A Label-Free Aptamer-Fluorophore Assembly for Rapid and Specific Detection of Cocaine in Biofluids

Abstract

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.

Specificity and Ligand Affinities of the Cocaine Aptamer: Impact of Structural Features and Physiological NaCl

“…These results are consistent with the reports of others. 5,7,8 To test for independent effects of cocaine on 2AP fluorescence, the structures of the 38COC1-2AP derivatives were distorted by annealing them with a complementary 20 nt oligonucleotide (cDNA (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)) that covers the presumptive cocaine binding pocket. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 These hybrid molecules showed no change in 2AP fluorescence upon the addition of cocaine, cocaine metabolites, or neomycin-B ( Figure S5).…”

Prediction of potential barcoding sites on ITS1 by wavelet transform

“…The high content of CG is a limiting factor for experimental screening for two different reasons: (a) the difficulty to design efficient amplification probes; and (b) the possibility that a probe with high content of GC could trigger cellular responses. In order to develop molecular screening tools that overcome the constrains above, we reckon that our proposed approach can be a valuable support to design synthetic oligomers (aptamers) (Roncancio et al, 2014) for different biotechnological applications, such as screening of OGM, food quality control, pathogen identification, and human non-infectious diseases.…”

Electrochemical assay of α-glucosidase activity and the inhibitor screening in cell medium

“…The maximum inhibition of gallic acid is 84.59% with reported previously (Minh Anh Thu et al, 2013). Different from gallic acid, quercetin can noncompetitively and anticompetitively inhibit α-glucosidase activity (Li et al, 2009;Roncancio et al, 2014). With the increase of quercetin concentrations from 0.05 to 10 μM, the inhibition ratios gradually increase and then level off when the inhibitor concentration reaches a certain level (Fig.…”