Magnetic susceptibility as a 1-year predictor of outcome in familial cerebral cavernous malformations: a pilot study.

OBJECTIVES: To test whether quantitative susceptibility mapping (QSM) of cerebral cavernous malformations (CCMs) assessed at baseline may predict the presence or absence of haemorrhagic signs at 1-year follow-up. METHODS: Familial CCM patients were enrolled in the longitudinal multicentre study Treat-CCM. The 3-T MRI scan allowed performing a semi-automatic segmentation of CCMs and computing the maximum susceptibility in each segmented CCM (QSMmax) at baseline. CCMs were classified as haemorrhagic and non-haemorrhagic at baseline and then subclassified according to the 1-year (t1) evolution. Between-group differences were tested, and the diagnostic accuracy of QSMmax in predicting the presence or absence of haemorrhagic signs in CCMs was calculated with ROC analyses. RESULTS: Thirty-three patients were included in the analysis, and a total of 1126 CCMs were segmented. QSMmax was higher in haemorrhagic CCMs than in non-haemorrhagic CCMs (p < 0.001). In haemorrhagic CCMs at baseline, the accuracy of QSMmax in differentiating CCMs that were still haemorrhagic from CCMs that recovered from haemorrhage at t1 calculated as area under the curve (AUC) was 0.78 with sensitivity 62.69%, specificity 82.35%, positive predictive value (PPV) 93.3% and negative predictive value (NPV) 35.9% (QSMmax cut-off ≥ 1462.95 ppb). In non-haemorrhagic CCMs at baseline, AUC was 0.91 in differentiating CCMs that bled at t1 from stable CCMs with sensitivity 100%, specificity 81.9%, PPV 5.1%, and NPV 100% (QSMmax cut-off ≥ 776.29 ppb). CONCLUSIONS: The QSMmax in CCMs at baseline showed high accuracy in predicting the presence or absence of haemorrhagic signs at 1-year follow-up. Further effort is required to test the role of QSM in follow-up assessment and therapeutic trials in multicentre CCM studies. KEY POINTS: • QSM in semi-automatically segmented CCM was feasible. • The maximum magnetic susceptibility in a single CCM at baseline may predict the presence or absence of haemorrhagic signs at 1-year follow-up. • Multicentric studies are needed to enforce the role of QSM in predicting the CCMs' haemorrhagic evolution in patients affected by familial and sporadic forms.

Quantification of heterogeneity to classify benign parotid tumors: a feasibility study on most frequent histotypes.

Aim: To differentiate Warthin tumors (WTs) and pleomorphic adenomas (PAs) measuring heterogeneity of intravoxel incoherent motion (IVIM) and dynamic-contrast enhanced-magnetic resonance imaging biomarkers. Methods: Volumes of interest were traced on 18 WT and 18 PA in 25 patients. For each IVIM and dynamic-contrast enhanced biomarker, histogram parameters were calculated and then compared using the Wilcoxon-signed-rank test. Receiver operating characteristic curves and multivariate analysis were employed to identify the parameters and their pairs with the best accuracy. Results: Most of the biomarkers exhibited significant difference (p < 0.05) between PA and WT for histogram parameters. Time to peak median and skewness, and D* median and entropy showed the highest area under the curve. No meaningful improvement of accuracy was obtained using two features. Conclusion: IVIM and dynamic-contrast enhanced histogram descriptors may help in the classification of WT and PA.

Organically modified silica nanoparticles doped with new acridine-1,2-dioxetane analogues as thermochemiluminescence reagentless labels for ultrasensitive immunoassays.

