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A facile and rapid colorimetric approach was described for selective and sensitive determination of adenosine triphosphate (ATP) based on a hairpin aptamer probe and the anti-aggregation of AuNPs. Poly(diallyldimethylammonium chloride) (PDDA) can induce the aggregation of AuNPs due to the electrostatic interaction causing a red to blue color change. Upon the addition of ATP, aptamer-based hairpin probe is opened and releases flexible ssDNA ends. The released flexible ssDNA ends can interact with PDDA and prevent PDDA-induced AuNPs aggregation. Thus, a visible color change from blue to red and a decrease in the absorption ratio (A 610 /A 520) are observed. Under the optimal conditions, the hairpin aptamer-based colorimetric assay exhibits high sensibility and selectivity for the detection of ATP with a detection limit of 1.7 nM. Moreover, this assay is successfully used in the rapid determination of ATP in spiked human serum samples with good recoveries in the range of 102.88 to 104.07%. Schematic illustration of the colorimetric assay based on the anti-aggregation of AuNPs for the ATP detection. Flexible ssDNA ends were released due to the forming of the aptamer-ATP complexes, which electrostatically hybridize to PDDA to form a duplex and prevents the PDDA-induced aggregation of AuNPs with a color change from blue to red. Unlabelled Image • It has a high sensitivity with a detection limit of 1.7 nM for ATP. Furthermore, the coupling a hairpin-like DNA probe and anti-aggregation of AuNPs guarantees the good selectivity of the assay to ATP and eliminates the effect of non-specific interference. • It is a more universal method that can be used to study other targets by simply changing the sequences of the corresponding aptamers of the hairpin-like DNA probe. [ABSTRACT FROM AUTHOR]