Screen-printed biosensor based on electro-polymerization of bio-composite for nitrate detection in aqueous media

Bacillus sp. possessing a periplasmic nitrate reductase was used as a recognition element to develop a nitrate biosensor. The bacteria was embedded within a polyaniline (PANI) electro-conductive matrix via electro-polymerization on miniaturized carbon screen-printed electrodes (SPE) at 100 mV/s and scan rate from -0.35 V to + 1.7 V. Surface medication of SPE was verified via Fourier Transform Infrared spectroscopy (FTIR) and Scanning Electron Microscopy (SEM). The optimal bacterial density was OD₆₀₀ 1.2. To enhance the biosensors performance, Bacillus sp. was (1) grown in riboflavin (RF) inducing media as an endogenous redox mediator and (2) exposed to different gamma radiation doses as a physical method to increase electron transfer. Results show a link between exposing cells to gamma irradiation stress, this was evident by electron spin resonance (ESR) and changes in FTIR spectrum, in addition to the increase in catalase enzyme. The nitrate limit of detection (LOD) was 0.5-25 mg/L for non-irradiated RF induced immobilized cells and LOD was 0.5-75 mg/L nitrate for 2 kGy gamma irradiated cells. The prepared biosensor showed acceptable reproducibility and multiple usages after storage at 4°C over 3 months. Low cost and simple preparation allow the biosensor to be mass-produced as a disposable device. Bacillus sp. and its endogenous redox mediator immobilized within polyaniline are good candidates for the improvement of amperometric biosensors for the quantification of nitrate in aqueous solutions.