Micro Beads-based Fluorescence Enriching Sensors For The Detection Of DNA And Protein Kinase Activity

The properties of high sensitivity, easy readout, low sample volume and handy separationmake the homogeneous fluorescent sensors widely used in the specific detections of metalions, nucleic acids and other bio-molecules. However, a majority of the homogeneousfluorescent sensors are not suitable for the analysis of complex biological samples due to thestrong light scattering and autofluorescence background in the complex matrix such as serum;besides, the practical application of this kind of sensors is also limited by difficulties in devicemaking, disposable using and easy to be contaminated. In this regard, bio-sensors made byfunctionalized micro beads coupled with fluorescence labeling have become a fascinatingchoice for the detection of many kinds of bio-molecules, allowing extraction of the signalsfrom the sample matrix filled up with interference. This manner has become one of the mostimportant technologies for the detection of bio-molecules in complex matrix. In this thesis,micro beads are used as the fluorescence enriching matrix to investigate their applications inDNA and protein kinase activity detection.We established a new DNA detection method by combining the superparamagnetic beadsfor easy separation and signal collection, hybridization chain reaction (HCR) for enzyme-freeamplification and flow cytometer for powerful detection. In this design, thesuperparamagnetic streptavidin (STV)-functionalized micro beads Dynabeads M-270areapplied as the carrier of the fluorescence signals. The hairpin biotin-modified probe DNABio–H1could bind to the surface of the M-270via specific STV-biotin interaction. Upon theexistence of target, it will hybridize with the sticky end of Bio-H1to open the hairpinstructure. After the magnetic separation, the newly released sticky end of Bio-H1on the beadswill further induce the hybridization chain reaction (HCR) on the addition of two fluorescencelabeled hairpin structures. In this way, the fluorescence signals enriched on the micro beadsnumerously, the purpose of signal amplification achieved. The fluorescence signals of M-270are analyzed by flow cytometry. It is tested by flow cytometry that the detection limit of thissensor is0.5pmol/L, which is about3orders of magnitude higher than the tradition beadsbased sensors. The specificity of this sensor has also been evaluated as well as the application in complex matrix.Protein kinases play important roles in intracellular signal transduction pathways.Hyperactivity of some kinases and the following abnormal protein phosphorylation may leadto various human diseases including cancer. Therefore, protein kinases are regarded as afamily of extremely important molecular target for drug therapy. In this article, wesuccessfully design a protein kinase activity detection sensor by Zr4+functionalized SiO2micro beads. The Zr4+on the surface of the micro beads could specifically bind thephosphorylated peptides, and does not affect on the non-phosphorylated ones. Hence, there isa positive correlation between the activity of the protein kinase and the fluorescence signalsenriched on the micro beads. The quantitative assay of the protein kinase activity could bedetermined by the elution of the micro beads. This sensor is of easy operation, high specificityand feasible in complex biological samples, providing a new way in protein kinase detectionand screening of potential protein kinase inhibitors.