A microfluidic DNA computing processor for gene expression analysis and gene drug synthesis

Boolean logic performs a logical operation on one or more logic input and produces a single logic output. Here, we describe a microfluidic DNA computing processor performing Boolean logic operations for gene expression analysis and gene drug synthesis. Multiple cancer-related genes were used as input molecules. Their expression levels were identified by interacting with the computing related DNA strands, which were designed according to the sequences of cancer-related genes and the suicide gene. When all the expressions of the cancer-related genes fit in with the diagnostic criteria, positive diagnosis would be confirmed and then a complete suicide gene (gene drug) could be synthesized as an output molecule. Microfluidic chip was employed as an effective platform to realize the computing process by integrating multistep biochemical reactions involving hybridization, displacement, denaturalization, and ligation. By combining the specific design of the computing related molecules and the integrated functions of the microfluidics, the microfluidic DNA computing processor is able to analyze the multiple gene expressions simultaneously and realize the corresponding gene drug synthesis with simplicity and fast speed, which demonstrates the potential of this platform for DNA computing in biomedical applications.     

Study of flow behaviors on single-cell manipulation and shear stress reduction in microfluidic chips using computational fluid dynamics simulations

Various single-cell retention structures (SCRSs) were reported for analysis of single cells within microfluidic devices. Undesirable flow behaviors within micro-environments not only influence single-cell manipulation and retention significantly but also lead to cell damage, biochemical heterogeneity among different individual cells (e.g., different cell signaling pathways induced by shear stress). However, the fundamentals in flow behaviors for single-cell manipulation and shear stress reduction, especially comparison of these behaviors in different microstructures, were not fully investigated in previous reports. Herein, flow distribution and induced shear stress in two different single-cell retention structures (SCRS I and SCRS II) were investigated in detail to study their effects on single-cell trapping using computational fluid dynamics (CFD) methods. The results were successfully verified by experimental results. Comparison between these two SCRS shows that the wasp-waisted configuration of SCRS II has a better performance in trapping and manipulating long cylinder-shaped cardiac myocytes and provides a safer “harbor” for fragile cells to prevent cell damage due to the shear stress induced from strong flows. The simulation results have not only explained flow phenomena observed in experiments but also predict new flow phenomena, providing guidelines for new chip design and optimization, and a better understanding of the cell micro-environment and fundamentals of microfluidic flows in single-cell manipulation and analysis.     

Label-free isolation of a prostate cancer cell among blood cells and the single-cell measurement of drug accumulation using an integrated microfluidic chip

Circulating tumor cells (CTCs) are found in the blood of patients with cancer. Although these cells are rare, they can provide useful information for chemotherapy. However, isolation of these rare cells from blood is technically challenging because they are small in numbers. An integrated microfluidic chip, dubbed CTC chip, was designed and fabricated for conducting tumor cell isolation. As CTCs usually show multidrug resistance (MDR), the effect of MDR inhibitors on chemotherapeutic drug accumulation in the isolated single tumor cell is measured. As a model of CTC isolation, human prostate cancer cells were mixed with mouse blood cells and the label-free isolation of the tumor cells was conducted based on cell size difference. The major advantages of the CTC chip are the ability for fast cell isolation, followed by multiple rounds of single-cell measurements, suggesting a potential assay for detecting the drug responses based on the liquid biopsy of cancer patients.     

Aortic heterogeneity across segments and under high fat/salt/glucose conditions at the single-cell level

The aorta, with ascending, arch, thoracic and abdominal segments, responds to the heartbeat, senses metabolites and distributes blood to all parts of the body. However, the heterogeneity across aortic segments and how metabolic pathologies change it are not known. Here, a total of 216 612 individual cells from the ascending aorta, aortic arch, and thoracic and abdominal segments of mouse aortas under normal conditions or with high blood glucose levels, high dietary salt, or high fat intake were profiled using single-cell RNA sequencing. We generated a compendium of 10 distinct cell types, mainly endothelial (EC), smooth muscle (SMC), stromal and immune cells. The distributions of the different cells and their intercommunication were influenced by the hemodynamic microenvironment across anatomical segments, and the spatial heterogeneity of ECs and SMCs may contribute to differential vascular dilation and constriction that were measured by wire myography. Importantly, the composition of aortic cells, their gene expression profiles and their regulatory intercellular networks broadly changed in response to high fat/salt/glucose conditions. Notably, the abdominal aorta showed the most dramatic changes in cellular composition, particularly involving ECs, fibroblasts and myeloid cells with cardiovascular risk factor-related regulons and gene expression networks. Our study elucidates the nature and range of aortic cell diversity, with implications for the treatment of metabolic pathologies.     

