Double inner standard plot model of an electrophoresis titration chip for a portable and green assay of protein content in milk.

High portability and environmental safety ("green") are two of the most important objectives pursued by microfluidic methods. However, there remain many challenges for the design of portable and visual microfluidic methods (e.g., chip electrophoresis) due to use of a cumbersome pump, power supply and detector. Herein, a facile double inner standard plot (DISP) model of electrophoresis titration (ET) was proposed for portable and visual assay of proteins in test milk samples without use of a pump, power supply or detector based on a moving reaction boundary (MRB) chip. The DISP-ET model predicted that: (i) by setting the upper limit (UL) and lower limit (LL) of double inner standard milk protein contents, points U and L were, respectively, achieved in the relationship D = -aC + b (D: MRB motion distance; C: protein content); and (ii) the two points divided both the C-axis and D-axis into "poor", "eligible" and "superior" rulers scaled for quantitative assay of test samples. To demonstrate the model of DISP-ET, an original portable device (120 mm × 78 mm × 30 mm, 341 g) was designed, which had a chip (25 mm × 25 mm × 4 mm) of three channels (15 mm × 200 µm × 80 µm), platinum electrodes, a lithium cell and touch screen. A series of experiments were undertaken based on the developed portable device. The relevant experiments demonstrated systemically the validity of the DISP-ET model, theory and method. In particular, the experiments clearly showed the advantages of the DISP-ET chip: portability, visuality, green use, rapidity, and flexibility for real-life use. Finally, the device was applied for a portable and visual assay of fresh milk from a cow on a dairy farm. The DISP-ET model opens a window for designing portable and visual quantitative methods of food-safety control and clinical diagnoses.

Characterization of biomechanical properties of cells through dielectrophoresis-based cell stretching and actin cytoskeleton modeling.

BACKGROUND: Cytoskeleton is a highly dynamic network that helps to maintain the rigidity of a cell, and the mechanical properties of a cell are closely related to many cellular functions. This paper presents a new method to probe and characterize cell mechanical properties through dielectrophoresis (DEP)-based cell stretching manipulation and actin cytoskeleton modeling. METHODS: Leukemia NB4 cells were used as cell line, and changes in their biological properties were examined after chemotherapy treatment with doxorubicin (DOX). DEP-integrated microfluidic chip was utilized as a low-cost and efficient tool to study the deformability of cells. DEP forces used in cell stretching were first evaluated through computer simulation, and the results were compared with modeling equations and with the results of optical stretching (OT) experiments. Structural parameters were then extracted by fitting the experimental data into the actin cytoskeleton model, and the underlying mechanical properties of the cells were subsequently characterized. RESULTS: The DEP forces generated under different voltage inputs were calculated and the results from different approaches demonstrate good approximations to the force estimation. Both DEP and OT stretching experiments confirmed that DOX-treated NB4 cells were stiffer than the untreated cells. The structural parameters extracted from the model and the confocal images indicated significant change in actin network after DOX treatment. CONCLUSION: The proposed DEP method combined with actin cytoskeleton modeling is a simple engineering tool to characterize the mechanical properties of cells.

High-throughput, low-loss, low-cost, and label-free cell separation using electrophysiology-activated cell enrichment.

Currently, cell separation occurs almost exclusively by density gradient methods and by fluorescence- and magnetic-activated cell sorting (FACS/MACS). These variously suffer from lack of specificity, high cell loss, use of labels, and high capital/operating cost. We present a dielectrophoresis (DEP)-based cell-separation method, using 3D electrodes on a low-cost disposable chip; one cell type is allowed to pass through the chip whereas the other is retained and subsequently recovered. The method advances usability and throughput of DEP separation by orders of magnitude in throughput, efficiency, purity, recovery (cells arriving in the correct output fraction), cell losses (those which are unaccounted for at the end of the separation), and cost. The system was evaluated using three example separations: live and dead yeast; human cancer cells/red blood cells; and rodent fibroblasts/red blood cells. A single-pass protocol can enrich cells with cell recovery of up to 91.3% at over 300,000 cells per second with >3% cell loss. A two-pass protocol can process 300,000,000 cells in under 30 min, with cell recovery of up to 96.4% and cell losses below 5%, an effective processing rate >160,000 cells per second. A three-step protocol is shown to be effective for removal of 99.1% of RBCs spiked with 1% cancer cells while maintaining a processing rate of ∼170,000 cells per second. Furthermore, the self-contained and low-cost nature of the separator device means that it has potential application in low-contamination applications such as cell therapies, where good manufacturing practice compatibility is of paramount importance.

