Xiaojun Feng

Microfluidic chip: next-generation platform for systems biology.

Systems biology advocates the understanding of biology at the systems-level, which requires massive information of correlations among individual components in complex biological systems. Such comprehensive investigation entails the use of high-throughput analytical tools. Microfluidic technology holds high promise to facilitate the progress of biology by enabling miniaturization and upgrading of current biological research tools due to its advantages such as low sample consumption, reduced analysis time, high-throughput and compatible sizes with most biological samples. In this article, we documented the recent applications of microfluidic chips in biological researches at the molecular level, cellular level and organism level, serving the purpose for systems-level understanding of biology.

Rapid, highly efficient extraction and purification of membrane proteins using a microfluidic continuous-flow based aqueous two-phase system

Membrane proteins play essential roles in regulating various fundamental cellular functions. To investigate membrane proteins, extraction and purification are usually prerequisite steps. Here, we demonstrated a microfluidic aqueous PEG/detergent two-phase system for the purification of membrane proteins from crude cell extract, which replaced the conventional discontinuous agitation method with continuous extraction in laminar flows, resulting in significantly increased extraction speed and efficiency. To evaluate this system, different separation and detection methods were used to identify the purified proteins, such as capillary electrophoresis, SDS-PAGE and nano-HPLC-MS/MS. Swiss-Prot database with Mascot search engine was used to search for membrane proteins from random selected bands of SDS-PAGE. Results indicated that efficient purification of membrane proteins can be achieved within 5-7s and approximately 90% of the purified proteins were membrane proteins (the highest extraction efficiency reported up to date), including membrane-associated proteins and integral membrane proteins with multiple transmembrane domains. Compared to conventional approaches, this new method had advantages of greater specific surface area, minimal emulsification, reduced sample consumption and analysis time. We expect the developed method to be potentially useful in membrane protein purifications, facilitating the investigation of membrane proteomics.

Environmentally friendly surface modification of PDMS using PEG polymer brush

A PEG‐NH2‐based environmentally friendly surface modification strategy was developed for PDMS microchips to prevent protein adsorption and to enhance separation performance. PEG‐NH2 was synthesized using a modified synthesis procedure. A two‐step grafting method was used for PDMS modification. FTIR absorption by attenuated total reflection and contact angle measurements verified the successful grafting of PEG‐NH2 onto the PDMS surface. Subsequent EOF Measurements and protein adsorption studies of PEG‐modified PDMS microchips revealed noticeable EOF suppression and resistance to nonspecific protein adsorption for more than 30 days. Separation of four FITC‐labeled amino acids was further demonstrated with high repeatability and reproducibility. Comparison of electrophoresis of 3‐(2‐furoyl)quinoline‐2‐carboxaldehyde‐labeled BSA using PDMS microchips before and after surface modification resulted in significantly improved electrophoretic performance of the PEG‐modified PDMS microchips, suggesting that our PEG grafting method successfully modified PDMS surface property and prevented adsorption of proteins. We expect that this environmentally friendly surface modification method will be useful for future protein separations with long‐term surface stability.

Hydrodynamic gating valve for microfluidic fluorescence-activated cell sorting.

Microfluidic cell sorter allows efficient separation of small number of cells, which is beneficial in handling cells, especially primary cells that cannot be expanded to large populations. Here, we demonstrate a microfluidic fluorescence-activated cell sorter (muFACS) with a novel sorting mechanism, in which automatic on-chip sorting is realized by turning on/off the hydrodynamic gating valve when a fluorescent target is detected. Formation of the hydrodynamic gating valve was investigated by both numerical simulation and flow visualization experiment. Separation of fluorescent polystyrene beads was then conducted to evaluate this sorting mechanism and to optimize the separation conditions. Isolation of fluorescent HeLa-DsRed cells was further demonstrated with high purity and recovery rate. Viability of the sorted cells was also examined, suggesting a survival rate of more than 90%. We expect this sorting approach to find widespread applications in bioanalysis.

Microfluidic worm-chip for in vivo analysis of neuronal activity upon dynamic chemical stimulations.

Conventional neuronal analysis at the single neuron level usually involves culturing of neurons in vitro and analysis of neuronal activities by electrophysiological or pharmacological methods. However, the extracellular environments of in vitro neuronal analysis cannot mimic the exact surroundings of the neurons. Here, we report a microfluidic worm-chip for in vivo analysis of neuronal activities upon dynamic chemical stimulations. A comb-shaped microvalve was developed to immobilize whole animal for high-resolution imaging of neuronal activities. Using a sequential sample introduction system, multiple chemical stimuli were delivered to an individual Caenorhabditis elegans nose tip based on programmed interface shifting of laminar flows. ASH sensory neuron responses to various stimuli in individual C. elegans were quantitatively evaluated, and mutants were significantly defective in neuronal responses to certain stimulus in comparison to others. Sensory reduction in the magnitude of the response to repetitive chemical stimulation with different durations was also found. Our study explored the possibility of real-time detection of neuronal activities in individual animals upon multiple stimulations.

