Quantitative Detection Of Single Biomolecules By Employment Of DNA Tetrahedron Nanostructure

Single-molecule detection (SMD), is a research method can obtain the information on single molecule level. It can reveal subtle differences between molecules which may conceal by macroscopic though study single molecules. SMD can study the conformational change of biomolecules, dynamics, interaction between molecules, single-molecule manipulation and single-molecule quantitative detection. Single-molecule quantitative detection is the ultimate goal in analytical chemistry. Single-molecule counting is the most common used method in single-molecule detection. This method possesses the advantages of high sensitivity, high resolution. Compared with traditional methods that rely on detection the intensity, single-molecule counting is relying on counting the number of targets, which is more suitable for the detection of biomolecules under ultra-low concentration.Expression of microRNA (miRNA) has been associated with the regulation and progression of numerous cancers such as breast, thyroid, colorectal, prostate, lung, and ovarian. Monitoring changes in miRNA biomarkers can be used to signify the early onset of disease. However, quantitative detection of miRNAs has always been a tough call that is attributed to their small size, vulnerable degradability, similarities of the sequences, and relatively low expression levels in cells. Thus, to develop a miRNA detection method with high sensitivity and specificity is in great demand.Recently, the DNA nanotechnology has a great development. Researcher used DNA strands as materials to build many two-dimension (2D) and three-dimension (3D) structure DNA nanomaterials based on the complementary pairing between bases. These materials were employed to biosensors and nanoinstrument. The development of structure DNA nanomaterials provides powerful tools for analytical chemistry. Our group has already employed DNA tetrahedron to construct substrate for single-molecule detection.Based on the above review, in this work, we assembled a new fluorescent nanolabel based DNA tetrahedron for detection single protein molecule. And we also constructed a substrate decorated with DNA tetrahedra that have a toehold hairpin for sensitive and specific detection of single miRNA.The main contents are as follows:Chapter one is an introduction section to summarize the backgrounds, significance, principles and common used methods of SMD and quantitative SMD. The disadvantages of fluorescence probes that frequently-used in fluorescence imaging and the affection of nonspecific adsorption on substrate during quantitative SMD are pointed out. What is more, several common used methods for miRNA detection and the development of structure DNA nanomaterials and the application in biology are also introduced in this chapter.In Chapter two, we assembled a new nanolabel based on DNA tetrahedron for the detection of single protein molecules. The use of DNA tetrahedron nanolabels in SMD can avoid the shortcoming of QDs and overcome the limitations of organic dyes by preventing self-quenching and increasing the fluorescence intensity and photostability. By combine the DNA tetrahedron nanolabel with single-molecule counting, we detected single human IgG molecule. The linear relationship between the human IgG concentration and the number of molecules in the range of3.0x10-14to1.0×10-12mol L-1, the coefficient correlation was0.9997. The relative standard deviation (R.S.D.) of this method was5.09%(n=3). This method also showed an excellent specificity and a low matrix effect.In Chapter three, we constructed a substrate decorated with DNA tetrahedral that have toehold hairpin for sensitive and specific detection of single miRNA molecules. After added the target miRNA on the substrate, the hairpin was opened through the toehold-mediated strands displaced reaction and hybridized with reporter probe for single-molecule counting. The strands with single base mispaire may open the hairpin slowly or even could not open it. Thus, this method can detect miRNA with high sensitivity and specificity. In this work, we characteristic the substrate and optimized the reaction time and the number of toehold base (5nt,6nt,7nt,8nt). When there are6bases at toehold, this method had a best specificity for the detection of miRNA.