The Design And Application Of Fluorescent Nanoprobes Based On Functional Nucleic Acid And Luminescent Nanomaterials

With rapid development of fluorescence technology, fluorescent probes, possess such advantages as simple design, fast response and capable of in situ detection, have made great progress in a wide variety of fields, including food safety, environmental monitoring, disease diagnosis, etc. Utilizing fluorescent probes to monitor intracellular biological molecules, is of great significance to early diagnosis and timely treatment of diseases. Although great improvement and wide applications have been made in fluorescent probe technology, several disadvantages of conventional fluorescent probes still restricted their application at the cellular level, including photobleaching, delivery inefficiency, autofluorescence interference in cells and tissues and shallow penetration depth. In order to meet the increasing requirements for biomedical research and clinical diagnosis, the development excellent fluorescent probes are still challenging.In recent years, the emergence of novel nanomaterials and fluorescent technique provides new insight to solve the above-mentioned problem. The discovery of functional nucleic acids（FNAs） has provided a new direction to improve sensitivity and specificity of fluorescent probes. FNAs possess the advantages of easy synthesis and modification, high specificity, affinities and biocompatibility. Functional nucleic acids mainly include two categories of nucleic acid molecules with specific molecular recognition function, one is DNAzyme, or deoxyribozyme, which resembles protein enzyme, performing catalytic reactions in the presence of specific cofactors. The other one is aptamer, which can bind target molecules with high affinities like antibodies. On the other hand, the luminescent nanomaterials, including silica nanoparticles, quantum dots, upconversion nanoparticles, have many virtues such as good luminescent performance, strong cell-penetrating ability, biocompatibility, easy modification and stability. Incorporation of these luminescent nanomaterials in fluorescence probes could reduce the cost of labelling. Besides, luminescent nanomaterials also can deliver dyes and drugs into target cells, increasing in vivo circulation time of their cargo, making them great candidates in biological applications. Based on the low excitation energy of two photon imaging, two-photon microscopy imaging（TPM） can realize deep-tissue imaging, prolonged imaging time, minimized fluorescence background, less light scattering, better three-dimensional spatial localization and less tissue injury, which is favorable for complex biological systems.In this thesis, we combined functional nucleic acids with luminescent nanomaterials, and took advantage of two-photon fluorescence imaging to build a series of fluorescent nanoprobes for quantitative detection of ions and small molecules that plays vital roles in life activities. Details are as follows:（1） In the second chapter,we develop a kind of simple, fast, highly selective probe for detection ATP and NAD, by making use of the characteristic that T4 DNA ligase has high dependence on ATP and E.Coli DNA ligase has high dependence on NAD, and employing streptavidin- biotin complex as fluorescence anisotropy quality amplifier. This fluorescent probe could effectively distinguish ATP and its analogues（AMP and ADP）, NAD and its analogs（NADH）, the limit of detection are 50 n M and10 n M, respectively.（2） In the third chapter, we develop a DNAzyme-based label-free fluorescent probe for the detection of DNAzyme cofactors such as Pb²⁺ and L-histidine. The introduction of target cofactor could trigger enhanced fluorescence of Ag NCs, providing a “turn-on” fluorescence response to target biomolecule. By employing the multiple enzymatic turnover of DNAzyme for cycle amplifying, the proposed sensing system shows high sensitivity to specific cofactor. In addition, the design is simple, easy operation, and inexpensive. Since numerous DNAzymes have been selected to bind a wide range of targets, the label-free catalytic and molecular beacon provides a general platform for sensitive detection of various targets and could find wide applications in the environmental and biomedical fields.（3） Based on the advantages of two-photon fluorescence imaging, in Chapter 4, we developed a ratiometric two-photon fluorescent nanoprobe of Au NCs decorated silica nanoparticles to monitor HOCl levels in cells. In this nanosystem, Au NCs with their fluorescence quenched in response to HOCl were employed as recognition units and fluorescence reporters, while two-photon silica nanoparticles were employed as an internal reference, providing intrinsic built-in correction to the environment interference and thus improving sensitivity and accuracy. Compared with previously reported fluorescent probes for HOCl detection, this nanoprobe combines the advantages of TP fluorescent sensors and ratiometric sensors, making it suitable for fluorescence imaging of HOCl at the cellular level.（4） To further develop the two-photon fluorescent probe, in the fifth chapter, we combined the two-photon nanoparticles（TPNPs） with CoOOH nanosheets to developed a two-photon fluorescence nanoprobe（TPNPs@CoOOH） for monitoring ascorbic acid（AA） in living systems. The nanosystem exhibits high sensitivity towards AA, with a LOD of 170 n M. It also shows high selectivity toward AA over potential interfering species. The nanoprobe possessed the advantages of TP imaging, and has been successfully applied in TP-excited imaging of AA in living cells and tissues.（5） Taking advantage of passive targeting effect of human serum albumin on the surface of cancer cells, in Chapter 6, we developed a multifunctional nanoparticle for the delivery of anti-cancer drug and genes to multi-drug resistant cancer cells. In this chapter, three kinds of m RNAs related to multi-drug resistance were selected as the target genes, the delivery of corresponding antisense oligonucleotides by nanogel inhibit the expression of m RNAs, successfully overcomes drug resistance and increase drug’s intracellular concentration. Through cell proliferation toxicity studies, it can be confirmed that this nanodrug carrier system can effectively inhibit the proliferation of multi-drug resistant cancer cells MCF-7 / ADR, and realize effective synergistic therapy.