A hallmark of multicellularism is the diversity of specialized cell types with specific gene expression patterns. Dynamic control of gene expression involves many layers of regulations including epigenetic factors such as DNA methylation. In view of the complexity of gene regulation, individual data type alone cannot fully depict the developmental stages or cellular subtypes of a sample. Integrating data of multiple “omic” dimensions, including DNA methylation, gene expression, and genotype information from the same biological sample can collectively provide a more comprehensive picture of genome regulation during normal development and pathogenesis.

Here, we present a method to simultaneously interrogate DNA methylation state, gene expression and gene mutation at particular loci in single cells in an automated, high throughput microfluidic platform. We applied this platform to profile cellular heterogeneity in single cells from human fibroblast cell population undergoing reprogramming to induced pluripotent stem cell, and show that we can simultaneously capture the gene expression and DNA methylation dynamics of the single cells at various stages of reprogramming. We then applied this platform to interrogate cell-to-cell variability in primary lung adenocarcinoma. We detected substantial cell type heterogeneity in the primary tumor based on gene expression of marker genes and a subpopulation of epithelial cells were found to be hypermethylated in a set of tumor associated loci. This result reflects the rich microenvironment of the tumor, and also a unique epigenetic and gene expression profile of the tumor subpopulation that distinguish them from the stromal cells. In conclusion, single cell analysis uncovers important molecular variations in cellular subpopulations that would guide disease diagnosis and shed light on fundamental biological processes.

Cellular microenvironment, such as physical and chemical properties in the neighborhood, could strongly affect cellular activities, such as morphology, proliferation, differentiation. One powerful tool to control the microenvironment and affect cellular behaviors is microfluidic device, which is frequently developed based on PDMS. By controlling PDMS surface structures at different scales, we studied the cellular responses to the mechanical cues after a long term culture in term of adhesion, proliferation, differentiation for MSC. On the other hand, PDMS structures with various curvatures and angles were designed and fabricated to study responses of neurons, such as neurite morphology as well the formation of cultured network. 

The human eye is an important and yet difficult organ to study in situ. Microfluidic technologies provide the tool to model the human eye and allow the simultaneous observation of cellular behaviors under different physiological conditions. In this talk, I will share some of the conditions that are needed for constructing a physiologically relevant in vitro eye model. The model will be used to study the effects of saccadic eye movement and hydrostatics on the fluidic and cellular behaviors on-chip. The ability to control, observe, measure and monitor the relevant variables on a microfluidic chip enables the potential to understand the physo-chemical and fluid mechanical origins of conditions often observed in the human eye, and suggest new therapeutic and surgical strategies.

Fluorescence Activated Cell Sorting (FACS) is an essential technique widely used in biomedical analyses. Microfluidics, benefiting from its low power and sample consumption, has enabled miniaturization of the existing bulky and complex FACS equipment into cost-effective and benchtop devices. However, these devices still have limited efficiency and biocompatibility with sorting mechanisms based on dielectrophoresis, optical tweezers and gas valve. Acoustophoresis, the migration of particles in an acoustic field, has recently emerged as a promising method to manipulate microscale particles in aqueous solutions, thanks to the rapid response and good biocompatibility. In this work, we present a new microfluidic FACS system that makes use of a highly focused traveling surface acoustic wave (FTSAW) for highly accurate sorting. The ~30 µm wide FTSAW is produced by a set of circular-arc interdigital transducers (IDTs) patterned on a piezoelectric substrate. Since the aperture of the FTSAW field becomes comparable to the size of individual biological cells, it enables single particle level sorting even in a high concentration particle suspension upon the activation of fluorescence detection. Separation of tumor cells from white blood cells will be demonstrated to showcase the single cell sorting capability of the developed acoustic FACS system with sub-ms acoustic actuation.

Gold nanostructures are a highly attractive class of materials with unique electrochemical and optical sensing properties. Recent developments have greatly improved the sensitivity of optical sensors based on metal nanostructured arrays. We introduce the localized surface plasmon resonance (LSPR) sensors and describe how its exquisite sensitivity to size, shape and environment can be harnessed to detect molecular binding events. We then describe recent progress in three areas representing the most significant challenges: integration of LSPR with complementary electrochemical techniques, long term live-cell biosensing and practical development of sensors and instrumentation for routine use and high-throughput detection. As an example we will demonstrate a novel refractive index and charge sensitive device integrated with nanoplasmonic islands to develop nano-metal-insulator-semiconductor (nMIS) junctions. The developed sensor facilitates simultaneous detection of charge and mass changes on the nanoislands due to biomolecule binding. A brief insight on microcontact printing to functionalize proteins on nanoplasmonic sensors will also be discussed. The developed nanosensors can readily be adopted for multiplexed and high throughput label-free immunoassay systems, further driving innovations in biomedical and healthcare research.

