Study On The Raman Biosensor Based On The Mesoporous Titaniumdioxide

Surface-enhanced Raman spectroscopy（SERS） has been widely applied in the detection of small bioactive molecules, nucleic acid, proteins, bacteria and cells. In order to detect small amounts of biomolecules, additional signal enhancement is necessary for the SERS method. In this thesis, a series of novel biosensing assay systems were developed for sensitive SERS detection based on mesoporous titaniumdioxide SERS probe, aptamer-target recognition, enzyme assisted cycle amplification strategy and self-assembly of DNA-gold nanoaggregates. The detailed content was described as follows:1. A novel Raman probe was prepared based on mesoporous titaniumdioxide tags. Transmission electron microscope（TEM）, X-ray Diffractometer（XRD）, Raman Spectrum and N₂ adsorption-desorption were used to characterize. Through the amide bond（amino and carboxyl reaction）, mesoporous titaniumdioxide and Au nanoparticles were connected. DNA modified by thiol could be connected on Au nanoparticles through Au-S bond. In the presence of target DNA, target DNA could hybridize with DNA that modified magnetic beads（MB） and Raman marker respectively, and then, MBs-target DNA-Raman marker sanwich structure was formed.After magnetic separation, the Raman markers that uncombined were removed. The Raman probes labeled with mesoporous titaniumdioxide tags were used in a“sandwich-type”strategy to detect the target DNA. The detection limit of 0.77× 10-15mol/L could be achieved. This detection method exhibited impressive simplicity,convenience, and rapid without multiple separation and washing steps.2. In this paper, a padlock probe-based exponential rolling circle amplification（P-ERCA） assay was developed for highly specific and sensitive detection of DNA and Ramos cells. The padlock probe was composed of a hybridization sequence to DNA and a nicking site for nicking endonuclease. Using the target DNA as a template,specific ligation to the padlock probe and linear rolling circle reaction（LRCA） were achieved under isothermal conditions. After multiple nicking reactions, many copiesof short DNA products were successively produced and then were used as triggers in next circle amplification. Thus, a small amount of target DNA were converted to a large number of triggers to initiate the rolling circle amplification reaction, and Raman signal was significantly amplfied, with the Raman probe based the mesoporous titaniumdioxide, the detection limit of 3.6×10-18 mol/L might be achieved.The DNA and Ramos cell analysis were performed with this method. The result indicated this highly sensitive P-ERCA strategy would become a promising DNA and Ramos cell quantification method in early clinical diagnostics.3. An ultrasensitive detection assay method was developed based on gold nanoparticle（AuNP） hybrid probes and surface enhanced Raman scattering method.In this work, a signal amplification strategy for the detection of thrombin, target DNA and Ramos cells were designed by combining the hybridization-inducing aggregate of DNA-functionalized gold nanoparticles（AuNPs） and mesoporous titaniumdioxide（MTiO2）. The multiprobes containing hybridization DNA and aptamers were anchored onto the gold nanoparticles. After a sandwich-type reaction, two kinds of DNA-modified AuNPs/MTiO2 were simultaneously formed the AuNPs/ MTiO2 aggregate caused by in situ hybridization of DNA. The assay was prepared through the immobilization of capture aptamer that specifically recognizes thrombin, target DNA and Ramos cells on magnetic beads（MB）, the sandwich structure would be formed when the target molecules were added. Compared to the signal-labeled tag,the tracing aggregate tags showed a strong signal enhancement. By using the hybridization-inducing aggregate as Raman marker, the sensor showed low detection limit, the limit of detection of thrombin was 4.78×10-16 mol/L, target DNA was4.01×10-15 mol/L and Ramos cell was 5, respectly. This assay represents a novel technique holded great promise for the broad applications in the field of medical research.