Team:Edinburgh/PMABiosensor
Banned drugs are unregulated and can contain various contaminants and cutting agents. The past several years have seen a rise in the use of MDMA, more commonly known as ecstasy. During the production of MDMA, either MDMA or PMA will be produced1. PMA is pharmacologically very similar to MDMA, however it is far more toxic to the human body2. Professor David Nutt suggested we should focus on the detection of PMA after there was a spate of deaths attributed to PMA.
PMA and MDMA have very similar chemical structures, with one key difference. PMA contains a primary amine group, rather than a secondary amine as is the case in MDMA. Monoamine oxidase A can differentiate between the two as it is specific for primary amines3. Monoamine oxidase A is a copper/topaquinone containing enzyme and will convert primary monoamines to their corresponding aldehydes4. This reaction also results in the production of ammonia and hydrogen peroxide, which is crucial to the detection element of our biosensor.
When a reaction that produces hydrogen peroxide is coupled with horseradish peroxidase and Amplex red dye, resorufin is produced which has a red colour5. The colour can be detected by eye, but for accuracy, the fluorescence can be measured at 585 nm. The appearance of this red colour will show if PMA is present in a binary way.
For our biosensor, it was essential to experimentally determine the optimal amount of laccase and ABTS to freeze-dry onto the paper, Monoamine oxidase A is encoded by the structural gene MaoA. It was isolated from Klebsiella pneuomiae6 and the sequence was codon optimized for E. coli. The illegal RFC25 restriction sites were taken out and the RFC25 prefix and suffix sequences were added.
Since the production of MDMA is a chemical process either PMA or MDMA is produced at the end 1. That means that while MDMA does inhibit Monoamine oxidase A7 it should not be that much of an issue.
References
1Kraner, J.C., McCoy, D.J., Evans, M.A., Evans, L.E. & Sweeney, B.J. (2001) Fatalities caused by the MDMA-related drug Paramethoxyamphetamine (PMA). Journal of Analytical Toxicology, 25, 645-648.
2Independent Scientific Comitee on Drugs (2013). PMA Warning for Ecstasy Users
3Sabol, S. Z., Hu, S., & Hamer, D. (1998). A functional polymorphism in the monoamine oxidase A gene promoter. Human genetics, 103(3), 273-279.
4McIntire, W. S., & Hartmann, C. (1993). Copper-containing amine oxidases. Principles and applications of quinoproteins Marcel Dekker, Inc.
5Zhou, M. & Panchuk-Voloshina, N. (1997). A one-step fluorometric method for the continuous measurement of monoamine oxidase activity. Analytical Biochemistry, 253, 169-174.
6Sugino, H., Sasaki, M., Azakami, H., Yamashita, M., & Murooka, Y. (1992). A monoamine-regulated Klebsiella aerogenes operon containing the monoamine oxidase structural gene (maoA) and the maoC gene. Journal of bacteriology, 174(8), 2485-2492.
7Gough, B., Imam, S.Z., Blough, B., Slikker, W.Jr. & Ali, S.F. (2002) Comparative effects of substituted amphetamines (PMA, MDMA, and METH) on monoamines in rat caudate. Annals of the New York Academy of Sciences, 965, 410-420.