WormSpace µ-TAS enabling automated on-chip multi-strain culturing and multi-function imaging of Caenorhabditis elegans at the single-worm level on the China Space Station.

As a model organism for space biology experiments, Caenorhabditis elegans (C. elegans) has low demand for life support and strong resistance to unfavorable environments, making experimentation with C. elegans relatively easy and cost-effective. Previously, C. elegans has been flown in several spaceflight investigations, but there is still an urgent need for analytical platforms enabling on-orbit automated monitoring of multiple phenotypes of worms, such as growth and development, movement, changes of biomarkers, etc. To solve this problem, we presented a fully integrated microfluidic system (WormSpace µ-TAS) with an arrayed microfluidic chip (WormChip-4.8.1) and a replaceable microfluidic module (WormChip cartridge), which was compatible with the experimental facility on the China Space Station (CSS). By adopting technologies of programmed fluid control based on liquid medium CeMM as well as multi-function imaging with a camera mounted on a three-dimensional (3D) transportation stage, automated and long-term experimentation can be performed for on-chip multi-strain culturing and bright-field and fluorescence imaging of C. elegans at the single-worm level. The presented WormSpace µ-TAS enabled its successful application on the CSS, achieving flight launch of the sample unit (WormChip cartridge) at low temperature (controlled by a passive thermal case at 12 °C), automated 30-day cultivation of 4 strains of C. elegans, on-orbit monitoring of multiple phenotypes (growth and development, movement, and changes of fluorescent protein expression) at the single worm-level, on-chip fixation of animals at the end of the experiment and returning the fixed samples to earth. In summary, this study presented a verified microfluidic system and experimental protocols for automated on-chip multi-strain culturing and multi-function imaging of C. elegans at the single-worm level on the CSS. The WormSpace µ-TAS will provide a novel experimental platform for the study of biological effects of space radiation and microgravity, and for the development of protective drugs.

Automated and parallel microfluidic DNA extraction with integrated pneumatic microvalves/pumps and reusable open-channel columns.

A novel microfluidic DNA extraction protocol based on integrated diaphragm microvalves/pumps and silica-deposited open-channel columns was developed specifically for automated and parallel DNA solid-phase extraction (SPE). The method uses microfluidic chips with a sandwiched structure containing three layers, which are the upper fluidic layer with surface-deposited silica on glass open channels as the extraction phase, the lower actuation layer with valve actuation channels on a glass wafer, and the middle poly(dimethylsiloxane) (PDMS) membrane for reversible bonding of the two glass substrates. These two glass substrates can be reused after thoroughly cleaning and the PDMS membrane can be replaced conveniently, which could effectively decrease the time and cost of chip manufacturing. The normally closed microvalves/pumps were used to automatically control all processes of the on-chip DNA SPE without cross-contamination and leakage, enabling the processing of multiple samples in parallel without changing the microvalve control module. Using the microchip device with integrated microvalves/pumps, automated, programmable, and simultaneous λ-DNA extractions from different samples could be attained, even from complex solutions such as human blood, and the silica-deposited open-channel columns could be reused stably and reliably. Results have demonstrated that most of the eluted λ-DNA was recovered in the second 2 µL of elution buffer with high-purity suitable for successful polymerase chain reaction amplification, making it possible for further integration into microfluidic devices for fully functional and high-throughput genetic analysis.

A real-time isothermal amplification based portable microfluidic system for simple and reliable detection of Vibrio splendidus.

