Microchimica Acta

, Volume 183, Issue 10, pp 2791–2797 | Cite as

Aptamer based fluorescent cocaine assay based on the use of graphene oxide and exonuclease III-assisted signal amplification

  • Yulin Zhang
  • Zhongyue Sun
  • Lina Tang
  • Hong ZhangEmail author
  • Guo-Jun ZhangEmail author
Original Paper


The article reports an aptamer based assay for cocaine by employing graphene oxide and exonuclease III-assisted signal amplification. It is based on the following scheme and experimental steps: (1) Exo III can digest dsDNA with blunt or recessed 3-terminus, but it has limited activity to ssDNA or dsDNA with protruding 3-terminus; (2) GO can absorb the FAM-labeled ssDNA probe and quench the fluorescence of probe, while the affinity between FAM-labeled mononucleotide and GO is negligible; (3) Cocaine aptamer can be split into two flexible ssDNA pieces (Probe 1 and Probe 2) without significant perturbation of cocaine-binding abilities; (4) The triple complex consisting of Probe 1, Probe 2 and cocaine can be digested by Exo III with the similar efficiency as normal dsDNA. Cocaine aptamer is split into two flexible ssDNA pieces (Probe 2 and 3′-FAM-labeled Probe 1). Cocaine can mediate the cocaine aptamer fragments forming a triplex. The triple complex has unique characteristic with 3′-FAM-labeled blunt end at the Probe 1 and 3′-overhang end at Probe 2. If exonuclease III is added, it will catalyze the stepwise removal of fluorescein (FAM) labeled mononucleotides from the 3-hydroxy termini of the special triplex complex, resulting in liberation of cocaine. The cocaine released in this step can produce a new cleavage cycle, thereby leading to target recycling. Through such a cyclic bound-hydrolysis process, small amounts of cocaine can induce the cleavage of a large number of FAM-labeled probe 1. The cleaved FAM-labeled mononucleotides are not adsorbed on the surface of graphene oxide (GO), so a strong fluorescence signal enhancement is observed as the cocaine triggers enzymatic digestion. Under optimized conditions, the assay allows cocaine to be detected in the 1 to 500 nM concentration range with a detection limit of 0.1 nM. The method was applied to the determination of cocaine in spiked human plasma, with recoveries ranging from 92.0 to 111.8 % and RSD of <12.8 %.

Graphical abstract

Aptamer based fluorescent cocaine assay based on graphene oxide and exonuclease III-assisted signal amplification


Drug analysis Cocaine aptamer fragment Exo III triggered amplification Enzymatic digestion Plasma analysis 



The authors acknowledge the support of Natural Science Foundation of China (Nos. 21275040 and 21475034).

Compliance with ethical standards

The author(s) declare that they have no competing interests.

Supplementary material

604_2016_1923_MOESM1_ESM.doc (1008 kb)
ESM 1 (DOC .98 mb)


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Copyright information

© Springer-Verlag Wien 2016

Authors and Affiliations

  1. 1.School of Laboratory MedicineHubei University of Chinese MedicineWuhanPeople’s Republic of China
  2. 2.Hubei Provincial Collaborative Innovation Center of Preventive Treatment by Acupuncture and MoxibustionWuhanPeople’s Republic of China
  3. 3.Teaching and Research Office of Forensic MedicineHubei University of Chinese MedicineWuhanPeople’s Republic of China

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