Dai Zong, Yan Feng, Chen Jin, Ju Huangxian
Department of Chemistry, Institute of Analytical Science, State Key Laboratory of Coordination Chemistry, Nanjing University, Nanjing 210093, P. R. China.
Anal Chem. 2003 Oct 15;75(20):5429-34. doi: 10.1021/ac034213t.
A novel strategy for immunoassay and the preparation of reagentless immunosensors was proposed. This strategy was based on the immobilization of antigen and the direct electrochemistry of horseradish peroxidase (HRP) that was labeled to an antibody. A reagentless immunosensor for carcinoma antigen-125 (CA 125) determination was developed. The immunosensor was prepared by immobilizing CA 125 with titania sol-gel on a glassy carbon electrode by the vapor deposition method. The incubation of the immunosensor in phosphate buffer solution (PBS) including HRP-labeled CA 125 antibody led to the formation of a HRP-modified surface. The immobilized HRP displayed its direct electrochemistry with a rate constant of 3.04 +/- 1.21 s(-1). With a competition mechanism, a differential pulse voltammetric determination method for CA 125 was established by the peak current decrease of the immobilized HRP. The current decrease resulted from the competitive binding of the CA 125 in sample solution and the immobilized CA 125 to the limited amount of HRP-labeled CA 125 antibody. Under optimal conditions, the current decrease was proportional to CA 125 concentration ranging from 2 to 14 units mL(-1) with a detection limit of 1.29 units mL(-1) at a current decrease by 10%. The CA 125 immunosensor showed good accuracy and acceptable precision and fabrication reproducibility with intraassay CVs of 8.7 and 5.5% at 8 and 14 units mL(-1) CA 125 concentrations, respectively, and interassay CV of 19.8% at 8 units mL(-1). The storage stability was acceptable in a pH 7.0 PBS at 4 degrees C for 15 days. The proposed method provided a new promising platform for clinical immunoassay.
提出了一种免疫分析和无试剂免疫传感器制备的新策略。该策略基于抗原的固定以及标记在抗体上的辣根过氧化物酶（HRP）的直接电化学。开发了一种用于测定癌抗原125（CA 125）的无试剂免疫传感器。通过气相沉积法将CA 125与二氧化钛溶胶 - 凝胶固定在玻碳电极上制备免疫传感器。将免疫传感器在含有HRP标记的CA 125抗体的磷酸盐缓冲溶液（PBS）中孵育，形成HRP修饰的表面。固定化的HRP表现出其直接电化学，速率常数为3.04 +/- 1.21 s(-1)。基于竞争机制，通过固定化HRP的峰电流降低建立了CA 125的差分脉冲伏安测定方法。电流降低是由于样品溶液中的CA 125与固定化的CA 125竞争结合有限量的HRP标记的CA 125抗体所致。在最佳条件下，电流降低与CA 125浓度在2至14单位mL(-1)范围内成正比，在电流降低10%时检测限为1.29单位mL(-1)。CA 125免疫传感器在CA 125浓度为8和14单位mL(-1)时，批内CV分别为8.7%和5.5%，批间CV为19.8%（8单位mL(-1)），显示出良好的准确性、可接受的精密度和制备重现性。在4℃的pH 7.0 PBS中储存稳定性在15天内是可接受的。该方法为临床免疫分析提供了一个新的有前景的平台。
