The Experts below are selected from a list of 2442 Experts worldwide ranked by ideXlab platform
Susan M. Lunte - One of the best experts on this subject based on the ideXlab platform.
-
Monitoring carnosine uptake by RAW 264.7 macrophage cells using Microchip Electrophoresis with fluorescence detection
2019Co-Authors: Claudia G. Fresta, Michael L. Hogard, Giuseppe Caruso, Elton Melo E. Costa, Giuseppe Lazzarino, Susan M. LunteAbstract:In this report, a Microchip Electrophoresis system with fluorescence detection was used for the quantitation of intracellular carnosine in untreated and stimulated macrophage cell lysates. Carnosine was derivatized with NDA/CN and separated from other endogenous amine reported in macrophage cells. Based on ME-LIF with standard addition, macrophages were estimated to contain a basal intracellular concentration of carnosine (0.079 ± 0.02 nmol per million cells). Carnosine is readily taken up by macrophages in cell culture. Incubation with 20 mM carnosine led to a 600-fold increase in intracellular carnosine compared to basal levels. Furthermore, we have shown that under pro-inflammatory conditions using LPS and IFN-gamma stimulation there is a further 3-fold increase in carnosine uptake in macrophage cells. This suggests that there is a mechanism through which macrophages increase the usage of carnosine during oxidative stress
-
a review of microdialysis coupled to Microchip Electrophoresis for monitoring biological events
Journal of Chromatography A, 2015Co-Authors: Rachel A Saylor, Susan M. LunteAbstract:Microdialysis is a powerful sampling technique that enables monitoring of dynamic processes in vitro and in vivo. The combination of microdialysis with chromatographic or electrophoretic methods yields along with selective detection methods yields a “separation-based sensor” capable of monitoring multiple analytes in near real time. Analysis of microdialysis samples requires techniques that are fast (<1 min), have low volume requirements (nL–pL), and, ideally, can be employed on-line. Microchip Electrophoresis fulfills these requirements and also permits the possibility of integrating sample preparation and manipulation with detection strategies directly on-chip. Microdialysis coupled to Microchip Electrophoresis has been employed for monitoring biological events in vivo and in vitro. This review discusses technical considerations for coupling microdialysis sampling and Microchip Electrophoresis, including various interface designs, and current applications in the field.
-
evaluation of in channel amperometric detection using a dual channel Microchip Electrophoresis device and a two electrode potentiostat for reverse polarity separations
Electrophoresis, 2015Co-Authors: Diogenes Meneses, Dulan B Gunasekara, Pann Pichetsurnthorn, Jose Alberto Fracassi Da Silva, Fabiane Caxico De Abreu, Susan M. LunteAbstract:In-channel amperometric detection combined with dual-channel Microchip Electrophoresis is evaluated using a two-electrode isolated potentiostat for reverse polarity separations. The device consists of two separate channels with the working and reference electrodes placed at identical positions relative to the end of the channel, enabling noise subtraction. In previous reports of this configuration, normal polarity and a three-electrode detection system were used. In the two-electrode detection system described here, the electrode in the reference channel acts as both the counter and reference. The effect of electrode placement in the channels on noise and detector response was investigated using nitrite, tyrosine, and hydrogen peroxide as model compounds. The effects of electrode material and size and type of reference electrode on noise and the potential shift of hydrodynamic voltammograms for the model compounds were determined. In addition, the performance of two- and three-electrode configurations using Pt and Ag/AgCl reference electrodes was compared. Although the signal was attenuated with the Pt reference, the noise was also significantly reduced. It was found that lower LOD were obtained for all three compounds with the dual-channel configuration compared to single-channel, in-channel detection. The dual-channel method was then used for the detection of nitrite in a dermal microdialysis sample obtained from a sheep following nitroglycerin administration.