The spread of infectious diseases among aquaculture species has a serious impact on the aquaculture industry. Simple, specific and low-cost detection methods are urgently needed for early diagnosis and timely treatment, particularly for on-site identifying and tracking of pathogens. Vibrio splendidus (V. splendidus) is regarded as one of the main pathogenic bacteria causing skin ulcerative syndrome in cultured sea cucumbers, leading to massive mortality and severe economic losses. We herein present a microfluidic-based real-time fluorogenic loop-mediated isothermal amplification (LAMP) system for simple and reliable detection of V. splendidus. A LAMP primer set with six primers (arsB1) specifically targeting the arsB gene of V. splendidus was successfully designed and tested on the portable microfluidic system for the first time. Only a single step of sample loading using a pipette is required to fill an array of reaction wells (with 10 or 18 wells) in a disposable chip for multiplex detection. A dedicated plastic shell is then utilized to tightly seal the openings of the chip by buckling to prevent contamination and evaporation. Up to four chips (one sample per chip) can be held in the stand-alone and inexpensive microdevice simultaneously, enabling on-demand detection of multiple samples in a single run. Reproducible (relatively low intra- and inter-chip variability) and sensitive (as few as ∼20 CFU, Colony-Forming Units, per reaction well) on-chip arsB1-LAMP assay was demonstrated by using diluted lysate of V. splendidus. A linear standard curve (R2 > 0.98) was attained over the template concentration range of 5 × 103 to 5 × 106 CFU mL-1. V. splendidus can be detected in samples containing different bacteria, indicating the feasibility of the portable microfluidic LAMP system for parallel detection of multiple bacterial pathogens. The proposed on-chip LAMP assay is simple to operate, reliable for amplification, flexible in detection and cost-effective in instrumentation and testing, holding great potential for on-site rapid detection and routine monitoring of aquaculture pathogens.

iso-µmGene: an isothermal amplification-based portable microfluidic system for simple, reliable and flexibly multiplexed genetic identification and quantification.

Simple, reliable and flexibly multiplexed genetic identification and quantification of microbial pathogens is in urgent need for early disease diagnosis and timely treatment. This study presented an isothermal amplification-based portable microfluidic system (iso-µmGene) with features of multi-well chips for convenient filling and reliable sealing, flexible detection throughput, and a stand-alone and well-performing point of care (POC) genetic testing device. Using disposable chips with two kinds of reaction wells (eighteen and ten wells) and a device prototype with independent four chip holders, the iso-µmGene enables on-demand analysis of different target genes in one sample per chip and one to four samples (chips) per run, requiring only a single pipetting step for dispensing per chip with dehydrated primers. To completely seal the loop-mediated isothermal amplification (LAMP) reaction system to minimize the risk of amplicon escape, a dedicated plastic shell is used to assemble the array-type chip and reliably close its openings. Meanwhile, to enhance the precision for flexibly multiplexed detection and decrease the size and cost of the device, we designed a thermoelectric cooler (TEC)-based temperature-control module including two separate units and a CCD-based fluorescence imaging module containing a linear translation stage for real-time LAMP assay. This work demonstrated applications for the parallel detection of 2-2000 CFU (colony forming units) per reaction well with good intra- and inter-chip reproducibility using the crude lysates of two aquaculture pathogens Edwardsiella tarda and Vibrio harveyi. Overall, the iso-µmGene presented here possesses both a sophisticated instrument's functionality and performance and POC device's portability and cost.

A 3D mixing-based portable magnetic device for fully automatic immunofluorescence staining of γ-H2AX in UVC-irradiated CD4+ cells.

Immunofluorescence (IF) is a common method used in cell biology. The conventional protocol for IF staining is time and labor-intensive, operator dependent and reagent-consuming. Magnetic Bead (MB)-based microdevices are frequently utilized in cellular assays, but integration of simple and efficient mixing with downstream multi-step manipulation of MBs for automatic IF staining is still challenging. We herein present a portable, inexpensive and integratable device for MB-based automatic IF staining. First, a front-end cell capture step is performed using a 3D-mixing module, which is built upon a novel mechanism named ec-2MagRotors and generates periodically changing 3D magnetic fields. A 5-fold enhancement of cell capture efficiency was attained even with a low bead-to-cell concentration ratio (5 : 1), when conducting magnetic 3D mixing. Second, a 1D-moving module is employed downstream to automatically manipulate MB-cell complexes for IF staining. Further, a simplified protocol for staining of γ-H2AX, a biomarker widely used in evaluation of cell radiation damage, is presented for proof-of-principle study of the magnetic device. Using UVC-irradiated CD4+ cells as samples, our device achieved fully automatic γ-H2AX staining within 40 minutes at room temperature and showed a linear dose-response relationship. The developed portable magnetic device is automatic, efficient, cost-effective and simple-to-use, holding great potential for applications in different IF assays.

