Get to Understand More from Single-Cells: Current Studies of Microfluidic-Based Techniques for Single-Cell Analysis

This review describes the microfluidic techniques developed for the analysis of a single cell. The characteristics of microfluidic (e.g., little sample amount required, high-throughput performance) make this tool suitable to answer and to solve biological questions of interest about a single cell. This review aims to introduce microfluidic related techniques for the isolation, trapping and manipulation of a single cell. The major approaches for detection in single-cell analysis are introduced; the applications of single-cell analysis are then summarized. The review concludes with discussions of the future directions and opportunities of microfluidic systems applied in analysis of a single cell.     

Flow control in paper-based microfluidic device for automatic multistep assays: A focused minireview

Although lateral flow tests (LFTs) are easy-to-use diagnostics, they have fundamental limitations for sequential multistep assay that can be reduced to a single chemical reaction step. Paper-based microfluidic devices have attracted considerable attention for use in automatic multi-step assays because paper can be an excellent platform to control sequential fluid flow without external equipment. This review focuses on recent developments on how to control flow rate in paper-based microfluidic devices for automating sequential multi-step assays. The aim of this review is to discuss the limitations of LFTs and potential paper-based microfluidic devices for automated sequential multi-step assays in developing countries; and the existing fluidic control technologies for sequential multi-step assays. In addition, we present future challenges for commercialization of paper-based microfluidic devices to perform automatic multi-step assays.     

Single Cell Electrical Characterization Techniques

Electrical properties of living cells have been proven to play significant roles in understanding of various biological activities including disease progression both at the cellular and molecular levels. Since two decades ago, many researchers have developed tools to analyze the cell’s electrical states especially in single cell analysis (SCA). In depth analysis and more fully described activities of cell differentiation and cancer can only be accomplished with single cell analysis. This growing interest was supported by the emergence of various microfluidic techniques to fulfill high precisions screening, reduced equipment cost and low analysis time for characterization of the single cell’s electrical properties, as compared to classical bulky technique. This paper presents a historical review of single cell electrical properties analysis development from classical techniques to recent advances in microfluidic techniques. Technical details of the different microfluidic techniques are highlighted, and the advantages and limitations of various microfluidic devices are discussed.     

Single-Circulating Tumor Cell Whole Genome Amplification to Unravel Cancer Heterogeneity and Actionable Biomarkers

The field of single-cell analysis has advanced rapidly in the last decade and is providing new insights into the characterization of intercellular genetic heterogeneity and complexity, especially in human cancer. In this regard, analyzing single circulating tumor cells (CTCs) is becoming particularly attractive due to the easy access to CTCs from simple blood samples called “liquid biopsies”. Analysis of multiple single CTCs has the potential to allow the identification and characterization of cancer heterogeneity to guide best therapy and predict therapeutic response. However, single-CTC analysis is restricted by the low amounts of DNA in a single cell genome. Whole genome amplification (WGA) techniques have emerged as a key step, enabling single-cell downstream molecular analysis. Here, we provide an overview of recent advances in WGA and their applications in the genetic analysis of single CTCs, along with prospective views towards clinical applications. First, we focus on the technical challenges of isolating and recovering single CTCs and then explore different WGA methodologies and recent developments which have been utilized to amplify single cell genomes for further downstream analysis. Lastly, we list a portfolio of CTC studies which employ WGA and single-cell analysis for genetic heterogeneity and biomarker detection.     

Analysis of CGF Biomolecules,Structure and Cell Population: Characterization of the Stemness Features of CGF Cells and Osteogenic Potential

Concentrated Growth Factors (CGF) represent new autologous (blood-derived biomaterial), attracting growing interest in the field of regenerative medicine. In this study, the chemical, structural, and biological characterization of CGF was carried out. CGF molecular characterization was performed by GC/MS to quantify small metabolites and by ELISA to measure growth factors and matrix metalloproteinases (MMPs) release; structural CGF characterization was carried out by SEM analysis and immunohistochemistry; CGF has been cultured, and its primary cells were isolated for the identification of their surface markers by flow cytometry, Western blot, and real-time PCR; finally, the osteogenic differentiation of CGF primary cells was evaluated through matrix mineralization by alizarin red staining and through mRNA quantification of osteogenic differentiation markers by real-time PCR. We found that CGF has a complex inner structure capable of influencing the release of growth factors, metabolites, and cells. These cells, which could regulate the production and release of the CGF growth factors, show stem features and are able to differentiate into osteoblasts producing a mineralized matrix. These data, taken together, highlight interesting new perspectives for the use of CGF in regenerative medicine.     