-
Optimization of the Separation of NDA-Derivatized Methylarginines by Capillary and Microchip Electrophoresis:
Journal of Laboratory Automation, 2012Co-Authors: Thomas H. Linz, Christa M. Snyder, Susan M. LunteAbstract:The methylated arginines (MAs) monomethylarginine (MMA), asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA) have been shown to be independent predictors of cardiovascular disease. This article describes progress regarding the development of an analytical method capable of rapidly analyzing MAs using capillary Electrophoresis (CE) and Microchip Electrophoresis (MCE) with laser-induced fluorescence (LIF) detection. Several parameters including buffer composition and separation voltage were optimized to achieve an ideal separation. The analytes of interest were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA) to produce fluorescent 1-cyanobenz[f]isoindole (CBI) derivatives and then subjected to CE analysis. Baseline resolution of SDMA, ADMA, MMA, and arginine was achieved in less than 8 min. The limits of detection for SDMA, ADMA, MMA, and arginine were determined to be 15, 20, 25, and 5 nM, respectively, which are well below the expected plasma concentrations. The CE separation method was then transferred to a glass MCE device with LIF detection. MAs were baseline resolved in 3 min on-chip using a 14 cm separation channel with detection limits of approximately 10 nM for each species. To the best of the authors’ knowledge, this is the first report of the separation of MAs by MCE.
-
in channel amperometric detection for Microchip Electrophoresis using a wireless isolated potentiostat
Electrophoresis, 2011Co-Authors: Dulan B Gunasekara, Matthew K Hulvey, Susan M. LunteAbstract:The combination of Microchip Electrophoresis (ME) with amperometric detection leads to a number of analytical challenges that are associated with isolating the detector from the high voltages used for the separation. While methods such as end-channel alignment and the use of decouplers have been employed, they have limitations. A less common method has been to utilize an electrically isolated potentiostat. This approach allows placement of the working electrode directly in the separation channel without using a decoupler. This paper explores the use of Microchip Electrophoresis and electrochemical detection (ME-EC) with an electrically isolated potentiostat for the separation and in-channel detection of several biologically important anions. The separation employed negative polarity voltages and tetradecyltrimethylammonium bromide (TTAB, as a buffer modifier) for the separation of nitrite (NO2-), glutathione (GSH), ascorbic acid (AA), and tyrosine (Tyr). A half-wave potential (E½) shift of approximately negative 500 mV was observed for NO2- and H2O2 standards in the in-channel configuration compared to end channel. Higher separation efficiencies were observed for both NO2- and H2O2 with the in-channel detection configuration. The limits of detection were approximately two-fold lower and the sensitivity was approximately two-fold higher for in-channel detection of nitrite when compared to end-channel. The application of this microfluidic device for the separation and detection of biomarkers related to oxidative stress is described.
Robert T Kennedy - One of the best experts on this subject based on the ideXlab platform.
-
droplet sample introduction to Microchip gel and zone Electrophoresis for rapid analysis of protein protein complexes and enzymatic reactions
Analytical and Bioanalytical Chemistry, 2019Co-Authors: Claire M Ouimet, Cara I Damico, Robert T KennedyAbstract:Electrophoresis has demonstrated utility as tool for screening of small molecule modulators of protein-protein interactions and enzyme targets. Screening of large chemical libraries requires high-throughput separations. Such fast separation can be accessed by Microchip Electrophoresis. Here, Microchip gel Electrophoresis separations of proteins are achieved in 2.6 s with 1200 V/cm and 3-mm separation lengths. However, such fast separations can still suffer from limited overall throughput from sample introduction constraints. Automated introduction of microfluidic droplets has been demonstrated to overcome this limitation. Most devices for coupling microfluidic droplets to Microchip Electrophoresis are only compatible with free-solution separations. Here, we present a device that is compatible with coupling droplets to gel and free-solution Electrophoresis. In this device, automated sample introduction is based on a novel mechanism of carrier phase separation using the difference in density of the carrier phase and the running buffer. This device is demonstrated for Microchip gel Electrophoresis and free-solution Electrophoresis separations of protein-protein interaction and enzyme samples, respectively. Throughputs of about 10 s per sample are achieved and over 1000 separations are demonstrated without reconditioning of the device. Graphical abstract.
-
Recent advances in protein analysis by capillary and Microchip Electrophoresis
The Analyst, 2017Co-Authors: Mohamed Dawod, Natalie E Arvin, Robert T KennedyAbstract:This review article describes the significant recent advances in the analysis of proteins by capillary and Microchip Electrophoresis during the period from mid-2014 to early 2017. This review highlights the progressions, new methodologies, innovative instrumental modifications, and challenges for efficient protein analysis in human specimens, animal tissues, and plant samples. The protein analysis fields covered in this review include analysis of native, reduced, and denatured proteins in addition to Western blotting, protein therapeutics and proteomics.
