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基于适配体探针的微芯片电泳无标记通用抗生素检测平台。

A label-free and universal platform for antibiotics detection based on microchip electrophoresis using aptamer probes.

机构信息

Faculty of Material Science and Chemical Engineering, Ningbo University, Ningbo 315211, China.

Faculty of Material Science and Chemical Engineering, Ningbo University, Ningbo 315211, China.

出版信息

Talanta. 2017 May 15;167:544-549. doi: 10.1016/j.talanta.2017.02.061. Epub 2017 Feb 28.

Abstract

A novel label-free, universal, and high throughput aptasensor was developed based on a microchip electrophoresis (MCE) platform for automatic detection of antibiotic residues in food. Firstly, chloramphenicol (CAP) was employed as a model to be captured by its aptamer probe (Apt). Then, the partial complementary oligonucleotide of CAP's aptamer (C-DNA) was introduced into the reaction system. Because the Apt-CAP complex can't further hybrid with free C-DNA, the amount of hybrid Apt-C-DNA double strand DNA (dsDNA) was less than that without adding the target. Finally, the above mixture was introduced into the microchip electrophoresis (MCE) platform for detection, both dsDNA and Apt-CAP can be separated and produce different fluorescence signals in the MCE. In a certain concentration range, the ratio of signal between dsDNA and Apt-CAP (I/I ) was proportional to the concentration of targets. Under the optimum conditions, the ratio showed a satisfactory linearity range from 0.008 to 1ng/mL of CAP with a detection limit of 0.003ng/mL. Thus, a universal MCE-based assay was developed for quantifying CAP automatically. The method was also successfully applied in the different food samples for CAP detection, which showed a good recovery (Milk: 91.1-108%, Fish: 86.1-114%) and the results were consistent with that of ELISA. This method owned many merits as follows: firstly, MCE was a high throughput screening platform and the detection time is limited to 3min for each sample. Secondly, the aptamer probes can be directly used for detection without labeling any signal tag which can facilitate the preparation procedures of probes. Thirdly, the operation was easy just by the following steps: firstly, the mixture of aptamer probes were incubated followed adding C-DNA; then measurement was performed. Moreover, the assay with MCE platform can be used to detect other targets just by changing the corresponding aptamer probe; it can even realize simultaneous detection when the targets have aptamers with different number of base pairs. Above all, it's a high- throughput and prospective method which can be applied in high throughput screening of antibiotics in food safety.

摘要

一种新型的无标记、通用、高通量适体传感器,基于微芯片电泳(MCE)平台,用于自动检测食品中的抗生素残留。首先,氯霉素(CAP)被用作模型,由其适体探针(Apt)捕获。然后,将 CAP 适体的部分互补寡核苷酸(C-DNA)引入反应体系。由于 Apt-CAP 复合物不能进一步与游离 C-DNA 杂交,因此没有添加目标的情况下,杂交 Apt-C-DNA 双链 DNA(dsDNA)的量少于添加目标的情况下。最后,将上述混合物引入微芯片电泳(MCE)平台进行检测,dsDNA 和 Apt-CAP 都可以分离,并在 MCE 中产生不同的荧光信号。在一定的浓度范围内,dsDNA 和 Apt-CAP 的信号比(I/I)与目标物的浓度成正比。在最佳条件下,该比率在 0.008 至 1ng/mL 的 CAP 浓度范围内呈现出令人满意的线性范围,检测限为 0.003ng/mL。因此,开发了一种通用的基于 MCE 的自动定量测定 CAP 的方法。该方法还成功地应用于不同食品样品中 CAP 的检测,回收率良好(牛奶:91.1-108%,鱼:86.1-114%),与 ELISA 结果一致。该方法具有许多优点:首先,MCE 是一种高通量筛选平台,每个样品的检测时间限制在 3 分钟内。其次,适体探针可以直接用于检测,无需标记任何信号标签,这有利于探针的制备。第三,操作简单,只需以下步骤:首先,孵育适体探针混合物,然后加入 C-DNA;然后进行测量。此外,使用 MCE 平台的测定法可以通过改变相应的适体探针来检测其他目标物;甚至当目标物具有不同碱基对数的适体时,还可以实现同时检测。总之,这是一种高通量、有前途的方法,可应用于食品安全中抗生素的高通量筛选。

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