采用化学共沉淀法制备四氧化三铁(Fe_3O_4)磁性纳米粒子(MNPs),依次用3-氨基丙基三乙氧基硅烷(APTS)、丁二酸酐(SAH)对Fe_3O_4MNPs表面进行修饰,得到羧基功能化的核壳型磁性纳米粒子(Fe_3O_4@APTS·SAH MNPs),分别采用透射电镜(TEM)、磁滞回线、X射线光电子能谱(XPS)和傅里叶红外光谱(FTIR)对其进行了表征。将此纳米粒子修饰在自制的磁性玻碳电极(MGCE)表面,用1-(3-二氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺(NHS)活化纳米粒子表面的羧基,通过与氨基的共价交联,将抗微囊藻毒素-(亮氨酸-精氨酸)(MCLR)抗体(anti-MCLR)固定于该修饰电极上,用牛血清白蛋白(BSA)封闭非特异性吸附位点,构建了一种检测MCLR的电流型免疫传感器。采用直接竞争免疫反应模式,在标记物辣根过氧化物酶(HRP)的MCLR(MCLR-HRP)存在下,利用差分脉冲伏安法(DPV)测定溶液中的微囊藻毒素。在优化的实验条件下,免疫传感器对MCLR的线性测定范围为0.05~100μg/L,检出限为0.01μg/L(S/N=3)。构建的免疫传感器呈现出良好的重现性、稳定性和特异性。将本传感器用于实际水样的测定,加标回收率为94.3%~99.5%。
An immunosensor for detection of microcystin-(leucine-arginine) (MCLR) was constructed. Chemical co-precipitation method was used to synthesize ferriferrous oxide magnetic nanoparticles (Fe304 MNPs). The Fe3 04 MNPs were modified with 3-aminopropyltriethoxysilane (APTS) and succinic anhydride (SAH) in turn to generate carboxyl-functionalized core-shell MNPs ( F%O4@ APTS · SAH MNPs), which were characterized by transmission electron microscope (TEM), hysteresis loop, X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR) , respectively. The Fe3 04 @ APTS · SAH MNPs were modified onto the surface of a home-made magnetic glassy carbon electrode (MGCE). Anti- MCLR antibody (anti-MCLR) was immobilized on the modified MGCE by coupling reaction with ethyl-3- (dimethyl aminopropyl ) carbodiimide ( EDC ) and N-hydroxysuccinimide ( NHS ). Subsequently, bovine serum albumin (BSA) was used to block the non-specific adsorption sites of the electrode. Direct competitive immunoassay format was adopted. In the presence of horseradish peroxidase-conjugated MCLR (MCLR- HRP), MCLR in solution was determined by differential pulse vohammetry (DPV). Under the optimized conditions, MCLR could be detected within a wide linear range of 0.05-100 μg/L with a detection limit of 0.01 μg/L (S/N=3). Moreover, the immunosnssoe exhibited good reproducibility, stability and selectivity. The immunosensor was applied for the determination of MCLR in real water samples with the recoveries from 94.3% to 99.5%.