化学学报 ›› 2014, Vol. 72 ›› Issue (9): 1029-1035.DOI: 10.6023/A14030234 上一篇    下一篇

研究论文

基于磁纳米颗粒的二段对称分裂式G-四分体DNA酶生物传感器用于Hg2+的快速检测

张佳佳, 代佩卿, 李超, 李南忘, 程圭芳, 何品刚, 方禹之   

  1. 华东师范大学化学系 上海 200241
  • 收稿日期:2014-03-31 出版日期:2014-09-14 发布日期:2014-08-27
  • 通讯作者: 程圭芳 E-mail:gfcheng@chem.ecnu.edu.cn
  • 基金资助:

    项目受国家自然科学基金(No. 21175045)和国家重大科学仪器设备开发专项(No. 2011YQ150078)资助.

A Symmetrically Split G-quadruplex DNAzymes Biosensor Based on Magnetic Nanoparticles for the Rapid Detection of Hg2+

Zhang Jiajia, Dai Peiqing, Li Chao, Li Nanwang, Cheng Guifang, He Pingang, Fang Yuzhi   

  1. Chemistry Department, East China Normal University, Shanghai 200241
  • Received:2014-03-31 Online:2014-09-14 Published:2014-08-27
  • Supported by:

    Project supported by the National Natural Science Foundation of China (No. 21175045) and National Key Foundation for Exploring Scientific Instrument (No. 2011YQ150078).

提出了一种可用于Hg2+快速检测的基于磁纳米颗粒与二段对称分裂式G-四分体DNA酶的生物传感器. 分别用紫外-可见光谱法, 圆二色光谱法和荧光显微镜成像技术对实验设计的DNA酶传感器进行了表征. 传感器中磁纳米颗粒的应用不仅可以直接从水样中通过磁分离方法分离和富集被测物Hg2+, 并且还能将游离的未与Hg2+结合的DNA酶和hemin等除去, 有效地提高检测灵敏度和降低背景信号; 此外, 二段对称分裂式G-四分体DNA酶的运用还可增强传感器的灵活性和选择性. 传感器对Hg2+检测的线性范围为0.8~20 nmol/L, 检出限为0.3 nmol/L. 当水体中的共存离子大量存在时, 传感器对Hg2+的检测仍具有高度特异性. 对实际水样的检测回收率在95.3%~104.4%之间. 实验设计的DNA酶传感器操作简便, 费用低廉, 具有良好的再生能力. 可用于天然水体和饮用水样品中痕量Hg2+的检测.

关键词: 磁纳米颗粒, 二段对称分裂式G-四分体, DNA酶, 生物传感器, 比色法, Hg2+

A biosensor composed of magnetic nanoparticles and symmetrically split G-quadruplex DNAzymes was proposed for the rapid and sensitive detection of Hg2+. It was mainly based on the formation of a special thymine-Hg2+-thymine (T-Hg-T) structure by T-riched nucleic acid sequences and Hg2+, and then G-quadruplex DNAzymes catalyzed the oxidation of H2O2 and 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), so that the trace amount of Hg2+ in water can be successfully detected by colorimetry. The assembly of the biosensor was characterized by using of UV-Visible spectroscopy, CD spectroscopy and fluorescence microscopy imaging techniques respectively. The magnetic nanoparticles coated with T-riched short oligonucleotide (DNA1) via streptavidin-biodin interaction were employed as the capture elements. The long oligonucleotides (DNA2) consisting of the T-riched sequence in the middle and two G-riched fragments at the both ends were used as the sensing elements. The procedure of the protocol was divided into three steps. First: the magnetic nanoparticles covered with DNA1 (MNPs/DNA1) mixed with DNA2 were added into a water sample to capture the target Hg2+ by forming the special T-Hg-T structure. Incubating 2 h at 37 ℃, two G-riched fragments at the ends of DNA2 were close to each other assembling the symmetrically G-quadruplex DNAzymes in the presence of hemin. Then, the supernatant was discarded by magnetic separation, and the residues were washed several times to resuspend in the mixture solution containing definite amount of H2O2 and ABTS. The catalytic signal of absorbance of ABTS+ was recorded by UV-Visible spectrophotometer. The utilization of MNPs can greatly reduce the background signal, and the employment of symmetrically split DNAzymes made the design of the experiment more flexible and selectable. The biosensor was sensitive, and the calibration curve was identified in the range from 0.8 nmol/L to 20 nmol/L with a detection limit of 0.3 nmol/L. And the biosensor can detect Hg2+ without interference in the presence of a large number of competing ions. This sensor was simple, inexpensive, and renewable. Satisfactory values between 95.3% and 104.4% were obtained for the recovery experiments, and it can meet the requirement of detection of trace Hg2+ in natural water especially in drinking water.

Key words: magnetic nanoparticles, symmetrically split G-quadruplex, DNAzymes, biosensors, colorimetry, mercury ions