title: DNA aptamers selection and characterization for development of label-free impedimetric aptasensor for neurotoxin anatoxin-a.
abstract: High affinity DNA aptamers against anatoxin-a (ATX), the smallest potent neurotoxin (Mol. Wt, 165.23 Da) were selected and identified in vitro using the systematic evolution of ligands by exponential enrichment (SELEX) approach. Aptamers with dissociation constants (Kd) of nanomolar range were isolated. The aptamer sequence of highest affinity was used to design a label-free impedance based aptasensor to assay ATX, for which there are no reported biosensors so far. The aptamer self assembled monolayer is formed on a gold electrode using the disulfide modified aptamer. The assembly process of the aptasensor was characterized using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Upon ATX binding to the immobilized aptamer, a significant decrease in the electron-transfer resistance was observed as a result of the aptamer conformation change, which is used as the sensor signal. The aptasensor showed a limit of detection of 0.5 nM and a wide linear range for ATX concentrations between 1 nM and 100 nM. The Kd of anti-ATX aptamer was calculated by electrochemical methods as well as the fluorescence. Interestingly, the Kd that was calculated from the aptasensor signal showed a lower value implying that the anchoring of the aptamer on the Au surface enhanced its affinity to ATX. The ATX aptasensor showed high stability as well as high specificity against common cynaobacterial toxins. Further development of biosensors that use anatoxin-a binding aptamers as a new recognition receptors could provide potential alternatives to the traditional assays for fast and simple monitoring of anatoxin-a.
authors.affiliation: Institut National de la Recherche Scientifique, Centre - Énergie, Matériaux et Télécommunications, 1650, Boul. Lionel Boulet, Varennes, Québec, Canada J3X 1S2.
authors.forename: Reda
authors.initials: R
authors.lastname: Elshafey
authors.affiliation: Département de Chimie et Biochimie, Université du Québec à Montréal, Montréal, Québec, Canada H3C 3P8.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Siaj
authors.affiliation: Centre of Biomedical Engineering, School of Engineering, Cranfield University, Cranfield, Bedfordshire MK43 0AL, UK; Department of Chemistry, Alfaisal University, Riyadh 11533, Kingdom of Saudi Arabia. Electronic address: m.zourob@cranfield.ac.uk.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Zourob
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry*; D015374:Biosensing Techniques / Q000295:instrumentation*; D000458:Cyanobacteria / Q000737:chemistry*; D000087522:Cyanobacteria Toxins; D058266:Dielectric Spectroscopy; D017097:Electric Impedance; D055664:Electrochemical Techniques / Q000295:instrumentation*; D004566:Electrodes; D004867:Equipment Design; D006046:Gold / Q000737:chemistry; D057230:Limit of Detection; D009498:Neurotoxins / Q000032:analysis*; D052156:SELEX Aptamer Technique; D014326:Tropanes / Q000032:analysis*
chemical_list: D052157:Aptamers, Nucleotide; D000087522:Cyanobacteria Toxins; D009498:Neurotoxins; D014326:Tropanes; D006046:Gold; C509783:anatoxin a
keywords: Anatoxin-a; Aptamer; Aptasensor; Impedance; Label-free; SELEX