thrombin-aptamer and betadex

An ultrasensitive label-free electrochemiluminescence (ECL) aptasensor for the detection of thrombin was developed based on the specific recognition between tris(bipyridine)ruthenium(II)-β-cyclodextrin (tris(bpyRu)-β-CD) and the anti-thrombin aptamer (aptamer). The NH2-aptamer was first immobilized on the activated glassy carbon electrode (GCE) by coupling interaction. By use of the specific recognition between tris(bpyRu)-β-CD and aptamer, tris(bpyRu)-β-CD was then attached on the surface of GCE. Resulting from the outstanding photoactive properties of tris(bpyRu)-β-CD, the fabricated GCE performed strong ECL signal with the coreactant of 2-(dibutylamino)ethanol (DBAE). However, in the presence of thrombin, aptamer-thrombin bioaffinity complexes were formed, which restricted the recognition activities between aptamer and tris(bpyRu)-β-CD. Thus, fewer tris(bpyRu)-β-CD could be attached on the surface of GCE and led to an obvious decrease of ECL signal. Fortunately, the difference of ECL intensity before and after combination with thrombin was logarithmically linear with the concentration of thrombin in a wide range of 10 nM-1 pM. Meantime, a detection limit of 0.1 pM without any other signal labeling or amplifying procedures indicated that the biosensor performed excellent sensitivity, operability and simplicity.

Topics: 2,2'-Dipyridyl; Aptamers, Nucleotide; beta-Cyclodextrins; Biosensing Techniques; Coordination Complexes; Electrochemical Techniques; Humans; Luminescent Measurements; Sensitivity and Specificity; Thrombin

A novel fluorescent thrombin binding aptamer (TBA), conjugated with the environmentally sensitive dansyl probe at the 3'-end and a β-cyclodextrin residue at the 5'-end, has been efficiently synthesized exploiting Cu(I)-catalyzed azide-alkyne cycloaddition procedures. Its conformation and stability in solution have been studied by an integrated approach, combining in-depth NMR, CD, fluorescence, and DSC studies. ITC measurements have allowed us to analyze in detail its interaction with human thrombin. All the collected data show that this bis-conjugated aptamer fully retains its G-quadruplex formation ability and thrombin recognition properties, with the terminal appendages only marginally interfering with the conformational behavior of TBA. Folding of this modified aptamer into the chairlike, antiparallel G-quadruplex structure, promoted by K(+) and/or thrombin binding, typical of TBA, is associated with a net fluorescence enhancement, due to encapsulation of dansyl, attached at the 3'-end, into the apolar cavity of the β-cyclodextrin at the 5'-end. Overall, the structural characterization of this novel, bis-conjugated TBA fully demonstrates its potential as a diagnostic tool for thrombin recognition, also providing a useful basis for the design of suitable aptamer-based devices for theranostic applications, allowing simultaneously both detection and inhibition or modulation of the thrombin activity.

Topics: Aptamers, Nucleotide; beta-Cyclodextrins; Dansyl Compounds; DNA; Fluorescence; G-Quadruplexes; Humans; Models, Molecular; Molecular Structure; Nucleic Acid Conformation