Thrombin-linked aptamer assay for detection of platelet derived growth factor BB on magnetic beads in a sandwich format

Abstract

a b s t r a c tHere we describe a thrombin-linked aptamer assay (TLAA) for protein by using thrombin as an enzyme label, harnessing enzyme activity of thrombin and aptamer affinity binding. TLAA converts detection of specific target proteins to the detection of thrombin by using a DNA sequence that consists of two aptamers with the first aptamer binding to the specific target protein and the second aptamer binding to thrombin. Through the affinity binding, the thrombin enzyme is labeled on the protein target, and thrombin catalyzes the hydrolysis of small peptide substrate into product, generating signals for quantification. As a proof of principle, we show a sandwich TLAA for platelet derived growth factor BB (PDGF-BB) by using anti-PDGF-BB antibody coated on magnetic beads and an oligonucleotide containing the aptamer for PDGF-BB and the aptamer for thrombin. The binding of PDGF-BB to both the antibody and the aptamer results in labeling the complex with thrombin. We achieved detection of PDGF-BB at 16 pM. This TLAA contributes a new application of thrombin and its aptamer in bioanalysis, and shows potentials in assay developments.

Determination of the platelet-derived growth factor BB by a competitive thrombin-linked aptamer-based Fluorometric assay

“…The use of amplification strategy through rolling circle amplification (RCA) or highly sensitive techniques can provide extremely high sensitivity for detection of PDGF-BB [28-31, 33, 35], better than our results. Compared with the sandwich TLAA assay [13], the competitive TLAA has lower sensitivity. Although many sensitive aptamer-based assay have been reported for the detection of PDGF-BB [28-31, 33, 35], here we demonstrate the competitive TLAA for protein detection is feasible by using the detection of PDGF-BB as an example.…”

“…A DNA probe with the sequence, 5′-TAC TCA GGG CAC TGC AAG CAA TTG TGG TCC CAA TGG GCT GAG TA TTTTTT AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-3′, was synthesized and purified by Sangon Biotech (Shanghai, China, http://www.sangon.com). In the sequence, the boldface portion was the aptamer for PDGF-BB, and the underlined portion was the aptamer for thrombin [13]. A polyT sequence was used as a linker between these two aptamer sequences.…”

“…Taking advantage of affinity capture and enzyme activity of thrombin in catalyzing cleavage of small peptide substrates, we developed aptamer capture-based assays for thrombin detection [11,12]. Using thrombin as an enzyme label, recently we have reported a thrombin-linked aptamer assay (TLAA) for non-thrombin protein detection in a sandwich format [13], which relies on thrombin-binding aptamer, enzyme activity of thrombin, and affinity aptamer for protein targets. In the TLAA strategy, the target protein is sandwiched by the antibody on solid surface and a DNA probe that consists of an aptamer sequence for protein target and an aptamer sequence for thrombin.…”

Programming Affinity for Precise Tumor Recognition with Allosteric Nanosensing-Circles

“…To address that, we designed several PDGF-NSCs stimulated by PDGF-BB. First, a PDGF-BB-binding aptamer (PBBA) 46 was embedded into PDGF-NSCs with the aim of preventing the formation of Sgc8c by steric hindrance and thus restraining recognition affinity (Figure 4A). On the other hand, the configuration of PDGF-NSCs transformed from the random intrastrand hybridization to the ordered state with assistance of PDGF-BB, thus restoring the conformation and recognition function of Sgc8c in PDGF-NSCs.…”

Thrombin-linked aptamer assay for sensitive detection of immunoglobulin E in sandwich format

“…Taking advantage of aptamer binding affinity and the enzymatic activity of thrombin in cleaving peptide substrates, we previously reported thrombin linked aptamer assays (TLAA) for the detection of platelet derived growth factor-BB (PDGF-BB). 16,17 This strategy converted detection of PDGF-BB to the measurement of thrombin by using a DNA sequence that consists of two aptamers with the first aptamer binding to the PDGF-BB and the second aptamer binding to thrombin, the thrombin was labeled on the affinity complex, and thrombin catalyzed the peptide substrate into product, generating signals for final quantification of PDGF-BB. This TLAA contributes a new application of thrombin and its aptamer in bioanalysis, and provides a new way for signal generation.…”