Recent advances in electric analysis of cells in microfluidic systems

Microfluidics offers an ideal platform to integrate cell-based assays with electric measurements. The technological advances in microfluidics, microelectronics, electrochemistry, and electrophysiology have greatly inspired the development of microfluidic/electric devices that work with a low number of cells or single cells. The applications of these microfluidic systems range from the detecting of cell culture density to the probing of cellular functions at the single-cell level. In this review, we introduce the recent advances in the electric analysis of cells on a microfluidic platform, specifically related to the quantification and monitoring of cells in static solution, on-chip patch-clamp measurement, and examination of flowing cells. We also point out future directions and challenges in this field. Figure Different microfluidic devices applied to electrical analysis of cells     

Capillary-based microfluidic analysis systems

An overview of the recent development of high-throughput capillary-based microfluidic analysis systems (CBMAS) is presented. The advantages and applications of these systems are discussed and illustrated by recent results obtained in the authors’ group and other groups. A possible outlook for CBMAS is discussed.     

Complex three-dimensional microparticles from microfluidic lithography

Complex 3D microparticle, as an emerging and attractive field, has attracted more and more attention due to its versatile morphologies and broad range of applications. In this review, we provide an overall recent progress in 3D microparticles fabricated by microfluidic lithography. This review will focus on the synthesis mechanisms, synthesis process, the resultant 3D microparticles, and their applications. Finally, we will look into the future trends in complex 3D microparticles. This review will be beneficial for researchers in numerous fields, including functional materials, sensors, encryption, and biomedical engineering.     

New advances in microchip fabrication for electrochromatography

There is a great demand in separation technologies for faster and more effective analysis processes. Miniaturization is a suitable technique for satisfying this demand as reduction in size gives increased separation speed with higher efficiency. CEC is an electric-field-mediated separation technique where the liquid flow is generated by the electric field itself. The main advantage of using electric field over pressure for flow generation is the flat flow profile of the EOF; thus, CEC is one of the best candidates to construct a novel and high-efficiency microanalytical device. The aim of the present paper is to review the basic fabrication and bonding principles, as well as connection and system integration options for microfluidics-based electrochromatography. The physical structure and fluidic channel formation are critically evaluated, including glass microstructuring and fusion bonding. Recent developments in nanoflow measurements and the application of various flow control units are also extensively discussed.     

Impedance‐based viscoelastic flow cytometry

Elastic nature of the viscoelastic fluids induces lateral migration of particles into a single streamline and can be used by microfluidic based flow cytometry devices. In this study, we investigated focusing efficiency of polyethylene oxide based viscoelastic solutions at varying ionic concentration to demonstrate their use in impedimetric particle characterization systems. Rheological properties of the viscoelastic fluid and particle focusing performance are not affected by ionic concentration. We investigated the viscoelastic focusing dynamics using polystyrene (PS) beads and human red blood cells (RBCs) suspended in the viscoelastic fluid. Elasto‐inertial focusing of PS beads was achieved with the combination of inertial and viscoelastic effects. RBCs were aligned along the channel centerline in parachute shape which yielded consistent impedimetric signals. We compared our impedance‐based microfluidic flow cytometry results for RBCs and PS beads by analyzing particle transit time and peak amplitude at varying viscoelastic focusing conditions obtained at different flow rates. We showed that single orientation, single train focusing of nonspherical RBCs can be achieved with polyethylene oxide based viscoelastic solution that has been shown to be a good candidate as a carrier fluid for impedance cytometry.     

Recent advances in miniaturized microfluidic flow cytometry for clinical use

This article provides an overview of recent research achievements in miniaturized flow cytometry. The review focuses on chip-based microfluidic flow cytometers, classified by cell transport method, detection technology, and biomedical application. By harnessing numerous ideas and cutting-edge microfabrication technologies, microfluidic flow cytometry benefits from ever-increasing functionalities and the performance levels achieved make it an attractive biomedical research and clinical tool. In this article, we briefly describe an update of recent developments that combine novel microfluidic characteristics and flow cytometry on chips that meet biomedical needs.     

