Sensitive electrogenerated chemiluminescence biosensors for protein kinase activity analysis based on bimetallic catalysis signal amplification and recognition of Au and Pt loaded metal-organic frameworks nanocomposites
Introduction
Protein phosphorylation catalyzed by protein kinase and dephosphorylation catalyzed by phosphatase play important roles in intracellular signaling pathways (Hunter, 2000). In principle, protein kinase can catalyze the phosphorylation of protein through transferring phosphate groups from nucleoside triphosphates (adenosine-5′-triphosphate, ATP) to the OH group of an amino acid side chain in peptides or protein substrate. As is well known, protein kinase is involved in a variety of cellular activities such as cell surface signal transduction (Nishizuka, 1986), gene transcription (Whitmarsh, 2007), metabolism (Whiteman et al., 2002), cellular proliferation (Zhang et al., 1990). Thus, the level of protein kinase activity can be considered as an indicator of many human diseases, such as diabetes (Evcimen and King, 2007), Alzheimer's disease (Salminen et al., 2011), metabolic syndrome and heart disease (Hardie, 2008). In addition, kinase inhibitors can regulate down the activity of protein kinase and have been widely investigated in medicinal research and clinical treatment (Hughes et al., 2006). Therefore, it is highly desirable to develop a sensing platform for the accurate protein kinase activity analysis and potential inhibitors screening.
Up to now, several methods have been developed to detect protein kinase activity such as isotope labeling technique (Hennrich et al., 2013), fluorescence (Colombo et al., 2017, Shen et al., 2015), colorimetry (Zhou et al., 2014) and electrochemical based analytical systems (Shen et al., 2016a), etc. (Liu et al., 2014). Among these methods, electrogenerated chemiluminescence (ECL) technique exhibits unique advantages (Ji et al., 2017, Zhu et al., 2017), such as low background, easy temporal and spatial controllability due to the intrinsic light-generation process of ECL on the electrode surface (Meng et al., 2016). For instance, an ECL biosensor for protein kinase A (PKA) activity detection was reported based on double-quenching of graphene quantum dots ECL by G-quadruplex-hemin DNAzyme and gold nanoparticles (AuNPs) (Liu et al., 2015a). AuNPs mediated dual-potential ECL ratiometric approach based on the simultaneous decrease of cathodic ECL from graphene quantum dots and enhancement of anodic ECL from luminol was reported for protein kinase activity assay (Zhao et al., 2015). Ruthenium complex functionalized protein A and specific peptides were utilized as ECL probe and recognition substrates respectively in an ECL strategy for cyclic adenosine monophosphate-dependent protein kinase and casein kinase II detection (Liu et al., 2016). However, because of the limited phosphate group recognition sites, the metal ion chelation and antibody recognition can suffer the poor recognition efficiency, multistep operations and cross-linking reactions. Therefore, novel strategies with efficient phosphate groups recognition and intensified ECL generation are still in high demand.
Bimetallic nanoparticles are critically important in catalysis and electrocatalysis based systems (He et al., 2017, Joseph et al., 2007). They show superior electronic and catalytic properties due to synergistic effects compared to the corresponding homologous monometal nanoparticles (Chen et al., 2012). Amongst various bimetal nanoparticles, Au and Pt bimetallic nanoparticles exhibit outstanding performance in sensing fields, ascribed to their extraordinary catalytic activities, good conductivity and favorable biocompatibility, etc (Qi et al., 2016, Zhang et al., 2017). For instance, bimetallic Au and Pt nanoparticles were investigated in detection of H2O2 (Yu et al., 2015), Nitrite (Yang et al., 2016), metal ions (Bu et al., 2015), small organic molecules (Liu et al., 2015b), etc (Wang et al., 2016). Moreover, bimetallic Au and Pt nanoparticles were utilized in an ECL sensor for their ability to enhance the ECL signals of luminol (Shan et al., 2016). However, metal nanoparticles are prone to aggregation during catalytic reactions because of their high surface energy. As a class of crystalline porous materials, metal-organic frameworks (MOFs) featured ordered structures, controllable porosities and large internal surface areas are appropriate candidate to prevent the agglomeration of nanoparticles (Meilikhov et al., 2010). For example, Pd nanoparticles have been loaded into alkyne functionalized MOFs for heterogeneous catalysis (Gole et al., 2016). Pt-Co bimetallic nanoparticles encapsulated within MOFs with controllable size and spatial distribution have been synthesized (Chang and Li, 2017). Besides, the pores inside MOFs provide numerous channels to improve the species diffusion (Yang et al., 2017), affording great potential in sensing applications. Particularly, MOFs hybrids that decorate with metal nanoparticles (NPs@MOFs) can offer great possibilities in designing new signal transductions due to their exceptional tunability (Lei et al., 2014). For instance, AuNPs decorated Ce based MOFs were designed as nanocarriers in an electrochemical strategy, during which AuNPs were utilized to capture SH terminated probes while Ce based MOFs acted as catalysts (Shen et al., 2016b). Besides the ultrahigh porosities and large internal surface areas, NPs@MOFs based sensing systems can benefit from their numerous reactive sites for guest species adsorption or reaction (Wu et al., 2013), thus improve the performances.
