基于纳米金辅助信号生成顺序堆积的毛细管电泳免疫分析研究

Capillary Electrophoresis Immunoassay by Gold Nanoparticles Assisted Signal Generation and Sequential Stacking

短裸甲藻毒素(BTX)是具有极强毒性的生物毒素,能够通过食物链传递引起人类中毒.由于该毒素没有光学和电化学信号,检测十分困难.本工作利用纳米金作为载体,将辣根过氧化物酶(HRP)和毒素抗体同时固定到纳米金表面,通过HRP催化H_2O_2氧化邻氨基酚(OAP)产生的电化学信号检测样品中的毒素,增大纳米金表面HRP和抗体的物质的量比使电化学信号得到极大增强.免疫反应样品电动进样引入分离毛细管中,在毛细管入口端进行顺序堆积在线富集,使检测灵敏度进一步提高.该方法通过纳米金辅助信号生成和顺序堆积在线富集技术实现了对扇贝样品中BTX-B的快速灵敏检测,线性范围为0.1~120 ng/mL,检出限为26 ng/L,检出限比常规酶联免疫分析(ELISA)法低365倍.

Brevetoxins （BTXs） are highly toxic biotoxin and can cause human intoxication through food chain. The detec- tion of brevetoxins is very difficult due to lack of optical and electrochemical （EC） signal. In this work, we developed an ultrasensitive capillary electrophoresis （CE） immunoassay and EC method for the determination of BTX-B by gold nanopar- ticles （AuNPs） assisted signal generation and sequential stacking concentration. The AuNPs were synthesized by sodium citrate reduction of HAuCl4 in water. The AuNPs were conjugated with horseradish peroxidase （HRP） and antibody （Ab） to immobilize the HRP and Ab on the AuNPs surface with the molar ratio of HRP/Ab of 9/1. The Ab conjugated on the AuNPs surface incubated with limited amount of BTX-B in standard solution or shellfish samples to produce immunocomplex on the basis of the noncompetitive irnmunoreactions. Before sample injection, a NaOH plug with I 0 cm height difference for 150 s was hydrodynamically injected into the separation capillary. After incubation for 40 min at room temperature, the immune sample was then electrokinetically injected into the capillary at 10 kV for 330 s. The positively charged analytes migrated rapidly into the capillary and were neutralized and stacked at the boundary between sample and NaOH plug, which led to the first preconcentration. After sample loading, the capillary inlet vial was changed to low-pH buffer solution, and H＋ in the buffer solution moved rapidly into the capillary toward cathode across the neutral analytes zone. The neutralized analytes were positively charged again and the injected analytes were further condensed. Next, the formed immunocomplex, unbound HRP-Au-Ab probe and the excess HRP were separated by CE and sensitively detected by EC detection. AuNPs were used as carriers of HRP and Ab in order to carry out EC detection with the EC signals derived from catalytic reaction of the carried HRP to the H202/o-aminophenol system. Simultaneously, the EC signal was highly amplified by improving the HRP/Ab molar ratio on the surface of AuNPs. The proposed method by AuNPs assisted signal generation and on-line sequential con- centration realized the sensitive and rapid determination of BTX-B in shellfish samples. In the range between 0.1 and 120 ng/mL, the assay allowed quantitative determination of BTX-B and the limit of detection （LOD） was 26 ng/L. The LOD was 365-fold lower than ELISA method. The amplified sensitivity was enhanced by high HRP/Ab ratio at AuNPs surface and sequential preconcentration. The proposed method provides a convenient and sensitive analytical approach for the determina- tion of trace BTX in complex samples.