Study On Novel Functionalized Fluorescent Probes For Biosensing And Celluar Anaysis

Optical probes attract more and more interests because of their superiority of sensitivity and in situ imaging. The principle is generally elaborated as follows:upon the interaction with the target, the optical properties (especially the fluorescence) of the probe is usually changed, which can be measured through proper instrument for the quantitative or qualitative information, and even can be used for visual imaging analysis in living cells or in vivo. Combining with the nanomaterials of diverse structure and property, this project is dedicated to bio-functionalized optical nanoprobes in biosensing and celluar analysis.1. Stepwise chemical reaction strategy for highly sensitive electrochemiluminescent detection of dopamineA stepwise chemical reaction strategy based on the specific recognition of boronic acid to diol, and N-hydroxysuccinimide (NHS) ester to amine group, was designed to construct a "signal on’" electrochemiluminescence (ECL) platform for highly sensitive detection of dopamine. A boronic acid-functionalized pyrene probe was synthesized and was self-assembled on the sidewalls of carbon nanotubes via π-π stacking interactions as capture probes on a glassy carbon electrode. Meanwhile, 3,3’-dithiodipropionic acid di(N-hydroxysuccinimide ester) (DSP)-functionalized CdTe quantum dots (QDs) were designed as signal probes and characterized with transmission electron microscopy and spectroscopic techniques. Upon stepwise chemical reaction of dopamine with boronic acid and then DSP-QDs, the QDs were captured on the electrode as ECL emitters for signal readout, leading to an ultralow background signal. By using O2 as an endogenous coreactant, the "signal on" ECL method was employed to quantify the concentration of dopamine from 50 pM to 10 nM with a detection limit of 26 pM. Moreover, the stepwise chemical reaction-based biosensor showed high specificity against cerebral interference and was successfully applied in the detection of dopamine in cerebrospinal fluid samples. The stepwise chemical reaction strategy should be a new concept for the design of highly selective analytical methods for the detection of small biomolecules.2. Self-assembled DNA hydrogel as switchable material for aptamer-based fluorescent detection of proteinThe methodology based on target-responsive structural switching is powerful in bioanalysis with the controllability and sensitivity. In this paper, an aptamer-functionalized DNA hydrogel was designed as a specifically target-responsive switchable material for protein detection. This pure DNA hydrogel was constructed by using a Y-shaped DNA and an aptamer linker through a DNA self-assembly without synthetic polymer backbone. With use of thrombin as the model analyte, the DNA hydrogel was first applied to visual detection with the entrapped Au nanoparticles (AuNPs) as indicating agent. Furthermore, the positively charged quantum dots (QDs) as the fluorophore were synthesized by using polyethyleneimine (PEI) as wrapper and characterized with spectroscopy, transmission electron micrograph, ζ potential, and dynamic laser scattering techniques. Along with a gel-to-sol transition in the presence of the target, the released negatively charged AuNPs from the hydrogel could approach the positively charged QDs. Due to the electrostatic interaction, fluorescence resonance energy transfer between PEI-QDs and AuNPs therefore occurred and quenched the fluorescence signal for the sensitive detection of thrombin. This assay for the detection of thrombin showed a good linear relationship in a range of 0.075 to 12.5 μM with a detection limit of 67 nM at 3σ, and demonstrated excellent feasibility in complex serum matrixes. The biocompatible DNA hydrogel provides a universal switchable material for signal transduction and significantly demonstrates proof-of-concept for the detection of proteins.3. Persistent luminescence nanoprobe for biosensing and lifetime imaging of cell apoptosis via time-resolved fluorescence resonance energy transferTime-resolved fluorescence technique can reduce the short-lived background luminescence and auto-fluorescence interference from cells and tissues by exerting the delay time between pulsed excitation light and signal acquisition. Here, we prepared PLNP probes to design a universal TR-FRET platform for biosensing, lifetime imaging of cell apoptosis and in situ lifetime quantification of