Sensitive detection of atrazine in tap water using TELISA.
Sensitive detection of atrazine in tap water using TELISA.
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DOI：
10.1039/c5an00636h
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发表时间：
2015-07
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通讯作者：
Zhiwei Qie;Jialei Bai;B. Xie;Lin Yuan;Nan Song;Yuan Peng;Xianjun Fan;Huanying Zhou;Fengchun Chen;Shuang Li;Baoan Ning;Zhixian Gao
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作者：
Zhiwei Qie;Jialei Bai;B. Xie;Lin Yuan;Nan Song;Yuan Peng;Xianjun Fan;Huanying Zhou;Fengchun Chen;Shuang Li;Baoan Ning;Zhixian Gao
建立了一种高灵敏度的流动注射热酶联免疫分析法（TELISA），用于自来水中阿特拉津（ATZ）的快速检测。ATZ和β-内酰胺酶标记的ATZ用于使用单克隆抗体（mAb）的竞争性免疫测定。柱外液相竞争后，将mAb捕获在Protein G Sepharose™ 4 Fast Flow（PGSFF）柱支持材料上。注射的β-内酰胺酶底物氨苄青霉素被柱结合的ATZ-β-内酰胺酶降解，产生可检测的热信号。优化了几个测定参数，包括底物浓度、流速和再生条件，以及mAb和ATZ-β稀释比和浓度。测定线性范围为0.73-4.83 ng/mL，检测限为0.66 ng/mL。一个完整的热信号需要10分钟的生成，周期时间小于40分钟。结果是可重复的和稳定的。ATZ加标自来水样品的回收率为103%-116%，与UHPLC-MS/MS测量值相关。我们将灵敏度的显著提高归因于以下因素：（i）通过固定化蛋白G在柱上捕获已经形成的免疫复合物，这消除了抗体的化学固定;（ii）离柱预孵育允许在接近理想的条件下形成免疫复合物;和（iii）多种缓冲液可用于在一种情况下增强免疫复合物的形成和在另一种情况下使酶活性最大化。此外，该方案创建了一个通用的测定平台，其中在酶热敏电阻（ET）检测器中捕获后，在离柱孵育和检测中进行传感，这开辟了检测抗体可用的任何抗原的可能性。
A highly sensitive flow injection analysis (FIA)-based thermal enzyme-linked immunoassay, TELISA, was developed for the rapid detection of atrazine (ATZ) in tap water. ATZ and β-lactamase-labeled ATZ were employed in a competitive immunoassay using a monoclonal antibody (mAb). After the off-column liquid-phase competition, the mAb was captured on the Protein G Sepharose™ 4 Fast Flow (PGSFF) column support material. Injected β-lactamase substrate ampicillin was degraded by the column-bound ATZ-β-lactamase, generating a detectable heat signal. Several assay parameters were optimized, including substrate concentration, flow rates and regeneration conditions, as well as the mAb and ATZ-β dilution ratios and concentrations. The assay linear range was 0.73-4.83 ng mL(-1) with a detection limit of 0.66 ng mL(-1). An entire heat signal requires 10 min for generation, and the cycle time is less than 40 min. The results were reproducible and stable. ATZ-spiked tap water samples exhibited a recovery rate of 103%-116%, which correlated with the UHPLC-MS/MS measurements. We attributed this significant increase in sensitivity over our previously published work to the following factors: (i) the capture of already-formed immune complexes on the column via immobilized Protein G, which eliminated chemical immobilization of the antibody; (ii) off-column preincubation allows the formation of immune complexes under nearly ideal conditions; and (iii) multiple buffers can be used to, in one case, enhance immune-complex formation and in the other to maximize enzymatic activity. Furthermore, the scheme creates a universal assay platform in which sensing is performed in the off-column incubation and detection after capture in the enzyme thermistor (ET) detector, which opens up the possibility of detecting any antigen for which antibodies were available.