Electrochemical Biosensor Arrays Utilising Bacteria and Aptamer Nano-bioreceptors for Toxic Chemicals Detection

This work was dedicated to development of novel biosensing technologies for detection of toxic chemicals, such as heavy metals, pesticides and petrochemicals, which possess a serious threat to humans and all living organisms in our planet nowadays. This was the main motivation for research in such important field. In the present work a novel approach in detection of heavy metal salts (HgCl2, PbCl2, ZnCl2 and CdCl2), pesticides (atrazine, simazine, DDVP), and petro-chemicals (hexane, octane, pentane, toluene, pyrene and ethanol) dissolved in water was proposed. It is based on a concept of inhibition sensor array utilising different whole bacteria cells. The main aim of this project is to develop novel, simple and cost-effective biosensing technologies for in-field detection of the above pollutants in water which effectively reduce the time and cost of analysis. Electrochemical detection appeared to be the most suitable for such task. In this project, three types of bacteria, e.g. Escherichia coli, Methylococcus capsulatus (Bath) or Methylosinus trichosporium (OB3b) and Shewanella oneidensis, were selected because of their different inhibition patterns. The concentration of live bacteria (which is an indicator of the presence of pollutants) was first characterised by the optical analytical methods of optical density OD600, fluorescence microscopy and flow cytometry. The main findings of this study were the facts that E. coli (K12 strain, gram-negative bacteria) are very sensitive to all above mentioned pollutants; methanotrophic bacteria (Mc. capsulatus Bath & Ms. trichosporium OB3b) appeared to be more resistant to petrochemicals; while S. oneidensis (MR-1 strain, gram negative bacteria) are more tolerant to heavy metals. A series of AC and DC electrochemical measurements were carried out on the same bacteria samples. As a first step, a correlation between optical and electrochemical characteristics of bacteria concentration in solution was established. The study of the effect of heavy metals, pesticides and petrochemicals on DC and electrical characteristics of bacteria in suspension revealed a similar inhibition pattern as was found in optical study. Then a similar study was carried out on samples of bacteria immobilized on the surface of screen-printed electrodes, which is more suitable for sensing applications. The results of DC (cyclic voltammograms) and AC (impedance spectroscopy) measurements were consistent with previous studies. A possibility of pattern recognition of pollutants by their inhibition effects on the selected bacteria was found. The classes of pollutants, e.g. heavy metals, pesticides, and petrochemicals, can be identified from pseudo-3D graphs of responses of the three sensing channels, e.g. electrodes with different immobilized bacteria. Much more accurate assessment of pollutants was achieved with Artificial Neural Network (ANN) software which was developed using MatLab. ANN programme was capable of both the identification of pollutants with 91% accuracy and rough estimation of their concentrations in five bands from 0.01 ng/ml to 1000 ng/ml (ppb). The developed bacteria sensor array could be suitable for simple, inexpensive, and quick preliminary in-field detection (screening) of water samples. The suspected highly contaminated samples could be easily identified and passed to specialized laboratories for further more detailed testing. In such way, the time and cost of analysis could be substantially reduced. In addition to the inhibition sensor array utilising non-specific bio-receptors such as bacteria, the electrochemical detection of heavy metal ions (Hg2+ and Pb2+) was attempted using novel highly specific aptamer bio-receptors labelled with redox groups. Such experiments were successful; the above metal ions in very low concentrations down to 1 pg/ml (or 1 ppt) were detected using both cyclic voltammograms and impedance spectroscopy. The affinity of the aptamers used was found to be very high and similar to that of antibodies. Additional advantages of aptamers were their high stability and simple recovery by thermo-cycling. Considering fast evolvement of aptamer research, their advantages and low cost, the development of aptasensor arrays for accurate detection of large number of pollutants is possible in near future.