2,4-Dinitrophenol (DNP) is commonly used as a dye, wood preservative and pesticide 1. One of the effects on humans after oral exposure of DNP is weight loss and it has therefore been used in diet pills2, however, it has multiple negative effects, such as nausea, vomiting, sweating and dizziness, leading to death3. A Global Health Warning has been issued after several deaths have occurred in the UK caused by DNP overdose3.
Our biosensor for DNP relies on the actions of laccase, an enzyme that can degrade DNP. The low redox potential of laccase, along with the use of a mediator, can expand the range of substrates oxidised by the enzyme4. In our biosensor, ABTS is used as a mediator, which is also a substrate for laccase5. When laccase is paired with ABTS, laccase activity causes the production of ABTS radicals which have a blue colour which in turn indicates laccase activity. When there is another substrate present that can compete with ABTS for binding laccase, fewer ABTS radicals are produced. Therefore, the blue colour produced is less intense in the presence of DNP due to competitive inhibition of the two substrates.
This detection method requires two biosensors, one control biosensor to which only water will be applied and another for the actual sample. Our DNP biosensor uses crude cell lysate from cells expressing the sequence confirmed laccase CBD fusions which is then washed with PBS to purify the enzyme. A solution containing 0.2mM ABTS and 100mM sodium acetate buffer at pH5 is then added to the biosensor and is freeze-dried onto paper. A dark blue colour in the control will appear as soon as the biosensor is rehydrated. A difference in the blue colour between the two biosensors will indicate the presence of DNP. In the presence of DNP, the colour change will be slower and produce a much lighter blue, which when read with a spectrophotometer at 420 nm has a lower absorbance than the control. This is also a binary detection which should be visible with the naked eye.
For our biosensor, it was essential to experimentally determine the optimal amount of laccase and ABTS to freeze-dry onto the paper, as well as the concentration of enzyme which would produce a colour change visible enough for our app to detect. Immobilised laccase shows better activity and it is more stable than free enzyme over a time period5.
The laccase we are using is a copper-dependent tvel5 laccase from Trametes versicolor encoded by the gene lccl. To make our fusions compatible, we used biobrick assembly standard RFC25 6. The RFC25 prefix and suffix were added to the gene and all illegal restriction sites (EcoRI, SpeI, AgeI, NotI, NgoMIV and XbaI) were removed. The sequence was codon optimised and ordered as a gBlock. It was then digested and ligated into the pSB1C3 backbone and transformed into an Escherichia coli chassis.
1Agency for Toxic Substances and Disease Registry (ATSDR). Toxicological Profile for Dinitrophenols. Public Health Service, U.S. Department of Health and Human Services, Atlanta, GA. 1995.
2Grundlingh, J., Dargan, P. I., El-Zanfaly, M., Wood, D. M., (2011). 2,4-Dinitrophenol (DNP): A Weight Loss Agent with Significant Acute Toxicity and Risk of Death. Journal of Medical Toxicology.7:205-212 DOI 10.1007/s13181-011-0162-6
3National Research Council. Drinking Water and Health. Volume 4. National Academy Press, Washington, DC. 1982.
4Chandra, R. (2015). Advances in Biodegradation and Bioremediation of Industrial Waste. CRC Press, Taylor & Francis Group.
5Dehghanifard, E., Jafari, A. J., Kalantary, R. R., Mahvi, A. H., Faramarzi, M. A., Esrafili, A. (2013). Biodegradation of 2,4-dinitrophenol with laccase immobilized on nano-porous silica beads. Iranian Journal of Environmental Health Science and Engineering, 10(1), 25.
6Anderson, J., Dueber, J. E., Leguia, M., Wu, G. C., Goler, J. A., Arkin, A. P., & Keasling, J. D. (2010). BglBricks: A flexible standard for biological part assembly. Journal of biological engineering, 4(1), 1-12.