Detection Of Caspase-3by The Fluorescence Quenching Effect Of A Self-assembly Nanoparticle

The cysteine proteases are widely present in biological cells with a similar structure, generally containing the active site cysteine. Since they are possible to specifically cleave aspartic acid residue, they are also known as caspases. Caspase is one of the main effectors of apoptosis process and plays an important role in many disorders such as Alzheimer. However, caspase plays different roles, express different concentrations in the tumor cells inside different tissues or during different stages of apoptosis. Caspase-3, one of the well-studied proteases in the caspase family, can specifically cleave Asp-Glu-Val-Asp (DEVD). The detection of caspase-3activity can help with our understanding of tumor cell growth and provide effective means for early diagnosis and targeted therapy.Self-assembly is a chemical, biological process of small molecules aggregate into larger molecules even supramolecular, such as the double helix structure of DNA as well as cell membrane in the animal and plant cells. Traditional self-assembly reaction controlled by the protease requires need high concentrations of the reaction substrate or the protease. However, the reaction in recent discovery generated by CBT cyano and cysteine1,2-aminothiol, can be accomplished by only a few units of caspase-3enzyme and controlled by pH or by reducing conditions. The condensation products can self-assemble to form nanoparticles and if properly fluorophore is connected on the reactants, it will bring certain chemical changes such as fluorescence intensity, This provides us a powerful tool to measure low levels of concentration of the enzyme caspase-3.We report a new probe Ac-Asp-Glu-Val-Asp-Cys(StBu)-Lys(FITC)-CBT (1) which is able to be reduced by TCEP and cleaved by caspase-3to yield the amphiphilic dimer (1-D).1-D can quickly self-assemble into nanoparticles to induce a clear fluorescence quenching effect because of the Aggregation-caused quench. The nanoparticles were characterized with Dynamic Light Scattering (DLS), transmission electron microscope (TEM), and HPLC. As a result, the fluorescence intensity has a linear correlation with the concentration of caspase-3, which could be applied for sensing caspase-3activity in vivo in the future.