Organ-on-a-Chip: New Platform for Biological Analysis.

Direct detection and analysis of biomolecules and cells in physiological microenvironment is urgently needed for fast evaluation of biology and pharmacy. The past several years have witnessed remarkable development opportunities in vitro organs and tissues models with multiple functions based on microfluidic devices, termed as "organ-on-a-chip". Briefly speaking, it is a promising technology in rebuilding physiological functions of tissues and organs, featuring mammalian cell co-culture and artificial microenvironment created by microchannel networks. In this review, we summarized the advances in studies of heart-, vessel-, liver-, neuron-, kidney- and Multi-organs-on-a-chip, and discussed some noteworthy potential on-chip detection schemes.

A rapid, straightforward, and print house compatible mass fabrication method for integrating 3D paper-based microfluidics.

Three-dimensional (3D) paper-based microfluidics, which is featured with high performance and speedy determination, promise to carry out multistep sample pretreatment and orderly chemical reaction, which have been used for medical diagnosis, cell culture, environment determination, and so on with broad market prospect. However, there are some drawbacks in the existing fabrication methods for 3D paper-based microfluidics, such as, cumbersome and time-consuming device assembly; expensive and difficult process for manufacture; contamination caused by organic reagents from their fabrication process. Here, we present a simple printing-bookbinding method for mass fabricating 3D paper-based microfluidics. This approach involves two main steps: (i) wax-printing, (ii) bookbinding. We tested the delivery capability, diffusion rate, homogeneity and demonstrated the applicability of the device to chemical analysis by nitrite colorimetric assays. The described method is rapid (<30 s), cheap, easy to manipulate, and compatible with the flat stitching method that is common in a print house, making itself an ideal scheme for large-scale production of 3D paper-based microfluidics.

Automatic extraction and processing of small RNAs on a multi-well/multi-channel (M&M) chip.

The study of the regulatory roles in small RNAs can be accelerated by techniques that permit simple, low-cost, and rapid extraction of small RNAs from a small number of cells. In order to ensure highly specific and sensitive detection, the extracted RNAs should be free of the background nucleic acids and present stably in a small volume. To meet these criteria, we designed a multi-well/multi-channel (M&M) chip to carry out automatic and selective isolation of small RNAs via solid-phase extraction (SPE), followed by reverse-transcription (RT) to convert them to the more stable cDNAs in a final volume of 2 µL. Droplets containing buffers for RNA binding, washing, and elution were trapped in microwells, which were connected by one channel, and suspended in mineral oil. The silica magnetic particles (SMPs) for SPE were moved along the channel from well to well, i.e. in between droplets, by a fixed magnet and a translation stage, allowing the nucleic acid fragments to bind to the SMPs, be washed, and then be eluted for RT reaction within 15 minutes. RNAs shorter than 63 nt were selectively enriched from cell lysates, with recovery comparable to that of a commercial kit. Physical separation of the droplets on our M&M chip allowed the usage of multiple channels for parallel processing of multiple samples. It also permitted smooth integration with on-chip RT-PCR, which simultaneously detected the target microRNA, mir-191, expressed in fewer than 10 cancer cells. Our results have demonstrated that the M&M chip device is a valuable and cost-saving platform for studying small RNA expression patterns in a limited number of cells with reasonable sample throughput.

Simply and reliably integrating micro heaters/sensors in a monolithic PCR-CE microfluidic genetic analysis system.

