Enzyme catalyzed reaction detection systems will be developed for incorporation into analytical flow systems, in order to achieve enhancement in both selectivity and sensitivity in the detection methods.
Biosensors have been developed which are based on carbon paste electrodes modified with tyrosinase. One of the major problems with the electrodes was initially that a stable analytical signal was not obtained. When working with the same enzyme preparation but immobilised on solid graphite electrodes, this stability problem was not encountered. A major investigation was therefore performed to improve both the stability and activity of the enzyme in the carbon paste environment.
The addition of a non ionic detergent, Tween 20, to the eluent buffer in the flow system resulted in a substantial increase of the stability but also on the activity of tyrosinase in the carbon paste environment. The immobilisation of the catalytic inactive protein, bovine serum albumin (BSA) before the immobilisation of tyrosinase was performed as a target to counteract any kind of chemical or electrostatic interactions with the electrode material which could be responsible of the poor stability of the tyrosinase carbon paste electrodes BSA was found to have a slightly positive effect on the stability, but more so on the activity of tyrosinase in the carbon paste. It was found that the admixing of a positively charged or neutral monomer, oligomer or polymer to the enzyme modified carbon paste had a positive influence on the observed kinetics and stability of tyrosinase. Experiments performed with chronoamperometry indicate that these additives enhances the available catalytic surface which might be one reason for the improved performance of the biosensors.
Biosensors based on carbon paste electrodes modified with laccase from Trichoderma are also being developed. In addition, a polymer based composite electrode configuration is under study. This type of biosensor yielded somewhat lower catalytic currents but resulted in much more stable enzyme electrodes compared to the carbon paste electrodes.
Liquid chromatography-mass spectrometry (LC-MS) has been used for the characterization of phenolic compounds. The coupling of the 3 types of biosensors to online sample clean up and chromatographic separation has been performed.
A biosensor based on commercially available phenol oxidases and solid graphite electrodes has been developed for the analysis of phenol and related hazardous compounds. The detection system couples enzymatic oxidation of phenolic of compounds and electrochemical reduction of enzyme products. Selectivity of the amperometric biosensor relies on catalytic molecular recognition and low working potentials minimizing the risk of interferences from electroactive compounds in the sample. The inherent sensitivity to electrochemical transduction is enhanced by a recycling catalytic and electrochemical system. The analytical performance of the biosensor in a flow injection system was determined. Low detection limits and high sensitivity for phenol have been achieved.
The study of this biosensor demonstrated the viability for the selective recognition of phenolic compounds. Hydroxylation of monophenols appeared to be the limiting step of this detection system. Study of new electrode materials and their pretreatment, different immobilization procedures, as well as alternative catalytic reactions are in progress with the aim of an extended selectivity and a better implementation of sample handling techniques.
Biosensor technology has been combined with sample pretreatment techniques to increase sensitivity and allow automation of the screening of phenolic compounds. The online sample pretreatment systems combine solid phase extraction techniques with biosensor detection and the systems are centred around the ASPEC sample handling unit (Gilson). The interface technology allows the use of optimum biosensor and sample enrichment conditions. While using a very hydrophobic support for solid phase extraction, organic solvents used to desorb the phenols are excluded from the biosensor detection system. The system can work in a parallel mode. During the time a sample is analyzed by the biosensor, the next sample can already be processed with the sample pretreatment unit. By using the entire sample rack space, 70 samples can be screened automatically and the maximum sample volume which can be processed within a reasonable period of time is 20 ml.
Sample handling biosensor detection systems are being developed in which high sample volumes (20-1000 ml) can be processed. The higher enrichment factors governed by these systems will provide a higher sensitivity of the screening unit.
The following studies have been carried out:
Comparison of online solid phase disk extraction to liquid liquid extraction for the determination of phenolic compounds from environmental water samples:
Empore extraction disks of 2 different types, carbon-18 and styrene divinylbenzene were placed in a disk holder and were coupled on-line with liquid chromatography (LC) and ultraviolet (UV) and/or fluorescence detection. The results obtained with such a system were compared with dichloromethane liquid liquid extraction (LLE) procedures. The main advantages of the online system were the use of less water, 150 ml versus 1 litre in the LLE, better limits of detection (due to the use of the direct analysis of the whole extract instead of an aliquot) and more automation, with better coefficients of variation.
Use of liquid chromatography-mass spectrometry (LC-MS) with thermospray interface for the characterization of various phenolic compounds:
A variety of phenolic derivatives (ie vanilin, 4-hydroxybenzoic acid, 7-hydroxycoumarin, hydroxybenzaldehydes, m-coumaric acid, 2,5-dimethoxy phenols) were characterized by using positive and negative ion mode thermospray LC-MS. Most of these compounds could be characterized by their molecular weight information either by the formation of adducts with ammonia or formate in the positive and negative ion modes, respectively. The information obtained was useful for the unequivocal determination of m-coumaric acid in fractionated waste water samples.
Development of offline and online solid phase extraction protocols and stability studies of 4 phenolics which can be used in the biosensor studies:
An offline solid phase extraction protocol involving Empore extraction disks of carbon-18 and styrene divinylbenzene material was developed for the isolation and trace enrichment of 10 phenolics from water samples at pH 2.5. It was found that when using styrene divinylbenzene material the recoveries were higher than 65% for all the phenolics except for phenol, which had a recovery of 51%. When using carbon-18 material the recoveries were much lower.
Two of the groups (Lund and Madrid) will develop biosensors through the study of immobilized enzyme systems according to fundamental studies based on electrochemically modified carbon paste electrodes, including enzymatic reaction mechanisms.
The research will focus on optimizing the factors affecting the enzymatic activity within the carboneous electrode materials used.
The developed biosensors will be used as detector unit in analytical flow systems. In combination with the flow system coupled pre-column systems will be developped for sample clean-up and preconcentration of the analytes present in the sample. Such a pre-column will eliminate matrix components thus making a simultaneous concentration of the sample solutes. This will be applied to the detection of phenolic compounds.
The dutch group, in combination with the group in Sweden will combine the enzyme base detection systems with the pre-column flow systems and will develop its incorporation into conventional liquid chromatographic systems. Such a systems will be used for the determination of phenolic compounds, including pesticide degradation products of phenolic structure. The environmental samples will be provided and applied in the laboratory of Barcelona. Coupling with mass spectrometric techniques will be used in order to avoid false positives of the water samples analyzed containing the phenolic metabolites.
As a final step, validation of the proposed method will be carried out by the different groups thus comparing the proposed analytical method development based on biosensor with the conventional methods of analysis of phenolics in water, including liquid-liquid extraction and liquid-solid extraction procedures.
Funding SchemeCSC - Cost-sharing contracts
221 00 Lund
2300 RA Leiden
28801 Alcalá De Henares - Madrid