Colorimetric aptamer-based sensors – IP 1846

Small molecules are importanttargets with the potential of clinical or commercial applications such asmedical diagnostics, environmental monitoring, and forensic science. Thus,efforts to develop methods for portable, low-cost, and quantitative on-sitedetection of a broad range of small molecules are gaining momentum. Various immunoassays have also beendeveloped for the detection of small molecules such as cocaine and/or its majormetabolite benzoylecgonine in biofluids, including the enzyme-linkedimmunosorbent assay (ELISA).Unfortunately, the use of immunoassays for the detection of designerdrugs is often limited because of the high cost of generating new antibodiesand issues with narrow target binding-spectrum and poor specificity, and mostof these assays offer only limited capabilities for naked-eye detection,because the resulting absorbance changes can only be detected by instruments. FIU inventors have developed aptamersensors that report the presence of small-molecule target such as cocaine andsynthetic cathinones via a sensitive colorimetric signal for naked-eyedetection. The aptamer sensors are CBSAzyme-based sensors having both target-mediated cooperative behavior of theCBSA and peroxidase-mimicking catalytic activity of DNAzyme. The CBSAzyme-based sensors comprise a longfragment and a short fragment, the long fragment comprising a first segment ofa split DNAzyme and a long fragment of a CBSA, the short fragment comprising asecond segment of the split DNAzyme and a short fragment of the CBSA. The two fragments of the CBSAzyme remainseparate in the absence of the small-molecule target, but effectively assemblein the presence of the small-molecule target. The assembly of the two fragments of theCBSAzyme activates the DNAzyme that subsequently catalyzes the oxidation of2,2′-azinobis(3-ethylbenzthiazo-line)-6-sulfonic acid (ABTS), producing avisible color change from colorless to dark green that reveals the presence ofthe target within minutes. In an initialdemonstration, the inventors have generated a cocaine-binding CBSAzyme thatenables naked-eye detection of this drug at concentrations as low as 10 uMwithin 15 minutes. They subsequently demonstrated the generality of this assaystrategy by coupling the same split DNAzyme into a CBSAzyme that responds tothe designer drug methylenedioxypyrovalerone (MDPV). This CBSAzyme enabledvisual detection of MDPV and 11 other synthetic cathinones at concentrations aslow as 30 uM within 15 minutes, but did not respond to five common cuttingagents. Anne Laure Schmitt Olivier aschmitt@fiu.edu 305-348-5948

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