Biopolymer system for cell recovery from microfluidic cell capture devices

Microfluidic systems for affinity-based cell isolation have emerged as a promising approach for the isolation of specific cells from complex matrices (i.e., circulating tumor cells in whole blood). However, these technologies remain limited by the lack of reliable methods for the innocuous recovery of surface captured cells. Here, we present a biofunctional sacrificial hydrogel coating for microfluidic chips that enables the highly efficient release of isolated cells (99% ± 1%) following gel dissolution. This covalently cross-linked alginate biopolymer system is stable in a wide variety of physiologic solutions (including EDTA treated whole blood) and may be rapidly degraded via backbone cleavage with alginate lyase. The capture and release of EpCAM expressing cancer cells using this approach was found to have no significant effect on cell viability or proliferative potential, and recovered cells were demonstrated to be compatible with downstream immunostaining and FISH analysis.     

微流体通道循环肿瘤细胞检定系统研究

提出在微流体框架上搭建电阻抗型循环肿瘤细胞检测计数和引入光学元件搭建病变循环肿瘤细胞检测的双重荧光激发传感器系统.前者提出了两种不同结构,在直流电路型中得到细胞体积越大峰值电压越高,并且两者有很好的线性关系,相关系数可以达到0.998,也可以得到细胞体积越大,细胞移位时间越长;在交流电路型中,创新性提出印刷电路板和聚二甲基硅氧烷结合作为衬底的思想,并和商用流式细胞仪的测量结果进行了对比,验证了这种结构的可行性.后者用特定病变肿瘤被荧光标记物标记后,受激光激发出的荧光颜色不同而可以被有效地区分开,并拿商用流式细胞仪结论进行对比,结果吻合.两种传感器都用COMSOL软件进行了理论模拟,便于确定结构参数的同时也能与实验结果对比从而优化结论.     

Versatile, fully automated, microfluidic cell culture system

There is increasing demand for automated and quantitative cell culture technology, driven both by the intense activity in stem cell biology and by the emergence of systems biology. We built a fully automated cell culture screening system based on a microfluidic chip that creates arbitrary culture media formulations in 96 independent culture chambers and maintains cell viability for weeks. Individual culture conditions are customized in terms of cell seeding density, composition of culture medium, and feeding schedule, and each chamber is imaged with time-lapse microscopy. Using this device, we perform the first quantitative measurements of the influence of transient stimulation schedules on the proliferation, osteogenic differentiation, and motility of human primary mesenchymal stem cells.     

Electrophoretic cell manipulation and electrochemical gene-function analysis based on a yeast two-hybrid system in a microfluidic device

A novel microfluidic device with an array of analytical chambers was developed in order to perform single-cell-based gene-function analysis. A series of analytical processes was carried out using the device, including electrophoretic manipulation of single cells and electrochemical measurement of gene function. A poly(dimethylsiloxane) microstructure with a microfluidic channel (150 microm in width, 10 microm in height) and an analytical chamber (100 x 20 x 10 microm (3)) were fabricated and aligned on a glass substrate with an array of Au microelectrodes. Two microelectrodes positioned in the analytical chamber were employed as a working electrode for the electrophoretic manipulation of cells and electrochemical measurements. A yeast strain ( Saccharomyces cerevisiae Y190) carrying the beta-galactosidase reporter gene was used to demonstrate that the device could detect the enzyme. Target cells flowing through the main channel were introduced into the chamber by electrophoresis using the ground electrode laid on the main channel. When the cell was treated with 17beta-estradiol, gene expression was triggered to produce beta-galactosidase, catalyzing the hydrolysis of p-aminophenyl-beta- D-galactopyranoside to form p-aminophenol (PAP). The enzymatically generated PAP was detected by cyclic voltammetry and amperometry at the single-cell level in the chamber of the device. Generator-collector mode amperometry was also applied to amplify the current response originating from gene expression in the trapped single cells. After electrochemical measurement, the trapped cells were easily released from the chamber using electrophoretic force.     

Improved experimental time of ultra‐large bioassays using a parallelised microfluidic biochip architecture/scheduling

Digital microfluidic is an emerging technology to reduce the cost and time of experiments and improve the flexibility, automate‐ability and correctness of biochemical assays. In many of applications such as drug discovery and DNA profiling, a large number of bio‐operations (e.g. the chemical operations used in biology applications) must be done. In these applications, parallelising the operations will be critical in accuracy and cost of the process and digital microfluidic biochips can be considered as a reasonable platform. In this study, a new microfluidic architecture and the corresponding CAD flow is introduced to parallelise the assays on this platform. The authors implemented the proposed architecture and evaluated it using the large real bioassays. The authors’ simulations show that the degree‐of‐parallelism and speed of bioassays are increased more than 4× and the improvements will be better for larger assays. This contribution can open new horizons in drug testing, biology experiments and medical diagnosis operations that contain iterative, time‐consuming and labour experiments.Inspec keywords: microfluidics, lab‐on‐a‐chip, biochemistry, bioMEMS, biomedical equipment, CADOther keywords: biochemical assays, medical diagnosis operations, biology experiments, drug testing, CAD flow, parallelised microfluidic biochip architecture‐scheduling, ultralarge bioassays     

