DNA Aptamer–Cyanine Complexes as Generic Colorimetric Small‐Molecule Sensors

Abstract

Aptamers are promising biorecognition elements for sensors. However, aptamer‐based assays often lack the requisite levels of sensitivity and/or selectivity because they typically employ structure‐switching aptamers with attenuated affinity and/or utilize reporters that require aptamer labeling or which are susceptible to false positives. Dye‐displacement assays offer a label‐free, sensitive means for overcoming these issues, wherein target binding liberates a dye that is complexed with the aptamer, producing an optical readout. However, broad utilization of these assays has been limited. Here, we demonstrate a rational approach to develop colorimetric cyanine dye‐displacement assays that can be broadly applied to DNA aptamers regardless of their structure, sequence, affinity, or the physicochemical properties of their targets. Our approach should accelerate the development of mix‐and‐measure assays that could be applied for diverse analytical applications.

Near-Infrared Dye-Aptamer Assay for Small Molecule Detection in Complex Specimens

“…For dye-displacement assays, the LOD is determined by the binding affinity of the aptamer and sensitivity of the instrumentation to minute changes in dye absorbance/concentration. Typically, aptamers with nanomolar K D have LODs around the same range, while aptamers with micromolar K D can exhibit LODs 2–10-fold lower than the K D . This is primarily because of instrumental limitations in detecting absorbance changes of dye at nanomolar levels.…”

“…Typically, aptamers with nanomolar K D have LODs around the same range, while aptamers with micromolar K D can exhibit LODs 2−10-fold lower than the K D . 7 This is primarily because of instrumental limitations in detecting absorbance changes of dye at nanomolar levels. In this work, the LODs we observed for all three tested targets (DHEAS, phenylalanine, and quinine) were 2−10-fold lower than the K D of the employed aptamers, and sufficient for the detection of targets at clinically relevant concentrations in biological samples.…”

“…They have been applied to several aptamer-target combinations to date, and we have recently demonstrated the broad applicability of this approach with cyanine dyes and a variety of small-molecule-binding aptamers. 7 However, it remains challenging to perform such assays in biologically relevant specimens such as blood and urine, which contain interferents that absorb and scatter light and can also fluoresce in the UV and visible spectrum. 8−10 One effective solution would be to employ far-red and near-infrared (NIR) signaling probes that absorb or emit light in the range of 700− 900 nm, where the absorption coefficients are low for most interferents in biological matrices, minimizing interference with assay performance.…”

“…These assays are label-free, rapid, sensitive, specific, and simple to performrequiring only a single mix-and-read step. They have been applied to several aptamer-target combinations to date, and we have recently demonstrated the broad applicability of this approach with cyanine dyes and a variety of small-molecule-binding aptamers …”

Ultrasensitive Automatic Detection of Small Molecules by Membrane Imaging of Single Molecule Assays

“…On the other hand, the DNA labels can also be easily edited into specially designed sequences involving aptamers, which can bind to targets with high specificity and affinity through their unique 3D conformation . In recent years, an increasing number of aptamer-based biosensors , or aptasensors have been developed for ultrasensitive small molecule detection. By selecting a suitable readout method, these small molecule detection approaches based on aptamer sensing and nucleic acid amplification are expected to achieve “single-molecule sensitivity”.…”