Highly sensitive two-photon sensing of thrombin in serum using aptamers and silver nanoparticles

Thrombin plays an important role in pathological conditions. It is important, however challenging, to detect thrombin in complex biological media for clinical practice and diagnostic applications. Here we demonstrate a label-free, fast, highly sensitive and selective two-photon sensing scheme for detection of thrombin on the picomolar level. The assay is based on interactions between thrombin and a DNA aptamer, which induce aggregation of silver nanoparticles to display significantly enhanced two-photon photoluminescence. The limit of detection (LOD) of this two-photon sensing assay is as low as 3.1 pM in the buffer solution, more than 400 times lower than that of the extinction method (1.3 nM). The dynamic range of this method covers more than 4 orders of magnitude. Furthermore, this two-photon sensing assay can be applied to detection of thrombin in 100% fetal bovine serum with LOD of 1.8 nM. In addition to the unique advantages of two-photon sensing such as deep penetration and localized detection, this method could be potentially integrated with two-photon microscopy to offer additional advantages of 3D detection and mapping for potential in vivo applications.