Construction of microscale structures in enclosed microfluidic networks by using a magnetic beads based method

A large number of microscale structures have been used to elaborate flowing control or complex biological and chemical reaction on microfluidic chips. However, it is still inconvenient to fabricate microstructures with different heights (or depths) on the same substrate. These kinds of microstructures can be fabricated by using the photolithography and wet-etching method step by step, but involves time-consuming design and fabrication process, as well as complicated alignment of different masters. In addition, few existing methods can be used to perform fabrication within enclosed microfluidic networks. It is also difficult to change or remove existing microstructures within these networks. In this study, a magnetic-beads-based approach is presented to build microstructures in enclosed microfluidic networks. Electromagnetic field generated by microfabricated conducting wires (coils) is used to manipulate and trap magnetic beads on the bottom surface of a microchannel. These trapped beads are accumulated to form a microscale pile with desired shape, which can adjust liquid flow, dock cells, modify surface, and do some other things as those fabricated microstructures. Once the electromagnetic field is changed, trapped beads may form new shapes or be removed by a liquid flow. Besides being used in microfabrication, this magnetic-beads-based method can be used for novel microfluidic manipulation. It has been validated by forming microscale dam structure for cell docking and modified surface for cell patterning, as well as guiding the growth of neurons.     

Emerging trends in miniaturized and microfluidic electrochemical sensing platforms

Electrochemical sensing has established a strong presence in diverse areas. The conventional electrochemical sensing approach consumes large sample volumes and reagents and requires bulky potentiostat, macro-electrodes, and other equipment. The synergistic integration of electrochemical sensing systems with miniaturized or microfluidic electrochemical devices and microelectrodes in a single platform provides rapid analysis with a disposable, reusable, and cost-effective platform for multiplexed point-of-care detections. Such microdevices have created scope for using several materials as electrodes and sensing platforms by using appropriate fabrication techniques. One of the most recent advancements in miniaturized devices includes the integration of automation and Internet of Things to realize fully automated and robust electrochemical microdevices. The review summarizes the emerging trends in fabrication methods of miniaturized and microfluidic devices, their multiple applications in real-time, integration of Internet of Things, automation, identifying research gaps with strategies for bridging these gaps, future outlook, and recent approaches to intelligent electrochemical sensing.     

Microfluidic flow focusing: drop size and scaling in pressure versus flow-rate-driven pumping

We experimentally study the production of micrometer-sized droplets using microfluidic technology and a flow-focusing geometry. Two distinct methods of flow control are compared: (i) control of the flow rates of the two phases and (ii) control of the inlet pressures of the two phases. In each type of experiment, the drop size l, velocity U and production frequency f are measured and compared as either functions of the flow-rate ratio or the inlet pressure ratio. The minimum drop size in each experiment is on the order of the flow focusing contraction width a. The variation in drop size as the flow control parameters are varied is significantly different between the flow-rate and inlet pressure controlled experiments.     

Polymeric microfluidic system for DNA analysis

The application of micro total analysis system (μTAS) has grown exponentially in the past decade. DNA analysis is one of the primary applications of μTAS technology. This review mainly focuses on the recent development of the polymeric microfluidic devices for DNA analysis. After a brief introduction of material characteristics of polymers, the various microfabrication methods are presented. The most recent developments and trends in the area of DNA analysis are then explored. We focus on the rapidly developing fields of cell sorting, cell lysis, DNA extraction and purification, polymerase chain reaction (PCR), DNA separation and detection. Lastly, commercially available polymer-based microdevices are included.     

Polymer microfabrication technologies for microfluidic systems

Polymers have assumed the leading role as substrate materials for microfluidic devices in recent years. They offer a broad range of material parameters as well as material and surface chemical properties which enable microscopic design features that cannot be realised by any other class of materials. A similar range of fabrication technologies exist to generate microfluidic devices from these materials. This review will introduce the currently relevant microfabrication technologies such as replication methods like hot embossing, injection molding, microthermoforming and casting as well as photodefining methods like lithography and laser ablation for microfluidic systems and discuss academic and industrial considerations for their use. A section on back-end processing completes the overview.     

