Laura Micheli

Development of a bio-electrochemical assay for AFB1 detection in olive oil.

A novel biosensor assay format for aflatoxin based on acetylcholinesterase (AChE) inhibition by aflatoxin B(1) (AFB(1)) is proposed. The AChE was present in solution and an amperometric choline oxidase biosensor was used for monitoring its residual activity. To create the biosensor, the choline oxidase was immobilized by cross-linking onto screen-printed electrodes modified with Prussian Blue (PB) and these were used to detect the H(2)O(2) at low potential (-0.05V versus a screen-printed internal silver pseudoreference electrode). For the development of the AFB(1) assay, several parameters such as AChE and substrate concentration, the methanol effect, and pH were evaluated and optimized. The linear working range was assessed to be 10-60ppb. Concentrations as low as 2ppb, which correspond to the legal limit of AFB(1) in food for humans, were detected after a pre-concentration step. The suitability of the method was evaluated using commercial olive oil samples. A recovery equal to 78+/-9% for 10ppb of AFB(1) in olive oil samples was obtained.

Development of an Electrochemical Immunosensor for Ochratoxin A

Abstract A direct, competitive electrochemical enzyme‐linked immunosorbent assay (ELISA) has been developed for the quantitative determination of ochratoxin A (OTA) using polyclonal antibodies. The assay is carried out on carbon‐based screen printed electrodes (SPE). Optimisation of the ELISA competitive conditions allowed us to realise an assay with improved analytical behaviour compared to the classical spectrophotometric ELISA based assay. The performance was comparable to a published monoclonal based assay. The assay gave a detection limit of 180 pg mL−1 and sensitivity of 6.1 ± 0.1 ng mL−1. The immunosensor was challenged with wine to assess a matrix effect. Recoveries obtained were in the 70–118% range. The method appears to be suitable for OTA contamination screening in food samples.

Antimicrobial and Biosensing Ultrasound-Responsive Lysozyme-Shelled Microbubbles

Air-filled lysozyme microbubbles (LSMBs) were engineered as a support for the immobilization of gold nanoparticles and an enzyme, alkaline phosphatase, in order to develop micro-antimicrobial and biosensing devices. Gold nanoparticles immobilized on LSMBs significantly improved the antimicrobial efficacy of the microbubbles against M. lysodeikticus. The surface functionalization of the microbubbles with gold nanoparticles did not affect their echogenicity when exposed to an ultrasound imaging probe. Alkaline phosphatase was conjugated on the surface of microbubbles without compromising its enzymatic activity. The functionalized microbubbles were used for the detection of paraoxon in aqueous solutions.

Rapid Screening Electrochemical Methods for Aflatoxin B1 and Type‐A Trichothecenes: A Preliminary Study

Abstract In this work are presented methods for detection of aflatoxin B1 and type‐A trichothecenes, based on the use of indirect competitive ELISA format coupled with a 96‐well screen‐printed microplate. Electrochemical immunoassays for AFB1, T‐2, and HT‐2 were performed and the activity of the alkaline phosphatase or horseradish peroxidase labeled enzymes were measured using intermittent pulse amperometry (IPA) as electrochemical technique. Using standard solutions of the target analyte the LOD of the assays were 0.3 and 0.2 ng ml−1 for T‐2 and AFB1 respectively, while the sensitivity was 1.2 ng ml−1 for both. For Aflatoxin B1, a stability study of electrochemical plate was also performed. Moreover, the matrix effect was evaluated using two different extraction treatments from corn.

Gellan hydrogel as a powerful tool in paper cleaning process: a detailed study.

HYPOTHESIS Wet cleaning of ancient papers is one of the most critical steps during a conservation treatment. It is used to improve the optical qualities of a graphic work and remove dust and by-products resulting from cellulose degradation. Nevertheless, washing treatment usually involves a substantial impact on the original morphological structure of paper and can sometimes be dangerous for water sensitive inks and pigments. EXPERIMENTS The use of rigid hydrogel of Gellan gum as an alternative paper cleaning treatment is developed. The application of a rigid hydrogel minimizes damages caused by the use of water, and therefore is much more respectful for the original integrity of ancient paper. FINDINGS Gellan hydrogel has been used to clean paper samples belonging to different centuries (from XVI to XIX) and therefore, characterized by a different story in terms of degradation condition and paper composition. Several techniques, such as high-performance liquid chromatography, Fourier transform infrared spectroscopy, scanning electron microscopy and pH measurements, has been employed to assess the effectiveness and safety of the proposed cleaning method.

