Development of electrochemical (bio)sensors and microanalytical systemsapplication to the wine industry /

Resumen

The growing wine market needs to guarantee quality standards and the consumer protection by monitoring several parameters along the winemaking processes. Application of electrochemical (bio)sensors in this field is promising due to their simplicity, fast response, low-cost and easy miniaturization for on-site detection. Besides, biosensors are a good alternative to expensive and tedious traditional methods applied in specialized laboratories. In this Thesis, the development of electrochemical (bio)sensors fabricated with microelectronic technology and its implementation in fast-prototyped flow systems for the monitoring of chemical parameters of interest in the winemaking industry, is reported. Ion-Sensitive-Field-Effect-Transistors (ISFETs) and Platinumt/Gold microelectrodes as amperometric bienzymatic biosensors, have been used. This manuscript is the result of five papers published in international journals and contains five chapters. In the introduction chapter, the theoretical principles and the state-of-the-art of the ISFETs and amperometric biosensors are described. The methods for enzyme immobilization on sensors and in particular those based on polypyrrole polymers are explained. Finally, the implementation of electrochemical sensors in electronic tongues and in flow analytical systems is reviewed. In the experimental chapter is described in detail the fabrication of all the (bio)sensors and microanalytical flow systems used and their characterization methodology, including the procedure for wine measurements. Regarding Results and discussion chapter, describe the specific goals achieved. Firstly, ISFETs were applied for the determination of free and total sulfur dioxide in wine samples. A simple microanalytical flow system based on the separation of the analyte from the sample with a gas diffusion membrane and its indirect detection with a pH-ISFET, was proposed. The validation of the system was carried out using wine samples and two standard methods, the Ripper and the Paul method. The good results obtained with this system encouraged us to develop a more compact flow system for the simultaneous determination of free sulfur dioxide and acetic acid. This small-sized microanalytical flow system integrated in only one module the gas-diffusion membrane, the ISFET and the reference electrode, accomplishing the requirements of measurement in a barrel. This system was also evaluated by comparing the results obtained for wine samples with those obtained with the official methods. On the other hand, the development of amperometric enzymatic biosensors for the determination of L-lactate and L-malate was carried out. They were fabricated by modifying the metal surface of the microelectrodes with polymeric membranes of polypyrrole synthetized electrochemically. These three-dimensional matrices allowed the entrapment of the biochemical reagents involved in the bienzymatic reaction. The amperometric biosensors showed an excellent working stability, retaining more than 90 % of its original sensitivity after 40 days. This allowed their application to monitor the malolactic fermentation of red wines, showing a good agreement with the standard colorimetric method. Once both amperometric biosensors were optimized, a new silicon-chip was designed and fabricated for simultaneous determination of both parameters. This chip was assembled in a small-size flow system for on-line analysis of L-lactate and L-malate. Finally, the microanalytical flow system was successfully evaluated with wine samples. The application of a portable electronic tongue based on microsensors for the analysis of Cava wine was also described. The system contained an array of microsensors formed by six ISFETs sensitive to pH, Na+, K+, Ca2+, Cl− and CO32-, one conductivity sensor, one redox potential sensor and two amperometric gold microelectrodes. This system, combined with chemometric tools, was applied to the analysis of 78 Cava wine samples, classifying them according to the ageing time (using a Linear Discriminant Analysis) and quantifying the total acidity, pH, volumetric alcoholic degree, potassium, conductivity, glycerol and methanol parameters (using Partial Least Squares regressions).