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PubMed2002

Chemical approaches to the investigation of cellular systems.

Biochemistry in the context of a living cell or organism is complicated by many variables such as supramolecular organization, cytoplasmic viscosity, and substrate heterogeneity. While these variables are easily excluded or avoided in reconstituted systems, they must be dealt with in cellular environments. New developments have allowed researchers to begin probing the inner workings of the cell to gain new insight into cell function and metabolism. Advances in cellular imaging and in small molecule-controlled gene expression, signal transduction and cell surface modification are discussed in this review. These techniques have permitted the study of molecular components within the context of living cells.

Animals↗

PubMed2002

Neurochemistry and electroanalytical probes.

Electroanalytical techniques have been applied to monitoring chemical events including neurotransmitter release during rodent behaviour and the release of zeptomoles of molecules from single cells. Transgenic mice models have been developed and studied to identify specific cell types in vitro. Characterization and surface modification of electroanalytical probes has enhanced the selectivity and sensitivity of measurements.

Animals↗

PubMed2002

Protein adsorption onto auto-assembled polyelectrolyte films.

Surface modification by deposition of ordered protein systems constitutes one of the major objectives of bio-related chemistry and biotechnology. In this respect a concept has recently been reported aimed at fabricating multilayers by the consecutive adsorption of positively and negatively charged polyelectrolytes. We investigate the adsorption processes between polyelectrolyte multilayers and a series of positively and negatively charged proteins. The film buildup and adsorption experiments were followed by Scanning Angle Reflectometry (SAR). We find that proteins strongly interact with the polyelectrolyte film whatever the sign of the charge of both the multilayer and the protein. When charges of the multilayer and the protein are similar, one usually observes the formation of protein monolayers, which can become dense. We also show that when the protein and the multilayer become oppositely charged, the adsorbed amounts are usually larger and the formation of thick protein layers extending up to several times the largest dimension of the protein can be observed. Our results confirm that electrostatic interactions dominate protein/polyelectrolyte multilayer interactions.

Adsorption↗

PubMed2003

Is reduced systemic heparinization justified with heparin-bonded bypass circuits in cardiac surgery?--Experience with and without aprotinin.

The effects of aprotinin combined with heparin-bonded bypass circuits and reduced systemic heparinization on haemostasis and inflammatory reactions were measured in patients with elective CABG operation. Patients were randomized to be operated on either without aprotinin (NOAPRO, n=15) or with aprotinin (APRO, n=15) at a low dose of 2 Mio KIU in the priming volume. Activated clotting time was adjusted to 400 +/- 50 s during cardiopulmonary bypass (CPB). Haemostasis (fibrinopeptide A (FPA), thrombin-antithrombin complex (TAT), D-Dimer, plasmin-antiplasmin (PAP), plasminogen-activator inhibitor (PAI)), inflammatory reaction (lactoferrin, IL-6, sTNF-IIR, SC5b-9) and clinical data were evaluated perioperatively. Perioperative clinical and laboratory data including mediastinal drainage volume, postoperative morbidity and mortality were comparable for patients in both groups. FPA was elevated in the APRO group during CPB (P=0.001), D-Dimer in the NOAPRO group after CPB (P=0.002). No differences were seen for TAT, PAP or PAI between the groups. Lactoferrin was elevated in NOAPRO at the end of CPB (P=0.01) and after heparin reversal with protamine sulphate (P=0.02). No intergroup differences were seen for IL-6, sTNF-IIR or SC5b-9 between the groups. In association with reduced heparinization, pump prime aprotinin retains its antifibrinolytic effect in modified bypass equipment with a heparin surface besides an anti-inflammatory effect in terms of inhibition of leukocyte activation. However, thrombin activation may be increased with aprotinin. We therefore recommend sufficient systemic heparinization despite heparin surface modification of bypass equipment.

Aged↗

PubMed2002

Surface modified microelectrodes for selective electroanalysis of metal ions in environmental components.

