Since microdroplets are able to be generated rapidly in large amount and each droplet can be well controlled as an independent micro-cultivator, droplet microfluidic technology can be potentially used in the culture of microorganisms, and provide the microbial culture with high throughput manner. But its application mostly stays in the laboratory-level building and using for scientific research, and the wide use of droplet microfluidics in microbial technology has been limited by the key problems that the operation for microdroplets needs high technical requirements with wide affecting factors and the difficulties in integration of automatic microdroplet instrumentation. In this study, by realizing and integrating the complicated operations of droplet generation, cultivation, detection, splitting, fusion and sorting, we design a miniaturized, fully automated and high-throughput microbial microdroplet culture system (MMC). The MMC can be widely used in microbial growth curve test, laboratory adaptive evolution, single factor and multi-level analysis of microbial culture, metabolite detection and so on, and provide a powerful instrument platform for customized microbial evolution and screening aiming at efficient strain engineering.
Industrial Development ; Microfluidics

Paper-based analytical devices are fluidic chips fabricated with extremely inexpensive materials, namely paper, thereby allowing their use as a zero-cost analytical device in third-world countries that lack access to expensive diagnostic infrastructures. The aim of this review is to discuss: (1) microfluidic paper-based analytical devices (microPADs) for quantitative analysis, (2) fabrication of two- or three-dimensional microPADs, (3) analytical methods of microPADs, and (4) our opinions regarding the future applications of microPADs for quantitative urinalysis.
Developing Countries ; Methods ; Microfluidics ; Urinalysis*

Microalgae are a group of photosynthetic microorganisms, which have the general characteristics of plants such as photosynthesis, and some species have the ability of movement which resembles animals. Recently, it was reported that microalgae cells can be engineered to precisely deliver medicine-particles and other goods in microfluidic chips. These studies showed great application potential in biomedical treatment and pharmacodynamic analysis, which have become one of the current research hotspots. However, these developments have been rarely reviewed. Here, we summarized the advances in manageable movement exemplified by a model microalgae Chlamydomonas reinhardtii based on its characteristics of chemotaxis, phototaxis, and magnetotaxis. The bottlenecks and prospects in the application of microalgae-based tactic movement were also discussed. This review might be useful for rational design and modification of microalgal manageable movement to achieve targeted transport in medical and other fields.
Chlamydomonas reinhardtii ; Microalgae ; Microfluidics ; Photosynthesis

We developed a high-efficiency microfluidic chip for extracting exosomes from human plasma. We collected peripheral blood from normal human, designed and fabricated a microfluidic chip based on nanoporous membrane and agarose gel electrophoresis to isolate exosomes. The extracted exosomes were characterized by transmission electron microscopy, nanosight and Western blotting, the morphology, concentration and particle size of exosomes were identified and analyzed. Meanwhile, we used ultracentrifugation and microfluidic chip to isolate exosomes separately. The particle size and concentration of the exosomes extracted by two methods were compared and analyzed, and their respective extraction efficiency was discussed. Finally, the expression level of miRNA-21 in exosomes was analyzed by RT-PCR. The microfluidic chip isolated (in 1 hour) high-purity exosomes with size ranging from 30-200 nm directly from human plasma, allowing downstream exosomal miRNA analysis. By comparing with ultracentrifugation, the isolation yield of microfluidic chip was 3.80 times higher than ultracentrifugation when the volume of plasma sample less than 100 μL. The optimized parameters for exosome isolation by gel electrophoresis microfluidic chip were: voltage: 100 V; concentration of agarose gel: 1.0%; flow rate of injection pump: 0.1 mL/h. The gel electrophoresis microfluidic chips could rapidly and efficiently isolate the exosomes, showing great potential in the research of exosomes and cancer biomarkers.
Exosomes ; Humans ; MicroRNAs/genetics* ; Microfluidics ; Plasma ; Ultracentrifugation

Recently, the droplet microfluidic system attracts interests due to its high throughput and low cost to detect and screen. The picoliter micro-droplets from droplet microfluidics are uniform with respect to the size and shape, and could be used as monodispensed micro-reactors for encapsulation and detection of single cell or its metabolites. Therefore, it is indispensable to characterize micro-droplet and its application from droplet microfluidic system. We first constructed the custom-designed droplet microfluidic system for generating micro-droplets, and then used the micro-droplets to encapsulate important amino acids such as glutamic acid, phenylalanine, tryptophan or tyrosine to test the droplets' properties, including the stability, diffusivity and bio-compatibility for investigating its application for amino acid detection and sorting. The custom-designed droplet microfluidic system could generate the uniformed micro-droplets with a controllable size between 20 to 50 microm. The micro-droplets could be stable for more than 20 h without cross-contamination or fusion each other. The throughput of detection and sorting of the system is about 600 micro-droplets per minute. This study provides a high-throughput platform for the analysis and screening of amino acid-producing microorganisms.
Amino Acids ; isolation & purification ; Microfluidic Analytical Techniques ; Microfluidics ; instrumentation

