Alginate-based hydrogel platform embedding silver nanoparticles and cisplatin: characterization of the synergistic effect on a breast cancer cell line.

Introduction: Breast cancer is a significant cause of mortality in women globally, and current treatment approaches face challenges due to side effects and drug resistance. Nanotechnology offers promising solutions by enabling targeted drug delivery and minimizing toxicity to normal tissues. Methods: In this study, we developed a composite platform called (Alg-AgNPs-CisPt), consisting of silver nanoparticles coated with an alginate hydrogel embedding cisplatin. We examined the effectiveness of this nanocomplex in induce synergistic cytotoxic effects on breast cancer cells. Results and Discussion: Characterization using various analytical techniques confirmed the composition of the nanocomplex and the distribution of its components. Cytotoxicity assays and apoptosis analysis demonstrated that the nanocomplex exhibited greater efficacy against breast cancer cells compared to AgNPs or cisplatin as standalone treatments. Moreover, the nanocomplex was found to enhance intracellular reactive oxygen species levels, further validating its efficacy. The synergistic action of the nanocomplex constituents offers potential advantages in reducing side effects associated with higher doses of cisplatin as a standalone treatment. Overall, this study highlights the potential of the (Alg-AgNPs-CisPt) nanocomplex as a promising platform embedding components with synergistic action against breast cancer cells.

Focal adhesion kinase activation is involved in contractile stimulation-induced detrusor muscle contraction in mice.

Recent studies suggested smooth muscle contraction may involve mechanisms besides the myosin regulatory light chain (MLC) phosphorylation-induced actomyosin crossbridge cycling. This study aims to determine if focal adhesion kinase (FAK) activation is involved in mouse detrusor muscle contraction. The mouse detrusor muscle strips were preincubated with PF-573228 (2 µM), latrunculin B (1 µM), or the same volume of vehicle (DMSO) for 30 min. The contractile responses to KCl (90 mM), electrical field stimulation (EFS, 2-32 Hz), or carbachol (CCh, 10-7.5-10-4.5 M) were measured. In a separate experiment, the phosphorylated FAK (p-FAK) and MLC (p-MLC) levels were measured in the detrusor strips stimulated with CCh (10 µM) after incubation with PF-573228 or vehicle (DMSO) compared to those with vehicle incubation but without CCh stimulation. KCl-induced contractile responses decreased significantly after incubation with PF-573228 or latrunculin B compared to the corresponding vehicle-treated strips (p < 0.0001). The contractile responses induced by EFS were markedly inhibited by preincubation with PF-573228 at 8, 16, and 32 Hz (p < 0.05) or latrunculin B at 16 and 32 Hz (p < 0.01). Following the application of PF-573228 or Latrunculin B, CCh-induced dose-response contractions were lower than the corresponding vehicle group (p = 0.0021 and 0.0003, respectively). Western blot examination showed that CCh stimulation enhanced the expression of p-FAK and p-MLC, while preincubation with PF-573228 prevented the increase of p-FAK but not p-MLC. In conclusion, FAK activation involves tension development induced by contractile stimulation in the mouse detrusor muscle. This effect is likely caused by promoting actin polymerization rather than elevating MLC phosphorylation.

Microspheres with 2D rGO/Alginate Matrix for Unusual Prolonged Release of Cefotaxime.

A synergistic interaction between reduced graphene oxide (rGO) and a biodegradable natural polymer, sodium alginate, was developed to create unique microspheres with protruding spiky features at the surface (spiky microspheres) that act as a super encapsulation and sustained release system for the highly effective antibiotic cefotaxime. Three forms of microspheres, namely alginate (Alg), alginate-cefotaxime (Alg-CTX), and alginate-cefotaxime-reduced graphene (Alg-CTX-rGO) composites, were prepared using calcium chloride as a cross-linking agent. The microspheres were characterized using field emission scanning electron microscopy (FESEM), Fourier-transform infrared (FT-IR) spectroscopy, and X-ray diffraction to investigate their pores, roughness, surface morphology, functional groups, phase formation, purity, and structural properties. The membrane diffusion method was employed to determine the release profile of Cefotaxime from the fabricated microspheres. The antibacterial activities of CTX solution, Alg microspheres, Alg-CTX microspheres, and Alg-CTX-rGO microspheres were investigated against gram-negative bacteria (Escherichia coli) using the agar diffusion method on Muller-Hinton agar. The prepared samples exhibited excellent results, suggesting their potential for enhanced antibiotic delivery. The results demonstrated the potential of the microsphere 2D rGO/alginate matrix for enhancing cefotaxime delivery with an unusual, prolonged release profile.