The surface modification of electrodes was achieved by the Langmuir-Blodgett technique. The monolayers of laponite clay and polythiophene were formed at the air-water interface and these films were then transferred onto carbon microelectrodes. The behaviour of both untreated and coated electrodes was tested by originally developed double-step voltcoulometry (DSVCM). The dependence of charge response on the concentration of Cu(2+) species was investigated. Straight calibration curves were obtained and enhanced sensitivity of coated electrodes was documented. It is shown that the accumulation of Cu ions into laponite clay was maintained even after transferring the electrode into a pure water. The characteristic features of the "memory effect" are discussed.

Aluminum Silicates↗

PubMed2005

Pedobarometric evaluation of equinus foot disorder after injection of botulinum toxin A in children with cerebral palsy: a pilot study.

The purpose of this pilot study was to evaluate the effect of treatment with botulinum toxin A (BTX-A) injections on function in children with cerebral palsy (CP) using pedobarometric data collected in a standing position. Ten children with CP (seven males, three females) participated, five with diplegia and five with hemiplegia. All children exhibited signs of unilateral dynamic equinus foot disorder and were community ambulators. Age range at the time of treatment was 6 to 13 years (mean age 9 y, SD 2.5). A comprehensive clinical examination and a pedobarometric evaluation were performed before and after BTX-A injection. All children received at least one injection in each spastic muscle. BTX-A was given to eight children unilaterally and to two children bilaterally. A maximum dose of 4 to 8 units of BTX-A per kilogram body weight was administered. In comparison with pretreatment values, the pedobarometric evaluation revealed a significant increase in the whole plantar surface area on the affected side (p < 0.05), an increase in the peak pressure value at the hindfoot on the affected side (p < 0.05), and a significant shift in the body centre of mass to the affected limb (p < 0.05) 1 month after treatment with BTX-A. A significant decrease in the peak pressure value at the hindfoot on the unaffected side was also observed. Similarly, a decrease in the modified Ashworth scale and an increase in the range of motion were observed. Thus, BTX-A produces a significant plantar surface modification and a significant shift of the body centre of mass which represents an indirect measure of postural attitude. Pedobarometric evaluation can also be useful in quantifying clinical changes after treatment with BTX-A.

Adolescent↗

PubMed2000

Uniform-sized molecularly imprinted polymers for 2-arylpropionic acid derivatives selectively modified with hydrophilic external layer and their applications to direct serum injection analysis.

Uniform-sized molecularly imprinted polymers (MIPs) for (S)-naproxen and -ibuprofen selectively modified with hydrophilic external layer, restricted access media (RAM)-MIPs, have been prepared. First, the MIP for (S)-naproxen or -ibuprofen was prepared using 4-vinylpyridine and ethylene glycol dimethacrylate as a functional monomer and cross-linker, respectively, by a multistep swelling and thermal polymerization method. Next, a 1:1 mixture of glycerol monomethacrylate and glycerol dimethacrylate was used for hydrophilic surface modification, and it was added directly to the MIP for (S)-naproxen or -ibuprofen 4 h after the start of molecular imprinting. The obtained RAM-MIP material for (S)-naproxen or -ibuprofen was applied for direct serum injection assays of the drug by a column-switching system, consisting of a RAM-MIP material and conventional C18-silica column. However, leakage of the imprint molecule prevented accurate and precise assays of the drug. This problem has been overcome by using the RAM-MIP for (S)-naproxen for the assays of ibuprofen in rat plasma. The optimized column-switching system was applied successfully to the assay of ibuprofen in rat plasma after oral administration.

Animals↗

PubMed2003

Chemicofunctional membrane for integrated chemical processes on a microchip.

Here we report a design and synthesis of a chemically functional polymer membrane by an interfacial polycondensation reaction and multilayer flow inside a microchannel. Single and parallel dual-membrane structures are successfully prepared by using organic/aqueous two-layer flow and organic/aqueous/organic three-layer flow inside the microchannel followed by an interfacial polycondensation reaction. By using the inner-channel membrane, permeation of ammonia species through the inner-channel membrane is successfully achieved. Furthermore, horseradish peroxidase is immobilized on one side of the membrane surface to integrate the chemical transform function onto the inner-channel membrane. Here substrate permeation through the membrane and subsequent chemical transformation at the membrane surface are realized. The polymer membrane prepared inside the microchannel has an important role in ensuring stable contact of different phases such as gas/liquid or liquid/ liquid and the permeation of chemical species through the membrane. Furthermore, membrane surface modification chemistry allows chemical transformation of permeated chemical species. These methods are expected to lead to development of complicated and sophisticated chemical systems involving membrane permeation and chemical reactions.

