Development of Biosensor for Heavy Metal Detection

Abstract

In the present study bacteria were isolated from soil on the basis of urease and alkaline phosphatase activities. Enzyme activities of the bacterial isolates were monitored in free and immobilized state and effect of three metal ions Cu, Zn and Cd was observed to determine the threshold concentrations of the metal ions. Two transducer systems potentiometric and amperometric were used to assemble the whole cell and electrode system to monitor the enzyme reactions and inhibition by metal ions electrochemically in order to develop a biosensor for heavy metal detection.Two urease producing strains were isolated from soil and identified as Bacillus sp. MD028 (FJ005050) and Bacillus subtilis strain MD008 (EU780733) using 16S rDNA sequencing. Bacillus sp. MD028 (FJ005050) showed good urease activity(12 U/mg (Dry cell weight)) as compared to Bacillus subtilis strain MD008(EU780733) [9 U/mg (Dry cell weight)].The effect of three heavy metals viz., Cu, Cd and Zn on the alkaline phosphatase activity of free and immobilized P. striata cells showed an inhibition trend ofZn>Cd>Cu. The inhibitory response of heavy metals was linear in the concentration range of 0.01-1 mgl-1 for Zn, 0.1-4 mgl-1 for Cd and 1-30 mgl-1 for Cu.This work presented a novel and efficient strategy for immobilization of lyophilizedcell mass of P. striata in poly vinyl chloride matrix which upon immobilization retained more than 85 % of the alkaline phosphatase activity which indicated thatthe protein structure was not damaged after immobilization. An equally efficient and novel strategy of immobilization was used for the immobilization of lyophilized biomass of Bacillus sp. MD028 (FJ005050) by covalently attaching the bacteria cells on the conducting polymer matrix of PPy-PVS/ITO electrodes. The Bacillus sp. MD028 cells retained approximately 98 % of the urease activity after immobilization. Both these immobilization techniques are novel and are being reported for the first time for the immobilization of bacterial cells. The potentiometric bioelectrode fabricated by immobilizing Pseudomonas striatacells in PVC was found to be fairly selective and sensitive for the Zn ions. The present work is the first report on the use of bacterial cell as metal ligands in a sensing device. The potentiometric response generated by the zinc selectiveelectrode can be attributed to the selective binding of the zinc ions to the zinc ligating sites present in the bacterial biomass immobilized in the PVC membrane. This zinc bioelectrode exhibited a linearity range of 10-1 to 10-5 M for zinc with near nernstian slope of 26.2 mV per decade. The membrane electrode showed a sharp response time of 6-10 sec and detection limit of 5×10-5 M at 25±1ºC in the pH optima of 3-5. The interference was found to arise only from few transition metals such as Hg+2, Ag+ and Pb+2. For alkali and alkaline earth metal ions, the pot Ag B K , log  values are quite small. Also the membrane electrode did not show any serious interference from Cu2+ ions.