Investigation of solid oxide fuel cell short stacks for mobile applications by electrochemical impedance spectroscopy

Solid oxide fuel cells (SOFC) for mobile applications are developed and investigated at the German Aerospace Center (DLR) in Stuttgart. Therefore a light-weight stack design was developed in cooperation with the automotive industry (BMW/Munich, Elring-Klinger/Dettingen, ThyssenKrupp/Essen) and the Research Center Jülich (FZJ). This concept is based on the application of stamped metal sheet bipolar plates, into which the SOFC cells are integrated by brazing technology. For the development and the investigation of the SOFC cells and short stacks, the electrochemical impedance spectroscopy (EIS) is an important and useful characterization method. The paper concentrates on the investigation and on the electrochemical testing of the SOFC short stacks with sintered anode-supported cells (ASC). The short stacks were electrochemically characterized mainly by electrochemical impedance spectroscopy, by current-voltage measurements and by long-term measurements. The cells and stacks were operated at different temperatures, varying fuel gas compositions, different fuel gas flow rates and at different electrical current loads. The influence of these operating conditions on the electrochemical performance of the short stacks is outlined. The nature of losses, e.g. ohmic and the polarization resistances of the electrodes were examined and determined by fitting of the impedance spectra to an equivalent circuit.     

Continuous and segmented flow microfluidics: applications in high-throughput chemistry and biology

This account highlights some of our recent activities focused on developing microfluidic technologies for application in high-throughput and high-information content chemical and biological analysis. Specifically, we discuss the use of continuous and segmented flow microfluidics for artificial membrane formation, the analysis of single cells and organisms, nanomaterial synthesis and DNA amplification via the polymerase chain reaction. In addition, we report on recent developments in small-volume detection technology that allow access to the vast amounts of chemical and biological information afforded by microfluidic systems.     

Fully automated on-chip imaging flow cytometry system with disposable contamination-free plastic re-cultivation chip

We have developed a novel imaging cytometry system using a poly(methyl methacrylate (PMMA)) based microfluidic chip. The system was contamination-free, because sample suspensions contacted only with a flammable PMMA chip and no other component of the system. The transparency and low-fluorescence of PMMA was suitable for microscopic imaging of cells flowing through microchannels on the chip. Sample particles flowing through microchannels on the chip were discriminated by an image-recognition unit with a high-speed camera in real time at the rate of 200 event/s, e.g., microparticles 2.5 μm and 3.0 μm in diameter were differentiated with an error rate of less than 2%. Desired cells were separated automatically from other cells by electrophoretic or dielectrophoretic force one by one with a separation efficiency of 90%. Cells in suspension with fluorescent dye were separated using the same kind of microfluidic chip. Sample of 5 μL with 1 × 10(6) particle/mL was processed within 40 min. Separated cells could be cultured on the microfluidic chip without contamination. The whole operation of sample handling was automated using 3D micropipetting system. These results showed that the novel imaging flow cytometry system is practically applicable for biological research and clinical diagnostics.     

NUMERICAL SIMULATION OF ELECTROKINETIC MICROFLUIDICS IN COLLOIDAL SYSTEMS

A numerical scheme is presented for simulating electrokinetic microfluidics in systems with arbitrary morphology. This scheme is based on a numerical solution of the coupled Poisson, Nernst-Planck, and Navier-Stokes equations. While traditional finite-difference methods were used to resolve the first two problems, the lattice Boltzmann method was applied to the latter. The developed numerical approach was used for the simulation of electroosmotic flow through a simple cubic array of hard (impermeable, nonconducting) micro-sized spheres. Volumetric electroosmotic flow was studied for dependence on electrical field strength, ζ-potential at the solid-liquid interface, electrical double layer interaction, and numerical grid resolution. Colloid stability and electrokinetics in microfluidic devices with particulate or monolithic fixed-bed elements represent two potential applications of this work.     

Electrochemical surface plasmon spectroscopy—Recent developments and applications

A survey is given on recent developments and applications of electrochemical techniques combined with surface plasmon resonance (SPR) spectroscopy. Surface plasmon spectroscopy (SPS) and optical waveguide mode spectroscopy make use of evanescent waves on metal-dielectric interfaces and can be conveniently combined with electrochemical methods. Selected examples of applications of high-pressure surface electrochemical plasmon resonance spectroscopy to study supramolecular architectures such as layer-by-layer films of conducting polymers or thin composite films will be presented. Then a combination of SPS with the electrochemical quartz crystal microbalance (EQCM) will be introduced and illustrated with a study on doping/de-doping process of a conducting polymer. This combination allows for simultaneous electrochemical, optical and microgravimetric characterization of interfaces. Finally, new technical developments including integration of SPS into microfluidic devices using a grating coupler and surface plasmon enhanced diffraction will be discussed.     

