Thrombin binding to its aptamer is the most frequently used model system to demonstrate the proof-of-concept of the aptamer-based affinity assays. The advantages of specific binding affinity, various binding sites, the availability of two aptamers each targeting a specific site, switching of aptamer structures triggered by binding to thrombin, and the catalytic activity of thrombin are the major reasons why thrombin is often selected as a test model. Keeping pace with the market, Creative Biolabs provides one-stop anti-thrombin aptamer development, maturation, and modification services for our honor clients. Aptamer binding assays are also available for you.
Thrombin is a serine protease that selectively cleaves peptide substrates at Arg residues. With vital roles in physiological and pathological coagulation, thrombin is involved in different diseases. Thrombin is not present in blood under normal conditions. Only after vascular injury, thrombin is rapidly generated from inactive zymogen prothrombin by a series of enzyme cleavages. During coagulation, the concentration of thrombin may vary from pM to μM levels. Therefore, sensitive and specific detection of thrombin assays is important for related disease diagnostics. Immunoassays, clotting-based assays, and enzymatic activity-based assays have been developed for the detection and quantification of thrombin in the blood. Most of the assays for human thrombin deal with the intact α-thrombin which can be proteolyzed to form β-thrombin and γ-thrombin.
Fig.1 Role of thrombin in the pathogenesis of neurodegenerative diseases. (Ebrahimi, 2017)
Two DNA aptamers are widely used in aptamer-based assays for thrombin. Apt-15 (5’-GGTTGGTGTGGTTGG-3’) binds with the fibrinogen binding site of thrombin with a Kd around 100 nM, and Apt-29 (5’-AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3’) can bind to the heparin binding site of thrombin with a Kd around 0.5 nM.
Fig.2 Thrombin (A) and two frequently used aptamers (B and C). (Deng, 2014)
In recent years, different methods have been developed for thrombin detection such as aptamer-based strip biosensor, electrochemical aptasensor, homogeneous assays based on beacons, nanomaterial-assisted assays, and so forth. Besides the traditional strategy using intact aptamer, split aptamer fragments could also be used for thrombin detection. One part is labeled with fluorescein and another part is oligonucleotide sequence. When thrombin was introduced into the system, a target-induced G-quadruplex forms with two split aptamer fragments and thrombin. Moreover, label-free aptasensor system could also be used for thrombin detection.
The coupling of thrombin aptamer to different transduction principles has demonstrated the wide applicability of aptamers as bioreceptors in bioanalytical assays. If you are interested in aptamers for thrombin detection, please feel free to contact us for more information or a detailed quotation.
References
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