作　　者： ; ; ; ; ; ; ;

机构地区： 宁波大学

出　　处： 《分析化学》 2013年第6期828-834,共7页

摘　　要： 制备了基于酶-抗体共固定二氧化锆(ZrO2)纳米探针进行信号放大的盐酸特伦克罗瘦肉精(CLB)检测安培免疫传感器。首先,利用ZrO2纳米粒子负载葡萄糖氧化酶(Glucose oxidase,GOD)和CLB抗体(Clen-buterol antibody,anti-CLB),制得纳米探针(ZNPs/GOD-anti-CLB signal probe,简称ZNPG)。同时将聚二烯丙基二甲基氯化铵[Poly(diallyldimethylammonium chloride),PDDA]、氯化血红素(Hemin)和纳米金(AuNPs)层层组装到多壁碳纳米管(MWCNTs)修饰的丝网印刷电极(SPCE)表面,制得SPCE│MWCNTs/(PDDA/Hemin/AuNPs)n电流型传感器,可对H2O2产生还原催化电流。检测CLB时,将样品和ZNPG一起加入预先包埋好CLB的96孔板底部。样品CLB与板底CLB共同竞争结合孔溶液中的纳米探针ZNPG。反应结束后加入葡萄糖,板底ZNPG-CLB免疫复合物上的GOD催化葡萄糖氧化产生H2O2,可通过传感器检测。电流下降值(ΔI)与样品CLB成反比,可用于CLB的定量分析。由于ZNPG上具有高的GOD酶标记密度,故可以显著放大检测信号,提高检测灵敏度。在最佳条件下(pH 7.0,温育时间30 min,温育温度37℃),此传感器对CLB的检测范围为0.003～100μg/L,检测下限为1 ng/L,比酶联免疫分析法(ELISA)法灵敏度提高2个数量级,在猪饲料样品中进行加标回收实验,平均回收率达93.6%,相对标准偏差(RSD)小于2.5%,精密度好。同时该传感器具有可重复使用、灵敏快速,适合于食物样品中痕量CLB现场检测。 A novel amperometic immunosensor for clenbuterol（CLB） detection based on enzyme-antibody co-immobilized zirconium dioxide（ZrO2） nanoprobes for signal amplification was developed.First,ZrO2 nanoparticles（ZNPs） were used to adsorb glucose oxidase（GOD） enzyme and CLB antibody（anti-CLB） to prepare the signal tag（ZNPs/GOD-anti CLB）.Poly（diallyldimethylammonium chloride）（PDDA）,hemin and Au nanoparticles（AuNPs） were assembled layer by layer on the surface of multi-walled carbon nanotubes（MWCNTs）,which was modified by screen-printed electrodes（SPCE）.By this means,the sensor（SPCE │ WCNTs/PDDA/hemin/AuNPs）,which could catalyze H2O2 into generate reduction current,was obtained.When CLB was detected,samples and ZNPG were embedded to the bottom of the 96-well plate in advance.Based on the reaction mechanism of competitive enzyme-linked immunoassay,CLB in samples would compete with slab CLB for the combination with the nanoprobe ZNPG.After reaction,glucose（Glu） was added to the 96-well plate,then the GOD catalyzed Glu to generate H2O2,which could be detected by the immunosensor for quantification.Current decline in value（ΔI） was inversely proportional to a certain concentration range of CLB and under the optimal conditions（pH 7.0,incubation at 37 ℃ for 30 min）,the immunosensor provided an excellent response for CLB ranging from 0.003 μg/L to 100 μg/L with a detection limit of 1 ng/L（3σ）.The sensitivity of this method was 2 orders of magnitude higher than that of the ELISA method due to the high density of GOD on the signal tag,which greatly amplify the detection signal.The average recovery of the standard addition was 93.6% and the relative standard deviation was less than 2.5%,which confirmed that prepared had a high precision.The results showed that the amperometic immunosensor was reusable,sensitive and rapid for on-the site determination of ultra trace amounts of CLB in food samples.

关 键 词： 竞争性酶联免疫分析 瘦肉精 酶 抗体共固定二氧化锆纳米探针 信号放大 安培免疫传感器

领　　域： [理学] [理学]