Development of MIP-based assays for drugs of abuse

在这个项目中，我们建议开发一种新的分析方法来测量滥用药物和设计药物，这将是完全非生物的。该试验将不依赖于天然抗体和酶，它们将被磁性和荧光分子印迹聚合物纳米颗粒（MIP NPs）所取代。建议的分析不需要冷藏或额外的程序，如洗涤，洗脱或添加试剂，它将具有较长的保质期和使用寿命，多路复用能力，因此适合警察部队在现场使用。目前，这类设备在市场上存在空白，特别是在检测苯二氮卓类药物方面。针对这些化合物的基于抗体的检测显示出不可接受的假阳性水平。此外，由于相关抗体的生产和验证需要很长时间，因此开发用于设计药物的基于抗体的传感器是不切实际的。如果成功，我们的检测方法可以作为开发其他重要目标化合物的检测方法和传感器的蓝图，行业（MIP Diagnostics和检测方法制造商）将从其通用平台性质中获益良多。拟议的检测将基于由莱斯特生物技术集团（GB1114231.2和US 20150119274 A10）开发的配备磁性插入物的新型微量滴度板。这项技术已经被证明在实验室规模上起作用，因此，我们认为现在是将这项技术转化为工业用途并将其应用于检测具有实际重要性的化合物的好时机。测试系统的目标化合物将从苏格兰警察和犯罪部门提供的优先目标列表中选择（地西泮、安替唑仑、二氮西泮、氯硝唑仑、非那西泮、U-47700、5-Fluoro-AKB48、fu - amb、AB-FUMINACA、JWH-018、5-Fluoro-ADB、5-F-MDMB-PIINACA、MMB2201、芬太尼）。苏格兰警方已经同意测试和验证开发的分析。该项目涉及使用分子建模来选择对模板具有高亲和力的功能单体。MIPs的分子建模是由莱斯特生物技术集团（LBG）首创的，依靠Sybyl 7.3软件和Leapfrog算法（Tripos， USA）。模拟的结果将用于聚合物的制备。Kal Karim博士将为学生提供使用分子建模的培训。荧光MIP纳米颗粒的合成和表征将按照Canfarotta F., Poma A., Guerreiro A., Piletsky A.（2016）所述进行。用于诊断应用的分子印迹纳米颗粒（MIP NPs）固相合成。Nature Protocols, 11, 443-455)。荧光MIP纳米颗粒将被用作莱斯特集团（US 20150119274 A10）开发的新型磁性分析格式抗体的替代品。开发的分析方法将根据分析金标准- HPLC ms进行验证，LBG成员将提供使用设备的培训。

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.