- the linearity, with values for the linearity coefficients of 0.9998 for the case of potassium nitrite calibration (10-100 mg/L) and 0.9952 (5-125 mg/L) for potassium nitrate calibration.
The literature studies and the preliminary investigations proved the feasibility of the submitted theme.
2nd Phase summary
Nitrates and nitrites are largely used as preservatives and fertilizing agents. However, a continuous exposition of these ions to humans can have serious health implications. Particularly, nitrites can react irreversibly with hemoglobin to produce methemoglobin, therefore reducing blood capacity to transport oxygen. The disease is known as "blue baby syndrome". Also, the nitrite can form nitrosoamines in the digestive tract, with cancer risk.
The European Community by the European Drinking Water Directive from 1998 has established the maximum admissible levels of nitrate and nitrite in drinking water at 50 and 0.1 mg/L, respectively. This level is now imposed also in Romania, by the law 458 from 8 July 2002.
The detection methods based on enzymes are preferred towards others due to selectivity, sensibility and an increased accuracy. Working with free enzymes for detection has big disadvantages, like a high cost of one-time using of enzyme and low stability, things that were resolved by immobilization of enzyme on insoluble support.
The researches done in the 2nd phase of the BIOENZINIT project by the coordinator, INCDCP-ICECHIM Bucharest had as main objective the obtaining of electroconductive polymers. For this, 2 methods for polypyrrole synthesis were elaborated: first one with FeCl3 initiation and the second one with aluminium persulfate as initiator. With these methods were obtained the quantities of polypyrrole needed for subsequent performing the acetalization and covalent immobilization researches, but didnít allow the deposition of polypyrrole on screen printed sensors, with carbon electrode.
The acetalization and peroxidase covalent immobilization studies had the purpose to indicate the working range and the reaction conditions and methods, both for polypyrrole powder and polypyrrole electrochemically deposited on the central coal sensor. The acetalyzed and enzyme containing polypyrrole samples were forwarded to project partners for a detailed characterization.
The electrochemical behavior of [Fe(CN)6]3- was analyzed, on different types of electrodes (vitreous carbon, gold, platinum, DropSens mini-electrodes and electrodes modified by electrodeposition of polypyrrole films). Using planar graphite DropSens electrodes, modified with peroxidase entrapped in polypyrrole films, the presence of interactions between immobilized enzyme and the electroactive species of paracetamol (N-acetyl-p-benzo-quinone imine - NAPQI ) was proved. The method for these measurements was chronoamperometry.
Cyclic voltametry studies were conducted in order to determine the influence of various parameters (electrolytic environment, electrode material, pH) on the electrochemical reduction of nitrates, respectively electrochemical oxidation of nitrites, and to establish the optimal working conditions.
As perspectives, it is intended the elaboration of miniaturized biosensors based on polymer immobilized enzymes for qualitative and quantitative determination of nitrates and nitrites from human consumption waters.
Analysis on phreatic waters from Giurgiu and Teleorman were achieved, in order to establish the content in nitrates and nitrites. The classical methods were used: HPLC and UV-VIS spectroscopy. The result showed that in some rustic regions the contamination with nitrites reaches dangerous high values, needing the monitoring of nitrates and nitrites in these areas.
The structural characterization of polymer composites with the SEM method showed morphological changes due to the acetalization process and the covalent immobilization of enzyme.
X ray detections on untreated polypyrrole samples, acetalyzed polypyrrole and polypyrrole with enzyme showed an amorphous structure of all products, and in the same time, structural changes are present, proved by the diffraction spectra.
The physical-chemical analysis on polymers and film nanocomposites containing enzymes, the FTIR spectroscopy and nitrogen content proved that the acetalization of polypyrrole with glutaraldehyde occurs and that on this modified polymer, the covalent immobilization of horseradish peroxidase takes place.
3rd Phase summary
The researches developed in the 3rd phase of the project were oriented in two directions:
- obtaining polypyrrole based biosensors by chemical polymerization of polypyrrole (oxidative), when the enzyme was covalently immobilized after functionalizing the polymer with glutaraldehide;
- obtaining biosensors by enzyme's entrapment into the polymer film on the electrode's surface, the film being obtained by electrochemical polymerization of polypyrrole.
1. The researches performed after first direction aimed the optimization of conditions for polypyrrole functionalizing by studying the influence of various parameters: glutaraldehide: polypyrrole ratio, catalyze concentration, solid: liquid ratio, temperature and reaction time. The studies continued with covalent immobilization of enzyme on polymer, by reacting the free aldehyde's groups with enzyme's NH or COOH groups.
FTIR spectra proved that by functionalizing the polypyrrole, the chemical structure is changing, because peaks characteristic for -OH groups appear. Moreover, specific peaks, around ~2850-2920 cm-1, associated to streching vibration of CH2 groups become visible, proving the new formed bonds with glutaraldehide. FTIR spectra did confirm that the covalent immobilization of Polyphenol oxidase (PPOx) on PPy occurs.
2. The second direction, obtaining biosensors by enzyme entrapment into the electroconductive polymer film, was oriented onto entrapment of nitrate reductase into the polypyrrole film. The researches were performed on various types of sensors: coal past, vitreous carbon and DropSens graphite electrode. Different methods for the polymer deposition and enzyme entrapment were tried, in the presence of various mediators.
The 3rd phase main objective was represented by the electrochemical behavior of electrodes modified with conductive polymer films, like polypyrrole, aiming the elaboration of some biosensors configurations for nitrates detection and quantification from various matrices.
The permeability of the prepared polymer films was tested in the presence of potassium ferrocyanide/ferricyanide system.
The electrochemical behavior of nitrates/nitrites during the oxidation/reduction processes was studied on different electrodes and pH values. Both processes, oxidation and reduction, were positively influenced by small pH values.
In order to obtain some biosensors configurations for nitrates detection and quantification, the influence of various parameters (electrolytic environment, electrode material, pH) was studied in order to establish the optimal experimental conditions.
In the same time, complex analyses on water samples taken from 3 plain villages in Teleorman County were effectuated. The influence of other compounds was taken into account (dissolved oxygen, chlorate pesticides). After one year study period, the conclusion was that the upper level of nitrates and nitrites in waters for human consumption is almost permanently exceeded, exception only for deep waters (>50 m). This situation justifies the researches aiming for a fast method to evaluate waters for human consumption.
The physical-chemical analysis by FTIR spectroscopy on polypyrrole, functionalized and non-functionalized, and film containing the enzyme, showed changes on adsorption band, proving the functionalizing and the covalent immobilization. Proofs regarding functionalizing of polypyrrole and enzime immobilizing were also obtained by Raman spectroscopy and DSC, and were completed with X ray and SEM results.