An automated microfluidic system for single-stranded DNA preparation and magnetic bead-based microarray analysis

We present an integrated microfluidic device capable of performing single-stranded DNA (ssDNA) preparation and magnetic bead-based microarray analysis with a white-light detection for detecting mutations that account for hereditary hearing loss. The entire operation process, which includes loading of streptavidin-coated magnetic beads (MBs) and biotin-labeled polymerase chain reaction products, active dispersion of the MBs with DNA for binding, alkaline denaturation of DNA, dynamic hybridization of the bead-labeled ssDNA to a tag array, and white-light detection, can all be automatically accomplished in a single chamber of the microchip, which was operated on a self-contained instrument with all the necessary components for thermal control, fluidic control, and detection. Two novel mixing valves with embedded polydimethylsiloxane membranes, which can alternately generate a 3-μl pulse flow at a peak rate of around 160 mm/s, were integrated into the chip for thoroughly dispersing magnetic beads in 2 min. The binding efficiency of biotinylated oligonucleotides to beads was measured to be 80.6% of that obtained in a tube with the conventional method. To critically test the performance of this automated microsystem, we employed a commercial microarray-based detection kit for detecting nine mutation loci that account for hereditary hearing loss. The limit of detection of the microsystem was determined as 2.5 ng of input K562 standard genomic DNA using this kit. In addition, four blood samples obtained from persons with mutations were all correctly typed by our system in less than 45 min per run. The fully automated, “amplicon-in-answer-out” operation, together with the white-light detection, makes our system an excellent platform for low-cost, rapid genotyping in clinical diagnosis.     

Effect of a dual inlet channel on cell loading in microfluidics

Unwanted sedimentation and attachment of a number of cells onto the bottom channel often occur on relatively large-scale inlets of conventional microfluidic channels as a result of gravity and fluid shear. Phenomena such as sedimentation have become recognized problems that can be overcome by performing microfluidic experiments properly, such as by calculating a meaningful output efficiency with respect to real input. Here, we present a dual-inlet design method for reducing cell loss at the inlet of channels by adding a new “ upstream inlet ” to a single main inlet design. The simple addition of an upstream inlet can create a vertically layered sheath flow prior to the main inlet for cell loading. The bottom layer flow plays a critical role in preventing the cells from attaching to the bottom of the channel entrance, resulting in a low possibility of cell sedimentation at the main channel entrance. To provide proof-of-concept validation, we applied our design to a microfabricated flow cytometer system (μFCS) and compared the cell counting efficiency of the proposed μFCS with that of the previous single-inlet μFCS and conventional FCS. We used human white blood cells and fluorescent microspheres to quantitatively evaluate the rate of cell sedimentation in the main inlet and to measure fluorescence sensitivity at the detection zone of the flow cytometer microchip. Generating a sheath flow as the bottom layer was meaningfully used to reduce the depth of field as well as the relative deviation of targets in the z-direction (compared to the x-y flow plane), leading to an increased counting sensitivity of fluorescent detection signals. Counting results using fluorescent microspheres showed both a 40% reduction in the rate of sedimentation and a 2-fold higher sensitivity in comparison with the single-inlet μFCS. The results of CD4⁺ T-cell counting also showed that the proposed design results in a 25% decrease in the rate of cell sedimentation and a 28% increase in sensitivity when compared to the single-inlet μFCS. This method is simple and easy to use in design, yet requires no additional time or cost in fabrication. Furthermore, we expect that this approach could potentially be helpful for calculating exact cell loading and counting efficiency for a small input number of cells, such as primary cells and rare cells, in microfluidic channel applications.     

