Electrochemical and Colorimetric Aptamer-Based Sensors for Rapid On-site Screening of Synthetic Cannabinoids in Seized Substances and Biological samples.

As submitted by the applicant: Synthetic cannabinoids (SCBs) belong to a class of designer drugs with more than 1000 distinct chemicals, and are wildly abused as an alternative to marijuana. However, compared to tetrahydrocannabinol (THC), SCBs are much more potent and can induce many severe adverse effects, even deaths. Most of SCBs are structurally different from natural cannabinoids along with significant inherent structural diversity. Current cannabinoid color tests based on the identification of one or two functional groups failed to detect all SCBs. Meanwhile, any minimal structural modifications on SCBs can greatly impair the antibody binding, generating false-negatives in existing immunoassays. With thousands of SCBs on the market and new compounds continuously emerging, the process of generating new antibodies is clearly too slow and costly to keep pace with designer drug evolution. The lack of reliable screening methods greatly hinders the regulation of SCBs. Here we propose the use of DNA aptamers as recognition elements to develop new electrochemical and colorimetric on-site screening methods for one of seven classes of SCBs (tetramethylcyclopropylketone indoles) in both seized substances and biological samples. Aptamers are single-stranded RNA- or DNA- oligonucleotides isolated in vitro that specifically bind with a wide range of targets. Compare with antibodies, aptamers can be isolated with good cross reactivity for a class of targets, and used as a good DNA substrate for nuclease-assisted signal amplification to achieve ultra-sensitive target detection. We chose two of the most abused SCBs, (1-pentylindol-3-yl)-(2,2,3,3-tetramethylcyclopropyl)methanone (UR-144) and [1-(5-Fluoropentyl)-1H-indol-3-yl](2,2,3,3-tetramethylcyclopropyl)methanone (XLR-11) that belong to tetramethylcyclopropylketone indoles, as the targets to demonstrate our proposed strategy. Specifically, we will isolate new aptamers using a novel NA-SELEX technique with two differently designed library pools. The isolated aptamers will then respectively used to fabricate an interference-free, electrochemical aptamer-based sensor for rapid and quantitative field screening for the targets in seized substances, and a colorimetric sensor platform based on target-induced exonuclease-assisted target recycling for a qualitative, ultra-sensitive, naked-eye screening of the targets in saliva. We believe that the proposed strategy provides an alternative to immunoassays with the ability to use a much shorter sensor development period to fabricate sensors that can detect a class of SCBs in various sample matrices, with high sensitivity and specificity. Upon the pilot success of this work, this technology can be adopted to sensor development for other classes of SCBs, which will be a significant step in regulating SCBs as well as other rapidly evolving designer drugs on the market. Note: This project contains a research and/or development component, as defined in applicable law. nca/ncf.

As submitted by the proposer: Synthetic cannabinoids (SCBs) belong to a class of designer drugs with more than 1000 distinct chemicals, and are wildly abused as an alternative to marijuana. However, compared to tetrahydrocannabinol (THC), SCBs are much more potent and can induce many severe adverse effects, even deaths. Most of SCBs are structurally different from natural cannabinoids along with significant inherent structural diversity. Current cannabinoid color tests based on the identification of one or two functional groups failed to detect all SCBs. Meanwhile, any minimal structural modifications on SCBs can greatly impair the antibody binding, generating false-negatives in existing immunoassays. With thousands of SCBs on the market and new compounds continuously emerging, the process of generating new antibodies is clearly too slow and costly to keep pace with designer drug evolution. The lack of reliable screening methods greatly hinders the regulation of SCBs. Here we propose the use of DNA aptamers as recognition elements to develop new electrochemical and colorimetric on-site screening methods for one of seven classes of SCBs (tetramethylcyclopropylketone indoles) in both seized substances and biological samples. Aptamers are single-stranded RNA- or DNA- oligonucleotides isolated in vitro that specifically bind with a wide range of targets. Compare with antibodies, aptamers can be isolated with good cross reactivity for a class of targets, and used as a good DNA substrate for nuclease-assisted signal amplification to achieve ultra-sensitive target detection. The researcher chose two of the most abused SCBs, (1-pentylindol-3-yl)-(2,2,3,3-tetramethylcyclopropyl)methanone (UR-144) and [1-(5-Fluoropentyl)-1H-indol-3-yl](2,2,3,3-tetramethylcyclopropyl)methanone (XLR-11) that belong to tetramethylcyclopropylketone indoles, as the targets to demonstrate our proposed strategy. Specifically, we will isolate new aptamers using a novel NA-SELEX technique with two differently designed library pools. The isolated aptamers will then respectively used to fabricate an interference-free, electrochemical aptamer-based sensor for rapid and quantitative field screening for the targets in seized substances, and a colorimetric sensor platform based on target-induced exonuclease-assisted target recycling for a qualitative, ultra-sensitive, naked-eye screening of the targets in saliva. We believe that the proposed strategy provides an alternative to immunoassays with the ability to use a much shorter sensor development period to fabricate sensors that can detect a class of SCBs in various sample matrices, with high sensitivity and specificity. Upon the pilot success of this work, this technology can be adopted to sensor development for other classes of SCBs, which will be a significant step in regulating SCBs as well as other rapidly evolving designer drugs on the market. This project contains a research and/or development component, as defined in the applicable law. nca/ncf