基于上转换发光技术的尿液中吗啡及甲基苯丙胺快速定量检测方法研究

摘要

目的：利用上转换发光免疫层析技术（UPT-LF）建立一种尿液中吗啡（MOP）及甲基苯丙胺（MET）快速定量检测方法并对其进行系统评价。方法以上转换发光纳米颗粒（UCP-NPs）作为生物示踪物，竞争模式免疫层析作为检测平台，建立可对尿液中 MOP 及 MET 进行定量检测的 UPT-LF，即 MOP-UPT-LF、MET-UPT-LF。以 MOP-UPT-LF 为代表，评价 UPT-LF 对痕量毒品检测极限，通过系列浓度标准品测定，评价定量检测能力。根据常规检测阈值，调整 MOP-UPT-LF 及 MET-UPT-LF 检测敏感性及线性范围，并评价其定量检测能力。以 LC-MS、GC-MS 分别作为 MOP、MET 检测的金标准，以胶体金免疫层析作为对照，对执法现场收集尿样进行检测，确定 UPT-LF 定性检测性能。对系列浓度 MOP 模拟阳性样本同时进行LC-MS 及 MOP-UPT-LF 定量检测，对系列浓度 MET 模拟阳性样本同时进行 GC-MS 及 MET-UPT-LF 定量检测，评价 UPT-LF 定量检测性能。结果在痕量检测条件下，MOP-UPT-LF 敏感性可达1ng/mL，线性范围为1~5000ng/mL（r=-0.98172，P<0.0005）。在常规检测条件下，MOP-UPT-LF 敏感性为50ng/mL，线性范围调整为50~3000ng/mL（r=-0.98464，P<0.0005）；MET-UPT-LF 敏感性为100ng/mL，线性范围为100~5000ng/mL（r=-0.99964，P<0.0005）。就定性检测而言，MOP-UPT-LF 及 MET-UPT-LF 均较优，灵敏度及特异度均为100%，与胶体金结果一致。就定量检测而言 MOP-UPT-LF 及 MET-UPT-LF 与定量确证方法 LC-MS 及 GC-MS 无显著差异。结论本研究建立 MOP-UPT-LF、MET-UPT-LF 方法，在满足快筛试剂快速简便的基础上，进一步实现了现场快速定量检测，为尿液中毒品的现场快速定量检测提供了技术保障。%Objective To develop and evaluate an up-converting phosphor technology based on lateral flow assay (UPT-LF) for qualitative and quantitative detection of morphine (MOP) and methamphetamine (MET) in urine. Methods With up-converting phosphor nano-particles (UCP-NPs) as the biological tracer, two competitive mode-based LF strips, MOP-UPT-LF and MET-UPT-LF were developed for quantitative detection of MOP and MET in urine. The comprehensive performances of MOP-UPT-LF and MET-UPT-LF were evaluated systematically. In order to explore the detection limit of UPT-LF for trace analysis, MOP-UPT-LF strips were used to test standard samples with series of concentrations, and then the detection limit and ability of quantitative detection were determined. According to the standard of detection threshold for MOP and MET, the quantitative detection performances (including detection sensitivity and linear range) of MOP-UPT-LF and MET-UPT-LF were optimized and re-evaluated. For the evaluation of qualitative detection ability, the results of MOP-UPT-LF and MET-UPT-LF for on-site urine samples were compared with those of colloidal gold based LF (CG-LF), and LC-MS and GC-MS were used as the gold standard for the detection of MOP and MET, respectively. For the evaluation of quantitative detection ability, the results of MOP-UPT-LF and MET-UPT-LF for simulated positive urine samples were compared with those of LC-MS and GC-MS, respectively. Results For trace analysis, the detection limit of MOP-UPT-LF could reach 1ng/mL with a linear range from 1ng/mL to 5000ng/mL (r = -0.98172,P < 0.0005). For routine detection with the threshold of standard, the detection limit of MOP-UPT-LF was 50ng/mL with a linear range from 50ng/mL to 3000ng/mL (r = -0.98464,P < 0.0005). The detection limit of MET-UPT-LF was 100ng/mL with a linear range from 100ng/mL to 5000ng/mL (r = -0.99964, P <0.0005). According to the detection of urine samples, the performance of qualitative and quantitative detection of MOP-UPT-LF and MET-UPT-LF could meet the need of the on-site rapid detection of MOP and MET in urine. The qualitative detection results of MOP-UPT-LF and MET-UPT-LF for on-site urine samples were consistent with those of colloidal gold and no false-positive and false-negative results observed. The ROC area of MOP-UPT-LF and MET-UPT-LF reached 1.000±0.000 (95%CI). For quantitative detection of simulated positive urine samples, the recovery rate was 77%~133% with the mean of 109% and CV of 21% for MOP-UPT-LF, for MET-UPT-LF the recovery rate was 80%~131% along with the mean of 112%and CV of 17%. After statistical analysis, there was no significant difference (P>0.05) between MOP-UPT-LF/MET-UPT-LF and LC-MS/GC-MS for quantitative detection of MOP and MET in urine. Conclusions With the novel optical nano-particle (UCP-NPs), the traditional LF assay was integrated with biosensor based on automated analysis and two kinds of UPT-LF strip were developed to meet the need of on-site qualitative and quantitative detection of MOP and MET. With GC-LF as the reference for qualitative detection and LC-MS/GC-MS for quantitative detection, the comprehensive performances (including detection limit, qualitative accuracy, and quantitative ability) were evaluated systematically. The good qualitative and quantitative detection performance of MOP-UPT-LF and MET-UPT-LF offers a new choice for on-site drug screening.