Fluorescence Assay For Biological Macromolecules And Hg²⁺ Based On Fluorescence Signal Amplifying Strategy

In recent years,with the development of Genome Project and Protein Project, in clinical diagnostics, food safety, environmental protection and other social areas, more sensitive, accurate, economic and simple detection methods for biological macromolecules, have been the focus of Life Analytical Chemistry. Signal amplification detection technologies is a cutting-edge analytical techniques which based on various enzymes and nanoparticles, have realized highly sensitive detections of biological macromolecules.In this thesis, we developed a variety of novel fluorescence signal amplifying detection technology platforms which based on nucleic acid enzymes, nanoparticles and fluorescent nucleic acid probes. The developed methods were demonstrated to have very high sensitivity for quantitative analysis of protein and DNA detection. The main researches are presented as following: 1. A label-free fluorescence assay for thrombin based on aptamer exonuclease pro tection and exonuclease III-assisted recycling amplificationIn this chapter, we have developed a simple and sensitive homogeneous fluorescence protein assay by Exo III-assisted recycling amplification-responsive cascade Zn PPIX/GQuadruplex supramolecular fluorescent labels coupled with aptamer exonuclease protection. First, this novel sensing system is simple in design and can be easily carried out by only simple mixing and incubation,thereby avoiding the complex handling procedures. Also, the assay eliminates the washing and separation steps by utilizing exonuclease, which saves time. Secondly, the aptamer probe without any modification and specific label is involved, which decreases the experimental cost and the design complexity, and retains the high target binding activity. Third, this assay provides high sensitivity and selectivity. It permits the detection of as low as 0.2 p M human thrombin. In addition, our strategy can be widely applied for the detection of other types of biomolecules by changing the corresponding aptamer. Thus, the proposed sensing system is important for its application in biological or clinical target detection. 2. Ultrasensitive fluorescence detection of nucleic acids using exonuclease III-induced cascade two-stage isothermal amplification and magnetic nanoparticlesIn this chapter, a cascadic sensing system was developed for detection of DNA target at ultralow concentration by a combination of magnetic nanoparticles(MNPs) and exonu clease III(Exo III)-induced cascade two-stage isothermal amplification. A ingeniously designed capture hairpin probe(CHP) that integrates target-binding and signal transduction sequences within one multifunctional design was assembled on MNPs. Upon sensing of the analyte nucleic acid, the hairpin probe on MNPs could beopened and stepwise removed by Exo III accompanied by the releasing of target DNA for the successive hybridization and cleavage process and the generation of bare signal transduction sequences of CHP as a new trigger for next circular reaction. The new DNA triggers initiate hybridizing with hairpin DNA probe, Then, Exo III digests the resulting duplex domain, leading to the recycling of new DNA trigger and simultaneously generating numerous Zn PPIX/Gquadruplex supramolecular complexes with the help of the zinc(II)-protoporphyrin IX(Zn PPIX). This newly designed protocol provides a high sensitivity with a detection limit of 0.75 f M, and give slow matrix effect due to using MNPs as the separation and amplification elements in the real samples. Therefore, it holds great potential for early diagnosis in gene-related diseases. 3. A DNA nanomachine based on rolling circle amplification-bridged two-stage exonuclease III-assisted recycling strategy for label-free multi-amplified biosensing of nucleic acidIn this chapter, an autonomous DNA nanomachine based on rolling circle amplification(RCA)-bridged two-stage exonuclease III(Exo III)-induced recycling amplifi cation(Exo III-RCA-Exo III) was developed for label-free and highly sensitive homo geneous multi-amplified detection of DNA combined with sensitive fluorescence detection technique. First, the target-binding of UMB in duplex DNA removed step wise by Exo III accompanied by the releasing of target DNA for the successive hybridization and cleavage process and autonomous generation of the primer. Second, the primer initiate RCA process with a rational designed padlock DNA. The RCA products generating remarkable Zn PPIX/G-quadruplex fluorescence signals with the help of zinc(II)-protoporphyrin IX(Zn PPIX). The proposed strategy showed a wide dynamic range over 7 orders of magnitude with a low limit of detection of 0.51 a M. In addition, this designed protocol holds a great potential for early diagnosis in gene-related diseases. 4. Highly sensitive fluorescence assay of DNA methyltransferase activity by methyl ation-sensitive cleavage-based primer generation exponential isothermal amplifica-tionSite-specific identification of DNA methylation and assay of MTase activity are imperative for determining specific cancer types, provide insights into the mechanism of gene repression, and develop novel drugs to treat methylation-related diseases. Herein, we developed a highly sensitive fluorescence assay of DNA methyltransferase by methylationsensitive cleavage-based primer generation exponential isothermal amplification(PGEXPA) coupled with supramolecular fluorescent Zinc(II)-protopor-phyrin IX(Zn PPIX)/Gquadruplex. In the presence of DNA adenine methylation(Dam) MTase, the methylation responsive sequence of hairpin probe is methylated and cleaved by the methylationsensitive restriction endonuclease Dpn I. The cleaved hairpin probe then initiate the exponential isothermal amplification reaction(EXPAR). The proposed method exhibits a wide dynamic range from 0.0002 to 20 U/ml and an extremely low detection limit of 8.6×10-5 U/ml, which is superior to most conventional approaches for the MTase assay. This novel method not only provides a promising platform for monitoring activity and inhibition of DNA MTases, but also shows great potentials in biological process researches, drugs discovery and clinical diagnostics. 5. Target-responsive dumbbell probe-mediated rolling circle amplification strategy for highly sensitive Hg2+detectionIn this chapter, a novel label-free amplified fluorescent sensing scheme based on target-responsive dumbbell probe-mediated rolling circle amplification(D-RCA) has been developed for sensitive and selective detection of mercuric ions. In this strategy, we reported an ingeniously designed dumbbell-shaped DNA probe(DDNA) that integrates target-binding, amplification and signaling within one multifunctional design. Stable T-Hg2+-T can be formed in the presence of target Hg2+, thus it induces the elongation and amplification reaction.resulting in numerous cascade dumbbell probes forming duplex G-rich quadruplex oligomeric structures. Upon addition of N-methyl mesoporphyrin IX(NMM), the signal reporter, a strong interaction between the G-quadruplex and NMM brings about a great fluorescence enhancement. This approach can detect 80 f M of mercuric ions, much lower levels than previously reported biosensors, and exhibits high discrimination ability. More significantly, the dynamic range of D-RCA is extremely large, covering 5 orders of magnitude. We also demonstrate Hg2+ quantification with this highly sensitive and selective D-RCA strategy in real samples.