Chiroplasmonic DNA Scaffolded "Fusilli" Structures.

DNA is an ideal template for the design of nanoarchitectures with molecular-like features. Here, we present an optimized assembly strategy for the concatenation of DNA quasi-rings into long scaffolds. Ionic strength, which played a major role during self-assembly, produced the expected high quality only at 15 mM MgCl2. Atomic force microscopy (AFM) characterization showed several micrometer long tubular structures that were used as templates for the positioning of plasmonic nanoparticles (NPs) along a three-dimensional helical path using DNA tethers. As imaged by high-resolution scanning transmission electron microscopy (HR-STEM) and modeled by theoretical calculations, the NPs distributed into a "fusilli" fashion (i.e., a helical pasta shape), displaying chiroptical activity as revealed by a bisignated CD absorption, centered at the plasmon resonance wavelength. The present structures contribute to enrich the ever-developing arena of chiroplasmonic DNA-based nanomaterials and demonstrate that large assemblies are attainable for their future application to develop metamaterials.

Rational design of hybrid DNA-RNA triplex structures as modulators of transcriptional activity in vitro.

Triplex nanostructures can be formed in vitro in the promoter region of DNA templates, and it is commonly accepted that these assemblies inhibit the transcription of the downstream genes. Herein, a proof of concept highlighting the possibility of the up- or downregulation of RNA transcription is presented. Hybrid DNA-RNA triplex nanostructures were rationally designed to produce bacterial transcription units with switchable promoters. The rate of RNA production was measured using the signal of a transcribed fluorescent RNA aptamer (i.e. Broccoli). Indeed, several designed bacterial promoters showed the ability of induced transcriptional inhibition, while other properly tailored sequences demonstrated switchable enhancement of transcriptional activity, representing an unprecedented feature to date. The use of RNA-regulated transcription units and fluorescent RNA aptamers as readouts will allow the realization of biocomputation circuits characterized by a strongly reduced set of components. Triplex forming RNA oligonucleotides are proposed as smart tools for transcriptional modulation and represent an alternative to current methods for producing logic gates using protein-based components.

Marine Microbial Fibrinolytic Enzymes: An Overview of Source, Production, Biochemical Properties and Thrombolytic Activity.

Cardiovascular diseases (CVDs) have emerged as a major threat to global health resulting in a decrease in life expectancy with respect to humans. Thrombosis is one of the foremost causes of CVDs, and it is characterized by the unwanted formation of fibrin clots. Recently, microbial fibrinolytic enzymes due to their specific features have gained much more attention than conventional thrombolytic agents for the treatment of thrombosis. Marine microorganisms including bacteria and microalgae have the significant ability to produce fibrinolytic enzymes with improved pharmacological properties and lesser side effects and, hence, are considered as prospective candidates for large scale production of these enzymes. There are no studies that have evaluated the fibrinolytic potential of marine fungal-derived enzymes. The current review presents an outline regarding isolation sources, production, features, and thrombolytic potential of fibrinolytic biocatalysts from marine microorganisms identified so far.

Industrial applications of immobilized nano-biocatalysts.

Immobilized enzyme-based catalytic constructs could greatly improve various industrial processes due to their extraordinary catalytic activity and reaction specificity. In recent decades, nano-enzymes, defined as enzyme immobilized on nanomaterials, gained popularity for the enzymes' improved stability, reusability, and ease of separation from the biocatalytic process. Thus, enzymes can be strategically incorporated into nanostructured materials to engineer nano-enzymes, such as nanoporous particles, nanofibers, nanoflowers, nanogels, nanomembranes, metal-organic frameworks, multi-walled or single-walled carbon nanotubes, and nanoparticles with tuned shape and size. Surface-area-to-volume ratio, pore-volume, chemical compositions, electrical charge or conductivity of nanomaterials, protein charge, hydrophobicity, and amino acid composition on protein surface play fundamental roles in the nano-enzyme preparation and catalytic properties. With proper understanding, the optimization of the above-mentioned factors will lead to favorable micro-environments for biocatalysts of industrial relevance. Thus, the application of nano-enzymes promise to further strengthen the advances in catalysis, biotransformation, biosensing, and biomarker discovery. Herein, this review article spotlights recent progress in nano-enzyme development and their possible implementation in different areas, including biomedicine, biosensors, bioremediation of industrial pollutants, biofuel production, textile, leather, detergent, food industries and antifouling.

