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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.

The cellular response to the complex extracellular microenvironment is highly dynamic in time and type of extracellular matrix. Accurately reconstructing this process and analyzing the changes in receptor conformation on the cell membrane surface and intracellular or intercellular signaling has been a major challenge in analytical chemistry and biophysical methodology. In this paper, a time-coded multi-concentration microfluidic chemical waveform generator was developed for the dynamic signaling probing with single-cell array of high temporal resolution, high throughput, and multi-concentration combination stimulation. Based on innovative microchannel structure, sophisticated external control methods and multiplexing technology, the system not only allowed for temporally sequential permutations of the four concentrations of stimuli (time code), but also generated pulsed and continuous waveforms at different frequencies in a highly controllable manner. Furthermore, the single-cell trap array was set up to efficiently capture cells in suspension, dramatically increasing throughput and reducing experiment preparation time. The maximum frequency of the platform was 1 Hz, and one cell could be stimulated at multiple frequencies. To show the ability of the system to investigate rapid biochemical events in high throughput, pulse stimulation and continuous stimulation of different frequencies and different time codes, combined with four concentrations of histamine (HA), were generated for probing G protein-coupled receptor (GPCR) signaling in HeLa cells. Then, statistical analysis was performed for the mean peak height and mean peak area of the cellular response. We believe that the time-coded multi-concentration microfluidic chemical waveform generator will provide a novel strategy for analytical chemistry, biophysics, cell signaling, and individualized medicine applications.

Point-of-care testing (POCT) has shown great advantages for public health monitoring in resource-limited settings. However, developing of POCT tools with automated and accurate quantitative dispensing of multiple reagents and samples is challenging. Here, we demonstrate a novel multi-reagents dispensing centrifugal microfluidics (MDCM) that allows rapid and automated dispensing of multiple reagents and samples with high throughput and accuracy. The MDCM was designed with multiple aliquoting units with the hydrophobic valve at different radial positions. All reagents and samples were loaded simultaneously, dispensed in parallel by centrifugation at low speed, and then introduced into the reaction chamber sequentially by centrifugation at high speed. Two MDCM chips are demonstrated, including a uniform concentration generator and a gradient concentration generator. The concentration coefficient of variation (CV) among the independent reaction chambers was lower than 0.56%, and the theoretical quantitative concentration gradient was strongly correlated with the actual concentration gradient (R

Biomolecular phase separation is currently emerging in both the medical and life science fields. Meanwhile, the application of liquid-liquid phase separation has been extended to many fields including drug discovery, fibrous material fabrication, 3D printing, and polymer design. Although more than 8600 proteins and other synthetic macromolecules are capable of phase separation as recently reported, there is still a lack of a high-throughput approach to quantitatively characterize its phase behaviors. To meet this requirement, here, we proposed fast and high-resolution acquisition of biomolecular phase diagrams using microfluidic chips. Using this platform, we demonstrated the phase behavior of polyU/RRASLRRASLRRASL in a quantitative manner. Up to 1750 concentration conditions can be generated in 140 min. The detection limitation of our device to capture the saturation concentration for phase separation is about 5 times lower than that of the traditional turbidity method. Thus, our results provide a basis for the rapid acquisition of phase diagrams with high-throughput and pave the way for its wide application.

Cell signaling greatly affected by complicated and temporally dynamic extracellular microenvironments controls most of the physiological functions in vivo. To reconstruct or simulate such microenvironments in vitro represents a fundamental approach for revealing the underlying mechanisms of those sophisticated processes. Recent advances in microfluidics have added a new dimension to cell signaling analysis, for example, concentration gradient generators (amplitude aspect) or hydrodynamic gating strategy (frequency aspect), but it is still challengeable to capture single-cell dynamic signaling in response to a mimicked extracellular microenvironment with varied stimuli waveforms of different amplitude and frequency in a high-throughput manner. In this article, we proposed a novel microfluidic strategy coupling multichannel synchronous hydrodynamic gating with microfluidic concentration gradient generators (μMHG-CGG) to probe dynamic signaling of single cells with high throughput. The μMHG-CGG allows rapid delivery of dynamic chemical signals in both high frequency (as high as 670 mHz) and multiple amplitude domains at the same time and simultaneously high-throughput probing cell dynamics at single-cell resolution in real time. By applying the proposed system, the mechanisms for encoding/decoding systems (termed "frequency coding" or "amplitude coding") via GPCRs-mediated signaling pathways responding to histamine (HA) and adenosine triphosphate (ATP) in single HeLa cells were investigated. The optimal drug concentrations of single cells responses to HA and ATP individually or in combination were also successfully discussed, allowing us to obtain both single-cell heterogeneity and statistics from the cell population.

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.

