Metal-Organic Framework MIL-101 Enhanced Fluorescence Anisotropy For DNA Analysis

Fluorescence anisotropy (FA) assay possesses many advantages and plays an important role in biochemical analysis and clinical diagnosis. Yet it is difficult for FA aplied to the reaction with small molecular change before and after recognition, for the induced anisotropy difference is too small to be detected. To solve this problem, protein, gold nanaparticle and graphene oxide et al were used as FA amplification materials. Metal-organic frameworks (MOFs) possess diverse topological structure, large specific surface area, open metal sites, adjustable particle size, morphology and excellent hydrothermal stability and other excellent properties, so they have been widely used in the fields of gas adsorption and separation, chromatographic separation, catalysis, drug delivery and chemical analysis. But, the research on biochemistry of MOFs is very few. However, there are also some reports about MOFs interacted with DNA. Some MOFs present different affinity to ssDNA and dsDNA, based on this principle MOFs can be used for DNA hybridization mediated target assay. In view of this, we employed MIL-101 as FA amplification platform for DNA detection. MIL-101 possesses large mass and volume, which meet the requirements for FA amplification in principle. In addition, we synthesized size-controled MIL-101, which have obvious amplification ability on FA and eliminated the scattering problem of nanomaterial at the same time. The specific researches displayed as follows:(1) Metal-organic framework MIL-101 enhanced fluorescence anisotropy for sensitive detection of DNA. MIL-101 possesses different affinity to single stranded DNA (ssDNA) and double stranded DNA (dsDNA). ssDNA can be absorbed and twined onto the surface of MIL-101 through electrostatic interaction and π-π stacking interaction. After the addition of target DNA, the formed dsDNA which with stable rigidity formation can not conjuncted with MIL-101. Thus different FA values were induced according to the Perrin eqution. We applied this principle to the analysis of HIV-1 DNA (Human immunodeficiency virus-1). Without MIL-101, the molecular mass change of fluorophore on probe DNA before and after hybridization is too small to generate detectable FA value variation. However, with the involvement of MIL-101, P was absorbed and twined onto the surface of MIL-101. As a result, the rotation of fuorophore was restricted manifestly, exhibiting a large FA value (r1). If P hybridized with target DNA(T) first, the formed dsDNA of P/T kept away from MIL-101, resulting in lower FA value (r2). Thus, a larger FA change was observed before and afer the hybridization. The mechanism was further proved by gel electrophoresis. The experimental data show that, the obtained FA value change in the prescence of MIL-101 is 24 times higher than that without MIL-101. And there is a good linear relationship between |△r| and concentration of T in the range of 0.3-12 nM with linear regression equation of |△r|=0.012+0.020 cT (R=0.992) and the detection limit of 0.2 nM (S/N=3). Contrastively, without MIL-101, there is no obvious FA change with the increasing concentration of T under the same conditions. The method has high sensitivity and good selectivity. In principle, this strategy can be used to detect different analytes, such as metal ions, by introducing probes that selectively bind to the analytes. This is the first report about MOFs to enhance FA for analytical assay.(2) Dual amplifying fluorescence anisotropy for detection of Respiratory syncytial virus DNA fragment with size-controled metal-organic framework MIL-101 In order to eliminate the scattered light induced by the signal amplification material in FA assay, nanosized MOF MIL-101, ranging from 500-80nm, has been synthesized through hydrothermal method with the additive of glycerol. We chose the 100 nm MIL-101 to amplify FA for lable-free detection of Respiratory syncytial virus DNA based on the different affinities of ssDNA and dsDNA toward MIL-101 and DNA-intercalating dye SYBR Green I (SGI) as the fluorophore. In the prescence of MIL-101, the Ar is 7-fold higher than that without MIL-101. The nanosized MIL-101 possess negligible scattering effect owning to its smaller particle size, which guarantee FA values were smaller than the maximum initial anisotropy of 0.4. As a specific advantage, a dual amplification phenomenon of not only increased the FA value of SGI/ssDNA (r1) but also decreased the FA value of SGI/dsDNA (r2) was presented. As a result, a larger FA value change △r(△r=r1-r2) was obtained and contribute to improve the sensitivity, which is superior to other amplification material such as graphene oxide. This phenomenon may contribute to the specific structures and metal ions of MOFs. There is a good liner relationship between △r and the concentration of target DNA (T) in the range of 1-20 nM. The quantitative detection result of T was good and the sensitive and selective detection of RSV DNA was achieved.