Hydrodynamic flow is an essential stimulus in many cellular functions, regulating many mechanical sensitive pathways and closely associating with human health status and diseases. The flow pattern of blood in vessels is the key factor in causing atherosclerosis. Hemodynamics has great effect on endothelial cells’ gene expression and biological functions. There are various tools that can be used for studying flow-induced cellular responses but most of them are either bulky or lack precise controllability. We develop an integrated microfluidic device that can precisely generate different flow patterns to human endothelial cells cultured on-chip. We monitored cell morphology and used small-input RNA-seq technology to depict the transcriptome profiles of human umbilical vein endothelial cells under uni- or bidirectional flow. Such integrated and miniatured device has greatly facilitated our understanding of endothelial functions with shear stimulus, not only providing new data on the transcriptomic scale but also building the connection between cell phenotypic changes and expression alternations.

We develop a novel single-cell-based platform through digital counting of amplified genomic DNA fragments, named multifraction amplification (mfA), to detect the copy number variations (CNVs) in a single cell. Amplification is required to acquire genomic information from a single cell, while introducing unavoidable bias. Unlike prevalent methods that directly infer CNV profiles from the pattern of sequencing depth, our mfA platform denatures and separates the DNA molecules from a single cell into multiple fractions of a reaction mix before amplification. By examining the sequencing result of each fraction for a specific fragment and applying a segment-merge maximum likelihood algorithm to the calculation of copy number, we digitize the sequencing-depth-based CNV identification and thus provide a method that is less sensitive to the amplification bias. In this paper, we demonstrate a mfA platform through multiple displacement amplification (MDA) chemistry. When performing the mfA platform, the noise of MDA is reduced; therefore, the resolution of single-cell CNV identification can be improved to 100 kb. We can also determine the genomic region free of allelic drop-out with mfA platform, which is impossible for conventional single-cell amplification methods.Keywords: copy number variation; digital counting; DNA amplification; genomic sequencing; maximum likelihood; single-cell;

AbstractDevelopment of Raman-active materials with enhanced and distinctive Raman vibrations in the Raman-silent region (1800–2800 cm−1) is highly required for specific molecular imaging of living cells with high spatial resolution. Herein, water-soluble cationic conjugated polymers (CCPs), poly(phenylene ethynylene) (PPE) derivatives, are explored for use as alkyne-state-dependent Raman probes for living cell imaging due to synergetic enhancement effect of alkyne vibrations in Raman-silent region compared to alkyne-containing small molecules. The enhanced alkyne signals result from the integration of alkyne groups into the rigid backbone and the delocalized π-conjugated structure. PPE-based conjugated polymer nanoparticles (CPNs) were also prepared as Raman-responsive nanomaterials for distinct imaging application. This work opens a new way into the development of conjugated polymer materials for enhanced Raman imaging.

Stable water-in-oil emulsion is essential to digital PCR and many other bioanalytical reactions that employ droplets as microreactors. We developed a novel technology to produce monodisperse emulsion droplets with high efficiency and high throughput using a bench-top centrifuge. Upon centrifugal spinning, the continuous aqueous phase is dispersed into monodisperse droplet jets in air through a micro-channel array (MiCA) and then submerged into oil as a stable emulsion. We performed dPCR reactions with a high dynamic range through the MiCA approach, and demonstrated that this cost-effective method not only eliminates the usage of complex microfluidic devices and control systems, but also greatly suppresses the loss of materials and cross-contamination. MiCA-enabled highly parallel emulsion generation combines both easiness and robustness of picoliter droplet production, and breaks the technical challenges by using conventional lab equipment and supplies.

Single-cell genome, DNA methylome, and transcriptome sequencing methods have been separately developed. However, to accurately analyze the mechanism by which transcriptome, genome and DNA methylome regulate each other, these omic methods need to be performed in the same single cell. Here we demonstrate a single-cell triple omics sequencing technique, scTrio-seq, that can be used to simultaneously analyze the genomic copy-number variations (CNVs), DNA methylome, and transcriptome of an individual mammalian cell. We show that large-scale CNVs cause proportional changes in RNA expression of genes within the gained or lost genomic regions, whereas these CNVs generally do not affect DNA methylation in these regions. Furthermore, we applied scTrio-seq to 25 single cancer cells derived from a human hepatocellular carcinoma tissue sample. We identified two subpopulations within these cells based on CNVs, DNA methylome, or transcriptome of individual cells. Our work offers a new avenue of dissecting the complex contribution of genomic and epigenomic heterogeneities to the transcriptomic heterogeneity within a population of cells.

