Biomedical subjects

Alexander Muck

Publications and source records attributed to Alexander Muck.

14 recordsLinked to original sources

PubMed2026

Revealing Hidden Variables in DESI-Based Spatial Metabolomics: Solvent Composition and Tissue Type as Critical Drivers.

In the development of a desorption electrospray ionization (DESI) workflow for spatial metabolomics, we investigated the impact of two commonly used solvent systems, 90% acetonitrile (ACN) and 90% methanol (MeOH), on the spatial metabolomic profiling of various murine tissues. The performance of both solvents was evaluated across several metabolite classes (central carbon metabolites, amino acids, and fatty acids). Although the ACN-based solvent system led to higher signal intensities for small polar metabolites involved in glycolysis, the tricarboxylic acid (TCA) cycle, and amino acid metabolism, the MeOH-based solvent system provided superior signal intensities for fatty acids. These findings demonstrate that the solvent composition differentially influences metabolite extraction and ionization processes in DESI and should be carefully matched to the biological questions and metabolite classes of interest. As a proof-of-principle, the ACN solvent system was applied to a pilot study based on a rat model of renal ischemic injury, revealing region-specific metabolic changes between normoxic and ischemic conditions. Together, these results demonstrate the importance of solvent selection in DESI-based spatial metabolomics and showcase the ability of this approach to uncover spatially resolved metabolic adaptations associated with tissue injury.

Animals↗

PubMed2006

Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. VII. Changes in the plant's proteome.

When Manduca sexta attacks Nicotiana attenuata, fatty acid-amino acid conjugates (FACs) in the larvae's oral secretions (OS) are introduced into feeding wounds. These FACs trigger a transcriptional response that is similar to the response induced by insect damage. Using two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization-time of flight, and liquid chromatography-tandem mass spectrometry, we characterized the proteins in phenolic extracts and in a nuclear fraction of leaves elicited by larval attack, and/or in leaves wounded and treated with OS, FAC-free OS, and synthetic FACs. Phenolic extracts yielded approximately 600 protein spots, many of which were altered by elicitation, whereas nuclear protein fractions yielded approximately 100 spots, most of which were unchanged by elicitation. Reproducible elicitor-induced changes in 90 spots were characterized. In general, proteins that increased were involved in primary metabolism, defense, and transcriptional and translational regulation; those that decreased were involved in photosynthesis. Like the transcriptional defense responses, proteomic changes were strongly elicited by the FACs in OS. A semiquantitative reverse transcription-PCR approach based on peptide sequences was used to compare transcript and protein accumulation patterns for 17 candidate proteins. In six cases the patterns of elicited transcript accumulation were consistent with those of elicited protein accumulation. Functional analysis of one of the identified proteins involved in photosynthesis, RuBPCase activase, was accomplished by virus-induced gene silencing. Plants with decreased levels of RuBPCase activase protein had reduced photosynthetic rates and RuBPCase activity, and less biomass, responses consistent with those of herbivore-attacked plants. We conclude that the response of the plant's proteome to herbivore elicitation is complex, and integrated transcriptome-proteome-metabolome analysis is required to fully understand this ubiquitous ecological interaction.

Animals↗

PubMed2006

Atmospheric molding of ionic copolymer MALDI-TOF/MS arrays: a new tool for protein identification/profiling.

An atmospheric molding protocol has been used to prepare an ionic methacrylate-based copolymer sample support chips for MALDI (pMALDI)-MS by targeting selected groups of various monomers copolymerized during molding, namely, carboxy, sulfo, dimethylalkyamino, and trimethylalkylammonium groups. The new disposable array chips provide analyte-oriented enhancement of protein adsorption to the modified substrates without requiring complicated surface coating or derivatization. The MALDI-MS performance of the new ionic copolymer chips was evaluated for lysozyme, beta-lactoglobulin A, trypsinogen and carbonic anhydrase I using washing with solutions prepared in pH or ionic strength steps. On cationic chips, the proteins are washed out at pH lower than their p/ values, and on anionic chips at pH higher than their p/ values. The ability of the microfabricated pMALDI chip set to selectively adsorb different proteins from real samples and to significantly increase their MS-signal was documented for the transmembrane photosystem I protein complex from the green alga Chlamydomonas reinhardtii. The proteins were almost exclusively adsorbed according to calculated pI values and grand average of hydropathy (GRAVY) indexes. The new disposable chips reduce manipulation times and increase measurement sensitivity for real-world proteomic samples. The simple atmospheric molding procedure enables additional proteomic operations to be incorporated on disposable MALDI-MS integrated platforms.

