Alessandro Poscia

Direct electrochemistry of membrane-entrapped horseradish peroxidase.: Part II: Amperometric detection of hydrogen peroxide

Abstract Direct (unmediated) electrochemistry of horseradish peroxidase (HRP) immobilized within a polymeric film is investigated at a pyrolytic graphite electrode by dc cyclic voltammetry, in the absence and in the presence of hydrogen peroxide. Under the latter condition, a reduction wave centered at approx. −280 mV (vs. SCE) is observed, the intensity of which is strictly dependent on the hydrogen peroxide concentration. This permits a voltammetric investigation of the electrocatalytic reduction of hydrogen peroxide. Flow and flow–injection measurements carried out at constant potential under the same conditions, support voltammetric data. The suitability of the immobilized HRP-based electrodic system to monitor the presence of important analytes, such as glucose or choline, in solution, is also discussed. To this issue, suitable amounts of HRP and glucose oxidase (or, in turn, choline oxidase) were simultaneously entrapped in the polymer. The results obtained are of potential value for basic and applied biochemistry and represent a first step for construction of a mediator-free (third-generation) biosensor which may find application in the biosensoristic area.

An electrochemical multienzymatic biosensor for determination of cholesterol.

This paper describes an electrochemical biosensor for free cholesterol monitoring. The sensor is a multienzymatic electrodic system in which horseradish peroxidase and cholesterol oxidase are simultaneously immobilized within a polymeric film, on the surface of a pyrolitic graphite electrode. From voltammetric and amperometric (flow-injection) data obtained, the efficiency, reproducibility and stability of the system are discussed. Results obtained, of interest for basic and applied biochemistry, represent a first step for construction of a mediator-free biosensor with potentialities for a successful application in the biosensor area.

Direct electrochemical evidence for an equilibrium intermediate in the guanidine-induced unfolding of cytochrome c.

This paper reports a voltammetric and spectroscopic investigation of the guanidine-induced unfolding of cytochrome c at neutral pH and 25 degrees C. Electrochemical data provide direct evidence for the presence of an equilibrium intermediate (form I) strictly dependent on the denaturant concentration. The midpoint potential of form I has been determined (E1/2 = +0.010 V vs. NHE) and its structural features defined from analysis of the circular dichroism and absorbance spectroscopy data obtained under the same experimental conditions. From the correlation of electrochemical and spectroscopic data, we propose that the features detected by the intermediate conform to the molten globule state.

Influence of glycerol on the structure and redox properties of horse heart cytochrome c. A circular dichroism and electrochemical study

The effect of glycerol on the structure and redox properties of horse heart cytochrome c was investigated by absorption spectroscopy, circular dichroism, and dc cyclic voltammetry techniques. The results show that the organic solvent increases the α-helix structure of the protein and induces slight changes at the active-site environment; however, the overall tertiary structure does not appear to be significantly perturbed. Glycerol stabilizes cytochrome c, the free energy of denaturation (ΔG0) being approximately 0.7 kcal/mol larger than that determined in phosphate buffer under the same conditions, and influences the heterogeneous electron transfer kinetics at a chemically modified gold electrode; on the other hand, the redox potential of the protein is unaltered. On the whole, the results obtained indicate that glycerol acts as a suitable stabilizing agent of cytochrome c, which is of interest for application in biotechnology; the organic solvent does not alter the tertiary structure significantly or the redox properties of the protein. This has to be interpreted not only in terms of the glycerol-induced solvent ordering around the protein surface, but also as due to the specific features of the protein matrix.

Nocturnal hypoglycaemia in type 1 diabetes - consequences and assessment

Hypoglycaemia is inevitable when striving for low HbA1c values. Nocturnal hypoglycaemia often occurs without symptoms, but results in diminished next day well‐being and hypoglycaemia unawareness. Frequency of nocturnal hypoglycaemia was first assessed in research ward settings, but suffered from insufficient glucose sampling frequency. This may have resulted in overestimation of the duration of hypoglycaemic episodes. The advent of the first continuous glucose sensor, the needle‐type MedtronicMiniMed Continuous Glucose Measurement System, revolutionized the assessment of glucose values. However, on scrutiny, the first version of this sensor showed a drift into the hypoglycaemic area and delayed recovery from hypoglycaemia. Using the microdialysis‐based GlucoDay system, our group reported a lower frequency of nocturnal hypoglycaemia in type 1 diabetes patients using an insulin pump, than that expected from the existing literature. Today, more than 80 years after the introduction of insulin for the treatment of type 1 diabetes, the associated frequency of nocturnal hypoglycaemia still awaits its definitive assessment. Copyright