Study And Application Of Fluorescent Biochemical Sensing Based On Copper Nanoparticles And Graphene Materials

Due to its high sensitivity, good selectivity, convenient and fast characteristics, fluorescence analysis method has a wide range of applications in biochemical sensing and analysis. Conventional fluorescence analysis method mainly based on organic dye, which has high fluorescence quantum yield, small molecular weight, easy synthesis and modification. However, it also exists some defects, such as poor photo-bleaching resistance and short fluorescence lifetimes, which restrict its application in the detection of complex system and long-term tracking and imaging of cells and in vivo. In recent years, with the development of nanotechnology, a series of new fluor escent nanomaterials with special physical and chemical properties have emerged, such as dye-doped nanoparticles, semiconductor quantum dots, upconversion fluorescent nanomaterials, fluorescent metal nanoclusters（or nanoparticles）, fluorescent carbon nanomaterials. Fluorescent nanomaterials not only overcome d the defects of traditional organic dyes, but also have a series of excellent physical and chemical properties, such as large surface-area, easy modification, controllable size and adjustable fluorescence, etc.. The emergence of different fluorescent nanomaterials has promoted the development of fluorescence sensing. And in order to meet the needs of sensing in actual samples and biological complex samples, the fluorescent nanomaterials should have properties of excellent fluorescence, good biocompatibility and water solubility, and so on. Fluorescent metal nanoclusters（or nanoparticles） and fluorescent carbon nanomaterials are not only satisfied with the development trend of fluorescent nanomaterials, but also have the advantages of easy preparation and low cost. In this thesis, we mainly focus on improving the fluorescence stability of fluorescent copper nanoparticles and exerting the superiority of graphene materials. And a series of new fluorescent s ensors for nucleic acid（micro RNA）, enzyme, small molecule and heavy metal ions have been developed. The detailed description is listed as followed:1. Concatemeric ds DNA-templated copper nanoparticles strategy based on rolling circle amplification and its application in micro RNA detectionds DNA-templated fluorescent copper nanoparticles（ds DNA-Cu NPs） have emerged as promising fluorescent probes for biochemical assays, but the reported monomeric Cu NPs remain problematic because of weak fluorescence and poor stability. To solve this problem, a novel concatemeric ds DNA-templated Cu NPs strategy was proposed by introducing the rolling circle amplification（RCA） technique into Cu NPs synthesis. In this strategy, a short oligonucleotide primer could trigger RCA and be further converted to a long concatemeric ds DNA scaffold through hybridization. After the addition of copper ions and ascorbate, concatemeric ds DNA-Cu NPs could effectively form and emit intense fluorescence in the range of 500-650 nm under a 340 nm excitation. In comparison with monomeric ds DNA-Cu NPs, the sensitivity of concatemeric ds DNA-Cu NPs was greatly improved with ～10 000 folds amplification. And their fluorescence signal was detected to reserve ～60% at 2.5 h after formation, revealing ～2 times enhanced stability. On the basis of these advantages, micro RNA let-7d was selected as the model target to testify this strategy as a versatile assay platform. By directly using let-7d as the primer in RCA, the simple, low-cost, and selective micro RNA detection was successfully achieved with a good linearity between 10 and 400 p M and a detection limit of 10 p M. The concatemeric ds DNA-Cu NPs strategy might be widely adapted to various analytes that can directly or indirectly induce RC A.2. “Super-long” poly（thymine）-templated copper nanoparticles strategy based on terminal deoxynucleotidyl transferase extention and its application in cyclic amplified detection of micro RNACompared with ds DNA-Cu NPs, poly T ss DNA-templated fluorescent copper nanoparticles（poly T-Cu NPs） have more convenient in the probe design. However, for the poly T-Cu NPs, the polymerization of long poly T in the strategy of RCA is limited. To slove this defect, a “super-long” poly T template Cu NPs was developed by using terminal deoxynucleotidyl transferase（Td T）. Td T is a template independent DNA polymerase that catalyzes the addition of deoxynucleotides to the 3’ hydroxyl terminus of DNA molecules, and the sequence is dependent on the d NTPs. Herein, we take advantage of Td T’s characteristic to tail a “super-long” poly T（>>500 mer）, and the poly T was used as template for formation