Chen Jinri, Chen Xin, Zhang Yuting, Wang Xiaoli, Zhou Nandi
The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
Jiangsu Key Laboratory of Marine Bioresources and Environment, Jiangsu Ocean University, 59 Cangwu Road, Haizhou 222005, China.
J Agric Food Chem. 2023 Feb 8;71(5):2628-2636. doi: 10.1021/acs.jafc.2c07784. Epub 2023 Jan 26.
A single-stranded DNA (ssDNA) aptamer specific for 6'-sialyllactose (6'-SL) was screened through magnetic separation-based SELEX and post-SELEX truncation and used to construct unique aptamer bio-dots for sensitive detection of 6'-SL. Eighteen rounds of screening were conducted during the SELEX process. The ssDNA aptamer Apt9 ( = 152.3 nM) with a length of 79 nucleotides (nt) was demonstrated as the optimal aptamer candidate after affinity and specificity evaluation. Then, Apt9 was truncated and optimized according to secondary structure and molecular docking. A 35 nt truncated aptamer Apt9-1 ( = 91.75 nM) with higher affinity than Apt9 was finally obtained. Furthermore, Apt9-1 was used to synthesize bio-dots as a new recognition element of 6'-SL, and the aminobenzene boric acid functionalized carbon dots were employed as the other recognition element. With the respective fluorescent characteristics, the two quantum dots (QDs) were made a pair to construct a 6'-SL fluorescent biosensor. The linear detection range of the biosensor is 10 μM to 5 mM, and the detection limit is 0.9 μM. With the advantages of time-saving, high efficiency, and simplicity in the actual sample detection, the screened aptamer and dual-QD-based biosensor have broad application prospects in 6'-SL detection.
通过基于磁分离的指数富集配体系统进化技术（SELEX）以及SELEX后截短筛选出了一种对6'-唾液酸乳糖（6'-SL）具有特异性的单链DNA（ssDNA）适配体，并将其用于构建独特的适配体生物点，以灵敏检测6'-SL。在SELEX过程中进行了18轮筛选。经过亲和力和特异性评估，长度为79个核苷酸（nt）的ssDNA适配体Apt9（解离常数Kd = 152.3 nM）被证明是最佳的适配体候选物。然后，根据二级结构和分子对接对Apt9进行截短和优化。最终获得了一个35 nt的截短适配体Apt9-1（Kd = 91.75 nM），其亲和力高于Apt9。此外，Apt9-1被用于合成生物点，作为6'-SL的一种新的识别元件，氨基苯硼酸功能化的碳点被用作另一种识别元件。利用各自的荧光特性，将这两种量子点（QD）配对构建了一种6'-SL荧光生物传感器。该生物传感器的线性检测范围为10 μM至5 mM，检测限为0.9 μM。筛选出的适配体和基于双量子点的生物传感器在实际样品检测中具有省时、高效、简便的优点，在6'-SL检测中具有广阔的应用前景。
