54 research outputs found
Continuous monitoring of adenosine and its metabolites using microdialysis coupled to microchip electrophoresis with amperometric detection
Rapid monitoring of concentration changes of neurotransmitters and energy metabolites is important for understanding the biochemistry of neurological disease as well as for developing therapeutic options. This paper describes the development of a separation-based sensor using microchip electrophoresis (ME) with electrochemical (EC) detection coupled to microdialysis (MD) sampling for continuous on-line monitoring of adenosine and its downstream metabolites. The device was fabricated completely in PDMS. End-channel electrochemical detection was accomplished using a carbon fiber working electrode embedded in the PDMS. The separation conditions for adenosine, inosine, hypoxanthine, and guanosine were investigated using a ME-EC chip with a 5 cm long separation channel. The best resolution was achieved using a background electrolyte consisting of 35 mM sodium borate at pH 10, 15% dimethyl sulfoxide (DMSO), and 2 mM sodium dodecyl sulphate (SDS), and a field strength of 222 V cm−1. Under these conditions, all four purines were separated in less than 85 s. Using a working electrode detection potential of 1.4 V vs. Ag/AgCl, the limits of detection were 25, 33, 10, and 25 μM for adenosine, inosine, hypoxanthine, and guanosine, respectively. The ME-EC chip was then coupled to microdialysis sampling using a novel all-PDMS microdialysis–microchip interface that was reversibly sealed. This made alignment of the working electrode with the end of the separation channel much easier and more reproducible than could be obtained with previous MD-ME-EC systems. The integrated device was then used to monitor the enzymatic conversion of adenosine to inosine in vitro
Recent trends in microdialysis sampling integrated with conventional and microanalytical systems for monitoring biological events: A review
Microdialysis (MD) is a sampling technique that can be employed to monitor biological events both in vivo and in vitro. When it is coupled to an analytical system, microdialysis can provide near realtime information on the time-dependent concentration changes of analytes in the extracellular space or other aqueous environments. Online systems for the analysis of microdialysis samples enable fast, selective and sensitive analysis while preserving the temporal information. Analytical methods employed for online analysis include liquid chromatography (LC), capillary (CE) and microchip electrophoresis and flow-through biosensor devices. This review article provides an overview of microdialysis sampling and online analysis systems with emphasis on in vivo analysis. Factors that affect the frequency of analysis and, hence, the temporal resolution of these systems are also discussed
Separation and Detection of Tyrosine and Phenylalanine-derived Oxidative Stress Biomarkers Using Microchip Electrophoresis with Electrochemical Detection
This is the peer reviewed version of the following article: D. B. Weerasekara, S. M. Lunte, Electroanalysis 2022, 34, 1913, which has been published in final form at https://doi.org/10.1002/elan.202100580. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited.A method for the determination of selected aromatic amino acid biomarkers of oxidative stress using microchip electrophoresis with electrochemical detection is described. The separation of the major reaction products of phenylalanine and tyrosine with reactive nitrogen and oxygen species was accomplished using ligand exchange micellar electrokinetic chromatography with a PDMS/glass hybrid chip. Electrochemical detection was achieved using a pyrolyzed photoresist film working electrode. The system was evaluated for the analysis of the products of the Fenton reaction with tyrosine and phenylalanine, and the reaction of peroxynitrite with tyrosine
Determination of Methylarginines in Infant Plasma by CE-LIF
Methylarginines (MAs) are a class of nitric oxide synthase inhibitors that have been implicated in respiratory complications of critically ill infants. This paper describes the development of an analytical method to measure these compounds in the plasma of newborns using capillary electrophoresis (CE). The CE separation method was optimized to enable complete baseline resolution of the four MA analogues of interest. Sample preparation concerns for infant-derived samples were addressed by validating a heat-assisted extraction method for the analysis of low volume (≤100 µL) samples from a plasma matrix. It was determined that the sample matrix (plasma versus serum) did not affect the measured MA concentrations, while extracting smaller volumes of plasma that underwent heat-induced gelation afforded higher MA recoveries than larger volume samples. These methods were then applied to blood samples collected from infants housed in the neonatal intensive care unit. It was discovered that these newborns had significantly elevated concentrations of MAs at younger ages (< 6 months) while amounts were similar between infants 6 months old and adults. The data are preliminary, but demonstrate an interesting age dependence on the concentrations of these nitric oxide inhibitors, which has not been previously reported
A review of microdialysis coupled to microchip electrophoresis for monitoring biological events
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
Optimization of the Separation of NDA-Derivatized Methylarginines by Capillary and Microchip Electrophoresis
