Preechaworapun Anchana, Dai Zong, Xiang Yun, Chailapakul Orawon, Wang Joseph
Department of Chemical and Material Engineering, Chemistry and Biochemistry, Biodesign Institute, Arizona State University, Tempe, AZ 85287-5801, USA.
Talanta. 2008 Jul 15;76(2):424-31. doi: 10.1016/j.talanta.2008.03.025. Epub 2008 Mar 25.
In this paper, we have critically evaluated the electrochemical behavior of the products of seven substrates of the enzyme label, alkaline phosphate, commonly used in electrochemical immunosensors. These products (and the corresponding substrates) include indigo carmine (3-indoyl phosphate), hydroquinone (hydroquinone diphosphate), 4-nitrophenol (4-nitrophenol phosphate), 4-aminophenol (p-aminophenyl phosphate), 1-naphthol (1-naphthyl phosphate), phenol (phenyl phosphate), and L-ascorbic acid (2-phospho-L-ascorbic acid). Cyclic voltammetry and amperometry of these products were carried out at glassy carbon (GC), screen-printed carbon (SPC) and gold (Au) electrodes, respectively. Among the products, L-ascorbic acid showed the most sensitive (24.8 microA cm(-2), 12.0 microA cm(-2), and 48.0 microA cm(-2) of 100 microM ascorbic acid at GC, SPC, and Au electrodes, respectively) and well-defined amperometric response at all electrodes used, making 2-phospho-l-ascorbic acid the best substrate in electrochemical detection involving an alkaline phosphatase (ALP) enzyme label. The 2-phospho-L-ascorbic acid is also commercially available and inexpensive. Therefore, it was the best choice for electrochemical detection using ALP as label. Using mouse IgG as a model, an ALP enzyme-amplified sandwich-type amperometric immunosensor was constructed. The immunosensor was designed by electropolymerization of o-aminobenzoic acid (o-ABA) conductive polymer on the surface of GC, SPC, and Au electrodes. The anti-mouse IgG was subsequently attached on the electrode surface through covalent bonding between IgG antibody and the carboxyl groups from poly(o-ABA). Using 2-phospho-L-ascorbic acid as a substrate, the poly(o-ABA)/Au immunosensor produced the best signal (about 297 times of current density response ratio between 1000 ng mL(-1) and 0 ng mL(-1) of mouse IgG), demonstrating that amperometric immunosensors based on a conducting polymer electrode system were sensitive to concentrations of the mouse IgG down to 1 ng mL(-1), with a linear range of 3-200 ng mL(-1) (S.D.<2; n=3), and very low non-specific adsorption.
在本文中，我们严格评估了常用于电化学免疫传感器的酶标记物碱性磷酸酶的七种底物的产物的电化学行为。这些产物（以及相应的底物）包括靛蓝胭脂红（3-吲哚基磷酸酯）、对苯二酚（对苯二酚二磷酸酯）、4-硝基苯酚（4-硝基苯磷酸酯）、4-氨基苯酚（对氨基苯磷酸酯）、1-萘酚（1-萘基磷酸酯）、苯酚（苯磷酸酯）和L-抗坏血酸（2-磷酸-L-抗坏血酸）。分别在玻碳（GC）电极、丝网印刷碳（SPC）电极和金（Au）电极上对这些产物进行循环伏安法和安培法检测。在这些产物中，L-抗坏血酸表现出最灵敏的安培响应（在GC电极、SPC电极和Au电极上，100 μM抗坏血酸的响应电流分别为24.8 μA cm⁻²、12.0 μA cm⁻²和48.0 μA cm⁻²），并且在所有使用的电极上响应都很明确，这使得2-磷酸-L-抗坏血酸成为涉及碱性磷酸酶（ALP）酶标记的电化学检测中最佳的底物。2-磷酸-L-抗坏血酸在市场上也可买到且价格便宜。因此，它是使用ALP作为标记物进行电化学检测的最佳选择。以小鼠IgG为模型，构建了一种ALP酶放大夹心型安培免疫传感器。该免疫传感器是通过在GC电极、SPC电极和Au电极表面电聚合邻氨基苯甲酸（o-ABA）导电聚合物来设计的。随后，通过IgG抗体与聚（o-ABA）的羧基之间的共价键合，将抗小鼠IgG附着在电极表面。以2-磷酸-L-抗坏血酸为底物，聚（o-ABA）/Au免疫传感器产生了最佳信号（小鼠IgG浓度在1000 ng mL⁻¹和0 ng mL⁻¹之间时，电流密度响应比约为297倍），表明基于导电聚合物电极系统的安培免疫传感器对低至1 ng mL⁻¹的小鼠IgG浓度敏感，线性范围为3 - 200 ng mL⁻¹（标准偏差<2；n = 3），且非特异性吸附非常低。