Inducible Nitric Oxide Synthase Embedded in Alginate/Polyethyleneimine Hydrogel as a New Platform to Explore NO-Driven Modulation of Biological Function.

Nitric oxide (NO), a small free radical molecule, turned out to be pervasive in biology and was shown to have a substantial influence on a range of biological activities, including cell growth and apoptosis. This molecule is involved in signaling and affects a number of physiologic functions. In recent decades, several processes related to cancer, such as angiogenesis, programmed cell death, infiltration, cell cycle progression, and metastasis, have been linked with nitric oxide. In addition, other parallel work showed that NO also has the potential to operate as an anti-cancer agent. As a result, it has gained attention in cancer-related therapeutics. The nitric oxide synthase enzyme family (NOS) is required for the biosynthesis of nitric oxide. It is becoming increasingly popular to develop NO-releasing materials as strong tumoricidal therapies that can deliver sustained high concentrations of nitric oxide to tumor sites. In this paper, we developed NO-releasing materials based on sodium alginate hydrogel. In this regard, alginate hydrogel discs were modified by adsorbing layers of polyethyleneimine and iNOS-oxygenase. These NO-releasing hydrogel discs were prepared using the layer-by-layer film building technique. The iNOS-oxygenase is adsorbed on the positively charged polyethyleneimine (PEI) matrix layer, which was formed on a negatively charged sodium alginate hydrogel. We show that nitric oxide is produced by enzymes contained within the hydrogel material when it is exposed to a solution containing all the components necessary for the NOS reaction. The electrostatic chemical adsorption of the layer-by-layer process was confirmed by FTIR measurements as well as scanning electron microscopy. We then tested the biocompatibility of the resulting modified sodium alginate hydrogel discs. We showed that this NOS-PEI-modified hydrogel is overall compatible with cell growth. We characterized the NOS/hydrogel films and examined their functional features in terms of NO release profiles. However, during the first 24 h of activity, these films show an increase in NO release flux, followed by a gradual drop and then a period of stable NO release. These findings show the inherent potential of using this system as a platform for NO-driven modulation of biological functions, including carcinogenesis.

Fast and Reliable Synthesis of Melanin Nanoparticles with Fine-Tuned Metal Adsorption Capacities for Studying Heavy Metal Ions Uptake.

PURPOSE: Adsorption and uptake of heavy metals by polymeric nanoparticles is driven by a variety of physicochemical processes. In this work, we examined heavy metal uptake by synthetic melanin nanoparticles and analyzed physicochemical properties that affect the extent of metal uptake by the nanoparticles. METHODS: Eumelanin nanoparticles were synthesized in a one-pot fast process from a 5,6-diacetoxy indole precursor that is hydrolyzed in situ into dihydroxy indole (DHI). The method allows the possibility of changing the level of sodium ions that ends up in the nanoparticles. Two variants of synthetic DHI-melanin (low-sodium and high sodium variants) were evaluated and demonstrated different relative adsorption efficiencies for heavy metal cations. RESULTS AND DISCUSSION: For the low-sodium DHI-melanin and in terms of percentages of metal ion removal, the relative order of extraction from 50 ppm solutions was Zn2+ > Cd2+ > Ni2+ > Co2+ > Cu2+ > Pb2+, with the extraction percentages ranging from 90% down to 76%, for a 30-minute adsorption time before equilibrium. The lower-sodium DHI-melanin consistently removed more Zn2+ than the higher-sodium variant. Electron microscopy (SEM) showed an increase in melanin particle size after metal ions uptake. In addition, X-ray photoelectron spectroscopy (XPS) of DHI-melanin particles with depth profiling after Zn ions uptake supported particle swelling and ion transport within the particles. CONCLUSION: These initial studies showed the potential of this straightforward synthesis to obtain synthetic DHI-melanin nanoparticles similar to those from biological sources with the possibility to fine-tune their metal adsorption capacity. These synthetic nanoparticles can be used either for the removal of a variety of metal ions or to mimic and study mechanisms of metal uptake by melanin deriving from biological sources, with the potential to understand, for instance, differential heavy metal uptake by various melanic pigments.

