Analysis Of Sugar And Nucleic Acid Kinase Activity Based On Novel Fluorescence Sensing Material

Kinases are a class of enzymes, which can catalyze substrate phosphorylation so as to regulate the function of the substrate molecules. It can transfer the phosphate groups of high-energy donor molecules (such as ATP) to specific target molecules, such as protein, lipid, sugar, nucleosides and so on, which plays important roles in cell signaling transduction and metabolism. The aberrant kinase activities are closely associated with many human diseases, such as cancer, diabetes, heart disease, Alzheimer’s disease and so on. Therefore, designing effective, sensitive and simple kinase assays are of great significance in clinical diagnosis, drug screening and targeted therapy of related diseases. In this thesis, based on the distinct features of upconversion nanophosphors (UCNPs) and cationic conjugated polymer, we aim to develop sensitive methods for detection of hexokinase (HK) activity and T4 polynucleotide kinase (T4 PNK) activity. The main contents are as follows:1. Novel Fluorescent Biosensor for Sensitive Detection of Hexokinase (HK) Activity Based on Zr4+-Coated Magnetic Beads (ZrMBs) Coupled with Phenylboronic Acid-Functionalized Upconversion Nanophosphors (PBA-UCNPs)A new fluorescent biosensor has been designed for sensitive detection of HK activity by use of ZrMBs coupled with PBA-UCNPs. HK can catalyze the conversion of glucose to glucose-6-phosphate (G-6-P) in the presence of ATP. ZrMBs can selectively capture the G-6-P catalyzed by HK. Besides, PBA-UCNPs can specifically react with the G-6-P enriched on the ZrMBs. Ultimately, a sandwich structure compound PBA-UCNPs/G-6-P/ZrMBs was formed through the above reaction, and the UCNPs eventually accumulated on the ZrMBs are proportional to the HK activity. The PBA-UCNPs/G-6-P complexes can be dissociated from ZrMBs when treated with 0.5% NH3·H2O solution. The activity of HK can be quantitatively determined by measuring the fluorescence signal of UCNPs in the reaction solution. In this assay, the use of magnetic ZrMBs greatly simplifies the separation and purification of the sample, and reduces the interferences. What’s more, owing to the NIR-excitation characteristic of UCNPs, the autofluorescence and light scattering interferences of the system can be significantly reduced, which can greatly improve the signal-to-background ratio. As a consequence, the ultrahigh sensitivity for the detection of HK is achieved. The HK activity can be quantitatively detected over a wide concentration range from 5×10-9 U/μL to 1×10-4 U/μL. In addition, the abnormal HK activity is closely associated with many human diseases, and effective HK inhibitors may become the potential targeted drugs for the treatment of diseases. Therefore, we have also successfully studied the inhibition effect of xylose on HK activity through this sensing system. By combining with the properties of UCNPs and ZrMBs, we have designed a new sensitivity strategy, which not only can be used for the detection of HK activity, but also show great potential in HK-related drug screening.2. Detection of T4 Polynucleotide Kinase (T4 PNK) Activity Based on Cationic Conjugated Polymer-Mediated Fluorescence Resonance Energy TransferBased on the cationic conjugated polymer PFP mediated fluorescence resonance energy transfer (FRET) coupled with a DNA phosphorylation-accelerated λ exonuclease (X. exo) cleavage reaction, we have designed a new homogeneous, simple and sensitive biosensing strategy for the detection of T4 PNK activity. Cationic conjugated polymer PFP is a kind of fluorescent material containing a large number of absorbing units. So PFP has strong light-harvesting and fluorescence amplification properties. In addition, SYBR Green I (SG I) is a double-stranded DNA-specific dye, which can emit strong fluorescence only when intercalated into the small groove of double stranded DNA. When mixed with PFP, due to the electrostatic attraction, the SG I-binding hairpin DNA will be brought in close proximity with the PFP and efficient FRET occurs from PFP to SG I. In the presence of T4 PNK, the 5’-OH terminus of the hairpin DNA can be phosphorylated and thus can be quickly recognized by λ exo as the initial cleave site of λ exo. The single-stranded DNA, generated after the cleavage reaction, cannot combine with SG I, and FRET phenomenon between the PFP and SG I will not occur. Thus, the FRET efficiency is inversely proportional to the activity of T4 PNK. Ultimately, we can easily detect the activity of T4 PNK in a mix-and-read manner. This assay can measure activity of T4 PNK down to 0.001 U/mL. By combining the PFP-mediated FRET with the λ exonuclease cleavage reaction, we have proposed a simple, cost-effective and easy readout sensing strategy, which not only can be used for the detection of T4 PNK activity, but also show great potential in the study of polynucleotide kinase-related biological processes as well as drug screening.