Construction, Application, And Comparative Study Of Nanoprobes For Biodetection Based On Three Types Of Sensing Strategies

Different types of sensing strategies have achieved rapid development and shown great potential in recent years.The single-molecule sensing strategy based on specific recognition has the advantages of better specificity,strong anti-interference ability,good selectivity.Although the strategies have found widespread applications in cellular imaging and medical diagnosis,a limitation of this technology is the need to use a specific probe for each analyte,which restricts multiplex analytes detection.A variety of non-specific sensing units with cross-sensitivity were utilized to construct a sensor array,which realized the simultaneous recognition of multiple components in a complex system.However,unlike specific molecule-based probes,the lack of physical integration of the different molecular components prevent them from operating in confined microscopic environments.To make it more powerful and widely used,a single-molecule probe with pattern identification ability was constructed by integrating a variety of recognition units on the same molecular skeleton.It integrated multiple signal transduction mechanisms and multiple non-specific recognition capabilities.However,it was difficult to integrate and ensure that output channels do not interfere.The emergence of nanoprobe provided a new way to improve the sensitivity and accuracy of the probe.Because of its large specific surface area,good optical performance,strong cell penetration,good biocompatibility,simple preparation,easy modification and functionalization,nanoprobes were widely used in biochemical detection.Herein,we prepared several different nanoprobes with different sensing strategies and investigated their applications in biological detection,biological imaging,and disease diagnosis.Furthermore,the detection principle,application score,and application scenarios of these sensing strategies were discussed in detail and compared their advantages and disadvantages.It is expected to provide reference and guidance for the design of later experiments,the selection of detection objects,and the determination of research fields.The details are as follows:（1）The research on single-molecule sensing strategies based on specific recognition.Two optical nanoprobes based on specific recognition strategies were prepared and further applied to detect specific biomolecules in cells.1)In chapter 1,inspired by a large number of hypochlorous acid molecular probes containing selenium,HClO fluorescent probe without any modification was prepared.Compared with molecular probes,this probe displayed improved performance in HClO detection,such as the low limit of detection（1.1 n M）,high selectivity,fast response,good aqueous solubility,friendly operation,excellent photo-stability,and p H-independent response.Furthermore,the probes were successfully used for the detection of exogenous and endogenous HClO in living cells.Meanwhile,this probe was applied to discriminate normal cells and cancerous cells on the basal of their different HClO levels.We anticipate that the as-prepared nanoprobe should have broad applications in chemical analysis and revealing the physiological function of HClO in a biological system.2)In chapter 3,in order to endow specific nanoprobes with versatility,a chiral nanoprobe with optical reversible activity was prepared and successfully used to indicate the dynamic changes of intracellular redox state.The nanoprobe could respond specifically to cellular redox states with excellent anti-interference capability and high stability.Furthermore,it has been used to indicate the trend of redox state over time in drug-induced glutathione and ROS imbalance cell models.Besides excellent indicating ability,the as-prepared chiral probes displayed good glutathione peroxidase-like activity to regulate intracellular redox state by catalytically reducing toxic H₂O₂ in vitro and in vivo.（2）The research on array sensing strategy based on combinatorial part recognition.Although the specific recognition probes mentioned above are good at detecting a kind of analyte in cells with high sensitivity and high selectivity,in practice,multiple target analytes often coexist and simultaneously control a recognition event.In chapter 4,a fluorescent array consisted of saccharide-functionalized quantum dots and 4-mercaptophenyl boronic acid-functionalized MoS₂ nanosheets were constructed and used for multiple identification and quantitation of lectins and bacteria.Compared to the specific lectin probe,the fluorescent displayed better performance in lectin detection,such as multiple lectins detection and lower detection.The fluorescent array has further been evidenced to be potent for distinguishing and quantifying different bacterial species by recognizing their surface lectins.The detection limits of Escherichia coli and Enterococcus faecium are 87 CFU/m L^-1 and 66 CFU/m L^-1,respectively.（3）The research on single-molecule pattern recognition strategy.As mentioned above,the fluorescent arrays consisted of different fluorescent probes that could simultaneously recognize multiple molecules.However,each probe unit still needs to detect repeatedly on parallel samples many times,which is time-consuming and consumption.More importantly,the fluorescence array does not allow in situ monitoring of many intracellular analytes.We designed and constructed a nanoprobe based on a single DNA tetrahedron,which for the first time realized the recognition of multiple micro RNAs in cells,in vivo,and even in serum samples.1)In chapter 5,a pattern-identification nanoprobe was constructed by integrating multiple fluorophores on the same DNA tetrahedron.This probe displayed good aqueous solubility and nuclease resistance.Five let-7 micro RNAs with highly similar sequences were identified effectively,and the recognition accuracy of both the training set and the unknown samples was 100%.Moreover,the nanoprobe has further been evidence to be potent for quantifying different let-7 families,the detection limits of let-7a,let-7b,let-7c,let-7e,and let-7f are 0.059,0.104,0.156,0.074,and 0.067 n M,respectively.2)In chapter 6,the DNA tetrahedron-based pattern-recognition nanoprobe was further applied to recognize different kinds of cells.The nanoprobe could enter the cells due to its rigid structure.According to the differences of let-7 family types and expression levels among different cells,six cancerous cells（Hela,MDA-MB-231,MCF-7,Hep G2,Hep3B,and Bx PC-3）and a normal hepatocyte（L02）were identified effectively with 100%accuracy for both the training set and the unknown samples.Furthermore,in situ pattern recognition of the nanoprobe has been demonstrated in living cells and mice.The single-structure pattern-recognition nanoprobe can accurately identify subtle differences without the tedious steps of RNA extraction and signal amplification,which has a better prospect in complex biological samples.3)In chapter 7,in light of its accurate and sensitive analysis performance,DNA tetrahedron-based nanoprobe was further used for clinical serum samples analysis.According to the difference in the let-7 family members and the expression levels among different serum samples,seven cancerous samples were identified effectively with 93.9%accuracy.And it has been successfully used for the diagnosis and prognosis of breast cancer.Thus,the emergence of this clinically feasible and economically applicable single-structure pattern recognition nanoprobe provided a possibility for early cancer screening and classification and even clinical monitoring.This thesis provided new design concepts for developing novel nanoprobe based on three types of sensing strategies,including specific recognition,combinatorial array,and single-molecules pattern-recognition strategy.And it provided a guiding ideology for achieving the identification of different analytes under different environmental conditions.