Aptamer-Based, Exonuclease-Amplified, Paper Device for Point of Collection Screening of Cocaine and Methamphetamine in Oral Fluid

The need for rapid reliable detection of intoxicating controlled or other substances in their parent and/or metabolized forms is an issue at the forefront of concerns affecting public health1. Immunoassay-based oral fluid onsite devices are currently used to screen for the presence of drugs in biological samples3,4. Although they provide fast and sensitive screening of parent drugs and/or their metabolites5, the specificity and accuracy of immunoassays varies depending on the assay principle and the drug or metabolite used to develop anti-bodies for the assay and most importantly the unknown chemicals or drugs that are commonly found co-ingested by subjects under investigation. There is an urgent need for a rapid, non-invasive, noninstrumental screening assay/onsite device that addresses these issues.We propose a colorimetric detection platform with a low cost, portable, paper-based microfluidic device to simultaneously detect trace amounts of drug molecules in oral fluid with high specificity. Cocaine and methamphetamine are chosen as targets because they are of particular interest for those who work in the health and criminal justice fields. In the proposed platform, new aptamers will be generated and used to specifically bind to the targeted drugs, achieving a better specificity than that of immunoassays. Then, exonuclease III (Exo III) will be used to digest the target/aptamer complex and recycle the target, generating high sensitivity and a reduction in false negatives. Detection will occur through the use of gold nanoparticles (AuNPs) and their aggregation based color change. By transferring this amplified platform into a portable, paper-based device, we expect to perform simultaneous screening and detection of cocaine and methamphetamine at low ng/mL concentrations in microliter (μL) volumes of oral fluid on this postage stamp sized paper device by a visible color change without requirement of any additional devices. In this project, our initial goal will be to use a known cocaine binding aptamer to develop a specific, exo-amplified and AuNP-reported screening platform for cocaine. This sensor platform will be developed for the specific and amplified detection of cocaine in the range of 0.1 -1.0 ng/mL using a known cocaine binding aptamer along with Exo III associated probe digestion and target recycling. We will then utilize Nuclease Assisted Microfluidic-SELEX to rapidly isolate new binding aptamers for benzoylecgonine and methamphetamine. The selected aptamers possess high affinity, specificity and a new function that can form double-stranded target/aptamer complexes. Finally, we will transfer this amplified, colorimetric drug screening platform into a single paper-based microfluidic device to permit the rapid detection of low ng/mL benzoylecgonine and methamphetamine in oral fluid within 5 minutes. To do so, we will fabricate a single paper device with four reaction chambers containing benzoylecognine and methamphetamine binding aptamers and two controls with threshold cut-off values for each target. The successful outcomes of this work will provide 1) an aptamer-based, exo-amplified and AuNP-reported colorimetric method for multiple drug screening with high sensitivity and specificity; 2) a set of newly generated aptamers to specifically bind to cocaine, benzoylecgonine or methamphetamine; and 3) a paper-based, portable device to sensitively perform point of collection and or at roadside drug screening in oral fluid within minutes. Upon the success of this pilot study, we will generalize this platform/device for simultaneous screening of six different drugs of abuse in oral fluid including THC, heroin (6-AM), cocaine, hydrocodone, methamphetamine and MDMA. ca/ncf