Coupling Fluorescent Silver Nanoclusters And DNA Amplification Platforms For The Detection Of Biomolecules

Fluorescent Ag nanoclusters (<2nm) are emerging as a new nanomaterial with potential applications in fluorescence imaging/sensing, data storage devices and biological labeling. Several scaffolds have been demonstrated to aid the synthesis of fluorescent Ag nanoclusters such as dendrimers, polymers, small molecular ligands, DNA, proteins and peptides, among which the DNA-hosted Ag nanoclusters are receiving considerable attention. DNA-hosted Ag nanoclusters have several outstanding spectral and photophysical properties. First, the fluorescence of these Ag nanoclusters can be tuned from the visible to the near-IR as the fluorescence is largely dependent on the specific sequences and structures of the DNA templates. Secondly, the high quantum yield and the photostability make Ag nanoclusters good candidates as fluorescent labels for biological applications, where the targets include metal ions, small molecules, proteins, DNA/RNA.While great advancements in DNA-hosted Ag nanoclusters-based methods have been made, the use of fluorescent Ag nanoclusters for biological or chemical sensing is still in its infancy, as the sensitivities of these methods are relatively low and unsatisfying compared with that of other sensors based on the well-developed fluorophores such as organic dyes and quantum dots, so we need to combine with DNA-amplifying strategies to improve the detection sensitivities of these methods. Description of research in the thesis is presented as follows:Chapter1:We first summarized recent FL-based sensing applications of NPs. The types of NPs described in this section included quantum dots (QD), metal NPs, rare earth doped NPs (up-converting NPs), dye-doped silica NPs, carbon NPs, and polymeric NPs. Sencondly, we review several recent examples of fluorescent silver nanoclusters based bio-sensing systems. Thirdly, we briefly summarized recent nucleic amplification strategies such as polymerase chain reaction, rolling circle amplification, strand-displacement polymerization reaction and hybridization chain reaction used in analytical measurements, lastly, we introduced two new kinds of fluorescent molecules which could insert into nucleic acids and their applications.Chapter2:Silver (Ag) nanoclusters have received considerable interests due to their unique properties and potential applications in numerous fields, and herein we report a simple miRNA-detection system which combines the amplification capability of hybridization chain reaction (HCR) with highly fluorescent DNA-hosted Ag nanoclusters. A multifunctional hairpin DNA structure with a poly-C loop integrates with in situ synthesis of an Ag nanoclusters for a sensitive fluorescent diagnostic of the let-7miRNA family. The linear range for the target DNA is12.5nM-500nM and the lower limit is12.5nM. The linear range for the let-7a is2.5nM-80nM and the lower limit is2.5nM. This label-free, enzyme-free and nanoparticle-free method allows a simple "mix and measure" assay to discriminate one-base differences in the let-7miRNA family. Owing to its good sensitivity and specificity as well as easy operation without the use of enzyme and tedious labeling, it is expected that the proposed technique would find promising applications in practical detection of single nucleotide polymorphism and provide a tool for early diagnosis and risk assessment of malignancy.Chapter3:We develop a new NAD+detection strategy based on the ligation-triggered hairpin DNA probe formation and fluorescent Ag nanoclusters, where the hairpin DNA (with a poly-C loop) structure serves as a template for the formation of Ag nanoclusters to obtain a high fluorescence signal and then detect NAD+without any labeling or modification. The DNA probe was designed as a single-strand molecule that formed dumbbell structure with self-complementary structure at both ends and a CCCCCC sequence was designed as the loop close to5’-end. The5’-end was modified with a phosphate group and the3’-end was exposed. When the system only contained E. coli DNA ligase, Klenow fragment DNA polymerase and dNTPs, the extension reaction can be performed from the3’-end, and instantly opened the hybridized structure at the5’-end and make the6C structure be locked. Thus, the6C loop of DNA probe is completely hybridized with the6G sticky end of MB2, and no fluorescent Ag nanoclusters are formed. However, in the presence of E. coli DNA ligase, together with the cofactor NAD+, the ends of DNA probe can be ligated to block the extension reaction from the3’-end and ensure the quadruplex-forming sequence reserved. In this state, highly fluorescent Ag nanoclusters can be synthesized after the addition of AgNO3and reduction with NaBH4. Compared to traditional NAD+assays, this unique strategy offers a highly specific and sensitivity to NAD+, which makes it a great potential for biomedical applications with feasible purification. Furthermore, this new strategy allows a label-free and simple "mix and measure" assay of NAD+without any expensive equipment or reagents.Chapter4:A fluorescence turn-on detection of Hg2+based on inhibition of RCA by Hg2+is developed. The original RCA products can cross-link with each other in the presence of Hg2+ion as the Hg2+ions invade T-T base pair to form a stable T-Hg2+-T complex, which inhibits the processability of phi29DNA polymerase and ends the subsequent process of RCA. A new fluorescent probe, i.e. perylene tetracarboxylic acid diimide (PTCDI) derivative which can quench the fluorescence of itself when changing from free monomeric to aggregated forms after interacting with DNA, is used to reverse the inhibition to generate a turn-on signal. The system allows ultrasensitive, selective and label-free determination of Hg2+, achieving improved assay characteristics ((e.g., wide linear response range (5orders of magnitude), low assay limit (0.37nM)). In addition, this molecular architecture can also be used to produce switching and logic gates which are operated using Hg2+and GSH as the inputs and DNA amplification as the outputs. This is first example of switching and logic operations based on the inhibition (or reverse inhibition) of RCA trigged by Hg2+ions (or GSH).