Doped organically modified silica nanoparticles (ORMOSIL NPs) with luminescent molecules represent a potent approach to signal amplification in biomolecule labeling. Herein, we report the synthesis of new ORMOSIL NPs incorporating thermochemiluminescent (TCL) 1,2-dioxetane derivatives to prepare TCL labels for ultrasensitive immunoassay, displaying a detectability comparable to those offered by other conventional luminescence-based systems. Amino-functionalized ORMOSIL NPs were synthesized for inclusion of acridine-containing 1,2-dioxetane derivatives with a fluorescence energy acceptor. The doped ORMOSIL NPs were further functionalized with biotin for binding to streptavidin-labeled species to be used as universal detection reagents for immunoassays. A quantitative non-competitive immunoassay for streptavidin has been developed by immobilizing anti-streptavidin antibody to capture streptavidin, then the antibody-bound streptavidin was detected by the biotinylated TCL ORMOSIL NPs. The analytical performance was similar to that obtained by chemiluminescent (CL) detection using horseradish peroxidase (HRP) as label, being the limits of detection 2.5-3.8 and 0.8 ng mL(-1) for TCL and CL detection, respectively. In addition, since the TCL emission is simply initiated by thermolysis of the label, chemical reagents were not required, thus allowing reagentless detection with a simplification of the analytical protocols. A compact mini dark box device based on the use of a cooled charge-coupled device (CCD) and a miniaturized heater has been developed and used to quantify the light emission after heat decomposition of the label at a temperature of 90-120 °C. These characteristics make TCL-doped ORMOSIL NPs ideal universal nanoprobes for ultrasensitive bioassays such as immuno- and DNA-based assay.

Preparation and characterization of thermochemiluminescent acridine-containing 1,2-dioxetanes as promising ultrasensitive labels in bioanalysis.

Thermochemiluminescence is the luminescence process in which a thermodynamically unstable molecule decomposes with light emission when heated above a threshold temperature. We recently reported the thermochemiluminescence properties of an acridine-containing 1,2-dioxetane, which emits at relatively low temperatures (i.e., below 100 °C). Herein, we explored the effect of the introduction of methyl substituents in the acridine system. The methyl group did not determine an excessive destabilization of 1,2-dioxetane ring nor significantly affect the general physical properties of the molecule. Monosubstituted methyl derivatives and a series of derivatives bearing several combinations of two, three, and four methyl groups were prepared. The rate of formation of 1,2-dioxetane derivatives 1b-k strongly depended on the methyl substitution pattern. All members of this library of mono-, di-, tri-, and tetramethyl-substituted derivatives were characterized in terms of photophysical and thermochemiluminescence properties. The introduction of methyl groups into the acridine ring caused a marked decrease in the activation energy of the thermochemiluminescent reaction. Tri- and tetramethyl-substituted acridones had the highest fluorescence quantum yields, in the range 0.48-0.52, and the corresponding 1,2-dioxetanes 1h and 1j showed in thermochemiluminescence imaging experiments limit of detection values more than ten times lower with respect to the unsubstituted derivative.

Dioxetane-doped silica nanoparticles as ultrasensitive reagentless thermochemiluminescent labels for bioanalytics.

Thermochemiluminescence (TCL; the light emission originating by the thermally triggered decomposition of a molecule) was proposed in the late 1980s as a detection technique for immunoassays. However, after little pioneering work, this technique was abandoned because of the high temperatures required and the poor detectability in comparison to other labels. Here we describe for the first time a thermochemiluminescent acridine-based 1,2-dioxetane with a remarkably low (i.e., below 100 °C) emission-triggering temperature, which made it possible to obtain light emission even in an aqueous environment, as well as amino-functionalized silica nanoparticles loaded with this compound and the fluorescent energy acceptor dipyridamole. Thanks to the signal amplification due to the large number of 1,2-dioxetane molecules in each nanoparticle (about 10(4)) and the increased emission efficiency due to energy transfer to the fluorescent acceptor, the doped nanoparticles could be revealed with a detectability close to that of chemiluminescent enzyme labels (the limit of detection of doped nanoparticles by TCL imaging was 1 × 10(-16) mol mm(-2), thus approaching the value of 5 × 10(-17) mol mm(-2) obtained for the enzyme label horseradish peroxidase with chemiluminescence detection). They could thus be used as highly detectable labels in the development of sensitive TCL-based immunoassays and nucleic acid hybridization assays, in which the detection step does not require any additional chemical reagent. We believe that these doped silica nanoparticles could pave the way for the revival of TCL detection in bioanalytics, taking advantage of the reagentless detection and the high signal/noise ratio in comparison with conventional luminescence detection techniques.

