Development of MIP-based assays for drugs of abuse

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In this project we propose developing a novel assay for the measurement of the drugs of abuse and designer drugs which will be completely abiotic. This assay will not rely on natural antibodies and enzymes which would be replaced by magnetic and fluorescent molecularly imprinted polymer nanoparticles (MIP NPs). The proposed assay will not require refrigeration or additional procedures such as washing, elution or adding reagents, it will have long shelf and operational life, multiplexing capabilities and thus be suitable for use in the field by police force. Currently there is a gap in the market for this type of devices, especially in relations to detecting benzodiazepines. The antibodies-based assays for these compounds have shown unacceptable level of false positives. In addition development of antibodies-based sensors for designer drugs is impractical due to long period required for the production and validation of relevant antibodies. Our assay, if successful, could be used as a blueprint for the development of assays and sensors for other important target compounds and industry (MIP Diagnostics and assay manufacturers) will benefit strongly from its generic platform nature. The proposed assay will be based on a new format of the microtiter plate equipped with magnetic inserts which was developed by Leicester Biotechnology Group (GB1114231.2, and US 20150119274 A10). This technology has already been shown to work at lab scale, therefore, we believe that it is a good time to translate this technology for industrial use and apply it for detection of the compound of practical importance. The target compound for the test-system will be selected from the list of priority targets provided by Scottish Police and Crime Unit (Diazepam, Etizolam, Diclazepam, Clonazolam, Phenazepam, U-47700, 5-Fluoro-AKB48, FUB-AMB, AB-FUMINACA, JWH-018, 5-Fluoro-ADB, 5-F-MDMB-PIINACA, MMB2201, Fentanyl). The Scottish police authorities have kindly agreed to test and validate developed assay.The project involves use of molecular modelling for selecting functional monomers which possess high affinity towards the template. Molecular modelling of MIPs is pioneered by Leicester Biotechnology Group (LBG) and relies on Sybyl 7.3 software and Leapfrog algorithm (Tripos, USA). The results of modelling will be used in polymer preparation. The training in the use of molecular modelling will be provided to student by Dr. Kal Karim.Synthesis and characterisation of fluorescent MIP nanoparticles will be performed as described in (Canfarotta F., Poma A., Guerreiro A., Piletsky A. (2016). Solid-phase synthesis of molecularly imprinted nanoparticles (MIP NPs) for diagnostic applications. Nature Protocols, 11, 443-455).Fluorescent MIP nanoparticles will be used as a replacement for antibodies in novel magnetic assay format developed by Leicester group (US 20150119274 A10).The developed assay will be validated against analytical gold standards - HPLC MS. Training in using equipment will be provided by LBG members.