微孔板生物芯片测定蜂蜜中四环素残留方法的研究及应用

目的: 采用微孔板生物芯片可视化分析技术建立一种内标法蛋白免疫分析新方法,实现单孔内测定蜂蜜中四环素的残留定量. 方法: 研究中根据间接竞争法反应原理,将四环素人工抗原和纳米银标记的羊抗兔IgG以微阵列形式固定于微孔板底部,制备成微孔板生物芯片阵列,并依次与四环素单克隆抗体、纳米银标记羊抗鼠IgG反应,最后用纳米增强法显色,并采用可视化芯片扫描仪对微孔板显色结果进行快速扫描成像,图像采用相关软件处理. 结果: 该法的检测限可达0.8 ng · mL^-1,线性范围为0.8~19.2 ng · mL^-1,R²=0.971,回收率达80%~120%,RSD<10% . 结论: 本法简便快速,易操作,高通量,能满足日常大规模样品的初筛.

Objective: To develop an internal standard protein immunoassay methods with visual microplate biochip,so as to achieve the quantitative determination of residues of tetracycline in honey. Methods: Artificial antigen of tetracycline and AgNPs(silver nanoparticles)labeled goat anti-rabbit IgG were respectively immobilized as arrayed spots on the bottom of each wells in the microplate.Monoclonal antibodies against tetracycline,second antibody(AgNPs labeled goat anti-mouse IgG )and silver enhancer were added in the reaction wells of the microplate in turn.Second antibody(AgNPs labeled goat anti-mouse IgG)recognizing the monclonal antibodies that coupled with the antigen spots,and spoted AgNPs(silver nanoparticles)labeled goat anti-rabbit IgG was employed,which could generate gray signals after the addition of the silver enhancer.The signals were detected with visual scanner. Results: According to the correlation relationship between the concentration of free tetracycline and the gray of signal as well as the internal standard curve,the tetracycline could be quantitatively assayed in the range from 0.8 to 19.2 ng · mL^-1 in each detection well(R²=0.971),and the detection limit was 0.8 ng · mL^-1.The sample average recovery was 80%-120%,with the RSD<10%. Conclusion: The method is simple,accurate and enjoys high throughput,which can be applied for the routing screen.