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
Separation and Detection of Peroxynitrite Using Microchip Electrophoresis with Amperometric Detection
Peroxynitrite (ONOO-) is a highly reactive species implicated in the pathology of several cardiovascular and neurodegenerative diseases. It is generated in vivo by the diffusion-limited reaction of nitric oxide (NO•) and superoxide anion (•O2-) and is known to be produced during periods of inflammation. Detection of ONOO- is made difficult by its short half-life under physiological conditions (∼1 s). Here we report a method for the separation and detection of ONOOfrom other electroactive species utilizing a microchip electrophoresis device incorporating an amperometric detection scheme. Microchip electrophoresis permits shorter separation times (∼25 s for ONOO-) and higher temporal resolution than conventional capillary electrophoresis (several minutes). This faster analysis allows ONOO- to be detected before substantial degradation occurs, and the increased temporal resolution
permits more accurate tracking of dynamic changes in chemical systems
Microdialysis Sampling Coupled to Microchip Electrophoresis with Integrated Amperometric Detection on an All Glass Substrate
This is the peer reviewed version of the following article: Scott, D. E., Grigsby, R., & Lunte, S. M. (2013). Microdialysis Sampling Coupled to Microchip Electrophoresis with Integrated Amperometric Detection on an All Glass Substrate. Chemphyschem : A European Journal of Chemical Physics and Physical Chemistry, 14(10), 2288–2294. http://doi.org/10.1002/cphc.201300449, which has been published in final form at doi.org/10.1002/cphc.201300449. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.The development of an all-glass separation-based sensor using microdialysis coupled to microchip electrophoresis with amperometric detection is described. The system includes a flow-gated interface to inject discrete sample plugs from the microdialysis perfusate into the microchip electrophoresis system. Electrochemical detection was accomplished with a platinum electrode in an in-channel configuration using a wireless electrically isolated potentiostat. To facilitate bonding around the in-channel electrode, a fabrication process was employed that produced a working and a reference electrode flush with the glass surface. Both normal and reversed polarity separations were performed with this sensor. The system was evaluated in vitro for the continuous monitoring of the production of hydrogen peroxide from the reaction of glucose oxidase with glucose. Microdialysis experiments were performed using a BASi loop probe with an overall lag time of approximately five minutes and a rise time of less than 60 seconds
Recent advances in the analysis of therapeutic proteins by capillary and microchip electrophoresis
The development of therapeutic proteins and peptides is an expensive and time-intensive process. Biologics, which have become a multi-billion dollar industry, are chemically complex products that require constant observation during each stage of development and production. Post-translational modifications along with chemical and physical degradation from oxidation, deamidation, and aggregation, lead to high levels of heterogeneity that affect drug quality and efficacy. The various separation modes of capillary electrophoresis (CE) are commonly utilized to perform quality control and assess protein heterogeneity. This review attempts to highlight the most recent developments and applications of CE separation techniques for the characterization of protein and peptide therapeutics by focusing on papers accepted for publication in the in the two-year period between January 2012 and December 2013. The separation principles and technological advances of CE, capillary gel electrophoresis, capillary isoelectric focusing, capillary electrochromatography and CE-mass spectrometry are discussed, along with exciting new applications of these techniques to relevant pharmaceutical issues. Also included is a small selection of papers on microchip electrophoresis to show the direction this field is moving with regards to the development of inexpensive and portable analysis systems for on-site, high-throughput analysis
Investigation of the metabolism of Substance-P at the blood-brain barrier using LC-MS/MS
Please note that this is an author-produced PDF of an article accepted for publication following peer review. The publisher version is available on its site.Substance P (SP) has been associated with pain, depression as well as neurodegenerative diseases. Many of these diverse actions of SP can potentially be attributed to SP metabolites generated at the blood-brain barrier (BBB). In these studies, the metabolism of SP was investigated using an in vitro model of the BBB and LC-MS/MS. Substance P metabolism was found to be non-saturable in the concentration range of 100 nM to 10 μM, with approximately 70% of the peptide remaining intact after 5 hrs. The major metabolites of SP were identified by MS to be 3-11 and 5-11. Two previously unreported metabolites, 5-11 and 6-11 were also found in our studies. Several additional minor SP metabolites including 1-9 and 2-11 were also identified. A profile of the SP metabolites generated by the BBB overtime was obtained. The results from the present study provide us a better understanding of the role of blood-brain barrier in the pharmacology of SP
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