Pichia pastoris is one of the most convenient and widely used heterologous protein expression systems. To further improve its ability to express heterologous proteins, we developed a high-throughput P. pastoris screening method based on droplet microfluidic and demonstrated the method by screening and obtaining mutants with enhanced xylanase expression and secretion abilities. We used PCR (Polymerase Chain Reaction) amplification to obtain a fusion fragment of xylanase xyn5 gene and green fluorescent protein gfp gene, and cloned this fragment into pPIC9K, the expression vector of Pichia pastoris, to construct the plasmid pPIC9K-xyn5-gfp that recombined the DNA fragments of xylanase and green fluorescent protein. After this plasmid entered P. pastoris GS115 by electroporation, the P. pastoris SG strain that could express xylanase and green fluorescent protein was obtained. The above-said strains were then mutagenized by atmospheric room temperature plasma and subsequently encapsulated to form single-cell droplets. After 24-hour cultivation of the droplets, microfluidic screening was carried out to obtain the mutant strain with high xylanase expression for further construction and screening of the next mutagenesis library. After five rounds of droplet microfluidic screening, a highly productive strain P. pastoris SG-m5 was obtained. The activity of the expressed xylanase was 149.17 U/mg, 300% higher than that of those expressed by the original strain SG. This strain's ability to secrete heterologous protein was 160% higher than that of the original strain. With a screening throughput of 100 000 strains per hour, the high-throughput P. pastoris screening system based on single-cell droplet microfluidic developed by the present study screens a library with million strains in only 10 hours and consumes only 100 μL of fluorescent reagent, thus reducing the reagent cost by millions of times compared with the traditional microplate screening and more importantly, providing a novel method to obtain P. pastoris with high abilities to express and secret heterologous proteins by efficient and low-cost screening.
Microfluidics ; Mutagenesis ; Pichia ; Plasmids ; Polymerase Chain Reaction ; Recombinant Proteins

BACKGROUND: Whole blood viscosity (WBV) refers to the internal resistance that occurs when blood flows through blood vessels. WBV is known to be related to many diseases including cardiovascular and neurovascular diseases. We have investigated the analytical performance and established reference intervals for a newly developed microfluidic viscometer, Viscore-300 (NanoBiz, Korea), used for the measurement of WBV. METHODS: We performed a precision test of 240 measurements over 20 days using three control materials. For evaluation of repeatability, a total of 60 WBV measurements were made in 3 whole blood samples 20 times a day. A total of 100 whole blood samples were used to evaluate the accuracy of the Viscore-300 in comparison to a rotating viscometer, DV3T (Brookfield, USA), in accordance with the the Clinical and Laboratory Standards Institute's guidelines. To establish the reference intervals, 122 healthy individuals were enrolled in this study. RESULTS: The precision and repeatability results showed that the CV was less than 5% for three samples and two shear rates. In the accuracy test, the mean differences between two viscometers were 0.09 cP (0.9%) and −0.07 cP (−1.4%) at shear rates of 10 s−1 and 300 s−1, respectively. The reference intervals of WBV for men were 6.88–13.52 cP at 10 s−1 and 4.32–6.43 cP at 300 s−1; those of women were 5.74–13.29 cP at 10 s−1 and 3.60–6.12 cP at 300 s−1. CONCLUSIONS: Viscore-300 showed excellent precision and accuracy and it might be a good instrument for reporting WBV quickly and accurately.
Blood Vessels ; Blood Viscosity ; Female ; Humans ; Male ; Microfluidics

Microfluidics technology may be an effective method to solve some problems in cryopreservation. This review presents the research progress of microfluidics technology in the field of cell membrane transport properties, cryoprotectant addition and washout and the vitrification for cryopreservation of biological materials. Existing problems of microfluidics technology in the application of cryopreservation are summarized and future research directions are indicated as well.
Cell Membrane ; Cryopreservation ; Cryoprotective Agents ; Membrane Transport Proteins ; Microfluidics

In recent 10 years, microfluidic technology has developed rapidly. Hence the speed of analysis can be upgraded, the performance be improved and the consumption of sample and reagent be reduced. In this review, we introduce the design, fabrication and application of microfluidic immunoassay chip.
Humans ; Immunoassay ; methods ; Microfluidic Analytical Techniques ; trends ; Microfluidics ; trends

In vitro compartmentalization (IVC) links genotype and phenotype by compartmentalizing individual genes (including expression system) or cells into a micro-droplet reaction region. Combined with fluorescence-activated cell sorting (FACS), it can detect and separate single droplets in ultra-high throughput. IVC-FACS screening method has been widely used in protein engineering, enzyme directed evolution, etc. However, it is difficult to control the homogeneity of droplet size by mechanical dispersion method in previous studies, which seriously affects the quantitative detection of droplets and reduces the efficiency and accuracy of this screening method. With the rapid development of microfluidic chip manufacturing technology, the microfluidic chip-based methods for droplet generation are becoming more efficient and controllable. In this study, firstly, the water-in-oil (W/O) single-layer droplet generation chip was used to prepare single-layer monodisperse W1/O droplets at a high generation frequency, and then the W1/O droplets were reinjected into water-in-oil-in-water (W/O/W) double-layer droplet generation chip to prepare uniform W1/O/W2 double-layer emulsion droplets. By optimizing the flow rate and ratio of the oil and water phases, a single-layer micro-droplet can be generated with a diameter range from 15.4 to 23.2 μm and remain stable for several days under normal incubation. Then the single-layer droplets were reinjected into the double emulsion generation chip. By adjusting the flow rate of drop phase, oil phase and water phase, the double-layer emulsion droplets with a diameter range from 30 to 100 μm at a rate of 1 000 droplets/s could be obtained. Escherichia coli embedded in the double-layer emulsion droplets could be cultured and induced for protein expression. This study lays a foundation for the establishment of a high-throughput screening method based on the droplet and flow cytometry.
Emulsions ; Flow Cytometry ; High-Throughput Screening Assays ; Microfluidics ; methods