Animals↗

PubMed2006

Residues of diflubenzuron on horse chestnut (Aesculus hippocastanum) leaves and their efficacy against the horse chestnut leafminer, Cameraria ohridella.

Residues of the insect growth regulator diflubenzuron were quantified on horse chestnut (Aesculus hippocastanum L.) leaves treated with a diflubenzuron 480 g litre(-1) SC, Dimilin. To analyse the samples, an analytical procedure was developed involving a simple extraction step followed by high-performance liquid chromatography on an octadecyl-modified silica column with methanol + 0.01 M ammonium acetate mobile phase. The results showed diflubenzuron to be highly stable on horse chestnut leaves; more than 4 months (127 days) after application, 38% (on average) of the insecticide still remained on/in the leaves. The data confirmed biological observations showing diflubenzuron's long-term efficacy against the horse chestnut leafminer, Cameraria ohridella Deschka and Dimić, which is the most important pest of the horse chestnut in Europe. The hypothesis of possible penetration of diflubenzuron into the leaf mass is explored and discussed.

Aesculus↗

PubMed2005

Fast prototyping of hydrophobic disposable polymer support arrays for matrix-assisted laser desorption/ionization-time of flight-mass spectrometry of proteins by atmospheric molding.

A fast protocol for prototyping hydrophobic disposable poly(alkyl methacrylate-co-methyl methacrylate) copolymer sample support arrays for matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) of proteins by atmospheric molding is introduced. The sample support arrays were replicated by molding prepolymer alkyl methacrylate solutions into sandwich molds containing a micromachined silicon master, an aluminum spacer, and glass cover plates, followed by UV-initiated in situ polymerization under atmospheric pressure. The fabrication procedure enables a simultaneous fabrication/modification of single-use polymer arrays by a targeted selection of functional groups of the copolymerized monomers during molding. The one-step modification during the fabrication is demonstrated for enhanced protein adsorption to the modified materials by introduction of hydrophobic butyl-, dodecyl-, and octadecyl groups to the polymer backbone without a need for additional surface coating or derivatization. The MALDI-MS performance of the new polymer chips was tested for spectral measurements of bovine pancreas insulin, horse heart myoglobin, and bovine serum albumin. The protein adsorption to the new hydrophobic copolymer chips was studied for bovine pancreas trypsinogen; the sample desalting parameters, such as time and volume, were optimized for myoglobin as model proteins. A significant signal increase was achieved after efficient desalting of an insect Delta11-desaturase membrane protein fragment from a complex elution buffer (100 mM phosphate, 10 mM tris(hydroxyethyl)aminomethane, 0.5 M NaCl, and 10 mM ethylenediamine tetraacetic acid) on the poly(butyl methacrylate-co-methyl methacrylate) copolymer chip (monomer ratio 8:2 v/v) by simply washing the target zones. The new chips offer reduced sample manipulation and device fabrication times as well as simple operation.

Atmosphere↗

PubMed2004

Bulk modification of polymeric microfluidic devices.