Experimental validation of numerical study on thermoelectric-based heating in an integrated centrifugal microfluidic platform for polymerase chain reaction amplification

A comprehensive study involving numerical analysis and experimental validation of temperature transients within a microchamber was performed for thermocycling operation in an integrated centrifugal microfluidic platform for polymerase chain reaction (PCR) amplification. Controlled heating and cooling of biological samples are essential processes in many sample preparation and detection steps for micro-total analysis systems. Specifically, the PCR process relies on highly controllable and uniform heating of nucleic acid samples for successful and efficient amplification. In these miniaturized systems, the heating process is often performed more rapidly, making the temperature control more difficult, and adding complexity to the integrated hardware system. To gain further insight into the complex temperature profiles within the PCR microchamber, numerical simulations using computational fluid dynamics and computational heat transfer were performed. The designed integrated centrifugal microfluidics platform utilizes thermoelectrics for ice-valving and thermocycling for PCR amplification. Embedded micro-thermocouples were used to record the static and dynamic thermal responses in the experiments. The data collected was subsequently used for computational validation of the numerical predictions for the system response during thermocycling, and these simulations were found to be in agreement with the experimental data to within ∼97%. When thermal contact resistance values were incorporated in the simulations, the numerical predictions were found to be in agreement with the experimental data to within ∼99.9%. This in-depth numerical modeling and experimental validation of a complex single-sided heating platform provide insights into hardware and system design for multi-layered polymer microfluidic systems. In addition, the biological capability along with the practical feasibility of the integrated system is demonstrated by successfully performing PCR amplification of a Group B Streptococcus gene.     

城镇职工医疗保险统筹住院次均费用增长分析和对策

次均费用反映了当今社会普遍关注的看病贵难题,体现医疗保险制度效率。次均费用受多种因素影响,经济、人口结构和质量、医药卫生体制和新诊疗技术的使用是主要影响因素。增加过度治疗的机会成本,当边际成本超过边际收益时定点机构才会加强自律,规范治疗。引导就医流向可以降低来自参保人行为导致的费用增长。医保管理者在控制次均费用增长问题上责任重大,并大有作为。实行按病种支付、对三级医院重点检查、增加平均住院日指标、规定大型设备阳性检查率、充分利用信息平台加强管理等有利于控制次均费用不合理增长。     

A microfluidic device for on-chip agarose microbead generation with ultralow reagent consumption

Water-in-oil microdroplets offer microreactors for compartmentalized biochemical reactions with high throughput. Recently, the combination with a sol-gel switch ability, using agarose-in-oil microdroplets, has increased the range of possible applications, allowing for example the capture of amplicons in the gel phase for the preservation of monoclonality during a PCR reaction. Here, we report a new method for generating such agarose-in-oil microdroplets on a microfluidic device, with minimized inlet dead volume, on-chip cooling, and in situ monitoring of biochemical reactions within the gelified microbeads. We used a flow-focusing microchannel network and successfully generated agarose microdroplets at room temperature using the “push-pull” method. This method consists in pushing the oil continuous phase only, while suction is applied to the device outlet. The agarose phase present at the inlet is thus aspirated in the device, and segmented in microdroplets. The cooling system consists of two copper wires embedded in the microfluidic device. The transition from agarose microdroplets to microbeads provides additional stability and facilitated manipulation. We demonstrate the potential of this method by performing on-chip a temperature-triggered DNA isothermal amplification in agarose microbeads. Our device thus provides a new way to generate microbeads with high throughput and no dead volume for biochemical applications.     

Microfluidic-driven viral infection on cell cultures: Theoretical and experimental study

Advanced cell culture systems creating a controlled and predictable microenvironment together with computational modeling may be useful tools to optimize the efficiency of cell infections. In this paper, we will present a phenomenological study of a virus-host infection system, and the development of a multilayered microfluidic platform used to accurately tune the virus delivery from a diffusive-limited regime to a convective-dominated regime. Mathematical models predicted the convective-diffusive regimes developed within the system itself and determined the dominating mass transport phenomena. Adenoviral vectors carrying the enhanced green fluorescent protein (EGFP) transgene were used at different multiplicities of infection (MOI) to infect multiple cell types, both in standard static and in perfused conditions. Our results validate the mathematical models and demonstrate how the infection processes through perfusion via microfluidic platform led to an enhancement of adenoviral infection efficiency even at low MOIs. This was particularly evident at the longer time points, since the establishment of steady-state condition guaranteed a constant viral concentration close to cells, thus strengthening the efficiency of infection. Finally, we introduced the concept of effective MOI, a more appropriate variable for microfluidic infections that considers the number of adenoviruses in solution per cell at a certain time.     

