A microfluidic generator of dynamic shear stress and biochemical signals based on autonomously oscillatory flow

Biological cells in vivo typically reside in a dynamic flowing microenvironment with extensive biomechanical and biochemical cues varying in time and space. These dynamic biomechanical and biochemical signals together act to regulate cellular behaviors and functions. Microfluidic technology is an important experimental platform for mimicking extracellular flowing microenvironment in vitro. However, most existing microfluidic chips for generating dynamic shear stress and biochemical signals require expensive, large peripheral pumps and external control systems, unsuitable for being placed inside cell incubators to conduct cell biology experiments. This study has developed a microfluidic generator of dynamic shear stress and biochemical signals based on autonomously oscillatory flow. Further, based on the lumped-parameter and distributed-parameter models of multiscale fluid dynamics, the oscillatory flow field and the concentration field of biochemical factors has been simulated at the cell culture region within the designed microfluidic chip. Using the constructed experimental system, the feasibility of the designed microfluidic chip has been validated by simulating biochemical factors with red dye. The simulation results demonstrate that dynamic shear stress and biochemical signals with adjustable period and amplitude can be generated at the cell culture chamber within the microfluidic chip. The amplitudes of dynamic shear stress and biochemical signals is proportional to the pressure difference and inversely proportional to the flow resistance, while their periods are correlated positively with the flow capacity and the flow resistance. The experimental results reveal the feasibility of the designed microfluidic chip. Conclusively, the proposed microfluidic generator based on autonomously oscillatory flow can generate dynamic shear stress and biochemical signals without peripheral pumps and external control systems. In addition to reducing the experimental cost, due to the tiny volume, it is beneficial to be integrated into cell incubators for cell biology experiments. Thus, the proposed microfluidic chip provides a novel experimental platform for cell biology investigations.     

Integrated microfluidic array plate (iMAP) for cellular and molecular analysis

Just as the Petri dish has been invaluable to the evolution of biomedical science in the last 100 years, microfluidic cell assay platforms have the potential to change significantly the way modern biology and clinical science are performed. However, an evolutionary process of creating an efficient microfluidic array for many different bioassays is necessary. Specifically for a complete view of a cell response it is essential to incorporate cytotoxic, protein and gene analysis on a single system. Here we present a novel cellular and molecular analysis platform, which allows access to gene expression, protein immunoassay, and cytotoxicity information in parallel. It is realized by an integrated microfluidic array plate (iMAP). The iMAP enables sample processing of cells, perfusion based cell culture, effective perturbation of biologic molecules or drugs, and simultaneous, real-time optical analysis for different bioassays. The key features of the iMAP design are the interface of on-board gravity driven flow, the open access input fluid exchange and the highly efficient sedimentation based cell capture mechanism (～100% capture rates). The operation of the device is straightforward (tube and pump free) and capable of handling dilute samples (5-cells per experiment), low reagent volumes (50 nL per reaction), and performing single cell protein and gene expression measurements. We believe that the unique low cell number and triple analysis capabilities of the iMAP platform can enable novel dynamic studies of scarce cells.     

Generation of dynamic temporal and spatial concentration gradients using microfluidic devices

This paper describes a microfluidic approach to generate dynamic temporal and spatial concentration gradients using a single microfluidic device. Compared to a previously described method that produced a single fixed gradient shape for each device, this approach combines a simple "mixer module" with gradient generating network to control and manipulate a number of different gradient shapes. The gradient profile is determined by the configuration of fluidic inputs as well as the design of microchannel network. By controlling the relative flow rates of the fluidic inputs using separate syringe pumps, the resulting composition of the inlets that feed the gradient generator can be dynamically controlled to generate temporal and spatial gradients. To demonstrate the concept and illustrate this approach, examples of devices that generate (1) temporal gradients of homogeneous concentrations, (2) linear gradients with dynamically controlled slope, baseline, and direction, and (3) nonlinear gradients with controlled nonlinearity are shown and their limitations are described.     

