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There remains an unmet need for a fully integrated microfluidic platform that can automatically perform multistep and multireagent immunoassays. Here, we proposed a novel online dual-active valve-based centrifugal microfluidic chip, termed DAVM, for fully automatic point-of-care immunoassay. Practically, the puncture valve, one of the dual active valves, is capable of achieving precise, on-demand, sequential release of prestored reagents, while the other valve-reversible active valve enables controlled retention and drainage of the reaction solutions. Thereby, our technology mitigates the challenges of hydrophilic/hydrophobic modifications and unstable valve control performance commonly observed in passive valve controls. As a proof of concept, the indirect enzymatic immunoblotting technique was employed on DAVM for fully automated immunological analysis of eight targets, yielding outcomes within an hour. Furthermore, we conducted a comparative analysis of 28 clinical samples with autoimmune diseases. According to 224 clinical data, the sample testing concordance rate between DAVM and the traditional instrument was 82%, with a target compliance rate of 97%. Therefore, our DAVM system has powerful potential for fully automated immunoassays.

Microfluidic paper-based analytical devices (μPADs) are emerging as powerful analytical platforms in clinical diagnostics, food safety, and environmental protection because of their low cost and favorable substrate properties for biosensing. However, the existing top-down fabrication methods of paper-based chips suffer from low resolution (>200 μm). Additionally, papers have limitations in their physical properties (e.g., thickness, transmittance, and mechanical flexibility). Here, we demonstrate a bottom-up approach for the rapid fabrication of heterogeneously controlled paper-based chip arrays. We simply print a wax-patterned microchip with wettability contrasts, enabling automatic and selective assembly of cellulose microfibers to construct predefined paper-based microchip arrays with controllable thickness. This paper-based microchip printing technology is feasible for various substrate materials ranging from inorganic glass to organic polymers, providing a versatile platform for the full range of applications including transparent devices and flexible health monitoring. Our bottom-up printing technology using cellulose microfibers as the starting material provides a lateral resolution down to 42 ± 3 μm and achieves the narrowest channel barrier down to 33 ± 2 μm. As a proof-of-concept demonstration, a flexible paper-based glucose monitor is built for human health care, requiring only 0.3 μL of sample for testing.

Traditional diagnostic strategies for infectious disease detection require benchtop instruments that are inappropriate for point-of-care testing (POCT). Emerging microfluidics, a highly miniaturized, automatic, and integrated technology, are a potential substitute for traditional methods in performing rapid, low-cost, accurate, and on-site diagnoses. Molecular diagnostics are widely used in microfluidic devices as the most effective approaches for pathogen detection. This review summarizes the latest advances in microfluidics-based molecular diagnostics for infectious diseases from academic perspectives and industrial outlooks. First, we introduce the typical on-chip nucleic acid processes, including sample preprocessing, amplification, and signal read-out. Then, four categories of microfluidic platforms are compared with respect to features, merits, and demerits. We further discuss application of the digital assay in absolute nucleic acid quantification. Both the classic and recent microfluidics-based commercial molecular diagnostic devices are summarized as proof of the current market status. Finally, we propose future directions for microfluidics-based infectious disease diagnosis., (© 2022. The Author(s).)

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., Competing Interests: Declaration of competing interest There are no conflicts to declare., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Microfluidic chip electrophoresis has been widely employed for separation of various biochemical species owing to its advantages of low sample consumption, low cost, fast analysis, high throughput, and integration capability. In this article, we reviewed the development of four different modes of microfluidics-based electrophoresis technologies including capillary electrophoresis, gel electrophoresis, dielectrophoresis, and field (electric) flow fractionation. Coupling detection schemes on microfluidic electrophoresis platform were also reviewed such as optical, electrochemical, and mass spectrometry method. We further discussed the innovative applications of microfluidic electrophoresis for biomacromolecules (nucleic acids and proteins), biochemical small molecules (amino acids, metabolites, ions, etc.), and bioparticles (cells and pathogens) analysis. The future direction of microfluidic chip electrophoresis was predicted., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

