High flux novel polymeric membrane for renal applications.

Biocompatibility and the ability to mediate the appropriate flux of ions, urea, and uremic toxins between blood and dialysate components are key parameters for membranes used in dialysis. Oxone-mediated TEMPO-oxidized cellulose nanomaterials have been demonstrated to be excellent additives in the production and tunability of ultrafiltration and dialysis membranes. In the present study, nanocellulose ionic liquid membranes (NC-ILMs) were tested in vitro and ex vivo. An increase in flux of up to two orders of magnitude was observed with increased rejection (about 99.6%) of key proteins compared to that of polysulfone (PSf) and other commercial membranes. NC-ILMs have a sharper molecular weight cut-off than other phase inversion polymeric membranes, allowing for high throughput of urea and a uremic toxin surrogate and limited passage of proteins in dialysis applications. Superior anti-fouling properties were also observed for the NC-ILMs, including a > 5-h operation time with no systemic anticoagulation in blood samples. Finally, NC-ILMs were found to be biocompatible in rat ultrafiltration and dialysis experiments, indicating their potential clinical utility in dialysis and other blood filtration applications. These superior properties may allow for a new class of membranes for use in a wide variety of industrial applications, including the treatment of patients suffering from renal disease.

Development of an Integrated Salt Cartridge-Reverse Electrodialysis (Red) Device to Increase Electrolyte Concentrations to Biomedical Devices.

Emerging technologies in nanotechnology and biomedical engineering have led to an increase in the use of implantable biomedical devices. These devices are currently battery powered which often means they must be surgically replaced during a patient's lifetime. Therefore, there is an important need for a power source that could provide continuous, stable power over a prolonged time. Reverse electrodialysis (RED) based biopower cells have been previously used to generate continuous power from physiologically relevant fluids; however, the low salinity gradient that exists within the body limited the performance of the biopower cell. In this study, a miniaturized RED biopower cell design coupled with a salt cartridge was evaluated for boosting the salt concentration gradient supplied to RED in situ. For the salt cartridge, polysulfone (PSf) hollow fibers were prepared in-house and saturated with NaCl solutions to deliver salt and thereby enhance the concentration gradient. The effect of operational parameters including solution flow rate and cartridge salt concentration on salt transport performance was evaluated. The results demonstrated that the use of the salt cartridge was able to increase the salt concentration of the RED inlet stream by 74% which in turn generated a 3-fold increase in the open circuit voltage (OCV) of the biopower cell. This innovative adaptation of the membrane-based approach into portable power generation could help open new pathways in various biomedical applications.

Effects of Resin Chemistries on the Selective Removal of Industrially Relevant Metal Ions Using Wafer-Enhanced Electrodeionization.

Wafer-enhanced electrodeionization (WE-EDI) is an electrically driven separations technology that occurs under the influence of an applied electric field and heavily depends on ion exchange resin chemistry. Unlike filtration processes, WE-EDI can be used to selectively remove ions even from high concentration systems. Because every excess ion transported increases the operating costs, the selective separation offered by WE-EDI can provide a more energy-efficient and cost-effective process, especially for highly concentrated salt solutions. This work reports the performance comparison of four commonly used cation exchange resins (Amberlite IR120 Na+, Amberlite IRP 69, Dowex MAC 3 H+, and Amberlite CG 50) and their influence on the current efficiency and selectivity for the removal of cations from a highly concentrated salt stream. The current efficiencies were high for all the resin types studied. Results also revealed that weak cation exchange resins favor the transport of the monovalent ion (Na+) while strong cation exchange resins either had no strong preference or preferred to transport the divalent ions (Ca2+ and Mg2+). Moreover, the strong cation exchange resins in powder form generally performed better in wafers than those in the bead form for the selective removal of divalent ions (selectivity > 1). To further understand the impact of particle size, resins in the bead form were ground into a powder. After grinding the strong cation resins displayed similar behavior (more consistent current efficiency and preference for transporting divalent ions) to the strong cation resins in powder form. This indicates the importance of resin size in the performance of wafers.

Capillary electrophoresis for the analysis of the effect of sample preparation on early stages of Aß1-40 aggregation.