The surface properties of microfluidic devices play an important role in their flow behavior. We report here on an effective control of the surface chemistry and performance of polymeric microchips through a bulk modification route during the fabrication process. The new protocol is based on modification of the bulk microchip material by tailored copolymerization of monomers during atmospheric-pressure molding. A judicious addition of a modifier to the primary monomer solution thus imparts attractive properties to the plastic microchip substrate, including significant enhancement and/or modulation of the EOF (with flow velocities comparable to those of glass), a strong pH sensitivity and high stability. Carboxy, sulfo, and amino moieties have thus been introduced (through the incorporation of methylacrylic acid, 2-sulfoethyl-methacrylate and 2-aminoethyl-methacrylate monomers, respectively). A strong increase in the electroosmotic pumping compared to the native poly(methylmethacrylate)(PMMA) microchip (ca. electroosmotic mobility increases from 2.12 to 4.30 x 10(-4) cm(2) V(-1) s(-1)) is observed using a 6% methylacrylate (MAA) modified PMMA microchip. A 3% aminoethyl modified PMMA microchip exhibits a reversal of the electroosmotic mobility (for example, -5.6 x 10(-4) cm(2) V(-1) s(-1) at pH 3.0). The effects of the modifier loading and the pH on the EOF have been investigated for the MAA-modified PMMA chips. The bulk-modified devices exhibit reproducible and stable EOF behavior. The one step fabrication/modification protocol should further facilitate the widespread production of high-performance plastic microchip devices.

Journal Article↗

PubMed2004

Fabrication of poly(methyl methacrylate) microfluidic chips by atmospheric molding.

A greatly simplified method for fabricating poly(methyl methacrylate) (PMMA) separation microchips is introduced. The new protocol relies on UV-initiated polymerization of the monomer solution in an open mold under ambient pressure. Silicon microstructures are transferred to the polymer substrate by molding a methyl methacrylate solution in a sandwich (silicon master/Teflon spacer/glass plate) mold. The chips are subsequently assembled by thermal sealing of the channel and cover plates. The new fabrication method obviates the need for specialized replication equipment and reduces the complexity of prototyping and manufacturing. Variables of the fabrication process were assessed and optimized. The new method compares favorably with common fabrication techniques, yielding high-quality devices with well-defined channel and injection-cross structures, and highly smoothed surfaces. Nearly 100 PMMA chips were replicated using a single silicon master, with high chip-to-chip reproducibility (relative standard deviations of 1.5 and 4.7% for the widths and depths of the replicated channels, respectively). The relatively high EOF value of the new chips (2.12 x 10(-4) cm(2) x V(-1) x s(-1)) indicates that the UV polymerization process increases the surface charge and hence enhances the fluidic transport. The attractive performance of the new CE microchips has been demonstrated in connection with end-column amperometric and contactless-conductivity detection schemes. While the new approach is demonstrated in connection with PMMA microchips, it could be applied to other materials that undergo light-initiated polymerization. The new approach brings significant simplification of the process of fabricating PMMA devices and should lead to a widespread low-cost production of high-quality separation microchips.

Journal Article↗

PubMed2004

Microchip capillary electrophoresis with a boron-doped diamond electrode for rapid separation and detection of purines.

Microchip capillary electrophoresis (CE) coupled with a boron-doped diamond (BDD) electrode has been employed for the separation and detection of several purines and purine-containing compounds. The BDD end-channel amperometric detector offers favorable signal-to-noise (S/N) characteristics at the high detection potential (+ 1.3 V) essential for detecting purine-related compounds. Factors influencing the separation and detection processes were examined and optimized. Five purines (guanine, hypoxanthine, guanosine, xanthine, and uric acid) have been separated within 6 min at a separation voltage of 1000V using a borate/phosphate run buffer (pH 8.2). Linear calibration plots are observed for micromolar concentrations of the purine compounds. Good stability and reproducibility (R.S.D. < 5%) are obtained reflecting the minimal adsorption of purines at the BDD surface. Applicability for the detection of nucleosides, nucleotides, and oligonucleotides is illustrated. The new microchip protocol offers great promise for a wide range of bioanalytical applications involving assays of purines and purine-containing compounds.

Diamond↗

PubMed2004

Microchip capillary electrophoresis with a boron-doped diamond electrochemical detector for analysis of aromatic amines.