Modeling and validation of autoinducer-mediated bacterial gene expression in microfluidic environments

Biosensors exploiting communication within genetically engineered bacteria are becoming increasingly important for monitoring environmental changes. Currently, there are a variety of mathematical models for understanding and predicting how genetically engineered bacteria respond to molecular stimuli in these environments, but as sensors have miniaturized towards microfluidics and are subjected to complex time-varying inputs, the shortcomings of these models have become apparent. The effects of microfluidic environments such as low oxygen concentration, increased biofilm encapsulation, diffusion limited molecular distribution, and higher population densities strongly affect rate constants for gene expression not accounted for in previous models. We report a mathematical model that accurately predicts the biological response of the autoinducer N-acyl homoserine lactone-mediated green fluorescent protein expression in reporter bacteria in microfluidic environments by accommodating these rate constants. This generalized mass action model considers a chain of biomolecular events from input autoinducer chemical to fluorescent protein expression through a series of six chemical species. We have validated this model against experimental data from our own apparatus as well as prior published experimental results. Results indicate accurate prediction of dynamics (e.g., 14% peak time error from a pulse input) and with reduced mean-squared error with pulse or step inputs for a range of concentrations (10 μM–30 μM). This model can help advance the design of genetically engineered bacteria sensors and molecular communication devices.     

Improved single-cell culture achieved using micromolding in capillaries technology coupled with poly (HEMA)

Cell studies at the single-cell level are becoming more and more critical for understanding the complex biological processes. Here, we present an optimization study investigating the positioning of single cells using micromolding in capillaries technology coupled with the cytophobic biomaterial poly (2-hydroxyethyl methacrylate) (poly (HEMA)). As a cytophobic biomaterial, poly (HEMA) was used to inhibit cells, whereas the glass was used as the substrate to provide a cell adhesive background. The poly (HEMA) chemical barrier was obtained using micromolding in capillaries, and the microchannel networks used for capillarity were easily achieved by reversibly bonding the polydimethylsiloxane mold and the glass. Finally, discrete cell adhesion regions were presented on the glass surface. This method is facile and low cost, and the reagents are commercially available. We validated the cytophobic abilities of the poly (HEMA), optimized the channel parameters for higher quality and more stable poly (HEMA) patterns by investigating the effects of changing the aspect ratio and the width of the microchannel on the poly (HEMA) grid pattern, and improved the single-cell occupancy by optimizing the dimensions of the cell adhesion regions.     

Fabrication of microfluidic devices using polydimethylsiloxane

Polydimethylsiloxane (PDMS) is nearly ubiquitous in microfluidic devices, being easy to work with, economical, and transparent. A detailed protocol is provided here for using PDMS in the fabrication of microfluidic devices to aid those interested in using the material in their work, with information on the many potential ways the material may be used for novel devices.     

农民信息技术水平影响因素分析

＂十一五＂以来,各地深入推进农业农村信息化建设,农民的信息技术能力有了很大提高,湖南省农村信息化水平处于一个什么样的状况,有哪些影响因素,本文在调查的基础上发现影响湖南省农村信息化水平有基础设施不足、农民的文化素质不高、经济状况偏低等因素,提出通过帮助农民提高文化和信息素质,大力发展农村经济,加快基础设施建设,建立完善的涉农信息服务体系,提供多种多样的培训方式,以此来提高农村的信息化进程。     

Review of microfluidic microbioreactor technology for high-throughput submerged microbiological cultivation

Microbial fermentation process development is pursuing a high production yield. This requires a high throughput screening and optimization of the microbial strains, which is nowadays commonly achieved by applying slow and labor-intensive submerged cultivation in shake flasks or microtiter plates. These methods are also limited towards end-point measurements, low analytical data output, and control over the fermentation process. These drawbacks could be overcome by means of scaled-down microfluidic microbioreactors (μBR) that allow for online control over cultivation data and automation, hence reducing cost and time. This review goes beyond previous work not only by providing a detailed update on the current μBR fabrication techniques but also the operation and control of μBRs is compared to large scale fermentation reactors.     