Journal Article↗

PubMed2004

Preparation, characterization, and time-resolved fluorometric application of silica-coated terbium(III) fluorescent nanoparticles.

Novel silica-coated terbium(III) chelate fluorescent nanoparticles have been prepared and characterized as a new type of fluorescence probe for highly sensitive time-resolved fluorescence bioassay. The preparation was carried out in a water-in-oil microemulsion containing a strongly fluorescent Tb(3+) chelate, N,N,N(1),N(1)-[2,6-bis(3'-aminomethyl-1'-pyrazolyl)-phenylpyridine]tetrakis(acetate)-Tb(3+), Triton X-100, hexanol, and cyclohexane by controlling hydrolysis of tetraethyl orthosilicate. The nanoparticles are spherical and uniform in size, 42 +/- 3 nm in diameter, strongly fluorescent, and highly photostable and have enough of a long fluorescence lifetime (1.52 ms) for time-resolved fluorescence measurement. A stable and nontoxic method was developed for the surface modification and protein immobilization of the nanoparticles. As a model of application, the nanoparticle-labeled streptavidin was prepared and used in a sandwich-type time-resolved fluoroimmunoassay of human prostate-specific antigen (PSA) by using a 96-well microtiter plate as the solid-phase carrier. The method gives a detection limit of 7.0 pg/mL for the PSA assay.

Journal Article↗

PubMed2004

Direct analysis of oxidizing agents in aqueous solution with attenuated total reflectance mid-infrared spectroscopy and diamond-like carbon protected waveguides.

A novel approach for the direct detection of oxidizing agents in aqueous solution is presented using diamond-like carbon (DLC) protected waveguides in combination with attenuated total reflectance (ATR) mid-infrared spectroscopy. Pulsed laser deposition was applied to produce high-quality DLC thin films on ZnSe ATR crystals with thicknesses of a few 100 nm. Scanning electron microscopy and X-ray photoelectron spectroscopy has been used to investigate the surface properties of the DLC films including the sp(3)/sp(2) hybridization ratio of the carbon bonds. Beside excellent adhesion of the DLC coatings to ZnSe crystals, these films show high chemical stability against strongly oxidizing agents. IR microscopy was utilized to compare differences in the chemical surface modification of bare and protected ATR waveguides when exposed to hydrogen peroxide, peracetic acid, and peroxydisulfuric acid. The feasibility of DLC protected waveguides for real-time concentration monitoring of these oxidizing agents was demonstrated by measuring calibration sets in a concentration range of 0.2-10%. Additionally, principal component regression has been applied to analyze multicomponent mixtures of hydrogen peroxide, acetic acid, and peracetic acid in aqueous solution. Due to high chemical stability and accurate monitoring capabilities, DLC protected waveguides represent a novel approach for directly detecting oxidizing agents in aqueous solution with promising potential for industrial process analysis.

Journal Article↗

PubMed2005

Radio frequency plasma polymer coatings for affinity capture MALDI mass spectrometry.

Surface modification of MALDI probes is an attractive approach for combining bioaffinity isolation of targeted biomolecules with mass spectrometric analysis of the captured species. In this work, we demonstrate that a polymer thin film, produced by pulsed rf plasma polymerization of allylamine and deposited directly on a MALDI probe, can be subsequently biotinylated to develop a bioaffinity capture MALDI probe. The synthesis and characterization of the probe by XPS, FT-IR, and AFM is described, and the selective isolation of avidin from a three-component mixture of avidin, lysozyme, and cytochrome c is presented. These initial results offer encouragement for the further exploration of rf plasma polymer deposition as a novel approach for the development of on-probe affinity capture MALDI probes.

Biotinylation↗

PubMed2004

Liquid membrane operations in a microfluidic device for selective separation of metal ions.