Continuous and precise particle separation by electroosmotic flow control in microfluidic devices

A new scheme has been described for continuous particle separation using EOF in microfluidic devices. We have previously reported a method for particle separation, called "pinched flow fractionation (PFF)", in which size-dependent and continuous particle separation can be achieved by introducing pressure-driven flows with and without particles into a pinched microchannel. In this study, EOF was employed to transport fluid flows inside a microchannel. By controlling the applied voltage to electrodes inserted in each inlet/outlet port, the flow rates from both inlets, and flow rates distributed to each outlet could be accurately tuned, thus enabling more effective separation compared to the pressure-driven scheme. In the experiment, the particle behaviors were compared between EOF and pressure-driven flow schemes. In addition, micrometer- and submicrometer-sized particles were accurately separated and individually collected using a microchannel with multiple outlet branch channels, demonstrating the high efficiency of the presented scheme.     

The good,the bad,and the tiny: a review of microflow cytometry

Recent developments in microflow cytometry have concentrated on advancing technology in four main areas: (1) focusing the particles to be analyzed in the microfluidic channel, (2) miniaturization of the fluid-handling components, (3) miniaturization of the optics, and (4) integration and applications development. Strategies for focusing particles in a narrow path as they pass through the detection region include the use of focusing fluids, nozzles, and dielectrophoresis. Strategies for optics range from the use of microscope objectives to polymer waveguides or optical fibers embedded on-chip. While most investigators use off-chip fluidic control, there are a few examples of integrated valves and pumps. To date, demonstrations of applications are primarily used to establish that the microflow systems provide data of the same quality as laboratory systems, but new capabilities-such as automated sample staining-are beginning to emerge. Each of these four areas is discussed in detail in terms of the progress of development, the continuing limitations, and potential future directions for microflow cytometers.     

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.     

A microfluidic biosensor based on nucleic acid sequence recognition

The development of a generic semi-disposable microfluidic biosensor for the highly sensitive detection of pathogens via their nucleic acid sequences is presented in this paper. Disposable microchannels with defined areas for capture and detection of target pathogen RNA sequence were created in polydimethylsiloxane (PDMS) and mounted onto a reusable polymethylmethacrylate (PMMA) stand. Two different DNA probes complementary to unique sequences on the target pathogen RNA serve as the biorecognition elements. For signal generation and amplification, one probe is coupled to dye encapsulated liposomes while the second probe is coupled to superparamagnetic beads for target immobilization. The probes hybridize to target RNA and the liposome–target-bead complex is subsequently captured on a magnet. The amount of liposomes captured correlates directly to the concentration of target sequence and is quantified using a fluorescence microscope. Dengue fever virus serotype 3 sequences and probes were used as a model analyte system to test the sensor. Probe binding and target capture conditions were optimized for sensitivity resulting in a detection limit of as little as 10 amol L^–1 (10 pmol L^–1) . Future biosensors will be designed to incorporate a mixer and substitute the fluorescence detection with an electrochemical detection technique to provide a truly portable microbiosensor system.     

Application of flow cytometry for rapid detection ofLactococcus garvieae

Flow cytometry (FCM) technique was applied to rapid determination of cell number ofLactococcus garvieae. An antiserum againstL. garvieae was prepared and its immunological property was examined. The present antibody would recognize some epitopes ofL. garvieae with a high specificity. The optimum conditions for the FCM assay were as follows: discriminate value, 60; dilution ratio of the antiserum, 1.0 X 10⁴. Calibration curve forL. garvieae cells was linear, in the range of 2.4 X 10⁴-1.5 X 10⁷ cells/mL. The detection ofL. garvieae in cell suspensions contaminated withEscherichia coli was carried out. A good correlation was observed in the range of 20–90% for the mixing ratio ofL. garvieae. One FCM assay could be completed within 2 min, and the total assay time, including the preparation of bacterial sample, was within 3 h.     

