Microfluidic whole genome amplification device for single cell sequencing.

We developed a microfluidic device to perform multiplex single-cell whole-genome amplification (WGA) using multiple annealing and looping-based amplification cycles (MALBAC). This device, made of polydimethylsiloxane (PDMS), allows us to monitor the whole process of cell loading and single-cell WGA for sequencing. We show that the genome coverage of MALBAC amplifications is reproducible between chambers on a single chip and between different chips, which enables data normalization using standard samples to accurately identify copy number variations (CNVs). This device provides an easy-to-operate approach to perform single cell sequencing library preparation with minimum hands-on time. It reduces the requirement of manual expertise as well as the risk of contamination, which is essential in future applications especially the medical diagnosis.

Liposomes physically coated with peptides: preparation and characterization.

Physically coating liposomes with peptides of desirable functions is an economic, versatile, and less time-consuming approach to prepare drug delivery vehicles. In this work, we designed three peptides-Ac-WWKKKGGNNN-NH2 (W2K3), Ac-WWRRRGGNNN-NH2(W2R3), Ac-WWGGGGGNNN-NH2(W2G3)-and studied their coating ability on negatively charged liposomes. It was found that the coating was mainly driven by the electrostatic interaction between the peptides' cationic side groups and the acidic lipids, which also mediated the "anchoring " of Trp residuals in the interfacial region of lipid bilayers. At the same conditions, the amount of the coated W2R3 was more than that of W2K3, but the stability of the liposome coated with W2R3 was deteriorated. This was caused by the delocalized charge of the guanidinium group of arginine. The coating of the peptide rendered the liposome pH-responsive behavior but did not prominently change the phase transition temperature. The liposome coated with peptides displayed appropriate pH/temperature dual responsive characteristics and was able to release the content in a controlled manner.

Highly reproducible and cost-effective one-pot organoid differentiation using a novel platform based on PF-127 triggered spheroid assembly.

Organoid technology offers sophisticatedin vitrohuman models for basic research and drug development. However, low batch-to-batch reproducibility and high cost due to laborious procedures and materials prevent organoid culture standardization for automation and high-throughput applications. Here, using a novel platform based on the findings that Pluronic F-127 (PF-127) could trigger highly uniform spheroid assembly through a mechanism different from plate coating, we develop a one-pot organoid differentiation strategy. Using our strategy, we successfully generate cortical, nephron, hepatic, and lung organoids with improved reproducibility compared to previous methods while reducing the original costs by 80%-95%. In addition, we adapt our platform to microfluidic chips allowing automated culture. We showcase that our platform can be applied to tissue-specific screening, such as drug toxicity and transfection reagents testing. Finally, we generateNEAT1knockout tissue-specific organoids and showNEAT1modulates multiple signaling pathways fine-tuning the differentiation of nephron and hepatic organoids and suppresses immune responses in cortical organoids. In summary, our strategy provides a powerful platform for advancing organoid research and studying human development and diseases.

Openly accessible microfluidic liquid handlers for automated high-throughput nanoliter cell culture.

Cell culture is typically performed in Petri dishes, with a few million cells growing together, or in microwell plates with thousands of cells in each compartment. When the throughput of each experiment, especially of screening based assays, is increased, even using microliter solution per well will cost a considerable amount of cells and reagents. We took a rational approach to reduce the volume of each cell culture chamber. We designed and fabricated a poly(dimethylsiloxane) based liquid pipet chip to deliver and transfer nanoliter (50-500 nL) samples and reagents with high accuracy and robustness. A few tens to a few hundreds of cells can be successfully seeded, transferred, passaged, transfected, and stimulated by drugs on a microwell chip using this pipet chip automatically. We have used this system to test the cell growth dynamically, observed the correlation between the culture conditions and cell viabilities, and quantitatively evaluated cell apoptosis induced by cis-diammineplatinum(II) dichloride (cisplatin). This system shows great potential to facilitate large-scale screening and high-throughput cell-array based bioassays with the volume of each individual cell colony at the nanoliter level.

Digital polymerase chain reaction in an array of femtoliter polydimethylsiloxane microreactors.

We developed a simple, compact microfluidic device to perform high dynamic-range digital polymerase chain reaction (dPCR) in an array of isolated 36-femtoliter microreactors. The density of the microreactors exceeded 20000/mm(2). This device, made from polydimethylsiloxane (PDMS), allows the samples to be loaded into all microreactors simultaneously. The microreactors are completely sealed through the deformation of a PDMS membrane. The small volume of the microreactors ensures a compact device with high reaction efficiency and low reagent and sample consumption. Future potential applications of this platform include multicolor dPCR and massively parallel dPCR for next generation sequencing library preparation.

Copy number alteration profiling facilitates differential diagnosis between ossifying fibroma and fibrous dysplasia of the jaws.