To investigate rapid suspension cell signaling, a microfluidic platform was urgently needed for flexibly manipulation of single cells and simultaneous generation of controllable chemical signals to stimulate single cells. In this paper, a microfluidic biosensor was developed to monitor intracellular calcium signal, integrated with single-cell trapping, chemical stimulation and releasing. Selective entrapment and discharge of individual cell were achieved by controlling the deformable membrane with pneumatic traps. The activation of intracellular calcium signal was qualitatively and quantitatively investigated by high-controllable chemical single-cell stimulation based on flexible hydrodynamic gating. And performing chemical stimulation and control assay in the same channel would improve the experimental robustness and effectiveness. Further investigation of the cellular responses to ATP pulses of varying concentrations and durations indicated that 20 μM ATP pulses with duration as short as 200 ms resulted in the same level of Ca 2+ response induced by sustained stimulations. Washing with buffer for 30 s was sufficient for single cell to recover from receptor desensitization caused by ATP stimulation. In addition, the responses of cells to ATP stimulation were heterogeneous. The developed microfluidic method opens up a new avenue for intracellular signaling studies and drug screening., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Random compartmentalization of cells by common droplet formation methods, i.e., T-junction and flow-focusing, results in low occupancy of droplets by single cells. To resolve this issue, a fluorescence-activated droplet formation method was developed for the on-command generation of droplets and encapsulation of single cells. In this method, droplets containing one cell were generated by switching on/off a two-phase hydrodynamic gating valve upon optical detection of single cells. To evaluate the developed method, flow visualization experiments were conducted with fluorescein. Results indicated that picoliter droplets of uniform sizes (RSD<4.9%) could be generated. Encapsulation of single fluorescent polystyrene beads demonstrated an average of 94.3% droplets contained one bead. Further application of the developed methods to the compartmentalization of individual HeLa cells indicated 82.5% occupancy of droplets by single cells, representing a 3 fold increase in comparison to random compartmentalization., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Active control over the cell gradient is essential for understanding biological systems and the reconstitution of the functionality of many types of tissues, particularly for organ-on-a-chip. Here, we propose a three-dimensional (3D) microfluidic strategy for generating controllable cell gradients. In this approach, a homogeneous cell suspension is loaded into a 3D stair-shaped PDMS microchannel to generate a cell gradient within 10 min by sedimentation. We demonstrate that cell gradients of various profiles (exponential and piecewise linear) can be achieved by precisely controlling the height of each layer during the fabrication. With sequential seeding, we further demonstrate the generation of two overlapping cell gradients on the same glass substrate with pre-defined designs. The cell gradient-based QD cytotoxicity assay also demonstrated that cell behaviors and resistances were regulated by the changes in cell density. These results reveal that the proposed 3D microfluidic strategy provides a simple and versatile means for establishing controllable gradients in cell density, opening up a new avenue for reconstructing functional tissues.

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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.

Digital single-cell assays hold high potentials for the analysis of cell apoptosis and the evaluation of chemotherapeutic reagents for cancer therapy. In this paper, a microfluidic hydrodynamic trapping system was developed for digital single-cell assays with the capability of monitoring cellular dynamics over time. The microfluidic chip was designed with arrays of bypass structures for trapping individual cells without the need for surface modification, external electric force, or robotic equipment. After optimization of the bypass structure by both numerical simulations and experiments, a single-cell trapping efficiency of ∼90 % was achieved. We demonstrated the method as a digital single-cell assay for the evaluation of five clinically established chemotherapeutic reagents. As a result, the half maximal inhibitory concentration (IC50) values of these compounds could be conveniently determined. We further modeled the gradual decrease of active drugs over time which was often observed in vivo after an injection to investigate cell apoptosis against chemotherapeutic reagents. The developed method provided a valuable means for cell apoptotic analysis and evaluation of anticancer drugs.

Preconcentration of pathogens from patient samples represents a great challenge in point-of-care (POC) diagnostics. Here, a low-cost, rapid, and portable agarose-based microfluidic device was developed to concentrate biological fluid from micro- to picoliter volume. The microfluidic concentrator consisted of a glass slide simply covered by an agarose layer with a binary tree-shaped microchannel, in which pathogens could be concentrated at the end of the microchannel due to the capillary effect and the strong water permeability of the agarose gel. The fluorescent Escherichia coli strain OP50 was used to demonstrate the capacity of the agarose-based device. Results showed that 90% recovery efficiency could be achieved with a million-fold volume reduction from 400 μL to 400 pL. For concentration of 1 × 10(3) cells mL(-1) bacteria, approximately ten million-fold enrichment in cell density was realized with volume reduction from 100 μL to 1.6 pL. Urine and blood plasma samples were further tested to validate the developed method. In conjugation with fluorescence immunoassay, we successfully applied the method to the concentration and detection of infectious Staphylococcus aureus in clinics. The agarose-based microfluidic concentrator provided an efficient approach for POC detection of pathogens.

