As is well recognized that microRNAs and protein kinases are important biomarkers for early diagnosis as well as targeted-treatment of many human diseases including cancers. Ultrasensitive detection of these biomarkers with vanishingly small copy numbers in cells, even at the single-molecule level, is of great significance for both fundamental biochemistry studies and clinical diagnostics. At present, total internal reflection fluorescence microscopy (TIRFM) and fluorescence correlation spectroscopy are the most powerful tools for single molecule detection. Nevertheless, these techniques rely heavily on use of expensive and sophisticated instruments by specialized and skilled technicians, which are only available in specialized labs. What’s more, they are also limited by the extremely low interrogation volume allowed for laser scanning because it is difficult to concentrate the real sample in such a tiny volume.
In recent years, the Bioanalytical Chemistry and Molecular Diagnosis team led by Prof. Li Zhengping from School of Chemistry and Chemical Engineering has committed to finding solutions for the ultrasensitive detection of nucleic acid and protein kinase biomarkers by using simple and low-cost techniques. Recently, they have developed a novel single microbead-based sensing platform for the ultrasensitive detection of genetic and enzyme biomarkers. They innovatively manipulated a single functionalized microbead as the carrier for signal amplification and signal enrichment, and achieved accurate detection of microRNAs and protein kinase activities at the single-cell and single-molecule level with a common fluorescence microscope.
Taking the single-molecule microRNA analysis for an example, the single microbead-based sensing strategy works by triggering an isothermal exponential amplification reaction (EXPAR) to the presence of a particular microRNA target. The microbead is covered by EXPAR template and the target microRNA will specifically initiate efficient EXPAR on the surface of the single microbead. This will incorporate biotinylated nucleosides – that will subsequently be tagged with fluorophores – all over the bead at an exponential rate. Therefore, when the target microRNA is present, the fluorophores will be highly concentrated on the surface of the microbead. By simply measuring the fluorescence signals of the microbead under a common fluorescence microscope, the amount of microRNA in a single cell can be accurately determined. This work was published in Chem. Sci., 2015, 6, 6213-6218 as a hot article, and is also highlighted by Chemistry World as well as the Chemical Science Blog.
The Chemistry World comments that: “An elegant strategy for detecting minuscule quantities of microRNA using just a single functionalised microbead could be an important innovation for biomedical research and molecular diagnostics”; “Traditional single molecule detection methods rely heavily on sophisticated and expensive instruments, and are only available in specialised labs. This research enables detection on a single microbead, and might provide a simple and powerful tool for the early diagnosis of human diseases”.
Further more, by innovatively integrating the advantages of the single microbead-based fluorescence imaging platform and rare earth ion-based highly specific phosphopeptide recognizing, the team has developed a protein kinase assay by using the rare earth ion-functionalized single microbead, which allows direct kinase analysis in a single-cell. This work may help us better understand the kinase-related disease initiation, progression, and therapeutic responses at the single-cell level. This work was published in Angew. Chem.Int. Ed., 2015, 54, 15186-15190.
Text by Liu Chenghui
Photo by Feng Wei