Impact of Different Measures of Body Size on the Radiation Dose During Coronary Angiography and Percutaneous Coronary Intervention: Results from a Large Single Center Cohort.

Efforts to reduce and optimize the radiation exposure during coronary angiography and intervention have pointed at patients' body size as a major determinant of irradiation for the patients and operators. We aimed at comparing body weight and body mass index (BMI) among consecutive patients undergoing angiographic procedures (coronary angiography and/or interventions) in a single center. Patients were divided in normal weight (NW, BMI <25 Kg/m2) and overweight (OW, BMI ≥25 Kg/m2). Patients' dose exposure was evaluated as dose area product (DAP), time of exposure (fluoroscopy duration), and relative DAP (DAP/minutes of exposure). We included 748 patients, 61.6% undergoing percutaneous coronary interventions and 56.8% classified as OW. OW patients were more often men (P < .001), with history of hypertension (P < .001) and diabetes (P = .001). Mean DAP and relative DAP were significantly higher among OW compared with NW patients (P < .001). DAP and relative DAP were directly related with body weight (both r = .22, P < .001); a similar linear association was also described for BMI (r = .18, P < .001 and r = .19, P < .001, respectively). At multivariate analysis, however, body weight, but not BMI, independently predicted the DAP. Therefore, body weight should be considered as the preferred indicator of body size in the setting and optimization of radiation exposure during coronary diagnostic and intervention procedures.

Characterization and application of a diamine oxidase from Lathyrus sativus as component of an electrochemical biosensor for the determination of biogenic amines in wine and beer.

In this work, we have characterized a diamine oxidase (DAO) from Lathyrus sativus and evaluated its use, for the first time, as biocatalytic component of an electrochemical biosensor for the determination of biogenic amines index in wine and beer samples. Firstly, DAO was electrokinetically characterized free in solution by means of a platinum electrode and then immobilized by using polyazetidine prepolimer on the surface of screen-printed electrodes constituted of two gold working electrodes. The amperometric measurements were carried out by using a flow system at a fixed potential of +600 mV vs the internal silver pseudo reference in phosphate buffer solution (0.1 mol l(-1), pH = 7.4). The analysis of wine and beer samples were performed in flow injection system using the dual channel transducer providing simultaneous detection of sample and blank signal, and the resulting signal (after subtraction of the blank signal) was referred to that of putrescine. The results were compared with those obtained using a modified reference method based on gas chromatography-mass spectrometry analysis on the same samples. The results obtained in the analysis of Italian wines shows the better suitability of DAO-based biosensor in the determination of the biogenic amines (BAs) index expressed as putrescine equivalent in both red and white wines, being less efficient in beer samples where it underestimates by about 50% the BAs content.

The FAST-STEMI Network in Biella From 2013 to 2019: Impact of the Delocalization of the Hospital Facilities on Ischemia Time and In-hospital Outcomes.

