A peculiar construct of lab on chip scaffold of bioluminescent bacteria based cellulosic nano-structured biosensor for luciferase based detection of organophosphates

Background: Organophosphorus compounds are compounds that contain phosphorus, which have been applied as war gases and pesticides since their synthesis (1937). The lungs, skin, gastrointestinal tract, and conjunctiva instantly absorb most organophosphorus compounds. These chemicals are metabolized by cytochrome P450 isozymes in the liver and generate metabolites that are more harmful than the parent compounds.

Objective: The purpose of the current research was to fabricate a micropad biosensor (using bioluminescent bacteria) for the detection of organophosphates, possessing attributes of lower cost, high sensitivity, and instant response for commercial applications, particularly in underdeveloped countries, where expensive disease diagnosis may lead to death for poor people.

Methods: Paper-based scaffold for biosensor was manufactured using discs of filter paper (Whatmann Chromatography No. 42) with a pore size and thickness of 2.5µm and 200µm, respectively. The biosensor was fabricated via bioluminescent bacterial cells (crude extract) and two different types of nanoparticles. The trichlorfon pesticide as a source of organophosphates, the bioluminescent bacteria for detection of organophosphates, and nanoparticles to enhance the binding of bacterial cells and organophosphates were used. The luminescence property of bacteria was confirmed via UV-transilluminator during growth curve analysis. Results were analyzed by using two types of carbohydrate-based nanoparticles: iron and silver nanoparticles (green synthesis). Firstly, 6.93×106 bioluminescent bacterial cells were fixed on a filter paper disc and then immobilized by coating them with nanoparticles. After that, the disc was treated with pesticide. Quenching effects of different concentrations of pesticide and nanoparticles were analyzed by using Image J software for analyzing the intensity of fluorescence.

Results: The result indicated minimum and maximum fluorescence values with a pesticide concentration of 0.66 M and 0.38 M, respectively, with iron nanoparticles. On the other hand, when the concentration of iron nanoparticles was varied, the minimum and maximum fluorescence were observed at 50µl and 10µl, respectively. By using silver nanoparticles minimum and maximum fluorescence were observed using pesticide concentrations of 5 mg/ml and 1mg/ml, respectively. Similarly, minimum and maximum fluorescence were observed by using silver nanoparticles with concentrations of 50µl and 10µl, respectively. The bioluminescent-based biosensor showed varying degrees of fluorescence by using different concentrations of both nanoparticles and pesticide. Our results depicted the high quenching effect of bioluminescent bacteria with increasing concentration of pesticide and nanoparticles.

Conclusion: After optimization of both pesticide and nanoparticles, our results depicted the minimum fluorescence using the maximum concentration of pesticide and nanoparticles. However, as compared to silver nanoparticles, better binding of bacterial cells was observed with iron nanoparticles.

To be updated shortly..