A cross-domain charge interaction governs the activity of NO synthase.

NO synthase (NOS) enzymes perform interdomain electron transfer reactions during catalysis that may rely on complementary charge interactions at domain-domain interfaces. Guided by our previous results and a computer-generated domain-docking model, we assessed the importance of cross-domain charge interactions in the FMN-to-heme electron transfer in neuronal NOS (nNOS). We reversed the charge of three residues (Glu-762, Glu-816, and Glu-819) that form an electronegative triad on the FMN domain and then individually reversed the charges of three electropositive residues (Lys-423, Lys-620, and Lys-660) on the oxygenase domain (NOSoxy), to potentially restore a cross-domain charge interaction with the triad, but in reversed polarity. Charge reversal of the triad completely eliminated heme reduction and NO synthesis in nNOS. These functions were partly restored by the charge reversal at oxygenase residue Lys-423, but not at Lys-620 or Lys-660. Full recovery of heme reduction was probably muted by an accompanying change in FMN midpoint potential that made electron transfer to the heme thermodynamically unfavorable. Our results provide direct evidence that cross-domain charge pairing is required for the FMN-to-heme electron transfer in nNOS. The unique ability of charge reversal at position 423 to rescue function indicates that it participates in an essential cross-domain charge interaction with the FMN domain triad. This supports our domain-docking model and suggests that it may depict a productive electron transfer complex formed during nNOS catalysis.

Functional Layer-by-Layer Thin Films of Inducible Nitric Oxide (NO) Synthase Oxygenase and Polyethylenimine: Modulation of Enzyme Loading and NO-Release Activity.

Nitric oxide (NO) release counteracts platelet aggregation and prevents the thrombosis cascade in the inner walls of blood vessels. NO-release coatings also prevent thrombus formation on the surface of blood-contacting medical devices. Our previous work has shown that inducible nitric oxide synthase (iNOS) films release NO fluxes upon enzymatic conversion of the substrate l-arginine. In this work, we report on the modulation of enzyme loading in layer-by-layer (LbL) thin films of inducible nitric oxide synthase oxygenase (iNOSoxy) on polyethylenimine (PEI). The layer of iNOSoxy is electrostatically adsorbed onto the PEI layer. The pH of the iNOSoxy solution affects the amount of enzyme adsorbed. The overall negative surface charge of iNOSoxy in solution depends on the pH and hence determines the density of adsorbed protein on the positively charged PEI layer. We used buffered iNOSoxy solutions adjusted to pHs 8.6 and 7.0, while saline PEI solution was used at pH 7.0. Atomic force microscopy imaging of the outermost layer shows higher protein adsorption with iNOSoxy at pH 8.6 than with a solution of iNOSoxy at pH 7.0. Graphite electrodes with PEI/iNOSoxy films show higher catalytic currents for nitric oxide reduction mediated by iNOSoxy. The higher enzyme loading translates into higher NO flux when the enzyme-modified surface is exposed to a solution containing the substrate and a source of electrons. Spectrophotometric assays showed higher NO fluxes with iNOSoxy/PEI films built at pH 8.6 than with films built at pH 7.0. Fourier transform infrared analysis of iNOSoxy adsorbed on PEI at pH 8.6 and 7.0 shows structural differences of iNOSoxy in films, which explains the observed changes in enzymatic activity. Our findings show that pH provides a strategy to optimize the NOS loading and enzyme activity in NOS-based LbL thin films, which enables improved NO release with minimum layers of PEI/NOS.

Endothelial nitric oxide synthase oxygenase on lipid nanodiscs: A nano-assembly reflecting native-like function of eNOS.

