Free Flow Electrophoresis Separation of Proteins and DNA Using Microfluidics and Polycarbonate Membranes (original) (raw)
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Analytical and Bioanalytical Chemistry, 2011
Free-flow electrophoresis techniques have been applied for separations in various areas of chemistry and biochemistry. Here we focus on the generation of a free-flow electrophoresis chip and direct monitoring of the separation of different molecules in the separation bed of the miniaturized chip. We demonstrate a fast and efficient way to generate a low-cost micro-free-flow electrophoresis (μFFE) chip with a filling capacity of 9.5 μL based on a multi-lamination technique. Separating webs realized by two transfer-adhesive tapes avoid the problem of gas bubbles entering the separation area. The chip is characterized by isoelectric focusing markers (IEF markers). The functionality of the chip is demonstrated by free-flow isoelectric focusing (FFIEF) of the proteins BSA (bovine serum albumin) and avidin and a single-stranded DNA (ssDNA) fragment in the pH range 3 to 10. The separation voltage ranges between 167 Vcm −1 and 422 V cm −1 , depending on the application.
Current advances and challenges in microfluidic free-flow electrophoresis—A critical review
Analytica Chimica Acta, 2017
The research field on microfluidic free-flow electrophoresis has developed vast amounts of devices, methods, applications and raised new questions, often in analogy to conventional techniques from which it derives. Most efforts have been employed on device development and a myriad of architectures and fabrication techniques have been reported using simple proof-of-principle separations. As technological aspects reach a quite mature state, researchers' new challenges include the development of protocols for the separation of complex mixtures, as required in the fields of application. The success of this effort is extremely dependent on the capability to transfer the device's fabrication to an industrial setting as well as to ensure interfacing simplicity, namely at the solutions' supply and collection, and actuation such as electric potential application and temperature control. Other advanced applications such as direct interfacing to downstream systems such as mass spectrometry, integration of sensing and feedback controls will require further development in the laboratory. In this review we provide an overview on the field, from basic concepts, through advanced developments both in the theoretical and experimental arenas, and addressing the above details. A comprehensive survey of designs, materials and applications is presented with particular highlights to most recent developments, namely the integration of electrodes, flow control and hyphenation of microfluidic free-flow electrophoresis with other techniques.
Injection and Separation Evaluation for Microfluidic Protein and DNA Separation
2018
Electrophoresis is the separation of charged particles under an applied electric field and is applied for macromolecules such as DNA, proteins, RNA and peptides through a solvent. It has been one of the most widely used analytical separation techniques since the early 1900s. Traditionally, capillary zone electrophoresis and gel electrophoresis have been used in laboratories, however, with the advent of the Lab-on-a-Chip (LOC) and the Micro Total Analysis System (TAS) concepts in the early 1990s, the focus has been on shrinking the entire laboratory with all its functions onto a microchip. The miniaturization of traditional, cumbersome laboratory equipment onto microchip devices offers the potential for decreased analysis times, reduced sample volumes, reduced operating and manufacturing costs, as well as portability. However, such downsizing necessitates a fundamental study of microscale fluid transport, microchip design, channel geometries, and sample manipulation and detection methods. This thesis reports theoretical and numerical investigations into microfluidic transport in protein and DNA separation. The thesis begins with a background chapter about electrophoresis, reviewing the fundamentals concepts of operation in microchannels. Different chip designs and electric potential configurations are discussed as well as the concepts of electroosmotic and electrophoretic velocities, diffusivity, and separation resolution. Next, the dimensional problem formulation is provided for a basic cross-linked chip, including an injection and a separation channel by assuming slip boundary conditions on the channel walls. This assumption is made after considering the details of the ion distribution in the electric double layer (EDL) adjacent to the walls as provided by the detailed derivation of the Poisson-Boltzmann equation from a statistical approach as presented in Appendix A. The non-dimensional formulation of the problem, as determined in Appendix B, was used to guide the numerical simulations and further analysis, using two numerical tools: Ansys CFX 15.0 and Matlab R2017. With the significant role that sample plug shape and size play, with regard to separation, the quality of the sample was investigated. A model was developed for evaluating the sample shape and size at the intersection of the injection and separation channels of cross-linked microchannels. A shape factor was developed to quantify the sample plug shape and size. A variety of sample plug shapes was analyzed and criteria to assess the sample plug were identified. Higher quality sample plugs result in increased separation resolution and this is predominantly possible when a rectangular sample plug is achieved at injection.