Aggregation of the amyloid-ß protein (Aß) contributes to the neurodegeneration characteristic of Alzheimer's disease. Of particular importance are the early stages of aggregation, which involve the formation of soluble oligomers and protofibrils. In these studies, we demonstrate the potential for CE with UV detection using a polyethylene oxide separation matrix to identify the evolution of various oligomeric species of Aß1-40 . To demonstrate the efficacy of this technique, UV-CE was utilized to compare two methods commonly used to prepare Aß for aggregation experiments and their effect on the formation of early aggregates. SEC-purified Aß1-40 initially contained more small species, including monomer, than did freshly dissolved Aß1-40 pretreated with hexafluoroisopropanol. Strikingly, the lag time to oligomer formation for SEC-isolated Aß1-40 samples was ∼23 h shorter compared to freshly dissolved Aß1-40 samples. Furthermore, oligomers formed from the aggregation of SEC-purified Aß1-40 persisted within solution for a longer period of time. These results indicate that the initial sample preparation has a drastic influence on the early stages of Aß1-40 aggregation. This is the first report of the use of UV-CE with a separation matrix to study the effect of sample preparation on early aggregation of Aß1-40 . UV-CE was also used in parallel with dot blot analysis and inhibitory compounds to discern structural characteristics of individual oligomer peaks, demonstrating the capacity of UV-CE as a complimentary technique to further understand the aggregation process.

Capillary electrophoresis-single strand conformation polymorphism for the detection of multiple mutations leading to tuberculosis drug resistance.

Drug resistant tuberculosis (TB) is a major health problem in both developed and developing countries. Mutations in the Mycobacterium (M.) tuberculosis bacterial genome, such as those to the rpoB gene and mabA-inhA promoter region, have been linked to TB drug resistance in against rifampicin and isoniazid, respectively. The rapid, accurate, and inexpensive identification of these and other mutations leading to TB drug resistance is an essential tool for improving human health. Capillary electrophoresis (CE) single strand conformation polymorphism (SSCP) can be a highly sensitive technique for the detection of genetic mutation that has not been previously explored for drug resistance mutations in M. tuberculosis. This work explores the potential of CE-SSCP through the optimization of variables such as polymer separation matrix concentration, capillary wall coating, electric field strength, and temperature on resolution of mutation detection. The successful detection of an rpoB gene mutation and two mabA-inhA promoter region mutations while simultaneously differentiating a TB-causing mycobacteria from a non-TB bacteria was accomplished using the optimum conditions of 4.5% (w/v) PDMA in a PDMA coated capillary at 20°C using a separation voltage of 278 V/cm. This multiplexed analysis that can be completed in a few hours demonstrates the potential of CE-SSCP to be an inexpensive and rapid analysis method.

Unraveling the early events of amyloid-ß protein (Aß) aggregation: techniques for the determination of Aß aggregate size.

The aggregation of proteins into insoluble amyloid fibrils coincides with the onset of numerous diseases. An array of techniques is available to study the different stages of the amyloid aggregation process. Recently, emphasis has been placed upon the analysis of oligomeric amyloid species, which have been hypothesized to play a key role in disease progression. This paper reviews techniques utilized to study aggregation of the amyloid-ß protein (Aß) associated with Alzheimer's disease. In particular, the review focuses on techniques that provide information about the size or quantity of oligomeric Aß species formed during the early stages of aggregation, including native-PAGE, SDS-PAGE, Western blotting, capillary electrophoresis, mass spectrometry, fluorescence correlation spectroscopy, light scattering, size exclusion chromatography, centrifugation, enzyme-linked immunosorbent assay, and dot blotting.

Blinded study determination of high sensitivity and specificity microchip electrophoresis-SSCP/HA to detect mutations in the p53 gene.

Knowledge of the genetic changes that lead to disease has grown and continues to grow at a rapid pace. However, there is a need for clinical devices that can be used routinely to translate this knowledge into the treatment of patients. Use in a clinical setting requires high sensitivity and specificity (>97%) in order to prevent misdiagnoses. Single-strand conformational polymorphism (SSCP) and heteroduplex analysis (HA) are two DNA-based, complementary methods for mutation detection that are inexpensive and relatively easy to implement. However, both methods are most commonly detected by slab gel electrophoresis, which can be labor-intensive, time-consuming, and often the methods are unable to produce high sensitivity and specificity without the use of multiple analysis conditions. Here, we demonstrate the first blinded study using microchip electrophoresis (ME)-SSCP/HA. We demonstrate the ability of ME-SSCP/HA to detect with 98% sensitivity and specificity >100 samples from the p53 gene exons 5-9 in a blinded study in an analysis time of <10 min.

Monitoring insulin aggregation via capillary electrophoresis.

