Novel biosensor chip for simultaneous detection of DNA-carcinogen adducts with low-temperature fluorescence
Novel biosensor chip for simultaneous detection of DNA-carcinogen adducts with low-temperature fluorescence
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DOI：
10.1016/s0956-5663(03)00274-4
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发表时间：
2004-01-15
影响因子：
12.6
通讯作者：
Small, GJ
中科院分区：
文献类型：
--
作者：
Grubor, NM;Shinar, R;Small, GJ
本文介绍了一种单克隆抗体-金生物传感器芯片低温激光诱导荧光检测法，用于分析DNA-致癌物加合物。MAb-金生物传感器芯片的检测限、动态范围和生物传感适用性的优化通过以下方式实现：（1）使用二硫代双（琥珀酰亚胺基丙酸酯（DSP））作为蛋白质接头和（2）使用重组蛋白A来提供MAb的定向固定。使用DSP，其具有短的亚甲基链长度，导致更快的蛋白质结合动力学和更高的蛋白质表面密度比更长的二硫代双（琥珀酰亚胺十一烷酸酯）（DSU）接头。重组蛋白A的掺入增加了定向MAb结合的分析物和金表面之间的距离。增加的距离使荧光猝灭最小化，导致与没有蛋白A的芯片相比荧光信号增加约10倍。改进的芯片结构用于证明两种结构相似的苯并[a]芘（BP）衍生的DNA加合物BP-6-N7 Gua和BP-diolepoxide-10-N（2）dG的生物传感是可行的。这些诱变加合物通过单电子氧化和单加氧途径形成，分别是脱嘌呤和稳定的DNA加合物。结果表明，DNA加合物可以很容易地确定在同一地址使用时间分辨，低温激光为基础的荧光光谱。检测的电流极限在低飞摩尔范围内。这些结果表明，一个单一的生物传感器芯片组成的Au/DSP/蛋白质A/单克隆抗体纳米组装，与分析物特异性单克隆抗体和低温荧光检测应适用于同时检测和定量上述加合物，以及发光抗原的选择性单克隆抗体存在。(C)2003 Elsevier B. V.保留所有权利。
A monoclonal antibody (MAb)-gold biosensor chip with low-temperature laser-induced fluorescence detection for analysis of DNA-carcinogen adducts is described. Optimization of the detection limit, dynamic range, and biosensing applicability of the MAb-gold biosensor chip was achieved by: (1) using dithiobis(succinimidyl propionate (DSP)) as a protein linker and (2) employing recombinant protein A to provide oriented immobilization of the MAbs. The use of DSP, which has a short methylene chain length, led to faster protein binding kinetics and higher protein surface density than a longer dithiobis(succinimidyl undecanoate) (DSU) linker. The incorporation of recombinant protein A increased the distance between the oriented MAb-bound analytes and the gold surface. The increased distance minimized fluorescence quenching, resulting in about a 10-fold increase in the fluorescence signal in comparison with a chip without protein A. The improved chip architecture was used to demonstrate that biosensing of two structurally similar benzo[a]pyrene (BP)-derived DNA adducts, BP-6-N7Gua and BP-diolepoxide-10-N(2)dG, bound to two specific MAbs immobilized from a mixture at the same address on the chip, is feasible. These mutagenic adducts are formed by one-electron oxidation and monooxygenation pathways, and are depurinating and stable DNA adducts, respectively. It is shown that the DNA adducts can be easily identified at the same address using time-resolved, low-temperature laser-based fluorescence spectroscopy. The current limit of detection is in the low femtomole range. These results indicate that a single biosensor chip consisting of a Au/DSP/protein A/MAb nanoassembly, with analyte-specific MAbs and low-temperature fluorescence detection should be suitable for simultaneous detection and quantitation of the above adducts, as well as the luminescent antigens for which selective MAbs exist. (C) 2003 Elsevier B.V. All rights reserved.