A microfluidic platform for size-dependent generation of droplet interface bilayer networks on rails

Droplet interface bilayer (DIB) networks are emerging as a cornerstone technology for the bottom up construction of cell-like and tissue-like structures and bio-devices. They are an exciting and versatile model-membrane platform, seeing increasing use in the disciplines of synthetic biology, chemical biology, and membrane biophysics. DIBs are formed when lipid-coated water-in-oil droplets are brought together—oil is excluded from the interface, resulting in a bilayer. Perhaps the greatest feature of the DIB platform is the ability to generate bilayer networks by connecting multiple droplets together, which can in turn be used in applications ranging from tissue mimics, multicellular models, and bio-devices. For such applications, the construction and release of DIB networks of defined size and composition on-demand is crucial. We have developed a droplet-based microfluidic method for the generation of different sized DIB networks (300–1500 pl droplets) on-chip. We do this by employing a droplet-on-rails strategy where droplets are guided down designated paths of a chip with the aid of microfabricated grooves or “rails,” and droplets of set sizes are selectively directed to specific rails using auxiliary flows. In this way we can uniquely produce parallel bilayer networks of defined sizes. By trapping several droplets in a rail, extended DIB networks containing up to 20 sequential bilayers could be constructed. The trapped DIB arrays can be composed of different lipid types and can be released on-demand and regenerated within seconds. We show that chemical signals can be propagated across the bio-network by transplanting enzymatic reaction cascades for inter-droplet communication.     

Design and integration of an all-in-one biomicrofluidic chip

We demonstrate a highly integrated microfluidic chip with the function of DNA amplification. The integrated chip combines giant electrorheological-fluid actuated micromixer and micropump with a microheater array, all formed using soft lithography. Internal functional components are based on polydimethylsiloxane (PDMS) and silver∕carbon black-PDMS composites. The system has the advantages of small size with a high degree of integration, high polymerase chain reaction efficiency, digital control and simple fabrication at low cost. This integration approach shows promise for a broad range of applications in chemical synthesis and biological sensing∕analysis, as different components can be combined to target desired functionalities, with flexible designs of different microchips easily realizable through soft lithography.     

Rapid microfluidic solid-phase extraction system for hyper-methylated DNA enrichment and epigenetic analysis

Genetic sequence and hyper-methylation profile information from the promoter regions of tumor suppressor genes are important for cancer disease investigation. Since hyper-methylated DNA (hm-DNA) is typically present in ultra-low concentrations in biological samples, such as stool, urine, and saliva, sample enrichment and amplification is typically required before detection. We present a rapid microfluidic solid phase extraction (μSPE) system for the capture and elution of low concentrations of hm-DNA (≤1 ng ml⁻¹), based on a protein-DNA capture surface, into small volumes using a passive microfluidic lab-on-a-chip platform. All assay steps have been qualitatively characterized using a real-time surface plasmon resonance (SPR) biosensor, and quantitatively characterized using fluorescence spectroscopy. The hm-DNA capture/elution process requires less than 5 min with an efficiency of 71% using a 25 μl elution volume and 92% efficiency using a 100 μl elution volume.     

A “twisted” microfluidic mixer suitable for a wide range of flow rate applications

This paper proposes a new “twisted” 3D microfluidic mixer fabricated by a laser writing/microfabrication technique. Effective and efficient mixing using the twisted micromixers can be obtained by combining two general chaotic mixing mechanisms: splitting/recombining and chaotic advection. The lamination of mixer units provides the splitting and recombination mechanism when the quadrant of circles is arranged in a two-layered serial arrangement of mixing units. The overall 3D path of the microchannel introduces the advection. An experimental investigation using chemical solutions revealed that these novel 3D passive microfluidic mixers were stable and could be operated at a wide range of flow rates. This micromixer finds application in the manipulation of tiny volumes of liquids that are crucial in diagnostics. The mixing performance was evaluated by dye visualization, and using a pH test that determined the chemical reaction of the solutions. A comparison of the tornado-mixer with this twisted micromixer was made to evaluate the efficiency of mixing. The efficiency of mixing was calculated within the channel by acquiring intensities using ImageJ software. Results suggested that efficient mixing can be obtained when more than 3 units were consecutively placed. The geometry of the device, which has a length of 30 mm, enables the device to be integrated with micro total analysis systems and other lab-on-chip devices.     