A novel fabrication process was presented to construct a monolithic integrated PCR-CE microfluidic DNA analysis system as a step toward building a total genetic analysis microsystem. Microfabricated Titanium/Platinum (Ti/Pt) heaters and resistance temperature detectors (RTDs) were integrated on the backside of a bonded glass chip to provide good thermal transfer and precise temperature detection for the drilled PCR-wells. This heater/RTD integration procedure was simple and reliable, and the resulting metal layer can be easily renewed when the Ti/Pt layer was damaged in later use or novel heater/RTD design was desired. A straightforward "RTD-calibration" method was employed to optimize the chip-based thermal cycling conditions. This method was convenient and rapid, comparing with a conventional RTD-calibration/temperature adjustment method. The highest ramping rates of 14 degrees C/s for heating and 5 degrees C/s for cooling in a 3-microL reaction volume allow 30 complete PCR cycles in about 33 min. After effectively passivating the PCR-well surface, successful lambda-phage DNA amplifications were achieved using a two- or three-temperature cycling protocol. The functionality and performance of the integrated microsystem were demonstrated by successful amplification and subsequent on-line separation/sizing of lambda-phage DNA. A rapid assay for Hepatitis B virus, one of the major human pathogens, was performed in less than 45 min, demonstrating that the developed PCR-CE microsystem was capable of performing automatic and high-speed genetic analysis.

Microfluidic device for integrated restriction digestion reaction and resulting DNA fragment analysis.

A microfluidic system combining temperature-controlled reactor, analyte delivery, chip electrophoresis (CE) separation, and fluorescence detection was developed, in which the heaters, resistance temperature detectors, enzymatic reactors, CE channels, and pneumatic valves/pumps were integrated onto a single glass-PDMS chip. The microdevice was used to perform the digestion reaction, followed by on-line electrophoresis separation and detection of the resulting fragments with endonuclease BamHI and FokI as models. Pneumatic valves/pumps served not only for isolating the reaction region from the separation medium to prevent contamination, but also for delivering and quantitatively diluting the fluid from the reaction chamber to the CE section. Thus enzymatic reaction and electrophoresis separation could be insulated and connected as needed. A dynamic coating procedure with the use of PVP and mannitol was firstly adopted for glass-PDMS hybrid chip-based DNA separations, leading to an improved separation efficiency with reproducible migration time and theoretical plates. The expected 263- and 287-bp digestion products of BamHI and FokI were definitely verified by the size-based electrophoretic separation and detection. The whole integrated reaction-CE system can be manipulated in a simple manner with good reproducibility, which is expected to be applied in other on-line analysis of various biochemical reactions.

Parallel microfluidic networks for studying cellular response to chemical modulation.

A microfluidic chip featuring parallel gradient-generating networks etched on glass plate was designed and fabricated. The dam and weir structures were fabricated to facilitate cell positioning and seeding, respectively. The microchip contains five gradient generators and 30 cell chambers where the resulted concentration gradients of drugs are delivered to stimulate the on-chip cultured cells. This microfluidics exploits the advantage of lab-on-a-chip technology by integrating the generation of drug concentration gradients and a series of cell operations including seeding, culture, stimulation and staining into a chip. Steady parallel concentration gradients were generated by flowing two fluids in each network. The microchip described above was applied in studying the role of reduced glutathione (GSH) in MCF-7 cells' chemotherapy sensitivity. The parental breast cancer cell line, MCF-7 and the derived adriamycin resistant cell line MCF-7(adm) were treated with concentration gradients of arsenic trioxide (ATO) and N-acetyl cysteine (NAC) for GSH modulation, followed by exposure to adriamycin. The intracellular GSH level and cell viability were assessed by fluorescence image analysis. GSH levels of both cell lines were down-regulated upon ATO treatment and up-regulated upon NAC treatment. For both cell lines, suppression of intracellular GSH by treatment with ATO has been shown to increase chemotherapy sensitivity; conversely, elevation of intracellular GSH by treatment with NAC leads to increased drug resistance. The results indicated that high intracellular GSH level has negative effect on chemotherapy sensitivity, while depletion of cellular GSH may serve as an effective way to improve chemotherapy sensitivity. The integrated microfluidic chip is able to perform multiparametric pharmacological profiling with easy operation, thus, holds great potential for extrapolation to the high-content drug screening.

Fabrication of two-weir structure-based packed columns for on-chip solid-phase extraction of DNA.

