DNA-aptamers for thrombin detection by quartz crystal microbalance.

Maja Snejdarkova 1Alexandra Poturnayová 2Isabel Neundlinger Andreas Ebner Tibor Hianik 3

1. Institute of Animal Biochemistry and Genetics, Slovak Academy of Science (IBAG-SAS), Moyzesova 61, Bratislava 90028, Slovakia (Slovak Rep.)
2. Institute of Animal Biochemistry and Genetics SASc, Moyzesova 61, Ivanka pri Dunaji 90028, Slovakia (Slovak Rep.)
3. Comenius University, Department of Biophysics and Chemical Physics, Mlynská dolina F1, Bratislava 842 48, Slovakia (Slovak Rep.)

Abstract

Thrombin is a highly specific serine protease involved in the coagulation cascade, which converts soluble fibrinogen into insoluble strands of fibrin which is a matrix of the blood clot. The thrombin is not present in the blood under physiological conditions but appears in pathological processes including deep vein thrombosis, myocardial infarction, stroke and in central nervous system injury. Under coagulation the concentration of the thrombin in blood varied from nM to µM levels. Development of the sensitive method of the thrombin detection in nanomole level is important for clinical practice. The traditional methods of thrombin detection are based on the antigen-antibody immunoassay, clotting – based assay or synthetic-substrate-based enzymatic assays. Alternative method of thrombin detection is based on biosensors using DNA aptamers as receptors.

In this work we used thickness shear mode method to study the interaction between thromnin and DNA aptamers immobilised at the surface of quartz crystal transducer. We used 30-mer biotinylated DNA aptamer (BF) of followinh composition: 5´-GGT TGG TGT GGT TGG TTT TTT TTT TTT TTT - 3´- biotin. The immobilization of the aptamers to the surface is crucial and we compared two types of the surfaces formed on the gold electrode. First surface was formed from neutravidine on which BF was attached. Second surface was formed from the nanostructure tetrahedron DS3BT1 for oriented attachemnt of neutravidine and than for BF binding. We studied the kinetics of the changes of the series resonant frequency, fs, and the motional resistance, Rm, of the quartz crystal transducer following addition of thrombin. We observed decrease of fs and increase of Rm, following addition of thrombin. The changes of both parameters started already at 50 nM of thrombin.The increase of Rm suggests on increase of surface viscosity. The kinetics analysis of aptamer-thrombin system was performed. Binding experiments carried out using BF immobilized on neutravidin layer at gold surface showed that constant of dissociation is Kd = 114 nM. This constant is measure of the affinity of the interactions. The lower Kd, the higher affinity. However, lower affinity of thrombin to the aptamer was observed when DS3BT1 structures were used for aptamer immobilization (Kd =151 nM). The reasons of these differences are discussed.

Acknowledgments. This work was financially supported by Slovak Research and Development Agency (Projects VVCE-0064-07, APVV-0410-10) and by project MNT-ERA.NET II project IntelliTip (ID 431, AN 234989 to M.Š. and FFG project 823980 I.N to A.E.).

 

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Presentation: Poster at SMCBS'2011 International Workshop, by Maja Snejdarkova
See On-line Journal of SMCBS'2011 International Workshop

Submitted: 2011-09-16 10:12
Revised:   2011-09-21 17:19