Ding Ying, Yuan Jing, Wang Lei, Jin Nana, Wang Siyuan, Li Yanbin, Lin Jianhan
Key Laboratory of Agricultural Information Acquisition Technology, Ministry of Agriculture and Rural Affairs, China Agricultural University, Beijing, 100083, China.
Department of Biological and Agricultural Engineering, University of Arkansas, Fayetteville, AR, 72701, USA.
Biosens Bioelectron. 2023 Jun 1;229:115230. doi: 10.1016/j.bios.2023.115230. Epub 2023 Mar 16.
Magnetic separation was often applied to isolate and concentrate foodborne bacteria using immunomagnetic nanobeads before downstream bacterial detection. However, nanobead-bacteria conjugates (magnetic bacteria) were coexisting with excessive unbound nanobeads, limiting these nanobeads on magnetic bacteria to further act as signal probes for bacterial detection. Here, a new microfluidic magnetophoretic biosensor was elaboratively developed using a rotated high gradient magnetic field and platinum modified immunomagnetic nanobeads for continuous-flow isolation of magnetic bacteria from free nanobeads, and combined with nanozyme signal amplification for colorimetric biosensing of Salmonella. First, the platinum modified immunomagnetic nanobeads were mixed with the bacterial sample to form the magnetic bacteria, and magnetically separated to eliminate non-magnetic background. Then, the mixture of free immunomagnetic nanobeads and magnetic bacteria was injected with sheath flow (PBS) at higher flowrate into the semi-circle magnetophoretic separation channel under rotated magnetic field, which was generated by two repulsive cylindric magnets and their in-between ring iron gear, leading to continuous-flow isolation of magnetic bacteria from free immunomagnetic nanobeads because they suffered from different magnetic forces and thus had different deviating positions at the outlet. Finally, the separated magnetic bacteria and unbound magnetic nanobeads were respectively collected and used to catalyze coreless substrate into blue product, which was further analyzed using the microplate reader to obtain bacterial amount. This biosensor could determinate Salmonella as low as 41 CFU/mL in 40 min.
在进行下游细菌检测之前，磁分离常用于使用免疫磁性纳米珠分离和浓缩食源细菌。然而，纳米珠 - 细菌结合物（磁性细菌）与过量未结合的纳米珠共存，限制了这些磁性细菌上的纳米珠进一步作为细菌检测的信号探针。在此，精心开发了一种新型微流控磁泳生物传感器，该传感器使用旋转高梯度磁场和铂修饰的免疫磁性纳米珠，用于从游离纳米珠中连续流分离磁性细菌，并结合纳米酶信号放大用于沙门氏菌的比色生物传感。首先，将铂修饰的免疫磁性纳米珠与细菌样品混合形成磁性细菌，然后进行磁分离以消除非磁性背景。接着，将游离免疫磁性纳米珠和磁性细菌的混合物以较高流速与鞘流（磷酸盐缓冲液）一起注入由两个排斥圆柱形磁体及其之间的环形铁齿轮产生的旋转磁场下的半圆形磁泳分离通道中，由于它们受到不同的磁力作用，因此在出口处有不同的偏离位置，从而实现从游离免疫磁性纳米珠中连续流分离磁性细菌。最后，分别收集分离出的磁性细菌和未结合的磁性纳米珠，并用于催化无芯底物生成蓝色产物，进一步使用酶标仪进行分析以获得细菌数量。该生物传感器能够在40分钟内检测低至41 CFU/mL的沙门氏菌。