Monitoring induced gene expression of single cells in a multilayer microchip

We present a microfluidic system that facilitates long-term measurements of single cell response to external stimuli. The difficulty of addressing cells individually was overcome by using a two-layer microfluidic device. The top layer is designed for trapping and culturing of cells while the bottom layer is employed for supplying chemical compounds that can be transported towards the cells in defined concentrations and temporal sequences. A porous polyester membrane that supports transport and diffusion of compounds from below separates the microchannels of both layers. The performance and potential of the device are demonstrated using human embryonic kidney cells (HEK293) transfected with an inducible gene expression system. Expression of a fluorescent protein (ZsGreen1-DR) is observed while varying the concentration and exposure time of the inducer tetracycline. The study reveals the heterogeneous response of the cells as well as average responses of tens of cells that are analyzed in parallel. The microfluidic platform enables systematic studies under defined conditions and is a valuable tool for general single cell studies to obtain insights into mechanisms and kinetics that are not accessible by conventional macroscopic methods.     

A time-coded multi-concentration microfluidic chemical waveform generator for high-throughput probing suspension single-cell signaling

The cellular response to the complex extracellular microenvironment is highly dynamic in time and type of extracellular matrix. Accurately reconstructing this process and analyzing the changes in receptor conformation on the cell membrane surface and intracellular or intercellular signaling has been a major challenge in analytical chemistry and biophysical methodology. In this paper, a time-coded multiconcentration microfluidic chemical waveform generator was developed for the dynamic signaling ...     

Microfluidic array cytometer based on refractive optical tweezers for parallel trapping, imaging and sorting of individual cells

Analysis of genetic and functional variability in populations of living cells requires experimental techniques capable of monitoring cellular processes such as cell signaling of many single cells in parallel while offering the possibility to sort interesting cell phenotypes for further investigations. Although flow cytometry is able to sequentially probe and sort thousands of cells per second, dynamic processes cannot be experimentally accessed on single cells due to the sub-second sampling time. Cellular dynamics can be measured by image cytometry of surface-immobilized cells, however, cell sorting is complicated under these conditions due to cell attachment. We here developed a cytometric tool based on refractive multiple optical tweezers combined with microfluidics and optical microscopy. We demonstrate contact-free immobilization of more than 200 yeast cells into a high-density array of optical traps in a microfluidic chip. The cell array could be moved to specific locations of the chip enabling us to expose in a controlled manner the cells to reagents and to analyze the responses of individual cells in a highly parallel format using fluorescence microscopy. We further established a method to sort single cells within the microfluidic device using an additional steerable optical trap. Ratiometric fluorescence imaging of intracellular pH of trapped yeast cells allowed us on the one hand to measure the effect of the trapping laser on the cells' viability and on the other hand to probe the dynamic response of the cells upon glucose sensing.     

Multiport flow-control system for lab-on-a-chip microfluidic devices

A multiport system suitable for pressure control on a lab-on-a-chip microfluidic device is described. An algorithm and a strategy for calculating pressures were developed to control the flow from multiple reservoirs for the microfluidic devices. Dye mixing and enzyme assay titration experiments were performed using pressure-driven flow only. Results show a good linear response over two orders of dynamic range.     

A microfluidic system enabling Raman measurements of the oxygenation cycle in single optically trapped red blood cells

Using a lab-on-a-chip approach we demonstrate the possibility of selecting a single cell with certain properties and following its dynamics after an environmental stimulation in real time using Raman spectroscopy. This is accomplished by combining a micro Raman set-up with optical tweezers and a microfluidic system. The latter gives full control over the media surrounding the cell, and it consists of a pattern of channels and reservoirs defined by electron beam lithography that is moulded into rubber silicon (PDMS). Different buffers can be transported through the channels using electro-osmotic flow, while the resonance Raman response of an optically trapped red blood cell (RBC) is simultaneously registered. This makes it possible to monitor the oxygenation cycle of the cell in real time and to investigate effects like photo-induced chemistry caused by the illumination. The experimental set-up has high potential for in vivo monitoring of cellular drug response using a variety of spectroscopic probes.     

Newly developed chemical probes and nano-devices for cellular analysis.

A cellular analyzing system including a "real-time cellular imaging system" and a "comprehensive analyzing system for cellular responses" was developed. A "real-time cellular imaging system" is a system used to measure real-time imaging of multiple phenomena of a single cell with high special and temporal resolutions for the purpose to understand the pathology and physiology in a single cell and realize to single cell level diagnosis. A "real-time cellular imaging system" includes multi-probe imaging with AFM (atomic force microscopy), optical and SECM (scanning electrochemical microscopy) modes, which provides us with topological information and biochemical reactions at the local area of the interior and exterior of a cell. Scanning electrochemical/optical microscopy was applied to image PC12 cells. On the other hand, cells respond to their specific substances via their ligands. Therefore, the comprehensive analysis of protein-protein interaction is the important issue to determine the functions of cells. For this purpose, a "comprehensive analysis system for cellular responses" was developed. This system is based on SPR (surface plasmon resonance) and MS (mass spectrometry) using a nano-fabricated substrate. The interaction between IL-1 beta and anti-IL-1 beta antibodies was detected.     

