Elisabetta Martini

Determination of Lactoferrin and Immunoglobulin G in Animal Milks by New Immunosensors

Two different immunosensors, recently developed for the determination of antibacterial proteins (lactoferrin and immunoglobulin G) in buffalo milk and in other commercial animal milks samples, were used in the present study. The aim was to propose these immunosensor methods for routine control of important diet products, such as cow and goat milks, and in particular buffalo milk. To this end we employed two different kinds of immunosensors: one for the analysis of immunoglobulin G (IgG), the other was a new amperometric immunosensor for lactoferrin analysis. Lactoferrin and IgG immunosensors were also used for the determination of lactoferrin and immunoglobulin G in buffalo milk on different days of lactation.

Antioxidant capacity of the algae using a biosensor method

Three different methods, i.e. a biosensor method, a voltammetric method and a spectrophotometric method, have been used to evaluate the total antioxidant capacity of certain types of algae. In the final evaluation of the data also the variation in time of the antioxidant capacity of cultivated algae was considered and some experimental factors, such as the use of different solvent mixtures to extract the antioxidant substances contained in the algae, were discussed.

A new surface plasmon resonance immunosensor for triazine pesticide determination in bovine milk: a comparison with conventional amperometric and screen-printed immunodevices

A detailed comparison was made of the analytical features of a new Surface Plasmon Resonance (SPR) immunodevice for triazine pesticide determination with those of two other amperometric (conventional and screen-printed) immunosensors and the advantages and disadvantages of the SPR method were thoroughly investigated. For conventional amperometric and screen-printed devices, “competitive” assays were used; conversely, the SPR transduction technique allowed a “direct” measurement format to be used. As far as the main analytical data are concerned, the SPR method does not seem to offer substantial advantages. Nevertheless the measurement time is much shorter and the measurement itself much easier to perform. Lastly several applications and recovery tests were carried out on bovine milk samples, before and after spiking, to check for triazine pesticides in the samples, obtaining satisfactory results.

Agent orange herbicides, organophosphate and triazinic pesticides analysis in olive oil and industrial oil mill waste effluents using new organic phase immunosensors.

New immunosensors working in organic solvent mixtures (OPIEs) for the analysis of traces of different pesticides (triazinic, organophosphates and chlorurates) present in hydrophobic matrices such as olive oil were developed and tested. A Clark electrode was used as transducer and peroxidase enzyme as marker. The competitive process took place in a chloroform-hexane 50% (V/V) mixture, while the subsequent enzymatic final measurement was performed in decane and using tert-butylhydroperoxide as substrate of the enzymatic reaction. A linear response of between about 10nM and 5.0μM was usually obtained in the presence of olive oil. Recovery tests were carried out in commercial or artisanal extra virgin olive oil. Traces of pesticides were also checked in the oily matrix, in pomace and mill wastewaters from an industrial oil mill. Immunosensors show good selectivity and satisfactory precision and recovery tests performed in olive oil gave excellent results.

Immunoglobulin G Determination in Human Serum and Milk Using an Immunosensor of New Conception Fitted with an Enzyme Probe as Transducer

To completely overcome the problem of the presence of urea in the serum, which can be the cause (especially at low immunoglobulin G concentrations) of a small but non negligible interference in the enzyme reaction of the enzymatic marker, when the measurement was performed by a potentiometric immunosensor that we constructed and characterized in previous work, and which used urease as marker, we have now constructed an entirely different and highly innovative immunosensor. This new device uses the enzyme alkaline phosphatase as marker, sodium phenylphosphate as substrate but above all, a tyrosinase biosensor obtained by coupling a Clark type gas diffusion amperometric electrode and the tyrosinase enzyme, immobilized in a cellulose triacetate membrane, as transducer. After optimizing the ‘competitive’ measurement procedures, the new immunosensor was used to determine both HIgG and the anti-HIgG, with a limit of detection (LOD) of the order of 3×10-11 M. Clearly this highly innovative construction geometry makes the immunosensor extremely selective. This makes it possible to determine immunoglobulin G both in human serum and milk without the slightest interference by any urea present in these biological matrixes.

Determination of traces of several pesticides in sunflower oil using organic phase immuno electrodes (OPIEs).

Testing for traces of different pesticides (triazinic, organophosphates and chlorurates), present in hydrophobic matrices such as sunflower oil was checked using new immunosensors working in organic solvent mixtures (OPIEs). The competitive process took place in an n-hexane-chloroform 75% (V/V) mixture, while the subsequent final enzymatic measurement was performed in decane using tert-butylhydroperoxide as substrate of the enzymatic reaction. A Clark electrode was used as transducer and peroxidase enzyme as marker. A linear response of between about 10 nM and 4 μM was usually obtained in the presence of sunflower oil. Immunosensors show satisfactory selectivity and precision and recovery tests carried out on commercial sunflower oil samples gave excellent results. Lastly, theoretical confirmation of the possibility that immunosensors can act positively in organic solvent mixtures was discussed on the basis of Hill׳s coefficient values.