The attractive features of a boron-doped diamond (BDD) thin-film detector for microchip capillary electrophoretic (CE) separations of dye-related amino-substituted aromatic compounds are described. The diamond electrode was employed in the end-column amperometric detection of 4-aminophenol (4-AP), 1,2-phenylenediamine (1,2-PDA), 2-aminonaphthalene (2-AN), 2-chloroaniline (2-CA), and o-aminobenzoic acid (o-ABA), and its attractive behavior was compared to commonly used screen-printed carbon and glassy-carbon electrodes. These conventional electrode materials exhibit a significant degree of passivation and low sensitivity to the above-mentioned environmental pollutants. The diamond-based electrochemical detection system displayed a favorable analytical performance, including lower noise levels, higher peak resolution with enhanced sensitivity, and improved resistance against electrode passivation. Factors influencing the on-chip analysis were assessed and optimized. The diamond detector displayed detection limits of 2.0 and 1.3 microM for 4-AP and 2-AN, respectively, and a wide linear response for these compounds over the 2-50 microM range. The enhanced stability was demonstrated by relative standard deviation (RSD) values of 1.4% and 4.7% for 100 microM 1,2-PDA and 200 microM 2-CA, respectively, for repetitive detections (n = 7). Besides, the simultaneously observed current decrease was 2.4 and 9.1% for 1,2-PDA and 2-CA, respectively (compared to 21.8 and 41.0% at the screen-printed carbon electrode and 28.3 and 34.1% at the glassy carbon electrode, respectively). The favorable properties of the diamond electrode indicate great promise for environmental applications in CE and other microchip devices.

Amines↗

PubMed2004

Atmospheric molded poly(methylmethacrylate) microchip emitters for sheathless electrospray.

Disposable poly(methylmethacrylate) (PMMA) sheathless electrospray microchip emitters were prepared for the first time using the atmospheric molding fabrication protocol. A sheathless electrospray from uncoated channel outlets, machined to cone-shaped three-dimensional tips, is demonstrated utilizing a simple cross design with an on-chip liquid junction to obviate the need for external unions to voltage electrodes, thus reducing the dead volume effects as well as the complexity of fabrication. The fast replication of microchip emitters was performed by molding prepolymeric methylmethacrylate solutions into silicon-master/aluminum-spacer/glass-plate molds followed by UV-initiated free radical polymerization. The performance of the new microchip emitters was demonstrated for mass spectral measurements of methionine enkephalin, adrenocorticotropic hormone and insulin peptide/protein mixtures. The samples were infused through capillary connections using hydrodynamic pumping. The polymeric emitters prepared by this flexible fabrication route offer an easy way of operation and high stability, without a need for attachment of external voltage unions or metallizing the emitter tips. The new approach should provide a useful low-cost tool for widespread coupling of mass spectrometry to chip systems.

Adrenocorticotropic Hormone↗

PubMed2003

Movable contactless-conductivity detector for microchip capillary electrophoresis.

A new movable contactless-conductivity detection system for microchip capillary electrophoresis is introduced. Such a versatile system relies on positioning the detector at different points along the separation channel via "sliding" the electrode holder. The new movable microchip detection system offers distinct improvements compared to common fixed-location conductivity detectors. For example, placing the detector at different locations along the microchannel offers useful insights into the separation process. Three-dimensional plots of resolution/channel length/separation voltage can be used for optimizing the separation process and selecting the analysis time. The system enables rapid switching between "total" (unresolved) and "individual" (resolved/fingerprint) signals on the basis of placing the detector at the beginning and end of the separation channel, respectively. By moving the detector to a shorter effective separation length, after eluting fast-migrating ions, shorter analysis times can be achieved (through faster detection of late-eluting analytes). These and other improvements in the analytical performance and insights into the separation process are illustrated in connection with the detection of low-energy ionic explosives and nerve agent degradation products.

Journal Article↗

PubMed2003

Boron-doped diamond microelectrodes for use in capillary electrophoresis with electrochemical detection.