Biological & Pharmaceutical Bulletin, 2006
Microchip electrophoresis has widely grown during the past few years, and it has showed a significant result as a strong separation tool for genomic as well as proteomic researches. To enhance and expand the role of microchip electrophoresis, several studies have been proposed especially for the low viscous separation media, which is an important factor for the success of microchip with its narrow separation channels. In this paper we show an overview for the done researches in the field of low viscous media developed for the use in microchip electrophoresis. For genomic separation studies polyhydroxy additives have been used enhance the separation of DNA at low polymer concentration of HPMC (Hydroxypropylmethyl cellulose) which could keep the viscosity low. Mixtures of poly(ethylene oxide) as well as Hydroxyporpyl cellulose have been successfully introduced for chip separation. Furthermore high molecular mass polyacrylamides at low concentrations have been studied for DNA separation. A mixture of polymer nanoparticle with conventional polymers could show a better resolution for DNA at low concentration of the polymer. For the proteomic field isoelectric focusing on chip has been well overviewed since it is the most viscous separation media which is well used for the protein separation. The different types of isoelectric focusing such as the ampholyte-free type, the thermal type as well as the ampholyte-depended type have been introduced in this paper. Isoelectric focusing on chip with its combination with sodium dodecyl sulfate (SDS) page or free solution could give a better separation. Several application for this low viscous separation medias for either genomic or proteomic could clearly show the importance of this field.
Microchannel networks for electrophoretic separations
…, 1999
UV excimer laser photoablation was used to micro-machine polymer substrates not only to drill microchannel structures but also to change the surface physical properties of the substrates. We first describe how UV laser photoablation can be used for the patterning of biomolecules on a polymer and discuss parameters such as surface coverage of active antibodies and equilibration time. Secondly, we show how to design a single-use capillary electrophoresis system comprising an on-chip injector, column and electrochemical detector. The potential of this disposable plastic device is discussed and briefly compared to classical systems. Finally, preliminary results on protein separation by isoelectric focusing on a disposable microchip are presented.
Simulation of 1d and 2d electrophoretic separations in microfluidics chips
2009
Electrophoretic separations comprise a group of analytical techniques such as capillary zone electrophoresis (CZE), isoelectric focusing (IEF), isotachophoresis (ITP) and free flow electrophoresis (FFE). In all cases, separation is based on the dissimilar mobility of ionic species under the action of an external electric field. These techniques, which are widely used in chemical and biochemical analysis, have been miniaturized in the last years and now represent one of the most important applications of the lab-on-a-chip technology. In a previous work, a generalized numerical model of electrophoresis on microfluidic devices was presented. The model is based on the set of equations that governs electrical phenomena (Poisson equation), fluid dynamics (Navier-Stokes equations), mass transport (Nerst-Planck equation) and chemical reactions. Also the relationship between the buffer characteristics (ionic strength, pH) and surface potential of channel walls is taken into consideration. In thi...
Microchannel protein separation by electric field gradient focusing
Lab on a Chip, 2005
A microchannel device is presented which separates and focuses charged proteins based on electric field gradient focusing. Separation is achieved by setting a constant electroosmotic flow velocity against step changes in electrophoretic velocity. Where these two velocities are balanced for a given analyte, the analyte focuses at that point because it is driven to it from all points within the channel. We demonstrate the separation and focusing of a binary mixture of bovine serum albumin and phycoerythrin. The device is constructed of intersecting microchannels in poly(dimethylsiloxane)(PDMS) inlaid with hollow dialysis fibers. The device uses no exotic chemicals such as antibodies or synthetic ampholytes, but operates instead by purely physical means involving the independent manipulation of electrophoretic and electroosmotic velocities. One important difference between this apparatus and most other devices designed for field-gradient focusing is the injection of current at discrete intersections in the channel rather than continuously along the length of a membrane-bound separation channel.