国家层面信息技术价值及“生产率悖论”研究

本文在生产理论的基础上,将国家特征作为生产效率的影响因素引入传统的随机生产边界模型,建立了基于柯布-道格拉斯生产函数的二个方程随机生产边界模型,实证检验了我国1997-2010年IT资本对经济增长及生产效率的影响。研究发现:IT资本对我国经济增长和生产效率具有显著的促进作用,从国家层面证明了"生产率悖论"并不是全球化的现象;IT资本弱化了物质资本对于经济增长的贡献,强化了人力资本对于经济增长的贡献;国家特征促进了生产效率的提高,但是并没有促进IT价值;我国经济在发展过程中呈现规模报酬递减。基于实证研究结果,提出了实现我国IT投资价值的相关政策建议。     

环境科学专业开设微流控分析技术课程的必要性与可行性

微流控分析技术已成为生物、化学、流体物理学等多学科的基本研究手段,并有力地促进了这些学科的发展。近年来,国际上将该分析技术应用于环境分析监测领域已成为一种趋势。本文探讨了在这种形势下为环境科学专业学生开设微流控分析课程的必要性、可行性,并对如何将该课程导入环境分析课程板块给出了一些具体的设想。     

High-throughput sorting of drops in microfluidic chips using electric capacitance

We analyze a recently introduced approach for the sorting of aqueous drops with biological content immersed in oil, using a microfluidic chip that combines the functionality of electrowetting with the high throughput of two-phase flow microfluidics. In this electrostatic sorter, three co-planar electrodes covered by a thin dielectric layer are placed directly below the fluidic channel. Switching the potential of the central electrode creates an electrical guide that leads the drop to the desired outlet. The generated force, which deflects the drop, can be tuned via the voltage. The working principle is based on a contrast in conductivity between the drop and the continuous phase, which ensures successful operation even for drops of highly conductive biological media like phosphate buffered saline. Moreover, since the electric field does not penetrate the drop, its content is protected from electrical currents and Joule heating. A simple capacitive model allows quantitative prediction of the electrostatic forces exerted on drops. The maximum achievable sorting rate is determined by a competition between electrostatic and hydrodynamic forces. Sorting speeds up to 1200 per second are demonstrated for conductive drops of 160 pl in low viscosity oil.     

On estimating a knowledge production function at the firm and sector level using patent statistics

This paper proposes a definition of the knowledge base of an agent using only patent statistics. It then develops a model of a knowledge production function that can be estimated at the firm level and the sector level using the knowledge base matrix. It identifies the impact of own knowledge base, absorptive capacity to exploit intersectoral spillovers, and absorptive capacity to exploit intrasectoral spillovers, on new technology generation. It permits a study of the dynamics of knowledge generation without having to resort to additional information on the R&D activities of firms. Finally, the paper illustrates the method with the case study of new biotechnology-based knowledge creation by firms in the foods sector.     

A practical guide for the fabrication of microfluidic devices using glass and silicon

This paper describes the main protocols that are used for fabricating microfluidic devices from glass and silicon. Methods for micropatterning glass and silicon are surveyed, and their limitations are discussed. Bonding methods that can be used for joining these materials are summarized and key process parameters are indicated. The paper also outlines techniques for forming electrical connections between microfluidic devices and external circuits. A framework is proposed for the synthesis of a complete glass/silicon device fabrication flow.     

