New advances in microfluidic flow cytometry

In recent years, researchers are paying the increasing attention to the development of portable microfluidic diagnostic devices including microfluidic flow cytometry for the point‐of‐care testing. Microfluidic flow cytometry, where microfluidics and flow cytometry work together to realize novel functionalities on the microchip, provides a powerful tool for measuring the multiple characteristics of biological samples. The development of a portable, low‐cost, and compact flow cytometer can benefit the health care in underserved areas such as Africa or Asia. In this article, we review recent advancements of microfluidics including sample pumping, focusing and sorting, novel detection approaches, and data analysis in the field of flow cytometry. The challenge of microfluidic flow cytometry is also examined briefly.     

Single channel layer, single sheath-flow inlet microfluidic flow cytometer with three-dimensional hydrodynamic focusing

Flow cytometry is a technique capable of optically characterizing biological particles in a high-throughput manner. In flow cytometry, three dimensional (3D) hydrodynamic focusing is critical for accurate and consistent measurements. Due to the advantages of microfluidic techniques, a number of microfluidic flow cytometers with 3D hydrodynamic focusing have been developed in recent decades. However, the existing devices consist of multiple layers of microfluidic channels and tedious fluidic interconnections. As a result, these devices often require complicated fabrication and professional operation. Consequently, the development of a robust and reliable microfluidic flow cytometer for practical biological applications is desired. This paper develops a microfluidic device with a single channel layer and single sheath-flow inlet capable of achieving 3D hydrodynamic focusing for flow cytometry. The sheath-flow stream is introduced perpendicular to the microfluidic channel to encircle the sample flow. In this paper, the flow fields are simulated using a computational fluidic dynamic (CFD) software, and the results show that the 3D hydrodynamic focusing can be successfully formed in the designed microfluidic device under proper flow conditions. The developed device is further characterized experimentally. First, confocal microscopy is exploited to investigate the flow fields. The resultant Z-stack confocal images show the cross-sectional view of 3D hydrodynamic with flow conditions that agree with the simulated ones. Furthermore, the flow cytometric detections of fluorescence beads are performed using the developed device with various flow rate combinations. The measurement results demonstrate that the device can achieve great detection performances, which are comparable to the conventional flow cytometer. In addition, the enumeration of fluorescence-labelled cells is also performed to show its practicality for biological applications. Consequently, the microfluidic flow cytometer developed in this paper provides a practical platform that can be used for routine analysis in biological laboratories. Additionally, the 3D hydrodynamic focusing channel design can also be applied to various applications that can advance the lab on a chip research.     

Velocity-independent microfluidic flow cytometry

Pressure-driven flow in microfluidic channels is characterized by a distribution of velocities. This distribution makes it difficult to implement conventional flow cytometry data analysis. We have demonstrated a method to measure velocity as an independent parameter when performing microfluidic flow cytometry. This method allows velocity-independent analysis of particles such as beads or cells, and allows flow cytometry analysis of extended objects, such as long DNA molecules. It allows accurate flow cytometry in transient and nonuniform flows. This general measurement method could be used in the future to measure the velocity of particles in a variety of existing microfluidic devices without the need for changes in their design.     

Multiple functions of microfluidic platforms: Characterization and applications in tissue engineering and diagnosis of cancer

Microfluidic system, or lab-on-a-chip, has grown explosively. This system has been used in research for the first time and then entered in the clinical section. Due to economic reasons, this technique has been used for screening of laboratory and clinical indices. The microfluidic system solves some difficulties accompanied by clinical and biological applications. In this review, the interpretation and analysis of some recent developments in microfluidic systems in biomedical applications with more emphasis on tissue engineering and cancer will be discussed. Moreover, we try to discuss the features and functions of microfluidic systems.     

Design of pressure-driven microfluidic networks using electric circuit analogy

This article reviews the application of electric circuit methods for the analysis of pressure-driven microfluidic networks with an emphasis on concentration- and flow-dependent systems. The application of circuit methods to microfluidics is based on the analogous behaviour of hydraulic and electric circuits with correlations of pressure to voltage, volumetric flow rate to current, and hydraulic to electric resistance. Circuit analysis enables rapid predictions of pressure-driven laminar flow in microchannels and is very useful for designing complex microfluidic networks in advance of fabrication. This article provides a comprehensive overview of the physics of pressure-driven laminar flow, the formal analogy between electric and hydraulic circuits, applications of circuit theory to microfluidic network-based devices, recent development and applications of concentration- and flow-dependent microfluidic networks, and promising future applications. The lab-on-a-chip (LOC) and microfluidics community will gain insightful ideas and practical design strategies for developing unique microfluidic network-based devices to address a broad range of biological, chemical, pharmaceutical, and other scientific and technical challenges.     

