Microfluidic Platforms for Yeast‐Based Aging Studies

The budding yeast Saccharomyces cerevisiae has been a powerful model for the study of aging and has enabled significant contributions to our understanding of basic mechanisms of aging in eukaryotic cells. However, the laborious low‐throughput nature of conventional methods of performing aging assays limits the pace of discoveries in this field. Some of the technical challenges of conventional aging assay methods can be overcome by use of microfluidic systems coupled to time‐lapse microscopy. One of the major advantages is the ability of a microfluidic system to perform long‐term cell culture under well‐defined environmental conditions while tracking individual yeast. Here, recent advancements in microfluidic platforms for various yeast‐based studies including replicative lifespan assay, long‐term culture and imaging, gene expression, and cell signaling are discussed. In addition, emerging problems and limitations of current microfluidic approaches are examined and perspectives on the future development of this dynamic field are presented.     

Signalling boosts the evolution of cooperation in repeated group interactions

Many biological and social systems show significant levels of collective action. Several cooperation mechanisms have been proposed, yet they have been mostly studied independently. Among these, direct reciprocity supports cooperation on the basis of repeated interactions among individuals. Signals and quorum dynamics may also drive cooperation. Here, we resort to an evolutionary game-theoretical model to jointly analyse these two mechanisms and study the conditions in which evolution selects for direct reciprocity, signalling, or their combination. We show that signalling alone leads to higher levels of cooperation than when combined with reciprocity, while offering additional robustness against errors. Specifically, successful strategies in the realm of direct reciprocity are often not selected in the presence of signalling, and memory of past interactions is only exploited opportunistically in the case of earlier coordination failure. Differently, signalling always evolves, even when costly. In the light of these results, it may be easier to understand why direct reciprocity has been observed only in a limited number of cases among non-humans, whereas signalling is widespread at all levels of complexity.     

Microfluidic approach to genotyping human platelet antigens

Centralised laboratories routinely determine blood types by serological and molecular methods. Current practices have limitations in terms of cost, time and accessibility. Miniaturised microfluidic platforms offer an alternative to conventional genotyping methods, since they consume fewer reagents, provide faster analysis and allow for complete integration and automation. As these 'lab-on-a-chip' devices have been used for bacterial and viral detection, the authors investigated blood group genotyping as a novel application of microfluidic technology. To demonstrate the feasibility of microfluidic chip-based genotyping, the authors compared human platelet antigen 1 (HPA-1) genotype results from conventional and chip-based analysis for 19 blood donor specimens. DNA purification was performed with ChargeSwitch? magnetic beads, DNA amplification (PCR), restriction length polymorphism (RFLP) and capillary electrophoresis (CE) for identification of the DNA on microfluidic chips. It was found that nine donors were HPA-1a/1a and ten were HPA-1a/1b. Concordance between the conventional and on-chip methods was achieved for all but one sample. All the steps were demonstrated for complete blood group genotyping analysis of patient whole blood specimens on separate microfluidic chips. Future work will focus on integration of all the genotyping protocols on a single microfluidic chip.     

Construction of Microfluidic Chip Structure for Cell Migration Studies in Bioactive Ceramics

Cell migration is an essential bioactive ceramics property and critical for bone induction, clinical application, and mechanism research. Standardized cell migration detection methods have many limitations, including a lack of dynamic fluid circulation and the inability to simulate cell behavior in vivo. Microfluidic chip technology, which mimics the human microenvironment and provides controlled dynamic fluid cycling, has the potential to solve these questions and generate reliable models of cell migration in vitro. In this study, a microfluidic chip is reconstructed to integrate the bioactive ceramic into the microfluidic chip structure to constitute a ceramic microbridge microfluidic chip system. Migration differences in the chip system are measured. By combining conventional detection methods with new biotechnology to analyze the causes of cell migration differences, it is found that the concentration gradients of ions and proteins adsorbed on the microbridge materials are directly related to the occurrence of cell migration behavior, which is consistent with previous reports and demonstrates the effectiveness of the microfluidic chip model. This model provides in vivo environment simulation and controllability of input and output conditions superior to standardized cell migration detection methods. The microfluidic chip system provides a new approach to studying and evaluating bioactive ceramics.     

