4 种硝基呋喃代谢物复合荧光免疫层析试纸条的制备

为建立一种基于量子点荧光免疫层析技术快速检测4 种硝基呋喃类代谢物的方法，采用N-羟基琥珀酰亚胺（N-hydroxysuccinimide，NHS）、1-乙基-3-（3-二甲氨基丙基）碳二亚胺盐酸盐（1-ethyl-3-（3-dimethylaminopropyl）carbodiimide hydrochloride，EDC）法合成4 种硝基呋喃类代谢物的人工抗原，并融合制备对应的单克隆抗体，通过EDC-NHS一步法将量子点微球（quantum dot submicrobeads，QBs）分别与3-氨基-2-恶唑烷酮（3-amino-2-oxazolidinone，AOZ）、5-甲基吗啉-3-氨基-2-唑烷基酮（5-morpholine-methyl-3-amino-2-oxazolidinone，AMOZ）、1-氨基-2-乙内酰（1-aminohydantoin，AHD）和氨基脲（semicarbazid，SEM）偶联，得到相应的QBs探针，在此基础上制备复合荧光免疫层析试纸条。结果表明：AOZ、AHD、SEM和AMOZ的检出限分别为0.4、0.4、0.5、0.5 μg/L；通过加标回收实验对批内和批间的重复性和准确度进行评价，得出该试纸条的批内检测回收率为80%～110%，批内变异系数在15%以内，批间回收率为80%～100%，批间变异系数在15%以内；且AOZ、AHD、SEM和AMOZ之间的交叉反应率小于0.1%，说明成功将荧光免疫层析技术和多元检测技术相结合，应用于同时检测4 种硝基呋喃类代谢物，且试纸条的灵敏度和准确性高、特异性好。

Preparation of a Fluorescent Immunochromatographic Strip for Simultaneous Detection of Four Nitrofuran Metabolites

This research was done in order to establish a rapid quantum dot (QD)-based immunochromatographic assay (ICA) for the simultaneous detection of four nitrofuran metabolites (3-amino-2-oxazolidinone (AOZ), 5-morpholine-methyl- 3-amino-2-oxazolidinone (AMOZ), 1-aminohydantoin (AHD) and semicarbazid (SEM)). Immunogens were synthesized by N-hydroxysuccinimide (NHS)/ 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) method, and monoclonal antibodies were prepared by cell fusion. QD microspheres were coupled with AOZ, AHD, SEM and AMOZ, respectively by one-step method using EDC and NHS to obtain quantum dot submicrobeads (QBs) probes. On this basis, a fluorescent immunochromatographic strip was prepared. The results showed that the detection limits of this test strip for AOZ, AHD, SEM and AMOZ were 0.4, 0.4, 0.5 and 0.5 μg/L, respectively. The repeatability and accuracy within and between batches were evaluated by spiked recovery experiments, and the results showed that the recovery rates were 80%–110% and 80%–100% with coefficient of variations of less than 15% for intra- and inter-batch assays, respectively. The cross-reactivity among AOZ, AHD, SEM and AMOZ was less than 0.1%. In conclusion, the combination of fluorescent immunochromatography and multivariate detection technique can be successfully applied to simultaneously detect four nitrofuran metabolites with high sensitivity, accuracy and specificity.