Kara Pinar, Erdem Arzum, Girousi Stella, Ozsoz Mehmet
Analytical Chemistry Department, Faculty of Pharmacy, Ege University, 35100 Bornova-Izmir, Turkey.
J Pharm Biomed Anal. 2005 Jun 1;38(1):191-5. doi: 10.1016/j.jpba.2004.12.011. Epub 2005 Feb 17.
Electrochemical biosensor for the detection of DNA hybridization using the reduction signal of alpha-naphthol is described. A pencil graphite electrode was used as a working electrode. Capture probes were covalently attached on to the pencil graphite electrode surface (PGE) at the 5' end amino group by using N-(dimethylamino)propyl-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS) as a coupling agent on to PGE. After capture probe immobilization on to PGE surface; probe was hybridized with complementary biotinylated oligonucleotide. Alkaline phosphatase labeled with extravidin (Ex-AP) binds to biotinylated hybrid via biotin-avidin interaction. alpha-Naphthyl phosphate (alpha-NAP) was added and the reaction between alkaline phosphatase (AP) and alpha-NAP was occurred consequently as a substrate of AP, alpha-NAP reduction signal was obtained from this reaction, at -0.100 V by using differential pulse voltammetry (DPV). Other experimental parameters were studied such as; optimizations of hybridization time, and the concentrations of capture probe, biotinylated oligonucleotide and enzyme.
描述了一种利用α-萘酚还原信号检测DNA杂交的电化学生物传感器。使用铅笔石墨电极作为工作电极。通过使用N-(二甲基氨基)丙基-N'-乙基碳二亚胺盐酸盐（EDC）和N-羟基磺基琥珀酰亚胺（NHS）作为偶联剂，将捕获探针共价连接到铅笔石墨电极表面（PGE）的5'端氨基上。在将捕获探针固定在PGE表面后，将探针与互补的生物素化寡核苷酸杂交。用抗生物素蛋白（Ex-AP）标记的碱性磷酸酶通过生物素-抗生物素蛋白相互作用与生物素化杂交体结合。加入α-萘基磷酸酯（α-NAP），碱性磷酸酶（AP）与α-NAP之间发生反应，作为AP的底物，通过差分脉冲伏安法（DPV）在-0.100 V下从该反应中获得α-NAP还原信号。还研究了其他实验参数，如杂交时间的优化以及捕获探针、生物素化寡核苷酸和酶的浓度。