BACKGROUND: The optimization of the strategies for myocardial revascularization has improved the outcomes of patients with ST-segment elevation myocardial infarction. In Piedmont, the FAST-STEMI regional network was created for improving the management and transportation of ST-segment elevation (STEMI) patients to primary percutaneous coronary intervention facilities, reducing the time to reperfusion. Within this network, the Hospital of Biella was delocalized in December 2014 to a new suburban structure designed for an easier access, which might have shortened the duration of patients' transportation and ischemia, with potential positive prognostic effects. The aim of the present study was to define the impact of the decentralization of the hospital structure on the time to reperfusion and in-hospital outcomes among STEMI patients admitted to the Hospital of Biella. METHODS: We included STEMI patients admitted to our urban hospital between 2013 and 2019 and included in the FAST-STEMI database. The primary endpoint was the duration of ischemia, defined as pain to balloon (PTB). The primary outcome endpoint (PE) was in-hospital mortality. RESULTS: We included 276 consecutive patients with STEMI undergoing primary percutaneous coronary intervention between 2016 and 2019 in the new hospital facility, which were compared with 170 patients treated between 2013 and June 2014 in the prior structure. Patients' characteristics included a mean age of 67.5 ± 12.5 years, 72.1% males and 18.7% patients with diabetes. In the new facility, the median PTB was 188 minutes [interquartile range: 125-340 min], reduced as compared with the period 2013-2014 [215 (128.5-352 min), P = 0.002]. The median in-hospital stay was also shorter (P = 0.004), whereas a nonsignificant improvement was noted for ejection fraction (EF) at discharge (P = 0.14). A linear relationship was demonstrated between PTB and the EF (r = -0.183, P = 0.003) in patients treated between 2016 and 2019 while not affecting the length of hospitalization or in-hospital outcomes. In fact, in-hospital death occurred in 36 patients, 8% in the new structure versus 7.7% in 2013-2014 [hazard ratio (HR) (95% confidence interval [CI]) = 1.20 (0.59-2.42), P = 0.62]. The independent predictors of mortality were patients' age and EF at discharge (age ≥ 75 y: adjusted HR [95% CI] = 6.75 [1.51-30.1], P = 0.01; EF: adjusted HR [95% CI] = 0.91 [0.88-0.95], P < 0.001). CONCLUSIONS: The present study shows that, among the STEMI patients treated in our center, the delocalization of the hospital facilities and the optimization of the FAST-STEMI network reduced the duration of ischemia, with positive effects on left ventricular function at discharge. However, this did not translate into a significant benefit in survival, which was instead conditioned by the aging of the population.

Electron-transfer properties of short-lived N-oxyl radicals. Kinetic study of the reactions of benzotriazole-N-oxyl radicals with ferrocenes. Comparison with the phthalimide-N-oxyl radical.

A kinetic study of the one-electron oxidation of a series of substituted ferrocenes (FcX: X = H, COCH3, CO2Et, CH2OH, Et, and Me2) by the benzotriazole-N-oxyl radical (BTNO) and of ferrocene (FcH) by a series of ring-substituted benzotriazole-N-oxyl radicals (Z-BTNO: Z = H, 6-CF(3), 6-Cl, 6-Me, 6-MeO) has been carried out in CH3CN. N-Oxyl radicals were produced by hydrogen abstraction from 1-hydroxybenzotriazoles (Z-HBT) by the cumyloxyl radical produced after 355 nm laser flash photolysis of a solution of dicumyl peroxide in CH3CN. In both systems, the rate constants exhibited a satisfactory fit with the Marcus equation allowing us to determine self-exchange reorganization energy values for the BTNO/BTNO- couple, which resulted in good agreement: 34.7 kcal mol(-1) from the oxidation of ferrocenes by BTNO and 30.5 kcal mol(-1) from the oxidation of ferrocene by aryl-substituted Z-BTNO. From the average value of 32.6 kcal mol(-1) it is possible to calculate a self-exchange rate for the BTNO/BTNO- couple of 7.6 x 10(5) M(-1 )s(-1), which is 3 orders of magnitude higher than that determined for the PINO/PINO- couple. The difference in the intrinsic barrier between the two oxidants is so large that it overcomes the thermodynamic factor and the oxidation of ferrocene by BTNO results significantly faster than that by PINO in spite of the higher reduction potential of the latter N-oxyl radical. The higher reactivity of BTNO with respect to PINO in an electron-transfer process contrasts with what is observed in hydrogen atom transfer processes where PINO is always more reactive than BTNO due to the higher NO-H bond dissociation energy in N-hydroxyphthalimide (HPI) than in HBT (88 vs. 85 kcal mol(-1), respectively). Thus, the relative reactivity of PINO and BTNO radicals might represent a criterium to help in the distinction of ET and HAT reactions promoted by these transient N-oxyl radicals.

Efficacy of a titanium dioxide nanoparticles - based indoor anti-odor product as assessed by electronic nose and gaschromatography-mass spectrometry.

