Study On Nanomaterials-Based Biosensing And The Application For Calmodulin And Cell Surface Glycan Analysis

Biosensor is an advanced device based on interdiscipline, which comprises a biological sensing layer with a physicochemical transducer and signal amplifier. On the basis of molecular level, this monitoring assay is a rapid and microanalysis method, which is one of the most widely applied assays in analytical research. Nowadays, biosensors not only can be used in the laboratorial studies, but also play an important role in clinical diagnosis, environmental monitoring, drug development, food safety inspection and other fields. This advancement mostly attributed to the involvement of nanomaterials, which is now opening new horizons for biomacromolecule analysis and has brought huge momentum to the biosensor development. Compared with traditional biosensors, nanomaterials based novel biosensors shows their own attractive advantages:much higher sensitivity, inherent miniaturization, the ability of in vivo monitoring and real-time determination. With the development of nanotechnology and molecular biological technique, nanomaterials based novel biosensors have developed rapidly and have been successfully applied to various biomacromolecule analysis as a highly selective and sensitive device. Therefore, the application of nanomaterials in biosensor development is of great promising and will surely continue to expand the realm.In this dissertation, we laid our emphasis on the development of biosensors based on the fabrication of multi-functional nanocomposite combining with fluorescent analysis and electrochemical technology. We have applied these novel biosensors to the determination of calmodulin and cancer cell surface glycan. The dissertation includes six chapters:Chapter1. OverviewIn this chapter, detailed outlines and reviews related with the development of biosensor were given, which introduced the concepts, fundamental principles and applications of the biosensors. Biosensors are classified into several types, and the advantages of each type have been exploited. Afterwards, the preparation and characterization of nanomaterials in the biosensor development were highlighted. In addition, electrochemistry was outlined with regard to concepts, mechanism, and application in biosensors. We also briefly reviewed the current development of sensitive detection methods of two types of significant proteins, and introduced neurodegenerative disorders. Finally, we emphatically indicated the purpose and significance of the dissertation, innovation spot and content as well.Chapter2. A highly sensitive immunosensor for calmodulin assay based on enhanced biocatalyzed precipitation adopting a dual-layered enzyme strategyCalmodulin (CaM) is a ubiquitous protein in eukaryotic cells, and it plays an important role in cancer progression. In this chapter, a highly sensitive immunosensor adopting a dual-layered enzyme strategy was proposed for electrochemical detection of CaM. This immunosensor was constructed by introducing honeycomb-like mesoporous carbon (HMPC) as a sensor platform to sequentially immobilize antibody (Ab1), CaM and a multi-functionalized label. The label (HRP-PAupc-Ab1) was synthesized by covalently binding Ab1and horseradish peroxidase (HRP) to poly(acrylic acid)-functionalized Au popcorn (PAupc) nanoparticles. A novel dual-layered enzyme strategy was employed by incubating HRP-secondary antibody (HRP-Ab2) onto the label surface and the enhanced biocatalyzed precipitation was therefore induced. This immunosensor exhibited satisfactory analytical performances for CaM detection with a linear response ranging from5.0pg mL-1to100ng mL-1and a detection limit of1.5pg mL-1. The immunosensor has also been successfully applied to the CaM analysis in two cancer cells (HepG2and MCF-7) with high sensitivity, which has shown great potency for cancer study.Chapter3. Amplified Electrochemical Immunosensor for Calmodulin Detection Based on Gold-Silver-Graphene Hybrid Nanomaterials and Enhanced Gold Nanorods LabelsIn this chapter, a new electrochemical immunosensing protocol for sensitive detection of CaM was developed by using gold-silver-graphene (AuAgGP) hybrid nanomaterials as protein immobilization matrices and gold nanorods (GNRs) as enhanced electrochemical labels. Electrode was first modified with thionine-chitosan film to provide an immobilization support for gold-silver-graphene hybrid nanomaterials. The hybrid materials formed an effective matrix for binding of CaM with high density and improved the electrochemical responses as well. Gold nanorods were prepared for the