Prevalent single cell RNA amplification and sequencing chemistries mainly focus on polyadenylated RNAs in eukaryotic cells by using oligo(dT) primers for reverse transcription. We develop a new RNA amplification method, “easier-seq”, to reverse transcribe and amplify the total RNAs, both with and without polyadenylate tails, from a single cell for transcriptome sequencing with high efficiency, reproducibility, and accuracy. By distributing the reverse transcribed cDNA molecules into 1.5 × 105 aqueous droplets in oil, the cDNAs are isothermally amplified using random primers in each of these 65-pL reactors separately. This new method greatly improves the ease of single-cell RNA sequencing by reducing the experimental steps. Meanwhile, with less chance to induce errors, this method can easily maintain the quality of single-cell sequencing. In addition, this polyadenylate-tail-independent method can be seamlessly applied to prokaryotic cell RNA sequencing.

Quantitative characterization of a single-cell phenotype remains challenging. We combined a scalable microfluidic array of parallel cell culture chambers and stimulated Raman scattering (SRS) microscopy to quantitatively characterize the response of lipid droplet (LD) formation to free-fatty-acid stimuli with single-LD resolution at the single-cell level. By enabling the systematic live-cell imaging with SRS microscopy in a microfluidic device, we were able to quantify the morphology of over a thousand live cells in 10 different chemical environments and with 8 replicates for each culture condition, in a single experiment, and without relying on fluorescent labeling. We developed an image processing pipeline for cell segmentation and LD morphology quantification using dual-channel SRS images. This allows us to construct distributions of the morphological parameters of LDs in the cellular population and expose the vast phenotypic heterogeneity among genetically similar cells. Specifically, this approach provides an analytical tool for quantitatively investigating LD morphology in live cells in situ. With this high-throughput, high-resolution, and label-free method, we found that LD growth dynamics showed considerable cell to cell variation. Lipid accumulation in nonadipocyte cells is mainly reflected in the increase of LD number, as opposed to an increase in their size or lipid concentration. Our method allows statistical single-cell quantification of the LD distribution for further investigation of lipid metabolism and dynamic behavior, and also extends the possibility to couple with other “omics” technologies in the future.

Despite advances in DNA methylome analyses of cells and tissues, current techniques for genome-scale profiling of DNA methylation in circulating cell-free DNA (ccfDNA) remain limited. Here we describe a methylated CpG tandems amplification and sequencing (MCTA-Seq) method that can detect thousands of hypermethylated CpG islands simultaneously in ccfDNA. This highly sensitive technique can work with genomic DNA as little as 7.5 pg, which is equivalent to 2.5 copies of the haploid genome. We have analyzed a cohort of tissue and plasma samples (n = 151) of hepatocellular carcinoma (HCC) patients and control subjects, identifying dozens of high-performance markers in blood for detecting small HCC (≤ 3 cm). Among these markers, 4 (RGS10, ST8SIA6, RUNX2 and VIM) are mostly specific for cancer detection, while the other 15, classified as a novel set, are already hypermethylated in the normal liver tissues. Two corresponding classifiers have been established, combination of which achieves a sensitivity of 94% with a specificity of 89% for the plasma samples from HCC patients (n = 36) and control subjects including cirrhosis patients (n = 17) and normal individuals (n = 38). Notably, all 15 alpha-fetoprotein-negative HCC patients were successfully identified. Comparison between matched plasma and tissue samples indicates that both the cancer and noncancerous tissues contribute to elevation of the methylation markers in plasma. MCTA-Seq will facilitate the development of ccfDNA methylation biomarkers and contribute to the improvement of cancer detection in a clinical setting.

