A label-free aptamer-fluorophore assembly for rapid and specific detection of cocaine in biofluids.

We report a rapid and specific aptamer-based method for one-step cocaine detection with minimal reagent requirements. The feasibility of aptamer-based detection has been demonstrated with sensors that operate via target-induced conformational change mechanisms, but these have generally exhibited limited target sensitivity. We have discovered that the cocaine-binding aptamer MNS-4.1 can also bind the fluorescent molecule 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND) and thereby quench its fluorescence. We subsequently introduced sequence changes into MNS-4.1 to engineer a new cocaine-binding aptamer (38-GC) that exhibits higher affinity to both ligands, with reduced background signal and increased signal gain. Using this aptamer, we have developed a new sensor platform that relies on the cocaine-mediated displacement of ATMND from 38-GC as a result of competitive binding. We demonstrate that our sensor can detect cocaine within seconds at concentrations as low as 200 nM, which is 50-fold lower than existing assays based on target-induced conformational change. More importantly, our assay achieves successful cocaine detection in body fluids, with a limit of detection of 10.4, 18.4, and 36 μM in undiluted saliva, urine, and serum samples, respectively.

[1]  K. Nakatani,et al.  Ligand-assisted complex formation of two DNA hairpin loops. , 2011, Angewandte Chemie.

[2]  N. Goeders,et al.  Cortical dopaminergic involvement in cocaine reinforcement. , 1983, Science.

[3]  R. Wise,et al.  Fluctuations in nucleus accumbens dopamine concentration during intravenous cocaine self-administration in rats , 1995, Psychopharmacology.

[4]  Ming Zhou,et al.  G-Quadruplex-based DNAzyme for colorimetric detection of cocaine: using magnetic nanoparticles as the separation and amplification element. , 2011, The Analyst.

[5]  M. Kuhar,et al.  Cocaine receptors on dopamine transporters are related to self-administration of cocaine. , 1987, Science.

[6]  P. Craig,et al.  Cocaine and benzoylecgonine in saliva, serum, and urine. , 1993, Clinical chemistry.

[7]  Xinhui Lou,et al.  Label-free fluorescent detection of ions, proteins, and small molecules using structure-switching aptamers, SYBR Gold, and exonuclease I. , 2012, Analytical chemistry.

[8]  Yi Lu,et al.  Quantum dot encoding of aptamer-linked nanostructures for one-pot simultaneous detection of multiple analytes. , 2007, Analytical chemistry.

[9]  S. Jayasena.  Aptamers: an emerging class of molecules that rival antibodies in diagnostics. , 1999, Clinical chemistry.

[10]  Juewen Liu,et al.  Fast colorimetric sensing of adenosine and cocaine based on a general sensor design involving aptamers and nanoparticles. , 2005, Angewandte Chemie.

[11]  L. Gold,et al.  Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. , 1990, Science.

[12]  S. Goldberg,et al.  Accelerating cocaine metabolism as an approach to the treatment of cocaine abuse and toxicity. , 2012, Future medicinal chemistry.

[13]  V. Thongboonkerd,et al.  Systematic evaluation of sample preparation methods for gel-based human urinary proteomics: quantity, quality, and variability. , 2006, Journal of proteome research.

[14]  Guang-Jiu Zhao,et al.  Ultrafast hydrogen bond strengthening of the photoexcited fluorenone in alcohols for facilitating the fluorescence quenching. , 2007, The journal of physical chemistry. A.

[15]  Guo-Li Shen,et al.  A novel label-free fluorescence aptamer-based sensor method for cocaine detection based on isothermal circular strand-displacement amplification and graphene oxide absorption , 2013 .

[16]  E. F. Ullman,et al.  Homogeneous enzyme immunoassay for opiates in urine. , 1973, Clinical chemistry.

[17]  M. Neri,et al.  Side effects of cocaine abuse: multiorgan toxicity and pathological consequences. , 2012, Current medicinal chemistry.

[18]  A. Heeger,et al.  An electronic, aptamer-based small-molecule sensor for the rapid, label-free detection of cocaine in adulterated samples and biological fluids. , 2006, Journal of the American Chemical Society.

[19]  M. Nilsen-Hamilton,et al.  Aptamer functionalized microcantilever sensors for cocaine detection. , 2011, Langmuir : the ACS journal of surfaces and colloids.

[20]  Milan N Stojanovic,et al.  Fluorescent Sensors Based on Aptamer Self-Assembly. , 2000, Journal of the American Chemical Society.

[21]  Guo-Li Shen,et al.  Fluorescence aptameric sensor for strand displacement amplification detection of cocaine. , 2010, Analytical chemistry.

[22]  J. Segura,et al.  Immunological screening of drugs of abuse and gas chromatographic-mass spectrometric confirmation of opiates and cocaine in hair. , 1999, Journal of chromatography. B, Biomedical sciences and applications.