Ossifying fibroma (OF) and fibrous dysplasia (FD) are two fibro-osseous lesions with overlapping clinicopathological features, making diagnosis challenging. In this study, we applied a whole-genome shallow sequencing approach to facilitate differential diagnosis via precise profiling of copy number alterations (CNAs) using minute amounts of DNA extracted from morphologically correlated microdissected tissue samples. Freshly frozen tissue specimens from OF (n = 29) and FD (n = 28) patients were obtained for analysis. Lesion fibrous tissues and surrounding normal tissues were obtained by laser capture microdissection (LCM), with ~30-50 cells (5 000-10 000 µm2) per sample. We found that the rate of recurrent CNAs in OF cases was much higher (44.8%, 13 of 29) than that in FD cases (3.6%, 1 of 28). Sixty-nine percent (9 of 13) of the CNA-containing OF cases involved segmental amplifications and deletions on Chrs 7 and 12. We also identified eight CNA-associated genes (HILPDA, CALD1, C1GALT1, MICALL2, PHF14, AIMP2, MDM2, and CDK4) with amplified expression, which was consistent with the copy number changes. We further confirmed a jaw lesion with a previous uncertain diagnosis due to its ambiguous morphological features and the absence of GNAS mutation as OF based on the typical Chr 12 amplification pattern in its CNA profile. Moreover, analysis of a set of longitudinal samples collected from an individual with a cellular lesion in suspicion of OF at the first surgery, recurrence and the latest malignant transformation revealed identical CNA patterns at the three time points, suggesting that copy number profiling can be used as an important tool to identify borderline lesions or lesions with malignant potential. Overall, CNA profiling of fibro-osseous lesions can greatly improve differential diagnosis between OF and FD and help predict disease progression.

Solvent resistant microfluidic DNA synthesizer.

We fabricated a microfluidic DNA synthesizer out of perfluoropolyether (PFPE), an elastomer with excellent chemical compatibility which makes it possible to perform organic chemical reactions, and synthesized 20-mer oligonucleotides on chip.

WormFarm: a quantitative control and measurement device toward automated Caenorhabditis elegans aging analysis.

Caenorhabditis elegans is a leading model organism for studying the basic mechanisms of aging. Progress has been limited, however, by the lack of an automated system for quantitative analysis of longevity and mean lifespan. To address this barrier, we developed 'WormFarm', an integrated microfluidic device for culturing nematodes. Cohorts of 30-50 animals are maintained throughout their lifespan in each of eight separate chambers on a single WormFarm polydimethylsiloxane chip. Design features allow for automated removal of progeny and efficient control of environmental conditions. In addition, we have developed computational algorithms for automated analysis of video footage to quantitate survival and other phenotypes, such as body size and motility. As proof-of-principle, we show here that WormFarm successfully recapitulates survival data obtained from a standard plate-based assay for both RNAi-mediated and dietary-induced changes in lifespan. Further, using a fluorescent reporter in conjunction with WormFarm, we report an age-associated decrease in fluorescent intensity of GFP in transgenic worms expressing GFP tagged with a mitochondrial import signal under the control of the myo-3 promoter. This marker may therefore serve as a useful biomarker of biological age and aging rate.

A compact optofluidic cytometer with integrated liquid-core/PDMS-cladding waveguides.

We developed a simple method to construct liquid-core/PDMS-cladding optical waveguides through pressurized filling of dead-ended micro-channels with optical fluids. The waveguides are in the same layer as microfluidic channels which greatly simplifies device fabrication. With proper contrast between the refractive index of the core and cladding, the transmission loss of the waveguides is less than 5 dB cm(-1). We also developed a method to create flat and optically clear surfaces on the sides of PDMS devices in order to couple light between free-space and the waveguides embedded inside the chip. With these newly developed techniques, we make a compact flow cytometer and demonstrate the fluorescence counting of single cells at a rate of up to ~50 cell s(-1) and total sample requirement of a few microlitres. This method of making liquid-core optical waveguides and flat surfaces has great potential to be integrated into many PDMS-based microsystems.

High-throughput immunoassay through in-channel microfluidic patterning.

We have developed an integrated microfluidic immunoassay chip for high-throughput sandwich immunoassay tests. The chip creates an array of reactive patterns through mechanical protection by actuating monolithically embedded button valves. We have demonstrated that this chip can achieve highly sensitive immunoassay tests within an hour, and requires only microliter samples.

Discretely tunable optofluidic compound microlenses.

We report a novel method to fabricate high zoom-ratio optofluidic compound microlenses using poly(dimethylsiloxane) with multi-layer architecture. The layered structure of deformable lenses, biconvex and plano-concave, are self-aligned as a group. The refractive index contrast of each lens, which is controlled by filling the chambers with a specific medium, is the key factor for determining the device's numerical aperture. The chip has multiple independent pneumatic valves that can be digitally switched on and off, pushing the liquid into the lens chambers with great accuracy and consistency. This quickly and precisely tunes the focal length of the microlens device from centimetres to sub-millimetre. The system has great potential for applications in portable microscopic imaging, bio-sensing, and laser beam configuration.