Pulsed laser irradiation induces the generation of alloy cluster ions for the screening of protease activity

Pulsed laser irradiation can cause the fragmentation of nanoparticles, which generates cluster ions. This allows nanoparticles to be adopted as mass tag/signal amplifiers in laser desorption/ionization mass spectrometry (LDI-MS) bioassays. Herein, we demonstrate the potential of using the signal from alloy cluster ions in bioassays through a fibrin clot model to determine the activity of thrombin. A mixed solution of silver and gold nanoparticles functionalized with fibrinogen (Fg‒Ag NPs/Fg‒Au NPs) treated with thrombin can form clots composed of aggregated fibrin-Au NPs/Ag NPs. These clots analyzed with LDI-MS are noted to form intense Ag–Au alloy cluster ions, especially [Ag₂Au]⁺, which were used to detect thrombin concentration with a dynamic range of 2.5–50 pM in human plasma. This sensing platform was further employed for the screening of direct thrombin inhibitors. This work developed a novel bioassay utilizing metallic gas-phase reactions generated from pulsed laser irradiation of aggregated nanoparticles to monitor enzymatic activity and to screen inhibitors. We believe that LDS-MS can serve as a new platform for gas-phase reaction-based bioassays.