The propagation of intercellular calcium wave (ICW) is essential for coordinating cellular activities in multicellular organisms. However, the limitations of existing analytical methods hamper the studies of this biological process in live animals. In this paper, we demonstrated for the first time a novel microfluidic system with an open chamber for on-chip microinjection of C. elegans and investigation of ICW propagations in vivo. Worms were long-term immobilized on the side wall of the open chamber by suction. Using an external micro-manipulator, localized chemical stimulation was delivered to single intestinal cells of the immobilized worms by microinjection. The calcium dynamics in the intestinal cells expressing Ca(2+) indicator YC2.12 was simultaneously monitored by fluorescence imaging. As a result, thapsigargin injection induced ICW was observed in the intestinal cells of C. elegans. Further analysis of the ICW propagation was realized in the presence of heparin (an inhibitor for IP3 receptor), which allowed us to investigate the mechanism underlying intercellular calcium signaling. We expect this novel microfluidic platform to be a useful tool for studying cell-cell communication in multicellular organisms in vivo., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Encapsulation of single cells is a challenging task in droplet microfluidics due to the random compartmentalization of cells dictated by Poisson statistics. In this paper, a microfluidic device was developed to improve the single-cell encapsulation rate by integrating droplet generation with fluorescence-activated droplet sorting. After cells were loaded into aqueous droplets by hydrodynamic focusing, an on-flight fluorescence-activated sorting process was conducted to isolate droplets containing one cell. Encapsulation of fluorescent polystyrene beads was investigated to evaluate the developed method. A single-bead encapsulation rate of more than 98 % was achieved under the optimized conditions. Application to encapsulate single HeLa cells was further demonstrated with a single-cell encapsulation rate of 94.1 %, which is about 200 % higher than those obtained by random compartmentalization. We expect this new method to provide a useful platform for encapsulating single cells, facilitating the development of high-throughput cell-based assays.

Genomic DNA damage was generally identified with a "comet assay" but limited by low throughput and poor reproducibility. Here we demonstrated an ultrahigh-throughput approach with a microfluidic chip to simultaneously interrogate DNA damage conditions of up to 10,000 individual cells (approximately 100-fold in throughput over the conventional method) with better reproducibility. For experiment, agarose was chosen as the chip fabrication material, which would further act as an electrophoretic sieving matrix for DNA fragments separation. Cancer cells (HeLa or HepG2) were lined up in parallel microchannels by capillary effect to form a dense array of single cells. After treatment with different doses of hydrogen peroxide, individual cells were then lysed for subsequent single-cell gel electrophoresis in the direction vertical to microchannel and fluorescence detection. Through morphological analysis and fluorescent measurement of comet-shaped DNA, the damage conditions of individual cells could be quantified. DNA repair capacity was further evaluated to validate the reliability of this method. It indicated that the agarose-based microfluidic comet array electrophoresis was simple, highly reproducible, and of high throughput, providing a new method for highly efficient single-cell genomic analysis.

Crowding stress has been reported to play an important role in affecting physiological behaviour. To study this process, a reliable analytical method under confined space is essential. In this work, we demonstrated a microfluidic approach for investigating physiological responses of C. elegans to confined spaces. The PDMS microfluidic chip consisting of arrays of micro-columns enabled us to mimic different crowding conditions by changing the intervals among micro-columns. C. elegans were transferred into this micro-column array and the subcellular distribution of DAF-16, which is a well-known transcription factor regulating different stress responses, was monitored for analysing the physiological responses to the confined spaces. We found that the worms exhibited a gradual increase in DAF-16 nuclear localization in the micro-column array with intervals from 200 μm to 40 μm. Moreover, the results showed that the absence of food and crowding stress could cooperate to promote increased DAF-16 nuclear localization. Finally, loss-of-function mutations in mec-4 and mec-10, which are amiloride-sensitive Na(+) channel genes expressed in all six gentle touch neurons, accelerated the velocity of DAF-16 nuclear localization, induced by confined space, revealing that mec-4/mec-10 were not required for this stress response. Thus, this device will provide a versatile, reliable and controllable platform for crowding stress studies.