fabrication of enhanced labels (HRP-Ab2-GNRs), which provided a large capacity for HRP-Ab2immobilization and a facile pathway for electron transfer. With two-step immunoassay format, the HRP-Ab2-GNRs labels were introduced onto the electrode surface, and produced electrochemical responses by catalytic reaction of HRP toward enzyme substrate of hydrogen peroxide (H2O2) in the presence of thionine. The proposed immunosensor showed an excellent analytical performance for the detection of CaM ranging from50pg mL-1to200ng mL-1with a detection limit of18pg mL-1. The immunosensor has also been successfully applied to the CaM analysis in two cancer cells (HepG2and MCF-7) with high sensitivity, which has shown great potency for improving clinic diagnosis and treatment for cancer study.Chapter4. Dendrimer modified carbon nanotube and its application in electrochemical detection of CaMAn electrochemical immunosensor was fabricated based on the dendrimer modified carbon nanotube (PCNT), and applied to the CaM analysis. The immunosensor was fabricated by introducing the poly(acrylic acid)-functionalized Au nanorods (PAuNRs) as a sensing platform to successively incubated antibody (Ab1), CaM and the multi-functionalized probe (HRP-PCNT-Ab1). HRP-PCNT-Ab1was synthesized by covalent binding HRP and Ab1to the PCNT, which could be linked to the HRP-Ab2and therefore enhance the enzyme attachment. The enhanced enzyme attachment increases the detection sensitivity. By applying the immunosensor to the CaM detection, the results show a good analytical performance:a linear response ranging from0.51ng mL-1to382.5ng mL-1, the detection limit was estimated as0.1ng mL-1. The synthesized PCNT presented an effective matrix for the immobilization of biomolecules with high stability and bioactivity, and the developed assay method offered a new approach for CaM assay, which is of great benefit to understand the relationship between CaM and cancer cell growth.Chapter5. A highly sensitive immunosensor for the electrochemical analysis of calmodulin coupled with a global mesoporous carbon based multifunctional biolabelIn this chapter, a highly sensitive immunosensor using a multi-functionalized biolabel (HRP-GMC-Ab1) based on the polyelectrolyte layer by layer assembling of global mesoporous carbon (GMC). This immunosensor was constructed by introducing reduced graphene (rGO)/Au popcorn nanoparticles (Aupcs) as a sensor platform to immobilize CaM. HRP-GMC-Ab1could specially recognize the immobilized CaM and the carried HRP could generate the catalyze current as the analytical data for CaM analysis. The HRP-PAu-Ab2was then caught on the surface of the biolabel, which has further increase the sensitivity. This immunosensor has been applied to the detection of CaM and received a satisfactory result:the linear response ranging from5.0pg mL-1to115ng mL-1with a detection limit of1.5pg mL-1. The proposed polyelectrolyte layer by layer assembling provides a general method for the modification of various materials, and the immunosensor based on this assembling method is a promising assay for the analysis of many other significant proteins.Chapter6. Fluorescence assay for glycan expression on living cancer cells based on competitive strategy coupled with dual-functionalized nanobiocompositesCell surface glycans are a class of sophisticated biomolecules being related to cancer development and progression, and their analysis is of great significance for early cancer diagnosis and treatment. In this chapter, we proposed a fluorescence assay to evaluate the glycan expression on living cancer cells based on competitive strategy coupled with dual-functionalized nanobiocomposites. The competitive assay was conducted between living cancer cells and thiomannosyl derivatives using concanavalin A (Con A)-modified electrode as the interaction platform. To impart the fluorescence signaling ability to the competitive derivatives, quantum dots (QDs) were anchored on the BSA-protected Au nanoparticles, and then thiomannosyl derivatives were immobilized on the nanoparticle surface through Au-S binding. Due to the spacing between QDs and Au nanoparticles by BSA, the {QDs-Au-BSA-mannose} nanobiocomposites maintained the fluorescence of QDs and meanwhile showed the binding ability with Con A-modified electrode. Au nanorods (AuNRs)-modified electrode was used as an effective substrate to immobilize Con A. This assay was successfully applied to quantify cancer cells and evaluate the average amount of mannose on signal cell surface. The method is simple and promising in studying and elucidating the glycan expression on living cancer cells.