Monti, Patrizia (2016) Sviluppo e studio di biosensori sostenibili e performanti dedicati alla determinazione di composti nutraceutici e contaminanti di interesse agroalimentare. Doctoral Thesis.
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The ability of a biosensor to respond to an analyte lies in the nature of the biological component. Based on the evidence that aflatoxin is able to inhibit the enzymatic activity of the acetyl cholinesterase enzyme (ACE) (Arduini et al., 2007), an amperometric biosensor based on enzymes ACE and choline oxidase has been developed. The biosensor was tested with different mixtures of mycotoxins (aflatoxin, patulin, type B trichothecene and ochratoxin) and reported the presence of aflatoxins and patulin in a biological matrix (Chapter 1), showing different sensitivity to the two mycotoxins.
In parallel, new chemical components from sustainable sources were synthesized and evaluated in order to improve the performance of the biosensor. To solve the problem of the interfering signals present in the matrix, such as ascorbic acid (AA) and dopamine (DA), the transducer is commonly covered with polymeric films derived from orthofenilendiammine (PPD) (Killoran and O'Neill, 2008), a monomer toxic and mutagenic (Murata et al., 2006).
In a first step, the monomers chosen as candidates for obtaining new permselctive films were represented by non-toxic phenols, naturally occurring in nature, such as eugenol, isoeugenol, magnolol and dehydrodieugenol. Among these, for practical reason, dehydrodieugenol was obtained through chemical synthesis starting from eugenol. Molecules were elettropolymerized using two techniques: constant potential amperometry and cyclic voltammetry. The polymers were characterized through permselectivity studies and Scanning Electron Microscopy (SEM); the studies were repeated over time to assess the stability of obtained polymers. High electropolymerization potentials were used, in order to overoxidate polymers and enhance charge repulsion characteristics of the polymer. Through the calculation of the permselectivity parameter (S%), permselective properties of the polymer films were quantified. The poly-magnolol proved the best alternative for PPD and it was included in the construction of a biosensor based on the enzyme glucose oxidase (Chapter 2).
The next step’s study of alternative polymers from natural monomers comprised the addition of a further molecule, guaiacol. This compound presents a simpler structure compared to the other molecules studied, being constituted of one guaiacyl unit. Taking into account the significant changes in permselectivity imparted by the polymerization conditions applied, in this part of the work the different phenols were polymerized by constant potential amperometry exploring a discrete range of electropolymerization potentials. Each phenol was subject to four different potentials and the characteristics of the resulting film were observed compared to H2O2, ascorbic acid and dopamine, and accompanied by images obtained by scanning electron microscopy (SEM). The experiments showed that the permeability characteristics are strongly related to the potential applied during polymerization; also permselectivity values were improved compared to previously reported work (Chapter 3).
Several molecules, that can alter the characteristics of βCD, have been selected in order to strengthen and widen βCD use in biosensors. The introduction of a bridge, created by a covalent bond between a linker molecule and two βCD, can enhance the ability of inclusive power of macrocycle; also the nature of the linker may make new functional groups and / or charges that help the interaction with the enzyme. A good attachment site is represented by the primary OH present on C6 of βCD, where, under certain conditions, can take place an esterification reaction between linker and βCD. As linker molecules have been used the polycarboxylic acid (PCA), the glyceroldiglycidilether (GDGE) and dimethyl carbonate (DMC). The products of the reactions were examined by means of nuclear magnetic resonance (NMR). Under the experimental conditions used, the reaction between the anhydride of 1,2,3,4-butanetetracarboxylic acid (BTCAa) and the βCD shows the formation of a mixture of different complexes. The complex was tested with carvacrol, a molecule that easily forms stable complexes with the βCD. The strong interaction BTCA-βCD has prevented the formation of the βCD-carvacrol complex and esterification between BTCAa and βCD (Chapter 4).
To include the βCD during electropolymerization various procedures were developed to obtain complexes between βCD and phenols. The βCD-eugenol complex, obtained in 85% of yield, was characterized by NMR spectroscopy; the analysis in D2O show a modification of the signal of the H5 βCD and the presence of well resolved eugenol signals in solution. Also the formation of βCD-guaiacol complex was confirmed by NMR with a yield of 47%. Repeated NMR analysis have confirmed a long-term stability for at least 2 weeks. Different conditions of complexation, all sustainable, including the technology to microwaves, have not been successful in obtaining a βCD- magnolol inclusion complex (Chapter 4).
The properties of a sulfur-containing phenol (S-PhOH) derivative product from Chemiplastica Specialties SpA were evaluated. The formation of strong complexes between S-PhOH and the βCD was studied by NMR spectroscopy under different experimental conditions, such as microwave, coprecipitation, sonication and solvent evaporation under vacuum (Rotavapor). These complexes are highly soluble and stable in water. The electro-deposition of S-PhOH is able to modify a platinum electrode imparting interesting characteristics towards ascorbic acid presence (Chapter 5).
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