Urraca Javier L, Moreno-Bondi María C, Orellana Guillermo, Sellergren Börje, Hall Andrew J
Department of Analytical and Organic Chemistry, Universidad Complutense de Madrid, E-28040 Madrid, Spain.
Anal Chem. 2007 Jul 1;79(13):4915-23. doi: 10.1021/ac070277i. Epub 2007 Jun 6.
An automated molecularly imprinted sorbent based assay (MIA) for the rapid and sensitive analysis of penicillin-type beta-lactam antibiotics (BLAs) has been developed and optimized. The polymers were prepared using penicillin G procaine salt as template (PENGp) and a stoichiometric quantity of a urea-based functional monomer to target the single oxyanionic species in the template molecule. Highly fluorescent competitors (emission quantum yields of 0.4-0.95), molecularly engineered to contain pyrene labels while keeping intact the 6-aminopenicillanic acid moiety for efficient recognition by the cross-linked polymers, have been tested as analyte analogues in the competitive assay. Pyrenemethylacetamido penicillanic acid (PAAP) was the tagged antibiotic providing for the highest selectivity when competing with PenG for the specific binding sites in the molecularly imprinted polymer (MIP). Upon desorption from the MIP, the emission signal generated by the PAAP was related to the antibiotic concentration in the sample. The 50% binding inhibition concentration of penicillin G standard curves was at 1.81 x 10(-6) M PENG, and the detection limit was 1.97 x 10(-7) M. The sensor showed a dynamic range (normalized signal in the 20 to 80% range) from 6.80 x 10(-7) to 7.21 x 10(-6) M (20-80% binding inhibition) PENG in acetonitrile:HEPES buffer 0.1 M at pH 7.5 (40:60, v/v) solutions. Competitive binding studies demonstrated various degrees of cross-reactivity with penicillin-type beta-lactam antibiotics such as ampicillin (71%), oxacillin (66%), penicillin V (56%), amoxicillin (13%), and nafcillin (46%) and a lower response to other isoxazolyl penicillins such as cloxacillin (27%) and dicloxacillin (16%). The total analysis time was 14 min per determination, and the MIP reactor could be reused for more than 150 cycles without significant loss of recognition. The automatic MIA has been successfully applied to the direct analysis of penicillin G in spiked urine samples with excellent recoveries (mean value 92%). Results displayed by comparative analysis of the optimized MIA with a chromatographic procedure for penicillin G showed excellent agreement between both methods.
已开发并优化了一种基于自动分子印迹吸附剂的分析方法（MIA），用于快速、灵敏地分析青霉素类β-内酰胺抗生素（BLAs）。以普鲁卡因青霉素G盐为模板（PENGp），使用化学计量的基于尿素的功能单体来靶向模板分子中的单氧阴离子物种制备聚合物。经过分子工程改造，含有芘标签同时保持6-氨基青霉烷酸部分完整以实现交联聚合物高效识别的高荧光竞争物（发射量子产率为0.4 - 0.95），已在竞争分析中作为分析物类似物进行测试。芘甲基乙酰氨基青霉烷酸（PAAP）是标记抗生素，在与青霉素G竞争分子印迹聚合物（MIP）中的特异性结合位点时具有最高的选择性。从MIP上解吸后，PAAP产生的发射信号与样品中的抗生素浓度相关。青霉素G标准曲线的50%结合抑制浓度为1.81×10⁻⁶ M PENG，检测限为1.97×10⁻⁷ M。该传感器在乙腈：0.1 M HEPES缓冲液（pH 7.5，40:60，v/v）溶液中显示的动态范围（归一化信号在20%至80%范围内）为6.80×10⁻⁷至7.21×10⁻⁶ M（20 - 80%结合抑制）PENG。竞争结合研究表明，该方法与青霉素类β-内酰胺抗生素如氨苄西林（71%）、苯唑西林（66%）、青霉素V（56%）、阿莫西林（13%）和萘夫西林（46%）存在不同程度的交叉反应，而对其他异恶唑青霉素如氯唑西林（27%）和双氯西林（16%）的响应较低。每次测定的总分析时间为14分钟，MIP反应器可重复使用超过150次而识别能力无明显损失。自动MIA已成功应用于加标尿液样品中青霉素G的直接分析，回收率极佳（平均值92%）。将优化后的MIA与青霉素G的色谱分析方法进行对比分析，结果显示两种方法具有极好的一致性。