The fabrication and characterization of boron-doped diamond microelectrodes for use in electrochemical detection coupled with capillary electrophoresis (CE-EC) is discussed. The microelectrodes were prepared by coating thin films of polycrystalline diamond on electrochemically sharpened platinum wires (76-, 25-, and 10-microm diameter), using microwave-assisted chemical vapor deposition (CVD). The diamond-coated wires were attached to copper wires (current collectors), and several methods were explored to insulate the cylindrical portion of the electrode: nail polish, epoxy, polyimide, and polypropylene coatings. The microelectrodes were characterized by scanning electron microscopy, Raman spectroscopy, and cyclic voltammetry. They exhibited low and stable background currents and sigmoidally shaped voltammetric curves for Ru(NH3)6(3+/2+) and Fe(CN)6(3-/4-) at low scan rates. The microelectrodes formed with the large diameter Pt and sealed in polypropylene pipet tips were employed for end-column detection in CE. Evaluation of the CE-EC system and the electrode performance were accomplished using a 10 mM phosphate buffer, pH 6.0, run buffer, and a 30-cm-long fused-silica capillary (75-microm i.d.) with dopamine, catechol, and ascorbic acid serving as test analytes. The background current (approximately 100 pA) and noise (approximately 3 pA) were measured at different detection potentials and found to be very stable with time. Reproducible separation (elution time) and detection (peak current or area) of dopamine, catechol, and ascorbic acid were observed with response precisions of 4.1% or less. Calibration curves constructed from the peak area were linear over 4 orders of magnitude, up to a concentration between 0.1 and 1 mM. Mass limits of detection for dopamine and catechol were 1.7 and 2.6 fmol, respectively (S/N = 3). The separation efficiency was approximately 33,000, 56,000, and 98,000 plates/m for dopamine, catechol, and ascorbic acid, respectively. In addition, the separation and detection of 1- and 2-naphthol in 160 mM borate buffer, pH 9.2, was investigated. Separation of these two analytes was achieved with efficiencies of 118,000 and 126,000 plates/m, respectively.

Ascorbic Acid↗

PubMed2003

Electrophoretic microchip with dual-opposite injection for simultaneous measurements of anions and cations.

A novel dual-injection poly(methylmethacrylate) (PMMA) microchip electrophoretic system has been designed and fabricated for simultaneous measurements of anions and cations using a single channel and detection device. It consists of two sample reservoirs, on both sides of a common separation channel. Anions and cations can be simultaneously electrokinetically injected into both ends of the separation channel. Due to lower electroosmotic flow in polymer channels compared to glass ones, the cations and anions migrate in opposite directions and can be separated from each other and detected using a movable contactless conductivity detector (MCCD) positioned around the center of the separation channel. The effects of the detector position and of the separation voltage on the response and resolution have been studied and optimized for simultaneous determination of six low-energy explosive-related ions, including ammonium, methyl ammonium, sodium, chloride, nitrate, and perchlorate in a single analytical run (of ca. 3 min). Simultaneous detection of nerve-agent degradation products along with explosive-related anions and cations is also demonstrated. The versatile system can also be used for separately measuring anions or cations. The attractive behavior of the dual-opposite injection microchip offers great promise for a wide range of applications, including "total ion analysis" of various samples.

Anions↗

PubMed2002

Photodegradation of 1-nitropyrene in solution and in the adsorbed state.

The photodegradation of the 1-nitropyrene (NPy) has been studied using conventional (Xe and medium pressure Hg lamps) and laser sources (XeCl excimer and Nd-YAG UV). Low energy monochromatic light sources were used to study the early stages of degradation (up to 50% conversion). The medium pressure Hg lamp was used for longer periods of irradiation (up to 6h) and for greater degradation of NPy. The results of our work confirm that degradation occurs by radical mechanism. Aromatic hydroxymethyl, methoxy, hydroxy and nitroso derivatives of pyrene (Py) are created by low energy UV irradiation. After a massive UV irradiation, the Py aromatic system is destroyed and more polar low-molecular compounds are generated. The photochemical method described in this paper, based on irradiation by UV lamp, is therefore, suitable for degradation of fused benzene ring(s) and thus should also be effective for degradation of other nitrated polycyclic aromatic compounds.

Adsorption↗