBB is multiplied by the number of available cells in comparison to that reported previously. We expect the developed method to be potentially useful to enhance the usability of CBB for biological and biomedical analysis. [ABSTRACT FROM AUTHOR]

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 . [ABSTRACT FROM AUTHOR]

In this work, we propose a microfluidic in-gel loop-mediated isothermal amplification (gLAMP) containing preloaded reagents as a self-contained microdevice for simultaneous detection of multiple pathogenic bacterial DNA in near point of care settings. The microchip consists of multiple channels with reaction chambers accommodating low-melting-point agarose and all LAMP reagents except the DNA samples, which can be kept at 4 °C for long-term storage. The current microchip enables simultaneous tests of four different bacterial DNA targets. After DNA samples were introduced into the microchip, gLAMP reactions were carried out on a hot plate at 65 °C. The gLAMP reaction conditions including the concentrations of the fluorescence indicator, Bst DNA polymerase, primers, and Mg 2+ ions were further optimized. Under the optimized conditions, detection of foodborne bacteria in serum samples, including Escherichia coli, Proteus hauseri, Vibrio parahaemolyticus, and Salmonella subsp. Enterica were demonstrated with high selectivity and sensitivity (as low as 3 copies/μL). The gLAMP microchip provides a simple and easy-to-operate platform for LAMP-based pathogen detection, which holds high potential for future applications in point-of-care settings. [ABSTRACT FROM AUTHOR]

In this paper, a new microfluidic approach, termed as gated pinched-flow (GPF), was developed for investigating cell-to-cell signaling dynamics coupling with optical imaging. It takes both advantages of conventional hydrodynamic gating and focusing, enabling precise on-chip chemical stimulation or perfusion upon targeted single cells among cells populations with high temporal (<50 ms) and spatial resolution. Theoretical model for GPF was first established and further validated by both numerical simulations and experiments. Stimulation of single HeLa cells was investigated to demonstrate the capability of the GPF for localized chemical stimulations of target single cells without interfering with adjacent cells. Guided by GPF, ATP-activated propagation of intercellular calcium waves (ICWs) among the seeded NIH-3T3 cells in the microchannel was then imaged with high repeatability. Inhibition investigations verified that those cell-to-cell calcium signals depended upon direct cytosolic transfer of molecules via gap junctions. The developed microfluidic method opens up a new avenue for cell-to-cell signaling studies and drug screening. [ABSTRACT FROM AUTHOR]

A rotate & react SlipChip (RnR-SlipChip) was developed for simultaneous visual detection of multiple bacterial pathogens by loop-mediated isothermal amplification (LAMP). The SlipChip system consists of two round PDMS-glass hybrid chips that are assembled coaxially by a plastic screw-nut suite. After sample loading, one-step rotation allowed immediate mixing and reaction of multiple bacteria samples with LAMP reagents on the chip. A home-built peltier heating device was used to implement the isothermal control by an advanced RISC machines (ARM) processor. Under the optimized LAMP conditions, a fluorescent signal-to-noise ratio of about 5-fold and a detection limit of 7.2 copies/μL genomic DNA were achieved. Furthermore, five common digestive bacterial pathogens including Bacillus cereus, Escherichia coli, Salmonella enterica, Vibrio fluvialis and Vibrio parahaemolyticus , were visually identified using the established method in 60 min with a success rate of 100%. The developed RnR-SlipChip provided a rapid, simple-to-use and cost-effective solution for pathogenic bacteria detection, which holds high potential for clinical diagnosis especially in resource-limited areas. [ABSTRACT FROM AUTHOR]

Multidrug resistance analysis represents a great challenge in cancer chemotherapy, drug development and pathological study. In this paper, multifunctional gradients-customizing microfluidic devices were developed for high-throughput single-cell multidrug resistance (MDR) analysis. The gradient profile was determined by the lengths of the distribution microchannels regardless of flow rates and pressure, which provided good stability and remarkably reduced redundancy of microfluidic architecture. The drug of gradient concentrations consecutively stimulate upon the cells in the downstreaming cell cultivation chamber. Time-dependent drug efflux kinetics of HepG2 cells were firstly investigated on our device using both the different-single-cell and the same-single-cell strategies. Furthermore, hepatic polarized HepG2 cells, which collected the secreted cholephilic substances in the apical vacuoles, were used as model to investigate the inhibition of MDR-associated protein with secretion inhibitor cyclosporine A of varied concentrations on single organelle level. Finally, a high-throughput drug screening experiment was conducted to examine both the chemo-sensitizing effect and the cytotoxity of the potential chemo-sensitizing agents. Conclusively, the results confirmed that our method was a highly efficient way to analyze multidrug resistance (MDR) at single-cell or even single-organelle level with advantages of high-throughput, flexibility, stability and low sample consumption. [ABSTRACT FROM AUTHOR]