Endothelial nitric oxide synthase (eNOS) is a membrane-anchored enzyme. To highlight the potential role and effect of membrane phospholipids on the structure and activity of eNOS, we have incorporated the recombinant oxygenase subunit of eNOS into lipid nanodiscs. Two different size distribution modes were detected by multi-angle dynamic light scattering both for empty nanodiscs, and nanodiscs-bound eNOSoxy. The calculated hydrodynamic diameter for mode 1 species was 9.0 nm for empty nanodiscs and 9.8 nm for nanodisc bound eNOSoxy. Spectroscopic Griess assay was used to measure the enzymatic activity. Remarkably, the specific activity of nanodisc-bound eNOSoxy is ∼65% lower than the activity of free enzyme. The data shows that the nano-membrane environment affects the catalytic properties of eNOS heme domain.

L-carnitine is a calcium chelator: a reason for its useful and toxic effects in biological systems.

BACKGROUND: Investigation of the direct link between l-carnitine (LC), a quaternary ammonium compound that facilitates the passage of unsaturated fatty acids into the mitochondrial matrix, and free calcium (Ca2+) is needed to explain a number of varying results obtained from different in vitro and in vivo studies of LC as a supplement. METHODS: The chemical structure of LC, which contains oxygen ligand atoms, prompted to measure its activity asa Ca2+ chelator. The measurement was carried out spectrophotometri cally by measuring the reduction in the formation of Ca2+-o-cresolphthalein complexone (Ca-CPC) in the presence of different doses of LC (0.075, 0.75, and 7.5 mM) compared to the control (0.0 mM LC). RESULTS: The effect of LC was measured as a free entity in solution and when added to human serum. Our results showed a significant decrease (p < 0.05) in the average absorbance of Ca-CPC in the presence of LC compared to the control. CONCLUSIONS: In conclusion, LC exhibits a significant Ca2+ chelating activity. As Ca2+ is vital in the biochemical and physiological processes of living cells, LC could be affecting the calcium-dependent biological systems by limiting the levels of free Ca2+. Examples include decelerating the blood clotting process, amplifying the effect of anticoagulants, reducing nitric oxide synthase activity, inhibiting

Distinct conformational behaviors of four mammalian dual-flavin reductases (cytochrome P450 reductase, methionine synthase reductase, neuronal nitric oxide synthase, endothelial nitric oxide synthase) determine their unique catalytic profiles.

Multidomain enzymes often rely on large conformational motions to function. However, the conformational setpoints, rates of domain motions and relationships between these parameters and catalytic activity are not well understood. To address this, we determined and compared the conformational setpoints and the rates of conformational switching between closed unreactive and open reactive states in four mammalian diflavin NADPH oxidoreductases that catalyze important biological electron transfer reactions: cytochrome P450 reductase, methionine synthase reductase and endothelial and neuronal nitric oxide synthase. We used stopped-flow spectroscopy, single turnover methods and a kinetic model that relates electron flux through each enzyme to its conformational setpoint and its rates of conformational switching. The results show that the four flavoproteins, when fully-reduced, have a broad range of conformational setpoints (from 12% to 72% open state) and also vary 100-fold with respect to their rates of conformational switching between unreactive closed and reactive open states (cytochrome P450 reductase > neuronal nitric oxide synthase > methionine synthase reductase > endothelial nitric oxide synthase). Furthermore, simulations of the kinetic model could explain how each flavoprotein can support its given rate of electron flux (cytochrome c reductase activity) based on its unique conformational setpoint and switching rates. The present study is the first to quantify these conformational parameters among the diflavin enzymes and suggests how the parameters might be manipulated to speed or slow biological electron flux.

Salvia fruticosa reduces intrinsic cellular and H2O2-induced DNA oxidation in HEK 293 cells; assessment using flow cytometry.

