Rational design of an allosteric G-quadruplex aptamer probe for ultra-sensitive detection of melamine in milk.

Efficient and accurate detection of melamine in dairy products remains a crucial yet challenging task. Herein, an allosterically modulated G-quadruplex-integrated aptamer is rationally designed with thymine-rich recognition termini for melamine binding. The detection process is facile by simply introducing the analyte into the mixture consisting of G-quadruplex aptamer probes, exonuclease III, and thioflavin T (ThT). The detection feasibility is confirmed by the polyacrylamide gel electrophoresis and fluorescence measurement results. This exonuclease III-assisted signal amplifiable approach works well in a linear range from 0.1 nM to 0.1 μM. Moreover, a detection limit as low as 83 pM is easily achieved, which is almost five orders of magnitude smaller than the maximum allowable melamine levels (about 8 μM) defined by many countries all over the world. The whole assay time for each test is no longer than 1 h. Additionally, the scheme is highly specific and satisfactory recovery rates (from 91% to 104%) are readily obtained when challenged with melamine-spiked milk samples. Therefore, the label-free, turn-on, low-cost, and time-efficient method can be used for reliable detection of melamine in an easily manipulated and ultra-sensitive manner, which may find its utilization in the field of food safety, biomedical engineering, and clinical diagnosis.