Mehdi SABERIAN, Davoud ASGARI, Yadollah OMIDI, Jaleh BARAR

Establishment of an electrochemical RNA aptamer-based biosensor to trace nanomolar concentrations of codeine

Codeine is an opium alkaloid with a great potential of abuse among opioid consumers. Detection of codeine is a routine procedure in the military and government of some countries for personnel recruitment. Therefore, a specific, selective, and easy to use method would be an important improvement in such detection procedures. According to previous reports, short single-stranded DNA or RNA sequences with high affinity and specificity to their targets, aptamers, could be used for designing an accurate and specific biosensor for codeine. This study introduces an aptamer-based biosensor for codeine detection in nanomolar concentrations by an electrochemical method, and ferrocene carboxylic acid was used as the redox molecule. The data show an improvement in codeine detection in comparison with the previous reports by other methods. The fabricated aptasensor offers a simpler and faster method with a lower limit of detection for codeine. The results demonstrate the reliability and robustness of the constructed aptasensor in codeine detection and measurement.

Anahtar Kelimeler:

Aptasensor, aptamer, SELEX, codeine

Establishment of an electrochemical RNA aptamer-based biosensor to trace nanomolar concentrations of codeine

Keywords:

Aptasensor, aptamer, SELEX, codeine,

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Capillary electrophoresis 5 × 10 −8 M Flow injection analysis Miniaturized devices 2 × 10 −5 M 8 × 10 −7 M SIA 1 × 10 −8 M 5 × 10 −10 M The specificity studies showed that the agonist of opioid receptors, tramadol, did not intervene in the process of codeine detection by the fabricated aptasensor. This was predictable because the chemical structure of tramadol is completely different from that of codeine. 28 In the case of naloxone, an antagonist for the opioid receptors, although its chemical structure is very similar to codeine, 15,28 this molecule did not interact with the aptasensor, either. However, in the case of morphine, an opioid receptor agonist with a very similar structure to codeine, the sensor was affected partially. In brief, the fabricated sensor was partially influenced by morphine and could not distinguish between morphine and codeine as we expected. Although morphine and codeine are only different in a methyl group, a very specific aptamer should be able to distinguish between them in theory. 7,29 A specific aptamer for theophylline is the best example for this case. Theophylline and caffeine are only different in a methyl group, but the aptamer was claimed to have about 10,000-fold more affinity to theophylline. 30 Since codeine is metabolized to morphine in the body, 28 the response of the sensor in the body fluids and urine sample would be relatively unspecific. However, it is valuable in the abuse detection point of view. Thus, the fabricated aptasensor is not 100% specific for codeine measurements in the presence of morphine. This problem probably originates from the counter selection stage against morphine in the SELEX process, which may have been done relatively poorly. 18 To solve this problem, the SELEX process should be done using another pool of RNA library. The data in the related paper 18 show that the aptamer has 16% affinity to morphine. As reported before, the response of a regenerated sensor should be at least 95% of the first detection for introducing it as a multiple use sensor. 19,21,26 However, the regeneration studies showed that the response of the fabricated aptasensor to its target reduced after each regeneration step. The obtained data represented only 69% recovery for the first stage of regeneration and the proportion of recovery for the 2nd to 4th stages of regeneration were 57%, 39%, and 26 %, respectively. Despite working under RNase free conditions, this problem may have been raised because of the susceptibility of the sensor to degradation by remaining trace nucleases. Therefore, the fabricated sensor could not be reused and is only appropriate for single-use applications. Since response of the aptamer to the target is very quick, the fabricated aptasensor is comparable with the antibody-based rapid strips. Although the instrumentation for the electrochemical aptasensors is more complicated, progress in the aptamer-based strip construction may solve this problem 31,32 and there will certainly be a possibility to introduce the new generation of aptamer-based rapid strips, i.e. aptastrips. The aptastrips would be more accurate than the antibody-based strips for of several reasons. The aptamers are much smaller than antibodies and can be immobilized more densely than antibodies on the surface. Moreover, lack of batch-to-batch variation, longer shelf lives, and possibility of aptamer selection for a wide range of targets such as ions, toxic compounds, and even a whole cell are some of the advantages of aptastrips. 