The Research And Application Of DNA-templated Click Chemistry In Biosensor

Copper(I)-catalyzed alkyne-zide Huisgen cycloaddition, the best example of "Click Chemistry", was used as a powerful linking reaction in biomacromolecule functionalization because of its high yielding capability under mild conditions with little by-product. In addition, this click reaction was intended for the development of copper ion sensors owing to its great specificity to the catalysis of copper(I). However, these methods were based on conventional organic reaction resulting in long reaction time and relatively high detection limit.DNA-templated organic synthesis (DTS) is an innovative technology capable of significantly increasing effective molarity of reactants and reaction specificity as well as remarkably accelerating reaction rate through DNA-encoded smallmolecule and hybridization-mediated proximity. The introduction of DNA into organic reaction allows non-natural synthetic events to be recognized, programmed, selected and amplified by DNA-based technology. Because of these features, DTS has become a promising and potent method in the construction of small molecular library, bioactive molecule screening, and homogenous peptide synthesis. However, the application of DTS in the bio-analytical field is still in its infant stage. Although there are a few pioneer works, the intrinsic advantages and powerful function of DTS warrant further efforts to develop its biosensing applications.Based on the above considerations and relevant reports in the literature previously, we intigrated the click reaction with the DTS reaction, and thus developed a series of new chemo/biosensors by taking advantage of DNA-templated click chemistry. In addition, this click cycloaddition was intended for the detection of biomacromolecules free raddicals owing to its powerful linking ability.(1) A novel fluorescent strategy has been developed for sensitive turn-on detection of Cu2+based on the high efficiency of DNA-templated organic synthesis, great specificity of alkyne-azide click reaction to the catalysis of copper ions, the sequential strand displacement for signal transduction and high sensitive SYBR Green I dye as the signal output. In comparison with the previously reported Cu2+assays using conventional organic click reaction, this DTS-based strategy has multifaceted advantages including higher sensitivity and lower detection limit (290nM). Moreover, this strategy can be readily expanded to other sensing applications by facilely changing the DNA-functionalized reactants capable for covalent bond formation. Therefore, taking this method as an example, we demonstrate that the rational design of DTS reaction is a promising platform for developing biosensors.(2) In order to improve the shortcomings of above-mentioned method, such as the heterogenous detection as well as the requirement of labeling biotin and megnatic separation, a novel fluorescent biosensing strategy has been developed for sensitive turn-on detection of copper ions based on the DNA-templated click chemistry induced generation of intramolecular G-quadruplex structure. Compared with the above DTS-based biosensors requiring separation, this approach provides a straightforward and homogenous fluorogenic strategy to achieve "mix-and-read" detection. Crystal violet (CV) is chosen as a signal reporter because its fluorescence can be enhanced greatly via binding to G-quadruplexe. Inspired by CV’s fluorogenic property susceptible to G-quadruplex structure switch, we exploited CV to identify the integration of split-G quadruplex by DTS-caused chemical DNA ligation. This sensor shows high sensitivity and excellent selectivity because of the high yielding capability of DTS and great specificity of click chemistry. Since glutathione is abundant in living cells, this method has potential for detection of biological CuT. Therefore, this method as a representative demonstrates the great promise and potency of DTS-mediated G-quadruplex formation in biosensors development.(3) Simple and on-site application are the future development trends of analysis and detection methods, and the colorimetric assay appears to fulfill the necessary criteria. Herein, we developed a novel colorimetric copper(Ⅱ) biosensor based on the high specificity of alkyne-azide click reaction and unmodified gold nanoparticles (AuNPs) as the signal reporter. The clickable DNA probe consists of two parts:an azide group-modified double-stranded DNA (dsDNA) hybrid with an elongated tail and a short alkyne-modified single-stranded DNA (ssDNA). Because of low melting temperature of the short ssDNA, these two parts are separated in the absence of Cu. Copper ion-induced azide-alkyne click ligation caused a structural change of probe from the separated form to entire dsDNA form.This structural change of probe can be monitored by the unmodified AuNPs via mediating their aggregation with a red-to-blue colorimetric read-out because of the differential ability of ssDNA and dsDNA to protect AuNPs against salt-induced aggregation. Under the optimum conditions, this biosensor can sensitively and specifically detect Cu2+with a low detection limit of250nM and a linear range of0.5-10μM. The method is simple and economic without dual-labeling DNA and AuNPs modification. It is also highly selective for Cu2+in the presence of high concentrations of other environmentally relevant metalions because of the great specificity of the copper-caused alkyne-azide click reaction, which potentially meets the requirement of the detection in real samples.(4) The click reaction was introduced to the detection of biomacromolecules free raddicals owing to its powerful linking ability in biomacromolecule functionalization. We proposed a new concept of "Click-Trap". In this study, several kinds of cyclic-nitrone spin traps have been synthesized and characterized by ESI/MS,’H-NMR,13C-NMR and so on. These novel free radical trapping agents have been used to study small molecule free radicals including oxygen and carbon centered radicals. In addition, the alkyne functional spin trap (Click-DMPO) has also been used in situ to capture protein oxidative damged by reactive oxygen species (ROS). In contrast to immuno-spin trapping technology, the present method is simple and does not require complicated operations. Owing to the excellent performance of the click reaction, it is very convenient to further modify and detect the spin adduct of Click-DMPO and protein.On the other hand, DNA is a potent material for self assembly because of its precise base pairing, highly controllable and addressable assembly, ease for pattern fabrication and the labeling of functional cargos. DNA nanostructures formed by self-assembly are highly ordered and possessDNA nanostructures formed by self-assembly, have good biocompatibility. As a new type of bio-nano-materials, DNA nanostructures have shown wide application prospects in analytical chemistry. Therefore, the development of robust biosensors based on DNA nanostructures is also significant and challenging work.(5) We developed a DNA-nanotube-basd mass amplifying probe for sensitive fluorescense anisotropy detection of ATP. A long ssDNA probe consists of a half of targeting aptamer domain against ATP and molecular mass amplifying domain for the self-assembly of DNA nanotube. The other ssDNA probe, bearing a fluorescent molecule, is the other half of ATP aptamer sequences. When ATP binds to the probe, the molecular mass and FA value of the probe/target complex will significantly increase. This method shows high sensitivity (the detection limit is0.5μM) and excellent selectivity. The results indicate that DNA nanostructure mass amplifying strategy can be used to design aptamer probes for rapid, sensitive, and selective detection of small molecules by means of FA in complex biological samples.