Affordable Access

deepdyve-link
Publisher Website

Nano-Affi: a solution-phase, label-free, colorimetric aptamer affinity assay based on binding-inhibited aggregation of gold nanoparticles.

Authors
  • Wan, Yuan1
  • Zhao, Jiaxing
  • He, Junlin
  • Lou, Xinhui
  • 1 Department of Chemistry, Capital Normal University, Beijing, 100048, China. [email protected] , (China)
Type
Published Article
Journal
The Analyst
Publisher
The Royal Society of Chemistry
Publication Date
Jun 21, 2020
Volume
145
Issue
12
Pages
4276–4282
Identifiers
DOI: 10.1039/d0an00827c
PMID: 32478774
Source
Medline
Language
English
License
Unknown

Abstract

The ideal way to assess aptamer affinity is when both aptamer and target are in a native state, without the unpredictable interference associated with labelling and surface immobilization. However, most current aptamer affinity assays need aptamer (or target) immobilization on surface and/or labelling. Ideally, such a solution-phase assay should also be high-throughput, in order to accelerate aptamer identification, binding site study, and engineering for various downstream applications. So far, only isothermal titration calorimetry (ITC) enables label-free solution-phase affinity measurements, but with low-throughput and the need of large amount of samples. Here, we report a solution-phase, label-free, colorimetric gold nanoparticle (AuNP)-based affinity assay (Nano-Affi) that addresses this need. Nano-Affi is based on kinetically-favoured, adsorbate charge-tuned aggregation of AuNPs, wherein positively-charged or near-neutral proteins induce instantaneous aggregation of negatively-charged AuNPs at the pH below or near the isoelectric point of the target protein. In contrast, protein-aptamer complexes possess a greater negative charge than free targets, and thus induce little or no aggregation of AuNPs due to electrostatic repulsion. The higher an aptamer's affinity for the protein, the less AuNP aggregation occurs. We demonstrate here that Nano-Affi enables the reliable aptamer screening and dissociation constant determination for diverse protein targets, as well as binding site identification, with readouts based on colour observation or absorbance or dynamic light scattering size measurements. Nano-Affi possesses sub-nanomolar sensitivity and can be performed with nanogram amounts of protein in less than half an hour with minimal training and minimal instrument requirements.

Report this publication

Statistics

Seen <100 times