Microfluidic synthesis of multifunctional Janus particles for biomedical applications

Multifunctional Janus particles have a variety of applications in a wide range of fields. However, to achieve many of these applications, high-throughput, low-cost techniques are needed to synthesize these particles with precise control of the various structural/physical/chemical properties. Microfluidics provides a unique platform to fabricate Janus particles using carefully controlled liquid flow in microfluidic channels to form Janus droplets and various types of solidification methods to solidify them into Janus particles. In this Focus article, we summarize the most recent representative works on Janus particle fabrication in microfluidics. The applications of Janus particles in biomedical areas are emphasized. We believe that microfluidics-enabled multifunctional Janus particles could resolve multiple prevalent issues in biomedicine (e.g., disease monitoring at an early stage, high-throughput bioassays, therapeutic delivery) if persistent effort and collaboration are devoted to this direction.     

Automated analysis of single stem cells in microfluidic traps

We report a reliable strategy to perform automated image cytometry of single (non-adherent) stem cells captured in microfluidic traps. The method rapidly segments images of an entire microfluidic chip based on the detection of horizontal edges of microfluidic channels, from where the position of the trapped cells can be derived and the trapped cells identified with very high precision (>97%). We used this method to successfully quantify the efficiency and spatial distribution of single-cell loading of a microfluidic chip comprised of 2048 single-cell traps. Furthermore, cytometric analysis of trapped primary hematopoietic stem cells (HSC) faithfully recapitulated the distribution of cells in the G1 and S/G2-M phase of the cell cycle that was measured by flow cytometry. This approach should be applicable to automatically track single live cells in a wealth of microfluidic systems.     

Microfluidics technology for manipulation and analysis of biological cells

Analysis of the profiles and dynamics of molecular components and sub-cellular structures in living cells using microfluidic devices has become a major branch of bioanalytical chemistry during the past decades. Microfluidic systems have shown unique advantages in performing analytical functions such as controlled transportation, immobilization, and manipulation of biological molecules and cells, as well as separation, mixing, and dilution of chemical reagents, which enables the analysis of intracellular parameters and detection of cell metabolites, even on a single-cell level. This article provides an in-depth review on the applications of microfluidic devices for cell-based assays in recent years (2002–2005). Various cell manipulation methods for microfluidic applications, based on magnetic, optical, mechanical, and electrical principles, are described with selected examples of microfluidic devices for cell-based analysis. Microfluidic devices for cell treatment, including cell lysis, cell culture, and cell electroporation, are surveyed and their unique features are introduced. Special attention is devoted to a number of microfluidic devices for cell-based assays, including micro cytometer, microfluidic chemical cytometry, biochemical sensing chip, and whole cell sensing chip.     

Enhancing signals of microfluidic impedance cytometry through optimization of microelectrode array

Microfluidic impedance cytometry shows a great value in biomedical diagnosis. However, the crosstalk between neighboring microelectrodes strongly weakens the impedance signal. Hereby, we demonstrate a novel microfluidic impedance cytometer consisted of sensing electrodes and ground electrodes (GNDs). The simulation reveals a signal enhancement by more than five times with GNDs compared to that without ones. We also found that the linear correlation between the impedance at a high frequency and that at a low frequency varies as microparticle size changes, which can be used for microparticle classification. The study can help with microelectrode optimization and signal processing for microfluidic impedance analysis.     

Toward low-voltage dielectrophoresis-based microfluidic systems: A review

Dielectrophoretically driven microfluidic devices have demonstrated great applicability in biomedical engineering, diagnostic medicine, and biological research. One of the potential fields of application for this technology is in point-of-care (POC) devices, ideally allowing for portable, fully integrated, easy to use, low-cost diagnostic platforms. Two main approaches exist to induce dielectrophoresis (DEP) on suspended particles, that is, electrode-based DEP and insulator-based DEP, each featuring different advantages and disadvantages. However, a shared concern lies in the input voltage used to generate the electric field necessary for DEP to take place. Therefore, input voltage can determine portability of a microfluidic device. This review outlines the recent advances in reducing stimulation voltage requirements in DEP-driven microfluidics.     

