The Study, Based On Functional Nano-materials, Bio-sensors / Reactor

With the development of biomedicine, higher requests are advanced for the science of analytical chemistry. Different kinds of biomolecules and cells should be screened in the microscopic scale due to the request of high sensitivity, selectivity and high throughput. The advent of nanotechnology and nanomaterials has brought about great advances in this effort. Especially, the two powerful approaches of biosensor and microfluidic chip that correlate to various nanomaterials have been generally recognized as essential technologies for diagnosis and life process research.In this thesis, we try to develop novel biosensors and microfluidic chips for the screening of proteins and cells based on the functional nanomaterials.The introduction in chapter 1 provides background information on the development and applications of nanomaterials in analytical chemistry. And then we summarize the recent progress on biosensors and micofluidic chips, including fabrication techniques, analytical applications and microreactors for proteins. Finally, we outline the experimental ideas and the research purposes of this thesis.The concrete research work of this dissertation can be divided into three chapters as follows:In chapter 2, a novel kinase biosensor was developed based on the labeling of nanoparticle to build new method for early disease diagnosis and the exploration of related medicines. Kemptide, the substrate of protein kinase A (PKA), was immobilized on the gold electrode surface. After the PKA-catalyzed reaction in the presence of ATP, the phosphate groups were transferred to the serine residues of Kemptide on the electrode. The surface characteristics were recorded by FT-IR. The peaks at 923.80 cm-1 and 1268.23 cm-1 were attributed to phosphates and peptides respectively. The phosphorylated peptides were labeled by TiO2 nanoparticles based on the specifically bound between TiO2 and phosphates. The modified electrode was immersed into the AgNO3 solution, and then the silver ions were adsorbed on the surface of TiO2 nanoparticle. Under a UV light at 365 nm, the silver ions were photocatalytically reduced to silver nanopaticles by the excited electrons of titanium oxides. The signal of phosphorylated peptides was transformed through a signal transforming medium and enhanced by silver nanoparticles. The deposited silver nanoparticles acting as the detector target could be analyzed by differential plus voltammetry (DPV). The bioactive unit of PKA was detected down to a limit of 0.2 U mL-1. To investigate its quantitative inhibition assay, ellagic acid was employed as a model inhibitor. The inhibition of PKA activity on Kemptide using ellagic acid was monitored and an IC50 was measured to be about 5.5μM. This biosensor is a general approach which can be readily transferable to the other substrates kinases.In chapter 3, an on-chip microreactor was proposed toward the acceleration of protein digestion through the construction of a nanozeolite-assembled network. The nanozeolite microstructure was assembled using a layer-by-layer technique based on poly(diallyldimethylammonium chloride)(PDDA) and zeolite nanocrystals. The assembly of PDDA/Nanozeolites to the microchannel and its side face were monitored by Scanning Electron Micrograph (SEM). The activity of the encapsulated enzyme in microchip with different layers of (PDDA/Nanozeolite)N, (N=1,2,3,4) was quantitatively testified by the values of the maximum velocity (Vmax), which is derived from a linearized form of the Michaelis-Menten equation. The maximum proteolytic rate of the adsorbed trypsin was measured to be 350 mM min-1μg-1, much faster than that in solution. The adsorption of trypsin in the (PDDA/Nanozeolite)3 was theoretically studied using a quartz crystal microbalance and theΓmax was derived from Langmuir adsorption equation (Γmax=1.4×10-6 mol m-2). This result clearly shows that the nanozeolite-assembled network provides a large surface-to-volume ratio favoring the high enzyme loading. Standard proteins (BSA and Cyt-c) were incubated through the miniaturized microchip reactor and the time-consuming sample digestion procedure was significantly shortened. The detection limit can reach low femtomole amounts,0.5 ng/μL (16 fmol) cyt-c is successfully identified per analysis and 11 peptides with the sequence coverage of 47% were still obtained.The on-chip approach was further demonstrated in the identification of complex extracts from mouse macrophages integrated with two-dimensional liquid chromatography-electrospray ionization-tandem mass spectrometry. And a total of 584 unique peptides were identified, which corresponds to 191 distinct proteins. This research exhibits a simple protocol for microreactor in enzyme immobilization and efficient protein digestion.With the development of different kinds of microfluidic technologies, the research objects of microchip are not only molecules. Microchip has played an important role in cell studies because of its advantages including easy operation, the potential for integration with other miniaturized devices and the potential for a close mimic system in vivo conditions. In chapter 4, a capillary was modified by assembling using a layer-by-layer technique based on poly(diallyldimethylammonium chloride)(PDDA)/PSS/poly(L-lysine)(PLL). The cells were immobilized on the surface of the capillary with the static adsorption on the surface between PLL and cells. The cells were successfully immobilized in the microchannel and can retain at least for 48 hours. The proposal of cell immobilization is simple and can be further used for general cell researches.Finally, we summarized and pointed out the shortcomings in this dissertation. And then, we proposed the objects and schemes of further research.