Epigenetic regulation is crucial to the establishment and maintenance of the identity of a cell. Recent studies suggest that transcription is implemented amongst a mixture of various histone modifications1. It has also been recognized that to interrogate function of genetic information, comprehensively systematic profiling of the epigenome in multiple cell stages and types is required2. Chromatin immunoprecipitation (ChIP) has become one of the most critical assays to investigate the complex DNA-protein interactions3. Combined with profiling technologies such as microarrays (ChIP-on-chip) or high-throughput sequencing (ChIP-Seq), this assay becomes a great tool to study the epigenetic regulatory networks in cells4,5,6. However, the ChIP process produces limited amount of DNA due to the low yield of antibody pull-down, DNA damage during fragmentation and cleavage of DNA-protein complex, and complicated downstream analysis7. The conventional approaches have to consume a considerable amount of samples, typically 106 - 107 cells, to overcome this low-yield issue and obtain reliable results5. This limitation also restricts ChIP applications from precious primary tissue samples such as early embryonic cells or rare tumor stem cells.ChIP-Seq, compared with ChIP-on-Chip, deeply sequences the target DNA fragments and generates highly comprehensive data with higher resolution, fewer artifacts, greater coverage and larger dynamic range6. Although recent application of automated microfluidic ChIP (AutoChIP) was successfully performed using 2 000 cells through locus-specific analysis by qPCR8, such assays do not achieve the comprehensiveness afforded by DNA sequencing approaches. Recently, several approaches have been developed to perform ChIP-Seq using as low as 10 000 or even only 5 000 cells7,9,10,11,14. However, all of these methods rely on ChIP reactions in tens of microliters and preamplification of ChIP product before sequencing library preparation, either through linear amplification (by in vitro transcription) or exponential amplification (by PCR), both of which potentially introduce significant bias. Adli et al.7 reported a modified protocol to realize the ChIP-Seq using 10 000 cells by revising the random primers used in amplification to reduce the primer self-annealing, with an optimized PCR condition to cover the GC-rich regions. Ng et al.14 developed another protocol to perform ChIP-Seq of H3K4me3 modification using 10 000 mouse primordial germ cells, requiring pre-amplification before the sequencing library preparation. Sachs et al.15 reported a chromatin immunoprecipitation study with low number of cells without pre-amplification, however, it needs at least 50 000 cells as starting material. Here we present a new method that implements a microfluidic device to facilitate the ChIP process, providing a technology to obtain the high-quality ChIP-Seq data from merely 1 000 mammalian cells, with no need of pre-amplification. The whole ChIP process has been greatly shortened to 8 h. Through this method, we have accomplished, for the first time, a rapid, semi-automated, and highly sensitive ChIP assay to investigate the genome-wide landscape of histone modification H3K4me3 using 1 000 mouse epiblast cells at E6.5, and found that the H3K4me3 landscape of post-implantation epiblast is more similar to that of the mEpiSCs than that of mESCs.Microfluidic devices are the ideal reaction systems for handling small number of cells. We fabricated our PDMS-based device to treat four samples in parallel on a single chip (Figure 1A). Each reaction pipeline could accept no more than 1 200 cells. We first performed the whole ChIP assay on a microfluidic device, including trapping the magnetic beads that pre-incubated with antibody, binding of the sonicated chromatin fragments with H3K4me3 to the beads, and washing away the chromatin fragments without H3K4me3 modification. Alternatively, the micro-device can be used for fragmentation of chromatins through micrococcal nuclease (MNase) treatment on chip, after the cell permeabilization. After the enrichment we collected the DNA out of the device and finished the sequencing library construction in microcentrifuge tubes. We eliminated preamplification of the ChIP product. Instead, we combined the end-repair, adenylation, and ligation steps in a one-tube reaction and then used carrier DNA to facilitate high-efficiency purification of the ligated DNA product. Then we used PCR to amplify these ligated DNAs to get nanogram amount of DNA for sequencing. Conventionally, the experimental process is tedious, taking at least two days to complete the sample treatment3. However, our new protocol can greatly accelerate the whole process; the complete experimental procedure, including microfluidic-based cell permeabilization, chromatin fragmentation, antibody pull-down, and many washing steps, can be completed within 8 h.We compared two approache for chromatin fragmentation, ultrasonic shearing and MNase digestion, and mainly used the former approach in our experiments since the result was more robust. The fragmentation step could be performed off-chip using the probe-free sonicator. 10 μl formaldehyde-crosslinked cell suspension was fragmented by the ultrasonic. Then the fragmented chromatin suspension was concentrated from 10 μl to 1 μl by evaporation.Since the starting material is very limited and the reaction yield is intrinsically low, even losing a small fraction of the target DNA8 would potentially compromise sequencing efforts. We hence carefully designed the microfluidic channels to sequentially perform the necessary reactions without losing target DNA. We implement the dead-end filling method12 to transfer the chromatin fragments into a ring-shape chamber to react with antibody-coated beads (Figure 1B). Dead-end filling was practical because PDMS was gas permeable; the air in the ring-chambers was expelled and replaced by liquid within a few minutes. Integrated 3-valve peristaltic pumps circulate the liquid in the ring chambers, facilitating the immunoprecipitation. This step is the most challenging practice in traditional ChIP assays. The challenges come from the incomplete crosslinking, nonspecific adsorption, low-efficiency chromatin fragmentation, and low specificity of binding between the antibodies and histone. However, with reduced reaction volume and active mixing, the efficiency of the whole process has been greatly improved. The semi-automated microfluidic control improved the precision and synchronization among all reaction pipelines in the same chip, ensuring the high reproducibility of the assay. We found that the amount of the beads used in an individual assay was also a critical factor. Excess beads elevated the background signals while insufficient beads would decrease the enrichment efficiency.The chromatin-antibody-bead complex was then flushed out of the chip and