Development of a low-cost magnetic microfluidic chip for circulating tumour cell capture

The authors have developed a novel fabrication process for a selective micro-magnetic activated cell sorting (MACS) chip based on ferromagnetic material encapsulated micropillars (FMEMs), which is technically simple and low cost. The FMEM produces a high field gradient to magnetically attract, capture and hold cells on its interface. System test simulations were carried out to predict the efficacy of target capture and verify that the actual magnetic particles behaviour agreed well with model predictions. To determine the ability of the novel microMACS chip to capture circulating tumour cells (CTCs), SW620 human colon cancer cells were used in an in vitro flow model system and were able to be captured with the efficiency of 72.8%. The obvious accumulation of CTCs at a certain location on the chip suggested shear stress events at the pillar boundary may influence efficacy, and should be considered in further optimisation efforts.     

Generating nonlinear concentration gradients in microfluidic devices for cell studies

We describe a microfluidic device for generating nonlinear (exponential and sigmoidal) concentration gradients, coupled with a microwell array for cell storage and analysis. The device has two inputs for coflowing multiple aqueous solutions, a main coflow channel and an asymmetrical grid of fluidic channels that allows the two solutions to combine at intersection points without fully mixing. Due to this asymmetry and diffusion of the two species in the coflow channel, varying amounts of the two solutions enter each fluidic path. This induces exponential and sigmoidal concentration gradients at low and high flow rates, respectively, making the microfluidic device versatile. A key feature of this design is that it is space-saving, as it does not require multiplexing or a separate array of mixing channels. Furthermore, the gradient structure can be utilized in concert with cell experiments, to expose cells captured in microwells to various concentrations of soluble factors. We demonstrate the utility of this design to assess the viability of fibroblast cells in response to a range of hydrogen peroxide (H(2)O(2)) concentrations.     

Infrared temperature control system for a completely noncontact polymerase chain reaction in microfluidic chips

A completely noncontact temperature system is described for amplification of DNA via the polymerase chain reaction (PCR) in glass microfluidic chips. An infrared (IR)-sensitive pyrometer was calibrated against a thermocouple inserted into a 550-nL PCR chamber and used to monitor the temperature of the glass surface above the PCR chamber during heating and cooling induced by a tungsten lamp and convective air source, respectively. A time lag of less than 1 s was observed between maximum heating rates of the solution and surface, indicating that thermal equilibrium was attained rapidly. Moreover, the time lag was corroborated using a one-dimensional heat-transfer model, which provided insight into the characteristics of the device and environment that caused the time lag. This knowledge will, in turn, allow for future tailoring of the devices to specific applications. To alleviate the need for calibrating the pyrometer with a thermocouple, the on-chip calibration of pyrometer was accomplished by sensing the boiling of two solutions, water and an azeotrope, and comparing the pyrometer output voltage against the known boiling points of these solutions. The "boiling point calibration" was successful as indicated by the subsequent chip-based IR-PCR amplification of a 211-bp fragment of the B. anthracis genome in a chamber reduced beyond the dimensions of a thermocouple. To improve the heating rates, a parabolic gold mirror was positioned above the microfluidic chip, which expedited PCR amplification to 18.8 min for a 30-cycle, three-temperature protocol.     

A microfluidic system for large DNA molecule arrays

Single molecule approaches offer the promise of large, exquisitely miniature ensembles for the generation of equally large data sets. Although microfluidic devices have previously been designed to manipulate single DNA molecules, many of the functionalities they embody are not applicable to very large DNA molecules, normally extracted from cells. Importantly, such microfluidic devices must work within an integrated system to enable high-throughput biological or biochemical analysis-a key measure of any device aimed at the chemical/biological interface and required if large data sets are to be created for subsequent analysis. The challenge here was to design an integrated microfluidic device to control the deposition or elongation of large DNA molecules (up to millimeters in length), which would serve as a general platform for biological/biochemical analysis to function within an integrated system that included massively parallel data collection and analysis. The approach we took was to use replica molding to construct silastic devices to consistently deposit oriented, elongated DNA molecules onto charged surfaces, creating massive single molecule arrays, which we analyzed for both physical and biochemical insights within an integrated environment that created large data sets. The overall efficacy of this approach was demonstrated by the restriction enzyme mapping and identification of single human genomic DNA molecules.     