Digital Microfluidics for Manipulation and Analysis of a Single Cell

The basic structural and functional unit of a living organism is a single cell. To understand the variability and to improve the biomedical requirement of a single cell, its analysis has become a key technique in biological and biomedical research. With a physical boundary of microchannels and microstructures, single cells are efficiently captured and analyzed, whereas electric forces sort and position single cells. Various microfluidic techniques have been exploited to manipulate single cells through hydrodynamic and electric forces. Digital microfluidics (DMF), the manipulation of individual droplets holding minute reagents and cells of interest by electric forces, has received more attention recently. Because of ease of fabrication, compactness and prospective automation, DMF has become a powerful approach for biological application. We review recent developments of various microfluidic chips for analysis of a single cell and for efficient genetic screening. In addition, perspectives to develop analysis of single cells based on DMF and emerging functionality with high throughput are discussed.     

Microfluidic extraction using two phase laminar flow for chemical and biological applications

We review the state of the art in microfluidic separation technique based two-phase laminar flow with an application focus on chemical and biological sample. As we describe herein, two-phase laminar flow in the microfluidic extraction has several biological and engineering advantages over other methods including high reproducibility, biocompatibility, and selectivity. We review advances in applications of two-phase laminar flow and examine key parameters such as flow rate, phase composition, and surface charge property, how these can affect extract performance with the technology including microfluidic separation system. A special technology focus is given to emerging novel integrative microfluidic extraction, which aims to merge aqueous phase laminar flow and electric field technologies into simple packages. We conclude with a brief discussion of some of the emerging challenges in the field and some of the approaches that are likely to enhance their application.     

Polymeric proton conducting systems based on commercial elastomers. III. Microstructural and electrical characterization of films based on HSBS/EPDM/PP/PS/silica

This study goes on with the microstructural and electrical characterization of the films, object of our study, containing hydrogenated poly(butadiene‐styrene) block copolymer (HSBS). Ethylene–propylene terpolymer (EPDM), and a third component, polypropylene (PP), polystyrene (PS), or silica, crosslinked with peroxides and heterogeneously sulfonated. This process pursues the obtaining of materials with high proton conductivity and great dimensional stability, suited for application in a variety of electronic devices. The structural characterization consists of the study of the dynamic mechanical properties of all the samples, before and after the sulfonation, and also the checking of their structure by infrared spectroscopy. The analysis of the electrical properties of the films hydrated is achieved by electrochemical impedance spectroscopy. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 102: 13–21, 2006     

Effects of shear and electrical properties on flow characteristics of pharmaceutical blends

This article examines the effects and interactions of shear rate, shear strain on electrical and flow properties of pharmaceutical blends. An unexpectedly strong relation between the flow and passive electrical properties of powders is observed to depend on the shear history of the powder bed. Charge density, impedance, dielectrophoresis, flow index, and dilation were measured for several pharmaceutical blends after they were subjected to a controlled shear environment. It was found that the increase in the shear strain intensified the electrical properties for blends that did not contain MgSt. The opposite effect was found in blends lubricated with MgSt. Different shear conditions resulted in different correlations between flow index and dilation. Flow properties of powders were found to improve with continuous exposure to shear strain. It was also found that flow properties correlated to charge acquisition and impedance for different shear treatments. © 2009 American Institute of Chemical Engineers AIChE J, 2010     

Hybrid membranes based on block co-polymer ionomers and silica gel. Synthesis and characterization

This work reports on the synthesis (via heterogeneous sulfonation) structural and electrical characterization of hybrid membranes based on block co-polymer ionomers (HSBR and EPDM) and silica gel prepared by sol-gel reaction.The structural characterization consisted of the analysis of their thermal and mechanodynamical transitions by DSC and DMA, respectively. The ion-exchange capacity of each polymer was determined by both titration and elemental analysis (EA). The introduction of sulfonic groups was verified by means of infrared spectroscopy (ATR). The electrical characterization was made using ac impedance at different hydration times, the conductivity being calculated from the corresponding impedance spectra. Finally, methanol crossover through the membranes was carried out, comparing the results obtained with Nafion 117.The results indicate the existence of a complex microstructure formed by different phases corresponding to both ionic and non-ionic blocks of the co-polymer as well as the aggregates or clusters owed to the electrostatic interaction among ion pairs. Conductivity values are similar to Nafion and they improve with hydration time for hybrid membranes due to the absorbent nature of the inorganic filler. Likewise, methanol crossover is lower than in Nafion, probably due to the barrier effect exerted by the non-sulfonated blocks of the co-polymer.     