collected by microcentrifuge tubes. The complex was incubated with proteinase K at 68 °C for 2 h to release the DNA, which was then purified by phenol-chloroform-isoamyalcohol extraction. Precipitated DNA was re-suspended in 10 μl RNase-free H2O, and analyzed by qPCR using the specific primers (Supplemental methods) and by high-throughput sequencing.To quantitatively assess the sensitivity and accuracy of this new protocol, we performed ChIP-Seq of H3K4me3 for mEpiSCs and mESCs from both a bulk amount (106) of cells and from 1 000 cells and compared the results. We found that for mEpiSCs, the ChIP-seq of the two 1 000-cell samples recovered 16 351 and 16 245 out of the 16 929 enriched transcription start site (TSS) regions with H3K4me3 peaks from the bulk sample, and for mESCs, the two 1 000-cell samples recovered 18 256 and 18 206 out of the 18 367 enriched TSS regions from the bulk sample, demonstrating the high sensitivity (on average 96.3% for mEpiSCs and 99.3% for mESCs) of our method. Moreover, 98.3% and 98.4% of the 16 636 and 16 516 enriched TSS regions from the 1 000 mEpiSCs overlapped with those from bulk sample, and 91.0% and 91.7% of the 19 917 and 19 733 enriched TSS regions from the 1 000 mESCs overlapped with those from bulk sample (Figure 1C and Supplementary information, Table S1), exhibiting the high accuracy of our method. The mean correlation coefficient is 0.94 when comparing the H3K4me3 profile of 1 000 mEpiSCs and 106 mEpiSCs, and 0.76 of 1 000 mESCs and 106 mESCs (Supplementary information, Figure S1J), proving the general agreement of the profile based on 1 000 cells of our method and that of standard ChIP-Seq. We plot the receiver operating characteristic (ROC) curves using the P-value ranked peaks from the 1 000-cell experiments against the positive TSS from the bulk samples (Figure 1D and Supplementary information, Figure S1G), and then calculated the area under curve (AUC) of mEpiSC and mESC as 0.949 and 0.923, indicating our method a good classifier to distinguish positive TSSs from negative ones. These results clearly prove that our method is able to recover a majority of H3K4me3 peaks from as low as 1 000 mammalian cells with a very low false positive rate.Next we tested the robustness of our 1 000-cell ChIP-Seq method. Besides mEpiSCs and mESCs, we also performed ChIP-Seq on two biological replicates of the H3K4me3 marker from the 1 000 epiblast cells of E6.5 mouse embryos. We found that most of the TSS regions that have enrichment peaks are overlapped between these duplicates (Figure 1E). The correlation coefficients (r) of these replicates are 0.884 (epiblast cells of E6.5 mouse embryos), 0.971 (mEpiSCs), and 0.973 (mESCs), which are comparable to the previous report that required 10 000 cells7.Since pluripotent mEpiSC cells were derived from epiblast cells of E6.5 mouse embryos, we asked whether the H3K4me3 landscape of mEpiSCs in vitro was similar to that of E6.5 epiblast in vivo. We found that, in general, the H3K4me3 pattern of mEpiSCs was very similar to that of epiblast from E6.5 embryos. Among the top 10 000 P-value ranked peaks around the TSS regions, 7 000 in mEpiSCs overlapped with those in E6.5 epiblast (Figure 1C). When we compared the mESCs and the E6.5 epiblast cells, the overlapped peak number was 6 680. These proved that the H3K4me3 epigenetic landscape of epiblast cells of E6.5 mouse embryos is more similar to that of mEpiSCs than to that of mESCs as expected.Since H3K4me3 was an active marker for gene expression, we analyzed the transcriptome of mEpiSCs and E6.5 epiblast cells by RNA-Seq. We found that the gene expression profiles of mEpiSCs and E6.5 epiblast cells were very similar as well (r = 0.940) for transcripts with FPKM ≥ 0.1 in at least one of the samples. We compared, in-depth, the ChIP-Seq result at some important gene loci for early embryonic development and found high similarity among epiblast cells of E6.5 mouse embryos, mEpiSCs, and mESCs (Figure 1F). At the same time, we also found the specific gene loci only enriched in mESCs (Figure 1G). Gene ontology terms showed that, compared with mESCs, both epiblast cells of E6.5 mouse embryos and mEpiSCs enriched for the ectodermal differentiation-related characteristics such as neural tube development and neuronal differentiation (Figure 1H). Furthermore, the RNA expression level of the genes clearly correlated with the enrichment of H3K4me3 around their TSS regions both in mEpiSCs and E6.5 epiblast cells (Figure 1I and Supplementary information, Figure S1M). This correlation verifies the previous assumption that EpiSCs are a reliable in vitro model for post-implantation epiblast cells13.In summary, we have developed a highly sensitive ChIP-Seq method by combining microfluidic chip-based chromatin immunoprecipitation with one-tube carrier sequencing library preparation. The integrated device is able to finish the major steps of ChIP, including concentration of the cells from tens of microliters to nanoliters, fixation and permeabilization of the cells, fragmentation of chromatins, binding of the target chromatin fragments onto the beads, as well as the elution of enriched chromatin fragments. Subsequently, without any preamplification, the purified DNA fragments were converted into a sequencing library by a one-tube reaction containing end-repair, adenylation, and ligation followed by carrier PCR. We have demonstrated that this microfluidic-assisted ChIP-Seq method works robustly for as low as 1 000 mammalian cells. We have shown that the quality of H3K4me3 profile acquired by our method from 1 000 cells is comparable to that of traditional approach using bulk materials. Moreover, our method is highly reproducible with the correlation coefficient of the two biological replicates of E6.5 epiblast cells, mEpiSCs and mESCs as high as 0.884, 0.971, and 0.973, respectively. Finally we have demonstrated that the H3K4me3 epigenetic landscape of mEpiSCs is very similar to that of epiblast cells from E6.5 mouse embryos, validating that mEpiSC is an appropriate in vitro model to study the epigenetic regulation of post-implantation epiblast cells in vivo. Our method will permit thorough analysis of the epigenomic landscape of early embryos or other situations in which only a very limited amount of materials are available.We thank Prof Azim Surani for kindly giving us the mEpiSC cell line, and thank Tao Chen, Dr Yongfan Men, Zhilong Yu, Zitian Chen, Dr Liang Zhao, Haiwei Qiu, Lu Yang and Dr Yun Zhang for discussion and experimental help. This work was supported by the Ministry of Science and Technology of China (2011CB809106, 2012CB966704) and the National Natural Science Foundation of China (21222501, 21327808, 31271543, 91313302). AMS was supported by the Whitaker International Biomedical Engineering Fellowship.(Supplementary information is linked to the online version of the paper on the Cell Research website.)