Immunochemical Potentiometric Method for HIgG and Anti‐HIgG Determination, Using a NH3 Probe and Immunoprecipitation as Preconcentration Procedure

Abstract With the aim of improving the LOD value of a previous method, able to determine HIgG using a classic immunosensor, a pretreatment involving immunoprecipitation of the urease enzyme marked immunocomplex was introduced. By new method we determined HIgG using anti‐HIgG conjugated with urease for immunocomplex formation, and then protein G‐agarose was added for the immunoprecipitation process, which was able to concentrate the immunocomplex (and thus also the HIgG) up to 10,000‐fold. In this way the marked immunocomplex was separated from the marked anti‐HIgG excess, subsequently detected after urea addition in solution, by a gas diffusion potentiometric electrode for NH3. The formation of the marked antigen–antibody complex was the first step of the assay, the immunoprecipitation of the complex; the second and third step consisted first in removal of the nonspecific binding protein and then in eluting the precipitated immunocomplex from the microcolumn. The last step was the enzymatic measurement as checked by NH3 sensor. In addition, using the same procedure, namely, using the same conjugated urease and the same sensor for the NH3, it was possible to determine also the anti‐HIgG, in any case preceding the process of preconcentration by immunoprecipitation by means of a classical competitive process in homogeneous solution.

Investigation of Interfering Species in Phytodrug Analysis Using an Inhibition Tyrosinase Enzyme Electrode Working Both in Water and in Organic Solvent

Abstract In recent years several inhibition biosensors have been proposed for the analysis of aqueous solutions of phytodrugs. We recently built an inhibition OPEE (organic phase enzyme electrode) based on the inhibition of tyrosinase for the analysis of triazine, carbamate, and organophosphate pesticides operating in water-saturated chloroform. It was possible to determine the concentration of these pesticides contained in lipophilic or aqueous samples by relating it to the inhibiting action measured directly in water, or in water-saturated chloroform, after using the same solvent to extract the pesticides themselves. In the present investigation, attention was focused above all on two points of particular interest: on the study of potential interferents, i.e., of other inhibitors of the tyrosinase enzyme consisting above all, when operating in aqueous solution, of different heavy metal ions or several carboxylic acids, such as cinnamic, sorbic, or benzoic acids, which can apparently interfere in inhibition analysis of pesticides in aqueous matrixes; in the second place, on a detailed comparison of the results of the analysis of triazine, organophosphate, and carbamate pesticides in the presence of the above-mentioned interferents operating both in aqueous solution and in water-saturated chloroform.

New immunosensors for 2,4-D and 2,4,5-T pesticides determination

New immunosensors for 2-(2, 4-dichlorophenoxy) acetic acid (i.e. 2,4-D) and (2, 4, 5-trichlorophenoxy) acetic acid (i.e. 2,4,5-T) pesticide determination were developed using a non commercial antibody, hydrogen peroxide transducer and horseradish peroxidase as enzyme marker. The results show the full validity of these immunosensor devices, which were optimized using a ‘competition’ separation procedure. These immunosensors were also used to test pesticide recovery in common real matrices such as field grass, maize and wheat samples, for which good results were obtained. The immunosensors developed demonstrated a good selectivity versus different kinds of pesticides and may thus be considered as suitable devices for application to real matrices (LOD = 8.0 × 10−11 mol L−1; RSD% = 5.2 for 2,4-D and LOD = 2.8 × 10−9 mol L−1; RSD% = 6.1 for 2,4,5-T).

Determination of polyphenol ‘pool’ in olive oil mill waste water using a tyrosinase biosensor operating in aqueous solution or in organic solvent

Biosensors are very versatile devices that can be used to solve various kinds of problem that are increasingly found in the various branches of chemistry, particularly in the fields of foodstuffs and the environment. In recent years, there has been considerable development in a new biosensor sector, that of OPEEs (organic phase enzyme electrodes). These are biosensors able to function also in organic solvents or in mixtures of several different organic solvents. One of these enzymatic biosensors that has recently proved to be particularly versatile is the tyrosinase biosensor, of which several different versions have been developed. Our group in particular has in recent years developed both a version that operates in aqueous solutions and one suitable for organic solvents. These tyrosinase biosensors are essentially made up of an amperometric transducer for oxygen (Clark type), coupled with the tyrosinase enzyme, which is suitably immobilized according to the solvent in which it must operate. In this article the possibility was assessed of using them to determine the polyphenol ‘pool’ in olive oil mill wastewater. The method has been optimized as regards both the solvent and the type of enzymatic immobilization to be used. Results have been compared with those obtained using the Folin–Ciocalteau method, which is chosen as reference method.