Analysis Of MicroRNA And Protein Kinase Activity Based On Single Microsphere Fluorescence Signal Enrichment Mechanism

MicroRNA (miRNA) and protein kinases are two important biomarker molecules, which play a key role in the regulation of cell growth, differentiation, apoptosis and other important life processes. Nearly half a century, a large number of medical studies have shown:there is a close link between the biomarkers of cellular-level abnormal expression and the major diseases that threaten human health. Today, tissue sample based detection of related biomolecular has become the basis of the disease diagnosis. In order to get the vital information of cell mutation in the early period of disease and to diagnosis major diseases more in advance. For the first time, we proposed a new single microsphere-based fluorescence signal enrichment strategy for ultrasensitive detection of various biomolecules. As a versatile experimental strategy, this new method could be divided into two parts:1. A well-designed signal amplification/enrichment strategy on the surface of a single microsphere according to the properties of the target molecules.2. A practical operation of single microspheres manipulation and fluorescence imaging. Accordingly, the target-responsive fluorescence signals can be highly concentrated on the surface of the single microspheres and then single microspheres imaging was conducted to read the fluorescence information on the single beads.In the second chapter, we have established a single microsphere-based miRNA assay by nucleic acids amplification and fluorescence accumulation mechanism. Firstly, we take advantage of the newly created single microsphere technique to enrich miRNA onto the surface of the microspheres, but experimental results show that the simple accumulation of trace to a single target molecule is not sufficient to achieve high sensitivity for miRNA detection. Therefore, we use the surface based nucleic acid amplification technology to in situ enlarge the signal of pre-enriched miRNAs. The newly designed exponential amplification template has a structure of Spacer-X-Y-Nicking Site-Y. Wherein, Spacer is a sequence of 20T, which make the amplification reaction keep a distance from surface of the microspheres, and increase the accessibility of miRNA and enzyme to the region of amplification. X is the anti-sequence of miRNA for capturing free miRNA. Nicking Site is the recognition sites of nicking endonuclease. MiRNA initiated polymerase extension products in the 5'end of template is nicked by nicking endonuclease, and the template remains intact. Y is a newly designed repeat amplification fragment. In the proposed miRNA assay, the sequence X capture target miRNA firstly, after that, miRNA will initiate the extension reaction under the catalysis of DNA polymerase by using dNTPs along with an appropriate portion of Biotin-dATP. During the surface exponential amplification, a large number of biotin molecules will be integrated into the newly synthesized DNA fragments and retained in the surface of the microspheres. After the Alexa Fluor 546 streptavidin-biotin staining, the single microsphere immediately detected by the Laser-Confocal with fluorescence imaging model. By using let-7a as a model target, miRNA let-7a can be clearly detected (-5 ?L reaction system containing 3 miRNA molecules). The ultrahigh sensitivity also enables the miRNA detection in a single HCT-116 cell.In the third chapter, we established single microsphere-based protein kinase assay by fluorescent signal accumulation mechanism. The latest research shows that there is a strong interaction between the lanthanide and phosphorylated peptides. Hence, we firstly screened the binding capacity between rare earth elements and phosphate groups in biomolecules, and found that Dy (No.66) is the excellent phospho-recognition element. Its binding strength to the phosphorylated peptide is stronger than conventional TiO2, Zr4+-based material. After selected the optimal material, we introduced the single microsphere-based fluorescence signal enrichment strategy to further improve the rare earth-based protein kinase activity analysis. For the detection of PKA (a model protein kinase), the newly established protocols has achieved an ultralow detection limit of 0.12 ?U/?L, which is the lowest value up to now as far as we know. Due to its high sensitivity, this strategy was successfully applied to the single MCF-7 cells'protein kinase assay.