<i>S</i>-Nitrosothiol Detection via Amperometric Nitric Oxide Sensor with Surface Modified Hydrogel Layer Containing Immobilized Organoselenium Catalyst^†

A novel electrochemical device for the direct detection of S-nitrosothiol species (RSNO) is proposed by modifying an amperometric nitric oxide (NO) gas sensor with thin hydrogel layer containing an immobilized organoselenium catalyst. The diselenide, 3,3‘-dipropionicdiselenide, is covalently coupled to primary amine groups in polyethylenimine (PEI), which is further cross-linked to form a hydrogel layer on a dialysis membrane support. Such a polymer film containing the organoselenium moiety is capable of decomposing S-nitrosothiols to generate NO(g) at the distal tip of the NO sensor. Under optimized conditions, various RSNOs (e.g., nitrosocysteine (CysNO), nitrosoglutathione (GSNO), etc.) are reversibly detected at ≤0.1 μM levels, with sensor lifetimes of at least 10 days. The presence of reducing agents (e.g., glutathione) added to the test solution enhances the amperometric dynamic range output to ∼25 μM levels of RSNO species. Sensitivities observed for different small molecule RSNO species are nearly equivalent, in sharp contrast to the behavior observed previously for a similar RSNO sensing configuration based on an immobilized Cu(I/II) catalytic layer. It is further shown that the new RSNO sensors can be used to assess the “NO-generating” ability of fresh blood samples by effectively detecting the total level of reactive low molecular-weight RSNO species present in such samples

Pyrroloquinoline Quinone-Doped Polymeric Nanospheres as Sensitive Tracer for Binding Assays

The preparation and analytical characteristics of novel prosthetic group loaded polymeric nanospheres for use in high-sensitivity bioaffinity assays is reported. Pyrroloquinoline quinone (PQQ), a prosthetic group for apoglucose dehydrogenase (apo-GDH), is loaded into poly(methyl methacrylate) (PMMA) nanospheres in the presence of methanol. PQQ released from the nanospheres in the presence of 40% acetonitrile is capable of reconstituting apo-GDH and triggers the enzymatic reaction with excess glucose. The two electrons generated are transferred from a reduced PQQ to a redox dye reagent, e.g., 2,6-dichloroindolphenol (DCPIP). The decrease in absorbance of DCPIP is observed visually or spectrophotometrically to assess the number of particles present. As initial model systems, this concept is applied to develop a microtiter plate assay to detect biotin (as a model for low molecular weight species) and C-reactive protein (CRP). For the CRP assay, neutravidin-coated PQQ-doped PMMA nanospheres are used to bind with a biotinylated reporter antibody directed toward CRP. Detection limits to CRP at subnanogram per milliliter levels are demonstrated. The advantage of this type of assay is that excess apoenzyme can be added, with detection capability dependent on the number of encapsulated PQQ species that can be readily released from the surface-bound nanospheres (ca. 20 000 PQQ molecules/PMMA particle)

Modeling the Effect of Oxygen on the Amperometric Response of Immobilized Organoselenium-Based <i>S</i>-Nitrosothiol Sensors

Amperometric detection of S-nitrosothiols (RSNOs) at submicromolar levels in blood samples is of potential importance for monitoring endothelial function and other disease states that involve changes in physiological nitric oxide (NO) production. It is shown here that the elimination of dissolved oxygen from samples is critical when covalently attached diselenocystamine-based amperometric RSNO sensors are used for practical RSNO measurements. The newest generation of RSNO sensors utilizes an amperometric NO gas sensor with a thin organoselenium modified dialysis membrane mounted at the distal sensing tip. Sample RSNOs are catalytically reduced to NO within the dialysis membrane by the immobilized organoselenium species. In the presence of oxygen, the sensitivity of these sensors for measuring low levels of RSNOs (<μM) is greatly reduced. It is demonstrated that the main scavenger of the generated nitric oxide is not the dissolved oxygen but rather superoxide anion radical generated from the reaction of the reduced organoselenium species (the reactive species in the catalytic redox cycle) and dissolved oxygen. Computer simulations of the response of the RSNO sensor using rate constants and diffusion coefficients for the reactions involved, known from the literature or estimated from fitting to the observed amperometric response curves, as well as the specific geometric dimensions of the RSNO sensor, further support that nitric oxide and superoxide anion radical quickly react resulting in near zero sensor sensitivity toward RSNO concentrations in the submicromolar concentration range. Elimination of oxygen from samples helps improve sensor detection limits to ca. 10 nM levels of RSNOs

