Aptamer binding assays for proteins: The thrombin example—A review

Abstract

Experimentally selected single-stranded DNA and RNA aptamers are able to bind to specific target molecules with high affinity and specificity. Many analytical methods make use of affinity binding between the specific targets and their aptamers. In the development of these methods, thrombin is the most frequently used target molecule to demonstrate the proof-of-principle. This paper critically reviews more than one hundred assays that are based on aptamer binding to thrombin. This review focuses on homogeneous binding assays, electrochemical aptasensors, and affinity separation techniques. The emphasis of this review is placed on understanding the principles and unique features of the assays. The principles of most assays for thrombin are applicable to the determination of other molecular targets.

Microplate based assay for thrombin detection using an RNA aptamer as affinity ligand and cleavage of a chromogenic or a fluorogenic peptide substrate

“…The specific binding of aptamers to thrombin also allows the use of aptamers as affinity ligands in assay developments for thrombin detection [7]. Many aptamer based assays for thrombin used DNA aptamers, either Apt-15 or Apt-29 [7]. Apt-15 or Apt-29 can also be used as a pair of affinity ligands in sandwich assays as they bind to different binding sites of thrombin [7].…”

“…The specific binding of aptamers to thrombin also allows the use of aptamers as affinity ligands in assay developments for thrombin detection [7]. Many aptamer based assays for thrombin used DNA aptamers, either Apt-15 or Apt-29 [7].…”

“…Many aptamer based assays for thrombin used DNA aptamers, either Apt-15 or Apt-29 [7]. Apt-15 or Apt-29 can also be used as a pair of affinity ligands in sandwich assays as they bind to different binding sites of thrombin [7]. Apt-15 has lower binding affinity than Apt-29, so Apt-29 usually gives better sensitivity in homogenous assays for thrombin [7].…”

“…Aptamers are the artificially generated single-stranded DNA and RNA by an in vitro process called SELEX (systematic evolution of ligands by exponential enrichment), specifically binding to targets with high affinity [5,6]. They have some unique analytical features in assay developments [7][8][9][10][11][12], such as ease of generation, synthesis, and labeling with functional groups, and they have been applied in many assays for a variety of targets [7][8][9][10][11][12][13][14][15][16]. Two DNA aptamers are widely used in these aptamer-based assays for thrombin.…”

“…Since the discovery of aptamers [5,6], many aptamer based assays have been developed for thrombin [7]. Aptamers are the artificially generated single-stranded DNA and RNA by an in vitro process called SELEX (systematic evolution of ligands by exponential enrichment), specifically binding to targets with high affinity [5,6].…”