Oxidative species and S-glutathionyl conjugates in the apoptosis induction by allyl thiosulfate

Natural allyl sulfur compounds show antiproliferative effects on tumor cells, but the biochemical mechanisms underlying the antitumorigenic properties of the organ sulfur compounds are not yet fully understood. Sodium 2‐propenyl‐thiosulfate is a garlic water‐soluble organo‐sulfane sulfur compound able to promote apoptosis in cancer cells, affecting the ‘managing’ of the redox state in the cell. Our studies show that sodium 2‐propenyl‐thiosulfate reacts spontaneously with reduced glutathione at physiological pH, leading to the formation of S‐allyl‐mercapto‐glutathione, radicals and peroxyl species, which are able to induce inhibition of enzymes with cysteine in the catalytic site, such as sulfurtransferases. S‐Allyl‐mercapto‐glutathione was purified and characterized by NMR and MS, and its cytotoxic effect at 500 μm on HuT 78 cells was analyzed, showing activation of the p38–MAPK pathway. Many allyl sulfur compounds are also able to promote chemoprevention by induction of xenobiotic‐metabolizing enzymes, inducing down‐activation or detoxification of the carcinogens. Thus, the effects of the S‐allyl‐mercapto‐glutathione on proteins involved in the cellular detoxification system, such as glutathione S‐transferase, have been evaluated both in vitro and in HuT 78 cells. Although the antitumor properties of water‐soluble sulfur compounds may arise from several mechanisms and it is likely that more cellular events occur simultaneously, a relevant role is played by the formation of both reduced glutathione conjugates and radical species that affect the activity of the thiol‐proteins involved in fundamental cellular processes.

Combining a hydrogel and an electrochemical biosensor to determine the extent of degradation of paper artworks

AbstractPaper-based artworks are among the most valuable assets for transmission of knowledge. Historical paper is composed of different polysaccharides (e.g. cellulose), binders, and glues. During aging all of these components undergo several degradation processes, as a result of external and intrinsic causes, and these can compromise the state of conservation of the document. In this work, application of a new biotechnological strategy for paper artefact preservation is reported. By making use of innovative and non-invasive materials, for example appropriate hydrogels, in combination with selective electrochemical biosensors, it is possible to simultaneously verify the degradation condition of the paper artwork and then to efficiently clean it, while monitoring the process of removal of both pollution and degradation products. In this paper, we focus on specific examples in which such techniques have been applied to paper artworks and that illustrate the advantages and potential of this biotechnology compared with the traditional paper-cleaning methods currently in use. FigureScheme of cleaning treatment of old paper and determination of the interested analyte using Flow Injection Analysis system (FIA) with integrated electrochemical biosensor

Cleaning of paper artworks: Development of an efficient gel-based material able to remove starch paste

The removal of old glue from paper artworks is of paramount importance for the preservation of its integrity during the restoration process. Wet cleaning is one of the traditional methods, although it may cause damages on artworks. In this work, an advantageous alternative method, based on the use of a rigid hydrogel, for a simple and localized removal of starch paste from paper supports is presented. The use of an appropriate hydrogel allows to overcome many of the problems faced by restorers minimizing damages, through a controlled release of water to the artwork, and a simple and not invasive application and removal. At the same time, the specific and targeted enzyme activity leads to a significant reduction in the application time of the cleaning procedure. In this context, experiments were carried out applying Gellan hydrogel carrying α-amylase enzyme on several paper samples soiled with starch paste. To assess the cleaning efficacy of the proposed hydrogel, we have used a multidisciplinary approach, by means of spectroscopic techniques, scanning electron microscopy, chromatographic analysis, and pH investigations.

Development of a competitive immunoassay for the determination of cortisol in human saliva.

Two new protein conjugates were prepared and studied to develop and compare two (direct and indirect) competitive enzyme-linked immunosorbent assay (ELISA) formats for the determination of cortisol in human saliva. Toward this goal, ovalbumin was conjugated to cortisol and used for developing an indirect competitive ELISA, while alkaline phosphatase was coupled with the same analyte for a direct competitive assay. The yield of the conjugation reactions was evaluated. The results obtained show that the indirect and direct ELISA formats developed for cortisol had working ranges of 0.5-70 and 2-330 ng/ml and detection limits of 0.5 and 1.2 ng/ml, respectively. Artificial and real saliva samples were spiked with cortisol to study the matrix effect of saliva. The suitability of the assays for quantification of cortisol in saliva was also studied.

Rheoreversible hydrogels in paper restoration processes: a versatile tool

BackgroundPaper based artworks are probably ones of the most difficult materials to restore, because of their complexity and fragile structure. Cleaning of paper artifacts, one of the process commonly carried out during restoration, usually involves the use of solvents (organic or not), that may cause several troubles, like swelling and dissolution of some components, and may also be harmful to the users.ResultsInnovative procedure for cleaning paper artworks is reported in this paper. It is based on the use of rheoreversible, biocompatible hydrogels containing poly(ethylene oxide) or poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) and α-cyclodextrin. We have studied two types of polymer with different hydrophobic properties in order to obtain two different hydrogels with slightly different cleaning capabilities. Our overall strategy has been to develop innovative systems based on these hydrogels so as to better confront the problems that a restorer faces during the cleaning of paper samples. Rheoreversible hydrogels are intriguing materials because their application and removal is not invasive and does not require a liquid treatment that could induce damage to the paper.ConclusionsThese hydrogels have been applied in the cleaning of both new and aged paper samples and their cleaning efficiency has been established. Moreover, by comparison with traditional methods, the greater efficacy of the proposed procedure has been demonstrated.To assess the cleaning efficacy of these hydrogels, a multidisciplinary approach, combining non-invasive spectroscopic infrared techniques together with scanning electron microscopy, chromatographic (HPLC) analysis and pH investigations has been used. Near infrared spectroscopy spectra were coupled with a chemometric analysis to achieve a better interpretation of data.This work constitutes a preliminary step towards focused study in the development of α-cyclodextrin/polymer hydrogel family which will allow cleaning of paper artifacts with peculiar characteristics.