Modular fluidic resistors to enable widely tunable flow rate and fluidic switching period in a microfluidic oscillator

Microfluidic systems with modular components are attractive alternatives to monolithically integrated microfluidic systems because of their flexibility. In this study, we apply the modular concept on a water‐head‐pressure‐driven microfluidic oscillator and obtain a widely tunable flow rate and fluidic switching period. Modular fluidic resistors can be easily mounted onto and demounted from a main chip by means of plastic male connectors. The connectors enable a leak‐free connection between the modular resistors and main chip (leakage pressure > 140 kPa). With modular resistors, we show independent control of the flow rate and flow switching period of the oscillator system in a wide range (2.5 s–6.4 h and 2 μL/min–2 mL/min). This modular approach can be used to enhance the flexibility of instruction‐embedded microfluidic circuits in which their operational range is limited.     

Spreadsheet analysis of the field-driven start-up flow in a microfluidic channel

We discuss, in this article, the solution method of the unsteady electroosmotic flow of Newtonian fluid in a square microfluidic channel cross-section in the framework of spreadsheet analysis. We demonstrate the implementation of the finite difference scheme, which is used for the discretization of the transport equations governing the flow dynamics of the present problem, in the spreadsheet tool. Also, we have shown the implementation details of different boundary conditions, which are typically used for the underlying electrohydrodynamics in a microfluidic channel, in the spreadsheet analysis tool. We show that the results obtained from the spreadsheet analysis match accurately with the numerical solutions for both the electrostatic potential distribution and the flow velocity. Our results of this analysis justify the credibility of the spreadsheet tool for capturing the intricate details of the electrically actuated microflows during the initial transiences, that is, for the start-up flows and the phenomenon due to the electrical double layer effect, quite effectively. The inferences of this analysis will open up a new research paradigm of microfluidics and microscale transport processes by providing the potential applicability of the spreadsheet tools to obtain the flow physics of our interest in a very intuitive and less expensive manner.     

AAO-CNTs electrode on microfluidic flow injection system for rapid iodide sensing

In this work, carbon nanotubes (CNTs) nanoarrays in anodized aluminum oxide (AAO-CNTs) nanopore is integrated on a microfluidic flow injection system for in-channel electrochemical detection of iodide. The device was fabricated from PDMS (polydimethylsiloxane) microchannel bonded on glass substrates that contains three-electrode electrochemical system, including AAO-CNTs as a working electrode, silver as a reference electrode and platinum as an auxiliary electrode. Aluminum, stainless steel catalyst, silver and platinum layers were sputtered on the glass substrate through shadow masks. Aluminum layer was then anodized by two-step anodization process to form nanopore template. CNTs were then grown in AAO template by thermal chemical vapor deposition. The amperometric detection of iodide was performed in 500-μm-wide and 100-μm-deep microchannels on the microfluidic chip. The influences of flow rate, injection volume and detection potential on the current response were optimized. From experimental results, AAO-CNTs electrode on chip offers higher sensitivity and wider dynamic range than CNTs electrode with no AAO template.     

Facile microfluidic fabrication of monodispersed self-coupling microcavity with fine tunability

Whispering gallery mode (WGM) resonators have received extensive attention because of their nonlinear optical application in lasers and sensors. Optical microcavities are excellent candidates for constructing powerful microlasers and label-free biosensors, owing to their low optical losses and small size. However, most of these microcavity syntheses rely on sophisticated fabrication methods and cannot be manipulated easily. To achieve facile and versatile microcavity fabrication, we present a robust microfluidics method for monodispersed self-coupling optical microcavity fabrication with a fine tunability. The microcavity polydispersity was less than 3%. The optical microcavity size could be varied from 10 to 30 µm with a steady quality factor (Q) of approximately 1000. The lowest laser threshold that we obtained was 0.82 µJ with a microcavity size of 20 µm. The doped fluorescent dye concentration can be tuned precisely from 0.001 to 0.05 wt% to explore an optimized fluorescent background. The experimental results and theoretical simulation match well in terms of Q and the electrometric resonance field intensity. Compared with previous precise and practical fabrication methods, we have demonstrated a facile approach for versatile optical microcavity fabrication. This method can vary the microcavity materials, size, doped fluorescent dye concentration, WGM resonance spectrum, Q factor, and laser threshold easily to adapt to various circumstances and specific applications.