Integrated system for rapid PCR-based DNA analysis in microfluidic devices

An integrated system for rapid PCR-based analysis on a microchip has been demonstrated. The system couples a compact thermal cycling assembly based on dual Peltier thermoelectric elements with a microchip gel electrophoresis platform. This configuration allows fast (approximately 1 min/ cycle) and efficient DNA amplification on-chip followed by electrophoretic sizing and detection on the same chip. An on-chip DNA concentration technique has been incorporated into the system to further reduce analysis time by decreasing the number of thermal cycles required. The concentration injection scheme enables detection of PCR products after performing as few as 10 thermal cycles, with a total analysis time of less than 20 min. The starting template copy number was less than 15 per injection volume.     

Measurement of enzyme kinetics using a continuous-flow microfluidic system

This paper describes a microanalytical method for determining enzyme kinetics using a continuous-flow microfluidic system. The analysis is carried out by immobilizing the enzyme on microbeads, packing the microbeads into a chip-based microreactor (volume approximately 1.0 nL), and flowing the substrate over the packed bed. Data were analyzed using the Lilly-Hornby equation and compared to values obtained from conventional measurements based on the Michaelis-Menten equation. The two different enzyme-catalyzed reactions studied were chosen so that the substrate would be nonfluorescent and the product fluorescent. The first reaction involved the horseradish peroxidase-catalyzed reaction between hydrogen peroxide and N-acetyl-3,7-dihydroxyphenoxazine (amplex red) to yield fluorescent resorufin, and the second the beta-galactosidase-catalyzed reaction of nonfluorescent resorufin-beta-D-galactopyranoside to yield D-galactose and fluorescent resorufin. In both cases, the microfluidics-based method yielded the same result obtained from the standard Michaelis-Menten treatment. The continuous-flow method required approximately 10 microL of substrate solution and 10(9) enzyme molecules. This approach provides a new means for rapid determination of enzyme kinetics in microfluidic systems, which may be useful for clinical diagnostics, and drug discovery and screening.     

Development of a multichannel microfluidic analysis system employing affinity capillary electrophoresis for immunoassay

A six-channel microfluidic immunoassay device with a scanned fluorescence detection system is described. Six independent mixing, reaction, and separation manifolds are integrated within one microfluidic wafer, along with two optical alignment channels. The manifolds are operated simultaneously and data are acquired using a singlepoint fluorescence detector with a galvano-scanner to step between separation channels. A detection limit of 30 pM was obtained for fluorescein with the scanning detector, using a 7.1-Hz sampling rate for each of the reaction manifolds and alignment channels (57-Hz overall sampling rate). Simultaneous direct immunoassays for ovalbumin and for anti-estradiol were performed within the microfluidic device. Mixing, reaction, and separation could be performed within 60 s in all cases and within 30 s under optimized conditions. Simultaneous calibration and analysis could be performed with calibrant in several manifolds and sample in the other manifolds, allowing a complete immunoassay to be run within 30 s. Careful chip conditioning with methanol, water, and 0.1 M NaOH resulted in peak height RSD values of 3-8% (N = 5 or 6), allowing for cross-channel calibration. The limit of detection (LOD) for an anti-estradial assay obtained in any single channel was 4.3 nM. The LOD for the cross-channel calibration was 6.4 nM. Factors influencing chip and detection system design and performance are discussed in detail.     

Lysing bacterial spores by sonication through a flexible interface in a microfluidic system

Cell disruptions using ultrasonic energy transmitted through a flexible interface into a liquid region has limitations because the motion of the vibrating tip is not completely transferred into the liquid. To ensure that some degree of contact will be maintained between the ultrasonic horn tip and the flexible interface, the liquid must be pressurized. The pressure conditions that yield consistent coupling between the ultrasonic horn tip and the liquid region were explored in this study by using an analytical model of the system and test fixture experiments. The nature of the interaction between the horn tip and the flexible interface creates pulses of positive pressure rises, increase in temperature, streaming flow, and almost no cavitation in the liquid. There was sufficient energy to create a cloud of microspheres, or beads, that maintain a consistent pattern of ballistic motion in the liquid. The sonication was found to be repeatable by studying video recordings of bead motion and was shown to be statistically consistent using measurements of temperature rise. Sonication of bacterial spores to obtain measurements of released nucleic acid and SEM images of damaged spores were used to verify the effects of liquid pressure on the horn-interface-liquid coupling.     