Microfluidics has often been used for chemical or biological sensing, but rarely for mechanical sensing. Furthermore, conventional lab-on-chip platforms require discrete patient samples, and rely on bulky equipment such as microscope and syringe pump. Here, we revolutionized the microfluidics regime into elements compatible to our skin to enable continuous and unobtrusive biomonitoring. The aim of this presentation is to introduce the mechanosensing principle using flexible microfluidics and demonstrate its potential in disease sensing, rehabilitation monitoring, and artificial sensing.

To achieve this, we combined the principles of material science and deterministic geometry to develop a microfluidic liquid-based tactile sensor that is soft, thin, flexible, stretchable, and cost-effective to produce. The sensor comprises a unique combination of soft silicone rubber substrate and conductive fluid which forms the active liquid-based sensing element. These liquids assume the shape of the microchannels within the soft silicone elastomer due to the weak intermolecular forces of attraction between particles. As such, their shape is highly reconfigurable. Essentially, the conductive fluid is displaced in proportion to the mechanical forces exerted by the user, which corresponded to a change in its electrical resistance.The combination of a highly deformable elastomer and conductive liquid of low viscosity forms an important aspect in our approach in transducing mechanical forces to electrical changes. Microfluidic channels form the geometrical pattern that enables efficient liquid movement, realizing a sensor with high responsiveness, low creep, and low hysteresis. 

Furthermore, by modifying the contents of the silicone elastomer and conductive fluid, the device sensitivity, material hardness, viscoelasticity, and stretchability may be further tuned for different applications. We have demonstrated the utility of our sensor in numerous healthcare applications, including disease sensing, rehabilitation monitoring, and even artificial sensing. For example, we designed an elastomeric structure with a microfluidic protrusion capable of sensing surface roughness, and even recognizing Braille elements. In another application, we developed a microfluidic sensor the size of a strand of hair that can be attached to the wrist to monitor pulse pressure continuously and unobtrusively. Importantly, the possibility of imperceptible monitoring is set to create a paradigm shift in traditional clinical healthcare assessment, allowing access to the patient’s vital signs anytime, anywhere. 

The concentration of various gases plays an essential role for mammalian cells. For instance, oxygen is an important modulator of cellular function in both normal physiology and disease states. Cells respond to oxygen over a wide range of oxygen tensions, from hypoxia to hyperoxia. Oxygen affects cellular responses in various ways, including metabolic pathways and plasma membrane integrity. Oxygen gradients in physiologic systems also play a critical role in maintaining homeostasis and inducing acute cellular response. For example, angiogenesis in development, tissue repair, tumor growth, and vascular remodeling is potentiated by spatial oxygen gradients and the expression of oxygen-responsive genes. Due to the high diffusivity of gases in an aqueous solution, controlling gaseous microenvironments has been a challenging task for biologists. Studies of cellular responses to gases in cellular microenvironments would benefit from a reliable platform that is capable of robustly controlling the gaseous concentration in both spatial and temporal domains. However, existing microfluidic cell culture devices face several challenges that hinder their practical usage in biological labs. First, directly using gases for concentration and gradient generation requires precise flow control instruments, tedious interconnections, and bulky gas cylinders used to store compressed gases. Moreover, the gas can easily penetrate through the permeable membrane and may cause media evaporation and bubble generation inside the cell culture channel. As a result, the entire setup is unreliable for long-term studies, and cannot be directly implemented in conventional cell incubators. To overcome these challenges, my lab develops microfluidic cell culture platforms capable of controlling various gaseous microenvironments. The platforms take advantage of the spatially confined chemical reactions to generate or scavenge gases inside cell culture microfluidic channels or wells without direct chemical contact. The flow rates of chemicals can be precisely controlled by syringe pumps that are commonly used in biological labs. By confining the areas for chemical reactions, the device can control the gas concentrations and gradients, efficiently using minimal chemicals without altering the surrounding gaseous compositions. Furthermore, without tedious and unreliable gas interconnections, the entire microfluidic setup can be directly implemented into conventional cell incubators for optimized temperature and humidity control without additional instrumentation. Here, we demonstrate three different cell studies taking advantages of our microfluidic devices, including: study of cell proliferation and migration under oxygen gradients, study of cell migration and drug efficiency under combinations of chemical and oxygen gradients, and characterization of cell responses under nitric oxide gradients. With the demonstrated results, the developed microfluidic devices show great promise and advantages for various in vitro cell biology studies for biomedical applications.

