Non-enzymatic detection of urea using unmodified gold nanoparticles based aptasensor

• We report FluMag SELEX method to isolate urea specific DNA aptamer.
• We demonstrate a novel, non-enzymatic, easy-to-use, dual readout aptasensor for urea.
• This method is free from complicated sample processing and labeling steps.
• The urea aptasensor reliably detects urea adulteration in milk.
• The results obtained are comparable to the other urease based detection methods.

Biosensing nitrogenous compounds like urea is required to control the incidents of Economically Motivated Adulteration (EMA). In this study, we report the FluMag Systematic Evolution of Ligands by Exponential Enrichment (FluMag-SELEX) method to isolate a urea specific DNA aptamer with a dissociation constant (Kd) of 232 nM. The interaction of DNA aptamer with urea has been confirmed by affinity assay, CD analysis, melting curve analysis and truncation studies. Unlike other urea sensing methods reported so far, using this urea aptamer, we demonstrate a simple, ‘non-enzymatic’ easy-to-use, dual readout aptasensor that exploits unmodified gold nanoparticles (AuNPs) to transduce the signals of aptamer binding to urea in terms of intrinsic fluorescence differences and color changes simultaneously. This method is free from complicated sample processing and labeling steps. The urea aptasensor displays high selectivity for urea and is free from interference from common milk adulterants. The developed aptasensor reliably detects urea adulteration in milk. The response signals linearly correlate with the increasing concentrations of urea in milk ranging from 20 mM to 150 mM with detection limit of 20 mM. We also show that this aptasensor can also be used as a simple fluorescence based “turn-on” sensor. The results obtained in this study are comparable to the commercial urease based detection methods.