Department of Chemistry, Alfaisal University, Al Zahrawi Street, Al Maather, Al Takhassusi Rd, Riyadh 11355, Saudi Arabia.
Department of Parasitology and Medical Entomology, Faculty of Medicine, Universiti Kebangsaan Malaysia, 56000 Cheras, Kuala Lumpur, Malaysia; Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800 Penang, Malaysia.
Int J Biol Macromol. 2024 May;267(Pt 2):131509. doi: 10.1016/j.ijbiomac.2024.131509. Epub 2024 Apr 11.
Giardia intestinalis is one of the most widespread intestinal parasites and is considered a major cause of epidemic or sporadic diarrhea worldwide. In this study, we aimed to develop a rapid aptameric diagnostic technique for G. intestinalis infection. First, the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process generated DNA aptamers specific to a recombinant protein of the parasite's trophozoite. Ten selection rounds were performed; each round, the DNA library was incubated with the target protein conjugated to Sepharose beads. Then, the unbound sequences were removed by washing and the specific sequences were eluted and amplified by Polymerase Chain Reaction (PCR). Two aptamers were selected, and the dissociation constants (K), were determined as 2.45 and 16.95 nM, showed their high affinity for the G. intestinalis trophozoite protein. Subsequently, the aptamer sequence T1, which exhibited better affinity, was employed to develop a label-free electrochemical biosensor. A thiolated aptamer was covalently immobilized onto a gold screen-printed electrode (SPGE), and the binding of the targeted protein was monitored using square wave voltammetry (SWV). The developed aptasensor enabled accurate detection of the G. intestinalis recombinant protein within the range of 0.1 pg/mL to 100 ng/mL, with an excellent sensitivity (LOD of 0.35 pg/mL). Moreover, selectivity studies showed a negligible cross-reactivity toward other proteins such as bovine serum albumin, globulin, and G. intestinalis cyst protein.
贾第虫是最广泛分布的肠道寄生虫之一，被认为是全球流行或散发性腹泻的主要原因。在本研究中，我们旨在开发一种用于贾第虫感染的快速适配体诊断技术。首先，通过指数富集的配体系统进化（SELEX）过程生成针对寄生虫滋养体重组蛋白的 DNA 适配体。进行了十轮选择；每轮，将 DNA 文库与琼脂糖珠偶联的靶蛋白孵育。然后，通过洗涤去除未结合的序列，通过聚合酶链反应（PCR）洗脱和扩增特异性序列。选择了两个适配体，解离常数（Kd）分别为 2.45 和 16.95 nM，表明它们对贾第虫滋养体蛋白具有高亲和力。随后，选择具有更好亲和力的适配体序列 T1 来开发无标记电化学生物传感器。巯基化的适配体被共价固定在金丝网印刷电极（SPGE）上，使用方波伏安法（SWV）监测靶向蛋白的结合。开发的适配体传感器能够在 0.1 pg/mL 至 100 ng/mL 的范围内准确检测贾第虫重组蛋白，具有出色的灵敏度（LOD 为 0.35 pg/mL）。此外，选择性研究表明，对其他蛋白质如牛血清白蛋白、球蛋白和贾第虫囊蛋白的交叉反应性可以忽略不计。
