Bionanomaterials-based Biosensor For Protein Kinase And Direct Electrochemistry Of Protein

Protein is the executor of physiological function. Study on the protein structure and its function is the research hotspot in the field of life science. Protein phosphorylation by kinase involves many physiological pathology processes, such as cell signaling transmission, muscle contraction, neural activity, growth and differentiation of cells. Aberrant phosphorylation and expression of kinase has been implicated in the pathogenesis of many diseases including cancer, diabetes and Alzheimer’s disease. Biosensor for protein kinase is widely used in medicine and biology.On the other hand, studies on the direct electrochemistry of redox protein on the electrode interface play a significant role in understanding their electronic transfer mechanism and physiological action in vivo. The direct electron transfer is difficult because the redox sites of the protein are deeply sited in the protein shell. It has been focused on exploring new electron accelerator in order to realize the reversible electrochemical behavior of redox protein on electrode.Based on the above considerations and reports in the literature previously, we took advantage of the unique characteristics of nanomaterials (nanoparticles, quantum dots, graphene, etc.) and developed several methods for rapidly lable-free detecting kinase. We treated glassy carbon electrode (GCE) or carbon nanotube (CNT) with cathodical modification, and realized the direct electrochemistry of redox enzyme. The details are summarized as follow:(1) We present here a novel label-free fluorescent assay for monitoring the activity and inhibition of protein kinases based on the aggregation behavior of unmodified CdTe quantum dots (QDs). In this assay, cationic substrate peptides induce the selective aggregation of unmodified QDs with anionic surface charge, whereas phosphorylated peptides do not. Phosphorylation by kinase alters the net charge of peptides and subsequently inhibits the aggregation of unmodified QDs, causing an enhanced fluorescence with a45nm blue-shift in emission and a yellow-to-green emission color change. Hence the fluorescence response allows this QD-based method to easily probe kinase activity by spectrometer or even by naked eye. The feasibility of the method has been demonstrated by sensitive measurement of the activity of cAMP-dependent protein kinase (PKA) with a low detection limit (0.47mU μL-1). Based on the fluorescence response of QDs to the concentration of PKA inhibitor H-89, IC50value was estimated, which was in agreement with the literature value. Moreover, the system can be applicable to detect the Forskolin/IBMX-stimulated activation of PKA in cell lysate. Unlike the existing QD-based enzyme activity assays where the modification process of QDs is essential, this method relies on unmodified QDs without the requirement of peptide labeling and QDs’modification, presenting a promising candidate for cost-effective kinase activity and inhibitor screening assays.(2) A novel label-free electrochemical strategy for monitoring the activity and inhibition of protein kinase is developed, based on the stable linkage between the phosphorylated peptide and DNA functionalized Au nanoparticles (DNA-AuNPs) by Zr4+. On the phosphorylation site, redox probe [Ru(NH3)6]3+could absorb onto DNA-AuNPs containing abundant negative charges. Hence the PKA activity could be monitored through the chronocoulometric interrogation of [Ru(NH3)6]3+IC50value of inhibitor H-89for PKA was estimated to be33nM, which was in agreement with the literature value.(3) A novel one-step strategy for rapidly detecting protein kinase based on quartz crystal microbalance (QCM) was developed to monitor the interaction of aptamer-mimicking peptide and kinase. PKA was used as model kinase and IP20was exploited as the aptameric peptide against PKA. The immobilization strategy of IP20on Au coated quartz crystal relied on the specific interaction of polyhistidine tag with the metal ion, producing a highly oriented peptide assembly and avoiding nonspecific protein adsorption. The frequency of the QCM crystal sensitively responds to the amount of kinase bound to the surface, allowing us to monitor the binding of PKA in real time and to quantitatively determine the concentration of PKA. Unlike previously reported kinase assays, the aptamer-mimcking peptide-kinase recognition does not involve phosphorylation process, thus it is rapid and reagentless to obtain the detection results in one-step (less than10min). The feasibility of this novel strategy for cellular catalytic subunit of PKA was also demonstrated in cell lysates.(4) A fluorescent principle for kinase activity assay based on grapheme/peptide complex and peptide digestion was presented. FITC-labled substrate peptide for kinase CKII (S-peptide, FITC-RRRADDSDDDDD) is efficiently quenched when it is mixed with GO. Carboxypeptidase Y (CPY) is an attractive enzyme to nonspecifically cleave all residues from the C-terminus. CPY removes amino acids from S-peptide, resulting in the production of highly fluorescent FITC. The phosphorylated peptide (P-peptide, FITC-RRRADDpSDDDDD) catalyzed by CKII and ATP, however, is resistant to digestion by the protease CPY and is facile to bind GO, resulting in the immediate fluorescence quenching of P-peptide. Thus, the fluorescence intensity measured in the assay is inversely correlated with kinse CKII activity. Principles based on the fluorescence intensity change of grapheme/peptide complex induced by kinase, will provide new strategies to detect protein kinase and inhibition with high-throughput.(5) The surface nanocrystallization of glassy carbon (GC) electrode was carried out using cyclic voltammetry in anhydrous dimethylformamide containing0.05M tetra-n-butylammonium bromide (TBAB), and carbon nanoparticles with diameter of10-40nm were formed on the electrode surface. Comparing with the conventional GC electrode, the surface-nanocrystalline GC (SNGC) electrode showed higher electrocatalytic activity for direct electrochemistry of glucose oxidase (GOD) due to higher proportion of edge sites on the surface of the SNGC electrode. Because of the surface nanocrystallization of the electrode, a pair of well-defined and quasi-reversible redox peaks of the immobilized GOD was observed for the first time on the GC electrode.(6) A new kind of modified carbon nanotubes (CNT) was created by an economical and convenient way for direct electrochemistry of redox protein. CNT-modified electrode is cathodically polarized in dimethyformamide (DMF) containing0.05M tetra-n-butyammonium bromide (TBAB) by cyclic voltammetry from0to-2.8V for10segments, to get the cathodically modified carbon nanotubes (CM-CNTs). It is found that CM-CNTs have many defects on tube wall by transmission electron microscopy (TEM), thus more edge atoms are obtained and better electrical conductance properties are achieved. Glucose oxidase (GOD) on CM-CNTs modified electrode displays direct electron transport process and good electrocatalytic activity for oxygen, glucose, etc.