基于微流控芯片技术的秀丽隐杆线虫研究进展

秀丽隐杆线虫具有体积小、生命周期短、结构简单和高基因保守性等特点,是生命科学研究领域中的一种重要模式生物。微流控芯片的通道尺寸与线虫大小相匹配,并可实现灵活集成的线虫操控,为线虫研究提供了一种全新的平台。在微流控平台上,线虫长期培养、固定、分选、精确刺激传递和单线虫包裹等单元操作已经实现,并被应用于线虫神经生物学、行为学、衰老及发育、药物筛选等研究中。该文着重介绍近几年基于微流控芯片技术的线虫研究最新进展,并对其应用前景予以展望。     

Brain slice on a chip: opportunities and challenges of applying microfluidic technology to intact tissues

Isolated brain tissue, especially brain slices, are valuable experimental tools for studying neuronal function at the network, cellular, synaptic, and single channel levels. Neuroscientists have refined the methods for preserving brain slice viability and function and converged on principles that strongly resemble the approach taken by engineers in developing microfluidic devices. With respect to brain slices, microfluidic technology may 1) overcome the traditional limitations of conventional interface and submerged slice chambers and improve oxygen/nutrient penetration into slices, 2) provide better spatiotemporal control over solution flow/drug delivery to specific slice regions, and 3) permit successful integration with modern optical and electrophysiological techniques. In this review, we highlight the unique advantages of microfluidic devices for in vitro brain slice research, describe recent advances in the integration of microfluidic devices with optical and electrophysiological instrumentation, and discuss clinical applications of microfluidic technology as applied to brain slices and other non-neuronal tissues. We hope that this review will serve as an interdisciplinary guide for both neuroscientists studying brain tissue in vitro and engineers as they further develop microfluidic chamber technology for neuroscience research.     

On the recent developments of insulator‐based dielectrophoresis: A review

Insulator‐based dielectrophoresis (iDEP), also known as electrodeless DEP, has become a well‐known dielectrophoretic technique, no longer viewed as a new methodology. Significant advances on iDEP have been reported during the last 15 years. This review article aims to summarize some of the most important findings on iDEP organized by the type of dielectrophoretic mode: streaming and trapping iDEP. The former is primarily used for particle sorting, while the latter has great capability for particle enrichment. The characteristics of a wide array of devices are discussed for each type of dielectrophoretic mode in order to present an overview of the distinct designs and applications developed with iDEP. A short section on Joule heating effects and electrothermal flow is also included to highlight some of the challenges in the utilization of iDEP systems. The significant progress on iDEP illustrates its potential for a large number of applications, ranging from bioanalysis to clinical and biomedical assessments. The present article discusses the work on iDEP by numerous research groups around the world, with the aim of proving the reader with an overview of the state‐of‐the‐art in iDEP microfluidic systems.     

Current Advances in Highly Multiplexed Antibody‐Based Single‐Cell Proteomic Measurements

Single‐cell measurements have played a critical role in revealing the complex signaling dynamics and heterogeneity present in cells, but there is still much to learn. Measuring samples from bulk populations of cells often masks the information and dynamics present in subsets of cells. Common single‐cell protein studies rely on fluorescent microscopy and flow cytometry but are limited in multiplexing ability owing to spectral overlap. Recently, technology advancements in single‐cell proteomics have allowed highly multiplexed measurement of multiple parameters simultaneously by using barcoded microfluidic enzyme‐linked immunosorbent assays and mass cytometry techniques. In this review, we will describe recent work around multiparameter single‐cell protein measurements and critically analyze the techniques.     

Review on recent advances in the analysis of isolated organelles

The analysis of isolated organelles is one of the pillars of modern bioanalytical chemistry. This review describes recent developments on the isolation and characterization of isolated organelles both from living organisms and cell cultures. Salient reports on methods to release organelles focused on reproducibility and yield, membrane isolation, and integrated devices for organelle release. New developments on organelle fractionation after their isolation were on the topics of centrifugation, immunocapture, free flow electrophoresis, flow field-flow fractionation, fluorescence activated organelle sorting, laser capture microdissection, and dielectrophoresis. New concepts on characterization of isolated organelles included atomic force microscopy, optical tweezers combined with Raman spectroscopy, organelle sensors, flow cytometry, capillary electrophoresis, and microfluidic devices.     

