Electrochemical detection of tobramycin based on enzymes-assisted dual signal amplification by using a novel truncated aptamer with high affinity

An aptamer with the length of only 15 nucleotides specific for tobramycin was obtained through rationally designed truncation from a previously reported long sequence. The structural and binding properties of the aptamer were characterized. The dissociation constant (Kd) was determined to be 42.12 nM, indicating high affinity of the aptamer for tobramycin. Then an electrochemical sensor based on this aptamer was developed, which employed an enzymes-assisted dual signal amplification cycle through target recycling and strand-displacement DNA polymerization. A hairpin probe containing the aptamer sequence was designed and used to start the production cycle of a short ssDNA fragment in the presence of tobramycin, with the help of phi29 DNA polymerase and nicking endonuclease Nt.AlwI. The ssDNA fragment was captured by a signal transduction probe modified on gold electrode to form a triple-helix structure. With the help of [Ru(NH3)6]3+, a significant electrochemical signal was observed in differential pulse voltammetry (DPV). Under the optimal conditions, the current in DPV is linearly related with the concentration of tobramycin in the range of 10-200 nM, and the detection limit is 5.13 nM. The electrochemical sensor showed high specificity for tobramycin when it was challenged by other antibiotics. In addition, the constructed sensor was used to detect tobramycin in milk and water samples, and showed satisfactory performance. Therefore, the screened aptamer as well as the developed sensor has great application prospects in the fields of food safety control, medical test and environment monitoring.