Laboratorio de Biosensores, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile; Santos Dumont N° 964, Independencia, Santiago 8380492, Chile.
Laboratorio de Nanobiotecnología y Nanotoxicología, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile; Santos Dumont N° 964, Independencia, Santiago 8380492, Chile.
Sensors (Basel). 2020 Oct 31;20(21):6240. doi: 10.3390/s20216240.
Prothrombin-related thrombophilia is a genetic disorder produced by a substitution of a single DNA base pair, replacing guanine with adenine, and is detected mainly by polymerase chain reaction (PCR). A suitable alternative that could detect the single point mutation without requiring sample amplification is the surface plasmon resonance (SPR) technique. SPR biosensors are of great interest: they offer a platform to monitor biomolecular interactions, are highly selective, and enable rapid analysis in real time. Oligonucleotide-based SPR biosensors can be used to differentiate complementary sequences from partially complementary or noncomplementary strands. In this work, a glass chip covered with an ultrathin (50 nm) gold film was modified with oligonucleotide strands complementary to the mutated or normal (nonmutated) DNA responsible for prothrombin-related thrombophilia, forming two detection platforms called mutated thrombophilia (MT) biosensor and normal thrombophilia (NT) biosensor. The results show that the hybridization response is obtained in 30 min, label free and with high reproducibility. The sensitivity obtained in both systems was approximately 4 ΔμRIU/nM. The dissociation constant and limits of detection calculated were 12.2 nM and 20 pM (3 fmol), respectively, for the MT biosensor, and 8.5 nM and 30 pM (4.5 fmol) for the NT biosensor. The two biosensors selectively recognize their complementary strand (mutated or normal) in buffer solution. In addition, each platform can be reused up to 24 times when the surface is regenerated with HCl. This work contributes to the design of the first SPR biosensor for the detection of prothrombin-related thrombophilia based on oligonucleotides with single point mutations, label-free and without the need to apply an amplification method.
凝血酶原相关血栓形成倾向是一种由单个 DNA 碱基对替换引起的遗传疾病，由腺嘌呤取代鸟嘌呤，主要通过聚合酶链反应 (PCR) 检测。一种合适的替代方法是表面等离子体共振 (SPR) 技术，它不需要样本扩增就能检测到单点突变。SPR 生物传感器非常有趣：它们提供了一个监测生物分子相互作用的平台，具有高度选择性，并能够实时进行快速分析。基于寡核苷酸的 SPR 生物传感器可用于区分互补序列与部分互补或非互补链。在这项工作中，一个覆盖有超薄（50nm）金膜的玻璃芯片用与导致凝血酶原相关血栓形成倾向的突变或正常（未突变）DNA 互补的寡核苷酸链进行修饰，形成两个检测平台，分别称为突变型血栓形成倾向 (MT) 生物传感器和正常型血栓形成倾向 (NT) 生物传感器。结果表明，杂交反应在 30 分钟内完成，无需标记且具有高重现性。两种系统获得的灵敏度约为 4ΔμRIU/nM。计算得出的解离常数和检测限分别为 MT 生物传感器的 12.2 nM 和 20 pM（3 fmol），NT 生物传感器的 8.5 nM 和 30 pM（4.5 fmol）。两个生物传感器在缓冲溶液中选择性地识别其互补链（突变或正常）。此外，当用 HCl 再生表面时，每个平台最多可重复使用 24 次。这项工作为设计基于具有单点突变的寡核苷酸的凝血酶原相关血栓形成倾向的第一个 SPR 生物传感器做出了贡献，该生物传感器无需标记且无需应用扩增方法。
