Fluorescent Graphene Oxide-based Optical Biosensors for Detection of Disease-related Protease

Abstract

Graphene oxide (GO) have attracted considerable attention as a fluorescent probe for biosensors due to its unique and fascinating properties such as strong UV absorption, stable fluorescence in biological environment, and fluorescence quenching ability as well as preferential interaction with biomolecules. Although many attempts have shown promising results by using GO as a quencher for fluorescent dye in fluorescence resonance energy transfer (FRET) sensor, there are still several challenging issues including the use of unstable organic dyes. Therefore, various optical biosensors were designed by using GO as a fluorescent probe to enhance its performance for the detection of target biomolecule. In the first part, GO PL-based optical biosensor consisting of a luminescent GO donor covalently linked with a peptide-quencher complex was described for a simple, rapid, and sensitive detection of proteases. To this end, the quenching efficiency of various candidate quenchers of GO fluorescence, such as metalloprotoporphyrins and QXL570, were examined and QXL570 was found to be much more effective for quenching the intrinsic fluorescence of GO than other charge transfer-based quenchers. The designed GO-peptide-QXL system was able to sensitively detect specific proteases, chymotrypsin and matrix metalloproteinase-2, via turn-on response of quenched GO fluorescence after proteolytic cleavage of the quencher. Finally, the GO–peptide–QXL hybrid successfully detected MMP-2 secreted from living cells, human hepatocytes HepG2, with high sensitivity. In the second part, a new type of GO optical turn-on biosensor for the detection of MMP-2 was fabricated by means of of a peptide-induced assembly of fluorescent GO. Functionalization of GO with a peptide substrate for MMP-2 bearing a thiol group led to its self-assembly via disulfide bonding, accompanied by selfquenching of GOs fluorescence. This peptide-induced GO assembly was then disassembled by proteolytic cleavage in the presence of MMP-2, thereby restoring the level of self-quenched GO fluorescence. With this approach, we were able to detect MMP-2 and to investigate the kinetic parameters of MMP-2 activity. The GO–peptide assembly was successfully applied to the selective and sensitive detection of MMP-2 secreted by living cells, human hepatocytes HepG2, at a concentration of 2 ng mL−1. In the third part, cysteine-modified GO (Cys-GO) doped with nitrogen and sulfur atoms was described. Cys-GO was simply synthesized by reacting with cysteine in the acidic water/dimethylformamide cosolvent system via ultra-sonication method under mild reaction conditions. The N and S contents in Cys-GO dramatically increased to 10.4 and 23.3 wt%. As-synthesized Cys-GO exhibited highly fluorescent properties compared to pristine GO, which could be attributed to the synergistic effective on radiative emission from heteroatoms of Cys-GO. Cys-GO was further found to be suitable as fluorescent probe for more sensitive detection of MMP-2. These studies suggest that GO PL-based optical biosensors could be a promising platform for sensitive and selective biomolecule detection in the fields of medicine and pharmaceutical industry.