Single cell omics analysis aims to collect all molecule information in single cells in the human body, but still lacks appropriate and efficient tools. Nanofluidics is promising for single cell omics analysis at the single molecule level. However, many challenges have impeded the nascent nanofluidics to enable the goal. A most critical one is the lack of methodology for the high-throughput capture and detection of single molecules in tiny nanofluidic channels. By using the nano-in-nano integration technology which previously developed by us [1-4], we established a nanofluidic nanoarray methodology allowing the high-throughput capture and detection of single molecules in samples with single cell volumes. In this presentation, the nanofluidic nanoarrays for single-cell omics with single-molecule sensitivity will be presented.

[1] Xu Y., et al., Lab Chip, 2015, 15, 1989-1993.
[2] Xu Y., et al., Lab Chip, 2015, 15, 3856-3861.
[3] Xu Y., et al., Small, 2015, 11, 6165-6171.
[4] Xu Y., et al., Adv. Mater., 2016, 28, 2209-2216.

It is well known that analyzing the dynamic behavior of reversible gels is a tough job, as it requires a detailed control of geometry, bond lifetimes, etc… .  In this context, we use an optofluidic microrehometer to investigate the properties of a system composed by DNA nanostars.

The device, allowing to test samples with volume smaller than 1 uL, consists in a square section microchannel realized in a glass substrate and having a couple of facing waveguides, realized by fs-laser inscription technique, on the two sides of the channel. Using the optical-shooting technique (T. Yang, et al. Scientific Reports 6, 23946 2016; T. Yang et al. Micromachines 8, 65, 2017.), we investigated the system viscosity as a function of the temperature and of the applied optical force, observing the transition from Newtonian to shear-thinning behavior while lowering the temperature below the gelation threshold.

Stress-strain curves analysis allowed assessing the system activation energy, which is in good agreement with that obtained by dynamic light scattering measurements.

Biography

Aoqun Jian received the B.Eng. degree in electronic and information technology and the M.Eng. degree in microelectronics and solid electronics from the North University of China, Taiyuan, China, in 2005 and 2008, respectively. He received the Ph.D. degree in the Department of Applied Physics, The Hong Kong Polytechnic University, Hong Kong, in 2013. He is currently an associate professor with College of Information Engineering, Taiyuan University of Technology. He has published more than 10 journal papers and has co-authored two books. His current research interests include optofluidics, photonics and sensing devices.

Optofluidics biosensor based on speckle focusing

Although the lab-on-a-chip system has been successfully applied in a wide variety of fields, a biosensor which has a simple structure, low cost and high flux still attracts continuous research efforts. Here, the authors explore a biosensor based on the speckle focusing to detect the concentration of the T lymphocyte. Firstly, several optical fibers are embedded in the trench fabricated previously by dry etching in the silicon substrate as the incident light source. The gold pattern with lens shape obtained by typical lift-off technology is modified with the anti-CD3 which can specific bind the T lymphocyte suspended in the solution. The light emits from the optical fibers are collimated by the collimating lens formed in the polydimethylsiloxane (PDMS). The T lymphocyte bound on the gold pattern acts as a convex lens，focusing the collimated light propagates through it. The focused light is collected by an optical fiber embedded at the other side of the device, and the intensity of the light indicates the concentrations of the T lymphocyte. This sensor is particularly useful for drug screening, cell pathology analysis and cancer pre-diagnosis.

Acknowledgement

This work was financially supported by National Natural Science Foundation of China (No. 61501316), Taiyuan University of Technology, Basic Research Program of Shanxi for Youths (No. 2012021013-1), The Shanxi Provincial Foundation for Returned Scholars (2015-047) and 863 project (2015AA042601).