3D Printed Micro Free-Flow Electrophoresis Device.

The cost, time, and restrictions on creative flexibility associated with current fabrication methods present significant challenges in the development and application of microfluidic devices. Additive manufacturing, also referred to as three-dimensional (3D) printing, provides many advantages over existing methods. With 3D printing, devices can be made in a cost-effective manner with the ability to rapidly prototype new designs. We have fabricated a micro free-flow electrophoresis (µFFE) device using a low-cost, consumer-grade 3D printer. Test prints were performed to determine the minimum feature sizes that could be reproducibly produced using 3D printing fabrication. Microfluidic ridges could be fabricated with dimensions as small as 20 µm high × 640 µm wide. Minimum valley dimensions were 30 µm wide × 130 µm wide. An acetone vapor bath was used to smooth acrylonitrile-butadiene-styrene (ABS) surfaces and facilitate bonding of fully enclosed channels. The surfaces of the 3D-printed features were profiled and compared to a similar device fabricated in a glass substrate. Stable stream profiles were obtained in a 3D-printed µFFE device. Separations of fluorescent dyes in the 3D-printed device and its glass counterpart were comparable. A µFFE separation of myoglobin and cytochrome c was also demonstrated on a 3D-printed device. Limits of detection for rhodamine 110 were determined to be 2 and 0.3 nM for the 3D-printed and glass devices, respectively.

An optical relay approach to very low cost hybrid polymer-complementary metal-oxide semiconductor electrophoresis instrumentation.

Electrophoresis is an integral part of many molecular diagnostics protocols and an inexpensive implementation would greatly facilitate point-of-care (POC) applications. However, the high instrumentation cost presents a substantial barrier, much of it associated with fluorescence detection. The cost of such systems could be substantially reduced by placing the fluidic channel and photodiode directly above the detector in order to collect a larger portion of the fluorescent light. In future, this could be achieved through the integration and monolithic fabrication of photoresist microchannels on complementary metal-oxide semiconductor microelectronics (CMOS). However, the development of such a device is expensive due to high non-recurring engineering costs. To facilitate that development, we present a system that utilises an optical relay to integrate low-cost polymeric microfluidics with a CMOS chip that provides a photodiode, analog-digital conversion and a standard serial communication interface. This system embodies an intermediate level of microelectronic integration, and significantly decreases development costs. With a limit of detection of 1.3±0.4nM of fluorescently end-labeled deoxyribonucleic acid (DNA), it is suitable for diagnostic applications.

Cost-effectiveness and efficacy of spa, SCCmec, and PVL genotyping of methicillin-resistant Staphylococcus aureus as compared to pulsed-field gel Electrophoresis.

Pulsed-field gel electrophoresis (PFGE) is a valuable molecular typing assay used for methicillin-resistant Staphylococcus aureus (MRSA) surveillance and genotyping. However, there are several limitations associated with PFGE. In Alberta, Canada, the significant increase in the number of MRSA isolates submitted to the Provincial Laboratory for Public Health (ProvLab) for PFGE typing led to the need for an alternative genotyping method. In this study, we describe the transition from PFGE to Staphylococcus protein A (spa), Staphylococcal cassette chromosome (SCCmec), and Panton-Valentine leukocidin (PVL) typing. A total of 1915 clinical MRSA isolates collected from 2005 to 2009 were used to develop and validate an algorithm for assigning PFGE epidemic types using spa, SCCmec, and PVL typing and the resulting data was used to populate a new Alberta MRSA typing database. An additional 12620 clinical MRSA isolates collected from 2010 to 2012 as part of ongoing routine molecular testing at ProvLab were characterized using the new typing algorithm and the Alberta MRSA typing database. Switching to spa, SCCmec, and PVL from PFGE typing substantially reduced hands-on and turn-around times while maintaining historical PFGE epidemic type designations. This led to an approximate $77,000 reduction in costs from 2010 to 2012. PFGE typing is still required for a small subset of MRSA isolates that have spa types that are rare, novel, or associated with more than one PFGE epidemic type.

Qualitative Analysis To Ascertain Genotypic Identity of or Differences between Mycobacterium tuberculosis Isolates in Laboratories with Limited Resources.

