上转发光技术快速检测尿液中氯胺酮
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摘要
目的利用上转发光免疫层析技术(UPT-LF)建立一种可在现场快速定量检测尿液中氯胺酮(Ketamine,KET)的方法并对其进行系统评价。方法利用竞争法免疫层析原理,以上转发光材料(UCP)作为生物示踪物,建立快速检测尿液中KET含量的上转发光免疫层析定量检测方法(KET-UPT-LF),并通过专用的便携UPT生物传感器分析进而计算出氯胺酮浓度。通过系列浓度标准品检测评价其敏感性、精密性及线性响应。收集经LC-MS鉴定的现场尿液样本,同时进行KET-UPT-LF及胶体金的检测,一方面通过与胶体金定性检测结果的比较来评价KET-UPT-LF对于实际尿液样本中KET的定性检测能力,另一方面通过与LC-MS的定量检测结果比较来评价KET-UPT-LF定量检测性能。结果通过对系列浓度标准品检测结果分析,KET-UPT-LF敏感性为93.75 ng/m L,线性范围为93.75~6 000 ng/m L(r=-0.99448,P<0.0005)。在现场样品检测评价中,同一样本的KET-UPT-LF和胶体金结果在定性分析,其结果是一致的。KET-UPT-LF与定量确证方法 LC-MS的定量检测结果经t检验无显著差异。结论本研究建立的氯胺酮上转发光免疫层析定量检测方法(KET-UPT-LF),在满足胶体金等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,且用户友好性优于LC-MS等定量确证方法,为现场快速定量检测尿液中的毒品提供了技术保障。
Objective To develop and evaluate an up-converting-phosphor-technology-based lateral flow(UPT-LF) assay for fast quantitative detection of Ketamine(KET) in urine. Methods KET-UPT-LF was established with the up-converting phosphor(UCP) nano-particles as the bio-label of competitive-mode-based LF assay. The results were analyzed by a specific portable UPT-based biosensor with which the concentration of ketamine was measured. The detectivity, precision and linearity were evaluated by testing the samples with serial standard concentrations. The urine samples were used to assess the KETUPT-LF's both qualitative and quantitative detection performance with immune colloidal gold technique and LC-MS as the respective reference. Results According to the analysis of urine samples detection, the sensitivity of KET-UPT-LF was 93.75 ng/m L and linearity range was 93.75-6000 ng/m L(r =-0.99448,P < 0.0005). The performance of qualitative and quantitative detection could meet the requirements of the on-site rapid detection of KET in urine. There was no significant difference between KET-UPT-LF and immune colloidal gold technique for qualitative detection and neither for quantitative detection between KET-UPT-LF and LC-MS. Conclusion The method of KET-UPT-LF for urine test is not only as quick and easy as colloidal gold technique but also provides quantitative detection with a more user-friendly feature compared to LC-MS, offering an alternative to realize on-site rapid detection of KET in urine quantitatively.
Objective To develop and evaluate an up-converting-phosphor-technology-based lateral flow(UPT-LF) assay for fast quantitative detection of Ketamine(KET) in urine. Methods KET-UPT-LF was established with the up-converting phosphor(UCP) nano-particles as the bio-label of competitive-mode-based LF assay. The results were analyzed by a specific portable UPT-based biosensor with which the concentration of ketamine was measured. The detectivity, precision and linearity were evaluated by testing the samples with serial standard concentrations. The urine samples were used to assess the KETUPT-LF's both qualitative and quantitative detection performance with immune colloidal gold technique and LC-MS as the respective reference. Results According to the analysis of urine samples detection, the sensitivity of KET-UPT-LF was 93.75 ng/m L and linearity range was 93.75-6000 ng/m L(r =-0.99448,P < 0.0005). The performance of qualitative and quantitative detection could meet the requirements of the on-site rapid detection of KET in urine. There was no significant difference between KET-UPT-LF and immune colloidal gold technique for qualitative detection and neither for quantitative detection between KET-UPT-LF and LC-MS. Conclusion The method of KET-UPT-LF for urine test is not only as quick and easy as colloidal gold technique but also provides quantitative detection with a more user-friendly feature compared to LC-MS, offering an alternative to realize on-site rapid detection of KET in urine quantitatively.
引文
[1]杨瑞琴.液相色谱-质谱联用技术在滥用药物检验中的应用.中国人民公安大学学报(自然科学版)2007,(4):8-11.
[2]陈礼莉,廖林川,李雯佳,等.血液、尿液中氯胺酮及其代谢物去甲氯胺酮的HPLC分析[J].法医学杂志,2008,(1):38-42.
[3]贾娟,王玉瑾,陈朝阳,等.氯胺酮薄层色谱扫描检测研究[J].山西医科大学学报,2005,36(1):69-70.