Whole-genome amplification (WGA) for next-generation sequencing has seen wide applications in biology and medicine when characterization of the genome of a single cell is required. High uniformity and fidelity of WGA is needed to accurately determine genomic variations, such as copy number variations (CNVs) and single-nucleotide variations (SNVs). Prevailing WGA methods have been limited by fluctuation of the amplification yield along the genome, as well as false-positive and -negative errors for SNV identification. Here, we report emulsion WGA (eWGA) to overcome these problems. We divide single-cell genomic DNA into a large number (105) of picoliter aqueous droplets in oil. Containing only a few DNA fragments, each droplet is led to reach saturation of DNA amplification before demulsification such that the differences in amplification gain among the fragments are minimized. We demonstrate the proof-of-principle of eWGA with multiple displacement amplification (MDA), a popular WGA method. This easy-to-operate approach enables simultaneous detection of CNVs and SNVs in an individual human cell, exhibiting significantly improved amplification evenness and accuracy.

Gold nanorods (AuNRs) have shown great potential as bio-compatible imaging probes in various biological applications. Probing nanomaterials in live cells is essential to reveal the interaction between them. In this study, we used a transient absorption microscope to selectively image AuNRs in live cells. The transient absorption signals were monitored through lock-in amplification. This provides a new way of observing AuNRs with no interference from background autofluorescence.

