9th July 2019

Traditionally, immuno-fluorescence polarization-based assays are used to detect small molecules by using antibodies and fluorescently labelled small-molecules. However, the assays do meet limitations such as complex antibody preparation, cost, and most importantly stability. Also the preparation of fluorescently labelled small molecules is challenging. The use of aptamers overcomes the limitations and brings new and diverse fluorescence anisotropy (FA) assays for small molecule detection with great improvement and advantages. Moreover, aptamers can be easily synthesised at low cost with good thermostability and specific fluorophore labelling.

Taking advantage of aptamers and this technique, Li and Zhao, 2019 described a simple and general aptamer structure switch FA assay for small molecule detection. The assay used streptavidin (SA) as a signal amplifier taking advantage of a proximity effect which reduces local rotation of fluorophore thereby enhancing the FA signal upon target binding (Figure 1).

Figure 1. A schematic diagram of binding-induced aptamer structure switch fluorescence anisotropy assay for small molecules using streptavidin as an effective signal amplifier based on proximity effect.

In this design, a FAM labelled aptamer was used as an affinity ligand and the SA labelled cDNA was employed as the signal enhancer. The SA-labelled cDNA hybridised with the FAM labelled aptamer in the absence of the target thereby drawing FAM close to SA and resulting in a much higher FA value due to restricted local rotation of FAM. Once the target molecule was introduced the high FA molecule displaced apart significantly reducing the overall signal. Therefore, FA detection of small molecules with higher sensitivity was achieved. The FA approach was used to detect aflatoxin B1 (AFB1), ochratoxin (OTA) and Adenosine triphosphate (ATP).

Figure 2. Detection of AFB1, OTA and ATP using aptamer structure switch FA assay via FAM labelled aptamer and SA-labelled cDNA.

The results gave a limit of detection of 60 pM for AFB1, 1nM for OTA and 500 nM for ATP which were lower than the regulation limits of the maximum target level in food set by European Union and United States. The results look very promising for developing highly sensitive assays for the detection of small molecules. Overall, the aptamer FA method combines the benefits of aptamers with FA analysis, and it is promising in applications such as affinity binding studies, drug discovery, clinical diagnosis, food safety and environmental monitoring.

At Aptamer Group, we are using a structure-switch based selection approach which is very advantageous for numerous applications, and could be easily transferred to this one. To know more about our small molecule detection methods and how the methods can help in your research, please contact us using the form below.

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Reference: Li Y & Zhao Q (2019). Aptamer Structure Switch Fluorescence Anisotropy Assay for Small Molecules Using Streptavidin as an Effective Signal Amplifier Based on Proximity Effect. Anal Chem, 91 (11): 7379-7384.