College of Food Science and Technology, Henan Key Laboratory of Cereal and Oil Food Safety Inspection and Control, Henan University of Technology, Zhengzhou, 450001, Henan, China.
Henan Province Food Inspection Research Institute, 8 Jinger Road, Zhengzhou, 450008, Henan, China.
Anal Bioanal Chem. 2022 Feb;414(5):1899-1907. doi: 10.1007/s00216-021-03825-3. Epub 2022 Jan 7.
As a common heavy metal ion with strong toxicity and wide distribution, lead ions (Pb) had great harm to the human body. In this work, a low-noise ratiometric fluorescence biosensor was developed based on Pb-dependent DNAzyme and exonuclease III (Exo III)-assisted cascade signal amplification. Firstly, the substrate chain of DNAzyme (S-DNA) was modified on the surface of magnetic beads (MBs) through the combination of biotin and streptavidin, and then the enzyme chain of DNAzyme (E-DNA) was connected to the MBs by forming a double-stranded DNA (dsDNA) with S-DNA. A hairpin DNA (HP) labelled with Cy3 and Cy5 respectively at both ends was used as a fluorescence probe. The emission peaks of Cy3 and Cy5 can appear at 562 nm and 665 nm respectively, and their fluorescence intensity ratio (F/F) was chosen as the acquisition signal. The ratiometric sensor can reduce the interference of detection environment and avoid false positive reactivity. Due to the cleavage of DNAzyme and the release of single-stranded DNA (ssDNA) in the presence of Pb, the hairpin structure of HP was opened and the FRET between two fluorophores disappeared, resulting in the strengthened signal of Cy3 and the weakened signal of Cy5. Furthermore, the ratio [Formula: see text] signal increased gradually with the increase of Pb concentration. When the concentration of Pb was in the range of 0.1-1000 nM, [Formula: see text] had a good linear relationship with [Formula: see text], the correlation coefficient (R) was 0.997, and the limit of detection (LOD) was 77 pM. The presented ratiometric fluorescence biosensor had lower LOD and wider detection range via comparing with other methods. At the same time, the sensor also obtained the satisfactory results for detection of Pb in tap water, tea, and rice flour samples. The provided ratiometric biosensor has great potential in the monitoring of various targets. A low-noise ratiometric fluorescence biosensor based on the FRET between two fluorophores was developed, and the DNAzyme and exonuclease III-assisted cascade signal amplification was used to improve the sensitivity of the method. The biosensor had a detection limit as low as 77 pM.
作为一种具有强毒性和广泛分布的常见重金属离子，铅离子 (Pb) 对人体有很大的危害。在这项工作中，基于依赖 Pb 的 DNA 酶和外切酶 III (Exo III)-辅助级联信号放大，开发了一种低噪声比率荧光生物传感器。首先，通过生物素和链霉亲和素的结合，将 DNA 酶的底物链 (S-DNA) 修饰在磁珠 (MBs) 的表面上，然后通过与 S-DNA 形成双链 DNA (dsDNA) 将 DNA 酶的酶链 (E-DNA) 连接到 MBs 上。分别在两端标记有 Cy3 和 Cy5 的发夹 DNA (HP) 用作荧光探针。Cy3 和 Cy5 的发射峰分别可以出现在 562nm 和 665nm，选择它们的荧光强度比 (F/F) 作为采集信号。比率传感器可以减少检测环境的干扰，避免假阳性反应。由于存在 Pb 时 DNA 酶的切割和单链 DNA (ssDNA) 的释放，HP 的发夹结构被打开，两个荧光团之间的 FRET 消失，导致 Cy3 的信号增强和 Cy5 的信号减弱。此外，[Formula: see text]信号随着 Pb 浓度的增加逐渐增加。当 Pb 浓度在 0.1-1000 nM 范围内时，[Formula: see text]与 [Formula: see text] 之间具有良好的线性关系，相关系数 (R) 为 0.997，检测限 (LOD) 为 77 pM。与其他方法相比，所提出的比率荧光生物传感器具有更低的 LOD 和更宽的检测范围。同时，该传感器在自来水中、茶叶和米粉样品中检测 Pb 也取得了令人满意的结果。所提供的比率生物传感器在监测各种目标方面具有很大的潜力。基于两个荧光团之间的 FRET，开发了一种低噪声比率荧光生物传感器，并利用 DNA 酶和外切酶 III 辅助级联信号放大来提高方法的灵敏度。该生物传感器的检测限低至 77 pM。
