Selection of ssDNA aptamers and construction of an aptameric electrochemical biosensor for detecting Giardia intestinalis cyst protein

Abstract

Giardia intestinalis, an intestinal protozoan parasite, is one of the potentially severe parasitic infections, especially in children. Rapid and simple diagnostic tools are highly desired to prevent the potential outbreak of G. intestinalis infection. The life cycle of Giardia species is quite simple and consists of trophozoite and cystic forms. This report presents the selection of ssDNA aptamers with high binding affinity to a G. intestinalis cyst recombinant protein using the SELEX process (systematic evolution of ligands by exponential enrichment). The process is based on incubating a random DNA library with the targeted protein, and the bound sequences are recovered and amplified by polymerase chain reaction (PCR). The generated pool of aptamer sequences is used in the subsequent selection round. After ten selection cycles, three sequences were isolated with low dissociation constants (K_d) of 7.98, 21.02, and 21.86 nM. Subsequently, the aptamer with the best affinity was integrated into a label-free electrochemical biosensor to detect G. intestinalis cyst protein. The developed aptasensor accurately detected the G. intestinalis recombinant cyst protein within the range of 0.1 pg mL⁻¹ to 1000 ng mL⁻¹, and a low detection limit of 0.0026 pg mL⁻¹. Furthermore, a selectivity study showed insignificant cross-reactivity against other proteins such as bovine serum albumin and globulin, and no reactivity against G. intestinalis trophozoite recombinant protein. Finally, the aptasensor was tested using G. intestinalis-spiked tap water samples and showed good recovery rates.