Behavior of liquid plugs at bifurcations in a microfluidic tree network

Flows in complex geometries, such as porous media or biological networks, often contain plugs of liquid flowing within air bubbles. These flows can be modeled in microfluidic devices in which the geometric complexity is well defined and controlled. We study the flow of wetting liquid plugs in a bifurcating network of micro-channels. In particular, we focus on the process by which the plugs divide as they pass each bifurcation. The key events are identified, corresponding to large modifications of the interface curvature, the formation of new interfaces, or the division of a single interface into two new ones. The timing of the different events and the amplitude of the curvature variations are analyzed in view of the design of an event-driven model of flow in branching micro-networks. They are found to collapse onto a master curve dictated by the network geometry.     

On-chip recalcification of citrated whole blood using a microfluidic herringbone mixer

In vitro assays of platelet function and coagulation are typically performed in the presence of an anticoagulant. The divalent cation chelator sodium citrate is among the most common because its effect on coagulation is reversible upon reintroduction of divalent cations. Adding divalent cations into citrated blood by batch mixing leads to platelet activation and initiation of coagulation after several minutes, thus limiting the time blood can be used before spontaneously clotting. In this work, we describe a herringbone microfluidic mixer to continuously introduce divalent cations into citrated blood. The mixing ratio, defined as the ratio of the volumetric flow rates of citrated blood and recalcification buffer, can be adjusted by changing the relative inlet pressures of these two solutions. This feature is useful in whole blood assays in order to account for differences in hematocrit, and thus viscosity. The recalcification process in the herringbone mixer does not activate platelets. The advantage of this continuous mixing approach is demonstrated in microfluidic vascular injury model in which platelets and fibrin accumulate on a collagen-tissue factor surface under flow. Continuous recalcification with the herringbone mixer allowed for flow assay times of up to 30 min, more than three times longer than the time achieved by batch recalcification. This continuous mixer allows for measurements of thrombus formation, remodeling, and fibrinolysis in vitro over time scales that are relevant to these physiological processes.     

Optofluidic tunable microlens by manipulating the liquid meniscus using a flared microfluidic structure

We have designed, demonstrated, and characterized a simple, novel in-plane tunable optofluidic microlens. The microlens is realized by utilizing the interface properties between two different fluids: CaCl(2)solution and air. A constant contact angle of ～90° is the pivotal factor resulting in the outward bowing and convex shape of the CaCl(2) solution-air interface. The contact angle at the CaCl(2) solution-air interface is maintained by a flared structure in the polydimethylsiloxane channel. The resulting bowing interface, coupled with the refractive index difference between the two fluids, results in effective in-plane focusing. The versatility of such a design is confirmed by characterizing the intensity of a traced beam experimentally and comparing the observed focal points with those obtained via ray-tracing simulations. With the radius of curvature conveniently controlled via fluid injection, the resulting microlens has a readily tunable focal length. This ease of operation, outstandingly low fluid usage, large range tunable focal length, and in-plane focusing ability make this lens suitable for many potential lab-on-a-chip applications such as particle manipulation, flow cytometry, and in-plane optical trapping.     

Probing the mechanical properties of brain cancer cells using a microfluidic cell squeezer device

Despite being invasive within surrounding brain tissues and the central nervous system, little is known about the mechanical properties of brain tumor cells in comparison with benign cells. Here, we present the first measurements of the peak pressure drop due to the passage of benign and cancerous brain cells through confined microchannels in a “microfluidic cell squeezer” device, as well as the elongation, speed, and entry time of the cells in confined channels. We find that cancerous and benign brain cells cannot be differentiated based on speeds or elongation. We have found that the entry time into a narrow constriction is a more sensitive indicator of the differences between malignant and healthy glial cells than pressure drops. Importantly, we also find that brain tumor cells take a longer time to squeeze through a constriction and migrate more slowly than benign cells in two dimensional wound healing assays. Based on these observations, we arrive at the surprising conclusion that the prevailing notion of extraneural cancer cells being more mechanically compliant than benign cells may not apply to brain cancer cells.     

浅谈基层科研单位科学基金的后期管理

本文探讨了科学基金后期管理的作用和重要性,并强调提高管理者的自身素质。