Toward a modular,integrated, miniaturized,and portable microfluidic flow control architecture for organs-on-chips applications

Microfluidic organs-on-chips (OoCs) technology has emerged as the trend for in vitro functional modeling of organs in recent years. Simplifying the complexities of the human organs under controlled perfusion of required fluids paves the way for accurate prediction of human organ functionalities and their response to interventions like exposure to drugs. However, in the state-of-the-art OoC, the existing methods to control fluids use external bulky peripheral components and systems much larger than the chips used in experiments. A new generation of compact microfluidic flow control systems is needed to overcome this challenge. This study first presents a structured classification of OoC devices according to their types and microfluidic complexities. Next, we suggest three fundamental fluid flow control mechanisms and define component configurations for different levels of OoC complexity for each respective mechanism. Finally, we propose an architecture integrating modular microfluidic flow control components and OoC devices on a single platform. We emphasize the need for miniaturization of flow control components to achieve portability, minimize sample usage, minimize dead volume, improve the flowing time of fluids to the OoC cell chamber, and enable long-duration experiments.     

An integrated, multiparametric flow cytometry chip using ��microfluidic drifting�� based three-dimensional hydrodynamic focusing

In this work, we demonstrate an integrated, single-layer, miniature flow cytometry device that is capable of multi-parametric particle analysis. The device integrates both particle focusing and detection components on-chip, including a “microfluidic drifting” based three-dimensional (3D) hydrodynamic focusing component and a series of optical fibers integrated into the microfluidic architecture to facilitate on-chip detection. With this design, multiple optical signals (i.e., forward scatter, side scatter, and fluorescence) from individual particles can be simultaneously detected. Experimental results indicate that the performance of our flow cytometry chip is comparable to its bulky, expensive desktop counterpart. The integration of on-chip 3D particle focusing with on-chip multi-parametric optical detection in a single-layer, mass-producible microfluidic device presents a major step towards low-cost flow cytometry chips for point-of-care clinical diagnostics.     

复杂系统视角下次发达地区科技金融创新体系构建研究——以河北省为例

基于复杂系统理论,以河北省为例分析我国次发达地区科技金融创新体系主体结构、合作过程和体系演化的复杂性,并分别针对上述三方面问题探索科技金融对接途径。设计多主体科技金融创新服务平台,建立主体间合作的收益分配、信息共享和风险管理机制,并提出有助于科技金融创新体系演化的政策建议,以实现金融服务与科技创新的深度融合,推进次发达地区经济结构转型和创新驱动发展。     

Magnetophoretic-based microfluidic device for DNA isolation

This paper presents a continuous flow microfluidic device for the separation of DNA from blood using magnetophoresis for biological applications and analysis. This microfluidic bio-separation device has several benefits, including decreased sample handling, smaller sample and reagent volumes, faster isolation time, and decreased cost to perform DNA isolation. One of the key features of this device is the use of short-range magnetic field gradients, generated by a micro-patterned nickel array on the bottom surface of the separation channel. In addition, the device utilizes an array of oppositely oriented, external permanent magnets to produce strong long-range field gradients at the interfaces between magnets, further increasing the effectiveness of the device. A comprehensive simulation is performed using COMSOL Multiphysics to study the effect of various parameters on the magnetic flux within the separation channel. Additionally, a microfluidic device is designed, fabricated, and tested to isolate DNA from blood. The results show that the device has the capability of separating DNA from a blood sample with a purity of 1.8 or higher, a yield of up to 33 μg of polymerase chain reaction ready DNA per milliliter of blood, and a volumetric throughput of up to 50 ml/h.     