Microfluidics: Accelerated Biofluid Filling in Complex Microfluidic Networks by Vacuum‐Pressure Accelerated Movement (V‐PAM) (Small 33/2016)

Rapid fluid transport and exchange are critical operations involved in many microfluidic applications. However, conventional mechanisms used for driving fluid transport in microfluidics, such as micropumping and high pressure, can be inaccurate and difficult for implementation for integrated microfluidics containing control components and closed compartments. Here, a technology has been developed termed Vacuum–Pressure Accelerated Movement (V‐PAM) capable of significantly enhancing biofluid transport in complex microfluidic environments containing dead‐end channels and closed chambers. Operation of the V‐PAM entails a pressurized fluid loading into microfluidic channels where gas confined inside can rapidly be dissipated through permeation through a thin, gas‐permeable membrane sandwiched between microfluidic channels and a network of vacuum channels. Effects of different structural and operational parameters of the V‐PAM for promoting fluid filling in microfluidic environments have been studied systematically. This work further demonstrates the applicability of V‐PAM for rapid filling of temperature‐sensitive hydrogels and unprocessed whole blood into complex irregular microfluidic networks such as microfluidic leaf venation patterns and blood circulatory systems. Together, the V‐PAM technology provides a promising generic microfluidic tool for advanced fluid control and transport in integrated microfluidics for different microfluidic diagnosis, organs‐on‐chips, and biomimetic studies.     

The importance of structured noise in the generation of self-organizing tissue patterns through contact-mediated cell�Ccell signalling

Lateral inhibition provides the basis for a self-organizing patterning system in which distinct cell states emerge from an otherwise uniform field of cells. The development of the microchaete bristle pattern on the notum of the fruitfly, Drosophila melanogaster, has long served as a popular model of this process. We recently showed that this bristle pattern depends upon a population of dynamic, basal actin-based filopodia, which span multiple cell diameters. These protrusions establish transient signalling contacts between non-neighbouring cells, generating a type of structured noise that helps to yield a well-ordered and spaced pattern of bristles. Here, we develop a general model of protrusion-based patterning to analyse the role of noise in this process. Using a simple asynchronous cellular automata rule-based model we show that this type of structured noise drives the gradual refinement of lateral inhibition-mediated patterning, as the system moves towards a stable configuration in which cells expressing the inhibitory signal are near-optimally packed. By analysing the effects of introducing thresholds required for signal detection in this model of lateral inhibition, our study shows how filopodia-mediated cell–cell communication can generate complex patterns of spots and stripes, which, in the presence of signalling noise, align themselves across a patterning field. Thus, intermittent protrusion-based signalling has the potential to yield robust self-organizing tissue-wide patterns without the need to invoke diffusion-mediated signalling.     

A methodology for global-sensitivity analysis of time-dependent outputs in systems biology modelling

One of the main challenges in the development of mathematical and computational models of biological systems is the precise estimation of parameter values. Understanding the effects of uncertainties in parameter values on model behaviour is crucial to the successful use of these models. Global sensitivity analysis (SA) can be used to quantify the variability in model predictions resulting from the uncertainty in multiple parameters and to shed light on the biological mechanisms driving system behaviour. We present a new methodology for global SA in systems biology which is computationally efficient and can be used to identify the key parameters and their interactions which drive the dynamic behaviour of a complex biological model. The approach combines functional principal component analysis with established global SA techniques. The methodology is applied to a model of the insulin signalling pathway, defects of which are a major cause of type 2 diabetes and a number of key features of the system are identified.     

