High throughput particle analysis: combining dielectrophoretic particle focussing with confocal optical detection

A microflow cytometer has been fabricated that detects and counts fluorescent particles flowing through a microchannel at a high speed based upon their fluorescence emission intensity. Dielectrophoresis is used to continuously focus particles within the flowing fluid stream into the centre of the device, which is 40 μm high and 250 μm wide. The method ensures that all the particles pass through an interrogation region approximately 5 μm in diameter, which is created by focusing a beam of light into a spot. The functioning of the device was demonstrated by detecting and counting fluorescent latex particles at a rate of up to 250 particles/s. A mixture of three different populations of latex particle was used, each sub-population with a distinct level of fluorescent intensity. The device was evaluated by comparison with a conventional fluorescent activated cell sorter (FACS) and numerical simulation demonstrated that for 6 mico m beads, and for this design of chip the theoretical throughput is of the order of 1000 particles/s (corresponding to a particle velocty of 1 mm/s)

Rapid cell separation with minimal manipulation for autologous cell therapies

The ability to isolate specific, viable cell populations from mixed ensembles with minimal manipulation and within intra-operative time would provide significant advantages for autologous, cell-based therapies in regenerative medicine. Current cell-enrichment technologies are either slow, lack specificity and/or require labelling. Thus a rapid, label-free separation technology that does not affect cell functionality, viability or phenotype is highly desirable. Here, we demonstrate separation of viable from non-viable human stromal cells using remote dielectrophoresis, in which an electric field is coupled into a microfluidic channel using shear-horizontal surface acoustic waves, producing an array of virtual electrodes within the channel. This allows high-throughput dielectrophoretic cell separation in high conductivity, physiological-like fluids, overcoming the limitations of conventional dielectrophoresis. We demonstrate viable/non-viable separation efficacy of > 98% in pre-purified mesenchymal stromal cells, extracted from human dental pulp, with no adverse effects on cell viability, or on their subsequent osteogenic capabilities

Micro/Nano-Chip Electrokinetics

Micro/nanofluidic chips have found increasing applications in the analysis of chemical and biological samples over the past two decades. Electrokinetics has become the method of choice in these micro/nano-chips for transporting, manipulating and sensing ions, (bio)molecules, fluids and (bio)particles, etc., due to the high maneuverability, scalability, sensitivity, and integrability. The involved phenomena, which cover electroosmosis, electrophoresis, dielectrophoresis, electrohydrodynamics, electrothermal flow, diffusioosmosis, diffusiophoresis, streaming potential, current, etc., arise from either the inherent or the induced surface charge on the solid-liquid interface under DC and/or AC electric fields. To review the state-of-the-art of micro/nanochip electrokinetics, we welcome, in this Special Issue of Micromachines, all original research or review articles on the fundamentals and applications of the variety of electrokinetic phenomena in both microfluidic and nanofluidic devices

Investigating the Use of Streaming and Robotic Dielectrophoresis to Enable Continuous Cell Sorting and Automatic Cell Transfer in Sample Preparation

The sorting of targeted cells or particles from a sample is a crucial step in the sample preparation process used in medical diagnosis, environmental monitoring, bio-analysis and personalized medicine. Current cell sorting techniques can be broadly classified as label based or label-free. Label-based techniques mostly rely on fluorophores or magnetic nanoparticles functionalized to bind with targeted cells. Although highly specific, this approach can be expensive and suffers from limitations in the availability of suitable markers. Label-free techniques exploit properties inherent to the cell, such as density and size, to simplify the sorting protocol and reduce cost by eliminating the need to incubate samples with labels. However, the specificity of separation is low due to minor differences between the density and size of many cells of interest. In this work, the use of dielectrophoresis (DEP) is emphasized as a label-free technique that exploits the combination of size and membrane capacitance of a cell as a marker. DEP is the movement of dielectric particles in the presence of a non-uniform electric field, which can be towards the electrode (positive DEP) or away from electrode (negative). The cell membrane capacitance used in this project can distinguish between cells based on their type, age, fate, and circadian rhythm to provide higher specificity than other label free sorting techniques. DEP has been demonstrated to separate various bio particles including viruses, plant and animal cells, biomolecules as well as stem cells. The work presented here integrates fabrication, numerical simulations, analysis and experimentation to focus on three main objectives 1) addressing the gaps of knowledge in electrode fabrication 2) developing analytical system for rapid cell sorting of cell population 3) demonstrating feasibility of automated single cell sorting. These are addressed in the following paragraphs in that order. Carbon electrodes are excellent alternatives for DEP because of the ease of fabrication of 3D electrode geometries and low voltages involved. Previous works have used these electrodes for applications like DEP, electrochemical bio sensing applications, fuel cells and micro-capacitors. The fabrication process involves photo patterning of SU-8 posts followed by carbonization in an inert atmosphere. During pyrolysis, the structures retain their shape, but show shrinkage. Though the fabrication process is reproducible, limited knowledge is available about the shrinkage process. Shrinkage affects the design of devices where these structures are used because the electrode dimensions after pyrolysis vary from the design and resulting electric field in the domain is affected. Previous works observed dependence of shrinkage on structure height and width, but a defining relation between shrinkage and the geometry was lacking. In this work, shrinkage is studied as an effect of degassing through the lateral and top surface area of the electrodes. Empirical relations are to enable prediction of shrinkage in the design stage StreamingDEP refers to focusing particles into narrow streams with a proper play of positive DEP and drag force. This is important in continuous sorting because of the high throughput, limited exposure of cells to electric field and ability of integration to further analysis steps. Though streamingDEP has been demonstrated previously, the dependence of the particle focusing on system parameters has not been studied. In this work, an analytical model is built to study the effect of electrode geometry, flow and electric field parameters as well as cell properties. The analytical expression developed here is validated by experiments and simulations. Robotic transfer is required for efficient handling of cells and integration with analysis steps. Liquid handling robots are currently used in laboratories to transfer cells between different steps. Though they have precise control over the transfer of cells, the sorting ability is limited. To address these limitations, a proof-of-concept of roboticDEP device was innovated to enable transfer of targeted cells. The development of this system required studying the influence of DEP parameters in pick up and transfer of cells. The device was studied for elimination of contamination by using flow and electric field. The robotic DEP platform demonstrates a novel and unique approach to automated cell sorting with potential applications for single cell analysis, cell sorting and cell patterning

