Advances in point-of-care testing can provide direct and immediate information on a patient's status, leading to faster stabilization of life-threatenin crises and more effective patient triage at the site of accidents and in emergency rooms. The need for detection of drug substances in their parent and/or metabolized forms has become an issue at the forefront of public health awareness. Immunoassay-based, on-site devices are currently used to quickly screen for the presence of drugs in biological samples. Although they provide fast and sensitive screening results, the specificity and sensitivity of immunoassays varies depending on the assay principle, the employed antibodies and the detected substance. They often generate false-positives and false-negatives. The overall goal of this proposal is to develop a non-invasive, non-instrumental colorimetric assay that accurately screens drugs in saliva at point-of-care testing with high sensitivity and specificity. The investigators propose a colorimetric detection platform with a portable paper-based microfluidic device to simultaneously screen trace amounts of drugs in saliva with high specificity. Methylenedioxypyrovalerone (MDPV) and Mephedrone 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, aptamers will be generated through Microfluidic-SELEX process and used to specifically bind MDPV or Mephedrone, achieving a better specificity than that of immunoassay-based assays to minimize false-positives. Then, exonuclease III will digest the target/aptamer bound complex and recycle the target, generating superior sensitivity to minimize false-negatives. Screening result will be reported through the use of gold nanoparticles and their aggregation based color change. By transferring this platform into a paper-based device, the investigators expect to perform simultaneous screening of two drug molecules at a few mg/L concentrations in mL volumes of saliva within 10 minutes by the naked eye. Upon the success of this pilot study, the investigators will generalize the platform for the simultaneous screening of almost any designer drug molecules newly appeared on the market in ml oral fluid within minutes in a single paper- based, disposable device. This project will promote the participation of undergraduate and graduate students to interdisciplinary research in chemistry, biochemistry, forensic and biomedical sciences while helping the PI to establish new collaborations, enhance the research environment at Florida International University and obtain strong preliminary results for future R01 competition.

Public Health Relevance

Advances in point-of-care testing are making it possible to directly collect information on a patient's health status, leading to faster stabilization of life-threatening crises and more effective patient triage at the site of accidents and in emergency rooms. Although current immunoassay-based, on-site devices can provide fast and sensitive screening of drugs, the specificity and accuracy of these immunoassays needs to be improved to minimize current problems with false-positives and false negatives. The overall goal of the proposed project is to develop a paper-based microfluidic device for a non-invasive, non- instrumental, point of care drug screen that utilizes DNA sequences that specifically bind to target drugs achieving better specificity to minimize false positives and uses exonuclease III to digest the DNA/target complex and recycle the target and thus delivering greater sensitivity to minimize false negatives.

National Institute of Health (NIH)
National Institute on Drug Abuse (NIDA)
Academic Research Enhancement Awards (AREA) (R15)
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Nanotechnology Study Section (NANO)
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Hillery, Paul
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Florida International University
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Yu, Haixiang; Canoura, Juan; Guntupalli, Bhargav et al. (2018) Sensitive Detection of Small-Molecule Targets Using Cooperative Binding Split Aptamers and Enzyme-Assisted Target Recycling. Anal Chem 90:1748-1758
Liang, Pingping; Canoura, Juan; Yu, Haixiang et al. (2018) Dithiothreitol-Regulated Coverage of Oligonucleotide-Modified Gold Nanoparticles To Achieve Optimized Biosensor Performance. ACS Appl Mater Interfaces 10:4233-4242
Canoura, Juan; Wang, Zongwen; Yu, Haixiang et al. (2018) No Structure-Switching Required: A Generalizable Exonuclease-Mediated Aptamer-Based Assay for Small-Molecule Detection. J Am Chem Soc 140:9961-9971
Yu, Haixiang; Yang, Weijuan; Alkhamis, Obtin et al. (2018) In vitro isolation of small-molecule-binding aptamers with intrinsic dye-displacement functionality. Nucleic Acids Res 46:e43
Yu, Haixiang; Xu, Xiaowen; Liang, Pingping et al. (2017) A Broadly Applicable Assay for Rapidly and Accurately Quantifying DNA Surface Coverage on Diverse Particles. Bioconjug Chem 28:933-943