Polymethacrylate-Based Nitric Oxide Donors with Pendant <i>N</i>-Diazeniumdiolated Alkyldiamine Moieties: Synthesis, Characterization, and Preparation of Nitric Oxide Releasing Polymeric Coatings

A series of new nitric oxide (NO) releasing copolymers have been prepared by covalently anchoring alkyldiamine side chains onto a polymethacrylate-based polymer backbone, followed by NO addition to form the desired pendant diazeniumdiolate structures. The resulting diazeniumdiolated copolymers were characterized via UV spectroscopy, and their proton-driven decomposition to release NO was also examined by UV and FTIR as well as chemiluminescence. Polymers with up to 22.1 mol % of incorporated amine sites that can be converted to corresponding diazeniumdiolates could be prepared, and such polymers release up to 0.94 μmol/mg of NO. Further, novel NO releasing polymeric coatings were formulated by doping one of the new polymethacrylate-based NO donors within inert polymeric matrixes. Biodegradable poly(lactide-co-glycolide) was employed as a film additive to greatly prolong the NO release of such coatings by continuously generating protons within the organic phase of the polymeric films, thereby driving decomposition of the diazeniumdiolates

Polymethacrylate-Based Nitric Oxide Donors with Pendant <i>N</i>-Diazeniumdiolated Alkyldiamine Moieties: Synthesis, Characterization, and Preparation of Nitric Oxide Releasing Polymeric Coatings

A series of new nitric oxide (NO) releasing copolymers have been prepared by covalently anchoring alkyldiamine side chains onto a polymethacrylate-based polymer backbone, followed by NO addition to form the desired pendant diazeniumdiolate structures. The resulting diazeniumdiolated copolymers were characterized via UV spectroscopy, and their proton-driven decomposition to release NO was also examined by UV and FTIR as well as chemiluminescence. Polymers with up to 22.1 mol % of incorporated amine sites that can be converted to corresponding diazeniumdiolates could be prepared, and such polymers release up to 0.94 μmol/mg of NO. Further, novel NO releasing polymeric coatings were formulated by doping one of the new polymethacrylate-based NO donors within inert polymeric matrixes. Biodegradable poly(lactide-co-glycolide) was employed as a film additive to greatly prolong the NO release of such coatings by continuously generating protons within the organic phase of the polymeric films, thereby driving decomposition of the diazeniumdiolates

Gold-Coated Magnetic Particles for Solid-Phase Immunoassays: Enhancing Immobilized Antibody Binding Efficiency and Analytical Performance

The preparation and characterization of gold-coated magnetic particles are described for use as more efficient solid-phase materials in immunoassay development. A thin gold coating on commercial tosylated magnetic polystyrene particles (4.5 μm) is achieved via an electroless plating method involving initial reaction of the particles with Sn(II), followed by redox deposition of Ag0, that serves as a catalytic site for the subsequent reduction of Na3Au(SO3)2 in the presence of formaldehyde to yield the adhered gold layer. Scanning electron microscopy, energy-dispersive X-ray analysis, and X-ray photoelectron spectroscopy indicate the presence of the desired Au0 outer layer. To characterize the improved yield of antibody binding sites on such gold-coated phases, the modified particles are reacted with the free thiols of Fab‘ fragments of an anti-alkaline phosphatase (ALP) antibody to orient all the antigenic binding sites in a favorable direction. After equilibration with ALP, the amount of ALP bound to the surface of such particles is nearly 2.5-fold greater than on non-gold-coated particles possessing the same amount of immobilized anti-ALP Fab‘, but oriented randomly on the surface. The new gold-coated magnetic particles are further used as a solid phase for developing a sandwich-type enzyme immunoassay to detect C-reactive protein (CRP) using horseradish peroxidase as the enzyme label. The gold-coated magnetic particles with anti-CRP monoclonal Fab‘ reagents provide assays with enhanced assay slope (1.8-fold), lower nonspecific adsorption, and a detection limit improvement of nearly 10-fold (0.14 vs 1.9 ng/mL) compared to the same Fab‘ anti-CRP immobilized on the initial tosylated polystyrene magnetic particles. The improved assay performance is attributed to the more favorable binding orientation of the self-assembled monolayer of Fab‘ fragments on the gold-coated particles compared to the random orientation on the non-gold-coated surfaces