Microchip-based packed column SPE of DNA was performed using the microfabricated two-weir structure within a microchannel. We developed two methods to fabricate the two-weir structured glass chips: a "two-side etching/alignment" method and a simplified "one-side/two-step etching" method. The former method required a straightforward alignment step, while the latter approach comprised a simplified wet etching process using paraffin wax as the temporary protective layer. Both methods were convenient and rapid as compared to the previous approaches. Through a reversibly sealed bead-introduction channel, beads can be fed into or out of the chip columns, thus enabling refreshment of the packing materials. Using the proposed chip columns, highly efficient lambda-DNA extractions (average recovery >80%) were performed with good chip-to-chip reproducibility (RSD <10%). The on-chip SPE procedure was completed within 15 min at the flow rate of 3 microL/min and the bulk of the loaded DNA was eluted within a small volume of approximately 8 microL. Application of the microchip-based packed columns was demonstrated by purifying PCR-amplifiable genomic DNA from human hepatocellular carcinoma (HepG2) cells and human whole blood samples.

Analysis of multiplex PCR fragments with PMMA microchip.

Microchip electrophoresis is a promising technique for analysis of bio-molecules. It has the advantages of fast analysis, high sensitivity, high resolution and low-cost of samples. Plastic chip has the potential of mass production for clinical use for its advantages in biocompatibility and low cost. In this work, the method for fabrication of poly(methyl methacrylate) (PMMA) chip was described, and conditions for DNA separation were investigated with the chip. The PMMA microchip was used for detection of multiplex PCR products of 18 and 36 cases with SARS and hepatitis B virus infection under optimized separation conditions. Microchip electrophoresis showed higher sensitivity, higher resolution and less time consumption when compared with gel electrophoresis. The microchip electrophoresis with PMMA chip provided a rapid, sensitive and reliable method for analysis of multiplex PCR products.

Highly sensitive determination of the methylated p16 gene in cancer patients by microchip electrophoresis.

The p16 tumor suppressor gene is inactivated by promoter region hypermethylation in many types of tumor. Recent studies showed that aberrant methylation of the p16 gene is an early event in many tumors, especially in lung cancer, and may constitute a new biomarker for early detection and monitoring of prevention trials. We detected tumor-associated aberrant hypermethylation of the p16 gene in plasma and tissue DNA from 153 specimens using a modified semi-nested methylation-specific PCR (MSP) combining plastic microchip electrophoresis or slab gel electrophoresis, respectively. Specimens were from 79 lung cancer patients, 15 abdominal tumor patients, 30 positive controls and 30 negative controls. The results showed that the positive rate obtained by microchip electrophoresis was more than 26.6% higher and the same specificity was kept when compared with slab gel electrophoresis. The microchip electrophoresis can rapidly and accurately analyze the PCR products of methylated DNA and obviously improve the positive rate of diagnosis of cancer patients when compared with gel electrophoresis. This method with the high assay sensitivity might be used for detection of methylation of p16 gene and even to facilitate early diagnosis of cancer patients.

Determination of SARS-coronavirus by a microfluidic chip system.

We have developed a new experimental system based on a microfluidic chip to determine severe acute respiratory syndrome coronavirus (SARS-CoV). The system includes a laser-induced fluorescence microfluidic chip analyzer, a glass microchip for both polymerase chain reaction (PCR) and capillary electrophoresis, a chip thermal cycler based on dual Peltier thermoelectric elements, a reverse transcription-polymerase chain reaction (RT-PCR) SARS diagnostic kit, and a DNA electrophoretic sizing kit. The system allows efficient cDNA amplification of SARS-CoV followed by electrophoretic sizing and detection on the same chip. To enhance the reliability of RT-PCR on SARS-CoV detection, duplex PCR was developed on the microchip. The assay was carried out on a home-made microfluidic chip system. The positive and the negative control were cDNA fragments of SARS-CoV and parainfluenza virus, respectively. The test results showed that 17 positive samples were obtained among 18 samples of nasopharyngeal swabs from clinically diagnosed SARS patients. However, 12 positive results from the same 18 samples were obtained by the conventional RT-PCR with agarose gel electrophoresis detection. The SARS virus species can be analyzed with high positive rate and rapidity on the microfluidic chip system.