Acidic Shift of Optimum pH of Bovine Serum Amine Oxidase upon Immobilization onto Nanostructured Ferric Tannates.

Protein-nanoparticle hybrids represent entities characterized by emerging biological properties that can significantly differ from those of the parent components. Herein, bovine serum amine oxidase (i.e., BSAO) was immobilized onto a magnetic nanomaterial constituted of surface active maghemite nanoparticles (i.e., SAMNs, the core), surface-modified with tannic acid (i.e., TA, the shell), to produce a biologically active ternary hybrid (i.e., SAMN@TA@BSAO). In comparison with the native enzyme, the secondary structure of the immobilized BSAO responded to pH variations sensitively, resulting in a shift of its optimum activity from pH 7.2 to 5.0. Conversely, the native enzyme structure was not influenced by pH and its activity was affected at pH 5.0, i.e., in correspondence with the best performances of SAMN@TA@BSAO. Thus, an extensive NMR study was dedicated to the structure-function relationship of native BSAO, confirming that its low activity below pH 6.0 was ascribable to minimal structural modifications not detected by circular dichroism. The generation of cytotoxic products, such as aldehydes and H2O2, by the catalytic activity of SAMN@TA@BSAO on polyamine oxidation is envisaged as smart nanotherapy for tumor cells. The present study supports protein-nanoparticle conjugation as a key for the modulation of biological functions.

Toward the Specificity of Bare Nanomaterial Surfaces for Protein Corona Formation.

Aiming at creating smart nanomaterials for biomedical applications, nanotechnology aspires to develop a new generation of nanomaterials with the ability to recognize different biological components in a complex environment. It is common opinion that nanomaterials must be coated with organic or inorganic layers as a mandatory prerequisite for applications in biological systems. Thus, it is the nanomaterial surface coating that predominantly controls the nanomaterial fate in the biological environment. In the last decades, interdisciplinary studies involving not only life sciences, but all branches of scientific research, provided hints for obtaining uncoated inorganic materials able to interact with biological systems with high complexity and selectivity. Herein, the fragmentary literature on the interactions between bare abiotic materials and biological components is reviewed. Moreover, the most relevant examples of selective binding and the conceptualization of the general principles behind recognition mechanisms were provided. Nanoparticle features, such as crystalline facets, density and distribution of surface chemical groups, and surface roughness and topography were encompassed for deepening the comprehension of the general concept of recognition patterns.

Enzyme self-assembly on naked iron oxide nanoparticles for aminoaldehyde biosensing.

The preservation of enzymatic activity is a fundamental requirement for exploiting hybrid nano-bio-conjugates, and the control over protein-nanoparticle interactions, leading to stable and catalytically active hybrids, represents the key for designing new biosensing platforms. In this scenario, surface active maghemite nanoparticles (SAMNs) represent a new class of naked magnetic nanoparticles, displaying peculiar electrocatalytic features and the ability to selectively bind proteins. Recombinant aminoaldehyde dehydrogenase from tomato (SlAMADH1) was used as a model protein, and successfully immobilized by self-assembly on the surface of naked SAMNs, where its enzymatic activity resulted preserved for more than 6 months. The hybrid nanomaterial (SAMN@SlAMADH1) was characterized by UV-Vis spectroscopy, mass spectrometry, and TEM microscopy, and applied for the development of a biosensor for the determination of aminoaldehydes in alcoholic beverages. Measurements were carried out in a low volume electrochemical flow cell comprising a SAMN modified carbon paste electrode for the coulometric determination of the NADH produced during the enzymatic catalysis. The present findings, besides representing the first example of an electrochemical biosensor for aminoaldehydes in an alcoholic matrix, open the door to the use of immobilized enzymes on naked metal oxides nanomaterials for biosensing.

Stealth Iron Oxide Nanoparticles for Organotropic Drug Targeting.