Microfluidic flow rate detection based on integrated optical fiber cantilever

We demonstrate an integrated microfluidic flow sensor with ultra-wide dynamic range, suitable for high throughput applications such as flow cytometry and particle sorting/counting. A fiber-tip cantilever transduces flow rates to optical signal readout, and we demonstrate a dynamic range from 0 to 1500 microL min(-1) for operation in water. Fiber-optic sensor alignment is guided by preformed microfluidic channels, and the dynamic range can be adjusted in a one-step chemical etch. An overall non-linear response is attributed to the far-field angular distribution of single-mode fiber output.     

A review on continuous-flow microfluidic PCR in droplets: Advances,challenges and future

Significant advances have been made in developing microfluidic polymerase chain reaction (PCR) devices in the last two decades. More recently, microfluidic microdroplet technology has been exploited to perform PCR in droplets because of its unique features. For example, it can prevent crossover contamination and PCR inhibition, is suitable for single-cell and single-molecule analyses, and has the potential for system integration and automation. This review will therefore focus on recent developments on droplet-based continuous-flow microfluidic PCR, and the major research challenges. This paper will also discuss a new way of on-chip flow control and a rational design simulation tool, which are required to underpin fully integrated and automated droplet-based microfluidic systems. We will conclude with a scientific speculation of future autonomous scientific discoveries enabled by microfluidic microdroplet technologies.     

Polyimide-based microfluidic devices

This paper describes the development of polyimide-based microfluidic devices. A layer transfer and lamination technique is used to fabricate flexible microfluidic channels in various shapes and with a wide range of dimensions. High bond strengths can be achieved by cure cycle adaptation and surface treatment of the polyimide layers prior to bonding. The polyimide microchannels can be combined with metallization layers to fabricate electrodes inside and outside channels. The resulting devices can be used for flexible fluidic and electrical connectors, implantable fluid delivery devices, microelectrodes with embedded fluidic channels, chip-based flow cytometry and for a great variety of other applications in medical, chemical or biological research.     

Detection of membrane biointeractions based on fluorescence superquenching

Assays for biointeractions of molecules with supported lipid bilayers using fluorescence superquenching are described. A conjugated cationic polymer was adsorbed on to silica microspheres, which were then coated with an anionic lipid bilayer. The lipid bilayer attenuated superquenching by acting as a barrier between the conjugated polymer and its quencher. Biointeractions of the lipid bilayer with a membrane lytic peptide, melittin, were detected and quantitated by superquenching of the conjugated polyelectrolyte in flow cytometric and microfluidic bioassays. A higher sensitivity for detecting melittin lysis of the lipid bilayer at lower concentrations and shorter times for melittin action was found using flow cytometry in this study in comparison to other existing methods. This study combined the sensitivity of superquenching and flow cytometry to detect biointeractions with a lipid bilayer, which serves as a platform for developing functional assays for sensor applications, lipid enzymology, and investigations of molecular interactions. In addition, this study demonstrated proof-of-concept for using superquenching detected as a result of lipid bilayer disruption in a microfluidic format.     

Continuous flow separations in microfluidic devices

Biochemical sample mixtures are commonly separated in batch processes, such as filtration, centrifugation, chromatography or electrophoresis. In recent years, however, many research groups have demonstrated continuous flow separation methods in microfluidic devices. Such separation methods are characterised by continuous injection, real-time monitoring, as well as continuous collection, which makes them ideal for combination with upstream and downstream applications. Importantly, in continuous flow separation the sample components are deflected from the main direction of flow, either by means of a force field (electric, magnetic, acoustic, optical etc.), or by intelligent positioning of obstacles in combination with laminar flow profiles. Sample components susceptible to deflection can be spatially separated. A large variety of methods has been reported, some of these are miniaturised versions of larger scale methods, others are only possible in microfluidic regimes. Researchers now have a diverse toolbox to choose from and it is likely that continuous flow methods will play an important role in future point-of-care or in-the-field analysis devices.