OBJECTIVE: To investigate the role of water-soluble extract of Salvia fruticosa (Greek sage) (S. fruticosa) leaves in reducing both intrinsic cellular and H2O2-induced DNA oxidation in cultured human embryonic kidney 293 cells. S. fruticosa, native to the Eastern-Mediterranean basin, is widely used as a medicinal herb for treatment of various diseases. METHODS: Dried leaves of S. fruticosa were extracted in phosphate buffer saline and purified using both vacuum and high pressure filtrations. Each mL of the preparation contained (7.1±1.0) mg of extract. HEK-293 cells were incubated in one set with S. fruticosa extract in the presence of 0.1 mmol/L H2O2, and in the other set with the addition of the extract alone. The DNA oxidation was measured using fluorescence upon fluorescein isothiocyanate derivatization of 8-oxoguanine moieties. The fluorescence was measured using flow cytometry technique. RESULTS: Cells incubated 3 h with 150 µL extract and exposed to 0.1 mmol/L H2O2 showed lower intensity of fluorescence, and thus lower DNA oxidation. Moreover, cells incubated 3 h with 100 µL of the extract showed lower intensity of fluorescence, and thus lower intrinsic cellular DNA oxidation compared to control (without S. fruticosa). CONCLUSIONS: The results from this study suggest that the water-soluble extract of S. fruticosa leaves protects against both H2O2-induced and intrinsic cellular DNA oxidation in human embryonic kidney 293 cells.

Salvia fruticosa reduces intrinsic cellular and H2O2-induced DNA oxidation in HEK 293 cells;assessment using flow cytometry

Objective:To investigate the role of water-soluble extract of Salvia fruticosa (Greek sage) (S. fruticosa) leaves in reducing both intrinsic cellular and H2O2-induced DNA oxidation in cultured human embryonic kidney 293 cells. S. fruticosa, native to the Eastern-Mediterranean basin, is widely used as a medicinal herb for treatment of various diseases. Methods: Dried leaves of S. fruticosa were extracted in phosphate buffer saline and purified using both vacuum and high pressure filtrations. Each mL of the preparation contained (7.1±1.0) mg of extract. HEK-293 cells were incubated in one set with S. fruticosa extract in the presence of 0.1 mmol/L H2O2, and in the other set with the addition of the extract alone. The DNA oxidation was measured using fluorescence upon fluorescein isothiocyanate derivatization of 8-oxoguanine moieties. The fluorescence was measured using flow cytometry technique. Results:Cells incubated 3 h with 150 μL extract and exposed to 0.1 mmol/L H2O2 showed lower intensity of fluorescence, and thus lower DNA oxidation. Moreover, cells incubated 3 h with 100 μL of the extract showed lower intensity of fluorescence, and thus lower intrinsic cellular DNA oxidation compared to control (without S. fruticosa). Conclusions: The results from this study suggest that the water-soluble extract of S. fruticosa leaves protects against both H2O2-induced and intrinsic cellular DNA oxidation in human embryonic kidney 293 cells.

Thermodynamic characterization of five key kinetic parameters that define neuronal nitric oxide synthase catalysis.

NO synthase (NOS) enzymes convert L-arginine to NO in two sequential reactions whose rates (k(cat1) and k(cat2)) are both limited by the rate of ferric heme reduction (k(r)). An enzyme ferric heme-NO complex forms as an immediate product complex and then undergoes either dissociation (at a rate that we denote as k(d)) to release NO in a productive manner, or reduction (k(r)) to form a ferrous heme-NO complex that must react with O2 (at a rate that we denote as k(ox)) in a NO dioxygenase reaction that regenerates the ferric enzyme. The interplay of these five kinetic parameters (k(cat1), k(cat2), k(r), k(d) and k(ox)) determines NOS specific activity, O2 concentration response, and pulsatile versus steady-state NO generation. In the present study, we utilized stopped-flow spectroscopy and single catalytic turnover methods to characterize the individual temperature dependencies of the five kinetic parameters of rat neuronal NOS. We then incorporated the measured kinetic values into computer simulations of the neuronal NOS reaction using a global kinetic model to comprehensively model its temperature-dependent catalytic behaviours. The results obtained provide new mechanistic insights and also reveal that the different temperature dependencies of the five kinetic parameters significantly alter neuronal NOS catalytic behaviours and NO release efficiency as a function of temperature.

Peroxynitrite activity of hemin-functionalized reduced graphene oxide.

Conducting interfaces modified with reduced graphene oxide (rGO) have shown improved electrochemical response for different analytes. The efficient formation of functionalized rGO based materials is thus of current interest for the development of sensitive and selective biosensors. Herein, we report a simple and environmentally friendly method for the formation of a hemin-functionalized rGO hybrid nanomaterial that exhibits remarkable sensitivity to peroxynitrite (ONOO(-)) in solution. The hemin-functionalized rGO hybrid nanomaterial was formed by mixing an aqueous solution of graphene oxide (GO) with hemin and sonicating the suspension for 5 h at room temperature. In addition to playing a key role in biochemical and electrocatalytic reactions, hemin has been proven to be a good reducing agent for GO. The sensitivity of the peroxynitrite sensor is ≈7.5 ± 1.5 nA mM(-1) with a detection limit of 5 ± 1.5 nM.