Indoor air pollutants and odorants may have psychological and physical impact on exposed individuals and the unpleasant room air is considered as one of the factors associated with sick building syndrome comprising general symptoms such as headache and lethargy. Approaches for improving the quality of indoor air are thus important as support for human health and well-being. Photo-oxidation catalyzed by titanium dioxide (TiO2), is one of the methods used for elimination of volatile organic compounds, which are the cause of odor nuisance in indoor and outdoor air. In the present investigation, the efficacy of an experimental anti-odor air freshener based on TiO2 nanoparticles was estimated by testing its ability in removing from a small air chamber (200mL) the odor of triethylamine solutions (50µL at concentrations between 0.700 to 700mM), used as a model volatile molecule for simulating fish-like unpleasant indoor environment. The evaluation was performed by electronic nose which provided a holistic and objective data on the efficacy of the product, demonstrating that the effects of triethylamine even at the highest tested concentrations can be completely removed by application of 3.0g of the product at 25% TiO2 nanoparticles concentration. The obtained results were confirmed by gaschromatography-mass spectrometry (GC-MS) analysis addressed to the quantitative determination of residual triethylamine in the environment after treatment by the anti-odor product.

Progress in chemical luminescence-based biosensors: A critical review.

Biosensors are a very active research field. They have the potential to lead to low-cost, rapid, sensitive, reproducible, and miniaturized bioanalytical devices, which exploit the high binding avidity and selectivity of biospecific binding molecules together with highly sensitive detection principles. Of the optical biosensors, those based on chemical luminescence detection (including chemiluminescence, bioluminescence, electrogenerated chemiluminescence, and thermochemiluminescence) are particularly attractive, due to their high-to-signal ratio and the simplicity of the required measurement equipment. Several biosensors based on chemical luminescence have been described for quantitative, and in some cases multiplex, analysis of organic molecules (such as hormones, drugs, pollutants), proteins, and nucleic acids. These exploit a variety of miniaturized analytical formats, such as microfluidics, microarrays, paper-based analytical devices, and whole-cell biosensors. Nevertheless, despite the high analytical performances described in the literature, the field of chemical luminescence biosensors has yet to demonstrate commercial success. This review presents the main recent advances in the field and discusses the approaches, challenges, and open issues, with the aim of stimulating a broader interest in developing chemical luminescence biosensors and improving their commercial exploitation.

Electronic nose and chiral-capillary electrophoresis in evaluation of the quality changes in commercial green tea leaves during a long-term storage.

Electronic nose and capillary electrophoresis were applied in quality control of green tea samples subjected to long-term storage. Twelve representative green teas were considered, available as an "aged" (tea leaves stored during a long-term period of two years) and/or "not aged" (fresh products) samples. Their infusions were analyzed by an electronic nose, equipped with an array of six metal oxide semiconductor (MOS) sensors to obtain olfactive fingerprints of the volatile compounds in the infusions headspace. Upon training and chemometric analysis of acquired data (linear discriminant analysis), the electronic nose was found to be able in correctly classifying unknown samples as "aged" or "not aged". Concomitantly, the infusion samples were analyzed by Cyclodextrin-modified Micellar Electrokinetic Chromatography (CD-MEKC) for determination of catechins. The analysis of seven most represented catechins and the methylxanthines theobromine and caffeine revealed a general loss of the polyphenols in each of the considered aged samples (up to 45%, w/w). In addition, the applied enantioselective method based on (2-hydroxypropyl)-ß-cyclodextrin (HP-ßCD) as chiral selector, was exploited for the estimation of (+)-Gallocatechin in the presence of (-)-Gallocatechin; the latter, as the non-native enantiomer, can be associated to the epimerisation of (-)-Epigallocatechin and was assumed as a marker occurring in case of uncorrected storage conditions of tea leaves. Interestingly, it was observed that epimerization did not significantly occur during aging. The application of CD-MEKC and electronic nose allowed for a fast characterization of green teas taking into account that the aroma is a decisive parameter for the acceptance of the product, whereas the catechins content is associated to the biological value.

Several approaches for vitamin D determination by surface plasmon resonance and electrochemical affinity biosensors.