of poly T-Cu NPs. This strategy can be directly used for DNA detection, which is a simple and sensitive and also have no limit on sequence and length of DNA. However, due to the restriction of primer to TDT, this strategy cannot be used for RNA detection. Based on the Td T-“super long” poly T strategy, a cycle amplification strategy for micro RNA detection by combination with duplex-specific nuclease（DSN） was developed. In this strategy, DSN cleaves the DNA in DNA/RNA duliexes and the RNA could be released and be cycled. It is a more effective and convenient platform for nuclear acid detection to the concatemeric Cu NPs strategy.3. Masking agent-free and channel-switch-mode simultaneous sensing of Fe³⁺ and Hg²⁺ using dual-excitation graphene quantum dotsSimultaneous fluorescent sensing of different heavy metal ions is significant for cost saving, operation simplification and efficiency improvement, but the existing probes are still problematic due to complex design or synthesis as well as unavoidable use of masking agents. Here, for the first time, a novel channel-switch-mode strategy for dual-ion simultaneous sensing has been developed based on graphene quantum dots（GQDs） with dual-excitation and single-emission fluorescence. The GQDs were synthesized through a simple citric acid pyrolysis method, which could emit intense blue fluorescence（⁴70 nm） under either 310 nm excitation（Channel-310） or 420 nm excitation（Channel-420）. These two different excitation channels were found to be specifically sensitive to Fe³⁺ and Hg²⁺ respectively; that is, the fluorescence in Channel-310 could be effectively quenched by Fe³⁺, while the fluorescence in Channel-420 could be exclusively quenched by Hg²⁺. Utilizing the dual-channel fluorescence response performance, Fe³⁺ and Hg²⁺ both were detected showing the linear relationship range of 10-200 μM and the detection limit of 10 μM, respectively. And thereupon, just by simply shifting th e excitation wavelength, this strategy realized a facile, low-cost, masking agent-free, quantitative and selective assay of Fe³⁺ and Hg²⁺ even in mixed ion samples and practical water samples. The unique channel switch mode might provide a novel design pat tern for multi-target bioanalysis.4. Cu²⁺/ascorbic acid reaction mediated fluorescence quenching of graphene quantum dots and its application in biochemical sensingIn the last chapter, we developed a channel-switch-mode simultaneous sensing of Fe³⁺ and Hg²⁺ by using graphene quantum dots. However, the poor detection sensitivity limits it in practical application. In order to develop a more sensitive detection for metal ion, a Fenton-like reaction was introduced in detection, which copper ion could generate highly reactive hydroxyl radical（·OH） in the presence of reducing ascorbate. And ·OH can damage virtually graphene quantum dot and cause fluorescence quenching, which could be used for sensitive detection of Cu²⁺. Furthermore, it also could be used for some biomaterial assay, such as ascorbic acid（Vitamin C）, dopamine and uric acid. And used ascorbic acid as model, with the optimization of the related conditions, the detection of ascorbic acid was carried out, and the method was also applied to detect the content of ascorbic acid in lake water, fruit juice and tablets.5. A facile graphene oxide-based DNA polymerase assayThe DNA polymerase assay is fundamental for related molecular biology investigation and drug screening, however, the commonly used radioactive method is laborious and restricted. Herein, we report a novel, simple and cost-effective fluorometric DNA polymerase detection method by utilizing graphene oxide（GO） as a signal switch. In this strategy, in the absence of DNA polymerase, the fluorophore-labeled template ss DNA could be strongly adsorbed and almost entirely quenched by GO. However, as DNA polymerase exists, the polymerized ds DNA product might lead to a much lower quenching efficiency after addition of GO due to the much weaker interaction of ds DNA with GO than ss DNA, thus resulting in a much higher fluorescence signal detected. As proof of concept, the quantitative DNA polymerase activity assay was performed using the Klenow fragment exo-（KF-） as a model. It was confirmed that, after optimization of detection conditions, KF- activity could be sensitively detected through facile fluorescence measurements, with a detection limit of 0.05 U/m L and a good linear correlation between 0.05 –2.5 U/m L（R2 = 0.9928）. In addition, this GO-based method was further inspected to evaluate the inhibitive behaviors of several drugs toward KF- activity, the result of which firmly demonstrated its potential application in polymerization-targeted drug screening.