Novel Quantum Dots-based Technologies For The Detection Of Nucleic Acid And Protein

Living organisms, bacteria, viruses and bacteria has the sequence-specific DNA or RNA, which has been became increasingly important for the diagnosis and treatment of genetic diseases, the detection of infectious agents, and forensic analysis. In addition, the concentration of some protein in blood is the indication of certain diseases. Therefore, it is highly important to establish some sensitive, accurate methods for biomacromolecules detection. Various techniques have been developed for the detection of DNA and protein. Among all of the analysis techniques, fluorescence has been widely used due to the advantages of simple operation, no substrates and the high sensitivity advantages. Semiconductor quantum dots (QDs) has been intensely developed as a new class of fluorescent label. In compare with traditional fluorescent label, QDs have unique chemical and physical properties, which accompany with recently improving understanding on QDs surface chemistry for biocompatibility and bioconjugation, lead QDs labeling to be a promising method for the detection of various biological analytes, liking DNA, protein, cells, and small organic molecules.In consider the significance of biomacrolecules, althrough the great advances of t echno-logy on detecting biomacromolecules,the development of new method is still e xtremely significant. Herein, this subject developed several highly sensitive, specific, and novel techn-ologies by the excellent properties of QDs, which provide a new versi on for nucleic acid and protein detection. The experimental content contains three sections:1A new quantum dot-based method to detect specific sequences of DNA has been developed. The capture and reporter probes did not hybridize to each other, but in the presence of a template they could anneal to each other via the formation of a stable ternary complex. Because of the specific design of the capture and reporter probes, the5’end of the template target DNA remained free to hybridize with another reporter. In this way, each capture DNA was an initiator strand that triggers a cascade of hybridization events between the target DNA and the reporter probe. This formed a superstructure, enhanced base stacking, and produces a strong fluorescent signal. The introduction of T4DNA ligase further stabilized the superstructure and greatly increased the fluorescence intensity, and the detection limit was as low as10fM. This fluorescence method is advantageous over conventional techniques because of its excellent ability to discriminate single base-pair mismatches and single nucleotide gap or flap.On the basis of detecting DNA by hybridization chain reaction (HCR), we expanded this technology for the detection of PDGF-BB based on aptameric system, where stable DNA monomers assembled only upon exposure to a target PDGF-BB aptamer. In this process, two complementary stable species of biotinylated DNA hairpins coexisted in solution until the introduction of initiator aptamer strands trigger a cascade of hybridization events that yielded nicked double helices analogous to alternating copolymers. In details, the aptamer firstly opened the hairpins in the solution, created long concatamers, and then reacted with the antibody captured PDGF-BB on the well surface. Our results showed that, the coupling HCR to aptamer triggers for the amplification detection of PDGF-BB achieved a better performance in the fluorescence detection of PDGF-BB as compared to the traditional antibody-antigen-aptamer assays. The proposed method was also implemented in the analysis of human serum specimens.2A novel multiplexed method for short RNA detection has been developed that employed a design strategy in which capture and reporter probes did not hybridize to each other and could anneal to each other in the presence of a short RNA target via the formation of a stable three-component complex. QDs functionalized with reporter DNA were used to capture a short single-stranded RNA (ssRNA) sequence from a target solution and then to specifically adsorb onto a common capture probe-modified96-well plate via a one-step template-dependent, surface hybridization for simultaneous fluorescence detection. This novel QD-based, template-dependent, surface hybridization showed several advantages compared with previous detection formats:(ⅰ) the use of reporter DNA-modified QD conjugates increased the melting temperature in comparison with conventional organic dye-modified reporter probes, leading to the detection of short RNA without the need for a ligation reaction; and (ii) QDs properties allowed multiple short RNA sequences to be simultaneously and homogeneously determined via a rapid and simple one-step hybridization, as exemplified herein.In order to improve the sensitivity, we developed an amplification assay for multiplex detection of HIV-1and HIV-2based on QDs layer-by-layer (LBL) assembled polystyrene microsphere (PS) composite in a homogeneous format. Based on the high affinity between streptavidin and biotin, QDs were adsorbed on the surface of PS layer by layer. Biotinylated reporter then bound to the PS-QDs conjugates and was hybridized with target DNA which had been immobilized on the96-well surface. Through this way, each target DNA referred to a large number of QDs and the fluorescence signal was greatly enhanced. This PS-QDs-based sensor possesses the advantages of a simple’mix and detection’assay with enzyme-free, extremely low sample consumption, high sensitivity, and short analysis time.3In the system of fluorescence resonance energy transfer (FRET), QDs has been always used as the donor or acceptor due to its excellent properties. In this part, we chose QDs as donor and Cy5as acceptor to form FRET for the detection of DNA. Firstly, the FRET between QDs and Cy5was investigated in a96well plate and homogeneous format respectively. Later, we used Y-shaped junction DNA which consisted of three complementary oligonucleotide branches to detect DNA. The probe was then cleaved by an endonuclease and the target was released, while the probe and the target were regenerated and attended another cleavage cycle to realize the signal amplification.