Single cell analysis has aroused great interest with its remarkable ability to investigate cell-to-cell heterogeneity in large populations. However, probing protein information at single cells resolution including quantity, interactions, and dynamics have been great challenges as a result of the small size of cells, the complexity and a large concentration range of the protein and the lack of genome-wide amplification method. Fortunately, microfluidics capable of high throughput, high reproducibility, large parallelization, easy operability and low-cost, has been emerging as a powerful platform for the analysis of single cell proteins. In this review, we focus the recent advances of microfluidics in single cell resolution protein analysis, particularly covering the following aspects: (1) microfluidic electrophoresis (capillary electrophoresis and gel electrophoresis); (2) microfluidic cytometry (microfluidic flow cytometry, droplet based cytometry and image cytometry); (3) microfluidic array (micro wells, micro chambers, valve-based microfluidics and static droplet array microfluidics); (4) microfluidic probe and (5) microfluidics based mass spectrometry.

Spatiotemporal manipulation of extracellular chemical environments with simultaneous monitoring of cellular responses plays an essential role in exploring fundamental biological processes and expands our understanding of underlying mechanisms. Despite the rapid progress and promising successes in manipulation strategies, many challenges remain due to the small size of cells and the rapid diffusion of chemical molecules. Fortunately, emerging microfluidic technology has become a powerful approach for precisely controlling the extracellular chemical microenvironment, which benefits from its integration capacity, automation, and high-throughput capability, as well as its high resolution down to submicron. Here, we summarize recent advances in microfluidics manipulation of the extracellular chemical microenvironment, including the following aspects: i) Spatial manipulation of chemical microenvironments realized by convection flow-, diffusion-, and droplet-based microfluidics, and surface chemical modification; ii) Temporal manipulation of chemical microenvironments enabled by flow switching/shifting, moving/flowing cells across laminar flows, integrated microvalves/pumps, and droplet manipulation; iii) Spatiotemporal manipulation of chemical microenvironments implemented by a coupling strategy and open-space microfluidics; and iv) High-throughput manipulation of chemical microenvironments. Finally, we briefly present typical applications of the above-mentioned technical advances in cell-based analyses including cell migration, cell signaling, cell differentiation, multicellular analysis, and drug screening. We further discuss the future improvement of microfluidics manipulation of extracellular chemical microenvironments to fulfill the needs of biological and biomedical research and applications.

Saccharomyces yeasts harbor many different viruses and parasitic genetic elements, including double-stranded RNA (dsRNA) viruses from the family Totiviridae. The identification of novel dsRNA viruses in yeasts has been constrained by the lack of effective protocols for the unbiased preparation and sequencing of dsRNAs. We have developed a next-generation sequencing method that enables the amplification and sequencing of yeast dsRNAs. Using this method, we have performed a metagenomic screen of more than 600 strains of Saccharomyces cerevisiae for the presence of novel dsRNA viruses. Surprisingly, we have identified several novel bipartite dsRNA viruses from the family Partitiviridae within different strains of S. cerevisiae. Partitiviruses have never been described within Saccharomyces yeasts or within the wider Saccharomycotina taxonomic subdivision of the Ascomycota phylum. We confirmed the presence of these partitivirus dsRNAs using reverse transcriptase-PCR and have been successful in purifying viral particles from infected yeasts. After deep sequencing of purified dsRNAs, we have identified three species of virus that have weak homology (20-35% amino acid identity) to viruses of the genus Cryspovirus, which replicate within the protozoan pathogen Cryptosporidium parvum. Partitiviruses can modulate the virulence and fecundity of plant and human pathogens, respectively. The discovery of partitiviruses in S. cerevisiae will enable the study of how partitiviruses interact with the host cell environment and alter cellular physiology.

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.

The integration of gel-based loop-mediated isothermal amplification (gLAMP) and finger-actuated microfluidic chip (μFAchip) was developed for the simultaneous detection of various different types of bacterial pathogens. The developed μFAchip consisted of three PDMS layers attached together by two adhesive tapes. Multiple chambers in the top PDMS layer were used for sample preparation, and the corresponding chambers in the bottom PDMS layer was used for long-term storage of LAMP reagents without DNA templates. The thin PDMS layer in the middle contained cross-shaped cuts as finger-actuated valves for fluid control. To reduce operation steps on the chip, such as pipetting and manipulation of samples, Whatman CloneSaver card was pre-embedded in the top chambers for on-chip DNA extraction and purification. Upon a simple press on the top layer, the finger-actuated valve was opened up, allowing DNA samples on the top layer flow into the bottom reaction chambers for gLAMP reaction. For POCT applications, on-chip LAMP reaction and imaging were conducted on a miniaturized peltier heater and a portable fluorescence imaging system respectively. Under the optimized condition, multiple pathogens were detected simultaneously with high selectivity and sensitivity (as low as 1.6 cells). The developed μFAchip provided a rapid and easy-to-operate platform for gLAMP-based pathogen detection, with the potential for in-field detection, especially in areas with limited resources.