Nanoprobe-Enhanced, Split Aptamer-Based Electrochemical Sandwich Assay for Ultrasensitive Detection of Small Molecules

Abstract

It is quite challenging to improve the binding affinity of antismall molecule aptamers. We report that the binding affinity of anticocaine split aptamer pairs improved by up to 66-fold by gold nanoparticles (AuNP)-attached aptamers due to the substantially increased local concentration of aptamers and multiple and simultaneous ligand interactions. The significantly improved binding affinity enables the detection of small molecule targets with unprecedented sensitivity, as demonstrated in nanoprobe-enhanced split aptamer-based electrochemical sandwich assays (NE-SAESA). NE-SAESA replaces the traditional molecular reporter probe with AuNPs conjugated to multiple reporter probes. The increased binding affinity allowed us to use 1,000-fold lower reporter probe concentrations relative to those employed in SAESA. We show that the near-elimination of background in NE-SAESA effectively improves assay sensitivity by ∼1,000-100,000-fold for ATP and cocaine detection, relative to equivalent SAESA. With the ongoing development of new strategies for the selection of aptamers, we anticipate that our sensor platform should offer a generalizable approach for the high-sensitivity detection of diverse targets. More importantly, we believe that NE-SAESA represents a novel strategy to improve the binding affinity between a small molecule and its aptamer and potentially can be extended to other detection platforms.

X-Aptamer Technology Identifies C4A and ApoB in Blood as Potential Markers for Schizophrenia

“…X-aptamers are chemically modified DNA affinity agents that employ a wide range of chemical modifications such as amino acid-like side chains, phosphorodithioates, and small molecules, which allow X-aptamers to bind to target proteins with high specificity and affinity. As novel elements for molecular recognition, X-aptamers possess specific recognition properties toward low-molecular-weight substrates or macromolecules [10]. Due to their affinity and specificity for target molecules, high stability, and facility of synthesis and modification, aptamers are feasible and practical alternatives to antibodies for many applications including research, diagnostics, and therapeutics [7,11,12].…”

A Novel Biosensor Based on Terminal Protection and Fluorescent Copper Nanoparticles for Detecting Potassium Ion

“…[6][7][8] Nucleic acid-based aptamers that are generated by a systematic evolution of ligands with an exponential enrichment (SELEX) process and obtained through selections, 9 are artificial single-stranded DNA (ssDNA) or RNA sequences that can recognize specific biological molecules with a wide range, such as small molecules, 10 organic compound, 11 proteins 12 or even whole cells. 13 With outstanding stability, high affinity, excellent selectivity, easy modification, as well as low immunogenesis, nucleic acid-based aptamers have successfully attracted the increasing attention of researchers, and have been widely used in food safety, diagnosis, cell imaging and drug delivery.…”

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“…131 This MNS-4.1 aptamer in theory undergoes a conformational change in the presence of cocaine, however the equilibrium between the folded and the unfolded states found in practice led towards higher background signals, limiting the sensitivity of the aptamer. 131,226 To address this issue, MNS-4.1 was subsequently split into multiple aptamer fragments (2 130 or 3 143,144 ) that remain separated in the absence of cocaine, however come together in the presence of cocaine, thereby reducing the background. Although the background is reduced with the split aptamer the sensitivity remained limited owing to the low binding affinity with single binding domain.…”

“…To address this issue, MNS-4.1 was subsequently split into two 131 or three 143,144 smaller aptamer fragments that remain separated in the absence of cocaine but assemble in the presence of cocaine, thereby reducing the background. Stojanovic's group used this split aptamer for fluorescence detection of cocaine, with the two fragments tagged with a fluorophore and quencher respectively.…”