DNA Assay And Cell Assay Based On Novel Bio-Functionalized DNA/Nanoparticles Probe

Biosensors are developed for the study of the structure, the function and the molecular interaction of molecules in organisms, which plays important roles in the development of clinical, pharmaceutical, and environmental applications. Combined DNA recognition, aptamer recognition with nanoparticles (NPs) with excellent properties, several novel bio-functionalized DNA/nanoparticles probes were constructed for the specific recognition and detection of target DNA and cancer cell. The experimental protocols could be summarized as follows:1. Acted as a cage-type cellular probe, an extracellular supramolecular reticular DNA/quantum dot (QD) sheath was developed for the high-intensity fluorescence microscopy imaging of Ramos cells. The extracellular supramolecular reticular DNA/QD sheath was constructed from layer-by-layer self-assembly of DNA/CdTe QD probes and DNA nanowire frameworks functionalized with Ramos cell-binding aptamer. The DNA/QD sheath formed specifically and quickly on the surface of Ramos cells at physiological temperature. And the assembly of large numbers of DNA/CdTe QD probes on the surface of Ramos cells produced exceedingly high fluorescence intensity. Using the extracellular supramolecular reticular DNA/QD sheath as the cellular probe, Ramos cells could be distinctly observed and easily distinguished from a mixture of multiple cancer cells by fluorescence microscopy imaging. This strategy presents a promising platform for convenient evaluation of cancer cell.2. Based on the extracellular supramolecular reticular DNA/QD sheath probe, a sandwich-type electrochemical cellular sensor was developed for the accurate quantitative analysis of Ramos cells. The electrochemical cellular sensor was constructed using Ramos cell-binding aptamer-immobilized polystyrene microwell plates to capture Ramos cells and then assembling the supramolecular reticular DNA/QD sheath on the surface of Ramos cells. A sensitive differential pulse anodic stripping voltammetry (DPASV) detection system was used to quantify the concentration of Cd2+ produced by the acid dissolution of DNA/CdTe QD probes. Under the optimized conditions, Ramos cells could be detected quantitatively in the range from 10 to 1000 cells with a detection limit of 10 cells. This strategy can be widely used in cell recognition, cancer imaging and detection, and also shows applied potential in cell surface engineering.3. A high-intensity polystyrene microbead probe that was functionalized with supramolecular DNA/CdTe QD nanowires was developed for the direct fluorescence microscopy imaging of DNA hybridization event. The supramolecular DNA/CdTe QD nanowires capable of loading a large number of CdTe QDs were formed on the surface of polystyrene microbeads through hybridization of DNA molecules and produced exceedingly high fluorescence intensity so that single polystyrene microbead probe could be easily observed by fluorescence microscopy imaging. Using the high-intensity polystyrene microbead probe, target DNA as low as 50 fM could be recognized. In addition, combined cell-specific aptamer recognition element with the probe, cancer cell could be easily distinguished by fluorescence microscopy imaging. The proposed method provided an alternative strategy for the rapid, visible determination of DNA hybridization and cancer cell.4. A new kind of one-to-one recognition DNA probe based on triple Au nanpparticles (Tri-AuNPs) was developed. The DNA biosensor based on the one-to-one recognition Tri-AuNPs DNA probe was constructed by immobilizing capture DNA on the gold electrode and hybridization with target DNA, which further hybridized with the only one single strand DNA on one terminal of the DNA probe. Electrochemical signals of hexaammineruthenium(Ⅲ) ([Ru(NH3)6]3+) bound to the reporter DNA via electrostatic interactions were measured by chronocoulometry (CC). The sensitivity of the DNA biosensor was achieved through the signal amplification by one-to-one recognition Tri-AuNPs DNA probe. Under the optimized experimental conditions, target DNA could be determined in a linear range from 1×10-16 M to 1×10"14 M. The detection limit of 53 aM of target DNA was obtained. The resulting DNA biosensor exhibited ultrasensitivity, high selectivity, and good reusability as a promise alternative technique for other bioassays.