A digital microfluidic system for the investigation of pre-steady-state enzyme kinetics using rapid quenching with MALDI-TOF mass spectrometry

A digital microfluidic system based on electrowetting has been developed to facilitate the investigation of pre-steady-state reaction kinetics using rapid quenching and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The device consists of individually addressable electrodes arranged to allow the combination of liquid droplets at well-defined time intervals and an integrated, electrohydrodynamically driven mixer. The device combines two droplets to initiate a reaction, then, with precise timing, combines a third droplet to quench the reaction, and finally combines a fourth droplet to form a matrix. Improvements to throughput when compared to traditional laboratory-scale methods, and previous MALDI-TOF MS digital microfluidic systems, were made. The device was tested against a model protein tyrosine phosphatase system, and results agreed well with published data. The system therefore allows for the analysis of reaction kinetics that were previously too rapid to analyze using MALDI-TOF MS.     

Honest signalling with costly gambles

Costly signalling theory is commonly invoked as an explanation for how honest communication can be stable when interests conflict. However, the signal costs predicted by costly signalling models often turn out to be unrealistically high. These models generally assume that signal cost is determinate. Here, we consider the case where signal cost is instead stochastic. We examine both discrete and continuous signalling games and show that, under reasonable assumptions, stochasticity in signal costs can decrease the average cost at equilibrium for all individuals. This effect of stochasticity for decreasing signal costs is a fundamental mechanism that probably acts in a wide variety of circumstances.     

Role of cell polarity dynamics and motility in pattern formation due to contact-dependent signalling

A key challenge in biology is to understand how spatio-temporal patterns and structures arise during the development of an organism. An initial aggregate of spatially uniform cells develops and forms the differentiated structures of a fully developed organism. On the one hand, contact-dependent cell–cell signalling is responsible for generating a large number of complex, self-organized, spatial patterns in the distribution of the signalling molecules. On the other hand, the motility of cells coupled with their polarity can independently lead to collective motion patterns that depend on mechanical parameters influencing tissue deformation, such as cellular elasticity, cell–cell adhesion and active forces generated by actin and myosin dynamics. Although modelling efforts have, thus far, treated cell motility and cell–cell signalling separately, experiments in recent years suggest that these processes could be tightly coupled. Hence, in this paper, we study how the dynamics of cell polarity and migration influence the spatiotemporal patterning of signalling molecules. Such signalling interactions can occur only between cells that are in physical contact, either directly at the junctions of adjacent cells or through cellular protrusional contacts. We present a vertex model which accounts for contact-dependent signalling between adjacent cells and between non-adjacent neighbours through long protrusional contacts that occur along the orientation of cell polarization. We observe a rich variety of spatiotemporal patterns of signalling molecules that is influenced by polarity dynamics of the cells, relative strengths of adjacent and non-adjacent signalling interactions, range of polarized interaction, signalling activation threshold, relative time scales of signalling and polarity orientation, and cell motility. Though our results are developed in the context of Delta–Notch signalling, they are sufficiently general and can be extended to other contact dependent morpho-mechanical dynamics.     

协议分析仪2Mb/s高速信令处理模块

为了满足2 Mb/s高速信令的测试要求，开发了2 Mb/s高速信令协议分析仪信令处理模块。信令处理模块分为硬件系统和软件系统两部分。介绍了开发2 Mb/s高速信令协议分析仪的意义，使用2 Mb/s高速信令链路的优势，信令处理模块的设计原理和功能。介绍嵌入式处理器和实时操作系统的相关内容。     

Overcoming the limitations of COVID-19 diagnostics with nanostructures,nucleic acid engineering,and additive manufacturing

