Graphene oxide-based chemiluminescent sensing platform for label-free detection of trypsin and its inhibitors

Abstract

In this study, a novel label-free chemiluminescent (CL) sensing platform for trypsin and trypsin inhibitor screening was developed by taking advantage of a luminol–NaIO₄-(cysteine (Cys)-containing peptide) system and graphene oxide (GO). The assay was based on the different catalytic activities of a Cys-containing peptide and a Cys-containing peptide–GO complex in a luminol–NaIO₄ CL reaction. A strong CL emission was obtained when the Cys-containing peptide was mixed with NaIO₄ and basic luminol, while a much lower CL signal was observed when the Cys-containing peptide–GO complex was injected into a mixture of NaIO₄ and basic luminol instead of the Cys-containing peptide. Based on a room-temperature electron spin-resonance spectroscopy (ESR) study, we speculated that the catalytic activity of Cys-containing peptide in the luminol–NaIO₄ CL system may be inhibited when the peptides were adsorbed on the GO surface. Based on the above observation, a label-free CL sensing platform for trypsin was developed. In the absence of trypsin, the Cys-containing peptide was adsorbed on the GO surface via electrostatic interactions resulting in a low CL signal. In contrast, in the presence of trypsin, the peptide can be catalytically hydrolyzed at the C-terminus of arginine (Arg), resulting in the release of Cys-containing residues from the surface of GO and subsequent CL recovery with the addition of luminol and NaIO₄. The proposed method enabled the determination of trypsin with a detection limit of 7.3 pM, and can also be employed for screening of trypsin inhibitors. The method can be easily generalized for monitoring the hydrolysis activity of other proteases by simply changing the peptide substrate sequence.