Abstract
In whole-cell based biosensors, spectrophotometry is one of the most commonly used methods for detecting organophosphates due to its simplicity and reliability. The sensor performance is directly affected by the cell immobilization method because it determines the amount of cells, the mass transfer rate, and the stability. In this study, we demonstrated that our previously-reported microbe immobilization method, a microbe-attached single-walled carbon nanotube film, can be applied to whole-cell-based organophosphate sensors. This method has many advantages over other whole-cell organophosphate sensors, including high specific activity, quick cell immobilization, and excellent stability. A device with circular electrodes was fabricated for an enlarged cell-immobilization area. Escherichia coli expressing organophosphorus hydrolase in the periplasmic space and single-walled carbon nanotubes were attached to the device by our method. Paraoxon was hydrolyzed using this device, and detected by measuring the concentration of the enzymatic reaction product, p-nitrophenol. The specific activity of our device was calculated, and was shown to be over 2.5 times that reported previously for other whole-cell organophosphate sensors. Thus, this method for generation of whole-cell-based OP biosensors might be optimal, as it overcomes many of the caveats that prevent the widespread use of other such devices.
Highlights
Organophosphates (OPs) are widely employed in the agricultural industry as insecticides, their long-term toxicity toward both humans and the ecosystem is an important issue worldwide.OP pesticides deactivate the enzyme AChE, which is essential for nerve function in insects and animals, including humans [1,2]
We previously reported a microbe immobilization method, microbe-attached single-walled carbon nanotube (SWNT) film, for microelectromechanical system (MEMS)-based chips to apply microbes in a microfluidic system [33]
We demonstrate that microbe-attached SWNT film can be applied to whole-cell optical OP sensors and exhibits better performance compared to other whole-cell OP sensors
Summary
Organophosphates (OPs) are widely employed in the agricultural industry as insecticides, their long-term toxicity toward both humans and the ecosystem is an important issue worldwide. Because the rate of product formation is proportional to the concentration of OP compound, the OP concentration can be determined by measuring the hydrolysis product [13] This method has the following advantages over inhibition-based methods: (1) it does not require a continuous supply of the enzyme substrate, because the analyte itself is the substrate; and (2) enzymes such as AChE are inhibited by both OP and carbamate pesticides, rendering OPH more selective, as it only detects. The device, which has a circular comb drive-shaped electrode, was fabricated for a large area of cell immobilization, and microbe-attached SWNT film was formed between electrodes using E. coli that express OPH in the periplasmic space. 400 nm, and the advantages of this method were demonstrated by comparing it to other whole-cell OP sensors
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