The COVID-19 pandemic revealed fundamental limitations in the current model for infectious disease diagnosis and serology, based upon complex assay workflows, laboratory-based instrumentation, and expensive materials for managing samples and reagents. The lengthy time delays required to obtain test results, the high cost of gold-standard PCR tests, and poor sensitivity of rapid point-of-care tests contributed directly to society’s inability to efficiently identify COVID-19-positive individuals for quarantine, which in turn continues to impact return to normal activities throughout the economy. Over the past year, enormous resources have been invested to develop more effective rapid tests and laboratory tests with greater throughput, yet the vast majority of engineering and chemistry approaches are merely incremental improvements to existing methods for nucleic acid amplification, lateral flow test strips, and enzymatic amplification assays for protein-based biomarkers. Meanwhile, widespread commercial availability of new test kits continues to be hampered by the cost and time required to develop single-use disposable microfluidic plastic cartridges manufactured by injection molding. Through development of novel technologies for sensitive, selective, rapid, and robust viral detection and more efficient approaches for scalable manufacturing of microfluidic devices, we can be much better prepared for future management of infectious pathogen outbreaks. Here, we describe how photonic metamaterials, graphene nanomaterials, designer DNA nanostructures, and polymers amenable to scalable additive manufacturing are being applied towards overcoming the fundamental limitations of currently dominant COVID-19 diagnostic approaches. In this paper, we review how several distinct classes of nanomaterials and nanochemistry enable simple assay workflows, high sensitivity, inexpensive instrumentation, point-of-care sample-to-answer virus diagnosis, and rapidly scaled manufacturing.     

Phase change microvalve for integrated devices

An active microvalve that uses a meltable piston in place of a conventional solid material to obstruct fluid flow in a microfluidic channel has been developed. This phase change valve is simple to operate and requires no additional fabrication steps. The valve is inherently latched, reusable, and leak-proof (to at least 250 psi) and can be electronically addressed using resistive heaters. The valve has been characterized for a range of operational parameters that will serve as a design guide. For the designs tested, piston displacements of 5 mm or more in 1 s have been achieved. Valves 1.4 mm in length in a 50 microm x 200 microm channel have been integrated on a biochemical reaction device, and successful DNA amplification using PCR has been achieved. The phase change valve can be easily implemented in an array format that can be used to realize complex microfluidic circuits.     

Fracture‐Based Fabrication of Normally Closed,Adjustable, and Fully Reversible Microscale Fluidic Channels

Adjustable fluidic structures play an important role in microfluidic systems. Fracture of multilayered materials under applied tension has been previously demonstrated as a convenient, simple, and inexpensive approach to fabricate nanoscale adjustable structures; here, it is demonstrated how to extend this concept to the microscale. This is achieved by a novel pairing of materials that leverages fracture mechanics to limit crack formation to a specified region, allowing to create size‐controllable and adjustable microfluidic structures. This technique can be used to fabricate “normally closed” microfluidic channels that are completely reversible, a feature that is challenging to achieve in conventional systems without careful engineering controls. The adjustable microfluidic channels are then applied to mechanically lyse single cells, and subsequently manipulate the released nuclear chromatin, creating new possibilities for epigenetic analysis of single cells. This simple, versatile, and robust technology provides an easily accessible pathway to construct adjustable microfluidic structures, which will be useful in developing complex assays and experiments even in resource‐limited settings.     

Steady-state invariant genetics: probing the role of morphogen gradient dynamics in developmental patterning

Morphogen-mediated patterning is the predominant mechanism by which positional information is established during animal development. In the classical view, the interpretation of positional signals depends on the equilibrium distribution of a morphogen, regardless of the dynamics of gradient formation. The problem of whether or not morphogen dynamics contribute to developmental patterning has not been explored in detail, partly because genetic experiments, which selectively affect signalling dynamics while maintaining unchanged the steady-state morphogen profile, are difficult to design and interpret. Here, I present a modelling-based approach to identify genetic mutations in developmental patterning that may affect the transient, but leave invariant the steady-state signalling gradient. As a case study, this approach is used to explore the dynamic properties of Hedgehog (Hh) signalling in the developing wing of the fruitfly, Drosophila melanogaster. This analysis provides insights into how different properties of the Hh gradient dynamics, such as the duration of exposure to the signal or the maximum width of the transient gradient, can be genetically perturbed without affecting the steady-state distribution of the Hh concentration profile. I propose that this method can be used as an experimental design tool to investigate the role of transient morphogen gradients in developmental patterning and discuss the generality of these ideas in other problems.     

