电化学生物传感器 2012

A novel sensitive DNA-biosensor for detection of a carcinogen, Sudan II, using electrochemically treated pencil graphite electrode by voltammetric methods.

Talanta Ensafi AA, Rezaei B, Amini M, Heydari-Bafrooei E
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组成图示

A novel sensitive DNA-biosensor for d... 传感器构成示意图

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传感器类型

电化学生物传感器

检测对象

苏丹红II(Sudan II);样品基质为辣椒酱、番茄酱等食品样品(乙醇提取液)及醋酸/磷酸缓冲溶液。

检测原理

DNA传感器中,Sudan II的平面芳香环嵌入ds-DNA碱基对之间,使DNA结构改变并屏蔽/损伤鸟嘌呤(G)和腺嘌呤(A)氧化位点。DPV扫描时,G(约+1.3 V)和A(约+1.0 V)氧化峰电流随Sudan II浓度增加而降低,峰电流变化量ΔI与浓度成正比,实现定量。溶液相中,Sudan II先与ds-DNA结合,再吸附到预处理PGE上,同样通过G/A信号差检测。PPGE分支中,PGE经循环伏安预处理(−0.20~+2.00 V)引入表面官能团,增强Sudan II吸附;在PBS(pH 4.0)中开电路搅拌富集300 s后,DPV扫描产生阳极剥离氧化峰(约+0.095 V),峰电流随浓度线性增加。该方法依靠DNA嵌入识别和表面吸附富集放大,无HCR/RCA等核酸扩增。

检测灵敏度

ds-DNA修饰PGE(鸟嘌呤): 线性范围 0.5–6.0 μg mL−1;斜率 −0.098 μA mL μg−1;R^2 = 0.9958;LOD 0.43 μg mL−1。ds-DNA修饰PGE(腺嘌呤): 线性范围 0.5–6.0 μg mL−1;斜率 −0.1021 μA mL μg−1;R^2 = 0.9823;LOD 0.48 μg mL−1。溶液相(鸟嘌呤): 线性范围 0.5–4.0 μg mL−1;斜率 −0.1504 μA mL μg−1;R^2 = 0.9918;LOD 0.40 μg mL−1。溶液相(腺嘌呤): 线性范围 0.5–4.0 μg mL−1;斜率 −0.1641 μA mL μg−1;R^2 = 0.9952;LOD 0.42 μg mL−1。PPGE: 线性范围 0.0015–0.30 μg mL−1;斜率 115.88 μA mL μg−1;R^2 = 0.9962;LOD 0.00007 μg mL−1。

效应效果

重现性:ds-DNA修饰PGE对3.0、5.0 μg mL−1 Sudan II的G信号RSD为4.2%、3.9%(n=10),A信号4.9%、4.0%;溶液相2.0 μg mL−1 G信号RSD 4.8%。抗干扰:辣椒红素、辣椒素、β-胡萝卜素、隐黄质耐受100倍(w/w),Ca2+、Mg2+、Fe3+、Al3+、Zn2+、Cu2+、SO4^2−、CO3^2−、NO3−耐受500倍。辣椒酱/番茄酱加标回收率:PPGE法97.0%–102.5%(RSD 2.7%–3.5%),DNA/溶液法95.0%–106.0%(RSD 3.5%–4.8%)。PPGE法LOD 0.00007 μg mL−1优于MWNTs-IL-Gel/GC(0.001 μg mL−1)和汞电极(0.082 μg mL−1),作者认为其快速、廉价、选择性好,适合在线/原位食品安全检测。

传感器的构成

  • 基底/换能器电极:铅笔石墨电极(PGE),可再生一次性碳电极,经电化学预处理形成PPGE,提供导电与吸附/氧化平台。
  • 识别元件:鲑鱼精双链DNA(ds-DNA),在+0.50 V、200 s下吸附固定于PGE表面,作为识别Sudan II的生物分子层。
  • 被测物结合层:苏丹红II(Sudan II),与ds-DNA发生嵌入/沟槽结合,改变电极表面DNA的氧化行为。
  • 信号换能层:鸟嘌呤(G)和腺嘌呤(A)碱基氧化信号,DPV读取其峰电流变化,用于定量Sudan II。
  • 支持电解质:醋酸缓冲液(pH 4.8,0.5 mol L−1,含0.02 mol L−1 NaCl)用于DNA固定与测量;Tris缓冲液(pH 7.0)用于Sudan II孵育。
  • PPGE吸附层:预处理PGE表面直接吸附Sudan II,PBS(0.1 mol L−1,pH 4.0)中开电路富集300 s,实现痕量富集。
  • 电化学测量体系:Ag/AgCl参比电极(3 mol L−1 KCl)、Pt对电极和Autolab PGSTAT 12,完成DPV/AdSDPV信号读出。

中文摘要

本文报道了一种简单、廉价的新型电化学生物传感器,用于测定致癌染料苏丹红II(Sudan II)。作者以铅笔石墨电极(PGE)为基底,将鲑鱼精双链DNA(ds-DNA)固定于电极表面,利用差分脉冲伏安法(DPV)研究Sudan II与ds-DNA在电极表面及溶液相中的相互作用。Sudan II与ds-DNA作用前后,鸟嘌呤(G)和腺嘌呤(A)氧化信号的变化量与Sudan II浓度成正比,从而实现定量检测。在ds-DNA修饰PGE上,Sudan II在0.5–6.0 μg mL−1范围内呈线性,相关系数R^2=0.9958;溶液相中线性范围为0.5–4.0 μg mL−1,检出限为0.4 μg mL−1。此外,作者还采用吸附剥离差分脉冲伏安法(AdSDPV)在预处理PGE(PPGE)上测定Sudan II,峰电流在0.0015–0.30 μg mL−1范围内线性,检出限达0.00007 μg mL−1。两种方法均用于辣椒酱和番茄酱等实际食品样品中Sudan II的检测。

英文摘要

A simple and inexpensive methodology was used to develop a novel electrochemical sensor for the determination of Sudan II. The interaction of Sudan II with salmon sperm ds-DNA on the surface of salmon sperm ds-DNA-modified pencil graphite electrode (PGE) and in solution phase was studied, using differential pulse voltammetry. The difference between adenine and guanine signals of the ds-DNA after and before interaction with Sudan II was directly proportional to Sudan II concentration, which used for quantitative inspections. Using PGE, a linear calibration curve (R(2)=0.9958) was observed with 0.5-6.0 μg mL(-1) Sudan II. Furthermore, the LOD of 0.4 μg mL(-1) and linear range between 0.5 and 4.0 μg mL(-1) were achieved in solution phase. In the second part, Sudan II was determined on a pretreated pencil graphite electrode by means of adsorptive stripping differential pulse voltammetry. The peak current was linearly dependent on Sudan II concentration over the range of 0.0015-0.30 μg mL(-1), with a detection limit of 0.00007 μg mL(-1) Sudan II. Both ds-DNA-modified PGE and PPGE were applied to analyze Sudan II in real samples.