Preparation Of Nucleic Acid Nanoprobes And Their Applications

Nucleic acid nanoprobe is one of the most important tools in DNA detection. In this thesis, the preparation and application of nucleic acid nanoprobes were investigated, among which the factors affect the stability of gold nanoparticles, DNA detection strategies based on nucleic acid nanoprobes, the new method for preparing nucleic acid nanoprobe and the preparation of novel nanoparticles used for bio-label as well as its optical properties were studied.Gold nanoparticle is the most commonly used label reagents for preparing nucleic acid nanoprobes. Meanwhile, the method of trisodium citrate deoxidizing hydrogen tetrachloroaurate, well known for its concentration and diameter of gold nanoparticle can be regulated by changing concentration and molar ratio of trisodium citrate and hydrogen tetrachloroaurate, is generally used for preparing water soluble gold nanoparticle. However, the stability of gold nanoparticles prepared by this method can be affected by solution environment, especially pH and ion strength. Acid distribution coefficient equation(ADCE) and electrical double layer theory(EDLT) were used to deduce the influence of pH and ion strength on the stability of gold nanoparticle. Experiments were carried out to confirm the results of ADCE and EDLT which indicate the optimal pH for reserving gold nanoparticle is 9.0 and the proper concentration of hydrogen tetrachloroaurate for preparing gold nanoparticle is among 0.25~1.0mmol/L.The density of gold is high, so the quality signal of target DNA can be amplified by nucleic acid nanoprobes which contain gold nanoparticles and this amplified signal can be easily detected by quartz crystal microbalance (QCM). Based on this theory, nucleic acid nanoprobe was used as"nanoamplicon"and an innovative scheme was designed to detect DNA on QCM. The key of this method is that large numbers of nanoamplicons could be integrated onto a single target, providing much greater amplification than the larger nanoparticles usually adopted. This concept is shown to be a feasible approach to detecting 0.17amol/L DNA without target amplification, based on QCM detection. To our knowledge, this method has a sensitivity that is close to that of PCR and superior to those of nanoparticle-based methods reported previously. Additionally, this novel nanoamplicon can be used for other diagnostic tests, such as mutation and SNPs.Gold nanoparticle is well known for its higher quenching efficiency than any other organic acceptor molecules in fluorescence resonance energy transfer(FRET). In this thesis, we introduce gold nanoparticle as fluorescence quencher to construct a new kind of duplex nucleic acid nanoprobe and demonstrate its possible application in DNA detection.The proposed duplex nucleic acid nanoprobe is composed of two complementary oligonucleotides of different lengths. The longer capture strand is labeled with a gold nanoparticle at the 5'-end and designed to hybridize with the target DNA, whereas the shorter signal strand is labeled with a fluorophore (FAM) at the 3'-end and designed to be released off. Thus, in the absence of target DNA, the duplex nucleic acid nanoprobe is non-fluorescent due to the close proximity between the gold nanoparticle and the fluorophore. In the presence of a target DNA, the signal strand can be displaced which lead to the release of fluorescence signal. Meanwhile, the gold nanoparticle making the excess signal strand be easily separated by centrifugation. Experimental results indicate that the fluorescence of the signal strand was almost totally quenched by the probe-bearing gold nanoparticle. This simple strategy provided a new way for fast DNA detection.Nucleic acid nanoprobe is generally prepared by linking thiol modified oligonucleotide to the surface of gold nanoparticle through Au-S bound. However, the problem of this method is that the density of DNA on the surface of gold nanoparticle will affect the hybridization efficiency. Both higher and lower density of DNA on the surface of gold nanoparticle will depress the hybridization reaction. The ligand-exchange strategy was used to regulate the self-assembly of Oligonucleotide on gold nanoparticles with 4-mercaptobenzoic acid (MBA) and 6-mercapto-1-hexanol (MCH) as the regulating molecule. The results show that the naked gold nanoparticles modified with MCH lead to irreversible aggregation, while the gold nanoparticles modified with DNA and MBA are stable in solution, implying that the negative charge of DNA and MBA molecules is critical in stabilizing gold nanoparticles. Both MBA and MCH can replace DNA on the surface of gold nanoparticles. Otherwise, the difference is that MCH can thoroughly replace DNA on the surface of gold nanoparticles but MBA could not. At the molar ratio of MCH/gold nanoparticles=2400/1, the aggregation of gold nanoparticles is observed. It was also found that the ligand-exchange reaction of using MBA as in-coming ligands and DNA as out-coming ligands is much more difficult than using DNA as in-coming ligands and MBA as out-coming ligands. The principle of ligand-exchange on gold nanoparticles provide a new way for preparing nucleic acid nanoprobes.In the last part of this thesis, the synthesis of innovative nanoparticle, gold-polyaniline nano-composite (PAn@AuNPs) used for bio-label was studied. The effection of molar ratio between aniline and hydrogen tetrachloroaurate as well as reaction temperature on PAn@AuNPs were investigated. It was found that the optimal molar ratio of aniline/hydrogen tetrachloroaurate is 3.0:1.0, and the proposed temperature is 100℃. This new kind of nano-composite can be used as bio-label reagent which is useful in biomedicine.