Compartmentalized Jet Polymerization as a High‐Resolution Process to Continuously Produce Anisometric Microgel Rods with Adjustable Size and Stiffness

In the past decade, anisometric rod‐shaped microgels have attracted growing interest in the materials‐design and tissue‐engineering communities. Rod‐shaped microgels exhibit outstanding potential as versatile building blocks for 3D hydrogels, where they introduce macroscopic anisometry, porosity, or functionality for structural guidance in biomaterials. Various fabrication methods have been established to produce such shape‐controlled elements. However, continuous high‐throughput production of rod‐shaped microgels with simultaneous control over stiffness, size, and aspect ratio still presents a major challenge. A novel microfluidic setup is presented for the continuous production of rod‐shaped microgels from microfluidic plug flow and jets. This system overcomes the current limitations of established production methods for rod‐shaped microgels. Here, an on‐chip gelation setup enables fabrication of soft microgel rods with high aspect ratios, tunable stiffness, and diameters significantly smaller than the channel diameter. This is realized by exposing jets of a microgel precursor to a high intensity light source, operated at specific pulse sequences and frequencies to induce ultra‐fast photopolymerization, while a change in flow rates or pulse duration enables variation of the aspect ratio. The microgels can assemble into 3D structures and function as support for cell culture and tissue engineering.     

Rapid prototyping of arrayed microfluidic systems in polystyrene for cell-based assays

Microfluidic cell-based systems have enabled the study of cellular phenomena with improved spatiotemporal control of the microenvironment and at increased throughput. While poly(dimethylsiloxane) (PDMS) has emerged as the most popular material in microfluidics research, it has specific limitations that prevent microfluidic platforms from achieving their full potential. We present here a complete process, ranging from mold design to embossing and bonding, that describes the fabrication of polystyrene (PS) microfluidic devices with similar cost and time expenditures as PDMS-based devices. Emphasis was placed on creating methods that can compete with PDMS fabrication methods in terms of robustness, complexity, and time requirements. To achieve this goal, several improvements were made to remove critical bottlenecks in existing PS embossing methods. First, traditional lithographic techniques were adapted to fabricate bulk epoxy molds capable of resisting high temperatures and pressures. Second, a method was developed to emboss through-holes in a PS layer, enabling creation of large arrays of independent microfluidic systems on a single device without need to manually create access ports. Third, thermal bonding of PS layers was optimized in order to achieve quality bonding over large arrays of microsystems. The choice of materials and methods was validated for biological function in two different cell-based applications to demonstrate the versatility of our streamlined fabrication process.     

A Polymer Microfluidic Chip With Interdigitated Electrodes Arrays for Simultaneous Dielectrophoretic Manipulation and Impedimetric Detection of Microparticles

This paper presents the design, fabrication, and characterization of a polymer microfluidic biochip with integrated interdigitated electrodes arrays (IDAs) used to simultaneously separate, manipulate, and detect microparticles using dielectrophoresis (DEP) and electrochemical impedance spectroscopy (EIS) methods. The DEP response of silica microspheres has been characterized, and microspheres of different sizes (1.8 and 3.5 in diameter) have been DEP flow separated and individually trapped in different microchambers by IDAs in a single run. Simultaneously, the impedance change caused by microspheres captured on IDAs has been analyzed for quantification. High-throughput polymer microfabrication techniques such as micro injection molding were used in this work, so that the polymer microfluidic chip can be produced in a low-cost, disposable platform. This low-cost microfluidic chip provides a generic platform for developing multifunctional lab-on-a-chip devices that require the ability to handle and sense microparticles.