| Silver nanoclusters (Ag NCs) are aggregations of several to tens of silver atoms and their size is less than 2 nm, which is close to the electronic Fermi wavelength. When the size of the metal material is comparable to the electronic Fermi wavelength, the migration of the electrons in metal is limited and the obvious quantum size effect is generated, which result in breaking up the continuous density of states into discrete energy levels, somewhat similar as the energy levels of molecules. Thus, the Ag NCs exhibit unique molecule-like optical properties, such as size-dependent light absorption and emission. Ag NCs have been widely applied in field of bioanalysis due to their ultrasmall size, unique photophysical properties and good biocompatibility. Up to now, several analytical techniques based on Ag NCs have been built, which all employ the fluorescence emission properties of Ag NCs, and they respond to the target because the fluorescence of Ag NCs is impacted by the introduction of target. In general, the developed analytical techniques based on Ag NCs do not take full advantage of the unique physicochemical properties of Ag NCs which limit the wide application and long-term development of Ag NCs. Thus, it is necessary to develop new analytical techniques based on Ag NCs and construct novel probes which response to the target with various forms. Ag NCs have attracted extensive interest in the past decade because of their unique optical and electronic properties. However, many properties of Ag NCs, such as molecule-like photophysical properties, which are resulted by discrete energy levels, still have not been studied fully. Those uncovered properties provide the possibility to broaden the application of Ag NCs. According to the mentioned above, this dissertation is designed to explore the new ways for constructing probes with Ag NCs and broaden their applications in bioanalysis. We have successfully constructed several novel fluorescence probes based on Ag NCs with the use of molecule-like properties of photoabsorption and light emission of Ag NCs. The main contents are as follows:1. Novel and label-free hairpin DNA probes based on target-induced in situ generation of luminescent Ag NCs were developed to detect DNA and thrombin. The probe was designed to contain three segments:a segment for formation of Ag NCs, i.e., the nucleation sequence, a target recognition segment and a blocking segment. In absence of the targets, the blocking segment hybridized with the nucleation sequence and thus locked the probe, so that the growth of Ag NCs on the template was prohibited. When target was introduced, the specific combination of the target with the recognition sequence released the Ag NC nucleation sequence, where fluorescent Ag NCs could be generated providing readout signal for the sensing of the target. This design is relatively simple and flexible without any label or pre-modification to the DNA chain. It not only can maintain the binding affinity and specificity of the probe but also preclude possible false-positive responses due to environmental alterations of the fluorophores in target sensing processes and hence increases the robustness of sensing. By embedding different recognition sequences, the probes can be conveniently used for specific and versatile detection of diverse targets.2. To expand the application of unique optical properties of Ag NCs in biosensing, the energy accepting capability of Ag NCs was exploited. The energy transfer from fluorescent energy donors (JOE) to Ag NCs generated on DNA scaffolds was studied by a hybridized DNA duplex model and a novel fluorescence probe was developed based on the energy transfer properties of Ag NCs. By changing the DNA duplex model and the number of hybridized pairs, the separation distance between JOE and Ag NCs was adjusted. Through investigating the functional relationship of energy transfer efficiency and the separation distance between the energy donor and acceptor, the energy transfer mechanism between JOE and Ag NCs can be assigned to Forster resonance energy transfer (FRET). Taking Ag NCs as energy acceptors, a probe for S1 nuclease was developed based on JOE-Ag NCs FRET system. Upon the catalytic cleavage property of S1 nuclease, the ssDNA strand functioning as the scaffold of Ag NCs would be broken into small fragments, which results that the energy acceptors are no longer connected with the donor. Thus, the energy transfer process was blocked and the fluorescence of JOE was recovered. The changes of fluorescence intensity were proportional to the S1 nuclease, which can be used to detect S1 nuclease.3. The sensing performance of the probe with Ag NCs as the acceptors for S1 nuclease was improved. Taking tryptophan (Trp) as energy donor and Ag NCs as energy acceptors, Trp-Ag NCs FRET system was constructed and typical dual emission signals of donor-acceptor pair was achieved, which can be used to develop a ratiometric fluorescence probe. A short peptide chain composed of two Trp residues as the energy donor and a Cys residue as the connection site for conjugation with the Ag NCs nucleation sequence (ssDNA) was designed. After covalently linking the peptide with the ssDNA following an coupling protocol, Ag NCs were then produced in situ on the peptide-ssDNA complex, thereby enabling FRET from Trp to Ag NCs and the quenching and increasing of the fluorescence of Trp and Ag NCs, respectively. The introduction of S1 nuclease broke the connection of energy donor and acceptor, resulting in the fluorescence recovery of donor and the fluorescence decrease of the acceptor. The ratio of donor-to-acceptor emission can be used for S1 nuclease determination.4. The application of the energy transfer properties of Ag NCs was expanded to biological imaging. Ag NCs were used as the effective energy acceptors for upconversion phosphors (UCPs) and a novel nanoprobe based on upconversion fluorescence resonance energy transfer (UC-FRET) from UCPs to Ag NCs was developed for biothiols. The constructed nanoprobe has been successfully used for intracellular detection and tissue imaging. Oleic acid-capped UCPs were synthesized by a solvothermal method. Then the originally synthesized hydrophobic UCPs were modified with PEI by a ligand-exchange procedure to obtain water solubility. Dimercaptosuccinic acid (DMSA)-stabilized Ag NCs were synthesized though a solid-state method. Under the condition of pH neutral, the positively charged PEI-UCPs and the negatively charged DMSA-Ag NCs were assembled by an electrostatic interaction, which brought the energy donor UCPs and energy acceptor Ag NCs in close proximity and trigged the energy transfer from UCPs to Ag NCs. The upconversion fluorescence of UCPs was quenched. When the biothiols were introduced into the system, the reaction of Ag NCs with thiols should alter the light absorption of Ag NCs and lead to inhibition of the energy transfer from UCPs to Ag NCs. The fluorescence of UCPs was therefore recovered. The degree of fluorescence restoration linearly depended on concentration of biothiols. |
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