Mycobacterium tuberculosis is currently genotyped using mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing, although the high cost of this technique restricts its implementation in resource-limited settings. We designed a MIRU-VNTR format, MLP3 (MIRU-VNTR length polymorphism triplex), that is based on the qualitative comparison of 5 nonfluorescent 3-band fingerprints in conventional electrophoresis and minimizes costs and technical demands. MLP3 successfully resolved cross-contamination alerts, discriminated reinfections from reactivations, clarified suspected microepidemics, and tracked transmission events of high epidemiological interest.

Digital electrophoresis of charged droplets.

A digital microfluidic system based on a direct electric charging and subsequent electrophoretic manipulation of droplets is made by simple fabrication at low cost. Digitally controlled two-dimensional droplet motions are realized by digital polarity control of an array of electrodes. By independent control of droplets and colorimetric detection, the coalescence and mixing of droplets is analyzed quantitatively. The gelation of sodium alginate and the crystallization of calcium carbonate by multiple droplet translations and coalescence and the actuation of glassy carbon beads are demonstrated to show the versatile manipulation capability of the proposed technology. Finally, we discuss the implications and potentials of the present technology.

Quantitative investigation of resolution increase of free-flow electrophoresis via simple interval sample injection and separation.

Interval free-flow zone electrophoresis (FFZE) has been used to suppress sample band broadening greatly hindering the development of free-flow electrophoresis (FFE). However, there has been still no quantitative study on the resolution increase of interval FFZE. Herein, we tried to make a comparison between bandwidths in interval FFZE and continuous one. A commercial dye with methyl green and crystal violet was well chosen to show the bandwidth. The comparative experiments were conducted under the same sample loading of the model dye (viz. 3.49, 1.75, 1.17, and 0.88 mg/h), the same running time (viz. 5, 10, 15, and 20 min), and the same flux ratio between sample and background buffer (= 10.64 × 10⁻³). Under the given conditions, the experiments demonstrated that (i) the band broadening was evidently caused by hydrodynamic factor in continuous mode, and (ii) the interval mode could clearly eliminate the hydrodynamic broadening existing in continuous mode, greatly increasing the resolution of dye separation. Finally, the interval FFZE was successfully used for the complete separation of two-model antibiotics (herein pyoluteorin and phenazine-1-carboxylic acid coexisting in fermentation broth of a new strain Pseudomonas aeruginosa M18), demonstrating the feasibility of interval FFZE mode for separation of biomolecules.

Conic electrophoretic concentrator for charged macromolecules.

A simple, rapid, and highly effective technique for concentrating charged macromolecules is described which employs electrophoresis in a conic cell made of a dialysis membrane. The cell is partly submerged in electrolyte solution, and the level of solution slowly moves down during the process. The electric field within the cell is at its maximum in the area that is level with the surface of the external solution. This maximum value increases and its location moves downward following the decreasing level of external solution carrying downward and concentrating charged macromolecules. It has been demonstrated that proteins can be concentrated within 12-15 min by a factor of ∼100,000 with the total yield of 60-80%. Concentrated proteins can be harvested from the nanoliter-sized cul-de-sac of the conic concentrator using chemically activated magnetic beads. The presence of certain protein molecules linked to the bead's surface can be further revealed by specific reaction with a microarray of antibody molecules. Such "reversed magnetic array" format was applied to a cone-concentrated exhaled breath condensate (EBC) to reveal the presence of human immunoglobulin in the EBC and to estimate its concentration. The technique may be used for concentrating and detecting trace amounts of pathogens and toxins, in protein crystallization, and in many other applications.

Whole blood assay for trypsin activity using polyanionic focusing gel electrophoresis.

The measurement of trypsin activity directly in blood is important for the development of novel diagnostics and for biomedical research. Presently, most degradative enzyme assays require sample preparation, making them time consuming, costly, and less accurate. We recently demonstrated a simple and rapid electrophoretic assay for the measurement of trypsin activity directly in whole blood. This assay utilizes a charge-changing fluorescent peptide substrate that produces a positively charged fluorescent product fragment upon cleavage by the target enzyme. This fragment is then rapidly separated from whole blood by electrophoresis and quantified with a fluorescent detector. In this study, we demonstrate that polyanionic poly-L-glutamic acid-doped polyacrylamide gels can focus the fluorescent cleavage product and markedly improve the LODs of the assay. A LOD of 2 pg in 6 microL (0.3 ng/mL) in whole human blood was achieved after a 1-h reaction of enzyme and substrate followed by 10 min of electrophoresis. This is 50- to 200-fold better than the estimated reference levels for trypsin (15-60 ng/mL) in blood. This straightforward technique now allows for the rapid measurement of clinically relevant levels of trypsin activity in microliter volumes of whole blood, providing a useful tool for the development of novel point-of-care diagnostics.