The ability of peculiar iron oxide nanoparticles (IONPs) to evade the immune system was investigated in vivo. The nanomaterial was provided directly into the farming water of zebrafish ( Danio rerio) and the distribution of IONPs and the delivery of oxytetracycline (OTC) was studied evidencing the successful overcoming of the intestinal barrier and the specific and prolonged (28 days) organotropic delivery of OTC to the fish ovary. Noteworthy, no sign of adverse effects was observed. In fish blood, IONPs were able to specifically bind apolipoprotein A1 (Apo A1) and molecular modeling showed the structural analogy between the IONP@Apo A1 nanoconjugate and high-density lipoprotein (HDL). Thus, the preservation of the biological identity of the protein suggests a plausible explanation of the observed overcoming of the intestinal barrier, of the great biocompatibity of the nanomaterial, and of the prolonged drug delivery (benefiting of the lipoprotein transport route). The present study promises novel and unexpected stealth materials in nanomedicine.

Metabolites from Marine Microorganisms, Micro, and Macroalgae: Immense Scope for Pharmacology.

Marine organisms produce a large array of natural products with relevance in drug discovery. These compounds have biological activities such as antioxidant, antibacterial, antitumor, antivirus, anticoagulant, anti-inflammatory, antihypertensive, antidiabetic, and so forth. Consequently, several of the metabolites have made it to the advanced stages of clinical trials, and a few of them are commercially available. In this review, novel information on natural products isolated from marine microorganisms, microalgae, and macroalgae are presented. Given due research impetus, these marine metabolites might emerge as a new wave of promising drugs.

Endogenous and food-derived polyamines: determination by electrochemical sensing.

Polyamines (PAs) are involved in a variety of fundamental physio-pathologic processes. The concentration of these polycations in organs and tissues depends on their endogenous production and oxidation rates, and on their intake from foods. Besides being largely accepted as markers for the progress of several pathologies, PAs may exert themselves different effects on humans, ranging from being positive to be drastically detrimental depending on the organism conditions. Thus, if the determination of polyamines content in tissue samples is of great importance as they could be indicators of several diseases, their quantification in food is fundamental for modulating the diet to respond to a specific human health status. Thus, the determination of PA content in food is increasingly urgent. Standard analytical methods for polyamine quantification are mainly based on chromatography, where high-performance liquid chromatography and gas chromatography are the most often used, involving pre-column or post-column derivatization techniques. Driven by the growing need for rapid in situ analyses, electrochemical biosensors, comprising various combinations of different enzymes or nanomaterials for the selective bio-recognition and detection, are emerging as competitors of standard detection systems. The present review is aimed at providing an up-to-date overview on the recent progresses in the development of sensors and biosensors for the detection of polyamines in human tissues and food samples. Basic principles of different electrochemical (bio)sensor formats are reported and the applications in human tissues and in foods was evidenced.

Analysis of hard protein corona composition on selective iron oxide nanoparticles by MALDI-TOF mass spectrometry: identification and amplification of a hidden mastitis biomarker in milk proteome.

Surface active maghemite nanoparticles (SAMNs) are able to recognize and bind selected proteins in complex biological systems, forming a hard protein corona. Upon a 5-min incubation in bovine whey from mastitis-affected cows, a significant enrichment of a single peptide characterized by a molecular weight at 4338 Da originated from the proteolysis of aS1-casein was observed. Notably, among the large number of macromolecules in bovine milk, the detection of this specific peptide can hardly be accomplished by conventional analytical techniques. The selective formation of a stable binding between the peptide and SAMNs is due to the stability gained by adsorption-induced surface restructuration of the nanomaterial. We attributed the surface recognition properties of SAMNs to the chelation of iron(III) sites on their surface by sterically compatible carboxylic groups of the peptide. The specific peptide recognition by SAMNs allows its easy determination by MALDI-TOF mass spectrometry, and a threshold value of its normalized peak intensity was identified by a logistic regression approach and suggested for the rapid diagnosis of the pathology. Thus, the present report proposes the analysis of hard protein corona on nanomaterials as a perspective for developing fast analytical procedures for the diagnosis of mastitis in cows. Moreover, the huge simplification of proteome complexity by exploiting the selectivity derived by the peculiar SAMN surface topography, due to the iron(III) distribution pattern, could be of general interest, leading to competitive applications in food science and in biomedicine, allowing the rapid determination of hidden biomarkers by a cutting edge diagnostic strategy. Graphical abstract The topography of iron(III) sites on surface active maghemite nanoparticles (SAMNs) allows the recognition of sterically compatible carboxylic groups on proteins and peptides in complex biological matrixes. The analysis of hard protein corona on SAMNs led to the determination of a biomarker for cow mastitis in milk by MALDI-TOF mass spectrometry.