[23]  G. Di Chiara,et al.  Drugs abused by humans preferentially increase synaptic dopamine concentrations in the mesolimbic system of freely moving rats. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[24]  L. Hillis,et al.  Cardiovascular complications of cocaine use. , 2001, The New England journal of medicine.

[25]  S. Amara,et al.  Cloning and expression of a cocaine-sensitive rat dopamine transporter. , 1991, Science.

[26]  Keitaro Yoshimoto,et al.  Influence of substituent modifications on the binding of 2-amino-1,8-naphthyridines to cytosine opposite an AP site in DNA duplexes: thermodynamic characterization , 2009, Nucleic acids research.

[27]  Jaroslav Kušnír,et al.  Concentration Matrices—Solutions for Fluorescence Definition of Urine , 2005 .

[28]  Philip E. Johnson,et al.  Defining a stem length-dependent binding mechanism for the cocaine-binding aptamer. A combined NMR and calorimetry study. , 2010, Biochemistry.

[29]  K. Farmer,et al.  Commonly prescribed medications and potential false-positive urine drug screens. , 2010, American journal of health-system pharmacy : AJHP : official journal of the American Society of Health-System Pharmacists.

[30]  Philip E. Johnson,et al.  Defining the secondary structural requirements of a cocaine-binding aptamer by a thermodynamic and mutation study. , 2010, Biophysical chemistry.

[31]  C. Zhan,et al.  Mechanism for cocaine blocking the transport of dopamine: insights from molecular modeling and dynamics simulations. , 2009, The journal of physical chemistry. B.

[32]  N K Mello,et al.  Management of cocaine abuse and dependence. , 1996, The New England journal of medicine.

[33]  M. Stojanović,et al.  Aptamer-based folding fluorescent sensor for cocaine. , 2001, Journal of the American Chemical Society.

[34]  Ľ. Podracká,et al.  Diagnostic monitoring of urine by means of synchronous fluorescence spectrum. , 2003, Journal of biochemical and biophysical methods.

[35]  H. Krebs.  Chemical composition of blood plasma and serum. , 1950, Annual review of biochemistry.

[36]  Arica A Lubin,et al.  Continuous, real-time monitoring of cocaine in undiluted blood serum via a microfluidic, electrochemical aptamer-based sensor. , 2009, Journal of the American Chemical Society.

[37]  Chunhai Fan,et al.  Visual cocaine detection with gold nanoparticles and rationally engineered aptamer structures. , 2008, Small.

[38]  Milan N Stojanovic,et al.  Aptamer-based colorimetric probe for cocaine. , 2002, Journal of the American Chemical Society.

[39]  S. Toennes,et al.  Studies on metabolic pathways of cocaine and its metabolites using microsome preparations from rat organs. , 2003, Chemical research in toxicology.

[40]  N. Teramae,et al.  Improvement of base selectivity and binding affinity by controlling hydrogen bonding motifs between nucleobases and isoxanthopterin: application to the detection of T/C mutation. , 2007, Bioorganic & medicinal chemistry letters.

[41]  Shuyan Niu,et al.  A Novel Fluorescence Sensor for Cocaine with Signal Amplification through Cycling Exo-Cleaving with a Hairpin Probe , 2012 .

[42]  Chun-Yang Zhang,et al.  Single quantum-dot-based aptameric nanosensor for cocaine. , 2009, Analytical chemistry.

[43]  K. Hall,et al.  2-Aminopurine fluorescence quenching and lifetimes: role of base stacking. , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[44]  Michel H M Eppink,et al.  Alternative affinity tools: more attractive than antibodies? , 2011, The Biochemical journal.

[45]  K. Nakatani,et al.  The SPR sensor detecting cytosine-cytosine mismatches , 2004 .

[46]  Z. Cheaib,et al.  Role of amylase, mucin, IgA and albumin on salivary protein buffering capacity: A pilot study , 2013, Journal of Biosciences.

[47]  O. Wolfbeis,et al.  The total fluorescence of human urine , 1987 .

[48]  G. Huang,et al.  Site-selective hydrogen-bonding-induced fluorescence quenching of highly solvatofluorochromic GFP-like chromophores. , 2012, Organic letters.

[49]  K. Nakatani,et al.  N,N'-Bis(3-aminopropyl)-2,7-diamino-1,8-naphthyridine stabilized a single pyrimidine bulge in duplex DNA. , 2005, Bioorganic & medicinal chemistry.

[50]  A. Leon,et al.  Fatal injuries after cocaine use as a leading cause of death among young adults in New York City. , 1995, The New England journal of medicine.

[51]  S. Sigurdsson,et al.  Folding of the cocaine aptamer studied by EPR and fluorescence spectroscopies using the bifunctional spectroscopic probe Ç , 2009, Nucleic Acids Research.

[52]  Stacy E F Melanson,et al.  The utility of immunoassays for urine drug testing. , 2012, Clinics in laboratory medicine.