Polymerized hemin as an electrocatalytic platform for peroxynitrite's oxidation and detection.

Peroxynitrite (ONOO(-)) constitutes a major cytotoxic agent, implicated in a host of pathophysiological conditions, thereby stimulating a tremendous interest in evaluating its role as an oxidant in vivo. Some of the detection methods for peroxynitrite include oxidation of fluorescent probes, EPR spectroscopy, chemiluminescence, immunohistochemistry, and probe nitration; however, these are more difficult to apply for real-time quantification due to their inherent complexity. The electrochemical detection of peroxynitrite is a simpler and more convenient technique, but the best of our knowledge there are only few papers to date studying its electrochemical signature, or reporting amperometric microsensors for peroxynitrite. Recently, we have reported the use of layered composite films of poly(3,4-ethylenedioxythiophene) (PEDOT) and hemin (iron protoporphyrin IX) as a platform for amperometric measurement of peroxynitrite. The main goal herein is to investigate the intrinsic catalytic role of hemin electropolymerized thin films on carbon electrodes in oxidative detection of peroxynitrite. The electrocatalytic oxidation of peroxynitrite is characterized by cyclic voltammetry. The catalytic current increased as a function of peroxynitrite's concentration, with a peak potential shifting positively with peroxynitrite's concentration. The catalytic efficiency decreased as the scan rate increased, and the peak potential of the catalytic oxidation was found to depend on pH. We show that optimized hemin-functionalized carbon electrodes can be used as simple platforms for peroxinitrite detection and quantification. We report dose-response amperometry as an electroanalytical determination of this analyte on hemin films and we contrast the intrinsic hemin catalytic role with its performance in the case of the PEDOT-hemin as a composite matrix. Finally, we include some work extending the use of simple hemin films for peroxynitrite determination on carbon microfiber electrodes in a flow system.

Charge-pairing interactions control the conformational setpoint and motions of the FMN domain in neuronal nitric oxide synthase.

The NOS (nitric oxide synthase; EC 1.14.13.39) enzymes contain a C-terminal flavoprotein domain [NOSred (reductase domain of NOS)] that binds FAD and FMN, and an N-terminal oxygenase domain that binds haem. Evidence suggests that the FMN-binding domain undergoes large conformational motions to shuttle electrons between the NADPH/FAD-binding domain [FNR (ferredoxin NADP-reductase)] and the oxygenase domain. Previously we have shown that three residues on the FMN domain (Glu762, Glu816 and Glu819) that make charge-pairing interactions with the FNR help to slow electron flux through nNOSred (neuronal NOSred). In the present study, we show that charge neutralization or reversal at each of these residues alters the setpoint [Keq(A)] of the NOSred conformational equilibrium to favour the open (FMN-deshielded) conformational state. Moreover, computer simulations of the kinetic traces of cytochrome c reduction by the mutants suggest that they have higher conformational transition rates (1.5-4-fold) and rates of interflavin electron transfer (1.5-2-fold) relative to wild-type nNOSred. We conclude that the three charge-pairing residues on the FMN domain govern electron flux through nNOSred by stabilizing its closed (FMN-shielded) conformational state and by retarding the rate of conformational switching between its open and closed conformations.

Probe functionalization with a Rhop-3 antibody: toward a Rhop-3 antigen immunosensor for detection of malaria.

The antibody specific for the malaria protein, Rhop-3, and FL-Rhop-3, were immobilized on the surface of a gold electrode modified with cysteamine. Colloidal gold was used to enhance the detection signal for Rhop-3 antigens. The Rhop-3 antibody was also immobilized on gold electrodes preactivated with dithiobis(succinimidyl proprionate) (DSP). Immobilization was performed at room temperature and at 37 °C. Cyclic voltammetry (CV) was used to monitor the interaction between the immobilized antibody and its cognate antigen in solution, using ferricyanide, K3Fe(CN)6, as reporting electroactive probe. Tests indicate recognition of Rhop-3 protein by the immobilized antibody. Antigen recognition was enhanced by incubation at 37 °C compared with room-temperature incubation. Our results suggest that an immunosensor can be developed and optimized to aid detection of Rhop-3 antigens in samples from malaria patients. As far as we are aware, this is the first amperometric immunosensor targeting Rhop-3 antigen as a malaria biomarker.