This work has been focused on the development of novel optical (Surface Plasmon Resonance) and electrochemical based biosensors for the determination of 25-OH vitamin D (25OHD) which is an important factor involved in avoiding both skeletal damage and a variety of pathological conditions, and to evaluate their potential use in clinical practice. Different approaches to the determination of vitamin D using affinity based biosensors, are described herein; firstly, an immunosensor based on SPR transduction was realized for direct determination of vitamin D, obtaining a LOD of 2 µg/ml which unfortunately is too far from the needs in clinical analysis. In order to enhance the sensitivity, the vitamin D was modified with gold nanoparticles (AuNPs): the binding of 25OHD with AuNPs determines the amplification of SPR signal, allowing to lower the LOD down to 1 µg/ml, doubling the sensitivity. An alternative SPR method, based on the indirect determination of vitamin D by means of Vitamin D Binding Protein (VDBP), led to a further sensitivity increase reaching a LOD of 45 ng/ml which is really close to the fixed accomplishment. Finally, an electrochemical transduced biosensor has been realized, based on the reaction of vitamin D with 4-ferrocenylmethyl-1,2,4-triazoline-3,5-dione (FMTAD): once derivatized, the determination of 25OHD was possible in the range 20-200 ng/ml with a LOD of 10 ng/ml. The latter proposed system fits the requirement of determining vitamin D in a concentration range which is of significance for clinical applications; moreover, since a screen printed electrode has been used, this opens the possibility to miniaturize the sensor and developing a portable and easy-to-automate point-of-care testing device. The proposed devices provide an improvement with respect to traditional methods that are time and reagents consuming and require radioactive compounds, pretreatment procedures and expensive instrumentation.

Polyazetidine-coated microelectrodes: electrochemical and diffusion characterization of different redox substrates.

The present paper reports on the diffusion characteristics and electron transfer properties of a membrane obtained from polyazetidine prepolymer (PAP) consisting of repeating units of 1-(aminomethyl)-1-{2-[(6-oxyhexane)amino]ethyl}-3-hydroxyazetidinium chloride studied in the presence of seven simple redox electroactive molecules: ABTS, catechol, dopamine, ferrocenecarboxylic acid, ferricyanide, ferrocyanide, and the osmium complex bis(2,2-bipyridyl)-4-aminomethylpyridine chloride hexafluorophosphate (Os[(bpy)(2) 4-AMP Cl](+)). Using water as medium, the apparent diffusion coefficients (D(app)), the concentrations of the compounds in the membrane, and the heterogeneous rate constants (k(s)) were calculated as a function of temperature, and the influence thereof on these parameters was evaluated. Even if D(app) and k(s) values in the presence of PAP are smaller than in solution, this decrease is small enough to indicate that the PAP membrane shows excellent diffusion and electron-exchange properties with respect to other commonly used membranes reported in the literature.

The structure of maize polyamine oxidase K300M mutant in complex with the natural substrates provides a snapshot of the catalytic mechanism of polyamine oxidation.

Polyamine oxidases are FAD-dependent enzymes catalyzing the oxidation of polyamines at the secondary amino groups. Zea mays PAO (ZmPAO) oxidizes the carbon on the endo-side of the N5-nitrogen of spermidine (Spd) and spermine (Spm). The structure of ZmPAO revealed that the active site is formed by a catalytic tunnel in which the N5 atom of FAD lies in close proximity to the K300 side chain, the only active-site residue conserved in all PAOs. A water molecule, (HOH309), is hydrogen-bound to the amino group of K300 and mutation of this residue results in a 1400-fold decrease in the rate of flavin reduction. The structural studies on the catalytically impaired ZmPAO-K300M mutant described here show that substrates are bound in an 'out-of-register' mode and the HOH309 water molecule is absent in the enzyme-substrate complexes. Moreover, K300 mutation brings about a 60 mV decrease in the FAD redox potential and a 30-fold decrease in the FAD reoxidation rate, within a virtually unaltered geometry of the catalytic pocket. Taken together, these results indicate that the HOH309-K300 couple plays a major role in multiple steps of ZmPAO catalytic mechanism, such as correct substrate binding geometry as well as FAD reduction and reoxidation kinetics.

