利用树枝状分子-金纳米粒子复合物修饰电极和金纳米粒子标记物构建电化学免疫传感器,用于污泥中大肠杆菌的检测。首先在玻碳电极表面电聚合对氨基苯甲酸,通过共价作用结合第Ⅳ代氨基末端的树枝状分子(G4-PAMAM),并在其内部载入金纳米粒子,制备修饰电极(GCE/p-ABA/PAMAM(AuNPs)),用于固定大肠杆菌。采用硫堇作为电活性物质包被金纳米粒子,用于标记二抗制备金纳米粒子标记物(Ab2-Au-Th)。通过抗原-抗体之间的特异性识别作用,将一抗、金纳米粒子标记物依次修饰在电极表面,用差分脉冲伏安法测定硫堇产生的电流信号,实现对大肠杆菌的检测。在优化的实验条件下,响应电流与大肠杆菌浓度的对数在1.0×102~1.0×106 cfu/mL范围内呈线性关系,检出限为70 cfu/mL(S/N=3)。利用本方法检测污水处理厂的不同污泥样品中的大肠杆菌,回收率为89.4%~105.8%。
A novel electrochemical immunosensor was developed for detection of E. coli in urban sludge based on dendrimer-encapsulated Au and enhanced gold nanoparticle labels. p-Aminobenzoic acid (p-ABA) was electropolymerized on glassy carbon electrode (GCE) surface to introduce abundant carboxyl groups. G4-polyamidoamine dendrimers (PAMAM) were covalently attached onto electrode surface through the formation of amide bonds between amino groups of dendrimer and carboxyl groups of poly-p-ABA. Subsequently, Au(Ⅲ) ions were coordinated in the interior of dendrimer and then reduced to form gold nanoparticles (AuNPs). The resulting electrode (GCE/p-ABA/PAMAM(AuNPs)) provided numerous amino groups to allow highly dense immobilization of E. coli, and facilitated the improvement of electrochemical responses. The rabbit anti-E. coli polyclonal antibody (Ab1) was captured by the electrode surface-confined E. coli, followed by the attachment of the enhanced gold nanoparticle labels (Ab2-Au-Th) featuring thionine (Th) as signal-generating molecule. The analysis of E. coli was performed by electrochemical detection of Th in the bound labels on the electrode surface. Under the optimal experimental conditions, a linear relationship between the peak current of Th and the logarithmic value of E. coli concentration ranging from 1.0×102 cfu/mL to 1.0×106 cfu/mL was obtained with a detection limit of 70 cfu/mL (S/N=3). The proposed strategy was also used to determine E. coli in samples obtained from waste water treatment plant, and the recoveries of standard additions were in the range of 89.4% to 105.8%. The results confirmed that the electrochemical immunoassay gave a useful protocol for E. coli analysis with high sensitivity, specificity and acceptable accuracy, and thus could potentially become a promising technique for estimating the feasibility of sludge recycling.
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