Sánchez Martín Darío, Oropesa-Nuñez Reinier, Zardán Gómez de la Torre Teresa
Department of Material Sciences and Engineering, Division of Nanotechnology and Functional Materials, Ångström Laboratory, Uppsala University, Box 534, SE-751 21 Uppsala, Sweden.
Department of Material Sciences and Engineering, Division of Solid-State Physics, Ångström Laboratory, Uppsala University, Box 534, SE-751 21 Uppsala, Sweden.
ACS Omega. 2023 Jan 18;8(4):4391-4397. doi: 10.1021/acsomega.2c07992. eCollection 2023 Jan 31.
Detection of pathogens has become increasingly important, especially in the face of outbreaks and epidemics all over the world. Nucleic acid detection techniques provide a solid base to detect and identify pathogens. In recent years, magnetic sensors and magnetic labels have become of more interest due to their simplicity of use, low cost, and versatility. In this work, we have used the isothermal DNA amplification technique of rolling circle amplification (RCA) in combination with oligo-functionalized magnetic nanoparticles. Detection of RCA products takes place through specific binding between magnetic nanoparticles and RCA products. Upon binding, the relaxation frequency of the nanoparticle changes. This change was measured using an AC susceptometer. We showcase that the RCA time can be reduced for a quicker assay when performing the RCA on the surface of micrometer-sized beads, which consequently increases the hydrodynamic volume of the RCA products. This, in turn, increases the Brownian relaxation frequency shift of the nanoparticles upon binding. We performed optimization work to determine the ideal quantity of micrometer-sized particles, oligo-functionalized nanoparticles, and the amplification time of the RCA. We show that the detection of 0.75 fmol of preamplification synthetic target is possible with only 20 min of amplification time. Finally, we showcase the high specificity of the assay, as the functionalized nanoparticles are unable to bind to amplified DNA that does not match their labels. Overall, this paves the way for a simple bioassay that can be used without expensive laboratory equipment for detection of pathogens in outbreak settings and clinics around the world.
病原体的检测变得越来越重要，尤其是在面对全球范围内的疫情和流行病时。核酸检测技术为检测和鉴定病原体提供了坚实的基础。近年来，磁传感器和磁性标签因其使用简单、成本低和多功能性而受到更多关注。在这项工作中，我们将滚环扩增（RCA）的等温DNA扩增技术与寡功能化磁性纳米颗粒相结合。通过磁性纳米颗粒与RCA产物之间的特异性结合来检测RCA产物。结合后，纳米颗粒的弛豫频率会发生变化。使用交流磁化率仪测量这种变化。我们展示了在微米级珠子表面进行RCA时，可以减少RCA时间以实现更快的检测，这相应地增加了RCA产物的流体动力学体积。反过来，这又增加了结合时纳米颗粒的布朗弛豫频率偏移。我们进行了优化工作，以确定微米级颗粒、寡功能化纳米颗粒的理想数量以及RCA的扩增时间。我们表明，仅需20分钟的扩增时间就可以检测到0.75 fmol的预扩增合成靶标。最后，我们展示了该检测方法的高特异性，因为功能化纳米颗粒无法与与其标签不匹配的扩增DNA结合。总体而言，这为一种简单的生物检测方法铺平了道路，该方法无需昂贵实验室设备即可用于全球疫情爆发地区和诊所的病原体检测。