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.     

Rapid spatial and temporal controlled signal delivery over large cell culture areas

Controlled chemical delivery in microfluidic cell culture devices often relies on slowly evolving diffusive gradients, as the spatial and temporal control provided by fluid flow results in significant cell-perturbation. In this paper we introduce a microfluidic device architecture that allows for rapid spatial and temporal soluble signal delivery over large cell culture areas without fluid flow over the cells. In these devices the cell culture well is divided from a microfluidic channel located directly underneath the chamber by a nanoporous membrane. This configuration requires chemical signals in the microchannel to only diffuse through the thin membrane into large cell culture area, rather than diffuse in from the sides. The spatial chemical pattern within the microfluidic channel was rapidly transferred to the cell culture area with good fidelity through diffusion. The cellular temporal response to a step-function signal showed that dye reached the cell culture surface within 45 s, and achieved a static concentration in under 6 min. Chemical pulses of less than one minute were possible by temporally alternating the signal within the microfluidic channel, enabling rapid flow-free chemical microenvironment control for large cell culture areas.     

Microfluidic lithography of SAMs on gold to create dynamic surfaces for directed cell migration and contiguous cell cocultures

A straightforward, flexible, and inexpensive method to create patterned self-assembled monolayers (SAMs) on gold using microfluidics-microfluidic lithography-has been developed. Using a microfluidic cassette, alkanethiols were rapidly patterned on gold surfaces to generate monolayers and mixed monolayers. The patterning methodology is flexible and, by controlling the solvent conditions and thiol concentration, permeation of alkanethiols into the surrounding PDMS microfluidic cassette can be advantageously used to create different patterned feature sizes and to generate well-defined SAM surface gradients with a single microfluidic chip. To demonstrate the utility of microfluidic lithography, multiple cell experiments were conducted. By patterning cell adhesive regions in an inert background, a combination of selective masking of the surface and centrifugation achieved spatial and temporal control of patterned cells, enabling the design of both dynamic surfaces for directed cell migration and contiguous cocultures. Cellular division and motility resulted in directed, dynamic migration, while the centrifugation-aided seeding of a second cell line produced contiguous cocultures with multiple sites for heterogeneous cell-cell interactions.     

Microfluidic techniques for dynamic single-cell analysis

Dynamic single-cell analysis is a very important and frontier research field of single-cell analysis. Microfluidic techniques have become new and effective tools for precise, high-throughput, automatic analysis of single-cell dynamic process. This review aims to give an overview of dynamic single-cell analysis methods based on microfluidic platforms, with emphasis on the recent developments of microfluidic devices and its application to real-time dynamic monitoring of the signal molecules release from single living cell with temporal and spatial resolution, dynamic gene expression in single cells, the cell death dynamic events at the level of a single cell, and direct cell—cell communication between individual cell pairs.     

Characterization of a microfluidic dispensing system for localised stimulation of cellular networks

We present a 3-D microfluidic device designed for localized drug delivery to cellular networks. The device features a flow cell comprising a main channel for nutrient delivery as well as multiple channels for drug delivery. This device is one key component of a larger, fully integrated system now under development, based upon a microelectrode array (MEA) with on-chip CMOS circuitry for recording and stimulation of electrogenic cells (e.g. neurons, cardiomyocytes). As a critical system unit, the microfluidics must be carefully designed and characterized to ensure that candidate drugs are delivered to specific regions of the culture at known concentrations. Furthermore, microfluidic design and functionality is dictated by the size, geometry, and material/electrical characteristics of the CMOS MEA. Therefore, this paper reports on the design considerations and fabrication of the flow cell, including theoretical and experimental analysis of the mass transfer properties of the nutrient and drug flows, which are in good agreement with one another. To demonstrate proof of concept, the flow cell was mounted on a dummy CMOS chip, which had been plated with HL-1 cardiomyocytes. A test chemical compound was delivered to the cell culture in a spatially resolved manner. Envisioned applications of this stand-alone system include simultaneous toxicological testing of multiple compounds and chemical stimulation of natural neural networks for neuroscience investigations.     