Microfluidic platforms for single-cell analysis.

Microfluidics, the study and control of the fluidic behavior in microstructures, has emerged as an important enabling tool for single-cell chemical analysis. The complex procedures for chemical cytometry experiments can be integrated into a single microfabricated device. The capability of handling a volume of liquid as small as picoliters can be utilized to manipulate cells, perform controlled cell lysis and chemical reactions, and efficiently minimize sample dilution after lysis. The separation modalities such as chromatography and electrophoresis within microchannels are incorporated to analyze various types of intracellular components quantitatively. The microfluidic approach offers a rapid, accurate, and cost-effective tool for single-cell biology. We present an overview of the recent developments in microfluidic technology for chemical-content analysis of individual cells.

High-throughput profiling of formalin-fixed paraffin-embedded tissue using parallel electrophoresis and matrix-assisted laser desorption ionization mass spectrometry.

Analysis of formalin-fixed paraffin-embedded tissues (FFPE) is increasingly recognized as a strategy for the discovery and validation of clinically useful biomarker candidates. Large tissue collections including tissue microarrays (TMAs) are available, but current analytical strategies for their characterization have limited throughput. In this report, we describe a workflow for rapid analysis of hundreds of FFPE tissue specimens. The strategy combines parallel sample processing and on-chip electrophoresis with automated matrix-assisted laser desorption ionzation mass spectrometry (MALDI MS) analysis. The method is optimized for small quantities of clinically valuable tissues allowing detection of hundreds of peptides from a single core in a TMA section. We describe results from the optimization of the method and apply it for the analysis of tissue microarrays containing formalin fixed tissue specimens from human kidney.

Comparison of fast protein liquid chromatography (FPLC) with HPLC, electrophoresis & microcolumn chromatography techniques for the diagnosis of beta-thalassaemia.

BACKGROUND & OBJECTIVE: beta-thalassaemia is a genetic disorder and an important health problem around the world. Quantitative haemoglobin A(2) (HbA(2)) levels are used for the diagnosis of beta-thalassaemia. The conventional methods are high performance liquid chromatography (HPLC), electrophoresis, and microcolumn chromatography techniques. We established a fast protein liquid chromatography (FPLC) method, to measure quantitatively of HbA(2) levels, and compared its efficacy with conventional methods. METHODS: The FPLC method, using a DEAE Sepharose, Hi Trap anion-exchange column chromatography technique was set up for HbA(2) measurement. In this study, 220 blood samples were screened for haemoglobin type by FPLC technique and also using HPLC, microcolumn chromatography and electrophoresis. RESULTS: The FPLC results were highly correlated (r = 0.985, P<0.001) with those of HPLC for quantification of HbA(2) as well as cellulose acetate electrophoresis (r = 0.977) and microcolumn chromatography (r = 0.980). The FPLC method showed 100 per cent sensitivity and specificity, positive and negative predictive value for beta-thalassaemia diagnosis. In addition, the FPLC method was simple, rapid, low cost and reproducible. The HbA(2)/E range of FPLC for beta-thalassaemia was 6-10 per cent, HbE trait was 10-40 per cent, beta-thalassaemia/HbE was 40-60 per cent and homozygous HbE was more than 60 per cent. INTERPRETATION & CONCLUSION: Our findings suggested that FPLC method could be used as a cost-effective method for routine beta-thalassaemia diagnosis.

Interactions of carbohydrates and proteins by fluorophore-assisted carbohydrate electrophoresis.

A sensitive,specific, and rapid method for the detection of carbohydrate-protein interactions is demonstrated by fluorophore-assisted carbohydrate electrophoresis (FACE). The procedure is simple and the cost is low. The advantage of this method is that carbohydrate-protein interactions can be easily displayed by FACE, and the carbohydrates do not need to be purified.

Electrophoresis: the march of pennies, the march of dimes.

