Investigation Of Novel Biosensing Technology Based On Surface Plasmon Resonance

Biosensors serve as a method for direct or indirect detection of biomolecules and correlative parameters of physiology and biochemistry process due to their specifically recognizing function and perfect selectivity. They have also been integrated with modern physical and chemical methods to produce a kind of sensitive, fast and accurate analytical methods. Therefore, biosensors have widely been applied in biomedicine, environment of detection, food and drug. This thesis focuses on the development and preparation novel biosensors based on surface plasmon resonance (SPR) and DNAzymes. In detail, the thesis includes the following parts: Part I: Investigation of biosensors base on surface plasmon resonance 1.Colloidal Au-enhanced surface plasmon resonance immunosensing Traditional SPR sensing technology has some advantages, such as real-time monitoring, free label and no purification of samples, so it has already been applied in life science widely. However its sensitivity is limited by instrument itself and the application of this technology is limited. Here, colloidal Au was incorporated into SPR biosensors, resulting in larger change of SPR signal of protein-protein interactions when a gold film-immobilized antigen and colloidal Au antibody comprise the binding pair. This enhanced change is a combined result of greatly increased surface mass, high dielectric constant of colloidal Au, and electromagnetic coupling between colloidal Au and the Au film. Compared to unlabelled antibody, the signal is 20-fold higher. 2. Detection of antibody using colloidal enhanced surface plasmon resonance Colloidal Au is biocompatibility, stable and has unique optical properties. So it has been used as a marker in molecular biology for many years. In this part, we proposed a new optical method to detect macrobiomolecules on the surface of an optically transparent substrate. This method relied on the enhanced surface plasmon resonance signal between protein coated colloidal Au and monolayer colloidal Au. Various concentration of goat-anti-huamn IgG were detected and the results showed that the size of probe and target molecules did not affect its sensitivity. This simple low-cost method has the potential to be constructed as array-chips for high-throughput screening of biomolecules. Part II: Lead ions fluorescence sensor based on DNAzymes With the development of macromolecular chemistry, a series lead ions chemical were designed by using organic ionophores such as crown ethers, calixarene, peptide. These sensors can provide rapid and real-time detection of lead ions. But the synthesis and purification of these organic ionophores are usually time consuming. In addition, the sensitivity and selectivity are not always satisfactory. In vitro selection of DNAzymes from a library of 1014-1015 random DNA sequences offers considerable opportunity for obtaining good metals ions sensors. The catalytic activity of DNAzymes is as good as enzymes of proteins and RNA. DNAzymes are composed of bases of DNA, so the synthesis simple and low cost. Here we report a special DNAzyme, in which two strands were linked together, i.e. quencher modified enzyme strand and fluorophore modified substrate strand. Similar to molecular beacon, the fluorescence of fluorophore was quenched by the intramolecular quencher, since the substrate strand was hybridized to the enzyme strand. In the presence of Pb2+, the substrate strand was cleaved under the catalysis of enzyme strand, and the fluorophore modified fragment was released, which resulted in the increase of the fluorescence intensity. Compared to the unlinked DNAzyme and substrate-strand this method showed similar high specificity detection range (10nM-40μM) and response rate, however, it is more convenient because preparation of enzyme-substrate complex is not necessary and the fluorescence increase is 8-times faster.