Construction Of Luminescent Nanoprobes For Peroxynitrite

Reactive oxygen species (ROS) widely exist in the nature and organisms, which are "stealthy" biological oxidants. Accordingly, they have been perceived as chief culprits in a growing list of diseases and organism aging. Monitoring of ROS contributes to a better understanding of their biological roles in the pathophysiological process. Although peroxynitrite has a longer life than free radicals among the ROS, its life is only about 10 ms in biological systems. In addition, there are complex reaction between and among ROS. Therfore, it remains challenging to selectively detect one individual ROS in vivo. Especially, organic fluorescent probes have been commonly designed for the detection of individual ROS, which are based on the changes of their optical behavior upon oxidation. It is hard to differentiate between analytes with similar oxidbillity. Compared with the fluorescence analysis method, the most significant advantage of chemiluminescence (CL) analysis is the low level of background signal by virtue of the absence of excitation light source. Based on the CL behavior of peroxynitrite, new types of nanoprobes for peroxynitrite, including montmorillonite (MMT)nanosheets, semiconductor quantum dots and carbon dots, have been developed in this paper. Sensitive and selective determination of peroxynitrite in living cells has been achieved by static injection CL analysis. In addition, ultra-stable fluorescent gold quantum dots have been designed and synthesized, which is expected to solve the problem that most of the luminescent nanoprobes are easy to be oxidized by ROS,leading to the quenching of luminescence and a decrease in sensitivity of detection. This strategy may offer valuable guidance to develop luminescent nanoprobes for the specific detection of ROS. The main contributions of this paper are as follows:(1) MMT nanosheet has been found as a novel catalyst for the ultweak CL of peroxynitrte. Natural MMT nanoparticles were exfoliated into MMT nanosheets in water. The esearch results showed that MMT nanosheets were able to amplify the ultra-weak CL signal from the peroxynitrite system. The morphology of MMT nano sheets was characterized by transmission electron microscope (TEM) and atomic force microscope (AFM). ROS scavengers, electron spin resonance (ESR)spectra, and CL spectra were used to confirm the intermediates and emitters in the chemiluminescence system. A possible chemiluminescence mechanism was proposed. The origin of CL enhancement was ascribed to the catalytic effects of MMT nanosheets on the decomposition of hydrogen peroxide via iron species in MMT nanosheets, resulting in the formation of more abundant hydroxyl radicals ('OH). Such 'OH radicals could immediately react with peroxynitrite to generate singlet oxygen(1O2), which could emit light when it returned to its ground state.(2) Based on the direct CL behaviors of semiconductor quantum dots induced by ROS, a CL nanoprobe for selective determination of peroxynitrite has been developed. Peroxynitrite can decompose into oxidizing ·OH radical and reducing superoxide anion (O2·-) radical. In principle, ·OH radical is capable of injecting a hole into the quantum dots,and O2·- radical is able to inject an electron into the quantum dots. The interaction between quantum dots and the radical pair could produce the CL emission by electron-transfer annihilation. The proposed nanoprobe exhibited good selectivity towards peroxynitrite among ROS. The CL intensity was found to be linear with the concentration of peroxynitrite in the range from 0.46 to 46 ?M, and the detection limit (S/N = 3) was 0.1?M. The proposed nanoprobe has been successfully applied to detect exogenous peroxynitrite in living cells with an ideal result.(3) A highly sensitive peroxynitride CL nanoprobe has been developed on the base of the surface emission mechanism of carbon dots.Tunable surface emission of carbon dots was achieved by altering the ratio of citric acid: urea during the facile microwave process. The surface states of carbon dots were characterized by Fourier transform infrared spectrometry (FT-IR) and X-ray photoelectron spectroscopy (XPS). It was found that the surface-state emission of carbon dots was attributed to the surface C-O group. The CL response of carbon dots towards peroxynitrite increased with an increase in C-O content. The response mechanism was investigated by evaluating energy levels of carbon dots using cyclic voltammetry. The specific nanoprobe can monitor peroxynitrite in a linear range from 0.01 to 3.0 ?M with a detection limit of 5.0 nM (S/N = 3). The proposed nanoprobe showed good biocompatibility and low cytotoxicity. It has been successfully applied to monitor exogenous and endogenous release of peroxynitrite in living cells with simple procedure, good selectivity and high sensitivity.(4) Ultra-stable luminescent gold quantum dots have been synthesized by tuning the structure of the surface ligands. They can act as promising fluorescent labels especially for ROS-containing cells and long-term imaging. Here we provided such a strategy by incorporating the red-emitting dihydrolipoic acid (DHLA)-capped gold quantum dots into the blue-emitting bovine serum albumin (BSA)-capped gold quantum dots via carbodiimide-activated coupling, leading to the formation of gold quantum dot pair inside the cross-linked BSA molecule. On one hand,fluorescence resonance energy transfer (FRET) occurs between the two kind of gold quantum dots inside the cross-linked BSA, leading to an obvious enhancement in fluorescence intensity of the synthesized gold quantum dots. On the other hand, covalent linking between BSA and DHLA decreased the helix structures of BSA on the surface of gold quantum dots. A polymer-like shielding layer was formed around the gold core, effectively emproving the oxidative and enzymatic resistance of the gold quantum dots. This strategy could be used to control the stability of the gold quantum dots towards ROS, and construct stable and specific luminescent nanoprobes for the determination of individual ROS.