Microdevices and Microsystems for Cell Manipulation

Microfabricated devices and systems capable of micromanipulation are well-suited for the manipulation of cells. These technologies are capable of a variety of functions, including cell trapping, cell sorting, cell culturing, and cell surgery, often at single-cell or sub-cellular resolution. These functionalities are achieved through a variety of mechanisms, including mechanical, electrical, magnetic, optical, and thermal forces. The operations that these microdevices and microsystems enable are relevant to many areas of biomedical research, including tissue engineering, cellular therapeutics, drug discovery, and diagnostics. This Special Issue will highlight recent advances in the field of cellular manipulation. Technologies capable of parallel single-cell manipulation are of special interest

High spatial and temporal resolution cell manipulation techniques in microchannels

The advent of microfluidics has enabled thorough control of cell manipulation experiments in so called lab on chips. Lab on chips foster the integration of actuation and detection systems, and require minute sample and reagent amounts. Typically employed microfluidic structures have similar dimensions as cells, enabling precise spatial and temporal control of individual cells and their local environments. Several strategies for high spatio-temporal control of cells in microfluidics have been reported in recent years, namely methods relying on careful design of the microfluidic structures (e.g. pinched flow), by integration of actuators (e.g. electrodes or magnets for dielectro-, acousto- and magneto-phoresis), or integrations thereof. This review presents the recent developments of cell experiments in microfluidics divided into two parts: an introduction to spatial control of cells in microchannels followed by special emphasis in the high temporal control of cell-stimulus reaction and quenching. In the end, the present state of the art is discussed in line with future perspectives and challenges for translating these devices into routine applications

Label-free cell separation and sorting in microfluidic systems

Cell separation and sorting are essential steps in cell biology research and in many diagnostic and therapeutic methods. Recently, there has been interest in methods which avoid the use of biochemical labels; numerous intrinsic biomarkers have been explored to identify cells including size, electrical polarizability, and hydrodynamic properties. This review highlights microfluidic techniques used for label-free discrimination and fractionation of cell populations. Microfluidic systems have been adopted to precisely handle single cells and interface with other tools for biochemical analysis. We analyzed many of these techniques, detailing their mode of separation, while concentrating on recent developments and evaluating their prospects for application. Furthermore, this was done from a perspective where inertial effects are considered important and general performance metrics were proposed which would ease comparison of reported technologies. Lastly, we assess the current state of these technologies and suggest directions which may make them more accessible

A concise review of microfluidic particle manipulation methods

Particle manipulation is often required in many applications such as bioanalysis, disease diagnostics, drug delivery and self-cleaning surfaces. The fast progress in micro- and nano-engineering has contributed to the rapid development of a variety of technologies to manipulate particles including more established methods based on microfluidics, as well as recently proposed innovative methods that still are in the initial phases of development, based on self-driven microbots and artificial cilia. Here, we review these techniques with respect to their operation principles and main applications. We summarize the shortcomings and give perspectives on the future development of particle manipulation techniques. Rather than offering an in-depth, detailed, and complete account of all the methods, this review aims to provide a broad but concise overview that helps to understand the overall progress and current status of the diverse particle manipulation methods. The two novel developments, self-driven microbots and artificial cilia-based manipulation, are highlighted in more detail