Interference of the Hedgehog (Hh) signaling pathway by cyclopamine leads to abnormal embryonic development. We monitor this dynamical drug effect in zebrafish embryos with highly precise microenvironment control using an integrated microfluidic device. This chip-based platform, which is programmable and automated, greatly facilitates the accuracy and reproducibility of the in vivo assays.

Single-cell whole-transcriptome analysis is a powerful tool for quantifying gene expression heterogeneity in populations of cells. Many techniques have, thus, been recently developed to perform transcriptome sequencing (RNA-Seq) on individual cells. To probe subtle biological variation between samples with limiting amounts of RNA, more precise and sensitive methods are still required. We adapted a previously developed strategy for single-cell RNA-Seq that has shown promise for superior sensitivity and implemented the chemistry in a microfluidic platform for single-cell whole-transcriptome analysis. In this approach, single cells are captured and lysed in a microfluidic device, where mRNAs with poly(A) tails are reverse-transcribed into cDNA. Double-stranded cDNA is then collected and sequenced using a next generation sequencing platform. We prepared 94 libraries consisting of single mouse embryonic cells and technical replicates of extracted RNA and thoroughly characterized the performance of this technology. Microfluidic implementation increased mRNA detection sensitivity as well as improved measurement precision compared with tube-based protocols. With 0.2 M reads per cell, we were able to reconstruct a majority of the bulk transcriptome with 10 single cells. We also quantified variation between and within different types of mouse embryonic cells and found that enhanced measurement precision, detection sensitivity, and experimental throughput aided the distinction between biological variability and technical noise. With this work, we validated the advantages of an early approach to single-cell RNA-Seq and showed that the benefits of combining microfluidic technology with high-throughput sequencing will be valuable for large-scale efforts in single-cell transcriptome analysis.

We directly observe non-fluorescent nanodiamonds in living cells using transient absorption microscopy. This label-free technology provides a novel modality to study the dynamic behavior of nanodiamonds inside the cells with intrinsic three-dimensional imaging capability. We apply this method to capture the cellular uptake of nanodiamonds under various conditions, confirming the endocytosis mechanism.

Notochord is a rod-shaped axial supporting structure unique only to chordates. In this study, we use cephalochordate amphioxus (Branchiostroma belcheri, a living basal chordate) and zebrafish (Danio rerio), a vertebrate, as model animals and employ stimulated Raman microscopy (SRS), a newly developed label-free technique, to investigate notochord structure and chemical composition in live animals. Coherent anti-Stokes Raman scattering (CARS) images have also been acquired for comparison. Both CARS and SRS images can construct the detailed three-dimensional structure of the notochord with resolution better than 1 μm. Label-free live imaging allows us to obtain the whole animal's intact internal morphology, which is difficult to obtain through other mechanical or optical sectioning methods. Intrinsically, chemical sensitive SRS images, with a simple data processing procedure, show that the amphioxus notochord is protein-rich. Our result agrees well with that drawn from the labeling and cryo-sectioning observation, verifying that the coherent Raman scattering techniques are useful to examine the structure and chemical composition of live animals without labels in a fast, simple and accurate way.