-
multiplexed western blotting using Microchip Electrophoresis
Analytical Chemistry, 2016Co-Authors: Michael D Furtaw, Don T Lamb, Stephen A Ferguson, Natalie E Arvin, Mohamed Dawod, Huaxian Chen, Robert T KennedyAbstract:Western blotting is a commonly used protein assay that combines the selectivity of electrophoretic separation and immunoassay. The technique is limited by long time, manual operation with mediocre reproducibility, and large sample consumption, typically 10–20 μg per assay. Western blots are also usually used to measure only one protein per assay with an additional housekeeping protein for normalization. Measurement of multiple proteins is possible; however, it requires stripping membranes of antibody and then reprobing with a second antibody. Miniaturized alternatives to Western blot based on microfluidic or capillary Electrophoresis have been developed that enable higher-throughput, automation, and greater mass sensitivity. In one approach, proteins are separated by Electrophoresis on a Microchip that is dragged along a polyvinylidene fluoride membrane so that as proteins exit the chip they are captured on the membrane for immunoassay. In this work, we improve this method to allow multiplexed protein det...
-
Multiplexed Western Blotting Using Microchip Electrophoresis
2016Co-Authors: Shi Jin, Michael D Furtaw, Don T Lamb, Stephen A Ferguson, Natalie E Arvin, Mohamed Dawod, Huaxian Chen, Robert T KennedyAbstract:Western blotting is a commonly used protein assay that combines the selectivity of electrophoretic separation and immunoassay. The technique is limited by long time, manual operation with mediocre reproducibility, and large sample consumption, typically 10–20 μg per assay. Western blots are also usually used to measure only one protein per assay with an additional housekeeping protein for normalization. Measurement of multiple proteins is possible; however, it requires stripping membranes of antibody and then reprobing with a second antibody. Miniaturized alternatives to Western blot based on microfluidic or capillary Electrophoresis have been developed that enable higher-throughput, automation, and greater mass sensitivity. In one approach, proteins are separated by Electrophoresis on a Microchip that is dragged along a polyvinylidene fluoride membrane so that as proteins exit the chip they are captured on the membrane for immunoassay. In this work, we improve this method to allow multiplexed protein detection. Multiple injections made from the same sample can be deposited in separate tracks so that each is probed with a different antibody. To further enhance multiplexing capability, the Electrophoresis channel dimensions were optimized for resolution while keeping separation and blotting times to less than 8 min. Using a 15 μm deep × 50 μm wide × 8.6 cm long channel, it is possible to achieve baseline resolution of proteins that differ by 5% in molecular weight, e.g., ERK1 (44 kDa) from ERK2 (42 kDa). This resolution allows similar proteins detected by cross-reactive antibodies in a single track. We demonstrate detection of 11 proteins from 9 injections from a single Jurkat cell lysate sample consisting of 400 ng of total protein using this procedure. Thus, multiplexed Western blots are possible without cumbersome stripping and reprobing steps
-
continuous operation of microfabricated Electrophoresis devices for 24 hours and application to chemical monitoring of living cells
Analytical Chemistry, 2009Co-Authors: Kendra R Reid, Robert T KennedyAbstract:Microchip Electrophoresis is an emerging analytical technology with several useful attributes including rapid separation time, small sample requirements, and automation. In numerous potential applications, such as chemical monitoring or high-throughput screening, it may be desirable to use a system for many analyses without operator intervention; however, long-term operation of Microchip Electrophoresis systems has received little attention. We have developed a Microchip Electrophoresis system that can automatically inject samples at 6 s intervals for 24 h resulting in collection of 14 400 assays in one session. Continuous operation time of a prototype of the device was limited to 2 h due to degradation of reagents and Electrophoresis buffers on the chip; however, modification so that all reagents were continuously perfused into reservoirs on the device ensured fresh reagents were always used for analysis and enabled extended operating sessions. The Electrophoresis chip incorporated a cell perfusion chamb...
Adam T. Woolley - One of the best experts on this subject based on the ideXlab platform.
