Shandong Provincial Key Laboratory of Biochemical Analysis, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, China.
Biosens Bioelectron. 2013 Jan 15;39(1):145-51. doi: 10.1016/j.bios.2012.07.005. Epub 2012 Jul 17.
In this work, a new signal amplified strategy based on target-induced strand release coupling cleavage of nicking endonuclease for the ultrasensitive detection of ochratoxin A (OTA) is reported. OTA aptamer (DNA1) and OTA aptamer complementary (DNA2) were immobilized onto a magnetic bead (MB). In the presence of OTA, DNA2 was dissociated and released from the MB. The released DNA2 then hybridized with DNA3, which was linked at the 5' terminus of the amplification template and can extend along the template in the presence of Phi 29 DNA polymerase. The formed double-stranded DNA was cleaved by nicking endonuclease Nb.BbvCI and produced a short single-stranded DNA. The cleaved DNA strand generated a new site by Phi 29 DNA polymerase and the process of extension and cleavage was cyclical. Thus, a amount of the short single-stranded DNA were produced. Using DNA and ABEI labeled carboxylic silica nanoparticles chemiluminescence (CL) probe, the short single-stranded DNA could be sensitively detected. The CL intensity (ΔI) versus the concentration of OTA was linear in the range from 1.0×10(-12) to 5.0×10(-8)g mL(-1). The detection limit was 3.0×10(-13)g mL(-1), and the RSD was 3.4% at 1.0×10(-10)g mL(-1) (n=7). The developed method has been applied to detect OTA in naturally contaminated wheat samples. Due to its simplicity, sensitivity and no need of specific recognition of aptamer for cleavage, this CL bioassay offers a promising approach for the detection of OTA and other biomolecules.
在这项工作中，报道了一种新的信号放大策略，该策略基于目标诱导的链释放，结合切口内切酶用于超灵敏检测赭曲霉毒素 A（OTA）。OTA 适体（DNA1）和 OTA 适体互补（DNA2）固定在磁珠（MB）上。在 OTA 存在的情况下，DNA2 从 MB 上解离并释放出来。释放的 DNA2 然后与 DNA3 杂交，DNA3 连接在扩增模板的 5'端，并且在 Phi 29 DNA 聚合酶的存在下可以沿着模板延伸。形成的双链 DNA 被切口内切酶 Nb.BbvCI 切割，并产生短的单链 DNA。切割的 DNA 链在 Phi 29 DNA 聚合酶的作用下产生新的位点，并且延伸和切割的过程是循环的。因此，产生了大量的短单链 DNA。使用 DNA 和 ABEI 标记的羧酸硅纳米粒子化学发光（CL）探针，可以灵敏地检测短的单链 DNA。CL 强度（ΔI）与 OTA 的浓度在 1.0×10(-12)至 5.0×10(-8)g mL(-1)范围内呈线性关系。检测限为 3.0×10(-13)g mL(-1)，在 1.0×10(-10)g mL(-1)时的 RSD 为 3.4%（n=7）。该方法已用于检测天然污染小麦样品中的 OTA。由于其简单性、灵敏度和不需要适体特异性识别用于切割，这种 CL 生物测定为 OTA 和其他生物分子的检测提供了一种很有前途的方法。