Versatile nano-platform for tailored immuno-magnetic carriers.

Custom immuno-magnetic devices are desirable tools for biomedical and biotechnological applications. Herein, surface active maghemite nanoparticles (SAMNs) are proposed as a versatile platform for developing tailored immuno-magnetic nano-carriers by simple wet reactions. Two examples for conjugating native and biotinylated antibodies were presented along with their successful applications in the recognition of specific foodborne pathogens. Nanoparticles were functionalized with rhodamine B isothiocyanate (RITC), leading to a fluorescent nano-conjugate, and used for binding anti-Campylobacter fetus antibodies (SAMN@RITC@Anti-Cf). The microorganism was selectively captured in the presence of two other Campylobacter species (C. jejuni and C. coli), as verified by PCR. Alternatively, SAMNs were modified with avidin, forming a biotin-specific magnetic nano-carrier and used for the immobilization of biotinylated anti-Listeria monocytogenes antibodies (SAMN@avidin@Anti-Lm). This immuno-magnetic carrier was integrated in piezoelectric quartz crystal microbalance (QCM) sensor for the detection of L. monocytogenes in milk, showing a detection limit of 3 bacterial cells. The present work presents a new category of customized immuno-magnetic nano-carriers as a competitive option for suiting specific applications. Graphical abstract ᅟ.

Study of diagnostic accuracy of Fagan's two-step nomogram in increasing the value of predictive tools for prostate cancer: application of specific spatial distribution of positive/negative bioptic cores to predict extracapsular extension.

BACKGROUND: Prostate cancer (PC) represents the second most frequent cancer in the male population worldwide. It is mandatory to have a very accurate staging to choice the best possible treatment. AIMS: To test the possibility of improving the performance of Partin's tables in predicting the pathological staging of PC by introducing bioptic parameters through an innovative statistic tool (Fagan's two-step nomogram). METHODS: We prospectivelly collected data of all 1048 consecutive patients undergoing saturation 24-core transrectal prostate biopsy. Then, in eligible 94 patients, we compared the prediction of presence/absence of extracapsular extension of neoplasm (EPE+/-), with pathological assessment of invasion through (pseudo)capsule in the prostatectomy specimens. Starting from the probability of EPE- (pre-test probability, calculated with formula "100%-risk of EPE+"), we used Fagan's nomogram to examine the diagnostic sensitivity (DSe) and specificity (DSp) of negative "lateral" bioptic cores. RESULTS: We specifically analyzed the status of "lateral" cores in each side (94 patients × 2 sides = 188 sides). "Lateral" cores were negative in 42.5% of sides (80/188) with a DSe and DSp of 91.7 and 45.4%, respectively. In these sides, the mean probability of EPE+ according to Partin's tables was 21.6%. With Fagan's nomogram, the post-test probability of EPE+ when all "lateral" cores were negative was 14.1%, with a substantial gain of 7.5%. DISCUSSION: The spatial distribution of bioptic positive cores allowed us to demonstrate the role Fagan's nomogram in increasing the accuracy of already existing, predictive tools for PC. CONCLUSIONS: This pioneering study may justify the use of the above nomogram in testing "local" predictive parameters in combination with pre-existing nomograms.

Effect of different home-cooking methods on textural and nutritional properties of sweet potato genotypes grown in temperate climate conditions.

BACKGROUND: The European Union (EU) market for sweet potato is small but is growing considerably and and has increased by 100% over the last 5 years. The cultivation of sweet potato in temperate climate conditions has not considered extensively and could be a new opportunity for the EU market. Healthy and qualitative traits of different sweet potato cultivars grown in temperate climate conditions were evaluated in accordance with four cooking methods. RESULTS: Traditional cultivars showed high hardness and adhesiveness values. The highest concentrations of sugars (especially maltose) and phenolic acids (caffeic and chlorogenic) were found in samples treated by boiling and steaming. High antioxidant activity was found in fried potatoes. Qualitative traits of sweet potatoes treated by microwaves did not report any significant variation compared to the control. CONCLUSION: Traditional and new sweet potato cultivars can be cultivated in temperate climate conditions and show interesting qualitative properties, especially as a result of the presence of antioxidant compounds. Concerning global quality, colored varieties expressed a better profile than traditional Italian ones and they are suitable for the European market, giving new opportunities for consumers and producers. © 2017 Society of Chemical Industry.