A three-phase flow, water/n-heptane/water, was constructed in a microchannel (100-microm width, 25-microm depth) on a glass microchip (3 cm x 7 cm) and was used as a liquid membrane for separation of metal ions. Surface modification of the microchannel by octadecylsilane groups induced spontaneous phase separation of the three-phase flow in the microfluidic device, which allows control of interfacial contact time and off-chip analysis using conventional analytical apparatus. Prior to the selective transport of a metal ion through the liquid membrane in the microchannel, the forward and backward extraction of yttrium and zinc ions was investigated in a two-phase flow on a microfluidic device using 2-ethylhexyl phosphonic acid mono-2-ethylhexyl ester (commercial name, PC-88A) as an extractant. The extraction conditions (contact time of the two phases, pH, extractant concentration) in the microfluidic device were examined. These investigations demonstrated that the conventional methodology for solvent extraction of metal ions is applicable to solvent extraction in a microchannel. Finally, we employed the three-phase flow in the microchannel as a liquid membrane and observed the selective transport of Y ion through the liquid membrane. In the present study, we succeeded, for the first time, in the selective separation of a targeted metal ion from an aqueous feed solution to a receiving phase within a few seconds by employing a liquid membrane formed in a microfluidic device.

Cations↗

PubMed2006

Enzyme-nanoparticle functionalization of three-dimensional protein scaffolds.

Various surface modification techniques have been developed for patterning functional biomolecules in two dimensions, allowing enzymes, antibodies, and other compounds to be localized for applications in bioanalysis and bioengineering. Here, we report a strategy for extending high-resolution patterning of biomolecules to three dimensions. In this approach, three-dimensional protein scaffolds are created by a direct-write process in which multiphoton excitation promotes photochemical cross-linking of protein molecules from aqueous solution within specified volume elements. After scaffold fabrication, protein microstructures are functionalized with enzyme-gold nanoparticle conjugates via a targeting process based in part on electrostatic attraction between the low-isoelectric-point enzyme and the microstructure, fabricated from high-isoelectric-point proteins. High signal-to-background ratios (approximately 20:1) are demonstrated for fluorescent product streams created by dephosphorylation of the fluorogenic compound, fluorescein diphosphate, at microstructures decorated with alkaline phosphatase-gold nanoparticle conjugates. We also demonstrate feasibility for using such structures to quantify substrate concentrations in flowing streams with low-micromolar detection limits and to create sensor suites based on both enzyme-nanoparticle functionalization and intrinsic enzymatic activity of protein scaffolds. These topographically complex sensors and dosing sources have potential applications in microfluidics, sensor array fabrication, and real-time chemical modification of cell culture environments.

Enzymes↗

PubMedDate not supplied

Synthesis of the first ferrocene-labeled dideoxynucleotide and its use for 3'-redox end-labeling of 5'-modified single-stranded oligonucleotides.

The target ferrocene-labeled dideoxynucleotide compound 5-[N-(beta-ferrocenyl-propanoyl)3-amino-propyn-1-yl]-2',3'-dideoxyuridine 5'-triphosphate, Fc-ddUTP, was synthesized and tested with terminal deoxynucleotidyl transferase for enzymatic 3'-redox-active end-labeling of 5'-phosphorylated single-stranded oligodeoxynucleotides. Starting from readily available 5-iodouridine and 3-ferrocenylpropanoic acid, the synthetic strategy elaborated here follows a mild multistep route. Each step involves reliable methods, and all ferrocene intermediates can be easily purified. Enzymatic 3'-ferrocene end-labeling of 5'-phosphorylated oligonucleotides is remarkably efficient, and 3'-ferrocene-labeled oligonucleotides can thus be prepared in sufficient amounts for further use in surface modifications.

Animals↗

PubMedDate not supplied

Different strategies for formation of pegylated EGF-conjugated PEI/DNA complexes for targeted gene delivery.