Laccase-based biosensor for the determination of polyphenol index in wine.

In this work we have developed and characterized the use of Laccases from Trametes versicolor (TvL) and Trametes hirsuta (ThL) as biocatalytic components of electrochemical biosensors for the determination of polyphenol index in wines. Polyazetidine prepolimer (PAP) was used as immobilizing agent, multi-walled and single-walled carbon nanotubes screen-printed electrodes as sensors (MWCNTs-SPE and SWCNTs-SPE) and gallic acid as standard substrate. The amperometric measurements were carried out by using a flow system at a fixed potential of -100 mV vs. silver/silver chloride electrode in Britton-Robinson buffer 0.1 mol L(-1), pH 5. The results were compared with those obtained with the Folin-Ciocalteau reference method. The results obtained in the analysis of twelve Italian wines put in evidence the better suitability of ThL-MWCNTs-based biosensor in the determination of the polyphenol index in wines. This biosensor shows fast and reliable amperometric responses to gallic acid with a linear range 0.1-18.0 mg L(-1) (r(2)=0.999). The influence of the interferences on both spectrophotometric and electrochemical measurements have been carefully evaluated.

Polyazetidine-based immobilization of redox proteins for electron-transfer-based biosensors.

A highly stable functional composite film was prepared using polyazetidine prepolymer (PAP) with peroxidase from horseradish (HRP) and/or glucose oxidase (GOx). The good permeability of the PAP layer to classical electrochemical mediators, as evaluated by the determination of the diffusion coefficient of different redox molecules, is of great importance in view of the use of PAP as an immobilizing agent in second-generation biosensor development. Cyclic voltammetry of the HRP-PAP layer on a glassy carbon electrode (GCE) showed a pair of stable and quasi-reversible peaks for the HRP-Fe((III))/Fe((II)) redox couple at about -370 mV vs. Ag/AgCl electrode in pH 6.5 phosphate buffer. The electrochemical reaction of HRP entrapped in the PAP film exhibited a surface-controlled electrode process. This film and the successive modifications (HRP-PAP self-assembled monolayer (SAM) modified Au electrode) were used as a biological catalyst (hydrogen peroxide transducers) for glucose biosensors, after coupling to GOx. Both HRP/GOx-PAP and HRP/GOx-PAP SAM third generation biosensors were prepared and characterized. The use of PAP as immobilizing agent offers a biocompatible micro-environment for confining the enzyme and foreshadows the great potentiality of this immobilizing agent not only in theoretical studies on protein direct electron transfer but also from an applications point of view in the development of second- and third-generation biosensors.

A kinetic study of the electron-transfer reaction of the phthalimide-N-oxyl radical (PINO) with ferrocenes.

A kinetic study of the one-electron oxidation of a series of ferrocenes (FcX: X = H, CO2Et, CONH2, CH2CN, CH2OH, Et, and Me2) by PINO generated in CH3CN by reaction of N-hydroxyphthalimide (NHPI) with the cumyloxyl radical produced by 355 nm laser flash photolysis of dicumyl peroxide has been carried out. Ferrocenium cations were formed, and the reaction rate was determined by following the decay of PINO radical at 380 nm as a function of the FcX concentration. Rate constants were very sensitive to the oxidation potential of the substrates and exhibited a good fit with the Marcus equation, from which a lambda value of 38.3 kcal mol(-1) was calculated for the reorganization energy required in the PINO/ferrocenes electron-transfer process. Knowing the ferrocene/ferrocenium self-exchange reorganization energy it was possible to calculate a value of 49.1 kcal mol(-1) for the PINO/PINO- self-exchange reaction in CH3CN. Moreover, from the Marcus cross relation and the self-exchange rates of ferrocene and dimethylferrocene, the intrinsic reactivity of PINO in electron-transfer reactions has been calculated as 7.6 x 10(2) M(-1) s(-1). The implications of these values and the comparison with the electron-transfer self-exchange reorganization energies of peroxyl radicals are briefly discussed.