-
analysis of thrombin antithrombin complex formation using Microchip Electrophoresis and mass spectrometry
Electrophoresis, 2019Co-Authors: Jacob B Nielsen, Anna V. Nielsen, Richard H Carson, Robert L Hanson, Mukul Sonker, Daniel N Mortensen, John C Price, Adam T. WoolleyAbstract:: Preterm birth (PTB) related health problems take over one million lives each year, and currently, no clinical analysis is available to determine if a fetus is at risk for PTB. Here, we describe the preparation of a key PTB risk biomarker, thrombin-antithrombin (TAT), and characterize it using dot blots, MS, and Microchip Electrophoresis (µCE). The pH for fluorescently labeling TAT was also optimized using spectrofluorometry and spectrophotometry. The LOD of TAT was measured in µCE. Lastly, TAT was combined with six other PTB risk biomarkers and separated in µCE. The ability to make and characterize TAT is an important step toward the development of an integrated microfluidic diagnostic for PTB risk.
-
3d printed microfluidic devices for Microchip Electrophoresis of preterm birth biomarkers
Analytical Chemistry, 2019Co-Authors: Michael J Beauchamp, Anna V. Nielsen, Hua Gong, Gregory P. Nordin, Adam T. WoolleyAbstract:This work demonstrates for the first time the creation of Microchip Electrophoresis devices with ∼50 μm cross-sectional dimensions by stereolithographic 3D printing and their application in the analysis of medically significant biomarkers related to risk for preterm birth (PTB). We determined that device current was linear with applied potential up to 800 V (620 V/cm). We optimized device and separation conditions using fluorescently labeled amino acids as a model system and compared the performance in our 3D printed microfluidic devices to that in other device materials commonly used for Microchip Electrophoresis analysis. We demonstrated for the first time Microchip Electrophoresis in a 3D printed device of three PTB biomarkers, including peptides and a protein, with suitable separation characteristics. Limits of detection for Microchip Electrophoresis in 3D printed microfluidic devices were also determined for PTB biomarkers to be in the high picomolar to low nanomolar range.
-
3D Printed Microfluidic Devices for Microchip Electrophoresis of Preterm Birth Biomarkers
2019Co-Authors: Michael J. Beauchamp, Anna V. Nielsen, Hua Gong, Gregory P. Nordin, Adam T. WoolleyAbstract:This work demonstrates for the first time the creation of Microchip Electrophoresis devices with ∼50 μm cross-sectional dimensions by stereolithographic 3D printing and their application in the analysis of medically significant biomarkers related to risk for preterm birth (PTB). We determined that device current was linear with applied potential up to 800 V (620 V/cm). We optimized device and separation conditions using fluorescently labeled amino acids as a model system and compared the performance in our 3D printed microfluidic devices to that in other device materials commonly used for Microchip Electrophoresis analysis. We demonstrated for the first time Microchip Electrophoresis in a 3D printed device of three PTB biomarkers, including peptides and a protein, with suitable separation characteristics. Limits of detection for Microchip Electrophoresis in 3D printed microfluidic devices were also determined for PTB biomarkers to be in the high picomolar to low nanomolar range
Detlev Belder - One of the best experts on this subject based on the ideXlab platform.
-
label free fluorescence detection in capillary and Microchip Electrophoresis
Analytical and Bioanalytical Chemistry, 2009Co-Authors: Detlev Belder, Philipp SchulzeAbstract:Herein, we summarize the current status of native fluorescence detection in microchannel Electrophoresis, with a strong focus on chip-based systems. Fluorescence detection is a powerful technique with unsurpassed sensitivity down to the single-molecule level. Accordingly fluorescence detection is attractive in combination with miniaturised separation techniques. A drawback is, however, the need to derivatize most analytes prior to analysis. This can often be circumvented by utilising excitation light in the UV spectral range in order to excite intrinsic fluorescence. As sensitive absorbance detection is challenging in chip-based systems, deep-UV fluorescence detection is currently one of the most general optical detection techniques in Microchip Electrophoresis, which is especially attractive for the detection of unlabelled proteins. This review gives an overview of research on native fluorescence detection in capillary (CE) and Microchip Electrophoresis (MCE) between 1998 and 2008. It discusses material aspects of native fluorescence detection and the instrumentation used, with particular focus on the detector design. Newer developments, featured techniques, and their prospects in the future are also included. In the last section, applications in bioanalysis, drug determination, and environmental analysis are reviewed with regard to limits of detection.