Intercellular Ca(2+) waves are propagation of Ca(2+) transients among cells that could be initiated by chemical stimulation. Current methods for analyzing intercellular Ca(2+) waves are difficult to realize localized chemical stimulations upon the target cell without interfering with adjacent contacting cells. In this paper, a simple and flexible microfluidic method was developed for investigating the intercellular communication of Ca(2+) signals. A cross-patterned microfluidic chip was designed and fabricated with polydimethylsiloxane as the structural material. Localized chemical stimulation was achieved by a new strategy based on hydrodynamic gating technique. Clusters of target cells were seeded at the location within 300 μm downstream of the intersection of the cross-shaped microchannel. Confined lateral molecular diffusion largely minimized the interference from diffusion-induced stimulation of adjacent cells. Localized stimulation of the target cell with adenosine 5'-triphosphate successfully induced the propagation of intercellular Ca(2+) waves among a population of adjacent contacting cells. Further inhibition studies verified that the propagation of calcium signals among NIH-3 T3 cells was dependent on direct cytosolic transfer via gap junctions. The developed microfluidic method provides a versatile platform for investigating the dynamics of intercellular communications.

Tracking the folding kinetics of macromolecules under molecular crowding conditions represents a tremendous challenge due to the high viscosity of the solution. In this paper, we report a unique T-type microfluidic mixer with seven consecutive ω-shaped baffles for fast mixing of high-viscosity fluids. Numerical simulations and experimental characterizations proved that the micromixer could achieve a mixing time of 579.4 μs for solutions with viscosities about 33.6 times that of pure water. Over a 1000-fold improvement in mixing dead time was accomplished in comparison to those reported previously. We further used this highly efficient micromixer to track the early folding kinetics of human telomere G-quadruplex under molecular crowding conditions. Results indicated an exponential process in the initial folding phase of G-quadruplex, and the G-quadruplex formed a more compact structure under higher degrees of molecular crowding conditions.

The propagation of intercellular calcium signals provides a mechanism to coordinate cell population activity, which is essential for regulating cell behavior and organ development. However, existing analytical methods are difficult to realize localized chemical stimulation of a single cell among a population of cells that are in close contact with one another for studying the propagation of calcium wave. In this work, a microfluidic method is presented for the analysis of contact-dependent propagation of intercellular calcium wave induced by extracellular ATP using multiple laminar flows. Adjacent cells were seeded ∼300 μm downstream the intersection of a Y-shaped microchannel with negative pressure pulses. Consequently, the lateral diffusion distance of the chemical at cell locations was limited to ∼26 μm with a total flow rate of 20 μL min(-1), which prevented the interference of diffusion-induced cellular responses. Localized stimulation of the target cell with ATP induced the propagation of intercellular calcium wave among the cell population. In addition, studies on the spread of intercellular calcium wave under octanol inhibition allowed us to characterize the gap junction mediated cell-cell communication. Thus, this novel device will provide a versatile platform for intercellular signal transduction studies and high throughput drug screening., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Analysis of fast biochemical reactions requires rapid mixing of solutions. Micromixers can achieve uniform mixing of solutions in a short time and have been recognized as an attractive tool to analyze fast reactions. However, it is still a challenge to design mixers with simple structure and short dead time. Here, a zigzag turbulent micromixer was developed with a rapid mixing time of 16 μs at sample consumption of 10 μL/s. Numerical simulations and confocal imaging validated this result. Application of the chemiluminescence (CL) reaction demonstrated the use of this mixer in analyzing the kinetic process of the CL reaction. In comparison to the turbulent micromixers reported previously, this zigzag mixer has advantages of short dead time, simple structure and low sample consumption. We anticipate the developed mixer to be a useful tool in studying biochemical kinetics or be integrated to Lab-on-a-chip device as a pretreatment functional unit., (Copyright © 2011 Elsevier B.V. All rights reserved.)

