The Application Of Electrochemiluminescence Of Quantum Dots And Near-Infrared Quantum Dots In Biosensing

In recent years, the synthesis and functional modification of inorganic fluorescent nanomaterial- Quantum Dots(QDs) has become a hot research area of analytical chemistry which is based on the specific chemical identification. As the chemical and biotical sensing are appealing the sustaining attentions in the clinical diagnosis, drug development and environmental protection, the combination between new analytical techniques and QDs with excellent optical properties has also made a great progress. Among them, the electrochemiluminescence(ECL) of QDs possessing high sensitivity, convenient operation and controllable features has provided many strategies and methods for the design of QDs based sensors. Through the reasonable design of nanomaterial modified electrode, researchers proposed several coreactants of QDs ECL, or found some small molecules which could affect the ECL intensity of QDs, the application area of ECL has been constantly expanded. On the other hand, by introducing the enzyme amplification reaction, the QDs ECL sensors with lower background signal and high target signal have been constructed. Near-Infrared(NIR) light has served as the “diagnostic windows” in bioimaging and biosensing due to its deep tissue penetration, weak scattering and background fluorescence. Thus, the synthesis of novel NIR QDs, the design of bioimaging in vitro and in vivo and the development of sensing strategy of NIR fluorescence detection have always been worth exploring. The incorporation of QDs and novel chemical/biotical sensing techniques could improve the recognition ability to target, decrease limit of detection, which is beneficial to the application in living analysis and disease diagnosis.However, in the existing researches on QDs ECL, there are only limited species of detection targets, most of which are the coreactant related small molecules, enzymes and other metal ion. Although the synthesis technology of NIR QDs in organic phase is relatively mature, the synthesis in aqueous phase is still in the stage of development, and the detection method utilizing NIR QDs is also rare. To address these issues, the most important of this thesis is focused in researching the relationship between the deoxyribonucleotide and the ECL of QDs. As the deoxyribonucleotide is the main component of DNA, a series of DNA related targets could be detected based on this phenomenon. In addition, a counterpart fluorescent nanomaterial was introduced to the NIR QDs, and a NIR based ratiometric sensor could be designed, which then was used to the detection of a model target. The main research content is as follows:(1) Versatile electrochemiluminescent biosensor for protein-nucleic acid interaction based on the unique quenching effect of d GMP on electrochemiluminescence of Cd Te/Zn S Quantum Dots. The efficient quenching effect of deoxyguanosine-5’-phosphate(d GMP) on anodic electrochemiluminescence(ECL) of the Cd Te/Zn S QDs is reported for the first time. This ECL quenching was found to be specific for free d GMP and not observed for d GMP residues in different DNA structures. The unique d GMP-based QDs ECL quenching was then utilized to develop a versatile biosensing strategy to determine various protein-DNA interactions with the assistance of exonuclease, Exo I, to hydrolyze DNA and liberate d GMP. Taking single-stranded DNA binding protein(SSB) and thrombin as examples, two novel detection modes have been developed based on d GMP-QDs ECL strategy. The first method used hairpin probes and SSB-promoted probe cleavage by Exo I for facile signal-off detection of SSB, with a wide linear range of 1-200 n M and a low detection limit of 0.1 n M. The second method exploited aptamer-thrombin binding to protect probes against Exo I degradation for sensitive signal-on detection of thrombin, giving a linear response over a range of 1-150 n M and a detection limit as low as 0.1 n M. Both methods were homogenous and label-free without QDs or DNA modification. Therefore, this d GMP-specific QD ECL quenching presents a promising detection mechanism suitable for probing various protein-nucleic acid interactions.(2) A co-template synthesis of near-infrared dual-emission Quantum Dots-gold nanoclusters nanohybrid for target-responsive detection of ascorbic acid. We proposed a protein-directed co-template strategy to synthesize NIR based dual-emission fluorescent nanohybrid(DEFN) constructed from far-red gold nanoclusters and NIR Pb S QDs(Au NCs-Pb S-QDs). The dual-emission signals of Au NCs-Pb S-QDs exhibit two well-resolved emission peaks separated by 173 nm which can eliminate environmental interferences by the built-in correction of ratiometric signal, resulting in a more favorable system for bioimaging and biosensing. Additionally, the convenient protein-directed co-template synthesis avoids the tedious chemical coupling and modification required in conventional preparation approaches of DEFNs. Moreover, the target-responsive capability of this NIR-based DEFN to ascorbic acid(AA) was discovered, enabling the proposed DEFN to ratiometrically detect AA with high sensitivity and selectivity, rapid response, and excellent photostability.(3) Ascorbic acid is rich in the fresh fruits and vegetables, especially in oranges, tomatoes, kiwi fruit and so on. Thus, the content of ascorbic acid in fruit is an important part of the detection of fruit internal quality. In view of this point, we exploit Au NCs-Pb S-QDs to acquire the fruit internal quality assay, and fluorescence spectrum and fluorescence imaging are used to compare the content of AA in different kinds of fruits. Experiment results revealed that the AA content calculated by the method was along with reported results. Ascorbic acid is the necessary nutrient in human body and could not be provided by itself, AA can promote the synthesis of collagen protein and polysaccharide, and increase the density of capillaries, the lacking of AA can cause the hematopoietic function obstacle, muscles bleeding and even death, these symptoms in clinic is often referred to as scurvy. In these years, some references have reported that AA has a positive effect on the treatment of cancer. We have carried out the in vitro cell imaging and in vivo imaging by Au NCs-Pb S-QDs. In the experiment of in vitro imaging, the proportion of the fluorescence imaging of two channels has the different results with the addition of AA. And through the in vivo imaging, we have demonstrated the unique advantages of NIR light in the process of deep tissue imaging.(4) Hydrogen peroxide(H2O2) plays a very important role in the human life. As a kind of high efficiency, stable oxidant and crosslinking agent, it has been widely used in electronic, environmental, chemical, pharmaceutical and military industry and other fields. At the same time, H2O2 is the main product of glucose oxidation. Detection of glucose in the process of food analysis and diagnosis of metabolites is very important. We have built a new kind of water-soluble cationic conjugated polymers(CCP)- NIR fluorescence resonance energy transfer between quantum dot systems, with CCP as a donor, and NIR QDs as a receptor. The addition of H2O2 would have an etching effect on the QDs, both the fluorescence intensity at 710 nm and the absorbance from 400 nm to 500 nm decreased, thus the fluorescence of CCP was recovered. This sensor of H2O2 has a linear range of 0.2 m M-4 m M, the detection limit was 0.1 m M. As the H2O2 is the main product of glucose catalyzed by glucose oxidase(GOD), we next realized the detection of glucose with high selectivity and high sensitivity from 0.1 to 5 m M, and the limit of detection was 0.05 m M.