An observed correlation between flow and electrical properties of pharmaceutical blends

We study the relation between flow and electrical conductivity of multiple formulations of pharmaceutical powder blends. Ten formulations were tested, consisting of two excipient sets, two active preparations, and a variety of food-grade additives including magnesium stearate (MgSt), and ionic and conductive materials such as ascorbic acid, talc, sodium carbonate, colloidal silica and TiO₂. Electrical impedance, flow index and dilation were independently measured for all of the blends, and a strong correlation was found between every pair of these three properties. The relation between flow and dilation has been observed before; we find for the first time that there is an exponential relationship between flow index or dilation and impedance. This indicates that cohesive powder behavior depends on powder electrical properties, raising the questions of whether additives such as MgSt affect friction and conductivity per se and what mechanism and phenomenon links cohesion and conductivity.     

An electrochemical impedance model for integrated bacterial biofilms

Bacterial cells attachment onto solid surfaces and the following growth into mature microbial biofilms may result in highly antibiotic resistant biofilms. Such biofilms may be incidentally formed on tissues or implanted devices, or intentionally formed by directed deposition of microbial sensors on whole-cell bio-chip surface. A new method for electrical characterization of the later on-chip microbial biofilm buildup is presented in this paper. Measurement of impedance vs. frequency in the range of 100 mHz to 400 kHz of Escherichia coli cells attachment to indium-tin-oxide-coated electrodes was carried out while using optical microscopy estimating the electrode area coverage. We show that impedance spectroscopy measurements can be interpreted by a simple electrical equivalent model characterizing both attachment and growth of the biofilm. The correlation of extracted equivalent electrical lumped components with the visual biofilm parameters and their dependence on the attachment and growth phases is confirmed.     

Multiwalled nanotube-coated mesophase carbon microbeads for use as anode material in lithium ion batteries

The recent developments in lithium ion secondary batteries (LIBs) have been achieved by using selected carbon materials as the anode. Mesophase carbon microbead (MCMB) anode materials have stable Li intercalation and de-intercalation characteristics, making them a good anode material for use in LIBs. However, batteries with pure MCMB anodes are known to have a low power density. Multiwalled carbon nanotubes (MWNTs) are one of the most promising materials for improving a range of electrochemical energy conversion and storage devices because of their unique physical properties, including high electrical conductivity and superior chemical and mechanical stability. Therefore, in this study, MWNTs were deposited on the surface of MCMB anodes to improve their electrical conductivity. The anode materials were separately functionalized using carboxylic acid and amine groups to form MWNT-COOH and MCMB-NH₂, respectively, providing them with surfaces of opposite charge. The surface morphology was assessed using scanning electron microscopy, and the electrochemical characteristics were analyzed by cyclic voltammetry and AC impedance measurements in a coin cell. The AC impedance and cyclic voltammetry measurements indicated that MCMBs with MWNTs deposited on their surfaces are promising electrode materials, providing high power density for LIBs.     

Residence-time distribution as a measure of mixing in T-junction and multilaminated/elongational flow micromixers

The ineffective mixing in microchannel mixers or reactors, primarily due to the laminar flow behavior in such microfluidic devices, has become an issue of significant interest to many researchers working in the field of microreaction engineering and related disciplines. The present study describes the numerical and experimental investigation of mixing performance in a proposed multilaminated/elongational flow micromixer (herein referred to as MEFM-4) and a standard T-junction micromixer (TjM). These two micromixers that employ different mixing enhancement strategies were fabricated from silicon using micro-electromechanical systems (MEMS) technology. Computational fluid dynamics (CFD) approach was first used to establish the experimental platform for the mixing study. Tracer experiment utilizing UV–vis absorption spectroscopy detection technique was used to obtain the required concentration data for residence-time distribution (RTD) analysis. The RTD and its coefficient of variation (CoV) were used for indirect characterization of flow and mixing behavior in the micromixers. Using this measure, the proposed MEFM-4, as expected, exhibits a better mixing performance (with its narrower RTD and lower CoV values) than the standard TjM. The comparison of results from the CFD simulation and the experiment shows very good agreement, especially in the low Reynolds number flow regime (Re<29). In combination with matching experiment and advanced microfabrication techniques, CFD simulation is a powerful tool for effective design and evaluation of simple to complex microfluidic devices for useful applications in chemical analysis and synthesis.