Characterization of a membrane-based gradient generator for use in cell-signaling studies

This paper describes a method to create stable chemical gradients without requiring fluid flow. The absence of fluid flow makes this device amenable to cell signaling applications where soluble factors can impact cell behavior. This device consists of a membrane-covered source region and a large volume sink region connected by a microfluidic channel. The high fluidic resistance of the membrane limits fluid flow caused by pressure differences in the system, but allows diffusive transport of a chemical species through the membrane and into the channel. The large volume sink region at the end of the microfluidic channel helps to maintain spatial and temporal stability of the gradient. The chemical gradient in a 0.5 mm region near the sink region experiences a maximum of 10 percent change between the 6 and 24 h data points. We present the theory, design, and characterization of this device and provide an example of neutrophil chemotaxis as proof of concept for future quantitative cell-signaling applications.     

Generation of concentration gradient by controlled flow distribution and diffusive mixing in a microfluidic chip

We have developed a simple method to generate a concentration gradient in a microfluidic device. This method is based on the combination of controlled fluid distribution at each intersection of a microfluidic network by liquid pressure and subsequent diffusion between laminas in the downstream microchannel. A fluid dynamic model taking into account the diffusion coefficient was established to simulate the on-chip flow distribution and diffusion. Concentration gradients along a distance of a few hundred micrometers were generated in a series of microchannels. The gradients could be varied by carefully regulating the liquid pressure applied to the sample injection vials. The observed concentration gradients of fluorescent dyes generated on the microfluidic channel are consistent with the theoretically predicted results. The microfluidic design described in this study may provide a new tool for applications based on concentration gradients, including many biological and chemical analyses such as cellular reaction monitoring and drug screening.     

微流控芯片实验室

以作者所在课题组近年来的研究工作为基础,就芯片实验室平台建设及相应的以系统生物学为最终目标的功能化研究作一说明,对在分子和细胞层面,甚至是单分子、单细胞水平上实现以规模集成为特征的临床诊断和药物筛选的努力予以特别的关注。     

Rapid measurement of sphingolipids from Arabidopsis thaliana by reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry

Changes in sphingolipids have been associated with profound effects in cell fate and development in both plants and animals. Sphingolipids as a group consist of a large number of different compound classes of which numerous individual species may vary in response to environmental stimuli to affect cellular responses. The ability to measure all sphingolipids simultaneously is, therefore, essential to an understanding of the biochemical regulation of sphingolipid metabolism and signaling molecules derived from it. In the model plant Arabidopsis thaliana, the major sphingolipid classes are glycosylinositolphosphoceramides, glucosylceramides, hydroxyceramides and ceramides. Other minor but potentially important sphingolipids are free long-chain bases and their phosphorylated derivates. By using a single solvent system with reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry detection we have been able to separate and measure 168 sphingolipids from a crude sample. This greatly speeds up and simplifies the analysis of plant sphingolipids and should pave the way for a better understanding of their role in plant performance.     

Polymeric materials that convert local fleeting signals into global macroscopic responses

We report a general design strategy for creating polymeric materials that are capable of providing global, macroscopic changes in their properties in response to specific local and fleeting stimuli. In a proof-of-concept demonstration, a single polymer is used, yet it enables selective responses to specific stimuli, and then internally drives a macroscopic change in the material (even in locations not exposed to the stimulus), where the magnitude of change is independent of the intensity of the applied stimulus.     

微流控芯片系统在单细胞研究中的应用

微流控芯片具有网络式通道结构,扩展了在细胞和亚细胞水平进行生命科学研究的能力,为单细胞研究提供了一个新的平台.在微流控芯片通道中,人们利用气压、液压和电压,或利用介电电泳、光学陷阱、行波介电电泳以及磁场等技术,可以操纵细胞通过或驻留在通道内的任意位置,从而使单细胞计数、筛选以及胞内组分分析等操作大大简化.本文对微流控芯片系统在血液流变学、单细胞操纵与计数以及单细胞胞内组分分析中的应用进行了综述,介绍了用于单细胞研究的多种微芯片系统,讨论了芯片上进行单细胞操纵的各种方法