To investigate rapid cell signaling, analytical methods are required that can generate repeatable chemical signals for stimulating live cells with high temporal resolution. Here, we present a chemical signal generator based on hydrodynamic gating, permitting flexible stimulation of single adherent cells with a temporal resolution of 20 ms. Studies of adenosine triphosphate (ATP)-induced calcium signaling in HeLa cells were demonstrated using this developed method. Consecutive treatment of the cells with ATP pulses of 20 or 1 s led to an increase of latency, which might be another indicator of receptor desensitization in addition to the decrease in the amplitude of calcium spikes. With increasing duration of ATP pulses from milliseconds to a few seconds, the cellular responses transitioned from single calcium spikes to calcium oscillation gradually. We expected this method to open up a new avenue for potential investigation of rapid cell signaling.

The use of cell-based biosensors is usually limited by agonist-induced desensitization of cell-surface receptors. In this work, a microfluidic cell-based biosensor (μCBB) was developed for the detection of ATP in liquid environments. It consists of a millisecond chemical pulse generator for sample introduction in a pulsatile manner and a single NIH-3T3 cell expressing endogenous P2Y receptors as the sensing element. ATP solutions were used to simulate input signals for investigating the μCBB. By controlling negative pressures on two outlets of a cross-shaped microfluidic chip, pulses of ATP solutions were generated based on hydrodynamic gated injection. With ATP pulses of 100 ms every 50s, the amplitude of the resulting calcium spikes maintained at a similar level, suggesting that the receptor desensitization was minimized. Consequently, the developed μCBB could be used for detecting pulsatile samples with extended use times. The sensitivity of the μCBB for detecting ATP was further determined and the cellular responses to millisecond ATP pulses were investigated in comparison to long-term stimulations., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Membrane proteins play essential roles in regulating various fundamental cellular functions. To investigate membrane proteins, extraction and purification are usually prerequisite steps. Here, we demonstrated a microfluidic aqueous PEG/detergent two-phase system for the purification of membrane proteins from crude cell extract, which replaced the conventional discontinuous agitation method with continuous extraction in laminar flows, resulting in significantly increased extraction speed and efficiency. To evaluate this system, different separation and detection methods were used to identify the purified proteins, such as capillary electrophoresis, SDS-PAGE and nano-HPLC-MS/MS. Swiss-Prot database with Mascot search engine was used to search for membrane proteins from random selected bands of SDS-PAGE. Results indicated that efficient purification of membrane proteins can be achieved within 5-7s and approximately 90% of the purified proteins were membrane proteins (the highest extraction efficiency reported up to date), including membrane-associated proteins and integral membrane proteins with multiple transmembrane domains. Compared to conventional approaches, this new method had advantages of greater specific surface area, minimal emulsification, reduced sample consumption and analysis time. We expect the developed method to be potentially useful in membrane protein purifications, facilitating the investigation of membrane proteomics., (Copyright © 2010 Elsevier B.V. All rights reserved.)

We present a microfluidic sample introduction approach based on a novel flow control mechanism, hydrodynamic gated injection. It has the advantages of easy and flexible flow control, similar to its analog, electrokinetic gated injection, but exhibits the unique properties of strong driving force and good biocompatibility, ideal for applications involving live biological samples. Theories for hydrodynamic gating were proposed and validated by both numerical simulations and flow visualization experiments. An investigation with fluorescein revealed that pico-liter samples can be injected with high repeatability (RSD <1.9%). Selective injections of GFP-transfected nematode eggs were demonstrated with a survival rate of >95%. We expect the developed method to be potentially useful for microfluidic cell and organism analysis, either as a sample introduction module or a stand-alone analyzer.

Systems biology advocates the understanding of biology at the systems-level, which requires massive information of correlations among individual components in complex biological systems. Such comprehensive investigation entails the use of high-throughput analytical tools. Microfluidic technology holds high promise to facilitate the progress of biology by enabling miniaturization and upgrading of current biological research tools due to its advantages such as low sample consumption, reduced analysis time, high-throughput and compatible sizes with most biological samples. In this article, we documented the recent applications of microfluidic chips in biological researches at the molecular level, cellular level and organism level, serving the purpose for systems-level understanding of biology.