-
deep uv laser induced fluorescence detection of unlabeled drugs and proteins in Microchip Electrophoresis
Analytical Chemistry, 2005Co-Authors: Philipp Schulze, Martin Ludwig, Frank Kohler, Detlev BelderAbstract:Deep UV fluorescence detection at 266-nm excitation wavelength has been realized for sensitive detection in Microchip Electrophoresis. For this purpose, an epifluorescence setup was developed enabling the coupling of a deep UV laser into a commercial fluorescence microscope. Deep UV laser excitation utilizing a frequency quadrupled pulsed laser operating at 266 nm shows an impressive performance for native fluorescence detection of various compounds in fused-silica microfluidic devices. Aromatic low molecular weight compounds such as serotonin, propranolol, a diol, and tryptophan could be detected at low-micromolar concentrations. Deep UV fluorescence detection was also successfully employed for the detection of unlabeled basic proteins. For this purpose, fused-silica chips dynamically coated with hydroxypropylmethyl cellulose were employed to suppress analyte adsorption. Utilizing fused-silica chips permanently coated with poly(vinyl alcohol), it was also possible to separate and detect egg white chicken proteins. These data show that deep UV fluorescence detection significantly widens the application range of fluorescence detection in chip-based analysis techniques.
-
Surface modification in Microchip Electrophoresis
Electrophoresis, 2003Co-Authors: Detlev Belder, M LudwigAbstract:Different approaches and techniques for surface modification of microfluidic devices applied for Microchip Electrophoresis are reviewed. The main focus is on the improved electrophoretic separation by reducing analyte-wall interactions and manipulation of electroosmosis. Approaches and methods for permanent and dynamic surface modification of microfluidic devices, manufactured from glass, quartz and also different polymeric substrates, are described.
Dulan B Gunasekara - One of the best experts on this subject based on the ideXlab platform.
-
evaluation of in channel amperometric detection using a dual channel Microchip Electrophoresis device and a two electrode potentiostat for reverse polarity separations
Electrophoresis, 2015Co-Authors: Diogenes Meneses, Dulan B Gunasekara, Pann Pichetsurnthorn, Jose Alberto Fracassi Da Silva, Fabiane Caxico De Abreu, Susan M. LunteAbstract:In-channel amperometric detection combined with dual-channel Microchip Electrophoresis is evaluated using a two-electrode isolated potentiostat for reverse polarity separations. The device consists of two separate channels with the working and reference electrodes placed at identical positions relative to the end of the channel, enabling noise subtraction. In previous reports of this configuration, normal polarity and a three-electrode detection system were used. In the two-electrode detection system described here, the electrode in the reference channel acts as both the counter and reference. The effect of electrode placement in the channels on noise and detector response was investigated using nitrite, tyrosine, and hydrogen peroxide as model compounds. The effects of electrode material and size and type of reference electrode on noise and the potential shift of hydrodynamic voltammograms for the model compounds were determined. In addition, the performance of two- and three-electrode configurations using Pt and Ag/AgCl reference electrodes was compared. Although the signal was attenuated with the Pt reference, the noise was also significantly reduced. It was found that lower LOD were obtained for all three compounds with the dual-channel configuration compared to single-channel, in-channel detection. The dual-channel method was then used for the detection of nitrite in a dermal microdialysis sample obtained from a sheep following nitroglycerin administration.
-
in channel amperometric detection for Microchip Electrophoresis using a wireless isolated potentiostat
Electrophoresis, 2011Co-Authors: Dulan B Gunasekara, Matthew K Hulvey, Susan M. LunteAbstract:The combination of Microchip Electrophoresis (ME) with amperometric detection leads to a number of analytical challenges that are associated with isolating the detector from the high voltages used for the separation. While methods such as end-channel alignment and the use of decouplers have been employed, they have limitations. A less common method has been to utilize an electrically isolated potentiostat. This approach allows placement of the working electrode directly in the separation channel without using a decoupler. This paper explores the use of Microchip Electrophoresis and electrochemical detection (ME-EC) with an electrically isolated potentiostat for the separation and in-channel detection of several biologically important anions. The separation employed negative polarity voltages and tetradecyltrimethylammonium bromide (TTAB, as a buffer modifier) for the separation of nitrite (NO2-), glutathione (GSH), ascorbic acid (AA), and tyrosine (Tyr). A half-wave potential (E½) shift of approximately negative 500 mV was observed for NO2- and H2O2 standards in the in-channel configuration compared to end channel. Higher separation efficiencies were observed for both NO2- and H2O2 with the in-channel detection configuration. The limits of detection were approximately two-fold lower and the sensitivity was approximately two-fold higher for in-channel detection of nitrite when compared to end-channel. The application of this microfluidic device for the separation and detection of biomarkers related to oxidative stress is described.