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.

Epigenetic regulation plays an important role in cell migration. Although many methods have been developed to measure the motility of mammalian cells, accurate quantitative assessments of the migration speed of individual cells remain a major challenge. It is difficult for conventional scratch assays to differentiate proliferation from migration during the so-called wound-healing processes because of the long experimental time required. In addition, it is also challenging to create identical conditions for evaluating cell migration by conventional methods. We developed a microfluidic device with precisely created blanks allowing for robust and reproducible cell migration inside accurately-controlled microenvironments to study the regulatory effect of the epigenetic regulator histone deacetylase 7 (HDAC7) on cell migration. Through analyzing time-lapse imaging of the cells migrating into individual blank regions, we can measure the migration speed parameter for human primary cells within a few hours, eliminating the confounding effect of cell proliferation. We also developed an automatic image analysis and a numeric model-based data fitting to set up an integrated cell migration analysis system at single-cell resolution. Using this system, we measured the motility of primary human umbilical vein endothelial cells (HUVECs) and the migration speed reduction due to the silencing of HDAC7 and various other genes. We showed that the migration behaviour of these human primary cells are clearly regulated by epigenetic mechanisms, demonstrating the great potential of this accurate and robust assay in the fields of quantitatively migration studies and high-throughput screening.

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.

We designed and fabricated a novel microfluidic device that can be operated through simple finger squeezing. On-chip microfluidic flow control is enabled through an optimized network of check-valves and squeeze-pumps. The sophisticated flow system can be easily constructed by combining a few key elements. We implemented this device to perform quantitative biochemical assays with no requirement for precision instruments.

Immunofluorescence (IF) is a common method to observe protein distribution and localization at the single-cell level through wide-field fluorescence or confocal microscopy. Conventional protocol for IF staining of cells typically requires a large amount of reagents, especially antibodies, and noticeable investment in both labor and time. Microfluidic technologies provide a cost-effective alternative: it can evaluate and optimize experimental conditions, and perform automatic and high-throughput IF staining on-chip. We employed this method to analyze lysosomal storage disorders (LSDs) based on the expression and morphological distribution of LAMP1 and LC3 in starving cells. With pneumatic valves integrated on-chip, the parallel staining process can be completed within a few hours. The total consumption of each antibody solution for the whole experiment is merely 0.3 μL. This device provides a promising tool for automated high-throughput molecular imaging at cell level that can be applied for diagnostic analysis.

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 20 000/mm2. 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.

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.

The interaction between tumor and endothelial cells is crucial to cancer metastasis and angiogenesis. We developed a novel microfluidic device to assess the cell–cell interaction quantitatively at the single cell resolution. This integrated chip offers 16 coculture experiments in parallel with controllable microenvironments to study interactions between cells dynamically. We applied this approach to model the tumor invasion using Hela cells and human umbilical vein endothelial cells (HUVECs) and monitored the migration of both. We observed the retreatment of HUVECs upon the approach of Hela cells during coculture, indicating that the interaction between two cells was mediated by soluble factors. This interaction was further analyzed through quantitatively processing the phase-contrast microscopic time-lapse images of each individual coculture chamber. We also confirmed this paracrine effect by varying the frequency of medium change. This microfluidic technique is highly controllable, contamination free, fully automatic, and inexpensive. This approach not only offers a unique way to quantitatively study the interaction between cells but also provides accurate spatial-temporal tunability of microenvironments for cell coculture. We believe this method, intrinsically high-throughput and quantitative, will greatly facilitate the study of cell–cell interactions and communications.

Most current methods for analyzing the growth rate of plant seedlings are limited to low-throughput experimental configurations. We have developed an automatic system to investigate the dynamics of the growth of hypocotyls using Arabidopsis as model. This system is able to capture time-lapse infrared images of 24 seedlings automatically, with a spatial resolution of 2 μm per pixel and temporal interval of 5 min. Seedling length is rapidly calculated using automated geometric image-processing algorithms. With this high-throughput platform, we have investigated the genotype dependent difference of growth patterns, as well as the response to plant hormone – ethylene. Our analyses suggest that cytoskeleton function is not required in ethylene-induced hypocotyl inhibition. This novel integrative method can be applied to large-scale dynamic screening of plants, as well as any other image-based biological studies related to dynamic growth.