Nanostructured poly(3,4-ethylenedioxythiophene)-metalloporphyrin films: improved catalytic detection of peroxynitrite.

We investigated in this paper the sensing performance of inherently conductive polymer, poly(3,4-ethylenedioxythiophene) (PEDOT), functionalized with hemin (iron protoporphyrin) as an electrocatalytic reporter. The sensing platform is prepared by electrodeposition of a composite film of hemin-PEDOT on a 30-microm diameter carbon fiber electrode (CFE). The polymerized films were characterized by field emission scanning electron microscopy (FESEM), which pointed to nanostructured films with tortuous pores. The electrocatalytic oxidation of peroxynitrite was characterized by cyclic voltammetry as well as other electrochemical methods. The catalytic current is proportional to the analyte's concentration. Optimized hemin-PEDOT modified CFEs were utilized for the first time to detect ONO2(-), with a response time down to 5 s and a limit of detection as low as 200 nM as evidenced by amperometry. Our hemin-PEDOT modified CFEs have a sensitivity of 13 nA/microM, ca.130 times higher than the bare CFE. More work is underway using other metalloporphyrins as electrocalalysts to improve the detection limit, the selectivity, and to further miniaturize these hemin-PEDOT modified electrodes.

Reductive decomposition of a diazonium intermediate by dithiothreitol affects the determination of NOS turnover rates.

Accurate determination of nitrite either as such or as the breakdown product of nitric oxide (NO) is critical in a host of enzymatic reactions in various settings addressing structure-function relationships, as well as mechanisms and kinetics of molecular operation of enzymes. The most common way to quantify nitrite, for instance in nitric oxide synthase (NOS) mechanistic investigations, is the spectrophotometric assay based on the Griess reaction through external standard calibration. This assay is based on a two-step diazotization reaction, in which a cationic diazonium derivative of sulfanilamide is formed as intermediate before the final absorbing azo-product. We show that this intermediate is very sensitive to reducing agents that may be transferred from the reaction media under investigation. The interaction of this vital intermediate with the reducing agent, dithiothreitol (DTT), which is widely used in NOS reactions, is characterized by both electrochemical and spectroscopic means. The effect of DTT on the performance of external calibration, both in sample recovery studies and in actual NOS reactions, is presented. Finally an alternative method of standard additions, which partially compensates for the accuracy and sensitivity problems of external calibration, is proposed and discussed.

Layer-by-layer fabrication and characterization of DNA-wrapped single-walled carbon nanotube particles.

Carbon nanotubes have been proposed as support materials for numerous applications, including the development of DNA sensors. One of the challenges is the immobilization of DNA or other biological molecules on the sidewall of carbon nanotubes. This paper introduces a new fabrication of DNA-carbon nanotubes particles using the layer-by-layer (LBL) technique on single-walled carbon nanotubes (SWCNTs). Poly(diallyldimethylammonium) (PDDA), a positively charged polyelectrolyte, and DNA as a negatively charged counterpart macromolecule are alternatively deposited on the water-soluble oxidized SWCNTs. Pure DNA/PDDA/SWCNTs particles can be prepared and separated by simple unltracentrifugation. The characterization of DNA/PDDA/SWCNTs particles was carried out by scanning electron microscopy (SEM), transmission electron microscopy (TEM), UV-visible spectroscopy, Raman spectroscopy, and thermogravimetric analysis (TGA). An electrode modified by the DNA/PDDA/SWCNTs particles shows a dramatic change of the electrochemical signal in solutions of tris(2,2'-bipyridyl)ruthenium(II) ((Ru(bpy)(3)2+) as a reporting redox probe. A preliminary application of the DNA-modified carbon nanotubes in the development of DNA sensors used in the investigation of DNA damage by nitric oxide is presented.