Enlightening mineral iron sensing in Pseudomonas fluorescens by surface active maghemite nanoparticles: Involvement of the OprF porin.

BACKGROUND: Mineral iron(III) recognition by bacteria is considered a matter of debate. The peculiar surface chemistry of novel naked magnetic nanoparticles, called SAMNs (surface active maghemite nanoparticles) characterized by solvent exposed Fe(3+) sites on their surface, was exploited for studying mineral iron sensing in Pseudomonas fluorescens. METHODS: SAMNs were applied for mimicking Fe(3+) ions in solution, acting as magnetically drivable probes to evaluate putative Fe(3+) recognition sites on the microorganism surface. Culture broths and nano-bio-conjugates were characterized by UV-Vis spectroscopy and mass spectrometry. RESULTS: The whole heritage of a membrane porin (OprF) of P. fluorescens Ps_22 cells was recognized and firmly bound by SAMNs. The binding of nanoparticles to OprF porin was correlated to a drastic inhibition of a siderophore (pyoverdine) biosynthesis and to the stimulation of the production and rate of formation of a secondary siderophore. The analysis of metabolic pathways, based on P. fluorescens Ps_22 genomic information, evidenced that this putative secondary siderophore does not belong to a selection of the most common siderophores. CONCLUSIONS: In the scenario of an adhesion mechanism, it is plausible to consider OprF as the biological component deputed to the mineral iron sensing in P. fluorescens Ps_22, as well as one key of siderophore regulation. GENERAL SIGNIFICANCE: The present work sheds light on mineral iron sensing in microorganisms. Peculiar colloidal naked iron oxide nanoparticles offer a useful approach for probing the adhesion of bacterial surface on mineral iron for the identification of the specific recognition site for this iron uptake regulation in microorganisms.

Covalently bound DNA on naked iron oxide nanoparticles: Intelligent colloidal nano-vector for cell transfection.

BACKGROUND: Conversely to common coated iron oxide nanoparticles, novel naked surface active maghemite nanoparticles (SAMNs) can covalently bind DNA. Plasmid (pDNA) harboring the coding gene for GFP was directly chemisorbed onto SAMNs, leading to a novel DNA nanovector (SAMN@pDNA). The spontaneous internalization of SAMN@pDNA into cells was compared with an extensively studied fluorescent SAMN derivative (SAMN@RITC). Moreover, the transfection efficiency of SAMN@pDNA was evaluated and explained by computational model. METHODS: SAMN@pDNA was prepared and characterized by spectroscopic and computational methods, and molecular dynamic simulation. The size and hydrodynamic properties of SAMN@pDNA and SAMN@RITC were studied by electron transmission microscopy, light scattering and zeta-potential. The two nanomaterials were tested by confocal scanning microscopy on equine peripheral blood-derived mesenchymal stem cells (ePB-MSCs) and GFP expression by SAMN@pDNA was determined. RESULTS: Nanomaterials characterized by similar hydrodynamic properties were successfully internalized and stored into mesenchymal stem cells. Transfection by SAMN@pDNA occurred and GFP expression was higher than lipofectamine procedure, even in the absence of an external magnetic field. A computational model clarified that transfection efficiency can be ascribed to DNA availability inside cells. CONCLUSIONS: Direct covalent binding of DNA on naked magnetic nanoparticles led to an extremely robust gene delivery tool. Hydrodynamic and chemical-physical properties of SAMN@pDNA were responsible of the successful uptake by cells and of the efficiency of GFP gene transfection. GENERAL SIGNIFICANCE: SAMNs are characterized by colloidal stability, excellent cell uptake, persistence in the host cells, low toxicity and are proposed as novel intelligent DNA nanovectors for efficient cell transfection.

Cooking processes increase bioactive compounds in organic and conventional green beans.

