Evaluation and validation of latent fingerprints as a non-invasive drug screening matrix for the detection of MDMA using lateral flow technology

Introduction: Lateral flow immunoassays (LFA) are routinely used to detect illicit drugs in biological matrices. Most recently, latent fingerprints (LFPs) have been analysed using this method. There is a lack of screening tests that identify MDMA(ecstasy) despite it being used by 2.6 million people in Europe and 524,000 in England and Wales. MDMA (pKa 9.9) has been shown to be excreted in sweat. This thesis explored the utility of LFPs as a biological matrix for screening for MDMA in the field via a LFA as a Point-of-Care test (POCT). Method: The Ridgeway (Intelligent Fingerprinting Ltd., UK) was employed to quantify LFPs and they were further characterised using ATR-FTIR to measure LFP mass and composition. The Ridgeway was used to investigate fingermarks sampling methods and variables known to influence sweat production including deposition pressure,environmental conditions and certain drugs. An optimised plate immunoassay was used to establish a suitable binding pair (Anti-MDMA and BSA-MDMA). Further characterisation included establishing an ideal Alexa Fluor 488 TFP:Anti-MDMA ratio and confirming cross-reactants. Results were validated with Surface Plasmon Resonance (SPR). A LFA suitable for MDMA detection using LFPs was achieved by testing different antibody binding concentrations against different extraction buffer compositions. Optimisation of the LFA identified: cross-reactants, cartridge run-time and cut-off level. Fluorescent LFA signals were measured using Intelligent Fingerprinting Reader 1000 (Intelligent Fingerprinting Ltd., UK). Results: A significant positive correlation was calculated between LFP mass and Ridgeway score [p=0.02] advising a relationship between the two measurements. Higher Ridgeway scores corresponded with sebaceous LFPs after ATR-FTIR analysis. An optimal sampling method was determined as: cumulative deposition of10 LFPs [p<0.1] and either hand can be used. To collect eccrine only LFPs, Carex soap with lint free tissue were most suitable [p<0.01] compared to antibacterial handgel. Different environmental climates did not change the sample quantity deposited for both natural and eccrine LFPs. Drug use, including methadone, significantly increased Ridgeway scores in the addict population versus the control population[p<0.01]. Comparable LFPs were collected via consistent deposition pressure (300 -400 g) and glass deposition surface. Both plate assay and SPR results confirmed the binding pairs' suitability for the MDMA LFA. Monoclonal Anti-MDMA detected MDMA at 0.15 ng/mL and KD values of 0.3 and 0.5 nM were calculated, respectively. A ratio of 7.3:1 was calculated as the ideal labelling ratio. A sensitive LFA, which produced a sharp-drop in signal, was designed using an extraction buffer coupled with 20 ng Anti-MDMA and 120 µg/mLBSA-MDMA. This established a cut-off value of 60 pg in 10 µL which generated excellent accuracy (0.98), sensitivity (1.0) and specificity (0.95). Immunoassay methods recognised Methylenedioxy-N-ethylamphetamine and para-Methoxy-Nmethylamphetamine as key cross-reactants at 55 pg in 10 µL. Conclusion: A sensitive MDMA LFA was successfully designed to identify MDMA inLFPs at a cut-off value of 60 pg in 10 µL for use in drug screening. For this purpose,an optimal LFP collection method was also established.