We developed an integrated microfluidic chip for long-term culture of isolated single cells. This polydimethylsiloxane (PDMS) based device could accurately seed each single cell into different culture chambers, and isolate one chamber from each other with monolithically integrated pneumatic valves. We optimized the culture conditions, including the frequency of medium replacement and the introduction of conditioned medium, to keep the single cells alive for 4 days. We cultured a few hundred cells in a separated chamber on the same chip to continuously supply the conditioned medium into the culture chambers for single cells. This approach greatly facilitated the growth of single cells, and created a suitable microenvironment for observing cells’ autonomous process in situ without the interference of other adjacent cells. This single cell colony assay is expandable to higher throughput, fitting the needs in the studies of drug screening and stem cell differentiation.

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.

A high-throughput microfluidic device is developed to handle liquid dispensation in nanoliter range. The dispenser system shows no cross-contamination between the microwells, indicating its great potential in large-scale screening experiments. An array of 115 nl PCR reactions, as well as the single channel addressable chip demonstrate the high flexibility and wide applications of this novel system.

•Single-molecule (SM) analysis is critical for studying heterogeneous biological systems.•Microfluidics offers a wide range of tools for precise and efficient sample handling.•The combination of SM detection and microfluidics has facilitated many areas of life science research.•Applications of microfluidic SM detection are reviewed.•Topics range from macromolecule biophysics to genomic analysis.Single-molecule approaches in biology have been critical in studies ranging from the examination of physical properties of biological macromolecules to the extraction of genetic information from DNA. The variation intrinsic to many biological processes necessitates measurements with single-molecule resolution in order to accurately recapitulate population distributions. Microfluidic technology has proven to be useful in the facilitation and even enhancement of single-molecule studies because of the precise liquid handling, small volume manipulation, and high throughput capabilities of microfluidic devices. In this review we survey the microfluidic “toolbox” available to the single-molecule specialist and summarize some recent biological applications of single-molecule detection on chip.Download high-res image (226KB)Download full-size image

We described a novel single-cell RNA-seq technique called MR-seq (measure a single-cell transcriptome repeatedly), which permits statistically assessing the technical variation and identifying the differentially expressed genes between just two single cells by measuring each single cell twice. We demonstrated that MR-seq gave sensitivity and reproducibility similar to the standard single-cell RNA-seq and increased the positive predicate value. Application of MR-seq to early mouse embryos identified hundreds of candidate intra-embryonic heterogeneous genes among mouse 2-, 4- and 8-cell stage embryos. MR-seq should be useful for detecting differentially expressed genes among a small number of cells.

Notochord is a rod-shaped axial supporting structure unique only to chordates. In this study, we use cephalochordate amphioxus (Branchiostroma belcheri, a living basal chordate) and zebrafish (Danio rerio), a vertebrate, as model animals and employ stimulated Raman microscopy (SRS), a newly developed label-free technique, to investigate notochord structure and chemical composition in live animals. Coherent anti-Stokes Raman scattering (CARS) images have also been acquired for comparison. Both CARS and SRS images can construct the detailed three-dimensional structure of the notochord with resolution better than 1 μm. Label-free live imaging allows us to obtain the whole animal's intact internal morphology, which is difficult to obtain through other mechanical or optical sectioning methods. Intrinsically, chemical sensitive SRS images, with a simple data processing procedure, show that the amphioxus notochord is protein-rich. Our result agrees well with that drawn from the labeling and cryo-sectioning observation, verifying that the coherent Raman scattering techniques are useful to examine the structure and chemical composition of live animals without labels in a fast, simple and accurate way.

Interference of the Hedgehog (Hh) signaling pathway by cyclopamine leads to abnormal embryonic development. We monitor this dynamical drug effect in zebrafish embryos with highly precise microenvironment control using an integrated microfluidic device. This chip-based platform, which is programmable and automated, greatly facilitates the accuracy and reproducibility of the in vivo assays.