With the aim of generating gene delivery systems for tumor targeting, we have synthesized a conjugate consisting of polyethylenimine (PEI) covalently modified with epidermal growth factor (EGF) peptides. Transfection efficiency of the conjugate was evaluated and compared to native PEI in three tumor cell lines: KB epidermoid carcinoma cells, CMT-93 rectum carcinoma cells, and Renca-EGFR renal carcinoma cells. Depending on the tumor cell line, incorporation of EGF resulted in an up to 300-fold increased transfection efficiency. This ligand-mediated enhancement and competition with free EGF strongly suggested uptake of the complexes through the EGF receptor-mediated endocytosis pathway. Shielded particles being crucial for systemic gene delivery, we studied the effect of covalent surface modification of EGF-PEI/DNA complexes with a poly(ethylene glycol) (PEG) derivative. An alternative way for the formation of PEGylated EGF-containing complexes was also evaluated where EGF was projected away from PEI/DNA core complexes through a PEG linker. Both strategies led to shielded particles still able to efficiently transfect tumor cells in a receptor-dependent fashion. These PEGylated EGF-containing complexes were 10- to 100-fold more efficient than PEGylated complexes without EGF.

Animals↗

PubMedDate not supplied

Functionalization and cellular uptake of boron carbide nanoparticles. The first step toward T cell-guided boron neutron capture therapy.

In this paper we present surface modification strategies of boron carbide nanoparticles, which allow for bioconjugation of the transacting transcriptional activator (TAT) peptide and fluorescent dyes. Coated nanoparticles can be translocated into murine EL4 thymoma cells and B16 F10 malignant melanoma cells in amounts as high as 0.3 wt. % and 1 wt. %, respectively. Neutron irradiation of a test system consisting of untreated B16 cells mixed with B16 cells loaded with boron carbide nanoparticles were found to inhibit the proliferative capacity of untreated cells, showing that cells loaded with boron-containing nanoparticles can hinder the growth of neighboring cells upon neutron irradiation. This could provide the first step toward a T cell-guided boron neutron capture therapy.

Animals↗

PubMedDate not supplied

Synthesis of water-soluble, nonimmunogenic polyamide cross-linking agents.

Novel polyamides were developed that can be used as cross-linking agents for proteins such as hemoglobin. Water-soluble, nonimmunogenic polyamides containing oxygen and sulfur atoms in the backbone were prepared by the polycondensation of the diacids bis(carboxymethyloxyacetyl)-1,4-diaminobutane (1a) or 3, 3'-thiodipropionic acid (1b) with diethylene glycol bis(3-aminopropyl) ether (2). The resulting alpha,omega-diacids were converted to the corresponding activated esters using any of a variety of carboxylic acid activating reagents including the novel reagent diphenyl(1-methylimidazol-2-thiyl)phosphonate (9). The resulting polyamides could be activated with a broad spectrum of groups that allow for the cross-linking and surface modification of proteins.

Chromatography, Gel↗

PubMedDate not supplied

Synthesis and characterization of a photoactivatable glycoaryldiazirine for surface glycoengineering.

Biological systems make considerable use of specific molecular interactions. Many biomolecules involved in biorecognition are glycosylated, the carbohydrate moiety playing an essential role. Controlled surface glycoengineering is thus of crucial importance in biosensing, cell guidance, and biomedical applications. This study describes the synthesis of an aryldiazirine-derivatized galactose and the functionalization of surfaces by carbohydrates using photochemical immobilization techniques. A photoactivatable glycosylated reagent was synthesized by addition of thiogalactopyranose to the maleimide group of N-[m-[3-(trifluoromethyl)diazirin-3-yl]phenyl]-4-maleimidobutyr amide (MAD) to give N-[m-[3-(trifluoromethyl)diazirin-3-yl]phenyl]-4-[3-thio (1-D-galactopyranosyl)succinimidyl]butyramide (MAD-Gal). The structure of the newly synthesized molecule was confirmed by UV spectroscopy, photoactivation, 1H NMR, and 13C NMR. MAD-Gal was immobilized on thin diamond films by photoactivation of the diazirine function (350 nm). Surface modification was investigated by XPS (X-ray photoelectron spectroscopy) and ToF-SIMS (time-of-flight secondary ion mass spectrometry). Imaging ToF-SIMS was applied to detect glycopatterns generated by mask-assisted lithography.

Glycosides↗