1,2,6 Conclusion The fabricated biosensor represents an improvement in the limit of detection of codeine regarding most of the previously reported methods. Rapidness and simplicity of the method can also be mentioned as an important advantage for this sensor. Although the sensor could not be regenerated, it may be useful in single-use stripbased aptasensor design (aptastrips). Aptastrips could be mentioned as good candidates for rapid and accurate detection of codeine in military, workplace, and medical services recruitments. Acknowledgment The authors would like to thank the Research Center for Pharmaceutical Nanotechnology and the vice chancellor of research of Tabriz University of Medical Sciences, Iran, for financially supporting this project as a part of a PhD thesis (Mehdi Saberian). The authors also thank Exir Pharmaceutical Co., Iran, for providing codeine phosphate and Ayoub Aghanejad for assisting in chemical synthesis procedures. References Nguyen, T. H.; Hilton, J. P.; Lin, Q. Microfluid. Nanofluid. 2009, 6, 347–362. Tombelli, S.; Minunni, M.; Mascini, M. Biosens. Bioelectron. 2005, 20, 2424–2434.
Stoltenburg, R.; Reinemann, C.; Strehlitz, B. Biomol. Eng. 2007, 24, 381–403.
Strehlitz, B.; Nikolaus, N.; Stoltenburg, R. Sensors 2008, 8, 4296–4307.
Rowe, W.; Platt, M.; Day, P. J. R. Integr. Biol. 2009, 1, 53–58.
Lee, J. O.; So, H. M.; Jeon, E. K.; Chang, H.; Won, K.; Kim, Y. H. Anal. Bioanal. Chem. 2008, 390, 1023–1032. Cho, E. J.; Lee, J. W.; Ellington, A. D. Annu. Rev. Anal. Chem. 2009, 2, 241–264.
Sassolas, A.; Blum, L. J.; Leca-Bouvier, B. D. Electroanal. 2009, 21, 1237–1250.
Hianik, T.; Wang, J. Electroanal. 2009, 21, 1223–1235.
Wei, F.; Ho, C. M. Anal. Bioanal. Chem. 2009, 393, 1943–1948.
Lai, R. Y.; Plaxco, K. W.; Heeger, A. J. Anal. Chem. 2007, 79, 229–233.
Hansen, J. A.; Wang, J.; Kawde, A. N.; Xiang, Y.; Gothelf, K. V.; Collins, G. J. Am. Chem. Soc. 2006, 128, 2228–229.
Hsu, A. F.; Liu, R. H.; Piotrowski, E. G. Phytochemistry 1985, 24, 473–476.
American Society of Health-System Pharmacists, AHFS Drug Information, Bethesda, MD, USA, 2010.
Sweetman, S. C. (ed). Martindale: The Complete Drug Reference, Pharmaceutical Press, London, 2011.
Kim, I.; Barnes, A. J.; Oyler, J. M.; Schepers, R.; Joseph, R. E. J.; Cone, E. J.; Lafko, D.; Moolchan, E. T.; Huestis, M. A. Clin. Chem. 2002, 48, 1486–1496.
Francis, P. S.; Adcock, J. L.; Costin, J. W.; Purcell, S. D.; Pfeffer, F. M.; Barnett, N. W. J. Pharmaceut. Biomed. 2008, 48, 508–518.
Win, M. N.; Klein, J. S.; Smolke, C. D. Nucleic Acids Res. 2006, 34, 5670–5682.
Xiao, Y.; Lai, R. Y.; Plaxco, K. W. Nat. Protoc. 2007, 2, 2875–2880.
El-Deab, M. S.; Ohsaka, T. Electrochim. Acta 2004, 49, 2189–2194.
Baker, B. R.; Lai, R. Y.; Wood, M. S.; Doctor, E. H.; Heeger, A. J.; Plaxco, K. W. J. Am. Chem. Soc. 2006, 128, 3138–3139.
Merrill, D. R.; Stefan, I. C.; Scherson, D. A.; Mortimer, J. T. J. Electrochem. Soc. 2005, 152, E212–E221.
Wink, T.; Van Zuilen, S. J.; Bult, A.; Van Bennekom, W. P. Analyst 1997, 122, 43R–50R.
March, G.; Noel, V.; Piro, B.; Reisberg, S.; Pham, M. C. J. Am. Chem. Soc. 2008, 130, 15752–15753.
Ferapontova, E. E.; Olsen, E. M.; Gothelf, K. V. J. Am. Chem. Soc. 2008, 130, 4256–4258.
Zhao, G. C.; Yang, X. Electrochem. Commun. 2010, 12, 300–302.
Thevenot, D. R.; Toth, K.; Durst, R. A.; Wilson, G. S. Pure Appl. Chem. 1999, 71, 2332–2348.
Fries, D. S. In Foye’s Principles of Medicinal Chemistry ; Lemarke, T. L.; Villiams, D. A., Eds.; Wolters Kluwer Health, Philadelphia, 2008.
Proske, D.; Blank, M.; Buhmann, R.; Resch, A. Appl. Microbiol. Biot. 2005, 69, 367–374.
Jenison, R. D.; Gill, S. C.; Pardi, A.; Polisky, B. Science 1994, 263, 1425–1429.
Liu, J.; Mazumdar, D.; Lu, Y. Angew. Chem. Int. Ed. 2006, 45, 7955–7959.
Mazumdar, D.; Liu, J.; Lu, G.; Zhou, J.; Lu, Y. Chem. Commun. 2010, 46, 1416–1418.
Barnett, N. W.; Hindson, B. J.; Lewis, S. W.; Purcell, S. D. Anal. Commun. 1998, 35, 321–324.
Barnett, N. W.; Bos, R.; Brand, H.; Jones, P.; Lim, K. F.; Purcell, S. D.; Russell, R. Analyst 2002, 127, 455–458. Brown, A. J.; Lenehan, C. E.; Francis, P. S.; Dunstan, D. E.; Barnett, N. W. Talanta 2007, 71, 1951–1957.
Barnett, N. W.; Hindson, B. J.; Lewis, S. W.; Jones, P.; Worsfold, P. J. Analyst 2001, 126, 1636–1639.
Barnett, N. W.; Hindson, B. J.; Lewis, S. W.; Purcell, S. D.; Jones, P. Anal. Chim. Acta 2000, 421, 1–6.
Qiu, B.; Chen, X.; Chen, H. L.; Chen, G. N. Luminescence 2007, 22, 189–194.
Adcock, J. L.; Francis, P. S.; Agg, K. M.; Marshall, G. D.; Barnett, N. W. Anal. Chim. Acta 2007, 600, 136–141. Costin, J. W.; Lewis, S. W.; Purcell, S. D.; Waddell, L. R.; Francis, P. S.; Barnett, N. W. Anal. Chim. Acta 2007, 597, 19–23.
L’Hostis, E.; Michel, P. E.; Fiaccabrino, G. C.; Strike, D. J.; Rooij, N. F.; Koudelka-Hep, M. Sensor. Acuat. B 2000, 64, 156–162.
Greenway, G. M.; Nelstrop, L. J.; Port, S. N. Anal. Chim. Acta 2000, 405, 43–50.
Gorman, B. A.; Barnett, N. W.; Bos, R. Anal. Chim. Acta 2005, 541, 119–124.