For the important impact of kinases in human health[1] and drug development[2], protein kinase has been used as one of the major drug targets against human diseases. Under the catalysis of protein kinase, one or more γ-phosphate groups are transferred to specific amino acids (i.e., serine, tyrosine, and threonine residues) of target proteins, and this phosphorylation process is one of the most important post-translational modifications (PTM) [3, 4]. Whereas, the overexpression and mutations of protein kinases and aberrant protein-phosphorylation states can result in cancer, heart dysfunctions and Alzheimer’s disease[5], and some other diseases, posing huge threats to human health. So, protein kinases are a group of especially significant targets for drug therapy. In this case, sensitive, accurate and widely applicable assays for monitoring protein kinase A (PKA) activity are beneficial to the development of protein kinase-related clinical diagnosis and kinase-targeted drug discovery, as well as for further understanding the signal transduction mechanism of molecular[6]. Simultaneously, the screening of inhibitors of protein kinases A are currently also important, and several protein kinase inhibitors have been FDA approved for targeted therapy. Real examples of kinase inhibitors (Pazopanib, Nilotinib, Sunitinib) provide a wealth of information about how tumors respond to such targeted therapy[7]. On the basis of above, it is also urgent that a fast pathway selectivity-based ECL-signal reading out the activity of different inhibitors is constructed.
The conventional approaches for the detection of PKA activities are almost based on the expensive and harmful radioactive labels with 32P[8]. In this regard, various alternative ways have been recently exploited for PKA analysis and inhibitor screening, such as colorimetric, quartz crystal microbalance (QCM), Raman Spectroscopy and fluorescent immuno-assay[9–10]. Although these designs are smart and antibody-antigen with high affinity have improved the selectivity, however, low accuracy due to false positive results, specialized instruments and tedious operations heavily hinder the real application. Recently, electrochemiluminescence (ECL), as a signal-emission process in a redox reaction of electrogenerated reactants, appears in scientific research. On the one hand, ECL technology is a potential analysis-method on account of its distinct merits lower background noise and cost, higher selectivity and sensitivity, as well as more convenient operation. In addition, ECL platform has also been increasingly employed in biomarkers detection based on the potential for rapid and sensitive diagnosis of human diseases[11]. Green nano materials have emerged as key components in most, if not all, optical and electrical sensing field. Unfortunately, the expensiveness, toxicity and hard to prepare limit the existing ECL luminophores[12] applications. Therefore, the synthesis of eco-friendly electro-chemiluminescent (ECL) nanomaterials becomes urgent needs to solve above key issues and relies greatly on advanced luminescent materials as well as the novel amplification technologies of the ECL-signal.
In recent years, some carbon-based materials (CMs) and their complexes have been successfully used to sensing-fields because of their high flexibility, good electrical properties and the harmless to environment, e.g., carbon dots (CDs)[13], carbon nanotube (CNT)[14], and graphene[15]. Among them, carbon dots (CDs) are the most popularly used light-responsive nanomaterial material in sensing-fields due to the good biocompatibility, photocatalysis and high energy-conversion efficiency. Fixing CDs directly on Au-electrode (AuE) has problems such as unstable response-signal and low ECL-intensity. Some attempts [16] to enhance their ECL signal focus on co-reactive reagents (H2O2) introduce or structure design. Studies have revealed that the cathodic ECL behavior of TiO2 is comparable to that of H2O2[17]. Therefore, TiO2 can act as co-reactants for CDs. In addition, TiO2-based nanomaterials with good biocompatibility and relative conductivity have been extensively certified to enhance ECL-performances cooperatively (such as TiO2@SnS2, TiO2-CNTs, ZnO/TiO2, CeO2@TiO2, etc.)[18]. However, only few researches focused on improving the ECL-properties of CDs, in which NH2-TiO2 acted as a signal-carrier for CDs-luminor in biosensor.
Semiconductor photocatalyst nano titanium oxide (TiO2) with the large surface area is considered as a promising transducing material due to its high chemical stability, low cost, and non-toxicity[19]. On the other hand, its affinity toward phosphoric group via Ti-O-P coordination has gained enormous application in phosphoprotein-enrichment. In addition, the lower Lewis acidity and higher hydrophilicity of amino-functionalized TiO2 (NH2-TiO2) than TiO2 show strong affinity with phosphoproteins, then modifying -NH2 onto TiO2 has gained enormous attraction[20]. Based on the prominent photocatalytic performance and strong specific recognition utility between NH2-TiO2 and phosphate group, if or not a label-free ECL- biosensor may be developed to detect the activity of PKA?
In light of these unique and outstanding properties of NH2-TiO2-doped CDs, herein, a robust ECL platform for the test of PKA activity was reported by using eco-friendly blue-emissive Ti@NMg-CDs. The Ti@NMg-CDs feature both one of the most protentional ECL-luminous known to date and as an ideal PKA-reporter and phosphopeptide-recognizing matrix. It was noteworthy that a single Mg@N-CDs could assemble multiple NH2-TiO2 to form the single-donor/multiple-acceptor configuration with an improved ECL intensity. In this alternative ECL-biosensor, the synthesis of the fluorescent nanoprobes Ti@NMgCDs increases the stability of the CDs and intensity of the ECL-signal. The in-situ phosphorylated Cys-kemptide-sensor could efficiently anchor the Ti@NMgCDs by Ti-O-P matching, achieving highly sensitive detection of PKA. To our knowledge, this is a new report on in-situ construction of Cys-kemptide and Ti@NMgCDs ECL-sensor on Au-electrode for PKA tests.