The present review encompasses ca. 65 years of history of developments in electrokinetic separations, taking as a starting point the year 1937, i.e. the official launching of Tiselius' moving boundary electrophoresis (MBE). The 1950s have been particularly rich in introducing novel methodologies in zone electrophoresis (ZE), thus bringing about the decline of MBE. Among them of extraordinary importance was the development of electrophoresis on agar gels coupled to immuno-diffusion at right angles, which brought a big revolution not only in biochemistry but also in clinical chemistry. Also the by now forgotten paper electrophoresis was a landmark in separation science, in that it implemented, in its "fingerprinting" version, the first genuine two-dimensional (2D) map, coupling orthogonally a charge to a hydrophobic scale separation, while permitting for the first time the detection of spot mutations, i.e. single amino acid replacements in a polypeptide chain, that paved the way to modern genetic analysis. Equally important was the introduction of starch-block electrophoresis, that brought about the notion of sieving and the first discontinuous buffers, refined, in the 1960s, by Ornstein and Davies with their classical papers combining multiphasic buffer systems to polyacrylamide gels, that went down to history as disc-electrophoresis. The 1960s also contributed with two fundamental techniques, isoelectric focusing (IEF) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) that permitted to discriminate proteins solely on the basis of surface charge and molecular mass, respectively. The 1970s gave other fundamental contributions, such as isotachophoresis, the first example of a fully instrumental approach to electrophoresis, both in its analytical and preparative version (Tachophor and Tachofrac), 2D maps combining IEF to SDS-PAGE at right angles and silver staining techniques, that incremented sensitivity by 3 orders of magnitude. The 1980s generated immobilized pH gradients and capillary zone electrophoresis (CZE), two big players that dominated the electrokinetic horizon for all the 1990s and still in vigorous use in present days. The review terminates with a glimpse, in the third millennium, onto microchip technology and hyphenated techniques, notably direct interfacing of various electrophoretic separation methods with mass spectrometry (MS).

Overview of digital electrophoresis analysis.

Gel electrophoresis has become a ubiquitous method in molecular biology for separating biomolecules. This prominence is the result of several factors, including the robustness, speed, and potentially high throughput of the technique. The results of this method are traditionally documented using silver halide-based photography followed by manual interpretation. While this remains an excellent method for qualitative documentation of single-gel results, digital capture offers a number of significant advantages when documentation requires quantitation and sophisticated analysis. Digital images of gel electropherograms can be obtained rapidly using an image-capture device, and the images can be easily manipulated using image analysis software. This overview presents reasons for digital documentation and analysis, defines some important key terms for imaging, explains the capture process and reviews the devices used for image capture, and provides an introduction to the software and methods used for one- and two-dimensional digital image analysis.

Digital electrophoresis analysis.

Digital recording of gel images offers several advantages over conventional photography. Capture of the image is quick and reliable, retaining the image in a medium that allows digital analysis of the image. These images are easy to handle, accurate, reproducible and less expensive to generate. This unit provides a guide to digital capture and analysis, discussing the equipment and methods for image capture and the process of image analysis for one- and two-dimensional gels.

Multistage magnetic and electrophoretic extraction of cells, particles and macromolecules.

Improved techniques for separating cells, particles, and macromolecules (proteins) are increasingly important to biotechnology because separation is frequently the limiting factor for many biological processes. Manufacturers of new enzymes and pharmaceutical products require improved methods for recovering intact cells and intracellular products. Similarly isolation, purification, and concentration of many biomolecules produced in fermentation processes is extremely important. Often such downstream processing contributes a large portion of the product cost. In conventional methods like centrifugation and even modern methods like chromatography, scale-up problems are enormous, making them uneconomical and prohibitively expensive unless the product is of very high value. Therefore there has been a need for efficient and economical alternative approaches to bioseparation processes to eliminate, reduce, or facilitate solids handling. Magnetic and electric field assisted separations may hold considerable potential for providing a future major improvement in bioseparation technology. In the present review the merits and demerits of the existing methods are discussed. We present mainly our own research on the development of unified multistage extraction processes that are versatile enough to handle cells and particles as well as macromolecules as described below. We describe multistage methods, namely ADSEP (Advanced Separator), MAGSEP (Magnetic Separator), and ELECSEP (Electrophoretic Separator), for quantitatively separating cells, particles, and solutes by using magnetically and electrophoretically assisted extraction processes. To the best of our knowledge, multistage magnetic and electrophoretic separations have not been reported in the earlier literature. The theoretical underpinnings of these separations are crucial to their success and to the identification of their advantages over other separation processes in particular applications. Hence mathematical modeling is stressed here, presenting our own models while also reviewing models reported in the literature. We also present suggestions for future work while analyzing the scale-up and economic aspects of these extraction processes. Commercial uses of the magnetic and electrophoretic processes, having both ground- and space-based research elements, also are presented in this review.