Controlled Photoinitiated Release of Nitric Oxide from Polymer Films Containing <i>S</i>-Nitroso-<i>N</i>-acetyl-dl-penicillamine Derivatized Fumed Silica Filler

This report describes the first hydrophobic nitric oxide (NO)-releasing material that utilizes light as an external on/off trigger to control the flux of NO generated from cured polymer films. Fumed silica polymer filler particles were derivatized with S-nitroso-N-acetyl-dl-penicillamine and blended into the center layer of trilayer silicone rubber films. Nitric oxide is generated upon irradiation with light, and fluxes increase with increasing power of incident light. The ability to precisely control NO generation from this material has the potential to answer fundamental questions about the levels of NO needed to achieve desired therapeutic affects in different biomedical applications

Spontaneous Catalytic Generation of Nitric Oxide from <i>S</i>-Nitrosothiols at the Surface of Polymer Films Doped with Lipophilic Copper(II) Complex

A new approach for preparing potentially more blood-compatible nitric oxide (NO)-generating polymeric materials is described. The method involves creating polymeric films that have catalytic sites within (lipophilic copper(II) complex) that are capable of converting endogenous S-nitrosothiols present in blood (S-nitrosoglutathione (GSNO), S-nitrosocysteine (CysNO), etc.) to NO. The catalytic NO generation reaction involves the initial reduction of Cu(II) to Cu(I) within the complex by appropriate reducing agents (e.g., thiolates or ascorbate), followed by the reduction of S-nitrosothiols to NO by the Cu(I) complex at the polymer/solution interface. The NO fluxes observed when PVC or polyurethane films containing the copper(II) complex are placed in solutions containing physiological levels of nitrosothiols (μM levels) reach ca. 8 × 10-10 mol cm-2 min-1, greater than that produced by normal endothelial cells that line all healthy blood vessels. It is thus anticipated that this spontaneous catalytic generation of NO from endogenous nitrosothiols will render such polymeric materials more thromboresistant when in contact with blood in vivo

Reversible Detection of Heparin and Other Polyanions by Pulsed Chronopotentiometric Polymer Membrane Electrode

The first fully reversible polymeric membrane-based sensor for the anticoagulant heparin and other polyanions using a pulsed chronopotentiometry (pulstrode) measurement mode is reported. Polymeric membranes containing a lipophilic inert salt of the form R+R− (where R+ and R− are tridodecylmethylammonium (TDMA+) and dinonylnaphthalene sulfonate (DNNS−), respectively) are used to suppress unwanted spontaneous ion extractions under zero-current equilibrium conditions. An anodic galvanostatic current pulse applied across the membrane perturbs the equilibrium lipophilic ion distribution within the membrane phase in such a way that anions/polyanions are extracted into the membrane from the sample. The membrane is then subjected to an open-circuit zero current state for a short period, and finally a 0 V vs reference electrode potentiostatic pulse is applied to restore the membrane to its initial full equilibrium condition. Potentials are sampled as average values during the last 10% of the 0.5 s open circuit phase of the measurement cycle. Fully reversible and reproducible electromotive force (emf) responses are observed for heparin, pentosan polysulfate (PPS), chondroitin sulfate (CS), and oversulfated chondroitin sulfate (OSCS), with the magnitude of the potentiometric response proportional to charge density of the polyanions. The sensor provides an emf response related to heparin concentrations in the range of 1−20 U/mL. The responses to variations in heparin levels and toward other polyanions of the pulstrode configuration are analogous to the already established single-use, nonreversible potentiometric polyion sensors based on membranes doped only with the lipophilic anion exchanger TDMA+

Highly Selective Optical Fluoride Ion Sensor with Submicromolar Detection Limit Based on Aluminum(III) Octaethylporphyrin in Thin Polymeric Film

A highly selective, sensitive, and reversible fluoride optical sensing film based on aluminum(III)octaethylporphyrin as a fluoride ionophore and a lipophilic pH indicator as the optical transducer is described. The fluoride optical sensing films exhibit a submicromolar detection limit and high discrimination for fluoride over several lipophilic anions such as nitrate, perchlorate, and thiocyanate