intracellular caspase-3. Three kinds of PLNP-based nanoprobes are assembled by covalently binding dye-labeled peptides or DNA to carboxyl-functionalized PLNPs for the efficient detection of caspase-3, microRNA and protein. The peptides-functionalized nanoprobe is also employed for fluorescence lifetime imaging to monitor cell apoptosis, which shows a dependence of cellular fluorescence lifetime on caspase-3 activity and thus leads to an in situ quantification method. This work provides a proof-of-concept for PLNPs-based TR-FRET bioanalysis and demonstrates its potential in exploring dynamical information of life process.4. Porphyrin photosensitized metal-organic framework for cancer cell apoptosis and caspase responsive theranosticsA multifunctional metal-organic framework (MOF) is designed as an efficient nanoprobe for selective photodynamic therapy against cancer cells and caspase responsive theranostics. The porphyrin derivative (TMPyP) as a photosensitizer is incorporated in the cage of a variant MOF by one-pot synthesis, which significantly increases the singlet oxygen quantum yield of TMPyP by 6.2 times due to the change of its excited level in MOF cage. By covalent co-assembly of H2N-PEG-folate and Cy3-labelled peptide on the MOF surface, the obtained nanoprobe can be selectively internalized into cancer cells to produce singlet oxygen in mitochondria under laser irradiation, which induces cell apoptosis and activates caspase-3 in cytoplasm to specifically cleave the peptide. The released Cy3 produces bright fluorescence for in situ monitoring of therapeutic effectiveness. The integration of theranostic functions in a single nanocarrier holds great promise in precision cancer diagnosis and treatment.5. In situ activating and monitoring the evolution of intracellular caspase familyThe evolution of intracellular caspase family is crucial in cell apoptosis. To evaluate this process, a universal platform of in situ activating and monitoring the evolution of intracellular caspase is designed. Using well-known gold nanostructure as a model of both nanocarrier and matter inducing the cell apoptosis for photothermal therapy, a nanoprobe is prepared by assembly of two kinds of dye-labelled peptides specific to upstream caspase-9 and downstream caspase-3 as the signal switch and folic acid as a targeting moiety. The energy transfer from dyes to the gold nanocarrier at two surface plasmon resonance absorption wavelengths leads to their fluorescence quenching. Upon endocytosis of the nanoprobe to perform the thearapy agnasit cancer cells, the peptides are successively cleaved by intracellular caspase activation with the evolution from upstream to downstream, which lights up the fluorescence of the dyes sequentially, and can be used to quantify both caspase-9 and caspase-3 activities in cancer cells and to monitor their evolution in living mice. The recovered fluorescence could also be used to assesse the therapeutic efficiency. This work provides a novel powerful tool for studying the evolution of intracellular caspase family and elucidating the biological roles of caspases in cancer cell apoptosis.6. Multi-assembled supercatalyst for ultrasensitive detection and cytosensingMetal porphyrins have catalytic properties of peroxidase, however, the catalytic capabilities are often limited by their poor solubility, the bridge μ-oxide dimers (hindered access to catalytic sites) and oxidative self-degradation. In this work, we design a new multi-assembled supercatalyst, which significantly enhance the catalytic performance of Hemin. The multi-assembly method is as follows:gold nanoparticle with amino group (N-AuNP) is firstly synthesized through pamidronic acid disodium as a new reducing agent, and covalently bond with Hemin as Au-Hemin via the EDC reaction. Then, a prepared imidazole derivative named as MPT can be axially coordinated to the metal center though the Fe-N interaction, leading to the formation of Au-Hem-MPT. In the presence of Hg2+, a supercatalyst is finally formed by the Hg2+-induced polymerization of MPT. The designed supercatalyst significantly improves the catalytic performance of Hemin on both the catalytic efficiency and the dynamics. In addition, the catalyst can achieve the ultrasensitive detection of Hg2+ down aM and the imaging of reactive oxygen species in live cells. The multi-assembled highly-efficient catalyst provides a new avenue for the catalytic mechanism research and ultrasensitive detection.