Ultrafast Microfluidic Mixer for Tracking the Early Folding Kinetics of Human Telomere G-Quadruplex

The folding of G-quadruplex is hypothesized to undergo a complex process, from the formation of a hairpin structure to a triplex intermediate and to the final G-quadruplex. Currently, no experimental evidence has been found for the hairpin formation, because it folds in the time regime of 10-100 μs, entailing the development of microfluidic mixers with a mixing time of less than 10 μs. In this paper, we reported an ultrarapid micromixer with a mixing time of 5.5 μs, which represents the fastest turbulent micromixer to our best knowledge. Evaluations of the micromixer were conducted to confirm its mixing efficiency for small molecules and macromolecules. This new micromixer enabled us to interrogate the hairpin formation in the early folding process of human telomere G-quadruplex. The experimental kinetic evidence for the formation of hairpin was obtained for the first time.

Rapid Assembly of Heterogeneous 3D Cell Microenvironments in a Microgel Array.

Heterogeneous 3D cell microenvironment arrays are rapidly assembled by combining surface-wettability-guided assembly and microdroplet-array-based operations. This approach enables precise control over individual shapes, sizes, chemical concentrations, cell density, and 3D spatial distribution of multiple components. This technique provides a cost-effective solution to meet the increasing demand of stem cell research, tissue engineering, and drug screening.

Microfluidic chip-based C. elegans microinjection system for investigating cell–cell communication in vivo

The propagation of intercellular calcium wave (ICW) is essential for coordinating cellular activities in multicellular organisms. However, the limitations of existing analytical methods hamper the studies of this biological process in live animals. In this paper, we demonstrated for the first time a novel microfluidic system with an open chamber for on-chip microinjection of C. elegans and investigation of ICW propagations in vivo. Worms were long-term immobilized on the side wall of the open chamber by suction. Using an external micro-manipulator, localized chemical stimulation was delivered to single intestinal cells of the immobilized worms by microinjection. The calcium dynamics in the intestinal cells expressing Ca(2+) indicator YC2.12 was simultaneously monitored by fluorescence imaging. As a result, thapsigargin injection induced ICW was observed in the intestinal cells of C. elegans. Further analysis of the ICW propagation was realized in the presence of heparin (an inhibitor for IP3 receptor), which allowed us to investigate the mechanism underlying intercellular calcium signaling. We expect this novel microfluidic platform to be a useful tool for studying cell-cell communication in multicellular organisms in vivo.

Click chemistry-based surface modification of poly(dimethylsiloxane) for protein separation in a microfluidic chip.

“Click” chemistry‐based surface modification strategy was developed for PDMS microchips to enhance separation performance for both amino acids and proteins. Alkyne‐PEG was synthesized by a conventional procedure and then “click” grafted to azido‐PDMS. FTIR absorption by attenuated total reflection and contact angle measurements proved efficient grafting of alkyne‐PEG onto PDMS surface. Manifest EOF regulation and stability of PEG‐functionalized PDMS microchips were illustrated via EOF measurements and protein adsorption investigations. The stability of nonspecific protein adsorption resistance property was investigated up to 30 days. Separation of fluorescence‐labeled amino acids and proteins was further demonstrated with high repeatability and reproducibility. Comparison of protein separation using PDMS microchips before and after surface modification suggested greatly improved electrophoretic performance of the PEG‐functionalized PDMS microchips. We expect the “click” chemistry‐based surface modification method to have wide applications in microseparation of proteins with long‐term surface stability.

Methylamidation for Isomeric Profiling of Sialylated Glycans by NanoLC-MS

The analysis of isomeric glycans is a challenging task. In this work, a new strategy was developed for isomer-specific glycan profiling using nanoLC-MS with PGC as the stationary phase. Native glycans were derivatized in the presence of methylamine and trispyrrolidinophosphonium hexafluorophosphate and reduced by the ammonia-borane complex. Methylamidation stabilized the retention time and peak width and improved the detection sensitivity of sialylated glycans to 2-80-fold in comparison to previous ESI-MS methods using the positive-ion mode. Up to 19 tetrasialylated glycan species were identified in the derivatized human serum sample, which were difficult to detect in the sample without derivatization. Furthermore, due to high detection sensitivity and chromatographic resolution, more isomeric glycans could be identified from the model glycoprotein Fetuin and the human serum sample. As a result, up to seven isomers were observed for the disialylated biantennary glycan released from Fetuin, and three of them were identified for the first time in this study. Using the developed analytical strategy, a total of 293 glycan species were obtained from the human serum sample, representing an increase of over 100 peaks in comparison to the underivatized sample. The strategy greatly facilitates the profiling of isomeric glycans and the analysis of trace-level samples.