In this work, a novel ECL strategy for PKA activity detection and inhibition screening based on Au&Pt@UiO-66 nanocomposites was developed, in which the bimetallic Au and Pt nanoparticles acted as electrocatalysts to improve the ECL signal and the UiO-66 was used as the carriers for bimetallic nanoparticles and the anchorages for phosphate groups. Using PKA as a model, Au&Pt@UiO-66 probes were assembled onto the phosphorylated kemptide modified electrode by forming Zr-O-P bonds between the Zr surface defects on UiO-66 and phosphate groups in phosphorylated kemptide. In addition, the synthesized Au&Pt@UiO-66 nanocomposites not only provided multiple electroactive sites to catalyze the luminol-H2O2 ECL reaction, but also afforded numerous Zr defect sites for high efficient phosphate groups recognition. Thus the ECL intensity of the sensing system was significantly improved, affording a sensitive, stable and universal platform for kinase activity analysis and inhibitor screening.
Section snippets
Materials and reagents
Cysteine-terminated kemptide (CLRRASLG) was obtained from GL Biochem (Shanghai, China). PKA (catalytic subunit from bovine heart), ATP and hemoglobin were obtained from Dingguo Biological Products Company (China). Luminol, 4,4′,5,5′,6,6′-hexahydroxydiphenic acid 2,6,2′,6′-dilactone (ellagic acid), N-(3-chlorophenyl)− 6,7-dimethoxy-4-quinazolinamine (Tyrphostin AG1478), anacradic acid, Forskolin and 3-isobutyl-1-methylxantine (IBMX) were purchased from Sigma. Zirconium chloride (ZrCl4),
Protein kinase activity evaluation
The proposed ECL strategy for PKA activity detection based on bimetallic catalysis signal amplification and recognition of Au&Pt@UiO-66 nanocomposites is demonstrated in Scheme 1. The cysteine-terminated kemptide was firstly assembled on the GO/GCE electrode through the reaction between carboxyl group of the GO and the amino group on the kemptide. Then the kemptide/GO/GCE was treated with 6-aminohexanoic acid for blocking blank binding sites. In the presence of PKA, the hydroxyl group of serine
Conclusions
In conclusion, a sensitive ECL biosensor for kinase activity evaluation based on one-step recognition and signal amplification strategy of Au&Pt@UiO-66 nanoprobes has been developed for kinase activity and inhibition assay. The majority of Zr surface defect sites afforded the high efficient recognition toward the phosphorylated kemptide. Moreover, the Au&Pt@UiO-66 not only provided multiple catalytic centers toward the luminol-H2O2 reaction but also accelerated the electron transfer rate on the
Acknowledgments
This work was financially supported by National Natural Science Foundation of China (No. 21622506, 21375073, 21621003, 21235004, 21475071), National Key Research and Development Program of China (No. 2016YFA0203101), Beijing Municipal Science and Technology Commission (Z171100001117135), Tsinghua University Initiative Scientific Research Program (2014z21027), China Postdoctoral Science Foundation (2017M610805), Beijing Postdoctoral Research Foundation (2017-22-077), and the Taishan Scholar
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