The influence of cooking methods on chlorophyl, carotenoids, polyamines, polyphenols contents and antioxidant capacity were analyzed in organic and conventional green beans. The initial raw material had a higher content of chlorophyl and total phenolics in conventional green beans, whereas organic cultive favored flavonoid content and antioxidant capacity. Polyamines and carotenoids were similar for the two crop systems. After the cooking process, carotenoids (ß-carotene, lutein and zeaxanthin) increased. Microwave heating favored the enhancement of some polar compounds, whereas pressure cooking favored carotenoids. When we used the estimation of the radical scavenging activity by electron spin resonance (ESR) spectroscopy, a reduction of the DPPH radical signal in the presence of green bean extracts was observed, regardless of the mode of cultivation. The highest reduction of the ESR signal ocurred for microwave cooking in organic and conventional green beans, indicating a higher availability of antioxidants with this type of heat treatment.

Colloidal Surface Active Maghemite Nanoparticles for Biologically Safe Cr(VI) Remediation: from Core-Shell Nanostructures to Pilot Plant Development.

The present study is aimed at the exploration of achievable improvements for Cr(VI) ex situ and in situ water remediation by using novel naked colloidal maghemite (γ-Fe2 O3 ) nanoparticles (surface active maghemite nanoparticles, SAMNs). The reliability of SAMNs for Cr(VI) binding and removal was demonstrated, and SAMN@Cr(VI) complex was characterized, as well as the covalent nature of the absorption was unequivocally proved. SAMNs were structurally and magnetically well conserved after Cr(VI) binding. Thus, in consideration of their affinity for Cr(VI) , SAMNs were exploited in a biological model system, mimicking a real in situ application. The assay evidenced a progressive reduction of revertant colonies of Salmonella typhimurium TA100 strain, as maghemite nanoparticles concentration increased, till the complete suppression of Cr(VI) mutagen effect. Finally, an automatic modular pilot system for continuous magnetic removal and recovery of Cr(VI) from water is proposed. SAMNs, thanks to their colloidal, binding, and catalytic properties, represent a promising tool as a reliable nanomaterial for water remediation by Cr(VI) .

Ternary Hybrid γ-Fe2 O3 /Cr(VI) /Amine Oxidase Nanostructure for Electrochemical Sensing: Application for Polyamine Detection in Tumor Tissue.

Dichromate binds to surface-active maghemite nanoparticles (SAMNs) to form a stable core-shell nanostructures (SAMN@Cr(VI) ). The hybrid was characterized by Mössbauer spectroscopy, high-angle annular dark-field imaging, electron energy-loss spectroscopy, and electrochemical techniques, which revealed a strong interaction of dichromate with the nanoparticle surface. Electrochemical characterization showed lower charge-transfer resistance, better electrochemical performance, and more reversible electrochemical behavior with respect to naked SAMNs. Moreover, SAMN@Cr(VI) is an excellent electrocatalyst for hydrogen peroxide reduction. Furthermore, an enzyme, namely, bovine serum amine oxidase (BSAO: EC 1.4.3.6), was immobilized on SAMN@Cr(VI) by self-assembly to give a ternary hybrid nanostructured catalyst for polyamine oxidation (SAMN@Cr(VI) -BSAO). SAMN@Cr(VI) -BSAO was applied for the development of a reagentless, fast, inexpensive, and interference-free polyamine biosensor, which was successfully exploited for the discrimination of tumorous tissue from healthy tissue in human crude liver extracts.

Electrocatalytic Nanostructured Ferric Tannates: Characterization and Application of a Polyphenol Nanosensor.

A novel core-shell hybrid nanomaterial composed of peculiar maghemite nanoparticles (surface-active maghemite nanoparticles (SAMNs)) as the core and tannic acid (TA) as the shell was developed by self-assembly of ferric tannates onto the surface of SAMNs by simple incubation in water. The hybrid nanomaterial (SAMN@TA) was characterized by using UV/Vis, FTIR, and Mössbauer spectroscopies, magnetization measurements, and X-ray powder diffraction, which provide evidence of a drastic reorganization of the iron oxide surface upon reaction with TA and the formation of an outer shell that consists of a cross-linked network of ferric tannates. According to a Langmuir isotherm analysis, SAMN@TA offers one of most stable iron complexes of TA reported in the literature to date. Moreover, SAMN@TA was characterized by using electrical impedance spectroscopy, voltammetry, and chronoamperometry. The nanostructured ferric tannate interface showed improved conductivity and selective electrocatalytic activity toward the oxidation of polyphenols. Finally, a carbon-paste electrode modified with SAMN@TA was used for the determination of polyphenols in blueberry extracts by square-wave voltammetry.