Early stages of insulin aggregation, which involve the transient formation of oligomeric aggregates, are an important aspect in the progression of Type II diabetes and in the quality control of pharmaceutical insulin production. This study is the first to utilize capillary electrophoresis (CE) with ultraviolet (UV) detection to monitor insulin oligomer formation at pH 8.0 and physiological ionic strength. The lag time to formation of the first detected species in the aggregation process was evaluated by UV-CE and thioflavin T (ThT) binding for salt concentrations from 100 mM to 250 mM. UV-CE had a significantly shorter (5-8 h) lag time than ThT binding (15-19 h). In addition, the lag time to detection of the first aggregated species via UV-CE was unaffected by salt concentration, while a trend toward an increased lag time with increased salt concentration was observed with ThT binding. This result indicates that solution ionic strength impacts early stages of aggregation and ß-sheet aggregate formation differently. To observe whether CE may be applied for the analysis of biological samples containing low insulin concentrations, the limit of detection using UV and laser induced fluorescence (LIF) detection modes was determined. The limit of detection using LIF-CE, 48.4 pM, was lower than the physiological insulin concentration, verifying the utility of this technique for monitoring biological samples. LIF-CE was subsequently used to analyze the time course for fluorescein isothiocyanate (FITC)-labeled insulin oligomer formation. This study is the first to report that the FITC label prevented incorporation of insulin into oligomers, cautioning against the use of this fluorescent label as a tag for following early stages of insulin aggregation.

Multiplexed p53 mutation detection by free-solution conjugate microchannel electrophoresis with polyamide drag-tags.

We report a new, bioconjugate approach to performing highly multiplexed single-base extension (SBE) assays, which we demonstrate by genotyping a large panel of point mutants in exons 5-9 of the p53 gene. A series of monodisperse polyamide "drag-tags" was created using both chemical and biological synthesis and used to achieve the high-resolution separation of genotyping reaction products by microchannel electrophoresis without a polymeric sieving matrix. A highly multiplexed SBE reaction was performed in which 16 unique drag-tagged primers simultaneously probe 16 p53 gene loci, with an abbreviated thermal cycling protocol of only 9 min. The drag-tagged SBE products were rapidly separated by free-solution conjugate electrophoresis (FSCE) in both capillaries and microfluidic chips with genotyping accuracy in excess of 96%. The separation requires less than 70 s in a glass microfluidic chip, or about 20 min in a commercial capillary array sequencing instrument. Compared to gel electrophoresis, FSCE offers greater freedom in the design of SBE primers by essentially decoupling the length of the primer and the electrophoretic mobility of the genotyping products. FSCE also presents new possibilities for the facile implementation of SBE on integrated microfluidic electrophoresis devices for rapid, high-throughput genetic mutation detection or SNP scoring.

The potential of electrophoretic mobility shift assays for clinical mutation detection.

As the understanding of the links between genetic mutations and diseases continues to grow, there is an increasing need for techniques that can rapidly, inexpensively, and sensitively detect DNA sequence alterations. Typically, such analyses are performed on PCR-amplified gene regions. Automated DNA sequencing by capillary array electrophoresis can be used, but is expensive to apply to large numbers of patient samples and/or large genes, and may not always reveal low-abundance mutations in heterozygous samples. Many different types of genetic differences need to be detected, including single-base substitutions and larger sequence alterations such as insertions, deletions, and inversions. Electrophoretic mobility shift assays seem well suited to this purpose and could be used for the efficient screening of patient samples for sequence alterations, effectively reducing the number of samples that must be subjected to full and careful sequencing. While there is much promise, many of the mobility shift assays presently under development have yet to be demonstrated to have the high sensitivity and specificity of mutation detection required for routine clinical application. Hence, further studies and optimization are required, in particular the application of these methods not only to particular genes but also to large numbers of patient samples in blinded studies aimed at the rigorous determination of sensitivity and specificity. This review examines the state-of-the-art of the most commonly used mobility shift assays for mutation detection, including denaturing gradient gel electrophoresis, TGGE, SSCP, heteroduplex analysis, and denaturing HPLC.

An optimized microchip electrophoresis system for mutation detection by tandem SSCP and heteroduplex analysis for p53 gene exons 5-9.

With the complete sequencing of the human genome, there is a growing need for rapid, highly sensitive genetic mutation detection methods suitable for clinical implementation. DNA-based diagnostics such as single-strand conformational polymorphism (SSCP) and heteroduplex analysis (HA) are commonly used in research laboratories to screen for mutations, but the slab gel electrophoresis (SGE) format is ill-suited for routine clinical use. The translation of these assays from SGE to microfluidic chips offers significant speed, cost, and sensitivity advantages; however, numerous parameters must be optimized to provide highly sensitive mutation detection. Here we present a methodical study of system parameters including polymer matrix, wall coating, analysis temperature, and electric field strengths on the effectiveness of mutation detection by tandem SSCP/HA for DNA samples from exons 5-9 of the p53 gene. The effects of polymer matrix concentration and average molar mass were studied for linear polyacrylamide (LPA) solutions. We determined that a matrix of 8% w/v 600 kDa LPA provides the most reliable SSCP/HA mutation detection on chips. The inclusion of a small amount of the dynamic wall-coating polymer poly-N-hydroxyethylacrylamide in the matrix substantially improves the resolution of SSCP conformers and extends the coating lifetime. We investigated electrophoresis temperatures between 17 and 35 degrees C and found that the lowest temperature accessible on our chip electrophoresis system gives the best condition for high sensitivity of the tandem SSCP/HA method, especially for the SSCP conformers. Finally, the use of electrical fields between 350 and 450 V/cm provided rapid separations (<10 min) with well-resolved DNA peaks for both SSCP and HA.

