Detection Of Protein Kinase Activity And Nucleic Acid Quantification Based On Biological Nanomaterials

Protein phosphorylation modification is an important type of chemicalmodification process in vivo, and it controls almost the whole life process. Abnormalphosphorylation process will lead to a lot of inflammation, and diseases such as cancer,diabetes, and so on. The sensitive detection of protein kinase is significantly importantfor early diagnosis of disease and searching for potential drug targets... Metalnanomaterials, such as magnetic nanoparticlesand gold nanoparticles, due to their highstability, good biocompatibility, and unique magnetic, optical, and electrical properties,are widely used in life sciences. Quantitative analysis of nucleic acid is of importancefor virus infection, pathogenic microorganism detection, genetic testing analysis, andother fields. Traditional methods of quantitative analysis of nucleic acids needexpensive instrument or complex manuscription. Therefore, developing a simple andsensitive method for nucleic acid quantitative analysis has great practical significance.Because of the unique optical characteristics and other properties such as heat-resisting,salt tolerance, and supercharged which obtained by artificial modification，supercharged fluorescence protein has a wide applicant potential in the field ofmolecular biology and medicine, etc.Here, we developed two kinds of fluorescence methods for protein kinase activitydetection and one fluorescence method to quantify nucleic acid. Specific work is asfollows:（1） Based on zirconium ions specificity combination with the phosphate groups,we put forward a new kind of fluorescence analysis method to detect protein kinaseactivity using zirconium ions immobilized magnetic nanoparticles andfluorescent-labeled phosphorylated substrates peptide,which was labeled by FITC.After phosphorylation, interact with zirconium ions immobilized magneticnanoparticles, and magnetic separation, the labled peptide was separated from thesolution. The fluorescent changes of solutions reflect the activity of PKA. This methodhas a low limit of detection of PKA (0.5mU μL^-1), and has been successfully appliedto the PKA activity inhibition experiment (H-89，IC₅₀=74.3nM). Furthermore, theactivity of PKA in cell lysis was successfully measured.（2） We designed a fluorescence sensor based on the charge change of substratepeptide before and after phosphorylation. This charge change of the peptide modulates the electrostatic interaction between positively charged gold nanoparticles andFITC-labeled substrate peptides. Positively charged gold nanoparticles combine withphosphorylated substrate peptides （product peptide） through electrostatic adsorption,which lead to the quenching of FITC fluorescence signal by gold nanoparticles. Thismethod has shorter reaction time, simple operation, sensitive signal, and has a lowlimit detection of PKA (0.5mU μL^-1), and has been successfully applied in theinhibitor experiment (H-89，IC₅₀=55nM).（3） Due to the positive charge and the nature of the fluorescence, a super chargedgreen fluorescent protein （scGFP） was used to quantitative detection of nucleic acidthrough replacing the quenching DNA, which binds to scGFP, by the amplificationproduct of target DNA. When scGFP combines with short chain DNA with quenchinggroup through electrostatic adsorption, fluorescence of scGFP is quenched. After theisothermal amplification reaction system is added, because of more negative charge,the long chain product DNA replaces the short chain quench DNA, and leads to thefluorescence recovery of scGFP. In a given condition, the amount of the template, thetarget DNA, determines the quantity of the product DNA, and indirectly related to thefluorescence recovery of scGFP. Our primary work has proved this principle, and hassuccessfully applied to detect the DNA quantity. The method is simple, fast inoperation, and no need of complex and expensive experiment instruments, and haspotential applificationin other nucleic acid quantitative experiments.