Novel Photochrome Aptamer Switch Assay (PHASA) for Adaptive Binding to Aptamers
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A novel Photochrome-Aptamer Switch Assay (PHASA) for the detection and quantification of small environmentally important molecules such as toxins, explosives, drugs and pollutants, which are difficult to detect using antibodies-based assays with high sensitivity and specificity, has been developed. The assay is based on the conjugation of a particular stilbene-analyte derivative to any aptamer of interest. A unique feature of the stilbene molecule is its reporting power via trans-cis photoisomerisation (from fluorescent trans-isomer to non-fluorescent cis-isomer) upon irradiation with the excitation light. The resulting fluorescence decay rate for the trans-isomer of the stilbene-analyte depends on viscosity and spatial freedom to rotate in the surrounding medium and can be used to indicate the presence of the analyte. Quantification of the assay is achieved by calibration of the fluorescence decay rate for the amount of the tested analyte. Two different formats of PHASA have been recently developed: direct conjugation and adaptive binding. New stilbene-maleimide derivatives used in the adaptive binding format have been prepared and characterised. They demonstrate effective binding to the model thiol compound and to the thiolated Malachite Green aptamer.
KeywordsStilbene Aptamer Fluorescence Photoisomerisation Maleimide
The authors wish to thank Dr. Vitali Lipik for his invaluable help with the MALDI-TOF analysis of the samples. This research is funded by the Singapore National Research Foundation and the publication is supported under the Campus for Research Excellence and Technological Enterprise (CREATE) programme (13-04-00364 А). The authors thank Huang Ruo Cheng, Tan Chong Yuan and Terence Mak of River Valley High School for their active participation in this project in the frame of the program for Singapore Science & Engineering Fair. In addition, the authors thank NTU for providing partial financial support to this program. Funding was also greatly appreciated from the Ministry of Education Tier 1 Grant: Photochrome aptamer switch assay: A universal bioassay device - RG54/13.
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