A multicolor fluorescent probe BNO for measuring intracellular pH is designed and synthesized, which is the first pH probe with a dual response mechanism combining an excited state intramolecular proton transfer (ESIPT) process and an intramolecular charge transfer (ICT) process. The probe shows negligible fluorescence at neutral and weak alkaline pH due to fluorescence quenching caused by ESIPT, and a sharp fluorescence enhancement in both acidic and strong alkaline environments. Meanwhile, it responds to pH changes with different emissions (green at acidic range, cyan at alkaline range). BNO is able to indicate pH variations in a wide range of 3.9–13.0. It is also capable of distinguishing intracellular pH values with high sensitivity. Furthermore, the probe exhibits favorable two-photon photophysics and high photostability, thus it is successfully applied to trace the dynamic change of intracellular pH with two-photon microscopy (TPM). The dual-mechanism strategy may be expanded to design other fluorescent probes. [ABSTRACT FROM AUTHOR]

It is a challenging task to realize gaseous stimulations on Caenorhabibditis elegans for in vivo neuronal analysis of chemo-sensing, due to the difficulties in gas control and worm manipulation. Here, we demonstrated an integrated microfluidic system that could accurately deliver the gaseous stimuli to the worms’ noses and track the in vivo neuronal activities simultaneously. The microfluidic chip consisted of a comb-shaped micro-valve for worm immobilization and a T-junction structure for precise gas delivery. Neuronal responses of C. elegans to polar and non-polar gases were both investigated. The microfluidic device clearly demonstrated that oxygen with increasing levels of 0–10% and 0–21% induced URX neuronal responses. BAG neuronal activities were inhibited by carbon dioxide, showing the symptom of anesthesia. The vapor of polar odorant 1-octonal evoked significant calcium transients in ASH neurons in the wild-type animals but weak signals in tph-1 and Ce-grk-2 mutants. These results indicated that the developed device could be useful to identify various odor-evoked neuronal activities. [ABSTRACT FROM AUTHOR]

Abstract: Conventional macro-scale methods are not appropriate for in vivo neuronal analysis at a single neuron level when experimental subject is small model organism Caenorhabditis elegans. In this paper, we demonstrate a powerful microfluidic device that allowed investigating in vivo chemo-sensing of intact C. elegans by integrating functions of automated manipulation of worms, effective immobilization without damages and well-controllable stimulations. Live C. elegans were effectively immobilized in microfluidic channels by a comb-shaped microvalve without physiological damages. A well-controlled sample introduction system was used to deliver chemicals to the chemosensory cilia at the worm''s nose tip. In vivo neuronal responses of the animal to the chemical stimulations were monitored using calcium fluorescence imaging. Dynamic calcium signals related to varying concentrations and durations of stimuli were investigated using this integrated microfluidic device. We anticipate this immobilization approaches can have an impact in a variety of applications, such as neuron responses investigation before and after laser ablation upstream or downstream neurons for studying olfactory or gustatory neural circuits in individual healthy worms, in this high-throughput system. [Copyright &y& Elsevier]

Smart light-driven machines that can be manipulated in a remote, contactless and controlled way are desired for many applications. Although considerable efforts have been devoted to the light-driven devices, challenges still remain in the multifunctional structure design, the development of patternable photothermal materials and precise control. In this report, we demonstrate a facile method to construct smart floating devices by the incorporation of candle soot with designable patterns into PDMS, which can be propelled by light-induced Marangoni effect. The device shows high photothermal conversion capability and photothermal stability. Meanwhile, it realizes multiple movement modes including linear, curvilinear movement and rotational movement by designing asymmetric structures or adjusting the illumination site of light sources. Moreover, the floating actuator can be driven not only by infrared (IR) light, but also by focused sunlight. This work will inspire us to explore great potentials in various fields such as soft robot, sensor, and electricity generator etc. [Display omitted] • A facile method to pattern candle soot was developed, which was applied to light-driven actuator via Marangoni effect. • The actuator exhibited high photothermal performance and photothermal stability. • Diverse movement modes with controllable direction and velocity could be easily realized. [ABSTRACT FROM AUTHOR]

• A low-cost and rapid agarose-based microfluidic chip for exosomes preconcentration and detection was proposed. • Detection sensitivity of a LOD of ∼103 exosomes along with the extremely low volume of samples (as low as 1 μL). • IIn situ concentration of different cell line's exosomes and detection of exosomal microRNA simultaneously. Exosomes, together with their accompanying proteins and nucleic acids have been increasingly recognized as biomarkers for non-invasive tumor diagnostic and prognostic. However, rapid and efficient detection of exosomes remains challenging, due to their small size (30−150 nm), and low concentration. Thus, to guarantee, especially when cancer screening requires detection limits lower than exosome concentrations, it not only needs to isolate and purify exosomes, but it needs to preconcentrate them as well. Here, a low-cost, rapid, and portable agarose-based microfluidic chip for exosome concentration and in situ exosomal microRNA detection was developed, termed isExoCD (in situ exosome concentration and detection). The target exosomes loaded at the entrance will be automatically enriched at the closed end of the microchannel by leveraging the capillary effect and the strong water permeability of the agarose gel. To detect the exosomal microRNA, we integrated the catalyzed hairpin assembly (CHA) based strategy into the isExoCD, allowing high sensitive detection of exosomes with a limit of detection (LOD) of ∼103. Using our method, we can successfully distinguish cancer cell-derived exosomes from other exosomes that obtained other cell types. Thus, our platform paves a new avenue to early cancer detection, liquid biopsy, and point-of-care diagnosis. [ABSTRACT FROM AUTHOR]