Single-Molecule Sandwich Aptasensing on Nanoarrays by Tethered Particle Motion Analysis

Abstract

High-throughput single-molecule techniques are expected to challenge the demand for rapid, simple, and sensitive detection methods in health and environmental fields. Based on a single-DNA-molecule biochip for the parallelization of tethered particle motion analyses by videomicroscopy coupled to image analysis and its smart combination with aptamers, we successfully developed an aptasensor enabling the detection of single target molecules by a sandwich assay. One aptamer is grafted to the nanoparticles tethered to the surface by a long DNA molecule bearing the second aptamer in its middle. The detection and quantification of the target are direct. The recognition of the target by a pair of aptamers leads to a looped configuration of the DNA–particle complex associated with a restricted motion of the particles, which is monitored in real time. An analytical range extending over 3 orders of magnitude of target concentration with a limit of detection in the picomolar range was obtained for thrombin.

Plasmonic Probing of Deoxyribonucleic Acid Hybridization at the Single Base Pair Resolution

“…In addition to the capability of label-free detection and easy handling, SPRM, due to its high spatial and temporal resolution, has been applied to probe the interaction of molecules at the single-particle or even single-molecule level. 28,29 Several studies have reported the application of plasmonic imaging for studying DNA hybridization adsorption behaviors and ultrasensitive detection of DNA analytes. 30,31 Combined with nanotechnology, singlemolecule sensing has been achieved using plasmonic imaging techniques.…”

“…By acquiring 2D images with high temporal resolution, SPRM realizes real-time monitoring and high-throughput analysis of molecular behaviors. In addition to the capability of label-free detection and easy handling, SPRM, due to its high spatial and temporal resolution, has been applied to probe the interaction of molecules at the single-particle or even single-molecule level. , Several studies have reported the application of plasmonic imaging for studying DNA hybridization adsorption behaviors and ultrasensitive detection of DNA analytes. , Combined with nanotechnology, single-molecule sensing has been achieved using plasmonic imaging techniques. , However, most of the research has focused on the kinetics of DNA hybridization behavior, ignoring how different complementary DNA sequences affect the DNA hybridization process, which is also challenging to detect for traditional surface plasmonic imaging.…”

Target-Synergized Biologically Mediated RAFT Polymerization for Electrochemical Aptasensing of Femtomolar Thrombin

“…Since its first discovery by Toole’s group in 1992, thrombin-binding aptamer (TBA) has been widely studied in therapeutic and diagnostic applications because of its high affinity and good specificity. − As aptamers are oligonucleotides screened from a random sequence library via systematic evolution of ligands by exponential enrichment (SELEX) and can be chemically synthesized in vitro, they offer the benefits of low cost, high thermal stability, long shelf life, and minimal interbatch production variability as molecular recognition receptors. − During the past few decades, a variety of aptamer-based fluorescent, colorimetric, electrochemiluminescent (ECL), , surface-enhanced Raman scattering (SERS), , chromatographic, nanopore, photoelectrochemical (PEC), , and electrochemical − methods have been put forward for thrombin detection. Relative to other methods, electrochemical aptasensors have drawn much more attention in thrombin detection, because of their high detection sensitivity, low matrix interference, high selectivity, and simple instrument. , On the basis of the significant change in redox current caused by the target-induced conformational change, for example, Plaxco et al illustrated a method for the electrochemical aptasensing of thrombin, using the methylene blue (MB)-tagged TBA as the recognition receptor.…”