Highly Sensitive Detection Of Protein Kinase Activity Based On Fluorescent Assay

Kinases play an important role in regulation of fundamental biological processes,including cell proliferation, differentiation and metabolism. Aberrations in kinase activitiescan result in a number of diseases. Therefore, sensitive and widely applicable assays formonitoring kinase activity are of great signifcance for further understanding the molecularmechanism in biochemical research, clinical diagnosis, and development of targeted therapy.In this study, we focus on the development of sensitive methods for detection of proteinkinase activity and hexokinase activity. This dissertation presents a series of new methods fordetection of kinase activity by using conjugated polymers, functionalized magneticmicrospheres and glucose meter technology integrated with molecular fluorescence andfluorescence images detection. The main contents are as follows:1. Anionic fluorescent conjugated polymer (PFPaa) coupled with the metal ion-mediatedFRET, can be used to design a versatile, homogeneous, and simple platform for detection ofprotein kinase activities. In conclusion, we have demonstrated that PFPaa can complex withZr4+and the complexed Zr4+can selectively recognize the phosphate group on the peptidesubstrates, resulting in e cient FRET from PFPaa to the fluorophores labeled on thephosphorylated peptides. Based on the light-harvesting property and amplification offluorescence signals, PFPaa can be used to design a versatile, homogeneous, robust platform todetect protein kinase activity with high sensitivity. Protein kinase A (PKA) activity can bequantitatively detected over a wide concentration range from0.0005U·μL-1~1U·μL-1. Thenew strategy provides a simple detection procedure, easy readout and cost e ective manner forprotein kinase assay, which shows great potential for high-throughput assay in clinicaldiagnostics and drug discovery applications.2. A simple, highly sensitive, and dual-readout fuorescent assay is developed for thedetection of protein kinase activity based on the specifc recognition utility of TiO2-coatedFe3O4/SiO2magnetic microspheres (TMSPs) for kinase-induced phosphopeptides. When thefuorophore-labeled substrate peptides are phosphorylated by the kinase reaction, they canbind specifcally to the TiO2layer of TMSPs by means of phosphate groups, resulting infuorophore enrichment on the TMSP surfaces. The accumulated fuorophores on the TMSPsare proportional to the kinase activity, and the fuorescence signal readout could be run through either direct fuorescent imaging of the TMSPs or measurement of the fuorescenceintensity by simply detaching the fuorescent phosphopeptides into the solution. The TMSPsexhibit extremely high selectivity for capturing phosphorylated peptides over thenonphosphorylated ones, resulting in an ultrahigh fuorescence signal-to-background ratio of42, which is the highest fuorescence change thus far in fuorescent assays for detection ofprotein kinase activities. Therefore, the proposed fuorescent assay presents high sensitivity,low detection limit of0.0001U·μL-1, and wide dynamic range from0.0005U·μL-1to0.5U·μL-1with protein kinase A (PKA) as a model target. Moreover, the TMSP-based fuorescentassay can simultaneously quantify multiple kinase activities with their specifc peptideslabeled with di erent dyes. This new strategy is also successfully applied to monitoringdrug-triggered PKA activation in cell lysates. Therefore, the TMSP-based fuorescent assay isvery promising in high-throughput screening of kinase inhibitors and in highly sensitivedetection of kinase activity, and thus it is a valuable tool for development of targeted therapy,clinical diagnosis, and studies of fundamental life science.3. We provide a simple, rapid, homogeneous method for measuring hexokinase activiteby quantitating the amount of glucose remaining in solution following a kinase reaction,which is monitored by the personal glucometer. The glucose level is correlated with theamount of glucose present and is inversely correlated with the amount of kinase activity. Thewide dynamic range was detected from10-6U·μL-1to10-2U·μL-1by using HK as a modeltarget. Moreover, this new method is also successfully applied to high-throughput screeningof HK inhibitors. Thus is a valuable tool for development of targeted therapy as well as thestudies of fundamental life science.