[4]林晓冬,黄向东,张敏,等.新型毒品“K”粉的检验[J].第三届全国毒物分析学术交流会论文选[C],北京:中国人民公安大学出版社,2000:176-177.
[5]曾立波,陈连康,胡小龙,等.胶体金标记苯丙胺单克隆抗体免疫试剂盒研究[J].中国司法鉴定,2007,(6):31-33.
[6]Pai-Sheng Cheng,Chien-Yu Fu,Choung-Huei Lee,et al.GCMS quantification of ketamine,norketamine,and dehydronorketamine in urine specimens and comparative study using ELISA as the preliminary test methodology[J].J Chromatogr B Analyt Technol Biomed Life Sci,2007,852(1-2):443-449.
[7]Zijlmans HJ,Bonnet J,Burton J,et al.Detection of cell and tissue surface antigens using up-converting phosphors:a new reporter technology[J].Anal Biochem,1999,267(1):30-36.
[8]Hampl J,Hall M,Mufti NA,et al.Upconverting phosphor reporters in immunochromatographic assays.Anal Biochem,2001,288(2):176-187.
[9]刘晓,林承喜,张平平,等.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[J].刑事技术,2015,40(1):28-34.
[10]Niedbala R.S.,Vail T.L.,Feindt H.Multiphoton up-converting phosphors for use in rapid immunoassays,in in-vitro diagnostic instrumentation[J].Editor,Proceedings of Spie,2000,3913:193-203.
[11]Kardos K.,Keith W.,Niedbala R S.Up-converting reporters for biological and other assays[P],2001,United States Patent:6312914
[12]周蕾.基于上转换发光技术生物传感器的研制[D]:[硕士学位论文].北京:中国人民解放军军事医学科学院,2004.
[13]Yan Z,Zhou L,Zhao Y,et al.Rapid quantitative detection of Yersinia pestis by lateral-flow immunoassay and up-converting phosphor technology-based biosensor[J].Sensors and Actuators b-Chemical,2006,119(2):656-663.
[14]张大雷,张吉林,才志成,等.GC/MS衍生化法检测血中4-溴-2,5-二甲氧基苯乙胺[J].刑事技术,2015,40(1):40-44.
[2]陈礼莉,廖林川,李雯佳,等.血液、尿液中氯胺酮及其代谢物去甲氯胺酮的HPLC分析[J].法医学杂志,2008,(1):38-42.
[3]贾娟,王玉瑾,陈朝阳,等.氯胺酮薄层色谱扫描检测研究[J].山西医科大学学报,2005,36(1):69-70.
[4]林晓冬,黄向东,张敏,等.新型毒品“K”粉的检验[J].第三届全国毒物分析学术交流会论文选[C],北京:中国人民公安大学出版社,2000:176-177.
[5]曾立波,陈连康,胡小龙,等.胶体金标记苯丙胺单克隆抗体免疫试剂盒研究[J].中国司法鉴定,2007,(6):31-33.
[6]Pai-Sheng Cheng,Chien-Yu Fu,Choung-Huei Lee,et al.GCMS quantification of ketamine,norketamine,and dehydronorketamine in urine specimens and comparative study using ELISA as the preliminary test methodology[J].J Chromatogr B Analyt Technol Biomed Life Sci,2007,852(1-2):443-449.
[7]Zijlmans HJ,Bonnet J,Burton J,et al.Detection of cell and tissue surface antigens using up-converting phosphors:a new reporter technology[J].Anal Biochem,1999,267(1):30-36.
[8]Hampl J,Hall M,Mufti NA,et al.Upconverting phosphor reporters in immunochromatographic assays.Anal Biochem,2001,288(2):176-187.
[9]刘晓,林承喜,张平平,等.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[J].刑事技术,2015,40(1):28-34.
[10]Niedbala R.S.,Vail T.L.,Feindt H.Multiphoton up-converting phosphors for use in rapid immunoassays,in in-vitro diagnostic instrumentation[J].Editor,Proceedings of Spie,2000,3913:193-203.
[11]Kardos K.,Keith W.,Niedbala R S.Up-converting reporters for biological and other assays[P],2001,United States Patent:6312914
[12]周蕾.基于上转换发光技术生物传感器的研制[D]:[硕士学位论文].北京:中国人民解放军军事医学科学院,2004.
[13]Yan Z,Zhou L,Zhao Y,et al.Rapid quantitative detection of Yersinia pestis by lateral-flow immunoassay and up-converting phosphor technology-based biosensor[J].Sensors and Actuators b-Chemical,2006,119(2):656-663.
[14]张大雷,张吉林,才志成,等.GC/MS衍生化法检测血中4-溴-2,5-二甲氧基苯乙胺[J].刑事技术,2015,40(1):40-44.