Article pubs.acs.org/ac
Using Graphene Quantum Dots as Photoluminescent Probes for Protein Kinase Sensing Ying Wang, Li Zhang, Ru-Ping Liang, Jian-Mei Bai, and Jian-Ding Qiu* Department of Chemistry, Nanchang University, Nanchang 330031, P. R. China S Supporting Information *
ABSTRACT: A simple and sensitive photoluminescence (PL) assay for the activity of a protein kinase based on the selective aggregation of phosphorylated peptide−graphene quantum dot (GQD) conjugates triggered by Zr4+ ion coordination has been established. With more sophisticated design of the peptide substrate sequences, detecting other enzymes could also be possible. Under optimal conditions, a linear relationship between the decreased PL intensity of peptide−GQD conjugates and the concentration of casein kinase II (CK2) in the range from 0.1 to 1.0 unit mL−1 with a detection limit of 0.03 unit mL−1 (3σ) was obtained. The EC50 value (i.e., the enzyme concentration producing 50% substrate conversion) for CK2 was evaluated to be 0.34 unit mL−1. The proposed method showed potential applications in kinase inhibitor screening. To demonstrate the potential of this GQD-based platform for screening of kinase inhibitors in real biological systems, the inhibition of CK2 phosphorylation activity by four different inhibitors (ellagic acid, 5,6-dichlorobenzimidazole-l-β-D-ribofuranoside, emodin, and quercetin) was tested in human serum by comparing signals from samples incubated with the inhibitors against that without any inhibitor. As expected, in the presence of inhibitors, the PL intensity increased with increasing inhibitor efficiency. The IC50 value (inhibitor concentration producing 50% inhibition) for ellagic acid was estimated to be 0.041 μM. The developed protocol provides a new and promising tool for the analysis of both the enzyme and its inhibitors with low cost and excellent performance.
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inchoate,15−21 and the obstacles mainly originate from the difficulties in constructing functionalized GQDs that can both selectively recognize a target and give a sensitive signal response. Protein kinases, which catalyze the phosphorylation of proteins by transferring phosphate groups to specific amino acids, make up a huge superfamily of enzymes (termed the “kinome”) that play a critical role in intracellular signal transduction and the regulation of cellular functions including cell growth, survival differentiation, and metabolism.22 With more than 300 protein substrates identified to date, the Ser/ Thr-specific protein kinase casein kinase II (CK2) is probably the most pleiotropic member of the human kinome. Abnormal expression of CK has been implicated in a number of diseases such as cancer23 and HIV and Alzheimer’s disease.24,25 The identification of kinase activities and their potential inhibitors is not only necessary for basic biology to clarify molecular mechanisms of signal transduction but also valuable for protein kinase-targeted drug discovery and therapy. Existing methods for the measurement of protein kinase activities include radioactive,26,27 electroactive,28,29 fluorescent,30−32 and biotin33,34 or thiol35,36 labeling techniques and phosphorylationspecific recognition protein (e.g., antibody or SH2 domain)based methods.37−41 Such assays are effective but require labor-
raphene, a two-dimensional carbon material, has sparked great excitement within the scientific community because of its fascinating and unusual physical and chemical properties.1,2 Nevertheless, as graphene is a zero-band-gap semiconductor, the possibility of observing its luminescence is highly unlikely,3 and its optoelectronic applications have been limited to date. To generate an electronic band gap in graphene, various methods such as opening the band gap through doping,4 manipulating graphene oxide (GO) through partial reduction and surface passivation,5,6 and cleaving GO materials into nanoscale graphene quantum dots (GQDs)7,8 have been used to induce photoluminescence (PL). Among them, fabrication of GQDs and tuning of their optical properties have become the most active area. In theoretical and experimental studies, GQDs with diameters of