农村科技信息平台多模式服务体系结合的研究——湖南省12396科技信息平台服务模式探索

本文从湖南省12396科技信息平台构架、多种农村服务体系模式结合方式及运行模式的实践中,研究探索"整合资源、集成优势、降低成本、优化服务、提高效能"的农村科技服务结合模式,并对该信息服务体系作出了"集合优势、互补优势、创新优势"的评价。为促进社会主义新农村建设,满足农业发展对科技信息需求,提出了可供借鉴的创新思路。     

Flow control concepts for thread-based microfluidic devices

The emerging concept of thread-based microfluidics has shown great promise for application to inexpensive disease detection and environmental monitoring. To allow the creation of more sophisticated and functional thread-based sensor designs, the ability to better control and understand the flow of fluids in the devices is required. To meet this end, various mechanisms for controlling the flow of reagents and samples in thread-based microfluidic devices are investigated in this study. A study of fluid penetration in single threads and in twined threads provides greater practical understanding of fluid velocity and ultimate penetration for the design of devices. “Switches” which control when or where flow can occur, or allow the mixing of multiple fluids, have been successfully prototyped from multifilament threads, plastic films, and household adhesive. This advancement allows the fabrication of more functional sensory devices which can incorporate more complex detection chemistries, while maintaining low production cost and simplicity of construction.     

基于DM642的混合高斯模型运动目标检测系统设计与实现

运动目标检测是智能视频监控技术的重要环节,精确的分割出运动目标是后续监控技术得以实现的重要基础。该文以强大运算能力的DM642为核心搭建了运动目标检测系统硬件平台,实现和优化了基于混合高斯模型的检测算法。实验表明,该系统能有效地检测出运动目标,实时性较高。     

Biofunctionalized nanoslits for wash-free and spatially resolved real-time sensing with full target capture

We propose biofunctionalized nanofluidic slits (nanoslits) as an effective platform for real-time fluorescence-based biosensing in a reaction-limited regime with optimized target capture efficiency. This is achieved by the drastic reduction of the diffusion length, thereby a boosted collision frequency between the target analytes and the sensor, and the size reduction of the sensing element down to the channel height comparable to the depletion layer caused by the reaction. Hybridization experiments conducted in DNA-functionalized nanoslits demonstrate the analyte depletion and the wash-free detection ∼10 times faster compared to the best microfluidic sensing platforms. The signal to background fluorescence ratio is drastically increased at lower target concentrations, in favor of low-copy number analyte analysis. Experimental and simulation results further show that biofunctionalized nanoslits provide a simple means to study reaction kinetics at the single-pixel level using conventional fluorescence microscopy with reduced optical depth.     

DNA capture-probe based separation of double-stranded polymerase chain reaction amplification products in poly(dimethylsiloxane) microfluidic channels

Herein, we describe the development of a novel primer system that allows for the capture of double-stranded polymerase chain reaction (PCR) amplification products onto a microfluidic channel without any preliminary purification stages. We show that specially designed PCR primers consisting of the main primer sequence and an additional “tag sequence” linked through a poly(ethylene glycol) molecule can be used to generate ds-PCR amplification products tailed with ss-oligonucleotides of two forensically relevant genes (amelogenin and human c-fms (macrophage colony-stimulating factor) proto-oncogene for the CSF-1 receptor (CSF1PO). Furthermore, with a view to enriching and eluting the ds-PCR products of amplification on a capillary electrophoretic-based microfluidic device we describe the capture of the target ds-PCR products onto poly(dimethylsiloxane) microchannels modified with ss-oligonucleotide capture probes.     

Efficient elusion of viable adhesive cells from a microfluidic system by air foam

We developed a new method for releasing viable cells from affinity-based microfluidic devices. The lumen of a microchannel with a U-shape and user-designed microstructures was coated with supported lipid bilayers functionalized by epithelial cell adhesion molecule antibodies to capture circulating epithelial cells of influx solution. After the capturing process, air foam was introduced into channels for releasing target cells and then carrying them to a small area of membrane. The results show that when the air foam is driven at linear velocity of 4.2 mm/s for more than 20 min or at linear velocity of 8.4 mm/s for more than 10 min, the cell releasing efficiency approaches 100%. This flow-induced shear stress is much less than the physiological level (15 dyn/cm²), which is necessary to maintain the intactness of released cells. Combining the design of microstructures of the microfluidic system, the cell recovery on the membrane exceeds 90%. Importantly, we demonstrate that the cells released by air foam are viable and could be cultured in vitro. This novel method for releasing cells could power the microfluidic platform for isolating and identifying circulating tumor cells.