SERS determination of protease through a particle-on-a-film configuration constructed by electrostatic assembly in an enzymatic hydrolysis reaction

We describe a simple and universal method for trypsin determination with the surface-enhanced Raman scattering (SERS) technique. This trypsin assay was carried out on a SERS sensing chip made by immobilizing an unlabeled double charged peptide on an Ag vapour-deposited film, while a Raman probe (4-mercaptobenzoic acid) was fixed together above the Ag film as the signal reporter. This designed peptide contains a specific amino acid (arginine) as the substrate domain of trypsin in the middle and two oppositely charged segments at two ends. When trypsin catalyzes the hydrolysis reaction of peptide at the location of arginine in a buffer solution, the segment that contains the negatively charged amino chain will leave from the Ag film. The remaining sequence makes positive charges on the Ag film surface increase, enabling the assembly of the citrate-stabilized Ag nanoparticles (negative charges) on the chip via an electrostatic interaction for the purpose of signal amplification. As a result, SERS signals of 4-mercaptobenzoic acid sharply increase due to the formation of the hotspot-like particle-on-a-film configuration, by which the concentration and activity of trypsin can be determined indirectly. Owing to the strong electromagnetic field enhancement in the gap region, the detection limit of trypsin down to 1.0 nM (the ratio of signal to noise of 3) can be reached. The specificity of this method can also be guaranteed due to the enzyme/substrate specific identification. The method can be extended to other enzyme-catalyzed systems and become a universal approach for other proteases.