Optimized sample preparation for tandem capillary electrophoresis single-stranded conformational polymorphism/ heteroduplex analysis.

Here we describe DNA sample preparation methods that allow the rapid, simultaneous generation of both single-stranded conformational polymorphism (SSCP) and heteroduplex DNA elements from a single sample in a single tube, which are suitable for direct injection into a capillary electrophoresis (CE) instrument with excellent sensitivity of genetic mutation detection. The p53 gene was used as a model DNA region for this study, which was performed on a high-throughput MegaBACE 96-capillary array electrophoresis instrument. We found that, contrary to the practice common in slab-gel SSCP analysis, denaturants such as formamide are incompatible with this novel technique because they result in homo- and heteroduplex peak broadening in CE (possibly as a result of incomplete dsDNA re-hybridization) that reduces the peak resolution and hence the sensitivity of mutation detection. We also have found that PCR buffers, which are typically used to suspend samples for slab-gel heteroduplex analysis (HA), but which are less suitable for CE because of the presence of extra salt that reduces the efficiency of electrokinetic injection, may be substituted with a 10 mM Tris-HCI buffer (pH 8.5). The use of this Tris-HCl buffer for sample preparation provides both a high sensitivity of mutation detection by tandem SSCP/HA and high efficiency ofelectrokinetic injection by CE. In a related study (published elsewhere), we have applied this optimized protocol to the screening of a set of 32 mutant DNA samples from p53 exons 7 and 8 and recorded 100% sensitivity of mutation detection for tandem CE-SSCP/HA, whereas each individual method yielded lower sensitivity on its own (93% for SSCP and 75% for HA).

Technical challenges in applying capillary electrophoresis-single strand conformation polymorphism for routine genetic analysis.

Recent and future advances in population genetics will have a significant impact on health care practices and the economics of health care provision only if a spectrum of patient-tailored, effective methods of DNA screening for sequence alterations has been developed. Genetic screening by capillary electrophoresis-single strand conformation polymorphism (CE-SSCP), which is based upon the differences in electrophoretic mobilities of wild-type and mutant DNA species, offers an important complement to other presently available techniques such as Sanger sequencing and DNA hybridization arrays due to its simplicity, versatility, and low cost of analysis. A two-part review of CE-SSCP that discusses its advantages and limitations is presented. Emphasis is placed on technological aspects of CE-SSCP (including such rarely addressed issues as sample preparation protocols and the nature of the polymeric DNA separation matrix) as well as on the potential of CE-SSCP for routine genetic analysis. An attempt is made to organize and present the information in sufficient detail to allow the use of SSCP for routine genetic screening even by those inexperienced in CE. Some discussion of CE-based heteroduplex analysis (HA) is also presented.

High-throughput, high-sensitivity genetic mutation detection by tandem single-strand conformation polymorphism/heteroduplex analysis capillary array electrophoresis.

We present the first optimization of linear polyacrylamide (LPA)-based DNA separation matrixes for an automated tandem microchannel single-strand conformation polymorphism (SSCP)/heteroduplex analysis (HA) method, implemented in capillary arrays dynamically coated with poly(N-hydroxyethylacrylamide) (polyDuramide). An optimized protocol for sample preparation allowed both SSCP and HA species to be produced in one step in a single tube and distinguished in a single electrophoretic analysis. A simple, two-color fluorescent sample labeling and detection strategy enabled unambiguous identification of all DNA species in the electropherogram, both single- and double-stranded. Using these protocols and a panel of 11 p53 mutant DNA samples in comparison with wild-type, we employed high-throughput capillary array electrophoresis (CAE) to carry out a systematic and simultaneous optimization of LPA weight-average molar mass (Mw) and concentration for SSCP/HA peak separation. The combination of the optimized LPA matrix (6% LPA, Mw 600 kDa) and a hydrophilic, adsorbed polyDuramide wall coating was found to be essential for resolution of CAE-SSCP/HA peaks and yielded sensitive mutation detection in all 11 p53 samples initially studied. A larger set of 32 mutant DNA specimens was then analyzed using these optimized tandem CAE-SSCP/HA protocols and materials and yielded 100% sensitivity of mutation detection, whereas each individual method yielded lower sensitivity on its own (93% for SSCP and 75% for HA). This simple, highly sensitive tandem SSCP/HA mutation detection method should be easily translatable to electrophoretic analyses on microfluidic devices, due to the ease of the capillary coating protocol and the low viscosity of the matrix.