Analysis of complex biological samples requires the use of high-throughput analytical tools. In this work, a microfluidic two-dimensional electrophoresis system was developed with mercury-lamp-induced fluorescence detection. Mixtures of 20 standard amino acids were used to evaluate the separation performance of the system. After fluorescent labeling with fluorescein isothiocyanate, mixtures of amino acids were separated by micellar electrokinetic chromatography in the first dimension and by capillary zone electrophoresis in the second. A double electrokinetic valve system was employed for the sample injection and the switching between separation channels. Under the optimized conditions, 20 standard amino acids were effectively separated within 20 min with high resolution and repeatability. Quantitative analysis revealed linear dynamic ranges of over three orders of magnitudes with detection limits at micromolar range. To further evaluate the reliability of the system, quantitative analysis of a commercial nutrition supplement liquid was successfully demonstrated.

Micro total analysis systems (microTAS) also referred to as "lab-on-a-chip" is one of the fastest progressing fields in biological and chemical analyses. In recent years, microTAS for single cell analysis has drawn the attention of researchers due to its significant advantages over traditional methods for single cell manipulation, fast cell sorting and integration of multiple functions. As the preliminary step for studying cells on chips, cell sorting using microfluidics have been investigated by researchers intensively. This article reviews the most recent advances on microfluidics-based cell sorting techniques including cell sorting principle, strategy, mechanism and procedure with emphases on the sorting mechanism and procedure. Furthermore, evaluation criteria for successful cell sorter are also discussed and future research directions are given.

In this article, a microfluidic platform integrating capillary electrophoresis and bioluminescence (BL) detection that was fabricated in poly(dimethylsiloxane) (PDMS) with lab-on-a-chip technology was demonstrated for cellular metabolic analyses. A microchannels network, "cross combining with Y", was designed to perform on-chip sample preparation, separation, and BL detection of ATP and ATP-conjugated metabolites, using firefly luciferin-luciferase BL system. A dynamic modification of the channel wall of PDMS proved to be crucial to reverse the direction of electroosmotic flow (EOF), which was uniquely achieved by a prewash cycle with a cationic surfactant didodecyldimethylammonium bromide. The influences of surfactant on the EOF and BL reaction were also investigated. Quantitative analyses revealed a dynamic linear range over 2 orders of magnitude for ATP, with a detection limit down to submicromolar (midattomole). The method was validated by measuring cellular ATP of E. coli. with direct on-chip cell lysis. Further work was emphasized on ATP-conjugated metabolite analysis, using galactose as an example. Assays of galactose in human urine samples confirmed the reliability of the protocol, which revealed good prospect of this platform for ATP-conjugated submetabolomic profiling.

Cell analysis at the single-cell level is of great importance to investigate the inherent heterogeneity of cell populations and to understand the morphology, composition, and function of individual cells. With the continuous innovation of analytical techniques and methods, single-cell analysis on microfluidic chip systems has been extensively applied for its precise single-cell manipulation and sensitive signal response integrated with various detection techniques, such as optical, electrical, and mass spectrometric analyses. In this review, we focus on the specific optical events in single-cell analysis on a microfluidic chip system. First, the four most commonly applied optical technologies, i.e., fluorescence, surface-enhanced Raman spectroscopy, surface plasmon resonance, and interferometry, are briefly introduced. Then, we focus on the recent applications of the abovementioned optical technologies integrated with a microfluidic chip system for single-cell analysis. Finally, future directions of optical technologies for single-cell analysis on microfluidic chip systems are predicted. [ABSTRACT FROM AUTHOR]

Kinetic measurement of biomacromolecular interaction plays a significant role in revealing the underlying mechanisms of cellular activities. Due to the small diffusion coefficient of biomacromolecules, it is difficult to resolve the rapid kinetic process with traditional analytical methods such as stopped-flow or laminar mixers. Here, we demonstrated a unique continuous-flow laminar mixer based on microfluidic dual-hydrodynamic focusing to characterize the kinetics of DNA–protein interactions. The time window of this mixer for kinetics observation could cover from sub-milliseconds to seconds, which made it possible to capture the folding process with a wide dynamic range. Moreover, the sample consumption was remarkably reduced to ＜0.55 μL min−1, over 1000-fold saving in comparison to those reported previously. We further interrogated the interaction kinetics of G-quadruplex and the single-stranded DNA binding protein, indicating that this novel micromixer would be a useful approach for analyzing the interaction kinetics of biomacromolecules. [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]

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]