Multiple open-channel electroosmotic pumping system for microfluidic sample handling

The development of a novel, fully integrated, miniaturized pumping system for generation of pressure-driven flow in microfluidic platforms is described. The micropump, based on electroosmotic pumping principles, has a multiple open-channel configuration consisting of hundreds of parallel, small-diameter microchannels. Specifically, pumps with microchannels of 1-6 microm in depth, 4-50 mm in length, and an overall area of a few square millimeters, were constructed. Flow rates of 10-400 nL/min were generated in electric-field-free regions in a stable, reproducible and controllable manner. In addition, eluent gradients were created by simultaneously using two pumps. Pressures up to 80 psi were produced with the present pump configurations. The pump can be easily interfaced with other operational elements of a micrototal analysis system (micro-TAS) device with multiplexing capabilities. A new microfluidic valving system was also briefly evaluated in conjunction with these pumps. The micropump was utilized to deliver peptide samples for electrospray ionization-mass spectrometric (ESI-MS) detection.     

Reproducibility and robustness of a real-time microfluidic cell toxicity assay

Numerous opportunities exist to apply microfluidic technology to high-throughput and high-content cell-based assays. However, maximizing the value of microfluidic assays for applications such as drug discovery, screening, or toxicity evaluation will require assurance of within-device repeatability, day-to-day reproducibility, and robustness to variations in conditions that might occur from laboratory to laboratory. This report describes a study of the performance and variability of a cell-based toxicity assay in microfluidic devices made of poly(dimethylsiloxane) (PDMS). The assay involves expression of destabilized green fluorescent protein (GFP) as a reporter of intracellular protein synthesis and degradation. Reduction in cellular GFP due to inhibition of ribosome activity by cycloheximide (CHX) was quantified with real-time quantitative fluorescence imaging. Assay repeatability was measured within a 64-chamber microfluidic device. Assay performance across a range of cell loading densities within a single device was assessed, as was replication of measurements in microfluidic devices prepared on different days. Assay robustness was tested using different fluorescence illumination sources and reservoir-to-device tubing choices. Both microfluidic and larger scale assay conditions showed comparable GFP decay rates upon CHX exposure, but the microfluidic data provided the higher level of confidence.     

Flow sensing of single cell by graphene transistor in a microfluidic channel

The electronic properties of graphene are strongly influenced by electrostatic forces arising from long-range charge scatterers and by changes in the local dielectric environment. This makes graphene extremely sensitive to the surface charge density of cells interfacing with it. Here, we developed a graphene transistor array integrated with microfluidic flow cytometry for the "flow-catch-release" sensing of malaria-infected red blood cells at the single-cell level. Malaria-infected red blood cells induce highly sensitive capacitively coupled changes in the conductivity of graphene. Together with the characteristic conductance dwell times, specific microscopic information about the disease state can be obtained.     

Real-time microfluidic system for studying mammalian cells in 3D microenvironments

We describe a microfluidic system that can control, in real time, the microenvironments of mammalian cells in naturally derived 3D extracellular matrix (ECM). This chip combines pneumatically actuated valves with an individually addressable array of 3D cell-laden ECM; actuation of valves determines the pathways for delivering reagents through the chip and for exchanging diffusible factors between cell chambers. To promote rapid perfusion of reagents through 3D gels (with complete exchange of reagents within the gel in seconds), we created conduits above the gels for fluid flow, and microposts to stabilize the gels under high perfusion rates. As a biological demonstration, we studied spatially segregated mouse embryonic stem cells and mouse embryonic fibroblasts embedded in 3D Matrigel over days of culture. Overall, this system may be useful for high-throughput screening, single-cell analysis and studies of cell-cell communication, where rapid control of 3D cellular microenvironments is desired.     

转子-轴承系统液体式在线自动平衡装置研究综述

针对自动平衡装置可在线调整转子-轴承系统不平衡状态、提高设备工作效率、延长设备使用周期,通过分析国内外已有液体式在线自动平衡装置,认为注液式、释液式及连续注排液式三种平衡装置均因注排液过程存在诸多缺陷。液体转移式平衡装置在平衡过程中无需注液、排液,可从根本上避免三种结构因注、排液造成的缺陷,是一种理想结构。在保持注液式平衡装置结构简单、旋转部分无可动部件等优点前提下研究开发实用的液体转移式平衡装置,是液体式平衡装置极具前途的发展方向。     

A ferrofluid guided system for the rapid separation of the non-magnetic particles in a microfluidic device

A microfluidic device was fabricated via UV lithography technique to separate non-magnetic fluoresbrite carboxy microspheres (approximately 4.5 microm) in the pH 7 ferrofluids made of magnetite nanoparticles (approximately 10 nm). A mixture of microspheres and ferrofluid was injected to a lithographically developed Y shape microfluidic device, and then by applying the external magnet fields (0.45 T), the microspheres were clearly separated into different channels because of the magnetic force acting on those non-magnetic particles. During this study, various pumping speeds and particle concentrations associated with the various distances between the magnet and the microfluidic device were investigated for an efficient separation. This study may be useful for the separation of biological particles, which are very sensitive to pH value of the solutions.